Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

RNA interference (RNAi)using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded proteinis very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A <b>poly-l-lysine</b> peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), <i>in vitro</i>. Light-triggered delivery resulted in ∼47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay

AZD6244 induced increase of <i>Bim</i> mRNA levels and Bim protein stabilization in lung cancer cells.

<p>(A) Total RNA was isolated in parallel. Expression of <i>Bim</i> was measured by real-time PCR, and normalized to the level of <i>GAPDH</i>. Data shown are representative of three independent experiments with similar results. <i>Columns</i>, mean; <i>bar</i>, SD. * , <i>P</i><0.05, compared with untreated cells. (B) Calu-6, H2347, H3122 and H196 cells were treated with 30 µM MG132 for 2, 4, 6, and 8 hours and Western blot analysis with Bim expression was performed. (C) Calu-6, H2347, H3122 and H196 cells were treated with DMSO, 3 µM AZD6244, or 30 µM MG132 for 6 hours, and then with 25 µg/ml of cycloheximide to block protein synthesis. Western blot analysis with Bim expression was performed. Data represent one of three independent experiments with similar results.</p

Effect of constitutively active AKT (caAKT) on FOXO3a-mediated Bim expression.

<p>(A) Lung cancer cell lines Calu-6 and H3122 were transfected with an empty retroviral vector or a caAKT-expressing vector. After a brief selection, AKT activity and p-Thr32-FOXO3a and p-Ser-FOXO3a expression were measured in caAKT transfected cells. α-Tubulin was used as a loading control. (B) Expression of Bim and p-FOXO3a were measured in vector- and caAKT-transfected cells after AZD6244 treatment. (C) Dose-response curves are shown for AZD6244 in vector-transfected and caAKT-transfected Calu-6 and H3122 cells. Cells were exposed to increasing concentrations of AZD6244 for 96 hours. Cell viability was determined by SRB. (D) Apoptosis induction by AZD6244. Cells were treated with 3 µM AZD6244 for 72 hours, and apoptosis was analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013026#pone-0013026-g003" target="_blank">Fig. 3C</a>. Numbers represent percentages of apoptotic sub-G₁–phase cells. Data represent one of three independent experiments with similar results. <i>Columns</i>, mean; <i>bar</i>, SD. *, <i>P</i><0.05, compared with the control vector transfected cells. (E) TUNEL assay were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013026#pone-0013026-g003" target="_blank">Fig. 3D</a>. The representative photographs of Calu-6 are shown.</p

The effect of Bim-specific small interfering RNA (siRNA) on AZD6244-induced apoptosis.

<p>(A) Calu-6 and H3122 cells were transfected with Bim-specific or control siRNA and then treated with 3 µM AZD6244 for 48 hours. Expression of Bim, PARP and Caspase-9 were analyzed by Western blotting. (B) Cells were cultured in medium containing various concentrations of AZD6244 for 96 hours. Cell viability was determined by sulforhodamine B, and relative cell viability was plotted as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013026#s2" target="_blank">Materials and Methods</a> section. Values represent mean ± SD of three independent triplicate assays. (C) Parallel cells were fixed with ethanol and stained with propidium iodide; DNA content was analyzed by flow cytometry. Numbers represent percentages of apoptotic sub-G₁–phase cells. Data represent one of three independent experiments with similar results. <i>Columns</i>, mean; <i>bar</i>, SD. * , <i>P</i><0.05, compared with the control siRNA transfected cells. (D) Parallel cells were also fixed for TUNEL and DAPI staining. Apoptotic cell nuclei in TUNEL staining were labeled with FITC and visualized under fluorescence microscopy. The relative apoptotic cells were determined by counting TUNEL positive cells in five random fields (at 100× magnification) for each sample. <i>Columns</i>, mean; <i>bar</i>, SD. *, <i>P</i><0.05, compared with the control siRNA transfected cells. The representative photographs of Calu-6 were shown in the upper panel and the percentage of apoptotic cells of both Calu-6 and H3122 were showed in the lower panel. (E) Calu-6 cells were transfected with BimEL expression vector and control vector for 48 hrs. Expression of Bim was analyzed by Western blotting and apoptotic cells were detected with TUNEL assay.</p

The expression of various Bcl-2 family proteins in lung cancer cells after AZD6244 treatment.

<p>Western blots of Bcl-2 family members after treatment with AZD6244. (A) Human lung cancer cell lines (Calu-6, H2347, and H3122) were treated with 3 µM AZD6244 for 4, 8, 24, 48, and 72 hours. (B) Human lung cancer cell lines (Calu-3, H196, H522, and HCC2450) were treated with 3 µM AZD6244 for 4, 24, and 72 hours. (C) Calu-6, H2347, H196 and H522 cells were treated with 0.03, 0.1, 0.3, 1 and 3 µM of AZD6244 for 24 hours. Data represent one of three independent experiments with similar results.</p

Model depicting the signaling pathways utilized by AZD6244 in lung cancer cells to induced Bim activation and subsequent apoptosis.

<p>Our results suggest that AZD6244-induced up-regulation of Bim is mediated by FOXO3a, which is regulated through both the MAPK/ERK and PI3K/AKT pathways. In sensitive cells, MEK inhibition is sufficient to induce expression of the downstream molecule, Bim, and to induce apoptosis. However, in resistant cells, in which the PI3K/AKT/FOXO3a pathway is constitutively activated, suppression of ERK is insufficient to induce apoptosis because of suppression of Bim expression.</p

Effective <i>in vivo</i> inhibition of tumor growth by TUSC2 systematic restoration and MK2206 combined treatment.

<p>A subcutaneous mouse model of human NSCLC H322 was used to evaluate the combined effect of systemic delivery of the DC–based TUSC2 nanoparticles and MK2206 treatment on tumor growth inhibition. A) Tumor volume was calculated, taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter, using the following formula: length × width²×0.52. Tumor growth inhibition rate was calculated as 100%× (tumor size_treated/tumor size_control) on each measurement day. Bars, SD; B) Tumors were resected, fixed with 4% paraformaldehyde, paraffin-embedded for immunohistochemistry staining with the indicated antibodies, and examined under a Nikon TC200 fluorescence microscope equipped with a digital camera.</p

Inhibition of tumor cell viability and colony formation by TUSC2 transfection and MK2206 combined treatment in LKB1-defective and wild type NSCLC cells.

<p>A) Forty-eight hours post-treatment, cells were assayed for viability as described in Materials and Methods. Cell viability was plotted against concentration of MK2206. B) Cells were transfected with DC-TUSC2. Twenty four hours post-transfection, cells were split, replated in triplicate, and grown in medium containing 400 µg/ml of the antibiotic G418 before treatment with 1 µM MK2206. Colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v), and counted using a stereomicroscope. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, <i>P</i><0.05, compared with EV control; **, <i>P</i><0.05, compared with EV+MK2206</p

Stimulation of AMPK phosphorylation and kinase activity by TUSC2 in LKB1-defective cells.

<p>A) HCC366, H322, and A549 were transfected with TUSC2 then either treated with 1 µM MK2206 for 24 hours or left untreated. AMPK kinase activity in the immunocomplexes was measured by phosphorylation of SAMS peptide as described in Materials and Methods. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, <i>P</i><0.05, compared with EV control; **, <i>P</i><0.05, compared with EV+MK2206. LKB1- defective cells HCC366 and H322 cells were co-transfected with 2 µg TUSC2 plasmid and 50 nM AMPK siRNA with Lipofectamine™ 2000. Twenty-four hours after transfection, cells were starved for 24 hours and treated with 1 µM MK2206 for an additional 48 hours. B) Cell lysis were collected for western blot analysis to assess levels of AMPK and p-AMPK proteins; or C) Cells were assayed for apoptosis as described in Materials and Methods. Columns, mean of three different experiments, each with duplicate samples; bars, SD. ^#, <i>P</i><0.05, compared with TUSC2; ^##, <i>P</i><0.05, compared with TUSC2+MK2206.</p

Inhibition of AKT and mTOR kinase activity by TUSC2 transfection and MK2206 combined treatment.

<p>LKB1-defective HCC366, H322, and A549 were transfected with TUSC2 for 24 hours, starved for 24 hours and either treated with 1 µM MK2206 for 24 hours or left untreated. Cell lysates were collected for western blot analysis for the levels of A) p-AKT(S473) and p-AKT(Th308); and B) p-mTOR(S2448). AKT and mTOR was precipitated from 200 ug cell lysis using A) AKT or B) mTOR antibodies. The kinase activity of AKT and mTOR were measured with KLISA AKT and mTOR assay kit, respectively, using GSK-3α and S6K GST fusion proteins as substrates, respectively. Kinase activities were determined by ELISA, as substrate absorbance was measured at 450 nm, and reference wavelengths were measured at 540/595 nm using a Synergy 2 Multi-detection microplate reader. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, <i>P</i><0.05, compared with EV control; **, <i>P</i><0.05, compared with EV+MK2206.</p