Cutaneous leishmaniasis (CL), a neglected tropical disease caused by protozoan parasites of the Leishmania genus, remains a significant global health challenge [1,2]. CL is a vector-borne disease spread through the bite of infected female sandflies, which transmit the parasite to reservoir hosts such as humans, rodents, and canines [3]. Visible CL lesions often cause psychological distress, as their appearance is linked to social stigma, potentially resulting in self-isolation, self-stigmatization, and broader psychosocial health challenges [2,4]. The disease is endemic in 89 countries, primarily concentrated in the Eastern Mediterranean, African, and Latin American regions, as well as the Indian subcontinent [2]. Annual global incidence is estimated at approximately 0.7–1 million new cases, with the 2019 Global Burden of Disease study reporting a CL prevalence of 4.6 million cases worldwide [2].

First-line therapy for CL relies on pentavalent antimonial compounds (e.g., Glucantime®, Pentostam). However, their efficacy is limited by slow therapeutic response, risk of incomplete healing, and potential disease relapse within six months. Treatment adherence is further complicated by the prolonged 30-day regimen of daily intramuscular injections, which is painful, traumatic, and associated with severe adverse effects. These challenges, coupled with limited healthcare access in rural areas, often lead to interrupted treatment cycles [5].

Second-line therapies, including amphotericin B, pentamidine (PTM), miltefosine, and paromomycin (PRM), present additional barriers. These alternatives are costly, exhibit significant toxicity profiles, and face formulation challenges due to the high hydrophilicity of active compounds, which cannot be easily mitigated by conventional excipients [5].

In recent years, nanotechnology-based drug delivery systems have emerged as transformative tools for addressing these challenges [[6], [7], [8]]. Among these, nanostructured lipid carriers (NLCs)—-second-generation lipid nanoparticles—offer distinct advantages over conventional formulations [5,9].

NLCs are colloidal systems composed of a hybrid solid-liquid lipid matrix stabilized by a solid lipid shell [10]. This blend creates a disordered lipid structure, enabling efficient drug encapsulation and prolonged shelf stability [10]. NLCs exhibit versatility in accommodating both hydrophilic and hydrophobic drugs [11], alongside advantages such as scalable production, biocompatibility, robust physical stability, high drug-loading capacity, minimized leaching, and enhanced solubility profiles, positioning them as superior to traditional drug delivery platforms [10]. Their biocompatibility allows administration via multiple routes, including oral, parenteral, topical, rectal, and pulmonary pathways [10].

Furthermore, NLCs leverage passive targeting by exploiting the innate propensity of macrophages to internalize these nanoparticles without requiring surface modifications [12]. Hence their lipidic nature facilitates passive targeting to the mononuclear phagocyte system, ensuring preferential accumulation in infected macrophages—a critical advantage for intracellular pathogens like Leishmania [10,12,13]. Furthermore, NLCs exhibit enhanced transdermal delivery capabilities due to their unique lipid matrix and reduced particle size. These properties promote stronger adhesion to the stratum corneum, facilitating improved drug permeation and absorption through the skin barrier [9].

These attributes have propelled NLCs to the forefront of research for parasitic diseases, with studies demonstrating their efficacy in improving drug delivery for malaria, visceral leishmaniasis, and other infections [9,14]. However, the application of NLCs for CL, particularly in co-delivering synergistic drug combinations, remains underexplored—a gap this study seeks to address.

Rifampicin (RIF) and PTM, two antimicrobial agents [15], have shown promise as antileishmanial candidates [16]. RIF targets parasite transcription [17,18], while PTM disrupts energy metabolism [19], potentially creating a synergistic effect to eradicate resistant strains. Although RIF has shown promise in leishmaniasis therapy through oral administration—demonstrated in recent studies including clinical cases [18,20]—its systemic use can lead to dose-limiting toxicity and hepatic side effects, especially in prolonged regimens required for intracellular pathogens like Leishmania [21]. Furthermore, PTM is associated with nephrotoxicity and requires parenteral administration [22]. The rationale for reformulating these established drugs using NLCs stems from the need to localize therapy, improve skin penetration, and mitigate systemic adverse effects [23]. Topical nanocarriers allow targeted delivery to dermal macrophages while minimizing off-target exposure [24]. Additionally, co-encapsulation of RIF and PTM within NLCs provides a unique advantage of synergistic antimicrobial action, sustained release, and enhanced drug solubility, which are not achievable with free drug application [12,16]. This formulation strategy represents a novel and clinically relevant approach to repurpose known agents for a safer, more effective treatment of CL. The sustained release profile of NLCs aligns with the prolonged drug exposure required to eliminate intracellular amastigotes, which often persist in host cells despite transient drug availability [12,25].

Previous studies have laid the groundwork for lipid-based nanocarriers in leishmaniasis therapy. For instance, artemether-loaded NLCs demonstrated enhanced efficacy against L. major promastigotes compared to free drugs [26], while miltefosine-loaded NLCs improved lesion resolution in murine models [12]. However, these efforts primarily focused on single-drug delivery. Combination therapies, which target multiple parasitic pathways, are increasingly recognized as essential for synergizing drug action, reducing effective dosages, and delaying resistance [27,28].

Despite this, few studies [29] have explored NLCs for co-delivering antileishmanial agents. A notable exception is the co-encapsulation of amphotericin B and PRM in a modified solid lipid nanoparticles, which showed superior efficacy in visceral leishmaniasis models [30]. For CL, however, such approaches remain scarce, highlighting a critical research gap.

This study introduces a novel NLC-based formulation co-loaded with RIF and PTM for topical CL therapy. The rationale for this approach is threefold: (1) NLCs enhance drug solubility and skin penetration, critical for topical applications; (2) dual-drug delivery may synergistically target Leishmania through complementary mechanisms; and (3) lipid encapsulation reduces cytotoxicity, preserving host macrophage viability. The objectives of this work include optimizing the NLC-RIF/PTM formulation, characterizing its physicochemical properties (size, polydispersity, encapsulation efficiency), evaluating in vitro antileishmanial activity against both promastigotes and intramacrophage amastigotes, and assessing therapeutic efficacy in a BALB/c mouse model of CL.

By integrating advanced nanocarrier technology with combinatorial drug delivery, this research aims to overcome the limitations of current CL treatments. The findings could pave the way for a safer, more effective topical therapy, reducing treatment duration and improving patient quality of life. Furthermore, the insights gained may extend to other parasitic infections, reinforcing the broader potential of NLCs in neglected tropical disease management.