Coencapsulated Doxorubicin and Bromotetrandrine Lipid

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Co-encapsulated Doxorubicin and Bromotetrandrine Lipid Nanoemulsions in Reversing Multidrug Resistance in Breast Cancer In Vitro and In Vivo Xi Cao, Jingwen Luo, Tao Gong, Zhirong Zhang, Xun Sun, and Yao Fu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500637b • Publication Date (Web): 03 Dec 2014 Downloaded from http://pubs.acs.org on December 6, 2014

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Molecular Pharmaceutics

Co-encapsulated Doxorubicin and Bromotetrandrine Lipid Nanoemulsions in Reversing Multidrug Resistance in Breast Cancer In Vitro and In Vivo Xi Cao, Jingwen Luo, Tao Gong, Zhi-Rong Zhang, Xun Sun, and Yao Fu* Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, Sichuan University, Sichuan, People’s Republic of China

ACS Paragon Plus Environment

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ABSTRACT: Multidrug resistance has remained a major cause of treatment failure in chemotherapy due to the presence of P-glycoproteins (P-gp) that actively pump drugs from inside the cell to the outside. P-gp inhibitors were developed and co-administered with chemotherapeutic drugs to overcome the effect of efflux pumps thus enhancing the chemosensitivity of therapeutics. Our study aimed at developing a lipid nanoemulsion system for the co-encapsulation of doxorubicin (DOX) and bromotetrandrine (W198) to reverse multidrug resistance (MDR) in breast cancer. W198 was a potent P-gp inhibitor, and DOX was selected as a model compound which is a common substrate for P-gp. Co-encapsulated DOX and W198 lipid nanoemulsions (DOX/W198-LNs) displayed significantly enhanced cytotoxicity in DOXresistant human breast cancer cells (MCF-7/ADR) compared with DOX loaded lipid nanoemulsions (DOX-LNs) (p < 0.05), which is due to the enhanced intracellular uptake of DOX in MCF-7/ADR cells. The biodistribution study was performed using a nude mice xenograft model, which demonstrates enhanced tumor uptake of DOX in the DOX/W198-LN treated group. Compared with DOX solution, DOX/W198-LNs showed reduced cardiac toxicity and gastrointestinal injury in rats. Taken together, DOX/W198-LNs represent a promising formulation for overcoming MDR in breast cancer. KEYWORDS:

multidrug

resistance,

P-glycoprotein

inhibitor,

doxorubicin,

lipid

nanoemulsions, pharmacokinetics, biodistribution


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INTRODUCTION The number of cancer-related deaths worldwide is on the rise despite the cutting-edge advances in drug discovery and drug delivery1. Among various treatment strategies, chemotherapy has remained the most predominant treatment option for the majority of cancer and tumor diseases2. However, resistance is a major cause of treatment failure in chemotherapeutic drugs due to the presence of efflux pumps on the cancer cell surface or nucleus membrane that actively pump drugs from inside the cell to the outside. Chemotherapeutic drugs such as doxorubicin (DOX) carry numerous dose-limiting normal tissue side effects and as a result, it is impractical to overcome drug resistance simply by increasing the drug dose3,4. P-glycoprotein (P-gp) encoded by MDR1 gene is a member of the ATP-binding cassette super family and has been found to be over expressed on cancer cells with multidrug resistance phenotype5. Thus, inhibition of the transporter function and expression of P-gp presents one of the most effective ways to reverse multidrug resistance (MDR) and improve the efficacy of chemotherapeutics. The combined administration of chemotherapeutics with small molecular P-gp inhibitors has appeared as a popular trend to reverse MDR and increase the sensitivity of tumor cells to chemotherapeutics in cancer therapy6. However, most attempts have been unsuccessful mainly due to a relatively low affinity for P-gp, low selectivity, inherent toxicity and pharmacokinetic interactions with anticancer drugs which inhibited drug metabolism and led to enhanced excretion of the anticancer drugs5,7. Tetrandrine (Tet), which is the main active component in the root Stephania tetrandra S. Moore, was proven a potential P-gp inhibitor with potent reversal effect on P-gp-mediated MDR in vitro and in vivo8,9. In 2004, Wang group from West China School of Pharmacy at Sichuan University reported the discovery of bromotetrandrine (W198) which is a novel derivative of Tet


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(Figure 1). Preliminary screening studies showed superior P-gp reversal activity of W198 compared with that of Tet which significantly potentiated the cytotoxicity of anti-cancer drugs in P-gp overexpressing cells but not in P-gp negative parental cells9. The reversal effect of W198 was attributed to the degradation of P-gp and decreased P-gp protein half-life. Moreover, W198 was proven less toxic than Tet, and had little effect on the pharmacokinetic profiles of DOX after co-administration10. A phase Ia clinical trial in healthy volunteers further demonstrated the safety of W198 in humans following intravenous administration11. Thus, W198 was selected as the Pgp inhibitor to be co-administered with DOX to multidrug resistant cancer cells in the following study.

Figure 1. Chemical structures of (A) tetrandrine and (B) bromotetrandrine (W198).

Free drugs enter cells most likely through passive diffusion across the cell membrane which can be easily pumped out by P-gp. In contrast, nanoscale carriers are internalized into cells via endocytosis which may bypass P-gp efflux and their internalization and retention are not negatively influenced by the overexpression of MDR proteins4,7. Here, we reported the fabrication of a phospholipid based lipid nanoemulsion (LN) for the co-encapsulation and


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concurrent delivery of DOX and W198 to multidrug resistant breast cancer cells. We first aimed to develop a neat nanoscale carrier system which can bypass P-gp efflux and achieve effective intracellular accumulation of drug loaded LNs. After systemic administration, LNs of concurrently loaded DOX and W198 were designed to achieve intratumoral accumulation via leaky vasculature and enhanced permeability and retention (EPR) effect. Next, W198 is expected to be released intracellularly and achieve inhibition of P-gp either on the cellular surface or the nucleus membrane. DOX is a common substrate for P-gp and its major target site of action is the cell nucleus. As a result, DOX can be specifically delivered to the cell nucleus in MDR cancer cells by efficiently inhibiting P-gp efflux pumps12,13. In our study, DOX was formulated with oleic acid to form a hydrophobic complex. DOX-oleic acid complex and W198 were then coencapsulated in phospholipid-based LNs (DOX/W198-LNs). The concurrent loaded LNs were optimized based on the entrapment efficiency of DOX and W198. The in vitro stability, release kinetics, cellular uptake behaviors, cell apoptosis, pharmacokinetics, biodistribution, and in vivo toxicities of DOX/W198-LNs were further investigated.

EXPERIMENTAL SECTION Materials and Animals. Doxorubicin hydrochloride was purchased from Huafenglianbo (Beijing, China). W198 was kindly gifted from Dr. Fengpeng Wang at the West China School of Pharmacy, Sichuan University (Chengdu, China). Soybean lecithin (Lipoid S100) was purchased from Lipoid (Germany). Soybean oil was provided by Beiya Medical oil Co, Ltd (Tieling, China). Pluronic F68 was kindly offered by BASF (Germany). Trypsin and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenltetrazolium bromide (MTT) were purchased from SigmaAldrich (USA). Rabbit anti-CD31 antibody was purchased from Abcam (Hong Kong, China).


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4’,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Haimen, China). Adhesion microscope slides were provided by Jiangsu CITOTEST Co., Ltd. (Haimen, China). Other chemical solvents and reagents were of analytical grade. Wistar rats (180 ± 20 g) and athymic nude mice (4 - 6 weeks) were purchased from Dashuo Biotechnology (Chengdu, China) and maintained under standard housing conditions. All animal experiments were approved by the ethics committee of Sichuan University and conducted in accordance with institutional guidelines. Cell Line and Cell Culture. Human breast cancer MCF-7 cells and an adriamycin resistant counterpart MCF-7/ADR cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100U/mL penicillin, and maintained at 37 °C in a humidified atmosphere containing 5% CO2. MCF-7/ADR cells were cultured in the same medium in the presence of 1 µg/mL DOX to maintain the multidrug resistance phenotype according to the manufacture’s instruction. Preparation of DOX/W198-LNs and DOX-LNs. The DOX-oleic acid (DOX-OA) complex was first prepared using a co-precipitation method as previously described14. DOX base was prepared by neutralizing doxorubicin hydrochloride solution with 50 mg/mL sodium bicarbonate solution. Then, 2 mL of DOX solution (5 mg/mL) in ddH2O and 800 µL of oleic acid ethanol solution (10% w/v) were mixed by vortex mixer for 5 min, the solution was then centrifuged and lyophilized to afford DOX-OA. Next, 30 mg of DOX-OA (containing about 10 mg of DOX), 2 mL of W198 ethanol solution (5 mg/mL), lipoid S100 and soybean oil were dissolved in ethanol in a round bottom flask. The organic phase was subsequently removed by rotary evaporation (Büchi, Switzerland) under reduced pressure at 37 ºC until a thin lipid film formed. The dried


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lipid film was rehydrated in 10 mL of water, followed by vigorous vortex-mixing. The predispersion was then passed through a high-pressure homogenizer (EmulsiFlex-C5, AVESTIN, Canada) for twelve cycles at an operating pressure of 96.4 - 115.6 MPa to obtain DOX/W198-LNs. Single loaded DOX LNs (DOX-LNs) were prepared similarly in the absence of W198. Characterization of DOX/W198-LNs. Mean diameter (nm) and polydispersity index values were determined by Malvern Zetasizer (Malvern, NanoZS90, UK). Analyses were performed in triplicate at 25 ºC and 90º angle. Zeta potentials of LN dispersions were determined to confirm the surface charge of the particles in triplicate at 25 ºC. The morphology of lipid nanoemulsions were investigated by transmission electron microscope (TEM) (JEM 100CX, JEOL, Japan) after negative staining with a uranyl acetate solution (1%, w/v). To determine the encapsulation efficiency (EE%), unentrapped DOX and W198 were separated from drug loaded LNs using a Sephadex G-75 mini-column. The concentrations of entrapped DOX and W198 were determined by fluorescence spectroscopy and HPLC, respectively. EE% was calculated by the following equations: EE% = (weight of the drug in lipid nanoemulsions/weight of the feeding drug) × 100% The concentration of W198 were assayed by Shimadzu LC-10AD high-performance liquid chromatography system (Tokyo, Japan) which consisted of a Shimadzu ultraviolet detector and an Agilent reversed-phase C18 column (5 µm, 20 mm × 4.6 mm) (Santa Clara, Ca, USA). The mobile phase employed for W198 quantification consisted of methanol/water (90/10, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 282 nm. The concentration of W198 was determined based on the peak area at the retention time of 4.6 min. The doxorubicin


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concentrations

were

determined

by

flurorescence

spectroscopy

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using

RF-5301PC

spectrofluorophotometer (Shimadzu, Japan) with an excitation wavelength of 499 nm and the emission wavelength of 592 nm. In Vitro Drug Release. The release profiles of DOX and W198 from DOX/W198-LNs were studied using a dialysis method. Briefly, DOX/W198-LNs containing 0.3 mg of DOX and 0.3 mg of W198 were transferred to a dialysis tube (MWCO = 4000 Da) and subjected to dialysis against 400 mL of saline containing 0.2% Tween 80 to maintain the sink condition and gently shaken at 100 rpm at 37 ºC. DOX+W198 solution were prepared as control. At predetermined time intervals, 4 mL of aliquot from the external medium was taken out and replenished with an equal volume of fresh medium. The concentrations of DOX and W198 at each time point were determined as previously described and the cumulative drug release percentage was calculated accordingly. Release profiles of DOX and W198 from DOX/W198-LNs were analyzed using classical pharmaceutical release models such as Zero-order, First-order, Higuchi, KorsmeyerPeppas and Weibull equations. Cytotoxicity. MCF-7 and MCF-7/ADR cells were treated with varying concentrations of DOX solution, DOX+W198 solution, DOX-LNs, DOX/W198-LNs and blank LNs, respectively and the cell viability was evaluated by MTT assay. Cells in logarithmic phase were seeded at a density of 1 × 104 cells/cm2 in 96 well plates (Corning, NY, USA). After 24 h, DOX solution, DOX+W198 solution, drug loaded LNs and blank LNs of predetermined concentrations were spiked in the culture medium and incubated for another 48 h. Then, 20 µL of MTT solution (5 mg/mL in PBS, pH 7.4) was added to each well, and incubated for 4 h at 37 ºC in dark. The medium was then removed and the formazan crystals were dissolved in 200 µL of DMSO. The absorbance at 570 nm was measured by Varioskan flash multimode plate reader (Thermo, NH,


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USA). The cell viability (%) was calculated and the 50% inhibitory concentration (IC50) was thus determined by plotting the dose-response curve using Origin 8.0 software (USA). Cellular Uptake. MCF-7 and MCF-7/ADR cells were seeded in 12-well plates at a density of 2 × 105 cells per well (Corning, NY, USA) and incubated for 24 h at 37 ºC. The cells were incubated for 1, 2, and 4 h with free DOX solution, DOX + W198 solution, DOX-LNs and DOX/W198-LNs at an equivalent dose of 5 µg/mL of DOX, respectively. At predetermined time points, cells were rinsed with 1X PBS twice and fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.5% Triton X-100 for 20 min, stained with 20 µg/mL DAPI for 5 min, and observed under confocal laser scanning microscope (CLSM) (Leica, Wetzlar, Germany). In parallel, cells were harvested, fixed, and stained accordingly for FACS analysis (Beckman Coulter, Fullerton, CA, USA). Apoptosis and Necrosis. MCF-7/ADR cells were seeded in 6-well plates at a density of 1 × 104 cells/cm2 and cultured overnight. Cells were then treated with different drug formulations at an equivalent concentration of 5 µg/mL of DOX. After 12 h incubation, cells were washed with 1X PBS twice and cultured in fresh medium for an additional 48 h. After treatment, cells were harvested and stained with Annexin V-FITC and propidium iodide (PI) following the manufacturer's instructions. Fluorescence was measured by FACS (Beckman Coulter, Fullerton, CA, USA). The numbers of cells undergoing necrosis (positive for PI), early apoptosis (positive for Annexin V-FITC), and late apoptosis (double-positive for Annexin V-FITC and PI) were quantified by FACS. Moreover, samples after similar treatment were washed with 1X PBS twice and the cell nuclei were stained with 1 µg/mL DAPI for 5 min. The morphology of cell nuclei was observed under a Zeiss fluorescence microscope (Germany). Apoptotic cells were evaluated


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based on the morphology of nucleus changes including chromatin condensation, fragmentation and apoptotic body formation. Pharmacokinetics in Rats. Female Wistar rats (200 ± 20 g) were fasted for 12 h before experiment. Rats were given DOX+W198 solution and DOX/W198-LNs at an equivalent dose of 6 mg/kg DOX via tail vein injection. About 0.3 mL of blood samples were collected at 15, 30, 60, 120, 240, 360, 480, 600, 720, and 1440 min post injection and centrifuged at 4500 rpm for 8 min. Then, 300 µL of acetonitrile was added to 100 µL of plasma sample and vortexed for 10 min and centrifuged at 10000 rpm for 5 min. The supernatant was collected for LC-MS/MS analysis. LC-MS/MS method was used to quantify the plasma concentrations of DOX and W198 in rats. The LC-MS/MS system consisted of an Agilent 1200 series RRLC, which comprised of an SL autosampler, SL binary pump, degasser and an Agilent triple-quadrupole MS (Agilent, USA). A Diamonsil ODS column (50 × 4.6 mm, 1.8 µm) with a corresponding guard column (ODS, 5 µm) was used for separation. HPLC grade acetonitrile/water (28:72, v/v) with 0.5% (v/v) formic acid was used as the mobile phase at 30 °C with a flow rate of 0.4 mL/min. Mass spectrometry operating conditions were established to detect DOX. Triple-quadrupole mass spectroscopy was carried out under positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. Nitrogen was used as the nebulizer gas. The gas flow was 10 mL/min (350 ºC) and the pressure was set at 40 psi. Voltages of fragmentor potential were 205eV (DOX) and 112 eV (W198) and collision energy were 72 eV (DOX) and 6 eV (W198), respectively. For DOX, ion recording used the parent ion at m/z 544.2 and the daughter ion at m/z 497.2, while for W198 ion recording used the parent ion at m/z 701 and the daughter ion at m/z 174.


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In Vivo and Ex Vivo Imaging. MCF-7/ADR cells with > 90% viability were harvested from subconfluent cultures after trpsinization and 1×107 cells were inoculated subcutaneously into the right flank of female nude mice (6 – 8 weeks). The length and width of the tumor were measured every other day using a caliper, and the tumor volume was calculated using following equation: volume = (length × width2)/2. When solid tumors reached approximately 200 mm3, mice were randomly divided into five groups (n = 3): DOX solution, DOX+W198 solution, DOX-LNs and DOX/W198-LNs at an equivalent dose of 6 mg/kg DOX, and empty LNs as control. After tail vein injection of each treatment, the whole body fluorescent images were obtained through an in vivo imaging system (IVIS Spectrum, Caliper, USA) 6 h after injection. The scanning parameters included:

excitation

wavelength = 500 nm,

emission

wavelength = 600 nm,

field

of

view = 13.5 cm, and fluency rate = 2 mW/cm2. The camera was set to maximum gain, a binning factor of 4, and a luminescent exposure time of 4 s. Then, the mice were sacrificed, and their tissues were subjected to ex vivo fluorescence imaging. Freshly isolated tumor tissues were fixed with 4% paraformaldehyde in PBS, and further dehydrated using 15% (w/v) sucrose followed with 30% (w/v) sucrose. Tissue blocks were embedded and completely covered by Tissue-Tek® OCT compound (SAKURA, USA), and stored at -80 ºC. Consecutive frozen sections (10 µmthick) were prepared using a cryotome cryostat (Leica, Germany) and placed on superfrost microscope slides. Cell nuclei were stained by 0.5 µg/mL DAPI for 5 min. Tumor neovessels were stained with rabbit anti-CD34 antibody (1:200) followed with AlexaFluor 594 conjugated donkey anti-rabbit antibody. DOX, CD34, and DAPI were visualized at 488 nm, 525 nm, and 460 nm, respectively, using a laser scanning confocal microscope (LSM710, Carl Zeiss, Germany).


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Safety Evaluation. Bone marrow suppression has been one of the most severe side effects associated with DOX. To assess the level of bone marrow suppression, DOX solution, DOX+W198 solution, DOX-LNs, DOX/W198-LNs, blank LNs were administered to female Wistar rats (200 ± 20 g). The rats were randomized into 5 groups with 10 rats in each group. DOX solution, DOX+W198 solution, DOX-LNs, DOX/W198-LNs at an equivalent dose of 6 mg/kg DOX or blank LNs were given via tail vein injection. About 0.4 mL of blood samples were collected in centrifuge tubes containing EDTA-2K+ salt on either the day before administration or day 3 after administration. White blood cells (WBC) were counted by MEK6318K automated hematology analyzer (Nijon-kohden, Shinjuku-ku, Japan) as an index of bone marrow suppression15. Female Wistar rats (200 ± 20 g) were used for assessing the cardiac and gastrointestinal toxicity of DOX solution, DOX+W198 solution, DOX-LNs and DOX/W198-LNs. The rats were fasted for 12 h before administration and were randomized into 5 groups with 5 rats in each group. The control group received saline, and treatment groups received DOX solution, DOX+W198 solution, DOX-LNs, and DOX/W198-LNs via tail vein injection at an equivalent dose of 6 mg/kg DOX once every two days for 3 times, respectively. All rats were sacrificed on day 6, and heart, stomach, duodenum, jejunum, ileum, and colon samples were collected, and processed for histological analysis. Statistical Analysis. Statistical analysis were performed by Student’s t-test for comparison between two groups and one-way ANOVA with Tukey post-hoc analysis for comparing means of multiple groups using Origin 8.0 software. P values of < 0.05 were considered indications of statistically significant differences.


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RESULTS AND DISCUSSION Preparation and Characterization of DOX/W198-LNs. Recently, LNs have emerged as an attractive nanoscale carrier system for the optimized delivery of drugs via various routes of administration such as parenteral, transdermal, oral and ocular16-24. LNs are oil-in-water dispersions covering a size range of 20 – 200 nm, and the structure of LNs has been described as a neutral lipid core stabilized by amphipathic lipids thus capable of retaining hydrophobic drugs within the lipid core14. To entrap both DOX and W198 in the lipid core, DOX hydrochloride was first neutralized with bicarbonate solution (5%, w/v) to afford DOX base under mild conditions and then complexed with OA via ion pair formation to improve the lipophilicity of DOX. In addition, W198 is a hydrophobic drug which is almost insoluble in water but can be solublized in ethanol. Thus, ethanol was selected as the solvent for DOX-OA and W198 for the preparation of DOX/W198-LNs by a simple lipid film hydration-high pressure homogenization method.

Figure 2. Size distribution and morphology of DOX/W198-LNs. (A) Size distribution of DOX/W198-LNs determined by dynamic light scattering (DLS). (B) Morphology of


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DOX/W198-LNs observed by transmission electron microscopy (TEM). Scale bar represents 100 nm.

DOX/W198-LNs of different DOX to W198 feeding ratios were successfully prepared. As determined by DLS measurement, DOX/W198-LNs showed a typical size distribution ranging from 99.5 ± 7.5 to 152.6 ± 4.3 nm with polydispersity index (PDI) values of less than 0.2 for all formulation groups under investigation (Table 1). Negative zeta potentials of less than -25.0 mV were observed for all formulations. Regarding the morphology of DOX/W198-LNs, the obtained LNs displayed a near spherical shape with uniform distributions as observed by TEM (Figure 2). DOX/W198-LNs with DOX to W198 feeding ratio of 1:1 displayed the highest EE for both DOX and W198 and was thus selected for the following studies (Table 1). Specifically, the DOX and W198 EE were determined as (82.22 ± 4.76)% and (93.44 ± 5.23)%, respectively. Additionally, DOX/W198-LNs dispersions were proven to maintain their stability over a 15-day period with slightly increased mean diameters and PDI values < 0.2 (Table 2). Table 1. Characterization of DOX/W198-LNs with varying DOX to W198 feeding ratios. Data represent mean ± S.D. (n = 3). Feeding ratio Average DOX:W198 particle (w/w) Size (nm) 1:3 152.60 ± 4.30 1:2 118.80 ± 5.70 1:1 103.60 ± 4.80 2:1 145.60 ± 3.30 3:1 99.50 ± 7.50 EE, entrapment efficiency.

Polydispersity index 0.14 ± 0.07 0.16 ± 0.08 0.14 ± 0.08 0.069 ± 0.01 0.15 ± 0.09

Zeta potential (mV)

EE of DOX (%)

EE of W198 (%)

-36.30 ± 2.30 -26.60 ± 3.50 -29.90 ± 4.20 -29.00 ± 2.80 -25.00 ± 3.20

59.34 ± 5.11 78.12 ± 5.72 84.22 ± 4.76 72.83 ± 7.21 73.22 ± 6.38

81.22 ± 5.89 90.38 ± 6.77 93.44 ± 5.23 87.45 ± 6.78 83.29 ± 7.49

Table 2. Stability of DOX/W198-LNs at room temperature. Data represent mean ± S.D. (n = 5). Time (day)

1

3

7

10

15

Mean particle size (nm)

103.60±4.80

108.40±3.50

109.70±5.30

113.80±3.70

120.40±5.60

Polydispersity index

0.14±0.08

0.14±0.06

0.16 ±0.07

0.15±0.05

0.14±0.09


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Figure 3. In vitro release profiles of DOX (A) and W198 (B) from DOX+W198 solution and DOX/W198-LNs in saline containing 0.2% w/v of Tween 80. Data represent mean ± S.D. (n = 3).


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In vitro Release Kinetics. Free DOX (Figure 3A) and free W198 (Figure 3B) released very rapidly from the DOX+W198 solution with over 95% drugs released cumulatively within 8 h. By co-encapsulation of DOX and W198 in the LNs, the release kinetics of DOX/W198-LNs displayed a dual phase profile for both DOX and W198 with more than 50% drugs released by 12 h. From 12 h to 120 h, both DOX and W198 were shown to release from LNs in a prolonged release manner. However, the release kinetics of DOX from single loaded DOX-LNs displayed a significant burst phase with more than 40% drugs released in the first 2 h (Figure 3A), which is most likely due to the rapid release of surface associated DOX. The dramatic differences in the release behavior of DOX from DOX-LNs and DOX/W198-LNs are likely due to the hydrophobic interaction and π-π stacking between DOX and W198 which appeared to retain DOX in the lipid core of the nanoemulsion. The release kinetics of DOX and W198 were further fitted to several mathematical models (Table 3) to elucidate the release mechanism of DOX and W198. For the first-order and Weibull functions, one additional parameter, a, was introduced taking into account the predicted asymptotic value for release, which may differ from 100% cumulative release25. For the regression of DOX release from either DOX-LNs or DOX/W198-LNs, Weibull function provided the best regression results followed by the Korsmeyer-Peppas function. Regarding the Weibull model, the parameter c is a shape parameter and thus, c < 1 indicates a parabolic release curve. However, the parameters in the Weibull model does not carry physical meanings. Thus, the empirical Peppas model was used to gain further insights into the release mechanisms. According to the Peppas model, 0.43 < b < 0.85 indicates an anomalous transport behavior from spheres, i.e., a superposition of both Fickian diffusion and Case II transport. Regarding


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DOX/W198-LNs, the release kinetics of DOX could be interpreted as both Fickian diffusion and Case II transport driven. However, the release kinetics of DOX from DOX-LN cannot be interpreted by this empirical model, which displayed a b-value of 0.31. For the regression of W198 release profile, first-order, Higuchi, Korsmeyer-Peppas and Weibull functions all achieved comparable level of performance. Based on the Peppas model, the release of W198 from DOX/W198-LNs was driven by both Fickian diffusion and Case II transport. Table 3. Regression parameters of DOX and W198 release behaviors from DOX-LNs and DOX/W198LNs via mathematical modeling. Data represent mean ± S.D. (n = 3). Function

Formulation

API

a

b

c

R2

DOX-LNs

DOX

3.65±1.02

–

–

0.59601

DOX

1.65±0.22

–

–

0.85017

W198

3.46±0.61

–

–

0.79851

DOX

68.70±8.27

0.54±0.19

–

0.72199

DOX

71.16±8.04

0.08±0.02

–

0.84662

W198

61.07±4.72

0.21±0.04

–

0.9618

DOX

19.48±1.98

–

–

0.66075

DOX

10.68±0.43

–

–

0.95025

W198

13.96±0.68

–

–

0.96621

DOX

30.96±2.32

0.31±0.03

–

0.93598

DOX

12.35±1.75

0.46±0.04

–

0.95054

W198

13.08±0.68

0.53±0.04

–

0.96517

DOX

119.10±2.51

0.06±0.13

0.44±0.12

0.93438

DOX

65.79±15.68

0.07±0.05

0.62±0.10

0.96663

W198

84.57±28.60

0.08±0.06

0.69±0.02

0.97148

Zero order: y = a·t

DOX/W198-LNs DOX-LNs

First order: y = a·(-exp(-b·t))


Higuchi: y = a·t0.5


Korsmeyer-Peppas: y = a·tb


Weibull: y = a·(1-exp(-b·tc)

DOX/W198-LNs

API, active pharmaceutical ingredient; –, not available.


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Figure 4. Viability of MCF-7/ADR (A) and MCF-7 (B) cells after 48 h of treatment of DOX solution, DOX+W198 solution, DOX-LN, DOX/W198-LNs, and blank LNs as control. Data represent mean ± S.D. (n = 3). Table 4. IC50 values of free DOX solution, free DOX+W198, DOX-LNs and DOX/W198-LNs on different cell lines. Data represent mean ± S.D. (n = 3) IC50 (µg/mL) DOX

DOX+W198

DOX-LNs

DOX/W198-LNs

MCF-7/ADR

> 100

3.4 ±0.12

> 100

1.2±0.27&

MCF-7

4.67±0.13

4.15±0.22

1.35±0.18*&

1.7±0.15*&

*, p < 0.05 vs. DOX solution group; ＆, p < 0.05 vs. DOX+W198 solution group.

Cytotoxicity. At the same concentration level, DOX+W198 and DOX/W198-LNs exhibited much greater inhibitory effect than DOX solution and DOX-LNs in MCF-7/ADR cells after 48 h of treatment (Figure 4A). Specifically, the 50% inhibitory concentration (IC50) values for DOX solution and DOX-LNs were above 100 µg/mL in MCF-7/ADR cells, while the IC50 values for DOX+W198 solution and DOX/W198-LNs were 3.4 ± 0.12 and 1.2 ± 0.27 µg/mL, respectively (Table 4). Thus, the presence of P-gp inhibitor, W198, successfully reversed MDR in MCF7/ADR cells. In contrast, free DOX, DOX+W198, DOX-LNs and DOX/W198-LNs showed


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concentration-dependent responses in MCF-7 cells which were not multidrug resistant (Figure 4B). Also, no statistical differences were observed between free DOX and DOX+W198 solution (p > 0.05) or between DOX-LNs and DOX/W198-LNs (p > 0.05) in MCF-7 cells, indicating the presence of W198 did not affect the efficacy or IC50 values of DOX in sensitive MCF-7 cells which is consistent with previous findings6. Regarding the effect of nanoscale carriers, drug loaded LNs appeared to be more potent against MCF-7 cells than drug solutions, which was likely due to the enhanced cellular uptake efficiency of LNs as discussed in the cell uptake section. In addition, blank LNs displayed no obvious cytotoxicity against MCF-7 and MCF7/ADR cells with relative cell viability around 100% at all concentrations under investigation, which indicated the excipients used to fabricate the LNs were of minimum cytotoxicity. Cellular Uptake. The cellular uptake study of different DOX formulations was conducted in both MCF-7/ADR and MCF-7 cells. In MCF-7/ADR cells, FACS analysis showed the intracellular DOX fluorescence intensity increased dramatically over time for DOX+W198 and DOX/W198-LNs groups while remaining slightly increased for DOX solution and DOX-LNs groups (Figure 5A), which suggests the presence of W198 efficiently reversed MDR by inhibiting P-gp and facilitating the cellular uptake of DOX in MCF-7/ADR cells. In comparison, all investigated treatment groups displayed an increasing trend over time in the cellular uptake efficiency in sensitive MCF-7 cells (Figure 5B). Interestingly, no significant differences were observed either between DOX solution and DOX+W198 solution or between DOX-LNs and DOX/W198-LNs (p > 0.05), indicating the presence of W198 did not impact the cellular uptake of DOX in sensitive MCF-7 cells. However, drug loaded LNs displayed significantly higher cellular uptake efficiency than the solution counterpart at each indicated time point in MCF-7 cells (p < 0.05). Thus, LNs were well proven to be more efficient in facilitating the


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internalization of therapeutics into cytoplasm possibly via endocytosis, while DOX or DOX+W198 solution was uptaken by MCF-7 cells mainly through the passive diffusion process.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Figure 5. Cellular uptake behavior of DOX solution, DOX+W198 solution, DOX-LNs, and DOX/W198-LNs in MCF-7/ADR and MCF-7 cells at 1, 2, and 4 h. Cell uptake efficiency in MCF-7/ADR (A) and MCF-7 cells (B) were quantified by FACS analysis. CLSM images of MCF-7/ADR cells (C) and MCF-7 cells (D) with cell nuclei stained with DAPI (blue) and DOX fluorescence displayed in red. Scale bar represents 50 µm. MCF-7/ADR: *, p < 0.01 vs. DOX group; $, p < 0.05 vs. DOX+W198 group; &, p < 0.01 vs. DOX group. MCF-7: **, p < 0.01 vs. DOX group; $$, p < 0.01 vs. DOX+W198 group. Consistent with quantitative analytical results, both DOX+W198 solution and DOX/W198-LNs groups displayed intracellular distribution of DOX and the DOX fluorescence intensity increased dramatically over time in MCF-7/ADR cells, while in the DOX solution treated group the fluorescence intensity remained extremely weak and no nucleus distribution was detectable by 4 h (Figure 5C). In DOX-LNs treated groups, red fluorescence was observed in the cytoplasm but not in the nucleus by 4 h in MCF-7/ADR cells (Figure 5C), which indicates DOX-LNs may partly bypass P-gp efflux pumps associated with cell surface. However, intracellularly released DOX could not reach cell nucleus due to the presence of efflux pumps on the nucleus membrane and the cell surface. In contrast, a cross comparison showed all DOX formulations were successfully uptaken and displayed nucleus distribution in sensitive MCF-7 cells (Figure 5D). Cell Apoptosis. To investigate the effect of DOX/W198-LNs on cell apoptosis, we quantified the proportion of apoptotic and necrotic cells by annexin V-FITC and PI double staining. MCF7/ADR and MCF-7 cells were further stained by DAPI to evaluate the morphological changes of cell nuclei. In Figure 6C, DOX/W198-LNs induced a significantly higher apoptotic rate compared with free DOX and DOX-LNs (p < 0.05) in MCF-7/ADR cells. The apoptotic rate of MCF-7/ADR cells treated with DOX+W198 solution increased by 20-fold compared to free


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DOX, while the apoptotic rate of cells treated by DOX/W198-LNs increased by 30-fold. As for MCF-7 cells, drug loaded LNs induced a much higher apoptotic rate compared with free DOX and DOX+W198 solution (Figure 6D). However, the apoptotic rate did not show significant differences between DOX-LNs and DOX/W198-LNs (p > 0.05) in MCF-7 cells despite that the necrotic rate of DOX-LNs was significantly higher than DOX/W198-LNs (Figure 6D). Regarding the nuclei morphology, the nuclei in the negative control groups remained in the regular round shape with homogeneous chromatin distributions in both MCF-7/ADR and MCF-7 cells (Figure 6E). In contrast, chromatin condensation and nuclear fragmentation were observed in DOX+W198 and DOX/W198-LNs treated MCF-7/ADR groups (Figure 6E), which indicates the formation of apoptotic bodies.


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Figure 6. Cell apoptosis and necrosis were analyzed by flow cytometry using Annexin V-FITC in combination with PI in MCF-7/ADR (A) and MCF-7 cells (B). The quantification of apoptotic


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and necrotic cell percentages after treatment with different formulations in MCF-7/ADR (C) and MCF-7 cells (D). Microscopic images of DAPI staining of fragmented chromatin and apoptotic bodies (E). MCF-7/ADR: *, p < 0.01 vs. DOX group; $, p < 0.01 vs. DOX+W198 group; MCF7: **, p < 0.01 vs. DOX group; $$, p < 0.01 vs. DOX+W198 group.

Figure 7. Plasma concentration-time profiles of (A) W198

and (B) DOX following i.v.

administration of DOX+W198 solution and DOX/W198-LNs at an equivalent dose of 6 mg/kg of DOX or W198. Data represent mean ± S.D. (n = 6).


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Table 5. Summary of pharmacokinetic parameters. Pharmacokinetic parameters were calculated for DOX and W198 after intravenous injection of DOX+W198 solution and DOX/W198-LNs in rats. Data represent mean ± S.D. (n = 6). (i) Pharmacokinetic parameters of DOX Parameters

DOX+W198 solution

DOX/W198-LNs

AUC0→24h (µg/L·min)

384.51 ± 49.98

1349.27 ± 195.10*

MRT (min)

2.43 ± 0.74

3.36 ± 2.36

CL (L/min/Kg)

71.17 ± 18.68

26.13 ± 9.86*

t1/2α (min)

1.224 ± 1.008

1.116 ± 1.349

t1/2β (min)

1.642 ± 1.334

3.36 ± 1.65*

Cmax (µg/L)

38.90 ± 12.99

83.27 ± 37.96*

Parameters

DOX+W198 solution

DOX/W198-LNs

AUC0→24h (µg/L·min)

9425.77 ± 1211.65

9824.43 ± 15.38

MRT (min)

400.11 ± 81.18

525.61 ± 142.59

CL (L/min/Kg)

2.54 ± 1.19

1.76 ± 0.44

t1/2α (min)

1.5364 ± 0.83

4.043 ± 1.169*

t1/2β (min)

10.893 ± 2.76

8.792 ± 2.655

Cmax (µg/L)

1.76 ± 0.44

2.54 ± 1.19*

＆

(ii) Pharmacokinetic parameters of W198

＆

*, p < 0.01 vs. DOX+W198 solution；&, p < 0.05 vs. DOX+W198 solution.

Pharmacokinetics. Intravenous injection of DOX+W198 solution resulted in the rapid clearance of both DOX and W198 (Figure 7). The intravenous injection of DOX/W198-LNs led to dramatically higher plasma concentrations of both DOX and W198 than the DOX+W198 solution at early time points. The concentration-time profiles of both DOX and W198 were fitted by the two-compartment pharmacokinetic model. The comparative pharmacokinetic parameters of DOX and W198 were summarized in Table 3. Specifically, DOX showed a MRT of 2.43 ±


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0.74 min and a clearance rate at 71.17 ± 18.68 L/min/Kg in the solution group, while DOX displayed a slightly higher MRT of 3.36 ± 2.36 min and a significantly lower clearance rate at 26.13 ± 9.86 L/min/Kg (p < 0.05) in the DOX/W198-LNs group. DOX/W198-LNs also displayed a significantly higher t1/2β for DOX than DOX+W198 solution group (p < 0.05) indicating a possible sustained therapeutic effect in rats. For the DOX/W198-LNs group, CmaxDOX displayed

a near two-fold increase than that of the solution, while Cmax-W198 showed a 2.75-

fold increase than that of the solution. The AUCDOX from DOX/W198-LNs group was about 3.5fold higher than that of the solution group (1349.27 ± 195.10 µg/L·min vs. 384.51 ± 49.98 µg/L·min) (p < 0.05), which is most likely due to the slower release of DOX from LNs than from solution group. In vivo and ex vivo Imaging. DOX has been used as the fluorescence probe to semiquantitatively study the biodistribution of DOX formulations by ex vivo imaging26-28. In our study, whole body fluorescence imaging was performed in MCF-7/ADR-bearing nude mice at 6 h after intravenous injection of DOX solution, DOX+W198 solution, DOX-LNs and DOX/W198-LNs. Among four treatment groups, extensive distribution of DOX throughout the mice body was observed compared with control (Figure 8A), and dramatic accumulation of DOX in liver and kidney was also observed by ex vivo imaging of excised organs (Figure 8B), which indicates either the co-administration of W198 with DOX or the carrier system did not change the overall biodistribution profile of DOX in mice. Regarding DOX distribution in tumor tissues, DOX/W198-LNs displayed remarkably higher accumulation of DOX than the other three treatment groups (Figure 8C), which is likely due to both the inhibitory effect of W198 on P-gp and the EPR effect of nanoscale carriers. Ex vivo imaging of control animals injected with vehicle produced weak background signal (Figure 8). The semi-quantitation results of


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fluorescence intensity revealed that DOX/W198-LNs achieved enhanced intra-tumor distribution despite the non-specific distribution of DOX in liver and kidney (Figure 8D and 8E). The distribution of DOX in liver and kidney was likely due to free DOX released from DOX/W198LNs. Moreover, biliary and urinary excretion are the major routes of elimination for DOX thus resulting in dramatic accumulation of DOX in liver and kidney29. Despite the lack of difference in biodistribution between DOX-LNs and DOX/W198-LNs, DOX/W198-LNs showed better in vivo antitumor efficacy in the MCF-7/ADR xenograft mice model than DOX solution and DOXLNs in our preliminary in vivo study (Figure S1). Additional evidence for enhanced uptake of DOX at solid tumors was obtained from resected tumor tissues (Figure 9). In the DOX/W198LN group, DOX was readily observed as green dots extensively distributed throughout the tumor tissue, while DOX solution group displayed less obvious distribution of DOX within the tumor tissue. DOX+W198 solution group and DOX-LN group displayed comparable level of DOX uptake in the tumor tissue, which further emphasized the critical roles of both P-gp inhibitors and nanoscale carriers in reversing tumor MDR. P-gp inhibitors, which specifically interact with Pgp, can reverse MDR effect and increase drug uptake in tumor cells. Nanoscale carriers can be endocytosed by tumor cells thus bypassing efflux pumps which also results in enhanced cellular uptake. CD-34 as an indicator for neovasculature within solid tumors did not show drastic differences among treatment groups (Figure 9).


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Figure 8. Biodistribution of DOX in MCF-7/ADR-bearing mice, 6 h after the injection of control (empty LNs), DOX solution, DOX+W198 solution, DOX-LNs, and DOX/W198-LNs. (A) Whole body fluorescent images of MCF-7/ADR-bearing mice. (B, C) Representative fluorescent images of excised organs and tumors after termination of treatments. (D, E) Semi-quantitative fluorescent intensity of excised organs and tumors. *, p < 0.05 vs. control.


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Figure 9. Laser scanning confocal microscopy images of tumor tissue sections. DOX is green. CD34 is red, representing neovessels. Cell nuclei are in blue (DAPI). Scale bar represents 100 µm.


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Toxicity. The suppression of bone marrow has been considered one of the most common side effects associated with intravenously administered DOX solution. To investigate the effect of bone marrow suppression, the number of WBC was quantified 3 days after drug administration. Changes in WBC count may reflect the occurrence of bone marrow suppression and the abnormality in the immune system. As shown in Figure 10, all treatment groups induced a significant reduction in WBC count compared to the WBC count before treatment indicating a strong bone marrow suppression effect of DOX. The control group (blank LNs) also displayed a minor inhibitory effect (~10% inhibition ratio) three days after treatment. DOX/W198-LNs and DOX-LNs treated groups displayed much higher WBC counts than that of free DOX and DOX+W198 groups, for example, DOX-LNs and DOX/W198-LNs displayed the WBC inhibition ratio of

(50 ± 3)% and (47 ± 4)% respectively, which indicates encapsulating

cytotoxic chemotherapeutics in LNs may help reduce the systemic toxicity in vivo.

Figure 10. (A) WBC cell count of free DOX, free DOX+W198, DOX-NP, DOX/W198-NP and free NP (control) groups before and three days after treatment. (B) WBC inhibition ratio. Each value represents the mean ± SD (n = 10). *, p < 0.01 vs. DOX group; $, p < 0.01 vs. DOX+W198 group; ＆, p < 0.05 vs. DOX group.


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A common problem of antitumor drugs in clinic is the injuries to normal tissues, which leads to multiple organ toxicity. DOX was reported to result in dose-limiting systemic toxicities such as congestive heart failure and gastrointestinal toxicity when administered in clinic30-32. Thus, it is critical to evaluating the damages of vital organs caused by DOX/W198-LNs and to further elucidate if the presence of W198 would increase cardiac and GI tract toxicity. As shown in Figure 11, both DOX solution and DOX+W198 solution displayed severe cardiac toxicity as observed by hyperemia, myocardial fiber breakage and necrosis. In contrast, the histological sections from DOX-LNs and DOX/W198-LNs treated groups did not exhibit obvious signs of cardiac toxicity and no necrosis, hyperemia or inflammation were visible (Figure 11). Therefore, LNs were proven effective in alleviating cardiac toxicity induced by free DOX. To confirm whether DOX, DOX+W198, DOX-LNs, DOX/W198-LNs treatment changes the histological structure of rat GI tracts, histological samples of stomachs and different intestinal sections were collected and processed to investigate gastrointestinal injury. In Fig. 9, DOX solution and DOX+W198 solution treated groups displayed gastric mucous atrophy, epithelial atrophy, intestinal expansion, intestinal villus congestion, intestinal villus atrophy, and a large quantity of cell debris in duodenum, jejunum, ileum, and colon. Intestinal epithelial cell degeneration was visible in the DOX-LNs and DOX/W198-LNs treated groups, but appeared to be much less obvious as compared with DOX solution and DOX+W198 solution treated groups. Overall, DOX/W198-LNs were proven to result in reduced cardiac toxicity and gastrointestinal injury.


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Figure 11. Heart and gastrointestinal morphology of DOX solution, DOX+W198 solution, DOXNP and DOX/W198-NP treated groups. Heart and gastrointestinal sections were isolated and stained with hematoxylin and eosin (H&E) for histopathological analysis. Scale bar represents 100 µm. These are representative sections from five rats analyzed for each condition.


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CONCLUSIONS In summary, co-encapsulating DOX and W198 in DOX/W198-LNs was demonstrated to successfully reverse MDR in MCF-7/ADR cells with significantly increased DOX cytotoxicity. Based on the inhibitory activity of W198 against P-gp and the nanoscale LN system which bypass P-gp efflux via endocytosis, DOX/W198-LNs achieved enhanced intracellular uptake of DOX in MCF-7/ADR cells and intratumoral distribution of DOX in a mice xenograft model. Moreover, DOX/W198-LNs did not result in severe cardiac and GI tract toxicity. Therefore, concurrent loading of DOX and W198 in LNs represents a promising strategy to deliver cytotoxic chemotherapeutics in MDR tumors with improved efficacy and safety.

ASSOCIATED CONTENT Supporting Information In vivo antitumor efficacy of DOX formulations in MCF-7/ADR tumor bearing mice. This material is free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *(Y.F.) Phone: 86-28-85503798. E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT


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The authors are grateful for the financial support from the National Natural Science Foundation of China (81273443) and Sichuan University Startup Foundation for Talents (2082204174131). The authors would also like to thank Mr. Shaobo Ruan for assistance with in vivo imaging analysis.

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12. Gottesman, M. M. Mechanisms of cancer drug resistance. Annu. Rev. Med. 2002, 53, (1), 615-627. 13. Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56, (2), 185-229. 14. Zhang, X.; Sun, X.; Li, J.; Zhang, X.; Gong, T.; Zhang, Z. Lipid nanoemulsions loaded with doxorubicin-oleic acid ionic complex: characterization, in vitro and in vivo studies. Die Pharmazie-An International Journal of Pharmaceutical Sciences 2011, 66, (7), 496-505. 15. Zhou, J.; Zhang, X.; Li, M.; Wu, W.; Sun, X.; Zhang, L.; Gong, T. Novel lipid hybrid albumin nanoparticle greatly lowered toxicity of pirarubicin. Mol. Pharm. 2013, 10, (10), 38323841. 16. Santos-Magalhães, N.; Pontes, A.; Pereira, V.; Caetano, M. Colloidal carriers for benzathine penicillin G: nanoemulsions and nanocapsules. Int. J. Pharm. 2000, 208, (1-2), 7180. 17. Zhao, D.; Gong, T.; Fu, Y.; Nie, Y.; He, L.-L.; Liu, J.; Zhang, Z.-R. Lyophilized Cheliensisin A submicron emulsion for intravenous injection: Characterization, in vitro and in vivo antitumor effect. Int. J. Pharm. 2008, 357, (1), 139-147. 18. Sonneville-Aubrun, O.; Simonnet, J.-T.; L'alloret, F. Nanoemulsions: a new vehicle for skincare products. Adv. Colloid Interfac. 2004, 108, 145-149. 19. Yilmaz, E.; Borchert, H.-H. Design of a phytosphingosine-containing, positivelycharged nanoemulsion as a colloidal carrier system for dermal application of ceramides. Eur. J. Pharm. Biopharm. 2005, 60, (1), 91-98. 20. Mou, D.; Chen, H.; Du, D.; Mao, C.; Wan, J.; Xu, H.; Yang, X. Hydrogel-thickened nanoemulsion system for topical delivery of lipophilic drugs. Int. J. Pharm. 2008, 353, (1), 270276. 21. Singh, K. K.; Vingkar, S. K. Formulation, antimalarial activity and biodistribution of oral lipid nanoemulsion of primaquine. Int. J. Pharm. 2008, 347, (1), 136-143. 22. Vyas, T. K.; Shahiwala, A.; Amiji, M. M. Improved oral bioavailability and brain transport of Saquinavir upon administration in novel nanoemulsion formulations. Int. J. Pharm. 2008, 347, (1), 93-101. 23. Bali, V.; Ali, M.; Ali, J. Study of surfactant combinations and development of a novel nanoemulsion for minimising variations in bioavailability of ezetimibe. Colloid. Surface. B. 2010, 76, (2), 410-420. 24. Sznitowska, M.; Zurowska-Pryczkowska, K.; Janicki, S.; Järvinen, T. Miotic effect and irritation potential of pilocarpine prodrug incorporated into a submicron emulsion vehicle. Int. J. Pharm. 1999, 184, (1), 115-120. 25. Costa, F.; Sousa, J.; Pais, A.; Formosinho, S. Comparison of dissolution profiles of ibuprofen pellets. J. Control. Release 2003, 89, (2), 199-212. 26. Lv, S.; Tang, Z.; Li, M.; Lin, J.; Song, W.; Liu, H.; Huang, Y.; Zhang, Y.; Chen, X. Codelivery of doxorubicin and paclitaxel by PEG-polypeptide nanovehicle for the treatment of nonsmall cell lung cancer. Biomaterials 2014, 35, (23), 6118-29. 27. Li, N.; Yi, Q.; Luo, K.; Guo, C.; Pan, D.; Gu, Z. Amphiphilic peptide dendritic copolymer-doxorubicin nanoscale conjugate self-assembled to enzyme-responsive anti-cancer agent. Biomaterials 2014, 35, (35), 9529-45. 28. Lee, H. J.; Liu, Y.; Zhao, J.; Zhou, M.; Bouchard, R. R.; Mitcham, T.; Wallace, M.; Stafford, R. J.; Li, C.; Gupta, S.; Melancon, M. P. In vitro and in vivo mapping of drug release


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For Table of Contents Use Only

Title: Co-encapsulated Doxorubicin and Bromotetrandrine Lipid Nanoemulsions in Reversing Multidrug Resistance in Breast Cancer In Vitro and In Vivo

Xi Cao, Jingwen Luo, Tao Gong, Zhi-Rong Zhang, Xun Sun, and Yao Fu*

TABLE OF CONTENTS GRAPHIC


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Figure 1. Chemical structures of (A) tetrandrine and (B) bromotetrandrine (W198). 124x53mm (300 x 300 DPI)



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Figure 2. Size distribution and morphology of DOX/W198-NP. (A) Size distribution of DOX/W198-NP determined by dynamic light scattering (DLS). (B) Morphology of DOX/W198-NP observed by transmission electron microscopy (TEM). Scale bar represents 100 nm. 70x82mm (300 x 300 DPI)


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Figure 3. In vitro release profiles of DOX (A) and W198 (B) from DOX+W198 solution and DOX/W198-LN in saline containing 0.2% w/v of Tween 80. Data represent mean ± S.D. (n = 3). 71x107mm (300 x 300 DPI)



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Figure 4. Viability of MCF-7/ADR (A) and MCF-7 (B) cells after 48 h of treatment of DOX solution, DOX+W198 solution, DOX-LN, DOX/W198-LN, and blank LN as control. Data represent mean ± S.D. (n = 3). 126x50mm (300 x 300 DPI)


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Figure 5. Cellular uptake behavior of DOX solution, DOX+W198 solution, DOX-LNs, and DOX/W198-LNs in MCF-7/ADR and MCF-7 cells at 1, 2, and 4 h. Cell uptake efficiency in MCF-7/ADR (A) and MCF-7 cells (B) were quantified by FACS analysis. CLSM images of MCF-7/ADR cells (C) and MCF-7 cells (D) with cell nuclei stained with DAPI (blue) and DOX fluorescence displayed in red. Scale bar represents 50 µm. MCF-7/ADR: *, p < 0.01 vs. DOX group; $, p < 0.05 vs. DOX+W198 group; &, p < 0.01 vs. DOX group. MCF-7: **, p < 0.01 vs. DOX group; $$, p < 0.01 vs. DOX+W198 group. 195x267mm (300 x 300 DPI)



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Figure 6. Cell apoptosis and necrosis were analyzed by flow cytometry using Annexin V-FITC in combination with PI in MCF-7/ADR (A) and MCF-7 cells (B). The quantification of apoptotic and necrotic cell percentages after treatment with different formulations in MCF-7/ADR (C) and MCF-7 cells (D). Microscopic images of DAPI staining of fragmented chromatin and apoptotic bodies (E). MCF-7/ADR: *, p < 0.01 vs. DOX group; $, p < 0.01 vs. DOX+W198 group; MCF-7: **, p < 0.01 vs. DOX group; $$, p < 0.01 vs. DOX+W198 group. 160x205mm (300 x 300 DPI)


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Figure 7. Plasma concentration-time profiles of W198 (A) and DOX (B) following i.v. administration of DOX+W198 solution and DOX/W198-NP at an equivalent dose of 6 mg/kg of DOX or W198. Data represent mean ± S.D. (n = 6). 84x113mm (300 x 300 DPI)



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Figure 8. Biodistribution of DOX in MCF-7/ADR-bearing mice, 6 h after the injection of control (empty LNs), DOX solution, DOX+W198 solution, DOX-LNs, and DOX/W198-LNs. (A) Whole body fluorescent images of MCF-7/ADR-bearing mice. (B, C) Representative fluorescent images of excised organs and tumors after termination of treatments. (D, E) Semi-quantitative fluorescent intensity of excised organs and tumors. *, p < 0.05 vs. control. 85x94mm (300 x 300 DPI)


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Figure 9. Laser scanning confocal microscopy images of tumor tissue sections. DOX is green. CD34 is red, representing neovessels. Cell nuclei are in blue (DAPI). Scale bar represents 100 µm. 86x115mm (300 x 300 DPI)



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Figure 10. WBC cell count of free DOX, free DOX+W198, DOX-NP, DOX/W198-NP and free NP (control) groups before and three days after treatment (A). WBC inhibition ratio three days after treatment compared to before treatment (B). Each value represents the mean ± SD (n = 10). *, p < 0.01 vs. DOX group; $, p < 0.01 vs. DOX+W198 group; &, p < 0.05 vs. DOX group. 139x59mm (300 x 300 DPI)


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Figure 11. Heart and gastrointestinal morphology of DOX solution, DOX+W198 solution, DOX-NP and DOX/W198-NP treated groups. Heart and gastrointestinal sections were isolated and stained with hematoxylin and eosin (H&E) for histopathological analysis. Scale bar represents 100 µm. These are representative sections from five rats analyzed for each condition. 157x146mm (300 x 300 DPI)



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For Table of Contents Use Only Title: Co-encapsulated Doxorubicin and Bromotetrandrine Lipid Nanoemulsions in Reversing Multidrug Resistance in Breast Cancer In Vitro and In Vivo Authors: Xi Cao, Jingwen Luo, Tao Gong, Zhi-Rong Zhang, Xun Sun, and Yao Fu* 86x34mm (300 x 300 DPI)


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Coencapsulated Doxorubicin and Bromotetrandrine Lipid

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