Ticarcillin, stearylamine, dicetylphosphate, cholesterol and egg lecithin was purchased from Sigma Chemical Company (St. Louis, USA) and chloroform, methanol, ammonium acetate and Muller-Hinton broth was purchased from Merck (Darmstadt, Germany).
P. aeruginosa (ATCC 29248) was purchased from American Type Culture Collection (Rockville, MD, USA). For experimentation, this strain was inoculated onto blood agar plates and incubated for 24 h at 37°C.
Preparation of nanoliposomes
Nanoliposomes were prepared by extrusion method as previously described. Briefly, egg lecithin and cholesterol in the molar ratio of 4:1 were dissolved in chloroform and dried to a lipid film with a rotary evaporator (Brinkman, Toronto, Canada) under N2 flow and vacuum at 30°C. The dried lipids were dispersed by agitation in 6 ml of an aqueous solution of ticarcillin (10 mg/ml in PBS, pH 7.4) and sonicated at 4°C in ultrasonic bath (Braun-sonic 2000, Burlingame, USA). At finally, ticarcillin-loaded neutral nanoliposomes were obtained by extruding of respective suspension through a polycarbonate membrane with 100 nm-sized pores for 12 times and separating excess free drug and larger lipid aggregation by centrifugation (100000 g for 30 min). Cationic and anionic ticarcillin-loaded nanoliposomes were prepared and optimized by added stearylamine and dicetylphosphate in nanoliposomal membrane formulations in molar ratios of 0.5, 1, 2 and 3, respectively. Control nanoliposomes were prepared similarly, but PBS (pH 7.4) was used instead of the ticarcillin solution.
Determination of encapsulation efficacy
The content of ticarcillin in prepared nanoliposomes was determined by HPLC as described previously[17
]. Then, the percentage of drug loading was calculated as:
Particle size, zeta-potential and polydispersity index determination
Mean particle size, zeta-potential and polydispersity index of nanoliposomes was evaluated by the reported method using Malvern zetasizer (Malvern instrument, Worcestershire, UK) apparatus.
Antimicrobial susceptibility testing
The MICs of free and variable ticarcillin-loaded nanoliposomes for P. aeruginosa (ATCC 29248) were determined by the broth dilution technique as recommended by CLSI (formerly NCCLS). Bacterial cell suspensions of ~ 5×105 cells/ml were diluted in Muller-Hinton broth and dispensed (100 μl) into a microtiter tray containing serial two-fold dilutions of ticarcillin. The tray was then incubated for 24 h at 37°C. The MIC was recorded to be the lowest concentrations of ticarcillin in free and nanoliposomal forms that prevented visible bacterial growth and expressed in μg/ml.
Time kill studies were preformed in triplicate in 10 ml tubes containing 2 ml of Mueller-Hinton broth as described previously. In brief, 100 μl of P. aeruginosa suspension were resuspended in 10 ml of Mueller-Hinton broth and incubated overnight at 37°C, and adjusted to a McFarland standard of 0.5. Then, 100 μl of this standardized inoculum were added to separate culture tubes containing 1 ml of Mueller-Hinton broth with 1 ml ticarcillin solutions in free and different nanoliposomal forms at 1, 2 and 4 times the MIC and incubated at 37°C. At finally, colony counts were performed onto Trypticase soy agar (TSA) plates at 0, 2, 4, 6 and 24 h and the results were expressed as log colony forming unit (CFU)/ml.
In vivo study
In vivo therapeutic efficacies of ticarcillin-loaded nanoliposomes were tested by a described method, with some modification. In brief, sixty male BALB/c mice (20–22 g) obtained from the National Institute of Pasture, Iran. Animals were handled according to the national guidelines of the laboratory animal and housed in separate and pathogen- free cages and received food and water ad libitum. All groups were anesthetized with ketamine-xylazine mixture (50 mg/kg each, given intramuscularly), and their backs were shaved. To induce burn in the backs of mice, a brass bar (10 by 10 by 100 mm) was heated in boiling water for 15 min and applied on the shaved back of the animals for 45 s. Then, 50 μl of the bacterial inoculum (containing 109 CFU of total bacteria) was applied subcutaneously into the sites of the burn on the animal’s back. The mice were divided into 5 groups. All groups were treated topically as follows: Group 1 received cationic ticarcillin-loaded nanoliposomes (75 mg/kg/12h); group 2 received neutral ticarcillin-loaded nanoliposomes (75 mg/kg/12h); group 3 received anionic ticarcillin-loaded nanoliposomes (75 mg/kg/12h); group 4 received anionic ticarcillin-loaded nanoliposomes (75 mg/kg/12h); group 5 received empty nanoliposomes (75 mg/kg/12h), and group 6 received physiological saline (1 ml/kg/12h); for 7 days starting from the 3rd day post infection. Two days after the last dose the surviving animals were anesthetized and sacrificed by cervical dislocation and the liver, kidney, spleen and skin of mice were removed under sterile conditions and homogenized for 5–10 min in PBS (2 ml/g). The homogenates were serially diluted and plated for growth in TSA. The inoculated plates were then incubated at 37°C for 24 h and the colony forming unit (CFU) was counted.
The results were expressed as means standard errors of means. The data of killing rate study were statistically evaluated by paired Student’s t-test, and P value of less than 0.05 was considered significant. The survival rates of control and treated mice were determined by using chi-squared with Yates correction and by Fisher’s exact test. Therefore, these data were used to prepare of cationic and anionic ticarcillin-loaded nanoliposomes.