Montmorillonite-Nasal-Gel, A Prophylactic Measure Against Covid-19

Tiankai Bai Shandong University of Traditional Chinese Medicine Jing Tian Shandong University of Traditional Chinese Medicine Xuan Zhai Shandong University of Traditional Chinese Medicine Xingyi Gao Shandong University of Traditional Chinese Medicine Kangmin Wang Shandong University of Traditional Chinese Medicine Zhiyong Zhang Shandong Academy of Medical Science, Shandong First Medical University Bin Yan (  robinyan2002@163.com ) Shandong University of Traditional Chinese Medicine


Introduction
The outbreak of coronavirus disease 2019 (Covid-19), which was caused by the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), has been declared a global pandemic in March 2020 by WHO 1 . As of October 7, 2021, Covid-19 has affected more than two hundred million people across 213 countries, areas or territories, and left more than four million death worldwide. To date, vaccines are applying through the world. But there are still so many people were infected by the virus and caused thousands of people to death. Moreover, small molecule drugs were expected to treat Covid-19, but only Remdesivir were approved by the FDA to treat Covid-19, and Molnupiravir is the next hopeful drug to treat Covid-19 2 .
Virus-neutralizing antibodies were proved effective through clinical trial, such as 47D11, S309 3,4 . Human immunoglobulin (pH4) for intravenous injection was approved for clinical trials in China on August 30,2021. However, physical protection is a useful way to ght against the epidemic. Montmorillonite-Nasal-Gel (MNG), a prophylactic preparation against Covid-19, had been approved by NMPA of China, can offer protection from Covid-19 virus.
MNG can adsorb pathogenic microorganisms because of negative charge on its crystal surface. It is a gel made of a mixture of 10% montmorillonite, 2% xylitol and distilled water. The active component of MNG is montmorillonite, came from Chifeng city, Nei Monggol Autonomous Region, China. Montmorillonite were found at Montmorillon, Vienne, France, Nouvelle-Aquitaine. Its formula is (Na,Ca) 0.33 (Al,Mg) 2 (Si4O 10 )(OH) 2 ·nH 2 O. The crystal structure of montmorillonite is composed of two layers of silicon-oxygen tetrahedron sheets and aluminum (magnesium)-oxygen (hydroxyl) octahedron sheet which are sandwiched between two layers of silicon-oxygen tetrahedra sheets 5 . The Si 4+ in the silicon-oxygen tetrahedron is often replaced by Al 3+ , and the Al 3+ in the aluminum oxygen octahedron is replaced by low-valence positive ion, such as Mg 2+ , Fe 2+ or other low-valence positive ion resulting in negative charges between the crystal layers (structural layers). In order to maintain electric neutrality, crystal layers are adsorbed positive ions, such as K + , Na + , Ca 2+ , Mg 2+ , Li + , H + , etc 5,6 . These ions appear in hydrated state and are inter-exchangeable, so that montmorillonite have characteristics such as ion exchange ability 7 . Because of negative charge on surface, montmorillonite crystal can adsorb positive charge pathogenic microorganisms or certain proteins 8 . Based on the pharmacology of montmorillonite and its unique high adsorption, it is mainly used in the elds of medicine, animal supplements and food additives 9,10 . The medicinal mechanism of montmorillonite mainly depends on its adsorption and gelation properties. Montmorillonite crystal has a lamellar structure and non-uniform charge distribution, which can anchor or inhibit viruses, germs and toxins in digestive tract 11 . It can repair and improve the defense function of intestinal mucosa against pathogens through binding with mucosa glycoproteins.
Montmorillonite rms colloidal state when added into water 12 . Colloidal montmorillonite can bind various pathogens such as bacteria and viruses, inhibiting their growth and infection. As Covid-19 is primarily transmitted via the respiratory route, we assume MNG can be used to prevent Covid-19 transmission.
Therefore, we designed an in vitro experiment to test its e cacy of anti-SARS-CoV-2. Also we did stimulation test of respiratory tract 13 .

Materials
The clinical isolate of SARS-CoV-2 was obtained from Institute of Pathogen Biology Chinese Academy of grease, high temperature resistance, high pressure resistance, used after sterilization.Drinking water meets GB 5749 requirements.

Anti-SARS-CoV-2 in vitro
We rst tested the effect of MNG on SARS-CoV-2 infection by using a SARS-CoV-2 S protein pseudovirus system. The lentiviral-based pseudovirus carrying the SARS-CoV-2 S protein (SARS-CoV-2pp) was prepared as previously described 14 . The pseudoyped SARS-CoV-2 (ppSARS-CoV-2) carrying a re y luciferase gene were treated with various concentrations (0%, 5%, 10% and 20%) of MNG at 37℃ for 1 h, followed by centrifugation at 3,000g for 10 min. Then the supernatant of each group was removed and used to inoculate 293T-ACE2/TMPRSS2 cells. After 48 h incubation, the cells were collected and subjected to luciferase assay.
To better evaluate the e cacy of MNG against SARS-CoV-2 infection, SARS-CoV-2 were treated with 50% MNG in BSL-3 laboratory. After 1 h treatment at 37℃ and centrifugation, the supernatants were removed, serially diluted and subjected to infect Vero cells for 2 h. Subsequently, the inoculum were replaced by fresh medium, and cells were incubated at 37℃ for 48 h. The viral yield was determined by quantifying the genome copies of SARS-CoV-2 in 1µL culture medium using qRT-PCR as described previously 15 .

Respiratory Stimulation Test
In addition to the study of MNG against SARS-CoV-2 infection, we also did respiratory mucosal stimulation test 16 .
Thirty male New Zealand rabbits were randomly divided into normal control group, high-dose group and low-dose group, and the doses administered are shown in Table 1. The animals were executed under overdose anesthesia 2 h after the last administration, and the nasal mucosa and tracheal tissues were xed with 10% formalin, then para n-embedded, sectioned and stained with HE, and the histological changes were observed microscopically 13 .

Anti-SARS-CoV-2 in vitro
We tested the effect of MNG on SARS-CoV-2 infection by using a SARS-CoV-2 S protein pseudovirus system. As shown ( Figure 1A), upon treatment with 5%, 10% and 20% MNG, the infection of ppSARS-CoV-2 were drastically decreased. In contrast, MNG treatment didn't interfere with the cell viability ( Figure 1B). These data clearly demonstrated that MNG can signi cantly block SARS-CoV-2 infection. In order to evaluate the e cacy of MNG against SARS-CoV-2 infection, we did the experiments of montmorillonite against SARS-CoV-2 in vitro. As shown in Table 2, MNG treatment greatly reduced the infection of infectious SARS-CoV-2, emphasizing the potency of MNG as a prophylactic drug to combat the Covid-19 pandemic.

Respiratory Stimulation Test
Microscopic observation showed that the mucosa of the nasal vestibule was well arranged with no interruption, and the ciliated columnar epithelium of the intrinsic nasal mucosa was neatly arranged with clear cilia. The mucosa and submucosal loose connective tissue were not congested and edematous, and no in ammatory cell in ltration or lymphoid hyperplasia was seen. Mucosal and submucosal layers of the trachea and bronchi were clear. The ciliated columnar epithelium was clearly arranged without interruptions. No exudates were seen in the lumen or submucosa, and occasionally exfoliated epithelium or a few lymphocytes (normal) were seen. No signi cant differences were seen between groups (Figures 2).
Through the mucous membrane stimulation test of New Zealand rabbits, it was found that the gel had no stimulation to the nasal mucosa of the rabbits.

Discussion
Montmorillonite becomes a hydrated gel by adding water, which has remarkable adhesion property. It enters the nasal cavity by spraying, and can combine with the mucus on the surface of the upper respiratory tract. It rmly covers the surface of the nasal mucosa and forms a protective barrier lm. Therefore, signi cantly enhances the cohesion and viscoelasticity of the mucus. It effectively blocks the invasion of viruses into the nasal mucosa cells acting like an "invisible mask". After entering the upper respiratory tract, it can adsorb or x the viruses, germs and toxins, and then be discharged out of the body with the nasal mucus. As a result, it reduces pathogenic effects. Even if the viruses are discharged with nasal mucus, it will not be transmitted to other people with droplets due to the viscoelasticity of the gel.
The surface of montmorillonite crystal appears negative charge 8 . The isomorphic substitution is the origin of the permanent charges that exist on the surface of montmorillonite crystal 8 , so it can adsorb certain viruses and bacteria. We assume that montmorillonite can physically adsorb SARS-CoV-2 either.
Speci cally, it may interact electrochemically with the cationic residue of spike protein RBD 17 . Its adsorption ability is related to the charge density. The gel also inhibits the replication of the virus and prevents the virus from spreading to the lower respiratory tract. The adsorption and immobilization of the virus by montmorillonite are physical effects that does not cause drug resistance. According to theory mentioned above, we assume montmorillonite will be equally effective against mutated coronavirus strains. MNG has been approved by NMPA of China and is available in market. It can be proposed that MNG can greatly reduce the risk of SARS-CoV-2 infection. MNG, in combination with the routine personal protective equipment, can provide further protection to all groups.

Figure 2
Nasal and tracheal tissues of each group

Supplementary Files
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