In vivo antimalarial efficacy test

ICR mice with parasitaemia percentage of 50–60% were used as donors. Blood samples were collected by the orbital plexus and put into heparinized tubes, then diluted it with physiological saline to gain an inoculum containing 5 × 10⁷ infected erythrocytes per millilitre. Each mouse was intraperitoneally injected with 0.2 mL of inoculum blood.

All formulations of liposomes, including ADLs, DHALs, artelinic acid liposomes (AALs), choline derivative liposomes (CDLs) and blank liposomes were prepared by thin-film hydration method, and AALs + CDLs isomolar mixture was an equimolar mixture of AALs and CDLs.

For the antimalarial activity studies, infected mice were randomly assigned to six groups. DHALs group and blank liposomes group were served as positive control and negative control, respectively. The treatment groups were administrated with the prepared formulations (ADLs, AALs, AALs + CDLs isomolar mixture or CDLs) through intravenous injection for 4 consecutive days.

For the intravenous injection, acute toxicity studies of ADLs, 20 male ICR mice (weight 18–22 g) were randomly divided into two groups (ten mice per group), which were treated with ADLs at the doses of 70.4 and 140.8 µmol kg⁻¹, respectively.

The suppression percentage of ADLs, DHALs, AALs, AALs + CDLs isomolar mixture and CDLs against PyBY265 were assessed by Peters 4-day suppression test (Sarkar et al., 2016). On the first day (D1), ICR mice were inoculated intraperitoneally with 0.2 mL inoculum. At 2 h after inoculation, the mice in treatment groups were intravenously treated with ADLs, DHALs, AALs, AALs + CDLs isomolar mixture or CDLs, respectively, at five different doses [4.4, 8.8, 17.6, 35.2, 70.4 µmol (kg·d)⁻¹, and six mice per dose for each treatment group] for 4 consecutive days (D1 to D4). The negative control group was treated with isometric blank liposomes intravenously for 4 days. At 24 h after the last treatment (D5), a thin smear was obtained from the tail vein blood of each mouse and dyed with Giemsa stain to measure parasitaemia percentage (by counting the number of Plasmodium-infected erythrocytes per 1000 erythrocytes in random fields). The mean suppression percentage was obtained using the average value of the parasitaemia percentage observed from an individual mouse. The 4-day suppression test was conducted twice.

The suppression percentage (D5), negative conversion fraction (D5) and recrudescence fraction (D6–D32) in each group were used as indices of antimalarial efficacy, which were all calculated as follows:

where A was the mean parasitaemia percentage in the various treatment groups on D5, B was the mean parasitaemia percentage in the negative control group on D5, C was the mice that had not been detected for parasitaemia on D5 in the group, D was the total number of alive mice in the group, E was the mice that had not been detected for parasitaemia on D5, but parasitaemia detected again within D6–D32.