(558e) Engineering the Molecular Architectures of Antimicrobial Peptide (AMP)-Polymer Conjugates | AIChE

(558e) Engineering the Molecular Architectures of Antimicrobial Peptide (AMP)-Polymer Conjugates

Authors 

Cui, Z. - Presenter, Carnegie Mellon University
Hawkins, K., University of Virginia, Department of Chemical Engineering
Crawford, M., University of Virginia
Fisher, D., University of Virginia
Hughes, M., University of Virginia
Letteri, R., University of Virginia
While antimicrobial peptides (AMPs) are promising treatments for infectious diseases caused by multi-drug resistant bacteria, they are limited by toxicity to mammalian cells and instability in biological environments. Linear AMP-polymer conjugates, having a single AMP attached to a linear hydrophilic polymer, overcome the above challenges but markedly reduce antimicrobial activity. Hypothesizing that multivalent AMP-polymer conjugates will circumvent this tradeoff by improving antimicrobial activity relative to linear analogues, we prepared conjugates of two AMPs (L8 and P9) with linear, star-shaped, and comb-like architectures. Both L8 and P9 are derived from chemokines: L8 is a cysteine-containing short, unstructured AMP, whereas P9 has a helical structure more typical of AMPs. While the reactive cysteines in L8 lead to side reactions during conjugation, we found replacing either of these with alanine or protected thiol groups made the peptide less active. Therefore, to improve the conversion of the conjugation while maintaining the activity of L8, we developed a protection-conjugation-deprotection strategy to attach the cysteine-rich L8 to a neutral, hydrophilic polymer. Using a cysteine derivative with a removable acetamidomethyl (Acm) protecting group for the synthesis of L8, the conjugation of the protected L8 to a linear, hydroxysuccinimide (NHS)-functionalized polyethylene glycol (PEG) proceeded to >70% conversion and was followed by a dithiothreitol-assisted deprotection to reveal the thiol groups. This strategy allowed us to generate 4- and 8-arm star-shaped L8-PEG conjugates with L8 peptide attached from either the N- or C-terminus. For the helical AMP P9, we synthesized conjugates with linear, star-shaped, and comb-like architectures. Comb-like conjugates were prepared by reversible addition−fragmentation chain-transfer (RAFT) polymerization of the neutral hydrophilic monomer 2-hydroxypropyl methacrylamide (HPMA) and methacrylamide-functionalized P9 using two trithiocarbonate chain transfer agents (CTAs). While the CTA with a dodecyl group and a cyano group yielded copolymers with a broad molecular weight distribution (Р= 1.8), the CTA with two carboxylic acid end groups better controlled the polymerization (Р= 1.2), a result we attribute to the higher solubility of the latter CTA. Compared to grafting AMPs to pre-formed polymers, this graft through method for preparing comb-like AMP-polymer conjugates may improve control and efficiency of conjugation. Together, these materials and associated synthetic methods will enable a comparison of conjugates with similar compositions and distinct molecular architectures to determine the effects of molecular architecture on antimicrobial performance (i.e., antibacterial activity, stability, and cytotoxicity). By studying this set of materials, we aim to inform the design and accelerate the clinical implementation of AMP-polymer conjugates. Beyond antimicrobial applications, we envision that these synthetic strategies and findings will contribute to the development of peptide-polymer conjugate design for a variety of drug delivery pursuits.