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GE11-modified liposomes for non-small cell lung cancer targeting: preparation, ex vitro and in vivo evaluation.

Cheng L, Huang FZ, Cheng LF, Zhu YQ, Hu Q, Li L, Wei L, Chen DW - Int J Nanomedicine (2014)

Bottom Line: Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX).Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis.It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, College of Pharmaceutical Science, Soochow University, Suzhou, Jiangsu Province, People's Republic of China.

ABSTRACT
Non-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG2000-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.

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Effects of low temperature (A) and ATP depletion (B) on cellular uptake of liposomes.Notes: The low temperature experiment was performed by preincubating A549 cells at 4°C for 30 minutes. The ATP depletion experiment was performed by preincubating A549 cells with sodium azide (10 mM) and 2-deoxy-d-glucose (6.5 mM) at 37°C for one hour. The data are shown as the mean ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.
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f6-ijn-9-921: Effects of low temperature (A) and ATP depletion (B) on cellular uptake of liposomes.Notes: The low temperature experiment was performed by preincubating A549 cells at 4°C for 30 minutes. The ATP depletion experiment was performed by preincubating A549 cells with sodium azide (10 mM) and 2-deoxy-d-glucose (6.5 mM) at 37°C for one hour. The data are shown as the mean ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.

Mentions: To elucidate whether the uptake of GE11-LP/DOX by A549 cells is an active energy-dependent or energy-independent process, the effect of low temperature and ATP depletion on cellular uptake was assessed. For comparison, cellular uptake of PEG-LP/DOX was also investigated under the same conditions. As shown in Figure 6A, low temperature markedly reduced cellular uptake of PEG-LP/DOX and GE11-LP/DOX to 61.5% and 41.3% of the control value, respectively. In the ATP depletion experiment (Figure 6B), cellular uptake of PEG-LP/DOX and GE11-LP/DOX was decreased to 68.9% and 57.0% of the control value after treatment with sodium azide and 2-deoxy-d-glucose, both of which are agents causing depletion of the intracellular ATP pool.33,34 These results show that cellular uptake of GE11-LP/DOX and PEG-LP/DOX involves an active energy-dependent process.


GE11-modified liposomes for non-small cell lung cancer targeting: preparation, ex vitro and in vivo evaluation.

Cheng L, Huang FZ, Cheng LF, Zhu YQ, Hu Q, Li L, Wei L, Chen DW - Int J Nanomedicine (2014)

Effects of low temperature (A) and ATP depletion (B) on cellular uptake of liposomes.Notes: The low temperature experiment was performed by preincubating A549 cells at 4°C for 30 minutes. The ATP depletion experiment was performed by preincubating A549 cells with sodium azide (10 mM) and 2-deoxy-d-glucose (6.5 mM) at 37°C for one hour. The data are shown as the mean ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928463&req=5

f6-ijn-9-921: Effects of low temperature (A) and ATP depletion (B) on cellular uptake of liposomes.Notes: The low temperature experiment was performed by preincubating A549 cells at 4°C for 30 minutes. The ATP depletion experiment was performed by preincubating A549 cells with sodium azide (10 mM) and 2-deoxy-d-glucose (6.5 mM) at 37°C for one hour. The data are shown as the mean ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.
Mentions: To elucidate whether the uptake of GE11-LP/DOX by A549 cells is an active energy-dependent or energy-independent process, the effect of low temperature and ATP depletion on cellular uptake was assessed. For comparison, cellular uptake of PEG-LP/DOX was also investigated under the same conditions. As shown in Figure 6A, low temperature markedly reduced cellular uptake of PEG-LP/DOX and GE11-LP/DOX to 61.5% and 41.3% of the control value, respectively. In the ATP depletion experiment (Figure 6B), cellular uptake of PEG-LP/DOX and GE11-LP/DOX was decreased to 68.9% and 57.0% of the control value after treatment with sodium azide and 2-deoxy-d-glucose, both of which are agents causing depletion of the intracellular ATP pool.33,34 These results show that cellular uptake of GE11-LP/DOX and PEG-LP/DOX involves an active energy-dependent process.

Bottom Line: Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX).Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis.It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics, College of Pharmaceutical Science, Soochow University, Suzhou, Jiangsu Province, People's Republic of China.

ABSTRACT
Non-small cell lung cancer (NSCLC) is a serious threat to human health, and 40%-80% of NSCLCs express high levels of epidermal growth factor receptor (EGFR). GE11 is a novel peptide and exhibits high affinity for EGFR binding. The aim of this study was to construct and evaluate GE11-modified liposomes for targeted drug delivery to EGFR-positive NSCLC. Doxorubicin, a broad-spectrum antitumor agent, was chosen as the payload. GE11 was conjugated to the distal end of DSPE-PEG2000-Mal by an addition reaction with a conjugation efficiency above 90%. Doxorubicin-loaded liposomes containing GE11 (GE11-LP/DOX) at densities ranging from 0% to 15% were prepared by combination of a thin film hydration method and a post insertion method. Irrespective of GE11 density, the physicochemical properties of these targeted liposomes, including particle size, zeta potential, and drug entrapment efficiency, were nearly identical. Interestingly, the cytotoxic effect of the liposomes on A549 tumor cells was closely related to GE11 density, and liposomes with 10% GE11 had the highest tumor cell killing activity and a 2.6-fold lower half maximal inhibitory concentration than that of the nontargeted counterpart (PEG-LP/DOX). Fluorescence microscopy and flow cytometry analysis revealed that GE11 significantly increased cellular uptake of the liposomes, which could be ascribed to specific EGFR-mediated endocytosis. It was found that multiple endocytic pathways were involved in entry of GE11-LP/DOX into cells, but GE11 assisted in cellular internalization mainly via the clathrin-mediated endocytosis pathway. Importantly, the GE11-modified liposomes showed enhanced accumulation and prolonged retention in tumor tissue, as evidenced by a 2.2-fold stronger mean fluorescence intensity in tumor tissue than the unmodified liposomes at 24 hours. In summary, GE11-modified liposomes may be a promising platform for targeted delivery of chemotherapeutic drugs in NSCLC.

Show MeSH
Related in: MedlinePlus