Photostable and Biocompatible Fluorescent Silicon Nanoparticles for Imaging-Guided Co-Delivery of siRNA and Doxorubicin to Drug-Resistant Cancer Cells
A novel all-in-one fluorescent nanomedicine platform based on silicon nanoparticles (SiNPs) was developed for imaging-guided co-delivery of short interfering RNA (siRNA) and doxorubicin (DOX).
The intracellular time-dependent release behaviors of siRNA and DOX were visually monitored by tracking the strong and stable fluorescence of SiNPs.
The SiNPs-based nanocarriers displayed pronounced therapeutic efficiency on drug-resistant breast cancer cells.
KeywordsFluorescent silicon nanoparticles Drug resistance Gene therapy Bioimaging
Despite great progress in cancer treatment, multidrug resistance (MDR), which can lead to high recurrence rates and treatment failures, remains a tremendous challenge in cancer chemotherapy . Generally, the abnormal expression of related genes on drug efflux, metabolism and targets, or survival/death signaling pathways accounts for the genetic basis of drug resistance [1, 2, 3]. For example, multidrug resistance protein 1 (MDR 1), also known as P-glycoprotein (P-gp) and ABCB1, is a member of the ATP-binding cassette (ABC) transporter protein family, which is overexpressed in many types of cancer cells . Crucially, the overexpression of P-gp promotes the efflux of various hydrophobic chemotherapeutics from cancer cells. This results in an extremely low accumulation of therapeutic agents inside cells and reduces chemotherapeutic efficiency. Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans and mammalian cells, synthetic small interfering RNA (siRNA) consisting of approximately 21 to 23-base-pair double-stranded RNA has emerged as a potential therapeutic agent for treatment of various diseases, including cancers [4, 5, 6, 7]. The ability of siRNA to specifically and efficiently silence nearly any target gene of interest could be valuable in suppressing the expression of MDR-related proteins, such as P-gp. The combination of chemotherapeutics and siRNA has been recognized as an attractive option for overcoming drug resistance [3, 8, 9].
In seeking to realize such a combination therapy, a critical challenge is the development of effective and safe vehicles to deliver chemotherapeutics and siRNA . Nanomaterials have been extensively explored as an RNAi-based delivery platform to treat cancer cells because of their ability to circumvent drug resistance mechanisms and protect siRNA from biodegradation [11, 12, 13, 14, 15, 16, 17, 18, 19]. Nowadays, to further optimize the RNAi nanotherapeutic approach and shorten drug development time, it is important to incorporate imaging modalities into therapy [20, 21, 22]. By visualizing, characterizing, and quantifying the biological process (e.g., cellular uptake, subcellular dissociation, and stability) of genes and drugs, the therapeutic effects can be monitored in real time. However, organic dyes (e.g., carboxyfluorescein (FAM) and Alexa Fluor 647) suffer from severe photobleaching, while heavy metal-containing quantum dots might pose potential safety hazards [13, 16]. Consequently, novel, fluorescent, all-in-one nanocarriers with superior optical properties (e.g., high and stable fluorescence) and excellent biocompatibility are needed to facilitate the development of imaging-guided RNAi-based combination therapy in drug-resistant cancer cells.
Recently, fluorescent silicon nanoparticles (SiNPs) that are extremely photostable and possess relatively strong fluorescence have emerged as a novel and promising fluorescent bioprobe in a wide range of optical applications [23, 24, 25]. For example, by modifying SiNPs with targeted peptides, the resulting SiNPs bioprobe featuring strong/stable fluorescence (photoluminescence quantum yield, PLQY, of approximately ~ 28%) and small size (< 10 nm) were applied for real-time and long-term imaging of cancer cells . Biosensors based on fluorescent SiNPs with negligible cytotoxicity were developed for the specific and sensitive detection of lysosomal pH fluctuation by the conjugation of pH-sensitive compounds (i.e., dopamine) to SiNPs . In particular, one of our recent studies demonstrated that doxorubicin (DOX) can be loaded on SiNPs to produce SiNPs-based nanocarriers with pronounced fluorescence and robust photostability . The prepared SiNPs-based nanocarriers with adjustable drug-loading capacity were very suitable for optical imaging-guided cancer therapy because of their high fluorescence and robust photostability. However, it remains unknown whether these nanocarriers are available for imaging-guided co-delivery of siRNA and chemotherapeutic agents, facilitating the enhancement of the therapeutic efficacy in MDR cancer cells.
Herein, the fluorescent SiNPs-based nanocarriers were used for MDR cancer cells via the co-delivery siRNAs and DOX. The strong and stable fluorescence signals of SiNPs allowed the long-term fluorescence tracking of the intracellular transport of siRNA and DOX and revealed their time-dependent and dual-responsive release behaviors. Remarkably, the successful MDR1 gene silencing (approximately 80%) by dissociated siRNA enhanced the accumulation of DOX molecules in drug-resistant MCF-7 cells (MCF-7/ADR), which decreased the half maximal inhibitory concentration (IC50) of DOX by over 35-fold. Our results suggest that SiNPs-based fluorescent nanocomposites can be used as imaging-guided RNAi-based co-delivery nanoagents for the treatment of MDR cancer cells.
2.1 Preparation and Characterization of SiNP-DOX/siRNA Nanocomposites
Fluorescent SiNPs were synthesized through a photochemical method as described in our previous work . In short, the precursor solution was first prepared by adding 100 mL (3-aminoprophyl) trimethoxysilane (APTES, 97%; SigmaAldrich, USA) containing 20 g 1,8-naphthalimide (SigmaAldrich) to 900 mL Milli-Q water. After a thorough 10-min stirring, the mixture was allowed to react for 40 min at room temperature by exposure to ultraviolet light at 365 nm (Spectroline, USA). To purify the as-prepared SiNPs, the solution was carefully dialyzed in de-ionized water in dialysis bags with a molecular weight cutoff of 1 kDa (Biotopped Life Sciences, China). Thereafter, DOX molecules (Huafeng United Technology Co. Ltd., China) were loaded onto purified SiNPs to prepare SiNP-DOX conjugates as we previously detailed . Excess DOX molecules were removed by ultrafiltration using 10 kDa Nanosep centrifugal devices (Pall Life Sciences, USA). SiNP-DOX/siRNA nanocomposites were further prepared by mixing the resultant SiNP-DOX with 176 ng of P-gp siRNA (GenePharma Co. Ltd., China) (Table S1) with vigorous stirring at different SiNPs/siRNA (w/w) ratios of 30, 90, 150, 210, and 270. The resultant mixtures were analyzed by 1.5% agarose gel electrophoresis. To visualize siRNA patterns, 1% Gel Red (Biotium, USA) was added in the gel. The siRNA patterns were imaged by Imager 600 (Amersham, UK) and quantified by Image J software (NIH, USA) .
Transmission electronic microscopy (TEM), high-resolution TEM (HRTEM, CM 200 electron microscope; Philips, USA), dynamic light scattering (DLS, ZEN3690; Malvern Corp, U.K.), ultraviolet–visible (UV–Vis)–near-infrared (NIR) absorption (Lambda 750 spectrophotometer; PerkinElmer, USA) and photoluminescence (PL, FLUOROMAX-4 spectrofluorimeter; HORIBA Jobin Yvon SAS, France) were utilized to characterize the resultant SiNPs, SiNPs-DOX, and SiNP-DOX/siRNA nanocomposites.
2.2 Stability Evaluation
To test the stability of SiNP-DOX/siRNA in the culture media, the as-prepared SiNP-DOX/siRNA nanocomposites (SiNPs/siRNA (w/w) ratio of 210) were first incubated in RPMI-1640 medium for 24 and 48 h at 37 °C. Before the samples were analyzed by 1.5% agarose gel electrophoresis, the siRNA was released from the nanocomposites using 1% heparin. For the RNase A protection assay, SiNP-DOX/siRNA nanocomposites were incubated with 1 ng RNase A at 37 °C for 1 h. The nuclease activity of RNase A was terminated by treatment with 25 mM sodium dodecyl sulfate (SDS, 99%; J&K Scientific Ltd., China) at 60 °C for 5 min. The same analyses as described in Sect. 2.2 were performed on the samples.
2.3 Dual-Responsive Release Behavior
To investigate the release of siRNA, SiNP-DOX/siRNA nanocomposites were incubated in phosphate-buffered saline (PBS) containing high concentrations of phosphate groups (5–40 mM) for 12 h at 37 °C. Next, the samples were analyzed by agarose gel electrophoresis. The release of DOX was further studied by incubating SiNP-DOX/siRNA nanocomposites at pH 5.0, 7.4, and 8.4 for 3, 6, 12, 24, 36, 48, 60, and 72 h. After each treatment, the samples were subjected to ultrafiltration to collect the released DOX, which was quantified using the determined UV–Vis–NIR absorbance spectra. To assess the effect of encapsulated siRNA on DOX release, SiNP-DOX/siRNA nanocomposites pretreated by 1% heparin were set as the control group. SiNP-DOX/siRNA (containing heparin) was then treated as described above.
2.4 Intracellular Distribution
MCF-7 and drug-resistant MCF-7/ADR cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin antibiotics. MCF-7/ADR cells were cultured in wells of 24-well plates on cover slips at a density of 1.2 × 105 cells/well for 24 h and then incubated with nanocomposites for 3, 6, 12, and 24 h. To determine the intracellular localization of SiNP-DOX/siRNA nanocomposites, cells were stained with LysoTracker Green DND-26 (125 nM; Invitrogen, USA) for 40 min. Afterward, the samples were mounted on microscope slides using Fluoromount (F4680; SigmaAldrich), examined by laser scanning confocal microscopy (LSCM) using a model TCS-SP5 microscope (Leica, Germany), and quantified with LAS AF Lite software (Leica). SiNPs and LysoTracker Green DND-26 were excited by 405 and 476 nm; corresponding emission windows were 420 to 480 nm and 500 to 550 nm, respectively.
2.5 Intracellular Release
To study the intracellular release of siRNA and DOX from SiNP-DOX/siRNA nanocomposites, FAM-labeled siRNA (siRNAFAM, purchased from GenePharma Co. Ltd., China) was used to fabricate SiNP-DOX/siRNAFAM nanocomposites. MCF-7/ADR cells were incubated with SiNP-DOX/siRNAFAM nanocomposites (ASiNP = 1, DOX = 5 μg mL−1, and 100 nM siRNAFAM) for 3, 6, and 12 h. After treatment, cells were examined using LSCM. The excitation wavelengths for SiNPs, siRNAFAM, and DOX were 405, 476, and 488 nm, and the emission windows were 420 to 480 nm, 500 to 550 nm, and 560 to 650 nm, respectively. All images were captured using the same instrument settings.
2.6 In vitro Gene-Silencing Efficiency
To evaluate the gene-silencing efficiency of SiNP-DOX/siRNA nanocomposites in vitro, quantitative real-time transcription polymerase chain reaction (qRT-PCR) was first employed to quantify intracellular P-gp expression at the mRNA level. MCF-7-/ADR cells were seeded in wells of 24-well plates at a density of 1.5 × 105 cells/well overnight, and then treated with SiNPs, SiNP-DOX, SiNP-DOX/NC siRNA (scrambled siRNA), or SiNP-DOX/siRNA (three strands of P-gp siRNA). After a 24 h incubation, cells were cultured in fresh medium and allowed to grow for another 24 h. After that, the total RNA in each sample was collected according to the established Trizol reagent protocol (Invitrogen, USA)  and corresponding cDNA was obtained using PrimeScript® RT reagent kit (Takara Biotechnology Co. Ltd, Japan). The mixture of cDNA, forward and reverse primers (designed by Primer Bank, Table S2) and the SYBR Green Master Mix (Biotool, USA) was run on the CFX96 Real-Time PCR Detection System (Bio-Rad, USA). β-actin was used as an internal loading control (Table S2).
Immunofluorescent staining was utilized to visually evaluate P-gp expression at the protein level. Briefly, MCF-7/ADR cells were treated as described above, fixed with 4% paraformaldehyde containing 4% sucrose for 20 min, and blocked with 4% BSA containing 0.1% Triton X-100 for 40 min. The cells were incubated with P-gp primary mouse monoclonal antibody (1:300, Santa Cruz Biotechnology, USA) for 2 h, followed by washing three times with PBS containing 0.1% Tween 20. Fluorescein isothiocyanate (FITC)-labeled secondary goat anti-mouse antibody (1:300, Santa Cruz Biotechnology) and Hoechst 33258 (3 μg mL−1; Beyotime Biotechnology, China) were used to stain cells. Finally, the samples were imaged by LSCM. For Hoechst 33258 staining, the excitation wavelength was 405 nm and the emission window was 420 to 480 nm. The excitation wavelength and corresponding emission window for FITC were 488 nm and 500 to 550 nm.
2.7 MTT Assay
A standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, SigmaAldrich) assay was carried out to evaluate the in vitro therapeutic effect. Cells were seeded in wells of a 96-well plate at a density of 0.8 × 104 cells/well. Different concentrations of free DOX (0.63–10 μg mL−1), pure SiNPs (ASiNP: 0.125–2), SiNP-DOX (DOX: 0.6310 μg mL−1, ASiNP: 0.125–2) conjugates, SiNP-DOX/NC siRNA and SiNP-DOX/siRNA (DOX: 0.63–10 μg mL−1, ASiNP: 0.125–2, siRNA: 12.5–200 nM) nanocomposites were used to treat the cells. After a 72-h incubation, cells were treated with MTT (20 μL, 5 mg mL−1) for 4 h and then lysed by 10% acidified SDS. To determine the cell viability, absorbance at 570 nm of each well was determined using the model 680 microplate reader (Bio-Rad). Three independent assays were performed in triplicate for all measurements. SPSS Statistics 17.0 software (SPSS Inc., USA) was used to calculate IC50 values.
3 Results and Discussion
3.1 Fabrication and Characterization of SiNP-DOX/siRNA Nanocomposites
The capability of SiNP-DOX/siRNA to protect siRNA from nuclease degradation is evaluated by incubating SiNP-DOX/siRNA with culture medium (RPMI-1640) or RNase A. As depicted in Figs. 1g and S5, the band intensity of naked siRNA decreased rapidly with time; only ~ 37% and 19% of the siRNA maintained its integrity after incubation with medium for 24 and 48 h, respectively (Fig. 1g, lane 2 and 5). In sharp contrast, ~ 100% siRNA loaded onto SiNP-DOX/siRNA nanocomposites was released by heparin, even after a 48-h incubation with medium (Fig. 1g, lane 7), suggesting that the loaded siRNAs were effectively protected from nuclease degradation. This protection could be attributed to steric hindrance on surfaces, consistent with several previous studies [15, 32]. Additionally, following the incubation of the SiNP-DOX/siRNA nanocomposites with RNase A for 1 h, the loaded siRNA was hardly degraded, consistent with its protection by the SiNP-DOX/siRNA nanocomposites (Fig. S6). Moreover, the SiNP-DOX/siRNA nanocomposites had extremely high storage or photostability (Fig. S7). The fluorescence intensity remained stable in different incubation conditions (i.e., PBS and RPMI-1640 medium) during 1 week at 37 °C. These results suggested the potential feasibility for the long-term analysis of the intracellular behavior of SiNPs-based nanocarriers via tracking of their fluorescence signals.
3.2 Dual-Controlled Release of siRNA and DOX
3.3 Intracellular Trafficking
To further demonstrate the controlled release behavior of SiNP-DOX/siRNA nanocomposites inside cells, we first investigated their subcellular localization. LysoTracker Green DND-26 was used to label lysosomes. After a 3-h incubation, the fluorescence signals of SiNPs were clearly observed as blue fluorescent dots (Fig. 2c, left panel). As shown in the merged confocal microscopic images, cyan dots were distinctly found, indicating the colocalization of SiNPs (blue signals) with lysosomes (green signals). The value of the Pearson’s correlation coefficient (Rr, one standard measure for analyzing colocalization) was calculated as 0.45, providing a quantitative confirmation of the good colocalization between SiNPs and lysosomes. At incubation times of 12 and 24 h, the Rr values increase to 0.61 and 0.75, respectively. These results clearly demonstrated that the co-delivered SiNPs-based nanocomposites (i.e., SiNP-DOX/siRNA) can be retained in lysosomes after cellular internalization. DOX and siRNA may be released from nanocomposites responsive to acidic (pH 5.0–5.5) and phosphate-enriched environment of lysosomes [15, 33, 34].
3.4 In Vitro Gene-Silencing Efficiency
3.5 Reversal of Drug Resistance of MCF-7/ADR Cells
We developed a novel fluorescent SiNPs-based nanomedicine platform, which is useful for imaging-guided co-delivery of siRNA and doxorubicin, enabling the enhancement of therapeutic efficacy in drug-resistant cancer cells. The nanomedicine platform (SiNP-DOX/siRNA nanocomposites) displayed dual-controlled release of siRNA and DOX molecules, which could be analyzed for prolonged periods in live cells by tracking fluorescence signals of SiNPs. The disassociated siRNA from SiNP-DOX/siRNA nanocomposite obviously down-regulated the expression of P-gp at the mRNA and protein levels (~ 80%), thus ensuring the sustained retention of released DOX in MCF-7/ADR cells. The SiNP-DOX/siRNA nanocomposites potently induced MCF-7/ADR cell death, as evident from the 36.5-fold decrease in IC50 (3 μg mL−1) compared to that of the free DOX group (112 μg mL−1), which overcame drug resistance. The results demonstrate the effectiveness of fluorescent SiNPs for the imaging-guided co-delivery of siRNA and DOX for therapy of MCF-7/ADR cells, and pave the way for the development of nanomedicines for MDR cancer cells. Of note, although silicon is distinguished by its low- or non-toxicity, numerous pioneering studies conducted by Professor Leong have revealed that nanoparticles may induce endothelial leakiness [37, 38, 39, 40]. Therefore, further understanding of the behavior of SiNPs and SiNP-DOX/siRNA nanocomposites in vivo requires further investigation for their potential clinical application.
We appreciate financial support from the National Basic Research Program of China (973 Program, 2013CB934400), the National Natural Science Foundation of China (Nos. 21825402, 31400860, 21575096, and 21605109), the Natural Science Foundation of Jiangsu Province of China (BK20170061), and a Project funded by Collaborative Innovation Center of Suzhou Nano Science & Technology (NANO-CIC), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the 111 Project as well as Joint International Research Laboratory of Carbon-Based Functional Materials and Devices.
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