MPEG-PLA block copolymer conjugate

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Biomaterials 26 (2005) 2121–2128

Synthesis and characterization of the paclitaxel/MPEG-PLA block copolymer conjugate Xuefei Zhanga, Yuxin Lib, Xuesi Chena, Xiuhong Wangb, Xiaoyi Xua, Qizhi Lianga, Junli Hua, Xiabin Jinga,* a State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun 130022, PR China b Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, PR China

Received 29 April 2004; accepted 15 June 2004 Available online 20 July 2004

Abstract A paclitaxel/MPEG-PLA block copolymer conjugate was prepared in three steps: (1) hydroxyl-terminated diblock copolymer of monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA) was synthesized by ring-opening polymerization of l-lactide using MPEG as a maroinitiator; (2) it was converted to carboxyl-terminated MPEG-PLA by reacting with mono-t-butyl ester of diglycolic acid and subsequent deprotecting the t-butyl group with TFA; (3) the latter was reacted with paclitaxel in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine. Structures of the polymers synthesized were confirmed by 1H NMR, and their molecular weights were determined by gel permeation chromatography. The antitumor activity of the conjugate against human liver cancer H7402 cells was evaluated by MTT method. The results showed that paclitaxel can be released from the conjugate without losing cytotoxicity. r 2004 Elsevier Ltd. All rights reserved. Keywords: Paclitaxel; Prodrug; Ring-opening polymerization; Diglycolic anhydride

1. Introduction Paclitaxel is one of antineoplastic agents, originally extracted from the bark of the Pacific yew, Taxus brevifolin [1]. It has been shown to exhibit a significant activity against various solid tumors, including advanced ovarian carcinoma, metastatic breast cancer, non-small cell lung cancer, head and neck carcinomas [2–5]. It has a unique mechanism of action different from other anticancer agents [6]. It involves polymerization of tubulin dimmers to form microtubules and subsequent stabilization of the microtubules by preventing their depolymerization. The microtubules formed due to paclitaxel action are stable and thus dysfunctional, leading to cell death by disrupting the normal tubule dynamics required for cell division and vital interphase process [7]. *Corresponding author. Tel.: +86-431-526-2112; fax: +86-431-5685653. E-mail address: [email protected] (X. Jing). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.06.024

Because of its high activity against many kinds of cancers, great efforts have been devoted to development of new delivery systems for paclitaxel with the aim of overcoming the main problems encountered in its use, such as very low water solubility and hypersensitivity reactions associated with its current formulation which contains Chremophor EL and ethanol. The systems that have been studied include parenteral emulsions [8], liposomes [9], micelles [10], polymeric micro/nanoparticles [11–14], and water-soluble prodrugs [15]. Among these new formulations, water-soluble prodrugs seem to be the most promising, because they provide an approach to aqueous solubilization and reformulation of paclitaxel [16]. Prodrug is a chemical derivative of an active parent drug which has modified physicochemical properties such as aqueous solubility and biodistribution, and keeps the inherent pharmacological properties of the drug intact [17]. The structure-activity relationships of paclitaxel has been explored extensively with the aim of preparing water-soluble prodrugs. It has been

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established that the 20 - or 7-hydroxy group of paclitaxel is suitable for structure modification. A lot of attempts have been made to connect low-molecular-weight solubilizing moieties at the C20 or C7 position. These prodrugs are mainly ester derivatives including succinate, sulfonic acid, and amino acid and phosphate derivatives [15,18–21]. Although these derivatives possess adequate aqueous solubility, some of them have no antitumor activity because they are too stable to release the parent drug, and several of them are not suitable for i.v. injection because of their instability in aqueous solution at neutral pH. Conjugation of paclitaxel to water-soluble macromolecular carriers, such as PEG [16], PG [22] and HMPA [23], recently attracted more and more attention. Not only it could improve solubility of the drug but also produce desirable pharmacokinetics and enhance antitumor activity. However, some of those conjugates released the parent drug too quickly. To overcome this disadvantage, an amino acid [24] or polypeptide spacer [23] was introduced between the paclitaxel and watersoluble polymer to adjust the release rate of the parent drug, and enhanced antitumor activity and therapeutic index were observed. But to our knowledge, few polyester spacers of low molecular weight have been reported. In this paper, we report a new paclitaxel conjugate, monomethoxy-poly(ethylene glycol)-b-poly(lactide) (MPEG-PLA)-paclitaxel, in which paclitaxel is covalently connected to block copolymer MPEG-PLA. The latter is amphiphilic and can form micelles in an aqueous system with PLA block in the core and MPEG in the shell. Therefore they are expected to be dispersed in aqueous media easily. As paclitaxel is highly hydrophobic, MPEG-PLA-paclitaxel is expected to form the same micelles with paclitaxel in the cores. Because paclitaxel is capsulated by PLA and covalently connected to PLA, its release rate is expected to be slower than that in the case of PEGpaclitaxel. Furthermore, owing to the biodegradability of PLA, the release of paclitaxel will be caused not only by the hydrolysis of the ester linkage directly formed by paclitaxel with PLA, but also by the biodegradation of PLA block itself. Therefore, the release kinetics of paclitaxel may be adjusted to certain extent. To prepare the conjugate MPEG-PLA-paclitaxel, a hydroxyl-terminated diblock copolymer MPEG-PLA was first synthesized by ring-opening polymerization of lactide with stannous octoate (Sn(Oct)2) as a catalyst and in the presence of monomethoxy-terminated PEG as a macroinitiator. The hydroxyl group of MPEG-PLA was then converted into a carboxyl group and further reacted with paclitaxel. The chemical structure and the cytotoxicity of the conjugate were experimentally investigated.

2. Experimental section 2.1. Material MPEG with a molecular weight of 5000 was obtained from Aldrich. Prior to use, MPEG was dried by an azeotropic distillation in toluene. l-lactide (LLA) was purchased from PURAC and recrystallized from ethyl acetate for several times. Paclitaxel purchased from Xian Baosai Biotechnology Inc., diglycolic anhydride (97%) and dimethylaminopyridine (DMAP, 99%) purchased from Acros, and dicyclohexylcarbodiimide (DCC) supplied by Chengdu Tenglong Corporation in China were used as received. t-Butyl alcohol and methylene dichloride were refluxed over CaH2 and distilled under argon. Other reagents were commercially available and used as received. 2.2. Synthesis of hydroxyl-terminated MPEG-PLA In a flame-dried and argon-purged flask, 10 g MPEG (Mn=5000), 4 g l-lactide, 30 ml toluene and Sn(Oct)2 were added under Ar stream, and the sealed flask was maintained at 110 C for 24 h, The synthesized polymer was recovered by dissolving in methylene chloride followed by precipitation in ice-cooled diethyl ether. The resultant precipitate was filtered and dried at room temperature in vacuum. Yield: 11.5 g (82%). 1H NMR (CDCl3): d ¼ 5:17 (dd. CH in PLA). 3.65 (s. methylene group of PEG). 3.38 (s. end group (CH3O) of PEG). 1.73 (d. methyl group of PLA). 2.3. Synthesis of mono-t-butyl ester of diglycolic acid A 100 ml flask with three necks, equipped with a magnetic stirrer and a condenser, was flame-dried and argon-purged three times. Two grams of diglycolic anhydride, 10 ml dry t-butyl alcohol, 2.1 g DMAP and 50 ml chloroform were added under argon stream. The solution was stirred at reflux temperature for 24 h. The solvent was evaporated under reduced pressure and the resultant residue was dissolved in 100 ml dilute HCl solution, and extracted with methylene chloride three times. The product was obtained by removal of the solvent from the dried extracts. Yield: 2.84 g (87%). 1H NMR (CDCl3): d ¼ 4:26 (s. methylene group of –CH2– O–CH2–COOH). 4.13 (s. methylene group of –CH2–O– CH2–COOH). 1.48 (s. t-butyl group). 2.4. Synthesis of carboxyl-terminated MPEG-PLA One gram of MPEG-PLA-OH and excessive mono-tbutyl ester of diglycolic acid were dissolved in 50 ml of anhydrous methylene chloride at room temperature. After the solution was chilled to 0 C, 50 mg DCC and 50 mg DMAP were added. The reaction was carried out

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at 0 C under stirring for 24 h. The dicyclohexylurea precipitate was filtered and washed with CH2Cl2. The filtrate was washed successively with water, saturated aqueous sodium bicarbonate, 0.1 mol/l HCl and again with water. The organic solution was dried with anhydrous MgSO4, concentrated to 10 ml, and then poured into ice-cooled diethyl ether. After drying in vacuum, the precipitate was dissolved in 50 ml anhydrous CH2Cl2. The solution was chilled to 0 C and treated with 25 ml trifluoroacetic acid (TFA). After stirring for 2 h, TFA and CH2Cl2 were removed under reduced pressure. The residue was dissolved in 50 ml CH2Cl2. The solution was washed with saturated aqueous sodium bicarbonate and water, and then dried with anhydrous MgSO4. The product was precipitated by pouring the concentrated solution into ice-cooled diethyl ether, and was dried under reduced pressure. Yield: 0.91 g (89%). 2.5. Synthesis of the MPEG-PLA-paclitaxel conjugate In a dried flask, 0.1 g carboxyl-terminated MPEGPLA and 30 mg paclitaxel were dissolved in 50 ml anhydrous methylene chloride. Five milligrams of DCC and 3 mg DMAP were added at 0 C. The reaction was continued under stirring for 24 h at 0 C. The precipitate was filtered out and the filtrate was washed with 0.1 mol/l HCl and water. The organic phase was dried with anhydrous MgSO4, condensed and poured into ice-cooled diethyl ether to precipitate the final product. Yield: 0.087 g (78%). 2.6. Characterizations 1

H NMR spectra were measured on a Bruker Unity400 NMR spectrometer at room temperature, with CDCl3 as solvent and TMS as internal reference. The gel permeation chromatography (GPC) measurements were conducted at 25 C with a Waters 410 GPC instrument equipped with two Waters Styragel columns (HT6E, HT3) and a differential refractometer detector. THF was used as eluent at a flow rate of 1.0 ml/min. 2.7. In vitro cytotoxicity assay The antitumor activity of the paclitaxel conjugate synthesized was evaluated by MTT method [25]. Human liver cancer H7402 cells were chosen as target cells. They were cultured in the growth medium DMEM containing 10% fetal bovine serum (FBS), 2.0 mmol/l glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, and the cell density of the cell suspension obtained was adjusted to 5  104 cells/ml. One hundred and fifty microlitres of aliquots of this suspension were added to the wells in a 96-well plate and incubated for 4 h in a humidified atmosphere containing 5% CO2 at 37 C. The conjugate

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used was made of MPEG(Mn=2000)-PLA(Mn=2000) and its weight content of paclitaxel was 10%. It was dissolved in DMSO at a proper concentration, diluted 100 folds with PBS buffer solution and added into the wells. After 56 h incubation, a 20 ml MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution (5 mg/ml) was added to each well of the plate. The incubation was continued for another 4 h. Then the MTT derivative was dissolved with DMSO and the optical density of the solution was determined by a Microplat Reader (Bio-Rad Model 680) at 570 nm. The relative cell viability was calculated and averaged.

3. Results and discussion A lot of work has been done to prepare paclitaxel prodrugs. It has been established that the 20 -and 7hydroxyl groups of paclitaxel are suitable for its structure modification. To synthesize the MPEG-PLApaclitaxel conjugate, it is necessary to functionalize the hydroxyl terminated MPEG-PLA with an active group which can react with the 20 - and 7-hydroxyl groups of paclitaxel. Carboxyl group was chosen for this purpose. The classic method for converting a terminal hydroxyl group into a terminal carboxyl group is to react the hydroxyl group with either succinic or maleic anhydride under basic condition. Instead, a more reactive anhydride, diglycolic anhydride, was employed in this paper to ensure high enough reaction conversion and to simplify subsequent purification. Therefore the preparation of the paclitaxel conjugate consists of following 3 steps: (1) a hydroxyl-terminated diblock copolymer, MPEG-PLA-OH, was synthesized by ring-opening polymerization of l-lactide using MPEG as a maroinitiator; (2) it was converted to a carboxyl-terminated copolymer MPEG-PLA-COOH by reacting with diglycolic anhydride; (3) the latter was reacted with paclitaxel. The detailed synthesis process will be discussed as follows. 3.1. Synthesis of MPEG-PLA-OH MPEG-PLA-OH was synthesized by ring-opening polymerization of l-lactide using MPEG-PLA as a macroinitiator and stannous octoate as a catalyst in toluene solution (Scheme 1). The block lengths of MPEG and PLA could be adjusted by changing the molecular weight of MPEG and the molar ratio of LA to MPEG. The average molecular weight of the MPEGPLA was calculated to be 6800 by comparing the integrated-area of the peak at 5.17 ppm, (CH in PLA) with that of the peak at 3.65 ppm (CH2CH2O in MPEG), which is consistent with the molecular weight determined by GPC (Table 1 and Fig. 1). In the GPC curve of MPEG-PLA-OH, a single and sharp peak was

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O CH3O

CH2CH2 O

n

H+

Sn(Oct)2

O

o

O

110 C

CH3O

O CH2CH2O C CH O H n m CH3

O

MPEG

MPEG-PLA-OH Scheme 1. Synthesis route of MPEG-PLA-OH.

Table 1 Molecular weight and molecular weight distribution of MPEG-PLA and its derivatives Polymera

Mn by 1H NMR

Mn by GPC

Mw/Mn by GPC

MPEG-PLA-OH MPEG-PLA-COOH MPEG-PLApaclitaxel

6800 7000 8100

7100 7300 8900

1.27 1.20 1.42

a

The molecular weight of MPEG is 5000.

was synthesized by opening diglycolic anhydride with excessive t-butyl alcohol in the presence of DMAP (Scheme 2). Successful synthesis was confirmed by 1H NMR results of the resultant compound. There appeared the characteristic peak at 1.48 ppm belonging to the t-butyl group and the peaks at 4.13 and 4.26 ppm corresponding to the –CH2–O–CH2–COOH and –CH2– O–CH2–COOH protons, respectively. Furthermore the integrated-area ratios of the above peaks were close to 9:2:2, indicating that mono-t-butyl ester of diglycolic acid had been obtained. 3.3. Synthesis of MPEG-PLA-COOH

(c)

(b) (a) 22

23

24

25 26 27 Elution time (min)

28

29

30

Fig. 1. GPC traces of MPEG-PLA-OH (a), MPEG-PLA-COOH (b) and MPEG-PLA-paclitaxel (c).

shown with the polydispersity of 1.27. These results convinced that the MPEG had reacted with LA successfully, and no homopolymerization of LA occurred during the process of reaction. 3.2. Synthesis of mono-t-butyl ester of diglycolic acid Diglycolic anhydride has been employed to prepare the water-soluble prodrug of paclitaxel [26], but obvious improvement in the water solubility of paclitaxel was not achieved. Here, mono-t-butyl ester of diglycolic acid

MPEG-PLA-COOH was obtained by the reaction of MPEG-PLA-OH with mono-t-butyl ester of diglycolic acid in the presence of DCC, and subsequently by deprotecting the t-butyl with trifluoroacetic acid under anhydrous condition (Scheme 3). In the 1H NMR spectrum of MPEG-PLA–OOC–CH2–O–CH2–COOH (Fig. 2), the characteristic peak corresponding to t-butyl (1.48 ppm) disappeared completely, demonstrating complete elimination of the t-butyl group. The peaks a and b related to the –CH2–O–CH2–COOH still existed, furthermore, their integrated-area ratio to the end group (c, CH3O) is approximately 2:2:3, implying that the hydroxyl group of MPEG-PLA had been converted to carboxyl group completely. The molecular weight of MPEG-PLA-COOH determined by 1H NMR (Table 1) or by GPC (Fig. 1) was a little bit higher than MPEGPLA-OH but in the range of error, indicating that the polymer backbone was not ruptured during the reaction. Therefore, the carboxyl-terminated MPEG-PLA was successfully synthesized. 3.4. Synthesis of MPEG-PLA-paclitaxel As mentioned earlier, the most suitable position in paclitaxel for structure modification is the 20 - or 7hydroxyl. Usually the 20 -hydroxyl is more active than the 7-hydroxyl because of space hindrance, and thus esterification often takes place preferentially with the 20 -hydroxyl. Therefore, MPEG-PLA-paclitaxel was prepared by reacting paclitaxel with MPEG-PLACOOH in the presence of DCC and DMAP at 0 C

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O

O

CH3

O CH3

CH3

O

O

DMAP

CO H

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t

CHCl3 Reflux

Bu

O

O C CH2 O CH2 C OH

Scheme 2. Synthesis route to mono-t-butyl ester of diglycolic acid.

O O Bu O C CH2 O CH2 C OH + MPEG-PLA-OH

t

TFA, CH2Cl2 o

MPEG-PLA-O

DMAP, DCC o

0C

O O C CH2 O CH2 C OH

0C (MPEG-PLA-COOH) Scheme 3. Synthesis scheme of carboxyl-terminated MPEG-PLA.

Fig. 2. 1H NMR spectrum of MPEG-PLA-COOH.

MPEG-PLA-O

O O C CH2 O CH2 C OH

MPEG-PLA-O

+ Paclitaxel

DMAP, DCC o

0C

O O C CH2 O CH2 C O Paclitaxel

MPEG-PLA-Paclitaxel Scheme 4. Synthesis scheme of MPEG-PLA-paclitaxel.

(Scheme 4). The 1H NMR spectra of MPEG-PLApaclitaxel and pure paclitaxel are shown in Fig. 3. Obviously, the characteristic peaks of paclitaxel can all be found in MPEG-PLA-paclitaxel, indicating successful preparation of the conjugate. The integrated-area ratio of the peak at 8.14 ppm which was assigned to the

two hydrogens on the phenyl ring of paclitaxel at the C2 position [27] to that of the end group (CH3O, 3.38 ppm) is approximately 1:2.1 and theoretically it should be 2:3, implying that the molar ratio of paclitaxel conjugated to the MPEG-PLA chain was about 70%. Furthermore, the GPC curve of MPEG-PLA-paclitaxel (Fig. 1)

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Fig. 3. 1H NMR spectra of paclitaxel (a) and MPEG-PLA-paclitaxel (b).

exhibits a single and sharp peak, and is left-shifted compared to that of MPEG-PLA. All these results support the conclusion that the paclitaxel had been conjugated with MPEG-PLA successfully.

tration of the drug. 20 ng/ml is a proper concentration under the test condition.

4. Conclusion 3.5. In vitro cytotoxicity assay of paclitaxel conjugate The antitumor activity of paclitaxel conjugate against the human liver cancer H7402 cells was evaluated using MTT method. Fig. 4(A) shows the cell viability after 56 h incubation for the pure paclitaxel, the conjugate and the control (pure copolymer without drug, 180 ng/ ml). At the same drug content (20 ng/ml), the conjugate exhibits almost the same antitumor activity as the pure paclitaxel. It implies that the conjugate exhibited obvious cytotoxicity against H7402 cells, i.e., the paclitaxel is released from MPEG-PLA-paclitaxel without losing cytotoxicity. In Fig. 4(B), the cell viability is plotted against paclitaxel concentration used. It can be seen that the cytotoxicity is dependent on the concen-

We reported a novel paclitaxel conjugate and its facile synthetic route. Paclitaxel was covalently connected to the chain end of an amphiphilic diblock copolymer MPEG-PLA via an ester linkage. Its synthesis consisted of three steps: (1) hydroxyl-terminated diblock copolymer MPEG-PLA-OH was synthesized by ring-opening polymerization of l-lactide using MPEG as a maroinitiator; (2) it was converted to carboxyl-terminated copolymer MPEG-PLA-COOH by reacting with mono-t-butyl ester of diglycolic acid and subsequent deprotecting the t-butyl group with TFA; (3) the latter was reacted with paclitaxel in the presence of DCC and DMAP. The antitumor activity of MPEG-PLA-paclitaxel against human liver cancer H7402 cells was evaluated by MTT method. The results indicated that

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Fig. 4. In vitro cytotoxicity of MPEG-PLA-paclitaxel against human liver cancer H7402 cells. The general test procedures are seen in the text. (A) Cell density: 1.5  105 cells/ml; (a) pure paclitaxel, 20 ng/ml; (b) the conjugate, paclitaxel concentration 20 ng/ml; (c) copolymer MPEG-PLA-OH concentration 180 ng/ml. The control was the 1 wt% solution of DMSO in PBS. Its cell viability was taken as unity. (B) Cell density: 5  104 cells/ml; paclitaxel concentrations as shown in the figure. The control was the 1 wt% solution of DMSO in PBS. Its cell viability was taken as unity.

paclitaxel can be released from the conjugate without losing cytotoxicity.

Acknowledgements The project was financially supported by the National Natural Science Foundation of China. (Project No: 20274048, 50373043) and by the ‘‘863 project’’(Project No. 2002AA326100) from the Ministry of Science and Technology of China.

References [1] Wang J, Li LS, Feng YL, Yao HM, Wang XH. Permanent hepatic artery embolization with dextran micropheres in 131 patients with unresectable hepatocellular carcinoma. Chin Med J 1993;106:441–5. [2] Spencer CM, Faulds D. Paclitaxel—a review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer. Drugs 1994;48:794–847. [3] Thigpen JT. Chemotherapy for advanced ovarian cancer: overview of randomized trials. Semin Oncol 2000;27(3 Suppl 7):11–6.

2127

[4] Chang AY, Rubins J, Asbury R, Boros L, Hui LF. Weekly paclitaxel in advanced non-small cell lung cancer. Semi Oncol 2001;28(4 Suppl.14):10–3. [5] Ishitobi M, Shin E, Kikkawa N. Metastatic breast cancer with resistance to both anthracycline and docetaxel successfully treated with weekly paclitaxel. Int J Clin Oncol 2001;6:55–8. [6] Panchagnula R. Review: pharmaceutical aspects of paclitaxel. Int J Pharm 1998;172:1–15. [7] Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm 2002;235:179–92. [8] Kan P, Chen ZB, Lee CJ, Chu IM. Development of nonionic surfactant/phospholipid o/w emulsion as a paclitaxel delivery system. J Control Release 1999;58:271–8. [9] Sharma A, Straubinger RM. Novel taxol formulations: preparation and characterization of taxol-containing liposomes. Pharm Res 1994;6:889–96. [10] Liggins RT, Burt HM. Polyether-polyester diblock copolymers for the preparation of paclitaxel loaded polymeric micelle formulations. Adv Drug Deliv Rev 2002;54:191–202. [11] Kim SY, Lee YM. Taxol-loaded block copolymer nanospheres composed of methoxy poly(ethylene glycol) and poly(e-caprolactone) as novel anticancer drug carriers. Biomaterials 2001;22: 1697–704. [12] Ruan G, Feng SS. Preparation and characterization of poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid)(PLA-PEG-PLA) microspheres for controlled release of paclitaxel. Biomaterials 2003;24:5037–44. [13] Feng SS, Mu L, Win KY, et al. Nanoparticles of biodegradable polymers for clinical administration of paclitaxel. Curr Med Chem 2004;11:413–24. [14] Dong YC, Feng SS. Methoxy poly (ethylene glycol)-poly (lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials 2004;25:2843–9. [15] Deutsch HM, Glinski JA, Hermandez M, Haugwitz RD, Narayanan LV, Suffness M, Zalkow LH. Synthesis of Congeners and Prodrugs. 3. Water-soluble prodrugs of taxol with potent antitumor activity. J Med Chem 1989;32:788–92. [16] Greenwald RB, Gibert CW, Pendri A, Conover CD, Xia J, Martinez A. Drug delivery systems: water soluble taxol 20 -poly (ethylene glycol) ester prodrugs-design and in vivo effectiveness. J Med Chem 1996;39:424–31. [17] Oliyai R, Stella VJ. Prodrugs of peptides and proteins for improved formulation and delivery. Annu Rev Pharmacol Toxicol 1993;32:521–44. [18] Mathew AE, Mejillano MR, Nath JP, Himes RH, Stella VJ. Synthesis and evaluation of some water-soluble prodrugs and derivatives of paclitaxel with antitumor activity. J Med Chem 1992;35:145–51. [19] Zhao Z, Kingston DG. Modified taxols. 6. Preparation of water-soluble prodrugs of taxol. J Nat Prod (Lloydia) 1991; 54:1607–11. [20] Vyas DM, Ueda Y, Wong H, Matiskella JD, Hauck S, Mikkilineni AB, Farina V, Rose WC, Casazza AM. Phosphatase-activated prodrugs of paclitaxel. In: Georg GI, Chen TT, Ojima I, Vyas DM, editors. Taxane anticancer agents: basic science and current status. Washington, DC: American Chemical Society; 1995. p. 124–37. [21] Rose WC, Clark JL, Lee FYF, Casazza AM. Preclinical antitumor activity of water-soluble paclitaxel derivatives. Cancer Chemother Pharmacol 1997;39:486–92. [22] Li C, Yu DF, Newman RA, Cabral F, Stephen LC, Hunter N, Milas L, Wallace S. Complete regression of well-established tumors using a Novel Water-soluble poly (l-glutamic acid)paclitaxel conjugate. Cancer Res 1998;58:2404–9. [23] Mongell N, Pesenti E, Suarato A, Biasoli G. Polymer-bound paclitaxel derivatives. US Patent 1994;5(362):831.

ARTICLE IN PRESS 2128

X. Zhang et al. / Biomaterials 26 (2005) 2121–2128

[24] Pendri A, Conover CD, Greenwald RB. Antitumor activity of paclitaxel-20 -glycinate conjugated to poly (ethylene glycol): a water-soluble prodrug. Anti-Cancer Drug Design 1998;13:387–95. [25] Kim SY, Lee YM, Baik DJ, Kang JS. Toxic characteristics of methoxy poly(ethylene glycol)/poly(e-caprolactone) nanospheres; in vitro and in vivo studies in the normal mice. Biomaterials 2003;24:55–63.

[26] Nicolaou KC, Rlemer C, Ker MA, Rideout D, Wrasidlo W. Design, synthesis and biological activity of protaxols. Nature 1993;364:464–6. [27] Shi Q, Wang HK, Bastow KF, Tachibana Y, Chen K, Lee FY, Lee KH. Antitumor agents 210. synthesis and evaluation of taxoid-epipodophyllotoxin conjugates as novel cytotoxic agents. Bioorg Med Chem 2001;9:2999–3004.