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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) Confocal images of HeLa cells transfected with nanoparticles (green) for 6 hours showing the intracellular localization. The nuclei are stained with DAPI and the endosomes are marked with LysoTracker Red DND-99. Merged images (yellow) show a perfect colocalization with the endosomes proving its uptake via endosomal pathway. (B) Images of HeLa cells after incubation with Cy3-labelled NP-siRNA complex at 37°C and 4°C. Cells incubated at 4°C show a complete loss of uptake proving its pathway via endosomes. (C) After pretreatment with NaN3. Showing a complete loss of uptake confirming the mechanism is energy-dependent. (D) Cellular internalization of cy3-labelled siRNA with NP complexes pretreated with 0.45 M sucrose, and K+-depleted by reconfirming the same pathway. Merged image in yellow clearly show formation of the siRNA-NP complexes. Nuclei were stained with DAPI. Scale bar 20 μm.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; DAPI, 4,6-diamidino-2-phenylindole.
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f4-ijn-10-6411: (A) Confocal images of HeLa cells transfected with nanoparticles (green) for 6 hours showing the intracellular localization. The nuclei are stained with DAPI and the endosomes are marked with LysoTracker Red DND-99. Merged images (yellow) show a perfect colocalization with the endosomes proving its uptake via endosomal pathway. (B) Images of HeLa cells after incubation with Cy3-labelled NP-siRNA complex at 37°C and 4°C. Cells incubated at 4°C show a complete loss of uptake proving its pathway via endosomes. (C) After pretreatment with NaN3. Showing a complete loss of uptake confirming the mechanism is energy-dependent. (D) Cellular internalization of cy3-labelled siRNA with NP complexes pretreated with 0.45 M sucrose, and K+-depleted by reconfirming the same pathway. Merged image in yellow clearly show formation of the siRNA-NP complexes. Nuclei were stained with DAPI. Scale bar 20 μm.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; DAPI, 4,6-diamidino-2-phenylindole.

Mentions: Next we examined the pathway of uptake of the NPs and the NP-siRNA complexes. The mechanism by which cells take up NPs and NP-siRNA complexes has been shown to involve endocytosis via the endosomal pathway.36 To confirm this pathway of uptake, we performed a series of experiments by incubating HeLa cells under different conditions using NPs followed by endosomal staining with LysoTracker Red-DND99. We observed colocalization (indicated by yellow color) of the red and green fluorescence signals in cells treated with NPs (Figure 4A). Of note is that incubation of the NPs not only produced red fluorescence with the LysoTracker Red probe, but also exhibited a diffuse pattern of localization within the cytoplasm. We can therefore hypothesize that NPs first accumulate in the lysosomes, and, as time elapses, they are released into the cytoplasm (Figure 4A). To substantiate our results further, we incubated the cells at 4°C and 37°C; endocytosis is a temperature (energy)-dependent uptake pathway, so uptake was negligible at 4°C (Figure 4B).39 To further verify the mechanism of energy-dependent uptake, we depleted the cellular ATP using NaN3, which is known to disturb the production of ATP in cells, then transfection with nanoplex. The uptake was much less in treated cells when compared with the control, confirming energy-dependent uptake (Figure 4C).40 To assess the role of clathrin in the internalization of the siRNA-nanoplexes, we carried out incubations under conditions that are known to disrupt the formation of clathrin-coated vesicles on the cell membrane. Here the treatment consisted of pretreating the cells with either sucrose (hypertonic treatment) or a K+-depleted medium prior to exposure to the NPs or nanoplexes.41 These pretreatments dramatically reduced the level of cellular uptake of nanoplexes as deduced from confocal images (Figure 4D), suggesting involvement of the clathrin pathway in the endocytotic cellular uptake of NPs and nanoplexes. The results confirmed an endosome-mediated mechanism (Figure 4E).


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) Confocal images of HeLa cells transfected with nanoparticles (green) for 6 hours showing the intracellular localization. The nuclei are stained with DAPI and the endosomes are marked with LysoTracker Red DND-99. Merged images (yellow) show a perfect colocalization with the endosomes proving its uptake via endosomal pathway. (B) Images of HeLa cells after incubation with Cy3-labelled NP-siRNA complex at 37°C and 4°C. Cells incubated at 4°C show a complete loss of uptake proving its pathway via endosomes. (C) After pretreatment with NaN3. Showing a complete loss of uptake confirming the mechanism is energy-dependent. (D) Cellular internalization of cy3-labelled siRNA with NP complexes pretreated with 0.45 M sucrose, and K+-depleted by reconfirming the same pathway. Merged image in yellow clearly show formation of the siRNA-NP complexes. Nuclei were stained with DAPI. Scale bar 20 μm.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; DAPI, 4,6-diamidino-2-phenylindole.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ijn-10-6411: (A) Confocal images of HeLa cells transfected with nanoparticles (green) for 6 hours showing the intracellular localization. The nuclei are stained with DAPI and the endosomes are marked with LysoTracker Red DND-99. Merged images (yellow) show a perfect colocalization with the endosomes proving its uptake via endosomal pathway. (B) Images of HeLa cells after incubation with Cy3-labelled NP-siRNA complex at 37°C and 4°C. Cells incubated at 4°C show a complete loss of uptake proving its pathway via endosomes. (C) After pretreatment with NaN3. Showing a complete loss of uptake confirming the mechanism is energy-dependent. (D) Cellular internalization of cy3-labelled siRNA with NP complexes pretreated with 0.45 M sucrose, and K+-depleted by reconfirming the same pathway. Merged image in yellow clearly show formation of the siRNA-NP complexes. Nuclei were stained with DAPI. Scale bar 20 μm.Abbreviations: NPs, nanoparticles; siRNA, short interfering RNA; DAPI, 4,6-diamidino-2-phenylindole.
Mentions: Next we examined the pathway of uptake of the NPs and the NP-siRNA complexes. The mechanism by which cells take up NPs and NP-siRNA complexes has been shown to involve endocytosis via the endosomal pathway.36 To confirm this pathway of uptake, we performed a series of experiments by incubating HeLa cells under different conditions using NPs followed by endosomal staining with LysoTracker Red-DND99. We observed colocalization (indicated by yellow color) of the red and green fluorescence signals in cells treated with NPs (Figure 4A). Of note is that incubation of the NPs not only produced red fluorescence with the LysoTracker Red probe, but also exhibited a diffuse pattern of localization within the cytoplasm. We can therefore hypothesize that NPs first accumulate in the lysosomes, and, as time elapses, they are released into the cytoplasm (Figure 4A). To substantiate our results further, we incubated the cells at 4°C and 37°C; endocytosis is a temperature (energy)-dependent uptake pathway, so uptake was negligible at 4°C (Figure 4B).39 To further verify the mechanism of energy-dependent uptake, we depleted the cellular ATP using NaN3, which is known to disturb the production of ATP in cells, then transfection with nanoplex. The uptake was much less in treated cells when compared with the control, confirming energy-dependent uptake (Figure 4C).40 To assess the role of clathrin in the internalization of the siRNA-nanoplexes, we carried out incubations under conditions that are known to disrupt the formation of clathrin-coated vesicles on the cell membrane. Here the treatment consisted of pretreating the cells with either sucrose (hypertonic treatment) or a K+-depleted medium prior to exposure to the NPs or nanoplexes.41 These pretreatments dramatically reduced the level of cellular uptake of nanoplexes as deduced from confocal images (Figure 4D), suggesting involvement of the clathrin pathway in the endocytotic cellular uptake of NPs and nanoplexes. The results confirmed an endosome-mediated mechanism (Figure 4E).

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