Nanoparticle delivery of pooled siRNA for effective treatment of non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death. To explore the potential of small interfering RNA (siRNA) therapy for NSCLC, we have developed anisamide-targeted LCP to efficiently deliver siRNA into the cytoplasm of sigma receptor-expressing NSCLC cells. Targeted LCP demonstrated a 9-fold higher siRNA delivery efficiency compared to nontargeted LCP in A549 cells in vitro. To simultaneously target multiple oncogenic mechanisms, we coformulated three siRNA sequences targeting HDM2, c-myc and VEGF oncogenes, and investigated their efficacy of cell-killing in A549 and H460 cells in vitro. The results indicated that the pooled siRNA codelivered by the targeted LCP could effectively and simultaneously knock down HDM2, c-myc and VEGF expressions and significantly inhibit tumor cell growth. After iv injection of mice bearing A549 xenografted tumor with Texas Red-labeled siRNA formulated in the targeted LCP, siRNA was successfully delivered to and concentrated in the tumor cells. Repeated intravenous injections of mice with pooled siRNA formulated in the targeted LCP significantly impaired NSCLC growth in vivo (p < 0.01) for both A549 and H460 tumors, demonstrating an ED50 for the treatment of ∼ 0.2 mg/kg in A549 tumors. The enhanced antitumor activity is due to the fact that the silencing of HDM2/c-myc/VEGF could inhibit tumor proliferation and angiogenesis and also simultaneously induce tumor apoptosis. Our results demonstrate that the targeted LCP is a promising vector to deliver pooled siRNA into tumors and to achieve multiple target blocking. This is potentially a valid therapeutic modality in the gene therapy of human NSCLC.

Figure 1

(a) Illustration of non-targeted and targeted LCP; (b) TEM images of LCP coated with DOTAP and DSPE-PEG.

Figure 2

Cellular uptake of siRNA in vitro. (a) Fluorescence photographs of cultured A549 cells after treatment with 5′-FAM-labeled oligo in non-targeted or targeted LCP for 4 h. (b) Quantitative measurement of mean fluorescence intensity of cell lysate from A549 cells treated with 5′-FAM-labeled oligo formulated in non-targeted or targeted LCP. A549 cells were incubated at 37 °C for 4 h in the absence or presence of 50 μM haloperidol. Columns, mean (n = 3); bars, SD. *, p < 0.05.

Figure 3

In vitro oncogene silencing. (a) Relative mRNA level after transfection of A549 cells with pooled siRNA in targeted LCP. (b) Comparison of each oncogene silencing efficiency for single siRNA formulated LCP versus pooled siRNA formulated LCP. (c) Western blot analysis of Hdm2 and c-myc after treatment of A549 cells with siRNA in different LCP formulations: 1) untreated; 2) control siRNA in targeted LCP; 3) pooled siRNA in non-targeted LCP; 4) pooled siRNA in targeted LCP. (d) ELISA analysis of VEGF in the A549 cell supernatant after treatment with pooled siRNA in different LCP formulations. Columns, mean (n = 3, in triplicate); bars, SD. *, p < 0.05.

Figure 4

In vitro oncogenes silencing inhibits NSCLC proliferation and induces apoptosis. (a) Cell viability was measured by MTT assay after treatment with different LCP for 48 h. (b) Histogram showing the quantification of apoptotic cells treated with different LCP for 48 h. (c) Representative colony formation assay of NSCLC cells treated with different formulation. 1) untreated; 2) control siRNA in targeted LCP; 3) pooled siRNA in non-targeted LCP; 4) pooled siRNA in targeted LCP. All colonies were stained with crystal violet. (d) Histogram showing the quantification of colony formation efficiency. Columns, mean (n = 3); bars, SD. *, p < 0.05; **p < 0.01.

Figure 5

In vivo cellular uptake of siRNA. (a) In vivo biodistribution of Texas Red-siRNA formulated in non-targeted LCP or targeted LCP; (b) Tumor uptake of Texas Red-labeled siRNA in the non-targeted LCP or targeted LCP. Magnification = ×400.

Figure 6

In vivo oncogenes silencing inhibits NSCLC tumor growth. (a) Tumor growth curve of H460 subcutaneous model. (b) Tumor growth curve of A549 subcutaneous model. (c) Dose response curve on A549 subcutaneous model. ED50=205 μg/kg. Data = mean ± SD, n = 5. *, p < 0.05; **, p < 0.01 comparing to the 5% GS group. H460 tumors were treated every 3 day starting at day 7 for 5 times and A549 tumors was treated every 5 days starting at day 11 for 6 times.

Figure 7

Effects of oncogene silencing on the cell apoptosis, angiogenesis and cell proliferation in vivo. (a) Relative HDM2, c-my and VEGF mRNA level in the H460 tumor tissues. Columns, mean (n = 5); bars, SD; (b) CD31 staining of microvessels in H460 tumor treated with different formulations (original magnification × 200). (c) PCNA-positive nuclei in H460 tumor treated with different formulations (original magnification × 200). (d) TUNEL assay of H460 tumor treated with different formulations (original magnification × 100). Quantitation of the image data is shown in the histograms on the right. *, p < 0.05; **, p < 0.01; ***, p < 0.001.