High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics

Abstract

Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

Figure 1

Characterization of PCPS as a delivery carrier for gene silencing agents. (a) SEM images of oxidized porous silicon (pSi) as the starting material and PCPS as the final product. (b) Changes in zeta potential of particles at various stages of fabrication. Results are presented as the mean of five measurements ± standard deviation. (c) Confocal microscopic images of PCPS/siRNA (left panel) and the released Arg-PEI-siRNA polyplex nanoparticles. Red fluorescence was from the Alexa555-conjugated siRNA. (d) Size distribution of released siRNA polyplex nanoparticles measured by dynamic light scattering. (e) Atomic force microscopic image for size analysis of siRNA polyplex nanoparticle.

Figure 2

Loading of siRNA into PCPS and release of siRNA polyplex nanoparticle. (a) Time-dependent loading of siRNA into PCPS. Scramble siRNA was used to test time-dependent loading, and UV absorbance of the supernatant at 260 nm was measured to assess loading capacity. (b) Dose-dependent loading of siRNA into PCPS. (c) Zeta potential changes during siRNA loading into PCPS. (d) Release of siRNA nanoparticle from the carrier. PCPS/Alexa555-siRNA was incubated in PBS with 10% fetal bovine serum. Supernatant was separated from the particles, and was used to measure fluorescent intensity at Ex543/Em590. Results are presented as the mean of five measurements ± standard deviation.

Figure 3

Cellular uptake of PCPS/siRNA and intra-cellular trafficking of Arg-PEI/siRNA polyplex nanoparticles. (a) Time-dependent release of Alexa555-siRNA inside tumor cells. PCPS/Alexa555 siRNA particles (in red) were added into MDA-MB-231 cell culture, and fluorescence from particles was monitored with a confocal microscope on days 1, 7, and 12 after incubation. (b) SEM images of cellular internalization of PCPS/siRNA. Multiple particles were at various stages of cellular entry. (c) Intra-cellular trafficking of Arg-PEI/siRNA polyplex nanoparticles. Arg-PEI/FAM-siRNA polyplex nanoparticles (in green) were added into MDA-MB-231 cell culture. Cells were harvested at the indicated time points, and stained with Lysotracker for late endosomes/lysosomes (in red) and DAPI for nuclei (in blue). Fluorescent images was captured with a confocal microscope.

Figure 4

Knockdown of gene expression in vitro and in murine model of MDA-MB-231 primary tumor. (a) Western blot analyses on knockdown of STAT3 and GRP78 expression in MDA-MB-231 cells following siRNA treatment. Cells were treated with the indicated agents, and harvested 72 hours later for protein analysis. The β-actin level indicates equal protein loading. (b) Knockdown of ATM expression in MCF-7 cells. (c) Accumulation of PCPS/siRNA in primary MDA-MB-231 tumor. Mice bearing MDA-MB-231 primary tumor were administrated with 150 × 10⁶ PCPS/Alexa555 siRNA by tail vein injection. They were sacrificed 6 hours later, and tumor samples were processed for confocal analysis. PCPS/siRNA particles were in red (Alexa555) and highlighted by the white arrows, and nuclei of tumor cells were stained in blue by DAPI. (d) SEM image of PCPS/siRNA in tumor tissue. (e) Knockdown of STAT3-α expression in vivo. Each mouse bearing MDA-MB-231 primary tumor received 150×10⁶ PCPS loaded with 15 μg siRNA by i.v. on day 1. Mice were sacrificed 6 or 10 days later for expression analysis by Western blot. (f) Mammosphere formation efficiency (MSFE) in MDA-MB-231 primary tumor cells treated with PCPS/siRNA. The inserted Western blot result shows gene expression levels in the cells used for the MSFE assay. MSFE results were average from 3 mice/group.

Figure 5

In vitro and in vivo acute toxicity. (a) Raw-264.7 mouse macrophage cells were incubated with the indicated agents for 24 hours, and TNF-α and IL-6 levels in supernatant were measured by ELISA. Results are presented as the mean of three measurements ± standard deviation. b) Changes in levels of selected serum cytokine/chemokine/colony-stimulating factors in post-treatment mice. Blood samples were collected 2 or 24 hours after intravenous dosing of treatment agents. A multiplexed bead-based immunoassay was used to measure levels of the cytokines/chemokines/colony-stimulating factors. Results were the average from 3 mice/group. *p <0.05. **p <0.01.

Figure 6

Analysis on subacute toxicity from PCPS/STAT3 siRNA. Mice (n = 3/group) were treated once a week for four weeks with the indicated agents. They were sacrificed 24 hours after the final treatment, and (a) hematological analysis, (b) blood chemistry analysis, (c) histological analysis were performed to evaluate potential toxicity. ALT, alanine aminotransferase; ALB, albumin; ALKP, alkaline phosphatase; Arg, arginine; AST, aspartate aminotransferase; BUN, blood urea nitrogen; GRAN, granulocytes; HCT, hematocrit; HGB, hemoglobin; LDH, lactate dehydrogenase; LYMPH, lymphocytes; MCH, Mean Corpuscular Hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MONO, monocytes; MPV, mean platelet volume; PBS, phosphate buffer saline; PLT, platelet count; RBC, red blood cells; RDW, red blood cell distribution width; WBC, white blood cells.

Figure 6

Analysis on subacute toxicity from PCPS/STAT3 siRNA. Mice (n = 3/group) were treated once a week for four weeks with the indicated agents. They were sacrificed 24 hours after the final treatment, and (a) hematological analysis, (b) blood chemistry analysis, (c) histological analysis were performed to evaluate potential toxicity. ALT, alanine aminotransferase; ALB, albumin; ALKP, alkaline phosphatase; Arg, arginine; AST, aspartate aminotransferase; BUN, blood urea nitrogen; GRAN, granulocytes; HCT, hematocrit; HGB, hemoglobin; LDH, lactate dehydrogenase; LYMPH, lymphocytes; MCH, Mean Corpuscular Hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MONO, monocytes; MPV, mean platelet volume; PBS, phosphate buffer saline; PLT, platelet count; RBC, red blood cells; RDW, red blood cell distribution width; WBC, white blood cells.

Scheme 1

Schematic illustration of fabrication of PCPS as a delivery carrier for gene silencing agents.