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Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles.

Singh J, Michel D, Chitanda JM, Verrall RE, Badea I - J Nanobiotechnology (2012)

Bottom Line: Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles.Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant.A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.

ABSTRACT

Background: Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency.

Results: Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression.

Conclusion: Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

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Effect of endocytic inhibitors on gene expression of P/G/L nanoparticles. Interferon (IFN)-γ expression was measured at 72 hours in cells incubated with P/G/L nanoparticles in the presence of endocytic inhibitors. In the P/12-7NH-12/L-treated cells, gene expression in cells pre-treated with caveolae-mediated uptake inhibitors (genistein, filipin) was significantly lower than the untreated (no inhibitors) cells, whereas gene expression was significantly higher on inhibiting clathrin-mediated uptake (chlorpromazine). For the P/12-7NGK-12/L nanoparticles, gene expression was significantly lower in presence of methyl-β-cyclodextrin (clathrin- and caveolae-mediated uptake inhibitor). * represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NH-12/L nanoparticles (No inhibitors black bar). $ represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NGK-12/L nanoparticles (No inhibitors dashed-line bar).
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Figure 6: Effect of endocytic inhibitors on gene expression of P/G/L nanoparticles. Interferon (IFN)-γ expression was measured at 72 hours in cells incubated with P/G/L nanoparticles in the presence of endocytic inhibitors. In the P/12-7NH-12/L-treated cells, gene expression in cells pre-treated with caveolae-mediated uptake inhibitors (genistein, filipin) was significantly lower than the untreated (no inhibitors) cells, whereas gene expression was significantly higher on inhibiting clathrin-mediated uptake (chlorpromazine). For the P/12-7NGK-12/L nanoparticles, gene expression was significantly lower in presence of methyl-β-cyclodextrin (clathrin- and caveolae-mediated uptake inhibitor). * represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NH-12/L nanoparticles (No inhibitors black bar). $ represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NGK-12/L nanoparticles (No inhibitors dashed-line bar).

Mentions: In the absence of any inhibitor treatment to the cells, the gene expression in the P/12-7NH-12/L transfected cells was 183 ± 17.5 pg IFNγ/20000 cells (Figure 6). Gene expression after P/12-7NH-12/L nanoparticle transfection was 84 ± 9.4 pg IFNγ/20000 cells and 31 ± 14.9 pg IFNγ/20000 cells in the presence of genistein and filipin, respectively, significantly lower (p < 0.05) compared to the cells treated with the P/G/L in the absence of the inhibitors.


Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles.

Singh J, Michel D, Chitanda JM, Verrall RE, Badea I - J Nanobiotechnology (2012)

Effect of endocytic inhibitors on gene expression of P/G/L nanoparticles. Interferon (IFN)-γ expression was measured at 72 hours in cells incubated with P/G/L nanoparticles in the presence of endocytic inhibitors. In the P/12-7NH-12/L-treated cells, gene expression in cells pre-treated with caveolae-mediated uptake inhibitors (genistein, filipin) was significantly lower than the untreated (no inhibitors) cells, whereas gene expression was significantly higher on inhibiting clathrin-mediated uptake (chlorpromazine). For the P/12-7NGK-12/L nanoparticles, gene expression was significantly lower in presence of methyl-β-cyclodextrin (clathrin- and caveolae-mediated uptake inhibitor). * represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NH-12/L nanoparticles (No inhibitors black bar). $ represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NGK-12/L nanoparticles (No inhibitors dashed-line bar).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Effect of endocytic inhibitors on gene expression of P/G/L nanoparticles. Interferon (IFN)-γ expression was measured at 72 hours in cells incubated with P/G/L nanoparticles in the presence of endocytic inhibitors. In the P/12-7NH-12/L-treated cells, gene expression in cells pre-treated with caveolae-mediated uptake inhibitors (genistein, filipin) was significantly lower than the untreated (no inhibitors) cells, whereas gene expression was significantly higher on inhibiting clathrin-mediated uptake (chlorpromazine). For the P/12-7NGK-12/L nanoparticles, gene expression was significantly lower in presence of methyl-β-cyclodextrin (clathrin- and caveolae-mediated uptake inhibitor). * represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NH-12/L nanoparticles (No inhibitors black bar). $ represents significant difference, p < 0.05, compared to 'No inhibitors' control cells treated with P/12-7NGK-12/L nanoparticles (No inhibitors dashed-line bar).
Mentions: In the absence of any inhibitor treatment to the cells, the gene expression in the P/12-7NH-12/L transfected cells was 183 ± 17.5 pg IFNγ/20000 cells (Figure 6). Gene expression after P/12-7NH-12/L nanoparticle transfection was 84 ± 9.4 pg IFNγ/20000 cells and 31 ± 14.9 pg IFNγ/20000 cells in the presence of genistein and filipin, respectively, significantly lower (p < 0.05) compared to the cells treated with the P/G/L in the absence of the inhibitors.

Bottom Line: Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles.Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant.A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.

ABSTRACT

Background: Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency.

Results: Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression.

Conclusion: Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

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