Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines

Bilayer fluidity
The bilayer fluidity, dependent on the lipid gel-liquid 
crystal transition temperature and its effects on immune 
responses, is of important interest when designing effica-
cious vaccines. Many published works have evaluated and 
described this phenomena, including the earliest work 
by Yasuda et al. [
49
]. The effects on immune responses 
of liposomes prepared with phospholipids of PC with 
different transition temperatures containing the hapten 
Dnp-Cap-PE were measured. DMPC, DPPC (1,2-dipal-
mitoyl-sn-glycero-3-phosphocholine) and DSPC (all with 
high transition temperatures T
m
> 20 °C) were favorable 
in eliciting antibodies to the hapten, contrasting to results 
obtained with DOPC (1,2-dioleoyl-sn-glycero-3-phos-
phocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phos-
phocholine) and EPC (T
m
< 0 °C). Another research 
team followed a similar experimental design preparing 
liposomes with low- (DOPC and DLPC, − 20–0 °C), 
intermediate- (DPPC and sphingomyelin, 25–40 °C) and 
high-transition temperature (DSPC, > 50 °C) lipids [
50
]. 
DMPC, DPPC and sphingomyelin induced immune 
responses as per a plaque-forming assay, but DSPC was 
a poor immunogen. Cholesterol was added to the liposo-
mal formulations, inducing significant humoral immune 
responses. The results from these two groups produced 
different outcomes: Yasuda et al. [
49
] presenting that less 
fluid lipids are better immunogens than fluid phospholip-
ids and van Houte et al. [
50
] establishing that intermedi-
ately fluid phospholipids (which have a phase transition 

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De Serrano and Burkhart  J Nanobiotechnol (2017) 15:83 
temperature of 25–40 °C) are better immunostimulatory 
agents. This discrepancy might be attributed to other fac-
tors, like particle size and Zeta potential, but such factors 
were not reported or analyzed to determine their role in 
these studies.
Additionally, Mazumdar et al. revealed that liposome 
composition may have an effect on immune responses 
[
51
]. Here, researches incorporated a leishmanial antigen 
(LAg) in liposomes containing DMPC, DPPC or DSPC 
and described the immunization process and results in 
a hamster model. No significant delayed hypersensitiv-
ity was detected in DMPC- or DPPC-containing lipids, 
which contrasted with DSPC-containing lipids. Moreo-
ver, DSPC-containing lipids protected up to 95% of the 
hamsters against a leishmanial infection. Recently, Kaur 
et al. presented results on how cholesterol influences 
the bilayer fluidity [
52
]. For instance, a direct correla-
tion of cholesterol and membrane fluidity was observed 
in DDA:TDB (trehalose dibehenate) liposomal formu-
lations. However, less IgG was detected as cholesterol 
increased in the system after 12 days of immunization 
in mice. This effect might be due to the loss of antigen 
in more fluid (high cholesterol) liposomes as the authors 
pointed out. The cytokine IFN-γ was at elevated lev-
els when cholesterol was not present in the lipid bilayer. 
Even with the compelling evidence of how transition tem-
perature and lipid bilayer composition affects immune 
responses, we can find conflicting results in published 
data. For example, Hampl et al. found no significant influ-
ence of immune response induction based on liposomes 
containing phospholipids with different transition tem-
peratures [
53
]. Although the data presented does not 
follow a clear pattern, the general conclusion is that the 
more fluid a liposome is the less immune response it will 
generate when administered in different animal models. 
Likely behind this phenomenon is that fluidity tends to 
increase the release of the antigen from the liposomes, 
affecting antigen presentation and consequently the 
strength of immune responses. We need to point out 
that cholesterol also participates as a bilayer stabilizer, 
increasing rigidity in biological membranes [
54
]. There-
fore, we might consider that the increase in bilayer fluid-
ity by cholesterol observed in the studies discussed above 
may occur in synthetic bilayers only. Further research 
must account for such correlation due to the complexities 
of biological membranes.