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Cysteine-functionalized nanostructured lipid carriers for oral delivery of docetaxel: A permeability and pharmacokinetic study Guihua Fang, Bo Tang, Yanhui Chao, Helin Xu, Jingxin Gou, Yu Zhang, Hui Xu, and Xing Tang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00081 • Publication Date (Web): 14 May 2015 Downloaded from http://pubs.acs.org on May 16, 2015

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Molecular Pharmaceutics

Cysteine-functionalized nanostructured lipid carriers for oral delivery of docetaxel: A permeability and pharmacokinetic study Guihua Fang1, Bo Tang1, Yanhui Chao1, Helin Xu1, Jingxin, Gou1, Yu Zhang1, Hui Xu1, Xing Tang 1

※

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016,

China

※

Corresponding author: Professor Xing Tang (X. Tang)

Tel: +86 24 23986343 fax: +86 24 23911736. E-mail: [email protected]

Table of contents graphic

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Abstract Here we report the development and evaluation of cysteine-modified nanostructured lipid carriers (NLCs) for oral delivery of docetaxel (DTX). The NLCs ensure high encapsulation efficiency of docetaxel, while the cysteine bound the NLCs with PEG2000-monostearate (PEG2000-MSA) as a linker, and allowed a specific interaction with mucin of the intestinal mucus layer and facilitated the intestinal transport of docetaxel. The cysteine-modified NLCs (cNLCs) had a small particle size (< 100 nm) and a negative zeta potential (-13.72 ± 0.07 mV), which was lower than that of the unmodified NLCs (uNLCs) (-6.39 ± 0.07 mV). This correlates well with the location of the cysteine group on the NLCs surface obtained by X-ray photoelectron spectroscopy (XPS). The cNLCs significantly improved the mucoadhesion properties compared with uNLCs. The intestinal absorption of cNLCs in total intestinal segments was greatly improved in comparison with uNLCs and docetaxel solution (DTX-Sol), and the In Vivo Imaging System captured pictures also showed not only increased intestinal absorption, but improved accumulation in blood. The cNLCs could be absorbed into the enterocytes via both endocytosis and passive transport. The results of the in vivo pharmacokinetic study indicated that the AUC0-t of cNLCs (1533.00 ng/mL.h) was markedly increased 12.3-fold, and 1.64-fold compared with docetaxel solution and uNLCs, respectively. Overall, the cysteine modification makes nanostructured lipid carriers more suitable as nanocarriers for oral delivery of docetaxel.

Key words: cysteine, nanostructured lipid carriers, mucoadhesion, intestinal absorption, docetaxel

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1. Introduction In recent years, an increasing number of oral anticancer drugs have been used clinically or are in the development stage. Compared with intravenous administration, oral delivery of anticancer drugs has many attractive benefits, including improving patient acceptance and reducing financial costs.1 Despite the above-mentioned advantages, oral delivery of anticancer drugs is severely hampered due to poor aqueous solubility, poor intestinal permeability and, in particular, the low bioavailability obtained after oral administration.2 Therefore, it is very important to develop effective approaches to improve the bioavailability of anticancer drugs. Docetaxel is a potent anticancer drug that displays a broad spectrum of anti-tumor activity.3 However, it is poorly water soluble (4.93 µg/mL in purified water) and highly lipophilic (LogP = 4.1).4, 5 Also, it is extremely sensitive to the P-gp efflux pump and cytochrome P450 and so the docetaxel exhibits very low bioavailability.6 The advances in the development of new drug delivery systems had led researchers to design a variety of nanocarriers for the oral delivery of docetaxel. Among these different drug delivery systems, nanostructured lipid carriers have been found to be a promising vehicle for the oral delivery of drugs not only because of their excellent biocompatibility and biodegradability, but also because they have the ability to provide high drug loading, and improve drug bioavailability and stability.7, 8 However, their ability to produce mucus anchorage is still quite weak and, after oral administration, the majority of nanoparticles exhibit rapid intestinal transit, resulting in insufficient uptake at the absorption site.9 In order to overcome these drawbacks, two strategies have recently been applied to improve the residence time of nanoparticles within the gastrointestinal tract. One approach involves the use of positively charged polymer materials.

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Many researchers have attempted to coat the nanoparticles with chitosan or its derivatives to increase mucoadhesion.10-12 Positively-charged chitosan can adhere to negatively charged sialic and sulfonic acids in the mucus layer via electrostatic interaction.13 Yet another approach involves thiolated polymers have been developed to improve the mucoadhesive properties of nanoparticles, and these are often termed “thiomers”.14-17 Thiomers are polymers with sulfhydryl groups, which are able to form covalent disulfide bonds between the sulfhydryl groups of thiomers and the cysteine-rich subdomains of the mucus layer. The underlying mechanism is based on thiol/disulphide exchange reactions and a simple oxidation process.18 In comparison with a physical electrostatic interaction, mucoadhesion of thiomers is based on chemical covalent bonding. Hence, they make the nanocarriers remain in the gut longer, and reduce the effect of gastrointestinal peristalsis and the stomach contents.19 In general, thiomers have been shown to exhibit several advantages regarding drug delivery. Firstly, thiomers can prolong the residence time of nanoparticles at the site of drug absorption, causing a local drug concentration gradient, resulting in a marked increase in absorption.20 Secondly, thiomers produced increased permeation, which can improve drug transport across the intestinal epithelial cells.21 Thirdly, they can inhibit the P-gp activity in the intestinal epithelium, due to the interaction of the thiol groups with the transmembrane domain of the efflux pump,22, 23 which is very important for the influx of the majority of anticancer drugs into intestinal epithelium cells. Encouraged by the advantages of NLCs and thiomers mentioned above, we have attempted to develop thiomer-coated nanostructured lipid carriers for oral delivery poorly water-soluble drugs. In the present study, bioadhesive amphiphilic thiomers (Cy-PEG-MSA) were obtained by conjugating cysteine (Cy) with the amphiphilic polymer polyethylene glycol monostearate

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(PEG-MSA). NLCs acted as a docetaxel loading carrier, and Cy-PEG-MSA was introduced to NLC via hydrophobic monostearate incorporated into the lipid core of the NLCs, and hydrophilic PEG ensures that the thiols were present on the external surface. The docetaxel-loaded lipid carriers were characterized by X-ray photoelectron spectroscopy (XPS), and their particle size, encapsulation efficiency, surface morphology, were investigated along with differential scanning calorimetry (DSC) and the in vitro release behavior was also investigated. The mucoadhesion properties were evaluated by mucus glycoprotein assay. The in situ single-pass perfusion technique was used to study the permeability of docetaxel. The endocytosis pathways of docetaxel-loaded lipid carriers were investigated using the everted gut sac model. Finally, pharmacokinetic studies of docetaxel-loaded lipid carriers were carried out in rats after oral administration

using

UPLC-MS/MS.

In

all,

we

attempted

to

discover

whether

cysteine-functionalized NLC could increase the bioavailability of docetaxel.

2. Materials and Methods 2.1 Materials Polyethylene glycol monostearate (PEG2000-MSA, Mw = 2000) was supplied by Tci Development Co., Ltd. (Shanghai, China). 4-Nitrophenyl chloroformate (4-NP) was obtained from Hatch Chemical Co., Ltd. (Shanghai, China), L-cysteine (L-Cys), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB, Ellman’s reagent) and Porcine stomach mucin (type III) were purchased from Sigma (St. Louis, MO, USA). Docetaxel and paclitaxel were purchased from Shanghai sanwei Pharma Co., Ltd. (Shanghai, China). Precifac ATO 5 (glyceryl palmitostearate) was kindly donated by Gattefosse (Lyon, France). Medium chain triglycerides (MCT) and soybean lecithin (S

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75) were obtained from Lipoid KG (Ludwigshafen, Germany) and Tween 80 was purchased from BASF (Ludwigshafen, Germany). Tert-butyl methyl ether (TBME) was supplied by Sinopharm Chemical Reagent Co., Ltd. (Shenyang, China ), formic acid by Dima Technology Inc., (Richmond Hill, USA), and anhydrous pyridine, tetrahydrofuran, methanol, acetonitrile and dehydrated alcohol by Concord (Tianjin, China), All other chemicals and reagents were of analytical grade or chromatographic grade. DTX solution was prepared according to market Taxotere®. 2.2 Synthesis of thiolated polyethylene glycol monostearate24, 25 Thiolated polyethylene glycol monostearate (Cy-PEG-MSA) was synthesized by (1) activating PEG-MSA with 4-NP in the presence of pyridine, followed by (2) reacting with L-Cys. In detail, PEG-MSA (1.127 g, 0.5 mmol) was dissolved in 40 mL dried THF, in an argon atmosphere , then a solution of anhydrous pyridine (0.198 g, 2.5 mmol) was added, followed by 20 mL dried THF solution containing 4-NP (0.504 g, 2.5 mmol) added dropwise at 4 °C. Upon completion of the addition, the mixture was stirred for about 24 h at room temperature then the salt was removed by filtration, the filtrate was concentrated, followed by precipitation in cold diethylether and drying in vacuo (40 °C) to obtain the polymer, pNP-PEG-MSA. Next, the purified pNP-PEG-MSA (0.480 g, 0.2 mmol) and L-Cys (0.121 g, 1 mmol) were dissolved in 3 mL pH 5.0 ammonium acetate buffer, and the pH of the reaction solution was then adjusted to about 8.0 by adding 4 mol/L NaOH. The reaction proceeded for about 2 h without exposure to light in an argon atmosphere. To terminate the reaction, the pH was then adjusted to 5.0 by adding acetic acid. To remove the unreacted reagents, the resulting polymer solution was transferred to a dialysis bag (MWCO: 1000) and dialyzed at 4 °C against pH 5.0 acetate acid solution. Finally, the dialyzed product was lyophilized

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and stored at -20 °C for further use.

2.3 Characterization of Cy-PEG-MSA polymer 2.3.1 Nuclear magnetic resonance spectroscopy (NMR) of Cy-PEG-MSA The 1H-NMR spectra of all the polymers were acquired on a Bruker spectrometer (Bollerica, MA) operated at a frequency of 400 MHz for protons with CDCl3 as the solvent.

2.3.2 Fourier transform infrared spectroscopy (FTIR) of Cy-PEG-MSA The FTIR spectra of all the polymers were obtained on a Bruker ISF 55 spectrometer, and samples were prepared as KBr disks.

2.3.3 Degree of polymer thiolation The amount of thiol groups on Cy-PEG-MSA was quantified using Ellman’s reagent.26 First, 10 mg Cy-PEG-MSA polymer was hydrated in 5 mL deionized water. Then, 500 µL aliquots were withdrawn, and mixed with 2 mL 0.5 mol/L phosphate buffer (pH 8.0) and 1 mL Ellman’s reagent ( 25 mg in 25 mL of 0.5 mol/L phosphate buffer, pH 8.0). The samples were incubated for 2 h at room temperature, protected from light. The supernatant was obtained after centrifuging at 14,000 rpm for 10 min and the absorbance was determined at 412 nm on an UV-vis spectrophotometer. The amount of free thiol groups was calculated from a standard curve.

2.4 Preparation of unmodified NLCs (uNLCs) and cysteine modified NLCs (cNLCs) The uNLCs and cNLCs were prepared as follows. Docetaxel, Precifac ATO 5, MCT, Tween

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80 and soybean lecithin (1:24:6:24:6, w/w) were dissolved in ethanol by heating to 75 °C. After removal of the ethanol by magnetic stirring for about 30 min, the aqueous solution (7 mL) was added dropwise to the melted lipid under magnetic stirring at 400 rpm in a water bath maintained at 75 °C. Then, the primary emulsion was obtained after stirring for 5 min, and ultrasonicated using a probe sonicator for 3 min at 400 W. Subsequently, the obtained nanoemulsion was mixed with aqueous solution (3 mL) containing PEG-MSA or Cy-PEG-MSA at 75 °C and incubated for 10 min under magnetic stirring. Finally, the resultant nanoemulsion was cooled in an ice bath for 1 h to form uNLCs and cNLCs.

2.5 Characterization of uNLCs and cNLCs 2.5.1

Surface analysis using X-ray photoelectron spectroscopy (XPS)

To confirm that the thiol was present on the cNLCs surface, X-ray photoelectron spectroscopy was applied using an ESCALAB 250 instrument (Thermo VG Corporation) with monochromated AlKα radiation (1486.6 eV). The calibration was performed by referring the C1s to the binding energy at 285 eV. The pressure in the XPS analysis chamber was 6.0 × 10-8 mba with an analyzer pass energy of 50 eV. The concentration of each element was calculated from the area of the corresponding peak.

2.5.2 Measurement of particle size and zeta potential The average particle size and size distribution of uNLCs and cNLCs were measured by dynamic light scattering using a Nicomp™ 380 submicron particle sizer (Santan Barbara, CA, USA) at room temperature. The zeta potential of uNLCs and cNLCs was measured by

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electrophoretic light scattering using the same equipment.

2.5.3 Particle Morphology Samples of uNLCs and cNLCs were dropped onto a copper grid and then negatively stained with 2% phosphotungstic acid. The samples were air-dried then examined by transmission electron microscopy.

2.5.4 Encapsulation Efficiency (EE) and Drug-Loading Content (DL) The DTX content was measured by high performance liquid chromatography (HPLC) as previously described.27 The encapsulation efficiency (EE) of uNLCs and cNLCs was determined by measuring free DTX in the aqueous phase. Ultrafiltration tubes (MWCO:100 kDa) were used to separate the free DTX from uNLCs and cNLCs by centrifuging at 3000 rpm for 10 min. The DTX in the ultrafiltrate was determined by HPLC. The amount of DTX incorporated into the uNLCs and cNLCs was determined after ethanol dilution and ultrasonic disruption by HPLC. The entrapment efficiency (EE, %) and drug loading (DL, %) of DTX in the uNLCs and cNLCs were then calculated from eqs 1 and 2, respectively.

EE % =

DL% =

Wtotal drug − W free drug Wtotal drug

Wtotal drug − W free drug Wtotal lipid

× 100% (1)

× 100% (2)

where Wtotal drug, Wfree drug and Wtotal lipids represent the total amount of DTX in the uNLCs or cNLCs, the amount of free drug in the aqueous phase and the amount of lipid added to the uNLCs

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or cNLCs, respectively.

2.5.5 Drug state in the uNLCs and cNLCs

To evaluate the physical state of docetaxel in the uNLCs and cNLCs, freshly prepared samples were lyophilized before the measurements. The thermal behaviors of the samples were examined by differential scanning calorimeter (Mettler-Toledo, Switzerland). Lyophilized samples (4 mg) were accurately weighed, sealed in aluminum crimped cells, and then analyzed while heating from -10 °C to 200 °C at a heating rate of 10 °C per minute.

2.5.6 Stability study The freshly prepared uNLCs and cNLCs were stored in vials at 4 °C. The particle size, drug content and organoleptic features, such as aggregation and precipitation, was measured at predetermined times.

2.6 In vitro release The determination of the drug release behavior from uNLCs and cNLCs was performed by the dialysis bag method.28 Phosphate-buffer saline (PBS, pH 6.8) containing 0.1% Tween-80 was used as the release medium. A sample of uNLCs or cNLCs containing 100 µg docetaxel was added to the dialysis bag (MWCO: 14 KDa) which was then placed in a vial containing 20 mL release medium. The vial was then transferred to a shaking bath maintained at 37 ± 0.5 °C with a shaking speed of 100 rpm. At predetermined time intervals, the medium in the vial was completely

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removed and replaced immediately with the same volume of fresh release medium. The released DTX content was determined by HPLC.

2.7 In vitro bioadhesion Mucoadhesion studies of uNLCs and cNLCs were performed by mucus glycoprotein assay.29 The suspensions of uNLCs and cNLCs (2.0 mL) were mixed with 2.0 mL mucin aqueous solution (0.5 mg/mL), and shaken at 37 °C for 1 h. The mixture was then centrifuged at 50,000 rpm for 2 h, then the free mucin in the supernatant was obtained and measured using a periodic acid/Schiff colorimetric method. The amount of mucin absorbed by the uNLCs and cNLCs was determined by subtracting the concentration of residual mucin in the suspension after adsorption from the total amount added.

2.8 Permeation study using an in situ single-pass intestinal perfusion model The in situ single-perfusion experiments were performed according to the method described previously.30 Male Sprague-Dawley (SD) rats (200-250 g) were fasted for 24 h before the experiment with free access to water. The rats were anesthetized with 20% urethane solution (1g/kg, ip) and kept in a supine position under an infrared lamps to maintain normal body temperature. A midline longitudinal incision was carefully made in the abdomen, the sections about 8-12 cm long in the duodenum, jejunum and ileum were exposed and cannulated with perfusion tubing. The drug perfusion solution containing 20 µg/mL DTX and 20 µg/mL phenol red, was pumped into the intestinal segments at a constant flow rate of 0.22 mL/min. The perfusate sample was collected every 15 min for a 120 min perfusion period. The length and perimeter of

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the three segments were measured following the last collection. Then, all samples were diluted 10-fold volume with methanol. Then, the samples were vortex mixed for 10 min and centrifuged at 12,000 rpm for another 10 min, and the obtained supernatant containing unabsorbed DTX was analyzed by HPLC as described above. In addition, the phenol red concentration was determined after diluting 10-fold with NaOH (0.2 mol/L) followed by UV spectrophotometer at 558 nm. The absorption rate constant (Ka) and effective permeability (Peff) were calculated using the following equation:

 C PR in K a = 1 - out ×  Cin PR out Peff = -

 Q  × 2  πr l

Q  Cout PR in × ln 2πrl  Cin PR out

  

where Cin and Cout are the concentrations of DTX in the perfusion solution and collected perfusate samples, respectively, PRin and PRout are the concentrations of phenol red in the perfusion solution and collected perfusate samples, respectively. Q is the flow rate (mL/min) through the intestinal segment, and l and r are the length and radius of the perfused intestinal segment (cm).

2.9 The biodistribution of uNLCs and cNLCs in intestines and blood All the animal studies were conducted according to the guidelines of the local Institutional Animal Ethical Care Committee (IAEC). In order to observe the absorption of uNLCs and cNLCs in the intestine and blood, both NLCs were fluorescently labeled with a hydrophobic dye DiR. The preparation method for DiR-labeled NLCs was the same as that for docetaxel-loaded NLCs, except that DTX was replaced with DiR. Mice were given oral doses of DiR-labeled uNLCs and DiR-labeled cNLCs involving 3.5 mg/kg. After 1 h and 4 h, blood samples of about 0.3 mL were

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collected from the mice into tubes, and then the mice were sacrificed and samples of intestine from the duodenum to the ileum were removed. The intestine and blood were immediately visualized under an In Vivo Imaging System (Carestream FX Pro, USA) with an excitation wavelength of 720 nm and an emission wavelength of 750 nm.

2.10 Absorption mechanism of uNLCs and cNLCs Everted intestinal rings were used to explore the uptake mechanism of uNLCs and cNLCs. Briefly, male SD rats (200-250 g) were anesthetized by intraperitoneal injection of 20% urethane solution (1 g/kg). Then after a small midline laparotomy, intestinal segments (20-25 cm) were removed, rinsed and washed in cold KRB solution. The intestinal ring was then everted, cut into 60-100 mg rings and placed in the wells of a 24-well plate. To clarify the uptake mechanism, the everted gut sacs were pre-incubated with specific endocytic inhibitors like chlorpromazine (10 µg/mL), indomethacin (20 µg/mL), colchicine (5 µg/mL) and quercetin (5 µg/mL) at 37 °C for 30 min. Meanwhile, the everted intestinal rings were incubated at 4 °C or in the absence of inhibitors as controls. Subsequently, the incubation medium was discarded, and the uNLCs or cNLCs (20 µg/mL) were added to the plate. After incubation for another 30 min, the everted rings were removed and rinsed with cold KRB solution to stop uptake. After blotting with filter paper, the everted rings were accurately weighed and the DTX in the intestinal rings was extracted with acetonitrile and determined by HPLC as described above.

2.11 In vivo pharmacokinetics Eighteen male SD rats were divided randomly into three groups, with six rats per group. After

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fasting for 18 h, the rats were orally administered with DTX solution, uNLCs and cNLCs at 15 mg/kg. Blood samples (about 0.3 mL) were withdrawn from rats at predetermined times. Plasma was obtained by centrifugation (8000 rpm, 10 min) and analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To determine the docetaxel concentration in each plasma sample, paclitaxel was used as the internal standard (IS). Briefly, 20 µL IS was added to 100 µL plasma, followed by vortexing for 1 min. Then, the mixture was extracted with 2.5 mL tert butyl methyl ether for 10 min. After centrifugation (4000 rpm, 10 min), 2.0 mL of the upper organic phase was transferred to another clean tube and dried under a steam of air at 35 °C. The residue was then reconstituted in 100 µL methanol and centrifuged (12 000 rpm, 10 min) before analysis. The plasma samples were separated under reversed-phase gradient elution using a C18 column (Waters, Milford, USA). The mass spectrometric determination was performed in positive ESI mode with the chromatographic system, and quantification was carried out by multiple reaction monitoring (MRM) with transitions from m/z 808.68 to 527.44 for docetaxel and 854.64 to 569.40 for paclitaxel, respectively. The pharmacokinetic data was performed using the statistical moment method with DAS 2.0 software.

2.12 Statistical analysis All data were presented as means ± SD. Statistical analysis was performed using one way ANOVA with a pairwise test. A p value of less than 0.05 was considered to be significant, and a p value of less than 0.01 considered to be very significant.

3. Results

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3.1 Synthesis and characterization of Cy-PEG-MSA Cy-PEG-MSA was synthesized with a two-step procedure. The first step is the synthesis with 4-nitrophenyl carbonate substituted PEG-MSA (pNP-PEG-MSA) by activating the hydroxyl groups of PEG-MSA with 4-nitrophenyl chloroformate (4-NP). Then, the aminolysis of pNPPEG-MSA in pH 8.0 aqueous buffer was carried out by adding 5-fold amount of L-Cys to obtain Cy- PEG-MSA. The obtained polymers appeared to be white, odourless powders of fibrous structure. Figure 1ii shows the 1H-NMR spectra of the PEG-MSA, pNP-PEG-MSA and CyPEG-MSA . The successful synthesis of pNP-PEG-MSA was confirmed by the 1H-NMR (CDCl3) spectra as shown in Figure 1iiB. The resonances at δ 7.4 and 8.3 ppm corresponded to the aromatic protons of the nitrophenyl group. The resonances at δ 8.0, 8.5 and 8.9 ppm are from the pyridinium chloride that was formed during the synthesis of pNP-PEG-MSA, and it is hard to eliminate pyridinium chloride from the system by precipitating the reaction mixture in diethylether. Fortunately, the pyridinium chloride in pNP-PEG-MSA has no effect on the following reaction and can be eliminated completely from the final polymer Cy- PEG-MSA by dialysis. Furthermore, compared with PEG-MSA, the characteristic peak at 1768 cm-1 corresponding to the C=O stretching vibration of p-nitrophenylcarbonyl was observed in the spectrum of pNP-PEG-MSA, indicating that 4-NP was combined with PEG-MSA. Compared with the 1H-NMR (CDCl3) spectrum of pNP-PEG-MSA, in the 1H-NMR (CDCl3) spectrum of Cy-PEG-MSA, the resonances at δ 7.4 and 8.3 ppm disappeared completely and a new resonance at δ 3.0, characteristic of the methylene group of –CH2-SH, was detected. In addition, an amide I band at 1632 cm-1 and an amide II band at 1528 cm-1 were observed, confirming the formation of a new amido link. All

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above results clearly confirmed the success of the synthesis of Cy-PEG-MSA. Ellman’s test was used to determine the amount of thiol groups attached to the PEG-MSA. The results showed that, on average, 299.2 ± 14.0 µmol of thiol groups were immobilized per gram PEG-MSA.

3.2 Preparation and characterization of uNLCs and cNLCs In this study, the docetaxel: Precifac ATO 5: MCT: Tween 80: soybean lecithin weight ratio was 1:24:6:24:6 to ensure the higher encapsulation of docetaxel and better stability of nanostructured lipid carriers (See Supporting Information). The post-insertion technique was used to prepare thiolated nanostructured lipid carriers. Compared with the one-step preparation process, the post-insertion technique could reduce the surface thiol oxidation or crosslinking caused by the higher temperature and ultrasonication. Surface modification of uNLCs was confirmed using XPS. The uNLCs did not have any sulfur on the surface of the nanoparticles, but the cNLCs had a 0.13% atomic concentration of sulfur on their surface (Table 1). Based on these results, it was concluded that the sulfur present on the surface of cNLCs belonged to cysteine. The average particle size, PI, zeta potential, DL(%), EE(%) of uNLCs and cNLCs are summarized in Table 2. The mean particle size of uNLCs and cNLCs was less than 100 nm with a low PI (< 0.25). The EE(%) of the uNLCs and cNLCs was above 99%. The surface modification of cysteine had no significant effect on the size and EE (p < 0.05). The cNLCs showed a statistically significantly decrease in zeta potential (-13.72 ± 0.07 mV) in comparison with uNLCs (-6.39 ± 0.07 mV), which could be attributed to the carboxyl of cysteine on the surface of the

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nanoparticles. Transmission electron microscopy (TEM) has been widely applied as an effective method for observing the surface morphology of nanoparticles. The TEM images of uNLCs and cNLCs are shown in Figure 2, indicating that both NLCs were spherical and about 100 nm in size, similar to the results obtained by DLS. Differential scanning calorimetry was used to characterize the physical status of DTX present in uNLCs and cNLCs. As shown in Figure 3, the melting endothermic peak of pure DTX appeared at 175.8 °C. However, the endothermic peak of DTX was no longer presented in the curve of uNLCs and cNLCs, implying that DTX was encapsulated in the NLCs mostly in a molecular or amorphous state. Over a period of ten days, the organoleptic features of uNLCs and cNLCs remained unchanged, and the particle size and drug content were not altered significantly (Figure 4). 3.3 In vitro release The in vitro drug release profiles of uNLCs and cNLCs are shown in Figure 5. In contrast with the DTX solution, there was a prolonged release of DTX from the uNLCs and cNLCs. Approximately 91% of the cumulative amount of DTX was released from DTX solution within 24 h. In the case of uNLCs and cNLCs, the percentage cumulative release of DTX was about 75% within 24 h, and this remained constant up to 48 h. Five different release models were used to predict the drug release kinetics, and the regression results for the release of drug from both nanostructured lipid carriers are shown in Table 3. The in vitro release of DTX from uNLCs and cNLCs corresponds to the Ritger-Peppas model with r values of 0.969 and 0.961, respectively. In addition, the rate constant (k) and release exponent (n) of cNLCs and uNLCs did not show any

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obvious differences, therefore, the cysteine could be considered not to interfere significantly with the drug release.

3.4 In vitro bioadhesion

The intestinal epithelium is covered by a protective mucus layer composed of glycoprotein chains with cysteine-rich subdomains. The improved mucoadhesion of thiomers is due to an interaction between the thiol groups of polymers and the cysteine-rich subdomains of mucus. The evidence for formation of disulfide bridges has been reported previously.31 Both uNLCs and cNLCs were evaluated for mucoadhesion by incubating the NLCs with mucin (from porcine stomach mucosa) and quantifying its adsorption onto their surface. The mucin adsorption on uNLCs and cNLCs was found to be 51.9% and 81.6%, respectively, which suggests that conjugation of cysteine on the uNLCs significantly improved their mucoadhesion. The uNLCs exhibited mild mucoadhesion, due to the weak physical hydrogen bonds formed with functional groups such as hydroxyl and other hydrogen bond-forming functional groups.32 In contrast to this, the cNLCs exhibited a stronger mucoadhesion by formation of chemical disulfide bonds between the thiol groups of Cy-PEG-MSA and the cysteine groups of mucin.

3.5 Intestinal absorption by in situ single-pass intestinal perfusion The absorption of cNLCs in the intestine was examined by the in situ single-pass intestine perfusion method in Sprague-Dawley rats. For three intestinal segments (duodenum, jejunum, ileum), the absorption rate (Ka) and effective permeability (Peff) are shown in Figure 6, The Ka of

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docetaxel when formulated as a solution was found to be very low (about 0.72 × 10-4 min-1). For the control formulation of uNLCs, the Ka (about 1.04 × 10-4 min-1) was slightly higher than for DTX solution in duodenum. However, when DTX was encapsulated in the thiolated cNLCs formulation, the absorption rate of DTX in the three intestinal segments was improved between 3and 5-fold compared with that of DTX solution. In addition, the Peff of cNLCs was significantly improved in the total intestinal segments in comparison with that of DTX solution and uNLCs (p < 0.05). As a result, it could be concluded that cNLCs could markedly increase the permeability of DTX in the total intestinal segments.

3.6 The biodistribution of uNLCs and cNLCs in intestines and blood The intestinal absorption of uNLCs and cNLCs was investigated using an In Vivo Imaging System after oral administration. Figure 7 shows the intestine and blood accumulation of fluorescent DiR after the mice were treated with DiR-uNLCs and DiR-cNLCs. The results obtained showed that a more intense fluorescence was found in the intestine and blood after administration of DiR-cNLCs compared with administration of DiR-uNLCs at each time point. It appears that thiolated NLCs increase the interaction with the intestinal tract by forming disulfide bonds, and increase the intestinal absorption, resulting in a higher drug concentration in blood.

3.7 The uptake mechanism of uNLC and cNLCs in intestine Exposure of the intestine to different inhibitors was used to explore the possible permeation route of uNLCs and cNLCs. Generally, chlorpromazine is used for clathrin-mediated endocytosis, indomethacin as a caveolae-mediated endocytosis inhibitor, colchicines as a macropinocytosis

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inhibitor, and quercetin as a caveolae- and clathrin-independent endocytosis inhibitor.33 As shown in Figure 8, after treatment with different inhibitors, both NLCs had significantly reduced uptake. These results indicated that the absorption of uNLCs and cNLCs was controlled by clathrin-mediated, caveolae-mediated, macropinocytosis and caveolae- and clathrin-independent endocytosis. In fact, on the basis of the results of the in vitro release (Table 3), it was found that the drug release from the nanostructured lipid carriers followed the Ritger-Peppas model, the release exponent of uNLCs and cNLCs were 0.423 and 0.391, respectively, indicated that Fickian diffusion was the drug release mechanism. When the drug was released from the NLCs by diffusion, this providing a concentration gradient across the intestinal epithelial cells, and the free drug could be absorbed into the intestinal by passive diffusion. Therefore, both endocytosis and passive transport appeared to be involved in the absorption of uNLCs and cNLCs in the intestine.

3.8 In vivo pharmacokinetic behaviors To further verify the in vivo absorption, the pharmacokinetic behavior of uNLCs and cNLCs was evaluated using male SD rats. As illustrated in Figure 9, in the case of oral administration of DTX solution, the DTX plasma concentration at 12 h post-administration was very low, under the quantification limit of the UPLC-MS/MS (5 ng/mL). However, in the case of uNLCs and cNLCs, sustained plasma levels of DTX were observed. Table 4 summarizes the pharmacokinetic parameters, the Cmax of cNLCs was 98.20 ± 17.40 ng/mL, a 2.80-fold increase compared with DTX solution and a 1.71-fold increase compared with uNLCs. Moreover, the AUC0-t of cNLCs was 1.64-fold and 12.3-fold higher than for uNLCs and DTX solution. These results indicate that the oral bioavailability of DTX could be significantly increased by cysteine-functionalized

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nanostructured lipid carriers.

4. Discussion Docetaxel, classified as a class IV drug by the biopharmaceutical classification system, exhibits a very low bioavailability when administered by the oral route. The low aqueous solubility and low intestinal permeability are considered as the two major reasons for the low oral bioavailability.6 Recently, several groups have reported that thiomers could be an effective approach to improve the oral administration of anticancer drugs. For example, Mazzaferro et al.34 reported that mucoadhesive thiolated chitosan-coated nanoparticles could increase the intestinal permeability of docetaxel. Iqbal et al.35 designed a poly(acrylic acid)-cysteine in combination with reduced glutathione for paclitaxel to increase its oral bioavailability. However, there are no reports that the oral absorption of docetaxel could be increased by nanostructured lipid carriers modified with thiolated polymers. The main aim of the present study is to investigate the possibility of improving the oral bioavailability of by mucoadhesive thiolated nanostructured lipid carriers. In order to avoid free thiol crosslinking induced by high temperature ultrasonication, thiolated NLCs were prepared by emulsification-ultrasonication combined with the post-insertion method. Firstly, docetaxel nanoemulsion was formed by emulsification-ultrasonication, and the lipid nanocarrier ensures high encapsulation efficiency of docetaxel. Then, the formed nanoemulsion was incubated with thiolated polymers, with the hydrophobic end of the thiolated polymers inserted into the lipid core of nanoemulsion. Finally, the low temperature helps to solidify the lipid core and form thiolated NLCs. Bernkop-Schnurch et al.36 reported that the free thiol groups could

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undergo oxidation to inter- or intramolecular disulfide bonds in aqueous medium above pH 5.0. Hence, in our case, the pH of the aqueous phase was set at 4.5, to maintain the stability of the free thiol groups and retard the oxidation process. The XPS results suggested that thiols were present on the surface of NLCs. From the results of the mucoadhesion study, it was found that cysteine could significantly increase the mucoadhesion of nanostructured lipid carriers. Increased mucoadhesion of the drug carrier can prolong its residence time by improving the association with the mucus layer, and increase drug uptake into intestinal epithelial cells, thereby increasing the intestinal absorption and systemic bioavailability.37 In addition, the physiological turnover time of intestinal mucus is between 50 and 270 min,38 and it is expected that mucoadhesive nanostructured lipid carriers can remain in contact with the intestinal mucus layer for a period (about 4 h) long enough to ensure drug systemic absorption. To further investigate the possible effect of mucoadhesion on DTX intestinal permeation, an in situ single-pass intestine perfusion experiment was conducted. The results obtained indicate that thiolated NLCs can improve the docetaxel intestinal permeability in total intestinal segments (duodenum, jejunum and ileum). The absorption rate (Ka) and effective permeability (Peff) were markedly higher than those of unmodified NLCs and DTX solution. These results confirmed that mucoadhesion made a great contribution to improving the intestinal permeability of DTX. The thiolated NLCs could be immobilized at the mucosal surface for a prolonged period by forming disulfide bonds with the cysteine-rich sub-domains of glycoproteins in the mucus layers. On the basis of the results of the uptake mechanism study, the thiolated NLCs were absorbed into intestinal cells by both endocytosis and passive transport. Hence, the thiolated nanostructured lipid

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carriers increased the intestinal absorption of docetaxel by two reasons. One hand, more thiolated NLCs could be internalized into intestinal cells by endocytosis. On the other hand, the free drug released from mucoahesive nanoparticles at the intestinal mucosal surface produced a higher drug concentration gradient to increase drug transport across the intestinal cells. Considering the in vivo pharmacokinetic results, it is worth noting that the thiolated NLCs could prolong the docetaxel plasma level for at least for 24 h, and, during this period, they maintained relatively high and stable drug blood levels. It has been reported that surface modification of nanoparticles with hydrophilic polymers like PEG, commonly known as PEGylation, reduces their uptake by the reticuloendothelial system (RES).39 In our present study, thiolated NLCs became attached to the mucus layer to increase the intestinal permeability of docetaxel, then the thiolated NLCs were absorbed into intestinal cells by endocytosis. When intact thiolated NLCs were absorbed into blood, PEG acted as a stealth coating to help the NLCs bypass monocytic macrophages upon systemic absorption, and then prolong the DTX plasma level. In a word, the oral bioavailability could be significantly increased after docetaxel was encapsulated into thiolated NLCs. The AUC0-t of docetaxel was clearly higher in the thiolated NLCs compared with DTX solution and unmodified NLCs.

5. Conclusion In summary, we developed cysteine-functionalized nanostructured lipid carriers and showed that the combined advantages of cysteine and NLCs offer a promising strategy for improved oral delivery of docetaxel. Owing to the introduction of cysteine into the NLCs, the mucoadhesion of cNLCs was significantly increased. The absorption of cNLCs was greatly improved in total

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intestinal segments compared with uNLCs and DTX solution. Both endocytosis and passive transport appeared to be involved in the absorption of cNLCs across the intestinal membrane. After oral administration, the relative bioavailability of cNLCs was markedly increased 12.3-fold, 1.64-fold compared with docetaxel solution and uNLCs. All of these results showed that nanostructured lipid carriers modified with cysteine had a great potential for improving oral absorption of anticancer drugs.

Acknowledgement This work is supported by National High-tech R&D Program of China 863 Program (2012AA020305), and the National Basic Research Program of China (973 Program) No. 2009CB930300.

Supporting Information The formulation development included the selection of lipids, emulsifiers and ratios. This information is available free of charge via the Internet at http://pubs.acs.org/.

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(33) Wang, J. L.; Sun, J.; Chen, Q.; Gao, Y.; Li, L.; Li, H.; Leng, D. L.; Wang, Y. J.; Sun, Y. H.; Jing, Y. K.; Wang, S. L.; He, Z. G. Star-shape copolymer of lysine-linked di-tocopherol polyethylene glycol 2000 succinate for doxorubicin delivery with reversal of multidrug resistance. Biomaterials 2012, 33, 6877-6888. (34) Mazzaferro, S.; Bouchemal, K.; Skanji, R.; Gueutin, C.; Chacun, H.; Ponchel, G. Intestinal permeation

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methyl-beta-cyclodextrin/poly(isobutylcyanoacrylate) nanoparticles coated with thiolated chitosan. J. Control. Release 2012, 162, 568-574. (35) Iqbal, J.; Sarti, F.; Perera, G.; Bernkop-Schnurch, A. Development and in vivo evaluation of an oral drug delivery system for paclitaxel. Biomaterials 2011, 32, 170-175. (36) Bernkop-Schnürch, A.; Clausen, A. E.; Hnatyszyn, M. Thiolated polymers: synthesis and in vitro evaluation of polymer–cysteamine conjugates. Int. J. Pharm. 2001, 226, 185-194. (37) Shahbazi, M. A.; A Santos, H. Improving oral absorption via drug-loaded nanocarriers: absorption mechanisms, intestinal models and rational fabrication. Curr. Drug Metab. 2013, 14, 28-56. (38) Lehr, C. M.; Poelma, F. G.; Junginger, H. E.; Tukker, J. J. An estimate of turnover time of intestinal mucus gel layer in the rat in situ loop. Int. J. Pharm. 1991, 70, 235-240. (39) Gref, R.; Domb, A.; Quellec, P.; Blunk, T.; Müller, R.; Verbavatz, J.; Langer, R. The controlled intravenous delivery of drugs using PEG-coated sterically stabilized nanospheres. Adv. Drug Delivery Rev. 1995, 16, 215-233.

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Table 1. Summary of peak binding energy and element ratio in XPS. Peak bind energy (eV)

C

Element ratio (%)

uNLCs

cNLCs

uNLCs

cNLCs

278.5

278.4

84.32

85.98

N

392.5

391.8

0.53

0.57

O

525.8

525.4

14.69

12.9

P

126.2

126.1

0.45

0.43

S

157.8

156.5

0

0.13

Table 2. Physicochemical characteristics of uNLCs and cNLCs (n = 3)a. NLC

Size (nm)

PI

ξ (mV)

EE (%)

DL (%)

uNLC cNLC

90.4 ± 3.2 96.6 ± 8.0

0.202 ± 0.002 0.196 ± 0.004

-6.39 ± 0.07 -13.72 ± 0.07

99.34 ± 0.33 99.27 ± 0.44

3.23 ± 0.04 3.26 ± 0.02

a

PI: polydipersity index; ζ: zeta potential; EE: entrapment efficiency; DL: drug loading.

Table 3. Release kinetics of uNLCs and cNLCs.

Formulation

uNLCs cNLCs

Zero order Q versus t

First order

Higuchi

Hixon-crowell

Ritger-Peppasa

ln (1 − Q) versus t

Q versus t1/2

(1-Q)1/3 versus t

lnQ versus lnt

y = 0.012x + 0.327 r = 0.854 y = 0.011x + 0.327 r = 0.831

y = -0.030x - 0.399 r = 0.919 y = -0.026x - 0.444 r = 0.895

y = 0.108x + 0.148 r = 0.939 y = 0.100x + 0.182 r = 0.923

y = -0.007x + 0.874 r = 0.899 y = -0.006x + 0.863 r = 0.875

y = 0.423x - 1.673 r = 0.969 y = 0.391x - 1.579 r = 0.961

a

Ritger-Peppas equation: lnQ = nlnt + lnk, Q is the fractional drug release, n is the release exponent, t is the release time and k is a rate constant.

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Table 4. The pharmacokinetic parameters of docetaxel solution, uNLCs and cNLCs after oral administration to rats at 15 mg/kg (n = 6)a.

parameters Cmax (ng/mL) Tmax (h) AUC0-t (ng/mL.h) MRT (h) CL (L/h/kg) a

DTX-Sol

uNLCs

cNLCs

35.02 ± 12.43 0.667 ± 0.204 124.35 ± 27.87 5.64 ± 0.46 72.10 ± 25.29

57.47 ± 12.68 7.55 ± 9.96 933.44 ± 142.01** 12.70 ± 0.80** 4.48 ± 1.08**

98.20 ± 17.40## 4.50 ± 3.87 1533.00 ± 100.91## 12.18 ± 0.54 6.12 ± 3.51

Cmax : peak plasma DTX concentration. Tmax: the time when peak plasma concentration was

reached. AUC0-t : area under the concentration-time curve. MRT: the mean residence time. CL: Clearance. **p < 0.01 versus docetaxel solution. ##p < 0.01 versus uNLCs.

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Figure Captions Figure 1. (i) chemical structure of pNP-PEG-MSA and Cy-PEG-MSA; (ii) 1H-NMR spectra (400 MHz, CDCl3) of PEG-MSA (A), pNP-PEG-MSA (B) and Cy-PEG-MSA (C); (iii) FTIR spectra of PEG-MSA (A), pNP-PEG-MSA (B) and Cy-PEG-MSA (C). Figure 2. TEM micrographs of uNLCs (a) and cNLCs (b). Figure 3. DSCs of docetaxel (a), blank NLCs (b), cNLCs (c) and uNLCs (d). Figure 4. Effect of storage on the physicochemical characteristics of uNLCs (A)and cNLCs (B). Figure 5. In vitro release profiles of docetaxel from docetaxel solution, uNLCs and cNLCs (n = 3). Figure 6. The absorption rate (Ka) (A) and effective permeability (Peff) (B) obtained by in situ single-pass intestinal perfusion comparison among docetaxel solution, unmodified NLCs and cysteine-modified NLCs in different intestinal segments (n = 6). *p < 0.05, **p < 0.01 versus docetaxel solution. #p < 0.05 versus uNLCs. Figure 7. The ex vivo optical images of intestine and blood at 1 h and 4 h after administration of DiR-labeled uNLCs (A, C) and DiR-labeled cNLCs (B,D). Figure 8. The intestine uptake efficiency of unmodified NLCs (A) and cysteine-modified NLCs (B) after incubation with different endocytosis inhibitors at 37 °C or 4 °C (n = 3). The intestine uptake of uNLCs and cNLCs without any treatment was used as a control. *p < 0.05, **p < 0.01. Figure 9. Plasma concentration profiles of DTX after oral administration of docetaxel, unmodified NLCs and cysteine modified NLCs in SD rats (n = 6).

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Figure 1. (i) chemical structure of pNP-PEG-MSA and Cy-PEG-MSA; (ii) 1H-NMR spectra 31



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(400 MHz, CDCl3) of PEG-MSA (A), pNP-PEG-MSA (B) and Cy-PEG-MSA (C); (iii) FTIR spectra of PEG-MSA (A), pNP-PEG-MSA (B) and Cy-PEG-MSA (C).

Figure 2. TEM micrographs of uNLCs (a) and cNLCs (b).

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Figure 3. DSCs of docetaxel (a), blank NLCs (b), cNLCs (c) and uNLCs (d).

Figure 4. Effect of storage on the physicochemical characteristics of uNLCs (A)and cNLCs (B).

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Figure 5. In vitro release profiles of docetaxel from docetaxel solution, uNLCs and cNLCs (n = 3).

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Figure 6. The absorption rate (Ka) (A) and effective effective permeability (Peff) (B) obtained by in situ single-pass intestinal perfusion comparison among docetaxel solution, unmodified NLCs and cysteine modified NLCs in different intestinal segments (n = 6). *p < 0.05, **p < 0.01 versus docetaxel solution. #p < 0.05 versus uNLCs.

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Figure 7. The ex vivo optical images of intestine and blood at 1 h and 4 h after administration of DiR-labeled uNLCs (A, C) and DiR-labeled cNLCs (B,D).

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Figure 8. The intestine uptake efficiency of unmodified NLCs (A) and cysteine-modified NLCs (B) after incubation with different endocytosis inhibitors at 37 °C or 4 °C (n = 3). The intestine uptake of uNLCs and cNLCs without any treatment was used as a control. *p < 0.05, **p < 0.01.

Figure 9. Plasma concentration profiles of DTX after oral administration of docetaxel, unmodified NLCs and cysteine-modified NLCs in SD rats (n = 6).

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Cysteine-Functionalized Nanostructured Lipid ... - ACS Publications

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