Preparation of Surfactant-Free and Core-Shell Type Nanoparticles of Methoxy Poly (ethylene Glycol)-b-Poly(ε-caprolactone-co-d,l-lactide) Diblock Copolymers

MATERIALS AND METHODS

Materials
Methoxy poly(ethylene glycol) (MPEG) with a molecular weight of 5,000 g mol^-1 (Fluka, Germany) was used after it was dried in a vacuum oven at 120 °C for 4 h. The ε-caprolactone (CL) monomer (99%, Acro, USA) was purified by drying with CaH₂ followed by distillation under reduced pressure before storage over molecular sieves in a refrigerator. D,L-lactide (DLL) was synthesized by well-established procedures from D,L-lactic acid (90%, Fluka, Switzerland). It was purified by repeated recrystallization from distilled ethyl acetate and dried in a vacuum oven at 50 °C for 48 h before used. The stannous octoacte (Sn(Oct)₂, 95%, Sigma, USA), acetone (AR, Merck, Germany) and ethanol (AR, Merck, Germany) were used without further purification.

METHODS

Synthesis of MPEG-b-P(CL-co-DLL)
The diblock copolymers with CL:DLL monomer feed mole ratios of 100:0, 90:10, 80:20 and 50:50 mol% were synthesized in bulk at 130 °C for 48 h under a dry nitrogen atmosphere. Polymerization reaction is presented in Scheme 1. The calculated molecular weights of the P(CL-co-DLL) blocks from feed ratios were approximately 60,000 g mol^-1. The MPEG and the Sn(Oct)₂ were used as the initiating system. The Sn(Oct)₂ concentration was kept constant at 0.04 mol%. The as-polymerized diblock copolymers were purified by dissolving in chloroform before being precipitated in cool n-hexane. Finally, they were dried to constant weight in a vacuum oven at room temperature before characterization and nanoparticle preparation.

Characterization of MPEG-b-P(CL-co-DLL)
Copolymer composition and number-average molecular weight () of the MPEG-b-P(CL-co-DLL) were determined by ¹H-NMR spectrometry using Bruker Advance DPX 300 ¹H-NMR spectrometer. CDCl₃ was used as a solvent at room temperature and tetramethylsilane was used as the internal standard. The thermal properties of polymers were characterized by non-isothermal Differential Scanning Calorimetry (DSC) using Perkin-Elmer Pyris Diamond DSC. For DSC, the sample (~10 mg) was heated at the rate of 10 °C min^-1 under helium flow to observe thermal transition temperatures.

Scheme 1:	Polymerization reaction of MPEG-b-P(CL-co-DLL)

Preparation of MPEG-b-P(CL-co-DLL) nanoparticles
The nanoparticles of MPEG-b-P(CL-co-DLL) without the addition of surfactant were prepared according to the modified-SESD method (Murakami et al., 1999). This procedure was explained as follows.

The diblock copolymer (~0.4 g) was dissolved in 20 mL acetone/ethanol [1/1 (v/v)] organic mixture. The solution was added dropwise into 160 mL distilled water in a 250 mL beaker with stirring at 600 rpm. Organic solvents were evaporated at room temperature for 6 hrs in a fume hood. Then, the nanoparticle colloid was obtained.

Characterization of MPEG-b-P(CL-co-DLL) Nanoparticles
Particle sizes and size distributions of the colloidal nanoparticles were determined by light-scattering analysis using Coulter LS230 light-scattering particle size analyzer at 25 °C. Shape and surface of the nanoparticles were investigated by Scanning Electron Microscopy (SEM) and transmission electron microscopy (TEM) using JEOL JSM-6460LV scanning electron microscope and JEOL JEM 1230 transmission electron microscope, respectively. For SEM, the nanoparticle colloid was dropped on the stab before freeze-dried for over night. Prior to examination, the samples were sputter-coated with gold for enhanced surface conductivity. For TEM, a drop of nanoparticle colloid was placed on a formvar film coated on the copper grid. The specimen on the copper grid was not stained.

Core-shell structure of the nanoparticles was studied by ¹H-NMR spectrometry using Bruker Advance DPX 300 ¹H-NMR spectrometer. For ¹H-NMR analysis, the nanoparticles were prepared in D₂O instead of distilled water.

RESULTS AND DISCUSSION

Characterization of MPEG-b-P(CL-co-DLL)
All purified diblock copolymers had percentage yields higher than 90%.

Table 1:	Copolymer compositions and of the diblock copolymers
aCalculated from comonomer feed ratios, bCalculated from 1H-NMR spectra

Fig. 1:	1H-NMR spectrum of MPEG-b-P(CL-co-DLL) with CL:DLL ratio of 50:50 mol% in CDCl3 (peak assignments as shown)

The copolymer compositions of diblock copolymers were determined from the ¹H-NMR spectra by ratioing the peak areas corresponding to the ethylene oxide (EO, repeating units of MPEG) methylene protons at δ = 3.4-3.6 ppm, the CL ε-methylene protons at δ = 3.9-4.2 ppm and the DLL methine protons at δ = 5.0-5.3 ppm. The ¹H-NMR spectrum of MPEG-b-P(CL-co-DLL) diblock copolymer with CL:DLL ratio of 50:50 mol% is shown as example in Fig. 1. The calculated compositions of CL:DLL and EO:CL:DLL ratios (mol%) are given in Table 1. As would be expected, the copolymer compositions are very similar to the comonomer feed ratios, indicating that the synthesis reactions proceeded to near-quantitative conversion.

The of diblock copolymers was carried out by mean of EO:CL:DLL ratio from ¹H-NMR spectra and MPEG molecular weight (5,000 g mol^-1). The calculated values are also summarized in Table 2. It was found that the of all diblock copolymers obtained from ¹H-NMR are similar to that obtained from the feed ratios (65,000 g mol^-1) suggested that the degradation side-reactions were small occurred.

The chain microstructures of P(CL-co-DLL) blocks are reflected in the fine structures of the ¹H-NMR spectra. The appearance of multiple resonances for the same proton can be attributed to the presence of difference monomer sequences and therefore slightly different chemical environments in the copolymer chain.

Table 2:	Thermal properties and particle size of the diblock copolymers
aDetermined from DSC thermograms, bDetermined from light-scattering analysis

Fig. 2:	DSC thermograms of the MPEG-b-P(CL-co-DLL) with CL:DLL ratios of (a) 100:0, (b) 90:10, (c) 80:20 and (d) 50:50 mol%

The α-CH₂ and ε-CH₂ protons in the CL units are seen to be particularly sensitive to this. The bands at 2.4 and 4.1 ppm, corresponding to the α-CH₂ and ε-CH₂ protons in the CL units, respectively are split into to quit distinct triplets adjacent to one another suggesting randomization of the CL units in the copolyester blocks (Baimark and Molloy, 2004).

Thermal analysis of the diblock copolymers was carried out by means of differential scanning calorimetry (DSC). DSC thermograms of the diblock copolymers are shown in Fig. 2 and the thermal properties are summarized in Table 2. The T_m and ΔH_m of diblock copolymer decreased when the DLL units were copolymerized. In addition, the T_m of diblock copolymer with CL:DLL ratio of 50:50 mol% can not be observed indicated that it is an complete amorphous polymer. The results suggested that the crystallizability of PCL block was suppressed when the DLL ratio was increased up to 50 mol%.

Characterization of MPEG-b-P(CL-co-DLL) Nanoparticles
All surfactant-free nanoparticle colloids of the diblock copolymers are clear aqueous suspensions. The nanoparticle sizes obtained from light-scattering analysis are shown in Table 2. The nanoparticle sizes were in the range of 84-639 nm. The results can be proposed that the MPEG block of diblock copolymer can be formed as the shell of nanodroplets during the solvent diffusion stage and its protective effect is adequate, then colloidal nanoparticles were formed after solidification. This suggested that the mechanism of modified-SESD method can be successfully used to prepare surfactant-free nanoparticles of the hydrophilic-hydrophobic diblock copolymers without any surfactant use. The size of the nanoparticles was increased as decreasing the DLL ratio. This may be explained that the diblock copolymers with higher crystalline PCL content give faster solidification of nanodroplets during solvent diffusion stage. From proposed mechanism of the modified-SESD method (Murakami et al., 1999), the droplet sizes of copolymer solution were reduced during the solvent diffusion stage. Therefore, if the diblock copolymer nanodroplets are faster solidified during the solvent diffusion stage, the nanoparticles with larger size are obtained.

Fig. 3:	SEM micrograph of the MPEG-b-P(CL-co-DLL) with CL:DLL ratio of 50:50 mol% (bar = 1 μm)

Fig. 4:	TEM micrograph of the MPEG-b-P(CL-co-DLL) with CL:DLL ratio of 50:50 mol% (bar = 100 nm)

It can be concluded that the diblock copolymer with higher crystalline CL content gave larger nanoparticle size. In addition, the particle sizes of nanoparticles seem to large distribution. However, these nanoparticles may be separated into similar size group by syringe filter (Zhang and Zhuo, 2005) and fractional centrifugation techniques (Kim et al., 1999). From SEM micrographs, it was found that the resulted nanoparticles were spherical shapes with smooth surfaces, as example of which is shown in Fig. 3. Figure 4 shows TEM micrograph of MPEG-b-P(CL-co-DLL) with CL:DLL ratio of 50:50 mol% confirmed that the resulted nanoparticles were spherical in shape with smooth surface according to SEM result.

Core-shell structure of the nanoparticles was demonstrated by comparing the ¹H-NMR spectra of the MPEG-b-P(CL-co-DLL) in CDCl₃ (Fig. 1) and the MPEG-b-P(CL-co-DLL) nanoparticles suspended in D₂O (Fig. 5). In CDCl₃ (Fig. 1), peaks of MPEG (peak a) and P(CL-co-DLL) (peaks b-h) blocks are observed. In contrast, only the MPEG peak (peak a) is found in D₂O (Fig. 5) while the P(CL-co-DLL) peaks are not detected. The results clearly indicate the core-shell structure of MPEG-b-P(CL-co-DLL) nanoparticles, according with literature reports base on similar ¹H-NMR data (Hrkach et al., 1997; Kim et al., 1999, 2000; Kim and Lee, 2001; Heald et al., 2002; Shuai et al., 2004; Zhang and Zhuo, 2005).

Fig. 5:	1H-NMR spectrum of nanoparticles of MPEG-b-P(CL-co-DLL) with CL:DLL ratio of 50:50 mol% suspended in D2O (peak assignments as shown)

It was concluded that core-shell type MPEG-b-P(CL-co-DLL) nanoparticles composed of hydrophilic outer shell of MPEG and hydrophobic core of P(CL-co-DLL) were successfully prepared by the modified-SESD method. It should be note that this method is easy for larger scale preparation of surfactant-free and core-shell type nanoparticles of biodegradable polymers than the other methods, especially dialysis method.

CONCLUSION

The MPEG-b-P(CL-co-DLL) diblock copolymers with different CL:DLL ratios were successfully synthesized by ring-opening polymerization of CL and DLL monomers by using MPEG with molecular weight of 5,000 g mol^-1 and stannous octoate as the initiating system at 130 °C for 48 h. The values of diblock copolymers from ¹H-NMR spectra were in the range of 62,300-65,600 g mol^-1. The thermal properties of diblock copolymers were strongly depended upon the CL:DLL ratios. The surfactant-free nanoparticles of diblock copolymers with particle sizes in the range of 84-639 nm were prepared by the modified-SESD method. The nanoparticle size decreased with increasing the DLL ratio. From SEM and TEM images, the obtained nanoparticles were spherical in shape and smooth surface. The structure of the nanoparticles studied from ¹H-NMR is of the core-shell type, with the P(CL-co-DLL) block forming the inner core and the anchored MPEG blocks on the nanoparticle surface. These surfactant-free and core-shell type biodegradable nanoparticles might be of interest for used as drug delivery systems.

ACKNOWLEDGMENTS

The work was supported by the Research Development and Support Unit, Mahasarakham University, fiscal year 2008 and the Center for Innovation in Chemistry: Postgraduate Education and Research Program in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand.