Biology Reference
In-Depth Information
antibiotics display higher reduction in CFU of endogenous
P. aeruginosa
in sputum
compared to the free antibiotic suggesting its potency in CF lung infections.
MATERIALS AND METHODS
F-actin and Other Chemicals
Human placental DNA, G-actin,
Escherichia coli
(O111:B4) lipopolysaccharide
(LPS), and
Staphylococcus aureus
LTA were purchased from Sigma Chemicals Co
(St. Louis, MO, USA). Monomeric G-actin was prepared from an acetone powder of
rabbit skeletal muscle in a non-polymerizing buffer (10 mM TRIS, pH 7.4, 0.2 mM
CaCl
2
, 0.2 mM ATP, 1 mM Dithiothreitol). The G-actin was then polymerized to F-
actin with the addition of 2 mM MgCl
2
and 150 mM KCl and gently shaken for 1 hr at
room temperature. Depending on the experiment, DNA, LPS, and LTA were dissolved
in double distilled H
2
O or in cation-adjusted MuellerHinton (CAMH) broth. Synthet-
ic DMPC, Chol, and DPPC were obtained from Northern Lipids Inc (Burnaby, BC,
Canada). Polymyxin B (Alexis Biochemicals, Burlington, NC, USA), and tobramycin
(Sandoz Laboratories, Boucherville, QC, Canada), were diluted in Phosphate Buffered
Saline solution (PBS: 160 mM NaCl, 10 mM KH
2
PO
4
, pH 7.4).
Organisms
Reference strains
P. aeruginosa
(ATCC 27853) were purchased from PML Microbio-
logicals (Mississauga, ON, Canada). Clinical isolate strains PA-48912-1, PA-48912-2,
and PA-48913 were kindly obtained from the Clinical Microbiology Laboratory of
Memorial Hospital (Sudbury, ON, Canada) and grown to form biofilm as described
elsewhere [56]. The strains were inoculated onto CAMH agar plates and incubated
for 18 hr at 37°C before any experiments. For any bactericidal experiment involving
ATCC 27853, single colonies were suspended to a concentration of 1×10
6
cfu/ml in
CAMH broth before addition to 96-well plates.
Preparation and Characterization of Liposomal Antibiotics
Liposome-entrapped tobramycin or polymyxin B was prepared from a lipid mixture
of either DMPC or DPPC and Chol (molar ratio of 2:1), respectively, by dehydration-
rehydration method as described previously with slight modifications [57, 58]. In
brief, lipids were dissolved in chloroform and removed under vacuum at 53°C using
a rotary evaporator (Buchi-Rotavapor R205, Brinkmann, Toronto, ON, Canada). 2 ml
of an aqueous solution of tobramycin or polymyxin B at a concentration of 10 mg/
ml were added to the thin dry lipid film and hand shaken in a warm water bath for 1
min. The lipid suspensions were sonicated in a round-bottom Erlenmeyer flask for
5 min (Sonic Dismembrator Model 500, Fischer Scientific, USA) while submerged
in an ice-bath. The sonicator was not in direct contact with the liposome suspension
at any time. The suspension was freeze-dried overnight for preservation and higher
entrapment (Labconco model 77540, USA). At the time of experiment, dehydrated
liposomes were rehydrated in PBS above the phase transition temperature of lipids
(DMPC T
c
= 23°C; DPPC T
c
= 41°C), for 2 hr and unentrapped drug was washed off
twice by ultracentrifugation at 62,000 g. This step ensures that the unentrapped drug
