Biomacromolecules 2002, 3, 1115-1119
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Biodegradable Polymeric Nanospheres Formed by Temperature-Induced Phase Transition in a Mixture of Poly(lactide-co-glycolide) and Poly(ethylene oxide)-Poly(propylene oxide)-Poly(ethylene oxide) Triblock Copolymer Ka Eul Lee, Byoung Ki Kim, and Soon Hong Yuk* Department of Polymer Science and Engineering, Hannam University, 133 Ojeong Dong, Daedeog Ku, Taejeon 306-791, Korea Received June 5, 2002
The mixture of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (F-127) and PLGA (poly(lactide-co-gycolide)) forms a liquid state above their phase transition temperatures, and the phase-separated state is induced by decreasing the temperature below the phase transition temperature. On the basis of the temperature-induced phase transition behavior in the mixture of F-127 and PLGA, a novel method for the preparation of drug-loaded PLGA nanospheres was designed and characterized by measuring the loading amount, the encapsulation efficiency, and the drug release pattern. Paclitaxel, used as a potent anticancer drug, was selected as a model drug. Introduction Polymeric nanospheres have attracted a lot of attention because of their scientific and technological interest.1,2 Especially, biodegradable polymeric nanospheres have recently attracted considerable attention as a drug delivery vehicle. Numerous scientists have reported on the formation of biodegradable polymeric nanospheres composed of poly(lactide-co-glycolide) (PLGA)3,4 and microparticles containing protein with a form of PLGA/Pluronic (poly(ethylene oxide) (PEO)-polypropylene oxide (PPO)-poly(ethylene oxide) (PEO) triblock copolymer) blend to achieve uniform release characteristics and improved protein delivery.5 In the conventional preparation of PLGA nanospheres or microspheres based on the emulsification/solvent evaporation technique,6,7 methylene chloride, ethyl acetate, or a mixture of methylene chloride and methanol (or acetone) was used to form the oil phase containing PLGA. Although the solvent is evaporated during the preparation, special care should be taken to remove the residual solvent completely for the clinical application of PLGA nanospheres. In this study, a novel method for the preparation of PLGA nanospheres has been designed without using solvents and characterized based on the temperature-induced phase transition in the liquidized mixture of PLGA and Pluronic. PLGA with molecular weight of 100 000 or less exhibits the glass transition between 50 and 90 °C depending on the molecular weight and changes into liquid state above its glass transition temperature. Pluronic also exhibits the melting transition in the similar temperature range depending on PEO/PPO ratio. On the basis of these characteristics, the liquidized mixtures of PLGA and F-127 (one of the Pluronic copolymers) were * To whom all correspondence should be addressed.
prepared above the transition temperatures and the formation of microstructures of F-127/PLGA mixtures were investigated during the temperature-induced phase transition. We have identified that the microstructure of F-127/PLGA mixture can lead to the formation of PLGA nanospheres. For the application as a drug delivery vehicle, paclitaxel, used as a potent anticancer drug, was loaded into the PLGA nanospheres and the loading amount, the encapsulation efficiency, and the release pattern from the nanospheres were measured. Materials and Methods Materials. F-127 was obtained as a gift from BASF Corp., Korea, and used as received. F-127 can be represented by the formula (EO)100(PO)65(EO)100 on the basis of its nominal molecular weight of 12 600 and 75% PEO content. DL-Poly(glycolide-co-lactide) (PLGA) (75 mol % of lactide, molecular weight 90 000) was purchased from Boelinger Ingelheim (Germany). Tween 80 and paclitaxel were purchased from Sigma Co. (USA). Differential Scanning Calorimetry (DSC) Measurements. DSC experiments were performed using a DSC-2910 (TA Instruments, USA). Nitrogen was used as the purge gas at a flow rate of 50 mL/min for the DSC cell. All experiments were performed using nonhermetic aluminum pans. The DSC parameters were a heating rate of 2.5 °C/min, an amplitude of (1 °C, and a period of 60 s. Prior to the start of the DSC run, an equilibration step at 120 °C for 10 min was included to remove any residual moisture in the sample. Experiments were performed in duplicate. Preparation of PLGA Nanoshperes. F-127 is in a flaky state and PLGA is in a powdery state at room temperature.
10.1021/bm020066h CCC: $22.00 © 2002 American Chemical Society Published on Web 07/13/2002
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Table 1. Preparation and Properties of PLGA Nanospheresa
sample (3/7) (w/w) F-127/PLGA mixture (5/5) (w/w) F-127/PLGA mixture (6/4) (w/w) F-127/PLGA mixture (7/3) (w/w) F-127/PLGA mixture (8/2) (w/w) F-127/PLGA mixture (9/1) (w/w) F-127/PLGA mixture a
loading amount (wt %)
encapsulation efficiency (wt %)
PLGA nanospheres were not formed PLGA nanospheres were not formed 5.7 ( 0.9 20.7 ( 1.4 9.1 ( 1.4 21.2 ( 0.9 10.8 ( 1.2 28.4 ( 1.2 7.4 ( 1.1 12.3 ( 1.7
The experiments were performed three times.
As presented in Table 1, the mixtures were prepared individually by weighing 85 mg of polymers with various ratios of F-127 and PLGA, 6 mg of paclitaxel, and 3 mg of Tween 80 into 20 mL vials, which were immediately put into a vacuum oven at 60 °C. Within 30 min, both polymers were liquidized completely to form a transparent polymer solution. The melted mixtures were transferred to a vacuum oven at 25 °C to induce the phase transition (the solidification of liquidized polymer mixture). With the phase transition, PLGA formed the phase-separated domain with spherical form and F-127 formed the continuous phase. Following the equilibration period for 3 h, the solidified mixture was withdrawn from the vacuum oven and immersed in distilled and deionized water for 20 h to solubilize F-127 using a dialysis bag (molecular weight cutoff range 12000-14000, Fisher Scientific, USA), replacing the distilled and deionized water every hour. With solubilization of the F-127 phase in the water, paclitaxel-loaded PLGA nanospheres were suspended into the aqueous medium containing solubilized F-127 and unloaded paclitaxel. This polymer aqueous solution was centrifuged for 20 min and filtered through a 0.45 µm filter membrane. The obtained PLGA nanospheres were freeze-dried. Figure 1 describes the preparation method of nanospheres schematically. Particle size distribution of PLGA nanospheres was analyzed using electrophoretic light scattering 8000 (Otsuka Electronics). All measurements were repeated three times. Scanning Electron Microscope (SEM) Measurements. The phase-separated state of F-127/PLGA mixture and PLGA nanospheres were examined by scanning electron microscopy (SEM) (model 2250N, Hitachi, Japan). The phase-separated F-127/PLGA mixtures were prepared by inducing the temperature-induced phase transition (solidification) of liquid mixture of F-127/PLGA in a vacuum oven at 25 °C. The PLGA nanospheres were freeze-dried for SEM measurement. The samples were gold deposited in a vacuum and examined with a tilt angle of 45°. Drug Loading Amount and Release Pattern. For the determination of loading amount, 10 mg of freeze-dried polymeric nanospheres was dissolved in 20 mL of methylene chloride. One milliliter of solution was withdrawn and immediately filtered through a 0.45 µm membrane filter. Subsequently, this was added to 3 mL of a mixture of acetonitile-water (50/50, v/v). Evaporation of methylene chloride continued until a clear solution was carried out under a stream of nitrogen. Paclitaxel was determined by reversedphase high-performance liquid chromatography (HPLC) using a symmetry C18 column and an acetonitrile-water (50/ 50 v/v %) mobile phase over 20 min at a flow rate of 1
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mL/min. The elute was monitored by UV absorption at 228 nm. Drug loading amount is defined as the ratio of the amount of drug in the nanospheres/the total weight of nanospheres and encapsulation efficiency is defined as the ratio of the amount of drug in the nanospheres/the total amount of drug used in the preparation of nanospheres. For measurement of the release pattern of paclitaxel from the nanospheres, 20 mg of freeze-dried polymeric nanospheres was put into dialysis bag, which was immersed in 500 mL of phosphate-buffered solution (PBS, pH 7.4) containing 0.1% (w/v) Tween 80. Temperature was maintained at 37 °C, and stirring was maintained at 600 rpm. At given time intervals, 5 mL aliquots were withdrawn from release medium (PBS) and 0.5 mL of methylene chloride was added to the aliquot for extraction followed by adding 1.5 mL of the mixture of acetonitile-water (50/50, v/v). Evaporation of methylene chloride continued until a clear solution was carried out under a stream of nitrogen. HPLC analysis can then be done as previously described. To maintain the sink condition in the release medium, 5 mL of fresh PBS was added to release medium after sampling. Results and Discussion Differential scanning calorimetry was used to observe the transition of polymers and polymer mixture as shown in Figure 2. PLGA used in this study exhibited the glass transition at around the 49.95 °C and changed into liquid state above its transition temperature. F-127 in this study exhibited the melting transition at around the 57.05 °C, which was presented previously.8 The DSC curve for the mixture ((8/2) (w/w) F-127/PLGA) showed the small melting point depression expected for a polymer/polymer mixture. For inducing the phase separation between PLGA and F-127, the temperature of liquid mixture was decreased to 25 °C and the solidification of liquid mixture was observed within 3 h. This is the expected result of crystallization of PEO block of F-127.9,10 In the course of the melting and solidification of mixture, external agitations such as sonication or homogenization were not applied to the mixture for homogeneous mixing. To verify the phase separation between PLGA and F-127, the solidified (8/2) (w/w) F-127/ PLGA mixture was examined by scanning electron microscope. The separated phase of PLGA with spherical shape was observed as shown in Figure 3. As presented in Table 1, the formation of phase-separated state became dominant above the 60 wt % of F-127 content in the F-127/PLGA mixture and the maxima in the loading amount and encapsulation efficiency were achieved at 80 wt % of F-127 content. Therefore, an (8/2) (w/w) F-127/ PLGA mixture was used in the preparation of nanospheres throughout the experiment. According to the preparation method of PLGA nanospheres, the solidified mixtures were solubilized in the distilled and deionized water and filtered. As shown in Figure 4, PLGA nanospheres with diameters of 200-500 nm were obtained from the (8/2) (w/w) F-127/PLGA mixture. This indicates that the microstructure of the (8/2) (w/w) F-127/ PLGA mixture plays an important role in the formation of nanospheres.
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Biodegradable Polymeric Nanospheres
Figure 1. Schematic description of preparation method of PLGA nanosphere.
Figure 2. DSC heat flow curves.
Figure 3. SEM picture of phase-separated (8/2) (w/w) F-127/PLGA mixture.
On the basis of the temperature-induced phase behavior in the liquidized mixture of F-127 and PLGA, a novel method for the preparation of PLGA nanospheres has been designed and the formation of nanospheres has been demonstrated. For application as drug delivery vehicle, paclitaxel was loaded into the nanospheres. For drug loading, the drug was mixed with a polymer mixture in the powdery state and the transparent liquid state was prepared by temperature-induced
Figure 4. (a) SEM picture of PLGA nanosphere prepared from (8/2) (w/w) F-127/PLGA mixture. (b) Size distribution of PLGA nanosphere prepared from (8/2) (w/w) F-127/PLGA mixture.
phase transition. Paclitaxel was loaded efficiently with approximately 10 wt % of drug loading amount. Zhang et al. reported that the ability to solubilize paclitaxel using polylactide-poly(ethylene oxide) dilblock copolymer was decreased with the increase of poly(ethylene oxide) content.11
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Figure 5. Size distribution of PLGA nanosphere suspended in the PBS for 7 days.
This indicates that paclitaxel interacts strongly with the polylactide segment. PLGA used in this study contains 75 mol % of lactide and is very hydrophobic. F-127 is a watersoluble polymer and contains 75 mol % of poly(ethylene oxide) in its backbone. Although paclitaxel exists both in the PLGA and F-127 phase in the liqudized state of polymer mixture, paclitaxel preferably partitions into more hydrophobic phase of PLGA nanospheres up to 10 wt % because of its hydrophobic character of paclitaxel (the solubility in water is 5.11 µg/mL). The generally accepted dose of paclitaxel is 200-250 mg and is given as an infusion for 3 and 24 h.12-14 Terminal half-life was found to be in the range of 1.3-8.6 h (mean 5 h).12-14 Because of low solubility in the aqueous media, the various approaches have been made to improve the solubility. Recently, it has been formulated in a vehicle composed of a 1:1 blend of Cremophor (polyethoxylated caster oil) and ethanol which is diluted with 5-20-fold in normal saline or dextrose solution (5 wt %) for administration. But the ethanol/Cremophor vehicle is reported to be toxic and has only short-term physical stability as some particles slowly tend to precipitate out of the aqueous media.14 The paclitaxel-loaded nanospheres in this study can be suspended in the aqueous media (4.54 mg/mL) by sonication for 10 s. The particle size analysis was performed to observe the stability of nanospheres in the aqueous media using the nanospheres suspended in the PBS for 7 days. As shown in Figure 5, there was a minimal change in the particle size and the formation of aggregation was not observed. These indicate that the nanospheres in this study can provide a long circulating carrier for paclitaxel delivery without using organic solvent. However, it was hard to detect a few large paclitaxel particles. For clinical application, a microscopic evaluation is required. Figure 6 shows the release pattern of paclitaxel from the nanospheres, which are formed from a (8/2) (w/w) F-127/ PLGA mixture. An almost zero-order release pattern was observed for 60 days, and more than 80 wt % of loaded paclitaxel was released. The release rate and percent of released drug in this study are rather higher than those in
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Figure 6. The release pattern of paclitaxel from the nanosphere. (The number of experiments is three.)
the previous report.15 There are three primary mechanisms for the loaded drug to be released from PLGA nano- or microspheres: swelling, diffusion, and degradation.16 Because of the nanosize of the delivery vehicle in this study, swelling by nanospheres after being exposed to release medium took place much faster and this led to the acceleration in the diffusion of paclitaxel and erosion of PLGA matrix in the nanospheres. Conclusions With the temperature-induced phase transition from the liquidized mixture of F-127 and PLGA to a solidified one, the phase-separated state was observed. On the basis of this characteristic, the formation of PLGA nanospheres was demonstrated without using a toxic organic solvent, which was used in the conventional method. For application as a drug delivery carrier, a potent anticancer drug, paclitaxel, was loaded into the nanospheres. Because of favorable hydrophobic interaction between paclitaxel and PLGA, paclitaxel was loaded efficiently into the PLGA nanospheres. Using paclitaxel-loaded nanospheres, the stability of paclitaxel in the aqueous medium was improved significantly. By consideration of the size of nanospheres, the possibility of the passive targeting to the specific tumor cells is under study based on the enhanced permeation and retention (EPR) effect.17-19 Acknowledgment. This work was supported in part by a grant from the Korea Research Foundation (2000-041E00573) and the Ministry of Commerce, Industry and Energy of Korea. References and Notes (1) Henselwood, F.; Liu, G. Macromolecules 1997, 30, 488. (2) Wang, G.; Henselwood, F.; Liu, G. Langmuir 1998, 14, 1554. (3) Coombes, A. G.; Scholes, P. D.; Davies, M. C.; Illum, L.; Davis, S. S. Biomaterials 1994, 15, 673. (4) Birnbaum, D. T.; Kosmala, J. D.; Henthorn, D. B.; Brannon-Peppas, L. J. Controlled Release 2000, 65, 375.
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Biodegradable Polymeric Nanospheres (5) Yeh, M. K.; Davis, S. S.; Coombes, A. G. A. Pharm. Res. 1996, 13, 1693. (6) Gautier, S.; Grudzielski, N.; Goffinet, G.; de Hassonville, S. H.; Delatti, J. R. J. Biomater. Sci., Polym. Ed. 2001, 12, 429. (7) Carrasquillo, K. G.; Stanley, A. M.; Aponte-Carro, J. C.; De Jesus, P.; Costantino, H. R.; Bosques, C. J.; Griebenow, K J. Controlled Release 2001, 76, 199. (8) Miyazaki, S.; Yokouchi, C.; Nakamura, T.; Hashiguchi, N.; Hou, W.-M. Chem Pharm. Bull. 1986, 34, 1801. (9) Fairclough, J. P. A.; Yu, G.-E.; Mai, S.-M.; Crothers, M.; Mortensen, K.; Ryan, A. J.; Booth C. Phys. Chem., Chem. Phys. 2000, 2, 1503. (10) Hamley, I. W.; Castello, V.; Yang, Z. Price, C,; Booth, C. Macromolecules 2001, 34, 4079. (11) Zhang, X.; Jackson, J. K.; Burt H. M. Int. J. Pharm. 1996, 132, 195. (12) Singla, A. K.; Garg, A.; Aggarwal, D. Int. J. Pharm. 2002, 235, 179.
(13) (14) (15) (16)
Rowinsky, E. K.; Donhover, R. C. Semin. Oncol. 1993, 20, 16. Dorr, R. T. Ann. Pharmacother. 1994, 28, 511. Mu, L.; Feng, S. S. J. Controlled Release 2001, 76, 239. Schwendeman, S. P.; Cardamore, M.; Klibanov, A.; Langer, R. In Microparticulate Systems For the DeliVery of Proteins and Vaccines; Cohen, S., Bernstein, H., Eds.; Marcel Dekker: New York, 1996, pp 1-49. (17) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. J. Controlled Release 1993, 24, 119. (18) Maeda H.; Ueda M.; Morinag T.; Matsumoto T. J. Med. Chem. 1985, 28, 455. (19) Maeda H.; Matsumura Y. CRC Crit. ReV. Ther. Drug Carrier Syst. 1989, 6, 193.
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