(161ae) Broad-Spectrum Antimicrobial Polymers to Prevent the Spread of Infectious Pathogens

Adherence of pathogens such as bacteria, viruses and fungi on various surfaces routinely leads to subsequent transmission to new hosts, significantly promoting the proliferation of potentially harmful organisms. This scenario can happen anywhere, but it is observed to be most prolific in healthcare setting such as hospitals, nursing homes and clinics. The infections caused by pathogens residing in such environments are termed as nosocomial or hospital-acquired infections (HAIs). The pathogens adhere to surfaces such as linens, drapes, counter-tops, door handles, monitory equipment and sanitation equipment, and infect subsequently on contact with personnel. Furthermore, due to compromised immune systems, the patients are more susceptible to such kind of infections. Previously treatable infections have now become fatal. This sequence is particularly troublesome in the case of antibiotic-resistant pathogens, which are becoming a global threat to human health.¹ With only a few novel antibiotics identified in recent times (often termed the "discovery void"), antibiotic-resistance has been rising at an alarming rate. Over-prescription of antibiotics, misdiagnosis and improper disinfection protocols have only accelerated the rise of antibiotic-resistance. According to a report² published by Centers for Disease Control and Prevention in April 2019, more than 2.8 million people are affected by antibiotic-resistant infections, causing 35,000 deaths annually in the U.S. alone. The number of deaths rises to ~50,000 if causalities by Clostridium difficile are included. Such antibiotic-resistant bacteria, popularly referred to as “nightmare bacteria” or “superbugs” with highly elevated resistance to last-resort antibiotics, wreak a greater havoc, resulting in about 700,000 deaths worldwide. A recent survey³ sponsored by the British government suggests that if immediate measures are not taken to mitigate this rampant problem, most bacterial infections will become resistant to antibiotics and lead to 10 million deaths, exceeding the number of deaths caused by cancer, every year. In addition to tragic loss of life, hospital-acquired infections (including antibiotic-resistant infections) promote a heavy economic burden of $28-45 billion on the healthcare industry for treatment of such infections.⁴ Since antibiotic-based treatments are anticipated to become ineffective unless novel antibiotics are discovered, a more prevention-based approach must be sought to tackle the issue of drug-resistant infections. Although many disinfections methods have been conducted to eradicate pathogens on surfaces, each has a limitation. For example, chemical disinfectants and ionizing radiation are not always safe for use in all environments. While UV radiation is effective in eliminating pathogens, it can harm healthy cells as well. Similarly, a few other strategies involve incorporation of antimicrobial agents such as metal nanoparticles or metal oxides such as silver,⁵ copper⁶ and titanium oxide⁷ into polymeric materials. Even these techniques suffer from drawbacks. Nanoparticles are known to be hazardous, posing additional health concerns when they leach into the environment. Metals and metal oxides do not have the ability to kill viruses, and bacteria can develop metal resistance through various mechanisms rendering them ineffective over time. Moreover, they need to come in direct contact with the pathogen to electrostatically interact with bacterial cell membrane. To address these issues and tackle the problem of hospital-acquired infections, we have developed two preventive routes to eliminate antibiotic-resistant/susceptible pathogens.

The first method employs the mechanism of antimicrobial photo­dynamic inactivation (aPDI), which relies on using a photo­sensitizing agent. Upon illumina­tion with noncoherent visible light (as depicted in Figure 1), the photosensitizer is activated. In the presence of molecular oxygen, the photosensitizer generates singlet oxygen (¹O₂), which is cytotoxic towards pathogens and reacts non-specifically with various constituents of the cell wall in pathogens. It is highly improbable that the pathogens can develop resistance to such multimodal non-specific attack. We incorporated a photosensitizer, zinc-tetra(4-N-methylpyridyl)porphine (ZnTMPyP⁴⁺, Figure 2), capable of producing ¹O₂ into various polymeric substrates such as a polyethylene-based multiblock polymer, styrenic triblock copolymers, polylactic acid, and nylon-6 microfibers. In the work with the multiblock polymer,⁸ which is discussed below, the photo­sensitizer was incorporated into the polymer matrix by co-dissolution in a toluene/2-propanol co-solvent, followed by melt-pressing several times to form films. The surface distribution of the photosensitizer was determined by scanning electron microscopy and energy dispersive x-ray spectroscopy, which together revealed that, in addition to a finer distribution throughout the film, the photosensitizer occasionally formed aggregates measuring ~2 m in diameter. Time-of-flight secondary-ion mass spectrometry indicated a higher concentration of photosensitizer on the polymer surface relative to the bulk, consistent with blooming. The photosensitizer-containing films were tested against pathogens from the ESKAPEfamily of infectious bacteria (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacterspecies). An antibacterial efficacy of >99.89% was achieved in all bacterial strains tested. In addition, the films were tested against two enveloped viruses (vesicular stomatitis virus, VSV, and Influenza A virus) and a non-enveloped virus (human adenovirus-5, HAd-5). The antiviral efficacy was measured as >99.96% against all the strains.

The second method involves the use of sulfonated block polymers for antimicrobial anionic inactivation (aAI). In this case, disinfection occurs by a drastic change in pH of aqueous media near the surface of the polymer (cf. Figure 3).⁹ An abrupt drop in pH across the pathogenic cell membrane ruptures the cell wall, as well as promotes protein denaturation and enzyme damage, which subsequently induce cell death. Several sulfonated styrenic block polymers including poly[tert-butylstyrene-b-(ethylene-co-propylene)-b-(styrene-co-styrenesulfonate)-b-(ethylene-co-propylene)-b-tert-butylstyrene] (TESETx), poly[styrene-b-(ethylene-co-butyl­ene)-b-styrene] (SEBSx) and poly[tert-butylstyrene-b-(styrene-co-styrenesulfonate)-b-tert-butylstyrene] (TSTx) (where x is the degree of sulfonation, DOS) have been investigated. These anionic block polymers were sulfonated to various degrees (TESET: x = 26 - 52% DOS, TST: x = 17 - 63% DOS, SEBS: x =

10 - 40% DOS) and cast from tetrahydrofuran. The solvent was allowed to evaporate over 2-4 days to obtain films. These films were tested against the ESKAPE family of bacterial pathogens for exposure times ranging from 5 to 10 min. An antibacterial efficacy of 99.9999% was achieved within 5 min for all block polymers at the highest DOS (cf. Figure 4). In similar fashion as the aPDI antiviral studies, only the TESET copolymer (the chemical structure of which is included in Figure 4) was tested against enveloped viruses (VSV and Influenza A) and a non-enveloped virus (HAd-5). The enveloped viruses were eliminated to below the minimum detection limit (99.9999%), whereas HAd-5 was reduced to 99.997% with TESET52 (TESET26 was ineffective over this exposure time). A recent study¹⁰ focusing on the survivability of the SARS-CoV-2 virus on a variety of surfaces suggests that the virus can remain alive on inanimate objects for at least several hours. This observation, combined with a current lack of antivirals/vaccines, makes this virus a serious global threat, resulting in considerable worldwide proliferation and a mindboggling number of human fatalities. To tackle the ongoing tragedy of this pandemic and provide a preventative solution to thwart the spread of virus through surface contact, we have tested TESET films against human

coronavirus 229E (an alpha coronavirus strain that commonly infects humans and serves as a proxy for SARS-CoV-2) at several exposure times. We have found that, after a relatively short exposure time, TESET52 inactivates the virus to below the minimum detection limit of 10^-4%, thus providing evidence for an effective new addition to the arsenal being developed to mitigate the present and possibly upcoming spread of COVID-19, a highly contagious and dangerous viral disease.

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