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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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Influence of galactose (A) or an antibody against ASGP-R (B) on the biological activity of nanosystems in HepG2 cells.Notes: Nanosystems were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Luciferase gene expression is presented as a percentage of control (cells transfected with the same formulations in the absence of antibody). Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose or antibody. Data are presented as mean ± SD obtained from triplicates and are representative of three independent experiments.Abbreviations: ASGP-R, asialoglycoprotein receptor; HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; ns, not significant.
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f4-ijn-9-4979: Influence of galactose (A) or an antibody against ASGP-R (B) on the biological activity of nanosystems in HepG2 cells.Notes: Nanosystems were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Luciferase gene expression is presented as a percentage of control (cells transfected with the same formulations in the absence of antibody). Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose or antibody. Data are presented as mean ± SD obtained from triplicates and are representative of three independent experiments.Abbreviations: ASGP-R, asialoglycoprotein receptor; HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; ns, not significant.

Mentions: As opposed to what was observed with HepG2 cells (Figure 1A), the results presented in Figure 3 showed no increase in the biological activity of lipoplexes prepared with lactosyl-PE in MDA-MB-231 cells, when compared to that obtained with the corresponding plain lipoplexes for both charge ratios. On the other hand, as illustrated in Figure 4A, the results obtained in the transfection assays performed in culture medium containing 40 mg/mL of galactose revealed that the presence of free galactose promoted a strong reduction in the biological activity of the best formulation (EPOPC:Chol:lactosyl-PE [15%]/DNA [+/−] 2/1). In contrast, the biological activity of lipoplexes prepared without lactosyl-PE was not inhibited by the presence of 40 mg/mL of galactose (Figure 4A). Although several concentrations of galactose were tested in the transfection studies, the concentration of 40 mg/mL of galactose was selected for these studies since it was found to be the concentration at which a maximum inhibition of biological activity occurred without affecting cell viability (data not shown). Moreover, transfection studies performed in HepG2 cells treated with an antibody against ASGP-R showed that its presence induced a substantial inhibition of the biological activity of the EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipoplexes, but not the transfection activity of the corresponding plain lipoplexes (Figure 4B).


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)

Influence of galactose (A) or an antibody against ASGP-R (B) on the biological activity of nanosystems in HepG2 cells.Notes: Nanosystems were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Luciferase gene expression is presented as a percentage of control (cells transfected with the same formulations in the absence of antibody). Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose or antibody. Data are presented as mean ± SD obtained from triplicates and are representative of three independent experiments.Abbreviations: ASGP-R, asialoglycoprotein receptor; HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; ns, not significant.
© Copyright Policy
Related In: Results  -  Collection

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

f4-ijn-9-4979: Influence of galactose (A) or an antibody against ASGP-R (B) on the biological activity of nanosystems in HepG2 cells.Notes: Nanosystems were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Luciferase gene expression is presented as a percentage of control (cells transfected with the same formulations in the absence of antibody). Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose or antibody. Data are presented as mean ± SD obtained from triplicates and are representative of three independent experiments.Abbreviations: ASGP-R, asialoglycoprotein receptor; HepG2, human hepatocellular carcinoma; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; ns, not significant.
Mentions: As opposed to what was observed with HepG2 cells (Figure 1A), the results presented in Figure 3 showed no increase in the biological activity of lipoplexes prepared with lactosyl-PE in MDA-MB-231 cells, when compared to that obtained with the corresponding plain lipoplexes for both charge ratios. On the other hand, as illustrated in Figure 4A, the results obtained in the transfection assays performed in culture medium containing 40 mg/mL of galactose revealed that the presence of free galactose promoted a strong reduction in the biological activity of the best formulation (EPOPC:Chol:lactosyl-PE [15%]/DNA [+/−] 2/1). In contrast, the biological activity of lipoplexes prepared without lactosyl-PE was not inhibited by the presence of 40 mg/mL of galactose (Figure 4A). Although several concentrations of galactose were tested in the transfection studies, the concentration of 40 mg/mL of galactose was selected for these studies since it was found to be the concentration at which a maximum inhibition of biological activity occurred without affecting cell viability (data not shown). Moreover, transfection studies performed in HepG2 cells treated with an antibody against ASGP-R showed that its presence induced a substantial inhibition of the biological activity of the EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) 2/1 lipoplexes, but not the transfection activity of the corresponding plain lipoplexes (Figure 4B).

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