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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.

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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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DNA-gemini surfactant binding properties. An ethidium bromide dye exclusion assay was performed to determine the binding efficiency of 12-7NH-12 and 12-7NGK-12 with plasmid DNA in the presence of DOPE and heparin (5 U/ml, 10 U/ml and 20 U/ml). At lower concentration of heparin (polyanions), amino-acid substituted gemini exhibited stronger DNA binding compared to unsusbtituted gemini surfactant. At higher concentration (20 U/ml heparin), a significant increase in fluorescence was observed, indicating dissociation of DNA and 12-7NGK-12.
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Figure 9: DNA-gemini surfactant binding properties. An ethidium bromide dye exclusion assay was performed to determine the binding efficiency of 12-7NH-12 and 12-7NGK-12 with plasmid DNA in the presence of DOPE and heparin (5 U/ml, 10 U/ml and 20 U/ml). At lower concentration of heparin (polyanions), amino-acid substituted gemini exhibited stronger DNA binding compared to unsusbtituted gemini surfactant. At higher concentration (20 U/ml heparin), a significant increase in fluorescence was observed, indicating dissociation of DNA and 12-7NGK-12.

Mentions: At the 1:10 charge ratio of DNA and gemini surfactant in the presence of DOPE, a small increase in fluorescence (8.9 ± 0.8%) was observed in P/12-7NGK-12/L nanoparticles upon addition of polyanions (5 U/ml heparin), exhibiting a stronger protection of DNA compared to the parent gemini surfactant which had an increase of 52.3 ± 2.3% fluorescence (Figure 9). A similar trend occurred at higher concentrations of 10 U/ml and 20 U/ml of heparin. Interestingly, a steady increase in fluorescence was observed in P/12-7NGK-12/L nanoparticles with a significant spike in fluorescence (32.9 ± 5.7%) occurring in the presence of 20 U/ml heparin. This indicates that 12-7NGK-12 has balanced DNA binding properties which ensures protection of DNA as well as an ability to interact with a highly negative charged membrane, such as the inside of caveolae endosomes. These balanced binding properties (protection and release) may be instrumental in efficient endosomal escape leading to higher transfection efficiency of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles (Figure 6).


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)

DNA-gemini surfactant binding properties. An ethidium bromide dye exclusion assay was performed to determine the binding efficiency of 12-7NH-12 and 12-7NGK-12 with plasmid DNA in the presence of DOPE and heparin (5 U/ml, 10 U/ml and 20 U/ml). At lower concentration of heparin (polyanions), amino-acid substituted gemini exhibited stronger DNA binding compared to unsusbtituted gemini surfactant. At higher concentration (20 U/ml heparin), a significant increase in fluorescence was observed, indicating dissociation of DNA and 12-7NGK-12.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298462&req=5

Figure 9: DNA-gemini surfactant binding properties. An ethidium bromide dye exclusion assay was performed to determine the binding efficiency of 12-7NH-12 and 12-7NGK-12 with plasmid DNA in the presence of DOPE and heparin (5 U/ml, 10 U/ml and 20 U/ml). At lower concentration of heparin (polyanions), amino-acid substituted gemini exhibited stronger DNA binding compared to unsusbtituted gemini surfactant. At higher concentration (20 U/ml heparin), a significant increase in fluorescence was observed, indicating dissociation of DNA and 12-7NGK-12.
Mentions: At the 1:10 charge ratio of DNA and gemini surfactant in the presence of DOPE, a small increase in fluorescence (8.9 ± 0.8%) was observed in P/12-7NGK-12/L nanoparticles upon addition of polyanions (5 U/ml heparin), exhibiting a stronger protection of DNA compared to the parent gemini surfactant which had an increase of 52.3 ± 2.3% fluorescence (Figure 9). A similar trend occurred at higher concentrations of 10 U/ml and 20 U/ml of heparin. Interestingly, a steady increase in fluorescence was observed in P/12-7NGK-12/L nanoparticles with a significant spike in fluorescence (32.9 ± 5.7%) occurring in the presence of 20 U/ml heparin. This indicates that 12-7NGK-12 has balanced DNA binding properties which ensures protection of DNA as well as an ability to interact with a highly negative charged membrane, such as the inside of caveolae endosomes. These balanced binding properties (protection and release) may be instrumental in efficient endosomal escape leading to higher transfection efficiency of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles (Figure 6).

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.

Show MeSH
Related in: MedlinePlus