Promising therapeutic efficacy of nitazoxanide-loaded zinc oxide nano-formula against intestinal and muscular phases of experimental trichinellosis

Abstract

Trichinellosis is a ubiquitous parasitic infection caused by a zoonotic nematode known as Trichinella spiralis (T. spiralis). It starts with the adult worm in the intestinal phase and ends up with the larva reaching the muscles. The disease generally manifests with acute gastroenteritis; however, it may regrettably lead to life-threatening myositis, myocarditis and seizures. The commercially existing chemotherapeutic regimens have numerous drawbacks including severe adverse effects, high resistance rate, poor bioavailability and low efficiency towards the muscular stage. Consequently, the current study targeted the evaluation of nitazoxanide-loaded zinc oxide nanoparticles (NTZ-loaded ZnO NPs) used for the first time in the treatment of both the intestinal and muscular phases of trichinellosis in mice. Swiss Albino mice were orally infected by 250 T. spiralis larvae. The experimental animals were treated with the gold standard albendazole, NTZ, ZnO NPs as well as NTZ-loaded ZnO NPs. Parasitological, biochemical (creatine kinase, alanine transaminase, aspartate transaminase, alkaline phosphatase, malondialdehyde and nitric oxide), immunological (interleukins 2 and 4) and histopathological assessments were conducted. The parasitological results denoted that the mice treated with NTZ-loaded ZnO NPs revealed the uppermost significant drug efficacy (>97%) in both the intestinal and muscular phases indicating efficacious tissue penetration. Additionally, this group revealed the most profound amelioration of the biochemical and immunological markers as well as restoration of the histopathological picture. Conclusively, the present work implied a bird's eye view on the promising effectiveness of NTZ-loaded ZnO NPs in the treatment of murine trichinellosis relying on the anti-parasitic safe nature of the formulation.

Fig 1. TEM micrographs of prepared NPs showing spherical and hexagonal shapes with weak agglomeration and increasing the size after NTZ loading.

(A) Blank ZnO NPs (scale bar of 50 nm). (B) NTZ-loaded ZnO NPs (scale bar of 200 nm).

Fig 2. FTIR micrographs of the prepared NPs.

(A) Blank ZnO NPs. (B) NTZ-loaded ZnO NPs.

Fig 3. X.ray diffraction pattern of the prepared NPs.

(A) Blank ZnO NPs. (B) NTZ-loaded ZnO NPs.

Fig 4. Counts of T. spiralis.

(A) Mean count of T. spiralis adult worms in the intestine. (B) Mean count of T. spiralis larvae in muscles.

Fig 5. Biochemical changes in the sera of various subgroups with statistical comparison.

Fig 6. Biochemical changes in the muscle tissues of various subgroups.

(A) Mean MDA (nmol/g tissue) among various subgroups. (B) Mean NO (μmol/g tissue) among various subgroups.

Fig 7. Immunological changes in the intestinal phase among various subgroups.

(A) Mean IL-2 (pg/mg protein) among various subgroups. (B) Mean IL-4 (ng/mg protein) among various subgroups.

Fig 8. Immunological changes in the muscular phase among various subgroups.

(A) Mean IL-2 (pg/mg protein) among various subgroups. (B) Mean IL-4 (ng/mg protein) among various subgroups.

Fig 9. Intestinal histopathologic photomicrographs (H&E).

(A) Intestinal mucosa of normal uninfected control representing normal villous architecture with minimal chronic inflammatory cell infiltrate (x100). (B) and (C) Intestine of infected untreated control shows moderate to marked mixed inflammatory infiltrate, villous broadening with focal fusion (x100). (D) Blank ZnO NPs treated mice reveals moderate villous broadening with infiltration by moderate mixed inflammation (x100). (E) Albendazole treated subgroup displays intestine with mild inflammation and focal villous fusion (x100). (F) Mice treated with NTZ demonstrate focal villous broadening with mild mixed inflammation (x100). (G) Well-formed villi with minimal inflammation are noticed in mice treated with NTZ-loaded ZnO NPs (x100).

Fig 10. Muscular histopathologic photomicrographs (H&E).

(A) Skeletal muscle tissue of normal uninfected control representing normal architecture (x100). (B) Muscle of infected untreated control reveals encysted larva with surrounding thick capsule and mild inflammatory reaction (x200). (C) Blank ZnO NPs treated mice displays viable larva surrounded by thick muscular capsule with mild inflammatory reaction (x200). (D) Mice treated with albendazole show larva surrounded by a thin capsule with moderate mixed inflammation (x100). (E) and (F) NTZ treated subgroup demonstrates some degenerated larvae with surrounding disrupted capsules attacked by moderate to marked inflammation (x200 and x100 respectively). (G) and (H) NTZ-loaded ZnO NPs with the mostly degenerated larvae, evidently disrupted capsules that are strongly attacked by marked mixed inflammatory infiltrate (x200 and x100 respectively).