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. SNA-NCs 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.

Fig. 1.

Uptake, safety, and gene suppression efficacy of SNA-NCs in hKCs. (A) Uptake of Cy3-labeled nonsense SNA-NCs (red) was noted in the cytoplasm of approximately 100% of the primary hKCs after 24 h incubation. Blue, Hoechst 33343-stained nuclei. Scale bar, 20 μm. (B) KCs exhibit dose-dependent SNA uptake that is higher than the uptake in the HeLa and HaCaT cell lines. (C) Cellular proliferation during 96 h of treatment with nonsense SNA-NCs is similar to that of control treatment with PBS only. (D) Heat map of cellular gene array study (Dataset S1). (E) Western blot showing EGFR protein levels. hKCs were treated for 48 h with SNA-NCs or DharmaFECT1-delivered siRNA, or with controls of PBS and/or nonsense SNA-NCs. After treatment, growth medium was replaced with fresh medium, and the cells were incubated for an additional 12 h. Cell lysates were then harvested for blotting. Note the greater suppression by 0.01 nM EGFR SNA-NCs (equivalent to 0.3 nM siRNA) as compared with the 30 nM EGFR siRNA delivered with DharmaFECT1 at 60 h. (F) EGFR mRNA suppression after 48 h treatment with EGFR SNA-NCs or DhamaFECT1-delivered EGFR siRNA (treatment terminated after 48 h). The decrease and eventual loss of gene suppression during the subsequent 48 h after termination of treatment was evaluated by RT-qPCR. By 48 h after removal of SNA-NCs from the medium, 50% of the maximum suppression is still observed in EGFR SNA-NC—treated KCs, whereas suppression by DharmaFECT1-delivered EGFR siRNA is no longer evident. All data are expressed as mean ± SD.

Fig. 2.

Penetration and clearance of SNA-NCs in hairless mouse skin. (A) Mouse (SKH1-E) skin treated topically with 1∶1 Aquaphor only (Left) or with 50 nM Cy5-labeled (red) SNA-NCs dispersed in the 1∶1 Aquaphor (Right); 3 h after application, the SNA-NCs are seen in the cytoplasm of epidermal cells and the dermis as well. Blue, DAPI-stained nuclei. Scale bars, 100 μm. (B) Mouse skin was treated daily for 3 d with 50 nM nonsense SNA-NCs; samples were harvested at 24 h to 10 d after treatment discontinuation and analyzed by ICP-MS for gold content. The gold content in mouse skin progressively decreases after cessation of topical treatment; 10 d after the final treatment, only 2% of the original gold content remains (n = 3 at each time point). (C) ELISA cytokine assays for TNF-α, IFN-α, IFN-β, IL-6, and CXCL10 in serum show no stimulation of innate immune responses after 3 wk of thrice-weekly treatment (every other day but Sunday; n = 3 per treatment group). (D) EGFR SNA-NC treatment does not induce the cytokine expression locally in the treated skin site after 3 wk of treatment. Results are presented in pg per 10 mg biopsy.

Fig. 3.

Targeted gene knockdown by SNA-NCs correlates with biological effects in vivo. (A) Studies were performed to assess EGFR knockdown after 3 wk of treatment with EGFR SNA-NCs (three times per week). RT-qPCR analysis showed a 65% reduction in mRNA expression. Hairless mice, untreated or similarly treated with PBS:Aquaphor, nonsense SNA-NCs, or free siRNAs, showed no significant EGFR mRNA knockdown (n = 5 mice in each group at each time point). (B) The protein expression of EGFR was nearly eliminated in the EGFR SNA-NC—treated group, whereas the downstream phosphorylation of ERK was inhibited by 74%; total ERK expression remained constant. (C) The mean thickness of EGFR SNA-NC—treated skin was 40% less than that of control-treated skin (P < 0.001), as measured by computerized morphometry. Epidermal thickness was measured from the top of the stratum granulosum to the basement membrane (arrows) at three equidistant sites. (D) EGFR SNA-NC—treated skin samples show approximately 40% reduction in the percentage of basal layer KCs that stain positively for Ki-67 antigen, a marker of cellular proliferation, in comparison with controls. Scale bars, 100 μm. Data in all figures are expressed as mean ± SD.

Fig. 4.

Penetration and gene knockdown in human skin equivalents. (A) Skin equivalents (EpiDerm; MatTek) treated with a single application of 25 nM Cy5-labeled (red) SNA-NCs or PBS for up to 48 h. Blue, Hoechst 33343-stained nuclei. Note the presence of SNA-NCs throughout the stratum corneum and nucleated epidermis. Scale bar, 50 μm. (B) ICP-MS measurements of gold show time-dependent uptake of the SNA-NCs in the epidermis. (C) The amount of gold in the cell culture medium, representing the number of particles passing through skin equivalents as measured by ICP-MS, increases with time. EGFR mRNA expression measured by RT-qPCR (D) and EGFR protein expression measured by immunoblotting (E) in skin equivalents treated with a single application of 25 nM EGFR SNA-NCs for 60 h demonstrate effective gene knockdown in human skin. Each study was performed at least three times in triplicate. Data are expressed as mean ± SD.