Biology Reference
In-Depth Information
and PBS or CAMH alone were used as positive (no antibiotic) and negative (no bac-
teria) controls, respectively. Following incubation for 18 hr, aliquots (100 μl) were
aspirated from each well and subcultured on CAMH agar plates overnight. The MBC
was defi ned as the lowest concentration of the antibiotic that resulted in less than 30
cfu live bacteria/Petri dish.
Expectorated Sputum
The sputum samples were collected by spontaneous expectoration from nine CF pa-
tients following informed consent and a protocol approved by the Research Ethics
Committee (Sudbury Regional Hospital, Sudbury, Ontario, Canada). Patients' age,
name, treatments, and exacerbation records were kept confidential. Sputum samples
colonized with moderate to heavy growth of
P. aeruginosa
were pooled and frozen at
−80°C in aliquots. To measure the effects of the formulations on endogenous
P.
aeruginosa
, aliquots of sputum samples were also stored at 4°C and used within 24
hr of collection. At the time of the experiment, the sputum samples were diluted 1:10
(w/v) in CAMH broth and mixed with the antibiotic formulations to achieve concen-
trations ranged 1 to 512 mg/l. The mixtures were then incubated for 18 hr at 37°C and
the cfu/ml of live bacteria was determined according to the aforementioned protocol.
The dilution of the samples should have affected the viscoelastic properties of the
sputum, and this dilution was only done for easier handling and measurement of the
sputum samples.
Data Analysis
All results were expressed as mean ± SEM obtained from three trials. Comparisons be-
tween free and liposomal formulations were made by ANOVA one-way post t-test, and
P-values were considered significant when (*) p<0.05, (**) p<0.01, (***) p<0.001.
DISCUSSION
Polycationic antibacterial agents, like aminoglycosides and polymyxins, require self-
promoted uptake pathways for entry and eradication of gram-negative bacteria [60].
The cationic antibiotics increase bacterial outer membrane permeability by displacing
magnesium ions and binding to LPS [41, 60]. In the highly ionic CF sputum, however,
the high affinity of excreted polyanionic bacterial endotoxins and glycoproteins from
lysed white blood cells towards cationic antibiotics decreases their overall interaction
with the bacteria in the lungs [46, 62]. Liposomes may create a protective environment
for antibacterial agents to minimize such interactions and subsequently maintain a
steady drug concentration in the lungs. Our data on the stability of the liposomal for-
mulations displays that tobramycin leakage was at equilibrium after 3 hr, while poly-
myxin B leakage continued up to half its concentration over 18 hr. This suggests that
these nanoparticles are effective in protecting the antibiotics in the CF sputum
in vitro
.
The stability will ensure a continuous presence of the antibiotic at the site of infection,
and improves antibiotic bioavailability and biodistribution
in vivo
[63].
Polyanions like DNA and F-actin have strong affi nity for their multivalent coun-
terions and tend to aggregate (form bundles) in the presence of cationic antibiotics
