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Improving effectiveness of (-)-epigallocatechin gallate (EGCG) against rabbit atherosclerosis by EGCG-loaded nanoparticles prepared from chitosan and polyaspartic acid Zhiyong Hong, Yong-Quan Xu, Jun-Feng Yin, Jianchang Jin, and Qizhen Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504603n • Publication Date (Web): 08 Dec 2014 Downloaded from http://pubs.acs.org on December 16, 2014

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Journal of Agricultural and Food Chemistry

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Improving effectiveness of (-)-epigallocatechin gallate (EGCG) against rabbit atherosclerosis by

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EGCG-loaded nanoparticles prepared from chitosan and polyaspartic acid

3 Zhiyong Hong†,§, Yongquan Xu‡,§, Jun-Feng Yin‡, Jianchang Jin†, Yongwen Jiang‡, Qizhen Du*,†

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†

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China.

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‡

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Resources Utilization, Ministry of Agriculture, 9 South Meiling Road, Hangzhou 310008, China.

Institute of Food Chemistry, Zhejiang A&F University, 88 Huanbei Road, Hangzhou, Zhejiang 311300,

Tea Research Institute Chinese Academy of Agricultural Sciences, Key Laboratory of Tea Biology and

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Corresponding Authors

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*Tel: +86 571 88218710. Fax: +86 571 63741276. E-mail: [email protected]

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§

These authors contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT

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(-)-Epigallocatechin gallate (EGCG) is the major bioactive compound in green tea. Its effect is limited

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by the harsh environment of the gastrointestinal tract. The present study investigates how the

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effectiveness of EGCG is influenced by its encapsulation into self-assembled nanoparticles of chitosan

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(CS) and aspartic acid (PAA). Blank nanoparticles with mean diameter of ca. 93 nm were prepared

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from 30–50 KDa PAA and 3–5 KDa CS with a mass rate of 1:1. EGCG was loaded in the nanoparticles

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to yield EGCG-CS-PAA nanoparticles with an average diameter of 102 nm, which were pH-responsive

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and demonstrated different EGCG release profiles in simulated gastrointestinal tract media. The

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average ratio (%) of lipid deposition for EGCG-CS-PAA nanoparticles administered orally to rabbits

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was 16.9 ± 5.8%, which was close to that of oral simvastatin (15.6 ± 4.1%). Orally-administered EGCG

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alone yielded an average ratio of lipid deposit area of 42.1 ± 4.0%, while this value was 65.3 ± 10.8%

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for the blank nanoparticles. The effectiveness of EGCG against rabbit atherosclerosis was significantly

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improved by incorporating EGCG into the nano-formulation.

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KEYWORDS: (-)-epigallocatechin gallate; nanoparticles; effectiveness; atherosclerosis

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INTRODUCTION

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Atherosclerosis is a progressive disease characterized by lipid plaque formation in arteries, resulting in

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insufficient blood supply to heart muscle, brain, or peripheral tissues, and it causes about half of the

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cardiovascular disease deaths1,2. Epigallocatechin gallate (EGCG) is a polyphenolic compound

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abundant in green tea, and its vasculoprotective effects have been demonstrated by animal and cell

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models.3 For example, EGCG improved endothelial function and lowerrd blood pressure in

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hypertensive rats.4 In humans, EGCG reversed endothelial dysfunction and improved brachial artery

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flow-mediated dilation in patients with coronary artery disease.5 The beneficial effects of EGCG on

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vascellum presumably result from its antioxidative and hypolipidemic activity. Emerging evidence

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from molecular biology shows that EGCG may exert vascular effects through other mechanisms.6

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However, most of the reported studies used EGCG at doses that are far beyond levels that are

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physiologically achievable (0.6 – 1.8 µM) in either humans or animals through dietary consumption.4-7

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Moreover, the evidence is inconclusive regarding the effectiveness for cardiovascular disease

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prevention or treatment for human being.8,9 The major problems are its low stability and bioavailability

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in humans.10−12 The oral bioavailability of EGCG after drinking tea containing catechins at 10 mg/kg

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body weight is about 0.1% in humans.11,13 EGCG is unstable in water and physiological fluid in vitro

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since it readily undergoes oxidation, degradation and polymerization.13-15. For example, studies have

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shown that EGCG is unstable in sodium phosphate buffer (pH 7.4), where 80% of it is lost in only 3

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h.16-18

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Encapsulation has been extensively investigated as a strategy to enhance the stability and

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effectiveness of EGCG. For example, chitosan-tripolyphosphate nanoparticles could enhance the

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plasma concentration of EGCG in mice by improving its stability in the jejunum,19,20

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chitosan-tripolyphosphate nanoparticles of EGCG showed a several-fold dose advantage over native

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EGCG to inhibit the cancer cell growth,21 and chitosan-coated nanoencapsulation enhanced EGCG

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stability and its antiatherogenic bioactivities in macrophages.22 In the present study we report the

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preparation of EGCG nanoparticles using chitosan and polyaspartic acid, and the effectiveness of the

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nanoparticles against rabbit atherosclerosis.

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MATERIALS AND METHODS

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Materials. EGCG (98%) was purchased from Taiyo Green Power Co., Ltd. (Wuxi, China).

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Polyaspartic acid (PAA) with molecular weight of 5–150 KDa was purchased from Zhangjiagang

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Xingyu Technology Co., Ltd (Zhangjiagang, China). The PAA product can be considered as food

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safety since its LD50 was greater than 5 g/kg body weight of mice in our experiment of acute toxicity.

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Chitosan (CS) with molecular weight of 3–5 KDa and 500–1500 KDa was provided by Zhejiang

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Aoxing Biological Technology Co., Ltd. (Yuhuan, China). CS with molecular weight of 180–220 KDa

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was purchased from Sigma-Aldrich (Shanghai, Shanghai). Bovine bile salt was purchased from Beijing

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Solarbio Science & Technology Co., Ltd. (Beijing China). Pepsin from porcine gastric mucosa

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(enzymatic activity of 800 to 2500 U/mg of protein) and pancreatin from porcine pancreas (4 × USP

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specifications) were obtained from Sigma-Aldrich (Shanghai Branch, Shanghai).

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Isolation of polyaspartic acid according to molecular weight. Polyaspartic acid (PAA) (50 g) with

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molecular weight of 5–150 KDa was dissolved in 5 L water. From this aqueous solution, PAA samples

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of 10–30 KDa, 30–50 KDa and 50–100 KDa were isolated. 400 ml of the aqueous solution was filtered

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using a 400 ml Millipore/Stirred Cell 8400 (Millipore, MA, USA) equipped with a 10 KDa

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ultrafiltration membrane to remove the molecules less than 10 KDa, yielding PAA of 10–150 KDa (3.4

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g, dry weight). The PAA (10– 150 KDa) was dissolved with 400 ml of water, and filtered through a 30

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KDa ultrafiltration membrane to afford PAA of 30–150 KDa (2.7 g, dry weight), and a solution of

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10-30 KDa PAA which was evaporated and dried under reduced pressure at 60 °C to give 0.6 g of

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10–30 KDa PAA. The PAA of 30–150 KDa was subjected to the above procedure, using 50 and 100

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KDa ultrafiltration membranes orderly, to produce 1.2 g of 30–50 KDa PAA and 0.8 g of 50–100 KDa

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PAA.

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Preparation of CS-PAA nanoparticles. CS-PAA nanoparticles were prepared from chitosan (CS) of

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molecular weight 3–5, 14–22, or 50–150 KDa and polyaspartic acid (PAA) with molecular weight of

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10–30, 30–50 or 50–100 KDa. The molecular weight of each component was varied to find the

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composition that would obtain the ideal particle size. The weight ratio of CS to PAA was 1:1. CS-PAA

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nanoparticles were prepared by mixing a positively charged CS/acetic acid solution with a negatively

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charged PAA solution at room temperature. Briefly, PAA (20 mg) was thoroughly dissolved in 10 ml

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of deionized water, and CS (20 mg) was fully dissolved in 10 ml of 1% acetic acid solution by

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magnetic stirring. The CS solution (10 ml) was added dropwise to the PAA solution (10 ml) at a flow

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rate of 1 ml/min using an injection pump, and mixed by magnetic stirring at a speed of 500 rpm to form

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a dispersion of CS-PAA nanoparticles. After the optimal molecular weight of CS and PAA was

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determined, the effect of the CS/PAA ratio on the particle size and zeta potential of the nanoparticles

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was investigated.

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Trial preparation of EGCG-CS-PAA nanoparticles. EGCG CS–PAA nanoparticles were prepared

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by mixing a positively charged CS acetic acid solution containing EGCG (2 mg/ml) and a negatively

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charged PAA solution at room temperature. The preparation process was similar to that of CS-PAA

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nanoparticles. To harvest the nanoparticles, the nanoparticle solution was centrifuged under 15000 × g

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for 20 min, following which the precipitate was immediately dispersed in 0.5% aqueous acetic acid (10

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ml) and freeze-dried. The freeze-dried powder was tested to determine the EGCG loading amount,

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release and stability in simulated body fluids.

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Characterization of the EGCG-CS-PAA nanoparticles. The average diameter and size distribution

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of particles were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS analyzer

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(Malvern Instruments, Worcestershire, U.K). All DLS measurements were done at 25 ± 0.1 °C and at a

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scattering angle of 90°. The particle size distribution was produced from the volume measurement, in

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which the relative percentage of particles in each size class is based on the volume they occupy. The

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zeta potential was measured with laser Doppler velocimetry (LDV) using the same Zetasizer Nano-ZS

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analyzer. The morphology of the EGCG-CS-PAA nanoparticles was performed on a transmission

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electron microscope (TEM, Hitachi H-600) after mounting the nanoparticles onto a carbon-coated

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copper grid.

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Determination of entrapment efficiency and loading amount of EGCG in the nanoparticles. The

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solution of EGCG-CS-PAA nanoparticles (2 mL) was transferred into an Amicon Ultra-3K centrifugal

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filter device (Millipore Corp. Billerica, MA, USA) made up of a centrifuge tube and a filter unit with a

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low-binding Ultracel membrane (3000 MWCO). After centrifugation at 4000 × g for 30 min,

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EGCG-CS-PAA nanoparticles remained in the filter unit and free EGCG and salts penetrated through

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the Ultracel membrane into the centrifuge tube. The free EGCG was determined by high performance

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liquid chromatography (HPLC). The EGCG-CS-PAA nanoparticles in the filter unit were freeze-dried,

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and part of the lyophilized powder was extracted with methanol for EGCG determination by HPLC.

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The HPLC analysis were performed on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) composed

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of two LC-10A pumps, an SIL-10Avp autosampler, a SPD-M10Avp UV detector and a

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Symmetry®C18 (5 µm, 4.6 mm × 250 mm) column. The elution employed a linear gradient system

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with mobile phase A (water:acetic acid 98:2) and mobile phase B (acetonitrile) at a flow rate of 1

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mL/min at 40 °C. The chromatogram was recorded at 280 nm. The entrapment efficiency (%) of EGCG

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in the nanoparticles and the loading amount (mg/g) was calculated from the results of HPLC analysis.

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Stability of EGCG-CS-PAA nanoparticles at different pH. EGCG CS–PAA nanoparticles were

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prepared by mixing a solution of positively charged CS in acetic acid, containing EGCG, with a

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solution of negatively charged PAA at room temperature. The nanoparticle dispersion was centrifuged

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at 15000 × g to harvest the nanoparticles. The collected nanoparticles were dispersed in 0.5% aqueous

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acetic acid and freeze-dried to form a lyophilized powder. This powder was tested to determine the PDI

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and particle size of the EGCG-CS-PAA nanoparticles in different pH (2.5, 3.5, 4.5, 6.6, 7.0 and 7.4), to

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evaluate the stability of the nanoparticles.

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Release of EGCG from EGCG-CS-PAA nanoparticles. The release profile of EGCG from test

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nanoparticles was investigated in different media (pH 2.5, 4.0, 6.6, 7.0 and 7.4), in the presence of the

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reducing agents ascorbic acid (20 mM) and tris[2-carboxyethyl] phosphine hydrochloride (13 mM) to

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prevent EGCG from oxidation in alkaline environments.23 Briefly, 5 mg of the EGCG-CS-PAA

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nanoparticles was dispersed in 20 ml pure water by ultrasonic process, and then incubated in a water

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bath at 37 °C under magnetic agitation (100 rpm). At given time intervals, samples (1.0 ml) were taken

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out and centrifuged, and the supernatants were analyzed by HPLC. The amount of EGCG released was

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expressed as a decrease in the percentage of EGCG in the nanoparticles, which was obtained from the

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EGCG concentration in the supernatants after centrifugation of the nanoparticle solutions.

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Stability evaluation of EGCG in nanoparticles under simulated digestion and adsorption

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conditions in vitro. The simulated digestion condition was PBS buffer with pH 2.1 containing pepsin

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(simulated gastric fluid, SGF), and the simulated adsorption condition was PBS buffer with pH 7.2

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containing pancreatin-bile salts (simulated intestinal fluid, SIF).24 The amount of EGCG remaining in

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EGCG-CS-PAA nanoparticles in SGF and SIF after 0–4 h was determined according to the study of

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Krook and Hagerman12. Briefly, EGCG-CS-PAA nanoparticles were added to SGF or SIF, to a final

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EGCG concentration of 0.25 mg/mL (0 h), and then incubated in a water bath at 37 °C under magnetic

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agitation (100 rpm). After 1, 2, 3 and 4 h of incubation, 1 mL of the incubated solution was removed

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and the EGCG concentration in this aliquot was determined by HPLC as described above. The amount

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of EGCG remaining after the incubation reflects the stability of EGCG in nanoparticles during

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digestion and adsorption in vitro.

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Preparation of EGCG-CS-PAA nanoparticles for animal experimentation. PAA (2 g) was

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thoroughly dissolved in 1 L of deionized water. CS (2 g) was fully dissolved in 1 L of 1% acetic acid

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solution by stirring; 2 g of EGCG was then added to this solution. The CS-EGCG solution was added

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to the PAA solution (1 L) at a flow rate of 10 ml/min using a BT100-1F peristaitc pump

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(Konap Peristaltic Pump Co., Ltd., Chongqing, China) and mixed by an ultrasonic processor

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(FS-1800N, Shanghai Sonxi Ultrasonic Instrument Company, Shanghai, China) to form a solution of

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EGCG-CS-PAA nanoparticles. The sizes of the nanoparticles in the solution and in the trial preparation

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were compared by DLS. This solution was centrifuged under 13000 × g for 20 min to harvest the

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nanoparticles; the pellet was dispersed in 0.5% aqueous acetic acid (500 ml), ultrasonicated, and

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freeze-dried. The freeze-dried powder was used in the animal experiments.

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Effectiveness of EGCG-CS-PAA nanoparticles on rabbit atherosclerosis. Thirty male New Zealand

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white rabbits (Animal Center of Zhejiang Academy of Medical Sciences) weighing 1.92–2.11 kg were

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randomly divided into six experimental groups. They were housed individually in metal cages in an

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air-conditioned room (22 ± 2 °C, 55 ± 5% humidity), and maintained on a 12 h light/12 h dark cycle

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with free access to food and water. Water was allowed ad libitum, and 150 g/day of food was provided.

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One group of rabbits was used as a control. Experimental rabbits (5 groups) were fed for 5 weeks on a

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high-fat diet (HFD) containing 93.75% standard rabbit chow (Jiangsu Synergistic Bioengineering Co.

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Ltd, Nanjing, China) supplemented with 1.0% cholesterol, 5% lard oil and 0.25% sodium cholate to

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provoke atherosclerosis. Four of the groups were orally treated with EGCG-CS-PAA nanoparticles

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(EGCG-NP), EGCG, blank CS-PAA nanoparticles (Blank-NP) or simvastatin (positive control). In the

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EGCG-NP-fed group, each rabbit was daily given 350 mg EGCG-NP (7 ml of 0.5% aqueous acetic

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acid) containing 100 mg of EGCG, while each rabbit in the EGCG group was given 100 mg EGCG (7

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ml of 0.5% aqueous acetic acid). Each rabbit in the Blank-NP-fed group was given 250 mg of

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Blank-NP (7 ml of 0.5% aqueous acetic acid) per day, and each rabbit in the positive control was given

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5 mg simvastatin (5 mg/tablet, Shanghai Xinyi Wanxiang Pharmaceutical Co. Ltd., Shanghai, China)

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per day. For EGCG-NP and Blank-NP, the sample was dispersed in water solution via ultrasonication,

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and then the solution was provided to the rabbit by oral administration.

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The daily EGCG dose was selected according to the suitable pharmacological dose for humans. All

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animal experiments were performed according to the protocols approved by the Animal Care Center of

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the Zhejiang Academy of Medical Sciences, and complied with the guidelines for the care and use of

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laboratory animals. Five weeks post-commencement of the HFD, the rabbits were sacrificed by

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exsanguination via the marginal ear vein after deep anesthesia with pentobarbital (30 mg/kg i.v.).

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Serum was stored at −80 °C until serum lipid analysis and measurements of serum values were

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conducted. The thoracic arteries with arches were carefully excised to avoid damaging the endothelial

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lining, and the adhering soft tissue was trimmed off. Thoracic arteries with arches were rapidly

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dissected, opened longitudinally and stained with Oil Red. Photographs of the inner surface were taken

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and copied onto graph paper with magnification (× 2) and athermanous plaques were delineated. The

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number of small squares surrounded by the line was counted on the graph paper and the percentage of

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the area of athermanous plaque was calculated. Serum and lipoprotein levels of cholesterol and

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triglyceride were measured by enzymatic colorimetry using commercial kits (Boehringer, Mannheim,

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Germany). Quantitative data was expressed as mean ± SD and analyzed by one-way ANOVA. Multiple

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comparisons between the groups were performed using the Student–Newman–Keuls (SNK) method.

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Statistical significance was set at a level of P < 0.05.

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RESULTS AND DISCUSSION

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Formation of chitosan-polyaspartic acid nanoparticles. Nanoparticles can form from self-assembly

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of polyelectrolytes such as chitosan (CS) and poly γ-glutamic acid.25 In the present study, we tested the

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preparation of nanoparticles by self-assembly of chitosan and polyaspartic acid (PAA) because PAA is

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less expensive than poly γ-glutamic acid. Table 1 shows the mean diameter, polydispersity index (PDI)

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and zeta potential of the CS-PAA nanoparticles prepared with CS-PAA (1:1, w/w) prepared from

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various molecular weights of CS and PAA. The zeta potential and particle size increased as the

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molecular weights of CS and PAA increased, which suggests that long-chain CS and PAA form large

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particles with high stability, and short chain CS and PAA form small particles with low stability.

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Furthermore, the values of PDI increased when the difference in the molecular weights of CS and PAA

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molecules was increased. Considering that the parameters for best stability and uptake in cellular

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systems are zeta potential > 30 mV or < −30 mV

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nanoparticles in the present study were self-assembled from PAA 30–50 KDa and CS 3–5 KDa, and

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had a diameter of 92.6 nm and a zeta potential of 31.5 mV. These nanoparticles were selected for

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further experiments.

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Further study showed that changing the CS/PAA ratio in the self-assembled nanoparticles could change

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their size and stability (Table 2). As the CS/PAA ratio increased, the values of both nanoparticle size

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and zeta potential enhanced. When the CS/PAA ratio was 0.75, the particle size decreased to 84.7 nm,

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but the particles (zeta potential 22.3 mV) were less stable than those in which the ratio of CS/PAA was

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1.0. On the basis of the above results, we decided to use the nanoparticles which were self-assembled

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from PAA 30–50 KDa and CS 3–5 KDa at a ratio of 1:1 for loading EGCG.

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EGCG-CS-PAA nanoparticles. EGCG was loaded into self-assembled CS-PAA nanoparticles by

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adding EGCG into a CS solution which was mixed with a PAA solution under magnetic stirring at

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room temperature. The mean particle diameter, PDI and entrapment efficiency (EE) of nanoparticles

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prepared with a 1:1 ratio of CS/PAA were determined. The prepared EGCG-CS-PAA nanoparticles

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and particle size near 75 nm,27 the optimal

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exhibited particle size 102.4 ± 5.6 nm, PDI 0.224, and EE 25.0 ± 2.1%. The EE value was greater than

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that of nanoparticles self-assembled from CS and the same mass amount of poly γ-glutamic acid as

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PAA25. Because the PAA molecule is shorter than γ-glutamic acid, PAA contains more carboxyl groups

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for forming hydrogen bonds with EGCG. The zeta potential of the nanoparticle solution was 33.3 ± 0.2,

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which indicates excellent stability.

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TEM images (Fig. 1) showed that the EGCG-CS-PAA nanoparticles were spherical and about 100 nm

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in diameter (Fig. 1). The diameters determined by TEM were consistent with those determined via DLS.

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This demonstrated that DLS was an effective method to determine the size of the prepared

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nanoparticles. After the nanoparticles were harvested by centrifuging and freeze-drying, HPLC showed

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that the concentration of EGCG in the nanoparticles was 34.4%. Dispersing the freeze-dried

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nanoparticles in 0.5% aqueous acetic acid yielded a sample whose particle size was similar to that of

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the original system before freeze-drying (Fig. 2).

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Stability of EGCG-CS-PAA nanoparticles in the solutions at different pH. The stability of the

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EGCG-CS-PAA nanoparticles in the solutions at different pH was evaluated from the PDI and particle

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diameter (Table 3). At pH 3.5, the original pH when the nanoparticles were prepared, the

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EGCG-CS-PAA nanoparticles were stable after 2 h at room temperature. At pH 2.5, no significant

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change in the nanoparticles was observed after 2 h. As the pH was increased from 4.5 to 7.4, the PDI

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and particle diameter rapidly increased, which means that small nanoparticles aggregated or

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re-assembled into larger nanoparticles. This result is similar to that obtained via TEM observation of

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CS/poly γ-glutamic acid nanoparticles.25

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Release of EGCG from EGCG-CS-PAA nanoparticles at different pH. Release of EGCG from

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EGCG-CS-PAA nanoparticles at different pH was investigated because the pH in the digestive system

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varies widely between gastric and intestinal fluid. The typical pH in the stomach is from 2.0 to 4.0

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depending on the species and food intake.28 The pH in the duodenum is 6.0–6.6 and in the jejunum and

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proximal ileum the pH is 6.6–7.0, while the pH is about 7.4 in the distal ileum and in the body fluid

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filling intercellular spaces between enterocytes.29 The present study determined the release of EGCG

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from EGCG-CS-PAA nanoparticles in dispersions at pH 2.5 (0–1 h), 4.0 (1–2 h), 6.6 (2–3 h), 7.0 (3–4 h)

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and 7.4 (4–5 h), conditions similar to those encountered in food ingestion. Fig. 3 shows the release of

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EGCG from the nanoparticles at the various pH conditions. At pH 2.5 and 4.0 (simulating the stomach)

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the release of EGCG in 0–2 h was about 25% of the total EGCG amount in the nanoparticles. At pH 6.6

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(simulating the duodenum), the release rate increased and the amount released reached 41% of the total.

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At pH 7.0 (simulating the jejunum and the proximal ileum) and pH 7.4 (simulating the distal ileum and

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the intercellular body fluid), the release rate showed a rapid increase, which implies that the

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nanoparticles became unstable and quickly disintegrated to release EGCG. The stability of the

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nanoparticles in the stomach, combined with the rapid release in the intestine, may improve the uptake

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of EGCG in intestine.

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The stability of EGCG-CS-PAA nanoparticles in simulated gastric and intestinal conditions.

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Loading EGCG into nanoparticles improves its stability in gastric and intestinal conditions. In SGF (pH

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2.1), the loss of EGCG in EGCG-CS-PAA nanoparticles was about 10–15% less than that of the free

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EGCG regardless of the presence of pepsin (Fig. 4). In SIF (pH 7.2) EGCG was more unstable than in

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SGF. Encapsulation with CS and PAA clearly protected EGCG. Upon adding the nanoparticles to SIF

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in the presence of pancreatin-bile salts, the amount of EGCG lost was 41%, 71%, 92% and 93% after

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1h, 2h, 3h and 4h, respectively. For nanoparticles in SIF without pancreatin-bile salts, a smaller amount

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of EGCG was lost at the same time intervals: 39%, 81%, 83% and 90%, respectively. Increasing the

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stability of compounds in the human stomach and intestine can improve their uptake. Therefore,

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loading EGCG in CS-PAA nanoparticles may improve the effectiveness of EGCG against diseases such

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as atherosclerosis.

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EGCG-CS-PAA nanoparticles (EGCG-NP) for animal experiments. EGCG-NP for animal

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experiments were prepared by a modified process in which ultrasonication replaced magnetic agitation

281

because the amount was scaled up from mg to grams. The product exhibited the same properties, i.e.

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particle size 100.6 ± 4.2 nm，PDI 0.215 ± 0.008，zeta potential 33.9 ± 0.5 mV and EGCG loading

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amount 348.1 ± 12.4 mg/g, dry weight.

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Effectiveness of EGCG-NP against atherosclerosis. The inner surface of the rabbit arteries all

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showed in Figure S1. The control rabbit artery was covered with opaque atherosclerotic lesions in its

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inner surface (Fig. 5a). The HFD-fed rabbits showed severe lipid deposition in their inner surface artery

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on which the average ratio (%) of lipid deposit area to intimal surface area (without artery arch) was

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69.0 ± 9.6% (Fig. 5b). The Blank-NP-fed rabbits exhibited an average ratio (%) of lipid deposit area of

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65.3 ± 10.8% (Fig. 5c), which suggests no significant difference in lipid deposition between the

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HFD-fed rabbits and the Blank-NP-fed rabbits. Compared to the HFD-fed rabbits, the EGCG-NP-fed

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rabbits and EGCG-fed rabbits exhibited significantly less lipid deposition. The average ratio (%) of

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lipid deposit area for EGCG-NP-fed rabbits and EGCG-fed rabbits was 16.9 ± 5.8% and 42.1 ± 4.0%,

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respectively, which showed that the EGCG-NP were much more effective against atherosclerosis

294

compared with EGCG alone (p < 0.01). The effect of EGCG-NP against rabbit atherosclerosis was

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close to that of simvastatin (Fig. 5d, % average ratio of lipid deposit area, 15.6 ± 4.1%), which is a

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clinical treatment for atherosclerosis. It should be mentioned that no difference in the body weight of

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the EGCG-NP-fed group and the normal-fed group (Table S1), which suggests that the

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nano-formulation EGCG did not trigger toxic reactions in the rabbits.

299

The effect of EGCG-NP and EGCG on blood lipid levels. The serum levels of total cholesterol (TC)

300

and LDL cholesterol (LDL-C) of HFD-fed rabbits were 10–20 times those of normal rabbits, while the

301

levels of triglyceride (TG) and HDL cholesterol (HDL-C) were increased but to a lesser degree (Table

302

4). After administration of EGCG-NP and EGCG, the levels of TG, TC, HDL-C and LDL-C decreased

303

by 52%, 55%, 27% and 65%, and 19%, 26%, 23% and 33%, respectively. It is clear that EGCG-NP

304

decreased the blood lipid levels of rabbits to a much greater extent than EGCG. Meanwhile, no

305

significant changes in the levels of TG, TC, HDL-C and LDL-C were found between the HFD-fed and

306

the Blank-NP-fed groups. The results demonstrate that the reduction of the blood lipid levels in the

307

EGCG-NP and EGCG groups fully corresponds to the reduction in lipid deposition.

308

The effectiveness of EGCG in humans is relatively low because EGCG is unstable at pH

309

conditions of body fluids.18 It is known that EGCG undergoes significant metabolism and conjugation

310

during absorption in the small intestine and the colon. In the small intestine EGCG is degraded to

311

smaller phenolic acids and valerolactones.30 To improve the effectiveness of EGCG, various methods

312

have been investigated by loading EGCG in vehicles such as liposomes, ethosomes, or other

313

nanoparticles, which can protect EGCG by delayed release, act to target the release of EGCG, or protect

314

it from unstable circumstances such as body fluids with various pH.31 The present study provides a

315

nano-formulation of EGCG to improve its effectiveness.

15



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316

In conclusion, EGCG-loaded nanoparticles with mean diameter ca. 100 nm were prepared from

317

30–50 KDa PAA and 3–5 KDa CS. The nanoparticles were pH-responsive and demonstrated different

318

EGCG release profiles in simulated gastrointestinal tract media. Compared to EGCG alone, this

319

nano-formulation of EGCG demonstrated higher stability in body fluids and showed greater activity

320

against atherosclerosis.

321 322

AUTHOR INFORMATION

323

Corresponding Author

324

*Telephone: +86-571-88218710 (Q.D.); E-mail: [email protected] (Q.D.)

325 326

Funding

327

This research was supported by a fund of the National Natural Science Foundation of China (Grant No.

328

31270724), a fund of the National Key Technology R&D Program (2012BAD36B06), and a fund of

329

Natural Science Foundation of Zhejiang Province (Grant No. LZ12C16004).

330 331

REFERENCES

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Fig. 1. TEM micrographs of CS-PAA-EGCG nanoparticles. (A) 50,000× magnification; (B) 150,000× magnification; (C) particle size determination by DLS. 137x118mm (300 x 300 DPI)



Fig. 2. DLS measurement of the particle size in the dispersion prepared by reconstituting the freeze-dried nanoparticles. 46x18mm (300 x 300 DPI)


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Fig. 3. The EGCG release profiles of EGCG-CS-PAA nanoparticles at pH 2.5, 4.0, 6.6, 7.0 and 7.4. 119x98mm (300 x 300 DPI)



Fig. 4. The amount of EGCG remaining in SGF and SIF after 0, 1, 2, 4, and 4 h. NP+Pep, EGCG-CS-PAA nanoparticles in the presence of pepsin; NP, EGCG-CS-PAA nanoparticles alone; EGCG+Pep, EGCG in the presence of pepsin; EGCG alone; NP+Pan, in the presence of pancreatin-bile salts; EGCG+Pan, EGCG in the presence of pancreatin-bile salts. 51x16mm (300 x 300 DPI)


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Fig. 5. Extent of atherosclerosis lesion development in rabbits after feeding with the experimental diets. (a) normal group; (b) HFD-fed group; (c) Blank-NP-fed group; (d) simvastatin-fed group (2.5 mg/kg); (e) EGCG-NP-fed group, 175 mg/kg (corresponding to 50 mg EGCG/kg); (f) EGCG-fed group (50 mg/kg). 93x73mm (300 x 300 DPI)



Table of Contents Graphic 79x45mm (300 x 300 DPI)


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