Biology Reference
In-Depth Information
(in the supernatant) is separated from the liposomal pellet and is aspirated from the
formulation. The liposomal suspensions were diluted at room temperature and size
and polydispersity index was automatically determined with the use of a NICOMP
270/autodilute Submicron Particle Sizer according to manufacturer instructions (Santa
Barbara, CA, USA). The content of antibiotic entrapped in liposomes (after disruption
with 0.2% Triton X-100) was measured by an established method as described previ-
ously for tobramycin and polymyxin B [51, 58]. Encapsulation efficiency (EE) was
calculated as follows:
EE
(%)
=
(
concentration of antibiotic released
)/(
concentration of initial antibiotic
)
×
100%
Stability of Liposomes Loaded with Antibiotics
The stability of antibiotics in the formulations was examined according to Mugabe et al.
[59] at 37°C for 18 hr in the presence of PBS, CAMH broth, supernatant of biofilm
forming
P. aeruginosa
(PA-48912-1, PA-48912-2, and PA-48913), a combination of
DNA, F-actin, LPS, and LTA at a concentration of 1,000 mg/l, and intact or autoclaved
sputum. In experiments involving sputum, pooled CF sputum was either kept intact
and diluted 1:10 (w/v), or autoclaved for 10 min before mixing with CAMH broth.
After incubation, aliquots of the mixtures were removed and centrifuged. Antibiotic
presence in the pellet was assayed by the microbiological assay as described above,
and the amount of antibiotic released from the liposomes was expressed as a percent-
age of the total antibiotic concentration at 0 hr.
Bacterial Killing Assays in Presence of Polyanions
Antibacterial activity of the formulations was measured in the presence or absence of
polyanions found in the CF lung.
P. aeruginosa
(ATCC 27853) was grown on CAMH
agar overnight at 37°C. Single colonies were diluted and suspended in CAMH broth
alone or with 2-fold dilutions of LPS, LTA, DNA, and F-actin (125 to 1,000 mg/l);
2-fold dilutions of DNA and F-actin (125 to 1,000 mg/l); and 10-fold dilutions of LPS
and LTA (1 to 1,000 mg/l). Equal volumes of 100 μl were added to a 96-well plate to
a final concentration of 1×10
6
cfu/ml. To each well, 100 μl of the free or liposome-
entrapped antibiotic (0.125-256 mg/l; final concentration) was added and the plates
were incubated for 3 hr at 37°C. The incubation period and concentration chosen were
adequate to allow liposome or free antibiotic-bacteria interaction and eradication. Af-
ter incubation, the suspensions were kept cool on ice and bacterial suspensions were
diluted 10-10,000 folds in PBS. Wells treated in the absence of polyanions were plated
as is, that is without any dilutions. Aliquots (100 μl) of each dilution were plated on
CAMH agar and incubated overnight at 37°C. The cfu/ml values were then determined
for each of the three independent experiments.
To determine the ability of liposomes to retain their antibiotic activity, the MBC
of the antibiotic formulations were determined in an 18 hr period by a standard mi-
crobroth dilution assay in CAMH broth alone or with a mixture of LPS, LTA, DNA,
and F-actin at a fi xed concentration of 1,000 mg/l. The MBC assay was performed as
mentioned above with addition of 2-fold dilutions of the free or liposomal antibiotic
formulations added to the 96-well plates. The fi nal volume in each well was 200 μl,
