Improved Oral Absorption of Doxorubicin by Amphiphilic Copolymer of

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Improved oral absorption of doxorubicin by amphiphilic copolymer of lysine-linked di-tocopherol polyethylene glycol 2000 succinate: In vitro characterization and in vivo evaluation Jinling Wang, Lin Li, Yuqian Du, Jin Sun, Xiaopeng Han, Cong Luo, Xiaoyu Ai, Qi Zhang, Yongjun Wang, Qiang Fu, Zhifu Yang, and Zhonggui He Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500833m • Publication Date (Web): 12 Jan 2015 Downloaded from http://pubs.acs.org on January 18, 2015

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Molecular Pharmaceutics

Research Paper

Title Improved oral absorption of doxorubicin by amphiphilic copolymer of lysine-linked di-tocopherol polyethylene glycol 2000 succinate: In vitro characterization and in vivo evaluation

Authors: Jinling Wang a, c, 1, Lin Li a, 1, Yuqian Du a, Jin Sunb, *, Xiaopeng Han a, Cong Luo a, Xiaoyu Ai a, Qi Zhanga , Yongjun Wang a, Qiang Fu a

Zhifu Yangd, Zhonggui Hea,*

Affiliation: a

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical

University, Wenhua Road, Shenyang 110016, China b

Municipal Key Laboratory of Biopharmaceutics, School of Pharmacy, Shenyang

Pharmaceutical University c

Beijing University of Chinese Medicine

d

Department of Pharmacy, Xijing Hospital, Fourth Military Medical University,

Beijing China

No.15 Changle West Road, Xi’an, Shanxi 710032, China 1

These authors contributed equally

*Corresponding Author: Prof. Zhonggui He Ph D; Prof. Jin Sun Ph D Mailbox 59#, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, 110016, China; Tel/Fax: +86-24-23986321; E-mail: [email protected] [email protected]

ACS Paragon Plus Environment


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Abstract Graphic


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ABSTRACT In the previous study, we have synthesized an amphiphilic copolymer of nanostructure-forming material and P-glycoprotein (P-gp) inhibitor, lysine-linked di-tocopherol polyethylene glycol 2000 succinate (PLV2K). The cytotoxicty in vitro and anticancer efficacy in vivo after intravenous administration of DOX loaded PLV2K micelles (PLV2K-DOX) was found more effective than DOX solution (DOX-Sol). However, its performance and mechanism on oral absorption of doxorubicin are not well understood yet. PLV2K-DOX was spherical micelles with a narrow size distribution of 20.53±2.44 nm. In situ intestinal perfusion model, the intestinal absorption potential of PLV2K-DOX was evaluated in comparison with DOX-Sol. PLV2K-DOX was specifically absorbed in duodenum and ileum sites of rats after oral administration. The intestinal absorption rate (Ka) of PLV2K-DOX is 3.19-, 1.61- and 1.80-fold higher than that of DOX-Sol in duodenum, jejunum and ileum, respectively. In Caco-2 uptake studies, PLV2K-DOX micelles significantly improve the internalized amount of DOX by P-gp inhibition of free PLV2K copolymer and endocytosis of DOX-loaded nanoparticles. Moreover, PLV2K-DOX micelles improve the membrane permeability of DOX by multiple transcytosis mechanisms, including caveolin-, clathrin-dependent, and caveolin- and clathrin-independent transcytosis in Caco-2 transport studies. However, the transepithelia electrical resistance (TEER) of Caco-2 cellular monolayer is not changed, suggesting no involvement of paracellular transport of PLV2K-DOX. In vivo pharmacokinetics in rats following oral administration demonstrated that PLV2K-DOX demonstrates higher AUC (5.6-fold)



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and longer t1/2 (1.2-fold) than DOX-Sol. The findings suggest the new PLV2K micelles might provide an effective nanoplatform for oral delivery of anticancer drugs with poor membrane permeability and low oral bioavailability.

Keywords:

amphiphilic copolymer; micelles; Doxorubicin; Oral absorption;

Vitamin E succinate; P-glycoprotein


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1. Introduction Oral delivery of anticancer agents is preferred by patients for its convenience, cost-effectiveness and favorable adaptability. Nevertheless, expected pharmacological activities of anticancer drugs rely on good intestinal absorption and high bioavailability after oral administration. Unfortunately, a majority of anticancer drugs are not orally available due to low water-solubility and poor permeability across intestinal epithelia. In addition, oral absorption of anticancer drugs is also limited by a series of bio-barriers in gastrointestinal tract, including multidrug efflux proteins (such as P-glycoproteins (P-gp), breast cancer resistance protein (BCRP)), enzyme degradation and mucus layers entrapment, etc. Therefore, low and variable oral bioavailability greatly limits the progress of oral chemotherapy. Consequently, to increase oral bioavailability of anticancer drugs is a prerequisite for successful development of oral chemotherapy. Doxorubicin (DOX) is one of the anthracycline antibiotic anticancer agents and widely used for tumor therapy, including lung, breast, ovarian cancers and multiple myeloma etc. Until now, DOX is mainly administered intravenously with the trade name of Adriamycin R (trade name of doxorubicin) , Mycoet R (doxorubicin liposome) , Doxil

R

( pegylated liposomal doxorubicin) and Caelyx

R

(pegylated liposomal

doxorubicin HCl) clinically 1. However, DOX is failed for oral administration because of its relatively low and variable oral bioavailability (only about 5%). The underlying reasons include intrinsic low intestinal permeability, hydrolysis in stomach, high affinity to P-gp pump and cytochrome P450 metabolic enzymes (CYP450) 2. Several



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strategies have been adopted to overcome these bio-barriers to improve the bioavailability of DOX. For instance, co-administration of inhibitors of P-gp or CYP450, such as cyclosporine A or quercetin, could improve the oral absorption of DOX to some extent, but these inhibitors can cause serious side-effects, such as the depression of immune system3-5. With the rapid development of nanotechnology, nanoscale drug delivery systems, such as micelles, lipid based nanoparticles and nanosuspensions, provide a favorable platform for oral chemotherapy 6. Nowadays, much effort has been made to enhance the oral bioavailability and to decrease the side effects of DOX by drug delivery nanoparticles. DOX-PAMAM dendrimer polyelectrolyte-stabilized liposomes

7

4

, lipid-based nanoparticles

5

and

have been reported to increase the oral

absorption of DOX to a different extent. Polymeric micelles (PMs) are attractive and effective nanoscale vehicles to enhance the bioavailability of oral chemotherapy. Core-shell structured PMs can improve the solubility, stability and permeability across the intestinal barriers of anticancer drugs by encapsulation into the inner core. Moreover, PMs can protect drugs from degradation in gastrointestinal tract, increase permeability in intestine by P-gp inhibition and reduce first-pass metabolism in liver by escaping the recognition of CYP450 enzymes. In recent years, various PMs are extensively studied for improving oral chemotherapy8-10. Lysine-linked di-tocopherol polyethylene glycol 2000 succinate (PLV2K), an amphiphilic copolymer, has been successfully synthesized based on TPGS structure


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by our group

11

. Compared with TPGS, PLV2K has a lower critical micelle

concentration (CMC) of 1.14 µg/mL, and the self-assembled PMs of PLV2K is more stable than that of TPGS. Moreover, PLV2K copolymer can inhibit the P-gp efflux pump through inhibition of P-gp ATPase. Based on the P-gp inhibitory effects in enterocytes and strengthening the micelles stability in GI tract by PLV2K, the bioperformance and mechanism of DOX-loaded PLV2K micelles (PLV2K-DOX) on oral absorption of DOX are evaluated in vitro and in vivo in the present study. With this in mind, the oral absorption characteristics of PLV2K-DOX is conducted by intestinal perfusion in situ in comparison with DOX solution (DOX-Sol), and then visualized under laser confocal scanning microscopy. Moreover, uptake and transport performances on Caco-2 cell monolayer of PLV2K-DOX and relevant mechanism studies are investigated. The in vivo pharmacokinetic studies of PLV2K-DOX micelles following oral administration are finally evaluated in rats.



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2. Materials and methods 2.1. Materials and reagents mPEG-2000 was purchased from Sigma-Aldrich (St. Louis). Vitamin E succinate was obtained from Wuhang Yuancheng Gongchuang Technology Co., Ltd. (China). H-lys-Obzl·HCl·TosOH was gained from GL Biochem Ltd. (Shanghai, China). Lysine-linked di-tocopherol polyethylene glycol 2000 succinate (PLV2K) was synthesized as described by our group 11. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing Huafeng United Technology CO., Ltd. Dulbecco’s modified Eagle medium (DMEM, high glucose) and fetal bovine serum were acquired from Gibco (Beijing, China). Penicillin-streptomycin solution, Hank’s buffered salt solution (HBSS) and 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Hyclone (Logan, US). All solvents used in the investigation were of analytical reagent grade. 2.2. Preparation and characterization of PLV2K-DOX micelles PLV2K-DOX micelles were prepared via solvent evaporation method as described previously11. Briefly, 5mg DOX·HCl was dissolved in methanol containing two molar triethylamine for 1h to remove HCl. Then DOX was added to chloroform solution of PLV2K nanocarrier (100mg) and evaporated to form the film by rotary vacuum evaporation. The film of drug-polymer was hydrated with Hepes at 37

. The

micelles were acquired after centrifugation at 13,000 rpm for 20 min to remove the unencapsulated drug. The morphology of PLV2K-DOX was observed by transmission electron


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microscopy (TEM) (H-600, Hitachi, Tokyo, Japan) after negative staining with 2% (w/v) phosphotungstic acid. The size distribution and zeta potential of PLV2K–DOX were evaluated by Zetasizer (Nano ZS, Malvern Co., UK). The dilution stability of micelles was evaluated by Zetasizer. For the stability of micelles in SGF and SIF, 100 µL of micelles was diluted with 1mL simulated gastric fluid (SGF) or simulated intestinal fluid (SIF), and then the particle size was measured by Zetasizer at defined intervals. The release studies of DOX from PLV2K-DOX were performed by dialysis method at 37 R in phosphate buffer (PBS, pH 6.8). Briefly, 1 mL of DOX-Sol (1 mg/mL) and PLV2K-DOX (1 mg/mL) were put into the dialysis bags (molecular weight cutoff 14,000) and then immersed in 30 mL of the release medium (PBS) in a conical flask, respectively. The flasks were placed in a constant shaking water bath with a speed of 100 rpm at 37

. At predetermined time intervals, an aliquot of 1 mL

of release medium in every flask was withdrawn, replenished with same volume of fresh medium, and analyzed by HPLC at 258 nm. 2.3. In situ intestinal perfusion of PLV2K-DOX micelles in rats All animals investigated in this research were executed according to the Guidelines for the Care and Use of Laboratory Animals approved by the Ethics Committee of Animal Experimentation of Shenyang Pharmaceutical University. Prior to the experiments, the rats were deprived of food overnight for 12 h with free access to water. After anesthetized with 20% urethane (1.0 g/kg) by intraperitoneal injection, the anesthetized rats were constrained on an operating table in a supine position, with



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infrared lamps to maintain normal temperature of the rats. The abdomen was opened with a 3-4 cm midline longitudinal incision; three intestinal segments including duodenum, jejunum and ileum were exposed about 10 cm in length respectively. Each selected intestinal segment was cannulated and ligated at both sides. After rinsed with 37

saline solution gently, the selected segments were rinsed with pre-warmed

Kerbs Ringer’s buffer (KRB, 7.8 g NaCl, 0.35 g KCl, 1.37 g NaHCO3, 0.02 g NaH2PO4, 1.48 g glucose in 1000 mL purified water). Afterwards, the intestinal segments were perfused with PLV2K-DOX micelles perfusion fluid (containing 20 µg/mL of DOX and 20 µg/mL phenol red

the phenol red was added as a

non-absorbable marker to measure the change of water in the perfusate) for 30 min at a constant flow rate of 0.22 mL/min to reach a steady state. Samples were collected in tubes for analysis and replaced by an empty receptor every 15 min. The experiment was not stopped until the intestine was perfused for 120 min. At the end, the length and diameter of each segment were measured. Meanwhile, 20 µg/mL DOX solution diluted with KRB was acted as control. 200 µL collected perfusate and equal volume of methanol were mixed, vortexed, and centrifuged at 13,000 rpm for 10 min. An aliquot of 20µL supernatant was analyzed by a validated HPLC method. As for phenol red, 500 µL perfusate after filtration through 0.45µm filter membrane and 5 mL NaOH (0.2 M) were mixed, and the concentration of phenol red was detected by ultraviolet spectrophotometer (λmax= 557 nm). The absorption rate (Ka) and effective permeability (Peff) of PLV2K-DOX micelles in each intestinal segment were calculated based on following equations:


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Ka =

Q  Cout PRin  ×  1 − 2πrL  Cin PRout 

Peff = −

Q Cout PRin ln( ) × 2πrL Cin PRout

where Cin is concentration of DOX in donor fluid, Cout is the concentration of DOX in receptor tube, PRin and PRout are the concentration of phenol red in the donor fluid and receptor tube, Q is the flow rate (0.22 mL/min), L and r refer to the radius and length of the selected intestinal segments.

2.4. Biodistribution of PLV2K-DOX micelles in rat small intestine The absorption of PLV2K-DOX and DOX-Sol in intestine was observed by laser confocal scanning microscopy (LCSM). Briefly, PLV2K-DOX and DOX-Sol were administered to rats by oral gavage (18 mg/kg). After 0.5 h, rats were sacrificed, and duodenum, jejunum and ileum segments were taken, carefully everted, and frozen in cryoembedding media. The selected segments were sectioned at 10 µm (CM 1900, LEICA, Germany), and placed on glass sides, then fixed with 4 % paraformaldehyde for 10 min. After rinsed with PBS, the actin filaments were stained with FITC-Phalloidin (Alexis Biochemicals, Lausen), and the nuclei were counterstained with 4´, 6-diamidino-2-phenylindole (DAPI). Subsequently, the counterstained sections were visualized under laser confocal scanning microscopy (LCSM, TCS SP2/AOBS, LEICA, and Germany).

2.5. Cellular uptake of PLV2K-DOX micelles on Caco-2 cells 2.5.1. Cell culture Caco-2 cells were seeded at a density of 1×105 cells per well in 24-well cell culture plate (Corning, USA) and cultured in DMEM medium supplemented with



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10% (v/v) fetal bovine serum, 20 mmol/L HEPES, 1 % (v/v) nonessential amino acid, 4 mmol/L L-Glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin in a 5 % CO2 humidified atmosphere (90 % humidity) at 37

for 15 days. Moreover, the

cells were fed every two days in the first week and everyday afterwards before cellular uptake experiments.

2.5.2. Uptake studies The cultured Caco-2 cells were washed with HBSS thrice and pre-incubated at 37

for 30 min. Then, DOX-Sol or PLV2K-DOX micelles were incubated with

different DOX concentration of 5-90 µg/mL on Caco-2 cells. After 1h incubation, the supernatants were removed and the cellular monolayers were washed with ice-cold PBS, lysed with 300 µL Triton 100 (0.1%, v/v), scrapped and homogenized with acetonitrile. The concentrations of DOX in cell lysates were measured by a validated UPLC-MS-MS method. A BCA protein assay reagent method was employed for the determination of protein content in cell lysates. In addition, to investigate the effects of P-gp efflux pumps on cellular uptake, DOX-Sol, DOX-Sol mixed with Cys A (20 µg/mL), DOX-Sol mixed with PLV2K nanomaterial (1 µg/mL) and PLV2K-DOX were added on cell monolayer, respectively. After incubation for 45 min, the cell monolayer was rinsed with cold PBS. The cell monolayer was processed as previously mentioned and assayed by UPLC-MS-MS. The protein content was determined by BCA protein assay reagent method. Caco-2 cells were seeded at a density of 1×10-5 cells in 6-well plate. After one week attachment, cells were washed and incubated with DOX-Sol or PLV2K-DOX


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with equivalent concentration of DOX (10 µg/mL) for 1 or 3 h at 37

. Meanwhile,

DOX-Sol mixed with Cys A or PLV2K nanomaterial were also added to incubated for 3h at 37 R to investigate P-gp inhibition. Then cellular monolayers were washed with 4

PBS thrice and fixed with 4% paraformaldehyde for 10 min. The nuclei were

stained with 4’6-diamidino-2-phenylindole (DAPI). Subsequently, the cells were observed by confocal laser scanning microscope (LCSM, TCS SP2/AOBS, LEICA, and Germany).

2.5.3. Endocytosis pathways of PLV2K-DOX micelles To study the endocytosis pathways by PLV2K-DOX, different specific inhibitors were applied in uptake experiments. Initially, the cell monolayers were pre-incubated with 20 µg/mL chlorpromazine, sodium azide, colchicine, quercetin and indomethacin for 1 h at 37

, respectively. After that, the cells were treated with PLV2K-DOX in the

presence of corresponding endocytosis inhibitors (20 µg/mL) with the concentration of 50 µg/mL DOX for further 1h at 37

. Then Caco-2 cells were treated and

analyzed by UPLC-MS-MS as directed under 2.5.2.

2.6. Transport studies of PLV2K-DOX micelles across Caco-2 cells Caco-2 cells were seeded at a density of 2×105 cells per well on 12-well Transwell R inserts ( 0.4 µm pore size, 12 mm diameter, Conining Costar, USA) for 21 days. The culture medium was exchanged every two day within one week and every day thereafter. The integrity of the cell monolayer was evaluated by measuring the transepithelial electrical resistance (TEER). The transport studies were started when the TEER value exceeded 300 Ω·cm2, indicating that the cellular monolayers



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were integrated and the tight junction of Caco-2 cells was built. In the transport trials, the culture medium was withdrawn and the cellular monolayers were washed with 400 µL and 600 µL 37

HBSS to apical (AP) and to

basal (BL) chambers three times, respectively. After the cells incubated for 30 min at 37

, the medium was removed and 400 µL test solutions were added to the AP side

while 600 µL blank HBSS were replenished to the BL side. The test solutions included DOX-Sol, mixed solution of DOX and PLV2K (1 µg/mL) and PLV2K-DOX micelles with equivalent DOX concentration of 50 µg/mL. The TEER values were assayed and 200 µL aliquots were sampled from the BL side while replenished the same volume of fresh HBSS every 20 min in a period of 2 h after incubation. DOX concentrations were determined by UPLC-MS-MS. The apparent permeability coefficients (Papp) for DOX were calculated according to the following equation: Papp =

dCr 1 1 × Vr × × dt A C0

where dCr/dt is the change of DOX concentration in the receiver solution, Vr is the volume of the receiver compartment, A is the area of the inserts, and C0 is the initial concentration of DOX at AP side. Paracelluar transport of micelles was determined by detection the change of TEER value. In brief, PLV2K-DOX and mixed solutions of PLV2K nanocarrier and DOX-Sol were added to the AP side, incubated at 37

. The TEER values were tested

every 20 min in 2 h. To investigate the transport mechanisms of PLV2K-DOX micelles, the culture medium of the AP side was replaced by 50 µg/mL chlorpromazine, sodium azide,


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colchicine, quercetin and indomethacin for 1h incubation, respectively. Then the inhibitors were displaced with mixed solutions of PLV2K-DOX micelles (containing 50 µg/mL DOX) with corresponding endocytosis inhibitors (50 µg/mL) for another 1h at 37

. The TEER values were evaluated and 200 µL solutions at BL side were

sampled every 20 min within 2 h. Finally, DOX concentrations were determined by the UPLC-MS-MS method.

2.7. Pharmacokinetic studies in rats Sprague-Dawley rats (Laboratory Animal Center of Shenyang Pharmaceutical University, Shenyang, Liaoning, China) weighing 220 g ± 20 g ( mean±standard deviation) were randomly divided into two groups (n=6). All animal experiments were performed in accordance with guidelines for Use and Care of Animals approved by Shenyang Pharmaceutical University Committee of Ethics. The rats were fasted 12-18 h with free access to water ad libitum before dosing. DOX-Sol and PLV2K-DOX were administered orally at a dose of 80 mg/kg and 18 mg/kg, respectively. Blood samples (0.25 mL) were collected into heparin tubes by puncture of the retro-orbital sinus at 0.08, 0.17, 0.33, 0.50, 0.75, 1.0, 3.0, 6.0, 8.0, 10, 12, 14, 24 h post-dosing via oral gavage. The plasma was obtained by centrifugation at 12,000×g for 10 min immediately and stored at -80

until analysis.

Meanwhile, in order to investigate the oral absolute bioavailability (Fabs), the solutions of doxorubicin and pegylated micelles were intravenous administered to every six rats at 5 mg/kg. Then blood samples (approximately 300 µL) were obtained by puncture of the retro-orbital sinus at 0.08, 0.17, 0.33, 0.50, 1.0, 2.0, 4.0, 6.0, 8.0,



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10, 12, 24, 36, 48, 96, 120, 144, 168, 192, 216, 240 h after injection and then collected into heparinized cryotubes. The blood samples were centrifuged immediately at 14,000×g for 10 min to obtain the plasma samples, stored at -80

until analysis.

2.8. Quantification of DOX by UPLC-MS/MS 2.8.1 Cellular sample preparation After spiking 200 µL methanol, 100 µL internal standard (IS) solution (glimepiride, 200 ng/ml) into 50 µL cellular samples, the mixed samples were vortexed for 3 min and centrifuged at 14,000×g for 10 min. The supernatant was transferred into a sample vial of autosampler at 7 , and an aliquot of 5 µL was injected into the UPLC/MS/MS for analysis.

2.8.2 Plasma sample preparation The concentration of DOX in rat plasma was determined after liquid-liquid extraction by ethyl acetate-2-propanol. Briefly, 50µL IS solution (glimepiride, 200 ng/ml) and 50 µL borax - sodium carbonate buffer (pH 10.8) were added to 50 µL plasma samples and mixed for 3 min. The mixed samples were extracted with 1 ml ethyl acetate-2-propanol (20:1, v/v) and vortexed for 3 min, before being centrifuged at 12,000×g for 10 min. The upper organic layers were evaporated to dryness under a nitrogen flow at 37

and then be reconstituted in 100µL mobile phase and

centrifuged for another 10 min at 12,000×g. An aliquot of 5 µL was injected into the UPLC/MS/MS for analysis.

2.8.3 UPLC-MS/MS analysis and quantification for plasma and cell samples An ACQUITY UPLCTM system (Waters Corp., Miford, MA, USA) equipped with


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a cooling auto-sampler and a column oven was applied for analysis. An ACQUITY UPLCTM BEH C18 column (50 mm × 2.1 mm, 1.7 µm; Waters Corp, Milford, MA, USA) was used for chromatographic separations. A gradient elution program was performed with a mobile phase A (methanol) and mobile phase B (0.1% glacial acetic acid–water) as follows: 0 min (45% A), 0.4 min (60% A), 0.8 min (95%A), 2.2min (45% A). The column temperature was set at room temperature and the flow rate was 0.2 mL/min. The temperature of auto-sampler was set at 7

and the injection volume

was conditioned at 5µL. Mass spectrometry was performed with a Waters Tandem Quadrupole (TQ) Detector (Waters) and was operated by positive electrospray ionization (ESI). The optimal ionization source conditions were as follows: capillary voltage 2.8 KV, cone voltage 22 V, source temperature 120 temperature 350

, desolvation

. The cone and desolvation gas flow rate were 50 and 550 L/h,

respectively. The collision energy was 12 and 18 V for doxorubicin and glimepiride. And cone voltage was set to 22 and 16 V for doxorubicin and glimepiride. Multiple reaction monitoring (MRM) was used for quantification to monitor the ion transitions of m/z 544→397 for doxorubicin and 491→352 for the internal standard of glimepiride, respectively. Data were acquired using Masslynx 4.1 software.

2.9. Statistical analysis Results were expressed as meas±SD (standard devitation). A student’s t-test or one-way ANOVA was applied to test the significance in the experiments. Statistical differences were significant at P

Improved Oral Absorption of Doxorubicin by Amphiphilic Copolymer of

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