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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.


Related in: MedlinePlus

EGF targeting of payload bearing nanoparticles. a Schematic figure of EGF-HPPS. EGF-HPPS consists of phospholipids, peptides, cholesteryl oleate (CO), DiR-BOA cargo, and EGF. b Three-dimensional imaging of uptake of EGF(DiR-BOA) HPPS by EGFR-GFP-A549 cells
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Fig3: EGF targeting of payload bearing nanoparticles. a Schematic figure of EGF-HPPS. EGF-HPPS consists of phospholipids, peptides, cholesteryl oleate (CO), DiR-BOA cargo, and EGF. b Three-dimensional imaging of uptake of EGF(DiR-BOA) HPPS by EGFR-GFP-A549 cells

Mentions: A schematic depiction of EGF-HPPS is shown in Fig. 3a. EGF was conjugated to the surface of HPPS and the fluorescent cargo DiR-BOA was loaded into the core of HPPS. Three-dimensional and dual-color confocal imaging was used to evaluate the EGFR-mediated nanoparticle endocytosis. Upon incubation with EGFR-GFP-A549 cells, DiR-BOA from EGF-HPPS co-localized with the GFP signal in EGFR-GFP-A549 cells (Fig. 3b). This confirmed the uptake and co-localization of EGF-HPPS by EGFR. We next used EGFR-GFP-LDLA7 cells to evaluate the target specificity of EGF-HPPS (Fig. 4). When incubated with the unconjugated HPPS nanoparticle, cells had no DiR-BOA uptake. In contrast, incubation EGF-HPPS with EGFR-GFP-LDLA7 cells revealed significant DiR-BOA uptake (white arrow). Importantly, cells without any EGFR-GFP expression (internal EGFR negative control) did not display any detectable DiR-BOA uptake (blue arrows). This demonstrates that EGF-HPPS specifically delivered its cargo (DiR-BOA) to target cells via EGFR-mediated endocytosis. To rule out the potential interference of native HDL on the EGF-HPPS targeting, the same cells were incubated with EGF-HPPS, along with excess HDL and/or EGF. Confocal images illustrated that there was no change to the specific uptake of EGF-HPPS in the presence of HDL alone. However, the specific uptake of EGF-HPPS was inhibited by addition of excess EGF. These data confirmed the EGFR-targeting ability of EGF-HPPS.Fig. 3


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)

EGF targeting of payload bearing nanoparticles. a Schematic figure of EGF-HPPS. EGF-HPPS consists of phospholipids, peptides, cholesteryl oleate (CO), DiR-BOA cargo, and EGF. b Three-dimensional imaging of uptake of EGF(DiR-BOA) HPPS by EGFR-GFP-A549 cells
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Related In: Results  -  Collection

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Fig3: EGF targeting of payload bearing nanoparticles. a Schematic figure of EGF-HPPS. EGF-HPPS consists of phospholipids, peptides, cholesteryl oleate (CO), DiR-BOA cargo, and EGF. b Three-dimensional imaging of uptake of EGF(DiR-BOA) HPPS by EGFR-GFP-A549 cells
Mentions: A schematic depiction of EGF-HPPS is shown in Fig. 3a. EGF was conjugated to the surface of HPPS and the fluorescent cargo DiR-BOA was loaded into the core of HPPS. Three-dimensional and dual-color confocal imaging was used to evaluate the EGFR-mediated nanoparticle endocytosis. Upon incubation with EGFR-GFP-A549 cells, DiR-BOA from EGF-HPPS co-localized with the GFP signal in EGFR-GFP-A549 cells (Fig. 3b). This confirmed the uptake and co-localization of EGF-HPPS by EGFR. We next used EGFR-GFP-LDLA7 cells to evaluate the target specificity of EGF-HPPS (Fig. 4). When incubated with the unconjugated HPPS nanoparticle, cells had no DiR-BOA uptake. In contrast, incubation EGF-HPPS with EGFR-GFP-LDLA7 cells revealed significant DiR-BOA uptake (white arrow). Importantly, cells without any EGFR-GFP expression (internal EGFR negative control) did not display any detectable DiR-BOA uptake (blue arrows). This demonstrates that EGF-HPPS specifically delivered its cargo (DiR-BOA) to target cells via EGFR-mediated endocytosis. To rule out the potential interference of native HDL on the EGF-HPPS targeting, the same cells were incubated with EGF-HPPS, along with excess HDL and/or EGF. Confocal images illustrated that there was no change to the specific uptake of EGF-HPPS in the presence of HDL alone. However, the specific uptake of EGF-HPPS was inhibited by addition of excess EGF. These data confirmed the EGFR-targeting ability of EGF-HPPS.Fig. 3

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