Polymeric micelles containing reversibly phospholipid-modified anti-survivin siRNA: a promising strategy to overcome drug resistance in cancer

Abstract

The discovery that survivin, a small anti-apoptotic protein, is involved in chemoresistance, opens a new scenario to overcome the drug resistance in cancer. It was shown that siRNA can efficiently inhibit the expression of survivin in cancer cells. However, the clinical use of siRNA is still hampered by an unfavorable pharmacokinetic profile. To address this problem, earlier we developed a novel system to deliver siRNA into cancer cells. Namely, we reversibly modified the survivin siRNA with a phosphothioethanol (PE) portion via a reducible disulfide bond and incorporated the resulting siRNA-S-S-PE conjugate into nanosized polyethyelene glycol 2000-phosphatidyl ethanolamine (PEG2000-PE)-based polymeric micelles (PM), obtaining survivin siRNA PM. The activity of these nanopreparations was evaluated by survivin protein down-regulation, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to paclitaxel (PXL). We found a significant decrease of cell viability and down-regulation of survivin protein levels after treatment with survivin siRNA PM in several cancer cell lines. In addition, the down-regulation of survivin by treating cells with survivin siRNA PM, elicited a significant sensitization of the cells to PXL, in both sensitive and resistant cancer cell lines. Finally, we demonstrated successful co-delivery of PXL and survivin siRNA in the same PM leading to superior therapeutic activity compared to their sequential administration. Our results support the use of this new platform for the treatment of the most aggressive tumors.

Keywords: Co-delivery; Multidrug resistance; Paclitaxel; Polymeric micelles; Survivin siRNA.

Figure 1

Incorporation efficiency of the modified siRNA into PM determined by SEC. The degree of the incorporation efficiency was measured by ratio of the area under the peak of free survivin siRNA-S-S-PE, not incorporated in PM, and the siRNA-S-S-PE initially added to PM at the same retention time (tr ca. 10 min). As a reference, plain PM were used.

Figure 2

Viability of different cancer cells. Cells were treated with survivin siRNA PM at a final siRNA concentration of 200 nM in the serum-containing medium for 6 h. Cell viability in the presence of survivin siRNA PM, free survivin siRNA-S-S-PE conjugate, PM containing scrambled siRNA-S-S-PE, and plain PM was followed by Cell Titer Blue assay after 48 h of incubation. **p < 0.01, and ***p < 0.001 values were obtained by comparing survivin siRNA PM to all the other treatments. Results were obtained from three independent experiments in triplicate (n = 9). Mean ± SD.

Figure 3

Survivin protein levels in A2780, MDA-MB231, SKOV3, and SKOV3-tr cells after different treatments. Survivin siRNA was quantified 48 h after the treatment by the ELISA method. The data are expressed as ng of survivin protein per mg of protein. Data = mean ± SD (n =3). **p < 0.01, and ***p < 0.001 values were obtained by comparing survivin siRNA PM to all the other treatments. Results were obtained from three independent experiments in triplicate (n = 9). Mean ± SD.

Figure 4

Chemosensitization of MDA MB-231(A), SKOV3 (B), and SKOV3-tr (C) cells mediated by the pre-treatment with survivin siRNA PM. Cells were incubated with survivin siRNA PM (range of siRNA concentrations from 200 to 17.5 nM) for 6 h. Forty eight hours later, cells were challenged with various concentrations of PXL. The viability of cells was measured 24 h later by the CTB assay. Data = mean ± SD (n =3).**p < 0.01, and ***p < 0.001 values were obtained by comparing survivin siRNA PM pre-treated cells (survivin siRNA PM) to PXL no pre treated cells (no pre-treatment).

Figure 5. Effect of PXL on microtubule stabilization after survivin down-regulation in SKOV3-tr cells

SKOV3-tr cells were pre-treated with 200 nM survivin siRNA PM for 48 h followed by the treatment with 40 nM of PXL for 24 h. Cells were then stained for β-tubulin (green). The nuclei (blue) were stained with DAPI. A–D: Representative images of three independent experiments showing organization of microtubules. Untreated cells (A); cells treated with free PXL for 24h (B); cells treated with survivin siRNA PM for 72 h (C); cells pre-treated with survivin siRNA PM for 48 h followed by treatment with PXL for 24 h (D). Data = mean ± SD (n =3).

Figure 6

The viability of SKOV3-tr cancer cells. The cells were treated with the different formulations at 37°C for 6 h. After 72 h, the cell viability was measured by the Cell Titer Blue assay. Data = mean ± SD (n =3). ***p < 0.001 values were obtained by comparing each treatment to survivin siRNA/PXL PM treated cells.