Phosphate buffer

For film preparation: collagen (Coll) was isolated in our laboratory from the fish skin of Salmo salar [42 ]. Chitosan (CTS, low molecular weight, deacetylation degree (DD) = 77%), tannic acid (TA, M_w = 1701.2 g/mol), diiodomethane (99%), acetic acid (CH₃COOH, ≥99.8%), phosphate buffer (PBS) solution, and absolute ethanol (>99.8%, EtOH) were supplied by Sigma Aldrich (Poznań, Poland). Potassium silicate (PS, anhydrous, −48 Mesh, SiO₂:K₂O 2.5:1 wt%) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH, free radical, 95%) were obtained from Alfa Aesar (Karlsruhe, Germany). Glycerine (pure for analysis) was purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).
For in vitro studies: phosphate-buffered saline (PBS) tablets were from BioShop Canada Inc. (Burlington, ON, Canada). Alpha- Minimum Essential Medium Eagle (MEM) was from Gibco by Thermo Fisher Scientific (Warsaw, Poland), Fetal Bovine Serum Qualified (FBS) from Biological Industries (Cromwell, CT, USA), and ZellShield from Minerva Biolabs (Berlin, Germany). MTS assay (CellTiter 96^® AQueous One Solution Cell Proliferation Assay) was from Promega (Madison, WI, USA).

TEM was used to observe the extracellular vesicles by providing high-resolution images of their ultrastructure after isolation and purification, followed by fixation with 2% glutaraldehyde (Sigma) in phosphate buffer (pH 7.4, Sigma) to preserve their structure. A small drop (3 µL) of the fixed extracellular vesicle suspension was applied to a carbon-coated TEM grid (697745, Sigma) and then washed and stained with a contrasting agent: 1% uranyl acetate (21447-25, Polysciences, Warrington, PA, USA). After air-drying, the grid was placed in the TEM (HITACHI H-7500, Tokyo, Japan), where an electron beam generated detailed images of the extracellular vesicles, typically appearing as round, cup-shaped vesicles with a 30–150 nm diameter. The exosomal morphology, size, and structural features were recorded.

All chemicals were used without further purification. G5 was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd. (Weihai, China). γ-PGA was purchased from Shanghai Yika Biotechnology Co., Ltd. (Shanghai, China). Dopamine hydrochloride, doxorubicin hydrochloride, EDC, and NHS were purchased from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium chloride was purchased from Greagent. Phosphate buffer was purchased from Sigma-Aldrich (Shanghai, China). CBS was purchased from Shanghai Titan Technology Co., Ltd. (Shanghai, China). Mouse fibroblasts and CT26 were purchased from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). Modified Eagle medium (DMEM) was purchased from Corning Incorporated (Shanghai, China). CCK-8 and live/dead cell staining kit were purchased from Shanghai Weiao Biotechnology Co., Ltd. (Shanghai, China). KM mice and BALB/c-Nude nude mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China). The Institutional Review Board of Changhai Hospital Affiliated with Naval Medical University (SYXK (Shanghai) 2020–0033) approved the animal study protocol.

ChemicalsDOX (Ebewe Pharma co. Austria), nZnO (Sigma-Aldrich, particle size<50 nm [TEM], purity >97%), Ham’sF10, NaHCO3, eosin-Y, ethanol, formalin, hematoxylin, paraffin, Carnoy’s fixative (methanol/acetic acid; 1/3), glutaraldehyde, acridine orange, 1,1,3,3,-tetra ethoxy-propane, trichloroacetic acid, n-butanol, 2,4,6-tripyridyl-s-triazine (TPTZ), 2- thiobarbituric acid (TBA), acetic acid, and phosphate buffer (Merck Chemical Co. Darmstadt, Germany) were used in this study.
Preparation of nZnO suspension nZnO particles were suspended in 1% sdium carboxy methyl cellulose as stabilizer or surfactant, stirred with magnetic stirrer for 5 minutes and then dispersed by ultrasonic vibration for 15 min (21 -23 (link)). In order to avoid the aggregation of the particles fresh suspension was prepared before every use.
Animal treatmentsIn this experimental study, 24 adult sexually mature male (4 months old weighing 220-250 g) Wistar rats were obtained from Razi Vaccine and Serum Research Institute (Tehran). They were kept under standard conditions of temperature (23±2^oC), and 12h light/dark period, and fed with a standard pellet diet and water ad libitum. Animal handling and care were performed in accordance with the guidelines established by the Canadian Council on Animal Care. In this study, four groups each containing six male rats were used.
Treatment groups were as follows: group 1 received normal saline by injection (ip) daily, group 2 received DOX (6 mg/kg/day) dissolved in normal saline, group 3 received nZnO (5 mg/kg/day) dissolved in normal saline by ip injection, and group 4 received DOX (6 mg/kg/day) and nZnO (5 mg/kg/day) following pretreatment with nZnO one day before. All groups were treated for 3 days.
SamplingAfter 28 days, the animals were euthanized by CO₂ exposure and were killed by decapitation. Blood samples were collected in vials containing heparin.
The plasma was separated and kept at -80^oC until analysis of LH, FSH, testosterone, and toxic stress markers including cellular lipid peroxidation (LPO) and total antioxidant power (TAP). Epididymes were removed, cleaned of adhering connective tissue, weighed and perfused with cold (0.9%) NaCl. Radioimmunoassay kits were used to determine concentrations of LH, FSH, and testosterone. The study was approved by the ethic committee of the Razi Institute.
LPO and TAPConcentration of LPO in plasma was determined by measurement of malonedialdehyde and other lipid peroxide aldehydes that react with TBA known as TBA-reactive substances (TBARS). The absorption of the TBARS was deterImined spectrophotometrically at 532 nm using 1, 1, 3, 3-tetraethoxypropan as standard (24 (link)). TAP of plasma and testis was determined by measuring their ability to reduce Fe³⁺ to Fe²⁺. The complex between Fe²⁺ and TPTZ gives a blue color with absorbency at 593 nm (25 ).
Sperm characteristicsEpididymal sperms were collected by slicing the epididymes in 5 mL of Ham’s F10 and incubating for 5 min at 37^oC in an atmosphere of 5% CO₂ to allow sperm to swim out of the epididymal tubules. One drop of sperm suspension was placed on a microscope slide, and a cover slip was placed over the droplet. At least 10 microscopic fields were observed at 400× magnification using a phase contrast microscope, and the percentage of motile sperm was evaluated microscopically within 2-4 min of their isolation from the epididymes and was considered as a percentage of motile sperm of the total sperm counted.
Epididymal sperm counts were obtained by the method described in the WHO Manual (1999). Briefly, 5 μl aliquot of epididymal sperm was diluted with 95 μl of diluent (0.35% formalin containing 5% NaHCO₃ and 0.25% trypan blue) and approximately 10 μl of this diluted specimen was transferred to each of the counting chambers of the hemocytometer and was allowed to stand for 5 min in a humid chamber to prevent drying. The cells sediment during this time and were counted with a light microscope at 400× (26 ).
A 20 μl of sperm suspension was mixed with an equal volume of 0.05% eosin-Y. After 2 mins incubation at room temperature, slides were viewed by bright-field microscope with magnification of 400×. Dead sperms appeared pink and live sperms were not stained. Two hundred sperms were counted in each sample and viability percentages were calculated. For analysis of morphological abnormalities, sperm smears were drawn on clean and grease-free slides, and allowed to air dry overnight. The slides were stained with 1% eosin-Y/5% nigrosin and examined at 400× for morphological abnormalities such as amorphous, bicephalic, coiled, or abnormal tails (26 , 27 (link)).
Staining of spermatozoa with acridine orangeAcridine orange staining was used to monitor the effects of DOX on cauda epididymal sperm. To perform this assay with fluorescent microscope, thick smears were fixed in Carnoy’s fixative (methanol: acetic acid 1: 3) for at least 2 h. The slides were stained for 5 min and gently rinsed with deionized water. Two-hundred sperms from each staining protocol were evaluated and graded as normal DNA (green) or damaged DNA (yellow to red) (28 (link)).
Sample preparation for light microscopy and histopathological analysisAfter fixation of epididymes in a 10% formalin solution, they were directly dehydrated in a graded series of ethanol and embedded in paraffin. Thin sections (4-5 μm) were cut using a microtome and stained with hematoxylin and eosin and examined using a light microscope. The qualitative changes of epididymes were recorded (26 ).
Statistical analysisValues are reported as mean±SEM. Statistical significance between groups was computed by analysis of variance and Tukey multiple comparison post hoc tests. Data was analyzed with SPSS-14 and one way ANOVA test. P<0.05 was considered significant.

Frozen samples of frontal and cerebellar cortex of patients were homogenized in phosphate buffer (pH 7.4, Sigma) at 10% (weight/volume) and were centrifuged (Eppendorf Centrifuge) at 4 °C, 800×g, for 1 min in order to remove cellular debris. Ten microliters of brain homogenates were treated with 50 μg/mL of proteinase K (PK) (Invitrogen) for 1 h at 37 °C under shaking (500 rpm). Digestion was stopped by the addition of LDS-PAGE loading buffer (Life Technologies), samples were then heated at 100 °C for 10 min and loaded into 12% Bolt Bis-Tris Plus gels (Life Technologies). Proteins were separated by means of SDS-PAGE, transferred onto Polyvinylidene difluoride (PVDF, Millipore) membrane and incubated with 5% (wt/vol) dry nonfat milk in 0.05% (vol/vol) Tween-20 (prepared in Tris-HCl) for 1 h at room temperature with shaking. PVDF membranes were finally incubated with anti-PrP antibodies 6D11 (epitopes 93–109, Covance) or 3F4 (epitopes 109–112, Dako) and developed with chemiluminescent system (ECL Prime, GE Healthcare Amersham).

A modified FRAP assay was used to assess the ferric reducing capacity of plant extracts [65 (link)]. The reduction of ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) by antioxidants present in the samples is how the assay determines the antioxidant potential. Blue coloration results from the conversion of ferric iron (Fe³⁺) to ferrous iron (Fe²⁺).
Equal amounts of 0.1 g dry extracts were dissolved in 100 mL 50% ethanol for every plant extract used in our study. Eight corresponding volumes of each obtained solution were brought into volumetric flasks and adjusted to 10 mL by adding the same solvent as above. An amount of 2.5 mL of each diluted solution was mixed with phosphate buffer (pH 6.6, Sigma–Aldrich, Hamburg, Germany) and 2.5 mL K₃(FeCN)₆ 1% (Sigma–Aldrich, Hamburg, Germany) before being heated to 50 °C for 20 min. 2.5 mL trichloroacetic acid (Sigma–Aldrich, Hamburg, Germany) was added to each sample. Furthermore, 2.5 mL of distilled water and 0.5 mL FeCl₃ 0.1% (Sigma–Aldrich, Hamburg, Germany) were added to 2.5 mL of each of the resulting solutions, the samples being left thereafter idle for 10 min. The change in the absorbance at 700 nm was measured relative to a blank sample obtained by mixing 5 mL distilled water with 0.5 mL FeCl₃ 0.1%.
The antioxidant capacity was calculated using the IC50 (half of the antioxidant effect—IC—effective concentration) value (mg/mL), which represents the solution concentration for which the absorbance has a value of 0.5.
Different extract volumes were tested in order to reach the absorbance value of 0.5, due to the variability of plant characteristics and the nonuniformity of phytochemical profiles of plant extracts (experimental values closer to the target value result in more accurate approximation—IC50 for y = 0.5). The optimized values have been set as mentioned above in order to conduct an appropriate comparative study within the same technique and between other methods of assessing the antioxidant activity.