HEPARIN-MIMETIC ALGINATE SULFATE/POLYCAPROLACTONE DOUBLE EMULSION NANOPARTICLES FOR ENHANCED INSULIN-LIKE GROWTH FACTOR (IGF-1) DELIVERY AND DIABETIC WOUND HEALING APPLICATIONS

Abstract

Introduction: Diabetic foot ulcers (DFUs) are a complication to diabetes mellitus that cause 85% of all amputations worldwide and pose an annual economic burden of $15 billion. The wound healing process is altered in DFU patients partly due to the deficiency of growth factors (GF) such as insulin-like growth factor-1 (IGF-1). To address the problem of GF deficiency in DFU, we engineered double emulsion nanoparticles (NPs) using heparin-mimetic alginate sulfate (AlgSulf) for the encapsulation and sustained release of IGF-1 to promote wound repair and decrease inflammation in the wound microenvironment. Methods: NPs were synthesized with an AlgSulf core and polycaprolactone (PCL) shell via the solvent evaporation technique. The NPs were characterized for their size, zeta potential, and polydispersity index using dynamic light scattering, and morphology using scanning electron microscopy. IGF-1 encapsulation efficiency (EE) and release rate were analyzed using enzyme-linked immunosorbent assay (ELISA). The NPs’ cytotoxicity was assessed on human keratinocyte (HaCaT) cell line using trypan blue and MTT assays. Cellular uptake of the NPs was evaluated by assessing the fluorescence intensity of HaCaT cells treated with fluorescently labeled Alg or AlgSulf loaded NPs. The ability of NPs to reduce inflammation was measured by quantifying the mRNA and protein expression levels of IL-6 inflammatory marker using qRT-PCR and ELISA. Results: NPs with AlgSulf of a degree of sulfation (DS=2) AlgSulf2.0/PCL had the smallest average size and lowest zeta potential (p<0.05). IGF-1 EE was higher in AlgSulf NPs compared to non-sulfated Alg-based NPs (98.59% ± 0.48 and 97.43% ± 2.83 AlgSulf0.8/PCL and AlgSulf2.0/PCL NPs, respectively compared to 95.56% ± 1.89 for AlgSulf0.0). Bare AlgSulf2.0/PCL NPs showed no cytotoxic effect up to 100 µg/mL after 72 hours as shown by trypan blue and up to 100 µg/mL after 48 hours as shown by MTT. The cellular uptake was significantly increased with time up to 24 hours (p<0.001) for Alg/PCL NPs and showed an increasing trend over 24 hours for AlgSulf2.0/PCL NPs (p=0.352). All NPs showed a similar trend in reducing IL-6 in the inflamed media. Conclusion: Heparin-mimetic IGF-1 loaded AlgSulf NPs represent a novel approach for improving DFUs healing. The developed NPs may also be used for the delivery of other heparin-binding GFs exhibiting broad applications in the therapeutics domain.