Mechanism of Antimicrobial Action
The physiochemical reactions, cycles, and pathways pertaining to biocidal action of different classes of antimicrobials are an interesting phenomenon and worthy of consideration for a comprehensive understanding of antimicrobial classes of polymer nanocomposites. In the present section, we will focus on the toxic mechanism of various types of active species that are presently used in fabricating polymer nanocomposites. Since the majority of the research is on metal and metal oxide ions/NPs with or without substrates, the action pathways of these classes are discussed in detail. Nonetheless, mechanisms concerning natural active nanostructures, such as clays and neem extracts, have also been brought into consideration for the development of a broader picturization and sense.
In a polymer nanocomposite system, the chief antimicrobial action occurs due to interaction of the active species with the pathogenic microbe, and as such, in most cases, the polymer remains inert to their interaction [9]. However, the hydrophilicity of the polymer affects the kinetics of migration of the active species from the bulk to the surface. Indeed, the higher amount of active species on the surface of the composite system does hasten the antimicrobial process and increase the biocidal behavior. But, in reality, bulk portions of the polymer nanocomposites contain higher antimicrobial activity than the surface, as confirmed by XPS analysis [203]. This would indicate that diffusion is a rate determining step in bringing out the active species. Generally, water molecules or moisture in air with dissolved oxygen penetrates into the intricate polymer microstructure over time through Knudsen diffusion, causing corrosion of active species present in the core. The corroded active species are further transported by dissolution in the water molecule to the surface of the polymer. This becomes more pronounced in nanocomposite systems compared to their pristine counterparts due to the presence of micron-sized voids and local polymer/particle interface regions. Even the most polar polymers, such as polyolefins, allow the passage of water molecules through diffusion, although understandably their extent of water uptake is vastly different from that of hydrogel polymers, which have larger free volume and water affinity [217]. The dissolution phenomenon is accelerated in the presence of a foreign body or a microbe that has a different protein chemistry component, high surface affinity, and a different pH value. Henceforth, it is established that the active species’ rate of dissolution and diffusion is critical to antimicrobial behavior, which is linked to the nature of the polymer matrix.
Mechanism in Metal-Based Antimicrobials
Metal- and metal oxide-based ions/NPs exert high toxicity levels on microorganisms at low concentrations, a characteristic highly desirable, yet sufficient care must be taken to ascertain their threshold ranges as they commence to exert cytotoxicity and genotoxicity on mammalian cells at high concentrations [218-220]. Although the biocidal effects of metal classes of antimicrobials have been widely reported over decades, the paradigm of sequential reactions and pathways leading to cell apoptosis is only vaguely understood [221-224]. Metal salts, colloidal solutions, and NPs consist of the subclasses of this group of antimicrobials, and there is a general consensus that the active species are only those released because of the ionization, dissolution, and instability of these active nanostructures [Fig. 7.6). As matter of fact, to elucidate the mechanism, most researchers quantify the metal ion release as a function of time instead of the release of the nanostructure employed. However, some recent articles show ТЕМ imaging evidence of NP uptake by endocytosis and adhesion on the cell wall of prokaryotes. Internalized NPs affect vital cell functions via the Trojan horse mechanism, in which large amounts of metal ions are released and dissolved intracellularly from unstable NPs and cause massive reactive oxygen species (ROS) generation and oxidative stress. Catalyzed free radical generation can additionally take place in NPs adhering to the cell wall, inducing pits and causing leakage.
In most cases, owing to the presence of telechoic acids interspersed with anionic biopolymers the electronegative nature of the bacterial cell wall leads to the development of an electrostatic attraction to any positively charged moieties. Metal ions, therefore, present excellent binding characteristics to the cell wall through the formation of coordination complexes. Ag and Zn disturb the chemiosmotic potential of the membrane, causing leakage. Strong
Figure 7.6 (a) Transmission electron micrograph of an E. coli cell treated with 50 pg cirr3 of silver nanoparticles in liquid Luria broth medium for 1 h and (b) enlarged view of the membrane of this cell. Reproduced with permission from Elsevier [223].
evidence suggests that Cu induces lipid peroxidation in membrane phospholipids, disrupting the intricate microstructure [225]. It is initially the ions that damages the membranes, post which both ions and NPs are taken up in the intracellular region.
External metal ions are capable of releasing iron from the iron coordination bonded cellular ligands of iron-dehydrogenase (Fe-S) clusters, which form integral parts of biomolecules like proteins. As a consequence, free iron, which is Fenton active, are released in the intracellular matrix. Consequently, high amounts of H202, generated by cells as part of anaerobic respiration, start participating in Fenton reaction catalyzed by metal ions.
The standard electrode potential of the metal is directly correlated to the generation of highly reactive radicals broken from molecular oxygen (02), such as hydroxides (H20«) and superoxides (02*), as products of Fenton reaction. Therefore, the higher the electrode potential of the metal ion, the higher is the rate of catalysis in Fenton reaction forming large doses of damaging ROS. The ROS radicals formed are highly reactive and react instantaneously with biological macromolecules of proteins, lipids, etc., inflicting fatal oxidative damage. Additionally, ROS generation is catalyzed through binding with thiol groups via the generation of reactive S# radical.
The most popular hypothesis regarding what inflicts biocidal damage—accepted unanimously by the present scientific community—is the excessive generation of ROS. However, selectivity and/or speciation of the metal donor atom is also widely theorized. 0, N, and S atoms of ligands also present excellent binding characteristics to external metals, which replace the original metals through molecular mimicry (for metal complexes) or ionic mimicry (for metal ions). This is also the basis for the replacement for Fe in Fe-S clusters. The functional groups inside the cell with the capability to donate electrons, such as thiols, phosphates, hydroxyls, imidazoles, indoles, and amines, are targeted. Hence, oxidative phosphorylation and osmotic imbalance fatally alter the typical conformational structure of biomolecules such as nucleic acids and proteins, programming the cell's apoptosis.
Mechanism of Antimicrobial Activity in Other Systems
Commercially available organically modified nanoclay consists of a tethered long chain terminated with a quaternary ammonium ion. The antimicrobial behavior is conferred from the positively charged ammonium ion, which behaves like cationic biocides, similar to metals in contact with microbes. Explanations for its mechanism, namely adsorption and penetration, leakage, and lysis, have been proposed in literature [226, 227]. However, ROS generation is not a plausible mechanism in the case of tethered cationic surfactants. Release of some surfactants on polymer chain intercalation is also reported. The released moieties through hydrophobic interaction cause alteration in cell membrane permeability and leakage of cytoplasmic fluid.
Contributing toward a green and sustainable earth, our group put in effort to utilize the antimicrobial properties of the Indian neem tree. The neem tree has been known for its medicinal properties and therapeutic applications since prehistoric times. Neem tree parts are utilized in different forms, like oil, seed extract, and powder, and it continues to be an important part of parallel medicine and antimicrobial applications as it contributes toward a sustainable and green environment. Active compounds, such as azadirachtin, nimbolinin, nimbin, nimbidin, nimbidol, salannin, and quercetin, isolated from the plant show antimicrobial behavior, although the majority is believed to be contributed through azadirachtin, a complex tetranortriterpenoid limonoid present in seeds [228]. Apoptosis of the microbial cell in the presence of azadirachtin takes place through bcl2, bax signaling pathways.
Conclusions
Antimicrobial polymer nanocomposites in a filament form or any other form represent an integral part of the versatile applications of functional polymer nanocomposites, and the volume of literature available on the same further emphasizes its significance in the modern society. Until now, silver-based active ingredients embedded in polymers freely or in an immobilized state have represented the research area toward which maximum efforts have been directed. The high potency, high thermos-oxidative stability, and resistant nature to microbes are identified as the chief factors while selecting an antimicrobial in a polymeric matrix. Copper, zinc, and titania NPs are also in fair usage in polymeric systems. Modified clays with super adsorptive capacity signify another emerging antimicrobial carrier for active species. However, nanotoxicological studies of polymer nanocomposites as well as their active species are still in their infancy. Reports about their cytotoxic and genotoxic behavior range from being scary to stating nil cytotoxic and genotoxic behavior. Future research efforts should be made in the systematic analysis of toxicological properties for real therapeutic applications. Other research directions include scaling-up processes for the development of NPs, nanoclays, and nanomaterial-filled polymer nanocomposites. These thrust areas would be instrumental in bringing the "nanoeffect" in polymers from the confined laboratory space to a full-blown commercial setup with monetary implications.
