Article pubs.acs.org/bc
PolyMPC−Doxorubicin Prodrugs Xiangji Chen,† Sangram S. Parelkar,† Elizabeth Henchey,‡ Sallie Schneider,‡ and Todd Emrick*,† †
Polymer Science & Engineering Department, 120 Governors Drive, University of Massachusetts, Amherst, Massachusetts 01003, United States ‡ Pioneer Valley Life Sciences Institute, 3601 Main Street, Springfield, Massachusetts 01199, United States S Supporting Information *
ABSTRACT: We demonstrate the conjugation of the cancer drug doxorubicin (DOX) to poly(methacryloyloxyethyl phosphorylcholine) (polyMPC), linked by hydrazone groups, using (1) a one-pot ATRP/click sequence, and (2) a post-polymerization conjugation strategy. While the one-pot method gave polyMPC−DOX conjugates in a facile single step, post-polymerization conjugation gave higher-molecular-weight polymers with very high DOX loadings. DOX release from the polyMPC backbone was pHdependent (faster at pH 5.0 than at pH 7.4) owing to the hydrazone linkage. Half-life values of DOX release ranged from 2 to 40 h at pH 5.0. Cell culture experiments showed that highly loaded polyMPC− DOX conjugates exhibited higher intracellular drug accumulation and lower half-maximal inhibitory concentration (IC50) values, while a polymer with 30 wt % drug loading showed a maximum tolerated dose in the range of 30−50 mg/kg DOX equivalent weight in healthy mice.
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poly(methacryloyloxyethyl phosphorylcholine) (polyMPC),22 has been used extensively in blood-contacting medical devices that require a high level of biocompatibility and resistance to protein adsorption.22−28 PolyMPC has recently been considered as an alternative to PEG, and studied in conjugation chemistry with therapeutic proteins such as erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), and interferon. 29,30 For example, 20 kDa PEG equivalent polyMPC−interferon was seen to have a longer pharmacokinetic (PK) profile (absorption t1/2 = 7.3 h) than the 20 kDa disulfide PEGylated analogue (absorption t1/2 = 3.3 h).30 With regard to cancer therapeutics, we demonstrated the conjugation of camptothecin (CPT) to polyMPC using azide/alkyne click cycloaddition, in a one-pot polymerization/conjugation procedure of alkyne-substituted polyMPC with CPT-azide.31 This approach provided polyMPC−CPT prodrugs with much higher drug loading than the PEGylation approach, due to the inherent restriction of PEG that limits drug conjugation only to the chain-ends. Doxorubicin (DOX) is a DNA intercalating drug with dosedependent cytotoxicity affecting a broad range of DNA processes. DOX and its derivatives show pronounced anticancer activity that unfortunately is accompanied by undesirable side effects. Doxil, a PEGylated liposome encapsulant of DOX, shows favorable pharmacokinetics compared to free DOX, with significantly reduced dose-limiting
INTRODUCTION Polymer−drug conjugates as nanoscale therapeutics1,2 are creating new opportunities for improved cancer therapy.3−5 Conjugating anticancer drugs to water-soluble synthetic polymers improves drug solubility, prolongs in vivo circulation half-life (t1/2), and reduces undesired side effects.6,7 The large size of polymer molecules, relative to small molecule drugs, affords the drug with a large (several nanometers to tens-ofnanometers) hydrodynamic volume, which slows renal clearance and improves drug uptake into tumors (relative to healthy tissue) by the enhanced permeability and retention (EPR) effect.8−10 The EPR effect benefits from the leaky vasculature and poor lymphatic drainage surrounding the growing neoplasms. As a result, polymer−drug conjugates have exhibited an increased therapeutic index (LD50 /ED50 ) compared to free drugs. Numerous synthetic polymers have been conjugated covalently to different anticancer drugs to give structures that fall into the classification of polymer prodrugs.11 Prominent examples of polymers used in cancer prodrug compositions include poly(ethylene glycol) (PEG),12 poly-N(2-hydroxypropyl)methacrylamide (HPMA),13−15 poly(L-glutamic acid) (PG),16,17 cyclodextrin-based polymers,18,19 dextran,20 and polyacetal poly(1-hydroxymethylethylene hydroxymethylformal) (PHF).21 Effective polymer therapeutics requires the use of watersoluble, biocompatible polymers as starting materials. The water solubility and low toxicity and low immunogenicity of PEG have made it attractive for conjugation to numerous therapeutic proteins and peptide drugs to enhance therapeutic efficacy.7 However, the zwitterionic water-soluble polymer, © 2012 American Chemical Society
Received: December 20, 2011 Revised: July 17, 2012 Published: August 10, 2012 1753
dx.doi.org/10.1021/bc200667s | Bioconjugate Chem. 2012, 23, 1753−1763
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Scheme 1
Figure 1. (a) One-pot ATRP/click cycloaddition synthesis of polyMPC−DOX; (b) aqueous GPC traces of conjugates 4-A and 4-B; (c) polyMPC− DOX conjugates prepared by one-pot ATRP/click method; NMPC, NTMS‑PgMA, NDOX‑N3 are the molar equivalent to initiator EBiB; Conv (%) represents monomer conversion determined by 1H NMR spectroscopy; DOX wt % is obtained by UV−vis spectroscopy at 488 nm.
into the polyMPC structure. In one approach, DOX-azide 3 was synthesized and used for the preparation of polyMPC− DOX conjugates by a one-pot simultaneous ATRP polymerization and DOX grafting by azide−alkyne click chemistry. In a second approach, polyMPC with pendant acyl hydrazides was synthesized by copolymerization, and DOX was incorporated into the polymer by hydrazone formation. Using the latter method, high DOX loading on the polyMPC backbone was achieved (30 wt % and greater), as characterized by UV/vis spectroscopy. The pH dependence of DOX release from the polyMPC backbone was revealed using HPLC, and subcellular localization, intracellular abundance, and cytotoxicity profiles were determined in human breast and colon adenocarcinoma cells. The maximum tolerated dose (MTD) of polyMPC−DOX prodrug was also evaluated in normal mice.
toxicities associated with cardiomyopathy and myelosuppresion.32,33 However, Doxil has its own dose-limiting toxicities, including palmer-planter erythrodysesthesia (hand-foot syndrome) and mucositis/stomatitis34−36 due to its accumulation in the skin.37 Moreover, acquired cellular resistance to DOX from overexpression of P-glycoprotein encoded by the MDR1 (multidrug resistance 1) gene has been documented.38,39 Polymer−drug conjugation can alter the cellular internalization pathway of a drug, and circumvent P-glycoprotein associated multidrug resistance.40,41 For example, DOX conjugation to a PEGylated dendrimer42 at a drug loading of 10 wt % proved promising in preclinical studies. In this case, DOX was conjugated through its ketone group (at the 13 position carbon) by reaction with an acyl hydrazine of the dendrimer. HPMA,43−45 poly(ethylene oxide)-block-poly(allyl glycidyl ether),46 and degradable dendrimers47 were also conjugated to DOX through hydrazones, showing improved efficacy in preclinical studies. The acid-sensitive hydrazone linker enables DOX release either extracellularly in the slightly acidic environment of tumor tissue48,49 or intracellularly in the acidic environment of endosomal (pH ∼5.5−6.0) and lysosomal (pH ∼4.5−5.0) compartments.50 Here, we describe the preparation of polyMPC−DOX prodrugs that integrate the pH-responsive hydrazone linkages
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RESULTS AND DISCUSSION Synthesis of polyMPC−DOX by One-Pot Polymerization. The preparation of hydrazine-containing DOX-azide 3 is shown in Scheme 1, accomplished by first reacting methyl 6bromohexanoate with sodium azide in DMF to give methyl 6azidohexanoate 1 in 98% yield. Refluxing compound 1 in hydrazine monohydrate afforded the desired linker, compound 2, in 60% yield after crystallization in methanol/ethyl ether 1754
dx.doi.org/10.1021/bc200667s | Bioconjugate Chem. 2012, 23, 1753−1763
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Figure 2. (a) ATRP copolymerization of MPC and TBOEMA, followed by acylhydrazine formation (7) and DOX conjugation (8). (b) Aqueous GPC trace of polyMPC−DOX conjugate 8-B. (c) Overlay of UV/vis spectra of conjugates 8 in water at concentration of 0.1 mg/mL. (d) Conjugates 8-A, 8-B, and 8-C are soluble in methanol and water, while 8-D and 8-D2 are not soluble in methanol but are soluble in water.
Table 1. Poly(MPC-co-TBOEMA) (6) and the Corresponding Acyl Hydrazide Polymers (7) polymer
target TBOEMA (mol %)a
conv (%)b
Mn (kDa)
PDI
TBOEMA (mol %)c
polymer
Mn (kDa)
PDI
hydrazine (mol %)d
6-A 6-B 6-C 6-D
10 10 20 40
90 70 50 53
16 25 28 22
1.54 1.32 1.43 1.50
10 12 19 38
7-A 7-B 7-C 7-D
16 25 29 31
1.56 1.34 1.49 1.45
9 13 21 37
a
1
Based on monomer feed ratio. bMonomer conversion measured by 1H NMR spectroscopy. cEstimated by 1H NMR spectroscopy. dEstimated by H NMR spectroscopy.
percentage of DOX in the conjugate was calculated based on its UV/vis absorption and the molar extinction coefficient of DOX·HCl (ε = 7654 L mol−1 cm−1 in water) at 488 nm. The drug loading was determined from the DOX-to-conjugate concentration, giving 3−5 wt % DOX by this one-pot method (Figure 1). PolyMPC−DOX Synthesis by Post-Polymerization Conjugation. While the one-pot strategy represents a simple method for obtaining polyMPC−DOX conjugates, we turned to post-polymerization conjugation in an effort to optimize drug loading on the polyMPC platform. We found that copolymerization of MPC and monomer 5 gave copolymer 6 in excellent yield, and ester-to-acyl hydrazine conversion, using hydrazine hydrate, gave polyMPC-hydrazine 7. Polymer 7 was then used for conjugation of DOX, again with DOX hydrochloride, as shown in the reaction sequence of Figure 2a. We note that the methyl and ethyl ester versions of monomer 5 were also employed successfully in polymerization and DOX-conjugation processes. This two-step polymerization/conjugation approach proved straightforward and effective, and superior to the one-pot method with respect to obtaining high drug loading of the conjugates that was not achievable with the one-pot method. Monomer 5, 2-tert-butoxy-2-oxoethyl methacrylate (TBOEMA), was synthesized according to the literature51 with some modification. The sodium salt of methacrylic acid was esterified with tert-butyl bromoacetate in acetonitrile, using tetra-n-
mixtures. Azide 2 was then coupled to DOX hydrochloride in methanol to give the desired DOX-azide 3. FT-IR spectroscopy of 3 showed the expected azide signal at 2098 cm−1, while 1H NMR spectroscopy showed the alkyl chain (linker) signals from 1.1 to 1.3 ppm, and the phenyl protons of DOX at 7.95, 7.93, and 7.68 ppm. By 13C NMR spectroscopy, the original DOX ketone carbon resonance at 214 ppm was absent, and a new resonance for the CN of the hydrazone was seen at 187 ppm. High-resolution mass spectroscopy (HRMS) confirmed the successful isolation of DOX-azide 3 (Calcd: 697.2833; Found: m/z 697.2822 [M+H]). One-Pot ATRP/Click Chemistry. DOX-azide 3 was introduced into the ATRP polymerization reaction with 1 molar equiv trimethylsilyl-protected propargyl methacrylate (TMS-PgMA), as shown in Figure 1. TMS-PgMA was copolymerized with MPC by ATRP, incorporating DOX into the structure by azide/alkyne cycloaddition as the polymerization proceeded. Monomer conversion of ∼70% was achieved over a 20 h period, and the polyMPC−DOX conjugates were isolated in ∼60% yield following precipitation into THF and column chromatography. The polyMPC−DOX conjugates were characterized by aqueous GPC (0.1 M NaNO3, PEO standards) as shown in Figure 1. The PDI values of the conjugates resulting from this one-pot method were in the range 1.23−1.40, and the molecular weights obtained (6700− 12 400 g/mol) confirm good compatibility of DOX with the click and ATRP conditions and mechanisms. The weight 1755
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based on the hydrazine loadings in copolymer 7 in Table 1. The molecular weight of DOX hydrochloride conjugated at this level gives a theoretical DOX loading as high as 43 wt %. These values compared closely to the values obtained by UV−vis characterization, as given in Table 2. We recognize that these DOX loadings are high relative to other reported polymer− DOX prodrugs (typically ∼10−20 wt %).42,46,52,53 Nonetheless, even with such high weight percent DOX, the conjugates maintained good solubility in water, owing to the extreme hydrophilicity of the polyMPC backbone. Such high drug loadings may represent an additional advantage of polyMPC− DOX for parenteral applications. PolyMPC−DOX conjugates containing high DOX loadings presented interesting solubility properties. During the course of conjugation reaction, solubility in methanol was maintained. However, conjugates 8-D and 8-D2 were found to be insoluble in methanol following purification and freeze-drying. Conjugates with lower DOX loading (
