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High Serum Stability of Collagen Hybridizing Peptides and Their Fluorophore Conjugates.

ABSTRACT: Collagen hybridizing peptides (CHPs) have a great potential for use in targeted drug delivery, diagnostics, and regenerative medicine due to their ability to specifically bind to denatured collagens associated with many pathologic conditions. Since peptides generally suffer from poor enzymatic stability, resulting in rapid degradation and elimination in vivo, CHP's serum stability is a critical parameter that may dictate its pharmacokinetic behavior. Here, we report the serum stability of a series of monomeric CHP derivatives and establish how peptide length, amino acid composition, terminal modification, and linker chemistry influence their availability in serum. We show that monomeric CHPs comprised of the collagen-like Gly-Pro-Hyp motif are resistant to common serum proteinases and that their stability can be further increased by simple N-terminal labeling which negates CHP's susceptibility to proline-specific exopeptidases. When fluorescent dyes are conjugated to a CHP via maleimide-thiol reaction, the dye can transfer from CHP onto serum proteins (e.g., albumin), resulting in an unexpected drop in signal during serum stability assays and off-target accumulation during in vivo tests. This work is the crucial first step toward understanding the pharmacokinetic behavior of CHPs, which can facilitate the development of CHP-based theranostics.

SUBMITTER: Bennink LL

PROVIDER: S-EPMC8063002 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Degenerative disc disease (DDD) is accompanied by structural changes in the intervertebral discs (IVD). Extra-cellular matrix degradation of the annulus fibrosus (AF) has been linked with degeneration of the IVD. Collagen is a vital component of the IVD. Collagen hybridizing peptide (CHP) is an engineered protein that binds to degraded collagen, which we used to quantify collagen damage in AF. This method was used to compare AF samples obtained from donors with no DDD to AF samples from patients undergoing surgery for symptomatic DDD. Fresh AF tissue was embedded in an optimal cutting temperature compound and cryosectioned at a thickness of 8 μm. Hematoxylin and Eosin staining was performed on sections for general histomorphological assessment. Serial sections were stained with Cy3-conjugated CHP and the mean fluorescence intensity and areal fraction of Cy3-positive staining were averaged for three regions of interest (ROI) on each CHP-stained section. Increases in mean fluorescence intensity (p = 0.0004) and percentage of positively stained area (p = 0.00008) with CHP were detected in DDD samples compared to the non-DDD samples. Significant correlations were observed between mean fluorescence intensity and percentage of positively stained area for both non-DDD (R = 0.98, p = 5E-8) and DDD (R = 0.79, p = 0.0012) samples. No significant differences were detected between sex and the lumbar disc level subgroups of the non-DDD and DDD groups. Only tissue pathology (non-DDD versus DDD) influenced the measured parameters. No three-way interactions between tissue pathology, sex, and lumbar disc level were observed. These findings suggest that AF collagen degradation is greater in DDD samples compared to non-DDD samples, as evidenced by the increased CHP staining. Strong positive correlations between the two measured parameters suggest that when collagen degradation occurs, it is detected by this technique and is widespread throughout the tissue. This study provides new insights into the structural alterations associated with collagen degradation in the AF that occur during DDD.

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High Serum Stability of Collagen Hybridizing Peptides and Their Fluorophore Conjugates.

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