Targeted cancer therapy with novel high drug-loading nanocrystals

Targeted Cancer Therapy With Novel HighDrug-Loading Nanocrystals

FENG LIU, JI-YOUNG PARK, YONG ZHANG, CHRISTINE CONWELL, YANG LIU,SURENDAR REDDY BATHULA, LEAF HUANG

Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill,Chapel Hill, North Carolina 27599-7360

Received 19 November 2009; revised 16 January 2010; accepted 20 January 2010

Published online 5 April 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22112

Corresponde919-966-0197; Ephone: 919-843-

Journal of Pharm

� 2010 Wiley-Liss

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ABSTRACT: A novel nanocrystal formulation of hydrophobic drugs has been developed forcancer therapy. The new method, called a three-phase nanoparticle engineering technology(3PNET), includes three phases: phase 1, amorphous precipitate; phase 2, hydrated amorphousaggregate; and phase 3, stabilized nanocrystal. The 3PNET has been applied to two anticancerdrugs, paclitaxel (PTX) and camptothecin (CPT), using Pluronic F127 (F127) polymer as a singleexcipient. The nanocrystals encapsulated over 99% of the drug with a high ratio of drug toexcipient. The nanocrystal formulation of PTX did not induce hemolysis at pharmacologicallyrelevant concentrations. Antitumor activity in two tumor models, human lung cancer andmurine breast cancer, demonstrated that intravenously injected nanocrystals significantlyinhibited the tumor growth. The nanocrystals also showed significant therapeutic effects viaoral administration. In addition, the nanocrystals could be further modified for targeted deliveryof PTX by conjugating a folate ligand to F127. The new nanomedicine formulations show clearpotential for clinical development because of the excellent antitumor activity, low toxicity, andthe ease of scale-up manufacture. The formulation method may apply to other hydrophobicdrugs. � 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3542–3551, 2010

Keywords: target; nanocrystal; high drug
loading; cancer therapy
INTRODUCTION

Recent advances in nanotechnology have made itpossible to design drug carriers with nanometer-scalefeatures (e.g., nanoparticles) that have the potentialto deliver drugs to specific tissues such as tumor.Nanoparticles represent a broad range of materials,including liposomes, micelles, dendrimers, nano-crystals (NCs), metal colloids, fullerenes, etc.,1 manyof which have been successfully used as platforms orcarriers for insoluble or poorly soluble drugs toimprove their pharmacokinetics (PK) and dispositionprofile. Currently, a few nanoparticle products arealready on the market and many more novelnanoparticles are being designed and characterizedby different laboratories.

Several variables, however, determine whether theproduct obtained in the laboratory can be translatedinto opportunities for the consumer.2 Even whenwell-established methodologies are employed, nano-

nce to: Feng Liu (Telephone: 919-843-2277; Fax:-mail: [email protected]) or Leaf Huang (Tele-0736; Fax: 919-966-0197; E-mail: [email protected])

aceutical Sciences, Vol. 99, 3542–3551 (2010)

, Inc. and the American Pharmacists Association

AL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 8, AUGUST

particle therapeutics pose some challenges.3 Onechallenge is the high production cost. Recently, ananoparticle albumin-bound paclitaxel (PTX) formu-lation (AbraxaneTM) has been approved by the FDAfor cancer therapy. This formulation is prepared byhigh-pressure homogenization of PTX in the presenceof serum albumin into a nanoparticle colloidalsuspension, which is the first Cremophor-EL–freePTX formulation on the market. AbraxaneTM hasseveral practical advantages over the traditionalPTX/Cremophor-EL formulation, many of whichreduce the toxicities commonly associated withPTX-based therapy and the increase of PTX loadingefficiency, with 10% (w/w) PTX and 90% (w/w)albumin.4 However, AbraxaneTM is very expensive,costing several thousand dollars per dose such thatAbraxane is generally not replacing Taxol in oncolo-gical practice.5 The drug industry is currently facingincreasing pressure to keep healthcare costs afford-able and still meet the demand of patients andhealthcare providers for added value.3 High produc-tion cost must be avoided in designing and developingnew nanoparticle formulations. Another challengeis to improve the efficiency of drug loading, sincenanodrug carriers are often characterized with a lowdrug to carrier ratio. Therefore, many efforts have

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NANOCRYSTALS FOR DELIVERY OF HYDROPHOBIC DRUGS 3543

been made to increase the drug loading. For example,Wang et al.6 have reported a micelle-based nanopar-ticle using the Pluronic block copolymer (P105) aloneas an excipient for the delivery of PTX, with a ratio of1/20 (PTX/P105, w/w). Torchilin’s group has success-fully developed PEG-PE/TPGS-based nanoparticlesand PEG-PE/phosphatidylcholine mixed immunomi-celle nanoparticles, in which the ratios of PTX to theexcipient(s) (w/w) were 1:20 and 1:15, respectively.7,8

An ideal nanoparticle should possess a high ratioof the carried drug to excipients, particularly fornanoparticles designed to target cancer cells. It wouldbe advantageous if a large amount of drug is deliveredfor each targeting event.

This study addresses the challenges of improvingthe drug-loading efficiency and decreasing the highproduction costs of nanoparticles. A stabilized NCformulation was developed for the delivery of PTXusing a triblock copolymer, Pluronic F127, as theexcipient. This NC provided several advantagessuch as very high efficiency of drug loading, easyscale-up for manufacture, and relatively low cost forpreparation. Most importantly, it showed muchless toxicity than that of the commercially availablePTX/Cremophor-EL. Tested in two animal cancermodels, namely, human lung and mouse breastcancers, the NC resulted in a significant tumorregression. Interestingly, the use of the NC alsoshowed a very encouraging anticancer effect when itwas administered orally.

MATERIALS AND METHODS

Animals and Materials

Female BALB/c and nude BALB/c nude mice (5 weeksold) were purchased from National CancerInstitute, U.S. National Institute of Health (NCI).Human lung cancer cell line, NCI-H460, was obtainedfrom American Type Culture Collection. Murinebreast cancer cell line, 4T1, was kindly provided byDr. Mark Dewhirst (Department of Radiation Oncol-ogy, Duke University Medical Center). All workperformed on animals were in accordance with andpermitted by the University of North CarolinaInstitutional Animal Care and Use Committee.PTX was bought from LC Laboratories (Woburn,MA) and Pluronic F-127 (F127), Pluronic F-68 (F 68),Cremophor-EL, and camptothecin (CPT) were pur-chased from Sigma-Aldrich Corp. (St. Louis, MO).

Preparation of the Nanocrystal

The NCs were prepared by a three-phase nano-particle engineering technology (3PNET), whichincludes phase 1, amorphous precipitate; phase 2,hydrated amorphous aggregate; and phase 3, stabi-lized NCs. Briefly, 0.5 mg PTX and F127 were first

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dissolved in chloroform (in a glass tube) with weightratios of 1/3, 1/4, and 1/5 (PTX/F127,) and thencoprecipitated by evaporating the chloroform with asteady stream of nitrogen gas, and the traces ofchloroform were removed under a vacuum withdesiccators for 2–4 h. Following 1 h hydration (in1 mL of 5% dextrose) and vortexing, the suspensions(4 mg/mL PTX was prepared for the dose of 60 mg/kgin vivo) were sonicated for 10–15 min by a bath-typesonicator (output 80 kc, 80 W) to form NCs. The CPTand F127 were dissolved in chloroform/methanol (4:1)and then formulated to the NCs as described above.When the ratio of PTX to F127 was lower than 1/4 (w/w), such as 1/3, the NCs could not be formed. Thus,our maximum drug-loading ratio was found to be ashigh as 20%.

Characterization of the Nanocrystals

Transmission electron microscopy (TEM) images ofthe resulting NCs were acquired using a PhillipsCM12 (FEI, Hillsboro, OR). Freshly prepared nano-particle samples, 5mL (0.1 mg PTX/mL, PTX/F127/1/5, w/w), were dropped onto a 300-mesh carbon-coatedcopper grid (Ted Pella, Inc., Redding, CA) and alloweda short incubation (5 min) at room temperature. Gridswere then stained with 1% uranyl acetate (40mL)and wicked dry. All images were acquired at anaccelerating voltage of 100 kV. Gatan Digital Micro-graph software was used to analyze the images. Toassess the encapsulation efficiency of PTX, the NCsuspension was kept at room temperature for 1 h,then loaded into 0.45 and 0.22mm, CentrifugalFilter Devices and a 3000 MWCO microcon (MilliporeCo., Bedford, MA), respectively, and centrifuged at16,000g for 20 min. NC PTX and free PTX in thefiltrate were quantified by HPLC. The distribution ofparticle size in the NC formulation was measuredusing a submicron particle sizer (NICOMP particlesizing systems, AutodilutePAT Model 370, SantaBarbara, CA) in the NICOMP mode. The zetapotential of the NCs diluted in 1 mM KCl wasdetermined by the Zeta Plus zeta potential analyzer(Brookhaven Instruments Corporation, Holtsville, NY).

Evaluation of Hemolytic Activity

Blood was collected from the nude BALB/c mice andcentrifuged at 48C. The centrifuged blood was washedtwice with PBS (pH 7.4) to isolate erythrocytes.Erythrocytes (2� 1010) were resuspended in 100mL ofPBS containing either the NCs or Cremophor-ELwith different concentrations of PTX. The aboveprocedures were performed on ice. After the sampleswere incubated at 378C for 1 h and then centrifugedfor 10 min at 16,000g, 70mL supernatants werediluted with 100mL PBS and measured for the opticaldensities (ODs) at 570 nm. The percentage of hemo-lysis was calculated as a function of OD measured

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according to the equation: hemolysis %¼OD(sample)/OD (total hemolysis)� 100%. The OD (totalhemolysis) was measured from samples that weresonicated for 5 min before the measurement.

Animal Model and Treatments

The NCI-H460 cells (5� 106) were subcutaneouslyinjected into the right flanks of the nude mice. The4T1 cells (105) were subcutaneously injected into thehair-trimmed belly of BALB/c mice. The mice, five ineach group, were intravenously administered withPTX in different formulations. Tumor volumes of thexenograft mice were monitored every other day andwere calculated as (a2� b)/2, where a and b were thelong and short dimensions of the tumor, respectively.9

Mice were sacrificed when the long dimension of thetumor reached 2 cm. In the oral administrationstudies, the lyophilized and reconstituted NCs wereadministered to mice-bearing 4T1 tumor by oralgavage. A PTX suspension that was freshly preparedby sonication of PTX in 10% sucrose was used as acontrol.

Conjugation of Folic Acid to Pluronic F127 andPreparation of the Folate-Receptor (FR)Targeted Nanocrystals

Folate-conjugated F127 was synthesized in two steps(Fig. 5). Step 1 was the addition of an amino functionto the ends of the Pluronic: a 100-mL three neck roundbottom flask was fitted with a nitrogen balloon and amixture of anhydrous chloroform (50 mL), Pluronic(1.26 g), NaH (0.24 g), and 2-bromoethylamine hydro-bromide (0.20 g) were added and allowed to reactfor 48 h at room temperature. After completion ofthe reaction, excess NaH in the reaction mixturewas quenched by methanol (2.03 mL). The modifiedPluronic was purified by dialysis against deionizedwater, followed by lyophilization. Step 2 was theconjugation of folic acid to the modified Pluronic:the mixture of amino Pluronic (0.28 g), folic acid(0.19 g), 1-ethyl-3-(3-dimethylaminopropyl) carbodii-mide hydrochloride (EDC, 0.42 g), 4-dimethylamino-pyridine (DMAP, 0.03 g), triethylamine (0.06 mL),and N-hydroxysuccinimide (NHS, 0.25 g) wasstirred for 12 h at room temperature in a 50-mLdry chloroform solution. The reaction mixture waschromatographed in a size exclusion column andthe void volume eluent containing F127-folate wascollected, and then dialyzed (dialysis membrane>10,000 Da) against deionized water and lyophilized.For the preparation of the FR targeted NCs, PTX wasdissolved in chloroform with varied amount of F127,F127-folate. The preparation of the targeted NCs wasthe same as that described above.


Evaluation of the Targeted Nanocrystals in aFR-Positive Tumor Cell Line

The targeted NCs was evaluated in a FR-positivehuman oral carcinoma cell line (KB),10 for theircytotoxicity determined by MTT assay, as describedpreviously11 with minor modifications. The KB cellline was cultured as a monolayer in folate-free5% FBS DMEM media for 5 weeks. The cells weresuspended by brief treatment with 10 mM EDTA andtransferred to 96-well tissue culture plates at 1� 104

cells per well 24 h prior to drug addition. The culturemedium was then replaced with 100mL of normalmedium containing serial dilutions of PTX inFR-targeted, nontargeted control NCs and Cremo-phor-EL. Following 2 h incubation at 378C, the cellswere washed twice with PBS and cultured in 200mLfresh medium and the MTT assay was performedafter incubation for additional 48 h.

Statistical Analysis

All statistical analyses were performed by a two-tailed Student’s t-test. Data were considered statis-tically significant when the p-value was less than0.05.

RESULTS

Currently, PTX is one of the most effective anticancerdrugs in the clinic. The clinical dosage form ofPTX commonly used is PTX/Cremophor-EL, whichis associated with serious side-effects such ashypersensitivity, nephrotoxicity, and neurotoxicityin some patients.12 The objective of this study is todevelop a Cremophor-EL-free nanoparticle formula-tion with decreased toxicity. The design of thenanoparticles is based on the hypothesis that asurfactant molecule included in a NC of PTX canstabilize the nanoparticles as they are suspended inan aqueous solution. Our initial experiment was toselect the excipients to formulate the PTX NCs, whichinclude two poloxamers, Pluronic F68 (F68) andPluronic F127 (F127), that have different physicalproperties.13 TEM demonstrated that rod-shapedNCs formulated by the 3PNET method using F127as the surfactant (Fig. 1a). The NCs could belyophilized to powder, then reconstituted to regainthe rod-shaped NC morphology (Fig. 1b) with a sizedistribution of 146� 44 nm (measured by a submicronparticle sizer) so that the storage stability can begreatly improved with the lyophilized powder. On theother hand, using F68 as a surfactant resulted inunstable aggregates (data not shown). Comparedwith F68, F127 consists of a longer hydrophobic (poly-propylene oxide) and two hydrophilic (poly-ethyleneoxide) chains, both of which may play an importantrole in forming and stabilizing the NCs. The longer

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Figure 1. Transmission electron microscopy (TEM)images. (a) Nanocrystals of PTX; (b) the lyophilized andreconstituted nanoparticles of PTX; (c) nanocrystals ofcamptothecin (CPT).

Figure 2. Turbidity study of the nanoparticles (a) andtransmission electron microscopy images of the nano-particles examined in the biological fluid (b). (a) Theturbidity was examined at an absorbance of 600 nm forthe PTX nanocrystals (PTX/F127) and CPT nanocrystals(CPT/F127). (b) The EM pictures were taken for the PTXnanocrystals incubated with serum for 4 h at 378C.


hydrophobic domain may favor the interaction ofF127 with the surface of the PTX crystal. Thehydrophilic domains of F127 may prevent thecrystals from aggregation and growth into largercrystals. When the ratio of PTX to F127 was lowerthan 1/4 (w/w), such as 1/3, the NCs could not beformed; thus, our maximum drug-loading ratio wasfound to be as high as 20%.

To test whether the methodology described abovecan be used to formulate other hydrophobic drugs to aNC form, CPT was investigated. TEM, image shownin Figure 1c, demonstrated that CPT could beformulated to cubic NCs with a drug to excipientratio of 1/5 (CPT/F127, w/w). It is important to notethat both PTX and CPT by themselves withoutany surfactant also assumed the same crystalline

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morphology as in the presence of F127 under theexperimental condition. In other words, pure PTXwas rod-shaped crystals and pure CPT was cubiccrystals (data not shown); however, these purecrystals aggregated and precipitated from thesuspension shortly after the preparation. Thus, itappears that the inclusion of F127 into a drugformulation did not change the drug’s crystallinestructure; it only stabilizes the crystals in theirnanosized physical state.

Characterization of the Nanocrystals

Additional evidence that the F127 included into PTXand CPT could stabilize the NCs was shown in theresults of our turbidity study. When F127 wasincorporated into either PTX or CPT, as shown inFigure 2, both NCs were well suspended and nochange in turbidity was observed for these formula-tions. In contrast, PTX and CPT alone (without F127)showed high absorption value (OD 600 nm) attime zero because of aggregation. With time, theaggregates precipitated and the turbidity of thesuspension decreased. In addition, we examined thezeta potential, the distribution of NC sizes, andencapsulation efficiency of PTX (Tab. 1). The NCsencapsulated over 99% of PTX. The encapsulationefficiency was measured using HPLC. It is importantto note that stable NCs could only be prepared whenboth PTX and F127 were in physical contact at the


Table 1. Characterization of PTX Nanocrystals

Zeta Potential (mV) Size (nm)

Recovery% in Filtrate

0.22mma 0.44mma MW 3000b

PTX nanocrystals 0.82 122� 35 96.5 100 <0.3PTXþF127 N/A N/A <0.3 <2 <0.3

N/A, the formulation was a precipitate such that no reproducible measurements could be made.aThe samples passed through 0.22 or 0.44mm Ultrafree-MC (Millipore Co.).bThe samples passed through a 3000 MWCO Microcon (Millipore Co.).

Figure 3. Hemolysis activity of the PTX/F127 nano-crystals and PTX/Cremophor-EL. The hemolytic activitieswere detected in erythrocyte suspensions obtained frommouse blood (n¼ 3, mean�SD). ��p< 0.005.

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beginning of solidification as the organic solventevaporated. If PTX alone was precipitated thenhydrated with F127 solution and sonicated, largeparticles (>1mM) were formed and the particlesprecipitated. This observation was demonstrated bythe data in Table 1. These large crystals, even aftermixing with F127 and sonication, could not passthrough either a 0.44- or 0.22-mm filter. In contrast,96.5% and 100% PTX formulated with F127 into theNCs was recovered in the 0.22 and 0.44mm filtrate,respectively, under the same experimental condition.As anticipated, neither NC preparation could passthrough a 3000 MWCO Microcon filter; therefore,the NCs encapsulated over 97% of PTX. The NCs(Tab. 1) exhibited a zeta potential close to neutral(0.8 mV), with a size distribution of 122� 35 nm(measured by a submicron particle sizer). Theseparameters remained unchanged for at least 12 h instorage at room temperature, which provides enoughtime to complete the lyophilization process for long-term storage. Next, the stability of the NCs in serumwas examined. NCs containing 0.72 mg PTX wereincubated in 250mL solution of 45% mouse serum at378C for 4 h before it was diluted and examined byTEM. As shown in Figure 2b, the many rod-shapedNCs were observed after 4 h incubation in the serumsolution. We conclude that the NCs were largelystable in the presence of a biological fluid.

Evaluation of the Toxicity

Low toxicity and biocompatibility of Pluronic F127has made it an attractive candidate in designing apharmaceutical vehicle to deliver drugs via differentroutes of administration.14 The PTX NCs developedhere only consisted of a small amount of F127, whichis expected to have much lower toxicity than PTX/Cremophor-EL. The toxicity of the NCs was firstevaluated in mice for the maximum tolerated dose.When female nude mice (18–20 g) were intravenouslyinjected with PTX in Cremophor-EL, six out of sixinjected mice died as the dose reached 30 mg/kg;however, none of six mice injected with PTX NCs died,when the dose was increased to even as high as60 mg/kg.


Surfactants are known to cause hemolysis of thered blood cells. The hemolytic property of PTXadministered in the commercial Cremophor-ELvehicle has already been reported because it isformulated with a high amount surfactant, Cremo-phor-EL (polyoxyethylated castor oil).15 In order todetermine that the NCs are safe for intravenous (i.v.)administration, the hemolytic potential of the for-mulations was evaluated. Drug-induced hemolysisin vitro is a simple and reliable estimation ofthe membrane damage caused by the drug or itsformulation in vivo.16 The concentration rangestudied was between 0 and 0.8 mM. Figure 3 showsthe hemolytic activity of the NCs and PTX/Cremo-phor-EL in mouse erythrocyte suspension. The NCswere significantly less hemolytic than the PTX/Cremophor-EL at a PTX concentration of 0.2 mM(p< 0.005). If a mouse (20 g) is intravenously injectedwith 20 mg/kg PTX, the blood concentration ofPTX is approximately 0.32 mM (the blood volumeis estimated to be 7.3% of the body weight for mice17).As shown in Figure 3, Cremophor-EL with aPTX concentration around 320mM PTX inducedabout 40% hemolysis, but this was not true for theNCs. As the concentration of F127 increases to0.8 mM, no hemolysis was detected in the nano-particles, but 100% hemolysis in PTX/Cremophor-ELwas observed. Taken together, the results indicatedthat the PTX NCs were much safer than PTX/Cremophor-EL.

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Figure 4. In vivo antitumor activity with the nanocrys-tals against H460 human lung (a) and 4T1 murine breastcancers (b and c). PTX in the nanocrystals or PTX/Cremophor-EL was intravenously injected into the nude(a) or BALB/c (b) tumor-bearing mice 7 days after the tumorinoculation (drug administrations indicated by arrows). Thelyophilized and reconstituted nanocrystals were injected inthe groups, 20 and 60 mg/kg (b). BALB/c tumor-bearing (c)were administered orally with the lyophilized and recon-stituted nanocrystals or PTX suspension that was freshlyprepared by sonication of PTX in 10% sucrose. The micewere first administered with two doses of 80 mg/kg by oralgavage in every other day and then decreased to 60 mg/kgthereafter (indicated by arrows).


Inhibition of Tumor Growth

PTX is highly efficacious in the treatment of nonsmallcell lung cancer, breast cancer, ovarian carcinoma,and head and neck cancers.18 In this study, the tumorgrowth inhibition effect of the NCs was evaluated intwo tumor models, H460 human lung cancer in axenograph model and murine 4T1 breast cancer in asyngeneic model. For the H460 tumor model shown inFigure 4a, the untreated tumors grew rapidly,reaching 1676� 238 mm3 in volume 27 days afterthe tumor cell inoculation. In contrast, the tumors inthe group of mice intravenously injected with the NCs(20 mg/kg PTX) had reached only 144� 35 mm3 involume, which was significantly smaller than that ofthe untreated group (p< 0.001). The tumor growthinhibition activity was similarly observed in thegroup treated with 20 mg/kg PTX in Cremophor-EL(p> 0.5 compared to NC group). The low toxicityof the NCs allowed us to examine the tumor growthinhibition using a higher dose of this formulation,that is, 60 mg/kg. As shown in Figure 4a, thehigher dose treatment could further inhibit thetumor growth and the tumor size was as small as100.2� 46 mm3 51 days after the tumor inoculation,but the tumor reached 1689� 538 and 1599�413 mm3 in the groups treated with the lowerdose (20 mg/kg) of Cremophor-EL and the NCs,respectively.

Next, tumor growth inhibition was studied usinglyophilized and reconstituted PTX NCs in the murinebreast cancer model (Fig. 4b). The lyophilized andreconstituted F127 NCs demonstrated a similartherapeutic pattern in the 4T1 breast tumor modelas seen in the H460 lung cancer model, that is, theNCs were as efficient as Cremophor-EL formulationin tumor growth inhibition at a low dose (20 mg/kg)but had significantly increased efficacy at a higherdose of 60 mg/kg.

The stability of the NCs prompted us to examinethe tumor growth inhibition via an oral administra-tion route. Currently, PTX is only marketed as i.v.formulations. Oral administration of PTX is attrac-tive because it may enable the development ofchronic treatment schedules, resulting in plasmaconcentrations at a pharmacologically relevant levelfor a prolonged period of time.19 Oral administrationis more convenient and more compliant than i.v.administration. However, PTX is a high affinitysubstrate for the drug efflux transporter P-glycopro-tein (P-gp) in the epithelial cells of gastrointestinaltract.20 We assumed that the PTX may be stabilizedin the GI track by the NCs or the NCs may protectPTX from recognition by P-gp such that the drug canbe transported into the blood circulation. Thisassumption was tested in the 4T1 tumor-bearingmice (Fig. 4c) that were administered orally with

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either PTX suspension or the lyophilized andreconstituted NCs. Indeed, no antitumor activitywas observed in the mice treated with the PTXsuspension; there was no significant difference intumor growth between the untreated and treated


Figure 5. Schematic representation of synthesis of folic acid conjugated PluronicF127 (F127-folate).

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(PTX suspension) groups (p> 0.5). The oral adminis-tration of the lyophilized and reconstituted NCs,however, significantly inhibited the tumor growth(p< 0.01). The tumor volume in the mice treated withthe NCs was three times smaller than that of micetreated with PTX suspension. Oral administration,however, resulted in less efficacy compared to the i.v.injection. The oral dose (Fig. 4c) required a total of760 mg of PTX per mouse (in 10 administrations),whereas the i.v. injection group only required 80 mgPTX per mouse (in 4 administrations) (Fig. 4b), whichindicates that the bioavailability of oral NCs has to befurther improved. It needs to be noted that Taxol wasoriginally included as a control in the oral study. Wefound that the mice lost more than 20% of their bodyweight after they were administered multiple oraldoses of Taxol (2� 80 and 5� 60 mg/kg); however, notumor regression was observed in this group duringthe observed time course.

Folate-Receptor Targeted Nanocrystals (NCs)

Selectively targeting the tumor can increase theefficacy and decrease the toxicity associated withanticancer drugs. Many efforts, therefore, havefocused on developing targeted nanoparticles, whichare formulated by modifying nanoparticle surfaceswith targeting molecules, such as antibody, peptide,small molecule ligand, and oligonucleotide. Thefolate receptor, overexpressed in a broad spectrumof malignant tumors, is an attractive target forselective delivery of anticancer drugs to tumor cells.21


Folic acid, therefore, was conjugated to Pluronic F127(Fig. 5) and folate-receptor targeted NCs wereprepared using a protocol identical to that of thenontargeted formulation. Cytotoxicity of FR-targetedand nontargeted NCs was determined in FR-positiveKB cells using the MTT assay. The results inFigure 6a showed that the targeted NCs containing10% (F127-folate/F127, w/w) of F127-folate resultedin significantly lower viability (p< 0.001) comparedwith the nontargeted NCs as the concentration ofPTX varied from 0.08 to 10mM. As shown inFigure 6b, the cytotoxicity decreased when theF127-folate decreased from 10% to 5% (p< 0.05).No further enhancement of the cytotoxicity wasobserved at high concentration of F127-folate(>10%). The differential cytotoxicity was abolishedin the presence of 1 mM free folate as a competitor(Fig. 6b), indicating the FR-specific cytotoxicity of theFR-targeted NCs. The nontargeted NCs exhibited thesame cytotoxicity as PTX in Cremophor-EL (data notshown).

Mechanism of the Nanocrystals (NCs) Formation

To elucidate the mechanism by which the NCs weregenerated, kinetic morphology changes wereobserved for PTX/F127 and PTX alone. Chloroformcontaining PTX with or without F127 was placed ontoa glass slide. After drying and desiccating, a dropof water was added to each sample, the slides wereplaced into a humidified chamber at room tem-perature for various hydration times and pictures

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Figure 6. Viability of human FR-positive oral carcinomacell line in the presence of the targeted (PTX/F127-folate)and nontargeted (PTX/F127) nanocrystals after incubationfor 48 h. The targeted nanocrystals contained 10% F127-folate with different concentrations of PTX (a) and variedamount of F127-folate with 2mM PTX. (b) Untreated cellswere taken as control with 100% viability and data werepresented as the mean�SD (n¼ 8). ��p< 0.01.


were taken. The hypothesized mechanism, accordingto the imaging (Fig. 7), includes three phases: phase 1,amorphous precipitate; phase 2, hydrated amorphousaggregate; and phase 3, stabilized NC (F127 coatedNC). The amorphous solid in phase 1 was formedwhen PTX and F127 coprecipitated as the chloroform

Figure 7. Images of amorphous precipitate. The leftpanel (a, c, and e) was from PTX/F127 and the right panel(b, d and f) was from PTX alone.

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was evaporated. PTX without F127 also formed theamorphous solid. The amorphous structure stillexisted after 2 min hydration for both PTX/F127and PTX alone (Fig. 7a and b). When the amorphousprecipitate was hydrated for a period of time (i.e.,30 min) in phase 2, the PTX/F127 formulation formedhydrated amorphous aggregates (Fig. 7c). For thePTX sample without F127, however, the hydrationresulted in the formation of crystals (Fig. 7d). If thehydration was for a longer period of time (i.e., 4 h)partial amorphous aggregates (PTX/F127) started toform small crystals and the crystals formed from PTXwithout F127 grew even larger (Fig. 7e and f). Uponnanonization (sonication for 10 min) of the hydratedamorphous aggregate, the NCs (F127 coated NC) wasformed (Fig. 1). Sonication performed before theformation of the hydrated amorphous aggregate (nohydration or short time hydration) required anincrease in sonication time (>10 min) in order toform the NCs. Heterogeneous NCs were observed assonication was performed after the formation of thecrystalline (extensive hydration, Fig. 7e), which maybe due to the fact that the nanonization cannot breakdown the crystalline PTX into NCs. Therefore, theNCs must be generated within the phase 2.

DISCUSSION

We reported here that NCs of at least two hydro-phobic drugs (PTX and CPT) can be formulated by asimple method of 3PNET using F127 as the soleexcipient. In the development of the new formulation,we took advantage of the minimal energy require-ment to form the amorphous, noncrystalline forms,specifically, that the formation of the amorphous formrequires much less energy than crystal formation,which maximizes our advantage in the generation ofthe NC.22 PTX is relatively complex organic drughaving several solid structures such as crystal andamorphous.23 Although, PTX is water insoluble, theamorphous form of PTX has higher solubility and ahigher dissolution rate than the crystal.24 Amorphoussolids, however, are thermodynamically unstable andtend to convert to a crystalline state,25 which limitstheir extensive commercialization.24 Actually, thephysical instability of the amorphous form may becritical for transition of the particles, upon nanoniza-tion, from the hydrated amorphous aggregates (phase2, Fig. 7c) to the rod-shaped NCs (phase 3). In fact, inphase 1, the PTX and F127 coprecipitate, and thestabilized amorphous aggregates play an importantrole in the 3PNET. First, the coprecipitation processprovides a dynamic force that increases the incor-poration efficiency of F127 into the amorphous PTX/F127, thereby requiring minimal F127 such that ahigh ratio of drug to excipient can be achieved.


3550 LIU ET AL.

Second, the hydrated amorphous aggregate is stabi-lized by F127, which prevents the aggregate fromforming crystals before nanonization (Fig. 7). Thecrystals that have higher packing energy compared toamorphous solid are difficult to be reformulated toform the stabilized NCs.22

We have discovered a simple way to stabilize thecrystals when they are still in the nanosize range. Theformulation exhibited a high efficiency of drugloading, low toxicity, and antitumor activity. Thesurfactant F127 is one of the least toxic, commerciallyavailable, block copolymers.14 Consequently, it hasbeen used extensively in a variety of pharmaceuticalformulations including low molecular mass drugs andpolypeptides. A formulation containing doxorubicinand Pluronic mixture (L61 and F127), SP1049C, hasbeen evaluated in clinical trials, which consists of0.25% (w/v) L61 and 2% (w/v) F127 in normal salinefor i.v. administration of doxorubicin hydrochlor-ide.26,27 While doxorubicin HCL is a water solubledrug, the ratio of drug to excipient in this formulation,1/11, is still lower than those of the NCs reportedhere. Since the NCs were formulated with a singleexcipient of F127 with a high ratio of drug to F127, theNCs exhibit the capability to attenuate toxicities,enhance delivery of hydrophobic drugs to the desiredbiological site (such as the gastric epithelium), andimprove the therapeutic efficacy.

In summary, the results of this study strengthenthe validity of the novel NCs as a drug delivery vehiclefor cancer therapy. This formulation has greatpotential for marketability due to the followingadvantages. First, the NCs achieved the efficiencyof drug loading (PTX/F127, w/w) as high as 20%.Second, the methodology (3PNET) for the preparationof the NCs is simple and without any chemicalmodifications for the drug and the excipient. Theprocess can be easily scaled up for manufacturing.Furthermore, the 3PNET, we believe, is suitable toformulate other hydrophobic drugs as NCs for thetreatment of different diseases. Third, it is cost-effective to prepare the NCs because the commonlyused F127 is the only excipient in this formulationand the price of F127 is comparable to Cremophor-EL.Lastly, the NCs can be further modified for targeteddelivery of anticancer drugs to increase theirtherapeutic efficacy.

ACKNOWLEDGMENTS

This work was supported by institutional funds at theUniversity of North Carolina at Chapel Hill. Wewould also like to thank Wallace Ambrose atthe CHANL facility for his assistance in the TEMimaging.


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Targeted cancer therapy with novel high drug-loading nanocrystals