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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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Biological activity (A) and cytotoxicity (B) of nanosystems prepared with or without lactosyl-PE in HepG2 cells. Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 5%, 10%, 15%, 20%, or 40% of lactosyl-PE.Notes: (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Cell viability is expressed as a percentage of untreated control cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared without lactosyl-PE. Data are presented as mean ± SD obtained from triplicates and are representative of at least three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); HepG2, human hepatocellular carcinoma; RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.
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f1-ijn-9-4979: Biological activity (A) and cytotoxicity (B) of nanosystems prepared with or without lactosyl-PE in HepG2 cells. Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 5%, 10%, 15%, 20%, or 40% of lactosyl-PE.Notes: (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Cell viability is expressed as a percentage of untreated control cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared without lactosyl-PE. Data are presented as mean ± SD obtained from triplicates and are representative of at least three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); HepG2, human hepatocellular carcinoma; RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.

Mentions: Since the ASGP-R is overexpressed in HCC cells, such as the HepG2 cell line, and specifically binds galactose, we evaluated what effect incorporating lactosyl-PE (which has a galactose terminal residue) into the cationic liposomes has on the biological activity of the generated nanosystem in HepG2 cells. For this purpose, we prepared several EPOPC:Chol cationic liposome formulations containing different amounts of lactosyl-PE (5%, 10%, 15%, 20%, and 40%) in order to verify how this lipid affects the biological activity of lipoplexes prepared at 2/1 and 4/1 (+/−) charge ratios. As illustrated in Figure 1A, the incorporation of lactosyl-PE into the EPOPC:Chol cationic liposomes could significantly increase the biological activity of the generated nanosystems, this being particularly evident for EPOPC:Chol:lactosyl-PE/DNA lipoplexes prepared with cationic liposomes containing 15% of lactosyl-PE at the 2/1 (+/−) charge ratio. For this formulation, a 50-fold increase in the transfection activity was observed when compared to that obtained with plain lipoplexes prepared at the same charge ratio with EPOPC:Chol cationic liposomes without lactosyl-PE. This level of potentiation was not verified for any of the other tested formulations. The effect of lactosyl-PE incorporation was evaluated only for lipoplexes prepared at 2/1 and 4/1 (+/−) charge ratios, since these formulations were the ones that presented the highest levels of biological activity (data not shown). On the other hand, results illustrated in Figure 1B show that the viability of HepG2 cells was not significantly affected (P >0.05) by any of the tested lipoplex formulations.


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)

Biological activity (A) and cytotoxicity (B) of nanosystems prepared with or without lactosyl-PE in HepG2 cells. Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 5%, 10%, 15%, 20%, or 40% of lactosyl-PE.Notes: (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Cell viability is expressed as a percentage of untreated control cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared without lactosyl-PE. Data are presented as mean ± SD obtained from triplicates and are representative of at least three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); HepG2, human hepatocellular carcinoma; RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.
© Copyright Policy
Related In: Results  -  Collection

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

f1-ijn-9-4979: Biological activity (A) and cytotoxicity (B) of nanosystems prepared with or without lactosyl-PE in HepG2 cells. Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 5%, 10%, 15%, 20%, or 40% of lactosyl-PE.Notes: (A) Luciferase gene expression is presented as RLU/mg of total cell protein. (B) Cell viability is expressed as a percentage of untreated control cells. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared without lactosyl-PE. Data are presented as mean ± SD obtained from triplicates and are representative of at least three independent experiments.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); HepG2, human hepatocellular carcinoma; RLU, relative light unit; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.
Mentions: Since the ASGP-R is overexpressed in HCC cells, such as the HepG2 cell line, and specifically binds galactose, we evaluated what effect incorporating lactosyl-PE (which has a galactose terminal residue) into the cationic liposomes has on the biological activity of the generated nanosystem in HepG2 cells. For this purpose, we prepared several EPOPC:Chol cationic liposome formulations containing different amounts of lactosyl-PE (5%, 10%, 15%, 20%, and 40%) in order to verify how this lipid affects the biological activity of lipoplexes prepared at 2/1 and 4/1 (+/−) charge ratios. As illustrated in Figure 1A, the incorporation of lactosyl-PE into the EPOPC:Chol cationic liposomes could significantly increase the biological activity of the generated nanosystems, this being particularly evident for EPOPC:Chol:lactosyl-PE/DNA lipoplexes prepared with cationic liposomes containing 15% of lactosyl-PE at the 2/1 (+/−) charge ratio. For this formulation, a 50-fold increase in the transfection activity was observed when compared to that obtained with plain lipoplexes prepared at the same charge ratio with EPOPC:Chol cationic liposomes without lactosyl-PE. This level of potentiation was not verified for any of the other tested formulations. The effect of lactosyl-PE incorporation was evaluated only for lipoplexes prepared at 2/1 and 4/1 (+/−) charge ratios, since these formulations were the ones that presented the highest levels of biological activity (data not shown). On the other hand, results illustrated in Figure 1B show that the viability of HepG2 cells was not significantly affected (P >0.05) by any of the tested lipoplex formulations.

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