Chitosan-caseinate-dextran ternary complex nanoparticles for potential oral delivery of astaxanthin with significantly improved bioactivity

Journal Pre-proof Chitosan-caseinate-dextran ternary complex nanoparticles for potential oral delivery of astaxanthin with significantly improved bioactivity

Qiaobin Hu, Siqi Hu, Erika Fleming, Ji-Young Lee, Yangchao Luo PII:

S0141-8130(20)30666-8

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.170

Reference:

BIOMAC 14787

To appear in:

International Journal of Biological Macromolecules

Received date:

20 January 2020

Revised date:

15 February 2020

Accepted date:

15 February 2020

Please cite this article as: Q. Hu, S. Hu, E. Fleming, et al., Chitosan-caseinate-dextran ternary complex nanoparticles for potential oral delivery of astaxanthin with significantly improved bioactivity, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.02.170

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© 2020 Published by Elsevier.

Journal Pre-proof

Chitosan-Caseinate-Dextran Ternary Complex Nanoparticles for Potential Oral Delivery of Astaxanthin with Significantly Improved Bioactivity Qiaobin Hu a, b, Siqi Hu b, Erika Fleming b, Ji-Young Lee b, Yangchao Luo b,* a

College of Food Science and Engineering, Nanjing University of Finance and Economics,

Nanjing, Jiangsu Province 210003, China Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA

Mailing Address:

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*Corresponding author.

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Dr. Yangchao Luo Assistant Professor, Department of Nutritional Sciences, University of Connecticut 27 Manter Road, U-4017, Storrs, CT 06269-4017, USA. Phone: (860) 486-2186. Fax: (860) 486-3674. Email: [email protected]

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Journal Pre-proof Abstract Astaxanthin (ASTX) has been reported as a potential therapeutic agent for hepatic fibrosis treatment. However, its therapeutic effect is limited due to low bioavailability and poor aqueous solubility. In this study, biopolymer-based nanoparticles were fabricated using stearic acidchitosan conjugate (SA-CS) and sodium caseinate (NaCas) via ionic gelation. Its nanostructure was cross-linked using oxidized dextran (Odex) via Schiff base reaction. Concentration of cross-

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linker, cross-linking temperature and time were systematically optimized by response surface

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methodology (RSM) to achieve superior particulate properties and colloidal stability. The

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optimized nanoparticles exhibited a diameter of 120 nm with homogeneous size distribution. A good ASTX encapsulation capacity with up to 6% loading ratio and high encapsulation

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efficiency was obtained. The final ASTX concentration in nanoparticles was 140 μM. The

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aqueous dispersibility of encapsulated ASTX was greatly improved, which was confirmed by

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significantly increased ABTS radical scavenging capacity. Compared to anti-fibrogenic effects of free ASTX in LX-2 cells, the encapsulated ASTX demonstrated dramatically enhanced cellular

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bioactivity, as evidenced by significantly lower TGFβ1-induced fibrogenic gene (ACTA2 and

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COL1A1) expression level, as well as α-SMA and COL1A1 protein levels. This study suggests that the as-prepared biopolymer nanoparticles hold promising features as an oral delivery vehicle for lipophilic bioactives.

Keywords: surface response methodology; complex nanoparticles; encapsulation; astaxanthin; anti-fibrogenic.

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Journal Pre-proof 1.

Introduction The biopolymer-based nanoparticles have attracted a great interest for use in the delivery of

bioactive compounds since the 1990s [1, 2]. Food proteins, including animal and plant proteins, have been widely used in preparation of biopolymer-based nanoparticles [3-5]. In particular, sodium caseinate (NaCas) has drawn great attention due to its amphiphilicity and high affinity for both hydrophilic and hydrophobic cargos. Besides proteins, various polysaccharides, such as

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chitosan (CS), alginate, and pectin, as well as protein-polysaccharide complex have also been

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applied in the fabrication of nanoparticles for drug/nutrients delivery [6-9]. Since CS is the only

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polysaccharide with a positive surface charge, it has been widely used to prepare colloidal nanoparticles via electrostatic interactions with negatively charged polyelectrolytes. In addition,

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studies have reported that CS derivatives with improved water solubility could be also used as a

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better coating material to form protein-polysaccharide hybrid nanoparticles [10-11]. In our

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previous study, biopolymer-based nanoparticles were successfully prepared using stearic acidchitosan conjugate (SA-CS) and NaCas for encapsulation and delivery of lipophilic bioactive

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compounds [12]. To maintain the integrity of the nanostructure in GI conditions, chemical cross-

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linking and biopolymer coating have been widely applied [13-16]. Oxidized dextran (Odex), a non-toxic cross-linker prepared by periodate oxidation [17, 18], can react with the amino group of amino acids via Schiff base formation, further stabilizing the formed nanostructure [19-21]. Thus, we aimed to develop a novel biopolymer-based nanoparticle oral delivery system via a simple and toxic crosslinker-free fabrication method. ASTX is a keto-carotenoid that presents in great quantity in marine animals, such as salmon and shrimp [2]. Studies have shown that ASTX has an anti-fibrotic effect on the liver by preventing the transforming growth factor β1 (TGFβ1) induced fibrogenic responses in activated

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Journal Pre-proof hepatic stellate cells (aHSC) [22]. However, the application of ASTX is currently limited due to its low bioavailability, poor water solubility, and instability when exposed to high temperature, oxygen, light and pH extremes [23, 24]. In recent years, various nanoparticle systems have been studied to improve the bioavailability of ASTX [21, 25, 26]. Wang et al. prepared an ASTXloaded DNA/chitosan colloidal system with a maximal ASTX concentration of 65 μg/mL [25]. Shen et al. fabricated an ASTX nanodispersion with a small particle size of 120 nm using whey

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protein isolates via an emulsification-evaporation technique [26]. The bioavailability was

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evidenced by 10-fold higher apparent permeability coefficient of Caco-2 cells than that of free

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ASTX. In another study, ASTX was encapsulated by solid lipid polymer hybrid nanoparticles prepared from glyceride, bovine serum albumin and oxidized dextran [21]. The ASTX showed

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dramatically enhanced antioxidant activity and sustained release profile in simulated GI fluids

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after encapsulation. In order to further enhance the therapeutic effect of lipophilic bioactive

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compounds, it is critical to develop a delivery system that is able to improve its aqueous dispersibility, and therefore, increase ASTX’s anti-fibrotic activity.

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Therefore, in the present study, the biopolymer-based nanoparticles were prepared and

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optimized using response surface methodology (RSM). The structure was confirmed by Fouriertransform infrared spectroscopy (FT-IR) and transmission electron microscope (TEM). The in vitro stability in simulated GI fluids was systematically investigated. In vitro cell studies were also conducted to evaluate the cytotoxicity and anti-fibrogenic effect using LX-2 cells which are a typical cell model to study the antifibrotic mechanism of bioactive compounds on hepatic fibrogenesis. 2.

Materials and methods

2.1.

Materials

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Journal Pre-proof CS (low molecular weight with deacetylation degree of 82.6% and viscosity of 62 cps at 1% w/w in 1% acetic acid), NaCas and ASTX were purchased from Sigma-Aldrich Corp (St. Louis, MO, USA). Stearic acid (SA) and dextran (molecular weight 40,000 Da) were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), 100× non-essential amino acids, 100× penicillin and streptomycin, 100× vitamins, and Dulbecco's phosphate buffer saline (PBS) were all purchased from HyClone

Preparation and characterization of SA-CS and Odex

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2.2.

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purchased from Acros Organics (Pittsburgh, PA, USA).

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(Thermo Fisher Scientific, Logan, UT, USA). Other chemicals were of analytical grade and

SA-CS with 8.4% degree of substitution and Odex were synthesized according to our

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previous study, and their structures were confirmed by FT-IR and 1H NMR [12].

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2.3. Preparation and optimization of nanoparticles

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The SA-CS/NaCas/Odex nanoparticles were prepared according to a modified method of Hu et al. [27] based on ionic gelation between SA-CS and NaCas, followed by cross-linking

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process using Odex. The stock solutions of SA-CS and NaCas were separately prepared by

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dissolving in DI water at 1.00 and 0.33 mg/mL, respectively. To prepare nanoparticles, 5 mL of NaCas solution was added dropwise to 10 mL of SA-CS aqueous solution to reach the SA-CS to NaCas mass ratios of 3:1. The mixture was stirred gently for 30 min at room temperature, followed by the addition of Odex with various concentrations and heating in a water bath. To optimize the formulation, Box-Behnken quadratic design was conducted with 15 randomized experiments and 3 center point replicates using Design-Expert Software Version 8. The bestfitted models for statistical analysis were considered significant, with probability limits of the p value < 0.05. The model is described by the following quadratic equation:

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Journal Pre-proof Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b11X12 + b22X22 + b33X32 where Y is the measured response associated with factor level combinations; b0 is the intercept and b1 to b33 are the regression coefficients computed from observed value of Y; X1, X2, and X3 are the coded levels of independent variables. XiXj (i= 1, 2 or 3 and j= 1, 2 or 3) and Xi2 (i= 1, 2 or 3) are the interaction and quadratic terms, respectively. The independent factors (X) selected were mass ratio of SA-CS to Odex (X1), heating

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temperature (X2), and heating time (X3), with three levels being low (-1), medium (0), and high

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(+1) for each independent factor (Table S1). The dependent responses (Y) were particle size

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(Y1), polydispersity index PDI (Y2), Δ particle size in SGF (Y3) and Δ particle size in SIF (Y4). The experiment design matrix is shown in Table S2. The optimal formulation was based on the

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criteria of particle size at 100 to 130 nm, the minimum PDI, minimum Δ particle size in SGF,

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and minimum Δ particle size in SIF (i.e. the smallest variation in particle size before and after

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incubation). At the end, a new batch of nanoparticles with the predicted levels of formulation

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factors was prepared to confirm the validity of the optimization procedure.

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2.4. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectra of SA-CS, NaCas, Odex, and spray dried nanoparticles were recorded on a Fisher Scientific Nicolet Is5 Spectrometer equipped with an iD7 ATR accessory (Thermo Fischer Scientific Inc., Waltham, MA, USA) by scanning from 4000-500 cm-1 at a resolution of 4 cm-1 and analyzed using OMNIC software version 8.0. 2.5. Determination of encapsulation efficiency of ASTX ASTX was used as a model bioactive to investigate the encapsulation capacity of nanoparticles. Prior to preparation of nanoparticles, ASTX stock solution was prepared by

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Journal Pre-proof dissolving in dimethyl sulfoxide (DMSO) at 4 mg/mL. Then, ASTX with different concentrations was pre-mixed with NaCas under mild stirring at room temperature for 30 min. Then the ASTX-loaded nanoparticles were prepared in the same process as described in section 2.2. Afterwards, the ASTX-loaded nanoparticles were centrifuged at 5,000 g for 10 min, and free ASTX in precipitates was collected and redissolved in acetone. The concentration of free ASTX was measured using high performance liquid chromatography (HPLC).

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The HPLC consisted of a modular system SPD-20AV (SHIMADZU, Japan) with diode

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array detector. A 150 × 4.6 mm i.d. 3 μm C8 analytical column (YMC, Japan) was used at 25 °C.

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A mobile phase of 100% methanol was used for astaxanthin analysis. The flow rate was 1 mL/min, and the injection volume was 20 μL. The peak area of astaxanthin was measured at a

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wavelength of 474 nm. The quantification used an external standard method, linear regression

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equation for astaxanthin at 474 nm is: A = 3E+08C - 43154, R2=0.9993, where C is the ASTX

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concentration (mg/mL), A is the peak area of ASTX. The encapsulation efficiency (EE) and loading capacity (LC) were calculated according to the equations, respectively. 𝑇𝑜𝑡𝑎𝑙 𝐴𝑆𝑇𝑋 − 𝐹𝑟𝑒𝑒 𝐴𝑆𝑇𝑋 × 100% 𝑇𝑜𝑡𝑎𝑙 𝐴𝑆𝑇𝑋

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EE (%) =

LC (%) =

𝑇𝑜𝑡𝑎𝑙 𝐴𝑆𝑇𝑋 − 𝐹𝑟𝑒𝑒 𝐴𝑆𝑇𝑋 × 100% 𝑇𝑜𝑡𝑎𝑙 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

where “Total mass of nanoparticles” refers to the total mass of SA-CS, NaCas, Odex, and ASTX used in the preparation of nanoparticles. 2.6. Morphology of nanoparticles TEM was employed to observe the morphology of empty nanoparticles, ASTX-loaded nanoparticles, and to monitor the morphological changes of nanoparticles during stability testing in simulated GI fluids. Briefly, 3 μL of each diluted sample (0.5 mg/mL) was deposited on a 7

Journal Pre-proof plasma cleaned carbon-coated TEM grid (CF400-CU, Electron Microscopy Science) for 2 min. Then, the grid was rinsed off by 100 μL of 0.5% uranyl acetate stain solution and air dried completely. Finally, the grid was examined using a TEM (FEI, Tecnai 12 G2, Spirit, BioTWIN, Netherlands) and images were obtained by a CCD camera (AMT 2k XR40). The spray-dried powders were obtained using a Nano Spray Dryer B-90 (BÜCHI Labortechnik AG, Flawil, Switzerland). Then, spray-dried powders were deposited onto double adhesive carbon conductive

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tape and observed under SEM (JSM-6330F, JEOL Ltd., Tokyo, Japan).

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2.7. ABTS radical scavenging assay

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The ABTS radical scavenging activity of ASTX-loaded nanoparticles was carried out in a 96-well microplate according to the method that described in previous study [28]. The ASTX-

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loaded nanoparticles were tested at a wide range of equivalent ASTX concentrations, i.e., 0.5, 1,

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2.5, 5, 10, 20, and 40 μg/mL. Free ASTX was dissolved in DMSO and then diluted with DI water

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with the same range of concentrations. The control was prepared by replacing sample with PBS. Meanwhile, the sample background (sample added in PBS without ABTS) was also measured to

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avoid any interference of background color.

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ABTS free radical scavenging activity (%) = (1 −

𝐴𝑠 − 𝐴𝑏 ) × 100 𝐴𝑐

where As, Ab, and Ac are the absorbance of the sample, background, and control, respectively. 2.8. Cytotoxicity of ASTX-loaded nanoparticles LX-2 cells were a generous gift from Dr. Scott Friedman (Icahn School of Medicine at Mount Sinai, New York, NY), and they were maintained as we previously described [29, 30]. All cell culture supplies were purchased from HyClone (Thermo Scientific, Logan, UT). LX-2 cells were incubated with free or ASTX-loaded nanoparticles at 10 μM ASTX equivalent concentrations for 48 h. Subsequently, cell viability was determined using a Cell 8

Journal Pre-proof Counting Kit-8 (Dojindo Inc., Rockville, MD) according to the manufacturer’s instructions. Sodium dodecyl sulfate (0.5 mM) served as a positive control to validate the assay. Cell viability was expressed as a percentage of the control. 2.9. Anti-fibrogenic activity of ASTX-loaded nanoparticles LX-2 cells were pretreated with 10 and 20 μM of free or ASTX-loaded nanoparticles for 24 h, followed by stimulation with 4 ng/mL transforming growth factor β1 (Peprotech, Rocky Hill,

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NJ) for another 24 h for real-time PCR and Western blot analysis. DMSO and nanoparticles

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vehicle were run in parallel.

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2.10. Quantitative real-time PCR (qRT-PCR)

Total RNA extraction from cells and cDNA synthesis and qRT-PCR were conducted as

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previously described [31]. Gene sequences were acquired from the GenBank database, and

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primers were designed by a Beacon Designer software (Premier Biosoft, Palo Alto, CA). The

2.11.Western blot analysis

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primer sequences will be provided upon request.

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Whole-cell lysates were prepared as previously reported to perform Western blot analysis

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[32]. Antibodies against alpha-smooth muscle actin (αSMA) (Sigma, St. Louis, MO) and collagen type I alpha 1 chain (COL1A1) (Sigma) were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a loading control. 2.12. Statistical analysis All experiments were conducted in triplicate and data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed with the Tukey’s multiple-

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Journal Pre-proof comparison test to compare significant differences between samples, using SPSS package (SPSS 13.0 for windows, SPSS Inc., Chicago, IL, USA). The significance level p was set as 0.05. 3.

Results and discussion

3.1. Preparation and optimization of nanoparticles To optimize the fabrication process, a total of 15 experimental runs were performed for three levels and three factors according to the RSM using the Box-Behnken design. The variables

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used in the study were selected based on the preliminary experiments. The summary of analysis

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results for observed response is shown in Table S3. The positive coefficient of a factor in the

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above quadratic equation indicates the enhancement or a synergistic effect of that particular

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response, and vice versa. The lack-of-fit was not significant in all the responses at 95% confidence level. The values of various statistical parameters, including mean square values

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(MS), F-value, p-value, multiple correlation coefficients (R2), standard deviation (SD),

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coefficient of variation (C.V%), and predicted residual sum of squares are shown in Table S4. The results indicated that the quadratic model was best-fitted for all the responses studied.

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The particle size showed a wide variation from 91.7 to 148.8 nm with the selected levels of

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variables. The most significant factors affecting the particle size are mass ratio of SA-CS to Odex (X1), heating temperature (X2), heating time (X3), the interaction between mass ratio of SA-CS to Odex and heating time (X1X2), as shown in Table S3. The particle size significantly increased with increases in Odex concentration and heating temperature, as well as the elongation of Schiff base reaction time (Fig. 1A1). In our previous study [12], it has been proven that the main driving force to form polymeric nanoparticles is the electrostatic attraction between the negatively charged NaCas core and positively charged SA-CS coating, as well as hydrophobic interactions between SA segments in SA-CS and hydrophobic residues of NaCas.

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Journal Pre-proof The aldehyde groups of Odex are able to react with amino groups of both NaCas and SA-CS via Schiff base reaction, resulting in the formation a SA-CS/Odex conjugate coating on the surface of protein molecules [33]. Therefore, the larger particle size may be attributed to thicker SACS/Odex coating, caused by the higher concentration of Odex and temperature. Meanwhile, the reaction time on different heating temperature also had a significant influence on the particle size, which was corroborated with previous literature [21].

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In the current study, the PDI of nanoparticles ranged from 0.11 to 0.2 for various factor

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level combinations. The most significant factors affecting the PDI are heating temperature (X2),

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heating time (X3), and the interaction between heating temperature and heating time (X2X3). As shown in Fig. 1B1, the PDI of nanoparticles was decreased from 0.16 to 0.11 with the increase of

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heating temperature when Odex concentration was 1.2 mg/mL and the mass ratio of SA-CS to

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Odex was 1:2, which could be attributed to the formation of more homogenous SA-CS/Odex

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coating. A similar trend was also observed in Fig. 1B2, indicating that both reaction temperature and time have significant negative effect on PDI. Our results were supported by the literature that

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Odex could not be absorbed onto the surface of NaCas core at the initial stage of heating due to

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the kinetic and thermodynamic process of Schiff-base formation [21]. However, with more Odex reacting with NaCas and SA-CS and less free Odex being available in the aqueous phase, the formation of uniform coating layer took place and thus more ordered structure of complex nanoparticles were formed with narrow size distribution. Nevertheless, the highest PDI was observed when nanoparticles were heated at 80 oC for 45 min. It could be ascribed to the aggregation caused by intermolecular conjugation among nanoparticles. The colloidal stability in simulated GI fluids is an important parameter used to indicate the applicability of the nanoparticles as an oral delivery system. In this experiment, the colloidal

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Journal Pre-proof stability was evaluated by measuring the change in particle size before and after incubation of nanoparticles in SGF and SIF, which was expressed as Δ particle size in SGF and Δ particle size in SIF, respectively. The results indicated that stability of nanoparticles in SGF was significantly affected by heating temperature and heating time. The smaller Δ particle size in SGF was observed for the nanoparticles prepared under higher heating temperature and longer heating time (Fig. 1C3). This could be explained by the formation of well-ordered and uniform coating

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layer, resulting in better protection of NaCas against enzymatic degradation under gastric fluid

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containing pepsin. This result was consistent with that from the influence of heating temperature

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and time on PDI of nanoparticles. However, the particle size significantly increased under high heating temperature, especially when the temperature was raised to 85 oC. Therefore, the optimal

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combination of heating temperature and heating time was required to achieve a good stability of

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nanoparticles in SGF while maintaining the small particle size.

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As described in our previous study [12], the protonation level of SA-CS was minimal in SIF where the neutral pH is close to the pKa value of amino groups on SA-CS. As a result, without

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the cross-linked complex nanostructure, the SA-CS coating was disassociated and severe

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aggregation was formed. Therefore, the Odex plays an important role in stabilizing the complex nanoparticles and maintaining their structural integrity in SIF. In this experiment, most factors did not dramatically affect the Δ particle size in SIF, as evidenced by the small values of coefficient with insignificance. The only factor that significantly affected Δ particle size in SIF was the mass ratio of SA-CS to Odex (X1). As shown in Fig. 1D1 & D2, the Δ particle size in SIF was significantly decreased with increasing Odex concentration at different heating temperature and heating time. At low level of X1, severe aggregation was observed after the incubation in SIF. This aggregation indicated that when the Odex concentration was insufficient,

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Journal Pre-proof it failed to form a protective coating layer to stabilize SA-CS/NaCas complex nanoparticles from dissociation in SIF. Nevertheless, at high level of X1, in which the concentration of Odex reached to 1.25 mg/mL, the lower Δ particle size in SIF was obtained. It is noteworthy that the lowest Δ particle size in SIF was -8.2%, which refers to the decreased particle size after the incubation of nanoparticles in SIF. The three-dimensional (3D) response surface plots generated by the Design-Expert software

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was used to acquire the optimized formulation of the SA-CS/NaCas/Odex nanoparticles. The

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response-fitting results indicated that optimized formulation with small particle size, low PDI

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value, and small Δ particle size in SGF and SIF, were obtained at the mass ratio of SA-CS to Odex of 1:1.95, heating temperature of 76 oC with a heating time of 43 min. Based on these

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calculations from the software, the nanoparticles with the predicted levels of formulation factors

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were prepared to validate the optimization procedure. The optimized formulation had particle

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size of 119.7 nm, PDI of 0.11, Δ particle size in SGF of 4.85%, and Δ particle size in SIF of 3.29%. Table S5 showed that the average experimental values of the three batches of

Fourier transform infrared (FT-IR) spectroscopy

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3.2.

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nanoparticles prepared using the optimized range were very close to the predicted values.

The FT-IR spectra of SA-CS, NaCas, pristine dextran, Odex, and optimized nanoparticles were presented in Fig. 2. SA-CS exhibited two major characteristic peaks for amide bonds including primary amine (N–H bending) at 1617 cm-1 and amide I (C=O stretching) at 1617 cm-1 due to its nature as an aminopolysaccharide as well as the new peak for carbonyl groups from ester bonds (C=O stretching) at 1692 cm-1. The strong amide I and amide II absorptions, and O– H stretching at 1637, 1514, and 3281 cm-1 was observed from NaCas, as well as C–H stretching at the region of 2920–2960 cm-1, indicating its strong amphiphilicity [34]. The band in the region

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Journal Pre-proof of 3320 cm-1 from pristine dextran was due to the hydroxyl stretching vibration of the polysaccharide. The band in the region of 2905 cm-1 was due to C–H stretching vibration. In the spectrum of Odex, the absorption peak at 1732 cm-1 that corresponding to the stretching of the carbonyl (C=O stretching) from an aldehyde group was detected in Odex [35]. Such band was not observed in the pristine dextran, indicating the successful oxidation of dextran. The chemical structure of SA-CS and Odex was also confirmed by nuclear magnetic resonance spectroscopy as

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reported in our recent work [12].

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The complex nanoparticles shared a high similarity with SA-CS and NaCas in their

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characteristic peaks including amide I and amide II absorptions at 1647 and 1530 cm-1, while the shifts of wavenumbers at these peaks in the region of amide bonds clearly indicated the

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electrostatic interactions between two biopolymers [36]. Likely, the O–H and C–H stretchings

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were observed in the complex nanoparticles with significant shifts in their wavenumbers, which

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evidenced that hydrophobic interactions also played a role in the formation of such complex. Noteworthy, the nanoparticles only showed one band at 1727 cm -1, which may be attributed to

3.3.

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in Odex.

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the overlapping of carbonyl stretching from ester bond in SA-CS and unreacted aldehyde groups

Encapsulation of ASTX Due to its poor water-solubility, it is necessary to select an appropriate organic solvent to

solubilize ASTX for encapsulation. According to previous literature, DMSO appears to be a good solvent to aid the encapsulation of ASTX in a liposomal formulation [37]. Thus, in the current study, DMSO was adopted to prepare ASTX stock solution, facilitating encapsulation within the core of the complex nanoparticles. Prior to preparation of nanoparticles, ASTX stock solution was mixed with NaCas which has the high affinity to lipophilic bioactive compounds. The

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Journal Pre-proof proposed scheme of nanoparticles preparation and ASTX encapsulation is presented in Fig. 3. Three batches of nanoparticles with different ASTX loading ratios were tested, i.e. 1%, 3%, and 5%. As depicted in Fig. 4A, compared to the empty nanoparticles, the particle size of ASTXloaded nanoparticles was in a range of 145-198 nm. This may be explained by the expansion of nanoparticles due to ASTX loading into the NaCas hydrophobic core. The same trend was observed from PDI results, indicating that the nanoparticles became more heterogeneous with

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increases in ASTX loading concentrations. Encapsulation of ASTX did not alter the surface

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charge of nanoparticles, as no significant change of zeta potential was detected among three

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loading ratios (Fig. 4B). Nevertheless, the amount of loaded ASTX exhibited negative impact on its encapsulation efficiency by the nanoparticles. With the increase of loaded ASTX from 1% to

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5%, the encapsulation efficiency decreased from 81.5% to 65.3%, indicating that increasing

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ASTX loading gradually compromised the encapsulation capability of nanoparticles. In addition,

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the cytotoxicity of nanoparticles with high ASTX loading ratio may increase as more DMSO was introduced into the composition. Due to these important factors, the ASTX-loaded nanoparticles

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with 3% loading ratio was selected for subsequent study. The encapsulation efficiency of as-

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prepared nanoparticles was 70.2%, which equaled to 72.1 μM (84 μg/mL) of ASTX in the SACS/NaCas/Odex complex nanoparticles. The loading ratio of ASTX in current study is higher than that in DNA/chitosan colloidal system (65 μg/mL) in literature [25]. 3.4.

Morphological observation The complex nanoparticles were negatively stained with uranyl acetate and observed under

TEM. As shown in Fig. 5A, the dimension of freshly prepared nanoparticles was in the range of 80-100 nm with a spherical shape and smooth surface, suggesting the monodispersity and wellcontrolled particle size. In order to further validate the stability of nanoparticles in simulated GI

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Journal Pre-proof fluids, the morphological observation using TEM was carried out after the nanoparticles were incubated in SGF (pH 2), and SIF (pH 7) individually. Unlike the rough and eroded surface of nanoparticles cross-linked by glutaraldehyde/Odex observed in previous study [12], the particle size, smooth surface and monodispersity of as-prepared nanoparticles were maintained after the incubation in SGF (Fig. 5B). This result revealed better stabilization effect by SA-CS/Odex coating layer formed via Schiff base reaction than that of glutaraldehyde in harsh acidic

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environment. Besides, the swelling and expansion of particle size as well as heterogeneous

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distribution of nanoparticles were not observed after the incubation in SIF (Fig. 5C), indicating

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that the cross-linked SA-CS/Odex coating layer was able to prevent deprotonation-induced dissociation SA-CS under neutral pH. The morphology of optimized ASTX-loaded nanoparticles

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was also tested. Compared to the empty nanoparticles, the ASTX-loaded nanoparticles exhibited

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larger particle size and relatively less uniform size distribution (Fig. 5D), which was consistent

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with DLS results. Interestingly, a distinct and brighter field in the core of nanostructure was

lipophilic ASTX.

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observed in ASTX-loaded nanoparticles, which might be ascribed to the encapsulation of

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In addition, the SEM images of nano spray dried nanoparticles were presented in Fig. 6. Both empty nanoparticles and ASTX-loaded nanoparticles showed spherical shape but wide size distribution. Some particles exhibited a size of 500 nm with smooth surface, while other particles had dramatically larger size (around 1 μm) with dented surface. It could be attributed to the aggregation during the spray drying process. 3.5.

Bioactivity of ASTX-loaded nanoparticles

3.5.1. ABTS radical scavenging ability of 3% ASTX-loaded nanoparticles

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Journal Pre-proof The antioxidant activity of ASTX has been associated with the beneficial effects on chronic diseases such as macular degradation, cardiovascular diseases, and cancer [38, 39]. Compared to other carotenoids, such as zeaxanthin, lutein and β-carotene, ASTX has been reported to show 10 times stronger antioxidant activity [40]. However, as a lipophilic compound, the potency of its antioxidant activity may be compromised if ASTX is suspended in aqueous condition. The contact probability of ASTX to free radicals in aqueous phase becomes a limiting factor to its

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bioactivity in a food system. As shown in Fig. 7A, the scavenging capacity against ABTS

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radicals of free ASTX, empty nanoparticles and 3% ASTX-loaded nanoparticles were tested and

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compared. At a wide range of concentrations studied (0.5–40 μg/mL), free ASTX showed low scavenging activity due to poor dispersibility in PBS and thus limited contact with ABTS

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radicals. The scavenging activity of empty nanoparticles slightly increased with increasing

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concentration. Nevertheless, encapsulation of ASTX into as-prepared biopolymer nanoparticles

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significantly improved its antioxidant activity, compared to free ASTX. At the concentration of 40 μg/mL, the ABTS radicals scavenging capacity of ASTX-loaded nanoparticlex was as high as

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85.6%. The concentration of ASTX (in nanoparticles) required to achieve 50% inhibition of

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ABTS radicals (IC50) was calculated as 0.081 mol ASTX/mol ABTS. This result was comparable with that of similarly-sized ASTX colloidal particles (0.076 mol ASTX/mol ABTS) prepared with polysorbate 20, NaCas, and gum Arabic in another study [41]. 3.5.2. Cytotoxicity and anti-fibrogenic activity of 3% ASTX-loaded nanoparticles The cytotoxicity was expressed as % viability compared with control cells. As shown in Fig. 7B, after incubation with empty nanoparticles, free ASTX, 3% ASTX-loaded nanoparticles (at equivalent ASTX concentration of 10 μM) and DMSO control, the cell viability of LX-2 were all

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Journal Pre-proof above 90%, indicating that toxicity of nanoparticles vehicle and ASTX encapsulated nanoparticles was minimal. To evaluate the anti-fibrogenic activity of ASTX-loaded nanoparticles, LX-2 cells were used as an HSC cell model which is a suitable model for HSC due to their similar cytokine signaling and fibrogenesis to human HSC [42]. TGFβ1, a potent fibrogenic cytokine, can induce the expression of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) proteins, such

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as procollagen type I, alpha 1 (COL1A1) [43]. After treated with 4 ng/mL TGFβ1 for 24 h,

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ACTA2 and COL1A1 expression was significantly increased, as shown in Fig. 7C&D. We

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previously showed that ASTX inhibits the induction of fibrogenic genes (ACTA2 and COL1A1) by TGFβ1 at the minimum concentration of 10 μM [44]. Therefore, LX-2 cells were pretreated

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with 10 μM free ASTX as a positive control prior to the incubation with TGFβ1. Both ACTA2

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and COL1A1 were significantly inhibited by free ASTX consistent with our previous finding

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[29]. Interestingly, both empty and 3% ASTX-loaded nanoparticles exhibited comparable inhibitory effects on ACTA2 expression (Fig. 7C), and they elicited stronger repression of

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COL1A1 expression by ~2-fold than free ASTX (Fig. 7D). It is of interest that the empty

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nanoparticles significantly inhibited the expression of both fibrogenic genes, with higher efficacy than free ASTX. A plausible explanation may be related to a hepato-protective effect of chitosan. The administration of chitosan exhibited antioxidative, anti-inflammatory and anti-fibrogenic effects in rats with cholestatic liver injury induced by bile duct ligation [45]. Therefore, the antifibrogenic effects of 3% ASTX-loaded nanoparticles may be attributed to chitosan present in SACS. As the internalized encapsulated ASTX in LX-2 cells was unable to further inhibit the expression of ACTA2 and COL1A1, we investigated the anti-fibrogenic effect of 6% ASTXloaded nanoparticles.

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Journal Pre-proof 3.5.3. Anti-fibrogenic activity of 6% ASTX-loaded nanoparticles As shown in Fig. 8A&B, the empty nanoparticles suppressed TGFβ1-induced fibrogenic gene expression to a basal level. In addition, the 6% ASTX-loaded nanoparticles (at equivalent ASTX concentration of 20 μM) significantly reduced the expression of ACTA2 and COL1A1 by ~3-fold, compared to that of free ASTX. This may be due to improved dispersibility of ASTX in aqueous conditions via encapsulation in biopolymer-based nanoparticles. Crystallized free ASTX

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was seen in the medium during treatment in LX-2 cells, which was not observed with ASTX-

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loaded nanoparticles. Thus, improved dispersibility of encapsulated ASTX in aqueous medium

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may facilitate the cellular uptake by LX-2 cells, resulting in higher inhibition of fibrogenic genes. The stronger anti-fibrogenic activity of ASTX-loaded nanoparticles was further confirmed

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by Western blot analysis, showing marked inhibition of α-SMA and COL1A1 protein levels upon

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TGFβ1 stimulation (Fig. 8C). Although the present study demonstrated promising delivery

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efficacy of as-prepared biopolymer-based nanoparticles, how the nanoparticles are taken up by LX-2 cells remains elusive. Also, whether the integrity of cellular membranes is maintained after

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the internalization of nanoparticles is still unknown. Future studies are warranted to further

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investigate cellular uptake and release mechanisms of encapsulated ASTX in the cytoplasm of LX-2 cells, as well as cell-type specificity via conjugation with HSC-targeting ligand [46]. 4.

Conclusions and future studies In summary, the biopolymer-based complex nanoparticles were successfully fabricated

using SA-CS, NaCas and Odex, and the formulations and preparation parameters were comprehensively optimized via RSM. The optimized nanoparticles demonstrated promising GI stability due to the strong covalent bond formed by Schiff base reaction between aldehyde group of Odex and amino groups of SA-CS and NaCas. Encapsulation of ASTX into nanoparticles was

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Journal Pre-proof achieved and confirmed by visualizing a distinct morphology under TEM. The ABTS radical scavenging capacity of ASTX was dramatically enhanced. The in vitro cell study revealed noncytotoxicity of nanoparticles and good delivery efficiency for ASTX, evidenced by improved anti-fibrogenic effects. Although controlled release study was not tested in the current study, our previous work demonstrated the efficacy of Odex cross-linking on the release profile of curcumin from the

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same nanoparticles, showing that less than 15% cumulative release under the simulated GI

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conditions. Since exceptional GI-stability (particularly in the stomach condition) was achieved in

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current study through heat-induced Schiff base reaction of polymer network structure, we anticipated a similar, if not slower, sustained release profile of ASTX from as-prepared complex

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nanoparticles. The ultimate goal of such nanoparticles is to conjugate with HSC-targeting ligand

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for cell-type specific delivery. Dextran-1,6-glucosidase, a dextran-splitting enzyme, can be

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produced in the liver, and thus the main release may happen in the liver, triggered by the breakdown of Odex and subsequent dissociation of nanostructure. The present study primarily

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focused on the formulation of nanoparticles and the anti-fibrogenic activity of encapsulated

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ASTX. While future studies are warranted to illustrate the underlying mechanism, this work provides a novel strategy to fabricate biopolymer-based nanoparticles that hold promising potential as an oral delivery system of lipophilic bioactives for liver fibrosis treatment. Conflicts of interest There are no conflicts of interest to declare. Acknowledgement The TEM study was performed in part at the Biosciences Electron Microscopy Facility of the University of Connecticut (UConn). The SEM studies were performed using the facilities in the UConn/FEI Center for Advanced Microscopy and Materials Analysis (CAMMA). 20

Journal Pre-proof References

Jo

ur

na

lP

re

-p

ro

of

[1] M. Bodnar, J.F. Hartmann, J. Borbely, Preparation and characterization of chitosan-based nanoparticles, Biomacromolecules 6(5) (2005) 2521-2527. [2] S. Choi, S. Koo, Efficient syntheses of the keto-carotenoids canthaxanthin, astaxanthin, and astacene, J. Org. Chem. 70(8) (2005) 3328-3331. [3] H. Zhang, Y. Fu, Y. Xu, F. Niu, Z. Li, C. Ba, B. Jin, G. Chen, X. Li, One-step assembly of zein/caseinate/alginate nanoparticles for encapsulation and improved bioaccessibility of propolis, Food Funct. 10(2) (2019) 635-645. [4] Z.-L. Wan, J. Guo, X.-Q. Yang, Plant protein-based delivery systems for bioactive ingredients in foods, Food Funct. 6(9) (2015) 2876-2889. [5] W.-J. Cao, S.-Y. Ou, W.-F. Lin, C.-H. Tang, Food protein-based phytosterol nanoparticles: fabrication and characterization, Food Funct. 7(9) (2016) 3973-3980. [6] C. Yang, S. Gao, F. Dagnæs-Hansen, M. Jakobsen, J. Kjems, Impact of PEG chain length on the physical properties and bioactivity of PEGylated chitosan/siRNA nanoparticles in vitro and in vivo, ACS Appl. Mater. Interfaces 9(14) (2017) 12203-12216. [7] S.A. Sande, Pectin-based oral drug delivery to the colon, Expert Opin. Drug Deliv. 2(3) (2005) 441-450. [8] N. Lin, A. Gèze, D. Wouessidjewe, J. Huang, A. Dufresne, Biocompatible double-membrane hydrogels from cationic cellulose nanocrystals and anionic alginate as complexing drugs codelivery, ACS Appl. Mater. Interfaces 8(11) (2016) 6880-6889. [9] Wusigale, L. Li, Y. Luo, Casein and pectin: Structures, interactions, and applications, Trends Food Sci. Technol. 97 (2020) 391-403. [10] Q. Hu, Y. Luo, Polyphenol-chitosan conjugates: Synthesis, characterization, and applications, Carbohydr. Polym. 151 (2016) 624-639. [11] Y. Luo, Z. Teng, Q. Wang, Development of zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3, J. Agric. Food Chem. 60(3) (2012) 836-843. [12] Q. Hu, M. Bae, E. Fleming, J.-Y. Lee, Y. Luo, Biocompatible polymeric nanoparticles with exceptional gastrointestinal stability as oral delivery vehicles for lipophilic bioactives, Food Hydrocoll. 89 (2019) 386-395. [13] C. Chang, T. Wang, Q. Hu, Y. Luo, Zein/caseinate/pectin complex nanoparticles: Formation and characterization, Int. J. Biol. Macromol. 104 (2017) 117-124. [14] C. Chang, T. Wang, Q. Hu, Y. Luo, Caseinate-zein-polysaccharide complex nanoparticles as potential oral delivery vehicles for curcumin: Effect of polysaccharide type and chemical cross-linking, Food Hydrocoll. 72 (2017) 254-262. [15] C. Chang, T. Wang, Q. Hu, M. Zhou, J. Xue, Y. Luo, Pectin coating improves physicochemical properties of caseinate/zein nanoparticles as oral delivery vehicles for curcumin, Food Hydrocoll. 70 (2017) 143-151. [16] A.O. Elzoghby, W.S.A. El-Fotoh, N.A. Elgindy, Casein-based formulations as promising controlled release drug delivery systems, J. Control. Release 153(3) (2011) 206-216. [17] R. Cortesi, E. Esposito, M. Osti, E. Menegatti, G. Squarzoni, S.S. Davis, C. Nastruzzi, Dextran cross-linked gelatin microspheres as a drug delivery system, Eur. J. Pharm. Biopharm. 47(2) (1999) 153-160. [18] U.V. Sojitra, S.S. Nadar, V.K. Rathod, Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker, Carbohydr. Polym. 157 (2017) 677-685. [19] S.H. Jung, S.J. Choi, H.J. Kim, T.W. Moon, Molecular characteristics of bovine serum albumin-dextran conjugates, Biosci. Biotechnol. Biochem. 70 (2006) 2064-2070. [20] N.J. Rodriguez, Q. Hu, Y. Luo, Oxidized Dextran as a Macromolecular Crosslinker Stabilizes the Zein/Caseinate Nanocomplex for the Potential Oral Delivery of Curcumin, Molecules 24(22) (2019) 4061. [21] T. Wang, Q. Hu, J.-Y. Lee, Y. Luo, Solid Lipid–Polymer Hybrid Nanoparticles by In Situ Conjugation for Oral Delivery of Astaxanthin, J. Agric. Food Chem. 66(36) (2018) 9473-9480. [22] M. Bae, Y.-K. Park, J.-Y. Lee, Food components with antifibrotic activity and implications in prevention of liver disease, J. Nutr. Biochem. 55 (2018) 1-11. [23] F. Tamjidi, M. Shahedi, J. Varshosaz, A. Nasirpour, Design and characterization of astaxanthin-loaded nanostructured lipid carriers, Innov. Food Sci. Emerg. Technol. 26 (2014) 366-374. [24] N. Cheeveewattanagul, K. Jirasripongpun, N. Jirakanjanakit, W. Wattanakaroon, Carrier design for astaxanthin delivery, Adv. Mat. Res. 93 (2010) 202-205. [25] Q. Wang, Y. Zhao, L. Guan, Y. Zhang, Q. Dang, P. Dong, J. Li, X. Liang, Preparation of astaxanthin-loaded DNA/chitosan nanoparticles for improved cellular uptake and antioxidation capability, Food Chem. 227 (2017) 9-15.

21

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

[26] X. Shen, C. Zhao, J. Lu, M. Guo, Physicochemical properties of whey-protein-stabilized astaxanthin nanodispersion and its transport via a Caco-2 monolayer, J. Agric. Food Chem. 66(6) (2018) 1472-1478. [27] Q. Hu, T. Wang, M. Zhou, J. Xue, Y. Luo, Formation of redispersible polyelectrolyte complex nanoparticles from gallic acid-chitosan conjugate and gum arabic, Int. J. Biol. Macromol. 92 (2016) 812-819. [28] Q. Hu, T. Wang, M. Zhou, J. Xue, Y. Luo, In vitro antioxidant-activity evaluation of gallic-acid-grafted chitosan conjugate synthesized by free-radical-induced grafting method, J. Agric. Food Chem. 64(29) (2016) 5893-5900. [29] Y. Yang, B. Kim, Y.-K. Park, S.I. Koo, J.-Y. Lee, Astaxanthin prevents TGFβ1-induced pro-fibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells, Biochim. Biophys. Acta, Gen. Subj. 1850(1) (2015) 178-185. [30] M. Bae, Y. Lee, Y.-K. Park, D.-G. Shin, P. Joshi, S.-H. Hong, N. Alder, S.I. Koo, J.-Y. Lee, Astaxanthin attenuates the increase in mitochondrial respiration during the activation of hepatic stellate cells, J. Nutr. Biochem. 71 (2019) 82-89. [31] Y.K. Park, H.E. Rasmussen, S.J. Ehlers, K.R. Blobaum, F. Lu, V.L. Schlegal, T.P. Carr, J.Y. Lee, Repression of proinflammatory gene expression by lipid extract of Nostoc commune var sphaeroides Kutzing, a blue-green alga, via inhibition of nuclear factor-kappaB in RAW 264.7 macrophages, Nutr. Res. 28(2) (2008) 83-91. [32] H.E. Rasmussen, K.R. Blobaum, Y.K. Park, S.J. Ehlers, F. Lu, J.Y. Lee, Lipid extract of Nostoc commune var. sphaeroides Kutzing, a blue-green alga, inhibits the activation of sterol regulatory element binding proteins in HepG2 cells, J. Nutr. 138(3) (2008) 476-81. [33] M. Fuentes, R.L. Segura, O. Abian, L. Betancor, A. Hidalgo, C. Mateo, R. Fernandez-Lafuente, J.M. Guisan, Determination of protein-protein interactions through aldehyde-dextran intermolecular cross-linking, Proteomics 4(9) (2004) 2602-2607. [34] H. Chen, Q. Zhong, Processes improving the dispersibility of spray-dried zein nanoparticles using sodium caseinate, Food Hydrocoll. 35 (2014) 358-366. [35] J. Maia, L. Ferreira, R. Carvalho, M.A. Ramos, M.H. Gil, Synthesis and characterization of new injectable and degradable dextran-based hydrogels, Polymer 46(23) (2005) 9604-9614. [36] T. Wang, Q. Hu, M. Zhou, Y. Xia, M.-P. Nieh, Y. Luo, Development of “all natural” layer-by-layer redispersible solid lipid nanoparticles by nano spray drying technology, Eur. J. Pharm. Biopharm. 107 (2016) 273-285. [37] S. Hama, K. Takahashi, Y. Inai, K. Shiota, R. Sakamoto, A. Yamada, H. Tsuchiya, K. Kanamura, E. Yamashita, K. Kogure, Protective effects of topical application of a poorly soluble antioxidant astaxanthin liposomal formulation on ultraviolet-induced skin damage, J. Pharm. Sci. 101(8) (2012) 2909-2916. [38] P. Palozza, C. Torelli, A. Boninsegna, R. Simone, A. Catalano, M.C. Mele, N. Picci, Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells, Cancer Lett. 283(1) (2009) 108-117. [39] R.G. Fassett, J.S. Coombes, Astaxanthin: a potential therapeutic agent in cardiovascular disease, Mar. Drugs 9(3) (2011) 447-465. [40] W. Miki, Biological functions and activities of animal carotenoids, Pure Appl. Chem. 63(1) (1991) 141-146. [41] N. Anarjan, C.P. Tan, I.A. Nehdi, T.C. Ling, Colloidal astaxanthin: preparation, characterisation and bioavailability evaluation, Food Chem. 135(3) (2012) 1303-1309. [42] L. Xu, A. Hui, E. Albanis, M. Arthur, S. O’byrne, W. Blaner, P. Mukherjee, S. Friedman, F. Eng, Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis, Gut 54(1) (2005) 142-151. [43] J. Gauldie, P. Bonniaud, P. Sime, K. Ask, M. Kolb, TGF-β, Smad3 and the process of progressive fibrosis, Biochem. Soc. Trans. 35 (2007) 661-664. [44] Y. Yang, B. Kim, Y.-K. Park, S.I. Koo, J.-Y.J.B.e.B.A.-G.S. Lee, Astaxanthin prevents TGFβ1-induced profibrogenic gene expression by inhibiting Smad3 activation in hepatic stellate cells, Biochim. Biophys. Acta, Gen. Subj. 1850(1) (2015) 178-185. [45] M. Dudea, S. Clichici, D.E. Olteanu, A. Nagy, M. Cucoş, S.J.U.i.m. Dudea, biology, Usefulness of real-time elastography strain ratio in the assessment of bile duct ligation-induced liver injury and the hepatoprotective effect of chitosan: an experimental animal study, Ultrasound Med. Biol. 41(1) (2015) 114-123. [46] Q. Hu, J.-Y. Lee, Y. Luo, Nanoparticles targeting hepatic stellate cells for the treatment of liver fibrosis, Eng. Sci. 6 (2019) 12-21.

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Figure 1. Surface response plots showing the effect of mass ratio of SA-CS to Odex, heating temperature and heating time on particle size (A1-A3), PDI (B1-B3), Δ particle size in SGF (C1-C3), and Δ particle size in SIF (D1-D3), respectively. X1, mass ratio of SA-CS to Odex; X2, heating temperature; X3, heating time. Figure 2. FT-IR spectra of SA-CS, NaCas, pristine dextran, Odex, and optimized nanoparticles. Figure 3. Proposed mechanism of fabrication of ASTX loaded SA-CS/NaCas/Odex nanoparticles. Figure 4. Characteristics of ASTX-loaded SA-CS/NaCas/Odex nanoparticles: (A) particle size; (B) PDI; (C) zeta potential; (D) EE. Bars sharing different letters are statistically different at p < 0.05. PDI: polydispersity index; EE: encapsulation efficiency. Figure 5. TEM images of nanoparticles as (A) freshly prepared; (B) incubated in SGF (pH 2.0); (C) incubated in SIF (pH 7.0); (D) ASTX-loaded nanoparticles. HV = 80.0 kV. Figure 6. SEM images of (A) spray dried empty nanoparticles and (B) spray dried ASTX-loaded nanoparticles. Figure 7. Biological function of 3% ASTX-loaded nanoparticles: (A) Scavenging activity against ABTS radical; (B) viability of treated LX-2 cells with 3% ASTX-loaded nanoparticles. Control: LX-2 cells were treated with 0.2% DMSO, empty nanoparticles, and 10 μM of free ASTX in 0.2% DMSO. Data are presented as means ± SD, n=5; Anti-fibrogenic effects of 3% ASTX-loaded nanoparticles. LX-2 cells were pretreated with empty nanoparticles, 10 μM of free ASTX and equivalent ASTX encapsulated nanoparticles for 24h, after which they were exposed to 4 ng/mL TGFβ1 for another 24h. qRT-PCR was conducted to measure (C) ACTA2 and (D) COL1A1 transcription levels. Data are presented as means ± SD, n=5. Bars sharing different letters are statistically different at p < 0.05. Figure 8. Anti-fibrogenic effects of 6% ASTX-loaded nanoparticles. LX-2 cells were pretreated with empty nanoparticles, 20 μM of free ASTX and equivalent ASTX encapsulated nanoparticles for 24h, after which they were exposed to 4 ng/mL TGFβ1 for another 24h. qRT-PCR was conducted to measure (A) ACTA2 and (B) COL1A1 transcription levels. Data are presented as means ± SD, n=5. (C) Western blot analysis was conducted to measure α-SMA and COL1A1 protein levels. Bars sharing different letters are statistically different at p < 0.05.

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CRediT authorship contribution statement

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Qiaobin Hu: Methodology; Investigation; Formal analysis; validation; Data Curation; Writing – Original Draft Siqi Hu: Methodology; Investigation; Writing – Original Draft Erika Fleming: Writing – Review & Editing; Ji-Young Lee: Resources; Writing – Review & Editing; Yangchao Luo: Conceptualization; Resources, Writing – Review & Editing; Supervision; Project administration; Funding acquisition.

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Journal Pre-proof Highlights Biopolymer ternary complex nanoparticles were developed for lipophilic bioactives

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The formulation was optimized via response surface methodology

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The aqueous dispersibility of encapsulated astaxanthin was enhanced

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The anti-fibrogenic activity of encapsulated astaxanthin was significantly improved

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Figure 1

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