Molecular Insights into the Binding Behavior of Imidazolium Ionic Liquids to the Receptor Binding Domain of the SARS-CoV-2 Spike Protein.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is considered as a key target for the design and development of COVID-19 drugs and inhibitors. Due to their unique structure and properties, ionic liquids (ILs) have many special interactions with proteins, showing great potential in biomedicine. Nevertheless, few research studies have been carried out on ILs and the spike RBD protein. Here, we explore the interaction of ILs and the RBD protein through large-scale molecular dynamics simulations (4 µs in total). It was found that IL cations with long alkyl chain lengths (nchain) could spontaneously bind to the cavity region of the RBD protein. The longer the alkyl chain is, the stabler the cations bind to the protein. The binding free energy (ΔG) had the same trend, peaking at nchain = 12 with -101.19 kJ/mol. The cationic chain lengths and their fit to the pocket are decisive factors that influence the binding strength of cations and proteins. The cationic imidazole ring has a high contact frequency with phenylalanine and tryptophan, and the hydrophobic residues phenylalanine, valine, leucine, and isoleucine are the most interacting residues with side chains of cations. Meanwhile, through analysis of the interaction energy, the hydrophobic and π-π interactions are the main contributors to the high affinity between cations and the RBD protein. In addition, the long-chain ILs would also act on the protein through clustering. These studies not only provide insights into the molecular interaction between ILs and the RBD of SARS-CoV-2 but also contribute to the rational design of IL-based drugs, drug carriers, and selective inhibitors as a therapeutic for SARS-CoV-2.

Cationic crosslinked carbon dots-adjuvanted intranasal vaccine induces protective immunity against Omicron-included SARS-CoV-2 variants.

Mucosal immunity plays a significant role in the first-line defense against viruses transmitted and infected through the respiratory system, such as SARS-CoV-2. However, the lack of effective and safe adjuvants currently limits the development of COVID-19 mucosal vaccines. In the current study, we prepare an intranasal vaccine containing cationic crosslinked carbon dots (CCD) and a SARS-CoV-2 antigen, RBD-HR with spontaneous antigen particlization. Intranasal immunization with CCD/RBD-HR induces high levels of antibodies with broad-spectrum neutralization against authentic viruses/pseudoviruses of Omicron-included variants and protects immunized female BALB/c mice from Omicron infection. Despite strong systemic cellular immune response stimulation, the intranasal CCD/RBD-HR vaccine also induces potent mucosal immunity as determined by the generation of tissue-resident T cells in the lungs and airway. Moreover, CCD/RBD-HR not only activates professional antigen-presenting cells (APCs), dendritic cells, but also effectively targets nasal epithelial cells, promotes antigen binding via sialic acid, and surprisingly provokes the antigen-presenting of nasal epithelial cells. We demonstrate that CCD is a promising intranasal vaccine adjuvant for provoking strong mucosal immunity and might be a candidate adjuvant for intranasal vaccine development for many types of infectious diseases, including COVID-19.

Derivatization of sialylated glycopeptides plus based sialoglycopeptides enrichment using cation exchange media.

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate <1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate >100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.

Comparison of rainwater quality before and during the MCO using chemometric analyses.

This study aimed to classify the spatiotemporal analysis of rainwater quality before and during the Movement Control Order (MCO) implementation due to the COVID-19 pandemic. Chemometric analysis was carried out on rainwater samples collected from 24-gauge stations throughout Malaysia to determine the samples' chemical content, pH, and conductivity. Other than that, hierarchical agglomerative cluster analysis (HACA) and discriminant analysis (DA) were used to classify the quality of rainwater at each location into four clusters, namely good, satisfactory, moderate, and bad clusters. Note that DA was carried out on the predefined clusters. The reduction in acidity levels occurred in 11 stations (46% of overall stations) after the MCO was implemented. Chemical content and ion abundance followed a downward trend, indicating that Cl- and Na+ were the most dominant among the anions and cations. Apart from that, NH4+, Ca2+, NO3-, and SO42- concentrations were evident in areas with significant anthropogenic activity, as there was a difference in the total chemical content in rainwater when compared before and during the MCO. Based on the dataset before the MCO, 75% of gauge stations were in the good cluster, 8.3% in the satisfactory cluster, 12.5% in the moderate cluster, and 4.2% in the bad cluster. Meanwhile, the dataset during the MCO shows that 72.7% of gauge stations were in the good cluster, 9.1% in the satisfactory cluster, 9.1% in the moderate, and 4.5% in the bad cluster. From this study, the chemometric analysis of the year 2020 rainwater chemical composite dataset strongly indicates that reduction of human activities during MCO affected the quality of rainwater.

A cationic lipid with advanced membrane fusion performance for pDNA and mRNA delivery.

The success of mRNA vaccines for COVID-19 prevention raised global awareness of the importance of nucleic acid drugs. The approved systems for nucleic acid delivery were mainly formulations of different lipids, yielding lipid nanoparticles (LNPs) with complex internal structures. Due to the multiple components, the relationship between the structure of each component and the overall biological activity of LNPs is hard to study. However, ionizable lipids have been extensively explored. In contrast to former studies on the optimization of hydrophilic parts in single-component self-assemblies, we report in this study on structural alterations of the hydrophobic segment. We synthesize a library of amphiphilic cationic lipids by varying the lengths (C = 8-18), numbers (N = 2, 4), and unsaturation degrees (Ω = 0, 1) of hydrophobic tails. Notably, all self-assemblies with nucleic acid have significant differences in particle size, stability in serum, membrane fusion, and fluidity. Moreover, the novel mRNA/pDNA formulations are characterized by overall low cytotoxicity, efficient compaction, protection, and release of nucleic acids. We find that the length of hydrophobic tails dominates the formation and stability of the assembly. And at a certain length, the unsaturated hydrophobic tails enhance the membrane fusion and fluidity of assemblies and thus significantly affect the transgene expression, followed by the number of hydrophobic tails.

R-DOTAP Cationic Lipid Nanoparticles Outperform Squalene-Based Adjuvant Systems in Elicitation of CD4 T Cells after Recombinant Influenza Hemagglutinin Vaccination.

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. Prototypic pathogens of this type are influenza and SARS-CoV-2, where the receptor-binding protein exhibits extremely high variability in its receptor-binding regions. T cells, known to target many viral proteins, and within these, highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms but are often poorly recruited by current vaccine strategies. Here, we have studied a promising novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which was previously recognized for its ability to generate anti-tumor immunity through the induction of potent cytotoxic CD8 T cells. Using a preclinical mouse model, we have assessed an R-DOTAP nanoparticle adjuvant system for its ability to promote CD4 T cell responses to vaccination with recombinant influenza protein. Our studies revealed that R-DOTAP consistently outperformed a squalene-based adjuvant emulsion, even when it was introduced with a potent TLR agonist CpG, in the ability to elicit peptide epitope-specific CD4 T cells when quantified by IFN-γ and IL-2 ELISpot assays. Clinical testing of R-DOTAP containing vaccines in earlier work by others has demonstrated an acceptable safety profile. Hence, R-DOTAP can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines.

Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice.

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

Development of Lipid Nanoparticles for the Delivery of Macromolecules Based on the Molecular Design of pH-Sensitive Cationic Lipids.

Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.

Development of lipid nanoparticles and liposomes reference materials (II): cytotoxic profiles.

Lipid based nanocarriers are one of the most effective drug delivery systems that is evident from the recent COVID-19 mRNA vaccines. The main objective of this study was to evaluate toxicity of six lipid based formulations with three surface charges-anionic, neutral or cationic, to establish certified reference materials (CRMs) for liposomes and siRNA loaded lipid nanoparticles (LNP-siRNA). Cytotoxicity was assessed by a proliferation assay in adherent and non-adherent cell lines. High concentration of three LNP-siRNAs did not affect viability of suspension cells and LNP-siRNAs were non-toxic to adherent cells at conventionally used concentration. Systematic evaluation using multiple vials and repeated test runs of three liposomes and three LNP-siRNA formulations showed no toxicity in HL60 and A549 cells up to 128 and 16 µg/mL, respectively. Extended treatment and low concentration of LNPs did not affect the viability of suspension cells and adherent cells at 96 h. Interestingly, 80% of A549 and HL60 cells in 3D conditions were viable when treated with cationic LNP-siRNA for 48 h. Taken together, anionic, cationic and neutral lipid formulations were non-toxic to cells and may be explored further in order to develop them as drug carriers.

Enzyme-Catalyzed One-Step Synthesis of Ionizable Cationic Lipids for Lipid Nanoparticle-Based mRNA COVID-19 Vaccines.

Ionizable cationic lipid-containing lipid nanoparticles (LNPs) are the most clinically advanced non-viral gene delivery platforms, holding great potential for gene therapeutics. This is exemplified by the two COVID-19 vaccines employing mRNA-LNP technology from Pfizer/BioNTech and Moderna. Herein, we develop a chemical library of ionizable cationic lipids through a one-step chemical-biological enzyme-catalyzed esterification method, and the synthesized ionizable lipids were further prepared to be LNPs for mRNA delivery. Through orthogonal design of experiment methodology screening, the top-performing AA3-DLin LNPs show outstanding mRNA delivery efficacy and long-term storage capability. Furthermore, the AA3-DLin LNP COVID-19 vaccines encapsulating SARS-CoV-2 spike mRNAs successfully induced strong immunogenicity in a BALB/c mouse model demonstrated by the antibody titers, virus challenge, and T cell immune response studies. The developed AA3-DLin LNPs are an excellent mRNA delivery platform, and this study provides an overall perspective of the ionizable cationic lipids, from aspects of lipid design, synthesis, screening, optimization, fabrication, characterization, and application.

Investigation of the ionic conditions in SiRNA-mediated delivery through its carriers in the cell membrane: a molecular dynamic simulation.

SiRNA is a new generation of drug molecules and a new approach for treating a variety of diseases such as cancer and viral infections. SiRNA delivery to cells and translocation into cytoplasm are the main challenges in the clinical application of siRNA. Lipid carriers are one of the most successful carriers for siRNA delivery. In this study, we investigated the interaction of siRNA with a zwitterionic bilayer and how ion concentration and lipid conjugation can affect it. The divalent cation such as Mg2+ ions could promote the siRNA adsorption on the bilayer surface. The cation ions can bind to the head groups of lipids and the grooves of siRNA molecules and form bridges between the siRNA and bilayer surface. Our findings demonstrated the bridges formed by divalent ions could facilitate the attachment of siRNA to the membrane surface. We showed that the divalent cations can regulate the bridging-driven membrane attachment and it seems the result of this modulation can be used for designing biomimetic devices. In the following, we examined the effect of cations on the interaction between siRNA modified by cholesterol and the membrane surface. Our MD simulations showed that in the presence of Mg2+, the electrostatic and vdW energy between the membrane and siRNA were higher compared to those in the presence of NA+. We showed that the electrostatic interaction between membrane and siRNA cannot be facilitated only by cholesterol conjugated. Indeed, cations are essential to create coulomb repulsion and enable membrane attachment. This study provides important insight into liposome carriers for siRNA delivery and could help us in the development of siRNA-based therapeutics. Due to the coronavirus pandemic outbreak, these results may shed light on the new approach for treating these diseases and their molecular details.

A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.

Electrostatic Map of the SARS-CoV-2 Virion Specifies Binding Sites of the Antiviral Cationic Photosensitizer.

Electrostatics is an important part of virus life. Understanding the detailed distribution of charges over the surface of a virus is important to predict its interactions with host cells, antibodies, drugs, and different materials. Using a coarse-grained model of the entire viral envelope developed by D. Korkin and S.-J. Marrink's scientific groups, we created an electrostatic map of the external surface of SARS-CoV-2 and found a highly heterogeneous distribution of the electrostatic potential field of the viral envelope. Numerous negative patches originate mainly from negatively charged lipid domains in the viral membrane and negatively charged areas on the "stalks" of the spike (S) proteins. Membrane (M) and envelope (E) proteins with the total positive charge tend to colocalize with the negatively charged lipids. In the E protein pentamer exposed to the outer surface, negatively charged glutamate residues and surrounding lipids form a negative electrostatic potential ring around the channel entrance. We simulated the interaction of the antiviral octacationic photosensitizer octakis(cholinyl)zinc phthalocyanine with the surface structures of the entire model virion using the Brownian dynamics computational method implemented in ProKSim software (version r661). All mentioned negatively charged envelope components attracted the photosensitizer molecules and are thus potential targets for reactive oxygen generated in photosensitized reactions.

Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells.

Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.

Infectivity and Morphology of Bovine Coronavirus Inactivated In Vitro by Cationic Photosensitizers.

Bovine coronaviruses (BCoVs), which cause gastrointestinal and respiratory diseases in cattle, and are genetically related to the human coronavirus HCoV-OC43, which is responsible for up to 10% of common colds, attract increased attention. We applied the method of photodynamic inactivation with cationic photosensitizers (PSs) to reduce the titers of BCoV and studied the morphological structure of viral particles under various modes of photodynamic exposure. The samples of virus containing liquid with an initial virus titer of 5 Log10 TCID50/mL were incubated with methylene blue (MB) or octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+) at concentrations of 1-5 µM for 10 min in the dark at room temperature. After incubation, samples were irradiated with LED (emission with maximum at 663 nm for MB or at 686 nm for Zn-PcChol8+) with light doses of 1.5 or 4 J/cm2. Next, the irradiation titrated virus containing liquid was studied using negative staining transmission electron microscopy. MB and Zn-PcChol8+ at concentrations of 1-5 µM, in combination with red light from LED sources in the low doses of 1.5-4.0 J/cm2, led to a decrease in BCoV titers by at least four orders of magnitude from the initial titer 5 Log10 TCID50/mL. Morphological changes in photodamaged BCoVs with increasing PS concentrations were loss of spikes, change in shape, decreased size of virus particles, destruction of the envelope, and complete disintegration of viruses. BCoV has been found to be sensitive to MB, which is the well-known approved drug, even in the absence of light.

Optimization of DOTAP/chol Cationic Lipid Nanoparticles for mRNA, pDNA, and Oligonucleotide Delivery.

Lipid nanoparticles (LNPs) can be used as delivery vehicles for nucleic acid biotherapeutics. In fact, LNPs are currently being used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. Cationic LNPs composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) LNPs have been classified as one of the most efficient gene delivery systems and are being tested in numerous clinical trials. The objective of this study was to examine the effect of the molar ratio of DOTAP/chol, PEGylation, and lipid to mRNA ratio on mRNA transfection, and explore the applications of DOTAP/chol LNPs in pDNA and oligonucleotide transfection. Here we showed that PEGylation significantly decreased mRNA transfection efficiency of DOTAP/chol LNPs. Among non-PEGylated LNP formulations, 1:3 molar ratio of DOTAP/chol in DOTAP/chol LNPs showed the highest mRNA transfection efficiency. Furthermore, the optimal ratio of DOTAP/chol LNPs to mRNA was tested to be 62.5 µM lipid to 1 µg mRNA. More importantly, these mRNA-loaded nanoparticles were stable for 60 days at 4 °C storage without showing reduction in transfection efficacy. We further found that DOTAP/chol LNPs were able to transfect pDNA and oligonucleotides, demonstrating the ability of these LNPs to transport the cargo into the cell nucleus. The influence of various factors in the formulation of DOTAP/chol cationic LNPs is thus described and will help improve drug delivery of nucleic acid-based vaccines and therapies.

In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression.

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.

Targeting of Silver Cations, Silver-Cystine Complexes, Ag Nanoclusters, and Nanoparticles towards SARS-CoV-2 RNA and Recombinant Virion Proteins.

Background: Nanosilver possesses antiviral, antibacterial, anti-inflammatory, anti-angiogenesis, antiplatelet, and anticancer properties. The development of disinfectants, inactivated vaccines, and combined etiotropic and immunomodulation therapy against respiratory viral infections, including COVID-19, remains urgent. Aim: Our goal was to determine the SARS-CoV-2 molecular targets (genomic RNA and the structural virion proteins S and N) for silver-containing nanomaterials. Methods: SARS-CoV-2 gene cloning, purification of S2 and N recombinant proteins, viral RNA isolation from patients' blood samples, reverse transcription with quantitative real-time PCR ((RT)2-PCR), ELISA, and multiplex immunofluorescent analysis with magnetic beads (xMAP) for detection of 17 inflammation markers. Results: Fluorescent Ag nanoclusters (NCs) less than 2 nm with a few recovered silver atoms, citrate coated Ag nanoparticles (NPs) with diameters of 20-120 nm, and nanoconjugates of 50-150 nm consisting of Ag NPs with different protein envelopes were constructed from AgNO3 and analyzed by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), ultraviolet-visible light absorption, and fluorescent spectroscopy. SARS-CoV-2 RNA isolated from COVID-19 patients' blood samples was completely cleaved with the artificial RNase complex compound Li+[Ag+2Cys2-(OH-)2(NH3)2] (Ag-2S), whereas other Ag-containing materials provided partial RNA degradation only. Treatment of the SARS-CoV-2 S2 and N recombinant antigens with AgNO3 and Ag NPs inhibited their binding with specific polyclonal antibodies, as shown by ELISA. Fluorescent Ag NCs with albumin or immunoglobulins, Ag-2S complex, and nanoconjugates of Ag NPs with protein shells had no effect on the interaction between coronavirus recombinant antigens and antibodies. Reduced production of a majority of the 17 inflammation biomarkers after treatment of three human cell lines with nanosilver was demonstrated by xMAP. Conclusion: The antiviral properties of the silver nanomaterials against SARS-CoV-2 coronavirus differed. The small-molecular-weight artificial RNase Ag-2S provided exhaustive RNA destruction but could not bind with the SARS-CoV-2 recombinant antigens. On the contrary, Ag+ ions and Ag NPs interacted with the SARS-CoV-2 recombinant antigens N and S but were less efficient at performing viral RNA cleavage. One should note that SARS-CoV-2 RNA was more stable than MS2 phage RNA. The isolated RNA of both the MS2 phage and SARS-CoV-2 were more degradable than the MS2 phage and coronavirus particles in patients' blood, due to the protection with structural proteins. To reduce the risk of the virus resistance, a combined treatment with Ag-2S and Ag NPs could be used. To prevent cytokine storm during the early stages of respiratory infections with RNA-containing viruses, nanoconjugates of Ag NPs with surface proteins could be recommended.