Biomacromolecules 2005, 6, 1846-1850
1846
Direct, Controlled Synthesis of the Nonimmunogenic, Hydrophilic Polymer, Poly(N-(2-hydroxypropyl)methacrylamide) via RAFT in Aqueous Media† Charles W. Scales,‡ Yulia A. Vasilieva,‡ Anthony J. Convertine,‡ Andrew B. Lowe,§ and Charles L. McCormick*,‡,§ Department of Polymer Science and Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406 Received April 29, 2005; Revised Manuscript Received June 1, 2005
Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is a nonimmunogenic, neutral-hydrophilic polymer currently employed in the delivery of anticancer drugs. Herein, we report conditions that facilitate the direct, controlled RAFT polymerization of HPMA in aqueous media. We demonstrate that the use of 4-cyanopentanoic acid dithiobenzoate and 4,4′-azobis(4-cyanopentanoic acid) as the chain transfer agent (CTA) and initiating species, respectively, in the presence of an acetic acid buffer solution at 70 °C is a suitable condition leading to controlled polymerization. The “living” nature of these polymerizations is demonstrated via chain-extension of an HPMA macroCTA to yield the corresponding poly(HPMA-b-HPMA) “homopolymer”. Introduction Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) as a vehicle in drug delivery has been well studied over the past few decades.1-8 Much of the work with this uncharged, hydrophilic, biocompatible polymer has focused on the delivery of anticancer drugs,4,5,7,8 site specific delivery in the GI tract,9,10 tumor-specific delivery of antisense oligonucleotides to their target mRNAs,11,12 and hydrogels.9,13-15 As with other successful delivery vehicles, drug conjugates of PHPMA are well suited for pharmaceutical applications because they exhibit the enhanced permeability and retention (EPR) effect, therefore, increasing the concentration of an active drug in tumor cells (i.e., 10-100 times higher than that of the free drug) and decreasing the dose limiting toxicity.16,17 The limiting factor to date in the synthesis of well-defined polymer-drug conjugates of HPMA has been the ability to control the free-radical polymerization of the monomer and, therefore, the molecular weight and molecular weight distribution of the resulting polymer. The first attempts to control the polymerization of HPMA were reported by Matyjaszewski et al.18-20 and employed atom transfer radical polymerization (ATRP) in organic media (1-butanol or ethanol) with two different ligand-initiating systems. However, these systems suffered from elevated polydispersities (Mw/Mn) and, in some cases, low conversions. An indirect method for the preparation of well-defined polymers of HPMA and PHPMA-drug conjugates was demonstrated by Godwin et al.21 in which N-methacryloxysuccinimide was †
Paper number 114 in a series entitled Water Soluble Polymers. * To whom correspondence should be addressed. E-mail:
[email protected]. ‡ Department of Polymer Science. § Department of Chemistry and Biochemistry.
first polymerized via ATRP followed by reaction of the succinimidyl-ester side-chains with stoichiometric amounts of 1-amino-2-propanol and an amino-terminal, peptide-linked “model drug”, glycine-glycine-β-naphthylamide. In recent years, we have focused on the controlled polymerization of water-soluble, acrylamido monomers via reversible addition-fragmentation chain transfer (RAFT) polymerization. To date, the controlled RAFT polymerization of anionic,22-24 zwitterionic,25,26 and neutral25-46 acrylamido monomers has been reported in both organic and aqueous media employing a variety of chain transfer agents (CTAs) including xanthates,44 dithiocarbamates,28,37 trithiocarbonates,29,36,46 and dithioesters.22-27,30-35,38-43,45 All of these CTAs can afford control over the molecular weight and yield (co)polymers with low Mw/Mn values under appropriate conditions. We have recently turned our attention to methacrylamido monomers and reported the first RAFT polymerization of a cationic methacrylamido species, namely N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA), mediated by 4-cyanopentanoic acid dithiobenzoate (CTP) in aqueous media.47 DMAPMA was polymerized with good control over the molecular weight and polydispersity in an acetic acid/ sodium acetate buffer solution (Mn ) 47 100 g/mol, Mw/Mn ) 1.08), whereas the corresponding polymerization in water alone exhibited a loss of control due to thiocarbonylthio hydrolysis (Mn ) 44 500 g/mol, Mw/Mn ) 1.62). More recently, we reported the controlled CTP-mediated polymerization of methacrylamide (MAM) in the same acidic buffer (Mn ) 23 800 g/mol, Mw/Mn ) 1.08).48 Once again, the use of an aqueous buffer yielded polymers of high conversion and low polydispersities. Retention of the dithioester end group functionality was demonstrated by efficient chainextension of a poly(MAM) (PMAM) macroCTA. Furthermore, a macroCTA of MAM was chain-extended with
10.1021/bm0503017 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/17/2005
Biomacromolecules, Vol. 6, No. 4, 2005 1847
Communications
acrylamide (AM) to form the corresponding block-copolymer, with a final Mn of 33 800 g/mol and Mw/Mn ) 1.25. Building on these earlier reports, we describe herein the first example of the direct, controlled polymerization of HPMA in aqueous media via RAFT. We show that the R-methyl substitution allows for a sufficiently fast rate of monomer conversion relative to hydrolysis of the thiocarbonylthio end groups, thus affording control of molecular weight and Mw/Mn. Additionally, an HPMA macroCTA has been synthesized and chain extended to further demonstrate the living nature of these polymerizations. Experimental Section Materials. All chemicals were purchased from Aldrich at the highest available purity and used as received unless otherwise noted. 4,4′-Azobis(4-cyanopentanoic acid) (V-501, a gift from Wako Pure Chemicals Industries, Ltd.) was recrystallized from methanol. 4-Cyanopentanoic acid dithiobenzoate (CTP) was synthesized according to the literature procedure.49 Deionized water (DI H2O) was obtained from a Barnstead NANO-Pure reverse osmosis/filtration unit (resistivity of 18.0 MΩ). HPMA was synthesized according to a literature procedure reported by Kopecˇek et al.50,51 Briefly, freshly distilled methacryloyl chloride (one equivalent) was diluted in acetonitrile and added dropwise to a solution of 1-amino-2-propanol (2 equivalents) in acetonitrile at 0 °C. Precipitated 2-hydroxypropylammonium chloride was removed by filtration, and HPMA was isolated by precipitation from acetonitrile, recrystallization from a methanol/ether (3/1) cosolvent, and a subsequent recrystallization from acetone. The crystalline monomer (mp ) 67 °C) was dried in vacuo and stored at -10 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) 1.15 (d, 3H), 1.42 (s, 3H), 3.10 (m, 1H) 3.45 (m, 1H) 3.90 (m, 1H,) 5.30 (s, 1H), 5.19 (s, 1H). 13 C NMR (500 MHz, CDCl3): δ (ppm) 19.0 (CH), 21.0 (CH), 47.0 (CH), 67.0 (CH), 120.0 (CH), 140.0 (C), 170.0 (CdO). Polymerizations. Polymerizations were conducted at 70 °C employing V-501 as the primary radical source and CTP as the RAFT CTA. All polymerizations were performed directly in an aqueous acetic buffer (pH ) 5.2, 0.27 mol/L acetic acid and 0.73 mol/L sodium acetate) with an initial monomer concentration ([M]o) of 1 M in a septa-sealed, round-bottom flask that was purged with nitrogen for 30 min prior to polymerization. The initial monomer to CTA ratio ([M]o/[CTA]o) was held constant at 780/1 and two different CTA to initiator ratios ([CTA]o/[I]o) of 5/1 and 3/1 were evaluated. Polymerization kinetics and absolute molecular weights were determined by extracting aliquots (0.5 mL) from the polymerization solution at predetermined time intervals which were immediately quenched by immersion in liquid nitrogen. HPMA macroCTA was isolated by dialysis in acidic conditions (pH ) 3-4) at 4 °C followed by lyophilization. Subsequent chain-extension experiments were conducted under the same conditions described above with a [CTA]o/[I]o of 5/1. Characterization. Polymerization aliquots were analyzed directly by aqueous size exclusion chromatography (ASEC) using an eluent of 20% acetonitrile/80% 0.05 M Na2SO4(aq)
Table 1. Conversion, Molar Mass, and Polydispersity Data for the Aqueous Polymerization of HPMA Mediated by CTPa entry no.
time (min)
[CTA]o/[I]o
conversion (%)b
Mn (theory)
Mn (expt)c
Mw/Mnc
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
60 120 180 240 360 480 720 1440 60 120 180 240 360 480 720 1440
5 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3
6 18 27 34 46 55 66 75 13 26 39 46 59 70 78 88
6900 19 500 29 700 38 400 51 600 61 400 73 200 84 200 13 700 29 400 43 300 51 500 65 800 77 300 87 500 98 200
15 800 30 400 40 800 50 500 62 900 70 800 80 400 88 300 21 500 39 300 51 200 62 800 72 400 83 000 89 500 97 000
1.09 1.05 1.04 1.05 1.06 1.08 1.08 1.07 1.05 1.03 1.03 1.03 1.06 1.05 1.07 1.07
a Polymers synthesized at 70 °C at 1 M monomer in acetic buffer ([CTA]o/[M]o ) 1/780 and [CTA]o/[I]o ) 5/1 and 3/1) under a nitrogen atmosphere with V-501 as the initiator. b Conversions were determined by comparing the area of the UV signal of the monomer at to to that at tx. c As determined by ASEC (0.5 mL/min, 25 °C, Tosoh Biosciences TSKgel columns (G3000 PWXL (
