The safety and efficacy
of hemoglobin vesicles (HbVs) as artificial
oxygen carriers encapsulating a purified and concentrated Hb solution
in liposomes have been studied extensively. The HbV surface, modified
with PEG by incorporating a PEG-conjugated phospholipid, is beneficial
for storage and biocompatibility. However, it might be possible that
interaction of PEG and the pre-existing anti-PEG antibody in the bloodstream
causes acute adverse reaction. This study used two sets of experiments
with rats and guinea pigs to ascertain whether the anti-PEG antibody
generated by the PEG-modified HbV injection can induce anaphylactic
reactions. SD rats received repeated intravenous injection of HbV
at a dose rate of 16 or 32 mL/kg three times. Not anti-PEG IgG but
anti-PEG IgM was detected. Nevertheless, no anaphylactic reaction
occurred. Guinea pigs were used to study the presence of active systemic
anaphylaxis further after injections of the PEG-modified liposomes
used for HbV. The animals were sensitized by three repeated subcutaneous
injections of PEG-modified liposomes (PEG–liposome) along with
adjuvant at 1 week intervals. For comparison, unmodified liposomes
(liposome) and 10 times excessively PEG-modified liposomes with ionizable
lipid (10PEG–DODAP–liposome) were used. Inclusion of
PEG modification induced not only anti-PEG IgM but also anti-PEG IgG.
Three weeks after the final injection, intravenous injection of both
PEG–liposome and liposome (1 mL/kg) induced no anaphylactic
reaction. However, the injection of 10PEG–DODAP–liposome
showed one lethal anaphylaxis case and one mild anaphylaxis case.
Antisera obtained from the animal sensitized as described above were
inoculated (0.05 mL) intradermally into fresh guinea pigs. The presence
of passive cutaneous anaphylaxis was evaluated after intravenous injections
(1 mL/kg) of three liposomes with Evans blue. No dye leakage was detected
at any inoculated skin point for PEG–liposome or liposome,
but a slight leakage was detected in one inoculated skin point for
10PEG–DODAP–liposome. These results indicate the absence
of acute allergic reactions at repeated injections of HbVs despite
the anti-PEG antibody induction. Not all the PEG-modified liposomes
show anaphylaxis, and it may depend on the amount of PEGylated phospholipid
and lipid composition of PEG-modified liposomes.