Biomacromolecules 2005, 6, 1085-1096
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Synthesis of Novel Folic Acid-Functionalized Biocompatible Block Copolymers by Atom Transfer Radical Polymerization for Gene Delivery and Encapsulation of Hydrophobic Drugs M. Licciardi,* Y. Tang, N. C. Billingham, and S. P. Armes* Department of Chemistry, University of Sussex, Falmer, Brighton, BN1 9QJ East Sussex, United Kingdom
A. L. Lewis Biocompatibles UK Ltd., Chapman House, Farnham Business Park, Weydon Lane, Farnham, Surrey GU9 8QL, United Kingdom Received November 16, 2004; Revised Manuscript Received December 21, 2004
Two synthetic routes to folic acid (FA)-functionalized diblock copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and either 2-(dimethylamino)ethyl methacrylate [DMA] or 2-(diisopropylamino)ethyl methacrylate [DPA] were explored. The most successful route involved atom transfer radical polymerization (ATRP) of MPC followed by the tertiary amine methacrylate using a 9-fluorenylmethyl chloroformate (Fmoc)-protected ATRP initiator. Deprotection of the Fmoc groups produced terminal primary amine groups, which were conjugated with FA to produce two series of novel FA-functionalized biocompatible block copolymers. Nonfunctionalized MPC-DMA diblock copolymers have been previously shown to be effective synthetic vectors for DNA condensation; thus, these FA-functionalized MPC-DMA diblock copolymers appear to be well suited to gene therapy applications based on cell targeting strategies. In contrast, the FA-MPC-DPA copolymers are currently being evaluated as pH-responsive micellar vehicles for the delivery of highly hydrophobic anticancer drugs. Introduction The use of colloidal drug and gene delivery systems such as copolymer-DNA complexes (polyplexes) and block copolymer micelles1,2 constitutes a promising approach for the treatment of cancer. Such nanosized particles can provide efficient stabilization and delivery of either complexed or encapsulated actives. In principle, polyplexes can overcome many of the problems associated with the use of viral vectors in gene therapy,3 including immunogenicity, infections, and oncogenesis. Similarly, block copolymer micelles of less than 100 nm are potentially ideal drug delivery vehicles for avoiding renal exclusion and the reticulo-endothelial system,4 as well as offering enhanced vascular permeability. However, the main obstacle for the clinical application of these systems for cancer therapy is undoubtedly their poor tumor selectivity.5 In recent years targeted gene and drug delivery via cellular receptors has emerged as a novel approach to enhance the efficacy of tumor-selective strategies. The folate receptor (FR), which is absent in most normal tissues, has been widely used in this context. The receptor for folic acid (FA; vitamin M) is overexpressed by a number of human tumors, including cancer of the ovaries6 (in over 90% of ovarian carcinoma), kidney, uterus, testis, brain, colon, lung, and myelocytic * To whom correspondence should be addressed. (M.L.) Permanent address: Dpt. Chimica e Tecnologie Farmaceutiche, University of Palermo, Via Archirafi 32, 90123 Palermo, Italy; e-mail
[email protected]. (S.P.A.) New address: Department of Chemistry, Sheffield University, Sheffield S3 7HF, UK; e-mail
[email protected].
blood cells.7 When attached via its γ-carboxyl site, folate retains its normal receptor-binding affinity and can, therefore, enter receptor-decorated cells by endocytosis.8 The covalent attachment of FA to a wide range of biomedically relevant molecules is an established methodology that has been exploited for the selective delivery of imaging agents,9 gene carriers,10,11 therapeutic agents,12 enymes,13 liposomes,8,14 block copolymer micelles,15 and other macromolecular complexes16 to tumor tissues. These developments have been the subject of a number of recent reviews.17,18 In the present study a series of structurally related diblock copolymers comprising 2-methacryloyloxyethyl phosphorylcoline (MPC) and either 2-(dimethylamino)ethyl methacrylate (DMA) or 2-(diisopropylamino)ethyl methacrylate (DPA) were synthesized by atom transfer radical polymerization (ATRP) in protic media using a protected primary amine-based initiator. After deprotection, FA was chemically conjugated via the primary amine terminus of the MPC block to obtain a series of FR-targeted copolymers. MPC is a biomimetic monomer that is known to confer clinically proven biocompatibility to various surface coatings, soft contact lenses, and medical implants such as ear grommets and coronary stents.19 The FA-MPC-DMA diblock copolymers were designed to form compact, colloidally stable polyplexes with DNA,20-22 and the FA-MPC-DPA diblock copolymers were expected to form micelles (with the MPC block forming the highly biocompatible micelle coronas and the DPA block forming the hydrophobic micelle cores) for the encapsulation of hydrophobic drugs. The DPA blocks
10.1021/bm049271i CCC: $30.25 © 2005 American Chemical Society Published on Web 01/27/2005
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Licciardi et al.
Scheme 1. Synthesis of the FA-Functionalized ATRP Initiator (3)
are pH-sensitive with a pKa of around 6.2; highly hydrophobic micelles are obtained at pH 7.4, whereas spontaneous release of the drug payload should occur below pH 6.0, which is typical of the intracellular environment.23 In both cases the chemical attachment of FA to the ends of the MPC blocks suggests that this targeting group should be exposed at the surface of the DNA polyplexes or drug-loaded micelles, thus, conferring potential cell targeting (e.g., tumor selectivity) to these colloidal delivery systems via a FR targeting mechanism. Experimental Section Materials. MPC monomer (99.5% purity) was obtained from Biocompatibles UK Ltd. The Cu(I)Br, 2,2′-bipyridine (bpy), 9-fluorenylmethyl chloroformate (Fmoc), 5-amino-1pentanol, 2-bromoisobutyryl bromide, 1,8-diazabicyclo(5.4.0)undecen-7-ene (DBU), N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbodiimide (DCC), FA (98% purity), DMA methanol, and 2-propanol were all purchased from Aldrich and used as received. N-(3-Dimethylaminopropyl)N′-ethyl-carbodiimide hydrochloride (EDC‚HCl) was purchased from Fluka. The silica used for removal of the ATRP copper catalyst was column chromatography grade silica gel 60 (0.063-0.200 mm) purchased from E. Merck (Darmstadt, Germany). DPA was purchased from Scientific Polymer Products. The water used in all experiments was deionized and doubly distilled prior to use. 1. Synthesis of Fmoc-Protected 5-Amino-1-pentanol (1). Fmoc chloride (2.2 g, 8.50 mmol, 1 equiv) was dissolved in
anhydrous dioxane (10 mL). After cooling this solution in an ice bath, 20 mL of 5 wt % NaHCO3 aqueous solution and 5-amino-1-pentanol (1.32 g, 12.8 mmol, 1.5 equiv) was added to the stirred solution. The reaction mixture gradually formed a suspension, indicating that the Fmoc protection reaction was proceeding. After approximately 20 h, water was added (50 mL) and the precipitate was extracted with ethyl acetate. The organic solution was dried with magnesium sulfate and evaporated under vacuum; the solid residue was finally purified using a silica column with dichloromethane as the eluent, gradually increasing the solvent polarity with ethyl acetate. The final product was characterized by both 1 H NMR (see Scheme 1) and high resolution mass spectroscopy (Bruker Daltonics APE III ESI spectrometer operating in ionization mode). The calculated mass for the M- + Na+ parent ion was 348.410; found, 348.157. This confirmed the structure and purity of the target molecule, which was isolated in 87% yield. 2. Synthesis of Fmoc-Protected Initiator (2). Fmoc-5amido-1-pentanol (1; (2.145 g, 7.42 mmol, 1 equiv) was dissolved in anhydrous tetrahydrofuran (20 mL) followed by the addition of excess triethylamine (1.4 mL). The resulting solution was cooled in an ice bath, and 2-bromoisobutyryl bromide (1.24 mL, 9.70 mmol, 1.35 equiv) was added to the stirred solution using a dropping funnel. The reaction was quite rapid, and a white precipitate of triethylammonium hydrobromide was formed. After stirring the mixture for 20 h, the white precipitate was removed by filtration, and 100 mL of a 3% NaHCO3 aqueous solution was added to the purified solution. The product was extracted
FA-Functionalized Biocompatible Block Copolymers
with ethyl acetate, and the organic solution was dried with magnesium sulfate and concentrated under vacuum; the oil residue was finally purified using a silica column using dichloromethane as the eluent, gradually increasing the solvent polarity with ethyl acetate. The final product was characterized by both 1H NMR (see Scheme 1) and high resolution mass spectroscopy (Bruker Daltonics APE III ESI spectrometer operating in ionization mode). The calculated mass for the M- + Na+ parent ion was 496.405; found, 496.110. This confirmed the structure and purity of the target molecule, which was isolated in 97% yield. 3. Conjugation of FA to Fmoc-Protected Initiator. Fmoc-protected initiator (2; 1.00 g, 2.10 mmol, 1 equiv) was dissolved in dimethylsulfoxide (DMSO; 20 mL) followed by the addition of DBU (0.50 mL, 3.34 mmol, 1.6 equiv). After stirring the mixture for 30 min, FA (0.927 g, 2.10 mmol, 1 equiv), DCC (0.520 g, 2.52 mmol, 1.2 equiv), and NHS (0.290 g, 5.20 mmol, 1.2 equiv) were added. After stirring the mixture for 20 h, the FA-initiator conjugate (3) was precipitated into excess dichloromethane and the resulting yellow solid was washed several times with dichloromethane and acetone and dried under vacuum. The final product was characterized by both 1H NMR (see Scheme 1) and high resolution mass spectroscopy (Bruker Daltonics APE III ESI spectrometer operating in ionization mode; calculated mass for the M- + Na+ parent ion was 698.555; found, 698.105) confirmed the structure and purity of the target molecule, which was isolated in 85% yield. 4. General Polymerization Protocol. ATRP of MPC Using the Fmoc-Protected Initiator. Fmoc-protected initiator 2 (0.210 g, 0.442 mmol, 1 equiv) was dissolved in methanol (5 mL). After purging with nitrogen for 30 min, the MPC monomer (4.00 g, 13.56 mmol) was added to the stirred solution under nitrogen. Cu(I)Br catalyst (0.0645 g, 0.449 mmol, 1 equiv) and bpy ligand (0.141 g, 0.902 mmol, 2 equiv) were then added to the reaction mixture under nitrogen. The reaction mixture immediately became dark brown and progressively more viscous. After 1 h, 1H NMR analysis indicated that more than 99% MPC had been polymerized (disappearance of vinyl signals at δ 5.5-6.0). On exposure to air, the reaction solution turned blue, indicating aerial oxidation of the Cu(I) catalyst. The resulting Fmoc-MPC30 homopolymer was passed through a silica column to remove the spent ATRP catalyst and dried under vacuum. Aqueous gel permeation chromatography (GPC) analysis indicated a Mn of 6000 and a Mw/Mn of 1.30 as determined by aqueous GPC at pH 8 using poly(ethylene oxide) standards. 5. Synthesis of MPC-DMA Diblock Copolymer. MPC (3.00 g, 10.16 mmol) was homopolymerized in methanol (3 mL) using [MPC]/[2]/[CuBr]/[bpy] ) 30:1:1:2. After 1 h, over 99% conversion was achieved and the isolated homopolymer had a Mn of 6000 and a Mw/Mn of 1.30 as determined by aqueous GPC at pH 8 using poly(ethylene oxide) standards. DMA monomer (3.17 g, 20.16 mmol, target Dp ) 60) was then added as a solution in methanol (3 mL) to this polymerizing solution. After 24 h, a Fmoc-MPC30DMA55 diblock copolymer was obtained at an overall conversion of more than 96%, as indicated by the 1H NMR
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spectrum (less than 4% residual vinyl bonds at δ 5.5-6.0). The reaction solution was then passed through a silica gel column to remove the copper catalyst. The diblock copolymer was washed with n-hexane and dried under vacuum. The diblock copolymer had a Mn of 19 500 and a Mw/Mn of 1.33 as determined by aqueous GPC at pH 2 using poly(2vinylpyridine) standards. 6. Synthesis of MPC-DPA Diblock Copolymer. MPC (2.00 g, 6.78 mmol) was homopolymerized in a 3:2 methanol/2-propanol solvent mixture (5 mL) using [MPC]/ [2]/[CuBr]/[bpy] ) 30:1:1:2 under a nitrogen atmosphere at 20 °C. After 2 h, over 95% conversion was achieved and the isolated homopolymer had a Mw/Mn of 1.24 and a Mn of 5100 as determined by aqueous GPC at pH 8 using poly(ethylene oxide) standards. DPA monomer (2.94 g, 13.74 mmol, target Dp ) 60) was then added to this polymerizing solution. After 24 h, a Fmoc-MPC30-DPA50 diblock copolymer was obtained at an overall conversion of 96%, as indicated by the 1H NMR spectrum (around 4% residual vinyl bonds at δ 5.5-6.0). The reaction solution was then passed through a silica gel column to remove the copper catalyst. The diblock copolymer was washed with n-hexane and dried under a vacuum. The resulting Fmoc-MPC30DPA50 diblock copolymer had a Mn of 16 200 and a Mw/Mn of 1.39 as determined by aqueous GPC at pH 2 using poly(2-vinylpyridine) standards. 7. General Protocol for Removal of the Fmoc Protecting Group. The Fmoc-MPC homopolymer, Fmoc-MPCDMA, or Fmoc-MPC-DPA diblock copolymers (1.00 g) were dissolved in methanol (5 mL), and DBU (0.5 mL, 3.34 mmol, 20-50 equiv) was added to the stirred solution. After 3 h, the resulting primary amine functionalized polymer (H2N-MPC30, H2N-MPC30-DMA55, or H2N-MPC30DPA50) was precipitated into n-hexane and washed with n-hexane. The dried copolymer was dissolved in water, purified by exhaustive dialysis against water [using cellulose membrane dialysis tubing (Sigma) with a 1200 molecular weight cutoff (for MPC homopolymer) or 12 400 molecular weight cutoff (for MPC-DMA and MPC-DPA diblock copolymers)], and finally freeze-dried from water overnight. The successful removal of the Fmoc protecting group was confirmed by the positive reaction of the amine-functionalized copolymers with a 2 wt % ethanol solution of ninhydrin. The H2N-MPC30 homopolymer had a Mn of 6400 and a Mw/ Mn of 1.26 as determined by aqueous GPC at pH 8 using poly(ethylene oxide) standards. The H2N-MPC30-DMA55 diblock copolymer had a Mn of 22 400 and a Mw/Mn of 1.30, and the H2N-MPC30-DPA50 diblock copolymer had a Mn of 17 100 and a Mw/Mn of 1.35 as determined by aqueous GPC at pH 2 using poly(2-vinylpyridine) standards. 8. Conjugation of FA to the H2N-MPC30 Homopolymer. H2N-MPC30 homopolymer (1.00 g, 0.110 mmol) was dissolved in water (5 mL), and FA (65 mg, 0.146 mmol, 1.3 equiv) was added to the stirred solution. If necessary, the solution pH was adjusted to 7.5 with 1 M HCl or NaOH and then EDC (56 mg, 0.292 mmol, 2.65 equiv) and NHS (34 mg, 0.292 mmol, 2.65 equiv) were added to the mixture. After stirring the reaction mixture for 20 h, HCl was used to lower the solution pH to pH 3 and the precipitated free
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FA was removed by filtration. The remaining copolymer solution was purified by exhaustive dialysis against water, using cellulose membrane dialysis tubing (Sigma) with a 1200 molecular weight cutoff, and finally freeze-dried from water overnight (yield ∼90%). Aqueous GPC analysis indicated a Mn of 7000 and a Mw/Mn of 1.28 for the resulting FA-MPC30 conjugate, as determined by aqueous GPC at pH 8 using poly(ethylene oxide) standards. The amount of free FA was estimated to be ∼0.1%, as determined using a UV GPC detector operating at 360 nm. 9. Conjugation of FA to the H2N-MPC30-DMA55 Diblock Copolymer. H2N-MPC30-DMA55 (1.00 g, 0.055 mmol) was dissolved in water (4 mL), and a solution (5 mL) containing FA (32.0 mg, 0.0715 mmol, 1.3 equiv), EDC‚ HCl (28.0 mg, 0.143 mmol, 2.65 equiv), and NHS (16.5 mg, 0.143 mmol, 2.65 equiv) in a 3:2 water/DMSO mixture was added to the aqueous block copolymer solution. If necessary, the solution pH was adjusted to 7.5 using either HCl or NaOH. After stirring the mixture for 20 h, the pH was lowered to 3 using HCl and the precipitated free FA was removed by filtration. The remaining solution was purified by exhaustive dialysis against water, using cellulose membrane dialysis tubing (Sigma) with a 12 400 molecular weight cutoff, and finally freeze-dried from water overnight (yield ∼90%). Aqueous GPC analysis indicated a Mn of 20 800 and a Mw/Mn of 1.30 for the resulting FA-MPC30-DMA55 conjugate, as determined by aqueous GPC at pH 2 using poly(2-vinylpyridine) standards. The amount of free FA was negligible (
