Sorvall mtx 150

To collect the exosomal fraction, we carried out fractionation using an ultracentrifuge (Sorvall MTX150, Thermo Scientific, Waltham, MA, USA) according to general methods for the exosomal fractionation [66] . Briefly, the culture medium, supplemented with exosomal free FBS, 24 hours after medium change was collected and centrifuged at 3,000×g for 10 min at 4°C. The supernatant was carefully transferred into a new tube and centrifuged at 10,000×g for 20 min at 4°C. The resulting supernatant was the carefully transferred into a new tube for ultracentrifuge and centrifuged at 100,000×g for 90 min at 4°C. The supernatant was carefully removed and pellet was re-suspended with sample buffer for western blotting and used as the exosomal fraction.

The encapsulation efficiency was determined by using ultracentrifugation method. Briefly, the liposomal solutions were centrifuged at 155,000 G for an hour using a benchtop Sorvall MTX 150 ultracentrifuge (Thermo Scientific, USA). The liposomal pellet obtained was broken using 1% Triton X-100 and ranibizumab was detected using ELISA (Enzyme-linked immunosorbent assay, SuperSignal® ELISA Pico Chemiluminescent Substrate 250 ml Kit) (see Section 2.6).

SARS-CoV-2 isolate (hCoV-19/Egypt/NRC-03/2020) with accession number EPI_ISL_430820 (available at https://www.gisaid.org/, accessed on 1 August 2021) was propagated at Vero-E6 cells (ATCC, CRL-1586) in T-175 flask with DMEM media containing 1% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO) at 37 °C in 5% CO₂. The cell culture supernatant after 5 days post infection was been harvested and centrifuged at 1800× g for 10 min to remove cell debris.
Inactivation of cell culture clarified harvest using β-propiolactone 0.1% v/v in a shaking incubator for 48 h at 4 °C. A total 15 mL inactivated batch mixed with 6 mL sucrose 20% then ultracentrifuged at 50,000 rpm/min for 1 h at 4 °C (Sorvall MTX 150, Thermo Fisher Scientific, Waltham, MA, USA) then eluted in 1 mL 1× PBS. The protein content was measured using PierceTM BCA Protein Assay kit (Thermo) following the manufacturer’s instructions.

Exosomes were isolated using a multi-step process. Large vesicles were eliminated by sequential centrifugation up to 50,000g as described^{12 (link)}. The remaining particles were pelleted by ultracentrifugation (Sorvall mTx 150, Thermo Scientific, Springfield, NJ) at 100,000g for 18 h. Vesicles expressing CD63 were immunoselected using CD63 magnetic bead isolation. The recovered particle size was verified by nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Amesbury, UK) as described^{11 (link)}. The data were analyzed with the NTA software (NANOSight version 2.3) using dilutions with deionized water. Three videos at a minimum of 200 completed tracks were collected at 30-s time intervals/video per sample.
Exosomes were further distinguished by flow cytometry with magnetic beads coupled to anti-CD9-PE, anti-CD63-FITC and anti-ALIX-APC (BD Biosciences).

Exosomes were isolated using a multi-step process. First, large particles were removed by sequential centrifugation up to 50,000 g (Lim et al., 2011 (link)). The remaining particles were pelleted by ultracentrifugation (Sorvall mTx 150, Thermo Fisher Scientific, Springfield, NJ) at 100,000 g for 18 h. The recovered vesicles were analyzed for tetraspaninins (CD63 and CD81) by western blot and flow cytometry. The latter method used CD63 magnetic bead isolation. The exosomes were captured onto the beads and then labeled with CD63-FITC and anti-CD81-APC (BD Biosciences).
The recovered particle size was verified by Nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument (Amesbury, United Kingdom) as described (Bliss et al., 2016 (link)). The data were analyzed with the NTA software (NANOSight version 2.3) using dilutions with deionized water.

MRPL was prepared by the thin-film ultrasonic method described by Isailović et al. [16 (link)] with moderate modification. In brief, 1 g of soybean lecithin and 0.2 g of ultra-pure cholesterol were dissolved in 100 mL of chloroform, and 4 mg of MRPs was mixed subsequently. The mixture was transferred into a 250 mL round flask to completely remove the chloroform, using a R-3 rotary vacuum evaporator (rotate at 100 rpm) (Buchi Laboratory Equipment Trading Co., Ltd., Shanghai, China) at 37 °C. After the thin lipid film was formed, 100 mL of distilled water was added to hydrate with the lipid film by rotating for 30 min at 200 rpm in a 37 °C water-bath. After that, the solution was treated with ultrasound (JY92-IIN, biotechnology Co., Ltd., Ningbo, China) at 400 W for 4 min with 3 s pulse-on and 3 s pulse-off in an ice-bath to transform liposomes from MLV to UV, and then successively filtered through 0.45 μm and 0.22 μm water microfiltration membranes to obtain MRPL suspension [12 (link),18 (link)]. The suspension was centrifugated at 45,000 rpm for 30 min at 4 °C using a Sorvall MTX 150 ultracentrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA) to collected MRPL precipitate and to remove the unencapsulated MRPs; the encapsulation rate of MRPs was 49% ± 2.6%.