Development of in vitro gene delivery system using ORMOSIL nanoparticle: Analysis of p53 gene expression in cultured breast cancer cell (MCF-7)
© Springer-Verlag 2012
Received: 10 March 2012
Accepted: 9 July 2012
Published: 29 July 2012
This article reports on the application of organically modified silica (ORMOSIL) nanoparticles as an efficient in vitro gene delivery system in the recent years. Based on that prime objective, the present study addresses the possible ways to reduce cancers incidence at cellular level. In this context, ORMOSIL nanoparticles had been synthesized and incubated along with pCMV–Myc (3.8 kb) plasmid vector construct carrying p53gene, and transfected into the breast cancer cell line MCF-7 cells. Western blot analysis showed that the p53 protein was significantly expressed in breast cancer cell upon transfection. The confocal and electron microscopic studies further confirmed that the nanoparticles were accumulated in the cytoplasm and the nucleus of the cancer cells transfected with p53 gene. Interesting agarose gel electrophoresis studies revealed that the nanoparticles efficiently complex with pCMV–Myc vector. The anti-cancer properties of p53 were demonstrated by assessing the cell survival and growth rate which showed a positive linear correlation in cancer cells. Whereas, the growth rate was significantly reduced in ORMOSIL/p53/pCMV–Myc transfected breast cancer cells compared to the growth rate of non-transfected cells. The results of this approach using ORMOSIL nanoparticles as a non-viral gene delivery platform have a promising future for use as effective transfection agent for therapeutic manipulation of cancer cells and targeted cancer gene therapy in vivo.
Nanomedicine is an emerging new field created by the combination of nanotechnology and medical sciences (Roy et al. 2005). The application of nanotechnology to biomedical research is expected to have a major impact in leading the development of new types of diagnostic and therapeutic tools (Prasad 2003, 2004). One focus in nanobiotechnology is the development and use of non-viral vectors for safe and efficient gene delivery (Davis 1997; Anderson 1992, 1998). With the potential of gene transfer as a therapeutic tool, the application of nanotechnology in the development of non-viral transfection agents for gene therapy is need of the hour (Bharli et al. 2005). The chances of using viral vectors as gene carriers is limited because of the risk factors such as pathogenicity, immunogenicity etc., and there is a need to supply a sufficient amount of DNA to the surface of the cells for effective gene transfer (Luo and Saltzman 2000). In spite of these limitations, the research focus has shifted towards the development of nanoparticle dependent gene delivery system. The other major advantages of nanoparticle vectors were the absence of limitations on the size and number of gene inserts. Ultrafine silica nanoparticles, with surfaces functionalized by cationic-amino groups, have been shown to not only bind and protect plasmid DNA from enzymatic degradation but also to transfect efficiently cultured cells and express encoded proteins (Allister et al. 2002; Kneuer et al. 2000a, b). Silica nanoparticles modified with aminosilanes [either N-(2 aminoethyl)-3 aminopropyltrimethoxysilane or N-(6-aminohexyl)-3 aminopropyltrimethoxysilane] are able to condense and deliver DNA, very much like cationic polymers (He et al. 2003; Kneuer et al. 2000a, b), without the addition of other cationic transfection reagents (Kneuer et al. 2000a, b). Other cationic silica nanoparticles with surfaces modified by aminohexyl-aminopropyltrimethoxysilane (AHAPS) are also reported to be successful transfection reagents (Ravi Kumar et al. 2004). Organically modified silica (ORMOSIL) nanoparticles have a potential to overcome many limitations of their “unmodified” silica counterparts. The presence of both hydrophobic and hydrophilic groups on the precursor alkoxyorganosilane helps them to self-assemble both as normal micelles and reverse micelles under appropriate conditions. The resulting micelles (and reverse micelles) cores can be loaded with bio-molecules such as drugs, proteins, etc. (Bharali et al. 2003). Such a system has number of advantages: (1) they can be loaded with either hydrophilic or hydrophobic drugs/dyes; (2) they can be precipitated in oil-in-water microemulsions in which corrosive solvents such as cyclohexane and complex purification steps such as solvent evaporation, ultracentrifugation, etc. can be avoided; (3) their organic groups can be modified further for attachment of targeting molecules; and (4) they can be possibly biodegraded through the biochemical decomposition of the Si–C bond (Bacskai et al. 2003). The presence of the organic group also imparts some degree of flexibility to the otherwise rigid silica matrix, which is expected to enhance the stability of such particles in an aqueous system against precipitation. In the present work, the hybrid amino-functionalized ORMOSIL nanoparticles have been synthesized by a synchronous hydrolysis of vinyltriethoxysilane (VTES) and 3-aminopropyltriethoxysilane (APTES). By varying the concentrations of Aerosol-OT and VTES, monodispersed nanoparticles of the mean diameter of the ORMOSILNs were under 60 nm which are highly stable in aqueous conditions (Das et al. 2002). Furthermore, the nanoparticles have been characterized by zeta potential measurement, XRD, and FTIR. The amino-functionalized nanoparticles were able to electrostatically condense DNA (both plasmid and genomic) and protect it from enzymatic degradation. Using confocal microscopy, it was found that the fluorescently labeled nanoparticles extensively accumulated in the cytoplasm and the nucleus of cancer cell lines used for the study (Jain et al. 1998). It was found that the nanoparticles release the genetic material inside the cytoplasm, which diffuses to the nucleus, a prerequisite for successful gene therapy. The present study clearly demonstrates the transfection efficiency of ORMOSIL nanoparticles as non-viral vectors, which are shown by the expression of p53 genes in the breast carcinoma cell lines.
ORMOSIL precursors are surfactant (Aerosol-OT 98 %) and co-surfactant (n-butanol 99.8 %), vinyltriethoxysilane (VTES 97 %), 3-aminopropyltriethoxysilane (APTES 99 %), and dimethyl sulfoxide (DMSO 99.5 %). The human cancer cell lines such as MCF-7 were generously donated by King Institute of Preventive Medicine Chennai (Dr. P. Gunasekaran’s laboratory). DMEM was purchased from Sigma, USA. The cells were seeded into 25-mm culture flasks and incubated at 37°C, in 5 % CO2. pCMV plasmid construct carrying anti-cancer protein p53 was purchased from Clontech. GFP was purchased from Invitrogen (Bangalore, India).
2.2 Preparation of ORMOSIL nanoparticles
The nanoparticles were synthesized in the non-polar core of Aerosol-OT/DMSO/water micelles as reported by Roy et al. and is briefly outlined as follows: Typically, the micelles were prepared by dissolving 0.44 g Aerosol-OT and 800 μl n-butanol in 20 ml of double-distilled water by vigorous magnetic stirring. One hundred microliters of DMSO was added. After that, 200 μl of neat VTES was added to the micelles system and the resulting solution was stirred for ~30 min. Furthermore, the ORMOSIL nanoparticles were precipitated by adding aqueous ammonia solution or APTES and stirring for ~20 h at room temperature using a magnetic stirrer. Formation of the nanoparticles is indicated by a white blue color. Surfactant Aerosol-OT and co-surfactant n-butanol were removed by dialyzing the aqueous solution against distilled water in a 12- to 14-kDa cut-off cellulose membrane for 50 h. The dialyzed solution was filtered through a 0.2-μm cut-off membrane filter and used straightway for further experimentation.
2.3 Characterization of ORMOSIL nanoparticles
Transmission electron microscopy was used to determine the morphology and the size of the aqueous dispersion of nanoparticles with a JEOL JEM 2020 electron microscope operating at an accelerating voltage of 200 kV. X-ray diffraction using Cukα radiation (PAN analytical X’pert Pro MPD diffractometer) was used to determine the amorphous structure of ORMOSIL nanoparticles. Powder X-ray analysis was carried out using a Philips Model PW 1050/37 diffractometer, operating at 40 kV and 30 mA, with a step size of 0.02° (2θ). Dried and powdered samples of colloids with the stabilizer before and after heat treatment were used for the measurements. The surface groups of the nanoparticles were qualitatively confirmed using FTIR spectroscopy. FTIR spectra were recorded on a Perkin-Elmer spectrum 2000 FTIR spectrophotometer. The pellets of the lyophilized powder were made with dried KBr. The size distribution of ORMOSILNs alone and their complexes with various amounts of DNA was measured by quasi-elastic light scattering (QLS) using Nicomp-370 (Nicomp Particles Sizing System, Santa Barbara, USA). For particle size analysis of the complexes, the complexes were diluted with phosphate-buffered saline (PBS) just prior to size measurement. The change in the particle diameter of complexes was measured for 3 min using Gaussian analysis. The zeta potential of complexes was analyzed using an electrophoretic light scattering spectrophotometer (ELS-8000, OTSUKA Electronics Co. Ltd., Japan) at room temperature to monitor the electrophoretic mobility of transfected complexes.
2.4 Nano-DNA complex preparation
We examined the complex formation of plasmid DNA with nanoparticles by agarose gel electrophoresis. DNA loading of ORMOSIL nanoparticles was accomplished by incubation with pCMV/p53 for 30 min at room temperature. The resulting ORMOSIL/pCMV/p53 nanoparticles were suspended in phosphate-buffered saline (PBS; 140 μg/ml DNA). The complex formation were run on 1 % agarose at 100 V for 1.5 h, subsequently stained with ethidium bromide, and documented by using a gel documentation system. Genei Ultraviolet Benchtop transilluminator was used in conjugation with an Olympus Digital Camedia C-4000 Zoom color camera with a UV filter and lens. The documentation was completed by using the DOC-IT system software.
2.5 In vitro transfection
MCF-7 (human breast carcinoma) cells were seeded in 24-well tissue culture plates at a density of 2 × 105 cells/well. The cells were allowed to adhere overnight as a monolayer and to achieve 70–80 % confluence. Transfection complexes were prepared by mixing the plasmid DNA (p53–pCMV) with ORMOSILNs and were incubated for 15 min at room temperature before in vitro studies. Prior to transfection, 800 μl of serum-free DMEM was added to each well; 100 μl of transfection complexes mixed with 2 μg of the plasmid DNA and various amounts (6–48 μg) of ORMOSILNs were then added to each of the wells. After 5 h of incubation at 37°C under 5 % CO2, cells were rinsed and cultured for 24 h in 1 ml of medium containing 10 % (FBS).
2.6 Western blot analysis
The levels of p53 protein expression in transfected cells with the plasmid DNA/ORMOSILNs complexes were determined by Western blot analysis. The transfected cells were harvested and lysed in lysis buffer (150 mM NaCl; 20 mM Tris base, pH 7.5; 1 mM PMSF; 1 mM Na3VO4; 25 mM NaF; 1 % aprotinin; 10 μg/ml leuprotinin; 1 % Triton X-100; 1 % NP-40) on ice for 30 min. After samples were centrifuged at 12,000 rpm for 10 min, the protein concentrations of supernatants were determined by Bio-Rad DC (detergent-compatible) microprotein assay using bovine serum albumin (BSA) as a protein standard. Then 15-μg aliquots of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose filters. The nitrocellulose membrane was incubated for 1 h in a blocking buffer (5 % non-fat milk in PBS) followed by incubation with the mouse anti-human p53 antibody (Pab240, Santa Cruz, CA, USA).
2.7 Cell viability
The effects of the plasmid DNA/ORMOSILNs complexes on growth inhibition were determined using a cell growth assay. The MCF-7 cells were seeded in six-well plates at a density of 1 × 104 cells/well. After 24 h, the cells were transfected with the plasmid DNA alone, DNA/ORMOSILNs, or DNA/Lipofectin® complexes, respectively. After transfection, the amounts of viable adherent cells were determined by trypan blue exclusion assay performed every other day. Untreated cells were used as a control. Growth assays were performed in triplicate.
2.8 Statistical analysis
Statistical analysis of data was performed using Student’s t test and analysis of variance (ANOVA). A p value less than 0.05 were considered significant.
3 Results and discussion
3.1 Physicochemical characteristics of ORMOSILNs
The mean diameters and zeta potentials of ORMOSILNs components (n = 3)
Zeta potential (mV)
6.5 ± 1.2
20.5 ± 2.1
3.2 X-ray diffraction analysis
3.3 FTIR analysis
IR peak (cm−1) of VTES and APTES, ORMOSIL nanoparticles present in chemical compounds
Peaks values (cm−1)
Name of the compound and functional groups
1,046, 773, 467
3.4 Examination of the complex formation of plasmid DNA with nanoparticles
3.5 Western blot analysis for p53 expression
3.6 In vitro growth assay
Efficient inhibition of cell growth by p53–pCMV/ORMOSILNs complexes indicates that these complexes would be efficient not only in increasing the expression of p53 proteins but also in inhibiting the growth of breast cancer cells.
3.7 Gene transfer efficiency of ORMOSIL nanoparticles
The present study shows that the nanomedicine approach using ORMOSIL nanoparticles provides a promising direction for non-viral gene delivery. In the present context, the use of DAPI, together with fluorescence imaging, established DNA delivery to the cell nucleus, whereas the use of pCMV/p53 western blot provided evidence for the functionality of delivered DNA. Our findings provide additional information regarding the delivery of p53 gene into a selected breast cancer cell line which is perfectly expressed in the cells. Thus, the ORMOSILNs are recommended. In particular, the effective indicated that the ORMOSILNs-mediated delivery of p53 gene might show potential for clinical use in non-viral vector mediated breast cancer therapies.
We thank R. Paulmurugan, Department of Radiology–Diagnostic Radiology, Stanford University, for useful discussion and All India Institute of Medical Sciences New Delhi for their excellent technical support for taking confocal, electron, and fluorescent microscopy results. Other technical support was provided by Centre for Life Science DRDO Bharathiar University Coimbatore. This work is financially supported by the DST-Nanomission Division, Ministry of Science and Technology, Government of India (Ref. DST/SR/NM/NS-60/2010).
The authors declare that they have no competing interests
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