**5. Restorative mechanism of nanoformulations against leishmaniasis**

The protozoan parasite *Leishmania* spp. causes cutaneous and visceral leishmaniasis. Depending on the immune responses induced by the diseased host, several clinical investigations show the development of self-curable to adverse situations [42]. Pentavalent antimonials (such as sodium stibogluconate or meglumine antimoniate) and other antileishmanial medications (amphotericin B, fluconazole, pentamidine, and miltefosine) are the most effective treatments for leishmaniasis. However, undesirable effects, high costs, complicated infusion routes, low cure rates, and rising resistance are all major concerns when it comes to developing more effective leishmaniasis treatments. Furthermore, the efficacy of the medicine employed in treatment differs per leishmanial species [28, 40, 42]. In self-treatment, phagocytes recognize and devour the causative agents, causing *Leishmania* assassination by releasing reactive oxygen species, nitric oxide, and tumor necrosis factors [43]. Following innate immune responses, TH1 immunity activates and produces CD8+, NK, and IFN cells, which kills the *Leishmania* parasites [42]. In sensitive situations, the defense system fails to overcome infections, resulting in erroneous TH2 immune responses as well as antibody responses, which is the main factor in developing new parasite elimination methods. Infected cells' proliferation and viability are inhibited by metal nanoparticles, which is dependent on the NP strength and exposure period [44, 45]. Several in vitro and in vivo data imply that bio-Ag-NPs have leishmanicidal actions via direct (non-inflammatory) or indirect (immunomodulatory) mechanisms [40, 45]. Metal-NPs destroy parasitic cells directly by producing vacuolation within the parasites and disruption to the cellular membrane, without generating immunomodulatory intermediaries such as reactive oxygen species (ROS), nitric oxide (NO), and apoptotic and necrotic factors [45]. Nanoformulations aid site-specific delivery and accumulation of medicines, which is responsible for parasite killing, when *Leishmania* parasites override the oxidative burst inside phagocytic cells and dwell in phagolysosomes [46]. According to Fanti et al. (2018) [45], Ag-NPs are oxidized by acidic conditions within the phagolysosomes following passage through the cellular membranes, and the release of free Ag + ions induces parasite assassination. The indirect strategy, on the other hand, includes inducing immunomodulatory responses at infection sites. Other methods of providing leishmanicidal effects include activating immune response mediators and reducing cell viability and proliferation as a result of metallic nanoparticles. NPs cause a variety of morphological changes, including distorted membrane integrity, cytotoxicity, mitochondrial destruction, cell cycle arrest (G1), increased/decreased ROS and NO production, altered enzymatic activity, and the release of apoptotic or necrotic components [47–49]. As a result of mitochondrial disintegration, ATP production is harmed, which leads to cytotoxic effects and, in turn, impairs infection growth [50]. Furthermore, NP exposure results in a lower parasitic load and a decrease in the trypanothione reductase system, which is an important parasitic enzyme [45].
