Development of peg-mimetic peptides by a combined experimental and computational approach
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Authors
Yang, Jasmine, Rose
Issue Date
2024-12
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
PEG is widely used in drug delivery as a “stealth” polymer, where it acts as a protective coating for biologics and drug nanocarriers to improve stability and blood circulation time. It is typically considered to have high biocompatibility and virtually no immunogenicity. However, studies in the past decade have shown that administering PEG in vivo can cause anti-PEG immunity which can lead to hypersensitivity and anaphylactic shock. Another consequence of this immune response is accelerated blood clearance of PEGylated drugs with repeated use, which actually decreases the efficacy of PEGylation in prolonging circulation time. With the increased use of complex biologics and mRNA vaccines, the problem of anti-PEG immunity is expected to grow. Therefore, our main goal is to develop a new class of biocompatible, biodegradable peptide-based materials that can be used as an alternative to PEG. Recent work in our lab has demonstrated that eADF4(C16), a recombinant protein inspired by spider dragline spidroin, exhibits hydrogel-like properties that shield the surface against bacterial attachment. Thus, we hypothesize that spidroin-inspired sequences can be the intellectual foundation for PEG-mimetic peptides. However, the role of specific sequence motifs and the mechanisms of PEG-like behavior in spidroins is currently unknown, hindering rational design of PEG-mimetic peptides. This project will utilize solid phase peptide synthesis, high throughput assays, and machine learning in an integrated, iterative approach. However, this document will focus on the creation of the peptide library and the development of a high-throughput antifouling assay. This is critical to the overall goal, as antifouling will be used as the metric for PEG-mimetic behavior. In addition, due to the large data set needed to train the machine learning algorithm, high-throughput method needs to be developed to evaluate the peptides for antifouling performance.
Peptides were designed based on 10 amino acids (G, A, L, Q, Y, S, P, R, F, N) using 5 amino acids motifs resulting a sequence space of 105 unique peptides. After filtering sequences, 1000 sequences were selected to be synthesized. Further analysis of GRAVY Index, isoelectric point and amino acid positional preferences confirmed there were no bias between the selected list and filtered lists. For the antifouling assay, several plate-based assays were tested using both CelluSpot Membranes and peptide synthesis resins; however, we were unable to measure fouling using those methods. The imaging-based methods were able demonstrate fouling in both the CelluSpot microarrays and the resin-based systems, however, because the base substrates seem to be anti-fouling, further optimizations need to be conducted for this assay, including finding a higher fouling substrate. The use of kosmotropic and neutral salts had a minimal effect on the fouling, in both plate-reader and image-based systems. Additionally, imaging-based methods demonstrated that sequence charge can significantly impact fouling and thus, this will need further consideration in the assay development and sequence design.
Description
December 2024
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY