(23c) Enhancing Cell-Based Therapies with Peptide-Modified Thermo-Responsive Citrate-Based Biomaterials for Treatment of Critical Limb Ischemia

Critical limb ischemia (CLI) represents a severe manifestation of lower extremity peripheral artery disease, characterized by inadequate blood flow to the extremities. Conventional treatment approaches, while providing temporary symptom relief, often fail to address the underlying vascular pathology, leading to high rates of morbidity and mortality. Despite the promise of cell-based therapy, the efficacy of such treatments is hindered by challenges like poor cell survival and engraftment during and after cell delivery.

This study delves into the potential of peptide-modified thermo-responsive citrate-based biomaterials as carriers for endothelial cell delivery, aimed at promoting vascular regeneration in CLI. Specifically, we synthesized thermoresponsive poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) and conjugated it with laminin-derived peptide A5G81 or VEGF-mimic peptide QK. Human umbilical vein endothelial cells (HUVECs) were incorporated into these modified polymers, and their in vitro performance was assessed for cell spreading, proliferation, and transcriptome. For in vivo evaluation, HUVECs expressing human sodium iodine symporter (hNIS) were mixed with the polymers and intramuscularly injected into nude mice hindlimbs. Single Photon Emission Computed Tomography with Computed Tomography (SPECT/CT) tracked the survival and distribution of the injected cells using radioisotope 99Tc. Subsequently, a hindlimb ischemia model (male and female nude mice, 12-week old, n=6) gauged the efficacy of cell delivery in the biomaterial carriers by evaluating limb perfusion, tissue loss, functional impairment, and histological examination.

PPCN's unique thermoresponsive behavior allows for easy cell encapsulation and retention at the delivery site. Injection of cells through PPCN, with and without peptide modification, improved cell survival compared to traditional injection methods due to the shear-thinning property of the polymer. Incorporation of A5G81 and QK peptides into PPCN promoted HUVEC spreading and proliferation in vitro, revealing distinct transcriptome profiles compared to cells encapsulated in unmodified PPCN. SPECT/CT imaging demonstrated robust cell survival for up to four weeks when delivered 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, cells delivered with PPCN-A5G81 and PPCN-QK exhibited significantly improved blood perfusion (Figure 1c-e), tissue salvage (Figure 1f,h) and motor functions (Figure 1g,i) compared to controls. Histological analysis confirmed skeletal muscle preservation and vascular regeneration in limbs treated with these modified polymers. Furthermore, endothelial cell delivery with PPCN-A5G81 and PPCN-QK also facilitated positive skeletal muscle remodeling following ischemic injury, indicating their potential to address multifaceted aspects of CLI pathogenesis. These findings underscore the promise of bioactive materials as innovative cell delivery carriers for CLI therapy, with far-reaching implications for advancing regenerative therapeutics and revolutionizing CLI treatment strategies.

Figure 1. Evaluation of cell survival following the intramuscular injection of hNIS-transduced HUVECs combined with PPCN-peptide hydrogels, and examination of limb perfusion after cell implantation with PPCN-peptide hydrogels in immunocompetent mice following femoral artery ligation. (a) Representative SPECT/CT images of immunocompetent mice (n= 4 per group) after intramuscular injection of hNIS-transduced HUVECs mixed with PBS, PPCN, PPCN-A5G81 and PPCN-QK. (b) Quantification of SPECT signal at different timepoints. (c) Representative laser Doppler images for PBS with cells, PPCN with cells, PPCN-A5G81 with cells and PPCN-QK with cells treatment groups in male mice. (d-e) Quantification of blood flow in male (d) and female (e) mice as determined by laser Doppler imaging. (f-i) Quantification of the percentage of tissue damage (f, h) and motor function (g,i) in male (f,g) and female (h,i) mice. The results are presented mean ± SD. (n=6, half male per group, * p<0.05; ** p<0.01; *** p<0.001; ****p<0.0001).