The effect of Niosomes-Loaded Rapamycin against Leishmania major: An in vitro and in vivo study

Technical Summary: The Effect of Niosomes-Loaded Rapamycin against Leishmania major: An in vitro and in vivo study

Problem Statement
Cutaneous leishmaniasis (CL), caused by Leishmania parasites, remains a significant public health challenge with limited therapeutic options. Current first-line treatments, such as pentavalent antimonials (e.g., Glucantime), are associated with severe toxicity, the need for painful parenteral administration, and emerging drug resistance. While topical formulations offer a route to avoid systemic toxicity, they often fail due to poor drug penetration through the stratum corneum and inadequate retention within dermal macrophages, the primary host cells for the parasite. Additionally, the immunomodulatory potential of rapamycin (an mTOR inhibitor) to shift the host response toward a protective Th1 phenotype has been noted, but its clinical application is hindered by poor aqueous solubility and low bioavailability. This study addresses the need for a stable, effective topical delivery system capable of targeting intracellular Leishmania major amastigotes while leveraging the immunomodulatory properties of rapamycin.

Methodology
The study employed a comprehensive approach involving formulation development, physicochemical characterization, and biological evaluation:

• Formulation: Rapamycin-loaded niosomes were prepared using the ethanol injection method. The formulation consisted of rapamycin, cholesterol, Polysorbate 80 (hydrophilic surfactant), and Span 20 (lipophilic surfactant). The organic phase was injected into a heated aqueous phase (60°C) to induce self-assembly.
• Characterization: The resulting niosomes were analyzed for particle size, polydispersity index (PDI), zeta potential, and morphology (via TEM). Encapsulation efficiency (EE%) was determined using HPLC after ultracentrifugation. Physical stability was assessed over 90 days at refrigerated (4–8°C) and room temperatures.
• In Vitro Assays:
  • Promastigote Susceptibility: L. major promastigotes were treated with free rapamycin, niosomal rapamycin, and Glucantime. Metabolic activity was measured via optical density (OD) at 540 nm after 72 hours to determine IC₅₀ values.
  • Amastigote Susceptibility: Murine peritoneal macrophages were infected with stationary-phase promastigotes. Infected cells were treated with the test formulations for 72 hours. Amastigote burden was quantified by counting parasites in Giemsa-stained slides.
• In Vivo Study: A murine model using female BALB/c mice was established by subcutaneous inoculation of L. major into the right hind footpad. After 4 weeks of infection, mice were divided into four groups (n=5):
  1. Topical Rapamycin-Loaded Niosomal gel.
  2. Topical Free Rapamycin gel.
  3. Topical Empty Niosomal gel (vehicle control).
  4. Intraperitoneal (IP) Glucantime (positive control).
     Lesion size (footpad thickness) was measured weekly for 28 days.

Key Contributions and Results

• Physicochemical Properties: The niosomal formulation was successfully optimized, yielding spherical bilayer vesicles with a mean particle size of approximately 220 nm, a low PDI (<0.3), and an exceptionally high encapsulation efficiency (≥99.99%). The formulation demonstrated robust physical stability over 90 days with no significant changes in size, phase separation, or drug crystallization.
• In Vitro Efficacy:
  • Amastigotes: Both free and niosomal rapamycin exhibited significant activity against intracellular amastigotes. Notably, the niosomal formulation showed efficacy comparable to the standard drug Glucantime, with IC₅₀ values around 14–15 µg/mL.
  • Promastigotes: Glucantime demonstrated significantly higher potency against extracellular promastigotes compared to rapamycin formulations. While niosomal rapamycin showed a slight numerical improvement over free rapamycin, the difference was not statistically significant.
• In Vivo Efficacy: Despite promising in vitro results, the topical application of Rapamycin-Loaded Niosomes failed to reduce lesion size in the murine model. There was no statistically significant difference in lesion progression between the niosomal rapamycin group, the free rapamycin group, and the untreated control group over the 4-week treatment period. In contrast, the intraperitoneal administration of Glucantime resulted in a consistent and significant reduction in lesion size.

Significance and Claims
The paper claims that while the niosomal formulation successfully achieved optimized physicochemical properties and demonstrated potent in vitro activity against the pathogenic amastigote stage (comparable to Glucantime), this efficacy did not translate to in vivo therapeutic success via the topical route.

The study highlights a critical disparity between in vitro potency and in vivo outcome. The authors conclude that the failure of the topical formulation is likely due to insufficient drug penetration into the deep dermal tissue or inadequate drug concentration at the site of infection to exert a therapeutic or immunomodulatory effect. The results underscore that for Leishmania major infection, systemic delivery (as demonstrated by IP Glucantime) remains the gold standard, and topical therapies face substantial challenges in delivering sufficient drug concentrations to combat deep-seated intracellular parasites. The paper suggests that future work must focus on enhancing drug penetration strategies or exploring systemic administration of niosomal rapamycin rather than relying solely on topical application for this specific formulation.