Self-Assembled Micelles of Biodegradable Triblock Copolymers

Dec 15, 2007 - Figure 3B shows the fluorescence–excitation spectra of pyrene in PEEP44-PCL27-PEEP44 micelles at different concentrations. A red shif...
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Biomacromolecules 2008, 9, 388–395

Self-Assembled Micelles of Biodegradable Triblock Copolymers Based on Poly(ethyl ethylene phosphate) and Poly(E-caprolactone) as Drug Carriers Yu-Cai Wang,† Ling-Yan Tang,‡ Tian-Meng Sun,‡ Chang-Hua Li,† Meng-Hua Xiong,† and Jun Wang*,‡ Department of Polymer Science and Engineering, and Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, P.R. China Received July 2, 2007; Revised Manuscript Received September 12, 2007

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.

Introduction Biodegradable nanoparticles such as micelles and vesicles self-assembled by amphiphilic block copolymers in aqueous solution have been widely studied as drug carriers due to their distinctive characteristics including thermodynamic stability, long-circulation potential following intravenous injection, and passive targeting ability to tumor tissues.1–6 Polymeric nanoparticles are reported to accumulate in solid tumor tissues through the enhanced permeability and retention effect, which results from a combination of the increased permeability of tumor blood vessels and the decreased rate of clearance caused by the lack of functional lymphatic vessels in the tumor.7 It supports the use of polymeric nanoparticles in therapy as drug carriers by taking the advantage of the unique pathophysiology of tumor vasculature. At the mean time, the hydrophilic surface or shell of those self-assembled nanoparticles, such as polyethylene glycol (PEG), helps nanoparticles escape from renal exclusion and the reticuloendothelial system, allowing prolonged periods of circulation and passive accumulation in the tumors of nanoparticles, further resulting in enhanced drug efficacy.8 Solubilization and stabilization of active agents, especially poorly water soluble anticancer agents, is a problem for drug delivery system. One of the resolutions is to load the drug molecules into the core of amphiphilic block copolymer micelles to increase the solubility.9 Among a variety of choices of hydrophobic components of amphiphilic copolymers as the core of micelles, biodegradable aliphatic polyesters, including poly* To whom correspondence should be addressed. Fax: +86 551 360 0402. Email: [email protected]. † Department of Polymer Science and Engineering. ‡ Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences.

(lactide acid), poly(D,L-lactide-coglycolide), poly(-caprolactone), and poly(hydroxybutyrate), are dominant and have been investigated most extensively.10–13 The intensive hydrophobicility of these aliphatic polyesters contributes to the tendency of polymer chain association for hydrophobic core formation. The crystalline or semicrystalline nature of some aliphatic polyesters may also enhance the micelle stability against dilution. On the other hand, among the choices of hydrophilic components as the outer shell of nanoparticles, PEG, also known as poly(ethylene oxide) has been most widely used due to its superior biocompatibility and hydrophilicity. PEG segments are also known to prevent plasma protein adhesion and increase the circulation time of nanoparticles following intravenous injection.14 In the present study, we reported self-assembled polymeric micelles, where poly(-caprolactone) (PCL) was used as the hydrophobic segments of the block copolymers, while polyphosphoester (PPE) was used as the hydrophilic segment. As a class of biodegradable polymers with repeated phosphoester linkage in the backbone, PPE has been used for many biomedical applications including drug, gene delivery, and tissue engineering due to its favorable biocompatibility and biodegradability.15–17 The pentavalent nature of phosphorus in the backbone of PPE polymers provides more structural flexibility. In addition, the physical and chemical properties of the polymers may also be adjusted through changing the side group conjunct to phosphorus. For example, Wang et al. prepared a series of cationic PPEs with different side charge groups, which served as effective, nontoxic, and biodegradable gene carriers.18 Li et al. synthesized multifunctional polyphosphate macromers with different weight fractions of acrylate, which were further crosslinked to obtain hydrogels with a tunable swelling ratio and mechanical strength.19 In addition, well-controlled ring-opening

10.1021/bm700732g CCC: $40.75  2008 American Chemical Society Published on Web 12/15/2007

Self-Assembled Micelles of PEEP and PCL

polymerization of cyclic phosphoester monomers initiated by Al(Oi-Pr)3 or alcohol/tin(II) octoate allows defined molecular weight control of PPE and presents functional hydroxyl end groups, which can be further modified for bioactive molecules conjugation.20,21 Nevertheless, very limited work has been done to explore the potential of PPE-based polymeric nanoparticles for drug delivery, while hydrophilic PPEs may be an interesting alternative for reporting hydrophilic components used as the outer shell of self-assembled nanoparticles. We reported here the syntheses and characterization of novel amphiphilic triblock copolymers of PPE and PCL. In this study, we aimed at understanding the micellization and in vitro degradation properties of such block copolymers as well as the potential of these polymeric micelles as drug carriers in vitro.

Experimental Section Materials. Ethyl ethylene phosphate, also known as 2-ethoxy-2oxo-1,3,2-dioxaphospholane (EEP), was synthesized, as previously described in literature,20 and purified with two consecutive vacuum distillations. -Caprolactone (CL; Acros Organics, 99%) was dried over calcium hydride for 48 h at room temperature, followed by distillation under reduced pressure just before use. Ethylene glycol (Sinopharm Chemical Reagent Co., Ltd., China) was freshly distilled under reduced pressure prior to use. Stannous octoate (Sn(Oct)2, Sinopharm Chemical Reagent Co., Ltd., China) was purified according to a method described in literature.22 Tetrahydrofuran (THF) was refluxed over potassium–sodium alloy under a N2 atmosphere and distilled out just before use. Spectra/Por membranes (Spectrum Laboratories, Inc., Rancho Dominguez, CA, U.S.A.) were employed for dialysis. Paclitaxel (PTX), phosphodiesterase I from Crotalus atrox, Pseudomonas lipase, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical Co. Pyrene (Acros Organics) and all other solvents and reagents were used as received. Synthesis of Dihydroxyl Poly(E-caprolactone) Macroinitiator. Dihydroxyl poly(-caprolactone) (denoted further as HO-PCL-OH) was synthesized through ring-opening polymerization of CL in bulk using ethylene glycol as an initiator and Sn(Oct)2 as the catalyst. Typically, CL (17.35 g, 0.15 mol), ethylene glycol (0.38 g, 6.0 mmol), and Sn(Oct)2 (0.025 g, 0.06 mmol) were added into a fresh flamed and nitrogen purged round-bottomed flask in a glovebox with H2O and O2 contents less than 0.1 ppm. The mixture was maintained at 110 °C for 12 h. The product was dissolved in chloroform and precipitated in cold n-hexane twice. The precipitate was dried under vacuum to a constant weight at room temperature. Synthesis of PEEP-PCL-PEEP Triblock Copolymer. Block copolymerization was carried out in THF at 35 °C using HO-PCL-OH as an initiator and Sn(Oct)2 as a catalyst. In a typical polymerization, HO-PCL-OH (Mn ) 4100, 2.51 g, 0.6 mmol), EEP (4.58 g, 32.2 mmol), and Sn(Oct)2 (0.49 g, 1.2 mmol) were dissolved in THF in a fresh flamed and nitrogen-purged round-bottomed flask at 35 °C. The mixture was further reacted for 3 h, concentrated, and precipitated in cold ethyl ether/methanol (10/1, v/v) twice. The product was filtrated and dried under vacuum to a constant weight at room temperature to obtain the product. The yield was approximately 85%. Preparation of Micelles. Micelles were prepared by a dialysis method. Briefly, copolymer (50 mg) was dissolved in 5 mL of THF, and this solution was added dropwise to 50 mL of Milli-Q ultrapurified water under gentle stirring. After standing for 3 h at room temperature, THF was removed by dialysis against water for 24 h to obtain the micelles. In Vitro Enzymatic Degradation of Micelles. The in vitro enzyme catalyzed degradation of micelles was performed at 37 °C with Pseudomonas lipase (0.2 g L-1) in phosphate buffer (0.05 mol L-1, pH 7.0) or phosphodiesterase I (0.2 g L-1) in Tris HCl buffer at pH 8.8. The concentration of micelle was set at 1.0 g L-1. At predetermined time intervals, samples were taken out and freeze–dried for GPC

Biomacromolecules, Vol. 9, No. 1, 2008 389 analysis. The degradation products were also dialyzed against water (MWCO 3500) for 72 h at room temperature and further freeze–dried for 1H NMR analysis in CDCl3. Paclitaxel Loading into PEEP-PCL-PEEP Micelles. PTX was loaded into micelles by the dialysis method. In a typical procedure, the block copolymer (10 mg) was dissolved in 1.0 mL of THF, and to this solution was subsequently added PTX dissolved in DMSO. Ultrapurified water was then added dropwise to this solution. The mixture was stirred at room temperature for 3 h and then dialyzed against 5 L of water for 24 h using Spectra/Por dialysis membrane (MWCO 15000). The solution was filtered through a 0.45 µm filter and freeze–dried. The loading amount of PTX was determined by HPLC after dissolving PTX-loaded micelles with acetonitrile–water (50:50, v/v). The drug-loading content (DLC%) and efficiency (DLE%) were calculated by the following equations:

DLC% ) DLE% )

amount of PTX in micelle × 100% amount of PTX-loaded micelle

(1)

amount of PTX in micelle × 100% amount of PTX used for micelle preparation (2)

In Vitro Paclitaxel Release from Micelles. In vitro release profiles of PTX from micelles were investigated in PBS (0.01 mol L-1, pH 7.4) using a dialysis bag diffusion technique. Micelles (1.5 mL) were introduced into a dialysis membrane tubing (Spectra/Por, Float-A-Lyzer, MWCO 25000) and incubated in 25 mL of buffer at 37 °C with stirring. At predetermined intervals, buffer were drawn and replaced with an equal volume of fresh medium. The concentration of PTX in the solution was measured by HPLC, as described below. Characterization of Polymers. Brucker AV300 NMR spectrometer (300 MHz) was used for 1H, 13C, and 31P NMR spectra to determine the structure and composition of the PEEP-PCL-PEEP triblock copolymers. Deuterated chloroform (CDCl3) containing 0.03% tetramethylsilane (TMS) was used as the solvent for NMR measurements. Phosphoric acid (85%) was used as external reference for 31P NMR analyses. Fourier transform infrared spectrometer (FT-IR) spectra were recorded on a Bruker Vector 22 Fourier transform infrared spectrometer at wavenumbers 400–4000 cm-1, with a resolution of 2 cm-1, using the KBr disk method. Number and weight average molecular weights (Mn and Mw) and molecular weight distributions (polydispersity index, PDI ) Mw/Mn) were determined by gel permeation chromatography (GPC) measurements on a Waters GPC system, which was equipped with a Waters 1515 HPLC solvent pump, a Waters 2414 refractive index detector, and four Waters styragel high resolution columns (HR4, HR2, HR1, HR0.5, effective molecular weight range 5000–500000, 500–20000, 100–5000, and 0–1000, respectively). Chloroform (HPLC grade, J.T. Baker, stabilized with 0.75% ethanol) was used as eluent at 40 °C, delivered at a flow rate of 1.0 mL min-1. Monodispersed polystyrene standards with a molecular weight range of 1310–5.51 × 104 were used to generate the calibration curve. Critical Micelle Concentrations (CMCs) of Micelles. To estimate the CMCs of the micelles, pyrene was used as the fluorescence probe.23 A predetermined amount of pyrene in acetone was added into a series of ampules, and the acetone was then removed first by gently flowing N2 and then by vacuum. A predetermined volume of copolymer solutions and ultrapurified water were added into the ampules consecutively to get solutions of different micelle concentrations ranging from 1.0 × 10-5 to 0.5 g L-1, while the concentration of pyrene in each flask was fixed at 6.0 × 10-7mol L-1, slightly lower than the saturation solubility of pyrene in water. Fluorescence spectra were recorded on a Shimadzu RF-5301PC spectrofluorophotometer at 373 nm excitation wavelength and 5 nm slit width. Particle Size and Zeta Potential Measurements. The size and size distribution of micelles in aqueous solution were measured by dynamic light scattering carried out on a Malvern Zetasizer Nano ZS90 with a He-Ne laser (633 nm) and 90° collecting optics. All samples were prepared in aqueous solution at a concentration of 0.2 g L-1 and filtered

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Scheme 1. Synthesis Pathway of Triblock Copolymer PEEP-PCL-PEEP

through Millipore 0.45 µm filter prior to measurements. All measurements were carried out at 25 °C, and data were analyzed by Malvern Dispersion Technology Software 4.20. The zeta potential measurements were performed using an aqueous dip cell in automatic mode using Malvern Zetasizer Nano ZS90. Transmission Electron Microscopy (TEM). TEM was performed on a Hitachi model H-800 transmission electron microscope with an accelerating voltage of 200 KV. The samples were prepared by pipetting a drop of the micelle solution (1 g L-1) onto 230 mesh copper grids coated with carbon and allowing the sample to dry in air before measurement. Methyl Tetrazolium (MTT) Assay. The relative cytotoxicity of micelles was assessed with a MTT viability assay against HEK293 cells. The cells were seeded in 96-well plates at 20000 cells per well in 100 µL of complete DMEM containing 10% fetal bovine serum, supplemented with 50 units/mL penicillin and 50 units/mL streptomycin, and incubated at 37 °C in a 5% CO2 atmosphere for 24 h, followed by removing culture medium and adding micelle solutions (100 µL in complete DMEM medium) at different concentrations (0∼1 g L-1). After a 24 h incubation, 25 µL of MTT stock solution (5 g L-1 in PBS) was added to each well to achieve a final concentration of 1 g L-1, with the exception of the wells as blank, to which 25 µL of PBS was added. After incubation for an additional 2 h, 100 µL of the extraction buffer (20% SDS in 50% DMF, pH 4.7, prepared at 37 °C) was added to the wells and incubated overnight at 37 °C. The solution was mixed, and the absorbance of the solution was measured at 570 nm using a Bio-Rad 680 microplate reader. The cell viability was normalized to that of HEK293 cells cultured in the culture medium without polymer micelle. HPLC Analysis of Paclitaxel. HPLC was performed on Waters HPLC system consisting of Waters 1525 binary pump, Waters 2487 2-channel UV–vis detector, 1500 column heater, and a Symmetry C18 column. Acetonitrile–water (50:50, v/v) was used as the mobile phase at 30 °C with a flow rate of 1.0 mL min-1. UV-vis detector was set at 227 nm and linked to Breeze software for data analysis.

The degree of polymerization (DP) of PCL was calculated from its 1H NMR (not shown) based on the integration ratio of triplet at 4.07 ppm and singlet at 3.66 ppm, assigned to methylene protons beside the oxygen of the CL units and methylene protons conjoint with hydroxyl end groups, respectively. HO-PCL-OH macrointiators with DP of 27–186 were used in this study. NMR, GPC, and FTIR spectra were employed to demonstrate the successful syntheses of the block copolymers. As shown in Figure 1A, resonances at 1.37 (d), 4.18 (c), and 4.26 (a + b) ppm were newly appeared compared with 1H NMR spectrum of HO-PCL-OH, which are characteristic signals from protons of the PEEP block and assigned to pendent methyl (-CH2CH3) and methylene (-OCH2CH3) protons and methylene protons (-POCH2CH2O-) from PEEP backbone, respectively. It is worth noting that no signal at 3.66 ppm was found in 1H NMR spectra of triblock copolymers, which should be assigned to methylene protons conjoint to hydroxyl end groups of HO-PCL-OH. Instead, a newly appeared peak at 3.82 ppm demonstrated the formation of PEEP blocks with hydroxyl end groups, indicating that HO-PCL-OH macroinitiators were completely involved in the copolymerization. This result is similar with what we observed in the PCL-PPE block copolymer synthesis using Al(iOPr)3 as the initiator.21 The DP of PEEP was calculated from the integrated peak area of 4.18 and 4.26 ppm (6H), assigned to the methylene group of PEEP block, by the integrated peak of the triplets at 2.35 ppm (2H), assigned to the methylene group of PCL block (Figure 1A). 13 C NMR spectrum of the triblock copolymer shown in Figure 1B demonstrated the blocky structure. A single peak at 173.6 ppm in the carbonyl region is in agreement with the block structure of the copolymer, as reported previously.21 Unlike

Results and Discussion Synthesis and Characterization of Triblock Copolymers. We synthesized a series of PEEP-PCL-PEEP triblock copolymers by ring-opening polymerization of EEP using the dihydroxyl-terminated PCL as the initiator and stannous octoate as the catalyst. Stannous octoate has been the most often used catalyst for ring-opening polymerization of lactone and lactide because of its high catalytic activity, as well as U.S. FDA approval as a food additive.22 In our earlier study, we reported PPE with a linear molecular structure was synthesized through ring-opening polymerization of EEP in THF under co-initiation of dodecanol and Sn(Oct)2.20 Instead of dodecanol, in this study, we prepared dihydroxyl-terminated PCL (HOPCL-OH) by ring-opening polymerization of CL in bulk using ethylene glycol as initiator and further used HO-PCL-OH as macroinitiator for EEP polymerization to obtain the triblock copolymers. The synthesis pathway is depicted in Scheme 1.

Figure 1. 1H NMR (A) and 13C NMR (B) spectra of typical PEEPPCL-PEEP triblock copolymer (in CDCl3).

Self-Assembled Micelles of PEEP and PCL

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Figure 2. GPC chromatograms of the PCL macroinitiator HO-PCL36OH (A) and block copolymer PEEP49-PCL36-PEEP49 (B).

resonance at 13.6 ppm of the EEP monomer, 31P NMR spectrum of block copolymer (data not shown) gave a strong resonance at -5.30 ppm, assigned to the phosphorus atoms in PEEP block, while the weak signal at -4.10 ppm was likely due to the ends of PEEP block. In the FTIR spectra (data not shown), absorbances at 1730 cm-1 and 1045 cm-1 are characteristic absorptions of the CdO stretching and CsO stretching due to the presence of the PCL block. Absorptions of asymmetrical and symmetrical PdO stretchings occurred at 1276 cm-1 and 1160 cm-1, respectively, while PsOsC stretching appeared at 984 cm-1, which demonstrated the presence of the PEEP block.21 The molecular weight and the molecular weight distribution were measured by GPC, and the typical overlaid GPC chromatograms of HO-PCL-OH and the triblock copolymer were shown in Figure 2. The molecular weight distribution for the macroinitiators was around 1.20, as summarized in Table 1. The unimodal peak of PEEP-PCL-PEEP with decreased retention time demonstrated the formation of the block polymer. The PDIs of those triblock copolymers were from 1.38 to 1.50, which were slightly increased. The basic data of the resultant triblock copolymers, including molecular weight, polydispersity index, and compositions, are summarized in Table 1. The nomenclature used for the triblock copolymers displays the DP. For example, PEEP44-PCL27PEEP44 represents the triblock copolymer composed of PCL with a DP of 27 in the middle block and PEEP with an average DP of 44 at each side block, respectively. Micellization of PEEP-PCL-PEEP Block Copolymers. Amphiphilic block copolymers consisting of hydrophobic block and water-soluble hydrophilic block have been widely studied as carriers for controlled drug delivery.24,25 When a block copolymer is dissolved in a solvent that is a thermodynamically good solvent for one block and a nonsolvent for the other block(s), the polymer chains can reversibly self-assemble into aggregates, and the covalent bond between the blocks preventing macrophase separation.26 Once PEEP-PCL-PPEP triblock copolymer solution in THF was mixed with water, the diffusion of THF into water induced microphase separation of PCL and PEEP blocks, and then self-assembly occurred. Figure 3A gave the typical 1H NMR spectrum of micelles in D2O, which was prepared by lyophilization of block copolymer micelles in aqueous solution following resuspension in D2O. It is obvious that signals assigned to protons of the PCL block disappeared, while signals at 1.30, 4.18, and 4.26 ppm assigned to protons of the PEEP block were still prominent, indicating the limited molecular motion of the PCL block surrounded by the solvated PEEP segments due to their hydrophicility. It also indicated the microphase separation of PCL and PEEP blocks in D2O was thermodynamically stable.

Figure 3. (A) 1H NMR spectrum of triblock copolymer micelle in D2O; (B) excitation spectra of pyrene in aqueous solution of PEEP44-PCL27PEEP44 at various concentrations (λem ) 373 nm).

The micelle formation of the triblock copolymers was also confirmed by fluorescence technique using pyrene as a probe. Figure 3B shows the fluorescence–excitation spectra of pyrene in PEEP44-PCL27-PEEP44 micelles at different concentrations. A red shift of (0,0) absorption band from 335.5 to 339.0 nm was observed when the concentration of copolymer was increased from 1.0 × 10-5 to 0.5 g L-1. This red shift results from the transfer of pyrene molecules from a water environment to the hydrophobic micellar core and thus provides information on the location of the pyrene probe in the system, in fact, indicating the formation of micelles.23 Figure 4 shows the TEM micrograph of copolymer micelles for PEEP49-PCL186-PEE49. The micelles took an approximately spherical shape, and the images were typical among those obtained from block copolymers listed in Table 1, confirming that PEEP-PCL-PEEP copolymers formed discrete particles in aqueous medium. CMC Values of the PEEP-PCL-PEEP Copolymers. The CMC of a copolymer gives an indication of the stability of a micellar formulation upon dilution in body fluids following intravenous administration. We further evaluated the CMC of amphiphilic triblock copolymers using a fluorescence spectroscopic method based on the preferential partition of the pyrene probe in the hydrophobic core against an aqueous environment, as described above. Figure 5A,B shows the intensity ratio of the bands at 339.0 and 335.5 nm (I339.0/I335.5) as a function of the logarithm of the copolymer concentrations for copolymers with different compositions. All curves exhibited sigmoid shapes.

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Table 1. Composition and Molecular Weight Distribution of PEEP-PCL-PEEP Triblock Copolymer Macroinitiator HO-PCL-OH

a

a

b

Triblock copolymer PEEP-PCL-PEEP c

block copolymer

Mn

Mn

PDI

PEEP44-PCL27-PEEP44 PEEP7-PCL36-PEEP7 PEEP21-PCL36-PEEP21 PEEP49-PCL36-PEEP49 PEEP45-PCL63-PEEP45 PEEP48-PCL94-PEEP48 PEEP49-PCL186-PEEP49

3080 4100 4100 4100 7180 10720 21200

5830 7090 7090 7090 12920 19830 40280

1.20 1.18 1.18 1.18 1.16 1.21 1.22

Determined by 1H NMR.

b

Mna 6690–3080–6690 1060–4100–1060 3190–4100–3190 7450–4100–7450 6840–7180–6840 7290–10720–7290 7450–21200–7450

Mnb

PDIc

15900 11030 16700 24700 35380 39660 55090

1.38 1.42 1.42 1.46 1.46 1.50 1.49

Determined by GPC. c Determined by GPC.

Figure 4. Typical TEM image of PEEP49-PCL186-PEEP49 micelles.

The I339.0/I335.5 ratio kept constant as the concentrations were below a certain value. When the concentrations were above the value, it generally increased. The fluorescence intensity ratio reached a plateau with a further increase of polymer concentration. The rapid increase of the ratio indicated that the triblock copolymers in water automatically assembled, and the pyrene probe preferentially partitioned from the aqueous environment into the hydrophobic core of the micelle. The CMC value was taken as the intersection of the tangents to the horizontal line of intensity ratio with relatively constant values and the diagonal line with rapid increased intensity ratio. CMC values were plotted against the DP of the PCL and PEEP block of the triblock copolymers and shown in Figure 5C. When the DP of PEEP block increased from 7 to 49, while the DP of PCL was fixed at 36, the CMC values increased from 1.45 to 3.31 × 10-3 g L-1. This trend is in agreement with the reported result that a block copolymer with a rather long PEG segment tended to increase the CMC value in aqueous media.23,27 When the DP of PEEP was similar from 44 to 49, but the DP of PCL was varied from 27 to 186 units, the CMC decreased sharply from 10.2 to 0.55 × 10-3 g L-1, indicating that the hydrophobic length plays a relatively important role on determining the thermodynamic stability of micelles. The low CMC values of PEEP-PCL-PEEP micelles may be due to the slight hydrophobicility of the PEEP block compared to PEG. Therefore, it is believed that PEEP-PCL-PEEP micelles would be stable thermodynamically in aqueous media and lower CMC values are surely a favorable indication when such micelles are used for systemic drug delivery. Thus, in this sense, PEEP-PCL-PEEP micelles should be suitable for pharmaceutical application. Size and size distribution of micelles was measured by dynamic light scattering. When the initial copolymer concentration was 0.2 g L-1 in THF, the average diameter of micelles

increased from 35 to 186 nm as the PCL length increased from DP ) 27 to DP ) 186 when PEEP block length remained nearly unchanged (DP ) 44–49), indicating that a longer PCL block can enhance its assembly and lead to a larger core. On the contrary, the diameter decreased from 78 to 47 nm as the PEEP length increased from DP ) 7 to DP ) 49, while the PCL block length remained unchanged (DP ) 36). Micelles were found negatively charged, with an average zeta potential value of around -20 mV, which indicates a high electric charge on the surface of micelles and may cause strong repellent forces among particles to prevent aggregation. In Vitro Degradation of PEEP-PCL-PEEP Triblock Copolymer Micelles. The in vitro degradation behavior of PEEP-PCL-PEEP micelles was evaluated at 37 °C at neutral pH in the presence of Pseudomonas lipase, which is a wellknown enzyme to accelerate degradation of PCL. The molecular weights of degradation products were analyzed by GPC. As an example, GPC chromatograms of the degradation products of PEEP49-PCL36-PEEP49 were shown in Figure 6. The molecular weights of degraded products decreased significantly with the incubation up to 18 h. Molecules with low molecular weights were detected and their peak intensities increased gradually with longer incubation. The well-resolved peaks that eluted at 35.41, 33.43, and 32.28 min, corresponding to peak molecular weights (Mp) of 110, 310, and 530, were believed from 6-hydroxycaproic acid, dimer and trimer, respectively.28 It is also worth noting that the peak eluted at 27.20 min, corresponding to a peak molecular weight of 5070, which was kept unchanged during degradation. This peak is likely due to the generation of PEEP polymer. To further demonstrate this hypothesis, the degradation products after a 48 h incubation with enzyme were dialyzed and analyzed by 1H NMR. The results shown in Figure 7A revealed that signals assigned to protons of the PCL block almost disappeared, while signals assigned to protons of PEEP remained prominent, demonstrating that PEEP blocks could not be degraded by Pseudomonas lipase enzyme catalysis, as we reported earlier.29 An important advantage of PPE over conventionally used PEG is that PPE is biodegradable, while the degradation rate of PPE may be adjusted by controlling the chemical structure of the backbone and side chain. Moreover, by choosing biocompatible building blocks of the polymer, degradation products of PPE can have minimal toxic effects and good biocompatibility. In this study, we investigated the degradation of micelles accelerated by phosphodiesterase I, an enzyme present in cytosome or subcellular regions of human cells.30 The micelles with 0.2 g L-1 of phosphodiesterase I at pH ) 8.8 turned turbid after one day of incubation at 37 °C. It is believed that degraded residues were mainly composed of PCL, therefore, it was no longer thermodynamically stable in aqueous solution. 1H NMR analysis of the degradation products extracted by CDCl3 shown in Figure 7B revealed the sharply reduced

Self-Assembled Micelles of PEEP and PCL

Biomacromolecules, Vol. 9, No. 1, 2008 393

Figure 6. GPC chromatograms of PEEP49-PCL36-PEEP49 at different degradation times cultured with Pseudomonas lipase (0.2 g L-1) at 37 °C in phosphate buffer (0.05 M, pH 7.0).

Figure 7. 1H NMR spectra of degradation products of PEEP49-PCL36PEEP49: (A) Pseudomonas lipase (0.2 g L-1) in phosphate buffer (0.05 mol L-1, pH 7.0) at 37 °C for 2 days; (B) phosphodiesterase I (0.2 g L-1) in Tris-HCl (0.02 mol L-1, pH 8.8) at 37 °C for 7 days.

Figure 5. Intensity ration (I339.0/I335.5) as a function of concentration of PEEP-PCL-PEEP with different DP of PCL (A) or DP of PEEP (B) and the relationship between the DP of PCL, PEEP block, and CMC values (C).

proton signals of PEEP blocks, demonstrating the degradation of PPE catalyzed by phosphodiesterase I enzyme. In Vitro Cytotoxicity. The cytotoxicity of micelles to HEK293 cells was evaluated using the MTT method. Figure 8A shows the cell viability after a 24 h incubation with micelles of PEEP44-PCL27-PEEP44 at different concentrations. Sodium dodecyl sulfate was used as the control. It was demonstrated that cells cultured with PEEP44-PCL27-PEEP44 micelles remained viable when the concentration was up to 125 µg/mL. Slight decrease of cell viability was only observed when the micelle concentration was higher than 125 µg/mL. With the highest micelle concentration at 1.0 mg/mL, about 90% cell

viability was observed. Figure 8B shows that no significant cytotoxicity effects are associated with copolymer micelles at a concentration of 1.0 mg/mL, suggesting these polymer micelles have good biocompatibility and low cytotoxicity to HEK293 cells. Paclitaxel Loading into PEEP-PCL-PEEP Micelles. PTX, a chemotherapy anticancer drug, is most commonly used to treat a variety of cancers, especially ovarian, breast, and nonsmall cell lung cancers.31 Due to the high hydrophobicity of PTX, Cremopho EL (polyethoxylated caster oil) is used as the vehicle for clinical application. However, Cremophor EL may cause serious adverse effects, like nephrotoxicity and neurotoxicity.32,33 Therefore, it is indeed necessary to explore new vehicles for PTX or other hydrophobic drugs, which can otherwise increase the solubility of the drugs and reduce the toxicity of the formulation. In this study, we loaded PTX into PEEP-PCL-PEEP micelles using the dialysis method. The loading amounts and efficiencies were measured by HPLC after dissolving PTXloaded micelles with acetonitrile–water (50:50, v/v). The results summarized in Table 2 indicated that PEEP-PCL-PEEP micelles made microreservoirs that efficiently solubilized PTX. Such micelles solubilized PTX at a level of 0.56–3.16% (wt/wt) under our testing conditions, depending on the composition of block copolymers. The drug loading efficiency (DLE%) of the PTXincorporating micelles increased with increasing the molecular weight of the hydrophobic segment poly(-caprolactone). This result implies that the increase of the hydrophobic chain length

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Figure 9. Release profile of paclitaxel from PEEP-PCL-PEEP triblock micelle in PBS (pH 7.4) at 37 °C.

Figure 8. Cytotoxicity of micelles to HEK293 cells determined by MTT viability assay: (A) cell viability after 24 h incubation with PEEP44PCL27-PEEP44 micelle at different concentrations; and (B) cell viability after a 24 h incubation with various micelles of PEEP-PCL-PEEP at 1.0 mg/mL.

of the block copolymer may enhance the interaction with hydrophobic PTX, leading to an increase in the drug loading amount. However, the loading efficiency of PEEP49-PCL186PEEP49 with the longest PCL length was even lower is likely due to the strong interaction between the polymer and the PTX as well as the severe precipitation during dialysis. The drug loading efficiencies and amounts into these PEEP-PCL-PEEP micelles listed in Table 2 were relatively lower than that of other micellar systems34,35 It is believed that various factors, including physiochemical properties of polymers, organic

solvent used, and conditions for micelle preparation, such as dialysis time, temperature, and medium, would affect the drug loading.36 To achieve a higher loading efficiency and amount, the process to load PTX into these PEEP-PCL-PEEP micelles still remained to be optimized. It is worth noting that the average size of micelles was only slightly affected by loading PTX. Such an effect is a little pronounced when PCL blocks are longer, while it is conservative with increasing PEEP block length. In Vitro Drug Release Behavior. In vitro release behavior of PTX was carried out in PBS (0.02 M, pH ) 7.4) at 37 °C. As shown in Figure 9, after a burst release of PTX, primarily attributed to the diffusion of PTX located close to the surface of particles, a sustained release lasting for about 1 week from these micelles was observed. About 45–60% of the initial loading amount was eventually released from these micelles. Polymer structure was found to be correlative to the drug release kinetics. For example, PEEP49-PCL36-PEEP49 micelles gave a faster release, while PTX release from PEEP49-PCL186-PEEP49 micelles was slower, which may be due to a stronger interaction between PTX with a longer length of hydrophobic core. Additionally, it is possible that an increased length of hydrophobic block would increase the polymer aggregation number per micelle and decrease surface area, resulting in a relatively slower release.37,38 It is also observed that about 25–55% of loaded PTX were not released from the micelles, which may be associated with the hydrophobic core of micelles, as observed by other groups.37

Conclusion A series of biodegradable triblock copolymers with varied PCL and PEEP block lengths were synthesized through ringopening polymerization of cyclic phosphate monomer co-

Table 2. Properties of Micelles Pre- or Post-Paclitaxel-Loading Micelles before paclitaxel-loading block copolymer

Dh (nm)

PEEP44-PCL27-PEEP44 PEEP7-PCL36-PEEP7 PEEP21-PCL36-PEEP21 PEEP49-PCL36-PEEP49 PEEP45-PCL63-PEEP45 PEEP48-PCL94-PEEP48 PEEP49-PCL186-PEEP49

35 ( 1.2 78 ( 6.5 57 ( 1.4 47 ( 2.5 90 ( 3.2 108 ( 7.1 186 ( 10.8

polydispersity 0.12 ( 0.01 0.25 ( 0.04 0.09 ( 0.01 0.18 ( 0.02 0.16 ( 0.02 0.20 ( 0.02 0.25 ( 0.03

Micelles after paclitaxel-loading Dh (nm) 36 ( 2.5 88 ( 9.9 65 ( 3.2 49 ( 1.1 104 ( 5.7 126 ( 6.3 224 ( 22.5

polydispersity 0.15 ( 0.01 0.33 ( 0.03 0.17 ( 0.02 0.16 ( 0.02 0.12 ( 0.01 0.23 ( 0.02 0.39 ( 0.07

DLC%

DLE%

0.56 ( 0.03 0.92 ( 0.12 1.56 ( 0.04 1.32 ( 0.57 2.46 ( 0.64 3.16 ( 0.33 1.81 ( 0.15

5.6 ( 0.3 9.2 ( 1.22 15.6 ( 0.42 13.2 ( 5.73 24.6 ( 6.40 31.6 ( 3.34 18.1 ( 1.47

Self-Assembled Micelles of PEEP and PCL

initiated with Sn(Oct)2 and PCL with dihydroxyl end groups. The structure of the copolymers was confirmed by NMR, GPC, and FT-IR. These block copolymers formed nanosized micellar structures in aqueous solution spontaneously, while CMC and particle size can be tailored. In vitro cytotoxicity and degradation studies revealed that these triblock micelles are biocompatible and biodegradable. As a model hydrophobic drug, PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable copolymers are believed to be suitable for hydrophobic drug delivery. Acknowledgment. This work is partly supported by grants from the National Natural Science Foundation of China (NSFC; 20774089), the Knowledge Innovation Program of the Chinese Academy of Sciences (KJCX2-YW-M02), the Chinese Ministry of Sciences 973 Project (2006CB933300), and “Bairen” Program of Chinese Academy of Sciences.

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