Mimicking Complex Biological Membranes and Their Programmable

Review pubs.acs.org/CR

Mimicking Complex Biological Membranes and Their Programmable Glycan Ligands with Dendrimersomes and Glycodendrimersomes Samuel E. Sherman, Qi Xiao, and Virgil Percec* Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, United States ABSTRACT: Synthetic vesicles have been assembled and coassembled from phospholipids, their modified versions, and other single amphiphiles into liposomes, and from block copolymers into polymersomes. Their time-consuming synthesis and preparation as stable, monodisperse, and biocompatible liposomes and polymersomes called for the elaboration of new synthetic methodologies. Amphiphilic Janus dendrimers (JDs) and glycodendrimers (JGDs) represent the most recent self-assembling amphiphiles capable of forming monodisperse, stable, and multifunctional unilamellar and multilamellar onion-like vesicles denoted dendrimersomes (DSs) and glycodendrimersomes (GDSs), dendrimercubosomes (DCs), glycodendrimercubosomes (GDCs), and other complex architectures. Amphiphilic JDs consist of hydrophobic dendrons connected to hydrophilic dendrons and can be thought of as monodisperse oligomers of a single amphiphile. They can be functionalized with a variety of molecules such as dyes, and, in the case of JGDs, with carbohydrates. Their iterative modular synthesis provides efficient access to sequence control at the molecular level, resulting in topologies with specific epitope sequence and density. DSs, GDSs, and other architectures from JDs and JGDs serve as powerful tools for mimicking biological membranes and for biomedical applications such as targeted drug and gene delivery and theranostics. This Review covers all aspects of the synthesis of JDs and JGDs and their biological activity and applications after assembly in aqueous media.

CONTENTS 1. Introduction 1.1. Background 1.2. Scope of the Review 1.3. Nomenclature 2. Amphiphilic Janus Dendrimers (JDs) 2.1. Twin−Twin Design 2.1.1. Convergent−Divergent 2.1.2. Convergent−Convergent 2.2. Single−Single Design 2.2.1. Convergent−Divergent 2.2.2. Convergent−Convergent 2.3. Twin−Single Design 2.4. Fluorinated JDs 2.5. Hyperbranched JDs 2.6. Fluorescence and Gadolinium-Labeled JDs 3. Self-Assembly of Amphiphilic JDs 3.1. Methods for the Synthesis of Dendrimersomes (DSs) and Other Complex Architectures 3.2. Nanoscale Visualization of Morphologies Self-Assembled from JDs 3.2.1. Unilamellar DSs 3.2.2. Onion-Like DSs 3.2.3. Dendrimercubosomes (DCs) and Other Complex Architectures 3.3. Predictable Thickness, Diameter, and Number of Bilayers © 2017 American Chemical Society

3.4. Molecular Simulation 3.5. Optical Imaging of Giant DSs 3.6. Mechanical Properties 3.7. Dye Encapsulation and Release 3.8. Drug Encapsulation and Release 3.9. Stability 3.10. Toxicity 3.11. Applications 3.11.1. Hydrogels 3.11.2. Drug Delivery 3.11.3. Nucleic Acid Delivery 3.11.4. Magnetic Resonance Imaging (MRI) and Theranostics 4. Amphiphilic Janus Glycodendrimers (JGDs) 4.1. Glycosylation and Stereochemistry 4.2. Twin−Twin Design 4.3. Single−Single Design 4.4. Twin−Mixed Design 4.5. Sequence-Defined JGDs 5. Self-Assembly of Amphiphilic JGDs 5.1. Method of Preparing Glycodendrimersomes (GDSs) and Other Complex Architectures 5.2. Nanoscale Visualization of Morphologies Self-Assembled from JGDs 5.2.1. Soft Unilamellar GDSs 5.2.2. Onion-Like GDSs

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Received: February 18, 2017 Published: April 18, 2017

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DOI: 10.1021/acs.chemrev.7b00097 Chem. Rev. 2017, 117, 6538−6631

Chemical Reviews 5.2.3. Glycodendrimercubosomes (GDCs) 5.2.4. Solid GDSs and Other Complex Architectures 5.3. Predictable Diameter and Number of Bilayers 5.4. Mechanical Properties, Confocal Microscopy, and Stability 6. Bioactivity of GDSs 6.1. Reactivity of Mannose (Man)-Presenting GDSs with Man-Specific Lectins (ConA) 6.2. Reactivity of Galactose (Gal)-Presenting GDSs with Gal-Specific Lectins (PA-IL, VAA, and GSL 1) 6.3. Reactivity of Lactose (Lac)-Presenting GDSs with Lac-Specific Lectins (Galectins, VAA) 6.3.1. Galectin-1 (Gal-1) 6.3.2. Galectin-3 (Gal-3) and Galectin-4 (Gal-4) 6.3.3. Galectin-7 (Gal-7) 6.3.4. Galectin-8 (Gal-8) 6.3.5. VAA 6.4. Reactivity of Lac-Presenting Sequence-Defined GDSs with Gal-8 6.5. Reactivity of Man-Presenting Sequence-Defined GDSs with ConA 6.6. Reactivity of GDCs with ConA and Banana Lectin (BanLec) 6.7. GDS Stimulation of Killer T Cells 6.8. Future Directions 7. Coassembly of DSs and GDSs with Other Components 7.1. Coassembly with Phospholipids 7.2. Coassembly with Block Copolymers 7.3. Coassembly with Membrane Proteins 7.4. Coassembly with Bacterial Cell Membranes 7.5. Coassembly and Self-Sorting of Hydrogenated, Fluorinated, and Hybrid JDs 8. Conclusion and Outlook Associated Content Special Issue Paper Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

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6596 6598 6598 6600 6601 6601 Figure 1. Biological membranes consist of a mosaic of glycolipids, proteins, and cholesterol embedded in phospholipid bilayers. They may be unilamellar (single layer) (left) or multilamellar (multiple layers) (right).

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Figure 2. Three major classes of synthetic vesicles include (a) stealth liposomes, polymersomes, and, most recently, (b) dendrimersomes, which self-assemble from Janus dendrimers.

development of synthetic analogues has become a major area of research. In particular, the use of synthetic vesicles as models for cell membranes and as vehicles for drug delivery has flourished. Early attempts at developing synthetic vesicles involving the self-assembly of phospholipids alone into liposomes1 resulted in unsatisfactory results because of their instability due to rapid oxidation. Stealth liposomes (Figure 2a) were developed as a means of extending the lifetime of synthetic phospholipid vesicles by the coassembly of phospholipids with phospholipids conjugated with poly(ethylene glycol) (PEG), cholesterol, and antioxidants.2,3 However, these vesicles are generally polydisperse and thus require additional time-consuming fractionation to prepare them with specific dimensions and narrow polydispersity.4 Despite these difficulties, they have been successfully implemented in many applications including as clinically approved drug delivery devices.3 Polymersomes (Figure 2a), consisting of amphiphilic diblock copolymers, were developed as an alternative to phospholipid-based synthetic vesicles.5,6 Although polymersomes have favorable stability and mechanical properties, they are polydisperse, their membrane bilayer is significantly thicker than that of biological membranes, and in addition they are sometimes toxic.7,8

1. INTRODUCTION 1.1. Background

Vesicles are a biologically important class of microscopic structures consisting of fluid-filled sacs delineated by thin membranes. These membranes result from the self-assembly of amphiphilic molecules into bilayers with hydrophobic sections isolated in the interior and hydrophilic sections interacting with the surrounding aqueous media. Assembled from phospholipids, biological vesicles, such as cell membranes (Figure 1) and the envelopes of organelles such as lysosomes, peroxisomes, and endosomes, are complex structures stabilized by cholesterol, proteins, and carbohydrates. They serve as barriers between cells and the outside environment, containers for transport, and vehicles for communication within and between cells, among other essential functions. As such, the 6539


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Figure 3. A variety of synthetic glycan-presenting molecules and membrane mimics have been synthesized including (left to right) glycopolymers, glycodendrimers, glycoliposomes, and most recently glycodendrimersomes.9 Reprinted with permission from ref 9. Copyright 2013 Springer.

(Figure 1). The glycans of glycoconjugates, such as lipids and proteins, serve as recognition sites for sugar-binding proteins (lectins), which play an essential role in cell recognition and adhesion, signaling, routing, differentiation, proliferation, immune response, and growth regulation.38−47 Individual sugar−protein interactions are weak, so complex multivalent glycan displays are necessary for selective recognition of lectins.44 The desire to understand the relationship between glycan topologies and lectin recognition has resulted in the development of various synthetic platforms48 for glycan multivalent display including glycopeptides,49−51 glycopolymers,52−58 glycodendrimers,38,59−61 glycoliposomes,62−65 cyclic clusters such as cyclophanes,66,67 and glycodynamers68,69 (Figure 3). Although glycopolymers, glycodendrimers, and glycodynamers provide some viable mimics for the biological display of glycans, they do not exhibit the vesicular architecture of biological systems, and they can be toxic. Carbohydrate-presenting vesicles such as glycoliposomes are more similar to biological systems, but their preparation by coassembly results in uncontrollable glycan topologies. Thus, it is difficult to control the sequence and density of their sugar displays, hampering their utility as probes for lectin recognition. Furthermore, time-consuming methods are required to prepare these vesicles because they are generally polydisperse, necessitating fractionation.4,70 In response to these difficulties, Percec and co-workers developed several libraries of amphiphilic Janus glycodendrimers (JGDs), which are amphiphilic JDs conjugated to carbohydrates at their hydrophilic dendrons.71 Many of these JGDs self-assemble in aqueous solution by simple injection to form monodisperse vesicles denoted glycodendrimersomes (GDSs). GDSs exhibit controllable sugar sequence and topology, serving as powerful tools to unpack the complexities of lectin recognition. The second half of this Review will focus on the syntheses of these JGDs and their characterization and biological functionality after self-assembly in aqueous solution.

To develop monodisperse, biologically compatible, and easily preparable vesicles, Percec and co-workers pioneered a third class of synthetic vesicles denoted dendrimersomes (DSs) (Figure 2b). They are self-assembled into monodisperse suspensions from bilayered amphiphilic Janus dendrimers (JDs) by simple injection into water or buffer.9,10 JDs consist of two chemically distinct low generation (usually first or second) dendritic (or tree-like) building blocks10,11 that are connected directly or through a core as schematized in Figure 2b, in which the two different dendrons appear blue and green and the core appears red. In the case of amphiphilic JDs, one side is hydrophilic and the other is hydrophobic. The name JD was coined by the Percec laboratory in 2005 when nonamphiphilic fluorinated−hydrogenated and hydrogenated−hydrogenated molecules created from two different hydrogenated dendrons were shown to selforganize in bulk into bilayered supramolecular columns.9−12 Related molecules were reported earlier in 1991 when Frechet and co-workers developed the first multifaced globular dendrimers based on poly(benzyl ether) repeat units.13 Later, the same group reported the synthesis of amphiphilic globular dendrimers called globular amphiphiles.14 In 2003, Goodby and co-workers reported Janus liquid crystals derived from rod-like mesogenic groups also attached in a branched architecture.15−17 Tomalia and co-workers and Wegner and co-workers also reported early examples of JDs but employed different nomenclature.18−20 Amphiphilic dendrimers and JDs are synthesized via convergent and divergent methodologies,11 and have also been prepared by liquid phase organic synthesis.21 A wide range of nonamphiphilic JDs, amphiphilic JDs, and amphiphilic dendrimers have since been synthesized for a multitude of applications including as fluorescent probes,22,23 agents for gene transfection, 24−27 scaffolds for the attachment of therapeutics,28,29 and liquid crystals.18−20,30 Amphiphilic JDs have also been studied for self-assembly at the air−water interface.31,32 Additionally, Janus-type dendronized polymers have been prepared and their structure analyzed.33,34 The Percec laboratory prepared and characterized in bulk state polymers dendronized with semifluorinated JDs.35 This work along with the previously reported synthesis of an amphiphilic JD with pentaerythritol core36 served as inspiration for the discovery of numerous libraries of amphiphilic JDs that self-assemble into DSs and other complex architectures in aqueous solution.37 Syntheses of these molecules, the characterization of their supramolecular structures in aqueous solution, and their application will be the focus of the first half of this Review. Along with developing stable and biologically compatible vesicles, another key area of research is the biological functionalization of synthetic vesicles by their decoration with carbohydrates, a major component of biological cell membranes

1.2. Scope of the Review

The scope of this Review encompasses all literature on the field of self-assembling amphiphilic JDs and JGDs in aqueous media published after Percec and co-workers’ May 2010 Science research article37 and before February 2017. A handful of publications of significant relation to the field dating prior to 2010 are also discussed. During the preparation of this manuscript, a review on amphiphilic JDs was published, which provides a brief introduction to the syntheses and applications of some of these molecules.72 This Review herein aims to provide a much more detailed view of the field. With that in mind, a comprehensive set of 57 libraries has been constructed consisting of every amphiphilic JD that has been synthesized for the purpose of self-assembly in aqueous media. These libraries were prepared 6540


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Scheme 1. Synthesis of Twin−Twin JDs37 by Convergent−Divergent Synthetic Strategiesa

a Reagents and conditions: (i) benzaldehyde, HCl, water, (25 °C); (ii) DCC, DPTS, CH2Cl2 (DCM) (25 °C); (iii) H2, Pd/C, MeOH−DCM (25 °C); (iv) HCl, THF (25 °C); (v) Dowex, DCM−MeOH; (vi) 12, DMAP, py-DCM (25 °C); (vii) 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid, DCC, DPTS, DCM; (viii) 13, DMAP, py-DCM; (ix) allyl bromide, K2CO3, acetone, reflux; (x) KOH, EtOH−water (80 °C); (xi) NMO, K2OsO4 (cat.), tBuOH−water (25 °C); (xii) bromopropionyl bromide, pyridine; DCM; (xiii) 1-thioglycerol, Et3N, MeCN-DCM; (xiv) SOCl2; MeOH (25 °C); (xv) 32, NaHCO3, DMSO−THF, reflux; (xvi) LiAlH4, THF; (xvii) allyl bromide, 50% NaOH, TBABr, DMSO−THF; (xviii) NMO, K2OsO4 (cat.), acetone−water−tBuOH.

specifically for this Review and do not always correspond to previously reported libraries in the literature. The syntheses of

each of the libraries will be discussed, as well as detailed characterizations of their aqueous self-assembly and applications 6541


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Figure 4. Twin−twin JDs from convergent−divergent synthetic strategies.37

been synthesized as presented in libraries 1−43 shown in Figures 4−11. The syntheses of these molecules will be reviewed in detail in this section. The JDs have been divided into groups on the basis of their structures including twin−twin designs (Figures 4 and 5), which exhibit two hydrophobic dendrons connected to two hydrophilic dendrons, twin−single designs (Figure 8), which exhibit two hydrophobic dendrons connected to one hydrophilic dendron, and single−single designs (Figures 6 and 7), which exhibit one hydrophobic dendron connected to one hydrophilic dendron. Each of the JDs in these groups has been further divided into libraries on the basis of synthetic methodology involving a combination of convergent strategies, in which dendrons are constructed from outside to inside, and divergent strategies, in which dendrons are constructed from inside to outside. The convergent−divergent methodology (Figures 4 and 6) involves synthesis of one side of the molecule with a convergent methodology followed by the divergent growth of the other side of the molecule on the first half. On the other hand, the convergent−convergent methodology (Figures 5, 7, and 8) involves coupling both sides of the molecule together in the final step. Thus, although most commonly all dendrons in convergent−convergent methodologies are constructed convergently before coupling, it is also possible for a JD to be included in this category even if one-half or all of its dendrons are constructed divergently but not connected to each other until a final coupling step. Additionally, to highlight their more unusual structures, some JDs have been placed into separate libraries regardless of their structure or synthetic methodology including fluorinated JDs, hyperbranched JDs, and fluorescent and gadolinium-labeled JDs (Figures 9−11). The syntheses of these molecules will be discussed separately as well.

if available in the literature. This is thus the most comprehensive review on the field yet published. 1.3. Nomenclature

Nomenclature of JDs and JGDs is based on that provided in the literature with most following a general naming scheme that lists the hydrophobic dendrons, the core structure if one is present, and the hydrophilic dendrons in that order.11 When names in the literature were not sufficiently specific, new ones were assigned in this Review. The nomenclature for each library is provided underneath the generic structure of the molecules. When reference is made to a specific JD or JGD in this Review, the library number is provided along with any necessary information to identify the specific molecule of interest in that library such as substitution patterns and, when required, the full name. When discussing syntheses, it is important to note that the numbering of molecules in this Review is not unique from one scheme to the next such that molecules must be referred to by their scheme number and their number within the scheme. For instance, compound 3 in Scheme 1 is referred to as compound 1.3 (Scheme 1, compound 3).

2. AMPHIPHILIC JANUS DENDRIMERS (JDs) Amphiphilic JDs are the first class of molecules that will be discussed in this Review. A multitude of diverse structures have

2.1. Twin−Twin Design

2.1.1. Convergent−Divergent. A total of four libraries of twin−twin amphiphilic JDs have been synthesized via convergent−divergent strategies (Figure 4).37 Library 1 consists of amphiphilic JDs with 2,2-bis(hydroxymethyl)propionic acid

Figure 5. Twin−twin JDs from convergent−convergent synthetic strategies.37

Figure 6. Single−single JDs from convergent−divergent synthetic strategies.37,73,74 6542


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Figure 7. Single−single JDs from convergent−convergent synthetic strategies.37,76−78,84−87,89

Synthesis of the Percec-type hydrophobic dendrons was conducted by Williamson ether synthesis of methyl esterprotected gallic acid or its (3,4)- and (3,5)-disubstituted analogues and halogenated alkyl chains. By a convergent strategy, esterification of acid 1.5, which is the hydrophobic dendron, and the hydroxyl groups of 1.4 was carried out via N,N′dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)-pyridinium-p-toluenesulfonate (DPTS) to yield 1.6, the amphiphilic JD with protected polyester dendrons. The final step of the synthesis of the first generation library 1 JD 1.7 was deprotection of the polyester dendrons in 1.6.

(bis-MPA)-based polyester hydrophilic dendrons connected through a pentaerythritol core to hydrophobic dendrons composed of alkyl aryl ethers, which are referred to as Percectype dendrons.11 The generic name for library 1 JDs is (3,4,5)nG1-PE-BMPA-G(m+1)-(OH)y. The synthesis of the library originated with pentaerythritol 1.1 (Scheme 1, compound 1), which was monoprotected by benzylidene, to give diol 1.2 as shown in Scheme 1. The diol was then esterified with acetonideprotected anhydride 1.12 to give 1.3 with protected firstgeneration polyester dendrons. The benzylidene was then selectively removed to give 1.4. 6543


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Scheme 2. Synthesis of Twin−Twin JDs37 by Convergent−Convergent Synthetic Strategiesa

Reagents and conditions: (i)TsCl, NaOH, THF−water; (ii) DHP, p-TsOH, DCM; (iii) 50% NaOH, BnBr (100 °C); (iv) 2 or 5 or 8, K2CO3, DMF (70 °C); (v) KOH, EtOH−water; (vi) DCC, DPTS, DCM (25 °C); (vii) TsOH, MeOH/DCM (25 °C); (viii) H2, Pd/C, MeOH/DCM (25 °C). a

(3,4,5)nG1-PE-G-G1-(OH)12.37 The synthesis of library 2 was carried out starting again with 1.2, benzylidene-protected pentaerythritol. Acid 1.5, the hydrophobic dendron, was esterified with the hydroxyl groups of 1.2 via DCC and DPTS to yield 1.14, which was deprotected to give 1.15. In a convergent step, another esterification was performed on 1.15 and acid 1.18, which is a first generation allyl-functionalized dendron synthesized from 1.16 via a protection-coupling-deprotection method. The resulting JD 1.19 was oxidized with osmium tetroxide to give amphiphilic JD 1.20. As a result of the cost and toxicity of osmium tetroxide, only a few of the possible compounds in library 2 were made.

The second and third generation library 1 JDs (1.9 and 1.11, respectively) were synthesized from 1.7 via a divergent approach. The hydroxyl groups of diol 1.7 were esterified with anhydride 1.12, to give the protected second generation JD, which was subsequently deprotected to form 1.9. A similar approach was taken to make 1.11 from 1.9, except that benzylidene rather than acetonide was employed as the protecting group via esterification with anhydride 13. This change in protecting groups was made to avoid slow reaction rates and significant cleavage of the ester groups during deprotection. Library 2 consists of JDs with glycerol-based hydrophilic dendrons connected through a pentaerythritol core to Percectype hydrophobic dendrons and is generically denoted as 6544


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Scheme 3. Synthesis of Library 737 by Convergent−Divergent Strategya

Reagents and conditions: (i) C12H25Br, 50% NaOH, TBABr (cat.) (80 °C); (ii) 10, DMAP, py-DCM (25 °C); (iii) H2, Pd/C, MeOH−DCM (25 °C). a


The JDs constituting library 3 consist of thioglycerol-based hydrophilic dendrons connected through a pentaerythritol core to Percec-type hydrophobic dendrons and are generically denoted (3,4,5)nG1-PE-TP-G(m+1)-(OH)y.37 This library was synthesized starting with diol 1.15, containing the hydrophobic dendrons. 1.15 was acylated with bromopropionyl bromide in the presence of pyridine to give dibromo 1.21. The first generation library 3 JD 1.22 was achieved after nucleophilic substitution of dibromo 1.21 with thioglycerol. The corresponding second and third generation JDs (1.23 and 1.24, respectively) were synthesized divergently from 1.22 by repetition of the prior two steps. Library 4 replaces the pentaerythritol core of library 2 with 2(dibenzylamino)propane-1,3-diol and is generically denoted (3,4,5)12G1-APD-G-Gm-(OH)y.37 Synthesis began with conversion of amine 1.25 to 1.27, through ammonium salt 1.26 via substitution with the brominated hydrophobic dendron 1.32. Compound 1.27 was reduced to give diol 1.28, which was treated with allyl bromide in the presence of K2CO3 base and tetra-nbutylammonium bromide (TBABr) to yield the first generation amphiphilic JD 1.30 after oxidation with osmium tetroxide. Synthesis of the second generation JD (1.31) was carried out divergently by repetition of the prior two steps, although the yield was low as the result of significant cleavage of the benzyl arms during oxidation. 2.1.2. Convergent−Convergent. Two libraries of twin− twin amphiphilic JDs as shown in Figure 5 have been synthesized via convergent−convergent synthetic strategies.37 These libraries consist of oligo(ethylene oxide) (mEO)-based hydrophilic dendrons connected through a pentaerythritol core to Percectype hydrophobic dendrons. Library 5, which is generically denoted (3,4,5)nG1-PE-(3,4,5)-mEO-G1-(OCH3)y, differs from library 6, which is generically denoted (3,4,5)12G1-PE-(3,4,5)mEO-G1-(OH)y, only in the presence of terminal methyl groups as opposed to hydroxyl groups on the hydrophilic dendrons. The syntheses of these libraries are outlined in Scheme 2. Synthesis of library 5 began with the tosylation of terminally methylated ethylene glycol, diethylene gycol, or triethylene glycol (TEG) as represented by compounds 2.1.37 Resulting compounds 2.2 were then reacted with methyl ester-protected gallic acid 2.9 by Williamson ether synthesis to give the methyl ester-protected hydrophilic dendron 2.10 where “R” is methyl. Hydrolysis followed to provide the corresponding acid 2.11. The syntheses of similar hydrophilic dendrons 2.11b,c that are (3,4)and (3,5)-mEO disubstituted are not shown but were conducted

a

Reagents and conditions: (i) ethylene glycol, DCC, DPTS; (ii) 2,2,5trimethyl-1,3-dioxane-5-carboxylic acid, DCC, DPTS; (iii) THF, 6 M HCl.


a

Reprinted with permission from ref 74. Copyright 2014 Royal Society of Chemistry.

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Scheme 6. Synthesis of Library 1037 by Convergent−Convergent Strategya

a

Reagents and conditions: (i) LiAlH4, THF; (ii) Dess−Martin periodinane, DCM; (iii) pentaerythritol, TsOH, benzene/DMF, reflux; (iv) TsOH, benzene/DMF, reflux.

Scheme 7. Synthesis of Constitutional Isomeric Libraries of L-1,2-Propanediol Ester and Benzyl Ester Containing Amphiphilic JDs (Libraries 11−1476)a

Reagents and conditions: (i) SOCl2, DCM, 2 h (0−60 °C); (ii) (S)-2-(benzyloxy)-1-propanol, DCC, DPTS, DCM, 8 h (23 °C); (iii) Pd/C, H2, MeOH/DCM = 1:2, 4 h (23 °C); (iv) DCC, DPTS, DCM, 8 h (23 °C). Reprinted with permission from ref 76. Copyright 2011 American Chemical Society.

a

similarly with corresponding methyl ester-substituted benzenediols.37 The Percec-type hydrophobic dendrons were synthesized by the same method as was discussed in the previous section and then connected to the pentaerythritol core to give 2.12a−c. Convergent coupling of the hydrophobic dendrons with the hydrophilic dendrons was then performed via orthogonal synthesis, in which all possible hydrophilic dendrons were connected to all possible hydrophobic dendrons as shown, for instance, at the center of Scheme 2. Thus, three different hydrophilic dendrons could combine with three different hydrophobic dendrons to yield a total of nine amphiphilic JDs. In this case, esterification via DCC and DPTS of diols 2.12a−c

and acids 2.11a−c was performed to yield compounds 2.13a−i, which constitute the whole of library 5, in accordance with the orthogonal methodology. Synthesis of library 6 proceeded similarly except that tosylated ethylene glycol, diethylene glycol, and TEG were protected with either tetrahydropyran (THP) or benzyl ether (Bn) (compounds 2.5 and 2.8, respectively).37 These protected hydrophilic chains were then incorporated into the hydrophilic dendrons via Williamson ether synthesis to give the corresponding compounds 2.11 after hydrolysis where “R” is THP or Bn. Orthogonal synthesis by esterification then followed to connect the hydrophilic dendrons to the hydrophobic scaffolding. Finally, 6546


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Scheme 8. Synthesis of L-Alanine and Glycine Amide Containing Amphiphilic JDs (Libraries 17 and 1876) and Hydrophilic and Hydrophobic Dendrons in Libraries 15 and 16a

a Reagents and conditions: (i) C12H25Br, K2CO3, DMF, KI, 8 h (80 °C); (ii) TsO(CH2CH2O)3CH3, K2CO3, DMF, KI, 8 h (80 °C); (iii) KOH, EtOH/H2O, reflux, 1−4 h; (iv) CH3CH(NH2)COOCH3·HCl, CDMT, NMM, THF, 6−8 h (23 °C); (v) KOH, EtOH/H2O, reflux, 1−4 h; (vi) LiAlH4, THF, 4−6 h (0−23 °C); (vii) SOCl2, DCM, 2 h (0−23 °C); (viii) potassium phthalimide, THF/DMF, 4 h (0 °C); (ix) NH2NH2·H2O, EtOH/THF = 2:1, reflux, 8 h; (x) CDMT, NMM, THF, 23 °C, 8 h; (xi) NH2CH2COOCH3·HCl, CDMT, NMM, THF, 8 h (23 °C); (xii) KOH, EtOH:H2O, reflux, 4 h; (xiii) CDMT, NMM, THF, 8 h (23 °C). Reprinted with permission from ref 76. Copyright 2014 American Chemical Society.

Scheme 9. Synthesis of Libraries 15−1877 by Convergent−Convergent Strategya

a Reagents and conditions: (i)RCH(NH2)COOCH3·HCl, CDMT, NMM, THF, 6−8 h (23 °C); (ii and iv) KOH, EtOH:H2O, reflux, 1−4 h; (iii and v) CDMT, NMM, THF, 8 h (23 °C). Reprinted with permission from ref 77. Copyright 2014 National Academy of Sciences USA.

as shown at the bottom of Scheme 2, the THP or Bn groups were removed from the hydrophilic dendrons to give the mEO amphiphilic JDs 2.16. 6547


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a Reagents and conditions: (i) (Boc)2O, NaOH, water (25 °C); (ii) 2, K2CO3, DMF (75 °C); (iii) NaOH, dioxane−MeOH (25 °C); (iv) citric acid; (v) C12H25Br, K2CO3, DMF (75 °C); (vi) HNO3, SiO2, DCM; (vii) NH2NH2, H2O, graphite, reflux; (viii) CDMT, NMM, THF (25 °C); (ix) 2 N HCl, Et2O (25 °C); (x) LiAlH4, THF; (xi) SOCl2, DCM; (xii) NaN3, DMF.

Scheme 11. Synthesis of Libraries 24 and 2578 by Convergent−Convergent Strategya

Reagents and conditions: (i) ICl, 10% H2SO4 (80 °C); (ii) AcOH, NaNO2, H2SO4; (iii) KI, H2O; (iv) NaH (0 °C); (v) Pd(PPh3)2Cl2, CuI, Et3N (80 °C); (vi) H2, Pd/C, ethanol, 24 h (23 °C); (vii) Zn(OAc)2, imidazole, 6 h (140 °C); (viii) triethylamine, THF, methylacryloyl chloride (0 °C). a

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Reagents and conditions: (i) TsOH, acetone, 2 h (23 °C); (ii) DCC, DPTS, DCM, 12 h (0−23 °C); (iii) Dowex, methanol, 4 h (23 °C); (iv) stearic acid, DCC, DPTS, DCM, 12 h (0−23 °C); (v) 3, DCC, DPTS, 12 h (0−23 °C); (vi) CuSO4·5H2O, sodium ascorbate, DCM/DMF/water (8:1:1), 48 h (70 °C). a

Scheme 13. Synthesis of Library 2784 by Convergent− Convergent Strategya

cane. Acylation of the free hydroxyl on 3.2 was then carried out by reaction with benzylidene-protected anhydride 3.10, affording 3.3. Deprotection followed to yield the first generation amphiphilic JD 3.4. The corresponding second and third generation JDs (3.7 and 3.9, respectively) could be prepared from 3.4 via a divergent methodology that amounts to the repetition of the prior two steps. Library 8, reported by Terreno and co-workers73 and generically denoted (3,5)12G1-2-BMPA-G2-(OH)4, can be thought of as the single−single analogue of the twin−twin amphiphilic JDs that make up library 1. The synthesis of library 8 is outlined in Scheme 4. The acid-presenting Percec-type hydrophobic dendron was coupled with ethylene glycol by DCC and DPTS to give 4.1. The polyester hydrophilic dendron was then constructed divergently on this scaffolding by successive iterations of esterification and deprotection in a method similar to that described previously in Scheme 1. It should be noted that rather than using an anhydride for growing the polyester dendron as was done in Scheme 1, acetonideprotected bis-MPA (see compound 1 in Scheme 12) was employed for the synthesis of library 8. Gillies and co-workers developed the JDs with TEG-based hydrophilic dendrons and photodegradable polyester hydrophobic dendrons that comprise library 9 and are generically denoted (acetonide) y -BnNO 2 -G(m+1)-(3,4,5)-3EO-G1(OCH3)3.74 The synthesis of this library is outlined in Scheme 5. Hydrophilic dendron 5.1 was linked to the acetonideprotected hydrophobic dendron 5.275 via esterification by 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC·HCl) in the presence of 4-(dimethylamino)pyridine (DMAP) to afford

a

Reprinted with permission from ref 84. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

2.2. Single−Single Design

2.2.1. Convergent−Divergent. Three libraries of single− single amphiphilic JDs have been synthesized by convergent− divergent methodologies as shown in Figure 6. Library 7 consists of trisdodecyloxy-substituted pentaerythritol bis-MPA amphiphilic JDs generically denoted tris12-PE-BMPA-G(m+1)(OH)y.37 Outlined in Scheme 3, this library was synthesized starting from pentaerythritol 3.1, which was trisubstituted with alkyl chains by TBABr-catalyzed SN2 attack on 1-bromodode6549


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Reprinted with permission from ref 85. Copyright 2012 American Chemical Society.

the first generation photodegradable polyester amphiphilic JD 5.3. In accordance with a divergent methodology, the third and fourth generation JDs (5.5 and 5.7, respectively) were prepared by successive repetitions of deprotection and esterification. 2.2.2. Convergent−Convergent. The synthesis of single− single amphiphilic JDs by convergent−convergent methodologies has resulted in a diverse set of 24 libraries as shown in Figure 7. The amphiphilic JD constituting library 10 consists of a TEG-based hydrophilic dendron connected by a diacetal core to a Percec-type hydrophobic dendron and is denoted (3,5)12G1dAC-(3,4,5)-3EO-G1-(OCH3)3, and its synthesis is outlined in Scheme 6.37 The methyl ester-substituted hydrophobic dendron 6.1 was reduced to the benzyl alcohol hydrophobic dendron 6.2 and then oxidized to the corresponding aldehyde 6.3. The same two steps were then carried out on the methyl ester-substituted hydrophilic dendron 6.4 to yield the corresponding aldehyde 6.6. The hydrophobic dendron 6.3 was then attached as an acetal moiety to the pentaerythritol core to afford 6.7, which was then reacted with the hydrophilic dendron 6.6 to form a second acetal moiety, yielding the amphiphilic JD 6.8 that comprises library 10. Libraries 11 and 12 (Figure 7), denoted generically as (3,4,5)12G1-CH2-PhE-(3,4,5)-3EO-G1-(OCH3)y and (3,4,5)-12G1PhE-CH2-(3,4,5)-3EO-G1-(OCH3)3, respectively, consist of constitutional isomers of benzyl ester containing amphiphilic JDs that differ only in the location of the benzyl ester core at either the TEG-based hydrophilic or the Percec-type hydrophobic dendron.37,76 The syntheses of these libraries are outlined in the bottom half of Scheme 7. In the case of library 12, the hydrophobic dendron acid 7.1b was connected to the benzyl

alcohol hydrophilic dendron 7.4f by esterification with DCC and DPTS to afford the amphiphilic JD 7.14bf. In contrast, library 11 was synthesized by the same esterification procedure but with the acid and benzyl alcohol groups on the opposite dendrons such that the benzyl alcohol hydrophobic dendron 7.4b reacted with the acid-substituted hydrophilic dendron 7.1f to afford the amphiphilic JD 7.15bf. Methods for the syntheses of the relevant hydrophobic and hydrophilic dendrons were discussed previously in Scheme 6. Libraries 13 and 14 (Figure 7), generically denoted ( 3 , 4 , 5 ) 1 2 G 1 - I - P P D - ( 3 , 4 , 5 ) -3 E O -G 1 -( O C H 3 ) 3 a n d (3,4,5)12G1-II-PPD-(3,4,5)-3EO-G1-(OCH3)3, respectively, are constitutional isomeric libraries of L-1,2-propanediol ester containing amphiphilic JDs with TEG-based hydrophilic dendrons and Percec-type hydrophobic dendrons.76 The syntheses of these libraries are outlined in the top half of Scheme 7. In the case of library 13, the TEG-containing hydrophilic dendron 7.1f was converted to an acid chloride and then coupled with (S)-2-(benzyloxy)-1-propanol in the presence of DCC and DPTS to afford 7.2f. Following deprotection of 7.2f to give the alcohol containing hydrophilic dendron 7.3f, the acid containing hydrophobic dendrons 7.1a−c were connected by esterification to afford the amphiphilic JDs 7.12af, 7.12bf, and 7.12cf that constitute library 13. Library 14 was synthesized identically except that the hydrophobic dendrons 7.1a−c rather than the hydrophilic dendrons were coupled with (S)-2(benzyloxy)-1-propanol to afford the alcohol containing hydrophobic dendrons 7.3a−c, which were subsequently linked to the 6550


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to produce the benzylamine hydrophilic dendron 8.8f from the corresponding ester 8.2f. Scheme 9 shows the final step to produce the amphiphilic JDs in both libraries.77 For library 15, amide coupling of the benzylamine hydrophobic dendron 9.8 (referred to as 8b in Scheme 8) and acid 9.1 (referred to as 3f in Scheme 8) was performed in the presence of 2-chloro-4,6dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) in THF. For library 16, the same procedure was followed to produce the desired amphiphilic JD except that benzylamine hydrophilic dendron 9.7 (referred to as 8f in Scheme 8) and acid 9.4 (referred to as 3b in Scheme 8) were used. Thus, essentially the same procedure for both libraries was followed but with the amine and acid functional groups synthesized on opposite dendrons.

acid-substituted hydrophilic dendron 7.1f. The resulting amphiphilic JDs 7.13af, 7.13bf, and 7.13cf constitute library 14. Similarly, libraries 15 and 16 (Figure 7), denoted (3,5)12G1CH2-PhA-(3,4,5)-3EO-G1-(OCH3)3 and (3,5)12G1-PhA-CH2(3,4,5)-3EO-G1-(OCH3)3, respectively, consist of constitutional isomers that differ only by the location of the amide bond between the TEG-based hydrophilic and Percec-type hydrophobic dendrons.77 The second line of Scheme 8 outlines the synthesis of the relevant hydrophobic and hydrophilic dendrons.76 For library 15, the benzylamine hydrophobic dendron 8.8b was synthesized from the corresponding ester 8.2b by reduction to alcohol 8.6b followed by phthalimidation to give 8.7b. Finally, the aminomethyl group was achieved via reaction with hydrazine to yield the desired hydrophobic dendron 8.8b. For library 16, the same procedure was followed 6551


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Scheme 16. Synthesis of Library 32 Hydrophobic Dendron87a

(3,4,5)3-G1-(NHBoc)3, and library 23, denoted (3,4,5)12G1BnA-(3,4,5)3-G1-(NH3Cl)3, is the salt version of library 21, denoted (3,4,5)12G1-BnA-(3,4,5)3-G1-(NHBoc)3. Library 21 differs from library 20 only in the presence of an additional methylene bridge in the core between the hydrophobic dendron and the amide bond. Library 19, denoted (3,4,5)12G1-PhA(3,4,5)-3EO-G1-(OCH3)3, is identical to library 20 except that its hydrophilic dendron consists of methyl-terminated TEG chains. The syntheses of libraries 20−23 are outlined in Scheme 10. Boc-protected terminal amine alkyl chains were prepared from alkylammonium chloride 10.1. These Boc-protected chains were then attached by Williamson ether synthesis to ester 10.3, which was hydrolyzed to the corresponding acid 10.5. For libraries 20 and 22, the amine-substituted hydrophobic dendron 10.9 was prepared from 10.6 by Williamson ether synthesis to attach the alkyl chains, followed by nitration and reduction to the desired amine. For libraries 21 and 23, the benzylamine hydrophobic dendron 10.15 was prepared from ester 10.12 by Williamson ether synthesis to attach the alkyl chains, followed by reduction of the ester to an alcohol and then substitution to afford the corresponding benzyl chloride, which was converted to an azide and then reduced to yield the desired benzylamine. For libraries 20 and 21, the amine-substituted hydrophobic dendrons (10.9 and 10.15, respectively) were connected to acid-substituted hydrophilic dendron 10.5 by amidation via CDMT and NMM to afford the desired amphiphilic JDs (10.10 and 10.16, respectively) that comprise the libraries. Compounds 10.10 and 10.16 could then be converted directly to their ammonium salt analogues by deprotection in acidic conditions, affording the constituents of libraries 22 and 23 (10.11 and 10.17, respectively). Library 19 was synthesized identically to library 20 except that the hydrophilic dendron is composed of methylterminated TEG chains. The synthesis of such a hydrophilic dendron was described previously in Scheme 2. Würthner and co-workers developed the amphiphilic perylene bisimide (PBI) containing JDs that comprise libraries 24 and 25 (Figure 7), denoted alkene-E-6-PBI-(3,4,5)-3-2EO-G1(OC2H5)3 and (3,4,5)12G1-PBI-(3,4,5)-3-2EO-G1-(OC2H5)3, respectively.78 The syntheses of these libraries are outlined in Scheme 11. The amine-substituted hydrophilic fragment 11.8 was prepared from p-nitroaniline (11.1) by successive iodination to form 3,4,5-triiodoaniline (11.3) followed by alkynylation with 11.6 and then reduction to the desired compound. Library 24, which has an alkene-functionalized hydrophobic tail, was prepared by combining hydrophilic dendron 11.8 and 6amino-1-hexanol (11.9) with perylene-3,4,9,10-tetracarboxylic dianhydride (11.10) in the presence of zinc acetate and imidazole to give 11.11. This was followed by reaction with methylacryloyl chloride, to complete the hydrophobic chain. Library 25 was synthesized similarly. Compound 11.8 and 3,4,5tridodecylaniline (11.12), a Percec-type hydrophobic dendron, were combined with 11.10 in the presence of zinc acetate and imidazole to afford the target compound after separation from byproduct. Libraries 26 and 27 (Figure 7) are structurally and synthetically related with both employing Cu(I)-catalyzed azide/alkyne cycloaddition (copper “click” chemistry)79,80 in their final steps. It is this final coupling step through the formation of a triazole ring that has resulted in their classification in the convergent− convergent category. However, it should be noted that divergent methodologies are also employed in the syntheses of these libraries for the growth of the dendrons prior to their coupling.

a


Libraries 17 and 18 (Figure 7), generically denoted (3,4,5)12G1- L -AAA-CH 2 -(3,4,5)-3EO-G1-(OCH 3 ) 3 and (3,4,5)12G1-L-CH2-AAA-(3,4,5)-3EO-G1-(OCH3)y, respectively, consist of amino acid containing amphiphilic JDs, in which the amino acid serves as the core of the JD, connecting the TEG-based hydrophilic and Percec-type hydrophobic dendrons by amide linkages.76,77 The amino acids are referred to by their three-letter abreviations (AAA) in the nomenclature. Libraries 17 and 18 differ only in whether the amino acid is first attached to the hydrophilic or hydrophobic dendron. The differing syntheses are outlined in the top half of Scheme 8 using an alanine (Ala) residue as an example.76 For library 17, the amino acid was first connected to the hydrophobic dendron via amidation. In the case shown in Scheme 8, L-alanine was linked to the acids 8.3a−c via CDMT and NMM to afford 8.5a−c. Subsequently, as second amidation was performed to connect the L-alanine containing hydrophobic dendrons 8.5a−c and the benzylamine hydrophilic dendrons 8.8d−f to give the relevant amphiphilic JDs 8.9. Library 18 was synthesized via the same methodology except that the hydrophilic dendron rather than the hydrophobic dendron was first connected to L-alanine. A second amidation again followed with these amino acid containing hydrophilic dendrons and benzylamine hydrophobic dendrons to afford the amphiphilic JDs as shown, for instance, in the fourth line of Scheme 8. The bottom portion of Schemes 8 and 9 outlines the same syntheses except employing different amino acids including glycine, valine, isoleucine, and phenylalanine.76,77 Libraries 19−23 (Figure 7) are structurally and synthetically related.37 Libraries 20−23 contain hydrophilic dendrons with either terminal t-butyloxycarbonyl (Boc)-protected amino groups or ammonium chloride salts. Library 22, generically denoted (3,4,5)nG1-PhA-(3,4,5)3-G1-(NH3Cl)3, is the salt version of library 20, generically denoted (3,4,5)nG1-PhA6552


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Scheme 17. Synthesis of Library 32 by Convergent−Convergent Strategy87a

a


Library 26, reported by Sierra and co-workers81 and generically denoted G(n+1)(C17)x-G(m+1)(OH)y, consists of bis-MPA-based polyester hydrophilic dendrons and alkylconjugated bis-MPA-based polyester hydrophobic dendrons. Scheme 12 outlines the methodology for the synthesis of this library. Both the hydrophilic and the hydrophobic dendrons were built from acetonide-protected bis-MPA (12.3). For the

hydrophobic dendron, 12.3 was coupled with propargyl alcohol (12.4) via esterification with DCC and DPTS to give 12.5 after deprotection. The generation of the dendron could then be expanded with additional coupling and deprotection steps with acetonide-protected bis-MPA. After the desired generation of the hydrophobic dendron was achieved, the periphery was then decorated with steric acid chains via esterification with DCC and 6553


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Reported by Kostiainen and co-workers,84 library 27, generically denoted (3,4,5)12G1-1-TRZ-1-BMPA-G(m+1)(OH)y, consists of bis-MPA-based polyester hydrophilic dendrons coupled to Percec-type hydrophobic dendrons. The hydrophilic dendrons were synthesized similarly to the hydrophilic dendrons in library 26 but functionalized with an alkyne rather than an azide. The final step in the synthesis of these JDs involved the copper-catalyzed click reaction of the alkynefunctionalized hydrophilic dendrons and the azide-functionalized hydrophobic dendrons as shown in Scheme 13. Kostiainen and co-workers also developed libraries 28−30 (Figure 7), which link spermine-modified Newkome-type hydrophilic dendrons with Percec-type hydrophobic dendrons.85 Syntheses of the amphiphilic JDs in libraries 28−30 are outlined in Scheme 14. For library 28, denoted (3,4,5)12G1-PhAspermine, the Percec-type hydrophobic dendron 14.3C12 was amide coupled with Boc-protected spermine 14.7 in the presence of DCC, 1-hydroxybenzotriazole (HOBT), and triethylamine to give the boc-protected amphiphilic JD 14.8, which was then deprotected under acidic conditions to give 14.3C12-G0, which constitutes library 28. For libraries 29 and 30, generically denoted (3,4,5)12G1-PhA-tris(2A-spermine)3 and (3,4,5-3,5)12G1PhA-tris(2A-spermine)3, respectively, apex-protected Newkome-type hydrophilic dendron 14.1 was decorated with Bocprotected spermine 14.7 to give 14.2, which was then deprotected at the apex to give amine 14.3. Library 29 was subsequently synthesized following the same steps as outlined for library 28 except employing 14.3 rather than 14.7. Library 30 was also synthesized analogously but with first generation Percectype hydrophobic dendron 14.6C12. Ling Peng and co-workers reported the synthesis of the compounds constituting libraries 31 and 32 (Figure 7) and generically denoted 2n-TRZ-1-PAMAM-G(m+1)-(EDA)y and (17)2-A-PAMAM-G1-spacer-TRZ-1-PAMAM-G3-(EDA)8, respectively.86,87 These libraries are composed of amphiphilic JDs consisting of poly(amidoamine) (PAMAM)-based hydrophilic dendrons coupled with alkyl-chain hydrophobic fragments. Although the synthesis of the PAMAM dendrons employs a divergent methodology, these libraries have been classified as convergent−convergent because the final step in their syntheses

Figure 8. Twin−single JD37 from convergent−convergent synthetic strategy.

Figure 9. Fluorinated JDs90 from convergent−convergent synthetic strategies.

DPTS to yield 12.6 and 12.7. The hydrophilic dendrons (12.9, 12.10) were built similarly except that 6-azido-1-hexanol (12.8) was attached in the first step rather than propargyl alcohol. The alkyne-functionalized hydrophobic dendrons could then be coupled with the azide-functionalized hydrophilic dendrons by copper click chemistry carried out with copper(II) sulfate and sodium ascorbate in a mixture of organic solvent and water, affording the compounds in library 26. A representative example of this click reaction is illustrated at the bottom of Scheme 12. It should also be noted that identical structures were synthesized but with the hydrophilic dendron decorated with a periphery of amines or ammonium salts (not shown).82,83 This was done by esterifying the hydrophilic dendron with Boc-Gly-OH before clicking it the hydrophobic dendron. Deprotection followed to give the amine- or ammonium-functionalized periphery. 6554


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Scheme 19. Synthesis of Fluorinated Library 3590 JDs by Convergent−Convergent Strategiesa

Reagents and conditions: (i) t-BuOK (cat.), DMF, 0 °C for 2 h, then 23 °C for 24 h; (ii) H2, Pd/C, DCM, methanol, 23 °C, 12 h; (iii) DCC, DPTS, DCM, 23 °C, 12 h. a

PAMAM dendron. The amine periphery of the third-generation JD was also decorated with argenine (not shown).88 The third generation hydrophilic PAMAM dendron in library 32 was constructed identically. Synthesis of the library 32 hydrophobic dendron is outlined in Scheme 16.87 TEG was tosylated then converted to diazide 16.3, which was reduced halfway to amine 16.4. A first generation PAMAM dendron was then synthesized on the amine as discussed previously to give 16.6. Coupling the peripheral amines with stearic acid via carbonyldiimidazole (CDI) completed the synthesis of hydrophobic dendron 16.7. Scheme 17 shows the last two steps of the synthesis of library 32, in which the azide-functionalized hydrophobic dendron was coupled to the alkyne-functionalized third-generation hydrophilic PAMAM dendron via copper click chemistry. In the final step, the peripheral esters of the PAMAM dendron were coupled

involves copper click chemistry to couple the hydrophilic and hydrophobic dendrons. Scheme 15 shows the synthesis of library 31. The first generation PAMAM hydrophilic dendron was constructed divergently from propargylamine by Michael addition onto methyl acrylate to give ester-terminated 15.2, which was then clicked with alkyl azide 15.1a (C18H37N3), affording 15.3. In the final step, the terminal esters were replaced with an amine periphery by coupling with ethylene diamine to give 15.4, one of the constituents of library 31. The higher generation JDs in library 31 were synthesized by first growing the PAMAM dendron to the desired generation via successive iterations of coupling with ethylene diamine followed by Michael addition onto methyl acrylate. These higher generation PAMAM dendrons were then clicked to the alkyl azide of desired chain length by the same procedure as used for the first generation 6555


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Scheme 20. Synthesis of Library 3690 by Convergent−Convergent Methodologya

a

Reagents and conditions: (i) DCC, DPTS, DCM, 23 °C, 12 h.

Denoted as polythioether-PEI1800, library 33 (Figure 7), synthesized by Ding et al.,89 consists of a low molecular weight branched polyethylenimine (PEI) hydrophilic fragment coupled to a polythioether hydrophobic dendron. The structure drawn in Figure 7 for this molecule, denoted in the literature as a Janus dendritic polymer, is only one possibility. It should be noted that PEI is a linear polymer with a few, random branches and thus is not a dendron. The polythioether dendron may be connected to any terminal amine of the PEI fragment, which may also exhibit less or different branching. The synthesis of this Janus dendritic polymer is outlined in Scheme 18. The polythioether was synthesized from dimercaprol by propargylation of the sulfur atoms followed by addition of ethanethiol across the triple bonds. The resulting polythioether alcohol was converted to an alkene by reaction with acryloyl chloride to afford the desired polythioether hydrophobic dendron. In the final step of the synthesis of library 33, the hydrophobic dendron was coupled with PEI1800, a readily commercially available polymer, via Michael addition to give the Janus dendritic polymer constituting library 33.

Figure 10. Hyperbranched JD92 with noncovalent linker. Reprinted with permission from ref 92. Copyright 2013 American Chemical Society.

2.3. Twin−Single Design

Only one twin−single amphiphilic JD denoted (3,4,5)12G1-PE(3,4,5)1-1EO-G1-(OH)3 has been prepared,37 and it constitutes library 34, shown in Figure 8. It has a single 1EO-based

with ethylene diamine to yield amphiphilic JD 17.4 that comprises library 32.

Scheme 21. Synthesis of Hydrophilic Hyperbranched Fragment for Library 3792a

a

Reprinted with permission from ref 92. Copyright 2013 American Chemical Society. 6556


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Scheme 22. Synthesis of Hydrophobic Hyperbranched Fragment for Library 3792a

a

Reprinted with permission from ref 92. Copyright 2013 American Chemical Society.

Figure 11. Fluorescence and gadolinium-labeled JDs.37,90,93,94

2.4. Fluorinated JDs

hydrophilic dendron connected through a pentaeryithritol core to two Percec-type hydrophobic dendrons. Research on this type of structure is thus extremely limited. Synthesis via a convergent−convergent strategy proceeded identically to library 6 (see section 2.1.2, Figure 5, and Scheme 2) until the coupling of the hydrophobic scaffolding (see compound 1.15 and its corresponding synthesis in Scheme 1) with the THP-protected hydrophilic dendron, which was carried out with DCC and DPTS in acetic acid and dichloromethane (DCM) rather than in just DCM. This gave the THP-protected twin−single amphiphilic JD, which was then deprotected via tosylic acid in methanol/DCM.

Percec and co-workers have prepared two libraries of fluorinated amphiphilic JDs (Figure 9), both of which employ twin−twin designs.90 Library 35, generically denoted (3,4,5)PPVEG1-PE(3,4,5)-3EO-G1-(OCH3)6, consists of two fluorinated Percectype hydrophobic dendrons connected to two TEG-based hydrophilic dendrons through a pentaerythritol core. Library 36, denoted [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1(OCH3)6, consists of one fluorinated and one hydrogenated Percec-type hydrophobic dendron connected to two TEG-based hydrophilic dendrons through a pentaerythritol core. 6557


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Scheme 23. Synthesis of Gadolinium-Labeled JDs (Library 39)93a

a


ization (ROMBP) as outlined in Scheme 21.92 β-Cyclodextrin (CD) serves as the apex of the dendron. In the first step of the synthesis, CD was deprotonated with potassium hydride in the presence of 18-crown-6. The hyperbranched polymer was then formed by slow addition of glycidol using the deprotonated CD as multihydroxyl initiator. Synthesis of the hydrophobic dendron, which is built on an azobenzene apex, was conducted in two steps.92 In the first step, 4-phenylazophenol was converted to 4-(3-hydroxypropyloxy)azobenzene (AZO-OH) via Williamson ether synthesis with 3bromo-1-propanol. In the second step, which is shown in Scheme 22, AZO-OH was used as the initiator for the cationic ringopening polymerization of 3-ethyl-3-oxetanemethanol in the presence of boron trifluoride. Upon addition of water dropwise to a solution of the two dendrons in DMF, the CD apex of the hyperbranched hydrophilic dendron was able to host the guest azobenzene apex of the hyperbranched hydrophobic dendron, thus forming the noncovalent hyperbranched JD that constitutes library 37.

The synthesis of library 35 is described in Scheme 19. The fluorinated hydrophobic dendrons (19.3, 19.5, 19.7) were constructed from esters 19.1a−c by reaction with perfluoropropyl vinyl ether (PPVE) in the presence of potassium tbutoxide as catalyst followed by deprotection to give the desired acids. The hydrophilic dendron 19.8 (synthesis described previously in Scheme 2) was connected to the benzylidenemonoprotected pentaerythritol core 19.9 (synthesis described previously in Scheme 1). The hydrophilic scaffolding 19.11 was afforded after deprotection of the core. Esterification of 19.11 and the various fluorinated hydrophobic dendrons (19.3, 19.5, 19.7) was then conducted via DCC and DPTS to yield the three amphiphilic JDs that constitute library 35. Scheme 20 outlines the synthesis of the hybrid fluorinated/ hydrogenated amphiphilic JD that comprises library 36. Compound 20.1, the same hydrophilic scaffolding from Scheme 19, was coupled with hydrogenated Percec-type hydrophobic dendron 20.2 via DCC and DPTS such that one free hydroxyl group remained on the scaffolding (20.3). This free hydroxyl group was then coupled with a fluorinated Percec-type hydrophobic dendron via DCC and DPTS to give the hybrid amphiphilic JD in library 36.

2.6. Fluorescence and Gadolinium-Labeled JDs

A variety of labeled amphiphilic JDs (libraries 38−43) have been reported as shown in Figure 11. Percec and co-workers reported the synthesis of the Texas Red-tagged amphiphilic JDs in libraries 38 and 39, which are analogues of libraries 22 and 1, respectively, and named accordingly.37 For library 38, synthesis proceeded from the dodecane (n = 12) amphiphilic JD in library 22 (Figure 7) denoted (3,4,5)12G1-PhA-(3,4,5)3-G1-(NH3Cl)3 by conjugation with Texas Red in the presence of DMAP. For the Texas Red-labeled amphiphilic JD in library 39, an identical synthesis proceeded from the library 1 JD with second generation bisMPA-based polyester dendrons and (3,5)-disubstituted Percectype hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2(OH)8 (Figure 4).

2.5. Hyperbranched JDs

Hyperbranched polymers (HPs) are extensively branched dendritic molecules with imperfect or irregular structures due to their one-pot syntheses, which facilitate random branching. A review has been published on the preparation and application of these molecules.91 Zhou and co-workers reported the synthesis of a hyperbranched JD, which constitutes library 37 (Figure 10).92 This molecule consists of a hydrophilic hyperbranched polyglycerol dendron noncovalently bonded to a hydrophobic hyperbranched poly(3-ethyl-3-oxetanemethanol) dendron. Synthesis of the hydrophilic dendron of the hyperbranched JD employed an anionic ring-opening multibranching polymer6558


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Scheme 24. Synthesis of Coumarin-Labeled JDs (Libraries 40 and 41)94

chelating agent was then deprotected with trifluoroacetic acid and treated with GdCl3 to form the desired NMR-labeled JD. Percec and co-workers developed the fluorescent-labeled amphiphilic JDs that make up libraries 40−43.90,94 The coumarin-labeled fluorescent amphiphilic JDs that constitute libraries 40 and 41, denoted (3,5)12-2G1-coumarin-tris(3,4,5)3EO-G2-(OCH3)6 and (3,5)12-G2-tris-coumarin-(3,4,5)-3EO2G1-(OCH3)6, respectively, are constitutional isomers. Their

As shown in Scheme 23, for the purpose of magnetic resonance imaging (MRI), Terreno and co-workers employed the same library 1 amphiphilic JD to synthesize the gadoliniumlabeled amphiphilic JD in library 39 by esterifying one of its peripheral hydroxyl groups with a t-butyl protected bifunctional chelating agent presenting an N-hydroxysuccinimide-activated carboxyl group (DOTAMA(tBu)3-C6-NHS).93 The conjugated 6559


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Scheme 25. Synthesis of RhB- and NBD-Labeled JDs (Libraries 42 and 43)90

amine 25.4 to give 25.5, which was clicked with alkynefunctionalized, fluorinated JD scaffolding 25.8 to give the desired NBD-labeled amphiphilic JD. Compound 25.8 was synthesized

syntheses, which are based on tris(hydroxymethyl)amminomethane (tris) cores, are shown in Scheme 24. For library 41, acid 24.1,95 which was synthesized from its constituent Percec-type hydrophobic dendrons by esterification with benzyl-protected bis-MPA via DCC and DPTS, was coupled with acetonide-protected tris (24.2) in the presence of CDMT and NMM to give compound 24.3. Coumarin was then coupled to the free hydroxyl group of the tris scaffold by esterification via DCC and DPTS to give the acetonide-protected diol 24.5. After deprotection under acidic conditions to give diol 24.6, the TEG-based hydrophilic dendrons 24.7 (see Scheme 2 for synthesis) were coupled to the tris core via esterification with DCC and DPTS to give the amphiphilic JD that constitutes library 41. The constitutional isomer that comprises library 40 was developed to make purification easier by introducing the Percec-type hydrophobic dendrons at the end of the synthesis rather than at the beginning, resulting in a dramatic difference in polarity of the product and its precursors. As such, the synthesis of library 40 followed the same general format as just described for library 41 except that the hydrophilic dendrons were incorporated into starting acid 24.8 rather than the hydrophobic dendrons, which were not introduced until the last step of the synthesis. Syntheses of the amphiphilic JDs in libraries 42 and 43, denoted (3,5)PPVEG1-tris(3,4,5)-3EO-G1-(OCH3)3-NBD and (3,5)12G1-tris(3,4,5)-3EO-G1-(OCH3)3-RhB, respectively, are shown in Scheme 25.90 For library 43, Rhodamine B (RhB) was coupled to the azide-functionalized TEG chain 25.1 to give 25.2, which was clicked with alkyne-functionalized JD scaffolding 25.3 (see Scheme 35 for synthesis) to yield the desired RhB-labeled amphiphilic JD. In the case of library 42, 4-chloro-7-nitrobenzofurazan (NBD-Cl) was coupled with azide-functionalized

Figure 12. (A) Cryo-TEM image of DSs from the library 5 JD (3,4)12G1-PE-(3,5)-3EO-G1-(OCH3)4 by ethanol injection in water and (B) their 3D intensity profile visualized using ImageJ software.37,105 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

from diol 25.6 by esterification with fluorinated hydrophobic dendron 25.7, the preparation of which was previously described in Scheme 19.

3. SELF-ASSEMBLY OF AMPHIPHILIC JDs Amphiphilic JDs have been reported to self-assemble into a wide range of morphologies in water including dendrimersomes (DSs), tubular vesicles, cubosomes, and micellar structures such as rods, disks, and helical ribbons.37 The methods used for selfassembly, the characterization of the assemblies, and basic 6560


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Figure 15. Cryo-TEM images of DSs self-assembled from (3,5)12G1-2BMPA-G2-(OH)4 (library 8).73 Reprinted with permission from ref 73. Copyright 2015 Royal Society of Chemistry.

Figure 13. Selected cryo-TEM images of DSs from single−single JDs in libraries 17 and 18. (a) (3,4,5)12G1-L-Ala-CH2-(3,4,5)-3EO-G1(OCH3)3; (b) (3,5)12G1-CH2-L-Ala-(3,4)-3EO-G1-(OCH3)2; (c) (3,5)12G1-L-CH2-Ala-(3,4,5)-3EO-G1-(OCH3)3; (d) (3,4,5)12G1-LAla-CH2-(3,4,5)-3EO-G1-(OCH3)3.76 Reprinted with permission from ref 76. Copyright 2014 American Chemical Society.

Figure 16. DLS distributions and TEM images of aggregates from (a,b) (acetonide)2-BnNO2-G2-(3,4,5)-3EO-G1-(OCH3)3 and DSs from (c,d) (acetonide)4-BnNO2-G3-(3,4,5)-3EO-G1-(OCH3)3 (library 9).74 Reprinted with permission from ref 74. Copyright 2014 Royal Society of Chemistry.

3.1. Methods for the Synthesis of Dendrimersomes (DSs) and Other Complex Architectures

The three main techniques by which dendrimersomes (DSs) and other complex architectures self-assembled from JDs have been prepared are the injection method, oil-in-water method, and thin-film hydration method.96,97 The injection method, which results in nanoscale selfassemblies, is performed by dissolving JDs in water miscible organic solvent such as ethanol, tetrahydrofuran (THF), acetone, or dimethyl sulfoxide (DMSO) to form a dilute solution. This dilute solution is then injected, most often rapidly, into water or buffer and vortexed, usually for 5−10 s.37,74,98 The injection method is often referred to as a kinetic trapping method because it results in the rapid transition of the molecular building blocks from organic to aqueous media, often affording the kinetically rather than the thermodynamically favored supramolecular asssemblies. Würthner and co-workers reported slow injection for the preparation of DSs coassembled from the PBI containing JDs that comprise libraries 24 and 25.78 Peng and co-workers reported a related procedure for the coassembly of JDs with siRNA into micellar architectures. Amphiphilic JDs and siRNA were added to separate aliquots of transfection medium, stirred, and then combined and vortexed to give the assemblies.86,87 Percec and co-workers reported a reverse injection method, in

Figure 14. TEM images before (a) and after (b) photopolymerization of DSs self-assembled from the PBI containing JDs in libraries 24 and 25 in an 8:1 molar ratio.78 Reprinted with permission from ref 78. Copyright 2007 American Chemical Society.

properties such as predictable size, mechanical properties, permeability, stability, and toxicity, as well as more advanced applications such as drug and nucleic acid delivery, will be reviewed in this section. 6561


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Figure 19. DSs from hyperbranched JDs (library 37) visualized by (a,c) SEM, (b) TEM, and (d) AFM. The inset in (a) shows SEM images of DSs with holes, and the inset in (b) shows a TEM image of freeze-dried DSs.92 Reprinted with permission from ref 92. Copyright 2013 American Chemical Society. Figure 17. (a,b) TEM images of DSs self-assembled in water from (17)2-A-PAMAM-G1-spacer-TRZ-1-PAMAM-G3(EDA)8 (library 32) and (c,d) complexed with siRNA.87 Reprinted with permission from ref 87. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

from the reverse injection method because the volume of aqueous media added to the organic solvent is small as compared to the volume of organic solvent, necessitating either evaporation or dialysis to remove the excess organic solvent. On the other hand, the reverse injection method involves a comparably large amount of water added to a small amount of organic solvent such that the organic solvent does not need to be removed in a separate step. The thin-film hydration method, which generally results in the formation of polydisperse self-assemblies ranging in diameter from several hundred nanometers to tens of micrometers, is performed by dissolving JDs in organic solvent, most often highly volatile solvents like DCM and chloroform, applying a layer of that solution to the bottom of a flask, followed by solvent removal by evaporation in air or under vacuum. Water or buffer is then added to the resulting dry film, and hydration occurs usually at 50−60 °C for several hours.37,81 Percec and co-workers have employed this method for the preparation of giant DSs, which have diameters of 2−50 μm, by evaporation of JD solution on a Teflon plate in a vial followed by hydration with water or buffer at 60 °C for 12 h.37 Smaller-sized DSs could be prepared by following a similar procedure and then conducting sonication for 30 or 60 min.37 Terreno and co-workers have used thin-film hydration for the preparation of monodisperse nanoscale DSs after extensive extrusion.73,93,99,100 Peng and co-workers,101 as well as Ding et al.,89 have also used thin-film hydration coupled with sonication for the preparation of nanomicelles selfassembled from JDs. To prepare micrometer-sized assemblies, Peng and co-workers have used the related electro-swelling method, in which indium−tin oxide (ITO)-coated glass plates are covered with a dry film of JDs after evaporation of the organic solvent and then placed in a Teflon chamber.87 The chamber is filled with aqueous media, and an AC voltage is applied to form vesicles.

Figure 18. Cryo-TEM images of DSs from hyperbranched JDs (library 37).92 Reprinted with permission from ref 92. Copyright 2013 American Chemical Society.

which aqueous media was injected into a water miscible organic solution of JDs.77 The oil-in-water method, which also results in nanoscale selfassemblies, generally involves dissolution of JDs in water immiscible organic solvent followed by addition of water or buffer. The resulting mixture is stirred rapidly until the organic solvent has completely evaporated.81 This method, which is significantly slower than the injection method, may result in the thermodynamically favored self-assembly rather than a kinetically trapped structure. A closely related procedure, which will also be classified as an oil-in-water method, involves the dissolution of JDs in a water miscible organic solvent followed by the addition of aqueous media and dialysis for the removal of organic solvent.92 It should be noted that this method is different 6562


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Figure 20. Representative cryo-TEM images of onion-like DSs self-assembled by injection of THF solution of JDs from libraries 15 and 18 including (A) (3,5)12G1-CH2-PhA-(3,4,5)-3EO-G1-(OCH3)3, (B) (3,5)12G1-CH2-L-Ala-(3,4,5)-3EO-G1-(OCH3)3, (C) (3,5)12G1-CH2-Gly-(3,4,5)-3EO-G1(OCH3)3, and (D) (3,5)12G1-CH2-L-Ile-(3,4,5)-3EO-G1-(OCH3)3 in water (1 mg/mL). Diameter (D, in nm) and polydispersity (PDI) were measured by DLS.77 Reprinted with permission from ref 77. Copyright 2014 National Academy of Sciences USA.

Figure 21. Cryo-TEM images of onion-like DSs self-assembled from (3,5)12G1-CH2-L-Ala-(3,4,5)-3EO-G1-(OCH3)3 (library 18) in water at concentrations of (a) 0.025 mg/mL, (b) 0.1 mg/mL, (c) 0.2 mg/mL, (d) 0.5 mg/mL, (e) 1 mg/mL, (f) 2 mg/mL, and (g) 2.5 mg/mL. The 3D intensity profiles under each cryo-TEM image were generated by ImageJ software.77,105 Reprinted with permission from ref 77. Copyright 2014 National Academy of Sciences USA.

3.2. Nanoscale Visualization of Morphologies Self-Assembled from JDs

average diameters of assemblies and their polydispersities via the polydispersity index (PDI). Given a single Gaussian distribution, the PDI is defined as the ratio of the squares of standard deviation (peak width, σ) and average diameter (ZD).

Visualization of the morphologies obtained after self-assembly of JDs into nanoscale assemblies has been done extensively with cryogenic transmission electron microscopy (cryo-TEM),102,103 in which samples are rapidly frozen to form vitreous ice, preserving the morphology of the self-assemblies in solution. Transmission electron microscopy (TEM) has also been widely employed for visualization of JD-based morphologies, although this method does not provide the same level of quality because it requires removal of suspension media, potentially impacting the observed morphologies. In addition to imaging, dynamic light scattering (DLS)104 has also been extensively used to assess the

PDI = σ 2/Z D2

(1)

3.2.1. Unilamellar DSs. Percec and co-workers have prepared soft, unilamellar, and often monodisperse DSs from a wide array of twin−twin and single−single JDs including many from libraries 1−3, 5−7, 10−12, 13−14, 16, 17−18 (R = CH3), and 20−23 (Figures 4−7).37,76,77 The injection method resulted in DSs with bilayer thickness of 5−8 nm and average radii ranging from 33 to 732 nm as determined by DLS.37 Polydispersity as 6563


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Figure 22. (a) Cryo-TEM images of DSs assembled from fluorinated library 35 and 36 JDs at 0.5 mg/mL. (b) Representative cryo-TEM images of onion-like DSs self-assembled from 0.5 mg/mL of the hybrid fluorinated-hydrogenated library 36 JD [(3,5)12G1+(3,5)PPVEG1]-PE-(3,4,5)-3EO-G1(OCH3)3 and their 3D intensity profiles (by imageJ software)105 with varying numbers of bilayers and diameters.90 Reprinted with permission from ref 90. Copyright 2016 American Chemical Society.

In nearly all cases, it is apparent that (3,4)-disubstituted Percectype hydrophobic dendrons are less suitable for the formation of soft, unilamellar DSs.37,76 Also, the greater flexibility and solubility that additional TEG chains confer to JDs apparently makes the incorporation of (3,4,5)-TEG trisubstituted hydrophilic dendrons particularly helpful for the formation of soft DSs.37,76 Würthner and co-workers reported the coassembly of the PBI containing JDs that comprise libraries 24 and 25 (see Figure 7) into unilamellar vesicles, which were visualized with TEM as shown in Figure 14.78,106,107 The wedge-shaped molecule that comprises library 24 (PBI 1 in Figure 14) formed micelles upon self-assembly because of its high curvature, but coassembly with the dumbbell shaped molecule that comprises library 25 (PBI 3 in Figure 14) reduced curvature, enabling the formation of vesicles with average diameter of 94 nm (Figure 14a). These vesicles were stabilized by in situ photopolymerization (Figure 14b). Terreno and co-workers prepared monodisperse, soft, unilamellar vesicles from both library 8 JDs.73 Cryo-TEM images of DSs with an average diameter of 157.4 nm self-assembled from the (3,5)-disubstituted library 8 JD denoted (3,5)12G1-2BMPA-G2-(OH)4 are shown in Figure 15. It should be noted that these DSs were prepared by coassembly with the pegylated phospholipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine ((DSPE)-PEG-COOH) via thin film hydration and extensive extrusion and dialysis (see section 3.9). Gillies and co-workers demonstrated self-assembly of the third generation (m = 2) library 9 JD denoted (acetonide)4-BnNO2G3-(3,4,5)-3EO-G1-(OCH3)3 (Figure 6) into unilamellar DSs.74 TEM images of these DSs, prepared by addition of

measured by the polydispersity index (PDI) mostly ranged from 0.02 to 0.20 with a value of 0 indicating a perfectly uniform size distribution.37 Micropipette aspiration experiments were employed to distinguish soft from hard vesicles (see section 5.4). Figure 12 shows a cryo-TEM image of unilamellar DSs prepared from the twin−twin library 5 JD with (3,4)-disubstituted hydrophobic dendrons and (3,5)-3EO disubstituted hydrophilic dendrons denoted (3,4)12G1-PE-(3,5)-3EO-G1-(OCH3)4 (Figure 5).37 In general, twin−twin JDs in libraries 5 and 6 (Figure 5) constructed from (3,5)- or (3,4,5)-substituted Percec-type hydrophobic dendrons and (3,4,5)-TEG trisubstituted hydrophilic dendrons were most likely to form soft, unilamellar vesicles. Furthermore, twin−twin library 1 JDs (see Figure 4) constructed from (3,5)- or (3,4,5)-substituted Percec-type hydrophobic dendrons and first or second generation bisMPA-based polyester hydrophilic dendrons exhibited particularly favorable soft, unilamellar vesicles. Figure 13 shows four cryo-TEM images of unilamellar DSs, although some multilamellar structures are present in panels (a) and (c), selfassembled from single−single JDs from libraries 17 and 18 that contain L-alanine residues (Figure 7).76 It should be noted that these DSs were prepared by injection of an ethanol solution of JDs into aqueous media. Different organic solvents, such as THF, may result in the formation of alternate morphologies (see next section).77 Although there are exceptions such as the DSs shown in Figure 13b that are self-assembled from JDs with (3,4)-TEG disubstituted hydrophilic dendrons, favorable soft, unilamellar DSs generally self-assemble from the single−single JDs in libraries 10−12, 13−14, 16, and 17−18 (R = CH3) (Figure 7) that have (3,5)- or (3,4,5)-substituted Percec-type hydrophobic dendrons and (3,4,5)-TEG trisubstituted hydrophilic dendrons. 6564


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Figure 23. Cryo-TEM and 3D intensity profiles of (A,D) polygonal DSs from (3,4)12G1-PE-(3,4)-3EO-G1-(OMe)4 (library 5). (B,E) DCs with a low concentration of spherical DSs from (3,5)12G1-PE-(3,4,5)-2EO-(OMe)6 (library 5). (C,F) Micelles from (3,4,5)12G1-PE-BMPA-G2-(OH)8 (library 1). (G,J) Tubular DSs from (3,5)12G1-PE-(3,4,5)-3EO-(OMe)6 (library 5). (H,K) Rod-like, ribbon-like, and helical micelles from tris-12-PE-BMPAG2-(OH)8 (library 1). (I,L) Disk-like micelles and toroids from (3,4,5)12G1-PE-(3,5)-3EO-(OMe)4 (library 5).37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

determined to be 7 nm thick, which is about twice the theoretical length of the constituent JD as expected. Upon addition of siRNA, a structural rearrangement of the DSs into smaller siRNA/JD complexes of about 100 nm in diameter occurred. These structures are shown in the TEM images in Figure 17c,d. The siRNA/JD complexes are composed of micelles that are 6−8 nm in diameter. Their application will be discussed in section 3.11.3. Zhou and co-workers prepared unilamellar DSs from the hyperbranched JD that comprises library 37 (Figure 10).92 Figure 18 shows cryo-TEM images of these vesicles, which were monodisperse, having a polydispersity (PDI) of 0.004, and were on average 220 nm in diameter. Vesicle wall thickness was measured to be about 9.8 nm, which is nearly double the theoretical length of the hyperbranched JD, indicating the

water to a solution of JDs in DMSO, are shown in Figure 16d. The polydispersity (PDI) as measured by DLS was 0.02, indicating monodisperse assemblies, and the average diameter was 158 nm. The sizes of the DSs could be altered by use of different solvents and different rates of removal of the organic solvent after addition of water. Irradiation with UV light resulted in the degradation of the vesicles as will be discussed in section 3.7. The first and second generation (m = 0 or 1) library 9 JDs (Figure 6) self-assembled into the solid, micellar aggregates shown in Figure 16b. Peng and co-workers reported the self-assembly of the library 32 JD denoted (17)2-A-PAMAM-G1-spacer-TRZ-1-PAMAMG3-(EDA)8 (Figure 7) into unilamellar DSs with an average diameter of 200 nm, as measured by DLS.87 These DSs are shown in the TEM images in Figure 17a,b. The bilayer was 6565


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Figure 24. Cryo-TEM images of third generation library 27 JDs. Fibers were obtained from (a) (3,4)12G1-1-TRZ-1-BMPA-G3-(OH)8 and (b) (3,5)12G1-1-TRZ-1-BMPA-G3-(OH)8, and a mixture of fibers and DSs was assembled from (c) (3,4,5)12G1-1-TRZ-1-BMPA-G3-(OH)8 at dilute concentrations (0.05 wt %) in water. The insets show 3D intensity profiles of selected regions.84 Reprinted with permission from ref 84. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

of these vesicles was achieved by UV irradiation, which induced isomerization of the AZO groups in the hyperbranched JDs from trans to cis, causing scission of the JDs, which are held together by a noncovalent guest−host interaction, into their hydrophobic and hydrophilic components. 3.2.2. Onion-Like DSs. Some biological systems exhibit more than one bilayer including Gram-negative bacteria108,109 (Figure 1) and the cell nucleus,110 which display two concentric bilayers. Additionally, multivesicular structures are encountered in organelles such as endosomes111 and the endoplasmic reticulum,112,113 as well as in myelin114,115 and multilamellar bodies.116,117 The development of synthetic multilamellar vesicles has thus been of significant interest for the modeling of complex biological systems and as multicompartmental drug delivery devices. Phospholipids and block copolymers have been employed for the development of multilamellar vesicles, but preparation is often time-consuming and the assemblies are frequently polydisperse and sometimes biologically incompatible in the case of polymersomes.118−120 Thus, the development of easily producible and predictably sized multilamellar vesicles with biocompatibility was in demand. In response, Percec and co-workers reported the self-assembly of single−single JDs into multilamellar vesicles termed onionlike DSs by simple injection of a THF solution of JDs into aqueous media.77 In particular, the amino-acid containing single−single JDs that constitute libraries 17 and 18, as well as the amide containing single−single JD in library 15 (Figure 7), tended to self-assemble into onion-like DSs. THF aside, acetone, acetonitrile, and 1,4-dioxane were also noted as organic solvents capable of producing onion-like DSs upon injection into water. Figure 20 shows cryo-TEM images of onion-like DSs selfassembled from the library 15 JD (Figure 20a) and several library 18 JDs (Figure 20b−d). Figure 21 shows cryo-TEM images of onion-like DSs self-assembled from the L-alanine-containing library 18 JD (also shown in Figure 20b) at different concentrations. As the concentration of JD increased, the number of bilayers increased from 2 to as many as 17. The specifics of this concentration dependence will be discussed in section 3.3. Polydispersity (PDI) was measured by DLS in the range of 0.14−0.18, indicating monodisperse assemblies. Average diameter was also measured by DLS in the range of 80−330 nm depending on concentration. This information is indicated for each JD that self-assembled into onion-like DSs in Scheme 9. These vesicular structures have been suggested for application as mimics for biological membranes and as timedependent drug delivery devices.77

Figure 25. Cryo-TEM images of CCMV mixed with the library 29 JD (3,4,5)12G1-PhA-tris(2A-spermine)3 in (a) 0 mM, (b) 250 mM, (c) 300 mM, and (d) 625 mM NaCl solutions (scale bar is 100 nm). (e) Integrated SAXS data collected from the CCMV−JD complexes in 250 mM NaCl solution with inset showing the 2D scattering pattern. For comparison, calculated curves from finite fcc and perfect calcium fluoride crystals are shown.85 Reprinted with permission from ref 85. Copyright 2013 American Chemical Society.

formation of a bilayer. The DSs were also imaged with scanning electron microscopy (SEM) (Figure 19a,c), TEM (Figure 19b), and atomic force microscopy (AFM) (Figure 19d). Disassembly 6566


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Figure 26. TEM images of assemblies from library 26 JDs. A molecular model is assigned to each compound that shows the balance between hydrophilic (red) and hydrophobic (blue) parts. In each case, morphologies are represented by an aggregation model.81 Reprinted with permission from ref 81. Copyright 2015 Royal Society of Chemistry.

assembled from the library 1 JD with (3,4,5)-trisubstituted Percec-type hydrophobic dendrons and second generation bisMPA-based polyester hydrophilic dendrons denoted (3,4,5)12G1-PE-BMPA-G2-(OH)8. The third generation library 7 JD denoted tris-12-PE-BMPA-G2-(OH)8 (Figure 6) selfassembled into rod-like, ribbon, and helical micelles as shown in Figure 23h,k. Thus, while systematic structural variations in JDs enable access to a variety of morphologies, predicting the exact morphology remains a challenge. Kostiainen and co-workers reported the self-assembly of amphiphilic JDs into hydrogels.84 The JDs in library 27 with third generation polyester bis-MPA-based hydrophilic dendrons (Figure 7) all demonstrated the ability to self-assemble into hydrogels at 0.2 wt % and above. The third generation library 27 JDs with (3,5)- and (3,4,5)-substituted Percec-type hydrophobic dendrons formed particularly robust hydrogels as compared to the JD with (3,4)-disubstituted Percec-type hydrophobic dendron. Small-angle X-ray scattering (SAXS) profiles of the hydrogels indicated that the JDs self-assembled into nanofibrils that bundled together to form thicker fibers with hexagonal structure. The self-assembled structures were observed by cryoTEM at dilute JD concentrations (0.05 wt %) as shown in Figure 24. The cyro-TEM image of the self-assemblies formed by the JD with (3,4)-disubstituted Percec-type hydrophobic dendron provided an especially clear view of the expected bundled fibers (Figure 24a). At higher concentrations, the hydrogels formed from the cross-linking of the bundled fibers into threedimensional networks with mesh size in the submicrometer to micrometer range. The mechanical properties and applications of these hydrogels will be discussed in section 3.11.1. Kostiainen and co-workers also demonstrated the coassembly of libraries 28, 29, and 30 (Figure 7) with Cowpea Chlorotic Mottle Virus (CCMV) in aqueous media.85 Under experimental conditions, the amine groups on the hydrophilic dendrons were protonated, enabling binding to the negatively charged surface of CCMV. All JDs in libraries 28, 29, and 30 were able to complex with CCMV. Because of a favorable balance of hydrophobic and

Percec and co-workers also prepared onion-like DSs from fluorinated JDs in libraries 35 and 36.90 In particular, the (3,5)disubstituted JD in library 35 denoted (3,5)PPVEG1-PE-(3,4,5)3EO-G1-(OCH3)3 (Figure 9) and the hybrid JD comprising library 36 (Figure 9) denoted [(3,5)12G1+(3,5)PPVEG1]-PE(3,4,5)-3EO-G1-(OCH3)3 formed onion-like DSs as shown in the cryo-TEM images at the top left and bottom right in Figure 22a. The hybrid JD (library 36) formed especially well-defined onion-like DSs with 2 to as many as 11 bilayers as shown in Figure 22b. 3.2.3. Dendrimercubosomes (DCs) and Other Complex Architectures. Amphiphilic JDs have also been observed to selfassemble into a variety of other complex architectures including cubosomes, 121−123 which are structures that contain a bicontinuous internal morphology, denoted dendrimercubosomes (DCs), tubular vesicles, disks, toroids, and helical ribbons. Percec and co-workers prepared a wide variety of morphologies self-assembled from JDs in libraries 5 and 6 (Figure 5), as well as library 7 (Figure 6).37 DCs were prepared by self-assembly of the library 5 JD with (3,5)-disubstituted Percec-type hydrophobic dendrons and (3,4,5)-2EO trisubstituted hydrophilic dendrons denoted (3,5)12G1-PE-(3,4,5)2EO-G1-(OMe)6. A cryo-TEM image of these DCs is shown in Figure 23b with the corresponding 3D-intensity profile in Figure 23e. Polygonal solid DSs were self-assembled from the library 5 JD with (3,4)-disubstituted Percec-type hydrophobic dendrons and (3,4)-3EO disubstituted hydrophilic dendrons denoted (3,4)12G1-PE-(3,4)-3EO-G1-(OMe)4 as shown in Figure 23a,d. The library 5 JD with (3,5)-disubstituted Percectype hydrophobic dendrons and (3,4,5)-3EO trisubstituted hydrophilic dendrons denoted (3,5)12G1-PE-(3,4,5)-3EO(OMe)6 self-assembled into tubular DSs as shown in Figure 23g,j. Disk-like micelles and toroids, as shown in Figure 23i,l, were self-assembled from the library 5 JD with (3,4,5)trisubstituted Percec-type hydrophobic dendrons and (3,5)3EO disubstituted hydrophilic dendrons denoted (3,4,5)12G1PE-(3,5)-3EO-(OMe)4. Micelles, as shown in Figure 23c,f, were 6567


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Figure 27. Chemical structures (a) and corresponding SAXS data collected in the lamellar phases of the indicated library 3 JDs (b). Reconstructed electron density maps (c) illustrating the change in thickness of the layers from less interdigitated (3,4)12G1-X and (3,4,5)12G1-X to more interdigitated (3,5)12G1-X JDs (d).124 Reprinted with permission from ref 124. Copyright 2011 American Chemical Society.

in Figure 26. These include the JD with first generation (n = 0) hydrophobic dendron and second generation (m = 1) hydrophilic dendron denoted G1(C17)2-G2(OH)4, the JD with first generation (n = 0) hydrophobic dendron and third generation (m = 2) hydrophilic dendron denoted G1(C17)2-G3(OH)8, and the JD with second generation (n = 1) hydrophobic dendron and third generation (m = 2) hydrophilic dendron denoted G2(C17)4-G3(OH)8. The JDs with equal generation hydrophilic and hydrophobic dendrons self-assembled into flexible bilayers and vesicles as shown in the leftmost two panels in Figure 26. These include the JD with second generation (n = 1 and m = 1) hydrophobic and hydrophilic dendrons denoted G2(C17)4G2(OH)4 and the JD with third-generation (n = 2 and m = 2) hydrophilic and hydrophobic dendrons denoted G3(C17)8G3(OH)8. Nanomicelles, which are generally 10−100 nm structures, exhibit hydrophobic cores and hydrophilic exteriors. They have been prepared from amphiphilic Janus dendrimers. In particular, the amphiphilic JDs in libraries 31 (n = 8−11, m = 2), 32, and 33 (see Figure 7) have been observed to self-assemble into nanomicelles.86,87,101

hydrophilic chains, the most efficient binding was observed with the library 29 JD with (3,4,5)-trisubstituted Percec-type hydrophobic dendron denoted (3,4,5)12G1-PhA-tris(2A-spermine)3. Its coassembly with CCMV is shown by cryo-TEM in Figure 25. Changing the concentration of sodium chloride in solution led to alterations in JD−CCMV interaction with concentrations below 120 mM yielding amorphous structures (Figure 25a), concentrations between 160 and 300 mM yielding highly ordered crystalline virus arrays (Figure 25b,c), and concentrations of 625 mM or greater yielding no binding due to loss of electrostatic interaction between viruses and JDs (Figure 25d). SAXS was used to analyze the crystalline arrays and demonstrated a face-centered cubic (fcc) structure (Figure 25e). The JD−CCMV crystalline arrays were composed of JD micellar structures occupying the tetrahedral voids between CCMV particles, and they were proposed as mimics of viral inclusion bodies. Sierra and co-workers examined the self-assembly of library 27 in water (Figure 7).81 All library 27 JDs self-assembled in water as shown in Figure 26 except for the JD with first generation hydrophobic and hydrophilic dendrons denoted G1(C17)2G1(OH)2. The JDs with hydrophilic dendrons of higher generation than their hydrophobic counterparts (m > n) all formed elongated micelles as shown in the three rightmost panels 6568


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Figure 28. Illustration of the self-assembly of twin−twin JDs into DSs. The alkyl substitution pattern determines the degree of interdigitation of the DS bilayer, which in turn controls the size and mechanical properties of the DS. The tendency to achieve the same local packing densities is the driving force behind the differing levels of interdigitation. For clarity, the alkyl chains of the inner and outer leaflets of the bilayer are depicted in yellow and green, respectively.124 Reprinted with permission from ref 124. Copyright 2011 American Chemical Society.

3.3. Predictable Thickness, Diameter, and Number of Bilayers

A series of equations were derived to quantify the relationship between DS thickness as determined by self-assembly in bulk (d001) and DS diameter (Dcalc).124 These equations were based on relating the mass of the vesicle bilayer (Massvesicle shell) to its volume (Volumevesicle shell) and then relating final JD concentration (c) in water to the mass of the bilayers as outlined in Figure 29b. Solving this system of equations enabled estimation of the diameter.

Amphiphilic JDs containing Percec-type hydrophobic dendrons can self-assemble into DSs with predictable thickness and diameter. This was shown first for twin−twin JDs with pentaerythritol cores (libraries 1, 2, and 3 in Figure 4 and libraries 5 and 6 in Figure 5).124 The basis of this predictability is that the bilayers observed upon self-assembly of amphiphilic JDs in bulk are about the same thickness as those formed upon selfassembly in water. It should be noted that 83.3% of twin−twin JDs tested that self-assembled into lamellar phases in bulk were also observed to form DSs in water, whereas more than one-half of the twin−twin JDs tested that self-assembled into columnar and cubic phases in bulk formed micellar structures in water. Thus, while most twin−twin JDs that form DSs also form lamellar phases in bulk, some do not, limiting the application of this prediction procedure. Self-assembly into lamellar phases in bulk of second generation library 3 JDs with (3,4)-, (3,5)-, and (3,4,5)-substituted Percec-type hydrophobic dendrons is characterized in Figure 27. As is apparent from SAXS, the (3,5)disubstituted analogue forms significantly thinner bilayers than the JDs with other hydrophobic substitution patterns. This is due to the ability of (3,5)-disubstituted Percec-type hydrophobic dendrons to interdigitate. Furthermore, the hydrophilic substitution pattern, for instance, in libraries 5 and 6, does not significantly affect bilayer thickness as compared to the effect of the hydrophobic substitution pattern. Thinner bilayers due to greater interdigitation are less flexible and thus have low curvatures, resulting in larger DSs. The inverse relationship between vesicle bilayer thickness and size is diagrammed in Figure 28.

Dcalc = d001 +

4 12d001δc − 3d001

(3d001)

(2)

In eq 2, the concentration proportion factor δ is specific to the JD. As shown in Figure 29a, this equation was applied successfully to model the experimental data relating diameter to concentration for the library 1 JD with second generation bisMPA-based polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1PE-BMPA-G2-(OH)8. Equations were also derived to predict the radius (Rcalc) of a DS based on the size of a reference structure at the same concentration. R calc =

d001 + 2

4 12d001Δ − 3d001

6d001

(3)

⎛ ref ⎞ ref str 3 ref str ref str 3 M wt ⎟⎟ Δ = [(RDLS ) − (RDLS − d001 ) ]⎜⎜ ⎝ M wt ⎠

(4)

The radius can be predicted with eq 3, which incorporates str parameter Δ, a value that can be calculated with eq 4. dref 001 and ref str RDLS are the thickness of the vesicle wall of the reference 6569


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In eq 5, p is a constant that depends only on the structure of the JD. This equation is particularly useful because it can predict the size of a DS at any concentration within the range of concentrations where monodisperse DSs form. An example of this concentration dependence is shown in Figure 31. As expected, a plot of the square of the experimental DS diameter versus concentration (Figure 31a) formed a nearly linear relationship (R2 = 0.97), which can be effectively used to predict size at any concentration. Accompanying cryo-TEM images (Figure 31b) at different concentrations corroborated the impact of concentration on size. The number of bilayers for onion-like DSs was also found to be predictable from final JD concentration in water and buffer.77 In particular, the amino-acid containing single−single JDs that constitute libraries 17 and 18, as well as the amide containing single−single JD in library 15 (Figure 7), tended to form onionlike DSs with predictable number of bilayers as shown in Figure 32. An equation was derived to estimate number of bilayers (n) from the DS radius (R), which can be predicted from concentration. n = R/σ

(6)

In eq 6, σ represents the average spacing between bilayers, which can be determined from cryo-TEM. This relationship is demonstrated in Figure 33a, which shows the high correlation between the actual number of bilayers as determined by cryoTEM and the calculated number of bilayers for various sized onion-like DSs self-assembled from the library 18 L-alanine containing JD denoted (3,5)12G1-CH2-L-Ala-(3,4,5)-3EO-G1(OCH3)3. Figure 33b graphs the relationship between the number of bilayers and the final JD concentration in water for the same library 18 JD. 3.4. Molecular Simulation Figure 29. (a) There is a clear dependence of the diameter determined by DLS (DDLS) of monodisperse DSs on concentration (c) from the library 1 JD (3,5)12G1-PE-BMPA-G2-(OH)8 in water. (b) Illustration of the DS model used to fit the experimental data. The inset in (a) shows an enlargement of the dependence at low concentrations. DSs were prepared by ethanol injection in water.124 Reprinted with permission from ref 124. Copyright 2011 American Chemical Society.

Molecular simulations of the self-assembly of JDs in water have been conducted. Percec and co-workers modeled JDs and their self-assembly with coarse-grained (CG) molecular dynamics.37 As shown in Figure 34, this was done for the library 1 JD with first generation bis-MPA-based polyester dendrons and (3,5)disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-(OH)4 (Figure 4). The JDs, modeled as shown in Figure 34a, begin in a disordered state (Figure 34b) and self-assemble on a nanosecond time scale into DSs (Figure 34d− f). CG was also used to demonstrate the ability of JDs to access other complex architectures such as cubosomes and disks. As shown in Figure 35, Peng and co-workers conducted a dissipative particle dynamics (DPD) simulation of the selfassembly of the library 32 JD (Figure 7).87 The simulation demonstrated the self-assembly of the JD into DSs (Figure 35a,b) and into micellar nanostructures in the presence of siRNA (Figure 35c). Self-assembly of the library 32 JD into micelles after encapsulation with doxorubicin (DOX) was also simulated.101 The same laboratory employed DPD to simulate the complexing of library 31 JDs and SiRNA into micelles.125 Zhou and co-workers also used DPD to simulate of the selfassembly of the hyperbranched JD comprising library 37 (Figure 10).92 Figure 36a shows the molecular model used to simulate the JD. Self-assembly into DSs was demonstrated as shown in panels b−h in Figure 36. The JDs begin in a disordered state, aggregate into a bilayer vesicular shape with holes, and then finally form a sealed DS after 4.160 μs. In a DPD study, Arai et al. employed a minimalist model of linear diblock oligomers to demonstrate the self-assembly of

structure and the radius of the reference structure, respectively. Mref wt and Mwt are the molecular weight of the reference JD and the molecular weight of the JD for which size of DS is predicted, respectively. d001 is the thickness of the vesicle wall of the DS for which the size is being predicted. Equations 2−4 were also found to apply to single−single JDs with Percec-type hydrophobic dendrons (e.g., libraries 11, 12, 13−14, and 16−18 in Figure 7).76 Aside from the impact on interdigitation of the substitution pattern on Percec-type hydrophobic dendrons, which follows the same trend as in twin−twin structures, single−single JDs with amino acid containing cores demonstrate reduced ability to interdigitate as the result of hydrogen bonding between core amide bonds as diagrammed in Figure 30. Thus, single−single JDs with amide containing cores tend to form smaller vesicles than related JDs with ester cores. For JDs with Percec-type hydrophobic dendrons that selfassemble into DSs, eq 2 can be simplified to relate the square of DS diameter (D2) to the final concentration (c) of JD in water.76 D2 = pc

(5) 6570


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Figure 30. Powder XRD data collected in the lamellar phases of the indicated library 13 and 17 JDs (a) with corresponding molecular models (b) and chemical structures (c). Blue represents the hydrophilic dendron, green represents the hydrophobic dendron, and red represents the JD core.76 Reprinted with permission from ref 76. Copyright 2014 American Chemical Society.

Figure 31. Concentration dependence of the diameter of DSs formed by (3,4)12G1-I-PPD-(3,4,5)-3EO-G1-(OCH3)3 (library 13). (a) The square of the diameter (DDLS2) of DSs has a linear dependence on the concentration of JDs. Representative cryo-TEM images of DSs with the indicated diameter (DDLS, in nm) and polydispersity (PDI, in parentheses) at final concentrations of (b) 0.25 mg/mL; (c) 1 mg/mL; (d) 2 mg/mL; and (e) 4 mg/mL.76 Reprinted with permission from ref 76. Copyright 2014 American Chemical Society.

onion-like vesicles.126 Simulation of weak hydrophilic character in oligomers with two hydrophilic beads and three hydrophobic beads resulted in the formation of onion-like vesicles. Snapshots

during simulated self-assembly are shown in Figure 37. Discharge of the contents of the vesicles was demonstrated to occur layerby-layer as if peeling an onion. 6571


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fluorinated Percec-type hydrophobic dendrons denoted (3,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH3)3 (RF in Figure 39), and the hybrid JD comprising library 36 (RHF in Figure 39) (see Figures 5 and 9 for structures).90 Fluorescence microscopy has also been extensively employed for the visualization of dye-containing giant DSs.37,90,94 Coassembly of (3,5)12G1-PE-BMPA-G2-(OH)8 with 1% of its Texas Red-tagged analogue in library 39 (Figure 11) enabled visualization of the resulting giant DS by fluorescence microscopy as shown in Figure 38b.37 The accompanying 3D intensity profile is shown in Figure 38c. Additional examples of fluorescence microscopy images are shown in the bottom part of each panel in Figure 39 for giant DSs coassembled from RH, RF, and RHF JDs and their respective dye-tagged analogues in libraries 42 and 43 (Figure 11).90 The red images result from coassembly with the rhodamine B (RhB)-tagged JDs, and the green images result from coassembly with 7-nitrobenzofurazan (NBD)-tagged JDs. Confocal fluorescence microscopy has also been utilized to visualize DSs coassembled with dye-tagged JDs.90 Dye encapsulation in both the hydrophobic bilayer and the aqueous cavity of giant DSs has been visualized as shown in Figure 40.37 The hydrophilic calcein dye was observed to localize in the cavity of the giant DSs, while the hydrophobic Nile red was observed to localize in the bilayer. Figure 40a shows this dual encapsulation for the giant DSs self-assembled from the library 6 JD with (3,4)-3EO disubstituted hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-(3,4)3EO-(OH)4 (Figure 5). The giant DS has a red rim with green interior as expected. Figure 40b shows Nile red encapsulated in the bilayer of the giant DS self-assembled from the library 1 JD with second generation bis-MPA-based polyester hydrophilic dendrons and (3,4)-disubstituted Percectype hydrophobic dendrons denoted (3,4)12G1-PE-BMPA-G2(OH)8 (Figure 4).

Figure 32. Linear dependence of the number of bilayers of individual onion-like DSs on diameter (nm) as determined by cryo-TEM. Onionlike DSs were self-assembled from the library 15 JD (3,5)12G1-CH2PhA-(3,4,5)-3EO-G1-(OCH 3) 3 (red), and the library 18 JDs (3,5)12G1-CH 2 - L -Ala-(3,4,5)-3EO-G1-(OCH 3 ) 3 (blue) and (3,5)12G1-CH2-L-Ile-(3,4,5)-3EO-G1-(OCH3)3 (green).77 Reprinted with permission from ref 77. Copyright 2014 National Academy of Sciences USA.

3.5. Optical Imaging of Giant DSs

Giant DSs, which have micrometer-scale diameters, have been prepared by thin film hydration. A variety of optical microscopy techniques have been employed to visualize these structures. Both differential interfering contrast (DIC) and phase-contrast microscopy have been utilized to view giant DSs without the aid of fluorescence.37,87,90,94 Figure 38a shows a DIC microscopy image of a giant DS self-assembled from the library 1 JD with second generation bis-MPA-based polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2-(OH)8 (Figure 4).37 The gray top panels of each part of Figure 39 show examples of phase-contrast microscopy images of giant DSs self-assembled from the library 5 JD with (3,4,5)-3EO trisubstituted hydrophilic dendrons and (3,5)-substituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-(3,4,5)-3EO-G1-(CH3)6 (RH in Figure 39), the library 35 JD with (3,5)-disubstituted

3.6. Mechanical Properties

DSs exhibit excellent mechanical properties that are comparable to those of biological membranes. Percec and co-workers performed micropipette aspiration experiments with giant DSs self-assembled from JDs in libraries 1 and 6 (Figures 4 and 5), as shown in Figure 41 and outlined in Table 1.37 The library 1 JD with second generation bis-MPA-based polyester hydrophilic

Figure 33. (A) Relationship between calculated and experimentally determined number of bilayers of onion-like DSs; and (B) concentration dependence of number of bilayers, diameter D (inset), and polydispersity (PDI) of onion-like DSs formed by the library 18 JD (3,5)12G1-CH2-L-Ala(3,4,5)-3EO-G1-(OCH3)3. PDI (yellow ▲), diameter (nm, blue ■), and number of bilayers (blue ●).77 Reprinted with permission from ref 77. Copyright 2014 National Academy of Sciences USA. 6572


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Figure 34. Molecular model (A) used for the self-assembly of library 1 JD (3,5)12G1-PE-BMPA-(OH)4. Self-assembly of the JDs was simulated using CG molecular dynamics. Spontaneous bilayer formation occurred on a multinanosecond time scale using a relatively small number of amphiphiles in the simulation box. The initial snapshot (B) shows isotropic mixing of JDs at t = 0 ns. A snapshot of lamellar structure formation (C) is shown at t = 20 ns. The snapshot (D) at t = 40 ns shows the spontaneous formation of a bilayer. The completed simulation (F) shows spontaneous vesicle formation at t = 80 ns. (F) The cut-away view of (E) shows the hollow interior of the vesicle. The solvent has been removed for clarity. Color code: red, hydroxyl; black, hydrophobic fragment of 2,2-bis(hydroxymethyl) propionate; dark purple, ester; magenta, neopentyl fragment; gray, benzene ring fragment; brown, dioxybenzene ring fragment; green, alkane fragment; blue, terminal alkyl.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

Figure 35. DPD simulation showing DSs formed from the library 32 JD (a) and a corresponding cross-section view (b). Simulations were also conducted with the same JD in the presence of siRNA (c) showing the formation of micelles. Color code: light green, hydrophilic units; dark green, hydrophobic units; orange sticks, siRNAs; light gray spheres, representative water molecules.87 Reprinted with permission from ref 87. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-(OH)8 exhibited an areal expansion modulus (Ka) of 976 mN/m, which is comparable to that of cholesterol-stabilized liposomes. Furthermore, tuning of JD structure can provide access to a wide range of Ka values. DSs also exhibited lipid-like critical areal strains (αc) between 0.03 and 0.06. The substitution pattern of Percec-type hydrophobic dendrons impacts DS toughness with (3,5)-disubstitution favoring the formation of stronger DSs due to greater interdigitation of hydrophobic chains in the bilayer.124

Figure 36. DPD simulations of the self-assembly of the hyperbranched JD (library 37). (a) The molecular model for one JD. Three-dimensional snapshots and corresponding cross sections of (b) the initial random distribution of JDs in solution, (c,d) the formation of an irregular aggregate with bilayer structures, (e) the formation of a vesicle with holes penetrating the membrane, and (f,g) the vesicle closing into the final structure (h). The solvent beads are removed for clarity. Color code: red, hydrophobic hyperbranched fragment; blue, hydrophilic hyperbranched fragment; gray, CD/AZO group.92 Reprinted with permission from ref 92. Copyright 2013 American Chemical Society.

3.7. Dye Encapsulation and Release

Dyes have been encapsulated in both the hydrophobic bilayer and the hydrophilic cavity of DSs. Encapsulation of a hydrophobic dye into the DS bilayer is generally done by addition of the dye to an organic solution of the JD, which can then be used as normal for thin film hydration or oil-in-water methods. Hydrophilic dyes can be added to the water or buffer solution that is used for hydration in the thin film method and for addition to the organic phase in the oil-in-water method, enabling encapsulation of the dye in the hydrophilic cavity.

Excess dye can be removed from the solution of DSs by dialysis or size-exclusion chromatography.37,74 Percec and co-workers encapsulated the hydrophilic dye calcein in the cavity of DSs self-assembled from the library 1 JDs 6573


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with first, second, and third generation bis-MPA-based polyester hydrophilic dendrons and (3,4,5)-trisubstituted Percec-type hydrophobic dendrons (Figure 4), demonstrating stability in time and impermeability.37 Fluorescent microscopy was used to image these DSs as was discussed in section 3.5. Addition of Triton X-100, a detergent that causes vesicles to rupture, resulted in the rapid release of the dye, as shown in Figure 42. Terreno and co-workers also demonstrated encapsulation and release by Triton X-100 of carboxyfluorescein in DSs coassembled from the library 1 JD with second generation bis-MPA-based polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-(OH)8 and the pegylated lipid (DSPE)-PEG-COOH.93 Gillies and co-workers showed the encapsulation and release of both hydrophilic and hydrophobic dyes in photodegradable DSs self-assembled from the third generation library 9 JD denoted (acetonide)4-BnNO2-G3-(3,4,5)-3EO-G1-(OCH3)3 (Figure 6).74 Upon irradiation with UV light, the hydrophobic dendrons of the JDs in the DSs decomposed into o-nitrobenzaldehyde products. Figure 43a provides evidence of the photodegradation of the DSs by showing a decrease in intensity of the peaks in the UV spectrum associated with o-nitrobenzyl groups (225 and 265 nm) and corresponding increases in intensity of peaks at 300 and 340 nm, which are signatures of the o-nitrobenzaldehyde photolysis product. Furthermore, DLS monitoring during UV irradiation (Figure 43b) showed a reduction in mean count rate and diameter, indicating degradation of DSs. After demonstrating the photodegradation properties of the DSs, the hydrophobic dye Nile red was loaded into the bilayer of the DSs. As shown in Figure 43c, after a period of 8 min of UV irradiation, fluorescence intensity of the loaded dye decreased to about 10%, indicating that it had been quenched as the result of release into the aqueous medium. The hydrophilic dye fluorescein was also encapsulated in the cavity of the DSs. Figure 43d demonstrates that, with UV irradiation, all of the fluorescein was released after 14 h, whereas without UV irradiation only 14% of the dye was released. The longer time scale of release as compared to Nile red can be attributed to the nature of the experiment, which employed lengthy dialysis to isolate the released dye. In related work, Würthner and co-workers developed PBI containing DSs (Figure 7) as fluorescent pH sensors.127 DSs coassembled from the library 24 and 25 JDs in a ratio of 20:1 were encapsulated with the hydrophilic bispyrene-based energy donor 1,7-bis (1-methyl-pyrenyl)-1,4,7-triazaheptane and then photopolymerized. As pH increases, the donor becomes deprotonated, increasing the wavelength of its emission after excitation with UV radiation. This shifted donor emission spectrum overlaps the absorption spectrum of the PBI

Figure 37. Self-assembly of 16 200 Janus oligomers into onion-like vesicles with snapshots at (a) 0 μs; (b) 2.1 μs; (c) 7.5 μs; (d) 24.5 μs; (e) 46.9 μs; (f) 110.9 μs; and (g) 122.0 μs. A cross-section view (g′) is shown of (g). Color code: red, hydrophobic fragment; blue, hydrophilic fragment.126 Reprinted with permission from ref 126. 2016 American Chemical Society.

Figure 38. (a) Differential interfering contrast microscopy (DIC) and (b) fluorescence images with (c) corresponding 3D intensity plot of giant DSs self-assembled from the library 1 JD (3,5)12G1-PE-BMPA-G2-(OH)8 and its library 39 Texas Red-tagged analogue.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science. 6574


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Figure 39. Representative fluorescence microscopy images of giant DSs assembled from the library 5 JD (3,5)12G1-PE-(3,4,5)-3EO-G1-(CH3)6 (RH), the library 35 JD (3,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH3)3 (RF), and hybrid RHF JD (library 36) with an additional 1% (w/w) RH-RhB (library 43) or RF-NBD (library 42).90 Reprinted with permission from ref 90. Copyright 2016 American Chemical Society.

Figure 40. (A) Fluorescence microscopy image of giant DSs assembled from the library 6 JD (3,5)12G1-PE-(3,4)-3EO-G1-(OH)4, encapsulating both hydrophobic Nile red and hydrophilic calcein dyes. (B) Microscopy image of a giant DS from the library 1 JD (3,4)12G1-PEBMPA-G2-(OH)8 visualized with Nile red.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

membrane of the DSs with increasing precision. When viewed under UV light, the resulting system consists of DSs that under acidic conditions exhibit blue-purple fluorescence due to the encapsulated donor. As pH increases, blue-purple fluorescence decreases and blue-green fluorescence increases due to the initial shifting of the donor emission spectrum toward longer wavelengths. Finally, as the emission spectrum of the donor overlaps the absorption spectrum of the PBI membrane, the blue-green fluorescence of the donor diminishes and the red fluorescence from the PBI membrane intensifies. Overall, the donor-loaded DSs act as an ultrasensitive pH probe by exhibiting fluorescence color changes covering the whole visible spectrum as a function of pH.

Figure 41. (A) Micropipette aspiration used to determine the mechanical strength by micro deformation under negative pressure of a giant DS from the library 1 JD (3,5)12G1-PE-BMPA-G2(OH)8. (B) The same DS under negative pressure showing deformation of the membrane. (C) Areal strain determined upon rupture of the same DS (αc). The asterisk indicates the point of membrane failure at the critical areal strain.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

drug doxorubicin (DOX) in DSs self-assembled from select JDs in libraries 1 and 5 (Figures 4 and 5).37 As shown in Figure 44a, at physiological temperature (37 °C) and pH 7.4, DSs loaded with DOX and self-assembled from the library 1 JD with second generation bis-MPA-based polyester hydrophilic dendrons and

3.8. Drug Encapsulation and Release

Drug encapsulation in DSs can be accomplished the same way as dye encapsulation (see section 3.7). Percec and co-workers demonstrated the encapsulation and release of the anticancer 6575


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Table 1. Mechanical Properties of DSs As Compared To Those of Polymersomes and Liposomes37a sample dendrimersomes

polymersomes

lipids

a

(3,4)12G1-PE-BMPA-(OH)8 (3,5)12G1-PE-BMPA-(OH)8 (3,4,5)12G1-PE-BMPA-(OH)8 (3,4,5)12G1-PE-(3,4,5)-3EO-G1-(OH)6 (3,5)12G1-PE-(3,4)-3EO-G1-(OH)4 OB2 (PEO-PBD) OE7 (PEO-PEE) OB18 (PEO-PEB) DAPC egg PC DMPC SOPC/% cholesterol 0% 28% 50% 78%

Ka (mN/m)

lysis tension τs (mN/m)

critical αc

energy stored at failure (mJ/m2)

961 976 42.44 267.5 582.5 100 140 109 57 140 234

20.4 15.5 0.88 12.7 15.11 14.0 18.0 33.0 2.3 4.0

0.04 0.03 0.06 0.04 0.06 0.21 0.19 0.40 0.04

0.42 0.69 0.11 0.27 0.73 2.20

5.7 12.6 19.7 28.0

0.03 0.05 0.03 0.02

0.10 0.33 0.34 0.31

193 244 781 1286

Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

Figure 42. Calcein release over time from selected DSs assembled from library 1 JDs after the addition of Triton X.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

(3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2-(OH)8 demonstrated less than 20% release of drug over 100 h, indicating good stability and impermeability. However, under physiological temperature and acidic conditions of pH 5.2, the same DOX loaded DSs released more than 60% of their payload over 100 h. This was due to the acid-catalyzed cleavage of the aromatic−aliphatic ester groups of the JDs composing the DSs, as well as the protonation of DOX, which enhances its solubility in the surrounding aqueous media. Figure 44b demonstrates the DOX releasing capability of DSs self-assembled from several other JDs in libraries 1 and 5. Peng and co-workers demonstrated DOX loading of micelles self-assembled from the library 32 JD (Figure 7).101 A maximum drug loading efficiency of 42% was achieved at a JD:DOX ratio of 15:10. At physiological temperature and pH 7.4, 15−20% DOX was released over 24 h, whereas at the more acidic pH of 5, more than 30% DOX was released in the same time period. The improved drug release under acidic conditions can be attributed to the proton-sponge effect, in which the tertiary amines of the PAMAM dendron of the library 32 JD are protonated. Under physiological conditions, only the terminal primary amines are protonated. The additional protonation under acidic conditions results in significant electrostatic repulsion between the branches

of the JDs, triggering drug release. A detailed discussion of in vitro and in vivo studies involving these DOX loaded micelles follows in section 3.11.2. Terreno and co-workers reported encapsulation of the glucocorticoid drug prednisolone phosphate (PLP) in DSs coassembled from the (3,5)-disubstituted library 8 JD denoted (3,5)12G1-2-BMPA-G2-(OH)4 (Figure 6), the lipid DSPEPEG2000-COOH, and an amphiphilic NMR contrast agent.100 PLP loading efficiency and retention were shown to be similar for the DS and a liposome analogue. In vitro and in vivo release of the drug was also described, as will be discussed in detail in section 3.11.4. Sierra and co-workers encapsulated the lipophilic anticancer drug Plitidepsin in the bilayers formed by the self-assembly of library 27 JDs (Figure 7).81 Of the library 27 JDs, the JD with second generation bis-MPA-based polyester hydrophilic and hydrophobic dendrons (m = n = 1) denoted G2(C17)4-G2(OH)4 demonstrated the highest drug loading capability, encapsulating more than one mole of drug per mole of dendrimer. The study indicated that a second generation or greater polyester hydrophobic dendron was required for optimal drug loading and that an equal ratio of hydrophobic and hydrophilic blocks was also beneficial. 6576


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3EO trisubstituted hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE(3,4,5)3EO-G1-(OCH3)6 (Figure 5). As described in the previous section, Percec and co-workers also demonstrated the ability of DOX loaded DSs to retain most of their payload under physiological temperature and pH 7.4 for over 100 h, further evidencing their stability. Zhou and co-workers demonstrated the excellent stability of DSs self-assembled from the hyperbranched JD constituting library 37 (Figure 10).92 After one-half a year at room temperature, the DSs exhibited no change in size or morphology. Furthermore, no significant changes were observed upon heating from 20 to 70 °C. The noncovalent guest−host linking of the hydrophobic and hydrophilic segments of the hyperbranched JD also exhibited stability even when competitive guest or host molecules were added to the suspension of DSs. Würthner and co-workers indicated that DSs coassembled from the library 24 and 25 PBI-containing JDs (Figure 7) exhibited good stability after photopolymerization.78,127 Addition of more of the library 25 JD to the suspension of photopolymerized DSs resulted in no change in morphology, indicating that the bilayers were locked.78 The DSs were also stable in acidic and basic conditions and could be prepared in buffer.127 Utilizing the library 1 JD with second generation polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2(OH)8 (Figure 4), Terreno and co-workers reported the aggregation of the DSs into structures with diameters greater than 1000 nm in buffer.93 To avoid this problem, the JDs were coassembled with 5% (by mole) DSPE-PEG-COOH, which is an anionic lipid that can reduce electrostatic forces and thereby stabilize vesicles, giving a homogeneous suspension of DSs with 100−130 nm diameters. Terreno and co-workers have since employed this coassembly stabilization method for all of their work with DSs. Terreno and co-workers also reported comparative stabilities of DSs in human serum at 37 °C as determined by their ability to retain the encapsulated magnetic resonance imaging (MRI) contrast agent Gadoteridol.73 Figure 47 shows that DSs selfassembled from the (3,5)-disubstituted library 8 JD (Figure 6, denoted JDG0G1(3,5) in Figure 47) demonstrated the best retention of Gadoteridol after dialysis of the tested JDs, indicating superior stability. In fact, these DSs exhibited payload retention and thus stability similar to that of conventional liposomes. On the other hand, the (3,4)-disubstituted library 8 JD (Figure 6, denoted JDG0G1(3,4) in Figure 47) and the

Figure 43. (a) UV−visible spectra from the library 9 JD (acetonide)4BnNO2-G3-(3,4,5)-3EO-G1-(OCH3)3 in THF over a period of 30 min of irradiation with UV light. (b) Photodegradation of DSs from the same JD as measured by DLS. (c) Normalized fluorescent emission intensity of the same DSs loaded with Nile red after exposure to UV light as compared to those kept in the dark. (d) Release profile of the same DSs loaded with fluorescein after exposure to UV light as compared to those kept in the dark.74 Reprinted with permission from ref 74. Copyright 2014 Royal Society of Chemistry.

3.9. Stability

DSs have been shown to possess excellent stability in time and in a variety of environments. Percec and co-workers demonstrated the stability over time of DSs at 25 °C in ultrapure water selfassembled from JDs in libraries 1, 5, 20, and 21 as shown in Figure 45.37 In almost all cases, DSs demonstrated good stability in the first 50 days after preparation. In some cases, DSs exhibited constant size for up to 244 days as was the case for the library 1 JD with second generation bis-MPA-based polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2-(OH)8 (Figure 4). In some cases, DSs were stable even upon annealing from 22 to 80 °C.37 As shown in Figure 46a, size remained nearly constant after heating for DSs self-assembled from (3,5)12G1-PE-BMPAG2-(OH)8, and polydispersity tended to decrease after annealing to higher temperatures. However, this was not true for all DSs as is apparent in Figure 46b, which shows a clear increase in size after heating to at least 50 °C for the library 5 JD with (3,4,5)-

Figure 44. (A) Release of DOX from DSs assembled from the library 1 JD (3,5)12G1-PE-BMPA-G2-(OH)8 showing excellent stability at physiological temperature and pH 7.4 and rapid release of the drug at physiological temperature and pH 5.2. (B) Comparative release of DOX from DSs assembled from JDs in libraries 1, 5, and 6 at pH 5.2 and 37 °C after addition of HCl.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science. 6577


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Figure 45. Stability in time of the size (in nm) and polydispersity (error bars) of DSs from JDs in libraries 1, 5, 20, and 21 obtained by ethanol injection at a final concentration of 0.5 mg/mL in water.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

Figure 46. Temperature dependence of size and polydispersity (PDI) by DLS of DSs from (A) the library 1 JD (3,5)12G1-PE-BMPA-G2-(OH)8 and from (B) the library 5 JD (3,5)12G1-PE-(3,4,5)-3EO-G1-(OCH3)6.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

retention, lacking stability in human serum at physiological temperature. 3.10. Toxicity

Many DSs have been reported to have minimal toxicity both in vitro and in vivo. As shown in Figure 48, Percec and co-workers compared cell viability against a control (bar A) in the presence of polymersomes (bars B and C) and DSs (bars D−F) selfassembled from libraries 5 and 6 (Figure 5).37 After 2 h, no significant reduction in cell viability was observed for any sample, and by 4 h both polymersomes and DSs showed similar levels of toxicity. Furthermore, it is expected that many JDs, including those in libraries 1, 5, and 6 and any others incorporating Percectype dendrons, in biological systems will ultimately break down into phenolic acids, which are known to be nontoxic.128,129 A study involving TEG-based dendrimers related to the JDs in libraries 5 and 6 also showed them to be nontoxic.130 Peng and co-workers have conducted extensive studies on the toxicity of library 31 JDs (Figure 7) in vitro and in vivo.86,125 No notable toxicity was observed for prostate cancer PC-3 and C4−2 cells after exposure to any library 31 JD over a range of concentrations. This is shown in Figure 49 specifically for the library 31 JD with third generation PAMAM hydrophilic dendron and hydrophobic C18 alkyl chain, which is denoted 1a in the figure.86 Moreover, micellar complexes of the same JD with siRNA showed no in vitro toxicity. The same JD and its siRNA complex were also tested for in vivo toxicity in mice.125 Figure 50, in which the JD is labeled 4, indicates no evident changes in blood chemistry (Figure 50c) and liver and kidney functions (Figure 50a and b, respectively) when the JD was

Figure 47. Concentration of Gd3+ in human serum suspension of Gadoteridol-loaded DSs self-assembled from the library 1 JDs (3,5)12G1-PE-BMPA-G2-(OH)8 (JDG1G2(3,4)) and (3,5)12G1PE-BMPA-G2-(OH)8 (JDG1G2(3,4)) and from the library 8 JDs (3,5)12G1-2-BMPA-G2-(OH)4 (JD0G1(3,5)) and (3,4)12G1-2BMPA-G2-(OH)4 (JD0G1(3,4)) before and after dialysis.73 Reprinted with permission from ref 73. Copyright 2015 Royal Society of Chemistry.

library 1 JDs with second generation bis-MPA-based polyester hydrophilic dendrons and (3,5)- or (3,4)-disubstituted Percectype hydrophobic dendrons (Figure 4, denoted JDG1G2(3,5) and JDG1G2(3,4) in Figure 47) formed DSs that exhibited poor 6578


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Figure 48. Cell viability study conducted on various DSs from libraries 5 and 6 with human umbilical vein endothelial cells (HUVEC) and CellTiterBlue cell viability assay after 1, 2, and 4 h from the moment the cells were incubated with DSs. (A) Control, EGM Endothelial Growth Media (LONZA); (B) polymersome 1, hydrogenated polybutadiene-bpoly(ethylene oxide); (C0 polymersome 2, polycaprolactone-b-poly(ethylene oxide); (D) DS 1, (3,4)12G1-PE-(3,4,5)-3EO-G1-(OMe)6 (library 5); (E) DS 2, (3,5)12G1-PE-(3,4,5)-3EO-G1-(OMe)6 (library 5); (F) DS 3, (3,4,5)12G1-PE-(3,4,5)3EO-G1-(OH)6 (library 6).37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

Figure 49. MTT assay of the toxicity of the library 31 JD 18-TRZ-1PAMAM-G3-(EDA)8 alone (1a) and complexed with siRNA (1a + Hsp27 siRNA) in prostate cancer PC-3 cells with increasing JD concentration using 50 nM siRNA.86 Reprinted with permission from ref 86. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA.

supplied alone or as complexes with siRNA. No pathological changes in the major organs of mice were observed as the result of treatment with the JD alone or with its siRNA complex as shown in Figure 51. Furthermore, evaluation of inflammatory cytokines revealed no significant inflammatory response to the JD or its siRNA complex. No significant animal weight loss was observed. Together, these results demonstrate the promising biocompatibility of the JD and its siRNA complexes. Additionally, Peng and co-workers demonstrated the excellent biocompatibility of the library 32 JD (Figure 7) in vitro and in vivo.87,101 No evident toxicity was noted for any tested cell types including PC-3, CEM, PBMC CD4+, CD4+, and PBT003 cells after exposure to the JD alone or its micellar complexes with siRNA.87 In vivo tests in mice demonstrated no identifiable toxicity as evidenced by the maintenance of normal liver and kidney functioning, blood chemistry, and major organ pathology after exposure to the JD alone or its siRNA complex.87,101 Furthermore, neither blood hemolysis nor weight loss was detected in the mice.101 Ding et al. reported minimal in vitro toxicity of micelles selfassembled from the library 33 Janus dendritic polymer (Figure 7) over a range of concentrations.89 Upon exposure to the micelles, 293T cells demonstrated no noticeable reduction in viability as will be discussed in section 3.11.3. This contrasts with polyethylenimine (PEI) alone, which at larger sizes (e.g., PEI25k) exhibits significant toxicity. Thus, incorporation of PEI into Janus dendritic polymers can significantly reduce cytotoxicity by restraining the polymer in stable micelles. Terreno and co-workers performed toxicity studies on DSs self-assembled from the (3,5)-disubstituted library 8 JD (Figure 6).73,99 No reduction in cell viability or proliferation was observed for RAW 254.7, J774A.1, or NIH/3T3 cells after

Figure 50. In vivo toxicity assessment of the library 31 JD 18-TRZ-1PAMAM-G3-(EDA)8 alone (4) and complexed with siRNA (4 + siRNA) in male C57BL/6 mice by (A) monitoring the hepatic enzyme level with the indicators of aspartate aminotransferase (AST), alanine transferase (ALT), and gamma glutamyl transferase (γ-GT), (B) examining the kidney function with the indicators of creatinine (CRE) and blood urea nitrogen (BUN), and (C) measuring the cholesterol (CHO) level in the blood following treatment.125 Reprinted with permission from ref 125. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

exposure, even at high concentrations, to the DSs alone or the DSs loaded with the MRI contrast agent Gadoteridol.73 Similar results were reported for the library 1 JD with second generation bis-MPA-based polyester hydrophilic dendrons and (3,5)disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2-(OH)8 (Figure 4).73 The DSs were also demonstrated to have minimal cytotoxicity comparable to conventional liposome formulations.99 This supports expectations that JDs incorporating Percec-type dendrons are likely to be nontoxic because they may break down into phenolic acids in biological systems.128−130 3.11. Applications

3.11.1. Hydrogels. Kostiainen and co-workers demonstrated the self-assembly into hydrogels of the library 27 JDs with 6579


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Figure 51. No notable pathology was observed in the major organs of male C57BL/6 mice treated with the library 31 JD 18-TRZ-1-PAMAMG3-(EDA)8 alone (4), complexed with siRNA (4 + siRNA), and scrambled with siRNA. PBS saline was used as a control. At the end of the experiment, mice were sacrificed, and sections of main organs were performed and stained by hematoxylin eosin saffron (HES).125 Reprinted with permission from ref 125. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

third generation bis-MPA-based polyester hydrophilic dendrons and (3,4)-, (3,5)-, and (3,4,5)-substituted Percec-type hydrophobic dendrons (Figure 7).84 The structure of these three hydrogels was discussed previously in section 3.2.3. A comparison of their mechanical properties is shown in Figure 52. The tube inversion test was used to measure the minimum gelling concentration, which was determined to be 0.2 wt % (Figure 52a). Mechanical properties were reported by rheology at this minimum concentration. As shown in Figure 52b, the three gels demonstrated invariance up to 1% strain after which structural breakdown occurred. In Figure 52c, frequency sweep analysis was performed with all samples showing gel-like responses over the full frequency range. The (3,5)- and (3,4,5)-substituted JDs had storage moduli (G′) of 1000−3000 Pa, which indicates extremely high gel strengths considering the low 0.2 wt %. In both tests, the gels assembled from (3,5)- and (3,4,5)-substituted JDs exhibited significantly greater mechanical robustness than the (3,4)-disubstituted analogue. This can be attributed to the efficient cross-linking of the fibers formed by the (3,5)- and (3,4,5)-substituted JDs. In temperature ramping experiments from 20−70 °C (Figure 52d), the gel assembled from the (3,4,5)-trisubstituted JD exhibited no sample collapse over the temperature range, whereas the other gels did at 50 °C. Thus, the (3,4,5)-trisubstituted JD formed a superior gel. The gel assembled from the (3,4,5)-trisubstituted JD was tested for its drug loading and release capability.84 The smallmolecule drug nadolol, the decapeptide gonadorelin, and the active enzyme horseradish peroxidase (HRP) were loaded into the gel and tested for release over time. Release followed firstorder kinetics with at least 50% release noted after 200 min for all samples. Payloads of higher molecular weight were released more slowly than those of lower molecular weight. Furthermore, RHP maintained its enzymatic activity throughout the load−release cycle. The advantageous mechanical and drug loading and release

Figure 52. Oscillatory rheological properties of 0.2 wt % hydrogels from third generation library 27 JDs with (3,4)-, (3,5)-, and (3,4,5)substituted Percec-type hydrophobic dendrons. (a) Images of the gel inversion test. (b) G′ and G″ values on strain sweep determined at a frequency of 1 Hz. (c) G′ and G″ values on frequency sweep at 0.1% strain. (d) G′ determined as a function of temperature (20−70 °C) at a shear stress of 0.15 Pa within the linear viscoelastic region (LVR) at a frequency of 1 Hz.84 Reprinted with permission from ref 84. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

properties of the gel make it a promising material for biomedical application. 3.11.2. Drug Delivery. Peng and co-workers prepared micelles containing the anticancer drug doxorubicin (DOX) from the library 32 JD (Figure 7) as described previously in section 3.8. These drug nanocarriers were tested in vitro and in vivo for their drug delivery capability.101 In vitro, the DOX loaded micelles were tested on DOX-sensitive breast cancer MCF-7S cells, DOX-resistant breast cancer MCF-7R cells, castration-resistant prostate cancer PC-3 cells, hepatoma HepG2 cells, and cervical cancer HeLa cells. In all cases, the DOX micelles exhibited an antiprolifation effect that was significantly 6580


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was able to effectively bypass P-glycoproteins in MCF-7R cell membranes, which efflux many anticancer drugs including DOX. Thus, the DOX micelles had promising anticancer potency even for drug-resistant cancer strains. In vivo experiments in mice bearing subcutaneous tumors derived from the resistant MCF-7R cells were also reported.101 Via the enhanced permeability and retention (EPR) effect, micelles loaded with a near-infrared fluorescent dye were shown to localize at tumors in mice. Furthermore, DOX delivered to mice via DOX micelles demonstrated significantly greater accumulation and penetration in tumors as compared to free DOX. Figure 54 demonstrates the anticancer potency of DOX loaded micelles as compared to free DOX in mice. The micelles are denoted as AmDM in Figure 54. Tumor volume increased significantly less after treatment by DOX micelles than after treatment with free DOX (Figure 54a). Relatedly, DOX micelles resulted in lower cell proliferation in tumors as compared to free DOX (Figure 54d). Furthermore, mice treated with DOX micelles did not exhibit the side effects that mice injected with free DOX did. Treatment with DOX micelles resulted in no significant change in body weight (Figure 54b). Additionally, while treatment with free DOX resulted in low survival rates over the 2-week testing period due to side effects, no mortality was observed over the same period for DOX loaded micelles (Figure 54c). This was further evidenced by the appearance of hyperemia and myocardial fiber breakage in mice treated with free DOX but not in mice treated with DOX micelles (Figure 54e). In summary, the high anticancer potency of the DOX micelles coupled with their low toxicity (see also section 3.10) makes them a promising cancer treatment. 3.11.3. Nucleic Acid Delivery. Ding et al. reported the selfassembly of the library 33 Janus dendritic polymer (Figure 7) into micelles capable of loading and delivering DNA (see also section 3.10).89 The micelles complexed with DNA to form polyplex-like assemblies131,132 with average diameter of 90 nm at the low nitrogen (on JD) to phosophate (on DNA) (N/P) ratio of 5. This diameter was significantly smaller than that of PEI1800/

Figure 53. Proposed mechanism by which DOX-loaded micelles (AmDM/DOX) from the library 32 JD avoid drug resistance in DOXresistant breast cancer MCF-7R cells.101 Reprinted with permission from ref 101. Copyright 2015 National Academy of Sciences USA.

higher than that of free DOX and Caelyx, a clinical DOX nanodrug. Especially in DOX-resistant cancer MCF-7R cells, cellular uptake of the DOX micelles was significantly faster and more effective than that of free DOX.101 A mechanism was proposed to explain this improved delivery as diagrammed in Figure 53. Cells internalized the DOX micelles by macropinocytosis followed by delivery to lysosomes. The acidic environment in lysosomes enabled release of DOX from the micelles by the proton-sponge effect (see section 3.8). The free DOX could then enter the nuclei, resulting in anticancer potency. This method of delivery

Figure 54. DOX-loaded micelles from the library 32 JD (AmDM/DOX) significantly enhanced anticancer activity and reduced toxicity in tumorxenograft mice as compared to free DOX. NSG mice were treated twice per week with free DOX and DOX-loaded micelles at DOX dose of 2.5 and 5.0 mg/kg via i.v. administration. PBS and JD (AmDM) were used as controls. (A) Tumor volume and (B) mice weight were measured twice per week. (C) Different mice survival curves were observed with different treatments. (D) Tumor proliferation was assessed with Ki-67 immunohistochemical (IHC) staining. (E) Toxicity was evaluated with histological analysis of heart tissue.101 Reprinted with permission from ref 101. Copyright 2015 National Academy of Sciences USA. 6581


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Figure 55. (A) GFP expressions in 293T cells transfected by PEI1800/DNA polyplexes, library 33 polythioether-PEI1800 micelles complexed with DNA, and PEI25k/DNA polyplexes at N/P ratios from 5 to 50. (B) Luciferase transfection efficiency of PEI1800/DNA polyplexes, polythioether-PEI1800 micelles complexed with DNA, and PEI25k/DNA polyplexes in 293T cells. (C) Cell viability of 293T cells in the presence of PEI1800, polythioether-PEI1800 micelles, and PEI25k.89 Reprinted with permission from ref 89. Copyright 2016 Royal Society of Chemistry.

polyplex refers mostly to complexes of cationic polymers with DNA and does not apply to complexes of JDs and DNA.131,132 Using Green Fluorescent Protein (GFP) and luciferase plasmids, transfection efficiency was tested on 293T cells. The DNA loaded micelles exhibited higher transfection efficiency than PEI1800/DNA polyplexes and even PEI25k/DNA polyplexes, which had previously been shown to exhibit high efficiency (Figure 55a,b). Furthermore, the low toxicity of the micelles as compared to PEI25k (see section 3.10 and Figure 55c) makes the micelle/DNA polyplexes particularly promising as gene delivery vectors. Small interfering RNA (siRNA) can efficiently silence complementary RNA transcripts, providing a powerful tool to treat disease. The ability of JD self-assemblies to load and deliver siRNA has been explored.86,87,125 Peng and co-workers determined that out of all library 31 JDs (Figure 7), only those with third generation PAMAM hydrophilic dendrons (m = 2) and C18, C20, or C22 alkyl chains could efficiently load siRNA on their micellar self-assemblies.77 Figure 56 shows the excellent transfection efficiency of the C18 JD (denoted 1a in the figure) when loaded with siRNA.86 After A549Luc cells were treated with GL3Luc siRNA loaded micelles assembled from the C18 JD, luciferase expression was nearly completely inhibited at a concentration of 50 nM siRNA (Figure 56a). Similarly, when loaded with siRNA targeting heat shock factor 1 (HSF1) and siRNA targeting heat shock protein 27 (Hsp27), micelles assembled from the C18 JD were able to significantly downregulate Hsp27 mRNA expression and Hsp27 protein levels in human prostate cancer PC-3 cells (Figure 56b,c). This transfection potency was significantly reduced for the analogues with C20 and C22 alkyl chains.125 As shown in Figure 57, experiments and simulations were conducted to explain the higher transfection efficiency of the C18 JD (denoted 4 in Figure 57) as compared to the C20 and C22 JDs (denoted 5 and 6, respectively, in Figure 57).125 Heparin,

Figure 56. Library 31 JD 18-TRZ-1-PAMAM-G3-(EDA)8 (1a)mediated siRNA delivery and gene silencing in A549Luc cells (A) and heat shock protein 27 (Hsp27) in human prostate cancer PC-3 cells (B,C).86 Reprinted with permission from ref 86. Copyright 2012 WileyVCH Verlag GmbH & Co. KGaA.

DNA complexes (more than 800 nm in diameter), suggesting its suitability for cellular uptake. It should be noted that the term 6582


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Figure 57. (A) siRNA release from complexes with library 31 JDs having C18 (4), C20 (5), and C22 (6) hydrophobic alkyl tails was assessed using heparin-coupled ethidium bromide fluorescent assays. (B) Molecular dynamics (MD) simulations of 4−6 were used to determine free energy of effective binding (ΔGbind,eff), number of effective charges (Neff), and effective-charge-normalized free energy of binding (ΔGbind,eff/Neff) for siRNA/JD complexes. (C) Atomistic MD simulations of JD self-assembly in the presence of siRNA. Water is omitted for clarity. Color code: dark green, JDs; light green sticks-and-balls, terminal charged amine groups of JDs; red ribbon, siRNA; light and dark gray spheres, Cl− and Na+, respectively.125 Reprinted with permission from ref 125. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 58. Library 31 JD 18-TRZ-1-PAMAM-G3-(EDA)8 (4)-mediated siRNA delivery and gene silencing in mice. Nude mice bearing prostate cancer PC-3 tumors were randomly selected for treatment with Hsp 27 siRNA/4 and with PBS, 4 alone, and scrambled siRNA/4 as controls (3 mg kg−1 siRNA and 4 at an N/P ratio of 5). Treatments were administered by intraperitoneal injection twice per work for a period of 4 weeks. (A) Western blotting was used to measure gene silencing of Hsp27 with quantification by ImageJ software.105 (B) Inhibition of tumor growth and (C) the antiproliferation activity in tumors was determined by measuring tumor size and by Ki-67 IHC staining, respectively. (D) During the treatment period, the body weight of the mice was recorded.125 Reprinted with permission from ref 125. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA.

which is a negatively charged polysaccharide, competes with siRNA for binding to the dendrimers. At high concentrations of

herapin, micelles assembled from the C18 JD exhibited greater release of siRNA (Figure 57a). This suggested that the C18 JD 6583


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energy of binding values (ΔGbind,eff/Neff) calculated for the C20 and C22 JD micelles demonstrate that those micelles are less capable of releasing their payload because they bind it so well. As a result, the C18 JD micelles form the best delivery system because they can efficiently bind siRNA, but not so tightly that they cannot release it. The siRNA loaded C18 JD micelles were also used for in vivo experiments with mice.86,125 As shown in Figure 58, which denotes the C18 JD as 4, treatment of prostate cancer xenographed nude mice with C18 micelles loaded with siRNA targeting Hsp27 resulted in significant reduction of Hsp27 expression (Figure 58a), as well as reduction in tumor growth and cell proliferation as compared to controls (Figure 58b,c).125 Furthermore, no toxicity was observed for the C18 JD micelles (see section 3.10, Figure 51, and Figure 58d). Thus, the C18 JD micelles hold great promise as an effective siRNA delivery system for treatment of disease. Additionally, the decoration of the amine-presenting hydrophilic dendron with argenine resulted in enhanced cellular uptake while also exhibiting no cytotoxicity.88 Peng and co-workers also demonstrated that the library 32 JD (Figure 7) could self-assemble into micelles capable of loading and delivering siRNA via macropinocytosis to cells in vitro and in vivo.87 For instance, in an in vivo experiment with prostate cancer xenographed mice, the library 32 JD micelles loaded with siRNA targeting Hsp27 effectively downregulated Hsp27 expression and reduced tumor growth as compared to controls. These micelles were also reported to show no evident toxicity (see section 3.10). 3.11.4. Magnetic Resonance Imaging (MRI) and Theranostics. Magnetic resonance imaging (MRI) has been a focus of the application of nanocarriers including liposomes and polymersomes.133 In this vein, Terreno and co-workers have explored DSs as tools for molecular imaging applications by loading them with MRI contrast agents.73,93,99,100 The library 1 JD with second generation polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-G2-(OH)8 (Figure 4) and its library 39 gadolinium(III) (Gd3+)-tagged analogue (Figure 11) were used in early investigations.93 As was previously described in section 3.9, Terreno and co-workers stabilized these DSs with a small amount of the lipid DSPE-PEG-COOH. The effectiveness of an MRI contrast agent can be measured by its relaxivity (r1), which is the longitudinal relaxation enhancement of water protons induced by a 1 mM solution of Gd3+ (see Figure 59), with higher values suggestive of better contrast. Coassembly of the library 1 JD and the library 39 JD resulted in DSs that exhibited a relaxivity of 15.6 mM−1 s−1, whereas coassembly of the library 1 JD with the lipophilic Gd-DOTAMA(C18)2 complex (see Figure 60 for structure) resulted in a higher relaxivity of 18.7 mM−1 s−1.93 Thus, coassembly of the library 1 JD with Gd-DOTAMA(C18)2 was advantageous as compared to coassembly with the Gdtagged library 39 JD. The laboratory also encapsulated the clinically approved MRI contrast agent Gadoteridol in the hydrophilic cavity of DSs self-assembled from the same library 1 JD, giving a relaxivity of 3.65 mM−1 s−1, which is only slightly lower than that of free Gadoteridol.93 In a comparative study, Terreno and co-workers tested the stability of Gadoteridol loaded DSs self-assembled from library 1 JDs with second generation bis-MPA-based polyester hydrophilic dendrons and (3,4)- or (3,5)-disubstituted Percec-type hydrophobic dendrons (Figure 4), as well as from both library 8 JDs (Figure 6).73 It was determined that the (3,5)-disubstituted library 8 JD denoted (3,5)12G1-2-BMPA-G2-(OH)4 produced

Figure 59. Normalized longitudinal relaxivity (r1p) calculated for Gadoteridol-loaded DSs from the library 8 JD (3,5)12G1--BMPA-G2(OH)4 and for Gadoteridol-loaded conventional liposomes.73 Reprinted with permission from ref 73. Copyright 2015 Royal Society of Chemistry.

Figure 60. Top: Chemical structures of amphiphilic Gd-chelates and the library 8 JD used for assembly of DSs. Bottom: Representative axial T1w images of melanoma tumors in mice immediately before and 10 min after treatment with GdDOTAGA(C18)2- or GdDOTAMA(C18)2DSs.99 Reprinted with permission from ref 99. Copyright 2015 Royal Society of Chemistry.

micelles were better transfection vectors because of their greater ability to release siRNA. Simulations by atomistic molecular dynamics (MD) provided further evidence for this hypothesis (Figure 57b,c). The higher effective-charge-normalized free 6584


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Figure 61. Representative axial T1w images showing evolution of T1 contrast over a 15-day period of monitoring the liver, spleen, and tumor in melanoma-bearing mice after treatment with PLP-loaded DSs coassembled from the library 8 JD (3,5)12G1-2-BMPA-G2-(OH)4 and GdDOTAGA(C18)2.100 Reprinted with permission from ref 100. Copyright 2017 Elsevier.

therapeutic capability. Given the effectiveness of DSs coassembled from the (3,5)-disubstituted library 8 JD and GdDOTAGA(C18)2 for contrast enhancement, as well as their lack of toxicity (see section 3.10), Terreno and co-workers tested the theranostic capability of the system by additionally loading the DSs with the anticancer drug prednisolone phosphate (PLP).100 Mice bearing melanoma tumors were injected with the DSs twice over 15 days. As demonstrated by Figure 61, the DSs significantly enhanced contrast for MRI imaging and sustained this enhancement over the 2-week period, enabling continuous monitoring of tumor growth. Additionally, as shown in Figure 62, tumor growth was significantly reduced for the drug loaded DSs. The use of DSs is associated with lower cost and simpler preparation as compared to liposomes. Thus, DSs that combine MRI contrast agents and drugs in the same system hold promise as theranostic cancer treatments.

Figure 62. Tumor growth in melanoma-bearing mice was reduced for PLP-loaded DSs coassembled from the library 8 JD (3,5)12G1-2BMPA-G2-(OH)4 and GdDOTAGA(C18)2 (PLP-Gd-DS) as compared to DSs without PLP (Gd-DS). PLP-loaded conventional liposomes coassembled with GdDOTAGA(C18)2 (PLP-Gd-lipo) also exhibited reduced tumor growth.100 Reprinted with permission from ref 100. Copyright 2017 Elsevier.

4. AMPHIPHILIC JANUS GLYCODENDRIMERS (JGDs) The second class of JDs that will be considered in this Review are those conjugated to sugars and denoted Janus glycodendrimers (JGDs). These molecules have been categorized into libraries 44−57 as shown in Figures 63−65 on the basis of their structure as twin−twin, single−single, or twin−mixed designs. Syntheses of JGDs follow mainly convergent methodologies and are detailed in this section. They were synthesized to provide programmable models of biological glycan multivalent display to uncover fundamental asepects of lectin recognition (see bottom of section 1.1). Prior platforms for the presentation of carbohydrates such as glycopolymers52−58 and glycodendrimers38,59−61 enable some control over sugar density but do not exhibit overall vesicular architectures consistent with most biological membranes. Glycoliposomes,62−65 which require coassembly from phospholipids, glycolipids, and other components for stability, exhibit architectures similar to biological systems but do not allow for control of sugar density and sequence. On the other hand, JGDs self-assemble into vesicles denoted glycodendrimersomes (GDSs) and other complex architectures consistent with biological systems while maintaining programmable sugar density and sequence, offering a superior platform for lectin recognition.

the most stable DSs under physiological conditions. Furthermore, as shown in Figure 59, these Gd loaded DSs exhibited significantly higher relaxivities over a range of temperatures as compared to conventional liposome analogues. This was attributed to the greater water permeability of the DSs as compared to liposomes, which reduces quenching of the contrast agent. As a result, DSs exhibit advantages over liposomes as tools for imaging. Both Gadoteridol-loaded DSs and liposomes exhibited similar blood circulation lifetimes of 70−80 min, which is significantly higher than that of free Gadoteridol, demonstrating the advantage of using nanoparticles as contrast probes. DSs coassembled from the (3,5)-substituted library 8 JDs (denoted JD0G1(3,5) in Figure 60) and the lipophilic contrast agent GdDOTAGA(C18)2 or GdDOTAMA(C18)2 (structures shown in Figure 60) were used to compare the effectiveness of the two probes in vivo.99 As is apparent from the MRI images of grafted melanoma tumors on mice shown in Figure 60, the DSs coassembled with GdDOTAGA(C18)2 exhibited superior contrast enhancement as a reflection of a more than doubled relaxivity as compared to the other probe. Theranostics has emerged as a promising method of cancer therapy, in which nanoassemblies combine diagnostic and

4.1. Glycosylation and Stereochemistry

The sugars D-galactose, D-mannose, and D-lactose have been extensively used in the preparation of JGDs. Syntheses of JGDs require stereoselective functionalization of the desired sugar, 6585


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Figure 63. Twin−twin JGDs.71,134

26.7 to yield the desired azide-functionalized sugar in excellent yield was accomplished with Zemplén de-O-acetylation with sodium methoxide in methanol. As shown in Scheme 28, an analogous synthesis was performed on lactose to give the β-Dlactosyl azide 28.27. Synthesis of the related α-D-mannosyl azide 27.17 is shown in Scheme 27.71,134 Mannose was first acetylated with acetic anhydride in the presence of iodine as catalyst to give the α- and β-anomer mixture 27.15, which was converted to the α-Dmannosyl azide 27.16 by reaction with trimethylsilyl azide in the presence of tin(IV) chloride. Subsequently, the desired azidefunctionalized mannose was achieved by deprotection with sodium methoxide in methanol. Scheme 28 also outlines the synthesis of the related azidoethyl lactoside 28.30. Acetylated lactose 28.24 was treated with 2bromoethanol in the presence of boron trifluoride in DCM to give 28.28, which was directly reacted without purification with sodium azide in DMF to give the protected azidoethyl lactoside 28.29. Standard Zemplén de-O-acetylation was performed to afford the desired compound 28.30. Azides have also been attached to sugars via oligo(ethylene) glycol linkers.71,134 As shown in Scheme 26, synthesis of the galactose analogue 26.11 began with tosylation of oligo-

most commonly with an azide or an alkyne, before conjugation to a JD scaffolding, usually by copper-catalyzed click chemistry. Sugars are notoriously difficult to work with because of their sensitivity to a variety of common reaction conditions. Percec and co-workers employed the methods outlined in Schemes 26−28 to prepare sugars for conjugation to JD scaffoldings.71,134 These procedures were chosen upon reviewing the literature.135−138 In general terms, glycosylation was conducted such that the substituent attached to the anomeric carbon was trans to the vicinal hydroxyl group of the sugar rather than cis, as this stereochemistry afforded the most stable reaction intermediates and was thus most accessible. This stereochemistry is also employed by biological membranes.139 A review of Oglycosylation methods has recently been published.140 Scheme 26 describes the synthesis of galactosyl azides and propargylated galactosides.71,134 Preparation of D-galactose by direct azide-functionalization of the anomeric carbon (compound 26.8) began with acetylation of galactose (26.4) with acetic anhydride at reflux in the presence of sodium acetate to give β-anomer 26.5. Subsequent bromination of the anomeric carbon with hydrogen bromide in acetic acid afforded α-anomer 26.6, which was treated directly with sodium azide in DMSO to give acetate-protected β-D-glycosyl azide 26.7. Deprotection of 6586


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related α-D-mannosyl oligo(ethylene) glycol azide 27.20 was also synthesized by the same procedure. Alkyne-functionalized sugars have also been prepared via oligo(ethylene) glycol linkers.71,134 As shown in Scheme 26, synthesis of the galactose analogue 26.13 began with the propargylation of the oligo(ethylene) glycol linker 26.1 by reaction with propargyl bromide in the presence of potassium tbutoxide in THF to give 26.3. Conjugation of 26.3 to the acetylated galactose β-anomer 26.5 was achieved with boron trifluoride in DCM to give the propargylated β-D-glycosyl oligo(ethylene) glycol 26.12, which was deprotected with Zemplén de-O-acetylation to afford the propargylated galactoside 26.13. The lactose analogue 28.35 was synthesized by the same method as illustrated in Scheme 28. Furthermore, as shown in Scheme 27, starting with acetylated mannose 27.15, the related propargylated α-D-mannoside 27.22 was also synthesized by the same procedure. Gillies and co-workers developed isothiocyanate-functionalized sugars for conjugation to amine-functionalized JDs.141 In particular, Scheme 29 outlines the synthesis of isothiocyanatefunctionalized α-galactoside 29.9.142 Acetylated α- and β-anomer mixture 27.1 was brominated at the anomeric carbon to afford the α-anomer 29.2, which was converted to alkene 29.3 by reaction with allyl phenyl sulfone. Ozonolysis of the alkene afforded the corresponding aldehyde 29.4.143 Reduction with sodium borohydride gave alcohol 29.5, which was mesylated and then converted to azide 29.7 with sodium azide.144 Deprotection via Zemplén de-O-acetylation gave 29.8, which was reduced by palladium-catalyzed hydrogenation to corresponding amine 29.9. Finally, the desired isothiocyanate 29.10 was obtained by reaction of the amine with thiophosgene.142

Scheme 26. Stereoselective Synthesis71,134 of Galactosyl Azides and Propargylated Galactosidesa

Reagents and conditions: (i) TsCl, pyridine, DCM (23 °C); (ii) propargyl bromide, tBuOK, THF (23 °C); (iii) AcONa, Ac2O, reflux; (iv) 33% HBr/AcOH (25 °C); (v) NaN3, DMSO (23 °C); (vi) 1 M MeONa in MeOH (23 °C); (vii) BF3·Et2O, DCM (0−23 °C); (viii) NaN3, DMF (23 °C). a

Scheme 27. Stereoselective Synthesis71,134 of Mannosyl Azides and Propargylated Mannosidesa

4.2. Twin−Twin Design

The first JGDs synthesized had twin−twin designs, in which two hydrophobic dendrons are connected through a pentaerythritol core to two glycosylated hydrophilic fragments. These JGDs constitute libraries 44−48 as shown in Figure 63, and their generic names are given under each structure. Each library is subdivided into two libraries denoted “a” and “b” with the difference being the respective absence or presence of oligo(ethylene) glycol spacers in the hydrophilic fragments of the JGDs.71 Syntheses of these molecules began with construction of the hydrophobic scaffoldings functionalized with azides or alkynes as shown in Scheme 30.71 For libraries 46 and 47, the hydrophobic scaffoldings 30.6 with alkyne-functionalization directly at the apex were synthesized from pentaerythritol by monoprotection with p-anisaldehyde to give methoxybenzylidene acetal 30.2. Subsequent etherification with propargyl bromide and deprotection gave diol 30.4, which underwent esterification with Percec-type hydrophobic dendrons 30.5 to give the desired hydrophobic scaffoldings. The related hydrophobic scaffoldings 30.13 with alkyne-functionalization via succinic ester spacers used for library 48 were synthesized by monoprotection of pentaerythritol with benzaldehyde to give benzylidene acetal 30.10. Subsequent esterification with Percec-type hydrophobic dendrons 30.5 afforded 30.12 after deprotection. A second esterification with alkyne-functionalized anhydride 30.14 was conducted to give the desired scaffoldings. For libraries 44 and 45, the azide-functionalized hydrophobic scaffoldings 30.9 were synthesized from pentaerythritol by bromination to give 30.7, which was converted to the corresponding azide 30.8 via sodium

Reagents and conditions: (i) TsCl, pyridine, DCM (23 °C); (ii) propargyl bromide, tBuOK, THF (23 °C); (iii) I2, Ac2O (23 °C); (iv) TMSiN3, SnCl4, DCM (23 °C); (v) 1 M MeONa in MeOH (23 °C); (vi) BF3·Et2O, DCM (0−23 °C); (vii) NaN3, DMF (23 °C). a

(ethylene) glycol to afford alcohol 26.2. Conjugation of 26.2 to the acetylated galactose β-anomer 26.5 was achieved with boron trifluoride in DCM to give the β-D-glycosyl oligo(ethylene) glycol tosylate 26.10, which was converted to the corresponding azide by treatment with sodium azide in DMF. In the final step, deprotection of the sugar was achieved with Zemplén de-Oacetylation. The lactose analogue 28.33 was synthesized by the same method as illustrated in Scheme 28. Furthermore, as shown in Scheme 27, starting with acetylated mannose 27.15, the 6587


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Scheme 28. Stereoselective Synthesis71,134 of Lactosyl Azides and Propargylated Lactosidesa

Reagents and conditions: (i) TsCl, pyridine, DCM (23 °C); (ii) propargyl bromide, tBuOK, THF (23 °C); (iii) AcONa, Ac2O, reflux; (iv) 33% HBr/AcOH (23 °C); (v) NaN3, Bu4N+HSO4−, DCM (23 °C); (vi) 1 M MeONa in MeOH (23 °C); (vii) 2-bromoethanol, BF3·Et2O, DCM (0−25 °C); (viii) NaN3, DMF (23 °C); (ix) BF3·Et2O, DCM (0−23 °C). a

Scheme 29. Stereoselective Synthesis142−144 of α-Galactosyl Isothiocyanatea

4.3. Single−Single Design

Several libraries of single−single JGDs, libraries 49−53 as shown in Figure 64 with generic names given under each structure, have been synthesized. Originally theorized by splitting twin−twin designs in half, single−single JGDs contain one hydrophobic dendron connected to a glycosylated hydrophilic fragment.134 They may also consist of one hydrophilic dendron coupled with a hydrophobic fragment as in the case of library 53.141 Percec and co-workers developed the syntheses for libraries 49−51.134 As shown in Scheme 32, the hydrophobic dendrons were synthesized with either alkyne- or azide-functionalized apexes. For the alkyne-functionalized hydrophobic dendron 32.8 used in library 51, 1,3-propanediol was reacted with propargyl bromide in the presence of potassium tert-butoxide in THF to give 32.3, which was coupled to the Percec-type hydrophobic dendron 32.7 by esterification via DCC and DPTS. Syntheses of the azide-functionalized hydrophobic dendrons 32.9 used in libraries 49 and 50 involved substitution of either 2chloroethanol or 3-bromo-1-propanol with sodium azide followed by esterification with Percec-type hydrophobic dendron 32.7 by DCC and DPTS. The syntheses of libraries 49−51 were completed by copper-catalyzed click chemistry of the appropriate azide- or alkyne-functionalized sugar (see section 4.1) with the corresponding alkyne- or azide-functionalized hydrophobic dendron. These accelerated orthogonal syntheses are shown in Scheme 33. Gillies and co-workers prepared libraries 52 and 53, denoted generically as L-Gn-Gal, which consist of α-galactose-function-

Reagents and conditions: (i) 30% HBr/AcOH (23 °C); (ii) allyl phenyl sulfone, bis(tributyltin) benzene, hν (23 °C); (iii) (1) O3, DCM (23 °C) and (2) PPh3; (iv) NaBH4, MeOH (0 °C); (v) MsCl, 0.2 M pyridine (0 °C); (vi) NaN3, DMF (23 °C); (vii) 1 M MeONa in MeOH (23 °C); (viii) H2/Pd, MeOH (23 °C); (ix) thiophosgene, DIPEA, EtOH/water (0−23 °C). a

azide. Finally, esterification with Percec-type hydrophobic dendrons 30.5 yielded the desired compounds. The JGDs were then completed by conjugation of azide- and alkyne-functionalized sugars (see section 4.1) to the alkyne- and azide-functionalized hydrophobic scaffoldings by copper-catalyzed click chemistry conducted with copper(II) sulfate in the presence of sodium ascorbate in THF/water. A representative example of this orthogonal synthesis is shown in Scheme 31. 6588


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Scheme 30. Synthesis71 of Twin-Hydrophobic Dendrons Functionalized with Alkyne and Azide Groups at Their Apexa

Reagents and conditions: (i) p-anisaldehyde, HCl, water (25 °C); (ii) propargyl bromide, NaH, DMF (0−25 °C); (iii) CH3CO2H−water (50 °C); (iv) DCC, DPTS, DCM (25 °C); (v) HBr, H2SO4−AcOH (reflux); (vi) NaN3, DMSO (110 °C); (vii) benzaldehyde, HCl, water; (viii) H2, Pd/C, MeOH−DCM (25 °C); (ix) 14, DMAP, pyridine, DCM (25 °C). Reprinted with permission from ref 71. Copyright 2013 American Chemical Society. a

alized, lipid-containing JGDs.141 A representative synthesis of one of the molecules that constitutes these libraries is shown in Scheme 34. Specifically, the synthesis of the library 53 molecule with second generation hydrophilic dendron denoted L-G2-Gal is outlined. The Boc-protected β-alanine-functionalized polyester hydrophilic dendrons (G2-NHBoc in Scheme 34) were synthesized divergently from propargyl alcohol.142 The azido diglyceride 34.2 was synthesized from racemic glycidol. The hydrophilic dendron was coupled with the hydrophobic lipid fragment by copper-catalyzed click chemistry. Deprotection by removal of the Boc-protecting groups was achieved with 1:1 trifluoroacetic acid (TFA):DCM to give L-G2-NH2 in Scheme 34. In the final step, isothiocyanate-functionalized α-galactoside 34.3 (see section 4.1) was coupled with L-G2-NH2 in the presence of N,N-diisopropylethylamine (DIPEA) in DMF to afford the desired JGD L-G2-Gal. The same procedure was used for the syntheses of the other molecules in library 53. The library 52 molecule was also synthesized similarly except that no hydrophilic dendron was incorporated. Instead, Boc-protected propargylamine was coupled directly with the azido diglyceride. It should be noted that this molecule is not a true JGD because it does not incorporate any dendrimers in its structure, but it can be thought of as the generation zero version of library 53 and is thus included.

The syntheses of twin−mixed designs originate with tris, which forms the core of these JGDs.134 The simplest twin−mixed design constitutes library 54, and its synthesis is outlined in Scheme 35. Tris (35.1) was first protected with acetonide to give acetal 35.2. The amine group on tris underwent amidation with alkyne-functionalized anhydride 35.7 in the presence of triethylamine in DCM to give alkyne 35.3. The free hydroxyl group on the tris core was then esterified with hydrophilic dendron 35.8 (see Scheme 2 for synthesis) to afford 35.4, which was deprotected to give diol 35.5. Subsequent esterification with Percec-type hydrophobic dendrons 35.9 afforded the desired JD scaffolding 13.6. The twin−mixed JGDs in library 54 were then completed as shown in Scheme 36.134 Copper-catalyzed click chemistry was utilized to couple azide-functionalized TEGlinked glycosides to the alkyne-presenting JD scaffolding to give the desired JGDs. Syntheses of libraries 55−57 were conducted with identical methodologies but utilized different hydrophilic and hydrophobic dendrons as schematized for the lactose analogues in Figure 66.95,145 4.5. Sequence-Defined JGDs

Percec and co-workers prepared a series of compounds with defined sugar sequence and density.95,145 The syntheses of these molecules have already been considered (see sections 4.2, 4.3, and 4.4). The short names of these molecules are shown above their structures in Figures 67 and 68. 1-Lac and 1-Man are single−single JGDs (taken from library 51), and 2-Lac and 2-Man are their twin−twin analogues (taken from library 46b). All of these structures exhibit 100% sugar coverage on their hydrophilic segments. The twin−mixed JGDs 3-Lac and 3-Man (taken from library 54) exhibit 25% sugar coverage on their hydrophilic segments with a 3:1 ratio of

4.4. Twin−Mixed Design

Libraries 54−57 as shown in Figure 65 have twin−mixed designs. Originally conceived by combining a single−single JD with a single−single JGD, twin−mixed designs incorporate twin hydrophobic dendrons coupled to a hydrophilic dendron and a glycosylated hydrophilic fragment.134 6589


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Scheme 31. Representative Example of Modular Synthesis134 of Twin−Twin JGDsa

a

Reagents and conditions: (i) CuSO4·5H2O, sodium ascorbate, THF/water (25 °C).

Scheme 32. Synthesis134 of Alkyne- and Azide-Functionalized Hydrophobic Dendronsa

a Reagents and conditions: (i) t-BuOK, THF (23 °C); (ii) NaN3, DMF (80 °C); (iii) DCC, DPTS, DCM (23 °C).

Figure 64. Single−single JGDs.134,141

3EO:sugar. The twin−mixed JGDs 4-Lac and 4-Man exhibit 14% sugar coverage of their hydrophilic segments with a 6:1 ratio of 3EO:sugar. The rest of the twin−mixed JGDs including 5aLac, 5a-Man, 5b-Lac, 5b-Man, 6a-Lac, 6a-Man, 6b-Lac, and 6bMan (taken from libraries 56 and 57) all exhibit 11% sugar coverage with an 8:1 ratio of 3EO:sugar. The difference between their designation as “5” or “6” is the respective absence or presence of an additional TEG-linker between the triazole ring and the sugar. Furthermore, designation as “a” or “b” is conferred on the basis of the sequence-defined location of sugar conjugation at the eighth or ninth TEG chain, respectively,

counting from the left. These molecules were used to assess the impact of sugar sequence and density on the bioactivity of selfasssemblies, as will be discussed in sections 6.4 and 6.5.

5. SELF-ASSEMBLY OF AMPHIPHILIC JGDs JGDs have been reported to self-assemble into a variety of structures in aqueous media including vesicles denoted glycodendrimersomes (GDSs), cubosomes denoted glycodendrimercubosomes (GDCs), micelles, and solid lamellae.71 The characterization of these assemblies and their basic properties 6590


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Scheme 33. Modular Synthesis134 of Single−Single JGDs in Libraries 49−51a

a

Reagents and conditions: (i) CuSO4·5H2O, sodium ascorbate, THF/water (25 °C).

Scheme 34. Representative Synthesis141 of an α-Gal-Functionalized JD (Library 53)a

a


the injection method generally by dissolving JGDs in either THF or ethanol followed by rapid injection into aqueous media and a few seconds of vortexing.71,134,145 Gillies and co-workers reported the preparation of submicrometer GDSs via the oilin-water method for the self-assembly of library 52 and 53 JGDs (Figure 64) into GDSs by dissolving the JGDs in THF or DMSO and adding water, stirring for 30 min, and finally dialyzing against 10 mM phosphate buffer for 24 h.141 Thin film hydration has

including predictable size, mechanical properties, and stability will be discussed in this section. 5.1. Method of Preparing Glycodendrimersomes (GDSs) and Other Complex Architectures

Glycodendrimersomes (GDSs) have been prepared by the same three methods as discussed for DSs in section 3.1, which include the injection, oil-in-water, and thin film hydration techniques. Percec and co-workers have prepared submicrometer GDSs via 6591


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Figure 65. Twin−mixed JGDs.95,134,145

Scheme 35. Synthesis134 of Twin-Hydrophobic−Single-Hydrophilic JD Scaffoldinga

Reagents and conditions: (x) p-TSA, DMP, acetone (23 °C); (xi) 7, Et3N, DCM (0 °C); (xii) 8, DCC, DPTS, DCM (23 °C); (xiii) Amberlite (acidic), MeOH (45 °C); (xiv) 9, DCC, DPTS, DCM (23 °C). Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA. a

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Scheme 36. Representative Example of Modular Synthesis134 of Twin−Mixed JGDsa

been used to prepare giant GDSs having diameters from single micrometers to several tens of micrometers. This has generally been accomplished by dissolving JGDs in dichloromethane and depositing the solution onto the bottom of a glass vial or onto a Teflon sheet in a vial and letting it evaporate. The dried film can then be hydrated at 60 °C for 24 h and vortexed for 5 s to give giant GDSs.71 5.2. Nanoscale Visualization of Morphologies Self-Assembled from JGDs

As with JDs, cryo-TEM has been used primarily for the characterization of morphologies self-assembled from JGDs at the submicrometer scale. Cryo-TEM gives superior results as compared to traditional TEM, which has also but more rarely been employed to image JGD assemblies, because it preserves the assemblies in their aqueous suspensions. 5.2.1. Soft Unilamellar GDSs. Out of the 62 twin−twin JGDs in libraries 44a−48b (Figure 63) that Percec and coworkers synthesized, 26 self-assembled into soft unilamellar GDSs.71,134 Figures 6971 and 70134 show the structures of these JGDs with yellow highlighting indicating stability in both water and buffer and blue highlighting indicating stability only in water. A total of 19 of these JGDs demonstrated stability in buffer, including in either or both phosphate buffered saline (PBS) and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES). The diameter and PDI of the GDSs, as measured by DLS, in water and buffer are indicated under the structure of each JGD. Polydispersities (PDI) ranged from 0.03 to 0.30, which falls in the range of monodisperse vesicles, and diameters ranged from as small as 36 nm to as large as 201 nm at concentrations of 0.5 mg/mL. Generally GDSs self-assembled in PBS were smaller than those self-assembled in HEPES. It was observed that JGDs with greater solubility and flexibility were more likely to form soft GDSs. Thus, branched-alkyl chains in the hydrophobic dendrons, which confer greater solubility, and TEG-spacers in the hydrophilic fragments, which increase flexibility, were advantageous for the formation of GDSs. As such, libraries 44b, 45b, 46b, 47b, and 48b proved to be particularly suitable for self-assembly into soft vesicles. Moreover, it was noted that any twin−twin JGDs containing TEG or tetra(ethylene glycol) spacers with (3,5)-disubstituted linear or branched hydrophobic dendrons are most likely to form GDSs. Other hydrophobic substitution patterns, particularly (3,4)-disubstitution, were less successful. Representative cryo-TEM images of soft GDSs from these libraries are shown in Figure 71.71 It should be noted that the GDSs self-assembled from TEG-containing JGDs (Figure 71b− d), including the galactose (Gal)-presenting (3,4)-, (3,5)-, and (3,4,5)-substituted library 45b JGDs, exhibited wall thicknesses of about 7 nm, which corresponds to twice the length of a single JGD. On the other hand, the JGD without TEG (Figure 71a), which corresponds to the mannose (Man)-presenting (3,4)disubstituted library 45a JGD, self-assembled into GDSs with walls almost 3 times thicker (∼20 nm). The thick membrane could be attributed to disordered aggregation of JGDs (thicker and lighter outer portion of wall in cryo-TEM image) surrounding a more ordered and densely packed vesicle wall (thinner and darker inner portion of the wall). These results are consistent with JGD rigidity associated with lack of TEG, which can result in kinetically trapped intermediates such as the one observed. Figure 72 illustrates the method by which GDS wall thickness was measured with cryo-TEM.71 ImageJ software105 was used to

a Reagents and conditions: (i) CuSO4·5H2O, sodium ascorbate, THF/ water (25 °C).

Figure 66. Summary of the accelerated iterative modular synthetic strategy used for the preparation of the JGDs 4-Lac, 5a-Lac, 5b-Lac, 6aLac, and 6b-Lac. “A” represents the structure between the triazole ring and the tris framework, “B” represents a second generation hydrophilic dendron, “C” represents a second generation hydrophobic dendron, and “S” represents the Lac group.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society. 6593


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Figure 67. Summary of amphiphilic JDs with different density and sequence-defined arrangement of Lac in the hydrophilic segment. The diameter (DDLS, in nm) and polydispersity (in parentheses) were determined by DLS at 0.1 mM of Lac in PBS.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society.

Gillies and co-workers reported the self-assembly of the single−single JGDs in libraries 52 and 53 (Figure 64).141 The JGD that comprises library 52 and the first and second generation JGDs (m = 0, 1) in library 53 were determined to self-assemble into unilamellar GDSs with diameters ranging from about 200 to 300 nm. As determined from DLS, a polydispersity (PDI) of 0.08 was reported for the library 52 JD, whereas the library 53 JDs resulted in higher polydispersities of 0.36−0.39. These findings were corroborated with TEM images. It should be noted that these GDSs were self-assembled from JGDs containing significantly greater carbohydrate to alkyl chain ratios as compared to previously prepared JGDs. It is possible that the greater hydrophobicity of the bis-MPA dendrons as compared to the previously used TEG chains could compensate for the greater hydrophilic character imparted by higher sugar density, enabling formation of GDSs.

calculate gray scale line profile intensity as shown in the graphs underneath the images. The distinct drop in intensity across the membrane enabled calculation of the width. GDS membrane width was generally found to be much narrower than that of polymersomes and only slightly thicker than that of DSs. On the basis of the principles learned from the self-assembly of twin−twin JGDs, Percec and co-workers synthesized the single− single JGDs that comprise libraries 49−51 (Figure 64).134 All of these molecules contain TEG spacers and (3,5)-disubstituted Percec-type hydrophobic dendrons, both of which are features that had been observed to facilitate formation of soft vesicles. As such, self-assembly of all JGDs in these libraries gave soft, unilamellar GDSs in both water and buffer (HEPES and PBS). As measured by DLS, polydispersities (PDI) ranged from 0.13 to 0.35 with most near 0.2, and diameters ranged from 40 to 145 nm at concentrations of 0.5 mg/mL. Representative cryo-TEM images of these GDSs are shown in Figure 73. 6594


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Figure 68. Summary of Man-presenting amphiphilic JGDs. Their diameter (DDLS, in nm) and polydispersity (in parentheses) were measured by DLS at 0.1 mM of Man in HEPES.145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

Soft unilamellar GDSs stable in water and PBS buffer were also prepared from all twin−mixed lactose (Lac)-presenting JDs in Figure 65, including libraries 54−57.95,134 Representative cryoTEM images of these GDs are shown in Figure 74 for the Laccontaining JDs in libraries 55−57. A representative cryo-TEM image of the Lac-presenting library 54 JGD is shown in Figure 75c. As will be discussed in more detail in the next section, Lac apparently confers the ability of these JGDs to self-assemble into unilamellar GDSs because the same JD scaffoldings presenting other sugars form onion-like GDSs. Figure 67 gives the diameters and polydispersities (PDI) of the GDSs prepared in PBS under each corresponding JGD structure. As determined by DLS, diameters ranged from 51 to 151 nm, and polydispersities ranged from 0.14 to 0.24 at concentrations of 0.1 mM Lac in PBS. 5.2.2. Onion-Like GDSs. Percec and co-workers have reported several examples of multilamellar vesicles denoted onion-like GDSs self-assembled from JGDs in buffer, all of which have twin−mixed designs.134,145 The biological significance of such vesicles was previously discussed in section 3.2.2. The first onion-like GDSs were prepared from Man- and Gal-presenting library 54 JGDs. Representative cryo-TEM images of these

onion-like GDSs are shown in Figure 75a,b for the Man and Gal analogues, respectively. All of the Man-presenting JGDs in libraries 56−57 also self-assembled into onion-like GDSs as evidenced by representative cryo-TEM images in Figure 76. The Man-presenting library 55 JGD formed cubosomes, which will be discussed in detail in the next section. As measured by DLS and shown in Figure 68, diameters ranged from 150 to 242 nm, and polydispersities ranged from 0.10 to 0.17 at concentrations of 0.1 mM Man in HEPES. Depending on concentration, the GDSs could exhibit 2 to as many as 12 or more bilayers as shown in cryo-TEM images and corresponding 3D intensity plots of the Man-presenting library 56a JGD (Figure 77). A more detailed description of this concentration dependence will be provided in section 5.3. It should be noted that the Lac analogues of the library 54−57 JGDs strictly form unilamellar GDSs. Thus, it is apparently the structure of the conjugated sugar that determines whether these JGDs form unilamellar or onion-like GDSs. This represents a newly discovered and not yet fully understood influence of JGD headgroup on the structure of self-assemblies.145 6595


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Figure 69. Part I: Twin−twin JGDs that self-assemble into stable GDSs in water (blue highlighting) and both water and buffer (yellow highlighting). GDS diameter and polydispersity are given for each suspension medium.71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

5.2.3. Glycodendrimercubosomes (GDCs). Cubosomes, which exhibit bicontinuous cubic phases as discussed in section 3.2.3, self-assembled from JGDs denoted glycodendrimercubosomes (GDCs) have been prepared from only two JGDs. These

include the Man-presenting twin−twin library 44b JGD with ethylene glycol spacers (m = 1) and (3,5)-disubstituted Percectype hydrophobic dendrons denoted (3,5)12G1-PE-TRZi1EOMan2 (short name 2i-Man),71 as well as the Man-presenting 6596


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Figure 70. Part II: Twin−twin JGDs that self-assemble into stable GDSs in water (blue highlighting) and both water and buffer (yellow highlighting). GDS diameter and polydispersity are given for each suspension medium.134 Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

cubic symmetry, was conducted with 3D cryo-electron tomography (Figure 80).145,146 This investigation revealed that the interior of the GDCs exhibits two sets of water channels arranged in a double diamond network. More importantly, as is apparent from the cryo-TEM images, the morphology of the outer membrane is modulated by the interior network of cubically arranged water channels, resulting in an undulating contour with variable surface curvature. This architecture gives rise to a unique sugar topology as compared to those generated by the smooth surface contour of uni- and multilamellar GDSs. The biological activity and relevance of this unusual architecture will be discussed in detail in section 6.6.

library 55 JGD denoted 3EO(1,2,3,4,5,6)-3EOMan(7) (short name 4-Man) (Figures 63 and 65).145,146 Cryo-TEM images of GDCs self-assembled from 2i-Man are shown in Figure 78 at a JGD concentration of 1.0 mg/mL in water. Analysis of the Fourier transform of these cryo-TEM images indicated that the GDCs have Pn3̅m cubic symmetry. At lower concentration, 2iMan self-assembled into GDCs with a diameter of 222 nm and a polydispersity of 0.05. These GDCs were not stable in buffer. Cryo-TEM images of GDCs self-assembled from 4-Man, which are the first reported synthetic sugar-presenting cubosomes stable in water and buffer, are shown in Figure 79.146 At a concentration of 1.0 mM in HEPES, they exhibited a diameter of 242 nm and a polydispersity (PDI) of 0.10. A detailed structural analysis of this GDC, which also exhibits Pn3̅m 6597


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81a,c), solid GDSs (Figure 81b), and polygonal GDSs (Figure 81d) self-assembled from select library 44a and 46a JGDs. Solid lamellae are 2D sheet-like morphologies consisting of crystalline or amorphous membranes below their glass transition temperatures, preventing the formation of closed 3D vesicles. Forming as the result of trapped kinetics, solid (or hard) vesicles are similar to soft unilamellar GDSs but have sharp edges and corners indicative of inflexible, rigid membranes. The Manpresenting twin−twin library 46a JGD with (3,4,5)-trisubstituted Percec-type hydrophobic dendrons denoted (3,4,5)12G1-PETRZ-Man2 formed tubular GDSs at low concentration and polygonal GDSs resembling icosahedra at concentrations above 0.5 mg/mL (Figure 81d). Additional representive cryo-TEM images of solid lamellae, solid GDSs, and tubular GDSs self-assembled from twin−twin library 48a JGDs are shown in Figure 82. A particularly interesting case is the bundles of tubular GDSs self-assembled from the Man-presenting twin−twin library 48a JGD with (3,4,5)-trisubstituted Percec-type hydrophobic dendrons denoted (3,4,5)12G1-PE-spacer-TRZ-Man2 shown in Figure 82c. At concentrations below 0.25 mg/mL, single tubes were observed, whereas at high concentrations bundles of tubes formed. The hexagonal arrangements seen in the cryo-TEM image are the result of tubular bundles positioned with their long axis perpendicular to the film. The tubes had uniform diameter and length. Solid lamellae could be transformed into soft, unilamellar GDSs by annealing to 60 °C for 30 min as exhibited in Figure 83.71 At higher temperature, the JGDs went above their glass transition temperatures, resulting in the transition of solid lamellae into flexible membranes that subsequently closed up to form GDSs. Even after cooling, the GDSs were not observed to transition back to solid lamellae. Thus, GDSs are the thermodynamic product of self-assembly in water, whereas solid lamellae are a kinetically trapped morphology. Gillies and co-workers reported the self-assembly of the third and fourth generation (m = 2, 3) library 53 JDs (Figure 64) into micelles.141 These JGDs did not form GDSs because of the increased curvature of their higher generation hydrophilic dendrons. By DLS and TEM, the diameter of these micelles was determined to be 10 nm.

Figure 71. Representative cryo-TEM images of GDSs self-assembled from twin−twin JGDs including (a) (3,4)2Et8G1-PE-TRZi-Man2 (library 45a), (b) (3,4)2Et8G1-PE-TRZi-3EOGal2 (library 45b), (c) (3,5)2Et8G1-PE-TRZi-3EOGal2 (library 45b), and (d) (3,4,5)2Et8G1PE-TRZi-3EOGal2 (library 45b).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

5.3. Predictable Diameter and Number of Bilayers

As was demonstrated in detail for DSs in section 3.3, GDSs also exhibit the same predictability of diameter size from final concentration of JGDs in aqueous media.95,145 In particular, the square of the diameter (D2) of GDSs is directly proportional to the JGD final concentration (c).

Figure 72. Illustration of the method used to determine the thickness of GDS bilayers. Measurement for (a) thick walled GDSs assembled from (3,4)2Et8G1-PE-TRZi-Man2 (library 45a) and (b) normal GDSs assembled from (3,5)2Et8G1-PE-TRZi-3EOGal2 (library 45b). Wall thickness was measured by utilizing the distinct drop in gray scale intensity across membrane edges.71,103 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

D2 = pc

(7)

In eq 7 (identical to eq 5 in section 3.3), p is a constant that depends only on the structure of the JGD. This relationship has been applied to all sequence-defined JDs, including but not limited to libraries 54−57 (Figure 65) in the range of concentrations that resulted in stable, monodisperse assemblies. For instance, Figure 84 shows the relationship between concentration and diameter (Figure 84a) and the linear dependence of the square of the diameter on concentration (Figure 84b) for GDSs self-assembled from the Lac-containing sequence-defined JGDs shown in Figure 67.95 Using the linear relationship, it is trivial to determine the appropriate final concentration of JGD for the preparation of any desired size to a high level of accuracy.

5.2.4. Solid GDSs and Other Complex Architectures. Many JGDs have been reported to self-assemble into solid GDSs, tubular GDSs, solid lamellae, spherical and rod-like micelles, and hard nonmicellar droplets denoted glycodendrimer aggregates.71 With few exceptions, the twin−twin JGDs comprising libraries 44a, 45a, 46a, 47a, and 48a (top row of Figure 63) self-assemble into these structures as opposed to soft GDSs because they have more rigid structures and lower solubility.71 The structures generally exhibit narrow polydispersity and range in dimensions from several tens to several hundreds of nanometers. Figure 81 shows representative cryo-TEM images of solid lamellae (Figure 6598


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Figure 73. Representative cryo-TEM images of GDSs self-assembled by injection of THF solutions of single−single JGDs including (a) (3,5)12G1-2TRZi-3EOMan (library 49) in PBS (0.5 mg/mL), (b) (3,5)12G1-3-TRZi-3EOMan (library 50) in HEPES (0.5 mg/mL), (c) (3,5)12G1-3-TRZ3EOMan (library 51) in HEPES (0.5 mg/mL), (d) (3,5)12G1-3-TRZi-3EOGal (library 50) in PBS (0.5 mg/mL), (e) (3,5)12G1-3-TRZi-3EOLac (library 50) in HEPES (0.5 mg/mL), and (f) (3,5)12G1-3-TRZ-3EOLac (library 51) in PBS (0.5 mg/mL).134 Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 74. Representative cryo-TEM images of GDSs self-assembled from twin−mixed JGDs including (a) 4-Lac 3EO(1,2,3,4,5,6)-3EOLac(7) (library 55), (b) 5a-Lac 3EO(1,2,3,4,5,6,7)-3EOLac(8)-3EO(9) (library 56a), (c) 5b-Lac 3EO(1,2,3,4,5,6,7,8)-3EOLac(9) (library 56b), (d) 6a-Lac 3EO(1,2,3,4,5,6,7)-6EOLac(8)-3EO(9) (library 57a), and (e) 6b-Lac 3EO(1,2,3,4,5,6,7,8)-6EOLac(9) (library 57b) at 0.1 mM in PBS.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society.

Figure 75. Representative cryo-TEM images of GDSs self-assembled by injection of THF solutions of library 54 JGDs including (a) 3EO(1,2,3)3EOMan(4) in PBS (0.5 mg/mL), (b) 3EO(1,2,3)-3EOGal(4) in HEPES (0.5 mg/mL), and (c) 3EO(1,2,3)-3EOLac(4) in water (1.0 mg/mL).134 Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

In eq 8 (identical to eq 6 in section 3.3), σ represents the average spacing between bilayers, which can be determined from cryoTEM. As shown in Figure 85, this equation was applied to all onion-like GDSs obtained from Man-presenting sequencedefined JGDs. Included in the graph are all JGDs in Figure 68 except for 3EO(1,2,3,4,5,6)-3EOMan(7) (short name 4-Man),

As was shown for onion-like DSs in section 3.3, the number of bilayers (n) for onion-like GDSs is also predictable from GDS radius (R).145 n = R/σ

(8) 6599


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Figure 76. Representative cryo-TEM images of onion-like GDSs self-assembled from twin−mixed JGDs including 5a-Man 3EO(1,2,3,4,5,6,7)3EOMan(8)-3EO(9) (library 56a), 5b-Man 3EO(1,2,3,4,5,6,7,8)-3EOMan(9) (library 56b), 6a-Man 3EO(1,2,3,4,5,6,7)-6EOMan(8)-3EO(9) (library 57a), and 6b-Man 3EO(1,2,3,4,5,6,7,8)-6EOMan(9) (library 57b) at 0.1 mM of Man in HEPES.145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

After converting the radius to the square of the diameter, eq 5 then could be used to determine the appropriate concentration. 5.4. Mechanical Properties, Confocal Microscopy, and Stability

The mechanical properties of GDSs were tested by micropipette aspiration experiments with giant GDSs prepared via thin film hydration.71 Images of these experiments are shown in Figure 86a for soft unilamellar GDSs self-assembled from the Manpresenting twin−twin library 44b JGD with 2EO-spacers and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-TRZi-2EOMan2 (51bc in Figure 86) and from the Lac-presenting twin−twin library 46b JGD with 4EO-spacers and (3,4,5)-trisubstituted Percec-type hydrophobic dendrons denoted (3,4,5)12G1-PE-spacer-TRZ-4EOLac2 (52dd in Figure 86), and for hard GDSs self-assembled from the Gal-presenting twin−twin library 48a JGD with (3,4)-disubstituted Percec-type hydrophobic dendrons denoted (3,4)12G1-PE-spacer-TRZGal2 (50aa in Figure 86) and from the Man-presenting twin− twin library 44b JGD with 1EO-spacer and (3,4)-disubstituted Percec-type hydrophobic dendrons denoted (3,4)12G1-PETRZi-1EOMan2 (51ad in Figure 86). The hard GDSs could not be aspirated and, as demonstrated in the image of 50aa, buckled in response to suction. On the other hand, the flexible membranes of the soft GDSs deformed in response to applied pressure, enabling determination of their mechanical properties. Figure 86b shows a comparison of the elastic moduli (Ka), which were calculated from the slope of membrane tension in response to increased area (Figure 86c), for GDSs self-assembled from 51bc, 52dd, and the Man-presenting twin−twin library 44b JGD with 3EO-spacers and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-TRZi-3EOMan2 (51bb in Figure 86).71 Higher elastic moduli are indicative of less flexible and thus tougher vesicles. GDSs self-assembled from 51bc exhibited the highest elastic modulus of the tested GDSs with a value of 163 ± 30 dyn/cm. Figure 86b also shows elastic moduli for polymersomes assembled from the diblock copolymer PEO30-b-PBD46 and for liposomes made from the lipid HSPC, both of which exhibited values comparable to those of GDSs. However, unlike polymersomes, GDSs demonstrated hysteresis as shown in Figure 85d. Confocal fluorescent microscopy was employed to image giant GDSs with diameters greater than 10 μm self-assembled with Nile red dye in their hydrophobic bilayers.71 These GDSs were prepared via thin film hydration by adding the dye to a dichloromethane solution of JGDs, which was then dried on the bottom of a vial to form the film for hydration. Figure 86e,f shows images of soft GDSs with uniform, smooth contours, whereas Figure 86g shows hard GDSs with uneven contours.

Figure 77. Representative cryo-TEM images of onion-like GDSs selfassembled from JGD 5a-Man 3EO(1,2,3,4,5,6,7)-3EOMan(8)-3EO(9) (library 56a) and their 3D intensity-plotting images with different numbers of bilayers and diameters at 0.1 mM in HEPES.145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

Figure 78. Cryo-TEM images of GDCs with Pn3̅m symmetry from the library 44b JGD (3,5)12G1-PE-TRZi-1EOMan2 at 1.0 mg/mL in water. The inset in (a) shows the Fourier transform of a particle with a hexagonal arrangement of reflections corresponding to {110} crystallographic planes, indicating orientation along the [111] direction. The Fourier transform in (b) shows the {111} reflections, indicating oriention along the [112] direction, which has about a 20° tilt with respect to (a).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

which formed GDCs. Because the size of the onion-like GDSs is predictable from concentration per eq 5, the number of bilayers is thus also predictable from concentration. For instance, to determine the final concentration of JGDs needed to form onionlike GDSs with a specific number of bilayers, the radius associated with that number of bilayers could first be determined using eq 6. 6600


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Figure 79. Representative cryo-TEM images of GDCs self-assembled from 4-Man 3EO(1,2,3,4,5,6)-3EOLac(7) (library 55) in HEPES demonstrate that the undulating surface of GDCs differs from the smooth, uniformly curved bilayers of vesicles.146 Reprinted with permission from ref 146. Copyright 2016 American Chemical Society.

linking and thus reactivity. Agglutination may also be monitored with DLS, which records in time the size of the increasingly large aggregates formed by cross-linking.71 Agglutination assays have most commonly been conducted in disposable semimicro cuvettes at 23 °C and with single wavelength λ = 450 nm.134 Immediately before placement in the spectrometer for at least 1000 s of monitoring, 100 μL of lectin solution is injected into 900 μL of GDS solution in a cuvette followed by 2 s of shaking. PBS or HEPES buffer is used depending on the identity of the lectin.

GDSs have also been reported to have high stability in water and buffer with no significant changes in size or polydispersity observed over 60 to as many as 140 days after preparation in most cases.71,134 Moreover, after self-assembly, suspensions of GDSs could be diluted with no observable change in size or polydispersity, demonstrating their irreversibility and nonequilibrium state.95,145

6. BIOACTIVITY OF GDSs Carbohydrates, the third alphabet of life,48,139 have an unmatched ability to code information as compared to proteins and nucleic acids. They serve as signals in many biological processes including cell recognition and adhesion, signaling, routing, differentiation, proliferation, immune response, and growth regulation.38−47 Lectins are the sugar recognition proteins that facilitate the reading of this poorly understood language, translating sugar presentation into action. These proteins require multivalent ligand displays for binding because of the weakness of interactions with individual carbohydrates alone. They also exhibit reactivity only in the presence highly specific cognate carbohydrates (e.g., Man or Lac). Analysis of the bioactivity of GDSs has centered on their reactivity to various lectins, unlocking new insights into biological functionality of lectins. Unlike previous synthetic platforms for multivalent display of carbohydrates including glycopolymers and carbohydrate-presenting liposomes, GDSs provide access to both programmable carbohydrate sequence and density while maintaining sufficient similarity to biological membranes to consider them model systems.71 Agglutination assays of GDSs incubated with lectins have generally been monitored with UV−vis spectroscopy.71,134 Addition of lectin to GDSs bearing the appropriate cognate sugar results in bridging between GDSs (trans-interactions) as opposed to contact on a single membrane (cis-interactions). The cross-linking of GDSs forms large aggregates, which absorb UVlight. Lectin reactivity with GDSs in time shows up on UV−vis spectra as curves extending from low absorbance values toward plateau values, with higher plateaus indicating greater cross-

6.1. Reactivity of Mannose (Man)-Presenting GDSs with Man-Specific Lectins (ConA)

Concanavalin A (ConA) (Figure 87) is a Man-specific tetrameric leguminous plant lectin commonly used in glycocluster research. It exhibits β-sandwich folding and two sets of bivalency on opposing sides located about 64 or 70 Å apart.147−149 Each of the four reactive sites binds Ca2+, which is necessary for proper functioning along with Mn2+. ConA shares similarities with certain Man-specific tissue lectins such as intracellular cargo transporters ERGIC-53, ERGL, VIP36, and VIPL, which also have β-sandwich folding and Ca2+ dependence.150−153 Agglutination assays with GDSs were performed in HEPES buffer, which contains Ca2+ and Mn2+ necessary for the saturation of ConA cation-binding sites. As shown in Figure 88, initial experiments were performed with GDSs self-assembled from the Man-presenting twin−twin library 44b JGD with (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-TRZi-3EOMan2 (51bb in Figure 88).71 The blue curve in Figure 88a demonstrates agglutination of ConA with the GDSs as compared to control experiments. Neither the GDSs alone nor the GDSs incubated with the Gal-specific lectin PA-IL exhibited increases in absorbance over time, ruling out any lectin-independent aggregation and sugar-independent reactivity. As shown in Figure 88b, increasing the concentration of 51bb increased agglutination with ConA. DLS was also used to monitor agglutination for GDSs of different sizes (Figure 88c). Most likely due to higher surface to volume ratios, GDSs of smaller size 6601


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Figure 80. Cryo-TEM images of a large particle prepared from 4-Man imaged with (a) 0° tilt and (b) 20° tilt. (c,d) The diffraction patterns of the fast Fourier transform (FFT) patterns of the regions denoted by a broken circle in (a) and (b). (e) Sequence of images by 3D cryo-electron tomography of GDCs assembled by 4-Man along the [111] direction. (f) Representative electron density map from slice 26 as shown in (e). (g) Schematic representation of bicontinuous channels in the cubic phase. Red, magenta, and blue hexagons represent different layers A, B, and C, as observed in (e). (h) Schematic representation of two independent networks of water channels (network 1 in green and network 2 in brown) connected in different layers. (i−k) Model of the cubic phase reconstructed from 3D cryo-electron tomogram presenting views from different angles, including (i) perspective view, (j) top view, and (k) side view.146 Reprinted with permission from ref 146. Copyright 2016 American Chemical Society.

tended to result in faster agglutination. Figure 88a also shows binding of PA-IL with the Gal-presenting analogue of 51bb denoted (3,5)12G1-PE-TRZi-3EOGal2 (51ba in Figure 88), which will be discussed in the next section. These results demonstrated that GDSs exhibit the multivalency necessary for agglutination with lectins. Increasing the concentration of ConA at constant concentration of 51bb also increased reactivity as shown in Figure 89a.71 Moreover, agglutination could be observed by cryo-TEM as shown in Figure 89b,c. With no ConA, separate GDSs are clearly visible (Figure 89b), whereas images taken after addition of ConA show the formation of large aggregates of bridged GDSs (Figure 89c). Man-presenting single−single GDSs also demonstrated agglutination with ConA.134 Figure 90 shows that GDSs selfassembled from the Man-presenting single−single library 49 JGD denoted (3,5)12G1-2-TRZi-3EOMan2 (48a in Figure 90), for instance, demonstrated increased reactivity at increased concentrations in the presence of a constant amount of ConA, as expected.

Given the capacity of Man-presenting GDSs self-assembled from structurally diverse JGDs for cross-linking with ConA, it was imperative to determine which design resulted in the greatest reactivity. Therefore, Man-presenting single−single, twin−twin, and twin−mixed designs were compared as shown in Figure 91.154 The single−single design is from library 49 and is denoted (3,5)12G1-2-TRZi-3EOMan2 (same as 48a in Figure 90), the twin−twin design is from library 46b and is denoted (3,5)12G1PE-TRZ-4EO-Man2, and the twin−mixed design is from library 54 and is denoted 3EO(1,2,3)-3EOMan(4). To ensure a valid comparison of reactivity, the concentration of Man was kept constant across all samples such that the twin−twin JGD, which presents two sugars per molecule, was kept at one-half the concentration of the single−single and twin−mixed JGDs. The single−single and twin−twin designs exhibited very similar reactivity, whereas the twin−mixed design exhibited much higher reactivity. This indicates that less densely sugar covered GDSs are more reactive toward lectins as compared to GDSs with high sugar density. The result can be explained by a reduction in steric hindrance between sugar and lectin for the less dense twin− 6602


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Figure 83. (a) Cryo-TEM image of (3,4,5)12G1-PE-TRZi-3EOMan2 (library 44b) showing solid lamellae at 0.5 mg/mL in water. (b) CryoTEM image of the same suspension after annealing at 60 °C for 30 min. The transition from solid lamellae to GDSs is observed.71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

The same Man-presenting twin−mixed design was also coassembled with its Lac analogue at various ratios.154 Although exact topology could not be controlled with this coassembly procedure, it still resulted in GDSs with different Man coverage. These different GDSs were reacted with ConA as shown in Figure 92. A large increase in reactivity was observed between 12.5% Man and 25% Man coverage. Thus, there is an apparent threshold coverage of sugar above which multivalency of GDSs increases significantly. It should be noted that in this specific case, maximum reactivity was observed for 100% Man, indicating that dilution of this structure did not yield improved binding.

Figure 81. Representative cryo-TEM images of assemblies from twin− twin JGDs including (a) solid lamellae from (3,4)12G1-PE-TRZ-Gal2 (library 46a), (b) solid GDSs assembled from (3,4)12G1-PE-TRZiMan2 (library 44a), (c) solid lamellae assembled from (3,4,5)12G1-PETRZ-Gal2 (library 46a), and (d) polygonal GDSs assembled from (3,4,5)12G1-PE-TRZ-Man2 (library 46a).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

6.2. Reactivity of Galactose (Gal)-Presenting GDSs with Gal-Specific Lectins (PA-IL, VAA, and GSL 1)

The Gal-specific bacterial lectin PA-IL comes from Pseudomonas aeruginosa, a bacterium that affects cystic fibrosis patients and immunocompromised individuals. In initial experiments, the multivalency of Gal-presenting GDSs was confirmed by agglutination with PA-IL as shown by the red curve in Figure 88a for GDSs self-assembled from the Gal-presenting twin−twin library 44b JGD with (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-TRZi-3EOGal2 (51ba in Figure 88).71 GDSs self-assembled from Gal-presenting single−single, twin−twin, and twin−mixed JGDs have all been tested for their reactivity toward PA-IL as shown in Figure 93.134 Gal concentration was held constant for all JGDs except for the twin−mixed design, which was also tested at one-half Gal concentration. GDSs self-assembled from the Man-presenting single−single library 49 JGD denoted (3,5)12G1-2-TRZi3EOMan showed no reactivity toward PA-IL, demonstrating the sugar specificity of the lectin and excluding noncarbohydrate mediated aggregation of GDSs. GDSs self-assembled from the Gal-presenting twin−twin library 44b JGD with (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1PE-TRZi-3EOGal2 (46 in Figure 93) and GDSs self-assembled from the Gal-presenting single−single library 49 JGD denoted (3,5)12G1-2-TRZi-3EOGal (50a in Figure 93) demonstrated similarly low reactivity with PA-IL (dark blue and brown curves, respectively). On the other hand, GDSs self-assembled from the Gal-presenting twin−mixed library 54 JGD denoted 3EO(1,2,3)3EOGal(4) (54 in Figure 93, which also has the alternative long name (3,5)12G1-Tris(3EO)3-TRZ-3EOGal) demonstrated much higher reactivity (pink curve) at the same Gal concentration. Even at one-half Gal concentration, GDSs self-

Figure 82. Representative cryo-TEM images of assemblies from library 48a twin−twin JGDs including solid lamellae and solid GDSs assembled from (a) (3,4)12G1-PE-spacer-TRZ-Man2 and (b) (3,4)12G1-PEspacer-TRZ-Gal 2; tubular GDSs bundles assembled from (c) (3,4,5)12G1-PE-spacer-TRZ-Man2; and a mixture of tubular and spherical GDSs assembled from (d) (3,4,5)12G1-PE-spacer-TRZGal2.71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

mixed design. It also suggests that there is an optimal sugar density and topology for maximum lectin reactivity, which was indeed discovered for sequence-defined JGDs as will be discussed in section 6.5. 6603


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Figure 84. Concentration dependence of the (a) diameter (DDLS, in nm) and (b) square of the diameter (DDLS2) of GDSs self-assembled from Laccontaining sequence-defined JGDs (refer to Figure 67 for structures) in PBS. R2 = coefficient of determination.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society.

assembled into GDSs with superior reactivity while also maintaining a controllable sugar topology. The highly toxic mistletoe lectin Viscum album agglutinin (VAA) was also employed in agglutination assays with Galpresenting GDSs.71 For instance, Figure 94 shows VAA agglutination with GDSs self-assembled from the same twin− twin library 44b JGD as in Figure 92 denoted (3,5)12G1-PETRZi-3EOGal2 (51ba in Figure 93). As expected, at higher VAA concentrations, reactivity increased. Gillies and co-workers utilized an alternative method for measuring lectin reactivity toward Gal-presenting GDSs and micelles self-assembled from library 52−53 JGDs (Figure 64), which have α-Gal-decorated hydrophilic polyester dendrons and hydrophobic lipid tails.141 The specific structures of these assemblies were discussed previously in sections 5.2.1 and 5.2.4. The α-Gal specific West African shrub lectin Griffonia simplicifolia Lectin I (GSL 1) was selected for testing. It is a tetramer with one binding site per subunit.155 Self-assemblies were prepared with Nile red dye in their hydrophobic bilayers and incubated with suspensions of GSL 1-coated agarose beads for 3 h at room temperature. The beads were then washed one or two times and viewed by fluorescence microscopy as shown in Figure 95a,b. A second wash was conducted to eliminate more weakly bound assemblies. On the basis of the observed fluorescence intensity, it was possible to compare the relative binding abilities of the various assemblies. Micelles self-assembled from the third generation library 53 JGD denoted L-G3-Gal were tested for binding to GSL 1 alone and in the presence of free Gal as inhibitor.141 As shown in Figure 95c, the micelles displayed extensive binding in the presence of GSL 1 but not when free Gal was also present, excluding carbohydrate-independent interactions between the assemblies and lectin. Furthermore, the micelles exhibited significantly lower reactivity toward the β-Gal specific lectin Jacalin, demonstrating preference for α-Gal specific lectins. Carbohydrate-independent binding was further ruled out by incubation of GSL 1 with aggregates self-assembled from the nonsugar presenting analogue of L-G3-Gal, which is denoted L-G3-OH and presents a periphery of hydroxyl groups. Only very limited binding was observed with assemblies from L-G3-OH as compared to micelles from L-G3-Gal. The relative binding abilities of assemblies from the five JGDs in libraries 52 and 53 are shown in Figure 95d.141 It should be noted that the lowest generation JGDs (L-G0-Gal, L-G1-Gal, LG2-Gal) formed GDSs, whereas the highest generation JGDs (L-

Figure 85. Relationship between diameter (nm) of individual onion-like GDSs and their corresponding number of bilayers determined by cryoTEM. Onion-like GDSs were self-assembled from 0.1 mM JGDs 3-Man 3EO(1,2,3)-3EOMan(4) (library 54), 5a-Man 3EO(1,2,3,4,5,6,7)3EOMan(8)-3EO(9) (library 56a), 5b-Man 3EO(1,2,3,4,5,6,7,8)3EOMan(9) (library 56b), 6a-Man 3EO(1,2,3,4,5,6,7)-6EOMan(8)3EO(9) (library 57a), and 6b-Man 3EO(1,2,3,4,5,6,7,8)-6EOMan(9) (library 57b). R2 = coefficient of determination. d = interbilayer distance (also referred to as σ).145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

assembled from the same twin−mixed JGDs demonstrated higher reactivity (light blue curve) than the GDSs from single− single and twin−twin designs. Thus, as was also true for Manpresenting GDSs, Gal-presenting GDSs from twin−mixed designs exhibit significantly higher reactivity. This is attributable to reduced sugar density that likewise reduces steric hindrance for lectin agglutination. Another method of reducing sugar density on GDSs to test lectin reactivity is by coassembly with JDs, which do not bear sugars. For instance, the Gal-presenting single−single JGD 50a was coassembled with the single−single library 11 JD with (3,4,5)-3EO substituted hydrophilic dendron and (3,5)disubstituted Percec-type hydrophobic dendron denoted (3,5)12G1-CH2-PhE-(3,4,5)-3EO-G1-(OCH3)3 (Figure 7).134 These coassembled GDSs were tested for their reactivity with PA-IL as shown in Figure 93 (gray curve). They exhibited significantly higher reactivity than GDSs from the JGD alone (brown curve). However, the twin−mixed JGD still self6604


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Figure 86. (a) Micropipet aspiration of soft GDSs from (3,5)12G1-PE-TRZi-2EOMan2 51bc (library 44b) and (3,4,5)12G1-PE-spacer-TRZ-4EOLac2 52dd (library 48b), and solid GDSs from (3,4)12G1-PE-spacer-TRZ-Gal2 50aa (library 48a) and (3,4)12G1-PE-TRZi-1EOMan2 51ad (library 44b); scale bar is 25 μm. (b) Comparison of elastic moduli of GDSs from 52dd, 51bc, and (3,5)12G1-PE-TRZi-3EOMan2 51bb (library 44b) with polymersomes from PEO30-b-PBD46 and liposomes from lipid HSPC. (c) Plot of tension versus areal strain for GDSs from 52dd, 51bc, and 51bb. (d) Comparison of tension versus areal strain plot for polymersomes from PEO30-b-PBD46 and GDSs from 52dd. In (d), filled circles are measurements taken during initial vesicle stressing, and open circles are measurements taken during vesicle relaxation. Confocal microscopy images of (e,f) giant soft GDSs and (g) giant hard GDSs encapsulated with Nile red dye in their bilayers.71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

6.3. Reactivity of Lactose (Lac)-Presenting GDSs with Lac-Specific Lectins (Galectins, VAA)

Galectins encompass a large set of animal lectins that exhibit specificity for β-galactosides such as Lac. They have been discovered in lower invertebrates such as sponges and nematodes as well as in higher vertebrates including humans.156,157 Galectins have been implicated in a diverse range of cellular processes including adhesion, growth, signaling, immune response, and metastasis.41−43,158 Because of their ubiquity and essential role in many fundamental cell processes, they have been extensively employed in agglutination assays with Lac-presenting GDSs. The highly toxic mistletoe lectin Viscum album agglutinin (VAA) has also been employed in agglutination assays with Lac-presenting GDSs. 6.3.1. Galectin-1 (Gal-1). Galectin-1 (Gal-1) is a homodimeric lectin that can be found in vertebrates including humans and functions in cell adhesion and growth control.159−161 It exhibits three types of topological display in vertebrates including homodimeric, tandem-repeat, and chimera-type designs as diagrammed in Figure 96. Artificial Gal-1 variants have been prepared by inserting linkers via cDNA engineering into the wildtype (WT) homodimeric form to give artificial tandem-repeat architectures.162 These include Gal-1(GG), which has a linker of minimal length consisting of two glycines, Gal-1(4), which incorporates the linker found in galectin-4, and Gal-(8S) and Gal(8L), both of which incorporate linkers of different lengths found in galectin-8.154 The human form (denoted hGal) of this

Figure 87. Representative conformation (A) of the tetrameric structure of ConA containing Ca2+ (green), Mn2+ (purple), and (B) its binding site loaded with methyl α-D-mannopyranoside (Protein Data Bank ID code 5CNA).145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

G3-Gal, L-G4-Gal) formed micelles. Out of all of the tested assemblies, micelles from the highest generation JGD (L-G4Gal) demonstrated by far the strongest binding after both the first and the second wash. This fourth generation JGD provided greater multivalency and as such yielded higher reactivity. In general, the micelles exhibited stronger binding as compared to the GDSs possibly due to increased flexibility and adaptability. It is important to note the particularly large decrease in binding observed for GDSs from L-G0-Gal after the second wash, which indicated their disassembly due to lack of stability. 6605


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Figure 88. (a) Agglutination of GDSs from twin−twin JGDs (3,5)12G1-PE-TRZi-3EOGal2 (51ba) (library 44b) and (3,5)12G1-PE-TRZi-3EOMan2 (51bb) (library 44b) in the presence of ConA and PA-IL. [51ba] = 0.5 mg/mL (900 μL), [51bb] = 0.5 mg/mL (600 μL), [Con A] = 0.3 mg/mL (100 μL), [PA-IL] = 0.0625 mg/mL (100 μL) in HEPES (1.0 mM MnCl2 and 1.0 mM CaCl2). (b) Agglutination of GDSs from 51bb at different concentrations (0−0.25 mg/mL, 900 μL) in the presence of ConA (0.125 mg/mL, 100 μL) in 10 mM HEPES (1.0 mM MnCl2 and 1.0 mM CaCl2). (c) Agglutination of GDSs from 51bb of different sizes in the presence of ConA recorded by DLS. [51bb] = 0.0625 mg/mL (400 μL), [ConA] = 0.5 mg/mL (100 μL) in HEPES (1.0 mM MnCl2 and 1.0 mM CaCl2).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

Figure 90. ConA-dependent agglutination of GDSs self-assembled from single−single JGD (3,5)12G1-2-TRZi-3EOMan 48a (library 49) at indicated concentrations in 10 mM HEPES buffer (1.0 mM MnCl2 and 1.0 mM CaCl2). [ConA] = 0.5 mg/mL.134 Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 89. (a) Agglutination of GDSs from twin−twin JGD (3,5)12G1PE-TRZi-3EOMan2 (51bb) (library 44b) in the presence of different concentrations of ConA. [51bb] = 0.5 mg/mL (900 μL), [Con A] = 0− 0.5 mg/mL (100 μL) in 10 mM HEPES (1.0 mM MnCl2 and 1.0 mM CaCl2) (a). Corresponding Cryo-TEM images at indicated ConA concentrations. Aggregation due to lectin-mediated cross-linking can clearly be observed (b,c).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

mixed design was incubated with hGal-1 as a control and demonstrated no reactivity, excluding carbohydrate-independent agglutination. As was found previously for Man- and Galpresenting GDSs, Lac-presenting GDSs self-assembled from the twin−mixed design demonstrated the highest reactivity likely due to relief of steric hindrance. These results indicate that there may be an optimal sugar density and topology for maximum lectin reactivity, which was indeed realized for sequence-defined JGDs and will be discussed in section 6.4. As illustrated in Figure 98a, it should be noted that after reaching a maximum value, a decrease in agglutination was observed over time for the twin− mixed GDS, indicating dissociation of the homodimer into monomers due to mechanical disruption.

panel of artificial tandem-repeats and the WT homodimer were tested for their reactivity with GDSs in PBS buffer. Agglutination assays of WT hGal-1 with Lac-presenting GDSs self-assembled from single−single, twin−twin, and twin−mixed JGDs are shown in Figure 97.154 The single−single JGD comes from library 50 and is denoted (3,5)12G1-3-TRZi-3EOLac, the twin−twin JGD comes from library 46b and is denoted (3,5)12G1-PE-TRZ-3EOLac 2, and the twin−mixed JGD comes from library 54 and is denoted 3EO(1,2,3)-3EOLac(4). The concentration of Lac was held constant to ensure valid comparisons between the GDSs. The Man analogue of the twin− 6606


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Figure 91. Agglutination with ConA (0.5 mg/mL) and Man-presenting GDSs (in mM) displaying different sugar topologies in HEPES buffer (10 mM; with 1.0 mM MnCl2 and CaCl2). The single−single JGD is (3,5)12G1-2-TRZi-3EOMan (library 49), the twin−twin JGD is (3,5)12G1-PE-TRZ4EOMan2 (library 46b), and the twin−mixed JGD is 3EO(1,2,3)-3EOMan(4) (library 54).154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 92. Agglutination assays between ConA (0.5 mg/mL) and GDSs coassembled from Man- and Lac-presenting twin−mixed library 54 JGDs at different ratios ([Man+Lac] = 0.2 mmol/L) in HEPES (10 mM) buffer (with 1.0 mM MnCl2 and 1.0 mM CaCl2).154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 93. PA-IL-dependent agglutination of GDSs self-assembled from JGDs at indicated concentrations in 10 mM HEPES buffer (1.0 mM MnCl2 and 1.0 mM CaCl2). [PA-IL] = 0.5 mg/mL. GDSs are from the twin−twin library 44b JGD (3,5)12G1-PE-TRZi-3EOGal2 (46), the single−single library 49 JGD (3,5)12G1-2-TRZi-3EOGal (50a), and the twin−mixed library 54 JGD (3,5)12G1-Tris(3EO)3-TRZ-3EOGal (54). The Man-presenting single−single library 49 JGD (3,5)12G1-2-TRZi3EOMan (50a) was used as a control.134 Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

As diagrammed in Figure 98b, the formation of stable aggregates was achieved in agglutination assays with the covalently connected tandem-repeat variants, which are not capable of dissociation into monomers.154 Agglutination assays were performed with the same library 54 twin−mixed design as shown in Figure 99. All of the artificial tandem-repeat variants exhibited significantly higher reactivity toward the GDSs and formed stable aggregates in time as compared to WT hGal-1. Longer linkers were associated with higher reactivity. These results demonstrated the utility of tandem-repeat designs for the stable associations necessary in cell−cell/cell−matrix interactions and transport processes.

To investigate the impact of further Lac dilution on the surface of GDSs, the Lac-presenting library 54 twin−mixed JGD was coassembled with its noncognate Man-presenting analogue at various ratios.154 Agglutination assays of these GDSs with WT hGal-1 are shown in Figure 100. Unlike in the case of ConA with Man-presenting GDSs (see section 6.1 and Figure 92), GDSs self-assembled from 100% Lac JGDs did not exhibit the highest reactivity. Rather, the diluted GDSs with 75% Lac exhibited the 6607


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Figure 96. Homodimeric (a), tandem-repeat (b), and chimera-type (c) galectin designs. Engineered proteins can be generated by turning the prototype (a) into artificial tandem-repeat designs (X, with linker lengths of n = 2 (GG), n = 33 (8S), n = 42 (4), and n = 75 (8L)), enabling functional comparison of natural and engineered variants.154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

observed between 10% Lac and 25% Lac, indicating the existence of a threshold concentration necessary for significant reactivity with the homodimer. On the other hand, while the advantages of further Lac dilution were observed for the artificial tandemrepeat variants, no apparent threshold concentration of Lac was necessary for agglutination. Significant levels of reactivity were observed at only 5% Lac. This behavior was also verified in cell systems by cytofluorometry with fluorescent galectin. Therefore, at low sugar density, the insertion of a linker into the WT homodimeric form of hGal-1 enhances cross-linking capacity. 6.3.2. Galectin-3 (Gal-3) and Galectin-4 (Gal-4). Galectin3 (Gal-3) and galectin-4 (Gal-4) are two members of the galectin family that are found in humans. They have important differences in structure with monomeric Gal-3 exhibiting a collagenasesensitive N-terminal tail that encourages aggregation and bivalent Gal-4 having two carbohydrate recognition domains (CRDs) connected by a linker. Gal-3 has been implicated in cell proliferation, differentiation, growth and cell cycle regulation, transport, immune response, and metastasis.163 Gal-4 also has a diverse set of functions including cell adhesion, growth regulation, transport, and signaling.164,165 Agglutination assays of these two galectins with GDSs were compared to investigate differences in aggregation associated with monomeric versus bivalent lectins.71 As shown in Figure 101, GDSs self-assembled from the Lacpresenting twin−twin library 46b JGD with 4EO-spacers and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-TRZ-4EOLac2 (52bd in Figure 101) were reacted with various concentrations of Gal-3 and Gal-4 in HEPES.71 Although similar plateau levels were reached for both lectins, very different kinetics were observed with Gal-4 demonstrating significantly slower reactivity than Gal-3. These results highlight the influence of lectin structure on reactivity with GDSs. When combined with findings from the previous section indicating the important effect of ligand density on reactivity, the collective conclusion is that sugar topology and lectin design team up to deliver high levels of sensitivity and selectivity in binding. 6.3.3. Galectin-7 (Gal-7). Galectin-7 (Gal-7), which has been associated with cell growth suppression and apoptosis, is a member of the galectin family and is found in humans (hGal7).166 Its expression has been shown to reduce cancer cell growth and proliferation in some cases.167 As shown in Figure 102, in the presence of hGal-7, agglutination assays were performed with Lac-presenting GDSs self-assembled from single−single, twin− twin, and twin−mixed JGDs in PBS buffer.134 The single−single JGD comes from library 50 and is denoted (3,5)12G1-3-TRZi3EOLac (55 in Figure 102), the twin−twin JGD comes from library 46b and is denoted (3,5)12G1-PE-TRZ-3EOLac2 (45 in Figure 102), and the twin−mixed JGD comes from library 54 and is denoted 3EO(1,2,3)-3EOLac(4) (55 in Figure 102). To

Figure 94. Agglutination of GDSs prepared from the twin−twin library 44b JGD (3,5)12G1-PE-TRZi-3EOGal2 (51ba) with different concentrations of VAA. [(3,5)12G1-PE-TRZi-3EOGal2] = 0.5 mg/mL (900 μL), [VAA] = 1.25, 2.5, 5.0, and 10.0 mg/mL (100 μL) in 10 mM HEPES buffer (1.0 mM MnCl2 and 1.0 mM CaCl2).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

Figure 95. (a,b) Fluorescence microscopy images of GSL 1-coated agarose beads. Image (a) shows a highly fluorescent sample (L-G3-Gal, library 53), and image (b) shows a weakly fluorescent sample (L-G3OH) indicating poor binding to the bead. (c) Normalized relative fluorescence of L-G3-Gal assemblies bound to GSL 1- or Jacalin-coated agarose beads in the absence or presence of excess free Gal. L-G3-OH assemblies bound to the beads are also included for comparison. (d) Relative fluorescence of GSL 1-coated beads incubated with varying generations of α-Gal-functionalized library 52 and 53 JGDs and then subjected to one or two washings. Error bars represent the standard error on the mean of the measurements.141 Reprinted with permission from ref 141. Copyright 2014 Royal Society of Chemistry.

highest reactivity. Moreover, GDSs with 50% Lac also exhibited a similarly high reactivity. Dilution of Lac to only 7.5% resulted in almost no aggregation. A large increase in agglutination was 6608


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Figure 97. Agglutination with WT hGal-1 (2 mg/mL) and Man-presenting GDSs (in mM) displaying different sugar topologies in PBS. The single− single JGD is (3,5)12G1-3-TRZi-3EOLac (library 50), the twin−twin JGD is (3,5)12G1-PE-TRZ-4EOLac2 (library 46b), and the twin−mixed JGD is 3EO(1,2,3)-3EOLac(4) (library 54).154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 98. Illustration of (a) disreputable agglutination between noncovalently linked homodimeric WT hGal-1 and twin−mixed Lac-presenting GDSs and (b) firm agglutination between hGal-1 variants with peptide linkers and twin−mixed Lac-presenting GDSs. Amino acid sequences are shown for each peptide linker (GG, 8S, 4, and 8L).154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

(3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-CH2-PhE-(3,4,5)-3EO-G1-(OCH3)3 (Figure 7), which does not bear a sugar headgroup. Reactivity of these coassembled GDSs (black line in Figure 102) was markedly higher than for GDSs assembled from the JGD alone (green line in Figure 102), providing further evidence of the advantage of sugar coverage dilution. However, unlike the twin−mixed design, the sugar topology of coassembled GDSs is uncontrollable, making them inferior models as compared to GDSs from twin− mixed designs. 6.3.4. Galectin-8 (Gal-8). Galectin-8 (Gal-8) is a member of the galectin family with tandem-repeat carbohydrate recognition domains (CRD) as shown in Figure 103. A peptide linker of variable length connects the two CRDs. Gal-8 facilitates cell adhesion and regulates cell growth, and its overexpression has been observed in various cancers.168,169 In humans, it occurs in

ensure valid comparisons, Lac concentration was held constant except for the twin−mixed JGD, which was also tested at half-Lac concentration. GDSs from the Man-analogue of the Lacpresenting twin−mixed JGD (53 in Figure 102) were incubated with hGal-7 as a control, excluding carbohydrate-independent agglutination. In agreement with previously described results for Gal-1 (see section 6.3.1), GDSs self-assembled from the Lac-presenting twin−mixed design were most reactive with hGal-7.134 The single−single and twin−twin designs showed almost identical reactivity, indicating that the single−single design is equivalent to one-half the potency of the twin−twin design as expected. The advantage of diluted sugar coverage on the surface of GDSs was also investigated by coassembling GDSs from the same Lacpresenting single−single JGD and from the single−single library 11 JD with (3,4,5)-3EO substituted hydrophilic dendron and 6609


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Figure 101. Agglutination of GDSs from the twin−twin library 46b JGD (3,5)12G1-PE-TRZ-4EOLac2 (52bd) in the presence of different concentrations of Gal-3 (a) and Gal-4 (b). [52bd] = 1.0 mg/mL (900 μL), [Gal-3] = 0−4.0 mg/mL (100 μL), [Gal-4] = 0−4.0 mg/mL (100 μL) in 10 mM HEPES buffer (1.0 mM MnCl2 and 1.0 mM CaCl2).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

Figure 99. Agglutination of Lac-presenting GDSs from the library 54 JGD 3EO(1,2,3)-3EOLac(4) (0.2 mM) with different hGal-1 variants (1.0 mg/mL) in PBS.154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 100. Agglutination with WT hGal-1 (1.5 mg/mL) and GDSs coassembled from Man- and Lac-presenting twin−mixed library 54 JGDs at different ratios ([Man + Lac] = 0.2 mM) in PBS.154 Reprinted with permission from ref 154. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 102. Agglutination assays with Lac-containing GDSs selfassembled from twin−mixed library 54 JGD 3EO(1,2,3)-3EOLac(4) (55), single−single library 50 JGD (3,5)12G1-3-TRZi-3EOLac (51), twin−twin library 46b (3,5)12G1-PE-TRZ-3EOLac2 (45), and twin− mixed 3EO(1,2,3)-3EOMan(4) (53) (in μmol/mL) in the presence of hGal-7 (0.5 mg/mL) in PBS buffer. GDSs coassembled from 51 and JD (3,5)12G1-CH2-PhE-(3,4,5)-3EO-G1-(OCH3)3 (library 11) were also tested.134 Reprinted with permission from ref 134. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

two isoforms, one with a shorter linker (8S) and one with a longer linker (8L), as the result of alternative splicing.170,171 Other species also exhibit Gal-8 but with different linker lengths including the chicken (CG-8S), which exhibits a linker of 9 amino acids.172 The two CRDs of Gal-8 can be separated by proteolytic cleavage into two separate proteins. Moreover, Gal-8 exhibits a single-amino acid polymorphism implicated in rheumatoid arthritis.173 The F19Y substitution results in a 1.5 Å displacement of the N-terminal amino acids 11−15 and a shift of the β-strand F0 near the linker.174 Thus, Gal-8 is a fruitful platform with which to test the reactivity and sensitivity of GDSs. Agglutination assays in the presence of Gal-8S were performed with Lac-presenting GDSs self-assembled from single−single, twin−twin, and twin−mixed JGDs in PBS buffer as shown in

Figure 104.175 As with previous investigations with galectins, the single−single JGD comes from library 50 and is denoted (3,5)12G1-3-TRZi-3EOLac, the twin−twin JGD comes from library 46b and is denoted (3,5)12G1-PE-TRZ-3EOLac2, and the twin−mixed JGD comes from library 54 and is denoted 3EO(1,2,3)-3EOLac(4). The concentration of Lac was kept constant across all samples to ensure valid comparisons. GDSs from the Man analogue of the Lac-presenting twin−mixed JGD 6610


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Figure 103. Three physiological forms of hGal-8 differing in linker length (hGal-8L and hGal-8S) and the presence of a single-site mutation at position 19 (labeled with an asterisk). Cartoons of the protein designs with two different CRDs (A), their folded structure (B), and their sequences (C). Arrow in (B) indicates a ligand (Gal) contact site. A schematic of the chicken galectin (CG-8S) is also shown in (A).175 Reprinted with permission from ref 175. Copyright 2015 National Academy of Sciences USA.

Figure 104. Agglutination assays between hGal-8S (2 mg/mL in 100 μL) and Lac-containing GDSs (mmol/L in 900 μL) displaying different sugar topologies in PBS. The single−single JGD is (3,5)12G1-3-TRZi-3EOLac (library 50), the twin−twin JGD is (3,5)12G1-PE-TRZ-4EOLac2 (library 46b), and the twin−mixed JGD is 3EO(1,2,3)-3EOLac(4) (library 54).175 Reprinted with permission from ref 175. Copyright 2015 National Academy of Sciences USA.

were incubated with Gal-8S as a control, excluding carbohydrateindependent binding. As was demonstrated for Gal-1 and Gal-7 (see sections 6.3.1 and 6.3.3), GDSs self-assembled from the Lacpresenting twin−mixed JGD demonstrated the highest reactivity in the presence of Gal-8S due to reduced steric hindrance. Notably, when the CRDs were cleaved into separate proteins, no aggregation was observed (data not shown).

The effect of reduction of GDS sugar density on reactivity with hGal-8S was also investigated by coassembling GDSs from the same Lac-presenting twin−mixed JGD and the noncognate Man analogue at various ratios as shown in Figure 105.175 At 10% Lac no agglutination was observed, and only low reactivity was noted for 15% Lac. However, at 25% Lac reactivity increased significantly, indicating the presence of a threshold sugar density for lectin reactivity. Furthermore, 50% Lac exhibited nearly 6611


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at various ratios. Even at only 15% Lac, Gal-8L showed a plateau level comparable to its agglutination with 100% Lac albeit at a much slower rate. In a more time-consuming process, longer linkers apparently allow the lectin to sense sugars even at low density. As shown in Figure 106, reactivities of hGal-8S, hGal-8L, the chicken galectin CG-8S, and the naturally occurring mutant hGal-8S F19Y in the presence of GDSs self-assembled from the same Lac-containing twin−mixed JGD were compared.175 Both hGal-8S and hGal-8L exhibited similar reactivities, although hGal-8S showed a slight advantage. Reactivity of the chicken galectin CG-8S was significantly lower than the reactivity of the human galectins. Most remarkably, the mutant hGal-8S F19Y exhibited about 50% lower reactivity than that of hGal-8S. Additionally, in dilution experiments with GDSs coassembled from twin−mixed Lac- and Man-presenting JGDs at various ratios, the mutant variant required a significantly higher threshold density of Lac for agglutination than hGal-8S did. Thus, a small structural change in hGal-8S can result in significant reduction of its activity, which is indicative of its implication in autoimmune disease. Moreover, GDSs provide a sufficiently sensitive platform on which to demonstrate changes in functionality associated with even slight structural alterations in lectins. These results highlight the value of GDSs as model systems for investigating fundamental biological questions. 6.3.5. VAA. The plant lectin VAA (see section 6.2) was also employed in agglutination experiments with Lac-presenting GDSs.71 As shown in Figure 107a, GDSs self-assembled from the Lac-presenting twin−twin library 46b JGD with 4EO-spacers and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-TRZ-4EOLac2 (52bd in Figure 107) were incubated with various concentrations of VAA. As expected, at higher concentrations of lectin, increased aggregation was observed. Cryo-TEM images of this process were also captured as shown in Figure 107b−e. Individual GDSs were present in the absence of VAA and formed progressively larger aggregates at higher VAA concentrations.

Figure 105. Agglutination assays between hGal-8S (2 mg/mL in 100 μL of PBS) and GDSs coassembled from Man- and Lac-presenting twin− mixed library 54 JGDs at different ratios ([Man + Lac] = 0.2 mmol/L in 900 μL of PBS).175 Reprinted with permission from ref 175. Copyright 2015 National Academy of Sciences USA.

6.4. Reactivity of Lac-Presenting Sequence-Defined GDSs with Gal-8

To determine an optimal sugar density and topology on GDSs for lectin binding, the Lac-presenting twin−mixed JGDs in libraries 55−57 were synthesized.95 These five JGDs along with the previously synthesized Lac-presenting twin−mixed library 54 JGD, the Lac-presenting single−single JGD in library 51, and a Lac-presenting twin−twin JGD in library 46b make up the panel of sequence-defined JGDs in Figure 67. These molecules will be referred to by their short names given in Figure 67. As discussed previously in section 4.5, the single−single and twin−twin designs have 100% sugar coverage, whereas the twin−mixed designs vary in sugar coverage due to dilution with TEG chains. Specifically, 3-Lac has 25% coverage, 4-Lac has 14% coverage, and the rest have 11% coverage. Unlike previous testing of the effect of sugar density on lectin agglutination, which involved coassembling GDSs from cognate and noncognate JGDs and JDs (see sections 6.1, 6.2, 6.3.1, and 6.3.4) and thus a random distribution of sugars on GDS surface, the synthesis of twin− mixed JGDs with more diluted sugar coverage (libraries 55−57) enabled the self-assembly of GDSs with defined topologies but with lower sugar density than had been previously obtained. Agglutination assays with GDSs self-assembled from this panel of Lac-presenting sequence-defined JGDs were performed using

Figure 106. Agglutination assays between Lac-presenting twin−mixed library 54 GDSs (0.2 mmol/L in 900 μL of PBS) and different Gal-8 proteins (2.0 mg/mL in 100 μL of PBS).175 Reprinted with permission from ref 175. Copyright 2015 National Academy of Sciences USA.

identical reactivity as compared to 100% Lac, demonstrating that GDSs composed of 100% Lac-presenting twin−mixed JGDs do not display a significant advantage over further diluted topologies. Thus, there is also a Lac density threshold for GDSs self-assembled from twin−mixed JGDs above which further increases in Lac density yield negligible improvements in reactivity. Additionally, lectin linker length impacted sensitivity to the presence of Lac on GDSs.175 As compared to Gal-8S, Gal-8L, which bears the longer linker, exhibited significantly higher sensitivity to low Lac density in experiments with GDSs coassembled from twin−mixed Lac- and Man-presenting JGDs 6612


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Figure 107. Agglutination of GDSs prepared from the library 46b JGD (3,5)12G1-PE-TRZ-4EOLac2 (52bd) in the presence of different concentrations of VAA. [52bd] = 1.0 mg/mL (900 μL), [VAA] = 0−2.0 mg/mL (100 μL) in 10 mM HEPES buffer (1.0 mM MnCl2 and 1.0 mM CaCl2) (a). Corresponding cryo-TEM images at indicated VAA concentrations. Aggregation due to lectin-mediated cross-linking can clearly be observed (b−e).71 Reprinted with permission from ref 71. Copyright 2013 American Chemical Society.

Figure 108. Agglutination assays between different Lac-presenting GDSs from sequence-defined JGDs (see Figure 67 for structures) at identical concentrations of Lac and two natural variants of Gal-8. Lac-presenting GDSs (0.1 mM of Lac in 900 μL of PBS) were incubated with (a) Gal-8S or (b) Gal-8L (2 mg/mL in 100 μL of PBS). The molar attenuation coefficient, ε = A/(cl), was adapted from the Beer−Lambert law, where A = plateau OD value, c = molar concentration of Lac, and l = semimicro cuvette path length (0.23 cm). Control experiments were performed by incubating 3-Man 3EO(1,2,3)-3EOMan(4) and 0.1 mM Man in 900 μL of PBS with Gal-8S/8L (2 mg/mL in 100 μL of PBS).95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society.

Gal-8 (see section 6.3.4) in PBS.95 As was discussed previously, Gal-8 is a bivalent lectin with two naturally occurring isoforms in humans, including one with shorter linker (8S) and one with longer linker (8L) between CRDs. Concentrations of Lac and Gal-8S/L were kept constant for each sample in the agglutination assays shown in Figure 108. As a means of better quantifying the differences between GDS reactivity, the molar attenuation coefficient (ε) was calculated for each sample using the Beer− Lambert law (A = εcl), in which A, c, and l stand for the plateau absorbance value, the molar concentration of Lac, and the path length of light (cuvette width), respectively. As was reported previously for Gal-8S (see section 6.3.4), the single−single design (1-Lac), twin−twin design (2-Lac), and the first twin−mixed design (3-Lac) exhibited a grading of activity

with the twin−mixed design having the highest activity.95 However, all of these GDSs had much lower reactivity as compared to the more diluted twin−mixed designs, including 4Lac, 5-Lac, and 6-Lac. In fact, 4-Lac exhibited an almost 2-fold increase in reactivity as compared to 3-Lac. Although it was expected that an increase in flexibility associated with the longer spacer between the Lac headgroup and the JGD scaffolding in 6Lac would increase reactivity, this was not found. Rather, with longer spacer the same or slightly decreased reactivity was observed, which may be the result of the headgroups backfolding onto the GDS surface, restricting accessibility to the lectins. The structural changes among 5-Lac and 6-Lac JGDs had a greater impact on binding with Gal-8L than with Gal-8S. In fact, a marked difference in reactivity was observed even between the 6613


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JGD, with some GDSs exhibiting slightly higher reactivity for smaller sizes and others exhibiting slightly lower reactivity. Thus, vesicle size had to be controlled along with concentration. This was achieved by preparing all GDSs at identical sizes and then calculating the molar attenuation coefficient to control for differences in Lac concentration. The results are shown in Figure 110 and exhibit the same trends that were already revealed in Figure 108. As was previously discussed (see section 6.3.4), Gal-8S also has a variant with a single-site mutation (F19Y) that has been found to occur naturally and is associated with autoimmune disease. The activities of wild-type (WT) Gal-8S and this mutant variant were compared in agglutination assays with the GDSs as shown in Figure 111.95 As expected, activity of the GDSs was significantly reduced for Gal-8S F19Y in all cases. However, the GDSs showed different sensitivity to the mutant with 4-Lac retaining 85% reactivity and 6b-Lac only retaining 25% reactivity as compared to the WT variant. This result supports the conclusion that 4-Lac provides an optimal sugar density and topology for Gal-8. Collectively, these results demonstrate the significance of surface density and topology of sugars on lectin binding. Optimal topology was achieved for 4-Lac with further increases in density associated with increases in steric hindrance and further reduction in density associated with loss of activity insufficiently compensated for by reduced steric hindrance. In summary, relative to 1-Lac, GDSs self-assembled from 4-Lac saw reactivity increase by a factor of 6 (Gal-8S), 7 (Gal-8L), or 12 (Gal-8S F19Y) despite a Lac dilution factor of 1/7. Thus, 4-Lac exhibits a topology with an optimal combination of density and accessibility.95

Figure 109. Rate of change in turbidity, k, of Lac-presenting GDSs from sequence-defined JGDs (see Figure 67 for structures) incubated with Gal-8S (blue) and Gal-8L (red) calculated from the data in Figure 108 at t0.5, where t0.5 is the time at which the observed absorbance is equal to one-half of the plateau absorbance. Binding was too fast (t0.5 ≈ 5−20 s) to determine the initial rate such that the calculated values of k presented here represent an underestimate of the true initial rate.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society.

“a” and “b” forms of 5-Lac and 6-Lac, which differ only in the location of sugar attachment to JGD scaffolding. Thus, as was also found in previous experiments (see section 6.3.4), the longer linker associated with Gal-8L apparently confers greater sensitivity to this lectin by providing additional spatial adaptability. As shown in Figure 109, the rate of change of turbidity, k, was also measured for GDS agglutination with Gal-8S (blue) and Gal-8L (red).95 Values were calculated within the first 5−20 s of incubation. They demonstrate the same trends observed from the plateau levels of absorbance. It is important to note that at constant concentration of Lac, GDSs self-assembled from the various JGDs were of different sizes.95 Experiments were preformed to determine the effect of differently sized vesicles on agglutination at constant Lac concentration. The results showed dependence on the specific

6.5. Reactivity of Man-Presenting Sequence-Defined GDSs with ConA

As shown in Figure 68, an analogous panel of Man-presenting sequence-defined JGDs was synthesized to determine the optimal sugar density and topology for Man-presenting

Figure 110. Agglutination assays between identically sized Lac-presenting GDSs from sequence-defined JGDs (see Figure 67 for structures). (a) Gal-8S (2 mg/mL in 100 μL of PBS) or (b) Gal-8L (2 mg/mL in 100 μL of PBS) was incubated with Lac-containing GDSs (DDLS = 63 ± 3 nm, in 900 μL of PBS). The molar attenuation coefficient, ε = A/(cl), was adapted from the Beer−Lambert law, where A = plateau OD value, c = molar concentration of Lac, and l = semimicro cuvette path length (0.23 cm). The boxes divide the GDSs into two groups: small JGDs (top) and large JGDs (bottom). The blue boxes indicate relatively high sensitivity, and the red boxes indicate comparatively low sensitivity of lectins toward different glycan topologies.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society. 6614


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Figure 111. Agglutination assays between identically sized GDSs from sequence-defined JGDs (see Figure 67 for structures). (a) Gal-8S (2 mg/mL in 100 μL of PBS) or (b) Gal-8S F19Y (2 mg/mL in 100 μL of PBS) was incubated with Lac-containing GDSs (DDLS = 73 ± 5 nm, in 900 μL of PBS). The molar attenuation coefficient, ε = A/(cl), was adapted from the Beer−Lambert law, where A = plateau OD value, c = molar concentration of Lac, and l = semimicro cuvette path length (0.23 cm). The boxes divide the GDs into two groups: small JGDs (top) and large JGDs (bottom). The blue boxes indicate high sensitivity, and the red boxes indicate low sensitivity of lectins toward different glycan topologies.95 Reprinted with permission from ref 95. Copyright 2015 American Chemical Society.

Figure 112. Agglutination assays between different Man-presenting GDSs and GDCs from sequence-defined JGDs (see Figure 68 for structures) with ConA. (A) Man-presenting GDSs with identical concentrations (0.1 mM of Man in 900 μL of HEPES, 1.0 mM CaCl2, and 1.0 mM MnCl2), and (B) Man-presenting GDSs with identical sizes (145 ± 15 nm, in 900 μL of HEPES, 1.0 mM CaCl2 and 1.0 mM MnCl2) were incubated with ConA (0.5 mg/ mL in 100 μL of HEPES, 1.0 mM CaCl2, and 1.0 mM MnCl2). The molar attenuation coefficient, ε = A/(cl), was adapted from the Beer−Lambert law, where A = plateau OD value, c = molar concentration of Man, and l = semimicro cuvette path length (0.23 cm). Control experiments were performed by incubating GDSs assembled from 3-Lac 3EO(1,2,3)-3EOLac(4), 0.1 mM of Lac in 900 μL of HEPES (1.0 mM CaCl2 and 1.0 mM MnCl2) with ConA (0.5 mg/mL in 100 μL of HEPES, 1.0 mM CaCl2, and 1.0 mM MnCl2).145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

GDSs.145 These JGDs will be referred to by their short names listed in Figure 68. The impetus for synthesizing these molecules was discussed in the previous section and in section 4.5. Agglutination assays were carried out in HEPES with ConA, a tetrameric leguminous lectin discussed previously in section 6.1. It is important to note that 4-Man formed GDCs, whereas the other twin−mixed designs formed onion-like GDSs. The single− single and twin−twin designs self-assembled into unilamellar GDSs. In Figure 112a, concentrations of Man and ConA were held constant for each sample, and molar attenuation coefficients were

calculated to more easily compare relative reactivity (see previous section).145 As was observed previously (see section 6.1), GDSs from the single−single JGD (1-Man) and the twin− twin JGD (2-Man) exhibited lower reactivity than onion-like GDSs from the first twin−mixed JGD (3-Man). However, as was demonstrated in analogous experiments with Lac-presenting GDSs in the previous section, the other twin−mixed designs (4Man, 5-Man, and 6-Man) displayed significantly higher reactivity than 3-Lac. In particular, the GDC self-assembled from 4-Man exhibited superior reactivity. Additionally, reactivity differences between onion-like GDSs from 5-Man and 6-Man, 6615


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6.6. Reactivity of GDCs with ConA and Banana Lectin (BanLec)

The bioactivity of Man-presenting GDCs self-assembled from 4Man (see Figure 67) was compared to that of onion-like GDSs.146 Agglutination assays were performed with ConA, a tetrameric leguminous lectin (see section 6.1), in HEPES. As shown in Figure 114, at increasing concentrations of Man, GDCs self-assembled from 4-Man displayed significantly higher reactivity to ConA than GDSs self-assembled from 3-Man and 5a-Man. However, the GDCs exhibited reduced agglutination rate as compared to GDSs from 5a-Man despite having higher reactivity (Figure 114e). This trend of reduced rate at increased reactivity is different from GDSs, indicating that GDCs display unique agglutination behavior. Even more striking differences in agglutination behavior of GDCs were observed in the case of banana lectin (BanLec), a dimer that exhibits β-prism I folding and two adjacent contact sites in each subunit for a total of four as illustrated in Figure 115f. BanLec is a hemagglutinin that has been reported to inhibit viruses including HIV and influenza A.176,177 A variant of the wild-type (WT) BanLec has been engineered by replacing histidine 84 with threonine (H84T). The mutant maintains antiviral activity despite having reduced activity at one-half of its binding sites. Both BanLec variants were employed in agglutination assays with GDCs and GDSs at various concentrations in HEPES as shown in Figure 115. GDCs exhibited a drastic reduction in rate and reactivity at Man concentrations above a threshold of 0.07 mM. Furthermore, GDCs displayed significantly lower reactivity with BanLec H84T (broken lines) as compared to the WT variant (solid lines) at high Man concentrations. On the other hand, GDSs exhibited no decrease in reactivity at high Man concentration and could not clearly distinguish between the two variants. Thus, GDCs demonstrated remarkably more sensitive bioactivity profiles with BanLec as compared to GDSs. This additional sensitivity of GDCs was also observed in agglutination assays with BanLec performed in the presence of free Man as inhibitor.146 As shown in Figure 116a,b, GDCs demonstrated significantly reduced agglutination in the presence of free Man for the WT BanLec and almost no reactivity in the presence of free Man for the mutant BanLec H84T. On the other hand, GDSs showed only relatively minor reductions in reactivity in the presence of free Man and could not distinguish between the two variants. These data provide further evidence of the unmatched sensitivity and selectivity of GDCs as compared to GDSs for binding to lectins. Given the sharp contrast between the bioactivity of the cubic membrane of GDCs and the lamellar membrane of GDSs, it has been hypothesized that the transition from lamellar to cubic membranes observed in some diseased cells may be an act of defense.146 Cells may be able to utilize changes in membrane morphology as a regulatory switch from one sugar topology to another with very different affinities for lectins, which play important roles in immune response.

Figure 113. Rate of change in turbidity, k, of GDSs and GDCs from sequence-defined JGDs (see Figure 68) with ConA calculated from the curves of GDSs (0.1 mM of Man, 900 μL) with ConA (0.5 mg/mL, 100 μL) in HEPES (1.0 mM CaCl2 and 1.0 mM MnCl2) shown in Figure 112a at t1/2, where t1/2 is the time at which the observed absorbance is equal to one-half of the plateau absorbance (A). Each rate of GDS agglutination was the average value from triplicate measurements.145 Reprinted with permission from ref 145. Copyright 2016 National Academy of Sciences USA.

which exhibit more subtle structural alterations, were observable, indicating the sensitivity of the system. These results were confirmed when both vesicle size and Man concentration were controlled (see previous section) as shown in Figure 112b. It should be noted that both onion-like GDSs and GDCs maintained high reactivity with conA despite having a large portion of carbohydrate buried within the interior structure as compared to unilamellar GDSs. Thus, the data actually underestimate the efficiency of onion-like GDSs and GDCs as compared to unilamellar GDSs because it does not take into consideration the reduction in concentration of exposed carbohydrate in those structures. As illustrated in Figure 113, the rate of change of turbidity, k, was also measured.145 Despite having the highest reactivity with ConA, 4-Man exhibited slower kinetics than 5-Man and 6-Man. The tetrameric design of ConA results in the binding of two of its sites to each GDS during cross-linking. The incorporation of 4Man into GDCs may reduce the spatial adaptability for this rigid binding and thus result in a reduced rate of change in turbidity as compared to onion-like GDSs. The stability of aggregates formed by the Con-A-mediated agglutination of the GDSs was also investigated by addition of free Man, which can saturate ConA binding sites, and EDTA, a chelating agent that can remove Ca2+ from ConA binding sites rendering them inactive.145 While 1-Man, 2-Man, and 3-Man exhibited resistance to aggregate dissociation in the presence of inhibitors, the other twin−mixed JGDs (4-Man, 5-Man, 6-Man) were highly susceptible to aggregate dissociation. Thus, higher density sugar topologies tend to result in slow aggregate formation with low plateau levels of absorbance and high resistance to dissociation, whereas lower density sugar topologies result in fast aggregate formation with high plateau levels of absorbance and high susceptibility to dissociation. Sugar density may therefore serve as a molecular switch between lectin reactivity behaviors.

6.7. GDS Stimulation of Killer T Cells

Gillies and co-workers tested Gal-presenting GDSs and micelles for their ability to stimulate invariant natural killer T (iNKT) cells, a class of T cells that responds to antigens displayed by CD1d and that plays important immunoregulatory roles in diseases such as infections, diabetes, and cancer.141 The synthetic agonist KRN7000 is a commonly employed CD1d specific ligand for iNKT cells. Its capacity to stimulate iNKT cells was compared 6616


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Figure 114. Agglutination assays of GDCs from 4-Man and GDSs from 3-Man, and 5a-Man (see Figure 68 for structures) with ConA. (a−c) Change of turbidity over time in solutions of Man-presenting GDCs self-assembled from (a) 4-Man (0.0125−0.1 mM in 900 μL of 10 mM HEPES, 1.0 mM CaCl2, and 1.0 mM MnCl2), and Man-presenting onion-like GDSs self-assembled from (b) 3-Man and (c) 5a-Man (0.0125−0.1 mM in 900 μL of 10 mM HEPES, 1.0 mM CaCl2, and 1.0 mM MnCl2) with ConA (0.5 mg/mL in 100 μL of 10 mM HEPES, 1.0 mM CaCl2, and 1.0 mM MnCl2). (d) Molar attenuation coefficient, ε, and (e) rate constant of change in turbidity, k, of 4-Man, 3-Man, and 5a-Man with ConA. ε is adapted from the Beer−Lambert law, ε = A/(cl), where A = plateau value of absorbance, c = molar concentration of Man, and l = semimicro cuvette path length (0.23 cm). k is calculated from the curves in (a−c) in HEPES at t1/2, where t1/2 is the time at which the observed absorbance is equal to one-half of the plateau absorbance. (f) Representative illustration of the tetrameric lectin ConA, in which each binding site is loaded with the ligand methyl α-D-mannopyranoside (PDB 5CNA). The two cations Ca2+ (green sphere) and Mn2+ (purple sphere) that are essential for lectin activity are also highlighted. Orange arrows indicate Man-binding sites. The distances between binding sites are shown.146 Reprinted with permission from ref 146. Copyright 2016 American Chemical Society.

the assemblies displayed greater activity, demonstrating the corresponding benefit of greater multivalency and JGD size. As such, the micelles from L-G4-Gal were the most stimulating of the assemblies from JGDs. Preparations generated at initial concentrations of 10 μg/mL resulted in higher activity, indicating that individual JGDs rather than assemblies stimulated the cells. Furthermore, a final JGD concentration of 20 ng/mL was adequate for saturating TCR because increasing the final concentration did not significantly impact activity. Collectively (see also section 6.2), the results suggest the possible utility of these GDSs and micelles in applications involving specific receptor−ligand interactions.

to that of the GDSs and micelles self-assembled from libraries 52 and 53 (also see sections 5.2.1, 5.2.4, and 6.2). Mouse DN32.D3 cells were used in the study because of their ability to express both CD1d and the canonical T cell receptor (TCR) of iNKT cells, preventing any need for additional antigen-presenting cells. Assemblies from JGDs were prepared at a concentration of either 1 mg/mL or 10 μg/mL and then diluted to 1 μg/mL or 20 ng/ mL for the bioassay. Because of the low starting concentration, samples prepared at 10 μg/mL before dilution were unlikely to form supramolecular assemblies, whereas samples prepared at 1 mg/mL before dilution were. The samples were incubated with cells for 24 h prior to measuring the level of the cytokine interleukin-2 (IL-2), which indicated the degree of T cell stimulation. As shown in Figure 117, although exhibiting reduced activity as compared to KRN 7000, GDSs from the second generation JGD (L-G2-Gal) and micelles from the third and fourth generation JGDs (L-G3-Gal, L-G4-Gal) displayed greater activity than the media control and the nonsugar presenting analogue of L-G3Gal, denoted L-G3-OH.141 In general, as generation increased,

6.8. Future Directions

The studies accomplished so far with GDSs and GDCs have illuminated fundamental aspects of lectin recognition of multivalent displays. It has been repeatedly shown that reduced sugar density on the surface of vesicles promotes more efficient binding.95,134,145,154,175 However, when sugar density is reduced too much, the reduction in ligand concentration outweighs the 6617


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Figure 115. Agglutination assays of GDCs from 4-Man and GDSs from 3-Man, and 5a-Man (see Figure 68 for structures) with WT BanLec and its H84T variant. (a−c) Agglutination assays of Man-presenting GDCs self-assembled from (a) 4-Man (0.0125−0.1 mM in 900 μL of 10 mM HEPES), and Man-presenting onion-like GDSs self-assembled from (b) 3-Man and (c) 5a-Man (0.0125−0.1 mM in 900 μL of 10 mM HEPES) with WT BanLec (solid lines) and its H84T variant (broken lines) (0.5 mg/mL in 100 μL of 10 mM HEPES). (d,e) Molar attenuation coefficient, ε, of 4-Man, 3-Man, and 5a-Man with (d) WT BanLec or (e) its variant. ε is adapted from the Beer−Lambert law, ε = A/(cl), where A = plateau value of absorbance, c = molar concentration of Man, and l = semimicro cuvette path length (0.23 cm). (f) Crystallographic structure of the dimeric WT BanLec loaded with dimannose (PDB 4PIK). Orange arrows indicate Man-binding sites. Distance between binding sites is also shown.146 Reprinted with permission from ref 146. Copyright 2016 American Chemical Society.

benefit of reduced steric hindrance.95,145 Additionally, characteristics of various lectin architectures have been explored, revealing, for instance, the utility of a longer linker between CRDs for increased reactivity.95,154,175 GDSs and GDCs also exhibit a remarkable ability to distinguish subtle variations in lectin architecture including the presence of a single site mutation.95,175 These results highlight the sensitivity and selectivity of supramolecular structures derived from JGDs. An important challenge that must be overcome is determining the exact topology of the glycan surface on GDSs and other complex architectures. With more precise information on topology in hand, it will be possible to develop a more detailed view of the intricacies of lectin recognition. Future work will also entail the synthesis of JGDs with more complex sugar headgroups. In particular, the development of JGDs bearing linear and branched oligosaccharides will supply more complex carbohydrate displays capable of providing new insight into lection recognition. Additionally, GDSs bearing complex sugars may be utilized as inexpensive and efficient vaccines63 by exhibiting an optimal sugar density.

7. COASSEMBLY OF DSs AND GDSs WITH OTHER COMPONENTS DSs and GDSs have been coassembled with a variety of amphiphilic molecules including other bilayer building blocks such as fluorinated JDs, block copolymers, and phospholipids, as well as membrane proteins and bacterial cell membranes. These hybrid assemblies have granted access to unique morphologies with biological applications. Inspiration for much of this work, especially coassembly with phospholipids, membrane proteins, and bacterial cell membranes, originated from previous studies of the coassembly of block copolymers and natural phospholipids and membrane proteins into hybrid vesicles.178−183 It should be noted that this section does not review coassemblies of JDs and JGDs with each other, which have already been discussed in many of the preceding sections, except in the case of fluorinated JDs, which have not been considered in detail yet and will be discussed below. 6618


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Figure 117. Levels of murine IL-2 after exposure to α-Gal-presenting library 52 and 53 JGDs, prepared as diluted assemblies or via direct dissolution and dilution, at either 20 ng/mL or 1 μg/mL. The right column shows the positive control KRN 7000 at 100 ng/mL, as well as negative controls L-G3-OH at 1 μg/mL and media alone. Error bars represent the standard deviation of three measurements.141 Reprinted with permission from ref 141. Copyright 2016 Royal Society of Chemistry.

Figure 116. Agglutination assays of GDCs from 4-Man and GDSs from 3-Man, and 5a-Man (see Figure 68 for structures) with WT BanLec and its H84T variant. Man-presenting (a,b) GDCs self-assembled from 4Man, and onion-like GDSs self-assembled from (c,d) 3-Man and (e,f) 5a-Man, prepared with 0.05 mM of JGDs, 900 μL of HEPES were incubated with WT BanLec (a,c,e), or the H84T variant (b,d,f) (0.5 mg/ mL in 100 μL of HEPES) with 100 mM of Man (red line) or Lac (blue line). A high concentration (100 mM) of Man solution in HEPES (100 μL) was added at t = 50 s to the suspensions of GDCs or onion-like GDSs (900 μL of HEPES) with WT BanLec (a,c,e) or H84T (b,d,f) (black line).146 Reprinted with permission from ref 146. Copyright 2016 American Chemical Society.

7.1. Coassembly with Phospholipids

Figure 118. Giant vesicles obtained from the 1:1 mixture of SOPC: (3,4,5)12G1-BnA-(3,4,5)3-G1-(NH3Cl)3 (library 23) containing 1% Texas Red-(3,4,5)12G1-PhA-(3,4,5)3-G1-(NH3Cl)3 (library 38) by film hydration in water.37 Reprinted with permission from ref 37. Copyright 2010 American Association for the Advancement of Science.

In initial experiments, Percec and co-workers coassembled amine-presenting library 22 and 23 JDs with the phospholipid SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) at a ratio of 1:1 to form giant DSs by thin film hydration.37 Microscopy images of these DSs are shown in Figure 118. The Texas Red-tagged library 39 JD was also incorporated in the DSs to permit fluorescence microscopy imaging. As is apparent, spherical vesicles were obtained, demonstrating the ability of JDs to coassemble with phospholipids. As shown in Figure 119, giant DSs were also achieved by coassembling the Texas-Red tagged phospholipid TR-DHPE (Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) with fluorinated JDs and their hydrogenated analogues, as well as hybrid fluorinated-hydrogenated JDs.90 In particular, TR-DHPE was coassembled with the library 5 JD with (3,4,5)3EO substituted hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE(3,4,5)-3EO-G1-(CH3)6 (RH in Figure 119), the library 35 JD

with (3,5)-disubstituted fluorinated Percec-type hydrophobic dendrons denoted (3,5)PPVEG1-PE-(3,4,5)-3EO-G1-(OCH3)3 (RF in Figure 39), and the hybrid JD comprising library 36 (RHF in Figure 119) (see Figures 5 and 9 for structures). According to the confocal images and fluorescence intensity, hybrid RHF exhibited a superior ability to coassemble with phospholipids, whereas completely fluorinated RF was not very miscible with phospholipids. These results highlight the potential utility of completely fluorinated DSs for remote drug loading and the utility of hybrid fluorinated-hydrogenated DSs for targeted delivery. By thin film hydration, Cheng and co-workers demonstrated the coassembly of phospholipid POPC (palmitoyloleoylphosphocholine) with first, second, and third generation aminepresenting library 31 JDs with C18 hydrophobic tails as shown in Figure 120.184 Although the JDs alone formed micelles (see also section 3.11.3), when coassembled with POPC, vesicles were 6619


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7.2. Coassembly with Block Copolymers

Initial experiments determined that giant vesicles could be coassembled from amine-presenting library 22 and 23 JDs and the block copolymer OB-29 (poly(1,2 butadiene)-b-poly(ethylene oxide)).37 Microscopy images of these assemblies are shown in Figure 121. Further exploration of this field has yet to be considered. 7.3. Coassembly with Membrane Proteins

Percec and co-workers demonstrated the incorporation of the membrane protein Melittin, which forms pores in membranes, into DSs self-assembled from the library 1 JD with secondgeneration bis-MPA polyester hydrophilic dendrons and (3,5)disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-(OH)8 (Figure 4).37 Via thin film hydration, the DSs were encapsulated with the fluorescent dye ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) and quencher DPX (p-xylene-bis(N-pyridinium bromide)). At high concentrations, DPX quenches the fluorescence of ANTS such that no fluorescence is observed when ANTS and DPX are both encapsulated in the DSs. However, when the compounds are released from DSs, DPX concentration is too small to quench ANTS so fluorescence can be observed. Using this system, a large increase of fluorescence was observed after the addition of Melittin to the suspension of DSs, indicating that the protein was incorporated into the DS membranes and formed pores. Giustini and co-workers coassembled submicrometer DSs from the photosynthetic Reaction Centre (RC), which is an integral membrane protein found in photosynthetic bacteria, and the library 1 JD with first generation bis-MPA-based polyester hydrophilic dendrons and (3,5)-disubstituted Percec-type hydrophobic dendrons denoted (3,5)12G1-PE-BMPA-(OH)4 (Figure 4).98 An injection method was used to prepare the coassembled DSs, which exhibited smaller diameter and polydispersity than did DSs self-assembled from the JD alone. Furthermore, the RC was determined to retain its photosynthetic functionality when incorporated in DSs. As shown in Figure 122, the RC can either face toward the interior (Figure 122b) of the vesicle or face toward the exterior (Figure 122a). By adding the reducing agent cyt-c2, which can only bind to externally facing RCs, it was determined that over 90% of RCs incorporated in DSs faced the exterior. This preference was not observed in conventional liposomes.

Figure 119. (a) Chemical structure of Texas Red (TR)-labeled phospholipid TR-DHPE. (b−e) Representative confocal fluorescent microscopy images of giant unilamellar vesicles assembled from (b) RH (library 5), (c,d) RF (library 35), and (e) hybrid RHF (library 36) JDs with 1% (w/w) of TR-DHPE. The image in (d) shows the same area as the image in (a) but with a 5-fold increase in intensity to better show vesicle formation. (f) Fluorescence intensity of giant vesicles. Error bars indicate standard error (SEM) of the mean based on data derived from 25 vesicles of each sample. ***denotes p-value

Mimicking Complex Biological Membranes and Their Programmable

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