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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

Schematic of ORMOSIL nanoparticle compositions
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Fig4: Schematic of ORMOSIL nanoparticle compositions

Mentions: For non-viral gene delivery, we have developed and tested the formation of nanoparticles which consist of a stable aqueous dispersion of ultrafine ORMOSIL nanoparticles. ORMOSIL-based nanoparticles can be synthesized with varying surface change and are stable (non-aggregating) in aqueous environments. The genetic payload (negative charged DNA) is bound to the cationic group present on the surface of the nanoparticles. The non-aggregating potential, coupled with the designed ability to bind and protect DNA, provides a second platform from which a non-viral gene transfer vector will be produced and tested. The nanoparticles for DNA delivery are functionalized with amino group, which made it possible to electrostatically attach DNA molecules on the surface as shown schematically in Fig. 4. Nanoparticles were found to have high efficiency for the formation of ORMOSIL DNA complex. Figure 5 represents the results of agarose gel electrophoresis of plasmid DNA, free and complexes with ORMOSIL nanoparticles. It can be seen that with increasing amounts of amino groups (positive charge) on the nanoparticles, the mobility of the DNA complexes toward 50 μl, 30 μl, and 10 μl retards the positive terminal (lanes 3–5). This result suggests that the plasmid is no more able to move freely because the resulting nanoparticle–DNA complex has restricted the mobility in the gel. After treatment with DNase 1, the free plasmid is completely digested (Fig. 5, lane 6). The reason for this protection against enzymatic digestion is not yet fully understood. It was suggested recently that this behavior can be due to (1) repulsion of Mg2+ ions (which are necessary for the enzymatic reaction) by the amino groups, (2) a hindered access of the enzymes to the DNA that is immobilized on the nanoparticle surface, or (3) both (He et al. 2003). Note that the plasmid treated with the non-amino terminated particle (ORMOSIL) is also partially protected (Fig. 5, lane 2) because it has bands corresponding to both its non-enzymatically treated counterpart (Fig. 5, lane 3) and also some DNA fragments appearing at the bottom of the band. Therefore, these nanoparticles can also be considered as some kind of inhibitors toward the enzymatic action of DNase 1 on plasmid DNA. Alternatively, it is also possible that the interaction between the genetic material and the nanoparticle will not be entirely of electrostatic nature.Fig. 4


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)

Schematic of ORMOSIL nanoparticle compositions
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4544070&req=5

Fig4: Schematic of ORMOSIL nanoparticle compositions
Mentions: For non-viral gene delivery, we have developed and tested the formation of nanoparticles which consist of a stable aqueous dispersion of ultrafine ORMOSIL nanoparticles. ORMOSIL-based nanoparticles can be synthesized with varying surface change and are stable (non-aggregating) in aqueous environments. The genetic payload (negative charged DNA) is bound to the cationic group present on the surface of the nanoparticles. The non-aggregating potential, coupled with the designed ability to bind and protect DNA, provides a second platform from which a non-viral gene transfer vector will be produced and tested. The nanoparticles for DNA delivery are functionalized with amino group, which made it possible to electrostatically attach DNA molecules on the surface as shown schematically in Fig. 4. Nanoparticles were found to have high efficiency for the formation of ORMOSIL DNA complex. Figure 5 represents the results of agarose gel electrophoresis of plasmid DNA, free and complexes with ORMOSIL nanoparticles. It can be seen that with increasing amounts of amino groups (positive charge) on the nanoparticles, the mobility of the DNA complexes toward 50 μl, 30 μl, and 10 μl retards the positive terminal (lanes 3–5). This result suggests that the plasmid is no more able to move freely because the resulting nanoparticle–DNA complex has restricted the mobility in the gel. After treatment with DNase 1, the free plasmid is completely digested (Fig. 5, lane 6). The reason for this protection against enzymatic digestion is not yet fully understood. It was suggested recently that this behavior can be due to (1) repulsion of Mg2+ ions (which are necessary for the enzymatic reaction) by the amino groups, (2) a hindered access of the enzymes to the DNA that is immobilized on the nanoparticle surface, or (3) both (He et al. 2003). Note that the plasmid treated with the non-amino terminated particle (ORMOSIL) is also partially protected (Fig. 5, lane 2) because it has bands corresponding to both its non-enzymatically treated counterpart (Fig. 5, lane 3) and also some DNA fragments appearing at the bottom of the band. Therefore, these nanoparticles can also be considered as some kind of inhibitors toward the enzymatic action of DNase 1 on plasmid DNA. Alternatively, it is also possible that the interaction between the genetic material and the nanoparticle will not be entirely of electrostatic nature.Fig. 4

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