Characterization of cationic siRNA-liposome complexes. A, loading of fluorescently tagged siRNA into nanoliposomes. Fluorescently tagged siRNA was complexed with cationic nanoliposomes at ratios of 1:5, 1:10, or 1:15 for 0.5, 3, or 6 h and run on a 2% agarose gel to determine loading efficiency. Maximal loading was reached at a 1:10 ratio following a 0.5-h incubation (top left). siRNAs at a 1:10 ratio with nanoliposomes were sized using dynamic light scattering and similar size ranges were observed for ghost or nanoliposomes loaded with siRNA (top right). siRNA protection by cationic nanoliposomes was measured by complexing fluorescent siRNA with nanoliposomes overnight followed by exposure to serum for 10, 30, 60, 180, or 360 min. Free fluorescent siRNA alone was used as a control (bottom left and middle). Release of siRNA in nanoliposomes was accomplished by collapsing serum-treated siRNA-nanoliposomal complexes with SDS to release free siRNA, which was then run on an agarose gel (bottom right). B, siRNA-nanoliposomal complex is taken up into normal cells. Uptake of siRNA-nanoliposomal complex into normal cells was measured by adding fluorescently tagged siRNA-nanoliposomal complex (200 nmol/L) to fibroblasts, keratinocytes, and melanocytes for 3 h followed by fixation and imaging by fluorescence microscopy (magnification, ×400). Ghost nanoliposomes lacking fluorescent siRNA were used as a control. C, siRNA-nanoliposomal complex is taken up into the cytoplasm of cells and is not merely surface bound. siRNA-nanoliposomal complex localization in cells following treatment with siRNA-nanoliposomal complexes (100 nmol/L) was ascertained by exposing 1205 Lu melanoma cells for 3 h, after which cells were trypsinized to remove surface-bound complex and replated overnight onto coverslips. Cells were fixed and imaged by fluorescence microscopy (magnification, ×400). D, ghost liposome or siRNA-nanoliposomal complex exerted negligible toxicity on normal or cancer cells. Cellular toxicity of ghost liposome and siRNA-nanoliposomal complex was measured by adding nanoliposomes (12.5, 25, and 50 μmol/L) to fibroblasts, keratinocytes, melanocytes, or melanoma cells for 24 h followed by MTS assay analysis. Untreated cells (−) served as controls for comparison. Columns, mean; bars, SE.
siMutB-Raf decreases protein expression of V600EB-Raf. A and B, siRNA can be designed to decrease V600EB-Raf expression but not normal protein expression. To verify specificity of siMutB-Raf for decreasing expression of mutant but not wild-type protein, melanoma cells containing mutant (UACC 903; A) or normal (C8161.Cl9; B) B-Raf protein were nucleofected with buffer, siScrambled, siC-Raf, siWTB-Raf, or siMutB-Raf siRNA; protein lysates were harvested 48 h later and analyzed by Western blot analysis for B-Raf and C-Raf knockdown. Erk2 served as a loading control. C, siMutB-Raf-nanoliposomal complex decreased the expression of V600EB-Raf protein in cells. 1205 Lu cells were exposed to siMutB-Raf or siScrambled-nanoliposomal complex; protein lysates were harvested at 18 and 32 h and analyzed by Western blot analysis. Erk2 served as a control for protein loading. Columns, mean; bars, SE. D, duration of V600EB-Raf protein knockdown following exposure to siRNA targeting mutant protein is beyond 8 d. Cells were nucleofected with C-Raf or V600EB-Raf siRNA and replated in culture dishes; protein was harvested 2, 4, 6, and 8 d later to measure duration of protein knockdown. Untreated cells or cells nucleofected with siRNA targeting C-Raf served as controls.
Ultrasound treatment permeabilizes skin, enabling melanocytic lesions to take up siRNA-nanoliposomal complexes. A, ultrasound assembly. A lightweight, low-profile ultrasound array was constructed using four-cymbal transducers, which were connected in parallel and encased in polymer. The temporal peak intensity was determined in a spatial plane 1 mm from the face of the transducer for exposure conditions. B, ultrasound treatment does not damage skin. Laboratory-generated skin was ultrasound treated followed by addition of ghost liposome, skin sectioned, and H&E stained. No changes in cellular structure or skin morphology were observed compared with untreated control skin (magnification, ×200). C and D, following ultrasound treatment of skin, siRNA-nanoliposomal complex is taken up by melanocytic lesions in the epidermis and at the epidermal-dermal junction. Following 20-min ultrasound treatment, fluorescent siRNA-nanoliposomal complex or ghost nanoliposomes were applied topically onto reconstructed skin. One hour later, skin was fixed and analyzed by fluorescence microscopy looking down at the skin (magnification, ×4; C) or by cross sections (magnification, ×400; D). Fluorescence (red) indicating the presence of siRNA-nanoliposomal complex was evident in melanocytic lesions in both epidermis and at epidermal-dermal junction (arrows).
Ultrasound treatment followed by topical application of siMutB-Raf-nanoliposomal complex inhibits melanocytic lesion development in reconstructed skin. A, schematic showing treatment regimen. Beginning on day 10 and on alternate days thereafter up to day 20, reconstructed skin was treated with ultrasound for 20 min followed by topical administration of siMutB-Raf-nanoliposomal complex (100 pmol) or ghost nanoliposomes. B and C, ultrasound followed by addition of siMutB-Raf-nanoliposomal complex decreases melanocytic lesion development in skin. Reconstructed skin containing UACC 903-GFP or WM35-GFP cells was treated with ultrasound for 20 min followed by topical administration of siMutB-Raf-nanoliposomal complex on alternate days from days 10 to 20. Skin was harvested on day 21, and the average area occupied by GFP-tagged tumors was calculated for each group. Ultrasound treatments followed by exposure to ghost nanoliposomes served as a control. Columns, mean; bars, SE.
siMutB-Raf inhibits melanocytic lesion growth in reconstructed skin. A, targeting melanocytic lesions using siRNA against V600EB-Raf decreases melanocytic lesion development in laboratory-generated skin. The effectiveness of siRNA targeting V600EB-Raf for decreasing cutaneous tumor development was established by nucleofecting GFP-tagged WM35 cells with buffer, scrambled siRNA, or siRNA targeting C-Raf or V600EB-Raf (100 pmol). Cells were then seeded into laboratory-generated skin at time of creation and, 10 d later, average area occupied by green melanocytic lesions was quantified. Top, a statistically significant reduction in green fluorescent lesions was observed following siMutB-Raf treatment (P < 0.05, one-way ANOVA). Columns, mean; bars, SE. Bottom, protein lysates harvested from cells were analyzed by Western blot for B-Raf, C-Raf, pMek1/2, pErk1/2, and cyclin D1 protein expression. Erk2 served as a control for protein loading. B, siRNA-mediated inhibition of V600EB-Raf protein expression in GFP-tagged UACC 903 cells decreases lesion formation in skin reconstructs. UACC 903-GFP cells were nucleofected with siScrambled or siB-Raf (12.5 or 50 pmol) and cells were seeded into laboratory-generated skin at time of creation. Reconstructed skin was analyzed by fluorescence microscopy 10 d later and area occupied by developing GFP lesions was quantified. Columns, mean; bars, SE. C, inhibition of V600EB-Raf decreased MAP kinase signaling in UACC 903-GFP cells. UACC 903-GFP cells were nucleofected with buffer, siScrambled, or siB-Raf (50 pmol) and harvested at 48 h for Western blot analysis. Westerns were probed with B-Raf, pMek1/2, pErk1/2, and cyclin D1 to show decreased MAP kinase pathway signaling. Erk2 served as a loading control. D, mechanistically, siRNA-mediated targeting of V600EB-Raf protein decreased the proliferative capacity of cells. Cultured UACC 903 melanoma cells treated with siMutB-Raf had an increased doubling time, indicating that cells were proliferating at a slower rate (left). Quantifying proliferating cells showed a 2- to 3-fold decrease following siMutB-Raf treatment of tumor cells compared with size- and time-matched tumor controls treated with siRNA to C-Raf (middle and right). Columns, mean; bars, SE.
Ultrasound treatment followed by topical application of siMutB-Raf-nanoliposomal complex alone or in combination with siAkt3-nanoliposomal complex inhibits melanocytic lesion development in animal skin. A, schematic showing treatment regimen. Ultrasound treatment followed by topical application of siMutB-Raf-nanoliposomal complexes that was started the day after injection of melanoma cells and continued on alternate days up to day 23. During the procedure, anesthetized mice were treated with ultrasound at the injection site for 15 min followed by topical application of siMutB-Raf-nanoliposomal complex. B, ultrasound treatment followed by topical application of siAkt3-liposomal complex + siMutB-Raf-liposomal complex decreased melanoma development in animal skin. UACC 903-GFP cells (1 × 106) were injected s.c. into nude mice and, after 24 h, tumors forming at injection sites were treated on alternate days with ultrasound for 15 min followed by topical administration of siMutB-Raf-liposomal complex, siAkt3-liposomal complex, or siAkt3-liposomal complex + siMutB-Raf liposomal complex. Tumors were measured on alternate days beginning on day 3. Control mice were ultrasound treated followed by addition of siScrambled-liposomal complex. Ghost liposomes were added to single treatments so that mice were treated with equivalent amounts of liposomal vehicle. Statistically significant differences between control and siMutB-Raf-liposomal complex + siAkt3-liposomal complex–treated tumors were observed beginning on day 11 (P < 0.05, two-way ANOVA). Columns, mean; bars, SE. Ultrasound treatment followed by topical application of siAkt3-liposomal complex + siMutB-Raf-liposomal complex does not cause a significant change in animal body weight. Animal weights were measured on alternate days beginning on day 1 to determine whether any weight-related toxicity occurred. No significant weight loss was observed between control and experimental groups (P > 0.05, two-way ANOVA; B, inset). Points, mean; bars, SE. C, siMutB-Raf and siAkt3 cooperate to reduce anchorage-independent growth in cell culture. UACC 903 cells were nucleofected with siAkt3 (200 pmol) and siMutB-Raf (1.5, 3, 6, or 12 pmol) in combination and compared with single siRNAs to siAkt3 (200 pmol), siMutB-Raf (1.5, 3, 6, or 12 pmol), siScrambled, or buffer only for the ability to inhibit anchorage-independent growth. D, siAkt3 and siMutB-Raf act additively to inhibit cell viability. Calculation of the combination index (CI) for the combination of siAkt3 and siMutB-Raf showed additive inhibition of cell viability with combination index values between 0.94 and 1.10.