Biomedical Engineering Reference
In-Depth Information
(1)
Liposomes with a composition of cholesterol/DOPC/maleimide-PEG-
DSPE/PEG-DSPE 35/50/5/10 by mol% is synthesized.
(2)
A 0.5 mol% of fluorescent rhodamine-labeled DOPE is also incorporated
for biodistribution experiments (histology and flow cytometry).
(3)
The lipids are vacuum-dried, rehydrated in PBS at 15 µmol/mL.
(4)
Sonicate the lipids for 4 min (alternating 5-7 Watts) using a Misonix probe-
tip sonicator (Qsonica, Newtown, CT) to form unilamellar liposomes.
(5)
Prepare the anti-CD40 with exposed free hinge region thiols by mixing
anti-CD40 (12-15 mg/mL) with a 25× molar excess of DTT for 20 min at
25 °C in the presence of 10 mm EDTA in PBS.
(6)
Pass the mildly reduced anti-CD40 through a desalting column to
remove DTT.
(7)
Immediately mix with maleimide-functionalized liposomes at a ratio of 1 mg
Ab:2.5 µmol liposomes for covalent coupling in the presence of 10 mm EDTA.
(8)
Allow the maleimide-thiol reaction to proceed for at least 10 h at 25 °C.
(9)
Centrifuge the resulting liposome aggregates and wash multiple times
with PBS to remove unbound antibody.
(10)
Syringe extrude the anti-CD40-liposomes 25× through 100 nm polycar-
bonate filter membranes (Avanti Polar Lipids).
Lipid-conjugated CpG can be prepared as previously described.
207
In this pro-
cess, the liposome phosphoramidite is added to the CpG during synthesis for
15 min, using the DNA synthesizer. After synthesis, CpG and CpG-PEG-lipid
conjugates are purified by reverse phase HPLC. The liposomes bearing anti-
CD40 + CpG are prepared by post-insertion approach to insert the purified
CpG-lipid conjugate onto the surface of anti-CD40-conjugated liposomes. This
process is as follows:
(1)
Mix ~3 nmol CpG-lipid with 1 umol anti-CD40-liposomes for 2 h at 25 °C.
(2)
Centrifuge the resulting combination liposomes and wash multiple times
with PBS.
(3)
Syringe extrude to remove unimer or micellar CpG-lipid through a 200 nm
polycarbonate filter membranes (Avanti).
(4)
Characterize the liposome size distributions by dynamic light scattering.
(5)
Store in PBS at 4 °C until use.
7.4.3.2 Quantification of Anti-CD40 and CpG on Liposomes115
115
An ELISA assay is used to quantify the amount of anti-CD40 or CpG on the
liposomes. To carry this out, the following protocol is followed.
(1)
Mix the anti-CD40-conjugated to liposomes with PBS containing 0.5%
Tween 20 surfactant to solubilize the lipids.
(2)
Coat standard 96-well plates with 2 µg/mL of goat anti-human IgG
antibody.
