Tri-component diblock copolymers of poly(ethylene glycol)–poly(ε-caprolactone-co-lactide): synthesis, characterization and loading camptothecin
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- Zhang, Y., Wang, C., Yang, W. et al. Colloid Polym Sci (2005) 283: 1246. doi:10.1007/s00396-005-1306-5
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Biodegradable tri-component diblock copolymer was synthesized by bulk copolymerization of ε-caprolactone (CL) and D, L-lactide (LA) in the presence of methoxy poly(ethylene glycol) (MePEG), using stannous octoate as catalyst. Their chemical structure and physical properties were investigated by GPC, NMR, DSC, TGAand XRD. The increase of CL/LA ratio in the diblock copolymer leads to lower Tg, higher decomposition temperature and crystallinity. Nanoparticles formulated from MePEG–poly(CL-co-LA) (PCAE) possess spherical structure, which was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The DLS results indicate that the particle size increased with the increase of CL/LA ratio and the hydrophobic fragment length in the copolymer. The drug encapsulation efficiency and the drug release behavior in vitro conditions of camptothecin were measured by high performance liquid chromatography (HPLC). The encapsulation efficiency can be achieved as high as 84.4% and the release behavior can be made well-controlled. MePEG–poly(CL-co-LA) nanoparticles might have a great potential as carriers for hydrophobic drugs.
KeywordsMethoxy poly(ethylene glycol)–poly(ε-caprolactone-co-lactide)Diblock copolymerNanoparticlesCamptothecinDrug carrier
Most conventional polymeric drug carriers (polymeric nanoparticles) are subjected to rapid elimination from the bloodstream through phagocytosis by cells of the reticuloendothelial system after intravenous administration and recognition by the macrophages of the mononuclear phagocyte system (MPS) [1, 2]. Long-circulating drug-loaded nanoparticles may be used to maintain a required level of a pharmaceutical agent in the blood for extended time intervals for better drug availability [3, 4]. Long-circulating polymer nanoparticles can be obtained from amphiphilic block copolymers modified with hydrophilic, flexible and non-ionic polymers, such as poly(ethylene glycol) (PEG). The non-toxic and non-immunogenic nature of PEG provides a great advantage in utilizing it as a component for medical purposes, and its strong hydration power contributes to regulation of the hydrophilicity of the materials [5–7].
Amphiphilic block copolymers are classified into several types according to sequential arrangement of component segment, such as AB-type diblock copolymer, ABA-type triblock copolymer, (AB)n type multiblock copolymer and star block copolymer. Compared with other type of amphiphilic block copolymers, AB type amphiphilic diblock copolymers are the most appropriate candidates for forming polymer nanoparticles in size design, aggregation number, and nanoparticle stability due to simple architecture of their molecules . Methoxy poly(ethylene glycol)–poly(lactic acid) (MePEG–PLA) copolymer can self-assemble into polymeric nanoparticles characterized by a core-shell architecture in aqueous systems, in which a segregated core of associated hydrophobic segments (PLA) is surrounded by a hydrophilic and sterically stabilized shell (PEG), which was used as drug carriers extensively [9–13].
As well known, polycarolactone is a biodegradable and biocompatible polymer with high permability to drugs . Methoxy poly(ethylene glycol)–poly(ε-caprolactone), was much attractive for drug-delivery applications [15–17]. However, it has a slow rate of biodegradation in human tissues due to the high hydrophobicity and crystallinity of poly(ε-caprolactone) fragment . For the controlled delivery of bioactive agents, it is necessary to adjust carefully both drug release rate and polymer degradation properties to achieve desired formulation properties. Copolymerization is a simple and most widely used approach for modifying polymer properties to meet specific requirements.
In previous studies, much attention was paid to the drug-loaded nanoparticles with different hydrophobic/hydrophilic ratio, different interactions between the drug and carriers, different drug-release behavior and triblock copolymers [18–22], few works were done to modify the hydrophobic segment of diblock copolymers. In this paper, AB type diblock copolymers composed of poly(CL-co-LA) (A) and MePEG (B) segments were synthesized, and a wide range of copolymers with various properties were obtained by adjusting the ratio of CL/LA in the hydrophobic block. A series of physico-chemical characterization was carried out in order to study the properties of the MePEG–poly(CL-co-LA) copolymers and confirm the formation of compolymer nanoparticles. Camptothecin (CPT), one of the best anticancer drugs [23–25] was encapsulated into MePEG–poly(CL-co-LA) copolymer nanoparticles as model drug.The release of CPT from these nanoparticles was investigated to identify that the nanoparticles could serve as useful carriers of hydrophobic drugs, and the drug-release behavior is releated to the composition and structure of the copolymers.
Methoxy poly(ethylene glycol) (MePEG, Mn=5,000) was supplied by Aldrich and dried under vacuum in a desiccator with P2O5 overnight before use. D, L-lactide (purity, 99.5%) was purchased from PURAC and purified by twice recrystallization from dried ethyl acetate. ε-caprolactone (ε-CL, Aldrich) was purified by drying over CaH2 and distilled under reduced pressure. Stannous octoate (stannous content, 26.5–27.5%) was supplied by Shanghai Chemical Reagent Company and distilled before use. CPT was purchased from Fudan Zishan New Technology Co. (Shagnhai, China). All other chemicals used were reagent grade and used without further purification. Cellulose dialysis bag (molecular weight cut off, 14,000) was supplied by Luniao Technology Co.
Synthesis of MePEG–poly(CL-co-LA) copolymer
Molecular weight and chemical composition of MePEG–poly(CL-co-LA) copolymers
Feed MePEG (wt%)
Preparation of polymeric nanoparticles
The PCAE copolymer nanoparticles were prepared by dialysis method. Copolymer (8 mg) was dissolved in 1 mL acetone, then 4 mL water was added to induce micellization under agitation. The aqueous solution was placed in a dialysis bag and dialyzed against doubly distilled water for 24 h to remove acetone.
To prepare the CPT-loaded PCAE nanoparticles, 100 mg PCAE copolymer and 5 mg CPT were dissolved in 5 mL tetrahydrofuran and dimethylsulfoxide (v/v, 4/1) co-solvent and then the organic phase was dropped into 30 mL water under moderate stirring. The organic solvent was removed by dialysis against doubly distilled water for 24 h.
In vitro release of CPT-loaded nanoparticles
The release test of the CPT in vitro was performed by incubating approximately 1 mL CPT-loaded nanoparticles in a dialysis bag and immersed in phosphate buffer solution (0.1 M, pH 7.4). The entire system was kept at 37±1 °C with horizontal shaking at about 60 rpm. At selected time intervals, 5 mL of the release medium was withdrawn from the media and the same volume of fresh dissolution medium was added. The aliquotos were acidified with 0.1 M HCl, and 20 μL was used to analyze by HPLC.
The molecular weight and molecular weight distribution of PCAE copolymers were characterized using GPC (HP1100, Hewlett Packard) with tetrahydrofuran as eluent at a flow rate of 1 mL/min. 1HNMR spectra of the copolymers in deuterated chloroform solution were recorded on a 500 MHz NMR (DMX-500, Bruker) with tetramethylsilane as internal standard to determine the chemical structure and molecular weight of the copolymers. DSC measurements were carried out under nitrogen atmosphere at a heating rate of 10 °C/min on a Perkin Elmer Instrument DSC7 thermal analyzer. Thermogravimetric analysis was run on Perkin Elmer Instrument (TGA, Pyrolysis-1, PE) from 40 °C to 500 °C at a heating rate of 10 °C/min, and the decomposition temperature (Td) was obtained. X-ray diffraction patterns of the copolymers were recorded on a D/max-rB X-ray diffractionmeter (Rigaku) equipped with a graphite monochromator-filtered Cu–Kα radiation.
Particle size and morphology of the nanoparticles
The hydrodynamic radii of nanoparticles were determined by dynamic light scattering (DLS) measurement at 25 °C using a light scattering spectrophotometer (Autosizer 4700, Malvern) with a vertically polarized incident beam at 532 nm supplied by an argon ion laser, and scattering angle 60°, 80°, 90°, 100° and 120° were used in this study. Before measurement, all samples were filtered through a 0.45 μm filter (Millipore). Nanoparticles morphology was performed on a transmission electron microscopy (TEM) (Hitachi H-600).
HPLC analysis of camptothecin
To determine the drug content, a predetermined aliquot of nanoparticles suspension was withdrawn and frozen, then lyophilized by freeze dryer system to obtain dried nanoparticle product. The CPT content of these nanoparticles was determined by HPLC analysis . HPLC was based on a Hypersil ODS (USA 5 μm), 150×4.6 mm column with isocratic elution (65:35 mixture of aqueous triethylamine-acetate buffer (pH 5.5) and acetonitrile at a flow rate of 0.55 ml/min) and detection of camptocin by a Shimadzu UV detector. CPT was monitored at 360 nm. Drug loading, content and encapsulation efficiency were obtained by Eqs. 1 and 2.
Results and discussion
The composition and molecular weight of MePEG–PLA–PCL block copolymers
Table 1 summarizes the molecular characteristics of these polymers. The Mn values calculated from GPC were lower than those from NMR spectra, which could be assigned to changes of hydrodynamic volume of MePEG–poly(CL-co-LA) copolymers bearing both hydrophilic PEG and hydrophobic PCL and PLA segments, as compared with the parent homopolymers .
Thermal properties of the copolymers
Thermal properties of MePEG–poly(CL-co-LA) block copolymers
X-Ray diffraction (XRD) analysis of the copolymers
Particle size and morphology
Hydrodynamic diameter of MePEG–poly(CL-co-LA) copolymer nanoparticles, scattering angle is 90°
Particle size (nm)
In vitro release behavior of CPT-loaded nanoparticles
Characteristics of the CPT-loaded nanoparticles
Biodegradable MePEG–poly(CL-co-LA) copolymers were synthesized by ring open polymerization in bulk, and stealthy nanoparticles were prepared by the dialysis method for controlled release of an anticancer drug CPT. The properties of the copolymers were determined by the chemical composition. The size and size distribution of the prepared nanoparticles and the drug-release behavior are affected significantly by the composition of the polymeric matrix. These results show that the copolymers of CL, D, L-lactide are promising materials for preparing nanoparticles as drug carriers and the release rate from nanoparticles can be adjusted by changing the composition of polymer matrix. This research may have potential to provide an alternative dosage form for CPT, one of the best anticancer drugs found from the nature in the past decades and commercially, the most successful anticancer agents.
Financial support from Association of Shanghai Science and Technology (No.034319242) is gratefully acknowledged.