Liposomes versus Lipid Nanoparticles Comparative Study of Lipid-Based Systems as Oryzalin Carriers for the Treatment of Leishmaniasis

Purpose: Current leishmaniasis chemotherapy lacks options that combine high activity, low toxicity, and affordable cost. Oryzalin (ORZ), a dinitroaniline with in vitro antileishmanial activity, is limited by poor water solubility and low accumulation in diseased organs. Lipid-based nanocarriers may overcome these limitations, but liposomes and solid lipid nanoparticles had not been systematically compared as ORZ carriers.

Hypothesis: Incorporating ORZ into liposomes or solid lipid nanoparticles (LNP) will improve its physicochemical properties, reduce cytotoxicity and haemolytic activity, enhance delivery to liver and spleen macrophages, and increase in vivo antileishmanial efficacy versus free ORZ. Liposomes and LNP may show different organ-specific activity profiles.

Aims: Prepare and characterize ORZ-loaded liposomes (Lip-ORZ) and LNP (LNP-ORZ) suitable for parenteral administration. - Evaluate in vitro haemolytic activity, cytotoxicity, and antileishmanial activity against L. infantum promastigotes and intracellular amastigotes. - Assess in vivo antileishmanial activity in a murine visceral leishmaniasis model: administration route, number of administrations, dose–response, and lipid excipient effects. - Systematically compare liposomes versus LNP as ORZ carriers and compare efficacy with Glucantime.

Delivery system: Platform A — Liposomes: DMPC:DMPG (7:3 molar ratio) with ORZ; prepared by dehydration–rehydration and extrusion. ORZ incorporated in the lipid bilayer. Size ~0.21–0.22 µm; zeta potential ~ −30 to −35 mV; ORZ incorporation efficiency 91–94%; loading ~32 g ORZ/mol lipid. - Platform B — Solid lipid nanoparticles (LNP): Lecithin:tripalmitin (7.3:2.4) with ORZ. ORZ incorporated in the lipid matrix and surfactant layer. Size ~0.16–0.17 µm; zeta potential ~ −22 to −24 mV; incorporation efficiency 95–97%; loading ~34 g ORZ/mol lipid. - Payload: Oryzalin, a dinitroaniline microtubule inhibitor. - Targeting ligand: None; passive macrophage uptake driven by size and negative surface charge. - Controls: Free ORZ, unloaded liposomes, unloaded LNP, Glucantime.

Approach: In vitro models: L. infantum promastigotes expressing GFP; THP-1 human monocytic/macrophage-like cells; human red blood cells. - In vivo model: BALB/c mice infected i.v. with 10⁶ L. infantum promastigotes. Treatment started day 7 post-infection; groups of five mice. - Treatment regimens: i.v. Lip-ORZ, LNP-ORZ, or free ORZ at 25 mg/kg/day for 5 or 10 days; dose–response at 6.25, 12.5, and 25 mg/kg/day; lipid excipient effect at 6.25 mg/kg ORZ with different lipid doses. - Comparison: Glucantime 45 mg/kg/day s.c. for 5 days. - Endpoint: Parasite burden in liver and spleen by limiting dilution assay; ED₅₀ and growth index calculated.

Key methods: HPLC for ORZ quantification. - Dynamic light scattering and zeta potential for particle size, PDI, and surface charge. - Haemolysis assay for HC₅₀. - Propidium iodide flow cytometry for cytotoxicity (CC₅₀) and antileishmanial activity (IC₅₀). - Intracellular infection assay in THP-1 macrophages using GFP-expressing parasites. - Limiting dilution assay for parasite burden in liver and spleen. - Statistical analysis: one-way ANOVA, Kruskal–Wallis with Dunn’s post-test, p < 0.05.

Key results: Formulation: Lip-ORZ ~0.21 µm, ζ ~ −30 to −35 mV, incorporation efficiency 91–94%. LNP-ORZ ~0.17 µm, ζ ~ −22 to −24 mV, incorporation efficiency 95–97%. - In vitro toxicity: Free ORZ: HC₅₀ 425 µM, CC₅₀ 20 µM. Lip-ORZ and LNP-ORZ: HC₅₀ > 500 µM, CC₅₀ > 25 µM. Both nanocarriers abolished haemolysis and reduced cytotoxicity. - In vitro antileishmanial activity: Free ORZ: promastigote IC₅₀ 17 µM, intracellular IC₅₀ 19 µM. Lip-ORZ: promastigote IC₅₀ > 25 µM, intracellular IC₅₀ 48 µM. LNP-ORZ: promastigote IC₅₀ > 25 µM, intracellular IC₅₀ 44 µM. Encapsulation reduced promastigote activity but retained intracellular activity. - In vivo efficacy (5 × 25 mg/kg i.v.): Lip-ORZ reduced liver parasite burden by 89% and spleen by 84%; LNP-ORZ reduced liver by 84% and spleen by 91% versus control. Free ORZ significantly reduced burden only in spleen. Lip-ORZ produced 97% liver reduction versus 81% for Glucantime, despite Glucantime being given at a ~2-fold higher dose. - Dose–response ED₅₀ reductions vs free ORZ: Liver — 65-fold for Lip-ORZ, 6-fold for LNP-ORZ; spleen — 3-fold for Lip-ORZ, 11-fold for LNP-ORZ. Plateaus: LNP-ORZ spleen at 6.25 mg/kg (86% reduction); Lip-ORZ spleen at 12.5 mg/kg (74%); Lip-ORZ liver at 6.25 mg/kg (95%); LNP-ORZ liver at 12.5 mg/kg (82%). Free ORZ max reduction: 73% spleen, 62% liver at 25 mg/kg. - Number of administrations: Increasing from 5 to 10 did not further reduce parasite burden. - Lipid excipient: Unloaded liposomes and LNP showed no activity; lipid dose did not significantly affect parasite burden.

Interpretation: The authors conclude that both ORZ nanoformulations improve in vivo antileishmanial efficacy, reduce toxicity and haemolysis, and are potential therapeutic candidates for visceral leishmaniasis. Lip-ORZ was more active in liver, while LNP-ORZ was more active in spleen; these differences may relate to carrier rigidity and splenic filtration. Biodistribution studies are needed to clarify the organ-specific behavior.

Limitations: No biodistribution profile was performed in this study; authors note this as future work. - Encapsulation reduced in vitro activity against promastigotes, and intracellular IC₅₀ values were higher than free ORZ. - Only one murine model (L. infantum in BALB/c mice) and small group sizes (n = 5) were used. - No survival study or long-term toxicity assessment. - Comparison with standard liposomal amphotericin B was not performed. - Free ORZ required Tween 80 vehicle, which may confound direct comparisons.