Biomacromolecules 2008, 9, 603–609
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Dendrimer-Epidermal Growth Factor Conjugate Displays Superagonist Activity Thommey P. Thomas,† Rameshwer Shukla,† Alina Kotlyar,† Bradley Liang,† Jing Yong Ye,‡ Theodore B. Norris,‡ and James R. Baker, Jr.*,† Michigan Nanotechnology Institute for Medicine and Biological Sciences, Department of Internal Medicine, Division of Allergy, University of Michigan, 9220 MSRB III, Box 0648, Ann Arbor, Michigan 48109, and Center for Ultrafast Optical Science, University of Michigan, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109 Received October 26, 2007
Binding of ligands on to epidermal growth factor receptor (EGFR) can stimulate cell growth; therefore, any application employing EGF as a targeting ligand for a “drug carrier” must evaluate the effect of the conjugate on cell growth. We report the synthesis and in vitro biological activity of EGF molecules coupled to a fluoresceinlabeled polyamidoamine dendrimer. The conjugate bound and internalized into several EGFR-expressing cell lines in a receptor-specific fashion. The conjugate effectively induced EGFR phosphorylation and acted as a superagonist by stimulating cell growth to a greater degree than free EGF. Concomitant administration of the chemotherapeutic drug methotrexate completely inhibited cell growth to a degree similar to its effect in the absence of the conjugate. Thus, dendrimer-EGF conjugates serve as EGFR superagonists, but this activity can be overcome by chemotherapeutic drugs. The agonist activity of these materials must be taken into consideration when using EGF conjugates for imaging applications.
Introduction The epidermal growth factor receptor (EGFR, ErbB) is a 170 kDa protein that is distributed randomly on the surface of cells, excluding hematopoietic cells.1,2 Its ligand, EGF, is a 53 amino acid peptide that mediates cellular signal events regulating cell proliferation, differentiation, cell cycle progression, adhesion, invasion, angiogenesis, and inhibition of apoptosis.3–5 Following binding of the EGF, the EGFR functions either as a homodimer through the complexation of two EGFR molecules or as a heterodimer by associating with one of the three other ErbB family members; in either case the resulting dimerization then initiates the cellular internalization.5 EGFR is known to be overexpressed in a variety of human carcinomas, including cancers of the head and neck, breast, colon, ovary, lung, prostate, and liver.6,7 Enhanced EGFR expression is associated with tumor invasiveness and resistance to chemotherapy and radiation therapy and clinically correlates with poor prognosis and lower patient survival.6,8,9 Squamous cell carcinoma of the head and neck is a cancer commonly associated with EGFR overexpression (>90%), which appears to play a role in the unregulated growth of these cells.10 EGF and anti-EGF antibody-based selective targeting of drugs, toxins, ribonuclease, and radionuclides has been shown to inhibit the growth of several EGFR expressing tumor cell types in vitro and in vivo.11–16 A number of synthetic and natural polymers have been evaluated as carriers for the targeted delivery of drugs into cancer cells.17–22 Several groups17,20,23–26 have utilized polyamidoamine (PAMAM) dendrimers as a drug delivery platform, given several attractive properties of these polymers including uniformity, biocompatibility, defined branched chain structure, * Corresponding author (
[email protected]). † Michigan Nanotechnology Institute for Medicine and Biological Sciences, Department of Internal Medicine, Division of Allergy. ‡ Center for Ultrafast Optical Science.
and the capability for chemically coupling multiple molecular entities to its surface amino groups. Importantly, biological molecules such as folic acid,23,27–29 antibodies,30–32 chemotherapeutic drugs,28,29 and apoptosis-sensing molecules33 covalently linked to the PAMAM dendrimer have been shown to retain their biological function. This should facilitate therapeutic targeting of dendrimer-couplexed therapeutics due to ligand binding to tumor cells. Prior work from our group using folate conjugated dendrimers showed cooperative binding of multiple ligands that yielded a high-avidity interaction resulting in effective tumor targeting.27–29 In addition, Barth et al. used human EGF conjugated to boronated generation 4-PAMAM dendrimer to target gliomas that overexpress the EGFR.34,35 Therefore, in the present study we attempted to synthesize a PAMAM dendrimer with multiple EGF conjugated to each dendrimer as a multivalent targeting agent with cooperative binding similar to our prior folate conjugates. We also sought to examine the applicability of the conjugate as a drug delivery platform by studying the kinetics of its cellular binding, signaling, and growth-regulating activity in comparison with that of free EGF. In order to maximize the biological activity of these conjugates, we charge-neutralized our dendrimers by partial acetylation of the surface primary amino groups to prevent nonspecific cellular binding36 and coupled mouse EGF lacking lysine residues that prevented the conjugation of EGF through any residue other than the amino terminus.
Experimental Procedures Materials. Generation 5 (G5)-PAMAM dendrimer was prepared at the Michigan Nanotechnology Institute for Medicine and Biological sciences, University of Michigan, and was analyzed extensively by 1H and 13C NMR, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, high-performance liquid chromatography, gel-permeation chromatography (GPC), and polyacrylamide
10.1021/bm701185p CCC: $40.75 2008 American Chemical Society Published on Web 01/15/2008
604 Biomacromolecules, Vol. 9, No. 2, 2008 gel electrophoresis. The molecular weight of synthesized dendrimer was measured by GPC to be 26530 g/mol, and the average number of primary amino groups was estimated by potentiometric titration36 to be 108. The A431, MCF7, and the SCC4 cell lines were obtained from ATCC (Rockville, MD). The SCC15 and Fadu cell lines were kindly provided by Dr. Brent Ward at the University of Michigan. TrypsinEDTA, Dulbecco’s phosphate-buffered saline (PBS), and Dulbecco’s modified Eagles medium (DMEM) were obtained from Gibco/BRL (Gaithersburg, MD). Mouse EGF was from Chemicon International (Temecula, CA), and FI-labeled mouse EGF (EGF-FI) was from Invitrogen (Eugene, OR). The FACE assay kit was from Active Motif (Carlsbad, CA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) and were used per manufacturer’s protocol. Synthesis and Purification of G5-FI-EGF. Synthesis of G5-Ac. G5 amine dendrimer (0.265 g, 0.0099 mmol) and triethylamine (0.088 g, 0.8635 mmol) were dissolved in 30 mL of anhydrous methanol (MeOH) and allowed to stir for 30 min. A solution of acetic anhydride (0.076 g, 0.744 mmol) in anhydrous MeOH (15 mL) was added dropwise while stirring. The reaction mixture was allowed to stir overnight at room temperature. After evaporation of the solvent, the residue was dissolved in H2O and dialyzed in 10000 MWCO regenerated cellulose dialysis bags initially against PBS buffer, followed by water. The partially acetylated dendrimer was lyophilized to give a colorless powder (0.270 g, 91.2%). The average number of acetyl groups (80) has been determined based on a 1H NMR calibration curve drawn by plotting a ratio of acetyl protons and the sum of all methylene protons versus the degree of acetylation. G5-Ac-FI. Fluorescein isothiocyanate (FITC, FI) (0.0058 g, 15.0 mmol) in DMSO (2 mL) was added dropwise to G5-Ac (0.102 g, 3.4 mmol) in DMSO (10 mL). The reaction was allowed to stir overnight. The reaction mixture was diluted 1:1 in PBS, and free dye was separated from the conjugate by gel filtration on Sephadex G-25 column. The eluted conjugate was concentrated using a “Centriprep” device (10000 MWCO) and was dialyzed against PBS and H2O before lyophilization. G5-Ac-FI-COOH. Glutaric anhydride (0.0029 g, 0.0257 mmol) dissolved in anhydrous MeOH (2 mL) was added dropwise while stirring to a solution of G5-Ac-Fl (0.0203 g, 0.0006 mmol) and TEA (0.0026 g, 0.0257 mmol) in anhydrous MeOH (18 mL), and the reaction mixture was allowed to stir for another 24 h at room temperature. The solvent was evaporated in a vacuum, and the residual material was dissolved in H2O, purified by extensive ultrafiltration against PBS and H2O using a Centricon device (10000 MWCO), and lyophilized. G5-FI-EGF. An active ester was prepared by reacting G5-Ac-FlCOOH (0.0017 g, 0.000053 mmol) in PBS buffer (2.0 mL, pH 7.4) with EDC (0.00012 g, 0.00533 mmol) for 3 h. Mouse EGF (0.001 g, 0.00016 mmol) in PBS buffer (0.5 mL) was added dropwise to the above solution and allowed to stir overnight. The product (G5-FI-EGF) was purified by extensive ultrafiltration against PBS buffer (pH 7.4) and H2O using a Centricon device (10,000 MWCO) and then lyophilized. Cell Culture and Treatment. All the cell lines were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were allowed to grow in a monolayer in tissue culture flasks incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Cells plated in 24-well plates (for flow cytometry), 35 mm dishes with glass coverslips in the bottom (for confocal microscopy), and 96-well plates (for “FACE” and “XTT” assays) were treated with the conjugate under the specified incubation conditions. Excess free EGF was added 30 min prior to the conjugates for blocking the binding of the latter. A competition assay with free EGF was conducted at 4 °C by simultaneously exposing the conjugate and free EGF. Flow Cytometry and Confocal Microscopy. The FITC fluorescence was quantified on a Beckman-Coulter EPICS-XL MCL flow cytometer, and the data were analyzed using Expo32 software (Beckman-Coulter, Miami, FL). The viable cells were gated, and the mean FL1-fluorescence of 10000 cells was quantified. For confocal microscopy experiments,
Thomas et al. the cells were seeded at a density of 5 × 105 cells/plate on glass-bottom culture dishes (Mattek, Ashland, MA) 2 days prior to the experiment. The cells were incubated with the conjugate in serum-free medium under the specified conditions and analyzed using an Olympus FluoView 500 laser scanning confocal microscope. FITC fluorescence was excited with a 488 nm blue argon laser, and the emission was measured through a 505–525 nm barrier filter. Samples were scanned on an Olympus IX-71 inverted microscope, using a 60× waterimmersion objective, and were magnified 2.5 times with FluoView version 4.3 software. TPOFF Analysis. The number of conjugate molecules bound on the cells was determined by a double-clad fiber-based two-photon optical fiber fluorescence (TPOFF) probe as described before.37 In this system we have utilized a novel solid double-clad fiber38 that is about 5-fold more sensitive than the single-mode fiber we have used previously.32,37 This fiber also has the advantage of not having any holes at the tip of the fiber in contrast to a previously described “double clad photonic crystal fiber”,39 thus preventing the problematic biological fluid suction by the latter. In 400 µL of DMEM medium containing 1% BSA, 5 × 106 cells were trypsinized and treated with different concentrations of G5-FI-EGF or G5-FI for 1 h at 4 °C. Cells were centrifuged, and the cell pellet obtained was dissolved in medium containing 0.5% Triton X-100. The fluorescence of the dissolved cell pellet was measured using the TPOFF probe with two-photon excitation by a Ti:sapphire laser at an 800 nm wavelength with a 50 fs pulse duration. The emitted fluorescence from the sample was recorded with a time-correlated single-photon counting system, and the integrated total fluorescence photon counts were used for quantifying the bound dendrimer conjugates. The number of molecules bound per cell was calculated based on the TPOFF measurement results of the standard conjugate solutions with different concentrations under the same excitation and detection conditions. FACE Assay. The fast activated cell-based ELISA (FACE) chemiluminescent assay for the quantification of EGFR phosphorylation was performed according to the manufacture’s instructions. Briefly, cells in serum-free medium containing 1% BSA were incubated with EGF or G5-FI-EGF for 5 min, rinsed, and fixed with p-formaldehyde. After nonspecific binding sites were blocked, the cells were incubated with specific rabbit antibodies against either phosphorylated EGFR (which recognizes residues surrounding the phosphorylated tyrosine 992) or total EGFR (recognizes EGFR protein regardless of its phosphorylation site). The amount of EGFR was determined using horseradish peroxidase-conjugated secondary antibody and using a chemiluminescent detection system. The values obtained were corrected for cell number using crystal violet staining by measuring absorbance at 595 nm. XTT Assay. For the cytotoxicity experiments, the cells (2500 cells/ well) were seeded in 96-well microtiter plates in DMEM containing 10% FCS, and the cells were treated with 100 nM each of the conjugates for 72 h. A colorimetric XTT assay was performed following the vendor’s protocol (Roche Molecular Biochemicals, Indianapolis, IN). After incubation with an XTT labeling mixture, microtiter plates were read on an ELISA reader (Synergy HT, BioTek) at 492 nm with the reference wavelength at 690 nm. Vehicle-treated cells were assigned a value of 100%.
Results Synthesis of the dendrimer conjugate is shown in Scheme 1. G5 PAMAM dendrimer was synthesized and characterized at the Michigan Nanotechnology Institute for Medicine and Biological Science. The amine-terminated dendrimer (G5-NH2) was partially acetylated by reacting with 80 mol equiv of acetic anhydride, as described previously.36 This was done in order to reduce the number of primary surface amines, which decreases the nonspecific charge interactions with biologic molecules. The purity of the partially acetylated compound and the extent of acetylation were evaluated by 1H NMR, which showed a
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Scheme 1
distinct signal for the terminal NHCOCH3 protons at δ 1.85 ppm. The degree of acetylation was determined by comparing the ratio of NHCOCH3 protons with the sum of all methylene protons in the dendrimer to a calibration curve as described previously. For targeting studies, acetylated G5 was fluorescently labeled by reacting with FITC in DMSO. The 1H NMR of the conjugate shows broad signals in the aromatic region corresponding to the FITC protons. The number of dye molecules attached to the dendrimer was calculated to be ∼3 based on UV-vis spectroscopy and 1H NMR. The remaining surface amines on the dendrimer were converted to carboxylic acid by reacting with excess glutaric anhydride in MeOH, in the presence of TEA as base. The loss of the peaks at δ 2.97 and 3.32 ppm and the emergence of two new peaks at δ 1.65 and 2.05 ppm (a 1:2 ratio) in the 1H NMR spectrum showed the presence of the glutarate moiety. The carboxyl groups of the purified G5-FI-COOH were activated by reacting with EDC, followed by the addition of 3 equiv of mouse EGF to give the dendrimer-EGF conjugate. The final conjugate was characterized by 1HNMR spectroscopy that showed peaks in the aliphatic region because of the presence of the EGF peptide. UV–visible spectral analysis showed absorbance at λmax 500 nm specific for FI (Figure 1). As EGF and FI absorb at 280 nm, the molar quantity of EGF present in the conjugate was difficult to ascertain by UV analysis. Comparison of the ratios of the 280 nm and 500 nm peaks confirmed the presence of EGF in the conjugate (Figure 1, inset). MALDI-TOF analysis showed the conjugate to contain an average of about 2 EGF per dendrimer. Since the A431 epidermoid tumor cell line expresses very high numbers of EGFR and has been an extensively studied model for EGFR regulation, we initially verified the binding of the conjugate using this cell line. The G5-FI-EGF conjugate bound to the A431 cell lines in a dose-dependent fashion, whereas the control dendrimer G5-FI that lacked EGF targeting ligands failed to bind even at the maximum concentration (300 nM) tested (Figure 2). The affinity for binding of the G5-FIEGF conjugate was compared to that of EGF-FI in the A431 cell line (Figure 3). Free EGF inhibited the binding of 100 nM of G5-FI-EGF and EGF-FI in a dose-dependent manner, with 50% inhibition occurring at 15 and 60 nM, respectively, suggesting that the affinity of G5-FI-EGF is about 4-fold lower than the affinity of EGF-FI and free EGF. The relative affinities for binding of G5-FI-EGF and EGF-FI were further examined by comparing the dose-dependent binding of G5-FI-EGF and EGF-FI. For this, the mean channel fluorescence obtained for the binding of different doses of the two conjugates was normalized for the fluorescence of the standard conjugates. Double reciprocal binding plots of the normalized values at different doses (Figure 3, inset) gave apparent KA values of 80 and 15 nM for G5-FI-EGF and EGF-FI, respectively. The double reciprocal plots showed similar Y-intercept values, indicating similar maximum binding for both EGF-FI and G5-FI-EGF.
Figure 1. UV–vis spectra of G5-FI-EGF, G5-FI, and EGF (2 µM each) in PBS. Inset: 280/500 nm absorbance ratios of G5-FI-EGF and G5FI. Note the increased 280/500 nm ratio for the G5-FI-EGF vs G5-FI due to the presence of EGF in the former.
Figure 2. Histogram showing the binding of different concentrations of G5-FI-EGF in A431 cells. Cells were incubated with different concentrations of G5-FI-EGF at 37 °C for 1 h, and the fluorescence was determined in a flow cytometer.
Figure 3. Inhibition of binding of G5-FI-EGF and EGF-FI by free EGF. A431 cells plated in 24-well plates were incubated with 100 nM of either EGF-FI or G5-EGF-FI and varying concentrations of free EGF for 1 h at 4 °C. The cells were rinsed, and the FL1-fluorescence of 10000 cells was measured in a flow cytometer. Inset: Double reciprocal plots for the dose-dependent binding of G5-FI-EGF and EGF-FI. A431 cells were incubated with different concentrations of G5-FI-EGF and EGF-FI, the mean fluorescence obtained by flow cytometric analysis was normalized for the fluorescence of the conjugates measured in a fluorometer.
As squamous cell carcinoma (SCC) of the head and neck is known to express very high levels of EGFR,10 we examined the binding and internalization of the conjugate in several human squamous cell carcinoma cell lines isolated from primary tumors (Figures 4, 5, and 6). The conjugate bound to the SCC4 cell line in a dose-dependent fashion, whereas it failed to bind to a control cell line, EGFR-negative MCF7 cells, at all concentrations tested (Figure 4). Saturation binding for this conjugate was also obtained with two well-defined squamous cell carcinoma cell lines, SCC15 and Fadu (data not shown).
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Figure 4. Comparison of binding of G5-FI-EGF in EGFR-positive SCC4 cells and EGFR-negative MCF7 cells. SCC4 (triangle symbols) and MCF7 (circle symbols) cells were incubated with different concentrations of G5-FI-EGF (filled symbols) and the control conjugate G5-FI (open symbols) at 37 °C for 1 h, and the mean fluorescence was determined in a flow cytometer.
Figure 5. Number of conjugate molecules bound per cell determined by a double-clad TPOFF probe. SCC15 cells in suspension were incubated with different concentrations of G5-FI-EGF or G5-FI at 4 °C for 1 h and the fluorescence of the bound conjugates was quantified using the TPOFF probe as described in Methods. The number of molecules bound per cell was calculated from the slope of the standard curves generated for different concentrations of the two conjugates using the TPOFF measurement performed under identical conditions.
The number of conjugate molecules bound to the SCC15 cells was quantified by a newly developed two-photon optical fiber method using a unique double-clad optical fiber.38 As shown in Figure 5, at saturation a single SCC15 cell bound about 4 million molecules of the conjugate. Internalization of the conjugate was demonstrated in all the EGFR cell lines tested by confocal microscopic analysis (Figure 6). The conjugate was localized primarily in the cytosolic compartment, although significant perinuclear and nuclear localization was also observed. A z-series analysis of the fluorescence of single SCC15 cells confirmed the presence of the conjugate in these intracellular compartments (data not shown). Because one of the earliest signal transduction events mediated by the binding of EGF on the EGFR is the tyrosine phosphorylation of the receptor, we checked if the G5-FI-EGF conjugate phosphorylates EGFR. As shown in Figure 7, the G5FI-EGF induced phosphorylation similarly to the free EGF molecule. The G5-FI-EGF (100 nM) induced about a 40% increase in cell growth over the control cells during a 3-day incubation period, although equimolar levels of free EGF failed to show any significant increase in cell growth (Figure 8). The chemotherapeutic drug methotrexate inhibited cell growth in the G5-FI-EGF-treated cells similarly to its inhibition of the control cells (Figure 8).
Thomas et al.
Figure 6. Internalization of G5-FI-EGF in SCC15 and SCC4 cells. The SCC cells and the EGFR-negative MCF7 cells grown on coverslips were incubated with 300 nM each of G5-FI or G5-FI-EGF at 37 °C for 1 h. Some cells were preincubated with 6 µM free EGF for 30 min prior to adding the conjugate (bottom middle panel). The green stain shows FI-fluorescence and the blue stain shows nuclei stained with DAPI. The arrows indicate the presence of the conjugate in perinuclear (white) and nuclear (red) compartments. Confocal microscopic analysis was done as given in Methods.
Figure 7. Phosphorylation of EGFR by G5-FI-EGF in SCC15 cells. Cells were incubated with free EGF (20 nM) or G5-FI-EGF (100 nM) for 5 min, and the amount of phosphorylated EGFR was determined by “FACE” assay, as described under Methods. *p < 0.05 vs the EGFR-phosphorylated vehicle-treated control cells. Similar results were obtained in the A431 cell line.
Figure 8. Reversal of G5-FI-EGF-mediated cell growth stimulation by the chemotherapeutic drug methotrexate. SCC15 cells were incubated with free EGF or G5-FI-EGF (100 nM each) in the presence or absence of methotrexate (100 nM) for 3 days, and the cell proliferation was determined by XTT assay as given in Methods. *p < 0.05 vs vehicle-treated control cells.
Discussion In these studies we have documented the synthesis of a novel generation 5 PAMAM dendrimer-Fl-EGF conjugate, characterized the kinetics of its cellular binding, and evaluated its cell-
Dendrimer-EGF Conjugate, Drug Delivery and Superagonist
growth-stimulating properties. We used mouse EGF that lacks lysine residues to ensure only N-terminal linkage with the dendrimer, thereby ensuring all conjugated EGF retain binding affinity to EGFR allowing for polyvalent interactions with EGFR expressing cells. This is important for comparing the biological activity of the conjugate with that of free EGF because the substitution of the lysines in human EGF results in a significant loss of receptor-binding activity.40 We have also utilized a fully acetylated and surface-charge-neutralized dendrimer to prevent nonspecific interactions with negatively charged cell membranes. Using FITC conjugated to this dendrimer, we detailed the in vitro binding and biological function of the conjugate in multiple EGFR-expressing squamous cell carcinoma cell lines. Unlike other dendrimer biological ligand conjugates, which either failed to activate receptors or served as receptor inhibitors, these conjugates stimulated EGFR and cell growth. We believe this is the initial report describing superagonist activity for dendrimer conjugates and suggests potential activity for similar types of conjugates in variety of therapeutic applications. As an example, EGFR activation has been implicated in the physiology of wound healing41 and the dendrimer-EGF conjugate might possibly serve as a topical agent to enhance wound healing. There has been active interest in engineering EGF to create highaffinity EGFR antagonists and affinity-matured agonists with limited success.42 The G5-FI-EGF conjugate clearly bound to EGFR-expressing cells in a receptor-mediated, dose-dependent fashion based on the findings that the control G5-FI dendrimer failed to bind to the EGFR-expressing cells, and the G5-FI-EGF failed to associate with the EGFR-negative MCF7 cells. The specificity of binding was also confirmed by competition between the conjugates and free EGF. As the affinity of growth factors for their receptors depends on the native conformation of the ligands, it is not surprising the G5-FI-EGF conjugate showed a 4-fold lower affinity when compared to free EGF. However, this is better than the 10-fold reduction observed in prior reports of boronated G4 dendrimer-EGF conjugates used for targeting brain cells.34,35 The improved activity of our conjugates is likely due to our use of mouse EGF that lacks lysine residues and the dendrimer surface acetylation. In addition, although the G5-FIEGF had a lowered affinity versus EGF-FI, the similar maximum binding predicts the full biologic activity of the conjugate when concentrations exceed the saturation concentration of 200–300 nM. The TPOFF analysis confirmed that the conjugate could achieve very high receptor occupation, enhancing its biological activity. Confocal microscopic analysis (Figure 6) showed cellular internalization of the G5-FI-EGF conjugate in a manner similar to other dendrimer conjugates using folic acid (FA)-targeted G5-FI-FA28 or the antibody conjugate, G5-Alexafluor-Herceptin.31 The conjugate was primarily localized in the cytosolic compartment of different EGFR-expressing cell lines. However, the confocal data also showed some nuclear uptake of the EGF conjugate, especially to the perinuclear region, demonstrating for the first time that a dendrimer can be targeted to the nucleus in a living cell. This last finding is supported by the biological activity of the conjugate, as the EGF/EGFR complex must enter the nucleus to activate cell growth. It also rules out the possibility that the EGF is released from the dendrimer, which is left behind in the cytoplasm and is consistent with previous studies using free or poly(ethylene glycol)-coupled EGF.40,43–45 The mechanism of EGF-mediated cell growth stimulation involves a series of signal transduction events following the binding of the growth factor, including autophosphorylation,
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dimerization, and internalization of the EGF-EGFR complex. The conjugate clearly displayed the ability to bind to the receptor, induce phosphorylation, and internalize with the EGFR. The G5-FI-EGF conjugate significantly increased cell growth, whereas free EGF under similar conditions failed to show significant cell growth stimulation (Figure 8). Previous studies have shown that the EGFR superagonist ligand “E4T”, which is a chimeric growth factor of EGF and the transforming growth factor R (TGFR), demonstrated increased mitogenic potential versus EGF in the EGFR-transfected NIH-3T3 cells through enhanced receptor cycling.46 The superagonistic mechanism of the dendrimer-EGF conjugate is presently unclear. It is possible that the conjugate favors cross-linking of the receptor, resulting in a sustained EGFR-mediated signal transduction and increased cell growth. It is interesting to note that the dendrimer-EGF conjugate acted as a superagonist to stimulate long-term cell growth despite its apparent lower affinity (Figure 3) and similar acute (5 min) receptor-phosphorylation (Figure 7), as compared to free EGF. Recent studies have shown that analogues of the thyrotropin-releasing hormone (TRH) with lower affinity than TRH can act as superagonists.47 EGFR is overexpressed in a wide variety of tumors and, therefore, is an attractive target for drug delivery. A number of strategies have been investigated for the targeting of the EGFR signaling pathway in cancer, including the usage of a small molecule tyrosine kinase inhibitor, monoclonal antibodies, EGF-toxin conjugates and liposomes, EGF vaccines, as well as antisense nucleotides and siRNAs for the EGFR.48–50 The applicability of this conjugate as a drug delivery agent will depend on how effective drug-induced cell growth inhibition can be achieved in the presence of the growth stimulatory activity. The observation that concomitant administration of the methotrexate significantly inhibited cell growth rate to a level similar to its inhibition of the control cells suggests that despite the activation of EGFR, the conjugate might still be used as a drug delivery platform, although its use in imaging raises concerns. In this regard, our previous studies have shown that a G5-folic acid-methotrexate (G5-FA-MTX) conjugate containing 4 to 5 FA and 10 to 11 MTX molecules has high tumoricidal activity and an increased chemotherapeutic index versus free MTX, despite the opposing growth effects of the FA and MTX present in the conjugate.29 Finally, although in this study we used mouse EGF, a genetically engineered lysinefree human EGF40 could potentially allow targeting human cancers without inducing an immune response. Acknowledgment. This project has been funded with Federal funds from the National Cancer Institute, National Institutes of Health, under awards: 1 R01 CA119409, 1 R33 CA112141, and 1 R21 RR021893. This work utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center funded by NIH5P60 DK20572 from the National Institute of Diabetes & Digestive & Kidney Diseases.
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