SPANosomes as delivery vehicles for small interfering RNA (siRNA)

Abstract

Nonionic surfactant vesicles, or SPANosomes (SPs), comprised of cationic lipid and sorbitan monooleate (Span 80) were synthesized and evaluated as small interfering RNA (siRNA) vectors. The SPs had a mean diameter of less than 100 nm and exhibited excellent colloidal stability. The SP/siRNA complexes possessed a slightly positive zeta potential of 12 mV and demonstrated a high siRNA incorporation efficiency of greater than 80%. Cryogenic transmission electron microscopy (cryo-TEM) imaging of the SP/siRNA indicated a predominantly core-shell structure. The SP/siRNA complexes were shown to efficiently and specifically silence expression of both green fluorescent protein (GFP) (66% knockdown) and aromatase (77% knockdown) genes in breast cancer cell lines. In addition, the cellular trafficking pathway of the SP/siRNA was investigated by confocal microscopy using molecular beacons as probes for cytosolic delivery. The results showed efficient endosomal escape and cytosolic delivery of the siRNA cargo following internalization of the SP/siRNA complexes. In conclusion, Span 80 is a potent helper lipid, and the SPs are promising vehicles for siRNA delivery.

Figure 1. Particle size and colloidal stability of SPs

The particle size (diamond) and size distribution width (error bar) of SPs remained stable for up to 3 weeks after synthesis and storage at 4°C.

Figure 2. Optimization of the SP formulation

SP with 1% TPGS was used to study the effect of nucleic acid/SPANosomes (NA/SP, w/w) ratios on (A) zeta potential ± S.D. (n=3) and particle size ± S.D. of size distribution, and (B) incorporation efficiency ± S.D. of SP/siRNA complexes (n=3). The effect of TPGS percentage on the (C) particle size, zeta potential, and (D) incorporation efficiency of SP/siRNA complexes.

Figure 3. Cryo-TEM photographs of SP/siRNA complexes

The SP with 1% TPGS was complexed with siRNA at the NA/SP ratio of 1/15 for cryo-TEM imaging. Bar=100 nm.

Figure 4. Cytotoxicity of SPs

Vehicle related cytotoxicity of SPs in MDA-MB-231 cells as a function of SP concentration. The cells were treated with increasing SP concentration for 24 h and cell viability was measured by the MTS assay. Equivalent siRNA concentration was calculated by SP concentration (g/L) × 15 / molecular weight of siRNA (g/mol). Values represent the mean ± S.D. (n=3).

Figure 5. GFP silencing activity of the SP/siRNA complexes

(A) GFP silencing activity of the SP/siGFP complexes at the 100 nM siRNA level with different NA/SP ratios. (B) GFP silencing activity of the SP/siRNA complexes with 1% and 5% TPGS in the SP formulation at the 100 nM siRNA level (NA/SP=1/15). (C) GFP silencing activity of the SP/siRNA and LF/siRNA complexes with different siRNA concentrations at the optimal NA/SP ratio (1/15). GFP expression was quantified by flow cytometry analysis. Values represent the mean ± S.D. (n=3).

Figure 6. Aromatase knockdown by the SP/siArom complexes

Aromatase silencing activity of SP and LF was measured in the SKBr-3 cell line at the 40 nM siRNA level. Aromatase activity was determined by ³H₂O release assay. The aromatase activity was normalized to the total amount of DNA to obtain the normalized aromatase level. Values are relative to untreated control and represent the mean ± S.D. (n=3). * p<0.05, ** p<0.01.

Figure 7. Subcellular localization of siRNA

MDA-MB-231 cells on glass coverslips were incubated with complexes containing 100 nM Cy3-siRNA and 2 µM LysoSensor Green DND-189 for 1 h at 37 °C. After treatment, the cells were washed three times with PBS before live-cell imaging on a confocal microscopy. Yellow dots (white arrow) indicated co-localization of Cy3-siRNA and LysoSensor, suggesting the complexes were trapped in lysosomal compartment. Red dots (purple arrow) indicated Cy3-siRNA that had escaped the lysosomes. LysoSensor Green DND-189 is a fluorescent pH indicator that partitions into lysosomes and is only fluorescent at low pH.

Figure 8. Flow cytometry analyses of SP and LF mediated uptake of Cy3-siRNA and MB

Cy3-siRNA and MB transfection were performed as described in Figure 7. After 2 h treatment, MDA-MB-231 cells were washed 3 times by PBS and fixed with 4% formalin. Fluorescent intensity in cells was measured by flow cytometry. (A) The MFI (mean fluorescent intensity) was used to quantify delivery of Cy3-siRNA and MB. (B) The ratio of the MFI of MB to that of Cy3-siRNA was used to determine the extent of cytosolic release of the siRNA by SP and LF. Values are represented as the mean ± S.D. (n = 3). * p < 0.05, ** p<0.01.

Figure 9. Mechanism of SP and LF mediated siRNA transfection

MDA-MB-231 cells were pre-incubated with or without an inhibitor of clathrin-mediated endocytosis (sucrose, 0.4 M), macropinocytosis (LY29004, 50 µM), and caveolae-mediated endocytosis (filipin III, 5 µg/ml) for 1 h at 37 °C. These cells were then transfected with 100 nM SP or LF/FAM-siRNA complexes in the absence or presence of the inhibitors at the same concentration for 1 h. After transfection, the cells were washed 3 times with PBS, fixed and analyzed by flow cytometry. MFI data was normalized to the cells treated without any inhibitor (100% relative MFI).