Fine Tuning Micellar Core-Forming Block of Poly(ethylene glycol

ARTICLE pubs.acs.org/Biomac

Fine Tuning Micellar Core-Forming Block of Poly(ethylene glycol)block-poly(ε-caprolactone) Amphiphilic Copolymers Based on Chemical Modification for the Solubilization and Delivery of Doxorubicin Jinliang Yan,† Zhaoyang Ye,*,‡ Min Chen,‡ Zhanzhan Liu,† Yan Xiao,† Yan Zhang,† Yan Zhou,‡ Wensong Tan,‡ and Meidong Lang*,† †

Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, 200237, China ‡ State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237, China ABSTRACT: This study aimed to optimize poly(ethylene glycol)-b-poly(εcaprolactone) (PEG-b-PCL)-based amphiphilic block copolymers for achieving a better micellar drug delivery system (DDS) with improved solubilization and delivery of doxorubicin (DOX). First, the FloryHuggins interaction parameters between DOX and the core-forming segments [i.e., poly(εcaprolactone) (PCL) and poly[(ε-caprolactone-co-γ-(carbamic acid benzyl ester)-ε-caprolactone] (P(CL-co-CABCL))] was calculated to assess the drugpolymer compatibility. The results indicated a better compatibility between DOX and P(CL-co-CABCL) than that between DOX and PCL, motivating the synthesis of monomethoxy-poly(ethylene glycol)-b-poly[(εcaprolactone-co-γ-(carbamic acid benzyl ester)-ε-caprolactone] (mPEG-b-P(CL-co-CABCL)) block copolymer. Second, two novel block copolymers of mPEG-b-P(CL-co-CABCL) with different compositions were prepared via ring-opening polymerization of CL and CABCL using mPEG as a macroinitiator and characterized by 1H NMR, FT-IR, GPC, WAXD, and DSC techniques. It was found that the introduction of CABCL decreased the crystallinity of mPEG-b-PCL copolymer. Micellar formation of the copolymers in aqueous solution was investigated with fluorescence spectroscopy, DLS and TEM. mPEGb-P(CL-co-CABCL) copolymers had a lower critical micelle concentration (CMC) than mPEG-b-PCL and subsequently led to an improved stability of prepared micelles. Furthermore, both higher loading capacity and slower in vitro release of DOX were observed for micelles of copolymers with increased content of CABCL, attributed to both improved drugcore compatibility and favorable amorphous core structure. Meanwhile, DOX-loaded micelles facilitated better uptake of DOX by HepG2 cells and were mainly retained in the cytosol, whereas free DOX accumulated more in the nuclei. However, possibly because of the slower intracellular release of DOX, DOX-loaded micelles were less potent in inhibiting cell proliferation than free DOX in vitro. Taken together, the introduction of CABCL in the core-forming block of mPEG-b-PCL resulted in micelles with superior properties, which hold great promise for drug delivery applications.

’ INTRODUCTION Polymeric micelles prepared from self-assembling amphiphilic block copolymers represent a very promising drug delivery system (DDS), especially for anticancer drugs.13 These nanosized carriers not only are able to enhance the apparent water solubility of hydrophobic drugs but also facilitate targeted delivery to solid tumors via either passive (i.e., enhanced permeability and retention (EPR) effect) or active (e.g., modified with tumor targeting ligands) means, thus achieving a great therapeutic efficacy with little systemic toxicity.2,4,5 Micelles generally exhibit a coreshell structure with a hydrophobic inner core as a depot for hydrophobic drugs and a hydrophilic outer shell as a protective interface between the hydrophobic core and external aqueous milieu.1,4 Hydrophobic interaction between drugs and r 2011 American Chemical Society

the micellar core is considered to be the main driving force for drug entrapment.6 However, owing to the distinct physicochemical properties of drug molecules, a universal DDS for all drugs is unlikely to be achievable, which calls for tailor-made polymeric micelles.4,7 To this end, recent research efforts have been concentrated on understanding the relationship between chemical compositions of polymers and performance-related properties of micelles including size, morphology, drug incorporation capacity, stability, release kinetics, circulation time, biodistribution, as well as subcellular trafficking.1,3,4,8 Received: March 17, 2011 Revised: May 16, 2011 Published: May 20, 2011 2562

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Biomacromolecules Among a large array of amphiphilic block copolymers, poly(ethylene glycol) (PEG)/poly(ε-caprolactone) (PCL) block copolymers have gained a great deal of interest and are applied for the delivery of diverse drugs.911 Whereas PEG forms the hydrophilic corona in the micellar structure, PCL constitutes the hydrophobic core. However, the main drawbacks of PEG-b-PCL copolymer as DDS are obvious, including poor drug loading content, low stability, and slow degrading rate in vivo, possibly due to the high crystallinity of PCL block and lack of functional groups in PCL that would otherwise provide favorable docking sites for drug molecules.8,12,13 Extending PCL block length could moderately improve both drug loading capacity and thermodynamic stability of PEG-b-PCL micelles, mainly attributed to hydrophobic interaction.6,9 However, longer PCL segments tend to have a higher crystallinity, which is not favorable for drug encapsulation.9,12 Shuai et al. prepared a toothbrush-like copolymer, PEG-b-P(HEMA-g-PCL), which resulted in an amorphous micellar core and showed an improved loading capability for doxorubicin (DOX) in comparison with PEG-b-PCL. However, faster release of DOX from these micelles was observed, which may lead to premature drug leaking upon in vivo administration.12 Hence, it becomes critical to achieve a delicate balance between amorphous and crystalline structure within micelles. The drugpolymer compatibility is emerging as a profound index that dictates the ultimate performance of a polymeric micelle and thus provides a valuable tool for designing new polymers and likely encompasses polar and nonpolar interactions as well as hydrogen bonding and can be quantitatively approximated using the FloryHuggins interaction parameter, χ.7,1316 Chemical modifications have been employed to modulate the drugpolymer compatibility with the objective to improve drug delivery performance of PEG-b-PCL-based micelles. A series of work by Lavasanifar and colleagues demonstrated that pendantly-linking carboxyl, benzyl, cholesteryl, stearyl, or palmitoyl groups on the PCL block differentially affected drug solubilization and release profiles for certain drug molecules such as cucurbitacin I (CuI) and amphotericin B (AmB).13,1719 In addition, conjugation of docetaxel (DTX) onto PCL termini was also demonstrated to achieve increased drug loading and retarded drug release of DTX in PEG-b-PCL micelles, possibly due to intermolecular interactions between physically entrapped DTX and covalently attached DTX (e.g., ππ interaction between aromatic groups in DTX).20 Similarly, Kim et al. reported a urea-functionalized PEG-b-polycarbonate block copolymer demonstrating great dynamic stability and drug loading capacity because of ureaurea or ureadrug hydrogen bonding interactions.21 We previously modified PCL block by introducing ketone groups on its backbone, thus conferring extra interactions (mainly hydrogen bonding) with DOX in PEG-b-PCL-based micelles.22 Whereas this modification indeed improved stability and release kinetics, drug loading capacity was dramatically compromised, which was possibly due to the high crystallinity of the ketone-bearing PCL block.23 As a continuing endeavor in developing micelles for drug delivery, the objective of the present study was to design and synthesize a novel family of amphiphilic copolymers based on PEG-b-PCL to improve both drug incorporation and release. Our strategy involved a ring-opening copolymerization of ε-caprolactone (CL) with a newly developed monomer (γ-(carbamic acid benzyl ester)-ε-caprolactone (CABCL)),24 using mPEG as macroinitiator to prepare mPEG-

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Figure 1. (A) Synthetic route to mPEG-b-P(CL-co-CABCL) copolymer. (B) Schematic illustration of DOX loading and probable noncovalent interactions between DOX and CAB.

b-PCL diblock copolymers bearing carbamic acid benzyl ester (CAB) group on the backbone of PCL block (Figure 1A). We hypothesized that the introduction of CAB groups was able to tune the properties of PEG-b-PCL micelles (mostly, the crystallinity and drugpolymer compatibility of the core-forming block) to achieve greater performance of DOX-loaded micelles. The CAB group had a number of drug-compatible groups such as carbamate and benzene ring (Figure 1B). In addition, CAB group as a bulk pendant group on PCL should be able to modulate the crystallinity of PCL block.24,25 Importantly, the content of CAB groups could be easily adjusted by varying the feed ratio of CABCL to CL during copolymerization, enabling a fine-tuning of the micellar core-forming block.

’ EXPERIMENTAL SECTION Materials. Monomethoxy poly(ethylene glycol) (Mn = 5000, mPEG113) was purchased from Aldrich (Milwaukee, WI) and dried by azeotropic distillation with anhydrous toluene prior to use. γ-(Carbamic acid benzyl ester)-ε-caprolactone (CABCL) was prepared as previously described.24 ε-Caprolactone (CL, 99%) from Acros (Geel, Belgium) was purified by vacuum distillation over calcium hydride. Doxorubicin hydrochloride (DOX 3 HCl, 99%) purchased from Wuhan 3B Pharmachem International (Hubei, China) was deprotonated to obtain the hydrophobic DOX according to the literature.26 40 ,60 -Diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 2563

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Table 1. Calculated Solubility Parameters of Doxorubicin, PCL, and P(CL-co-CABCL) partial solubility parameters (J/cm3)1/2 a drug/polymer

total solubility parameters (J/cm3)1/2 b χsmc

δd

δp

δh

Doxorubicin

17.02

11.97

17.69

27.31

PCL

18.74

5.64

6.40

20.59

5.25

P(CL-co-CABCL)

19.65

6.54

8.34

22.32

2.89

δd, δp, and δh are the partial solubility parameters estimated by the Hansen theory of solubility group contribution method (GCM) using Molecular Modeling Pro software. b Total solubility parameters were calculated using eq 2. c FloryHuggins interaction parameters between DOX and different polymers were calculated using eq 1. a

bromide (MTT) were obtained from Sigma (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). All other commercially available reagents and solvents were used as received without further purification unless otherwise mentioned.

Calculation of FloryHuggins Interaction Parameters (χsm) between DOX and Core-Forming Blocks. The Flory Huggins interaction parameter (χsm) between the micellar core and DOX was calculated using eq 113 χsm ¼

ðδs δm Þ2 Vs RT

ð1Þ

where δs and δm are solubility parameters for the drug and the micellar core, respectively; Vs is the molar volume of drug, R is the universal gas constant, and T is the Kelvin temperature. The solubility parameter was obtained by Hansen’s approach, which uses partial solubility parameters to calculate the total solubility parameters according to eq 213 δ ¼ ðδ2d þ δ2p þ δ2h Þ1=2

ð2Þ

where δd, δp, and δh refer to the partial solubility parameters accounting for van der Waals dispersion forces between atoms, dipoledipole interactions between molecules, and proclivity of hydrogen bonding between molecules, respectively. The partial solubility parameters for the drug (DOX) and the core-forming blocks (Table 1) were estimated by the Hansen theory of solubility group contribution method (GCM) using Molecular Modeling Pro software (Chem SW).27 Polymer Synthesis and Characterization. Synthesis of mPEGb-P(CL-co-CABCL) Block Copolymer. mPEG-b-P(CL-co-CABCL) block copolymers were synthesized by ring-opening polymerization. In a typical reaction, mPEG (0.5 g), CABCL (0.75 g), and CL (0.75 g) were added to a flame-dried and nitrogen-purged flask. After the reaction mixture became homogeneous at 50 °C, Sn(Oct)2 (0.001 equiv of monomer) was added. The reaction lasted for 24 h at 130 °C under vacuum. The crude product was dissolved in chloroform and purified by precipitation in cold diethyl ether. Similarly, mPEG-b-PCL was synthesized by homopolymerization of CL. 1 H NMR Spectroscopy. 1H NMR spectra were recorded using a Bruker Avance 400 spectrometer operating at 400 MHz with deuterated chloroform (CDCl3) as solvent and tetramethylsilane (TMS) as an internal standard. Gel Permeation Chromatography (GPC). A Waters GPC system equipped with a Waters 1515 HPLC solvent pump, Waters 2414 refractive index detector, and three Ultrastyragel columns in series was used to determine the molecular weight and polydispersity index of the copolymers. Tetrahydrofuran (THF) was used as eluent and delivered at a flow rate of 1.0 mL/min at 35 °C. The molecular weight was calibrated by polystyrene standards. Differential Scanning Calorimetry (DSC). DSC analysis was carried out on a TA Instruments modulated DSC2910 apparatus under a nitrogen flow (10 mL/min). Samples were first heated from 0 to

80 °C, followed by cooling to 0 °C to erase the thermal history and then heated to 80 °C at a rate of 10 °C/min. Wide-Angle X-ray Diffraction (WAXD). WAXD analysis was performed over the range 2θ from 10 to 40° on a Bruker AXS: D8 Focus equipped with graphite monochromatized Cu KR radiation (λ = 1.54056 Å). Fourier Transform Infrared Spectrometry (FT-IR). Polymer samples were dissolved in chloroform, and a film was cast on a KBr pellet by evaporation of the solvent. FT-IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer in the range of wavenumbers between 4000 and 500 cm1. Micelles Preparation and Characterization. DOX-Loaded and DOX-Free Micelles Preparation. DOX-loaded micelles were prepared by a dialysis method. Typically, copolymer (20 mg) and DOX (10 mg) were dissolved in 2 mL of DMSO. Under gentle stirring, the solution was added dropwise to 10 mL of double-distilled water. After stirring for 2 h at room temperature, the solution was transferred to a 7000 Da molecular weight cut-off dialysis bag and dialyzed for 48 h to remove the organic solvents and free DOX. The micellar solution was further filtered through a 0.45 μm syringe filter to remove the residual DOX aggregates. To obtain micelle powders, we froze and lyophilized the micellar solution. DOX-free micelles were prepared similarly without the addition of DOX. Critical Micelle Concentration (CMC). The CMC of the micelles was measured using fluorescent pyrene as a probe on a Fluorolog fluorescence spectrophotometer (Horiba Jobin Yvon). A predetermined amount of pyrene in acetone solution was added to volumetric flasks; then, the acetone was allowed to evaporate completely. Micellar solutions at different concentrations were added to pyrene and left to equilibrate with pyrene in an incubator overnight. The final concentration of pyrene was kept at 6.0 107 mol/L. The excitation spectra were scanned from 250 to 360 nm at a fixed emission wavelength of 390 nm with bandwidth 3 nm. The ratios of pyrene probe fluorescence intensity at 338 and 333 nm (I338/I333) were calculated and plotted against the concentration logarithm of micelles. CMC was obtained from the intersection of two tangent plots of intensity ratio I338/I333 versus the logarithm of polymer concentrations. Dynamic Light Scattering (DLS). DLS was performed on a zeta potential/particle sizer Nicomp 380 ZLS (Santa Barbara, CA) to determine the particle size and size distribution of prepared micelles. The micellar solutions (1 mg/mL) were passed through a 0.45 μm microfilter before measurement. Samples were measured at room temperature at a scattering angle of 90° to obtain the hydrodynamic diameter of the particles (Zave) and the particle size distribution (PDI). To evaluate the stability of prepared micelles, we incubated micellar solutions (1 mg/mL) in PBS (pH 7.4) at 37 °C for 0, 1, 3, 6, 11, and 20 days. Particle size (Zave) was monitored with DLS. All measurements were repeated three times, and the data were reported as the mean diameter ( standard deviation (SD). Transmission Electron Microscopy (TEM). For the preparation of TEM samples, a drop of micelle solution (1 mg/mL) was placed on a 2564


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copper grid coated with carbon film and dried at room temperature. The morphologies of micelles were observed on a TEM instrument (JEOL/ JEM-2000EXII) operated at an accelerating voltage of 60 kV.

Drug Loading Content (DLC) and Drug Loading Efficiency (DLE). To determine DOX loading level and encapsulation efficiency, a known amount of freeze-dried DOX-loaded micelles was dissolved in DMSO, and the absorbance at 485 nm was measured on a UVvis spectrophotometer (UVvis 8500 Techcomp, China). The weight of encapsulated DOX in the micelles was calculated with a standard curve obtained from DOX/DMSO solutions at a series of DOX concentrations. DLC and DLE were calculated as eqs 3 and 4 DLC ð%Þ ¼

weight of drug in micelles 100% weight of drug-loaded micelles

ð3Þ

weight of drug in micelles 100% weight of drug in feed

ð4Þ

DLE ð%Þ ¼

In Vitro Release of DOX. DOX-loaded micelles at a concentration of 1 mg/mL (3 mL) in a dialysis membrane tube (MWCO: 7000) were incubated in PBS (30 mL, pH 7.4) at 37 °C under stirring at a speed of 100 rpm. At specified time intervals, 2 mL of dialysate was withdrawn and replaced with fresh PBS. The DOX content in the samples was analyzed with a UVvis spectrophotometer at 485 nm. Three replicate measurements were carried out for each time point. In Vitro Cell Experiments. Cell Culture. HepG2 liver carcinoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin at 100 U/mL, and streptomycin at 100 mg/L at 37 °C in a humidified atmosphere of 5% CO2. MTT Assay. The cytotoxicity of DOX-free micelles, DOX-loaded micelles, and free DOX on HepG2 cells was examined by MTT assay. All sample solutions were filtered through a 0.22 μm microfilter and diluted with DMEM to obtain preset concentrations. The cells were seeded in a 96-well plate at a density of 1 104 cells/well. Twenty four hours after cell seeding, the culture medium was replaced with 100 μL of sample solutions in DMEM, and the cells were incubated for another 48 h. The culture medium was then replaced with fresh DMEM (100 μL), followed by the addition of 20 μL of MTT solution in PBS (5 mg/ mL), and incubated for 4 h. Afterward, the MTT-containing medium was aspirated, and 200 μL of DMSO was added to each well to extract the formazan products with gentle agitation for 10 min. The optical density (OD) of the extracts was measured at 570 nm on a Bio-Rad 550 microplate reader. The cell viability was calculated as follows: cell viability (%) = (ODtest)/(ODcontrol) 100, where ODtest was the absorbance at the presence of sample solutions and ODcontrol was the absorbance without treatment. All tests were performed in triplicate. Cellular Uptake Studies. The cellular uptake of DOX was studied with flow cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry studies, cells were seeded in six-well plates at a density of 1 106 cells/well and cultured for 24 h. Following this, the cells were treated with free DOX (10 μg/mL) or DOX-loaded micelles (10 μg/mL equivalent DOX concentration) for either 4 or 24 h. After the preset time intervals, the culture medium was discarded, and cells were washed three times with PBS and harvested with trypsinization. The cell pellets were resuspended in PBS and measured for the fluorescence intensity (excitation: 488 nm; emission: 575 nm) on a BD FACS Calibur flow cytometer (Beckton Dickinson), and Cell Quest software was used to analyze the data. For CLSM observation, cells were seeded onto glass coverslips in a six-well culture plate at a density of 5 104 cells/well and incubated for 24 h to allow the cells to attach. Following this, the cells were treated with free DOX (10 μg/mL) or DOX-loaded micelles (10 μg/mL

equivalent DOX concentration) for 4 or 24 h. At the end of incubation, the cells were washed with PBS three times and fixed with 4% paraformaldehyde for 30 min, and nuclei were stained with DAPI. Cells were mounted and observed with CLSM using an FV1000 apparatus (Olympus, Japan).

’ RESULTS AND DISCUSSION Design of PEG-b-PCL-Based Copolymer Based on Enhanced Compatibility Between DOX and Micellar CoreForming Block. Within recent years, polymerdrug compat-

ibility is considered to be one of the most important factors in dictating the effectiveness of a polymeric micelle-based DDS.7,13,14,16 Good compatibility is correlated with favorable performance-related properties of micelles including drug solubility, drug loading capacity, and drug release.7,15,16,27,28 The goal of the present study was to develop novel mPEG-b-PCL-based polymeric micelles with improved performance in the delivery of DOX based on an evaluation of the compatibility between DOX and the micellar core using the FloryHuggins interaction parameters (χsm). We proposed a novel modification on mPEG-b-PCL copolymers by introducing a functionalized monomer CABCL to the PCL block (Figure 1A). In comparison with CL, CABCL had extra drug-compatible groups such as secondary amine, carbonyl, and benzyl, which would induce more interactions between DOX and the micellar core through hydrogen bonding21 or ππ packing,29 as illustrated in Figure 1B. FloryHuggins interaction parameters between DOX and core-forming blocks (i.e., PCL and P(CL-co-CABCL)) were calculated with a GCM method, which has been successfully applied by others.13,27 Ideally, for achieving favorable drugpolymer interaction, the total solubility parameters for drug molecule and the core-forming block should be equal (i.e., in eq 1, when δs = δm, χsm = 0). Therefore, it is practically recognized that lower χsm value indicates more compatibility. Hence, the χsm values were used as a guide in predicting enhanced compatibility by comparing the trend of the computed interaction parameters.13,27 Table 1 listed the values of solubility parameters (δd, δp, and δh) obtained by Hansen’s approach for DOX, PCL, and P(CL-co-CABCL), which were then used to calculate total solubility parameter with eq 2. The value of χsm was then computed using eq 1 and also summarized in Table 1. It was found that the introduction of CABCL decreased χsm, suggesting that P(CL-co-CABCL) block copolymers would be more compatible with DOX and mPEG-b-P(CL-co-CABCL) block copolymers might be superior as DDS for DOX. Synthesis and Characterization of Block Copolymers. The synthesis of mPEG-b-P(CL-co-CABCL) diblock copolymers was carried out by ring-opening copolymerization of CL and CABCL in bulk using mPEG as a macroinitiator and Sn(Oct)2 as a catalyst at 130 °C (Figure 1A).30,31 The chemical structures of mPEG-b-PCL and mPEG-b-P(CL-co-CABCL) copolymers were characterized with 1H NMR, and typical spectra for both copolymers were shown in Figure 2. All of the peaks corresponding to characteristic hydrogen atoms in respective copolymers were labeled. Successful synthesis of mPEG-b-P(CL-co-CABCL) could be confirmed by comparing the 1H NMR spectra in Figure 2A,B. In addition, the unimodal peaks in the GPC traces for the three copolymers also confirmed that the copolymerization was successful (Figure 3). The molecular weights of prepared block copolymers were determined with 1H NMR and GPC analysis and summarized in Table 2. In the present 2565


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Figure 2. 1H NMR spectra of (A) mPEG113-b-P(CL106-co-CABCL11) and (B) mPEG113-b-PCL135 copolymers in CDCl3.

Figure 3. GPC traces of the macroinitiator mPEG with a molecular weight of 5000 (mPEG113) (I), mPEG113-b-P(CL67-co-CABCL27) (II), mPEG113-b-P(CL106-co-CABCL11) (III), and mPEG113-b-PCL135 (IV).

study, we chose mPEG with a molecular weight of 5000 Da (repeating units: 113), and the molecular weight of the coreforming block was preset to be 15 000 Da for all copolymers. The feed ratio of CL/CABCL was varied such that three different copolymers were prepared in this study (Table 2). On the basis of 1H NMR spectra, the molecular weights of the copolymers were calculated by comparing the integrated areas under characteristic peaks representing EG unit (CH2CH2O, δ 3.603.68), CL unit (CH2O, δ 1.301.43), as well as CABCL unit (C6H5, δ 7.317.40). It was found that for all three copolymers, the molecular weights determined by 1H NMR method agreed very well with the theoretical values (Table 2). However, the molecular weights measured with GPC analysis were obviously lower than the theoretical values for all three copolymers, which was possibly attributed to the difference in hydrodynamic volumes between block copolymers and polystyrene standards.32 These results demonstrated facile synthesis of the diblock copolymers with well-controlled chemical compositions, thus offering great advantages in tailoring the core structures of micelles. In the following studies, we compared these three copolymers, mPEG113-b-PCL135, mPEG113-b-P(CL106-co-CABCL11), and mPEG113-b-P(CL67-co-CABCL27), concerning the physicochemical properties as well as micellar formation, aiming at providing an insightful view on the effects of the core-forming block on the properties of micelles. Crystallinity Study. Physical state, especially the crystalline structure within the micellar core, has a great influence on drug incorporation, micelle stability, as well as drug release. Whereas an amorphous structure is favorable for drug loading,12 a crystalline structure provides good kinetic stability and sustained drug release of micelles.33,34 Therefore, the crystallinity of the copolymers was thoroughly investigated with WAXD, DSC, and FTIR measurements. As shown in Figure 4A, the WAXD patterns of PCL and mPEG homopolymers showed prominent diffraction peaks at 2θ = 21.4 and 23.8° and 2θ = 19.1 and 23.4°, respectively, suggesting the high crystallinity. For mPEG113-b-PCL135, the coexistence of the characteristic diffraction peaks for both PCL and mPEG were observed. It was also noted that the peaks at 2θ = 23.4° for mPEG and 23.8° for PCL overlapped with each other. These results suggested a microphase separation in the diblock copolymer.35

Importantly, it was further found that the introduction of CABCL units in the PCL block significantly decreased the crystallinity of PEG-b-PCL based on the fact of those diminishing characteristic peaks. Moreover, when the CABCL content increased to 30 mol % (i.e., mPEG113-b-P(CL67-co-CABCL27)), the diffraction peaks of the PCL block completely disappeared, indicating that the P(CL-co-CABCL) block became amorphous . DSC curves of polymers were shown in Figure 4B, and the melting temperature (Tm), and melting enthalpy (ΔHm) were summarized in Table 3. Whereas PCL and mPEG homopolymers showed a single melting peak with Tm of 58.8 and 53.2 °C, respectively, a bimodal endothermic peak appeared for mPEG113b-PCL135, suggesting that two distinct crystalline domains existed in the copolymer, which was consistent with the WAXD data. However, for both mPEG113-b-P(CL106-co-CABCL11) and mPEG113-b-P(CL67-co-CABCL27), only one single melting peak appeared, and Tm shifted down to 47.4 and 46.4 °C, respectively (Figure 4B). This was mainly attributed to the amorphization of P(CL-co-CABCL) block as well as the interactions at the molecular level between the two blocks, thus suppressing the mobility of molecular chain and decreasing the crystallinity of PEG block,35,36 which was in accordance with the gradually reduced diffraction peaks for mPEG in WAXD patterns, as shown in Figure 4A. On the basis of the results of WAXD and DSC studies, as shown in Figure 4A,B, for mPEG113-b-P(CL106-co-CABCL11), the single melting peak was ascribed to the overlapping of the melting peaks of the two blocks, whereas for mPEG113-b-P(CL67-co-CABCL27), the single melting peak was assigned to the melting peak of mPEG block only because of the completely amorphous P(CL67-coCABCL27) block. Meanwhile, the melting enthalpy (ΔHm) of copolymers decreased from 82.4 to 36.9 J/g with the increase in the content of CABCL from 0 to 30 mol %. The transition from crystalline structure to amorphous state of P(CL-co-CABCL) block in the block copolymers was further supported with FT-IR analysis. It has been established that the carbonyl absorption of PCL varies because of different physical states giving peaks at ∼1725 and ∼1735 cm1 within crystalline and amorphous microdomains, respectively.37 As shown in Figure 4C, in comparison with that of mPEG113-b-PCL135, the carbonyl absorption shifted toward ∼1735 cm1 for both mPEG113-b-P(CL106-coCABCL11) and mPEG113-b-P(CL67-co-CABCL27), indicating that 2566


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Table 2. Characterization of the Copolymers

polymersa mPEG113-b-PCL135

CABCL content (mol %)b

Mn,Th

c

1

feed 0

product

(g 3 mol )

Mn,NMRc 1

(g 3 mol )

Mn,GPCd 1

(g 3 mol )

polydispersity indexd

0

20 000

20 400

16 500

1.36

mPEG113-b-P(CL106-co-CABCL11)

10

9.2

20 000

20 000

15 100

1.70


30

29.2

20 000

19 700

13 200

1.88

Subscript refers to the degree of polymerization of each block determined by 1H NMR. b fCABCL = nCABCL/(nCL þ nCABCL) (mol/mol). c Determined by 1H NMR (CDCl3 as the solvent). d Determined by GPC (THF as the solvent). a

Table 3. DSC Analysis of the Copolymers.a polymers PCL mPEG113

Tm (°C) 58.8 53.2

ΔHm (J/g) 85.2 195.1

mPEG113-b-PCL135

54.2, 58.3

82.4


47.4

67.4


46.4

36.9

DSC analysis was carried out under N2 at 10 °C/min from 0 to 80 °C (second heating run).

a

Figure 4. WAXD patterns (A), DSC curves (B), and FT-IR spectra (C) of the copolymers: mPEG113-b-PCL135, mPEG113-b-P(CL106-coCABCL11), and mPEG113-b-P(CL67-co-CABCL27).

the P(CL-co-CABCL) block gradually turned amorphous. Hence, the crystalline property of PEG-b-PCL could be tuned by adjusting the content of CABCL units. Previously, the high crystallinity of PCL has been related to low drug loading content and poor degradation in vivo for micelles.12 By modulating the crystalline structure of the micellar core, it would possibly lead to a better performance.

Preparation and Characterization of Copolymer Micelles. The dialysis method was used to induce the self-assembly of the amphiphilic diblock copolymers into micelles in aqueous solution. The formation of micelles was first evaluated by measuring CMC with the fluorescence technique using pyrene as a probe. Pyrene was chosen because it preferentially partitions into the a hydrophobic microdomain accompanied by a change in the photophysical properties.38 With increased polymeric micelle concentration, a red shift from 333 to 338 nm of pyrene in the excitation spectrum happens, reflecting the change in environmental polarity. The ratio of pyrene fluorescence intensities excited at 338 and 333 nm (I338/I333) was plotted as a function of the logarithm of copolymer concentration for all three copolymers. As illustrated in Figure 5A, the intensity ratio remained almost unchanged at low copolymer concentrations and increased abruptly once the copolymer concentration reached the CMC, indicating the formation of micelles. The CMC values for these copolymers were listed in Table 4. Compared with the CMC of mPEG113-b-PCL135 (1.07 103 mg/mL), the CMC of mPEG113-b-P(CL106-co-CABCL11) (0.72 103 mg/mL) and mPEG113-b-P(CL67-co-CABCL27) (0.69 103 mg/mL) were lower regardless of their shorter hydrophobic blocks because of the smaller number of repeating units (Table 2). The lower CMC of mPEG113-b-P(CL106-coCABCL11) and mPEG113-b-P(CL67-co-CABCL27) indicated their higher thermodynamic stability, representing an advantage upon injection into body fluids.18,28 This was possibly because the introduction of CABCL unit provided more hydrophobicity in PCL block, thus showing a higher tendency to self-assembling in aqueous solution.18 In addition, possible intramolecular interactions between CAB groups (e.g., ππ interactions and hydrogen bonding) might also favor the self-association.21 Prepared micelles were also characterized with DLS and TEM for the size, size distribution, and morphology. As summarized in Table 4, the average diameter of the micelles decreased from 135.1 to 96.9 nm when the CABCL content increased from 0 to 30 mol % in the copolymers, suggesting that decreasing the hydrophobic block length led to a decreased micelle diameter.9 2567


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Figure 6A illustrated the size distribution of micelles prepared with mPEG113-b-P(CL67-co-CABCL27) with a narrow PDI (0.097) (Table 4), and a spherical morphology of the micelles was confirmed by TEM imaging (Figure 6C). A long-term stability study up to 20 days for the micelles was also performed in PBS (pH 7.4) at 37 °C by monitoring the particle size. As shown in Figure 5B, the size of all micelles decreased slightly during the first 3 days and then was maintained until the end of the study. The ratio of the particle size after 20 days to the initial size was 0.84, 0.89, and 0.88 for the three micelles, respectively, indicating that the micelles were stable under neutral conditions and those with modified micellar core were more stable.39 DOX Loading and In Vitro Drug Release. DOX was loaded into micelles via a membrane dialysis method. The characteristics of DOX-loaded micelles at the feed ratio of DOX to polymer of 10/20 (mg/mg) were also listed in Table 4. It was found that the sizes of DOX-loaded micelles were larger than those of the parent ones; however, all were below 200 nm, which was very crucial in avoiding the body defense mechanisms (e.g., reticuloendothelial system (RES)).21,40,41 In addition, the size distribution remained narrow with a PDI of 0.121 (Figure 6B and Table 4). As seen in Figure 6D, the TEM image of DOX-loaded micelles prepared with mPEG113-b-P(CL67-co-CABCL27) also had a spherical morphology. The DLC of mPEG113-b-PCL135, mPEG113-b-P(CL106-coCABCL11), and mPEG113-b-P(CL67-co-CABCL27) micelles was 10.1, 19.0, and 25.5%, respectively, corresponding to DLE of 22.5, 46.9, and 68.5%, respectively (Table 4). Obviously, the introduction of CABCL to the PCL block dramatically improved

both the DOX loading capacity and the efficiency more two-fold. Factors including core-forming block length and crystallinity, drug-core affinity, drug solubility in water, and micelles preparation method would affect drug encapsulation.6,8 Our crystallinity study for the copolymers demonstrated that with increased content of CABCL, the PCL block gradually became amorphous (Figure 4). In addition, similar results were found for the crystallinity study on prepared micelles (data not shown). Hence, the enhanced drug encapsulation ability of mPEG-b-P(CL-coCABCL) micelles could be attributed to the amorphous core structure providing more accessible volume for DOX.8,12 Furthermore, extra noncovalent interactions between DOX and P(CL-co-CABCL) block might also play an important role because there were several carbonyl groups, hydroxyl groups, benzene rings, and one primary amine group in one DOX molecule and one carbonyl group, second amine group, and benzene ring in mPEG-b-P(CL-co-CABCL) copolymer as schematically illustrated in Figure 1B. This was in agreement with a study by Kataoka et al., wherein enhanced noncovalent interactions between hydrophobic inner core and drug molecules improved the loading capacity of adriamycin (ADR).42 Lavasanifar et al. had also reported that the introduction carboxyl groups to the core forming block of PEO-b-PCL micelles led to efficient solubilization of Amphotericin B (AmB) because of the formation of hydrogen bonds between the carboxyl groups and hydroxyl groups of drug.18

Figure 5. Plots of intensity ratio (I338/I333) against log C of polymeric micelles (A) and the average diameter of micelles over time at 37 °C in PBS (pH 7.4) (B).

Figure 6. Size distribution determined with DLS (A,B) and TEM (C, D) for DOX-free micelles (A,C) and DOX-loaded micelles (B,D) prepared with mPEG113-b-P(CL67-co-CABCL27).

Table 4. Characteristics of Polymeric Micelles Before and After DOX Loading DOX-loaded micellesc

DOX-free micelles polymers

diameter (nm)a

PDIa

CMC (mg/mL)b

diameter (nm)a

PDIa

DLC (%)

DLE (%)

3

mPEG113-b-PCL135

135.1 ( 0.4

0.127 ( 0.012

1.07 10

169.6 ( 0.9

0.074 ( 0.006

10.1

22.5


122.8 ( 1.1

0.102 ( 0.007

0.72 103

191.0 ( 1.9

0.104 ( 0.021

19.0

46.9


96.9 ( 0.6

0.097 ( 0.017

0.69 103

186.7 ( 1.3

0.121 ( 0.005

25.5

68.5

Determined by DLS. b CMC was determined by a fluorescence spectroscopic method using pyrene as the fluorescence probe. c Feed ratio of DOX to polymers was 10/20 (mg/mg). a

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Biomacromolecules In vitro drug release of DOX from micelles was investigated in PBS (pH 7.4) at 37 °C and compared with that of free DOX. As shown in Figure 7, DOX release from micelles occurred in a controlled manner without a burst release compared with that of

Figure 7. In vitro release profiles of DOX from DOX-loaded micelles in PBS (pH 7.4) at 37 °C. Each data point represented mean ( SD (n = 3).

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free DOX, confirming that DOX molecules were well encapsulated in the inner core of micelles. Although a relative rapid release of DOX during the first 24 h for all three DOX-loaded micelles samples was shown, a very slow release over a prolonged time followed. In addition, the release of DOX from mPEG113-bP(CL106-co-CABCL11) and mPEG113-b-P(CL67-co-CABCL27) micelles was retarded in comparison with that from mPEG113b-PCL135 micelles, suggesting a more controlled drug release for mPEG-b-P(CL-co-CABCL) micelles. Within 72 h, the cumulative release of DOX was 32, 18, and 15% for mPEG113-b-PCL135, mPEG113-b-P(CL106-co-CABCL11), and mPEG113-b-P(CL67-coCABCL27) micelles, respectively. The release of a drug from polymeric micelles is a rather complicated process and is governed by many factors including polymer degradation, molecular weight, crystallinity, micelle stability, rate of drug diffusion from the micellar core, affinity between the micellar core-forming block and the drug, and so on.34,43,44 It is believed that the crystalline phase does not allow water to penetrate and acts as the tortuosity in the diffusional pathway.34 In other words, the higher the crystallinity, the slower the release rate. However, in this study, the slower release of DOX from polymeric micelles with more amorphous micellar core (mPEG-b-P(CL-co-CABCL) versus mPEG-b-PCL) was observed, possibly because of higher

Figure 8. CLSM-images of HepG2 cells incubated with free DOX for 4 (A) and 24 h (C), and with DOX-loaded mPEG113-b-P(CL67-co-CABCL27) micelles for 4 (B) and 24 h (D). For each panel, images from left to right were the cells with bright field, with DOX fluorescence, with nuclear staining with DAPI and overlays of images. Scale bar, 20 μm. 2569


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Figure 10. Cell viability of HepG2 cells after incubation for 48 h with DOX-free micelles at different concentrations of polymers (A) and with DOX-loaded micelles and free DOX at different concentrations of DOX (B).

Figure 9. Flow cytometry histogram profiles (A) and fluorescence intensity (B) of HepG2 cells after incubation with free DOX and DOX-loaded mPEG113-b-P(CL67-co-CABCL27) micelles for 4 and 24 h.

affinity between DOX molecules and P(CL-co-CABCL) block, which played a dominant role. Furthermore, the release of DOX from mPEG113-b-P(CL67-co-CABCL27) micelles was even slower than that from mPEG113-b-P(CL106-co-CABCL11) micelles, suggesting the possibility of fine-tuning the chemical composition of the core-forming block for better controlled drug release. The more controllable drug release for mPEG-b-P(CLco-CABCL) micelles implied that more DOX molecules could be retained in polymeric micelles during their in vivo transport before reaching target tissue/cells, thus attenuating side toxicity, providing higher therapeutic efficacy, and representing a beneficial factor for drug delivery.4,45 In Vitro Cellular Uptake of DOX-Loaded Micelles. Because DOX itself is red fluorescent, it allows us to monitor easily the cellular uptake and intracellular distribution of free DOX and DOX-loaded micelles. Free DOX and DOX-loaded micelles prepared with mPEG113-b-P(CL67-co-CABCL27) were incubated with HepG2 cells, followed by observation under CLSM at 4 and 24 h of post-treatment. As shown in Figure 8, after 4 h of incubation with the free DOX, strong fluorescence was observed mainly in the nuclei (stained as blue) of the cells (Figure 8A, Merge), indicating that DOX molecules entered the cells and rapidly accumulated in the nuclei. In contrast, when the cells were incubated with DOX-loaded micelles, the fluorescence was mainly observed in cytoplasm with a much weaker fluorescence in the nucleus (Figure 8B). When the incubation period increased to 24 h, cell death was obvious when treated with free

DOX (Figure 8C). For DOX-loaded micelles, the red fluorescence in both cytoplasm and nuclei increased with time, most likely resulting from the diffusion of released DOX from the micelles into nuclei (Figure 8D). These results suggested that DOX-loaded micelles might be internalized through the endocytic mechanism; then, the DOX molecules are released and diffused through endocytic compartments (i.e., endosomes and subsequently lysosomes) to the nuclei eventually.12,46 The cellular uptake of free DOX and DOX-loaded micelles into HepG2 cells was further quantitatively analyzed with flow cytometry. The fluorescence of DOX in cells at different time points after treatment (4 and 24 h) was recorded, and cells without treatment were used as a negative control. As shown in Figure 9, the uptake of DOX was much higher in the cells incubated with DOX-loaded micelles than that treated with free DOX for both 4 and 24 h. Moreover, the cellular uptake of free DOX and DOX-loaded micelles was time-dependent (Figure 9B). The enhanced cellular uptake of DOX by DOXloaded micelles could be attributed to the fact that micelles were more readily internalized by an endocytosis mechanism,47,48 whereas free DOX was transported into cells by a passive diffusion mechanism. Moreover, the multidrug resistance (MDR) effect might also play a role in reducing the accumulation of free DOX with cells because free DOX molecules transported within cells are likely to be pumped back out of the cytosol by the p-glycoprotein pump (Pgp) expressed on the membrane of cancerous cells, such as HepG2 cells, whereas amphiphilic copolymers have the potential to inhibit Pgp function.47,49 In Vitro Cytotoxicity of the Micelles. The cytotoxicity of micelles with and without DOX against HepG2 cells was determined by MTT assay. It is important to note that no obvious cytotoxicity against HepG2 cells was observed, even though the DOX-free polymeric micelles concentration reached 250 μg/mL, as shown in Figure 10A. Figure 10B showed the viability of HepG2 cells in the presence of free DOX and DOX-loaded 2570


Biomacromolecules micelles (mPEG113-b-PCL135, mPEG113-b-P(CL106-co-CABCL11) and mPEG113-b-P(CL67-co-CABCL27)). The cell viability was dose-dependent, and DOX-loaded micelles exhibited much lower cytotoxicity when compared with free DOX at the equivalent dose. The IC50 values, a concentration at which 50% of cells was killed, were 2.2, 7.9, 9.1, and 9.2 μg/mL for free DOX, mPEG113b-PCL135, mPEG113-b-P(CL106-co-CABCL11), and mPEG113-bP(CL67-co-CABCL27) micelles, respectively. The DOX-loaded micelles showed a much lower cytotoxicity than that of free DOX, which was mostly due to the slower release of DOX from micelles and the delayed the nuclear uptake of DOX in HepG2 cells, as confirmed with both in vitro DOX release and cellular uptake studies.12,50

’ CONCLUSIONS In this study, we presented a new approach for chemical tailoring of the micellar core of mPEG-b-PCL through the introduction of CAB groups onto PCL block. The incorporation of CAB functional groups significantly decreased the crystallinity and the CMC of mPEG-b-PCL block copolymers, enhanced the stability of micelles, improved the solubilization of hydrophobic drug DOX, which was consistent with the predication by calculated FloryHuggins interaction parameters, and also achieved much slower DOX release. The superior performance of mPEG-b-P(CL-co-CABCL) copolymers as DDS was attributed to both amorphous micellar core and noncovalent interactions between drugs and the core-forming block. Moreover, the DOX-loaded micelles were more efficiently internalized by HepG2 cells than free DOX; however, they exhibited lower cytotoxicity due to the slow release of encapsulated DOX from micelles. The present study highlighted the importance of chemical modification on the PEG-b-PCL micellar core for improved drug delivery. ’ AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected] (Z.Y.); [email protected]. cn (M.L.). Tel: þ86-21-64253916. Fax: þ86-21-64253916.

’ ACKNOWLEDGMENT This research was supported by the Fundamental Research Funds for the Central Universities, the National Natural Science Foundation of China (20804015, 31000424), Specialized Research Fund for the Doctoral Program of Higher Education (200802511021, 20100074120009), Shanghai Pujiang Program (10PJ1402200), “Shu Guang” Project of Shanghai Municipal Education Commission, the Natural Science Foundation of Shanghai (08ZR1406000), and Program for Changjiang Scholars and Innovative Research Team in University (IRT0825). ’ REFERENCES (1) Gaucher, G.; Dufresne, M.; Sant, V.; Kang, N.; Maysinger, D.; Leroux, J. J. Controlled Release 2005, 109, 169–188. (2) Torchilin, V. P. Pharm. Res. 2006, 24, 1–16. (3) Yokoyama, M. Expert Opin. Drug Delivery 2010, 7, 145–158. (4) Mikhail, A. S.; Allen, C. J. Controlled Release 2009, 138, 214–223. (5) Rapoport, N. Prog. Polym. Sci. 2007, 32, 962–990. (6) Yang, X.; Zhu, B.; Dong, T.; Pan, P.; Shuai, X.; Inoue, Y. Macromol. Biosci. 2008, 8, 1116–1125. (7) Liu, J. B.; Xiao, Y. H.; Allen, C. J. Pharm. Sci. 2004, 93, 132–143.

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Fine Tuning Micellar Core-Forming Block of Poly(ethylene glycol

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