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Postdoctoral Manuscripts

Yining Liu, Alejandra Suarez-Arnedo, Shamitha Shetty, Yaoying Wu, Michelle Schneider, Joel H. Collier, Tatiana Segura, Advanced Science 2023, 2204882

Microporous annealed particle scaffolds (MAPS) are a new class of granular materials generated through the interlinking of tunable microgels, which produce an interconnected network of void space. These microgel building blocks can be designed with different mechanical or bio-active parameters to facilitate cell infiltration and modulate host response. Previously, changing the chirality of the microgel crosslinking peptides from L- to D-amino acids led to significant tissue regeneration and functional recovery in D-MAPS-treated cutaneous wounds. In this study, the immunomodulatory effect of D-MAPS in a subcutaneous implantation model is investigated. How macrophages are the key antigen-presenting cells to uptake and present these biomaterials to the adaptive immune system is uncovered. A robust linker-specific IgG2b/IgG1 response to D-MAPS is detected as early as 14 days post-implantation. The fine balance between pro-regenerative and pro-inflammatory macrophage phenotypes is observed in D-MAPS as an indicator for regenerative scaffolds. The work offers valuable insights into the temporal cellular response to synthetic porous scaffolds and establishes a foundation for further optimization of immunomodulatory pro-regenerative outcomes.

Yaoying Wu; Hanning Wen; Zachary J Berstein; Kelly M Hainline; Tykia S Blakney; Kendra L Congdon; David J Snyder; John H Sampson; Luis Sanchez-Perez; Joel H Collier, Science Advances 2022, 8(29) eabm7833

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we employ a supramolecular a-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous  B-cell, CD8+ T-cell, and CD4+ T-cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8+ T cell-based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine-induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8+ and CD4+ responses, and they illustrate a multi-epitope cancer vaccine design approach using supramolecular nanomaterials.

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Jui-Lin Chen; Chelsea Fries; Stella Berendam; Nicole Rodgers; Emily Roe; Yaoying Wu; Shuk Hang Li; Rishabh Jain; Brian Watts; Joshua Eudailey; Richard Barfield; Cliburn Chan; M Moody; Kevin Saunders; Justin Pollara; Sallie Permar; Joel Collier; Genevieve Fouda, Science Advances 2022 , 8(38) eabq0273

To develop vaccines for certain key global pathogens such as HIV, it is crucial to elicit both neutralizing and non-neutralizing Fc-mediated effector antibody functions. Clinical evidence indicates that non-neutralizing antibody functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) contribute to protection against several pathogens. In this study, we demonstrated that conjugation of HIV Envelop (Env) antigen gp120 to a self-assembling nanofiber material named Q11 induced antibodies with higher breadth and functionality when compared to soluble gp120. Immunization with Q11-conjugated gp120 vaccine (gp120-Q11) demonstrated higher tier 1 neutralization, ADCP and ADCC as compared to soluble gp120. Moreover, Q11 conjugation altered the Fc N-glycosylation profile of antigen-specific antibodies, leading to a phenotype associated with increased ADCC in animals immunized with gp120-Q11. Thus, this nanomaterial vaccine strategy can enhance non-neutralizing antibody functions possibly through modulation of IgG Fc N-glycosylation.

Sean Kelly; Nicole Votaw; Benjamin Cossette; Yaoying Wu; Shamitha Shetty; Lucas Shores; Luqman Issah; Joel Collier, Science Advances 2022 8(47) eabq4120

Urinary tract infections (UTIs) are a major public health problem affecting millions of individuals each year. Recurrent UTIs are managed by long-term antibiotic use, making the alarming rise of antibiotic resistance a significant threat to future UTI treatment. Extended antibiotic regimens may also have adverse effects on the microbiome. Here, we report the use of a supramolecular vaccine to provide long-term protection against uropathogenic E. coli, which cause 80% of uncomplicated UTIs. We designed mucus-penetrating peptide-polymer nanofibers to enable sublingual (under the tongue) vaccine delivery and elicit antibody responses systemically and in the urogenital tract. In a mouse model of UTI, we demonstrate equivalent efficacy to high-dose oral antibiotics but with significantly less perturbation of the gut microbiome. We also formulate our vaccine as a rapid-dissolving sublingual tablet that raises response in mice and rabbits. Our approach represents a promising alternative to antibiotics for the treatment and prevention of UTIs.

Sean H Kelly; Emmanuel E Opolot; Yaoying Wu; Benjamin Cossette; Ajay K Varadhan; Joel H Collier, Advanced Healthcare Materials 2021, 10(6) p2001614

Widespread vaccination is essential to global health. Significant barriers exist to improving vaccine coverage in lower- and middle-income countries, including the costly requirements for cold-chain distribution and trained medical personnel to administer the vaccines. A heat-stable and highly porous tablet vaccine that can be administered sublingually via simple dissolution under the tongue is described. SIMPL tablet vaccines (Supramolecular IMmunization with Peptides subLingually) are produced by freeze-drying a mixture of self-assembling peptide-polymer nanofibers, sugars, and adjuvant. Sublingual immunization with SIMPL tablets raises antibody responses against both a model epitope from ovalbumin and a clinically relevant epitope from Mycobacterium tuberculosis. Further, sublingual antibody responses are not diminished after heating the tablets for 1 week at 45 °C, in contrast to a more conventional carrier vaccine (KLH). This approach directly addresses the need for a heat-stable and easily deliverable vaccine to improve equity in global vaccine coverage.

Sean H Kelly; Benjamin J Cossette; Ajay K Varadhan; Yaoying Wu; Joel H Collier, ACS Biomaterials Science & Engineering 2021, 7(5) p1876

Effective sublingual peptide immunization requires overcoming challenges of both delivery and immunogenicity. Mucosal adjuvants, such as cyclic-dinucleotides (CDN), can promote sublingual immune responses but must be codelivered with the antigen to the epithelium for maximum effect. We designed peptide–polymer nanofibers (PEG-Q11) displaying nona-arginine (R9) at a high density to promote complexation with CDNs via bidentate hydrogen-bonding with arginine side chains. We coassembled PEG-Q11 and PEG-Q11R9 peptides to titrate the concentration of R9 within nanofibers. In vitro, PEG-Q11R9 fibers and cyclic-di-GMP or cyclic-di-AMP adjuvants had a synergistic effect on enhancing dendritic cell activation that was STING-dependent and increased monotonically with increasing R9 concentration. The polyvalent display of R9 on assembled nanofibers was significantly more effective at promoting CDN-mediated DC activation in vitro than mixing nanofibers with an equimolar concentration of unassembled R9 peptide. The sublingual administration of nanofibers revealed a bell-shaped trend between increasing R9 concentration and enhancements to antigen trafficking and the activation of DCs in the draining lymph nodes. Intermediate levels of R9 within sublingually administered PEG-Q11 fibers were optimal for immunization, suggesting a balance between polyarginine’s ability to sequester CDNs along the nanofiber and its potentially detrimental mucoadhesive interactions. These findings present a potentially generalizable biomaterial strategy for enhancing the potency of CDN adjuvants and reveal important design considerations for the nascent field of sublingual biomaterial immunization.

Nicole L Votaw; Lauren Collier; Elizabeth J Curvino; Yaoying Wu; Chelsea N Fries; Madison T Ojeda; Joel H Collier, Biomaterials 2021, 273 p120825

Biomaterials capable of inducing immune responses with minimal associated inflammation are of interest in applications ranging from tissue repair to vaccines. Here we report the design of self-assembling randomized polypeptide nanomaterials inspired by glatiramoids, an immunomodulatory class of linear random copolymers. We hypothesized that peptide self-assemblies bearing similar randomized polypeptides would similarly raise responses skewed toward Type 2 immunity and TH2 T-cell responses, additionally strengthening responses to co-assembled peptide epitopes in the absence of adjuvant. We developed a method for synthesizing self-assembling peptides terminated with libraries of randomized polypeptides (termed KEYA) with good batch-to-batch reproducibility. These peptides formed regular nanofibers and raised strong antibody responses without adjuvants. KEYA modifications dramatically improved uptake of peptide nanofibers in vitro by antigen presenting cells, and served as strong B-cell and T-cell epitopes in vivo, enhancing immune responses against epitopes relevant to influenza and chronic inflammation while inducing a KEYA-specific Type 2/TH2/IL-4 phenotype. KEYA modifications also increased IL-4 production by T cells, extended the residence time of nanofibers, induced no measurable swelling in footpad injections, and decreased overall T cell expansion compared to unmodified nanofibers, further suggesting a TH2 T-cell response with minimal inflammation. Collectively, this work introduces a biomaterial capable of raising strong Type 2/TH2/IL-4 immune responses, with potential applications ranging from vaccination to tissue repair.

Sean H Kelly; Yaoying Wu; Ajay K Varadhan; Elizabeth J Curvino; Anita S Chong; Joel H Collier, Biomaterials 2020, 241 p119903

Short peptides are poorly immunogenic when delivered sublingually – under the tongue. Nanomaterial delivery of peptides could be utilized to improve immunogenicity towards designed sublingual vaccines, but nanomaterials have not been widely successful in sublingual vaccines owing to the challenges of transport through the sublingual mucosa. Here, we report that the sublingual immunogenicity of peptides is negligible, even in the presence of sublingual adjuvants or when PEGylated, but can be dramatically enhanced by assembly into supramolecular polymer-peptide nanofibers bearing low-molecular weight PEG, optimally between 2000 and 3000 Da. Neither PEGylation nor a sublingual adjuvant were capable of rendering peptides immunogenic without assembly into nanofibers. We found that PEG decreased nanofiber interactions with mucin and promoted longer residence time at the sublingual immunization site. Parallel investigations with shortened nanofibers indicated that the size of the assemblies had a surprisingly negligible influence over sublingual immunogenicity. In mice, optimized formulations were capable of raising strong and highly durable systemic antibody responses, antibodies in the upper respiratory and reproductive tracts, and systemic antigen-specific T-cell responses. These nanofiber-based sublingual vaccines were effective with both protein and nucleotide adjuvants and raised responses against both a model peptide epitope and a peptide epitope from M. tuberculosis. Further, PASylation (modification of nanofibers with peptide sequences rich in Pro, Ala, and Ser) could be substituted for PEGylation to also achieve sublingual immunogenicity. These findings indicated that surface properties supersede nanomaterial size in modulating sublingual nanomaterial immunogenicity, having important implications for the design of synthetic sublingual vaccines.

Yaoying Wu; Sean H Kelly; Luis Sanchez-Perez; John H Sampson; Joel H Collier, Biomaterials Science 2020, 8(12) p3522

Several different self-assembling peptide systems that form nanofibers have been investigated as vaccine platforms, but design principles for adjusting the character of the immune responses they raise have yet to be well articulated. Here we compared the immune responses raised by two structurally dissimilar peptide nanofibers, one a β-sheet fibrillar system (Q11), and one an α-helical nanofiber system (Coil29), hypothesizing that integrated T-cell epitopes within the latter would promote T follicular helper (Tfh) cell engagement and lead to improved antibody titers and quality. Despite significantly different internal structures, nanofibers of the two peptides exhibited surprisingly similar nanoscale morphologies, and both were capable of raising strong antibody responses to conjugated peptide epitopes in mice without adjuvant. Both were minimally inflammatory, but as hypothesized Coil29 nanofibers elicited antibody responses with higher titers and avidities against a conjugated model epitope (OVA323–339) and a candidate peptide epitope for vaccination against S. aureus. Subsequent investigation indicated that Coil29 nanofibers possessed internal CD4+ T cell epitopes: whereas Q11 nanofibers required co-assembly of additional CD4+ T cell epitopes to be immunogenic, Coil29 nanofibers did not. Coil29 nanofibers also raised stronger germinal center B cell responses and follicular helper T cell (Tfh) responses relative to Q11 nanofibers, likely facilitating the improvement of the antibody response. These findings illustrate design strategies for improving humoral responses raised by self-assembled peptide nanofibers.

Chelsea N Fries; Yaoying Wu; Sean H Kelly; Michelle Wolf; Nicole L Votaw; Stefan Zauscher; Joel H Collier, Advanced Materials 2020, 32(39) p2003310

Peptide nanofibers are useful for many biological applications, including immunotherapy, tissue engineering, and drug delivery. The robust lengthwise assembly of these peptides into nanofibers is typically difficult to control, resulting in polydisperse fiber lengths and an incomplete understanding of how nanofiber length affects biological responses. Here, rationally designed capping peptides control the length of helical peptide nanofibers with unique precision. These designed peptides bind the tips of elongated nanofibers to shorten and narrow their length distributions. Demonstrating their use as immunotherapies, capped nanofibers are preferentially cross-presented by dendritic cells compared to uncapped nanofibers. Due to increased cross-presentation, these capped nanofibers trigger stronger CD8+ T-cell responses in mice than uncapped nanofibers. This strategy illustrates a means for controlling the length of supramolecular peptide nanofibers to modulate their immunogenicity in the context of immunotherapies.

Christopher E Nelson; Yaoying Wu; Matthew P Gemberling; Matthew L Oliver; Matthew A Waller; Joel D Bohning; Jacqueline N Robinson-Hamm; Karen Bulaklak; Ruth M Castellanos Rivera; Joel H Collier; Aravind Asokan; Charles A Gersbach, Nature Medicine 2019, 25(3) p427

Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.

Kelly M Hainline; Fangqi Gu; Jacqueline F Handley; Ye F Tian; Yaoying Wu; Larischa de Wet; Donald J Vander Griend; Joel H Collier, Macromolecular Bioscience 2019, 19(1) p1800249

Progress in prostate cancer research is presently limited by a shortage of reliable in vitro model systems. The authors describe a novel self-assembling peptide, bQ13, which forms nanofibers and gels useful for the 3D culture of prostate cancer spheroids, with improved cytocompatibility compared to related fibrillizing peptides. The mechanical properties of bQ13 gels can be controlled by adjusting peptide concentration, with storage moduli ranging between 1 and 10 kPa. bQ13’s ability to remain soluble at mildly basic pH considerably improved the viability of encapsulated cells compared to other self-assembling nanofiber-forming peptides. LNCaP cells formed spheroids in bQ13 gels with similar morphologies and sizes to those formed in Matrigel or RADA16-I. Moreover, prostate-specific antigen (PSA) is produced by LNCaP cells in all matrices, and PSA production is more responsive to enzalutamide treatment in bQ13 gels than in other fibrillized peptide gels. bQ13 represents an attractive platform for further tailoring within 3D cell culture systems.

Yaoying Wu; Joel H Collier, Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology 2016

Self-assembling coiled coils, which occur commonly in native proteins, have received significant interest for the design of new biomaterials-based medical therapies. Considerable effort over recent years has led to a detailed understanding of the self-assembly process of coiled coils, and a diverse collection of strategies have been developed for designing functional materials using this motif. The ability to engineer the interface between coiled coils allows one to achieve variously connected components, leading to precisely defined structures such as nanofibers, nanotubes, nanoparticles, networks, gels, and combinations of these. Currently these materials are being developed for a range of biotechnological and medical applications, including drug delivery systems for controlled release, targeted nanomaterials, ‘drug-free’ therapeutics, vaccine delivery systems, and others.

Doctoral Manuscripts

Yaoying Wu; Adam E Smith; Theresa M Reineke, Bioconjugate Chemistry 2017, 28(8) p2035

A class of cationic poly(alkylamidoamine)s (PAAAs) containing lipophilic methylene linkers were designed and examined as in vitro plasmid DNA (pDNA) delivery agents. The PAAAs were synthesized via step-growth polymerization between a diamine monomer and each of four different diacid chloride monomers with varying methylene linker lengths, including glutaryl chloride, adipoyl chloride, pimeloyl chloride, and suberoyl chloride, which served to systematically increase the lipophilicity of the polymers. The synthesized polymers successfully complexed with pDNA in reduced serum medium at N/P ratios of 5 and greater, resulting in polyplexes with hydrodynamic diameters of approximately 1 μm. These polyplexes were tested for in vitro transgene expression and cytotoxicity using HDFa (human dermal fibroblast), HeLa (human cervical carcinoma), HMEC (human mammary epithelial), and HUVEC (human umbilical vein endothelial) cells. Interestingly, select PAAA polyplex formulations were found to be more effective than Lipofectamine 2000 at promoting transgene expression (GFP) while maintaining comparable or higher cell viability. Transgene expression was highest in HeLa cells (∼90% for most formulations) and lowest in HDFa cells (up to ∼20%) as measured by GFP fluorescence. In addition, the cytotoxicity of PAAA polyplex formulations was significantly increased as the molecular weight, N/P ratio, and methylene linker length were increased. The PAAA vehicles developed herein provide a new delivery vehicle design strategy of displaying attributes of both polycations and lipids, which show promise as a tunable scaffold for refining the structure–activity–toxicity profiles for future genome editing studies.

Zachary P Tolstyka; Haley Phillips; Mallory Cortez; Yaoying Wu; Nilesh Ingle; Jason B Bell; Perry B Hackett; Theresa M Reineke, ACS Biomaterials Science & Engineering 2016, 2(1) p43

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.

Yaoying Wu; Miao Wang; Dustin Sprouse; Adam E Smith; Theresa M Reineke, Biomacromolecules 2014, 15(5) p1716

A series of diblock glycopolycations were created by polymerizing 2-deoxy-2-methacrylamido glucopyranose (MAG) with either a tertiary amine-containing monomer, N-[3-(N,N-dimethylamino) propyl] methacrylamide (DMAPMA), or a primary amine-containing unit, N-(2-aminoethyl) methacrylamide (AEMA). Seven structures were synthesized via aqueous reversible addition–fragmentation chain transfer (RAFT) polymerization that varied in the block lengths of MAG, DMAPMA, and AEMA along with two homopolymer controls of DMAPMA and AEMA that lacked a MAG block. The polymers were all able to complex plasmid DNA into polyplex structures and to prevent colloidal aggregation of polyplexes in physiological salt conditions. In vitro transfection experiments were performed in both HeLa (human cervix adenocarcinoma) cells and HepG2 (human liver hepatocellular carcinoma) cells to examine the role of charge type, block length, and cell type on transfection efficiency and toxicity. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased in the glycopolycation-based polyplexes, toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Lastly, cell type played a major role in efficiency. These glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend, signifying that the MAG block could aid in hepatocyte transfection.

Antons Sizovs; Lian Xue; Zachary P Tolstyka; Nilesh P Ingle; Yaoying Wu; Mallory Cortez; Theresa M Reineke, Journal of the American Chemical Society 2013, 135(41) p15417

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or “poly(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.

Haibo Li; Mallory Cortez; Haley R Phillips; Yaoying Wu; Theresa M Reineke, ACS Macro Letters 2013, 2(3) p230

A series of nine poly(2-deoxy-2-methacrylamido glucopyranose)-b-poly(methacrylate amine) diblock copolycations has been synthesized as new colloidally stable polynucleotide vehicles. The cationic block was varied in length and in the degree of methyl group substitution (secondary, tertiary, quaternary) on the pendant amine in an effort to optimize the structure and activity for plasmid DNA (pDNA) delivery. Upon a thorough kinetic study of polymerization for each polymer, the glycopolymers were prepared with well-controlled Mn and Đ. The binding and colloidal stability of the polymer–pDNA nanocomplexes at different N/P ratios and in biological media have been investigated using gel electrophoresis and light scattering techniques. The toxicity and transfection efficiency of the polyplexes have been evaluated with Hep G2 (human liver hepatocellular carcinoma) cells; several polymers displayed excellent delivery and toxicity profiles justifying their further development for in vivo gene therapy.