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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) Concentration-dependent gel retardation assay NPs with siRNA (electrophoretic mobility of siRNA in the nanoparticle/siRNA complex). (B) Gel retardation assay (siRNA binding assay) of siRNA with our NPs at 1:20 (w/w). (C) RNase protection assay. Degradation of siRNA exposed to RNase, NP-siRNA complexes and compared with free siRNA. (D) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on HeLa cells. (E) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on MDA-MB-231/RFP cells. (F) Apoptotic analysis of HeLa after treatment with NPs (60 μg/mL).Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; RFP, red fluorescent protein.
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f2-ijn-10-6411: (A) Concentration-dependent gel retardation assay NPs with siRNA (electrophoretic mobility of siRNA in the nanoparticle/siRNA complex). (B) Gel retardation assay (siRNA binding assay) of siRNA with our NPs at 1:20 (w/w). (C) RNase protection assay. Degradation of siRNA exposed to RNase, NP-siRNA complexes and compared with free siRNA. (D) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on HeLa cells. (E) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on MDA-MB-231/RFP cells. (F) Apoptotic analysis of HeLa after treatment with NPs (60 μg/mL).Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; RFP, red fluorescent protein.

Mentions: To determine the amount of siRNA bound to the NPs, a series of dispersion was prepared using a fixed (1 μg) concentration of siRNA solution in water with increasing concentration of NPs (1–20 μg). A sample of siRNA (without NPs) served as the control (Figure 2A). To confirm the binding of NPs with siRNA, we a performed gel retardation assay. NP-siRNA complexes was prepared (1:20 w/w) and electrophoresed on 2% agarose gel containing ethidium bromide (Figure 2B). The gel was visualized using a Gel Doc system (Bio-Rad Laboratories Inc, Hercules, CA, USA). All the NPs showed significant interaction with siRNA. As expected, the siRNA migrated to the bottom of the gel. In contrast, all of the NPs complexed to fixed amounts of siRNA were blocked in the gel slots at the top, confirming that migration of siRNA was prevented due to formation of complexes with NPs (Figure 2A and 2B). We also quantified the amount of free (unbound) siRNA using Quant-IT Ribogreen reagent and found only trace amounts (<10%).37,38


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) Concentration-dependent gel retardation assay NPs with siRNA (electrophoretic mobility of siRNA in the nanoparticle/siRNA complex). (B) Gel retardation assay (siRNA binding assay) of siRNA with our NPs at 1:20 (w/w). (C) RNase protection assay. Degradation of siRNA exposed to RNase, NP-siRNA complexes and compared with free siRNA. (D) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on HeLa cells. (E) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on MDA-MB-231/RFP cells. (F) Apoptotic analysis of HeLa after treatment with NPs (60 μg/mL).Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; RFP, red fluorescent protein.
© Copyright Policy
Related In: Results  -  Collection

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

f2-ijn-10-6411: (A) Concentration-dependent gel retardation assay NPs with siRNA (electrophoretic mobility of siRNA in the nanoparticle/siRNA complex). (B) Gel retardation assay (siRNA binding assay) of siRNA with our NPs at 1:20 (w/w). (C) RNase protection assay. Degradation of siRNA exposed to RNase, NP-siRNA complexes and compared with free siRNA. (D) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on HeLa cells. (E) Cytotoxicity of NPs and NP-siRNA (scrambled) complex (siRNA concentration 100 nM) on MDA-MB-231/RFP cells. (F) Apoptotic analysis of HeLa after treatment with NPs (60 μg/mL).Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; RFP, red fluorescent protein.
Mentions: To determine the amount of siRNA bound to the NPs, a series of dispersion was prepared using a fixed (1 μg) concentration of siRNA solution in water with increasing concentration of NPs (1–20 μg). A sample of siRNA (without NPs) served as the control (Figure 2A). To confirm the binding of NPs with siRNA, we a performed gel retardation assay. NP-siRNA complexes was prepared (1:20 w/w) and electrophoresed on 2% agarose gel containing ethidium bromide (Figure 2B). The gel was visualized using a Gel Doc system (Bio-Rad Laboratories Inc, Hercules, CA, USA). All the NPs showed significant interaction with siRNA. As expected, the siRNA migrated to the bottom of the gel. In contrast, all of the NPs complexed to fixed amounts of siRNA were blocked in the gel slots at the top, confirming that migration of siRNA was prevented due to formation of complexes with NPs (Figure 2A and 2B). We also quantified the amount of free (unbound) siRNA using Quant-IT Ribogreen reagent and found only trace amounts (<10%).37,38

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