Functional Hydrophilic Polymers for Solution Assembly and Non-Viral Gene Therapy

This thesis examines functional hydrophilic polymers designed in linear and comb architectures and that carry functional moieties in the context of solution assembly and non-viral gene therapy. Specifically, polymers containing cations, zwitterions, and reactive groups are investigated as non-viral gene therapy reagents and at oil-water interfaces on droplets. Cations facilitate complexation of nucleic acids and interaction with cellular and nuclear membranes, while zwitterions impart stimuli-responsive solution properties and biocompatibility. Reactive groups, including alkenes, alkynes, and benzylic methylenes, permit post-polymerization modification leading to tunable polymer properties in solution and at interfaces. This work expands the knowledge base related to solution, interfacial, and DNA complexation properties imparted by the inclusion and arrangement of functional groups within hydrophilic polymers. Chapters 2-4 discuss polymers having a comb architecture designed for non-viral gene therapy. These polymers contain a polycyclooctene backbone and oligopeptide and zwitterionic pendent groups. Chapter 2 describes comb polymers with Simian Virus 40 nuclear localization sequence (SV40 NLS) pendent groups that interact with receptors on the nuclear membrane to provide enhanced nuclear uptake of complexed DNA. Cell culture experiments revealed a marked effect of oligopeptide orientation on the resulting gene expression levels provided by the NLS-containing comb polymers. Moreover, these polymers outperformed commercial transfection reagents, both in gene expression levels and cell viability. Chapter 3 describes the introduction of zwitterions into these comb polymers and investigates the impact of zwitterions on polymer-DNA complex (or ‘polyplex’) properties. Dispersing small amounts of zwitterion into comb polymers with SV40 NLS and oligolysine pendent groups improved gene expression levels. Chapter 4 describes the synthesis of comb polymers having oligoarginine sequences as pendent groups and properties of the corresponding polyplexes. Compared to oligolysine sequences, oligoarginine facilitated stronger polymer-DNA binding and allowed inclusion of higher loadings of biocompatible zwitterions without compromising the ability of the polymer to complex DNA. Chapters 5 and 6 investigate zwitterionic polymers at the oil-water interface of emulsion droplets. Experiments in Chapter 5 demonstrate the ability of zwitterionic sulfobetaine methacrylate polymers to stabilize oil-in-water droplets in pure water at room temperature, followed by coalescence upon increasing salt concentration or temperature. Integrating alkene and alkyne groups directly into the zwitterionic moiety allowed inclusion of high loadings of functional groups without interrupting the salt-triggered droplet coalescence exhibited by sulfobetaine homopolymers. Functional group placement was found to greatly impact interfacial properties. Chapter 6 describes the preparation of adhesive droplets from novel sulfur-based zwitterionic polymers. Droplet adhesion was modulated by the presence of salt and nucleophiles, and the resultant stability and adhesiveness of the droplets. The interdroplet interactions were found to dictate the mechanical properties of the emulsion and its stability upon application of centrifugal force

The Effect of Comb Architecture on Complex Coacervation

Complex coacervation is a widely utilized technique for effecting phase separation, though predictive understanding of molecular-level details remains underdeveloped. Here, we couple coarse-grained Monte Carlo simulations with experimental efforts using a polypeptide-based model system to investigate how a comb-like architecture affects complex coacervation and coacervate stability. Specifically, the phase separation behavior of linear polycation-linear polyanion pairs was compared to that of comb polycation-linear polyanion and comb polycation-comb polyanion pairs. The comb architecture was found to mitigate cooperative interactions between oppositely charged polymers, as no discernible phase separation was observed for comb-comb pairs and complex coacervation of linear-linear pairs yielded stable coacervates at higher salt concentration than linear-comb pairs. This behavior was attributed to differences in counterion release by linear vs. comb polymers during polyeletrolyte complexation. Additionally, the comb polycation formed coacervates with both stereoregular poly(L-glutamate) and racemic poly(D,L-glutamate), whereas the linear polycation formed coacervates only with the racemic polyanion. In contrast, solid precipitates were obtained from mixtures of stereoregular poly(L-lysine) and poly(L-glutamate). Moreover, the formation of coacervates from cationic comb polymers incorporating up to ~90% pendant zwitterionic groups demonstrated the potential for inclusion of comonomers to modulate the hydrophilicity and/or other properties of a coacervate-forming polymer. These results provide the first detailed investigation into the role of polymer architecture on complex coacervation using a chemically and architecturally well-defined model system, and highlight the need for additional research on this topic

Amphiphilic Cross-Linked Liquid Crystalline Fluoropolymer-Poly(ethylene glycol) Coatings for Application in Challenging Conditions: Comparative Study between Different Liquid Crystalline Comonomers and Polymer Architectures

Linear and hyperbranched poly­(ethylene glycol)-cross-linked amphiphilic fluoropolymer networks comprised of different liquid crystalline comonomers were developed and evaluated as functional coatings in extreme weather-challenging conditions. Through variation of the liquid-crystalline comonomer and hydrophilic:hydrophobic component ratios, several series of coatings were synthesized and underwent a variety of analyses including differential scanning calorimetry, water contact angle measurements and solution stability studies in aqueous media. These materials maintained an unprecedented reduction in the free water melting transition (<i>T</i>_m) temperature across the hyperbranched and linear versions. The coatings synthesized from hyperbranched fluoropolymers preserved the liquid crystalline character of the mesogenic components, as seen by polarized optical microscopy, and demonstrated stability in saltwater aqueous environments and in cold weather conditions

Erythrocyte-Membrane-Camouflaged Nanocarriers with Tunable Paclitaxel Release Kinetics via Macromolecular Stereocomplexation

Biomimetic-cell-membrane-camouflaged polymeric nanocarriers, possessing advantages related to the functional diversity of natural cell membranes and the physicochemical tailorability of synthetic polymers, serve as promising candidates for a therapeutic platform. Herein, we report a facile approach for the fabrication of erythrocyte (red blood cell, RBC)-membrane-camouflaged nanocarriers (RBC-MCNs) that exhibit tunable paclitaxel (PTX) release kinetics via altering macromolecular stereostructure. In this approach, biocompatible isotactic and atactic polylactides (PLAs) with similar molar masses (Mn = 8.2-8.9 kDa, as measured by NMR spectroscopy) and dispersities (&DStrok; < 1.1, as measured by size exclusion chromatography) were synthesized via organocatalyzed ring-opening polymerizations (ROPs), providing tunable crystalline structures via polymer tacticity, while RBC membranes provided biomimetic surfaces and improved colloidal stability of PLA nanoconstructs in phosphate-buffered saline (PBS, pH 7.4). Wide-angle X-ray diffraction (WAXD) and differential scanning calorimetry (DSC) analyses of the lyophilized nanoconstructs suggested significant retention of PLA stereocomplexation upon loading the hydrophobic anticancer drug PTX, enabling control over drug release kinetics. The structure-property relationships were maintained after the RBC coating, with 100% stereocomplexed PLA RBC-MCNs exhibiting the least PTX release during the first 12 h in PBS at 37 °C, compared to 2-, 3-, and 4-fold higher amounts of release for the 50% stereocomplexed, isotactic, and amorphous PLA counterparts, respectively. The extended release of PTX from the 100% stereocomplexed PLA RBC-MCNs resulted in an increased IC50 (0.50 μM) against SJSA osteosarcoma cells, relative to amorphous PLA RBC-MCNs (IC50 = 0.25 μM) or free PTX (IC50 = 0.05 μM). In contrast, non-PTX-loaded RBC-MCNs were not cytotoxic, and they also displayed lower immunotoxic responses against RAW 264.7 macrophage cells compared to RBC membrane vesicles. This work represents fundamental advances toward a potential personalized nanocarrier technology that would be capable of employing an individual's RBCs for membrane isolation, together with tuning of cargo loading and release simply via alteration of the biocompatible PLA stereoisomer feed ratio

Morphologic Design of Silver-Bearing Sugar-Based Polymer Nanoparticles for Uroepithelial Cell Binding and Antimicrobial Delivery

Platelet-like and cylindrical nanostructures from sugar-based polymers are designed to mimic the aspect ratio of bacteria and achieve uroepithelial cell binding and internalization, thereby improving their potential for local treatment of recurrent urinary tract infections. Polymer nanostructures, derived from amphiphilic block polymers composed of zwitterionic poly(d-glucose carbonate) and semicrystalline poly(l-lactide) segments, were constructed with morphologies that could be tuned to enhance uroepithelial cell binding. These nanoparticles exhibited negligible cytotoxicity, immunotoxicity, and cytokine adsorption, while also offering substantial silver cation loading capacity, extended release, and in vitro antimicrobial activity (as effective as free silver cations) against uropathogenic Escherichia coli. In comparison to spherical analogs, cylindrical and platelet-like nanostructures engaged in significantly higher association with uroepithelial cells, as measured by flow cytometry; despite their larger size, platelet-like nanostructures maintained the capacity for cell internalization. This work establishes initial evidence of degradable platelet-shaped nanostructures as versatile therapeutic carriers for treatment of epithelial infections