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Biodegradable stereocomplex micelles (SCMs) based on amphiphilic dextran-block-polylactide (Dex-b-PLA) were designed and used for efficient intracellu...
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Biodegradable Stereocomplex Micelles Based on Dextran-blockpolylactide as Efficient Drug Deliveries Ziwei Zhao,† Zhe Zhang,† Li Chen,*,† Yue Cao,‡ Chaoliang He,*,‡ and Xuesi Chen‡ †

Department of Chemistry, Northeast Normal University, Changchun 130024, P. R. China Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China

‡

S Supporting Information *

ABSTRACT: Biodegradable stereocomplex micelles (SCMs) based on amphiphilic dextran-block-polylactide (Dex-b-PLA) were designed and used for efficient intracellular drug deliveries. The Dex-b-PLA copolymers were successfully synthesized by click reaction. The structures of the resultant copolymers were verified by 1H NMR and FT-IR spectra. The formation of stable micelles through self-assembly driven by the stereocomplexation between enantiomeric L- and D-PLA blocks was characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), and fluorescence techniques. It was interesting to observe that the SCMs showed lower critical micelle concentration values (CMCs) because of the stereocomplex interaction between PLLA and PDLA. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis provided information on the thermal and crystal properties of the copolymers and SCMs. The improved stability of SCMs should be attractive for intracellular drug delivery. Thus, a model anticancer drug doxorubicin (DOX) was loaded into micelles, and the in vitro drug release in was also studied. The release kinetics of DOX showed DOX-loaded SCMs exhibited slower DOX release. Confocal laser scanning microscopy (CLSM) and flow cytometry studies also showed that the DOX-loaded SCMs exhibited a slower drug release behavior. Meanwhile, the MTT assay demonstrated that DOX-loaded SCMs show lower cellular proliferation inhibition against HepG2. In sum, the micelles through self-assembly driven by stereocomplex interaction would have great potential to be used as stable delivery vehicles for pharmaceutical and biomedical applications.

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INTRODUCTION Despite conventional chemotherapy proving partially successful in treatment and prolonging the lives of cancer patients, the clinical outcomes are dissatisfactory because of the lifethreatening side effects.1 The limited clinical success is mostly due to the lack of tumor selectivity of anticancer drugs, which results in severe side effects to normal tissues and low efficacy against multidrug resistant cancer cells.2 To reduce or minimize these side effects, tremendous effort has been centered on the development of various nanocarriers which can offer an opportunity to alter the pharmacokinetic profile of drugs, reduce off-target toxicity, and improve the therapeutic index.3 Among them, polymeric micelles prepared from self-assembly of amphiphilic copolymers have drawn a great deal of attention because of their many desirable features for targeted drug delivery. Micelles can not only accommodate a wide variety of compounds with a high drug loading capacity but also have the potential to accumulate preferentially at the target site because of the enhanced permeation and retention effect (EPR) and to release the payload in response to biological stimuli.4−8 However, for in vivo applications, the stability of polymeric micelles resulting from the large dilution volume and/or interactions with cells and biomolecules presented in the blood still remains a major concern, which often leads to premature © 2013 American Chemical Society

drug release and drug loss during storage because of demicellization. Therefore, the interest is high in developing cross-linked micelles for improved stability and circulation time.9 Usually, chemical cross-linking of either core or shell segments is the common strategy to retain the integrity of polymer micelles. However, the cross-linking may unfavorably affect the bioactivity of the encapsulated cargo and the biodegradability of the delivery system.10 Unlike chemical cross-linking that may decrease the structural biodegradability or affect the encapsulated molecules in some cases, introducing noncovalent interactions such as multiple hydrogen bonding,11 host−guest recognition,12 π−π stacking,13 metal−ligand coordination,14,15 and stereocomplexes interaction16 into polymeric assemblies could be a promising approach to improve the micelle stability. The most commonly studied polymers for stereocomplexation are PLLA and PDLA. When PLLA and PDLA are mixed at the ratio of 1:1, a unique composite is formed. Such mixtures display a melting point 50 °C higher than that of single PLLA Received: July 28, 2013 Revised: August 29, 2013 Published: September 26, 2013 13072

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or PDLA, and it shows absolutely different X-ray diffraction peaks due to the blend crystallization in a triclinic unit cell with distinctive 3/1 chain helical conformation. Moreover, this stereocomplex interaction can also occur in solution.17 Thus, the PLLA/PDLA segment contained with various architectures to form stereocomplex self-assemblies were extensively reported. For instance, enantiomeric mixtures of PLA-blockP(MA-POSS),18 polyhedral oligomeric silsesquioxane (POSS)star-PLA,19 PLA-g-P(NIPAM-co-MAA),20 and PEG-b-PLA block copolymers having various conformations, such as star (8-arms),21 H-shaped,22 and comb-shaped,23 have been reported to yield nanoparticles in aqueous solution via stereocomplexation. Taking advantage of the stereocomplexation in PLA, many researchers have designed stable polymeric micelles in aqueous solution which have potential for the transportation and delivery of biologically active agents.20,23−25 Because of the stereocomplexation, stereocomplex selfassemblies exhibit strong thermodynamic stability as well as kinetic stability, and stereocomplex micelles are less prone to aggregation during the lyophilization process. In addition, the drug release is slower from stereocomplex micelles than it is from corresponding conventional micelles without stereocomplexation.26 For any drug delivery to be useful practically, the nevertheless fundamental consideration is to tailor a safe biocompatible and biodegradable material. Dextran, a polysaccharide consisting of 1,6- and 1,3-glucosidic linkages, is a natural analogue to PEG and appears to be well promised for use as a polymeric carrier due to its biodegradability, wide availability, and nonfouling property.27−29 In contrast to PEG, dextran contains about 5% branching structure and abundant functional hydroxyl groups along the chain; it is convenient for chemical modification to endow materils with various desired functions.30 Some research groups have reported the synthesis of PLA grafted dextran copolymers and evaluation of their possibility as biomedical materials.31,32 Enantiomeric Dex-gPLLA and Dex-g-PDLA copolymers with well-defined compostion were obtained through the coupling reaction between dextran and PLA.33 Monodisperse stereocomplex nanogels were then obtained by the self-assembly of an equimolar mixture of Dex-g-PLA in a dilute aqueous solution. The stereocomplex nanogel can be expected to show an advantage requirement such as controlled drug release. In this article, we had exploited new biodegradable stereocomplex micelles (SCMs) based on amphiphilic dextran-block-poly(L-lactide) (Dex-b-PLLA) and dextran-blcokpoly(D-lactide) (Dex-b-PDLA) via stereocomplexation of enantiomeric poly(lactide) polymers by introducing dextran. DOX, as a model anticancer drug, was loaded into the SCMs by a simple dialysis technique (as shown in Scheme 1). Compared to the micelles of Dex-b-PLA, the SCMs showed lower CMC values and DOX-loaded SCMs exhibited slower release rate. The biocompatibilities of micelles and cellular proliferation inhibition of DOX-loaded micelles were also investigated with HepG2 cells.

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Scheme 1. Schematic Illustration of DOX Loading and Release from DOX-Loaded SCMs

(Sigma), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA, Sigma), and copper bromide (Sigma) were used as received. Toluene was dried by refluxing over Na metal under an argon atmosphere and distilled immediately before use. Dimethyl sulfoxide (DMSO) was dried over calcium hydride (CaH2) and purified by vacuum distillation with CaH2. Acetate buffer with a concentration of 0.2 mol L−1 was prepared by mixing acetic acid and sodium acetate at pH 5.0. Dry 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. Doxorubicin hydrochloride (DOX·HCl) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. Synthesis of Dex-b-PLLA and Dex-b-PDLA. Synthesis of Enantiomeric PLA (PLLA or PDLA). Enantiomeric PLA (PLLA or PDLA) was synthesized via the ring-opening polymerization of Llactide and D-lactide, respectively, with isopropyl alcohol as initiator and Sn(Oct)2 as catalyst, as shown in Scheme 2. Typically, lactide (5.0 g, 34.7 mmol) was dissolved in 30.0 mL of anhydrous toluene in a flame-dried flask at room temperature. And then, isopropyl alcohol (0.19 mL, 2.5 mmol) and Sn(Oct)2 (14.06 mg, 34.7 μmol 0.001 equiv) were added into the solution with stirring, and the ROP reaction was maintained at 85 °C for 4 days. After precipitating the solution into excessive diethyl ether, the crude product was further washed twice with diethyl ether and dried under vacuum at room temperature for 24 h to obtain resultant PLA (4.3 g; yield: 86%). The molecular weights of enanatiomeric PLAs from 1H NMR and GPC are listed in Table 1. Synthesies of α-Azido PLA (N 3 -PLLA or N 3 -PDLA). The introduction of azide group to PLA backbone was conducted similarly by two steps described as Scheme 2a. First, PLA (3.0 g, 1.5 mmol) was dissolved in methylene dichloride (30.0 mL) in a nitrogen atmosphere with vigorous stirring, followed by addition of triethylamine (3.14 mL, 1.5 mmol) at room temperature. After degassing, 1.78 mL of methanesulfonyl chloride was added dropwise, and the reaction was performed at room temperature for 24 h. The reaction mixture was washed three times by deionized water and then poured into 10-fold ethyl ether to yield the resultant methylsufonyl-PLA (1.86 g; yield: 62%). Second, sodium azide (0.488 g, 0.75 mmol) was added into a DMF (20.0 mL) solution of the obtained methylsufonyl-PLA (1.5 g, 0.75 mmol), and the reaction proceeded at 80 °C for 24 h. And then, the reaction mixture were cooled to room temperature and precipitated into excess deionized water. After drying under vacuum at 60 °C overnight, the resulting products N3-PLA were obtained with 0.8 g (yield: 53%). Synthesis of α-Alkyne Dextran (Alkyne-Dex). The modification of dextran was conducted as shown in Scheme 2b. Typically, dextran (2.0 g, 0.33 mmol) was dissolved in 2% (w/v) acetate buffer (pH 5.0) in a flask at 50 °C. Propargylamine (0.342 g, 4.95 mmol, 15 equiv) and sodium cyanoborohydride (0.314 g, 4.95 mmol, 15 equiv) were added under stirring. The mixture was allowed to stir at 50 °C for 96 h. The solution was then concentrated by a rotavapor and then dialyzed

EXPERIMENTAL SECTION

Materials. L-Lactide (L-LA, Purac) and D-lactide (D-LA, Purac) were recrystallized three times from ethyl acetate under an argon atmosphere. Dextran (Dex, Mn = 6 kDa, Sigma), isopropyl alcohol (Sigma), methanesulfonyl chloride (Sigma), sodium azide (Sigma), stannous octoate (tin(II) bis(2-ethylhexanoate) (Sn(Oct)2, 95%, Sigma), propargylamine (98%, Sigma), sodium cyanoborohydride 13073

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Scheme 2. Synthetic Routes of Dex-b-PLLA and Dex-b-PDLA Copolymers

freeze-dried stereocomplex micelles (SCMs of Dex-b-PLA) were obtained by lyophilization. Characterizations. 1H NMR spectra were recorded on a Bruker AV 400 NMR spectrometer in dimethyl-d6 sulfoxide (DMSO-d6) or chloroform-d1 (CDCl3-d1). FT-IR spectra were recorded on a Bio-Rad Win-IR instrument using the potassium bromide (KBr) method. Dynamic light scattering (DLS) measurements were performed on a WyattQELS instrument with a vertically polarized He−Ne laser (DAWN EOS, Wyatt Technology) and 90° collecting optics. All the samples were prepared in aqueous solution at a concentration of 0.25 mg mL−1. Transmission electron microscopy (TEM) measurement was performed on JEOL JEM-1011 transmission electron microscope with an accelerating voltage of 100 kV. A drop of the sample solution (0.25 mg mL−1) was deposited onto a 230 mesh copper grid coated with carbon and allowed to dry in air at 25 °C before measurement. Wide-angle X-ray diffraction (WAXRD) was carried out by a Rigaku X-ray diffractometer with a Ni-filtered Cu Kα radiation (λ = 0.154 nm) at room temperature. The scan rate was 5°/min from 5° to 40°. The selected voltage and current were 40 kV and 200 mA, respectively. Differential scanning calorimetry (DSC) was conducted under nitrogen using a heat/cool/heat cycle at a heating rate of 5 °C/min on a TA Instruments DSC Q100 with aluminum pan. Gel permeation chromatography (GPC) analyses were performed on a Polymer Laboratories PL-GPC 50 Plus integrated GPC system with two Jordi DVB mixed bed columns (300 × 7.5 mm), and absolute molecular weights were obtained using a refractive index detector coupled to a multiangle laser light scattering detector, the MiniDAWN Treos (Wyatt Technology). GPC analyses were carried out in HPLC-grade THF (flow rate: 1 mL/min) at 50 °C on samples of 1 mg/mL concentration. Critical Micelle Concentration (CMC) Measurements. The critical micelle concentration (CMC) of Dex-b-PLLA, Dex-b-PDLA, and an equimolar mixture of the two in aqueous media were confirmed by a fluorescence technique using pyrene as a probe. Steady-state fluorescence spectra were obtained by a Spex FluoroMax-2 fluorescence spectrophotometer at room temperature. The excitation spectra of pyrene were obtained at a fixed excitation wavelength of 390 nm. The spectra were accumulated with an integration of 1 s/nm. The concentration of polymer micelle solutions containing 6.0 × 10−7 M of

Table 1. Molecular Weights and Distributions of Enantiomeric PLA samples

monomer/initiator

Mn,tha

Mn,NMRb

Mn,GPCc

PDId

1-PLLA2000 2-PDLA2000 3-PLLA4000 4-PDLA4000

13.88 13.88 34.72 34.72

2000 2000 4000 4000

2200 2100 3900 3900

2500 2500 4100 4200

1.19 1.13 1.22 1.17

a

Theoretical molecular weight. bResultant molecular weights of PLLA and PDLA were calculated by 1H NMR. cFrom GPC using THF as the eluent. dPolydispersity index, Mw/Mn, from GPC.

against deionized water (MWCO 3.5 kDa) for 2 days, and the product was collected by lyophilization (1.4 g; yield: 70%).34 Synthesis of Dextran-block-polylactide (Dex-b-PLLA or Dex-bPDLA). Dextran-block-polylactide was synthesized by Huisgen’s 1,3dipolar cycloaddition (“click chemistry”) shown in Scheme 2c. A typical procedure for the preparation is briefly described as follows: N3-PLA (0.13 g, 0.06 mmol), α-alkyne-Dex (0.6 g, 0.1 mmol), and PMDETA (21 μL, 0.1 mmol) were dissolved in 30.0 mL of dried DMSO with stirring for 30 min. After being degassed by three freeze− thaw cycles, the mixture was transferred into another Schlenk flask containing CuBr (14.4 mg, 0.1 mmol) via N2-purged syringe in an oil bath at 60 °C for 72 h. After the reaction finished, the reaction medium was dialyzed against deionized water (MWCO 7 kDa) for 4 days, and the product was obtained by lyophilization (0.56 g; yield: 77%). Preparation of Polymer Micelles (PMs) and Stereocomplex Micelles (SCMs). Polymer micelles and stereocomplex micelles were prepared by a dialysis method.35 First, a copolymer (100 mg of Dex-bPLLA or Dex-b-PDLA) or an equimolar mixture of Dex-b-PLA (50 mg of Dex-b-PLLA and 50 mg of Dex-b-PDLA) was dissolved in 20 mL of DMSO. And then, 20 mL of deionized water was added dropwise into the copolymer solution of DMSO with vigorous stirring. After that, the mixed solutions were transferred into a dialysis bag (MWCO 7 kDa) against deionized water for 3 days to prepare polymer micelles aqueous solution by removing DMSO. For the thermal analysis the 13074

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pyrene was varied from 1.52 × 10−5 to 0.25 mg/mL. The samples were degassed by gentle bubbling of nitrogen for 30 min before measurements. The CMC value was obtained from the intersection of the tangent to the horizontal line of I337/I335 with relative constant value and the diagonal line with rapidly increased I337/I335 ratio.36 In Vitro Drug Loading and Release. Doxorubicin (DOX) was used as a model drug for in vitro drug loading and release. DOX-loaded micelles were prepared by a simple dialysis technique. Typically, SCMs (15 mg), DOX·HCl (3.0 mg), and triethylamine (0.9 mg) were mixed in 2.0 mL of DMSO. The mixture was stirred at room temperature for 24 h and then added dropwise into 20.0 mL of PBS at pH 7.4. The DMSO was removed by dialysis against deionized water for 24 h. The dialysis medium was refreshed five times, and the whole procedure was performed in the dark. Then, the solution was filtered and lyophilized. To determine the drug loading content (DLC) and drug loading efficiency (DLE), the drug-loaded micelles were dissolved in DMSO and analyzed by fluorescence measurement (PerkinElmer LS50B luminescence spectrometer) using a standard curve method (λex = 480 nm). The DLC and DLE of drug-loaded micelles were calculated according to eqs 1 and 2, respectively:

DLC (wt %) =

amount of drug in micelles × 100 amount of drug loaded micelles

DLC (wt %) =

amount of drug in micelles × 100 total amount of feeding drug

for 24 h. The culture medium was then removed, and micelle solutions in complete DMEM at different concentrations (0−10 g L−1) were added. The cells were subjected to MTT assay after being incubated for an additional 72 h. The absorbance of the solution was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was calculated based on eq 3:

cell viability (%) =

A sample Acontrol

× 100

(3)

where Asample and Acontrol represent the absorbances of the sample and control wells, respectively. The cytotoxicities of DOX-loaded micelles against HepG2 cells were also evaluated in vitro by a MTT assay. Similarly, cells were seeded into 96-well plates at 1 × 104 cells per well in 200.0 μL of complete DMEM and incubated for 24 h. After washing cells with PBS, 180.0 μL of complete DMEM and 20.0 μL of DOX-loaded micelle solutions in PBS were added to form culture media with different DOX concentrations (0−3.0 mg L−1 DOX). The cells were subjected to MTT assay after being incubated for 24, 48, and 72 h. The absorbance of the solution was measured on a Bio-Rad 680 microplate reader at 490 nm. Cell viability (%) was also calculated based on eq 3.

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(1)

RESULTS AND DISCUSSION Synthesis of Dex-b-PLA (Dex-b-PLLA or Dex-b-PDLA). The amphiphilic enantiomeric copolymers of Dex-b-PLA were synthesized by combining the clickable alkyne-Dex with azidoPLA precursor via click chemistry as depicted in Scheme 2. First, pairs of enantiomeric homopolymers of PLA listed in Table 1 were synthesized via the ring-opening polymerization of lactide by varying the ratio of lactide to isopropyl alcohol. 1H NMR spectra of PLLA and PDLA in CDCl3 were showed in Figure S1. Then, the end hydroxyl groups of PLA were further modified to azide group as shown in Scheme 2a. In the FT-IR spectrum of N3-PLA (in Figure 1) the new absorption peaks appearing at about 2100 cm−1 belonging to the azide groups strongly confirmed the successful modification.

(2)

In vitro drug release profiles of drug-loaded micelles were investigated in PBS at pH 7.4. The weighed freeze-dried DOX-loaded micelles were suspended in 10.0 mL of release medium and transferred into a dialysis bag (MWCO 3.5 kDa). The release experiment was initiated by placing the end-sealed dialysis bag into 50 mL of PBS at 37 °C with continuous shaking at 100 rpm. At predetermined time intervals, 2.0 mL of dialysate was taken out and an equal volume of fresh PBS was replenished. The amount of released DOX was determined by fluorescence measurement (λex = 480 nm). The release experiments were conducted in triplicate. Intracellular Drug Release. The cellular uptake and intracellular release behaviors of DOX-loaded micelles were assessed by confocal laser scanning microscopy (CLSM) and flow cytometry on HepG2 cells. Confocal Laser Scanning Microscopy (CLSM). For CLSM study, HepG2 cells were seeded in 6-well plates at a density of 2 × 105 cells per well in 2.0 mL of complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, supplemented with 50 IU mL−1 penicillin and 50 IU mL−1 streptomycin and cultured for 24 h. After the culture media were removed, the cells were incubated at 37 °C for an additional 3 h with DOX-loaded micelles at a final DOX concentration of 5.0 mg L−1 in complete DMEM. Then, the culture medium was removed, and cells were washed with PBS thrice. Thereafter, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and the cell nuclei were stained with 4′,6diamidino-2-phenylindole (DAPI, blue) for 20 min. CLSM images of cells were obtained through confocal microscope (Olympus FluoView 1000). Flow Cytometric Analyses. HepG2 cells were seeded in 6-well plates at 2 × 105 cells per well in 2.0 mL of complete DMEM and cultured for 24 h. The cells were then washed by PBS and incubated at 37 °C for an additional 3 h with DOX-loaded micelles at a final DOX concentration of 5.0 mg L−1 in complete DMEM. Thereafter, the culture medium was removed, and the cells were washed with PBS thrice and treated with trypsin. Then, 1.0 mL of PBS was added to each culture well, and the solutions were centrifuged for 4 min at 3000 rpm. After the removal of supernatants, the cells were resuspended in 0.3 mL of PBS. Data for 1 × 104 gated events were collected, and analysis was performed by flow cytometer (Beckman, Brea, CA). Cell Viability Assays. The relative cytotoxicities of micelles against HepG2 cells were evaluated in vitro by a standard MTT assay. The cells were seeded in 96-well plates at 1 × 104 cells per well in 200.0 μL of complete DMEM and incubated at 37 °C in 5% CO2 atmosphere

Figure 1. FTIR spectra of α-alkyne dextran, PLLA2000, CH3SO2OPLLA2000, N3-PLLA2000, and Dex-b-PLLA2000.

Second, in order to conduct the click reaction, an alkyne group was introduced to dextran by reductive amination with propargylamine in acetate buffer solution (pH 5.0). The chemical structure of alkyne-Dex was confirmed by FT-IR and 1 H NMR. As shown in Figure 1, the appearance of the α-alkyne peak at 2100 cm−1 suggested the successful synthesis of αalkyne dextran. Furthermore, the complete disappearance of the anomeric proton peaks of the reducing end group at 6.7 and 6.3 13075

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ppm was also a strong indication of the successful synthesis of the desired α-alkyne dextran in Figure S2. Lastly, the amphiphilic Dex-b-PLA was synthesized by click reaction. As shown in Figures 1 and 2, the disappearance of the

Figure 3. Intensity ratios of I337/I335 from pyrene excitation spectra as a function of concentration of Dex-b-PLLA2000 (PM-1), Dex-bPDLA2000 (PM-2), and SCM-1 + 2 in PBS at pH 7.4.

Figure 2. 1H NMR spectra of Dex-b-PLLA2000 (a) and Dex-bPDLA2000 (b) in DMSO-d6.

azide peak at 2100 cm−1 demonstrated that N3-PLA had been completely consumed during the reaction with alkyneterminated dextran, suggesting the successful synthesis of Dex-b-PLA. The appearance of the methine protons peaks at 5.15−5.25 ppm attributed to the characteristic signals in PLA units, and the peaks at 3−4 ppm assigned to the protons of the dextran further indicated the successful synthesis of amphiphilic Dex-b-PLA copolymers. Characterization of Polymer Micelles and Stereocomplex Micelles. It is well-known that the amphiphilic block copolymers can self-assemble into aggregates including micelles and vesicles in a selective solvent. In this study, the polymer micelles (PMs) and the stereocomplex micelles (SCMs) were prepared by a dialysis method. To demonstrate the formation of micelles, the critical micelle concentrations (CMCs) were investigated by a widely reported pyrene-probe-based fluorescence technique. Pyrene has been employed as an effective fluorescent probe for the micellization systems due to its photophysical sensitivity to the change of environmental polarity. The excitation spectra of pyrene with increased concentration of block copolymers were measured to demonstrate the self-assembly of block copolymers. Typically, a red-shift of absorption band was observed when the concentration of the copolymer increased as shown in Figure S3. This red-shift resulted from the transfer of pyrene molecules from a water environment to the hydrophobic micellar core, indicating the formation of micelles. In addition, from the plot of fluorescence intensity ratio of I337/I335 versus log C of the copolymers shown in Figures 3 and 4, the CMCs can be calculated, taken as the intersection of the tangents to the horizontal line of intensity ratio with relatively constant values and the diagonal line with rapidly increased intensity ratio. The CMCs (listed in Table 2) were affected by not only the length of PLA in the block copolymers but also the stereocomplex interaction. Compared with the corresponding single polymer micelles, SCMs showed lower CMCs due to their tightly packed isotactic hydrophobic core. The affection of stereo reaction on the micellization of this class of amphiphilic

Figure 4. Intensity ratios of I337/I335 from pyrene excitation spectra as a function of concentration of SCM-1 + 2 and SCM-3 + 4 in PBS at pH 7.4.

copolymers may be linked to the semicrystalline nature of isotactic PLA blocks. For SCMs, with the length of PLA chain increasing, the CMCs value became lower because the crystalline of PLA and the stereocomplex interaction increased. Usually, the self-assembly behavior of the amphiphilic polymer was driven by hydrophobic interactions between the PLA blocks and hydrophilic interactions between the dextran block and water. The hydrodynamic radii (Rh) of these amphiphilic aggregates were measured by DLS listed in Table 2 and Figure 5D. Similarly, SCMs exhibited a smaller size due to the formation of stereocoplex by mixing an equimolar Dex-bPLA. At the same time, with the molecular weight of PLA chains in the block copolymers becoming larger, the size of the micelles became larger which resulted in a bigger Rh value. The successful formations of SCMs were also confirmed by transmission electron microscopy (TEM) as shown in Figure 5A−C. The PMs and SCMs all showed spherical morphologies with a respective average diameters around 90, 90, and 70 nm, which were close to the results of DLS. Thermal and Crystal Properties of SCMs. To further confirm the successful stereocomplexation by mixing equimolar enantiomeric copolymers of Dex-b-PLA, the SCMs aqueous solution were lyophilized, and the thermal and crystal properties of freeze-dried SCMs were studied by DSC and XRD. The freeze-dried SCMs containing equimolar amounts of Dex-b-PLLA and Dex-b-PDLA displayed a higher melting points (listed in Table 2), 70 °C above that of Dex-b-PLLA or Dex-b-PDLA. This higher crystalline melting temperature 13076

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Table 2. DSC, DLS, CMC, DLC, and DLE Analyses of the Synthesized PMs and SCMs samples

entry

DSC (°C)

Dex-b-PLLA2000 Dex-b-PDLA2000 PM-1 + PM-2 Dex-b-PLLA4000 Dex-b-PDLA4000 PM-3 + PM-4

PM-1 PM-2 SCM-1 + 2 PM-3 PM-4 SCM-3 + 4

115.90 113.83 184.84 143.65 146.42 210.34

DLS (nm)

CMC (μg mL−1)

DLC (wt %)

DLE (wt %)

± ± ± ± ± ±

6.63 6.87 1.53 3.87 3.99 0.63

9.57 9.34 5.70 12.43 12.60 10.54

57.46 56.04 34.20 74.58 75.60 63.24

158 164 103 182 179 121

8.7 9.6 6.7 8.3 7.9 7.8

homo-PLAs, the block copolymer Dex-b-PLAs showed a weakened crystalline peak due to the introduction of dextran. In Vitro DOX Loading and Triggered Release. Doxorubicin is a widely used antineoplastic agent in the treatment of several adult and pediatric cancers, which interacts with DNA through the insertion and then inhibits the biosynthesis of bioactive macromolecules. In this current study, DOX was used as a model drug and loaded into the micelles. As shown in Table 2, the DLC of PMs and SCMs were in the range 5.7−12.6%, and the DLE were 34.2−75.6%. These dates indicated that stronger stereocomplex interaction of the core decreased the drug loading capacity DLC and DLE. The in vitro release behaviors were investigated at pH 7.4. The cumulative release percentages of DOX-loaded PMs and SCMs versus time are plotted in Figure 7; DOX-loaded SCMs

Figure 5. TEM micrographs of PM-1 (A), PM-2 (B), and SCM-1 + 2 (C) and hydrodynamic radii (Rh) of the PM-1, PM-2, and SCM-1 + 2 (D).

confirmed the presence of the stereocomplex crystallization between the two enantiomeric Dex-b-PLAs.37 The typical XRD patterns of PLAs, Dex-b-PLAs, and SCMs are illustrated in Figure 6. It is well-known that the diffraction peaks of homo-PLLA and homo-PDLA crystallites appear at 2θ =16.8°, 19.1°, and 22.5°.37 The equimolar mixtures of PLLA and PDLA yielded three peaks at 12.5°, 21.0°, and 24.0°, accompanied by the disappearing of the significant peak at 16.8°, which were characteristic crystalline structure of the enantiomeric stereocomplex PLA blocks. Compared to the

Figure 7. In vitro DOX release profiles for DOX-loaded PM-1, PM-2, SCM-1 + 2, PM-3, PM-4, and SCM-3 + 4 in PBS at 37 °C and pH 7.4.

exhibited a slower release rate compared to DOX-loaded PMs because of the physical cross-linking via stereocomplex interaction, which was the indication of the enhanced stability of the SCMs. Cell Viability Assays and Intracellular DOX Release. It is necessary to evaluate the potential toxicity of polymeric materials for drug delivery applications. The in vitro cytotoxicity of the SCMs to HepG2 cells was evaluated by a MTT assay. As shown in Figure 8A, the viabilities of HepG2 cells treated with SCMs for 72 h were over 85% at all test concentrations up to 3.0 mg L−1. The results suggested that PMs and SCMs all had low cytotoxicity and could be safely used as biocompatible carriers for drug delivery. The in vitro cellular proliferation inhibitions of DOX-loaded PMs and SCMs against HepG2 cells were also estimated by a MTT assay. As shown in Figure 8B as well as Figures S4 and S5, in contrast to DOX-loaded PMs, DOX-loaded SCMs exhibited significantly lower growth inhibition efficiency to HepG2 cells. The results revealed that the slower DOX release from DOX-loaded SCMs was attributed to the enhanced stability.

Figure 6. Wide-angle X-ray diffraction patterns of PLLA2000, PDLA2000, scPLA2000, Dex-b-PLLA2000, Dex-b-PDLA2000, and SC2000. 13077

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shown in Figure 10, the flow cytometric histogram for the cells incubated with DOX-loaded SCMs shifted clearly to the

Figure 10. Flow cytometric profiles of HepG2 cells incubated with DOX-loaded PM-1 (green), PM-2 (blue), and SCM-1 + 2 (red) micelles for 3 h.

direction of low fluorescence intensity as compared with that incubated with DOX-loaded PMs. Thus, the weaker fluorescence intensity in the HepG2 cells incubated with DOX-loaded SCMs should be attributed to the slower intracellular DOX release induced by stereocomplexes interaction.

Figure 8. Cytotoxicity of PM-1, PM-2, and SCM-1 + 2 toward HepG2 cells after incubation for 72 h (A). Cytotoxicities of DOX-loaded PM1, PM-2, SCM-1 + 2, and free DOX toward HepG2 cells after incubation for 72 h (B).

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CONCLUSION In summary, amphiphilic block copolymer based on dextran and enantiomeric PLA were successfully prepared by click reaction. The stereocomplexation of the equimolar mixture of Dex-b-PLA was evaluated by DSC and XRD analyses. DLS and TEM techniques verified that the equimolar mixture of copolymers could self-assemble into stable micelles by the

The cellular uptake and intracellular release behaviors of DOX-loaded PMs and SCMs were followed with CLSM and flow cytometry toward HepG2 cells. As expected, weaker intracellular DOX fluorescence was observed in the cells after incubation with DOX-loaded SCMs for 3 h in contrast to those incubated with DOX-loaded PMs (Figure 9A−C). A similar tendency was further confirmed by flow cytometric analyses. As

Figure 9. Representative CLSM images of HepG2 cells incubated with DOX-loaded PM-1 (A), DOX-loaded PM-2 (B), and DOX-loaded SCM-1 + 2 (C) for 3 h. For each panel, the images from left to right show a differential interference contrast (DIC) image, cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red), and overlays of the three images. 13078

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stereocomplexation. The CMCs were affected by not only the length of PLA in the block copolymers but also the stereocomplex interaction. The most significant aspect was that the SCMs showed lower CMC values due to the tightly packed structure of their isotactic hydrophobic core. DOXloaded SCMs exhibited a slower release rate compared to DOX-loaded PMs. Moreover, weaker intracellular proliferation inhibition efficacy and lower fluorescence intensity were also achieved. These results, therefore, demonstrated that the stereocomplex micelles based on block copolymers of Dex-bPLA provide favorable platforms to construct stable and excellent drug delivery systems for cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

Additional characterization data including 1H NMR spectra, fluorescence plots for the copolymers, and cytotoxicity of DOX-loaded PM-1, PM-2, SCM-1 + 2, and free DOX toward HepG2 cells after incubation for 24 and 48 h. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel +86 431 85099667; Fax +86 431 85099668; e-mail [email protected] (C.H.), [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (Projects 50903012, 51273037, 51003103, and 21174142), Jilin Science and Technology Bureau (International Cooperation Project 20120729, 20130206074GX), and Jilin Human Resources and Social Security Bureau (201125020).

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