Methoxy Poly(ethylene glycol)-block-Poly(Î´-valerolactone) Copolymer

Biomacromolecules 2005, 6, 3119-3128

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Methoxy Poly(ethylene glycol)-block-Poly(δ-valerolactone) Copolymer Micelles for Formulation of Hydrophobic Drugs Helen Lee,† Faquan Zeng,† Mike Dunne,‡ and Christine Allen*,†,‡ Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, and Faculty of Applied Science and Engineering, Division of Engineering Science, University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S 2S2 Received June 29, 2005; Revised Manuscript Received September 13, 2005

Six amphiphilic diblock copolymers based on methoxy poly(ethylene glycol) (MePEG) and poly(δvalerolactone) (PVL) with varying hydrophilic and hydrophobic block lengths were synthesized via a metalfree cationic polymerization method. MePEG-b-PVL copolymers were synthesized using MePEG with Mn ) 2000 or Mn ) 5000 as the macroinitiator. 1H NMR and GPC analyses confirmed the synthesis of diblock copolymers with relatively narrow molecular weight distributions (Mn/Mw ) 1.05-1.14). DSC analysis revealed that the melting temperatures (Tm) of the copolymers (47-58°C) approach the Tm of MePEG as the PVL content is decreased. MePEG-b-PVL copolymer aggregates loaded with the hydrophobic anticancer drug paclitaxel were found to have effective mean diameters ranging from 31 to 970 nm depending on the composition of the copolymers. A MePEG-b-PVL copolymer of a specific composition was found to form drug-loaded micelles of 31 nm in diameter with a narrow size distribution and improve the apparent aqueous solubility of paclitaxel by more than 9000-fold. The biological activity of paclitaxel formulated in the MePEG-b-PVL micelles was confirmed in human MCF-7 breast and A2780 ovarian cancer cells. Furthermore, the biocompatibility of the copolymers was established in CHO-K1 fibroblast cells using a cell viability assay. The in vitro hydrolytic and enzymatic degradation of the micelles was also evaluated over a period of one month. The present study indicates that the MePEG-b-PVL copolymers are suitable biomaterials for hydrophobic drug formulation and delivery. Introduction In recent years, block copolymer micelles have been investigated extensively as drug delivery vehicles with formulations based on this technology now in clinical trial development.1-3 The micelle systems explored for drug delivery are nanosized supramolecular assemblies of amphiphilic copolymers that consist of a hydrophobic core surrounded by a hydrophilic shell.4-6 These micelles have been shown to significantly enhance the solubility of highly lipophilic drugs such as paclitaxel and ellipticine.7,8 In some cases the aqueous solubility of the drug has been increased up to 30 000-fold.7 The lipophilic drugs partition into the hydrophobic core of the micelles, whereas the micelle shell provides a protective interface between the core and the external medium. The hydrophilic shell is usually formed from poly(ethylene glycol) (PEG) and has been shown to reduce secondary aggregation of the micelles and prevent their interactions with serum protein.9 Unlike the hydrophilic block, many different polymers have been explored as the hydrophobic core-forming block of the micelles. Examples of biocompatible and biodegradable core-forming polymers include poly(D,L-lactide) 8,10,11 (PDLLA), poly(β-benzyl-Laspartate),12-14 and poly(caprolactone) (PCL).15-19 * To whom all correspondence should be addressed. Phone: (416) 9468594. Fax: (416) 978-8511. E-mail: [email protected]. † Department of Pharmaceutical Sciences. ‡ Division of Engineering Science.

The extent to which the micelles are able to solubilize the drug or incorporate the drug within their core is highly dependent on the compatibility between the drug and the core-forming block.7,18 A high degree of compatibility between the drug and the core-forming block of the micelles has been shown to increase the drug loading capacity, drug loading efficiency, and stability of the formulation.7,13,20 Since each drug is unique in terms of physical and chemical properties, it is unlikely that any single core-forming block will allow for maximum solubilization of all drugs. Therefore, there is a need to continue to design new copolymer systems with different core-forming blocks in order to service the wide range of drugs that require formulation. To this point, the hydrolyzable polyesters PCL and PDLLA have been the most common hydrophobic building blocks for preparation of micellar delivery systems.7,8,10,15,16,19,21-23 Numerous reports have included the evaluation of PEG-b-PCL copolymer micelles for delivery of drugs such as paclitaxel, ellipticine, and doxorubicin.7,19,23 Yet to date, few studies have examined a structurally similar lactone derivative, δ-valerolactone, for use in drug delivery. Implants and microspheres formed from homopolymer or copolymers of poly(δ-valerolactone) (PVL) have been investigated.24-28 Lin et al. have also reported on the stability and drug release properties of a micelle system formed from PVL-b-PEG-b-PVL triblock copolymers.29 Yet micelle systems formed from PEG-b-PVL diblock copolymers have not

10.1021/bm050451h CCC: $30.25 © 2005 American Chemical Society Published on Web 10/05/2005

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been explored for drug delivery. The CMC values for diblock copolymers are predicted to be lower than that of triblock copolymers with the same total molecular weight and composition or hydrophobic hydrophilic balance (HLB).30 A lower CMC value for the diblock copolymers provides an indication that these micelles are thermodynamically more stable than triblock copolymer micelles, an attribute that is particularly advantageous for a delivery system to be employed for intraveneous (i.v.) administration of drugs.4,31 Diblock copolymer micelles are also expected to solubilize more drug than micelles formed from triblock copolymers of the same molecular weight and composition.30 Therefore, the focus of the present work is the synthesis of diblock copolymers of MePEG-b-PVL and the evaluation of MePEGb-PVL block copolymer micelles for solubilization of the hydrophobic drug paclitaxel. These studies include the metal-free synthesis of a series of MePEG-b-PVL copolymers wherein the length of the hydrophilic and hydrophobic blocks are varied. The synthesis and composition of the copolymers are confirmed by 1H NMR and gel permeation chromatography (GPC), whereas the thermal properties are evaluated using differential scanning calorimetry (DSC). In addition, the critical micelle concentrations (CMC) of the copolymers are evaluated in order to assess the thermodynamic stability of the micelles. The anticancer agent, paclitaxel, is incorporated into the micelles, and the drug loading efficiency is measured. The biological activity of paclitaxel formulated in the MePEGb-PVL micelles is assessed in two human cancer cell lines. Finally, the in vitro cytotoxicity of the MePEG-b-PVL copolymers is evaluated in CHO-K1 fibroblast cells, and the in vitro degradation of the micelles is measured in the absence and presence of lipase. Overall, it is found that the MePEG-b-PVL copolymers are suitable as a new biocompatible, biodegradable material for formulation of hydrophobic drugs. Experimental Section Materials. Methoxy-terminated poly(ethylene glycol) (MePEG, Mn ) 2000, Mw/Mn ) 1.06 and Mn ) 5000, Mw/ Mn ) 1.06 as determined by SEC) was obtained from SigmaAldrich (Oakville, Ontario) and dried twice by azeodistillation of toluene. Hydrogen chloride (1.0 M in diethyl ether), 1,6-diphenyl-1,3,5-hexatriene (DPH), and lipase candida rugosa were also purchased from Sigma-Aldrich and used without further purification. Dichloromethane obtained from Sigma-Aldrich and δ-valerolactone (VL, 99%) from Acros Organics were dried under calcium hydride and distilled before use. Other solvents such as ethyl ether, chloroform, and acetonitrile were used as received. Synthesis of MePEG-b-PVL Block Copolymers. The block copolymers of MePEG-b-PVL were synthesized following a reported procedure.32 In a typical experiment, 5.0 g of MePEG (2.5 mmol, Mn ) 2000, Mw/Mn ) 1.06) was added to a flame-dried flask and dried twice by toluene azeodistillation. Subsequently, 50 mL of dichloromethane (dried by calcium hydride) and 5.0 g of δ-valerolactone (50.0 mmol, distilled over calcium hydride) were added and cooled

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to 0 °C in an ice bath. A total of 7.5 mL of hydrogen chloride (7.5 mmol, 1.0 M in diethyl ether) was then added to the solution and maintained at this temperature for 3 h. A total of 0.5 mL of triethylamine was then added to terminate the reaction. The precipitate of triethylamine:hydrogen chloride was removed by filtration. Finally, the copolymer was collected by precipitation in diethyl ether and dried under vacuum at room temperature. The yield of the copolymer was found to be 9.5 g (i.e. 95%). Synthesis of MePEG-b-PCL Block Copolymer. The MePEG-b-PCL diblock copolymer was synthesized using a similar procedure to that employed for preparation of the MePEG-b-PVL copolymers. The detailed synthetic procedure and characterization is outlined elsewhere.9 The molecular weight of the MePEG block and the PCL block were both 2000 g/mol (i.e., 2k-MePEG-b-2k-PCL). Characterization of MePEG-b-PVL Copolymers. 1H NMR spectra were obtained on a Gemini 200 spectrometer (200 MHz for 1H) in CDCl3 solvent. Chemical shifts were reported in ppm with CHCl3 as the internal standard. GPC measurements were carried out at room temperature using a Waters 590 liquid chromatography system equipped with three Waters Styragel HR 4E columns and a 410 differential refractometer detector. THF with 1% triethylamine was used as the solvent at a flow rate of 1.0 mL/min at 40°C. Narrow polystyrene standards (Polysciences Inc., Warrington, PA) were used to generate a GPC calibration curve. The data obtained were recorded and manipulated using the Windowsbased Millenium 2.0 software package (Waters Inc., Milford, MA). Thermal analysis of the copolymers was performed on a DSC Q100 (TA Instruments Inc., New Castle, DE) under nitrogen at a heating rate of 5°C per minute. Measurement of Critical Micelle Concentrations. The CMCs of the MePEG-b-PVL copolymers were determined by an established fluorescence-based method.33 In short, an aliquot of a DPH stock solution prepared in chloroform was added into individual glass vials such that the final concentration of DPH in each solution was 1 mg/L. Subsequently, the MePEG-b-PVL copolymer stock solutions, also prepared in chloroform, were added to the DPH-containing vials resulting in copolymer concentrations that ranged from 0.125 to 625 mg/L. The samples were vortexed and dried thoroughly under nitrogen. The dried vials were then heated to 60 °C, and 1 mL of double distilled water was added to each vial. All hydrated samples were stirred in the dark for 2 h at 60 °C and then overnight at room temperature. The fluorescence emission of the samples was measured at 430 nm (λex ) 350 nm) with a dual-scanning microplate spectrofluorometer (Spectra GeminiXS, Molecular devices, Sunnyvale, CA). Preparation of Copolymer Micelles. Aliquots of a block copolymer stock solution in acetonitrile, or a copolymer and paclitaxel solution for preparation of the drug-loaded micelles, were added to glass vials and allowed to stir for 4 h at room temperature. The solvent was subsequently removed under nitrogen. The dried copolymer or paclitaxel-copolymer films were then heated to 60 °C and hydrated with 1 mL of phosphate buffer saline (0.01 M PBS, pH ) 7.4). The paclitaxel content and size distributions of the micelle

MePEG-b-PVL for Formulation of Hydrophobic Drugs

solutions were measured following rehydration using a UVbased assay and dynamic light scattering (DLS), respectively (methods described below). The micelle solutions were then centrifuged at 1000 rpm for 2 min (Eppendorf, Centrifuge 5804 R) in order to remove any drug that had precipitated. It had been determined that these conditions (i.e., 1000 rpm for 2 min) did not cause sedimentation of the larger copolymer aggregates present in some solutions. The paclitaxel content and size distribution of the supernatant were evaluated following centrifugation. In a separate series of studies, following preparation, the micelle solutions were filtered through a 0.22 µm Millipore filter in order to remove particles greater than 220 nm in diameter, and the paclitaxel content as well as size distribution were evaluated. Size of Paclitaxel-Loaded Micelles. The hydrodynamic diameter of micelles was determined by dynamic light scattering (DLS) at an angle of 90° and a temperature of 25 °C (90Plus Particle Size Analyzer, Brookhaven Instruments Corporation; Holtsville, NY). The sample solutions were diluted to a copolymer concentration of 8 mg/mL prior to analysis. The instrument was equipped with BIC Particle Sizing software, which determines the effective mean diameters of particles by cumulant analysis. Determination of Paclitaxel Loading Content. The paclitaxel loading content was determined by a UV assay. A linear calibration curve was obtained for paclitaxel in acetonitrile at concentrations ranging from 1.5 to 75 µg/mL. Drug-loaded micelle solutions were diluted in acetonitrile to concentrations within the linear region of the calibration curve. The absorbance was measured with a UV-visible spectrophotometer (Spectronic Unicam) at λ ) 225 nm. In Vitro Biological Activity of Free Paclitaxel and Paclitaxel Formulated in Micelles. MCF-7 breast cancer cells and A2780 ovarian cancer cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) and RPMI 1640 medium, respectively. Cells were allowed to grow in a monolayer in a tissue culture flask incubated at 37°C in 5% CO2 and 90% relative humidity. When cells were grown to 80% confluency, cells were counted and seeded in a 96well plate at a cell density of 5000 cells/well. The cells were incubated overnight prior to treatment with free paclitaxel or paclitaxel-loaded micelles for 24 h. Paclitaxel dissolved in sterile DMSO was added to the cells at a concentration of 3 mg/L in triplicate. The amount of DMSO in each well was kept constant at 1% (v/v). Paclitaxel-loaded 2k-MePEGb-2k-PVL micelles in PBS were filtered and added to the cells at a paclitaxel concentration of 3 mg/L and copolymer concentration of 30 mg/L. The amount of PBS in each well was also kept constant at 1% (v/v). Cell viability was evaluated using the MTT assay. Specifically, 10 µL of 5 mg/mL of thiazolyl blue tetrazolium bromide solution (in 0.01 M sterilized PBS, pH ) 7.4) was added to each well, and the samples were incubated for 4 h. Following incubation, 100 µL of cell extraction buffer (20% w/v SDS in a solution of 47.5% v/v DMF, 47.5% v/v H2O, 2.5% v/v of 80% acetic acid, and 2.5% v/v of 1N HCl) was added to each well to dissolve the MTT crystals. Cell viability was measured by optical absorbance at 570 nm using a Spectra Max plus microplate reader (Molecular devices, Sunnyvale,

Biomacromolecules, Vol. 6, No. 6, 2005 3121 Table 1. Molecular Weights, Thermal Properties, and the Critical Micelle Concentrations of the Six MePEG-b-PVL Diblock Copolymers that Were Synthesized with Different MePEG and PVL Block Lengths

Tg Tm CMC b entry macroinitiator Mn,GPC Mn,PVLa Mw/Mn (°C) (°C) (mg/L) 1 2 3 4 5 6

MePEG2k MePEG2k MePEG2k MePEG5K MePEG5K MePEG5K

3350 3550 5940 8540 12 400 15 200

550 950 2000 1100 2600 4880

1.05 1.10 1.12 1.08 1.14 1.12

-51

-50

50 49 47 58 56 53

176 80.4 23.3 161 73.3 35.3

a M 1 b n,PVL is determined from H NMR data. CMC is measured via a fluorescence-based method using DPH as the probe.

CA). The percent cell viability was then calculated relative to the cells that were treated with 1% DMSO or 1% PBS as the control. In Vitro Cytotoxicity of Copolymers. CHO-K1 fibroblast cells were maintained in DMEM. The DMEM was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (100 U/mL penicillin G and 100 µg/mL streptomycin). The cells were first seeded in a 96-well plate with a cell density of 10 000 cells/well. Following a 24-h incubation period, the growth media was removed and replaced with 150 µL of fresh media that contained 2k-MePEG-b-2k-PVL copolymer in concentrations ranging from 4 to 1000 mg/L in triplicate. In addition, a sterilized latex glove (2 mm × 2 mm) was used as the positive control.34 The cells were incubated for 24 h, and the cell viability was assessed using the MTT assay as described above. In Vitro Degradation of Micelles. The in vitro degradation of MePEG-b-PVL micelles was evaluated using a procedure described in detail elsewhere.35 Briefly, the block copolymer (Table 1, entry 3) was dissolved in DMF at a concentration of 100 mg/mL and then dried under nitrogen to form a copolymer film. The film was hydrated with warm PBS or PBS containing lipase at a concentration of 0.1 mg/ mL to give a final copolymer concentration of 10 mg/mL. The micelle solutions were incubated at 37 °C, and an aliquot of 200 µL was removed at designated time points for analysis of degradation. The aliquot of sample collected was lyophilized (FreeZone 6 L Freeze-Dry System, Labconco Corp., Kansas City, MO) and resuspended in THF (with 1% triethylamine) for GPC analysis. The in vitro degradation of MePEG-b-PCL copolymer micelles was also investigated in the absence of lipase. Degradation experiments were conducted in triplicate. Results Synthesis of MePEG-b-PVL Diblock Copolymers. Scheme 1 outlines the synthetic procedure employed for preparation of the MePEG-b-PVL copolymers. The resulting copolymers had controlled molecular weights and relatively narrow molecular weight distributions (Mw/Mn ) 1.05-1.14). Figure 1 includes the sample GPC traces for the macroinitiator (MePEG) and the copolymer (Table 1, entry 3). Figure 2 includes the 1H NMR spectrum for a MePEG-b-PVL

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Scheme 1. Synthesis of MePEG-b-PVL Using Hydrogen Chloride as the Catalyst in Dichloromethane at 0 °C

copolymer (Table 1, entry 3), and the assignments are as follows:32 The peaks at 1.63 ppm (4H, CO-CH2-CH2CH2-CH2-O), 2.32 ppm (2H, CO-CH2-CH2-CH2CH2-O), and 4.09 ppm (2H, CO-CH2-CH2-CH2-CH2O) were assigned to the PVL block, whereas the peaks at 3.40 ppm (CH3-O) and 3.61 ppm (4H, O-CH2-CH2-O) were assigned to the MePEG block. The composition of the block copolymers were calculated from the relative intensities of the PVL methylene proton signal at 4.09 ppm and the MePEG ethylene proton signal at 3.61 ppm. The compositions of the six MePEG-b-PVL diblock copolymers with varying MePEG and PVL block lengths are outlined in Table 1.

Figure 1. GPC traces for (trace a) 2k-MePEG (Mn ) 2000, Mw/Mn ) 1.06); (trace b) 2k-MePEG-b-2k-PVL block copolymer (Table 1, entry 3; MnGPC ) 5940, Mw/Mn ) 1.12).

Thermal Properties of MePEG-b-PVL Copolymers. The glass transition temperatures (Tg) and melting temperatures (Tm) of the copolymers as determined by DSC analysis are listed in Table 1. The Tg for both 2k-MePEG-b-2k-PVL (Table 1, entry 3) and 5k-MePEG-b-4.9k-PVL (Table 1, entry

Figure 2.

1H

6) copolymers was found to be approximately -50 °C, whereas Tgs for the other copolymers (Table 1, entries 1-2 and entries 4-5) were not observed. Figure 3 includes the thermograms of the copolymers (trace a-c, trace e-g) as well as the thermograms of 2k-MePEG (trace h) and 5kMePEG (trace d). Figure 3 and Table 1 illustrate that, as the PVL content of the 2k-MePEG and 5k-MePEG series of copolymers is decreased, the Tm’s of the copolymers increase and approach the Tm of MePEG of the same block length. Critical Micelle Concentrations of MePEG-b-PVL Copolymers. As listed in Table 1, the CMC values for the 2k-MePEG and 5k-MePEG series of copolymers range from 23.3-176 mg/L and 35.3-161 mg/L, respectively, depending on the PVL content. In general, the CMC values were found to increase with an increase in the MePEG content within the copolymer. Evaluation of MePEG-b-PVL Micelles for Formulation of Paclitaxel. Four MePEG-b-PVL copolymers (Table 1, entry 2-3 & entry 5-6) were selected for preparation of paclitaxel-loaded micelles based on their enhanced thermodynamic stability (i.e., lower CMC). Table 2 lists the effective mean diameters of the paclitaxel-loaded micelles and the extent to which paclitaxel was solubilized in each micelle formulation. The effective mean diameters and paclitaxel content of the solutions were first measured following centrifugation to remove the drug that had precipitated. As shown in Table 2, the 5k-MePEG series of copolymers and the 2k-MePEG-b-1k-PVL copolymer formed larger aggregates with effective mean diameters of 700 ( 40 nm (5k-MePEG-b-2.6k-PVL), 460 ( 60 nm (5k-MePEGb-4.9k-PVL), and 970 ( 180 nm (2k-MePEG-b-1k-PVL). By contrast, the 2k-MePEG-b-2k-PVL copolymer resulted in micelles having an effective mean diameter of 31 ( 1

NMR spectrum of 2k-MePEG-b-2k-PVL block copolymer (Table 1, entry 3).


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Figure 3. DSC thermograms for (a) 5k-MePEG-b-4.9k-PVL block copolymer (Table 1, entry 6); (b) 5k-MePEG-b-2.6k-PVL block copolymer (Table 1, entry 5); (c) 5k-MePEG-b-1.1k-PVL block copolymer (Table 1, entry 4); (d) 5k-MePEG (Tm ) 59°C); (e) 2k-MePEG-b-2k-PVL block copolymer (Table 1, entry 3); (f) 2k-MePEG-b-1k-PVL block copolymer (Table 1, entry 2); (g) 2k-MePEG-b-0.6k-PVL block copolymer (Table 1, entry 1); (h) 2k-MePEG (Tm ) 53°C). Table 2. Effective Mean Diameters of Paclitaxel-Loaded MePEG-b-PVL Micelles, and the Paclitaxel Content in the MePEG-b-PVL Micelle Solutions effective mean diameter of drug-loaded micelles (nm)

paclitaxel concentration (mg/mL)

entrya

copolymer

following centrifugationb

following filtrationc

following centrifugationb

following filtrationc

2 3 5 6

2k-MePEG-b-1k-PVL 2k-MePEG-b-2k-PVL 5k-MePEG-b-2.6k-PVL 5k-MePEG-b-4.9k-PVL

970 ( 180 31 ( 1 700 ( 40 460 ( 60

200 ( 4 31 ( 1 225 ( 3 138 ( 1

5.8 9.2 9.4 9.3

3.7 9.2 1.0 0.3

a The entry number corresponds to the entry number in Table 1. b The amount of paclitaxel solubilized by the MePEG-b-PVL micelles was measured following centrifugation at 1000 rpm for 2 min. The initial amount of paclitaxel added was 10 mg/mL and the copolymer concentration was 100 mg/mL for all micelle solutions. c The amount of paclitaxel solubilized by the MePEG-b-PVL micelles was measured following filtration through a 0.22 µm filter.

nm and a narrow size distribution. The paclitaxel content following centrifugation was approximately 9 mg/mL for formulations prepared from the 5k-MePEG series of copolymers and the 2k-MePEG-b-2k-PVL copolymer. The presence of precipitate was evidenced in the formulation prepared from the 2k-MePEG-b-1k-PVL copolymer, and following centrifugation, the paclitaxel content was found to be 5.8 mg/ mL. The effective mean diameters and paclitaxel content of the copolymer solutions was also measured following filtration through a 0.22 µm filter. In this way, the amount of paclitaxel incorporated in copolymer aggregates with diameters of approximately 200 nm or less (i.e., the suitable size range for particles to be employed for i.v. administration36) was determined. As shown in Table 2, following filtration, the effective mean diameters of aggregates present in the 5k-MePEG-b-2.6k-PVL, 5k-MePEG-b-4.9k-PVL, and 2kMePEG-b-1k-PVL copolymer solutions were 225 ( 3, 138 ( 1, and 200 ( 4 nm, respectively. However, the measured effective mean diameter for micelles formed from the 2kMePEG-b-2k-PVL copolymer, following filtration, was 31

( 1 nm. The filtration of the formulations prepared from 5k-MePEG-b-2.6k-PVL, 5k-MePEG-b-4.9k-PVL, and 2kMePEG-b-1k-PVL copolymers resulted in a decrease in the paclitaxel concentration to 1.0, 0.3, and 3.7 mg/mL, respectively. By contrast, the concentration of paclitaxel in the 2kMePEG-b-2k-PVL copolymer solution (i.e., 9.2 mg/mL) was not decreased following filtration. In Vitro Biological Activity of Paclitaxel Formulated in MePEG-b-PVL Copolymer Micelles. The in vitro biological activity of free paclitaxel and 2k-MePEG-b-2kPVL micelle formulated paclitaxel were evaluated in human MCF-7 breast and A2780 ovarian cancer cells. Following a 24-h incubation period, the percent viability was 40% and 38% for MCF-7 cells treated with free drug and micelle formulated drug, respectively. For the A2780 cells, the percent viability was 49% and 44% following treatment, for the 24-hour period, with free paclitaxel and micelle formulated paclitaxel, respectively. In Vitro Cytotoxicity of MePEG-b-PVL Copolymer. Figure 4 shows the in vitro cytotoxicity of the 2k-MePEGb-2k-PVL copolymer in the CHO-K1 fibroblast cells. The

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Figure 4. In vitro cytotoxicity of the 2k-MePEG-b-2k-PVL block copolymer (Table 1, entry 3). The cell viabilities are reported relative to controls and are expressed as a function of the copolymer concentration in CHO-K1 fibroblast cells.

2k-MePEG-b-2k-PVL copolymer was found to be biocompatible with relative cell viabilities above 75% for the copolymer concentrations investigated (4-1000 mg/L). The positive control (i.e., incubation with latex) resulted in a cell viability of 1.3% ( 0.6% at the CHO-K1 cell density employed for these studies. In Vitro Degradation of 2k-MePEG-b-2k-PVL and 2kMePEG-b-2k-PCL Copolymer Micelles. The in vitro degradation of the 2k-MePEG-b-2k-PVL micelles was evaluated in PBS in the absence and presence of lipase. The degradation of 2k-MePEG-b-2k-PVL micelles in the absence of lipase was also compared to the degradation of micelles formed from a MePEG-b-PCL diblock copolymer of similar hydrophilic and hydrophobic block lengths. Figure 5a illustrates the degradation in terms of the change in the apparent number average molecular weight as measured by GPC. In the absence of lipase, the molecular weight of the MePEG-b-PCL copolymer was decreased by approximately 15% over the 34-day period, whereas the molecular weight of the MePEG-b-PVL copolymer was reduced by 20%. In the presence of lipase, the MePEG-b-PVL copolymer had a 30% decline in molecular weight on day 34 relative to day 0. Figure 5b includes GPC traces of samples of the MePEGb-PVL copolymer collected on days 0, 4, and 34 of the degradation study carried out in the presence of lipase. The retention times were shifted to the right as the sample was incubated with lipase for longer periods of time. In addition, a second peak appeared on day 4, and the molecular weight distribution of the copolymer broadened over time. Discussion The diblock copolymers of MePEG-b-PVL were synthesized by a cationic ring opening polymerization method using MePEG as the monofunctional macroinitiator and hydrogen chloride-diethyl ether as the catalyst at 0 °C. MePEG was able to initiate VL polymerization in the presence of HCl, and following polymerization, as confirmed by GPC analysis, there was little unreacted residual MePEG. The copolymers were synthesized in a controlled manner resulting in copolymers with predicted compositions and relatively narrow molecular weight distributions. The results are agreeable with

that reported by Lou et al. for the synthesis of MePEG-bPVL copolymers.32 DSC analysis revealed that the thermal properties of the MePEG-b-PVL copolymers are largely dominated by the MePEG content. For instance, as the PVL content of the 5k-MePEG series of copolymers was decreased, corresponding to an increase in the MePEG content, the Tm of the copolymer increased from 53 to 58 °C, approaching the Tm of the 5k-MePEG (59°C). A similar trend was observed for the 2k-MePEG series of copolymers. Although the melting transition of PVL was not detectable for the diblock copolymers, the glass transition of PVL was detected in the copolymers where the ratio of the MePEG to PVL block lengths was equal to one. PVL is a semicrystalline polyester, and its Tg has been reported to range from -47 to -70 °C depending on the molecular weight and the architecture of the homopolymer.37 The two copolymers that show obvious glass transitions have similar Tg’s even though they vary in terms of the length of the PVL block (i.e., 2k vs 4.9k). These results indicate that the Tg of the diblock copolymers is dependent on the ratio of the MePEG to PVL block lengths rather than the specific length of the PVL block. The CMC of the copolymers was also found to depend on the ratio of the MePEG to PVL block lengths. The CMC is the copolymer concentration at which micellization occurs, and it provides an indication of the thermodynamic stability of the micelles. The existence of increasing hydrophobic interactions between amphiphilic copolymer chains tends to favor the formation of micelles at a lower concentration. In other words, copolymers that are more hydrophobic in nature, or equivalently with lower ratios for the hydrophilic to hydrophobic block lengths, have lower CMCs and therefore tend to form more thermodynamically stable micelles.4 The thermodynamic stability of the micelles is of importance if the system is to be used for the i.v. administration of drugs. Following i.v. administration, the micelles are subjected to significant dilution which may cause the copolymer concentration to fall below the CMC of the copolymer. In this way, it is advantageous to select micelles formed from copolymers having lower CMC values for use in drug delivery as this will enhance the stability of the formulation following administration. It should be noted that


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Figure 5. In vitro degradation of the diblock copolymers. (a) Degradation of 2k-MePEG-b-2k-PVL (Table 1, entry 3) and 2k-MePEG-b-2k-PCL block copolymer in the absence and presence of lipase. (b) GPC traces of the degradation products from 2k-MePEG-b-2k-PVL copolymer following incubation in the presence of lipase for 0 (trace a), 4 (trace b), and 34 (trace c) days.

there are a wide range of factors that influence the stability of micelle formulations in vivo and detailed discussions are provided in current reviews.4,36,38 The CMC values for the MePEG-b-PVL copolymers were found to decrease with a decrease in the ratio of the MePEG to PVL block lengths. Such a trend has been commonly observed for other diblock copolymer systems.3,4,39 In addition, for MePEG-b-PVL copolymers having similar PVL block lengths, an increase in the hydrophilic block length from 2k (Table 1, entry 2) to 5k (Table 1, entry 4) resulted in a 2-fold increase in the value for the CMC. This further confirms that the HLB of a diblock copolymer plays a significant role in determining the thermodynamic stability of micelles formed from that material. To develop a micelle formulation that would likely be stable following i.v. administration, the four MePEG-b-PVL copolymers having the lowest CMC values were selected for preliminary investigation of their ability to solubilize hydrophobic drugs. For this study, the hydrophobic anticancer agent paclitaxel, whose aqueous solubility has been reported to be approximately 1 µg/mL,40 was loaded into the MePEG-b-PVL copolymer micelles. The current commercially available formulation for paclitaxel, Taxol, consists of the drug dissolved in a Cremophor EL (CrEL) vehicle. In clinical settings, Taxol must be diluted

with buffer to a drug concentration of 0.3-1.2 mg/mL for i.v. administration.41 Due to drug dilution, a 3-h infusion is required for the administration of this formulation in order to achieve a therapeutically relevant dose. In addition, the maximum tolerated dose (MTD) of Taxol is largely attributed to the adverse effects caused by the presence of CrEL.1,42 Therefore, there is a need to explore new formulations for paclitaxel that can provide a more concentrated injectable dose, while also reducing the toxicity of the formulation. A number of different block copolymer micelle systems have been proposed for delivery of paclitaxel; yet, to date micelles formed from copolymers with PDLLA as the core-forming block have been investigated most extensively.1,8,10,11,23,43,44 The PEG-b-PDLLA micelle systems have been shown to function primarily as solubilizers for paclitaxel in that they provide a significant increase in the aqueous solubility of this drug.1,10,44 In order for the micelles to function as drug delivery vehicles they must retain the drug post-administration despite being subjected to significant dilution and exposure to the vast array of serum protein. Genexol-PM, a PEG-b-PDLLA micelle formulation of paclitaxel, is now in phase II clinical trial development.1 Paclitaxel administered to humans in this PEG-b-PDLLA formulation at a dose of 135 mg/m2 resulted in an AUC of 5473 ng‚h/mL and a t1/2β ) 12.7 h, whereas for paclitaxel administered as Taxol, the

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AUC is 9307.5 ng‚h/mL and the t1/2 ) 20.1 h.1,45 Thus, the Genexol-PM formulation acts primarily to solubilize paclitaxel, and following administration, the paclitaxel is released quite rapidly from the PEG-b-PDLLA micelles. Yet, the Genexol-PM formulation is advantageous as it provides a significant increase in the MTD for paclitaxel (i.e., MTD ) 390 mg/m2), when compared to the usual dose range of 135175 mg/m2 for paclitaxel administered as Taxol.1,45 The increase in the MTD is attributed to the better tolerability for the PEG-b-PDLLA copolymers relative to the primary excipient CrEL employed in Taxol. It is of significant interest to design copolymer micelle systems that are capable of acting as true delivery vehicles for paclitaxel and other hydrophobic drugs. The ability of micelles to act as delivery vehicles requires that they remain intact and retain their drug load following administration. The stability of the micelles depends on their thermodynamic stability, largely reflected by the CMC of the copolymer, and their kinetic stability.4 The thermodynamic stability of a micelle system may be increased by using a material that is more hydrophobic such as PVL or PCL compared to PLA of the same molecular weight.4 In addition, the kinetic stability of the micelles is influenced by both the hydrophobicity and state of the micelle core.4 The semicrystalline nature of materials such as PCL and PVL may result in micelles with enhanced kinetic stability when compared to micelles formed from copolymers with the amorphous polymer PDLLA as the core-forming block.4 In this particular series of studies, paclitaxel was selected as a suitable drug for loading into the MePEG-b-PVL micelles following consideration of the degree of compatibility or miscibility between paclitaxel and PVL. In a previous series of studies, our group had reported on calculation of partial and total solubility parameters as well as the enthalpy of mixing for polymer-drug pairs as a means to guide the design of micelle formulations.7 The enthalpy of mixing (∆Hm) for PVL and paclitaxel was calculated to be 4.5 J, whereas values for PCL-paclitaxel and PDLLApaclitaxel were 5.7 and 8.8 J, respectively. The miscibility of polymer and drug are considered to increase as the value for ∆Hm approaches zero.8 Since micelles formed from PDLLA-based copolymers had been shown to provide a significant increase in the aqueous solubility of paclitaxel, it was postulated that the PVL-based system would also enhance the solubility of this drug and may result in a more stable formulation. Indeed, evaluation of the MePEG-b-PVL copolymers for formulation of paclitaxel revealed that the 5k-MePEG series and the 2k-MePEG-b-2k-PVL copolymer could provide more than a 9000-fold increase in the apparent aqueous solubility of this drug resulting in paclitaxel concentrations of approximately 9 mg/mL. The solubilization capacity of the 2kMePEG-b-1k-PVL copolymer was limited, as some drug precipitated in solution, and the final paclitaxel concentration was only 5.8 mg/mL. As shown in Table 2, the 5k-MePEG series and the 2k-MePEG-b-1k-PVL copolymer formed large drug-containing aggregates and filtration (i.e., 0.22 µm filter) of these solutions resulted in a significant reduction in the paclitaxel concentration. For i.v. administration of drugs, it

Lee et al.

is advantageous to employ colloidal delivery systems with diameters of 200 nm or less as particles of this size have been shown to provide the most significant improvements in the circulation lifetime and degree of tumor accumulation of drugs.36 In this way, the 2k-MePEG-b-2k-PVL copolymer was found to be most suitable for preparation of an i.v. injectable formulation of paclitaxel. Following filtration, the 2k-MePEG-b-2k-PVL copolymer micelles had an effective mean diameter of 31 nm and the paclitaxel concentration was 9.2 mg/mL. In addition, it was confirmed in human MCF-7 breast and A2780 ovarian cancer cells that the biological activity of the drug was retained following formulation in the 2k-MePEG-b-2k-PVL micelles. The current dose of paclitaxel administered to patients, in the commercially available formulation Taxol, for treatment of ovarian cancer and Karposi’s sarcoma is 135-175 mg/ m2.1 In this way, administration of only 28 mL of the optimal MePEG-b-PVL micelle formulation, formed from 2k-MePEG-b-2k-PVL (10 wt/v %) with 9.2 mg/mL paclitaxel, would be required in order to provide this dose of paclitaxel to a typical 80 kg man. Furthermore, if we assume that the micelle formulation becomes homogeneously diluted in the entire blood volume then the total copolymer concentration following administration would be 440 mg/L which is at least 18-fold above the CMC of this copolymer. Therefore, On the basis of the consideration of results from evaluation of size and drug loading, the 2k-MePEG-b-2kPVL copolymer (Table 2, entry 3) was found to be the optimal material for formulation of paclitaxel and was further investigated in terms of other relevant properties for use in drug delivery. In developing a 2k-MePEG-b-2k-PVL copolymer micelle formulation, it is necessary to assess the cytotoxicity of the copolymer to ensure its suitability for biomedical use. Zeng et al. evaluated the biocompatibility of six-arm MePEG-b-PVL in CHO-K1 fibroblast cells and found that the star-shaped copolymers were indeed noncytotoxic.37 It was therefore anticipated that the MePEG-bPVL copolymers would also have no cytotoxic effect. However, several studies have shown that surfactants including linear amphiphilic copolymers can intercalate into cell membranes, increasing the membrane permeability, leading to subsequent cell death.46-48 In the present study, the 2kMePEG-b-2k-PVL copolymer did not result in any significant toxicity when incubated with the CHO-K1 cells at concentrations below and above the CMC. The cell density of 10 000 cells/well employed in these studies was shown to be sensitive to the MTT assay as demonstrated by the positive control (i.e., latex incubation). In this way, it is suggested that the 2k-MePEG-b-2k-PVL copolymer is an appropriate excipient for drug formulation and delivery. Finally, it is known that aliphatic polyesters including PCL and PVL are degraded via hydrolysis and that the rate of biodegradation is accelerated in the presence of lipase.24,26 The catalytic effect of lipase was also observed in the present study in which the degradation of the 2k-MePEG-b-2k-PVL copolymer was measurable as early as the second day of incubation in the presence of lipase, whereas the onset of degradation in the absence of lipase was not seen until the sixth day. In addition, results from the degradation study


indicate that MePEG-b-PCL and MePEG-b-PVL micelles are degraded in a similar manner except that the degradation of MePEG-b-PCL proceeds at a slower rate. The slower rate of degradation for MePEG-b-PCL was expected as it has been reported that PCL does not degrade as quickly as PVL both in vitro and in vivo.24,49 Throughout the degradation study, the main population of copolymers exists as micelles in solution, thus, the ester bond connecting the MePEG block to the PVL or PCL block is initially more susceptible to hydrolytic cleavage than the ester bonds in the PVL or PCL blocks. However, the micelles are dynamic systems with exchange occurring between the population of single copolymer chains and micellized copolymer. Also, MePEG is a non-hydrolytic polymer; therefore, some hydrolytic cleavage of the PVL or PCL blocks is also expected. In this way, the broadening of the GPC peaks in Figure 5b are likely a result of the simultaneous cleavage of the MePEG-PVL or MePEG-PCL junctions and the random polymer scission of the PVL or PCL blocks. The biodegradability of MePEGb-PVL copolymer suggests that the formulation should be lyophilized and stored in powder form in order to maintain its long-term stability. Conclusion Series of MePEG-b-PVL diblock copolymers having varied PVL block lengths were synthesized via a metal-free cationic polymerization method using 2k-MePEG and 5kMePEG as macroinitiators. The HLB of the copolymers was found to influence their thermal properties and micellization characteristics. These results suggest that the MePEG-b-PVL copolymers can be tailor-made to accommodate specific formulation requirements. The MePEG-b-PVL copolymers were able to form nanosized micelles and enhance the apparent aqueous solubility of paclitaxel by more than 9000fold and were shown to be biocompatible and biodegradable. Overall, these results demonstrate that these copolymers may be suitable for delivery of paclitaxel. In vivo evaluation of the pharmacokinetics and biodistribution of paclitaxel following administration in the MePEG-b-PVL micelles will reveal if this system is capable of acting as a true delivery vehicle for this drug. Acknowledgment. Dr. C. Allen is grateful to NSERC for the support of this research. The authors also thank Dr. M. V. Sefton for kindly donating the CHO-K1 fibroblast cells, Dr. J. P. Uetrecht for use of the fluorescence microplate reader, and Dr. Y. L. Cheng for use of the Particle Size Analyzer. References and Notes (1) Kim, T. Y.; Kim, D. W.; Chung, J. Y.; Shin, S. G.; Kim, S. C.; Heo, D. S.; Kim, N. K.; Bang, Y. J. Clin. Cancer Res. 2004, 10, 37083716. (2) Matsumura, Y.; Hamaguchi, T.; Ura, T.; Muro, K.; Yamada, Y.; Shimada, Y.; Shirao, K.; Okusaka, T.; Ueno, H.; Ikeda, M.; Watanabe, N. Br. J. Cancer 2004, 91, 1775-1781. (3) Alakhov, V.; Klinski, E.; Li, S. M.; Pietrzynski, G.; Venne, A.; Batrakova, E.; Bronitch, T.; Kabanov, A. Colloid Surf. B 1999, 16, 113-134. (4) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf. B: Biointerfaces 1999, 16, 3-27.

Biomacromolecules, Vol. 6, No. 6, 2005 3127 (5) Bae, J.; Kim, J. K.; Oh, N. K.; Lee, M. Macromolecules 2005, 38, 4226-4230. (6) Hwang, M. J.; Suh, J. M.; Bae, Y. H.; Kim, S. W.; Jeong, B. Biomacromolecules 2005, 6, 885-890. (7) Liu, J. B.; Xiao, Y. H.; Allen, C. J. Pharm. Sci. 2004, 93, 132-143. (8) Liggins, R. T.; Burt, H. M. AdV. Drug DeliVery ReV. 2002, 54, 191202. (9) Liu, J. B.; Zeng, F. Q.; Allen, C. J. Controlled Release 2005, 103, 481-497. (10) Burt, H. M.; Zhang, X. C.; Toleikis, P.; Embree, L.; Hunter, W. L. Colloids Surf. B: Biointerfaces 1999, 16, 161-171. (11) Zhang, X. C.; Jackson, J. K.; Burt, H. M. Int. J. Pharm. 1996, 132, 195-206. (12) La, S. B.; Okano, T.; Kataoka, K. J. Pharm. Sci. 1996, 85, 85-90. (13) Kataoka, K.; Matsumoto, T.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Fukushima, S.; Okamoto, K.; Kwon, G. S. J. Controlled Release 2000, 64, 143-153. (14) Yokoyama, M.; Satoh, A.; Sakurai, Y.; Okano, T.; Matsumura, Y.; Kakizoe, T.; Kataoka, K. J. Controlled Release 1998, 55, 219229. (15) Allen, C.; Han, J. N.; Yu, Y. S.; Maysinger, D.; Eisenberg, A. J. Controlled Release 2000, 63, 275-286. (16) Allen, C.; Yu, Y.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 1998, 9, 564-572. (17) Jette, K. K.; Law, D.; Schmitt, E. A.; Kwon, G. S. Pharm. Res. 2004, 21, 1184-1191. (18) Lim Soo, P.; Luo, L.; Maysinger, D.; Eisenberg, A. Langmuir 2002, 18, 9996-10004. (19) Shuai, X. T.; Ai, H.; Nasongkla, N.; Kim, S.; Gao, J. M. J. Controlled Release 2004, 98, 415-426. (20) Li, Y.; Kwon, G. S. Pharm. Res. 2000, 17, 607-611. (21) Adams, M. L.; Lavasanifar, A.; Kwon, G. S. J. Pharm. Sci. 2003, 92, 1343-1355. (22) Kang, N.; Leroux, J. C. Polymer 2004, 45, 8967-8980. (23) Shuai, X.; Merdan, T.; Schaper, A. K.; Xi, F.; Kissel, T. Bioconjug Chem. 2004, 15, 441-448. (24) Imasaka, K.; Yoshida, M.; Fukuzaki, H.; Asano, M.; Kumakura, M.; Mashimo, T.; Yamanaka, H.; Nagai, T. Int. J. Pharm. 1991, 68, 8795. (25) Lin, S. Y.; Chen, K. S.; Teng, H. H.; Li, M. J. J. Microencapsulation 2000, 17, 577-586. (26) Fukuzaki, H.; Yoshida, M.; Asano, M.; Kumakura, M.; Mashimo, T.; Yuasa, H.; Imai, K.; Yamanaka, H.; Kawaharada, U.; Suzuki, K. J. Controlled Release 1989, 10, 293-303. (27) Endoh, H.; Hagita, Y.; Seki, T.; Kawaguchi, T.; Juni, K. Yakuzaigaku 2000, 60, 119-127. (28) Imasaka, K.; Yoshida, M.; Fukuzaki, H.; Asano, M.; Kumakura, M.; Mashimo, T.; Yamanaka, H.; Nagai, T. Chem. Pharm. Bull. 1991, 39, 2096-2099. (29) Lin, W. J.; Juang, L. W.; Lin, C. C. Pharm. Res. 2003, 20, 668673. (30) Nagarajan, R.; Ganesh, K. J. Colloid Interface Sci. 1996, 184, 489499. (31) Allen, C.; Eisenberg, A.; Maysinger, D. S.T.P. Pharm. Sci. 1999, 9, 139-151. (32) Lou, X.; Detrembleur, C.; Jerome, R. Macromolecules 2002, 35, 1190-1195. (33) Zhang, X. C.; Jackson, J. K.; Burt, H. M. J. Biochem. Biophys. Methods 1996, 31, 145-150. (34) Lickorish, D.; Ramshaw, J. A. M.; Werkmeister, J. A.; Glattauer, V.; Howlett, C. R. J. Biomed. Mater. Res. A 2004, 68A, 19-27. (35) Zeng, F. Q.; Liu, J. B.; Allen, C. Biomacromolecules 2004, 5, 18101817. (36) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. ReV. 2001, 53, 283-318. (37) Zeng, F.; Lee, H.; Chidiac, M.; Allen, C. Biomacromolecules 2005, 6, 2140-2149. (38) Kwon, G. S.; Kataoka, K. AdV. Drug DeliVery ReV. 1995, 16, 295309. (39) Letchford, K.; Zastre, J.; Liggins, R.; Burt, H. Colloids Surf. B: Biointerfaces 2004, 35, 81-91. (40) Liggins, R. T.; Hunter, W. L.; Burt, H. M. J. Pharm. Sci. 1997, 86, 1458-1463. (41) Trissel, L. A. Handbook on injectable drugs, 11th ed.; American Society of Health-System Pharmacists: Bethesda, MD, 2001. (42) Hamaguchi, T.; Matsumura, Y.; Suzuki, M.; Shimizu, K.; Goda, R.; Nakamura, I.; Nakatomi, I.; Yokoyama, M.; Kataoka, K.; Kakizoe, T. Br. J. Cancer 2005, 92, 1240-1246.

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(43) Soga, O.; van Nostrum, C. F.; Fens, M.; Rijcken, C. J. F.; Schiffelers, R. M.; Storm, G.; Hennink, W. E. J. Controlled Release 2005, 103, 341-353. (44) Hamaguchi, T.; Matsumura, Y.; Suzuki, M.; Shimizu, K.; Goda, R.; Nakamura, I.; Nakatomi, I.; Yokoyama, M.; Kataoka, K.; Kakizoe, T. Br. J. Cancer 2005, 92, 1240-1246. (45) Gianni, L.; Kearns, C. M.; Giani, A.; Capri, G.; Vigano, L.; Locatelli, A.; Bonadonna, G.; Egorin, M. J. J. Clin. Oncol. 1995, 13, 180190.

Lee et al. (46) Batrakova, E.; Lee, S.; Li, S.; Venne, A.; Alakhov, V.; Kabanov, A. Pharm. Res. 1999, 16, 1373-1379. (47) Zastre, J.; Jackson, J.; Bajwa, M.; Liggins, R.; Iqbal, F.; Burt, H. Eur. J. Pharm. Biopharm. 2002, 54, 299-309. (48) Xia, W. J.; Onyuksel, H. Pharm. Res. 2000, 17, 612-618. (49) Lin, W.-J. J. Biomed. Mater. Res. 1999, 47, 420-423.

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