Designing Peptide-Polymer Conjugates for Cell-Mediated Scaffold Degradation

Introduction: Biodegradable polycaprolactone (PCL)-based scaffolds for tissue engineering do not degrade at the same rate as new tissue formation. For example, bone regeneration typically occurs within 6 months, while PCL persists for over 1-3 years in vivo. This significant mismatch can lead to voids because tissue formation is completed before the remainder of the scaffold degrades. Ideally, scaffold degradation occurs concurrently with and in response to biological events that occur during new tissue formation, such as cells secreting proteases to remodel the extracellular matrix (ECM). This project focuses on developing solid peptide-PCL conjugate scaffolds with protease-sensitive peptides within the conjugate backbone to direct cell-mediated degradation.

Materials and Methods: Peptide synthesis and conjugation—A fast-degrading peptide, (CKRVKRRLLMETK(biotin)C) and non-degrading scrambled peptide with the same amino acid residues (CLTRLMEKRRKVK(biotin)C) were synthesized using Fmoc solid phase peptide synthesis. Fmoc-Lysine(Mtt)-OH and biotin were manually coupled to the resin before adding the remaining amino acids using a microwave-assisted automated peptide synthesizer. Another sequence, CGGGRGDSK(biotin), was synthesized using the same techniques. Peptides were cleaved from the resin and purified using high-performance liquid chromatography. Conjugate synthesis—PCL (25 kDa) was modified with amine-poly(ethylene glycol)-maleimide using a carbodiimide reaction to form PCL-mal. The fast-degrading and scrambled peptides were reacted with the PCL-mal via Michael addition to make PCL-peptide-PCL. PCL (14 kDa) was modified with p-Maleimidophenylisocyanate to form PCL-mal. The RGDS peptide was reacted with the PCL(14kDa)-mal to make a peptide-PCL-peptide RGDS-PCL conjugate. Nuclear magnetic spectroscopy was used to confirm each conjugation step. Scaffold fabrication—The PCL-peptide-PCL conjugate and the RGDS-PCL conjugate were co-dissolved in hexafluoroisopropanol at a total of 200 mg/mL with 5.13% by weight RGDS-PCL conjugate, and cast into flat disks onto 12 mm diameter glass coverslips. Degradation study—Scaffolds were seeded with fibroblasts and incubated for up to 22 days at 37°C and 5% CO2 . Media was collected and replaced with fresh media every two days. A colorimetric biotin assay was used to measure the amount of biotin released from the scaffolds normalized to the PCL-RGDS group to quantify scaffold degradation.

Results: NMR confirmed successful PCL-peptide-PCL and peptide-PCL-peptide conjugate synthesis. Over the 22-day period, small holes formed in the center of the fast-degrading scaffolds while the scrambled and PCL-RGDS scaffolds were unaffected cumulative biotin release from PCL-peptide-PCL scaffolds normalized to PCL-RGDS showed that the fast-degrading scaffolds released more biotin compared to the non-degrading scrambled scaffolds. Together, these results demonstrate that fibroblasts degrade the fast-degrading scaffolds at a higher rate compared to the scrambled and control scaffolds.

Conclusion: The scaffold with a fast-degrading peptide sequence degraded more quickly in response fibroblasts compared to the scaffold with a scrambled version of the same peptide. These results demonstrated our ability to tune degradation response. This platform introduces a versatile approach to tailor peptide sequences and conjugates for specific cells and cellular events.