Limits...
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.

Show MeSH

Related in: MedlinePlus

Influence of galactose on the cell binding (A) and cell uptake (B) of nanosystems in HepG2 cells.Notes: Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Cell binding and (B) cell uptake are expressed as RFU/106 cells (cells were incubated with rhodamine-PE-labeled lipoplexes). Asterisks (**P<0.01, ***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose. Data are presented as mean ± SD obtained from triplicates and 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); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; RFU, relative fluorescence unit; ns, not significant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4216029&req=5

f5-ijn-9-4979: Influence of galactose on the cell binding (A) and cell uptake (B) of nanosystems in HepG2 cells.Notes: Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Cell binding and (B) cell uptake are expressed as RFU/106 cells (cells were incubated with rhodamine-PE-labeled lipoplexes). Asterisks (**P<0.01, ***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose. Data are presented as mean ± SD obtained from triplicates and 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); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; RFU, relative fluorescence unit; ns, not significant.

Mentions: In order to determine if the potentiation of the biological activity of the nanosystems, induced by the incorporation of lactosyl-PE, was due to the interaction of the galactose terminal residue of this glycolipid with ASGP-R, we evaluated the extent of cell binding and uptake of the generated formulations in HepG2 cells, in the presence or absence of galactose (40 mg/mL). For this purpose, cells were incubated with rhodamine-PE-labeled lipoplexes at 4°C (binding) or at 37°C (uptake). As illustrated in Figure 5, cell incubation with the best formulation (EPOPC:Chol:lactosyl-PE [15%]/DNA [+/−] 2/1) resulted in a much higher extent of cell binding and uptake than that obtained with the corresponding plain lipoplexes. This enhancing effect on cell binding and uptake promoted by lactosyl-PE was not observed for lipoplexes prepared at the 4/1 (+/−) charge ratio.


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 on the cell binding (A) and cell uptake (B) of nanosystems in HepG2 cells.Notes: Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Cell binding and (B) cell uptake are expressed as RFU/106 cells (cells were incubated with rhodamine-PE-labeled lipoplexes). Asterisks (**P<0.01, ***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose. Data are presented as mean ± SD obtained from triplicates and 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); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; RFU, relative fluorescence unit; ns, not significant.
© Copyright Policy
Related In: Results  -  Collection

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

f5-ijn-9-4979: Influence of galactose on the cell binding (A) and cell uptake (B) of nanosystems in HepG2 cells.Notes: Lipoplexes were prepared at 2/1 and 4/1 (+/−) charge ratios, either without or with 15% of lactosyl-PE. (A) Cell binding and (B) cell uptake are expressed as RFU/106 cells (cells were incubated with rhodamine-PE-labeled lipoplexes). Asterisks (**P<0.01, ***P<0.001) correspond to values that differ significantly from those obtained with the same nanosystem formulations in the absence of galactose. Data are presented as mean ± SD obtained from triplicates and 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); SD, standard deviation; EPOPC, palmitoyl-2-oleoyl-sn-glycene-3-ehylphosphocholine; Chol, cholesterol; RFU, relative fluorescence unit; ns, not significant.
Mentions: In order to determine if the potentiation of the biological activity of the nanosystems, induced by the incorporation of lactosyl-PE, was due to the interaction of the galactose terminal residue of this glycolipid with ASGP-R, we evaluated the extent of cell binding and uptake of the generated formulations in HepG2 cells, in the presence or absence of galactose (40 mg/mL). For this purpose, cells were incubated with rhodamine-PE-labeled lipoplexes at 4°C (binding) or at 37°C (uptake). As illustrated in Figure 5, cell incubation with the best formulation (EPOPC:Chol:lactosyl-PE [15%]/DNA [+/−] 2/1) resulted in a much higher extent of cell binding and uptake than that obtained with the corresponding plain lipoplexes. This enhancing effect on cell binding and uptake promoted by lactosyl-PE was not observed for lipoplexes prepared at the 4/1 (+/−) charge ratio.

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