Z216mk

Graphene Oxide was purchased from Graphenea (San Sebastián, Spain). GO solutions were subjected to sonication (Vibra cell sonicator VC505, Sonics and Materials, United Kingdom) and centrifugation (Hermle Z 216 MK, Hermle Labortechnik, Germany), to obtain large, medium and small GO nanoflakes. To prepare large nanoflakes, a GO solution was subjected to a sonication of 1 min at 125 W, followed by centrifugation at 14.850 RCF for 15 min; after that the supernatant was removed, and the pellet was resuspended. To prepare medium size nanoflakes, a GO solution was sonicated (10 min, 125 W) and centrifuged two times. The first centrifuge was performed at 7.700 RCF for 30 min, then the supernatant was recovered and centrifuged again for 30 min at 18,620 RCF. Finally, the supernatant was removed, and the pellet was resuspended. Lastly, small size GO nanoflakes were obtained by sonicating three times a GO solution for 180 min at 125 W. After each sonication, the resulting solution was centrifuged at 10,000 RCF for 15 min and the final supernatant was collected. GO concentration was estimated by UV-Vis spectrometry experiments using the characteristic GO absorption peak, which is located at 230 nm (Jasim et al., 2016 ; Di Santo et al., 2019 (link)).

Zymosan (5 mg in 1 mL HBSS) was opsonized with 500 μL pooled human or murine serum for 30 min at 37°C. For complement-free opsonization zymosan (5 mg in 1 mL PBS) was opsonized with 500 μL human serum pretreated with 20 mM EDTA. After opsonization zymosan was centrifuged (5,000g, 5 min, 4°C, Hermle Z216MK 45° fixed angle rotor), and washed once in HBSS.
Bacteria (OD₆₀₀ = 1.0 in 800 μL HBSS) were opsonized with 200 μL pooled human or murine serum or with EDTA pretreated human serum for 30 min at 37°C. After opsonization, bacteria were centrifuged (8,000 g, 5 min, 4°C, Hermle Z216MK 45° fixed angle rotor), and washed once in HBSS.

Blood and urine were collected each month from treated and control mice. Serum samples were collected from the retro-orbital sinus and centrifuged at 10,000 × g in a microcentrifuge (Z 216 MK; HERMLE) for 10 min at 4°C to obtain the serum. For urine collections, mice were put in metabolic cages for 24 hr. The urine samples were briefly centrifuged to remove debris and stored at −20°C. Mice were euthanized 5 or 6 months following the start of treatment. A cardiac perfusion with PBS was performed, and the liver, kidney, spleen, and heart were collected. Tissue samples were fixed in a methacarn solution (30% chloroform, 60% methanol, and 10% acetic acid) for 24 hr or frozen in dry ice (for ARSB activity and GAG quantitative assays).

The Met content in the dried samples was measured by spectrophotometry. For the investigations, a Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) was used. For the calibration curve, standard solutions were prepared in distilled water as follows: 0.025 g of Met was dissolved in 100 mL distilled water. 5 mL of this solution was diluted to 25 mL and further dilutions were made to concentrations of 2.5, 5, 10, 20, 30 and 40 µg/mL. The absorbance at 319 nm was measured. For the spray-dried sample, 20 mg product was dissolved in distilled water and diluted to a concentration of 80 µg/mL. After dissolution, the chitosan was centrifuged by Hermle Z216 MK centrifuge (15,000 rpm, 20 min, 20 °C, Hermle AG, Gosheim, Germany). The Met content of the dried sample was calculated using the following Equation (7): $$C_{Met}=\frac{\left({\mathrm{A}}_{319}+0.0019\right)/0.0518}{80}\times 100$$
where C_Met = Met content (m/m %) in the dried sample, A₃₁₉ = absorbance at λ = 319 nm.

PMNs (120 μL of 2.5*10⁷/mL) were added to 480 µl of EV samples at 37°C in a linear shaker (80 rpm) for 3 hours. Cells were centrifuged (500 g, Hermle Z216MK 45° fixed angle rotor, 10 min, 4°C) and supernatants were analyzed for IL-8, TGF-β, IL-1RA, IL-1α, IL-6, TNF-α with a human IL-8, TGF-β, IL-1RA, IL-1α, IL-6, TNF-α DuoSet sandwich ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA) in a microplate reader (Labsystems iEMS Reader MF, Thermo Scientific). We also prepared a control sample for the oZ-EV samples that contained the same amount of zymosan as the oZ-EV isolates. To achieve this, in half of the oZ-EV batch EVs were lysed whereas zymosan particles were left intact (16 (link)). We refer to this sample as “lysed oZ-EV”.

The cerebral cortex and hippocampus of PND15 (n = 5 pups in each group) were individually dissected and mechanically homogenized in PBS. Afterward, the homogenates were centrifuged at 12,500 rpm for 30 min at 4°C with a Z216MK microcentrifuge (HERMLE Labortechnik GmbH; Wehingen, Germany). Aliquots of the supernatants were used to quantify nitrites, lipid peroxidation, GSH and GSSG levels, and enzymatic activities of AST, ALT, LDH, SOD, CAT, and GPx. Protein content was measured in the supernatants using the Coomassie blue method [32 (link)] to normalize the biochemical results.