Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

Abstract

Topical application of nucleic acids offers many potential therapeutic advantages for suppressing genes in the skin, and potentially for systemic gene delivery. However, the epidermal barrier typically precludes entry of gene-suppressing therapy unless the barrier is disrupted. We now show that spherical nucleic acid nanoparticle conjugates (SNA-NCs), gold cores surrounded by a dense shell of highly oriented, covalently immobilized siRNA, freely penetrate almost 100% of keratinocytes in vitro, mouse skin, and human epidermis within hours after application. Significantly, these structures can be delivered in a commercial moisturizer or phosphate-buffered saline, and do not require barrier disruption or transfection agents, such as liposomes, peptides, or viruses. SNANCs targeting epidermal growth factor receptor (EGFR), an important gene for epidermal homeostasis, are >100-fold more potent and suppress longer than siRNA delivered with commercial lipid agents in cultured keratinocytes. Topical delivery of 1.5 uM EGFR siRNA (50 nM SNA-NCs) for 3 wk to hairless mouse skin almost completely abolishes EGFR expression, suppresses downstream ERK phosphorylation, and reduces epidermal thickness by almost 40%. Similarly, EGFR mRNA in human skin equivalents is reduced by 52% after 60 h of treatment with 25 nM EGFR SNA-NCs. Treated skin shows no clinical or histological evidence of toxicity. No cytokine activation in mouse blood or tissue samples is observed, and after 3 wk of topical skin treatment, the SNA structures are virtually undetectable in internal organs. SNA conjugates may be promising agents for personalized, topically delivered gene therapy of cutaneous tumors, skin inflammation, and dominant negative genetic skin disorders.T he recent development of small molecule inhibitors and antibodies that target components of signaling pathways has revolutionized the treatment of cancers, inflammatory diseases, and genetic disorders, including those that largely manifest in skin (1-3). These protein-based therapeutics, however, are costly, have limited targeting ability, and, when delivered through traditional intravenous or gastrointestinal routes, can lead to systemic toxicity (4, 5). Topical delivery is particularly attractive for the therapy of skin disorders. However, proteins larger than a few hundred daltons cannot easily enter the skin, and high concentrations of proteins must be applied for a cutaneous effect (6). An alternative to protein-based pathway inhibition involves the blocking and/or degradation of precursor mRNA before translation into protein. Gene silencing leads to down-regulation of protein expression and functions with greater specificity than inhibitors of protein function (7). In fact, targeted gene suppression by antisense DNA and siRNA has shown promising preclinical results, and/or is currently in clinical trials for a variety of diseases, including many forms of cancer (e.g., melanoma, neuroblastoma, and pancreatic adenocarcinoma), genetic disorders, and macular degeneration (8). For gene su...

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