Synthesis and characterization of amphiphilic poly(ɛ-caprolactone)-b-polyphosphoester diblock copolymers bearing multifunctional pendant groups

Polymer 53 (2012) 2854e2863

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Synthesis and characterization of amphiphilic poly(3-caprolactone)-bpolyphosphoester diblock copolymers bearing multifunctional pendant groups Haiyan Shao a, Mingzu Zhang a, Jinlin He a, Peihong Ni a, b, * a

Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University, Suzhou 215123, China b Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 March 2012 Received in revised form 4 May 2012 Accepted 7 May 2012 Available online 14 May 2012

A series of parent block copolyesters poly(3-caprolactone)-block-poly[2-(2-oxo-1, 3, 2dioxaphospholoyloxy)ethyl acrylate] (PCL-b-POPEA) with different block lengths have been synthesized by ring-opening polymerization (ROP) and four kinds of mercaptans were then used in the postpolymerization modification via Michael-type addition reaction, resulting in several block copolyesters with various functionalities (e.g., hydroxyl, carboxyl, amine, and amino acid) in their pendant groups. The chemical structures of these block copolymers were characterized by FT-IR, NMR spectroscopy and GPC analysis. The self-assembly behaviors of PCL-b-POPEA have been studied by fluorescence probe technique, transmission electron microscopy (TEM) and high-performance particle size (HPPS) instrument. In vitro cytotoxicity test indicated that the block copolymers possess good biocompatibility. Initial in vitro drug loading and release studies using Doxorubicin (DOX) as a model drug demonstrated a faster release in the presence of phosphodiesterase I as compared to the system without enzyme. Moreover, it was found that DOX-loaded nanoparticles displayed higher inhibition to KB cell proliferation in comparison with free DOX. Therefore, the combination of ROP and Michael-type addition reaction provides a general access to various types of multifunctional and biodegradable materials. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Drug delivery Polyphosphoester Poly(3-caprolactone)

1. Introduction In recent decades, amphiphilic copolymers have attracted growing interests for their unique ability of creating versatile selfassembled nanostructures. Since the pioneering works reported from the early 1990s [1e4], many different kinds of nanostructures, such as polymeric micelles [5e7], vesicles [8,9] and large compound micelles [10] have been studied. Amphiphilic copolymer micelles, with water-soluble shells for stabilizing nanoparticles and hydrophobic cores for accommodating water-incompatible molecules, have been widely used in many fields, such as antitumor drug or gene carriers [11,12], diagnostic imaging [6], nanoreactors [13], and so on. With respect to the drug delivery devices constructed from the self-assembly of amphiphilic copolymers, two important issues including biocompatibility and biodegradability have to be considered. Among these polymeric materials, biodegradable

* Corresponding author. Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University, Suzhou 215123, China. Tel.: þ86 512 65882047; fax: þ86 512 65880089. E-mail address: [email protected] (P. Ni). 0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2012.05.013

polymers are becoming one of the most attractive subjects because of their low immunogenicity, easily controlled degradation and mechanical properties. For example, polylactide (PLA) [10], polycaprolactone (PCL) [14] and polyglycolide (PGA) [15] are representative biocompatible and FDA-approved polyesters, and they can be degraded by enzymatic or hydrolytic degradation under physiological conditions. However, the limited structure and lack of functional groups for further modifications have restricted their applications to some extent in constructing complex and multifunctional biodegradable materials. Up to now, a great number of functional pendant groups (carboxyl, amino and hydroxyl etc.) have been introduced to polymer backbones [16e21], from which several advantages were obviously displayed, such as improved hydrophilicity and biodegradability, facilitated drug conjugation and further derivatization. However, it is worth noting that most of the modification methods involve multistep processes, for example, protection and deprotection procedures, and hence a more straightforward and efficient modification strategy is highly desirable. In recent years, the reactions of thiols with enes, whether proceeding by a radical (termed thioleene reaction) [22] or anionic chain (termed thiol-Michael addition) [23], are attractive methodologies for postpolymerization

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modifications. Several research groups have reported on degradable polymers possessing unsaturated carbonecarbon double bonds, like to acryloyl [24,25], allyl [26,27], and vinyl sulfone [28], which can be readily transformed into various functionalities through postpolymerization reactions. We have noted that the modifications of amphiphilic copolymers mainly focused on hydrophobic sections [29] whereas the functionalizations of hydrophilic chains are relatively rare. For the commonly used hydrophilic polymers such as poly(ethylene glycol) (PEG), poly(N-isopropyl acrylamide) (PNIPAAm) and poly(2hydroxypropyl methacrylate) (PHPMA), it is relatively difficult to impart further functionalities on these polymers. Moreover, although good biocompatibility has been well demonstrated, the biodegradability is another concern about these polymers. Polyphosphoesters (PPEs) have recently received considerable attentions in biomedical applications such as in drug and gene delivery, and tissue engineering due to their easy functionalization, favorable biocompatibility and biodegradability [30e32]. Wang et al. [33] utilized the protection/deprotection method to synthesize amphiphilic diblock copolymers of poly(3-caprolactone) and polyphosphoester bearing functional hydroxyl pendant groups, which could self-assemble into micellar or vesicular aggregates in aqueous solution. More recently, they reported on the synthesis and characterization of a series of biocompatible and biodegradable block copolymers of poly(3-caprolactone) with “Clickable” polyphosphoester (PPE) [34]. In addition, some linear or star-shaped block copolymers of poly(3-caprolactone) and poly(ethyl ethylene phosphate) have been synthesized and used as drug carriers for Doxorubicin or Paclitaxel delivery [35]. In this work, we propose a model of fully biodegradable amphiphilic copolymers, and report on the design, synthesis, and characterization of the amphiphilic block copolyesters with diverse functionalities. We aim to develop a general strategy for convenient

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introduction of various functional pendant groups covalently conjugated to polyphosphoester block by combination of ROP and Michael-type addition chemistry. Three key features of the amphiphilic and multifunctional polymers are reported: (i) the acryloyl groups on the polymer side chains allow to be quantitatively modified via Michael-type addition reaction, and biodegradable copolyesters with diverse functionalities would be prepared; (ii) the diblock copolyesters have good biocompatibility and are fully biodegradable; (iii) they can form stable micelles in aqueous solution and can be further used as drug delivery carriers. The parent diblock copolymer PCL-b-POPEA was synthesized via ROP of 3-caprolactone (CL) and cyclic phosphoester monomer [2(2-oxo-1, 3, 2-dioxaphospholoyloxy)ethyl acrylate] (OPEA). The pendant acryloyl groups in the copolymers could be modified via Michael-type addition reaction, and four mercaptans were selected for this reaction as shown in Scheme 1, resulting in a series of functionalized block copolymers bearing diverse pendant groups (e.g. hydroxyl, carboxyl, amine, and amino acid) in hydrophilic chains. On the other hand, on the basis of the amphiphilic structure, PCL-b-POPEA could self-assemble into micelles in aqueous solution and be further utilized as drug delivery carriers. Initial in vitro cytotoxicity test by MTT assay demonstrated that these block copolymers possess favorable biocompatibility. Moreover, it was found that DOX-loaded nanoparticles exhibited higher inhibition to KB cell proliferation in comparison with free DOX. 2. Experimental section 2.1. Materials 2-Chloro-2-oxo-1, 3, 2-dioxaphospholane (COP) was synthesized according to a method described previously and distilled under reduced pressure before use [36]. 2-Hydroxyethyl acrylate

Scheme 1. Schematic illustration of PCL-b-POPEA diblock copolymers with two functions: (I) extension to multifunctionalization via Michael-type addition reaction with various mercaptans; and (II) Self-assembly in aqueous solution and use as drug delivery carrier.

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(HEA, TCI), stannous octoate [Sn(Oct)2], 3-mercapto-propanoic acid (MPA, ACROS, 99%), 3-mercapto-1, 2-propaediol (TCI, 99%), cysteamine hydrochloride (ACROS, 98%), L-cysteine hydrochloride (TCI, 99%) were used as received. 3-Caprolactone (3-CL, Aldrich) was dried over calcium hydride (CaH2) for one day at room temperature and distilled under reduced pressure before use. Benzyl alcohol (BzOH), toluene, triethylamine (TEA) and N, N-dimethyl formamide (DMF) were purchased from Sinopharm Chemical Reagent Co. and distillated before use. Tetrahydrofuran (THF) was initially dried over potassium hydroxide for at least two days and then refluxed over sodium wire with benzophenone as an indicator until the color turned purple. KB cells were obtained from American Type Culture Collection (ATCC). 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenylte-trazolium bromide (MTT, Sigma), pyrene (Acros Organics), doxorubicin hydrochloride (99%, Beijing ZhongShuo Pharmaceutical Technology Development Co., Ltd.) and other chemicals were used as received. 2.2. Synthesis of monomer 2-(2-oxo-1, 3, 2-dioxaphospholoyloxy) ethyl acrylate (OPEA) Cyclic phosphoester monomer bearing acryloyl group was synthesized similar to the previously reported literature with some modifications [37]. In brief, HEA (10.0 g, 0.077 mol), TEA (7.8 g, 0.077 mol) were mixed in 100 mL of dry THF in a 250 mL freshly dried and argon-purged three-necked flask equipped with a dropping funnel and a magnetic bar. After cooling at 20  C for 15 min, 2-chloro-2-oxo-1, 3, 2-dioxaphospholane (COP, 11.0 g, 0.077 mol) in 30 mL of dry THF was added dropwise to the stirred solution over a period of 30 min. The mixture was maintained at 20  C overnight. The precipitate was filtered off and the filtrate was concentrated under reduced pressure, 50 mL of freshly dried diethyl ether was then added to precipitate a small amount of triethylammonium chloride. After removal of the solvent under vacuum, the colorless liquid OPEA was obtained (yield 70%). 2.3. Synthesis of poly(3 -caprolactone) macroinitiator Poly(3-caprolactone) with hydroxyl group in the chain end (PCLOH) was prepared by ring-opening polymerization of 3-CL in anhydrous toluene using benzyl alcohol as an initiator and Sn(Oct)2 as the catalyst. Briefly, 3-CL (28.5 g, 0.25 mol), benzyl alcohol (0.45 g, 4.2 mmol), and Sn(Oct)2 (0.42 g, 1 mmol) were dissolved in 25 mL of anhydrous toluene in a 50 mL of round-bottomed flask. After three times of degassingerefilling cycle, the solution was maintained at 90  C for 24 h. Toluene was evaporated under reduced pressure and the mixture was precipitated into 50 mL of cold diethyl ether twice. The white precipitate was collected and dried under vacuum at room temperature (yield 83%). 2.4. Synthesis of PCL-b-POPEA diblock copolymer PCL-b-POPEA diblock copolymers with different block lengths were synthesized through ring-opening polymerization of OPEA in anhydrous THF using PCL-OH as the macroinitiator and Sn(Oct)2 as the catalyst. A typical synthesis procedure for PCL70-b-POPEA23 is shown as follows: In one 50 mL of dry round-bottomed flask, PCL70OH (0.319 g, 0.04 mmol) and OPEA (0.267 g, 1.2 mmol) were dissolved in 7 mL of anhydrous THF. After stirring at 30  C for 10 min, Sn(Oct)2 (0.004 g, 0.01 mmol in 0.5 mL of THF) was added. The mixture was stirred for another 5 h and then concentrated under vacuum. The residue was precipitated in cold diethyl ether/methanol (10:1, v/v) and the diblock copolymer was obtained after drying under vacuum at room temperature (yield 75%).

2.5. Multifunctionalization of PCL-b-POPEA via Michael-type addition reaction In this part, four mercaptans including 3-mercapto-propanoic acid, cysteamine hydrochloride, L-cysteine hydrochloride and 3mercapto-1, 2-propaediol, were used to investigate the Michaeltype addition reaction. All the reactions were performed in DMF at room temperature in dark for three days with pyridine as the catalyst. The feeding molar ratio of acryloyl group (AC), mercaptan (ReSH), and pyridine (Py) (AC/ReSH/Py) was 1/10/10. The typical reaction procedure between PCL70-b-POPEA23 and 3-mercaptopropanoic acid was described here as an example, other reactions were conducted in a similar way. In a 50 mL of dry round-bottomed flask, PCL70-b-POPEA23 (0.1 g, 0.007 mmol), 3-mercapto-propanoic acid (0.17 g, 0.07 mmol) and pyridine (0.127 g, 0.07 mmol) were dissolved in 3 mL of anhydrous DMF, and the reaction was carried out at room temperature for three days in dark. The resulting mixture was precipitated into 50 mL of cold diethyl ether twice, and the precipitates were then collected and redissolved in 2 mL of DMF, which was placed in a dialysis bag (MWCO 3500) and dialyzed against water for three days to completely remove residual small molecules. The final product was obtained after freeze-drying. 2.6. Preparation of polymer films and contact angle measurements Thin films of PCL-OH, PCL-b-POPEA and four modified polymers were prepared by casting polymer solutions in DMF (0.5 wt%) on microscope slides. The films on the slides were dried by placing in a desiccator for 12 h followed by vacuum-drying for three days to remove DMF thoroughly. The static contact angle of the films was measured using a JC2000C1 contact angle goniometer (Shanghai Zhongchen Powereach Co., China). A droplet of Milli-Q water (2 mL) was placed on the surface of the tested films at room temperature, and the contact angle was measured at once when the water droplet touched the tested films. 2.7. Cell viability test The in vitro cytotoxicity of various polymer samples was measured using a standard MTT assay. KB cells were cultured in RPMI-1640 growth medium supplemented with 10% heatinactivated fetal bovine serum (FBS), 1% penicillin, and streptomycin, at 37  C in 5% CO2 atmosphere. For the toxicity test, cells were seeded in a 96-well plate at a density of about 10,000 cells per well in 100 mL of RMPI-1640 growth medium for 24 h. After incubating KB cells with various concentrations of block copolymer PCL70-b-POPEA23 micelles and the other four modified copolymers micelles for 24 h. 25 mL of MTT stock solution (5 mg mL1 in PBS) was added to each well and further incubated for 30 min. Finally, the absorbance at 490 nm of each well was measured using a BioRad model 680 microplate reader to obtain the optical density value. The cell viability (%), relative to that of control cells cultured without the addition of micelles, was calculated from [A]test/ [A]control  100%, where [A]test and [A]control are the absorbance values of the testing well (with the micelles) and the control well (without the micelles), respectively. Errors are based on the standard deviations of triplicate samples. 2.8. Formation of PCL-b-POPEA micelles and DOX-loaded micelles The PCL-b-POPEA micelles were prepared by a dialysis method. In a typical procedure, 25 mg of block copolymer was dissolved in 3 mL of DMF. After complete solubilization, 10 mL of Milli-Q water was added dropwise under moderate stirring. The mixture was left stirring for an additional 3 h and placed in a dialysis bag (MWCO

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3500) for three days to remove DMF. DOX-loaded micelles were prepared in the similar way. Briefly, 20 mg of PCL-b-POPEA was dissolved in 2 mL of DMF, followed by adding a predetermined amount of DOX$HCl and 2 equiv. of TEA, and then stirring at room temperature in dark for 2 h. Subsequently, 10 mL of Milli-Q water was added dropwise under moderate stirring. After stirring for an additional 2 h, the solution was dialyzed against Milli-Q water for three days (MWCO 3500), during which the water was renewed every 4 h. To determine the drug loading parameters, the DOXloaded micelles solution were lyophilized and then dissolved in DMF. The fluorescence absorbance at 480 nm was measured to determine the DOX concentration. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated by the following equations:

DLC% ¼

amount of DOX in micelles 100% amount of DOX  loaded micelles

DLE% ¼

amount of DOX in micelles  100% total amount of DOX for drug loading

2.9. In vitro drug release The DOX release study was separately performed in PBS buffer (pH 7.4, 0.01 M) and PBS buffer (pH 7.4, 0.01 M with 5 mM Mg2þ) with 0.25 mg mL1 phosphodiesterase I. Typically, 5 mL of DOXloaded PCL-b-POPEA micelles were transferred into a dialysis bag (MWCO 14,000), which was immersed into a tube containing 25 mL of PBS buffer. The tube was put in a shaking water bath at 37  C. At predetermined time intervals, 5 mL of the external buffer solution was withdrawn and replaced with 5 mL of fresh buffer solution. The fluorescence absorbance at 480 nm was measured to determine the DOX concentration. All release experiments were conducted in dark, and the results are the average data from triplicate samples with standard deviations. 2.10. In vitro enzymatic degradation of PCL-b-POPEA micelles The in vitro enzyme-catalyzed degradation of PCL-b-POPEA micelles was performed at 37  C with 0.2 mg mL1 phosphodiesterase I in TriseHCl buffer at pH 8.8. The concentration of PCL-bPOPEA micelles was set at 1.0 g L1, and the mixture solution was taken out three days later and freeze-dried. The obtained white powder was dissolved in chloroform, which was filtered to remove the insoluble salt and the filtrate was concentrated under reduced pressure. The degraded product was then dried under vacuum and analyzed by 1H NMR in CDCl3. 2.11. In vitro antitumor test The cytotoxicity of DOX-loaded micelles against KB cells was evaluated in vitro by MTT assay using free DOX and blank micelles as the controls, respectively. KB cells were cultured in RPMI-1640 growth medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin, and streptomycin, at 37  C in 5% CO2 atmosphere. For the cytotoxicity test, cells were seeded in a 96-well plate at a density of about 10,000 cells per well in 100 mL of RMPI1640 growth medium for 24 h. After incubating KB cells with various concentrations of DOX-loaded PCL70-b-POPEA23 micelles, blank PCL70-b-POPEA23 micelles, and free DOX for 24 h, 25 mL of MTT stock solution (5 mg mL1 in PBS) was added to each well and further incubated for 30 min. Finally, the absorbance at 490 nm of each well was measured by a Bio-Rad model 680 microplate reader

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to obtain the optical density value. The cell viability (%), relative to that of control cells without the addition of samples, was calculated from [A]test/[A]control  100%, where [A]test and [A]control are the absorbance values of the testing well (with the samples) and the control well (without the samples), respectively. Errors are based on the standard deviations of triplicate samples. 2.12. Characterizations 1

H NMR, 13C NMR, and 31P NMR spectra were performed on an INOVA-300 NMR spectrometer with CDCl3 as a solvent and TMS as an internal reference. Phosphoric acid (85%) was used as the external reference for 31P NMR analysis. FT-IR spectra were recorded on a Nicolet AVATAR 360 Fourier transform infrared spectrometer using the KBr disk method. The molecular weights and molecular weight distributions of polymers were measured by a Waters 1515 GPC instrument with a Waters 1515 Isocratic HPLC pump, a Waters 2414 refractive index detector, a Waters 2487 dualwavelength absorbance detector, and a set of Waters Styragel columns (HR3, HR4, and HR5, 7.8 mm  300 mm). GPC measurements were carried out at 35  C using DMF with 0.05 mol L1 LiBr as eluent and the flow rate was 1.0 mL min1. The calibration was carried out with polystyrene standards. The critical aggregation concentration (CAC) values of the diblock copolymers were determined by the fluorescence probe method using pyrene as hydrophobic probe on a FLS920 fluorescence spectrofluorometer (Edinburgh Co., UK), a predetermined amount of pyrene in acetone was added into a series of ampules, and the acetone was allowed to evaporate under vacuum. 10 mL of aqueous solutions at different concentrations of copolymers were then added to the ampules, and the concentration of pyrene was kept at 6  107 M. The aqueous solutions of copolymers were allowed to stir for 24 h at room temperature to reach the solubilization equilibrium of pyrene. Excitation was carried out at 335 nm, and emission spectra were recorded ranging from 350 nm to 500 nm. Both excitation and emission bandwidths were set at 1 nm. From the pyrene emission spectra, the intensity ratio (I3/I1) of the third band (383 nm, I3) to the first band (373 nm, I1) was analyzed as a function of polymer concentration. The CAC value was defined as the point of intersection of the two lines in the plot of fluorescence versus concentration. The experiments were conducted in triplicate, and the average values were reported. The morphologies of the diblock copolymer micelles were observed on a TEM instrument (TECNAI G2 20, FEI) at 200 kV. The micelles with a concentration of 1 mg mL1 were prepared by a dialysis method and each sample was vigorously stirred for three days before use. A drop of micelle solution was dript on an electron microscopy copper grid coated with carbon film, which was allowed drying at room temperature for 24 h before measurement. The particle sizes (Dz ) and size polydispersities (size PDI) of the diblock copolymer micelles were measured with a Malvern HPPS 5001 high-performance particle size instrument equipped with a 3.0 mW laser operating at l ¼ 633 nm. All the micelle solutions with a concentration of 1 mg mL1 were prepared as described in TEM analysis and then passed through 0.45 mm filter before measurements. All measurements were carried out at 25  C and data were analyzed by Malvern Dispersion Technology Software 3.0. 3. Results and discussion 3.1. Synthesis and characterization of PCL-b-POPEA diblock copolymer The amphiphilic polyesters PCL-b-POPEA with pendant acryloyl groups in polyphosphoester block were synthesized via ring-

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Scheme 2. Representative synthesis routes of diblock copolymer PCL-b-POPEA.

opening polymerization (ROP) as shown in Scheme 2. The cyclic phosphoester monomer OPEA bearing acryloyl group was first prepared through a reaction between 2-hydroxyethyl acrylate (HEA) and 2-chloro-2-oxo-1, 3, 2-dioxaphospholane (COP) in THF at 20  C according to the previous reports [29,36]. The chemical structure of OPEA was well characterized by NMR analysis (1H NMR, 13C NMR and 31P NMR) and the results are shown in Fig. S1 and Fig. S3(A) in the Supporting information. Poly(3-caprolactone) macroinitiator (PCL-OH) was obtained by ROP of 3-CL in toluene using benzyl alcohol as the initiator and Sn(Oct)2 as catalyst. Subsequently, PCL-OH was used to polymerize OPEA under the catalysis of Sn(Oct)2 in THF at 35  C, resulting in the diblock copolymer PCL-b-POPEA. Table 1 lists the characterization data of a series of PCL-OH and PCL-b-POPEA polymers, and the molecular weights of PCL and POPEA blocks can be controlled by simply changing the molar ratios of monomers and initiators. A typical GPC overlay of the macroinitiator and block copolymer is shown in Fig. 1, from which one can find the increasing molecular weight after block copolymerization, indicating the successful preparation of PCL-b-POPEA diblock copolymer. The FT-IR spectra of PCL70-OH macroinitiator and PCL70-bPOPEA23 diblock copolymers are shown in Fig. S2 in the Supporting information. The peak at 1730 cm1 can be ascribed to the characteristic C]O stretching vibration from PCL block, while the asymmetrical and symmetrical P]O stretching vibration are located at 1276 cm1 and 1158 cm1. Moreover, the PeOeC stretching vibration is also verified at 988 cm1 and the peak at 1635 cm1 is the characteristic signal of C]C stretching vibration. Fig. 2(A,B) displays the 1H NMR spectra of PCL70-OH macroinitiator and PCL70-b-POPEA45 diblock copolymer, respectively. And notably all the proton signals on the polymers can be found. The average

Fig. 1. GPC curves of PCL40-OH macroinitiator (M n; GPC ¼ 5.15  103 g mol1, PDI ¼ 1.10), and PCL40-b-POPEA20 diblock copolymer (M n; GPC ¼ 9.81  103 g mol1, PDI ¼ 1.23).

degree of polymerization of PCL-OH was estimated from 1H NMR spectrum on the basis of integration ratio of peak at d w5.10 ppm to that at d w4.05 ppm assigned to the methylene protons b and f in Fig. 2(A). After polymerization of the second monomer OPEA, the newly appeared resonances in Fig. 2(B) can be ascribed to the protons from POPEA block. Similarly, the number-average molecular weight of POPEA block can be also calculated from 1H NMR spectrum on the basis of integration ratio of peak c at d w 2.27 ppm to that of peak h at d w 6.18 ppm. The 13C NMR spectrum shown in Fig. 2(C) also confirms the chemical structure of PCL70-b-POPEA45 diblock copolymer. Furthermore, 31P NMR measurement was carried out to confirm the structure of polyphosphoester and the result is depicted in Fig. S3. One can find from Fig. S3(A) that the spectrum of monomer gives a strong resonance at d w 17.7 ppm, while for block copolymer, the strong resonance appear at about

Table 1 Characterization data of the compositions, number-average molecular weights and molecular weight distributions (PDI) of PCL-OH macroinitiator and PCL-b-POPEA diblock copolymers. Samples

M n; theor: a (g mol1)

PCL40-OH PCL40-b-POPEA20 PCL60-OH PCL60-b-POPEA25 PCL70-OH PCL70-b-POPEA23 PCL70-b-POPEA45 PCL96-OH PCL96-b-POPEA4

4.56 9.00 6.84 12.3 7.98 13.1 17.9 10.9 11.8

        

103 103 103 103 103 103 103 103 103

M n; NMR b (g mol1) 4.90 9.05 7.98 13.1 8.55 13.2 18.7 11.0 11.9

        

103 103 103 103 103 103 103 103 103

M n; GPC c (g mol1) 5.15 9.81 9.51 11.6 8.49 11.8 15.3 11.5 10.1

        

103 103 103 103 103 103 103 103 103

PDIc 1.10 1.23 1.05 1.05 1.17 1.12 1.05 1.16 1.24

a

Theoretical molecular weight. Calculated on the basis of 1H NMR analysis in CDCl3. Determined by GPC in DMF with 0.05 mol L1 LiBr, using polystyrene as standards. b c

Fig. 2. 1H NMR spectra of (A) PCL70-OH macroinitiator and (B) PCL70-b-POPEA45 diblock copolymer; and (C) 13C NMR spectrum of PCL70-b-POPEA45 diblock copolymer.

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Scheme 3. Extension of PCL-b-POPEA to multifunctionalization via Michael-type addition reaction, using four mercaptans: (A) 3-mercapto-propanoic acid, (B) cysteamine Hydrochloride, (C) L-cysteine hydrochloride, and (D) 3-mercapto-1, 2-propaediol.

d w 0.9 ppm assigning to the phosphorus atoms in polyphosphoester backbone except the weak signals generated at around d w 0.1 ppm, which can be ascribed to the phosphorous atom linked with PCL block. Therefore, judging together with the aforesaid analysis, we can conclude that the PCL-b-POPEA diblock copolymers have been successfully synthesized. 3.2. Multifunctionalization of PCL-b-POPEA via Michael-type addition reaction To construct a synthetic methodology for the preparation of multifunctional and amphiphilic copolyesters, four thiolcontaining molecules including 3-mercapto-propanoic acid, cysteamine hydrochloride, L-cysteine hydrochloride and 3-mercapto-1,

2-propaediol were selected to react with the acryloyl groups via Michael-type addition reaction, as shown in Scheme 3. All the reactions were performed in DMF with pyridine as the catalyst at room temperature in dark for three days. The feeding molar ratio of acryloyl group (AC), thiol-containing molecules (ReSH), and pyridine (Py) (AC/ReSH/Py) was 1/10/10. 1H NMR spectra from Fig. 3 reveal the complete disappearance of peaks assignable to acryloyl groups and the occurrence of new signals corresponding to cysteamine, L-cysteine, and 3-mercapto-1, 2-propaediol moieties, respectively, indicating 100% functionalization with these three molecules. While the system treated with 3-mercapto-propanoic acid yielded only ca. 60% conversion of acryloyl groups (Fig. 3(A)), which is in agreement with the previous reports that the reactivity of mercaptans was greatly weakened by the neighboring carboxylic

Fig. 3. 1H NMR spectra of functional diblock copolyesters derived from PCL70-b-POPEA23 modified respectively with (A) 3-mercapto-propanoic acid, (B) cysteamine hydrochloride, (C) L-cysteine hydrochloride, and (D) 3-mercapto-1, 2-propaediol.

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Fig. 4. Photographs of water droplets on various polymer films and the obtained contact angle values. (A) PCL70-OH as a control; (B) PCL70-b-POPEA45 diblock copolymer; (C)e(F) functionalized PCL70-b-POPEA45 diblock copolymers modified by 3-mercapto-propanoic acid (signated as COOH), cysteamine hydrochloride (NH2), L-cysteine hydrochloride (COOH, NH2), and 3-mercapto-1, 2-propaediol [(OH)2], respectively.

groups in Michael-type addition modification [25,38]. Furthermore, thin films of functionalized copolyesters were prepared by casting the polymer solutions onto microscope slides. The static contact angle measurements displayed in Fig. 4 demonstrate that after modification with the mercaptans, all the functionalized copolymers displayed the decrease of water contact angle and the increase of hydrophilicity as compared to the parent copolymer. These results indicate that various kinds of functional molecules can be efficiently incorporated onto the polymer chains in a relatively mild condition. Biocompatibility is one of the most important properties for polymer materials used in biomedical applications. It has been welldocumented that both polycaprolactone (PCL) and polyphosphoesters

(PPE) have low cytotoxicity and good biocompatibility [29,31,32,39]. In the present work, in vitro cytotoxicity of parent copolymer PCL70-b-POPEA23 and the other four functionalized copolymers against KB cells were evaluated using MTT assay. As shown in Fig. 5, the viabilities of KB cells incubated with each sample are all above 80% after 24 h of incubation up to the highest testing concentration of 400 mg mL1. The results demonstrate that the PCL70-b-POPEA23 has good biocompatibility against KB cells (see Fig. 5(A)), which is suitable for drug delivery vector and the detailed discussion will be shown later. Meanwhile, all the diblock copolymers modified with the four kinds of mercaptans show favorable cytocompatibility. The cytotoxicity tests demonstrate that the multifunctionalized diblock copolymers modified via Michael-type addition reaction maintain favorable biocompatibility. Furthermore, it is envisioned that these functional groups can be further conjugated with drug molecules or fluorescent agents, and the detailed investigations on this point are ongoing in our laboratory. 3.3. Micellization of PCL-b-POPEA diblock copolymers Amphiphilic diblock copolymer PCL-b-POPEA can self-assemble and readily form nanoparticles in water. We first used fluorescence probe method to determine the critical aggregation concentration (CAC) of PCL-b-POPEA diblock copolymers. Pyrene was used as the probe because the ratio of the intensity of the third and the first vibronic peaks at 383 nm and 373 nm (I3/I1) in pyrene fluorescence Table 2 Properties of the PCL-b-POPEA micelles.

Fig. 5. Cell viability test of KB cells incubated with (A) PCL70-b-POPEA23, and the modified copolymers with (B) 3-mercapto-propanoic acid, (C) cysteamine hydrochloride, (D) L-cysteine hydrochloride, and (E) 3-mercapto-1, 2-propaediol.

Samples

CAC (mg mL1)

PCL40-b-POPEA20 PCL60-b-POPEA25 PCL70-b-POPEA23 PCL70-b-POPEA45 PCL96-b-POPEA4

1.5 3.0 1.8 1.5 3.0

    

102 103 103 102 104

Dz (nm)

Size PDI

173 149 121 114 186

0.442 0.178 0.276 0.173 0.392

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Fig. 6. TEM images of (A) micelles obtained from PCL70-b-POPEA45, and (C) DOX-loaded micelles of PCL70-b-POPEA45 copolymer. (B) and (D) are the plots of particle size distributions corresponding to those samples in (A) and (C). In the top right of (C), an amplified TEM image of a DOX-loaded micelle (bar ¼ 20 nm).

the potential of PCL-b-POPEA micelles in biomedicine, an anticancer drug doxorubicin (DOX) was encapsulated into the core of the micelles using a dialysis method. Fig. 6(C and D) displays the TEM image of the drug-loaded micelles with a diameter around 50 nm and the curve of the size distribution of drug-loaded micelles, respectively. The drug loading content and drug loading efficiency into the micelles were determined by using fluorescence measurement with excitation at 480 nm, and estimated to be 10% and 33.3%, respectively. Phosphodiesterase I is a well-known enzyme to accelerate degradation of PPE and it is present in cytosome or subcellular regions of human cells [43]. Initial drug release experiments of DOX from PCL70-b-POPEA23 micelles were carried out in two kinds of media at 37  C, one was PBS solution (pH 7.4) and another was the same buffer with 0.2 mg mL1 phosphodiesterase I. The cumulative release profile of DOX from the micelles is

80

Cumulative release (%)

spectrum is sensitive to the polarity of medium, and the I3/I1 ratio was large in a less polar medium. A typical spectrum of I383/I373 as a function of polymer concentration is shown in Fig. S4 in the Supporting information, and the properties of micelles selfassembled from these diblock copolymers are summarized in Table 2. One can find that the CAC values decrease with the increase of PCL block length, which is due to the stronger interactions occurred in longer hydrophobic chains, resulting in a lower CAC value. The particle size and size distribution of the polymeric micelle are important parameters for drug delivery application. Small size micelles (<200 nm in diameter) are beneficial to maintain a lower level of reticuloendothelial system (RES) uptake [40,41], minimal renal excretion, and the ability to take advantage of the enhanced permeability and retention (EPR) effect for passive tumor targeting [42]. The morphology, particle size and size distribution of the selfassembled nanoparticles of PCL-b-POPEA block copolymers were determined by TEM and HPPS. As an example, Fig. 6(A) shows the typical TEM image of micelles self-assembled from PCL70-b-POPEA45 (1 mg mL1 in aqueous solution), from which one can find that they mainly formed the uniform micelles with an average size about 20 nm. The corresponding size distribution curve measured by HPPS for the micelles in aqueous solution displays a monomodal peak (see Fig. 6(B)) with an average diameter of 114 nm and size polydispersity index (size PDI) of 0.173. The large difference in particle size between the results of TEM and HPPS is maybe because the hydrophilic POPEA segment can extend into water phase in HPPS measurement, while in TEM analysis, the hydrophilic chains tend to collapse and it is very difficult to observe them from TEM images.

40

The PCL-b-POPEA micelles with good biocompatibility and appropriate sizes are suitable for drug delivery vehicles. To evaluate

Without Phosphodiesterase I

20

0 3.4. Initial loading and release of DOX

With Phosphodiesterase I

60

0

10

20

30

40

50

Time (h) Fig. 7. Cumulative release of DOX from PCL70-b-POPEA23 micelles in pH 7.4 PBS solution without (-) and with (C) 0.2 mg mL1 phosphodiesterase I at 37  C.

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Fig. 8. 1H NMR spectra of (A) PCL70-b-POPEA23, and (B) the enzymatic degradation product in the presence of phosphodiesterase I (0.2 g L1) in TriseHCl (0.02 mol L1, pH 8.8) at 37  C for 3 days.

depicted in Fig. 7, from which we can find that only 28% of drug was released after 48 h of incubation at pH 7.4 in the absence of phosphodiesterase I. However, the drug released about 62% in the first 10 h in the presence of phosphodiesterase I, which was much faster than that without enzyme. This could be ascribed to the fast degradation of polyphosphoester and the resulting instability of the micelles. This kind of enzymatic degradation behavior of PCL70-bPOPEA23 copolymer was also studied by analyzing the degradation products using 1H NMR analysis and the result is shown in Fig. 8, in which the chemical shifts attributed to the proton signals of POPEA block (peaks g, h and i) were completely disappeared after degradation process. 3.5. In vitro antitumor effect Here, the ability of DOX-loaded PCL70-b-POPEA23 micelles with different concentrations to inhibit proliferation of KB cells was investigated using MTT assay, in which free DOX and blank PCL70-bPOPEA23 micelles were used as the controls. The KB cells were treated with DOX-loaded micelles with DOX dosage ranging from

Fig. 9. In vitro inhibition to KB cell proliferation after 24 h incubation with DOX-loaded PCL70-b-POPEA23 micelles and free DOX at various DOX concentrations, PCL70-bPOPEA23 micelles were used as the control.

0.17 mg mL1 to 6 mg mL1. It can be found from Fig. 9 that the blank micelles show minimal cytotoxicity to the cells, whereas the DOXloaded micelles exhibit higher inhibition to KB cell proliferation after 24 h culture in comparison with free DOX. The DOX dosage required for 50% cellular growth inhibition (IC50) is about 1.4 mg mL1 in treatment with DOX-loaded micelles, which is much lower than that required for treatment with free DOX (4 mg mL1). It is conceivable that the free DOX molecules across the cell membrane directly is a less efficient means to deliver drugs into the cells, because the drug molecules diffuse in and out of the cell depending on the concentration gradient. However, the endocytotic uptake of cells for DOX-loaded micelles is unidirectional. Hence, the drug is not only more efficiently transported but also retained at the site of action inside the cells. Enhanced inhibition of DOX-loaded micelles to cancer cell growth and persistently intracellular release of DOX from the micelles inside the cells would be advantageous in lowering the dosage of DOX in applications [44].

4. Conclusions A general methodology for the preparation of fully biodegradable and multifunctional amphiphilic copolymers has been developed. The chemical structures and molecular weights of the obtained copolymers were confirmed by 1H NMR, 13C NMR, and 31P NMR, GPC and FT-IR measurements. The acryloyl groups in the hydrophilic polyphosphoester segments can be conveniently and efficiently modified via Michael-type addition reaction and various kinds of functionalities including hydroxyl, amine, carboxyl, and amino acid can be introduced to the polymers under mild conditions which do not involve any metal catalyst, protection/deprotection procedures, and toxic byproduct. The micellization of these amphiphilic copolymers were characterized by fluorescence probe technique, TEM, and HPPS. MTT assay against KB cells indicated that these polymeric micelles have low cytotoxicity and favorable biocompatibility. DOX was loaded into the micelles using the dialysis method and the release of DOX from the micelles was accelerated in the presence of phosphodiesterase I, which was also confirmed by the 1H NMR analysis of the degradation product. In addition, the DOX-loaded micelles exhibited higher inhibition to KB cell proliferation in comparison with free DOX. We are convinced

H. Shao et al. / Polymer 53 (2012) 2854e2863

that these multifunctionalized and fully biodegradable amphiphilic copolymers have broad potential in biomedical applications. Acknowledgements The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (21074078 and 20974074), the Natural Science Foundation of Jiangsu Province for Rolling Support Project (BK20110045), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Province Key Laboratory of Stem Cell Research, Soochow University. J. He would like to thank the financial support from the Innovative Graduate Research Program of Jiangsu Province, China (CX09B_021Z). The authors are also grateful to Prof. Bingyan Li, Prof. Zhuang Liu and Dr. Aiqing Wang for their valuable help and discussion in cytotoxicity tests. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.polymer.2012.05.013. References [1] Hawker CJ, Wooley KL, Frechet JMJ. J Chem Soc Perkin Trans 1 1993;97: 1287e97. [2] Thurmond II KB, Kowalewski T, Wooley KL. J Am Chem Soc 1996;118(30): 7239e40. [3] Zhang LF, Yu K, Eisenberg A. Science 1996;272(5269):1777e9. [4] Discher DE, Eisenberg A. Science 2002;297(5583):967e73. [5] Rösler A, Vandermeulen GWM, Klok HA. Adv Drug Deliv Rev 2001;53(1): 95e108. [6] Ghoroghchian PP, Frail PR, Susumu K, Blessington DB, Brannan AK, Bates FS, et al. Proc Natl Acad Sci USA 2005;102(8):2922e7. [7] Nie L, Liu SY, Shen WM, Chen DY, Jiang M. Angew Chem Int Ed 2007;119(33): 6437e40. [8] Nikolic MS, Olsson C, Salcher A, Kornowski A, Rank A, Schubert R, et al. Angew Chem Int Ed 2009;48(15):2752e4. [9] Wang XF, Zhang YF, Zhu ZY, Liu SY. Macromol Rapid Commun 2008;29(4): 340e6. [10] Yi Z, Liu XB, Jiao Q, Chen EQ, Chen YM, Xi F. J Polym Sci Part A Polym Chem 2008;46(12):4205e17. [11] Zhang WL, Li YL, Liu LX, Sun QQ, Shuai XT, Zhu W, et al. Biomacromolecules 2010;11(5):1331e8. [12] Zhang WL, He JL, Liu Z, Ni PH, Zhu XL. J Polym Sci Part A Polym Chem 2010; 48(5):1079e91. [13] Rossbach BM, Leopold K, Weberskirch R. Angew Chem Int Ed 2006;45(8): 1309e12. [14] Butsele KV, Jérôme R, Jérôme C. Polymer 2007;48(26):7431e43. [15] Baran J, Penczek S. Macromolecules 1995;28(15):5167e76. [16] Barrera DA, Zylstra E, Lansbury PT, Langer R. J Am Chem Soc 1993;115(23): 11010e1.

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