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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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Access of ethidium bromide (A) and resistance to DNase I (B) of DNA carried by nanosystems.Notes: Complexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Data (mean ± SD of triplicates) are presented as percentage of control (DNA in the absence of lipid) and are representative of three independent experiments. The asterisks (**P<0.01) correspond to values that differ significantly from those obtained with nanosystem formulations prepared at the same charge ratios without lactosyl-PE. (B) Lipoplexes and naked DNA were submitted to DNase I (inactive DNase I [I] or active DNase I [A]), followed by electrophoresis analysis. Data are representative of three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; EtBr, ethidium bromide.
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f7-ijn-9-4979: Access of ethidium bromide (A) and resistance to DNase I (B) of DNA carried by nanosystems.Notes: Complexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Data (mean ± SD of triplicates) are presented as percentage of control (DNA in the absence of lipid) and are representative of three independent experiments. The asterisks (**P<0.01) correspond to values that differ significantly from those obtained with nanosystem formulations prepared at the same charge ratios without lactosyl-PE. (B) Lipoplexes and naked DNA were submitted to DNase I (inactive DNase I [I] or active DNase I [A]), followed by electrophoresis analysis. Data are representative of three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; EtBr, ethidium bromide.

Mentions: In order to evaluate the capacity of the generated lipid-based nanosystems to condense and protect the carried DNA, we used the EtBr and DNase I assays. As illustrated in Figure 7A, nanosystems containing 15% of lactosyl-PE allowed much lesser access of EtBr to the carried DNA than that observed for the corresponding plain lipoplexes, at both 2/1 and 4/1 (+/−) charge ratios. The electrophoretic profile of lipoplexes submitted to DNase I assay (Figure 7B) showed that the intensity of the bands of EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipoplexes, incubated with inactive DNase I, was smaller than that observed with the corresponding plain lipoplexes (submitted to the same experimental conditions), showing a lesser access of EtBr to DNA, in agreement to what was observed in the EtBr assay (Figure 7A). Moreover, EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipo-plexes exhibited a smaller difference between the intensity of their bands (incubation with inactive DNase I versus active DNase I), than the corresponding plain lipoplexes, showing that this new nanosystem confers a higher DNA protection.


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)

Access of ethidium bromide (A) and resistance to DNase I (B) of DNA carried by nanosystems.Notes: Complexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Data (mean ± SD of triplicates) are presented as percentage of control (DNA in the absence of lipid) and are representative of three independent experiments. The asterisks (**P<0.01) correspond to values that differ significantly from those obtained with nanosystem formulations prepared at the same charge ratios without lactosyl-PE. (B) Lipoplexes and naked DNA were submitted to DNase I (inactive DNase I [I] or active DNase I [A]), followed by electrophoresis analysis. Data are representative of three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; EtBr, ethidium bromide.
© Copyright Policy
Related In: Results  -  Collection

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

f7-ijn-9-4979: Access of ethidium bromide (A) and resistance to DNase I (B) of DNA carried by nanosystems.Notes: Complexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Data (mean ± SD of triplicates) are presented as percentage of control (DNA in the absence of lipid) and are representative of three independent experiments. The asterisks (**P<0.01) correspond to values that differ significantly from those obtained with nanosystem formulations prepared at the same charge ratios without lactosyl-PE. (B) Lipoplexes and naked DNA were submitted to DNase I (inactive DNase I [I] or active DNase I [A]), followed by electrophoresis analysis. Data are representative of three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; EtBr, ethidium bromide.
Mentions: In order to evaluate the capacity of the generated lipid-based nanosystems to condense and protect the carried DNA, we used the EtBr and DNase I assays. As illustrated in Figure 7A, nanosystems containing 15% of lactosyl-PE allowed much lesser access of EtBr to the carried DNA than that observed for the corresponding plain lipoplexes, at both 2/1 and 4/1 (+/−) charge ratios. The electrophoretic profile of lipoplexes submitted to DNase I assay (Figure 7B) showed that the intensity of the bands of EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipoplexes, incubated with inactive DNase I, was smaller than that observed with the corresponding plain lipoplexes (submitted to the same experimental conditions), showing a lesser access of EtBr to DNA, in agreement to what was observed in the EtBr assay (Figure 7A). Moreover, EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipo-plexes exhibited a smaller difference between the intensity of their bands (incubation with inactive DNase I versus active DNase I), than the corresponding plain lipoplexes, showing that this new nanosystem confers a higher DNA protection.

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