Amanda C. Engler

Highly Efficient “Grafting onto” a Polypeptide Backbone Using Click Chemistry

A cells extracellular matrix consists of macromolecules, such as glycoproteins, proteoglycans, and collagen, that control both the mechanical structure and the microenvironment.[1] These properties provide physical cues that are necessary to induce various cell functions and morphologies. An important goal of tissue engineering is to mimic the environment of the extracellular matrix on several levels, mechanically, chemically, and architecturally.[2] To accomplish this goal, new synthetic methods are necessary in order to mimic the structure of these complex macromolecules. We have developed a synthetic method to form highly functionalized grafted polypeptides that can be made to mimic complex biomacromolecules such as glycoproteins and proteoglycans. While, these new synthetic polypeptides are much simpler than natural peptides, they still possess the α-helical conformation of natural polypeptides and various chemical moieties can be attached to mimic the microenvironment of the extracellular matrix. These polymers have several features that make them very attractive for biological applications including low toxicity, biodegradability, tunable structures, and well-controlled dimensions.

Ligand-Clustered “Patchy” Nanoparticles for Modulated Cellular Uptake and In Vivo Tumor Targeting†

Despite the evident success of using a multivalent approach to increase efficacy of targeted delivery, a clear understanding of how multiple ligands behave collectively to influence the uptake of nanoparticle cell-targeting agents has not been reached. Although when present in large quantity, multivalent ligands can increase binding avidities to cells, it is also conceivable, that the manner in which these ligands are presented to the cell may have a significant effect on uptake. Here we examine this parameter using a linear dendritic polymer construct that enabled us to pattern the surfaces of nanoparticles with variable sized ligand clusters in different spatial arrangements. We demonstrate for the first time the clear impact of folate presentation on intracellular uptake both in vitro and in vivo. The findings presented here suggest that the nature of ligand presentation on a nanoparticle surface may play an important role in drug targeting; the results suggest potential impact for other targeting moieties and provide a framework for further refinement of future multivalent targeting strategies.

Effects of Side Group Functionality and Molecular Weight on the Activity of Synthetic Antimicrobial Polypeptides

The rapid emergence of antibiotic-resistant bacteria along with increasing difficulty in biofilm treatment has caused an immediate need for the development of new classes of antimicrobial therapeutics. We have developed a library of antimicrobial polypeptides, prepared by the ring-opening polymerization of γ-propargyl-L-glutamate N-carboxyanhydride and the alkyne-azide cycloaddition click reaction, which mimic the favorable characteristics of naturally occurring antimicrobial peptides (AmPs). AmPs are known not to cause drug resistance as well as prevent bacteria attachment on surfaces. The ease and scale of synthesis of the antimicrobial polypeptides developed here are significantly improved over the traditional Merrifield synthetic peptide approaches needed for naturally occurring antimicrobial peptides and avoids the unique challenges of biosynthetic pathways. The polypeptides range in length from 30 to 140 repeat units and can have varied side group functionality, including primary, secondary, tertiary, and quaternary amines with hydrocarbon side chains ranging from 1 to 12 carbons long. Overall, we find these polypeptides to exhibit broad-spectrum activity against both Gram positive and Gram negative bacteria, namely, S. aureus and E. coli , while having very low hemolytic activity. Many of the polypeptides can also be used as surface coatings to prevent bacterial attachment. The polypeptide library developed in this work addresses the need for effective biocompatible therapeutics for drug delivery and medical device coatings.

Broad-spectrum antimicrobial and biofilm-disrupting hydrogels: stereocomplex-driven supramolecular assemblies.

Fighting the resistance: biodegradable and injectable/moldable hydrogels with hierarchical nanostructures were made with broad-spectrum antimicrobial activities and biofilm-disruption capability. They demonstrate no cytotoxicity in vitro, and show excellent skin biocompatibility in animals. These hydrogels have great potential for clinical use in prevention and treatment of various multidrug-resistant infections.

Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection

Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 μm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and ~100% substitution of primary amine groups leads to significantly lower gene transfection efficiency. These findings provide insights to modification of PEI for development of effective and non-cytotoxic non-viral vectors.

The synthetic tuning of clickable pH responsive cationic polypeptides and block copolypeptides

A series of pH responsive synthetic polypeptides has been developed based on an N-carboxyanhydride ring opening polymerization combined with a facile and versatile click chemistry. Poly(γ-propargyl L-glutamate) (PPLG) homopolymers and poly(ethylene glycol-b-γ-propargyl L-glutamate) (PEG-b-PPLG) block copolymers were substituted with various amine moieties that range in pKa and hydrophobicity, providing the basis for a library of new synthetic structures that can be tuned for specific interactions and responsive behaviors. These amine-functionalized polypeptides have the ability to change solubility, or reversibly self-assemble into micelles with changes in the degree of ionization; they also adopt an α-helical structure at biologically relevant pHs. Here we characterize the pH responsive behavior of the new polypeptides and the hydrolysis of the ester containing amine side chains. We examine the reversible micellization with block copolymers of the polypeptides and nucleic acid encapsulation that demonstrate the potential use of these materials for systemic drug and gene delivery.

Broad-spectrum antimicrobial supramolecular assemblies with distinctive size and shape

With the increased prevalence of antibiotic-resistant infections, there is an urgent need for innovative antimicrobial treatments. One such area being actively explored is the use of self-assembling cationic polymers. This relatively new class of materials was inspired by biologically pervasive cationic host defense peptides. The antimicrobial action of both the synthetic polymers and naturally occurring peptides is believed to be complemented by their three-dimensional structure. In an effort to evaluate shape effects on antimicrobial materials, triblock polymers were polymerized from an assembly directing terephthalamide-bisurea core. Simple changes to this core, such as the addition of a methylene spacer, served to direct self-assembly into distinct morphologies-spheres and rods. Computational modeling also demonstrated how subtle core changes could directly alter urea stacking motifs manifesting in unique multidirectional hydrogen-bond networks despite the vast majority of material consisting of poly(lactide) (interior block) and cationic polycarbonates (exterior block). Upon testing the spherical and rod-like morphologies for antimicrobial properties, it was found that both possessed broad-spectrum activity (Gram-negative and Gram-positive bacteria as well as fungi) with minimal hemolysis, although only the rod-like assemblies were effective against Candida albicans.

Organic Acid-Catalyzed Polyurethane Formation via a Dual-Activated Mechanism: Unexpected Preference of N-Activation over O-Activation of Isocyanates

A systematic study of acid organocatalysts for the polyaddition of poly(ethylene glycol) to hexamethylene diisocyanate in solution has been performed. Among organic acids evaluated, sulfonic acids were found the most effective for urethane formations even when compared with conventional tin-based catalysts (dibutyltin dilaurate) or 1,8-diazabicyclo[5.4.0]undec-7-ene. In comparison, phosphonic and carboxylic acids showed considerably lower catalytic activities. Furthermore, sulfonic acids gave polyurethanes with higher molecular weights than was observed using traditional catalyst systems. Molecular modeling was conducted to provide mechanistic insight and supported a dual activation mechanism, whereby ternary adducts form in the presence of acid and engender both electrophilic isocyanate activation and nucleophilic alcohol activation through hydrogen bonding. Such a mechanism suggests catalytic activity is a function of not only acid strength but also inherent conjugate base electron density.

pH-sensitive polycarbonate micelles for enhanced intracellular release of anticancer drugs: a strategy to circumvent multidrug resistance

In this study, we have synthesized two novel amphiphilic diblock copolymers with aldehyde groups via organocatalytic ROP of a functionalized cyclic carbonate monomer (MTC-Bz) using polyethylene oxide (PEG) as the macroinitiator. The polymers were covalently conjugated with an anti-tumor drug doxorubicin (DOX) via a pH-sensitive Schiff-base linkage. The resulting conjugates formed micelles in phosphate-buffered saline (PBS) (pH 7.4) with an average size of about 100 nm and narrow size distribution. The surface charge of the micelles was close to zero. The micelles were stable in both PBS and cell culture media containing 10% FBS up to 5 days. The results obtained from the in vitro release study indicated that DOX release from the micelles was pH-dependent, being faster at pH 5.0 (the endolysosomal environment) than pH 7.4 (the extracellular environment). Human breast cancer MCF-7 cells and DOX-resistant MCF-7/Adr cells were employed to investigate the cellular uptake and cytotoxicity of DOX-loaded micelles. The confocal microscopy and flow cytometry studies showed that the uptake of DOX-loaded micelles by MCF-7 cells was similar to that of free DOX. In sharp contrast, the uptake of DOX-loaded micelles by MCF-7/Adr cells was significantly higher than that of free DOX. The polymers showed no toxicity to MCF-7 and MCF-7/Adr cells. The DOX-loaded micelles killed the cells efficiently. In particular, they were more potent against drug-resistant MCF-7/Adr cells than free DOX due to the higher cellular drug accumulation and pH-triggered intracellular drug release, providing a strategy to navigate around drug resistance. These DOX-conjugated micelles can be a promising carrier for the delivery of anticancer drugs with amine functional groups.

Mitigated cytotoxicity and tremendously enhanced gene transfection efficiency of PEI through facile one-step carbamate modification.

Extremely efficacious gene transfection vector: The rapid and facile modification of PEI with commercially available TMC produces an extremely efficacious gene delivery vector with minimal cytotoxicity. Functionalization of PEI is easily controlled by PEI:cyclic carbonate feed ratios and allows for the addition of functionality. Modified PEIs hold great potential as gene delivery systems due to easy synthesis, scalability, low cost, low toxicity, and outstanding transfection capacity.