Abstract
Impaired wound healing in people with diabetes has multifactorial causes, with insufficient neovascularization being one of the most important. Hypoxia-inducible factor-1 (HIF-1) plays a central role in the hypoxia-induced response by activating angiogenesis factors. As its activity is under precise regulatory control of prolyl-hydroxylase domain 2 (PHD-2), downregulation of PHD-2 by small interfering RNA (siRNA) could stabilize HIF-1α and, therefore, upregulate the expression of pro-angiogenic factors as well. Intracellular delivery of siRNA can be achieved with nanocarriers that must fulfill several requirements, including high stability, low toxicity, and high transfection efficiency. Here, we designed and compared the performance of layer-by-layer self-assembled siRNA-loaded gold nanoparticles with two different outer layers—Chitosan (AuNP@CS) and Poly L-arginine (AuNP@PLA). Although both formulations have exactly the same core, we find that a PLA outer layer improves the endosomal escape of siRNA, and therefore, transfection efficiency, after endocytic uptake in NIH-3T3 cells. Furthermore, we found that endosomal escape of AuNP@PLA could be improved further when cells were additionally treated with desloratadine, thus outperforming commercial reagents such as Lipofectamine® and jetPRIME®. AuNP@PLA in combination with desloratadine was proven to induce PHD-2 silencing in fibroblasts, allowing upregulation of pro-angiogenic pathways. This finding in an in vitro context constitutes a first step towards improving diabetic wound healing with siRNA therapy.
Highlights
Diabetes mellitus (DM) is a metabolic chronic disease characterized by hyperglycemia, which results from defective insulin secretion, defective insulin action, or both [1–3]
The size increased to 88 ± 5 nm, while the zeta potential became strongly positive at 41 ± 3 mV
We demonstrate that small interfering RNA (siRNA) can be formulated into a tunable layer-bylayer platform around gold core particles, for which the outer layer can be conveniently modified, which allows tuning of cellular internalization and endosomal escape
Summary
Diabetes mellitus (DM) is a metabolic chronic disease characterized by hyperglycemia (high levels of glucose in the blood), which results from defective insulin secretion, defective insulin action, or both [1–3]. The degradation pathway of HIF-1α is suppressed, allowing dimerization with HIF-1β in the nucleus, and binds to the hypoxia response element (HRE), which promotes transcription of a cascade of genes that enhance oxygen delivery such as multiple angiogenic growth factors [36], cell metabolism [37], proliferation [38], and the recruitment of endothelial progenitor cells [39] (Scheme 1). Together, this indicates that HIF-1α plays a major role in the angiogenesis process, requiring high expression levels in normal wounds under hypoxic conditions for wound healing [40]. This can be accomplished by downregulation of PHD-2, which should result in stabilization, and upregulation of HIF-1α [45,46], which has been proven to improve wound healing in diabetic conditions [47–51]
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