Sonopuls mini20

40 mg of PMPs was dissolved in 1000 µL of PBS to prepare stock solution. The stock solution was subsequently treated with an ultrasonic homogenizer SONOPULS mini20 (Bandelin electronic, Berlin, Germany) for 2 min to reduce the aggregation, evolved during the storage of particles in a dried state. 50 µL of suspension was further mixed with 200 µL of PBS. To remove the undesired impurities, nanoparticles were washed with PBS and Britton-Robinson buffer (pH 2), using a permanent magnet (Chemagen, Baesweiler, Germany). Isolation of amino acids/peptide (100 µg·mL⁻¹ in all cases, diluted in phosphate buffered saline—PBS) was carried out according to the conditions, which we optimized in a previous study [25 (link)]. After isolation, the solution was removed and the remaining conjugate with amino acids was dissolved in 3 M HCl. The dissolved particles were quantitatively transferred into a 96-well evaporation plate (Deepwell plate 97, Eppendorf AG, Hamburg, Germany) and evaporated using the nitrogen blow-down evaporator (Ultravap with spiral needles, Porvair Sciences, Leatherhead, UK). Prior to IEC analyses, particles were re-suspended with a sodium dilution buffer.

Cells were disrupted by sonication with 15 one-second pulses separated by intervals of one second, on ice-water slurry, with an Ultrasonic homogenizer (SONOPULS mini20, Bandelin, equipped with a 2.5 mm-diameter microtip probe). Chlorophyll was extracted by the vigorous vortexing, for one minute, of 100 μl of cell lysate mixed with 900 μl of 90 % acetone, followed by centrifugation at 10,000 × g for 10 min. The absorption spectrum of the chlorophyll-containing supernatant was determined directly with a UV-2600 UV-visible light spectrophotometer (Shimadzu), over wavelengths of 600 to 750 nm.
For chlorophyll content determination, we used the equation developed by Porra et al. [56 (link)] (Chla (μg · ml⁻¹) = 12.25 A_663.6 – 2.55 A_646.6, where A_663.6 and A_646.6, are the absorbance values at wavelengths of 663.6 and 646.6 nm, respectively, minus the absorbance at a wavelength of 750 nm).

The tissue SOD activity was measured using a SOD kit (Cat No MBS162314, MyBioSource, Inc., San Diego, CA, USA). In brief, the rat hippocampal samples were weighed and homogenized in PBS (pH = 7.4) in the ratio of 300–500 mg tissue homogenized in 500 µL PBS, with the rotary homogenizer SONOPULS mini20 (BANDELIN electronic, Berlin, Germany). The samples were centrifuged for 20 min at 5000 pm, and the supernatants were carefully collected. Samples (40 μL) were mixed with 10 μL anti-SOD antibody, then 50 μL streptavidin-HRP was added to sample and standard wells. After incubation for 60 min at 37 °C, the plate was washed five times with wash buffer (1X). For color development, 50 μL of chromogenic solutions A and B were used. The addition of 50 μL of Stop Solution to all wells stopped the reaction and changed the color to yellow. SOD activity was measured by detecting the absorbance at 450 nm using a microplate reader (Tecan Infinite F200 PRO (Tecan Austria GmbH, Salzburg, Austria). The activity was calculated via 4PL regression and expressed as ng/mL.

Dispersions of aqueous GO solutions (or their derivatives) were accordingly done using a Sonopuls mini20 ultrasonic homogenizer coupled with a 2.5 mm diameter tip (Bandelin electronic GmbH & Co. KG, Berlin, Germany), for 15 min, 70% input and 0.5 Hz, at room temperature, whereas mixing of solutions was done with a standard vortex mixer. Dispersions of GO-g-AEMA in the DEAEMA monomer were performed in a model Sonorex Digitec ultrasonic bath (Bandelin electronic GmbH & Co. KG, Berlin, Germany), for 15 min. Thermal control of reactions was done with a model Pura 4 water bath (Julabo GmbH, Seelbach, Germany) coupled to a test tube rack.

Western blotting was performed as described previously [23 (link),26 (link),27 (link)]. Briefly, a cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and cOmplete™ Protease Inhibitor Cocktail tablet (Roche, Basel, Switzerland) per 25 mL buffer). Samples were incubated for 30 min on ice followed by 30 s sonication (Bandelin Sonopuls mini20 homogenizer). The samples were then centrifuged for 15 min (12,000× g at 4 °C). The cleared lysates were separated and analyzed by SDS-PAGE (NuPage^® 10% bis-tris gel) with subsequent blotting onto a PVDF-membrane. The blotted membrane was blocked using blocking buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 3% bovine serum albumin (BSA), 0.5% Tween 20) overnight at 4 °C. Immunodetection was conducted using mouse anti-His₆ and HRP-conjugated rabbit anti-mouse antibodies (IBA) (1:2500 dilution). Band quantification was performed using ImageJ [28 (link)].

A mixture of 10 mL of Nafion and 200 µL of trichloro(1H,1H,2H,2H‐perfluorooctyl)silane was sonicated for 15 min and stirred for 2 h to obtain a uniform suspension of F‐Nafion. The F‐Nafion was mixed with 10 mL of poly(diallyl dimethylammonium chloride) solution and stirred at 100 rpm for 6 h at room temperature. The mixture was further sonicated for 5 min at 150 kJ (SONOPULS mini20, BANDELIN, Schaffhausen, Switzerland) with a 5 s on/off cycle to obtain a uniform suspension of F‐Nafion (PDDA).

After treatment, cells were trypsinized, pelleted at 200 × g, and washed with PBS to remove extracellular ASC1R. Cells (5 × 10⁴) were lysed using 40 µL of lysis buffer containing RIPA (Serva, Heidelberg, Germany) and 10% SDS at 4 °C for 30 min, and then briefly sonicated using a Sonopuls Mini 20 instrument (Bandelin, Germany). A 10 µL sample of cell lysate was loaded onto a 6% polyacrylamide gel and subjected to electrophoresis (110 V, 40 min, room temperature). ASC1R was visualized at 680 nm, and the amount of ASC1R was normalized against a standard and expressed as the number of ASC1R molecules per single cell.