However, in order to target biofilm-associated chronic infections, medical devices, and food industries [110], the ChNPs must have the ability to block quorum sensing. It was revealed from various experimental observations that the positively charged ChNPs are usually loaded in Oxa or oxacillin and ChNP–DNase–Oxa or Deoxyribonuclease I [111]. The anti-biofilm activity is generally studied against the biofilm network formed by nosocomial bacterial species, such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilm structuring on silicone surfaces was checked and researched with the help of SEM or scanning electron microscopy [112]. Confocal laser scanning microscopy (CLSM) was used for looking upon alive or dead microorganisms inside the biofilm matrix, which revealed that ChNP–DNase–Oxa had a higher level of anti-biofilm activity than the Oxa-mixed nanoparticles, which is present without the ChNP–Oxa or the DNase and the summation of Oxa and DNase, which involves free Oxa [41,113]. Both the formation of new biofilms and the eradication of mature biofilms in vitro could be achieved with the help of the ChNP–DNase–Oxa. Actually, through the denaturation of eDNA, ChNP–DNase–Oxa can damage the biofilm matrix, decrease the width of the biofilm, and the number of viable cells on silicone. Back-to-back treating with the help of ChNP–DNase–Oxa over two days was seen to give a shocking and successful result of almost a 99% decrease in the biofilm [114]. Moreover, ChNP–DNase–Oxa was found to be effective against the biofilm of any type of normal and clinical strains of Staphylococcus aureus [113].

This shows the high potential and effectivity of nanoparticles for treating the infections associated with biofilms [115]. Attenuation of the signals of bacterial quorum sensing can inhibit infection and can also stop the generation of bacterial virulence. Many research works have been conducted and almost all of them showed that the natural compounds possess more effectiveness over artificially synthesized chemicals regarding their treatment of biofilms and establishing them as anti-quorum-sensing agents.

Especially, flavonoid compounds are highly efficient anti-microbial and antibiofilm compounds. However, due to the very low or no dissolution of the flavonoid molecules and the rare bioavailability, minimal application of flavonoids is found [116]. Experimental observations revealed that phytochemicals, when mixed with chitosan nanoparticles, significantly decreased the QS activity through the inactivation of AI molecules [117]. Kaempferol, a flavonoid, is known to possess high-anti-quorum-sensing activity [118]. The application of the kaempferol and chitosan nanoparticles was analyzed on the basis of their properties of hydrogen bonding, hydrodynamic diameter, antioxidant activity, and amorphous transformation. After this, the inhibition of the quorum-sensing molecules by the nanoparticles in a time-dependent pattern is usually studied [119]. This measurement is done in a violacein pigment with the help of a biosensor strain Chromobacterium violaceum CV026, which is again operated by an AI known as acylated homoserine lactone (AHL) [120]. Kaempferol-loaded sodium tripolyphosphate (TPP)on ChNPshave typical particle sizes and zeta potentials of 190 to 200 nm and +30 to +35 mV, respectively, and can be stored up to 30 days and still successfully inhibit the quorum-sensing molecules, namely, the violacein pigment, in Chromobacterium violaceum CV026 [121]. After the success of this method, attempts are being made to use it as a novel antimicrobial chemotherapy. In this process, the kaempferol-encapsulated chitosan nanoparticles play the role of a stable and effective quorum-sensing-dependent antimicrobial, antibacterial, and antibiofilm agent [122].

Quercetin (QUE), another flavonoid phytocompound that is found in many commonly used medicinal plants [123] holds strong potential for establishing itself as a QS-inhibiting agent against Staphylococcus aureus, Pseudomonas aeruginosa, etc. [124]. However, the effective laboratory application of quercetin alone has stopped because of its lesser solubility in physiological fluids [125]. Therefore, many research works convey a solubility increase strategy for quercetin, which is done in the form of an amorphous and stable complex of nanoparticles of quercetin and chitosan [126]. The preparation of this complex is done using an electrostatic method and it is performed to form a complex involving ionized quercetin components and oppositely charged ChNPs [127]. In optimal conditions, a quercetin and chitosan nanoparticle complex with a size of roughly 150 to 170 nm shows a payload of about 25 to 30% having 60 to 70% efficiency with a long storage ability. Due to the absence of any adverse side effects, the complex of quercetin and ChNPs can be used for various therapeutic purposes. Such a complex is found to be more effective in the inhibition of quorum sensing than quercetin alone. Although this complex could bring about the increased suppression of quorum-sensing-regulating genes, resulting in the haltingof swimming motility and formation of Pseudomonas aeruginosa biofilms, it could not suppress the formation of its virulence factor [46]. The prior inhibition of the production of the biofilm’s swimming motility using the quercetin and ChNPs complex revealed an almost five-fold increase in kinetic solubility [128].

A new type of ChNPsthat are dually crosslinked with genipin and sodium tripolyphosphate (TPP) display quorum quenching activity [129].

Trans-cinnamaldehyde (CA) is an intensively studied compound that was shown to inhibit QS activity by decreasing the DNA-binding ability of LuxR while inhibiting acyl-homoserine lactone production. In this work, chitosan-based nanocapsules laden with a high concentration of CA were applied to a transformed E. coli Top 10 strain fluorescence-based reporter [14,130].

A nanocapsule is a shell made from a nontoxic polymer that encapsulates an inner liquid core at the nanoscale. These have many uses, including promising medical applications for drug delivery, food enhancement, nutraceuticals, and self-healing materials. The benefits of encapsulation methods are the protection of the drug and/or allied substances from the adverse environment, controlled release, and precision targeting. Hence, chitosan in the form of nanoparticles can exert its antibiofilm activity in a more targeted way. TPP-crosslinked nanoparticles (ionically crosslinked; IC-NPs) show considerable anti-quorum-sensing activity despite their inherent colloidal instability in microbiological media.

It was found that nanocapsules can interact with bacteria via electrostatic interaction, thus effectively delivering the quorum-quenching compound CA to the bacteria. The electrostatic adsorption of the chitosan-coated nanocapsules to the bacterial cell envelope is the mechanism that underpins the observed enhancement of the QS inhibition activity [131].

The polycationic groups in organic nanoparticles that are used for antimicrobial activity cause cell damage, perhaps via an ion exchange interaction between bacteria and charged polymer surfaces, resulting in the disruption of cellular membranes [132]. Polycationic nanoparticles can enter into cells via endocytosis, followed by the formation of nanoscale membrane holes, which leads to a final membrane translocation. [133]. The mechanism of interaction of nanoparticles on the cell surface was also reported in terms of the adsorption and penetration (or disruption) of cell membranes, triggering NP-mediated toxicity. This may include steps such as nanoparticle adhesion at the membrane/water interface, passive membrane translocation, membrane restructuring and leakage, and adhesive lipid extraction [134]. Nanoparticle translocation into a cell is observed to occur via the outer wrapping, followed by free translocation and inner attachment and embedment [135]. The adsorption of NPs leads to cell wall depolarization, inducing cellular toxicity and degradation, which allows ions to enter the cytosol. Sometimes, NPs cause irregular pits on the cell wall surface, enabling ions to enter the cell [136]. The polysaccharides of EPS interact with SO₄ groups of functionalized polystyrene NPs via hydrophobic complexation, which disrupts bacterial biofilm formation [137].

Quaternary ammonium chitosan NPs can produce long cationic polymer chains that penetrate the cell membrane and can induce ion exchange, which disrupts biofilms [138].

The positive surface of QAS ciprofloxacin-loaded nanochitosan-coated Ti implants disintegrates the negatively charged bacteria, followed by the release of ciprofloxacin, which inhibits enzymes, including DNA gyrase, and topoisomerase causes bacterial disruption. Free radicals interact with endogenous molecular oxygen to produce ROS, superoxide hydroxyl radicals, and hydrogen peroxide, which damages the bacteria membrane integrity and causes irreparable bacteria lysis [139]. Quaternized-chitosan-loaded Ag NPs release Ag ions that disintegrate the bacteria and inhibit biofilm development [140] (Figure 2).