Poster N8 - Designing Peptide-Polymer Conjugates for Cell-Mediated Scaffold Degradation

Introduction: Biodegradable polymers are ideal materials for biomaterial scaffolds because they can degrade under physiological conditions. However, these scaffolds typically degrade too slowly or too quickly to adequately support complete tissue formation. Biomaterials for tissue engineering must have a degradation rate that matches the rate of new tissue growth. For example, materials that degrade too slowly can inhibit tissue growth and do not degrade until after the healing process has completed, leaving behind voids in the injury site that compromise long-term success. If the material degrades too fast, it fails to support newly formed tissue at early stages of repair, leading to incomplete tissue regeneration. In this project, we designed a new class of polymer-based conjugates where scaffold degradation is directly linked to new tissue formation via cell-mediated processes. We achieve this by fabricating scaffolds composed of peptide-polymer conjugates that incorporate enzyme-sensitive peptide sequences into the backbone of the polymer. Scaffold degradation is therefore a consequence of cell-mediated processes that occur during tissue formation and remodeling. Moreover, our design offers modularity where we can integrate different peptide sequences to vary degradation rates and cater to different cell types.

Materials and

Methods: Peptide Synthesis and Purification – A fast-degrading sequence (Fast; CK(Cy3)KRVKRRLLMETC) was synthesized following standard Fmoc solid phase peptide synthesis techniques. The first two amino acids, including an Mtt-protected lysine, were coupled manually. The Mtt group was removed to modify the epsilon amine in the lysine side chain with a water-soluble cyanine3 (Cy3) dye to track scaffold degradation. The remaining amino acids were coupled using a CEM Liberty Blue automated microwave peptide synthesizer. A scrambled version of the Fast peptide (ScrFast; CK(Cy3)LTRLMEKRRKVC) was also synthesized following the same procedure to highlight sequence specificity of the degradation process. Both peptides were purified using HPLC and characterized with MALDI-TOF MS to confirm the mass.
Peptide-PCL Conjugate Synthesis – Polycaprolactone (PCL; 25 kDa) was modified with amine-polyethylene glycol (PEG)-maleimide (mal) following an amide coupling process using O-(benzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate. PCL-peptide-PCL (PCL-pep-PCL) conjugates were synthesized via maleimide-thiol conjugation between PCL-mal and the thiol-terminated peptides. An RGDS-PCL conjugate was also prepared using established methods in our lab.[1] Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to confirm each step of the PCL-pep-PCL conjugation.
Scaffold Fabrication – Conjugate-containing inks were prepared by mixing each PCL-pep-PCL (195 mg/mL) with RGDS-PCL (5 mg/mL) conjugates in 1,1,1,3,3,3-hexafluoro-2-propanol. Inks were cast on a glass petri dish and left to dry overnight. Samples were obtained using a 10-mm biopsy punch.

Results, Conclusions, and Discussions: We successfully created PCL-pep-PCL conjugates containing an enzyme-degradable peptide sequence and fabricated them into scaffolds. Fast and ScrFast peptides were synthesized with the correct masses according to MALDI-TOF MS. All peptide-polymer conjugations were confirmed by 1H NMR (Figure 1). Notably, PCL-pep-PCL conjugates were designed with PEG linkers to increase hydrophilicity and improve peptide accessibility. The RGDS-PCL conjugate was also included during fabrication to enhance cell adhesion. Ongoing work involves in vitro studies to monitor scaffold degradation in the presence of collagenase or fibroblasts. Future work will expand this versatile strategy to incorporate different peptide sequences. For example, we will integrate peptides sequences that degrade by specific cell types during osteogenic differentiation to link scaffold degradation to bone formation. We will characterize scaffold degradation profiles in vitro with different cell types to demonstrate cell-specific degradation and optimize scaffold design.

Reference: [1] Camacho, P., Busari, H., Seims, K.B., Schwarzenberg, P., Dailey, H.L., & Chow, L.W. (2019). 3D printing with peptide-polymer conjugates for single-step fabrication of spatially functionalized scaffolds. Biomater. Sci., 7, 4237-4247.

Acknowledgements (Optional): The authors acknowledge Lehigh University’s NMR Facility. The Bruker Neo 500 MHz NMR was acquired through NSF-MRI-1725883, with additional support from Lehigh University. This work was generously supported by a Collaborative Research Grant from Lehigh University awarded to LWC and ETP.