RELEASE, STABILITY AND FUNCTIONALITY OF NANODELIVERED HYDROPHOBIC AND HYDROPHILIC ANTIOXIDANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1008750
• Project Start Date: Dec 23, 2015
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
Biological & Agricultural Engineering

Non Technical Summary
Engineered nanoparticles (NPs) exhibit great potential as smart delivery systems in foods and agriculture. It is the goal of this proposal to determine if NPs made from zein are able to protect the stability of hydrophobic and hydrophilic antioxidants, to control their release under GI conditions, and to enhance their antioxidant activity. Two antioxidants have been selected, lutein and folic acid, of great interest for human health. Lutein is known to prevent macular degeneration, while folic acid is associated with cell production and maintenance especially during infancy and pregnancy. If our results confirm that the functionality of the bioactives can be improved by nanoentrapment, new applications of zein nanoparticles loaded with antioxidants can be pursued by the functional foods industry.

Animal Health Component

90%

Research Effort Categories

Basic

10%

Applied

90%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
502	1510	2020	100%

Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1510 - Corn;

Field Of Science
2020 - Engineering;

Keywords

zein

nanoparticles

lutein

folic acid

release stability

antioxidant activity

Goals / Objectives
The goal of the proposed research is to assess the effect of nano-entrapment on the chemical stability, release kinetics and functionality of entrapped and chemically attached hydrophobic and hydrophilic antioxidants. The hypotheses of the study are 1. Nano-entrapment of antioxidants will improve their functionality by enhancing their stability, 2. Nanoparticles with surfactants will provide the controlled release mechanisms associated with improved functionality of the bioactive, 3. Covalent linking of hydrophilic antioxidant will provide the means to improve its loading and to sustain the bioactive release, 4. Hydrophobic antioxidants will be entrapped at higher entrapment efficiencies than hydrophilic bioactives and will release slower when physically entrapped in the nanoparticle. The knowledge generated in this study will aid in adoption and safe application of antioxidant loaded nanoparrticles in functional foods with the goal of improving human health.

Project Methods
The methodology is described under each specific objective, as follows:Objective 1: Synthesize and characterize zein (ZN) nanoparticles to deliver hydrophobic (lutein, LT) and hydrophilic (folic acid, FA) antioxidants Four different types of nanoparticles will be synthesized based on their ability to deliver hydrophobic and hydrophilic antioxidants. LT-loaded ZN nanoparticles will be synthesized with and without surfactants using liquid-liquid phase separation method. ZN nanoparticles will be synthesized with physically entrapped or covalently linked FA. FA-loaded ZN nanoparticles will be prepared by manipulating the pH for electrostatic binding of FA to the protein. FA-linked ZN nanoparticles will be made by covalently linking FA to ZN using carbodiimide chemistry conjugation prior to nanoparticle formation by nanoprecipitation. Average particle size, surface characteristics, morphology, entrapment efficiency, loading capacity of freshly made, loaded nanoparticles will be determined prior to proceeding to stability, release, and functionality studies.Objective 2: Elucidate the release mechanism of the entrapped antioxidants from nanoparticles in PBS as control and simulated GI tract environmentRelease kinetics of the entrapped antioxidants will be performed in phosphate-buffered saline (PBS), pH 7.4, at 37°C as control. The effect of simulated gastrointestinal (GI) tract environment at pH 1.2 (stomach) and 6.8 (small intestine) at 37°C will be performed to elucidate the release profiles of the antioxidants under GI environment.Objective 3: Study physical stability of nanoparticlesPhysical stability of nanoparticles in terms of average particle size, surface charge, and morphology will be tested in parallel with release testing under GI conditions. In addition, lyophilized nanoparticles will be stored in the dark in desiccators at 25°C and 4°C over a period of three months and monitored for changes in average particle size, surface characteristic, morphology, and entrapment efficiency.Objective 4: Study the effect of nanodelivery system on the chemical stability of the entrapped antioxidantsThe effect of nanodelivery systems on the chemical stability of antioxidant compounds will be tested to investigate the stability of lutein and folic acid under release, storage, and processing conditions. Chemical stability of the entrapped antioxidant will be assessed under GI conditions, and during storage at 4°C and 25°C. To test stability during processing conditions, photo and thermal stabilities of entrapped antioxidants will be performed as a function of time and compared with the free antioxidant at the same concentration. The photo stability test will be performed in a lightproof cabinet where particles will be exposed to two 352nm UV light bulbs (15W) to imitate natural light for 10 h. The thermal stability test will be performed in water bath at 40°C for 4 days. The entrapped antioxidants will be analyzed by standard chemical methods.Objective 5: Study the effect of nanodelivery system on the functionality of the entrapped antioxidants in terms of antioxidant activity (AOA)The effect of nanodelivery systems on the functionality of the antioxidants will be determined by measuring the antioxidant activity (AOA) of the entrapped antioxidants in comparison with pure antioxidants based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method, using UV-vis spectroscopy by TBARS (thiobarbituric acid assay), and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays.

Progress 12/23/15 to 09/30/19

Outputs
Target Audience:Research community, students, industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergraduate student, a graduate student and a visiting scientistwere trained on nanoparticle synthesis and characterization and actively participated in this study. How have the results been disseminated to communities of interest?The data was published and presented at various national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?This is the final year of the project.

Impacts
What was accomplished under these goals? Surfactants are used to stabilize nanoemulsions by protecting their physical stability and preventing deterioration of the entrapped bioactive during processing and storage. The effect of surfactant concentration on physical-chemical properties of nanoemulsions with entrapped curcumin, relevant to commercial applications, was addressed in this research. Furthermore, the functionality of nanoemulsified curcumin in terms of lipid oxidation inhibition was determined. Protection against varying pH and thermal treatments was more significant in the nanoemulsions at the elevated surfactant level;but at these high concentrations, the surface charges of the emulsions dramatically decreased under sodium salt addition, which may result in instability over time. Nanoemulsions showed the potential to inhibit malondialdehyde (MDA) formulation by protecting the entrapped curcumin and enhancingits antioxidant activity when added to milk. The fortified milk with added curcumin systems had a yellow color compared to the control. The results of the study are critical in choosing the surfactant concentration needed to stabilize emulsified curcumin. And to protect the entrapped curcumin under specific conditions of use to support the utilization of curcumin nanoemulsions as a food additive in different commercial products.

Publications

• Type: Book Chapters Status: Published Year Published: 2019 Citation: Sadeghi, R., T. Chuacharoen, C. M. Sabliov, C. Moraru, M. Karimi, and J. Kokini. 2019. Chapter 2. Advances in Nanotechnology of Food Materials for Food and Non-Food Applications. In: Handbook of Food Engineering. Publisher: CRC Press Taylor & Francis Group. Editors: Heldman, Lund and Sabliov.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chuacharoen, T., and C. M. Sabliov. 2019. Comparative effects of curcumin when delivered in a nanoemulsion or nanoparticle form for food applications: study on stability and lipid oxidation inhibition. LWT. 113:1-9. https://doi.org/10.1016/j.lwt.2019.108319.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chuacharoen, T., S. Prasongsuk, and C. M. Sabliov. 2019. Effect of Surfactant Concentrations on Physicochemical Properties and Functionality of Curcumin Nanoemulsions Under Conditions Relevant to Commercial Utilization. Molecules. 24(15): 2744. DOI: 10.3390/molecules24152744.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Kacso, T., I. O. Neaga, A. Erincz, C. E. Astete, C. M. Sabliov, R. Oprean, E. Bodoki. November 2018. Zein-based polymeric delivery systems with programmed wear down for sustainable agricultural applications. SNO Annual Conference. Washington DC, USA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Sabliov, C. M. October 2018. Cytotoxicity, hemotoxicity, and in-vivo toxicity of surface modified PLGA nanoparticles. NNI QEEN II: 2nd Quantifying Exposure to Engineered Nanomaterials from Manufactured Products Workshop Session B. Consumer Exposure: Food, Food Contact, and Personal Care Products, Washington, DC.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Prasad, A., C. E. Astete, A. E. Bodoki, M. Windham, E. Bodoki, and C. M. Sabliov. October 2018. Zein nanoparticles uptake and translocation in hydroponically grown sugarcane plants. 19th World Congress of Food Science and Technology. IuFoST 2018. Mumbai, India.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergraduate student, a graduate student and two visiting scientists were trained on nanoparticle synthesis and characterization and actively participated in this study. How have the results been disseminated to communities of interest?The data was published and presented at various national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?Given their ability to adhere to roots for extended periods of time, ZNPs are proposed as effective delivery systems for agrochemicals to plants, but more studies are needed to identify effect of nanoparticle exposure to health of the plant, as well as environmental degradation of ZNPs.

Impacts
What was accomplished under these goals? Zein was tagged with fluorescein isothiocyanate (FITC) and made into nanoparticles (135 ± 3 nm diameter, 0.202 ± 0.034 PDI and 81 ± 4 mV zeta-potential at pH 6) using an emulsion-diffusion method. After 10 days of hydroponic exposure, association between particles and roots of soybean plants was found to vary based on bulk nanoparticle concentration. While 0.37 mg NP/mg dry weight were detected in roots immersed in 0.88 mg NP/mL nanoparticle suspension, 0.58 mg NP/mg dry weight associated with roots immersed in a high dose nanoparticle suspension of 1.75 mg NP/mL at 10 days. Fluorescent measurement following digestion of sugarcane plant tissue indicated that roots had a significant uptake of ZNPs (0.11 mg NPs/mg dry weigh of root when exposed to a low dose nanoparticle suspension of 0.88 mg NP/ml, and 0.34 mg NPs/mg of dry root in a high dose nanoparticle suspension of 1.75 mg NP/ml). Sugarcane leaves contained only 0.005 mg NP/mg dry weight at the low, and 0.013 mg NP/mg dry weight at the high dose exposer. Confocal microscopy studies confirmed presence of fluorescent ZNPs in the epidermis and endodermis of the root system. These results confirmed that ZNPs can be used as effective delivery systems not only of antioxidants, but also agrochemicals, given their ability to adhere to roots for extended periods of time and to translocate to some extent to the leaves. To the authors' knowledge, this is the first study in which the uptake and time-dependent association between organic nanoparticles and plants were quantified.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Prasad, A., C. E. Astete, A. E. Bodoki, M. Windham, E. Bodoki, and C. M. Sabliov. 2017. Zein Nanoparticles Uptake and Translocation in Hydroponically Grown Sugar Cane Plants. J Agric Food Chem. Jul 5;66(26):6544-6551. doi: 10.1021/acs.jafc.7b02487. Epub 2017 Aug 17.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Ristroph, K. D., C. M. Astete, E. Bodoki, and C. M. Sabliov. June 2017. Zein nanoparticles uptake by hydroponically grown soybean plants. ISFANS 2017. Jeju, Korea.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: K. D. Ristroph, C. M. Astete, E. Bodoki, and C. M. Sabliov. 2017. Zein nanoparticles uptake by hydroponically grown soybean plants. Journal of Environmental Science and Technology. 51(24):14065-14071. doi: 10.1021/acs.est.7b03923. Epub 2017 Dec 4.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Sabliov, C. M. and C. E. Astete. July 2017. Polymeric nanoparticles for antioxidant delivery. ICCE-25. Rome, Italy.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Academia, pharmaceuticaland food companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A visiting scientist from China was hosted in the lab for one year. How have the results been disseminated to communities of interest?The data was published and presented at various national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, other types of antioxidant delivery systems will be studied with the same goal of determining the effect of nanoentrappment and delivery on the efficacy of the antioxidant.

Impacts
What was accomplished under these goals? Zein nanoparticles covalently linked to folic acid were hypothesized to sustain the release of the folic acid in addition to targeting cancer cells overexpressing folate-binding receptors, whereas zein nanoparticles with physically entrapped folic acid would only be able to control the release of the bioactive without targeting of cancer cells. The two types of particles, folic acid-covalently linked zein nanoparticles (ZN-FA nps) and zein nanoparticles with entrapped folic acid (ZN(FA) nps) were synthesized and characterized. The covalent link between the carboxyl group of folic acid and the primary amino group of zein was confirmed by fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H NMR). The release studies of the folic acid preformed in phosphate-buffered saline (PBS) at 37°C for 7 days, concluded that the release of the loaded folic acid was sustained over 7 days for both systems. The cytotoxicity was investigated using a methyl thiazolyl tetrazolium (MTT) assay, and the results showed that zein nanoparticles were biocompatible to HeLa (an over-expressing folate receptor cell type) and A549 (a deficient folate receptor cell type) cells and both systems were able to diminish the adverse toxic effect of folic acid to cells. The increased uptake of ZN-FA nps relative to ZN(FA) nps supported the use of ZN-FA nps as targeting nanoagents to cells over-expressing folate receptors.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Navarro, S. N., S. Swetledge, T. Morgan, C. E. Astete, R. Stout, D. Coulon and C. M. Sabliov. 2016. Biodistribution of orally administered poly(lactic-co-glycolic) acid nanoparticles for 7 days followed by 21 day recovery in f344 rats. NanoImpact. 5: 1-5. (http://dx.doi.org/10.1016/j.impact.2016.12.002)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chuacharoen, T. and C. M. Sabliov. 2017. Zein nanoparticles with covalently linked and physically entrapped folic acid  a focus on entrapment, controlled release, cytotoxicity and cellular uptake. Journal of Nanoparticle Research. 19:81. (DOI: https://doi.org/10.1007/s11051-017-3763-4)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ye, F., C. E. Astete, C. M. Sabliov Entrapment and delivery of a-tocopherol by a self-assembled, alginate-conjugated prodrug nanostructure. 2017. Food Hydrocolloids 72 (2017) 62-72. http://dx.doi.org/10.1016/j.foodhyd.2017.05.032.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Ye, F. and C. M. Sabliov. 2016. Entrapment and delivery of ?-tocopherol by a self-assembled, alginic acid-conjugated, prodrug nanostructure for effective inhibition of lipid oxidation Conference of Food Engineering. Columbus, OH.

Progress 12/23/15 to 09/30/16

Outputs
Target Audience:Industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student who was trained on this project and is a professor in Thailand, her home country. How have the results been disseminated to communities of interest?The data has been published and presented at national conferences. What do you plan to do during the next reporting period to accomplish the goals?Zein nanoparticles as a novel targeting nanocarrier will be developed using folic acid as a targeting ligand. Zein will be covalently linked with folic acid through the reaction between the carboxyl group of folic acid and the primary amino group of zein. Covalent linking of zein to folic acid should improvethe hydrophilic loading ability of zein nanoparticles. The release of the covalently linked folic acid will be measuredand cytotoxicity of the particles assessed. The goal is to develop an efficient delivery system for hydrophilic antioxidant bioactives such as folic acid.

Impacts
What was accomplished under these goals? Zein nanoparticles loaded with 7.5% lutein stabilized by the combined lecithin and pluronic F127 surfactants were successfully synthesized using a solvent-free liquid-liquid dispersion method. The addition of surfactants increased particle size and improved polydispersity index. Zeta potential slightly changed, and entrapment efficiency increased significantly. In the presence of the surfactants, the burst release decreased and the release kinetic was subsequently sustained. Zein nanoparticles showed a great ability to protect lutein from degradation under various storage conditions as compared to the emulsified lutein. The preferred storage condition of lutein-loaded zein nanoparticles with surfactants was 4°C for 30 days. In addition, this complex formulation providedgood protection against UV light for 10 hours. Based on characteristics, release, and stability data, it was suggested that with the addition of surfactants to improve entrapment efficiency of hydrophobic bioactives and to protect lutein against chemical degradation, zein nanoparticles could provide better protection against bioactive losses during thermal and neutral conditions than other delivery systems such as emulsions. The addition of nanoentrapped β-carotene in milk is proposed as a valid method to improve stability and enhance antioxidant activity of β-carotene during GI transit. This study focused on two types of delivery systems, nanoparticles and nanoemulsions, and compared their ability to enhance the physicochemical stability and antioxidant activity of β-carotene in the presence of milk under simulated GI conditions. β-Carotene-loaded zein nanoparticles and nanoemulsion, stabilized by pluronic F127 and lecithin, were synthesized and added to liquid milk. Degradation of β-carotene was spectrophotometrically analyzed, and antioxidant activity was measured using ABTS and TBARS assays in simulated gastric fluid (SGF) with pepsin for 2 h and in simulated intestinal fluid (SIF) with pancreatin for 24 h. The initial amount of β-carotene remained at 18.30±1.29% and 4.41±2.51% after a 26 h total time of GI exposure in nanoentrapped and emulsified forms, respectively. In both systems, nanoentrapment proved to be beneficial to beta-carotene stability, but this benefit was decreased in the presence of milk. The improvement in antioxidant activity of nanodelivered β-carotene was confirmed by ABTS and TBARS assays. In conclusion, zein nanoparticles improved chemical stability and antioxidant activity of entrapped beta-carotene versus emulsions in the presence of milk under simulated gastrointestinal environments.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chuacharoen, T. and C. M. Sabliov. 2016. Stability and Controlled Release of Lutein Loaded in Zein Nanoparticles with and without Lecithin and Pluronic F127 Surfactants. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 03: 1118.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Chuacharoen, T. and C. M. Sabliov. 2016. The Potential of Zein Nanoparticles to Protect Entrapped ?-Carotene in the Presence of Milk under Simulated Gastrointestinal (GI) Conditions. LWT. 72 (2016) 302-309.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chuacharoen, T. and C. M. Sabliov. 2015. Influence of combined lecithin and pluronic F127 surfactant on stability of lutein-loaded zein nanoparticles as a function of time and temperature. 250th American Chemical Society National Meeting and Exposition. Boston, MA.