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Development of in vitro gene delivery system using ORMOSIL nanoparticle: Analysis of p53 gene expression in cultured breast cancer cell (MCF-7).

Rejeeth C, Kannan S, Muthuchelian K - Cancer Nanotechnol (2012)

Bottom Line: Interesting agarose gel electrophoresis studies revealed that the nanoparticles efficiently complex with pCMV-Myc vector.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.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Molecular Cell Physiology Lab, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, 641 046, TN India.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Confocal and fluorescence images of cancer cells treated with ORMOSILNs previously incubated with DNA labeled with ethidium monoazide (EMA). a Confocal bioimaging of cancer cells demonstrating expression of green fluorescent protein following in vitro administration. b Fluorescent image of nano-DNA complex stained with 4′-6-diamidino-2-phenylindole (DAPI). c 2-h incubation of nano-DNA complex with cells shows that the nanoparticles are present outside the cells. d Transformed into cells after 4-h incubation
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Fig9: Confocal and fluorescence images of cancer cells treated with ORMOSILNs previously incubated with DNA labeled with ethidium monoazide (EMA). a Confocal bioimaging of cancer cells demonstrating expression of green fluorescent protein following in vitro administration. b Fluorescent image of nano-DNA complex stained with 4′-6-diamidino-2-phenylindole (DAPI). c 2-h incubation of nano-DNA complex with cells shows that the nanoparticles are present outside the cells. d Transformed into cells after 4-h incubation

Mentions: The efficiency of ORMOSIL nanoparticles mediated transformation was analyzed using a transmission electron microscope (TEM) at cellular level. Figure 8 shows that the ORMOSIL nanoparticles are highly dispersed in the cytoplasm of transformed breast cancer cell line MCF-7 cells. There was no cellular distortion present on TEM images of breast carcinoma cells exposed to ORMOSIL nanoparticles alone. All organelles are intact and the cells imaged are unchanged except for the presence of ORMOSIL nanoparticles. Normally for breast carcinoma cells, the nuclear cytoplasmic ratio is very low. Lower concentration of nuclear cytoplasm ratio in transformed breast carcinoma cells was also confirmed by TEM image. Furthermore, at ×100,000 magnification, ORMOSIL nanoparticles of various sizes that are dispersed in cytoplasm are clearly visible. A previous study reported that hydrophobic dye-doped ORMOSIL nanoparticles are actively taken up by tumor cells in vitro, as seen by fluorescence staining of the cytoplasm (Roy et al. 2003). This cytoplasmic staining is observed for several types of tumor cells that we have investigated, an example of which is shown for MCF-7 cells in (Fig. 9). Although the mechanism of this cellular uptake is not yet fully understood, the net positive charge on the nanoparticles has been thought to play a crucial role. A similar observation was found in a recent study that has demonstrated high non-specific uptake of anionic magnetic nanoparticles by cells in vitro (Wilhem et al. 2003). In gene delivery, the genetic material, as soon as it is released inside the cytoplasm of a cell, should migrate to the nucleus (Davis 1997). Again, optical imaging can play a significant role in studying this nuclear migration of DNA. In our experiment, we irreversibly labeled DNA with the fluorescent dye GFP and DAPI complexed with unlabeled ORMOSIL nanoparticles as described. The post-release nuclear trafficking of DNA is demonstrated in the confocal image of MCF-7 cells in Fig. 9a. After comparison of this image with the image of MCF-7 cells stained with DAPI encapsulated nanoparticles (Fig. 9b), it can be seen that in addition to the cytoplasmic staining, there is considerable staining inside the nucleus. Optimal transfection conditions were evaluated by varying the amounts of plasmid DNA at different ORMOSIL/DNA ratios, incubation times, and media in order to form the complexes. Figure 9c and d shows finally the expression of nano-DNA complex as confirmed by DAPI fluorescent stain with the help of fluorescent microscopy. The 4-h incubation of the nano-DNA complex with breast cancer cell line MCF-7 showed that the nano-DNA complex was taken up completely by the cell as compared to 2 h of incubation. We observed that pCMV–p53 was transfected successfully by using the ORMOSIL nanoparticles as the non-viral delivery vector.Fig. 8


Development of in vitro gene delivery system using ORMOSIL nanoparticle: Analysis of p53 gene expression in cultured breast cancer cell (MCF-7).

Rejeeth C, Kannan S, Muthuchelian K - Cancer Nanotechnol (2012)

Confocal and fluorescence images of cancer cells treated with ORMOSILNs previously incubated with DNA labeled with ethidium monoazide (EMA). a Confocal bioimaging of cancer cells demonstrating expression of green fluorescent protein following in vitro administration. b Fluorescent image of nano-DNA complex stained with 4′-6-diamidino-2-phenylindole (DAPI). c 2-h incubation of nano-DNA complex with cells shows that the nanoparticles are present outside the cells. d Transformed into cells after 4-h incubation
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Related In: Results  -  Collection

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Fig9: Confocal and fluorescence images of cancer cells treated with ORMOSILNs previously incubated with DNA labeled with ethidium monoazide (EMA). a Confocal bioimaging of cancer cells demonstrating expression of green fluorescent protein following in vitro administration. b Fluorescent image of nano-DNA complex stained with 4′-6-diamidino-2-phenylindole (DAPI). c 2-h incubation of nano-DNA complex with cells shows that the nanoparticles are present outside the cells. d Transformed into cells after 4-h incubation
Mentions: The efficiency of ORMOSIL nanoparticles mediated transformation was analyzed using a transmission electron microscope (TEM) at cellular level. Figure 8 shows that the ORMOSIL nanoparticles are highly dispersed in the cytoplasm of transformed breast cancer cell line MCF-7 cells. There was no cellular distortion present on TEM images of breast carcinoma cells exposed to ORMOSIL nanoparticles alone. All organelles are intact and the cells imaged are unchanged except for the presence of ORMOSIL nanoparticles. Normally for breast carcinoma cells, the nuclear cytoplasmic ratio is very low. Lower concentration of nuclear cytoplasm ratio in transformed breast carcinoma cells was also confirmed by TEM image. Furthermore, at ×100,000 magnification, ORMOSIL nanoparticles of various sizes that are dispersed in cytoplasm are clearly visible. A previous study reported that hydrophobic dye-doped ORMOSIL nanoparticles are actively taken up by tumor cells in vitro, as seen by fluorescence staining of the cytoplasm (Roy et al. 2003). This cytoplasmic staining is observed for several types of tumor cells that we have investigated, an example of which is shown for MCF-7 cells in (Fig. 9). Although the mechanism of this cellular uptake is not yet fully understood, the net positive charge on the nanoparticles has been thought to play a crucial role. A similar observation was found in a recent study that has demonstrated high non-specific uptake of anionic magnetic nanoparticles by cells in vitro (Wilhem et al. 2003). In gene delivery, the genetic material, as soon as it is released inside the cytoplasm of a cell, should migrate to the nucleus (Davis 1997). Again, optical imaging can play a significant role in studying this nuclear migration of DNA. In our experiment, we irreversibly labeled DNA with the fluorescent dye GFP and DAPI complexed with unlabeled ORMOSIL nanoparticles as described. The post-release nuclear trafficking of DNA is demonstrated in the confocal image of MCF-7 cells in Fig. 9a. After comparison of this image with the image of MCF-7 cells stained with DAPI encapsulated nanoparticles (Fig. 9b), it can be seen that in addition to the cytoplasmic staining, there is considerable staining inside the nucleus. Optimal transfection conditions were evaluated by varying the amounts of plasmid DNA at different ORMOSIL/DNA ratios, incubation times, and media in order to form the complexes. Figure 9c and d shows finally the expression of nano-DNA complex as confirmed by DAPI fluorescent stain with the help of fluorescent microscopy. The 4-h incubation of the nano-DNA complex with breast cancer cell line MCF-7 showed that the nano-DNA complex was taken up completely by the cell as compared to 2 h of incubation. We observed that pCMV–p53 was transfected successfully by using the ORMOSIL nanoparticles as the non-viral delivery vector.Fig. 8

Bottom Line: Interesting agarose gel electrophoresis studies revealed that the nanoparticles efficiently complex with pCMV-Myc vector.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.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Molecular Cell Physiology Lab, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore, 641 046, TN India.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus