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Increased gene delivery efficiency and specificity of a lipid-based nanosystem incorporating a glycolipid.

Magalhães M, Farinha D, Pedroso de Lima MC, Faneca H - Int J Nanomedicine (2014)

Bottom Line: In the presence of galactose, which competes with lactosyl-PE for the binding to the asialoglycoprotein receptor (ASGP-R), a significant reduction in biological activity was observed, showing that the potentiation of transfection induced by the presence of lactosyl-PE could be due to its specific interaction with ASGP-R, which is overexpressed in HCC.In addition, it was found that the incorporation of lactosyl-PE in the nanosystems promotes an increase in their cell binding and uptake.Regarding the physicochemical properties of lipoplexes, the presence of lactosyl-PE resulted in a significant increase in DNA protection and in a substantial decrease in their mean diameter and zeta potential, conferring them suitable characteristics for in vivo application.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal ; Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Hepatocellular carcinoma (HCC) is the third most common cause of death related to cancer diseases worldwide. The current treatment options have many limitations and reduced success rates. In this regard, advances in gene therapy have shown promising results in novel therapeutic strategies. However, the success of gene therapy depends on the efficient and specific delivery of genetic material into target cells. In this regard, the main goal of this work was to develop a new lipid-based nanosystem formulation containing the lipid lactosyl-PE for specific and efficient gene delivery into HCC cells. The obtained results showed that incorporation of 15% of lactosyl-PE into liposomes induces a strong potentiation of lipoplex biological activity in HepG2 cells, not only in terms of transgene expression levels but also in terms of percentage of transfected cells. In the presence of galactose, which competes with lactosyl-PE for the binding to the asialoglycoprotein receptor (ASGP-R), a significant reduction in biological activity was observed, showing that the potentiation of transfection induced by the presence of lactosyl-PE could be due to its specific interaction with ASGP-R, which is overexpressed in HCC. In addition, it was found that the incorporation of lactosyl-PE in the nanosystems promotes an increase in their cell binding and uptake. Regarding the physicochemical properties of lipoplexes, the presence of lactosyl-PE resulted in a significant increase in DNA protection and in a substantial decrease in their mean diameter and zeta potential, conferring them suitable characteristics for in vivo application. Overall, the results obtained in this study suggest that the potentiation of the biological activity induced by the presence of lactosyl-PE is due to its specific binding to the ASGP-R, showing that this novel formulation could constitute a new gene delivery nanosystem for application in therapeutic strategies in HCC.

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Transfection efficiency of nanosystems in HepG2 cells, evaluated by flow cytometry (A) and fluorescence microscopy (B).Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) Percentage of transfected cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with lipoplexes prepared at the same charge ratio, without lactosyl-PE. (B) Representative fluorescence microscopy images: panels (I) EPOPC:Chol/DNA (+/−) (2/1); (II) EPOPC:Chol/DNA (+/−) (4/1); (III) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1); and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (4/1). Data are representative of at least three independent experiments.Abbreviations: HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; GFP, green fluorescent protein.
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f2-ijn-9-4979: Transfection efficiency of nanosystems in HepG2 cells, evaluated by flow cytometry (A) and fluorescence microscopy (B).Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) Percentage of transfected cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with lipoplexes prepared at the same charge ratio, without lactosyl-PE. (B) Representative fluorescence microscopy images: panels (I) EPOPC:Chol/DNA (+/−) (2/1); (II) EPOPC:Chol/DNA (+/−) (4/1); (III) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1); and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (4/1). Data are representative of at least three independent experiments.Abbreviations: HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; GFP, green fluorescent protein.

Mentions: In order to assess the transfection efficiency of the generated nanosystems, which shows the amount of cells that could directly benefit from a gene therapy strategy, we prepared lipoplex formulations with the plasmid DNA encoding the GFP and analyzed the percentage of transfected cells by flow cytometry and fluorescence microscopy (Figure 2). The results obtained by flow cytometry (Figure 2A) showed that the percentage of transfected cells obtained with the best formulation, EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipoplexes, was approximately 40%, this being much higher than that observed with plain lipoplexes (without lactosyl-PE) prepared at the same charge ratio (EPOPC:Chol/DNA [+/−] 2/1). On the other hand, for lipoplexes prepared at the 4/1 (+/−) charge ratio, the incorporation of 15% of lactosyl-PE promoted only a slight increase in the percentage of transfected cells.


Increased gene delivery efficiency and specificity of a lipid-based nanosystem incorporating a glycolipid.

Magalhães M, Farinha D, Pedroso de Lima MC, Faneca H - Int J Nanomedicine (2014)

Transfection efficiency of nanosystems in HepG2 cells, evaluated by flow cytometry (A) and fluorescence microscopy (B).Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) Percentage of transfected cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with lipoplexes prepared at the same charge ratio, without lactosyl-PE. (B) Representative fluorescence microscopy images: panels (I) EPOPC:Chol/DNA (+/−) (2/1); (II) EPOPC:Chol/DNA (+/−) (4/1); (III) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1); and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (4/1). Data are representative of at least three independent experiments.Abbreviations: HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; GFP, green fluorescent protein.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4216029&req=5

f2-ijn-9-4979: Transfection efficiency of nanosystems in HepG2 cells, evaluated by flow cytometry (A) and fluorescence microscopy (B).Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) Percentage of transfected cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with lipoplexes prepared at the same charge ratio, without lactosyl-PE. (B) Representative fluorescence microscopy images: panels (I) EPOPC:Chol/DNA (+/−) (2/1); (II) EPOPC:Chol/DNA (+/−) (4/1); (III) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1); and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (4/1). Data are representative of at least three independent experiments.Abbreviations: HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; GFP, green fluorescent protein.
Mentions: In order to assess the transfection efficiency of the generated nanosystems, which shows the amount of cells that could directly benefit from a gene therapy strategy, we prepared lipoplex formulations with the plasmid DNA encoding the GFP and analyzed the percentage of transfected cells by flow cytometry and fluorescence microscopy (Figure 2). The results obtained by flow cytometry (Figure 2A) showed that the percentage of transfected cells obtained with the best formulation, EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipoplexes, was approximately 40%, this being much higher than that observed with plain lipoplexes (without lactosyl-PE) prepared at the same charge ratio (EPOPC:Chol/DNA [+/−] 2/1). On the other hand, for lipoplexes prepared at the 4/1 (+/−) charge ratio, the incorporation of 15% of lactosyl-PE promoted only a slight increase in the percentage of transfected cells.

Bottom Line: In the presence of galactose, which competes with lactosyl-PE for the binding to the asialoglycoprotein receptor (ASGP-R), a significant reduction in biological activity was observed, showing that the potentiation of transfection induced by the presence of lactosyl-PE could be due to its specific interaction with ASGP-R, which is overexpressed in HCC.In addition, it was found that the incorporation of lactosyl-PE in the nanosystems promotes an increase in their cell binding and uptake.Regarding the physicochemical properties of lipoplexes, the presence of lactosyl-PE resulted in a significant increase in DNA protection and in a substantial decrease in their mean diameter and zeta potential, conferring them suitable characteristics for in vivo application.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal ; Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Hepatocellular carcinoma (HCC) is the third most common cause of death related to cancer diseases worldwide. The current treatment options have many limitations and reduced success rates. In this regard, advances in gene therapy have shown promising results in novel therapeutic strategies. However, the success of gene therapy depends on the efficient and specific delivery of genetic material into target cells. In this regard, the main goal of this work was to develop a new lipid-based nanosystem formulation containing the lipid lactosyl-PE for specific and efficient gene delivery into HCC cells. The obtained results showed that incorporation of 15% of lactosyl-PE into liposomes induces a strong potentiation of lipoplex biological activity in HepG2 cells, not only in terms of transgene expression levels but also in terms of percentage of transfected cells. In the presence of galactose, which competes with lactosyl-PE for the binding to the asialoglycoprotein receptor (ASGP-R), a significant reduction in biological activity was observed, showing that the potentiation of transfection induced by the presence of lactosyl-PE could be due to its specific interaction with ASGP-R, which is overexpressed in HCC. In addition, it was found that the incorporation of lactosyl-PE in the nanosystems promotes an increase in their cell binding and uptake. Regarding the physicochemical properties of lipoplexes, the presence of lactosyl-PE resulted in a significant increase in DNA protection and in a substantial decrease in their mean diameter and zeta potential, conferring them suitable characteristics for in vivo application. Overall, the results obtained in this study suggest that the potentiation of the biological activity induced by the presence of lactosyl-PE is due to its specific binding to the ASGP-R, showing that this novel formulation could constitute a new gene delivery nanosystem for application in therapeutic strategies in HCC.

Show MeSH
Related in: MedlinePlus