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Para amino benzoic acid-derived self-assembled biocompatible nanoparticles for efficient delivery of siRNA.

Reddy TL, Krishnarao PS, Rao GK, Bhimireddy E, Venkateswarlu P, Mohapatra DK, Yadav JS, Bhadra U, Bhadra MP - Int J Nanomedicine (2015)

Bottom Line: A number of diseases can result from abnormal gene expression.Our findings indicated high gene transfection efficiency.These biocompatible nanoparticles allow targeted delivery of siRNA, providing an efficient vehicle for gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, India ; Academy of Scientific and Innovative Research, New Delhi, India.

ABSTRACT
A number of diseases can result from abnormal gene expression. One of the approaches for treating such diseases is gene therapy to inhibit expression of a particular gene in a specific cell population by RNA interference. Use of efficient delivery vehicles increases the safety and success of gene therapy. Here we report the development of functionalized biocompatible fluorescent nanoparticles from para amino benzoic acid nanoparticles for efficient delivery of short interfering RNA (siRNA). These nanoparticles were non-toxic and did not interfere with progression of the cell cycle. The intrinsic fluorescent nature of these nanoparticles allows easy tracking and an opportunity for diagnostic applications. Human Bcl-2 siRNA was complexed with these nanoparticles to inhibit expression in cells at both the transcriptional and translational levels. Our findings indicated high gene transfection efficiency. These biocompatible nanoparticles allow targeted delivery of siRNA, providing an efficient vehicle for gene delivery.

No MeSH data available.


Related in: MedlinePlus

(A) Real-time polymerase chain reaction analysis and (B) representative Western blot analysis of the Bcl-2 gene expression in HeLa cells after transfection with NP-Bcl-2-siRNA. (C) Densitometry graph demonstrating Bcl-2 protein expression in HeLa cells post transfection with NP-Bcl-2-siRNA. Untreated and transfected with non-silencing siRNA serves as controls. Cells transfected with Lipofectamine 2000 served as the positive control. Data represented in the graph are expressed as a ratio to the control. All the data are normalized to the house-keeping gene β-actin.Notes: Values are mean ± standard deviation; n=3; ***P<0.001.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA.
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f5-ijn-10-6411: (A) Real-time polymerase chain reaction analysis and (B) representative Western blot analysis of the Bcl-2 gene expression in HeLa cells after transfection with NP-Bcl-2-siRNA. (C) Densitometry graph demonstrating Bcl-2 protein expression in HeLa cells post transfection with NP-Bcl-2-siRNA. Untreated and transfected with non-silencing siRNA serves as controls. Cells transfected with Lipofectamine 2000 served as the positive control. Data represented in the graph are expressed as a ratio to the control. All the data are normalized to the house-keeping gene β-actin.Notes: Values are mean ± standard deviation; n=3; ***P<0.001.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA.

Mentions: To examine whether the NPs could deliver the siRNA to their target and silence the gene of interest at the mRNA level, we performed real time-PCR analysis after transfecting HeLa cells for 24 hours with NPs complexed with siRNA against Bcl-2 (conc. of siRNA 100 nM). Bcl-2 was chosen as it exhibits anti-apoptotic activity and is overexpressed in several types of cancer. Bcl-2 protein blocks the release of cytochrome-C, which in turn prevents the propagation of death signals and promotes cell survival. Earlier studies have shown that apoptosis in cancer cells can be induced by silencing this gene. Real time-PCR was performed and the amount of mRNA expression was calculated using the ΔΔCT method. Downregulation of Bcl-2 mRNA was determined by comparison of the ratio between Bcl-2 and endogenous control (β-actin) mRNA expression for the transfected samples against the non-transfected samples. NP-Bcl-2-siRNA complexes were able to silence Bcl-2 mRNA levels by 80%–90% of the normal expression level (G10–G18U) when compared with the control (Figure 5A). Simultaneous experiments with scrambled siRNA confirmed that the NPs themselves do not contribute to silencing the target gene because there was no significant change in expression of mRNA of the target gene when compared with untreated controls (Figure S3B).


Para amino benzoic acid-derived self-assembled biocompatible nanoparticles for efficient delivery of siRNA.

Reddy TL, Krishnarao PS, Rao GK, Bhimireddy E, Venkateswarlu P, Mohapatra DK, Yadav JS, Bhadra U, Bhadra MP - Int J Nanomedicine (2015)

(A) Real-time polymerase chain reaction analysis and (B) representative Western blot analysis of the Bcl-2 gene expression in HeLa cells after transfection with NP-Bcl-2-siRNA. (C) Densitometry graph demonstrating Bcl-2 protein expression in HeLa cells post transfection with NP-Bcl-2-siRNA. Untreated and transfected with non-silencing siRNA serves as controls. Cells transfected with Lipofectamine 2000 served as the positive control. Data represented in the graph are expressed as a ratio to the control. All the data are normalized to the house-keeping gene β-actin.Notes: Values are mean ± standard deviation; n=3; ***P<0.001.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608593&req=5

f5-ijn-10-6411: (A) Real-time polymerase chain reaction analysis and (B) representative Western blot analysis of the Bcl-2 gene expression in HeLa cells after transfection with NP-Bcl-2-siRNA. (C) Densitometry graph demonstrating Bcl-2 protein expression in HeLa cells post transfection with NP-Bcl-2-siRNA. Untreated and transfected with non-silencing siRNA serves as controls. Cells transfected with Lipofectamine 2000 served as the positive control. Data represented in the graph are expressed as a ratio to the control. All the data are normalized to the house-keeping gene β-actin.Notes: Values are mean ± standard deviation; n=3; ***P<0.001.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA.
Mentions: To examine whether the NPs could deliver the siRNA to their target and silence the gene of interest at the mRNA level, we performed real time-PCR analysis after transfecting HeLa cells for 24 hours with NPs complexed with siRNA against Bcl-2 (conc. of siRNA 100 nM). Bcl-2 was chosen as it exhibits anti-apoptotic activity and is overexpressed in several types of cancer. Bcl-2 protein blocks the release of cytochrome-C, which in turn prevents the propagation of death signals and promotes cell survival. Earlier studies have shown that apoptosis in cancer cells can be induced by silencing this gene. Real time-PCR was performed and the amount of mRNA expression was calculated using the ΔΔCT method. Downregulation of Bcl-2 mRNA was determined by comparison of the ratio between Bcl-2 and endogenous control (β-actin) mRNA expression for the transfected samples against the non-transfected samples. NP-Bcl-2-siRNA complexes were able to silence Bcl-2 mRNA levels by 80%–90% of the normal expression level (G10–G18U) when compared with the control (Figure 5A). Simultaneous experiments with scrambled siRNA confirmed that the NPs themselves do not contribute to silencing the target gene because there was no significant change in expression of mRNA of the target gene when compared with untreated controls (Figure S3B).

Bottom Line: A number of diseases can result from abnormal gene expression.Our findings indicated high gene transfection efficiency.These biocompatible nanoparticles allow targeted delivery of siRNA, providing an efficient vehicle for gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, India ; Academy of Scientific and Innovative Research, New Delhi, India.

ABSTRACT
A number of diseases can result from abnormal gene expression. One of the approaches for treating such diseases is gene therapy to inhibit expression of a particular gene in a specific cell population by RNA interference. Use of efficient delivery vehicles increases the safety and success of gene therapy. Here we report the development of functionalized biocompatible fluorescent nanoparticles from para amino benzoic acid nanoparticles for efficient delivery of short interfering RNA (siRNA). These nanoparticles were non-toxic and did not interfere with progression of the cell cycle. The intrinsic fluorescent nature of these nanoparticles allows easy tracking and an opportunity for diagnostic applications. Human Bcl-2 siRNA was complexed with these nanoparticles to inhibit expression in cells at both the transcriptional and translational levels. Our findings indicated high gene transfection efficiency. These biocompatible nanoparticles allow targeted delivery of siRNA, providing an efficient vehicle for gene delivery.

No MeSH data available.


Related in: MedlinePlus