Covalent Functionalization of Melt-Blown Polypropylene Filters with Diazirine-Photosensitizer Conjugates Producing Visible Light Driven Virus Inactivating Materials
The SARS-CoV-2 pandemic has highlighted the weaknesses of relying on single-use mask and respirator personal protective equipment (PPE) and the global supply chain that supports this market. There have been no major innovations in filter technology for PPE in the past two decades. Non-woven textiles used for filtering PPE are single-use products in the healthcare environment; use and protection is focused on preventing infection from airborne or aerosolized pathogens such as Influenza A virus SARS-CoV-2. Recently, C-H bond activation under mild and controllable conditions was reported for crosslinking commodity aliphatic polymers such as polyethylene and polypropylene.Significantly, these are the same types of polymers used in PPE filtration systems. In this report, we take advantage of this C-H insertion method to covalently attach a photosensitizing zinc-porphyrin to the surface of a melt-blow non-woven textile filter material. With the photosensitizer covalently attached to the surface of the textile, illumination with visible light was expected to produce oxidizing 1 O2/ROS at the surface of the material that would result in pathogen inactivation. The filter was tested for its ability to inactivate Influenza A virus, an enveloped RNA virus similar to SARS-CoV-2, over a period of four hours with illumination of high intensity visible light. The photosensitizer-functionalized polypropylene filter inactivated our model virus by 99.99% in comparison to a control.
Introduction<!>Functionalization of non-woven polypropylene filter with photoactive porphyrin<!>Conclusions
<p>The increase in global demand of filter materials for personal protective equipment (PPE) from the SARS-CoV-2 pandemic has highlighted the weakness of the global supply chain and presented the imminent possibility of a limited supply for those that require PPE to complete their job without an undue risk of (self-)infection. 1,2 Potential solutions have been explored to allow PPE reuse in circumstances where supply is not able to keep up with demand. Thus far, the main approaches have focused on treating contaminated materials to inactivate any pathogens captured by the filter. These solutions use available sterilization processes including heat, steam, ethylene oxide, hydrogen peroxide vapour, UV-C, microwaves, salt, and photosensitizers. [3][4][5][6][7][8] The research using currently available sterilization processes has focused on the number of cycles before device failure, a timely procedure for practicality, and additional health risks-which for sterilization using ethylene oxide is currently in conflict. 4,9 Developing safe, reliable, and reusable filtering materials for PPE remains a challenge. Ideally, sterilizing should not require specialized equipment in order to improve access for healthcare workers and remote communities during times of global supply chain disruption and PPE shortages. In addition, there is potential to decrease the environmental impact of this large industry producing billions of single-use products per year that cannot be recycled. Finally, reports suggest contaminated surfaces and materials are able to produce aerosolized fomites (non-respiratory particles aerosolized from virus-contaminated surfaces) that are capable of spreading disease; specifically, it was hypothesized the aerosolized fomites were originating during removal of contaminated PPE. [10][11][12] An alternative approach to PPE reuse is to modify the base materials. Ideally, the materials possess filtering, continuous pathogen inactivation, and the ability to selfsterilize when exposed to a plentiful and accessible stimulus. Light is a stimulus that is abundant from both natural and synthetic sources, accessible, and relatively low in cost.</p><p>Photodynamic therapy (PDT) is a technology that utilizes light (including within the visible spectrum) and photosensitizing (PS) molecules to eradicate pathogens, reduce infection, or eliminate unwanted or cancerous tissue through the production and reaction of singlet oxygen ( 1 O2) and/or reactive oxygen species (ROS). 13,14 PDT has been used extensively in vivo and can be used without causing undue bodily harm. 14 When illuminated with visible light the photosensitizer is excited and proceeds through an energy transfer process with abundant triplet oxygen ( 3 O2) to produce 1 O2 and ROS locally that can be used to eliminate pathogens. The activity is non-specific and can eliminate resistant pathogens that are typically difficult to eradicate. [15][16][17] As an added benefit, pathogens cannot evolve resistance to 1 O2 as easily as they can to small molecule inhibitors. 15,17 Polymer composites, natural and synthetic polymers, and silica particles have been covalently functionalized with photosensitizing molecules and used for microbial inactivation, with the majority of applications focusing on bacterial inactivation. 18 Noncovalent impregnation of photosensitizers into bulk polymer matrices has also been demonstrated to result in microbial inactivation, 19 but the lack of covalent attachment within these systems may lead to leaching of the photosensitizer into the environment.</p><p>As a result, larger concentrations of the additive will be required in order to avoid a decrease in surface concentration-and therefore activity-over time. Furthermore, the leaching of active components can impact human and environmental health.</p><p>Covalent attachment approaches have the advantage of reducing the possibility of active component release (leaching) which would inevitably result in a decrease in performance over time. In addition, covalent attachment of photosensitizers can allow the surface of a material to be modified without affecting the bulk. This places the photosensitizer where it is needed (since only the surface of a material can interact with pathogens), thereby requiring less agent to achieve a desired response. In addition, the bulk properties of the substrate material (e.g. tensile strength or glass transition temperature) will be left unperturbed.</p><p>Rose Bengal, methylene blue, ruthenium-based complexes, pthalocyanines, and porphyrins have been covalently attached to natural and synthetic polymers that bear suitable chemical functionality to support the occurrence of chemical reactions at their surface. [20][21][22][23][24][25][26][27][28][29] These covalent attachment methods have been specifically designed for high-functionality materials that are not typically used in PPE/healthcare settings, or else require material pre-functionalization to allow 'click chemistry' (i.e. azide-alkyne coupling through Huisgen cycloadditions). A general solution for attaching photosensitizing molecules onto non-functional materials, such as aliphatic polymers used in PPE for example, remains an obstacle. These types of materials are comprised entirely of C-C and C-H bonds, and therefore lack any chemical "handles" that can be used for derivatization with photosensitizers.</p><p>One of our research groups recently described a family of bis-diazirine reagents that can be used to crosslink simple aliphatic polymers like polyethylene and polypropylene. 30,31 The reagents work by expelling nitrogen gas (N2) from the tethered diazirine groups upon thermal or photochemical stimulation, to afford high-energy carbenes can then undergo rapid and nearly barrierless insertion any available C-H bonds. In principle, such a technique can also be used to add functionality to base polymeric materials: the desired functional group can simply be tethered to one or more diazirine units, and the resulting conjugate can then be irreversibly linked to the surface of any polymeric material through appropriate activation of the diazirine function.</p><p>Herein, we employ this C-H activation strategy to permit the covalent functionalization of non-woven melt-blown polypropylene textiles with a common photoactive molecule (Fig. 1). Melt-blown polypropylene (MBPP) is a low-functionality polymer that serves as the main filtering component of surgical masks and N95/PAPR respirators. The lack of functional groups in this material means that it cannot be derivatized by traditional covalent linking methods. As a proof of concept, we demonstrated the inactivation of influenza A virus (IFV) using a visible light stimulus. IFV was chosen as a model because IFV itself is a major threat as a potential pandemic virus, and because IFV's enveloped structure and RNA genome is similar to those of SARS-CoV-2-the organism that is the proximal cause of the current pandemic. Researchers have indicated previously the importance of developing and testing inactivation methods against enveloped viruses, given that a pandemic was expected long before the emergence of SARS-CoV-2.   The MBPP substrate was chosen because of the widespread use of the non-woven material in filtering PPE, specifically single-use surgical masks and N95/personal air purifying respirators. A commercially available photosensitizer (1) was selected, which possessed a high quantum yield for the production of singlet oxygen with visible light, and which possessed nucleophilic residues for easy derivatization. To facilitate insertion into the surface accessible C-H bonds of the MBPP, we utilized a 3-trifluoromethyl-3phenyl-3H-diazirine motif (TFMPD) that is known to generate carbenes that are capable of ready insertion into C-H bonds. 33 A commercially available benzyl bromide (2) that incorporates the TFMPD motif was reacted with the pyridyl groups of 1 to produce the desired tetrakis diazirinyl zinc-porphyrin 3 in near-quantitative yield (Fig. 2a). We confirmed 1 O2 production with fluorescence spectroscopy from the excitation of 3 and subsequent phosphorescence of 1 O2 at 1273 nm. The 1 O2 generation proceeds through the photoexcitation of ground state 3 (S0) to generate the singlet excited state of 3 (S1), intersystem crossing to a triple state 3 (T3), and then an energy transfer to the triplet ground state of oxygen ( 3 O2) to produce singlet oxygen ( 1 O2) which then undergoes phosphorescence back to the ground state observed as an emission at a wavelength of 1273 nm indicating that the zinc-porphyrin moiety still possessed activity when functionalized with four diazirine groups (Fig. 2b).</p><!><p>The surface of the MBPP was then covalently functionalized with 3 using a modified procedure from Lepage et al. 30 To facilitate this, the reactivity of the diazirines in 3 was first analyzed by differential scanning calorimetry (DSC). These data revealed an onset temperature of 108 °C (determined by extrapolation of the tangent of the upward slope to the fitted baseline of the plot) and a peak temperature of 137 °C (Fig. 2c). With this information in hand, we chose to react compound 3 with MBPP for 4 hours at 120 °C (using 10 wt% of 3 with respect to MBPP) to afford MBPP-3, a green-coloured textile. The intense green colour of 3 was advantageous for (1) ensuring coverage of MBPP with the solution of 3, and (2) monitoring the effectiveness of the diazirine C-H insertion reaction through the persistent green colour after washing (Fig. S5). Prior to virus inactivation testing we completed a robust washing protocol analyzing the wash fractions with UV-Vis spectroscopy to observe the removal of 3 that did not covalently react with the surface of MBPP. The washing process was initially completed over ~6 days with 12 changes in solvent (Fig. S4). It was crucial to ensure complete removal of any 3 that did not covalently bind to MBPP during the C-H insertion step to ensure that our subsequent virus inactivation testing focused on the abilities of MBPP-3 exclusively.</p><p>The efficacy of free antimicrobials is greater than that of an immobilized antimicrobial because of the ability to diffuse throughout a solution; in the case of an immobilized antimicrobial, the activity is reliant upon virus diffusion to within the proximity of the surface of the material where the interaction/reaction can occur. The lifetime of the 1 O2/ROS produced (µs to ms) limits the space in which inactivation may take place which we hypothesized would create layer at and above the material's surface that would inactivate any virus occupying that space. 34,35 The inactivation would then rely on pathogen diffusion in the transfer medium to within this concentrated 1 O2 area on the MBPP-3 surface and was therefore expected to present a time-dependence. Virus inactivation testing was then completed using a minimal volume of carrier medium to mimic aerosolized and droplet capture by the filter.</p><p>IFV (strain A/California/07/2009) was used as a model enveloped RNA virus. IFV would provide insight into the inactivation performance of our material against enveloped RNA viruses similar to the SARS-CoV-2, which continues to cause a worldwide pandemic in 2021. We used a high-intensity LED light capable of producing ~30,000 lux to excite MBPP-3 (see Supplementary Fig. 6 for manufacturer's provided UV-Vis profile) which would mimic the amount of light that may be found in a hospital operating theatre or emergency area. 36 The virus particles were exposed to the light for between 0.5 and 4 hours in 10 µL of tissue culture medium in a well of a 96-well plate that contained a test surface (see Supplementary Fig. 5). After 1 hour exposure the virus titer was reduced by 1.06 log with MBPP-3, compared with only a minor 0.13 log reduction on the MBPP control surface (Fig. 3a). Over the 4-hour testing period a logarithmic relationship between reduction of PFUs and exposure time was observed. At 4 hours of exposure, the treated surface resulted in a 4.07 log reduction (99.99%) in comparison to an empty well, a 3.38 log reduction (99.96%) in comparison to untreated MBPP and a 5.95 log reduction (99.9999%) in comparison to the starting virus PFU concentration. inactivated the virus with a 0.76 log reduction (Fig. 3b; MBPP-3 Dark), which was substantially less than MBPP exposed to light. These data indicated that immobilized 3 and visible light are required to achieve efficient virus inactivation, and that switchable on/off performance and lithography/patterning of virus inactivation may be possible with this system.</p><p>The ability to functionalize aliphatic polymers with diazirines is a relatively new methodology and has the potential to impact a large portion of the commodity polymer market. Low-functionality aliphatic polymers-such as the MBPP used in this research-account for the largest three (polypropylene, low-density and high-density polyethylene) volumes of thermoplastics in the world equating to ~36% or 23 million tons of polymers used worldwide each year. 34 With over 39% in packaging and 22% in other areas that include health and safety, the potential for improvement in product performance and safety across different markets that utilize aliphatic polymers is high.</p><p>Furthermore, this method of functionalization can be applied to different commodity polymers including polystyrene or polyethylene terephthalate and as such may allow recycled materials an avenue for use in high-performance and advanced material applications from renewable resources. The diazirine functionality is not limited to C-H bond activation and can also be used to functionalized more reactive O-H and N-H bonds in polyalcohols and polyamides which could be used for post-processing functionalization of different materials that would benefit from self-sterilization in healthcare settings. 30 Future testing will focus on the development of this technology to understand the performance limitations of the material with respect to repeated sequential virus inactivation, potential photobleaching, light intensity-dependent performance, response to washing and detergents, and performance against different types of pathogens. The optimization of inactivation materials should be focused on developing technology that is broadly applicable, robust, and possesses broad spectrum activity without the potential for developing resistance.</p><!><p>We have described the production of a polypropylene-based non-woven filter that was covalently functionalized with a zinc-porphyrin photosensitizer using diazirine C-H activation chemistry. The MBPP-3 material was tested against a model virus, Influenza A, to explore the virus inactivation abilities of the material when exposed to visible light.</p>