2.1. Chemicals and reagents
Powdered Spirulina platensis was purchased from the Noor Daro Gonbad Company (Gonbad Kavous, Golestan, Iran). ABTS (2,2′-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) diammonium salt), DPPH (2,2-diphenylpicrylhydrazyl), Alcalase, and Chitosan with a medium molecular weight were purchased from Sigma-Aldrich.
2.2. Preparation of Spirulina low-molecular-weight peptides (SPs)
Peptides with low molecular weights were extracted from Spirulina platensis powder using the method described by (Wang and Zhang, 2016). The preparation of the extract included the mixing of 20 g of Spirulina platensis powder into a 100 mL alkali solution (pH = 10), followed by centrifugation at 9000 g for 30 min at a temperature of 4 °C. The initial supernatant was collected, while the remaining pellet was subsequently re-dissolved in an alkali solution. This was followed by centrifugation at a force of 9000 g for 30 min, resulting in the second supernatant. The supernatants were then combined and subjected to freeze-drying using a vacuum freeze dryer, and the resulting spirulina platensis protein powder (3% of protein solutions) was further subjected to enzymolysis with alcalase. The ratio of enzyme to substrate, pH, temperature, and reaction time were set at 7% (w/w), 8.5, 55 °C, and 10 h, respectively. After hydrolysis, the enzyme activity was stopped by placing the reaction medium in a water bath (90 °C) for 20 min(Ma et al., 2021). The cooled hydrolysates were then subjected to centrifugation at 15000 g and 4 °C for 20 min and then fractioned with 10, 5, and 3 kDa MW cut-off polyethersulphone (PES) ultrafiltration membrane (Millipore Co., USA) in ultrafiltration centrifuge tubes at 6500 rpm at 4 °C for 30 min. Four fractions were obtained with a MW of < 3, 3–5, 5–10, and >10 kDa. The protein concentration was analyzed using the Bradford method assay (Bradford, 1976). Finally, the antioxidative peptides isolated from the most active fractions (< 3 kDa) were used for the preparation of liposomes and were then used for animal experiments (Supplementary Fig S2.1).
2.3. Preparation of Spirulina peptide (SPs)-loaded liposomes and chitosan-coated liposomes (SP-Ch-LP)
Spirulina peptide-loaded nanoliposomes (SP-LP) were prepared using the methods described by Saroglu et al., (2022)and Mohammadi et al., (2023). Briefly, soy lecithin (2% w/v), cholesterol (0.04 g), and Tween-80 (15 µL) were mixed into a total volume of 25 mL of absolute ethanol. The organic solvent was removed with a rotary evaporator (RotavaporR-300, BUCHI, New Castle, United States) at 80 rpm at 60 °C to form a thin lipid film. After the evaporation of organic solvents, the mixture was stirred at 55 °C for 4 minutes at room temperature. After that, spirulina peptides 4% v/v were added, and the mixture was homogenized for 5 min at 12,000g and then passed through a high-pressure homogenizer (HPH) (YC-BASIC series Yocell Biotechnology (Qingdao) Co., China) at 10,000 psi three times by high-pressure homogenization (M110Y, Microfluidics, USA). Chitosan-coated liposomes were prepared by mixing liposomes (SP-LP) with the chitosan solution (0.4% w/v chitosan in 1% v/v acetic acid) at a ratio of 1:1 under constant stirring (100 rpm) for 2 h at room temperature. The characterization of liposomal spirulina peptides (mean particle size, zeta potential, encapsulation efficiency (EE%), and storage stability) was performed as in our previous work (Karimzadeh et al., 2023).
2.4 Fourier-transform infrared spectroscopy
The Fourier transform infrared (FTIR) spectra of spirulina peptides, unloaded liposomes, and liposomal spirulina peptides were obtained using a Perkin-Elmer FTIR spectrometer (PerkinElmer, Waltham, MA, USA). The spectral measurements were recorded within the wavelength range of 400 to 4000 cm-1.
2.5. Transmission Electron Microscopy (TEM), polydispersity index, zeta potential, and Encapsulation efficiency
Additional information about the morphology of SP-Ch-LP was obtained using transmission electron microscopy (TEM, Zeiss– EM10C–100 kV, Germany) The mean particle diameter of nanoparticles, zeta potential (ZP), and polydispersity index (PDI) of samples (UN-LP, SP-LP, SP-Ch-LP) was determined using Zetasizer Nano zs (Malvern Instrument Ltd, Malvern, UK) at a wavelength of 633 nm with a fixed scattering angle of 90° and a power of 4 mV (Baxa, 2018). The encapsulation efficiency (EE) was determined following the method described by (Mohammadi et al., 2023).For that purpose, the encapsulation efficiency (EE) of the chitosan nanoparticles was measured by passing the sample through a filter (molecular weight cutoff of 30 kDa, Millipore Corp., Ireland), followed that centrifugation at 10000 rpm for 20 min at 4°C. The free peptide content (non-encapsulated) was then determined using the Bradford method (described previously). The EE was determined by the following formula:
EE = (Total protein content - The amount of free hydrolysate)/Total protein content × 100
2.6. Antioxidant activity
Antioxidant activity of Spirulina peptides (SPs), with different molecular weights (< 3, 3–5, 5–10, and >10 kDa and SPs-loaded liposomes, was were measured using methods of DPPH radical scavenging and ABTS activities. To determine DPPH radical scavenging activity(Shetta et al., 2019), briefly, 1 mL of SPs or SP-loaded nanoliposomes were mixed with 1 mL of DPPH solution (0.1 mM), and the absorbance was recorded at 517nm. DPPH radical scavenging activity was calculated using the following equation.
![](data:image/png;base64,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)
where A blank and A sample are the absorbance of the control and the sample
ABTS assay was done according to the method described by H. and Öner (2011). 20 μL of SPs or SP-loaded nanoliposomes were mixed with 4 mL of diluted ABTS solution and kept in the dark for 6 min. Trolox was used as the standard antioxidant, and the results were expressed as μmol Trolox equivalent (TE)/μmol sample. Trolox equivalent antioxidant capacity (TEAC) was obtained from the gradient of the plot of the percentage inhibition of absorbance versus sample concentration was divided by the gradient of the plot for Trolox to give TEAC at the specific time.
2.7. Release studies
The release properties of SPs, SP-LP, and SP-Ch-LP were carried out using the dialysis method that was previously described(Xu et al., 2021). Briefly, 2 mL of each sample was loaded separately into dialysis bags (MW 14K Da) (SERVA, GmbH, Germany) at 25 and 37°C for 24 h and the ends of the dialysis bag were tightened and then placed into a 30 ml PBS (pH 7.4) buffer under constant stirring at room temperature. At regular intervals, a definite volume (3 mL) was withdrawn and replaced with an equal volume of fresh phosphate buffer solution. The amount of SP release was measured using total phenolic content by UV-Vis spectrometer (Karimzadeh et al., 2023).
2.8. Cytotoxicity study
The MTT (methyl thiazolyl diphenyl-tetrazolium bromide conversion) method was used to determine the safe and nontoxic amount of SP and or SP-loaded nanoliposomes in mouse fibroblast cell lines L-929 (IBRC C10102, cell bank of the National Center for Biological and Genetic Reserves ,Tehran, Iran). Cells were first seeded in 96-well plates under DMEM growth medium containing FBS (10% (v/v), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C in a humid atmosphere with 5% CO2 concentration(Karimzadeh and Zahmatkesh, 2021). Upon reaching around 80% confluence, the cells were separated from the flask using trypsin–EDTA (0.25%) solution and centrifuged at 1500 rpm for 5 min. Cells at density 104 were cultured on the 96-well plates, and different concentrations of SPs, SP-LP, and SP-Ch-LP samples were distributed in freshly prepared DMEM medium and treated with seeded cells for 24h, 48h, and 72 h. After that, 20% MTT (5mg/mL) was added to each well for continuous incubation for 4 h. Finally, DMSO (150 µL) was added to each well, and the absorbance was measured at 570 nm using an ELISA reader. The cell viability percentage was calculated using the following equation:
% viability = [Mean OD sample / Mean OD blank] × 100
2.9.In vivo animal experimental design
Forty male Wistar rats with an average age of three months (221.14±7.2g) were obtained from the animal breeding facility center of the Pasteur Institute, Karaj, Iran. The animal trial was conducted in the department of biology at the Islamic Azad University, Lahijan Branch, Iran. The experiments involving rats followed animal protection guidelines and were approved by the Ethics Committee of the Islamic Azad University, Rasht Branch, Iran. lyAnimals were maintained under control conditions with a 12:12 h light/dark cycle and free access to standard laboratory water and food. Following one-week acclimatization, animals were divided into two groups: one fed regular chow (Behparvar Company, Tehran, Iran) for 12 weeks, and the NAFLD group, which received a high-fat diet (HFD), respectively. After eight weeks under this feeding regimen, NAFLD group rats were divided randomly into the following four groups (n = 8 rats/group): High-fat diet (HFD) fed group: containing 20% fat and equivalent to 38.9% of the total calories (Supplementary TableS1.1) (Ali et al., 2022; Bakir et al., 2019), HFD+SP: HFD diet feeding + 200 mg/kg body weight of SPs (Bin-Jumah et al., 2021; Brito et al., 2020); HFD+SP-LP: HFD + received 200 mg/kg body weight of SP-LP, and HFD+SP-Ch-LP received 200 mg/kg body weight of SP-Ch-LP. All supplementations were administered orally in a phosphate-buffered saline solution at 10:00 a.m. for four weeks, while the rats were kept on a high-fat diet (HFD).
2.10. Blood and tissue collection
At the end of the experiment, rats were kept fasting overnight, were weighed, and anesthetized with ketamine (100 mg/kg). Blood samples were collected, centrifuged at 3000 g for 15 min, and serum collected was stored at −80 °C until use. The liver of each rat was removed, weighed, and then sectioned into two different parts; one part was fixed in a 10% formalin solution for histopathological analysis, and the other part was homogenized in TBS buffer (50 mM Tris-HCl, 0.15 mM NaCl, pH 7.6, with a ratio of 1:2) (w/v) using a homogenizer (IKA T 10 Basic Ultra-Turax homogenizer, UK). Afterward, the homogenates were centrifuged at 14000 g for 15 min. The supernatants were collected and were used for biochemical analysis. All processes were carried out at 4 °C.
2.11. Serum biochemical and stress-oxidative analysis
Serum lipid profiles, including triglyceride (TG), total cholesterol (TC), low-density lipoprotein (HDL), and high-density lipoprotein (LDL), were measured using Boehringer Mannheim colorimetric kits (Mannheim, Germany). The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were spectrophotometrically determined (Randox colorimetric kit, Antrim, UK). Fasting blood sugar (FBS) was assayed with a commercial kit according to the manufacturer's instructions (Pars Azmoon glucose kit and Bio-system kit, Iran). The serum concentration of insulin was measured using an ELISA kit (Monobind, California, USA). The Homeostasis Model Assessment-Insulin Resistance Index (HOMA-IRI) was measured to examine individually the levels of insulin (Katsuki et al., 2001) using the following formula:
HOMA-IR = [insulin (μU/ml) × FBS (mg/dL)] /405
Protein carbonyls (PCO) were assayed in serum by the colorimetric method using a commercially available protein carbonyl colorimetric assay kit (Cayman Chemical Company, USA). In this assay, the reaction between 2,4-dinitrophenylhydrazine (DNPH) and carbonyl results in the creation of hydrazones, which are then detected spectrophotometrically at a wavelength of 360–385 nm. Advanced Oxidation Protein Products (AOPP) values were detected using an AOPP assay kit (Cell Biolabs, Inc., USA) following the manufacturer’s instructions. Advanced glycation end products (AGE) in the serum of rats were determined based on spectrofluorimetric detection according to the method described by Kalousová et al., (2002). The serum was diluted with phosphate-buffered saline (pH = 7.6, 1:50) and monitored for fluorescence intensity at 350 nm excitation and 440 nm emission. Fluorescence intensity was determined in arbitrary units.
2.12. Hepatic TG and TC levels, liver oxidative stress index, and pro-inflammatory factors
Triglyceride (TG) and total cholesterol (TC) contents were extracted in a chloroform-methanol mixture (2:1) and performed according to the method described earlier (Matthews et al., 1985). The malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) activities, as well as glutathione peroxidase (GPx) levels, were measured in the rat liver homogenates and measured using the specific assay kit according to the manufacturer’s instructions (Cayman, Ann Arbor, MI, USA). The levels of hepatic tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-I (L-Iβ) were determined by ELISA kits specified for rats (Boster Bio, Pleasanton, CA 94566, USA) according to the manufacturer’s instructions.
2.13. Hepatic phospho/AMPK ratio and phospho/ACC ratio
Hepatic contents of phospho/ AMPK ratio and phospho/ACC ratio were determined using sandwich enzyme-linked immunosorbent assay (ELISA), following the manufacturers’ instructions: AMPK (Indirect, Human/Mouse/Rat total AMPK α1, DuoSet IC, ELISA, R&D Systems, MN, USA), p-AMPK (Indirect, AMPK alpha pThr172 ELISA Kit, Abcam, Cambridge, UK), ACC, and p-ACC (RayBio, USA, Human and Rat Phospho-ACC1 (Ser79) and Total ACC1 ELISA Kit, Cat # PEL-ACC1-S79-T).
2.14. Histopathology of liver
Liver of rats were fixed in a 4% paraformaldehyde solution and were subjected to hematoxylin and eosin staining. The fat accumulation in liver sections was observed by the method of Brunt et al., (1999).