NOVEL ALGINATE AND ALGINATE SULFATE/POLYCAPROLACTONE NANOPARTICLES FOR ENHANCED GROWTH FACTORS DELIVERY IN WOUND HEALING APPLICATIONS

Abstract

Diabetes is a metabolic disorder characterized by hyperglycemia, affecting more than 460 million people worldwide. Uncontrolled diabetes can lead to secondary complications such as diabetic foot ulcers (DFUs) due to peripheral neuropathy and peripheral arterial disease. Non-healing DFUs can progress to gangrenes and may require amputations, affecting 11.2 per 1,000 patients per year and burdening the healthcare system with billions of dollars annually. DFUs are caused, in part, by the deficiency in growth factors (GFs), particularly connective tissue growth factor (CTGF), which disrupts efficient wound healing. Similarly, myocardial infarction (MI), the most severe and prevalent type of cardiovascular disease, is associated with an increased risk of heart failure and an overall worse prognosis in patients with low circulating levels of insulin-like growth factor 1 (IGF1). Exogenous delivery of CTGF and IGF1 can promote complete wound healing of DFUs and improve cardiac remodeling in MI. However, the delivery of GFs is limited by their low stability and short half-life, implicating the need for a nanocarrier to entrap and shield the GFs, promote their controlled release, and enhance their availability at the wound site.

In this thesis, we aimed to develop novel double-emulsion alginate (Alg) and heparin (HN)-mimetic alginate sulfate (AlgSulf2.0)/polycaprolactone (PCL) nanoparticles (NPs). These NPs were designed for the enhanced affinity binding and controlled delivery of CTGF and IGF1 with the ultimate aim of accelerating DFU healing and providing MI cardioprotection. First, we synthesized the NPs by the double emulsion solvent evaporation technique and assessed the NPs’ size, encapsulation efficiency (EE), cytotoxicity, and wound healing capacity in immortalized human adult epidermal cells (HaCaT). The sonication time and amplitude used for NP synthesis produced particles with a minimum size of 236 ± 25 nm. Treatment of HaCaT cells with up to 50 μg/mL of NPs showed no cytotoxic effects after 72 h. The highest bovine serum albumin EE (94.6 %, P = 0.028) and lowest burst release were attained with AlgSulf2.0/PCL NPs. Moreover, cells treated with AlgSulf2.0/CTGF exhibited the most rapid wound closure compared to controls while maintaining fibronectin synthesis. Second, based on the previous promising results, we explored the wound healing potential of CTGF-loaded Alg and AlgSulf2.0/PCL NPs in in vitro and in vivo settings. The NPs’ cytocompatibility, stability, and wound healing activity were assessed on HaCaT, primary human dermal fibroblasts (HDF), and a murine cutaneous wound model. The NPs were biocompatible, and their size was not affected by elevated temperatures, acidic pH, or protein-rich medium. We found that treatment of HaCaT and HDF cells with CTGF-loaded Alg and AlgSulf2.0/PCL NPs, respectively, induced rapid cell migration (76.12% and 79.49%, P < 0.05). Additionally, in vivo studies showed that CTGF-loaded Alg and AlgSulf2.0/PCL NPs resulted in the fastest and highest wound closure at early and late stages of wound healing, respectively (36.49%, P < 0.001 at day 1; 90.45%, P < 0.05 at day 10), outperforming free CTGF. Third, considering the previous encouraging findings, we were motivated to explore the versatile potential of the GF-affinity binding NPs. To this end, we examined the protective role of IGF1-loaded Alg and AlgSulf2.0/PCL NPs on an MI-mimetic cardiac hypoxia/reoxygenation (H/R) injury using rat neonatal cardiomyocytes (NCM). The NPs exhibited controlled release of IGF1 and did not induce any significant toxicity on NCM cells. Moreover, H/R injury led to a significant decrease in NCM cell viability (46.08%, P < 0.01) and metabolic activity (40.82%, P < 0.05), with significant 1.41- and 1.66-fold (P < 0.05) increase in the transcript levels of cell damage and cell survival markers. Conversely, treating NCM cells with IGF1-loaded Alg and AlgSulf2.0/PCL NPs after H/R resulted in more than 40% increase in cell viability outperforming free IGF1, implicating their ability to alleviate cell death post-MI.

Double-emulsion NPs based on Alg or the HN-mimetic AlgSulf represent a viable strategy for enhancing cutaneous wound healing in DFU and alleviating cell death post-MI. These novel polymeric NPs can be further expanded for the delivery of various HN-binding proteins, potentially aiding distinct pathological conditions characterized by GF deficiencies.