Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

Abstract

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

Fig 1. Sensitive profile of T. brucei bloodstream forms.

(a) IC₅₀ analysis. Pentamidine (grey column); pentamidine-loaded PEGylated chitosan nanoparticles (pentamidine-chNPs, red column) and nanobody-coated pentamidine-loaded PEGylated chitosan nanoparticles (NbAn33-pentamidine-chNPs, blue column). Errors bars indicate S.D. from 3–9 independent experiments. Statistical significance was ***, p<0.001. (b) Therapeutic effect in T. brucei acute infection mouse model. Survival (Kaplan-Meier plot) of female C57BL/6J mice infected with T. brucei AnT1.1 (1 x 10⁴ parasites). The treatment started once the parasites were detected in blood, at the 3rd day after inoculation and consisted in a daily dose in four consecutive days. Treatment with pentamidine, pentamidine-chNPs, NbAn33-pentamidine-chNPs, NbAn33-chNPs empty (nanobody-coated non pentamidine-loaded PEGylated chitosan nanoparticles) and vehicle (physiological saline solution). (c) Parasitemia in T. brucei acute infection mouse model. Treatment with vehicle (physiological saline solution), NbAn33-chNPs empty (nanobody-coated PEGylated-chitosan nanoparticles), free pentamidine, pentamidine-chNPs (pentamidine-loaded PEGylated chitosan nanoparticles), NbAn33-pentamidine-chNPs (nanobody-coated pentamidine-loaded PEGylated chitosan nanoparticles).

Fig 2. Circulation kinetics of fluorescent chitosan nanoparticles in mouse model.

Percentage of the initial dose in peripheral blood when injected via intravenous (i.v.) and intraperitoneal (i.p.) vs. time. Error bars represent the S.D. from 5 mice.

Fig 3. Endocytosis of NbAn33-chNPs.

(a) Bloodstream trypanosomes observed by fluorescence microscopy after incubation with NbAn33-chNPs-Alexa Fluor 594 (red) and tomato lectin-FITC (TL, green) as described in Materials and Methods. Samples were taken after 2 minutes (bottom panel) and 10 minutes (top panel) of incubation. DNA is stained with DAPI (blue). Regions of colocalization appear yellow in merged images. (b) Parasite viability after incubation with NbAn33-pentamidine-chNPs at 37° and 4°C for 2 h. Cell death was estimated by propidium iodide staining and FACS analysis at three time points. Error bars represent the S.D. from three independent experiments. Statistical significance was *, p<0.05; ***, p<0.001.

Fig 4. Characterization of the pentamidine resistant strain TbR25.

(a) Relative quantification (RQ) of TbAT1/P2 expression in wild type AnTat 1.1 and TbR25 strains estimated by qRT-PCR. (b) IC₅₀ value for diminazene aceturate in the same strains. Error bars indicate S.D. from 3 replicates. (c) Schematic illustration of the AQP2/AQP3 locus showing the heterozygote character of TbR25 strain with a chimeric gene in one allele and the complete deletion of AQP2 gene and the intergenic region in the other (deletion from position 3441867 to 3443663 in chromosome 10, -Tb927_10_v5).

Fig 5. Sensitive profile of the pentamidine resistant strain TbR25.

(a) IC₅₀ analysis. Free pentamidine (grey column); pentamidine-chNPs (red column) and NbAn33-pentamidine-chNPs (blue column). Errors bars indicate SEM from 3–9 independent experiments. Fold reductions are indicated in the graph. (b) Therapeutic effect in TbR25 acute infection mouse model. Survival (Kaplan-Meier plot) of female C57BL/6J mice infected with TbR25 (inoculum 2.5 x 10⁶ parasites).