Cisplatin-functionalized silica nanoparticles for cancer chemotherapy
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Cisplatin is used to treat a variety of tumors, but dose-limiting toxicities or intrinsic and acquired resistance limit its application in many types of cancer including breast. Cisplatin was attached to silica nanoparticles using aminopropyltriethoxy silane as a linker molecule and characterized in terms of size, shape, as well as the dissolution of cisplatin from the silica surface. The primary particle diameter of the as received silica nanoparticles ranged from 20 to 90 nm. The results show that adverse effects on cell function, as evidenced by reduced metabolic activity measured by the MTT assay and increased membrane permeability observed using the live/dead stain, can be correlated with surface area of the silica. Cisplatin-functionalized silica nanoparticles with the highest surface area incited the greatest response, which was almost equivalent to that induced by free cisplatin. Moreover, if verified by further studies, would indicate that cisplatin was attached to silica nanoparticles might prove to be useful in site-specific drug delivery.
KeywordsSilica Cisplatin TEM Nanoparticles MCF-7 cells Fluorescence
Worldwide, deaths: an estimated 39,920 breast cancer deaths (39,510 women, 410 men) are expected in 2012. Breast cancer ranks second as a cause of cancer death in women (after lung cancer) (American Cancer Society 2012). Cis-diamminedichloroplatinum (II), known as cisplatin, a platinum-based drug, is one of the most potent antitumor agents has been widely used in the clinic to treat a variety of cancers such as ovarian, breast, bladder, head and neck, and small cell lung cancer because of its potent activity to cross-link DNA upon entering the cells (Wang and Lippard 2005). Chemotherapy is the mainstay treatment for advanced and metastatic disease. DNA-damaging agents have a long and proven record as anticancer drugs (Decatris et al. 2004). It is proved that after both passive and active cellular uptake, cisplatin may react with the N7 atom of guanine in DNA to form adducts and causes cellular apoptosis (Giese and McNaughton 2003). However, chronic cisplatin usage results in resistance by several possible mechanisms including increased interactions with metal lothionein and glutathione as well as increased DNA repair (Reedijk 2003). To counteract resistance, which lowers the efficiency of cisplatin significantly, very high systemic doses of cisplatin are administered. Unfortunately, such high dose of cisplatin results in severe systemic toxicity and poor patient compliance, including nausea/vomiting, renal toxicity, gastrointestinal toxicity, peripheral neuropathy, asthenia, and ototoxicity, thus limiting its clinical use (Hill and Speer 1982; Rosenberg 1977). To improve the efficacy and safety of cisplatin, a variety of methods are applied to drug delivery system, which include particulate carriers, such as liposomes, polymers, and nanoparticles (NPs) (Bontha et al. 2006; Junior et al. 2007; Geng et al. 2004). The drug carriers may concentrate in the tumor because tumors exhibit a unique enhanced permeability and retention effect for 50–100 nm particles. As a result of this, a large increase in tumor drug concentrations (tenfold or higher) could be achieved relative to administration of the same dose of free drug, (Maeda et al. 2000) thereby decreasing the massive systemic side effects of conventional chemotherapy. In our previous study on modified silica nanoparticles, (ORMOSIL, LSN) however, it was shown that the cytotoxicity across the MCF-7 cells as measured by methyl-thiazol tetrazolium salts (MTT) assay was significantly reduced for the case of silica nanoparticles synthesized and p53 gene delivery successful in breast cancer cell line (Rejeeth et al. 2012a; Rejeeth et al. 2012b). In the case of materials in combination, the relevant question is whether apparently non-toxic particles are transformed into a potentially toxic material if surface attached with known toxic molecules. Three possibilities exist: the nanoparticle/toxic molecule combination becomes more toxic, the toxicity of the combination is unchanged, or the toxicity of the combination is reduced compared to the toxic molecule alone. One common chemical used for surface functionalization is aminopropyltriethoxysilane (APTES), which has the chemical formula NH2 (CH2)3–Si (OC2H5)3 (Gan et al. 2009; Liu et al. 2009; Jang and Liu 2009; Libertino et al. 2008). The silane end of the APTES molecule binds covalently to surface silicon atoms, and the amino end of the molecule increases protein adsorption on the surface by electrostatic interactions. The structural transition of a single dsDNA molecule immobilized on an APTES-treated substrate has been demonstrated. DNA binding to the APTES linker is much stronger than that on an alkylthiol/substrate (Nguyen et al. 2009). Silica–cisplatin system provides a unique opportunity to explore effects of surface area, in terms of the ability of nanoparticles to serve as carriers. The testable hypothesis herein is that nontoxic nanoparticles with a surface-attached toxic molecule would then adversely affect cell function, defined here by reduced metabolic activity and increased membrane permeability. Specifically, these adverse effects are expected to increase with increasing surface area. Results of physicochemical characterization of the nanoparticles and nanoparticle/cisplatin combination including surface area and cisplatin dissolution rates are presented. Effects on metabolic activity as measured via the MTT assay, and membrane permeability observed by fluorescence microscopy using the DAPI/JC-1 live/dead stain, we demonstrated that these silica-cisplatin prodrug conjugate NPs had well-controlled drug loading yield, excellent acid-responsive drug release characteristics, and potent cytotoxicity against breast cancer.
2 Materials and methods
The chemicals, i.e., APTES (3-aminopropyl) triethoxysilane, Triton X-100, Cisplatin, DAPI and JC-1 (Sigma-Aldrich) were used without further purification. All the glassware (glass bottle and small pieces of glass substrate) was cleaned and sonicated in ethanol for 5 min, rinsed with double-distilled water, soaked in a H2O/HNO3 (65 %)/H2O2 (1:1:1, v/v/v) solution, rinsed again with doubly distilled water, and finally dried in air. The MCF-7 cells were purchased from National Centre for Cell Sciences (NCCS), Pune, India.
2.2 Surface attachment of cisplatin to silica particles
Approximately, 0.64 g of APTES-functionalized Triton X-100, silica particles were kept in a three-necked flask respectively and dispersed in 50 mL distilled water with constant stirring for sample; 0.385 g of cisplatin was added for APTES-functionalized Triton X-100 samples of suspended silica particles, respectively. These amounts correspond to sufficient cisplatin to react with all available silanol groups (2.5 nm−2) on the surface of silica. The suspended particles were constantly stirred under nitrogen over-night at room temperature inside the fume hood. Cisplatin-functionalized silica particles were then collected by centrifugation at 3,500 rpm for 15 min. Harvested particles were dried at room temperature and stored in glass bottles for further analysis.
2.3 FT-IR of cisplatin-functional silica nanoparticles
Fourier transform infrared (FT-IR) spectroscopy of silica nanoparticle, cisplatin, and cisplatin-functionalized silica nanoparticles were performed by using Nicolet 5700 instrument (Nicolet Instrument, Thermo Company, USA) with KBr pellet method. Each KBr disk was scanned over a wave number region of 500–4,000 cm−1 with the resolution of 4.0 × 108 cm.
2.4 Cisplatin estimation
The platinum content of cisplatin-functionalized silica particles was measured by inductively coupled plasma-mass spectrometry (ICP-MS) measurements; 0.020 g of cisplatin functionalized silica particles was added to 25 mL of 2 % HNO3. The suspension was sonicated in an ultrasonic bath for 30 min. After centrifugation, a clear supernatant was obtained which was analyzed for total platinum (Pt). The platinum content of the sample was determined by comparison with a platinum standard which was prepared by dilution of a standard solution of platinum of defined concentration. The total amount of platinum measured from the sample was converted into the amount of cisplatin.
2.5 Transmission electron microscopy
Particle morphology was observed using a field emission transmission electron microscope (TEM). The selected area diffraction patterns of the samples were recorded using the same TEM instrument. The sample preparation procedure used for TEM analysis of the particles was as follows. The particles were first dispersed in ethanol 0.1 mg/mL suspension in a glass beaker and beaker was sonicated for 10 min. One drop of sample suspension was placed on a carbon-coated grid (Ted Pella, USA) and dried at room temperature overnight before analysis under TEM.
2.6 Particle size analysis with dynamic light scattering
Dynamic light scattering (DLS) was performed using a Photocor-FC light-scattering instrument employing a 5 mW laser light source at 633 nm and a logarithmic correlate instrument to determine the mean equivalent hydro-dynamic diameters of the samples. For the analysis, a suspension of ~0.1 mg/mL concentration of silica sample was made in ethanol. The suspension was sonicated for 30 min prior to analysis. The instrument measures the change of intensity of the laser light with time at 90° angles after interaction with spherically shaped particles suspended in liquid media. The diffusion coefficient was determined from the correlation function, and, assuming the Stokes–Einstein equation is valid, the equivalent hydrodynamic diameter distribution was obtained using the Dyna-LS software package supplied by Photocor.
2.7 Dissolution of cisplatin from silica particles
2.8 Cell culture
The breast cancer cells (MCF-7) were maintained in Dulbecco’s modified eagles medium (DMEM) supplemented with 2 mM l-glutamine and balanced salt solution adjusted to contain 1.5 g/L Na2CO3, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 1.5 g/L glucose, 10 mM (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid), and 10 % fetal bovine serum (GIBCO, USA). Penicillin and streptomycin (100 IU/100 μg) were adjusted to 1 mL/L. The cells were maintained at 37 °C with 5 % CO2 in a humidified CO2 incubator.
2.9 Metabolic activity assay
The mitochondrial activity of MCF-7 cells was measured after treatment with the cisplatin-functionalized silica samples using the MTT assay. This test was performed on MCF-7 cells after 24, 36, and 48 h of incubation with the samples, as described by (Mosmann 1983). Cells were grown in a 24-well plate in DMEM media with 10 % fetal bovine serum at 37 °C and 5 % CO2. In this study, silica/cisplatin particles were added with the weight ranging from 13 to 144 μg/mL corresponding to the amount needed to give a concentration of surface attached cisplatin equal to 5 μg/mL in each well of a single 24-well plate. Four replicates were performed for each sample. Positive and negative controls were included on each plate. The MTT assays involved the following steps: the growth medium was removed from the culture wells and washed with 300 μL PBS once. After washing, 300 μL of MTT solution (0.5 mg/mL) was added in each well of the plate. For the reduction of tetrazolium salt, plates were incubated at 37 °C for 2 h. The tetrazolium salt was reduced to a formazan (blue) product by the active mitochondria of the cells. In each well, 300 μL of solubilizing buffer (10 % Triton X-100 with 0.1 N HCl in anhydrous isopropanol) was added to dissolve the reduced tetrazolium salt. The color of reduced salt was measured at 570 nm using microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). The absorbance at 570 nm was taken as the index of mitochondrial activity.
2.10 Fluorescence microscopy
Cellular viability produced by the combination of cisplatin and silica particles was examined using fluorescence microscopy after staining the MCF-7 cell with the live/dead system (Molecular Probes, USA). After incubation with the samples in 96-well plates, cells were stained by adding DAPI and JC-1 to reach a final concentration in the well plates of 4 μM DAPI and 2 μM JC-1. DAPI undergoes fragment DNA conversion to fluorescent DAPI (blue, 340 nm wavelength), which is retained well within live cells. JC-1 enters cells with damaged membranes, and undergoes a fluorescence enhancement (brown, 635 nm wavelength) upon binding to DNA fragments. Cells were incubated with the dyes for 20 min in 100 μL PBS, and imaged under the fluorescence microscope (Nikon instrument Inc., USA) using an excitation wavelength of 488 nm.
2.11 Cell sectioning for electron microscopy
Cisplatin-functionalized silica-treated MCF-7 cell line was grown on the 24-well cell culture plates. MCF-7 cells exposed to silica (Triton X- 100) were used as the control. Cells were washed with PBS buffer and collected in centrifuge tubes after harvesting. Cells were washed with 0.13 M phosphate buffer three times for 10 min. At the end of the washing, cells were treated with a fixative solution (2 % glutaraldehyde) overnight at 4 °C. After fixation, cells were washed with 0.1 M phosphate buffer three times for 10 min to remove the excess glutaraldehyde. Cells were post-fixed with 1 % OsO4 in PBS for 60 min and washed with double-distilled water three times for 10 min. After washing, cells were dehydrated at room temperature in 30, 45, 65, 90, and 100 % ethanol for 10 min each. Cells were then infiltrated with propylene oxide and LR white resin mixture for 4 h. Cells were kept in fresh pure LR white resin overnight at room temperature. Finally, the cells were embedded in capsules (Electron Microscopy Sciences, USA) with freshly prepared LR white resin. Polymerization was carried out in an oven at 60 °C for 24 h. Sections from the embedded cells were cut on an Ultra cut microtome (Reichert, Wien, Austria) and collected on 100-mesh copper TEM grids (Ted Pella Inc.,). Ultrathin sections mounted on copper grids were stained with 1 % aqueous uranyl acetate (Ted Pella Inc.,) for 15 min and lead citrate (Electron Microscopy Sciences, USA) for 1.5 min.
2.12 Statistical analysis
Appropriate statistical procedures (Student t test for means, Stat graphics plus 3.3 software) were applied in the statistical analysis of the experimental data.
3 Results and discussion
3.1 Synthesis and characterization of cisplatin-functionalized silica particles
3.2 FT-IR characterization of cisplatin-functionalized silica nanoparticles
3.3 Amount of cisplatin attached to the silica surface
3.4 Dissolution of cisplatin from silica particles
3.5 In vitro effects on metabolic function
3.6 Cellular response to cisplatin-functionalized silica particles
TEM analysis of the MCF-7 cells can shed light in understanding the possible interaction mechanisms of cisplatin-functionalized silica particles with the cells. Cisplatin must be able to react with DNA to affect cell function, so it needs to reach the cell nucleus. In the TEM images, both extracellular and intracellular particles are visible suggesting two possibilities. The extracellular particles may be releasing cisplatin into solution, which is then taken up by the cells. Free cisplatin is believed to enter cells via both passive and active transport routes (Ohmori et al. 1993). Cisplatin-functionalized silica nanoparticles can enhance both possible interaction mechanisms by bringing high loadings of cisplatin near the cells for passive transport of free cisplatin to take place. The intracellular particles, on the contrary, may carry bound cisplatin into the MCF-7 cells, which is then released into the cytoplasm with higher surface area corresponding to greater rates of active transport.
In conclusion, successful attachment of a controlled amount of toxic cisplatin molecules on the surface of the nano-sized silica particles was demonstrated and the particles were characterized in terms of particle size and surface area. In this study, we have demonstrated that the dissolution rate of cisplatin from the surface of the particles to the solution phase depends on the surface areas of the particles. Biological studies have shown that, the impaired cellular function in the MCF-7 induced by cisplatin-functionalized silica particles, as indicated by the reduced metabolic activity and cell viability. These effects appeared to be correlated with the cisplatin functionalized surface areas of the particles. Apparent uptake of the particles by the cells and morphological differences after active interaction with the cisplatin-functionalized silica particles were observed by TEM. The importance of our study lies in showing the combinatorial effect of relatively high surface area nanoparticles and toxic molecules at the cellular level. In the wake of increasing use of nanoparticles, which may be either surface modified prior to end use in drug delivery constructs or consumer products, or upon release into the environment, this study issues a cautionary note that the properties of the nanoparticles, especially specific surface area, may affect biological response.
This work was financially supported by DST-NANOMISSION (DST No. SR/NM/NS-60/2010) Ministry of Science and technology and UGC-NON-SAP (G2/6966/UGC NON-SAP (Zoology)/2010) New Delhi, Govt. of India. The authors greatly acknowledged DRDO Centre for Life Sciences for nanoparticles characterization studies, Electron Microscopy Centre, AIIMS, New-Delhi and Sankara Nethralaya for their kind assistance with fluorescence microscopy studies. The authors are very much thankful to all faculty members of the Department of Zoology, Bharathiar University for their constant support and encouragement throughout this study.
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