Multifunctional Poly(methyl vinyl ether-co-maleic anhydride)-graft

Article pubs.acs.org/molecularpharmaceutics

Multifunctional Poly(methyl vinyl ether-co-maleic anhydride)-graf thydroxypropyl-β-cyclodextrin Amphiphilic Copolymer as an Oral High-Performance Delivery Carrier of Tacrolimus Dong Zhang,† Xiaolei Pan,†,‡ Shang Wang,†,§ Yinglei Zhai,*,∥ Jibin Guan,† Qiang Fu,† Xiaoli Hao,† Wanpeng Qi,† Yingli Wang,† He Lian,∥ Xiaohong Liu,† Yongjun Wang,† Yinghua Sun,† Zhonggui He,† and Jin Sun*,†,⊥ †

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China ‡ Department of Pharmaceutics, Virginia Commonwealth University, Richmond, Virginia 23284-2526, United States § Jiangsu Hengrui Medicine Co., Ltd., No. 7, Kunlunshan Road, Lianyungang Eco & Tech Development Zone, Jiangsu, 222047, China ∥ School of Medical Devices, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China ⊥ Municipal Key Laboratory of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China ABSTRACT: In order to improve oral bioavailability of tacrolimus (FK506), a novel poly(methyl vinyl ether-co-maleic anhydride)-graf t-hydroxypropyl-β-cyclodextrin amphiphilic copolymer (CD-PVM/MA) is developed, combining the bioadhesiveness of PVM/MA, P-glycoprotein (P-gp), and cytochrome P450-inhibitory effect of CD into one. The FK506-loaded nanoparticles (CD-PVM/MA-NPs) were obtained by solvent evaporation method. The physiochemical properties and intestinal absorption mechanism of FK506loaded CD-PVM/MA-NPs were characterized, and the pharmacokinetic behavior was investigated in rats. FK506-loaded CD-PVM/MA-NPs exhibited nanometer-sized particles of 273.7 nm, with encapsulation efficiency as high as 73.3%. FK506-loaded CD-PVM/MA-NPs maintained structural stability in the simulated gastric fluid, and about 80% FK506 was released within 24 h in the simulated intestinal fluid. The permeability of FK506 was improved dramatically by CD-PVM/MA-NPs compared to its solution, probably due to the synergistic inhibition effect of P-gp and cytochrome P450 3A (CYP3A). The intestinal biodistribution of fluorescence-labeled CD-PVM/MA-NPs confirmed its good bioadhesion to the rat intestinal wall. Two endocytosis pathways, clathrin- and caveolae-mediated endocytosis, were involved in the cellular uptake of CD-PVM/MA-NPs. The important role of lymphatic transport in nanoparticles’ access to the systemic circulation, about half of the contribution to oral bioavailability, was observed in mesenteric lymph duct ligated rats. The AUC0−24 of FK506 loaded in nanoparticles was enhanced up to 20-fold compared to FK506 solutions after oral administration. The present study suggested that the novel multifunctional CD-PVM/MA is a promising efficient oral delivery carrier for FK506, due to its ability in solubilization, inhibitory effects on both P-gp and CYP 3A, high bioadhesion, and sustained release capability. KEYWORDS: tacrolimus (FK506), nanoparticles, bioavailability, P-gp, CYP 3A

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25%).4 The major reasons responsible for low oral bioavailability include (i) its very low aqueous solubility (1.27 μg/ mL),5 (ii) cytochrome P450 3A (CYP3A)-mediated first-pass intestinal and hepatic metabolism,6 and (iii) P-glycoprotein (Pgp) mediated efflux transport in the intestinal epithelium.7 To address the issues mentioned above, various approaches have been investigated to improve oral bioavailability of FK506.

INTRODUCTION Tacrolimus (FK506, molecular weight of 803.03 Da), a 23member macrolide calcineurin inhibitor isolated from Streptomyces sukubaensis early in 1984,1 exerts potent immunosuppressive effects and is widely used as prophylaxis against organ transplant rejection reaction and treatment for T cell mediated autoimmune diseases.2 Compared with other immunosuppressants, FK506 is more potent (up to 100-fold)3 and elicits fewer adverse effects. However, FK506 belongs to BCS (biopharmaceutical classification system) class II drugs with narrow therapeutic index, and its efficacy is strongly restricted due to the low and highly variable oral bioavailability (4−89%, mean © 2015 American Chemical Society

Received: Revised: Accepted: Published: 2337

January 4, 2015 April 14, 2015 May 29, 2015 May 29, 2015 DOI: 10.1021/acs.molpharmaceut.5b00010 Mol. Pharmaceutics 2015, 12, 2337−2351

Article

Molecular Pharmaceutics

in rat intestinal tract was visualized by confocal laser scanning microscopy (CLMS). The possible role of mesenteric lymphatic transport on nanoparticles in accessing systemic circulation was also studied by mesenteric lymph duct ligation in rats. Finally, the pharmacokinetics and oral bioavailability of CD-PVM/MA-NPs at different dosages were investigated in rats, compared with FK506 solutions.

In the earlier years, the main work merely focused on the increase in drug solubility, such as preparation of highly watersoluble prodrug,8 inclusion complex with cyclodextrins,5 and solid dispersions,9 while the efficacy to enhance FK506 oral bioavailability was limited. Subsequently, more drug delivery systems were developed by considering the synergistic inhibitory effect of CYP3A and P-gp on oral bioavailability of FK506. In 2008, double-coated FK506-loaded nanocapsules were prepared, given that the nanocapsules could escape the recognition of P-gp and protect FK506 from CYP3A metabolism via cellular endocytosis pathway.10 Unfortunately, the protection provided by nanocapsules disappeared when FK506 was released as the free drug molecules.11 With this in mind, a new self-microemulsifying drug delivery system (SMEDDS) was developed and a significant improvement in bioavailability was achieved by the application of excipients with inhibition effects on P-gp and CYP3A.12 But SMEDDS suffered from limitations owing to the rapid release of drug, which increases the risk of causing toxicity. It is noticed that the capability to control drug release for maintaining a smooth blood concentration was also essential,13 especially for drugs with narrow therapeutic index. To some extent, all of the above strategies could improve the bioavailability of FK506, but suffered from different shortcomings. In this study, a new amphiphilic graft copolymer, poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) grafted with hydroxypropyl-β-cyclodextrin (HP-β-CD), was synthesized to develop an FK506-loaded nanoparticulate drug delivery system with an expectation of overcoming the above drawbacks. Apart from the ability of cyclodextrins to modify the solubility of hydrophobic drugs, their inhibitory effect on the activity of both P-gp14,15 and CYP3A16 has been confirmed recently. Besides, the choice of HP-β-CD was made in terms of its safety suggested by the Food and Drug Administration (FDA).17 Meanwhile, PVM/MA (Gantrez AN) is a biocompatible copolymer widely used in pharmaceutical applications. PVM/ MA belongs to GRAS (generally recognized as safe) excipients18 and is applied as denture adhesives, thickening and suspending agents, and transdermal adjuvants.19 Recently, it was found that nanoparticulate systems based on PVM/MA were suitable for drug20−22 or antigen23−25 oral delivery. First, bioadhesive properties provided by PVM/MA could extend the gastrointestinal retention of drug loaded nanoparticles effectively.26 Moreover, this capability of bioadhesion could be improved when particulate systems were coated with some excipients (bovine serum albumin,27 poly(ethylene glycol),28 or dextran29). Finally, the anhydride groups in PVM/MA showed a good amenability for multifunctional modifications with hydroxyl (polyethylene glycol21) or amino groups (polyethylenimine30). Therefore, with HP-β-CD grafted to PVM/MA, a new amphiphilic graft copolymer (CD-PVM/MA) was obtained with integrated functions of mucosa bioadhesion and inhibition effects of P-gp and CYP3A. The successful synthesis of CD-PVM/MA was confirmed by infrared (IR) spectra and 1H nuclear magnetic resonance (1H NMR) spectra. Then FK506-loaded CD-PVM/MA nanoparticles were prepared by the solvent evaporation method. The physicochemical properties of FK506-loaded nanoparticles were characterized, including morphology, size, zeta potential, drug loading, encapsulation efficiency, and release behavior in vitro. Effects of nanoparticles on the permeability of FK506 were investigated by in situ single-pass intestinal perfusion and everted gut sac methods. The biodistribution of nanoparticles

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EXPERIMENTAL SECTION

Materials. FK506 (purity: 99.8%) was purchased from Jinan Huifengda Chemical Industrial Co., Ltd. (Shandong, China). Glyburide (purity: 99.5%) was obtained from Tianjin Institute of Drug Pharmaceutical Co., Ltd. (Tianjin, China). Poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA, Gantrez AN 119; MW 200 kDa) was kindly gifted by ISP (Barcelona, Spain). Hydroxypropyl-β-cyclodextrin (HP-β-CD) was supplied by Shandong Xinda Chemical Co., Ltd. (Shandong, China). 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) was obtained from Zhejiang Pukang Pharm Co., Ltd. (Zhejiang, China). Verapamil and coumarin-6 were purchased from Sigma (St. Louis, MO, USA). Rhodamine-labeled phalloidin was obtained from Cytoskeleton (1830 S. Acoma St., Denver, CO, USA). Acetone, tetrahydrofuran (THF), and anhydrous ethanol of analytical grade were purchased from Shandong Yuwang Industrial Co., Ltd. (Shandong, China) and dehydrated by 4 Å molecular sieves. All other reagents used in this investigation were high performance liquid chromatography (HPLC) or reagent grade. Deionized−distilled water was used throughout this study. Synthesis of Amphiphilic CD-PVM/MA Graft Copolymer. CD-PVM/MA was synthesized via the formation of ester bonds between the hydroxyl groups in HP-β-CD with the anhydride groups in the PVM/MA backbone using EDC as coupling agent. Briefly, PVM/MA (100 mg, 0.5 μmol) was activated in 5 mL of mixed solution of anhydrous acetone and anhydrous tetrahydrofuran (THF) (3:2, v/v) with EDC (10 mg, 52.2 μmol) in ice bath under stirring condition for 2 h without exposure to light. HP-β-CD (25 mg, 17.9 μmol) was added to the activated reaction solution with stirring, followed by the reaction being carried out at ambient temperature (25 °C) for 3 h under the protection of nitrogen. Then the solution was filtered to remove the unreacted HP-β-CD. The filtrate was further purified with a dialysis bag (molecular weight from 12,000 to 14,000) in excess water for 3 days. The white graft copolymer powders were obtained by freeze-drying. Characterization of Amphiphilic CD-PVM/MA Copolymer. IR spectra of HP-β-CD, PVM/MA, a physical mixture of CD and PVM/MA (1:4, w/w), and CD-PVM/MA were recorded on an IFS55 infrared spectrophotometer (Bruker, Switzerland). Each sample was mixed with potassium bromide (KBr) and compressed into a KBr disk. The spectrum was recorded at wavenumber ranging from 4000 to 400 cm−1 and a resolution of 8 cm−1. The structure of CD-PVM/MA was further confirmed by 1H NMR, for which all samples were dissolved in DMSO-d6. 1H NMR spectra of the polymers were performed on a Bruker AVANCE III HD instrument operating at a frequency of 400 MHz. Besides, the degree of substitution (DS) of CD-PVM/ MA based on the data of 1H NMR was also calculated as follows: 2338

DOI: 10.1021/acs.molpharmaceut.5b00010 Mol. Pharmaceutics 2015, 12, 2337−2351

Article

Molecular Pharmaceutics DS (%) =

I1ppm I1.7ppm

×

a 0.45 μm syringe filter. Detection wavelength was 220 nm. Column temperature was maintained at 60 °C. The samples were delivered at a flow rate of 1.0 mL/min. The HPLC method was linear (r = 0.9995) in the concentration range of 10.0−200.0 μg/mL. The DL and EE were calculated according to the following formulas (eq 1, eq 2).

2 × 100% 21

where 1 ppm was the characteristic peak of HP-β-CD, 1.7 pmm was the characteristic peak of PVM/MA, 2 was the hydrogen atom number of −CH2− in PVM/MA, and 21 was the hydrogen atom number of −CH3 in HP-β-CD. Preparation of FK506-Loaded CD-PVM/MA-NPs. The solvent evaporation method was employed to prepare FK506loaded CD-PVM/MA nanoparticles, and the whole preparation process is shown in Figure 1. Briefly, CD-PVM/MA polymer

DL (%) =

amount of FK506 loaded in NPs × 100% total amount of NPs

(1)

EE (%) =

amount of FK506 loaded in NPs × 100% total amount of FK506

(2)

Microscopical Observation. The morphology of CD-PVM/ MA-NPs was observed by H-600 transmission electron microscope (TEM) (Hitachi, Japan). A drop of diluted nanoparticle solution was negatively stained with 2% phosphotungstic acid for 5 min on a copper grid and dried at room temperature before observation. Differential Scanning Calorimetry (DSC). DSC analysis was carried out using a TA-60 WS instrument (Shimadzu, Japan) to examine the status of FK506 in the CD-PVM/MA-NPs. For DSC measurement, samples, including FK506, blank nanoparticles, FK506 nanoparticles, and a physical mixture of FK506 and blank nanoparticles, were weighed accurately and sealed into an aluminum pan. The analysis was conducted with heating temperature ranging from 20 to 260 °C at a rate of 10 °C/min under nitrogen atmosphere. In Vitro Release of FK506 from CD-PVM/MA-NPs. The release tests were studied by the dialysis method. 1 mL of FK506-loaded CD-PVM/MA-NP suspension (1.0 mg/mL) was sealed in a dialysis membrane bag (molecular weight from 12,000 to 14,000) and was immersed in 50 mL of release medium (hydrochloric acid solution (pH 1.2, 0.5% sodium dodecyl sulfonate (SDS)) or phosphate buffer solution (PBS) (pH 6.8, 1% SDS). Besides, the release test of FK506 solutions was also carried out with PBS (pH 6.8, 1% SDS) as release medium. The solutions of FK506 were prepared according to a previous report:12 The desired amount of FK506 was added to a mixture consisting of PEG 400, ethanol, and water (1:1:3, v/ v/v) to obtain the drug solutions at a final concentration of 1.0 mg/mL. The samples were placed in a constant shaking water bath at 100 rpm at 37 °C. At predetermined time intervals, 5 mL samples were withdrawn and fresh media were simultaneously replenished to maintain a constant total volume. The FK506 content was determined under the same HPLC conditions as described above. Permeability across Enterocyte. Sprague−Dawley (SD) rats (male, 240−260 g, Central Animal Laboratory of Shenyang Pharmaceutical University, China) were employed for all animal studies. The animal experimental protocols described below 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. In Situ Single Pass Intestinal Perfusion (SPIP) Method in Rats. Before the SPIP experiment, FK506-loaded CD-PVM/ MA-NPs or solutions were diluted by Krebs Ringer (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 water) as perfusates. And the stability of FK506 nanoparticles in blank KR buffer solution was investigated by measuring the size of nanoparticles during 105 min at 37 °C.

Figure 1. Procedures for preparation of FK506 CD-PVM/MA-NPs (suspension and dried powders).

(100 mg) and FK506 were dissolved in 1 mL of mixed solvent of acetone and THF (2:1, v/v) and stirred for 5 min to mix uniformly. Then the mixture was slowly added dropwise into an ethanol/water mixture (2:1, v/v), stirring for 5 min. The resulting solution was then sonicated using a JY92-2D probetype sonifier (Ningbo Scientz Apparatus Research Institute, China) at 200 W for 10 min with the pulse turned off for 3 s at intervals of 2 s in an ice bath. After that, the organic solvents were removed by RE-52A rotary evaporation (Shanghai Yarong Biochemical Instrument Plant, China). Finally, the solution was centrifuged at 3500 rpm for 10 min to remove the free drug using LDZ5-2 low speed autobalancing centrifuge (Beijing Medical Centrifuge Factory, China), acquiring FK506-loaded CD-PVM/MA-NP solution. 7.5% mannitol and 5% maltose were added as freeze-dried protective agents, and vacuum freeze-drying was employed to obtain the freeze-dried powder for storage. Characterization of FK506-Loaded CD-PVM/MA-NPs. Size and Zeta Potential. The size, size distribution, and zeta potential of FK506-loaded CD-PVM/MA-NPs were evaluated by Nano ZS Zetasizer (Malvern, U.K.) based on the dynamic light scattering (DLS) method. The measurements were repeated in triplicate, and the results are shown as mean ± standard deviation (SD). Drug Loading (DL) and Encapsulation Efficiency (EE). The content of FK506 entrapped and loaded in CD-PVM/MA-NPs was determined by high performance liquid chromatography (HPLC) (Hitachi, Japan). A reverse-phase ODS Kromasil-C18 (Akzo Nobel, Sweden) column (200 mm × 4.6 mm, 5 μm) was used. In brief, 1 mL of FK506-loaded CD-PVM/MA-NP suspension was diluted with 25 mL of mobile phase (acetonitrile/water: 75/25), and sonication was applied to collapse the nanoparticles and dissolve the drug. 20 μL of the solution was then injected to HPLC after being passed through 2339


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(37 °C) was added to the sac. The vials with samples were accurately weighed again, and the samples were treated and determined by the UPLC−MS/MS method as described in Pharmacokinetic Studies in Rat. Finally, the length and radius of intestinal sac were measured accurately. Papp was calculated as follows:

The in situ SPIP method was used to evaluate the permeability of FK506 loaded in CD-PVM/MA-NPs and solutions in different intestinal segments of rats. It was performed as previously described with slight modification.31 Briefly, rats fasted for 12 h were anesthetized with an intraperitoneal injection of ethyl carbamate (1.0 g/kg of body weight), restrained on a warming pad under an infrared lamp to maintain a body temperature of 37 °C. The abdomen was opened with a midline incision of 3−4 cm. An intestinal segment of about 10 cm for testing (duodenum, jejunum, ileum, or colon) was carefully exposed and cannulated at both ends with two polypropylene tubes. First, the isolated segment was washed gently with 37 °C saline solution and equilibrated with perfusate (equivalent of 20 μg/mL FK506) at a flow rate of 0.2 mL/min. Then the perfusion experiment lasted for 105 min. Perfusate was collected every 15 min in a preweighed receptor vial; meanwhile, preweighed donor vial and the receptor vial were simultaneously replaced and accurately weighed again. At the end of experiment, the length and radius of infused intestinal segment was also measured accurately. The samples were diluted 2-fold with solvent (acetonitrile/water: 75/25) and centrifuged at 13000 rpm for 5 min to obtain supernatants for HPLC analysis. Additionally, three concentrations (10, 20, and 30 μg/mL) of FK506-loaded CD-PVM/MA-NPs or solutions were also infused at the optimal absorption segment to investigate the absorption patterns. The absorption rate (Ka) and apparent permeability (Papp) of FK506 in nanoparticles or solutions were calculated as follows:

Papp =

where dQ/dt was the permeability rate, C0 was the initial concentration of FK506 in donor solution, l was the intestinal sac length, and r was the intestinal sac radius. Biodistribution of CD-PVM/MA-NPs in Rat Intestinal Tract. To visualize the absorption of CD-PVM/MA-NPs after oral administration, coumarin-6 labeled nanoparticles were prepared as described in Preparation of FK506-Loaded CD-PVM/MANPs. Briefly,33 the obtained CD-PVM/MA-NPs were given to rats by oral gavage (1 mg/kg). After 45 min, the rats were sacrificed, and the selected intestinal segments (duodenum, jejunum, ileum, and colon) were gently removed, washed, 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 ambient temperature. Subsequently, the sections were washed with PBS (pH 7.4) twice, labeled with Rhodamine-labeled phalloidin for 90 min at 37 °C, and then incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min to stain the nucleus. Finally, the coverslips were added on microscope slides and observed under a FluoViewFV1000 CLSM (Olympus, Japan). In addition, coumarin-6 dissolved in ethanol was also administrated to rats for CLSM observation as control. Endocytosis Pathways of CD-PVM/MA-NPs in the Everted Intestinal Rings. To clarify the uptake mechanism of FK506 nanoparticles in rat intestine, the everted intestinal ring model34 was employed. Briefly, the ileum segment was removed, everted, and rinsed in 37 °C KR buffer solution and then cut into 80−100 mg pieces. The rings obtained were placed in the wells of a 24-well plate with 1 mL of KR buffer solution and washed twice. Then, the intestinal rings were incubated with different endocytosis inhibitors for 45 min at 37 °C (n = 3). The inhibitors included chlorpromazine (16 μg/mL), indomethacin (36 μg/mL), colchicine (4 μg/mL), quercetin (1.6 μg/ mL), and sodium azide (3 μg/mL). Then, the media were removed and FK506-loaded CD-PVM/MA-NPs (equivalent of 25 μg/mL FK506) diluted by KR buffer solution were added to the wells for a further incubation of 45 min. Finally, the uptake of nanoparticles was terminated by removing the drug containing media and adding cold KR buffer solutions rapidly. After washing and homogenizing, the amounts of FK506 absorbed into intestinal rings were analyzed by HPLC. In addition, the experiments were also performed at 4 °C or without specific inhibitors as controls. The results were expressed as relative absorption ratio (Rr), which was calculated as follows:

⎛ C V ⎞ Q K a = ⎜1 − out · out ⎟ · 2 C in Vin ⎠ πr l ⎝

(

−Q ·ln Papp =

Cout Vout · C in Vin

dQ 1 · dt 2πrlC0

)

2πrl

where Cout and Cin were the concentration of FK506 in the receptor vial and donor vial, respectively; Vout and Vin were the volume of perfusate in the receptor vial and donor vial, respectively (the density of perfusate was determined as 1 kg/ L); Q was the perfusion flow rate, and l was the intestinal length, while r was the intestinal radius. Everted Gut Sac Method in Rats. The everted gut sac method was used to verify the inhibition of CD-PVM/MA-NPs on P-gp. The operation process was conducted as previously described.32 Briefly, intestinal segments were identified and further washed as described in In Situ Single Pass Intestinal Perfusion (SPIP) Method in Rats. Then one end of the segment was tied with a 3−0 silk suture, and subsequently gently everted over using a glass rod to form a sac. The sac obtained was then tied to a hollow glass tube, which was further hung on an iron support with a silk suture, and then filled with blank KR buffer solution as the receptor system. Subsequently, the whole receptor system was incubated in different donor solutions saturated with 95% O2 and 5% CO2 at 37 °C. The donor solutions were prepared by dissolving FK506 solutions in the absence or presence of verapamil (VRP) or dispersing FK506-loaded CD-PVM/MA-NPs by KR buffer solution. The terminal concentration of FK506 was 25 μg/mL, and that of VRP was 100 μg/mL if added. At predetermined times (0.5, 1, 1.5, and 2 h), samples were drawn from the receptor system completely into a preweighed vial, and blank KR buffer solution

R r (%) =

A t /mt × 100% A s /ms

where At and As are the peak areas of samples treated with inhibitors and without inhibitors, respectively; mt and ms were the mass of intestinal rings in test groups and control groups. 2340


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Figure 2. (A) Synthetic route of CD-PVM/MA copolymer and (B) the color change of reaction solution: (B1) faint yellow at the initial addition of HP-β-CD turning to (B2) lavender at the end of reaction.

Mesenteric Lymph Duct Ligation Experiment. To verify the hypothesis that CD-PVM/MA-NPs underwent mesenteric lymphatic transport, mesenteric lymph duct ligation surgery was performed as previously reported with modifications.35 In brief, one-half hour prior to surgery, rats fasted for 12 h received 1 mL of olive oil by oral gavage. Then the rats were anesthetized with an intraperitoneal injection of 350 mg/kg chloral hydrate and placed on a warming pad under an infrared lamp to maintain a body temperature of 37 °C. Then the rats underwent a middle laparotomy from the xiphoid process caudally toward the right flank of the rat. The small intestine was removed from the abdominal cavity and covered with saline-saturated gauze, and the liver was displaced cephalad. The superior mesenteric lymph duct could be clearly visible, accompanying the mesenteric artery. Subsequently, it was isolated by blunt dissection and ligated with a 3−0 silk suture. The intestines were then replaced in the abdominal cavity, and the abdominal incision was closed by using a continuous running suture. In addition, the rats of the control group underwent the same surgery except lymph duct ligation. Three hours later, when rats recovered consciousness and moved freely, 10 mg/kg FK506-loaded CD-PVM/MA-NPs were given to the rats in the ligation group and the control group by oral gavage. 300 μL whole blood samples taken from the retro-orbital plexus were collected in a heparinized tube immediately at predose, 0.25, 0.5, 1, 1.5, 2, 3, 4, and 6 h after administration. The whole blood samples were kept frozen at −20 °C until UPLC−MS/MS analysis as described in Pharmacokinetic Studies in Rat. Rats were permitted free access to 5% glucose solution during the whole experimental process. Pharmacokinetic Studies in Rat. Rats deprived of food overnight but havinf free access to water were divided randomly into five groups. Group A was treated with FK506 solutions (10 mg/kg) by oral gavage; group B, group C, and group D were treated with FK506-loaded CD-PVM/MA-NPs (5, 10, and 15 mg/kg) respectively by oral gavage; group E was treated with FK506 saline solutions (FK506 solutions diluted by saline, 2 mg/kg) by intravenous (iv) administration. Whole blood samples were collected into heparinized tubes at 5, 10, 20, 30, 45, and 90 min and 2, 3, 4, 6, 8, 12, and 24 h and were frozen at −20 °C for UPLC−MS/MS analysis. The FK506

concentrations in whole blood were determined after liquid− liquid extraction using methylene dichloride and n-hexane (1:1) mixture with glibenclamide as internal standard (IS). The chromatographic separations were carried out on an ACQUITY UPLC system (Waters Co., Ltd., Milford, MA, USA) and BEH C18 column (50 mm × 2.1 mm, 1.7 μm; Waters Co., Ltd., Milford, MA, USA) using gradient elution with a mobile phase composed of acetonitrile and water containing 0.1% formic acid. Concretely, the initial mobile phase composition was maintained at 70% acetonitrile for 0.2 min, changed linearly to 80% acetonitrile (0.2−0.5 min), then changed linearly to 85% acetonitrile (0.5−2.8 min) followed by a return to the initial conditions within 0.2 min. The column temperature was maintained at 40 °C with the flow rate set at 0.2 mL/min. The mass spectrometer was operated with an electrospray ionization interface in positive ionization (ESI+) mode. The ionization parameters were as follows: the capillary voltage of 3.2 kV and cone voltage of 50 V; source temperature of 120 °C and desolvation temperature 350 °C; the cone gas flow rates of 50 L/h and desolvation gas flow rates of 500 L/h. The optimized collision energy was 34 and 19 V for FK506 and glibenclamide (IS), respectively. The compounds were analyzed by multiple reaction monitoring (MRM) of the transitions of m/z 826.3 [M + Na]+ → 616.5 [M + H]+ for FK506 and m/z 494.0 → 352.0 for glibenclamide (IS), respectively. The scan time was set at 0.2 s per transition. The maximum plasma concentration of FK506 (Cmax) and the time to reach Cmax (Tmax) were acquired directly from the plasma concentration−time profiles. The area under curve (AUC) was calculated using the linear trapezoidal rule up to the last data point. The elimination rate constant (ke) was calculated as −2.303 multiplied by the slope of the terminal linear phase, and the elimination half-life (t1/2) could obtained by the formula t1/2 = ln 2/ke. The relative bioavailability (Frel) and absolute bioavailability (Fabs) were calculated as follows: Frel % =

Fabs% = 2341

AUCnano /dosenano × 100% AUCsol /dosesol

AUCpo /dose po AUCiv /doseiv

× 100% DOI: 10.1021/acs.molpharmaceut.5b00010 Mol. Pharmaceutics 2015, 12, 2337−2351

Article

Molecular Pharmaceutics Statistical Analysis. The statistical analyses were performed using ANOVA and t test. Analyses were conducted in permeability across enterocyte, endocytosis pathways, mesenteric lymph duct ligation experiment, and pharmacokinetic study. The differences were considered significant at p < 0.05.

PVM/MA, the characteristic peaks of anhydride disappeared and a prominent peak at 1726.4 cm−1 corresponding to carboxyl CO stretch vibration remained, indicating that the opening of cyclic anhydride was conducive to the formation of ester linkages with HP-β-CD. Moreover, it was surprising that carboxylic acid dimer (928.1 cm−1) could not be found in the spectrum, which might be ascribed to the conjugation of HP-βCD containing abundant hydroxyl groups to PVM/MA, inhibiting the formation of carboxylic acid dimer by the hydrogen bond interaction. The chemical structure of CDPVM/MA was also confirmed by 1H NMR spectra (Figure 4). A broad peak at 3.0−3.7 ppm in PVM/MA was attributable to tertiary carbon of the main chain and the methyl group of methoxy group. The peak corresponding to methylene of the main chain was detected at about 1.7 ppm. For CD-PVM/MA, the peak shifts were similar to those of PVM/MA, except that the peaks at 3.0−3.7 ppm were partly overlapped with the characteristic resonances of the HP-β-CD (3.0−4.0 ppm, tertiary carbon of C2, C3, C4, C5 and methylene of C6 in the hydroxyl glucose ring as well as methylene of C7 and tertiary carbon of C8), and a group of evident peaks (4.5−5.0 ppm, hydroxyl groups) and a methyl group peak (1.0 ppm) belonging to HP-β-CD were also observed, demonstrating that HP-β-CD was grafted onto PVM/MA. Besides, a broader peak of carboxyl group (12.3 ppm) indicated that more anhydride ring opened, as could also be seen from the results of IR. These results were consistent with IR, confirming the successful synthesis of CD-PVM/MA. The degree of substitution (DS) of CD-PVM/MA by HP-β-CD was 2.3%, calculating from the 1H NMR spectroscopy based on the peak intensity of −CH3 (1.0 ppm) in HP-β-CD to that of −CH2− (1.7 ppm) in PVM/MA. Preparation and Characterization of CD-PVM/MANPs. To prepare FK506-loaded CD-PVM/MA-NPs, the solvent evaporation method was used without any surfactant, which could overcome the adverse allergic reactions induced by surfactants, such as Cremophor EL contained in Prograf (a marketed formulation of FK506),36 polysorbate 80 in Taxotere (a marketed formulation of docetaxel),37 and some others.38 Feeding ratios from 5:100 to 20:100 (w/w) of FK506 and CDPVM/MA were used to evaluate the effects of drug amount on DL and EE (Table 1). The DL increased with the feeding ratio and reached a plateau at 15:100 (about 9.9%). Meanwhile, the highest EE of 73.3% was also obtained at this ratio. Further increase in this ratio was incapable of enhancing DL of FK506; instead, a decrease of EE was observed. Due to the complex inclusion of lipophilic inner cavities of HP-β-CD and hydrophobic drug, FK506 could be effectively solubilized by CD-PVM/MA-NPs (Figure 5A), and the physical appearances of CD-PVM/MA-NPs are shown in Figure 5B. As shown in Table 1, the size of the nanoparticles of the optimal formulation was 273.7 ± 13.3 nm, with uniform size distribution (polydispersion index (PDI) as 0.081 ± 0.036). The zeta potential of nanoparticles was about −9.82 mV, beneficial to the physical stability of the nanosystem as a result of electrostatic repulsion.39 The morphological analysis of CD-PVM/MA-NPs by TEM is shown in Figure 5C. The nanoparticles were spherical with rough surfaces, contributing to higher bioadhesive interactions with the intestinal mucosa.40 Besides, a smaller size than that obtained by DLS measurements (Figure 5D) was observed, due to shrinkage of the hydration shells during the manufacturing procedure of TEM samples.41

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RESULTS AND DISCUSSION Synthesis and Characterization of Amphiphilic CDPVM/MA Copolymer. Grafting HP-β-CD onto PVM/MA

Figure 3. IR spectra of (A) HP-β-CD, (B) PVM/MA, (C) a physical mixture of HP-β-CD and PVM/MA (1:4, w/w), and (D) CD-PVM/ MA.

involves the esterification reaction of cyclic anhydride with hydroxyl groups in the linking sites (Figure 2A). The reaction condition was strictly water-free and oxygen-free with the use of EDC as a reaction coupling agent. A color change in the reaction solution could be observed (Figure 2B) due to insolubility of HP-β-CD in nonpolar solvent at the beginning of the reaction (faint yellow) and the reduction in its polarity when grafted to PVM/MA (lavender). Excessive HP-β-CD was then removed by filtration for the difference of solubility between HP-β-CD and CD-PVM/MA. The filtrate was dialyzed against an excess amount of water for further purification. The IR spectra of HP-β-CD, PVM/MA, a physical mixture of CD and PVM/MA (1:4, w/w), and CD-PVM/MA are shown in Figure 3. Both PVM/MA and its physical mixture with CD exhibited the characteristic peaks of PVM/MA: CO stretching peaks of anhydride at 1855.4 and 1780.4 cm−1; C− O−C stretching peaks of anhydride at 1224.1 cm−1; anhydride C−O−C/methyl ether C−O−CH3 deformation peaks at 1093.1 cm−1; carboxyl CO stretching peaks at 1729.1 cm−1; and the deformation peaks of carboxylic acid dimer at 928.7 cm−1. Besides, compared with PVM/MA, the physical mixture or CD-PVM/MA showed a broader characteristic peak at around 1093.1 cm−1, owing to the presence of the coupled (C−C/C−O/C−H) stretch vibration from HP-β-CD. For CD2342


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Figure 4. 1H NMR spectra of (A) HP-β-CD, (B) PVM/MA, and (C) CD-PVM/MA.

Table 1. Physicochemical Properties of CD-PVM/MA-NPs (Mean ± SD, n = 3) FK506:copolymer (charge ratio) 5:100 10:100 15:100 20:100 a

particle sizea (nm)

273.7 ± 13.3

PDI

zeta potential (mV)

0.081 ± 0.036

−9.82 ± 1.32

DL (%) 3.06 5.03 9.91 9.19

± ± ± ±

0.13 0.28 0.21 0.54

EE (%) 63.3 53.0 73.3 50.6

± ± ± ±

2.7 2.9 1.6 3.2

Measured by the intensity pattern.

To identify the physical existing status of FK506 in CDPVM/MA-NPs, DSC analyses of FK506, blank nanoparticles, FK506 nanoparticles, and a physical mixture of FK506 and blank nanoparticles were conducted. As shown in Figure 6, the melting peak of pure FK506 was about 136 °C, which was also detected in the physical mixture. However, the melting peak of FK506 was invisible in both blank nanoparticles and FK506loaded nanoparticles. The above results indicated that FK506

was encapsulated into CD-PVM/MA-NPs successfully and existed in the form of an amorphous status. In Vitro Release Study of FK506 from CD-PVM/MANPs. The in vitro release behaviors of FK506 solutions and CD-PVM/MA-NPs were studied in PBS (pH 6.8) and/or in HCl solution (pH 1.2) containing 1% or 0.5% SDS to maintain sink condition. Figure 7A shows the release profile of FK506 from nanoparticles at pH 1.2, with lower than 10% release 2343


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within 2 h, implying the structural stability of nanoparticles in gastric juice. For the pH 6.8 PBS (Figure 7B), the total release of FK506 from nanoparticles was less than 82% within 24 h, whereas it was over 90% within 2 h for FK506 solutions. More importantly, no initial burst release phenomenon was observed, suggesting that no free drug was absorbed onto the surface of nanoparticles and CD-PVM/MA-NPs possessed satisfactory sustained-release property. Membrane Permeability. The membrane permeability of FK506 when loaded in CD-PVM/MA-NPs was investigated in different rat intestinal segments by in situ SPIP and everted gut sac methods. Gravimetric method was used in both methods to calibrate the volume change of samples. Good stability of FK506 nanoparticles in blank KR buffer solution could be maintained as the change rate of particle size was less than 2%. For the SPIP method, higher values of Papp and Ka of FK506 were observed in ileum and colon when loaded in nanoparticles compared with solutions (Figure 8A,B). The Papp of nanoparticles was shown to be the highest in ileum, while the Papp of solutions was highest in duodenum, indicating that nanoparticles changed the major absorption site of FK506. Compared with the solutions, increased permeability of nanoparticles was found in ileum and colon (p < 0.05), however, no improvement was found in duodenum and jejunum. For the former, inhibition of P-gp, mainly expressed in the lower region of the intestine,42 by CD-PVM/MA probably played a dominant role; for the latter, the metabolism of FK506 by CYP3A, distributed mainly in the upper region of the intestine,43 resulted in a lower Cout and an overestimate44 for the intrinsic permeability of FK506 in solutions. The influence of drug concentrations on the permeability of FK506 was also studied (Figure 8C,D). Both Papp and Ka were not affected regardless of the concentrations of FK506 solutions, demonstrating that FK506 solutions were absorbed by passive diffusion. On the contrary, middle and high concentration nanoparticles produced a significant increase in membrane permeability compared with the low concentration (p < 0.05). Besides, the Papp of high concentration nanoparticles was not superior to that of middle concentration. This result suggested that FK506 nanoparticles were perhaps involved in a special saturation absorption mechanism. For the everted gut sac method, the permeability was enhanced in the whole intestine, especially in duodenum and ileum (p < 0.05), when FK506 was incorporated into nanoparticles (Figure 9). The major absorption site of FK506 for nanoparticles was in accordance with the above SPIP result, but the permeability in duodenum and jejunum was improved

Figure 5. (A) Schematic of FK506 loaded CD-PVM/MA-NPs (drug existed in both of lipophilic inner cavities of HP-β-CD and hydrophobic core of nanoparticles). (B) Physical appearance of CDPVM/MA-NPs before (B1) and after (B2) rotary evaporation. (C) TEM images of the CD-PVM/MA-NPs. Scale bar on TEM image is 200 nm. (D) Dynamic light scattering (DLS) analysis of CD-PVM/ MA-NPs.

Figure 6. DSC thermograms of (A) blank CD-PVM/MA-NPs, (B) FK506, (C) FK506 loaded CD-PVM/MA-NPs, and (D) physical mixture of blank CD-PVM/MA-NPs and FK506 (5:1, w/w).

Figure 7. In vitro release profiles of FK506 (A) from CD-PVM/MA-NPs in HCl solution (pH 1.2) containing 0.5% SDS and (B) from CD-PVM/ MA-NPs and FK506 solutions in PBS (pH 6.8) containing 1% SDS (mean ± SD, n = 3). 2344


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Figure 8. Single-pass intestinal perfusion studies (SPIP) in rats. (A) The apparent permeability coefficient (Papp) and (B) absorption rate constant (Ka) of duodenum, jejunum, ileum, and colon for FK506 nanoparticles and solutions at 20 μg/mL. (C) Papp and (D) Ka of duodenum and ileum for FK506 solutions and for FK506 nanoparticles at different concentrations (mean + SD, n = 3), p < 0.05 (*) and p < 0.01 (**).

CD-PVM/MA can enter intestinal epithelium cells by some uptake mechanisms. Biodistribution of CD-PVM/MA-NPs in Rat Intestinal Tract. The biodistribution of coumarin-6 labeled nanoparticles in rat intestinal tract was investigated, and coumarin-6 solutions were used as the negative control. Coumarin-6 was used as a marker for CLMS instead of FK506 to show the contribution by bioadhesion directly. A stronger fluorescence could be observed in the whole intestinal tract after loading in nanoparticles compared with the coumarin-6 solutions (Figure 10). The green signals not only were visible at the surface of intestinal villi but also appeared in myenteron of intestinal tract segments, especially duodenum and ileum. The results suggested bioadhesive properties and then cellular uptake of CD-PVM/MA-NPs. Endocytosis Mechanisms of CD-PVM/MA-NPs. It has been reported that polymeric nanoparticles transport across intestinal membrane by endocytosis process,46 and the endocytotic pathways dominate the intracellular fate of nanoparticles. To clarify the endocytosis mechanism and to investigate the intracellular fate of CD-PVM/MA-NPs, the uptake experiment was performed with different endocytosis inhibitors using the everted intestinal ring model. First, the effect of temperature (37 and 4 °C, a temperature at which the endocytosis process was blocked47) on the uptake of nanoparticles was studied. The results showed that the uptake efficiency of FK506 at 4 °C was reduced to about 20% of that at 37 °C (p < 0.01) (Figure 11). In combination with a significant decrease in uptake (p < 0.01) by sodium azide (energy inhibitor), it could be confirmed that energy-dependent endocytosis played a significant role in the absorption of CDPVM/MA-NPs. Then the effects of different endocytosis inhibitors on uptake were investigated at 37 °C. Compared with the control group without inhibitor, the uptake efficiency

Figure 9. Papp of duodenum, jejunum, ileum, and colon for FK506 nanoparticles and solutions (without or with verapamil) at 25 μg/mL obtained by the everted gut sac method in rats (mean + SE, n = 3), p < 0.05 (*) and p < 0.01 (**).

greatly in comparison with FK506 solutions. Combining both results into one, inhibition of CYP3A by CD-PVM/MA was probably responsible for the discrepancy of permeability in the intestinal upper region between the two methods. In addition, the presence of VRP, a P-gp inhibitor, increased the permeability of FK506 solutions in the intestinal distal region, despite extent less than nanoparticles. The absorption of FK506 was significantly affected by P-gp efflux mechanism, and CDPVM/MA produced a better P-gp inhibition. Finally, it is necessary to point out that CYP3A protein as other members of the CYP450 family was localized in the endoplasmic reticulum (ER).45 Therefore, the direct contact with CYP3A in cytoplasm was a precondition for CD-PVM/MA to exert the inhibition of CYP3A activity. We could postulate from this perspective that 2345


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Figure 10. Fluorescence micrographs of rat intestine (line 1, duodenum; line 2, jejunum; line 3, ileum; line 4, colon) 45 min after oral administration in rats with coumarin-6 solutions (A) and coumarin-6 loaded CD-PVM/MA-NPs (B). The histological sections were counterstained with DAPI (blue) and Rhodamine Phalloidin (red) and observed under CLSM (10×).

Figure 12. Mean concentration−time curves for FK506-loaded CDPVM/MA-NPs (10 mg/kg) groups (mesenteric lymph duct (MLD) ligated group and control group) by oral administration. Each point represents the mean + SE (n = 5).

Figure 11. Endocytosis pathways of FK506-loaded CD-PVM/MANPs after incubation with endocytosis inhibitors (a, chlorpromazine; b, indomethacin; c, colchicine; d, quercetin; e, sodium azide) for 45 min at 37 or 4 °C (mean + SD, n = 3), p < 0.05 (*) and p < 0.01 (**) versus the control.

of FK506 was reduced by 31% after the treatment with chlorpromazine (clathrin-mediated endocytosis inhibitor) (p < 0.05), and 27% when pretreated with indomethacin (caveolaemediated endocytosis inhibitor) (p < 0.01). But no inhibition in the uptake of FK506 was found when preincubated with colchicine (macropinocytosis inhibitor) and quercetin (caveolae- and clathrin-independent endocytosis inhibitor). Accordingly, clathrin-mediated endocytosis and caveolae-mediated endocytosis were involved in the internalization process of CDPVM/MA-NPs. Besides, the two endocytosis pathways implied different intracellular fates of nanoparticles. For the former, nanoparticles underwent the AEE (apical early endosome)/LE (late endosome)/lysosome route,46,48 resulting in the structural collapse of nanoparticles and drug release, while for the latter, nanoparticles could be transported to endoplasmic reticulum

Table 2. Pharmacokinetic Parameters Following Oral Administration of FK506-Loaded CD-PVM/MA-NPs in MLD Ligated and Nonligated Rats (Mean ± SE, n = 5) parameters dosage (mg/kg) Tmax (h) Cmax (ng/mL) k (h−1) t1/2 (h) AUC0−3 (ng/mL·h) a

MLD ligated rats (group F) 10 0.70 67.3 0.77 1.03 95.6

± ± ± ± ±

0.11 12.1*a 0.14* 0.15 11.4*

MLD nonligated rats (group G) 10 0.90 ± 0.09 145.8 ± 18.9 0.30 ± 0.07 3.10 ± 0.87 228.5 ± 24.2

(*) p < 0.05 versus MLD nonligated rats (group G) as the control. 2346


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Figure 13. (A) Mean concentration−time curves for FK506 solution group and FK506 nanoparticle (different dosages) groups by oral administration and (B) for FK506 saline solution group by intravenous injection. Each point represents the mean + SE (n ≥ 5).

Table 3. Pharmacokinetic Parameters Following Oral Administration of FK506 Solutions and Nanoparticles and Following Intravenous Injection of FK506 Saline Solutions in SD Rats (Mean ± SE, n ⩾ 5) po

iv

parameters

solutions (group A)

nanoparticles (group B)

nanoparticles (group C)

nanoparticles (group D)

dosage (mg/kg) Tmax (h) Cmax (ng/mL) k (h−1) t1/2 (h) AUC0−24(ng/mL·h) Frel% Fabs%

10 1.2 ± 0.3a,b 20.7 ± 4.2a,b,e 0.11 ± 0.03 5.76 ± 0.95 173.3 ± 10.8a,b,e 100 2.6 ± 0.2

5 1.0 ± 0.2c,d 120.9 ± 23.5f 0.14 ± 0.01 5.27 ± 0.45 638.4 ± 48.3f 737 ± 56 18.9 ± 1.4

10 2.4 ± 0.4 238.9 ± 95.8g 0.13 ± 0.02 4.93 ± 0.46 1648.0 ± 560.0h 951 ± 323 24.5 ± 8.3

15 3.6 ± 0.7 781.9 ± 149.0 0.21 ± 0.04 3.73 ± 0.72 5279.7 ± 954.1 2031 ± 335 52.2 ± 8.6

solutions (group E) 2

0.16 ± 0.01 4.60 ± 0.40 1347.6 ± 125.9 100.0

a

p < 0.05 compared with group C. bp < 0.01 compared with group D. cp < 0.01 compared with group C. dp < 0.05 compared with group D. ep < 0.01 compared with group B. fp < 0.01 compared with group D. gp < 0.01 compared with group D. hp < 0.05 compared with group D.

contribution of lymphatic transport for oral absorption of FK506-loaded CD-PVM/MA-NPs. Chloral hydrate was selected as the anesthetic to form a conscious rat model with normal circulation level for facilitating the oral administration.51 As shown in Table 2 and Figure 12, the Cmax and AUC0−3 were decreased by 54% and 58%, respectively, after MLD ligation, compared with the control group (p < 0.05). This result demonstrated that the lymphatic route played a dominant role in the oral absorption process for CD-PVM/MA-NPs, nearly half of the contribution to oral bioavailability. Bioavailability Study. The validated UPLC−MS/MS analysis method was used to investigate oral bioavailability of FK506 after administration of CD-PVM/MA-NPs and solutions in rats. The concentrations of FK506 were detected in whole blood samples rather than plasma samples due to its high binding fraction to erythrocytes.52 The mean FK506 blood concentration−time profiles of oral administration groups and intravenous injection group are shown in Figure 13, and the main pharmacokinetic parameters of FK506 are shown in Table 3. For Tmax, it was prolonged 2-fold and 3-fold in groups C (p < 0.05) and D (p < 0.01), respectively, compared with group A (solutions). For Cmax and AUC0−24, all of the nanoparticle groups (groups B, C, and D) were superior to the solution group (group A) with significant statistical difference. Besides, group D was also increased significantly compared with groups B and C (p < 0.01), with a nonlinear absorption characteristic for different doses of CD-PVM/MA-NPs (Figure 14). For k and t1/2, no statistically significant difference (p > 0.05) was found among the five groups (groups A−E), indicating that nanoparticles did not alter the elimination behavior of FK506 in vivo. The relative bioavailability of groups B, C, and D was

Figure 14. Nonlinear correlations between Cmax/AUC0−24 and oral dose for nanoparticles.

(ER) through the AEE/ER pathway,46,49 bypassing the degradation of lysosome. After assembling into chylomicrons in ER, similar to solid lipid nanoparticles,50 CD-PVM/MA-NPs underwent exocytosis through the ER/Golgi pathway and probably became available for lymphatic transport instead of blood capillary for the limited pore size (40−80 nm) of vascular epithelium.32 Thus, the roles played by CD-PVM/MA-NPs in two endocytosis pathways were also different: inhibition of Pgp and CYP3A by the carrier polymers and escapement of the recognition of P-gp and CYP3A. Contribution to Oral Absorption by Mesenteric Lymph Transport. In this study, mesenteric lymph duct (MLD) ligation experiment was used to quantify the potential 2347


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Figure 15. Illustration of the hypothetic oral absorption process for FK506 poly(anhydride) nanoparticles and solutions [reasons for drug loss (numbers in arrows): 1, free drugs or loaded drugs before absorption; 2, drugs released before absorption; 3, drugs untreated by GI clearance; 4, drugs untreated by P-gp and CYP3A; 5, drugs released and untreated by P-gp and CYP3A; 6, loaded drugs after release; 7, drugs released in lysosome; 8, loaded drugs bypassing lysosome; 9, drugs untreated by liver first-pass effect; 10, loaded drugs transported to lymph duct].

about 7.4-, 9.5-, and 20-fold larger than that of group A. For Fabs, the absolute bioavailability of groups A, B, C, and D was 2.6 ± 0.2%, 18.9 ± 1.4%, 24.5 ± 8.3%, and 52.2 ± 8.6%, respectively. From the above results, oral bioavailability was greatly improved after the incorporation of FK506 into CD-PVM/MANPs, superior to that of optimal double-coated nanocapsules (11.0%).10 Concerning the remarkable results, we surmised several contributions made by CD-PVM/MA-NPs throughout the whole absorption process of FK506 (Figure 15). First, most of CD-PVM/MA-NPs were stable in gastrointestinal fluid for at least 2 h, as shown in the stability tests. The inherent bioadhesive properties of Gantrez nanoparticles27 were beneficial to immobilize nanoparticles at the intestinal cell surface by the formation of hydrogen bonds between the carboxyl groups generated after hydrolysis and mucosa components. 53 Besides, the surface binding was also strengthened by the rough surfaces of nanoparticles observed by TEM. Then FK506-loaded nanoparticles entered enterocytes by clathrin- and caveolae-mediated endocytosis, during which the inhibition of CYP3A and P-gp and the protection by intact nanostructures ensured the improved permeability of FK506. Subsequently, more than half of FK506 in NPs were drained to mesenteric lymph duct in terms of the size about 300 nm, bypassing the liver first-pass effect, and directly entering systemic circulation. Besides, high concentration nanoparticles could promote the formation of chylomicron, leading to an increased proportion of FK506 transported via lymphatic pathway, which might explain the nonlinear drug

absorption for CD-PVM/MA-NPs. While a portion of FK506 was released before oral absorption, the amorphous form confirmed by DSC displayed the ability to reach a supersaturated state in liquor entericus,54 establishing a drug concentration gradient to facilitate the absorption by passive diffusion. The free drug also benefited from the inhibiting activity of CD-PVM/MA, avoiding the synergistic effects55 of metabolism and efflux. In summary, the molecular mechanism to increase bioavailability of FK506 by CD-PVM/MA-NPs could be described as follows. (i) Drug loaded nanoparticles: adhere to the surface of enterocytes → caveolae-mediated endocytosis (protection from intact nanostructures) → AEE/ER/Golgi → lymphatic capillary by exocytosis → systemic circulation. (ii) Drug loaded nanoparticles: adhere to the surface of enterocytes → clathrin-mediated endocytosis (protection from intact nanostructures) → AEE/LE/lysosome (drug release, inhibition of P-gp and CYP3A by carrier materials) → blood capillary by passive diffusion → liver → systemic circulation. (iii) Drug released from nanoparticles: transmembrane transport by passive diffusion (inhibition of P-gp and CYP3A by carrier materials) → intracellular transport → blood capillary by passive diffusion → liver → systemic circulation.

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CONCLUSIONS AND OUTLOOK In this study, the CD-PVM/MA-NPs were prepared by an amphiphilic graft copolymer (CD-PVM/MA) to enhance the oral bioavailability of FK506, and the oral absorption mechanism was also clarified. CD-PVM/MA-NPs could 2348


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Molecular Pharmaceutics maintianed structural integrity at the simulated gastric fluid for 2 h and had a delayed-release property over 24 h in the simulated intestinal fluid. The pharmacokinetics study in vivo demonstrated that the relative bioavailability of FK506 in rats was enhanced up to 20-fold compared to solutions, when incorporated into CD-PVM/MA-NPs with the benefit of high bioadhesion, improved permeability, decreased first-pass effect, and contribution of lymphatic transport. In conclusion, CDPVM/MA was evaluated as an oral high-performance carrier for insoluble drug, especially substrates of CYP3A and P-gp, i.e., FK506, to improve the oral delivery efficiency. FK506 is widely used to prevent the organ transplant rejection reaction. However, the therapeutic effect was limited due to the low bioavailability. Low aqueous solubility and the synergistic effects of metabolism and efflux are the main reasons responsible for this phenomenon. This study demonstrated that CD-PVM/MA as a high-performance oral carrier could enhance the bioavailability of FK506 remarkably with a relatively clear absorption mechanism. Future studies will be focused on the possible absorption of FK506 loaded CD-PVM/ MA-NPs by paracellular pathway and M cells and the relationship between concentrations of nanoparticles and the formation of chylomicron. Apart from FK506, nanoparticles composed of CD and PVM/MA have also been applied to several other drugs suffering from poor bioavailability, such as cyclosporin A,56 atovaquone,57 and paclitaxel.58 Although the oral bioavailability was enhanced for all the drugs, the degree of improvement was dependent on the drug’s properties. Atovaquone (a BCS II drug) loaded nanoparticles showed a 2.2-fold AUC compared to that of its methylcellulose suspension,57 whereas FK506 (a BCS II drug, cosubstrates of CYP3A and P-gp) loaded nanoparticles displayed a 20-fold higher AUC compared to the solutions in this study and the nanoparticles of paclitaxel (BCS IV and cosubstrates of CYP3A and P-gp) provided a 33-fold higher bioavailability than Taxol.59 From these results, it was clear that the superiority of CD-poly(anhydride) nanoparticles could be maximized when applied to the drug with limited solubility, limited permeation, and extensive metabolism and efflux. Therefore, further investigation about the application of CD-PVM/MA to delivery of other insoluble drugs, especially cosubstrates of CYP3A and P-gp, will be required.

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Doctoral Foundation Project (No. 20122134110004), Technology bureau in Shenyang (NO.ZCJJ2013402), and General Project of the Education Department of Liaoning Province (NO. L2013394).

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*No. 59 Mailbox, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103 of Wenhua Road, Shenyang 110016, China. Tel/fax: 86-2423986325. E-mail: [email protected]. *No. 129 Mailbox, School of Medical Devices, Shenyang Pharmaceutical University, No. 103 of Wenhua Road, Shenyang 110016, China. Tel/fax: 86-24-23986325. E-mail: zhaiyinglei@ aliyun.com. 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. 81273450, 81202480), National Basic Research Program of China (973 Program), No. 2015CB932100, Program for New Century Excellent Talents in University (No. NCET-12-1015), The Ministry of Education 2349


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