Chitosan-Based Nanomaterials as Valuable Sources of Anti-Leishmanial Agents: A Systematic Review

Background: The current chemotherapy agents against various forms of leishmaniasis have some problems and side effects, including high toxicity, high cost, and the emergence of resistant strains. Here, we aimed to review the preclinical studies (in vitro and in vivo) on the anti-leishmanial activity of chitosan and chitosan-based particles against Leishmania spp. Methods: This study was conducted based on the 06-PRISMA guidelines and registered in the CAMARADES-NC3Rs Preclinical Systematic Review and Meta-Analysis Facility (SyRF) database. Various English databases such as PubMed, Google Scholar, Web of Science, EBSCO, ScienceDirect, and Scopus were used to find the publications related to the anti-leishmanial effects of chitosan and its derivatives and other pharmaceutical formulations, without a date limitation, to find all the published articles. The keywords included “chitosan”, “chitosan nanoparticles”, “anti-leishmanial”, “Leishmania”, “leishmaniasis”, “cutaneous leishmaniasis”, “visceral leishmaniasis”, “in vitro”, and “in vivo”. The language for data collection were limited to English. Results: Of 2669 papers, 25 papers, including 7 in vitro (28.0%), 7 in vivo (28.0%), and 11 in vitro/in vivo (44.0%) studies conducted up to 2020 met the inclusion criteria for discussion in this systematic review. The most common species of Leishmania used in these studies were L. major (12, 48.0%), L. donovani (7, 28.0%), and L. amazonensis (4, 16.80%). In vivo, the most used animals were BALB/c mice (11, 61.1%) followed by hamsters (6, 33.3%) and Wistar rats (1, 5.5%), respectively. In vitro, the most used Leishmania form was amastigote (8, 44.4%), followed by promastigote (4, 22.2%), and both forms promastigote/amastigote (6, 33.3%). Conclusion: According to the literature, different types of drugs based on chitosan and their derivatives demonstrated considerable in vitro and in vivo anti-leishmanial activity against various Leishmania spp. Based on the findings of this review study, chitosan and its derivatives could be considered as an alternative and complementary source of valuable components against leishmaniasis with a high safety index. Nevertheless, more investigations are required to elaborate on this result, mainly in clinical settings.


Introduction
Leishmaniasis is a tropical and subtropical poverty-related disease caused by an intracellular parasite belonging to the genus Leishmania [1]. Humans are generally infected via the bite of a sandfly, mostly Phlebotomus and Lutzomyia, around the world [2]. According to reliable reports, 0.7-1 million new cases of the disease are notified annually, and 12-15 million people are now infected with the disease in different parts of the world [3,4]. Depending on the geographical distribution, various species of Leishmania such as L. tropica, L. major, L. donovani, L. infantum, L. mexicana, L. braziliensis, and L. amazonensis can cause different clinical forms of the disease [5,6]. Considering the classification and clinical picture of leishmaniasis in humans, the diseases are divided into four forms of cutaneous (CL), mucocutaneous (NCL), diffuse cutaneous (DCL), and visceral or kala-azar leishmaniasis (VL) [7,8].
There are a number of systemic and local therapeutic strategies for the treatment of various forms of leishmaniasis, including drugs (e.g., pentavalent antimony derivatives such as meglumine antimoniate (Glucantime ® ) and sodium stibogluconate (Pentostam ® ), miltefosine, pentamidine, amphotericin B (Amp B), and paromomycin), as well as physical treatments (e.g., cryotherapy, surgery, thermotherapy, and laser therapy) [9]. Based on recent studies, the current conventional chemotherapeutics generally have difficulty reaching the target tissues at the applied doses and are also linked to adverse side effects on healthy tissues [10], indicating that the drug delivery systems must improve the efficacy, tolerability, specificity, and therapeutic index of anti-leishmanial drugs. Moreover, unresponsiveness to these anti-leishmanial compounds, even to their higher doses, is regularly reported in some parts of the world [10,11]. These limitations motivate researchers to discover an effective alternative agent with low toxicity in natural compounds as a major source of medications with various therapeutics characteristics.
Chitosan (poly-(b-1/4)-2-amino-2-deoxy-D-glucopyranose) is the general name used for a group of natural polysaccharide polymers produced by deacetylation of chitin (Figure 1) [12][13][14]. In recent years, the use of chitosan and its derivatives has attracted the attention of many researchers in medical and pharmaceutical sciences [15] due to its unique properties such as potent biological properties, low toxicity, biocompatibility, biodegradability, immunomodulatory [16], and anti-cancer, anti-nociceptive, anti-oxidant, anti-inflammatory, and anti-microbial properties [17,18]. Recent studies have demonstrated that the preparation of chitosan-based biomedical drugs such as nanoparticles, hydrogels, coatings, suspensions, powders, membranes, and Recent studies have demonstrated that the preparation of chitosan-based biomedical drugs such as nanoparticles, hydrogels, coatings, suspensions, powders, membranes, and films can impact the pharmaceutical and biomedical effects of these agents [19,20]. Recently, the antimicrobial activities of chitosan and its derivatives have been reported against a wide range of pathogenic viruses, bacteria, filamentous and yeast-like fungi [21,22], and helminthic and protozoan parasites [23,24]. Considering the anti-parasitic properties of chitosan and its derivatives, several investigations have demonstrated their potent anti-parasitic effects against some pathogenic strains such as Cryptosporidium spp. [25], Echinococcus granulosus [26], Leishmania spp. [23,24], and Toxoplasma gondii [27,28]. The present study aimed to review the preclinical studies (in vitro and in vivo) on the antileishmanial activity of chitosan and chitosan-based particles against Leishmania spp.

Database Search
This investigation was performed based on the 06-PRISMA guideline and registered in the CAMARADES-NC3Rs Preclinical Systematic Review and Meta-Analysis Facility (SyRF) database [29]. English databases, including PubMed, Google Scholar, Web of Science, EB-SCO, ScienceDirect, and Scopus, were searched for publications related to anti-leishmanial effects of chitosan and its derivatives and other pharmaceutical formulations without a date limitation to identify all published articles. The keywords included "chitosan", "chitosan nanoparticles", "anti-leishmanial", "Leishmania", "leishmaniasis", "cutaneous leishmaniasis", "visceral leishmaniasis", "in vitro", and "in vivo". Moreover, the language for data collection was limited to English.

Quality Assessment and Article Selection
The studies evaluating the anti-leishmanial effects of chitosan and its derivatives were examined. First, the studies were imported into EndNote X9 (Thomson Reuters, New York, NY, USA) and the duplicates were removed. Afterwards, three authors independently examined the title and abstract of the studies, and the relevant documents about the in vitro and/or in vivo anti-leishmanial effects of chitosan, chitosan derivatives, and chitosanbased nanoparticles were included for further analysis. The same authors carefully read the studies and selected the eligible investigations that adequately met the inclusion criteria. The inclusion criteria were articles evaluating the anti-leishmanial effects of chitosan and its derivatives and emphasizing the design of various forms of nanoparticles containing chitosan and other pharmaceutical formulations against leishmaniasis.
A total of 2669 articles were identified through database searching. Among these articles, 227 articles were removed due to duplication. Of the remaining 2442 articles, 2385 studies were removed due to the inadequate information and the ones in which the abstract was submitted in congresses as preceding papers, conferences, and editorials without full text. One of the main limitations in such studies is the frame of differing access to full texts between different research locations of study, which prevents us from accessing the full-text of some articles. To solve this problem, we searched in various research websites such as ResearchGate and LinkedIn which suggest a choice of direct fulltext request from authors as well as exploring archives of wanted journals, or contacting principal investigator to purchase it if available.
From the remaining 57 articles which were assessed for eligibility, 32 articles were excluded due to some reasons such as inconsistency between methods with results, incorrect interpretation of the results, poor methodology, etc.; whereas 32 articles were finally included in this review ( Figure 2).

Data Extraction
Three authors independently extracted information from the selected articles and, if necessary, the differences were resolved upon discussions with the corresponding author. The extracted data included chitosan type, whether it was used in combination or loaded with other drugs, the type of study, animals, doses, time, important results, and references.

Data Extraction
Three authors independently extracted information from the selected articles and, if necessary, the differences were resolved upon discussions with the corresponding author. The extracted data included chitosan type, whether it was used in combination or loaded with other drugs, the type of study, animals, doses, time, important results, and references.

Results and Discussion
Chitosan as a natural agent with diverse biological activities is generally found in the shells of crustaceans, such as crab, shrimp, squid pen, and crawfish; however, recent investigations have reported that chitosan can be produced from some fungi [15][16][17]. Of 2669 papers, 25 papers, including 7 in vitro (28.0%), 7 in vivo (28.0%), and 11 in vitro/in vivo (44.0%) studies conducted up to 2020 met the inclusion criteria for discussion in this systematic review. Totally, the most common species of Leishmania used in these studies were L. major (12%, 48.0%), L. donovani (7%, 28.0%), and L. amazonensis (4%, 16.80%), respectively.

Results and Discussion
Chitosan as a natural agent with diverse biological activities is generally found in the shells of crustaceans, such as crab, shrimp, squid pen, and crawfish; however, recent investigations have reported that chitosan can be produced from some fungi [15][16][17]. Of 2669 papers, 25 papers, including 7 in vitro (28.0%), 7 in vivo (28.0%), and 11 in vitro/in vivo (44.0%) studies conducted up to 2020 met the inclusion criteria for discussion in this systematic review. Totally, the most common species of Leishmania used in these studies were L. major (12%, 48.0%), L. donovani (7%, 28.0%), and L. amazonensis (4%, 16.80%), respectively.

2017
Amastigotes L. donovani 50, 100, 250, 500, 1000 ng/mL 4 h The results showed that the highest effect of these nanoparticles was at a concentration of 1000 ng/mL, which killed more than 80% of amastigotes, while AmB alone reduced the parasitic load by about 60%. [34] Chitosan nanocapsules containing essential oil of The results showed that AmB-SCNPs and AmB-CNPs had a better effect in comparison with amphotericin B and more than 80% of their lethality was recorded, while for amphotericin B 70% lethality have been recorded. [36] Chitosan nanoparticles -2019 Amastigotes L. major 5-250 µL/mL 48 h Chitosan coupled with L. major secretory and excretory proteins can increase the ability of infected macrophages to remove parasites by reducing apoptosis.

[40]
Curcumin-loaded mannose-functionalized chitosan nanoparticles (Cur-MCN) The results showed that Cur-MCN at the concentration of 0.518 ± 0.01 mg/L reduced 50% of amastigotes; also, no toxic effect on macrophages was observed in the use of Cur-MCN. The results showed that this compound has no cytotoxicity on macrophages and at a dose of 5 µg/mL reduced more than 60% of the parasitic load inside macrophages.
[43] The IC 50 values of AmB-SAGCS-NP and AmB for amastigotes of L. donovani were 0.128 ± 0.024 µg/mL and 0.214 ± 0.06 µg/mL, respectively. [47] The results exhibited that the most used in vitro screening strategy to check the antileishmanial effects of chitosan and its derivatives were the intracellular living amastigote assay, followed by the extracellular promastigote assay. Now, various in vitro systems are used in the evaluation of agents, which show direct toxic action on the parasite [48]; however, for some agents that act through their metabolites and/or host defense system, it does not seem like a good option [49]. Some in vitro screening studies target the promastigote forms of Leishmania because of the simplicity of the cultivation and handling of parasites, the short time required and the low cost [50]; nevertheless, because of some weaknesses, for example, the lack of host cells in this method and the presence of this parasitic form in the invertebrate vector, it is not considered a reliable target to test anti-leishmanial compounds [51]. The use of intracellular amastigote forms in the in vitro system is also well-known as a more clinically relevant model for the assessment of anti-leishmanial compounds due to having some unique features such as acquiring important cell-health information, low cost, and no need for secondary assays [52,53].
Previously, Mohebali et al. [31] have reported that the chitosan at the doses of 100, 200 and 400 µg/mL completely killed the L. major promastigotes in vitro. In another in vitro study conducted by Feizabadi et al. [37], the results showed that chitosan coupled with L. major secretory and excretory proteins can increase the ability of infected macrophages to remove parasites by reducing apoptosis. Riberio et al. (2014) have reported that chitosan nanoparticles combined with AmpB significantly reduced the lesion size and parasite burden in all the evaluated organs of mice infected with L. amazonensis. They concluded that this compound controls leishmaniasis by increasing the cytokines of IFN-γ, IL-12, IL-4, and IL-10 in the infected mice [54].

Chitosan Treatments In Vivo
The obtained results showed that the most used animals in vivo were BALB/C mice (11%, 61.1%) followed by hamsters (6%, 33.3%) and Wistar rats (1%, 5.5%). In terms of the concentrations of chitosan and its formulations, the results demonstrated that, in vivo, they were used in the range of 0.005 mg/kg to 500 mg/kg. The most common species of Leishmania used in in vitro studies were L. major (9%, 44.4%), followed by L. donovani (7%, 33.3%) ( Table 2).  The results showed that the use of AmB-DOC and the chitosan platelets caused thickening and dry scales on the lesion, which indicated improvement; granuloma spread in these mice is more limited and the number of infected macrophages is less than the use of AmB-DOC.

[45]
Curcumin-loaded mannose-functionalized chitosan nanoparticles The results showed that Cur-MCN have more anti-leishmaniasis properties than curcumin alone and are also more efficient at drug delivery than Cur-CN (curcumin-loaded unconjugated chitosan nanoparticles). Cur-MCN were able to reduce the parasitic load in the spleen by 94.20% and the number of amastigotes from 1647 ± 125.2 in the control group to 112 ± 32.2 per 500 macrophages.
2018 [41] Nano chitosan Amphotericin B -Intralesional BALB/c mice Leishmania major 5, 7, 10 mg/kg 3 weeks The results showed that this compound improved the lesion and reduce its diameter to 0 mm and killed 81% of amastigotes; additionally, no mortality was reported in mice after using this compound; while using amphotericin B alone, 10% of mice died, and no toxicity or side effects were reported. The results showed that topical application of this compound with or without AmB-DOC on the skin of L. major mice could cause a slight improvement of the CL lesion; also, the collected skin samples showed that this combination reduces the parasitic load.
2019 [46]  The lesion size in positive control group (GUL200) was negligibly decreased to 1.2 mm; also, in B20 mg/kg and K12.5 mg/kg receiver mice, the lesion size was slightly decreased, while in the group of BK20 mg/kg, the lesion size was considerably decreased and reached to zero (p < 0.001)

[59]
Chitosan-based nano-scaffolds -Electrospinning method Topically BALB/c mice Leishmania major 20 wt% 28 days This compound significantly reduced skin ulcer diameter (p = 0.000), parasite burden (p = 0.003), changes in the epidermis (p = 0.023), and dermis (p = 0.032); indicated significantly strong effectiveness of the produced nano-scaffolds against Leishmania ulcers. 2015 [60] In vivo assay or using the standardized animal models in laboratories are considered as the best strategies to evaluate the anti-leishmanial drug compounds against various forms of leishmaniasis which has the closest resemblance to the human condition [61]. Although the predictive power of the in vivo assay is not very noticeable, however, the important points in this model are that (i) if a drug is not effective in vivo, there is no need to study further and that (ii) some useful information is obtained about the effective doses as well as the toxicity of the studied drugs [48]. Today, several in vivo experimental models are used to assess and test novel agents against various forms of leishmaniasis, e.g., (i) the BALB/C mice-L. major model considered as a validated model for human CL; (ii) C57BL/6, BALB/C, and CBA/J mice to induce L. amazonensis, L. mexicana infection; (iii) BALB/C mice and Syrian golden hamster as experimental models for L. infantum infection [48,49].
Mohebali et al. [30] have demonstrated that the chitosan at the doses of 100, 200 and 400 µg/mL reduced the size of the lesion from 10.7 ± 3.24 mm in the control group to 1.05 ± 1.02 mm on day 28 at a dose of 400 µg/mL, and also from 6.27 ± 1.23 mm to 2.07 ± 0.87 mm in the 200 µg/mL concentration in the mice infected with L. major. Mohammadi-Samani et al. [56] showed that superoxide dismutase B1 combined with chitosan nanoparticles is able to promote the immunogenicity to cell-mediated immunity (T(H)1 cells producing IgG2a in mice) that is effective in removing of Leishmania parasites and might be considered as a single-dose nanovaccine for leishmaniasis.

Treatments Using Chitosan as Vehicle
Nowadays, it has been demonstrated that chitosan, its derivatives, and chitosanbased nanomaterials are able to possibly remove barriers in the carrying of drugs, thus improving the efficacy of the drug and subsequently the targeted drug therapy [62]. The findings demonstrated that the most used synthetic drugs for combination therapy in vivo and in vitro were amphotericin B (14%, 70.0%), followed by miltefosine (3%, 15.0%) and doxorubicin hydrochloride (2%, 10.0%). Although most of the studies in this review use chitosan in combination with other drugs, however, chitosan and its derivatives without combination with common drugs have been considered in some studies.  have also demonstrated that the nanoparticles of poly (isobutylcyanoacrylate) coated with chitosan have potent anti-leishmanial effects on L. major promastigotes through morphological changes such as the aberrant shape and swelling of mitochondria and parasitic vacuoles [45]. In a study conducted by Feizabadi et al. (2019), it has been proven that chitosan combined with L. major secretory and excretory proteins can improve the ability of infected macrophages to remove parasites by decreasing apoptosis [37].
For example, Lima et al. [32] have reported that chitosan-silver nanoparticles have more anti-leishmanial activity than chitosan on L. amazonensis promastigotes with the IC 50 values of 1.69 and 7.81 µg/mL, respectively. In the study conducted by Seyyed Tabaei et al. [44] have showed that chitosan-polyethylene oxide-berberine nanofibers has potent therapeutic effects on healing of CL induced by L. major in BALAB/C mice through reducing the parasite burden, decreasing the lesion size as well as change in the epidermis and dermis.
In recent years, the anti-parasitic activities of chitosan and its various derivatives/formulations have been studied against several parasitic pathogens such as C. pavum [25], Echinococcus spp. [26], and T. gondii [27,28]. For example, Mammeri et al. (2018) demonstrated that chitosan significantly decreased the viability of Cryptosporidium parvum oocysts by >95% after 24 h of treatment with chitosan mix (C-Mix) and chitosan N-acetyl-D-glucosamine (CNAD). They also reported that C-Mix (34.5%) and CNAD (56%) significantly decreased the oocysts' shedding by 34.5% and 56% in newborn mice infected with cryptosporidiosis, respectively [25]. Torabi et al. (2018) have demonstrated that chitosan-praziquantel and chitosan-albendazole nanoparticles especially in combination at the doses of 1, 5, and 10 µg/mL significantly reduced the viability of microcysts, weight and number of cysts in vitro and in vivo [26]. In the study conducted by Teimouri et al. (2018), it has been proven that low molecular weight chitosan nanoparticles completely killed the tachyzoites at the concentration of 500 and 1000 ppm in vitro; they also showed that this compound considerably increased the survival time of infected mice with T. gondii RH strain from 6 to 8 days after infection [27].

Possible Antimicrobial Mechanisms of Chitosan
The precise antimicrobial mechanism of action of chitosan is yet to be fully understood; still, based on the literature, the most likely antimicrobial mechanisms of action of chitosan include the disruption of the cell wall and, consequently, an effect on the membrane's permeability, inhibition of DNA replication, cell death, and bindings to the trace metal elements resulting in toxin production and microbial growth inhibition [63].
Mohammadi-Samani et al. (2011) have reported that chitosan nanoparticles containing Leishmania superoxide dismutase could be considered a nano-vaccine for leishmaniasis eradication by promoting the immune response toward cell-mediated immunity (TH1 cells producing IgG2a in mice) [56].

Cytotoxicity Effects of Chitosan
With respect to the cytotoxic effects of chitosan and its various formulations, Karam et al. (2020) found that chitosan nanocapsules containing the essential oil of Matricaria chamomilla have no significant cytotoxicity against macrophage cells with a CC 50 (the 50% cytotoxic concentration) value of 207.92 ± 18.53 µg/mL compared to 19.71 ± 1.73 µg/mL for essential oil alone [35]. Another study conducted by Chaubey et al. (2018) indicated that the mannose-conjugated chitosan nanoparticles of curcumin had no significant cytotoxicity against the J774A.1 macrophage cell line with a CC 50 value of 26 ± 0.60 mg/mL [41]. Recently, Esfandiari et al. (2019) have reported that paromomycin-loaded mannosylated chitosan nanoparticles had no considerable cytotoxicity against the human monocyte cell line of THP-1 cells with a CC 50 value of 3911 µg/mL [43].

Conclusions
Studies in recent years revealed that chitosan, its derivatives, and chitosan-based nanomaterials are able possibly remove barriers in the carrying of drugs thus improving the efficacy of the drug and subsequently the targeted drug therapy. In the present review, based on the literature, various forms of drugs based on chitosan and their derivatives exhibited significant antileishmanial activity against various Leishmania spp, in vitro and in vivo. The results showed that chitosan and chitosan-based particles could be considered as an alternative and complementary source of valuable components against leishmaniasis with a high safety index. However, more studies are required to elucidate this finding, particularly in clinical settings.  Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare that they have no competing interests.