Evaluation of Polymeric Micelles from Brush Polymer with Poly (ε

Jul 8, 2009 - For a more comprehensive list of citations to this article, users are .... Based on Poly(ethylene glycol) and Poly(ε-caprolactone) as D...
0 downloads 0 Views 3MB Size
Download PDF
Biomacromolecules 2009, 10, 2169–2174

2169

Evaluation of Polymeric Micelles from Brush Polymer with Poly(ε-caprolactone)-b-Poly(ethylene glycol) Side Chains as Drug Carrier Jin-Zhi Du,† Ling-Yan Tang,‡ Wen-Jing Song,‡ Yue Shi,§ and Jun Wang*,‡ Department of Polymer Science and Engineering, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, and Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China Received March 26, 2009; Revised Manuscript Received June 7, 2009

Brush polymers PHEMA-g-(PCL-b-PEG) with poly(2-hydroxyethyl methacrylate) (PHEMA) as the backbone and poly(ε-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) block copolymers as side chains were synthesized and evaluated as drug delivery vehicles. Two brush polymers were synthesized, and their structures were confirmed by gel permeation chromatography analyses and 1H NMR measurements. The brush polymers self-assembled into micelles in aqueous solution, and the critical micellization concentrations of brush polymers were 2fold lower than that of the linear diblock copolymer PCL-b-PEG with structure similar to that of the grafted side chains of brush polymers, indicating the higher aqueous stability of brush polymer micelles. The micelles were spherical with average diameters below 100 nm. Brush polymer micelles exhibited higher loading doxorubicin capacity compared with micelles from linear PCL-b-PEG block copolymer by the dialysis method, and the burst doxorubicin release from the brush polymer micelles was significantly suppressed. Doxorubicin-loaded brush polymer micelles can be effectively internalized by A549 human lung carcinoma cells and slowly released the encapsulated drug molecules as demonstrated by the drug accumulation in cytoplasm, which was opposite to free doxorubicin, which accumulated rapidly in the cell nuclei.

Introduction Polymeric micelles self-assembled from amphiphilic copolymers have attracted significant attention in the fields of biomedical applications. They have been developed as delivery systems of drug1-5 as well as contrast agents in diagnostic imaging applications.6 In an aqueous environment, the hydrophobic component of the copolymer is expected to segregate into the core of the micelles, while the hydrophilic component forms the corona or outer shell. The hydrophobic micelle core serves as a microenvironment for incorporation of various therapeutic compounds, while the corona can act as a stabilizing interface between the hydrophobic core and the external medium. Although various micelles have been developed for in vitro and in vivo studies and applications, the majority of works have been focused on micelles formed by intermolecular aggregation of amphiphilic linear block copolymers.1,3,7-15 Micelles from polymers with other architectures as drug delivery vectors are less studied, especially brush-like macromolecules.16-19 However, it has been reported that the architecture of polymer may determine the static and dynamic stability, morphology, size and size distribution of the micelles, and further affect the performance of micelles including drug loading and release rate, even in vivo circulation and distribution.20,21 Cylindrical brush polymers have recently attracted considerable attention from polymeric chemists. They are a special type of graft polymer, in which multiple polymer chains are grafted to the polymer backbone. Various well-defined brush polymers * To whom correspondence should be addressed. Fax: +86 551 360 0402; E-mail: [email protected]. † Department of Polymer Science and Engineering. ‡ Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences. § Department of Chemistry.

have been synthesized by facile and well-controlled polymer synthesis methods, including atom transfer radical polymerization (ATRP), ring-opening polymerization (ROP), and click chemistry.22-26 Among those brush polymers, cylindrical brush polymers with block copolymers as side chains have been extensively studied, mainly on the intramolecular phaseseparation in selective solvent.24,26-28 To the best of our knowledge, however, the micelles from cylindrical brush polymers with amphiphilic block copolymer side chains have been merely studied as the carrier for drug delivery, while the unique architecture of cylindrical brush polymers may bring advantages in drug loading and release. In this work, we synthesized cylindrical brush polymers PHEMA-g-(PCL-b-PEG) with poly(2-hydroxyethyl methacrylate) (PHEMA) as the backbone and poly(ε-caprolactone)-bpoly(ethylene glycol) (PCL-b-PEG) amphiphilic block copolymers as the side chains. The micelle formation of these polymers was studied, and doxorubicin (DOX) was used to evaluate the drug loading and release behavior from the micelles. The internalization and cytotoxicity of drug-loaded micelles against A549 human lung carcinoma cells were also investigated.

Experimental Section Materials. 2-Hydroxyethyl methacrylate (HEMA, Acros Organics) was purified according to the literature.29 Ethyl 2-bromoisobutyrate (EBiB, Aldrich) was distilled just before use. Pentamethyldiethylenetriamine (PMDETA, Acros Organics) was stirred overnight over CaH2 and distilled under reduced pressure prior to use. CuBr was purified by stirring in acetic acid and washing with methanol, and then dried under vacuum. ε-Caprolactone (ε-CL, Acros Organics) was distilled under reduced pressure after being treated with CaH2. Stannous octoate (Sn(Oct)2, Sinopharm Chemical Reagent Co., Ltd., China) was purified according to a method described in the literature.30 Monomethoxy

10.1021/bm900345m CCC: $40.75  2009 American Chemical Society Published on Web 07/08/2009

2170

Biomacromolecules, Vol. 10, No. 8, 2009

poly(ethylene glycol) with Mn ) 2000 g/mol (mPEG45, Fluka) was dried by azeotropic distillation in the presence of toluene. N,N′-Dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) purchased from Shanghai Medpep Co., Ltd. were used as received. Succinic anhydride (Sinopharm Chemical Reagent Co., Ltd., China) was crystallized from acetic anhydride. N,N-Dimethylformamide (DMF) was dried over CaH2 and distilled before use. Linear PCL-b-PEG diblock copolymer was synthesized according to the literature using mPEG45 as the initiator, and the degree of polymerization (DP) of PCL was calculated to be 19 on the basis of its 1H NMR spectrum following the described method,31 and it is further denoted as PCL19-b-PEG45. Carboxyl-terminated mPEG (mPEG45-COOH) was prepared by reacting mPEG45 with succinic anhydride as described in the literature.26 Doxorubicin hydrochloride (DOX · HCl) was purchased from Zhejiang Hisun Pharmaceutical Co, Ltd. Synthesis of PHEMA. In a typical example, a dry glass tube was charged with freshly purified HEMA (13.0 g, 0.1 mol), PMDETA (42 µL, 0.2 mmol), and ethanol (24.4 mL). The tube was degassed with three freeze-thaw cycles, followed by addition of CuBr (28.8 mg, 0.2 mmol). After sealed under vacuum, the tube was immersed in a thermostatted oil bath at 70 °C and the polymerization lasted for 24 h. Upon cooling to room temperature, the tube was opened to air, and the mixture was stirred overnight. The crude product was diluted with ethanol and purified by passing through a column with Al2O3 to remove the copper catalyst. The solution was concentrated under vacuum and precipitated twice in water. The solid product was collected and dried under vacuum to a constant weight. Synthesis of PHEMA Grafted with PCL (PHEMA-g-PCL). In a typical procedure, PHEMA (0.22 g, 1.69 mmol -OH) was dissolved in 1 mL of freshly distilled anhydrous DMF in a flame-dried and nitrogen-filled glass flask. ε-CL (4.82 g, 42.28 mmol) was then added to the solution, and the tube was immersed into an oil bath at 115 °C with vigorous stirring. The polymerization was processed for 24 h after addition of Sn(Oct)2 (11.0 mg, 0.027 mmol). The resulted polymer was dissolved in dichloromethane, and it was precipitated twice from methanol, affording PHEMA-g-PCL, which was dried under vacuum to a constant weight. Synthesis of PHEMA-g-(PCL-b-PEG). PHEMA-g-(PCL-b-PEG) copolymers were synthesized by coupling mPEG45 to PHEMA-g-PCL. As an example, PHEMA70-g-PCL20 (0.10 g), mPEG45-COOH (92.0 mg), DCC (12.8 mg), and DMAP (5.1 mg) were dissolved in 10 mL of anhydrous dichloromethane. The reaction was performed at room temperature for 24 h under nitrogen atmosphere. After filtration to remove the dicyclohexylcarbodiurea, the filtrate was washed with water and dried over anhydrous Na2SO4. The product obtained by precipitation in diethyl ether was further dissolved in water and dialyzed against water for 48 h using dialysis membrane tubing with a molecular weight cut off (MWCO) of 15 000. Characterization of Polymers. 1H NMR spectra were recorded on a Bruker AV300 NMR spectrometer. Molecular weights and molecular weight distributions of PHEMA were determined by gel permeation chromatography (GPC) analyses with two Styragel columns (HT3 and HT4, Waters Co.) at 40 °C. DMF was delivered at a flow rate of 1.0 mL min-1. A Waters 1515 pump and a Waters 2414 differential refractive index detector were used. Molecular weights and molecular weight distributions of the PHEMA-g-PCL polymers were measured on a Waters 150C GPC system, equipped with three Ultrastyragel columns in series and a refractive index (RI) detector at 30 °C. Highperformance liquid chromatography (HPLC)-grade tetrahydrofuran was used as the eluent at a flow rate of 1.0 mL min-1. Monodispersed polystyrene polymers were used as calibration standards. Preparation of Micelles. Micelles were prepared by a dialysis method. Briefly, PHEMA-g-(PCL-b-PEG) or PCL-b-PEG (10 mg) was dissolved in 2 mL of dimethyl sulfoxide (DMSO) and stirred for 2 h at room temperature. Then, the polymer solution was added dropwise into 5 mL of ultrapurified water (Millipore Milli-Q Synthesis, 18.2 MΩ) under vigorous stirring. Two hours later, the solution was transferred

Du et al. into dialysis membrane tubing (MWCO 2000) and dialyzed for 24 h against Milli-Q water to remove the organic solvent. Fluorescence Measurements. The fluorescent probe method was employed to determine the critical micellization concentration (CMC) of the micelles as followed. A predetermined amount of pyrene solution in acetone was added into a series of volumetric flasks, and the acetone was then evaporated completely. A series of copolymer solutions at different concentrations ranging from 1.0 × 10-5 to 1.0 mg mL-1 were added to the flasks, while the concentration of pyrene in each flask was fixed at a constant value (6.0 × 10-7 mol L-1). The excitation spectra were recorded at 25 °C on a Shimadzu RF-5301PC spectrofluorophotometer with λem at 390 nm and a slit width of 3 nm. Laser Light Scattering (LLS) Measurements. Particle size of micelles was measured by dynamic light scattering (DLS) on a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., U.K.) with a He-Ne laser (633 nm) and 90° collecting optics. Samples were filtered through 0.45 µm aqueous membrane filter prior to measurements. Static light scattering (SLS) studies were conducted with a modified commercial LLS spectrometer (ALV/SP-125) equipped with an ALV-5000 multi-τ digital time correlator and a solid-state laser (ADLAS DPY42511, λ ) 632 nm) at scattering angles ranging from 15° to 135°. The dn/dc value of the micelles was determined to be 0.073 according to the reported method.32 The weight-average molecular weights (Mw) were obtained using standard Zimm plot analyses. Transmission Electron Microscopy (TEM). TEM was performed on a JEOL-2010 transmission electron microscope with an accelerating voltage of 200 kV. Samples were prepared by pipetting a drop of micelle solution (1 g L-1) onto a 230 mesh copper grid coated with carbon and allowing the sample to dry in air before measurements. Samples were stained with 0.1 wt % phosphotungstic acid before measurements. Preparation of DOX-Loaded Micelles. DOX-loaded micelles were prepared similarly to the blank micelles. Briefly, 10 mg of each polymer was dissolved in 2 mL of DMSO, followed by adding a predetermined amount of DOX · HCl and two molar equivalents of triethylamine (TEA) and stirred at room temperature for 2 h. Then, the mixed solution was added dropwise to 5 mL of Mill-Q water. After being stirred for an additional 2 h, the solution was dialyzed against water for 24 h (MWCO 2000). The solution was further centrifuged at 3000 g for 3 min to remove free DOX. Determination of DOX Loading Content (DLC) and Loading Efficiency (DLE). To measure the amount of DOX trapped in the micelles, an aliquot of the DOX-loaded micelle solution was lyophilized and dissolved in DMSO. The concentration of DOX was measured by HPLC analyses as described below. The percentages of DLC and DLE were calculated according to the following equations:

DLC% ) (weight of DOX in the micelle/ weight of DOX-loaded micelle) × 100%

DLE% ) (weight of DOX in the micelle/ weight of DOX for DOX-loaded micelle preparation) × 100% In Witro Drug Release. DOX-loaded micelle solution was diluted to 1 mg mL-1 in phosphate buffered saline (PBS, 0.01 M, pH 7.4), and transferred into a dialysis membrane tubing (Spectra/Por, Float-A-Lyzer, MWCO 15000). The tubing was immersed in 20 mL of PBS (0.01 M, pH 7.4) and shaken at 37 °C. At a predetermined time interval, the external buffer of the tubing was collected, and it was replaced by fresh PBS. The concentration of DOX was determined by HPLC analyses, which were performed on a Waters 1525 HPLC system with a Waters 2487 2-channel fluorescence detector, 1500 column heater, and a Symmetry C18 column. Acetonitrile-water (50:50, v/v, pH 2.7, adjusted by perchloric acid) was used as the mobile phase at 30 °C with a flow rate of 1.0 mL min-1. A fluorescence detector was set at 460 nm for excitation and 570 nm for emission and linked to Breeze software for data analysis.

Brush Polymer Micelles as Drug Carriers

Biomacromolecules, Vol. 10, No. 8, 2009

Table 1. Characterization of PHEMA Polymers sample PHEMA70 PHEMA200 b

feed ratio [HEMA]/[EBiB] DPa Mn (g/mol)b Mw (g/mol)b Mw/Mnb 200/1 500/1

70 200

9300 27 300

10 790 34 670

2171

Scheme 1. Synthetic Route of PHEMA-g-(PCL-b-PEG) Brush Polymers

1.16 1.27

a Calculated based on HEMA conversion from 1H NMR analyses. Determined by GPC analyses using DMF as the eluent.

Cell Culture. A549 human lung carcinoma cells (ATCCs) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, containing 10% Hyclone fetal bovine serum, 50 units mL-1 penicillin, and 50 units mL-1 streptomycin) at 37 °C and 5% CO2 atmosphere. Confocal Laser Scanning Microscopy (CLSM). A549 cells were seeded on coverslips in a 24-well tissue culture plate and incubated in complete DMEM at 37 °C in 5% CO2. Twenty-four hours later, free DOX (6 µM) and DOX-loaded micelles (final DOX concentration at 6 µM) were added to the cells, and the cells were incubated for 2 h. The cells were then washed twice with PBS and fixed with 4% formaldehyde. The slides were mounted and observed with a Zeiss LSM510 Laser Confocal Scanning Microscope imaging system. Cytotoxicity Study. Cytotoxicity of DOX-loaded micelles and free DOX were measured against A549 cells by MTT assay. The cells were seeded in a 96-well plate at the density of 5000 cells/well and incubated in DMEM at 37 °C in 5% CO2 for 24 h. Then, the medium was removed and replaced with 100 µL of free DOX or DOX-loaded micelles containing medium. The equivalent DOX concentrations of each formulation were prepared by serial dilution with DMEM medium, with a maximal DOX concentration of 10 µg mL-1. After treatment for 72 h, the medium was replaced by 100 µL of fresh DMEM, followed by adding 25 µL of MTT stock solution (5 mg mL-1 in PBS). After incubation for an additional 2 h, 100 µL of the extraction buffer (20% SDS in 50% DMF, pH 4.7) was added to the wells and incubated overnight at 37 °C. The absorbance was measured at 570 nm using a Bio-Rad 680 microplate reader, and the relative cell viability was calculated.

ratios of resonances d and d′ shown in Figure 1B. The molecular weights of the PHEMA-g-PCL summarized in Table 2 were also determined according to the equation Mn ) DPCL × 114 + Mn,PHEMA, where DPCL is the DP of CL in the polymer and Mn,PHEMA is the molecular weight of the PHEMA macroinitiator. The grafting efficiency of mPEG-COOH was calculated based on the integrals of resonances f of mPEG and d of PCL in the 1 H NMR spectrum. Preparation and Characterization of PHEMA-g-(PCL-bPEG) Micelles. PHEMA-g-(PCL-b-PEG) brush polymers with amphiphilic PCL-b-PEG side chains self-assembled into micelles in aqueous solution using the dialysis method. The formation of micellar nanoparticles was confirmed by TEM observations.

Results and Discussion Syntheses of PHEMA-g-(PCL-b-PEG) Copolymers. ATRP technique was chosen for the polymerization of HEMA in ethanol, due to its potency in the preparation of well-defined polymers.23,29,33 In the present work, the polymerization was performed in ethanol using the CuBr/PMDETA/EBiB system. As listed in Table 1, conversions of HEMA determined by 1H NMR were 35% and 40% for the two polymers, which are close to the conversion values reported in the literature.23,29,33 The DP of the PHEMA was thus calculated to be 70 and 200, and the polymers were further denoted as PHEMA70 and PHEMA200, respectively. Mw/Mn determined by GPC using DMF as the eluent was around 1.2 for both PHEMA70 and PHEMA200. As shown in Scheme 1, PHEMA-g-(PCL-b-PEG) copolymers were synthesized by a combination of ROP and coupling reaction according to the reported procedure.26 PHEMA-g-PCL was first synthesized using PHEMA as a macroinitiator for CL polymerization in dried DMF under the catalysis of Sn(Oct)2. The coupling reaction of mPEG-COOH to PHEMA-g-PCL was performed at room temperature in the presence of DCC and DMAP. In the synthesis, the same feed ratio of [CL] to [OH] of PHEMA at 25:1 was applied to obtain the grafted PCL chains with similar DP. A representative 1H NMR spectrum of PHEMA-g-(PCL-b-PEG) is showed in Figure 1, which is compared with the precursor polymers. Assuming that all of the hydroxyl groups of PHEMA have been involved in the polymerization of PCL, the average DP of PCL in PHEMAg-PCL polymers was then calculated on the basis of the integral

Figure 1. 1H NMR spectra of PHEMA70 in DMSO-d6 (A), and PHEMA70-g-PCL20 (B) and PHEMA70-g-(PCL20-b-PEG45) (C) in CDCl3 (ppm).

2172

Biomacromolecules, Vol. 10, No. 8, 2009

Du et al.

Table 2. Synthesis and Characterization of PHEMA-g-(PCL-b-PEG) Brush Polymers PHEMA-g-PCL parental polymer a

b

sample

feed ratio [CL]:[OH]

DP of PCL

Mn

PHEMA70-g-(PCL20-b-PEG45) PHEMA200-g-(PCL22-b-PEG45)

25:1 25:1

20 22

168 000 527 600

PHEMA-g-(PCL-b-PEG) Mn

c

276 280 789 340

c n

Mw/M

1.10 1.32

coupling efficiency (%)d

Mne

98 96

305 200 911 600

a Determined by 1H NMR. b Determined by 1H NMR. Mn ) DPCL × 114 + Mn,PHEMA, DPCL represents the DP of PCL and Mn,PHEMA is the molecular weight of PHEMA. c Determined by GPC. d Calculated on the basis of 1H NMR results. e Mn ) Mn,PHEMA-g-PCL + Mn, mPEG × coupling efficiency × DPPHEMA.

Figure 2. TEM images of micelles from PHEMA70-g-(PCL20-b-PEG45) (A) and PHEMA200-g-(PCL22-b-PEG45) (B) brush polymers.

As shown in Figure 2, micelles of PHEMA70-g-(PCL20-b-PEG45) and PHEMA200-g-(PCL22-b-PEG45) are well-dispersed and display spherical morphology. They are relatively uniform in size and the average diameters are approximately 45 nm for PHEMA70-g-(PCL20-b-PEG45) micelles. PHEMA200-g-(PCL22b-PEG45) micelles are relatively larger with diameter around 60 nm. The size and size distribution measured by DLS are listed in Table 3. It can be seen that cumulant diameters of the micelles are 61 and 91 nm for PHEMA70-g-(PCL20-b-PEG45) and PHEMA200-g-(PCL22-b-PEG45), respectively. It is noteworthy that the diameter determined by TEM is smaller than that obtained by DLS measurements. Such a difference is reasonable since DLS determined the hydrodynamic diameter or the “equivalent sphere diameter” in solution, while TEM images were obtained in the absence of the solvent. Nevertheless, sizes measured by both methods exhibited the same trend. The self-assembly of brush polymers was also characterized by measuring the CMC using pyrene as a fluorescent probe. The linear diblock copolymer PCL19-b-PEG45 with similar molecular structure to the grafted side chains were studied parallelly for comparison. With increasing the polymer concentration, several changes in the fluorescence spectra of pyrene were observed. For example, peak intensity was significantly increased with the increase of polymer concentration, and red shifts of the (0,0) band of pyrene from 335 to 338 nm were observed. The intensity ratio of bands at 338 and 335 nm (I338/ I335) was calculated and plotted against the polymer concentrations, giving a sigmoid curve as shown in Figure 3. I338/I335 remained nearly unchanged at low polymer concentrations and increased dramatically when the polymer concentration reached a certain value, exhibiting the characteristic of pyrene entirely in a hydrophobic environment, which is actually a reflection of micelle formation. From the sigmoidal curves, CMCs of PHEMA70-g-(PCL20-b-PEG45) and PHEMA200-g-(PCL22-bPEG45) were determined to be 2.70 ( 0.28 and 2.50 ( 0.14 µg mL-1, respectively, which are about half of the CMC of linear PCL19-b-PEG45 diblock copolymer (CMC ) 4.93 ( 0.04 µg mL-1). CMC is an effective parameter to evaluate the micelle stability in aqueous solution, and a lower value represents relatively higher stability. Thus, it can be concluded that the stability of micelles from PHEMA-g-(PCL-b-PEG) brush polymers is higher than that from linear block polymer with chemical

structure similar to that of grafted side chains. Two factors may contribute to the enhanced stability of the brush polymer micelles. Compared with linear polymer micelles, the hydrophobic interaction can be strengthened due to the intramolecular interaction of PCL side chains. On the other hand, the PCL segments from different brush polymer molecules may entangle each other, further enhancing the hydrophobic interaction. Such an enhanced stability will be beneficial to the micellar nanoparticles for systemic drug delivery considering the dilution of micellar nanoparticles by the body fluids upon injection. Micelles of brush polymers and the linear block polymer were further analyzed by SLS to determine their weight-average molecular weight (Mw) using standard Zimm plot analyses. The aggregation number was then calculated using equation Nagg ) Mw,micelle/Mw,unimer, where Mw,micelle and Mw,unimer are the micelle molar mass determined by SLS and the molar mass of the corresponding polymer, respectively. The aggregation number of micelles from PHEMA70-g-(PCL20-b-PEG45) and PHEMA200g-(PCL22-b-PEG45) are almost the same, which are 65 and 64, respectively. However, the aggregation number of linear PCL19b-PEG45 polymer in its micelle is ca. 2500, which is in the range of 103 to 104 orders of magnitude for most of the linear block copolymer micelles.34,35 The significant reduction of aggregation number of brush copolymer micelles should attribute to the unique molecular architecture. Loading and Release of DOX. To better understand the potential of micelles based on brush copolymers, DOX was selected to determine the drug loading and release properties. DOX was encapsulated into the micelles of brush copolymers by a dialysis method with comparison to the linear PCL19-bPEG45 micelles. Size and size distributions of DOX-loaded micelles by DLS measurements revealed that encapsulation of DOX into micelles increased the diameters of micelles, as summarized in Table 3. When the feed ratio of polymer to DOX was 18:1, the drug loading contents of PHEMA70-g-(PCL20-bPEG45) and PHEMA200-g-(PCL22-b-PEG45) micelles were 1.23% and 1.27%, respectively, corresponding to DLEs of 22.09% and 22.90%. Under the same loading conditions, the DLC and DLE of the linear PCL19-b-PEG45 micelles is only the half of the above values. For conventional linear polymers, the hydrophobic drug was surrounded in the core by individual polymer chains. However, both the inter- and intramolecular hydrophobic interactions in brush polymers may contribute to the drug encapsulation, leading to improved drug loading capacity and efficiency. When feed ratio of polymer to DOX was changed to 10:1, the DLC and DLE for the micelles of PHEMA70-g(PCL20-b-PEG45) and PCL19-b-PEG45 increased, but severe precipitation occurred when using PHEMA200-g-(PCL22-bPEG45). In order to investigate the influence of polymer architecture on the drug release behavior, DOX-loaded micelles from a linear diblock copolymer and two brush polymers were studied and compared. The in vitro drug release of DOX from the micelles was carried out in PBS (0.01 M, pH 7.4) at 37 °C. The DOX release profiles are presented in Figure 4, from which it can be

Brush Polymer Micelles as Drug Carriers

Biomacromolecules, Vol. 10, No. 8, 2009

2173

Table 3. Properties of Polymeric Micelles before and after DOX Encapsulation blank micelles sample PHEMA70-g(PCL20-b-PEG45) PHEMA200-g(PCL22-b-PEG45) PCL19-b-PEG45 a

DOX-loaded micelles at 18:1 (w/w)a

DOX-loaded micelles at 10:1 (w/w)a

cumulant diameter (nm)

PDI

cumulant diameter (nm)

PDI

61.5 ( 0.3

0.22 ( 0.01

67.1 ( 4.2

0.23 ( 0.01

1.23

22.09

72.0 ( 1.5

0.22 ( 0.01

2.68

26.80

91.1 ( 1.6

0.16 ( 0.01

134.0 ( 3.8

0.17 ( 0.03

1.27

22.90

-b

-b

-b

-b

0.09 ( 0.02

68.1 ( 5.1

0.25 ( 0.01

0.6

10.78

72.6 ( 2.0

0.10 ( 0.03

1.16

11.56

58.8 ( 1.5

Feed ratio of polymer to DOX.

b

cumulant DLC (%) DLE (%) diameter (nm)

PDI

DLC (%) DLE (%)

Not detected due to precipitation. All the measurements were performed in triplicate.

Figure 5. CLSM images of A549 cells after 2 h incubation with free DOX (A) or DOX-loaded PHEMA70-g-(PCL20-b-PEG45) micelles (B). Figure 3. Plot of the I338/I335 ratio against log C of polymeric micelles.

Figure 6. Cytotoxicity of free DOX and DOX-loaded micelles to A549 cells after 72 h incubation (n ) 3).

Figure 4. Cumulative DOX release from micelles in PBS (0.01 M, pH 7.4) at 37 °C.

found that DOX release kinetics was indeed affected by polymer architectures. Micelles of PHEMA70-g-(PCL20-b-PEG45) and PHEMA200-g-(PCL22-b-PEG45) displayed more controllable drug release behavior compared with micelles from linear PCL19-bPEG45 copolymer. PCL19-b-PEG45 micelles displayed severe burst release effect, with more than 50% of DOX released within the initial 10 h. In contrast, brush polymer micelles only released less than 20% of encapsulated DOX in the same period. The depressed DOX release from brush polymer micelles may be due to the enhanced hydrophobic interaction of drug and PCL segments. On the other hand, it is noted that the amount of released DOX from PHEMA200-g-(PCL22-b-PEG45) micelles was less that from PHEMA70-g-(PCL20-b-PEG45) micelles in the tested period, indicating that DOX release profile was also affected by the DP of the PHEMA backbone. Cellular Uptake and Cytotoxicity of DOX-Loaded Brush Copolymer Micelles. To determine the effect of brush polymer micelles carrying DOX in cellular level, we incubated DOX-

loaded PHEMA70-g-(PCL20-b-PEG45) micelles with A549 cells for 2 h. The cells were then observed by CLSM. As shown in Figure 5, cells incubated with free DOX showed strong fluorescence in cell nuclei, while very weak fluorescence was observed in cytoplasm, indicating DOX molecules entered the cells and rapidly accumulated in the nuclei. On the contrary, the intracellular distribution of DOX in the cells incubated with DOX-loaded brush copolymer micelles is significantly different. After 2 h incubation, intense DOX fluorescence was observed in the cytoplasm rather than in cell nuclei. It implies that DOXloaded brush copolymer micelles can be effectively internalized by A549 cells. The cytotoxicity of DOX-loaded brush polymer micelles compared to that of free DOX was determined by MTT assay against A549 cells. It should be mentioned that both the blank micelles of PHEMA70-g-(PCL20-b-PEG45) and PHEMA200-g(PCL22-b-PEG45) did not show significant cytotoxicity to A549 cells (data not shown). As shown in Figure 6, DOX-loaded brush polymer micelles exhibited relatively lower cytotoxicity to A549 cells when compared with free DOX at the same dose. The IC50 values, at which 50% cells proliferation was inhibited, were

2174

Biomacromolecules, Vol. 10, No. 8, 2009

ca. 0.3, 6.7, and 6.9 µg mL-1 for free DOX, DOX-loaded PHEMA70-g-(PCL20-b-PEG45) micelles, and DOX-loaded PHEMA200-g-(PCL22-b-PEG45) micelles, respectively. Similar results were also observed by other groups in terms that DOX-loaded micellar nanoparticles are less potent in cell proliferation inhibition than free DOX at the same dose.9,36 This may be due to the time-consuming DOX-release from micelles and delayed nuclear uptake, as demonstrated by the in vitro DOXrelease and internalization studies by CLSM.

Conclusions PHEMA-g-(PCL-b-PEG) brush polymers were synthesized by combination of ATRP, ROP, and coupling reactions. The brush polymers self-assembled into spherical micelles with the diameter less than 100 nm in aqueous solution. The brush polymer micelles showed enhanced aqueous stability than linear PCL-b-PEG diblock copolymer with similar structure to the side chains as determined by fluorescent probe method. When used as drug delivery carriers, the brush polymer micelles exhibited higher DOX loading capacity than the micelles from linear PCLb-PEG, and the burst drug release in the initial period was significantly suppressed. The micelles can be effectively internalized by A549 cells and slowly released the encapsulated drug molecules. They showed less potent but effective cell proliferation inhibition compared to free DOX. With these advantages, the brush polymer micelles are potential carriers for efficient drug delivery. Acknowledgment. This work was supported by grants from the National Natural Science Foundation of China (50733003, 20774089) and the Ministry of Sciences and Technology of the People’s Republic of China (2006CB933300, 2009CB930300).

References and Notes (1) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113–131. (2) Torchilin, V. P. Pharm. Res. 2007, 24, 1–16. (3) Savic, R.; Luo, L. B.; Eisenberg, A.; Maysinger, D. Science 2003, 300, 615–618. (4) Jabr-Milane, L.; van Vlerken, L.; Devalapally, H.; Shenoy, D.; Komareddy, S.; Bhavsar, M.; Amiji, M. J. Controlled Release 2008, 130, 121–128. (5) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res. 2000, 17, 113–126. (6) Torchilin, V. P. Colloids Surf. B 1999, 16, 305–319. (7) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119–132.

Du et al. (8) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Angew. Chem., Int. Ed. 2003, 42, 4640–4643. (9) Shuai, X.; Ai, H.; Nasongkla, N.; Kim, S.; Gao, J. J. Controlled Release 2004, 98, 415–426. (10) Velluto, D.; Demurtas, D.; Hubbell, J. A. Mol. Pharmaceutics 2008, 5, 632–642. (11) Lavasanifar, A.; Samuel, J.; Kwon, G. S. AdV. Drug DeliVery ReV. 2002, 54, 169–190. (12) Allen, C.; Yu, Y. S.; Maysinger, D.; Eisenberg, A. Bioconjugate Chem. 1998, 9, 564–572. (13) Wang, Y. C.; Tang, L. Y.; Sun, T. M.; Li, C. H.; Xiong, M. H.; Wang, J. Biomacromolecules 2008, 9, 388–395. (14) Zhang, Y.; Zhuo, R. X. Biomaterials 2005, 26, 2089–2094. (15) Kim, S. Y.; Lee, Y. M. Biomaterials 2001, 22, 1697–1704. (16) Kim, K. H.; Lee, J. C.; Lee, J. Macromol Biosci. 2008, 8, 339–346. (17) Arribade, A. V.; Savariar, E. N.; Thayumanavan, S. Mol. Pharmaceutics 2005, 2, 264–272. (18) Wei, H.; Zhang, X.; Cheng, C.; Cheng, S. X.; Zhuo, R. X. Biomaterials 2007, 28, 99–107. (19) Wang, F.; Bronich, T. K.; Kabanov, A. V.; Rauh, R. D.; Roovers, J. Bioconjugate Chem. 2008, 19, 1423–1429. (20) Qiu, L. Y.; Bae, Y. H. Pharm. Res. 2006, 23, 1–30. (21) Geng, Y.; Dalhaimer, P.; Cai, S. S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Nat. Nanotechnol. 2007, 2, 249–255. (22) Cheng, G. L.; Boker, A.; Zhang, M. F.; Krausch, G.; Muller, A. H. E. Macromolecules 2001, 34, 6883–6888. (23) Lee, H. I.; Jakubowski, W.; Matyjaszewski, K.; Yu, S.; Sheiko, S. S. Macromolecules 2006, 39, 4983–4989. (24) Sheiko, S. S.; Sumerlin, B. S.; Matyjaszewski, K. Prog. Polym. Sci. 2008, 33, 759–785. (25) Gao, H. F.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 6633– 6639. (26) Yuan, W. Z.; Yuan, J. Y.; Zhang, F. B.; Xie, X. M.; Pan, C. Y. Macromolecules 2007, 40, 9094–9102. (27) Zhang, M. F.; Breiner, T.; Mori, H.; Muller, A. H. E. Polymer 2003, 44, 1449–1458. (28) Ostmark, E.; Nystrom, D.; Malmstrom, E. Macromolecules 2008, 41, 4405–4415. (29) Beers, K. L.; Boo, S.; Gaynor, S. G.; Matyjaszewski, K. Macromolecules 1999, 32, 5772–5776. (30) Kricheldorf, H. R.; Kreiser-Saunders, I.; Stricker, A. Macromolecules 2000, 33, 702–709. (31) Bae, S. J.; Suh, J. M.; Sohn, Y. S.; Bae, Y. H.; Kim, S. W.; Jeong, B. Macromolecules 2005, 38, 5260–5265. (32) Wu, C.; Woo, K. F.; Luo, X. L.; Ma, D. Z. Macromolecules 1994, 27, 6055–6060. (33) Saito, R.; Iijima, Y.; Yokoi, K. Macromolecules 2006, 39, 6838–6844. (34) Carstens, M. G.; van Nostrum, C. F.; Ramzi, A.; Meeldijk, J. D.; Verrijk, R.; de Leede, L. L.; Crommelin, D. J. A.; Hennink, W. E. Langmuir 2005, 21, 11446–11454. (35) Sachl, R.; Uchman, M.; Matejicek, P.; Prochazka, K.; Stepanek, M.; Spirkova, M. Langmuir 2007, 23, 3395–3400. (36) Soppimath, K. S.; Liu, L. H.; Seow, W. Y.; Liu, S. Q.; Powell, R.; Chan, P.; Yang, Y. Y. AdV. Funct. Mater. 2007, 17, 355–362.

BM900345M