Diabetic foot ulcers (DFUs) that are not effectively treated might ultimately lead to partial or complete lower limb amputations;​ such losses take place every 30 seconds worldwide. DFU’s and related amputations together pose an annual economic burden of up to $15 billion on the global healthcare system. The lack of connective tissue growth factor (CTGF) and insulin-like growth factor (IGF-I) in DFU results in limited matrix deposition and consequently limited tissue repair. In the current thesis, we sought to address the lack of growth factors in DFUs by engineering double emulsion polymeric nanoparticles (NPs) with high affinity and sustained release of CTGF and IGF and study the effect of the synthesized growth factor (GF)-loaded NPs on DFU healing. The double emulsion NPs were made of an alginate (Alg) or the heparin-mimetic alginate sulfate (AlgSulf) core and a polycaprolactone (PCL) shell. The optimal NPs formulation was determined by investigating the effects of sonication time and amplitude, organic solvent evaporation rate and processed volume on the morphology, size, and polydispersity of the NPs using scanning electron microscopy (SEM) and dynamic light scattering (DLS). The protein encapsulation efficiency (EE) and release profile were assessed using bovine serum albumin (BSA) as a model drug via the Lowry protein assay. Toxicity of the synthesized NPs and their effect on signaling pathways were evaluated using trypan blue, MTT and immunoblotting assays. Finally, the effect of NPs on wound healing and the role of RhoA signaling were evaluated using a scratch assay.

The results showed that an increase in the sonication time and amplitude which correspond to an increase in the total delivered energy, significantly reduced the NPs diameter to a minimum of 235.5±25 nm. Similarly, increasing the organic solvent evaporation rate by which particles solidify or decreasing the total processed volume caused a significant decrease in the NPs diameter (P=0.002). The highest BSA (EE) based on Alg or AlgSulf in the inner core combined with PCL or poly(lactic-co-glycolic) (PLGA) in the shell was attained with PCL/AlgSulf (94.6 %, P=0.028). The unloaded PCL/Alg NPs showed no cytotoxic effect for concentrations below 100µg/ml after 72hr (P>0.213) as shown by trypan blue and including 100µg/ml by MTT (P=0.436), and immunoblotting (P>0.646) assays. Moreover, the treatment that caused the most rapid wound closure was the combination of CTGF (250 ng/ml) and highly sulfated AlgSulf (10µg/ml). Finally, western blot analysis of the wound healing assay after 72 hr showed that cells treated with alginates bound or unbound to GFs, expressed fibronectin unlike all other conditions which expressed it only for 24 hr implying that alginates prevented fibronectin degradation with time. Therefore, NPs based on heparin-mimetic sulfated alginates and loaded with CTGF represent a novel approach which can significantly enhance diabetic wound healing. The developed nanoparticles may also be tailored for the delivery of various drugs and growth factors especially given the known high affinity of AlgSulf to all heparin-binding GFs.