Supramolecular Protein Stabilization with Zwitterionic Polypeptide–Cucurbit[7]uril Conjugates

Abstract

Protein aggregation is an obstacle for the development of new biopharmaceuticals, presenting challenges in shipping and storage of vital therapies. Though a variety of materials and methods have been explored, the need remains for a simple material that is biodegradable, nontoxic, and highly efficient at stabilizing protein therapeutics. In this work, we investigated zwitterionic polypeptides prepared using a rapid and scalable polymerization technique and conjugated to a supramolecular macrocycle host, cucurbit[7]uril, for the ability to inhibit aggregation of model protein therapeutics insulin and calcitonin. The polypeptides are based on the natural amino acid methionine, and zwitterion sulfonium modifications were compared to analogous cationic and neutral structures. Each polymer was end-modified with a single cucurbit[7]uril macrocycle to afford supramolecular recognition and binding to terminal aromatic amino acids on proteins. Only conjugates prepared from zwitterionic structures of sufficient chain lengths were efficient inhibitors of insulin aggregation and could also inhibit aggregation of calcitonin. This polypeptide exhibited no cytotoxicity in human cells even at concentrations that were five-fold of the intended therapeutic regime. We explored treatment of the zwitterionic polypeptides with a panel of natural proteases and found steady biodegradation as expected, supporting eventual clearance when used as a protein formulation additive.

Activation of Upper Critical Solution Temperature Behaviors of Zwitterionic Poly(l-methionine-g-poly(sulfobetaine methacrylate)m) with a Bottlebrush Structure

“…In contrast, compared to linear MS-70, the degraded pap-MS-70 exhibits a reduction in the degree of aggregation in the aqueous solution but maintains weak UCST-type behaviors. Owing to the impact of abundant charges from side chains belonging to the SBMA units, the degradation of the PMet backbone by papain is incomplete . This conclusion is also supported by SEC traces in Figure S16 and 1 H NMR spectra in Figure S17.…”

“…Owing to the impact of abundant charges from side chains belonging to the SBMA units, the degradation of the PMet backbone by papain is incomplete. 45 This conclusion is also supported by SEC traces in Figure S16 and 1 H NMR spectra in Figure S17. The molecular weight distribution of pap-MS-70 becomes wider than that of MS-70, and the elution time is an intermediate between bottlebrush MS-70 and linear PSBMA-70.…”

“…Variable temperature nuclear magnetic resonance (VT-NMR) was utilized to investigate the chemical environments of the protons. Furthermore, to examine the effect of the zwitterionic bottlebrush structure on the MS-70 (m = 70) solution properties, we used papain to degrade MS-70, 45 and linear PSBMA-70 was synthesized, 11 both of which were used for the comparative study.…”

Noncovalent PEGylation of protein and peptide therapeutics

“…This dictates the need for studying a noncovalent approach with other promising stealth alternatives (Hoang Thi et al, 2020), such as polyoxazolines (Bludau et al, 2017; de la Rosa, 2014) and zwitterionic systems (Blackman et al, 2019; Qu et al, 2022). In fact, a recent publication already describes the attachment of zwitterionic polypeptide structures based on the naturally occurring amino acid methionine to the protein via cucurbit[7]uril conjugation (Clauss et al, 2023). Overall, given the rapid emergence of versatile noncovalent methodologies and their evolving roles in the field of drug delivery and biomaterials (Webber, Appel, Meijer, & Langer, 2016; Webber & Langer, 2017), it is reasonable to expect significant advances in the development of noncovalently enabled stealth technologies in the coming years.…”

Recent Progress in Site-Selective Modification of Peptides and Proteins Using Macrocycles

“…In addition to PEGylation, Kramer et al have illustrated an alternative avenue for achieving supramolecular stabilization of insulins using the conjugates of CB [7] and zwitterionic polypeptides (CB [7]−PMets, Figure 4). 31 Their investigation revealed that the degree of polymerization of the zwitterionic polypeptides played a pivotal role in influencing the inhibitory efficacy, with optimal outcomes achieved when the degree of polymerization exceeded 80-mer. Moreover, the zwitterionic polypeptides were nontoxic to human cells and could be degraded by nonspecific native protease.…”