Biomaterials 24 (2003) 55–63

Toxic characteristics of methoxy poly(ethylene glycol)/poly(e-caprolactone) nanospheres; in vitro and in vivo

studies in the normal mice

So Yeon Kima, Young Moo Leea,*, Doo J. Baikb, Ju S. Kangc

aDepartment of Biomedical Engineering, School of Chemical Engineering, College of Engineering, Hanyang University, Seoul 133-791, South KoreabDepartment of Anatomy, College of Medicine, Hanyang University, Seoul 133-791, South Korea

cDepartment of Pharmacology and Institute of Biomedical Science, College of Medicine, Hanyang University, Seoul 133-791, South Korea

Received 6 February 2002; accepted 10 June 2002

Abstract

Amphiphilic diblock polymeric nanospheres composed of methoxy poly(ethylene glycol) (MePEG) and poly(e-caprolacto-ne)(PCL) was prepared for application as a novel drug carrier. We could obtain the MePEG/PCL nanospheres that exhibited an

average diameter of less than 200 nm with narrow size distribution and a relatively high drug-loading efficiency of about 41.98% and

20.8% for indomethacin and paclitaxel, respectively. To estimate the toxicity of nanospheres, we investigated cytotoxicity using the

normal human fibroblast, the median lethal dose (LD50) and various organ toxicities using male ICR mice. The indomethacin-

loaded nanosphere showed higher cell viability than indomethacin in the cytotoxicity test using 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl tetrazolium bromide (MTT) assay. The LD50 of MePEG/PCL nanospheres determined by Litchfield–Wilcoxon method

was 1.47 g/kg. After the mice were intraperitoneally injected with MePEG/PCL nanospheres as a half-dose level of LD50 for 7 days,

no significant histopathologic changes were observed in MePEG/PCL nanospheres-treated mice compared with normal mice in the

light and electron microscopic observations of various organs such as heart, lung, liver and kidney. It was suggested that MePEG/

PCL nanospheres might be useful candidate as a novel injectable drug carrier for hydrophobic drugs such as indomethacin and

paclitaxel.

r 2002 Published by Elsevier Science Ltd.

Keywords: Methoxypolyethylene glycol/ploy(e-caprolactone) nanosphere; Drug delivery system; MTT assay; Median lethal dose (LD50); Organ

toxicity

1. Introduction

In recent years, colloidal drug carriers have been usedto develop the site-specific drug delivery systems andsignificant advances are being made in the design,formulation, and surface modification of colloids, whichimprove the prospects for site-specific drug deliveryfollowing intravenous administration [1–4]. Most of thestudies on these carriers were concerned with liposomes[4–5]. Fewer studies have been carried out on biocom-patible and biodegradable polymeric carriers than onliposome but these carriers are an alternative in anattempt to overcome the problem of stability of

phospholipid vesicles during storage in biological fluids[6–9]. Among polymeric drug delivery devices, nano-spheres represent a promising approach to decreaseuptake of drugs to reticuloendothelial system, especiallyliver and spleen after intravenous injection [10–12]. Theadvantages of the nanospheres are the improvement ofbody distribution, the reduced drug toxicity and theimproved drug efficacy.

Diblock copolymers composed of hydrophilic andhydrophobic segments have been studied in thesustained release system as an alternative drug carriersince they are known to form a micellar structure[13–16]. Hydrophilic–hydrophobic diblock copolymersexhibit amphiphilic behavior and form micelles withcore-shell architecture. These polymeric carriers havebeen used to solubilize hydrophobic drugs, to increaseblood circulation time and to decrease the liver uptake

*Corresponding author. Tel.: +82-2-2291-9683; fax: +82-2-2291-

5982.

E-mail address: ymlee@hanyang.ac.kr (Y.M. Lee).

0142-9612/02/$ - see front matter r 2002 Published by Elsevier Science Ltd.

PII: S 0 1 4 2 - 9 6 1 2 ( 0 2 ) 0 0 2 4 8 - X

of the nanospheres. A particular attention has beenpaid to the development of parenteral therapeuticsystems that are made of biodegradable polymers,as potential formulations for site-specific drug deliveryincluding drug targeting [17–18]. Although medianlethal dose value is of limited significance, acutetoxicity studies involve a qualitative and quantitativeevaluation of the toxic effects of a substance, andthe assessment of their time-related occurrence aftersingle administration [19–26]. Despite of the interestingphysicochemical characteristic results, data on in vivoand in vitro toxicity study of polymeric carriers arerare.

Therefore, we mainly focused on acute organ toxi-city of methoxy poly(ethylene glycol) (MePEG)/poly(e-caprolactone) (PCL) nanosphere system sincesuch polyesters were well tolerated and also known tobe biodegradable to metabolites of the Krebs cycle afterintravenous administration in this study. In addition topreparation of MePEG/PCL nanosphere-loading indo-methacin and paclitaxel and their physicochemicalcharacteristics as a drug carrier, this study describescytotoxicity tests using normal human fibroblast. Acutetoxicity studies have performed by determining medianlethal dose (LD50) and various organ toxicities innormal ICR mice.

2. Materials and methods

2.1. Materials

Methoxy poly(ethylene glycol) (MePEG, Mn=5.0� 103 by supplier, Mn=5.5� 103 by our gel permea-tion chromatography (GPC) measurements) was sup-plied by Fluka and purified by azeotropic distillationwith benzene (Junsei Chemical Co. Ltd.). e-caprolactonewas purchased from Tokyo Kasei Organic Chemicalsand recrystallized from ethyl acetate. Hanmi Pharma-ceutical Co. Ltd. in Seoul, Korea kindly supplied semi-synthetic paclitaxel. Indomethacin was obtained fromSigma Chemical Co. (St. Louis, MO). All otherchemicals used were of reagent grade and used aspurchased without further purification. Experimentalanimals (ICR mice, 22–25 g, Charles River Co., USA)received humane care and the Institutional AnimalCare. Use Committee approved this study. The animalswere housed in a ventilated (10 to 15 times/h) roomunder conditions of controlled temperature maintainedat 20–231C and humidity of 50–70% during theinvestigation. The noise and luminous intensity of thecontrolled room with a 12 h light and 12 h dark (6:00 to18:00) cycle were below 60 db and 200 lx duringexperiment, respectively. Free access to food and waterwas allowed.

2.2. Preparation and characterizations of hydrophobic

drug-loaded MePEG/PCL nanospheres

As previously reported in our papers [27–29],MePEG/PCL diblock copolymeric nanosphere copoly-mers composed of MePEG and PCL were synthesizedby the ring opening polymerization of PCL in thepresence of MePEG homopolymer without using anycatalysts. Drug-loaded MePEG/PCL nanospheres thatcontained hydrophobic drug such as indomethacin andpaclitaxel were prepared. The molecular weight andmolecular weight distribution of diblock copolymerswere measured with gel permeation chromatographyapparatus (GPC: Waters Model 510 HPLC pump,Milford, USA) with a Millennium software program.The composition and number-average molecular weightof each copolymer were also determined by a 500MHz1H NMR (Bruker AMX-500). Average size and sizedistribution of nanospheres were determined by adynamic light scattering using an argon ion laser (model95-2, Lexel Laser Inc., USA) at a wavelength of514.5 nm at 201C. The intensity of the scattered lightwas detected with a photomultiplier (BrookhavenInstrumental Co. EMI9863) at a scattering angle of901 and the digital photon correlator accumulated bythe time correlation function was fitted by the use of themethod of CONTIN programs [30].

2.3. Determination of drug-loading efficiency of

indomethacin and paclitaxel in the MePEG/PCL

nanosphere

The amount of drugs entrapped was determined bymeasuring the UV absorbance at 319 nm using theUV–visible spectrophotometer (Shimadzu Model UV-2101 PC). The drug content entrapped into the core ofnanospheres was calculated from the weight of initialdrug-loaded nanospheres and the amount of drugincorporated from the following equation:

Drug-loading efficiency ðDLEÞ ð%Þ

¼Amount of drug in nanosphere

Amount of drug� loaded nanosphere� 100

¼drug

drugþ polymer� 100: ð1Þ

2.4. In vitro cytotoxicity test of MePEG/PCL

nanospheres

The toxicological properties of MePEG/PCL nano-spheres were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test andthe cell viability was determined as cytotoxicity para-meter [24]. Because indomethacin is less toxic thanpaclitaxel, we choose the indomethacin as control drug

S.Y. Kim et al. / Biomaterials 24 (2003) 55–6356

in the in vitro cytotoxicity test. The cytotoxicity testwas carried out while the cells were in a phase ofexponential growth with a doubling time of 48 h, andstarting cell density in a 96-well microplate was 5� 103/well. Detailed description of the MTT test is givenelsewhere [29].

The cell viability was expressed as a percentagecompared to a control untreated with indomethacin-loaded MePEG/PCL nanospheres or indomethacin bythe following equation:

Cell viability ð%Þ ¼ ðNt=NcÞ � 100; ð2Þ

where Nt and Nc are the number of surviving cells in thetreated fibroblasts with indomethacin-loaded MePEG/PCL nanospheres or free indomethacin and in theuntreated fibroblasts, respectively.

2.5. Median lethal dose (LD50) and organ toxicity of

MePEG/PCL nanospheres

In order to investigate acute toxicity of MePEG/PCLnanospheres [19–21,24–25], we estimated the medianlethal dose (LD50) and observed the histopathologicalchanges of major organs using male ICR mice. Wedetermined the median lethal dose (LD50) using theLitchfield–Wilcoxon method [31,32]. Male ICR mice,weighing 22–25 g, were divided into six groups (n ¼ 10).Experimental animals were intraperitoneally injected byvarious doses of MePEG/PCL nanospheres from 0.8 to2.4 g/kg for six groups and lethal response wereobserved for 48 h after administration. In order toinvestigate the major organs toxicity of MePEG/PCLnanospheres, we observed the histopathological changesof major organs such as heart, lung, liver and kidney bylight and electron microscope in the mice. Mice wereintraperitoneally administered with MePEG/PCL nano-spheres as a half-dose level of LD50 for 7 days. The

histopathologic changes of each organ were observed bylight and electron microscope.

3. Results

The structure of MePEG/PCL nanospheres withvarious compositions was confirmed by a Fouriertransform infrared spectroscopy, wide angle X-raydiffraction pattern, GPC and 1H-NMR measurement.In addition, the critical micelle concentration, in vitrodrug release behavior and pharmacokinetics of indo-methacin-loaded MePEG/PCL nanospheres were pre-viously investigated in our papers [27–29,33]. Thecomposition and molecular weight of MePEG/PCLnanospheres measured by GPC and 1H NMR analysiswere summarized in Table 1. The average size of theMePEG/PCL nanospheres was smaller than 200 nm andthe size distribution exhibited a narrow and mono-disperse pattern. After the loading of drugs such asindomethacin and paclitaxel in MePEG/PCL nano-spheres, their size somewhat increased. However, thesize distribution of nanospheres was relatively identicalwith the narrow distribution to that before drug loading.

The amount of drugs introduced into the MePEG/PCL nanosphere by controlling the molecular weight ofblock copolymer and the weight ratio between polymerand drugs were investigated and listed in Table 2. Theloading efficiency of indomethacin in MePEG/PCLnanosphere increased in proportion to the molecularweight and the length of hydrophobic block ofcopolymer, and that of paclitaxel had maximal whenthe feed ratio of drug: polymer was 0.50:1.00 as shownin Table 2. In addition, we prepared the drug-loadednanospheres by varying the initial feed weight ratio ofdrug to copolymer. As shown in Table 2, the loadingamount of drug in nanosphere increased with the feed

Table 1

Compositions and molecular weight distribution of MePEG/PCL block copolymers

Sample Feed molar

ratioaMolar

compositionbComposition

(wt %)bComposition

(wt %)cNumber-average molecular weight Polydispersity

ð %Mw= %MnÞ

e-CL/MePEG e-CL/MePEG MePEG:e-CL MePEG:e-CL Calc.d Expt’l.e Expt’l.c

MePEG 0 0 100:0 100:0 5000 5541 5333 1.128

MEP35 35 21.8 68.9:31.1 66.9:33.1 8995 8037 7971 1.256

MEP50 50 40.1 54.8:45.2 51.7:48.3 10707 10116 10317 1.250

MEP70 70 54.2 47.2:52.8 39.9:60.1 12990 11734 13367 1.102

MEP100 100 81.5 37.3:62.7 37.1:62.9 16414 14839 14401 1.102

MEP150 150 109.9 30.6:69.4 29.3:70.7 22121 18085 18205 1.178

aDetermined on the basis of Mn of MePEG calculated in GPC experiments.bEstimated as the difference between the experimental total Mn of copolymer and MePEG homopolymer in GPC experiments.cDetermined by 1H NMR spectroscopy (CDCl3).dCalculated from MePEG (MW=5000, Fluka).eMeasured by GPC analysis.

S.Y. Kim et al. / Biomaterials 24 (2003) 55–63 57

weight ratio of drug to MePEG/PCL diblock copoly-mer. For the loading of paclitaxel, the maximal loadingefficiency of 20.79% could be obtained when the feedweight ratio of MePEG/PCL copolymer: paclitaxel was1.0:0.5 (TMC–0.50). The DLE of TMC-1.00 sample(feed weight ratio of polymer: paclitaxel=1.0:1.0)exhibited about 9.08%.

After 3-day incubations in the culture of normalhuman fibroblast cell, the cell viability of sample wasassayed. Fig. 1 exhibited the cell viability of indometha-cin-loaded MePEG/PCL nanosphere sample with41.98% DLE and free indomethacin. The free indo-methacin exhibited higher cytotoxic activity thanindomethacin-loaded MePEG/PCL nanospheres at the

same concentration of indomethacin. This tendency wasenhanced with the concentration of indomethacin.Particularly, while the indomethacin-loaded MePEG/PCL nanospheres showed high cell viability of about85%, the free indomethacin exhibited the viability of lessthan 50% at the concentration of 0.064 mg/ml. Itsviability gradually decreased in proportion to theconcentration of indomethacin.

To estimate the median lethal dose (LD50), the singleintraperitoneal injection with varying the doses ofMePEG/PCL nanosphere (0.8–2.4 g/kg) was performedfor six groups (n ¼ 10) and close observations weremade during the period of 48 h after nanosphereadministration. The median lethal dose (LD50) of

Table 2

Drug-loading contents of MePEG/PCL block copolymeric nanosphere

No. Sample MePEG/PCL

copolymer used

Drug feed weight ratio Drug-loading

efficiency (DLE) (%)a

Drug: Polymer

1 DIP25 MEP70 IMCb 0.25:1.00 16.33

2 DIP50 MEP70 IMC 0.50:1.00 20.99

3 DIP75 MEP70 IMC 0.75:1.00 31.96

4 DIP100 MEP70 IMC 1.00:1.00 41.98

5 DMEP35 MEP35 IMC 1.00:1.00 25.83

6 DMEP50 MEP50 IMC 1.00:1.00 34.08

7 DMEP70 MEP70 IMC 1.00:1.00 41.98

8 DMEP100 MEP100 IMC 1.00:1.00 42.03

9 TMC-0.05 MEP50 Taxolc 0.05:1.00 4.10

10 TMC-0.10 MEP50 Taxol 0.10:1.00 9.06

11 TMC-0.20 MEP50 Taxol 0.20:1.00 12.15

12 TMC-0.50 MEP50 Taxol 0.50:1.00 20.79

13 TMC-1.00 MEP50 Taxol 1.00:1.00 9.08

aðDLEÞ ð%Þ ¼Amount of drug in nanosphere

Amount of drug� loaded nanosphere� 100 ¼

drug

drugþ polymer� 100:

b Indomethacin.cPaclitaxel.

0.005 0.01 0.032 0.064

0

20

40

60

80

100

120

Concentration (µg/ml)

Indomethacin-loaded MePEG/PCL nanosphere

Free indomethacin

Cel

l Via

bilit

y (%

)

Fig. 1. Cytotoxicity assay of indomethacin-loaded MePEG/PCL nanosphere after 3-day incubation in normal human fibroblast cell cultures. Cell

viability (%)=(Nt=Nc)� 100, where Nt and Nc are the number of surviving cells in the treated group with indomethacin-loaded nanospheres or free

indomethacin and in the untreated group, respectively.

S.Y. Kim et al. / Biomaterials 24 (2003) 55–6358

MePEG/PCL nanosphere that was determined byLitchfield–Wilcoxon method was about 1.47 g/kg.

To investigate the histopathologic effect of MePEG/PCL nanosphere on the various organs such as heart,lung, liver and kidney, mice were intraperitoneallyadministered with MePEG/PCL nanosphere solutionas a half-dose level of LD50 for 7 days. The histopatho-logic changes of each organ were observed on the 8 daysafter treatment of the MePEG/PCL nanaospheres bylight and electron microscopic measurements. Fig. 2exhibited the light microscopic images of each organtreated with or without the MePEG/PCL nanaospheres.As shown in this figure, no histopathologic changes wereobserved in MePEG/PCL nanosphere-treated groupscompared with normal group as a control.

In addition, the electron microscopic appearances ofnormal tissue and tissue treated with MePEG/PCLnanosphere were shown in Figs. 3–5. Fig. 3(a)–(d) wereelectron micrographs of endothelial cells of proximalconvoluted tubule (PCT) and glomerulus of mousekidney. For epithelial cell images of PCT in normalmouse kidney as a control, there were abundantmicrovilli (Mv) at apical surface, and the nucleus (N)was located more toward bases than apical portion.Numerous mitochondria (Mt), Golgi complex (Go), andrough endoplasmic reticulum (RER) were distributed inapical zone of cytoplasm. The tubular epithelial cell wasattached to adjacent PCT cell by junctional complex(Jc). In the epithelial cell of PCT in mouse kidneytreated with nanosphere (Fig. 3(b)), significant histo-pathological changes were not observed, and round Nand cytoplasmic organelles such as Mt, pinocytoticvesicles (Pv), RER, and Go were intact.

As shown in Fig. 3(d), the glomerulus of mousekidney treated by nanosphere was observed similar tothat of normal (Fig. 3(c)). glomerular capillary (Ca),their endothelium (END) and endothelial N seemed tobe intact. Basement membrane (BM) of glomerularfiltration membrane was seen electrolucently. The podo-cytes (Po) and their pedicles (Pe) were well developed.The Bowman’s spaces (BS) were regular shape. The redblood cell (RBC) was within capillary lumen.

Fig. 4 exhibited the electron micrographs of hepato-cyte. In both normal and MePEG/PCL nanosphere-treated hepatocyte, RER was abundantly distributedand consisted of paralleled cisternae with ribosomegranule (R). Glycogen particle (Gly), Go, Mt, primarylysosomes (Ly) and lipid droplets (L) were observed.The hepatocyte was attached to adjacent cell by Jc.Also, bile canaliculi (BC) was located between hepato-cytes. Go was distributed adjacent to bile canaliculi(BS).

The ultrastructures of alveolar wall in lung wereshown in Fig. 5. For the MePEG/PCL nanosphere-treated lung tissue (Fig. 5(b)), no significant differencewas observed compared with normal tissue (Fig. 5(a)).The endothelial N, BM, and Mt of alveolar capillarywere seen. The alveolar spaces (lumen) (AL) of the lungwere separated by the elements of the interaveolarseptum (AS). The alveolar cell (AC) had Mv thatprotruded into the lumen of an alveolus and containround lipid inclusion (L).

Fig. 6(a) is electron micrograpic image of normalheart muscle. In the normal myofibrils (Mf), orderedmyofilament, M-line (M) and relatively dark Z-line (Z)were seen. Mt and ribosomes (R) were observed in theinterfibrillar space. Likewise, distinct M-, Z-line andnumerous Mt which occupied the interfibrilar sarco-plasm could be seen in nanosphere-treated heart muscletissue (Fig. 6(b)). In addition, aggregated glycogengranules (Gly) in the interfibrillar space were seen.

Fig. 2. Light micrographs of each organ in mouse treated with or

without the MePEG/PCL nanosphere (� 400): (a) normal heart

muscle, (b) nanosphere-treated heart muscle, (c) normal lung, (d)

nanosphere-treated lung, (e) normal kidney, (f) nanosphere-treated

kidney, (g) normal liver and (h) nanosphere-treated liver.

S.Y. Kim et al. / Biomaterials 24 (2003) 55–63 59

4. Discussion

The size of MePEG/PCL nanospheres increased withthe molecular weight of block copolymer and theloading amount of drug also influenced on the size ofMePEG/PCL nanosphere. In the case of indomethacin,the aggregation was found during the preparation ofnanospheres as we increased the feed ratio of indo-methacin to copolymer exceeding 1.0:1.0 becauseindomethacin had a hydrophobic character. When thefeed ratio of paclitaxel to copolymer exceeded 1.0:0.5, itwas found that unloaded paclitaxel significantly aggre-gated during the preparation of MePEG/PCL nano-sphere, resulting in the decrease of both the yield ofpaclitaxel-loaded nanosphere and the loading efficiencyof paclitaxel. It denoted that the hydrophobic interac-tion between drugs was greater than that between drugand MePEG/PCL copolymer due to the high lipophiliccharacter of paclitaxel as the feed ratio of paclitaxelincreased.

We could consider two factors that influence the cellviability from the results on MTT test such as theinteraction of indomethacin with cells and the releasecharacteristics of indomethacin from drug-loaded Me-PEG/PCL nanospheres. Firstly, the direct interactionsbetween indomethacin and cells could be reduced since

indomethacin was loaded in the core of poly-meric nanospheres. Furthermore, the outer shell com-posed of hydrophilic MePEG block could reduce theinteraction between nanospheres themselves and be-tween nanospheres and cells by forming the stealthsurface. Second, the viability of cells could be main-tained due to the sustained release characteristics ofindomethacin from drug-loaded MePEG/PCL nano-spheres.

Indomethacin is a non-steroidal antiinflammatoryand antipyretic drug, useful for the relief of symptomsof rheumatoid arthritis. Since the practical effective doseof indomethacin is about 25–75mg at once in human[34] and intraperitoneal LD50 of indomethacin in rat isabout 13mg/kg [35], the MePEG/PCL nanospheresystem which had DLE of more than 40% could beavailable as an injectable drug carrier. The maximaldoses of paclitaxel for promising anticancer activity arepractically 30mg/m2 per day for 5 days or 210–250mg/m2 given once every 3 weeks. If we consider the requiredamount of MePEG/PCL nanosphere with paclitaxel(DLE=20.79%) on the basis that average body surfacearea of adult with 60 kg in body weight is about 1.73m2,the amount of MePEG/PCL nanosphere needed isabout 4.33mg/kg that is much less than the LD50

(1.47 g/kg).

Fig. 3. Electron micrographs (� 10,000) of epithelial cell of PCT of: (a) normal mouse kidney and (b) MePEG/PCL nanosphere-treated mouse

kidney: Mv (microvilli), N (nucleus), Mt (mitochondria), RER (endoplasmic reticulum), Go (Golgi complex), Jc (Junctional complex) and Pv

(pinocytotic vesicles). Electron micrographs (� 15000) of glomerulus of (c) normal mouse kidney and (d) MePEG/PCL nanosphere-treated mouse

kidney: Ca (glomerular capillary), N (nucleus), END (endothelium), BM (basement membrane), Pe (pedicles), Po (podocytes), BS (Bowman’s spaces)

and RBC (red blood cell).

S.Y. Kim et al. / Biomaterials 24 (2003) 55–6360

From the results of histopathological analysis, itcould be confirmed that MePEG/PCL nanosphere didnot seriously damage the organ by accumulation withinorgan. Therefore, it can be concluded that the presentMePEG/PCL nanosphere is available as drug carriersystem for anticancerous paclitaxel.

5. Conclusions

The size of MePEG/PCL nanospheres was less than200 nm and their size distribution showed the narrowpattern. After the loading of drugs such as indomethacinand paclitaxel in MePEG/PCL nanospheres, the sizedistribution of nanospheres was relatively identical withthe narrow distribution to that before drug loading. In

addition, MePEG/PCL nanospheres having relativelyhigh drug loading efficiency (DLE) could be obtained.The DLE of more than about 40% was obtained forindomethacin-loaded nanosphere when the feed weightratio of indomethacin to polymer was 1:1. About 20%was determined for the DLE of paclitaxel when theinitial feed weight ratio of paclitaxel to polymer was0.5:1. The median lethal dose (LD50) of MePEG/PCLnanosphere determined by Litchfield–Wilcoxon methodwas 1.47 g/kg in the ICR mice and no histopathologicchanges were observed in MePEG/PCL nanosphere-treated group compared with untreated normal group asa control in the histopathologic study with light andelectron microscopies. From these results, we couldsuggest that the MePEG/PCL diblock copolymericnanosphere system was biocompatible drug vehicle

Fig. 5. Electron micrographs (� 10,000) of the alveolar wall structure

of: (a) normal mouse lung and (b) MePEG/PCL nanosphere-treated

mouse lung: N (nucleus), BM (basement membrane), M (mitochon-

dria), AC (alveolar cell), AL (alveolar spaces, lumen), AS (inter-

alveolar septum), Mv (microvilli) and L (lipid droplet).

Fig. 4. Electron micrographs (� 10,000) of hepatocyte of (a) normal

mouse and (b) MePEG/PCL nanosphere-treated mouse: RER

(endoplasmic reticulum), R (ribosome granule), Gly (glycogen

particle), Go (Golgi complex), Ly (primary lysosome), Mt (mitochon-

dria), L (lipid droplet), Jc (Junctional complex) and BC (bile

canaliculi).

S.Y. Kim et al. / Biomaterials 24 (2003) 55–63 61

candidates for hydrophobic drugs in injectable deliverysystems.

Acknowledgements

The Korea Research Foundation Grant No. 98-E00080 supported this research.

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