Aerosol capture and coronavirus spike protein deactivation by enzyme functionalized antiviral membranes

The airborne transmission of coronaviruses necessitates the development of barrier technologies that effectively reduce both aerosol and viral spread. Current respiratory face masks, such as N95 masks, while offering some protection, have limitations in capturing smaller particles (<300 nm) and maintaining their electrically charged properties after decontamination. Studies have shown inconsistencies in their effectiveness, with only 70% of tested N95 masks providing >90% rejection of coronavirus-sized particles. This research explores the potential of thin-film polymeric water-filtration membranes, specifically poly(vinylidene fluoride) (PVDF), as a superior alternative. PVDF membranes offer precise control over pore size and structure, enabling optimization of air permeability and pressure drop while maintaining high filtration efficiency. Furthermore, the addition of antiviral agents can further enhance protection. While various antiviral agents have been explored, including silver nanoparticles, copper sulfide, and salts, limitations exist regarding deactivation time and potential toxicity. Enzyme functionalization offers a promising solution, enabling rapid inactivation without the addition of toxic materials. Specific enzymes can denature proteins by causing small conformational changes, potentially disrupting the SARS-CoV-2 spike glycoprotein (SGP) and deactivating the virus. Subtilisin Carlsberg, a stable serine protease, is a suitable candidate for this purpose, given its stability in various conditions and its well-understood substrate-binding mechanisms. The addition of poly(methacrylic acid) (PMAA) can further enhance enzyme functionalization and longevity, providing a non-toxic and biocompatible polymer for water retention and electrostatic interaction with the enzyme. This study aims to develop a membrane filter that combines enhanced aerosol capture with antiviral enzyme functionalization for superior protection against coronavirus transmission.

Existing literature highlights the limitations of current respiratory face masks, particularly N95 masks, in consistently preventing coronavirus transmission due to their inability to effectively filter smaller particles and maintain their filtration efficiency after decontamination. Studies have shown that water-filtration membranes have demonstrated virus filtration capabilities, but their application to aerosol capture in respiratory protection remains largely unexplored. The research explores the use of PVDF membranes, known for their efficacy in capturing coronavirus-simulated aerosols, and demonstrates that membrane characteristics like thickness, porosity, and pore size can be precisely controlled to optimize air permeability while maximizing particle capture. The incorporation of antiviral agents, specifically enzymes, offers a promising avenue for enhanced virus inactivation, surpassing the limitations of other antiviral agents in terms of efficacy and toxicity. Studies on enzyme functionalization of membranes have shown its effectiveness in inactivating viruses, making it a compelling strategy for creating more effective and safer respiratory protection. The selection of subtilisin Carlsberg, known for its stability and well-understood properties, is discussed within the context of literature, and the addition of PMAA is justified based on its biocompatibility and ability to enhance enzyme longevity and loading.

The study involved a comprehensive investigation of PVDF400 membranes and their functionalization for enhanced aerosol capture and virus deactivation. Membrane characterization included SEM imaging to determine porosity, thickness, and pore size distribution, and contact angle measurements to assess hydrophilicity. Air permeability was assessed using both experimental measurements and theoretical models, accounting for compressible fluid flow. The relationship between membrane variables (porosity, pore radius, thickness) and air permeability was established experimentally using different commercial membranes and stacking techniques. The performance of PVDF400 membranes, polysulfone (PS35), and nanofiltration (NF270) membranes was compared to commercial N95 and surgical masks in terms of air permeability and pressure drop. Polystyrene latex (PSL) particles, simulating coronavirus-sized aerosols, were used to evaluate filtration efficiency and the dual-mode capture mechanism of PVDF400 membranes (normal and reverse orientation). PMAA functionalization of PVDF400 membranes was achieved through a polymerization process, followed by subtilisin enzyme immobilization using batch and convective methods. The effectiveness of the functionalization was assessed by measuring enzyme loading and the impact on air permeability. The ability of the enzyme-functionalized membranes to denature SARS-CoV-2 spike glycoprotein (SGP) was evaluated under low-hydration conditions using DSC and Sypro Orange fluorescent dye. SGP-functionalized PSL particles were used to mimic real coronavirus particles and evaluate the denaturation process under realistic conditions. Finally, ambient air filtration tests were performed to assess the longevity and fouling resistance of the PMAA-PVDF membranes compared to N95 masks.

The study found that thin, asymmetric PVDF400 membranes exhibit high filtration efficiency (>98.90%) for 100-nm PSL particles, exceeding the performance of commercial N95 masks with a protection factor of 540 ± 380. The relationship between membrane variables and air permeability was confirmed, demonstrating the potential to tailor membrane properties for optimal breathability while maintaining high filtration efficiency. PMAA functionalization significantly increased enzyme loading (84-125%) without dramatically reducing air permeability. Subtilisin-functionalized PMAA-PVDF membranes effectively denatured SARS-CoV-2 SGP in low-hydration environments (0.02-0.2% water content) within 30 seconds, as shown through DSC and Sypro Orange fluorescence assays. Ambient air filtration tests demonstrated significantly less fouling and greater longevity for PMAA-PVDF membranes compared to N95 masks. The cost-effectiveness of the functionalized membranes was also demonstrated, with an estimated cost of $0.63 per mask, comparable to surgical masks and significantly less than N95 masks, while offering superior protection.

The findings demonstrate the potential of enzyme-functionalized PVDF membranes as a superior alternative to existing respiratory face masks for coronavirus protection. The ability to tailor membrane properties for optimal breathability while maintaining high filtration efficiency addresses the limitations of current N95 masks. The effective denaturation of the SARS-CoV-2 SGP in low-hydration environments highlights the advantage of enzyme functionalization, overcoming the challenges of maintaining enzyme activity in dry conditions. The significantly reduced fouling and increased longevity of the PMAA-PVDF membranes compared to N95 masks further supports their viability for practical application. The cost-effectiveness of the proposed membranes makes them a compelling alternative to existing PPE options.

This research successfully developed a novel membrane-based respiratory filter system that offers superior protection against coronavirus transmission compared to existing N95 masks. The combination of high filtration efficiency, effective spike protein deactivation, and extended longevity makes this system a promising advancement in respiratory protection. Future research could focus on optimizing membrane design for even greater breathability, exploring other enzymes and functionalization strategies, and conducting in vivo studies to validate the protective efficacy of these membranes.

The study primarily utilized PSL particles to simulate coronavirus aerosols, which may not fully capture the complexity of real-world viral particles. The filtration efficiency tests were conducted with non-neutralized PSL particles. While the study demonstrated the denaturation of SGP under low-hydration conditions, further in vivo testing is needed to confirm the overall antiviral efficacy in a real-world setting. The cost analysis is based on material costs and may not include the costs associated with large-scale manufacturing and distribution.