Bioconjugate Chem. 1995, 6, 203-210
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Preparation and in Vitro Evaluation of Magnetic Microsphere-Methotrexate Conjugate Drug Delivery Systems Damayanthi Devineni,+ Charles D. Blanton,s a n d J a m e s M. Gallo*,+ Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and College of Pharmacy, University of Georgia, Athens, Georgia 30602. Received September 30, 1994@
Magnetic microsphere-methoxtrexate (MM-MTX) conjugates prepared by several different methods were analyzed for their suitability for in vivo use. MM-MTX were prepared by the following methods: (A) reaction of MTX with poly(ethy1ene glycol) 1500 (PEG) to form a poly(ethy1ene glycol)methotrexate conjugate (PEGMTX) which was then added to a ferrous/ferric ion salt solution to give MM-MTX I; (B) reaction of ferroudferric ion salts with PEG to give a ferromagnetic polymer complex which was then coupled with MTX to give MM-MTX 11;(C) MM-MTX IIIA were prepared by reacting MTX with amino-terminated magnetic microspheres, commercially available, in the presence of l-ethyl3,3-bis(methylamino)propylcarbodiimide(EDCI); (D) reaction of aminohexanol with di-tert-butyl dicarbonate to form a n [N-(tert-butoxycarbonyl)aminolhexanol(t-Boc-AH),which was then coupled with MTX in the presence of 1,3-dicyclohexylcarbodiimideand 4-pyrrolidinopyridine to give a t-BocAH-MTX conjugate, which was then saturated with hydrogen chloride to give a n aminohexanolmethotrexate (AH-MTX) conjugate. MM-MTX IIIB were then prepared by reacting AH-MTX with carboxyl-terminated magnetic microspheres, commercially available, in the presence of EDCI and 4-(dimethylamino)pyridine. The identity of MTX conjugates was confirmed using ultraviolet, infrared, and nuclear magnetic resonance spectroscopy. Drug content of the magnetic microsphere-methotrexate conjugates as determined by HPLC was 0.45% (w/w), 4.0% (w/w), and 6.3% (w/w)MTX for MMMTX I, MM-MTX 11, and MM-MTX IIIB, respectively. In vitro stability studies of MM-MTX in rat plasma revealed that approximately 97% (w/w) (MM-MTX I), 74% (w/w) (MM-MTX II), and 11% (w/w) (MM-MTX IIIB) of MTX was released from MM-MTX over a 24 h period. The ability to increase drug loading, compared to matrix microsphere systems, via an ester linkage offers another dimension to tumor delivery of chemotherapeutic agents.
Methotrexate, N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylaminolbenzoyllglutamicacid, MTX, is used for the treatment of lymphoreticular and other malignancies including metastic and recurrent primary brain tumors (1-3). A major drawback with MTX therapy is its poor ability to cross the blood-brain barrier (BBB) (4).Attempts have been made to improve transport of MTX across the BBB by (i) administering MTX directly into a brain tumor and (ii)osmotic BBB disruption techniques (5-6). Various macromolecules have been shown to localize in tumor cells in vivo and were suggested as possible carriers for MTX (7-9). A typical depot effect and prolonged plasma concentrations were demonstrated by Chu and Whiteley for MTX linked to albumin and dextran derivatives. Shen and Ryser (10)have reported a n increased cellular uptake in vitro in MTX-resistant cells, using a poly@-lysine)-MTX conjugate. Ghosh et al. (11)have reported an immunoglobin-MTX conjugate for targeting the drug to tumor-associated antigens. However, the use of drug-macromolecular conjugates as a vehicle for targeting drugs relies largely on the ability of the carrier to achieve either cell or organ specificity. In recent years the concept of using small colloidal particles for the selective drug delivery has been explored using a variety of different physical systems, such as liposomes and polymeric microspheres or nanoparticles (12-17). Although the surface properties of the colloids may alter the systemic distribution, intravascular ad-
* To whom correspondence and reprint requests should be addressed. Fox Chase Cancer Center. University of Georgia. Abstract published in Advance ACS Abstracts, February 15, 1995. +
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1043-180219512906-0203$09.00/0
ministration of such carriers results in their predominant uptake by the reticuloendothelial system. Magnetic microspheres were therefore designed to avoid rapid reticuloendothelial clearance that is problematic for other particulate carriers (18-19). Magnetic microspheres are usually injected into the arterial supply of the target organ to take advantage of first-pass organ extraction. Because these spheres are 1pm or smaller in diameter, they are able to pass through target capillaries, prior to systemic clearance. As the magnetic particles traverse the target organ capillaries, an external magnetic field can retain the particles in small arterioles and capillaries. Retained particles may undergo extravascular uptake which could ultimately lead to intracellular (i-e.,tumor cell) drug uptake. The original magnetic albumin microspheres contained approximately 1.0% (w/w) adriamycin. Widder et al. (19) were the first to demonstrate the utility of magnetic albumin microspheres (MM-ADR) in animal tumor models. Significantly greater responses, both in terms of tumor size and animal survival, were achieved with MM-ADR than adriamycin alone. Gupta et al. (20) demonstrated that the efficacy of magnetic microspheres in the targeted delivery of incorporated drug is predominantly due to the magnetic effects and not due to the particle’s size or nonmagnetic holding. The ultrastructural disposition of adriamycin-associated magnetic albumin microspheres was also demonstrated in normal rats by Gupta et al. (21). The transmission electron micrographs showed extravascular transport of microspheres as early as 2 h after dosing and were observed and remained in the extravascular tissue for up to 72 h. Since the drug delivery device was retained in the vascular endothelium of the target tissue for up to 72 h,
0 1995 American Chemical Society
204 Bioconjugate Chem., Vol. 6,No. 2, 1995
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h) for 24 h and then lyophilized and stored in a desiccator. The above procedure was repeated without adding EDCI to the reaction mixture. Step I I Preparation of M M - M T X I Conjugates. MMMTX I conjugates were prepared by mixing 15 mL of PEG-MTX conjugate (500 mg) with 2 mL of a n aqueous solution containing 300 mg of ferric chloride'and 120 mg of ferrous chloride. While stirring, the mixture was adjusted to pH 8.0-8.5 by the dropwise addition of 30% (w/v) aqueous ammonia solution. After the reaction the resulting magnetic material was kept under the electromagnet and was washed four times with 25 mL of water and then dried under nitrogen gas a t room temperature. The product was then stored in a desiccator until further use. Method I I Synthesis of MM-MTX II. Step I: Preparation of Ferromagnetic Polymer Complex. Ferromagnetic polymer complex was prepared by mixing 10 mL of 50% (w/w) poly(ethy1ene glycol) 1500 with 2.5 mL of an aqueous solution containing 375 mg of ferric chloride and 150 mg of ferrous chloride. This mixture was stirred at 500 rpm, the pH adjusted to 8.0-8.5 by the dropwise addition of 30% (w/v) aqueous ammonia solution, and the EXPERIMENTAL SECTION mixture heated to 60 "C for 10 min to remove excess Materials. Methotrexate was a gift from Lederle ammonia. The resultant ferromagnetic polymer complex Laboratories (Pearl River, NY). 6-Amino-1-hexanol,tertwas washed four times with 50 mL of water, being butyl alcohol, di-tert-butyl dicarbonate, 4-pyrrolidinopyseparated by a magnet after each wash. The resultant ridine (4-PP), dicyclohexylcarbodiimide (DCC), 44dimeferromagnetic polymer complex was sonicated for 2 min thy1amino)pyridine (4-DMAP), and hydrochloric acid a t 200 W with a n ultrasonic probe and then passed (anhydrous) were obtained from Aldrich Co. (Milwaukee, through a microfluidizer for 3 min to reduce the particle WI). 1-Ethyl-3-[3-(dimethylamino)propyllcarbodiimide size. The ferromagnetic polymer complex was readily (EDCI), ammonium hydroxide, ferrous chloride, and separated from the colloidal solution in a magnetic field ferric chloride were obtained from Sigma Chemicals (St. of 6000 G in 2-5 min. It was washed four times with 50 Louis, MO). Poly(ethy1ene glycol) 1500 was purchased mL of water, being separated by a magnet after each from Scientific Polymer Products, Inc. (Atlanta, GA). wash. The resultant magnetic colloid was lyophilized and Spectrapor cellulose dialysis tubing (MW cutoff 1000) was then stored in a desiccator. obtained from Fisher Scientific (Atlanta,GA). CarboxylStep I I Chemical Modification of Methotrexate with terminated biomag 4125 and amine-terminated biomag the Ferromagnetic Polymer Complex. MM-MTX I1 con4100 were purchased from Advanced Magnetics, Inc. jugate was prepared by reacting 6 mL of a methotrexate (Cambridge, MA). All analytical-grade reagents and solution (10 mg/mL) in the presence of EDCI (15 mg/mL) HPLC-grade solvents were obtained from J. T. Baker, dissolved in 4 mL of phosphate-buffered saline, pH 7.4 Inc. (Phillipsburg, NJ). (PBS), with 2 mL of ferromagnetic polymer complex (22.5 Equipment. An electromagnet was purchased from mg/mL in PBS) prepared as described in step I. The Applied Magnetics Laboratory (Baltimore, MD). The reaction mixture was sonicated in a water bath a t room ultrasonic water bath was a Bransonic 220 (Danbury, temperature for 10 min to yield a homogeneous system. CT); the ultrasonic probe was a Branson sonifier (WestThe mixture was stirred for 3 h a t room temperature and bury, N Y ) . A Model 110s microfluidizer obtained from stored overnight a t 4 "C and then purified by dialysis Microfluidics International Corp. (Newton, MA) was used using deionized distilled water (2000 mL exchanged every to reduce the particle size. A Nicomp submicron particle 1 2 h) for 24 h. Following dialysis, the contents of the sizer model 370 (Santa Barbara, CA) was used for dialysis bag were centrifuged a t 2000 rpm for 7 min, and particle size determinations. Ultraviolet spectra and the supernatant was decanted. The pellet, representing infrared scans were obtained with a Beckman Model DUMM-MTX 11, was washed four times with 50 mL of 70 spectrophotometer (Fullerton, CAI and a Nicolet 205 water and then dried under nitrogen a t room temperaFT-IR spectrometer (Norwalk, CT), respectively. The ture and stored in a desiccator. The above procedure was HPLC system consisted of a Waters (Milford, MA) 717 repeated without adding EDCI to the reaction mixture. pump, Lambda Max 486 variable wavelength detector, Method IIIA: Synthesis of MM-MTX IIIA. Amineand an Alltech (Deerfield, IL) Hypersil CIS reversed-phase terminated magnetic microspheres [about 240 pmol of column. amine groups per gram of microsphere (MM')] were Synthesis of Magnetic Microsphere-Methotrexsupplied in distilled water a t a concentration of 50 mg/ ate Conjugate (MM-MTX) Drug Delivery Systems. mL. One milliliter of M M was transferred to a scintilMethod I Synthesis of MM-MTX I . Step I Synthesis lation vial, 10 mL of pyridine buffer (pyridine buffer was of Poly(ethy1ene glycol) 2500-Methotrexate Conjugates prepared by dissolving 0.8 mL of pyridine in 1L of water (PEG-MTX). PEG-MTX conjugates were synthesized and adjusted to pH 6.0 with hydrochloric acid) was added, in a biphasic reaction by mixing 10 mL of poly(ethy1ene and the contents were shaken vigorously. The vial was glycol) 1500 (50% w/w) with 8 mL of methotrexate kept under an electromagnet with a field of 6000 G and solution (10 mg/mL) in the presence of EDCI (17.5 mgl the contents were aspirated, leaving the microspheres as mL) dissolved in 4 mL of PBS. The reaction mixture was a wet cake on the container wall. This washing procestirred for 3 h a t room temperature and stored overnight dure was repeated with three more additions of coupling a t 4 "C. The product was purified by dialysis using buffer. deionized distilled water (2000 mL exchanged every 12
it was suggested that the microspheres may act as a depot from which the drug is released. The disposition of magnetic microsphere drug delivery systems with brain tumors has been a focus of our labooratory (18). It has been demonstrated in normal rats that a magnetic cationic polysaccharide microsphere system, containing the anticancer drug oxantrazole, significantly increased total brain oxantrazole concentration compared to a conventional administration of oxantrazole. However, in this system and other matrix microsphere devices, only a small percentage (1-2% w/w) of the drug is physically entrapped, and drug release may be fast possibly preventing significant quantities of drug from reaching tumor cells. Low drug entrapment may limit the optimal delivery of cytotoxic drugs due to the potentially large amounts of carrier required. On the contrary, if MTX is covalently attached to a magnetic carrier, then high drug loading may be achieved and drug release may be prolonged and controlled. The objectives of this investigation, therefore, were to synthesize magnetic microsphere (MM)-methotrexate (MTX) conjugated systems and analyze their suitability for in vivo use.
Magnetic Microsphere-Methotrexate Conjugates MM-MTX IIIA was synthesized in a biphasic reaction by mixing 2 mL of MTX solution (8 mg/mL in PBS) in the presence of EDCI (40 mg) dissolved in 5 mL of coupling buffer with 1 mL of M M (10 mg/mL). The reaction mixture was sonicated in a water bath a t room temperature for 5 min to yield a homogeneous system and then stirred a t 475 rpm for 3 h, respectively. The reaction mixture was kept under the electromagnet to separate the MM-MTX IIIA. The unreacted MTX was aspirated, and 15 mL of wash buffer (prepared by dissolving 1.21 g of Tris, 8.7 g of sodium chloride, 1 g of bovine serum albumin, 1 g of sodium azide, and 0.37 g of ethylenediaminetetraacetic acid in 1 L of water, adjusted the pH to 7.4) was added to the MM-MTX IIIA. The contents were shaken vigorously and separated magnetically, and the unreacted drug was aspirated. This washing procedure was repeated for a total of three times prior to the storage of MM-MTX IIIA as a suspension in 10 mL of wash buffer a t 4 "C. Method IIIB: Synthesis of MM-MTX IIIB. Step I: Preparation of N-(tert-Btuoxycarbonyl)-6-amino-1 -hexanol (t-Boc-AH). In a 1 L round-bottomed flask containing sodium hydroxide (4.4 g) in 110 mL of water was added 10 g of 6-amino-1-hexanol while the mixture was stirred a t ambient temperature, and the resulting mixture was then diluted with 75 mL of tert-butyl alcohol. To the wellstirred clear solution, 22.5 g of di-tert-butyl dicarbonate was added dropwise within 10 min. A white precipitate appeared during addition of the di-tert-butyl dicarbonate. M e r a short induction period, the temperature increased to 30-35 "C. The reaction was brought to completion by further stirring overnight a t room temperature. At this time, the clear solution reached a pH of 7.5-8.5. The reaction mixture was extracted two times with 50 mL of ether followed by three extractions of the combined ether phases with 100 mL of saturated aqueous sodium bicarbonate solution. The combined aqueous layers were acidified to pH 1-1.5 by careful addition of a solution of 22.5 g of potassium hydrogen sulfate in 150 mL of water. The acidification was accompanied by copious evolution of carbon dioxide. The turbid reaction mixture was then extracted with two 50 mL portions of ether. The combined organic layers were washed with 25 mL of water, dried over anhydrous sodium sulfate, and filtered. The solvent was removed under reduced pressure using a rotary evaporator. The yellowish oil that remained was treated with 150 mL of hexane and placed in a freezer (-20 "C) overnight. A white precipitate was obtained from the yellow oil by collection on a Buchner funnel. Step 11 Direct Esterification of t-Boc-AH with Methotrexate. A 100 mL flask was charged with 80 mg of MTX in 5 mL of dimethylformamide (DMF), 217 mg of t-BocAH, and 50 mg of 4-pyrrolidinopyridine. The solution was stirred and cooled in a n ice bath to 0 "C while 133 mg of dicyclohexylcarbodiimide was added over a 5 min period. After a further 5 min a t 0 "C the ice bath was removed and the reaction mixture was stirred for 24 h a t room temperature. Dicyclohexylurea which had precipitated was removed by filtration through a fritted Buchner funnel, and the filtrate was diluted with 50 mL of methylene chloride. The filtrate was then washed with two 25 mL portions of 0.5 N hydrochloric acid and two 25-mL portions of saturated sodium chloride solution. During this procedure some additional dicyclohexyl urea was precipitated, which was removed by filtration of both layers to facilitate their separation. The organic solution was dried over anhydrous sodium sulfate and concentrated with a rotary evaporator. The concentrate was distilled under reduced pressure to give t-Boc-AH-MTX. Step III Removal of t-Boc Group from t-Boc-AH-MIX.
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A solution of t-Boc-AH-MTX (50 mg) in 10 mL of methylene chloride, cooled to 0 "C, was saturated with hydrogen chloride by passing the anhydrous gas through the solution with stirring for 20 min. The turbid reaction mixture was left for a n additional 1 h a t room temperature to precipitate a light-brown crystalline product, AH-MTXSHCl, that was removed by filtration. Step N : Free Ester. AH-MTX.HC1 (30 mg) was dissolved in 5 mL of DMF a t 0 "C, and 0.5 mL of anhydrous triethylamine was added in small portions. The mixture was stirred for 20 min a t 0 "C and filtered to remove triethylamineHC1. The product, AH-MTX, was precipitated with 50 mL of diethyl ether, filtered, washed extensively with water, and dried. Step V Synthesis of MM-MTX IIIB. Carboxylterminated magnetic microspheres (Biomag 4125) were supplied in distilled water a t a concentration of approximately 20 mg/mL and have about 4.8 pmol of carboxyl groups per mL (240 pmollg of biomag). One mL of the magnetic microspheres (MM) was transferred to a 20 mL scintillation vial, to which 10 mL of PBS was added, and the contents were shaken vigorously. The vial was placed between the poles of an electromagnet (6000 GI and the liquid aspirated, leaving the MM as a wet cake on the container wall. This washing procedure was repeated with three more additions of 10 mL of PBS. MM-MTX IIIB was prepared by reacting 1 mL of MM (20 mg/mL) in the presence of EDCI (40 mg) and 4-(dimethy1amino)pyridine (10 mg, 4-DMAP) dissolved in 3 mL of water with 3 mL of AH-MTX solution (10 mg). The reaction mixture was sonicated in a water bath a t room temperature for 5 min to yield a homogeneous system and then stirred for 3 h a t ambient temperature. After 3 h, the reaction mixture was placed in the electromagnet and the MM-MTX IIIB was separated magnetically. The unreacted MTX was aspirated, and 15 mL of wash buffer was added to the MM-MTX IIIB. The contents were shaken vigorously, separated magnetically, and the unreacted drug was aspirated. This washing procedure was repeated for a total of three times. The supernatants collected from each washing step were analyzed for MTX content using HPLC. The MM-MTX IIIB was stored as a suspension in 10 mL of wash buffer at 4 "C. Characterizationof MTX Conjugates. The identity of the conjugates was confirmed using thin-layer chromatography (TLC), ultraviolet (W),infrared (IR), and nuclear magnetic resonance (NMR) spectroscopy in combination with the control reaction, conducted in the absence of the cross-linking agent, EDCI. Thin Layer Chromatography. Precoated silica gel plates in saturated chambers were used with the solvent system methanol/acetone/ethyl acetate (1O:lO:l). Absorption was observed a t 254 and 366 nm. A ninhydrin spray reagent was used for t l e detection of free amino groups in AH-MTX conjugates. UVSpectroscopy. W spectra of PEG, t-Boc-AH, MTX, and its conjugates were recorded in PBS/methanol between 200 and 400 nm. IR Spectroscopy. IR spectra of PEG, MTX, magnetite, ferromagnetic polymer complex, PEG-MTX, t-Boc-AH, t-Boc-AH-MTX, AH-MTX conjugates, MM-MTX (I and 11) conjugates, a physical mixture of PEG and MTX, a physical mixture of ferromagnetic polymer complex and MTX, a physical mixture of PEG-MTX conjugates and magnetite, were recorded using potassium bromide (Kl3r) disks. KBr disks were prepared by grinding a sample (2 mg) with KBr powder (210 mg), placing the mixture between a punch and die, and applying a pressure of about 50 000 psi.
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PEG (KBr): 3432,2880,1653,1456,1352,1252,1105, 951, 841, 581 cm-l. MTX (KBr): 3357,1647,1605,1541,1507,1449,1404, 1368, 1254, 1209, 1101, 941, 833, 768, 745, 581 cm-l. Ferromagnetic polymer complex (KBr): 3430, 2917, 1653, 1558, 1509, 1456, 1400, 1352, 1300, 1252, 1100, 951, 590 cm-'. PEG-MTX conjugate (KBr): 3420, 2888, 2741, 2695, 1968, 1468, 1458, 1414, 1359, 1344, 1281, 1242, 1150, 1117, 1061, 964, 947,843, 530, 509 cm-'. MM-MTX I (KBr): 3144, 1404, 1111, 949, 843, 583 cm-l. MM-MTX I1 (KBr): 3436, 1636, 1559, 1507, 1457, 1208, 1090, 577 cm-l. t-Boc-AH (KBr): 3450, 3368, 3100, 2935, 2850, 1685, 1523, 1350, 1300, 1250, 1173, 1050, 1000, 600 cm-'. t-Boc-AH-MTX (KBr): 3450, 3350, 3200, 2950, 2800, 1701, 1650, 1600, 1550, 1525, 1450, 1350, 1250, 1200, 1150, 1100, 850, 800 cm-'. AH-MTX (KBr): 3400,3150,2950,2850,1729,1649, 1600, 1550, 1500, 1400, 1200 cm-l. NMR Spectroscopy. 'H NMR spectra of t-Boc-AH (CDC13),t-Boc-AH-MTX, and AH-MTX-HC1 [lo% PHs) DMSO] were recorded with a General Electric QE 300 MHz spectrometer. t-Boc-AH (lH-NMR, CDC13): 6 1.4 ppm (s, 9H, -C(CH3)3), 1.2-1.6 (m, 8H, 4 CHZ groups), 2.4 (s, lH, NH), 3.1 (m, 2H, -CH&HzNH-), 3.6 (t, 2H, -CHzCHzOH), 4.7 (b, lH, OH). t-Boc-AH-MTX (lH-NMR, DMSO-&): 6 1.3 ppm (s, 9H, -C(CH&), 1.2-1.8 (m, 8H, CHZgroups at positions 2-5 of hexane moiety), 1.9-2.3 (m, 4H, -CHZCHz- of glutamate), 3.2 (s, 3H, N10CH3 of MTX), 3.9-4.0 (m, 2H, -CHzOOC-), 3.8 (b, l H , -CHflHCOO-), 4.8 (s, 2H, CHZa t position 9 of MTX), 6.6 (b, 2H, 2 or 4 NHz of MTX), 6.7-6.9 (m, 2H, aromatic protons of MTX), 7.4-7.5 (b, 2H, 2 or 4 NHz of MTX), 7.6-7.7 (m, 2H, aromatic protons of MTX), 8.1-8.2 (b, lH, -CONH of glutamate), 8.6 (s, lH, CH a t position 7 of MTX), 10.7 (s, -COOH). AH-MTX ('H-NMR, DMSO-&): It is essential identical to t-Boc-AH-MTX but without the characteristic peak a t 6 1.3 ppm for the tert-butyl group (t-Boc). Particle Size. The size distribution of the MM-MTX conjugates (1-111) were determined with a submicron particle sizer at 23 "C assuming the viscosity and refractive index to be 0.933 centipoise and 1.333,respectively. About 2 mg of the conjugate was suspended in 1 mL of PBS and sonicated in an ultrasonic water bath for 2 min to prevent the formation of aggregates and then introduced into the particle sizer with an autodiluter. Two types of particle size analyses, a Gaussian or nicomp, were conducted and expressed as the volume-weighted distribution of particle diameters. Unlike the Gaussian analysis, the proprietary (nicomp) distribution analysis does not assume any particular shape for the particle size distribution. Drug Loading. MM-MTX conjugate (1-111, 4 mg) was suspended in 10 mL of PBS and sonicated in an ultrasonic water bath for 2 min. The contents were shaken vigorously and separated magnetically, and then the PBS was aspirated leaving the conjugate as a wet cake on the container wall. The washing procedure was repeated with two more additions of PBS. After the final wash, the conjugate was digested in 10 mL of 0.02 N NaOH for 3 h a t 50 "C to release MTX and then centrifuged a t 10 000 rpm for 10 min. An aliquot of the supernatant was analyzed by HPLC for MTX. Chromatographic separation was achieved using a flow rate of 1.5 m u m i n on a n Alltech Hypersil ODS Cl* reversedphase column with a mobile phase consisting of 20% (v/
v) methanol in water with 40 mM dibasic potassium phosphate, pH 7.0. UV detection was made a t 313 nm. In Vitro Release Study. The release of MTX from MM-MTX conjugates was monitored for 24 h in various test media (pH 7.4 buffer, pH 5.6 citrate buffer, rat plasma, and brain homogenate) a t 37 "C. The conjugates (4 mg) were suspended in 2 mL of the test medium and sonicated on a n ultrasonic water bath for about 2 min to yield a homogeneous system. The microspheres were placed in the test medium within dialysis tubing and dialyzed against 100 mL of PBS. Aliquots of PBS were collected periodically and analyzed for MTX using HPLC as described above. RESULTS AND DISCUSSION
Fine ferromagnetic particles have been coated with poly(ethy1ene glycol) (22)lamino or carboxyl groups to permit the covalent attachment of proteins, glycoproteins, and other ligands with the retention of biological activity. Ferromagnetic particles have also been used for various in vivo applications such as a tracer of blood flow, in radioneuclide angiography, and for use in inducing clotting in arteriovenous malformations. Zimmermann and Pilwat (23) were the first ones to propose that erythrocytes or lymphocytes containing fine ferromagnetic particles could be propelled to a desired site by an external magnetic field. It was demonstrated by Freeman et al. (24) that iron particles could pass through capillaries when properly conditioned and later confirmed by Meyers et al. (25)who showed that iron particles could be magnetically controlled in the vascular system of experimental animals. There have been no previous investigations to examine the ability of magnetic microspheres to deliver drugs to brain tumors; however, there have been two investigations in normal rats (18,26). Following administration of magnetic microspheres containing oxantraxole, the brain contained 100-400 times higher oxantrazole levels than those obtained after the solution dosage form, indicating the successfulness of drug delivery via magnetic microspheres. It was evident from these studies that under the proper conditions, magnetic microspheres were capable of enhancing total brain concentrations. Coupling MTX to a carrier must not result in permanent loss of structural features required for drug activity (e.g., an intact pteridine moiety). For this reason, logical linkage groups are the free carboxyl groups of the glutamate moiety and amino groups of the linker. The unusually high affinity of MTX for dihydrogen folate reductase (DHFR) depends upon its pteridine moiety with an amino group in position 4 (27). The glutamate residue a t the opposite end of the molecule has been modified to some extent without seriously impairing this strong interaction (28-31). Przybylski et al. (32) described the synthesis of polymeric [poly(L-lysine), poly(iminoethy1ene), poly(viny1 alcohol), and carboxymethyl cellulose] derivatives of MTX and were characterized by thin layer chromatography, UV, IR, and NMR spectra. Yeshwant et al. (33)described the synthesis and characterization ( U V , IR, and control reaction, in the absence of crosslinking agent EDCI) of an chitosan-MTX conjugate designed specifically to interact with the vascular endothelium and cross the BBB. The simplest way of coupling MTX to a free hydroxyl/ amino group of a carrier molecule would be through nonselective activation of the carboxyl groups in its glutamic acid moiety by the carbodiimide method. This method may lead to formation of two structural isomers, in which MTX is linked either through the a-carboxyl or the y-carboxyl group of its glutamic acid moiety (34,351.
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Magnetic Microsphere-Methotrexate Conjugates
I:
a .COORI
I
a, 2.0 CONJUGATE PEG-MTX
AH-MTX
-(CHA-OH
2a 2b
-(CH2)6-m2
- CH2 CNHI
Figure 1. Structures of PEG-MTX and AH-MTX conjugates. Synthesis of MM-MTX I and I1 was attempted to obtain a small magnetic colloid that had a high drug loading and controllable release rate of drug. In both methods I and 11, the reaction of ferrous (Fez-) and ferric (Fe3+)ions a t pH 8.0-8.5 in the presence of PEG-MTX or PEG yielded a colloidal solution of PEG-MTX or PEGcoated ferromagnetite. This adsorption may be attributed to the formation of a complex between the hydroxyl group of P E G - M W E G and iron on the magnetite particles. Cross-linking between the carboxyl group (a or y ) of MTX and the hydroxyl group of PEG or PEG-coated magnetite particles using a water soluble carbodiimide could result in the formation of esters la or lb (34, 35) (see Figure 1). In order to simplify the synthetic procedure of producing magnetic particles, commercially available amino or carboxyl-terminated magnetic microspheres were utilized in method I11 (A and B). Initially, direct covalent linkage (by the EDCI method) between MTX and magnetic microspheres coated with amino groups, MM', was attempted (MM-MTX IIIA). That this method yielded a covalent conjugate (MM-MTX IIIA) was confirmed with the control reaction, conducted in the absence of the cross-linking agent, EDCI. The MTX content of MMMTX IIIA as determined by HPLC was found to be 4.29% (0.5 h), 5.14% (1h), 7.19% (2 h), 8.5% (3 h), and 6.3%(17 h) depending on the reaction time. However, when MMMTX IIIA was digested in 0.02 N NaOH for 24 h a t 50 "C, only a small amount of MTX was released and suggested that the inability to cleave the amide linkage between MTX and M M under basic conditions would possibly lead to low drug release rates in vivo and ultimately minimal tumor cell cytotoxicity. In order to overcome this problem (viz. MM-MTX IIIB), a spacer molecule was used between MTX and carboxyl-terminated magnetic microspheres, MM, to increase the lability by formation of an accessable ester linkage. 6-Amino1-hexanol (AH) was chosen as a spacer to link MTX and MM, since it has both amino and hydroxy groups besides having an elongated hydrocarbon chain. Prior to the esterification of AH with MTX, the amino group was protected as a t-Boc derivative using di-tertbutyl dicarbonate (36)(method IIIB, step I). Di-tert-butyl dicarbonate is a highly reactive and safe reagent of the "ready-to-use" type which reacts under mild conditions with amino acids, peptides, hydrazine and its derivatives, amines, and CH-acidic compounds in aqueous organic solvent mixtures to form pure derivatives in very good yields (37, 38). The nonspecific activation of the a- or y-carboxyl groups in MTX by DCC may lead to formation of two structural isomers, 2a or 2b (see Figure 1). The reaction (method IIIB, step 11)is based on both DCC and 4-pyrrolidinopyridine catalyst. This reaction was applied to a wide variety of acids and alcohols, including polyols
0.4 A
200
I
1
I
250
300
350
400
Wavelength( in nm) Figure 2. LJV absorption spectra o f (A) PEG, (B)MTX, and ( C ) PEG-MTX conjugate in phosphate-buffered saline (PBS). (39),a-hydroxy carboxylic acid esters (40),and even very
acid labile alcohols like vitamin A. It has also been used for the esterification of urethane-protected a-amino acids with polymeric supports carrying hydroxy groups (411. It was also shown by Hassner et al. (42) that in the absence of 4-pyrrolidinopyridine, phenyl benzoate was formed in 10% instead of 94% yield, whereas in the absence of DCC no reaction occurs. The marked lability of tert-butyl esters toward anhydrous acids permits the facile cleavage of the carbo-tertbutoxy group in the t-Boc-AH-MTX (method IIIB, step 11). Treatment of a methylene chloride solution of t-BocAH-MTX with anhydrous hydrogen chloride a t 0 "C liberated the amino group to give AH-MTX*HCl (method IIIB, step 111). The UV spectra of PEG, MTX, and PEG-MTX in PBS are shown in Figure 2. The spectra were found to be identical, while PEG itself showed no U V absorption in this range. Since all free MTX had been previously removed by extensive dialysis, only MTX linked to PEG would lead to the identical spectra. The comparison of the UV spectra of MTX with t-Boc-AH-MTX and AHMTX conjugates in methanol showed identical maxima with A,, a t 208, 260, and 306 nm (Figure 3). Since no free MTX could be detected on thin-layer chromatography, only MTX conjugated to t-Boc-AH would lead to a n identical spectrum. IR spectra were used to establish that the MM-MTX (I and 11) conjugates were chemically distinct from a physical mixture of PEG and MTX and of PEG-MTX and magnetite. Computer additions of the individual spectra of PEG and MTX were found to be identical to a physical mixture of PEG and MTX but different from the PEGMTX conjugate scan. IR spectra of a physical mixture of PEG-MTX and magnetite were found to be identical with the computer additions of the individual spectra of PEG-MTX conjugates and magnetite but different from the MM-MTX conjugate scan. Computer additions of the individual spectra of ferromagnetic polymer complex and MTX were found to be identical to a physical mixture of ferromagnetic polymer complex and MTX but different from the MM-MTX conjugate scan. The synthetic reaction conducted in the absence of EDCI did not yield a stable conjugate but rather a mixture from which MTX was rapidly dialyzed into the bulk PBS. This fact along with the spectroscopic evidence established that the conjugates were chemically different. The bands a t 1701 cm-' and 1729 cm-l in the IR spectra of t-Boc-AH-MTX and AH-MTX were attributed
Devineni et al.
208 Bioconjugate Chem., Vol. 6, No. 2, 1995 3.0
2.4
a
0
C
a
1.8
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2
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20 0.0
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I
4.0
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8.0
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Time (h) A 01
200
1
240.0
I
I
I
280.0
320.0
360.0
400
Figure 4. Release of methotrexate from magnetic microPBS, sphere-methotrexate delivery system (MM-MTX I) in (0) (A)pH 5.6 citrate buffer, and (B) rat plasma.
Wave Length (in nm) Figure 3. UV absorption spectra of (A) t-Boc-AH, (B) MTX, (C) t-Boc-AH-MTX conjugate, and (D)AH-MTX conjugate in methanol.
to a n ester linkage formed during the reaction of the glutamic acid moiety of MTX with the hydroxyl group of t-Boc-AH. The proton NMR spectrum of t-Boc-AH,t-BocAH-MTX had a characteristic singlet at 6 1.4 ppm corresponding to the tert-butyl group of t-Boc. After the deprotection step (method IIIB, step 1111,this singlet was absent in the proton NMR spectrum of AH-MTX indicating the removal of t-Boc group from AH-MTX conjugate. The synthetic reaction (method IIIB, step V) conducted in the absence of EDCI and 44dimethylamino)pyridine (4-DMAP) did not yield a stable conjugate but rather a mixture, from which MTX was rapidly washed away. Thus, the formation of a covalent linkage between magnetite-COOH and AH-MTX when reacted in the presence of EDCI and 4-DMAF' was established from the control reaction. Table 1 shows the mean diameter, MTX content of MM-MTX (I, 11, and IIIB) and percent of MTX released in various test media over a 24 h period. Laser lightscattering particle size analyses indicated the mean diameter to be 700 & 50 nm (MM-MTX I), 580 f 40 nm (MM-MTX 11) and 808.1 f 39.4 nm (MM-MTX IIIB). MTX content of MM-MTX as determined by HPLC was 0.45% (wiw), 4.0% (w/w), and 6.3% (w/w) for methods I, 11, and IIIB, respectively. The lower MTX content obtained in method I compared to methods I1 and I11 may be ascribed to the plausible hydrolysis of the basesensitive ester linkage in PEG-MTX during the preparation of MM-MTX I conjugate (step 11, method I). Figures 4-6 show the percentage of MTX released as a function of time from MM-MTX suspended in various test media. Dynamic dialysis studies in PBS, pH 5.6 citrate buffer and rat plasma over a 24 h period revealed that MTX was released to a n extent of about 97% (w/w) from MM-MTX I and 69% (w/w) from MM-MTX 11. The
75
I
0
P
S
IS
10
20
Figure 5. Release of methotrexate from magnetic microsphere-methotrexate delivery system (MM-MTX 11) in (0) PBS, (e)pH 5.6 citrate buffer, and ( 0 )rat plasma.
lack of significant differences in the release of MTX from MM-MTX (I and 11) amongst the test media suggests that the drug may be released by hydrolysis rather than by enzymatic degradation. In vitro studies of MM-MTX IIIB in PBS, rat plasma, and brain homogenate revealed that 0% (w/w) (PBS), 11%(w/w) (rat plasma), and 3.2% (w/w) (brain homogenate) of MTX was released from MM-MTX IIIB over a 24 h period. The significant differences in release of MTX in the various test media suggest that the drug was released by enzymatic hydrolysis. The low percentage of MTX released from MMMTX IIIB in plasma and brain homogenate may be an advantage to maintain MTX concentrations in brain tumors via the MM-MTX IIIB system. Assuming cytotoxicity is determined by free MTX, then hydrolysis of the MM-MTX IIIB conjugate in brain tumors and specifically within tumors cells is requisite for anticancer activity. When compared to magnetic microsphere systems in which a drug is physically entrapped (43,441,MM-MTX
Table 1. Characteristics of MM-MTX Coniugates PreDared by Methods I-IIIB MM-MTX coniugate mean diameter (nm) 5%drug loading (wlw) ?E of MTX released in PBS at 24 h 7i of MTX released in pH 5.6 citrate buffer at 24 h % of MTX released in rat plasma at 24 h R of MTX released in brain homogenate at 24 h a
Below the limit of assay sensitivity.
25
Time (h)
method I 700 k 50 0.45 2C 0.2 92.7 95.2 97
method I1 580 rt 40 4.0 40.9 69 72.1 74.2
method IIIB 808 2C 39 6.3 2C 2.1 a 11
3.2
Bioconjugafe Chem., Vol. 6,No. 2, 1995 209
Magnetic Microsphere-Methotrexate Conjugates l2
I
0
0
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10
15 Time (h)
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Figure 6. Release of methotrexate from magnetic microsphere-methotrexate delivery system (MM-MTX IIIB) in ( 0 ) rat plasma and (0) brain homogenate.
conjugates were found to have equal or smaller particle sizes. The drug loading of MM-MTX I conjugate was approximately the same as traditional microsphere systems, and the release of MTX was fast from the conjugates (MM-MTX I and 11) and hence did not meet our initial objective. However, the MTX content of MMMTX IIIB was approximately 50% higher than that obtained with MM-MTX I1 conjugate and released MTX markedly slower than typically observed with magnetic microspheres in which a drug is physically entrapped (45, 46). The conjugation approach may allow for greater verstality in magnetic drug delivery system design through the use of different linker molecules. On the basis of the desirable properties of MM-MTX IIIB, further investigations were recently conducted in brain tumor bearing rats (47). LITERATURE CITED
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