*Corresponding author. Tel.: #1-82-2-2291-9683; fax: #1-82-2-2291-5982.E-mail address: ymlee@email.hanyang.ac (Y.M. Lee).

Biomaterials 22 (2001) 1697}1704

Taxol-loaded block copolymer nanospheres composedof methoxy poly(ethylene glycol) and poly(�-caprolactone)

as novel anticancer drug carriers

So Yeon Kim, Young Moo Lee*Department of Industrial Chemistry, College of Engineering, Hanyang University, Seoul, 133-791, South Korea

Received 7 January 2000; accepted 7 September 2000

Abstract

We prepared the methoxy poly(ethylene glycol) (MePEG)/poly(�-caprolactone) (PCL) amphiphilic block copolymeric nanospherescontaining taxol which has promising anticancer activity. MePEG/PCL block copolymeric nanospheres (MEP50) showed a narrowsize distribution and an average diameter of less than 100 nm. When the initial weight ratio of taxol to polymer was 0.5 : 1.0, we couldobtain the nanospheres having a relatively high drug-loading of more than about 20%. The size of theMePEG/PCL nanospheres alsoincreased according to the taxol loading. However, the nanospheres did not exhibit a signi"cant change in the size distribution andalso showed a size of less than 100nm for even that with drug-loading content (DLC) of about 20%. From the �H NMR analysis, weidenti"ed that the MePEG/PCL nanospheres prepared by dialysis procedure have core-shell structure consisting of the hydrophilicouter shell of MePEG and the hydrophobic inner core of PCL.We con"rmed the low toxicity of MePEG/PCL nanospheres (MEP70)in the acute toxicity study using male ICR mice. In addition, considering the extremely lipophilic characteristics of taxol, thisMePEG/PCL, nanosphere system with high taxol loading content and suspended properties in water could be useful for the deliveryof taxol. � 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Taxol; Methoxy poly(ethylene glycol); (�-Caprolactone; Nanosphere; Drug delivery system

1. Introduction

For optimal drug action it is necessary to deliver thedrug to the desired site of action in the body in the moste$cient way. There have been tremendous e!orts todevelop systems for site-speci"c drug delivery [1}5].

One possible means of reaching such goal may bea delivery via particulate drug delivery systems [6}11].Particulate drug delivery systems due to their small sizemay be useful as sustained-release injections or for thedelivery of a drug to a speci"c organ or target site. Inparticular, the most promising application of polymericnanoparticles is their use as carriers for anti-cancerdrugs. It has been found that the polymer}anticancerdrug conjugates in comparison with low-molecular-weight anticancer drugs were accumulated more in thetumor tissues than in the normal tissues due to the

enhanced permeability and retention (EPR) e!ect. Inaddition, polymer}anticancer drug conjugates can pro-long the antitumour activity by releasing the drug com-ponent at a controlled rate [6}11].

A major problem of drugs used in cancer chemother-apy is nonspeci"c toxicity against normal cell as well astumor cells. Such toxic action to normal cells limitsthe dose of the anticancer drugs to be administered topatients.

Taxol (Paclitaxel, tax-11-en-9-one, 5�,20-epoxy-1,2�,4,7�,10�,13�-hexahydroxy-4,10-diacetate-2-benzoate-13-(�-phenylhippurate)) isolated from the bark of the paci"cyew tree, Taxus brevifoli has promising anticancer activityagainst most solid tumors, and was approved as ananticancer agent by the US Food and Drug Administra-tion (FDA) in 1992 [12}18]. Currently, it is for clinicaluse formulated in a 50 : 50 mixture of Cremophore� EL(polyethoxylated caster oil) and ethanol due to the poorsolubility of taxol in water, which is diluted with normalsaline or dextrose solution (5%) prior to administration.However, since this clinical formulation, the mixture ofthe surfactant and ethanol is physically incompatible as

0142-9612/01/$ - see front matter � 2001 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 2 9 2 - 1

an intravenous infusion system; serious hypersensitivityreactions have occurred [12}18].

Generally, amphiphilic block or graft copolymerscomposed of a hydrophobic part and hydrophilic onecan self-assemble to form micelles in selective solventwhich is a good solvent for one component of copolymerand a precipitant for the other component [19}31],These core-shell type polymeric nanosphere systems con-sisting of hydrophobic inner core and hydrophilic outershell have predominant characteristics such as the reduc-tion of toxic side-e!ects of drug, selective targeting, thesolubilization of hydrophobic drugs, stable storage, longblood circulation, biodistribution, and lower interactionswith reticuloendothelial cell system.

Therefore, we intend to investigate the block copolymericnanosphere system to develop the aqueous-based formu-lation for taxol which could diminish a serious hypersen-sitivity, and to provide a more e!ective chemotherapy.Based on our previous studies on block copolymericmicelle containing drugs [28}31], in the present study,we prepared the novel taxol-loaded nanosphere com-posed of biodegradable hydrophobic poly(�-caprolac-tone) (PCL) and hydrophilic methoxy poly(ethyleneglycol) (MePEG). We determined their size distributionand the factors that in#uenced the size and the core-shellstructure. The amount of taxol incorporated intoMePEG/PCL nanospheres was also determined. Acutetoxicity of MePEG/PCL nanospheres was estimated bydetermining the LD

��of the nanosphere itself in ICR

mice.

2. Experimental

2.1. Materials

Methoxy poly(ethylene glycol) (MePEG, M�"5.0�10�

by supplier, M�"5.5�10� by our GPC measurements)

was purchased from Fluka and puri"ed by azeotropicdistillation with benzene (Junsei Chemical Co., Ltd.Tokyo, Japan). �-Caprolactone (Mw"114.14) wasobtained from Tokyo Kasei Organic Chemicals and rec-rystallized from ethyl acetate. Taxol was kindly suppliedby Hanmi Pharmaceutical Co., Ltd. in Seoul, Korea.Distilled-deionized water was prepared with a Milli-QPlus System (Waters, Millipore, USA). All other chem-icals used were reagent grade and used as purchasedwithout further puri"cation. Animals (male ICR mice,22}25g, Myungjin Co., Seoul, Korea) were housed ina ventilated (10}15 times/h) room under conditions ofcontrolled temperature maintained at 20}233C and hu-midity of 50}70% during the investigation. The noiseand luminous intensity of the controlled room with a12-h light and 12-h dark (6 : 00}18 : 00) cycle were below60db and 200 lux, respectively. Free access to food andwater was allowed.

2.2. Preparation of taxol-loaded MePEG/PCL nanosphere

We already reported that amphiphilic diblockcopolymers composed of MePEG and �-caprolactonewere synthesized by the ring-opening polymerization of�-caprolactone in the presence of MePEG homopolymerwithout any catalyst in our previous papers [25}27].Using the MePEG/PCL diblock copolymer, the nano-sphere containing hydrophobic drug, taxol, as preparedas follows: MePEG/PCL block copolymer (100mg) wasdissolved in 10ml of DMF (dimethylformamide) fol-lowed by adding taxol and stirred at room temperature.In order to form taxol-loaded micelle-like nanospheresand to remove free taxol and organic solvent, the solu-tion was dialyzed for 24 h against 3 l of ultrapure waterusing cellulose dialysis membranes (molecular weight cuto!: 6.0�10�}8.0�10�, and 12.0�10�}14.0�10�, size:21/35, Sigma, MO, USA). The nanosphere solution wassonicated using a sonicator (Branson Ultrasonics Co.,Danbury, CO, USA), and then centrifuged (JouanBP403, VA, USA) to eliminate unloaded taxol and ag-gregated particles. The supernatant, nanosphere solu-tion, obtained in this process, was frozen and lyophilizedby a freeze dryer system (Labconco, Kansas, MO, USA),to obtain dried nanosphere products.

2.3. Characterization of MePEG/PCL copolymer

The molecular weight and molecular weight distribu-tion of block copolymers were determined by gel per-meation chromatography apparatus (Waters Model 510HPLC pump, Milford, MA, USA). They were character-ized by relative elution time to polystyrene monodispersestandards from GPC with a Millenium software pro-gram. Three ultrastyragel� tetrahydrofuran (THF)columns (each 7.8mm inner diameter and 30 cm length,HR-0.5, HR-4, HR-5, all by Waters, Milford, MA, USA)and Waters R410 di!erential refractometeric detectorwere used. The mobile phase was THF with a #ow rateof 1ml/min. The injection volume was usually 100 �lof stock solutions (0.1}0.5w/v%). The calibrationcurve was prepared before measurements by usingstandard polystyrene (molecular weights: 1.28�10�,2.96�10�, 1.13�10�, 2.85�10� and 6.50�10�, respec-tively, Shodex Standard SM-105, Showa Denko, Tokyo,Japan).

In addition, the composition and the number-averagemolecular weight of each copolymer in CDCl

�(deuterated chloroform) solution were determined by500MHz �H NMR (Bruker AMX-500).

2.4. Size and size distribution

Average size and size distribution of nanospheres weredetermined by a dynamic light scattering (DLS) using anargon ion laser (model 95-2, Laxel Laser Inc., USA) at

1698 S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704

a wavelength of 514.5 nm at 203C. The intensity of thescattered light was detected with a photomultiplier(Brookhaven Instrumental Co. EMI9863) at a scatteringangle of 903. The signal from the photomultiplier wasdigitized via an ampli"er-discriminator and was fedinto a correlator (Brookhaven Instrumental Co. Bl-9000AT). The digital photon correlator which accumu-lated the time correlation function was "tted by the use ofthe method of CONTIN programs of Provencher et al.[32].

Samples for DLS measurement were prepared by thedilution of the nanosphere suspensions with distilled-deionized water "ltered through syringe "lter. Fromthese DLS measurements we obtained the diameter andstandard deviation of nanospheres.

2.5. Measurement of xeld emission scanningelectron microscopy

MePEG/PCL block copolymeric nanospheres ob-tained through dialysis method were observed witha "eld emission scanning electron microscopy (FE-SEM,Jeol Model JSM-6340F, Kyoto, Japan) measurement. Toprepare the sample for FE-SEMmeasurement, the micel-lar solution of nanospheres was placed onto the coppermount. Then the external solution was evaporated slowlyat room temperature and then the completely driedsample was coated with gold.

2.6. Acute toxicity of MePEG/PCL copolymeric material

In order to estimate the biocompatibility of MePEG/PCL block copolymeric nanosphere itself, we investi-gated the acute toxicity using male ICRmice. As an acutetoxicity study [33}35], we determined the median lethaldose (LD

��) of MePEG/PCL nanosphere using the Be-

hrens}Karber method [35]. Male ICR mice, weighing22}25g, were divided into groups of 10 animals. Theywere injected via the tail vein by varying the dose levels ofMePEG/PCL nanosphere solution (20}60mg/25 g) fordivided groups. Animals were observed for 48 h aftersingle administration.

2.7. Determination of taxol loading content

The nanospheres suspension was sonicated and centri-fuged to remove an unbound taxol and aggregated tax-ol-loaded nanospheres, then they were lyophilized. Thenanospheres obtained by freeze-drying were dissolvedby the addition of methanol and DMF (1 : 1, v/v); theamount of taxol entrapped was determined by measuringthe UV absorbance at 261 nm using the UV-visible spec-trophotometer (Shimadzu Model UV-2101 PC). Thecontent of entrapped taxol into the core of nanosphereswas calculated from the weight of initial drug-loadednanospheres and the amount of drug incorportated from

the following equation:

Drug loading content (DLC)

"

Amount of taxol in nanospheres

Amountof taxol-loadednanospheres�100

"Taxol/(Taxol#polymer)�100. (1)

3. Results and discussion

3.1. Characterization of MePEG/PCL nanospheres

In our previous studies [25}27], we already reportedon the synthesis of amphiphilic block copolymer com-posed of methoxy poly(ethylene glycol) and �-caprolac-tone and the preparation of the MePEG/PCL blockcopolymeric nanospheres containing hydrophobic drugs.MePEG/PCL diblock copolymer could be prepared byring-opening polymerization mechanism of �-caprolac-tone in the presence of MePEG homopolymer, contain-ing hydroxyl functional group at one end of the chain,without any catalysts [25}27]. The structure of MePEG/PCL diblock copolymers synthesized was identi"ed byFourier-transform infrared (FT-IR) spectroscopy, wide-angle X-ray di!raction (WAXD) pattern, gel permeationchromatography (GPC) and �H-NMR measurement inour previous papers [23,24].

The composition and molecular weight of MePEG/PCL block copolymer by GPC and �H NMR analysisare summarized in Table 1. In order to obtain a satisfac-tory formula, copolymers with di!erent compositionwere tested as shown in Table 1. `MEP seriesa has a "xedmolecular weight of 5.0�103 in the MePEG chain, andPCL portion varies from M/l"35 to 150.

3.2. Size and size distribution of MePEG/PCL nanospheres

Typical size distributions of MePEG/PCL blockcopolymeric nanospheres (MEP50) before and afterloading of taxol are exhibited in Fig. 1. Fig. 1(a) showedthe size distribution of taxol-unloaded MePEG/PCLnanospheres prepared with MEP50 copolymer (M

�mea-

sured by GPC analysis"10,116, as shown in Table 1),and (b) was the size distribution pro"le of taxol-loadednanospheres (TMC - 0.50; DLC"20.79%, as shown inTable 2). Their average sizes were smaller than 100nm,and the size distribution showed a narrow pattern fromthe DLS measurements. After the taxol loading inMePEG/PCL nanosphere, their size increased. However,the size distribution of nanosphere was relatively identi-cal to that before taxol loading, maintaining the narrowdistribution as shown in Fig. 1(b).

In addition, we investigated the size change ofMePEG/PCL nanospheres according to the molecularweights of block copolymers used and the loading

S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704 1699

Table 1Compositions and molecular weight distribution of MePEG/PCL block copolymers

Sample Feed molarratio�

Molar com-position�

Compositionweight%�

Compositionweight%�

Number-averagemolecularweight

Polydispersity(MM

�/MM

�)

�-CL/MePEG

�-CL/MePEG

MePEG:�-CL

MePEG:�-CL

Calc.� Expt'l.� Expt'l�

MePEG 0 0 100 : 0 100 : 0 5000 5541 5333 1.128MeP35 35 21.8 68.9 : 31.1 66.9 : 33.1 8995 8037 7971 1.256MeP50 50 40.1 54.8 : 45.2 51.7 : 48.3 10,707 10,116 10,317 1.250MeP70 70 54.2 47.2 : 52.8 39.9 : 60.1 12,990 11,734 13,367 1.102MeP100 100 81.5 37.3 : 62.7 37.1 : 62.9 16,414 14,839 14,401 1.102MeP150 150 109.9 30.6 : 69.4 29.2 : 70.7 22,121 18,085 18,205 1.178

�Determined on the basis of MM�of MePEG calculated in GPC experiments.

�Estimated as the diference between the experimental total MM�of copolymer and MePEG homopolymer in GPC experiments.

�Determined by �HNMR spectroscopy (CDCl�).

�Calculated from MePEG (MW"5000 Fluka).�Measured by GPC analysis.

Fig. 1. Typical size distribution pro"le of MePEG/PCL diblock copolymeric nanospheres (MEP50) measured by dynamic light scattering method: (a)before loading taxol and (b) after loading taxol (TMC-0.50; DLC"20.79%).

content of taxol. As shown in Fig. 2(a), the size ofMePEG/PCL nanospheres gradually increased with theincrease in the weight ratio ([M]/[I], where [M]"�-caprolactone units and [I]"MePEG homopolymer)used in the preparation of block copolymer. Namely, theincrease of the molecular weight of block copolymerproduced larger nanospheres. They are 54, 77, 114 and130nm for MEP35, MEP50, MEP70 and MEP100 asdescribed in Table 1, respectively. These results agree wellwith those by other researchers [36,37]. They reportedthat the aggregation number and the size of the micelleincreased with the solvophobicity of the unfavorable partfor surrounding solvent of amphiphilic block copolymerssince the solvent repulsion force of the solvent-unfavor-able block and the interfacial tension between the solvent

and the surfaces of the micellar core increased. Parti-cularly, the theories by Nagarajan et al. [36] and White-more et al. [37] suggested the scaling relations for thesemicellar parameters which were proportional to N�

�N�

�,

where NA is the length of the insoluble block and N�, the

soluble block and � and �, the exponents of the scalingrelation, and represented the dependence of the micellarparameters on the block lengths.

In the case of MEP150, however, nanospheres werenot detected. A possible reason for not forming a core-shell type micelle for amphiphilic block copolymerMEP150 is due to the fact that it contains a long hydro-phobic chain in the backbone of a block copolymer.

The amount of taxol entrapped in the nanospheresin#uenced the size of the MePEG/PCL nanospheres as

1700 S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704

Fig. 2. Change in the size of MePEG/PCL nanospheres depending on the molecular weight of block copolymer (a) and the loading content of taxol innanosphere (b).

Table 2Taxol loading contents of MePEG/PCL block copolymer nanospheres

No. Sample MePEG/PCLcopolymer used

Weight ratio Drug loading content(DLC) (%)�

Taxol� : Polymer

1 TMC-0.05 MEP50 0.05 : 1.00 4.102 TMC-0.10 MEP50 0.10 : 1.00 9.063 TMC-0.20 MEP50 0.20 : 1.00 12.154 TMC-0.50 MEP50 0.50 : 1.00 20.795 TMC-1.00 MEP50 1.00 : 1.00 9.08

�Taxol.

�DLC(%)"Amountof taxol in nanospheres

Amountof taxol-loaded in nanospheres�100"

Taxol

(Taxol#Polymer)�100.

shown in Fig. 2(b). As the loading content of taxol in-creased, the size of MeEG/PCL nanosphere increased.However, even MePEG/PCL nanosphere (TMC - 0.50,in Table 2) with DLC of about 20.79% exhibited the sizeof less than 100nm, which was considered appropriatefor passive targeting carriers for solid tumors.

Furthermore, we observed the MePEG/PCL nano-spheres with "eld emission scanning electron microscopy(FE-SEM). Fig. 3 exhibited the taxol-loaded MePEG/PCLnanospheres prepared with MEP50 block copolymer(TMC - 0.50; DLC"20.79%, as shown in Table 2). Ascan be seen from these FE-SEM images, theMePEG/PCL block copolymeric nanosphere has thespherical shape and sub-micron size.

3.3. Core-shell structure of MePEG/PCL nanosphere

In addition, we estimated the core-shell structure ofMePEG/PCL block copolymeric nanospheres using�HNMR analysis. High-resolution �H NMR spectro-scopy has often been employed to analyze the micro-structure of polymer chains in solution. Liu, Ando and

others have investigated the interactions between poly-mer chains and low-molecular weight materials in solu-tion [38,39]. Nakamura et al. reported the molecularmotion of block copolymers in solution by high-resolu-tion �HNMR study [40].

Fig. 4 showed the �HNMR spectra of taxol-loadedMePEG/PCL block copolymeric nanospheres in D

�O

(a), taxol in CDCl�

(b) and taxol-loaded MePEG/PCLblock copolymeric nanospheres in CDCl

�(c). Taxol-

loaded MePEG/PCL block copolymeric nanosphereswere completely dissolved in CDCl

�solvent. Therefore,

in the spectrum (Fig. 3(c)) of IMC-loaded Pluronic/PCLnanosphere in CDCl

�, we could clearly observe the peaks

at 4.13 ppm assigned to methylene proton of poly(�-caprolactone) units, peak at 3.65 ppm due to the ethyleneproton in PEO units, and peaks assigned to taxol. Forthe �H NMR spectrum of taxol-loaded MePEG/PCLblock copolymeric nanospheres in D

�O (c), however,

only chemical shift due to ethylene protonin PEO block was resolved at around 3.65 ppm. Fromthese results, it is presumed that the conformation of theamphiphilic diblock copolymer in CDCl

�is of the

S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704 1701

Fig. 3. Taxol-MePEG/PCL nanospheres images observed by "eld emission scanning electron microscopy: (a) TMC-0.50 nanospheres(DLC"20.79%) (�25,000) and (b) TMC-0.50 nanospheres (�50,000).

Fig. 4. �H NMR spectra of (a) taxol-loaded MePEG/PCL nanospheres (TMC-0.50) in D�O, (b) free taxol unloaded in CDCl

�and (c) taxol-loaded

MePEG/PCL nanospheres (TMC-0.50) in CDCL�.

mixed-microphase form in which both MePEG and poly(�-caprolactone) blocks are dissolved. In D

�O, however,

the amphiphilic block copolymer is in a separated-micro-phase form consisting of a soluble MePEG block partand insoluble poly(�-caprolactone) block part. It was

concluded that taxol-loaded MePEG/PCL blockcopolymeric nanospheres existed as a core-shell-likestructure composed of hydrophobic core of poly(�-cap-rolactone) and hydrophilic outer shell of MePEG inwater.

1702 S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704

3.4. LD50 of MePEG/PCL block copolymeric material

From the results of the acute lethal toxicity studies, themedian lethal dose (LD

��) of MePEG/PCL nanosphere

was determined by Behrens}Karber method [35]. TheLD

��of MePEG/PCL nanosphere (MEP70) was

1.343g/kg. Practically, the maximal doses of taxol are30mg/m� per day for 5 days or 210}250mg/m�

given once in every 3 weeks [41]. If we consider therequired amount of this MePEG/PCL nanosphere withtaxol loading content of about 20% on the basisthat body surface area of average adult with 60 kgweight is about 1.73m�, it can be concluded that thisMePEG/PCL nanosphere is suitable as carrier for taxoldelivery.

3.5. Taxol loading content in MePEG/PCL nanosphere

Table 2 shows the amount of taxol introduced into theMePEG/PCL nanosphere by controlling the weightratio between polymer and the drug. We preparedthe taxol-loaded nanosphere with varying initial weightratio of taxol to copolymer from 0.05 : 1.0 to 1.0 : 1.0. Asshown in this table, the drug loading content increasedwith the ratio of drug to polymer. Especially, for theinitial weight ratio of taxol to MePEG/PCL copolymerwas 0.5 : 1.0 (TMC-0.050), the drug loading content was20.79%.

When the initial weight ratio of taxol to copolymerexceeded 0.5 : 1.0 it was found that the aggregation ofunloaded taxol signi"cantly occurred during the prep-aration of MePEG/PCL nanosphere, resulting in thedecrease of both the yield of taxol-loaded nanosphereand the loading content of taxol. As described in Table 2,TMC-1.00 sample (initial weight ratio of taxol to poly-mer"1.0 : 1.0) exhibited the DLC of 9.08%. It indicatedthat the hydrophobic interaction between drug moleculeswas greater than that between drug and MePEG/PCLcopolymer due to the high lypophilic character of taxolas the amount of taxol increased. Therefore, in the drug-loading experiment, the initial weight ratio of taxol tocopolymer was "xed to 0.5 : 1.0 for all the samples.

It is known that the solubility of taxol itself is lowerthan 0.001mg/ml in PBS solution of pH 7.4. [16]. Be-cause of these extremely hydrophobic properties of taxol,the commercially available injection is a sterile solutionof the drug in Cremophor EL (polyethoxylated casteoroil) and dehydrated alcohol. Since the clinical formula-tion such as the mixture of the surfactant and ethanol isphysically incompatible as an intravenous infusion sys-tem, serious hypersensitivity reactions occur. Consider-ing the poor aqueous solubility of taxol, it could beconcluded that the MePEG/PCL nanospheres systemimproved the dispersal property of taxol in aqueousmedia through the formation of core-shell type nano-spheres.

4. Conclusions

As a novel drug carrier for taxol, we prepared diblockcopolymeric nanospheres composed of methoxypoly(ethylene glycol) (MePEG) and �-caprolactone(PCL). MePEG/PCL nanosphere containing the hydro-phobic drug taxol in their inner core were formed asa result of the solution behavior of amphiphilic blockcopolymer in selective solvents. MePEG/PCL nano-sphere (MEP50) exhibited the average size of less than100nm with narrow size distribution pattern. Their sizewas in#uenced by the molecular weight of MePEG/PCLblock copolymer used and the amount of incorporateddrug. After the loading of taxol, the size of MePEG/PCLnanosphere did not considerably increase, and the size ofless than 100nm and narrow size distribution were main-tained. From the acute toxicity study, we could con"rmthe biocompatibility of nanosphere itself. In addition, wecould obtain the MePEG/PCL nanospheres having rela-tively high DLC of more than about 20%. From theseresults, we could conclude that the present MePEG/PCLdiblock copolymeric nanospheres system could be poten-tially useful as a novel delivery system for anticancerdrug taxol.

Acknowledgements

SYK is grateful to the Graduate School of AdvancedMaterials and Chemical Engineering at Hanyang Uni-versity for a fellowship. Thanks are due to Hanmi Phar-maceutical Co., Ltd. in Korea for donating Taxol.

References

[1] Dunn RL, Ottenbrite RM. Polymeric drug and drug deliverysystems. Washington, DC: American Chemical Society, 1991.

[2] El-Nokaly MA, Piatt DM, Charpentier BA. Polymericdelivery systems. Washington DC: American Chemical Society,1993.

[3] Park K. Controlled drug delivery, ACS professional referencebook. Washington, DC: American Chemical Society, 1997. 49}67[Chapter 4].

[4] Chasin M, Langer R. Drug and the pharmaceutical sciences,vol, 3: biodegradable polymers as drug delivery system. NewYork, USA: Marcel Dekker, Inc., 1990, 71}120.

[5] Puisieux F, Barratt G, Couarraze G, Couvreur P, Devissaguet JP,Dubernet C, Fattal E, Fessi H, Vauthier C. Polymeric bi-omaterials. New York: Marcel Dekker, Inc., 749}794.

[6] AlonsoMJ, Drug and the pharmaceutical sciences, vol. 77: micro-particulate systems for the delivery of proteins and vaccines. NewYork, USA: Marcel Dekker, Inc., 1996, 203}242.

[7] Peppas LB. Recent advances on the use of biodegradable micro-particles and nanoparticles in controlled drug delivery. IntJ Pharm 1995;116:1}9.

[8] Jalil R, Nixon JR. Biodegradable poly(lactic acid), poly(lactide-co-glycolide) microcapsules: problems associated with pre-parative techniques, release properties. J Microencapsulation1990;7:297}325.

S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704 1703

[9] Uchida T, Yoshida K, Ninomiya, A, Goto S. Optimization ofpreparative conditions for polylactide (PLA) microspheres con-taining ovalbumin. Chem Pharm Bull 1995;43:1569}73.

[10] Kreuter J. Nanoparticle-based drug delivery systems. J Contr Rel1991;16:169}76.

[11] Seijo B, Fattal E, Treupel LR, Couvreus P. Design of nanopar-ticles of less than 50nm diameter: preparation, characterizationand loading. Int J Pharm 1990;62:1}7.

[12] Panchagnula R. Pharmaceutical aspects of paclitaxel. IntJ Pharm 1998;172:1}15.

[13] Dhanikula AB. Panchagnula R. Localized paclitaxel delivery. IntJ Pharm 1999;183:85}100.

[14] Coudore F, Authier N, Guillaume D, Beal A, Duroux E, Fialip J.High performance liquid chromatographic determination of pac-litaxel in rat serum: application to a toxicokinetics study.J Chromatogr B 1999;721:317}20.

[15] Zhang X, Jackson JK, Wong W, Min W, Cruz T, Hunter WL,Burt HM. Development of biodegradable polymerics paste for-mulations for taxol: an in vitro and in vivo study. Int J Pharm1996;137:199}208.

[16] Miwa A, Ishibe A, Nakano M, Yamahira T, Itai S, Jinno S,Kawahara H. Development of novel chitosan derivatives asmicellar carriers of taxol. Pharm Res 1998;15:1844}50.

[17] Winternitz CI, Jackson JK, Oktaba AM, Burt HM. Developmentof a polymeric surgical paste formulation for taxol. Pharm Res1996;13:368}75.

[18] Lee SH, Yoo SD, Lee KH. Rapid and sensitive detemination ofpaclitaxel in mouse plasma by high-performance liquid chromato-graphy. J Chromatogr B 1999;724:357}63.

[19] Gao Z, Eisenberg A. A model of micellization for blockcopolymers in solutions. Macromolecules 1993;26:7353}60.

[20] Quintana JR, Villacampa M, Munoz M, Andrio A, Katimc A.Micellization of a polystyrene-block-poly(ethylene/propylene)copolymer in n-alkanes. 1. Thermodynamic study. Macro-molecules 1992;25:3125}8.

[21] Munk P, Qin A, Tian M, Ramireddy C, Webber SE, Tuzar Z,Prochazka K. Characterization of block copolymer micelles inaqueous media. J Appl Polym Sci: Appl Polym Symp 1993;52:45}54.

[22] Hamad E, Qutubuddin S. Theory of micelle formation by am-phiphilic side chain polymers. Macromolecules 1990;23:4185}91.

[23] Malmsten M, Lindman B. Self-assembly in aqueous blockcopolymer solutions. Macromolecules 1992;25:5440}5.

[24] Price C, Stubbers"eld RB, Kafrawy SE, Kendall KD. Thermo-dynamics of micellization of polystyrene-block-poly(ethylene/propylene) copolymers in decane. Briti Polym J 1989;21:391}4.

[25] Shin IG, Kim SY, Lee YM, Cho CS, Sung YK. Methoxy poly(ethylene Glycol)/�-caprolactone amphiphilic block copolymericmicelle containing indomethacin: I. preparation and characteriza-tion. J Contr Rel 1998;51:1}11.

[26] Kim SY, Shin IG, Lee YM, Cho CS, Sung UK. Methoxypoly(ethylene glycol)/�-caprolactone amphiphilic block copolymeric

micellc containing indomethacin: II. Micelle formation and drugrelease behaviours. J Contr Rel 1998;51:13}22.

[27] Kim SY, Lee YM. Methoxy poly(ethylene glycol)/poly(�-cap-rolactone) amphiphilic block copolymeric nanosphere containingindomethacin: III. Pharmacokinetic study in rat. J Contr Rel.Submitted for publication.

[28] Kim SY, Shin IG, Lee YM. Preparation and characterization ofbiodegradable nanoshperes composed of methoxy poly(ethyleneglycol) and D,L-lactide block copolymer as novel drug carriers.J Contr Rel 1998;56:197}208.

[29] Kim SY, Shin IG, Lee YM. Amphiphilic diblock copolymericnanospheres composed of methoxy poly(ethylene glycol) andglycolide: properties, cytotoxicity and drug release behavior. Bio-mater 1999;20:1033}42.

[30] Ha JC, Kim SY, Lee YM. Poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) (Pluronic)/poly(�-caprolactone) (PCL)amphiphilic block copolymeric nanospheres: I preparation andcharacterization. J Contr Rel 1999;62:381}92.

[31] Kim SY, Ha JC, Lee YM. Poly(ethylene oxide)-poly(propyleneoxide)-poly(propylene oxide) (Pluronic)/poly(�-caprolactone) (PCL)amphiphilic block copolymeric nanospheres: II Thermo-respon-sive drug release behaviors. J Contr Rel 2000;65:345}58.

[32] Provencher SW, Hendrixs J. Direct determination of molecularweight distributions of polystyrene in cyclohexane with photoncorrelation. J Chem Phys 1978;69:4273}6.

[33] Guzman A, Garcia C, Demestre I. Acute and subchronic toxicitystudies of the new quinolone antibacterial agent irloxacin inrodents. Drug Res 1999;49(1):448}56.

[34] Souza LC, Campa A. Pharmacological parameters of intra-venously administered amphotericin B in rats: comparison of theconcentional formulation with amphotericin B associated witha triglyceride-rich emulsion. J Antimicrob Chemother 1999;44:77}84.

[35] Klaassen C. Casarett and Doull's toxicology: the basic science ofpoisons, 5th ed. New York: McGraw-Hill, 1996.

[36] Nagarajan R, Ganesh K. Block copolymer self-assembly in selec-tive solvents: spherical micelles with segregated cores. J ChemPhys 1989;90:5843}56.

[37] Whitemore MD, Noolandi J. Theory of micelle formation inblock copolymer-homopolymer blends. Macromolecules 1985;18:657}65.

[38] Liu K, Burlant W. High-resolution nuclear magnetic resonance ofcrosslinked polymers: e!ects of crosslinked density and solventinteraction. J Polym Sci, Part A-1 1967;5:1407}13.

[39] Ando I, Nishioka A. Magnetic anisotropy e!ect on the chemicalshift in linear hydrocarbons. Macromol Chem 1972;152:7}14.

[40] Nakamura K, Endo R, Takeda M. Study of molecular motion ofblock copolymers in solution by high-resolution proton magneticresonance. J Polym Sci Polym Phys Ed 1977;15:2095}101.

[41] Marcus R, Coulston AM. In: Gilman AG, Rall TW, Niles AS,Taylor P, editors. The pharmacological basis of therapeutics. vol.2, New York: Pergamon Press, 1991. p. 1239.

1704 S.Y. Kim, Y.M. Lee / Biomaterials 22 (2001) 1697}1704