Enhanced Topical Treatment of Cutaneous Leishmaniasis Using Niosomal Gel of Tioxolone-Benzoxonium Chloride: In silico, in vitro, and in vivo Evaluation

Abstract

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease that poses significant therapeutic challenges owing to its toxicity and low compliance with existing treatments. In this study, we formulated and evaluated niosomal gels containing tioxolone (NT), benzoxonium chloride (NB), and their combination (NTB) for topical application against CL.

Methods

Various in silico methods, including homology modeling and molecular docking, were used to assess pharmacological potential. In vitro anti-leishmanial activity was tested against Leishmania major promastigotes and amastigotes, whereas in vivo efficacy was evaluated in a BALB/c mouse model.

Results

Docking studies showed strong interactions between NT, NB, and NTB and the target proteins. The ADMET analysis indicated that NTB had favorable aqueous solubility (LogS = −3.71) and low toxicity (2.88 mol/kg). In vitro, NTB demonstrated the lowest IC₅₀ values (79.0±3.2 μg/mL for promastigotes and 44.4±0.8 μg/mL for amastigotes) against both parasite forms and the highest selectivity index (9.8), outperforming the standard drug meglumine antimoniate (MA). In vivo tests revealed that NTB significantly reduced lesion size and splenic parasite burden compared with placebo, drug-blank niosome, and MA (p < 0.05).

Conclusion

Overall, these findings suggest that the NTB formulation exhibits enhanced pharmacological properties and effective antileishmanial activity, indicating the potential for further clinical research.

Introduction

Leishmaniasis, a neglected tropical disease (NTD) and a significant public health issue, is caused by over 20 species of protozoan parasites belonging to the genus Leishmania. This vector-borne disease primarily affects individuals with low socioeconomic status. It has been designated by the World Health Organization (WHO) as an emerging, uncontrolled, and severe disease. With an estimated 700000–1 million new cases occurring annually and approximately 211000 new cutaneous and 13000 new visceral cases reported to WHO in 2024, leishmaniasis remains a major neglected tropical disease. Globally, 99 countries and territories are endemic, and more than one billion people living in endemic regions remain at risk of infection.¹ Transmission occurs through the bite of infected female phlebotomine sand flies.²

In 2014, the WHO’s slogan, small bite, big threat, poignantly illustrated the considerable impact of vector-borne diseases, particularly leishmaniasis, on human populations. There are three key clinical manifestations of leishmaniasis: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL). Effective strategies for managing leishmaniasis face numerous hurdles, especially in vector control and management of animal reservoirs. However, treatment remains a critical concern. Untreated VL and MCL were associated with high mortality rates. While CL lesions may be painless and self-resolve within 3–18 months, persistent lesions and severe scarring can cause significant distress and stigma.³ Consequently, timely and effective intervention is necessary to reduce the risk of complications.⁴

Historically, pentavalent antimonial compounds have been the preferred chemotherapeutic agents for leishmaniasis.^5,6 Despite their benefits, these therapies have several drawbacks, including low effectiveness, uncomfortable parenteral administration, lengthy treatment times, recurrent infections, potential drug resistance, and serious adverse reactions.^7,8

Cutaneous leishmaniasis management often involves local treatment, favoring improved patient acceptance, lower costs, and reduced risk of systemic toxicities. Effective topical antileishmanial drug delivery requires optimal absorption and retention of active substances within the dermis, a challenge addressed by employing advanced drug delivery systems such as niosomes.⁹

Niosomes, formed from non-ionic surfactants, cholesterol, and hydrated bases, encapsulate drugs and offer stability, biodegradability, and biocompatibility.¹⁰ In addition, niosomal gel is suitable for this drug delivery model because of its excellent skin-preservation properties, which can facilitate prolonged drug release at the lesion site, as well as its dispersibility, a critical factor that ensures continuous distribution of the drug to the target area.¹¹ Niosomal gels have shown promising progress in the treatment of leishmaniasis, especially CL. These gels enhance drug delivery by improving skin penetration and providing sustained drug release. Recent studies have demonstrated the efficacy of niosomal formulations, such as those combining amphotericin B and pentamidine, which have shown significant antileishmanial activity and high entrapment efficiency.¹² Furthermore, niosomal dapsone gel has been shown to achieve high complete response rates in clinical trials, indicating its potential as a safe alternative to traditional therapies.¹³ The use of niosomal gels in combination with other treatments, such as intralesional injections, has shown improved efficacy in clinical settings.¹³

Niroumand et al (2024) examined artemether (ART)-loaded niosomes and evaluated their antileishmanial effects against L. major. The results of the in vitro and in vivo studies revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and a higher selectivity index (SI).¹⁴ In a study by Ardeshiri et al (2025), the potential of amphotericin B (AmB) niosomal hydrogel alone and in combination with Siberian Artemisia annua essential oil (ASEO) as a treatment for cutaneous leishmaniasis was investigated. In vivo results in BALB/c mice showed a significant reduction in lesion size in the AmB niosomal hydrogel and AmB + ASEO niosomal hydrogel.¹⁵

Although niosomal gels offer significant benefits in the treatment of leishmaniasis, challenges remain in their commercial application and widespread adoption in clinical practice. Further research is needed to optimize these formulations and validate their efficacy in different patient populations.

Tioxolone (T) and benzoxonium chloride (B), which are known for their antimicrobial and anti-inflammatory properties, have shown promise in CL treatment.^16,17 Daie Parizi et al (2015) demonstrated that a combination of T and B (Tioben, TB) with cryotherapy was as effective as meglumine antimoniate (MA, Glucantime^®) with cryotherapy, with fewer side effects.¹⁸ Hakimi Parizi et al (2019) further explored NT, NB, and NTB against L. tropica in vitro, revealing lower IC₅₀ values and reduced toxicity compared to their simple forms and MA.^19–21 Subsequent studies by the same research group have also demonstrated the efficacy of other niosomal formulations against Leishmania, including hydroxychloroquine-loaded niosomes combined with photodynamic therapy²² and the co-administration of silver- and dapsone-loaded niosomes.²³

IL-10 and IL-12 were selected because of their central and opposing roles in the host immune response to Leishmania. Overexpression of the anti-inflammatory cytokine IL-10 promotes parasite survival by suppressing macrophage activation and Th1 responses,^24,25 whereas IL-12 drives protective Th1 immunity and is essential for parasite clearance.^24–26 Metacaspase-5, a cysteine protease that regulates programmed cell death in the parasite, was chosen because its modulation can promote apoptosis of infected cells and thereby facilitate parasite elimination.^27–29 Targeting these three molecules therefore allows simultaneous evaluation of both direct anti-parasitic activity and host immunomodulatory potential of the niosomal formulations.^25–27

In silico approaches, including molecular docking and ADMET profiling, were employed to investigate the interactions of TB, NB, NT, and NTB with immune proteins, such as IL-10, IL-12, and metacaspase-5. Homology modeling was used to predict the 3D structure of metacaspase-5, enabling molecular docking studies. These in silico studies aimed to understand the molecular mechanisms and potential therapeutic benefits of these formulations.

Given that L. tropica, because of its anthropic nature, does not induce lesions in mouse models, L. major is used for in vivo studies. This study investigated the anti-leishmanial effects of NT, NB, and NTB on L. major lesions in experimental mouse models, bridging in vitro findings with in vivo validation. The objective of this research was to develop a topical treatment for CL that is more effective and has fewer side effects than the current options.

Materials and Methods

Preparation of Drugs

NT, NB, and NTB were prepared using the thin-film hydration method described by Hakimi Parizi et al.^19–21 In brief, non-ionic surfactants (Span 60 and Tween 60) and cholesterol (35:35:30 mol%) were dissolved in chloroform and mixed at different ratios in a round-bottom flask. Tioxolone was dissolved in chloroform and was added to the surfactant mixture in the flask. The solvent was evaporated using a rotary evaporator at 180 rpm and 70 °C for 15 min, until a thin, dry lipid film formed on the wall of the round-bottom flask. The thin lipid film was then hydrated with benzoxonium chloride dissolved in deionized water at 70 °C. The final concentration of tioxolone and/or benzoxonium chloride in both niosomal lotion and gel was 0.1 and 0.3 w/w %, respectively.

In silico Study

Homology Modeling, Structure Analysis, and Model Validation

Metacaspase-5 was chosen as the receptor in this study because of its crucial role in regulating apoptosis and immune responses, making it an essential target for exploring the therapeutic efficacy of formulations against cutaneous leishmaniasis.^27–30

Sequence alignment and homology modeling for metacaspase-5 began by retrieving the FASTA sequence from the UniProt database (https://www.uniprot.org/). The BLAST tool from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify a suitable template, and metacaspase-4 (PDB ID: 8w8t) was found to be the closest match. Homology modeling was conducted using the SWISS-MODEL server (https://swissmodel.expasy.org/),³¹ and the generated models were assessed for accuracy. The selected model was evaluated based on geometric and 3D alignment analyses using 3Drefine (https://3drefine.mu.hekademeia.org/).³² The stereochemical feasibility was checked using the Ramachandran plot and MolProbity for Z-scores (https://molprobity.biochem.duke.edu/)³³

Target/Receptor Preparation

The design of niosome structures involved obtaining conformers of Span 60, Tween 60, Cholesterol, B, and T from the NCBI PubChem database and downloading them as SDF files. Geometry optimization was performed using HyperChem with the MM+ force field and the AM1 semi-empirical method,³⁴ resulting in Mol2 files. Molecular quantum designs for NB, NT, and NTB were conducted using the DMol3 module of Materials Studio,³⁵ including energy calculations for the HOMO and LUMO levels. The most stable configuration was selected and saved in the SDF format. Figure 1a–c show the ligand structures prepared for molecular docking.

Figure 1.

Molecular structures designed of (a) NB, (b) NT, and (c) NTB by Materials Studio software.

This study focused on the IL-10 and IL-12 receptors because of their crucial roles in regulating immune responses, making them key targets for assessing the effectiveness of niosomal formulations against cutaneous leishmaniasis.²⁶ The structures were obtained from the RCSB protein database (PDB IDs: 4×51 and 1F45).

Molecular Docking (MVD) Process

The MVD software achieved 87% precision in molecular docking. Proteins were prepared and relevant binding poses were identified³⁶ using a 0.30 Å grid and 1500 iterations, and interactions for IL-10, IL-12, and metacaspase-5 were noted. Energy minimization was performed using the Nelder-Mead simplex method, and the results were analyzed using Discovery Studio to select the best composite.

ADMET and Drug-Likeness

AdmetSAR is a versatile platform that assesses ADMET properties and drug-likeness of chemical compounds, helping researchers predict ADMET characteristics for drug discovery. It is freely available and offers a quick and accurate way to evaluate pharmacokinetic properties and optimize drug candidates for clinical trials (http://lmmd.ecust.edu.cn/admetsar2).³⁷

In vitro Assays

Promastigote Survival Assay

In each well of a 96-well plate, 1×10⁵ logarithmic-phase promastigotes of L. major (MRHO/IR/76/ER) were cultured in RPMI-1640 medium supplemented with 10% FBS. Then, a serial dilution was prepared from the niosomal forms of the drugs (at doses between 1.0 and 300.0 µg/mL) using RPMI medium, and the mixture was added to the wells. Incubation of the plates was carried out at 26 °C ± 1 for a duration of 72 h, followed by evaluation using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Different concentrations of MA, promastigotes without niosomes, and complete medium with no promastigotes and niosomes were used as positive controls, untreated controls, and blanks, respectively. Each experiment was performed in triplicate, and absorbance readings were recorded at 490 nm.³⁸

Amastigote Survival Assay

One hundred microliters of macrophage J774A (1 × 10⁶/mL) were seeded on each slide (75 × 25 mm) in a sterile Petri dish and incubated for 6 h at 37 °C, 5% CO₂, and 85% relative humidity. The macrophages were subsequently infected at a 1:10 ratio (macrophages to promastigotes in the stationary phase of L. major) and incubated for 24 h. Once the infection levels in the macrophages were confirmed to be over 80% via light microscopy, 100 µL of niosomal drug concentrations, comparable to those used in the promastigote assay, were added to each slide.³⁹

Macrophages containing amastigotes without any drug treatment were designated as the untreated control, whereas complete medium without macrophages or parasites served as the blank. Each experiment was performed in triplicate. After incubation for 72 h at 37 °C, the slides were stained with Giemsa and the number of amastigotes per 100 macrophages was counted by direct observation under a microscope. The dose-response curves and 50% inhibitory concentration (IC₅₀) values were determined using the probit test in the SPSS software.⁴⁰

In vivo Assay

Preparation of Niosomal Gel

Carbopol was chosen to prepare the niosomal gel in this study because of its hydrophilic nature and bioadhesive properties, which may increase the residence time of the drug at the site of absorption by interacting with the skin. Therefore, the niosomal formulation was incorporated into Carbopol 934 gel at 1% (w/v) to make it suitable for topical application, and smooth homogeneous niosomal gels were prepared using a glass rod. Finally, 2–3 drops of triethanolamine (C₆H₁₅NO₃), an alkalyzing agent, were added to adjust the pH of the formulation and gel formation. Finally, a homogeneous gel was prepared by continuously stirring the mixture for 2 h. The optimal niosomal concentration for in vivo testing was selected based on the best results from the in vitro experiments. No preservative was added, and the gel was stored at 4 °C.⁴¹ The consistency and spreadability of the final T, B, and TB niosomal gels were evaluated qualitatively by tactile examination (pressing a small amount of the gel between the thumb and index finger), as a preliminary sensory assessment of the formulations.⁴²

Animals and Ethics Statement

Thirty BALB/c mice (6–8 weeks old) were obtained from the Center for Research and Training in Skin Diseases and Leprosy (Tehran University of Medical Sciences and Health Services). The animals were housed in a colony room with a 12:12 h light/dark cycle at 21 ± 2 °C and handled according to the Guide for the Care and Use of Laboratory Animals (8th edition, National Research Council, 2011) and the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). The Committee on the Ethics of Animal Experiments of Kerman University of Medical Sciences (permission number: IR.KMU.AH.REC.1396.130) approved the protocol. Every effort was made to minimize suffering.

BALB/c Infection and Treatment

To establish CL, healthy BALB/c mice received subcutaneous injections in the left hind footpad of 2×10⁶ stationary-phase L. major promastigotes in 40 μL of sterile PBS. Four weeks post-infection, the animals were randomly divided into six groups of five mice each: Group 1, infected and untreated (placebo); Group 2, infected and treated with blank niosome (N); Group 3: Infected and treated with NT; Group 4: Infected and treated with NB; Group 5: Infected and treated with NTB; Group 6: Infected and treated with MA 20 mg/kg/day; intraperitoneally.⁴³ Niosomal gel was applied to the lesions twice daily (250 mg of each niosomal gel formulation, 12 h apart) for 4 weeks using a sterile medical cotton applicator.

Measurement of Lesion Size

Lesion development and progression were monitored using a Vernier caliper to measure the thickness (D) and width (F) of the infected left hind footpad and compare them with those of the non-infected right hind footpad at weekly intervals for 4 weeks after the challenge by an investigator blinded to the treatment groups. Lesion size (mm) was determined based on the formula S = (D + F)/2.⁴⁴

Parasite Burden Determination

The number of viable L. major parasites in the spleens of the mice was assessed using a limiting dilution assay. The mice were euthanized 12 weeks post-infection in accordance with the American Veterinary Medical Association (AVMA Guidelines for the Euthanasia of Animals: 2020 Edition) using the cervical dislocation method performed by a highly trained and technically skilled expert. Subsequently, the spleens were aseptically removed and homogenized in 2 mL RPMI-FBS using a sterile syringe piston. The homogenate was diluted with the same medium in eight serial 10-fold dilutions in each well of flat-bottom 96-well microtiter plates containing a solid layer of rabbit blood agar in triplicate, and the plates were incubated at 25 °C for seven days. Positive wells (which contained motile parasites) and negative wells (which did not contain motile parasites) were identified using an inverted microscope. The data were reported as the calculated mean and standard error of the mean of the last positive well multiplied by the dilution factor.⁴⁵

Statistical Analysis

Data were entered into SPSS version 20 (Chicago, IL, USA). ANOVA and t-tests were used to define the possible relationships between the treatment groups. IC₅₀ values were analyzed using probit analysis in the SPSS software.⁴⁰ Statistical significance was set at p < 0.05. All experiments were performed in triplicate. Graphs were constructed using GraphPad PRISM 6 (GraphPad Software Inc., San Diego, CA, USA). To determine the effect of niosomal formulations on lesion size in BALB/c mice, two-way repeated-measures analysis of variance was used to assess the significance of differences between the treatment and control groups. When the p-value was significant, the multiple-comparison Bonferroni test was used to compare the means of the different treatment groups, and p < 0.05 was considered significant.

Results

Physicochemical Characterization of the Formulations

All niosomal formulations were comprehensively characterized for morphology, particle size distribution, in vitro release and encapsulation efficiency.^19–21 Morphological examination confirmed the formation of spherical, unilamellar vesicles. Particle-size analysis showed mean diameters in the nanometer range with acceptable polydispersity. Encapsulation efficiencies were high, reaching 96% for NB and more than 97% for the Span/Tween 60 NTB formulation. In vitro release studies demonstrated a controlled, gradual release profile without a burst effect. Span/Tween 60 niosomes exhibited the best physical stability, highest drug entrapment, and most favorable sustained-release behavior, and were therefore selected for subsequent biological evaluation.

Validation of Homology Modeling

To develop a metacaspase-5 model, the sequence (accession number: O64518) was compared with the PDB database, revealing that metacaspase-4 (PDB ID: 8w8t) provided the highest query coverage and sequence identity (77.36%). Using the SWISS-MODEL program, metacaspase-4 served as a template for the model. Evaluation via the MolProbity server showed that 90.4% of residues were in favored regions and 98.3% were in allowed regions, confirming the model’s quality. The Z-score was −3.36, indicating similarity to native proteins of the same size. Figure 2 shows the 3D structure and Ramachandran plots used for validation.⁴⁶ These values supported the validity of the metacaspase-5 model.

Figure 2.

Results of the homology modeling: (a) 3D structure of the metacaspase-4 (PDB ID:8w8t) (b) 3D structure and (c) Ramachandran plot of the metacaspase-5 homologized model.

Molecular Docking Study

Molecular docking was performed to evaluate the binding affinities of TB, NB, NT, and NTB toward IL-10, IL-12, and metacaspase-5. The results are summarized in Table 1. Negative MolDock scores indicate spontaneous binding, with more negative values reflecting stronger interactions.

Table 1.

Calculated Docking Score by MVD

Compound	IL-10	IL-12	metacaspase-5
TB	−176.31	−185.89	−191.99
NB	−107.91	−127.42	−116.09
NT	−64.10	−70.66	−80.76
NTB	−174.29	−210.63	−205.29

The MolDock scores of TB, NB, NT, and NTB for IL-10 were −176.316, −107.91, −64.10, and −174.289, respectively. For IL-12, the corresponding scores were −185.89, −127.42, −70.66, and −210.63. Against metacaspase-5, the scores were −191.99, −116.09, −80.76, and −205.29. NTB consistently exhibited the strongest binding affinity among the four compounds, followed closely by TB, while NB and especially NT showed considerably weaker interactions.

For the IL-10 receptor (PDB: 4×51), the interaction patterns of TB, NB, and NT are shown in Figures 3a–c. NTB was stabilized through van der Waals interactions, conventional and carbon–hydrogen bonds, amide–Pi stacking, and Pi-alkyl contacts with key residues including Met(A)77, Gly(A)81, Gly(A)95, and Glu(A)96 (Figure 3d).

Figure 3.

Representation of the best-scoring docking solution of the (a) TB, (b) NB, (c) NT, and (d) NTB with the IL-10 receptor with the selected crystal structure of 4X51, along with the ligand map with various chemical bonds of Discovery Studio.

Against the IL-12 receptor (PDB: 1F45), the corresponding binding modes of TB, NB, and NT are presented in Figure 4a–c. NTB formed an extensive interaction network comprising van der Waals forces, hydrogen bonds, Pi-Sigma, Pi-sulfur, Pi-donor hydrogen bonds, alkyl, Pi-alkyl, Pi–Pi T-shaped, and amide–Pi stacked interactions involving residues such as Ile(A)89, Pro(A)101, Lys(A)102, Asn(A)103, Lys(A)104, Thr(A)105, and Tyr(A)114 (Figure 4d).

Figure 4.

Representation of the best-scoring docking solution of the (a) TB, (b) NB, (c) NT, and (d) NTB with the IL-12 receptor with the selected crystal structure of 1F45, along with the ligand map with various chemical bonds of Discovery Studio.

Docking to metacaspase-5 revealed that the binding poses of TB, NB, and NT are illustrated in Figure 5a–c. NTB interacted mainly via van der Waals forces, conventional hydrogen bonds, alkyl, and Pi-alkyl contacts with residues Gln(A)151, Pro(A)160, Leu(A)170, Val(A)180, Ala(A)183, Lys(A)195, Thr(A)341, Ala(A)343, Ser(A)392, and Gln(A)393 (Figure 5d).

Figure 5.

Representation of the best-scoring docking solution of (a) TB, (b) NB, (c) NT, and (d) NTB with metacaspase-5 receptor, along with the ligand map with various chemical bonds of Discovery Studio.

Overall, the superior binding affinity of NTB toward all three targets supports its potential dual role in immunomodulation and direct antiparasitic activity.

ADMET and Drug-Likeness Predictions

ADMET is a vital tool for predicting the pharmacokinetics of molecules and for assessing Human Intestinal Absorption (HIA).⁴⁷ Our findings indicate that both NTB and B were predicted as HIA⁻, while T was predicted as HIA⁺. Notably, NTB was predicted not to penetrate the Blood-Brain Barrier (BBB⁻) and to act as a P-glycoprotein (P-gp) substrate (both NTB and B were substrates). Regarding metabolism, NTB acts as a substrate but not an inhibitor of most CYP450 microsomal enzymes. Using the AMES test for drug toxicity, we found that NTB, B, and T were neither mutagenic nor carcinogenic. NTB exhibited lower oral toxicity than B and T, as evidenced by the higher LD₅₀ value (2.88 mol/kg). Although NTB showed the lowest aqueous solubility (LogS = −3.71), its overall toxicity profile was more favorable. In conclusion, our study underscores the importance of ADMET in drug development decisions, as summarized in Table 2.⁴⁸

Table 2.

ADMET Predictions of B, T, and NTB Calculated by ADMET SAR Tool

Model	Result
	NTB			B			T
Absorption
Blood-Brain Barrier	BBB−*	0.7027		BBB+		0.6525	BBB+	0.9188
Human Intestinal Absorption	HIA−	0.6144		HIA−		0.8273	HIA+	1.0000
Caco-2 Permeability	Caco2−	0.6406		Caco2+		0.6619	Caco2+	0.5000
P-glycoprotein Substrate	Yes	0.9399		Yes		0.7058	No	0.7345
P-glycoprotein Inhibitor	No	0.6133		No		0.9777	No	0.9477
	No	0.6368		No		0.8230	No	0.9777
Renal Organic Cation Transporter	No	0.8392		No		0.6665	No	0.9088
Distribution
Subcellular localization	Mitochondria	0.4821		Lysosome		0.8148	Mitochondria	0.5892
Metabolism
CYP450 2C9 Substrate	No	0.7634		No		0.7773	No	0.7585
CYP450 2D6 Substrate	No	0.7885		No		0.6795	No	0.8684
CYP450 3A4 Substrate	Yes	0.7242		No		0.6772	No	0.7743
CYP450 1A2 Inhibitor	No	0.7645		No		0.8246	Yes	0.6802
CYP450 2C9 Inhibitor	No	0.7428		No		0.8843	No	0.8538
CYP450 2D6 Inhibitor	No	0.8383		Yes		0.5455	No	0.8146
CYP450 2C19 Inhibitor	No	0.6708		No		0.8858	No	0.5000
CYP450 3A4 Inhibitor	Yes	0.6585		No		0.7368	No	0.8309
CYP Inhibitory Promiscuity	Low	0.7187		Low		0.9270	Low	0.8050
Excretion
Toxicity
Human Ether-a-go-go-Related Gene Inhibition	Weak		0.7107		Weak	0.5890	Weak	0.9446
	Yes		0.5391		Yes	0.5998	No	0.9797
AMES Toxicity	No		0.6132		No	0.8382	No	0.8418
Carcinogens	No		0.8285		No	0.6786	No	0.9109
Acute Oral Toxicity	III		0.5879		III	0.7915	III	0.7981
Absorption
Aqueous solubility, LogS	−3.7077				−1.9524		−2.2202
Rat Acute Toxicity, LD50, mol/kg	2.8837				2.1834		2.4553

Notes: *Superscripts “+” and “−” indicate positive and negative qualitative predictions generated by the admetSAR server, respectively

In vitro Assay

Anti-Leishmanial Activity Against Promastigote and Intramacrophage Amastigote of L. major

The IC₅₀ values of NT, NB, and NTB were 322.7±10.0 μg/mL, 101.8±1.9 μg/mL, and 79.0±3.2 μg/mL against promastigotes of L. major, respectively. However, the IC₅₀ value of MA was higher (673.0±17.3 μg/mL) than that of the positive control drug. The IC₅₀ values of NT, NB, and NTB were significantly lower than that of MA (p < 0.001).

The antileishmanial activities of NT, NB, and NTB in the macrophage model were evaluated by counting the number of amastigotes in 100 macrophages in triplicate. The IC₅₀ values of NT, NB, NTB, and MA were 83.9±3.1 μg/mL, 62.2±3.2 μg/mL, 44.4±0.8 μg/mL, and 103.6±8.5 μg/mL against amastigotes of L. major, respectively. The selectivity index [SI = CC₅₀ macrophage/IC₅₀ ≥1, non-toxic for amastigote forms] was also calculated for the medications, and the highest level was obtained for NTB (9.8) (Table 3).

Table 3.

Anti-Leishmanial Activity and 50% Inhibitory Concentration (IC₅₀) Values and Selectivity Index (SI) Were Calculated for NT, NB, NTB, and MA on Promastigotes and Amastigotes of Leishmania major

Compounds	IC50 Promastigote Mean ± SD (µg/mL)	IC50 Amastigote Mean ± SD (µg/mL)	SI
NT	322.7±10.0	83.9±3.1	1.6
NB	101.8±1.9	62.2±3.2	5.1
NTB	79.0±3.2	44.4±0.8	9.8
MA	673.0±17.3	103.6±8.5	6.9

In vivo Assay

Effect of Niosomal Formulations on Lesion Size in BALB/c Mice

All therapeutic groups showed a significant reduction in lesion size compared with the placebo group (p < 0.001) and the blank niosome group (N) (p < 0.05). At the end of the 4-week treatment period, mean lesion sizes (± SD) were as follows: placebo 8.6 ± 0.9 mm, blank niosome 8.3 ± 1.1 mm, NT 3.1 ± 0.5 mm, NB 2.9 ± 0.6 mm, NTB 1.2 ± 0.1 mm and MA 3.3 ± 0.5 mm. NTB was more effective than the other formulations and MA in reducing the size of the lesion, and these differences were significant (p < 0.05) (Figure 6). After cessation of treatment, a partial increase in lesion size was observed in all groups; however, lesions in the NTB-treated animals remained significantly smaller than those in the placebo group (p < 0.001).

Figure 6.

In vivo efficacy of placebo, topical blank niosome (N), NT, NB, NTB, and injectable MA on the course of disease in BALB/c mice infected with Leishmania major. Values are the mean of lesion sizes in five mice in each group; bars represent the standard deviation.

Representative photographs of the footpad lesions after 4 weeks of treatment are shown in Figure 7. Mice in the placebo and blank niosome groups exhibited severe ulcerative lesions with marked swelling and necrosis. Treatment with meglumine antimoniate (MA) resulted in a moderate reduction in lesion size, although residual inflammation was still evident. The NT and NB niosomal gels produced partial healing with noticeably smaller lesions and reduced erythema compared with the control groups. The most pronounced therapeutic effect was observed in the NTB-treated group, in which the lesions showed almost complete resolution with minimal residual swelling, consistent with the quantitative lesion-size measurements.

Figure 7.

Representative photographs of footpad lesions in BALB/c mice infected with Leishmania major after 4 weeks of treatment. (a) Placebo (untreated control) showing a severe ulcerative lesion with necrosis and extensive swelling. (b) Meglumine antimoniate (MA, systemic positive control) showing moderate reduction in lesion size with residual inflammation. (c) Blank niosome (N) showing a persistent lesion similar to the placebo group. (d) Tioxolone niosomal gel (NT) showing partial healing with reduced swelling and mild residual erythema. (e) Benzoxonium chloride niosomal gel (NB) showing further improvement with smaller lesion size and reduced inflammation. (f) Combined tioxolone–benzoxonium chloride niosomal gel (NTB) showing the most pronounced healing, with almost complete resolution of the lesion and minimal residual swelling.

Splenic Parasite Burden

Eight weeks after the start of treatment, the spleen parasite burden in mice treated with topical niosomal gels of NT, NB, and NTB, and with injectable MA, showed a more substantial reduction relative to the placebo group and the blank niosomes (p < 0.001). There were statistically significant differences between the NTB group and the other niosomal drug groups and the MA group (p < 0.05) (Figure 8).

Figure 8.

Splenic parasite burdens in BALB/c mice treated with placebo, topical blank niosome, NT, NB, NTB, and injectable MA. Data represent the mean ± SE for 5 animals per group. *p < 0.05 compared with the placebo and blank niosome groups (one-way ANOVA).

Discussion

Cutaneous leishmaniasis causes skin lesions that develop weeks to months after a female sandfly bite and, if left untreated, can leave permanent scars, grave stigmatization, and significant disabilities.⁴⁹ In developing and third-world countries, CL is still endemic because only a limited number of drugs are available to patients. In addition, most of these treatment regimens require systemic administration, which causes serious discomfort, poor patient compliance, high treatment costs, and poor treatment outcomes. Various topical treatments have long been studied to overcome these problems, as they are more convenient dosage forms for CL that deliver high drug concentrations at the site of infection.

Although no definitive cure for CL has been found to date, pentavalent antimonials remain the first-line treatment for most leishmaniasis forms. The need for intralesional or systemic administration by physicians in specialized centers, the possibility of treatment failure, drug resistance in parasite species, painful injection, toxicity, and other side effects, especially at high doses of pentavalent antimonials, have led to the search for new alternatives that are worthwhile. Therefore, over the past few years, efforts have been made to replace systemic or injectable drugs with topical treatment. In addition, the topical application of niosomal drugs increases patient compliance and completes the treatment course, which can reduce the incidence of resistance.^10,50

Many topical treatments, such as topical miltefosine, paromomycin, imiquimod skin cream, fluconazole, ketoconazole, and topical liposomal amphotericin B, have been studied and proposed for the treatment of CL.^51–56

In this study, we used in silico techniques to simulate virtual expression data analysis, hypothesis testing, molecular models, and design to uncover the core concepts related to immune response pathways. In comparison to laboratory results, computation-based investigations or in silico strategies can reveal the molecular interactions between IL-10, IL-12, and metacaspase-5 with TB, NB, NT, and NTB. In silico techniques simulate virtual expression data, molecular models, and designs, allowing researchers to evaluate the need for specific research or immunoassays through computational simulation. In addition, it is essential to study the structural attributes of the metacaspase-5 receptor. In this study, we predicted the 3D structure of metacaspase-5 using homology modeling and performed molecular docking analysis of TB, NB, NT, and NTB with IL-10, IL-12, and metacaspase-5. We also conducted in silico ADMET and drug-likeness profiling to identify a potential therapeutic alternative for Leishmania. This study demonstrated that the NTB formulation exhibited a lower LogS value, indicating reduced aqueous solubility, together with lower predicted oral toxicity than the individual components, while showing superior efficacy compared with the other niosomal formulations.^{19–21,37,47}

The NTB formulation showed strong binding affinity for key immunomodulatory proteins, including IL-10, IL-12, and metacaspase-5, as demonstrated by molecular docking. IL-10 is an anti-inflammatory cytokine that, when overexpressed, can promote parasite survival by suppressing macrophage activation and Th1 immune responses; NTB’s interaction with IL-10 suggests it may help inhibit this immunosuppressive effect. Conversely, IL-12 plays a crucial role in promoting the Th1 immune response, which is essential for effective clearance of Leishmania parasites; therefore, NTB’s enhancement of IL-12-related pathways could help boost host defense. The balancing effect of NTB on the IL-10/IL-12 ratio helps to reduce local inflammation while enhancing parasite killing, thereby preventing tissue damage from excessive inflammation.²⁵ Additionally, the influence of NTB on metacaspase-5, an apoptosis-regulating enzyme, may facilitate programmed death of infected cells, contributing to parasite clearance and controlled immune responses. These combined immunomodulatory effects of NTB underpin its potential as a topical treatment that directly targets the parasite while modulating the host immune environment to favor healing and reduce pathology.

In our in vitro studies, the IC₅₀ of niosomal formulations of both promastigotes and amastigotes against L. major showed significant differences compared to MA (standard treatment) (p < 0.05). In addition, a comparison between the IC₅₀ values obtained for L. tropica and L. major showed that these formulations had a similar effect on promastigotes and amastigotes of both parasite species, with no significant difference in IC₅₀ values. In addition, in previous in vitro studies, we showed that the niosomal forms of these drugs performed much better than their simple forms in inhibiting L. tropica, modulating the immune response, and inhibiting the parasite. The results of this study, which were conducted as in silico and in vivo studies, confirm previous results.^19,20

Sharifi et al (2024) recently reported that Gossypium hirsutum niosomes produced greater therapeutic outcomes than the conventional formulation in a murine model and evaluated their effectiveness by assessing the parasite burden, immunomodulatory gene expression, and histopathological profile.⁵⁷ Similar results were obtained in studies by Mostafavi et al (2019) on amphotericin B, selenium, and MA.^58–61 The results of these studies are fully consistent with ours.

To evaluate the safety and therapeutic window of each formulation, the selectivity index (SI) was calculated as the ratio of the CC₅₀ value for macrophages to the IC₅₀ for amastigotes. An SI ≥ 1 is generally considered an indication of selective toxicity towards the parasite rather than the host cells.⁶² NTB exhibited the highest SI (9.8), indicating potent anti-leishmanial activity and low cytotoxicity, which is consistent with our in silico results. In comparison, the SI values for NB (5.1) and MA (6.9) were moderate, whereas NT had a relatively low SI (1.6), suggesting limited selectivity and a narrower therapeutic margin.

The enhanced efficacy and selectivity of NTB may be attributed to multiple factors: the synergistic pharmacological action of its two active components, the ability of the niosomal system to facilitate cellular uptake and drug accumulation at the site of infection, and the improved stability and controlled-release properties of the formulation.^18,20 These results strongly support the potential of NTB as a promising and safe topical alternative for cutaneous leishmaniasis treatment.

In our in vivo studies, topical niosomal formulations were evaluated for their efficacy in treating ulcers induced by L. major in BALB/c mice. The results of the in vivo study with BALB/c mice infected with L. major showed that the topical niosomal formulations induced the cure of the lesions in susceptible BALB/c mice, and the treated mice had significantly lower parasite burdens in the spleen than the placebo and blank niosome groups at week 12 post-infection. We sought a combined formulation (NTB) that significantly reduced the size of footpad swelling and the number of parasites in the spleen of mice compared with other groups.

After topical application of the niosomal gel to the lesions, the niosomes are thought to pass through the subcutaneous tissues and reach the epidermal and dermal layers. In the deep dermis, infected macrophages may take up the niosomes by endocytosis, after which the drug is released following lysosomal degradation of the vesicles, as has been reported in similar niosomal delivery studies.⁶³

The superior performance of NTB may be attributed to the synergistic action of tioxolone and benzoxonium chloride as well as the enhanced skin penetration and sustained release properties afforded by the niosomal delivery system. The ability of NTB to reduce lesion size more effectively than MA, a widely used first-line treatment, suggests its potential as a viable topical alternative to current systemic therapy. Following treatment cessation, a partial increase in lesion size was observed across all groups, indicating the degree of disease relapse. However, lesions in the NTB-treated group remained significantly smaller than those in the placebo group (p < 0.001), suggesting a more durable therapeutic effect compared with other formulations.

The benzoxonium chloride component of NTB exhibits strong antimicrobial properties, particularly against bacterial biofilms.⁶⁴ Biofilms are complex bacterial communities that protect pathogens from antibiotics and immune responses, often leading to persistent infections at wound sites. By disrupting the bacterial biofilm matrix, benzoxonium chloride can help reduce biofilm formation and facilitate bacterial eradication, thereby controlling secondary infections that commonly complicate cutaneous leishmaniasis wounds. This antibiofilm activity of NTB contributes to improved wound healing, reduces inflammation driven by bacterial colonization, and enhances overall treatment outcomes, supporting its use as a promising topical formulation.⁶⁵

Although niosomal delivery systems have previously been explored for the topical treatment of cutaneous leishmaniasis, the present study provides additional value by evaluating the specific combination of tioxolone and benzoxonium chloride in a niosomal gel, supported by integrated in silico, in vitro and in vivo data. The superior efficacy of the NTB formulation compared with both its individual components and systemic meglumine antimoniate in the murine model further supports its potential as a promising topical candidate.

This study has several limitations that should be acknowledged. First, the in vivo experiments were conducted with a relatively small sample size (n = 5 mice per group), which may limit the statistical robustness and generalizability of the findings. Second, although in silico docking suggested possible interactions with IL-10 and IL-12, actual cytokine levels (eg, IL-10, IL-12) were not measured in the treated animals, meaning the proposed immunomodulatory mechanism remains a hypothesis to be validated experimentally. Third, pharmacokinetic, skin-penetration, and rheological characterizations of the niosomal gels were not performed; therefore, the extent of local drug delivery, systemic exposure, and the physical flow behavior and stability of the formulations remain to be determined. Finally, a formal toxicity evaluation (biochemical, histopathological, or dermal safety assessment) was not conducted. These aspects should be addressed in future studies to strengthen the translational potential of the NTB niosomal gel.

Conclusion

In conclusion, this study demonstrated that NTB exhibited superior in silico binding affinity to key leishmaniasis-related proteins, favorable ADMET properties, and enhanced in vitro and in vivo anti-leishmanial efficacy compared to its individual components and meglumine antimoniate. While niosomal approaches for cutaneous leishmaniasis have been investigated previously, the specific drug combination and the comprehensive multi-level evaluation presented here highlight NTB as a promising topical candidate that warrants further clinical investigation.

Funding Statement

This work was supported by the Vice-Chancellor for Research, Kerman University of Medical Sciences, Kerman, Iran [grant number 98000424]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abbreviations

NTD, neglected tropical disease; WHO, World Health Organization; CL, cutaneous leishmaniasis; VL, visceral leishmaniasis; MCL, mucocutaneous leishmaniasis; ART, artemether; AmB, amphotericin B; SI, selectivity index; ASEO, Siberian Artemisia annua essential oil; T, Tioxolone; B, benzoxonium chloride; TB, Tioben; MA, meglumine antimoniate; N, niosome; NT, niosomal of T; NB, niosomal of B; NTB, niosomal of TB; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; D, thickness; F, width; IM, intraperitoneally; IC₅₀, 50% inhibitory concentration; CC_50, 50% cytotoxicity concentration.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report that there are no competing interests in this work.