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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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Flow cytometry studies of cellular uptake of liposomes showing histogram profiles and mean fluorescence intensities of doxorubicin in A549 cells treated with PEG-LP/DOX, GE11-LP/DOX, and GE11-LP/DOX preincubated with free GE11 (A and B) and the results of cellular uptake of PEG-LP/DOX and GE11-LP/DOX by K562 cells (C).Notes: Before determination by flow cytometry, A549 cells were rinsed three times in cold phosphate-buffered saline to remove the absorbed liposomes on the cell surface. In the competition experiments, free GE11 (20 μg/mL) was preincubated with A549 cells for 30 minutes, followed by continued incubation with GE11-LP/DOX solution. The data are shown as the mean ± standard deviation (n=3). **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.
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f5-ijn-9-921: Flow cytometry studies of cellular uptake of liposomes showing histogram profiles and mean fluorescence intensities of doxorubicin in A549 cells treated with PEG-LP/DOX, GE11-LP/DOX, and GE11-LP/DOX preincubated with free GE11 (A and B) and the results of cellular uptake of PEG-LP/DOX and GE11-LP/DOX by K562 cells (C).Notes: Before determination by flow cytometry, A549 cells were rinsed three times in cold phosphate-buffered saline to remove the absorbed liposomes on the cell surface. In the competition experiments, free GE11 (20 μg/mL) was preincubated with A549 cells for 30 minutes, followed by continued incubation with GE11-LP/DOX solution. The data are shown as the mean ± standard deviation (n=3). **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.

Mentions: Quantitative analysis of cellular uptake of the liposomes was performed by flow cytometry. As shown in Figure 5B, A549 cells consistently took up more GE11-LP/DOX and the mean fluorescence intensities were about 1.7-fold and 1.9-fold stronger than that for PEG-LP/DOX at 1 hour and 2 hours, respectively. Moreover, after treatment with free GE11, the mean fluorescence intensity for GE11-LP/DOX decreased significantly. These results agree fairly well with the observations made by fluorescence microscopy.


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)

Flow cytometry studies of cellular uptake of liposomes showing histogram profiles and mean fluorescence intensities of doxorubicin in A549 cells treated with PEG-LP/DOX, GE11-LP/DOX, and GE11-LP/DOX preincubated with free GE11 (A and B) and the results of cellular uptake of PEG-LP/DOX and GE11-LP/DOX by K562 cells (C).Notes: Before determination by flow cytometry, A549 cells were rinsed three times in cold phosphate-buffered saline to remove the absorbed liposomes on the cell surface. In the competition experiments, free GE11 (20 μg/mL) was preincubated with A549 cells for 30 minutes, followed by continued incubation with GE11-LP/DOX solution. The data are shown as the mean ± standard deviation (n=3). **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

f5-ijn-9-921: Flow cytometry studies of cellular uptake of liposomes showing histogram profiles and mean fluorescence intensities of doxorubicin in A549 cells treated with PEG-LP/DOX, GE11-LP/DOX, and GE11-LP/DOX preincubated with free GE11 (A and B) and the results of cellular uptake of PEG-LP/DOX and GE11-LP/DOX by K562 cells (C).Notes: Before determination by flow cytometry, A549 cells were rinsed three times in cold phosphate-buffered saline to remove the absorbed liposomes on the cell surface. In the competition experiments, free GE11 (20 μg/mL) was preincubated with A549 cells for 30 minutes, followed by continued incubation with GE11-LP/DOX solution. The data are shown as the mean ± standard deviation (n=3). **P<0.01; ***P<0.001.Abbreviations: DOX, doxorubicin; PEG, polyethylene glycol; LP, liposomes; NS, not statistically significant.
Mentions: Quantitative analysis of cellular uptake of the liposomes was performed by flow cytometry. As shown in Figure 5B, A549 cells consistently took up more GE11-LP/DOX and the mean fluorescence intensities were about 1.7-fold and 1.9-fold stronger than that for PEG-LP/DOX at 1 hour and 2 hours, respectively. Moreover, after treatment with free GE11, the mean fluorescence intensity for GE11-LP/DOX decreased significantly. These results agree fairly well with the observations made by fluorescence microscopy.

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