Pentavalent antimony is used as the first–line conventional drug for the treatment of leishmaniasis in endemic areas; however, it is associated with severe consequences, and in 10–25% of cases, there is a possibility of disease recurrence (Horber et al. 1991; Sundar and Chakravarty 2010). These drugs have shown toxic effects on the heart, liver, and kidney, and the number of reports regarding resistance to these drugs is increasing (Özbilgin et al. 2020). Consequently, many researchers have focused on finding alternative, cheaper, safer, and more accessible drugs for the poorest population on Earth and less complicated to use (Gervazoni et al. 2020; Rahimi-Esboei et al. 2018). Natural compounds extracted from plants are potent for these objectives. According to the WHO, about 80% of the world’s population now uses herbal remedies for the treatment of various infectious and non-infectious diseases (Zhang et al. 2019). Numerous studies have been conducted on the effectiveness of medicinal plant extracts against leishmaniasis (Brito et al. 2013; Elmi et al. 2021; Raeisi et al. 2020). Medicinal plants and their secondary metabolites-such as terpenes, phenolic compounds, and nitrogen/sulfur-containing compounds-exhibit many therapeutic effects against leishmania spp. However, most of these products are not reproducible in in vivo experiments. Consequently, many, such as Ligustim chuanxiong, Menta villosa, Artemisia annua, and Croton argyropylloides are still under pre-clinical trials (Da Silva et al. 2012; Raj et al. 2020).

In the present study, commercial chitosan at concentrations of 100, 200, and 400 µg/ml was used to assess its anti-leishmanial activity in vitro and in vivo against CL caused by L. major. The obtained results were compared to the amphotericin B as a positive control. Lesion sizes in different groups was calculated before treatment and at 7, 14, 21, and 28 days after treatment. Chitosan demonstrated a time- and dose-dependent manner, and the combination of chitosan and amphotericin B showed better effectiveness than either amphotericin B or chitosan alone. The results also indicated that the leishmanicidal effects of all groups were significantly better than those of glucantime (P = 0.003).

The chitosan in current study in combination to the Amphotricine B showed 70–100% anti-leishmanial effects. Similarly to our study, Rahimi-Esboei et al. (2018) reported that Chitosan at concentrations of 200 and 400 µg/mL after 180 min had 100% lethality effectiveness against L. major promastigotes and chitosan reveals as a strong anti-leishmanial agent, achieving complete parasite elimination (Rahimi-Esboei et al. 2018). Chitosan-Based Silver Nanoparticles (2017) showed a potent activity against both promastigote and amastigote stages of L. amazonensis, with IC50 values ranging from 0.422 to 2,120 µg/mL and revealed a synergistic effect of silver nanoparticles that enhances the antimicrobial efficacy of chitosan (Lima et al. 2017). In the current study, the IC50 of chitosan on L. major after 48 h was calculated as 71.63 µg/ml that was more effective than Chitosan-Based Silver Nanoparticles, while the IC50 of amphotricine B was calculated as 255.88 µg/ml. Rahimi et al. (2020) assessed the anti-leishmanial effects of Chitosan-Polyethylene Oxide Nanofibers against L. major and the IC50 values on promastigotes and amastigotes was ranged from 0.197 to 1.023 µg/mL. The reported effectiveness of chitosan in the present study aligns well with results from other studies, which also establish high anti-leishmanial activity against various Leishmania species. The efficiency varieties from complete lethality to significant reductions in parasite viability, depending on the formulation.

Chitosan at different molecular weightsshowed excellent antibacterial effects against Pseudomonas fluorescens, Escherichia coli, Vibrio parahaemolyticus, and Salmonella typhimurium as Gram-negative bacteria, as well as Bacillus megaterium, Lactobacillus monocytogenes, L. bulgaricus, L.plantarum, L. brevis, Staphylococcus aureus, and B. cereus as Gram-positive bacteria (No et al. 2002). Furthermore, various studies have reported the anti-parasitic effects of chitosan with promising results. Fungal chitosan extracted from Penicillium viridicatum and P. aurantiogriseum exhibited better scolicidal effects against the protoscolices of Echinococcus granulosus in vitro (Rahimi-Esboei et al. 2013). A similar study evaluated the anti-giardia activity of chitosan on the cyst stage of Giardia lamblia (G. lamblia) and reported that chitosan at a concentration of 400 µg/ml killed 100% of cysts after 180 min of exposure (Yarahmadi et al. 2016). Elmi et al. (2020) assessed the anti-protozoan effects of chitosan and nano- chitosan against Plasmodium falciparum, G. lamblia, and Trichomonas vaginalis, which are amongthe most important protozoa affecting. The results indicated that nano- chitosan at a concentration of 50 µg/ml inhibited 59.5%, 99.4%, and 31.3% of the growth rates of P. falciparum, T. vaginalis, and G. lamblia, respectively (Elmi et al. 2021). Najm et al. (2021) indicated that the methanolic extracts of Artimisia persica, A. fragrance, and A. spicigera inhibited the growth of the Leishmania parasite, with the IC50 values of 51 µg/ml, 400 µg/ml, and 200 µg/ml, respectively (Najm et al. 2021).

In this study, a chitosan–amphotericin B combination was also used, and surprising results were obtained. The combination of chitosan–amphotericin B had a synergistic effect in vitro and in vivo, and the growth inhibition effectiveness of the drug in the combined form was much better than when the drugs were used separately.

In the present study, we used chitosan as a nanocarrier to deliver amphotericin B, and the results revealed that chitosan can potentially reduce toxicity and increase the efficacy of amphotericin B. It can also facilitate stable drug delivery at the site of action and prevent drug degradation. Our study found that the combination of chitosan with amphotericin B was more stable and effective than amphotericin B alone, which is consistent with the findings of Riezk et al. (Riezk et al. 2020). The liposomal structures used to formulate amphotericin B are both costly and require a cold chain for stability; thus, we have largely overcome this issue by using chitosan (Frézard et al. 2022).

To maximize the effect of the synthesized nanocomposite on Leishmania inside the cell, we synthesized nanoparticles smaller than 100 nm. The study by Wijnant et al. showed that nanoparticles less than 100 nm were more effectively adsorbed at the site of infection. Additionally, Danaei et al. demonstrated that small nanoparticles have greater penetration into the dermis upon topical application, which can be effective in treating CL; this finding aligns with our present study (Danaei et al. 2021).

One of the most significant challenges in drug use is toxicity, where sometimes the side effects outweigh their effectiveness. In this study, we evaluated the toxicity of all drugs using the MTT method, and no toxicity was observed in test groups on the L929 cell line or human red blood cells. Most toxins in the human body are metabolized in the liver, where the first signs of toxicity typically occur. After 28 days of outweigh, blood samples were taken from mice, and the levels of AST and ALT enzymes were measured. The results showed that, except for amphotericin B, no toxicity was observed in the studied groups. The highest drug toxicity was associated with amphotericin B; low levels of toxicity were noted in the combination forms. The increase in leishmanicidal effect and reduction in toxicity on human cell lines in the combination form of the drugs represent a significant finding in our current study.

Amphotericin B in this study exhibited less cell toxicity when used with chitosan compared to when it was used alone. Therefore, this combination could reduce the side effects of amphotericin B, which is consistent with findings by Ribeiro et al. (2014). Furthermore, it also reduced the hemolysis rate of red blood cells. Ribeiro et al. (2014) showed that the EC50 of chondroitin sulfate-labeled amphotericin B had a greater efficacyin eliminating the Leishmania parasites compared to amphotericin-B alone, which aligns with our present study. The effects of the synthesized nanocomposite on the Leishmania parasites in thisstudy may also be attributed to chitosan’s properties in activating immune system mechanisms against the parasite. A study by Khademi et al. (2016), showed that nano-chitosan can enhance the Th1 response, an active mechanism in eliminating leishmaniasis. Another study found that chitosan can increase superoxide dismutase activity in the body, seving as a barrier against free radicals and reducing clinical symptoms of the disease while also enhancing macrophage anti-parasitic activity (Wang et al. 2013).

The toxicity of the drugs studied in curremt study was lower than cisplatin (positive control groups) and amphotericin B, even at the highest concentration. Many drugs that are evaluated in various studies have good antimicrobial effects, but in most cases, due to high toxicity, they are not approved for use in humans. Therefore, assessing the toxicity level is of great importance. However, the use of natural ingredients often has fewer side effects, which was also confirmed in this study. Chitosan revealed remarkable cytotoxicity effects against HepG2 (liver cancer), HeLa (cervical cancer) and MCF-7 (breast cancer) human cancer cell lines but the effectiveness of chitosan towards normal fibroblast cells was negligible (Frigaard et al. 2022).

Findings from both in vitro and in vivo aspects of this study demonstrated that chitosan possesses highly effective leishmanicidal effects, which are notably heightened in the presence of amphotericin B. This study also investigated the toxicity of chitosan in vitro on cell lines and in vivo on liver enzymes, showing that chitosan is not toxic. Therefore, results obtained from this study, combined with findings from other research endeavors, suggest that chitosan can be considered a natural anti-parasitic drug pending further clinical trials. According to the results of this study, it can be suggested to use combined drugs in different ratios for the treatment of cutaneous leishmaniasis. Considering the role of time, it is possible to use forms of medicine that are exposed to the wound for a longer period of time. Also, along with anti-parasitic compounds, it is appropriate to use supplements to enhance the growth and proliferation of skin cells.

For future investigations, it would be advantageous to explore the optimal dosages and treatment durations of chitosan and chitosan–amphotericin B to maximize efficacy while minimizing potential side effects. Examining the synergistic effects of combination thrapies with other anti-leishmanial agents could also improve healing consequences. Furthermore, organize mechanistic studies to comprehend how chitosan and chitosan–amphotericin B interact with L. major at the molecular level could provide valuable insights. Furthermore, extending the in vivo studies to include different animal models and assessing the long-term safety and efficacy of these treatments are crucial steps towards clinical translation. Finally, evaluating the stability, bioavailability, and pharmacokinetics of chitosan–amphotericin B formulations would be essential for developing an alternative therapeutic products.