Mucoadhesive chitosan-gum arabic nanoparticles enhance the

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Mucoadhesive chitosan-gum arabic nanoparticles enhance the absorption and antioxidant activity of quercetin in the intestinal cellular environment Eun Suh Kim, Da Young Kim, Ji-Soo Lee, and Hyeon Gyu Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00008 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Journal of Agricultural and Food Chemistry

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(To be submitted to the Journal of Agricultural and Food Chemistry)

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Mucoadhesive chitosan-gum arabic nanoparticles

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enhance the absorption and antioxidant activity of

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quercetin in the intestinal cellular environment

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†

†

†

†, *

Eun Suh Kim , Da Young Kim , Ji-Soo Lee , and Hyeon Gyu Lee

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†

Department of Food and Nutrition, Hanyang University,

222, Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea

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Running title: Nanoencapsulation of quercetin

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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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Quercetin (QUE)-loaded nanoparticles (QCG–NPs) were fabricated by ionic

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gelation between chitosan (CS) and gum arabic (GA) at pH 3.5. At constant CS (0.5

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mg/mL) and QUE (60 μM) concentrations, QCG–NPs (260–490 nm) were prepared

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uniformly with 0.8–2.2 mg/mL of GA and exhibited high QUE encapsulation efficiency

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(94.8–98.0%) and sustained QUE release (4.42–8.89% after 8 h). Due to the

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electrostatic interaction between QCG–NPs and mucin layer, in vitro mucin and cell

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adhesion of QUE were significantly (p250 kDa) seem to have

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increased mucoadhesion with increasing GA concentration.

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The results were verified in the cell experiments using a small intestinal cell

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model (Figure 2B). When bioactive compounds are orally administered, the compounds

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migrate along the GI tract to the small intestine and are metabolized after passing

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through the small intestinal cell monolayer40. The small intestinal surface consists of an

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upper mucin layer covering the small intestine tissue and small intestinal cells of the

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lower layer, and thus bioactive compounds that enter the small intestine first come into

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contact with the mucin layer. The small intestinal cell model in this study consisted of

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Caco-2 cells, an absorptive type cell that exhibits a variety of small intestinal cell

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functions, and HT-29 cells, which are a goblet type cell that excretes mucus upon

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differentiation; this model is recognized as an effective system for evaluating intestinal

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cell adhesion and permeation28. Since administered compounds move continuously

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during the digestion process, the absorption rate of bioactive compounds can be

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increased when the residence time at the small intestine site is prolonged due to

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adhesiveness with the mucin layer. Therefore, due to the absorption mechanism of the

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material described above, the bioactive substances with higher in vitro mucus and cell

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adhesiveness can be deduced to have higher intestinal absorption. Likewise, it can be


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assumed that the comprehensive cellular absorption of QUE is also increased by QCG-

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NPs, since in vitro mucus and cell attachment of QUE was increased by encapsulation

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in QCG-NPs, in this study. This hypothesis was verified by the study evaluating the

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amount of permeated QUE through intestinal cell monolayer.

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Transcellular transport across a co-culture cell monolayer The amount of

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transported QUE through the cell monolayer formed by co-culturing Caco-2 and HY-29

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cells gradually increased with time in all QCG–NP groups (Figure 3A). In the first 30

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min, free QUE showed generally higher amounts of transported QUE (0.16 ± 0.01

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μmol/L) compared with the QCG–NP groups (0.09–0.18 μmol/L). However, after 120

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min, the permeation of QUE was increased in the experimental group with higher

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mucoadhesive properties, and the transport flux was higher in the following order: QCG

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2.0 (0.0122), 2.2 (0.0100), 1.6 (0.0097), 0.8 (0.0076), and free QUE (0.0071).

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Moreover, Papp values which calculated using the transport flux showed the same

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tendencies (Figure. 3B). The Papp of free QUE (11.90 ± 1.07 cm-6/sec) was increased

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to 20.59 ± 0.86 cm-6/sec, 16.84 ± 0.94 cm-6/sec, 16.27 ± 0.49 cm-6/sec, and 12.85 ± 1.66

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cm-6/sec in QCG–NPs 2.0, 2.2, 1.6, and 0.8, respectively.

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Substances can transport into cells through two distinct mechanisms: active

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transport, which involves a carrier, or passive transport such as diffusion, which is

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driven by the concentration gradient40. Moreover, substances less than 700 Da in MW

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usually can be transported through passive transport which can be classified into simple

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diffusion, restricted diffusion, and facilitated diffusion41. Specifically, restricted

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diffusion which is transmitted through tight junctions (0.4 nm) and the facilitated

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diffusion which is transmitted by carrier occurs mainly in substances with less than 200



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Da of MW. In contrast, most physiologically active substances in the 300–400 MW

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range, including QUE (MW 302.236), are absorbed through simple diffusion42. The

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QCG–NPs developed in this study were prepared using polymeric polysaccharides CS

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(50–190 kDa) and GA (>250 kDa), and the particle size of resultant NPs ranged from

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267–493 nm. Therefore, the increased permeation of QUE may be due to QCG–NPs

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allowing QUE to be stably transported through simple diffusion in the mucosa-adherent

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state due to increased retention time in the intestinal environment by mucoadhesion

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properties22.

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However, the permeability of QUE in QCG–NPs 2.2 was significantly lower

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than that of QCG–NPs 2.0, unlike the results of adhesion studies. This may be due to a

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decrease in surface area from the increased particle size of QCG–NPs that reduced the

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cellular contact efficiency of QUE released from QCG–NPs36. Thus, QCG–NPs 2.2

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with higher particle size than that of QCG–NPs 2.0 had a practically lower cell

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permeability of QUE, although the cell attachment characteristics were similar.

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Antioxidant activity The CAA unit of free QUE (115.8 ± 4.4) was

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significantly increased by encapsulation within QCG–NPs (Figure 4A). QCG–NPs 2.0

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showed significantly the highest CAA unit (191.3 ± 13.8), followed by QCG 2.2 (174.1

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± 18.9), 1.6 (165.7 ± 15.8), and 0.8 (134.8 ± 12.5). Moreover, the in vivo FRAP assay

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results were in good agreement with the CAA results (Figure 4B). Since previous

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studies reported that the in vivo FRAP value rapidly increased to its maximum within

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the initial 2 h of ingestion and then sharply decreased, the blood collection time was

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determined to be 2 h in this study. After 2 h of ingestion of free QUE or QCG–NPs, the

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FRAP value was the highest in QCG–NPs 2.0 (142.1 ± 22.4 μmol/L), followed by 2.2


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(130.8 ± 23.6 μmol/L), 1.6 (104.4 ± 8.0 μmol/L), 0.8 (87.3 ± 24.0 μmol/L), and free

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QUE (79.8 ± 7.8 μmol/L).

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CAA results can be influenced by various factors including the interaction

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between bioactive compounds and targeted cells, because antioxidant activity of

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bioactive compounds can be expressed following after permeation in to the cells in

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CAA assay43. Moreover, in vivo experiments are recognized as useful models that can

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reflect various biological mechanisms that are not present in in vitro or cell studies. As

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shown in the cell adhesion and permeation studies, QCG–NPs showed high

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mucoadhesive properties following interactions with the mucosal layer, resulting in

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improved permeation of QUE through epithelium cells by simple diffusion. Therefore,

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the increased cell adhesion and absorption properties of QCG–NPs seemed to result in

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higher antioxidant activities expression of QUE after permeation into targeted cells or

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oral administration.

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In this study, we encapsulated QUE within QCG–NPs prepared by ionic

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gelation between CS and GA with the aim of improving its bioavailability. QCG–NPs

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were effectively prepared using constant concentrations of CS (0.5 mg/mL) and QUE

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(60 μM) and 0.8–1.6 mg/mL GA concentrations that showed suitable PDI, and particle

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size varied from 267.3 to 493.2 nm. QCG–NPs prepared with all GA concentrations

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showed significantly higher in vitro mucin and intestinal cell adhesion compared with

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free QUE due to interactions between positively charged QCG–NPs and the negatively

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charged mucin layer. Moreover, the adhesion properties were increased with increasing

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concentration of GA. Based on the combined MW of QUE (302.236), CS (50–190

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kDa), and GA (>250 kDa) constituting the QCG–NPs and the particle size of the



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resultant NPs, QUE encapsulated within QCG–NPs could pass through the intestinal

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cell by simple diffusion. Because QCG–NPs cannot migrate directly into intestinal cells,

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QCG–NPs could enhance the cell permeation of QUE through its increased adhesion

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properties in the intestinal cell monolayer. Therefore, QCG–NPs with higher adhesion

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properties exhibited higher cell permeability of QUE in the permeation studies. In

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contrast, comparison between QCG–NPs with similar levels of adhesion showed that as

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the particle size was smaller, QUE absorption on the cell monolayer was effective due

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to the increased surface area. Furthermore, the results of the permeation experiments

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were also verified by the CAA and in vivo FRAP assays, which evaluated the

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antioxidant activities of QUE that was substantially absorbed into cells or rat blood.

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This study is significant, as it has partially demonstrated that the substantial

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absorption of bioactive compounds using NPs in the food field is due to mucus and cell

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adhesion properties. Further validation of our results is needed in an experimental

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model using more practical small intestinal environment, and future studies should also

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be examined the absorption mechanism in more detail.

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ACKNOWLEDGEMENT

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This research was supported by the Basic Science Research Program through

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the National Research Foundation of Korea (NRF), which is funded by the Ministry of

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Science, ICT, & Future Planning (No. 2014M3A7B4051898).

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NOTES

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The authors declare no competing financial interest. REFERENCES


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619



620

Table 1. Preparation conditions and characteristics of quercetin-loaded chitosan-gum arabic nanoparticles. Types of nanoparticles

Chitosan (mg/mL)

Quercetin Gum Arabic (μM) (mg/mL)

Particle size (nm)

Polydispersity index

Zeta potential (mV)

Encapsulation efficiency (%)

Loading efficiency (%)

QCG NPs 0.8

0.8

493.2 ± 28.1a

0.481 ± 0.057a

31.5 ± 3.6a

94.8 ± 0.8b

1.31 ± 0.01a

QCG NPs 1.6

1.6

267.3 ± 43.6d

0.234 ± 0.022b

24.7 ± 1.9b

97.3 ± 0.8a

0.83 ± 0.01b

QCG NPs 2.0

2.0

332.7 ± 31.3c

0.219 ± 0.014b

19.9 ± 0.7c

98.0 ± 1.3a

0.71 ± 0.01c

QCG NPs 2.2

2.2

427.1 ± 11.3b

0.200 ± 0.016b

18.6 ± 1.0c

97.3 ± 1.7a

0.65 ± 0.01d

0.5

621

Page 30 of 36

a–dDifferent

60

letters in the same column indicate significant differences (p < 0.05).


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622


Figure captions

623 624

Figure 1. In vitro QUE release from QCG NPs. Data are the mean ± SD of three replicate

625

experiments.

626 627

Figure 2. Adhesion of free QUE and QCG NPs to (A) mucin and (B) co-cultured Caco-2 and

628

HT-29 cell monolayer. Data are the mean ± SD of three replicate experiments.

629

letters indicate significant differences (p

Mucoadhesive chitosan-gum arabic nanoparticles enhance the

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