Enhanced activity of Ellagic acid in lipid nanoparticles (EA-liposomes) against Acinetobacter baumannii in immunosuppressed mice

Abstract

Acinetobacter baumannii infections have come to the surface in huge numbers in the recent decades. Furthermore, A. baumannii has adopted great ability to nullify the majority of currently available antibiotics. With the purpose of finding a nontoxic and efficient therapeutic agent, we analyzed the activity of Ellagic acid (EA) against the multidrug-resistant A. baumannii. EA not only demonstrated its activity against A. baumannii, but also inhibited the biofilm formation. Since EA shows poor solubility in an aqueous environment, a lipid nanoparticle-based (liposomal) formulation of EA (EA-liposomes) was prepared and its effectiveness was assessed to treat bacterial infection in the immunocompromised murine model. Therapy with EA-liposomes imparted greater protection to infected mice by increasing the survival and decreasing the bacterial load in the lungs. A. baumannii infected mice treated with EA-liposomes (100 mg/kg) showed 60% survival rate as compared to 20% of those treated with free EA at the same dose. The bacterial load was found to be 32778 ± 12232 in the lungs of EA-liposomes (100 mg/kg)-treated mice, which was significantly lower to 165667 ± 53048 in the lung tissues of free EA treated mice. Likewise, EA-liposomes also restored the liver function (AST and ALT) and kidney function parameters (BUN and creatinine). The broncho-alveolar fluid (BALF) from infected mice contained greater quantities of IL-6, IL-1β and TNF-α, which were significantly alleviated in EA-liposomes treated mice. These findings together support the possible implication of EA-liposomes to treat A. baumannii infection, especially in immunocompromised mice.

Keywords: Acinetobacter baumannii; Ellagic acid; Immune-suppression; Liposomes.

Fig. 1

In vitro antibacterial activity of Ellagic acid (EA) against A. baumannii by (A) Microscopic analysis (B) Agar well diffusion method, and (C) Time-kill assay.

Fig. 2

EA-liposomes significantly inhibited the formation of the biofilm. *** (p < 0.001), ** (p < 0.01).

Fig. 3

Treatment with EA-liposomes resulted in greater recovery of leukocytes on days (A) 5 and (B) 10 post-CYP injection. *** (p < 0.001), ** (p < 0.01).

Fig. 4

EA-liposomes showed superior anti-A.baumannii activity in immunocompromised mice. (A) Mice were challenged with bacteria and were observed for 30 days. PBS vs EA-50 (p = 0.009), PBS vs EA-100 (p = 0.0027), PBS vs EA-liposomes-50 (p = 0.0002), PBS vs EA-liposomes-100 (p = 0.0001), EA-50 vs EA-liposomes-50 (p = 0.0214), EA-100 vs EA-liposomes-100 (p = 0.0259). (B) The CFUs of A. baumannii were assessed as revealed in the methodology part. *** (p < 0.001).

Fig. 5

A. baumannii infected mice treated with EA-liposomes showed improved liver and kidney functioning parameters. (A) AST, (B) ALT, (C) BUN and (D) Creatinine. *** (p < 0.001), ** (p < 0.01), * (p < 0.05).

Fig. 6

Therapy with EA-liposomes significantly improved the status of inflammatory cytokines in the BALF of A. baumannii infected immunocompromised mice. (A) IL-6, (B) IL-1β and (C) TNF-α. *** (p < 0.001), ** (p < 0.01), * (p < 0.05).