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Colloids and Surfaces B: Biointerfaces 81 (2010) 530–536

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

haracterization and preparation of core–shell type nanoparticle forncapsulation of anticancer drug

i-Kyeong Jang, Young-Il Jeong, Jae-Woon Nah ∗

epartment of Polymer Science & Engineering, Sunchon National University, 315 Maegok, Suncheon, Jeonnam 540-742, Republic of Korea

r t i c l e i n f o

rticle history:eceived 15 October 2009eceived in revised form 22 July 2010ccepted 23 July 2010vailable online 3 August 2010

eywords:MWSC

a b s t r a c t

The aim of this study is to prepare delivery vehicles of paclitaxel using low molecular weight water-soluble chitosan (LMWSC) and evaluate them as an anticancer drug delivery system. LMWSC wasmodified with methoxy polyethylene glycol (LMWSC-MPEG, ChitoPEG), and then it was conjugatedwith cholesterol (LMWSC-MPEG-Chol). Core–shell type LMWSC-MPEG-Chol nanoparticles (LMWSC-NPs)were prepared by the dialysis method, and the core–shell structure was confirmed by 1H NMR analysis. Tothis polymer, paclitaxel was encapsulated and core–shell type nanoparticles were prepared. The releasetests indicated that release of paclitaxel from the core–shell type nanoparticles and its transport across

anoparticleaclitaxelydrophobic anticancer drugumor inhibition

the dialysis membrane was slower than dialysis of free paclitaxel. In a cytotoxicity study using CT26 cell,the paclitaxel-encapsulated core–shell type nanoparticles (LMWSC-NPs) showed a toxicity against tumorcells similar to paclitaxel itself. The results of a tumor inhibition test with CT26 implanted upon mousetumor models in vivo indicated that the application of a dose of 10 mg/kg of LMWSC-NPT showed a supe-rior survival rate, and a slower tumor growth than when paclitaxel alone was administered, although the

al rawed a

tumor growth and survivdose above 10 mg/kg sho

. Introduction

Paclitaxel, attested by FDA in 1992, has been known to showignificant anticancer activity to a wide range of tumors such asefractory ovarian cancer, non-small cell lung cancer, metastaticreast cancer, head and neck malignancies, AIDS-related Kaposi’sarcoma, etc. [1–5]. Despite its excellent anticancer activity, pacli-axel has serious problems including toxic side-effects and poorolubility in the conventional aqueous injection solution. Espe-ially, low therapeutic index of paclitaxel is attributed to its toxicide-effects [6]. Because of low solubility of paclitaxel [7], it has toe dispersed in a mixed solution of cremophor EL (polyethoxylatedil) and ethanol (50:50), which is diluted 5- to 20-fold in normalaline or in 5% dextrose solution for intravenous injection. Variousystems have been proposed to make paclitaxel formulations fornjection such as parenteral emulsion [8–10], mixed micelles [11],

ater-soluble prodrugs [12], polymer micelles [13,14], core–shellype nanoparticles [15], albumin-bound nanoparticles [16], and

iodegradable polymeric nanoparticles [17].

Application of nanoparticles based on biodegradable polymersre also a useful tool for intravenous introduction of anticancerrug into the body in order to effectively deliver the sustained

∗ Corresponding author. Tel.: +82 61 750 3566; fax: +82 61 750 5423.E-mail address: jwnah@sunchon.ac.kr (J.-W. Nah).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.07.053

te were not significantly changed at a dose of 2 mg/kg. The LMWSC-NPTsuperior antitumor activity.

© 2010 Elsevier B.V. All rights reserved.

drug action, reduce side-effects, facilitate extravasations into thetumor, increase the capability to cross various physiological bar-riers as well as control and target the delivery [18–20]. To date,nanoparticles for formulation of anticancer agent have frequentlybeen prepared using poly lactic-co-glycolic acid (PLGA) [21], poly-lactic acid [22], poly ethylene oxide-co-poly(lactic acid) [23],poly(�-caprolactone)[24,25] and poly-propylene oxide [26]. Thesepolymeric nanoparticles offer a number of advantages for drugdelivery to tumor: enhanced loading efficiency of drug by thehydrophobic core, prevention of burst effects, easy modificationof targeting ligands for site specificity, and exact targeting atthe desired site by ligand. In spite of these advantages, syntheticpolymers have many obstacles such as the complexity of prepa-ration process, the remnants of surfactant used for preparation ofnanoparticle, their own cytotoxicity in the body. Natural polymers,however, can overcome the problems of synthetic polymers.

Chitosan has been extensively investigated as biomaterials andcarriers of anticancer drug. The structure of chitosan is a longunbranched polysaccharide similar to that of a cellulose derivativein which amino groups have replaced the hydroxyl group at theC2 position [27]. It is biodegradable and non-toxic and has reac-

tive hydroxyl and amino groups that can be modified chemicallyfor various applications [28]. Therefore, chitosan will emerge as animportant biomedical material of the 21st century. However, appli-cations of chitosan are limited by the poor water solubility and thesalts introduced to solve this problem, which result in blocking the

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eactivity of amine group. High molecular weight chitosan (HMWC)as poor solubility in water or organic solvents, and that is one ofhe severe disadvantages that are blocking its actual use.

In this study, we prepared core–shell type nanoparticles usingydrophobically modified and PEGylated LMWSC (low molecu-

ar weight water-soluble chitosan) as an anticancer drug delivery

ystem. We have previously reported on the potentials of self-ssembling nanoparticles using LMWSC [29,30]. LMWSC is auperior property that can enhance the solubility of hydropho-ic anticancer drug and give passive targeting potential. MPEGmethoxy polyethylene glycol) chains introduced as a hydrophilicroup can prevent cell adhesion by entropically driving stericepulsion and by increasing the hydrophilicity of carrier surfaces.urthermore, introduction of cholesterol as a hydrophobic groupan enhance the association behavior of LMWSC, and the stablectivity of the hydrophobic drug can be enhanced by forming aydrophobic core. We expect that hydrophobically modified Chi-oPEG (LMWSC-MPEG) may be used as carriers of hydrophobicnticancer drug.

. Materials and methods

.1. Materials

Low molecular weight water-soluble chitosan (LMWSC,8 kDa) was supplied by KITTOLIFE Co., Seoul, Korea. Methoxyoly(ethylene glycol) N-hydroxysuccinimide (MPEG-NHS, 5 kDa)as purchased from SunBio Co., Korea. Cholesteryl chloroformatesere purchased from Aldrich Chem. Co. Ltd., USA. Paclitaxel wasurchased from Sigma Co., USA. BALA/c mice (20 g, 5 weeks) wereurchased from Damul Science, Co. Dialysis tubing (MWCO 12,000)as commercially obtained from Spectrum. Dimethylformamide

DMF), dimethylsulfoxide (DMSO) was purchased from Sigma Co.,SA and used without further purification.

.2. Synthesis of LMWSC-MPEG copolymer

Synthesis of LMWSC-MPEG graft copolymer was performed asollows: 100 mg of LMWSC was dissolved in 0.2 ml of deionizedater and diluted with 9.8 ml of DMSO. To this solution, MPEG-HS dissolved in 2 ml of DMSO was added and reacted for overnightt nitrogen atmosphere. After that, the resulting solution was dia-yzed extensively against deionized water for 2 days followed byts lyophilization. The lyophilized solid was resuspended into alenty of DCM (dichloromethane) to remove unreacted MPEG-NHShree times and fractionated into deionized water followed by itsyophilization. The substitution (DS) ratio of PEG was calculated asreviously reported [31]. 1H NMR spectra were measured to eval-ate DS (data not shown). DS of PEG was found to be 10 wt.% by theatio of methyl group of PEG/proton of C1 position of chitosan.

S = Proton integration ratio of methyl g(Proton integration of C1 position of chitosan + Proton integr

.3. Synthesis of LMWSC-MPEG-Cholesterol copolymer

DS = Proton integrati(Proton integration of C1 position of ch

Synthesis of LMWSC-MPEG-Cholesterol (LMWSC-MPEG-Chol)opolymer was performed as follows [30]: cholesterol chlorofor-ate and LMWSC-MPEG copolymer was dissolved in DMSO and

tirred magnetically for 6 h at room temperature (25 ◦C). After that,he resulting solution was precipitated to cold ether. This process

: Biointerfaces 81 (2010) 530–536 531

of MPEG/3ratio of acetyl group of chitosan)/3

× 100

was repeated three times. Unreacted cholesterol chloroformatewas removed because cholesterol chloroformate is soluble in etherwhile synthesized copolymer is not soluble in it. Precipitants wereobtained by filtration and then dried under vacuum for 3 days. Thesubstitution ratio of cholesterol using 1H NMR was calculated usingthe following equation:

tio of methyl group of cholosterol/3n + Proton integration ratio of acetyl group of chitosan)/3

× 100

In this equation, each peak was as follows: methyl group ofcholesterol, 2 ppm; C1 position of chitosan, 6.1–6.5 ppm of Fig. 2(a);acetyl peak of LMWSC, 1.9–2.0 ppm of Fig. 2(b) [29–31].

The result indicated that the DS value of cholesterol is 3.8 wt.%.

2.4. Preparation of core–shell type LMWSC nanoparticles(LMWSC-NPs)

LMWSC-MPEG-Chol (25 mg) was dispersed in 10 ml of PBS (pH7.4) and magnetically stirred for 30 min. Paclitaxel (4 mg) was dis-solved in 2 ml of ethanol, and it was slowly dropped into the abovesolution while sonication was performed using a bath type soni-cator. This solution was sonicated again using a bar type sonicatorfor 20 s (2 s × 10 times). This solution was introduced into dialysistube (dialysis membrane, molecular weight cut-off: 12,000 g/mol).Dialysis was performed in PBS (pH 7.4). To harvest paclitaxel-encapsulated LMWSC nanoparticles (LMWSC-NPTs), dialysis wasperformed in distilled water for 12 h, followed by centrifugation.

Then, LMWSC-NPTs were redistributed into deionized waterand filtered by 0.45 �m syringe filter. After that, LMWSC-NPT solu-tion was lyophilized and analyzed. Experimental drug contentswere 10% (w/w) (theoretical drug contents: 13.8% (w/w) and load-ing efficiency was 69.4% (w/w).

Drug contents = Amount of drug in the nanoparticlesTotal weight of nanoparticles

× 100

Loading efficiency = Residual amount of drug in the nanoparticlesFeeding amount of drug

× 100

2.5. Characterization of core–shell type LMWSC-NPs andLMWSC-NPT

To characterize paclitaxel and LMWSC-NP, paclitaxel-encapsulated LMWSC nanoparticles (LMWSC-NPT) were quantifiedby high performance liquid chromatography (HPLC) using phe-nomenex sphereclone 5 micro ODS(2) 250 mm × 4.6 mm columnand 75% methanol as a moving phase. At this point, the flowrate was set to 1.5 ml/min, and the temperature at 50 ◦C. The UVdetector (226 nm) was used for the measurement. To prepare thesample for the measurement of HPLC, paclitaxel was dissolved inethanol, making 20 �l for injection.

For the drug release test, 10 mg of LMWSC-NPT was recon-stituted in 5 ml of phosphate-buffered saline (PBS, pH 7.4,0.1 M), and then this solution was introduced to dialysis bag

(molecular weight cut-off (MWCO): 12,000 g/mol). This dialysisbag was introduced into a bottle with 95 ml of PBS–SDS (pH 7.4,0.1 M, SDS 2.0% (w/v)). For comparison, 1.0 mg of paclitaxel was

suspended in 5 ml of PBS–SDS (pH 7.4, 0.1 M, SDS 2.0% (w/v)) andmagnetically stirred until paclitaxel was completely dissolved. Thiswas introduced into dialysis bag, and then the dialysis bag wasintroduced into a bottle with 95 ml of PBS–SDS. At predeterminedtime intervals, the whole media was taken and replaced with fresh

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BS–SDS media. The released amount of paclitaxel from nanopar-icles was evaluated by HPLC method described above.

.6. Analysis of core–shell type LMWSC-NPs

Particle size of polymeric micelle was measured with a dynamicaser scattering spectrophotometer (DLS-7000, Otsuka Electronicso., Japan). A sample solution prepared by the dialysis method wassed for particle size measurement (concentration: 0.1 wt.%).

The 1H nuclear magnetic resonance (NMR) spectra of the copoly-er and core–shell type nanoparticles were measured in D2O

r D2O + pyridine using a 400 MHz NMR spectrometer (AVANCE00FT-NMR 400 MHz, Bruker).

.7. Cytotoxicity of paclitaxel and LMWSC-NP against tumor celline in vitro

To test the anti-proliferation effect of empty LMWSC-NP,aclitaxel and LMWSC-NPT, CT26 colon carcinoma cells were main-ained under 5% CO2 incubator at 37 ◦C. The effect of emptyMWSC-NP, free paclitaxel and LMWSC-NPT on the tumor cell pro-iferation was determined using the MTT cell proliferation assay.mpty LMWSC-NP was reconstituted in DMEM (supplementedith 10% FBS) with a concentration of 1 mg/ml and then diluted itith DMEM (10% FBS) for an appropriate concentration. Paclitaxelas dissolved in 100% DMSO and diluted 100 times using DMEM

supplemented with 10% serum) and diluted it to an appropriateoncentration. LMWSC-NPTs were dissolved in DMEM (supple-ented with 10% serum) and diluted to adjust the equivalent

oncentration of the free paclitaxel. The tumor cell lines wereeeded at a density of 5 × 103 per well in 96-well plates using 100 �lf DMEM supplemented with 10% serum in a CO2 incubator (5%O2 at 37 ◦C) for 12 h. After that, 100 �l of DMEM (supplementedith 10% serum) containing empty LMWSC-NP or free paclitaxel

r LMWSC-NPT was added. After 1 or 2 days of incubation, MTTas added to the 96 wells and incubated for 4 h in a CO2 incubator

5% CO2 at 37 ◦C). Then, the supernatant was discarded, and 100 �lf DMSO was added to each of the 96 wells. The absorbance waseasured at 560 nm using a microtiter plate reader (Thermomaxicroplate reader, Molecular Devices).

.8. Antitumor activity of LMWSC-NPT at in vivo with CT26ouse tumor models

CT26 murine tumor cells (5 × 104 cells/mice) were implantedubcutaneously into the back of BALB/c mice (Female, averageody weight was 20 g). When tumors were grown to approxi-ately 3 mm × 3 mm (approximately day 14), the animals were

ivided into treatment and control groups. Each group consisted oftumor-bearing mice that were ear-tagged and followed-up indi-

idually throughout the study. The intravenous administration ofrugs or vehicle began on day 15. For this in vivo test, the pacli-axel dissolved in cremophor vehicle and LMWSC-NPT dissolved inistilled water were used. Each drug was administered at doses ofmg/kg as a low dose and 10 mg/kg as a high dose 4 times for 12ays at intervals of 3 days. The control group received the vehiclecremophor:dehydrated ethyl alcohol, 1:1, v/v). The mortality was

onitored daily, and the tumor growth was measured at two orhree day intervals by calliper measurement. Tumor volume wasalculated using the following formula:

umor volume (mm3) = length × width2

2

8 mice were used for each group.

Fig. 1. Chemical structure of LMWSC-MPEG-Chol (a) and schematic illustration ofcore–shell type LMWSC-NP (b).

3. Results

3.1. Characterization of paclitaxel-encapsulated core–shell typeLMWSC nanoparticles (LMWSC-NPTs)

Core–shell type LMWSC nanoparticles (LMWSC-NPs) have aunique chemical structure as shown in Fig. 1(a), and we presume,after NMR analysis, that it forms a core–shell structure in theaqueous environment as shown in Fig. 1(b), i.e., the cholesterol isconjugated in the LMWSC main chain and forms a hydrophobiccore of the core–shell, and PEG has formed a hydrated outer shell.To prove this hypothesis, the core–shell type nanoparticles wereprepared by sonication and the dialysis method, and its structurewas confirmed using 1H NMR as shown in Fig. 2. Specific peaksof LMWSC (1–6), MPEG (11–15), and cholesterol (a–d) were con-firmed by D2O + pyridine solution. When these nanoparticles werein D2O, the specific peaks of cholesterol disappeared while thepeaks of MPEG and LMWSC were still shown. These results provedthat cholesterol formed the hydrophobic inner core and the MPEGformed the hydrated outer shell.

As an anticancer drug, paclitaxel was encapsulated intocore–shell type LMWSC-NPs. Drug content was about 10% (w/w).The size of empty LMWSC-NP was around 270 nm, and the parti-cle size of LMWSC-NPT was not significantly changed compared toempty nanoparticles as shown in Table 1. However, LMWSC-NPTshowed a slightly broad distribution pattern compared to emptynanoparticles as shown in Fig. 3. Release of paclitaxel was mon-itored by HPLC as shown in Fig. 4(b). The result of HPLC revealedthat the retention time (RT) of paclitaxel is 3.4 min. Paclitaxel in theLMWSC-NPT was released over 1 month continuously while pacli-taxel itself was released fast in several days (Fig. 4(a)). These resultsindicated that LMWSC-NPs are acceptable vehicles for sustainedrelease of paclitaxel. Furthermore, the release rate of paclitaxel wasslower than that we had expected. It was suggested that the lowrelease rate was due to the strong hydrophobicity of paclitaxel.

3.2. Cytotoxicity of paclitaxel-encapsulated core–shell typenanoparticles

To examine the cytotoxicity of LMWSC-NPT, paclitaxel andLMWSC-NP were treated to CT26 colon carcinoma cells. Fig. 5shows cell survivability against drug concentration. Survivabil-ity of tumor cells gradually decreased as the drug concentrationincreased with paclitaxel treatment. Treatment of LMWSC-NPT

M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530–536 533

Fig. 2. 1H NMR spectra of core–shell type LMWSC-NP. Core–shell type nanoparticles in D2O + pyridine (a) and D2O (b).

Table 1Characterization of core–shell type nanoparticles.

Drug contents (%, w/w) Particle size (nm)

Intensity Volume Number

Empty LMWSC-NP –Paclitaxel-encapsulated nanoparticles (LMWSC-NPT) 10.0

Drug contents = [drug weight in the nanoparticle/total weight of nanoparticle] × 100.

Fig. 3. Particle size distribution of empty (a) and paclitaxel-encapsulated core–shelltype nanoparticles (b).

304.9 ± 56.3 274.6 ± 49.9 254.0 ± 40.5330.7 ± 82.3 274.6 ± 68.8 234.8 ± 51.4

showed an almost similar trend of cell survivability in proportion tothe drug concentration. As can be seen in Fig. 5, the empty LMWSC-NP did not have significant cytotoxicity against tumor cells, i.e.,higher than 80% of tumor cells survived at the highest concen-tration of nanoparticles (1000 �g/ml). These results indicate thatpaclitaxel-encapsulated core–shell type LMWSC-NP have a potencysimilar to that of paclitaxel itself.

3.3. In vivo, antitumor activity of LMWSC-NPT

For in vivo test of paclitaxel carriers, paclitaxel was dissolvedin cremophor vehicle, but LMWSC-NPT was dispersed in distilledwater because of the enhanced water solubility. Approximately2 weeks after tumor implantation into the back of the mice, anadvanced tumor appeared (approximately 3–5 mm in diameter),the mice received a vehicle (control group) or drug via tail vein.Fig. 6(a) shows the survival rate of the mice treated with pacli-taxel and LMWSC-NPT. The control group showed the fastest deathrate among all the groups. At a high dose of paclitaxel 10 mg/kg,the mice died more rapidly than other three groups until 40 days.The survival rate of the groups of paclitaxel 2 mg/kg, 10 mg/kg, andLMWSC-NPT 2 mg/kg were higher than the control group. Practi-cally, the survival rate between the groups of paclitaxel 2 mg/kg,10 mg/kg, and LMWSC-NPT 2 mg/kg were not significantly differ-

ent. At a dose of 10 mg/kg of LMWSC-NPT, the highest survival ratewas maintained throughout all the experimental period among allof the test groups.

The in vivo antitumor activity of paclitaxel and LMWSC-NPTagainst CT26 murine tumor was tested for tumor volume as shown

534 M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530–536

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ig. 4. Release of paclitaxel from core–shell type LMWSC-NP (a). For comparison, 1.nto dialysis bag and 10 mg of LMWSC-NPT (equivalent concentration of 1.0 mg pacliaclitaxel (b).

n Fig. 6(b). Tumor volume (mm3) was measured from 15 days afterhe tumor cell implantation. When paclitaxel and LMWSC-NPT arenjected at a low dose of 2 mg/kg, mice did not show any significanthanges against control group as a tumor volume.

At high doses of 10 mg/kg, paclitaxel showed a decreased tumorrowth compared to the control group, paclitaxel (2 mg/kg), andMWSC-NPT (2 mg/kg). At a dose of 10 mg/kg of paclitaxel, theumor growth was favorably suppressed, but the tumor growthestarted after 30 days. As shown in Fig. 6(b), LMWSC-NPT at aose of 10 mg/kg showed superior antitumor activity among allhe groups. In the case of LMWSC-NPT, the tumor growth was welluppressed until 38 days and restarted after 40 days. As shown inig. 6(c), the body weight changes among control groups, paclitaxel2 mg/kg), and LMWSC-NPT (2 mg/kg) were not significantly dif-erent. At high doses of 10 mg/kg, LMWSC-NPT showed decreasedody weight changes. These results indicated that tumor volume ofMWSC-NPT at 10 mg/kg was smaller than the other group, induc-ng decreased body weight changes.

Although tumor growth and survival rate did not significantlyhange at low dose of 2 mg/kg of LMWSC-NPT, LMWSC-NPT atoses of 10 mg/kg showed a superior survival rate and a decreasedumor growth compared to paclitaxel. LMWSC-NPT at the dose of0 mg/kg seems to offer a superior antitumor activity.

. Discussion

Chitosan, a (1–4) linked-2-amino-2-deoxy-�-d glucan, isiodegradable, non-cytotoxic, and has some biologically activeharacteristics. For its cationic properties, chitosan is extensivelynvestigated as a gene or drug carriers, and its amine group

ay support easy modification for drug delivery vehicles [32–34].specially, unlike HMWC (high molecular weight chitosan) chi-ooligosaccharide itself such as LMWSC are known to have an

etastatic activity to tumor cells [35]. Furthermore, LMWSC is

haracterized as water-soluble chitosan having low moleculareight and high density of free-amine group. Enhanced mucosalelivery and enhanced efficiency of transfection are the advantagesf LMWSC which give the increased free-amine group and cationicroperties [36,37].

f paclitaxel distributed in PBS–SDS (pH 7.4, 0.1 M, SDS 2.0% (w/w)) was introducedwas introduced into dialysis bag. Typical HPLC characteristic peak of the quantified

We previously reported on self-assembling chitosan nanopar-ticles having a core–shell structure [30]. Self-assembled LMWSCnanoparticles are composed of cholesterol-conjugated LMWSCbackbone and MPEG side chain. They have a core–shell structurein aqueous environment, i.e., the cholesterol that makes up thehydrophobic inner core is conjugated with the MPEG that makesup the hydrophilic outer shell to form the LMWSC nanoparticle.

Hydrophobic inner core is composed of cholesterol-conjugatedLMWSC, and the outer shell of the nanoparticles is composed ofMPEG. These nanoparticles have particle size around 30–150 nmaccording to the composition ratio [30]. After lyophilization ofthese nanoparticles, they can easily reconstitute into aqueoussolution such as distilled water or phosphate-buffered saline (pH7.4, 0.1 M) (data not shown). Miwa et al. [38] have reportedthat paclitaxel-entrapped micelle carriers are prepared from N-lauryl-carboxymethyl-chitosan with a maximum of 2.37 mg/mland their particle sizes were less than 100 nm. However, theirmicellar solution was maintained in an aqueous solution ratherthan in lyophilized form. For long-term storage, lyophilized form ofmicellar solution or nanoparticle solution is preferred to aqueoussolution because the lyophilized form has some advantages such asmaintenance of peculiar properties of drug, avoidance of aggrega-tion or precipitation of carriers and therapeutical activity of drugwhen compared to aqueous solution [39–41]. At this point, recon-stitution of lyophilization is an important factor for clinical usage.As shown in Fig. 3, nanoparticles with empty LMWSC-NP and theLMWSC-NPT are completely reconstituted in the aqueous solution,and no precipitants or large aggregates were observed. Hydropho-bically modified chitosan as a carrier of paclitaxel has been reportedby Kim et al. [32] and their self-assembled nanoparticles showed0.94–9.0% (w/w) of loading contents. Also, they reported that thesize of their nanoparticles increased up to 400 nm according to thedrug contents. However, their carriers of paclitaxel have drawbacksin the surface properties. The surfaces of our nanoparticles arecovered with PEG, which gave the barrier properties of nanopartic-

ulate, but their nanoparticles are not covered with PEG. That is whytheir nanoparticles gave no protection. As shown in Fig. 2, strongPEG peaks were observed at D2O while the peaks of cholesterol moi-ety were not observed. However, when the nanoparticle broke upin D2O + pyridine, the peaks of cholesterol moiety were observed.

M.-K. Jang et al. / Colloids and Surfaces B: Biointerfaces 81 (2010) 530–536 535

Fig. 5. Cell cytotoxicity of LMWSC-NPT against CT26 tumor cells. CT26 cells (5 × 103

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Fig. 6. Antitumor activity of LMWSC-NPT against CT26 colon carcinoma xenograftmodel. Each drug was administered at doses of 2 mg/kg as a low dose and10 mg/kg as a high dose for 4 times for 12 days at intervals of 3 days (arrows).The control group received the vehicle (cremophor:dehydrated ethyl alcohol, 1:1,v/v). 8 mice were used for each group: (a) Survival ratios of mice were moni-tored daily. LMWSC-NPT (paclitaxel-encapsulated core–shell type nanoparticles,10 mg/kg) showed increased survival time compared to all other treatment (P < 0.01vs. paclitaxel 10 mg/kg); (b) Tumor growth after drug injection was measured attwo or three day intervals. LMWSC-NPT (paclitaxel-encapsulated core–shell typenanoparticles, 10 mg/kg) effectively suppressed tumor growth (P < 0.01 vs. paclitaxel10 mg/kg); and (c) Body weight changes after tumor implantation following drugadministration. LMWSC-NPT (paclitaxel-encapsulated core–shell type nanoparti-

ells/well) were treated with paclitaxel and LMWSC-NPT for 1 day (top) and 2 daymiddle). Empty LMWSC-NP was also employed for comparison of cell cytotoxicitybottom). Empty LMWSC-NP was treated to CT26 cells (5 × 103 cells/well).

hese results indicate that the outer shell of nanoparticles is com-osed of PEG.

Currently, nanoparticles as colloidal drug carriers have beenxtensively investigated for tumor targeting applications [18,42].uring the past decade, the core–shell type nanoparticles or poly-eric micelles have been in the spotlight for drug targeting to

umor since their core–shell structure is suitable to solubilizeydrophobic anticancer drug [12,14,43]. Furthermore, their stealthroperties by surface-enriched PEG encourage long-circulationf drug vehicles, avoidance of reticuloendotherial system (RES)

cles, 10 mg/kg) showed decrease to all other treatment (P < 0.01 vs. paclitaxel10 mg/kg).

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ptake, and passive targeting of tumor [12,15,43]. Especially, theassive targeting of these drug vehicles can be described by EPRenhanced permeation and retention) effect to solid tumor [38]. Itseduced particle size (10–200 nm) enables intravenous injection ofydrophobic drug and passive targeting to a specific organ [16,17].

The loading of paclitaxel in the core–shell type nanoparticlesf LMWSC-MPEG-Chol was identified using HPLC (Fig. 4(b)). Theesult of HPLC revealed that the retention time (RT) of paclitaxel is.4 min. As shown in Fig. 4(a), paclitaxel release from the core–shellype LMWSC-NPT continued over 1 month while paclitaxel releasedtself from the dialysis membrane for 10 days.

Feng and Huang have reported the paclitaxel-encapsulatedanospheres [22]. In their report, nanospheres released paclitaxel

ess than 60% in 80 days. They reported that the duration of dif-usion was 2 months. Cho et al., reported that sodium salicylate isffected to the dissolution and release of paclitaxel from the poly-eric micelle [44]. We used SDS to increase the dissolution and

elease rate of paclitaxel from nanoparticles. However, paclitaxelelease took longer than expected. There are some experimentalactors that affect the release rate of paclitaxel from the nanopar-icles. For example, dialysis membrane itself can act as a barrier ofrug transportation. Other researchers have also reported that freeaclitaxel was released very slow [44]. Furthermore, the hydropho-ic core environment can increase the duration of drug release.

The antitumor efficacy of core–shell type LMWSC-NPT is shownn Fig. 6(a)–(c). The core–shell type LMWSC-NPT was most effec-ive to suppress tumor growth at 10 mg/kg paclitaxel dose whileaclitaxel at equivalent dose was less effective to suppress tumorrowth. Although the tumor growth of both core–shell type andaclitaxel (LMWSC-NPT) treatment at 2 mg/kg paclitaxel doseas not significantly different, the core–shell type LMWSC-NPT

t 10 mg/kg paclitaxel dose showed a higher survival rate upo 50 days as shown in Fig. 6(a). Furthermore, body weighthanges were also smaller than paclitaxel treatment at 10 mg/kgaclitaxel dose. These results indicated that the core–shell typeMWSC-NPT is suitable paclitaxel carriers for inhibition of tumorrowth in vivo with minimized side-effects. Antitumor effects ofanoparticulate-paclitaxel were also reported by other researchers32,45]. Polymeric micelle based on PEG-polylactide copolymerlso showed effectiveness in inhibiting tumor growth but their tar-eting mechanism was not fully understood [45]. Hydrophobicallyodified glycol chitosan was also available to treat solid tumor

32].Our results demonstrated that the core–shell type LMWSC-NPT

s a useful vehicle to increase the solubility of paclitaxel and deliveryo the tumor since the core–shell type LMWSC-NPT showed excel-ent antitumor activity against CT26-bearing mice. Furthermore,he core–shell type of LMWSC-NPT has an effective targeting prop-rty to tumor, thanks to its enhanced antitumor activity whereasaclitaxel itself has no selectivity.

. Conclusion

LMWSC was modified with MPEG (LMWSC-MPEG) andhen cholesterol was conjugated to them (LMWSC-MPEG-Chol).ore–shell type LMWSC-NPs were prepared by the dialysis method,nd the core–shell structure was confirmed from the results ofH NMR. To this polymer, paclitaxel was encapsulated and theore–shell types LMWSC-NPs were prepared. The release test indi-ated that paclitaxel in the LMWSC-NPT was sustained release from

anoparticles but paclitaxel itself was more rapidly released out. Aytotoxicity study using colon carcinoma cells, CT26, paclitaxel-ncapsulated core–shell type LMWSC-NPT showed an almostimilar toxicity against tumor cells as paclitaxel itself. The resultsf the tumor inhibition test with CT26 mouse tumor models in vivo

[

[[

: Biointerfaces 81 (2010) 530–536

indicated that the LMWSC-NPT at a dose of 10 mg/kg has a superiorsurvival rate, and decreasing the tumor growth to the paclitaxel;however, the tumor growth and survival rate were not significantlydifferent from each other at a dose of 2 mg/kg of LMWSC-NPT.LMWSC-NPT above the dose of 10 mg/kg seems to have a superiorantitumor activity.

Acknowledgment

This work was partially supported by KITTOLIFE Co. andResearch Foundation of Engineering College, Sunchon National Uni-versity.

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