CYTOTOXICITY STUDIES OF NANOLIPOSOMES AND NANOEMULSIONS OBTAINED FROM CHIA SEED LIPIDS IN LIVER AND BRAIN CELLS

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CHIA SEED OIL COMPARED TO OTHER VEGETABLE OILS RICH IN UNSATURATED FATTY ACIDS

In terms of nutritional value, there are a number of sources rich in omega-3 FA on the market, including menhaden fish, salmon, a number of algae, flax seeds. Chia seeds have some advantages including ease in transport as compared with marine sources, and absence of undesirable effects like allergy, and can be consumed in its raw form with no limitations (Sosa et al., 2016).
One of the main potential competitors for chia seed oil as a source of ALA is flaxseed (Línum usitatíssimum) oil. Also, a number of researchers are studying the oil of the seeds of perilla (Perilla frutescens) plant, which belongs to the same family, like the Spanish sage and grows in Southeast Asia.
In a study by Ciftci et al. (2012) compares the lipid component of chia, perilla and flax seeds. Lipid extraction was carried out by the Folch method using a mixture of chloroform and methanol (2:1). According to the authors, the oil yield was almost 45% for flax seeds, 40% for perilla seeds and 35% for chia seeds. In general, the oil content of flax, as well as perilla, varies depending on the region and can range from 29% to 46%, on average about 40%, and for perilla from 25% to 52%. The content of ALA was 58.2%, 60.9% and 59.8% for flax, perilla and chia, respectively. It was noted that the amount of ALA in linseed oil can vary from 52% to 64%, in perilla seed oil from 57% to 64%, and in chia seed oil it can reach 65.1%.
Interestingly, in the composition of flax and perilla seeds, more oleic acid was identified (18.1% and 16.21%) than LA (15.3% and 14.72%), and the inverse relationship was observed in chia seeds (10.53 and 20.27%), however, in the other studies mentioned there is no such dependence (Ciftci et al., 2012).
The total content of PUFA in flaxseed oil was 73.6%, in chia and perilla 80.5% and 74.6%, respectively, while the content of MUFA in chia seeds is lower (10.95%) than in other types of seeds (18.50% for flax and 16.57% for perilla). The ratio of omega-6 to omega-3 FA was significantly higher in chia (0.35) and flax seed oil (0.28) than in perilla oil (0.22), but higher values were noted in previous studies by other authors. According to the data obtained in the study, chia seeds contain significantly fewer total tocopherols and fewer varieties of sterols with a sufficiently high total content (Ciftci et al., 2012).
Another study compared the characteristics, oxidative stability, and antioxidant potential of cold pressed chia seed and flax seeds oils. The authors note that the obtained data on the content of ALA are consistent with previously known data on chia seeds, as the richest source of omega-3 plant origin. In addition, according to the study, chia seed oil is more prone to oxidation due to greater unsaturation, however, it had more antioxidant compounds and the total phenol content in the oil also exceeded this indicator in flaxseed oil (319.12 mg / g and 244.65 mg / g, respectively) (de Souza et al., 2019). According to Saphier et al. (2017) chia seeds contain 42% more polyphenols than flax seeds.

LIPID-BASED MICRO- AND NANOVESICLES AND DRUG DELIVERY

The high levels of unsaturated FA and the peculiarity of the chemical structure of PUFA contribute to the instability of chia seed oil to external stress factors. Today, an urgent issue is both the preservation of the properties of vegetable oils, as well as the development of systems for delivery of essential lipid components while maintaining their quality.
According to a review by Timilsena et al. (2017 and 2020), today 2 main approaches are used to minimize the processes of deterioration of vegetable oils – the introduction of natural or synthetic antioxidant systems or microencapsulation of oils to minimize direct contact with environmental stress factors. In the latter approach, wall materials used include proteins, lipids (including phospholipids) and polysaccharides, or a combination of these components. In addition, with this method, characteristics of the encapsulated substance can be hidden such as an undesirable taste, color, or odor. The type of encapsulation can be used to ensure controlled delivery and/or to increase bioavailability of the molecule of interest. The first successful encapsulation was reported in 1927, using acacia gum and spray drying. The main methods for obtaining microcapsules include spray and freeze drying, fluidized bed coating, centrifugal extrusion, inclusion complexation, complex coacervation, ionotropic gelation, electrospray, and liposomal capture.
The use of these and other techniques has been studied in recent years in relation to chia seed oil. An example of this is a recent study by Lehn et al. (2018), chia seed oil was microencapsulated using spray drying. The emulsions were formed using different wall materials, including pure whey, whey permeate, gum arabic and soy lecithin. The resulting emulsions were dried, and he obtained microcapsules of chia seed oil had different sizes particles with hemispherical shapes without agglomeration with sizes ranging from 15 to 50 μm. According to the authors, the effectiveness of encapsulation depends on the type of oil used and is also related to the degree of unsaturation of the oil. For chia seed oil, the figure ranged from 43% to almost 57%, depending on the wall material used. With higher encapsulation values, the amount of free oil is reduced, which has a positive effect on storage, as indicated by lower peroxide levels even after 30 days of storage at room temperature (25°C).
Microparticles with chia seed oil have been obtained using Maillard reaction products with different protein / carbohydrate ratios (Copado et al., 2017). In this study, the particles were obtained by freeze-drying an oil-in-water emulsion containing sodium caseinate and lactose and chia seed oil at various concentrations. A number of emulsions were heated to 60°C at the stage of preparing a protein-carbohydrate mixture to obtain the products of the Maillard reaction. The encapsulation efficiency in the study ranged from 41 to 84%; the particle size varied depending on the method used from 0.1 to 10 μm in the case of heating and up to 239 μm without preliminary heat treatment. Heat treatment of the samples increased the oxidative stability. In addition, the microcapsules were similar in composition to the untreated oil. It is noted that all the formulations obtained have high rehydration properties.
In a study by Encalada et al. (2020) considered a method for preparing an emulsion based on chia seed oil and using a pectin-rich food additive from carrots as a source of antioxidants (carotenoids) and a gelling agent. The resulting emulsion showed high storage and oxidative stability at 25°C for 45 days.
Conventional oil-in-water emulsions were one of the first systems believed to deliver biologically active lipids due to their relative ease of preparation and low cost. The question of formulating microemulsions using the lipid fraction of Spanish sage seeds and various materials as an emulsifier and microcapsule wall material, as well as several techniques used, has been studied quite extensively (Julio et al., 2015; González et al., 2016; Julio et al., 2016; Timilsena et al., 2017b; Guimarães-Inácio et al., 2018; Julio et al., 2018; Us-Medina et al., 2018; Alcântara et al., 2019). However, the use of chia seed oil for particles of nanoscale remains yet almost unexplored (de Campo et al., 2017; Teng et al., 2018), despite the increased interest in nanotechnology in recent years.
Research by de Campo et al. (2017) focused on chia seed oil nanocapsules using chia seed mucilage as wall material. Nanoparticles with high stability for 28 days at 40°C were obtained using the surfactant Tween 80 and subsequent homogenization. Interestingly, the experiment used unrefined mucilage, which also included minor components such as proteins, lipids, and inorganic elements. The authors believe that the presence of proteins in the composition facilitate the emulsification process, and the high percentage of dietary fiber in the mucilage increases the efficiency of encapsulation and the stability of the system. The average particle size of these particles was 205 nm and the charge was in the range of -12 mV, with an encapsulation efficiency of about 83%. The authors note that the resulting nanoparticles showed good thermal stability at 300°C, and a higher oxidation stability as compared to unencapsulated chia seed oil.

TOTAL DIETARY FIBER CONTENT

Total dietary fiber (TDF) content in chia solid residue obtained after Folch-based extraction were determined using a Megazyme Total Dietary Fiber Kit according to detailed manufacturer’s instructions. Due to this manual, assay procedure based on AOAC 991.43, AOAC 985.29, AACC 32-07.01 methods. Used technique is applicable to cereal grains, fruit and vegetables, cereal and fruit products and foods. Determination of TDF was implemented by few steps. Main principle describes quantification on duplicate samples of dried and defatted material. In our case we used chia lipid solid residue for analysis directly. Briefly, samples (1 g) (completely dispersed in earlier prepared Megazyme buffer (pH 8.2)) and blanks (control) (with buffer) were maintained with 50 μL of heat-stable α-amylase 30 min at 100°C in water bath to provide gelatinization, hydrolysis and depolymerization of starch. Then they were incubated 30 min at 60°C in water bath with 100 μL protease to solubilize and depolymerize proteins. After this step 5 mL of 0.561N HCl was added and pH checked (4.1-4.8), pH was adjusted if needed using 5% NaOH or 5% HCl. Then 200 μL amyloglucosidase were added to hydrolyze starch fragments to glucose and incubated 30 min at 60°C in water bath. At the next step samples were treated with 4 volumes (225 mL) of pre-heated 60°C ethanol during 60 min at room temperature to precipitate soluble fiber and remove depolymerized protein and glucose from starch. The rest was vacuum filtered (Pyrex N2), washed with 78% ethanol, 95% ethanol and acetone, dried (103°C, 12 h) and weighted. One duplicate was analyzed for protein content by Kjeldahl method as described in section 2.2.1.3 and another one was analyzed for ash content using 5 h incubation at 525°C. Calculation of TDF was done by using the Megazyme Mega-CalcTM, downloadable from where the product appears on the Megazyme web site (www.megazyme.com). In general, TDF is the weight of filtered and dried rest excepting ash and protein content.

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FATTY ACIDS COMPOSITION AND CHARACTERIZATION OF LIPID CLASSES

Fatty acid composition and lipid classes were characterized as described previously (Gbogouri et al., 2011; Arab-Tehrany et al., 2012; Hasan et al., 2014; Hasan et al., 2019).
Briefly, fatty acid methyl esters (FAMEs) were prepared by the Ackman method (1998). First, nonadecanoic acid (19:0) internal standard solution with concentration 1 mg/mL was prepared using chloroform as solvent. Before samples esterification 1mL of this solution was added in a glass tube as an internal standard (IS) for quantification of FA and totally evaporated by N2(g). For FAME preparation, tested sample (100 mg) was weighted in glass tube with pre-added IS, with following addition of 1.5 mL hexane and 1.5 mL of boron trifluoride (14% in methanol). Samples were vortexed 60 s and incubated 60 min at 100°C under N2(g). After tube cooling, 1 mL of hexane and 2 mL of distilled water were added, vortexed 60 s and incubated 1-2 min for phases separation. Supernatant was transferred to the second glass tube by Pasteur pipette. The rest in fist tube was washed with 1mL of hexane, vortexed, supernatant transferred, and washing repeated one more time. Next, second tube washed with 2 mL of water, vortexed and supernatant transferred to the third glass tube, which was pre-weighted with the lid. All steps were implemented under N2(g) to preserve lipids degradation. Liquid part in third tube was evaporated during 5-10 min by N2(g). The rest FAMEs were weighted, hexane was added in proportion 100 mg FAMEs to 1000 μL of hexane, samples stored under N2(g) at 4°C, covered with aluminum. Before analysis samples were diluted 10 times using hexane.
The separation of FAMEs was carried out by gas chromatography on a Shimadzu GS 2010-Plus (Perichrom, Saulx-lès-Chartreux, France) equipped with a flame-ionization detector. A fused silica capillary column was used (60 m, 0.25 mm i.d. × 0.2 μm film thicknesses, SPTM2380 Supelco, Bellfonte, PA, USA). The column temperature was set initially at 120°C for 3 min, then increased to 180°C at a rate of 2°C min−1 and maintained at 220°C for 25 min. Injection and detection temperatures were set at 250°C. Standard mixtures (PUFA1 from marine source and PUFA2 from vegetable source; Supelco, Sigma–Aldrich, Bellefonte, PA, USA) were used to identify fatty acids in the elution profile. The results are shown as means of triplicate determinations.
Lipid classes of chia seed, rapeseed and flax seed extracts were determined by thin layer chromatography (Iatroscan MK-5 TLC-FID, Iatron Laboratories Inc.,Tokyo, Japan) as shown on the Figure 9.

PHOSPHOLIPID ANALYSIS

Qualitative and semi-quantitative analyses of chia seeds phospholipids were performed on an HPLC-MS system consisting of an UltiMate 3000TM quaternary solvent delivery pump connected to a linear ion trap mass spectrometer (LTQ) equipped with an atmospheric pressure ionization interface operating in electrospray negative ion mode (ESI-) (ThermoFisher Scientific, San Jose, CA, USA).
Extracts (16 μL) were injected on a LiChroCART (250mm × 4mm-5μm) LiChrospher 100 DIOL column (Merck, Darmstadt, Germany). The flow rate was set at 300μl.min-1 and the column temperature at 30°C. Mobile phase A consisted of methanol with 0.1% (v:v) formic acid, ammonia added to pH 5.3 (approx. 0.05%, v:v of ammonia) and 0.05% (v:v) triethylamine; mobile phase B was chloroform. Lipids were eluted using a first linear gradient from 95% to 70% of B for 11 min, a second linear gradient to 20% of B for 3 min and an isocratic step at 20% B for 4 min. Mass spectrometric conditions were as follows for ESI- mode: spray voltage was set at -4.5 kV; source gases were set (in arbitrary units min-1) for sheath gas, auxiliary gas and sweep gas at 40, 5 and 5, respectively; capillary temperature was set at 230°C; capillary voltage at -36 V; tube lens, split lens and front lens voltages at -133 V, 70 V and 6.25 V, respectively. Ion optics parameters were optimized by automatic tuning using a standard solution of a phospholipids mixture at 0.1 g L-1 infused in mobile phase (A/B: 5/95) at a flow rate of 5 μL min-1.
Full scan MS spectra (500 to 2000 m/z) allowed us to detect parent ions of general form [M-H]- for all the phospholipids of interest except for phosphatidylcholine (PC) and lysophosphatidyl-choline (lyso-PC) classes for which the parent ion is [M+HCOO]- due to ionic complex formation between choline group and formiate.
Data dependent MS2 an MS3 scans were carried out automatically in order to obtain fatty acid composition for phospholipid classes phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylglycerol (PG), PC and lyso-PC. In the case of PC and lyso-PC phospholipids, MS3 scans were necessary to obtain the same level of information because MS2 events were only informative of their class (through characteristic neutral loss of 60 corresponding to simultaneous departure of formiate and a methyl group of choline).
A semi-quantitative analysis of the samples was carried out for each class of phospholipids using MS screening by grouping together the main species identified in the class.
Two phospholipid standard solutions were used (1 g/L). Phosphatidylcholine and phosphatidylglycerol were selected as standards. Since the phospholipids PG, PA, PI and PE give rise to a parent ion [M-H]- of similar structure, we estimated that the MS response of these four classes was similar and we used the standard phospholipid PG for their semi-quantitative analysis. The same approach was used for the phospholipids PC and lyso-PC classes, both giving rise to a similar parent ion of structure [M+HCOO]- due to the presence of the choline group. The standard phospholipid PC was therefore used for semi-quantitative analysis of these phospholipids.

Table of contents :

CHAPTER I. LITERATURE REVIEW
1.1 ROLE OF LIPIDS IN BIOLOGICAL PROCESSES
1.2 PLANT AND SEEDS OF SALVIA HISPANICA L. AS SOURCES OF BIOLOGICALLY ACTIVE SUBSTANCES
1.3 COMPOSITION OF CHIA SEEDS
1.4 CHIA SEEDS LIPID FRACTION
1.5 CHIA SEED OIL COMPARED TO OTHER VEGETABLE OILS RICH IN UNSATURATED FATTY ACIDS
1.6 EFFECT OF CHIA SEED LIPIDS ON HEALTH AND DISEASE
1.7 METHODS OF CHIA SEED LIPIDS EXTRACTION
1.8 LIPID-BASED MICRO- AND NANOVESICLES AND DRUG DELIVERY
1.9 MAIN OBJECTIVE AND SPECIFIC AIMS OF THESIS RESEARCH
CHAPTER II. MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 CELLS
2.2 METHODS
2.2.1 PHYSICO-CHEMICAL ANALYSIS OF ORURO CHIA SEEDS
2.2.2 CHARACTERIZATION OF CHIA SEED LIPID FRACTION
2.2.3 NANOPARTICLES PREPARATION
2.2.4 NANOPARTICLES SIZE, POLYDISPERSITY INDEX AND ZETA-POTENTIAL MEASUREMENTS
2.2.5 TRANSMISSION ELECTRON MICROSCOPY
2.2.6 STABILITY OF NANOPARTICLES
2.2.7 LONG-TERM STUDIES OF OBTAINED NANOPARTICLES
2.2.8 PHOSPHOLIPID DETERMINATION IN NANOLIPOSOMES AND NANOEMULSIONS
2.2.9 СYTOTOXICITY OF OBTAINED CHIA-BASED NANOLIPOSOMES AND NANOEMULSIONS
2.2.10 ANTIOXIDANT STUDIES OF CHIA-DERIVED NANOPARTICLES
2.2.11 STATISTICAL ANALYSIS
CHAPTER III. NANOLIPOSOMES AND NANOEMULSIONS BASED ON CHIA SEED LIPIDS: PREPARATION AND CHARACTERIZATION
ABSTRACT
3.1 INTRODUCTION
3.2 RESULTS
3.2.1 CHIA SEED LIPID EXTRACTION
3.2.2 CHARACTERIZATION OF CHIA SEED LIPID FRACTION
3.2.3 PHYSICOCHEMICAL CHARACTERIZATION OF NANOEMULSIONS AND NANOLIPOSOMES
3.3 DISCUSSION
3.4 CONCLUSIONS
CHAPTER IV. CYTOTOXICITY STUDIES OF NANOLIPOSOMES AND NANOEMULSIONS OBTAINED FROM CHIA SEED LIPIDS IN LIVER AND BRAIN CELLS
4.1 INTRODUCTION
4.2 RESULTS AND DISCUSSION
4.2.1 HEPA 1-6 CELL LINE
4.2.2 ASTOCYTES CELL LINES
4.2.3 PRIMARY CULTURE OF CORTICAL NEURONS
4.3 CONCLUSIONS
CHAPTER V. ADDITIONAL STUDIES
5.1 LONG-TERM STORAGE OF CHIA-DERIVED NANOLIPOSOMES AND NANOEMULSIONS
5.2 ANTIOXIDATIVE PROPERTIES OF NANOLIPOSOMES AND NANOEMULSIONS OBTAINED FROM CHIA SEED LIPIDS
CHAPTER VI. THESIS RESEARCH CONCLUSIONS AND PERSPECTIVES 
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