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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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Transmission electron microscopy (A) and zeta potential (B) of the generated nanosystem.Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) TEM analysis: panels (I) and (II) EPOPC:Chol/DNA(+/−) (2/1); (III) and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1). (B) Zeta potential (mV) is presented as mean ± SD of triplicates and is representative of at least three independent experiments. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared at the same charge ratios without lactosyl-PE.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); TEM, transmission electron microscopy; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.
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f6-ijn-9-4979: Transmission electron microscopy (A) and zeta potential (B) of the generated nanosystem.Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) TEM analysis: panels (I) and (II) EPOPC:Chol/DNA(+/−) (2/1); (III) and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1). (B) Zeta potential (mV) is presented as mean ± SD of triplicates and is representative of at least three independent experiments. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared at the same charge ratios without lactosyl-PE.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); TEM, transmission electron microscopy; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.

Mentions: Since the efficacy of the nanocarriers to deliver genetic material into target cells is strongly dependent on their physicochemical properties, we determined their size/structure by TEM and surface charge by zeta potential. TEM analysis (Figure 6A) demonstrated that the lipoplexes prepared at 2/1 (+/−) charge ratio and containing 15% of lactosyl-PE (panels III and IV) presented considerably reduced mean diameters, approximately 200 nm, when compared to those obtained for the corresponding plain lipoplexes (panels I and II), approximately 800 nm. Moreover, TEM analysis showed that lipoplexes containing 15% of lactosyl-PE consisted of clusters of small nanostructures with sizes around 50 nm (panel III) that formed the observed 200 nm nanosystems (panels III and IV). The results obtained from zeta potential measurements (Figure 6B) showed that the nanosystems containing 15% of lactosyl-PE prepared at both 2/1 and 4/1 (+/−) charge ratios exhibited a positive zeta potential that is much lower than that observed for the corresponding plain 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)

Transmission electron microscopy (A) and zeta potential (B) of the generated nanosystem.Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) TEM analysis: panels (I) and (II) EPOPC:Chol/DNA(+/−) (2/1); (III) and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1). (B) Zeta potential (mV) is presented as mean ± SD of triplicates and is representative of at least three independent experiments. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared at the same charge ratios without lactosyl-PE.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); TEM, transmission electron microscopy; 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

f6-ijn-9-4979: Transmission electron microscopy (A) and zeta potential (B) of the generated nanosystem.Notes: Lipoplexes, either containing or not containing 15% of lactosyl-PE, were prepared at 2/1 and 4/1 (+/−) charge ratios. (A) TEM analysis: panels (I) and (II) EPOPC:Chol/DNA(+/−) (2/1); (III) and (IV) EPOPC:Chol:lactosyl-PE (15%)/DNA (+/−) (2/1). (B) Zeta potential (mV) is presented as mean ± SD of triplicates and is representative of at least three independent experiments. Asterisks (***P<0.001) correspond to values that differ significantly from those obtained with complexes prepared at the same charge ratios without lactosyl-PE.Abbreviations: lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); TEM, transmission electron microscopy; SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol.
Mentions: Since the efficacy of the nanocarriers to deliver genetic material into target cells is strongly dependent on their physicochemical properties, we determined their size/structure by TEM and surface charge by zeta potential. TEM analysis (Figure 6A) demonstrated that the lipoplexes prepared at 2/1 (+/−) charge ratio and containing 15% of lactosyl-PE (panels III and IV) presented considerably reduced mean diameters, approximately 200 nm, when compared to those obtained for the corresponding plain lipoplexes (panels I and II), approximately 800 nm. Moreover, TEM analysis showed that lipoplexes containing 15% of lactosyl-PE consisted of clusters of small nanostructures with sizes around 50 nm (panel III) that formed the observed 200 nm nanosystems (panels III and IV). The results obtained from zeta potential measurements (Figure 6B) showed that the nanosystems containing 15% of lactosyl-PE prepared at both 2/1 and 4/1 (+/−) charge ratios exhibited a positive zeta potential that is much lower than that observed for the corresponding plain 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