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Investigating the specific uptake of EGF-conjugated nanoparticles in lung cancer cells using fluorescence imaging.

Jin H, Lovell JF, Chen J, Ng K, Cao W, Ding L, Zhang Z, Zheng G - Cancer Nanotechnol (2010)

Bottom Line: Furthermore, specific EGFR-mediated uptake of the EGF-HPPS nanoparticle was confirmed using human non-small cell lung cancer A549 cells.Subsequent confocal microscopy and flow cytometry studies delineated how secondary targeting mechanisms affected the EGFR targeting.Together, this study confirms the EGFR targeting of EGF-HPPS in lung cancer cells and provides insight on the potential influence of unintended targets on the desired ligand-receptor interaction.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute and Campbell Family Cancer Research Institute, University of Toronto, Toronto, Canada ; Department of Medical Biophysics, University of Toronto, Toronto, Canada ; Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, China.

ABSTRACT

Targeted nanoparticles have the potential to deliver a large drug payload specifically to cancer cells. Targeting requires that a ligand on the nanoparticle surface interact with a specific membrane receptor on target cells. However, the contribution of the targeting ligand to nanoparticle delivery is often influenced by non-specific nanoparticle uptake or secondary targeting mechanisms. In this study, we investigate the epidermal growth factor (EGF) receptor-targeting specificity of a nanoparticle by dual-color fluorescent labeling. The targeted nanoparticle was a fluorescently labeled, EGF-conjugated HDL-like peptide-phospholipid scaffold (HPPS) and the cell lines expressed EGF receptor linked with green fluorescent protein (EGFR-GFP). Using LDLA7 cells partially expressing EGFR-GFP, fluorescence imaging demonstrated the co-internalization of EGFR-GFP and EGF-HPPS, thus validating its targeting specificity. Furthermore, specific EGFR-mediated uptake of the EGF-HPPS nanoparticle was confirmed using human non-small cell lung cancer A549 cells. Subsequent confocal microscopy and flow cytometry studies delineated how secondary targeting mechanisms affected the EGFR targeting. Together, this study confirms the EGFR targeting of EGF-HPPS in lung cancer cells and provides insight on the potential influence of unintended targets on the desired ligand-receptor interaction.

No MeSH data available.


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A partial transfection assay reveals EGRF-GFP and EGF uptake and co-localization. a Real-time visualization of co-internalization of AF555-EGF and EGFR-GFP by EGFR-GFP-LDLA7 cells. b Visualization of cellular uptake of AF555-EGF by EGFR-GFP-A549 cells. c Analysis of GFP expression versus AF555-EGF uptake from confocal images. Data was acquired from 16 cells
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Fig1: A partial transfection assay reveals EGRF-GFP and EGF uptake and co-localization. a Real-time visualization of co-internalization of AF555-EGF and EGFR-GFP by EGFR-GFP-LDLA7 cells. b Visualization of cellular uptake of AF555-EGF by EGFR-GFP-A549 cells. c Analysis of GFP expression versus AF555-EGF uptake from confocal images. Data was acquired from 16 cells

Mentions: To understand the EGFR specificity of EGF-HPPS nanoparticles, two cell lines were chosen based on their endogenous expression of EGFR (the receptor specific for the EGF ligand) and SR-BI (the receptor with affinity for the HPPS nanoparticle). Immunoblotting analysis showed that LDLA7 cells expressed low levels of both EGFR and SR-B1 (EGFR−, SR-BI−) whereas A549 cells expressed high levels of EGFR and moderate levels of SR-B1 (EGFR+, SR-BI+; data not shown). To quantify the correlation between EGF uptake and EGFR expression level, a fluorescent surrogate of EGF, AF555-EGF (biotinylated EGF conjugated with an Alexa Fluor 555-streptavidin) was used to enable sensitive detection of EGFR. Meanwhile, we transfected both LDLA7 and A549 cells with a plasmid encoding an EGFR-GFP. The EGFR-GFP-LDLA7 cells were established with only partial EGFR-GFP expression on LDLA7 cells with no endogenous EGFR expression. This is a useful model as the fluorescence of the EGFR-GFP served as a convenient measure of EGFR expression and the partial transfection created internal EGFR positive and negative controls. Once the EGFR-GFP-LDLA7 cell line was established, it was characterized using AF555-EGF together with real-time confocal microscopy. Upon incubation with AF555-EGF, confocal fluorescence imaging revealed the EGFR-mediated, time-dependent formation of endocytotic vesicles and their subsequent internalization (Fig. 1a). The highly co-localized fluorescent signals from AF555 and GFP also confirmed the intact EGFR function of EGFR-GFP expressed on the LDLA7 cell surface (Fig. 1a). As shown in Fig. 1b, cells that had high expression levels of EGFR-GFP took up the most AF555-EGF. A strong positive linear correlation (R2 = 0.98) was found between the expression level of EGFR-GFP and the uptake of AF555-EGF (Fig. 1c). Thus, the correlation between the differential receptor expression levels in the partially transfected LDLA7 cells and the ligand uptake pattern in those same cells are useful to confirm EGFR-specific uptake. We next examined the A549 lung cancer cells expressing EGFR-GFP. The stably expressing EGFR-GFP-A549 cell line was established through cell sorting where higher expression of EGFR than native A549 cells was induced with EGFR-GFP. As expected, AF555-EGF was internalized with strong co-localization with the EGFR-GFP (Fig. 2a). Correlation between EGFR-GFP expression levels and AF555-EGF uptake in A549 cells was also observed using dual channel analysis using flow cytometry (Fig. 2b). The co-localization of AF555 and GFP signals clearly indicated the internalization of AF555-EGF through EGFR-mediated endocytosis pathway. Thus, two cell lines, EGFR-GFP-LDLA7 and EGFR-GFP-A549, were successfully constructed and validated for specific EGF uptake. These cell models provide a solid framework to further analyze and validate nanoparticle targeting of EGFR expressing cells.Fig. 1


Investigating the specific uptake of EGF-conjugated nanoparticles in lung cancer cells using fluorescence imaging.

Jin H, Lovell JF, Chen J, Ng K, Cao W, Ding L, Zhang Z, Zheng G - Cancer Nanotechnol (2010)

A partial transfection assay reveals EGRF-GFP and EGF uptake and co-localization. a Real-time visualization of co-internalization of AF555-EGF and EGFR-GFP by EGFR-GFP-LDLA7 cells. b Visualization of cellular uptake of AF555-EGF by EGFR-GFP-A549 cells. c Analysis of GFP expression versus AF555-EGF uptake from confocal images. Data was acquired from 16 cells
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Fig1: A partial transfection assay reveals EGRF-GFP and EGF uptake and co-localization. a Real-time visualization of co-internalization of AF555-EGF and EGFR-GFP by EGFR-GFP-LDLA7 cells. b Visualization of cellular uptake of AF555-EGF by EGFR-GFP-A549 cells. c Analysis of GFP expression versus AF555-EGF uptake from confocal images. Data was acquired from 16 cells
Mentions: To understand the EGFR specificity of EGF-HPPS nanoparticles, two cell lines were chosen based on their endogenous expression of EGFR (the receptor specific for the EGF ligand) and SR-BI (the receptor with affinity for the HPPS nanoparticle). Immunoblotting analysis showed that LDLA7 cells expressed low levels of both EGFR and SR-B1 (EGFR−, SR-BI−) whereas A549 cells expressed high levels of EGFR and moderate levels of SR-B1 (EGFR+, SR-BI+; data not shown). To quantify the correlation between EGF uptake and EGFR expression level, a fluorescent surrogate of EGF, AF555-EGF (biotinylated EGF conjugated with an Alexa Fluor 555-streptavidin) was used to enable sensitive detection of EGFR. Meanwhile, we transfected both LDLA7 and A549 cells with a plasmid encoding an EGFR-GFP. The EGFR-GFP-LDLA7 cells were established with only partial EGFR-GFP expression on LDLA7 cells with no endogenous EGFR expression. This is a useful model as the fluorescence of the EGFR-GFP served as a convenient measure of EGFR expression and the partial transfection created internal EGFR positive and negative controls. Once the EGFR-GFP-LDLA7 cell line was established, it was characterized using AF555-EGF together with real-time confocal microscopy. Upon incubation with AF555-EGF, confocal fluorescence imaging revealed the EGFR-mediated, time-dependent formation of endocytotic vesicles and their subsequent internalization (Fig. 1a). The highly co-localized fluorescent signals from AF555 and GFP also confirmed the intact EGFR function of EGFR-GFP expressed on the LDLA7 cell surface (Fig. 1a). As shown in Fig. 1b, cells that had high expression levels of EGFR-GFP took up the most AF555-EGF. A strong positive linear correlation (R2 = 0.98) was found between the expression level of EGFR-GFP and the uptake of AF555-EGF (Fig. 1c). Thus, the correlation between the differential receptor expression levels in the partially transfected LDLA7 cells and the ligand uptake pattern in those same cells are useful to confirm EGFR-specific uptake. We next examined the A549 lung cancer cells expressing EGFR-GFP. The stably expressing EGFR-GFP-A549 cell line was established through cell sorting where higher expression of EGFR than native A549 cells was induced with EGFR-GFP. As expected, AF555-EGF was internalized with strong co-localization with the EGFR-GFP (Fig. 2a). Correlation between EGFR-GFP expression levels and AF555-EGF uptake in A549 cells was also observed using dual channel analysis using flow cytometry (Fig. 2b). The co-localization of AF555 and GFP signals clearly indicated the internalization of AF555-EGF through EGFR-mediated endocytosis pathway. Thus, two cell lines, EGFR-GFP-LDLA7 and EGFR-GFP-A549, were successfully constructed and validated for specific EGF uptake. These cell models provide a solid framework to further analyze and validate nanoparticle targeting of EGFR expressing cells.Fig. 1

Bottom Line: Furthermore, specific EGFR-mediated uptake of the EGF-HPPS nanoparticle was confirmed using human non-small cell lung cancer A549 cells.Subsequent confocal microscopy and flow cytometry studies delineated how secondary targeting mechanisms affected the EGFR targeting.Together, this study confirms the EGFR targeting of EGF-HPPS in lung cancer cells and provides insight on the potential influence of unintended targets on the desired ligand-receptor interaction.

View Article: PubMed Central - PubMed

Affiliation: Ontario Cancer Institute and Campbell Family Cancer Research Institute, University of Toronto, Toronto, Canada ; Department of Medical Biophysics, University of Toronto, Toronto, Canada ; Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, Wuhan, China.

ABSTRACT

Targeted nanoparticles have the potential to deliver a large drug payload specifically to cancer cells. Targeting requires that a ligand on the nanoparticle surface interact with a specific membrane receptor on target cells. However, the contribution of the targeting ligand to nanoparticle delivery is often influenced by non-specific nanoparticle uptake or secondary targeting mechanisms. In this study, we investigate the epidermal growth factor (EGF) receptor-targeting specificity of a nanoparticle by dual-color fluorescent labeling. The targeted nanoparticle was a fluorescently labeled, EGF-conjugated HDL-like peptide-phospholipid scaffold (HPPS) and the cell lines expressed EGF receptor linked with green fluorescent protein (EGFR-GFP). Using LDLA7 cells partially expressing EGFR-GFP, fluorescence imaging demonstrated the co-internalization of EGFR-GFP and EGF-HPPS, thus validating its targeting specificity. Furthermore, specific EGFR-mediated uptake of the EGF-HPPS nanoparticle was confirmed using human non-small cell lung cancer A549 cells. Subsequent confocal microscopy and flow cytometry studies delineated how secondary targeting mechanisms affected the EGFR targeting. Together, this study confirms the EGFR targeting of EGF-HPPS in lung cancer cells and provides insight on the potential influence of unintended targets on the desired ligand-receptor interaction.

No MeSH data available.


Related in: MedlinePlus