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vitamin E TPGS copolymer for cancer chemotherapy: Synthesis, formulation, characterization and in vitro drug release
ARTICLE IN PRESS
Biomaterials 27 (2006) 262–270 www.elsevier.com/locate/biomaterials
Nanoparticles of poly(lactide)/vitamin E TPGS copolymer for cancer chemotherapy: Synthesis, formulation, characterization and in vitro drug release Zhiping Zhanga, Si-Shen Fenga,b, a
Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore b Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 3 April 2005; accepted 31 May 2005 Available online 18 July 2005
Abstract Paclitaxel is one of the best anticancer drugs, which has excellent therapeutic effects against a wide spectrum of cancers. The formulation of paclitaxel used in its currently clinical administration includes Cremophor EL, which has been found to cause serious side effects. Nanoparticle formulation of paclitaxel may provide an ideal solution for this problem and achieve a sustained chemotherapy. A novel copolymer, poly(lactide)-vitamin E TPGS (PLA-TPGS), was synthesized from lactide and d-a-tocopheryl polyethylene glycol 1000 succinate by bulk polymerization for nanoparticle formulation of anticancer drugs. 1H NMR, FTIR and GPC were used to detect molecular structure of the copolymer. Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation technique with or without emulsifier involved, which were characterized by laser light scattering for size and size distribution; field emission scanning electron microscopy for surface morphology; zeta potential for surface charge; X-ray photoelectron spectroscopy for surface chemistry. The drug encapsulation efficiency and the in vitro drug release kinetics were measured by high-performance liquid chromatography. Formulation optimization was pursued. The particles were found of around 300 nm in size and narrow size distribution. Of all, 89% drug encapsulation efficiency has been achieved for nanoparticles of 5% drug loading. The drug release from PLA-TPGS nanoparticles was found to be biphasic with an initial burst of 17% in the first day, followed by a sustained pattern with 51% release after 31 days. r 2005 Elsevier Ltd. All rights reserved. Keywords: Biodegradable polymers; Cancer nanotechnology; Chemotherapy; Paclitaxel; Taxols
1. Introduction Nanoparticle formulation of anticancer drugs has become an important research area in cancer nanotechnology, which can provide a way of sustained, controlled and targeted drug delivery to improve the therapeutic effects and reduce the side effects of the formulated drugs. Such drug delivery systems are Corresponding author. Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore. Tel.: +65 6874 3835; fax: +65 6779 1936. E-mail address: [email protected] (S.-S. Feng).
0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.05.104
usually restricted by biocompatibility of the polymeric matrix material and the surfactant used in the formulation process. Poly (lactide) (PLA), poly (d,l-lactide-coglycolide) (PLGA), and poly (caprolactone) (PCL) are FDA-approved biodegradable polymers, which are used most often in the literature of drug delivery. These polymers were originally synthesized to be used as surgical sutures, which thus have disadvantages to be used for drug formulation such as too high hydrophobicity and too slow degradation. Novel biodegradable polymers/copolymers with desired hydrophobic/ hydrophilic balance and desired degradation rate are thus needed. In the literature, PLGA nanoparticles were usually prepared by using chemical emulsifiers such as
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poly (vinyl achohol) (PVA), which has been found of disadvantages including low emulsification efficiency, side effects and difficulties to wash away in the formulation process. Instead, d-a-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS) has high emulsification efficiency (67 times higher than PVA). It can also greatly improve the drug encapsulation efficiency (up to 100% EE achieved) and enhance cellular uptake of nanoparticles and thus increase the cancer cell mortality [1–5]. TPGS is a water-soluble derivative of natural vitamin E. Its hydrophile–lipophile balance is 13. The chemical structure of TPGS is similar to other amphiphiles comprising lipophilic alkyl tail and hydrophilic polar head portion. Its bulky structure and large surface area characteristics make it an excellent emulsifier. Moreover, it has been found that co-administration of vitamin E TPGS could enhance cytotoxicity, inhibit Pglycoprotein mediated multi-drug resistance, and increase the oral bioavailability of anticancer drugs [6–8]. This triggered us to take such advantages of TPGS to synthesize PLA-TPGS copolymers for nanoparticle formulation of anticancer drugs, which can be expected to have self-emulsification effects (no emulsifiers are needed for the nanoparticles formulation) and achieve high drug encapsulation efficiency and desired drug release profiles. In this research, PLA-TPGS copolymers were synthesized by ring-opening polymerization. The synthesized copolymer was characterized by Fourier transform infrared spectroscopy (FTIR) for its molecular structure. The TPGS content and number average molecular weight of the synthesized PLA-TPGS copolymers were determined by 1H NMR. The weight-averaged molecular weight and its distribution was determined by gel permeation chromatography (GPC). Paclitaxel, one of the best antineoplastic drugs found from nature in past decades, was chosen as a prototype drug in this research due to its excellent therapeutic efficacy against a wild spectrum of cancers and its great success as the best seller among various anticancer drugs. Like many other anticancer drugs, paclitaxel has formulation problem, which is caused by its high hydrophobicity (water solubility less than 0.5 mg/L). The popular dosage form used in its current clinical administration is Taxols formulated in ajuvant Cremophor EL, which has been found to be responsible for serious side effects including hypersensitivity reactions, nephrotoxicity, cardiotoxicity and neurotoxicity [9–13]. Paclitaxel-loaded nanoparticles of the PLA-TPGS copolymer were fabricated by a modified solvent extraction/evaporation technique, which were then characterized by various state-of-theart techniques such as laser light scattering (LLS) for size and size distribution; field emission scanning electron microscopy (FESEM) for surface morphology; zeta potential for surface charge and X-ray photoelec-
263
tron spectroscopy (XPS) for surface chemistry of the nanoparticles. High-performance liquid chromatography (HPLC) was used to measure the drug encapsulation efficiency (EE) and the in vitro drug release kinetics.
2. Materials and methods 2.1. Materials Lactide (3,6-dimethyl-1,4-dioxane-2,5-dione, C6H8O4) was purchased from Aldrich. It was recrystallized twice from ethyl acetate before reaction. Vitamin E TPGS (d-a-tocopheryl polyethylene glycol 1000 succinate, C33O5H54 (CH2CH2O)23) was from Eastman chemical company (USA). It was freezedried for two days before use. Stannous octoate (Sn(OOCC7H15)2) was purchased from Sigma and was used as 1% distilled toluene solution. Paclitaxel of purity 99.8% was purchased from Dabur India Ltd. (India). PLGA (50:50, MW 40–75,000) was also purchased from Sigma. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Breford, USA). All chemicals including acetonitrile, dichloromethane (DCM) and ethyl acetate were HPLC grade. They were used without further purification. 2.2. Synthesis of PLA-TPGS copolymer PLA-TPGS copolymers were synthesized by ring-opening bulk polymerization of lactide monomer with vitamin E TPGS in presence of stannous octoate as catalyst. In brief, weighted amounts of lactide, TPGS and 0.5 wt% stannous octoate (in distilled toluene) were added in an ampoule. The ampoule was evacuated in liquid nitrogen for 45 min. After that the ampoule was sealed by butane burner and reacted in silicone oil bath at 145 1C. After 12 h, the reaction product was dissolved in DCM and then precipitated in excess cold methanol to remove unreacted lactide monomers and TPGS. The final product was collected by filtration and vacuum dried at 45 1C for two days. 2.3. Characterization of PLA-TPGS copolymer The molecular structure of PLA-TPGS copolymer was investigated by FTIR (Shimadzu, Japan). The samples for FTIR analysis were prepared by grinding 99% KBr with 1% copolymer and then pressing the mixture into a transparent tablet. The TPGS content and number-averaged molecular weight of the copolymer was determined by 1H NMR in CDCl3 at 300 Hz (Bruker ACF300). The weight-averaged molecular weight and molecular weight distribution was determined by gel permeation chromatography (GPC, Agilent 1100 series GPC analysis system with RI-G1362A refractive index detector). Agilent PLgel 5 mm mixed-C 300 mm 7.5 mm column was used. The mobile phase was chloroform delivered at a flow rate of 1 ml/min. The injection volume was 50 ml sample solution (0.5% w/v copolymer in mobile phase). The calibration curve was established by using green standard polystyrene standard sample (molecular weights: 6.95 105, 5.04 104, 2.96 103 and 162 respectively).
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2.4. Preparation of paclitaxel-loaded PLA-TPGS nanoparticles Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation method [3]. A given amount of paclitaxel and 50 mg PLA-TPGS copolymer were dissolved in 4 ml methylene chloride. The formed solution was poured into 60 ml 0.03% TPGS Millipore water solution under gentle stirring. The mixture was sonicated for 120 s at 20 W. The emulsion was then evaporated overnight under magnetic stirring to remove the organic solvent. The particle suspension was centrifuged at 11,500 rpm for 15 min and then washed three times to remove the unloaded drug and surfactant. The resulted particles were resuspended in 10 ml water and freeze-dried for two days. 2.5. Characterization of drug-loaded PLA-TPGS nanoparticles 2.5.1. Particle size analysis Size and size distribution of the drug-loaded PLA-TPGS nanoparticles were measured by the LLS (Brookhaven Instruments Corporation 90-PLUS analyzer). The samples were prepared by diluting the nanoparticles suspension with deionized water until the counter rate was less than 1.5 Mpcs. 2.5.2. Surface morphology The nanoparticles were imaged by a field emission scanning electron microscopy (FESEM) system at an accelerating voltage of 10 kV. To prepare samples for FESEM, the particles were fixed on the stub by a double-sided sticky tape and then coated with platinum layer by JFC-1300 automatic fine platinum coater (JEOL, Tokyo, Japan) for 50 s. 2.5.3. Surface charge Zeta potential of the drug-loaded PLA-TPGS nanoparticles was detected by the laser Doppler anemometry (Zeta Plus zeta potential analyzer, Brookhaven corporation). The particles (2 mg) were suspended in deionized water before measurement. The data were obtained with the average of seven measurements. 2.5.4. Drug encapsulation efficiency The paclitaxel entrapped in the drug-loaded PLA-TPGS nanoparticles was measured by HPLC (Agilent LC1100). A reverse-phase Inertsils ODS-3 column (150 mm 4.6 mm, pore size 5 mm, GL science Inc, Tokyo, Japan) was used. Briefly, 3 mg nanoparticles were dissolved in 1 ml DCM and then extracted by 3 ml mobile phase (50/50(v/v) acetonitrile/ water solution). DCM was evaporated in nitrogen atmosphere and the clear solution was obtained for HPLC analysis. The solution was transferred into HPLC vial after filtered through 0.22 mm syringe filter. The flow rate of mobile phase was 1 ml/ min. Retention time was controlled at 15 min. The column effluent was detected at 227 nm with a UV/VIS detector. The calibration curve was linear in the range of 50–50,000 ng/ml with a correlation coefficient of R2 ¼ 0:9999. The drug encapsulation efficiency was defined as the ratio between the amount of paclitaxel encapsulated in nanoparticles and that added in the process.
2.5.5. Surface chemistry The XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu, Japan) was used to analyze surface chemistry of the drugloaded PLA-TPGS nanoparticles. The fixed transmission mode was utilized with pass energy of 80 eV for the survey spectrum covering a binding energy from 0 to 1100 eV. Data were analyzed with the software provided by the manufacturer. 2.5.6. In vitro drug release In all, 2 mg drug-loaded PLA-TPGS nanoparticles were put in a centrifuge tube and dispersed in 10 ml phosphate buffer solution (PBS) of pH 7.4 containing 0.1% w/v Tween 80. Tween 80 was used to increase the solubility of paclitaxel in the buffer solution and avoid the binding of paclitaxel to the tube wall. The tube was put in an orbital water bath shaking at 120 rpm at 37.2 1C. At given time intervals, the tube was taken out and centrifuged at 11,500 rpm for 15 min. The supernatant was transferred into a test tube and the pellet was resuspended in 10 ml fresh PBS solution and put back to the shaker for continuous measurement. The supernatant was extracted with 2 ml DCM and reconstituted in 1 ml mobile phase. The DCM was evaporated by nitrogen stream. The analysis procedure was similar as for the measurement of EE.
3. Results and discussions 3.1. Synthesis of PLA-TPGS copolymer The PLA-TPGS copolymer was synthesized in this research by using the stannous octoate as catalyst. The mechanism of synthesis is schematically described in Fig. 1, which is similar to that in synthesis of the MPEG-PLA copolymers [14]. The hydroxyl end of TPGS served as initiator to selectively cleave acyloxygen chain of lactide. The molecular structure of vitamin E TPGS and the PLA-TPGS copolymer are shown in Fig. 2. Fig. 3 shows the FTIR spectra of the PLA-TPGS copolymer and TPGS. The carbonyl band of TPGS appears at 1730 cm1. For the synthesized copolymer, the carbonyl band was shifted to 1755 cm1. Overlapping of the CH stretching band of PLA at 2945 cm1 and that of TPGS at 2880 cm1 was observed. In the PLA-TPGS copolymer, the CH stretching band of TPGS was decreased. The absorption band at 3400–3650 cm1 is attributed to the terminal hydroxyl group and that at 1050–1250 cm1 is due to the C–O stretching. 3.2. Molecular weight and molecular weight distribution The structure of the synthesized PLA-TPGS copolymer was detected by 1H NMR in CDCl3. Fig. 4 shows a typical 1H NMR spectroscopy of the
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3.3. Size, size distribution and drug encapsulation efficiency
Cat.
O CH3 O
O
R O H
+ R-OH O
CH3 O
Catalyst
CH3
O CH3 O O
Lactide monomer
Cat.
O
OOR TPGS
R ( O
C
HC O
O
CH3
initiation
O
CH3
C
CH ) OH
+
H CH3
O
O
CH3
CH3
+
O
(m-1)
O O
CH3 O O R ( O
C
HC O
O
CH3
C
CH )m
OH
265
where R-OH:TPGS
The size and size distribution of the paclitaxel-loaded PLA-TPGS nanoparticles prepared in this research are shown in Table 1. The drug-loaded nanoparticles can be prepared with or without extra TPGS added as surfactant in the fabrication process. This is because the TPGS component in the copolymer has a selfemulsifying function. This is an advantage of the PLATPGS copolymer in nanoparticle formulation, which avoids the side effects of the traditional chemical emulsifiers such as PVA. The particle size for all samples was found around 300 nm with narrow size distribution of less than 0.2 polydispersity. It can be observed from Table 1 that addition of TPGS in the process as emulsifier slightly increased the particle size. This is because the coating effects of TPGS on the particle surface and extra TPGS on the particle surface may cause aggregation of the formed nanoparticles.
Propagation CH3
Fig. 1. Schematic description of the synthesis mechanism of PLATPGS copolymer.
PLA-TPGS copolymer and monomers. The signals at 5.2 and 1.69 ppm were assigned to the CH protons and methyl protons CH3 of PLA segment, respectively. The peak at 3.65 ppm was assigned to the CH2 protons of PEO part of TPGS. The lower peaks in the aliphatic region belong to various moieties of vitamin E tails [15–19]. Lactide monomer peak at 5.1 ppm was not detected. The precipitation process in treatment of the copolymer can remove the TPGS and lactide monomer thoroughly. The molecular weight of the PLA-TPGS was calculated by using the ratio between the peak areas at 5.2 and 3.65. The number-averaged molecular weight of the PLA-TPGS copolymer of 12% TPGS weight content was determined to be 12,700. In GPC analysis, we confirmed that the PLA-TPGS copolymer was synthesized by the ring-opening polymerization. The product was not a physical mixture of TPGS with lactide. Fig. 5 shows that the peak for TPGS appeared at 8.9 min. Instead, the peak of the copolymer shifted to 7.6 min. There was just one narrow peak for the copolymer product and the TPGS peak could not be detected. The polydispersity of the copolymer molecular weight was narrow, around 1.31. The number-averaged molecular weight calculated from the GPC chromatograph was 13,600. It seemed that the molecular weight detected from GPC and NMR can confirm each other.
3.3.1. Drug encapsulation efficiency It has been shown that the surfactant used in the fabrication process and the drug loading level are important factors to influence the particle size and size distribution and the drug encapsulation efficiency in the nanoparticles [1,2], which then determine the drug release kinetics, cellular uptake and thus the therapeutic effects of the drug-loaded nanoparticles [20]. Table 1 also shows the drug loading and surfactant effects on size and size distribution and the drug loading efficiency. It can be seen that the PLA-TPGS nanoparticles prepared with no emulsifier added can result in a satisfactory drug EE, which was found to increase from 61.9% for 5% drug loading to 91.5% for 1% drug loading. This is natural since the higher the drug loading is, the lower the EE would be. For the high drug loading of 5%, however, the EE of the paclitaxel-loaded PLATPGS nanoparticles can be improved by adding extra TPGS in the process as emulsifier. Nevertheless, the amount of TPGS to be added should be carefully figured out. The amphipathic surfactants align themselves at the oil–water interface to promote the stability of the particles by lowering the surface energy and thus resist coalescence and flocculation of the particles. Too little emulsifier would not be enough to cover the interface and too much emulsifier would cause particle aggregation [1]. It was suggested that an appropriate amount of TPGS for PLGA nanoparticle formulation of paclitaxel would be 0.03% w/w, which achieved 100% EE [2]. Indeed, it can be seen from Table 1 that 0.03% TPGS used as an emulsifier did improve the EE of the PLATPGS nanoparticles from 61.9% to 89.1% for a high drug loading of 5%. Size and surface coating play an important role in determining the mechanism and efficiency of nanoparticle
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266 COO(CH2CH2O)nH
(CH2)2
O CH3
C O
CH3
CH3 CH3
CH3 CH3
O
CH3
CH3
(a)
1.69ppm
O 3.65ppm
CH3
C
CH ) m OH
5.2ppm
COO(CH2CH2O)n ( C
(CH2)2
O CH
O
CH3
O CH3
C O
CH3
CH3 CH3
CH3 H3C
O
CH3
CH3
(b)
Fig. 2. Molecular structure of (a) vitamin E TPGS and (b) PLA-TPGS copolymer.
and small particles (o500 nm) can cross the membrane of epithelial cells through endocytosis [21–23]. TPGS coating could greatly enhance the cellular uptake of the nanoparticles [20]. tranmittance
PLA-TPGS copolymer
TPGS
4000
3500
3000
2500 2000 1500 wave number (1/cm)
1000
500
Fig. 3. FTIR spectra of PLA-TPGS copolymer and TPGS.
absorption in the GI tract for oral chemotherapy [20]. It was suggested that large particles (o5 mm) would be taken up via the lymphatics (M Cells of Peyer’s patches)
3.3.2. Effects of polymeric matrix materials Table 2 shows the results of paclitaxel-loaded nanoparticles fabricated from the blends of the PLATPGS copolymer and PLGA with various PLATPGS:PLGA ratios. It can be found that the drug encapsulation efficiency increased with increasing the blend ratio of PLA-TPGS copolymer to PLGA. It may be attributed to the core–shell structure of the nanoparticles as found from those of the MPEG-PLA/PLGA blends [24]. The PLA-TPGS copolymer has lower molecular weight (Mn ¼ 12,700) than that of the PLGA. The mixture of the copolymer with the PLGA can decrease the viscosity of the oil phase and thus result in better emulsification, which leads to a better drug encapsulation efficiency and smaller particle size [4]. Similar results were also found for the nanoparticles of
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267
Lactide
7
6
5
4 ppm
3
2
1
0
6
5
4
3
2
1
0
Vitamin E TPGS
7
ppm
CH3
CH2
PLA-TPGS CH
7
6
Fig. 4. Typical
5 1
4 ppm
3
2
1
0
H-NMR spectra of monomers and PLA-TPGS copolymer in CDCl3.
encapsulation efficiency and particle size (data not shown). The optimal sonication time seemed to be 120 s, which resulted in smallest particle size and highest drug encapsulation efficiency. Further increase of the sonication time may cause particle aggregation and thus was not preferred. The particle size was found to be increased by 45 nm after sonication for 180 s.
8.9min
3.4. Surface morphology TPGS 7.6min PLA-TPGS copolymer
2
4
6 retention time (min)
8
10
Fig. 5. Typical GPC chromatograms of PLA-TPGS copolymer and TPGS.
Surface morphology of the drug-loaded PLA-TPGS nanoparticles was examined by FESEM. Fig. 6 shows the FESEM images of the PLGA and PLA-TPGS (12%) nanoparticles. The particles seemed to have smooth surface within the FESEM resolution level. The smooth surface may lead to a slower drug release from the nanoparticles than that from those of rough surface. The FESEM images further confirmed the particle size detected from the LLS. 3.5. Zeta potential
other blends such as that of PLLA-g-oligoEG copolymer with the PLGA [25]. 3.3.3. Effects of mechanical mixing strength Sonication strength was also found to play an important role for the nanoparticle formulation, which can significantly affect the particle recovery, drug
Zeta potential determines the particle stability in dispersion. High absolute value of the zeta potential indicates high surface charge of the nanoparticles, which leads to strong repellent interactions among the nanoparticles in dispersion and thus high stability. The zeta potential of the TPGS emulsified, paclitaxel-loaded PLA-TPGS nanoparticles was found to be about
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Table 1 Effects of drug loading and surfactant on size, size distribution and drug encapsulation efficiency of paclitaxel-loaded nanoparticle Polymer
Drug loading
Surfactant
Size
PI
EE
PLA-TPGS PLA-TPGS PLA-TPGS PLA-TPGS PLA-TPGS
1% 2.5% 5% 5% 5%
No No No 0.03%TPGS 0.06%TPGS
293.4 290.5 293.1 304.5 333.3
0.141 0.134 0.124 0.185 0.246
91.5% 74.4% 61.9% 89.1% 87.9%
*
PI ¼ Polydispersity Index, EE ¼ Drug encapsulation efficiency.
Table 2 Effects of polymeric matrix materials on size, size distribution and drug encapsulation efficiency of paclitaxel-loaded nanoparticle Polymer/5% drug
Surfactant
Size
PI
ZP
EE
PLGA PLGA:PLA-TPGS ¼ 1:1 PLA-TPGS
0.03% TPGS 0.03% TPGS 0.03% TPGS
336.8 302.9 304.5
0.248 0.105 0.185
32.7 30.6 26.3
79.9% 83.9% 89.1%
*
Drug loading: 5%.
30 mV. Similar results were found for the paclitaxelloaded PLGA nanoparticles [2,3]. 3.6. Surface chemistry The atomic composition of the PLA-TPGS copolymer and the drug-loaded PLA-TPGS nanoparticles was determined by XPS. XPS can quantitatively determine the chemical composition of the particle surface in 5–10 nm depth. The XPS spectrum of the paclitaxelloaded PLA-TPGS nanoparticles is shown in Fig. 7. It can be seen that no nitrogen element signal was detected, which means that paclitaxel was mainly distributed inside the nanoparticles. The C 1s XPS spectra of the PLA-TPGS copolymer and the paclitaxelloaded PLA-TPGS nanoparticles are shown in Fig. 8. The peak at the binding energy 286.1 eV is regarded as the indicator of the PEG component of the PLA-TPGS copolymer. From Fig. 8, the presence of TPGS on the particle surface can be evidenced by an increasing C–O–C peak ratio from 18.5% for the pure PLA-TPGS copolymer to 27.8% for the paclitaxel-loaded PLATPGS nanoparticles with no surfactant used in fabrication. 3.7. In vitro drug release Fig. 9 shows the in vitro drug release profile of the paclitaxel-loaded PLA-TPGS nanoparticles, which look biphasic with a initial burst followed by a sustained release. The drug release from the PLA-TPGS nanoparticles was found to be 17% and 51% of the encapsulated drug in the first day and after 31 days, respectively, which was much faster than the paclitaxel-
loaded PLGA nanoparticles, which is only 7% and 19%, respectively, in the same periods. This is another advantage of the PLA-TPGS nanoparticles versus the traditional PLGA nanoparticles, which were found to release the drug too slowly to meet the therapeutic needs. The faster drug release of the PLA-TPGS nanoparticles may be caused by the lower molecular weight and the higher hydrophilicity of PLA-TPGS copolymer in comparison with the PLGA nanoparticles, which causes the copolymer swell and degrade faster and thus promote the drug release from the nanoparticles. The drug release mechanism could be referred to drug diffusion, polymer matrix swelling and the polymer erosion or degradation.
4. Conclusion A novel copolymer, PLA-TPGS, was synthesized in the present study for nanoparticle formulation of anticancer drugs. Paclitaxel was used as a prototype drug due to its excellent therapeutic effects against a wide spectrum of cancers and its great commercial success as the best seller among various anticancer drugs. The paclitaxel-loaded PLA-TPGS nanoparticles were prepared by a modified solvent extraction/evaporation technique with or without assistance of emulsifiers. Paclitaxel-loaded nanoparticles can also be formulated with blends of PLA-TPGS and PLGA in a similar way. It was found that PLA-TPGS copolymer can greatly increase the drug encapsulation efficiency and achieve much faster drug release than the PLGA nanoparticles to meet the therapeutic needs. PLA-TPGS nanoparticle formulation can avoid using toxic adjuvant Cremophor
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269
1200
500 counts
N 1s
1000
450 400
counts
800
390
600
C1s
395
400
405
410
O 1s
400 N 1s
200 0 0
200
400
600
800
1000
1200
binding energy (ev) Fig. 7. XPS spectrum of paclitaxel-loaded PLA-TPGS nanoparticles. The insert shows the nitrogen signal at high resolution.
C-C/C-H
O-C=O
292
290
(a)
C-O-C=O
C-O-C
288 286 binding energy (ev)
284
282
C-C/C-H
Fig. 6. FESEM images of paclitaxel-loaded nanoparticles with 0.03% TPGS as surfactant and 5% drug loading. (a) PLGA (b) PLGA:PLATPGS ¼ 50:50 (c) PLA-TPGS.
EL. It may have great potential to enhance the cellular uptake of the drug-loaded nanoparticles as well as to promote oral chemotherapy. The technology can also be applied to other anticancer drugs.
C-O-C O-C=O
292
(b)
290
C-O-C=O
288
286
284
282
binding energy (ev)
Fig. 8. XPS spectra of (a) the PLA-TPGS copolymer and (b) the paclitaxel-loaded PLA-TPGS nanoparticles.
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270 60 55
PLA-TPGS(12%)
50
paclitaxel released (%)
45 40 35 30 25
PLGA50/50
20 15 10 5 0 0
5
10
15
20
25
30
35
released time (days)
Fig. 9. In vitro drug release profile of paclitaxel-loaded PLA-TPGS nanoparticles prepared with 0.03% TPGS as surfactant and 5% drug loading.
Acknowledgement This research was supported by NUS Grant R-379000-014-112, National University of Singapore (SS Feng, PI) and Singapore Cancer Syndicate (SCS) Grant UU0028 (NUS 397-000-606-305 in 2004 and NUS R-279-000-187-305 in 2005. SS Feng, PI). ZP Zhang is grateful of the National University of Singapore for the financial support for his PhD study.
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[6] Fischer JR, Harkin KR, Freeman LC. Concurrent administration of water-soluble vitamin E can increase the oral bioavailability of cyclosporine A in healthy dogs. Vet Tech: Res Appl Vet Med 2002;3:465–73. [7] Dintaman JM, Silverman JA. Inhibition of P-glycoprotein by Dalpha-tocopheryl polyethylene glycol 1000 succinate (TPGS). Pharm Res 1999;16:1550–6. [8] Yu L, Bridgers A, Polli J, et al. Vitamin E-TPGS increases absorption flux of an HIV protease inhibitor by enhancing its solubility and permeability. Pharm Res 1999;16:1812–7. [9] Gelderblom H, Verweij J, Nooter K, et al. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 2001;37(13):1590–8. [10] Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm 2002;235(1–2):179–92. [11] Dorr RT. Pharmacology and toxicology of Cremophor EL diluent. Ann Pharmacother 1994;28:S11–4. [12] Fjallskog ML, Frii L, Bergh J. Is Cremophor El, Solvent for Paclitaxel, Cytotoxic. Lancet 1993;342:873. [13] Weiss RB, Donehower RC, Wiernik PH, et al. Hypersensitivity Reactions from Taxol. J Clin Oncol 1990;8:1263–8. [14] Ha JC, Kim SY, Lee YM. Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronic)/poly(epsilon-caprolactone) (PCL) amphiphilic block copolymeric nanospheres—I. Preparation and characterization. J Control Release 1999;62:381–92. [15] Mosmann T. Rapid calorimetric assay for cellular growth and survival: application of proliferation and cytotoxicity assay. J Immunol Methods 1983;65:55–63. [16] Neuzil J. Vitamin E succinate and cancer treatment: a vitamin E prototype for selective antitumour activity. Br J Cancer 2003;89: 1822–6. [17] Birringer M, EyTina JH, Salvatore BA, Neuzil J. Vitamin E analogues as inducers of apoptosis: structure–function relation. Br J Cancer 2003;88:1948–55. [18] Momot KI, Kuchel PW, Chapman BE, et al. NMR study of the association of propofol with nonionic surfactants. Langmuir 2003;19:2088–95. [19] Schroder H, Netscher T. Determination of the absolute stereochemistry of vitamin E derived oxa-spiro compounds by NMR spectroscopy. Magn Reson Chem 2001;39:701–8. [20] Win KY, Feng SS. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005;26:2713–22. [21] Savic R, Luo LB, Eisenberg A, Maysinger D. Micellar nanocontainers distribute to defined cytoplasmic organelles. Science 2003;300:615–8. [22] Luo LB, Tam J, Maysinger D, Eisenberg A. Cellular internalization of poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymer micelles. Bioconjugate Chem 2002;13:1259–65. [23] Lefevre ME, Vanderhoff JW, LaIssue JA, Joel DD. Accumulation of 2-micron latex particles in mouse Peyer’s patches during chronic latex feeding. Experimentia 1978;34:120–2. [24] Dong YC, Feng SS. Methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials 2004;25:2843–9. [25] Cho KY, Choi SH, Kim CH, et al. Protein release microparticles based on the blend of poly(D,L-lactic-co-glycolic acid) and oligoethylene glycol grafted poly(L-lactide). J Control Release 2001; 76:275–84.
Biomaterials 27 (2006) 262–270 www.elsevier.com/locate/biomaterials
Nanoparticles of poly(lactide)/vitamin E TPGS copolymer for cancer chemotherapy: Synthesis, formulation, characterization and in vitro drug release Zhiping Zhanga, Si-Shen Fenga,b, a
Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore b Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 3 April 2005; accepted 31 May 2005 Available online 18 July 2005
Abstract Paclitaxel is one of the best anticancer drugs, which has excellent therapeutic effects against a wide spectrum of cancers. The formulation of paclitaxel used in its currently clinical administration includes Cremophor EL, which has been found to cause serious side effects. Nanoparticle formulation of paclitaxel may provide an ideal solution for this problem and achieve a sustained chemotherapy. A novel copolymer, poly(lactide)-vitamin E TPGS (PLA-TPGS), was synthesized from lactide and d-a-tocopheryl polyethylene glycol 1000 succinate by bulk polymerization for nanoparticle formulation of anticancer drugs. 1H NMR, FTIR and GPC were used to detect molecular structure of the copolymer. Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation technique with or without emulsifier involved, which were characterized by laser light scattering for size and size distribution; field emission scanning electron microscopy for surface morphology; zeta potential for surface charge; X-ray photoelectron spectroscopy for surface chemistry. The drug encapsulation efficiency and the in vitro drug release kinetics were measured by high-performance liquid chromatography. Formulation optimization was pursued. The particles were found of around 300 nm in size and narrow size distribution. Of all, 89% drug encapsulation efficiency has been achieved for nanoparticles of 5% drug loading. The drug release from PLA-TPGS nanoparticles was found to be biphasic with an initial burst of 17% in the first day, followed by a sustained pattern with 51% release after 31 days. r 2005 Elsevier Ltd. All rights reserved. Keywords: Biodegradable polymers; Cancer nanotechnology; Chemotherapy; Paclitaxel; Taxols
1. Introduction Nanoparticle formulation of anticancer drugs has become an important research area in cancer nanotechnology, which can provide a way of sustained, controlled and targeted drug delivery to improve the therapeutic effects and reduce the side effects of the formulated drugs. Such drug delivery systems are Corresponding author. Department of Chemical & Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore. Tel.: +65 6874 3835; fax: +65 6779 1936. E-mail address: [email protected] (S.-S. Feng).
0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.05.104
usually restricted by biocompatibility of the polymeric matrix material and the surfactant used in the formulation process. Poly (lactide) (PLA), poly (d,l-lactide-coglycolide) (PLGA), and poly (caprolactone) (PCL) are FDA-approved biodegradable polymers, which are used most often in the literature of drug delivery. These polymers were originally synthesized to be used as surgical sutures, which thus have disadvantages to be used for drug formulation such as too high hydrophobicity and too slow degradation. Novel biodegradable polymers/copolymers with desired hydrophobic/ hydrophilic balance and desired degradation rate are thus needed. In the literature, PLGA nanoparticles were usually prepared by using chemical emulsifiers such as
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poly (vinyl achohol) (PVA), which has been found of disadvantages including low emulsification efficiency, side effects and difficulties to wash away in the formulation process. Instead, d-a-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply, TPGS) has high emulsification efficiency (67 times higher than PVA). It can also greatly improve the drug encapsulation efficiency (up to 100% EE achieved) and enhance cellular uptake of nanoparticles and thus increase the cancer cell mortality [1–5]. TPGS is a water-soluble derivative of natural vitamin E. Its hydrophile–lipophile balance is 13. The chemical structure of TPGS is similar to other amphiphiles comprising lipophilic alkyl tail and hydrophilic polar head portion. Its bulky structure and large surface area characteristics make it an excellent emulsifier. Moreover, it has been found that co-administration of vitamin E TPGS could enhance cytotoxicity, inhibit Pglycoprotein mediated multi-drug resistance, and increase the oral bioavailability of anticancer drugs [6–8]. This triggered us to take such advantages of TPGS to synthesize PLA-TPGS copolymers for nanoparticle formulation of anticancer drugs, which can be expected to have self-emulsification effects (no emulsifiers are needed for the nanoparticles formulation) and achieve high drug encapsulation efficiency and desired drug release profiles. In this research, PLA-TPGS copolymers were synthesized by ring-opening polymerization. The synthesized copolymer was characterized by Fourier transform infrared spectroscopy (FTIR) for its molecular structure. The TPGS content and number average molecular weight of the synthesized PLA-TPGS copolymers were determined by 1H NMR. The weight-averaged molecular weight and its distribution was determined by gel permeation chromatography (GPC). Paclitaxel, one of the best antineoplastic drugs found from nature in past decades, was chosen as a prototype drug in this research due to its excellent therapeutic efficacy against a wild spectrum of cancers and its great success as the best seller among various anticancer drugs. Like many other anticancer drugs, paclitaxel has formulation problem, which is caused by its high hydrophobicity (water solubility less than 0.5 mg/L). The popular dosage form used in its current clinical administration is Taxols formulated in ajuvant Cremophor EL, which has been found to be responsible for serious side effects including hypersensitivity reactions, nephrotoxicity, cardiotoxicity and neurotoxicity [9–13]. Paclitaxel-loaded nanoparticles of the PLA-TPGS copolymer were fabricated by a modified solvent extraction/evaporation technique, which were then characterized by various state-of-theart techniques such as laser light scattering (LLS) for size and size distribution; field emission scanning electron microscopy (FESEM) for surface morphology; zeta potential for surface charge and X-ray photoelec-
263
tron spectroscopy (XPS) for surface chemistry of the nanoparticles. High-performance liquid chromatography (HPLC) was used to measure the drug encapsulation efficiency (EE) and the in vitro drug release kinetics.
2. Materials and methods 2.1. Materials Lactide (3,6-dimethyl-1,4-dioxane-2,5-dione, C6H8O4) was purchased from Aldrich. It was recrystallized twice from ethyl acetate before reaction. Vitamin E TPGS (d-a-tocopheryl polyethylene glycol 1000 succinate, C33O5H54 (CH2CH2O)23) was from Eastman chemical company (USA). It was freezedried for two days before use. Stannous octoate (Sn(OOCC7H15)2) was purchased from Sigma and was used as 1% distilled toluene solution. Paclitaxel of purity 99.8% was purchased from Dabur India Ltd. (India). PLGA (50:50, MW 40–75,000) was also purchased from Sigma. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Breford, USA). All chemicals including acetonitrile, dichloromethane (DCM) and ethyl acetate were HPLC grade. They were used without further purification. 2.2. Synthesis of PLA-TPGS copolymer PLA-TPGS copolymers were synthesized by ring-opening bulk polymerization of lactide monomer with vitamin E TPGS in presence of stannous octoate as catalyst. In brief, weighted amounts of lactide, TPGS and 0.5 wt% stannous octoate (in distilled toluene) were added in an ampoule. The ampoule was evacuated in liquid nitrogen for 45 min. After that the ampoule was sealed by butane burner and reacted in silicone oil bath at 145 1C. After 12 h, the reaction product was dissolved in DCM and then precipitated in excess cold methanol to remove unreacted lactide monomers and TPGS. The final product was collected by filtration and vacuum dried at 45 1C for two days. 2.3. Characterization of PLA-TPGS copolymer The molecular structure of PLA-TPGS copolymer was investigated by FTIR (Shimadzu, Japan). The samples for FTIR analysis were prepared by grinding 99% KBr with 1% copolymer and then pressing the mixture into a transparent tablet. The TPGS content and number-averaged molecular weight of the copolymer was determined by 1H NMR in CDCl3 at 300 Hz (Bruker ACF300). The weight-averaged molecular weight and molecular weight distribution was determined by gel permeation chromatography (GPC, Agilent 1100 series GPC analysis system with RI-G1362A refractive index detector). Agilent PLgel 5 mm mixed-C 300 mm 7.5 mm column was used. The mobile phase was chloroform delivered at a flow rate of 1 ml/min. The injection volume was 50 ml sample solution (0.5% w/v copolymer in mobile phase). The calibration curve was established by using green standard polystyrene standard sample (molecular weights: 6.95 105, 5.04 104, 2.96 103 and 162 respectively).
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2.4. Preparation of paclitaxel-loaded PLA-TPGS nanoparticles Paclitaxel-loaded PLA-TPGS nanoparticles were fabricated by a modified solvent extraction/evaporation method [3]. A given amount of paclitaxel and 50 mg PLA-TPGS copolymer were dissolved in 4 ml methylene chloride. The formed solution was poured into 60 ml 0.03% TPGS Millipore water solution under gentle stirring. The mixture was sonicated for 120 s at 20 W. The emulsion was then evaporated overnight under magnetic stirring to remove the organic solvent. The particle suspension was centrifuged at 11,500 rpm for 15 min and then washed three times to remove the unloaded drug and surfactant. The resulted particles were resuspended in 10 ml water and freeze-dried for two days. 2.5. Characterization of drug-loaded PLA-TPGS nanoparticles 2.5.1. Particle size analysis Size and size distribution of the drug-loaded PLA-TPGS nanoparticles were measured by the LLS (Brookhaven Instruments Corporation 90-PLUS analyzer). The samples were prepared by diluting the nanoparticles suspension with deionized water until the counter rate was less than 1.5 Mpcs. 2.5.2. Surface morphology The nanoparticles were imaged by a field emission scanning electron microscopy (FESEM) system at an accelerating voltage of 10 kV. To prepare samples for FESEM, the particles were fixed on the stub by a double-sided sticky tape and then coated with platinum layer by JFC-1300 automatic fine platinum coater (JEOL, Tokyo, Japan) for 50 s. 2.5.3. Surface charge Zeta potential of the drug-loaded PLA-TPGS nanoparticles was detected by the laser Doppler anemometry (Zeta Plus zeta potential analyzer, Brookhaven corporation). The particles (2 mg) were suspended in deionized water before measurement. The data were obtained with the average of seven measurements. 2.5.4. Drug encapsulation efficiency The paclitaxel entrapped in the drug-loaded PLA-TPGS nanoparticles was measured by HPLC (Agilent LC1100). A reverse-phase Inertsils ODS-3 column (150 mm 4.6 mm, pore size 5 mm, GL science Inc, Tokyo, Japan) was used. Briefly, 3 mg nanoparticles were dissolved in 1 ml DCM and then extracted by 3 ml mobile phase (50/50(v/v) acetonitrile/ water solution). DCM was evaporated in nitrogen atmosphere and the clear solution was obtained for HPLC analysis. The solution was transferred into HPLC vial after filtered through 0.22 mm syringe filter. The flow rate of mobile phase was 1 ml/ min. Retention time was controlled at 15 min. The column effluent was detected at 227 nm with a UV/VIS detector. The calibration curve was linear in the range of 50–50,000 ng/ml with a correlation coefficient of R2 ¼ 0:9999. The drug encapsulation efficiency was defined as the ratio between the amount of paclitaxel encapsulated in nanoparticles and that added in the process.
2.5.5. Surface chemistry The XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu, Japan) was used to analyze surface chemistry of the drugloaded PLA-TPGS nanoparticles. The fixed transmission mode was utilized with pass energy of 80 eV for the survey spectrum covering a binding energy from 0 to 1100 eV. Data were analyzed with the software provided by the manufacturer. 2.5.6. In vitro drug release In all, 2 mg drug-loaded PLA-TPGS nanoparticles were put in a centrifuge tube and dispersed in 10 ml phosphate buffer solution (PBS) of pH 7.4 containing 0.1% w/v Tween 80. Tween 80 was used to increase the solubility of paclitaxel in the buffer solution and avoid the binding of paclitaxel to the tube wall. The tube was put in an orbital water bath shaking at 120 rpm at 37.2 1C. At given time intervals, the tube was taken out and centrifuged at 11,500 rpm for 15 min. The supernatant was transferred into a test tube and the pellet was resuspended in 10 ml fresh PBS solution and put back to the shaker for continuous measurement. The supernatant was extracted with 2 ml DCM and reconstituted in 1 ml mobile phase. The DCM was evaporated by nitrogen stream. The analysis procedure was similar as for the measurement of EE.
3. Results and discussions 3.1. Synthesis of PLA-TPGS copolymer The PLA-TPGS copolymer was synthesized in this research by using the stannous octoate as catalyst. The mechanism of synthesis is schematically described in Fig. 1, which is similar to that in synthesis of the MPEG-PLA copolymers [14]. The hydroxyl end of TPGS served as initiator to selectively cleave acyloxygen chain of lactide. The molecular structure of vitamin E TPGS and the PLA-TPGS copolymer are shown in Fig. 2. Fig. 3 shows the FTIR spectra of the PLA-TPGS copolymer and TPGS. The carbonyl band of TPGS appears at 1730 cm1. For the synthesized copolymer, the carbonyl band was shifted to 1755 cm1. Overlapping of the CH stretching band of PLA at 2945 cm1 and that of TPGS at 2880 cm1 was observed. In the PLA-TPGS copolymer, the CH stretching band of TPGS was decreased. The absorption band at 3400–3650 cm1 is attributed to the terminal hydroxyl group and that at 1050–1250 cm1 is due to the C–O stretching. 3.2. Molecular weight and molecular weight distribution The structure of the synthesized PLA-TPGS copolymer was detected by 1H NMR in CDCl3. Fig. 4 shows a typical 1H NMR spectroscopy of the
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3.3. Size, size distribution and drug encapsulation efficiency
Cat.
O CH3 O
O
R O H
+ R-OH O
CH3 O
Catalyst
CH3
O CH3 O O
Lactide monomer
Cat.
O
OOR TPGS
R ( O
C
HC O
O
CH3
initiation
O
CH3
C
CH ) OH
+
H CH3
O
O
CH3
CH3
+
O
(m-1)
O O
CH3 O O R ( O
C
HC O
O
CH3
C
CH )m
OH
265
where R-OH:TPGS
The size and size distribution of the paclitaxel-loaded PLA-TPGS nanoparticles prepared in this research are shown in Table 1. The drug-loaded nanoparticles can be prepared with or without extra TPGS added as surfactant in the fabrication process. This is because the TPGS component in the copolymer has a selfemulsifying function. This is an advantage of the PLATPGS copolymer in nanoparticle formulation, which avoids the side effects of the traditional chemical emulsifiers such as PVA. The particle size for all samples was found around 300 nm with narrow size distribution of less than 0.2 polydispersity. It can be observed from Table 1 that addition of TPGS in the process as emulsifier slightly increased the particle size. This is because the coating effects of TPGS on the particle surface and extra TPGS on the particle surface may cause aggregation of the formed nanoparticles.
Propagation CH3
Fig. 1. Schematic description of the synthesis mechanism of PLATPGS copolymer.
PLA-TPGS copolymer and monomers. The signals at 5.2 and 1.69 ppm were assigned to the CH protons and methyl protons CH3 of PLA segment, respectively. The peak at 3.65 ppm was assigned to the CH2 protons of PEO part of TPGS. The lower peaks in the aliphatic region belong to various moieties of vitamin E tails [15–19]. Lactide monomer peak at 5.1 ppm was not detected. The precipitation process in treatment of the copolymer can remove the TPGS and lactide monomer thoroughly. The molecular weight of the PLA-TPGS was calculated by using the ratio between the peak areas at 5.2 and 3.65. The number-averaged molecular weight of the PLA-TPGS copolymer of 12% TPGS weight content was determined to be 12,700. In GPC analysis, we confirmed that the PLA-TPGS copolymer was synthesized by the ring-opening polymerization. The product was not a physical mixture of TPGS with lactide. Fig. 5 shows that the peak for TPGS appeared at 8.9 min. Instead, the peak of the copolymer shifted to 7.6 min. There was just one narrow peak for the copolymer product and the TPGS peak could not be detected. The polydispersity of the copolymer molecular weight was narrow, around 1.31. The number-averaged molecular weight calculated from the GPC chromatograph was 13,600. It seemed that the molecular weight detected from GPC and NMR can confirm each other.
3.3.1. Drug encapsulation efficiency It has been shown that the surfactant used in the fabrication process and the drug loading level are important factors to influence the particle size and size distribution and the drug encapsulation efficiency in the nanoparticles [1,2], which then determine the drug release kinetics, cellular uptake and thus the therapeutic effects of the drug-loaded nanoparticles [20]. Table 1 also shows the drug loading and surfactant effects on size and size distribution and the drug loading efficiency. It can be seen that the PLA-TPGS nanoparticles prepared with no emulsifier added can result in a satisfactory drug EE, which was found to increase from 61.9% for 5% drug loading to 91.5% for 1% drug loading. This is natural since the higher the drug loading is, the lower the EE would be. For the high drug loading of 5%, however, the EE of the paclitaxel-loaded PLATPGS nanoparticles can be improved by adding extra TPGS in the process as emulsifier. Nevertheless, the amount of TPGS to be added should be carefully figured out. The amphipathic surfactants align themselves at the oil–water interface to promote the stability of the particles by lowering the surface energy and thus resist coalescence and flocculation of the particles. Too little emulsifier would not be enough to cover the interface and too much emulsifier would cause particle aggregation [1]. It was suggested that an appropriate amount of TPGS for PLGA nanoparticle formulation of paclitaxel would be 0.03% w/w, which achieved 100% EE [2]. Indeed, it can be seen from Table 1 that 0.03% TPGS used as an emulsifier did improve the EE of the PLATPGS nanoparticles from 61.9% to 89.1% for a high drug loading of 5%. Size and surface coating play an important role in determining the mechanism and efficiency of nanoparticle
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266 COO(CH2CH2O)nH
(CH2)2
O CH3
C O
CH3
CH3 CH3
CH3 CH3
O
CH3
CH3
(a)
1.69ppm
O 3.65ppm
CH3
C
CH ) m OH
5.2ppm
COO(CH2CH2O)n ( C
(CH2)2
O CH
O
CH3
O CH3
C O
CH3
CH3 CH3
CH3 H3C
O
CH3
CH3
(b)
Fig. 2. Molecular structure of (a) vitamin E TPGS and (b) PLA-TPGS copolymer.
and small particles (o500 nm) can cross the membrane of epithelial cells through endocytosis [21–23]. TPGS coating could greatly enhance the cellular uptake of the nanoparticles [20]. tranmittance
PLA-TPGS copolymer
TPGS
4000
3500
3000
2500 2000 1500 wave number (1/cm)
1000
500
Fig. 3. FTIR spectra of PLA-TPGS copolymer and TPGS.
absorption in the GI tract for oral chemotherapy [20]. It was suggested that large particles (o5 mm) would be taken up via the lymphatics (M Cells of Peyer’s patches)
3.3.2. Effects of polymeric matrix materials Table 2 shows the results of paclitaxel-loaded nanoparticles fabricated from the blends of the PLATPGS copolymer and PLGA with various PLATPGS:PLGA ratios. It can be found that the drug encapsulation efficiency increased with increasing the blend ratio of PLA-TPGS copolymer to PLGA. It may be attributed to the core–shell structure of the nanoparticles as found from those of the MPEG-PLA/PLGA blends [24]. The PLA-TPGS copolymer has lower molecular weight (Mn ¼ 12,700) than that of the PLGA. The mixture of the copolymer with the PLGA can decrease the viscosity of the oil phase and thus result in better emulsification, which leads to a better drug encapsulation efficiency and smaller particle size [4]. Similar results were also found for the nanoparticles of
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267
Lactide
7
6
5
4 ppm
3
2
1
0
6
5
4
3
2
1
0
Vitamin E TPGS
7
ppm
CH3
CH2
PLA-TPGS CH
7
6
Fig. 4. Typical
5 1
4 ppm
3
2
1
0
H-NMR spectra of monomers and PLA-TPGS copolymer in CDCl3.
encapsulation efficiency and particle size (data not shown). The optimal sonication time seemed to be 120 s, which resulted in smallest particle size and highest drug encapsulation efficiency. Further increase of the sonication time may cause particle aggregation and thus was not preferred. The particle size was found to be increased by 45 nm after sonication for 180 s.
8.9min
3.4. Surface morphology TPGS 7.6min PLA-TPGS copolymer
2
4
6 retention time (min)
8
10
Fig. 5. Typical GPC chromatograms of PLA-TPGS copolymer and TPGS.
Surface morphology of the drug-loaded PLA-TPGS nanoparticles was examined by FESEM. Fig. 6 shows the FESEM images of the PLGA and PLA-TPGS (12%) nanoparticles. The particles seemed to have smooth surface within the FESEM resolution level. The smooth surface may lead to a slower drug release from the nanoparticles than that from those of rough surface. The FESEM images further confirmed the particle size detected from the LLS. 3.5. Zeta potential
other blends such as that of PLLA-g-oligoEG copolymer with the PLGA [25]. 3.3.3. Effects of mechanical mixing strength Sonication strength was also found to play an important role for the nanoparticle formulation, which can significantly affect the particle recovery, drug
Zeta potential determines the particle stability in dispersion. High absolute value of the zeta potential indicates high surface charge of the nanoparticles, which leads to strong repellent interactions among the nanoparticles in dispersion and thus high stability. The zeta potential of the TPGS emulsified, paclitaxel-loaded PLA-TPGS nanoparticles was found to be about
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268
Table 1 Effects of drug loading and surfactant on size, size distribution and drug encapsulation efficiency of paclitaxel-loaded nanoparticle Polymer
Drug loading
Surfactant
Size
PI
EE
PLA-TPGS PLA-TPGS PLA-TPGS PLA-TPGS PLA-TPGS
1% 2.5% 5% 5% 5%
No No No 0.03%TPGS 0.06%TPGS
293.4 290.5 293.1 304.5 333.3
0.141 0.134 0.124 0.185 0.246
91.5% 74.4% 61.9% 89.1% 87.9%
*
PI ¼ Polydispersity Index, EE ¼ Drug encapsulation efficiency.
Table 2 Effects of polymeric matrix materials on size, size distribution and drug encapsulation efficiency of paclitaxel-loaded nanoparticle Polymer/5% drug
Surfactant
Size
PI
ZP
EE
PLGA PLGA:PLA-TPGS ¼ 1:1 PLA-TPGS
0.03% TPGS 0.03% TPGS 0.03% TPGS
336.8 302.9 304.5
0.248 0.105 0.185
32.7 30.6 26.3
79.9% 83.9% 89.1%
*
Drug loading: 5%.
30 mV. Similar results were found for the paclitaxelloaded PLGA nanoparticles [2,3]. 3.6. Surface chemistry The atomic composition of the PLA-TPGS copolymer and the drug-loaded PLA-TPGS nanoparticles was determined by XPS. XPS can quantitatively determine the chemical composition of the particle surface in 5–10 nm depth. The XPS spectrum of the paclitaxelloaded PLA-TPGS nanoparticles is shown in Fig. 7. It can be seen that no nitrogen element signal was detected, which means that paclitaxel was mainly distributed inside the nanoparticles. The C 1s XPS spectra of the PLA-TPGS copolymer and the paclitaxelloaded PLA-TPGS nanoparticles are shown in Fig. 8. The peak at the binding energy 286.1 eV is regarded as the indicator of the PEG component of the PLA-TPGS copolymer. From Fig. 8, the presence of TPGS on the particle surface can be evidenced by an increasing C–O–C peak ratio from 18.5% for the pure PLA-TPGS copolymer to 27.8% for the paclitaxel-loaded PLATPGS nanoparticles with no surfactant used in fabrication. 3.7. In vitro drug release Fig. 9 shows the in vitro drug release profile of the paclitaxel-loaded PLA-TPGS nanoparticles, which look biphasic with a initial burst followed by a sustained release. The drug release from the PLA-TPGS nanoparticles was found to be 17% and 51% of the encapsulated drug in the first day and after 31 days, respectively, which was much faster than the paclitaxel-
loaded PLGA nanoparticles, which is only 7% and 19%, respectively, in the same periods. This is another advantage of the PLA-TPGS nanoparticles versus the traditional PLGA nanoparticles, which were found to release the drug too slowly to meet the therapeutic needs. The faster drug release of the PLA-TPGS nanoparticles may be caused by the lower molecular weight and the higher hydrophilicity of PLA-TPGS copolymer in comparison with the PLGA nanoparticles, which causes the copolymer swell and degrade faster and thus promote the drug release from the nanoparticles. The drug release mechanism could be referred to drug diffusion, polymer matrix swelling and the polymer erosion or degradation.
4. Conclusion A novel copolymer, PLA-TPGS, was synthesized in the present study for nanoparticle formulation of anticancer drugs. Paclitaxel was used as a prototype drug due to its excellent therapeutic effects against a wide spectrum of cancers and its great commercial success as the best seller among various anticancer drugs. The paclitaxel-loaded PLA-TPGS nanoparticles were prepared by a modified solvent extraction/evaporation technique with or without assistance of emulsifiers. Paclitaxel-loaded nanoparticles can also be formulated with blends of PLA-TPGS and PLGA in a similar way. It was found that PLA-TPGS copolymer can greatly increase the drug encapsulation efficiency and achieve much faster drug release than the PLGA nanoparticles to meet the therapeutic needs. PLA-TPGS nanoparticle formulation can avoid using toxic adjuvant Cremophor
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269
1200
500 counts
N 1s
1000
450 400
counts
800
390
600
C1s
395
400
405
410
O 1s
400 N 1s
200 0 0
200
400
600
800
1000
1200
binding energy (ev) Fig. 7. XPS spectrum of paclitaxel-loaded PLA-TPGS nanoparticles. The insert shows the nitrogen signal at high resolution.
C-C/C-H
O-C=O
292
290
(a)
C-O-C=O
C-O-C
288 286 binding energy (ev)
284
282
C-C/C-H
Fig. 6. FESEM images of paclitaxel-loaded nanoparticles with 0.03% TPGS as surfactant and 5% drug loading. (a) PLGA (b) PLGA:PLATPGS ¼ 50:50 (c) PLA-TPGS.
EL. It may have great potential to enhance the cellular uptake of the drug-loaded nanoparticles as well as to promote oral chemotherapy. The technology can also be applied to other anticancer drugs.
C-O-C O-C=O
292
(b)
290
C-O-C=O
288
286
284
282
binding energy (ev)
Fig. 8. XPS spectra of (a) the PLA-TPGS copolymer and (b) the paclitaxel-loaded PLA-TPGS nanoparticles.
ARTICLE IN PRESS Z. Zhang, S.-S. Feng / Biomaterials 27 (2006) 262–270
270 60 55
PLA-TPGS(12%)
50
paclitaxel released (%)
45 40 35 30 25
PLGA50/50
20 15 10 5 0 0
5
10
15
20
25
30
35
released time (days)
Fig. 9. In vitro drug release profile of paclitaxel-loaded PLA-TPGS nanoparticles prepared with 0.03% TPGS as surfactant and 5% drug loading.
Acknowledgement This research was supported by NUS Grant R-379000-014-112, National University of Singapore (SS Feng, PI) and Singapore Cancer Syndicate (SCS) Grant UU0028 (NUS 397-000-606-305 in 2004 and NUS R-279-000-187-305 in 2005. SS Feng, PI). ZP Zhang is grateful of the National University of Singapore for the financial support for his PhD study.
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