Enhancing Cell-Based Therapies with Peptide-Modified Thermo-responsive Citrate-Based Biomaterials for Treatment of Critical Limb Ischemia

Introduction: Critical limb ischemia (CLI) poses a significant health challenge, marked by severe morbidity and limited treatment options leading to high mortality rates. Cell-based therapies for CLI have undergone extensive clinical investigation, but acute donor cell death has limited their clinical translation. Citrate-based biomaterials (CBBs) show promise for therapeutic cell delivery, and extracellular matrix-memetic peptides such as fibronectin-derived RGD and laminin-derived A5G81 can support cell adhesion and proliferation. In this study, we modified PPCN with a selection of bioactive peptides with the goal of optimizing endothelial cell delivery for vascular regeneration in CLI. Through a series of in vitro and in vivo experiments, we identified peptides with significant potency to facilitate endothelial cell survival and promote vascular regeneration in a 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. After 3 days and 6 days of incubation, the PicoGreen dsDNA assay was employed to measure the cell number. 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. To evaluate treatment efficacy in vivo, hindlimb ischemia was induced in immunocompetent NU/NU nude mice and materials and cells were intramuscularly injected into the injured area. Following surgery, the blood flow was monitored by laser Doppler imaging (LDI). Tissue loss, functional impairment, and histological examination were also assessed. Tissue samples from the injured area were collected after 6 weeks for histology and immunohistochemistry analysis.

Results, Conclusions, and Discussions: Injection of cells through PPCN, with and without peptide modification, improved cell survival compared to traditional methods due to the polymer's shear-thinning property. Incorporating A5G81 and QK peptides into PPCN promoted HUVEC spreading and proliferation in vitro, revealing distinct transcriptome profiles compared to cells in unmodified PPCN. SPECT/CT imaging showed robust cell survival for up to four weeks with PPCN-A5G81 and PPCN-QK, while PBS and unmodified PPCN showed over 90% cell loss by day 7 (Figure 1a, b). In the hindlimb ischemia model, PPCN-A5G81 and PPCN-QK improved blood perfusion (Figure 1c-e), tissue salvage (Figure 1f, h), and motor functions (Figure 1g, i) compared to controls.
Histological examination of the tibialis anterior (TA) muscle, which experienced severe ischemic injury, showed that female mice had more pronounced damage than males at 36 days post-injury. Severe damage, characterized by significant loss of muscle fibers replaced with adipose-like and fibrotic tissues, was evident in the PBS with cells-treated groups (Figure 2a, b). In contrast, endothelial cell delivery with all hydrogels (PPCN, PPCN-A5G81, and PPCN-QK) improved muscle recovery post-ischemic injury, with the PPCN-QK endothelial cell delivery group showing the most robust recovery. This was marked by the emergence of larger, more mature muscle fibers, a rightward shift in the histogram of muscle fiber size distribution (Figure 2c, d), increases in average muscle fiber diameter (Figure 2e, g), and reduced centralized nuclei, indicating superior muscle regeneration (Figure 2f, h). Additionally, fibrosis (Figure 2i, j) and adipose-like tissue infiltration (Figure 2k, l) were notably reduced in PPCN-A5G81 and PPCN-QK groups compared to PBS groups.
In summary, peptide-modified PPCN biomaterials enhance cell engraftment for CLI therapy. Integrating antioxidant thermoresponsive citrate-based biomaterials with pro-survival peptides, specifically PPCN-A5G81 and PPCN-QK, enhanced endothelial cell spreading, proliferation, and prolonged survival in vivo. These biomaterials protected against cell death during injection and regulated endothelial cell behavior post-injection, resulting in significant improvements in blood perfusion and regenerative outcomes. PPCN-A5G81 and PPCN-QK also promoted skeletal muscle recovery following severe injury, highlighting their potential in comprehensive tissue regeneration.

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