Prolonged survival of injected cells via peptide-modified thermoresponsive citrate-based biomaterials

Introduction:: Atherosclerotic peripheral arterial disease (PAD) affects over 200 million people globally, with at least 8.5 million affected in the US. Critical limb ischemia (CLI), a severe form of PAD, is associated with high rates of limb amputation and cardiovascular events.^[1] Cell-based therapies for PAD and CLI have undergone extensive clinical investigation, but acute donor cell death has limited their clinical translation.^[2] Citrate-based biomaterials (CBBs) show promise for therapeutic cell delivery,^[3] and extracellular matrix-memetic peptides such as fibronectin-derived RGD and laminin-derived A5G81 can support cell adhesion and proliferation.^[4] This study aims to engineer delivery vehicles for cell-based therapies to enhance blood perfusion and promote muscle regeneration in PAD. This will be achieved through synthesis and characterization of various peptide-modified, citrate-based thermoresponsive hydrogels and evaluate their chemical composition, rheological properties, and capacity to promote endothelial cell proliferation, angiogenesis, and inhibit oxidative stress in the hindlimb ischemia model.

Materials and Methods:: Poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) was synthesized by reacting citric acid with polyethylene glycol, glycerol 1,3-diglycerolate diacrylate via thermal condensation, and subsequent free radical polymerization with N-isopropylacrylamide. Various pro-survival peptides, such as fibronectin-derived peptide RGD, laminin-derived peptide A5G81, YIGSR, IKVAV, and VEGF-mimic peptide QK, were conjugated to the PPCN scaffold by functionalizing PPCN with N-β-maleimidopropionic acid hydrazide (BMPH) and reacting it via “Click” chemistry with a peptide that contains a terminal cysteine. The materials were characterized with ¹H NMR and FT-IR spectroscopy. Tubulogenic properties were evaluated by tubule formation assay. HUVECs were seeded on the surface of peptide-modified PPCN hydrogel. After 4 hours of incubation, the cell morphology was visualized by fluorescence microscopy. Endothelial cell viability and proliferation were studied by encapsulating HUVECs in hydrogel scaffold and incubated at 37°C in 5% CO₂. After 3 days and 6 days of incubation, the PicoGreen dsDNA assay was employed to measure the cell number. To evaluate efficacy and safety in vivo, hindlimb ischemia was induced in immunocompetent NU/NU nude mice and material was intramuscularly injected into the injured area using 28G insulin syringe. Following surgery, the blood flow was monitored by laser Doppler imaging (LDI). To monitor the survival of transplanted cells in living mice, human sodium iodide symporters (hNIS)-transduced endothelial cells were encapsulated in peptide-modified PPCN hydrogels, and the cell viability was tracked using Single Photon Emission Computed Tomography (SPECT/CT) imaging in NU/NU nude mice. Tissue samples from the injured area were collected after 6 weeks for histology and immunohistochemistry analysis.


Results, Conclusions, and Discussions:: We first aimed to assess various PPCN-peptide hydrogels on endothelial cell survival and proliferation. The cell morphology was visualized by seeding HUVECs on the surface of different hydrogels. HUVECs exhibited a well-spread morphology on the surface of PPCN-A5G81 and PPCN-QK hydrogels after 4 hours of incubation, whereas they appeared round on the surface of PPCN, PPCN-RGD, PPCN-IKVAV and PPCN-YIGSR (Figure 1a). HUVECs were further encapsulated in PPCN-peptide hydrogels and incubated in 3D for 3 and 6 days. The enhanced cell proliferation was observed in PPCN-A5G81 and PPCN-QK groups compared to PPCN, PPCN-RGD, PPCN-IKVAV and PPCN-YIGSR hydrogels (Figure 1b and 1c). These results suggest that PPCN-A5G81 and PPCN-QK promote endothelial cell spreading and proliferation in 3D culture. We next evaluate the effect of PPCN-A5G81 and PPCN-QK alone in a murine model of hindlimb ischemia, with PPCN and PBS as negative controls. Mice that received material alone did not show significant improvement in blood perfusion but also did not experience any negative effects, indicating the biocompatibility of these materials in the hindlimb ischemia model (Figure 2). The in vivo cell survival was evaluated by SPECT/CT imaging. HUVECs delivered with PBS, PPCN and scrambled PPCN-A5G81 and PPCN-QK demonstrated poor cell survival, with over 90% cell loss by day 7. In contrast, cells mixed with PPCN-A5G81 and PPCN-QK showed robust levels of SPECT signal for up to four weeks following delivery in immunocompetent mice, suggesting the pro-survival effect of PPCN-A5G81 and PPCN-QK (Figure 3).

The peptide-modified PPCN pro-survival biomaterials were designed to enable cell engraftment in vivo by combining the advantages of antioxidant thermoresponsive citrate-based biomaterials with the immobilization of pro-survival peptides. Our findings demonstrate that PPCN-A5G81 and PPCN-QK promote endothelial cell spreading and proliferation as well as prolonged survival of transplanted endothelial cells in vivo. These results suggest that further investigation of cell delivery in a hindlimb ischemia model is warranted, which could lead to the development of more effective cell-based therapies for PAD.


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