PEG-Stabilized Bilayer Nanodisks As Carriers for Doxorubicin Delivery

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PEG-Stabilized Bilayer Nanodisks As Carriers for Doxorubicin Delivery Wenping Zhang,†,‡ Jin Sun,*,† Yan Liu,† Mengying Tao,† Xiaoyu Ai,† Xiaonan Su,† Cuifang Cai,† Yilin Tang,†,§ Zhi Feng,† Xiaodan Yan,† Guoliang Chen,# and Zhonggui He*,† †

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016, P. R. China ‡ Institute of Clinical Pharmacology, Department of Pharmacy, General Hospital of Ningxia Medical University, No. 804 Shengli Street, Yinchuan 750004, P. R. China § School of Materials Science and Engineering, Xi’an Jiaotong University, Xi’an 710019, P. R. China # Key Laboratory of Structure Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016, P. R. China S Supporting Information *

ABSTRACT: Spherical nanoparticles as a classic delivery vehicle for anticancer drugs have been extensively investigated, but study on the shape of nanoparticles has received little attention until now. Here, a nonspherical poly(ethylene glycol) (PEG)-stabilized bilayer nanodisk consisting of 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC) and PEG5000-glyceryl distearate (PEG5K-GCDS) was prepared for doxorubicin delivery, called DOX-Disks. The prepared disks were open bilayer structures, with a hydrophobic discoid center built by DSPC and a hydrophilic PEG edge. Mean particle diameter of the disk was 80.14 nm, and the disk height was about 6 nm with aspect ratio about 12. Encapsulation efficiency of DOX-Disks was as high as 96.1%, and DOX release from DOX-Disks was pH-dependent (25.6% of total DOX released at 24 h in pH 7.4). The pharmacokinetic performances showed that DOX-Disks demonstrated long circulation time in blood and larger AUC (11.7-fold of t1/2 and 31.7-fold of AUC) in rats compared with DOX solutions (DOX-Sol). Tissue distribution in H22 tumor bearing mice demonstrated higher tumor accumulation (9.7-fold) and lower heart toxicities (25.7-fold) at 48 h after iv administration, in comparison with DOX-Sol. In addition, DOX-Disks exhibited much effectiveness in inhibiting tumor cell growth, and the IC50 values were 2.03, 0.85, and 0.86 μg/mL for DOX-Sol and 0.23, 0.24, and 0.20 μg/mL for DOX-Disks after treatment for 48, 72, and 96 h against MCF-7/Adr cells, respectively. DOX-Disks were taken up into MCF-7/Adr cells via energy-dependent endocytosis processes, involved in clathrin-mediated, macropinocytosis-mediated, and non-clathrin- and non-caveolae-mediated endocytosis pathways. In summary, such PEG-stabilized bilayer nanodisks could be one of the promising carriers for antitumor drugs via extended blood circulation and improved tumor distribution. KEYWORDS: drug delivery, bilayer nanodisks, doxorubicin, shape

1. INTRODUCTION Nanoparticles are helping address a variety of challenges faced by the delivery of modern as well as conventional anticancer drugs, due to the distinct advantages of spatial targeted location of nanoparticles and temporal sequencing on drug release. Design, research, and development of novel nanocarriers have always been a hot topic for anticancer drug delivery.1,2 The impact of size and surface chemistry on biodistribution and cellular internalization of nanoparticles has been elucidated to a great extent using spherically shaped particles.3−6 However, the importance of nanoparticle shape has only been recognized for drug delivery in recent years.7−11 Several studies have investigated how the shape of nanocarriers influences in vivo pharmacokinetic behavior and cellular internalization. Geng et al.12 found that the cylindrically shaped filamentous micelles (filomicelles, width 22−60 nm, length 8−18 mm) had persistent blood circulation for up to 1 week after intravenous injection, much longer than their spherical counterparts. A © 2014 American Chemical Society

study on the cellular localization of discoid and spherical particles showed that remarkable reduction of cellular internalization was observed to disks relative to spheres despite of the two particles sharing the same endocytosis pathway.13 Wormshaped polystyrene particles showed reduced phagocyosis compared with the spherical ones with the same volume.14 Moreover, wormlike micelles could increase the loaded dose of water-insoluble drugs such as paclitaxel and docetaxel.15 Further studies revealed that the needle-shaped particles induced disruption of cell membranes as indicated by the Special Issue: Recent Molecular Pharmaceutical Development in China Received: Revised: Accepted: Published: 3279

September 21, 2013 March 28, 2014 April 22, 2014 April 22, 2014 dx.doi.org/10.1021/mp400566a | Mol. Pharmaceutics 2014, 11, 3279−3290

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nanodisks were obtained by extrusion through a membrane of pore size 220 nm. Doxorubicin loaded bilayer nanodisks (DOX-Disks) were prepared by dissolution of DSPC/PEG5K-GCDS (75:25) in chloroform, addition of 2 mL of doxorubicin (1 mg/mL) in methanol (molar ratio of doxorubicin:triethylamine = 1:3), and mixing for 1 h. The rest of the procedure is the same as that for the blank disks. 2.2.2. Preparation of DOX-Loaded Micelles. As much as 200 mg of PEG5000-GCDS was dissolved in chloroform, and 10 mL of DOX (1 mg/mL) was added in methanol (stoichiometric molar ratio of doxorubicin:triethylamine = 1:3), followed by mixing for 1 h. The organic solvents were removed using a rotary evaporator to form the drug-containing lipid film and then hydrated with 5 mL of HBS buffer (10 mM HEPES buffered saline, pH 7.4) at 37 °C for 4 h. The DOX -loaded nanoassemblies (DOX-NAs) were obtained after extrusion through a membrane of pore size 220 nm. The final concentration of doxorubicin in micelles was diluted to 1.5 mg/mL as determined by HPLC−UV at 258 nm. The average size and zeta potential of the nanomicelles were determined by a Zetasizer 5000 (Malvern Instruments, Malvern Worcestershire, U.K.). All measurements were carried out in triplicate. The morphology of DOX-NAs was examined using transmission electron microscopy (H-600, Hitachi, Japan). A drop of nanoparticle suspension was visualized after staining with 2% (w/v) phosphotungstic acid for 30 s on a copper grid under TEM. 2.3. Physicochemical Characteristics of DOX-Loaded Bilayer Nanodisks (DOX-Disks). The morphology of blank disks and DOX-Disks was examined using transmission electron microscopy (H-600, Hitachi, Japan). A drop of nanoparticle suspension was visualized after staining with 2% (w/v) phosphotungstic acid for 30 s on a copper grid under TEM. Average size and zeta potential of DOX-Disks were determined by a Zetasizer 5000 (Malvern Instruments, Malvern Worcestershire, U.K.). All the analyses were carried out in triplicate. Encapsulation efficiency of DOX-Disks was determined applying the Sephadex G50 microcolumn centrifugation method. To separate the loaded DOX in micelles and free DOX, 0.5 mL of micelles in water with DOX concentration of 0.5 mg/mL was loaded into the Sephadex microcolumn and eluted by 1 mL of distilled water several times under pressure. The eluted fractions containing DOX-Disks and free DOX were collected respectively for determination of DOX by HPLC−UV at 258 nm. After filtration by 0.45 μm filter membrane, an aliquot of 20 μL of filtrate was analyzed by a validated HPLC−UV method for determination of DOX concentration. Chromatographic separation was carried out on a Diamonsil C18 column (200 mm × 4.6 mm, 5 μm; Dikma Technologies, China) using methanol−ammonium acetate (5 mM)−acetic acid (60:40:0.1, v/v/v) as mobile phase at detection wavelength of 258 nm. 2.4. In Vitro Release of DOX from DOX-Disks. Release of DOX from DOX-Disks was determined using a dialysis bag technique in different phosphate buffer saline or acetate buffer (pH 7.4, pH 6.8, pH 5.5, and pH 4.5). An aliquot of 1 mL of nanomicelles in water with DOX concentration of 1.0 mg/mL was sealed in a dialysis tube (molecular weight cutoff 14,000 Da) and immersed in 150 mL of preheated release medium. The release was conducted in an incubator shaker set at 100

release of lactate dehydrogenase and uptake of extracellular calcein.16 In summary, the shape of nanoparticles does have a great effect on the pharmacokinetic and pharmacodynamic performances of nanomedicine for anticancer drug delivery. Compared with extensive research focused on particle size and surface chemistry of nanoparticles, there is still a lacking of studies on understanding the effect of particle shape on the in vivo pharmacokinetic behavior and cellular internalization. Recently, a promising nonspherical vehicle, PEG-stabilized bilayer nanodisk, formed by carefully adjusted amounts of PEGsubstituted lipids into a mixture of phospholipids and cholesterol has received significant attention.17 The ordered hydrophilic and hydrophobic regions of PEG-stabilized bilayer nanodisk exhibited better biomembrane-mimetic properties. They are commonly used as model biomembranes to study drug partitioning into biomembrane and structure/function of membrane-associated proteins18,19 and introduced as a pseudostationary phase in electrokinetic chromatography.20−22 They are characterized by excellent long-term stability, controllable size, and biocompatible components. Noteworthy, such desired nanodisks remained silent regarding applications for drug delivery carriers. But just recently, the nanodisks were reported as a carrier for antimicrobial peptides, which showed a significant cell-killing effect upon a second exposure to bacteria due to an extended release of peptide from the lipid disks.23 In consideration of distinct characteristics of PEG-stabilized bilayer nanodisks and no appreciation of efficacy of the nanodisks in drug targeted delivery, the novel bilayer nanodisks, consisting of DSPC and PEG5K-GCDS, were first employed for doxorubicin delivery into tumor. Herein, doxorubicin-encapsulated bilayer nanodisks (DOX-Disks) were prepared and characterized by morphology, size, encapsulation efficiency, and in vitro release behavior. In addition, pharmacokinetics behavior, tissue distribution in tumor-bearing mice, cytotoxicity, cellular uptake, and endocytosis mechanism were systematically examined to exploit the potential use of the nonspherical nanodisks as delivery system of anticancer drugs. This preliminary study attempts to provide more promising insights that the novel PEG-stabilized bilayer nanodisks might act as a potential carrier for anticancer drugs.

2. EXPERIMENTAL SECTION 2.1. Materials. Reference standard doxorubicin hydrochloride (99.4% purity) was provided by Beijing Huafenglianbo Tech. Co., Ltd. DSPC was purchased from Avanti Polar Lipids (Sigma-Aldrich). PEG5K-GCDS was self-made in our lab. Doxorubicin hydrochloride liposome injection was purchased from Shanghai Fudan-zhangjiang Bio-Pharmaceutical Co., Ltd. Trypsin, DMEM (low glucose), and fetal bovine serum (FBS) were purchased from Gibco BRL, USA. DAPI and other materials were supplied by Sigma (Germany). All solvents used in this study were of HPLC grade. 2.2. Preparation of Nanopreparations. 2.2.1. Preparation of Blank and PEG-Stabilized Bilayer Nanodisks. Blank disks were prepared by different molar ratio of DSPC/PEG5KGCDS (70:30 mol %, 75:25 mol %, and 85:15 mol %). Lipids were dissolved in chloroform and mixed for 1 h. The organic solvents were removed using a rotary evaporator to form the drug-containing lipid film. Then 4 mL of HBS buffer (10 mM HEPES buffered saline, pH 7.4) was added and sonicated for 45 min at 200 W using an ultrasonic cell disrupter (Ningbo xinzhi, China). During sonication, DSPC/PEG-GCDS based samples were kept in ice-cold water. The blank PEG-stabilized bilayer 3280

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density of 3 × 104 cells/mL and incubated for 24, 48, and 72 h, respectively. Following attachment, the cells were then exposed to a series of concentrations of blank micelles, free doxorubicin, or DOX-Disks for 24, 48, and 72 h at 37 °C. Medium without DOX was added as control. After that, 50 μL of MTT (2 mg/ mL) was added to every well for additional incubation. Then, the medium was removed and 200 μL of dimethyl sulfoxide was added to dissolve the crystals formed by living cells. The absorbance of the wells was measured by the microplate reader at a wavelength of 570 nm. The viability of cells was measured using the MTT method as previously described. Sigmoidal dose−response curves for inhibition rate vs logarithm of MTO concentration were plotted using Origin software. The values of IC50 were calculated as the concentration of DOX producing a 50% inhibition rate. The cytotoxicity of DOX-loaded micelles was evaluated by the MTT method. MCF-7/Adr cells were seeded in a 96-well plate at a density of 3 × 104 cells/mL and incubated for 72 h. Following attachment, the cells were then exposed to a series of concentrations of blank micelles and DOX-loaded micelles for 72 h at 37 °C. Medium without micelles was added as control. After that, 50 μL of MTT (2 mg/mL) was added to every well for additional incubation. Then, the medium was removed and 200 μL of dimethyl sulfoxide was added to dissolve the crystals formed by living cells. The absorbance of the wells was measured by the microplate reader at a wavelength of 570 nm. The viability of cells was measured using the MTT method as previously described. Sigmoidal dose−response curves for inhibition rate vs logarithm of MTO concentration were plotted using Origin software. The values of IC50 were calculated as the concentration of DOX producing a 50% inhibition rate. 2.8. Cellular Uptake. MCF-7/Adr cells were seeded into coverslips which were placed in 6-well plates at a density of 1 × 104 cells/mL and grown for 24 h at 37 °C. Then, the cells were washed carefully with PBS three times and incubated with 1 mL of the DOX-Sol and DOX-Disks at 8 nM for 1, 6, and 16 h at 37 °C in a shaking table, respectively. After incubation, 4 °C PBS was added to terminate uptake and washed three times. The cells were incubated with 95% ethanol for 10 min and then washed 3 times every 5 min. Then 0.5% Triton X-100 was added, incubated for 5 min, and washed 3 times every 5 min. After that, 1 mL of DAPI was added into the cells, placed in a shaker table at 37 °C for 1 h, and then washed 3 times every 5 min with PBS. 50 μL of cell sealing liquid (glycerol:carbonate 1:1) was dropped on a glass slide. A coverslip was carefully placed on the sealing liquid, to avoid air bubbles, dried for 30 min, and observed using confocal microscopy. MCF-7/Adr cells were seeded into 24-well plates at a density of 1 × 105 cells/mL and grown for 24 h, respectively. The cells were incubated with DOX-Sol and DOX-Disks, at a DOX concentration of 8 nM for 1 and 6 h at 37 °C, respectively. Then, the cells were washed carefully with 4 °C HBSS three times and lysed with 0.3 mL of HBSS containing 1% Triton X100 for 1 h. After centrifugation of the samples at 3000g for 10 min, the supernatant was collected and stored at −80 °C until analysis. The concentration of DOX in the cell supernatant was determined by a validated HPLC-FLD method. Cell uptake efficiency was expressed as the percentage of determined DOX content in the incubated cells versus the total amount of DOX in the feed solution. 2.9. Cellular Uptake Mechanisms. To investigate the underlying endocytotic mechanism that was responsible for the

rpm and 37 °C. At predetermined time intervals, 5 mL of sample was withdrawn and replaced with the same amount of fresh release medium. The amount of DOX released from the micelles was determined by HPLC at 258 nm. 2.5. Pharmacokinetics in Rats. Twelve healthy adult male Wistar rats (Laboratory Animal Center of Shenyang Pharmaceutical University, Shenyang, Liaoning, China) weighing 250 ± 20 g were housed in a room with controlled temperature and humidity and had free access to food and water. Before the day of administration via the tail vein, the rats were fasted for 24 h but allowed water ad libitum. All animal studies were performed according to the Guidelines for the Care and Use of Laboratory Animals that was approved by the Committee of Ethics of Animal Experimentation of Shenyang Pharmaceutical University with a performed number 2011011803. DOX-Sol and DOX-Disks (5 mg/kg) were administered by intravenous injection from caudal vein. Serial blood samples (300 μL) were collected into heparinized cryotubes by puncture of the retro-orbital sinus according to the following time schedule: 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120 h postdosing. The blood samples were centrifuged immediately at 15,000 rpm for 10 min to obtain plasma. The plasma samples were labeled and kept frozen at −80 °C until analysis. The DOX concentration in plasma was determined by a UPLC−MS−MS method. 2.6. Tissue Distribution in H22 Tumor-Bearing Mice. Thirty male mice (Laboratory Animal Center of Shenyang Pharmaceutical University, Shenyang, Liaoning, China) weighing 22 to 25 g (∼12 weeks of age) were housed in a room with controlled temperature and humidity and had free access to food and water. All animal studies were performed according to the Guidelines for the Care and Use of Laboratory Animals that were approved by the Committee of Ethics of Animal Experimentation of Shenyang Pharmaceutical University with a performed number 20110306-01. The mice were injected subcutaneously in the right flank with 0.2 mL of cell suspension containing 2 × 107 H22-H8D8 cells and maintained in DMEM medium. Solid tumor tissue was processed by mechanical and enzymatic digestion to generate single-cell suspensions. Tumors were allowed to grow for approximately 7 days to a volume of 100−200 mm3 measured using calipers before treatment. Tissue distribution of DOX-Sol and DOX-Disks in H22 tumor-bearing mice after intravenous administration in mice was investigated. Twenty tumor-bearing mice were given a single 5 mg/kg iv dose of DOX-Sol or DOX-Disks. At 2, 4, and 48 h, blood samples were collected and plasma was separated. Heart, liver, spleen, lungs, kidneys, brains, and tumors were rapidly excised and homogenized following blood collection. The concentration of doxorubicin was determined by the same method as for the pharmacokinetics study in rats. 2.7. Cell Culture and Cytotoxicity. MCF-7 (human breast cancer cells) and P-gp overexpressing MCF-7/Adr (multidrug resistant variant) were provided by Nanjing Kaiji Biotech. Ltd. Co. (China). Cells were cultured in DMEM medium (low glucose) containing 10% (v/v) fetal bovine serum (FBS) and 1% penicillin−streptomycin at 37 °C, 5% humidity atmosphere, and 5% CO2. Cells were subcultured regularly by trypsin/EDTA upon reaching 80% to 90% confluence. The cytotoxicity was evaluated by the MTT method. MCF-7 and MCF-7/Adr cells were seeded in a 96-well plate at a 3281

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seen that the discoid shape dominated in the pictures. The spherical shape in the background represented face-on or front side of the nanodisks while elongated shape stood for edge-on or flank side of them, depending on their orientation toward the incoming electron beam. Size distribution of the blank disks with different lipid ratios was determined by DLS (dynamic light scattering) as shown in Table 1. The particle size of lipid

internalization of DOX-Disks, uptake inhibition experiments were carried out in MCF-7/Adr cells which were seeded into 24-well plates at a density of 1 × 105 cells/mL and grown for 24 h. Cells were treated with 25 μM azide sodium, 50 μM chlorpromazine, 50 μM colchicine, 50 μM indomethacin, and 50 μM quercetin prior to 1 h incubation with DOX formulations for 1 h at 37 °C, respectively. Then, the cells were incubated with DOX-Disks, at a DOX concentration of 8 nM at 37 °C for 6 h. Then, the cells were washed carefully with PBS three times and carefully scraped to obtain cell suspension. After that, the cell suspension was sonicated for 10 min at 200 W and stored at −80 °C until analysis. The concentration of DOX in the cell supernatant was determined by a validated HPLC-FLD method. Cell uptake efficiency was calculated as described above. 2.10. Statistical Analysis. Two-sided, unpaired Student’s t test was applied to test the significance of differences between pharmacokinetic parameters or cellular uptake extent of the DOX-Sol, DOX-Disks, and DOX-Micelles group. The differences were considered to be significant at P < 0.05 and very significant at P < 0.01. All values were expressed as the mean value ± standard deviation (mean ± SD, n = 6 for pharmacokinetics and n = 3 for cellular uptake).

Table 1. Size Distribution of Three Lipid Mixture with Different Molar Ratio (n = 3) DSPC:polymer ratio particle size (nm) % identity PDI

85:15

75:25

65:35

135.4/539.2 93.5/4.4 0.348

81.91/105.3 97.9/2.1 0.262

62.6/129.1 75.8/23.2/ 0.478

mixtures with a 75:25 molar ratio was the most homogeneous, and PDI (polydispersity index) was the lowest among the three formulations. The disk-shaped aggregates were also found in lipid mixtures (85:15), but the size distribution was more or less irregular. It suggested that the lower molar ratio of DSPC to PEG5K-GCDS was not better and the optimized lipid ratio was 75:25. The mean disk diameter was about 81.91/105.3 nm, and the disk height was about 6 nm, like the cell membrane. 3.2. Physicochemical Characteristics of DOX-Loaded Bilayer Nanodisks (DOX-Disks). The DOX-loaded bilayer nanodisks (DOX-Disks) were prepared by thin-film dispersion in combination with the sonication hydration method. DOX was incorporated into the hydrophobic region of the disks. The nanostructure of the DOX-Disks is illustrated schematically in Figure 2A,B. The disks are open bilayer structures, with a hydrophobic discoid center built by DSPC and a hydrophilic PEG edge covering the hydrophobic rim by PEG5K-GCDS. As shown by the TEM image and diameter distribution diagram in Figure 2C and Figure 2D, the DOX-Disks were successfully prepared with essentially disk-shaped morphology. The mean particle diameter was 80.14 nm, and the disk height was about 6 nm with aspect ratio about 12. Zeta potential of the nanodisks was −1.84 mV. Encapsulation efficiency was as high as 96.1%, indicating strong binding affinity between hydrophobic DOX and the hydrophobic discoid area. 3.3. Release Performances of DOX-Disks. The release of DOX from DOX-Disks was performed in phosphate buffer or acetate buffer solutions at different pH values to simulate in vivo different conditions. It is reported that pH values in blood circulation, early endosomes, late endosomes, and lysosomes are about 7.4, 6.5, 5.5, and 4.5, respectively. The cumulative release profiles of DOX from DOX-Disks are shown in Figure 3. The DOX release was pH-dependent and increased with the decrease of pH in release media. At pH 7.4, less than 26% of total DOX was released from DOX-Disks at 24 h, suggesting that the nanodisks were stable at the normal physiological condition with less nonspecific release. That is, the nanodisks had the potential to enclose DOX before reaching the tumor site. Nevertheless, a rapid release of DOX from DOX-Disks was presented, reaching 56% at pH 5.5 and even up to 85% at pH 4.5 after 24 h. It indicated that DOX was released more rapidly from the nanodisks in endosomal/lyposomal compartments within tumor cells, which contributed to elicit desired antitumor efficacy. 3.4. Pharmacokinetics in Rats. The pharmacokinetic performances of doxorubicin in DOX-Disks and DOX solutions

3. RESULTS 3.1. Preparation of Blank PEG-Stabilized Bilayer Nanodisks. Here, the novel disks consisted of two different lipids, DSPC and PEG5K-GCDS with a carefully adjusted molar ratio. The morphology of the disks with different molar ratios of DSPC to PEG5K-GCDS is shown in Figure 1. Lipid mixtures

Figure 1. Transmission electron microscopy (TEM) images of blank disks containing different molar ratios of DSPC/PEG-GCDS: 70:30 mol % (A), 75:25 mol % (B), and 85:15 mol % (C). The black arrows and arrowheads in panels B and C denote disks observed face-on and edge-on, respectively. Scale bar on TEM image is 100 nm.

with a molar ratio of 70:30 DSPC/PEG5K-GCDS contained the majority of spherical nanostructure particles (Figure 1A). With the increased ratio of DSPC to PEG5K-GCDS, the flat, circular disk-shaped morphology of lipid mixtures was observed dominantly, suggesting that PEG5K-GCDS played an essential role in the nanostructure of disks. In Figures 1B and 1C, it was 3282

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Figure 2. Spatial (A) and planar (B) structures of doxorubicin loaded PEG-stabilized bilayer nanodisks (DOX-Disks). Lipids with light blue head groups symbolize phospholipids, those with dark blue head groups represent PEG-lipids, and the red parts represent doxorubicin. (C) TEM images of the DOX loaded nanodisks. The black arrows and arrowheads denote disks observed face-on and edge-on, respectively. The scale bar on the TEM image is 100 nm. (D) Dynamic light scattering analysis of DOX-Disks.

respectively. The tumor distribution at 2, 12, and 48 h following iv administration of DOX increased by 3.4-, 4.2-, and 9.7-fold in DOX-Disks compared with DOX-Sol. With the extension of time, doxorubicin accumulation in tumor was increased for DOX-Disks, but the reverse for DOX-Sol. In heart, the concentration of doxorubicin decreased by 1.7-, 4.9-, and 25.7-fold compared with DOX-Sol at 2, 12, and 48 h after iv administration, respectively. The biodistribution of doxorubicin in heart was significantly reduced by encapsulation in the nanodisks. Doxorubicin level in the liver was greatly decreased by 4.1-fold at 48 h compared with at 2 h, and DOX-Disks were also not trapped by spleen. The lung distribution at 2, 12, and 48 h following iv administration of DOX increased by 1.8-, 2.5-, and 9.3-fold in DOX-Disks compared with DOX-Sol. As for

(DOX-Sol) after iv administration (5 mg/kg) to SD rats are shown in Figure 4A. The pharmacokinetic parameters were analyzed by the noncompartmental model method. The plasma half-life (t1/2) of DOX-Disks was 11.7-fold (41.9 h) longer than that of DOX-Sol (3.5 h). Moreover, AUC for DOX-Disks was 17452.5 μg·h/L, and increased by 31.7-fold of 550.8 μg·h/L of DOX-Sol. It was inferred that DOX-Disks could achieve the desired superlong circulation property and better therapeutic effect, indicated by much larger AUC and longer half-life than those of DOX-Sol. 3.5. Tissue Biodistribution in H22 Tumor-Bearing Mice. The biodistribution of doxorubicin in H22 tumorbearing mice at 2, 12, and 48 h after iv injection (5 mg/kg) of DOX-Disks and DOX-Sol is shown in Figures 4B, 4C, and 4D, 3283

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accumulated in the perinuclear region. After 16 h of incubation, fluorescence was mainly localized in the nucleus where doxorubicin elicits its anticancer effect. Accordingly, cellular uptake of DOX-Sol and DOX-Disks nanoparticles was timedependent, and DOX-Disks could enhance DOX accumulation in the target nucleus. In addition to confocal microscopy, the uptake amount of doxorubicin in MCF-7/Adr cells was determined to calculate cell uptake efficiency and evaluate cellular uptake of DOX-Sol and DOX-disks. Here, cell uptake efficiency was defined as the percentage of determined DOX amount in the cells versus the total amount of DOX in the feed solution. Cellular uptake efficiency of DOX-Sol and DOX-Disks in MCF-7/Adr cells is shown in Figure 6D. The uptake efficiency was found to be 12.7% and 21.8% for DOX-Sol and 16.2% and 46.8% for DOXDisks after incubation for 1 and 6 h, respectively. When incubation time was short, DOX-Disks achieved slightly better penetration effect compared with DOX-Sol. However, DOXDisks increased by nearly 3-fold of cell uptake efficiency compared with DOX-Sol when cells were cultured for 6 h. 3.8. Cell Uptake Mechanisms. Endocytotic pathways dominate the intracellular fate of nanomicelles and its contained drugs. Generally speaking, endocytosis pathways are classified as macropinocytosis, clathrin-dependent endocytosis (CME), caveolin-dependent endocytosis, and clathrinand caveolin-independent pathways. Thus, internalization mechanism of DOX-Disks was investigated with specific endocytosis inhibitors. MCF-7/Adr cells were preincubated with 25 μM azide sodium, 50 μM chlorpromazine, 50 μM colchicine, 50 μM indomethacin, and 50 μM quercetin for 1 h at 37 °C, respectively. Then, DOX-Disks were added with a DOX concentration of 8 nM and incubated for 6 h. Compared with the control group without inhibitor, the cellular uptake efficiency of DOX-Disks (Figure 7A) was significantly inhibited by sodium azide (energy inhibitor), choropromazine (clathrinmediated endocytosis inhibitor), colchicine (microtubuledependent macropinocytosis inhibitor), and quercetin (a nonclathrin-, non-caveolae-mediated endocytosis inhibitor) (P < 0.01), whereas it was not significantly changed by indomethacin (caveolae-mediated endocytosis inhibitor) (P > 0.05). The proposed endocytic mechanism of DOX-Disks is shown in Figure 7B. Endocytosis of DOX-Disks nanoassemblies was mediated by energy-dependent endocytosis processes and was involved in clathrin-mediated, macropinocytosis-mediated, and non-clathrin- and noncaveolae-mediated endocytosis pathways.

Figure 3. In vitro pH-dependent release profiles of DOX from DOXDisks in pH 4.5 acetate buffer and pH 5.5, pH 6.5, and pH 7.4 PBS. Data are represented as mean ± standard deviation (n = 3).

kidney, the difference between DOX-Disks and DOX-Sol was not significant at all time points (P > 0.05). Neither DOX-Sol nor DOX-Disks was detected in brain at all predetermined time points because the blood brain barrier (BBB) contributes to the obstacle between blood and brain. The concentration of doxorubicin in the heart of DOX-Disks and PEGylated liposomes in mice at 48 h after iv injection (5 mg/kg) was 0.05 μg/g and 0.33 μg/g, respectively. 3.6. Cytotoxicity. In vitro cytotoxicity of DOX-Disks was evaluated by MTT assay against MCF-7 cells and MCF-7/Adr cells, in comparison with DOX-Sol. The cells were treated with blank micelles, DOX-Disks, and DOX-Sol at different DOX concentrations from 0.001 to 100 μg/mL after 24, 48, and 72 h incubation, respectively. No significant growth inhibition effect was found after incubation with the blank micelles, indicating that PEG2K-GCDS were nontoxic as nanocarriers. The dose− response curves of DOX in DOX-Sol and DOX-Disks are profiled in Figure 5. The anticancer effects were further quantitated by IC50 (Table 2). The IC50 was found to be 0.51, 0.31, and 0.27 μg/mL for DOX-Sol, 0.69, 0.56, and 0.55 μg/mL for DOX-Disks in MCF-7 cells after treatment at 24, 48, and 72 h, respectively. IC50 values of DOX-Sol were slightly higher than those of DOX-Disks. On the other hand, for P-gp overexpressing MCF-7/Adr cells, the IC50 was 2.03, 0.85, and 0.86 μg/mL for DOX-Sol, 0.23, 0.24, and 0.21 μg/mL for DOX-Disks after treatment for 48, 72, and 96 h, respectively. The IC50 of doxorubicin was decreased by 8.8-, 3.5-, and 4.1fold compared with DOX-Sol at 24, 48, and 72 h in MCF-7/ Adr cells, respectively. The cytotoxicity of DOX was significantly enhanced in DOX-Disks compared with DOXSol. The IC50 was found to be 0.43 μg/mL for DOX-loaded micelles in MCF-7/Adr cells after treatment at 72 h. 3.7. Cellular uptake. Cellular uptake and intracellular accumulation of DOX-Sol and DOX-Disks against MCF-7/Adr cells were investigated by confocal microscopy (CLSM). MCF7/Adr cells were cultured at 37 °C for 1, 6, and 16 h, respectively, after addition of DOX-Sol and DOX-Disks in culture medium to maintain DOX concentration of 8 nM. Then the nucleus was stained with DAPI and the pretreated cells were observed under CLSM shown in Figure 6A−C. DOX fluorescence was observed mainly in the cytoplasm when the cells were incubated with DOX-Sol and DOX-Disks for 1 and 6 h. It was evident that the fluorescence intensity of DOX was stronger in DOX-Disks than DOX-Sol. With the incubation time increased, DOX fluorescence in DOX-Disks primarily

4. DISCUSSION Bilayer disks, so-called bicelles, can be prepared from a mixture of medium- and short-chain phospholipids and other lipids,24,17 such as DHPC/DMPC and PEG-DSPE (5000)/DSPC/ cholesterol. It is reported that the amount of PEG-lipid mainly influences the phase behavior and aggregate structure of the nanodisks. A very similar phase behavior was observed containing DSPE-PEG (2000) and DSPE-PEG (5000).25 Lipids in lipid/PEG-lipid systems were used to simulate different biomembranes for different studies, which did not have an effect on the formation structure of the nanodisks.19 PEG-lipid concentration in the range of 15−25 mol % is required to produce nanodisk preparations without further purification.26,27 Here, PEG-lipid concentrations of both 15 and 25 mol % were used to prepare nanodisks. The size distribution was not regular in the DSPS/PEG5K-GCDS system with 85:15 mol % (Figure 1b) whereas the particle size of the same lipid 3284

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Figure 4. Pharmacokinetics and distribution of doxorubicin formulated in the DOX-Disks in comparison with those of DOX-Sol. (A) DOX level vs time curves after iv administration to SD rats (n = 6). (B, C, D) Tissue distribution of DOX-Sol and DOX-Disks in H22 tumor-bearing mice at 2, 24, and 48 h after iv injection (n = 5). Doxorubicin dose was 5 mg/kg.

mixtures with 75:25 mol % (Figure 1c) was more homogeneous and with lower PDI (polydispersity index). Herein, a PEG-lipid concentration of 25 mol % was chosen to produce the desired disk preparations for in vitro and in vivo studies without purification. PEG-lipids, which favor the rim of the disks, offer steric protection against fusion and self-closure and maintain their stability in terms of lowering the edge energy.19 Based on our data from dynamic light scattering and electron microscopy and theoretical calculations, the prepared nanodisks were open bilayer structures, with a hydrophobic discoid center built by

DSPC and a hydrophilic PEG edge, according to refs 18 and 25, by cryo-TEM and DLS. The structural evolution of the aggregates formed in lipid/PEG-lipid mixture with increasing PEG-lipid concentration can be summarized as liposomes → discoidal micelles → spherical micelles. It is important to note that a complete conversion of all liposomes into nanodisks seems to occur at concentrations close to 20 mol % of lipidPEG. PEG-lipid concentration was used to adjust the diskshaped morphology of the DSPC/PEG5K-GCDS mixture. Besides, high-density lipoprotein (HDL) is a structurally and functionally fascinating class of lipoproteins that encompass 3285

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Figure 5. Dose−response curves of DOX-Sol and DOX-Disks against human breast cancer cells MCF-7 (A) and resistant human breast cancer cells MCF-7/Adr (B) at 24, 48, and 72 h post-treatment, respectively (n = 3).

with maximum antitumor efficacy and minimum side effects in normal tissues. When DOX was encapsulated into DOX-Disks, some improvements were exhibited, such as sustained plasma profile and delayed retention time in systemic circulation. Plasma half-life of DOX-Disks is approximately 42 h, which provided ample time to enter tumor tissue from the vasculature. The circulation time of the disks was also longer than that of the spherical PEGylated nanoparticles and liposomes of doxorubicin with t1/2 about 21.9−34.3 h33,34 The outer steric hydrophilic PEG rim could prevent the rapid uptake of nanoparticles by mononuclear phagocyte systems and prolong their half-life in the blood circulation.35 Prolonged circulation of the DOX-Disks might also be attributed to their discoid shape. It was reported that filomicelles can reduce phagocytosis by macrophage cells because of their filamentous shape. The decreased clearance by phagocytosis could be due to the elongated lifetime of filomicelles in blood circulation.36 In addition, hydrodynamic features of disks in the bloodstream, such as alignment with blood flow, might prolong circulation time by diminishing nonspecific collisions with the vascular walls. This paradigm was illustrated by long-circulating discoid red blood cells circulating in the blood, and also by recent findings that elongated liposomes and polymer filomicelles

Table 2. IC50 for DOX-Sol and DOX-Disks To Inhibit Growth of MCF-7 and MCF-7/Adr Cells after 24, 48, and 72 h Incubation (n = 3) IC50 (μg/mL) MCF-7

MCF-7/Adr

time (h)

DOX-Sol

DOX-Disks

DOX-Sol

DOX-Disks

24 48 72

0.51 0.31 0.27

0.69 0.56 0.55

2.03 0.85 0.86

0.23 0.24 0.20

nanoparticles with distinct sizes (6−13 nm) and discoidal shape.28 Discoidal HDL-mimicking peptide−phospholipid nanocarrier can mimic the behavior of plasma-derived HDL in both structural and functional properties, such as monodisperse size, long circulation half-life, and excellent biocompatibility.30,31 They have been successfully used to attenuate toxicity of anticancer drugs to nontargeted cells, improve cancer theranostics, and efficiently deliver siRNA in tumor.32,33 Desirable pharmacokinetics characteristics in vivo are required for achieving effective delivery of cytotoxic agents 3286

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Figure 7. Effect of endocytosis inhibitors on cellular uptake in resistant MCF-7/Adr cells treated with DOX-Disks. (A) DOX-Disks with 50 μM sodium azide (energy inhibitor), 50 μM chloropromazine (clathrin inhibitor), 50 μM indomethacin (caveolae inhibitor), 50 μM colchicine (macropinocytosis inhibitor), and 50 μM quercetin (nonclathrin and non-caveolae inhibitor). The concentration of DOX was 8 nM (n = 3). (B) Schematic illustration of the proposed endocytic mechanism of DOX-Disks.

mice was 0.05 μg/g at 48 h after iv administration of nanodisks. However, doxorubicin levels achieved in the heart of PEGylated liposomal doxorubicin formulations were 0.33 μg/g. Doxorubicin levels in heart of nanodisks was decreased by 6.6-fold compared with PEGylated liposome. Biodistribution of doxorubicin in heart was significantly reduced by encapsulation in the nanodisks. It was well-known that doxorubicin was widely used in the treatment of various cancers, but cardiotoxic effects significantly limited its clinical application. Doxorubicin encapsulated in DOX-Disks could achieve the desired cancer treatment efficacy and reduce the cardiac toxicity concurrently. Shape can affect particle velocity, diffusion, and adhesion to walls in blood vessels in a complex way.40 Mathematical models and in vitro flow chamber experiments have demonstrated that the discoid particles can drift laterally toward the wall41 and can more avidly adhere to the vascular walls under blood flow,10,42 compared to spherical and quasi-hemispherical particles. Diskshaped, flexible red blood cells with diameters of 10 μm routinely pass through the spleen. Movement of nonspherical particles was not easier to predict due to the presence of blood flow, but escape of DOX-Disks from liver and spleen was really relevant to their disk shape. Moreover, the local shape of the particle at the point where the cell attached determined whether or not a macrophage began internalization.43 It was reported that a macrophage attached to an ellipse at the pointed end internalized it in a few minutes while a macrophage attached to a flat region of the same ellipse did

Figure 6. Cellular uptake in MCF-7/Adr cells 7 cells treated with DOX-Sol and DOX-Disks. Confocal laser scanning microscopy (CLSM) images of treated with DOX-Sol and DOX-Disks after 1 h (A), 6 h (B), and 16 h (C) with DOX concentration of 8 nM. From left to right, the images show cell nuclei stained by DAPI (blue), DOX fluorescence (red), and the merging of the two images. (D) Cellular uptake efficiency of DOX was calculated after incubation for 1 and 6 h in MCF-7/Adr cells (n = 3).

circulated for a prolonged time period due to flow alignment.14,37 The results indicated that superlong circulation property of the disk may result from PEG rim, discoid shape, and hydrodynamic features of disks in the bloodstream, and therefore the shape of nanoparticles played an important role in their pharmacokinetic performances. It was reported that tumor tissue was characterized by hypervascular permeability and impaired lymphatic drainage to facilitate accumulation of nanoparticles (