Enzymatically partial hydrolyzed Î±-lactalbumin peptides for self

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Bioactive Constituents, Metabolites, and Functions

Enzymatically partial hydrolyzed #-lactalbumin peptides for self-assembled micelles formation and their application for co-encapsulation of multiple anti-oxidants Ping Jiang, Jing Huang, Cheng Bao, Lulu Jiao, Huiying Zhao, Yizheng Du, Fazheng Ren, and Yuan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03798 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Journal of Agricultural and Food Chemistry

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Enzymatically partial hydrolyzed α-lactalbumin peptides for self-assembled micelles formation and their application for co-encapsulation of multiple anti-oxidants

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Ping Jiang1,2,#, Jing Huang1,# , Cheng Bao1, Lulu Jiao1, Huiying Zhao2,Yizheng Du1, Ren

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Fazheng1, Yuan Li 1,*

1 2 3

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1

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of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural

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University, 100083, China;

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Key Laboratory

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2

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100029, Beijing, China;

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#

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*Corresponding author: [email protected]; Fax: +86-10-62736344

College of Life Science and Technology, Beijing University of Chemical Technology,

All authors contributed equally

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ACS Paragon Plus Environment


ABSTRACT

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The co-delivery system for multiple anti-oxidants such as anthocyanins (Ant) and curcumin

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(Cur) of synergistic action may effectively enhance their stability and cellular absorption. We

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have reported that amphiphilic peptides obtained from enzymatic partial hydrolysis of α-

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lactalbumin (α-lac) can self-assemble into 20 nm monodispersed nanomicelles in aqueous

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solution. Cur and Ant could be co-loaded into the micelles sequentially via hydrophobic and

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electrostatic interactions, which are proved by fluorescence quenching experiment for the Cur-

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micelle and Ant-micelle interactions. Circular dichroism spectra proved that the Cur and Ant

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binding did not affect their structure confirmation. Both Cur and Ant loaded micelles showed

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improved stability and also exhibited an intestinal pH-responsive release property in simulated

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gastrointestinal fluid. Besides, the nanomicelles exhibited an advanced cellular uptake and

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transmembrane permeability based on Caco-2 cell monolayer models. Finally, the co-loaded

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micelles possessed a synergistic efficiency that cellular anti-oxidant activity (CAA) for Cur

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and Ant was markedly improved.

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Key words: α-lactalbumin micelles; delivery; curcumin; anthocyanins; bioavailability

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INTRODUCTION

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Currently, nanocarriers, such as nanoparticles, micelles, vesicles and liposomes, belong to

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nanoscaled delivery systems. They are usually made of synthetic or natural polymers and are

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used to transport valuable ingredients such as bioactive compounds or drugs to specific sites1.

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Nanocarriers, as targeted delivery systems, have been extensively studied for the encapsulation

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of anti-cancer drugs2, bioactive ingredients3, pathogen detection agents4 and disease

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theragnostic agents5 etc. In recent decades, there is an increasing demand for delivering food-

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sourced bioactive compounds due to the unpleasant flavor, poor water solubility and chemical

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instability which cause poor stability and bioavailability6,7. Nanocarrier is reported to be a

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potential strategy that enables a monodispersed size distribution with controlled release kinetics,

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enhances mucus barrier permeability and intestinal epithelial cellular uptake8. Various

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nanocarriers are fabricated from generally recognized as safe (GRAS) food-grade natural

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polymers, including oxidized starch nanoparticles9, nanoassemblies of hydroxyethyl cellulose

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polymer micelles10, sodium caseinate-stabilized oil-in-water nanoemulsions11 and gum arabic

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aldehyde-gelatin nanogels12. Nanocarriers of GRAS biopolymers present unique advantages

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for delivering functional ingredients in complex food systems13. Firstly, the food-sourced

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natural biopolymers such as polysaccharides and proteins that are used for preparing

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nanocarriers have excellent biocompatibility and low toxicity compared with synthetic

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polymers14. Secondly, nanoscaled carriers possess improved dispersibility and colloidal

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stability, which are important for maintaining the stability and acceptable quality of food

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systems15. In addition, the aqueous solubility of hydrophobic ingredients can be greatly

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enhanced by incorporating hydrophobic ingredients into the hydrophobic core of amphiphilic



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nanocarriers16. Moreover, nanocarriers can overcome the mucus barrier and improve

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transmembrane permeability and bioavailability of bioactive ingredients8. Besides,

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nanocarriers conjugated with targeting ligands can arrive at specific sites under controlled

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release manner17. An efficient and enhanced absorption can be achieved via smart nanocarriers

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with multiple functions mentioned above.

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Recently, plant-derived bioactive compounds, such as unsaturated olefin compounds,

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flavonoids, polyphenols, bioactive polysaccharides and other phytochemicals, have garnered

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much attention due to their strong anti-oxidation, anti-inflammation, anti-cancer and immune-

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regulating activity18. Cur has obviously anti-inflammation and anti-tumor properties19, and Ant

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present effective anti-oxidants and anti-inflammatory activity20. Although anthocyanins are

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water soluble, they are sensitive to various environmental stimuli, such as pH, light, oxygen

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and high temperatures21. The encapsulation of sensitive bioactive compounds has been

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considered as an effective approach to improve their stability, aqueous solubility and

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bioavailability22. Nanocarriers formed by self-assembly have attracted great attention in

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targeted drug delivery and nutraceutical transportation in recent years. For instance,

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nanocapsules prepared by self-assembled whey protein nanoparticles can substantially improve

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the solubility, stability and transmembrane permeability of β-carotene23. The aqueous solubility

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of Cur can be improved by incorporating into the core of polymeric micelles through self-

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assembly via

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bioavailability24. Li et al. reported that the encapsulated Cur inside the tumor-targeting peptide

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conjugated micelles can respond to the tumor microenvironment. The results demonstrated that

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Cur can be specifically delivered to tumor sites with a high therapeutic efficacy and low side

hydrophobic

interactions,

while

simultaneously improved


its

oral

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effects25. Bile-fatty acid mixed micelles encapsulation has accelerated the permeation of Cur

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into Caco-2 cell monolayers26. The clinic trial showed that the silicon dioxide encapsulated Cur

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has a high oral bioavailability and safety on human27. Oxidized starch microgel has been used

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to encapsulate Ant, offering controlled uptake and release properties at different pH values and

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salt concentrations. The stability of Ant against pH and light is enhanced via encapsulation28.

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Various studies indicated that compared with single compound, the combination of several

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bioactive compounds can better facilitate synergistic action29. For instance, co-encapsulation of

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the multiple anti-oxidants gallic acid/Cur or ascorbic acid/quercetin multiple anti-oxidants in

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Tween 60 niosome vesicles present synergic capability on reducing free radicals30.

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Encapsulation of Cur and catechin by W/O/W double emulsion, in which Cur loaded in the oil

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phase and catechin in the water phase, resulted in improved stability and bioaccessibility31.

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However, both Tween 60 niosome vesicles and double emulsions are microscaled and have

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poor physicochemical stability, which are easily susceptible to disruption and phase migration.

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Therefore, novel nanoscale carrier systems need to be developed to further improve the

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synergistic activity for the combination delivery of multiple anti-oxidants.

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We have discovered that amphiphilic peptides could be produced after enzymatic partial

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hydrolysis of α-lactalbumin. The amphiphilic peptides can self-assemble into nanomicelles in

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aqueous conditions25. α-Lac accounts for 20 % of the whey protein in milk which is considered

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to be GRAS biopolymers, and it was reported to have a strong anti-tumor activity32. The α-lac

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micelles have high encapsulation ability for hydrophobic ingredients. Cur can be loaded into

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the core of the α-lac micelles via a hydrophobic interaction, and the positively charged Ant can

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be absorbed onto the negatively charged α-lac micelles. In this way, both Cur and Ant can be



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co-loaded into the α-lac micelles with a high loading efficiency. In this study, Ant and Cur were

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selected as model hydrophilic and hydrophobic antioxidants. The synergistic efficiency of co-

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delivery system of α-lac micelles for delivering multiple antioxidants was investigated.

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MATERIALS AND METHODS

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Materials and Cell Culture. α-Lactalbumin (≥85 %), Tris (hydroxymethyl) aminomethane

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(≥99 %), 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA), Curcumin (≥99 %) and

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2,2-azobis (2-amidinopropane) dihydrochloride (ABAP) were supplied by Sigma-Aldrich,

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USA. Bacillus licheniformis protease (BLP) was a kind gift from Novozymes, Denmark.

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Blueberry Anthocyanins was purchased from Jinghuakewei company (Beijing, China).

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Gibco® Minimum Essential Media, Gibco® fetal bovine serum (FBS), 0.05 % (w/v) trypsin-

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EDTA, penicillin and streptomycin (P/S) were purchased from Gibco (Gibco, NY, USA). Alexa

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Fluor™ 488 Phalloidin was obtained from Life technologies (CA, USA). Lipid soluble

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Cyanine 5 (Cy5), water soluble Cy5, Hoechst 33342 were purchased from Fanbo

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Biomachemical company (Beijing, China). Acetonitrile and formic acid were purchased from

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Fisher Scientific (GA, USA). Quercetin (≥97 %) was purchased from J&K company (CA,

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USA).Caco-2 cells were purchased from the Cell Resource Center (Peking Union Medical

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College Headquarters of National Infrastructure of Cell Line Resource, NSTT) and cultured in

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MEM media supplemented with 10 % FBS and 1 % P/S at 5 % CO2, 37 ℃. All other chemicals

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used were of analytical grade.

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Preparation of α-Lactalbumin Micelles and Cur, Ant, Cy5 Loaded Micelles. α-

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Lactalbumin (1 g/L) and BLP (BLP to α-lactalbumin weight ratio, 1:70) were dissolved in

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75 mM pH 7.5 Tris-HCl buffer. The reaction mixture was then passed through the 0.22 μm


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filter and heated at 50 °C for 30 min, allowing the partial hydrolysis of the α-Lactalbumin.

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The amphiphilic peptides were obtained after hydrolysis. Then 1 g/L amphiphilic peptides

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were self-assembled into micelles with 5 mins gentle rotation on the vertical mixer. Cur (1

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g/L) dissolved in ethanol was added in the micelles reaction mixture with a volume ratio

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1:6; Hydrophobic Cy5 (1 mg/mL) and micelles (1 g/L) in Tris buffer were mixed with a

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volume ratio 1:500. Then the mixtures were mildly rotated on the vertical mixer for 30 mins

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allowing Cur or Cy5 incorporation into the micelles core respectively during the self-

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assembly. Unabsorbed Cur or Cy5 was removed by dialysis. Later the positively charged

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Ant can be absorbed on Cur loaded micelles via electrostatic interaction at pH 6.

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Unabsorbed Ant were removed by dialysis. Then the dual components loaded micelles

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powder would be obtained under the freeze-drying process.

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Micelles Morphology Characterization by Transmission Electron Microscopy. α-Lac

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micelles were dispersed in ultrapure water (1 g/L) and one drop of the diluted dispersion was

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placed on a 200-mesh carbon copper grid, then they were stained with uranyl acetate. Surface

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morphology of micelles, Cur loaded micelles, Cur & Ant co-loaded micelles were observed

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under transmission electron microscopy (TEM) (JEM-1400, Japan), operating at 200 kV.

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Size Distribution and Zeta-potential of Micelles Determination. The average size and

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zeta-potential of micelles, Cur loaded micelles, Cur & Ant co-loaded micelles were determined

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by dynamic light scattering (Nano-ZS 2000, Malvern Instruments, UK) respectively. After

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appropriate dilution, the measurements were carried out at 25 °C. The average sizes and zeta-

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potentials of three independent measurements were gained respectively.

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Embedded Amount of Ant or Cur Inside Micelles. Cur was extracted from the α-lac



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micelles by methanol. First, the calibration curve of Cur in methanol was determined at 442

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nm excitation wavelength via fluorescence spectroscopy. Then 1 mg Cur loaded micelles was

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dissolved in 1 mL methanol and under the ultra-sonification for 30 s, the encapsulated Cur was

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extracted from the micelles. The extracted Cur-methanol solution was diluted 1,000 times and

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measured the fluorescence intensity at 442 nm wavelength. The amount of Cur embedded in

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the micelles, expressed in Cur mg/g dry micelles can be calculated according to the calibration

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curve.

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The Ant content was measured by a modified pH differential method refer to our previously

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study27 by UV spectroscopy. In brief, the Ant were dissolved in pH 6 HCl solution. The spectral

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absorbance was measured at 520 and 700 nm, respectively. A standard curve was made by

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plotting the absorbance (A) difference of A (520 nm) - A (700 nm) as a function of Ant concentration

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(mg/mL). The spectral absorbance of Ant embedded in the micelles was measured and

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calculated according to the calibration curve and expressed in Ant mg/g dry micelles can be

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calculated.

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Secondary Structure Characterized by Circular Dichroism (CD) Spectroscopy. Circular

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dichroism (CD) spectroscopy is applied for studying the secondary structural elements of α-

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lac. CD spectroscopy was measured by Jasco J-1500 spectrophotometer (Tokyo, Japan), the

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wavelength ranges from 185 to 260 nm with the interval of 0.5 nm, the scanning speed is 1000

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nm/min. As for this method, the concentration of micelles need to be kept in the range of 0.1

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to 0.5 mg/mL. Percentage of α-helix, β-sheet, and random coil were calculated using the

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Spectra Analysis software refer to the instruments.

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Protein-compounds Interaction Characterized by Fluorescence Spectroscopy.


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Fluorescence spectroscopy could demonstrate the binding interaction of Cur or Ant with

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micelles. The fluorescence spectra were measured by steady-state fluorescence measurements

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of PerkinElmer (MA, USA). We measured the fluorescence of micelles with a constant protein

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concentration of 1 mg/mL were encapsulated with different concentrations of Cur or Ant (0,

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0.02, 0.03, 0.05, 0.08, 0.1, 0.2 mg/mL). The emission spectra were recorded from 300 to 450

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nm with an excitation wavelength of 280 nm with the step of 3 nm. In this case, equivalent

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concentrations of free Cur or Ant were used as controls. The fluorescence quenching for protein

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binding sites and Cur or Ant incorporated inside the micellar core were observed respectively.

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In Vitro Release Kinetics of Ant and Cur from Micelles under Gastrointestinal pH

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Value. In vitro release of Cur and Ant from the micelles was performed in 10 mL PBS buffer

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of pH 2 mimicking the stomach pH value and pH 7 mimicking the intestinal pH condition. 10

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mg Ant or Cur loaded micelles powder were dissolved in 1mL ultrapure water respectively in

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centrifuge tube. The tube with the opening of the tube with semipermeable membrane and

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immersed in PBS buffer in a beaker upside down allowing the release. The released compounds

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were dialyzed out to the buffer over 30 h at 37 °C with continuous stirring. Then 200 μL buffer

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containing the released compounds at different time intervals (2, 4, 6, 8, 10,12, 14, 16, 18, 20,

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22, 24, 26, 28, 30 h) were measured by fluorescent spectroscopy or UV spectroscopy

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mentioned above. At the same time, 200 μL PBS buffer was re-added to the beaker every time

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to keep the volume unchanged.

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The Retention Rate of Co-loaded Micelles against Heat and UV Light. Free Ant, Cur

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and micelles loaded with equivalent amount of Ant and Cur were heated at 60 °C or under UV

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light of 254 nm at room temperature for 120 h and sampled at different time intervals (4, 8, 16,



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24, 48, 72, 96, 120 h). 2 mg micelles loaded with 0.18 mg Ant and 0.4 mg Cur were dissolved

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in pH 6 HCl solution. The amount of Ant and Cur would be degraded under heat and UV light

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stimuli. The remained amount for micelle-loaded and free formulations, before and after

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stimuli, were measured by fluorescence spectroscopy or UV spectroscopy mentioned above.

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The retention rate Aretent was calculated according to Equation (1). A0 is the absorbance /

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fluorescent intensity of initial sample before heating or lighting, At is the absorbance /

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fluorescent intensity of samples after heating or lighting for a certain time t.

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The retention rate Aretent can be calculated as: Aretent (% ) =

At  100% A0

(1)

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Qualitative Cellular Uptake of Co-loaded Micelles Measured by Confocal Laser

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Scanning Microscope. Caco-2 cells were seeded onto round glass plates put in 12-well culture

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plate at the density of 6×104 cells/cm2 , and then cultured for 3 days. Cells were rinsed with

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PBS for three times and equilibrated at 37 °C for 10 min. Because Cur and Ant have weak

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fluorescence, so the hydrophobic Cy5 and hydrophilic Cy5 were used to represent Cur and Ant

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for CLSM and flow cytometry determination. Cells incubated with free hydrophobic Cy5,

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hydrophilic Cy5 or micelles (500 µL, 200 µg/mL) encapsulated with equal amount of Cy5 at

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37 °C, 5 % CO2 for 2 h. Then cells were washed 3 times with PBS to remove the remaining

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micelles and Cy5. 200 µL PBS containing 2 µL Alexa Fluor™ 488 Phalloidin (1 mg/mL) was

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used to stain the cytomembrane of cells for 30 min. 200 µL Hoechst 33342 (4’6-diamidino-2-

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phenylindole) was used to stain the cell nucleus for 15 min. In the end, all the samples were

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washed by PBS, the confocal laser scanning microscope (CLSM) images were captured by

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exciting the Alexa Fluor™ 488 Phalloidin at 488 nm, Hoechst 33342 at 405 nm, Cy5 at 651


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nm, and emitted at 520 nm, 488 nm and 670 nm respectively.

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Quantitative Cellular Uptake of Co-loaded Micelles Measured by Flow Cytometer. The

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flow cytometer was used to quantify the number of free Cy5 and Cy5 loaded micelles uptake

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by Caco-2. Cells were seeded onto 6-well culture plate at the density of 1.0×105 cells/cm2, and

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cultured for 2 days. Cells were rinsed with PBS for three times, and equilibrated at 37 °C for

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10 min. Then cells were incubated with free hydrophobic Cy5, hydrophilic Cy5 or micelles

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(500 µL, 200 µg/mL) encapsulated with equal amount of Cy5 at 37 °C, 5 % CO2 for 2 h. Then,

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the cells were washed 3 times with PBS to remove the remaining micelles and Cy5. Then cells

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were collected in 200 µL PBS and measured under flow cytometer (FC500, Beckman) to

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quantify the Caco-2 uptake according to fluorescent intensity of each sample.

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The quantification of endocytosed Cur and Ant by HPLC. The quantification of

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endocytosed Cur and Ant in Caco-2 cells was detected by HPLC. The test conditions for

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analyzing endocytosed Cur and Ant were carried out according to Zhou et al. or Li et al.

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researches literatures31,32. In brief, Caco-2 cells (2×105 cells/well) were seeded in 6-well plates

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and treated with different free Cur or Ant formulations or micelles encapsulated with equal

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amount of Cur or Ant for 2 h. The medium was collected for measuring the unabsorbed amount

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of Cur or Ant by HPLC. In consideration of metabolism of Cur or Ant in cells, the endocytosis

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of Cur or Ant were calculated by total amount minus extracellular amount. Cur was extracted

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from the cell medium by acetonitrile, while Ant was extracted by acetonitrile with 5 % formic

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acid. Cur with different concentration dissolved in acetonitrile and Ant with different

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concentrations dissolved in acetonitrile with 5 % formic acid were prepared for standard

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calibration curve. Each samples (10 μL) were analyzed by HPLC system (LC-20AT, Shimadzu,



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Japan) fitted with a diode array detector (DAD). Cur detection was performed at 425 nm

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wavelength and anthocyanins detection was performed at 530 nm with a Diamonsil C18

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reversed phase column (5 μm, 4.6 mm×250 mm, Dikam Tech, CA, USA) at 30 °C. The flow

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rate was maintained at 1.0 mL/min. For Cur, elution solvents were A (water) and B

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(acetonitrile), maintaining 55 % A and 45 % B, for anthocyanins were A (1 % formic acid in

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water) and B (acetonitrile) with a gradient elution. Three replications were performed of each

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treatment.

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Transmembrane Permeability of α-Lac Micelles based on Caco-2 Cell Monolayer

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Model. Caco-2 cells were cultured on a polycarbonate filter (pore size of 0.4 µm, growth area

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0.33 cm2) in Costar Transwell 12 wells (Corning Costar Corp., NY) and were used for

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permeability experiments about 21 days for differentiating to monolayers (TEER values in the

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range of 600–800 Ω*cm2 and the permeation rate of lucifer yellow, a paracellular transport

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marker,

Enzymatically partial hydrolyzed Î±-lactalbumin peptides for self

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