Porous Silicon Microparticles for Delivery of siRNA Therapeutics

* These authors contributed equally
Bioengineering

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Summary

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

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Shen, J., Wu, X., Lee, Y., Wolfram, J., Yang, Z., Mao, Z. W., Ferrari, M., Shen, H. Porous Silicon Microparticles for Delivery of siRNA Therapeutics. J. Vis. Exp. (95), e52075, doi:10.3791/52075 (2015).

Abstract

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Introduction

Small interfering RNAs (siRNAs) are double-stranded RNA molecules that can suppress the expression of genes. In recent years, siRNAs have been developed as a new generation of biodrugs that show therapeutic potential for future use in clinical applications1-5. However, the successful implementation of siRNA therapy remains a considerable challenge, due to degradation by nucleases, poor intracellular uptake, low transfection efficiency and inefficient release from the endosome/lysosome5. Many of these hurdles can be overcome by the development of delivery platforms, which can safely and efficiently deliver siRNA to diseased tissue. Compared to viral carriers, non-viral platforms provide several advantages, such as safety, low cost and ease of tailoring. In particular, cationic nanoparticles, such as polymers and lipids, have proved useful for siRNA delivery3.

Previously, we have developed a discoidal drug delivery system, termed the multistage vector (MSV). This platform is based on sequential stages, in which one vehicle is released from another. The first stage vehicle is a microparticle made from biodegradable porous silicon (pSi), while the second stage vehicles are nanoparticles loaded with drugs or contrast agents6,7. The nanoparticles, which are embedded in the pSi material, are gradually released as the Si degrades8. A benefit of using Si particles is that the morphology and surface characteristics can easily be tailored to achieve optimal biodistribution and drug release. Recently, the successful use of the MSV platform for the delivery of siRNA liposomes to tumor tissue was shown in an ovarian and breast cancer mouse model9, 10.

In this work, we have fabricated a universal delivery system for siRNA based on the principals of the MSV platform. The efficacy of this delivery system has previously been demonstrated using different siRNA molecules11. The system is a polycation-functionalized porous silicon (PCPS) carrier, consisting of pSi grafted with polyethylenimine (PEI) and arginine (Arg). PEI can aid in forming electrostatic interactions with siRNA, while Arg and pSi can serve to reduce the toxicity of PEI, as previously demonstarted11. In addition, the presence of PEI can assist in intracellular uptake and endosomal escape, while the pSi microparticles enable siRNA protection and sustained release. The pSi particles gradually degrade under physiological conditions, thereby resulting in the formation of Arg-PEI/siRNA nanoparticles (Figure 1), which have a distinct morphology and a narrow size distribution11. For details regarding the stability of the PCPS/siRNA system, please refer to the study by Shen et al.11. This PCPS platform differs from the conventional MSV, since the second stage nanoparticles are not initially present in the carrier, but are formed over time as the first-stage carrier degrades11, 12. The siRNA loading efficiency, cytotoxicity and gene silencing efficiency of the PCPS system has been evaluated in vitro. Transfection efficiency was measured using siRNA against the ataxia telangiectasia mutated (ATM) oncogene, which is involved in DNA repair10. Previously, the suppression of ATM has been shown to decrease tumor growth in a breast cancer model10.

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Protocol

1. PCPS Particle Preparation

  1. Oxidize non-functionalized porous silicon particles in a 30% solution of hydrogen peroxide at 95 °C for 2 hr. Aminate the oxidized particles in 2% (3-​aminopropyl)​triethoxysilane solution in isopropyl alcohol for 2 days at 65 °C with gentle stirring.
  2. Centrifuge the solution for 30 min at 18,800 x g and wash the particles twice in isopropyl alcohol and three times in ethanol, using brief sonication to suspend the pellet. Leave the particles in ethanol solution when performing step 1.3 and 1.4.
  3. Add a known volume of particle suspension (e.g., 10 μl) to 10 ml isotone diluent and count the particles with a particle counting analyzer to determine the concentration of particles in the stock solution.
  4. Activate the acid group of L-arginine (0.1 nmol) with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 0.1 nmol)/N-hydroxysuccinimide (NHS, 0.1 nmol) in 20 ml of ethanol for 2 hr at RT with gentle stirring.
  5. Briefly sonicate the particle stock solution and add 1 billion particles to the L-arginine solution and leave the reaction for 18 hr at RT with gentle stirring.
  6. Activate the first aspartic acid group of N-(tert-Butoxycarbonyl)-L-aspartic acid (Boc-Asp-OH, 1 nmol) with EDC (0.1 nmol)/NHS (0.1 nmol) in 20 ml of ethanol for 4 hr at 4 °C with gentle stirring.
  7. Dissolve 50 mg polyethylenimine in 10 ml ethanol and add the solution to the Boc-Asp-OH mixture. Let the reaction proceed for 24 hr at RT with gentle stirring.
  8. Activate the second aspartic acid group of the Boc-Asp-OH/PEI solution with EDC (0.1 nmol)/NHS (0.1 nmol) at 4 °C for 6 hr with gentle stirring.
  9. To obtain PCPS particles, add the particle solution from step 1.5 into the Boc-Asp-OH/PEI solution from step 1.8. Allow the reaction to proceed for 18 hr at RT with gentle stirring.
  10. Centrifuge the solution for 30 min at 18,800 x g and wash the particle solution three times with ethanol, using brief sonication to suspend the pellet.

2. PCPS Particle Characterization

  1. Measure the size of the particles using a scanning electron microscope (SEM).
    1. Place a drop of particle suspension (10,000 particles/μl in ethanol) on a clean silica SEM sample stub and let dry at RT under vacuum.
    2. Measure SEM images at 8 kV with a 3–5 mm working distance using an in-lens detector.
  2. Measure the zeta potential of the particles using a particle analyzer system.
    1. Mix 10 μl of particle suspension (10,000 particles/μl in ethanol) with 1 ml of 10 mM phosphate buffer (pH 7.4).
    2. Load the sample into folded capillary cells and measure the zeta potential according to the manufacturer’s instructions.

3. Loading of siRNA into PCPS Particles

  1. Dry the PCPS particles (from PCPS particle preparation step 1.9) under vacuum O/N.
  2. Add siRNA (4 μg) in nuclease-free water (20 μl) to the dried PCPS particles and sonicate briefly. Use the following particle to siRNA ratios: 2 × 105 particles/0.2 μg siRNA, 4 × 105 particles/0.2 μg siRNA, 6 × 105 particles/0.2 μg siRNA, 8 × 105 particles/0.2 μg siRNA, 10 × 105 particles/0.2 μg siRNA and 12 × 105 particles/0.2 μg siRNA.
  3. Incubate for 3 hr at 4 °C on a shaker (1,000 rpm) to allow siRNA binding to the particles.

4. Optimization of siRNA/PCPS Particle Ratio

  1. Add DNA loading dye to 20 μl of the PCPS/control siRNA particles with different particle to siRNA ratios (see loading of siRNA into PCPS particles).
  2. Load the samples into a 2% agarose gel containing DNA gel stain.
  3. Perform electrophoresis at a constant voltage of 120 V for 20 min in running buffer.
  4. Analyze the gel with image acquisition and analysis software.

5. Release of siRNA from PCPS Particles

  1. Mix 20 μl of the PCPS/control siRNA particles with different particle to siRNA ratios (see step 3) in sodium dodecyl sulfate (SDS, 2%) and let stand for 1 hr at RT.
  2. Add DNA loading dye to the samples.
  3. Load samples into a 2% agarose gel containing DNA gel stain.
  4. Perform electrophoresis at a constant voltage of 120 V for 20 min in running buffer using DNA electrophoresis equipment and a power supply.
  5. Analyze the gel with image acquisition and analysis software.

6. Confocal Microscopy of PCPS Particles

  1. Add 5 μl of PCPS/fluorescent control siRNA particles (10 × 105 particles/0.2 μg siRNA/20 μl) to a glass cover slip.
  2. Visualize particle layers by confocal microscopy.

7. Characterization of Arg-PEI/control siRNA Nanoparticles

  1. To degrade the silicon material and form Arg-PEI/control siRNA nanoparticles, add PCPS/siRNA particles (10 × 106 PCPS particles/2 μg siRNA) to 100 μl of phosphate buffered saline and shake (1,000 rpm) at 37 °C for 2 days.
  2. Centrifuge the sample for 30 min at 18,800 x g and collect the supernatant.
  3. Measure the size of the formed Arg-PEI/siRNA nanoparticles with dynamic light scattering (DLS).
    1. Mix 10 μl of the supernatant with 1 ml 10 mM phosphate buffer (pH 7.4) and place into a plastic cuvette.
    2. Measure the size of the particles using a particle analyzer system according to the manufacturer’s instructions.
  4. Determine the size and morphology of the formed Arg/PEI-siRNA nanoparticles with atomic force microscopy.
    1. Take 10 µl of the supernatant and place on a silicon wafer.
    2. Visualize the particles with atomic force microscopy (AFM).

8. Cell Culture

  1. Culture MDA-MB-231 human breast cancer cells in cell culture media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 5% CO2, 95% humidity and 37 °C.

9. Confocal Microscopy of Live Cells with PCPS Particles

  1. Plate cells in 2-well culture slides at a seeding density of 3 x 105 cells/well for 24 hr.
  2. Add PCPS particles loaded with fluorescent control siRNA (10 × 105 particles/0.2 μg particle to siRNA ratio, 50 nM siRNA) to cells.
  3. Record a movie of the cells with a confocal microscope (supplied with a chamber, 5% CO2, 95% humidity and 37 °C) for 12 hr following particle exposure.

10. Confocal Microscopy of Fixed Cells with PCPS Particles

  1. Plate cells in 2-well culture slides at a seeding density of 3 x 105 cells/well for 24 hr.
  2. Add PCPS particles loaded with fluorescent control siRNA (10 × 105 particles/0.2 μg particle to siRNA ratio, 50 nM siRNA) to cells and incubate for 1 day, 7 days and 10 days.
  3. Wash the cells twice with phosphate buffered saline and then fix them with 4% paraformaldehyde solution for 10 min.
  4. Wash the cells with phosphate buffered saline.
  5. Permeabilize the cells with 0.1% octyl phenol ethoxylate for 10 min and then wash them three times with phosphate buffered saline.
  6. Block the cells with albumin from bovine serum (10 mg/ml) in phosphate buffered saline for 10 min at RT with gentle stirring.
  7. To visualize filamentous actin, incubate the cells with fluorescently labeled phalloidin (1 μl/40 μl blocking solution) for 20 min at RT with gentle stirring and then wash in phosphate buffered saline.
  8. Remove the slides from the frame and add antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) to visualize the nucleus.
  9. Add a cover glass on top and take images of the cells with confocal microscopy.

11. Flow Cytometry of Cells with PCPS/fluorescent Control siRNA Particles

  1. Plate cells in a 6-well plate at a seeding density of 3 x 105 cells/well for 24 hr.
  2. Add PCPS particles loaded with fluorescent control siRNA (10 × 105 particles/0.2 μg particle to siRNA ratio, 50 nM siRNA) to the cells and incubate for 24 hr.
  3. Wash the cells with phosphate buffered saline and scrape them with a cell scraper.
  4. Store the cells in phosphate buffered saline with 2% fetal bovine serum prior to analysis. Use untreated cells as a negative control.
  5. Perform flow cytometry.

12. Cell Viability of Cells with PCPS Particles and PCPS/control siRNA Particles

  1. Plate cells in a 96-well plate at a cell density of 3 × 103 cells/well for 24 hr.
  2. Treat the cells with PCPS particles (1.5 × 105/well and 6 × 105/well) or PCPS/control siRNA particles (10 × 105 particles/0.2 μg particle to siRNA ratio, 10 nM and 100 nM siRNA) for 48 hr and 72 hr. Use untreated cells and cells treated with phosphate buffered saline (same volume as the added particles) as controls. Assay each sample in triplicate.
  3. Perform a cell proliferation assay according to the manufacturer’s instructions.
  4. Represent data as the mean ± standard deviation.

13. Western Blot of Cells with PCPS/ATM Mutated siRNA Particles

  1. Plate cells in a 6-well plate at a cell density 2 × 105 cells/well for 24 hr.
  2. Incubate the cells with PCPS/control siRNA (50 nM) particles or PCPS/ATM siRNA (50 nM) particles for 72 hr. Use untreated cells as a control.
  3. Lyse the cells using a protein extraction reagent supplemented with a protease inhibitor cocktail.
  4. Centrifuge the cell lysates for 10 min at 14,000 x g and recover the supernatant.
  5. Determine the protein concentration with a protein quantification assay according to the manufacturer’s instructions.
  6. Add sample loading buffer (with 5 μl 2-mercaptoethanol/ml buffer) to the samples and heat them for 6 min at 99 °C.
  7. Load the protein samples (20 μg/μl) in a 12% SDS-polyacrylamide gel in running buffer and perform polyacrylamide gel electrophoresis (1 hr, 120 V) using electrophoresis equipment and a power supply.
  8. Transfer the gel in transfer buffer (with 20% methanol) to a nitrocellulose membrane (1 hr, 100 V) using electrophoresis equipment and a power supply.
  9. Block the membrane with 5% dry milk for 1 hr.
  10. Incubate the membrane with the ATM primary antibody (from rabbit) in blocking solution (from step 13.9) at a 1:1,000 dilution O/N.
  11. Wash the membrane with phosphate buffered saline containing 0.1% polyethylene glycol sorbitan monolaurate and then incubate it with the secondary antibody (anti-rabbit) in blocking solution (from step 13.9) at a 1:2,500 dilution for 1 hr.
  12. Wash the membrane with phosphate buffered saline containing 0.1% polyethylene glycol sorbitan monolaurate and detect the protein bands with Western blot detection reagent using image acquisition and analysis software.
  13. For the loading control, wash the membrane and repeat steps 13.9-13.12 using a β-actin primary antibody (from mouse, 1:10,000 dilution) and a secondary antibody (anti-mouse 1:4,000 dilution).

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Representative Results

This protocol describes the use of a non-viral delivery system for safe and efficient siRNA transfection. The SEM results reveal that the PCPS particles are cylindrical in shape and have a diameter of 2.6 μm (Figure 2A). The particles are positively charged with a zeta potential of approximately +8.21 (Figure 2B), thereby enabling electrostatic binding with negatively charged nucleotides. Confocal images of different layers of the PCPS particles demonstrate that fluorescent control siRNA is loaded inside the porous silicon particles (Figure 2C). The formation and release of Arg-PEI/siRNA nanoparticles from the pSi particles was confirmed with DLS and AFM. The size distribution of the particles ranged from 70-120 nm, with an average size of 94 nm (Figure 2D). AFM images illustrate that the nanoparticles have a spherical shape (Figure 2E).

The ratio of particles to siRNA was optimized by agarose gel electrophoresis to ensure high binding affinity (Figure 3A). A wide range of particle to siRNA ratios was used (2 × 105, 4 × 105, 6 × 105, 8 × 105, 10 × 105 and 12 × 105/0.2 μg siRNA). The results indicate that siRNA can bind tightly to the particles when the particle amount is above 8 × 105. A ratio of 10 × 105 PCPS/0.2 μg siRNA was selected for further experiments. Furthermore, siRNA was successfully released from the carrier when treated with SDS, as illustrated in Figure 3B.

Next, the cellar internalization of PCPS/fluorescent control siRNA particles was evaluated in MDA-MB-231 cells. Confocal images taken after 24 hr of treatment show that the particles are effectively internalized into cells (Figure 4). Similarly, Figure 5 demonstrates that 89% of cells have internalized PCPS/siRNA particles after 24 hr of incubation. Moreover, the internalization process was recorded for 12 hr (Video 1). These results indicate that the PCPS particles can efficiently deliver siRNA into cells. The long-term accumulation of siRNA inside the cells was also evaluated by confocal microscopy. At day 7 and day 10 the siRNA was still detectable inside the cells (Figure 6).

One of the most important factors to consider when developing a siRNA delivery system is the safety of the carrier13. PEI is known to form polyplexes with siRNA, aid in cellular uptake and trigger release from the endosome/lysosome. However, PEI can have toxic effects, due to the presence of positively charged primary amino groups in the backbone14,15. For instance, PEI binding to the glycocalyx on the cell surface can result in the formation of large clusters16. In order to eliminate this charge-induced toxicity, PEI was covalently conjugated to arginine through a bridge linker, to reduce the number of primary amino groups. The cell viability remained over 95% after 48 hr and 72 hr when particles and siRNA were used at a concentration of up to 6 × 105/well and 100 nM, respectively (Figure 7A). Next, the transfection efficacy of siRNA against the oncogene ATM was evaluated in MDA-MB-231 cells. Western blot results demonstrate that the protein levels of ATM are decreased after treatment with PCPS/ATM siRNA (Figure 7B). The results suggest the PCPS platform is a safe and efficient delivery system for siRNA.

Figure 1
Figure 1. Schematic representation of polycation-functionalized porous silicon (PCPS) particles. Arginine (Arg)-polyethylenimine (PEI)/small interfering RNA (siRNA) nanoparticles are formed following the degradation of silicon (Si).

Figure 2
Figure 2. Characterization of PCPS particles. (A) Scanning electron microscopy (SEM) image of PCPS particles. (B) Zeta potential of PCPS particles. (C) Confocal images of different layers of the PCPS/fluorescent control siRNA particles. (D) Size distribution of the arginine Arg-PEI/control siRNA nanoparticles released from the porous silicon microparticles. (E) Atomic force microscopy (AFM) images of Arg-PEI/control siRNA nanoparticles. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Agarose gel for optimization of the PCPS/siRNA delivery system. (A) Binding affinity between PCPS particles and control siRNA. The bands represent unbound siRNA. (B) siRNA release following incubation with 2% SDS for 1 hr. The bands represent total siRNA (unbound and bound). Sample 1: siRNA; sample 2: 2 × 105 PCPS particles/0.2 μg siRNA; sample 3: 4 × 105 PCPS particles/0.2 μg siRNA; sample 4: 6 × 105 PCPS particles/0.2 μg siRNA; sample 5: 8 × 105 PCPS particles/0.2 μg siRNA; sample 6: 10 × 105 PCPS particles/0.2 μg siRNA; sample 7: 12 × 105 PCPS particles/0.2 μg siRNA.

Figure 4
Figure 4. Confocal microscope images of PCPS/fluorescent siRNA particles (red) in MDA-MB-231 cells (24 hr incubation). The nucleus and filamentous actin was visualized with 4',6-diamidino-2-phenylindole (DAPI, blue) and phalloidin (green), respectively. Three different layers were imaged (top, mid and basal). Please click here to view a larger version of this figure.

Figure 5
Figure 5. Quantitative flow cytometry analysis of fluorescent MDA-MB-231 cells after incubation with PCPS/control fluorescent siRNA particles. Untreated cells were used as a negative control. 89% of cells had internalized the particles.

Figure 6
Figure 6. Confocal images of fluorescent control siRNA (red) inside MDA-MB-231 cells. Cells were incubated with PCPS/fluorescent control siRNA particles for 1 day, 7 days and 10 days. Cells were then visualized with confocal microscopy. The nucleus and filamentous actin was visualized with 4',6-diamidino-2-phenylindole (DAPI, blue) and phalloidin (green), respectively. Please click here to view a larger version of this figure.

Figure 7
Figure 7. Cell viability and gene silencing in vitro. (A) A cell viability assay of cells incubated with PCPS particles and PCPS/control siRNA particles (Scr). Experiment was performed in triplicate and results are presented as mean ± standard deviation. Untreated cells (blank) and cells incubated with PBS were used as controls. (B) Western blot of PCPS/ataxia telangiectasia mutated (ATM) siRNA particles. Cells were exposed to PCPS/control siRNA (Scr) particles and PCPS/ATM siRNA particles. Untreated cells (mock) were used as a control. β-actin was used as a loading control.

Video 1. Time-dependent uptake of PCPS/fluorescent control siRNA particles in live MDA-MB-231 cells. The video was recorded for 12 hr after exposing the cells to particles.

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Discussion

This protocol describes a method for the successful delivery and transfection of siRNA into cells. In particular, the delivery of siRNA is achieved by using a multifunctional platform consisting of polycation-functionalized pSi particles. The use of siRNA therapy has great potential, e.g., cancer treatment, as various oncogenes can be targeted with high specificity. Therefore, there exists a demand to develop siRNA delivery vehicles, which can mitigate the challenges of siRNA therapy. In conclusion, we have outlined a protocol that shows promise for the safe and efficient delivery of siRNA. However, there are some key factors that should be taken into account when performing the described technique. For instance, an important consideration when preparing the PCPS particles is to handle the siRNA carefully, in order to avoid degradation by nucleases. In particular, clean gloves and RNAse free tubes and water should be used at all times when working with siRNA. If the siRNA transfection efficacy is low, a spray that removes RNAse contamination can be used to spray gloves and working areas. In addition, if the transfection is unsuccessful, the siRNA should be tested with a commercial transfection reagent, to determine whether the problem is caused by the siRNA or the particles. Another critical consideration for successful implementation of the PCPS/siRNA delivery system is to take care when performing centrifugation and washing steps. Namely, the supernatant should be fully discarded to remove excess reagents that are required throughout the particle preparation steps.

An important advantage of the PCPS/siRNA delivery system is safety. Several siRNA transfection agents have a high cationic charge, which contributes to cellular toxicity13,15. Indeed, most commercial protocols indicate that the reagents should be incubated with the cells for only a few hours, to avoid cell death. On the contrary, cells can be exposed to the PCPS particles for several days without any signs of toxicity, as is evident from the cell viability results. The PCPS particles can bind siRNA with high affinity due to the presence of PEI. However the charge-induced toxicity of PEI is prevented by the unique setup of the delivery platform. Especially the covalent binding of Arg to PEI and the encapsulation of PEI inside the Si pores contribute to reduced toxicity. Another advantage of the PCPS platform is that siRNA transfection can take place in the presence of serum. Other existing methods usually require the use of serum-free cell culture media, thereby adding additional steps to the transfection process and potentially interfering with regular cell signaling pathways.

An additional benefit of the PCPS system is that it does not require any modification of the siRNA molecules. While some current methods require complicated conjugation steps to stabilize the siRNA or to enable cellular internalization13, the PCPS system relies on simple mixing for siRNA loading. The binding to PEI and the pores in the Si material provide a protective environment for the siRNA, thereby reducing contact with nucleases. The PCPS particles can be stored for prolonged periods of time as dried material or in isopropyl alcohol. Moreover, if the PCPS/siRNA particles are lyophilized they can be stored for at least three months at 4 °C. The lyophilized PCPS particles should be suspended in RNase free water just before transfection, and should not be stored in solution. Finally, the PCPS platform permits sustained release of siRNA, as previously reported11, consequently increasing the time period in which target genes remain suppressed. Accordingly, after cellular internalization, the Si particles gradually degrade, resulting in the formation of Arg-PEI/siRNA nanoparticles. Subsequently, the siRNA is slowly released in the cytoplasm, where it can bind to messenger RNA (mRNA), thereby exerting biological activity. Although, PCPS particles provide several advantages in comparison to existing methods for siRNA delivery, a limitation of the technique is that non-functionalized pSi microparticles are required as a starting material. While the particles can be synthesized using photolithography and electrochemical etching17, these techniques are not readily available at all institutions.

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgments

The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

Materials

Name Company Catalog Number Comments
Polyethylenimine (PEI), branched Sigma-Aldrich 408727 Average molecular weight ~25,000 Da
L-Arginine Sigma-Aldrich A5006 Reagent grade, ≥98%
Boc-Asp-OH Sigma-Aldrich 408-468 99%
Hydrogen peroxide solution Sigma-Aldrich 216763 30 wt. % in H2O
Sulfuric acid Sigma-Aldrich 339741 100.00%
Isopropyl alcohol Sigma-Aldrich W292907 ≥99.7%
Ethanol Sigma-Aldrich 459844 ≥99.5% 
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) Sigma-Aldrich 03449 ≥99%
Albumin from bovine serum Sigma-Aldrich A7030-10G Blocking agent
Ataxia-telangiectasia mutated siRNA  Sigma-Aldrich Designed in-house
Tris Acetate-EDTA buffer Sigma-Aldrich T9650 For DNA agarose gel electrophoresis
Penicillin-Streptomycin Sigma-Aldrich P4333
Fetal Bovine Serum Sigma-Aldrich F2442
TWEEN 20 Sigma-Aldrich P1379 Polyethylene glycol sorbitan monolaurate
2-Mercaptoethanol Sigma Aldrich M6250 For Western blot
Sodium dodecyl sulfate Sigma-Aldrich L3771
Sodium phosphate monobasic Sigma-Aldrich 71496 For making phosphate buffer 
Sodium phosphate dibasic Sigma-Aldrich 71640 For making phosphate buffer
Anti-Mouse IgG Sigma-Aldrich A4416 Secondary antibody (anti-mouse) for Western blot
N-Hydroxysuccinimide (NHS) Sigma-Aldrich 130672 98%
CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid Greiner Bio-One 655182 96-well plate
10x Tris-Glycine Liquid Li-Cor  928-40010 Transfer buffer for Western blot
Paraformaldehyde solution 4% in PBS  Santa Cruz Sc-281692 Fixation of cells
CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) Promega G5421 Proliferation assay
Phosphate buffered saline Fisher Scientific BP399-500 10x Solution
Corning cellgro Dulbecco's Modification of Eagle's (Mod.) Fisher Scientific  MT-15-017-CM Cell culture media, 1x solution
Triton X-100 Fisher Scientific AC21568-2500 Octyl phenol ethoxylate, permeabilization agent
Cover glass Fisher Scientific 12-530C 
Methanol Fisher Scientific  A412-1 For Western blot transfer buffer
Plastic Cuvettes Fisher Scientific  14-377-010  For size measurements using Zetasizer Nano ZS
Molecular BioProducts RNase AWAY Surface Decontaminant Fisher Scientific 14-754-34 Spray for removing RNAse contamination
Agarose Fisher Scientific BP165-25 Low melting point, for running RNA samples
ProLong Gold Antifade Reagent with DAPI  Invitrogen P36935 Antifade reagent with DAPI, nucelus detection
Alexa Fluor 488 Phalloidin Invitrogen A12379 Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin
SYBR Safe DNA Gel Stain Invitrogen S33102 Visualization of RNA
Negative Control siRNA Qiagen 1022076 Control siRNA
AllStars Neg. siRNA AF 555 Qiagen 1027294 Fluorescent control siRNA
Cell scraper Celltreat 229310
BioLite Multidishes and Microwell Plates Thermo Scientific 130184 6-well plate
Pierce LDS Sample Loading Buffer (4x) Thermo Scientific 84788 Sample loading buffer for Western blot
Pierce BCS Protein Assay Kit Thermo Scientific 23227 Protein quantification assay
Halt Protease Inhibitor Single-Use Cocktails (100x) Thermo Scientific 78430 Protease inhibitor cocktail, use at 1x
M-PER Mammalian Protein Extraction Reagent Thermo Scientific  78501 Protein extraction reagent
Sorvall Legend Micro 21R Thermo Scientific 75002440 Centrifuge
Beta Actin Antibody  Thermo Scientific  MA1-91399 β-actin primary antibody (from mouse) for western blot
6x TriTrack DNA Loading Dye Thermo Scientific R1161 DNA loading dye
Nuclease-Free Water  Life Technologies AM9938
Non-Fat Dry Milk Lab Scientific M0841 For Western blot
2-well BD Falcon culture slides  BD Biosciences 354102 2-well culture slides
Amersham ECL Western blot detection reagent GE Healthcare Life Sciences RPN2106 Western blot detection reagent
BA Membranes GE Healthcare Life Sciences 10402096 Nitrocellulose membrane for Wester blot
ATM (D2E2) Rabbit mAb Cell Signaling 2873S ATM primary antibody (from rabbit) for Western blot
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling  7074 Secondary antibody (anti-rabbit) for Western blot 
Folded capillary cells Malvern  DTS 1061 For zeta potentail measurements using Zetasizer Nano ZS
MDA-MB-231 cell line ATCC HTB-26 Mammary Gland/Breast
12% Mini-PROTEAN TGX Gel Bio-rad 456-1043  For Western blot
Biorad PowerPac HC Bio-rad 164-5052 Power supply for electrophoresis
10x Tris/Glycine/SDS Buffer Bio-rad 161-0732 Running buffer for Western blot
Wide Mini-Sub Cell GT Cell Bio-rad 170-4405 Electrophoresis equipment for DNA agarose gel 
Mini-PROTEAN Tetra cell Bio-rad 165-8000 Electrophoresis equipment for Western blot
ChemiDoc XRS+ System with Image Lab Software Bio-rad 170-8265 Image acquisition and analysis software for gels and blots
4" (10 cm) dia., 5 x 7 mm diced Silicon Wafer Ted Pella  16007 Silicon waferfor scanning electron microscopy and atomic force microscopy
Thermomixer R Eppendorf 22670107 Shaker 
Isoton II diluent Beckman Coulter 8546719 Isoton diluent
Multisizer 4 Coulter Counter Beckman Coulter A63076 Particle counting analyzer
Non-functionalized porous silicon particles  Microelectronics Research Center, University of Texas at Austin Dicoidal shape. 2.6 μm (diameter) x 0.7 μm (height), provided in isopropyl alcohol
Zetasizer Nano ZS Malvern Particle analyzer system for size and zeta potential 
Scanning Electron Microscope FEI Particle size and shape 
Atomic Force Microscope Bruker Particle size and shape
Fluo ViewTM 1000 Confocal Microscope Olympus Visualization of fixed and live cells

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References

  1. De Fougerolles, A., Vornlocher, H. P., Maraganore, J., Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov. 6, (6), 443-453 (2007).
  2. Rolland, A. Gene medicines: the end of the beginning. Adv Drug Deliv Rev. 57, (5), 669-673 (2005).
  3. Oh, Y. K., Park, T. G. siRNA delivery systems for cancer treatment. Adv Drug Deliv Rev. 61, (10), 850-862 (2009).
  4. Mintzer, M. A., Simanek, E. E. Nonviral vectors for gene delivery. Chem Rev. 109, (2), 259-302 (2009).
  5. Whitehead, K. A., Langer, R., Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov. 8, (2), 129-138 (2009).
  6. Decuzzi, P., et al. Size and shape effects in the biodistribution of intravascularly injected particles. J Control Release. 141, (3), 320-327 (2010).
  7. Decuzzi, P., Ferrari, M. Design maps for nanoparticles targeting the diseased microvasculature. Biomaterials. 29, (3), 377-384 (2008).
  8. Martinez, J. O., Evangelopoulos, M., Chiappini, C., Liu, X., Ferrari, M., Tasciotti, E. Degradation and biocompatibility of multistage nanovectors in physiological systems. J Biomed Mater Res A. 102, (10), 3540-3549 (2014).
  9. Shen, H., et al. Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery. Clin Cancer Res. 19, (7), 1806-1815 (2013).
  10. Xu, R., et al. Multistage vectored siRNA targeting ataxia-telangiectasia mutated for breast cancer therapy. Small. 9, (9-10), 1799-1808 Forthcoming.
  11. Shen, J., et al. High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics. ACS Nano. 7, (11), 9867-9880 (2013).
  12. Zhang, M., et al. Polycation-functionalized nanoporous silicon particles for gene silencing on breast cancer cells. Biomaterials. 35, (1), 423-431 (2014).
  13. Ozpolat, B., Sood, A. K., Lopez-Berestein, G. Nanomedicine based approaches for the delivery of siRNA in cancer. J Intern Med. 267, (1), 44-53 (2010).
  14. Philipp, A., Dehshahri, A., Wagner, A., E, Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity. Bioconjug Chem. 19, (7), 1448-1455 (2008).
  15. Hunter, A. C. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity. Adv Drug Deliv Rev. 58, (14), 1523-1531 (2006).
  16. Andrews, P. M., Bates, S. B. Dose-dependent movement of cationic molecules across the glomerular wall. Anat Rec. 212, (3), 223-231 (1985).
  17. Ananta, J. S., et al. Geometrical confinement of gadolinium-based contrast agents in nanoporous particles enhances T1 contrast. Nat Nanotechnol. 5, (11), 815-821 (2010).

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