- Email: [email protected]
Methotrexate-loaded biodegradable polymeric micelles: Preparation, physicochemical properties and in vitro drug release
Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
Methotrexate-loaded biodegradable polymeric micelles: Preparation, physicochemical properties and in vitro drug release Ying Zhang a,b , Tuo Jin a , Ren-Xi Zhuo b,∗ b
a School of Pharmacy, Shanghai JiaoTong University, Shanghai 200030, China Key Laboratory of Biomedical Polymers of the Ministry of Education, Wuhan University, Wuhan 430072, China
Received 11 May 2005; received in revised form 4 June 2005; accepted 16 June 2005
Abstract Polymeric micelles based on amphiphilic diblock copolymers methoxy poly(ethylene glycol)–polylactide with various hydrophobic lengths were designed as carriers of poorly water-soluble anticancer drug methotrexate (MTX). Relationship between physicochemical characteristics of micelles and release behavior was explored. The critical micelle concentration was determined by fluorescence spectroscopy using 9chloromethyl anthracene as fluorescence probe. Core-shell type polymeric micelles were prepared by free-surfactant dialysis technique. The mean size of micelles loaded with MTX was 50–200 nm with narrow polydispersity. Physicochemical properties of drug-loaded micelles were evaluated. In vitro release behavior of MTX was also investigated. MTX was continuously released from micelles and less than 50% MTX was released in 5 days. Release rate was dependent on chemical structures of micelles and enhanced by decreasing polylactide lengths. © 2005 Published by Elsevier B.V. Keywords: Amphiphilic diblock copolymers; Methoxy poly(ethylene glycol); Poly(d,l-lactide); Micelles; Methotrexate
1. Introduction Polymeric micelles formed by amphiphilic block copolymers demonstrate a series of attractive properties as drug vectors, such as high stability both in vitro and in vivo and good biocompatibility, and can be successfully used for the solubilization of various poorly water-soluble drugs [1–3]. These micelles can also be used as targeted drug delivery systems [4–6]. Methotrexate (MTX, as shown in Fig. 1) is a folate antimetabolite. Methotrexate has been used in the treatment of various malignancies including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin’s lymphoma, Hodgkin’s disease, head and neck cancer, lung cancer, and breast cancer [7]. However, methotrexate may cause some adverse effects, such as bone marrow suppression, acute and chronic hepatotoxicity, interstitial pneumonitis and chronic ∗
Corresponding author. Fax: +86 27 68754509. E-mail address: [email protected] (R.-X. Zhuo).
0927-7765/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.colsurfb.2005.06.004
interstitial obstructive pulmonary disease [8]. Therefore, it’s necessary to make some attempts to reduce its toxic and side effects and improve the specificity and selectivity. For example, Kwon et al. [9] conjugated MTX with PEG-b-PHEA by chemical bond linkage. Small size (10–30 nm) and stable (CMC value was 0.019–0.140 mg/mL) micelles were obtained. However, MTX was released very slowly and in 18 days only 20% MTX was released. On the other hand, it was difficult to modulate release rate. Kim and coworkers [10] encapsulated MTX into PHEA-C18 micelles. MTX release profile had obvious burst effect and in initial 1 h 40% MTX was released. Furthermore, 80% MTX was released from micelles in 5 h. Although polymeric micelles or nanoparticles loaded with MTX have been widely investigated, to our knowledge, previous work has undesired release behavior. Polylactide (PLA) is one of the most commonly used relatively hydrophobic and biodegradable polyesters. A number of polymeric micelles based on PLA-based amphiphilic block copolymers have been investigated in terms of various biomedical applications [11,12]. Poly(ethylene
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
105
cycles that also removed the solvent introduced in the catalyst solution. The flask was then sealed under reduced pressure. Copolymerization was carried out at 150 ◦ C for 16 h. The resulting polymer was dissolved in dichloromethane, precipitated into methanol, and then dried in vacuo at room temperature to constant weight. 2.3. Preparation of polymeric micelles
Fig. 1. Chemical structure of methotrexate (MTX).
glycol) (PEG) is frequently chosen as a hydrophilic segment to complement PLA because of its biocompatibility [13–16]. In this present work, amphiphilic diblock copolymers were synthesized by ring-opening polymerization of d,llactide (d,l-LA) initiated by methoxy poly(ethylene glycol) (mPEG). Polymeric micelles were prepared by freesurfactant dialysis technique. Anticancer drug MTX was encapsulated into the polymeric micelles. Physicochemical properties of the micelles were investigated and in vitro drug release behavior was evaluated.
Polymeric micelles were prepared by the dialysis method, which was free-surfactant [18,19]. Ten milligrams diblock copolymers were dissolved in 2 mL DMF, introduced into dialysis tube (MWCO = 8000–10,000 g/mol), and then dialyzed against 1.0 L distilled water, which was replaced every 3 h in the course of 24 h. The suspension was filtered through 0.45 m filter to remove aggregates and freezedried. To prepare drug-loaded micelles, 10 mg mPEG–PLA copolymers and 1 mg MTX were dissolved in 2 mL DMF, introduced into dialysis tube, and then dialyzed against 1.0 L distilled water as described above. For an evaluation of drug loading content and entrapment efficiency, polymeric micelles were dissolved in CHCl3 and then measured with ultraviolet–visible (UV–vis) spectrophotometer (Perkin-Elmer Lambda Bio 40) at 303 nm. 2.4. Diblock copolymers characterization 1H
2. Experimental 2.1. Materials d,l-lactic acid (≥85%) and Zn powder were purchased from Shanghai Chemical Reagents Co. Ltd. and d,llactide was synthesized according to the reference [17]. Poly(ethylene glycol) monomethylether (mPEG, molecular weight is 5000) was purchased from Alfa Aesar and purified by dissolving in chloroform, precipitated into diethyl ether and then dried in vacuo. Methotrexate was kindly provided by SuRi Biochem Co. Ltd., Suzhou. Stannous octoate (95%, Aldrich) was distilled under reduced pressure prior to use. 9-Chloromethyl anthracene was purchased from Acros Organics. Dialysis tube (molecular weight cut off = 8000–10000 g/mol) was purchased from Shanghai Chemical Reagent Co. Ltd. Other reagents were of analytical grade and used as received. 2.2. General procedure for copolymerization In a dried polymerization tube equipped with a magnetic stirring bar, given amounts of mPEG and d,l-LA was added. The system was dried under vacuum for 30 min and purged with nitrogen. Subsequently, 0.1 mol% Sn(Oct)2 in anhydrous toluene solution was charged into the vessel by a syringe. The flask was degassed by several vacuum-purge
NMR spectra were recorded on Mercury VX-300 spectrometer in CDCl3 with tetramethylsilane (TMS) as an internal reference. Number- and weight-average molec¯ n and M ¯ w , respectively) of the copolymers ular weight (M were determined by gel permeation chromatographic (GPC) system equipped with Waters 2690D separations module, Waters 2410 refractive index detector and Waters Styragel HR1DMF & Styragel HR4 DMF. DMF was used as the eluent at the flow rate of 0.3 mL/min. Waters Millennium module software was used to calculate molecular weight on the basis of a universal calibration curve generated by narrow molecular weight distribution poly(ethylene glycol) standard. 2.5. Fluorescence measurement In order to determine the critical micelle concentration (CMC) of diblock copolymers, 9-chloromethyl anthracene was used as fluorescent probe [20]. Stock solutions were prepared by dissolving 25 mg mPEG–PLA in 1 mL THF. Four milliliter distilled water was added with agitation, and THF was removed by rotary evaporator at 20 ◦ C. This solution was distilled to 25 mL. Subsequently, the 1.0 g/L stock solution was gradually diluted to ten concentration points and final polymeric solution was 1.0–10−5 g/L. To get sample solution, calculated amount of 9-chloromethyl anthracene in CH2 Cl2 solution was added to 10 mL volumetric flask and the solvent evaporated. The stock solution was added to the flask,
106
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
shaken for 3 h at 60 rpm at 37 ◦ C, and cooled overnight to 20 ◦ C. The final concentration of the probe was 6.0 × 10−6 M. Fluorescence spectra were recorded on Shimadzu RF-5301 PC spectrophotometer. Excitation wavelength was 370 nm, emission wavelength was 416 nm, and slit width was 5 nm for measurement. 2.6. Transmission electron microscopy measurement A drop of micelles suspension containing 2 wt.% phosphotungstic acid was placed on copper grid with Formvar® film and dried before measurement by JEOL JEM-100CXII transmission electron microscope (TEM) at an acceleration voltage of 80 keV. 2.7. Size distribution and zeta potential measurement Micelle size in aqueous solution was measured by dynamic light scattering at 90◦ to the incident beam and 20 ◦ C on a Beckman Coulter N4 Plus submicron Particle Size Analyzer. Zeta potential was determined by Zetasizer 3000HS (Malvern Instrument). 2.8. Wide-angle X-ray diffraction measurement Wide-angle X-ray powder diffraction was carried out on Shimadzu XRD-6000 X-ray diffractometer by Ni-filtered Cu K␣ radiation (40 kV, 30 mA) with 4◦ /min scanning rate at room temperature. 2.9. In vitro drug release studies Polymeric micelles 20 mg and 2 mL PBS (0.1 M, pH 7.4) were introduced into dialysis tube that immersed into 30 mL PBS solution. The system was shaken at 60 rpm at 37 ◦ C. At predetermined interval, 3 mL PBS solution was taken out and each sample was measured three times and their mean values were used. Accumulative release weight of MTX was calculated according to the calibration curve.
3. Results 3.1. Characterization of amphiphilic diblock copolymers Fig. 2 showed synthetic scheme and the structure of mPEG–PLA copolymers. Various chain lengths of PLA in the copolymers were obtained by modulating the feed ratio of mPEG and d,l-lactide. Copolymers structure was confirmed by 1 H NMR spectrum measurements (Fig. 3): the peaks at 3.63 and 3.48 ppm corresponded to methylene units and CH3 O- in the mPEG blocks, signals for PLA segments were 1.59 and 5.20 ppm for CH3 - and CH-groups, respectively. From the peak integrity ratio of their methylene and methyl groups, the molar ratio of repeating units in mPEG and PLA blocks can be calculated. The value was very close to that of feed composition (Table 1). Number- and weight¯ n and M ¯ w , respectively) of the average molecular weight (M copolymers were also listed in Table 1. 3.2. Critical micelle concentration Critical micelle concentration (CMC) of diblock copolymers was determined by fluorescence spectrum and 9chloromethyl anthracene was used as fluorescent probe. In the emission spectra (Fig. 4a, emission wavelength was 416 nm), fluorescence intensity was enhanced with increasing polymer concentration and red shift (from 410 to 416 nm) [21] was also present with increasing polymer concentration. As shown in Fig. 4b, at low concentration fluorescence intensity increased slowly and almost was a constant and then increased sharply due to the formation of micelles. 3.3. Preparation and characterization of polymeric micelles Core-shell type polymeric micelles were prepared by dialysis method. Size and size distribution of micelles were measured by dynamic light scattering. The mean diameters of polymeric micelles were 50–200 nm with narrow
Fig. 2. Synthetic scheme of mPEG–PLA amphiphilic diblock copolymers.
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
107
Fig. 3. 1 H NMR spectrum of PEL-2 in CDCl3 . Table 1 Copolymerization of d,l-LA with mPEG at various contents catalyzed by Sn(Oct)2 at 150 ◦ C for 16 h Sample
PLA PEL-1 PEL-2 PEL-3 PEL-4 a b c
mPEG (g)
– 0.0280 0.0787 0.1931 0.4377
d,l-LA (g)
mPEG content (wt.%)
0.4239 0.4326 0.4349 0.4321 0.4343
In feed
In the
– 6.1 15.3 30.9 50.2
– 5.0 17.7 35.7 53.4
MNMR a (×10−4 )
Mn b (×10−4 )
¯ w /M ¯ nb M
Yield (%)
– 10.1 2.8 1.4 0.9
6.2c 6.6 2.7 1.3 0.7
1.30 1.43 1.39 1.29 1.08
95.5 80.2 82.0 75.9 74.5
copolymera
Calculated by 1 H NMR. Determined by GPC with DMF as the eluent. Determined by GPC with chloroform as the eluent.
polydispersity (Table 2 ). Micelle sizes with the presence of MTX were bigger about 10–20 nm than that of the absence of MTX. Zeta potential was measured by Laser Doppler Anemometry and all micelles had negative zeta potential (from −15 to −7 mV). The morphology of micelles was observed by Transmission electron microscopy. As shown in Fig. 5 , these micelles were spherical in shape and the granulity was equal. Crystallization of MTX-loaded micelles was evaluated by X-ray powder diffraction. As shown in Fig. 6, the patterns
between micelles and MTX-loaded micelles were identical; however, physical mixture of MTX and micelles had extra peaks at 23◦ . Drug loading content and entrapment efficiency of polymeric micelles were calculated by absorbance of MTX at 303 nm from UV. The results were also summarized in Table 2. There was no interference from the copolymers at this wavelength. Drug loading content and entrapment efficiency depend on the composition of the mPEG–PLA copolymers and the highest loading efficiency was 47%.
Table 2 Micelle size, drug loading content, loading efficiency, and zeta potential of the polymeric micelles Sample
Loading content (%)
Loading efficiency (%)
Micelle size with the absence of MTX (nm)
PEL-1 PEL-2 PEL-3 PEL-4
12.8 6.4 4.2 3.7
47.3 34.8 22.2 17.4
180.1 121.4 71.3 42.6
± ± ± ±
16.7 19.8 22.5 13.3
Micelle size with the presence of MTX (nm) 200.6 134.7 86.0 50.1
± ± ± ±
40.6 21.2 17.8 19.7
Zeta potential (mV) −15.1 −12.3 −9.5 −7.0
± ± ± ±
1.7 1.9 1.3 1.5
108
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
Fig. 6. X-ray diffraction patterns for (a) PEL-2 micelles, (b) MTX-loaded micelles, (c) physical mixture of micelles with MTX (MTX 10 wt.%), and (d) MTX.
Fig. 4. (a) Fluorescence emission spectra of 9-chloromethyl anthracene with increasing concentrations of PEL-2. (b) Plot of intensity vs. log C for PEL-2.
3.4. In vitro MTX release behavior of polymeric micelles Fig. 7. Release curves of MTX from polymeric micelles.
In vitro release behavior of MTX was carried out in PBS (0.1 M, pH 7.4) at 37 ◦ C. The influence of various PLA length incorporated into the copolymers on release rate was evaluated. As shown in Fig. 7, there was no burst effect during
initial period and MTX was continuously released from these micelles. PEL-4 micelles had fastest release rate and less than 50% MTX was released in 5 days.
4. Discussion
Fig. 5. TEM image of PEL-4 micelles loaded with MTX.
A series of mPEG–PLA copolymers with various PLA chain lengths were synthesized. The relationship between chemical structure and physicochemical properties of obtained micelles and release behavior was investigated. Polymeric micelles were prepared by dialysis technique. In this method, polymer and drug are dissolved in the organic solvent, which is completely dissolved in the external aqueous phase, and then the solution diffuses instantaneously to the external aqueous phase, followed by precipitation of the polymer and drug. The advantage of this method is that no surfactant is employed. Initial diblock copolymeric concentration will influence micelle size. When polymeric concentration is bigger and the solution is more viscous,
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
macromolecule diffusion is restrained and larger micelles are obtained. In the case of our experiment, polymeric concentration during micelle preparation was 5 mg/mL. As shown in Table 2, when mPEG molecular weight was constant, polymeric micelle sizes enhanced as increasing PLA chain length. It is difficult to form compact polymeric micelles for amphiphilic copolymers with longer hydrophobic chain length. This result was in agreement with the characteristic of amphiphilic copolymer micelles that the fewer the hydrophobic component, the smaller the micelles. On the other hand, micelle sizes with the presence of MTX were bigger about 10–20 nm than that of the absence of MTX. The presence of PEG can shield surface ion charge of the micelles. All micelles had negative zeta potential. The absolute value of zeta potential reduced as increasing PEG content in the copolymers. Due to negative charge on the surface of the micelles, ion intensity and pH value of the medium during preparation process may influence micelle size. When ion intensity and acidity value of the medium were reduced, micelle size enhanced due to intermicellar aggregation. In our experiment, the aqueous medium was neutral. The physical state of both the drug and polymeric micelles can also influence release behavior of the drug. Interaction between MTX and hydrophobic chain in the core and crystallization of MTX in the micelles was evaluated by X-ray powder diffraction. From X-ray diffraction patterns (Fig. 6), it is suggested that MTX was totally encapsulated into polymeric micelles and dispersed molecularly. In vitro drug release behavior of polymeric micelles was also dependent on the chemical composition of polymeric micelles. As shown in Fig. 7, sustained release was obtained and release rate reduced as enhancing PLA chain length. It is stemming from stronger interaction between poorly watersoluble MTX and longer hydrophobic PLA chain length.
5. Conclusions A series of mPEG–PLA copolymers with various PLA chain lengths were synthesized. Micelles based on amphiphilic mPEG–PLA copolymers were prepared with free surfactant. Anticancer drug MTX was loaded into inner core of polymeric micelles due to hydrophobic interaction. Physicochemical characteristics of micelles were varying by changes in molecular weight of the copolymers. When molecular weight of mPEG was constant, changes in molecular weight of the copolymers can modulate
109
hydrophilic/hydrophobic block ratio. Relationship between physicochemical characteristics of micelles and release behavior was explored. mPEG–PLA micelles proposed in this paper had desired in vitro drug release kinetics. In initial period there was no burst effect and less than 50% MTX was released in 5 days.
Acknowledgments The authors are grateful for the financial support from the National Key Basic Research and Development Program (G1999064703) of China and a grant (29934060) from the National Natural Science Foundation of China.
References [1] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Science 263 (1994) 1600. [2] K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev. 47 (2001) 113. [3] G.S. Kwon, Crit. Rev. Ther. Drug Carrier Syst. 20 (2003) 357. [4] P.D. Scholes, A.G.A. Coombes, L. Illum, S.S. Davis, M. Vert, M.C. Davies, J. Control. Rel. 25 (1993) 145. [5] E.S. Lee, K. Na, Y.H. Bae, J. Control. Rel. 91 (2003) 103. [6] Y. Matsumura, M. Yokoyama, K. Kataoka, T. Okano, Y. Sakurai, T. Kawaguchi, T. Kakizoe, Jpn. J. Cancer Res. 90 (1999) 122. [7] P. Calabresi, R.E. Parks, The Pharmacological Basis of Therapeutics, Macmillan, New York, 1975, p. 1254. [8] P. Calabresi, B.A. Chabner, in: L.S. Goodman, A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, Macmillan, New York, 1991, p. 1202. [9] Y. Li, G.S. Kwon, Pharm. Res. 17 (2000) 607. [10] H. Kang, J.D. Kim, S.H. Han, I.S. Chang, J. Control. Rel. 81 (2002) 135. [11] C.L. Lo, K.M. Lin, G.H. Hsiue, J. Control. Rel. 104 (2005) 477. [12] H. Arimura, Y. Ohya, T. Ouchi, Biomacromolecules 6 (2005) 720. [13] Y. Dong, S.S. Feng, Biomaterials 25 (2004) 2843. [14] A. Vila, A. Sanchez, C. Evora, I. Soriano, J.L. Vila Jato, M.J. Alonso, J. Aerosol. Med. 17 (2004) 174. [15] Y. Nagasaki, K. Yasugi, Y. Yamamoto, A. Harada, K. Kataoka, Biomacromolecules 2 (2001) 1067. [16] K. Emoto, Y. Nagasaki, M. Iijima, M. Kato, K. Kataoka, Colloids Surf. B 18 (2000) 337. [17] X. He, X.K. Guo, D.S. Zheng, J.H. Zhang, Chin. Fine Chem. 21 (2004) 745. [18] Y. Yamamoto, K. Yasugi, A. Harada, Y. Nagasaki, K. Kataoka, J. Control. Rel. 82 (2002) 359. [19] C.L. Sang, K. Chulhee, C.K. Ick, C. Hesson, Y.J. Seo, J. Control. Rel. 89 (2003) 437. [20] C. Li, X. Liu, L.Z. Meng, Polymer 45 (2004) 337. [21] Y. Zhang, R.X. Zhuo, Biomaterials 26 (2005) 2089.
Methotrexate-loaded biodegradable polymeric micelles: Preparation, physicochemical properties and in vitro drug release Ying Zhang a,b , Tuo Jin a , Ren-Xi Zhuo b,∗ b
a School of Pharmacy, Shanghai JiaoTong University, Shanghai 200030, China Key Laboratory of Biomedical Polymers of the Ministry of Education, Wuhan University, Wuhan 430072, China
Received 11 May 2005; received in revised form 4 June 2005; accepted 16 June 2005
Abstract Polymeric micelles based on amphiphilic diblock copolymers methoxy poly(ethylene glycol)–polylactide with various hydrophobic lengths were designed as carriers of poorly water-soluble anticancer drug methotrexate (MTX). Relationship between physicochemical characteristics of micelles and release behavior was explored. The critical micelle concentration was determined by fluorescence spectroscopy using 9chloromethyl anthracene as fluorescence probe. Core-shell type polymeric micelles were prepared by free-surfactant dialysis technique. The mean size of micelles loaded with MTX was 50–200 nm with narrow polydispersity. Physicochemical properties of drug-loaded micelles were evaluated. In vitro release behavior of MTX was also investigated. MTX was continuously released from micelles and less than 50% MTX was released in 5 days. Release rate was dependent on chemical structures of micelles and enhanced by decreasing polylactide lengths. © 2005 Published by Elsevier B.V. Keywords: Amphiphilic diblock copolymers; Methoxy poly(ethylene glycol); Poly(d,l-lactide); Micelles; Methotrexate
1. Introduction Polymeric micelles formed by amphiphilic block copolymers demonstrate a series of attractive properties as drug vectors, such as high stability both in vitro and in vivo and good biocompatibility, and can be successfully used for the solubilization of various poorly water-soluble drugs [1–3]. These micelles can also be used as targeted drug delivery systems [4–6]. Methotrexate (MTX, as shown in Fig. 1) is a folate antimetabolite. Methotrexate has been used in the treatment of various malignancies including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin’s lymphoma, Hodgkin’s disease, head and neck cancer, lung cancer, and breast cancer [7]. However, methotrexate may cause some adverse effects, such as bone marrow suppression, acute and chronic hepatotoxicity, interstitial pneumonitis and chronic ∗
Corresponding author. Fax: +86 27 68754509. E-mail address: [email protected] (R.-X. Zhuo).
0927-7765/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.colsurfb.2005.06.004
interstitial obstructive pulmonary disease [8]. Therefore, it’s necessary to make some attempts to reduce its toxic and side effects and improve the specificity and selectivity. For example, Kwon et al. [9] conjugated MTX with PEG-b-PHEA by chemical bond linkage. Small size (10–30 nm) and stable (CMC value was 0.019–0.140 mg/mL) micelles were obtained. However, MTX was released very slowly and in 18 days only 20% MTX was released. On the other hand, it was difficult to modulate release rate. Kim and coworkers [10] encapsulated MTX into PHEA-C18 micelles. MTX release profile had obvious burst effect and in initial 1 h 40% MTX was released. Furthermore, 80% MTX was released from micelles in 5 h. Although polymeric micelles or nanoparticles loaded with MTX have been widely investigated, to our knowledge, previous work has undesired release behavior. Polylactide (PLA) is one of the most commonly used relatively hydrophobic and biodegradable polyesters. A number of polymeric micelles based on PLA-based amphiphilic block copolymers have been investigated in terms of various biomedical applications [11,12]. Poly(ethylene
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
105
cycles that also removed the solvent introduced in the catalyst solution. The flask was then sealed under reduced pressure. Copolymerization was carried out at 150 ◦ C for 16 h. The resulting polymer was dissolved in dichloromethane, precipitated into methanol, and then dried in vacuo at room temperature to constant weight. 2.3. Preparation of polymeric micelles
Fig. 1. Chemical structure of methotrexate (MTX).
glycol) (PEG) is frequently chosen as a hydrophilic segment to complement PLA because of its biocompatibility [13–16]. In this present work, amphiphilic diblock copolymers were synthesized by ring-opening polymerization of d,llactide (d,l-LA) initiated by methoxy poly(ethylene glycol) (mPEG). Polymeric micelles were prepared by freesurfactant dialysis technique. Anticancer drug MTX was encapsulated into the polymeric micelles. Physicochemical properties of the micelles were investigated and in vitro drug release behavior was evaluated.
Polymeric micelles were prepared by the dialysis method, which was free-surfactant [18,19]. Ten milligrams diblock copolymers were dissolved in 2 mL DMF, introduced into dialysis tube (MWCO = 8000–10,000 g/mol), and then dialyzed against 1.0 L distilled water, which was replaced every 3 h in the course of 24 h. The suspension was filtered through 0.45 m filter to remove aggregates and freezedried. To prepare drug-loaded micelles, 10 mg mPEG–PLA copolymers and 1 mg MTX were dissolved in 2 mL DMF, introduced into dialysis tube, and then dialyzed against 1.0 L distilled water as described above. For an evaluation of drug loading content and entrapment efficiency, polymeric micelles were dissolved in CHCl3 and then measured with ultraviolet–visible (UV–vis) spectrophotometer (Perkin-Elmer Lambda Bio 40) at 303 nm. 2.4. Diblock copolymers characterization 1H
2. Experimental 2.1. Materials d,l-lactic acid (≥85%) and Zn powder were purchased from Shanghai Chemical Reagents Co. Ltd. and d,llactide was synthesized according to the reference [17]. Poly(ethylene glycol) monomethylether (mPEG, molecular weight is 5000) was purchased from Alfa Aesar and purified by dissolving in chloroform, precipitated into diethyl ether and then dried in vacuo. Methotrexate was kindly provided by SuRi Biochem Co. Ltd., Suzhou. Stannous octoate (95%, Aldrich) was distilled under reduced pressure prior to use. 9-Chloromethyl anthracene was purchased from Acros Organics. Dialysis tube (molecular weight cut off = 8000–10000 g/mol) was purchased from Shanghai Chemical Reagent Co. Ltd. Other reagents were of analytical grade and used as received. 2.2. General procedure for copolymerization In a dried polymerization tube equipped with a magnetic stirring bar, given amounts of mPEG and d,l-LA was added. The system was dried under vacuum for 30 min and purged with nitrogen. Subsequently, 0.1 mol% Sn(Oct)2 in anhydrous toluene solution was charged into the vessel by a syringe. The flask was degassed by several vacuum-purge
NMR spectra were recorded on Mercury VX-300 spectrometer in CDCl3 with tetramethylsilane (TMS) as an internal reference. Number- and weight-average molec¯ n and M ¯ w , respectively) of the copolymers ular weight (M were determined by gel permeation chromatographic (GPC) system equipped with Waters 2690D separations module, Waters 2410 refractive index detector and Waters Styragel HR1DMF & Styragel HR4 DMF. DMF was used as the eluent at the flow rate of 0.3 mL/min. Waters Millennium module software was used to calculate molecular weight on the basis of a universal calibration curve generated by narrow molecular weight distribution poly(ethylene glycol) standard. 2.5. Fluorescence measurement In order to determine the critical micelle concentration (CMC) of diblock copolymers, 9-chloromethyl anthracene was used as fluorescent probe [20]. Stock solutions were prepared by dissolving 25 mg mPEG–PLA in 1 mL THF. Four milliliter distilled water was added with agitation, and THF was removed by rotary evaporator at 20 ◦ C. This solution was distilled to 25 mL. Subsequently, the 1.0 g/L stock solution was gradually diluted to ten concentration points and final polymeric solution was 1.0–10−5 g/L. To get sample solution, calculated amount of 9-chloromethyl anthracene in CH2 Cl2 solution was added to 10 mL volumetric flask and the solvent evaporated. The stock solution was added to the flask,
106
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
shaken for 3 h at 60 rpm at 37 ◦ C, and cooled overnight to 20 ◦ C. The final concentration of the probe was 6.0 × 10−6 M. Fluorescence spectra were recorded on Shimadzu RF-5301 PC spectrophotometer. Excitation wavelength was 370 nm, emission wavelength was 416 nm, and slit width was 5 nm for measurement. 2.6. Transmission electron microscopy measurement A drop of micelles suspension containing 2 wt.% phosphotungstic acid was placed on copper grid with Formvar® film and dried before measurement by JEOL JEM-100CXII transmission electron microscope (TEM) at an acceleration voltage of 80 keV. 2.7. Size distribution and zeta potential measurement Micelle size in aqueous solution was measured by dynamic light scattering at 90◦ to the incident beam and 20 ◦ C on a Beckman Coulter N4 Plus submicron Particle Size Analyzer. Zeta potential was determined by Zetasizer 3000HS (Malvern Instrument). 2.8. Wide-angle X-ray diffraction measurement Wide-angle X-ray powder diffraction was carried out on Shimadzu XRD-6000 X-ray diffractometer by Ni-filtered Cu K␣ radiation (40 kV, 30 mA) with 4◦ /min scanning rate at room temperature. 2.9. In vitro drug release studies Polymeric micelles 20 mg and 2 mL PBS (0.1 M, pH 7.4) were introduced into dialysis tube that immersed into 30 mL PBS solution. The system was shaken at 60 rpm at 37 ◦ C. At predetermined interval, 3 mL PBS solution was taken out and each sample was measured three times and their mean values were used. Accumulative release weight of MTX was calculated according to the calibration curve.
3. Results 3.1. Characterization of amphiphilic diblock copolymers Fig. 2 showed synthetic scheme and the structure of mPEG–PLA copolymers. Various chain lengths of PLA in the copolymers were obtained by modulating the feed ratio of mPEG and d,l-lactide. Copolymers structure was confirmed by 1 H NMR spectrum measurements (Fig. 3): the peaks at 3.63 and 3.48 ppm corresponded to methylene units and CH3 O- in the mPEG blocks, signals for PLA segments were 1.59 and 5.20 ppm for CH3 - and CH-groups, respectively. From the peak integrity ratio of their methylene and methyl groups, the molar ratio of repeating units in mPEG and PLA blocks can be calculated. The value was very close to that of feed composition (Table 1). Number- and weight¯ n and M ¯ w , respectively) of the average molecular weight (M copolymers were also listed in Table 1. 3.2. Critical micelle concentration Critical micelle concentration (CMC) of diblock copolymers was determined by fluorescence spectrum and 9chloromethyl anthracene was used as fluorescent probe. In the emission spectra (Fig. 4a, emission wavelength was 416 nm), fluorescence intensity was enhanced with increasing polymer concentration and red shift (from 410 to 416 nm) [21] was also present with increasing polymer concentration. As shown in Fig. 4b, at low concentration fluorescence intensity increased slowly and almost was a constant and then increased sharply due to the formation of micelles. 3.3. Preparation and characterization of polymeric micelles Core-shell type polymeric micelles were prepared by dialysis method. Size and size distribution of micelles were measured by dynamic light scattering. The mean diameters of polymeric micelles were 50–200 nm with narrow
Fig. 2. Synthetic scheme of mPEG–PLA amphiphilic diblock copolymers.
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
107
Fig. 3. 1 H NMR spectrum of PEL-2 in CDCl3 . Table 1 Copolymerization of d,l-LA with mPEG at various contents catalyzed by Sn(Oct)2 at 150 ◦ C for 16 h Sample
PLA PEL-1 PEL-2 PEL-3 PEL-4 a b c
mPEG (g)
– 0.0280 0.0787 0.1931 0.4377
d,l-LA (g)
mPEG content (wt.%)
0.4239 0.4326 0.4349 0.4321 0.4343
In feed
In the
– 6.1 15.3 30.9 50.2
– 5.0 17.7 35.7 53.4
MNMR a (×10−4 )
Mn b (×10−4 )
¯ w /M ¯ nb M
Yield (%)
– 10.1 2.8 1.4 0.9
6.2c 6.6 2.7 1.3 0.7
1.30 1.43 1.39 1.29 1.08
95.5 80.2 82.0 75.9 74.5
copolymera
Calculated by 1 H NMR. Determined by GPC with DMF as the eluent. Determined by GPC with chloroform as the eluent.
polydispersity (Table 2 ). Micelle sizes with the presence of MTX were bigger about 10–20 nm than that of the absence of MTX. Zeta potential was measured by Laser Doppler Anemometry and all micelles had negative zeta potential (from −15 to −7 mV). The morphology of micelles was observed by Transmission electron microscopy. As shown in Fig. 5 , these micelles were spherical in shape and the granulity was equal. Crystallization of MTX-loaded micelles was evaluated by X-ray powder diffraction. As shown in Fig. 6, the patterns
between micelles and MTX-loaded micelles were identical; however, physical mixture of MTX and micelles had extra peaks at 23◦ . Drug loading content and entrapment efficiency of polymeric micelles were calculated by absorbance of MTX at 303 nm from UV. The results were also summarized in Table 2. There was no interference from the copolymers at this wavelength. Drug loading content and entrapment efficiency depend on the composition of the mPEG–PLA copolymers and the highest loading efficiency was 47%.
Table 2 Micelle size, drug loading content, loading efficiency, and zeta potential of the polymeric micelles Sample
Loading content (%)
Loading efficiency (%)
Micelle size with the absence of MTX (nm)
PEL-1 PEL-2 PEL-3 PEL-4
12.8 6.4 4.2 3.7
47.3 34.8 22.2 17.4
180.1 121.4 71.3 42.6
± ± ± ±
16.7 19.8 22.5 13.3
Micelle size with the presence of MTX (nm) 200.6 134.7 86.0 50.1
± ± ± ±
40.6 21.2 17.8 19.7
Zeta potential (mV) −15.1 −12.3 −9.5 −7.0
± ± ± ±
1.7 1.9 1.3 1.5
108
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
Fig. 6. X-ray diffraction patterns for (a) PEL-2 micelles, (b) MTX-loaded micelles, (c) physical mixture of micelles with MTX (MTX 10 wt.%), and (d) MTX.
Fig. 4. (a) Fluorescence emission spectra of 9-chloromethyl anthracene with increasing concentrations of PEL-2. (b) Plot of intensity vs. log C for PEL-2.
3.4. In vitro MTX release behavior of polymeric micelles Fig. 7. Release curves of MTX from polymeric micelles.
In vitro release behavior of MTX was carried out in PBS (0.1 M, pH 7.4) at 37 ◦ C. The influence of various PLA length incorporated into the copolymers on release rate was evaluated. As shown in Fig. 7, there was no burst effect during
initial period and MTX was continuously released from these micelles. PEL-4 micelles had fastest release rate and less than 50% MTX was released in 5 days.
4. Discussion
Fig. 5. TEM image of PEL-4 micelles loaded with MTX.
A series of mPEG–PLA copolymers with various PLA chain lengths were synthesized. The relationship between chemical structure and physicochemical properties of obtained micelles and release behavior was investigated. Polymeric micelles were prepared by dialysis technique. In this method, polymer and drug are dissolved in the organic solvent, which is completely dissolved in the external aqueous phase, and then the solution diffuses instantaneously to the external aqueous phase, followed by precipitation of the polymer and drug. The advantage of this method is that no surfactant is employed. Initial diblock copolymeric concentration will influence micelle size. When polymeric concentration is bigger and the solution is more viscous,
Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 104–109
macromolecule diffusion is restrained and larger micelles are obtained. In the case of our experiment, polymeric concentration during micelle preparation was 5 mg/mL. As shown in Table 2, when mPEG molecular weight was constant, polymeric micelle sizes enhanced as increasing PLA chain length. It is difficult to form compact polymeric micelles for amphiphilic copolymers with longer hydrophobic chain length. This result was in agreement with the characteristic of amphiphilic copolymer micelles that the fewer the hydrophobic component, the smaller the micelles. On the other hand, micelle sizes with the presence of MTX were bigger about 10–20 nm than that of the absence of MTX. The presence of PEG can shield surface ion charge of the micelles. All micelles had negative zeta potential. The absolute value of zeta potential reduced as increasing PEG content in the copolymers. Due to negative charge on the surface of the micelles, ion intensity and pH value of the medium during preparation process may influence micelle size. When ion intensity and acidity value of the medium were reduced, micelle size enhanced due to intermicellar aggregation. In our experiment, the aqueous medium was neutral. The physical state of both the drug and polymeric micelles can also influence release behavior of the drug. Interaction between MTX and hydrophobic chain in the core and crystallization of MTX in the micelles was evaluated by X-ray powder diffraction. From X-ray diffraction patterns (Fig. 6), it is suggested that MTX was totally encapsulated into polymeric micelles and dispersed molecularly. In vitro drug release behavior of polymeric micelles was also dependent on the chemical composition of polymeric micelles. As shown in Fig. 7, sustained release was obtained and release rate reduced as enhancing PLA chain length. It is stemming from stronger interaction between poorly watersoluble MTX and longer hydrophobic PLA chain length.
5. Conclusions A series of mPEG–PLA copolymers with various PLA chain lengths were synthesized. Micelles based on amphiphilic mPEG–PLA copolymers were prepared with free surfactant. Anticancer drug MTX was loaded into inner core of polymeric micelles due to hydrophobic interaction. Physicochemical characteristics of micelles were varying by changes in molecular weight of the copolymers. When molecular weight of mPEG was constant, changes in molecular weight of the copolymers can modulate
109
hydrophilic/hydrophobic block ratio. Relationship between physicochemical characteristics of micelles and release behavior was explored. mPEG–PLA micelles proposed in this paper had desired in vitro drug release kinetics. In initial period there was no burst effect and less than 50% MTX was released in 5 days.
Acknowledgments The authors are grateful for the financial support from the National Key Basic Research and Development Program (G1999064703) of China and a grant (29934060) from the National Natural Science Foundation of China.
References [1] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Science 263 (1994) 1600. [2] K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev. 47 (2001) 113. [3] G.S. Kwon, Crit. Rev. Ther. Drug Carrier Syst. 20 (2003) 357. [4] P.D. Scholes, A.G.A. Coombes, L. Illum, S.S. Davis, M. Vert, M.C. Davies, J. Control. Rel. 25 (1993) 145. [5] E.S. Lee, K. Na, Y.H. Bae, J. Control. Rel. 91 (2003) 103. [6] Y. Matsumura, M. Yokoyama, K. Kataoka, T. Okano, Y. Sakurai, T. Kawaguchi, T. Kakizoe, Jpn. J. Cancer Res. 90 (1999) 122. [7] P. Calabresi, R.E. Parks, The Pharmacological Basis of Therapeutics, Macmillan, New York, 1975, p. 1254. [8] P. Calabresi, B.A. Chabner, in: L.S. Goodman, A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, Macmillan, New York, 1991, p. 1202. [9] Y. Li, G.S. Kwon, Pharm. Res. 17 (2000) 607. [10] H. Kang, J.D. Kim, S.H. Han, I.S. Chang, J. Control. Rel. 81 (2002) 135. [11] C.L. Lo, K.M. Lin, G.H. Hsiue, J. Control. Rel. 104 (2005) 477. [12] H. Arimura, Y. Ohya, T. Ouchi, Biomacromolecules 6 (2005) 720. [13] Y. Dong, S.S. Feng, Biomaterials 25 (2004) 2843. [14] A. Vila, A. Sanchez, C. Evora, I. Soriano, J.L. Vila Jato, M.J. Alonso, J. Aerosol. Med. 17 (2004) 174. [15] Y. Nagasaki, K. Yasugi, Y. Yamamoto, A. Harada, K. Kataoka, Biomacromolecules 2 (2001) 1067. [16] K. Emoto, Y. Nagasaki, M. Iijima, M. Kato, K. Kataoka, Colloids Surf. B 18 (2000) 337. [17] X. He, X.K. Guo, D.S. Zheng, J.H. Zhang, Chin. Fine Chem. 21 (2004) 745. [18] Y. Yamamoto, K. Yasugi, A. Harada, Y. Nagasaki, K. Kataoka, J. Control. Rel. 82 (2002) 359. [19] C.L. Sang, K. Chulhee, C.K. Ick, C. Hesson, Y.J. Seo, J. Control. Rel. 89 (2003) 437. [20] C. Li, X. Liu, L.Z. Meng, Polymer 45 (2004) 337. [21] Y. Zhang, R.X. Zhuo, Biomaterials 26 (2005) 2089.