Antimicrobial Properties of Polypeptide Based Materials

Abstract

Foot ulcers develop in 9.1 to 26.1 million diabetics annually worldwide, and 1 to 3 million in the U.S. alone have a history of foot ulcerations (22). More than 50% of foot ulcers become infected, many with antibiotic resistant infections, and of those infected ulcers, 20% lead to some level of amputation (3). Our novel matrix, a biosynthetic, elastin-like polymer (ELP) hydrogel that can be either a liquid, or semi-solid gel chip, offers a non-invasive potential therapeutic for these patients. We aimed to evaluate the safety and impact of the ELP matrix (“gel-chip”) itself both in vitro using cell culture, and when placed on the soles of human feet. The “gel-chip” also called “hydrogel” is a biosynthetic platform with a variety of uses in biomaterials applications, like stabilizing antimicrobials, particularly antimicrobial peptides (AMPs) that otherwise rapidly degrade, for topical application and drug delivery. We proceeded to both evaluate the ability of the ELP matrix to alter the microbiome of healthy human feet and, then tested the ELP matrix in its liquid form biosynthetically engineered with AMPs, to determine if it could alter the diabetic foot infection (DFI) microbiome in vitro. We demonstrated that the ELP matrix in solid form, itself captured a unique microbiome distinct from that obtained by the current standard of care, a dry culture swab. Of crucial importance was our finding that our ELP matrix (“gel-chip”) itself was well tolerated for over eight hours when placed on intact inner soles of human feet (n=11) without any notable skin irritation. Our studies revealed changes in the type and total amount of bacterial genera, as illustrated by number of reads, indicating how much genetic material was picked up and the diversity, as well as relative amounts of those genera, indicating if a sample is homogenous with a large amount of one or few types of bacteria, or heterogenous with smaller or more equal amounts of several types of bacteria. Further, we performed growth curve assays which allowed us to calculate growth inhibition of the DFI wound microbiome in the presence of an AMP either free or tethered within the ELP. Our studies revealed that the free AMP, LL37 (50uM) reduced the overall microbiome from two of the three patient samples we tested (“USC 1” and “USC 7”) between 45-65% after 12 hours of exposure. We also demonstrated that in liquid phase, 12.5 mg/ml ELP-Pex, our liquid matrix synthesized with an AMP, reduced bacterial growth of these same two subjects by 26-34%. These results show the ability of our novel biosynthetic ELP material, based on naturally occurring AMPs, to alter clinical sample microbial profiles, with varying levels of bacterial growth suppression and composition alteration. Our results suggest that our antimicrobial ELP matrix has potential as an alternative DFI therapeutic and may be preferable over antibiotics for the reduced risk of multi-drug resistance development, natural occurrence in a wide range of organisms, and little to no toxic side effects.