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Colloids and Surfaces B: Biointerfaces 64 (2008) 194–199

Preparation and characterization of Pluronic/TPGS mixedmicelles for solubilization of camptothecin

Yan Gao, Ling Bing Li ∗, Guangxi ZhaiDepartment of Pharmaceutics, School of Pharmacy, Shandong University,

Jinan 250012, China

Received 26 November 2007; received in revised form 5 January 2008; accepted 22 January 2008Available online 5 February 2008

bstract

To increase the solubility and cytotoxicity of poorly soluble anticancer drug camptothecin (CPT), mixed micelles made of Pluronic P105 (P105)nd d-�-tocopheryl polyethylene glycol 1000 succinate (TPGS) were prepared. The interaction of Pluronic and TPGS was studied and critical

icelle concentration (CMC) was used to evaluate the micellar stability towards dilution. Poorly soluble anticancer drug CPT was incorporated

nto the mixed micelles. The solubility of CPT by the mixed micelles was more than that of the free drug. The cytotoxicity of the CPT-loadedixed micelles against MCF-7 cancer cell in vitro was remarkably higher than that of the free drug.2008 Elsevier B.V. All rights reserved.

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eywords: Mixed micelles; Pluronic and TPGS; Solubilization and cytotoxicit

. Introduction

Polymeric micelles formed by amphiphilic copolymers areromising drug carriers. Polymeric micelles have a core–shelltructure and poorly soluble drugs can be solubilized withinhe hydrophobic inner core of a micelle. As a result, micellesan substantially improve solubility and bioavailability of var-ous hydrophobic drugs. The small size (<100 nm) of micellesllows for their efficient accumulation in pathological tissuesith permeabilized vasculature, via the enhanced permeability

nd retention (EPR) effect. Therefore, micelles are capable ofelivering drugs into poorly permeable tumors [1]. Polymericicelles have been evaluated in many pharmaceutical appli-

ations such as drug and gene delivery systems, as well as iniagnostic imaging as carriers for various contrasting agents2,3].

Pluronic block copolymer consists of ethylene oxide (EO)nd propylene oxide (PO) blocks that are arranged in a basic

Ox–POy–EOx structure. A prominent feature of Pluronicopolymer is the ability of individual block copolymerolecules to self-assemble into polymeric micelles in aqueous

∗ Corresponding author. Tel.: +86 531 88382015; fax: +86 531 88382548.E-mail address: cnlilingbing@yahoo.com (L.B. Li).

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927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2008.01.021

ptothecin

olutions, which has a hydrophobic core formed by PO chainsnd a hydrophilic corona formed by EO chains. The PO corean serve as a ‘pool’ and the hydrophobic drug can be incorpo-ated into the hydrophobic PO core, while the hydrophilic coronaaintains the dispersion stability of Pluronic micelles. Incor-

oration into micelles leads to increased solubility, metabolictability and circulation time of the drug [4].

Camptothecin (CPT), a plant alkaloid from Camptothecacuminate demonstrated strong antitumor activity against lung,varian, breast, pancreas, and stomach cancers by targeting intra-ellular topoisomerase I, a nuclear enzyme that reduces theorsional stress of supercoiled DNA [5]. However, CPT is insol-ble in water and exists in two forms depending on the pHalue: the active lactone forms at pH below 5 and the inactivepen ring-carboxylated CPT-Na+ forms, which is present at neu-ral pH values [6]. Although the lactone form of CPT is crucialor its anticancer activity, at physiological pH values most CPTolecules exist in the inactive carboxylated form. Thus, sparing

olubility and labile lactone ring hinder the clinical applicationf CPT.

Mixed micelles manifest synergistic properties, such as

ncreased micelle stability and drug loading efficiency, supe-ior to those of the individual components [7]. d-�-Tocopherylolyethylene glycol 1000 succinate (TPGS), a derivative ofhe natural Vitamin E (�-tocopherol) and polyethylene glycol

Y. Gao et al. / Colloids and Surfaces B:

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000, is used as solubilizer, absorption enhancer and a vehi-le for lipid-based drug delivery formulations [8], because itsipophilic portion is relatively bulky, which might allow it foretter drug solubilization (Fig. 1). It was also reported that suc-inate esters of Vitamin E behave as potent proapoptotic agentelective for cancer cells [9]. Therefore, we can hypothesize thatixed micelles made of Pluronic and TPGS may allow for their

etter drug encapsulation, more stability and higher anticancerfficiency. In present work CPT-loaded mixed Pluronic/TPGSicelles were prepared. Some of their properties and increased

n vitro cytotoxicity against MCF-7 cancer cell also weretudied.

. Materials and methods

.1. Materials

Pluronic P105 (P105) was purchased from Sigma–AldrichSt. Louis, MO, USA) and used without additional purification.-�-Tocopheryl polyethylene glycol 1000 succinate (TPGS)as purchased from Eastman Co. (USA). CPT was purchased

rom Sigma–Aldrich. All other reagents and buffer solutionomponents were analytical grade preparation. Distilled andeionized water were used in all experiments.

.2. Micelle preparation

To prepare empty mixed micelles, P105 or its mixture withPGS was dissolved in chloroform and the organic solventas subsequently removed by rotary vacuum evaporation. Thelm formed was additionally freeze-dried in vacuum, hydratedith a suitable amount of 5 mM HEPES-buffered saline (HBS).he resulting mixture was filtered through a 0.2 �m Nylon fil-

er.To obtain drug-loaded micelles, CPT dissolved in chloroform

nd adjusted with acetic acid was added to P105 solution in chlo-oform or P105 and TPGS solution in chloroform. The micellereparation steps described above were repeated.

.3. Critical micelle concentration (CMC) determination

CMC values were determined by the laser dynamic lightcattering method (DLLS) using a DynaPro instrument (Wyatt

echnology Corp. USA). Samples were diluted and the hydrody-amic radius of a micelle was determined. The changes in lightntensity were compared and a sharp increase in the scatteringntensity indicated the formation of micelles [10].

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Biointerfaces 64 (2008) 194–199 195

.4. Drug solubilization efficiency

To prepare drug-loaded micelles, different amount of CPT0.5–1.5 mg) in chloroform was added to 10 mM of P105 solu-ion in chloroform or P105 and TPGS solution in chloroform.he micelle preparation steps described above were repeated.on-incorporated CPT was separated by filtration of the micelle

uspension through a 0.2 �m Nylon filter.The amount of CPT in the micellar phase was deter-

ined by the reversed phase-HPLC. The LC-10ATVP-ODSPLC system equipped with a diode array and fluorescenceetector (Shimadzu, Japan) and Spherisorb ODS2 column,.6 mm × 250 mm (Analytical Cartridge waters, Ireland) wassed. After filtration of CPT-containing micelle preparationhrough a 0.2 �m nylon filter, the micelles were diluted by 200-old with acetonitrile (to destroy micelles and release CPT), andn aliquot of the diluted solution was injected into HPLC system.he determination of the micellar CPT was performed using thealibration curve of pure CPT. The mobile phase composed of6% (v/v) acetonitrile (HPLC grade) in water (pH 5.0 adjustedith acetic acid) was used with a flow rate of 1.1 ml/min. CPTas detected by a fluorescence spectrometer set at λex = 360 nm

nd λem = 430 nm.

.5. CPT release from micelle formulation

The in vitro CPT release from P105/TPGS micelles wasnvestigated with a hydrotropic agent, sodium salicylate, to cre-te pseudo-sink conditions according to the reported procedureith modifications [9]. Briefly, 1 ml of CPT-loaded micelles was

ntroduced into a dialysis membrane bag, MWCO = 5000 Da,nd the end-sealed dialysis bag was incubated in 20 ml 1 Modium salicylate at 37 ◦C. The dialysis solution was shakent a speed of 100 rpm. At predetermined time intervals, 2 mlliquots of the sodium salicylate solution were withdrawn andeplaced with an equal volume of the fresh 1 M Na salicylate.he concentration of CPT in a sample was measured by HPLCith the correction for the volume replacement. The mobilehase composed of 66% (v/v) acetonitrile (HPLC grade) in waterpH 5.0 adjusted with acetic acid) was used with a flow rate of.1 ml/min. CPT was detected by a fluorescence spectrometeret at λex = 360 nm and λem = 430 nm.

.6. Micelle stability

To test the storage stability, drug-loaded micelles were storedt room temperature for 3 months. The stability of the micellesas evaluated by monitoring the time-dependent changes in thehysical characteristics, like drug precipitation and change inicelle size.The effect of dilution on the stability of micelles was studied.

amples were diluted and the micelle hydrodynamic radii wereetermined. The changes in weight fraction were analyzed to

easure dissociation of micelles.To elucidate protective effects of mixed micelles on the lac-

one ring of CPT against its hydrolysis, CPT-loaded mixedicelles and Pluronic P105 micelles at the concentration of

196 Y. Gao et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 194–199

Table 1CMC values of P105/TPGS micelles (n = 3)

Concentration (M) Formulation [P105:TPGS (molar ratio)]

10:0 7:3 5:5 3:7 0:10

CMC (M) 2.5 × 10−4 2.0 × 10−4 1.25 × 10−4 8.0 × 10−5 2 × 10−4

CMC*(Cal. M) 4.6 × 10−4a 2.8 × 10−4 2.3 × 10−4 1.9 × 10−4 1.5 × 10−4a

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mM were incubated with 0.2% bovine serum albumin (BSA)t 37 ◦C for 72 h respectively. After filtration of CPT-containingicelles through a 0.2 �m nylon filter, the micelles were diluted

y 200-fold with acetonitrile and an aliquot of the diluted solu-ion was injected into HPLC system. The determination of the

icellar CPT was performed using the same method describedbove.

.7. Cell culture

Human breast adenocarcinoma MCF-7 cells were grown inMEM medium with 2 mM l-glutamine supplemented with0% (v/v) heat-inactivated fetal bovine serum, 100 units/mlenicillin G, 0.25 �g/ml amphotericin B, and 100 �g/ml strep-

caoc

ig. 2. The hydrodynamic radius of different formulations. Pure P105 micelles at 0..5 mM (molar ratio 5:5) (C) and mixed micelles at 0.5 mM (molar ratio 3:7) (D).

omicin. Cultures were maintained at 37 ◦C in a humidified 5%O2 sterile incubator.

.8. In vitro cytotoxicity

For the cytotoxicity analysis, MCF-7 cells were seeded athe density of 2.5 × 103 of cells per well in 96-well plates.fter 24 h, the medium was exchanged by a medium containing

ncreasing concentrations of each of the following substances:ree CPT drug, empty micelles and drug-loaded micelles. The

ells were reseeded with medium containing the CPT, micellesnd CPT-loaded micelles for 3 days. After 72 h exposure to vari-us substances, the cell viability was determined using the MTSolorimetric assay for the dehydrogenase activity of viable cells.

5 mM (A), mixed micelles at 0.5 mM (molar ratio 7:3) (B), mixed micelles at

Y. Gao et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 194–199 197

Table 2Drug loading of CPT in micelles at 10 mM copolymers (n = 3)

C 0 mM, 7:3) P105/TPGS (10 mM, 5:5) P105/TPGS (10 mM, 3:7)

D 0.0260 ± 0.002 0.0363 ± 0.0011

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ibttiwchment and could proceed via both the hydration of the interfacialdrug molecules and their passive diffusion. The drug incorpo-rated into the inner core compartment stayed firmly inside themicelles showing a very slow release even at sink conditions with

omposition (molar ratio) P105 (10 mM) P105/TPGS (1

rug loading (%) 0.0254 ± 0.0008 0.0278 ± 0.001

. Results and discussion

.1. Micelle size and CMC values

The hydrodynamic radii of different micelles were measuredy the laser dynamic light scattering (DLLS) method (Fig. 2).nly one peak showed up on DLLS, indicating that micellesere formed with a relative low polydispersity.CMC values were determined by the laser dynamic light scat-

ering method. The changes in light intensity were comparednd a sharp increase in the scattering intensity indicated theormation of micelles. This method perhaps was not accurateecause of sensitivity issues, but it could reflect the changes inhe system. The CMC values of different micelles are listed inable 1. The results showed that mixed micelles made of P105nd TPGS (molar ratio 3:7) had lowest CMC value, indicatinghat they were most stable towards dilution among micelles. Thiss because the aromatic ring of TPGS might lead to an increasen hydrophobic interactions between the polymer chains in the

icelle’s core and thus provide some advantages in particletability towards dilution compared with Pluronic polymers.

.2. Characteristics of mixed micelles

The CMC values for the mixed surfactant system can bealculated using the following equation [11]:

1

CMC∗ = X1

CMC1+ X2

CMC2, (1)

here CMC* is the theoretical CMC of mixed micelles, X1 and2 are the molar fractions of the component 1 and 2, and CMC1nd CMC2 are the CMC values of component 1 and 2. Thealculated CMC* values are listed in Table 1. Table 1 showshat CMC* values decreased slightly with the increase of theatios of TPGS. This trend was different from changes of CMCalues observed for Pluronic and TPGS mixed micelles. Thiss due to the aromatic ring of TPGS, which might lead to anncrease in hydrophobic interactions between the polymer chainsn the micelle’s core and thus provide some advantages in particletability towards dilution compared with Pluronic polymers.

.3. Drug solubilization efficiency

The amount of CPT in the micellar phase was determinedy the reversed phase-HPLC. The results showed that addingPGS could increase the drug loading. At 10 mM of copolymer,

he drug loading of mixed micelles made of P105/TPGS (3:7olar ratio) was about 0.0323 ± 0.0011% (w/w) compared to

.0254 ± 0.0008% (w/w) as were found for the micelle made ofure P105 (Table 2). This could be explained by more “loose”

Fm

Fig. 3. Schematic representation of mixed micelles.

nd “density” core structure because of the aromatic ring ofPGS (Fig. 3), which may cause stronger hydrophobic interac-

ion between drug and polymers. At 10 mM of copolymer, theicelle suspension was produced containing about 17 �g of drug

er 1ml of micelle suspension, which significantly increased theolubility of CPT compared to the free drug [12].

.4. CPT release from micelle formulation

The CPT release from P105/TPGS micelles (molar ratio 3:7)s presented in Fig. 4, which starts with an initial burst, followedy a very slow release phase. The CPT release behavior reflectedhe CPT incorporation stability and could be explained throughhe geometry of CPT location within the micelles (Fig. 4). Thenitial burst happened within the first few hours of incubationas attributed to the relatively small quantity of the drug asso-

iated on the interface of the micelle hydrophobic core andydrophilic corona, or even within the micelle corona compart-

ig. 4. Release profile of CPT from P105/TPGS micelles (�); CPT-loadedicelles made of P105 alone (�) were used for comparison.

198 Y. Gao et al. / Colloids and Surfaces B: Biointerfaces 64 (2008) 194–199

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Fig. 5. HPLC profile of CPT-loaded micelles. Pure P105 micelles at 4 mM

0–30% of the initially incorporated drug still being associatedith the micelles even after 72 h incubation at 37 ◦C.It looks like mixed Pluronic P105/TPGS micelles retain the

rug even better than “pure” Pluronic P105 micelles indicatinghat the presence of TPGS facilitates not only CPT-incorporationnto micelles but also the stability of its entrapment, probablyecause of the aromatic ring of TPGS, which may cause strongerydrophobic interaction between drug and polymers.

.5. Micelle stability

The drug-loaded Pluronic micelles and mixed micellesmolar ratio 3:7) were used in the stability study. The resultshowed that the micelles were stable during storage at roomemperature (20 ◦C) for 3 months. No precipitation of drug ando change in the micelle size and size distribution were founduring this period.

The effect of dilution on the stability of micelles was studiedFig. 2). The results showed that the weight fractions of differ-nt micelles were different, indicating that part of the micelles

egan to dissociate when they were diluted to 0.5 mM. Amonghe different formulations, the P105/TPGS mixed micelles (3:7

olar ratio) were most stable towards dilution, because leastarts of micelles dissociated when it was diluted to 0.5 mM.

cops

ig. 6. In vitro cytotoxicity of micelles against MCF-7 cell lines. (A) CPT-loaded m105/TPGS micelles). (B) CPT-free micelles. (�, empty P105 micelles; �, empty P1

; mixed micelles made of P105 and TPGS (molar ratio 3:7) at 4 mM (B).

To elucidate protective effects of mixed micelles on the lac-one ring of CPT against its hydrolysis, the reversed phase-HPLCas used to simultaneously separate the carboxylate and lactone

pecies of camptothecin according to literature with a modestodification [12]. The peaks on HPLC profile (Fig. 5) con-rmed that most of camptothecin existed in the active lactoneorm confirming stabilizing the lactone ring by the micelles.ig. 5 also showed that the intensity of Pluronic micelles wasmaller than that of mixed micelles at the same concentration ofmM of micelle, which indicated that drug loading of Pluronicicelles was smaller than that of mixed micelles at 4 mM oficelle.

.6. In vitro activity of CPT-loaded micelles

The cytotoxicity of CPT encapsulated into mixed micellesas investigated and compared with that of free camptothecinsing MCF-7 cells. As a negative control, empty mixed micellesere used. Cells were treated with the same concentrations of

ree or micellar CPT. Empty micelles were added in the same

oncentrations as the tested CPT-micelles (same concentrationf micelle-forming material) (Fig. 6B). After 3 days in theresence of micellar preparations, cells were analyzed for theirurvival using the MTS colorimetric assay for the dehydroge-

icelles. (�, free CPT solution; �, CPT-loaded P105 micelles; �, CPT-loaded05/TPGS micelles).

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ase activity of viable cell. For MCF-7 cell line, CPT-loadedixed micelles demonstrated a significantly superior cytotoxi-

ity compared to that of the free drug (Fig. 6A), which coulde explained by the enhanced solubility of the poorly solublePT in micelle solution, increased stability of the cytotoxic lac-

one form of CPT inside the micelle core, and better uptakef CPT-loaded micelles by the cells. The P105/TPGS (molaratio 3:7) micelles had a higher cytotoxicity than that of the105 micelles, which was, most probably, due to the interactionetween TPGS and MCF-7 cancer cell resulting in chemosen-itization of the cells with respect to anticancer agents andther effects [9]. Empty P105 micelles were shown not to haveignificant cytotoxity at used concentration, whereas empty105/TPGS micelles showed some inhibition of cell growthompared to that observed for the free CPT (Fig. 6B). This wasrobably caused by the cytotoxic property of TPGS unimers forpecial cells [13].

. Conclusion

Mixed micelles made of Pluronic P105 and TPGS could pro-ide a more stable system and more efficient solubilization of

he poorly soluble anticancer drug CPT than free drug whenhe molar ratio of Pluronic and PEG-PE was more than 3:7.his was because TPGS has an aromatic ring, which may causetronger hydrophobic interaction between drug and polymers.

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Biointerfaces 64 (2008) 194–199 199

dditionally, CPT encapsulation by mixed micelles also hadigher cytotoxicity.

cknowledgement

This work was supported by natural science funds fromdministration of science and technology of Shandong Province,hina (Y2005C65).

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