Novel Antimicrobial Surfaces to Defeat COVID-19 Transmission

Antimicrobial surface coatings function as a contact biocide and are extensively used to prevent the growth and transmission of pathogens on environmental surfaces. Currently, scientists and researchers are intensively working to develop antimicrobial, antiviral coating solutions that would efficiently impede/stop the contagion of COVID-19 via surface contamination. Herein we present a flavonoid-based antimicrobial surface coating fabricated by laser processing that has the potential to eradicate COVID-19 contact transmission. Quercetin-containing coatings showed better resistance to microbial colonization than antibiotic–containing ones.

the simultaneous action of the evaporation softly desorbs the composite material. The photon energy absorbed by the matrix is converted to thermal energy that causes the complex material heating and matrix vaporization. After this, composite material molecules are exposed at the gas-target matrix interface. The nanocomposite material molecules achieve sufficient kinetic energy through collective collisions with the evaporating matrix molecules to be transferred in the gas phase. By careful optimization of the MAPLE deposition conditions (laser wavelength, repetition rate, matrix type, concentration, target-substrate distance, target and substrate rotation speed values, substrate temperature, type and pressure of background gas), this process can occur without any major nanomaterial decomposition. The MAPLE process proceeds layer-bylayer, depleting the target of matrix and nanocomposite material compound in the same concentration as the initial matrix. The MAPLE set-up used in experiments is depicted schematically in Fig. 1. The precise thickness control at the nanometric scale produces a better control of drug content, which is crucial since the synthesis of sufficient quantities of an antimicrobial coating is challenging and expensive [10]. Extension of MAPLE to composite materials is interesting, owing to its reliability and good control over deposition process parameters. This way, MAPLE technique offers an effective way to integrate novel antimicrobial functionality into complex nanocomposite-based thin films that are difficult, if not impossible, to achieve otherwise.

EXPERIMENTAL DETAILS:
In our MAPLE experiments, we have chosen to combine the following categories of antimicrobial compounds: conventional large-spectrum antibiotics (norfloxacin and cefuroxime), systemic antifungal agents (amphotericin B and voriconazole), unconventional bioactive organic (vegetal flavonoids compounds: quercetin dihydrate and resveratrol), as well as inorganic molecules (silver nanoparticles). These active compounds will be embedded in polyvinylpyrrolidone biopolymer and assembled in composite forms. Thus, functionalized nanostructured composite coatings, specifically biopolymer -antibiotic, biopolymer -flavonoid, and biopolymer -flavonoidantibiotic have been synthesized as thin coatings deposited using MAPLE. The solvent 2841 used for MAPLE target preparation was dimethyl sulfoxide (DMSO). Thin composite coatings were deposited by MAPLE and drop-casting. Before each deposition, 3.5 mL of the newly prepared solution was placed using a syringe in a pre-cooled copper target holder that had a 3 cm diameter and a 5 mm height. The MAPLE cryogenic target was created by placing the target in a Dewar vessel that contains liquid nitrogen. After the freezing step, the target holder was quickly placed in the target position within the matrixassisted pulsed laser evaporation chamber. The MAPLE thin films were obtained onto optical glass slides and one-side polished Si <100> wafers. To get clean and sterilized substrates, we used both an ethanol ultrasonic bath and a UV lamp. A KrF* excimer laser source (λ = 248 nm@10 Hz, 25 ns pulse duration) that was operated at a fluence of 50-500 mJ/cm 2 a repetition rate of 10 Hz, and for 10,000-140,000 pulses was utilized for MAPLE deposition. The laser beam browsed the target surface at a 45° angle. A laser beam homogenizer was utilized to enhance the distribution of energy for the laser spot and increase the coated region on the substrate. During MAPLE experiments, the target and substrate were placed at 5 cm separation distance; both were rotated at a rate of 0.4 Hz. All of the depositions were conducted with a background pressure of (0.1-0.5) Pa and a substrate-to-target distance of (4-5) cm. The rotating target was kept in direct contact with a cooling device that contained a liquid nitrogen reservoir; it was connected to the target using copper pipes. Rapid evaporation of MAPLE target inside the deposition chamber is reduced using this setup.

RESULTS AND DISCUSSION:
In a first attempt, in view of obtaining improved coatings capable of modulating and controlling microbial biofilm behavior, we used MAPLE to fabricate thin composite coatings resistant to microbial colonization, containing natural (quercetin flavonoid) and synthetic (norfloxacin and cefuroxime antibiotics) compounds [11]. The optimum laser fluence for which we obtained high quality MAPLE-deposited composite coatings in terms of surface uniformity (a uniform surface formed by globular structures) and structural integrity preservation has been identified (300 mJ/cm 2 ) [11]. Both the antibiofilm efficiency of norfloxacin (Nor) and cefuroxime (Cef) antimicrobial agents embedded in the polyvinylpyrrolidone (PVP) biopolymer and prospective simultaneous activity of quercetin (Q) with the investigated antimicrobial agents have been evaluated. The anti-biofilm assays have been performed on two bacterial strains typical for Grampositive (Staphylococcus aureus (S. aureus ATCC 25923)) and Gram-negative (Pseudomonas aeruginosa (P. aeruginosa ATCC 27853)) microorganisms that are usually involved in the aetiology of biofilm-related infections. Microbial biofilm growth on optical glass slides coated with the experimental synthesis variants has been evaluated at 48 and 72 h, respectively. In the case of S. aureus biofilms (Fig. 2), the utmost number of microbial cells in biofilm has been registered at 48 h, while the number of biofilmsenclosed viable cells considerably diminished at 72 h. This outcome could be assigned to the choice of a persisting population, represented by microbial cells that were viable, but were not developed in the vicinity of the inhibitory agent. The polyvinylpyrrolidone (PVP) biopolymer itself has not presented any substantial antimicrobial activity in comparison to the uncoated optical glass slide (p > 0.05). The most effective antimicrobial combination against S. aureus biofilms of 48 h proved by far to be polyvinylpyrrolidonenorfloxacin (PVP-Nor) (generating a 9 log decreasing of colony-forming unit (CFU)number) (p < 0.001), followed by polyvinylpyrrolidonecefuroxime (PVP-Cef) (p < 0.001), polyvinylpyrrolidonequercetinnorfloxacin (PVP-Q-Nor) (p < 0.001), quercetinnorfloxacin (Q-Nor) (p < 0.001) and quercetincefuroxime (Q-Cef) (p < 0.001), revealing a 4 orders of magnitude decrease of biofilm embedded cells. At 72 h, under different experimental conditions, the biofilm reduction has been far lower (1-2 logs), while the most effective combinations have been confirmed to be polyvinylpyrrolidonecefuroxime (PVP-Cef), quercetincefuroxime (Q-Cef), and polyvinylpyrrolidonequercetin (PVP-Q) (p < 0.001). It has to be pointed out that polyvinylpyrrolidonequercetin (PVP-Q) has showed a very good anti-biofilm activity, much superior to the scrutinized antimicrobial agents. The dynamics of the P. aeruginosa biofilm has been distinct from that corresponding to S. aureus biofilm; the number of biofilm-enclosed cells continued to be relatively unchanged at 48 and 72 h, respectively (Fig. 3). It should be remarked that the polyvinylpyrrolidone (PVP) substrate alone facilitates bacterial biofilm proliferation at 48 h; yet, in combination with antibiotics, it ensures the antibiotic delivery in the active form, as shown by the decrease of the number of viable cells evaluated from CFU/ml data (p < 0.001). Like the case of S. aureus biofilms, polyvinylpyrrolidonequercetin (PVP-Q) exhibited an anti-biofilm activity that is comparable to that of polyvinylpyrrolidonequercetinnorfloxacin (PVP-Q-Nor) (p < 0.001), proving once more the anti-biofilm activity of quercetin. The result has been even more obvious in the case of 72 h biofilms, where the combination of quercetin with both antibiotics and polyvinylpyrrolidone radically diminished the number of viable cells within the biofilm (p < 0.001). Going further with our studies, we have used MAPLE to fabricate quercetin dihydrate (Q)-, resveratrol (R)-, and silver nanoparticle (AgNP)-polyvinylpyrrolidone (PVP) biopolymer thin films, characterized their chemical structure and morphology, and evaluated their antimicrobial activity against both Gram-positive (Staphylococcus aureus (S. aureus, ATCC 29213)) and Gram-negative (Escherichia coli (E. coli, ATCC 25922)) bacteria in view of the potential use of these combination systems for the development of novel antimicrobial approaches [12]. Gentamicin was used as positive control. The silver nanoparticle (AgNP) was dispersed in H2O or ethylene glycol (EG). The structural and morphological investigations revealed that the best quality coatings (chemical structure with a very close resemblance to that of the initial (drop-cast) materials and surface morphology with AgNPs homogeneously dispersed into the coating) have been obtained at the ~80 mJ/cm 2 optimum laser fluence value [12]. In order to enhance the activity of the anti-bacterial surfaces, a combination of contact-based (the inhibition of bacterial growth at the contact surface with the MAPLE-coated glass slide) and release-based (inhibition region occurred around the MAPLE-coated glass slide) bactericidal effect, followed by the withdrawal of treated bacteria would be beneficial to design [13]. In this research, the modified disk diffusion assay has been used to explore the contact-and release-based bactericidal effect of the MAPLE-deposited coatings, respectively.  The comparative study of the growth inhibition diameters has revealed that the most efficient antimicrobial combinations have been the films containing polyvinylpyrrolidone (PVP) and silver nanoparticles (AgNPs), and particularly polyvinylpyrrolidonesilver nanoparticles dispersed in ethylene glycol (PVP-AgNP-EG) (Fig. 6). In the case of S. aureus strain, the antibacterial activity of polyvinylpyrrolidone alone containing films has been similar or even higher than that of the polyvinylpyrrolidone -flavonoids combinations. These results could be correlated to the anti-fouling activity of the polyvinylpyrrolidone biopolymer that is able to confer a hydrophilic microenvironment, inhibiting the initial bacterial adherence that is mediated by the hydrophobic interactions with the substrate [14,15]. Additionally, the preservation of a hydrophilic environment could promote the activity of other antibacterials comprised within the thin films (such as silver nanoparticles). Polyvinylpyrrolidonequercetin (PVP-Q) thin films have demonstrated to be more effective against the E. coli strain, while polyvinylpyrrolidoneresveratrol (PVP-R) thin films showed activity against S. aureus (Fig. 6). In the latest study, we used MAPLE to prepare different biomimetic thin films containing the polyvinylpyrrolidone (PVP) biopolymer, flavonoids (quercetin dihydrate (Q) and resveratrol (R)) and/or systemic antifungal agents (amphotericin B (AmphB) and voriconazole (Vor)). The chemical structures and morphologies of thin films were examined. The antifungal activity of thin films against two yeast strains, Candida albicans (C. albicans, ATCC 90028) and Candida parapsilosis (C. parapsilosis, ATCC 22019), was investigated to assess the potential of these materials for the development of novel antimicrobial strategies [16]. The chemical structure studies have proved that highquality thin films with chemical structures similar to the drop-cast ones were MAPLEcoated using an optimum laser fluence of ~ 80 mJ/cm 2 . The surface morphology investigations demonstrated that there are no grain boundaries. Also, no large material agglomerates were observed in the scanned samples [16]. In order to explore the fungal cultures that have been grown in contact with MAPLE-coated thin films, a slight adjustment of the disk diffusion assay was performed. Regions of growth inhibition in the fungal cultures are characteristic of the antifungal activity of the assessed materials; the diameters of the regions were measured to the nearest millimeter, as previously described. Images of the investigated samples are provided in Figs. 7 and 8, while the diameter values of the growth inhibition regions are presented in Fig. 9.   In this research, the capacity of our biomimetic coatings to impede the growth of biofilms developed by C. parapsilosis and C. albicans, two fungal species frequently medical device-related infections, has been assessed under in vitro conditions. The achieved results have been substantially different for the two strains (p < 0.001). In the case of C. parapsilosis (Figs. 7 and 9), regions of growth inhibition have been examined for the positive controls (voriconazole (Vor), amphotericin B (AmphB)) and polyvinylpyrrolidonevoriconazole (PVP-Vor) MAPLE-coated samples. The incidence of growth inhibition regions suggests that these materials exhibit antifungal activity against C. parapsilosis, revealing an absence of detectable colony forming units (CFUs) in the regions surrounding the samples. This perception signifies that the antifungal activity of corresponding samples was not restricted to a surface interaction between the MAPLE-deposited coatings and fungal cells. The anti-biofilm activity of the coatings tested against C. albicans biofilms is visible in Fig. 8. The growth inhibition regions surrounding the samples were observed for the positive controls, polyvinylpyrrolidonevoriconazole (PVP-Vor), polyvinylpyrrolidonequercetin dihydrate -voriconazole (PVP-Q-Vor), and polyvinylpyrrolidoneresveratrolvoriconazole (PVP-R-Vor) samples (Fig. 9). The analysis of the coating efficacy against C. albicans versus C. parapsilosis biofilms suggests a statistically significant higher activity of voriconazole (Vor), polyvinylpyrrolidonevoriconazole (PVP-Vor), polyvinylpyrrolidonequercetin dihydratevoriconazole (PVP-Q-Vor), and polyvinylpyrrolidoneresveratrol -voriconazole (PVP-R-Vor) samples against C. albicans biofilms when compared to C. parapsilosis ones (p < 0.001). The coating consisting of voriconazole enclosed in polyvinylpyrrolidone (PVP-Vor) and polyvinylpyrrolidonequercetin dihydrate (PVP-Q-Vor) proved to be substantially more active than the voriconazole (Vor) itself against C. albicans biofilms (p < 0.05 and p < 0.0001, respectively). The coatings comprising both voriconazole and quercetin dihydrate have revealed a higher activity against C. albicans biofilms, as compared to the coatings consisting of either voriconazole or quercetin dihydrate alone (p < 0.001). For C. parapsilosis biofilms, the coatings that incorporate polyvinylpyrrolidone-voriconazole (PVP-Vor) have been found more active than the voriconazole (Vor) alone (p <0.001).
It is noteworthy that our findings are in good accordance with the literature that validate the synergistic combination therapy of polyvinylpyrrolidone or quercetin and azoles. For instance, the quercetin combined with fluconazole has evidenced their promising synergistic activity in the clinical management of vulvovaginal candidiasis caused by C. albicans biofilms [17]. In other case, the voriconazole pills may contain excipients such as polyvinylpyrrolidone biopolymer as granulation binder [18]. Other relevant studies have been focused on demonstrating the efficacy of combinational use of polyvinylpyrrolidone-coated silver nanoparticles and conventional azole antifungals (e.g., fluconazole, voriconazole) to combat the CA10 drug-resistant C. albicans strain infection [19].
As a whole, our results highlight that the polyvinylpyrrolidone biopolymer constitutes an interesting biomimetic matrix for targeted and controlled local release of antifungal voriconazole, but not for the delivery of amphotericin B or flavonoid bioactive compounds of plant origin taken apart. This drug release strategy could enhance the therapeutic efficiency and reduce the systemic toxic effects of voriconazole.

CONCLUSIONS:
The presented work provides a direct evidence for quercetin ability to target SARS-CoV-2 3CLpro main protease. Quercetin, a natural product with well-known pharmacokinetics, exhibits antioxidant, antiallergic, anti-inflammatory, and antiproliferative indications. Most important, quercetin can be directed for drug repositioning, which points to a potential repurposing for COVID-19 treatment. We have demonstrated that the MAPLE technique is an appropriate approach for obtaining biomimetic thin films containing combinations of antimicrobial compounds: conventional large-spectrum antibiotics (norfloxacin and cefuroxime), systemic antifungal agents (amphotericin B and voriconazole), unconventional bioactive organic (vegetal flavonoids compounds: quercetin dihydrate and resveratrol), as well as inorganic molecules (silver nanoparticles) that exhibit chemical structures with a very close resemblance to that of the initial materials and a uniform surface morphology. Agar diffusion assays has validated the antifungal activity of polyvinylpyrrolidone-voriconazole and polyvinylpyrrolidonequercetin dihydrate -voriconazole containing MAPLE-coated films against C. parapsilosis and C. albicans, respectively. These findings suggest the potential application of polyvinylpyrrolidone biopolymer and quercetin flavonoid combination for advanced voriconazole delivery systems that both enhance the therapeutic efficacy of the antifungal agent at the site of infection and reduce the side effects of voriconazole. Also, coatings containing both voriconazole and quercetin have revealed an improved efficiency against C. albicans biofilms. The thin films fabricated by MAPLE have shown antibacterial efficiency against Gram-positive and Gram-negative bacterial strains, substantially mediated by a contact-killing effect, demonstrating the potential application of these composite systems and deposition approach for the development of innovative, safe, green antimicrobial strategies. We emphasize that the quercetin used in our work exhibits a significant anti-biofilm activity similar to that of the investigated largespectrum antibiotics, in particular for the case of 72 h biofilms. These results could be important for the possible use of quercetin as a reliable substitute to antibiotics to inhibit mature biofilm growth on various substrates.
A medical device containing these MAPLE-deposited thin films may be evaluated by the US Food & Drug Administration as a combination product since the device includes features of devices and drugs [20]. The US Food & Drug Administration organized an Office of Combination Products in 2002 to enable suitable regulation of combination devices. The complexity of the approval process for a given combination device is governed by the regulatory status of the constituent drug(s) and device(s). The Investigational Device Exemption (IDE) process rules the regulatory processes for combination products that are supervised as medical devices and the Investigational New Drug (IND) process manages the regulatory processes for combination products that are regulated as drugs.
It is also important to note that the commercial production of the MAPLE thin film is required to follow the Good Manufacturing Practice (GMP) regulation of the US Food, Drug, and Cosmetic Act, which requires that medical devices complete specific quality assurance tasks, including (a) written manufacturing processes that confirm that manufacturing of the medical device matches that of the original design, (b) specifications for labelling of the medical device, (c) written protocols for process controls, (d) quality assurance activities, as well as (e) labelling specifications [21,22].