Poster N6 - Designing End-Functionalized Polymer Conjugates to Control the Spatiotemporal Presentation of Growth Factor-Mimetic Peptides

Introduction: The anisotropic and temporal presentation of growth factors (GFs) guides cell differentiation, migration, and proliferation during development of the native osteochondral (bone-cartilage) tissue.[1] Biomaterials-based approaches to deliver GFs have therefore been used to guide tissue regeneration. Though promising, current approaches for GF delivery have limited spatial and/or temporal control, which results in poor-quality tissue formation. For example, studies have shown that regulating osteogenic and chondrogenic GF signaling promoted stable, organized cartilage formation.[2,3] Delayed presentation of an osteogenic peptide and sustained presentation of an adhesion peptide also improved bone formation compared to other delivery combinations.[4] In addition, GFs have low stability, short half-lives, and rapidly diffuse from the delivery site, limiting the success of GF-based therapies. GF-mimetic peptides provide a more stable, cost-effective alternative to full GF proteins.[5] In this project, we exploited a 3D printing platform developed by our lab to spatially deliver multiple GF-mimetic peptides at desired timepoints in the presence of cells. We achieve this by 3D printing polymer conjugates modified with bioorthogonal groups to spatially functionalize the biomaterial surface during fabrication.[6] Bioorthogonal chemistries offer a powerful tool for spatiotemporal control due to their high efficiency and selectivity under physiological conditions.[7] Here, we introduced peptides that mimic transforming growth factor β-1 (TGFβ-1) and bone morphogenetic protein-2 (BMP-2), which have previously been shown to enhance chondrogenic and osteogenic differentiation, respectively.[8,4] This versatile strategy provides unprecedented control over the spatiotemporal organization of GF-mimicking cues to guide local hMSC response and osteochondral tissue formation.

Materials and

Methods: Peptide Synthesis. Peptides mimicking BMP-2 (BMP2pep; norbornene-GGGKIPKASSVPTELSAISTLYLK(FAM)) and TGFβ-1 (TGFB1pep; DBCO-CGGGLIANAKK(Cy3)) were synthesized using standard Fmoc-solid phase peptide synthesis techniques. Both peptide sequences contained a C-terminal lysine (K) modified with fluorophores (carboxyfluorescein (FAM), cyanine-3 (Cy3)) for tracking the peptides after delivery. Additionally, biorthogonal groups were added on the N-terminus. Norbornene was added to BMP2pep on resin, while TGFB1 was functionalized with dibenzocyclooctyne (DBCO) after cleaving via a thioether bond. Peptides were purified using HPLC. Masses were confirmed using MALDI.
Conjugate Synthesis. Azide- and methyltetrazine-modified conjugates were synthesized based on prior methods.[6] Briefly, poly(caprolactone) diol (PCL, 14 kDa) was reacted with p-maleimidophenyl isocyanate (PMPI) to form PCL-maleimide (PCL-mal). PCL-mal was reacted with azide-poly(ethylene glycol)-amine or methyltetrazine-amine to form PCL-azide and PCL-methyltetrazine, respectively. Synthesis steps were confirmed using 1H NMR.
3D Printing. PCL only inks were prepared by dissolving 370 mg/mL unmodified PCL (80 kDa) in hexafluoroisopropanol (HFIP). Conjugate inks were prepared by dissolving 20 mg/mL PCL-azide or PCL-methyltetrazine conjugates before adding unmodified PCL. Inks were solvent-cast 3D printed into cross-hatched 14-layer scaffolds with a programmed filament spacing of 260 µm and 45 µm between layers. To create multi-functionalized scaffolds, a dual printerhead setup was used in the 3D printer to fabricate scaffolds with alternating filaments printed with PCL-azide and PCL-methyltetrazine.
Fluorescent labelling. PCL and PCL-conjugate scaffolds were labeled by incubating with fluorescent probes (DBCO-Cy3, norbornene-FITC) or fluorophore-tagged peptides (BMP2pep(FAM), TGFB1pep(Cy3)) with and without cells. Fluorescence was compared between functionalized and non-functionalized scaffolds using microscopy and a plate reader.

Results, Conclusions, and Discussions: We successfully designed a biomaterials-based platform that allows us to spatially and temporally present multiple peptides within the same construct. Fluorescently tagged BMP2pep and TGFB1pep bearing biorthogonal groups were successfully synthesized. NMR confirmed successful synthesis of PCL-methyltetrazine and PCL-azide conjugates. We 3D printed PCL scaffolds presenting azide, methyltetrazine, or alternating filaments of each. Autofluorescence of azide and methyltetrazine groups demonstrated spatial control of multiple functional groups within the same construct (Figure 1A). Fluorescence microscopy also showed higher fluorescence intensity on conjugate-containing scaffolds compared to PCL only when scaffolds were tagged using either fluorescent probes or fluorophore-bearing peptides (Figure 1 B-C). Fluorophore-tagged peptides were also delivered in the presence of cells, confirming that we can temporally deliver GF-mimetic peptides during culture. Ongoing and future work includes investigating the effect of TGFB1pep and BMP2pep presentation on mesenchymal stromal cell (MSC) differentiation. Our biomaterial design allows for controlled spatiotemporal delivery of multiple GF-mimetic peptides, which will enable us to guide local cell behavior and regenerate native-like osteochondral tissue.

References. [1] Camacho+ Biomater Sci 9 (20): 6813–6829, 2021. [2] Occhetta+ PNAS 115(18): 4625-4630, 2018. [3] Ng+ PNAS 114(10): 2556-2561, 2017. [4] Fumasi+ Biomaterials Advances 157: 2024. [5] Seims+ Bioconjug Chem 32(5): 861–878, 2021. [6] Camacho+ Biomater Sci. 7(10):4237-4247, 2019. [7] Karver+ Bioconjug Chem 22: 2263-2270, 2011. [8] Beeren+ Biofabrication 15(1): 014106, 2023.

Acknowledgements (Optional): The authors acknowledge Lehigh’s Electron Microscopy and Nanofabrication Facility and Health Science and Technology Facilities. This work made use of the Lehigh University 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 the National Institutes of Health (NIH) through National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) 5R21AR079117.