Preparation and characterization of stereocomplex aggregates based on PLA–P188–PLA

Abstract

Novel dumbbell-shaped amphiphilic copolymers ((PLLA)₂–G1–P188–G1–(PLLA)₂ and (PDLA)₂–G1–P188–G1–(PDLA)₂) and linear-shaped amphiphilic copolymers (PLLA–P188–PLLA and PDLA–P188–PDLA) were synthesized by click chemistry and ring opening polymerization (ROP). The stereocomplexes (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA were prepared and confirmed by X-ray diffraction (XRD). The stereocomplex interaction between PLLA and PDLA was firstly analyzed using microscale thermophoresis (MST) technology and the dissociation constant (K_d) was obtained as 342 μM. The aggregation behaviors of these stereocomplexes were studied using fluorescence spectroscopy, transmission electron microscopy (TEM) and light scattering (LS). The critical aggregate concentration (CAC) obtained from fluorescence measurements was 0.021 mg mL⁻¹ for (scPLA)₂–G1–P188–G1–(scPLA)₂ and 0.042 mg mL⁻¹ for scPLA–P188–scPLA. These stereocomplexes can self-associate in aqueous solution into spherical aggregates with a diameter of 181 nm for (scPLA)₂–G1–P188–G1–(scPLA)₂ and 222 nm for scPLA–P188–scPLA. Furthermore, the biocompatibility of the stereocomplexes was evaluated with a relatively lower cytotoxicity. Finally, DOX was encapsulated into the stereocomplex aggregates to evaluate the drug release ability in phosphate buffer at a pH value of 7.4 or 5.4. The drug loading content and the encapsulation efficiency of the aggregates are 9.8%, 54% for (scPLA)₂–G1–P188–G1–(scPLA)₂ and 8.2%, 45% for scPLA–P188–scPLA. The release of DOX at pH 5.4 is faster than that at pH 7.4. The pH value has a greater effect on the release rate of DOX from dumbbell-shaped stereocomplex aggregates than that from the linear-shaped ones, which ensures the long blood circulation and the higher DOX-release surrounding the tumor site.

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Introduction

Poloxamers, which consist of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO) blocks arranged in a basic triblock A–B–A structure of PEO–PPO–PEO, have been extensively studied as potential drug delivery vehicles due to their excellent biocompatibility.^1–7 P188 is the only poloxamer approved by the US Food and Drug Administration for intravenous injection. However, the application of P188 micelles as a drug delivery vehicle is limited by its low drug loading and poor dilution stability, which was ascribed to its high critical micelle concentration (CMC).⁸ In order to overcome such drawbacks, chemical modifications have been applied to decrease the CMC of P188 in aqueous solution.^7,9

Poly(lactide) (PLA), an important kind of synthetic biodegradable polyester, represents an excellent hydrophobic block. It has been used widely in medicine and bioengineering due to its biodegradability, good biocompatibility and excellent shaping and modeling properties.^10–14 PLA has been successfully grafted to both ends of poloxamers to obtain amphiphilic PLA–poloxamer–PLA pentablock copolymers, such as PLA–F127–PLA,¹⁵ PLLA–P188–PLLA^16–18 and PLLA–F87–PLLA.¹⁹ The results reported by K. C. Tam for PLA–F127–PLA¹⁵ showed that the CMC values decrease with increasing PLA block length indicating an effective method by grafting PLA to the end of poloxamer to decrease their CMC values. Moreover, it is worth mentioning that PLA has three stereoisomers, that is, poly(D-lactide) (PDLA), poly(L-lactide) (PLLA) and poly(D,L-lactide) (PDLLA). It is well known that the molecular chirality is an important natural property that affects physiochemical properties and biological activities.^20,21 Since the polymeric stereocomplex between the equimolar PLLA and PDLA has been firstly reported by Ikada et al. in 1987,²² the stereocomplex interaction as an important physical cross-linking method was used to decrease the CMC and stabilize the micelles.²³ Such kind of stereocomplex interaction that assists polymeric micellar formation has attracted gradually increasing attentions in recent years.^24–31 To the best of our knowledge, there is no report on stabilizing the poloxamer micelle by stereocomplex interaction between the PLLA and PDLA. Furthermore, for deeply understanding the stereocomplex interaction between the homochiral PLLA and PDLA, a technology named microscale thermophoresis (MST)^32,33 was firstly used to characterize the stereocomplex interaction for PLLA complex with PDLA.

In this work, two novel dumbbell-shaped amphiphilic copolymers, (PLLA)₂–G1–P188–G1–(PLLA)₂ and (PDLA)₂–G1–P188–G1–(PDLA)₂, based on P188 and PLA were synthesized by click chemistry and ring opening polymerization (ROP) (Scheme 1). Their chemical structures were determined by nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). Their corresponding stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂ was prepared and confirmed by X-ray diffraction (XRD). In addition, two linear-shaped amphiphilic copolymers (PLLA–P188–PLLA and PDLA–P188–PDLA) with the same molecular weight, also based on P188 and PLA were synthesized by ROP (Scheme 2) and the stereocomplex scPLA–P188–scPLA was prepared for comparison with the dumbbell-shaped stereocomplex. Meanwhile the self-assembly behaviour in aqueous solution, the biocompatibility and drug release profile of the stereocomplexes were explored in details for the application in drug delivery.

Scheme 1 Synthesis of dumbbell-shaped amphiphilic copolymers, 5a: (PLLA)₂–G1–P188–G1–(PLLA)₂, 5b: (PDLA)₂–G1–P188–G1–(PDLA)₂.

Scheme 2 Synthesis of linear-shaped amphiphilic copolymers, 6a: PLLA–P188–PLLA, 6b: PDLA–P188–PDLA.

Experimental section

Materials

Tetrahydrofuran (THF) was dried by reflux over sodium with benzophenone as an indicator under nitrogen. N,N-Dimethylformamide (DMF) was distilled under the reduced pressure from calcium hydride. Copper(I) bromide, 3-bromo-1-propyne, N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) and tin octoate (Sn(oct)₂) were purchased from Aladdin Chemical Co., Ltd. L- and D-Lactides were purchased from Changchun SinoBiomaterials Co., Ltd. P188 was purchased from Sigma Co., Ltd. Compound 2-methyl-2-((prop-2-yn-1-yloxy)methyl)propane-1,3-diol (G1) (see Scheme 1) was synthesized according to previous reported procedures.³⁴

Measurements

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 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 R_g/R_h value. Toluene was used as a standard for SLS measurements. All of the used 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. 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.

The fluorescence spectroscopy (PL) was used to evaluate the aggregation behavior of the 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 dissolving equilibrium of pyrene in the aqueous phase before measuring by fluorescence.

The X-ray diffraction (XRD) were performed on a Bruker D8 Advance X-ray diffractometer, using Cu Ka 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.

The stereocomplex binding affinities between PLLA and PDLA were conducted on the Monolith NT.LabelFree microscale thermophoresis (MST) instrument (NanoTemper Technologies GmbH, Munich, Germany). The value of dissociation constant (K_d) was calculated by NT ANALYSIS SOFTWARE provided by NanoTemper Technologies GmbH. The solvent DMF was used in MST measurements and the selected capillaries are standard glass capillaries. The used concentration of the PLLA (2.8 K) is 1 μM. The initial concentration of the PDLA (2.8 K) is 1500 μM and then it was diluted into different concentration solutions with DMF. After mixing 10 μL PLLA (1 μM) with 10 μL PDLA with different concentrations, these mixtures were placed into the capillaries for determination.

Synthesis

Synthesis of Ms–P188–Ms (2). P188 (16.21 g, 1.70 mmol) was introduced into a 250 mL flame-dried Schlenk flask and dried under reduced pressure at 80 °C for 1 h. After cooling to room temperature under nitrogen atmosphere, 120 mL dichloromethane was added to dissolve the P188 and then followed by 6 mL triethylamine to the flask. Under stirring, 5 mL methanesulfonyl chloride was added dropwise to the mixture and the reaction temperature was kept at 0 °C. After the addition, the whole mixture was stirred for another 48 h at room temperature under nitrogen atmosphere. The final reaction mixture was concentrated and filtered. The filtrate was poured into an excess amount of diethyl ether and the precipitates 2 were obtained, further washed with diethyl ether for three times, and dried in vacuum at 35 °C. ¹H NMR (400 MHz, CDCl₃): δ = 1.13–1.15 (m, 96H, –O–CH(CH₃)–CH₂–), 2.90 (s, 6H, –(SO₂)–CH₃), 3.40–3.43 (m, 32H, –O–CH(CH₃)–CH₂–), 3.50–3.57 (m, 64H, –O–CH(CH₃)–CH₂–), 3.61–3.78 (m, 697H, –O–CH₂–CH₂–O–), 3.81–3.83 (m, 4H, –S–O–CH₂–CH₂–), 4.37–4.40 (m, 4H, –S–O–CH₂–CH₂–); M_n,NMR = 9720 g mol⁻¹, M_n,GPC = 9150 g mol⁻¹, M_w/M_n = 1.22.

Synthesis of N₃–P188–N₃ (3). Polymer 2 (10.09 g, 1.04 mmol) and sodium azide (0.79 g, 12.20 mmol) were added to a 250 mL flask containing 50 mL fresh distilled DMF. The reaction mixture was stirred at 50 °C for 24 h. After the completion of the reaction, the residue was filtered and the filtrate was poured into an excess amount of diethyl ether. The precipitates 3 obtained were filtered, washed with diethyl ether, and dried in vacuum at 35 °C. ¹H NMR (400 MHz, CDCl₃): δ = 1.13–1.15 (m, 96H, –O–CH(CH₃)–CH₂–), 3.38–3.43 (m, 32H, –O–CH(CH₃)–CH₂–), 3.47–3.58 (m, 64H, –O–CH(CH₃)–CH₂–), 3.60–3.69 (m, 697H, –O–CH₂–CH₂–O–), 3.81–3.83 (t, 4H, N₃–CH₂–CH₂–); M_n,NMR = 9610 g mol⁻¹, M_n,GPC = 9120 g mol⁻¹, M_w/M_n = 1.22.

Synthesis of G1–P188–G1 (4). Polymer 3 (5.01 g, 0.52 mmol) and G1 (0.42 g, 2.66 mmol) was introduced into a 100 mL flame-dried Schlenk flask containing 25 mL DMF and the mixture was bubbled with nitrogen at 35 °C with stirring for 2 h. Then CuBr (0.10 g, 0.69 mmol) and PMDETA (110 μL) were added under nitrogen atmosphere. The resulting mixture was stirred at 45 °C for 48 h. When the reaction was complete, the solution was added with EDTA (0.10 g) and subjected to dialysis against distilled water using a dialysis membrane tube (3.5 kDa MWCO) for 4 days to remove DMF and copper. After lyophilization, polymer 4 was obtained as white powder. ¹H NMR (400 MHz, CDCl₃): δ = 0.81 (s, 6H, CH₃–C(CH₂OH)₂–CH₂–), 1.14–1.15 (m, 96H, –O–CH(CH₃)–CH₂–), 3.22–3.24 (t, 4H, –C(CH₂OH)₂–CH₂–O–), 3.40–3.42 (m, 32H, –O–CH(CH₃)–CH₂–), 3.47–3.51 (m, 64H, –O–CH(CH₃)–CH₂–), 3.61–3.83 (m, 697H, –O–CH₂–CH₂–O–), 3.86–3.88 (m, 4H, –CH₂–CH₂–O–), 4.54–4.57 (t, 4H, –CH₂–CH₂–N), 4.68 (s, 4H, –O–CH₂–C(N)=CH–), 7.76 (s, 2H, –CH₂–CH=CH–). M_n,NMR = 9930 g mol⁻¹; M_n,GPC = 9190 g mol⁻¹, M_w/M_n = 1.23.

General procedure for the synthesis of (PLLA)₂–G1–P188–G1–(PLLA)₂ (5a) and (PDLA)₂–G1–P188–G1–(PDLA)₂ (5b). Polymer 4 (0.50 g, 0.05 mmol) and L-lactide (1.66 g, 11.51 mmol) were introduced into a flame-dried Schlenk flask and dried under reduced pressure at 40 °C for 1 h. Then Sn(Oct)₂ (3.0 mg, 7.4 × 10⁻³ mmol) was added to the flask and the whole mixture was heated up to 120 °C for 24 h with stirring under nitrogen atmosphere. The final reaction mixture was dissolved in 20 mL dichloromethane and poured into an excess amount of diethyl ether. The white precipitates 5a obtained were filtered, washed with diethyl ether, and dried in vacuum at 35 °C. ¹H NMR (400 MHz, CDCl₃): δ = 0.94 (s, 6H, CH₃–C(CH₂O–)₂–CH₂–), 1.13–1.15 (m, 96H, –O–CH(CH₃)–CH₂–), 1.49–1.61 (m, 317H, –C OCH(CH₃)), 3.36–3.42 (m, 32H, –O–CH(CH₃)–CH₂–), 3.53–3.57 (m, 68H, –O–CH(CH₃)–CH₂–), 3.59–3.83 (m, 693H, –O–CH₂–CH₂–O–), 3.87–3.89 (t, 4H, –O–CH₂–CH₂–N–), 3.98–4.11 (m, 8H, –C(CH₃)(CH₂–)–CH₂–O–), 4.34–4.39 (q, 4H, –CO–CH(CH₃)–OH), 4.53–4.55 (t, 4H, –CH₂–CH₂–N), 4.57 (s, 4H, –C(N)(=CH)–CH₂–O–), 5.14–5.19 (m, 108H, –CO–CH (CH₃)–O–), 7.70 (s, 2H, –CH₂–CH=CH–); M_n,NMR = 17 780 g mol⁻¹; M_n,GPC = 17 440 g mol⁻¹, M_w/M_n = 1.21.

The polymer (5b) was obtained according to the procedure similar to that of synthesizing 5a. For the 5b, ¹H NMR (400 MHz, CDCl₃):δ = 0.94 (s, 6H, CH₃–C(CH₂O–)₂–CH₂–), 1.13–1.15 (m, 96H, –O–CH(CH₃)–CH₂–), 1.49–1.61 (d, 327H, –COCH–(CH₃)), 3.36–3.43 (m, 32H, –O–CH(CH₃)–CH₂–), 3.53–3.57 (m, 68H, –O–CH(CH₃)–CH₂–), 3.59–3.82 (m, 693H, –O–CH₂–CH₂–O–), 3.87–3.89 (t, 4H, –O–CH₂–CH₂–N–), 3.98–4.11 (m, 8H, –C(CH₃)(CH₂–)–CH₂–O–), 4.35–4.39 (q, 4H, –CO–CH(CH₃)–OH), 4.53–4.55 (t, 4H, –CH₂–CH₂–N), 4.57 (s, 4H, –C(N)(=CH)–CH₂–O–), 5.14–5.19 (m, 111H, –CO–CH (CH₃)–O–), 7.70 (s, 2H, –CH₂–CH=CH–); M_n,NMR = 17 920 g mol⁻¹; M_n,GPC = 17 830 g mol⁻¹, M_w/M_n = 1.25.

The polymer (6a) was obtained according to the procedure similar to that of synthesizing 5a. ¹H NMR (400 MHz, CDCl₃): δ = 1.14–1.16 (m, 96H, –O–CH(CH₃)–CH₂–), 1.49–1.60 (m, 377H, –COCH–(CH₃)), 3.40–3.44 (m, 32H, –O–CH(CH₃)–CH₂–), 3.51–3.59 (m, 68H, –O–CH(CH₃)–CH₂–), 3.63–3.70 (m, 676H, –O–CH₂–CH₂–O–), 3.82–3.84 (t, 4H, –O–CH₂–CH₂–O–), 4.23–4.39 (m, 6H, –O–CH₂–CH₂–O–, –COCH(CH₃)–OH), 5.14–5.19 (m, 123H, –CO–CH(CH₃)–O–); M_n,NMR = 18 100 g mol⁻¹; M_n,GPC = 20 690 g mol⁻¹, M_w/M_n = 1.19.

The polymer (6b) was obtained according to the procedure similar to that of synthesizing 5b.¹H NMR (400 MHz, CDCl₃): δ = 1.13–1.17 (m, 96H, –O–CH(CH₃)–CH₂–), 1.49–1.60 (m, 379H, –COCH–(CH₃)), 3.38–3.43 (m, 32H, –O–CH(CH₃)–CH₂–),3.51–3.58 (m, 68H, –O–CH(CH₃)–CH₂–), 3.62–3.70 (m, 676H, –O–CH₂–CH₂–O–), 3.81–3.84 (t, 4H, –O–CH₂–CH₂–O–), 4.27–4.37 (m, 6H, –O–CH₂–CH₂–O–, –COCH(CH₃)–OH), 5.14–5.19 (m, 125H, –CO–CH(CH₃)–O–); M_n,NMR = 18 750 g mol⁻¹; M_n,GPC = 21 080 g mol⁻¹, M_w/M_n = 1.22.

The polymer (7a) was obtained according to the procedure similar to that of synthesizing 5a using n-pentanol as the initiator. ¹H NMR (400 MHz, CDCl₃): δ = 0.90 (t, 3H, CH₃–CH₂–), 1.27–1.35 (m, 4H, CH₃–CH₂–CH₂–CH₂–), 1.50 (t, 3H, –CO–CH(CH₃)–OH), 1.54–1.63 (m, 116H, CH₃–CH₂–CH₂–CH₂–, –CO–CH(CH₃)–O–), 4.08–4.18 (m, 2H, –CH₂–CH₂–O–CO–), 4.36 (q, 1H, –CO–CH(CH₃)–OH), 5.12–5.22 (m, 37H, –CO–CH(CH₃)–OH); M_n,NMR = 2760 g mol⁻¹, M_n,GPC = 2880 g mol⁻¹, M_w/M_n = 1.26.

The polymer (7b) was obtained according to the procedure similar to that of synthesizing 5b using n-pentanol as the initiator. ¹H NMR (400 MHz, CDCl₃): δ = 0.90 (t, 3H, CH₃–CH₂–), 1.26–1.36 (m, 4H, CH₃–CH₂–CH₂–CH₂–), 1.51 (t, 3H, –CO–CH(CH₃)–OH), 1.58–1.63 (m, 116H, CH₃–CH₂–CH₂–CH₂–, –CO–CH(CH₃)–O–), 4.07–4.18 (m, 2H, –CH₂–CH₂–O–CO–), 4.36 (q, 1H, –CO–CH(CH₃)–OH), 5.10–5.24 (m, 37H, –CO–CH(CH₃)–OH); M_n,NMR = 2760 g mol⁻¹, M_n,GPC = 2890 g mol⁻¹, M_w/M_n = 1.23.

Preparation of stereocomplex

The stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂ was prepared from these two synthesized polymers 5a and 5b. The polymer 5a (0.05 g) and 5b (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 scPLA–P188–scPLA was prepared as the same procedure as that of stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂.

The blank aggregate preparation

The dialysis method was employed to prepare micellar aggregates of the stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA.

Aggregates for stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂ were prepared as follows: (scPLA)₂–G1–P188–G1–(scPLA)₂ (5.0 mg) was dissolved in 5 mL DMF in a 100 mL round-bottom flask with a stirrer, and then 25 mL of the twice-distilled water was added dropwise with vigorously stirring 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.

Aggregates for stereocomplex scPLA–P188–scPLA were prepared according to the same procedure as that of the stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂ and the obtained micellar solution is 0.10 mg mL⁻¹.

Preparation of the DOX-loaded aggregates from the (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA

The DOX-loaded aggregates were also prepared by using the dialysis method. DOX·HCl (6 mg) and 3-fold molar triethylamine were dissolved in DMF (3 mL) and kept stirring for 0.5 h to remove hydrochloride. After this, the copolymer (scPLA)₂–G1–P188–G1–(scPLA)₂ (30 mg) or scPLA–P188–scPLA (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 (2.0 kDa MWCO) 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.

In vitro release of DOX from (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA aggregates

The in vitro DOX release properties from the drug-loaded (scPLA)₂–G1–P188–G1–(scPLA)₂ or scPLA–P188–scPLA aggregates 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 (2.0 kDa MWCO). 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 (V_e) 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 (E_r) was calculated based on the eqn (3). The in vitro DOX release properties in PBS (pH = 5.4) from the drug-loaded aggregates of these stereocomplexes were determined as the same procedure mentioned above.

where m_DOX represents the amount of DOX in the aggregate (mg), V_e is the volume taking from the release media (V_e = 4 mL), V₀ is the whole volume of the release media (V₀ = 40 mL), C₁ represents the concentration of DOX in the first sample (mg mL⁻¹), and C_n represents the concentration of DOX in the nth sample (mg mL⁻¹). 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 for 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 stereocomplexes with the DMEM as control group, and continued to culture another 72 h. After the required incubation periods, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent stock solution (20 μL, 5 mg mL⁻¹ in Phosphate Buffered Saline (PBS)) was added to each well and incubated for 4 h at 37 °C. Plates were centrifuged at 1200 rpm for 5 min. After the supernatant was discarded, DMSO (150 μL) was added to each well and followed by incubation for 10 min 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 = (A_test/A_control) × 100% (A_test is the absorbance of the experiment group and A_control 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 for 48 h at 37 °C in humidified air containing 5% CO₂ with DMEM solutions containing stereocomplexes (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 dumbbell-shaped miktoarm copolymers

In order to investigate the effect of the stereocomplex formed from dumbbell- or linear-shaped amphiphilic copolymers on the self-assembly behavior and drug release profile, the dumbbell-shaped miktoarm amphiphilic copolymers were designed and synthesized via click chemistry and ROP (Scheme 1). As shown in Scheme 1, the targeted polymers were synthesized through four steps using P188 as the starting material. The terminal hydroxyl groups of P188 were converted to azide groups via mesylation and azide displacement. The macroinitiator 4 was obtained by a copper(I)-catalyzed azide–alkyne cycloaddition between polymer 3 and G1. The targeted dumbbell-shaped copolymers were synthesized by ROP of D- or L-lactides using 4 as the initiator and Sn(Oct)₂ as the catalyst at 120 °C. Meanwhile, for comparison with dumbbell-shaped amphiphilic copolymers, the linear-shaped copolymer 6a and 6b with almost the same molecular weight were synthesized by ROP of D- or L-lactides using P188 as the initiator (Scheme 2).

As shown in Fig. 1(a), the peaks at 4.37–4.40 (m, 4H) and 2.90 (s, 6H) in ¹H NMR spectrum can be assigned to –S–O–CH₂–CH₂– and –(SO₂)–CH₃. While the same peaks don't appear in Fig. 1(b), which indicates that the mesyl groups were completely changed to azide groups. The ¹H NMR spectra (b) and (c) clearly show the new peak at 7.76 ppm appearing in ¹H NMR spectrum (c) corresponding to the proton of the triazole proton indicating the occurrence of the click reaction between polymer 3 and G1. Comparing the ¹H NMR spectra (d) with spectra (c), the new appeared peaks at 5.14–5.19 ppm and 1.49–1.60 ppm assigned to –CO–CH(CH₃)–O– and –COCH(CH₃) in PLLA indicate the success of the ROP. The detailed assignation of hydrogen signals was shown in the ESI (Fig. 1–7†).

Fig. 1 ¹H NMR spectra of (a) Ms–P188–Ms, (b) N₃–P188–N₃, (c) G1–P188–G1, (d) (PLLA)₂–G1–P188–G1–(PLLA)₂.

Furthermore, FT-IR technique was also used to verify the success of the reaction for the dumbbell-shaped miktoarm amphiphilic copolymers (Fig. 2). As seen from spectra (a) and (b), the new peak in IR spectrum (b) at about 2100 cm⁻¹ could be assigned to peak of N₃ in N₃–P188–N₃, which indicates that the mesyl groups in Ms–P188–Ms was replaced by azide groups. Meanwhile, the peak in IR spectrum (c) at about 2100 cm⁻¹ disappeared and the peak in IR spectrum (d) at about 1750 cm⁻¹ appeared, indicating the success of the click reaction and the ROP.

Fig. 2 FT-IR spectra of (a) Ms–P188–Ms, (b) N₃–P188–N₃, (c) G1–P188–G1, (d) (PLLA)₂–G1–P188–G1–(PLLA)₂.

Furthermore, the GPC traces of the synthesized copolymers shown in Fig. 3 are narrow and monomodal peaks. The GPC traces of the targeted dumbbell-shaped polymers shift to higher molecular weight region compared with that of the starting material (P188) and the intermediate (macroinitiator 4 G1–P188–G1). The GPC traces of the linear polymers show the same tendency. These results of ¹H NMR, IR and GPC show that those block copolymers with narrow molecular distribution were synthesized.

Fig. 3 GPC traces of (a) P188, (b) G1–P188–G1, (c) (PLLA)₂–G1–P188–G1–(PLLA)₂, (d) (PDLA)₂–G1–P188–G1–(PDLA)₂, (e) PLLA–P188–PLLA, (f) PDLA–P188–PDLA.

The XRD spectra were used to confirm the formation of these stereocomplexes. The typical XRD patterns of the synthesized copolymers and their stereocomplexes were illustrated in Fig. 4. It is obviously seen that (PLLA)₂–G1–P188–G1–(PLLA)₂, (PDLA)₂–G1–P188–G1–(PDLA)₂, PLLA–P188–PLLA and PDLA–P188–PDLA exhibit the marked peaks at 16.7° and 19.0°, corresponding to the homocrystallized PLA block.²² The stereocomplexes (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA yield three new peaks at 11.9°, 20.7° and 24.0°, which are in agreement with the pattern of the equimolar mixture of PLLA and PDLA.^35,36 This is a characteristic of the crystalline structure of the enantiomeric stereocomplex PLA blocks in the synthesized dumbbell- and linear-shaped copolymers. The XRD results confirmed the successful formation of the stereocomplex between the corresponding synthesized homochiral copolymers.

Fig. 4 X-ray diffraction spectra of (A): (a) (PLLA)₂–G1–P188–G1–(PLLA)₂, (b) (PDLA)₂–G1–P188–G1–(PDLA)₂, (c) (scPLA)₂–G1–P188–G1–(scPLA)₂; (B): (a) PLLA–P188–PLLA, (b) PDLA–P188–PDLA, (c) scPLA–P188–scPLA.

Furthermore, we presented a new method utilizing MST to analyze the stereocomplex interaction between the synthesized PLLA (7a 2.8 K) and PDLA (7b 2.8 K) (¹H NMR spectra shown in ESI Fig. 8 and 9†). MST instrument provides a quick quantification of the stereocomplex interactions by detection of even minute changes in the thermal diffusion coefficient. It allows the observation of stereocomplex interaction by thermophoresis, and it can concomitantly measure the variations in fluorescence intensity, as shown in Fig. 5. The obtained K_d between the PLLA and PDLA is 342 μM, which confirms the stereocomplex interaction between the PLLA and PDLA.

Fig. 5 MST experiments for the determination of the dissociation constant between PDLA (2.8 K) and PLLA (2.8 K).

Aggregation of stereocomplex in aqueous solution

The CAC is one of the most important parameters of the self-assembly process of amphiphiles in aqueous solution. The studied stereocomplexes were expected to be able to self-assemble into micelle-like nanoparticles in aqueous solution like other amphiphilic block copolymers.³⁷ Their CAC values were measured by fluorescence method with pyrene as the probe (ESI Fig. 10†). The obtained values of I₃₇₃/I₃₈₃ as a function of polymer concentration were shown in Fig. 6. The derived CAC values are 0.021 mg mL⁻¹ for the stereocomplex (scPLA)₂–G1–P188–G1–(scPLA)₂ and 0.042 mg mL⁻¹ for scPLA–P188–scPLA respectively, which are much lower than that of P188 (about 4.0 mg mL⁻¹).⁸

Fig. 6 Plot of I₃₇₃/I₃₈₃ of pyrene excitation spectra in water as a function of the concentration of (scPLA)₂–G1–P188–G1–(scPLA)₂ (A) and scPLA–P188–scPLA (B).

The aggregation morphologies of the stereocomplexes were further confirmed by TEM measurements (Fig. 7). For (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA, the spherical aggregates were visually observed and the diameters are 181 nm and 222 nm respectively, which are coincident with the results derived from the DLS (226 nm and 250 nm) (ESI Fig. 11†). Furthermore, the R_g/R_h values for (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA aggregates in aqueous solution were 0.92 and 0.89 respectively, meaning that the spheres with a relatively loose structures were formed.^38–40

Fig. 7 The representative TEM images for the self-assembly aggregates of (scPLA)₂–G1–P188–G1–(scPLA)₂ (A) and scPLA–P188–scPLA (B).

Cytotoxicity assay

In order to test biocompatibility of the obtained stereocomplexes (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA, the in vitro cytotoxicity was measured. Fig. 8 shows L929 cells proliferation in the presence of different concentrations of (scPLA)₂–G1–P188–G1–(scPLA)₂ or scPLA–P188–scPLA 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 polymers additive) (p < 0.05, n = 6). The event was confirmed by F-actin staining (Fig. 9). There was no significantly difference in cell density after 48 h cell culture. The effects on cell morphology were not found in the presence of different concentrations of (scPLA)₂–G1–P188–G1–(scPLA)₂ or scPLA–P188–scPLA. This result is in agreement with the previous relative growth rate (RGR) in the cytotoxicity testing, as a result, the studied stereocomplexes show better compatibility with L929 cell. Therefore, polymeric aggregates based on the biodegradable stereocomplexes may be suitable for the development of nanoscale drug delivery systems.

Fig. 8 Cell toxicity results of (scPLA)₂–G1–P188–G1–(scPLA)₂ (green) and scPLA–P188–scPLA (purple).

Fig. 9 Fluorescence microscope micrographs of mouse L929 fibroblasts grown on (scPLA)₂–G1–P188–G1–(scPLA)₂ [(A) (5 μg mL⁻¹), (B) (50 μg mL⁻¹)], on scPLA–P188–scPLA [(C) (5 μg mL⁻¹), (D) (50 μg mL⁻¹)] and DMEM (CK) expressing F-action.

In vitro release of DOX from (scPLA)₂–G1–P188–G1–(scPLA)₂ and scPLA–P188–scPLA aggregates

DOX, a hydrophobic anticancer drug, was encapsulated in (scPLA)₂–G1–P188–G1–(scPLA)₂ or scPLA–P188–scPLA aggregates to evaluate the drug release ability. The drug loading content was determined to be 9.8% for (scPLA)₂–G1–P188–G1–(scPLA)₂ and 8.2% for scPLA–P188–scPLA. The encapsulation efficiency was 54% for (scPLA)₂–G1–P188–G1–(scPLA)₂ aggregates and 45% for scPLA–P188–scPLA aggregates.

Subsequently, in vitro release of the drug from DOX-loaded aggregates was conducted under simulated physiological conditions (phosphate buffer pH 7.4 or 5.4, 37 °C) as shown in Fig. 10. As for DOX-loaded (scPLA)₂–G1–P188–G1–(scPLA)₂ aggregates, it could be seen from Fig. 10 that about 37% was released in PBS (pH = 5.4) in first 10 h, while for the case with pH = 7.4, it is about 19%. Then it slowly rose to 56% (pH = 5.4) and 25% (pH = 7.4) in the followed 88 h. As for DOX-loaded scPLA–P188–scPLA aggregates, it could be seen from Fig. 10 that about 31% was released in PBS (pH = 5.4), which is 7% higher than that (24%) in PBS (pH = 7.4) in first 10 h. Then it slowly rose to 39% (pH = 5.4) and 33% (pH = 7.4) in the followed 88 h. It was found that the release of DOX at a pH value of 5.4 was faster than that at a pH value of 7.4 for the same DOX-loaded aggregates. This pH-dependent release profile could be attributed to the re-protonation of the amino group of DOX and the faster degradation of the aggregate core at lower pH values.^41–43

Fig. 10 Release curve of DOX from (scPLA)₂–G1–P188–G1–(scPLA)₂ ((a) pH 7.4, (d) pH 5.4) and scPLA–P188–scPLA ((b) pH 7.4, (c) pH 5.4) aggregates at 37 °C in phosphate buffer.

It could be seen from the Fig. 10 that the pH value has a greater effect on the release rate of DOX from dumbbell-shaped stereocomplex aggregates than that from the linear-shaped ones. As obtained by DSC (ESI Fig. 12†), the crystallinity is 49% for (scPLA)₂–G1–P188–G1–(scPLA)₂ and 78% for scPLA–P188–scPLA. The core is loose for (scPLA)₂–G1–P188–G1–(scPLA)₂ aggregates due to the low crystallinity, where the aqueous solution is easy to permeate into stereocomplex aggregates and the degradation of the aggregate core for (scPLA)₂–G1–P188–G1–(scPLA)₂ is faster than that for scPLA–P188–scPLA, leading to a relatively faster release. Such release profile ensures the long blood circulation and the higher DOX-release surrounding the tumor site.

Conclusions

In this study, novel amphiphilic dumbbell- and linear-shaped copolymers were successfully synthesized. Their stereocomplexes have also been prepared by evaporation method. The stereocomplex interaction between the PLLA and PDLA was firstly analyzed by MST and the obtained K_d is 342 μM. Both the amphiphilic stereocomplexes could self-assemble into loose spherical aggregates in aqueous solution. The CAC value of the dumbbell-shaped stereocomplex aggregates is lower than that of the linear-shaped stereocomplex aggregates. The in vitro cytotoxicity investigation presented that both the stereocomplexes exhibit good compatibility with L929 cells. The dumbbell-shaped stereocomplex aggregates show higher drug loading content and encapsulation efficiency than that of the linear-shaped ones. The drug release ability in phosphate buffer at a pH value of 7.4 and 5.4 was evaluated. The in vitro drug release profile shows that the stereocomplex aggregates can release DOX in a controlled manner at different pH values. It is worth mentioning that the pH value has a greater effect on the release rate of DOX from dumbbell-shaped stereocomplex aggregates than that from the linear-shaped ones. Therefore, a highly desirable application of the dumbbell-shaped stereocomplex is their use as micellar vehicles for anticancer drugs delivery.

Acknowledgements

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

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra04537e

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