Preparation and Characterization of Mucoadhesive Buccal

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Preparation and Characterization of Mucoadhesive Buccal Nanoparticles Using Chitosan and Dextran Sulfate Ji Woon Suh, Ji-Soo Lee, Sanghoon Ko, and Hyeon Gyu Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00849 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 26, 2016

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Journal of Agricultural and Food Chemistry

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Preparation and Characterization of Mucoadhesive

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Buccal Nanoparticles Using Chitosan and Dextran

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Sulfate

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Ji Woon Suh,† Ji-Soo Lee,† Sanghoon Ko,‡ and Hyeon Gyu Lee*,†

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Department of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea

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‡

Department of Food Science and Technology, Sejong University, 98 Gunjadong, Gwangjin-gu, Seoul, 143-747, Republic of Korea

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*Corresponding author.

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Tel: +82-2-2220-1202; Fax: +82-2-2281-8285;

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E-mail: [email protected] (H.G. Lee).

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ACS Paragon Plus Environment


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ABSTRACT

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The aim of this study was to formulate buccal mucoadhesive nanoparticles

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(NPs) using the natural mucoadhesive polymers. The natural mucoadhesive

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polymers chitosan (CS) and dextran sulfate sodium salt (DS) were used to

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prepare mucoadhesive nanoparticles using ionic gelation method. As the

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molecular weight of DS decreased, the amount of mucin and the number of

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buccal cells adsorbed on DS increased. The CS/DS NPs ranged from 100-200

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nm in diameter. The adhesive interactions of CS/DS NPs with mucin were not

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significantly different from those of CS/sodium triphosphate pentabasic (TPP)

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NPs; however, CS/DS NPs exhibited 5 times greater mucoadhesive activity to

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buccal cells compared with control CS/TPP NPs in ex vivo adhesion tests.

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These results indicate that the buccal mucoadhesive properties of NPs can be

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improved by using natural mucoadhesive polymers.

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KEYWORDS: mucoadhesive; dextran sulfate; chitosan; nanoencapsulation

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INTRODUCTION

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Mucoadhesion is defined as the adhesive capacity of synthetic or natural

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polymers to the mucous gel layer covering mucosal membranes.1 It is generally

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believed that prolonging the residence time on the adsorbing membrane by

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mucoadhesion provides optimal conditions for effective delivery. Therefore,

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mucoadhesive systems have been studied as an approach to increase the

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residence time of the dosage form on the absorbing mucosal surface and to

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localize drugs or biological nutrients in a particular region.2 Mucoadhesion also

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allows the diffusion and penetration of mucoadhesive materials into the mucous

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layer, resulting in improved absorption and bioavailability.3

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Buccal delivery provides a number of unique advantages including

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excellent accessibility, bypass of the first pass metabolism, and avoidance of

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presystemic elimination within the gastrointestinal tract. However, the major

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limitations of absorption across the oral mucosa include exposure to salivary

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flow, shearing forces due to tongue movements and swallowing, and short

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residence time. These limitations could in principle be effectively alleviated by

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the use of a buccal mucoadhesive delivery system.

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Buccal mucoadhesive delivery systems have been fabricated by several

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different approaches. Most studies have focused on buccal mucoadhesive

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tablets, patches, films, gels, and ointments for drug delivery to treat oral disease;

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these patches are based on synthetic mucoadhesive polymers.4 Tiyaboonchai

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et al.5 used polyethylenimine–dextran sulfate nanoparticles for the buccal

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mucoadhesive delivery of Punica granatum peel extract. This strategy resulted

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in prolonged release and significant antibacterial activity against oral pathogens

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that cause plaque, cavities, and oral malodor. For a mucoadhesive delivery

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system to be used in food applications, an encapsulation technique is

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necessary to protect the cargo from potentially hostile characteristics of the

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external environment such as pH, temperature, and moisture. In addition,

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encapsulation can mask unwanted flavor and taste. Nanoparticles also have

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sensory advantages relevant to the food industry, because nano-sized particles

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cannot be felt by the mouth due to their reduced particle size; in contrast, micro-

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sized particles can be felt. 6, 7 However, only a few studies have used a buccal

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mucoadhesive nanoparticle system for biological nutrients such as curcumin

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and plant phenolic extracts and reported improved absorption and bioavailability.

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These studies were based on natural biopolymers for their application in the

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food industry.

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For optimal formulation of mucoadhesive delivery systems, appropriate

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mucoadhesive polymers are required. The binding capacity of polymers to the

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mucosal membrane has been reported to be influenced by ionic bonds,

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hydrophobic interactions, covalent bonds, and physical entanglement.2,

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Mucoadhesive properties have also been reported to be affected by a number

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of factors such as molecular weight, flexibility, hydrogen bonding capacity,

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cross-linking density, charge, concentration, and polymer swelling.4 Various

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polymers including chitosan (CS), alginate, dextran sulfate sodium salt (DS),

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and carrageenan have been reported as mucoadhesive biopolymers.10

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Particularly, mucoadhesive biopolymers, CS and DS can be also used for

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nanoencapsulation. The positive charge of CS binds to a negatively charged

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mucous surface due to ionic bonds. Müller et al.11 observed that CS-modified

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nanosuspensions exhibited increased retention time in the gastrointestinal tract.

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In addition, the sulfate functional groups of DS form strong hydrogen bonding

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interactions with mucin.12 Tiyaboonchai et al.5 demonstrated the ability of DS to

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adhere to the mucosal surface. Although nanoencapsulation using CS and DS

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could enhance cellular uptake of curcumin,13 the use of CS and DS to enhance

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the mucoadhesive activity of nanoparticles has not been fully explored.

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For a buccal mucoadhesive nanoparticle system for biological nutrient to

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be used in food industry, the natural mucoadhesive polymers is preferable to

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wall material of nanoparticle system with regard to safety. Hence, the aim of this

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study was to formulate buccal mucoadhesive nanoparticles using natural

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mucoadhesive polymers CS and DS. The physical properties of the

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nanoparticles, including particle size, polydispersity index (PDI), and derived

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count rate (DCR) were investigated. In addition, the effect of CS and DS

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concentration on the mucoadhesive activity of the nanoparticles was evaluated

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using in vitro and ex vivo adhesion tests.

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MATERIALS AND METHODS

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Materials. CS (water soluble, M.W. 1,000-3,000, 24 cps, 95% deacetylated)

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was obtained from Kittolife Co. (Seoul, Korea). DS (MW: 15,000, 40,000, and

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200,000 kDa), sodium triphosphate pentabasic (TPP), and magnesium chloride

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were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All

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other chemicals were of reagent grade and all solvents were of HPLC grade.

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Preparation of nanoparticles. Two types of nanoparticles, CS/DS and

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CS/TPP, were obtained by ionic gelation of CS with DS and TPP, respectively.14

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CS was dissolved in distilled water at a concentration of 1 - 2 mg/mL. The

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CS/DS nanoparticles were prepared by adding 3 mL of DS solution (0.2 – 0.6

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mg/mL) into CS solution for 10 min under magnetic stirring (1,000 rpm, WiseStir

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MS-MP8, Wise Laboratory Instruments, Wertheim, Germany). The CS/TPP

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nanoparticles (control group) were prepared by adding 1 mL of TPP solution

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(0.3 – 0.6 mg/mL) into CS solution for 10 min under magnetic stirring.

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Physical properties of the nanoparticles. The physical properties of

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the CS/DS and CS/TPP nanoparticles, including particle size, PDI, zeta

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potential, and DCR, were determined by dynamic light scattering with a Malvern

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Zetasizer

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Worcestershire, UK). All measurements were performed in triplicate at multiple

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narrow modes at 25 ± 1°C.

Nano

ZS

instrument

(Malvern

Instruments

Ltd.,

Malvern,

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Determination

of

in

vitro

mucoadhesive

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properties.

The


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mucoadhesive properties of the nanoparticles to mucin were investigated as

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described previously,15 but with minor modifications. Briefly, 0.6 mL of mucin

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solution (0.5 mg/mL) was mixed with 0.6 mL of nanosuspension and incubated

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at 37°C in a shaking water bath for 1 h. After centrifugation at 14,000xg for 40

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min, the supernatant was collected and the amount of free mucin was

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measured using the Bradford protein assay. The supernatant was incubated

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with Bradford reagent for 5 min, after which the absorbance was measured at

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595 nm with a spectrophotometer (Biomate 3S, Thermo Scientific, Waltham, MA,

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USA). The amount of mucin-adsorbed nanoparticles was calculated as the

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difference between the total amount of mucin added and the residual mucin in

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the supernatant. Mucin concentration was calculated from a standard curve of

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mucin in concentration from 0-5 mg/mL .16

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Determination of ex vivo mucoadhesive properties. The ex vivo

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mucoadhesive properties of the nanoparticles were determined by the methods

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of Kockkisch et al.17 and Patel et al.,18 with minor modifications. All procedures

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for collecting buccal cells were approved by the Hanyang University Institutional

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Review Board. Buccal cells were collected from 10 healthy male and female

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volunteers by gently scraping the oral cavity with a tongue depressor. Donors

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were prohibited from eating or drinking for 1 h prior to buccal cell collection.

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Cells were immediately suspended in 0.25 M aqueous sucrose solution and the

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cell suspension was then added to 0.5% (w/v) trypan blue solution. The cell

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concentration was determined using a hemocytometer and standardized to 48 x

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106 cells per test. All cells were stored at 4°C and used within 4 h of collecting.

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The cells were separated from supernatants by centrifugation at 700 x g

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for 5 min and then reacted with samples (polymers or nanoparticles) in

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phosphate buffer in a 30°C shaking incubator for 30 min. The 0.1% Alcian blue

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was added and incubation for 1 h at 30°C was carried out. The cells were then

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washed using 0.25 M sucrose solution. The complexed dye with polymer

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treated cells was eluted by immersion in 5 M magnesium chloride for 1 h at

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30°C. Cells were then spun by centrifugation at 700 x g for 15 min and the

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absorbance of the supernatant was measured at 605 nm using a

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spectrophotometer. A control was performed following the same procedure

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without a test sample. Results are expressed as a percentage reduction as

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compared with control.

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Statistical analysis. Statistical analyses were conducted using SPSS

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(Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA).

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One-way analyses of variance (ANOVA) were performed to investigate the

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significance of differences between conditions (p

Preparation and Characterization of Mucoadhesive Buccal

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