Versatile Functionalization of Polysaccharides via Polymer Grafts

Article pubs.acs.org/accounts

Versatile Functionalization of Polysaccharides via Polymer Grafts: From Design to Biomedical Applications Yang Hu,†,‡,§,∥ Yang Li,†,‡,§,∥ and Fu-Jian Xu*,†,‡,§ †

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China ‡ Key Laboratory of Carbon Fiber and Functional Polymers (Beijing University of Chemical Technology), Ministry of Education, Beijing 100029, China § Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, China CONSPECTUS: Because of their biocompatibility, biodegradability, and unique bioactive properties, polysaccharides have been recognized and directly applied as excellent candidates for various biomedical applications. In order to introduce more functionalities onto polysaccharides, various modification methods were applied to improve the physical-chemical and biochemical properties. Grafting polysaccharides with functional polymers with limited reaction sites maximizes the structural integrity. To the best of our knowledge, great efforts have been made by scientists across the world, including our research group, to explore different strategies for the synthesis and design of controllable polymer-grafted polysaccharides. By the application of some reasonable strategies, a series of polymer-grafted polysaccharides with satisfactory biocharacteristics were obtained. The first strategy involves facile modification of polysaccharides with living radical polymerization (LRP). Functionalized polysaccharides with diverse grafts can be flexibly and effectively achieved. The introduced grafts include cationic components for nuclei acid delivery, PEGylated and zwitterionic moieties for shielding effects, and functional species for bioimaging applications as well as bioresponsive drug release applications. The second synthetic model refers to biodegradable polymer-grafted polysaccharides prepared by ring-opening polymerization (ROP). Inspired by pathways to introduce initiation sites onto polysaccharides, the use of amine-functionalized polysaccharides was explored in-depth to trigger ROP of amino acids. A series of poly(amino acid)-grafted polysaccharides with advanced structures (including linear, starshaped, and comb-shaped copolymers) were developed to study and optimize the structural effects. In addition, biodegradable polyester-grafted polysaccharides were prepared and utilized for drug delivery. Another emerging strategy was to design polysaccharide-based assemblies with supramolecular structures. A variety of assembly techniques using non-covalent interactions were established to construct different types of polysaccharide-based assemblies with various bioapplications. On the basis of these strategies, polymer-grafted polysaccharides with controllable functions were reported to be well-suited for different kinds of biomedical applications. The exciting results were obtained from both in vitro and in vivo models. Viewing the rapid growth of this field, the present Account will update the concepts, trends, perspectives, and applications of functionalized polysaccharides, guiding and inspiring researchers to explore new polysaccharide-based systems for wider applications.

1. INTRODUCTION With the emergence of new materials to suit the rapid increase of bioapplications, polysaccharides have been comprehensively explored because of their unique features such as biocompatibility, degradability, and bioactivity. To impart polysaccharides with more functionalities, researchers have explored numerous methods to effectively manipulate desired functions.1 Conventional modification methods, i.e., direct immobilization of molecular functional groups, usually consume huge amounts of amino, carboxyl, or hydroxyl groups of polysaccharides and hence destroy the structure and functional integrity. Grafting polysaccharides with functional polymers, on the contrary, can solve this dilemma with limited reaction sites and maximize the structural integrity. In addition, modern polymerization © 2017 American Chemical Society

methods, such as living radical polymerization (LRP) and ringopening polymerization (ROP), have provided numerous advantages in the preparation of well-defined polymer grafts with facile reaction conditions, unrestrained solvents, a broad range of functional monomers, and flexible structures.2−5 In this Account, we mainly focus on the recent progress in synthetic methodologies of versatile types of polymer-grafted polysaccharides. Their biomedical applications, including gene/drug delivery, bioimaging, and multifunctional applications, will also be critically reviewed. Received: September 23, 2016 Published: January 9, 2017 281

DOI: 10.1021/acs.accounts.6b00477 Acc. Chem. Res. 2017, 50, 281−292

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Accounts of Chemical Research

Figure 1. Typical introduction of ATRP initiation sites onto polysaccharides.

2. POLYMER-GRAFTED POLYSACCHARIDES BY ATRP

2.1. Introduction of Initiation Sites onto Polysaccharides

LRP methods, such as atom transfer radical polymerization (ATRP) and reversible addition−fragmentation chain transfer polymerization (RAFT), have several advantages in constructing novel biomedical materials with specific compositions, architectures, and functions.3,4 Well-defined polymer-grafted polysaccharides composed of polysaccharide backbones and different polymer side chains can be prepared via LRP through “grafting onto” and “grafting from” methods. The “grafting onto” technique involves the conjugation of the reactive polymer chains onto the functional backbone. However, for such a technique, tedious procedures are involved because of the presynthesis of end-functionalized polymers. Moreover, the resultant grafting density is low because of steric hindrance. On the other hand, the “grafting from” technique is chemically versatile in preparing polymer side chains with high density.4 Thus, most research work has focused on “grafting from” for functionalization of polysaccharides. For the synthesis of polymer-grafted polysaccharides via LRP, both ATRP initiation sites and RAFT chain transfer agents were introduced onto polysaccharide backbones.4,6 In this Account, the ATRP technique is the predominant focus, as it does not require critical experimental conditions and can tolerate a wide range of functional monomers.

For the “grafting from” method, polymer side chains grow directly from the defined initiation sites of a prefunctionalized polysaccharide. It is essential to immobilize ATRP initiators on the polysaccharide backbone. Our group has developed many effective synthetic methods for introducing ATRP initiation sites onto polysaccharides (Figure 1). For nonionic polysaccharides, including cyclodextrins (CDs, cyclic oligosaccharides of six to eight glucose units), hydroxypropyl cellulose (HPC), and pullulan, their hydroxyl groups can be directly reacted with 2bromoisobutyrl bromide (BIBB) to produce alkyl halide initiation sites (macroinitiators).7−9 Such esterification reactions can be carried out in anhydrous organic solvents, such as dichloromethane and N,N-dimethylacetamide. However, dextran is hardly soluble in most traditional solvents, except for dimethyl sulfoxide (DMSO). A simple one-step strategy involving α-bromoisobutyric acid (BIBA) in DMSO was developed to fix ATRP initiation sites on dextran.10 The carboxylic group of BIBA can be activated by 1,1′-carbonyldiimidazole (CDI) to react with the hydroxyl groups of dextran. Anionic polysaccharides, such as heparin and hyaluronic acid (HA), are soluble only in water. This drawback limits the conversion of hydroxyl groups into ATRP initiation sites. Heparin and HA sodium salt can become soluble in organic solvents after ion exchange with tetrabutylammonium chloride 282


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Accounts of Chemical Research (TBA).11,12 Thus, the hydroxyl groups of anionic polysaccharides are able to react with BIBB or BIBA in organic solvents. The introduction of stimuli-responsive species into initiation sites can produce smart polymer grafts with specific applications. Disulfide linkages are responsive to reducing agents, making the immobilized initiation sites reducible. ATRP initiation sites containing disulfide bonds have been flexibly introduced onto polysaccharides, including β-CD, dextran, and pullulan (Figure 1).13−15 The hydroxyl groups of polysaccharides were first reacted with cystamine. The resultant disulfide-bond-linked amine groups were reacted with BIBA to produce alkyl halide. pH-responsive species, such as ketal groups, are readily hydrolyzed in acidic environments. Acid-labile ATRP initiation sites were conjugated onto β-CD via a two-step reaction: (1) 2(vinyloxy)ethanol was reacted with BIBB to produce vinethenecontaining ATRP agent (VEBB), and (2) VEBB was conjugated with the hydroxyl groups of β-CD through acetal group linkages.16 This method can be elegantly employed in the synthesis of versatile acid-labile polysaccharide initiators. With these functionalized polysaccharide macroinitiators, different kinds of polymer grafts are readily obtained via ATRP. It should be noted that when ATRP is initiated by a macroinitiator (with multiple initiation sites) with a high local concentration, gelation probably happens as a result of radical−radical chain coupling reactions. To avoid gelation, moderate alkyl halide substitutions are required for polysaccharide initiators.4

Figure 2. Star-shaped β-CD-PDs with different arm numbers (group I, β-CD-PDs with similar arm lengths; group II, β-CD-PDs with similar molecular weights). Reproduced with permission from ref 17. Copyright 2012 Elsevier Ltd.

decreased buffering capacity. Different polysaccharides possess varied constituent units and distinct bioactivities. Altering polysaccharide backbones with different stiffness/flexibility can affect the transfection behaviors of polysaccharide-PDs. For instance, as a result of the better water solubility and higher flexibility of dextran compared with HPC, Dextran-PD exhibited better DNA condensation ability and higher transfection ability than HPC-PD.8,10 Chitosan-PD exhibited degradability in the presence of lysozyme.18 By the use of stimuli-responsive polysaccharide initiators, polysaccharide-PD vectors with smart PDMAEMA grafts have been constructed for responsive transfection behaviors. A reducible comblike dextran-backboned vector (Dextran-SS-PD) was prepared via ATRP of DMAEMA from the disulfide-containing initiator (Figure 3a).13 Disulfide linkages between dextran and PDMAEMA side chains were readily cleavable by reducing reagents, for instance glutathione in cells. Such responsiveness facilitated DNA release in cells and contributed to the transfection efficiency (Figure 3b). Properly grafting cationic polymer chains from a polysaccharide backbone/core provides an effective means for designing polysaccharide-based gene vectors.

2.2. Cationic PDMAEMA Grafts for Gene Delivery

Poly(2-(dimethylamino)ethyl methacrylate)) (PDMAEMA), one typical nonviral polycationic gene vector, can facilitate cell transfection by protecting plasmids from degradation. Welldefined PDMAEMA can be easily synthesized via ATRP.3,4 By the use of polysaccharide macroinitiators, a series of polysaccharides have been flexibly grafted with PDMAEMA as new gene carriers via ATRP. The star-shaped polycations possess dense cationic arms with moderate flexibility. Such a unique architecture would contribute to the complexation process of polycations with DNA and produce stable polycation/DNA complexes (polyplexes) for the resultant good transfection.4 βCD-cored star-shaped polycations (β-CD-PDs) were prepared via direct ATRP of DMAEMA from the starting β-CD-Br initiators.7 Varying the polymerization time easily produced different arm lengths. Longer PDMAEMA arms can increase the DNA-condensing ability, leading to more stable polyplexes and higher transfection efficiency. For the optimization of starshaped β-CD-PDs, the arm number−property relationship was investigated (Figure 2).17 With a fixed arm length, the cytotoxicity of β-CD-PDs increased with the arm number, where β-CD-PD with a suitable arm number showed the best transfection efficiency. With a fixed molecular weight, β-CD-PD with 21 arms possessed the lowest toxicity. Thus, star-shaped gene carriers can be structurally tailored by the number and length of polycation arms. In comparison with CDs, long polysaccharide backbones, such as cellulose, dextran, and chitosan, can be grafted with more PDMAEMA side chains to construct high-molecular-weight comb-shaped gene vectors (polysaccharide-PDs).8,10,18 Polysaccharide-PDs exhibited higher gene transfection efficiencies than high-molecular-weight PDMAEMA homopolymers. Partial quaternization was used for further modification of the PDMAEMA side chains.8,10 However, this method enhances the DNA condensation ability but lowers the transfection efficiency, probably because of the increased cytotoxicity and

2.3. Aminated PGMA Grafts for Gene Delivery

In addition to direct polymerization of cationic monomers, postfunctionalization of well-defined active precursors can be utilized to produce functional polycations for gene delivery. Well-defined poly(glycidyl methacrylate) (PGMA) is readily prepared via ATRP, and its pendant epoxide groups can react with small amino molecules via a ring-opening reaction.4 The resultant aminated PGMA contains flanking amine and hydroxyl units. In particular, ethanolamine (EA)-functionalized PGMA (PGEA) with plentiful hydrophilic hydroxyl groups has been recognized as a low-toxicity vector.4 Several PGEA-grafted polysaccharides, including responsive PGEA-grafted β-CD copolymers and liver-targeted PGEA-grafted pullulan copolymers, have been prepared as low-toxicity, high-transfection vectors.9,14−16 Star-shaped CD-SS-PGEA (or A-CD-PGEA) vectors consisting of β-CD cores and disulfide-linked (or acteal-linked) PGEA arms were synthesized via ATRP from a reducible (or acid-labile) β-CD initiator. CD-SS-PGEA mediated highly efficient gene delivery due to the favorable properties of star-shaped architecture, low-toxicity PGEA, and responsive disulfide linkages.14 Acetal linkers endowed A-CD-PGEA with acid lability and degradation (Figure 4a).16 In weakly acidic endosome, the broken acetal linkers caused decomposition of ACD-PGEA. Meanwhile, the morphologies of the A-CD-PGEA/ 283


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Figure 3. (a) Schematic diagram illustrating the preparation processes of Dextran-SS-PD copolymers. (b) Responsive pDNA release and resultant gene expression. Reproduced with permission from ref 13. Copyright 2011 Elsevier Ltd.

Figure 4. (a) Structural illustration of A-CD-PGEA. (b) Acid-lability of A-CD-PGEA characterized by GPC and AFM. Reproduced from ref 16. Copyright 2015 American Chemical Society.

xenograft models (Figure 5a).9 Pu-PGEA mediated stronger P53 and MEG3 expression in liver than in other organs. Codelivery of P53 and MEG3 by Pu-PGEA effectively suppressed the growth of hepatocellular carcinoma without affecting the health of mice (Figure 5b). This work indicated that polymers can be grafted from functional polysaccharides, such as liver-targeting pullulan, to produce new gene/drug vectors with the bioactivity of polysaccharides. It should be noted that moderate substitution of functional polysaccharides is important to the bioactivity of polymer-grafted polysaccharides.

pDNA complexes were transformed from compact and spherical particles to loose and irregular particles, accelerating DNA release (Figure 4b). A-CD-PGEA demonstrated better transfection performance than its counterpart CD-PGEA without acetal linkers. Among polysaccharides, pullulan has been explored for livertargeting gene/drug delivery because of its specificity for liver. High-performance comb-shaped PGEA vectors composed of pullulan backbones and PGEA side chains (Pu-PGEA) were prepared via ATRP.15 Pu-PGEA exhibited obviously higher cellular uptake and transfection efficiency in HepG2 cells than in Hela cells. In particular, Pu-PGEA possessed satisfactory hemocompatibility without causing obvious hemolysis. PuPGEA was also used to codeliver antitumor pDNA (P53) and long noncoding RNA (MEG3) in hepatocellular carcinoma

2.4. PEGylated and Zwitterionic Grafts for Gene Delivery

For potential clinical applications, the stability of positively charged polycation/pDNA polyplexes in the bloodstream cannot be ignored. In the presence of platelets and proteins, polyplexes are easily cleared in the bloodstream as a result of colloidal 284


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Figure 5. (a) Illustration of the synthetic process of Pu-PGEA and codelivery of MEG3 and P53 in hepatocellular carcinoma. (b) MEG3/P53-loading nanocomplexes inhibit tumor growth in vivo. Adapted with permission from ref 9. Copyright 2016 Wiley-VCH.

MA were individually grafted from CNC surfaces with ATRP and RAFT initiation sites.19 As alternatives to PEG, carboxybetaine-, sulfobetaine-, and phosphorylcholine-based zwitterionic polymers also exhibit high resistance to protein adsorption. Versatile functionalization of Dextran-SS-PD was proposed by introducing different zwitterionic betaine species to improve the biophysical properties (Figure 6).20 Some DMAEMA units of Dextran-SS-PD were randomly converted into carboxybetaine methacrylate (CBMA) by the reaction with β-propionolactone (BPL), producing the Dextran-SS-PD-r-PCBMA copolymer. Different contents of CBMA can be introduced by varying the feed amounts of BPL. For the betaine-containing Dextran-SS-PD-based block copolymers (Dextran-SS-PD-b-PCBMA or Dextran-SS-PD-bPSBMA), Dextran-SS-PD was used as the ATRP macroinitiator to produce PCBMA or poly(sulfobetaine methacrylate)

aggregation. PEGylation of polycations can resist nonspecific protein adsorption of polyplexes and enhance the stability. As ATRP is a kind of LRP, the dormant alkyl halide chain ends of the polymers prepared by ATRP can be reactivated for subsequent polymerization. By a second ATRP from PDMAEMA, a PDMAEMA-grafted β-CD (or dextran) vector can easily be functionalized with poly(poly(ethylene glycol)ethyl ether methacrylate) (PPEGEEMA) blocks.7,13 The incorporation of PEGEEMA with good biocompatibility reduced the cytotoxicity. PEGEEMA monomer has an average of three EG units and a hydrophobic ethyl end. This unique structure imparts PPEGEEMA with slight hydrophobicity, which could enhance the interactions of the polyplexes with cells, cell uptake, and subsequent gene transfection (Figure 3b). Moreover, PPEGEEMA can also be directly grafted from polysaccharide backbones. Heterogeneous polymer brushes of PDMAEMA and PPEGEE285


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stability of the polyplexes in serum and reduced the cytotoxicity. More importantly, betaine-containing copolymers, particularly for Dextran-SS-PD-b-PCBMA and Dextran-SS-PD-b-PSBMA, exhibited higher transfection efficiencies than Dextran-SS-PD. 2.5. Functional Grafts for Drug Delivery

Some functional polymers were grafted onto polysaccharides for drug delivery, such as pH-sensitive and thermosensitive grafts. Among pH-sensitive polymers, poly(sodium methacrylate) (PMANa) with plentiful carboxyl groups shows pH-dependent solubility in an aqueous environment. PMANa side chains were grafted from the presynthesized HA macroinitiator by ATRP.21 The resultant copolymer, HA-PMANa, showed pH-sensitive behavior and became insoluble below pH 2.5. α-Chymotrypsin was effectively entrapped in HA-PMANa as a gastro-resistant excipient under simulated gastric conditions. Poly(2(diethylamino)ethyl methacrylate) (PDEAEMA) is another pH-sensitive polymer with a pKa of 6.5. PDEAEMA-grafted ethyl cellulose (EC-PDEAEMA) was prepared via ATRP for loading of rifampicin.22 Rifampicin-loaded EC-PDEAEMA micelles showed controlled and pH-responsive release behavior.

Figure 6. Schematic diagram illustrating the preparation processes of zwitterionic random- and block-copolymer-based gene vectors. Reproduced with permission from ref 20. Copyright 2013 Elsevier Ltd.

(PSBMA) blocks. For all three types of betaine-containing copolymers, the incorporated zwitterionic species enhanced the

Figure 7. (a) Illustration of the preparation processes of degradable polysaccharide-PAsp-ED grafts and (b) their degradable behavior and cytotoxicity. Reproduced with permission from ref 25. Copyright 2014 Elsevier Ltd. 286


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and poly(amino acid) were prepared by grafting prefunctionalized poly(amino acid)s onto monosubstituted polysaccharides using the click reaction.28 The reductive end of dextran was modified with one alkyne group via the reductive amination reaction with propargylamine. Click coupling of alkyneterminated dextran and poly(γ-benzyl L-glutamate) (PBLG) with terminal azide blocks produced Dex-b-PBLG copolymers. Dextran from Dex-b-PBLG was further cross-linked with 3,3′dithiodipropionic acid to prepare disulfide-cross-linked micelles.29 The shell cross-linked micelles can effectively load the typical anticancer drug doxorubicin (DOX) and mediate responsive drug release.

Among the thermosensitive polymers, poly(N-isopropylacrylamide) (PNIPAAm), with a lower critical solution temperature (LCST) of about 32 °C, is the most widely studied. Polysaccharides were grafted with PNIPAAm to prepare thermosensitive copolymers.12,23 HPC possesses an LCST of >40 °C, which is higher than body temperature (37 °C). The high LCST limited the applications in sustained drug release at body temperature. Comb-shaped copolymers with HPC backbones and PNIPAAm side chains (HPC-PNIPAAm) were prepared via ATRP from the prefunctionalized HPC initiator.23 The LCST of HPC-PNIPAAm decreased with increasing PNIPAAm content. HPC-PNIPAAm with an average PNIPAAm content of above 53 wt % exhibited an LCST below 37 °C. Stable HPC-PNIPAAm hydrogels were prepared via the cross-linking reaction of hydroxyl groups of the HPC backbones with divinylsulfone. Compared with pure HPC hydrogels, HPCPNIPAAm hydrogels provided long-term/sustained drug release at body temperature. In addition, PNIPAAm grafts imparted the hydrogels with higher swelling ratios and interconnected pore structures, resulting in higher drug loading ability.

3.2. Polyester Grafts

Because of their hydrophobic property, polyester grafts were introduced onto polysaccharides to obtain new amphiphilic materials for drug delivery. PCL-grafted chitosan (CS-PCL) was synthesized by ROP of caprolactone from chitosan.30 CS-PCL was fabricated into polymeric micelles to encapsulate the drug, and the drug loading/release property was well-controllable. It should be noted that the different hydroxyl groups of polysaccharides possess varied initiation activities. A block copolymer of dextran and PCL (Dex-b-PCL) was prepared by the coupling of amino-monosubstituted dextran and acryloylterminated polyester.31 The introduction of stimuli-responsiveness can enhance the drug delivery performance of Dex-b-PCL. A disulfide-connected block copolymer (Dex-SS-PCL) was constructed by the thiol−disulfide exchange reaction between orthopyridyl disulfide-monosubstituted dextran and thiolterminated PCL (Figure 8). Dex-SS-PCL was able to assemble into shell-sheddable micelles for drug loading and realize intracellular redox-responsive drug release.32

3. BIODEGRADABLE-POLYMER-GRAFTED POLYSACCHARIDES BY ROP Biodegradable polymers, mainly poly(amino acid) and polyester, are widely used in biomedical fields because of their biodegradability and low toxicity.5,24 Poly(amino acid)s are readily synthesized by ROP of N-carboxyanhydrides. Wellknown polyesters, such as poly(lactide) (PLA), polycaprolactone (PCL), and poly(glycolide), can be produced through ROP of hydroxyalkanoic acids. Degradable polymer-grafted polysaccharides were easily constructed via the introduction of biodegradable polymers through “grafting from” and “grafting onto” methods.

4. SUPRAMOLECULAR ASSEMBLIES INVOLVING POLYMER-GRAFTED POLYSACCHARIDES Supramolecular assembly, based on non-covalent interactions, provides a flexible platform for the design of functional

3.1. Poly(amino acid) Grafts

A simple and versatile “grafting from” method was proposed by our group for the preparation of poly(amino acid)-grafted polysaccharides via direct ROP from polysaccharides. Some hydroxyl groups of polysaccharides, such as β-CD and dextran, can be first activated by CDI and functionalized with ethylenediamine (ED) (Figure 7).25 The introduced amino groups serve as the initiation sites for the ROP of β-benzyl-L-aspartate Ncarboxyanhydride (BLA-NCA), producing star-/comb-shaped poly(β-benzyl-L-aspartate) (PBLA)-grafted polysaccharides. PBLA-grafted polysaccharides can be easily aminolyzed with versatile cationic species, such as primary-amine-terminated PDMAEMA, EA, and ED, to produce different types of degradable gene vectors.25,26 PBLA chains of CD-PBLA were aminolyzed with excess ED to produce cationic CD-PAsp-ED (Figure 7a). Similarly, comb-shaped Dex-PAsp-ED and CSPAsp-ED with dextran and chitosan backbones, respectively, were produced from the corresponding polysaccharide-PBLAs. Because of their degradability in physiological environments, three such polysaccharide-PAsp-ED vectors exhibited significantly low cytotoxicity (Figure 7b).25 Besides, as a result of their obviously higher molecular weights of PAsp-ED grafts, the DexPAsp-ED and CS-PAsp-ED vectors mediated higher gene transfection efficiencies than CD-PAsp-ED.25 Diethylenetriamine with a high buffering capacity can also be used in the aminolysis of CD-PBLA, producing CD-PAsp-DET vectors with the capability to facilitate endosome escape.27 Amphiphilic block copolymers have been reported to be ideal materials for drug delivery. Block copolymers of polysaccharide

Figure 8. Synthetic pathway of Dex-SS-PCL micelles and their reduction-responsive intracellular release of DOX. Adapted from ref 32. Copyright 2010 American Chemical Society. 287


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Figure 9. (a) Illustration of the preparation processes of supramolecular pseudocomb polycation/pDNA complexes. (b) Reducible responsiveness of Dex-SS-Ad/CD-SS-PDM/pDNA complexes and their resultant gene expression. Reproduced with permission from ref 39. Copyright 2014 The Royal Society of Chemistry.

biomaterials with optimized structures.33 Recently, supramolecular assemblies involving polymer-grafted polysaccharides have been increasingly constructed and explored for biomedical applications, such as drug/gene delivery and bioimaging.

constructed for drug delivery.36 PVP−PCL assembled into a novel kind of micelle, showed low protein adsorption and good stability, and achieved a satisfactory drug loading content and encapsulation efficacy. Recently, our group developed a series of pseudocomb polycations as novel gene vectors by utilizing the dynamic tunability of supramolecular polymers.27,37−39 A pseudocomb PGEA (l-PGEA-Ad/CD-PGEA) was readily prepared by assembling multiple star-shaped PGEA units with the Admodified linear PGEA backbone. l-PGEA-Ad/CD-PGEA provided markedly enhanced transfection efficiency. Furthermore, the transfection performance could be easily adjusted by the number of assembled star-shaped PGEA units.37 To introduce more functionalities, a novel strategy was proposed to prepare a PGMA-based supramolecular gene vector (PGEDGd@PGEA) with magnetic resonance imaging (MRI) function.38 An ethylenediamine-aminated PGMA (PGED) backbone was prefunctionalized with rich flanking β-CD species and gadolinium ion (Gd3+) and assembled with multiple Ad-headed star-shaped PGEA units to produce PGED-Gd@PGEA. The chelated Gd3+ imparted the PGED-Gd@PGEA vector with good MRI ability without obvious adverse effects on transfection performance. Stimuli-responsiveness can also be introduced to pseudocomb vectors for smart delivery. One kind of responsive supramolecular pseudocomb polycation (Dex-SS-Ad/CD-SSPDM) was constructed by assembling multiple reducible β-CDcored star PDMAEMA (CD-SS-PDM) vectors with disulfidelinked Ad-modified dextran (Figure 9).39 The disulfide linkages imparted Dex-SS-Ad/CD-SS-PDM with reducibility, resulting in better gene delivery performance than for Dex-Ad/CD-PDM without disulfide linkages.

4.1. Host−Guest Assemblies

Host−guest assembly has attracted tremendous interest in the construction of supramolecular structures because of the facile assembly process. The well-known host−guest assembly of βCD and adamantane (Ad) has been increasingly utilized to construct polysaccharide-based assemblies because of the biocompatibility and versatile functionalization of β-CD. A series of supramolecular pseudoblock copolymers, comprising βCD-cored star-shaped polymers and Ad-ended linear copolymers, have been constructed for gene and drug delivery. A reducible pseudoblock polycationic gene vector (β-CD-SSPDM/Ad-PPEG) was realized by assembling the β-CD-cored polycation with four disulfide-linked PDMAEMA arms (β-CDSS-PDM) and Ad-ended PPEGEEMA.34 β-CD-SS-PDM/AdPPEG integrated the advantages of the disulfide-based reducible property, the good DNA-condensing ability of the star-shaped polycation, and the shielding effect of biocompatible PPEGEEMA. β-CD-SS-PDM/Ad-PPEG effectively delivered P53 gene to inhibit tumor growth in mice. Because of the favorable properties of phosphorylcholine in extracellular stabilization and cell uptake, Ad-terminated zwitterionic phosphorylcholine was also assembled with β-CD-SS-PDM for pseudoblock gene vectors.35 This supramolecular gene carrier exhibited protein stability, serum tolerance, and intracellular DNA release properties. A pseudoblock copolymer of β-CD-cored poly(N-vinylpyrrolidone) (PVP) and Ad-terminated PCL (PVP−PCL) was 288


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initiate the ATRP of PEG methyl ether methacrylate to form a triblock copolymer.44 The amphiphilic copolymer was able to self-assemble into nanoparticles for high drug loading and sustained release. Cationic CD-based supramolecular PRs were developed as a new type of gene delivery vector. Multiple polyamidoaminegrafted α-CDs were threaded onto a PEG chain capped with Ad for a dilution-stable vector.45 Such a vector could condense DNA into very stable complexes that kept their initial sizes under ultrahigh dilution. Two novel types of PR-based polycations (SSPR and SS-PR-PDM) were proposed by our group for gene delivery (Figure 11).46 Multiple α-CDs were threaded on a prefunctionalized PEG backbone with two reducible bromoisobutylryl terminals (Br-SS-PEG-SS-Br) to initiate the in situ ATRP of DMAEMA, producing SS-PR with disulfide-linked PDMAEMA blocks. By the use of bromoisobutylryl-functionalized SS-PR as the macroinitiator, SS-PR-PDM was produced by consecutive ATRP of DMAEMA. The disulfide linkages in the PR backbone permitted SS-PR and SS-PR-PDM to be easily disassembled upon exposure to intracellular reductive stimuli. Compared with SS-PR, SS-PR-PDM with a higher cationic density exhibited improved DNA-condensing ability and cell uptake. In addition, the favorable property of the pseudocomb SS-PR-PDM benefited entry of pDNA into the nucleus for better transfection performance.

Other host−guest supramolecular assemblies with different topological architectures were also explored for gene/drug delivery.40−42 A supramolecular hyperbranched polycation (SACP) was skillfully proposed by the assembly of AB2 macromonomer (ACP) with one Ad group and two β-CDPGEA arms (Figure 10).40 S-ACP possessed better DNA-

4.3. Other Assemblies

Other kinds of polymer-grafted polysaccharide-based assemblies were also developed for biomedical applications. As a result of the electrostatic interactions of PDMAEMA grafts with negatively charged heparin, PDMAEMA-grafted heparin copolymers selfassembled into nanoparticles. Such positively charged nanoparticles could serve as effective gene vectors.11 On the basis of Pu-PGEA, composed of one pullulan backbone and PGEA side chains, two types of Pu-PGEA-based nanoparticles were prepared by assembling Pu-PGEA with aminophenylboronic acid-modified Gd-DTPA (GdL) or GdW10O369− (GdW) via the corresponding etherification or electrostatic interaction (Figure 12).47 Such pullulan-containing cationic nanoparticles showed high gene transfection performance in hepatocellular liver carcinoma cells as well as in vitro and in vivo MRI abilities. Cationic supramolecular nanoparticles with MRI function were also produced by the assembly of β-CD-cored star-shaped PGEA, Ad-modified bovine serum albumin (BSA-Ad), and Gd3+ through the synergistic actions of host−guest and electrostatic interactions.48 Such PGEA@BSA-Ad/Gd3+ nanoparticles demonstrated low cytotoxicity, high gene transfection, and efficient cell uptake. Moreover, their MRI performance was much better than that of commercial Magnevist.

Figure 10. Schematic diagram illustrating the preparation of PGMAbased supramolecular hyperbranched polycations and their resultant pDNA delivery process. Adapted with permission from ref 40. Copyright 2016 The Royal Society of Chemistry.

condensing ability and much higher transfection efficiency than its disassembled counterpart (D-ACP). Ad-functionalized lowmolecular-weight branched polyethylenimine (PEI-Ad4) and poly(β-CD) were assembled into cationic supramolecular complexes, which could be further PEGylated with Ad-PEG for increased salt and serum stability.41 For drug delivery, a new supramolecular hydrogel was obtained by assembling Adsubstituted hyperbranched polyglycerol with β-CD-modified dextran.42 The resultant drug release behavior could be wellcontrolled by modifying the compositions.

5. CONCLUSIONS AND PERSPECTIVES This Account has reviewed recent advances in functionalized polysaccharides via polymer grafts. The synthetic methodologies for polymer-grafted polysaccharides, including ATRP, ROP, and assembly, have been summarized. The corresponding designs of polymer-grafted polysaccharides as well as their biomedical applications have also been highlighted. The functionalization via versatile polymer grafts can effectively improve the properties of polysaccharides to extend their biomedical applications. To date, polymer grafting techniques have been well-developed and characterized. However, there are still several challenges. First, the interactions of polymer grafts and polysaccharide backbones need to be carefully evaluated in future studies. Second, the

4.2. Polyrotaxane-Based Assemblies

Polyrotaxane (PR) is an interlocked architecture in which several cyclic molecules are threaded onto a linear polymer chain (axis) that is end-capped by bulky groups (stoppers). CD-based supramolecular PRs have recently been explored as novel drug/ gene vectors. The anticancer drug paclitaxel was conjugated to a β-CD-based PR through a hydrolytically unstable ester linkage.43 The PR−paclitaxel conjugate retained the pharmacological activity of paclitaxel and showed high permeability and retention. A polypseudorotaxane consisting of a distal 2-bromopropionylterminated Pluronic F127 axis and multiple β-CDs was used to 289


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Figure 11. (a) Illustration of the preparation of SS-PR-PDM/pDNA complexes. (b) Enhancement of gene transfection ability by grafting polycation chains from threaded α-CDs. (c) Intracellular process of released pDNA entering the nucleus. Reproduced with permission from ref 46. Copyright 2015 Elsevier Ltd.

Figure 12. (a) Synthesis pathways of different pullulan-based cationic nanoparticles with gadolinium ions. (b) Transfection performance in HepG2 cells and (c) T1-weighted MR images of HepG2 and Hela cells treated with Pu-PGEA-based complexes. Reproduced from ref 47. Copyright 2016 American Chemical Society.

bioactivity of polysaccharides mostly relies on their unsubstituted regions. Thus, after modifications, the inherent functions of polysaccharides, such as targeting and immunogenicity, need to be critically investigated. Third, in the design of new polymer-

grafted polysaccharides, attention should be paid to the varied stability of glycosidic linkages in different polysaccharides. The advent of novel facile chemistries may produce huge potentials of polymer-grafted polysaccharides. Furthermore, the 290


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development of novel polymer grafts with therapeutic effects, such as polyphenols, can be directly conjugated onto polysaccharides to enhance the bioavailability for wider biomedical applications.49 As we expand the applications of functionalized polysaccharides, closer collaborations from a range of disciplines are urgently needed. The examples illustrated in this Account will inspire progressive research activities and bring many more scientists into this exciting field.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Fu-Jian Xu: 0000-0002-1838-8811 Author Contributions ∥

Y.H. and Y.L. contributed equally to this work.

Notes

The authors declare no competing financial interest. Biographies Yang Hu received his Ph.D. from Beijing University of Chemical Technology (BUCT) in 2016 under the supervision of Prof. Fu-Jian Xu. He then joined BUCT as a member of the research staff. His research interests focus on gene/drug delivery vectors. Yang Li received his Ph.D. from The University of New South Wales in 2014 under the supervision of Prof. Thomas P. Davis and Prof. Cyrille Boyer, after which he worked as a research fellow at Monash University. In 2016 he joined in BUCT as a lecturer. His research interests focus on multifunctional drug carriers. Fu-Jian Xu obtained his Ph.D. in 2006 from the National University of Singapore (NUS). He joined BUCT as a professor in 2009. His research interests focus on functional biomacromolecules. He has published over 140 peer-reviewed journal papers. He was the recipient of the National Science Foundation for Distinguished Young Scholars (NSFC, 2013) and Cheung Kong Distinguished Professor (Ministry of Education of China, 2014) awards.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 51325304 and 51521062). REFERENCES

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Versatile Functionalization of Polysaccharides via Polymer Grafts

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