Maximizing RNA Loading for Gene Silencing Using Porous Silicon

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22993−23005

Maximizing RNA Loading for Gene Silencing Using Porous Silicon Nanoparticles Terence Tieu,†,‡ Sameer Dhawan,§ V. Haridas,§ Lisa M. Butler,∥,⊥ Helmut Thissen,‡ Anna Cifuentes-Rius,*,† and Nicolas H. Voelcker*,†,‡,#

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†

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, 381 Royal Parade, Parkville, Victoria 3052, Australia ‡ CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168, Australia § Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India ∥ Adelaide Medical School & Freemasons Foundation Centre for Men’s Health, University of Adelaide, Adelaide, South Australia 5005, Australia ⊥ South Australian Health & Medical Research Institute, Adelaide, South Australia 5001, Australia # Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, 151 Wellington Road, Clayton, Victoria 3168, Australia S Supporting Information *

ABSTRACT: Gene silencing by RNA interference is a powerful technology with broad applications. However, this technology has been hampered by the instability of small interfering RNA (siRNA) molecules in physiological conditions and their inefficient delivery into the cytoplasm of target cells. Porous silicon nanoparticles have emerged as a potential delivery vehicle to overcome these limitations being able to encapsulate RNA molecules within the porous matrix and protect them from degradation. Here, key variables were investigated that influence siRNA loading into porous silicon nanoparticles. The effect of modifying the surface of porous silicon nanoparticles with various amino-functional molecules as well as the effects of salt and chaotropic agents in facilitating siRNA loading was examined. Maximum siRNA loading of 413 μg/(mg of porous silicon nanoparticles) was found when the nanoparticles were modified by a fourth generation polyamidoamine dendrimer. Low concentrations of urea or salt increased loading capacity: an increase in RNA loading by 19% at a concentration of 0.05 M NaCl or 21% at a concentration of 0.25 M urea was observed when compared to loading in water. Lastly, it was demonstrated that dendrimer-functionalized nanocarriers are able to deliver siRNA against ELOVL5, a target for the treatment of advanced prostate cancer. KEYWORDS: gene delivery, nanoparticles, porous silicon, RNA, siRNA oligonucleotides toward the target cell.6−8 Viral vectors have shown their proficiency to achieve gene silencing, although challenges such as mutagenesis and immunogenicity have limited their clinical translation.7 In contrast, nonviral vectors are considered safer than their viral counterpart but generally lack the high transfection efficiency of viruses.6 Thus, there is a considerable effort afoot to engineer nonviral delivery systems for the efficient delivery of oligonucleotides. Nonviral delivery systems that have been studied for gene delivery include lipidbased complexes,9,10 cationic polymers,11,12 and inorganic nanoparticles.13−16

1. INTRODUCTION RNA interference (RNAi)-based therapeutics has the intriguing potential to silence a gene of interest, including those deemed “undruggable” by small molecules or proteins.1 This has spurred research activity over the years in the design of RNAi therapeutics for the treatment of many types of disease, including cancer.2,3 However, clinical translation of RNAibased therapies such as small interfering RNA (siRNA) have faced physiological barriers in the delivery of oligonucleotides to the target site. The systemic administration of naked oligonucleotides leads to nonspecific distribution, rapid degradation, and renal clearance. Additionally, due to its molecular weight and polyanionic nature, naked oligonucleotides lack the ability to permeate the cell membrane.4,5 In order to overcome these obstacles, numerous delivery vehicles, both viral and nonviral, have been developed to deliver © 2019 American Chemical Society

Received: March 29, 2019 Accepted: June 5, 2019 Published: June 5, 2019 22993

DOI: 10.1021/acsami.9b05577 ACS Appl. Mater. Interfaces 2019, 11, 22993−23005

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ACS Applied Materials & Interfaces

Scheme 1. (A) Modification of the (i) Surface Chemistry of pSiNPs and the (ii) Composition of the Loading siRNA Solution To Maximize the Loading Capacity of pSiNP; (B) siRNA-Loaded pSiNPs Able To (a) Protect the Cargo from Degradation, (b) Facilitate Its Internalization and Delivery into the Cytoplasm of the Cell, and (c) Downregulate Expression of the Target

prostate tumors.27 In this regard, the elongation of very long chain fatty acids protein 5 (ELOVL5) is a highly expressed endogenous gene found in prostate cancer cells and has been identified as a novel potential target for prostate cancer therapy. Thus, we used siRNA targeted toward ELOVL5 to explore the potency/efficacy of nanocarrier-based gene silencing.

A range of nanoparticle-based delivery systems have been investigated in which the oligonucleotide is loaded by different mechanisms. For solid nanoparticles, oligonucleotides are conjugated or adsorbed onto the solid surface and, thus, are readily subjected to degradation by endogenous nucleases upon systemic administration.17 In contrast, when oligonucleotides are loaded into porous nanostructures, the payload is protected from enzymatic degradation.18 In this work, we utilize porous silicon nanoparticles (pSiNPs), which have been shown to be effective in delivering oligonucleotides and other biomacromolecules in vivo.19−23 pSiNPs have also been shown to protect siRNA from degradation by ribonucleases.20 The high drug loading capacity and ease of surface functionalization afford salient advantages to pSiNPs as drug delivery vehicles.24,25 Often confused with the more common counterpart mesoporous silica, a poorly degradable biomaterial, pSi is biodegradable by nature, hydrolyzing to the nontoxic byproduct orthosilicic acid.14 These characteristics make pSiNPs a highly suitable nanomaterial for siRNA delivery, and we are exploring a way to optimize this system in the present contribution. Here, we studied two variables for increasing the loading capacity of oligonucleotides in pSiNPs: (i) altering the surface chemistry of the nanoparticles and (ii) modifying the loading solution composition (Scheme 1A). We used a variety of aminegroup-containing surface modifiers of different sizes and architectures to take advantage of the favorable electrostatic interactions with anionic oligonucleotides.26 We evaluated the loading capacity and release kinetics of these various surface modifications. Additionally, we assessed the effects of different loading solution compositions, specifically the effects of salt and chaotropic agents. We investigated if siRNA-loaded pSiNPs protected the payload from degradation, assisted in its internalization to the cytoplasm, and exhibited high transfection efficiency (Scheme 1B). We validated the cytotoxicity and gene silencing capabilities of this nanocarrier for the treatment of prostate cancer, addressing the urgent need to develop precision nanomedicines for the treatment of intrinsically heterogeneous and complex

2. MATERIALS AND METHODS 2.1. Materials. Single-crystalline silicon wafers were purchased from Siltronix (Archamps, France). Hydrofluoric acid (HF; 49%) was purchased from J. T. Baker (Center Valley, PA, USA). Ethanol (EtOH), dimethylformamide (DMF), dichloromethane (DCM), and triethylamine (TEA) were purchased from Merck (Australia). RPMI-1640, penicillin−streptomycin, OptiMEM, RNase-free water, Block-iT Fluorescent Oligo (FITC-siRNA), Block-iT Alexa Fluor Red Fluorescent Control (AF555-siRNA), lipofectamine RNAiMAX, and 7-aminoactinomycin D (7-AAD) were purchased from Life Technologies. siRNA-targeting ELOVL5 (5′-CACCAGUGCGAGAGAGGAU-3′) and nontargeting siRNA were purchased from Dharmacon Inc. A generic strand of siRNA (5′-GCCAGAAUGUGGAACUCUUU-3′) was also purchased from Dharmacon for loading, release, and RNase treatment studies. Oligolysine dendrimers K1−K7 were synthesized by previously reported methods.28,29 Poly(ethylene glycol) bis(3-aminopropyl) terminated (Mn ∼ 1,500) and all other chemicals were purchased from Sigma-Aldrich unless stated otherwise. 2.2. Porous Silicon Nanoparticle Fabrication. pSiNPs were prepared by anodic electrochemical etching from p+-type (0.0055− 0.001 Ω cm) silicon wafers by periodically etching at 5 mA/cm2 for 20 s and 139 mA/cm2 for 0.2 s for 1000 cycles in a 3:1 HF (49%):EtOH solution. Afterward the pSi film was detached by etching at a constant current density of 139 mA/cm2 for 60 s in a 1:1 HF (49%):EtOH solution. The detached film was then sonicated in an ultrasonicator water bath in absolute EtOH for 20 h into NPs. pSiNPs were harvested by centrifugation in a multistep process. The particles were centrifuged at 2000g for 6 min, where the supernatant was collected. The supernatant was then centrifuged at 20000g for 10 min, and the pellet consisted of ∼160 nm NPs. 2.3. Functionalization of pSiNPs with Undecylenic Acid. A 5 mL aliquot of undecylenic acid (UA) was liquefied under a N2 atmosphere to remove all trace oxygen and water from the system for 20 min. A 5 mg amount of pSiNPs was then added to the solution and allowed to react for 24 h at 120 °C under N2 atmosphere. After the 22994


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6, 12, 24, 48, and 72 h), the particles were centrifuged and 5 μL of the supernatant was removed and replaced with fresh PBS. siRNA concentration in the supernatant was determined by their UV absorbance to determine the amount of siRNA released from the pSiNPs. 2.9. Dynamic Light Scattering and ζ Potential Measurements. The mean hydrodynamic diameter of pSiNPs, size distribution, polydispersity index (PDI), and ζ potential (ζ-pot) of pSiNPs were determined by means of dynamic light scattering (DLS) using a Zetasizer Nano ZS. A scattering angle of 173° and a temperature of 25 °C were used with pSiNPs dispersed in RNase-free water. 2.10. Transmission Electron Microscopy. pSiNPs were imaged by transmission electron microscopy (TEM; JEOL JEM-2100F) equipped with a field emission gun. pSiNP samples were deposited on Formvar film-coated copper grids (ProSciTech, Australia). Images were acquired at 200 kV accelerating voltage. 2.11. Fourier Transform Infrared Spectroscopy. A 1 μL aliquot of pSiNPs (1 mg/mL) in EtOH was spotted on a flat high resistivity (3−6 Ω cm) p-type silicon wafer and air-dried. Analysis was conducted on a Hyperion 1000 Fourier transform infrared spectroscopy (FTIR) microscope coupled to a Vertex 70 IR source (Bruker, Germany) and a liquid-N2-cooled MCT detector. Spectra were acquired between 650 and 4000 cm−1 at a resolution of 4 cm−1 for 64 scans. 2.12. Determination of pSi Concentration. A 1 mL aliquot of sample was pipetted into a Teflon beaker and heated to partially evaporate the EtOH in the solution. The sample that remained was then digested with 0.2 mL of 48% HF, 2 mL of 69% nitric acid (HNO3), and 2 mL of Milli Q water. Once the reaction had ceased, the solutions were then made up to 50 mL with Milli Q water in a plastic volumetric flask. The solutions were analyzed by the Varian 730-ES axial ICP-OES. Certified multielement solutions were used to check the accuracy of the calibration standard and the method used. 2.13. RNase Treatment and siRNA Stability Assay. PAMAM(G4)-pSiNPs and UA-pSiNPs (3 mg) were loaded with siRNA (5′GCCAGAAUGUGGAACUCUUU-3′) overnight under agitation at 4 °C. The NPs were then washed once in water to remove loosely bound siRNA. The supernatant was collected to determine the amount of siRNA loaded into the pSiNPs. RNase treatment and siRNA stability assay was adapted from a previously described method.20 The NPs were then separated and incubated in PBS containing various concentrations of RNase A (Thermo Fisher Scientific) ranging from 1 pg/mL to 10 μg/ mL for 1 h under agitation at RT. The RNase solution was removed via centrifugation and the remaining siRNA was released from the pSiNPs by treatment with aqueous potassium hydroxide (100 mM, 150 μL) to dissolve the silicon structure. The solution was then analyzed using a Quant-iT RNA assay kit (Thermo Fisher Scientific) following the manufacturer’s protocol to determine the amount of intact siRNA remaining. The evaluation of different amine-functionalized pSiNPs was conducted on the basis of the RNase A enzyme concentration curve, where concentrations of 0.1 and 1 ng/mL RNase A were selected to determine the difference in surface functionalization in shielding siRNA from ribonuclease degradation. Each different amine-modified pSiNP (30 μg per sample) was loaded overnight with siRNA (5′GCCAGAAUGUGGAACUCUUU-3′), incubated with RNase A for 1 h and then digested in potassium hydroxide to dissolve the silicon structure. The total remaining siRNA was analyzed using a Quant-iT RNA assay kit following the manufacturer’s protocol. 2.14. Cell Viability of UA-pSiNPs and Amine-Modified pSiNPs. C4−2B cells were seeded in a 96-well plate at 1 × 104 cells per well and allowed to attach overnight. Cell culture medium was replaced by 100 μL of different amine-modified pSiNPs in media at concentrations of 10, 50, and 100 μg/mL. After 24 h of incubation with particles, the number of living cells was determined using an ATP-based luminescent cell viability assay (CellTiter-Glo, Promega, Madison, WI, USA) according to the manufacturer’s protocol. Each experiment was performed in triplicate and compared to the negative (untreated cells) and positive control (1% Triton-X 100). Luminescence was measured on a PerkinElmer EnSpire multimode plate reader. 2.15. Cellular Association of Amine-Modified pSiNPs Measured via Flow Cytometry. Amine-modified pSiNPs were

reaction, UA-pSiNPs were washed 3× in absolute EtOH via centrifugation and stored in EtOH until further use. 2.4. Functionalization of pSiNPs with 11-Bromo-1-undecene. A 5 mL aliquot of 11-bromo-1-undecene (Br) was purged under N2 atmosphere to remove all trace oxygen and water from the system for 20 min. A 5 mg amount of pSiNPs was then added to the solution and allowed to react for 24 h at 95 °C under N2 atmosphere. After the reaction, Br-pSiNPs were washed 3× in absolute EtOH via centrifugation and stored in EtOH until further use. 2.5. Functionalization of UA-pSiNPs via EDC/NHS. UA-pSiNPs were washed twice in DMF to remove all trace EtOH from the NPs. A 40 mM solution of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC HCl) was prepared with 0.2 equiv of TEA in DMF. A 20 mM solution of N-hydroxysuccinimide (NHS) was prepared separately in DMF. pSiNPs were then resuspended for 5 min in a 1:1 ratio of EDC:NHS providing a final concentration of 20 mM EDC and 10 mM NHS. Afterward, either diethylenetriamine, tris(2aminoethyl)amine, poly(ethylene glycol) diamine (∼1500 Mn), or polyamidoamine (PAMAM) dendrimers generations 1−4 were added to the reaction mixture at a final concentration of 10 mM. The reaction was left under agitation, protected from light for 24 h at room temperature (RT). After 24 h, the EDC/NHS solution was removed via ultracentrifugation and the amine-modified pSiNPs were washed in ice cold water five times, then once in 70% EtOH, and finally once in EtOH. The NPs were stored in EtOH until further use. 2.6. Functionalization of Br-pSiNPs via Click Chemistry. BrpSiNPs were washed twice in DMF to remove all trace EtOH from the NPs. A 10% (w/v) sodium azide (NaN3) solution in 10 mL of DMF was prepared; 5 mg of pSiNPs were added and allowed to react for 24 h at 60 °C. After 24 h, the NPs were washed three times in Milli-Q water to remove free sodium bromide. The nanoparticles were then resuspended in Milli-Q water. To attach the oligolysine dendrons, click chemistry between the alkyne truncated dendrons and azide on the pSiNPs was employed based on a previously described method.30 To the mixture of 1 mg of pSiNPs in water (in 200 μL of H2O), 0.1 μM copper sulfate (CuSO4) (in 13 μL of H2O), 1.2 μM tris(hydroxypropyltriazolylmethyl)amine (THPTA; in 13 μL of DMSO), 1 μM sodium ascorbate (NaAc) (in 13 μL of H2O), and 0.5 mg of the oligolysine dendrons (K1, K3, or K7; in 130 μL of DMSO) was added and the total volume was made up to 1 mL with a final solution ratio of 1:1 dimethyl sulfoxide (DMSO):H2O. The reaction was left under agitation for 24 h at RT. Afterward, the dendron-functionalized pSiNPs were washed three times in Milli-Q water. To remove the tert-butyloxycarbonyl (BOC) groups, the NPs were washed twice in DCM. A 1 mg amount of pSiNPs was dispersed in 30% trifluoroacetic acid (TFA) in 1 mL of DCM. The reaction was left under agitation, protected from light for 1 h at RT. The final oligolysine dendrons-functionalized pSiNPs were then washed one time in the following order: DCM, water, 70% EtOH, and absolute EtOH. The NPs were stored in EtOH until further use. 2.7. Loading of pSiNPs with siRNA. siRNA (5′-GCCAGAAUGUGGAACUCUUU-3′) was loaded into the amine-modified pSiNPs (200 μg) by mixing with 200 μL of siRNA stock (280 μg/mL in water) solution. The concentration of siRNA in solution was determined by measuring UV absorbance at 260 nm using a spectrophotometer (Nanodrop 1000, Thermo Fisher Scientific). The dispersed particles were centrifuged to separate the free siRNA from the siRNA-loaded pellet. pSiNPs were allowed to load under agitation at 4 °C over 72 h. At specific time points (6, 12, 24, 48, and 72 h) the concentration of free siRNA was measured. Loading profiles were established for the various amine-modified pSiNPs. For the altered compositions of loading solutions and comparison of solid polystyrene nanoparticles (200 nm; Polysciences, Inc.), the time point taken was at 6 h. 2.8. Determination of siRNA Release Kinetics from pSiNPs. siRNA-loaded pSiNPs (200 μg) were pelleted from the supernatant by ultracentrifugation. The particles were washed twice in water to remove any free or loose surface bound siRNA, then resuspended in 200 μL of PBS and incubated at 37 °C. At specific time points over 72 h (1, 2, 3, 4, 22995


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Figure 1. Physicochemical characterization of pSiNPs by means of (A) TEM showing freshly etched pSiNPs, (B) particle size/DLS, and (C) ζ potential of different amine-modified pSiNPs. Data shown as mean ± S.D. (n = 3). (D) Chemical structures of various amine molecules used to functionalize pSiNPs. Generation 1 and 2 polyamidoamine (PAMAM) dendrimers are provided as an example. plate at 1 × 105 cells per well and allowed to attach overnight. The culture medium was replaced with amine-modified pSiNPs in OptiMEM at a concentration of 100 μg/mL and was incubated with the cells for 6 h. Afterward, the wells were washed six times with PBS to remove

loaded with Block-iT Fluorescent Oligo (Thermo Fisher Scientific) for 12 h overnight according to section 2.7. C4−2B prostate carcinoma cells were maintained in RPMI 1640 medium supplemented with 1% penicillin−streptomycin and 10% FBS. Cells were seeded in a 12-well 22996


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ACS Applied Materials & Interfaces pSiNPs and Block-iT Fluorescent Oligo. Cells were then detached with 0.01 M EDTA in PBS and resuspended in FACS buffer (1× PBS containing 10% FBS, 2 mM EDTA, and 0.1% NaN3) and kept on ice until analysis. Samples were analyzed by flow cytometry (BD FACS Canto II) for FITC fluorescence. 2.16. Cellular Association of Amine-Modified pSiNPs Measured via Confocal Microscopy. PAMAM(G4)-pSiNPs were labeled with an Alexa Fluor 488 (AF488) NHS ester (Thermo Fisher Scientific). A 1 mg amount of PAMAM(G4)-pSiNPs was washed 3× with 0.1 M NaHCO3 (pH 8.3). The NPs were then resuspended in 1 mL of NaHCO3 and reacted with 40 μM AF488 NHS ester. The reaction was left under agitation for 3 h at 4 °C protected from light. Afterward, the NPs were washed 3× with PBS to remove free dye. The NPs were then loaded with Block-iT Alex Fluor Red Fluorescent Control (AF555-siRNA) (Thermo Fisher Scientific) as per section 2.7 overnight under agitation at 4 °C protected from light. The loaded PAMAM(G4)-pSiNPs were then washed once in PBS and then resuspended in Opti-MEM. C4−2B cells were seeded on coverslips in a 6-well plate at 2 × 105 cells per well and allowed to attach overnight. The cells were washed twice in PBS and then treated with 100 μg/mL pSiNPs. At 1 and 6 h, coverslips were washed 2× in PBS and then fixed in 4% paraformaldehyde (PFA) for 15 min at RT. The coverslips were then washed 3× in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT. The coverslips were then washed 3× in PBS and incubated with Hoechst 33342 (1:2000, Thermo Fisher Scientific) and Phalloidin-iFluor 647 Reagent (1:300, Abcam) for 40 min. Afterward, the coverslips were washed 3× in PBS and then mounted onto glass slides with Prolong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were taken with a confocal fluorescence microscope (Leica TCS SP8, Leica Microsystems). 2.17. Knockdown of ELOVL5 Using Amine-Modified pSiNPs. C4−2B cells were seeded in a 6-well plate at 2 × 105 cells per well and allowed to attach overnight. The cells were then washed 2× in PBS and then incubated with 100 μg of ELOVL5-siRNA-pSiNPs and relevant controls (unloaded particles and scrambled siRNA-loaded particles) in 1 mL of OptiMEM for 6 h. Afterward, 1 mL of RPMI + 5% FBS was added to each treatment and allowed to incubate for a total time of 72 h. As a positive control, siRNA-lipofectamine RNAiMAX complexes for both ELOVL5-targeting and scrambled sequence (20 nM final siRNA concentration) were prepared according to the manufacturer’s protocol. After 72 h incubation, the cells were washed 2× with warm PBS and then lifted off using 0.01 M EDTA in PBS. Cells were then washed in cold PBS once, incubated with 7-AAD cell viability dye for 5 min, and then washed 2× in cold PBS to remove free dye. The cells were then fixed for 15 min in 2% PFA in PBS at RT. Cells were washed 2× in cold PBS and then permeabilized with 0.2% saponin in PBS for 15 min at RT. Afterward, cells were blocked in human IgG for 20 min on ice and then incubated with ELOVL5 primary antibody (ab205535, Abcam) for 30 min on ice. Afterward, the cells were washed with 3× in cold 0.2% saponin in PBS to remove free primary antibody. The cells were then incubated with a DyLight 650 secondary antibody (ab96894, Abcam) in 0.2% saponin for 20 min on ice and then washed 3× in 0.2% saponin in PBS. The cells were then resuspended in FACS buffer on ice until flow cytometry analysis. Samples were analyzed on a BD FACS Canto II flow cytometer. 2.18. Western Blotting. After treatment and 72 h of incubation, cells were washed 3× with warm PBS and then directly lysed in RIPA lysis buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Sigma, P8340). Protein concentration was quantified using a BCA assay kit (Alfa Aesar). The proteins were mixed with NuPAGE sample reducing agent (Thermo Fisher Scientific) and LDS sample buffer (Thermo Fisher Scientific) and heated at 70 °C for 10 min. To analyze for ELOVL5, proteins were electrophoresed in 4−12% Bis-Tris gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. Membranes were blocked with 3% filtered BSA in TBS-T (0.1% Tween 20 in TBS) for 1 h, followed by incubating with a primary antibody (ab205535, Abcam (1:5000)) in blocking buffer overnight at 4 °C. Membranes were washed with copious amounts TBS-T and then

incubated with a secondary antibody (goat antirabbit HRP, ab6721, Abcam (1:10000)) in PBS-T (0.1% Tween 20 in PBS) for 1 h. After washing, membranes were developed with Pierce ECL Plus solution (Thermo Fisher Scientific) and imaged using a ChemiDoc imaging system (Biorad). β-actin was used as a housekeeping control to ensure lanes were equally loaded. Primary antibody dilution was at 1:1500 (sc47778, Santa Cruz Biotechnology), and secondary antibody dilution was at 1:10000 (1705047, Bio Rad).

3. RESULTS AND DISCUSSION 3.1. Characterization of Amine-Modified pSiNPs. The fabrication of pSiNPs was adapted from a method previously described.25 After electrochemical etching, the porous film was electropolished from the Si wafer and fractured into particles by ultrasonication. To obtain uniform nanoparticles, size selection via ultracentrifugation allowed for the exclusion of ∼160 ± 3 nm particles in water, as confirmed by transmission electron microscopy (TEM) images (Figure 1A) and dynamic light scattering (DLS) measurements (Figure 1B). Pore size was measured to be 12.5 ± 3 nm as an average of 20 pores using ImageJ. Freshly anodized pSi remains highly reactive and unstable due to the reactive hydrogen terminated surface. In order to stabilize the surface and provide carboxyl groups for further modification, the pSiNPs were reacted with neat undecylenic acid (UA-pSiNPs) or 11-bromo-1-undecene (BrpSiNPs) via thermal hydrosilylation. The ζ potential value of pSiNP in water when functionalized with undecylenic acid or 11-bromo-1-undecene was −44 ± 7 and −42 ± 1 mV, respectively (Figure 1C). UA-pSiNPs or Br-pSiNPs were further modified with a suite of amine-containing molecules, including small diamine molecules (diethylenetriamine and tris(2-aminoethyl)amine), diamine poly(ethylene glycol) (PEG), polyamidoamine dendrimers (PAMAM) generations 1−4 (G1−4), and oligolysine dendrons (K1, K3, and K7) (Schemes S1 and S2). The structures of those molecules can be found in Figure 1D. Successful functionalization was verified via IR analysis (Figure S1). UA-pSiNPs were conjugated with the small diamine molecules, PEG and PAMAM dendrimers using EDC/NHS coupling, while the oligolysine dendrons were attached via click chemistry to the Br-pSiNPs. IR peaks at 1550 and 1650 cm−1 are key characteristics of N−H bending and CO stretching vibrations expected for these types of molecules. Changes in particle size and ζ potential were measured after the reactions. After the incorporation of amine molecules onto the pSiNP surface, the ζ potential changed from −44 mV (UA-pSiNPs) and −42 mV (Br-pSiNPs) to a range of +2 to +39 mV, depending on the amine molecule conjugated (Figure 1C). ζ potentials were measured in water, because the RNA loading solutions are predominantly water-based and the amine molecules conjugated to the pSiNP allow for favorable electrostatic interactions with the negatively charged phosphate groups on the RNA backbone. The particle size was measured by means of DLS after amine modification. In general, the immobilization of smaller amine molecules induced a higher increase in DLS size measurements most likely due to a lack of electrostatic repulsion between particles. However, the size for PEG- and generation 4 (G4) PAMAM-modified particles remained unaltered probably due to a combination of the high solubility of these molecules in water-based solvents and the increase of electrostatic repulsion between particles. 3.2. Loading and Protection Capacity of AmineModified pSiNPs. Amine-modified pSiNPs were evaluated 22997


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ACS Applied Materials & Interfaces for their capacity to load siRNA molecules. When the cationic particles are loaded with siRNA, there is a reversal of the ζ potential, due to the adsorbed siRNA molecules (Figure S2). To evaluate the effect of the siRNA concentration in solution to determine the loading efficiency of pSiNPs, PAMAM(G2)pSiNPs were incubated with siRNA solutions ranging from 95 to 950 ng/μL (Figure S3). There was no additional loading effect seen when the loading solution concentration was greater than 280 ng/μL; thus the concentration of siRNA was chosen for subsequent loading experiments between the different amine modifications. When incubated at a lower concentration of siRNA solution of 95 ng/μL, a loading efficiency of 58.8% can be observed, whereas when the concentration was >280 ng/μL, the loading efficiency decreases to 29%, despite more siRNA molecules being loaded into the same concentration of PAMAM(G2)-pSiNPs. Results show that, for small diamine molecules and oligolysine dendrons, loading remained relatively similar compared to the control UA-pSiNPs (Figure 2A). This suggests that the smaller amine molecules do not affect siRNA loading noticeably, possibly due to a lack of multivalency effects. In contrast, when functionalized with PAMAM dendrimers (Figure 2B), an increase in siRNA loading was observed with each successive generation. Information on the maximum loading capacity after 72 h can be found in the Supporting Information (Figure S4). PAMAM(G4)-pSiNPs were able to load 264 ± 4 and 413 ± 2 μg of siRNA/(mg of pSi) after 6 and 72 h, respectively. The loading efficiency of PAMAM(G4)pSiNPs was calculated to be 73.8%. The PAMAM(G4)-pSiNPs loading data obtained in this study showed higher loading compared with previous works including Joo et al.,20 where the authors obtained ∼120 μg/(mg of pSi). Additionally, similar work by Kang et al.21 and Kim et al.31 demonstrated ∼200 and ∼250 μg of siRNA/(mg of pSi), respectively. To confirm that the siRNA molecules were loaded into the porous structure and not simply adsorbed onto the aminefunctionalized surface of pSiNPs, the loading capacity of commercially available solid polystyrene nanoparticles of similar size was determined. Using the same surface functionalization carboxylic group and PAMAM(G4) coatingand loading conditionsincubation time and siRNA concentration, polystyrene and pSiNPs were compared for siRNA loading. When incubated with siRNA for 6 h, the UA-pSiNPs and PAMAM(G4)-pSiNPs were able to load 108 and 262 μg of siRNA/(mg of pSi), respectively. In comparison, the polystyrene nanoparticles with identical surface chemistry only loaded 3 and 53 μg of siRNA/(mg of polystyrene), respectively. While the surface chemistry of both materials was modified in the same way, the bulk material differed, which can lead to differences in behavior and adsorption of siRNA, and therefore, the results cannot be directly correlated with the amount of siRNA loaded within the pores. However, from the results obtained, we hypothesized that the majority (>80%) of siRNA molecules were loaded into the pores and not adsorbed onto the surface of the dendrimer surface (Figure S5). The porous structure of pSiNPs not only represents an ideal platform for high loading efficiencies but also has the potential to protect the payload from degradation. To confirm this protective effect of loading siRNA into porous nanoparticles, loaded UA-pSiNPs and PAMAM(G4)-pSiNPs were incubated with various concentrations of RNase A, a ribonuclease that hydrolyzes RNA molecules by endonuclease cleavage of the phosphodiester bonds.32 The pSiNPs were incubated with RNase A for 1 h, washed in PBS to remove RNase A, and

Figure 2. Loading profile (A and B) and ribonuclease degradation profile (C) of siRNA-loaded amine-modified pSiNPs. (A) Loading profile of small amine molecule and oligolysine dendron modified pSiNPs as a function of incubation time. (B) Loading profile of PAMAM-modified pSiNPs. (C) Percent of siRNA loaded in UApSiNPs and PAMAM(G4)-pSiNPs that is still intact after exposure to various concentrations of RNase A (0.001, 0.01, 0.1, 1, 10, 100, 1000, and 10000 ng/mL). Nanoparticle formulations loaded with siRNA were incubated in RNase A for 1 h, and the remaining amount of intact siRNA was quantified. Free siRNA was found to be degraded at RNase A concentrations > 0.01 ng/mL. When siRNA was loaded into UApSiNPs, intact siRNA was still detected at an RNase A concentration of 10 ng/mL, compared to PAMAM(G4)-pSiNPs where intact siRNA was still observed at 100 ng/mL. Data shown as mean ± S.D. (n = 4).

digested to recover the amount of intact siRNA that had remained in the particles. Exposure of naked siRNA to RNase A resulted in complete degradation after 1 h at RNase A concentrations as low as 0.01 ng/mL consistent with the results of Joo et al.20 At the same RNase A concentration, when loaded into different types of pSiNPs, no detectable siRNA degradation was observed (Figure 2C). At a 10× higher RNase A concentration of 0.1 ng/mL, 35% of the loaded siRNA had degraded in UA-pSiNPs, whereas all siRNA loaded into PAMAM(G4)-pSiNPs was recovered after RNase A incubationdemonstrating that modification with the globular dendrimer provided a better protective effect against ribonuclease degradation. An RNase A concentration of 1 μg/mL was required to degrade all loaded siRNA in PAMAM(G4)-pSiNPs. Additionally, we examined the protective effect for each aminemodified pSiNP at two different RNase A concentrations of 0.1 and 1 ng/mL. The general trend showed that, at both 22998


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their attraction to one another and hindering the loading. Results using ethanol as chaotropic agent showed that as the ethanol to water concentration increased, a decrease in the amount of siRNA loading was observed (Figure 3B). We also examined the effect of “fast” salt aging as described by Mirkin et al.36 Salt aging is a process in which the salt concentration is slowly increased every 20 min, for a total of 4 h. Previously, Mirkin et al. had reported that higher salt concentrations resulted in higher DNA loading.37 Here, we investigated the salt aging process in terms of loading of siRNA into the porous structure of pSiNPs (Figure 3C). We found that the addition of salt increased the amount of siRNA molecules loaded, plateauing at a salt concentration of 0.6 M. The 36% increase in siRNA loading at 0.6 M can be attributed to the reduction of repulsive forces between the RNA molecules as the concentration of counterions increases. Since salt aging involved the process of slowly introducing an increasing amount of counterions, we also investigated the effects of incubating the loading solutions with fixed salt concentrations (Figure 3D). Results obtained were similar to that of urea, low concentrations of salt such as 0.05 and 0.1 M increased siRNA loading by 19% and 14%, respectively, while, at concentrations above 0.5 M, a decrease in loading was detected when compared to a loading composition consisting of pure waterprobably due to the same interference phenomena discussed before when using urea. We hypothesize that, without the slow input of additional counterions by the salt aging process, the sudden influx of counterions promotes diffusion out of the nanoparticle pores. Additionally, to ensure that the increased loading amount did not originate from precipitation from nanoparticle aggregation in the presence of small quantities of urea or NaCl, the sizes of PAMAM(G2)-pSiNPs were measured via DLSresults showing that particle size remained unchanged (Figure S7). Finally, we investigated if a combination of salt and urea would produce an additive/synergistic effect resulting in an even higher siRNA loading. Because we previously observed that the highest increase in siRNA loading occurred in solutions when incubated with either 0.25 M urea or 0.05 M NaCl, we combined the two conditions to determine if these interactions would benefit each other. However, no enhancement in terms of siRNA loading was seen (Figure S8). It has been reported that NaCl and urea behave differently as salting agents for proteins.38 Whereas salting with NaCl disrupts the hydrogen-bonding network in proteins, urea molecules accumulate locally around the protein surface and replace water to initiate protein unfolding.38,39 Urea then is able to penetrate the interior of the protein in order to break the hydrophobic interactions. Computational models show that the addition of NaCl strengthens interactions between solute molecules and their surroundings causing an energy increase.38 However, in a urea solution, an energy decrease is predicted due to unfavorable solvent−solvent interaction.38 We hypothesize that these interactions may apply to nucleic acids, and as both chaotropic agents denature biological molecules differently, when added together, they cancel each other outwhich could explain the lack of additive effect toward siRNA loading. 3.4. siRNA Release Kinetics from Amine-Modified pSiNPs. The amount of siRNA released from the aminemodified pSiNPs was determined by incubating the particles in phosphate-buffered saline (PBS, pH 7.4, 37 °C). For all of the modified nanoparticles, an initial burst release was observed, ranging from approximately 8 to 20% depending on the

concentrations, larger amine molecules were able to protect the siRNA cargo better from degradation (Figure S6). These results confirm that pSiNPs are able to shield siRNA from RNase A degradation, and this protective effect was further enhanced when large amine molecules such as PAMAM(G4) are conjugated onto the surface. Therefore, the combination of physical propertiespore size, porosity, and nanoparticle sizeand chemical propertiessurface functionalization reported here have the potential to load an unprecedented amount of siRNA per particle. 3.3. Effects of Loading Solution Composition. Under native conditions, nucleic acids are covered by a hydrate shell consisting of water molecules.33 Chaotropic agents are known to disrupt the hydrogen-bonding network between water molecules while weakening the hydrophobic effect of RNA molecules. By weakening the hydrophobic interactions of the RNA molecules, more siRNA can potentially be loaded into the porous structure as the molecules are more tightly packed. Urea and ethanol both can behave as chaotropic agents,34,35 and thus their effect on RNA loading was examined for PAMAM(G2)pSiNPs. This surface modification was selected to analyze the effects of the loading solution composition, due to its ability to load and retain more siRNA molecules compared to unmodified pSiNPs. Higher generation PAMAM-modified pSiNPs were avoided as the high loading capacity may have masked any consequential loading due to solution composition. Our results showed that when PAMAM(G2)-pSiNPs were incubated with urea overnight, at low concentrations of 0.25 and 0.5 M, 21% and 19% increases in the amounts of siRNA were loaded into nanoparticles compared to a loading solution comprised purely of water (Figure 3A). When incubated with various concentrations of urea, ranging from 0.25 to 3 M, the amount of siRNA loaded decreased from 174 ± 16 to 126 ± 5 μg/(mg of pSi), respectively. We hypothesize that the excess of urea molecules interferes with the interaction of the positively charged pSiNP and the negatively charged siRNA, reducing

Figure 3. Effect of salt and chaotropic agents on the loading capacity of pSiNPs. PAMAM(G2)-pSiNPs were used for these loading profiles. The NPs were incubated with siRNA molecules in different loading solutions overnight (12 h), and the supernatant was measured for free siRNA to determine the effects of the loading composition on loading capacity. The effects of (A) urea incubation, (B) ethanol incubation, (C) salt aging, and (D) salt incubation were evaluated. Data shown as mean ± S.D. (n = 4). 22999


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Figure 4. (A) Release profile of siRNA from small amine molecule and oligolysine dendron modified pSiNPs when incubated in PBS under agitation at 37 °C. (B) Release profile of PAMAM-modified pSiNPs. (A, B) Data shown as mean ± S.D. (n = 4). (C) Cell viability results for C4−2B prostate cancer cells treated with different concentrations (10 (black), 50 (red), and 100 μg/mL (blue)) of amine-modified pSiNPs for 24 h and evaluated by an ATP-based luminescent cell viability assay. Cell viability was compared with the controls (untreated C4−2B cells or 1% Triton X-100 were the negative and positive controls, respectively). (D) Cellular association studies of amine-modified pSiNPs. C4−2B prostate cancer cells were incubated with FITC-siRNA-loaded amine-modified pSiNPs for 6 h and then examined using flow cytometry. Cellular association was compared to a negative control of cells without particles. (C−D) Data shown as mean ± S.D. (n = 3).

of dendrimers may help in trapping the RNA molecules within the pores for a prolonged period.43 An ∼54% release after 72 h was observed when coating the particles with PAMAM(G1), while, for the same time period, the release was decreased to 27% when modified with PAMAM(G4). However, although the release rate decreased with successive generations, a larger total amount of RNA was released from NPs modified with PAMAM(G4) due to the higher loading capacity compared to the lower generations (111.5 ± 2 μg of RNA/(mg of pSi) for PAMAM(G4), compared to 104.8, 83.1, and 96.4 μg/(mg of pSi) modified with G1, G2, and G3, respectively). In summary, we have demonstrated that when modified with large amine molecules such as PAMAM dendrimers, pSiNPs showed favorable loading and release kinetics for siRNA. We have defined favorable release kinetics as a slow sustained release rate as the slow release of siRNA into the cytoplasm may be more beneficial in order to prevent saturation of argonaute-2 (AGO2)avoiding competition between strands of siRNA.44 Thus, we established that a sustained siRNA release profile over 72 h was appropriate for gene silencing via intravenous delivery. The delivery of siRNA molecules into the cytoplasm is another crucial barrier to overcome in RNAi therapy; thus the transfection efficiency of amine-modified pSiNPs was investigated in prostate cancer cells. In this regard, their cytotoxicity, association to prostate cancer cells, and ability to silence a novel prostate cancer target (ELOVL5) was determined. 3.5. Cell Viability of Amine-Modified pSiNPs in C4−2B Cells. The in vitro cell viability of the amine-modified pSiNPs

conjugated amine molecules, followed by a general sustained release profile over the subsequent 72 h measured (Figure 4). We hypothesize that the initial burst release consists of the loosely bound or adsorbed siRNA molecules on the particle surfacewith the majority of the loaded siRNA molecules slowly diffusing out over time. A release profile of sustained siRNA release over a 3 day period is desirable for gene silencing.9,40−42 UA-pSiNPs released ∼20% of the payload after an hour and ∼70% by 12 h (Figure 4A). The pSiNPs modified with small amine molecules (PEG and tris(2-aminoethyl)amine) and oligolysine dendrons, with the exception of diethylenetriamine, showed less burst release (∼16%) in the first hour compared to the ∼20% released in the control UA-pSiNPs (Figure 4A). Over the first 12 h, oligolysine K3- and K7-functionalized pSiNPs showed ∼38% of the payload released, while ∼50% release was observed for PEG and tris(2-aminoethyl)amine-functionalized pSiNPs. In contrast, diethylenetriamine-modified pSiNPs showed that 72% of the cargo had been released in 12 h which was even higher than the UA-pSiNP control (∼70%). The PAMAM-functionalized pSiNPs, on the other hand, exhibited burst releasebetween 8 and 20% after an hour depending on the generation (Figure 4B). After this initial release, a sustained release rate of 0.48%/h of the payload was observed over the subsequent 72 h when modified with PAMAM(G1), while for the same time period, a release rate of 0.22%/h was seen for PAMAM(G4). This again highlights the effect of multivalency for these interactions. Furthermore, the compact globular shape 23000


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Figure 5. Confocal microscopy images of C4−2B prostate cancer cells treated for 1 and 6 h with PAMAM(G4)-pSiNPs. (A) The nuclei were stained with Hoechst 33342 (blue), the cytoskeleton was stained with Phalloidin-647 (red), PAMAM(G4)-pSiNPs were labeled with AF488 (green), and the siRNA was labeled with AF555 (yellow). (B) The orthogonal slices of the Z stacks compared the particle distribution between 1 and 6 h, where, after 6 h, the cargo had been internalized inside the cell (scale bar = 20 μm).

over all measured concentrations. Evidently, when C4−2B cells were treated with 100 μg/mL of siRNA-loaded PAMAM(G4)pSiNPs and stained with 7-AAD viability staining solution during ELOVL5 knockdown experiments, there was no difference in cell viability when compared to untreated cells (Figure S9). This could be attributed to the charge inversion when loaded with siRNA as shown in Figure S2, where when loaded with siRNA, PAMAM(G4)-pSiNPs, displayed a negative ζ potential of −21 mV. Thus, we concluded that amine-modified pSiNPs are biocompatible in vitro and suitable for gene delivery applications. 3.6. Cellular Association of Amine-Modified pSiNPs. Cellular association was evaluated for the various aminemodified pSiNPs (Figure 4D). The amine-modified pSiNPs were loaded with FITC-tagged siRNA overnight. C4−2B cells were incubated with the loaded NPs for 6 h and then washed copiously to remove unbound NPs. Cells were then sorted by

was evaluated. Cell viability was assessed on C4−2B cellsa clinically relevant cell derivative subline of the human prostate cancer cell line LNCaP,45 following incubation with different concentrations of UA-pSiNPs and amine-modified pSiNPs for 24 h using an ATP activity-based luminescence assay (Figure 4C). Cell viability was assessed over three concentrations, 10, 50, and 100 μg/mL. UA-pSiNPs showed no decrease in cell viability over the three concentrations of particles. For the amine-modified pSiNPs, no noteworthy cytotoxicity was noticed at 10 μg/mL. When particle concentration increased, cell viability fell between 85 and 100% at concentrations of 50 and 100 μg/mL, depending on the amine conjugated to the surface. The decrease in cell viability could be possibly attributed to the cationic charge of the amine-modified pSiNPs, where a higher surface charge led to a slight decrease in cell viability. However, as cell viability was >80%, we concluded that the amine-modified pSiNPs showed a good biocompatibility profile 23001


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Figure 6. (A, B) Flow cytometry data of C4−2B cells after incubation with pSiNP formulations. (A) Flow cytometry histogram evaluating the effect of PAMAM(G4)-pSiNPs on ELOVL5 protein expression after 72 h in C4−2B cells. (B) Percentage knockdown of ELOVL5 expression determined by flow cytometry for PAMAM(G2)-, PAMAM(G4)-, and K7-modified pSiNPs in different loading solutions consisting of only water or with 0.25 M urea. Data shown as mean ± S.D. (n = 3). (C, D) ELOVL5 expression in C4−2B cells exposed to lipofectamine or PAMAM(G4)-pSiNPs at different particle concentrations (25, 50, 75, and 100 μg/mL) carrying ELOVL5-targeting or scrambled siRNA, (C) measured by Western blotting and (D) quantified. Data shown as a mean ± S.D. (n = 4).

FACS for FITC fluorescence emitted by the siRNA molecules. Cells that interacted with pSiNPs showed an increase in fluorescence intensity due to the FITC-tagged RNA molecules. Since the negatively charged membrane would repel free siRNA and it would therefore be removed when the cells were washed, the fluorescence detected coming from the siRNA molecules confirm that they were either already internalized within the cell or still loaded in the pSiNPs attached to the cell membrane. FACS analysis showed that after 6 h of incubation, over 90% of cells had associated with the various amine-modified pSiNPs compared to 43.8% of cells interacting with bare UA-pSiNPs. When comparing different amine modifications, pSiNPs functionalized with tris(2-aminoethyl)amine, PAMAM(G1), and PEG modifications showed less cellular association (95, 94, and 88%, respectively) in comparison to the remaining amine modifications, which showed near 100% FITC-positive cells. For the Tris and PAMAM(G1) modifications, we suspect that, due to aggregation determined by a larger DLS size distribution, the particles may not be associating or internalizing with the cellular membrane as efficiently as the NPs modified with larger amine-containing molecules that avoid aggregation. The lower cellular association percentage of 88% for PEG-modified pSiNPs

is consistent with previous reports showing that PEG-coated nanoparticles show weak interactions with cells and a stealth effect.46 Confocal microscopy was used to further assess the cellular interactions between C4−2B cells and amine-modified pSiNPs (Figure 5A). PAMAM(G4)-pSiNPs were labeled with an Alexa Fluor 488 dye and subsequently loaded with fluorescently tagged Alexa Fluor 555 siRNA. The loaded PAMAM(G4)pSiNPs were incubated with C4−2B cells for 1 and 6 h. At 1 h, confocal images showed that the NPs interacted with the cell, where the orthogonal slice of the z-stack showed that the majority of the loaded particles rested on the cell surface. In contrast, at 6 h, confocal z-stacks showed that the NPs were more dispersed and were found to be inside the cytoplasm of the cell (Figure 5B and Figure S10). The data corroborate the potential of amine-modified pSiNPs as potent carriers for siRNA delivery. When taken together, larger amine molecules such as the higher generations of PAMAM dendrimers and oligolysine dendrons show favorable characteristics. We have shown that these favorable characteristics of amine-modified pSiNPs, especially those modified with PAMAM dendrimers (G2−G4) are able to (i) load more siRNA 23002


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Our results further advance the understanding of the parameters that govern the RNA loading capacity in porous nanomaterials and contribute to the development of a set of general design rules that are essential for achieving increased efficacy in gene therapy applications.

per milligram of pSi, have a sustained release over 72 h, and protect the siRNA cargo when compared to UA-pSiNPs; (ii) show low cell cytotoxicity to C4−2B prostate cancer cells, and (iii) have near 100% cellular interaction. The next step was to see if the amine-modified pSiNPs were able to provoke gene silencing. 3.7. Knockdown of ELOVL5 in C4−2B Cells. To evaluate the ability of amine-modified pSiNPs in mediating gene-specific knockdown, we used siRNA against ELOVL5, an endogenously expressed gene in humans. The levels of ELOVL5 protein expression in C4−2B cells were quantitatively determined by flow cytometry and Western blotting. Three different amine-modified pSiNPs were evaluated for their in vitro gene knockdown capabilities, namely, PAMAM(G2), PAMAM(G4), and K7 based on their favorable loading capacity, release kinetics, cell viability, and cellular association. The amine-modified pSiNPs were loaded with ELOVL5-siRNA in water or 0.25 M urea overnight and incubated with C4−2B cells for 72 h. Protein expression was then quantified by means of flow cytometry. Treatment with PAMAM(G2) and K7-modified pSiNPs loaded in water reduced ELOVL5 protein expression by 49% and 48%, respectively (Figure 6B)while when an increase in RNA was loaded into PAMAM(G2)-pSiNPs in the presence of 0.25 M urea, protein expression was reduced by 65%. In comparison, PAMAM(G4)-modified pSiNPs were able to reduce ELOVL5 protein expression by 85% when loaded in a solution of water and 93% when loaded in a solution of 0.25 M urea using FACScompared to the lipofectamine-based knockdown of 65% (Figure 6A,B). To eliminate the possibility of gene silencing caused by toxicity, amine-modified pSiNPs loaded with a scrambled sequence of siRNA and without siRNA payload were evaluated. No gene silencing or cytotoxicity was observed, validating that the downregulation of ELOVL5 was likely due to the combination of the amine-modified pSiNPs loaded with ELOVL5-targeting siRNA. Western blot data showed that the increase in concentration of PAMAM(G4)-pSiNPs, thus an increase in concentration of siRNA, correlates with an increase in ELOVL5 knockdown (Figure 6C,D). Western blots also confirmed the results obtained by FACS showing a knockdown of 93% in protein expression at a particle concentration of 100 μg/mL. Taken as a whole, we conclude that by altering the surface chemistry of pSiNPs with amine-containing surface modifiers, the nanocarriers are able to deliver siRNA into the cytoplasm, successfully escape endosomal degradation, and elicit a gene silencing effect with no cytotoxicity observed.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05577.

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Additional figures of characterization and further experimental results (PDF)

AUTHOR INFORMATION

Corresponding Authors

*(A.C.-R.) E-mail: [email protected]. *(N.H.V.) E-mail: [email protected]. ORCID

Helmut Thissen: 0000-0002-3254-6855 Anna Cifuentes-Rius: 0000-0002-9478-2239 Nicolas H. Voelcker: 0000-0002-1536-7804 Funding

This work was supported by the Prostate Cancer Foundation of Australia (Grant NCG 1816 to L.M.B., A.C.-R., and N.H.V.). T.T. and N.H.V. acknowledge support from the Office of the Chief Executive and CSIRO Manufacturing. T.T. acknowledges support of an Australian Government RTP scholarship. A.C.-R. acknowledges the National Health & Medical Research Council (NHMRC) of Australia (Grant GNT1112432). S.D. thanks the Department of Science & Technology (DST), New Delhi, India for the INSPIRE Fellowship. L.M.B. is supported by a Principal Cancer Research Fellowship produced with the financial and other support of the Cancer Council SA’s Beat Cancer Project on behalf of its donors and the State Government of South Australia through the Department of Health. L.M.B. acknowledges grant support from The Movember Foundation/Prostate Cancer Foundation of Australia (MRTA3). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge use of facilities within the Monash Centre for Electron Microscopy. This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF). We thank Winston Liew, Chris Sheedy, and the Biophysics group from CSIRO Manufacturing for performing ICP-OES analysis. We also thank MCIM arquitectura for providing us with the schematic representation of the porous silicon nanoparticle in Scheme 1,,.

4. CONCLUSION The intention of the study was to determine parameters that are able to facilitate maximum siRNA loading into pSiNPs. We have demonstrated that by modifying the surface chemistry and adding low concentrations of salt or urea in the oligonucleotide loading solution, we were able to maximize the amount of RNA molecules per nanoparticle. From the different amine-modified surfaces developed and tested in this study, PAMAM(G4)pSiNPs were able to load the highest amount of RNA molecules, displayed a sustained release over 72 h, protected the cargo from degradation, and delivered it into the cytoplasm effectively, and thus inhibited ELOVL5 expression by 93%. Although pSiNPs were used in this instance, these findings may also be adapted to different types of organic or inorganic porous nanoparticles, providing a platform and guideline for maximizing RNA loading.

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on June 24, 2019, with an incomplete Supporting Information file. The corrected version was reposted on July 3, 2019.

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Maximizing RNA Loading for Gene Silencing Using Porous Silicon

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