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DOI: 10.1039/C6RA00699J (Paper) RSC Adv., 2016, 6, 20761-20771

Stable stereocomplex micelles from Y-shaped amphiphilic copolymers MPEG–(scPLA)2: preparation and characteristics†

Weiwei Zhang, Delong Zhang, Xiaoshan Fan, Guangyue Bai*, Yuming guo and Zhiguo Hu*
Henan Engineering Laboratory of Chemical Pharmaceuticals & Biomedical Materials, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, P. R. China. E-mail: zghu@htu.cn; baiguangyue@htu.cn; Tel: +86-373-3326335 Tel: +86-373-3328622

Received 9th January 2016 , Accepted 12th February 2016

First published on 15th February 2016

Abstract

Four new Y-shaped miktoarm amphiphilic copolymers were synthesized by ring-opening polymerization (ROP) and click chemistry. The structure of these copolymers was determined by nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). The stereocomplexes were prepared by an evaporation method and confirmed by FT-IR, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Further the aggregation behaviors of these synthesized polymers and their stereocomplexes were studied by fluorescence spectroscopy (PL), transmission electron microscopy (TEM) and light scattering (LS). Their critical micelle concentrations (CMC) obtained by PL were 0.005 mg mL−1 for MPEG1.9k–(scPLA4.5k)2 and 0.039 mg mL−1 for MPEG5k–(scPLA4.5k)2. The aggregation morphologies of homochiral copolymers were worm-like aggregates, while for the stereocomplexes, the spherical micelles were visually observed. The biocompatibility of these copolymers and their stereocomplexes was evaluated with relatively lower cytotoxicity. Finally, the release of doxorubicin (DOX) encapsulated into the micelles in buffer at pH 5.4 was faster than that at pH 7.4. This study demonstrates that the DOX-loaded stereocomplex micelles could be a potential carrier for cancer treatments.

Introduction

Amphiphilic block copolymers have received much attention due to their spontaneous self-assembly in an aqueous environment1–3 into different morphologies like vesicles or micelles, which could be used as drug delivery vehicles.4–8 Such polymeric aggregations are fairly stable compared with low molecular weight ones, including greater thermodynamic and kinetic stabilities.1 However, it is still an equilibrium system between the polymeric aggregations and the corresponding polymers. Once being introduced into the bloodstream, polymeric aggregations might disassemble into free polymeric chains as being subjected to high dilution and other factors such as temperature, pH, ionic strength.9,10 For improving the stability of such aggregations, chemical cross-linking methods through either core11–14 or shell15–18 segments were usually used. However, the encapsulation, release or biodegradable properties of such aggregations might be altered by such chemical cross-linking procedures.

It is well known that molecular chirality is an important natural property that affects physiochemical properties and biological activities.19,20 Since the polymeric stereocomplex formed between the equimolar PLLA and PDLA has been firstly reported by Ikada et al. in 1987,21 the stereocomplex interaction as an important physical cross-linking method was used to stabilize the micelles.22 Such kind of stereocomplex interaction-assisted polymeric micelles have attracted increasing attention in recent years.23–28 The previous studies have mainly focused on linear PEG–PLA diblock polymers. In 2009, Y-shaped miktoarm copolymers including PEG5k–PLLA2k–PDLA2k, PEG5k–(PLLA2k)2 and PEG5k–(PDLA2k)2 were synthesized by J. L. Hedrick and co-workers.25 In aqueous solution, the reported stereocomplex mixture of PEG5k–(PLLA2k)2 and PEG5k–(PDLA2k)2, or the stereoblock PEG5k–PLLA2k–PDLA2k could form stabilized micelles with a significantly lower critical micelle concentration (CMC) than those derived from conventional stereo regular linear or Y-shaped amphiphiles. Among them, the lowest CMC (10 mg L−1) was found in the stereocomplex of PEG5k–(PLLA2k)2 mixed with PEG5k–(PDLA2k)2. The micelles derived from the stereocomplex could provide high capacity for loading of the drug and possess narrow size distribution as well as unique structure, which leads to sustained and near zero-ordered release of drug without significantly initial burst. These excellent properties were attributed to the special Y-shaped miktoarm structure and the stereocomplex interaction between PLLA and PDLA parts of the amphiphilic copolymer.

The self-assembly behaviors and morphologies depend on the structure and hydrophilic/lipophilic ratio of the amphiphilic copolymers. In the previous paper, the studied Y-shaped miktoarm copolymers PEG5k–(PLLA2k)2 and PEG5k–(PDLA2k)2 have a relatively high hydrophilic/lipophilic ratio.25 In this work, in order to deeply understand the dependence of the stereocomplex interaction and self-assembly behaviors on the hydrophilic/lipophilic ratio, four Y-shaped miktoarm amphiphilic copolymers having a relatively low hydrophilic/lipophilic ratio, MPEG1.9k–(PLLA4.5k)2, MPEG1.9k–(PDLA4.5k)2, MPEG5k–(PLLA4.5k)2 and MPEG5k–(PDLA4.5k)2, were designed.

Since the concept of the click chemistry was introduced in 2001 by Sharpless and coworkers,29 click chemistry was proven to be a powerful tool for the preparation of block copolymers7,30–35 due to its high selectivity and almost complete conversion obtained under mild reaction conditions.36,37 To the best of our knowledge, there are no reports on the synthesis of Y-shaped miktoarm copolymers based on PEG and PLA via click chemistry and until now the self-assembly of the stereocomplex mixture of Y-shaped miktoarm amphiphilic copolymers has been rarely studied.25 The aim of this work is to explore in detail the synthesis (Scheme 1) and the process of self-assembly of the stereocomplex mixture of Y-shaped miktoarm copolymers in aqueous solution, drug release profile and further to evaluate their biocompatibility.


image file: c6ra00699j-s1.tif
Scheme 1 Synthesis of Y-shaped miktoarm amphiphilic copolymers. 2a: alkyne–(PLLA4.5k)2, 2b: alkyne–(PDLA4.5k)2, 3a: MPEG1.9k–(PLLA4.5k)2, 3b: MPEG1.9k–(PDLA4.5k)2, 3c: MPEG5k–(PLLA4.5k)2 and 3d: MPEG5k–(PDLA4.5k)2.

Experimental section

Materials

Tetrahydrofuran (THF) was dried by reflux over sodium with benzophenone as an indicator under nitrogen. Copper(I) bromide, 3-bromo-1-propyne and N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) were purchased from Aladdin Chemical Co., Ltd. L- and D-lactides were purchased from Changchun SinoBiomaterials Co., Ltd. Poly(ethylene glycol) methyl ether (MPEG, Mn 1900 and 5000) and tin octoate (Sn(Oct)2) were purchased from Sigma-Aldrich. N,N-Dimethylformamide (DMF) was distilled under the reduced pressure from calcium hydride.

Measurement

The 1H NMR spectra were recorded on a Bruker-400 NMR instrument in CDCl3 solvents. FT-IR spectra were conducted on a FTS NEXUS FT-IR spectrometer using the KBr pellet method. Molecular weights and molecular weight distribution of copolymers were estimated by a gel permeation chromatography (GPC) system using a Waters 1515 apparatus equipped with three styragel columns (Styragel HR3, HR4, and HR5) and a Waters 2414 differential refractometer, and here DMF was used as eluent at a flow rate of 1.0 mL min−1 at 40 °C. Polystyrene standards with narrow molecular weight distribution were used for GPC calibration.

Dynamic light scattering (DLS) and static light scattering (SLS) were carried out on ALV/CGS-3 compact goniometer system with vertically polarized incident light of wavelength λ = 632.8 nm supplied by a HeNe laser operating at a maximum power of 22 MW. DLS measurements were made at 25.0 °C and an angle of 90°. All the solutions were filtered through 0.45 μm Millipore membrane filters. The autocorrelation functions from DLS were analyzed by using the photon cross-correlation spectroscopy (PCCS) method to obtain the diameter distributions. SLS was carried out with vertically polarized incident light of wavelength λ = 632.8 nm supplied by an a HeNe laser measurements were made at 13 different angles from 30° to 150° in angular step of 10°. The berry plot was used to obtain the Rg/Rh value. Toluene was used as a standard for SLS measurements. All of the solutions were filtered through 0.45 μm Millipore membrane water filters. Each experiment was repeated at least 3 times.

Transmission electron microscope (TEM) images were obtained using a JEM-2100 operating at an acceleration voltage of 200 kV. The concentration of dialysis solutions used for detection of TEM is 0.05 mg mL−1 (for MPEG1.9k–(PLLA4.5k)2, MPEG1.9k–(PDLA4.5k)2 and MPEG1.9k–(scPLA4.5k)2) and 0.20 mg mL−1 (for MPEG5k–(PLLA4.5k)2, MPEG5k–(PDLA4.5k)2 and MPEG5k–(scPLA4.5k)2). A drop of solution was placed onto TEM copper/carbon grid and the excess solution was blotted up using a strip of filter paper, then the sample was allowed to dry at room temperature before observation.

Fluorescence spectroscopy (PL) was used to evaluate the critical micelle concentration (CMC) of these stereocomplexes in aqueous solution. The fluorescence spectra were recorded by a JASCO FP-6500 fluorescence spectrophotometer using right angle geometry at 25 °C. Pyrene is excited with 336 nm light. The excitation and emission band passes are 2.5 nm. The polymer solutions were kept at room temperature for 24 h to reach solubility equilibrium of pyrene in the aqueous phase before measuring by fluorescence.

The thermal analyses for the synthesized Y-shaped miktoarm amphiphilic copolymers and their stereocomplexes were measured using a Netzsch 204F1 differential scanning calorimeter (DSC) equipped with an intercooler at a scan rate of 10 K min−1. The DSC was calibrated using the melting temperature and the enthalpy of indium. Approximately 5 mg of sample was placed in the aluminium pans for measurement under N2 atmosphere. The first DSC scan from 20 °C to 250 °C at speed of 10 °C min−1 to evaluate the crystallization and melting behavior of samples. Melting and crystallizing temperatures were determined from the maximum of the endothermic and the exothermic peaks, respectively.

The X-ray diffraction (XRD) morphological analyses were performed on a Bruker D8 Advance X-ray diffractometer, using Cu Kα radiation (λ = 0.154178 nm) at room temperature and the accelerating voltage was set at 40 kV with a 100 mA flux. The scattering angle ranged from 2θ = 5–35° at a speed of 3° min−1.

Synthesis

Compound initiator 1 (2-methyl-2-((prop-2-yn-1-yloxy) methyl)-propane-1,3-diol)38 and MPEG–N3 (ref. 30) were synthesized according to literature procedures.
Synthetic procedure for compound 1. Acetonide-protected bis-MPA (43.62 g, 0.27 mol) and NaH (48.84 g, 1.22 mol) were introduced into a round-bottom flask containing 500 mL THF under nitrogen atmosphere and stirred for 1 h at room temperature. Then 3-bromo-1-propyne (64.3 mL, 0.83 mol) was added dropwise to the solution, followed by stirring at room temperature until the reaction was complete. After neutralized by 1 M hydrochloric acid, the reaction mixture was filtered and evaporated under reduced pressure. The crude product obtained was dissolved in 500 mL methanol (containing 2.5 mL 1 M hydrochloric acid). After the reaction was complete, the mixture was neutralized with Na2CO3, filtered and evaporated under reduced pressure. Crude product was purified by column chromatography using SiO2 gel with hexane/ethyl acetate (5/1, v/v) as an eluent. Thus the obtained product, compound 1 has a status as yellow oil (yield = 36.63 g; 85%) (see ESI Fig. 1†).
General procedure for the synthesis of alkyne–(PLLA)2 (2a) and alkyne–(PDLA)2 (2b). L-Lactide (5.0117 g, 34.70 mmol) and initiator 1 (91.2 mg, 0.58 mmol) were introduced into a flame-dried Schlenk flask and dried under reduced pressure at 40 °C for 1 h. Then Sn(Oct)2 (15.0 mg, 0.04 mmol) were added to the flask and the whole mixture was heated up to 120 °C for 24 h with stirring under nitrogen atmosphere. The final alkyne–(PLLA)2 was dissolved in a small volume of dichloromethane and poured into an excess amount of diethyl ether for three times. The white precipitates were filtered, washed with diethyl ether, and dried in vacuum at 35 °C to obtain 2a, 91% yield.

The alkyne–(PDLA)2 (2b) was obtained in 93% yield according to the similar procedure to that of synthesizing alkyne–(PLLA)2.

General procedure for synthesis of MPEG1.9k–N3 and MPEG5k–N3. Both MPEG (Mn = 5000; 10.00 g, 2.0 mmol) and toluene-4-sulfonyl chloride (3.81 g, 20 mmol) were completely dissolved in CH2Cl2 (100 mL) under a N2 atmosphere. Triethylamine (2.78 mL, 20 mmol) was added dropwise to the above solution at ice-water bath, and then the resulting solution was stirred for 24 h at room temperature. The reaction solution was centrifuged and precipitated into 600 mL of diethyl ether, and then the powder was dried in vacuo at 35 °C to give the monotosylated MPEG (10.00 g, 97% yield). Thus, sodium azide (1.30 g, 20 mmol) was added to a solution of the obtained MPEG monotosylate (10.00 g, 2 mmol) in dry DMF (50 mL) under a N2 atmosphere, and the reaction mixture was stirred vigorously at room temperature for 24 h. The reaction mixture was filtered and the remaining DMF solvent was removed under reduced pressure, and then the product was dissolved in 400 mL of dichloromethane. The mixture was washed sequentially by NaCl (5 wt%) solution and distilled water, dried with anhydrous Na2SO4, and then precipitated in diethyl ether to yield 7.80 g of MPEG5k–N3 in 80% yield. MPEG1.9k–N3 was obtained as the same procedure as that of MPEG1.9k–N3 in 82% yield (see ESI Fig. 2 and 3†).
General procedure for synthesis of MPEG–(PLLA)2 or MPEG–(PDLA)2 (3a–3d). The synthesized compounds 2a (0.9145 g, 0.10 mmol), and MPEG1.9k–N3 (0.2738 g, 0.14 mmol) and PMDETA (0.1 mL) were dissolved in DMF (30 mL) under stirring at 35 °C. After flushing with N2 for 1 h, CuBr (0.0279 g, 0.20 mmol) was added and the resulting mixture was stirred at 45 °C for 24 h. When the reaction was complete, the solution was subjected to dialysis against distilled water using a dialysis membrane tube (3.5 kDa MWCO). After lyophilization, compound MPEG1.9k–(PLLA4.5k)2 (3a) was obtained as white powder in 95% yield.

Compounds 3b, 3c and 3d were all obtained in over 94% yield according to the similar procedure to that of synthesizing compound 3a.

For all the compounds, the corresponding data were summarized in Table 1.

Table 1 Characteristics of the synthesized polymers
Polymer Initiator 1 (g) Lactide (g) MPEG–N3 (g) Alkyne–(PLA)2 (g) CuBr (g) PMDETA (mL) Mn (NMR) Mn (GPC) Mw/Mn
Alkyne–(PLLA4.5k)2 (2a) 0.0912 5.0117 (L) — — — — 9372 8801 1.19
Alkyne–(PDLA4.5k)2 (2b) 0.0908 5.0315 (D) — — — — 9300 8799 1.20
MPEG1.9k–(PLLA4.5k)2 (3a) — — 0.2738 0.9145 0.0279 0.1 11[thin space (1/6-em)]343 10[thin space (1/6-em)]967 1.17
MPEG1.9k–(PDLA4.5k)2 (3b) — — 0.2894 0.9009 0.0301 0.1 11[thin space (1/6-em)]271 10[thin space (1/6-em)]958 1.19
MPEG5k–(PLLA4.5k)2 (3c) — — 0.6211 0.9164 0.0291 0.1 14[thin space (1/6-em)]445 14[thin space (1/6-em)]362 1.20
MPEG5k–(PDLA4.5k)2 (3d) — — 0.6176 0.8924 0.0286 0.1 14[thin space (1/6-em)]373 14[thin space (1/6-em)]235 1.21

Preparation of stereocomplex

The stereocomplex MPEG1.9k–(scPLA4.5k)2 was prepared from the pairs 3a/3b by evaporation from polymer solution in THF at room temperature. The polymer 3a (0.05 g) and 3b (0.05 g) were dissolved in THF (5 mL) respectively. The solutions were mixed at equimolar ratio. After evaporating the solvent, all precipitates were vacuum-dried to obtain the stereocomplexes.

The stereocomplex MPEG5k–(scPLA4.5k)2 was prepared as the same procedure of MPEG1.9k–(scPLA4.5k)2. And all the prepared stereocomplexes were used for DSC, FT-IR and XRD determination.

The blank micelle preparation

The dialysis method was employed to prepare the blank micelles of the synthesized Y-shaped miktoarm amphiphilic block copolymers 3a, 3b, 3c, 3d and their stereocomplexes derived from 3a/3b and 3c/3d.
General procedure for the micellar solution of 3a, 3b, 3c and 3d. Micellar solution for 3a or 3b: compound 3a or 3b (5.0 mg) was dissolved in 5 mL of DMF in a 100 mL round-bottom flask with a stirrer. With vigorously stirring, 25 mL of the twice-distilled water was added dropwise at room temperature. After stirring for 3 h, the solution was dialyzed against the twice-distilled water using a dialysis membrane tube (2.0 kDa MWCO) for 4 days to remove DMF. The polymer solution was transferred to a 100 mL volumetric flask. Then, the appropriate amount of twice-distilled water was added to obtain the micellar solution of 0.05 mg mL−1.

Micellar solution for 3c or 3d: the solution of 3c or 3d with 0.20 mg mL−1 was obtained according to the same procedure as that of compound 3a or 3b.

General procedure for the stereocomplex micellar solution of MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2. Two methods were used to prepare the stereocomplex micellar solution.
Method A. Micelles for stereocomplex MPEG1.9k–(scPLA4.5k)2 were obtained from 3a and 3b. Compounds 3a (2.5 mg) and 3b (2.5 mg) were dissolved in 5 mL of DMF in a 100 mL round-bottom flask with a stirrer for 2 h. With vigorously stirring, 25 mL of the twice-distilled water was added dropwise at room temperature. After stirring for 3 h, the solution was dialyzed against the twice-distilled water using a dialysis membrane tube (2.0 kDa MWCO) for 4 days to remove DMF. The polymer solution was transferred to a 100 mL volumetric flask. Then, the appropriate amount of twice-distilled water was added to obtain the micellar solution of 0.05 mg mL−1.

Micelles for stereocomplex MPEG5k–(scPLA4.5k)2 were obtained from 3c and 3d. The copolymer 3c (10.0 mg) and 3d (10.0 mg) were used to obtain the stereocomplex micelles (0.20 mg mL−1) according to the same procedure as that of micelles for stereocomplex MPEG1.9k–(scPLA4.5k)2.


Method B. Micelles for stereocomplex MPEG1.9k–(scPLA4.5k)2 were obtained from mixing micellar solution 3a and micellar solution 3b. Micellar solution 3a (50.0 mL, 0.05 mg mL−1) and micellar solution 3b (50.0 mL, 0.05 mg mL−1) were introduced in a 100 mL volumetric flask, shaked and kept for 24 h.

Micelles for stereocomplex MPEG5k–(scPLA4.5k)2 were obtained from mixing micellar solution 3c (50.0 mL, 0.20 mg mL−1) and micellar solution 3d (50.0 mL, 0.20 mg mL−1) according to the same procedure of micelles for stereocomplex MPEG1.9k–(scPLA4.5k)2.

Preparation of the DOX-loaded micelles from the MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2

The DOX-loaded micelles were also prepared by using the dialysis method. DOX·HCl (3 mg) and 3-fold molar triethylamine (TEA) were dissolved in DMF (3 mL) and kept stirring for 0.5 h to remove hydrochloride. After this, the copolymer MPEG1.9k–(scPLA4.5k)2 (30 mg) or MPEG5k–(scPLA4.5k)2 (30 mg) was added and stirred to form a homogeneous solution. After stirring for another 2 h, the solution was dialyzed against twice-distilled water using a dialysis tube (Mw cut off, 2.0 kDa) for 24 h and the solution outside the tube was replaced by fresh twice-distilled water every 4 h. And then, the solution in the tube was freeze-dried to obtain the final product which was stored at −20 °C until further experiments. The DOX loading content (LC) and encapsulation efficiency (EE) were determined by UV-vis spectrophotometer. 1 mg of the above product was dissolved in 5 mL of DMF. The concentration of DOX at 481 nm was recorded with reference to a calibration curve of pure DOX–DMF solution. The LC and EE of DOX were calculated using the following formulas, respectively.
image file: c6ra00699j-t1.tif

image file: c6ra00699j-t2.tif

In vitro release of DOX from MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2 micelles

The in vitro DOX release properties from the drug-loaded MPEG1.9k–(scPLA4.5k)2 or MPEG5k–(scPLA4.5k)2 self-assembled micelles were determined as follows: 3 mg of the freeze-dried product was suspended in 3 mL of PBS (pH = 7.4) in a dialysis membrane tube (Mw cut off 2.0 kDa). The dialysis tube was then immersed in 40 mL of PBS buffer at pH 7.4, and kept in a 37 °C water bath. At specific time intervals, a 4 mL (Ve) sample was taken out and replaced by 4 mL fresh PBS to maintain the total volume. The concentration of DOX in different samples was determined using UV-vis spectrophotometer with reference to a calibration curve of pure DOX–PBS solution. The cumulative drug release percent (Er) was calculated based on the equation. The in vitro DOX release properties in PBS (pH = 5.4) from the drug-loaded MPEG1.9k–(scPLA4.5k)2 or MPEG5k–(scPLA4.5k)2 self-assembled micelles were determined as the same procedure mentioned above.
image file: c6ra00699j-t3.tif
where mDOX represents the amount of DOX in the micelle (mg), V0 is the whole volume of the release media (V0 = 40 mL), and Cn represents the concentration of DOX in the nth sample (mg). The in vitro experiments were repeated three times, and all samples were analyzed in triplicate to get the final release curve.

Cell proliferation and morphology

In vitro cytotoxicity testing. Mouse L929 fibroblasts were seeded into 96-well tissue culture plates at a concentration of 1 × 104 cells per well and incubated 24 h at 37 °C in humidified air containing 5% CO2 with Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, Inc, Grand Island, NY, USA) containing 10% fetal calf serum (FCS, Gibco, USA). Then the medium was replaced with DMEM containing various concentrations (100 μg mL−1, 50 μg mL−1 and 5 μg mL−1) of extraction media of the synthesized polymers and their stereocomplexes with the DMEM as control group, and continued to culture another 72 h. After the required incubation periods, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent stock solution (5 mg mL−1 in Phosphate Buffered Saline (PBS)) was added to each well and incubated for 4 hours at 37 °C. Plates were centrifuged at 1200 rpm for 5 minutes. After the supernatant was discarded, DMSO (150 μL) was added to each well followed by incubation for 10 minutes with shaking. The supernatant was transferred into a new enzyme-linked immunosorbent assay (ELISA) plate. Its absorbance was measured at 570 nm with an ELISA reader (Bio-Tek, Winooski, Vermont, USA), where 630 nm was chosen as a reference wavelength. The optical density values obtained for cultures exposed to the extracts were normalized to untreated control cultures (corresponding to 100%). The relative growth rate (RGR) was calculated from the following equation: RGR = (Atest/Acontrol) × 100% (Atest is the absorbance of the experiment group and Acontrol is the absorbance of the control group. The final result was assumed to be the means of triplicate).
F-Actin staining. Mouse L929 fibroblasts were seeded on the glass slide into 24-well culture plates at a concentration of 2 × 104 cells per well and incubated 48 h at 37 °C in humidified air containing 5% CO2 with DMEM containing three dilute solutions containing various concentrations (100 μg mL−1, 50 μg mL−1 and 5 μg mL−1) of extraction media of the synthesized polymers and their stereocomplexes, DMEM as control group. Then L929 was processed for immune fluorescence detection. The cells were initially fixed with 2.5% paraformaldehyde (Sigma) at room temperature for 15 min, washed and incubated with 2% Bovine Serum Albumin (BSA) to block any unspecific binding. The cells were further incubated with phalloidin for 1 h at room temperature. Further, the cells were washed with PBS and incubated with DAPI for 5 min at room temperature for nuclear staining. Finally, images were obtained by fluorescence microscope (Nikon Eclipse80i).

Result and discussion

Synthesis of AB2 Y-shaped miktoarm copolymers

In order to investigate the effects of stereocomplex interaction on the stability and the morphology of micelles, the AB2 Y-shaped miktoarm amphiphilic copolymers were designed and synthesized via ROP and click chemistry. As shown in Scheme 1, the hydrophilic block is a monodisperse MPEG chain of 1900 or 5000 molecular weight, which was converted to MPEG–N3 as the terminally “clickable” part for the Y-shaped miktoarm amphiphilic copolymers. The hydrophobic block alkyne–(PLA)2 was synthesized by ROP of D- and L-lactides using 1 as initiator and Sn(Oct)2 as catalyst at 120 °C for 24 h. Finally, the AB2 Y-shaped miktoarm amphiphilic copolymers were obtained via click chemistry between MPEG–N3 and alkyne–(PLA)2. The characteristics for the synthesized polymers were summarized in Table 1. As a matter of convenience, the copolymer 3a was selected as the representative of the synthesized Y-shaped copolymer for detailed discussion in the following.

As shown in Fig. 1, the 1H NMR spectra clearly show that the peak at 2.43 ppm corresponding to the proton of the alkyne moieties of alkyne–(PLLA4.5k)2 in spectra b completely disappears in spectra c, and a new peak at 7.7 ppm corresponding to the triazole proton appeared in the 1H NMR spectra c, indicating the success of the click reaction. The chemical shift and assignment of proton signal were shown in the ESI (see ESI Fig. 4–9†).


image file: c6ra00699j-f1.tif
Fig. 1 1H NMR spectra of (a) MPEG1.9k–N3, (b) alkyne–(PLLA4.5k)2, (c) MPEG1.9k–(PLLA4.5k)2.

Furthermore, FT-IR was also used to verify the success of the click reaction for the AB2 Y-shaped miktoarm amphiphilic copolymers. The FT-IR spectra of MPEG1.9k–N3 (a), alkyne–(PLLA4.5k)2 (b) and MPEG1.9k–(PLLA4.5k)2 (c) were shown in Fig. 2. As could be seen from Fig. 2a, the peak at about 2100 cm−1 could be assigned to peak of N3 in MPEG1.9k–N3. The disappearance of the N3 peak and the creation of C[double bond, length as m-dash]O in ester at 1758 cm−1 in Fig. 2c indicates the success of the click reaction. The IR spectra for all the synthesized polymers were shown in ESI Fig. 10.†


image file: c6ra00699j-f2.tif
Fig. 2 FT-IR spectra of (a) MPEG1.9k–N3, (b) alkyne–(PLLA4.5k)2, (c) MPEG1.9k–(PLLA4.5k)2.

Meanwhile, the GPC traces of the AB2 Y-shaped copolymers MPEG1.9k–(PLLA4.5k)2 and MPEG5k–(PLLA4.5k)2 are narrow and monomodal peaks and shift to higher molecular weight region compared with that of the precursor MPEG and alkyne–(PLLA)2 (Fig. 3). These above results indicate that the polymerization is successful. The GPC traces of the D-type showed the same tendency (ESI Fig. 11†).


image file: c6ra00699j-f3.tif
Fig. 3 GPC traces of (a) MPEG1.9k, (b) MPEG5k, (c) alkyne–(PLLA4.5k)2, (d) MPEG1.9k–(PLLA4.5k)2 and (e) MPEG5k–(PLLA4.5k)2.

The formation of the stereocomplex MPEG1.9k–(scPLA4.5k)2 was investigated by FT-IR spectroscopy using dried samples (Fig. 4). The vibrational stretch of the carbonyl group of polylactide was found to shift to a lower wavenumber (from 1758 to 1750 cm−1) (Fig. 4B). Such a shift is attributed to the arrangement of the polylactide chains from a disordered state to an ordered one throughout the stereocomplex formation.39–41 The IR spectra for MPEG5k–(scPLA4.5k)2 were shown in ESI Fig. 12.†


image file: c6ra00699j-f4.tif
Fig. 4 FT-IR spectra of (A) (a) MPEG1.9k–(PLLA4.5k)2, (b) MPEG1.9k–(PDLA4.5k)2 and (c) MPEG1.9k–(scPLA4.5k)2; (B) partial enlarge part.

The thermal properties of the synthesized AB2 Y-shaped copolymers and their stereocomplexes were examined and DSC thermograms were shown in Fig. 5. It could be seen from Fig. 5 that the melting temperature of stereo-crystals displays 65–69 °C above that of homo-crystals reaching at 205 °C for MPEG1.9k–(scPLA4.5k)2 and 202 °C for MPEG5k–(scPLA4.5k)2, which are about 20 °C higher than that of the previous reported Y-shaped miktoarm stereocomplex.25 This higher crystalline melting temperature confirms the formation of the stereocomplex crystallization between the MPEG–(PLLA)2 and MPEG–(PDLA)2.42 Furthermore, it could be seen from Fig. 5B that in all the thermograms the melting temperature of MPEG5k could also be observed at about 50 °C, which could not be observed in the thermograms in Fig. 5A due to there relatively lower molecular weight of MPEG1.9k.


image file: c6ra00699j-f5.tif
Fig. 5 DSC heating curves of (A) (a) MPEG1.9k–(PLLA4.5k)2, (b) MPEG1.9k–(PDLA4.5k)2, (c) MPEG1.9k–(scPLA4.5k)2; (B) (a) MPEG5k–(PLLA4.5k)2, (b) MPEG5k–(PDLA4.5k)2, (c) MPEG5k–(scPLA4.5k)2.

The XRD was used to confirm the successful co-crystallization of the two enantiomeric PLA blocks in the synthesized AB2 Y-shaped copolymers. The typical XRD patterns of the synthesized AB2 Y-shaped copolymers and their stereocomplexes were illustrated in Fig. 6. As could been seen from Fig. 6A, MPEG1.9k–(PLLA4.5k)2 and MPEG1.9k–(PDLA4.5k)2 exhibited marked peaks at 16.7° and 19.0°, corresponding to the homocrystallized PLA block.21 The stereocomplex MPEG1.9k–(scPLA4.5k)2 yielded three new peaks at 11.9°, 20.7° and 24.0° while the significant peaks at 16.7° and 19.0° disappeared, which were a characteristic of the crystalline structure of the enantiomeric stereocomplex PLA blocks in the synthesized AB2 Y-shaped copolymers and in agreement with the pattern of the equimolar mixture of PLLA and PDLA.21,41,43 In Fig. 6B, MPEG5k–(PLLA4.5k)2, MPEG5k–(PDLA4.5k)2 and MPEG5k–(scPLA4.5k)2 all exhibited marked peaks at 19.2° and 23.4°, corresponding to the crystallized MPEG5k block.24,44 Both MPEG5k–(PLLA4.5k)2 and MPEG5k–(PDLA4.5k)2 exhibited marked peaks at 16.8°, corresponding to the homocrystallized PLA.45 The stereocomplex MPEG5k–(scPLA4.5k)2 yielded two new peaks at 11.9° and 20.8°, which were a characteristic of the crystalline structure of the enantiomeric stereocomplex PLA.46 The appeared peaks (19.2° and 23.4°) for MPEG5k in XRD is consistent to the foundation of the melting temperature of MPEG5k observed at about 50 °C in DSC curves. Both DSC and XRD results, together with the results from FT-IR, confirmed the formation of the stereocomplex between the corresponding synthesized AB2 Y-shaped copolymers.


image file: c6ra00699j-f6.tif
Fig. 6 X-ray diffraction spectra of (A) (a) MPEG1.9k–(PLLA4.5k)2, (b) MPEG1.9k–(PDLA4.5k)2, (c) MPEG1.9k–(scPLA4.5k)2; (B) (a) MPEG5k–(PLLA4.5k)2, (b) MPEG5k–(PDLA4.5k)2, (c) MPEG5k–(scPLA4.5k)2.

Aggregation of MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2 in aqueous solution

The CMC is one of the most important parameters for the self-assembly process of amphiphiles in solution. For MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2, their CMC values have been measured by fluorescence method with pyrene as the probe (ESI Fig. 13†). The obtained values of I373/I383 as a function of polymer concentration could be seen in Fig. 7. The derived CMC value for MPEG5k–(scPLA4.5k)2 is 0.039 mg mL−1, but for the case of MPEG1.9k–(scPLA4.5k)2, its CMC is 0.005 mg mL−1, which is much lower than the former's due to the lower molecular weight of MPEG part. It is worth to mention that the CMC value of MPEG1.9k–(scPLA4.5k)2 is relatively lower than the reported Y-shaped copolymers due to the higher molecular weight of PLA part.25
image file: c6ra00699j-f7.tif
Fig. 7 Plot of I373/I383 of pyrene excitation spectra in water as a function of the concentration of MPEG1.9k–(scPLA4.5k)2 (A) and MPEG5k–(scPLA4.5k)2 (B).

Due to the successful formation of the stereocomplex, DLS was further employed to characterize the size distribution of the micelles formed from the synthesized AB2 Y-shaped copolymers in aqueous solution. As shown in Fig. 8A and B, the size distribution of the micelles formed from MPEG1.9k–(PLLA4.5k)2 or MPEG1.9k–(PDLA4.5k)2 both are wide. The size distribution of the stereocomplex micelles formed from the equal molar mixture of MPEG1.9k–(PLLA4.5k)2 and MPEG1.9k–(PDLA4.5k)2 is in a narrow distribution (Fig. 8D). It is worth noting that when the MPEG1.9k–(PLLA4.5k)2 micelles solution was equally mixed with the MPEG1.9k–(PDLA4.5k)2 micelles solution for 24 h, the size distribution changed from wide range to narrow range (Fig. 8C) as that of the stereocomplex by method A. The same tendency could be obtained by mixing the MPEG5k–(PLLA4.5k)2 micellar solution with the MPEG5k–(PDLA4.5k)2 micellar solution for 24 h (see in ESI Fig. 14†). The occurrence could be attributed to the formation of the corresponding stereocomplex.


image file: c6ra00699j-f8.tif
Fig. 8 Size distribution of the self-assembled micelles determined by DLS: (A) MPEG1.9k–(PLLA4.5k)2 micelles; (B) MPEG1.9k–(PDLA4.5k)2 micelles; (C) MPEG1.9k–(scPLA4.5k)2 micelles (method B); (D) MPEG1.9k–(scPLA4.5k)2 micelles (method A).

For clearly observing the changing process, an investigation on the changes of the size distribution of the aggregates along with the time by DLS was carried out through mixing MPEG1.9k–(PLLA4.5k)2 micelles and MPEG1.9k–(PDLA4.5k)2 micelles. As shown in Fig. 9A, the tendency could be found that the range of the size distribution of the micelles became narrow along with the time. The same result could be obtained for MPEG5k–(PLA4.5k)2 series as shown in Fig. 9B. The proposed formation mechanism as shown in Fig. 10 is that there exists a dynamic equilibrium among the micelles of MPEG1.9k–(PLLA4.5k)2, MPEG1.9k–(PDLA4.5k)2 and their corresponding polymers. When the dissociative MPEG1.9k–(PLLA4.5k)2 meets with dissociative MPEG1.9k–(PDLA4.5k)2, the PLA parts of these two polymers form the corresponding stereocomplex, which is an irreversible process. Further the formed stereocomplex naturally assembles to the new stereocomplex micelles. Finally, if the time is over 12 h, the micelles derived from MPEG1.9k–(PLLA4.5k)2 or MPEG1.9k–(PDLA4.5k)2 disappear and change to the stereocomplex micelles.


image file: c6ra00699j-f9.tif
Fig. 9 Size distribution of the self-assembled micelles determined by DLS: (A) MPEG1.9k–(PLLA4.5k)2 micellar solution + MPEG1.9k–(PDLA4.5k)2 micellar solution; (B) MPEG5k–(PLLA4.5k)2 micellar solution + MPEG5k–(PDLA4.5k)2 micellar solution.

image file: c6ra00699j-f10.tif
Fig. 10 Proposed mechanism for forming stereocomplex micelles by mixing MPEG–(PLLA)2 micellar solution with MPEG–(PDLA)2 micellar solution.

The morphologies of the self-assembly aggregates were further confirmed by TEM (Fig. 11). For the MPEG1.9k–(PLLA4.5k)2, MPEG1.9k–(PDLA4.5k)2, MPEG5k–(PLLA4.5k)2 and MPEG5k–(PDLA4.5k)2, worm-like aggregates were obtained, which were different from the spherical micelles formed from the previous reported Y-shaped miktoarm copolymers in aqueous solution due to their different hydrophilic/lipophilic ratio.25 While for MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2, the spherical micelles were visually observed and the diameters are 160 nm and 222 nm respectively, which are coincident with the results derived from the DLS (207 nm and 230 nm). Furthermore, the Rg/Rh values for MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2 micelles in water were 0.84 and 0.93 respectively, which indicated that the spheres with a relatively loose structures were formed.47–49


image file: c6ra00699j-f11.tif
Fig. 11 The representative TEM images for the self-assembly aggregates of (A) MPEG1.9k–(PLLA4.5k)2; (B) MPEG1.9k–(PDLA4.5k)2; (C) MPEG1.9k–(scPLA4.5k)2 (method A); (D) MPEG5k–(PLLA4.5k)2; (E) MPEG5k–(PDLA4.5k)2; (F) MPEG5k–(scPLA4.5k)2 (method A).

Cytotoxicity assay

In order to test biocompatibility of the obtained AB2 Y-shaped copolymers and their stereocomplexes, the in vitro cytotoxicity was determined. Fig. 12 shows L929 cells proliferation in the presence of different concentrations of MPEG1.9k–(PLLA4.5k)2, MPEG1.9k–(PDLA4.5k)2, MPEG5k–(PLLA4.5k)2, MPEG5k–(PDLA4.5k)2, MPEG1.9k–(scPLA4.5k)2 or MPEG5k–(scPLA4.5k)2 assessed by MTT assay. The relative growth rate (RGR) in the MTT test was adopted to evaluate the cell toxicity at predetermined time. After co-culture, all of the experimental specimens at dilute concentration ranging from 5 μg mL−1 to 100 μg mL−1 were considered to be of no cytotoxicity compared with the control group (without any polymer additive) (p < 0.05, n = 6). The event was confirmed by F-actin staining (Fig. 13 and 14). There was no significant difference in cell density after 48 h cell culture. The effects on cell morphology were not found in the presence of different concentrations of AB2 Y-shaped copolymers and their stereocomplexes. This result is in agreement with the previous relative growth rate (RGR) in the cytotoxicity testing, indicating that the copolymers AB2 Y-shaped copolymers and their stereocomplexes show better compatibility with L929 cell. Therefore, polymeric micelles based on the biodegradable AB2 Y-shaped copolymers and their stereocomplexes may be suitable for the development of nanoscale drug delivery systems.
image file: c6ra00699j-f12.tif
Fig. 12 Cell toxicity results of left: MPEG1.9k–(PLLA4.5k)2 (blue), MPEG1.9k–(PDLA4.5k)2 (purple), MPEG1.9k–(scPLA4.5k)2 (green); right: MPEG5k–(PLLA4.5k)2 (blue), MPEG5k–(PDLA4.5k)2 (purple), MPEG5k–(scPLA4.5k)2 (green).

image file: c6ra00699j-f13.tif
Fig. 13 Fluorescence microscope micrographs of mouse L929 fibroblasts grown on MPEG1.9k–(PLLA4.5k)2 [(A) (5 μg mL−1), (B) (50 μg mL−1), (C) (100 μg mL−1)], on MPEG1.9k–(PDLA4.5k)2 [(D) (5 μg mL−1), (E) (50 μg mL−1), (F) (100 μg mL−1)], on MPEG1.9k–(scPLA4.5k)2 [(G) (5 μg mL−1), (H) (50 μg mL−1), (I) (100 μg mL−1)] and DMEM (CK) expressing F-action.

image file: c6ra00699j-f14.tif
Fig. 14 Fluorescence microscope micrographs of mouse L929 fibroblasts grown on MPEG5k–(PLLA4.5k)2 [(A) (5 μg mL−1), (B) (50 μg mL−1), (C) (100 μg mL−1)], on MPEG5k–(PDLA4.5k)2 [(D) (5 μg mL−1), (E) (50 μg mL−1), (F) (100 μg mL−1)], on MPEG5k–(scPLA4.5k)2 [(G) (5 μg mL−1), (H) (50 μg mL−1), (I) (100 μg mL−1)] and DMEM (CK) expressing F-action.

In vitro release of DOX from MPEG1.9k–(scPLA4.5k)2 and MPEG5k–(scPLA4.5k)2 micelles

DOX, a hydrophobic anticancer drug, was encapsulated into MPEG1.9k–(scPLA4.5k)2 or MPEG5k–(scPLA4.5k)2 micelles to evaluate the drug release ability. The drug loading content of micelles was determined to be 7.06% for MPEG1.9k–(scPLA4.5k)2 and 3.90% for MPEG5k–(scPLA4.5k)2. The encapsulation efficiency of the copolymer was 42.96% for MPEG1.9k–(scPLA4.5k)2 and 35.37% for MPEG5k–(scPLA4.5k)2. Subsequently, in vitro release of the drug from DOX-loaded micelles was conducted under simulated physiological conditions (phosphate buffer pH 7.4 or 5.4, 37 °C) as shown in Fig. 15. As for DOX-loaded MPEG1.9k–(scPLA4.5k)2 micelles, it is about 35% was released in PBS (pH = 5.4), which is 10% higher than that (25%) in PBS (pH = 7.4) in first 20 h. Then it slowly rose to 32% (pH = 7.4) and 44% (pH = 5.4) in the followed 88 h. As for DOX-loaded MPEG5k–(scPLA4.5k)2 micelles, it is about 51% was released in PBS (pH = 5.4), which is 18% higher than that (33%) in PBS (pH = 7.4) in first 20 h. Then it slowly rose to 37% (pH = 7.4) and 64% (pH = 5.4) in the followed 88 h. It clearly could be found that the release of DOX at a pH value of 5.4 was faster than that at a pH value of 7.4. This pH-dependent release profile could be attributed to the re-protonation of the amino group of DOX and the faster degradation of the micelle core at lower pH values, which could enhance the release rate at low local pH surrounding the tumor site.50–52 Furthermore, whether at a pH value of 7.4 or at a pH value of 5.4, the release of DOX from the MPEG5k–(scPLA4.5k)2 micelles is faster than that from the MPEG1.9k–(scPLA4.5k)2 micelles due to the relatively long MPEG part in MPEG5k–(scPLA4.5k)2.
image file: c6ra00699j-f15.tif
Fig. 15 Release curve of DOX from MPEG1.9k–(scPLA4.5k)2 ((a) pH 7.4; (c) pH 5.4) and MPEG5k–(scPLA4.5k)2 ((b) pH 7.4; (d) pH 5.4) micelles at 37 °C in phosphate buffer.

Conclusions

In this study, four new amphiphilic AB2 Y-shaped copolymers have been successfully synthesized by click chemistry. These amphiphilic stereocomplexes of the synthesized AB2 Y-shaped copolymers could stably self-assemble into spherical micelles in water. The CMC of MPEG1.9k–(scPLA4.5k)2 is much lower than that of MPEG5k–(scPLA4.5k)2. The micellar aggregate size was actually dependent on the hydrophobic block length of MPEG part. In particular, the in vitro cytotoxicity investigation presents that the synthesized AB2 Y-shaped copolymers and their stereocomplexes exhibits good compatibility with L929 cells. In addition, the in vitro drug release profile showed that the copolymers micelles could release DOX in a controlled manner. Therefore, a highly desirable application for these amphiphilic stereocomplex materials is their use as micellar vehicles for drugs delivery.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (NSFC No. 21271066, 21273061, 21327003 and U1504214).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra00699j
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