A new approach to extraordinary efficient protection against COVID 19 based on nanotechnology

A new approach to the protection against infections caused by bacteria and
 various viruses, including SARS-CoV-2 is described. In concrete example,
 protective filters and ALBO nanosilver masks showed extraordinary efficiency
 in protection against Staphylococcus aureus. These results show that it
 highly overcomes the efficiency of ordinary surgical masks. Besides,
 systematic meta-analysis given for ordinary surgical masks and filters N95
 for masks and respirators, showed no statistical difference between them in
 the case of SARS-CoV-2. On the base our experimental data and systemic
 meta-analysis given in this paper, it can be concluded that ALBO nanosilver
 masks have significant advantages, and show a very perspective concept of
 developing new protective gear.


Background about COVID-19 (disease caused by SARS-CoV-2)
At the end of 2019, three bronchi alveolar lavage samples were collected from a patient with pneumonia of unknown etiology and examined by Real-time PCR (RT-PCR) assays that were positive for pan-Beta coronavirus. Furthermore, using Illumina and nanopore sequencing, the whole genome sequences of the virus were determined, approving similarity between SARS and SARS-CoV-2 virus, which crown-like shape and its cytopathic effects (CPE) were confirmed 96 hours after inoculation. Investigations on transgenic human ACE2 mice and Rhesus monkey induced multifocal pneumonia with interstitial hyperplasia [1].
As it is now well known, COVID-19 is transmitted via droplets during close unprotected contact between an infector and infected, while the airborne spread has not been reported. Its human-to-human transmission is largely occurring in families. Testing for COVID-19 disease includes RT-PCR testing in influenza-like-illness (ILI) and severe acute respiratory infection (SARI), as well as testing of results among all visitors to fever clinics [1][2][3][4][5][6]. There is no pre-existing immunity for COVID-19 because it is caused by new human pathogen. That is the main risk factor of infection. Furthermore, its transmission dynamics is very fast, particularly during the epidemic growth phase and in the post-control period [1][2][3][4][5][6].
Approximately 80% of laboratory-confirmed cases have mild to moderate disease, which includes non-pneumonia and pneumonia cases, 13.8% have a severe disease (dyspnea, respiratory frequency ≥30/minute, blood oxygen saturation ≤93%, PaO2/FiO2 ratio <300, and/or lung infiltrates >50% of the lung area within 24-48 hours, whereby 6.1% are critical (respiratory failure, septic shock, and/or multiple organ dysfunction/failure). Individuals at the highest risk for severe disease and death are people aged over 60 years and those with underlying conditions such as hypertension, diabetes, cardiovascular disease, chronic respiratory disease, and cancer. Disease in children appears to be relatively rare and mild with approximately 2.4% of the total reported cases amongst individuals aged less than 19 years. A very small proportion of those aged less than 19 years have developed severe (2.5%) or critical disease (0.2%). Mortality increases with age, with the highest mortality among people over 80 years of age (CFR 21.9%) [1][2][3][4][5][6].
The CFR is higher among males compared to females (4.7% vs. 2.8%), while patients who reported comorbid conditions had much higher rates of COVID-19: 13.2% were with cardiovascular disease, 9.2% diabetes, 8.4% hypertension, 8.0% chronic respiratory disease, and 7.6% with cancer. This virus is highly contagious, can spread quickly, and must be considered capable of causing enormous health, economic and societal damage, on the global level. It is unique among human coronaviruses because it combines high transmissibility, substantial fatal outcomes in some high-risk groups, and the ability to cause huge societal and economic disruption [1][2][3][4][5][6].

Nano masks and respirators 95
After the appearance of COVID-19, the use of facemasks has become necessary. The surgical facemasks are widely used by medical workers for protection from infection in contact with patients with respiratory diseases. The used facemasks are often very low quality. In the last time, the best filter and masks among them, like N95 filters in respirators show very low protection efficiency against COVID 19. Besides, model simulations, using data relevant to COVID-19 dynamics in the USA, suggest that broad application of even so relatively ineffective facemasks may significantly reduce community transmission of COVID-19 and decrease patient hospitalizations and deaths. Medical masks (i.e., surgical masks and N95 respirators) have yielded more controversial results [7,8,9]. The traditional model for respiratory disease transmission infection via infectious droplets (generally 5-10 µm) that have a short lifetime in the air and infect the upper respiratory tract, or finer aerosols, which may remain in the air for many hours, was applied in the case of SARS-CoV-2 [10,11,12]. Although the N95 respirator vs. surgical mask offers better protection, great numbers of medical workers that use them are infected by COVID-19, because medical systems in different countries are generally jeopardized. It is well known that such masks may have only a limited effect (but still nontrivial, in terms of absolute lives saved) in more severe epidemics, such as the ongoing epidemic COVID-19.
Masks with HEPA filters are designed with a filter that can be inserted and replaced made of a double-layered HEPA air filter, such as the 3M Filtrete 2800 Ultrafine Filter. The efficacy of non-fit tested HEPA filter masks, such as the Totobobo mask, is still inferior compared to N95, and it should be used with great caution [9]. Additionally it has been noticed that 3M Filtrete MP 2800 filter out particles as small as 0.3 microns, while the size of some viruses, like SARS-CoV-2 is about 0.06-0.14 microns in size. Therefore, its relative efficacy to filter pathogens of the current pandemic is very suspicious. Besides, many researchers think that their efficiency significantly reduces after washing, even in the case of the polypropylene filters (similar to 3M Filtrates). A particular challenge is the maintenance of reusable filters on hospital premises and their alternative decontamination and sterilization with methods such as ultraviolet germicidal irradiation and autoclaving. N-95 respirator has 95% filtration efficiency, while surgical mask has < 50% filtration ability for small particles (0.1-0.4 µm in diameter). Corresponding studies showed that airflow through the commercial N-95 respirator was very poor, and inhaled air from the enclosed space of the mask was quite hypoxic, with a fraction of inspired oxygen [FIO2], about 16.4%, that is why it is not safe for patients who have pulmonary or cardiovascular diseases or sepsis and who require a good oxygen supply [10].
Future evaluation of masks should include not only their filtration efficiency and safety of their use but also its efficiency in preventing bacteria and viruses infections. Improved masks are urgently needed in order to help to prevent communicable infectious diseases. Finally, although filters in N-95 respirator and surgical masks can partially prevent inhalation of the nano-metric and submicronic airborne particles, they cannot protect us from viruses, like SARS-CoV-2. This fact is shown in one brilliant way given in the text of meta-analysis, which details are described in the Appendix of this paper [12,13].

Silver and silver compounds
In general, silver is the most efficient antimicrobial and antiviral metal, although some other metals such as zinc, copper, and cobalt have shown effective inhibition of microbes. It is very often used for preventing bacterial colonization of medical devices, as well as on other textile fabrics [12,14]. It is believed that heavy metals react with proteins by combining the thiol (-SH) groups, which leads to protein inactivation. In the presence of moisture (e.g. from the air), metal ions are formed and inhibit microbial replication. Proposed antimicrobial mechanism is that metal ions destroy or pass through the cell membrane, and bond to the -SH group of cellular enzymes. The consequent critical decrease of enzymatic activity causes microorganism's metabolisms to undergo change and their growth to be inhibited, up to the death of the cell [14]. As it is known, metal ions catalyze the production of oxygen radicals that interact with the molecular structure of bacteria. Then, silver ions lead to protein denaturation influencing cell death by their reaction with nucleophilic amino acid residues in proteins, and their attachments to sulphydryl, amino, imidazole, phosphate and carboxyl groups of membrane or enzyme proteins. Silver also inhibits a number of oxidative enzymes such as yeast alcohol dehydrogenase, influencing the uptake of succinate by membrane vesicles and respiratory chain of Escherichia coli, consequently causing metabolite efflux and interference with DNA replication [14,15] The actual mechanisms by which antimicrobial substances control microbial growth vary and depend on the type of agent used. Generally, they prevent cell reproduction, damage cell walls or cell permeability, denature proteins, block enzymes and make cell survival impossible [16,17], while polycationic antimicrobial compounds damage their cytoplasmic membranes following the mechanism adsorption and diffusion through the cell walls, binding to the cytoplasmic membrane and its disruption, releasing cytoplasmic constituents like K + ion, DNA and RNA, and finally causing the cells death. Antimicrobial agents act in two distinct ways: by contact and by diffusion [16,17].

Surgical face ALBO -nanosliver mask
For the first time, innovative company ALBOS doo, following the procedure that will be patented soon, started producing new protection facemasks. This concept is completely different from the recently used concepts of protection against viruses. Nanosilver masks are very safe, due to its high activity against viruses and bacteria, as it was shown in the example of the Staphylococcus aureus bacteria. This experiment, made in Laboratory for control food and drugs, of Scientific Veterinary Institute of Serbia, using so-called horizontal method for quantification of the number of positive staphylococci, showed that the concentration of the total number and coagulase-positive Staphylococcus bacteria measured in CFU/ml is less than 1. For comparison both of these values measured for surgical masks (masks without nanosilver) were higher: 1,500,000 CFU/ml, while the total number of bacteria was 18,180,000 CFU/ml. From these data it is clear that these differences are incredibly high, showing the extraordinary efficiency for the concept with nanosilver in antimicrobial protection, not only for health workers but also for general population.
Important question is why Staphylococcus aureus was chosen for the experiment. Knowing that they are the leading cause of both healthcare-and community-associated bloodstream infections in the industrialized world influencing significant morbidity and mortality, it was natural to choose such kind of bacteria. Namely, S. aureus is a pathogen, which frequently attacks the cardiovascular system inducing its serious complications, such as infective endocarditis or thrombophlebitis, and causing organ failure and death [18][19][20][21][22][23]. Although similar investigation but on SARS-CoV-2 virus was not done yet, based on the results for Staphylococcus aureus, it is possible to extrapolate results. Knowing that virus size is of the order of 100 nm (HIV size is about 120 nm and SARS virus size is also about 100 nm), probably these sizes of NSPs of about 10 nm and less are the most effective in interacting with the virus because the NSPs are significantly smaller than the virus. In the case of the ALBO nanosilver mask, the suitable sizes of NSPs are within the range of 3-10 nm, which are probably the most effective range of NSPs for the killing of any kind of viruses ( Figure 1).
In the case of HIV-1, it is approved that silver nanoparticles inhibit the initial stages of the HIV-1 infection cycle by blocking viral entry, blocking particularly the gp120-CD4 interaction. Besides, silver nanoparticles inhibit also post-entry stages of the HIV-1 life cycle, due to form complexes between silver ions and its donor groups containing sulfur, oxygen, or nitrogen, inside thiols or phosphates in viruses' amino acids and nucleic acids. As a consequence, reverse transcription by direct binding to the RNA or DNA is reduced, as it is shown in Figure 2 [18][19][20][21][22][23]. Similar kind of interactions can be expected even in the case of SARS-CoV-2.

CONCLUSION
Numerous disadvantages of protective surgical facemasks and masks and respirators with nanofillers N95 are presented. Mata analyses showed no statistically significant differences between them. Besides, it is discussed that a new approach with ALBO filters and masks on the base of nanosilver is new very promising approach. The extraordinary efficiency of these gears on the example of Staphylococcus aureus, indicates efficiency of this product  There are many conflicting and confusing recommendations for the severe acute respiratory syndrome (SARS) and pandemic influenza: the World Health Organization (WHO) recommends using masks in low risk situations and respirators in high risk situations, but the Centers for Disease Control and Prevention (CDC) recommends using respirators in both low and high risk situations. Besides, N95 respirators are frequently unaffordable [24,25]. Additionally, previous Meta analyses concluded that real efficiency of N95 respirators is not scientifically and statistically confirmed. More rigorous studies comparing N95 respirators with surgical masks against influenza published in the past decade were not included in previous Meta analyses [26,27]. However, systematic review and meta analysis on the effectiveness of filters N95 in respirators and surgical masks for prevention of` influenza are extraordinary significant.
One review that compared the efficiency of respirators and ordinary surgical masks was provided for the first time [24]. This review included: i) randomized controlled trial (RCT) study (including cluster randomized trial) and nonrandomized controlled study; ii) humans with influenza (including pandemic strains, seasonal influenza A or B viruses and zoonotic viruses such as swine or avian influenza), and other respiratory viral infections (as a proxy for influenza); iii) N95 respirators versus surgical masks, iv) primary outcome: laboratory confirmed influenza; v) secondary outcomes: laboratory confirmed respiratory viral infections, laboratory confirmed bacterial colonization, laboratory confirmed respiratory infection, and influenza-like illness; and vi) settings: hospital or community [24].
RCTs were selected due to the potential of high evidence level results. Search strategy included all corresponding topic data on PubMed, EMBASE, and The Cochrane Library databases from January 27, 2020; to identify all published papers with the subject related to evaluating the use of masks for preventing influenza. The strategy was adequately adjusted to use in other databases, which included all papers with this topic in the past five years from January 27, 2015, to January 27, 2020. A search also included ClinicalTrials.gov to obtain unpublished data, without publication status and language restrictions on selecting the studies [24][25][26][27].
Two reviewers were involved, who independently screened the articles based on the titles, abstracts, and full texts. Then, both of them independently extracted the data included in study like the first author, publication year, country, disease, details of study population and intervention, study design, sample size, settings, and results, while all disagreements were subjected to discussion. Both reviewers independently assessed the risk of bias of the selected RCTs using the Cochrane Risk of Bias tool, which included domains on random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessors, incomplete outcome data, and selective reporting [24][25][26][27].
For each RCT, every domain was judged among 3 levels: high risk, unclear risk, and low risk. Disagreements were resolved by discussion. All statistical analyses were performed using Review Manager (RevMan) version 5.3. Comparable data from studies with similar interventions and outcomes were pooled using forest plots. Relative risk (RR) with 95% confidence intervals (CIs) for dichotomous data was used as the effect measure. Between studies heterogeneity was assessed using the I2 for each pooled estimate [28].
It was adopted a random effects model for heterogeneity p < 0.10 and performed a subgroup analysis based on the settings (hospital, community) due to the opportunity of clinical heterogeneity. A sensitivity analysis was done to evaluate the robustness of the results by excluding individual studies for each forest plot. A total of 9,171 participants in Canada, Australia, China, or America were included, and the number of participants in each RCT ranged from 435 to 5180 patients. The follow up duration varied from 2 to 15 weeks. Five studies included participants in hospitals and one in households. Because of different definitions of outcome in included studies, it was redefined the laboratory confirmed respiratory infection as respiratory influenza, other viruses, or bacteria infection. Five RCTs involving 8,444 participants reported laboratory confirmed influenza. Meta analysis with fixed effects model revealed no statistically significant differences in preventing influenza using N95 respirators and surgical masks (RR = 1.09, 95% CI 0.92-1.28, p > 0.05) [24,29,30].
The results of subgroup analyses were consistent regardless of the observed hospital or the community. The results of the sensitivity analysis were not changed after exclusion of any trial. Four RCTs, involving 3,264 participants reported laboratory confirmed respiratory viral infections. Meta analysis with a fixed effects model revealed no statistically significant differences in preventing respiratory viral infections using N95 respirators and surgical masks (RR = 0.89, 95% CI 0.70-1.11, p > 0.05). The results of subgroup analyses were also consistent regardless of the hospital or the community [24,29,30]. Two RCTs involving 6,621 participants with reported laboratory confirmed respiratory infection. Meta analysis with random effects model revealed no statistically significant differences in preventing respiratory infection using N95 respirators and surgical masks in hospitals (RR = 0.74, 95% CI 0.42-1.29, p > 0.05) [24,29,30].
Five RCTs involving 8,444 participants reported influenza-like illness [24,29,30]. Meta analysis with the random effects model also revealed no statistically significant differences in preventing influenza-like illness using N95 respirators and surgical masks (RR = 0.61, 95% CI 0.33-1.14, p > 0.05). The sensitivity analysis showed results remained unchanged after excluding each trial [24,29,30]. This meta-analysis showed no statistically significant differences in preventing laboratory confirmed influenza, laboratory confirmed respiratory viral infections, laboratory confirmed respiratory infection, and influenza like illness using N95 respirators and surgical masks. In subgroup analysis, similar results could be found in the hospital and community for laboratory confirmed influenza and laboratory confirmed respiratory viral infections. However, sensitivity analysis showed unstable results for the prevention of laboratory confirmed respiratory viral infections and laboratory confirmed respiratory infection. Through the course of influenza pandemics, large numbers of facemasks may be required to use in long periods to protect people from infections [24,31].
Using N95 respirators is likely to result in discomfort, for example, headaches. A previous study reported that there was an inverse relationship between the level of compliance with wearing N95 respirator and the risk of clinical respiratory illness. It is difficult to ensure high compliance due to discomfort of N95 respirators in all studies [24,31]. The reason for the similar effects on preventing influenza with use of N95 respirators versus surgical masks may be related to low compliance to N95 respirators wear, which may lead to more frequent doffing compared to surgical masks [31]. Although N95 respirators in the routine use seem to be less acceptable than surgical masks due to more significant discomfort, it should be noted that surgical masks are primarily designed to protect the environment from the wearer, whereas the respirators are supposed to protect the wearer from the environment [24,32].
In conclusion, the current meta analysis shows the use of N95 respirators compared to surgical masks is not associated with lower risk of laboratory confirmed influenza. It suggests that N95 respirators should not be recommended for the general public and medical staff that are not in close contact with influenza patients or suspected patients [24,32]. Naučni institut za veterinarstvo Srbije, Zavod za kontrolu hrane i lekova, Beograd, Srbija