Investigating the specific uptake of EGF-conjugated nanoparticles in lung cancer cells using fluorescence imaging
© Springer-Verlag 2010
Received: 15 October 2010
Accepted: 27 October 2010
Published: 11 November 2010
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.
Nanoparticle-based drug delivery is on the verge of revolutionizing cancer therapy due to superior drug solubility, improved serum stability, longer circulation half-lives, better drug loading and shielding ability, and excellent drug accumulation in tumor through the enhanced permeability and retention effect (Brigger et al. 2002; Petros and DeSimone 2010). External ligands, including antibodies, proteins, peptides, aptamers, and other small molecule ligands can be attached to nanoparticle carriers (Brannon-Peppas and Blanchette 2004; Byrne et al. 2008; Lammers et al. 2008), resulting in targeted nanoparticles. When integrating a targeting ligand with a nanoparticle, the resulting targeting efficiency may be influenced by many factors, such as the ligand numbers (Jiang et al. 2008), the ligand coupling method and site of ligand coupling (Zheng et al. 2005), the interaction between the ligand and nanoparticle (Vincent et al. 2009), as well as non-specific binding caused by nanoparticle itself (Chen et al. 2010). Therefore, validation of targeting effects for a given ligand may be difficult to resolve with traditional competition–inhibition methods using an excess of free ligand or simple models via positive–negative cells (Yu et al. 2010). This is further complicated by the heterogeneous nature of tumor cells (Poste et al. 1982). Co-expression of different receptors or biomarkers on the cell surface may complicate the desired ligand–receptor interaction, which may lead to false-positive or negative results that obscure the understanding of how nanoparticle targeting works.
We recently reported a HDL-mimicking peptide–phospholipid scaffold (HPPS) nanocarrier for the delivery of diagnostic and therapeutic payloads (Zhang et al. 2009). HPPS closely resembles the structure of plasma-derived spherical HDL by replacing apoA-I protein with self-assembled apoA-I mimetic peptides on the phospholipid monolayer of the nanoparticle (Zhang et al. 2009). This results in nanoparticles of well-controlled, monodispersed, sub-30 nm size, that retain the HDL-like capacity to carry lipophilic payloads. Perhaps equally important, HPPS mimics the functions of plasma-derived HDL in its pharmacokinetics and targeting specificity against scavenger receptor type B1 (SR-BI), which permits excellent delivery of the nanoparticle cargo to the target cells. Furthermore, like HDL (Corbin et al. 2007), HPPS can be tailored to target a receptor of choice by introducing various targeting ligands for different cancer applications.
Epidermal growth factor receptor (EGFR), a member of the human epidermal growth factor receptor (HER)–ErbB family of receptor tyrosine kinases, represents an important target for non-small cell lung cancer diagnosis and treatment. Its activation stimulates key processes involved in tumor growth and progression, including proliferation, angiogenesis, invasion, and metastasis. Therefore, EGFR has become an attractive target for nanoparticle-based lung cancer detection and treatment (Doroshow 2005; Gatzemeier 2003). Many EGFR-specific targeting ligands (antibodies, single chain antibody fragments, affibodies, recombinant epidermal growth factor (EGF), EGF peptide mimetics, etc.) have been developed and used to target nanoparticle carriers to EGFR (Dechant et al. 2007; Nakamura et al. 2005; Reilly et al. 2000; Senekowitsch-Schmidtke et al. 1996; Tolmachev et al. 2009) with various degrees of success. Targeting nanoparticles to EGFR in lung cancer provides a desirable model to study the complexities of ligand/nanoparticle–receptor interactions. Previously, we have successfully developed an EGF-conjugated HPPS nanoparticle and tested its targeting to cancer cells in vitro and in vivo (Zhang et al. 2010). The objective of this study is to take advantage of this already established targeted nanoparticle model to systematically investigate the receptor specific uptake of EGF-conjugated nanoparticles in lung cancer cells, thus providing valuable insight on the potential impact of tumor cell heterogeneity on the targeting specificity of nanoparticle carriers.
2 Materials and methods
2.1 Preparation of EGF-conjugated HPPS nanoparticle
Human EGF was obtained from R&D Systems, Inc. (USA). EGF-HPPS was generated as previously described (Zhang et al. 2010). In brief, EGF was treated with Traut’s reagent to make sulfhydryl-activated EGF. HPPS containing DSPE-PEG (2000) maleimide and loaded with the near-infrared fluorescent dye DiR-BOA was then mixed with sulfhydryl-activated EGF at room temperature for 20 h. The EGF-conjugated HPPS particle, termed EGF-HPPS, was subsequently filtered (0.2 μm) and purified by gel filtration chromatography using the Akta FPLC system (Amersham Biosciences, USA) equipped with a HiLoad 16/60 Superdex 200 pg column.
2.2 Cell culture
Cell culture media RPMI 1640 and Hams F-12, along with fetal bovine serum (FBS) and Geneticin (G418) were purchased from Gibco-Invitrogen Co. (USA). LdlA7 cells, which minimally express EGFR and scavenger receptor class B type I (EGFR−, SR-BI−), were kindly provided by Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA). LdlA7 cells were cultured in Hams F-12 media with 2 mM L-glutamine, 100 U/ml penicillin–streptomycin, and 5% FBS. Human lung adenocarcinoma A549 cell line (EGFR+, SR-BI+) and human lung squamous cell carcinoma NCL-H520 cell line (SR-BI+, EGFR−) were purchased from the American Type Culture Collection. A549 cells and H520 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. All cells were grown at 37°C in a humidified atmosphere containing 5% CO2.
2.3 Plasmid transfection
Plasmid transfections of pCDNA-EGFR-EGFP (provided by Dr. Peter Verveer, Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Australia) were performed on LDLA7 and A549 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’ protocol. Transfected cells were then exposure to Geneticin (800 μg/ml) and sorted by the flow cytometry equipped with a Becton Dickinson FACS Aria cell sorter to select the cells with EGFR-GFP expressing.
2.4 Confocal microscopy and flow cytometry
Cells were seeded into 8-well cover glass-bottom chambers (Nunc Lab-Tek, Sigma-Aldrich, 2 × 104/well) for the confocal microscopy imaging and into 6-well cell culture plates (3 × 105/well) for the flow cytometry study. AF555-EGF (Alexa Fluor® 555 EGF complex, Molecular Probes, Inc., USA) was used as a standard probe to identify the EGFR positive cells. Final concentration (2.5 μg/ml) of AF555-EGF was used in confocal imaging and flow cytometry analysis. To evaluate the uptake specificity of HPPS particles, cells were incubated with HPPS or EGF-HPPS with DiR-BOA concentration at 1 μM in cell culture medium containing 10% FBS for 3 h at 37°C. The total volume of incubation medium was 300 μl/well for confocal imaging and 1 ml/well for flow cytometry. For the competition assay, cells were coincubated with an 800-M excess of HDL (1 mg/ml) or 5.6 μM EGF. Confocal imaging was taken by Olympus FV1000 laser confocal scanning microscopy (Olympus, Tokyo, Japan) with the excitation wavelength of 488 (GFP), 543 (AF555), and 633 nm (DiR-BOA). For the flow cytometry study, the fluorescence signal of cells were detected by a Dako Cytomation MoFlo 9-color cell sorter for AF555 (ex. 543 nm) and a Beckman Coulter FC500 5-color analyzer for GFP (ex. 488 nm) and DiR-BOA (ex. 633 nm).
3 Results and discussion
3.1 Establishing the correlation between EGFR expression in cells and EGF uptake using fluorescence imaging
3.2 Evaluating the specific uptake of EGF-HPPS using EGFR-GFP-LDLA7 and EGFR-GFP-A549 cells
We have validated the specific uptake of EGF-HPPS nanoparticles via the EGFR pathway as well as the secondary targeting via SR-BI pathway using dual fluorescent labeling approach (labeling both the nanoparticles and cell lines). The use of double negative LDLA7 cells (EGFR− and SR-BI−) established a clear baseline for EGFR-GFP partial transfection to create internal EGFR+ and EGFR− controls. On the other hand, the use of both EGFR-GFP-A549 cells (EGFR++) and wild-type A549 cells (EGFR+) together with the use of H520 cells (EGFR−) allowed us to analyze the EGF/EGFR response at different levels of EGFR expression in lung cancer cells.
In summary, this study confirmed the EGFR targeting of EGF-HPPS in lung cancer cells. More importantly, the dual fluorescent labeling approach (labeling both the ligand-conjugated nanoparticle carrier and the targeted receptors on the cells) developed in this study provides more insight on the potential influence of secondary targets on the intended ligand–receptor interaction. Furthermore, this fluorescent imaging strategy may be applied for target validation in other applications such as confirming the putative target receptor for a ligand of interest.
This study was conducted with the support of the Ontario Institute for Cancer Research through the Government of Ontario, the Canadian Institute of Health Research, the Natural Sciences and Engineering Research Council of Canada, the China-Canada Joint Health Research Initiative (CIHR CCI-102936, NSFC-30911120489), the Ontario Ministry of Health and Long Term Care, and the Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research.
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