Solid LipidâPolymer Hybrid Nanoparticles by In Situ Conjugation for

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Food and Beverage Chemistry/Biochemistry

Solid lipid-polymer hybrid nanoparticles by insitu conjugation for oral delivery of astaxanthin Taoran Wang, Qiaobin Hu, Jiyoung Lee, and Yangchao Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02827 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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Journal of Agricultural and Food Chemistry

Solid lipid-polymer hybrid nanoparticles by in-situ conjugation for oral delivery of astaxanthin

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Taoran Wang, Qiaobin Hu, Ji-Young Lee, Yangchao Luo*

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Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA

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*Corresponding author.

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Mailing address:

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Yangchao Luo, Ph.D.

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Assistant Professor

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Department of Nutritional Sciences

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University of Connecticut

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3624 Horsebarn Road extension, U-4017

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Storrs, CT 06269-4017, USA

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ACS Paragon Plus Environment


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Abstract

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Solid lipid-polymer hybrid nanoparticles (SLPN) are nanocarriers made from a combination of

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polymers and lipids, integrating the advantages of biocompatible lipid-based hydrophobic nanoparticles

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and gastrointestinal (GI)-stable polymeric nanoparticles. In this study, a novel preparation strategy was

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proposed to fabricate GI-stable SLPN through in-situ conjugation between oxidized dextran and BSA.

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Effects of molecular weight of dextran (20, 40, 75, and 150 kDa), conjugation temperature (65 °C, 75 °C,

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and 85 °C) and time (30, 60, 120 min) on the particulate characteristics and stability were

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comprehensively investigated and optimized. As heating temperature increased from 65 °C to 75 °C, the

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particle size of SLPN increased from 139 nm to 180 nm with narrow size distribution, but when the

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temperature reached 85 °C severe aggregation was observed after 60 min. SLPN prepared with 40 kDa

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oxidized dextran under 85 °C/30 min heating condition exhibited excellent GI stability with no significant

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changes in particle size and PDI after incubation in simulated GI fluids. The prepared SLPN were then

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used to encapsulate astaxanthin, a lipophilic bioactive compound, studied as a model nutrient. After

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encapsulation in SLPN, antioxidant activity of astaxanthin was dramatically enhanced in aqueous

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condition and a sustained release was achieved in simulated GI fluids. Therefore, the SLPN developed in

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this study are a promising oral delivery system for lipophilic compounds, such as astaxanthin.

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Keywords: nanoparticles; astaxanthin; encapsulation; controlled release; stability; antioxidant activity.


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1. Introduction

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Solid lipid nanoparticles (SLN) have been one of the most popular colloidal delivery systems for

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lipophilic bioactive compounds since introduced in early 1990s.1-2 The primary components in SLN

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include saturated fatty acid, glycerides, cholesterol, or their combinations. Given the fact that these lipids

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are solid at room temperature, the preparation of SLN usually involves the emulsification at high

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temperature above their melting points with the presence of highly concentrated surfactants to stabilize

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lipid droplets during subsequent cooling process. Although desirable colloidal stability under neutral

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condition (pH 6-8) can be achieved by using appropriate surfactants and emulsifiers during preparation,

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the gastrointestinal stability is a major challenge to explore SLN as oral delivery vehicles. Under acidic

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environment, protonation of carboxyl groups of lipids and neutralization of surface charge destabilize

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SLN, resulting in dissociation of lipids core from surrounding stabilizers and then precipitation due to

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strong hydrophobic interactions.3-4

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During the last decade, two major strategies have been reported to improve colloidal stability of SLN

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under gastrointestinal conditions, particularly the extreme acidic pH of gastric environment. The first and

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widely studied strategy is to take advantage of a blend of surfactants to offer synergistic stabilization

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effect to lipids. As the pioneer researchers, Zimmermann and Müller studied the influence of artificial GI

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media on the physical stability of SLN formulations consisting of different lipids and various

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surfactants/stabilizers.5 They concluded that the stabilization effect was highly dependent on the

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combination of lipids and surfactants. In other words, formulations must be carefully optimized when

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different lipids and surfactants are used to prepare SLN. Another strategy to stabilize SLN in GI

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conditions is the surface modification by means of incorporation of polyethylene glycol or its derivatives.

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This strategy is frequently referred as PEGylation, and it not only provides steric stabilization to SLN that

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are not affected by pH or ionic strength but also prolongs blood circulation time of SLN in vivo.6-8

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Nevertheless, PEGylation compromises the food-grade status of resultant nanoparticles, unsuitable for

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delivery of nutrients in functional foods.



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In recent years, our laboratory has been focusing on the development of polymer-coated SLN with

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exceptional stability under simulated GI conditions for potential oral delivery applications in foods. To

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simultaneously achieve desirable GI stability and maintain food-grade, we have designed a series of SLN

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formulations using natural biopolymers as food-grade emulsifiers and stabilizers during preparation.3, 9 In

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particular, the Maillard conjugates of bovine serum albumin (BSA) and dextran were tested in our

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previous study to fabricate highly stable solid lipid-polymer hybrid nanoparticles (SLPN) suitable for oral

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delivery.4 Although the Maillard conjugate was found to be effective, pectin coating was needed to

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stabilize SLPN in gastric condition. Pectin coating, however, significantly increased particle size from

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150 nm to 250-300 nm, which may affect delivery efficacy.

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Therefore, in the present study, we aimed to develop a novel strategy that can not only maintain the

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small size of SLPN but also achieve exceptional GI-stability. To accomplish this goal, dextran was first

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functionalized by oxidation through sodium periodate, and the resultant oxidized dextran (Ox-Dex)

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having multiple aldehyde groups is capable of forming covalent bond (imine bond or Schiff base) with

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polymers containing amino groups, i.e. BSA, under mild reaction condition. We hypothesized that SLPN

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could be prepared using BSA as a natural emulsifier and then Ox-Dex could act as a macromolecular

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crosslinker to covalently bind with BSA layer to further stabilize enveloped SLN in the core, forming

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stable SLPN. This process is therein referred as in-situ conjugation as the covalent bond is spontaneously

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formed during heating step within SLPN preparation. The physicochemical properties of obtained SLPN,

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including particulate characteristics, colloidal stability, and morphology, were comprehensively

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characterized. Astaxanthin (ASTN), a keto-carotenoid, was studied as a lipophilic model compound to

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explore the encapsulation and delivery potential of SLPN.

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2. Materials and methods

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2.1. Materials

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Precirol® ATO 5 was a kind gift from Gattefossé. Dextran (20, 40, 75, and 150 kDa), sodium

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periodate (NaIO4), bovine serum albumin (BSA) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic


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acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxylamine hydrochloride,

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hydrochloric acid, sodium hydroxide, acetone, and ethanol were obtained from Fisher Scientific Co.

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(Norcross, GA, USA). Astaxanthin was generously provided by Fuji Chemical Industry Co., Ltd.

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(Toyama, Japan) to Dr. Ji-Young Lee. Unless noted otherwise, all chemicals were of analytical grade and

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used without further purification.

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2.2. Preparation and characterization of oxidized dextran

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Oxidized dextran (Ox-Dex) was synthesized according to previous literature with some

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modifications.10 Dextran with molecular weights 20, 40, 75, and 150 kDa were used to prepare oxidized

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dextran and the oxidation procedure was carried out in a beaker protected from light. Briefly, native

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dextran solution (2.4 g/50 mL) was treated with 0.2 M sodium metaperiodate (50 mL) for 24 h at room

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temperature at pH 4. The mixture was then dialyzed against purified water for 24 h, followed by freeze

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drying for 48 h.

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The substitution degree of dextran was measured by titration method.11 Briefly, 100 mg of Ox-Dex

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powder was precisely weighed and dissolved in 25 ml of 0.25 M hydroxylamine hydrochloride solution.

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The obtained solution was stirred for 2 h in the dark at room temperature for the reaction to complete.

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Then, the mixture was titrated with standardized 0.1 M sodium hydroxide (NaOH) solution. The

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substitution degree of Ox-Dex was calculated by the following equation: Substitution degree % =

V × N × 10#$ Mol of CHO = × 100% Mol of dextran W&#'(& × MW'(&)*+

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where V is the volume of NaOH solution used for titration (mL), N is the NaOH concentration

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(mol/L), W&#'(& is the weight of Ox-Dex sample used (g), and MW'(&)*+ is molecular weight of

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dextran used for oxidation.

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Both Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy

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(NMR) was used to confirm the existence of aldehyde groups after oxidation. For FTIR spectrum analysis,

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freeze-dried Ox-Dex was mounted into ATR crystal for measurement using a NicoletTM iSTM5 FTIR



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spectrometer (Thermo Scientific, Waltham, MA, USA). The spectra were collected from the wave

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number of 500-4000 cm-1 at a resolution of 4 cm-1. For NMR, 1H spectra of native dextran and Ox-Dex

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(dissolved in D2O at concentration of 10 mg/mL) were acquired on an AVANCE 300 MHz NMR

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spectrometer.

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2.3. Preparation of SLPN with Ox-Dex and BSA

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SLPN were prepared through an organic solvent-free homogenization and sonication technique as

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described in our previous study.12 Briefly, 10 mg of Precirol® ATO 5 powder was heated to melt at 65 °C.

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Then, 10 mL of pre-heated (65°C) water phase, containing 1 mg/ml BSA and 1 mg/ml Ox-Dex solution,

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was mixed with melted lipid under homogenization at 25,000 rpm for 30 s to obtain coarse emulsion,

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followed by 3 min sonication by a probe-type sonicator (Misonix Sonicator® 3000, USA). Then, samples

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were incubated under 65, 75, or 85 °C for 30, 60, and 120 min to initiate conjugation reaction between

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BSA and Ox-Dex, forming Schiff-based complexes. After heating, samples were rapidly cooled down in

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ice bath to solidify the solid lipid core. As a control, SLPN composed of native dextran were prepared

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similarly but using native dextran instead of Ox-Dex.

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To encapsulate ASTN into SLPN, ASTN was first dissolved in acetone at a concentration of 0.2

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mg/ml. Then, 1 ml of ASTN solution and solid lipid were mixed and incubated together at 65 °C. Then,

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the same fabrication procedures followed as mentioned above.

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2.4. Characterization of SLPN

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Particle size and polydispersity index (PDI) of SLPN were measured by dynamic light scattering

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(DLS) using Zetasizer Nano ZS at 25 °C (Malvern Instruments Ltd, Worcestershire, UK). Zeta potential

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was measured using electrophoresis technique by the same instrument. Samples were diluted 10 times

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with buffer solution (same pH as samples) to avoid multiple scattering.

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The gastrointestinal stability of the SLPN samples was determined according to the method in our

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previous study.3 Briefly, 1 ml of SLPN sample was mixed with 9 ml of simulated gastric fluid (SGF, pH 2


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with 1 mg/ml pepsin) and incubated at 37 °C for 2 h. After SGF stage, 1 ml of above mixture was added

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into 9 ml of simulated intestinal fluid (SIF, pH 7 with 10 mg/ml pancreatin) and incubated at 37 °C for

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another 4 h. The pepsin and pancreatin were dissolved in SGF and SIF overnight respectively under 4 °C.

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Both SGF and SIF were filtered through 0.45 µm membrane to remove any impurities before use. At the

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end point of each incucation, particle size and PDI of SLPN were measured by DLS as previously

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described.

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2.5. Determination of encapsulation efficiency

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Encapsulation efficiency (EE) of ASTN-loaded SLPN was determined by measuring the

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concentration of free ASTN using extraction method. Briefly, 1 ml of freshly prepared ASTN-loaded

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SLPN was freeze-dried, and then 5 ml of acetone was added to extract the free ASTN from obtained

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SLPN powder. The concentration of ASTN in acetone was analyzed by a UV/Vis spectroscopy at 480 nm,

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with a pre-established ASTN standard curve. The EE of ASTN-loaded SLPN was calculated using

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following equation: EE % =

M- − M/*(( × 100% M-

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Where MT is the total mass of ASTN added during SLPN fabrication, and Mfree is mass of free ASTN

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in the extract.

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2.6. Spray drying process

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The SLPN samples were spray dried by a Nano Spray Dryer B-90 (Büchi Labortechnik AG, Flawil,

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Switzerland). The spray drying conditions were as follows: inlet temperature at 100 °C, flow rate at 120

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L/min, and mesh size is 4 µm.

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2.7. Morphological observation

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The morphology of freshly prepared colloidal SLPN was observed using a transmission electron

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microscopy (FEI, Tecnai 12 G2, Spirit, BioTWIN, Netherlands). Before observation, 3 µL of each diluted



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sample (6 times dilution, 0.5 mg/mL) was deposited on a plasma cleaned carbon-coated grid for 2 min.

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The grid was rinsed off by 100 µL of 0.5% uranyl acetate stain solution and air dried completely. The

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morphology of spray dried SLPN sample was analyzed using a scanning electron microscopy (SEM,

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JSM-6335F, JEOL Ltd., Tokyo, Japan) at an accelerated voltage of 10 KV. Spray-dried powder samples

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were directly placed on double-sided carbon tape pre-affixed on a specimen stub and coated with gold

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layer by a sputter coater before observation under SEM.

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2.8. ABTS radical scavenging assay

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ABTS assay was performed to determine the antioxidant activity and the detailed protocol was

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described in our previous study.8 The antioxidant activity was expressed as mg vitamin C equivalent

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antioxidant capacity per micro liter sample. The ASTN-loaded SLPN and free ASTN were tested at

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equivalent ASTN concentrations in a wide range, i.e. 0.25, 0.5, 2.5, 5 and 10 µg/mL. Free ASTN was

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dissolved in acetone at 0.5 mg/mL and then diluted with water to the appropriate concentration.

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2.9. In vitro controlled release study

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The in vitro release of ASTN from SLPN was conducted using the dialysis method reported by

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previous study.3 The free ASTN or ASTN-loaded SLPN was placed in a dialysis bag and the both sides of

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the bag were clipped and sealed. The bag was first placed into simulated gastric fluid (SGF, pH 2 with 0.5%

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v/v Tween 80, 37°C) for 2 h and then transferred into simulated intestinal fluid (SIF, pH 7 with 0.5 % v/v

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Tween 80, 37°C) for another 4 h. The release system was carefully sealed to prevent evaporation. During

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the experiment, 2 mL of release medium were withdrawn at predetermined time intervals (every 15 and

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30 min for SGF and SIF stage, respectively) with the replacement of the same volume of fresh medium.

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The collected release medium at each time point were then lyophilized for 24 h. After that, the released

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ASTN was extracted by 3 mL of acetone. The amount of released ASTN was determined by UV/vis

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spectroscopy at 480 nm using an ASTN standard curve.

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2.10. Statistical analysis


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All experiments were performed in triplicate at least and all data are presented as mean ± standard

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deviation (SD) form. Analysis of variance (ANOVA) with Tukey’s post hoc-test was performed to

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analysis the data. The level of significance was set at p < 0.05. Calculations were done with the software

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Minitab 18 and R.

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3. Results and discussion

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3.1. Characterization of oxidized dextran

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Native dextran was oxidized with sodium periodate (NaIO4). During oxidation process, the periodate

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ion attacks one of the hydroxyl groups (between C2–C3 or C3–C4) of dextran, resulting in break of C–C

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bond and yielding two aldehyde groups (Fig. 1A). Since the aldehyde group in C3 position has a vicinal

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hydroxyl group which is susceptible for further oxidation, it will undergo second oxidation and form a

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dialdehyde group.13-15 The oxidation degree of Ox-Dex was determined by hydroxylamine method and the

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results are displayed in Table S1. The number of aldehydes in native dextran and Ox-Dex was calculated

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and compared. The degree of oxidation was 21.7, 28.1, 25.2, and 25.3% for 20, 40, 75, and 150 kDa

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dextran, respectively. To confirm the oxidation reaction of dextran, the Ox-Dex (40 kDa) was analyzed by

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FTIR (Fig. 1B) and NMR (Fig. 1C) spectroscopy. In FTIR spectrum, the dialdehyde absorption peak

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(1730 cm-1) which is associated with the C=O group was detected in Ox-Dex.13 In the 1H NMR spectra of

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native dextran and Ox-Dex, there were peaks between δ 3.0 – 5.0 ppm which were assigned to the protons

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at six carbons in the glucose unit. The spectra of Ox-Dex exhibited several distinctive peaks in the range

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of 4.2 – 5.8 ppm, which were assigned to the protons from different hemiacetal structures.15-16 These

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results confirmed that oxidation of dextran was successful and dialdehyde groups were formed in dextran

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chain.

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3.2. Characterization and optimization of SLPN

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In our previous study, GI-stable SLPN were successfully prepared with BSA-dextran Maillard

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conjugate as a natural macromolecular emulsifier and pectin coating as a stabilizer.4 In that study, we



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proved that BSA-dextran conjugate alone was unable to provide sufficient stabilization against

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aggregation under gastric condition. Although pectin coating covering the surface of BSA-dextran

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emulsified SLN conferred satisfactory stability, the chemical nature of pectin limited further modification

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on its surface to fine-tune nanoparticles functionality, such as associating with ligand for target delivery.

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Therefore, in current study, an attempt was made to design a novel GI-stable formulation of SLPN

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without using pectin coating, and simultaneously a plenty of aldehyde functional groups were introduced

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on the nanoparticles surface via in-situ conjugation between Ox-Dex and BSA. Detail preparation

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procedure is illustrated in Fig. 2. Initially, native dextran was attempted in this design, as it also contains a

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few aldehyde groups in its open-chain structure, although very few, which may also cross-link with amino

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groups in BSA. Nevertheless, even though SLPN with small particle size and homogenous distribution

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could be successfully prepared with native dextran, the GI-stability of obtained SLPN was very poor that

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they precipitated instantly when incubating in simulated gastric fluid, due to weak and limited

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conjugation degree between native dextran and BSA (Fig. S1).

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In order to investigate optimal conjugation condition between BSA and Ox-Dex, effects of four types

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of Ox-Dex prepared from dextran with different molecular weight (20, 40, 75, and 150 kDa), three

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different heating temperatures (65 °C, 75 °C, and 85 °C) and three different heating durations (30, 60, and

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120 min) on the particulate characteristics were explored. Particulate characteristics including particle

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size, PDI and count rate were measured and the data were analyzed with the statistical significance at

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α=0.05 by variance analysis. Count rate is defined as the number of photons detected per second by the

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DLS instrument, which is often considered as an indicator of nanoparticles concentration and formation.

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The standardized Pareto chart for the three factors is presented in Fig. 3, where large effect indicates

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strong influences and a reference line was drawn to elucidate the statistical significance. It is notable that

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no association between molecular weight of dextran and particulate characteristics of SLPN, including

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particle size (p=0.78), PDI (p=0.76), and count rate (p=0.57). The mean particle size, PDI, and count rate

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of SLPN prepared using Ox-Dex with different molecular weight was found to be similar, around 158 nm,

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0.199, and 358.2 kcps, respectively (Fig. S2). Clearly, there is no evidence that low or high molecular


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weight of dextran could help produce SLPN with the best characteristics (smallest particle size and PDI,

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highest count rate). Nevertheless, SLPN prepared with 40 kDa Ox-Dex showed overall slightly superior

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properties with relatively small particle size, smallest PDI, and highest count rate. Therefore, SLPN

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prepared with 40 kDa Ox-Dex was selected in further studies. The information of particulate

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characteristics and GI-stability of SLPN prepared with 20, 75, and 150 kDa Ox-Dex can be found in Fig.

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S3-S6.

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From Fig. 4, it is clear that the characteristics of SLPN prepared with 40 kDa Ox-Dex, including

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particle size, PDI, and count rate, are mostly determined by the temperature and duration of heating and

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their interactions. In particular, as shown in Fig. 4A1 and 4A2, the particle size significantly increased

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from 150 nm to 800 nm by increasing heating temperature. Unlike conventional bifunctional reagents (e.g.

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glutaraldehyde) which are known to create intermolecular conjugation among protein molecules, Ox-Dex

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could instead rapidly cover the surface of BSA molecules and thus avoid inter-protein reactions during

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conjugation procedure.17 Thus, higher conjugation efficiency at elevated temperature may lead to thicker

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Ox-Dex layer covering the surface of SLPN, resulting in larger particle size. Meanwhile, the interaction

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between heating temperature and time had a significant impact on particle size. When the heating

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temperature was equal or higher than 75 °C, greater particle size was observed as the increase of heating

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time. In contrast, the effect of heating time on particle size was negligible when the heating temperature

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was 65 °C. This observation revealed that optimal combination of heating time and temperature is

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required to achieve high conjugation degree while maintaining original particle size. Our results were

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corroborated with previous literature that the reaction conditions (e.g. temperature, incubation times)

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could strongly influence the properties of final product produced by crosslinking between aldehydes and

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proteins.18-19 Notably, aggregation of SLPN (particle size > 1000 nm) was observed if being heated at

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85 °C for extended time, i.e. 60 and 120 min. Such precipitation of SLPN may be attributed to the loss of

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emulsification capability of BSA due to excessive conjugation and thermal denaturation under these two

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conditions.20 The effects of heating temperature and time on PDI of SLPN are shown in Fig. 4B1 and 4B2.

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Generally, the PDI ranged from 0.11 to 0.23 under various preparation conditions, suggesting that under



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studied preparation conditions all SLPN samples had a narrow distribution of particle size. The Pareto

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chart in Fig. 3B indicated that heating temperature, duration, and their interaction had remarkable

259

influence on the PDI values, while such influence was not noted until the temperature reached 75 °C. In

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particular, heating temperature played a predominant role and the lowest PDI was observed when SLPN

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was heated at 85 ºC for 30 min. Nevertheless, if SLPN were heated at 85 ºC for more than 30 min, the

262

theoretical PDI would have soared from 0.15 to 0.5-0.7, based on contour plot (Fig. 4B2). Smaller PDI of

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SLPN could be ascribed to the formation of more homogeneous Ox-Dex coating as the degree of

264

conjugation increased. Since the Schiff-base formation is a kinetic and thermodynamic process, Ox-Dex

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did not adsorb onto and conjugate with BSA layer at the initial stage of heating. But as the reaction

266

proceeded, more Ox-Dex conjugated with BSA and less free Ox-Dex remained in the aqueous phase, and

267

hence a more ordered structure was formed, resulting in the formation of uniform coating layer and thus

268

the SLPN with narrow size distribution. As shown in Fig. 4C1 and 4C2, the count rate varied from 295

269

to 475 kcps with different levels of variation. The count rate gradually and significantly increased as the

270

increase of both heating temperature and time. The significant augment in count rate evidenced that more

271

nanoparticles were formed during preparation when higher temperature and longer heating time were

272

involved. Nevertheless, count rate was significantly reduced when SLPN was heated at 85 ºC for 60 min

273

or longer, which could be due to the precipitation.

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3.3. Gastrointestinal stability

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GI-stability is an important parameter for measuring the capability of SLPN as a potential oral

276

delivery system. The stability of SLPN in GI tract was evaluated by incubating SLPN samples in either

277

simulated gastric condition (pH 2) with pepsin for 2 h or intestinal condition (pH 7) with pancreatin for 4

278

h. Generally speaking, SLPN prepared with higher temperature and longer heating time exhibited better

279

stability under both SGF and SIF conditions (Fig. 5). Comparing the three SLPN prepared under 65 ºC,

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their stability in gastric phase was more appreciably affected by heating time, while such heating time-

281

dependent effect was not noted in intestinal phase. However, once the heating temperature raised to 75 ºC,


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the GI-stability was greatly improved regardless of heating time. Apparently, with negligible changes in

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particle size and maintaining smallest PDI throughout GI incubation, the SLPN prepared with heating at

284

85 ºC for 30 min had the optimal GI stability among all samples.

285

To investigate whether heating time could be shortened when heated at high temperature, SLPN were

286

prepared by heating at 85 ºC for 10 and 20 min. The initial particulate characteristics and their GI-stability

287

data are shown in Fig. 6. Although shorter heating time was favorable to reduce particle size, these

288

samples had significantly greater PDI values under SGF phase, compared with the one prepared with 30

289

min heating. It turned out that 30 min heating time was necessary to induce complete conjugation

290

between BSA and Ox-Dex, otherwise the prepared SLPN had poor GI-stability. This confirmed that

291

heating at 85 ºC for 30 min was the optimal condition to prepare GI-stable SLPN, and thus this SLPN was

292

selected in following studies.

293

3.4. Morphological observation

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The morphology of freshly liquid SLPN sample (heating at 85 °C for 30 min) was observed using

295

TEM (Fig. 7A). The SLPN had spherical shape and narrow size distribution ranging from 120 – 150 nm.

296

Due to high vacuum condition in TEM and hydrodynamic and electrokinetic effects in DLS.21-22 the

297

observed particle size was smaller than the size measured by DLS. Fig. 7B illustrates a SEM image for

298

the spray-dried SLPN sample obtained by nano spray drying technology. These powders showed

299

spherical particles with a wide distribution of size ranging from 500 nm to 1.5 µm. The increased particle

300

size may be attributed to agglomeration of multiple nanoparticles during untrasonic vibration and

301

compression process of the mesh during spray drying.3, 9 According to previous studies, powders with

302

significant aggregation could be observed when spray-drying lipid nanoparticles without sufficient

303

protection such as addition of spacers or coating.9, 23 Thus, the conjugation procedure between BSA and

304

Ox-Dex prevented solid lipid core from severe aggregation and helped SLPN to from spherical, distinct

305

and separated particles during spray drying.

306

3.5. Encapsulation and delivery potential of astaxanthin



307

It is well known that ASTN has a very poor solubility in water, but it is soluble in organic solvents

308

such as acetone, chloroform, and DMSO. Thurs, in current study, ASTN was encapsulated into SLPN

309

with assistance of acetone. The ASTN-loaded SLPN (A-SLPN) were successfully prepared with 70%

310

encapsulation efficiency, which equaled to 0.14 mg of ASTN was encapsulated into the core of SLPN.

311

The characteristics of ASTN-loaded SLPN are shown in Fig. 8A. Compare to empty SLPN vehicle, the

312

particle size and PDI slightly increased to 197 nm and 0.119 from 167 nm and 0.111, respectively.

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Encapsulation of ASTN did not alter the GI-stability of SLPN, as no significant changes were detected for

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particle size and PDI of ASTN-SLPN during incubation under simulated digestive conditions. As

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indicated in Fig. 8B, the morphology of A-SLPN was consistent with original SLPN and the particle size

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estimated from TEM image well matched with DLS measurement.

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Potent antioxidant activity is one of the health-promoting properties that distinguishes ASTN from

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other carotenoids.24 Nevertheless, due to its poor water solubility, its antioxidant activity is specifically

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limited to the lipid oxidation and so its health benefits may be limited in aqueous phase. Encapsulation of

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lipophilic bioactives into nanoscale vehicles that can disperse well in aqueous condition has been

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demonstrated as a promising strategy to tackle the challenge of water solubility/dispersibility. The

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comparison of antioxidant activity between free ASTN and encapsulated ASTN in SLPN (A-SLPN) is

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presented in Fig. 8C. It is apparent that at a wide range of concentrations studied (0.25 – 10 µg/mL), A-

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SLPN exhibited significantly higher antioxidant activity in the aqueous condition-based ABTS assay.

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Especially, A-SLPN had strong activity at very low concentration (0.25 µg/mL, while free ASTN began

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to have similarly notable activity at 10 µg/mL. Free ASTN cannot be well dissolved thus separated out in

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aqueous media, resulting in limited contact probability with free radicals. However, SLPN with a

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hydrophobic lipid core and hydrophilic coating not only provided non-polar microenvironment for the

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encapsulated ASTN but also improved its dispersion in aqueous condition to scavenge the hydrophilic

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free radicals, such as ABTS.8

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The in vitro release profile of ASTN from A-SLPN was evaluated in SGF (pH 2, 2 h) and SIF (pH 7,

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4 h) consecutively (Fig. 8D). The free ASTN control group exhibited a triphasic diffusion pattern, with an


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initial burst diffusion of about 50% within 1 h and then a slower rate for remaining 1 h in SGF, followed

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by a slightly faster and constant diffusion rate in SIF. In contrast, the A-SLPN showed a similar triphasic

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pattern but with a significantly slower rate, with only about 40 and 55% of ASTN detected in release

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medium after SGF and SIF stage, respectively. The data for ASTN release from A-SLPN fitted well into

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the Higuchi model (R2 = 0.9434, y = -0.9795 + 3.3353x), which indicated that the release of ASTN

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followed a matrix diffusion-based kinetic.

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4. Conclusion

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In this study, Ox-Dex was first prepared by oxidizing native dextran to expose more aldehyde groups.

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Then, the prepared Ox-Dex, together with BSA, were used to stabilize Precirol® ATO 5 (glyceryl

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distearate) to produce SLPN through an in-situ conjugation technique. The formulations and preparation

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parameters during preparation procedure including molecular weight of native dextran and conjugation

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temperature and time were comprehensively optimized. High conjugation temperature (85 °C) and short

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incubation time (30 min) were found to be critical in producing small, homogenous, and GI stable SLPN.

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The optimized SLPN exhibited significantly improved GI stability due the strong covalent bond between

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aldehyde group of Ox-Dex and amino group of BSA. The optimized SLPN were later used to encapsulate

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ASTN and they were able to offer a good capacity for ASTN with 70% encapsulation efficiency.

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Encapsulation of ASTN in SLPN showed sustained release kinetics in simulated GI fluid compared with

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free ASTN. Our study demonstrated that the SLPN coated with covalently bonded Ox-Dex-BSA layer

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hold promising potential as an oral delivery system for lipophilic nutrients.

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Conflicts of interest The authors have declared no conflict of interest.

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Acknowledgement



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This work was supported by the USDA National Institute of Food and Agriculture grant

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(Award No. 2017-67018-26478). The TEM study was performed at the Biosciences Electron

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Microscopy Facility of the University of Connecticut UConn). The SEM study was performed

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using the facilities in the UConn/FEI Center for Advanced Microscopy and Materials Analysis

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(CAMMA).

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20. Borzova, V. A.; Markossian, K. A.; Chebotareva, N. A.; Kleymenov, S. Y.; Poliansky, N. B.; Muranov, K. O.; Stein-Margolina, V. A.; Shubin, V. V.; Markov, D. I.; Kurganov, B. I., Kinetics of thermal denaturation and aggregation of bovine serum albumin. PLoS One 2016, 11 (4), e0153495. 21. Wu, X.; Van de Ven, T., Characterization of hairy latex particles with colloidal particle scattering. Langmuir 1996, 12 (16), 3859-3865. 22. Min, G. K.; Bevan, M. A.; Prieve, D. C.; Patterson, G. D., Light scattering characterization of polystyrene latex with and without adsorbed polymer. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2002, 202 (1), 9-21. 23. Freitas, C.; Müller, R. H., Spray-drying of solid lipid nanoparticles (SLNTM). European Journal of Pharmaceutics and Biopharmaceutics 1998, 46 (2), 145-151.

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Fig. 1. (A) the corresponding reaction of preparing oxidized dextran; (B) FTIR spectra of native dextran and oxidized dextran; (C) 1H NMR spectra of native dextran and oxidized dextran

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Fig. 2. In-situ conjugation process and formation of SLPN

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Fig. 3. Pareto chart of the standardized effects on different responses: (A) particle size, (B) PDI and (C) count rate.

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Fig. 4. Particle size (A1), PDI (B1), and count rate (C1) of SLPN (40 kDa dextran used); Contour plot illustrating the effect of significant factors (time and temperature) to particle size (A2), PDI (B2), and count rate (C2). Under the same heating temperature, data not sharing the same upper letter were significantly different.

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Fig. 5. Stability of SLPN in simulated gastric (A) and intestinal (B) conditions. The “*” indicates the statistical difference compared to the original value of particle size (Fig. 4A1) and PDI (Fig. 4B1) for the same sample.

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Fig. 6. Particle size (A), PDI (B) and count rate (C) of SLPN (40 kDa dextran used) prepared under heating at 85 °C for different durations, as well as their respective stability in simulated gastric (SGF) and intestinal (SIF) fluids. In (A) and (B), the statistical differences between samples before (original) and after incubation in SGF or SIF were indicated by “*”. While in (C), data not sharing the same upper letter were significantly different.

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Fig. 7. TEM (A) of freshly prepared SLPN sample. SEM (B) of spray-dried SLPN sample.

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Fig. 8. (A) Particle size, PDI, and encapsulation efficiency (EE) of astaxanthin (ASTA)-loaded SLPN; (B) TEM image of ASTA-loaded SLPN; (C) The ABTS radical scavenging activity of free and encapsulated ASTN. (D) In vitro release profile of ASTA-loaded SLPN. In (C), at each concentration of ASTN, data not sharing the same upper letter were significantly different..

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Graphical Abstract

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Solid lipid-polymer hybrid nanoparticles prepared with food-derived biomaterials via an in-situ conjugation process are a promising oral delivery vehicle for astaxanthin.

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