Formation of nanoparticles in aqueous solution from poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone)

Dihydroxyl-terminated PEG with a molecular weight of 4000 ^{g mol −1} was obtained from Sigma-Aldrich Chemical Co. (MO, USA). Before use, this polymer was purified by extraction with ethyl ether, followed by drying through azeotropic distillation in toluene. CL and stannous 2-ethyl hexanoate (stannous octoate, ^Sn(^Oct)₂) were purchased from Sigma-Aldrich Chemical Co. and were used as received. All other chemicals were of analytical grade and used without further purification.

The PCL–PEG–PCL triblock copolymer was synthesized by a ring opening polymerization of PEG and CL in the presence of stannous octoate as catalyst, according to the previous literature [9] with some modifications (scheme 1). A typical procedure was carried out as follows. 4 g of PEG (1.0 mmol) was introduced to a three neck flask and a predetermined volume of CL monomer (22.8 g, 200 mmol) and stannous octoate 0.005 mol% were added to the reaction vessel. The resulting mixture was placed in an oil bath at 130 °^C and stirred for 24 h. After that time, the resulting copolymer was cooled to room temperature and dissolved in dichloromethane, and precipitated in an excess amount of cold ether: hexane (1:1, v/v) to remove residual CL monomers and PEG initiators. The triblock copolymer was collected by filtration. Purification of the copolymer was achieved by the dissolution/precipitation method with dichloromethane and ether/hexane (1:1, v/v), respectively, followed by filtration and drying in a vacuum.

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In order to determine the structure of the triblock copolymer, proton nuclear magnetic resonance (¹H NMR) spectra were obtained using Bruker 500 MHz with deuterated chloroform (^CDCl ₃) as the solvent. Proton assignments were determined from the ¹H NMR spectra. Fourier transform infrared (FT-IR) spectra were measured by a FT-IR spectrometer (JASCO Corp, Japan) and the block copolymer-KBr discs were prepared from the polymer-KBr mixtures. In addition, the melting temperatures of the polymers were measured by differential scanning calorimetry (PerkinElmer, USA) under a flow of nitrogen at a scanning rate of 10 °^{C  min −1}. Measurements were performed in the range from 5 to 120 °^C.

The nanoprecipitation method was used to prepare PCL–PEG–PCL nanoparticles [10]. In brief, triblock copolymer PCL–PEG–PCL (5 mg) was dissolved in 2 ml of various organic solvents that are miscible with water. The polymer solutions were added to 10 ml of Millipore water. The resulting solutions were stirred at room temperature for 3 h and transferred to a dialysis bag (MW cut off: 50 000), then dialyzed against Millipore water for 24 h. The solution was filtered through a 0.45 μ^m syringe filter. The nanoparticles were produced in four solvents: acetone, acetonitrile, N,N-dimethylformamide (DMF) and tetrahydrofuran (THF). The effects of the various solvents were assayed on the overall size of the nanoparticles.

The particle size distributions were measured by dynamic light scattering with a Zetasizer 3000 (Malvern instruments, England) at a wavelength of 633 nm and 25 °^C, and at a scattering angle of 90° at a concentration of approximately 0.4 ^{mg  ml −1} water. The Z average value was recorded as the average of ten measurements.

Viable cells were assessed using an MTT assay. The L929 cells were seeded at densities designated as 1×10⁵ cells per well into 24-well tissue plates using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 100 ^{units  ml −1} penicillin and 100 ^{mg  l −1} streptomycin. The plates were incubated at 37 °^C. After 24 h, the culture medium was replaced with DMEM medium plus 10% FBS, and polymeric nanoparticles of various concentrations were added into the culture medium. The DMEM medium was used as a control. The cells were incubated for 24 h and an MTT assay was performed to evaluate cell viability. Briefly, the culture medium was removed, and the cells were washed with PBS twice. About 160 μ^l of DMEM medium and 40 μ^l MTT solution (5 ^{mg ml −1} in PBS) were added to each well, followed by incubation at 37 °^C for 4 h to allow formazan formation. The medium and MTT were removed and 200 μ^l of dimethylsulfoxide was added to dissolve the formazan crystals. After 5 min, the optical density (OD) at 570 nm was determined using a microplate reader. The ratios of ^OD ₅₇₀ from each experimental group (n=4) to the ^OD ₅₇₀ of the control group were used to assess cell viability.

The amphiphilic triblock copolymer was prepared by ring opening polymerization of CL initiated by PEG diol in the presence of ^Sn(^Oct)₂ as catalyst, according to scheme 1. The molecular weight of triblock copolymer was calculated from ^{1 H NMR} (M _{ⁿ (^PCL-^PEG-^PCL)}=M ^_nPEG +M ^_nPCL ). The molecular weight of the obtained copolymer was 27 810 and similar to the molecular weight calculated from theory (M ^_n =26 820). The typical signals of ^{1 H NMR} spectra of both PEG and PCL were detected when ^CDCl ₃ was used as the solvent, thus confirming that the ring opening polymerization and coupling reaction were carried out. In the ^{1 H NMR} spectra of triblock copolymers dissolved in ^CDCl ₃, the characteristic chemical shifts corresponding to both PCL (1.40, 1.64, 2.28 and 4.04 ppm) and PEG (3.62 ppm) were observed.

Figure 1 shows the FT-IR spectra of PEG and the PCL–PEG–PCL triblock copolymer. A new strong carbonyl band appears at 1728 ^{cm –1}, which could be attributed to the formation of the block copolymer. In addition, the aliphatic CH stretching band of ε-caprolactone is shifted to 2954 ^{cm –1}, whereas the absorption band of CH stretching vibration of PEG at 2892 ^{cm –1}. The absorption band at 3447 ^{cm –1} is determined to be the terminal hydroxyl groups in the block copolymers. The absorption at 1184–1103 ^{cm –1} is due to C–O stretching. Moreover, a large absorption peak from the C–O–C stretching can be seen at 1184 ^{cm –1} in the FT-IR spectra of the triblock copolymer.

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DSC results are shown in figure 2. There are two kinds of thermal curves of PEG–PCL copolymers. The first one is the thermal curve showing a monomodal melting peak, and the second one is the thermal curve resulted in a bimodal melting peak. The higher temperature endotherm can be attributed to the melting of the PCL crystal phase. The low temperature peaks correspond to the melting of the PEG crystal phase. The bimodal melting peaks of the copolymer system were reported by Bogdanov et al [11]. The bimodal melting peak observed for PEG is again most likely due to the existence of folded-chain lamellae with different fold numbers. In our DSC results for the PCL–PEG–PCL triblock copolymer, the melting endotherm showed monomodal melting peaks at 53.6 °^C. These results may be attributed to the melting endotherm of the PEG block, which is shifted and less pronounced in the low molecular weight ranges.

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The amphiphilic PCL–PEG–PCL with a hydrophilic PEG block and hydrophobic PCL blocks can self-assemble to form nanoparticles in an aqueous environment. The formation of the hydrophobic PCL inner core and the PEG outer core in an aqueous solution has been reported elsewhere [12]. To investigate the size of nanoparticles of the triblock copolymer in an aqueous milieu, DLS was employed to evaluate the size and size distribution of the obtained nanoparticles. Various water-miscible solvents, such as acetonitrile, acetone, DMF and THF, were used for the preparation of core-shell type nanoparticles of the triblock copolymer. The nanoparticles sizes of the triblock copolymer were 70, 59.4, 72.6 and 92.2 nm for DMF, acetone, acetonitrile and THF, respectively.

As shown in figure 3, the size of the triblock copolymer and the organic solvent water miscibility are generally correlated: a decrease in water miscibility (as indicated by the arrow) led to an increase in the average nanoparticle size. Among these, THF and acetone resulted in the largest and smallest particle size with a narrow size distribution, and maintained a stable nanoparticle solution after the dialysis procedure. Two populations of nanoparticles were observed with DMF and acetonitrile, with a smaller one with DMF with sizes ranging from 20 to 40 nm and a larger one from 45 to 120 nm. In contrast, with acetonitrile, a larger one with sizes in the range of 40–120 nm and a smaller one from 120 to 180 nm (figure 4) were achieved. Similar changes were observed for other copolymers [10, 12]. Therefore, the nanoparticles are dynamic systems and are stable upon dilution and organic solvent water miscibility, which is of major interest for intravenous injection.

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The MTT assay was performed to evaluate the cytotoxicity of polymeric nanoparticles to L929 cells (figure 5). The results of MTT indicated that all concentrations did not significantly affect the cell viability after 24 h incubation with polymeric micelles. Furthermore, the cell viability of concentrations up to 0.25 ^{mg  ml −1} was similar to the control group, and 80% of cell viability was observed with a concentration of 1.0 ^{mg  ml -1}. The cell viability suggests that the polymeric micelle has generally low cytotoxicity to the L929 fibroblast cells with concentrations of up to 1.0 ^{mg  ml -1}.

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