Fluorescent carbon dots as an efficient siRNA nanocarrier for its interference therapy in gastric cancer cells
- Qing Wang†1, 2,
- Chunlei Zhang†2,
- Guangxia Shen2Email author,
- Huiyang Liu2,
- Hualin Fu2 and
- Daxiang Cui1, 2Email author
© Wang et al.; licensee BioMed Central. 2014
Received: 28 October 2014
Accepted: 5 December 2014
Published: 30 December 2014
Fluorescent carbon dots (Cdots) have attracted increasing attention due to their potential applications in sensing, catalysis, and biomedicine. Currently, intensive research has been concentrated on the synthesis and imaging-guided therapy of these benign photoluminescent materials. Meanwhile, Cdots have been explored as nonviral vector for nucleic acid or drug delivery by chemical modification on purpose.
We have developed a microwave assisted one-step synthesis of Cdots with citric acid as carbon source and tryptophan (Trp) as both nitrogen source and passivation agent. The Cdots with uniform size show superior water solubility, excellent biocompatibility, and high quantum yield. Afterwards, the PEI (polyethylenimine)-adsorbed Cdots nanoparticles (Cdots@PEI) were applied to deliver Survivin siRNA into human gastric cancer cell line MGC-803. The results have confirmed the nanocarrier exhibited excellent biocompatibility and a significant increase in cellular delivery of siRNA, inducing efficient knockdown for Survivin protein to 6.1%. In addition, PEI@Cdots complexes mediated Survivin silencing, the arrested cell cycle progression in G1 phase as well as cell apoptosis was observed.
The Cdots-based and PEI-adsorbed complexes both as imaging agents and siRNA nanocarriers have been developed for Survivin siRNA delivery. And the results indicate that Cdots-based nanocarriers could be utilized in a broad range of siRNA delivery systems for cancer therapy.
KeywordsCarbon dots siRNA interference therapy Gastric cancer Nanocarriers
Over recent decades, great advances have been made in the combination of nanotechnology and medicine, which is paving the way towards the goal of clinic application ,. A large amount of biocompatible fluorescence nanomaterials, such as quantum dots, metal nanoclusters, and fluorescent polymers, have been developed -. Carbon dots (Cdots) are currently emerging as a class of promising fluorescent probe on account of their low photobleaching, no optical blinking, tunable photoluminescence, versatile surfaces, and excellent biocompatibility ,. Therefore, fluorescent Cdots possess additional benefits over organic fluorophores and semiconductor quantum dot, which are more or less circumscribed by their photobleaching or intrinsic potential hazards of heavy metal elements (e.g. Cd and Pb) . These excellent properties of Cdots have made bright prospects in the applications of bioimaging, drug delivery, biochemical detection, and sensors -. Currently, intensive research has been focus on the synthesis of Cdots with high quantum efficiency and the construction of multifunctional systems based on Cdots ,,. Until now, various precursors including graphite, C60, citric acid, glucose, and silk have been developed for the preparation of Cdots with a wide variety of approaches, processes, and tools ,,-. Sun’s group has reported a new strategy to prepare core-shell dots based on Cdots doped with inorganic salts with quantum yield around 45% ~ 60%, but the preparation process is quite complicated .
Recently, our group has developed a green synthetic route for Cdots with high quantum yield around 24.2% using Ribonuclease A (RNase A) as an assisting and passivating reagent via microwave assisted one step procedure . Interestingly, the RNase A@Cdots can effectively inhibit the survival rate of cancer cells. But the price of RNase A is not economic enough. Taking into account the mechanism of the photoluminescence (PL) enhancement in RNase A@Cdots, electron-donating effect from neighbor amino acids especially those with benzene rings could play an important role. Therefore, we select tryptophan (Trp), a kind of amino acids with benzene ring while possesses a higher nitrogen content than tyrosine and phenylalanine, for the synthesis of Cdots with lower cost. Besides, we have ever developed a new theranostic platform based on photosensitizer-conjugated Cdots with excellent imaging and tumor homing ability for NIR fluorescence imaging guided photodynamic therapy . However, till now few reports are closely associated with the using of Cdots as gene transfection vector for cancer therapy.
RNA interference (RNAi) has emerged as a valuable research tool to downregulate the expression of specific target proteins in a wide variety of cells . RNAi is a biological process in which RNA molecules inhibit gene expression by causing the destruction of specific mRNA molecules. Viral delivery such as Lentivirus, Adenovirus and Adeno-Associated-Virus, has successfully been used for therapeutic applications, including cancer therapy ,. To deal with concerns over the potential risk of undesired immune and toxic side reactions in virus-mediated nucleic acid delivery systems, non-viral gene delivery systems based on inorganic nanoparticles, cationic liposomes, and cationic polyamidoamine dendrimers have been employed as carriers for gene silencing -. The nanomaterials-based non-viral gene delivery systems have benefited from many advantages over viral vectors, as they are simple to prepare, rather stable, easy to modify and relatively safe. Meanwhile, Cdots would be an ideal nanocarrier due to the high biological safety, well-defined structures together with their tunable surface functionalities.
Citric acid, L-tryptophan (99%) and polyethylenimine (PEI) with a molecular weight of 1800 Da were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was obtained from Sinopharm Chemical Reagent Co., Ltd, China. Hieff™ qPCR SYBR® Green Master Mix and Annexin V-FITC/PI Apoptosis Detection Kit were purchased from Yeasen Corporation (Shanghai, China). 3-[4, 5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). Random primer and M-MLV reverse transcriptase were purchased from Promega (Madison, WI, USA). RIPA lysis buffer and BCA (bicinchoninic acid) protein assay kit were purchased from Beyotime Biotechnology (Jiangsu, China). Monoclonal rabbit anti-Survivin antibody, polyclonal rabbit anti-β-actin antibody, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG were purchased from Epitomics (Burlingame, CA, USA). Complete protease inhibitor cocktail and BM Chemiluminescence Western Blotting Kit were obtained from Roche (Mannheim, Germany). Human gastric cancer MGC-803 cells and human gastric mucous epithelial GES-1 cells were available in the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Cell culture products and reagent were purchased from GIBCO. All the above chemicals were used without any further purification. Ultrapure water (Millipore Milli-Q grade) with a resistivity of 18.2 MΩ cm was used in all the preparations.
Synthesis and characterization of Cdots and Cdots@PEI
The luminescent Cdots was synthesized by a one-step green route of microwave assisted pyrolysis method. Briefly, 2 g citrate and 0.01 g L-Trp was dissolved in 30 ml of ultrapure water, and stirring for 1 h to form a homogeneous solution in a 100 ml beaker. Then the beaker containing clear transparent solution was placed at the center of the rotation plate of a domestic microwave oven (700 W) and heated for 3 minutes. When cooled down to room temperature, the Cdots were isolated from the opaque suspension via centrifugation with 10,000 rpm which aims at removing carbon residual. Excessive citric acid and L-Trp were removed via repeated dialysis against deionized water using a low molecular weight cut-off membrane (1000 Da) for 2 days. Finally, different concentration can be got through rotary evaporation, which is used to remove water as it depends. The morphologies of the Cdots were detected using a transmission electron microscope (TEM, 2100F, JEOL, Japan), operating at an accelerated voltage of 200 kV. Photoluminescence (PL) spectra were measured on a Hitachi FL-4600 spectrofluorometer. UV–vis spectra were recorded with a Varian Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA, USA). X-ray diffraction (XRD) measurement was performed with a D8 Advance (Bruker AXS Corporation, Germany). Fourier transform infrared (FTIR) spectra were conducted on a Nicolet 6700 spectrometer (Thermo Electron Corporation, Madison, WI) using KBr pellets. X-ray photoelectron spectrum (XPS) was acquired with a Kratos Axis UltraDLD spectrometer (AXIS Ultra, Kratos Analytical Ltd, Japan) using a monochromatic Al Kα source (1486.6 eV). Zeta potential was completed using a NICOMP 380 ZLS Zeta potential/Particle sizer (PSS Nicomp, Santa Barbara, CA, USA) equipped with a He-Ne laser (λ = 633 nm).
Quantum yield measurement
Where Φ std is the known quantum yield of the standard compound, F sample and F std are the integrated areas of fluorescence of the sample and standard in the emission region at 350–600 nm. A std and A sample are the absorbance of the standard and sample at the excitation wavelength (360 nm); n is the refractive index of solvent, for water the refractive index is 1.33, 0.1 M H2SO4 is 1.33. All samples were diluted to ensure the optical densities less than 0.10 measured by Varian Cary 50 UV–vis spectrophotometer to minimize re-absorption effects.
Preparation of siRNA-Cdots@PEI complexes and agarose gel electrophoresis
Three couples of siRNA oligonucleotides (noted as Surv-1, Surv-2 and Surv-3, respectively) and a couple of non-silencing-siRNA as negative control (NC) oligonucleotides were chemically synthesized by Shanghai Genechem Co. All siRNAs were annealed with complementary antisense strands with 3′-dTdT overhangs. The siRNA duplexes are as follows:
Surv-1: sense, 5′-CACCGCAUCUCUACAUUCATT (dTdT)-3′; antisense, 5′-UGAAUGUAGAGAUGCGGUGTT (dTdT)-3′.
Surv-2: sense, 5′-GAAGCAGUUUGAAGAAUUATT (dTdT)-3′; antisense, 5′-UAAUUCUUCAAACUGCUUCTT(dTdT)-3′.
Surv-3: sense, 5′-GGUCCCUGGAUUUGCUAAUTT (dTdT)-3′; antisense, 5′-AUUAGCAAAUCCAGGGACCTT(dTdT)-3′.
NC: sense, 5′-UUCUCCGAACGUGUCACGUTT(dTdT)-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT(dTdT)-3′.
The Cdots-based complexes were formed by electrostatic interactions between positively charged PEI and the negatively charged Cdots, and then the negatively charged phosphate backbone of siRNA attaching to the Cdots@PEI complexes, resulting the siRNA loaded siRNA-Cdots@PEI complexes. The Cdots@PEI and siRNA-Cdots@PEI complexes were purified by gel filtration over a Sephadex G-50 column equilibrated with 10 mM NaCl to remove un-adsorbed PEI or siRNA. The electrophoretic mobility of the siRNA-Cdots@PEI complexes was determined by 1% agarose gel in 1× TBE buffer with a constant voltage of 120 V for 20 min. The siRNA loading amount was determined by measuring the absorption at 260 nm using the relation 1OD duplex = 3.0 nmols after subtracting the absorbance contributed by Cdots@PEI at the same wavelength. In aqueous solution of pH 7.4, the concentration of siRNA was determined to be about 15 nM when the concentration of Cdots@PEI was 100 μg/ml. All data when used for siRNA-Cdots@PEI complexes were expressed as 100 μg/ml Cdots@PEI with about 15 nM siRNA, unless otherwise specified.
Cell culture and MTT assay
Human gastric cancer MGC-803 cells and human gastric epithelial GES-1 cells were available in the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% (vol/vol) fetal bovine serum (Gibco) and penicillin-streptomycin (100 U/ml to 0.1 mg/ml) and incubated in a humidified incubator containing 5% CO2 at 37°C. MTT assay was carried out to investigate the cytotoxicity of Cdots and Cdots@PEI. MGC-803 and GES-1 cells were first seeded to 96-well plates at a seeding density of 5 × 103 cells per well in 100 μl complete medium, which was incubated at 37°C for 24 h. Then the culture medium in each well was replaced by 100 μl fresh complete medium containing serial concentrations of Cdots and Cdots@PEI. After incubation for 24 h, the medium was replaced with 150 μL fresh medium containing 15 μl MTT (5 mg/ml in PBS) and incubated for another 4 h. Afterwards, the culture medium with MTT was removed and 150 μl/well of DMSO was added, followed by shaking for 10 min at room temperature. The absorbance of each well was measured at 490 nm using a standard micro plate reader (Scientific Multiskan MK3, thermo, USA). The cell viability was calculated according to the equation: cell viability = (OD490 nm of the experimental group/OD490 nm of the control group) × 100% and the cell viability of control group was denoted as 100%.
In vitro siRNA transfection and cellular uptake of siRNA-Cdots@PEI complexes
Before transfection, the MGC-803 cells were seeded in 6-well plates and the appropriate transfected number of cells is based on the fact that confluent of cells achieve to 30% to 50% at the time of transfection. Each sample prepared siRNA oligo-Cdots@PEI complexes are as follows: A. siRNA oligo stock solution was diluted to 1 μM before transfection. Then 110 μl 1 μM of siRNA oligo was added to 200 μl serum-free DMEM and mix gently at room temperature for 5 min. B. After incubated for 5 min, 100 μl Cdots@PEI are taken into diluted siRNA oligo (mentioned in a.) with immediate shaking (using a scroll instrument or pipetting more than 10 s). After mild centrifugation, the solution needs to stand still at room temperature for 10 min, to allow the effective formation of siRNA oligo-Cdots@PEI complexes. C. While it is incubated, the medium in the cell culture plates need to be refreshed. Each well was added 1.8 ml of complete medium (containing 10% serum and antibiotics). The siRNA oligo-Cdots@PEI complexes were dropped into each well containing cells and the medium. Gently shake the culture plate after mixing. Complete medium can be changed after 4–6 h transfection. Scrambled siRNA with the transfection reagent of Cdots@PEI was used as the nontargeting control. After incubation with different transfection complexes for 48 h, approximately 2 × 105 MGC-803 cells were collected from each sample and then subjected to quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis to determine the silencing efficiency against Survivin gene and Survivin protein expression. For evaluation of cellular uptake of siRNA-Cdots@PEI complexes, we tracked the cellular internalization of Cdots-PEI, Cy3-labelled siRNA, or Cy3-siRNA-Cdots@PEI in MGC-803 cells. The cells were plated on 14 mm glass coverslips and allowed to adhere for 24 h. After co-incubation with Cdots-PEI, Cy3-labelled siRNA, or Cy3-siRNA-Cdots@PEI for different times, the cells were washed twice with PBS sufficiently and fixed with 4% paraformaldehyde. Confocal fluorescence images were captured with a TCS SP5 confocal laser scanning microscopy (Leica Microsystems, Mannheim, Germany). Blue and red fluorescence images were acquired using DAPI-specific (excitation, 340–380 nm; emission, 450–490 nm) and Cy3-specific (excitation, 515–560 nm; emission, > 590 nm) sets of filters, respectively.
Survivin expression assay by qRT-PCR analysis
In qRT-PCR experiment, the total RNA was extracted from transfected MGC-803 cells using the TRIzol reagent (Invitrogen, America) according to the manufacturer’s instructions. A total of 1 μg of RNA was transcribed into cDNA using random primers and M-MLV reverse transcriptase (Promega). The cDNA templates were stored at −20°C. The qRT-PCR was performed in a final volume of 25 μl containing 12.5 μl of Hieff™ qPCR SYBR® Green Master Mix (Yeasen, Shanghai), and 1 μl of each 10 μM primer, and 1 μl of 1:10-diluted cDNA products. The PCR amplification was carried out in a Bio-Rad iQ5 with one cycle at 95°C for 5 min, followed by 30 cycles at 95°C for 30 sec, at 67°C for 30 sec, and at 72°C for 1 min, and finally at 72°C for 5 min. GAPDH was chosen as the endogenous control in the assay. The following PCR primers were used: GAPDH primers, forward: 5′-CCACCCATGGCAAATTCCATGGCA-3′, reverse: 5′-TCTATCTAGACGGCAGGTCAGGTCCACC-3′; Survivin primers, forward: 5′-GTGAATTTTTGAAACTGGACAG-3′, reverse: 5′-CCTTTCCTAAGACATTGCTAA-3′.
Western blot analysis
MGC-803 cells were lysed at 72 h after the transfection using RIPA lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3VO4, 0.5 μg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride) in the presence of complete protease inhibitor cocktail (Roche Diagnostics). The homogenate was then subjected to 10,000 rpm centrifugation for 10 min at 4°C. All the above procedures were performed in ice bath. The protein concentration was determined using BCA (bicinchoninic acid) protein assay kit (Beyotime Biotech, Jiangsu, China) and store at −20°C. The cell extracts (20 μg total proteins) were mixed with four times loading buffer (16% glycerol, 20% mercaptoethanol, and 2% SDS, and 0.05% bromophenol blue) (3:1, sample/loading buffer) and boiled for 5–7 min at 100°C. The samples were then subjected to 12% sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) at 120 V for 1 h and then transferred onto 0.45 mm polyvinylidene difluoride membrane (PVDF, Immobilon-P 0.45 μm, Millipore, Billerica, MA), using a semi-dry system (Biocraft, Tokyo, Japan) at 300 mA for 150 min. Membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 and 5% dry skim milk powder and then incubated with rabbit anti-human survivin antibody (1:1000, Epitomics) and rabbit anti-human β-actin antibody (1:2500, Epitomics) at 4°C overnight with a gentle shaking. The next day, after four 5 min washes with TBST buffer, the bolts were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG, 1:2500, Epitomics) for 1 h at room temperature. Antibody binding was detected by enhanced chemiluminescence (BM Chemiluminescence Western Blotting kit, Roche) and autora-diography (Kodak X-OMAT; Kodak, Rochester, NY).
Apoptosis assay by Annexin V-FITC and propidium iodide (PI) staining
The apoptotic and necrotic cells were analyzed by Annexin V/PI apoptosis detection Kit (Yeasen) according to the manufacturer’s protocol. In brief, MGC-803 cells were seed in 6-well plates at 5 × 104 cells/well for 24 h before co-incubated with Surv-1-Cdots@PEI, Surv-2-Cdots@PEI, Surv-3-Cdots@PEI, NC-Cdots@PEI, and Cdots@PEI complexes, respectively. The cells incubated with complete medium only were set as blank control. After 48 h incubation, the cells were harvested, washed with PBS and re-suspended in 200 μL of binding buffer containing 5 μL Annexin V and 10 μL PI. After incubation in dark at room temperature for 15 min, 400 μL of binding buffer was added to each sample, and the cells were immediately analyzed by FACSCalibur (BD Biosciences, Mountain View, CA). The data analysis was performed with Flow Jo 7.6 software. Positioning of quadrants on Annexin-V/PI plots was performed to distinguish living cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), late apoptotic/necrotic cells (Annexin V+/PI+).
Cell cycle analysis
Cell cycle analysis was performed using Flow cytometry. After the incubation of MGC-803 cells with Surv-1-Cdots@PEI, Surv-2-Cdots@PEI, Surv-3-Cdots@PEI, NC-Cdots@PEI, and Cdots@PEI complexes for 48 h, respectively, cells were harvested, washed with PBS and fixed overnight in 70% ethanol at −20°C. Then the cells were washed with PBS and stained with 50 μg/ml PI and 100 μg/ml RNase A for 30 min in the dark at room temperature. The cell cycle phase distribution was acquired by FACSCalibur and G1, S, and G2/M populations were quantified using FlowJo 7.6 software.
Result and discussion
Synthesis and characterization of Cdots
Formation and characterization of Cdots@PEI and siRNA-Cdots@PEI complexes
Intracellular delivery of siRNA by Cdots@PEI complexes
Gene silencing efficiency of siRNA-Cdots@PEI complexes
Apoptosis assay and cell cycle analysis
In summary, the Cdots-based and PEI-adsorbed complexes both as imaging agent and siRNA nanocarrier have been developed for Survivin siRNA delivery. The Cdots were prepared via one-step microwave assisted approach with citric acid as carbon source and tryptophan as passivation agent and nitrogen source. The as-synthesized Cdots exhibited excellent water dispersibility, biocompatibility, and high quantum yield. In the elaborately fabricated complexes of siRNA-Cdots@PEI, Cdots acted as a nanocarrier and a fluorescent indicator, while the positively charged PEI acted as the ties attaching negative charged siRNA to Cdots. Furthermore, the confocal fluorescence images indicted the cellular uptake of siRNA-Cdots@PEI complexes, and subsequently qRT-PCR and Western blot analysis confirmed the successfully entrance of siRNA into MGC-803 cells and superior gene silencing efficiency. Importantly, the siRNA-Cdots@PEI complexes, which target Survivin gene, can induce apoptosis and cell cycle arrest in G1 phase inhuman gastric cancer cells MGC-803. The resulting Cdots-based delivery system may be used to advance the field of siRNA therapeutics.
Supporting information is available from the XX Online Library or from the author.
This work was supported by Chinese Key Basic Research Program (973 Project) (No. 2010CB933901), the National Natural Scientific Foundation of China (Grant No. 31170961, 81225010, and81327002), and 863 project of China(no.2012AA022703 and 2014AA020700), Shanghai Science and Technology Fund (No.13NM1401500). Biomedical and Engineering Multidisciplinary Funding of SJTU(No. YG2012MS13), YG2014MS01.
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