Poster U11 - Extended delivery of UPI peptide via microparticles to enhance endothelization

Introduction: Tissue-engineered vascular grafts (TEVGs) are a promising alternative for coronary and peripheral artery bypass, but limited endothelialization and graft stenosis are still challenges. Our prior research showed that epsin-mimetic UPI peptides can promote endothelial cell (EC) proliferation by preventing vascular endothelial growth factor receptor 2 (VEGFR2) internalization, and incorporation within TEVGs allowed rapid endothelialization and 100% patency at 1 week. However, we hypothesize that local, more sustained release from microparticle carriers is important for longer-term grafting outcomes. Thus, the goals of this study are to develop microparticle-incorporated luminal coatings for more extended delivery of UPI peptides and to assess their effects on graft endothelialization and remodeling.

Materials and

Methods: The UPI peptide was entrapped within poly (DL-lactide-co-glycolide) (PLGA) microparticles using a water/oil/water double emulsion technique with 2 mg/g peptide/PLGA. After lyophilization, peptide release in saline was evaluated via a BCA assay. These microparticles were integrated into a luminal gelatin coating within electrospun polycaprolactone (PCL) conduits using a custom mold. SEM imaging characterized the individual microparticles and final graft structure. Human umbilical vein ECs (HUVECS) were used to assess the effects of exogenous UPI peptide on cell migration (scratch test, 0-18 h intervals) and on cell attachment to PCL spincoated films 1 day after seeding. Scrambled peptides (control group) were also tested to determine the specificity of the UPI response. Further, the specificity of the iRGD sequence of the peptide for ECs was tested by comparing the response of smooth muscle cells (SMC) and normal human dermal cells (NHDF). Immunofluorescence and real time-PCR was performed to characterize cell number and phenotype in vitro. Finally, end-to-end grafting in rat infrarenal aortae is being performed for 4 weeks prior to graft characterization with histology and immunofluorescence. One-way ANOVA and Tukey comparisons were performed to assess significance.

Results, Conclusions, and Discussions: HUVECs migrated significantly further with exogenous 100 µg/mL UPI peptide compared to scrambled peptide and culture media only (n=3), demonstrating specificity. Preliminary results also appear to show that incorporating UPI peptide improves EC organization at day 1 and 3 of culture compared to scrambled peptide (n=3), with dense areas of circular organization observed. For scrambled peptides, the individual cells appeared more rounded at this dose. Further, the 3 day culture appeared to show higher cell confluency for the UPI peptide condition. Spherical microparticles with/without peptide loading were prepared with an average diameter of 6.62±4.07 µm. UPI peptide exhibited an extended release from PLGA microparticles and continued to release over the 28 days tested so far (42% released). Microparticles were incorporated within the gelatin coating, and SEM images of the lumen showed a mostly consistent layer of gelatin with peptide loaded microparticles. This graft was viable in a preliminary results after 4 weeks in vivo with no stenosis, only a thin layer of intimal tissue, and cells and tissue throughout the graft wall. This study analyzing the in vivo impact of the gelatin/microparticle coated grafts both with and without UPI peptides is ongoing. Overall, this study showed that UPI peptides exhibit specific benefits for EC migration and organization not seen with scrambled peptides, which may explain how they improve in vivo TEVG endothelialization. Further, extended and controlled release of UPI peptides can be achieved for PLGA microparticles embedded conduits. This may be important clinically where graft endothelialization takes longer than in rodent models.

Acknowledgements (Optional): This project was funded by American Heart Association grants 18AIREA33960390 24AIREA1195356.