Comparative efficacy of amphotericin B-loaded chitosan nanoparticles and free amphotericin B drug against Leishmania tropica

The preparation of an effective drug delivery formulation is an urgent need to treat cutaneous leishmaniasis (CL). Pentavalent antimonials and Amphotericin B (AmB) are considered to treat leishmaniasis; however, their clinical usage is hampered by poor solubility, high cost, toxicity, and the emergence of drug-resistant Leishmania spp. The drug delivery systems (DDS) could be used as an alternative treatment option for the treatment of CL to circumvent these problems. We tested the antileishmanial efficacies of free AmB and amphotericin B-loaded chitosan nanoparticles (AmB-CNPs) under in vitro conditions. Chitosan nanoparticles (CNPs) were synthesized using the ionic gelation method with negatively charged tripolyphosphate (TPP). During the synthesis of CNPs, AmB was incorporated into the nanoparticles (NPs). The NPs were characterized for their size, surface morphology, encapsulation efficacy (EE), drug loading content (DLC), and surface charge using different techniques. Their efficacy was evaluated against promastigotes and axenic amastigotes forms of Leishmania tropica using MTT assay. The synthesized AmB-CNPs displayed a spherical shape with a mean particle size of 118 nm, a positive zeta potential of (+ 6.21 ± 2.02 mV), and an encapsulation efficacy of 88%. Dynamic light scattering technique (DLS) shows that the average size of prepared AmB-CNPs was 95.5 nm. Free AmB presented very low efficacy (only 65% and 67% inhibition of the promastigotes and axenic amastigotes parasite load), whereas AmB-CNPs exhibited 90% and 84% parasite inhibition after 72 h incubation. The AmB-CNPs exhibited significantly higher efficacy than free AmB in terms of reduction in parasite viability. Half-maximal inhibitory concentration (IC50) measured values of the AmB-CNPs were significant lowers than free AmB. The present data indicated that AmB-CNPs exhibited vigorous anti-leishmanial activity than free AmB by dose and time-dependent manner. This formulation can be used for local therapy of CL after in vivo efficacy conformational studies.

97 endemic countries, due to the absence of a vaccine and effective chemotherapy (Serban 2019). In terms of morbidity and mortality, leishmaniasis is the third most common parasitic disease among neglected tropical diseases (Reguera et al. 2019). The disease appears in three main infectious types cutaneous, mucocutaneous, and visceral leishmaniasis; among these, the cutaneous type is predominant (Oliveira et al. 2021). About 15 Leishmania species cause CL, mainly infecting the human body's exposed parts (Rather et al. 2021). Approximately 0.7 to 1.2 million cases of CL have been reported annually around the globe, with more than 90% of cases of CL are prevalent in Afghanistan, Algeria, Iran, Iraq, India, Pakistan, Saudi Arabia, Syria, Brazil, and Peru (Soltani et al. 2019). CL appears in different clinical appearances, including small self-healing lesions but scarring skin and even gross lesions leading to considerable disfigurements, such as diffused mucocutaneous leishmaniasis (DMCL) and mucocutaneous leishmaniasis (MCL) (Ballart et al. 2021). In Pakistan, CL caused by L. tropica is a significant health concern, especially in Khyber Pakhtunkhwa (KPK) province (Kämink et al. 2021). Until now, anti-leishmanial vaccines are not available, and chemotherapy is the only option to combat the disease (Ikeogu et al. 2020). The currently recommended antileishmanial drugs such as antimonials compounds, miltefosine, amphotericin B, pentamidine, and paromomycin have been associated with considerable disadvantages, including high costs outlay, toxicity, poor bioavailability, and the emergence of drug resistance Leishmania spp in recent years. Hence, there is an urgent need for safe, efficient, and low-cost novel alternative treatment options to cope with leishmaniasis (Garrido-Jareño et al. 2020). Nanomedicine is applying nano-scale medicine, which has recently attracted the attention of researchers to treat infectious diseases (Bezerra-Souza et al. 2021). Nanodrug delivery systems (NDDS) have been widely used in various fields as a vehicle for the targeted delivery of loaded drugs to minimize their toxicity and increase the therapeutic potential of the drugs. Drug delivery systems may also reduce the required therapeutic dosages of the loaded drugs to eradicate the intracellular parasites residing within the macrophages (Patra et al. 2018). Up till now, several drug-nanoparticle formulations have been investigated against leishmaniasis both in vitro and in vivo (Riaz et al. 2020;Rebouças-Silva et al. 2020). Polymeric nanoparticles synthesized by the ionic cross-linking technique are of great concern as NDDS because of their low cost, environmental and easy preparation, and long-term stability at room temperature (Krishnamurthy et al. 2015). Among various NDDS, Chitosan NPs have gained significant attention in biomedical sciences as they are non-toxic, biodegradable, and biocompatible (Saeed et al. 2020). The Food and Drug Administration (FDA) approved the chitosan polymer for wound dressing (Matica et al. 2019). The antibacterial, antiviral, and anti-leishmanial activity of the CNPs prepared by the ionic gelation method have been investigated previously. The antimicrobial activity of the chitosan and chitosan NPs is due to the interactions between the positively charged ammonia groups of the chitosan and the negatively charged cell membrane of microorganisms (Alqahtani et al. 2020;Ke et al. 2021).
Amphotericin B deoxycholate is used as a second-line anti-leishmanial agent for the treatment of leishmaniasis. Studies showed that AmB interferes with the synthesis of ergosterol present in the cell membranes of the Leishmania spp, resulting in membrane damage (Riezk et al. 2020). However, the use of AmB deoxycholate is restricted due to severe side effects, including nephrotoxicity, liver damage, hemolysis, nausea, and poor solubility. In order to overcome the toxicity and emergence of drug resistance issues associated with conventional AmB administration, a liposomal formulation was also prepared to cope with the event of drug intolerance or toxicity. The liposomal form of the AmB is widely used for the treatment of VL and is also reported to have good efficacy in CL patients. However, high cost, the requirement of the cold chain, and a change in drug content upon storage limited its clinical use (Silva-Carvalho et al. 2020).
In the present study, we report the synthesis, characterization, and in vitro antileishmanial effect of chitosancoated AmB formulation. We demonstrated substantial anti-leishmanial activity of AmB-CNPs than free AmB against promastigotes and axenic amastigotes forms of L. tropica in terms of in vitro parasite viability reduction.

Culture media and compound
RPMI 1640 and M199 media were acquired from Sigma-Aldrich, USA. Acetic acid, TPP, and low molecular weight chitosan polymer (MW, 120 kDa) were supplied by Merck, Germany. Amphotericin B, Dimethyl sulfoxide (DMSO), and MTT dye were from Sigma Aldrich, USA. Temperature deactivated fetal bovine serum (FBS) was attained from Thermo Fisher Scientific, US. The antibiotics penicillin/streptomycin was received from Scharlau, Spain. All the other solvents used were of analytical grade (AG) and purchased locally.

Preparation of AmB-CNPs
Chitosan NPs were formed by the ionic gelation method (Shafiei et al. 2019). Different concentrations of chitosan polymer (1, 2, 3 mg/mL) were dissolved in 1% v/v acetic acid solution to synthesize CNPs. NPs were formed by adding TPP solution (in the concentration of 0.75 mg/ mL) dropwise with a pipette to chitosan solution, stirred on a magnetic stirrer, and kept overnight at 25 ℃, followed by sonication for 15 min. AmB-CNPs were prepared spontaneously upon dropwise adding TPP solution to chitosan solution containing 4 mg/mL AmB drug, under magnetic stirring at room temperature. The solution was mechanically stirred for 4 h. The resulting NPs suspensions were centrifuged at 15,000 g for 1 h. In the end, nanoparticles were washed with ultrapure-water and dried.

Physicochemical characterization Scanning electron microscopy
The morphological analysis and size of solid-state AmB-CNPs were performed using a high-resolution scanning electron microscope (SEM) (TESCAN VEGA-3, New York, USA). The samples of dried particles were covered with a carbon coating, with an accelerating voltage of 20 kV and a counting time of 1 min to assess the morphology of AmB-CNPs. Then, the particle images were observed at 10,000 to 40,000× magnification power.

Determination of average size and surface charge
The dynamic light scattering (DLS) method was employed to determine the average size of AmB-CNPs. About 300 µL of nanoparticles suspension was filled directly into the cuvette of the Zetasizer instrument by placing it in the device. The zeta potential of the nanoparticles was assessed by using a Zetasizer Nano ZS apparatus (ZEN0040, Malvern Instruments, UK). Operating conditions during the experiment were as follows: temperature 25 ℃, with a scattering angle of 90°.

In vitro release of AmB from CNPs
Synthesized AmB-CNPs were examined for in vitro drug release activity. Prepared AmB-CNPs were dissolved in a beaker containing 5 mL Tris-HCL buffer solution.
The sample was sonicated at 100 rpm for 72 h at room temperature. At predetermined time periods, 0, 0.5, 1, 2, 4, 6, 12, 20, 24, and 48 h samples were centrifuged at 15,000 g for 40 min and the temperature was adjusted to 14 °C. The supernatant was discarded and replaced with an equal amount of fresh PBS solution. In the end, the amount of AmB released was observed by a UV-Vis spectrophotometer at 270 nm. The release concentration of the drug at each time interval was calculated using the cumulative calibration curve method.

Encapsulation efficacy and drug loading content
To observe the percentage of encapsulation efficacy (EE) and drug loading content (DLC) of AmB-CNPs, the NPs were centrifuged at 20,000 g for 30 min at a controlled temperature of 4 ℃. The experiment was performed in triplicate. The EE and DLC of the synthesized nano-formulation were then calculated by using the equations as follows: whereA is the total volume of AmB utilized to synthesize nanoparticles (mg) and B is equivalent to the free AmB calculated in the supernatant in mg.
where A is the total volume of AmB utilized to synthesize nanoparticles (mg) and B is equivalent to the volume of free AmB calculated in the supernatant in mg, while C is the mass of nanoparticles in the supernatant.

Cytotoxicity study on promastigotes
Cytotoxicity of AmB-CNPs on L. tropica promastigotes was investigated using the tetrazolium dye assay (MTT) as described earlier (Lima et al. 2017). Stationary-phase promastigotes (1 × 107 cells/mL) were seeded in 96 well plates and incubated with six different concentrations (50, 40, 30, 20, 10, and 5 µg/mL) of AmB-CNPs for 24 h, 48 h, and 72 h. Later on, 20 µL of MTT dye (5 mg/mL) was added to each well and incubated the plate for a further 4 h at 37 ℃. After incubation, the purple-colored formazan crystals were dissolved in 100 µL of dimethyl sulfoxide (DMSO) and centrifuged at 3000 g for 5 min. Optical density (OD) was evaluated at 570 nm using an ELISA reader (Thermo Scientific Microplate Reader). Miltefosine was used as a positive control. The IC50 values of AmB-CNPs and free AmB were calculated by ELISA machine and compared using the GraphPad Prism software (version 5.0). The experiments were performed in triplicate, and data are expressed as mean ± SD. The percentage of viability was calculated using the following formula: where AB is the absorbance of the blank sample, AC is the absorbance of the negative control, and AT is the absorbance of the treated samples. (1)

Cytotoxicity study on axenic amastigotes
In vitro, antileishmanial activities of AmB-CNPs and free AmB were assessed against axenic amastigotes of L. tropica (Dias-Lopes et al. 2021). Initially, the promastigotes were incubated at 37 ℃ in a 5% CO 2 incubator. This results in the conversion of promastigotes form into axenic amastigotes type. Afterward, the amastigotes were transferred into an ELISA plate and then exposed to the same concentrations of AmB-CNPs and free AmB as used for promastigotes.

Statistical analysis
The IC50 values were determined using sigmoid doseresponse curves using GraphPad Prism version 8.0 for Windows (GraphPad Software, USA). The IC50 value of AmB-CNPs was lower than the free AmB drug.

Preparation of nanoparticles
The synthesis of AmB-CNPs, chitosan polymer, TPP solution, and AmB drug was mixed using an ionotropic gelation method. Briefly, the aqueous solution of AmB was mixed with chitosan and 1% acetic acid solution; in last, the TPP was added dropwise to synthesized AmB-CNPs. This reaction results in a light yellow color solution of nanoparticles (Fig. 1a, b). The solution was then centrifuged, and d-Trehalose was added to the solution.

Characterization of AmB-CNPs
The physicochemical characterization of AmB-CNPs was evaluated using different methods, involving UVvisible spectroscopy, SEM, DLS, zeta potential, DLC, and encapsulation efficacy. The synthesis of NPs was demonstrated through UV-visible spectroscopy, which is the easiest method to confirm nanoparticles synthesis. The absorbance spectra were recorded at 320 nm. A broad absorption band intensity was examined for chitosan polymer, whereas CNPs show sharp intensity spectra as depicted in Fig. 2.
For the morphological studies of AmB-CNPs, SEM illustrations of the prepared NPS revealed spherical primary particles with a mean size of 118 nm, as shown in Fig. 3. The average size of NPs determined by DLS technique is shown in Fig. 4. Zeta potential or surface charge plays a vital role in the physical stability and interaction of nanoparticles with biological surfaces. AmB-CNPs possessed a stable positive surface charge of (+ 6.21 ± 2.02 mV) as represented in Fig. 5. The intense interaction between positively charged chitosan nanoparticles and negatively charged microbial membranes may be due to this surface charge.
The cumulative release concentration of AmB drug from the CNPs was divided into two stages (Fig. 6). In the first phase, at a pH of 7.4, the drug was immediately released from the nanoparticles in the first 6 h. This results in a 42% release of the drug. In the second stage, AmB released slowly from the NPs up to 48 h resulting in 68% of the drug.
DLC means an appropriate amount of drug present in a defined quantity of the NPs. At the same time, EE is the amount of drug in percentage that has been encapsulated into the NPs. EE can be computed by (initial amount of drug added-free non encapsulated drug) divided by the initial amount of drug added. The DLC and EE of the AmB-CNPs were 48% and 88%, respectively.

Anti-promastigotes cytotoxicity by MTT assay
The promastigotes viability assay was evaluated by using MTT colorimetric assay. Briefly, L. tropica promastigotes were incubated with six concentrations (50,40,30,20,10, and 5 µg/mL) of both AmB-CNPs and free AmB at 24, 48, and 72 h, respectively. Both the loaded and free drugs have time and dose-dependent parasite viability inhibition at different time intervals. However, AmB-CNPs show a maximum reduction of promastigotes viability rate (90%) at 50 µg/mL after 72 h of incubation (Table 1). The viability of promastigotes exposed to various   et al. Bull Natl Res Cent (2021) 45:187 dilutions of free AmB is also shown in Table 2. The IC50 values of AmB-CNPs and free AmB are shown in Table 3.

Leishmanicidal effects on axenic amastigotes
The in vitro axenic amastigotes effects of AmB-CNPs and free AmB were also evaluated against L. tropica at 37 ℃ in a 5% CO2 incubator. The axenic amastigotes culture was incubated with six dilutions (50, 40, 30, 20, 10, and 5 µg/mL) of AmB-CNPs and free AmB at 24, 48, and 72 h at 37 ℃ with a pH of 5.5. Table 4 represents the cytotoxicity of AmB-CNPs on L. tropica axenic amastigotes form at different incubation periods. The maximum dose and time-dependent parasite viability reduction for AmB-CNPs against amastigotes form was 84% after 72 h incubation. On the other hand, free AmB presents anti-axenic amastigotes viability inhibition rate of only 67% (Table 5). AmB-CNPs have potent anti-axenic amastigotes activity than free AmB in terms of reduction in parasites viability percentage.

Discussion
Nanotherapy can be used as a novel alternative treatment to overcome various problems related to leishmanicidal drugs, such as low solubility, painful parenteral administration, and adverse effects (Baranwal et al. 2018). In this study, we examined for the first time the anti-leishmanial effects of chitosan nanoparticles loaded with AmB Fig. 6 Amphotericin B release pattern from chitosan nanoparticles (chitosan 3 mg/mL, TPP 2 mg/mL, pH of the medium 7.4)      et al. Bull Natl Res Cent (2021) 45:187 and free AmB, one of the most potent and commercially available antileishmanial drugs on L. tropica promastigotes and amastigotes in vitro. Chitosan polymer, acquired from the deacetylation of chitin, is one of the most famous drug delivery carriers in nanotechnology. Previous studies have investigated its most potent antibacterial, antiviral, antileishmanial, and antifungal effects (Loiseau et al. 2020;Sudatta et al. 2020 (Mehrizi et al. 2019). Their results are practically in line with our results. Casa et al. investigated the inhibitory effects of AmB-loaded bovine serum albumin nanoparticles and free AmB drug in murine cutaneous leishmaniasis (Casa et al. 2018). Their findings proved that the loaded drug has more potency than the free drug against the Leishmania parasite. In a previous study, Ammar et al. also investigated the toxicity of nanoparticles loaded amphotericin b drug for the local treatment of cutaneous leishmaniasis using MTT assay (Ammar et al. 2019). They observed that NPs loaded AmB drug was more cytotoxic than free AmB drug. These results comply with our results.

Conclusion
The present study demonstrates that a chitosan-based drug delivery system is helpful for the in vitro clearance of L. tropica. Herein, Chitosan NPs were synthesized by the ionic gelation method, and AmB was encapsulated in CNPs during the synthesis process. The developed AmB-CNPs had a small size range of (118 nm), high encapsulation efficacy (88%), with a stable positive zeta potential of (+ 6.21 ± 2.0 2 mV), respectively. The average size of AmB-CNPs was 95.5 when analyzed by DLS. The synthesized nano-formulation exhibited strong antileishmanial efficacy on L. tropica promastigotes and axenic amastigotes. The obtained IC50 values of AmB-CNPs against promastigotes and amastigotes were significantly lower than conventional AmB and the control drug miltefosine. AmB-CNPs also presented a significant antileishmanial effect in reducing parasite viability compared to free AmB drugs. In conclusion, AmB-CNPs may be a suitable alternative treatment candidate for eradicating drugresistant Leishmania parasites.