Drug Delivery, 14:497–505, 2007Copyright c© Informa Healthcare USA, Inc.ISSN: 1071-7544 print / 1521-0464 onlineDOI: 10.1080/10717540701606418

Liposomal or Nanoparticle α-TEA Reduced 66cl-4 MurineMammary Cancer Burden and Metastasis

Pei Wang, Li Jia, and Bob G. SandersSection of Molecular Genetics and Microbiology in the School of Biological Sciences,University of Texas, Austin, Texas, USA

Kimberly KlineDivision of Nutrition, University of Texas, Austin, Texas, USA

Efficacy of α-TEA formulated in liposome or biodegradablepoly(D, L-lactide-co-glycolide) (PLGA) nanoparticle was evaluatedin a BALB/c clone 66cl-4 mammary cancer mouse model using oraldelivery. α-TEA-loaded liposome or nanoparticle at 5 mg, but not2.5 mg/day, significantly reduced tumor burden (p<0.001). Bothformulations at 5 mg significantly reduced visible lung metastasesand lymph node and lung micrometastatic tumor foci. Surpris-ingly, nanoparticle control mice exhibited significantly reduced tu-mor burden, lymph node, and lung micrometastatic tumor foci.Both formulations at 5 mg significantly enhanced tumor apoptosis.In summary, liposomes and nanoparticles are effective means foradministering the lipophilic anticancer drug α-TEA.

Keywords Liposome, Metastasis, Nanoparticle, PLGA, SyngeneicBALB/C Mouse Mammary Tumor Model, Vitamin EAnalog (A-Tea)

Received 20 December 2006; accepted 20 March 2007.This work was supported by Public Health Service Grant CA59739

and the Foundation for Research (to K. Kline B. G. Sanders) and bythe National Institute of Environmental Health Sciences Center GrantES07784 (of which they both are members).

We thank John Mendenhall (Institute of Cell and Molecular Biology,U.T. Austin, TX), John N. Wright (Institute of Biomedical Engineering,U.T. Austin, TX), and Dr. Ti Cao (Institute of Chemistry and Biochem-istry, U.T. Austin, TX) for help and advice in the development andcharacterization of nanoparticles. We thank Dr. LuZhe Sun (Univer-sity of Texas Health Science Center at San Antonio, TX) for providingthe 66cl-4-GFP murine mammary tumor cell line. We thank Dr. MarkMcArthur (University of Texas M.D. Anderson Cancer Center, Depart-ment of Veterinary Sciences, Bastrop, TX) for evaluating H&E-stainedtumor tissue sections for cellular infiltration. We thank the Universityof Texas MD Anderson Histology Core Facility (Science Park, Bas-trop, TX) for preparation of H&E and immunohistochemically stainedtissues. And we thank Dr. Howard Thames, director of the Biostatis-tics and Data Processing Core (University of Texas M.D. Anderson,Houston, TX) for help with statistical analyses.

Address correspondence to K. Kline at Division of Nutrition/A2703,University of Texas at Austin, Austin, TX 78712-1097, USA. E-mail:k.kline@mail.utexas.edu

To develop a stable and clinically useful vitamin E-basedchemotherapeutic agent, a nonhydrolyzable ether linked aceticacid analogue of RRR-α-tocopherol, namely, α-TEA, has beenproduced (Lawson et al. 2003). α-TEA has been shown to in-duce human breast, prostate, colon, lung, ovarian, cervical, andendometrial cancer cells to undergo apoptosis in vitro, but notnormal human mammary epithelial cells and normal prostateepithelial cells (Anderson et al. 2004b). α-TEA formulated inliposomes and delivered by aerosol exhibits antitumor activityin preclinical xenograft models using A2780/cp70 human ovar-ian cancer cells and human MDA-MB-435 breast cancer cells(Anderson et al. 2004a; Zhang et al. 2004). Also, α-TEA hasbeen shown to be a potent concentration- and time-dependentinducer of apoptosis of murine mammary 66cl-4-GFP tumorcells in vitro, and to significantly decrease primary tumor bur-den and lung metastasis of 66cl-4-GFP tumor cells transplantedinto syngeneic Balb/c mice when formulated in liposome andadministered by aerosol or gavage (Lawson et al. 2003, 2004).

Breast cancer is the most common cancer and the second lead-ing cause of cancer deaths among women in the United States(Smigal et al. 2006). Thus, there is a great need for effectivechemotherapy agents for breast cancer. Expectations are that α-TEA, a new, stable, nontoxic vitamin E analog with promisinganticancer properties, will prove to be an effective anticanceragent for human breast cancer. Since α-TEA is hydrophobic, anappropriate drug delivery system is needed for better therapeuticefficacy.

Biomedical research has shown the great need to control, reg-ulate, and target the release of drugs in the body to tissues or cellsof interest (Lee 2006). A primary goal is to use less frequent drugadministration to achieve a constant therapeutic level of drug inthe systemic circulation or at the specific target organ site to re-duce undesirable drug effects. Among the different strategies fordrug delivery, colloidal carriers were found to be effective. Themost popular and well-studied colloidal drug delivery systemsare the liposome systems that permit high drug load and are rel-atively easy to handle. However, they have some fundamental

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498 P. WANG ET AL.

problems, such as susceptibility to degradation by lysosomesand noncontrollable drug release rate (Hofheinz et al. 2005;Zamboni 2005). Studies in our lab have used α-TEA/liposomeformulations delivered by aerosol and gavage (Anderson et al.2004a; Lawson et al. 2003, 2004; Zhang et al. 2004). Recently,biodegradable polymeric nanoparticle systems have been devel-oped to improve drug delivery. Nanoparticles, in general, can beused to improve oral bioavailability, to sustain drug/gene effectin target tissue, and to improve the stability of therapeutic agentsagainst enzymatic or lysosomal degradation (Lee 2006; Panyamand Labhasetwar 2003a, 2003b).

Biodegradable polymers are natural or synthetic in originand are degraded in vivo to produce biocompatible, toxicologi-cally safe byproducts that are further eliminated via metabolicpathways (Jain 2000; Shive and Anderson 1997). Of these poly-mers, polylactides (PLA) and poly (D,L-lactide-co-glycolide)(PLGA) have been investigated the most. As reviewed by Jain(2000), PLGA has been used to form an effective nanoparticledelivery system, has been approved by the Food and Drug Ad-ministration, and has a history of safe use in humans. A GoodManufacturing Practices grade material is available from com-mercial sources, making it an ideal candidate for use in novelformulations for the poorly water-soluble vitamin E analog.

In this study, we used DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) as lipid for liposomes and PLGA as polymerfor nanoparticles. we compared the ability of these two α-TEAformulations in two drug dosages (5 mg/day or 2.5 mg/day) toreduce BALB/c 66cl-4 tumor burden and metastasis to lung andlymph nodes when delivered by gavage.

MATERIALS AND METHODSα−TEA (formula weight = 488.8) was prepared as described

previously (Lawson et al. 2003); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) was purchased from Avanti Polar-Lipids, (Alabaster, AL, USA); PLGA, (MW 143,900, copoly-mer ratio 50:50), Tween 80, and dichloromethane (HPLC grade)were purchased from Sigma Chemicals (St. Louis, MO, USA).

Preparation of Liposomal and Nanoparticle α-TEAAn α-TEA/DLPC ratio of 1:3 (w/w) has been determined

previously to be optimal (Lawson et al. 2003). To prepare theα-TEA/DLPC combinations, the components were first broughtto room temperature. The lipid DLPC, at a concentration of120 mg/ml, was dissolved in tertiary-butanol (Fisher Scientific,Houston, TX, USA) and then sonicated to obtain a clearsolution. α-TEA at 40 mg/ml was dissolved in tertiary-butanoland vortexed until all solids were dissolved. DLPC and α-TEAwere combined in equal amounts (v:v) to achieve the desiredratio of 1:3 α-TEA/liposome, mixed by vortexing, frozen at-80◦C for 1-2 hr, and lyophilized overnight to a dry powderprior to storing at −20◦C until needed. Particle size range ofα-TEA liposomes delivered orally was determined to be 4–10µm (Lawson et al. 2004).

α-TEA-loaded nanoparticles were prepared by an emul-sion/solvent evaporation method based on previous work (Davda

and Labhasetwar 2002; Feng et al. 2004; Sinha et al. 2002) andmodified for α-TEA. The main variables that influence the mi-croencapsulation process and the final product are, (a) the natureand solubility of the drug being encapsulated, (b) the polymerconcentration, (c) the drug/polymer ratio, (d) the organic solventused, (e) the concentration and nature of the emulsifier used, (f)the status of emulsifier in aqueous solution, (g) the temperatureand stirring speed of the emulsification process, (h) the evapora-tion time, and (i) the viscosities and volume ratio of the dispersedand continuous phases (Jain 2000). These factors were all takeninto consideration when modifying the published protocols. Inbrief, PLGA (200 mg) was first dissolved in a water immisci-ble, volatile organic solvent, dichloromethane (16 ml). α-TEAat 20 mg was added to the polymer solution. A 0.4% solution ofTween 80 was prepared in 250 ml of distilled water maintainedat 40◦C and then filtered through a 0.22 µm filter. The formedPLGA solution was subsequently added to the Tween 80 solu-tion. The resulting emulsion was sonicated with an energy outputof 50W in a pulse mode for 90 sec. The oil-in-water emulsionwas stirred for 3 hr at room temperature on a magnetic stir plateto allow the evaporation of dichloromethane and formation ofthe nanoparticles.

The suspension was transferred into ultracentrifuge tubes andcentrifuged at 12,000 rpm for 30 min at room temperature. Thepellet was washed with distilled water three times to removeresidual Tween 80. The supernatant and the washings werecollected and used to determine the amount of drug that wasnot trapped in the nanoparticles. The suspension was collectedand frozen at −80◦C overnight and subsequently lyophilizedfor 1 day. The lyophilized nanoparticles were stored desic-cated at −20◦C. The nanoparticles were stable for at least3 months.

Nanoparticle Size DistributionTo measure nanoparticle size distribution using dynamic laser

light scattering, nanoparticles were suspended in distilled waterand then subjected to particle size analysis using a Zeta PlusTM

particle size analyzer (Brookhaven Instruments, NY, USA). Thesize and morphology of the nanoparticles were further analyzedby transmission electron microscopy. Briefly, 2 µl of nanopar-ticle suspension was placed on a carbon film coated on a TEMcopper grid. The particles were immediately frozen with liquidnitrogen, dried by vacuum and visualized.

Nanoparticle Drug Encapsulation EfficiencyThe drug encapsulation efficiency (DEE) is calculated as

DEE= [(total amount of drug–free amount of drug)/total amountof drug] × 100. The supernatant and washings generated dur-ing the production of nanoparticles were collected and used todetermine the amount of α-TEA that was not trapped in thenanoparticles (free drug). Free α-TEA was extracted using 100%chloromethane. Reverse phase HPLC analyses were conductedas previously described (Tirmenstein et al. 1998). In this case,DEE = 99%. This indicates complete drug encapsulation can

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α-TEA-LOADED LIPOSOME OR POLYMERIC NANOPARTICLE 499

be achieved if the ratio of drug and polymer is appropriate (Linet al. 2000).

66cl-4-GFP Murine Mammary Tumor Cell Line66cl-4 cells were derived from a spontaneous mammary tu-

mor in a BALB/cfC3H mouse and isolated as a 6-thioguanine-resistant clone (Dexter et al. 1978; Miller et al. 1983). Thesecells were stably transfected with green fluorescence protein aspreviously described (Lawson et al. 2003). 66cl-4-GFP cells arehighly metastatic, 100% of animals develop micrometastatic le-sions detectable with fluorescent microscopy in the lungs 26days following subcutaneous injection of 2 × 105 tumor cellsinto the inguinal area (Lawson et al. 2003).

BALB/c MiceFemale BALB/c mice at 6 weeks of age (about 25 g body

weight) were purchased from Jackson Labs (Bar Harbor, ME,USA) and allowed to acclimate for 1 week. Mice were housed5/cage and given water and standard lab chow (Harlan Teklad#2018 Global 18% Protein Rodent Diet; Madison, WI, USA)ad libitum at the Animal Resource Center at the University ofTexas at Austin. Mice were maintained in an environment of74 ± 2◦F with 30%–70% humidity and a 12-hr alternating light-dark cycle. Guidelines for the humane treatment of animals werefollowed as approved by the University of Texas InstitutionalAnimal Care and Use Committee.

Tumor Cell Inoculation66cl-4-GFP cells were harvested by trypsinization, collected

by centrifugation, and resuspended at a density of 2 ×105

cells/100 µl in McCoy’s media, containing no supplements.Mice were injected with 2 × 105 cells/100 µl in the inguinalarea at a point equal distance between the fourth and fifth nip-ples on the right side using a 25-gauge needle.

Fully 60 mice, 10/group, were placed in 6 groups [(li-posome/gavage control, liposome α-TEA/gavage low dose(2.5 mg), liposome α-TEA/gavage high dose (5 mg), nanoparti-cle (NP) gavage control, NP α-TEA/gavage low dose (2.5 mg),and NP α-TEA/gavage high dose (5 mg)] such that the averagetumor volume for all groups was closely matched. Each grouphad an average tumor volume/group of 1.94 mm3, 1.91 mm3,1.84 mm3, 1.84 mm3, 1.88 mm3, and 1.84 mm3, respectively,at the start of treatments, that were begun 12 days followingtumor cell inoculation. Tumors were measured using calipersevery other day, and tumor volumes were calculated using thefollowing formula: volume (mm3) = [width (mm2) × length(mm)]/2 (Clarke 1997). Body weights were determined weekly.

Gavage DeliveryFor gavage delivery, lyophilized preparations of liposome

α-TEA, nanoparticle α-TEA, liposome only and nanoparticleonly, were brought to room temperature and reconstituted by

adding 1 ml (low dose groups) or 2 ml (high dose and controlgroups) distilled water to achieve the final desired concentrationof 25 mg/ml α-TEA. Treatments were vortexed vigorously im-mediately prior to administration by gavage. The total amountof α-TEA administered by gavage was 100 µl/mouse per dayfor low dose groups and 200 µl/mouse/day for high dose groups(final concentration, 2.5 or 5 mg α-TEA/mouse/day). High doseand control group treatments were given twice/day, approxi-mately 4 hrs apart. All treatments were given 7 days/week, fora total of 20 days.

Lung and Lymph Node MetastasisMacroscopic metastases in all 5 lung lobes were counted

visually at time of euthanasia. Fluorescent microscopic lunglesions were counted as described previously, using a Nikonfluorescence microscope [TE-200; 3200 magnification (Lawsonet al. 2003)]. Fluorescent microscopic lesions were scored bysize into four size groupings: <20 µm, 20–50 µm, 50-100 µm,and >100 µm. On the basis of a typical 66cl-4-GFP tumor cellsize of 10–20 µm in diameter, the <20 µm grouping is thoughtto represent solitary cells; the 20–50 µm grouping 2 to 5 cells;50-100 µm grouping 5 to 10 cells, and the > 100 µm groupingmicroscopic lesions of greater than 10 cells.

TUNEL Assay for Detection of Apoptosis In VivoDeparaffinized sections (5 µm) of tumor tissue were used to

assess apoptosis using reagents supplied in the ApopTag In SituApoptosis Detection kit (Intergen, Purchase, NY, USA) accord-ing to the manufacturer’s instructions. Nuclei that stained brownwere scored as positive for apoptosis, and those that stained bluewere scored as negative. At least 16 microscopic fields (×400)were scored per tumor. Data are presented as the mean ± SEnumber of apoptotic cells counted in 6 separate tumors fromeach group.

Detection of Immune Cell Infiltration in Tumor TissueDeparaffinized tumor sections (5 µm) were stained with an-

tibodies to CD45R or CD3 to determine B or T-cell infiltration.Briefly, endogenous peroxidase activity was blocked with 3%H2O2 and nonspecific antibody binding was blocked with 10%normal rabbit serum. Tumor sections were incubated with ei-ther CD45R or CD3 antibody (rat anti mouse antibody; Serotec,Raleigh, NC, USA; 1:200 dilution) overnight at 4◦C. Next, thetissue sections were incubated with biotinylated rabbit-antiratIgG (Vector Laboratories, Burlingame, CA, USA) at a 1:200dilution for 30 min at room temperature, washed, and then in-cubated with avidin-biotin complex (ABC-HRP, Vector Labo-ratories) for 30 min at room temperature. Immunoreactivity wasvisualized via incubation with diaminobenzidine dihydrochlo-ride. Slides were lightly counterstained with hematoxylin. Then16 or more microscopic fields (400×) were scored for positivestained cells/tumor. Data were collected using 3 separate tumorsfrom each group. In addition, H&E-stained tumor sections wereused for the detection of cellular infiltration.

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Statistical AnalysesTumor growth was evaluated by transforming volumes us-

ing a logarithmic transform (base 10) and analyzed usinga nested two-factor ANOVA with Tukey HSD, using SPSS(SPSS, Chicago, IL, USA). The differences in the number ofmacroscopic and microscopic metastatic lesions/group, T-andB-stained cells/group and TUNEL positive cells/group were an-alyzed by the two tailed Mann-Whitney rank test using SPSS.A level of p <0.05 was regarded as statistically significant.

FIG. 1. Nanoparticle size distribution. (A) and (B) Size distribution ofnanoparticles (PLGA-α-TEA) determined by dynamic light scattering, usinga neon laser of λ = 677.0 nm and measured at 90◦ degree. The effective diam-eter of nanoparticles = 272 nm, polydispersity = 0.313. Measurements wererepeated three times, each time with similar patterns. (C) Transmission electronmicroscopy of α-TEA-loaded nanoparticles (PLGA: α-TEA = 10:1). Nanopar-ticles were visualized directly after freeze-drying. The mean diameter was190 ± 65 nm. Bar represents 500 nm.

RESULTS

Nanoparticle Morphology, Size Determination, andDistribution

The mean size and distribution pattern of α-TEA loadednanoparticles was determined by dynamic light scattering (DLS)(De and Robinson 2004). The mean size diameter of the vesi-cle was found to be approximately 272 nm, and polydispersity,which is a measure of the distribution of molecular weights ina given polymer sample, was equal to 0.313 (Figure 1A). Thesize distribution pattern as determined by measuring the inten-sity of the nanoparticles by DLS is shown in Figure 1B. Thesize and morphology of the nanoparticles were further checkedby transmission electron microscopy (TEM) (Figure 1C). UsingTEM, the nanoparticles were found to be of spherical shape witha mean diameter of 190 ± 65 nm. The nanoparticle mean diam-eter measured using TEM was much smaller than the mean di-ameter obtained with the DLS method. This discrepancy in sizeof nanoparticles is because the DLS method gives the hydrody-namic diameter rather than the actual diameter of nanoparticles.

Liposomal α-TEA High Dose and All NanoparticleTreatments Significantly Suppressed Tumor Growth

Mean tumor volumes in mice receiving liposomal andnanoparticle α-TEA at 5 mg/mouse/day were significantly lowerthan liposome or nanoparticle controls, respectively, over 20days of treatment (p < 0.001; Figure 2). Mean tumor volumein mice receiving nanoparticle α-TEA at 2.5 mg/mouse/day wasnot significantly different from nanoparticle control, but it wassignificantly different from liposome control (p < 0.001; Figure2). Liposomal α-TEA at 2.5 mg/mouse/day reduced tumor bur-den but not significantly from liposome control. Mean tumor vol-umes in mice receiving nanoparticle α-TEA at 5 mg/mouse/day,in comparison to all the other treatment groups, was significantlylower over the 20 days of treatment (p < 0.001 in comparisonto liposomal α-TEA at 2.5 mg/ml; p < 0.002 in comparison toliposomal α-TEA at 5 mg/ml; and p < 0.017 in comparisonto nanoparticle α-TEA at 2.5 mg/ml). Surprisingly, data showthat the nanoparticle control, in comparison to liposome control,significantly reduced tumor burden to a degree comparable toliposomal α-TEA at 5 mg/mouse/day or nanoparticle α-TEA at2.5 mg/mouse/day treatment groups (Figure 2).

Reduced Visible Macroscopic Lung MetastasesAt sacrifice, all 5 lung lobes from each mouse were ex-

amined for visible metastases. All mice in the liposome andnanoparticle control groups as well as nanoparticle α-TEA at2.5 mg/mouse/day had visible lung tumor foci. But 4/10 mice inliposomal and nanoparticle high dose treatment groups did nothave visible lung tumor foci; but this was not significantly differ-ent from controls. However, the average number of macroscopiclung tumor foci was significantly reduced in all treatmentgroups when compared with their respective control group(Table 1); namely, in comparison to liposome control: liposomal

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FIG. 2. Effects of liposome- or nanoparticle-formulated α-TEA delivered by gavage on tumor burden. 66cl-4-GFP cells at 2 × 105/mouse were injected intothe inguinal area at a point equal distance between the fourth and fifth nipples. Then 12 days after tumor injection, mice were assigned to control and α-TEAtreatment groups such that the mean tumor volume of each group was closely matched (average tumor volume/group = 1.88 mm3). Mice (10/group) were treatedwith liposomal control, nanoparticle control, liposomal α-TEA, or nanoparticle α-TEA at high dose (5 mg/mouse/day) or low dose (2.5 mg/mouse/day) for 20days. Tumor volumes (mm3) are depicted as mean ± SE. (1) = significantly reduced in comparison to liposome control, (2) = significantly reduced in comparisonto nanoparticle control, and (3) = significantly reduced in comparison to liposomal α-TEA at 2.5 mg/day.

α-TEA at 2.5 mg/mouse/day, p < 0.011; liposomal α-TEA at5 mg/mouse/day, p < 0.002; and in comparison to nanoparticlecontrol: nanoparticle α-TEA at 2.5 mg/mouse/day, p < 0.028;nanoparticle α-TEA at 5 mg/mouse/day, p < 0.001.

Reduced Lung Micrometastatic Tumor FociGreen fluorescing microscopic lung lesions were classified

into four size groups: <20, 20-50, 50-100, and >100 µm. Themean total number of microscopic lung tumor foci in liposomaland nanoparticle treatment groups were significantly reduced incomparison to liposome control (p < 0.001 for all) or nanopar-ticle control (p < 0.001) for all except liposomal α-TEA at2.5 mg/mouse/day, in which p < 0.044 (Figure 3). The meantotal number of microscopic lung tumor foci in nanoparticle α-TEA at 2.5 mg/mouse/day were significantly reduced in compar-ison to liposomal α-TEA at 2.5 mg/mouse/day (p < 0.003). Thissignificant reduction appears to be attributable mainly to lesionsin the <20 µm size grouping. Although nanoparticle α-TEA at5 mg/mouse/day had a comparable total number of microscopiclung lesions as liposomal α-TEA at 5 mg/mouse/day, the mi-croscopic lung tumor foci in the 20–50 µm and 50–100 µmsize grouping were significantly lower (p < 0.024 for bothsize groupings). The mean total number of microscopic lunglesions in the nanoparticle control group itself was significantlyreduced in comparison to the liposome control group (p <0.005)(Figure 3A).

TABLE 1α-TEA formulated into liposome or nanoparticle reduced the

average number of visible macroscopic lung metastases.

Visible Lung Metastasis

No. Animals/group Average No.with visible lung visible lung

Treatmentsa metastasesb tumor focic

Liposome control 10/10 7.0 ± 1.1Liposome, 2.5 mg 9/10 3.0 ± 0.6∗∗∗

Liposome, 5 mg 6/10 1.1 ± 0.3∗∗∗

Nanoparticle control 10/10 8.3 ± 1.2Nanoparticle, 2.5 mg 10/10 4.7 ± 0.8∗∗

Nanoparticle, 5 mg 6/10 1.2 ± 0.4∗∗∗

aα-TEA was formulated into liposomes or nanoparticles and deliv-ered daily for 20 days by gavage.bMacroscopic metastases in all lung lobes in control and treatmentgroups were counted visually at the time of sacrifice.cData are expressed as the average number ± S.E. of macroscopic lungtumor foci observed in tumor bearing mice in control and treatmentgroups.∗Significantly different from liposome control.∗∗Significantly different from nanoparticle control.

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FIG. 3. Effects of liposome- and nanoparticle-formulated α-TEA delivered by gavage on micrometastatic tumor foci in lung and lymph node. (A) Using a Nikonfluorescence microscope, the number of fluorescent microscopic tumor foci on the surface (top and bottom) of flattened left lung lobes was determined for controland treatment groups. Micrometastatic tumor foci were grouped into four size groupings (<20 µm, 20-50 µm, 50–100 µm, or >100 µm). (B) Using a Nikonfluorescence microscope, the number of fluorescent microscopic metastases in brachial and axillary lymph nodes was determined for control and treatment groups.For both (A) and (B), average number of total lesions was calculated. Data are depicted as mean ± SE of total number of microscopic metastases. (1) = significantlyreduced in comparison to liposome control, (2) = significantly reduced in comparison to nanoparticle control, and (3) = significantly reduced in comparison toliposomal α-TEA at 2.5 mg/day.

Reduced Microscopic Lymph Node Tumor FociGreen fluorescent microscopic lymph node metastatic le-

sions, like the lung microscopic lesions, were classified intofour different size groups: <20, 20–50, 50–100, and >100 µm.Data are shown for the total number of microscopic tumorfoci. The mean total number of microscopic lymph node metas-tases in liposomal high dose and both of the nanoparticle treat-ment groups were significantly reduced in comparison to li-posome control (p < 0.001 for all) or nanoparticle control(liposomal α-TEA at 5 mg/mouse/day, p < 0.006; nanoparticleα-TEA at 2.5 mg/mouse/day, p < 0.048; and nanoparticle α-TEA at 5 mg/mouse/day, p < 0.005). The mean total num-ber of microscopic lymph node metastases in nanoparticle α-TEA at 2.5mg/mouse/day was significantly reduced in compar-ison to liposomal α-TEA at 2.5 mg/mouse/day (p < 0.002).The mean total number of microscopic lymph node metastasesin the nanoparticle control group itself was significantly re-

duced in comparison to liposome control group (p < 0.001)(Figure 3B).

Tumor Cells Undergo ApoptosisApoptosis was evaluated using TUNEL staining of 5 µm

tumor sections from control and treatment groups. The meannumber of TUNEL positive cells in the high dose liposomal andnanoparticle groups were significantly higher than their respec-tive controls (liposomal α-TEA at 5 mg/mouse/day, p < 0.009;nanoparticle α-TEA at 5 mg/mouse/day, p < 0.003). TUNELpositive cells in the liposome and nanoparticle α-TEA low dosegroups were not significantly different from their respective con-trols; however, the average number of TUNEL positive cells inthe low dose nanoparticle group was significantly higher thanthe liposomal control p < 0.016 (Figure 4). The average num-ber of TUNEL positive cells in the nanoparticle and liposomecontrol groups were not significantly different from each other.

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α-TEA-LOADED LIPOSOME OR POLYMERIC NANOPARTICLE 503

FIG. 4. α-TEA formulated into liposome or nanoparticle and delivered by gavage-induced 66cl-4-GFP cells to undergo apoptosis. Fully 5 µ micron tumor tissuesections from control and treatment groups were examined for levels of apoptosis by TUNEL. Data are depicted as mean ± SE of TUNEL positive-stained cells/field.(1) = significantly enhanced in comparison to liposome control, (2) = significantly enhanced in comparison to nanoparticle control, and (3) = significantly enhancedin comparison to liposomal α-TEA at 2.5 mg/day.

Cellular Infiltration in Tumor TissueNumber of immune cells infiltrating tumors based on anal-

yses of H&E stained tumor sections and immunohistochemicalanalyses of tumor sections stained specifically for B and T lym-phocytes from all control and treatment groups were low, withno significant differences among control and treatment groups(data not shown). Of special note, there was no difference innumber of intratumoral B and T lymphocytes in tumor sectionsfrom liposome and nanoparticle control groups.

Oral Deliver No Effect on Body WeightsAlthough the mean body weights of mice receiving nanopar-

ticle formulations were slightly less than body weights of micereceiving liposomes, there were no significant differences in themean body weights of mice among all the control and treatmentgroups (data not shown).

DISCUSSIONPrevious studies in our lab showed that α-TEA, a novel

acetic acid analog of vitamin E (RRR-α-tocopherol), when for-mulated in liposomes and delivered by either aerosol at 7.5mg/mouse/day or gavage at 6.4 mg/mouse/day had marked an-titumor efficacy and no evidence of toxicity in BALB-c mousemammary tumor model (Lawson et al. 2003; 2004). To conservethe amount of drug we used and to make it clinically friendlyfor future administration practice, we chose gavage method inthis study. Two levels of α-TEA, low (2.5 mg/mouse/day) andhigh (5 mg/mouse/day), were used for this comparison of lipo-some versus nanoparticle formulation effectiveness. The highdose α-TEA/liposome formulation was expected to produce a

superior anticancer efficacy than the low dose α-TEA/liposomeformulation. Effects of α-TEA formulated in nanoparticles wereunknown, but based on the literature, expectations were thatnanoparticle formulations would be as effective and perhapsmore effective as an anticancer agent than α-TEA formulated inliposomes.

This study used two control groups: liposome control groupand nanoparticle control group. Since previous work in our labshowed that the tumor volume of the liposome control groupwas not significantly different than the tumor volume in un-treated controls (Lawson et al. 2003), an untreated group wasnot included in this study. Since there was a substantial antitu-mor effect by the control nondrug-containing nanoparticles, theliposome control group was used for comparing the anticancerefficacy of all treatment groups.

Data showed α-TEA high dose formulated into either lipo-some or nanoparticle to be effective in reducing tumor volumewhen evaluated in comparison to liposome or nanoparticle con-trols, respectively. Low dose α-TEA formulated in liposomes ornanoparticles was significantly less effective than high dose α-TEA formulated by the two methods in reducing tumor burdenwhen compared with their respective controls. Both low and highdose α-TEA formulated in either liposomes or nanoparticles sig-nificantly reduced the average number of visible macroscopiclung metastases. High dose α-TEA formulated in liposomes aswell as both low and high dose α-TEA formulated in nanoparti-cles significantly reduced the total numbers of lung and lymphnode micrometastatic tumor foci. Low dose α-TEA formulatedin liposomes reduced the mean number of lymph node metastaticlesions but not significantly different from control.

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504 P. WANG ET AL.

Surprisingly, the nanoparticle control group significantly re-duced tumor burden in comparison to the liposome controlgroup, with the reduction in tumor volume being similar to the tu-mor volume reduction observed in the liposome high dose groupand nanoparticle low dose group. Furthermore, the nanoparticlecontrol group significantly reduced the total number of lung andlymph node micrometastatic lesions in comparison to the lipo-some control group. However, the number of mice with visibletumors as well as the average number of visible lung tumors inthe nanoparticle control group were not significantly reduced incomparison to the liposome control group. Thus, a substantialamount of the anticancer properties of the α-TEA low and highnanoparticle groups may reflect the anticancer properties of thenanoparticle vehicle.

This is the first report of which we are aware that showsthat PLGA polymer nanoparticles alone delivered by gavagecan significantly reduce tumor burden and lung and lymph nodemicrometastases. Although we do not know the mechanismswhereby PLGA polymers reduce tumor burden, it is possiblethat the PLGA polymer delivered at higher levels via gavagedelivery versus smaller amounts via IV delivery may (i) exhibittumor cell specific toxicity or may (ii) induce enhancement ofimmune killing of tumor cells. Regarding toxicity, based on bodyweights, none of the α-TEA treatment or control groups showedany overt toxicity. It is important to note that both chemicals usedin the nanoparticle formulation, namely, PLGA and surfactantTween 80, have FDA approval for use in drug delivery systems(Feng et al. 2004; Panyam et al. 2002; Shive and Anderson 1997).PLGA undergoes hydrolysis, forming biologically compatibleand metabolizable moieties (lactic acid and glycolic acid) thatare eventually removed from the body by the citric acid cycle(Shive and Anderson 1997). Tween 80 is a food-grade surfac-tant commonly used in dietary supplements, flavoring agents,whipped toppings and shortenings (Abriola and Pennell 1994).Therefore, nanoparticles made from these materials were ex-pected to be nontoxic.

Nevertheless, the nanoparticle control exhibited significantanticancer efficacy and these anticancer effects were not asso-ciated with increased ability to induce apoptosis. Some type oftumor specific toxicity is a possibility that warrants further in-vestigation. Support for the second possible explanation comesfrom studies showing enhancement of antitumor immunity byPLGA nanoparticles used for delivery of vaccine (Jilek et al.2004; Yoshida and Babensee 2004). These studies showed thatPLGA when formed into nanoparticles enhanced maturation ofmonocyte-derived dendritic cells. As antigen-presenting cells,mature dendritic cells could enhance antitumor immunity (Jileket al. 2004; McKenna et al. 2005; Yoshida and Babensee 2004).To investigate possible immune involvement, immune cell infil-tration of tumors from liposome and nanoparticle control groupswas evaluated. No detectable differences in intratumoral B or Tlymphocyte numbers between the liposome and nanoparticlecontrols at euthanasia were observed, providing no support forT and B cell involvement at this one time point.

CONCLUSIONα-TEA formulated in either nanoparticles or liposomes at

a dosage of 5 mg/day was very effective in inhibiting tumorgrowth and lung metastases. α-TEA at 2.5 mg/day formulatedin nanoparticles was more effective than α-TEA at 2.5 mg/dayformulated in liposome in reducing tumor burden and lymphnode and lung micrometastatic tumor foci. However, the dif-ference between the two formulations could be attributed to ananoparticle effect rather than an α-TEA effect since nanopar-ticles alone significantly reduced tumor burden and metastases.These studies show that oral delivery of α-TEA at an appropri-ate dosage formulated in either liposomes or nanoparticles tobe an effective, clinically relevant means for administrating thishydrophobic drug.

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