Methotrexate-Modified Superparamagnetic Nanoparticles and Their

Nathan Kohler, Conroy Sun, Jassy Wang, and Miqin Zhang* ...... Geta David , Gheorghe Fundueanu , Mariana Pinteala , Bogdan Minea , Andrei Dascalu ...
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Methotrexate-Modified Superparamagnetic Nanoparticles and Their Intracellular Uptake into Human Cancer Cells Nathan Kohler,† Conroy Sun,† Jassy Wang,‡ and Miqin Zhang*,† Department of Materials Science & Engineering, University of Washington, Seattle, Washington 98195-2120, and Union Chemical Laboratories, ITRI, 321 Kuang Fu-Rd, Hsinchu, Taiwan, Republic of China Received February 7, 2005. In Final Form: June 19, 2005 A magnetic nanoparticle conjugate was developed that can potentially serve both as a contrast enhancement agent in magnetic resonance imaging and as a drug carrier in controlled drug delivery, targeted at cancer diagnostics and therapeutics. The conjugate is made of iron oxide nanoparticles covalently bound with methotrexate (MTX), a chemotherapeutic drug that can target many cancer cells whose surfaces are overexpressed by folate receptors. The nanoparticles were first surface-modified with (3-aminopropyl)trimethoxysilane to form a self-assembled monolayer and subsequently conjugated with MTX through amidation between the carboxylic acid end groups on MTX and the amine groups on the particle surface. Drug release experiments demonstrated that MTX was cleaved from the nanoparticles under low pH conditions mimicking the intracellular conditions in the lysosome. Cellular viability studies in human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa) further demonstrated the effectiveness of such chemical cleavage of MTX inside the target cells through the action of intracellular enzymes. The intracellular trafficking model proposed was supported through nanoparticle uptake studies which demonstrated that cells expressing the human folate receptor internalized a higher level of nanoparticles than negative control cells.

1. Introduction Over the past several years, there has been a growing interest in the development of targeted nanoparticle-based probes for tumor diagnostics and therapeutics.1-3 It is recognized that with these targeted probes, tumors, or other lesions can be detected at the cellular or molecular level.4,5 Two major applications associated with these systems are magnetic resonance imaging (MRI) and controlled drug release (CDR).6,7 In MRI, nanoparticles with superparamagnetic properties serve as contrast enhancement agents, while in CDR, they function as drug carriers delivering and releasing the drugs into target * To whom correspondence may be addressed. E-mail: mzhang@ u.washington.edu. † University of Washington. ‡ Union Chemical Laboratories. (1) Knauth, M.; Egelhof, T.; Roth, S. U.; Wirtz, C. R.; Sartor, K. Monocrystalline iron oxide nanoparticles: possible solution to the problem of surgically induced intracranial contrast enhancement in intraoperative MR imaging. AJNR Am. J. Neuroradiol. 2001, 22, 99102. (2) Alexiou, C.; Arnold, W.; Klein, R. J.; Parak, F. G.; Hulin, P.; Bergemann, C.; Erhardt, W.; Wagenpfeil, S.; Lubbe, A. S. Locoregional cancer treatment with magnetic drug targeting. Cancer Res. 2000, 60, 6641-6648. (3) Bulte, J. W.; Hoekstra, Y.; Kamman, R. L.; Magin, R. L.; Webb, A. G.; Briggs, R. W.; Go, K. G.; Hulstaert, C. E.; Miltenyi, S.; The, T. H.; et al. Specific MR imaging of human lymphocytes by monoclonal antibody-guided dextran-magnetite particles. Magn. Reson. Med. 1992, 25, 148-157. (4) Jaffer, F. A.; Weissleder, R. Seeing within: molecular imaging of the cardiovascular system. Circ. Res. 2004, 94, 433-445. (5) Schellenberger, E. A.; Reynolds, F.; Weissleder, R.; Josephson, L. Surface-functionalized nanoparticle library yields probes for apoptotic cells. ChemBioChem. 2004, 5, 275-279. (6) Kircher, M. F.; Mahmood, U.; King, R. S.; Weissleder, R.; Josephson, L. A multimodal nanoparticle for preoperative magnetic resonance imaging and intraoperative optical brain tumor delineation. Cancer Res. 2003, 63, 8122-8125. (7) Alexiou, C.; Schmid, R.; Jurgons, R.; Bergemann, Ch.; Arnold, W.; Parak, F. G. Targeted Tumor Therapy with “Magnetic Drug Targeting”: Therapeutic Efficacy of Ferrofluid Bound Mitoxantrone. In Ferrofluids: Magnetically Controllable Fluids and Their Applications; Odenback, S., Ed.; Springer-Verlag: Berlin, 2002; pp 233-251.

cells. The combination of these two technologies may allow simultaneous diagnosis and treatment of the diseased tissues. Whether these nanoparticles are serving as contrast agents or drug carriers, both applications rely on the efficiency of specific targeting by the nanoparticle systems. In addition, the nanoparticle system in CDR is also required to have an effective mechanism of drug release within the target cells. An effective approach to improving the targeting capability and drug release efficiency is to conjugate nanoparticles with chemical or biological reagents including antibodies or low molecular weight targeting agents that have strong affinity for target cells and high efficiency for nanoparticle internalization. However, identification of specific targeting agents or drugs that can be effectively released from nanoparticles inside target cells remains a challenge and is the central focus of the current studies in the field.8-10 Folic acid (FA) is generally recognized as an effective tumor targeting agent to conjugate with nanoparticles. Folate receptors are overexpressed on the cell membranes of many cancer cells including ovarian, endometrial, colorectal, breast, lung, renal cell carcinomas, brain metastases derived from epithelial cancers, and neuroendocrine carcinomas.11-14 (8) Rapoport, N. Y.; Christensen, D. A.; Fain, H. D.; Barrows, L.; Gao, Z. Ultrasound-triggered drug targeting of tumors in vitro and in vivo. Ultrasonics 2004, 42, 943-950. (9) Di Stefano, G.; Kratz, F.; Lanza, M.; Fiume, L. Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier. Dig. Liver Dis. 2003, 35, 428-433. (10) Rihova, B.; Jelinkova, M.; Strohalm, J.; St’astny, M.; Hovorka, O.; Plocova, D.; Kovar, M.; Draberova, L.; Ulbrich, K. Antiproliferative effect of a lectin- and anti-Thy-1.2 antibody-targeted HPMA copolymerbound doxorubicin on primary and metastatic human colorectal carcinoma and on human colorectal carcinoma transfected with the mouse Thy-1.2 gene. Bioconjugate Chem. 2000, 11, 664-673. (11) Sudimack, J.; Lee, R. J. Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 2000, 41, 147-162. (12) Gabizon, A.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit, F.; Qazen, M. M.; Zalipsky, S. Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in vitro studies. Bioconjugate Chem. 1999, 10, 289-298.

10.1021/la0503451 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/12/2005

Nanoparticle Uptake in Cells

Compared with widely used antibodies which are bulky and difficult to cross the cell membrane, FA has short chains and a small size, and thus facilitates the internalization of nanoparticles. FA is stable, nonimmunogenic, inexpensive and, in addition, has a very high affinity for its cell surface receptors.14 The science and technology utilizing liposomes as drug carriers have recorded major advances in the past decade.15-17 Interest in liposomes is directed upon their lipid-bilayer vesicular structure capable of encapsulating drugs and interacting with living cells.18,19 Nanoparticles as drug carriers are more attractive in view of their high tissue permeability, high colloidal stability, and small size. Drugs can be grafted onto nanoparticles via physical adsorption,20 ionic bonding,2,7 and covalent bonding.21,22 Covalent bonding of drugs on nanoparticles is usually favored because the bond strength makes nanoparticledrug conjugates highly stable and therefore is most likely to be disrupted only under harsh environments inside lysosomes. Delivery of chemotherapeutic agents to target cells is not sufficient to induce cell death. Once the chemotherapeutic drug has been released inside the cell, it must retain within the cell at concentrations sufficient to inhibit cell growth and function such as biosynthesis of expressed proteins. Methotrexate (MTX), an analogue of folic acid, exhibits not only a targeting role as folic acid but also a therapeutic effect to many types of cancer cells that overexpress folate receptors on their surfaces.23 It has been utilized for the treatment of several forms of cancers for decades, including leukemias, breast cancer, head and neck cancer, lymphomas, and carcinomas.24 However, despite the value of MTX in the treatment of cancers, (13) Sirotnak, F. M.; Tolner, B. Carrier-mediated membrane transport of folates in mammalian cells. Annu. Rev. Nutr. 1999, 19, 91-122. (14) Stella, B.; Arpicco, S.; Peracchia, M. T.; Desmaele, D.; Hoebeke, J.; Renoir, M.; D’Angelo, J.; Cattel, L.; Couvreur, P. Design of folic acid-conjugated nanoparticles for drug targeting. J. Pharm. Sci. 2000, 89, 1452-1464. (15) Bazile, D.; Prud’homme, C.; Bassoullet, M. T.; Marlard, M.; Spenlehauer, G.; Veillard, M. Stealth Me. PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system. J. Pharm. Sci. 1995, 84, 493-498. (16) Kong, G.; Anyarambhatla, G.; Petros, W. P.; Braun, R. D.; Colvin, O. M.; Needham, D.; Dewhirst, M. W. Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release. Cancer Res. 2000, 60, 6950-6957. (17) Rivera, E.; Valero, V.; Syrewicz, L.; Rahman, Z.; Esteva, F. L.; Theriault, R. L.; Rosales, M. M.; Booser, D.; Murray, J. L.; Bast, R. C., Jr.; Hortobagyi, G. N. Phase I study of stealth liposomal doxorubicin in combination with gemcitabine in the treatment of patients with metastatic breast cancer. J. Clin. Oncol. 2001, 19, 1716-1722. (18) Fenart, L.; Casanova, A.; Dehouck, B.; Duhem, C.; Slupek, S.; Cecchelli, R.; Betbeder, D. Evaluation of effect of charge and lipid coating on ability of 60-nm nanoparticles to cross an in vitro model of the bloodbrain barrier. J. Pharmacol. Exp. Ther. 1999, 291, 1017-1022. (19) Avgoustakis, K.; Beletsi, A.; Panagi, Z.; Klepetsanis, P.; Karydas, A. G.; Ithakissios, D. S. PLGA-mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties. J. Controlled Release 2002, 79, 123-135. (20) Reszka, R.; Beck, P.; Fichtner, I.; Hentschel, M.; Richter, J.; Kreuter, J. Body distribution of free, liposomal and nanoparticleassociated mitoxantrone in B16-melanoma-bearing mice. J. Pharmacol. Exp. Ther. 1997, 280, 232-237. (21) Torchilin, V. P.; Levchenko, T. S.; Lukyanov, A. N.; Khaw, B. A.; Klibanov, A. L.; Rammohan, R.; Samokhin, G. P.; Whiteman, K. R. p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups. Biochim. Biophys Acta 2001, 1511, 397-411. (22) Zhang, Y.; Kohler, N.; Zhang, M. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 2002, 23, 1553-1561. (23) Duthie, S. J. Folic-acid-mediated inhibition of human coloncancer cell growth. Nutrition 2001, 17, 736-737. (24) Messmann, R.; Allegra, C. Antifolates. In Cancer Chemotherapy & Biotherapy, 3 ed.; Chabner, B., Longo, D., Eds.; Lippincott Williams & Wilkins: Philadelphia, 2001; pp 139-184.

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target cells may develop resistance to this drug due to cellular efflux of the molecule.25 To counteract this phenomenon, researchers have developed MTX conjugates consisting of poly(glutamic acid) or poly(ethylene glycol) which retains a higher concentration of MTX within the cell.26,27 These conjugates have been shown to increase cellular cytotoxicity and increase cellular mortality. Despite the success of these conjugates in vitro, there is still no clinical method of detecting the levels of MTX taken up by the target cells, which may reduce the efficacy of the treatment. MTX has been conjugated to large microparticles made of gelatin or polyglutaraldehyde to improve the level of MTX taken up by the tumor cells.28,29 These methods of drug delivery can provide sustained release of MTX and improve the efficacy of treatment. However, the large size of the drug conjugates does not facilitate intravenous drug delivery. To be effective, the drug carrier must be sufficiently small to perfuse out of the bloodstream to reach the target cell of interest.30 Thus, the large size of these conjugates limits the administration of the drug carriers only to direct injection into the tumor site. In this study, we developed a drug-nanoparticle conjugate by grafting MTX to the iron oxide nanoparticle surface. The design of this MTX-nanoparticle system has a number of combined advantages in view of its therapeutic functionality to treat tumors. This new nanoparticle system enables real-time monitoring of drug delivery to the target tumor through MRI, thus allowing physicians to access the efficacy of their treatment utilizing MRI. Further, by covalently modifying the surface of the nanoparticle via a peptide bond, MTX is not released from the surface of the nanoparticles under intravenous conditions. Instead, cleavage of the amide bond occurs under conditions present in the lysosomal compartment, namely, at low pH and in the presence of lysozymes, a typical environment inside the target cells. Due to the overexpressed folate receptor on target cells as opposed to healthy cells, this release mechanism will greatly reduce toxic effects of MTX to healthy tissues within the body. Finally, we demonstrated increased uptake of the MTX conjugated nanoparticles in tumor cells overexpressing the folate receptor. 2. Experimental Section 2.1. Surface Modification of Nanoparticles with (3Aminopropyl)trimethoxysilane and Methotrexate. Magnetite, Fe3O4, nanoparticles were synthesized by a coprecipitation method reported previously22 with minor modifications. The magnetite nanoparticles were surface-modified with MTX via a chemical scheme outlined in Scheme 1. The nanoparticles were first surface-modified with (3-aminopropyl)trimethoxysilane (APS) to form a self-assembled monolayer (SAM) and subse(25) Banerjee, D.; Mayer-Kuckuk, P.; Capiaux, G.; Budak-Alpdogan, T.; Gorlick, R.; Bertino, J. R. Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase. Biochim. Biophys Acta 2002, 1587, 164-173. (26) Riebeseel, K.; Biedermann, E.; Loser, R.; Breiter, N.; Hanselmann, R.; Mulhaupt, R.; Unger, C.; Kratz, F. Poly(ethylene glycol) conjugates of methotrexate varying in their molecular weight from MW 750 to MW 40000: synthesis, characterization, and structure-activity relationships in vitro and in vivo. Bioconjugate Chem. 2002, 13, 773785. (27) Piper, J. R.; McCaleb, G. S.; Montgomery, J. A. A synthetic approach to poly(gamma-glutamyl) congugates of methotrexate. J. Med. Chem. 1983, 26, 291-294. (28) Narayani, R.; Rao, K. P. Controlled release of anticancer drug methotrexate from biodegradable gelatin microspheres. J. Microencapsulation 1994, 11, 69-77. (29) Hung, C. M., A. D.; Gupta, P. K, Formulation and Characterization of Magnetic Polygluteraldehyde Nanoparticles as Carriers for PolyL-Lysine-Methtrexate. Drug Dev. Ind. Pharm. 1990, 16, 509-521. (30) Howard, C. V. Size matters. Occas. Pap. Ser. 2003, 7, 1-14.

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Scheme 1. Surface Modification of Magnetite Nanoparticles with MTX

quently conjugated with MTX through amidation between the carboxylic acid end groups on MTX and the amine groups on the particle surface. The MTX conjugation reaction may occur through either the R or β carboxylic acid groups on the glutamic acid residue. One milliliter of APS was added to a colloidal suspension of 200 mg of magnetite nanoparticles in 100 mL of toluene dried using molecular sieves. The nanoparticles were sonicated in a sonicating bath for 4 h at 60 °C. The resulting aminated nanoparticles were then isolated using a rare earth magnet and washed twice with 200 proof ethanol and twice with deionized water. To conjugate the nanoparticles with MTX, free MTX was dissolved in 17 mL of DMSO (10 mM) due to the limited solubility of MTX in water. The solution of MTX was then mixed with a 17 mL aqueous solution of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) (75 mM) and N-hydroxysuccinimide (NHS) (15 mM). The pH of the solution was adjusted to 8.2 by addition of 1.0 M NaOH. The resulting suspension was agitated overnight at 37 °C in the dark. Following MTX conjugation, the modified particles were again isolated with a rare earth magnet and washed 5 times with deionized (DI) water. 2.2. Characterization of MTX-Modified Nanoparticles with FTIR. Fourier transform infrared (FTIR) spectra were acquired using a Nicolet 5-DXB FTIR spectrometer with a resolution of 4 cm-1. To characterize the amine SAM and MTX on the nanoparticle surface, 2 mg of dried nanoparticles was added to 200 mg of KBr. The mixture was pressed into a pellet for analysis. 2.3. Analysis of Methotrexate Release from Magnetite Nanoparticles. To simulate intracellular lysosomal conditions, MTX-grafted nanoparticles at a concentration of 0.1 mg/mL were suspended in a solution of 0.1 mg/mL crude protease from bovine pancreas (Sigma) in 5 mL of phosphate-buffered saline (PBS) solution at 37 °C under constant stirring. The solution pH was adjusted by the titration of 1.0 M HCl and 1.0 M NaOH to achieve pH values of 2, 3, 4, 5.6, and 7.44, respectively. Following incubation for 8, 24, 48, and 72 h, the nanoparticle suspensions were centrifuged at 2000 rpm to isolate the nanoparticles from the cleaved MTX, PBS, and protease solutions. MTX cleaved from nanoparticles was then quantified with UV spectroscopy at wavelength of 304 nm. 2.4. Drug Efficacy of MTX-Grafted Nanoparticles in HeLa and MCF-7 Cells. Human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa) were obtained from the Fred Hutchinson Cancer Research Center. Cells were grown in T-75 flasks with RPMI media (Invitrogen) supplemented with 10% fetal calf serum, 25 mM HEPES, 2.05 mM L-glutamine, 50 µg/mL streptomycin, and 50 IU/mL penicillin. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 with the medium being changed every third day. The cells were transferred to RPMI-1640 folate-free medium (Invitrogen) 24 h prior to plating. The cells were then subcultured in 24-well plates at a concentration of 25 000 cells/mL in RPMI-1640 folate-free medium. Nanoparticles were mixed in RPMI-1640 folate-free media at iron concentrations of 0.01, 0.025, 0.05, 0.075, and 0.1 mg/mL. In addition, a control medium containing soluble MTX

Kohler et al. was prepared at a concentration of 2 µg/mL. Cells were also cultured in 1.0 mL of medium without nanoparticles as a control. The cell culture proceeded for 24, 48, 72, 96, and 120 h, at which times the cells were washed three times with 1.0 mL of Hank’s balanced salt solution (HBSS, Invitrogen), detached with 500 µL of 0.25% trypsin-EDTA (Sigma), and resuspended in 1.0 mL of PBS supplemented with 10% FBS. Cell viability was determined by cell count via a model Z1 Beckman particle counter. 2.5. Transmission Electron Microscopy (TEM) of Cells Exposed to MTX Nanoparticles. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. The medium was changed every third day. When the cells achieved confluence, they were incubated with 10 mL of folate free cellular medium (Invitrogen) containing 300 mg/L L-glutamine, 10% fetal calf serum, 50 IU/mL penicillin, and 50 µg/mL streptomycin. Following 24 h of incubation, MTX nanoparticles were introduced into the culture medium at an iron concentration of 0.1 mg/mL. Control cells were cultured in folate-free medium without nanoparticles. After 1 day in culture with nanoparticles, the cells were washed once with 5 mL of Versene (Invitrogen) and twice with HBSS, followed by detachment with trypsin. The cells were then centrifuged and resuspended in 5 mL of Karnovsky’s fixative for 24 h. Following fixation, the cells were processed in agar and embedded in epoxy for sectioning. Cell sections were stained with osmium tetroxide, lead citrate, and uranyl acetate for transmission electron microscopy (TEM) contrast enhancement. Cell and nanoparticle images were taken using a Phillips 420 TEM microscope operating at 100 kV. 2.6. Intracellular Uptake of Magnetite Nanoparticles. To quantify the cellular uptake of MTX nanoparticles by cancer cells, HeLa and MCF-7 cells were grown in T-75 flasks and rat cardiomyocyte cells (Cell Applications, Inc) on 12-well plates. HeLa and MCF-7 cells were cleaved using 0.25% trypsin-EDTA solution in HBSS and were seeded in 12-well tissue culture plates at a concentration of 106 cells/mL. Prior to the uptake experiments, all cells were cultured in RPMI-1640 folate-free medium in the 12-well plates for 24 h. The MTX nanoparticle stock solution in DI water was sonicated for 4 min and dispersed into RPMI1640 folate-free medium at a concentration of 0.1 mg/mL. The cells were then grown with 2 mL of the MTX nanoparticle medium for 2 h for particle internalization. Following culture with the nanoparticles, the cells were washed once with 1.0 mL of RPMI1640 folate-free medium, twice with 1.0 mL of Versene, and four times with HBSS. The cells were then cleaved with 0.25% trypsin-EDTA and resuspended in 1 mL of PBS supplemented with 10% FBS. A 100 µL aliquot of the cell suspension in PBS was then dispersed in 9.9 mL of Isoton solution, and cells were counted using a Beckman Z1 particle counter. To lyse the cells, 100 µL of concentrated HCl was added to the 900 µL of remaining cell suspension and incubated for 1 h at 70 °C. The resulting intracellular iron concentration was determined by inductively coupled plasmon resonance spectroscopy (ICP).

3. Results and Discussion 3.1. Characterization of Superparamagnetic Magnetite Nanoparticles. The TEM image in Figure 1A shows that the particles as synthesized are well dispersed and have a uniform shape and size distribution. The X-ray powder diffraction pattern shown in Figure 1B for the nanoparticles agrees with the pattern of magnetite nanoparticles listed in ASTM XRD standard card (190629). The nanoparticle size evaluated from the diffraction pattern using the Scherrer formula is about 10 nm, consistent with the TEM estimation shown in Figure 1A. 3.2. Surface Modification of Nanoparticles with Methotrexate. FTIR spectroscopy was used to confirm that MTX was successfully immobilized on the nanoparticles. FTIR spectra of unmodified and MTX modified iron oxide nanoparticles are shown in Figure 2. The unmodified iron oxide nanoparticles show a broad band at 3300 cm-1 indicative of the presence of -OH groups on the nanoparticle surface. For nanoparticles modified with APS, the peaks at 1550 and 1407 cm-1 indicate the presence of the primary amine on the nanoparticle surface. The peak

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Figure 1. (A) TEM image and (B) XRD pattern of superparamagnetic magnetite nanoparticles.

Figure 2. FTIR spectra of (A) unmodified iron oxide nanoparticles, (B) APS-modified nanoparticles, (C) MTX-modified nanoparticles, and (D) free MTX.

at 1100 cm-1 indicates Si-O bonding on the nanoparticle surface. Further, the peak at 2930 cm-1 indicates the -CH stretch present in the APS. Standard MTX shows the characteristic IR absorption peaks of 1644 and 1603 cm-1. Spectra of nanoparticles modified with MTX following APS immobilization show increased absorbance at 1606 cm-1 and appearance of a new peak at 1550 cm-1 indicative of the presence of MTX on the nanoparticle through amide bonds within the MTX structure and amide bonding between APS and MTX. 3.3. Drug Release. The mechanism of releasing MTX from the nanoparticle conjugate inside target cells is similar to folic acid. Figure 3 conceptually illustrates the intracellular trafficking model of the uptake of MTXmodified nanoparticles into target cells. Following their uptake via receptor-mediated endocytosis, nanoparticles are transported to early endosomes. The endosomes then fuse with low pH lysosomes containing proteases which during normal cellular metabolism are responsible for the breakdown of proteins and other exogenous materials brought into the cell. These proteases then cleave the peptide bond between the MTX and the nanoparticle, allowing the MTX to be released from the particle surface inside the target cell. Once the MTX is free from the nanoparticle surface, it may enter the cellular cytosol. It is then assumed that MTX will be free to inhibit dihy-

drofolate reductase and stop the folic acid cycle reducing cellular viability. The number of MTX molecules immobilized on each nanoparticle was quantified using UV-vis absorbance data and the particle concentration data determined by ICP. From this analysis, the average number of MTX molecules per particle for nanoparticles with a 10 nm diameter was determined to be ∼418.9. The release of MTX from the nanoparticle conjugate in simulated lysosomal conditions (i.e., acidic pH and in the presence of proteases) was studied by UV spectroscopy. The amide bonds formed during the surface modification with MTX are between the glutamic acid residues of the MTX molecule and the amino terminal SAM. It was theorized that the proteases found in the lysosomal compartment may be capable of hydrolyzing the peptide bond releasing free MTX into the cellular cytoplasm. To test this theory, the nanoparticles were incubated with crude protease solution at alternate pH conditions to facilitate hydrolysis, similar to conditions found in the lysosome. Results of MTX release from the nanoparticle surface under lysosomal conditions as measured by UV absorbance at 304 nm are shown in Figure 4. Using the standard protease solution in buffer as a blank, it is seen that UV absorption increases with decreasing pH. The greatest UV absorption occurs at pH 2 which is most likely due to the greatest concentration of active protease at this pH. Studies were conducted from 12 to 72 h to gain an understanding of the kinetics of the MTX release from the nanoparticles. However, the data suggest that MTX release occurred prior to the 12 h interval, indicating that the protease readily cleaves the peptide bond. This study also indicates that there is some release of MTX from the nanoparticle at pH 4-7.44, which may be due to the presence of a small amount of active protease capable of cleaving the amide bond of the nanoparticle. 3.4. Drug Release Efficacy. Once the release of MTX from the nanoparticle surface had been verified, the effectiveness of superparamagnetic nanoparticles to serve as drug carriers was evaluated in vitro. Human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa) were grown in the presence of free MTX and MTXmodified nanoparticles. Figures 5 and 6 show the cellular viability, in terms of surviving fraction of MCF-7 and HeLa cells grown in the presence of MTX nanoparticles and soluble MTX over time. After 120 h in culture, all cells demonstrated a notable downward trend in viability indicative of the cytotoxicity of both the MTX nanoparticles and soluble MTX over time. In both the MCF-7 cells and HeLa cells, the MTX-grafted nanoparticles and the soluble

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Figure 3. Schematic representation of the intracellular uptake of MTX modified nanoparticles into breast cancer cells.

Figure 4. Methotrexate release from nanoparticles in simulated lysozomal pH conditions, as measured by UV absorbance. Figure 6. Reduction in cellular viability for HeLa cells as a function of time in culture.

Figure 5. Reduction in cellular viability for MCF-7 cells as a function of time in culture.

MTX show a similar reduction in cell viability establishing the ability of the cells to cleave MTX in the lysosomes, thus allowing the freed MTX to reduce cellular viability.

In addition, a dose response for the MTX-grafted nanoparticles was elucidated through the viability results. For the MCF-7 cells, concentrations of MTX grafted nanoparticles at or above 0.025 mg/mL Fe showed a similar reduction in cellular viability. HeLa cells demonstrated a similar dose response; namely, for concentrations of MTX-grafted nanoparticles at or higher than 0.025 mg/ mL, the cells showed a statistically equivalent reduction in cellular viability. It should be noted that for the HeLa cells, 2 µg/mL of soluble MTX demonstrated a slightly higher cytotoxicity than the MTX-grafted nanoparticles. MTX is an analogue of folic acid, which contains an amino group at position 4 in the pteridine ring leading to a critical change in the structure of folic acid allowing for its tight binding to dihydrofolate reductase (DHFR), a critical enzyme in the folic acid cycle and key to regulating homeostasis. When delivered in high enough doses, MTX causes the toxic buildup of cellular intermediates, reducing cellular viability and ultimately causing cellular mortality.24 3.5. Cellular Uptake of Nanoparticle Conjugates. Cellular uptake by target cells measures the specificity of a nanoparticle conjugate for the target cells. ICP

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Figure 7. Preferential uptake of nanoparticle-MTX conjugate by breast cancer cells compared to cardiomyocytes.

spectroscopy was utilized to quantify the cellular uptake of nanoparticle conjugates into HeLa and MCF-7 cells in terms of iron concentration. The results shown in Figure 7 demonstrate the specificity of the MTX-grafted nanoparticles for the human folate receptor and reduced folate carrier. To demonstrate the specificity for cancer cells, rat primary cardiomyocyte (heart muscle) cells were used as a negative control. Following 2 h in culture, the HeLa cells demonstrated an uptake approximately 10 times higher than the negative control (Figure 7), while the MCF-7 cells demonstrated an uptake approximately 20 times higher than the primary cardiomyocyte cells in culture. This might be due to the high metabolic activity

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of HeLa and MCF-7 cells leading to the overexpression of the folate receptor on the cell surface and the low metabolic activity of cardiomyocyte cells. Both MCF-7 and HeLa cells have also been shown to be positive for the reduced folate carrier.31,32 The uptake of MTX is known to have at least two different carrier systems which include (1) the reduced folate carrier for which MTX and reduced folates have a higher affinity than folic acid and (2) the folate receptor for which folic acid has a higher affinity than that of MTX. Folic acid and MTX themselves are low molecular weight targeting molecules which have little ability to pass through the cellular membrane nonspecifically.13,24,33 To further confirm that the MTX-nanoparticle conjugates were indeed internalized by the target cells rather than simply bound to the surface of the cells, and to visualize the location of the nanoparticles inside the cells after the internalization, TEM images were taken both on MCF-7 and HeLa cells that were cultured with MTX nanoparticles and, for comparison, on their corresponding cells that were cultured without MTX nanoparticles. Figure 8 shows the images of MTX-nanoparticle treated MCF-7 (B) and HeLa (D) cells, and comparative untreated MCF-7 (A) and HeLa (C) cells. This comparison provides evidence that a large number of MTX-nanoparticle conjugates accumulated in both MCF-7 and HeLa cells treated with MTX-nanoparticle conjugates and appeared as black dots scattered in the cell cytoplasm but not in the nuclei. A closer look at the images reveals that the majority of the internalized MTX-nanoparticle conjugates resided in the lysosomes of the cells (insets of parts B and D of Figure 8), which supports the intracellular trafficking

Figure 8. TEM images of (A) MCF-7 cells, (B) MCF-7 cells cultured with MTX-nanoparticle conjugates, (C) HeLa cells, and (D) HeLa cells cultured with MTX-nanoparticle conjugates.

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model illustrated in Figure 3. Similar observations have been reported in the study of intracellular trafficking of polymeric and liposome nanoparticles as therapeutic drug carriers.34,35 4. Conclusions Superparamagnetic magnetite nanoparticles were successfully modified with methotrexate (MTX) and characterized with FTIR. Following 120 h in cell culture medium, MTX-modified nanoparticles showed a similar reduction in cellular viability in both human breast cancer (MCF-7) and human cervical cancer (HeLa) cells compared to soluble MTX at serum levels cited in the literature.36 TEM results of both MCF-7 and HeLa cells grown in the presence of 0.1 mg/mL MTX nanoparticles after 24 h show the successful internalization of MTX-modified nanoparticles into lysosomes, confirming the method of cellular internalization and MTX release. To quantify the uptake of MTX-modified nanoparticles by target cells, MCF-7, HeLa, and rat cardiomyocyte cells were grown in the (31) Trippett, T. M.; Garcia, S.; Manova, K.; Mody, R.; Cohen-Gould, L.; Flintoff, W.; Bertino, J. R. Localization of a human reduced folate carrier protein in the mitochondrial as well as the cell membrane of leukemia cells. Cancer Res. 2001, 61, 1941-1947. (32) Wang, Y.; Zhao, R.; Goldman, I. D. Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier. Clin. Cancer Res. 2004, 10, 6256-6264. (33) Jackson, R. C. Biological effects of folic acid antagonists with antineoplastic activity. Pharmacol. Ther. 1984, 25, 61-82. (34) Panyam, J.; Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Delivery Rev. 2003, 55, 329-347. (35) Tachibana, R.; Harashima, H.; Shono, M.; Azumano, M.; Niwa, M.; Futaki, S.; Kiwada, H. Intracellular regulation of macromolecules using pH-sensitive liposomes and nuclear localization signal: qualitative and quantitative evaluation of intracellular trafficking. Biochem. Biophys Res. Commun. 1998, 251, 538-544. (36) Metzger, R.; Deglmann, C. J.; Hoerrlein, S.; Zapf, S.; Hilfrich, J. Towards in-vitro prediction of an in-vivo cytostatic response of human tumor cells with a fast chemosensitivity assay. Toxicology 2001, 166, 97-108.

Kohler et al.

presence of MTX nanoparticles. Cells expressing the human folate receptor (MCF-7 and HeLa) took up a significantly higher concentration of MTX grafted nanoparticles than healthy primary cells (rat cardiomyocytes). These results indicate that MTX-grafted nanoparticles are taken up into cells to a great degree by the human folate receptor. This receptor is overexpressed on many types of cancer cells, which may provide a convenient route for cellular targeting. This drug delivery system is dependent on the release of the MTX molecule within the lysosomal compartment. Model studies demonstrated the release of MTX via peptide bond cleavage in the presence of crude protease and at low pH. These results suggest that if this system is delivered intravenously, MTX immobilized on nanoparticles will be released largely in lysosomes inside target cells following the particle internalization where pH is low and many enzymes exist. Covalently binding MTX onto nanoparticles reduces the possibility of MTX release until the nanoparticle conjugate has been internalized into the tumor cell and cleaved by intracellular enzymes, which may minimize the drug side effects to normal cells. This drug delivery system may allow real-time monitoring of drug delivery by MRI due to the superparamagnetic nature of the system. Acknowledgment. This work was supported by University Washington Royal Research Fund and TaiwanIndustrial Technology Research Institute (ITRI). Nathan Kohler acknowledges the fellowship from the Joint Institute for Nanoscience funded by the Pacific Northwest National Laboratory (operated by Battelle for the U.S. Department of Energy). Also acknowledged are the University of Washington Engineered Biomaterials (UWEB) Center for providing cell culture facilities, Dr. Andrew Hallahan for his thoughtful conversations on methotrexate and cellular biology, and Dr. Julian Simon for providing the MCF-7 cell line. LA0503451