**5.5 Nanoparticles**

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

Quorum sensing is a mechanism that enables bacteria to regulate the expression of genes in a manner based on cell density. To control virulence and biofilm formation, *P. aeruginosa* utilizes quorum sensing [107]. Las and Rhl are two major *P. aeruginosa* quorum-sensing systems responsible for the synthesis of the signal molecules of N-acyl homoserine lactone (AHL), N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL). 3O-C12-HSL and C4-HSL bind to and activate their LasR and RhlR cognate transcription factors, respectively, inducing the formation of biofilms and the expression of various virulence factors, including elastase, proteases, pyocyanin, lectins, rhamnolipids, and toxins [108]. The third *P. aeruginosa* quorum-sensing system, PQSMvfR, has been reported to facilitate the formation of biofilms in addition to the LasI-LasR and RhlI-RhlR systems. This mechanism regulates the development of the *Pseudomonas* quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, by the transcriptional regulator MvfR, also known as PqsR, by controlling the pqsABCDE operon. In addition, PqsA and PqsD proteins have been implicated in the development of

A promising technique for treating *P. aeruginosa* infections is known to be the inhibition of quorum sensing. This approach is capable of preventing or decreasing the formation of biofilms, reducing bacterial virulence and has a low risk of bacterial resistance growth. In addition, this strategy has a small scope, such that any unwanted inhibitory effects on beneficial bacteria are impossible. For the Las and Rhl systems, quorum sensing inhibitors may be either natural or synthetic and are capable of reducing the activity of AHL synthase, inhibiting the development of AHL, degrading AHLs or competing for AHL receptor binding [109]. In recent years, the use of quorum sensing inhibitors for the treatment of infections with *P. aeruginosa* has been intensively studied. The carotenoid zeaxanthin, typically found in plants, algae and lichens, for example, reduced the formation of biofilms in *P. aeruginosa* by binding to the signal receptors for quorum sensing, lasR and RhlR, and blocking the expression of virulence genes, lasB and rhlA [110]. Flavonoids are a class of naturally developed plant metabolites that have acted as LasR and RhlR antagonists and substantially decreased their ability to bind to the

Iron is important for bacterial growth and is involved in a number of cellular processes, such as the production of electricity, the replication of DNA and the transport of electrons [112]. Compared to healthy people, the iron content of human sputum was found to be substantially elevated in CF patients, indicating that an increased amount of iron promotes chronic CF lung infection [113]. *P. aeruginosa* utilizes pyoverdine and pyochelin siderophores to obtain iron from the extracellular environment [114]. Therefore, a technique to fight *P. aeruginosa* infections is to limit the concentration of extracellular iron or disrupt iron uptake by *P. aeruginosa*. Several studies have related iron metabolism to the pathogenesis of chronic infections, indicating that iron analogues and chelators may work against *P. aeruginosa* as potential therapeutic agents. For example, iron chelators, 2,2′ dipyridyl (2DP), diethylenetriaminepentacetic acid (DTPA) and EDTA, have been reported to impair growth and biofilm formation of *P. aeruginosa* and have been

Gallium is a nonredox iron III analog that disrupts the metabolism of bacterial iron by acting in several biological processes as an iron replacement, so it is a US

*P. aeruginosa* promoters of quorum sensing-regulated genes [111].

more effective under anaerobic conditions [115].

**5.3 Quorum sensing inhibition**

biofilms [82].

**5.4 Iron chelation**

**86**

Currently, a variety of diseases, including cancer and bacterial infectious diseases, have received significant attention from nanoparticles to treat them. Nanoparticles are small materials that have been used in a number of chemical, biological and biomedical applications, having a size of less than 100 nm and a large surface area to mass ratio [117]. The nanoparticles used for their antimicrobial activity are highly penetrable in the bacterial membranes, may interfere with the formation of biofilms, have several antimicrobial mechanisms, and are strong antibiotic carriers [118]. For the prevention of *P. aeruginosa* infections, metallic and antimicrobial agent-loaded nanoparticles have been extensively examined. Silver nanoparticles, for example, are powerful antimicrobial agents that generate silver ions responsible for the inhibition, like DNA synthesis, of bacterial enzymatic systems. Silver nanoparticles have shown important antimicrobial effects on the clinical strains of *P. aeruginosa*, killing *P. aeruginosa* effectively and inhibiting its in vitro growth. In addition, silver nanoparticles have demonstrated low mammalian cell cytotoxicity, although this requires more in vivo research [119].

Nanoparticles are capable of delivering antimicrobial agents such as antibiotics to bacteria, as described earlier. Kwon et al., developed porous silicon nanoparticles with a novel antimicrobial peptide fused with a synthetic bacterial toxin, containing membrane-interacting peptides. This engineered nanoparticle was discovered in a mouse model of *P. aeruginosa* lung infection to increase the survival rate and bacterial clearance. Moreover, it has been found that the binding of antibiotics to nanoparticle surfaces greatly improves the effectiveness of both antibiotics and nanoparticles. In this respect, silver ampicillin-attached nanoparticles have a higher rate of in vitro killing of ampicillin-resistant *P. aeruginosa* isolates compared to silver ampicillin-attached nanoparticles [120].
