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Biomacromolecules 2010, 11, 2478–2486
Polymeric Drugs Based on Random Copolymers with Antimitotic Activity M. L. Lo´pez-Donaire,†,‡ J. Parra-Ca´ceres,‡,§ M. Ferna´ndez-Gutie´rrez,†,‡ B. Va´zquez-Lasa,*,†,‡ and J. San Roma´n†,‡ Institute of Polymer Science and Technology, CSIC, Juan de la Cierva, 3, 28006 Madrid, Spain, CIBER-BBN, Ebro River Campus, Building R&D, Block 5, 1st Floor, c/Poeta Mariano Esquillor s/n, Zaragoza 50 018, Spain, and Associate Unit CSIC, Hospital Provincial de A´vila, CHA.SACyL, C/Jesu´s del Gran Poder 42, 05003 A´vila, Spain Received June 16, 2010; Revised Manuscript Received July 26, 2010
Polymeric drugs based on random copolymers with antimitotic activity were obtained by free radical copolymerization of oleyl 2-acetamido-2-deoxy-R-D-glucopyranoside methacrylate (OAGMA) and 2-ethyl-(2pyrrolidone) methacrylate (EPM) at low and high conversion and analyzed in terms of microstructure, physicochemical, and biological properties. Reactivity ratios of monomers were found to be rOAGMA ) 1.34 and rEPM ) 0.98, indicating the obtaining of statistical copolymers with random sequence distribution of the comonomeric units in the macromolecular chains. The glass transition temperature of the copolymers presents a negative deviation from the predicted values according to the Fox equation, suggesting a higher flexibility of the alternating diad. Copolymeric systems with OAGMA contents between 10-50 mol % presented thermosensitive behavior in a heating process showing cloud point temperatures (CPT) in the range 45-28 °C with increasing OAGMA content and hysteresis in one heating-and-cooling cycle. In vitro glycolipid release studies revealed the stability of the ester group in culture medium. The polymeric drugs with 30 and 50 mol % OAGMA presented antimitotic activity on a human glioblastoma line, but they were less toxic on normal human fibroblast cultures.
1. Introduction Polymers that are currently used in cancer therapy are defined under the umbrella term of “polymer therapeutics”.1 One category of them includes polymeric drugs. The structure of these systems is based on the Ringsdorf’s model2 in which solubilizing groups are attached to the polymer backbone to provide the bioavailability of the carrier system and moieties with biological or pharmacological activity are bound to the polymer backbone via spacers. Different studies have shown that polymeric drugs could be entrapped or accumulated in solid tumors and be retained there at high concentrations for prolonged periods. This phenomenon was identified by Matsumura et al.3 and it is known as the enhanced permeability and retention (EPR) effect of macromolecules and lipids in solid tumor.4,5 Based on these principles, some polymeric systems containing anticancer drugs, such as paclitaxel, doxorubicin, and so on, are currently being tested in clinical trials.6-9 In the frame of this strategy, our group is involved in a research project devoted to the preparation of polymeric drugs containing synthetic glycolipids with demonstrated antitumor activity.10 These glycolipids are based on the natural molecule neurostatin, a glycosphingolipid present in mammalian brain that inhibits the proliferation of primary astroblasts and glioma cells.11 Amphiphilic polymeric drugs were obtained by the radical copolymerization of a methacrylic derivative of oleyl 2-acetamido-2-deoxy-R-D-glucopyranoside (OAGMA) and vinyl pyrrolidone (VP).12 Resulting copolymers with therapeutic properties presented a specific microstructure derived from the * To whom correspondence should be addressed. Tel.: 34 915618806, Ext 321. E-mail:
[email protected]. † CSIC. ‡ CIBER-BBN. § ´ vila. Hospital Provincial de A
very different monomers reactivity, providing copolymers enriched in the glycomonomer with a distribution of the glycomonomer units in long sequence, so-called blocky sequences, that led to the formation of self-assembled nanoparticles. Further work presented in this paper has been focused on a new design of polymeric drugs with similar functional groups but possessing a random and homogeneous distribution of the bioactive glycoside in the macromolecular chains and in the study of the effect of monomeric sequence distribution on the physicochemical properties of these systems and its implication in the action of the anchored glycoside. To achieve this goal, a methacrylic derivative of pyrrolidone was selected as a comonomer for radical copolymerization reactions with the bioactive glycomonomer, considering that the reactivity of two methacrylates will be of the same order of magnitude as is documented in previous works.13 This paper describes the synthesis and characterization of the new polymeric drugs by radical copolymerization of OAGMA with 2-ethyl-(2-pyrrolidone) methacrylate EPM. Copolymerization parameters and microstructure of the copolymers were studied from reactions carried out at low conversion. High conversion copolymers were obtained under the same conditions and the main physicochemical properties analyzed. The biological effects of the polymeric drugs were assessed using human glioblastoma and human fibroblast cells.
2. Materials and Methods 2.1. Materials. The monomers oleyl 2-acetamido-2-deoxy-R-Dglucopyranoside methacrylate (OAGMA)12 and 2-ethyl-(2-pyrrolidone) methacrylate (EPM)13 were synthesized as it was described in previous papers. Azobisisobutyronitrile (AIBN; Merck) was recrystallized from methanol (mp 104 °C). The solvents dioxane and diethyl ether (Panreac) were purified by standard procedures. Phosphate buffered solution (PBS)
10.1021/bm100672c 2010 American Chemical Society Published on Web 08/10/2010
Polymeric Drugs Based on Random Copolymers of pH 7.4 (Sigma-Aldrich) was used as received. The culture medium Dulbecco’s modified Eagle’s medium enriched with 4500 mg/L of glucose and supplemented with 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES; DMEM) was supplied by Sigma. L-Glutamine, penicillin-streptomycin solutions, sodium pyruvate, trypsin-ethylenediaminetetraacetic acid (EDTA), and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were all purchased from SigmaAldrich. Foetal bovine serum (FBS; Gibco), tissue culture media, additives, trypsin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all purchased from Sigma. 2.2. Characterization Techniques. Proton (1H NMR) nuclear magnetic resonance spectra were recorded in an INOVA-400 spectrophotometer. The spectra were recorded in pyridine-d5 (10 wt/v) at 80 °C. Tetramethylsilane (TMS) was used as internal standard. Attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectra were recorded on a Perkin-Elmer-Spectrum One spectrophotometer, with an ATR attachment. Glass transition temperatures (Tg) were measured by differential scanning calorimetry (DSC) with a Universal V4 3A TA Instruments, DSC Q2000 V24.2 Build 107. The dry samples (2-5 mg) were placed in aluminum pans and heated from -30 to 150 °C at a constant rate of 10 °C/min. Tg was taken as the midpoint of the heat capacity transition. Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere with a TGA Q500 (TA Instruments) thermobalance at a heating rate of 5 °C/min and a temperature interval between 30 and 700 °C. The cloud point temperature (CPT) was determined by ultraviolet-visible (UV/vis) spectrophotometry with a Varian Cary 3 UV/vis apparatus equipped with a Peltier cell holder for temperature control, by monitoring the turbidity of aqueous solutions (0.5-3 wt %) as a function of temperature at 450 nm and under magnetic stirring. Distilled water solutions were placed in the cuvette and heating scans between 10 and 80 °C were undertaken at a constant rate of 1 °C/min. Cooling scans were performed immediately after heating at the same rate. The CPT was defined as the temperature at the inflection point in the normalized absorbance versus temperature curve. The thermoresponsive character of poly(2-ethyl-(2-pyrrolidone) methacrylate) (PEPM) was studied by dynamic light scattering (DLS) using a Malvern Nanosizer Nano S Instrument equipped with a 4 mW He-Ne laser (λ ) 633 nm) at an angle of 173°. The autocorrelation function was converted in a volume particle size distribution with Zetasizer Software 6.12 version to get the apparent hydrodynamic diameter (Dh) in terms of the mean obtained from volume-Dh distribution. The measurement was performed with a PEPM solution (0.1 wt %) in ultrapure water and filtered using a 0.2 µm PTFE filter (Symta). The temperature trend was carried out between 40 and 65 °C at intervals of 1 °C and an equilibration time of 2 min. Number and weight average molecular weights were determined by size exclusion chromatography (SEC) in a Perkin-Elmer apparatus with an isocratic pump serial 200 connected to a differential refractometric detector (serial 200a). Two Resipore columns (Varian) were conditioned at 70 °C and used to elute the samples (1 mg/mL concentration) at 0.3 mL/min HPLC-grade N,N′-dimethylformamide (DMF) supplemented with 0.1% v/v LiBr. Calibration of SEC was carried out with monodisperse standard poly(methyl methacrylate) samples in the range of 2.9 × 103 to 480 × 103 obtained from Polymer Laboratories. Release of the glycoside oleyl 2-acetamido-2-deoxy-R-D-glucopyranoside (OAG) from OAGMA/EPM-30 and OAGMA/EPM-50 copolymers in free-serum supplemented culture medium (DMEM) was analyzed by high performance liquid chromatography (HPLC) in an apparatus SHIMADZU SIL 20A, at 215 nm. First, a calibration curve (R ) 0.9997) with OAG was obtained previously using acetonitrile/water (60:40) as a mobile phase by measuring the integration of the peak at 13.93 ( 0.05 min obtained from solutions of known concentration (0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 mM). Afterward, copolymer solutions (1 mg/mL) were prepared in free-serum supplemented culture medium and incubated at 37 °C for 2, 4, 6, 8, 24, and 72 h. At the desired time, the corresponding copolymer solution was freeze-dried and subsequently
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dissolved in acetonitrile/water (60:40) for its analysis by HPLC. Three measurements were performed for each time and each copolymeric sample. 2.3. Copolymerization Reactions. Low conversion (
