Enhanced Oral Delivery of Paclitaxel Using Acetylcysteine

Aug 3, 2013 - Jinling Wang , Lin Li , Yuqian Du , Jin Sun , Xiaopeng Han , Cong Luo , Xiaoyu Ai ... chitosan as an efficiency oral delivery system of ...
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Enhanced Oral Delivery of Paclitaxel Using Acetylcysteine Functionalized Chitosan-Vitamin E Succinate Nanomicelles Based on a Mucus Bioadhesion and Penetration Mechanism He Lian,† Tianhong Zhang,‡ Jin Sun,*,†,§ Xiaohong Liu,† Guolian Ren,† Longfa Kou,† Youxi Zhang,† Xiaopeng Han,† Wenya Ding,† Xiaoyu Ai,† Chunnuan Wu,† Lin Li,† Yongjun Wang,† Yinghua Sun,† Siling Wang,† and Zhonggui He*,† †

Department of Biopharmaceutics and ‡Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, P. R. China § Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, No. 308, Western Anshan Road, Tianjin, 300193, P. R. China S Supporting Information *

ABSTRACT: In addition to being a physiological protective barrier, the gastrointestinal mucosal membrane is also a primary obstacle that hinders the oral absorption of many therapeutic compounds, especially drugs with a poor permeability. In order to resolve this impasse, we have designed multifunctional nanomicelles based on the acetylcysteine functionalized chitosan-vitamin E succinate copolymer (CS-VES-NAC, CVN), which exhibit marked bioadhesion, possess the ability to penetrate mucus, and enhance the oral absorption of a hydrophobic drug with a poor penetrative profile, paclitaxel. The intestinal absorption (Ka = 0.38 ± 0.04 min−1, Papp = 0.059 cm·min−1) of CVN nanomicelles was greatly improved (4.5-fold) in comparison with paclitaxel solution, and CLSM (confocal laser scanning microscope) pictures also showed not only enhanced adhesion to the intestinal surface but improved accumulation within intestinal villi. The in vivo pharmacokinetics indicated that the AUC0−t (586.37 ng/mL·h) of CVN nanomicelles was markedly enhanced compared with PTX solution. In summary, the novel multifunctional CVN nanomicelles appear to be a promising nanocarrier for insoluble and poorly permeable drugs due to their high bioadhesion and permeation-enhancing capability. KEYWORDS: chitosan-vitamin E succinate-N-acetyl-L-cysteine, mucoadhesion, permeation enhancer, oral absorption, paclitaxel

1. INTRODUCTION

have been found to overcome the above problems associated with the oral absorption of paclitaxel. Nanomicelles are currently receiving growing scientific attention as a drug delivery nanosystem.6−8 Nanomicelles have a variety of advantages, such as self-assembly capability, uniform size distribution, high solubilization capacity and stability, sustained release, and easy surface functionalization with active ligands. This allows the nanomicelles to be widely employed as a promising carrier of poorly soluble anticancer drugs.9−12 In our previous work,13 we showed that nanomicelles based on chitosan-vitamin E succinate (CS-VES) copolymer exhibited many attractive features in terms of their size distribution, zeta potential, encapsulation efficiency, and in vitro release profile.

Despite the rapid development of drug delivery systems, efficient oral administration of many anticancer drugs still poses many notable challenges. Their poor permeability and solubility and rapid passage through the intestinal tract are the main factors contributing to their low and variable bioavailability. This situation has greatly limited the efficacy and development of oral chemotherapy. Therefore, it is very important to develop an effective delivery platform to improve the oral bioavailability of anticancer drugs and macromolecules, such as the use of nanoparticles. Paclitaxel (PTX), an effective cancer chemotherapy agent, has been widely used for the treatment of various tumors, including ovarian, breast, non-small cell lung, and prostate tumors.1−3 However, its poor aqueous solubility, extensive metabolism, and active efflux by P-gp transporter have seriously impeded its application as oral chemotherapy since its oral availability is very low (about 1%).4,5 However, nanoparticles © 2013 American Chemical Society

Received: Revised: Accepted: Published: 3447

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permeation-enhancing nanomaterial to enhance the bioavailability of paclitaxel and to improve the efficacy of oral chemotherapy.

We successfully used this nanocarrier for selective tumor delivery of paclitaxel after intravenous administration. However, its limitations in terms of intestinal bioadhesion, especially mucus anchorage and penetration, have hampered its use as an effective nanocarrier for the oral absorption of paclitaxel. In this study, we describe the design of a novel multifunctional mucoadhesive and permeation-enhancing copolymer based on CS-VES for paclitaxel oral administration. Thiolated polymers (thiomers) have gradually become a “hot” topic as oral delivery nanocarriers.14,15 Thiolated chitosan is a readily soluble polymer that can be prepared by coupling free thiol agents with the amino groups of chitosan. This kind of polymer can tightly adhere to and anchor the mucus layer to prolong the gut residence time. Two factors contribute to the enhanced intestinal bioadhesion. First, an electrostatic interaction occurs between the positively charged chitosan and the negatively charged sialic and sulfonic acid of the intestinal mucin; then, the formation of covalent S−S bonds by free thiol groups of thiolated chitosan and the mucus cysteine domain results in the steady adhesion of the nanomaterial to the mucus layer.16−18 It has been reported that trimethyl chitosan-cysteine can increase insulin intestinal transport by 11.7-fold compared with insulin solution in rats.19 Also, compared with the original chitosan, the bioadhesion of chitosan-thioglycolic acid copolymer is increased by about 3−9-fold.20,21 Furthermore, the bioadhesion of chitosan-4-thiolbutylamidine was 140-fold higher than that of ordinary chitosan,22 and, in addition, thiolated chitosan exhibits a permeation-enhancing effect which is important for the transepithelial delivery of anticancer drugs. The mechanism of this action involves an interaction between the positively charged polymer and the tight junction-associated protein on the cell membrane, leading to reconstruction and opening of the tight junctions.23,24 Moreover, the hydrophilic polymers with free thiol groups can inhibit the activity of tyrosine phosphates, followed by the opening of the tight junctions.25 It has been proved that the chitosan-TBA/GSH system can effectively increase membrane permeability by suppressing the ATPase activity of P-gp in the intestinal epithelium.26 In spite of these advantages, the above thiolated chitosan derivatives are not suitable as a nanocarrier for poorly soluble drugs due to their lack of a hydrophobic region. Based on our information about the previously developed amphiphilic chitosan derivative, we conjugated the bioadhesive and permeation-enhancing N-acetyl-L-cysteine (NAC) and CSVES to develop a multifunctional nanomaterial for enhancing the oral absorption of insoluble drugs. The resulting copolymer CS-VES-NAC (CVN) could easily be self-assembled into core/ shell nanoassemblies in aqueous medium, and paclitaxel could be readily encapsulated in the hydrophobic core of the nanomicelles. The paclitaxel-loaded nanomicelles were characterized by their size distribution, encapsulation efficiency, appearance under transmission electron microscopy, and X-ray diffraction, and their in vitro release behavior was also investigated. The intestinal absorption of CVN nanomicelles was investigated by in situ single-pass perfusion and visualized by confocal laser scanning microscopy. The endocytosis pathways were explored to clarify the biological mechanisms governing the oral absorption of the nanomicelles. In addition, the pharmacokinetics and bioavailability of paclitaxel-loaded CVN nanomicelles were studied in rats after duodenal administration using UPLC−MS/MS. This study was carried out to explore the use of an intestinal mucoadhesive and

2. MATERIALS AND METHODS 2.1. Materials. Paclitaxel (PTX) was obtained from Xi’an Helin Biological Engineering Co. Ltd. (Xi’an, China). Chitosan (Mw = 20 kDa; degree of deacetylation = 92.5%) was purchased from Aoxing Biochemical Co. Ltd. (Zhejiang, China). Vitamin E succinate (VES) was purchased from Yuancheng Technology Development Co. Ltd. (Wuhan, China). N-Acetyl-L-cysteine (NAC) was obtained from Aladdin (Shanghai, China). 1Hydroxybenzotriazole (HOBT) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Zhejiang PuKang Pharm Co., Ltd. Coumarin-6, mucin type III, and 5,5′dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) were obtained from Sigma (St. Louis, MO, USA). Rhodamine-labeled phalloidin was purchased from Cytoskeleton (1830 S. Acoma St., Denver, CO, USA). Paclitaxel solution (PTX-sol) was prepared in our laboratory based on the commercially available product, Taxol. All other chemicals and reagents were of analytical grade, and used without further purification. 2.2. Synthesis of CS-VES-NAC Copolymer. In our previous work,13 CS-VES (CV) copolymer was synthesized by conjugating the carboxyl group of VES with the amino groups of chitosan in the presence of EDC. To synthesize the CS-VES-NAC (CVN) copolymer, NAC was initially activated by EDC in anhydrous ethanol and CS-VES underwent nucleophile catalysis by adding HOBt in deionized water. This was followed by dropwise addition of NAC solution to the CS-VES solution. The pH was then adjusted to 5.0 by adding NaOH, and the reaction was carried out at room temperature for 3 h without exposure to light. To remove unreacted reagents, the resulting copolymer conjugate was dialyzed at 4 °C, first against 5 mmol/L HCl, then once against 5 mmol/L HCl containing 1% NaCl, twice against 5 mmol/L HCl, and, finally, against 1 mmol/L HCl. Controls were prepared in the same way but without EDC and HOBt during the coupling reaction. Finally the aqueous polymer solution was lyophilized and stored at 4 °C for further use. 2.3. Characterization of CS-VES-NAC Copolymer. 2.3.1. Nuclear Magnetic Resonance Spectroscopy (NMR) of CS-VES-NAC. The chemical structure of CS-VES-NAC was analyzed by 1H NMR. The 1H NMR spectrum of the polymer was recorded on a Bruker spectrometer operated at a frequency of 300 MHz for protons with D2O as the solvent. 2.3.2. Energy Dispersive X-ray Spectroscopy (EDS) and Elemental (Combustion) Analysis of CS-VES-NAC. To eliminate any measurement error caused by the floccose copolymer gap, 30 mg CS-VES-NAC and CS-VES were pressed into thin tablets using a single punch tablet (6 mm) machine for use with X-ray energy-dispersive spectroscopy (OXFORD instruments, INCA software, U.K.). Also, 3 mg of CS-VESNAC and CS-VES copolymer was used for elemental analysis (Flash EA 1112 Series, Thermo Electron Corporation). The above techniques were applied to identify and semiquantitatively characterize the chemical elements present on the copolymer tablet surface.27 2.3.3. Thermogravimetric Analysis (TGA). Thermogravimetric analysis was carried out using a TA-60 WS instrument (Shimadzu, Japan) to examine the copolymer including CS, CS-VES, and CS-VES-NAC from 30 to 600 °C at a heating rate of 10 °C/min under nitrogen. 3448

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2.3.4. Degree of Thiolation of the Polymer Conjugates. The amount of thiol groups on modified chitosan was quantified using Ellman’s reagent as described previously.28 First, 5 mg of CS-VES-NAC copolymer was hydrated in 2.5 mL of deionized water. Then, the test solution was prepared by mixing 900 μL of 0.5 mol/L phosphate buffer (pH 8.0) and 1 mL of Ellman’s reagent (30 mg in 10 mL of 0.5 mol/L phosphate buffer, pH 8.0) and 100 μL of copolymer solution. The reaction was performed at room temperature for 2 h without light. After removal of the precipitated polymers by centrifugation (10000g, 10 min), the supernatant was obtained and the absorbance was measured at a wavelength of 412 nm with a UV−vis spectrophotometer (UV1800, RuiLi, China). NAC standard was used to calculate the amount of thiol groups from a standard curve. To measure the amount of disulfide bonds, the CS-VESNAC copolymer was reduced using sodium borohydride (NaBH4). After hydrating each copolymer, 250 μL of deionized water and 650 μL of 0.05 mol/L phosphate buffer (pH 6.8) were added to swell the copolymer followed by the addition of 1 mL of a freshly made 4% NaBH4 solution (m/v). The mixtures were incubated in a water bath at 37 °C for 1 h. Then, 200 μL of 5 mol/L HCl was added to the reaction solution to destroy the remaining sodium borohydride. Then, the solution was neutralized by adding 1 mL of 1 mol/L phosphate buffer (pH 8.5). Finally, 1 mL of Ellman’s reagent was added and the mixtures were agitated for 2 h at room temperature. The absorbance was determined using a UV−vis spectrophotometer (UV1800, RuiLi, China). The quantity of disulfide bonds was calculated by subtracting the amount of free thiol groups from the total amount of thiol groups on the copolymer. 2.4. Preparation of CS-VES-NAC Nanomicelles. Paclitaxel loaded CS-VES-NAC (CVN) nanomicelles were prepared by a probe-type ultrasonic method using a nanoprecipitation/ dispersion technique. For this, 6 mL of CS-VES-NAC copolymer solution was prepared at a concentration of 2.5 mg/mL, PTX was dissolved in dehydrated alcohol, and then the drug solution was added to the CS-VES-NAC solution with stirring. Then, the dehydrated alcohol was removed by rotary vacuum evaporation. The resulting mixture was sonicated by a probe-type sonifier (JY92-2D, Scientz, China) for 20 min in an ice bath to keep the temperature low. Then, the micelle solution was centrifuged (3500 rpm, 10 min) and passed through a 0.8 μm filter to remove the unloaded drug and other impurities. The obtained CS-VES-NAC nanomicelles were then kept at 4 °C until required. Coumarin-6 loaded micelles were prepared in the same way except that coumarin-6 was added to the copolymer solution instead of paclitaxel. 2.5. Characterization of CS-VES-NAC Nanomicelles. 2.5.1. Size and Zeta Potential. The particle size, size distribution, and zeta potential were measured using a Zetasizer (Nano ZS, Malvern Co., U.K.). The measurements were repeated in triplicate. 2.5.2. Particle Morphology. The morphology of the CSVES-NAC nanomicelles was visualized by transmission electron microscopy (TEM) (Tecnai G20, FEI, USA). A drop of micellar solution was deposited on a carbon-coated lacey support copper grid, stained with 1% phosphotungstic acid, and then examined under TEM. 2.5.3. Drug-Loading Content (DL) and Encapsulation Efficiency (EE). The EE and DL of paclitaxel-loaded CS-VESNAC nanomicelles were measured by high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan). Briefly,

drug-loaded micellar solution was diluted 25-fold with methanol and sonicated to collapse the micelles and dissolve the drug. The solution was then transferred to an HPLC vial after being passed through a 0.22 μm syringe filter. Chromatographic separation was achieved using a reverse-phase ODS Diamonsil-C18 column (250 mm × 4.6 mm, 5 μm) at 25 °C. The mobile phase consisted of deionized water and HPLC grade acetonitrile (40:60, v/v). The samples were delivered at a flow rate of 1.0 mL/min and detected at 227 nm using an ultraviolet−visible detector. The EE was calculated as the ratio of the drug encapsulated in nanomicelles to the amount of drug used in the fabrication. Also, DL was defined as the percentage of the drug encapsulated in nanomicelles to the total weight of PTX loaded nanomicelles. 2.5.4. X-ray Powder Diffraction. The status of PTX in CSVES-NAC nanomicelles was analyzed using a Japan D/Max 2500 PC X-ray diffractomer (Rigaku, Japan). Samples, including PTX powder, CS-VES-NAC copolymer, physical mixtures of PTX and CS-VES-NAC copolymer, and lyophilized PTX loaded CS-VES-NAC nanomicelles, were examined under graphite monochromatized Cu Kα radiation over the range 3− 45° (2θ) at 50 kV and 300 mA. 2.6. In Vitro Release. An in vitro release study was conducted by the dialysis method. The drug-loaded nanomicelles were dispersed in phosphate buffered saline (pH 6.8) containing 2% Cremophor EL, which can improve the solubility of paclitaxel in PBS. Then, a 1.5 mL dispersion was sealed in a dialysis bag (MWCO = 14 kDa, Spectrum Laboratories, USA) and incubated in 30 mL of release medium at 37 °C under orbital shaking (100 rpm). At designated intervals, 2 mL samples were removed for analysis and replaced with the same volume of fresh medium. The PTX content was determined under the same HPLC conditions as described above. The error bars were obtained from triplicate samples. 2.7. Mucin Adhesion. The mucoadhesive force was defined as the amount of mucin adsorbed by 2 mg of blank CS-VES-NAC nanomicelles in a certain time period. Micellar solutions (4 mg/mL) were mixed with a type III mucin (Sigma, St. Louis, MO, USA) solution (0.5 and 1 mg/mL), vortexed, and incubated for 6 h at 37 °C. After adsorption, the free mucin in the supernatant was obtained by centrifugation (10000 rpm, 10 min) and measured by a colorimetric method using periodic acid/Schiff (PAS) staining.29,30 The amount of mucin adsorbed by the CS-VES-NAC nanomicelles was calculated by subtracting the concentration of mucin in solution after adsorption from that before. A mucin standard calibration curve was prepared by measuring mucin standards (0.1, 0.25, 0.5 mL/mL) using the same procedure. To further confirm disulfide bond formation between CS-VES-NAC and mucin, the mixture of CS-VES-NAC solution and mucin solution was incubated at 37 °C for 6 h, and then lyophilized for differential scanning calorimetry (DSC) measurement using a thermal analyzer (TA-60 WS, Shimadzu, Japan). The samples were analyzed at a heating temperature ranging from 30 to 300 °C at a rate of 10 °C/min in nitrogen. The thermograms were compared with those obtained from mucin, CS-VES-NAC copolymer, and their physical blend. 2.8. In Situ Single-Pass Intestinal Perfusion of Paclitaxel-Loaded Nanomicelles 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. First, phenol red was 3449

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rats underwent a middle laparotomy, and the duodenum segment was removed and rinsed in 37 °C KRB solutions and then cut into 60−90 mg pieces. The everted intestine rings were placed in the wells of a 24-well plate with 1 mL of KRB solution and washed twice. Then, the intestinal rings were incubated for 45 min at 37 °C with a series of endocytic inhibitors, such as chlorpromazine (50 μmol/L), indometacin (100 μmol/L), colchicine (10 μmol/L), sodium azide (25 μmol/L), and quercetin (10 μmol/L). Then, the medium was removed and the CVN-PTX micellar solution was added to the plate at a terminal concentration of PTX 20 μg/mL and incubation allowed to take place for 45 min. Following this, the intestinal segments were washed and homogenized, and PTX was analyzed by HPLC. In addition, to further explain the absorption mechanism of CVN-PTX micelles, the experiments were performed at 4 °C or in the absence of specific inhibitors as controls. 2.11. Pharmacokinetic Profiles. Three groups of five rats were deprived of food overnight but had free access to water before the experiments and then were divided randomly into groups. Group 1 was treated with PTX solution (PTX dissolved in absolute alcohol and Cremophor EL, 6 mg/mL), which was diluted with sterile saline before use (15 mg/kg); groups 2 and 3 were treated with CV and CVN micellar solutions (7.5 mg/ kg), respectively. All the groups received duodenal administration. Blood samples were collected into heparinized tubes at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h and centrifuged immediately at 10000g for 10 min. Then, the plasma was frozen at −20 °C for analysis using ultraperformance liquid chromatography/tandem mass spectrometry (UPLC−MS/ MS, ACQUITY UPLC/tandem quadrupole detector (TQD), MassLynx V4.1 software, Waters, USA). To determine the level of total PTX in each plasma sample, docetaxel was used as the internal standard. Also, 100 μL of plasma was mixed with 100 μL of docetaxel solution and mobile phase for 2 min. Then, 3 mL of diethyl ether was added to the mixture, which was vortexed for 3 min, then centrifuged at 3500 rpm for 10 min, and dried under a stream of nitrogen. The residue was reconstituted in 100 μL of mobile phase (mobile phase A, acetonitrile containing 0.1% formic acid; mobile phase B, deinoized water containing 0.1% formic acid) and centrifuged at 10000g for 10 min before analysis. The separation was carried out under reversed-phase conditions using a C18 column (ACQUITY UPLC BEH, 50 mm × 2.1 mm, 1.7 μm, Waters Corp, Milford, MA, USA). The flow rate was maintained at 0.2 mL/min, and the column temperature was maintained at 40 °C. The mass spectrometer was operated in positive ESI mode, and quantification was carried out by multiple reaction monitoring with transitions from m/z 854.8 to 286.2 for paclitaxel and 830.65 to 304.35 for docetaxel, respectively.31,32 Samples were quantitatively tested against a calibration curve over the range 10−2000 ng/mL. The correlation coefficients were 0.992, indicating a very high specificity, linearity, and accuracy. The pharmacokinetic parameters were obtained using DAS 2.0 software. The relative bioavailability was calculated as the ratio of the AUC0−t of micellar solutions to that of PTX solution. 2.12. Statistical Analysis. Statistical analysis was applied using one-way ANOVA with a pairwise test to identify any significant differences in pharmacokinetic parameters. Also, Student’s t test was used in the other experiments. Statistical difference was set at P < 0.05, and a very significant difference was defined as P < 0.01.

dissolved in freshly prepared Krebs Ringer’s (KR) buffer solution (7.8 g of NaCl, 0.35 g of KCl, 1.37 g of NaHCO3, 0.02 g of MgCl2, 0.32 g of NaH2PO4, 1.4 g of glucose, and 0.32 g of CaCl2 in 1000 mL of purified water) as the indicator at a concentration of 20 μg/mL, and then the drug-loaded nanomicelles were dispersed in it to achieve a final concentration of 20 μg/mL. Sprague−Dawley rats weighing 200−250 g were fasted overnight before the perfusion experiment but had free access to water. Anesthesia was induced by intraperitoneal injection of 20% urethane (1 g/kg), and the rats then underwent a laparatomy (n = 3). Three intestinal segments (about 10 cm) including the duodenum, jejunum, ileum, and colon were first washed gently with 37 °C saline solution at a flow rate of 0.2 mL/min. Then, the intestinal segments were infused with the paclitaxel-loaded CVN micellar dispersion. Perfusate was collected every 15 min in tubes up to 105 min. Then, the length and radius of the infused intestinal segments were measured accurately. The samples were diluted five times with methanol and centrifuged at 10000 rpm for 10 min to determine the PTX content using the HPLC method described above. The phenol red content was determined after colorizing with NaOH using a UV−vis detector at 558 nm. The absorption rate (Ka) and apparent permeability (Papp) of the CVN-PTX nanomicelles in the intestinal segments are given by the following equations: ⎛ Cpout C PRin ⎞ v ⎟• ka = ⎜⎜1 − • Cpin C PRout ⎟⎠ πr 2l ⎝

Papp =

⎛ Cpout −v ln⎜ C • ⎝ pin

C PRin ⎞ ⎟ C PRout ⎠

2πrl

where Cpout is the PTX concentration in the receptor tube, Cpin is the PTX concentration in the donor solution, CPRout is the phenol red concentration in the receptor tube, CPRin is the phenol red concentration in the donor solution, and r is the intestinal radius, while v is the perfusion flow rate. PTX and PTX-loaded CS-VES micellar solutions were perfused as the control at a terminal PTX concentration of 20 μg/mL. 2.9. Biodistribution of Coumarin-6 Labeled Nanomicelles in the Gastrointestinal Tract. In order to investigate the absorption of nanomicelles in the gastrointestinal (GI) tract, the CVN and CV nanomicelles were fluorescently labeled by coumarin-6 as mentioned previously (see section 2.4). Briefly, the freshly prepared coumarin-6labeled nanomicelles were administered to SD rats by oral gavage (4 mg/kg). After 30 min, the rats were sacrificed, and the selected duodenum, jejunum, and ileum segments were carefully removed, everted, and frozen in cryoembedding media (OTC) at −80 °C. Then, the frozen intestines were sectioned at 10 μm intervals (CM 3050S, Leica) and fixed on cationic resinous slides with 4% formalin for 10 min at room temperature. Then, the sections were washed with PBS (pH 7.4) and labeled with Rhodamine-labeled phalloidin (Cytoskeleton, USA) for 90 min at 37 °C, and the nuclei were stained with DAPI for 5 min. Finally, the coverslips were mounted on microscope slides and visualized under a confocal laser scanning microscope (CLSM, FluoViewFV1000, Olympus, Japan). 2.10. Endocytosis Mechanism of CVN Nanomicelles in the Everted Intestinal Rings. To clarify the absorption mechanism of paclitaxel CV and CVN micelles in rat intestine, 3450

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Figure 1. Synthetic route of chitosan-vitamin E succinate-N-acetyl-L-cysteine (CS-VES-NAC) copolymer.

3. RESULTS 3.1. Synthesis and Characterization of CS-VES-NAC Copolymer. For thiolation of CS-VES, N-acetyl-L-cysteine (NAC) was used to react with the amino groups of chitosan to provide a physiologically stable amide linkage in the presence of EDC and HOBT (Figure 1). The remaining free NAC and other impurities were removed after dialysis. Following lyophilization, the CS-VES-NAC copolymer appeared as fibrous-structured powders, which were pale-yellow and highly soluble over a wide pH range (e.g., pH 1.0−7.0). The fibrousstructured lyophilizates were used, and no pulverization of the powders was carried out in any of the experiments. The synthesized CS-VES-NAC copolymer was confirmed by nuclear magnetic resonance spectroscopy (NMR). A typical 1H NMR spectrum of CS-VES-NAC is shown in Figure 2. The proton peaks from 0.73 ppm to 1.2 ppm were ascribed to the section of vitamin E succinate, and the peak at 2.5 ppm corresponded to the succinyl methylene group. The typical NAC peak at about 2.0 ppm was assigned to the methyl group (shown in Figure 1s

in the Supporting Information). The above results indicated that N-acetyl-L-cysteine was successfully conjugated to the CSVES backbone. Energy dispersive X-ray spectroscopy (EDS) was used to semiquantitatively characterize the chemical elements and further identify the CS-VES-NAC copolymer, with CS-VES conjugate as the control. EDS analysis (Figure 3) of the CS-

Figure 3. Energy dispersive X-ray spectroscopy (EDS) of CS-VESNAC copolymer showing the presence of carbon (C), oxygen (O), nitrogen (N), and chlorine (Cl) atoms and many sulfur (S) atoms.

VES-NAC copolymer tablet surface showed that it contained mainly carbon (C), oxygen (O), and nitrogen (N) atoms together with a great deal of sulfur (S) and chlorine (Cl) atoms, since no hydrogen (H) atoms could be detected. In contrast, a typical EDS spectrum of CS-VES copolymer is shown in Figure 2s in the Supporting Information, which included carbon (C), oxygen (O), nitrogen (N), and chlorine (Cl) atoms, but no sulfur (S) atoms. After analysis using INKA software, the weight proportions of the individual elements of carbon, oxygen, nitrogen, sulfur, and chlorine atoms in the CS-VESNAC polymer were 60.08%, 23.48%, 6.10%, 3.03%, and 7.31%, respectively. Also, the weight proportions of carbon, oxygen, nitrogen, and chlorine atoms in the CS-VES copolymer were 61.92%, 27.65%, 7.06%, and 3.37%, respectively. The high content of sulfur (S) in the CS-VES-NAC EDS spectrum

Figure 2. The 1H NMR spectrum of CS-VES-NAC copolymer in D2O. 3451

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3.2. Preparation and Characterization of CS-VES-NAC Nanomicelles. In our previous work, vitamin E succinate grafted chitosan could form core−shell micelles through a selfassembly process in aqueous solution. So, it was anticipated that the amphiphilic chitosan-vitamin E succinate backbone in the structure of CS-VES-NAC could form a hydrophobic core and spontaneously encapsulate insoluble paclitaxel via a hydrophobic interaction, while the grafted hydrophilic thiol NAC groups remained exposed outside. The self-assembly process would finally produce spherical nanomicelles with a hydrophobic compacted core surrounded by a hydrophilic NAC shell layer. Figure 5 illustrates the self-assembly process of paclitaxel-loaded CVN nanomicelles. We performed a dynamic light scattering (DLS) experiment to analyze the effect of various concentrations of paclitaxel on the size and surface charge of CVN-PTX nanomicelles in aqueous solution. As summarized in Table 2, CS-VES-NAC copolymer and various amounts of PTX could self-assemble to form nanoscale micelles in the size range of 220−250 nm. It was clearly observed that every formulation exhibited a narrow size distribution and the polydispersity indexes were all below 0.25, as given by the representative particle size graph shown in Figure 6A. The TEM images showed that the nanomicelles were spherical and about 200 nm in diameter (Figure 6B), consistent with the above DLS results. In addition, the nanoparticles exhibited a clear core−shell structure, with a core consisting of paclitaxel and hydrophobic vitamin E succinate chains, and a shell composed of chitosan and Nacetyl-L-cysteine. Regarding the zeta potential, PTX loading did not affect the surface electrical properties of NAC nanomicelles, with all values being about 50 mV. This indicated that PTX was encapsulated into the hydrophobic core and the NAC copolymer was able to easily self-assemble to form nanosized micelles with a highly positive surface charge, thereby playing an important role in the in vitro physicochemical stability of micellar solutions and in vivo bioadhesion and permeation enhancement. Using a combination of nanoprecipitation and probe sonication, PTX could be incorporated into polymeric nanomicelles induced by physical entrapment through hydrophobic interactions between PTX and vitamin E succinate grafts. With an increase in the amount of PTX, the drug loading of PTX in CS-VES-NAC nanomicelles ranged from 5.5% to 25%. The encapsulation efficiency of PTX was always more than 60%, reaching 80% in some cases, and this was attributed to the favorable solubilization of the CS-VES-NAC nanomicelles. X-ray powder diffraction was used to characterize the physical status of PTX present in nanomicelles. As shown in Figure 7, typical diffraction peaks of paclitaxel were visible between 5° and 25°, and these were also observed in the patterns obtained for the physical mixture of PTX and CS-VESNAC copolymer. The crystal peak characteristics for PTX in drug-loaded nanomicelles were clearly weakened, indicating that PTX was encapsulated in the polymeric nanomicelles mostly in a molecular or amorphous state and almost no free crystalline drug was present in the micelles. 3.3. In Vitro Release. The in vitro release experiments of paclitaxel from the CS-VES-NAC nanomicelles were performed in pH 6.8 PBS, and the results are presented in Figure 8. In contrast with the PTX solution, there was a markedly prolonged release of PTX from the CS-VES-NAC nano-

showed that this copolymer had been successfully prepared. Elemental (combustion) analysis also confirmed that large amounts of carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) atoms were present in the CS-VES-NAC copolymer, and the composition ratios were 51.52%, 8.31%, 5.02%, and 1.62%, respectively. However, in the case of the CS-VES copolymer, no sulfur atoms could be detected. The weight proportions were 56.00% for carbon (C), 8.73% for hydrogen (H), and 4.85% for nitrogen (N) atoms. Thermogravimetric analysis (TGA) was used to examine the crystallinity of the unreacted chitosan, CS-VES, and CS-VESNAC copolymers. As shown in Figure 4, there was a clear

Figure 4. TGA thermograms of CS, CS-VES, and CS-VES-NAC copolymers.

weight loss induced by glucosidic bond breakage between 180 and 320 °C for each polymer. The decomposition temperature of chitosan, CS-VES, and CS-VES-NAC copolymers was 299.2, 274.8, and 258.8 °C, respectively. The lower decomposition temperature of chitosan derivatives suggested that, after chemical modification, the molecular conformation is changed, and the intermolecular hydrogen bonds and crystallinity were reduced. The phenomenon of transit from crystallization to amorphism implied the formation of a grafted and thiolated chitosan copolymer. Ellman’s test was used to determine the amount of thiol groups attached to the polymer. The result obtained showed that, on average, 1 g of CS-VES-NAC copolymer contained about 182.6 μmol of thiol groups. Also, the amount of interand intramolecular disulfide bonds was 54 μmol per gram of conjugate (Table 1). Bernkop-Schnurch et al. reported that the free thiol groups could be oxidized to inter- or intramolecular disulfide bonds and the oxidation process accelerated with an increase in the pH of the aqueous medium.33 The pH value of the CS-VES-NAC copolymer solution in our experiment was as low as 3.70, which maintained the stability of the free thiol groups and retarded the oxidation process. Table 1. Synthesis of CS-VES-NAC Conjugate with a High Content of Thiol Groups (Mean ± SD, n = 3) copolymer CS-VESNAC

free thiol (μmol/g)

disulfide content (μmol/g)

total amt of sulfhydryl groups (μmol/g)

182.6 ± 10.8

54.0 ± 13.7

236.6 ± 7.1

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Figure 5. Schematic illustration of self-assembled paclitaxel-loaded CVN nanomicelles in aqueous medium.

Table 2. Physicochemical Characterization of CS-VES-NAC Nanomicelles with Different Amounts of PTX (Mean ± SD, n = 3) PTX (mg) 0 1 2 3 4 5 6

mean hydrodynamic diam (nm) 240.3 225.9 245.6 229.2 229.5 234.6 249.1

± ± ± ± ± ± ±

2.1 2.9 4.6 3.0 0.9 3.8 1.6

polydispersity index 0.169 0.161 0.219 0.179 0.178 0.202 0.168

± ± ± ± ± ± ±

0.022 0.017 0.007 0.012 0.021 0.007 0.004

zeta potentiala (mv) 59.4 56.3 50.2 53.0 59.1 58.3 56.1

± ± ± ± ± ± ±

1.3 0.9 0.7 1.0 0.2 1.4 1.7

DLb (%) 4.33 9.58 13.18 16.93 19.19 24.06

± ± ± ± ± ±

0.09 0.07 0.02 0.13 0.38 0.24

EEc (%) 64.66 77.88 76.16 76.03 71.67 78.67

± ± ± ± ± ±

1.44 0.66 0.10 0.69 1.75 1.02

a

Measurement was performed in deionized water. bDrug loading ratio = [(amount of drug loaded in nanomicelles)/(the total amount of drug in micelles and CS-VES-NAC copolymer in fabricate)] × 100. cDrug loading efficiency = [(amount of drug loaded in the nanomicelle)/(amount of drug added in the procedure)] × 100.

Figure 7. X-ray diffractions of: (A) paclitaxel-loaded nanomicelles; (B) CS-VES-NAC copolymer; (C) the mixture of PTX and the copolymer; (D) paclitaxel.

micelles. The dissolution of PTX solution was so fast that almost 80% of the total drug was released within 12 h, while only 35% of PTX was released from the micellar solution. In addition, the cumulative release percentage of CS-VES-NAC nanomicelles ranged from about 80% up to 168 h for PTX nanomicelles. It was obvious that paclitaxel-loaded CVN nanomicelles exhibited a delayed release pattern up to 168 h. 3.4. Mucin Adhesion. In the mucin adhesion experiment, the CS-VES nanomicelles exhibited mild mucoadhesion, due to the weak noncovalent bonds formed from hydrogen bonds, van der Waals forces, and ionic interactions. In contrast to this, the thiolated nanomicelles exhibited a stronger mucoadhesive force by formation of stable covalent disulfide bonds between the thiol groups of CS-VES-NAC and the cysteine groups of mucin. Therefore, the thiolated CS-VES-NAC nanomicelles exhibited a much more pronounced mucoadhesion force compared with

Figure 6. Characterization of self-assembled paclitaxel-loaded CVN nanomicelles. (A) Particle size distribution. (B) Transmission electron microscopy (TEM) image of paclitaxel-loaded CVN nanomicelles.

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Figure 8. In vitro release profiles of PTX from PTX solution and PTXloaded CS-VES-NAC nanomicelles in pH 6.8 phosphate buffer (containing 2% Cremophor EL, w/v).

the CS-VES nanomicelles. Figure 9A,B shows that CS-VESNAC nanomicelles exhibited about a 2-fold increase in mucin adhesion. As seen from the DSC thermograms (Figure 9C), the thermal melting peak of CS-VES-NAC copolymer was about 240 °C, and this was also seen in the curve of the physical blend without coincubation. However, no decomposition peak was observed in the thermogram of the coincubated mucin/CSVES-NAC copolymer. This indicated that almost all the CVN copolymer had been conjugated with mucin through covalent disulfide bonding. 3.5. In Situ Single-Pass Intestinal Perfusion of CVNPTX Nanomicelles in Rats. The oral absorption of CVNPTX nanomicelles in different rat intestinal segments was investigated using the in situ single-pass perfusion method. For each segment, the absorption parameter (Ka) and the apparent permeability (Papp) were using 20 μg/mL paclitaxel. Compared with PTX solution, PTX-loaded nanomicelles were absorbed in the whole intestine, with the duodenum producing the highest absorption (Figure 10). For PTX-loaded CVN nanomicelles, the absorption in the duodenum segment (Ka, 0.38 ± 0.04 min−1) was 1.1- and 4.5-fold higher, and the apparent permeability (Papp, 0.059 cm·min−1) was 1.5- and 4.6-fold higher than for CS-VES nanomicelles and PTX solution, respectively. There was also a significant increase in permeation produced by CVN-PTX nanomicelles in the jejunum, ileum, and colon segments. As a result, the thiolated nanomicelles effectively improved the absorption of insoluble paclitaxel in the whole intestinal segment, especially in the duodenum. This important finding also suggested that perhaps a special transporting mechanism was responsible for the oral absorption of PTX-loaded thiolated nanomicelles. 3.6. Biodistribution of Coumarin-6 Labeled Nanomicelles in the GI Tract. The biodistribution of coumarin-6 loaded nanomicelles in the GI tract of rats after oral distribution was investigated, and coumarin-6 solution was used as a negative control. A reduced fluorescence of free coumarin-6 solution was observed (Figure 11) in the duodenum, and decreased to the ileum. However, more coumarin-6 labeled CV nanomicelles were absorbed by the duodenum, jejunum, and ileum. Regarding CVN nanomicelles, a stronger fluorescence was observed not only at the surface of all the intestinal tract segments by S−S and electrostatic interaction, but many green signals were visible deep within the intestinal villi. So, it appears that the CVN nanomicelles could rapidly adhere to the intestine mucus layer, and then permeate through the intestinal epithelial cells into the systemic circulation. These observed

Figure 9. Adhesion kinetics of CS-VES-NAC in comparison with CSVES nanomicelles at different concentrations of mucin III: (A) at 0.5 mg/mL; (B) at 1 mg/mL. The amount of mucin adsorbed means the amount of mucin adsorbed by 2 mg of each kind of polymer. Data are shown as mean ± SD, n = 3. *, p < 0.05; **, p < 0.01 vs CS-VES group. DSC thermograms (C) of mucin, CS-VES-NAC polymer, physical mixture, and coincubated mucin/CS-VES-NAC nanomicelles.

images were in good agreement with the results obtained by the in situ single-pass intestinal perfusion experiment. 3.7. The Endocytosis Mechanism of Paclitaxel-Loaded CVN Nanomicelles in Everted Intestinal Rings. Endocytosis plays an important role in nanoparticle transport and absorption. To clarify the absorption mechanisms of CV and CVN nanomicelles, the absorption experiment was performed with different types of endocytosis inhibitors using the everted intestinal ring method. We used chlorpromazine as a clathrindependent endocytosis inhibitor, indomethacin as a caveolin3454

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Figure 10. The in situ absorption of CS-VES-NAC and CS-VES nanomicelles in rat intestinal segments compared with PTX solution. (A) The absorption rate (Ka). (B) The apparent permeability (Papp). Data are shown as mean ± SD, n = 3. *, p < 0.05; **, p < 0.01 vs CSVES-NAC group.

dependent endocytosis inhibitor, colchicine as a macropinocytosis inhibitor, quercetin as a caveolae- and clathrinindependent endocytosis inhibitor, and sodium azide as an energy inhibitor. As shown in Figure 12, the everted intestinal ring absorption of nanoparticles was involved in many endocytosis pathways. All the endosytosis inhibitors mentioned above had a clear effect on everted intestine ring absorption. First, 25 μmol/L sodium azide inhibited the absorption of the nanomicelles, suggesting that the endocytosis pathways were energy-dependent. The absorption of paclitaxel-loaded CV and CVN nanomicelles was controlled by clathrin-mediated, caveolae-mediated, and caveolae- and clathrin-independent endocytosis and macropinocytosis. This experiment was also performed at 4 °C, a temperature at which endocytosis is usually inhibited. The data obtained showed that the absorption of CV and CVN nanomicelles at 4 °C was reduced to below 10%. This result suggests that endocytosis plays a significant role in the absorption of CV and CVN nanomicelles. 3.8. Pharmacokinetics. To eliminate the gastric bioadhesion of CS-VES-NAC nanomicelles, and to correspond with the results of in situ single-pass perfusion, the pharmacokinetics was studied following duodenal administration in rats. The mean plasma concentration−time curves of PTX after administration of PTX solution at a dose of 15 mg/ kg and CV and CVN nanomicelles at a dose of 7.5 mg/kg, respectively, are shown in Figure 13, and the pharmacokinetic parameters are summarized in Table 3. The plasma concentration of PTX after administration of CVN nano-

Figure 11. The fluorescence micrographs of rat intestine (line 1, duodenum; line 2, jejunum; line 3, ileum) 30 min after oral administration of coumarin-6 labeled solution (A), CS-VES-NAC micelles (B), and CS-VES micelles (C) to rats. The histological sections were counterstained with DAPI (blue) and Rhodamine phalloidin (red) and observed under CLSM (20×). 3455

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mL, increased 1.42-fold compared with PTX-sol and 3.3-fold compared with CV nanomicelles (P < 0.05). Moreover, the AUC0−t of CVN nanomicelles was 586.37 ng/mL·h, an increase of 2.12-fold in comparison with that of PTX-sol. In addition, the relative bioavailability of thiolate CS-VES-NAC nanomicelles was 425.06% compared with that of PTX solution.

4. DISCUSSION Insolubility and poor permeability have been a major problem for the oral administration of BCS IV class drugs, such as paclitaxel. It is important to find an effective approach to enhance its oral absorption by overcoming absorption barriers. With the development of nanotechnology, nanomicelles had been widely used for anticancer drug delivery. For decades, it has been known that the cellular permeability of Caco-2 monolayers can be significantly increased by thiolated copolymer. Although this kind of polymer plays an important role in the delivery of hydrophilic drugs and macromolecules, it is unable to effectively improve the absorption and bioavailability of insoluble anticancer drugs. To our knowledge, there are few reports about how the absorption of hydrophobic drugs can be increased by nanomicelles based on thiolated polymers. Chitosan, a polysaccharide derived from chitin by incomplete or complete deacetylation, has been widely used in oral delivery nanosystems due to its positive charge, good biodegradability and biocompatibility, and long-term safety. VES, an esterified redox-silent analogue of vitamin E, has been reported to be a selective apoptosis inducer in cancer cells, rather than in normal cells.34,35 Therefore, we developed the new amphiphilic CSVES nanomicelles, in which chitosan acts as the hydrophilic backbone and VES as the hydrophobic chain. To further increase the bioadhesion and permeation, CS-VES nanomicelles were covalently modified by NAC molecules with free thiol groups to form disulfide bonds with intestinal mucin, since NAC possesses some advantages over other mercapto compounds, such as good thiol activity, suitable reactivity, and fewer reaction byproducts. In this work, the anticancer drug paclitaxel was successfully incorporated into nanomicelles based on a thiolated amphiphilic copolymer. We successfully synthesized an Nacetyl-L-cysteine modified CS-VES copolymer, which could form core−shell nanomicelles through a self-assembly process in aqueous solution. The prepared CVN nanomicelles had a high entrapment efficiency of up to 80.12%, and the drug loading was 24.18%. The positive zeta potential of CVN nanomicelles might be caused by the amino groups of chitosan on the surface of nanomicelles. The introduction of NAC into this system did not affect the particle size and size distribution. The nanoparticles were uniform and round-shaped as shown by their TEM image. In addition, PTX-loaded CVN nanomicelles exhibited a sustained release profile up to 168 h. The results of in situ single-pass perfusion indicated that the most effective absorption domain of CVN nanomicelles was the duodenum segment, followed by the jejunum and ileum. The absorption rate (Ka) and apparent permeability (Papp) were markedly higher than those of PTX solution. This enhancement in intestinal absorption was due to the following factors. First of all, the water-insoluble vitamin E succinate was located in the inner hydrophobic core as a drug reservoir, which facilitated the drug loading and solubility of paclitaxel. Second, the outer hydrophilic chitosan maintains the thermodynamic stability of nanomicelles. Third, when reaching the surface of the small intestine, the positive charged chitosan rapidly

Figure 12. The endocytosis pathways of CV and thiolated CVN nanomicelles after incubation with endocytosis inhibitors for 45 min at 37 or 4 °C. Data are shown as mean ± SD, n = 3. *, p < 0.05; **, p < 0.01 vs control group.

Figure 13. Mean plasma concentration−time curves of paclitaxel in rats after administration of PTX solution at a dose of 15 mg/kg and CV and CVN nanomicelles at a dose of 7.5 mg/kg, respectively (mean ± SD, n = 5).

Table 3. Pharmacokinetic Parameters of Paclitaxel in Rats after Duodenal Administration of PTX Solution (15 mg/kg) and CV and CVN Nanomicelles (7.5 mg/kg), Respectively (Mean ± SD, n = 5)a nanomicelles parameters

PTX solution

CV

CVN

Cmax (ng/mL) Tmax (h) AUC(0−t) (ng/ mL·h) AUC(0−∞) (ng/mL·h) t1/2 (h)

27.39 ± 10.98 5.20 ± 1.79 275.9 ± 150.78

11.81 ± 4.53 1.80 ± 2.0* 145.78 ± 64.84

38.96 ± 22.44 3.80 ± 2.68 586.37 ± 126.11

373.53 ± 163.1

234.58 ± 77.92

2257.90 ± 290.85

8.94 ± 6.54

11.93 ± 10.90

38.53 ± 23.3

a

Statistically significant difference between PTX solution and nanomicelles, *P < 0.05.

micelles was much higher than that of PTX solution and CV nanomicelles. The Cmax of CVN nanomicelles was 38.96 ng/ 3456

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Shenyang, 110016, China; tel, +86-24-23986325; e-mail, [email protected].

adheres to the negative glycoproteins through an electrostatic interaction. More importantly, the free thiol groups on the surface of CVN nanomicelles are able to form disulfide bonds with the cysteine-rich mucin layer, thereby being able to effectively penetrate into the mucus and be internalized by enterocytes. Suitable in vivo pharmacokinetic characteristics are fundamental for successful oral anticancer chemotherapy. This study focused on the development of CVN nanomicelles, which might exhibit suitable bioadhesion and increased permeation for improved oral absorption. The pharmacokinetic results obtained show that the oral bioavailability could be significantly increased after paclitaxel was encapsulated into CVN nanomicelles. The AUC0−t of paclitaxel was clearly higher in the new type of CVN nanomicelles compared with PTX solution and CV nanomicelles. In summary, the intestinal absorption of paclitaxel in rats is markedly increased by CVN nanomicelles in terms of bioadhesion and permeation enhancement. The image in the abstract graphic illustrates the mechanism of the absorption improvement: paclitaxel-loaded CVN nanomicelles stably adhere to the surface of the intestine, and rapidly penetrate through the mucus layer, allowing effective entry into epithelial cells, followed by transfer to the systemic circulation.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Nature Science Foundation of China (No. 81173008), the National Basic Research Program of China (973 Program) No. 2009CB930300, Project for Excellent Talents of Liaoning Province (No. LR20110028), and Program for New Century Excellent Talents in University (No. NCET-12-1015).

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5. CONCLUSION A new type of self-assembled CS-VES-NAC conjugate was synthesized by covalent modification of N-acetyl-L-cysteine with CS-VES. Paclitaxel was successfully incorporated into a CS-VES-NAC nanocarrier to form bioadhesive and permeation-enhancing nanomicelles. These novel nanomicelles exhibited many fascinating features in terms of their size distribution, zeta potential, drug loading, and sustained-release profile. Owing to the introduction of thiol groups into the nanocarrier, the absorption of CS-VES-NAC nanomicelles was greatly improved in the in situ single-pass perfusion experiment compared with PTX-sol. The CLSM experiment further confirmed the superiority of CS-VES-NAC nanomicelles with regard to their mucoadhesion and permeation-enhancing effects. The relative bioavailability of paclitaxel loaded CSVES-NAC nanomicelles was higher compared with that of PTX-sol and CS-VES nanomicelles. All of these results show that CS-VES-NAC nanomicelles have many promising features as nanocarriers for the oral absorption of anticancer drugs.

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ASSOCIATED CONTENT

S Supporting Information *

The 1H NMR spectrum of N-acetyl-L-cysteine (NAC) in D2O (Figure 1s). Energy dispersive X-ray spectroscopy (EDS) of CS-VES copolymer showing the presence of carbon (C), oxygen (O), nitrogen (N), and chlorine (Cl) atoms (Figure 2s). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Z.H.: Mailbox 59#, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China; tel/fax, +86-2423986321; e-mail, [email protected]. J.S.: Mailbox 32#, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, 3457

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