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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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Laser scanning confocal microscopy (LSCM) images of Sf 1 Ep cells. Cells were pretreated with genistein (B & G), chlorpromazine (C & H), methyl-β-cyclodextrin (D & I) and wortmannin (E & J). Cells (A to E) were incubated with P/12-7NGK-12/L and Cells (F to J) were incubated with P/12-7NH-12/L for 2 hours. DAPI was used for staining the cell nuclei. At the end of the study, cells were washed with PBS and images were recorded. DNA (red), DOPE (green) and cell nuclei (blue).
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Figure 5: Laser scanning confocal microscopy (LSCM) images of Sf 1 Ep cells. Cells were pretreated with genistein (B & G), chlorpromazine (C & H), methyl-β-cyclodextrin (D & I) and wortmannin (E & J). Cells (A to E) were incubated with P/12-7NGK-12/L and Cells (F to J) were incubated with P/12-7NH-12/L for 2 hours. DAPI was used for staining the cell nuclei. At the end of the study, cells were washed with PBS and images were recorded. DNA (red), DOPE (green) and cell nuclei (blue).

Mentions: P/G/L nanoparticles were prepared using TM-rhodamine (red) labeled plasmid in the presence of fluorescent (green) labeled helper lipid. DAPI (blue) was used as fluorescent stain for the cell nuclei. The objective was to determine, qualitatively, whether materials were transferred into the cells and whether the presence of inhibitors induced a difference in the cellular uptake of P/12-7NH-12/L and P/12-7NGK-12/L nanoparticles. Interestingly, we observed that the inhibition of caveolae-mediated (genistein) or clathrin-mediated (chlorpromazine) uptake pathway did not have any effect on cellular uptake of P/12-7NGK-12/L or P/12-7NH-12/L nanoparticles (Figure 5B &5G, 5C &5H). However, almost no particles were internalized by the cells treated with either P/12-7NGK-12/L or P/12-7NH-12/L nanoparticles when both clathrin-mediated and caveolae-mediated uptake pathways were inhibited by prior treatment with methyl-β-cyclodextrin (Figure 5D &5I). This suggests that both clathrin-mediated and caveolae-mediated pathways are playing an important role in uptake of these particles. Wortmannin (Figure 5E &5J) showed no difference in the cellular uptake of P/12-7NGK-12/L or P/12-7NH-12/L nanoparticles.


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)

Laser scanning confocal microscopy (LSCM) images of Sf 1 Ep cells. Cells were pretreated with genistein (B & G), chlorpromazine (C & H), methyl-β-cyclodextrin (D & I) and wortmannin (E & J). Cells (A to E) were incubated with P/12-7NGK-12/L and Cells (F to J) were incubated with P/12-7NH-12/L for 2 hours. DAPI was used for staining the cell nuclei. At the end of the study, cells were washed with PBS and images were recorded. DNA (red), DOPE (green) and cell nuclei (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3298462&req=5

Figure 5: Laser scanning confocal microscopy (LSCM) images of Sf 1 Ep cells. Cells were pretreated with genistein (B & G), chlorpromazine (C & H), methyl-β-cyclodextrin (D & I) and wortmannin (E & J). Cells (A to E) were incubated with P/12-7NGK-12/L and Cells (F to J) were incubated with P/12-7NH-12/L for 2 hours. DAPI was used for staining the cell nuclei. At the end of the study, cells were washed with PBS and images were recorded. DNA (red), DOPE (green) and cell nuclei (blue).
Mentions: P/G/L nanoparticles were prepared using TM-rhodamine (red) labeled plasmid in the presence of fluorescent (green) labeled helper lipid. DAPI (blue) was used as fluorescent stain for the cell nuclei. The objective was to determine, qualitatively, whether materials were transferred into the cells and whether the presence of inhibitors induced a difference in the cellular uptake of P/12-7NH-12/L and P/12-7NGK-12/L nanoparticles. Interestingly, we observed that the inhibition of caveolae-mediated (genistein) or clathrin-mediated (chlorpromazine) uptake pathway did not have any effect on cellular uptake of P/12-7NGK-12/L or P/12-7NH-12/L nanoparticles (Figure 5B &5G, 5C &5H). However, almost no particles were internalized by the cells treated with either P/12-7NGK-12/L or P/12-7NH-12/L nanoparticles when both clathrin-mediated and caveolae-mediated uptake pathways were inhibited by prior treatment with methyl-β-cyclodextrin (Figure 5D &5I). This suggests that both clathrin-mediated and caveolae-mediated pathways are playing an important role in uptake of these particles. Wortmannin (Figure 5E &5J) showed no difference in the cellular uptake of P/12-7NGK-12/L or P/12-7NH-12/L nanoparticles.

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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