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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-HPPS as nanoprobe for targeting lung cancer cells expressing high level of EGFR in vitro. Confocal imaging of distinguished uptake of EGF-HPPS between: a EGFR positive A549 cells and b EGFR negative H520 cells. c Quantification of cellular uptake of HPPS, HPPS with excess of HDL, EGF-HPPS, EGF-HPPS with excess of HDL or EGF-HPPS with excess of HDL and EGF by H520, A549, and EGFR-GFP-A549 cells within 3 h incubation. d Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells during 3, 6, and 24 h incubation by flow cytometry. e Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells with excess of HDL
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Fig5: EGF-HPPS as nanoprobe for targeting lung cancer cells expressing high level of EGFR in vitro. Confocal imaging of distinguished uptake of EGF-HPPS between: a EGFR positive A549 cells and b EGFR negative H520 cells. c Quantification of cellular uptake of HPPS, HPPS with excess of HDL, EGF-HPPS, EGF-HPPS with excess of HDL or EGF-HPPS with excess of HDL and EGF by H520, A549, and EGFR-GFP-A549 cells within 3 h incubation. d Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells during 3, 6, and 24 h incubation by flow cytometry. e Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells with excess of HDL

Mentions: In a more complex scenario, H520 cells (EGFR−, SR-B1+), A549 cells (EGFR+, SR-B1+), and EGFR-GFP-A549 (EGFR++, SR-B1+), with various EGFR expression levels from negative, positive to strongly positive, were used to quantify the specific uptake of EGF-HPPS. It should be noted that both A549 and H520 cells are positive for SR-B1 receptor (SR-B1+), which has natural affinity for HDL (Acton et al., 1996) and for apoA-1 mimetic helical peptides (Wool et al., 2008; Zhang et al., 2010). Therefore, the secondary targeting of SR-BI along with the targeting to EGFR was investigated using confocal microscopy and flow cytometry. First, EGFR-GFP-A549 cells were incubated with EGF-HPPS alone, with excess HDL or with excess of both HDL and EGF. As shown in Fig. 5a, the uptake of EGF-HPPS by EGFR-GFP-A549 cells was completely inhibited when both excess HDL and EGF were added but not with only HDL, which is indicative of EGFR targeting. Under the same condition when using H520 cells (EGFR−, SR-BI+), very weak fluorescent signal was detected for EGF-HPPS presumably due to the SR-BI pathway (Fig. 5b). This secondary targeting was further confirmed by the diminished signal in the presence of excess HDL (Fig. 5b). Further evidence on the influence of secondary targeting was obtained using flow cytometry. As shown in Fig. 5c, HPPS (in the absence of EGF ligand) was taken up via SR-BI pathway in H520, A549, and EGFR-GFP-A549 cells (Fig. 5c, 1st column), but their uptake were all inhibited by excess of HDL (Fig. 5c, 2nd column). The 6.3-fold difference in the uptake of EGF-HPPS between A549 cells and H520 cells (Fig. 5c, 3rd column) was probably due to differential EGFR expression levels and the shielding of SR-BI recognition by EGF conjugation, evidenced by a 2.2-fold increase in EGF-HPPS uptake by A549 cells but a 2.3-fold decrease by H520 cells (Fig. 5c, 3rd versus 1st column). Furthermore, HDL blocking enhanced the EGF-HPPS uptake contrast between A549 cells and H520 cells from 6.3-fold (Fig. 5c, 3rd column) to 9.7-fold (Fig. 5c, 4th column). Next, uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells with or without excess of HDL were evaluated at 3, 6, and 24 h after incubation (Fig. 5d and e). At all three time points, the difference of EGF-HPPS uptake between A549 (EGFR+) and EGFR-GFP-A549 (EGFR++) could not be distinguished without the excess of HDL which blocks secondary SR-BI targeting (Fig. 5d). Clear differences only appeared after adding excess of HDL, which revealed the pure EGFR targeting (Fig. 5e). Another interesting effect of this secondary targeting lies in the fact both EGFR and SR-BI contributed to the EGF-HPPS uptake. This observation could be explored for enhancing the uptake through dual receptor coordination.Fig. 5


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-HPPS as nanoprobe for targeting lung cancer cells expressing high level of EGFR in vitro. Confocal imaging of distinguished uptake of EGF-HPPS between: a EGFR positive A549 cells and b EGFR negative H520 cells. c Quantification of cellular uptake of HPPS, HPPS with excess of HDL, EGF-HPPS, EGF-HPPS with excess of HDL or EGF-HPPS with excess of HDL and EGF by H520, A549, and EGFR-GFP-A549 cells within 3 h incubation. d Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells during 3, 6, and 24 h incubation by flow cytometry. e Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells with excess of HDL
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Fig5: EGF-HPPS as nanoprobe for targeting lung cancer cells expressing high level of EGFR in vitro. Confocal imaging of distinguished uptake of EGF-HPPS between: a EGFR positive A549 cells and b EGFR negative H520 cells. c Quantification of cellular uptake of HPPS, HPPS with excess of HDL, EGF-HPPS, EGF-HPPS with excess of HDL or EGF-HPPS with excess of HDL and EGF by H520, A549, and EGFR-GFP-A549 cells within 3 h incubation. d Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells during 3, 6, and 24 h incubation by flow cytometry. e Quantification of cellular uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells with excess of HDL
Mentions: In a more complex scenario, H520 cells (EGFR−, SR-B1+), A549 cells (EGFR+, SR-B1+), and EGFR-GFP-A549 (EGFR++, SR-B1+), with various EGFR expression levels from negative, positive to strongly positive, were used to quantify the specific uptake of EGF-HPPS. It should be noted that both A549 and H520 cells are positive for SR-B1 receptor (SR-B1+), which has natural affinity for HDL (Acton et al., 1996) and for apoA-1 mimetic helical peptides (Wool et al., 2008; Zhang et al., 2010). Therefore, the secondary targeting of SR-BI along with the targeting to EGFR was investigated using confocal microscopy and flow cytometry. First, EGFR-GFP-A549 cells were incubated with EGF-HPPS alone, with excess HDL or with excess of both HDL and EGF. As shown in Fig. 5a, the uptake of EGF-HPPS by EGFR-GFP-A549 cells was completely inhibited when both excess HDL and EGF were added but not with only HDL, which is indicative of EGFR targeting. Under the same condition when using H520 cells (EGFR−, SR-BI+), very weak fluorescent signal was detected for EGF-HPPS presumably due to the SR-BI pathway (Fig. 5b). This secondary targeting was further confirmed by the diminished signal in the presence of excess HDL (Fig. 5b). Further evidence on the influence of secondary targeting was obtained using flow cytometry. As shown in Fig. 5c, HPPS (in the absence of EGF ligand) was taken up via SR-BI pathway in H520, A549, and EGFR-GFP-A549 cells (Fig. 5c, 1st column), but their uptake were all inhibited by excess of HDL (Fig. 5c, 2nd column). The 6.3-fold difference in the uptake of EGF-HPPS between A549 cells and H520 cells (Fig. 5c, 3rd column) was probably due to differential EGFR expression levels and the shielding of SR-BI recognition by EGF conjugation, evidenced by a 2.2-fold increase in EGF-HPPS uptake by A549 cells but a 2.3-fold decrease by H520 cells (Fig. 5c, 3rd versus 1st column). Furthermore, HDL blocking enhanced the EGF-HPPS uptake contrast between A549 cells and H520 cells from 6.3-fold (Fig. 5c, 3rd column) to 9.7-fold (Fig. 5c, 4th column). Next, uptake of EGF-HPPS by H520, A549, and EGFR-GFP-A549 cells with or without excess of HDL were evaluated at 3, 6, and 24 h after incubation (Fig. 5d and e). At all three time points, the difference of EGF-HPPS uptake between A549 (EGFR+) and EGFR-GFP-A549 (EGFR++) could not be distinguished without the excess of HDL which blocks secondary SR-BI targeting (Fig. 5d). Clear differences only appeared after adding excess of HDL, which revealed the pure EGFR targeting (Fig. 5e). Another interesting effect of this secondary targeting lies in the fact both EGFR and SR-BI contributed to the EGF-HPPS uptake. This observation could be explored for enhancing the uptake through dual receptor coordination.Fig. 5

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