Biomedical Engineering Reference
In-Depth Information
(3)
Add 100 ng/mL recombinant mouse CD40/human Fc fusion protein. Use
of recombinant CD40 as the capture agent for lipid-anchored anti-CD40
ensured that only bioactive anti-CD40 still capable of binding its target
receptor would be quantified.
(4)
Add the solubilized anti-CD40+liposomes with PBS containing 0.5%
Tween 20 and incubate for 2 h.
(5)
Wash three times with PBS containing 0.5% Tween 20.
(6)
Add 200 ng/mL of HRP-conjugated goat anti-rat IgG and incubate for 1 h.
(7)
Wash three times with PBS containing 0.5% Tween 20.
(8)
Place 100 uL HRP-anti goat IgG and incubate for 30 min or until color
development is complete.
(9)
Stop the reaction with 3 N HCl.
(10)
Measure the absorbance at 492 nm.
(11)
Determine the anti-CD40 concentration against a standard.
(12)
The CpG and CpG-lipid were both tracked using a fluorescent FAM label on
the 3′ end. The fluorescence was measure at 480ex and 520em wavelengths.
7.4.4 Release of Anti-CD40 and CpG from Liposomes In vitro
115
To measure the
in vitro
release of anti-CD40 or CpG-lipid from combination
liposomes, dialysis cassettes (1 mL capacity) with a 300 kD MWCO membrane
were used (Float-a-lyzer G2, Spectrum Labs, Rancho Dominguez, CA). 400 µL
samples were dialyzed against 20 mL PBS containing 10% fetal calf serum,
with gentle agitation, at 37 °C. Samples and dialysis buffers were collected at
each indicated time point; anti-CD40 was measured by sandwich ELISA and
CpG was measured by its fluorescent label FAM. All samples were mixed with
0.5% Tween 20 to disrupt intact liposomes prior to the anti-CD40 ELISA.
The stability of anti-CD40 and CpG complexed with PEGylated liposomes
were measured in terms of the release of the immunostimulatory ligands
in
vitro
in the presence of serum. The combination liposomes carrying anti-CD40
and CpG were dialyzed (300 kD MWCO membrane in PBS pH 7.4 containing
10% fetal calf serum) at 37 °C. The release of ligands into the dialysis buffer
indicated that anti-CD40 and CpG both diffused freely into the buffer with sub-
stantial release in <5 h. In contrast, the PEG-DSPE-anchored anti-CD40 only
showed <10% released over 7 days. The CpG-lipid inserted into combination
liposomes was retained by the vesicles inefficiently allowing ~80% release in
24 h that was complete in ~7 days. The release materials were confirmed with
gel electrophoresis.
When used with live animals, the anti-CD40-liposomes did not directly exert
cytotoxic effects on B16 tumor cells during
in vitro
culture
115
with minimal non-
specific binding of immunoliposomes to the tumor cells. During
in vivo
studies,
locally administered soluble anti-CD40 strongly inhibited B16 tumor growth,
while the anti-CD40-liposomes slowed tumor growth by ~50% as effectively as
the soluble antibody. In terms of systemic effects, the locally administered soluble
