N-Deoxycholic acid-N,O-hydroxyethyl Chitosan with a Sulfhydryl

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N-deoxycholic acid-N,O-hydroxyethyl chitosan with a sulfhydryl modification to enhance the oral absorptive efficiency of paclitaxel Yanfang Yu, Meirong Huo, Ying Fu, Wei Xu, Han Cai, Lingling Yao, Qinyu Chen, Yan Mu, Jianping Zhou, and Tingjie Yin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00662 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Molecular Pharmaceutics

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N-deoxycholic acid-N,O-hydroxyethyl chitosan with a sulfhydryl modification to enhance the oral absorptive

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efficiency of paclitaxel

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Yanfang Yu,1 Meirong Huo,1 Ying Fu, Wei Xu, Han Cai, Lingling Yao, Qingyu Chen,Yan Mu, Jianping Zhou*, Tingjie Yin*

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a

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Tongjiaxiang, Nanjing 210009, China

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b

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24

Department of Pharmacy, Shandong Provincial QianFoshan Hospital, Shandong University, Jinan 250014, China.

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ABSTRACT

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Currently, the most prominent barrier to the success of orally delivered paclitaxel (PTX) is the extremely limited

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bioavailability of delivered therapeutic. In light of this issue, an amphiphilic sulfhydrylated N-deoxycholic

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acid-N,O-hydroxyethyl chitosan (TGA-DHC) was synthesized to improve the oral bioavailability of PTX. Firstly,

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TGA-DHC demonstrated substantial loading of PTX into the inner hydrophobic core. A desirable enhancement in the

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bioavailability of PTX by TGA-DHC was verified by pharmacokinetic studies on rats against Taxol® and

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non-sulfhydrylated DHC micelles. Moreover, cellular uptake studies revealed significant accumulation of TGA-DHC

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micelles encapsulating PTX or rhodamine-123 into Caco-2 cells via clathrin/caveolae-mediated endocytosis and inhibition

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of P-gp efflux of substrates. The results of the Caco-2 transport study further confirmed the mechanistic basis of TGA-DHC

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efficacy; which was attributed to permeabilized tight junctions, clathrin-mediated transcytosis across the endothelium and

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inhibition of P-gp. Finally, in vitro mucoadhesion investigations on freshly excised rat intestine intuitively confirmed

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increased intestinal retention of drug-loaded TGA-DHC through thiol-mediated mucoadhesion. TGA-DHC has

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demonstrated the capability to overcome what is perhaps the most prominent barrier to oral PTX efficacy; low

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bioavailability, and serves as a prominent platform for oral delivery of P-gp substrates.

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KEYWORDS: paclitaxel, sulfhydrylated N-deoxycholic acid-N,O-hydroxyethyl chitosan, micelles, oral delivery,

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mucoadhesion

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1. INTRODUCTION

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Paclitaxel (PTX) is an extensively used, clinical front-line therapeutic against a wide spectrum of solid cancers.

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Mechanistically, PTX effects over-stabilization of cellular microtubules, preventing mitosis. Therefore, cell apoptosis occurs

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at the late G2/M phase.1 In its commercially available formulation, Taxol® (Bristol-Myers Squibb, New York, NY, USA),

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PTX is formulated in a 50:50 (v/v) mixture of Cremophor EL and dehydrated ethanol, further diluted in 0.9% saline solution 1

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forintravenous (i.v.) administration. However, any therapeutic delivered by i.v. administration comes with an increased risk

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of catheter-related infection, potential thrombosis, and extravasation. Furthermore, in the case of Taxol®, Cremophor EL has

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been known to be the cause of hypersensitivity, neurotoxicity, nephrotoxicity, and cardiotoxicity during the course of

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treatment.2 Consequently, the delivery of PTX through safer, alternate methods has remained an area of active investigation.

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Oral delivery of PTX has appealed over i.v. routes due to factors such as patient convenience, compliance, and the

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relative safety of the oral route in terms of minimizing infection. Yet, orally delivered Taxol® therapy is sparingly used due

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to its low oral bioavailability (< 10%).3 Biologically, the low availability is mainly attributed to the poor solubility and

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dissolution of PTX in gastrointestinal fluids. Subsequent hepatic metabolism of drug by cytochrome P450 metabolic

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enzymes (CYP450), rapid clearance by mucus, low permeability across the epithelium, and efflux of PTX by multidrug

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transporter P-glycoprotein (P-gp) all contribute to the insufficient bioavailability of orally delivered Taxol®.4,5

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The advent of nanotechnology has revealed a wide range of prospects for efficient oral drug delivery. Recently, numerous

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nanocarriers including liposomes, microemulsions, cyclodextrins, nanogels and polymeric micelles have been developed to

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improve oral PTX bioavailability.6,7 In particular, polymeric micelles (PMs) possess appealing properties such as nanoscopic

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dimensions and distinctive core-shell structures.8,9 Insoluble molecules such as PTX can be easily solubilized through an

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effective encapsulation into the hydrophobic inner core of PMs. In addition, the hydrophilic outer shell of PMs confers

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aqueous solubility and steric stability; protecting PTX from inactivation in gastrointestinal fluids or CYP450 recognition.

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Resultantly, PMs have attracted great attention as effective oral PTX delivery carriers. Yet, a PM designed for oral PTX

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delivery must still overcome PTX effluxby P-gp after release from the delivery carrier, rapid mucosal clearance and low

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epithelium permeability.10

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Chitosan (CS), a cationic polysaccharide derived from alkaline N-deacetylation of chitin, has garnered interest as an oral

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delivery carrier owning to its well-known biocompatibility, biodegradability and adhesion properties to mucosal

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membranes.11 CS and its derivatives were also reported to enhance oral drug absorption through opening of tight junctions

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(TJ) to mediate paracellular permeation and further promote P-gp inhibition through P-gp-independent internalization in the

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form of integral drug-loaded micelles and P-gp ATPase inhibition by free polymers.12 Therefore, chitosan based PMs offer a

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multitude of advantages for transport of PTX across the epithelium while overcoming the issue of P-gp based efflux.

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To further enhance the efficacy of orally administered PMs, the influence of particle clearance by the mucus lining must

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also be taken into consideration. The slippery lining of many epithelial tissues is quickly renewed, and functions to rapidly

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remove foreign matter.13 In the case of drug delivery, however, mucosal renewal presents a prominent pathway for reduced

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bioavailability. In light of this issue, thiolated polymers, often referred to as thiomers, have emerged as a promising mucosal

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adhesion-enhancing excipient for oral drug delivery.14 Such polymers are capable of adhering to the mucosa through

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formation of covalent disulfide linkage between their free sulfhydryl group and the cysteine-rich mucosal glycoprotein; 2


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leading to an enhanced mucus retention. Furthermore, thiomers demonstrate athiol mediated P-gp and CYP450 inhibitory

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activity.15,16 Investigations have confirmed that the absorption of the P-gp-substrate, Rhodamine 1223 (Rho-123), across the

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intestine was increased by 3-fold in the presence of CS-4-thiobutylamidine (CS-TBA) over an untreated

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control.17Additionally, poly (acrylic acid)-cysteine conjugated with reduced glutathione (GSH) prepared by Iqbal et al.

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exhibited a 7.4-fold increase in PTX oral absorption compared with the buffer solution control.18

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Considering the advancements offered by thiolated, chitosan based PMs for enhancing the bioavailability of orally

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delivered therapeutics, we herein proposed an amphiphilic sulfhydrylated N-deoxycholic acid-N,O-hydroxyethyl chitosan

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(TGA-DHC)-based polymeric micelle to improve the oral bioavailability of PTX. Through an effective solubilization by

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TGA-DHC, PTX was entrapped in the hydrophobic cores of TGA-DHC micelles and protected from CYP450 degradation

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in the gastrointestinal tract. Subsequently, PTX-loaded TGA-DHC micelles demonstrated thiol-mediated mucosal adhesion

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for increased intestinal retention, followed by a P-gp-independent internalization in their intact core/shell form by

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enterocytes or M cells. Furthermore, TGA-DHC polymers permitted opening of tight junctions for paracellular permeation

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while inhibiting P-gp efflux to enhance absorption of released PTX from TGA-DHC PMs (Scheme 1). The present study

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investigated the basic physicochemical characteristics of micelles with or without PTX encapsulation (TGA-DHC and

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PTX-TGA-DHC, respectively), compared the oral bioavailability of PTX-TGA-DHC micelles with non-sulfhydrylated

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PTX-DHC micelles and Taxol®. Finally, the mechanism of TGA-DHC on oral absorptive enhancement was studied.

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Scheme 1. Scheme of the construction of PTX-TGA-DHC and its effect on improving the oral absorption of PTX including

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mucous adhesion (A), transcytosis across endothelium (B), paracellular transport through the opening of tight junctions (C),

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and inhibition of PTX efflux by P-gP (D).

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2. MATERIAL AND METHODS

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2.1. Materials. Chitosan (100 kDa) with deacetylation degree of 90.75% was purchased from the Aoxing Biotechnology

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Co., Ltd. (Yuhuan, China). N-deoxycholic acid-N,O-hydroxyethyl chitosan (DHC) was synthesized as described in our

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previous article, and the substitution degree of deoxycholic acid and hydroxyethyl determined by elemental analysis was

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3.77% and 106.57%, respectively.19 Paclitaxel (PTX) was purchased from Shanghai Sanwei Pharmaceutical Co.Ltd.

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Verapamil was offered by Hengrui Pharmaceutical Co., Ltd. (Jiangsu, China). Cell Counting Kit-8 (CCK8) was purchased

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by Dojindo Molecular Technologies, Inc. (Shanghai, China). Rhodamine 123 (R-123) and BCA protein assay kit were

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purchased from Beyotime Institute of Biotechnology (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM, high

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glucose), trypsin-EDTA (0.25%), fetal bovine serum, penicillin-streptomycin solution, nonessential amino acid (NEAA)

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solution, Hank’s buffered salt solution (HBSS) and phosphate buffered saline (PBS), were provided by Thermo Fisher

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Scientific Inc. (Beijing, China). Chlorpromazine, amiloride and nystatin were purchased from Sigma-Aldrich Co. (Shanghai,

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China). All other chemicals and reagents were of analytical grade.

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2.2. Animals and Cell Lines. Sprague-Dawley (SD) rats (180-220g) were obtained from Origin Biosciences Inc.

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(Nanjing, China). All animals were received care incompliance with the National Institute of Health Guide for the Care and

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Use of Laboratory Animals.

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Caco-2 cells were obtained from the cell bank of Chinese Academy of Sciences, and frozen at -170 ºC in liquid nitrogen

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before use. The cells were cultured in complete DMEM with 1% (v/v) NEAA, 100 U/mL penicillin and 100 µg/mL

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streptomycin at 37 ºC in 5% CO2 atmosphere and 90% relative humidity. The cells were sub-cultivated every 5 days at a

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split ratio of 1:10.

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2.3. Synthesis of Sulfhydrylated N-deoxycholic acid-N,O-hydroxyethyl Chitosan (TGA-DHC). 250 mg of DHC was

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dissolved in 25 mL of distilled water with addition of EDC, NHS and thioglycolic acid (TGA) at different molar ratios

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(nDHC:nTGA:nEDC:nNHS). The reaction was stirred away from light at pH of 3, 4, 5, or 6. After 24 h, the mixture was dialyzed

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against 5 mM HCl using a dialysis membrane (MWCO: 12-14 kDa) for 72 h, 5 mM HCl containing 1% NaCl for 48 h, 1

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mM HCl for 28 h, and subsequently lyophilized. The TGA-DHC was obtained and stored at 4 ºC.

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2.4. Characterization of TGA-DHC. Ellman's method was used to estimate the number of thiol groups on TGA-DHC.20

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0.5 mg of TGA-DHC was dissolved in pH 8.0 PBS (0.5 M) with or without 4% NaBH4 and stirred at 37 ºC for 1 h.

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Subsequently, 200 µL of 5 M HCl was added to inactivate NaBH4, followed by addition of 100 µL Ellman's reagent. The

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amount of free and totalthiol groups was quantitated using a standard curve prepared from solutions with a known amount 4


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of thiol groups.

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2.5. Preparation of PTX-loaded Micelles. PTX-loaded micelles (PTX-DHC, PTX-TGA-DHC) were prepared by

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dialysis. PTX (10 mg) was dissolved in 300 µL of dehydrated ethanol while DHC/TGA-DHC was dissolved in 3 mL of

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distilled water. The PTX solution was added dropwise into the DHC or TGA-DHC solution under constant stirring for 0.5 h.

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The mixture was dispersed using a probe-type ultrasonicator (JY92-2D; Ningbo Scientz Biotechnology Co., Ltd., China) at

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100 W for 30 min and dialyzed against distilled water overnight. After filtration by a 0.45 µm filter, the DHC-PTX and

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TGA-DHC-PTX were obtained and stored at 4 ºC until use.

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The entrapment efficiency (EE) and drug-loading (DL) were calculated by the following formulas: EE % = DL % =

amount of PTX in micelles × 100 amount of PTX dissolved in ethanol

amount of PTX in micelles × 100 amount of PTX in micelles + amount of conjugates fed initially

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The amount of PTX was determined by high-performance liquid chromatography (HPLC, Shimadzu LC-2010 system,

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Kyoto, Japan) with UV detection at 227 nm using a LichrospheTM C18 column (5 µm particle size, 250 mm × 4.6 mm).

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2.6. Characterization of PTX-loaded Micelles. The particle size and zeta potential of PTX-DHC and PTX-TGA-DHC

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were measured using dynamic light scattering (DLS, Nano-ZS90, Malvern Instruments, UK). The morphology of

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TGA-DHC-PTX was observed by transmission electron microscopy (TEM, H-600, Hitachi, Japan). PTX-loaded micelles

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for TEM analysis were negatively stained by phosphotungstic acid (1%, v/v). The efficient encapsulation of PTX into

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TGA-DHC was demonstrated by wide-angle X-ray diffraction (WAXD) and differential scanning calorimetric (DSC)

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analysis. WAXD was performed using an XD-3A powder diffraction meter (Bruker, AXS, Germany) with Cu Ka radiation.

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DSC analysis was performed using NETZSCH DSC 204 equipment with the temperature and heating rate of 40-300 °C and

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10 °C/min, respectively.

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2.7. In Vitro Drug Release. The PTX release from Taxol®, PTX-DHC and PTX-TGA-DHC micelles was evaluated by

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dialysis. Taxol®, PTX-DHC and PTX-TGA-DHC micelles containing 1 mg of PTX were dissolved in 1 mL of distilled

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water and placed into a dialysis bag (MWCO = 12,000-14,000 Da). The whole bag was immersed in 150 mL of artificial

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gastric juice (pH 1.2) containing 0.1% (w/v) Tween 80 for 2 h, followed by another 24 h of incubation with artificial

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intestinal fluid (pH 6.8) containing 0.1% (w/v) Tween 80. The experiment was performed in an incubating shaker at 37 ºC

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and 100 rpm. At predetermined time intervals, 1 mL of release media was removed for HPLC analysis and the whole media

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was refreshed. All data points represent the average of six readings.

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2.8. Pharmacokinetic Studies. To investigate the enhancement on the oral absorption of PTX by PTX-TGA-DHC, nine

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rats were divided randomly into the following groups (n = 3): (1) Taxol® (p.o.); (2) PTX-DHC (p.o.); (3) PTX-TGA-DHC

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(p.o.); (4) Taxol® (i.v.). The intragastric administration dose of PTX was 20 mg/kg, while the i.v. administration dose of

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PTX was 7 mg/kg. After oral administration of PTX formulations at a total dose of 20 mg/kg, blood samples were collected

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into heparinized eppendorf tubes at 0.17, 0.33, 0.5, 1, 2, 4, 8, 12, 24, 48 h. Blood samples from animals receiving Taxol®

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administered intravenously were collected at 0.083, 0.17, 0.33, 0.67, 1, 2, 4, 8, 12 h. The collected samples were centrifuged

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at 9648 ×g for 10 min, and the plasma was obtained and stored at -20 ºC for HPLC analysis.

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Plasma concentration of PTX versus time in rats was analyzed using standard non-compartment analysis with the aid of

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PKSolver, an add-in program for pharmacokinetic and pharmacodynamic data analysis by our group.21 The main

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pharmacokinetic parameters used in this study were the area under the plasma concentration-time curve (AUC0-∞) for

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intravenous administration, the area under the concentration-time profilefrom time zero to 48 h (AUC0-48 h) for oral

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administration, total body clearance (CL), mean residence time (MRT), maximum concentration (Cmax ) and the time to Cmax

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(Tmax ).

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Furthermore, the relative bioavailability (F%) of PTX from these oral formulations was calculated as the following

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equation: F% = (AUC0-48 h × Di.v.)/(AUC0-∞ × Dp.o.) × 100%, where AUC0-48 h was the areaunder the concentration-time

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profile from time zero to 48 h from the oral administration, while AUC0-∞ was the area under the plasma concentration-time

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curve from the intravenous administration. Meanwhile, Di.v. and Dp.o. represented the dosage of PTX for the intravenous and

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oral administration, respectively.

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2.9. In Vitro Cytotoxicity. In vitro cytotoxicity of blank micelles and PTX-loaded micelles was evaluated in Caco-2 cells

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using CCK-8 assay.22,23 The cells were seeded in 96-well plates at a density of 1 × 104 cells per well. After 2 days of

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incubation, cells were incubated with 200 µL of test solutions diluted in fresh serum-free medium for 2 h at 37 ºC, using

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200 µL of non-complete DMEM as negative control. The test solutions were divided into five groups: (1) DHC (0.1

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mg/mL-1 mg/mL); (2) TGA-DHC (0.1 mg/mL-1 mg/mL); (3) Taxol® (PTX: 50 µg/mL-250 µg/mL); (4) PTX-DHC (PTX:

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50 µg/mL-250 µg/mL); (5) PTX-TGA-DHC (PTX: 50 µg/mL-250 µg/mL). Subsequently, medium was removed, and 100

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µL of serum-free DMEM with 10 µL of CCK-8 solution was added to the wells. The cells were then incubated for an

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additional 0.5 h and the absorbance of samples was measured at 450 nm using a microplate reader (MultiskanMk3, Thermo

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Labsystems, Beverly, MA, USA).

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2.10. Uptake Studies. To evaluate the enhancement of cellular uptake of PTX offered by sulfhydrylated micelles and the related mechanism, the uptake studies were divided into three parts as follows. 6


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2.10.1. Effect of PTX Concentration on Cell Uptake. Caco-2 cells were seeded in 24-well plates at a density of 1 × 105

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cells per well. After 18-21 days of incubation, the cells were washed twice with Hank’s balanced salt solutions (HBSS) and

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used for the uptake assays. 400 µL of Taxol® and PTX-loaded micelles diluted in DMEM (containing 50, 67, 100 µg/mL of

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PTX) were added into each well. Subsequently, the test solutions were removed after culture at 37 ºC for 2 h and the cells

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were washed three times with PBS at 4 ºC. The amount of PTX in Caco-2 cells was assayed by HPLC. Uptake ratio (U%)

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was expressed as following：U% = QPTX in cells/Qcell protein× 100%; where QPTX in cells and Qcell protein represented the amount of

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PTX in Caco-2 cells and cell protein respectively.

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2.10.2. Endocytosis Pathways of PTX-TGA-DHC. Caco-2 cells were cultured with various endocytosis inhibitors to

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demonstrate the pathways of cellular uptake. Herein, the cells were first incubated with 20 µg/mL of chlorpromazine

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(inhibitor of clathrin-mediated endocytosis),24 15 µg/mL of nystatin (inhibitor of caveolin-mediated endocytosis)25 or 550

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µM of amiloride (inhibitor of macropinocytosis)26. After incubation with the endocytosis inhibitors at 37 ºC for 1 h, the cells

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were further cultured with PTX-TGA-DHC or PTX-DHC at a PTX concentration of 50 µg/mL. Finally, the test solutions

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were removed and washed three times with PBS at 4 ºC. The amount of PTX in Caco-2 cells was assayed by HPLC.

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2.10.3. Inhibition of P-gp Efflux Pumps by TGA-DHC Polymer. Caco-2 cells were cultured with different PTX

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formulations (50 µg/mL of PTX) to evaluate the effect of TGA-DHC polymer on P-gp inhibition. After incubation with

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either Taxol®, Taxol® with 100 µM of verapamil, Taxol® with 0.12 mg/mL of DHC polymer, Taxol® with 0.12 mg/mL of

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TGA-DHC polymer, PTX-DHC, PTX-DHC with 100 µM of verapamil, PTX-TGA-DHC or PTX-TGA-DHC with 100 µM

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of verapamil at 37 ºC for 2 h, the cells were washed with PBS three times at 4 ºC and the amount of cellular PTX was

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assayed by HPLC.

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High content cellular imaging system and flow cytometry were also employed to confirm the effect of TGA-DHC on

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P-gp inhibition. R-123 loaded micelles (R-123-TGA-DHC, R-123-DHC) were prepared following the procedure for

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PTX-TGA-DHC preparation except by replacing PTX with R-123. Caco-2 cells were seeded in 6-well plates at a density of

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1 × 105 cells per well. The cells were washed twice with PBS before being incubated with free R-123 (5 µM), free R-123

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with 100 µM of verapamil, R-123-DHC, R-123-DHC with 100 µM of verapamil, R-123-TGA-DHC, or R-123-TGA-DHC

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with 100 µM of verapamil at 37 ºC for 2 h. Then, the cells were washed three times with 4 ºC PBS and observed by high

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content cellular imaging system (ImageXpress, Molecular Devices, USA). Subsequently, cells were trypsinized and

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collected through centrifugation for analysis by flow cytometry (BD FACSCantoTM, USA).

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2.11. Transport Studies of PTX-TGA-DHC across Caco-2 Cells. Caco-2 cells were seeded at a density of 1 × 105 cells

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per well in inserts containing permeable polycarbonate membrane (6.5 mm insert diameter, 3.0 µm pore size, 0.33 cm2

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growth area) in 24-well plates. The cells were fed every two days for the first week, and then fed daily until used for 7



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experiments 21 ~ 24 days after seeding. Transepithelial electrical resistance (TEER) values were measured with a Millicell®

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electrical resistance system (Millipore, USA) to evaluate the integrity of the cell monolayers. ATEER value exceeding 350

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Ω cm2 indicated a well-developed tight junction of the cells. In addition, the integrity of the cell monolayers was further

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confirmed through Transmission electron microscopy (TEM, H-600, Hitachi, Japan).

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Before the transport studies, the cell monolayers were washed twice with HBSS at 37 ºC for the apical (AP) and

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basolateral (BL) side. Subsequently, the monolayers were pre-incubated with HBSS at 37 ºC for 15 min. To estimate the

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transport from AP to BL side, 200 µL of test solutions was added to the AP side while 800 µL of blank HBSS was added to

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the BL side. Test solutions included Taxol® (37 ºC), Taxol® with 100 µM of verapamil (37 ºC), PTX-DHC (37 ºC),

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PTX-TGA-DHC (37 ºC), PTX-TGA-DHC (4 ºC), PTX-TGA-DHC with 20 µg/mL of chlorpromazine (37 ºC), and

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PTX-TGA-DHC with 15 µg/mL of nystatin (37 ºC). Samples of 100 µL were taken from the receiving chamber and

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supplemented with fresh HBSS at 0.5, 1, 1.5 and 2 h.Then samples were mixed with an equal volume of methyl cyanides,

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vortexed, separated by centrifugation and analyzed by HPLC. Apparent permeability coefficients (Papp) of PTX could be

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determined according to the equation:

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Papp = (dQ/dt)/(A × C0),

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where the dQ/dt (µg/s) was the drug permeation rate, A was the surface area of polycarbonate membrane (0.33 cm2) and C0

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(µg/mL) was the initial concentration of PTX in the donor compartment at 0 h.

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2.12. In Vitro Mucoadhesion Studies of TGA-DHC on Freshly Excised Rat Intestine. Rats were fasted for 12 hours

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with water, anesthetized, and incised along the abdominal midline. The intestine was washed by pre-warmed saline at 37 ºC

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until the effluent is clean. The intestinal segment of the rat was divided into four segments: duodenum, jejunum, ileum, and

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colon. The different segments of the intestine were separated and 6 cm of each segment was obtained. Coumadin-6 loaded

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micelles (C6-TGA-DHC, C6-DHC) were prepared following the procedure for PTX-TGA-DHC preparation except by

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replacing PTX with coumadin-6. 0.4 mL of C6-TGA-DHC and C6-DHC diluted by pH 6.8 PBS was injected into the

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separated intestinal segments with ligation on both sides and incubated at 37 ºC for 2 h. Micelles which were not absorbed

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were removed by two washes with 1 mL of PBS. Tissue sections were stored in formalin and 7 µm thick slices were

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obtained for florescent microscopy. In addition, after two washeswith PBS to remove the unabsorbed micelles, mucus layers

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covering the treated intestinal segments were isolated by scraping off with a scalpel.27 The corresponding intestinal

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segments minus the mucus layer and the obtained mucus samples were submerged into 4 mL of methanol and homogenized

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by ultrasonication to dissolve coumadin-6 internalized within the intestine or detained in the mucus layer. After

235

centrifugation at 603 ×g for 10 min, the concentration of coumadin-6 in the supernatant was detected.

236 8


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237 238

2.13. Statistical Analysis. All the experiments were repeated at least three times. Data were expressed as means ±

239

standard deviation (S.D.). All results were analyzed by 2-tailed Students t-test or one-way ANOVA, followed by the

240

Tukey-Kramer test. P < 0.05 was considered to be statistically significant.

241 242

3. RESULTS AND DISCUSSION

243

3.1. Synthesis and Characterization of Sulfhydrylated N-deoxycholic acid-N,O-hydroxyethyl Chitosan

244

(TGA-DHC). As shown in Scheme 2, the amphiphilic chitiosan-based conjugate, DHC, was synthesized and characterized

245

in our previous report.19 The DS of deoxycholic acid and hydroxyethyl in DHC determined by elemental analysis was 3.77%

246

and 106.57%, respectively. The objective sulfhydrylated micelle, TGA-DHC, was obtained through conjugating the

247

carboxyl of thioglycolic acid (TGA) to the free amino of DHC at different feeding ratios of reactants and various pH.

248

249 250

Scheme 2. Synthetic scheme of DHC and TGA-DHC conjugates.

251 252

The quantity of thiol in TGA-DHC was quantitatively determined by Ellman's method with or without NaBH4

253

pretreatment. Because thiol is susceptible to redox bylight, oxygen and metal ions into the disulfide form inner carriers,

254

reduced and oxidized thiol exist simultaneously in TGA-DHC.27 The quantity of thiol detected by Ellman's reagent without

255

pretreatment of NaBH4 represented the amount of free thiol, while those detected after reduction by NaBH4 represented both

256

the reduced and oxidized thiol groups (total amount). Results in Table 1 demonstrated that the total amount of thiol in

257

TGA-DHC increased as pH increased from 3 to 6. This trend may be attributed to the enhanced catalytic activity of EDC at

258

pH values close to, but less than 7.0. However, the pH-mediated change in the amount of free thiol in TGA-DHC was 9



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259

inconsistent with the measured total thiol content. It was observed that the quantity of free thiol increased as pH increased

260

from 3 to 5. However, a subsequent decline in the amount of free thiol was observed in pH values above 5. This observation

261

can be primarily attributed to the formation of disulfide bonds from thiol in the oxidative environment.28 Thus generating

262

TGA-DHC with the maximal quantity of free thiol for mucosal adhesion enhancement could be obtained at pH 5.

263

Furthermore, results indicated that the grafting rate of thioglycolic acid was not improved when feeding ratio was increased

264

to 0.5:1 (n(DHC):n(TGA)) at pH of 5. Resultantly, the optimal TGA-DHC conjugate synthesized at pH 5 with the molar ratio

265

n(DHC):n(TGA):n(EDC):n(NHS) of 1:1:2:1 was used in the following article. Table 1. DS of thiol group measured by Ellman’s reagent of a series of TGA-DHC.

266 No. 1 2 3 4 5 6

n(DHC):n(TGA) :n(EDC):n(NHS) 1:1:2:1 1:1:2:1 1:1:2:1 0.5:1:2:1 2:1:2:1 1:1:2:1

The free amount of thiol (µmol/g) 65.5 137.3 286.8 203.7 193.9 145.8

pH 3 4 5 5 5 6

The total amount of thiol (µmol/g) 186.4 354.3 697.9 601.5 517.9 729.1

267 268

3.2. Preparation and Characterization of PTX-loaded TGA-DHC and DHC Micelles. PTX was loaded into DHC and

269

TGA-DHC micelles thorough sonication and dialysis. As shown in Table 2, PTX-DHC and PTX-TGA-DHC exhibited an

270

average size of 150 nm, a cationic charge of around +18 mV. An excellent PTX-loading capability was observed with a drug

271

loading (DL) content of ~30 wt% and a drug encapsulation efficiency (EE) of ~75%. Modification of TGA showed no

272

significant effect on the particle size, zeta potential or drug-loading ability of micelles (P > 0.05). TEM was used to

273

visualize the size and morphology of PTX-loaded micelles. Results in Figure 1A and Figure S1 validated that

274

PTX-TGA-DHC and PTX-DHC micelles displayed similar spherical morphology at approximately 100 nm diameter with a

275

narrow size distribution. The difference in size observed by TEM from those obtained from DLS was attributed to micellar

276

collapse during the drying processes of TEM sampling.

277 278

Table 2. Loading capacity of TGA-DHC and DHC micelles for PTX (n = 3).

PTX-DHC PTX-TGA-DHC

Diameter (nm)(µ2/Г2)

Zeta Potential (mV)

DL (wt%)

EE (%)

148.11 ± 5.42（0.116） 156.32 ± 6.93（0.098）

19.23 ± 3.11 17.83 ± 2.13

30.21 ± 1.83 31.91 ± 3.13

74.87 ± 3.43 79.67 ± 5.84

279 280

Furthermore, to investigate the physical state of PTX in micelles, WAXD and DSC analysis were performed for

281

PTX-TGA-DHC with PTX, TGA-DHC, and physical mixture of PTX + TGA-DHC as controls. The WAXD diagrams were

282

illustrated in Figure 1B. PTX showed intense peaks at 2θ of 5-15º and small peaks between 15 and 30º. Meanwhile, 10


Page 11 of 32

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283

TGA-DHC gave a broad peak at 15º ~ 30º. The typical crystal peaks of PTX and TGA-DHC were both present in the

284

WAXD diagram representing a physical mixture of PTX + TGA-DHC. On the other hand, the PTX-TGA-DHC micelles

285

possessed negligible characteristic peaks of PTX and exhibited peaks of free TGA-DHC. This finding confirms that input

286

PTX was encapsulated in micelles with reduced crystallinity. The results of DSC in Figure 1C further confirmed the

287

existing state of PTX in PTX-TGA-DHC. The PTX in the crystalline state showed an endothermic peak at 220.0 ºC and an

288

exothermic peak at 238.1 ºC, while TGA-DHC possessed a single, endothermic peak at 58.4 ºC. Additionally, the

289

representative peaks of PTX were seen for the physical mixture of PTX + TGA-DHC, but almost absent for

290

PTX-TGA-DHC micelles, indicating a notably reduction in the crystalline state of PTX in PTX-TGA-DHC.

291

When administered, orally delivered nanoparticles firstly interact with the strong acidic environments in gastric juice

292

before passing in to the mildly acidic environment in intestinal juice; finally passing into blood circulation. To simulate this

293

change, Taxol®, PTX-DHC micelles and PTX-TGA-DHC micelles were incubated with artificial gastric juice (pH 1.2) for 2

294

h followed by incubation with artificial intestinal fluid (pH 6.8) for another 22 h to simulate the PTX release profiles after

295

oral administration of micelles before transport across the epithelium. As shown in Figure 1D, both PTX-DHC and

296

PTX-TGA-DHC exhibited a slow rate of drug release. It was found that only 1.5% of PTX was released within 2 h after

297

administration in artificial gastric juice (pH 1.2) and only around 5.0% was released within 22 h in artificial intestinal fluid

298

(pH 6.8). These results indicated that PTX-DHC and PTX-TGA-DHC micelles retained structural integrity in the

299

gastrointestinal tract, with only minimal drug leakage. This property allows the benefit of increased oral bioavailability of

300

PTX delivered by PTX-TGA-DHC for transcytosis across enterocytes. Compared with the two micelles, a similar but

301

slightly faster drug release was observed from Taxol®. Specifically, around 12% of PTX was released after 24 h incubation

302

in the simulated condition. This result suggested improved oral absorption of PTX-DHC and PTX-TGA-DHC over Taxol®

303

not resulting from different release kinetics.

11



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Page 12 of 32

304 305

Figure 1. (A) TEM images of PTX-TGA-DHC micelles. XRD (B) and DSC (C) spectrum of blank TGA-DHC micelles, the

306

physical mixture of PTX + TGA-DHC, PTX-loaded TGA-DHC micelles and free PTX. (D) In vitro release of

307

PTX-TGA-DHC micelle, PTX-TGA micelles and Taxol®: 0-2 h in simulated gastric fluid (pH 1.2), 2-24 h in simulated

308

intestinal fluid (pH 6.8) (n = 6).

309 310

3.3. Pharmacokinetic Studies. To directly verify the advancement of PTX oral absorptive efficiency when delivered by

311

TGA-DHC, pharmacokinetic analysis of PTX-TGA-DHC after oral administration into rats at a PTX dosage of 20 mg/kg

312

was performed alongside orally delivered PTX-DHC and Taxol® as controls. Rats treated with a single i.v. administration of

313

Taxol® at 7 mg/kg were set as the reference group. The plasma PTX concentration plotted against time for each of these

314

four groups were shown in Figure 2. As exhibited in Figure 2A, the plasma concentration-time curves of the three oral

315

administrated formulations all represented a prominent hepatobiliary circulation-mediated bimodal effect. However, the

316

PTX plasma concentration decreased rapidly when treated with Taxol®, and reached non-detectable level after just 24 hours.

317

In contrast, PTX plasma concentrations when delivered using PTX-DHC or PTX-TGA-DHC remained above the 0.1 µg/mL

318

threshold even after 48 hours. Furthermore, the PTX-TGA-DHC group possessed a higher PTX plasma concentration at all 12


Page 13 of 32

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319

measured time points compared with PTX-DHC. The corresponding pharmacokinetic parameters of PTX in these

320

formulations were summarized in Table 3. As depicted, the oral absorption of PTX was extremely limited when

321

administrated as Taxol® with a bioavailability of 18.14% and AUC0-∞ of 13.99 ± 1.62 µg·h· mL-1. Oral administration of

322

PTX using DHC micelles resulted in a 1.3-fold increase in Cmax and a 2.5-fold increase in bioavailability compared with

323

Taxol®. The oral absorptive enhancement of this chitosan derivative micelle has been demonstrated and reported by our

324

group.19 However, the Cmax and bioavailability were further improved when PTX was orally delivered by TGA-DHC.

325

Specifically, PTX-TGA-DHC possessed a 3.2-fold greater AUC0-∞ and a 1.8-fold higher Cmax than Taxol®. TGA-DHC

326

micelles increased the bioavailability of PTX to 57.52%; 3.2-fold and 1.3-fold higher when compared with Taxol® and DHC

327

respectively. This result demonstrates that TGA-DHC was able to further promote the oral absorption of PTX over DHC.

328

Advantages presented herein may be attributed to a two faceted system composed of the sulfhydryl-mediated mucosal

329

adhesion and the enhanced oral PTX absorption by chitosan-based micelles. Firstly, improved mucosal adhesion allowed for

330

reduced clearance by mucus and a subsequently prolonged retention in the gastrointestinal tract. Secondly, chitosan-based

331

micelles help PTX absorbed through transcytosis cross the endothelium. Additionally, PTX delivered from chitosan

332

derivative polymers also benefit from P-gp inhibition.

333

334 335

Figure 2. (A) Mean pharmacokinetic profiles of PTX-DHC, PTX-TGA-DHC micelles and Taxol® after an oral dose of 20

336

mg/kg in rats (n = 3). (B) Mean pharmacokinetic profiles of Taxol® after an i.v. administration at a dose of 7 mg/kg (n = 3). 13



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Page 14 of 32

337 338

Table 3. Comparison of the mean pharmacokinetic parameters of PTX-DHC, PTX-TGA-DHC and Taxol® after a single oral

339

dose of 20 mg/kg in rats (n = 3). Parameter

Unit

Taxol® (7 mg/kg i.v.)

Taxol® (20 mg/kg p.o.)

PTX-DHC (20 mg/kg p.o.)

PTX-TGA-DHC (20 mg/kg p.o.)

Cmax AUC0-t

µg·mL-1 µg·h·mL-1

16.74 ± 1.66 17.89 ±1.85

0.76 ± 0.13 9.77 ± 1.03

1.00 ± 0.07* 24.39 ± 1.49**

1.36 ± 0.13**† 30.98 ± 2.21**†

AUC0-∞

µg·h·mL-1

18.85 ± 1.83

13.99 ± 1.62

30.53 ± 2.04**

42.87 ± 3.11**†

CL

mL·h-1

71.77 ± 6.51

245.17 ± 28.94

112.42 ± 4.80**

85.38 ± 11.03**

MRT

h

3.42 ± 0.24

22.32 ± 5.76

31.08 ± 7.89

37.77 ± 7.89*

F

%

N/A

18.14 ± 1.85

45.28 ± 2.52*

57.52 ± 3.51**†

340

*significantly different fromTaxol® (P < 0.05)

341

**significantly different from Taxol® (P < 0.01)

342

†

significantly different from PTX-DHC (P < 0.05)

343 344

3.3. Cellular Evaluation for the Mechanism of Oral Absorptive Enhancement by TGA-DHC. Having confirmed the

345

enhancement of oral bioavailability with PTX delivered using TGA-DHC, the mechanism of oral absorptive enhancement

346

was investigated using the Caco-2 cell line.

347

3.3.1. Determination of the Tested Concentration of PTX in Micelles in Cell Experiments. To ensure the feasibility and

348

accuracy of cellular experiments on Caco-2, the highest possible concentration of PTX delivered using micelles, or Taxol®

349

with negligible toxicity should be investigated. Firstly, the cytotoxicity of blank DHC micelles, TGA-DHC micelles,

350

PTX-loaded micelles and Taxol® on Caco-2 after 2 h of incubation was investigated through quantifying cell viability using

351

a cell counting kit-8 (CCK-8) assay. Results in Figure 3A indicated that DHC and TGA-DHC exhibited negligible toxicities

352

on Caco-2 cells even with a concentration up to 1 mg/mL, demonstrating the advantage of chitosan-based polymers in

353

biocompatibility. The relative cell viability of PTX-DHC, PTX-TGA-DHC and Taxol® group were all well above 90% when

354

the PTX concentrations were below 100 µg/mL, implying no significant toxicity toward Caco-2 cells. Yet, toxicities against

355

Caco-2 increased notably when the PTX concentration was raised to 250 µg/mL delivered using Taxol®. As a result,

356

PTX-DHC, PTX-TGA-DHC and Taxol® with the PTX concentrations of 100, 67, 50 µg/mL were used as the test

357

formulations in the following Caco-2 experiments to comparatively evaluate the effect of TGA-DHC micelles on enhancing

358

the oral absorption of PTX.

359

3.3.2. Uptake Studies in Caco-2 Cells. The ability of the DHC and TGA-DHC micelles to improve the cellular uptake of

360

PTX into Caco-2 was determined in comparison with Taxol®. Results in Figure 3C showed ~0.90 µg/mg PTX

361

accumulation was seen in Caco-2 cell when delivered by Taxol® at PTX concentrations of 50, 75, 100 µg/mL. In contrast, 14


Page 15 of 32

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362

Caco-2 cells treated with PTX-DHC at 50, 75, 100 µg/mL showed PTX concentrations of 12.85 ± 1.73, 13.36 ± 3.08 and

363

13.96 ± 2.64 µg/mg respectively. Meanwhile PTX-TGA-DHC treatment at the same concentrations of 50, 75, 100 µg/mL

364

displayed a PTX internalization amount of 11.66 ± 3.46, 14.01 ± 2.14 and 14.24 ± 3.04 µg/mg respectively. The ~15.5-fold

365

increase in uptake of PTX by Caco-2 cells treated by micelles over Taxol® demonstrated the appealing effects of

366

chitosan-based micelles on improving drug internalization. This finding may be explained by the encapsulation of drug

367

molecules into hydrophobic cores and transcytosis across cells in a manner that protects PTX from P-gp efflux. It was worth

368

noting that PTX accumulation concentrations were nearly identical in Caco-2 cells when delivered by TGA-DHC even

369

when the PTX incubation concentration increased to 100 µg/mL. Additionally, sulfhydrylation did not remarkably enhance

370

the drug intercellular delivery capability of chitosan-based micelles into Caco-2 cells. This finding may be due to the

371

deficiency of cysteine-rich mucosal glycoprotein around Caco-2 cells secreted by goblet cells in the intestinal epithelium.29

372 373

3.3.3. Endocytosis Pathways of PTX-TGA-DHC. It has been confirmed that TGA-DHC and DHC micelles were capable

374

of improving PTX accumulation in Caco-2 cells. Next, the cellular endocytosis-mediated enhancement offered by these two

375

micelles was investigated with regards to the mechanism of endocytosis. Uptake studies on Caco-2 were performed in the

376

presence of chlorpromazine, nystatin, or amiloride which inhibited clathrin-mediated endocytosis, caveolin-mediated

377

endocytosis, or macropinocytosis respectively. As depicted in Figure 3D, incubation with chlorpromazine resulted in a

378

significant decrease (P < 0.05) of cellular uptake of PTX into Caco-2 cells, while amiloride and nystatin had no significant

379

effect on the Caco-2 uptake of PTX. This finding indicates that PTX-DHC is permeated into cells mainly through the

380

clathrin-mediated endocytotic pathway. However, the uptake of PTX-TGA-DHC into Caco-2 cells was remarkably

381

decreased by chlorpromazine and nystatin (Figure 3E), implying that clathrin and caveolin were both involved in the

382

Caco-2 cellular internalization of PTX-TGA-DHC.

15



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Page 16 of 32

383 384

Figure 3. (A) Cytotoxicity of DHC and TGA-DHC against Caco-2 cells at different concentrations (n = 6). (B) Cytotoxicity

385

of Taxol®, PTX-DHC and PTX-TGA-DHC to Caco-2 cells (n = 6). (C) Cellular uptake of Taxol®, PTX-DHC and

386

PTX-TGA-DHC at PTX concentrations of 50, 67, 100 µg/mL into Caco-2 cells (n = 6, **P < 0.01 PTX-loaded micelles vs

387

Taxol® at the same PTX concentration). Relative uptake efficiency of PTX-loaded micelles with 50 µg/mL PTX in absence

388

or presence of various endocytosis inhibitors: (D) PTX-DHC, (E) PTX-TGA-DHC (n = 3, *P < 0.05 vs control, **P < 0.01

389

vs control). (F) Cell uptake of PTX into Caco-2 cells: Taxol®, Taxol® with verapamil, Taxol® with DHC, Taxol® with

390

TGA-DHC, PTX-DHC, PTX-TGA-DHC, PTX-DHC with verapamil and PTX-TGA-DHC with verapamil diluted at 50

391

µg/mL PTX (n = 3, *P < 0.05 vs Taxol®, **P < 0.01 vs Taxol®). (G) High content cellular imaging and flow cytometry

392

results of R-123 accumulation in Caco-2 cells: (a): free R-123, (b): free R-123 with verapamil, (c): free R-123 with

393

TGA-DHC, (d): R-123-TGA-DHC, (e) R-123-TGA-DHC with verapamil. Scale bar represents 100 µm. 16


Page 17 of 32

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394 395

As reported, chitosan-based micelles exhibited a profound effect on P-gp inhibition.17 To determine whether the

396

TGA-DHC and PTX-DHC micellar polymers could positively impact the cellular accumulation of PTX (a typical

397

P-gpsubstrate) through P-gp inhibition, an uptake experiment of Taxol®, PTX-DHC and PTX-TGA-DHC on Caco-2 cells

398

was further performed with or without the addition of verapamil, a traditional P-gp inhibitor. As shown in Figure 3F, the

399

cellular internalization of Taxol® (50 µg/mL PTX), in which PTX mainly exist as free molecules, could be significantly

400

increased (P < 0.05) through co-incubation with 100 mM of verapamil. When PTX-TGA-DHC or PTX-DHC was diluted to

401

the PTX concentration of 50 µg/mL, the concentration of micellar polymer was around 0.12 mg/mL. The presence of 0.12

402

mg/mL of TGA-DHC or DHC also resulted in a remarkably improved uptake of Taxol® (P < 0.05), demonstrating the P-gp

403

inhibitive effect of these two micellar polymers. However, the presence of verapamil did not significantly increase the

404

uptake efficiency of PTX-TGA-DHC or PTX-DHC micelles; likely due to the P-gp-independent cellular internalization of

405

the integrate micellar particles.

406

To further intuitively observe the improvement of TGA-DHC on the uptake of PTX, R-123 which is a substrate of P-gp,

407

was selected as fluorescence probe and loaded into micelles to evaluate the P-gp activity in Caco-2 cells. The intracellular

408

fluorescence intensity of R-123 in Caco-2 cells was tested in various combinations: cells incubated with 5 mM of free

409

R-123 solution with or without verapamil, cells incubated with free R-123 with TGA-DHC, and cells incubated with

410

R-123-TGA-DHC with or without 100 mM of verapamil. All groups were imaged using a high content cellular imaging

411

system. Furthermore, the intracellular fluorescence intensity of R-123 was quantitatively detected by flow cytometry. As

412

shown in Figure 3G, both verapamil and TGA-DHC increased the amount of internalized R-123 over the free R-123 group,

413

confirming that TGA-DHC possessed an inhibitive effect on P-gp efflux of R-123. Moreover, the intracellular fluorescence

414

intensity of R-123 was further enhanced when encapsulated and delivered by TGA-DHC. Meanwhile, the presence of

415

verapamil did not significantly increase the cellular accumulation of R-123-TGA-DHC. This phenomenon resulted from the

416

P-gp-independent internalization of R-123-TGA-DHC into Caco-2 cells, during which R-123 was loaded firmly in the inner

417

cores of TGA-DHC micelles and protected from the P-gp recognition. Resultantly, R-123-TGA-DHC effectively bypassed

418

the P-gp efflux. All results of this study suggest that TGA-DHC offers a promising delivery vehicle for P-gp substrates,

419

including pharmaceutical therapeutics, for oral administration.

420 421

3.3.4. Transport Studies of PTX-TGA-DHC. Despite the demonstration that TGA-DHC can effectively improve PTX

422

accumulation in Caco-2 cells, determination of the drug’s apparent permeability coefficient (Papp) is necessary owning that

423

PTX absorption through oral administration requires successful transport of PTX across intestinal epithelial cells into blood

424

circulation. Consequently, Caco-2 monolayers with the TEER higher than 350 Ω·cm2 were used to stimulate intestinal 17



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Page 18 of 32

425

epithelial cells and PTX Papp across the Caco-2 monolayers was then detected.5,30 Herein, a higher Papp value represented

426

greater oral absorption efficiency. As shown in Figure 4A, the TEER of the Caco-2 cell monolayer was above 350 Ω·cm2

427

after 21 days of cultivation, indicating well-developed tight junctions between cells. This tight junction in Caco-2 cell

428

monolayers was also intuitively demonstrated by TEM images in Figure 4B. In this study, Caco-2 monolayers were treated

429

with PTX-TGA-DHC (50 µg/mL PTX), PTX-DHC (50 µg/mL PTX), Taxol® (50 µg/mL PTX) with or without 100 mM

430

verapamil, and Taxol® (50 µg/mL PTX) with 0.12 mg/mL TGA-DHC polymer to comparatively investigate the ability of

431

TGA-DHC to enhance PTX transport (Figure 4C). Consistent with previous reports, the Papp of Taxol® was low, ranging

432

around 2.13 ± 0.22 × 10-6 cm/s despite the presence of Cremophor EL; a surfactant speculated to inhibit P-gp efflux

433

pumps.31 Such a considerably low Papp of Taxol® was attributed to P-gp efflux as PTX existed as free molecules in the

434

formulation system of Taxol®. Thus, a poor oral absorption of Taxol® was observed, which has been illustrated in

435

pharmacokinetic studies. Yet, it was exhibited that 100 mM of verapamil significantly improved the Papp of Taxol® (P
0.05). Collectively, clathrin was implicated in both

463

the cellular endocytosis and intestinal transport mechanisms of PTX-TGA-DHC, whereas caveola was only associated with

464

the cellular endocytosis but not with the intestinal transcytosis.

465 466

Figure 4. (A) TEER of Caco-2 cell monolayersover 22 days (n = 3). (B) Electron microscopy of Caco-2 cell monolayers. (C)

467

Apparent permeability coefficients of PTX: Taxol®, Taxol® with verapamil, Taxol® with TGA-DHC, PTX-DHC and

468

PTX-TGA-DHC micelles diluted to 50 µg/mL PTX at 37 ºC (n = 3, *P < 0.05 vs Taxol® at 37 ºC, **P < 0.01 vs Taxol® at

469

37 ºC). (D) TEER at the beginning and end of PTX-DHC and PTX-TGA-DHC transport experiments (n = 3). (E) Apparent

470

permeability coefficients of PTX for PTX-TGA-DHC at 37 ºC, PTX-TGA-DHC at 4 ºC, PTX-TGA-DHC with

471

chlorpromazine at 37 ºC and PTX-TGA-DHC with nystatin at 37 ºC (n = 3, *P < 0.05 vs PTX-TGA-DHC at 37 ºC, **P

N-Deoxycholic acid-N,O-hydroxyethyl Chitosan with a Sulfhydryl

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