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

Critical limb ischemia (CLI) poses a significant health challenge, marked by severe morbidity and limited treatment options leading to high mortality rates. Despite the promise of cell-based therapy, challenges such as poor cell survival and engraftment during and after cell delivery hinder its efficacy. This study explores the potential of peptide-modified thermo-responsive citrate-based biomaterials as carriers for endothelial cell delivery to promote vascular regeneration in CLI. Specifically, various pro-survival peptides were covalently tethered to poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) to investigate their effect on the delivery of vascular endothelial cells to skeletal muscle tissue. After screening with in vitro and in vivo experiments, laminin-derived peptide A5G81 and VEGF-derived peptide QK were identified to promote endothelial cell spreading, proliferation, and prolonged cell survival when tethered onto PPCN The viscoelastic properties of PPCN provided protection against shear stress induced cell death during injection, while the peptides regulated endothelial cell behavior via distinct molecular pathways. Importantly, intramuscular delivery of endothelial cells with PPCN-A5G81 and PPCN-QK in a murine hindlimb ischemia model resulted in significant improvements in limb perfusion, tissue preservation and functional outcomes. Furthermore, endothelial cell delivery with PPCN-A5G81 and PPCN-QK also promoted positive skeletal muscle remodeling following ischemic injury. These findings underscore the potential of bioactive materials as novel cell delivery carriers for CLI therapy, with implications for advancing regenerative therapeutics and revolutionizing CLI treatment strategies. Overall design: Extraction of RNA was carried out utilizing the Aurum™ Total RNA Mini Kit (Bio-rad), following the manufacturer's protocol. The quality of the extracted RNA was assessed using the Agilent Bioanalyzer 2100, with acceptance criteria for RNA integrity numbers (RIN) set at > 7. Quantification was performed using Qubit. To construct RNA sequencing libraries, Illumina TruSeq mRNA Sample Preparation Kits were employed, adhering to the manufacturer's instructions. The procedure involved the capture of polyadenylated mRNAs from total RNA through oligo-dT selection. Subsequently, cDNA synthesis occurred through reverse transcription, and each sample was ligated to Illumina sequencing adapters featuring unique barcode sequences. Following this, barcoded samples underwent PCR amplification, and the resulting cDNA libraries were quantified using qPCR. To achieve a comprehensive overview, equimolar concentrations of each cDNA library were pooled and sequenced on the Illumina HiSeq 4000. Subsequently, the quality of reads in FASTQ format was evaluated using FastQC. Trimming of reads, specifically removing Illumina adapters from the 3' ends, was executed using cutadapt. Trimmed reads were aligned to the human genome (hg38) using STAR. Read counts per gene were computed utilizing htseq-count alongside a gene annotation file for hg38 obtained from Ensembl. Normalization and differential expression analyses were conducted using DESeq2 1.42.0, employing the Wald test. The criterion for identifying significantly differentially expressed genes was an FDR-adjusted p-value less than 0.05, employing the Benjamini-Hochberg method. Genes were annotated with biomaRt 2.58.0 using GRCh38.p14 human genome. Gene set enrichment analysis was done using clusterProfiler 4.10.0 with gene sets c2.all.v2023.2.Hs.symbols, h.all.v2023.2.Hs.symbols, and c5.all.v2023.2.Hs.symbols.