**1. Introduction**

The most recent global crisis caused by the SARS-CoV-2 pandemic demonstrated that it is vital to be prepared for emerging sanitary, biological, chemical, or environmental hazards. Decontamination has always represented a major challenge, but especially now, in the

**Citation:** Toader, G.; Diacon, A.; Rotariu, T.; Alexandru, M.; Rusen, E.; Ginghin ˘a, R.E.; Alexe, F.; Oncioiu, R.; Zorila, F.L.; Podaru, A.; et al. Eco–Friendly Peelable Active Nanocomposite Films Designed for Biological and Chemical Warfare Agents Decontamination. *Polymers* **2021**, *13*, 3999. https://doi.org/ 10.3390/polym13223999

Academic Editor: Eduardo Guzmán

Received: 26 October 2021 Accepted: 12 November 2021 Published: 19 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

current situation of the COVID-19 crisis, developing efficient methods for the neutralization and the removal of the contaminants should become a priority. Although chemical and biological weapons are forbidden by the Chemical Weapons Convention [1] and Biological and Toxin Weapons Convention [2], certain states are still suspected to currently possess chemical weapons, as well as biological weapons. Moreover, even if a virus, like SARS-CoV-2, is not on the list of biological weapons, it is still important to develop new methods for biohazards management. Considering the large number of nosocomial infections with pathogens resistant to classical methods of treatment and decontamination (with antibiotics and disinfectants), in recent years, the emphasis has been on the development of new, versatile products with antimicrobial properties. Another important threat is represented by the main ingredients used for the synthesis of chemical warfare agents (**CWA**): sulfur mustard, sarin, soman, tabun, Vx, etc., which are also found in the manufacturing processes of various chemical and pharmaceutical industries (chlorine, phosgene, and cyanides). The use of chemical and biological agents from military stockpiles or biological civilian applications drives the critical need to improve decontamination capabilities worldwide. Therefore, decontamination plays a crucial role in defense against biological and chemical warfare agents (BCWA). After a chemical or biological attack, decontamination is vital. This complex process converts hazardous materials into products that can be safely handled. The methods that are typically applied are nucleophilic reactions or oxidations [3]. Toxic chemicals or micro-organisms must be eliminated by the application of efficient decontamination methods as quickly as possible in order to be able to resume routine activities. For military purposes, decontamination is undertaken to restore the combat effectiveness of equipment and personnel as rapidly as possible [4]. Most current decontamination procedures are labor- and resource-intensive, require excessive amounts of water, are corrosive and/or toxic, and are not considered environmentally safe [4–7]. Current research and development are focused on developing decontamination systems that would overcome these limitations and effectively decontaminate a broad spectrum of chemical and biological agents (CB agents) from all surfaces and materials [6,8–11]. There is no single technology that will be applicable in all situations and all types of contaminations because the nature and extent of contamination are different in different places. Surface decontamination is very difficult to achieve as contaminants can be located within the pores and cracks of materials, which makes their removal more challenging [12]. Depending on the type of contaminating agent, many decontamination methods for surfaces can be found in the literature: aspiration [13], abrasion of the surface layer [14], rinsing with water or with solvents [15], foams [15,16], gels [16], polymeric coatings [17–19], etc. The main disadvantages and limitations of the existing surface-decontamination solutions are: most of them are corrosive and/or toxic, affecting the decontaminated substrate and also exposing the user to hazardous materials; most of the existing decontamination methods are not considered environmentally safe because they require excessive amounts of reactants, solvents, or water, generating enormous quantities of post-decontamination waste, which requires subsequent decontamination; the decontamination systems that require large amounts of water do not represent a feasible solution because water can often be difficult to find (for example, on the battlefield [4]), and the disposal of this contaminated water will also further represent a cumbersome problem. Recent trends in BCWA decontamination technologies involve the use of materials capable of neutralizing contaminants under atmospheric conditions via hydrolysis and/or oxidation routes, under mild conditions of the reaction. Besides the classical decontamination methods, the use of modern methods that imply using polymeric films/coatings seems to bring multiple advantages for BCWA removal [11,17,20]. In comparison with the traditional techniques, these decontamination methods usually consist of applying a smaller amount of material (containing the active ingredients) onto the contaminated surface, thus resulting in a coating that will entrap and neutralize the contaminant and can be easily removed and compactly stored at the end of the decontamination process. This new decontamination method can be found referred to in that literature as "stripping/peelable coating techniques" [5,11,17,18,20]. This technique applies

to a wide range of contaminants and surfaces/materials. Thus, polymeric peelable films represent a modern and versatile method for surface decontamination [6,10,14,21,22]. Various polymers can be listed as film-forming polymers: acrylates [17,21,23,24], silicones [25], vinyl polymers [12,26,27], polyurea [28–32], alginate derivates [17,33–35]. Film-forming materials are already commercially available as products comprising paint-like polymeric mixtures that can be applied by spray-on or roll-on/brush-on techniques, and they can form peelable coatings for decontamination/decommissioning purposes: CBI Polymers, New York, USA—DeconGel™ 1108, Instacote Inc Protective Coatings, Erie, USA—InstaCote™ CC Wet/CC Strip, or Bartlett Nuclear Inc., Plymouth, USA—StripCoat TLC Free™. Although these products do generate a smaller volume of secondary waste and they ensure reasonable DFs, their main disadvantage is represented by the toxicity and corrosivity of some of the active ingredients comprised in these commercial formulations.

The development of nanotechnology in various fields has experienced exponential growth over the last decade. In the biological and chemical decontamination fields, formulations based on nanoparticles and metal oxide nanoparticles have attracted a tremendous interest due to their remarkable properties. Small particle sizes and high specific surface areas bring multiple advantages and unique physicochemical properties that facilitate the adsorption and degradation of toxic compounds [36,37]. Advances in the preparation of metallic NPs and metallic oxides like ZnO, MgO, CaO, CeO2, ZrO2, TiO2, etc., have led to the development of a new class of antimicrobial materials and decontaminants for chemical warfare agents with a high stability under harsh process conditions [8,37]. Highly ionic metallic NPs (e.g., Cu-NPs, Ag-NPs) are of particular interest due to their numerous reactive surface sites with atypical crystal morphologies. Ag and Cu nanoparticles immobilized on metal-oxide substrate have been demonstrated to neutralize viruses, bacteria, and fungi [38]. Nano-scaled copper particles (Cu-NPs) have many applications in industry, such as in gas sensors, high-temperature superconductors, solar cells, and other applicatoin. Copper ions have demonstrated antimicrobial activity against a wide range of micro-organisms (*Staphylococcus aureus*, *Salmonella enteric*, *Campylobacter jejuni*, *Escherichia coli*, and *Listeria monocytogenes*) [39]. The antibacterial effect exhibited against bacterial cell functions can occur through various mechanisms, depending on the physicochemical properties of NPs and the type of interactions between bacterial cells (e.g., adhesion to s Gram-negative bacterial cell wall due to electrostatic interaction [39]). These interactions lead to a disruption of the integrity of the bacterial membrane and finally cause the death of the micro-organism. Copper NPs possess better properties in comparison with other expensive metals with antimicrobial activity, such as silver and gold [40]. Silver nanoparticles (Ag-NPs) are known to neutralize both bacteria and viruses through metal-ion binding. In 2003, during the first SARS outbreak, Al2O3-supported Ag was investigated for the neutralization of SARS coronavirus, *E. coli* (bacterium), and *Debaryomyces polymorfus* (fungi). After only five minutes of exposure to the Ag nanoparticles, the three pathogens were inactivated successfully. The mechanism was not investigated, but it is assumed that catalytic oxidation is responsible and not metal poisoning (Au and Cu inactivate bacteria, viruses, and fungi only under aerobic conditions) [38].

Metal NPs and metal oxide NPs are also efficient for the decontamination of chemical warfare agents [41,42]. Sulfur mustard (**HD**) can be decontaminated through dichlorination, oxidation, or hydrolysis mechanisms, thus being converted into non-toxic products [41]. From all the materials used for the chemical degradation of **HD**, it was demonstrated that nano-oxides can adsorb and degrade sulfur mustard to thiodiglycol and divinyl sulfide at room temperature. A disadvantage of this decontamination method is that requires several hours for full degradation [42]. There are also studies regarding metal-organic frameworks (MOFs) constructed from metal ions or clusters and multifunctional organic linkers through self-assembly, which have been reported as perfect candidates for chemical and biological decontamination. The most well-known is Cu-BTC MOF, and it was also demonstrated to be capable of hydrolyzing **HD** and nerve agents under ambient conditions via its coordination of water molecules, which have an important practical value [43,44]. Silver

nanoparticles (Ag-NPs) encapsulated in MOF were reported as efficient decontaminants for **HD** [43].

While the abovementioned BCWA decontamination methods offer promising possibilities, they also possess a series of disadvantages, such as high production costs, laborious production processes, toxicity and corrosivity of some of the active ingredients, generation of a large amount of post-decontamination waste, unsatisfying decontamination degrees, etc.

The novelty of this paper consists in the development of a new method of biological and chemical decontamination by employing non-toxic, film-forming, water-based biodegradable solutions using both neutralization and adsorption mechanisms for the removal of the contaminant from a surface by employing a nanosized reagent together with bentonite as trapping agents for BCWA contaminants. Once they are applied to the contaminated surface, the neutralization of the contaminants occurs, followed by their entrapment in the polymer-clay system. After drying, these solutions form strippable films that can be easily removed from the surface. Decontamination tests herein reported confirmed the antimicrobial activity of the decontamination solutions (DF ≥ 93%) and the successful neutralization and removal of chemical agents: up to 90% decontamination efficiency for **HD** and over 99% decontamination efficiency for dimethyl methylphosphonate. Therefore, this study reveals that BC contaminants were successfully neutralized and entrapped in the polymer matrix, demonstrating that this novel ecological approach towards obtaining innovative peelable active nanocomposite films for the removal of biological and chemical agents from contaminated surfaces could represent a powerful environmentally responsible tool for decontamination applications in the future.

#### **2. Materials and Methods**

#### *2.1. Materials*

Poly(vinyl alcohol) (**PVA**, 86.7–88.7% hydrolysis degree, Mw ≈ 130,000 Da, DP ≈ 2700, Sigma–Aldrich), hydrophilic bentonite (**BT**, Nanomer® PGV, Sigma–Aldrich, St. Louis, MO, USA), anhydrous glycerol (**GLY**, Sigma–Aldrich, St. Louis, MO, USA), copper(II) acetate monohydrate (≥98%, Sigma Aldrich, St. Louis, MO, USA), titanium(IV) oxide (nanopowder, <100 nm particle size, 99.5% trace metals basis, Sigma Aldrich, St. Louis, MO, USA), Triton™ X-100 solution (Sigma Aldrich, St. Louis, MO, USA), ascorbic acid (≥99%, Sigma Aldrich, St. Louis, MO, USA), Silver nitrate (≥99.0%, Sigma Aldrich, St. Louis, MO, USA), tri-sodium citrate dihydrate (Sigma Aldrich, St. Louis, MO, USA), and sodium borohydride (≥99.0%, Sigma Aldrich, St. Louis, MO, USA) were used as received. For the chemical decontamination tests, real chemical warfare agents (**CWA**) were used: bis(2-chloroethyl) sulfide (**HD,** sulfur mustard, purity: 95%, own synthesis), together with a chemical warfare simulant: dimethyl methylphosphonate (**DMMP**, as simulant for nerve agents, ≥97%, Sigma Aldrich). All the tests involving the decontamination of the toxic agents utilized in this study were performed at the Research and Innovation Center for CBRN Defense and Ecology in the 'Chemical Analysis Laboratory' from Bucharest, the only OPCW-designated laboratory in Romania.

### *2.2. Methods*

#### 2.2.1. Preparation of Decontamination Solutions

Solutions free of metal nanoparticles and bentonite were initially prepared to serve as reference points (Table 1). The BCWA decontamination solutions based on bentonitesupported metal nanoparticles were obtained as follows: the metallic salts were dissolved in water (according to Table 2), various amounts of bentonite and TiO2 were dispersed in these solutions (continuous magnetic stirring, 800 rpm), and the nanoparticle precursor and bentonite (or bentonite and TiO2) were kept in contact under stirring for 24 h. After this, the obtained dispersions were sonicated for 30 min while the corresponding reducing agents (according to Tables 2 and 3) were added. **PVA** was introduced next, and the dispersions were maintained at 95 ◦C under vigorous stirring until the complete dissolution of the

polymer. Finally, the glycerol was added last, while the dispersions were allowed to cool down.

**Table 1.** Composition of the reference samples.


**Table 2.** Composition of the solutions containing CuNPs.


**Table 3.** Composition of the solutions containing AgNPs.


### 2.2.2. Preparation of the Nanocomposite Films

The obtained solutions were further used for decontamination tests, but they were also employed for obtaining square-shaped, thin nanocomposite films that were useful for characterization through different analytic procedures. To obtain the nanocomposite films by the casting method, approximately 100 mL of each decontamination solution was introduced in a square (12 cm × 12 cm) glass mold, placed on a perfectly flat surface, and allowed to dry (at 25 ◦C, 50–55% relative humidity). Afterwards, the films obtained were peeled and employed for further investigation.
