In-depth study on the gene silencing capability of silica nanoparticles with different pore sizes: degree and duration of RNA interference

Abstract

The mesoporous silica nanoparticle (MSN) has been utilized for various drug delivery systems. For the efficient loading and functional delivery of bioactive molecules, the pore size should be finely controlled. Here, we synthesize MSNs having different pore sizes and investigate the relationship between the pore size of the MSN and the efficacy and kinetics of gene silencing induced by siRNA loaded MSNs. We found that MSNs with various pore sizes present great differences in gene down-regulation of green fluorescence protein (GFP), vascular endothelial growth factor (VEGF) and hepatitis C virus non-structural protein 3 (HCV NS3) as well as the endocytosis pathways of MSNs in cancer cells. In specific, MSNs with a 7 and 10 nm pore size show the most significant gene knockdown with a long-lasting effect and great protective effect of the cargo from nuclease-mediated degradation compared to MSNs with a 2, 4 and 23 nm pore size. In addition, although similar amounts of siRNA was delivered inside cells, the degree of gene knockdown was significantly different depending on pore size of the employed MSN. The present study indicates that the porosity in nanoparticles acts as a crucial factor in a potent biomolecule cargo delivery system and the optimized MSN based system should provide better opportunities in the application of porous nanoparticles in biomedical research and clinical applications in the future.

---

1. Introduction

RNA interference (RNAi) has attracted great interest as a useful research and therapeutic tool since its report demonstrating the sequence-specific target gene knockdown in mammalian cells by the treatment of exogenous synthetic siRNA in 2001.¹ The siRNA therapeutics are advantageous over conventional small molecule based drugs in terms of utilization of natural intracellular machinery and simplicity in the design of new drugs by adapting the gene sequences responsible for target disease.^2–6 However, the impermeability of siRNA through the cell membrane and the fast degradation of siRNA upon exposure to RNases in biological fluids necessitate the delivery vehicle for siRNA to reach into the cytoplasm where it initiates RNAi.^7,8 Although a lot of efforts have been devoted to develop the effective siRNA delivery system, there are still strong needs for clinically relevant, safe and efficient siRNA delivery vehicle. According to recent reports, various nanomaterials serve as valuable platforms for siRNA delivery due to their unique physicochemical properties.^9–18 Among them, mesoporous silica nanoparticle (MSN) is one of the attractive materials due to its biocompatibility, high porosity, structural stability and ease of surface functionalization. Since the first report that showed its potential as a drug delivery vehicle in 2001, MSN has been intensively studied to substantialize the smart drug delivery mainly utilizing its porosity.^19–27 However, the relatively small pore size (∼2 nm) in conventional porous silica nanoparticle has limited the application of MSN as a carrier of bio-molecular drugs such as nucleic acids and proteins. In this regard, there has been a strong need for MSN possessing pores larger than ∼5 nm in diameter. Several strategies have been developed to obtain MSN possessing large pores using specialized surfactant and polymer as structure directing agents that generate large pores.^28–30 However, it is still challenging to synthesize MSN having large pores with narrow size distribution. In addition, the cost effective and scalable fabrication process is required in the preparation of nanoparticle for successful industrial production and clinical translocation.

Recent several reports demonstrated that pore size plays a crucial role in drug loading and release rate for both small drugs and proteins.^31–33 Moreover, biomolecules such as proteins and nucleic acids exhibit various sizes ranging from several to a few tens of nanometers. Therefore, the pore size of the delivery carrier is needed to be customized for its specific purpose. For siRNA loading and delivery, the differences of pore size might affect the loading capacity, release profile, and protective effect for the loaded siRNA from nuclease mediated degradation, considering potential key factors of gene regulation.

Here we report a strategy on the facile pore size tuning of MSN, ranging from 2 to 23 nm in diameter, and its siRNA loading, delivery efficacy and duration of gene knockdown, targeting green fluorescence protein (GFP), vascular endothelial growth factor (VEGF) and hepatitis C virus non-structural protein 3 (HCV NS3) (Scheme 1). In addition, endocytosis route of each MSN is investigated by using inhibitors of each endocytosis pathway. We demonstrate that the control over siRNA release rate based on pore size tuned MSN is significantly related to not only the gene knockdown efficacy but also the time dependent changes in the degree of gene knockdown.

Scheme 1 Representative illustration of siRNAs loaded on MSNs. Pore size could be a key parameter in controlling the loading capacity of siRNA, the protective effect of MSNs for siRNA from being degraded by nucleases, the release kinetics of siRNA, and gene knockdown kinetics.

2. Experimental section

2.1. Materials

3-Aminopropyltriethoxysilane (APTES), tetramethyl orthosilicate (TMOS), toluene, dimethylsulfoxide (DMSO), and mesitylene (trimethyl benzene, TMB) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Ethanol was purchased from Merck (Darmstadt, Germany). Cetyl trimethyl ammonium bromide (CTAB) was purchased from Acros (New Jersey, USA). Sodium hydroxide was purchased from Junsei Chemical Co. (Tokyo, Japan). All the reagents were used as received without further purification. Phosphate buffered saline (PBS, pH 7.4), Dulbecco's Modified Eagle's Medium (DMEM), and fetal bovine serum (FBS) were purchased from WelGENE Inc. (Daegu, Korea). RNase and heparin were purchased from Sigma Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA). The siRNAs targeting human VEGF, hepatitis C virus (HCV) non-structural protein 3 (NS3) and green fluorescent protein (GFP) were obtained from Bioneer Inc. (Daejeon, Korea). All PCR primers were purchased from Genotech, Inc. (Daejeon, Korea).

2.2. Synthesis of mesoporous silica nanoparticles (MSNs) with different pore size

MSN2 and MSN23 were prepared according to our previous report.³⁴ MSN4, MSN7, and MSN10 were prepared as described below. For the synthesis of MSN containing template molecules, 3.94 g of CTAB and 2.28 mL of 1 M NaOH solution were dissolved in 800 g of methanol/water (0.4/0.6 = w/w). With vigorous stirring, 1.3 mL of TMOS was added to the reaction mixture under ambient condition. The obtained white suspension was aged overnight after stirring for 8 h. The resulting white precipitate was collected by centrifugation and filtration and washed with ethanol and water 5 times each. To prepare the pore size tuned MSNs, as-synthesized silica nanoparticles were dispersed in ethanol by sonication for 30 min, followed by addition of 20 mL of 1 : 1 mixture (v/v) of water and TMB. The amount of TMB was varied in this step—volumes of TMB were 1, 3 and 5 mL for MSN4, MSN7, and MSN10, respectively. The mixture was placed in an autoclave and kept at 100 °C for 4 days. The resulting white powder was washed with ethanol and water 5 times each. To extract out the template, the materials were dispersed in 2 M ethanolic HCl solution and the dispersion was placed in an autoclave, followed by heating at 140 °C for 20 h. The solutions were filtered, washed with ethanol and water extensively, and dried in a vacuum oven. The pore size, surface area, and pore volume of the prepared MSNs were analysed through nitrogen sorption experiments. Nitrogen adsorption isotherms were obtained at the temperature of liquid nitrogen (77 K) using a volumetric Micromeritics Tristar-II instrument. Prior to the measurements, the sample was degassed for 12 h at 573 K. The specific surface area was calculated by the Brunauer–Emmett–Teller (BET) equation using the adsorption data obtained in the pressure range P/P₀ = 0.05–0.2, whereas the pore size distribution was analyzed with the adsorption branch of the nitrogen adsorption isotherm according to the Barrett–Joyner–Halenda (BJH) algorithm.

2.3. Preparation of amine functionalized MSNs

The template extracted materials (100 mg each) were suspended in toluene (10 mL), followed by the addition of APTES (1 mL). The suspension was refluxed overnight, filtered out and washed with ethanol and water extensively. After removing moisture and ethanol by drying, the white powders were dispersed in distilled water to make a stock solution at 10 mg mL⁻¹ concentration and the solution was stored at 4 °C.

2.4. Cell culture

GFP expressing HeLa (GFP-HeLa) cells and MDA-MB-231 cells were cultured in DMEM containing 4.5 g L⁻¹ D-glucose and supplemented with 10% FBS, 100 units per mL penicillin, and 100 μg mL⁻¹ streptomycin. Cells were maintained in a humidified incubator under 5% CO₂ at 37 °C. Huh7 replicon (Huh7-rep) cells containing HCV gene were cultured in the same culture media containing 500 μg mL⁻¹ of G418 antibiotics (A.G. Scientific Inc., USA).

2.5. RNase protection assay

The siRNA loaded MSN (siRNA@MSN) complexes were prepared to the final concentration of 25 pmol of siRNA against GFP (siGFP) with 20 μg of particles in 5 μL of PBS. The complexes were incubated for 1 h at room temperature. After adding RNase at a final 0.05% concentration, samples were incubated for varying time period at 37 °C. The samples were treated with heparin and analysed by polyacrylamide gel electrophoresis (PAGE).

2.6. Cell viability test

The cytotoxicity of each particle was evaluated by measuring the viability of HeLa cells after the treatment of MSNs using CCK-8 assay. HeLa cells were seeded in a 96-well cell culture plate 24 h before MSN treatment at a density of 1 × 10⁴ cells per well. The cells were then treated with varying concentrations of the prepared MSNs in PBS and control cells were treated with equivalent volume of PBS. After 24 h, media was removed and 100 μL of serum-free media and 10 μL of CCK-8 stock solution were added to each well. The cells were further incubated for 1 h. The optical density of formazan salt at 450 nm wavelength and 670 nm were measured using a microplate absorbance reader (Molecular Devices, Inc., USA), and background absorbance of media was subtracted. Experiments were carried out in triplicate, and data were shown as mean ± SEM.

2.7. Cellular uptake study

Prior to the MSN treatment, HeLa cells were seeded in a 12-well plate and placed in an incubator. After 24 h, cells were treated with chlorpromazine (10 μg mL⁻¹) and genistein (200 μM) which are known to inhibit the clathrin- and caveolae-dependent endocytosis at 37 °C, respectively. To inhibit macropinocytosis, amiloride (50 μM) were treated at 37 °C. To inhibit energy dependent cellular uptake, the cells were incubated at 4 °C for 1 h in serum-free media. Those pre-treated cells were then incubated with 20 μg mL⁻¹ of MSNs for 1 h, and then washed with 2 mL of 1× PBS 5 times. For quantitative assessment of cellular uptake of MSNs by flow cytometry, cells were trypsinized and harvested. Each group of cells was centrifuged at 4 °C and washed with cold 1× PBS. After centrifugation, cells were re-suspended in 1× PBS containing 1% FBS. Fluorescence intensities of Cy5-labeled siRNA were measured using Aria III flow cytometer (Becton Dickinson, USA) equipped with an argon laser. Experiment was carried out in triplicate, and data were shown as mean ± SEM. To monitor the cellular uptake of MSN into Huh7-rep cells, HCV NS3 targeting siRNA@MSN complex were treated to 5 × 10⁴ of Huh7-rep cells in a 24-well culture plate with cell culture media omitting serum. The media was replaced with serum-containing media after 4 h incubation, followed by observation of fluorescence signals by using a Ti-inverted fluorescence microscope (Olympus, Japan) with 20× objective lens. Manders' overlap coefficient was calculated based on Image J software.

2.8. GFP knockdown

GFP-HeLa cells were seeded in a 24-well cell culture plate at a density of 2 × 10⁴ cells per well 24 h prior to transfection. The siRNA@MSN complexes were prepared as follows; 25 pmol of siGFP was incubated with 20 μg of MSNs in 1× PBS at room temperature for 1 h. Each mixture was treated to the cells in serum-free media and incubated for 4 h at 37 °C. After 4 h, cells were washed with 1× PBS and incubated for additional 44 h and 92 h in serum-containing media. Following incubation, gene knockdown and cellular uptake property were investigated by observing the optical images using a Ti-inverted fluorescence microscope with 10× objective lens. The flow cytometry analysis was performed to quantify the knockdown efficiency. The fluorescence intensity was measured with a FACS Aria I flow cytometer (BD bioscience, USA). Transfection efficiency was shown as mean fluorescence of GFP. Experiments were carried out in triplicate, and data were shown as mean ± SEM.

2.9. VEGF and HCV NS3 knockdown

MDA-MB-231 cells and Huh7-rep cells were seeded in a 12-well cell culture plate at a density of 4 × 10⁴ cells per well 24 h prior to transfection and then, siRNA@MSN (50 nM) complexes were treated to the cells in serum-free media. After incubation for 4 h, media was removed, washed with 1× PBS and then, replaced with serum-containing media. The cells were trypsinized and harvested after additional incubation for 44 h and 92 h. The expression level of VEGF and HCV NS3 mRNA was estimated by a reverse transcription polymerase chain reaction (RT-PCR) analysis. Total RNA was isolated by using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. RT-PCR was performed using a Super-Script™ II RT kit (Invitrogen, Carlsbad, CA). RT-PCR was carried out under following thermal cycling conditions: cDNA synthesis; 1 cycle 65 °C for 5 min, 42 °C for 2 min, 42 °C for 50 min, inactivation at 70 °C for 15 min, denaturation; 1 cycle 94 °C for 2 min, PCR amplification; 32 cycles at 94 °C for 20 s, at 60 °C for 30 s, and at 72 °C for 30 s, final extension; 1 cycle 70 °C for 5 min (for PCR amplification of HCV NS3, 2 min at 95 °C, (60 s at 95 °C, 60 s at 62 °C, 30 s at 72 °C) × 35 cycle, for PCR amplification of GAPDH (GDH), housekeeping gene, 5 min at 94 °C, (30 s at 94 °C, 30 s at 60 °C, 30 s at 72 °C) × 26 cycles). The PCR products were separated in a 0.9% agarose gel by electrophoresis and analyzed by using a Gel Doc (ATTO, Korea). Each band intensity was quantified by using Image J software, and both VEGF and HCV NS3 mRNA band intensities were normalized by comparing to GDH gene expression band intensities. The sequence information of primer is as follows.

(1) GDH forward primer:

5′-TTGTTGCCATCAATGACCCCTTCATTGACC-3′

(2) GDH reverse primer:

5′-CTTCCCGTTCTCAGCCTTGACGGTG-3′

(3) Human VEGF forward primer:

5′-AGGAGGGCAGAATCATCACG-3′

(4) Human VEGF reverse primer:

5′-CAAGGCCCACAGGGATTTTCT-3′

(5) HCV NS3 forward primer:

5′-CCTACTGGTAGCGGCAAGAG-3′

(6) HCV NS3 reverse primer:

5′-CTGAGTCGAAATCGCCGGTA-3′

3. Results and discussion

The pore size of MSN was successfully tuned by adjusting the amount of 1,3,5-trimethylbenzene (TMB) during pore expansion process under hydrothermal condition. By employing different hydrothermal conditions, we prepared MSNs having five different mean pore sizes—2, 4, 7, 10 and 23 nm in diameter. TEM images of the prepared MSNs showed different pore sizes with similar particle size of ∼200 nm (Fig. 1). According to the nitrogen sorption experiments, pore expansion process resulted in the increase of pore volume and the decrease of surface area (Table 1 in ESI†). Generally, customized polymers and/or surfactants are required for the synthesis of MSNs having large pores. The present pore expansion approach enabled the reproducible and scalable synthesis of MSN having relatively large pores with control over pore size by varying TMB amount and reaction temperature. The pore expanded silica nanoparticles were next modified with amine functional groups by treating 3-aminopropyltriethoxysilane (APTES) to generate positively charged pore surface needed for the loading of siRNA molecules into the pores of silica nanoparticles via electrostatic interaction. The degree of modification with amine moieties was revealed as 3.1–3.6 mmol g⁻¹ through elemental analysis. Loading capacity for siRNA was measured by gel electrophoresis utilizing siRNA against GFP. MSNs having 7, 10 and 23 nm pores showed similar siRNA loading capacity as 1.25 pmol μg⁻¹ whereas MSNs having 2 and 4 nm pores presented lower values as 0.55 and 0.8 pmol μg⁻¹, respectively.

Fig. 1 TEM images of the prepared silica nanoparticles having pores with varying sizes. The pore size of the porous silica nanoparticle was successfully tuned from 2 to 23 nm.

Prior to the investigation of siRNA delivery efficiency of MSNs, we evaluated the viability of mammalian cells after treatment of MSNs and the intracellular uptake mechanism of MSN depending on pore size. More than 90% of HeLa cells were viable under the concentration ranges of the applied MSNs with no significant cytotoxicity (Fig. S1†). Endocytosis pathway of nanoparticles is known to be affected by various physicochemical properties of nanomaterials including size, surface roughness and surface functional groups. Fig. 2a showed that intracellular uptake of MSN2, where number ‘2’ denotes for average pore diameter of 2 nm, was largely decreased by pretreatment of genistein and amiloride, which are inhibitors of the caveolae-dependent endocytosis and macropinocytosis, respectively.^35,36 The pore expanded MSNs—MSN4, 7, 10 and 23—showed decreased intracellular uptake under the pretreatment of genistein and chlorpromazine, inhibitors of the caveolae- and clathrin-dependent endocytosis, respectively.^37,38 Furthermore, MSN2 showed lower energy dependency in endocytosis compared to other MSNs as shown in the cells treated with MSNs at 4 °C. The data suggested that the cellular internalization pathway could be changed with very little modification like pore size in this study, even if its composition, particle size, and surface charge are similar.

Fig. 2 (a) Pore size dependent cellular uptake properties investigated by the treatment of each inhibitor that prevents clathrin-, caveolae-dependent endocytosis and macropinocytosis. (b) The protection of siRNA from the RNase mediated degradation. Top gel: siRNA was released from MSNs by the treatment of heparin in the absence of the RNase, bottom gel: the siRNA loaded MSNs and naked siRNA were incubated with RNase for 2 h. Gel analysis revealed that MSN7, 10 and 23 could provide better protection for the loaded siRNA compared to MSN2 and 4.

Next, we examined the protective effect of MSNs for the loaded siRNAs against the nuclease mediated degradation. The same amount of siRNA was first incorporated to each MSN having different pore size and the siRNA@MSN complex was treated with ribonuclease (RNase). In the gel retardation assay (Fig. 2b), we observed that the majority of siRNA molecules loaded to the nanoparticles exist as intact forms in case of MSN7, 10 and 23 even after the incubation with RNase for 2 h. In detail, the MSN7and MSN10 showed slightly better protection compared to MSN23. The siRNA loaded to MSN7 was not completely released out from the pores under the treatment of heparin, highly polyanionic polymer that is expected to disturb electrostatic interaction between siRNA and MSN pore surface, probably due to unfavorable trade between siRNA and heparin originated from very tight pore size adequate for siRNA rather than heparin. In general, the siRNA based therapy requires the repeated treatment of drugs due to the degradation of siRNA molecules by RNase and the dilution of intracellular drug concentration over time. The protection of siRNA from degradation could enhance the therapeutic efficacy of the delivered siRNAs inside cells. In this respect, it is expected that the enhanced cargo protection in the MSN based drug delivery system might elicit longer gene knockdown duration through the prolonged RNAi. Recently, Horcajada group reported that the pore size plays an important role in release kinetics.³⁹ According to the report, release rates of ibuprofen adsorbed on MCM-41 type mesoporous matrix are proportional to the pore size. In this regard, it is expected that the pore size may affect the release kinetics of the siRNA loaded on/in MSN, enabling the control of release pattern—burst or sustained manner. Furthermore, pore size might determine the local site where major binding events of siRNA happen on the surface of each silica particle. The major portion of siRNA molecules might interact with MSN2 on the outer surface rather than inside pore surface since the free accessibility of siRNA molecules into the pores of MSN2 might be restricted due to its pore size smaller than siRNA. On the other hand, the siRNA molecules could bind not only onto outer surface but also onto inner surface in case of MSNs having large pores than siRNA itself. One of the plausible evidence might be found by measuring the changes of zeta potential of MSNs before and after the siRNA loading. Fig. S2† showed the measured zeta potential values of MSNs before and after siRNA loading. In case of MSN2 and MSN4, the loading of siRNA molecules induced larger changes with decrease of charge in zeta potential values compared to that of MSN7, 10 and 23. The data indicates that the binding of siRNA molecules on MSN7, 10 and 23 results in smaller changes in the surface net charge than other MSNs, suggesting the more favorable interaction of siRNA with inner surface of MSN.

The control of release behavior may change incubation time required for maximum gene knockdown and the duration of the gene silencing as well. The cell proliferation rate is one parameter in determining apparent knockdown duration due to the dilution of concentration of siRNA by cell division and thus, we first investigated the proliferation of HeLa cells upon treatment of MSNs at our working concentration, 80 μg mL⁻¹. The result showed the similar proliferation of the MSN treated cells to the control group during 48 h with little cytotoxicity (Fig. S3†). Next, to investigate the properties of gene down-regulation depending on pore size, the knockdown efficiency was compared at 48 h and 96 h after treatment of the GFP targeting siRNA (siGFP) loaded MSN complex (siGFP@MSN). As shown in Fig. 3a, the notable decrease of green fluorescence in GFP-HeLa cells, HeLa cells constitutively expressing GFP, was observed after treatment of siGFP@MSN for different time period. As a control, green fluorescence decrease was not observed in the cells treated with MSN alone (Fig. S4†). Next, quantitative evaluation of GFP knockdown was performed by the analysis of green fluorescence in GFP-HeLa cells using flow cytometry. Fig. 3b showed the flow cytometry histograms of cell populations versus green fluorescence intensity. With increasing pore size from 2 to 23 nm, the mean green fluorescence gradually decreased in the cells treated with siGFP@MSN down to 89, 47, 24, 14, and 12% relative to control group at 48 h post-transfection. It is notable that MSN mediated siGFP delivery showed higher gene silencing of GFP than lipofectamine2000 (Lipo) mediated transfection and the expression of GFP was silenced almost perfectly in the siGFP@MSN10 and siGFP@MSN23 treated cells. At 48 h post-transfection, GFP gene down-regulation in HeLa cells treated with siGFP@MSN10 and siGFP@MSN23 showed little difference. However, the knockdown induced by siGFP@MSN10 was more effective after relatively longer incubation for 96 h. Collectively, MSN7, MSN10 and MSN23 showed outstanding efficacy in functional siRNA delivery in GFP-HeLa cells among the tested MSNs. In specific, for more effective gene knockdown at longer incubation such as 96 h, MSN7 would be better option than MSN23. On the other hand, to achieve similar degree of gene knockdown at 48 and 96 h, MSN10 would be the best siRNA delivery carrier.

Fig. 3 Regulation of GFP gene silencing by controlling pore size of MSN. (a) Fluorescence images obtained after the incubation of GFP expressing HeLa cells with siGFP@MSN complex for 48 h and 96 h. (b) Quantitative analysis of GFP gene knockdown in GFP-HeLa performed by using flow cytometry. Scale bar is 100 μm.

Next, as an important therapeutic target in tumor growth and metastasis, siRNA targeting VEGF (siVEGF) was chosen to further prove the gene delivery efficiency of pore size tuned MSNs. As shown in Fig. 4, consistent to the results in down regulation of GFP expression, the VEGF knockdown at 48 h was efficient in MDA-MB-231 cells (breast cancer cell line) treated with siVEGF@MSN10 and siVEGF@MSN23 complex. To assess the influence of the pore size on VEGF knockdown duration, the knockdown efficiency was examined at 96 h post-transfection. The result revealed that MSN7 and MSN10 served as efficient siRNA delivery vehicle for both GFP and VEGF in relatively long-term period in which MSN7 was more favorable for better gene knockdown at longer time period. Overall, the result suggests that the pore size influences on the knockdown duration and the incubation time required for maximum knockdown. One possible explanation for the differences in the knockdown efficiency in MSN10 and MSN23 is that the degradation of siRNA in intracellular environment by nuclease might be slower in MSN10, as shown in Fig. 2b, hindering the access of nuclease to the cargo loaded inside pores. In addition, the delayed knockdown tendency in MSN7 could be due to the delayed release of the loaded siRNA from particle as indicated in gel retardation assay in Fig. 2b. Furthermore, according to quantitative measurement of the amount of intracellularly uptaken siRNAs performed by flow cytometry after the treatment of the complex of Cy5-labeled siRNA (Cy5-siRNA) and MSNs, we found that MSN2 and MSN4 could deliver similar amount of siRNA molecules to the other MSNs across cellular membrane although they showed much lower knockdown efficacy than the other MSNs (Fig. S5†). The data emphasizes the fact that for efficient gene knockdown, the protection of siRNA from nuclease attack in cytoplasm is also very important in addition to efficient translocation of siRNA molecules across cell membrane with the release from the MSN vehicles inside cells.

Fig. 4 VEGF gene knockdown in MDA-MB-231 cells based on pore size tuned MSNs observed to investigate the relationship between pore size of MSN and VEGF gene knockdown properties depending on incubation time. (a) Gene expression levels were evaluated by RT-PCR and gel electrophoresis. (b) The band intensities of VEGF were normalized compared to expression level of GDH with bar graph by using Image J software.

Finally, we demonstrated target gene knockdown efficiency of MSN based siRNA delivery system in viral disease model since the disease-causing viral infection attracted significant attention recently. In the present study, hepatitis C virus (HCV) non-structural protein 3 (NS3) targeting siRNA was delivered by using MSN10 to prove the therapeutic effect through direct inhibition of HCV gene replication. The NS3 plays a key role having both serine protease and RNA helicase activity, which are essential for HCV replication in human liver cells and has been an important therapeutic target to treat HCV.^40–42 As shown in Fig. 5a, HCV NS3 targeting siRNA (siNS3) was successfully transfected to Huh7 replicon (Huh7-rep) cells mediated by MSN10 and significant down-regulation of NS3 gene expression was shown by RT-PCR and gel analysis (Fig. 5b). MSN mediated siNS3 transfection showed more effective knockdown down to 17.9% of NS3 expression than Lipo-mediated siNS3 transfection which resulted in 46.7%, with lower cytotoxicity of MSN (cell viability: 94.8%) compared to Lipo (cell viability: 81.6%) under the same condition. Collectively, the data demonstrated that MSN could be an attractive siRNA delivery carrier and pore size tuning of MSN showed an improved effect for RNAi system to silence therapeutically relevant HCV NS3 and VEGF gene expression in addition to knockdown of GFP expression.

Fig. 5 The siRNA targeting HCV NS3 gene (siNS3) delivered by using pore size optimized MSN10. (a) Distribution of fluorescent dye Cy5-labeled siNS3 in Huh7-rep cells. Red fluorescence of siNS3 was observed in cytoplasm, indicating successful intracellular uptake of siNS3 assisted by MSN. (b) Gene expression levels of HCV NS3 and GDH were evaluated by RT-PCR and gel electrophoresis. The band intensities of HCV NS3 were normalized compared to expression level of GDH by using Image J software. Scale bar is 20 μm.

4. Conclusions

In conclusion, we demonstrated the highly practical synthetic method for MSNs with tunable pore sizes through simple and scalable pore expansion procedure. The developed MSNs are expected to be applied for the loading and delivery of versatile bio-molecule drugs, from small to large size molecules. In this report, the synthesized MSNs were applied to siRNA delivery of which dimension is about 2 × 7.5 nm.⁴³ The result suggests that the pore size plays critical roles in the knockdown properties for a model gene (GFP) and therapeutic target genes (VEGF and HCV NS3) as well. Although the more detailed mechanistic study has to be carried out further, our results suggest and emphasize the significance of the pore size in porous nanomaterials used for drug delivery system. Using the pore size tuned MSN system, we successfully altered the knockdown duration and maximum knockdown time point by controlling the degradation and release kinetics of siRNA. The siRNA delivery strategy based on MSN with finely optimized pore size could be a very potent, carefully designed, attractive therapeutic system for the gene therapy to treat various diseases in the near future.

Acknowledgements

This work was supported by the Basic Science Research Program (2011-0017356, 2011-0020322), International S&T Cooperation Program (2014K1B1A1073716) and the Research Center Program (IBS-R008-D1) of IBS (Institute for Basic Science) through the National Research Foundation of Korea (NRF) and Research Program (S2255137) through the SMBA funded by the Korean government (MEST).

Notes and references

1. S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber and T. Tuschl, Nature, 2001, 411, 494.
2. A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello, Nature, 1998, 391, 806.
3. D. Bumcrot, M. Manoharan, V. Koteliansky and D. W. Sah, Nat. Chem. Biol., 2006, 2, 711.
4. J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, R. K. Pandey, T. Racie, K. G. Rageev, I. Röhl, I. Toudjarska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan and H. Vornlocher, Nature, 2004, 432, 173.
5. D. M. Dykxhoorn, D. Palliser and J. Lieberman, Gene Ther., 2006, 13, 541.
6. J. C. Burnett and J. J. Rossi, Chem. Biol., 2012, 19, 60.
7. D. M. Dykxhoorn and J. Lieberman, Annu. Rev. Biomed. Eng., 2006, 8, 377.
8. K. A. Whitehead, R. Langer and D. G. Anderson, Nat. Rev. Drug Discovery, 2009, 8, 129.
9. J. H. Lee, K. Lee, S. H. Moon, Y. Lee, T. G. Park and J. Cheon, Angew. Chem., Int. Ed., 2009, 48, 4174.
10. H. Lee, I. K. Kim and T. G. Park, Bioconjugate Chem., 2010, 21, 289.
11. H. Zhu, S. Zhang, Y. Ling, G. Meng, Y. Yang and W. Zhang, J. Controlled Release, 2015, 220, 529.
12. X. Yuan, S. Naguib and Z. Wu, Expert Opin. Drug Delivery, 2011, 8, 521.
13. W. H. Kong, K. H. Bae, S. D. Jo, J. S. Kim and T. G. Park, Pharm. Res., 2012, 29, 362.
14. K. Niikura, K. Kobayashi, C. Takeuchi, N. Fujitani, S. Takahara, T. Ninomiya, K. Hagiwara, H. Mitomo, Y. Ito, Y. Osada and K. Ijiro, ACS Appl. Mater. Interfaces, 2014, 6, 22146.
15. D. Zheng, D. A. Giljohann, D. L. Chen, M. D. Massich, X. Wang, H. Iordanov, C. A. Mirkin and A. S. Paller, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 11975.
16. C. Y. Sun, S. Shen, C. F. Xu, H. J. Li, Y. Liu, Z. T. Cao, X. Z. Yang, J. X. Xia and J. Wang, J. Am. Chem. Soc., 2015, 137, 15217.
17. A. Sengupta, R. Mezencev, J. F. McDonald and M. R. Prausnitz, Nanomedicine, 2015, 10, 1775.
18. W. Ngamcherdtrakul, J. Morry, S. Gu, D. J. Castro, S. M. Goodyear, T. Sangvanich, M. M. Reda, R. Lee, S. A. Mihelic, B. L. Beckman, Z. Hu, J. W. Gray and W. Yantasee, Adv. Funct. Mater., 2015, 25, 2646.
19. V. S. Y. Lin, C. Y. Lai, J. Huang, S. A. Song and S. Xu, J. Am. Chem. Soc., 2001, 123, 11510.
20. M. Vallet-Regí, A. Rámila, R. P. del Real and J. Pérez-Pariente, Chem. Mater., 2001, 13, 308.
21. J. L. Vivero-Escoto, I. I. Slowing, B. G. Trewyn and V. S. Y. Lin, Small, 2010, 6, 1952.
22. Q. He and J. Shi, J. Mater. Chem., 2011, 21, 5845.
23. Q. He and J. Shi, Adv. Mater., 2014, 26, 391.
24. C. Argyo, V. Weiss, C. Bräuchle and T. Bein, Chem. Mater., 2014, 26, 435.
25. Z. Li, D. L. Clemens, B. Y. Lee, B. J. Dillon, M. A. Horwitz and J. I. Zink, ACS Nano, 2015, 9, 10778.
26. J. L. Paris, M. V. Cabañas, M. Manzano and M. Vallet-Regí, ACS Nano, 2015, 9, 11023.
27. C. Yu, L. Qian, M. Uttamchandani, L. Li and S. Q. Yao, Angew. Chem., Int. Ed., 2015, 54, 10574.
28. F. Q. Tang, L. L. Li and D. Chen, Adv. Mater., 2012, 24, 1504.
29. Z. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart and J. I. Zink, Chem. Soc. Rev., 2012, 41, 2590.
30. J. L. Vivero-Escoto, R. C. Huxford-Phillips and W. Lin, Chem. Soc. Rev., 2012, 41, 2673.
31. S. Hudson and J. Cooney, Angew. Chem., Int. Ed., 2008, 47, 8582.
32. P. Yang, S. Gai and J. Lin, Chem. Soc. Rev., 2012, 41, 3679.
33. C. Xu, M. Yu, O. Noonan, J. Zhang, H. Song, H. Zhang, C. Lei, Y. Niu, X. Huang, Y. Yang and C. Yu, Small, 2015, 11, 5949.
34. H. K. Na, M. H. Kim, K. Park, S. R. Ryoo, K. E. Lee, H. Jeon, R. Ryoo, C. Hyeon and D. H. Min, Small, 2012, 8, 1752.
35. T. dos Santos, J. Varela, I. Lynch, A. Salvati and K. A. Dawson, PLoS One, 2011, 6, e24438.
36. D. Dutta and J. G. Donaldson, Cell. Logist., 2012, 2, 203.
37. J. Rejman, A. Bragonzi and M. Conese, Mol. Ther., 2005, 12, 468.
38. D. Vercauteren, R. E. Vandenbroucke, A. T. Jones, J. Rejman, J. Demeester, S. C. De Smedt, N. N. Sanders and K. Braeckmans, Mol. Ther., 2010, 18, 561.
39. P. Horcajada, A. Rámila, J. Pérez-Pariente and M. Vallet-Regí, Microporous Mesoporous Mater., 2004, 68, 105.
40. P. D. Bryson, N. J. Cho, S. Einav, C. Lee, V. Tai, J. Bechtel, M. Sivaraja, C. Roberts, U. Schmitz and J. S. Glenn, Antiviral Res., 2010, 87, 1.
41. H. Jang, S. R. Ryoo, Y. K. Kim, S. Yoon, H. Kim, S. W. Han, B. S. Choi, D. E. Kim and D. H. Min, Angew. Chem., Int. Ed., 2013, 52, 2340.
42. S. Kim, S. R. Ryoo, H. K. Na, Y. K. Kim, B. S. Choi, Y. Lee, D. E. Kim and D. H. Min, Chem. Commun., 2013, 49, 8241.
43. A. Schroeder, C. G. Levins, C. Cortez, R. Langer and D. G. Anderson, J. Intern. Med., 2010, 267, 9.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra27777a

‡ These authors contributed equally to this work.

§ Current address: Center for Nano-Bio Measurement, Korea Research Institute of Standards and Science, Daejeon 305-340, Republic of Korea.

---