Safety and Therapeutic Efficacy of Thymoquinone-Loaded Liposomes against Drug-Sensitive and Drug-Resistant Acinetobacter baumannii

Abstract

In the present study, we investigated the activity of free thymoquinone (TQ) or liposomal thymoquinone (Lip-TQ) in comparison to standard antibiotic amoxicillin (AMX) against the drug-sensitive and drug-resistant Acinetobacter baumannii. A liposomal formulation of TQ was prepared and characterized and its toxicity was evaluated by analyzing the hematological, liver and kidney function parameters. TQ was effective against both drug-sensitive and drug-resistant A. baumannii as shown by the findings of drug susceptibility testing and time kill kinetics. Moreover, the therapeutic efficacy of TQ or Lip-TQ against A. baumannii was assessed by the survival rate and the bacterial load in the lung tissues of treated mice. The mice infected with drug-sensitive A. baumannii exhibited a 90% survival rate on day 30 post treatment with Lip-TQ at a dose of 10 mg/kg, whereas the mice treated with AMX (10 mg/kg) had a 100% survival rate. On the other hand, the mice infected with drug-resistant A. baumannii had a 70% survival rate in the group treated with Lip-TQ, whereas AMX was ineffective against drug-resistant A. baumannii and all the mice died within day 30 after the treatment. Moreover, Lip-TQ treatment effectively reduced the bacterial load in the lung tissues of the mice infected with the drug-sensitive and drug-resistant A. baumannii. Moreover, the blood of the mice treated with Lip-TQ had reduced levels of inflammation markers, leukocytes and neutrophils. The results of the present study suggest that Lip-TQ may prove to be an effective therapeutic formulation in the treatment of the drug-sensitive or drug-resistant A. baumannii infection as well.

Keywords: Acinetobacter baumannii; drug-resistant; inflammation; liposome; thymoquinone.

Figure 1

TQ exhibits multitargeting behavior in the treatment of various diseases.

Figure 2

Antibiotic susceptibility screening of (A) drug-sensitive and (B) drug-resistant A. baumannii.

Figure 3

The activity of TQ or AMX against the drug-sensitive and drug-resistant A. baumannii. The activity of TQ (25, 50 and 100 µg/mL) against the (A) drug-sensitive and (B) drug-resistant A. baumannii. The activity of AMX (25, 50 and 100 µg/mL) against the (C) drug-sensitive and (D) drug-resistant A. baumannii. Negative controls (NC) contain 5% DMSO.

Figure 4

Microscopic analysis of the effect of TQ or AMX against the drug-sensitive and drug-resistant A. baumannii. The activity of TQ (1, 2.5 and 5 µg/mL) against the (A) drug-sensitive and (B) drug-resistant A. baumannii. The activity of AMX (1, 2.5 and 5 µg/mL) against the (C) drug-sensitive and (D) drug-resistant A. baumannii. Negative controls (NC) contain 5% DMSO.

Figure 5

Time-kill curves of TQ or AMX at the doses of 1, 2, 4 and 8 µg/mL against the drug-sensitive and drug-resistant A. baumanni. Time-kill curves of TQ against the (A) drug-sensitive and (B) drug-resistant A. baumannii. Time-kill curves of AMX against the (C) drug-sensitive and (D) drug-resistant A. baumannii. The data are represented as the means ± SD of three independent experiments. Control (♦), TQ—1 µg/mL or AMX—1 µg/mL (●), TQ—2 µg/mL or AMX—2 µg/mL (○), TQ—4 µg/mL or AMX—4 µg/mL (■), TQ—8 µg/mL or AMX—8 µg/mL (☐).

Figure 6

Characterization of liposomes. (A) A transmission electron microscopy (TEM) image of TQ liposomes, (B) size of TQ liposomes.

Figure 7

Stability and release kinetics of Lip-TQ. (A) Stability of Lip-TQ in deionized water, (B) Release kinetics of TQ from the liposomes into the serum.

Figure 8

Lip-TQ or AMX effectively eliminated the drug-sensitive A. baumannii infection in a murine model. (A) Each mouse was infected with 1 × 10⁷ CFUs of A. baumannii through an intravenous dose. After 12 h of the infection, the mice were treated with 1, 5 and 10 mg/kg of free TQ and Lip-TQ for seven consecutive days. The mice were monitored for 30 days for the survival. Saline (○), sham-Lip (●), free TQ—1 mg/kg (☐), free TQ—5 mg/kg (△), free TQ—10 mg/kg (◊), Lip-TQ—1 mg/kg (■), Lip-TQ—5 mg/kg (▲), Lip-TQ—10 mg/kg (♦). Saline vs. free TQ—1 mg/kg (p = 0.0271), sham-Lip vs. Lip-TQ—1 mg/kg (p = 0.009), free TQ—5 mg/kg vs. Lip-TQ—5 mg/kg (p = 0.0195), free TQ—10 mg/kg vs. Lip-TQ—10 mg/kg (p = 0.0195). (B) The bacterial load (CFUs) was determined as described in the methodology section. The data are represented as the means ± SD of three independent experiments. * p < 0.05, *** p < 0.001. (C) The mice infected with drug-sensitive A. baumannii were treated with AMX (1, 5 and 10 mg/kg) for seven days and their survival rate was observed on day 30 after the infection. Saline (○), AMX—1 mg/kg (●), AMX—5 mg/kg (☐), AMX—10 mg/kg (■). Saline vs. AMX—5 mg/kg (p = 0.0023), saline vs. AMX—10 mg/kg (p < 0.001). (D) The bacterial load (CFUs) was determined as described in the methodology section. The data are represented as the means ± SD of three independent experiments. ** p < 0.01, *** p < 0.001.

Figure 9

Treatment with Lip-TQ, not with AMX, was effective against the drug-resistant A. baumannii infection in a murine model. (A) Each mouse was infected with 1 × 10⁷ CFUs of drug-resistant A. baumannii and treated with 1, 5 and 10 mg/kg of free TQ or Lip-TQ for seven days as described in the methodology section. The survival rate of the mice was monitored for 30 days. Saline (○), sham-Lip (●), free TQ—1 mg/kg (☐), free TQ—5 mg/kg (△), free TQ—10 mg/kg (◊), Lip-TQ—1 mg/kg (■), Lip-TQ—5 mg/kg (▲), Lip-TQ—10 mg/kg (♦). Saline vs. free TQ—1 mg/kg (p = 0.0051), sham-Lip vs. Lip-TQ—1 mg/kg (p = 0.0368), free TQ—5 mg/kg vs. Lip-TQ—5 mg/kg (p = 0.0272), free TQ—10 mg/kg vs. Lip-TQ—10 mg/kg (p = 0.0295). (B) Three mice from each group were sacrificed on day 5 after thetreatment and equally weighed portions of the lung tissue were homogenized to determine the bacterial load. The data are represented as the means ± SD of three independent experiments. *** p < 0.001. (C) The mice infected with drug-resistant A. baumannii were treated with AMX (1, 5 and 10 mg/kg) for seven days and their survival rate was observed on day 30 post-infection. Saline (○), AMX—1 mg/kg (●), AMX—5 mg/kg (☐), AMX—10 mg/kg (■). (D) The bacterial load (CFUs) was determined as described in the methodology section. The data are represented as the means ± SD of three independent experiments.

Figure 10

Treatment with Lip-TQ, but not with AMX, alleviated the parameters of total leukocytes and neutrophils in the mice infected with drug-resistant A. baumannii. On day 5 post-treatment, the blood was sampled from three mice and the leukocyte and neutrophil counts were analyzed. (A) Leukocytes in the free TQ- or Lip-TQ-treated mice, (B) leukocytes in the AMX-treated mice, (C) neutrophils in the free TQ- or Lip-TQ-treated mice, (D) neutrophils in the AMX-treated mice. The data are represented as the means ± SD of three independent experiments. ^≠≠≠ p < 0.001. Normal control vs. saline-treated mice, * p < 0.05, ** p < 0.001, *** p < 0.001. Saline vs. free TQ or Lip-TQ treatment groups.

Figure 11

Treatment with Lip-TQ, but not with AMX, alleviated the parameters of the CRP and PCT in the mice infected with drug-resistant A. baumannii. On day 5 post-treatment, the blood was sampled from three mice and the levels of the CRP and PCT were analyzed. (A) The CRP in the free TQ- or Lip-TQ-treated mice, (B) the CRP in the AMX-treated mice, (C) PCT in the free TQ- or Lip-TQ-treated mice, (D) PCT in the AMX-treated mice. The data are represented as the means ± SD of three independent experiments. ^≠≠≠ p < 0.001. Normal control vs. saline-treated mice, * p < 0.05, ** p < 0.001, *** p < 0.001. Saline vs. free TQ or Lip-TQ treatment groups.