Microcrystalline cellulose

In the study following materials were used: pharmaceutical secondary standards (Certified Reference Material) of acetylsalicylic acid, salicylic acid and glycine (Fluka). Talc (Imifabi) and potato starch (Peepes) were used as excipients for dosage 100 mg ASA and 40 mg GLY, whereas microcrystalline cellulose (JRS Pharma) and maize starch (Roquette) were used as excipients for dosage 150 mg ASA and 40 mg GLY as well as 75 mg ASA and 40 mg GLY. Acetonitrile—gradient grade (Sigma-Aldrich) and orthophosphoric acid (Chempur). Deionized water was obtained by means of ELGA Purelab UHQ PS (High Wycombe, Bucks, UK). Syringe nylon filters (0.45 µm) (Agilent Technologies) were used.

We used the plaque reduction neutralization test (PRNT) as a reference for this study because neutralization assays are the standard for coronavirus serologic analysis. We tested serum samples for their neutralization capacity against SARS-CoV-2 (German isolate; GISAID ID EPI_ISL 406862; European Virus Archive Global #026V-03883) by using PRNT as described with some modifications (9 (link)). We 2-fold serially diluted heat-inactivated samples in Dulbecco modified Eagle medium supplemented with NaHCO₃, HEPES buffer, penicillin, streptomycin, and 1% fetal bovine serum, starting at a dilution of 1:10 in 50 μL. We then added 50 μL of virus suspension (400 plaque-forming units) to each well and incubated at 37°C for 1 h before placing the mixtures on Vero-E6 cells. After incubation for 1 h, we washed, cells supplemented with medium, and incubated for 8 h. After incubation, we fixed the cells with 4% formaldehyde/phosphate-buffered saline (PBS) and stained the cells with polyclonal rabbit anti-SARS-CoV antibody (Sino Biological, https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit IgG (Dako, https://www.agilent.com). We developed signal by using a precipitate forming 3,3′,5,5′-tetramethylbenzidine substrate (True Blue; Kirkegaard and Perry Laboratories, https://www.seracare.com) and counted the number of infected cells per well by using an ImmunoSpot Image Analyzer (CTL Europe GmbH, https://www.immunospot.eu). The serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of >50% (PRNT₅₀). We considered a titer >20 to be positive.
We performed the PRNT for serum samples from Germany by using Vero E6 cells, as described (R. Wölfel et al., unpub. data, https://doi.org/10.1101/2020.03.05.20030502) (10 (link)) and 24-well plates. Before the PRNT, we heat-inactivated patient serum samples at 56°C for 30 min. For each dilution step (in duplicate), we diluted patient serum samples in 200 μL of OptiPro serum-free medium (https://www.thermofisher.com) and mixed 1:1 with 200 μL of virus solution containing 100 PFUs. We vortexed the 400-μL serum–virus solution gently, incubated at 37°C for 1 h, and then incubated each 24-well plate with 200 μL serum–virus solution. After incubation for 1 h at 37°C, we discarded supernatants, washed cells once with PBS, and supplemented them with 1.2% microcrystalline cellulose solution in Dulbecco modified Eagle medium. After 3 days, we fixed and inactivated the plates by using a 6% formaldehyde/PBS solution and stained with crystal violet.

Twenty-two undergraduate students of the Leiden University (all females, mean age = 19.7 years, range 18–25; mean Body Mass Index = 21.5, range 18–24; all right-handed) with no cardiac, hepatic, renal, neurological or psychiatric disorders, personal or family history of depression, migraine, and medication or drug use participated in the experiment. Participants were selected individually via a phone interview by the same lab-assistant using the Mini International Neuropsychiatric Interview (M.I.N.I.; Sheehan et al., 1998 (link)). The M.I.N.I. is a well-established brief diagnostic tool in clinical and stress research that screens for several psychiatric disorders and drug use (Sheehan et al., 1998 (link); Elzinga et al., 2007 (link)). Psychoactive substance use was not assessed.
Written informed consent was obtained from all subjects; the protocol and the remuneration arrangements of 20 euro in cash payment were approved by the local ethical committee (Leiden University, Institute for Psychological Research).
In separate sessions participants were exposed to either an oral dose (powder) of 2.0 g of l-Tyrosine (TYR) (supplied by Bulkpowders Ltd.) or of 2.0 g of microcrystalline cellulose (Sigma-Aldrich Co. LLC), a neutral placebo, dissolved in 400 ml of orange juice. The two sessions were separated by 3–7 days. A double blind, placebo-controlled, randomized cross-over design with counterbalancing of the order of conditions was used to avoid expectancy effects.
Following Markus et al. (2008 (link)) women using contraception were tested when they actually used the contraception pill. On each experimental morning, participants arrived at the laboratory at 9:30 a.m. Participants had been instructed to fast overnight; only water or tea without sugar was permitted. In addition, subjects were not allowed to use any kind of drugs before and during the experiment or to drink alcohol the day before their participation and arrival at the laboratory. Thirty minutes after the administration of either TYR or the neutral placebo participants were allowed to eat an apple.

Microcrystalline cellulose (MC) (average particle size of 20 µm, purchased from Sigma-Aldrich, Leverkusen, Germany) was dissolved with the Ionic liquid (IL) 1-ethyl-3-methylimidazolium chloride (EMIMCl, >97%). The MCNTs (MWCNT, Baytubes^®; length > 1 µm, inner diameter 4 nm, and outer diameter 12 nm) were purchased from Bayer Material Science (BMS, Leverkusen, Germany) and used as supplied. After 12 h stirring at 85 °C, the MCNTs (MCNT, 50 wt.%) were added to the suspension and ultra-sonicated at room temperature for 30 min. The mixture was then placed in a syringe (760 µm inner diameter) and extruded in anti-solvent (Milli-Q+, Tallinn, Estonia) using a procedure that was shown in detail in a previous study [11 (link)]. To remove excess EMIMCl, the MC-MCNT fibers were washed with ethanol several times and then dried in an oven at 40 °C at 2 mbar. The MC-MCNT fiber had a cylindrical form with a diameter of 965 ± 75 µm and a length of 4.5 ± 0.3 mm. The diameter of the MC-MCNT fibers was measured with a screw gauge (Eisco Labs, Rochester, NY, USA) after they had been stored in different electrolytes for 24 h. The applied electrolytes (0.1 M) were dissolved in propylene carbonate (PC, 99%) with the chosen salts, 1-ethyl-2,3-dimethylimidazolium trifluoro-methanesulfonate (EDMICF₃SO₃, EDMITF, 95%), lithium triflouro-methanesulfonate (LiCF₃SO₃, LiTF, 99%), and tetrabutylammonium triflouro-methanesulfonate (TBACF₃SO₃, TBATF, 98%). The MC-MCNT fibers applied in this research weighed 1.2 ± 0.1 mg with an estimated MCNT weight of 500 ± 48 µg, resulting in a density of MC-MCNT fibers in the range of 0.33 ± 0.03 g cm⁻³. To ensure reproducibility, at least three fibers were formed independently of each other and investigated. The EMIMCl, PC, EDMITF, LiTF, TBATF, and ethanol (technical degree) were obtained from Sigma-Aldrich (Taufkirchen, Germany) and used as supplied.

Microcrystalline cellulose, sulfuric acid (H₂SO₄ 98%), and solvents were purchased from Merck (Germany), and nanocrystalline cellulose was obtained from Microcrystalline cellulose using the acid hydrolysis method.