Development of Yam Dioscorin-Loaded Nanoparticles for Paracellular

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Article

Development of yam dioscorin-loaded nanoparticles for paracellular transport across human intestinal Caco-2 cell monolayers Hung-Ling Hsieh, Chia-Hung Lee, and Kuo-Chih Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04150 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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Journal of Agricultural and Food Chemistry

TOC Graphic

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ACS Paragon Plus Environment

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1

Development of yam dioscorin-loaded nanoparticles for paracellular transport

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across human intestinal Caco-2 cell monolayers

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Hung-Ling Hsieh, Chia-Hung Lee, Kuo-Chih Lin*

5 6

Department of Life Science, National Dong Hwa University, Hualien County 974,

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Taiwan

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*Corresponding author; E-mail: [email protected].

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ABSTRACT

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Dioscorins, the major storage proteins of yam tubers, exert immunomodulatory

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activities. To improve oral bioavailability of dioscorins in the intestine, recombinant

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dioscorin (rDioscorin) was coated with N,N,N-trimethyl chitosan (TMC) and

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tripolyphosphate (TPP), resulting in the formation of TMC-rDio-TPP nanoparticles

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(NPs). The loading capacity and entrapment efficiency of rDioscorin in the NPs were

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26+0.7% and 61+1.4%, respectively. The NPs demonstrated a substantial release

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profile in the pH environment of the jejunum. The rDioscorin released from the NPs

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stimulated proliferation and phagocytosis of the macrophage RAW264.7 and activated

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the gene expression of IL-1β and IL-6. Incubation of the NPs in the Caco-2 cell

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monolayer led to a 5.2-fold increase of Papp compared with rDioscorin alone,

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suggesting that rDioscorin, with the assistance of TMC, can be promptly transported

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across the intestinal epithelia. These results demonstrate that the TMC-rDio-TPP NPs

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can be utilized for elucidating the immunopharmacological effects of dioscorins

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through oral delivery.

29 30 31

Key words: Dioscorin; Trimethyl chitosan; Nanoparticles; Oral delivery; Caco-2

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1. INTRODUCTION

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Yams (Dioscorea spp.) are an important staple in Africa and are also widely

35

consumed in Asia.1 Dried slices of yam tubers have been used in Chinese herbal

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medicines since the ancient times. It may strengthen the functions of the spleen,

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kidney, liver, and stomach, reduce phlegm, and heal fatigue, chronic diarrhea, and

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diabetes.2 Yam tubers comprise of approximately 6%–13% crude proteins on a dry

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weight basis.3 Dioscorins, the major storage proteins of yam tubers, account for

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approximately 85% of the total protein content in yam tubers. Several studies have

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demonstrated that both native and recombinant dioscorins (rDioscorin) of several yam

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species exhibit immunomodulatory activities by activating the Toll-like receptor 4

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(TLR4) signaling pathway and upregulating the expression of cytokine genes

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involved in pro-inflammatory, inflammatory, and anti-inflammatory responses.4–7

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Furthermore, both native and recombinant dioscorins inhibit ovalbumin-induced

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allergic reactions in the BALB/c mice by promoting T helper (Th) 1 cell responses

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and thus modulating the Th1/Th2 imbalance.8,9 Oral administration of dioscorins from

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D. alata to BALB/c mice may improve hypertension, stimulate splenocytes to secrete

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cytokines such as IFN-γ, IL-4, and IL-10, increase the subpopulation in NK and B

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cells and the numbers of Peyer’s patches, attenuate oxidative status and learning

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dysfunction, and improve the metabolic syndrome of diabetes mice.10–13 The feasible

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dosages for the dioscorins used in oral administration range from 50 to 80 mg/kg/dose.

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However, for a person with a 60-kg body weight, the dosage is approximately 0.5–0.8

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g/dose according to the body surface area scaling method for dose translation from

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animal to human.14

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Oral bioavailability of protein and peptide therapeutics has been demonstrated to

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be very poor, mainly because of protease digestion in the gastrointestinal (GI) track,

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the epithelial cells that prevent cellular uptake of hydrophilic macromolecules, and the


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tight junctions between epithelial cells that inhibit paracellular transport of protein

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and peptide therapeutics.15 To overcome these obstacles, many studies have focused

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on using chitosan as a delivery carrier of biomacromolecules for oral administration to

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enhance intestinal absorption and paracellular transport across mucosal epithelia to

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improve the oral bioavailability of biological macromolecules.16–18 Chitosan is a low

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toxic, biodegradable, and biocompatible muco-adhesive biopolymer. The US Food

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and Drug Administration (FDA) has approved the use of chitosan in the hemostatic

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ChitoFlex® PRO dressing (HemCon Medical Technologies Inc., OR, USA) for

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hemorrhage control.19 However, the low solubility of chitosan at neutral pH values

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restricts its use as an absorption enhancer at specific sites of the intestine, such as in

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the jejunum and ileum.20 To solve this problem, N,N,N-trimethyl chitosan (TMC), a

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quaternized derivative of chitosan, has been synthesized by adding methyl groups to

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the amine groups of chitosan molecules.20–24 Synthesis of TMC may produce

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O-methylation on C3 and C6 carbons, depending on the synthetic strategy used.25

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O-methylation lowers the solubility of TMC in aqueous solution, while O-methylated

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TMC displays reduced cytotoxicity comparing to O-methyl free TMC.23 TMC

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exhibits a permeation-enhancing trait, which is attributed to its positively charged

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ionic interaction with the tight junctions via the C-Jun NH2-terminal kinase-dependent

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pathway.20,26

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The TMC nanoparticles (NPs) have been widely explored for diverse

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pharmaceutical and biomedical applications such as drug delivery, protein and peptide

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delivery, gene delivery, vaccine delivery, absorption enhancement, diagnostic, and

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tissue engineering applications.22,24 TMC NPs can be prepared through ionic

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cross-linking with negatively charged multivalent anions or polyanions such as

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tripolyphosphate (TPP), poly(lactide-co-glycoside), and poly(γ-glutamic acid) to



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improve the packaging of therapeutic macromolecules and the stability of the NPs.27

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All of these cross-linking agents have been approved by the FDA for use in food or

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clinical applications.27–29 Of these polyanions, the TMC-TPP has been applied for

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encapsulating antigens such as bovine serum albumin and ovalbumin for nasal

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vaccination,

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neuroexcitation-associated diseases such as epilepsy and convulsions, vitamins as

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nutritional supplements in certain foods, paclitaxel against several types of cancers,

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diclofenac sodium for relieving ocular inflammation, and curcumin against tumor

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cells.30–36 All these reports demonstrate that the TMC-TPP NPs have the potential

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application in various pharmaceutical, biomedical, and biomaterials fields.

anti-neuroexcitation

peptide

for

the

treatment

of

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To protect the rDioscorin from degradation in strong acidic environment in the

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stomach, enhance intestinal absorption, and improve effective dose usage for

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elucidating the immunopharmacological effects of dioscorins through oral delivery,

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we aim to use TMC-TPP NPs for encapsulating rDioscorin and further evaluate the

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immunomodulatory effects of the encapsulated rDioscorin and the potential of

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TMC-rDio-TPP NPs for oral delivery. TMC was synthesized with a high degree of

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quaternization and rDioscorin coated with TMC-TPP to make TMC-rDio-TPP NPs.

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The quaternization of TMC on the formation of particles can increase the electrostatic

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attractions between the protein and TPP molecules, which may reduce the amount of

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protein-leaching that occurs during the delivery process and protect the rDioscorin

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from acid degradation and protease digestion in the GI tract. Various physicochemical

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properties of the TMC-rDio-TPP NPs such as particle size, zeta potential, loading

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capacity, entrapment efficiency, in vitro release profiles, and paracellular permeability

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were characterized and the immunomodulatory effects of released rDioscorin

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investigated. Finally, the effect of TMC-rDio-TPP NPs on the improvement of

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paracellular permeability of released rDioscorin was evaluated in vitro using Caco-2


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cell monolayers.

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2. MATERIALS AND METHODS

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Chitosan (50-80 kDa, 93% deacetylated), TPP, bovine serum albumin (BSA),

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and the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Sodium iodide (NaI) and 1-methyl-2-pyrrolidinone (NMP) were purchased from Alfa

116

Aesar (Heysham, UK) and methyl iodide (CH3I) from Kanto (Tokyo, Japan).

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2.1 Preparation of recombinant dioscorin

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The rDioscorin (pI 6.27) of Japanese yam was isolated from the cultures of E.

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coli BL21 (pDj-dioA3) and purified according to our previous study Jheng et al.6

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2.2 Preparation of TMC

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TMC was obtained from chitosan according to the methodology published by

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Sieval et al. with modification (Supplementary material).21

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2.3 Determination of the degree of quaternization of TMC

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We used nuclear magnetic resonance (NMR) to determine the degree of

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quaternization of TMC according to the method described by Sieval et al.21 Briefly,

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10 µg each of chitosan and TMC were lyophilized and subsequently dissolved in 500

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µL D2O (Sigma, MO, USA). The 1H-NMR spectrum of TMC was measured using a

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500 MHz spectrometer (Bruker-Spectro Spin 400 UltraShieldTM, Allemagne,

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Germany). The degree of quaternization was calculated from the following equation:

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Degree of quaternization = [(∫TM/∫H) x 1/9] x 100%

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∫TM: is the integral of chemical shift of the hydrogens of -N+(CH3)3 peak at 3.3 ppm.

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∫H is the integral of H1 peaks between 4.8 and 6.3 ppm, related to hydrogen atoms

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bound to carbon 1 of the chitosan molecule, which is taken as the reference signal.

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Fourier Transform Infrared Spectrometer (FTIR) analysis was performed by

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using potassium bromide (KBr) pellet method.37 Ten micrograms each of chitosan and

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TMC were lyophilized. One gram of KBr powder (Thermo, Waltham, USA) was

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heated in oven at 100 oC for 24 h and used as a blank. The TMC samples were mixed


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with KBr in a mass ratio of 1:99, compressed into a thin piece, and scanned for

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percent transmittance in the range of 500–4000 cm-1 using FTIR spectrophotometer

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(Alpha FT-IR Spectrometer, Bruker, USA).

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2.4 Preparation of TMC-rDio-TPP NPs and calculation of loading capacity and

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entrapment efficiency

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TMC-rDio-TPP NPs were prepared by ionic gelation process according to the

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methods described by Gan and Wang.38 Fifty micrograms of TMC were dissolved in

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50 mL of ddH2O (1 mg/mL, pH 6.5). A solution of TPP at the concentration of 0.6

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mg/mL was prepared with ddH2O (pH 6.5). Various amounts of rDioscorin were

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separately mixed with 0.2 mL TPP (0.6 mg/mL) at mass ratio ranged from 0.2:1 to 2:1

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for rDio:TPP, and the rDio-TPP mixtures were stored at 4 oC. The rDio-TPP mixtures

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were separately added dropwise to the 0.5 mL TMC solution (1 mg/mL) to form

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various ratios ranged from 4:0.2:1 to 4:2:1 (TMC:rDio:TPP). An opalescent

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suspension was formed spontaneously under the aforementioned conditions. The

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TMC-rDio-TPP NPs were separated by centrifugation at 20000 xg at 15 oC for 30 min.

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The supernatant was collected, and the amount of unincorporated rDioscorin was

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determined according to the absorbance at OD280. The pellet of TMC-rDio-TPP NPs

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was collected followed by freeze drying and stored at 4 oC for further studies. Both

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the loading capacity and entrapment efficiency were determined by indirect method:

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loading capacity (%) = (protein in the NPs/weight of the NPs) x 100%。

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entrapment efficiency (%) = (protein in the NPs/the feeding protein) x 100%。

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2.5 Determination of particle size, zeta potential, and polydispersity index

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The TMC-TPP NPs or TMC-rDio-TPP NPs were diluted to 0.1 mg/mL with

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ddH2O and adjusted to pH 6.3–6.4 by 1.0 mM HCl. The particle diameter, zeta

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potential, and polydispersity index (PDI) of NPs were determined by Laser diffraction

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submicron particle size analyzer (Zetasizer 3000HS, Malvern Instruments, UK)



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through the 90° scattering angle (Supplementary Figure). The samples were filled into

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a plastic cuvette with an electrode for sample analysis. The zeta potential was

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determined by the electrophoretic mobility of water-dispersed samples in the automatic

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mode. The measured conditions were: laser wavelength at 532 nm, temperature at 25

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°C, six consecutive measurements for the number of scan. Size distribution was

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measured by intensity analysis. The samples were detected in triplicate, and a total of

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three independent experiments were performed.

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2.6 Examination of the nanoparticles by scanning electron microscopy and

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atomic force microscope

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The morphology of the TMC-rDio-TPP NPs was assessed by scanning electron

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microscopy (SEM) and atomic force microscope (AFM). Accordingly, the

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nanoparticles were prepared in solution as described above at pH 6.5 at 37 oC for 1 h.

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The TMC-rDio-TPP NPs were washed with ddH2O (pH 6.5) for 4 times to eliminate

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the ions and then lyophilized. The lyophilized nanoparticles were coated with a gold

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layer under vacuum followed by SEM (S-3400N, HITACHI, Japan) and AFM

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(Caliber-Peak Force Tapping, Bruker, USA) examination. The NPs size from AFM or

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SEM analysis was obtained from the measurement of approximately 50 particles, and

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the representative image was presented in Figure 3.

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2.7 Evaluation of dioscorin released from nanoparticles in various pH

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environments

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Three hundred micrograms of TMC-rDio-TPP NPs (containing approximately 50

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µg of rDioscorin) were incubated in 1 mL each of hydrochloride solution at pH 2.5

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(simulating gastric fluid), 10 mM phosphate buffer saline (PBS) at pH 6.0 (simulating

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duodenal fluid), or 10 mM PBS at pH 7.4 (simulating jejunal fluid) at 37 oC, 50 rpm,

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and 150 µL of each sample was collected for various time intervals. The samples were

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centrifuged at 20000 xg for 30 min at 15 oC. The release of rDioscorin in the


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supernatant was determined according to the absorbance at OD280. The studies were

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performed in triplicate, and the values were collected from three separate

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experiments.

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2.8 Proliferation assay of macrophage RAW264.7 in response to TMC-rDio-TPP

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NPs

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Detection of the proliferation of macrophage cell line RAW264.7 in response to

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TMC-rDio-TPP NPs was performed according to our previous study.5 Briefly, the

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macrophage RAW264.7 of 1 x 104 cells/100 µL/well were treated with 10 µL each of

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10 mM PBS buffer (pH 7.4), lipopolysaccharide (LPS, from E. coli 0111:B4, Sigma,

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MO, USA)(5 µg/mL), rDioscorin (50 µg/mL), TMC-TPP NPs (500 µg/mL), or

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TMC-rDio-TPP NPs (600 µg/mL) (containing approximately 100 µg/mL rDioscorin).

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To eliminate any possible LPS contamination in the recombinant dioscorin extracts, the

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TMC-rDio-TPP NPs were predisposed to LPS inhibitor polymyxin B (10 µg/mL,

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Sigma, Missouri, USA) for 20 min. After incubation at 37°C in a humidified incubator

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containing 5% CO2 for 48 h, viability of the cells were determined by CellTiter 96

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AQuenous Non-Radioactive Cell Proliferation Assay (Promega, WI, USA) following

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the manufacture’s protocol. The values were calculated as relative intensity of the

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absorbance as compared to the vehicle control with PBS. All the studies were

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performed in triplicate, and the values were collected from three separate experiments.

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2.9 Phagocytic effect of TMC-rDio-TPP NPs on macrophage RAW264.7

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The effect of TMC-rDio-TPP NPs on phagocytosis of RAW264.7 was performed

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according to our previous study.5 Briefly, the macrophage RAW264.7 (1 x 105

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cells/100 µL/well) were cultured with 100 µL LPS (5 µg/mL), rDioscorin (50 µg/mL),

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TMC-TPP NPs (500 µg/mL), TMC-rDio-TPP NPs (600 µg/mL), or 10 mM PBS

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buffer (pH 7.4) as a control. The rDioscorin and TMC-rDio-TPP NPs were

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predisposed to LPS inhibitor polymyxin B (10 µg/mL, Sigma, Missouri, USA) for 20



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min to eliminate any possible LPS contamination in the recombinant dioscorin extracts.

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After incubation at 37°C, 5% CO2 for 4 h, 100 µL of fluorescein isothiocyanate

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(FITC)-labeled E. coli DH5α (1 x107 cfu/well) was added to each well to obtain an

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effector : target ratio of 100:1 for phagocytosis analyses. The fluorescence intensity of

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FITC was determined by using a microplate fluorometer (Fluoroskan Ascent FL,

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Lsbsystems, Finland) with an excitation wavelength at 480 nm and emission

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wavelength at 517 nm. The phagocytic effect of dioscorins on RAW264.7 is defined as

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relative intensity of FITC fluorescence as compared to the control. Each treatment was

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performed in triplicate, and the values were collected from three separate experiments.

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2.10 Stimulation of cytokine gene expression by TMC-rDio-TPP NPs

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The effect of TMC-rDio-TPP NPs on stimulating cytokine gene expression was

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performed according to our previous study.7 Briefly, the macrophage RAW264.7 of 1

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x 106 cells/500 µL/well were treated with 2 µL of 10 mM PBS buffer (pH 7.4), LPS (4

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ng/mL), rDioscorin (0.2 µg/mL), TMC-TPP NPs (1.0 µg/mL), or TMC-rDio-TPP NPs

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(1.2 µg/mL) (containing approximately 0.2 µg/mL rDioscorin) for 4 h. The rDioscorin

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and TMC-rDio-TPP NPs were predisposed to 10 µg/mL LPS inhibitor polymyxin B

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for 20 min to eliminate any possible LPS contamination in the recombinant dioscorin

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extracts. Real-time quantitative PCR (RT-qPCR) amplification of the cDNA clones

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were performed in thermocycling conditions of 95°C for 2 minutes followed by 39

235

cycles of 95°C for 15 sec, Tm°C for 1 min, and a final 1 min at 72°C on a CFX96TM

236

real-time PCR detection system (Bio-Rad, CA, USA). The amplified GAPDH gene

237

was used as an internal control. Relative gene expression levels were calculated with

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the 2-△△Ct method. All the qPCR were performed in triplicate, and the values were

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collected from three separate experiments.

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2.11 Test of paracellular transport of TMC-rDio-TPP NPs


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The transcellular investigation of TMC-Dio-TPP NPs through Caco-2 cell

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monolayers were carried out by the method described by Arshad et al.39 Two hundred

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microliters of Caco-2 cells (3x104 cells/insert) were seeded in the apical chamber

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(PET membrane, 0.4 mm in pore size, diameter 6.5 mm, 0.33 cm2 of cell growth area)

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of Costar Transwell 24 wells/plate (Corning Costar Corp, Cambridge, MA, USA)

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which made the cell number to be 8.9x104 cells/cm2. In addition, 0.8 mL of medium

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was added in the basolateral chamber. To test the confluent growth of Caco-2 cell

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monolayer, the PBS in the apical chamber were replaced with 0.2 mL of PBS (pH 7.4)

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containing 10 µg/mL of sodium fluorescein (Na-Flu; Sigma, MO, USA) as

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paracellular marker with or without 0.2 mL of 1.5 mg/mL TMC-TPP NPs. One

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hundred microliters of the solution was collected from the basolateral chamber at

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various time points, and the removed solution was replenished with 100 µL of fresh

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PBS. The amount of Na-Flu transported across the Caco-2 cell monolayer was

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determined by an ELISA plate reader (Multiskan Go, Thermo, USA) with λex at 460

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nm and λem at 515 nm. The Na-Flu of 0.01–5 µg/mL was used as a standard. The

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results were collected from three separate experiments.

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For transport study, the medium in the apical chamber were replaced with 0.2

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mL of FITC-rDioscorin (300 µg/mL) or TMC-FITC-rDio-TPP NPs (1750 µg/mL)

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which contains 300 µg/mL of FITC-rDioscorin based on the entrapment efficiency of

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60%. At predetermined time intervals, 0.1 mL of samples were collected from the

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basolateral chamber, and the removed solution was replenished with 0.1 mL of fresh

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PBS. The amount of FITC-rDioscorin transported across the Caco-2 cell monolayer

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was determined by an ELISA plate reader (Multiskan Go, Thermo, USA) with λex at

264

485 nm and λem at 535 nm. The amount of FITC-rDioscorin transported through the

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cell monolayers was calculated according to the standard FITC-rDioscorin (0.01–0.5

266

µg/mL). The results were collected from three separate experiments. The cumulative



267

amount of transported FITC-rDioscorin were determined. The apparent permeability

268

coefficients (Papp, cm/s) was calculated according to the following equation:

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Papp = dQ/dt x 1/A．C0

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Where Q is the total amount of FITC-rDioscorin permeated (ng), t is the total time of

271

the experiment, A is the diffusion area of the cell monolayer (cm2), and C0 is the

272

initial concentration of the FITC-rDioscorin in the insert compartment (ng/cm3).

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2.12 Statistical analysis

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Statistical analysis for two groups was performed using Student's t-test.

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Differences were considered to be statistically significant if P < 0.05 (*), P < 0.01

276

(**), or P < 0.001 (***). Comparison among multiple groups was performed by

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one-way ANOVA followed by Tukey’s honest significant different (HSD) test using

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Statistical Product and Service Solutions version 14.0 (SPSS, IL, USA).

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3. RESULTS AN DISCUSSION

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3.1 Preparation and characterization of TMC-rDio-TPP NPs

282

To improve the bioavailability of dioscorin in the intestine by oral administration,

283

avoid enzymatic (or acidic) degradation in the GI tract, and prevent poor GI

284

permeability, the TMC-rDio-TPP NPs were prepared through ionic gelation with the

285

positively charged TMC, the negatively charged TPP, and rDioscorin at pH 6.5. TPP

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was used to cross-link and stabilize the NPs. The TMC used in this study was

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synthesized by methylating chitosan according to the method described by Sieval et al.

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with modification.21 The results of the NMR that was used to analyze the modification

289

of the TMC are depicted in Figure 1A. The 1H-NMR spectrum of the TMC is similar

290

to that reported by Sieval et al.21 The peak at 3.0–3.1 ppm assigned to the hydrogen

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atoms of C2 in chitosan disappeared, while the peak at 3.35 ppm assigned to

292

-N(CH3)3+ in TMC increased substantially. In addition, the peaks at 3.6–3.9 ppm

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assigned to the hydrogen atoms of C3-C6 in chitosan disappeared, and at 3.4–3.6 ppm,

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new peaks appeared, which were assigned to the O-methylated site [3-OCH3] and

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[6-OCH3] in TMC. The percentage of O-methylation on [3-OCH3] and [6-OCH3] is

296

32.99% and 37.14%, respectively. The integral for the peak at 3.3–3.4 ppm is 9.0,

297

while it is 1.5 for the peak at 4.8–6.3 ppm (hydrogen of the anomeric carbon). The

298

degree of quaternization is 67.0%, which is within the range of high degree of

299

quaternization (60%–80%).24 FTIR spectroscopy was also performed for functional

300

group identification of the TMC (Figure 1B). In the FTIR spectrum, the peak at 1580

301

cm−1 of -NH2 on chitosan decreased in TMC. By contrast, strong absorption peaks at

302

2890 cm−1 (C-H stretch) and 1489 cm−1 (-CH3 asymmetric angular deformation) for

303

TMC were detected, compared with those for chitosan, indicating that the -NH2

304

functional group of chitosan had been quaternized to -N(CH3)3+, resulting in the

305

formation of TMC.



306

To determine the optimal ratio of TMC to TPP for preparing NPs, different

307

combinations of the two were tested. The results presented in Table 1 reveal that with

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a decreasing TMC:TPP ratio (from 6:1 to 2:1), the particle size first decreased

309

drastically from 428 nm to 235 nm and then increased to 304 nm. The TMC:TPP

310

mass ratio of 3:1 and 4:1 gave the smallest particle sizes (235–259 nm). With

311

increasing amounts of TPP in the NP preparation, the TPP anions bound to positively

312

charged TMC, resulting in an increase in the cross-linking density between TMC and

313

TPP, which may have compacted the NPs and therefore decreased the particle size.

314

However, when greater amounts of negatively charged TPP bound to TMC, it may

315

have neutralized the positive charge of TMC, decreasing the zeta potential, and

316

destabilizing the cross-linkage between TMC and TPP, resulting in the dilation of the

317

NPs. The results of this study are similar to those reported by Fan et al.,40 who studied

318

the effect of the mass ratio of chitosan to TPP on particle size and zeta potential and

319

found that with increasing mass ratio of TPP to chitosan, the particle size first

320

decreased and then increased drastically.

321

The zeta potential decreased from +24 mV to +14 mV in accordance with the

322

decreasing mass ratio of TMC to TPP (Table 1). The TMC:TPP mass ratios of 3:1 and

323

4:1 gave good zeta potential values (+19.7 mV to +23.3 mV). The polydispersity

324

index (PDI) of both ratios was less than 0.7. Since the TMC:TPP ratio of 4:1 gave the

325

smallest particle size (235 nm) and a high zeta potential (+23.3 mV), it was chosen as

326

the ratio for preparing the TMC-rDio-TPP NPs. Based on the 4:1 ratio, various

327

amounts of rDioscorin were tested for the NPs, ranging from 4:0.2:1 to 4:2:1 for the

328

TMC:rDio:TPP ratio. The particle sizes of the NPs for the various combinations were

329

between 250 nm and 284 nm (Figure 2A). The zeta potential decreased with

330

increasing amounts of rDioscorin added to the NPs (Figure 2B). The NPs with a

331

4:0.28:1 TMC:rDio:TPP ratio had the highest zeta potential (+33 mV). The more


Page 16 of 43

Page 17 of 43


332

rDioscorin that was added, the greater the quantity of proteins that was loaded into the

333

NPs (Figure 2C). The highest loading capacity was 26%, obtained from the NPs with

334

4:2:1 ratio. The entrapment efficiency ranged from 59% to 72% for the various

335

combinations, with the highest entrapment efficiency for NPs being at the 4:0.8:1

336

ratio (Figure 2D). The loading capacity and entrapment efficiency of TMC-rDio-TPP

337

NPs are in the range of those of TMC NPs encapsulated with various drugs or

338

proteins.22,24 Because the ratio of 4:2:1 gave the maximal loading capacity at 26% and

339

high entrapment efficiency at 61%, with similar particle size and zeta potential

340

compared with those of other ratios, the ratio of 4:2:1 for TMC:rDio:TPP was used for

341

preparing the NPs for the subsequent study experiments.

342

The morphology of the TMC-rDio-TPP NPs was examined by SEM and AFM

343

(Figure 3). The results of SEM and AFM reveals that the TMC-TPP NPs exhibited a

344

particle size in the range of 100–150 nm and 107–136 nm, respectively, while the

345

TMC-rDio-TPP NPs exhibited a spherical shape and a slightly larger particle size in

346

the range of 150–200 nm for SEM and of 158–235 nm for AFM. The particle sizes of

347

the TMC-rDio-TPP NPs analyzed by SEM coincided well with those determined by

348

AFM, ranging from 150 nm to 235 nm. However, the size of the TMC-rDio-TPP NPs

349

determined by a particle size analyzer was 284 + 6.6 nm, which was larger than that

350

determined by SEM and AFM. This discrepancy in the size of the TMC-rDio-TPP

351

NPs between the particle size analyzer and SEM and AFM could be due to the

352

swelling of the TMC-rDio-TPP NPs in aqueous media, which yields the

353

hydrodynamic diameter of nanoparticles, whereas SEM and AFM give the actual

354

diameter of nanoparticles in a dry state. Similar results were also observed for the

355

TMC-insulin polyelectrolyte complexes by Mao et al., QCS/AL NPs by Li et al., and

356

TMC-TPP NPs by Fan et al.40–42 Recently, Facchi et al. reported the use of

357

water/benzyl alcohol microemulsion method for preparing TMC-TPP NPs with an



358

average particle size of 98.8 nm and a zeta potential of 47.2 mV, suggesting that

359

microemulsion method provides a potential approach for synthesizing ultrafine

360

TMC-TPP NPs.36

361

3.2 In vitro release of rDioscorin from TMC-rDio-TPP NPs

362

To establish an in vitro–in vivo correlation and evaluate the in vitro release

363

profiles of rDioscorin from the TMC-rDio-TPP NPs in the GI tract, the

364

TMC-rDio-TPP NPs were incubated in buffers of pH 2.5, pH 6.0, or pH 7.4, which

365

reflect the pH environments of the stomach, duodenum, and jejunum, respectively,

366

and the amounts of rDioscorin released from the NPs were determined. The particles

367

sizes, zeta potential, and PDI of the TMC-rDio-TPP NPs in the various pH buffer

368

environments were examined. The results of Table 2 reveals that the particle sizes of

369

the TMC-rDio-TPP NPs in buffers of pH 2.5 and pH 6.0 were 237.7 nm and 231.3 nm,

370

respectively, whereas in the buffer at pH 7.4, they swelled to 457.3 nm. The zeta

371

potential of the TMC-rDio-TPP NPs at pH 2.5 and pH 6.0 were +18.5 mV and +13.1

372

mV, respectively, whereas at pH 7.4, it sharply decreased to +1.0 mV. The PDI were

373

similar among the TMC-rDio-TPP NPs in the different pH buffers.

374

The zeta potential decreased in both pH 2.5 and pH 6.0 solution, while the

375

particle size of TMC-rDio-TPP NPs in both the pH 2.5 and pH 6.0 solution was

376

similar to that of the TMC-rDio-TPP NPs prepared at pH 6.5 (Figure 2), suggesting

377

that the TMC-rDio-TPP NPs remain intact in both pH environments, although the

378

charge density over NP surface was affected in both pH solutions. By contrast, the

379

particle size of TMC-rDio-TPP NPs in the pH 7.4 solution swelled to almost twice its

380

size in both pH 2.5 and pH 6.0 solution, and the zeta potential sharply decreased to

381

+1.0 mV, indicating that the charge of the TMC-rDio-TPP NPs was neutralized at pH

382

7.4, thus leading to the dissociation of the ionic bonds among TMC, rDioscorin, and

383

TPP and subsequently, the swell of TMC-rDio-TPP NPs.43 The neutralization of


Page 18 of 43

Page 19 of 43


384

charge might be due to the following factors. First, with the increase of solution pH

385

from acidic to neutral condition, the negative charge numbers of TPP increase

386

accordingly.44 Secondly, in addition to TMC, N-monomethyl chitosan and

387

N,N-dimethyl chitosan may exist in the TMC polymers. With increasing pH value,

388

monomethylated and dimethylated amines of the TMC polymers could be

389

deprotonated.36 Thirdly, the rDioscorin, with a pI of 6.27, may become negatively

390

charged at pH 7.4.6 All these factors may contribute to the decrease of zeta potential

391

at pH 7.4 and lead to the intermolecular electrostatic repulsion, resulting in the swell

392

of the TMC-rDio-TPP NPs.

393

The results of in vitro release profiles of rDioscorin from the TMC-rDio-TPP

394

NPs reveals that in pH 2.5 buffer, 34.7% of the rDioscorin was released by 15 min,

395

and the release reached 48.6% at 8 h after treatment (Figure 4). In pH 6.0 buffer,

396

20.7% of the rDioscorin was released by 15 min, and the release gradually increased

397

to 38.7% at 8 h. By contrast, in pH 7.4 buffer, 19.0% of rDioscorin was released by 15

398

min, and the release increased to 65.0% at 8 h after treatment. A significant amount of

399

the rDioscorin (60.7%–65.0%) was released from the TMC-rDio-TPP NPs at pH 7.4

400

at 1–8 h after incubation compared with those at pH 2.5 (42.0%–48.6%) and pH 6.0

401

(30.0%–38.7%), indicating that the rDioscorin may release more efficiently from the

402

TMC-rDio-TPP NPs in jejunum.

403

Significant amount of rDioscorin released from the TMC-rDio-TPP NPs in the

404

pH environment of the jejunum demonstrated that the TMC-rDio-TPP NPs can be

405

used for studying the immunopharmacological effects of the oral administration of

406

dioscorins. However, at 15 min after treatment, 34.7% and 20.7% of the rDioscorin

407

had been released at pH 2.5 and pH 6.0, respectively (Figure 4). Because the particle

408

size did not vary substantially in these pH environments (Table 2), the burst release of

409

the rDioscorin from TMC-rDio-TPP NPs at pH 2.5 and pH 6.0 during the early period



Page 20 of 43

410

of treatment may be attributed to the diffusion of the rDioscorin that was absorbed on

411

the external surfaces of the TMC-rDio-TPP NPs rather than packaged inside the NPs

412

during NP preparation. The change in pH may have affected the electrostatic

413

interaction between the loosely bonded rDioscorin and the NPs, resulting in the quick

414

release of rDioscorin. Similar phenomena have been observed in TMC-TPP loaded

415

with bovine serum albumin or bovine hemoglobin, TMC-TPP loaded with paclitaxel,

416

a

417

methacrylamide copolymer) or TMC-cysteine encapsulated with insulin.34,45–47 In

418

these reports, an initial burst release of 20%–50% is observed in the first 15–30 min.

419

Chen et al. attempted to use alginate-modified TMC NPs to reduce burst release

420

without getting a satisfactory result.45

classical

microtubule

inhibitor,

and

TMC-HPMA

(N-(2-hydroxypropyl)

421

The amount of rDioscorin released from TMC-rDio-TPP NPs at pH 2.5

422

(42.0%–48.6%) and pH 6.0 (30.0%–38.7%) at 1–8 h after incubation remain high

423

(Figure 4). Further efforts must be taken to minimize the release of rDioscorin from

424

NPs at pH 2.5 and pH 6.0. Enteric-coated gelatin capsules or polymethacrylate-based

425

copolymers such as Eudragit® could be utilized to load such NPs to prevent the

426

release of rDioscorin from NPs in the gastric environment.48

427

3.3 Bioactivity assay of rDioscorin released from TMC-rDio-TPP NPs

428

Previous studies have demonstrated that both native and recombinant dioscorins

429

have stimulation effects on immune cell proliferation, phagocytic activity, and

430

expression of cytokine genes, such as IL-1β and IL-6.5 –7 To elucidate whether the

431

rDioscorin released from TMC-rDio-TPP NPs are bioactive and have a stimulation

432

effect on the immune cell proliferation, the macrophage cell line RAW264.7 was

433

treated with LPS, rDioscorin alone, TMC-TPP NPs, or TMC-rDio-TPP NPs for 48 h,

434

and cell proliferation was subsequently measured. The results of Figure 5 shows that

435

the rDioscorin released from TMC-rDio-TPP NPs had a similar effect on stimulation


Page 21 of 43


436

of RAW264.7 proliferation as LPS and rDioscorin alone. By contrast, TMC-TPP NPs

437

that did not contain rDioscorin did not stimulate cell proliferation. RAW264.7 cells

438

were then treated with LPS, rDioscorin alone, TMC-TPP NPs, or TMC-rDio-TPP NPs

439

for 4 h and the phagocytic activity of RAW264.7 macrophages against E. coli was

440

evaluated. The results of Figure 6 reveals that the rDioscorin released from the

441

TMC-rDio-TPP NPs, LPS, and rDioscorin alone stimulated RAW264.7 phagocytosis

442

2 to 3 times more than the control or TMC-TPP NPs. The effect of the rDioscorin

443

released from TMC-rDio-TPP NPs on stimulating cytokine gene expression was also

444

evaluated. RAW264.7 cells were treated with LPS, rDioscorin alone, TMC-TPP NPs,

445

or TMC-rDio-TPP NPs for 4 h, and the expression of IL-1β and IL-6 genes was

446

evaluated. As shown in Figure 7, similar to LPS and rDioscorin alone, the rDioscorin

447

released from the TMC-rDio-TPP NPs upregulated the expressions of the IL-1β and

448

IL-6 genes. Overall, this study demonstrated that the rDioscorin released from the

449

TMC-rDio-TPP NPs stimulated proliferation and phagocytic activity of RAW264.7

450

macrophages and activated cytokine gene expression (Figures 5, 6, 7), indicating that

451

the rDioscorin released from the TMC-rDio-TPP NPs remains bioactive.

452

The methodology used for TMC preparation might lead to the formation of

453

surface ionic pairs between TMC NPs and NaCl, resulting in shielding the NPs by

454

NaCl and preventing the interaction of TMC NPs with cell membranes.49 The surface

455

ionic pairs promote a Cl- excess on the TMC structure which may increase

456

cytotoxicity of the TMC NPs. The shielding effect can be eliminated by dialysis

457

process. Therefore, removal of surface ionic pairs by dialysis in future TMC

458

preparation may increase drug delivery performance by providing effective

459

interaction of TMC-rDio-TPP NPs with cells.

460

3.4 Paracellular transport of rDioscorin released from TMC-rDio-TPP NPs

461

across Caco-2 cells



462

Chitosan and TMC have been demonstrated to assist the transport of target

463

proteins across intestinal epithelium cells by opening the tight junctions.20 To evaluate

464

the effect of TMC-rDio-TPP NPs on the improvement of paracellular permeability of

465

released rDioscorin, transport of rDioscorin across a Caco-2 cell monolayer, a cell

466

line that has been widely used to determine drug permeability of intestinal epithelium

467

cells, was investigated.50 The monolayer integrity was evaluated by testing the

468

permeability of sodium fluorescein (Na-Flu), a paracellular marker. Caco-2 cells were

469

incubated with Na-Flu or Na-Flu plus TMC-TPP NPs for various time periods. The

470

results of Figure 8A reveals that the cumulative amount of Na-Flu transported across

471

the Caco-2 cell monolayer was 0.01 µg at 0.5 h and increased to only 0.06 µg at 3 h

472

after treatment. The apparent permeability coefficient (Papp) value for the

473

permeability of Na-Flu was only 1.63 + 0.2 × 10−6 cm/s at 3 h after incubation (Figure

474

8C). The low cumulative amount and Papp value for the permeability of Na-Flu

475

indicated that the Caco-2 cell monolayer was intact, and only a small amount of

476

Na-Flu was transported across the Caco-2 cell monolayer. However, in the presence

477

of TMC-TPP NPs, the cumulative amount of Na-Flu transported across the Caco-2

478

cell monolayer was significantly increased to 0.12 µg at 3 h after treatment, which is

479

2-fold higher than that of Na-Flu only. The Papp value also significantly increased to

480

3.37 + 0.08 × 10−6 cm/s in the presence of TMC-TPP NPs, which is more than 2-fold

481

faster than that of the Na-Flu only, suggesting that TMC-TPP caused the tight

482

junctions to open and assisted in the transport of Na-Flu across the Caco-2 cell

483

monolayer.

484

Next, the Caco-2 cells were incubated with FITC-rDioscorin (FITC-rDio) or

485

TMC-FITC-rDio-TPP NPs and the permeability of FITC-rDio across the Caco-2 cell

486

monolayer was tested. The cumulative amount of FITC-rDio transported across the

487

Caco-2 cell monolayer was 0.43 µg at 0.5 h and increased to 1.0 µg at 3 h after


Page 22 of 43

Page 23 of 43


488

treatment (Figure 8B). The Papp value for the permeability of FITC-rDio was only

489

0.97 + 0.36 × 10−6 cm/s at 3 h after incubation. By contrast, the cumulative amount of

490

FITC-rDio released from the TMC-FITC-rDio-TPP NPs and transported across the

491

Caco-2 cell monolayer was 1.86 µg at 0.5 h and increased to 5.52 µg at 3 h after

492

treatment, which was 5-fold more than that of the Caco-2 cell monolayer incubated

493

with FITC-rDio alone. The Papp value for the permeability of FITC-rDio released

494

from TMC-rDio-TPP NPs was 5.16 + 0.76 × 10−6 cm/s at 3 h after incubation, which

495

is more than 5-fold faster than that of the Caco-2 cell monolayer incubated with

496

FITC-rDio alone and is faster than the Papp values for the insulin released from the

497

TMC-cysteine-insulin NPs (4.0 × 10−6 cm/s), the TMC-insulin-HPMA NPs (3.7 ×

498

10−6 cm/s), and the TMC-insulin-PLGA NPs (4.48 × 10−6 cm/s).46,47,51

499

Overall, the results of this study indicate that TMC-TPP NPs can efficiently

500

encapsulate rDioscorin and, with the assistance of TMC, rDioscorin can be promptly

501

transported across the mucosal epithelia of the intestine and enter the bloodstream

502

through a paracellular pathway to improve the oral bioavailability. Therefore, the

503

TMC-rDio-TPP NPs can be orally administered and used for elucidating the

504

immunopharmacological effects of dioscorins in vivo.

505



506

ACKNOWLEDGEMENT

507

We are indebted to Dr. Chin-Piao Chen and Mr. Ting-Horng Cho of Department of

508

Chemistry of National Dong Hwa University for their assistance in operating NMR

509

spectrometer and Dr. Shien-Der Tzeng and Mr. I-Chih Ni of Department of Physics of

510

National Dong Hwa University in operating SEM and AFM. We thank Drs.

511

Ching-Feng Weng and Jung-Hsin Hsu of Department of Life Science of National

512

Dong Hwa University for kindly providing RAW264.7 and Caco-2 cells, and cell

513

culture facility, respectively. This project was funded by Ministry of Science and

514

Technology (MOST 103-2514-S-259 -003), Taiwan.

515


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Figure legends

672

Figure 1. 1H-NMR (A) and FTIR (B) spectra of chitosan (CS) and TMC.

673 674

Figure 2. Effect of mass ratio of TMC/rDio/TPP on particle size (A), Zeta potential

675

(B), loading capacity (C), and entrapment efficiency (D). The column not sharing a

676

letter in common differs significantly (P < 0.05) according to Tukey's HSD test.

677 678

Figure 3. SEM and AFM images of TMC-TPP (A) and TMC-rDio-TPP (B) NPs at pH

679

6.5. The scale bar in SEM image (left panel) is 200 nm.

680 681

Figure 4. Cumulative in vitro release profiles of rDioscorin from TMC-rDio-TPP NPs

682

at pH 2.5, 6.0, and 7.4. The column not sharing a letter in common differs

683

significantly (P < 0.05) according to Tukey's HSD test.

684 685

Figure 5. The effect of the rDioscorin released from TMC-rDio-TPP NPs on the

686

proliferation of macrophage RAW264.7. The proliferation index of each treatment

687

was calculated as compared to that in PBS treatment (control group). Asterisk (*)

688

represented a significant difference of the proliferation index between the

689

experimental groups and the control group if P < 0.05 (*) or P < 0.01 (**).

690 691

Figure 6. The effect of the rDioscorin released from TMC-rDio-TPP NPs on the

692

phagocytosis of macrophage RAW264.7. The phagocytosis index of each treatment

693

was calculated as compared to that in PBS treatment (control group). Asterisk (*)

694

represented a significant difference of the phagocytosis index between the

695

experimental groups and the control group if P < 0.05 (*) or P < 0.01 (**).

696



697

Figure 7. RT-qPCR amplification assay of the effect of the rDioscorin released from

698

TMC-rDio-TPP NPs on the stimulation of cytokine gene expression. The relative fold

699

increase of each gene was calculated as compared to that in PBS treatment (control

700

group). Asterisk (*) represented a significant difference of the relative expression

701

level of cytokine genes between the experimental groups and the control group if P

Development of Yam Dioscorin-Loaded Nanoparticles for Paracellular

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