Amphiphilic Toothbrushlike Copolymers Based on Poly(ethylene

Biomacromolecules 2010, 11, 1331–1338

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Amphiphilic Toothbrushlike Copolymers Based on Poly(ethylene glycol) and Poly(ε-caprolactone) as Drug Carriers with Enhanced Properties Wenlong Zhang,† Yanli Li,‡ Lixin Liu,*,‡ Qiquan Sun,§ Xintao Shuai,*,§ Wen Zhu,† and Yongming Chen*,† State Key Laboratory of Polymer Physics and Chemistry, Joint Laboratory of Polymer Sciences and Materials, Institute of Chemistry, The Chinese Academy of Sciences and Beijing National Laboratory for Molecular Science, Beijing 100190, China, College of Life Sciences, Graduate University of Chinese Academy of Sciences, Yuquanlu 19A, Beijing, 100049, China, and Center of Biomedical Engineering, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China Received February 2, 2010; Revised Manuscript Received April 7, 2010

Amphiphilic poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-g-poly(ε-caprolactone)) (PEG-b-P(HEMAg-PCL)) toothbrushlike copolymers were synthesized and evaluated as drug delivery carriers. Two toothbrushlike polymers were synthesized via ring-opening polymerization of ε-caprolactone (CL) initiated by poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate) (PEG-b-PHEMA) macromolecular initiators, and their molecular structures and physical properties were characterized using 1H NMR, gel permeation chromatography (GPC), and differential scanning calorimetric analysis (DSC). The melting points and crystallizable temperature have been decreased obviously, implying that the PCL cores of PEG-b-P(HEMA-g-PCL) toothbrushlike copolymer micelles with shorter PCL segments were unlikely to crystallize at room temperature for drug delivery application. Also the micellization properties of toothbrushlike copolymers in aqueous solution were investigated by fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Compared with the micelles from linear PEG-b-PCL block copolymers, the micelles of PEG-b-P(HEMA-g-PCL)s exhibited higher loading capacity to the anticancer drug, doxorubicin (DOX), and the drug-loaded micelles were highly stable in aqueous solution. In vitro DOX release data and confocal laser scanning microscopy (CLSM) studies showed that DOX-loaded toothbrushlike copolymer micelles could be effectively internalized by bladder carcinoma EJ cells, and the DOX could be released into endocytic compartments and finally transported to the nucleus. Such toothbrushlike copolymer micelles can be analogues of linear PEG-b-PCL diblock copolymers, but demonstrated better properties of loading and release due to their hydrophobic PCL cores do not crystallize at delivery conditions.

Introduction Over the past two decades, polymeric nanoparticles including micelles formed from amphiphilic block copolymers have received significant attention as nanocarriers to deliver drugs to the target sites via the enhanced permeability and retention effect.1-5 Lipophilic drug molecules can be incorporated into the hydrophobic core of polymeric micelles by physical entrapment, while the hydrophilic shell composed of flexible polymers provides steric protection and helps these nanoparticles to escape from the reticuloendothelial system (RES) uptake after intravenous administration.6,7 A wide variety of natural and synthetic polymers have been used in the drug delivery system.8 Among them, amphiphilic PEG-b-PCL block copolymers have been widely investigated as drug carriers due to their biocompatibility, biodegradability, and nonimmunogenicity.9-12 The polymeric nanoparticles prepared from the self-assembled PEG-b-PCLs exhibit a core-shell architecture, in which the biodegradable PCL segments aggregate to form a core to entrap hydrophobic drugs and the PEG segments used as hydrophilic shell to enhance the circulation time in blood. * To whom correspondence should be addressed. E-mail: [email protected] (L.L.); [email protected] (X.S.); [email protected] (Y.C.). † The Chinese Academy of Sciences and Beijing National Laboratory for Molecular Science. ‡ Graduate University of Chinese Academy of Sciences. § Sun Yat-Sen University.

Besides traditional linear block polymers, polymers with other architectures such as dendrimers,13 linear-b-dendritic block copolymers,14 star block copolymers,15-18 and brush copolymers19,20 have also investigated as carriers of drug delivery systems. Numerous studies have revealed that these branched polymers exhibited significantly different physical properties from linear polymers, such as melt rheology, mechanical behavior, and solution properties.21 The architecture of the polymers also has a great influence on the physicochemical properties of the polymer itself in vivo application as well as other aspects of the drug delivery, including drug loading efficiency, drug release kinetics, biodistribution, and even interaction with specific tissues or cells in vivo.22 Therefore, more extensive work is required to optimize polymer architecture to fabricate novel drug delivery systems. The performance of polymeric micelles as drug carriers is decided by the loading capacity, size, circulation time, stability, release kinetics, and biodistribution. Drug loading content (DLC) and drug encapsulation efficiency of polymeric micelles were affected by the affinity of the loaded drugs with the micellar cores, the volume of hydrophobic cores, and the solubility of drugs in water.23 The physical entrapment of hydrophobic drugs into polymeric micelles is driven by the hydrophobic interactions between the drugs and the hydrophobic segments of polymers. In general, the micelle cores constructed with longer PCL segments can encapsulate more drug molecules, so the DLC

10.1021/bm100116g  2010 American Chemical Society Published on Web 04/20/2010

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increased with an increase of the PCL length.24 However, longer PCL segments lead to a higher degree of crystallinity, resulting in low DLC and poor degradability in body because only the amorphous PCL phase is likely to accommodate drug molecules.25 So, the higher DLC may be expected by decrease of the crystallization ability of the PCL segments. However, as far as we know, little efforts have been paid, except by copolymerization of CL and lactide.26,27 It is known that the PCLs with either low polymerization degree or grafted structure show a decreased crystallization temperature.28-30 In this work, we described the synthesis and micellar characterization of novel amphiphilic toothbrushlike copolymers poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-g-poly(ε-caprolactone)) (PEG-b-P(HEMA-g-PCL). These toothbrushlike copolymers were synthesized by ring-opening polymerization (ROP) of ε-caprolactone initiated by PEG-bPHEMA. These toothbrushlike copolymers have the similar EG/ CL ratio to traditional linear PEG-b-PCL, however, the PCL blocks are composed of many densely grafted short PCL grafts, hence, their crystallization is restricted. Differential scanning calorimetric (DSC) analysis showed that these toothbrushlike copolymers had no crystallinity at room temperature. Compared to the linear PEG-b-PCL with comparable EG/CL ratio, micelles formed from the toothbrushlike copolymers exhibited higher DLC of anticancer doxorubicin (DOX), and the loaded drugs showed extraordinary stability in an aqueous solution. Meanwhile, both the number and the length of PCL segments of these toothbrushlike copolymers can be easily controlled during synthesis, thus, the crystallinity of PCL and DLC can be tuned. The cytotoxicity and cellular uptake of the DOX-loaded toothbrushlike copolymer micelles against bladder carcinoma EJ cells were also investigated using MTT assay and confocal laser scanning microscopy (CLSM).

Experimental Section Materials. 2-Hydroxyethyl methacrylate (HEMA; 99%, Aldrich) was purified by passing through a column filled with basic alumina to remove inhibitor. ε-Caprolactone (CL, Aldrich) was dried over CaH2 and distilled before use. Stannous octoate (SnOct2) and 2-bromoiosobutyryl bromide (BrBBr) were purchased from Sigma-Aldrich. Copper(I) bromide and 2,2′-bipyridine (bpy) were used as received. Monomethoxy poly(ethylene glycol) with Mn ) 5000 g/mol (PEG113, Fluka) was dried by azeotropic distillation in the presence of toluene. N,N-dimethylformamide (DMF) was distilled from CaH2. Doxorubincin hydrochloride (DOX · HCl) was purchased from Zhejiang Haizheng Co., China, and was deprotonated at pH 9.6 to obtain the hydrophobic DOX with sodium hydroxide solution. Toluene, diethyl ether, tetrahydrofuran (THF), dichloromethane, and triethylamine (TEA) were purchased from Beijing Corporation of Chemical Reagent and used as received. Linear PEG-b-PCL diblock copolymer was synthesized using PEG113 as initiator of ROP of CL, and the degree of polymerization (DP) of PCL was 102 calculated from its 1H NMR spectrum with the PCL weight fraction (fwPCL) of 0.70, and it was further named as PEG113-b-PCL102.31 PEG113-Br macroinitiator was synthesized by a reaction between PEG113 and BrBBr according the literature.32,33 Synthesis of PEG-b-PHEMA Diblock Polymer. A typical polymerization procedure was as follow. A dry Schlenk flask with a magnetic stirrer was charged with PEG113-Br (2.06 g, 0.4 mmol), HEMA (2.6 g, 20 mmol), bpy (124 mg, 0.8 mmol), and 5 mL of methanol as solvent. The flask was degassed with three freeze-evacuate-thaw cycles; CuBr (57.4 mg, 0.4 mmol) was added into the reaction system under the protection of nitrogen atmosphere. Next, the polymerization was performed at 40 °C for 1 h. After being rapidly cooled to room temperature, the reaction flask was opened to air, and the crude product was precipitated in an excess of diethyl ether, and then redissolved in

Zhang et al. 5 mL of methylene chloride and passed through a neutral oxide alumina column to remove the copper catalysts. The polymer was obtained by precipitation in diethyl ether and dried in vacuum for 48 h until constant weight. Synthesis of PEG-b-P(HEMA-g-PCL) Copolymers. A typical polymerization procedure was as follows. In a dried polymerization tube, PEG113-b-PHEMA13 (1.00 g, 1.86 mmol -OH), CL (1.92 g, 16.8 mmol), Sn(Oct)2 (12 mg, 0.03 mmol), and 1 mL of anhydrous DMF were added, and the air was exchanged with nitrogen three times. The tube was immersed into an oil bath at 115 °C with vigorous stirring for 24 h. The resulting product was dissolved in 5 mL of dichloromethane and precipitated twice from diethyl ether. PEG-b-P(HEMAg-PCL) copolymer was dried in vacuum for 48 h until constant weight. Preparation of DOX-Loaded Micelles and DOX-Free Micelles. DOX-loaded micelles were prepared by a dialysis method. Briefly, the copolymer (10 mg) and DOX (2 mg) were dissolved in a THF (1 mL) and DMSO (0.1 mL) mixed solvent in a glass vial at room temperature. After that, the polymer solution was added dropwise into 10 mL of deionized water under vigorous stirring. The beaker was then exposed to air for 12 h, allowing slow evaporation of THF and formation of micelles. The residual THF was then completely removed by vacuum distillation with a rotary evaporator. The micelle solution was transferred into dialysis bag (MWCO 14000, Viskase Co.) and dialyzed against deionized water for 48 h to remove free DOX dissolved in the micelle solution. DOX-free copolymer micelles were prepared in the similar procedure without using DOX. Characterization of Copolymers and Micelles. 1H NMR spectrum was recorded on a Bruker AV 400 MHz proton NMR spectrometer with DMSO-d6 or CDCl3 as solvent. Molecular weight distributions of copolymers were determined by gel permeation chromatography (GPC) using a series of three linear Styragel columns (HT2, HT4, HT5) calibrated by polystyrene standards. DMF with LiBr (1 g/L) was used as eluent solvent at a flow rate of 0.8 mL/min at 35 °C. DSC measurements were carried out on a DSC thermal analysis system (Diamond from Perkin-Elmer). Samples were first heated from room temperature to 100 °C to erase thermal history at a heating rate of 10 °C/min under nitrogen atmosphere, followed by cooling to -50 at 10 °C/min after stopping at 100 °C for 1 min and finally heating to 100 at 10 °C/min. Transmission electron microscopy (TEM) studies were carried out on Hitachi H-800 instrument microscope operating at an accelerating voltage of 100 kV. Samples were prepared by dropping a few microliters of the aqueous solution (1.00 g/L) onto the copper grids coated with a sustaining film and allowing the samples to dry in air before measurements. Samples were stained with 1 wt % phosphotungstic acid before measurements. The size of micelles was measured by dynamic light scattering (DLS) on ALV/DLS-5022F equipped with a multi-τ digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He-Ne laser source (λ0 ) 632 nm). The measurements were carried out at 90° scattering angle at 25 °C. The stock solutions were filtered through filters of 0.45 µm pore size into the scattering cells with diameter of 10 mm. The obtained results were analyzed by using the commercial CONTIN software provided by ALV. Determination of DOX DLC. The DLC was measured by determining the absorbance at 485 nm using a Shimadzu UV-1601 spectrophotometer. An aliquot of the DOX-loaded micelle solution was lyophilized to yield the solid micelle sample. Then the samples were redissolved in a mixture of chloroform and DMSO (1/1, v/v) for the UV measurement. DOX solutions with various concentrations were prepared, and the absorbances at 485 nm were used to generate a calibration curve for the DLC calculations from DOX-loaded micelles. In Vitro Drug Release. A total of 10 mg of DOX-loaded micelles were diluted to 1 mg/mL in phosphate buffered saline (PBS, pH 7.4) and transferred into a dialysis bag with a molecular weight cutoff of 3600 Da (Viskase Co.). The dialysis bag was then immersed into 90 mL PBS solution with gentle shaking (100 rpm) at 37 °C in a ZhiCheng ZHWY-200B shaker. At predetermined time intervals, 2.0 mL buffer solution outside the dialysis bag was extracted, and it was replaced by

Amphiphilic Toothbrushlike Copolymers


Scheme 1. Synthesis of Amphiphilic PEG-b-P(HEMA-g-PCL) Copolymers

fresh PBS to remain the sink condition. The DOX concentration was determine by UV spectrophotometer based on the absorbance intensity at 485 nm. Cytotoxicity Study. Cytotoxicities of DOX-loaded micelles and free DOX were measured against bladder carcinoma EJ cells by MTT assay. The cells harvested in a logarithmic growth phase were seeded in a 96-well plates with a density of 5000 cell per well. Cells were incubated in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C for 1 day in a humidified atmosphere with 5% CO2. Then the cells were incubated with the medium containing free DOX or DOX-loaded micelles. The DOX concentrations of each formulation were prepared by serial dilution with DMEM medium. After treatment for 48 h, 100 µL of fresh medium containing 10% of MTT of 5 mg/mL stock was replaced to each well. The plates were incubated for 4 h; then 150 µL of DMSO (Sigma) was added to each well to dissolve intracellular MTT formazan crystals, followed by absorbance at 570 nm using a microplate reader. Experiments were done in triplicate and were repeated at least twice. Confocal Laser Scanning Microscopy (CLSM). Bladder carcinoma EJ cells were seeded on coverslips in a 24-well plate, free DOX (3 µM) and DOX-loaded micelles (DOX concentration: 3 µM) were incubated with EJ cells for different times before CLSM measurement. Cells were stained with Hoechst 33342 to mark the nuclei. Before the measurement, the cells were washed three times with PBS and fixed with 4% formaldehyde. Images were obtained with an Olympus PV1000-IX81 Confocal Microscope. Hoechst 33342 and DOX were excited at 352 and 485 nm with emissions at 455 and 595 nm, respectively.

Results and Discussions Synthesis of PEG-b-PHEMA Diblock Polymers. ATRP was chosen to synthesize PEG-b-PHEMA block copolymers in this paper due to its well control over molecular weight and chain uniformity.34 As shown in Scheme 1, the monofunctionallized PEG113 macroinitiator was used for the ATRP of HEMA in methanol. The feed ratios of PEG-Br/HEMA were adjusted to achieve different chain length of PHEMA. The resulted PEGb-PHEMAs gave a monomodal GPC trace, indicating that the PEG-Br showed high efficiency of initiation. The composition and the molecular weight of the product were estimated by the 1 H NMR spectrum. Figure 1A shows the spectrum of the diblock copolymer PEG113-b-PHEMA13; the polymerization degree was

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calculated by comparing the signals at 3.9 ppm (COOCH2) of HEMA and the signals at 3.5 ppm (OCH2CH2) of EG unit. The peaks at 4.8 ppm (OH) represent the hydroxyl groups of HEMA units. The molecular weight obtained by 1H NMR agrees with the theoretical molecular weight calculated by monomer conversion. The DPs of the PHEMA blocks in two samples were calculated to be 13 and 26, and the block copolymers were further named as PEG113-b-PHEMA13 and PEG113-b-PHEMA26, respectively, to synthesize different PCL brushes. The condition of ATRP and the characterization of PEG-b-PHEMA block copolymer were listed in Table 1. Synthesis of PEG-b-P(HEMA-g-PCL) Copolymers. PEGb-P(HEMA-g-PCL) copolymers were synthesized by ROP using the hydroxyl groups in the side chains of PEG-b-PHEMA polymers to initiate the CL polymerization in dried DMF under the catalysis of Sn(Oct)2 at 115 °C for 24 h. To obtain toothbrushlike copolymers with the similar fwPCL of PEG113-bPCL102, the feed molar ratio of CL and hydroxyl groups of PEG113-b-PHEMA13 copolymer was 9, while the ratio of CL and hydroxyl groups of PEG113-b-PHEMA26 was 6 to synthesize the toothbrushlike copolymer with higher fwPCL. The monomer conversion was 81 and 78%, respectively. 1H NMR spectrum of one sample of PEG-b-P(HEMA-g-PCL) copolymer is shown in Figure 1B. Assuming that all of the hydroxyl groups of PHEMA chain took a part in initiation, the average DP of PCL in PEG-b-P(HEMA-g-PCL) copolymer can be calculated by comparing the proton signals at 2.3 ppm (OCOCH2) of PCL and the signals at 3.6 ppm (OCH2CH2) of PEG113. The average DPs of PCLs in the toothbrushlike copolymers were 7.7 and 5.4, respectively. As listed in Table 2, these toothbrushlike copolymers were named as PEG113-b-P(HEMA-g-PCL8)13 with fwPCL of 0.70 and PEG113-b-P(HEMA-g-PCL5)26 with fwPCL of 0.76. All of these results indicated that PEG-b-P(HEMA-g-PCL) toothbrushlike copolymers could be easily synthesized and tailored by varying the segment lengths of PHEMA in PEGb-PHEMA and the feed ratios of CL and hydroxyl groups of PEG-b-PHEMA. DSC Characterization. The melting and crystallization behaviors of these PEG-b-P(HEMA-g-PCL) toothbrushlike copolymers were investigated by DSC. The crystallization temperature (Tc) was obtained from the cooling run, and the melting temperature (Tm) was obtained from the second heating run. The obtained DSC curves were shown in Figure 2 and that of PEG113-b-PCL102 polymer was used for comparison. With the same fwPCL of 0.70, as listed in Table 2, Tc,PCL of the PEG113b-P(HEMA-g-PCL8)13 toothbrushlike copolymer was -7.0 °C, while Tc,PCL of the PEG113-b-PCL102 linear diblock polymer was 27.6 °C. An obvious decrease of Tc should be attributed to the crystalline imperfection of the short PCL grafts. Moreover, the toothbrushlike structure of these polymers should make another contribution to the imperfection. Further shortening the chain length of PCL segments, the Tc,PCL of PEG113-b-P(HEMA-gPCL5)26 toothbrushlike copolymer decreased to -17.4 °C even though it had a higher fwPCL of 0.76. These results suggested that crystallization properties of these PEG-b-P(HEMA-g-PCL) toothbrushlike copolymers could be easily adjusted with the number and length of PCL segments. Meanwhile, Tc,PEGs of

Table 1. Experimental Conditions and Characterization Results of PEG-b-PHEMA Polymers

a

polymers

conditionsa

time

conversion (%)

DPPHEMAb

PEG113-b-PHEMA13 PEG113-b-PHEMA26

25:1:1:2 50:1:1:2

0.5 h 1h

48.2 47.8

13 26

Feed: [HEMA]/[PEG-Br]/[CuBr]/[bpy], T ) 40 °C.

b

Mn

b

6840 8530

Mw/Mnc 1.11 1.13

Calculated based on 1H NMR. c Determined by GPC analyses using DMF as the eluent.

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Figure 1. 1H NMR spectra of (A) PEG113-b-PHEMA13 in DMSO-d6 and (B) PEG113-b-P(HEMA-g-PCL8)13 in CDCl3. Table 2. Synthesis and Characterization of PEG-b-P(HEMA-g-PCL) Polymers

a

polymers

[CL]/[OH]

DPPCLa

Mna

PEG113-b-P(HEMA-g-PCL8)13 PEG113-b-P(HEMA-g-PCL5)26 PEG113-b- PCL102

9:1 6:1 105

8 5 102

18200 24500 16700

Calculated based on 1H NMR.

b

PDI

b

1.21 1.26 1.24

fwPCLa

Tc,PCLc (°C)

0.70 0.76 0.70

-7 -17 27.6

Determined by GPC analyses using DMF as the eluent. c Determined by DSC analyses.

Figure 2. DSC curves of PEG113-b-PCL102, PEG113-b-P(HEMA-g-PCL8)13, and PEG113-b-P(HEMA-g-PCL5)26 copolymers in the cooling run (solid lines) and in the second heating run (dotted lines).

these PEG-b-P(HEMA-g-PCL)s also were greatly reduced, indicating that the brushlike blocks would also influence the crystallization behaviors of the PEG blocks. Generally, the micelles made from amphiphilic copolymers used in drug delivery were performed at room temperature. So the cores of these toothbrushlike copolymer micelles would have no crystallinity that would benefit drug loading and delivering and also adjust biodegradation behavior of PCL segments. Preparation and Characterization of Copolymer Micelles. The amphiphilic toothbrushlike copolymer self-assembled into micelles in aqueous solution using the dialysis method. The

critical micelle concentrations (CMC) of these toothbrushlilke copolymers were analyzed by fluorescence spectra using pyrene as a hydrophobic probe. In the excitation spectra, several aspects of the pyrene spectroscopic properties were observed as the copolymer concentration increased. The intensity ratios at band at 372 and 383 nm (I1/I3) of the pyrene excitation spectra versus the logarithm of copolymer concentration were shown in Figure 3. The I1/I3 remained almost constant at low copolymer concentrations and increased sharply when the copolymer concentrations reached a value, indicating formation of copolymer micelles and the pyrene was trapped into the micelle core



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Table 3. Influence of Copolymer Architecture and Compositions on Micellar Properties DOX-free micelles a

DOX-loaded micelles a

sample

diameter (nm)

PDI

PEG113-b-P(HEMA-g-PCL8)13 PEG113-b-P(HEMA-g-PCL5)26 PEG113-b- PCL102

46.9 ( 1.5 78.9 ( 2.4 44.2 ( 1.3

0.13 ( 0.01 0.32 ( 0.04 0.16 ( 0.02

a

diameter (nm) 132.2 ( 3.2 151.1 ( 4.3 90.6 ( 3.4

a

PDIa

DLC (%)

0.18 ( 0.01 0.17 ( 0.01 0.25 ( 0.03

7.9 6.9 6.2

Measured by DLS analyses, all the measurements were performed in triplicate.

Figure 3. Plot of the I372/I383 ratio against log C of polymeric micelles.

with more hydrophobic environment. From the sigmoidal curves, CMCs of PEG113-b-P(HEMA-g-PCL8)13 and PEG113-b-P(HEMAg-PCL5)26 were determined as 2.5 and 2.9 mg/L in aqueous solution, which were similar with the CMC of linear PEG113b-PCL102 block copolymer (2.5 mg/L).35 To further evaluate the properties of toothbrushlike copolymers micelles, both the morphology and the mean size of these PEG-b-P(HEMA-g-PCL) micelles were studied by TEM and DLS. As shown in Figure 4 and Table 3, the micelles of these toothbrushlike copolymers exhibited a uniform spherical morphology. From the DLS experiments, the average diameters of micelles were about 47 nm, with a polydispersity index of 0.13 for PEG113-b-P(HEMA-g-PCL8)13 and 79 nm with a polydispersity index of 0.32 for PEG113-b-P(HEMA-g-PCL5)26, respectively. The size of these micelles measured from TEM images were 54 and 98 nm that were comparable to that from DLS. Drug Loading and Encapsulation Efficiency. To assess the influence of toothbrushlike copolymer structure on drug incorporation, DOX was used as model anticancer drug to evaluate the drug loading and release properties. DOX-loaded PEG-bP(HEMA-g-PCL) micelles were prepared by the dialysis

method. The size and size distribution of DOX-loaded micelles measured by DLS showed that the diameters of these DOXloaded micelles were larger than their parent micelles without DOX as summarized in Table 3. The drug loading contents of PEG113-b-P(HEMA-g-PCL8)13 and PEG113-b-P(HEMA-gPCL5)26 were 7.9 and 6.9%, respectively. However, under the same fwPCL and loading conditions, the DLC of the linear PEG113b-PCL102 was 6.2%. The DLC of the PEG113-b-P(HEMA-gPCL8)13 copolymers increased about 27% compared to that of the linear PEG113-b-PCL102. It is well-known that physical trapping of hydrophobic drugs into polymeric micelles is driven by the hydrophobic interaction between the drug and the hydrophobic segments of copolymers. Generally, long PCL blocks with more hydrophobic favor DOX-encapsulating into the micelles. However, long PCL segments results in a higher crystallinity, which lead to less drug loading in the micelles. Compared with the linear PEG113-b-PCL102 diblock copolymer, these toothbrushlike copolymer analogues with shorter grafted PCL segments have no crystallinity at room temperature, so their micelles could entrap more drug molecules. In vitro release profiles of the loaded DOX in micelles of PEG-b-P(HEMA-g-PCL) copolymers were studied and compared to that of the linear PEG113-b-PCL102 diblock copolymer micelles under a simulated physiological condition PBS (0.01 M, pH 7.4) at 37 °C. As illustrated in Figure 5, the DOX release behaviors of the novel micellar carriers and their comparison had the similar typical two-phase release profile. In the first stage, about 19-28% DOX encapsulated in micelles released within 24 h, followed by a sustained and slow release over a prolonged time. It is expected that the most of DOX remains in the micelle cores for a considerable time when these micelles circulate in the plasma at normal physiological conditions. However, the faster releases of DOX from toothbrushlike copolymer micelles were observed. This is because PEG-b-P(HEMA-g-PCL) toothbrushlike copolymers with the amorphous short and grafted PCL branches under the delivery condition would facilitate the drug molecules diffusing out from the micelle cores. PEG113b-P(HEMA-g-PCL5)26 had shorter PCL segments than PEG113b-P(HEMA-g-PCL8)13 did, so the DOX in PEG113-b-P(HEMA-

Figure 4. TEM photographs of the self-assembled nanoparticles from (A) PEG113-b-P(HEMA-g-PCL8)13 and (B) PEG113-b-P(HEMA-g-PCL5)26 in aqueous solution: 1 mg/mL.

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Figure 5. In vitro DOX release profiles from DOX-load micelles in PBS (0.01M, pH 7.4) at 37 °C.

g-PCL5)26 micelles was released faster even though the PEG113-b-P(HEMA-g-PCL8)13 micelles had a higher DLC. All these results suggested that these toothbrushlike copoly-

Zhang et al.

mers with a different PCL number and length provide a straight way to adjust the DOX release kinetics. It is noteworthy that the DOX-loaded toothbrushlike copolymer micelles in PBS exhibited no deposits for 6 months, but the compared DOX-loaded PEG113-b-PCL102 micelles produced a lot of floccules in 4 days, as shown in Figure 6. This phenomenon should be contributed by the architecture of toothbrushlike copolymers. So the toothbrushlike copolymer micelles with drugs have enhanced stability in aqueous solution than that of the linear PEG-b-PCL polymer micelles. This is very important in practical application of drug delivery system. Cellular Uptake and Cytotoxicity. To investigate the mechanism of internalization of DOX-loaded toothbrushlike copolymers micelles and intracellular drug delivery, bladder carcinoma EJ cells were observed by CLSM microscopy at different time. As shown in Figure 7, intracellular distribution of the DOX-loaded micelles was different from that of free DOX. After 4 h of cell incubation with the free DOX, strong fluorescence was observed in the nucleus of the cell. In contrast, DOX was observed mainly in the cytoplasm of the cells instead of the nucleus when the cells were incubated with the DOXloaded toothbrushlike copolymer micelles for 4 and 10 h.

Figure 6. Stability properties of DOX-loaded micelles of (1) PEG113-b-P(HEMA-g-PCL8)13, (2) PEG113-b-P(HEMA-g-PCL5)26, and (3) PEG113-bPCL102 in PBS (0.01 M, pH 7.4) at 37 °C in 96 h.

Figure 7. CLSM images of EJ cells incubated with DOX-loaded PEG113-b-P(HEMA-g-PCL8)13 micelles for (A) 4 h and (B) 24 h, DOX-loaded PEG113-b-P(HEMA-g-PCL5)26 micelles for (C) 4 h and (D) 24 h, DOX-loaded PEG113-b-PCL102 micelles for (E) 4 h and (F) 24 h, and DOX for (G) 4 h and (H) 24 h. For each panel, images from left to right show the cells with nuclear staining by Hoechst 33342, with DOX fluorescence, and overlays of both images; scale bars correspond to 20 µm in all images.



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copolymers micelles exhibited much lower cytotoxicity when compared with free DOX at the same dose. The lower cytotoxicity of DOX in the micelles was likely due to a timeconsuming DOX release from micelles and delayed nuclear uptake in EJ cells, as shown by the in vitro DOX release and internalization studies by CLSM.

Conclusions

Figure 8. Viability of EJ cells as a function of polymer type and concentration after a 48 h incubation (n ) 3).

In conclusion, PEG-b-P(HEMA-g-PCL) toothbrushlike copolymers of different molecular weights and compositions were synthesized by a combination of ATRP and ROP. Because these short and grafted PCL branches were difficult to crystallize, the micelles prepared from these toothbrushlike copolymers possessed higher DOX loading capability than the micelles formed by linear PEG-b-PCL polymer did. The DOX-loaded toothbrushlike copolymer micelles also exhibited extraordinary stability in aqueous solution than that of DOX-loaded linear PEG-b-PCL polymer micelles. In vitro release data indicated that the DOX-release from toothbrushlike copolymer micelles was faster than that in linear block copolymer micelles. CLSM studies showed that DOX-loaded toothbrushlike copolymer micelles could be effectively internalized by bladder carcinoma EJ cells and DOX could be released in endocytic compartments and then reached to the nucleus. With these enhanced properties relative to that of linear PEG-b-PCL, the toothbrushlike copolymer micelles composed of PEG and PCL have potential application as a versatile nanocarrier of various liposolubility drugs. Acknowledgment. Financial support from NSF China (20534010, 20625412, and 50830107) and the Chinese Academy of Sciences (Knowledge Innovation Program, KJCX2-YW-H19) is gratefully acknowledged.

References and Notes Figure 9. Cytotoxicity of free DOX and DOX-loaded micelles to EJ cells after a 48 h incubation (n ) 3).

However, there was a significant DOX accumulation in the nucleus when the cells were incubated with the DOX-loaded PEG-b-P(HEMA-g-PCL) micelles for 24 h, which differed from DOX-loaded PEG113-b-PCL102 micelles that DOX still mainly stayed in the cytoplasm after 24 h incubation. These results suggested that the novel DOX-loaded micelles may have been taken up by the cells through a nonspecific endocytosis mechanism; and then the DOX molecules were released in endocytic compartments (i.e., endosomes and later lysosomes) and finally rapidly reached to the nucleus. To investigate cytotoxicity of the free blank micelles and DOX-loaded micelles, bladder carcinoma EJ cells were exposed to a series of different concentrations of the blank micelles, free DOX, and DOX-loaded micelles for 48 h, and the percentages of viable cells were quantified using the MTT method. As shown in Figure 8, these blank toothbrushlike copolymer micelles have the similar cytotoxicity to that of micelles formed by PEG113b-PCL102. No obvious cytotoxicity against EJ cells was observed even the copolymer micelles concentration reached 500 µg/mL. The cytotoxicity of DOX-loaded PEG113-b-P(HEMA-g-PCL8)13 and PEG113-b-P(HEM-g-PCL5)26 copolymer micelles compared with that of free DOX was shown in Figure 9. The concentration of DOX in micelles that cause 50% cell killing was relatively higher than that of free DOX. DOX in the toothbrushlike

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BM100116G

Amphiphilic Toothbrushlike Copolymers Based on Poly(ethylene

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