Abstract:

Objective To investigate the inhibitory effect of artemether liposome (ARM-Lip) on Toxoplasma gondii tachyzoite proliferation in vitro. Methods Human cervical cancer cells (Hela cells) were cultured in 96-well flat bottom cell culture plate, with 100 μl cell culture medium in each well containing 1 × 10⁴ Hela cells. Setting ARM-Lip groups with different concentrations (3.125, 6.250, 12.500, 25.000, 50.000, 100.000, 200.000, 400.000 and 800.000 μmol/L), a control group (only containing 1 × 10⁴ human cervical cancer cells), and a blank group (only containing culture medium). A total of 100 μl corresponding drugs were added to each well in the ARM-Lip groups after 24 h, and 10 μl CCK-8 solution was added to each well after 48 h. A microplate reader was used to detect the absorbance at 450 nm wavelength after 1 h (A-value) to calculate the cell survival rate and 50% inhibitory concentration (IC₅₀) of ARM-Lip. Hela cells were infected with T. gondii green fluorescence-expressing RH (RH-GFP) straintachyzoites, and co-cultivated with different concentrations of ARM-Lip to screen safe concentrations of ARM-Lip those rendered higher than 50% cell survival rate. Through setting of 400 μmol/L sulfadiazine (SDZ) group, and untreated group, the proliferations of tachyzoites were observed under fluorescence microscope after cultured for 24, 48, and 72 h. The average fluorescence gray scale values of each group at 48 h were measured and the group having optimal concentration of ARM-Lip was selected. After Giemsa staining, the parasitophorous vacuoles in Hela cells and the growth of T. gondii tachyzoites were observed and counted. One-way ANOVA was used for inter-group comparison, and the least significant difference (LSD) test was used for pairwise comparison. Results ARM-Lip at a concentration of 25 μmol/L or lower had little toxic effect on Hela cells, and its IC₅₀ was 285.1 μmol/L. Treated with 100.000, 200.000, 400.000, and 800.000 μmol/L ARM-Lip, the cell survival rate was 89.03%, 76.30%, 42.12% and 27.85%, respectively. ARM-Lip concentrations of 6.250, 12.500, 25.000, 50.000, and 100.000 μmol/L were selected to conduct subsequent experiments. Under a fluorescence microscope, a large amount of T. gondii RH-GFP strain tachyzoites were observed in the untreated group, with normal morphology and high intensity of green fluorescence. As the concentration of ARM-Lip was increased, the intensity of green fluorescence was gradually decreased. The optimal concentration for ARM-Lip to inhibit T. gondii growth in vitro was 100.000 μmol/L and the optimal action time was 48 h. After cultured for 48 h, the average gray scale values of the untreated group, ARM-Lip group (100.000 μmol/L) and SDZ group were 89.92 ± 1.12, 35.74 ± 1.55 and 7.34 ± 0.60 (F = 7 916.301, P < 0.01). Compared with the untreated control group, the average grey scale values of the ARM-Lip group and SDZ group were significantly decreased (t = 81.247, 123.831, both P < 0.01); compared with the ARM-Lip group, the average gray scale value of the SDZ group was significantly decreased (t = -42.584, P < 0.01). After Giemsa staining, the numbers of parasitophorous vacuoles of untreated group, ARM-Lip group, and SDZ group were 28.667 ± 2.658, 16.667 ± 0.817 and 12.667 ± 1.211 (F = 135.652, P < 0.01); the numbers T. gondii tachyzoites in each group were 176.167 ± 13.273, 105.333 ± 7.789 and 70.167 ± 5.947 (F = 192.763, P < 0.01). Compared with the untreated control group, the numbers of parasitophorous vacuoles of ARM-Lip group and SDZ group were significantly decreased (t = 0.083, 0.063, both P < 0.01), and the numbers of T. gondii tachyzoites of ARM-Lip group and SDZ group were significantly decreased (t = 0.014, 0.009, both P < 0.01). The numbers of parasitophorous vacuoles and T. gondii tachyzoites in the SDZ group were significantly fewer than in the ARM-Lip group (t = -0.500, -0.057, both P < 0.01). Conclusion ARM-Lip have relatively good safety and can inhibit the proliferation of T. gondii tachyzoites in vitro, whereas its effectiveness remains insufficient when compared with SDZ.

Key words: Toxoplasma gondii, Tachyzoite, Artemether liposome, Hela cell, Parasitophorous vacuole