Spatiotemporal Delivery of Growth Factor-Mimetic Peptides

Introduction: : The anisotropic and temporal presentation of growth factors (GF) directs cell differentiation, proliferation, and migration in the native extracellular matrix (ECM).[1] Spatiotemporal control of GF presentation directs organized ECM formation during development.[2,3] For example, cartilage-promoting and bone-promoting GFs guide the formation of osteochondral (OC) tissue, which contains physical and biochemical gradients necessary for joint function.[4] GFs like transforming growth factor β-1 (TGFβ-1) and bone morphogenetic protein (BMP-2) are widely used to induce human mesenchymal stromal cell (hMSC) chondrogenic and osteogenic differentiation, respectively. However, whole GFs have short half-lives and can cause unwanted off-target effects in vivo.[5] GF-mimetic peptides offer stable, low-cost alternatives that can be chemically modified for spatiotemporal control.[6] In this project, we propose a biomaterials approach to regulate the spatiotemporal delivery of TGFβ-1 and BMP-2 mimetic peptides. We achieved this by 3D printing a scaffold with two different end-functionalized polymer conjugates to spatially functionalize the scaffold surface with distinct chemistries during fabrication. The first conjugate included a TGFβ-1 mimetic peptide. The second conjugate was modified with a bioorthogonal group (methyltetrazine; MTz) to tether a BMP2 mimetic peptide bearing the complementary chemistry (trans-cyclooctene; TCO). Prior studies have shown that restricting BMP2 delivery during early stages of hMSC chondrogenesis supports stable cartilage formation.[2,7] Our approach enables us to deliver the BMP2 mimetic peptide at desired times and to specific locations to direct hMSC behavior and promote stable osteochondral tissue formation.

Materials and

Methods: : Peptide Synthesis. Peptides mimicking BMP-2 (BMP2pep; CGGGKIPKASSVPTELSAISTLYLK(biotin)) and TGFβ-1 (TGFB1pep; CYGGGLIANAKK(azide)), were synthesized using standard Fmoc solid phase peptide synthesis techniques. TGFβ1pep and BMP2pep design included a C-terminal lysine (K) modified with azide or biotin, respectively, to fluorescently label each peptide. Additionally, the N-terminal amine of BMP2pep was functionalized with TCO for delivery to the scaffold surface via bioorthogonal chemistries. Peptides were purified using HPLC. Masses were confirmed using MALDI.

Conjugate Synthesis. Poly(caprolactone) (PCL) was modified with MTz and TGFβ1pep based on prior methods.[8] Briefly, PCL diol (14 kDa) was reacted with p-maleimidophenyl isocyanate (PMPI) to form PCL-maleimide (PCL-mal). PCL-mal was reacted with MTz-amine or TGFβ1pep to form PCL-MTz and TGFβ1pep-PCL, respectively. Each synthesis step was confirmed using 1H NMR.

3D Printing Scaffolds. Conjugate inks were prepared by dissolving 20 mg/mL PCL-MTz or TGFβ1pep-PCL conjugates in hexafluoroisopropanol (HFIP) before adding unmodified PCL (80 kDa, 370 mg/mL). Inks were solvent-cast 3D printed into cross-hatched 14-layer scaffolds with a programmed filament spacing of 260 µm. Multi-functionalized scaffolds were created using a dual printerhead setup in which PCL-MTz and TGFβ1pep-PCL inks were extruded independently in alternating-filament patterns.

Fluorescent labelling. TFGβ1pep-containing scaffolds were labeled using a dibenzocyclooctyne (DBCO)-Cy5 probe. TCO and MTz are complementary bioorthogonal groups. TCO-BMP2pep was delivered into the scaffolds containing PCL-MTz by incubating the scaffolds in peptide solution. TCO-BMP2pep was then tagged using streptavidin-FITC. Peptides were delivered with or without cells, and fluorescence of functionalized and non-functionalized scaffolds was compared using fluorescence microscopy.

Results, Conclusions, and Discussions:: We successfully designed a biomaterials-based platform that allowed us to control the spatial and temporal presentation of multiple peptides within the same construct. Mass spec and NMR confirmed successful synthesis of both peptides and the PCL-MTz and TGFβ1pep-PCL conjugates. We 3D printed scaffolds presenting methyltetrazine, TGFβ1pep, or alternating filaments of each using inks containing PCL-MTz and TGFβ1pep-PCL (Figure 1). Fluorescent labeling of TGFβ1pep demonstrated spatial control of the two conjugates within the same construct (Figure 1B). TCO-BMP2pep was temporally delivered via bioorthogonal click chemistry to MTz-functionalized filaments and fluorescently labeled using streptavidin-FITC fluorophore (Figure 1C). We exploited bioorthogonal chemistries and a 3D printing platform developed by our lab to spatially deliver multiple GF-mimetic peptides in the same construct at desired timepoints in the presence of cells. Ongoing and future work includes demonstrating the ability to deliver TGFβ1pep and BMP2pep at different times in the presence of hMSCs to influence their differentiation. Our biomaterial design allows us to control spatiotemporal delivery of multiple GF-mimetic peptides to guide local cell behavior and promote the regeneration of native-like osteochondral tissue.

Acknowledgements and/or References (Optional):: Sources. [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] Lepage+ Tissue Eng Part B Rev. 2019, 25(2):114-125. [5] Seims+ Bioconjug Chem 32(5): 861–878, 2021. [6] Tzavlaki+ Biomolecules 2020, 10(3):487. [7] Fumasi+ Biomaterials Advances 157: 2024. [8] Camacho+ Biomater Sci. 7(10):4237-4247, 2019.

Acknowledgement. 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 made use of Lehigh’s Department of Biological Sciences’ Confocal Microscope. This work was generously supported by the Clare Boothe Luce Program and the National Institutes of Health (NIH) through National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) 5R21AR079117.