Nonviral Gene Delivery with Cationic Glycopolymers - American

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

pubs.acs.org/accounts

Nonviral Gene Delivery with Cationic Glycopolymers Craig Van Bruggen, Joseph K. Hexum,‡ Zhe Tan,‡ Rishad J. Dalal,‡ and Theresa M. Reineke*

Downloaded via 5.62.157.190 on April 17, 2019 at 19:58:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States

CONSPECTUS: The field of gene therapy, which aims to treat patients by modulating gene expression, has come to fruition and has landed several landmark FDA approvals. Most gene therapies currently rely on viral vectors to deliver nucleic acid cargo into cells, but there is significant interest in moving toward chemical-based methods, such as polymer-based vectors, due to their low cost, immunocompatibility, and tunability. The full potential of polymer-based delivery systems has yet to be realized, however, because most polymeric transfection reagents are either too inefficient or too toxic for use in the clinic. In this Account, we describe developments in carbohydrate-based cationic polymers, termed glycopolymers, for enhanced nonviral gene delivery. As ubiquitous components of biological systems, carbohydrates are a rich class of compounds that can be harnessed to improve the biocompatibility of non-native polymers, such as linear polyamines used for promoting transfection. Reineke et al. developed a new class of carbohydrate-based polymers called poly(glycoamidoamine)s (PGAAs) by step-growth polymerization of linear monosaccharides with linear ethyleneamines. These glycopolymers were shown to be both efficient and biocompatible transfection reagents. Systematic modifications of the structural components of the PGAA system revealed structure−activity relationships important to its function, including its ability to degrade in situ. Expanding upon the development of step-growth glycopolymers, monosaccharides, such as glucose, were functionalized as vinyl-based monomers for the formation of diblock copolymers via radical addition−fragmentation chain-transfer (RAFT) polymerization. Upon complexation with plasmid DNA, the glucose-containing block creates a hydrophilic shell that promotes colloidal stability as effectively as PEG functionalization. An N-acetyl-D-galactosamine variant of this diblock polymer yields colloidally stable particles that show increased receptor-mediated uptake by liver hepatocytes in vitro and promotes liver targeting in mice. Finally, the disaccharide trehalose was incorporated into polycationic structures using both step-growth and RAFT techniques. It was shown that these trehalose-based copolymers imparted increased colloidal stability and yielded plasmid and siRNA polyplexes that resist aggregation upon lyophilization and reconstitution in water. The aforementioned series of glycopolymers use carbohydrates to promote effective and safe delivery of nucleic acid cargo into a variety of human cells types by promoting vehicle degradation, tissue-targeting, colloidal stabilization, and stability toward lyophilization to extend shelf life. Work is currently underway to translate the use of glycopolymers for safe and efficient delivery of nucleic acid cargo for gene therapy and gene editing applications.

■

INTRODUCTION

macromolecules via engineered viruses due to their high delivery efficiency.2 There is a strong desire, however, for nonviral alternatives due to concerns with the high manufacturing cost, limited production scalability, small cargo capacity, and safety risks associated with using viruses in humans.3 Chemical-based nonviral methods for nucleic acid delivery include the use of lipids, polymers, and cellpenetrating peptides to bind and shuttle nucleic acids into

Recent FDA approvals for gene therapies that treat acute lymphoblastic leukemia, large B-cell lymphoma, and RPE65 mutation-associated retinal dystrophy have been significant breakthroughs in the field of gene therapy. By the end of 2017, almost 2600 gene therapy clinical trials were ongoing, completed, or approved worldwide.1 A significant stumbling block for this rapidly advancing field, however, is the difficulty in delivering nucleic acid payloads, including plasmid DNA, mRNA, and siRNA, into the cells of interest to modulate gene expression. Most current therapies rely on transporting these © XXXX American Chemical Society

Received: December 27, 2018

A

DOI: 10.1021/acs.accounts.8b00665 Acc. Chem. Res. XXXX, XXX, XXX−XXX

Article

Accounts of Chemical Research

Figure 1. Graphical illustration of glycopolymer architectures that can vary on the level of (A) repeat unit, (B) copolymer composition types (blue = carbohydrate and red = cationic monomer), and (C) higher-order assembly of polymer chains. (D) Architectural characteristics, such as the composition of the copolymer, can yield interpolyelectrolyte complexes (polyplexes) with varying morphologies and transfection properties.

Table 1. Key Glycopolymer Abbreviations and Compositions class

identifier

carbohydrate/neutral comonomer

PGAAs

D1−4

D-glucarate

PAAs

G1−4 M1−4 T1−4 O4

meso-galactarate D-mannarate L-tartarate oxalate

S4 A4 MAG

succinate adipate methylacrylamidoglucopyranose

MAGalNAc

methacrylamido N-acetyl-Dgalactosamine

Tr1−4

diazide-functionalized trehalose

MAT

methacrylamido trehalose

monosaccharide glycopolymers

trehalose glycopolymers

polymer type

refs

linear ethyleneamines (1−4 secondary amines)

cationic comonomer

step-growth (polycondensation)

11−19, 25, 26, 28−30

pentaethylenehexamine

step-growth (polycondensation)

15, 20, 21

N-(2-aminoethyl) methacrylamide) (AEMA) (N,N-dimethylamino)ethyl] methacrylamide (DMAEMA) (N,N-dimethylamino)propyl] methacrylamide (DMAPMA) N-(2-aminoethyl) methacrylamide) (AEMA) 3-guanidinopropyl methacrylamide (GPMA) linear dialkynyl-ethyleneamines (1−4 secondary amines) N-(2-aminoethyl) methacrylamide) (AEMA)

diblock (via RAFT)

35−41, 58

diblock (via RAFT)

33, 34, 42

step-growth (click)

28, 45−53

diblock (via RAFT)

55−57

target cells in a process known as transfection.4 These platforms boast advantages in their low cost of production, synthetic tunability, large cargo capacities, ability to target specific cell types, extended shelf life, and immunocompatibility.5 Despite these attractive features, most of these chemical systems are less efficient than viral vectors and can be cytotoxic.6 Therefore, significant effort has been put forth in the field to develop a nonviral delivery system that transfects with high efficiency while exhibiting minimal toxicity toward cells.

To achieve the aforementioned goal, many groups have looked toward polymer-based systems since they provide extensive synthetic and structural flexibility to overcome a range of extra- and intracellular barriers to transfection.7 These polymer systems typically contain amine groups with pKa values above or near physiological pH, which give the polymer a positive charge and the ability to electrostatically bind polyanions, such as DNA, to form complexes termed polyplexes.8 Polymer vectors can accommodate the integration of degradable components, targeting moieties, and stimuliresponsive functional groups to help overcome these barriers.9 B


Article


Figure 2. Chemical structures of (A) linear PEI, chitosan, and (B) PGAA analogues. (C) Each PGAA analogue was synthesized with oligoethylenamines that contained between 1 and 4 secondary protonatable amines. (D) PGAA hydrolysis helps unpackage the DNA cargo from the polyplex. (E) Cell viability of BHK-21 cells after transfection with PGAA D-analogues and PEI at increasing N/P ratios (adapted with permission from ref 11, Copyright 2004 American Chemical Society). (F) The level of luciferase expression in HeLa cells (RLU/mg = relative light units per mg of protein) varies significantly between the PGAA types and their PAA analogues (adapted with permission from ref 15, Copyright 2010 American Chemical Society).

well-defined architectures that allow for systematic modification of the polymer structure and the determination of structure−activity relationships. These structure−activity relationships provide guidance for the development of increasingly efficient polymer-based gene delivery vehicles that can be utilized for nonviral gene therapies and gene editing technologies such as CRISPR/Cas9.

The efficacy of the polymer can also be affected by placement of these components within several levels of its overall architecture (Figure 1a−c).7 In this Account, we report the efforts by Reineke et al. to advance the field of gene therapy through the development of novel carbohydrate-containing polymeric vectors, termed glycopolymers (see Table 1), which exhibit enhanced biocompatibility, tissue-specific targeting, and colloidal stability. Synthesis of these glycopolymers emphasizes C


Article


Figure 3. (A) Endocytosis of polyplexes and their trafficking pathways to the nucleus.27 (B) A three-dimensional confocal microscopy rendering of a HeLa cell nucleus (pseudocolored in yellow) with polyplexes (pseudocolored in blue), including a (i) zoomed out view, (ii) zoomed in view, and (iii) top view (adapted with permission from ref 28, Copyright 2013 American Chemical Society). (C) A confocal fluorescence micrograph showing colocalization of ER lumen protein BiP (red) in H9c2(2-1) cells and G4 polyplexes carrying FITC-ODN (green) as compared to PEI polyplexes. The Manders coefficient for green pixels overlapping with red was found to be 0.889 for Glycofect-transfected cells and 0.457 for PEItransfected cells (scale bar = 20 μm).27 (D) A zoomed in image of C showing colocalization of the ODN and ER antibody for G4 (top) and PEI (bottom) (scale bar = 2 μm).27 Panels A, C, and D adapted with permission from ref 27, Copyright 2013 American Chemical Society.

■

POLY(GLYCOAMIDOAMINE)S (PGAA)S: NONTOXIC AND DEGRADABLE CARBOHYDATE-BASED POLYMERS Polyethylenimine (PEI) (Figure 2a) is a commercially available polymeric transfection reagent that yields high transfection rates but is often severely cytotoxic. 10 The cationic polysaccharide chitosan (Figure 2a) is a nontoxic transfection reagent but is generally too inefficient to be practically useful.11 In order to obtain high transfection efficiencies comparable to PEI with a low-toxicity profile similar to chitosan, Liu et al. developed a polymer platform that melded structural features from both systems.11 Linear monosaccharides were copolymerized with linear ethyleneamines in an alternating fashion, thereby creating a PEI analogue with decreased charge density and increased hydrophilic character. In a series of studies, linear ethyleneamines containing between 1 and 4 secondary amines were copolymerized with esterified monosaccharides Dglucarate (D), meso-galactarate (G), D-mannarate (M), and Ltartarate (T) via a step-growth mechanism (Figure 2b). This facile synthesis yielded short polymers, called poly(glycoamidoamines) or PGAAs, with similar degrees of polymerization (n between 11 and 14).11−13 Dynamic light scattering (DLS), transmission electron microscopy (TEM),

and zeta potential measurements showed that the polymers compacted DNA to form positively charged polyplexes. Upon mixing in water at an N/P ratio of 30, where N is the concentration of protonatable polymer-based amines and P is the concentration of anionic phosphodiester groups in the DNA, the resulting polyplexes ranged between 54 and 625 nm in diameter and exhibited zeta potentials between ∼10 and 30 mV.11−13 These positively charged particles interact with negatively charged anionic proteins (proteoglycans) on the cell surface, which promotes endocytosis.14 PGAA polyplexes were readily internalized by a variety of cell types, including BHK-21 (baby hamster kidney), H9c2(2-1) (rat cardiomyoblast), HeLa (human cervical adenocarcinoma), and HepG2 (human hepatocellular carcinoma) cells, leading to the transgene expression of a luciferase reporter plasmid.11−13,15−18 For each of the PGAAs (D, G, M, and T), variants that contained 4 secondary amines per repeat unit (Figure 2c) gave the maximum luciferase expression per cell.11−13,15−17 Remarkably, the transfection efficiency of the PGAAs was comparable to PEI while the cytotoxicity of these polymers was far lower than PEI and comparable to chitosan (Figure 2e,f).11−13,16,17 Generally, among this series of PGAAs, T4 and G4 struck D


Article


commercialized and marketed as Glycofect Transfection Reagent. Another high-performing analogue, T4, was successfully utilized for subsequent in vivo therapy studies where it was used to deliver oligodeoxynucleotide (ODN) decoys to mouse heart tissue by injection into the pericardial sac.25 The T4mediated delivery of these ODN decoys blocked activation of the transcription factor NF-κB, which provided a significant reduction in myocardial infarction after ischemia−reperfusion injury. This work illustrates the potential of PGAAs for in vivo therapies.

the best balance of achieving high transfection efficiencies along with maintaining high cell viability. In addition to its status as a new class of efficient and nontoxic transfection agents, the PGAA platform allowed for careful tuning of its chemical structure to explore structure− activity relationships. Alterations to the carbohydrate type and amine number in the PGAA repeat unit were shown to impact the buffering capacity of the polymer.17 The “proton sponge” theory suggests that polymers that can better buffer against decreasing endosomal pH yield higher transfection efficiencies by increasing the osmotic swelling of the endosome, leading to vesicle rupture and release of the nucleic acid cargo into the cytoplasm.5 In congruence with the “proton sponge” theory, larger buffering capacities correlated positively with higher transfection efficiencies when comparing PGAA analogues with different carbohydrate monomers (e.g., D4 vs T4). When examining variants with different amine numbers (e.g., T3 vs T4), however, no correlation between buffering capacity and transfection efficiency was observed.17 As shown by this result, no single factor is fully predictive in determining the performance of these glycopolymers. Differences in the performance of PGAA analogues, including the exceptional performance of the T and G analogues, could be attributed to other properties such as their strength of binding with DNA.16 The concentration and stereochemistry of hydroxyl groups on the polymers influences the strength of binding to the DNA19 and the ability of the polymer to bind proteoglycans on the cell surface and promote particle uptake.14 It was later shown that another factor contributing to the effectiveness of the PGAAs was their ability to degrade at physiological pH to benign monomeric components, which may enhance gene expression by improving unpackaging and release of the nucleic acid cargo (Figure 2d).15 The hydrolysis rate of the PGAAs was shown to be minimal in the acidic environment of lysosomes (pH ≈ 5.0), with an approximate half-life of 40 h, prompting intracellular degradation of the polymer only after release into the cytoplasm.17 Degradation at physiological pH (7.4) occurs more rapidly, with an approximate half-life of 10 h.17 Therefore, premature release of the nucleic acid cargo with in vivo applications is a concern, so minimizing the transit time to target cells is a priority, which can be accomplished via direct injection of formulations to the tissue of interest. To examine the role of hydroxyl groups in polymer degradation, the linear monosaccharides in the PGAA analogues were replaced by adipate (A4), succinate (S4), and oxylate (O4) to yield PGAA variants called poly(amidoamine)s (PAAs) that did not contain hydroxyl groups. PAA variants A4 and S4 did not degrade appreciably and were far less efficient than the degradable polymers in promoting transfection (Figure 2F).15,20 Other modifications to the PGAA structure, such as repeat units with 5 or 6 secondary amines, the introduction of branching variants (Figure 1c),18 or increasing the number of methylene units between amines failed to improve transfection efficiencies without increasing toxicity.17 Additional studies aimed at improving the polymer system focused on tuning the lipophilicity of the structure,21 using ring-closed monosaccharides,22 using guanidium-based charge centers,23 and enhancing polyplex/cell contact through improved transfection methodology.24 Through the systematic modification of the PGAA platform, the structure−activity relationship of each polymer component was determined and used to identify the optimal polymer system. The PGAA variant G4 was eventually

■

INTRACELLULAR TRAFFICKING OF PGAAs As a model system, Glycofect (G4) and its PGAA analogues were used in a series of studies to better understand the cellular mechanisms that lead to successful transfections with polymerbased systems. Polyplexes can enter into the cell via energyindependent pathways, such as through direct cell penetration, or by energy-dependent endocytic processes such as clathrinmediated and caveolar endocytosis (Figure 3a).26 Using pharmacological endocytosis inhibitors and confocal microscopy analysis, McLendon et al. determined that PGAA polyplexes entered HeLa cells primarily through actin- and dynamin-dependent pathways such as a clathrin- and caveolaemediated endocytosis.26 This work concluded that caveolae/ raft-mediated endocytosis was the primary pathway leading to nuclear delivery and efficient expression for the cargo transported by both PEI and PGAA polymers.26 In addition, Fichter et al. determined that Glycofect tends to bypass acidic endocytic vesicles and is trafficked through the Golgi and endoplasmic reticulum (ER) to a larger extent than PEI (Figure 3c,d).27 Cellular imaging via 4D spatiotemporal confocal fluorescence microscopy of Glycofect polyplexes by Ingle et al. confirmed colocalization with caveolae vesicles, minimal colocalization with late endosomes, and rapid trafficking to the nucleus.28 Representative spatiotemporal confocal imaging is shown in Figure 3b. This cellular processing of Glycofect polyplexes resembles the trafficking of viral vectors to the nucleus.27 In addition, PGAA analogues with the best transfection efficiencies, such as Glycofect, show the highest aptitude for inducing nuclear envelope permeability and, thereby, assisting the entry of plasmids into the nucleus.29,30 Taken together, these studies elucidate the stepby-step process through which glycopolymers overcome the multitude of cellular barriers leading to successful transfection.

■

MAG AND GalNAc BLOCK COPOLYMERS: ENHANCED COLLOIDAL STABILITY AND TISSUE TARGETING Although the PGAA system yielded efficient and nontoxic delivery of nucleic acid acids both in vitro and in vivo, systemic injection into an organism ultimately requires colloidally stable complexes less than 100 nm in size that resist aggregation in the presence of salt and serum while maintaining efficacy.31 Many polycations, such as PEI, form large aggregates that are quickly cleared from the body by the reticuloendothelial system and can also be caught in lung capillaries.3 A common strategy to increase the colloidal stability of polyplexes is to prepare diblock copolymer architectures composed of both a cationic block, such as N-(2-aminoethyl) methacrylamide (AEMA), and a hydrophilic block, such as poly(ethylene glycol) (PEG).5 Such diblock systems form core−shell structures while complexing with nucleic acid macromolecules. E


Article


Figure 4. (A) Structure of MAG-based diblock glycopolymers with various amine-containing comonomers. (B) A TEM micrograph shows polyplexes formed with P(MAG46-b-AEMA21) and plasmid (N/P = 5) in water (adapted with permission from ref 35, Copyright 2011 American Chemical Society). (C) DLS data of MAG-diblock polyplexes in reduced serum media (Opti-MEM) shows that the copolymers resist aggregation compared to cationic homopolymers consisting of either AEMA (1) or DMAPMA (2).40 (D) A luciferase plasmid transfection in HepG2 cells shows comparable transfection efficiencies to cationic homopolymers.40 Asterisks denote measurements found to be statistically significant (p < 0.05) compared to cells only. Panels C and D are adapted with permission from ref 40, Copyright 2014 American Chemical Society.

diameters around 100 nm in both salt and serum over the time frame of the transfection (Figure 4b). With zeta potentials ranging between 15 and 40 mV in water, these polyplexes were readily internalized by HeLa cells.35 Similar to PEG, the MAG block provided a hydrated shell that effectively screened polyplexes from aggregation.36,37 Plasmid delivery yielded promising transgene expression in HeLa cells while siRNA delivery yielded significant luciferase gene knock-down in U-87 MG cells (glioblastoma). Interestingly, the shortest cationic blocks (n = 21) performed the best for plasmid delivery while the longest cationic (n = 48) blocks were more effective for siRNA delivery.35 This trend may be explained by the length dependence on the strength of polymer binding as shown by Jung et al.38 Different nucleic acid types and lengths require polymers with different binding strengths to effectively protect the cargo while still allowing for unpackaging.35 Further optimization studies of the MAG−AEMA copolymer system evaluated the effect of amine substitution, block length, composition, and architecture (Figure 4a) on gene delivery efficiency.39−41 Most of the polyplexes formed with MAG-

The cationic block compacts DNA in the core while the hydrophilic block forms a shell that provides steric repulsion to inhibit aggregation and resist clearance by the immune system. Unfortunately, the hydrophilic shell can hinder the interaction and binding of the core−shell polyplex to the cell membrane, leading to reduced cell entry and inefficient gene delivery.32 A carbohydrate block, however, can ameliorate this issue by serving as the hydrated shell for enhanced colloidal stability while allowing the polyplex to interact with native carbohydrate receptors on cell surfaces to promote tissue- and organspecific delivery.33,34 To create a carbohydrate-based block polymer with a welldefined molar mass and composition, our group utilized reversible addition−fragmentation chain transfer (RAFT) polymerization with vinyl-based monomers. Smith et al. used RAFT to copolymerize a glucose-containing monomer, 2deoxy-2-methacrylamido glucopyranose (MAG), with AEMA to form poly(MAG-b-AEMA) block copolymers with various AEMA chain lengths (Figure 4a).35 Importantly, polyplexes formed with these block copolymers maintained hydrodynamic F


Article


Figure 5. (A) Structure of MAGalNAc-based diblock glycopolymers with amine-containing comonomers. (B) Plasmid-based polyplexes formed with MAGalNAc diblock copolymers promote the transfection of mouse liver tissue via ASGPR-mediated endocytosis.34 (C) Twenty-four hours following systemic delivery via tail-vein injection, the biodistribution of pDNA delivered by MAGalNAc vehicles in mice was measured by qPCR and show preferential delivery to the liver.34 Panels B and C are adapted with permission from ref 34, Copyright 2016 American Chemical Society. (D) Incorporation of MAGalNAc in a GPMA polymer reduces GPMA-mediated membrane penetration by polyplexes containing Cy5-labeled plasmid (N/P = 5).33 The # denotes statistical difference between mean of two samples (p < 0.01) (adapted with permission from ref 33, Copyright 2017 American Chemical Society).

on their cell surface that selectively bind galactose and Nacetyl-D-galactosamine (GalNAc) terminal units on asialoglycoproteins. To target these receptors and promote hepatocyte uptake, Lee et al. conjugated azide-modified β-D-galactose units to a PGAA backbone through a copper(I)-catalyzed azide/ alkyne “click” reaction and studied the effect of galactose substitution density on ASGPR-mediated uptake in the hepatocyte cell line HepG2.42 Although this polymer system showed preferential hepatocyte delivery, maximum ligand density was difficult to achieve due to the constraints of postpolymerization modification. Another study by Dhande et al. overcame this limitation by synthesizing a methacrylamido GalNAc analog (termed MAGalNAc, Figure 5a).34 A homopolymer of MAGalNAc (n = 62) had an exceptionally strong binding affinity with ASGPR receptors (IC50 = 0.02 ± 0.005 μM), roughly 2 orders of magnitude higher than asialofetuin and five orders higher than monomeric GalNAc. MAGalNAc and AEMA (7) were copolymerized to form a series of diblock copolymers that functioned as liver-specific gene delivery vehicles (Figure 5b). Plasmid polyplexes formed with diblock MAGalNAc−AEMA copolymers were more colloidally stable than PEG-based analogues in cell culture media and showed enhanced luciferase expression in ASGPR-

based block copolymers showed far less aggregation in reduced serum media compared to their cationic homopolymer and statistical copolymer counterparts39−41 while maintaining similar levels of transfection efficiency (Figure 4c,d).40 Among the series of amines with varying levels of substitution (4−6), the secondary amine (5) gave the best balance of high transfection rates with minimal toxicity.41 A MAG diblock copolymer with the tertiary amine (3) gave high transfection efficiencies but also exhibited high toxicity levels likely due to the enhanced membrane penetration/disruption caused by the hydrophobicity of the tertiary amine. It was found that copolymerizing a primary amine (1) block linked to a short tertiary amine block (3) to form a triblock copolymer yielded an optimal balance of high transfection efficiency and low toxicity.39 These studies demonstrate how the architecture and composition of chain-growth glycopolymers can be tuned to optimize transfection properties. Tissue-specific targeting is a primary goal of the gene therapy field; to achieve this, the pendant carbohydrate was modified to enable liver targeting. The liver is an important target for gene therapy due to its ability to produce and secrete therapeutic serum proteins into the circulatory system. Hepatocytes express asialoglycoprotein receptors (ASGPRs) G


Article


Figure 6. Structure of (A) trehalose click copolymers and (B) MAT-AEMA diblock copolymers. (C) MAT-diblock copolymers form core−shell structures with siRNA (adapted with permission from ref 55, Copyright 2013 American Chemical Society). (D) After lyophilization and resuspension in water, MAT-diblock polyplexes retained colloidal stability as shown by TEM (scale bar = 100 nm).56 (E) Luminescence images of a hydrodynamically injected mouse with p(MAT-b-AEMA) and luciferase plasmid (left) show significant increase in luminescence as compared to control injected with 5% dextrose in water (right).56 Panels D and E are adapted with permission from ref 56, Copyright 2016 American Chemical Society.

expressing HepG2 cells.34 Systemic injection of MAGalNAc polyplexes in mice led to selective uptake of the particles by the liver. One day postinjection, qPCR revealed 70-fold higher accumulation in the liver than in the lungs (Figure 5c). The MAG-based analogue yielded no accumulation in either organ, while in vivo-jetPEI showed equal accumulation in both the liver and the lungs (Figure 5c).34 These results, in conjunction with imaging after delivery of fluorescently labeled plasmid, suggest that the MAGalNAc polyplexes undergo accumulation in the liver via a receptor-mediated process, while PEI passively accumulates in the liver and lungs due its large particle size.34,43 The MAGalNAc monomer was again utilized in the work of Tan et al.33 during a study of guanidinium-based polymers designed for enhanced cell penetration. In comparison to 3-guanidinopropyl methacrylamide (GPMA) homopolymers, p(MAGalNAc-b-GPMA) block copolymers (8) were significantly less toxic and demonstrated increased transfection efficiency in HepG2 cells.33 The conjugation of a MAGalNAc block altered the internalization pathway of polyplexes from guanidinium-promoted direct membrane penetration to ASGPR-promoted receptor-mediated endocy-

tosis (Figure 5d), exemplifying its ability to enhance liverspecific targeting.

■

TREHALOSE: ENHANCING COLLOIDAL STABILITY AND BIOCOMPATIBILITY The previously described chemistry has been further expanded upon to incorporate the disaccharide trehalose in order to improve colloidal stability and cryostability of gene delivery vehicles. Trehalose is an α-glucose dimer linked by a 1,1glycosidic bond and is synthesized by a wide range of organisms, including tardigrades, to survive desiccation and freezing.44 It was hypothesized that trehalose could be incorporated into a polycationic platform and provide enhanced stability to nucleic acid complexes in both colloidal and anhydrous conditions. Both polymerization techniques mentioned previously, including step-growth (e.g., PGAAs) and chain-growth (e.g., MAG and GalNAc), were utilized to develop new trehalose-containing gene delivery vehicles. Trehalose-based polymers were prepared via a step-growth mechanism by Srinivasachari et al. using “click” chemistry to achieve high degrees of polymerization and increase nucleic acid binding (promoted via interaction with the triazole ring). H


Article


encounters limitations in delivering large plasmids (9.5−14 kbp) that may be needed for gene editing applications, such as plasmids encoding for Cas9 or large donor genes, a problem shared among other nonviral delivery vehicles.51 It has been hypothesized that these large plasmids yield poor transfection results due to transport limitations into the cellular nuclei. Studies by Boyle et al. supported the existence of the nuclear barrier for large plasmid delivery and discovered methods to further improve delivery through the use of two separate small molecule additives, dexamethasone and thymidine.51 Dexamethasone enhances nuclear delivery by dilating nuclear pores, while thymidine achieves this goal by synchronizing cells into the S phase of mitosis where the nuclear membrane starts to break down. Dexamethasone was used to improve the delivery of a large plasmid encoding for a Cas9 derivative (dCas9VP64), which acts as a transcription activator. By targeting increased transcription activation of the collagen VII gene (COL7A1) in human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs), Boyle et al. aimed to provide a therapy that improves the strength of skin in patients with epidermolysis bullosa, a genetic disorder that causes skin to be extremely fragile. Plasmids encoding for dCas9-VP64 (13 kbp) and guide RNA (3 kbp) were complexed with Tr4-heparin and transfected in HDFs and iPSCs in vitro in the presence of dexamethasone and achieved an increase in collagen expression of 5- and 20-fold, respectively, compared to an untransfected control and outperformed the commercial reagent Lipofectamine 2000.51 Supplementing Tr4 with the additives heparin and dexamethasone shows great potential for the efficient delivery of Cas9 constructs for gene-based therapies. Along with this step-growth system, the incorporation of trehalose was achieved in block systems using a similar strategy to that of the MAG and MAGalNAc block copolymers. A methacrylamide-based monomer containing trehalose as a pendant group, termed methacrylamido-6-deoxytrehalose (MAT), was also synthesized to form a series of diblock copolymers with AEMA at several cationic chain lengths (Figure 6b).55 Polyplexes formed with siRNA (Figure 6c) and these diblock trehalose copolymers achieved efficient siRNAinduced luciferase knockdown (IC50 = 19 nM) in U-87 MG cells in the presence of serum.55 These polyplexes were both nontoxic to cells and colloidally stable in the presence of high salt and serum. Detailed lyophilization studies showed that the polyplexes could be fully lyophilized to dryness and reconstituted in water without any changes to their size, morphology, or potency, a trait not shared by the PEG-based control (Figure 6d). This ability to be stored as a powder would likely make gene therapies more practical and accessible as off-the-shelf treatments. To test the compatibility of these complexes in vivo, Tolstyka et al. introduced MAT-based polyplexes into mice via slow tail vein injection and showed no observed toxicity at elevated doses (19.6 mg/kg of polymer/ mouse).56 Statistically significant increases in luciferase expression over Glycofect and jetPEI were achieved with hydrodynamic injection of MAT polyplexes to the murine liver (represented by images in Figure 6e). This study illustrated the enhancement in colloidal stability and biocompatibility that trehalose provides in diblock polymer gene delivery systems.

The primary alcohols of trehalose were substituted with azides, which could then be polymerized with dialkynyl-ethyleneamines via a Cu(I)-catalyzed azide−alkyne cycloaddition (Figure 6a).45,46 These “trehalose click polymers” contained between 1 and 4 secondary amines in each repeat unit (abbreviated as Tr1−4). Similar to the PGAAs, plasmid transfection efficiency in vitro increased as the amine number was increased from 1 through 4. Prevette et al. determined that increasing the amine number correlates with decreasing electrostatic interaction with the plasmid but increasing hydrogen bonding between the polymer and the base pairs of the plasmid. This change in interaction may contribute to the observed trend in transfection efficiency between the variants.47 Kizjakina et al. examined trehalose click polymer analogues with 5 and 6 amines per repeat unit, and similarly to the PGAA system, it was found that the increase in amine density did not further enhance transfection efficiency, yet polymer molar mass did play a role in efficacy.48 The Tr4 derivative was prepared with four different degrees of polymerization, ranging from 35 to 100 repeat units, to examine the effect of chain length on polyplex stability and transfection. Upon complexation with a plasmid at N/P = 7, the zeta potential of the polyplex formulation in water ranged between 43 and 47 mV.46 In the presence of serum, Tr4 consisting of at least 53 repeat units maintained a hydrodynamic diameter below 400 nm, a significant decrease in aggregation compared to non-diblock systems such as the PGAAs.46 Tr4 analogues with repeat unit lengths between 99 and 123 showed an exceptional ability to transfect rat mesenchymal stem cells (RMSCs) in the presence of serum in comparison to commercial controls with approximately a 10-fold increase in %GFP+ cells (30−40%).48 A follow-up study by Anderson et al. found that capping the polymer ends with linear ethyleneamines (Figure 6a) yielded a significant improvement in transfection of HeLa cells in the presence of serum.49 In addition to plasmids, the trehalose click polymer system has been used to successfully deliver siRNA. A study by Xue et al. found that both the trehalose click polymers and analogues formed with β-cyclodextrin were more efficient than PGAAs in achieving siRNA-induced knockdown of luciferase expression (80−90% in Opti-MEM).52 Between the two click polymers tested, however, only the trehalose version was able to achieve both efficient siRNA and plasmid delivery, showing its compatibility with a range of nucleic acid cargo. To examine how trehalose affects nucleic acid packaging, decomplexation, and delivery, Xue et al. included lanthanide-chelate domains into the trehalose click polymer to quantify polyplex packaging of tetramethylrhodamine-labeled siRNA through lanthanide resonance energy transfer (LRET).53,54 This work showed that the incorporation of trehalose can help promote polymerbased siRNA delivery compared to polycations lacking the carbohydrate. Recently, it was found that certain macromolecule and small molecule additives can further improve transfections with Tr4 and can make the technology well suited for the application of CRISPR/Cas9-based therapies. Boyle et al. showed that adding heparin to the Tr4 polyplex to form a ternary complex can drastically improve plasmid-based transfection with this polymer in a variety of cell types for in vitro and ex vivo applications.50 Heparin was shown to both improve cell surface binding and allow for more efficient uptake and intracellular trafficking. This nonviral transfection technology, however,

■

SUMMARY AND OUTLOOK This Account has described collective contributions of Reineke et al. to the field of nonviral gene therapy through the development of several glycopolymer-based nucleic acid I


Article

Accounts of Chemical Research delivery platforms. Our group has incorporated carbohydrate moieties, including galactose, glucose, GalNAc, trehalose, and others, such as β-cyclodextrin, into polycationic structures that have helped polymer-based vehicles overcome several key hurdles in nonviral gene delivery. The architectures and compositions of these glycopolymers have been systematically modified to optimize their ability to efficiently transfect cells with limited cytotoxicity, remain colloidally stable in the presence of serum, resist aggregation during lyophilization, target liver tissue, and deliver a range of nucleic acids safely in vitro and in vivo. With recent advances in the field of gene editing, catalyzed by advanced nuclease systems such as CRISPR/Cas9, the demand for even more efficacious nonviral delivery systems has greatly expanded. Along with adapting the established glycopolymer systems for new gene editing applications, such as CRISPR/Cas9,51 and new cargo types, such as ribonucleoprotein complexes, we are developing new delivery platforms for these applications based upon polymer micelles with highly controlled structures and compositions. In-depth studies on the physics of glycopolymer-based micelles57,58 and micelle−polyelectrolyte complexation59,60 have set the foundation for utilizing these structures for welldefined, safe, and effective protein/nucleic acid delivery for gene editing. It is our hope that the structure−activity relationships and synthetic techniques developed through the establishment of new glycopolymer-based delivery vehicles will help those in the field to advance nonviral gene therapy.

■

Theresa M. Reineke received her B.S. Degree from the University of WisconsinEau Claire, an M.S. Degree from Arizona State University, and a Ph.D. from the University of Michigan. As a National Institutes of Health Postdoctoral Fellow, she performed research at the California Institute of Technology. She is currently a Distinguished McKnight University Professor in the Department of Chemistry at UMN.

■

ACKNOWLEDGMENTS The authors are grateful for the many students, postdoctoral associates, collaborators, and staff whose work supported the research described in this Account. The authors also acknowledge the outstanding and persistent research of the entire nonviral nucleic acid community that was unable to be discussed or referenced herein due to space constraints. The authors acknowledge funding by Limelight Bio, Inc., and the Defense Advanced Research Projects Agency (DARPA; contract no. N660011824041).

■

REFERENCES

(1) Ginn, S. L.; Amaya, A. K.; Alexander, I. E.; Edelstein, M.; Abedi, M. R. Gene Therapy Clinical Trials Worldwide To 2017: An Update. J. Gene Med. 2018, 20, No. e3015. (2) Hong, S. J.; Ahn, M. H.; Sangshetti, J.; Choung, P. H.; Arote, R. B. Sugar-Based Gene Delivery Systems: Current Knowledge and New Perspectives. Carbohydr. Polym. 2018, 181, 1180−1193. (3) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non-Viral Vectors for Gene-Based Therapy. Nat. Rev. Genet. 2014, 15, 541−555. (4) Miller, J. B.; Siegwart, D. J. Design of Synthetic Materials for Intracellular Delivery of RNAs: From siRNA-Mediated Gene Silencing to CRISPR/Cas Gene Editing. Nano Res. 2018, 11, 5310−5337. (5) Mintzer, M. A.; Simanek, E. E. Nonviral Vectors for Gene Delivery. Chem. Rev. 2009, 109, 259−302. (6) Lai, W.; Wong, W. Design of Polymeric Gene Carriers for Effective Intracellular Delivery. Trends Biotechnol. 2018, 36, 713−728. (7) Rinkenauer, A. C.; Schubert, S.; Traeger, A.; Schubert, U. S. The Influence of Polymer Architecture on In Vitro pDNA Transfection. J. Mater. Chem. B 2015, 3, 7477−7493. (8) Wang, H.; Li, M.; Lee, C. M.; Chakraborty, S.; Kim, H.; Bao, G.; Leong, K. W. CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery. Chem. Rev. 2017, 117, 9874−9906. (9) Li, Y.; Gao, J.; Zhang, C.; Cao, Z.; Cheng, D.; Liu, J.; Shuai, X. Stimuli-Responsive Polymeric Nanocarriers for Efficient Gene Delivery. In Polymeric Gene Delivery Systems; Cheng, Y., Eds.; Springer: Cham, 2017; pp 167−215. (10) Grandinetti, G.; Ingle, N. P.; Reineke, T. M. Interaction of Poly(Ethylenimine)-DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity. Mol. Pharmaceutics 2011, 8, 1709−1719. (11) Liu, Y.; Wenning, L.; Lynch, M.; Reineke, T. M. New Poly(DGlucaramidoamine)s Induce DNA Nanoparticle Formation and Efficient Gene Delivery into Mammalian Cells. J. Am. Chem. Soc. 2004, 126, 7422−7423. (12) Liu, Y.; Reineke, T. M. Hydroxyl Stereochemistry and Amine Number within Poly(Glycoamidoamine)s Affect Intracellular DNA Delivery. J. Am. Chem. Soc. 2005, 127, 3004−3015. (13) Liu, Y.; Wenning, L.; Lynch, M.; Reineke, T. M.; Svenson, S. Gene Delivery with Novel Poly(l-Tartaramidoamine)s. ACS Symp. Ser. 2006, 923, 217−227. (14) McLendon, P. M.; Buckwalter, D. J.; Davis, E. M.; Reineke, T. M. Interaction of Poly(Glycoamidoamine) DNA Delivery Vehicles with Cell-Surface Glycosaminoglycans Leads to Polyplex Internalization in a Manner Not Solely Dependent on Charge. Mol. Pharmaceutics 2010, 7, 1757−1768.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (T.M.R.). ORCID

Zhe Tan: 0000-0002-3518-0772 Theresa M. Reineke: 0000-0001-7020-3450 Author Contributions ‡

J.K.H., Z.T., and R.J.D. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies Craig Van Bruggen obtained his B.S. in Biochemistry with honors at the University of Puget Sound in 2012 under advisement by Prof. Johanna Crane and Prof. Eric Scharrer. He is currently a chemistry Ph.D. candidate in Prof. Theresa Reineke’s group at the University of Minnesota (UMN). Joseph K. Hexum obtained his B.S. in Chemistry at Hamline University in 2009 and his M.S. in Environmental Health Sciences at UMN in 2011. Prior to joining the Department of Chemistry at the UMN, he worked as a researcher in a medicinal chemistry lab for several years. Zhe Tan obtained her B.S. in Chemistry from Fudan University in China with Prof. Qiaowei Li in 2014. She is currently a chemistry Ph.D. candidate in Prof. Theresa Reineke’s group at the UMN. Rishad J. Dalal obtained his B.S. in Chemistry with honors from University of California, Irvine, while conducting research under Prof. Kenneth J. Shea. He is currently a chemistry Ph.D. candidate in Prof. Theresa Reineke’s group at the UMN. J


Article

Accounts of Chemical Research (15) Liu, Y.; Reineke, T. M. Degradation of Poly(Glycoamidoamine) DNA Delivery Vehicles: Polyamide Hydrolysis at Physiological Conditions Promotes DNA Release. Biomacromolecules 2010, 11, 316−325. (16) Liu, Y.; Reineke, T. M. Poly(Glycoamidoamine)s for Gene Delivery: Stability of Polyplexes and Efficacy with Cardiomyoblast Cells. Bioconjugate Chem. 2006, 17, 101−108. (17) Liu, Y.; Reineke, T. M. Poly(Glycoamidoamine)s for Gene Delivery. Structural Effects on Cellular Internalization, Buffering Capacity, and Gene Expression. Bioconjugate Chem. 2007, 18, 19−30. (18) Lee, C.; Liu, Y.; Reineke, T. M. General Structure-Activity Relationship for Poly(Glycoamidoamine)s: The Effect of Amine Density on Cytotoxicity and DNA Delivery Efficiency. Bioconjugate Chem. 2008, 19, 428−440. (19) Prevette, L. E.; Kodger, T. E.; Reineke, T. M.; Lynch, M. L. Deciphering the Role of Hydrogen Bonding in Enhancing pDNAPolycation Interactions. Langmuir 2007, 23, 9773−9784. (20) Prevette, L. E.; Lynch, M. L.; Reineke, T. M. Amide Spacing Influences sDNA Binding of Poly(Amidoamine)s. Biomacromolecules 2010, 11, 326−332. (21) Wu, Y.; Smith, A. E.; Reineke, T. M. Lipophilic Polycation Vehicles Display High Plasmid DNA Delivery to Multiple Cell Types. Bioconjugate Chem. 2017, 28, 2035−2040. (22) Lee, C. C.; Liu, Y.; Reineke, T. M. Glucose-Based Poly(Ester Amines): Synthesis, Degradation, and Biological Delivery. ACS Macro Lett. 2012, 1, 1388−1392. (23) Taori, V. P.; Lu, H.; Reineke, T. M. Structure-Activity Examination of Poly(Glycoamidoguanidine)s: Glycopolycations Containing Guanidine Units for Nucleic Acid Delivery. Biomacromolecules 2011, 12, 2055−2063. (24) Agrawal, P.; Ingle, N. P.; Boyle, W. S.; Ward, E.; Tolar, J.; Dorfman, K. D.; Reineke, T. M. Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension. ACS Appl. Mater. Interfaces 2016, 8, 8870−8874. (25) Tranter, M.; Liu, Y.; He, S.; Gulick, J.; Ren, X.; Robbins, J.; Jones, W. K.; Reineke, T. M. In Vivo Delivery of Nucleic Acids via Glycopolymer Vehicles Affords Therapeutic Infarct Size Reduction in Vivo. Mol. Ther. 2012, 20, 601−608. (26) McLendon, P. M.; Fichter, K. M.; Reineke, T. M. Poly(Glycoamidoamine) Vehicles Promote pDNA Uptake through Multiple Routes and Efficient Gene Expression via CaveolaeMediated Endocytosis. Mol. Pharmaceutics 2010, 7, 738−750. (27) Fichter, K. M.; Ingle, N. P.; McLendon, P. M.; Reineke, T. M. Polymeric Nucleic Acid Vehicles Exploit Active Interorganelle Trafficking Mechanisms. ACS Nano 2013, 7, 347−364. (28) Ingle, N. P.; Xue, L.; Reineke, T. M. Spatiotemporal Cellular Imaging of Polymer-pDNA Nanocomplexes Affords in Situ Morphology and Trafficking Trends. Mol. Pharmaceutics 2013, 10, 4120−4135. (29) Grandinetti, G.; Reineke, T. M. Exploring the Mechanism of Plasmid DNA Nuclear Internalization with Polymer-Based Vehicles. Mol. Pharmaceutics 2012, 9, 2256−2267. (30) Grandinetti, G.; Smith, A. E.; Reineke, T. M. Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity? Mol. Pharmaceutics 2012, 9, 523−538. (31) Crowley, S. T.; Rice, K. G. Evolving Nanoparticle Gene Delivery Vectors for the Liver: What Has Been Learned in 30 Years. J. Controlled Release 2015, 219, 457−470. (32) Mishra, S.; Webster, P.; Davis, M. E. PEGylation Significantly Affects Cellular Uptake and Intracellular Trafficking of Non-Viral Gene Delivery Particles. Eur. J. Cell Biol. 2004, 83, 97−111. (33) Tan, Z.; Dhande, Y. K.; Reineke, T. M. Cell Penetrating Polymers Containing Guanidinium Trigger Apoptosis in Human Hepatocellular Carcinoma Cells Unless Conjugated to a Targeting NAcetyl-Galactosamine Block. Bioconjugate Chem. 2017, 28, 2985− 2997. (34) Dhande, Y. K.; Wagh, B. S.; Hall, B. C.; Sprouse, D.; Hackett, P. B.; Reineke, T. M. N-Acetylgalactosamine Block-co-Polycations Form

Stable Polyplexes with Plasmids and Promote Liver-Targeted Delivery. Biomacromolecules 2016, 17, 830−840. (35) Smith, A. E.; Sizovs, A.; Grandinetti, G.; Xue, L.; Reineke, T. M. Diblock Glycopolymers Promote Colloidal Stability of Polyplexes and Effective pDNA and siRNA Delivery under Physiological Salt and Serum Conditions. Biomacromolecules 2011, 12, 3015−3022. (36) Jung, S.; Lodge, T. P.; Reineke, T. M. Structures and Protonation States of Hydrophilic-Cationic Diblock Copolymers and Their Binding with Plasmid DNA. J. Phys. Chem. B 2018, 122, 2449− 2461. (37) Buckwalter, D. J.; Sizovs, A.; Ingle, N. P.; Reineke, T. M. MAG versus PEG: Incorporating a Poly(MAG) Layer to Promote Colloidal Stability of Nucleic Acid/“Click Cluster” Complexes. ACS Macro Lett. 2012, 1, 609−613. (38) Jung, S.; Lodge, T. P.; Reineke, T. M. Complexation between DNA and Hydrophilic-Cationic Diblock Copolymers. J. Phys. Chem. B 2017, 121, 2230−2243. (39) Sprouse, D.; Reineke, T. M. Investigating the Effects of Block versus Statistical Glycopolycations Containing Primary and Tertiary Amines for Plasmid DNA Delivery. Biomacromolecules 2014, 15, 2616−2628. (40) Wu, Y.; Wang, M.; Sprouse, D.; Smith, A. E.; Reineke, T. M. Glucose-Containing Diblock Polycations Exhibit Molecular Weight, Charge, and Cell-Type Dependence for pDNA Delivery. Biomacromolecules 2014, 15, 1716−1726. (41) Li, H.; Cortez, M. A.; Phillips, H. R.; Wu, Y.; Reineke, T. M. Poly(2-Deoxy-2-Methacrylamido Glucopyranose)-b-Poly(Methacrylate Amine)s: Optimization of Diblock Glycopolycations for Nucleic Acid Delivery. ACS Macro Lett. 2013, 2, 230−235. (42) Lee, C.; Grandinetti, G.; McLendon, P. M.; Reineke, T. M. A Polycation Scaffold Presenting Tunable “Click” Sites: Conjugation to Carbohydrate Ligands and Examination of Hepatocyte-Targeted pDNA Delivery. Macromol. Biosci. 2010, 10, 585−598. (43) Lorenzer, C.; Dirin, M.; Winkler, A. M.; Baumann, V.; Winkler, J. Going Beyond the Liver: Progress and Challenges of Targeted Delivery of siRNA Therapeutics. J. Controlled Release 2015, 203, 1− 15. (44) Ramløv, H.; Westh, P. Survival of the Cryptobiotic Eutardigrade Adorybiotus Coronifer during Cooling to −196 °C: Effect of Cooling Rate, Trehalose Level, and Short-Term Acclimation. Cryobiology 1992, 29, 125−130. (45) Srinivasachari, S.; Liu, Y.; Zhang, G.; Prevette, L.; Reineke, T. M. Trehalose Click Polymers Inhibit Nanoparticle Aggregation and Promote pDNA Delivery in Serum. J. Am. Chem. Soc. 2006, 128, 8176−8184. (46) Srinivasachari, S.; Liu, Y.; Prevette, L. E.; Reineke, T. M. Effects of Trehalose Click Polymer Length on pDNA Complex Stability and Delivery Efficacy. Biomaterials 2007, 28, 2885−2898. (47) Prevette, L. E.; Lynch, M. L.; Kizjakina, K.; Reineke, T. M. Correlation of Amine Number and pDNA Binding Mechanism for Trehalose-Based Polycations. Langmuir 2008, 24, 8090−8101. (48) Kizjakina, K.; Bryson, J. M.; Grandinetti, G.; Reineke, T. M. Cationic Glycopolymers for the Delivery of pDNA to Human Dermal Fibroblasts and Rat Mesenchymal Stem Cells. Biomaterials 2012, 33, 1851−1862. (49) Anderson, K.; Sizovs, A.; Cortez, M.; Waldron, C.; Haddleton, D. M.; Reineke, T. M. Effects of Trehalose Polycation End-Group Functionalization on Plasmid DNA Uptake and Transfection. Biomacromolecules 2012, 13, 2229−2239. (50) Boyle, W. S.; Senger, K.; Tolar, J.; Reineke, T. M. Heparin Enhances Transfection in Concert with a Trehalose-Based Polycation with Challenging Cell Types. Biomacromolecules 2017, 18, 56−67. (51) Boyle, W. S.; Twaroski, K.; Woska, E.; Tolar, J.; Reineke, T. M. Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells. Bioconjugate Chem. 2019, 30, 418−431. K


Article

Accounts of Chemical Research (52) Xue, L.; Ingle, N. P.; Reineke, T. M. Highlighting the Role of Polymer Length, Carbohydrate Size, and Nucleic Acid Type in Potency of Glycopolycation Agents for pDNA and siRNA Delivery. Biomacromolecules 2013, 14, 3903−3915. (53) Xue, L.; Kelkar, S. S.; Wang, X.; Ma, J.; Madsen, L. A.; Reineke, T. M. A Theranostic Polycation Containing Trehalose and Lanthanide Chelate Domains for siRNA Delivery and Monitoring. RSC Adv. 2015, 5, 74102−74106. (54) Kelkar, S. S.; Xue, L.; Turner, S. R.; Reineke, T. M. LanthanideContaining Polycations for Monitoring Polyplex Dynamics via Lanthanide Resonance Energy Transfer. Biomacromolecules 2014, 15, 1612−1624. (55) Sizovs, A.; Xue, L.; Tolstyka, Z. P.; Ingle, N. P.; Wu, Y.; Cortez, M.; Reineke, T. M. Poly(Trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective Polyplex-Mediated siRNA Delivery. J. Am. Chem. Soc. 2013, 135, 15417−15424. (56) Tolstyka, Z. P.; Phillips, H.; Cortez, M.; Wu, Y.; Ingle, N.; Bell, J. B.; Hackett, P. B.; Reineke, T. M. Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and In Vivo pDNA Delivery. ACS Biomater. Sci. Eng. 2016, 2, 43−55. (57) Tale, S. R.; Yin, L.; Reineke, T. M. Trehalose-Functionalized Block Copolymers Form Serum-Stable Micelles. Polym. Chem. 2014, 5, 5160−5167. (58) Yin, L.; Dalsin, M. C.; Sizovs, A.; Reineke, T. M.; Hillmyer, M. A. Glucose-Functionalized, Serum-Stable Polymeric Micelles from the Combination of Anionic and RAFT Polymerizations. Macromolecules 2012, 45, 4322−4332. (59) Jiang, Y.; Reineke, T. M.; Lodge, T. P. Complexation of DNA with Cationic Copolymer Micelles: Effects of DNA Length and Topology. Macromolecules 2018, 51, 1150−1160. (60) Jiang, Y.; Lodge, T. P.; Reineke, T. M. Packaging pDNA by Polymeric ABC Micelles Simultaneously Achieves Colloidal Stability and Structural Control. J. Am. Chem. Soc. 2018, 140, 11101−11111.

L


Nonviral Gene Delivery with Cationic Glycopolymers - American

Recommend Documents