Monitoring of siRNA-Dox complex formation by fluorescence spectrometry

We employed fluorescence spectrometry to monitor the siRNA and doxorubicin complex formation based on previous studies in which doxorubicin (thereafter denoted as Dox) intercalated within nucleic acids (plasmid or aptamer) to generate the physical complex.^14–17 In general, different concentrations of Dox in a fixed volume (50 μL) were mixed with 100 μL siRNA water solution at a fixed concentration (1 mg/mL), and the fluorescence of Dox was then measured by LS 55 Luminescence Spectrometer (Perkin Elmer, USA) at excitation 480 nm, and emission data was obtained in the interval of 520–680 nm.

Preparation of siRNA-Dox-SiO₂, siRNA-SiO₂ and lipofectamine-siRNA-Dox

For the complexing of siRNA and Dox, 100 μL water solution of siRNA (1 mg/mL) was added to a 1.5 mL centrifuge tube and mixed with 50 μL Dox water solution (1 mg/mL). The mixture was dispersed by vortex for 1 min and 0.21 μL APTES and 0.23 μL TEOS (1:12.5:3:3.75; siRNA: Dox: APTES: TEOS, M/M) were successively added and immediately vortexed for 1 min at room temperature. The obtained sample was incubated in 40°C water bath for 8 mins. Finally, free siRNA and Dox in the supernatant were eliminated by centrifugation at 10,000 rpm, and the precipitation was further washed using deionized water. The product siRNA-Dox-SiO₂ was resuspended in deionized water for the following experiments. Similarly, siRNA-SiO₂ nanovector could be produced without complexing with Dox before the treatment of APTES and TEOS. Lipofectamine-siRNA-Dox was produced by mixing Lipofectamine^TM2000 with the prior formed siRNA-Dox complex at 2:1 weight ratio of siRNA to lipofectamine.

The hydrodynamic size distribution and zeta potential of siRNA-Dox-SiO₂ were determined by Dynamic light scattering (DLS) method and Zetasizer Nano ZS (Malvern, Britain), respectively. Samples were suspended in ethanol and dried on holey carbon-coated Cu grids, and their morphological images were obtained with a transmission electron microscopy (TEM, JEM-2010, Japan) at 200 kV acceleration voltage.

Doxorubicin releasing from the nanocarrier in vitro

To investigate pH-triggered Dox release, siRNA-Dox-SiO₂ was incubated in PBS buffers of different acidity (pH 7.4 and pH 4.5) for 96 hrs at 37. At different time points, samples were subjected to a centrifuge speed of 13,200 rpm, and 25 μL of the supernatant was taken out from the total volume (500 μL) to test the amount of released Dox by Nanodrop 1000 at 580 nm. Fresh buffer of 25 μL was then replenished to maintain a fixed volume (500 μL).

Cell lines and cultures

MCF-7 cells (a human breast adenocarcinoma cell line) were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China), and MCF-7/MDR cells (MCF-7 cells with multidrug resistance (MDR)) were purchased from Nanjing KeyGen Biotech. Co. Ltd, China, which was a kind gift from Dr. Xiaofang Hu, Laboratory of Heat Transfer and Proteomics, Shanghai Jiao Tong University. MCF-7 and MCF-7/MDR cell lines were cultured in RMPI-1640 supplemented with 10% fetal bovine serum (FBS; v/v) and 2% penicillin-streptomycin (PS; v/v). Cells were incubated at 37 with 5% CO₂ in a humid cell incubator.

Cytotoxicity assessment of NC siRNA-SiO₂

Cell viability of MCF-7 and MCF-7/MDR cells were measured using the CCK-8 assay. Cells were seeded at a density of 1×10⁴ cells per well on 96-well plates and grown until approximately 80% confluence and then the following experiments were conducted. After removing the culture medium, the fresh medium containing 10% FBS and various concentrations (0–600 nM, applied siRNA concentrations) of NC siRNA-SiO₂ were added. Cells in the treatment of an equivalent volume of culture medium were used as a negative control, and cells treated with 2% Triton X-100 were used as a positive control. Cells were incubated with nanoparticles for 24 hrs and subjected to CCK-8 assay following the vendor’s protocol. All experiments were performed in hexaplicate, and the averaged absorbance values were normalized to the negative control.

Gene knockdown efficacy of siRNA-Dox-SiO₂ in vitro

To evaluate gene silencing efficacy, MCF-7/MDR cells were grown in 6-well plates to about 80% confluence, and each well was treated with siRNA-Dox-SiO₂ containing siRNA dosage of 162.5 nM with a fixed Dox concentration (1000 ng/mL). After incubation with various siRNA vectors for 24 hrs, MCF-7/MDR cells were replenished with fresh medium and cultured for another 24 hrs. For testing the effectiveness of VEGF gene-silencing (by using VEGF specific siRNA), the conditioned medium was collected and subjected to centrifugation. An ELISA assay was then used to determine VEGF levels. For testing the silencing of the MDR-1 gene (by using MDR-1 specific siRNA), cells were collected and the total protein was extracted for measurement by BCA assay. The extracts containing 50 μg of total protein were separated by 10% SDS-PAGE, and the target protein was electro-transferred onto nitrocellulose membranes. The membrane was then blocked and successively incubated with primary antibody and corresponding HRPconjugated- secondary antibody. After washing, the ECL chemical reagents were added to the membrane, and their images were captured using ChemiDoc XRS (BioRad, Hercules, CA, USA).

Cytotoxicity of NC siRNA-Dox-SiO₂ and MDR-1 siRNA-Dox-SiO₂

For measuring the cytotoxicity produced by NC siRNA-Dox-SiO₂, MCF-7 cells were grown in 96-well plates to about 80% confluence, and each well was treated with siRNA-Dox-SiO₂ (containing 162.5 nM NC siRNA) that encapsulated Dox at a fixed concentration (1000 ng/mL). After incubation with various vectors for 24 hrs, cells were subjected to CCK-8 assay following the vendor’s protocol. All experiments were performed in hexaplicate, and the averaged absorbance values were normalized to negative control.

For measuring the cytotoxicity produced by MDR-1 siRNA-Dox-SiO₂, MCF-7/MDR cells were grown in 96-well plates to about 80% confluence, and each well was treated with MDR-1 siRNA-Dox-SiO₂ (containing 162.5 nM MDR-1 siRNA) that encapsulated Dox at various concentrations (500, 1000, 2000 ng/mL, respectively), Dox (2000 ng/mL) group, NC siRNA-SiO₂ (containing 162.5 nM NC siRNA) and NC siRNA-Dox-SiO₂ (containing 162.5 nM NC siRNA with Dox concentration at 2000 ng/mL). After incubation for 24 hrs, the wells were replaced with fresh culture medium and incubated for another 36 hrs, followed by CCK-8 assay. Control groups also included no treatment and Dox only. All experiments were performed in hexaplicate, and the averaged absorbance values were normalized to negative control (without treatment).

Intracellular localization of siRNA-Dox-SiO₂

To monitor the intracellular trafficking of siRNA-Dox-SiO₂, MCF-7/MDR cells were seeded onto glass-bottom dishes at a density of 2×10⁴ cells per dish and incubated at 37°C for 4 hrs and 24 hrs. The growth medium was then replaced with fresh medium containing siRNA-Dox-SiO₂ (siRNA modified with fluorescein amidite). Before imaging, cells were stained with LysoTracker® Deep Red for lysosome detection and treated with Hochest33342 for nucleus visualization. Confocal laser scanning microscopy (CLSM, Leica SP5 II, Germany) was used to estimate the intracellular distribution of siRNA, Dox and lysosome.

Preparation and characterization of siRNA-Dox-SiO₂ and determination of dox release from the complex

The synthesized complex of siRNA-Dox provided a convenient tool for targeted and combination cancer therapy. Inspired by previous studies in which anionic surfactants such as DNA or RNA could be used as templates to synthesize anionic-surfactant-templated mesoporous silicas mediated by CSDA,^19–21 we speculated that siRNA or siRNA-Dox complex could also be appropriate to serve as novel anionic surfactants for the synthesis of siRNA/siRNA-Dox-based silica nanoparticles, by the above-mentioned self-assembly method. To this end, we adopted a sequential assembly approach (Figure 1A): first the siRNA-Dox complex was produced via non-covalent physical interactions, electrostatic interactions between the complex and the positively charged amino site of APTES, and co-condensation with TEOS assembled subsequently to form the silica framework at an appropriate molar proportion (siRNA: Dox: APTES: TEOS=1: 12.5: 3: 3.75). Interestingly, the amount of encapsulated siRNA and Dox in this nanocarrier was determined by the reaction time. As shown in Figure 1C, loaded quantities of siRNA and Dox in the nanocarrier increased steadily over time: specifically at the 8-min timepoint, loaded siRNA and Dox reached about 70% and about 50% of the total capacity, respectively; over 15–30 mins, the loaded siRNA and Dox almost maintained at a steady level (~85% or higher). In parallel, the nanoparticle size increased as the siRNA and Dox-loaded volumes increased over reaction time (Figure 1C). Taken together, the loading capacities of siRNA and Dox and nanocarrier size could be readily controlled by reaction time. For the current study, we chose 8 mins as the reaction termination time point, producing nanoparticles that consistently contained siRNA (~75% of total) and Dox (~50% of total), with a uniform size of about 250 nm. We characterized the physical properties of these nanoparticles. TEM image of siRNA-Dox-SiO₂ showed the nanoparticle size to be about 220 nm (Figure 1D). The presence of Dox in this nanocarrier was evidenced by the red precipitation compared with that before centrifugation (Figure 1E). DLS analysis revealed the nanocarrier’s diameter at 240±1.5 nm, along with zeta potential of the nanoparticle that was approximately −38 mV (Figure 1F), which was generally regarded suitable for cellular endocytosis via the strong electrostatic interactions between silanol groups of silica nanoparticles and trimethyl-ammonium head groups of the cellular membrane^.22 In comparison with several published siRNA/Dox co-delivery systems,^4,13,23 our method has the ability to synthesize co-delivery vectors conveniently in about 10 mins without prior preparation of inorganic materials and eliminate drug encapsulation, leading overall to improved drug loading and decreased preparation time.

In vitro Dox release study was conducted at 37 in sterile PBS buffer at pH 4.5 and 7.4, simulating the acidic lysosomes/endosomes and the normal physiological environment, respectively. Results confirmed that Dox was slowly and steadily released (~8%) at pH 7.4, extending up to 96 hrs, whereas at pH 4.5, Dox was released more rapidly (>30%) within 96 hrs, suggesting Dox release from the nanoparticle was both time- and pH-dependent (Figure 1G-a and b). pH sensitivity of siRNA-Dox-SiO₂ was an advantage for drug delivery. During circulation or in the extracellular space with a pH of approximately 7.4, Dox was only released from the nanocarrier at a basal level. Contrarily, when siRNA-Dox-SiO₂ was internalized by tumor cells and entered endosomal/lysosomal compartments, Dox was quickly released from the particles to facilitate the killing of cancer cells. To test the stability of siRNA in siRNA-Dox-SiO₂ nanocarrier, siRNA-release behavior in PBS was monitored. Results reflected that the amount of siRNA was slowly released from the nanocarrier and eventually reached the plateau at 72 hrs with about 85% of the total loaded siRNA (^{Figure S2}A), as corroborated by the agarose gel electrophoresis result (^{Figure S2}B) that revealed the highly effective retardation and protection of siRNA by the nanocarrier.

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Figure S2

(A) siRNA release profiles of siRNA-Dox-SiO₂ nanocarrier in PBS at different time points (1, 12, 24, 48, 72 and 96 hrs). (B) Agarose gel electrophoretic analysis to evaluate the siRNA release at different time points. Lane 1: 1 hr; Lane 2: 12 hrs; Lane 3: 24 hrs; Lane 4: 48 hrs; 5: 72 hrs; Lane 6: 96 hrs; 7: free siRNA (equivalent to the total loaded siRNA in siRNA-Dox-SiO₂ nanocarriers); 8: DNA ladder (50 bp).

Abbreviations: siRNA, small interfering RNA; Dox, doxorubicin; SiO₂, silica; PBS, phosphate-buffered saline; DNA, deoxyribonucleic acid.

Cytotoxicity and gene silencing efficacy of siRNA-Dox-SiO₂

Although silica-based materials are generally regarded biocompatible and suitable for in vivo use,^24,25 the cytotoxicity of siRNA-SiO₂ delivery system (similarly prepared as siRNA-Dox-SiO₂ but without Dox) remains to be tested prior to further studies. The cell viability of MCF-7 and MCF-7/MDR incubated with different concentrations of NC siRNA-SiO₂ (applied siRNA concentration, from 0 to 600 nM) for 24 hrs were measured by the CCK-8 assay (Figure 2A). All concentrations exhibited negligible cytotoxicity even at 600 nM for both MCF-7 and MCF-7/MDR cell lines compared to that of nearly 100% cell death in treatment of 2% Triton X-100 that has widely been used as a positive control group,^26,27 indicating that siRNA-SiO₂-based nanocarrier presented great biocompatibility with cells, and possibly could be suitable for in vitro/in vivo biological applications.

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Figure 2

The biological function tests of siRNA-Dox-SiO₂ nanoparticles. (A) Cytotoxicity of NC siRNA-SiO₂ in MCF-7 and MCF-7/MDR cell lines with various nanocarrier concentrations (applied siRNA concentrations: 0, 38, 75, 150, 300, 450, 600 nM). (B) Cytotoxicity of MCF-7 cells treated with various formulations. (C) VEGF expressions in MCF-7/MDR cells regulated by various formulations. Lipo, Lipofectamine^TM2000; Lipo-NC siRNA, NC siRNA loaded Lipofectamine^TM2000; Lipo-VEGF siRNA, VEGF siRNA loaded Lipofectamine^TM2000. Data are represented as the means ± S.D. (**p<0.001; n=3; ***p<0.0001; n=3). (D) Evaluation of endosomal escapability of FAM-NC siRNA-Dox-SiO₂. Fluorescence images of MCF-7/MDR cells incubated with FAM-NC siRNA-Dox-SiO₂ vectors for 4 hrs and 24 hrs using confocal laser scanning microscopy (CLSM).

Abbreviations: NC siRNA, negative small interfering RNA; Dox, doxorubicin; SiO₂, silica.

Following biocompatible analysis of siRNA-SiO₂ nanoparticles, a similar study on siRNA-Dox complex that whether siRNA and Dox in the siRNA-Dox-SiO₂ nanoparticle could interfere with their own functions should be confirmed. We again measured the cytotoxicity of NC-siRNA-Dox in MCF-7 cells and chose the VEGF gene and tested for target gene silencing efficacy on MCF-7/MDR cells (to eliminate the toxic influence from Dox). In MCF-7 cells, NC siRNA-Dox-SiO₂ (applied 162.5 nM NC siRNA with Dox concentration at 1000 ng/mL) showed very similar cytotoxicity levels compared with an equivalent amount of free Dox, while the control groups presented undetectable toxicity (Figure 2B). In parallel, VEGF siRNA-Dox-SiO₂ (applied 162.5 nM VEGF siRNA with Dox concentration at 1000 ng/mL) caused significantly better VEGF silencing than Lipofectamine^TM2000-VEGF siRNA did at an equivalent siRNA dosage (Figure 2C). Five other groups (Control, Lipofectamine^TM2000, Lipofectamine^TM2000-NC siRNA, Dox, NC siRNA-SiO₂) showed no VEGF knockdown in MCF-7/MDR cells, as shown in Figure 2C. Interestingly, VEGF siRNA-Dox-SiO₂ showed better VEGF knockdown, plummeting to about 30% of the control, than VEGF siRNA-SiO₂ did in which VEGF level only dropped to about 85% of the control group (Figure 2C). This improved gene silencing capacity of VEGF siRNA-Dox-SiO₂ in comparison to VEGF siRNA-SiO₂ plausibly resulted from Dox intercalation that not only stabilized siRNA, but also mediated timely escape of siRNA from endosomes/lysosomes.^26,28,29 Initial evidence of this enhanced siRNA escape was provided by confocal microscopy visualizing the cellular localization FAM-labeled siRNA from the two types of nanoparticles siRNA-Dox-SiO₂ (Figure 2D) and siRNA-SiO₂ (^{Figure S3}), in which the most FAM-labeled siRNA carried by siRNA-SiO₂ nanoparticles were trapped in endosomes/lysosomes after transfection (^{Figure S3}), whereas the siRNA encapsulated by siRNA-Dox-SiO₂ nanoparticles were nearly completely separated from endosomes/lysosomes (Figure 2D). Taken together, these experiments demonstrated, in principle, siRNA-Dox-SiO₂ as a novel co-delivery system to simultaneously deliver Dox and siRNA into cancer cells. Such a system could produce effective target gene silencing and induce marked cytotoxicity, suggesting its potential use for combination cancer therapy, especially when the target gene was related to MDR.

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Figure S3

Evaluation of endosomal escapability of FAM-NC siRNA-SiO₂. Fluorescence images of MCF-7/MDR cells incubated with FAM-NC siRNA-SiO₂ vectors for 4 hrs and 24 hrs using confocal laser scanning microscopy (CLSM).

Abbreviations: NC siRNA, negative small interfering RNA; Dox, doxorubicin; SiO₂, silica.

This study is supported by grants from National Natural Science Foundation of China [No. 81701828;], Natural Science Foundation of Zhejiang Province [No. LY16H180008;], Wenzhou Science and Technology Bureau Foundation [No. Y20150082].