Immunomodulatory and Physical Effects of Phospholipid Composition in Vaccine
Christopher B. Fox,1,4Susan L. Baldwin,1Malcolm S. Duthie,1,2Steven G. Reed,1,3and Thomas S. Vedvick1
Received 9 December 2011; accepted 28 February 2012; published online 14 March 2012
Abstract. Egg phosphatidylcholine is commonly used as an emulsifier in formulations administered paren-
terally. However, synthetic phosphatidylcholine (PC) emulsifiers are now widely available and may be
In earlier work,we demonstrated that a squalene–1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
emulsion provided equivalent physical stability compared to a squalene–egg PC emulsion. In the present
manuscript, we evaluate the physical stability of vaccine adjuvant emulsions containing a range of other
synthetic phosphatidylcholine emulsifiers. Besides the POPC emulsion, the 1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC) emulsion showed good particle size and visual stability compared to emulsions
emulsions employing various synthetic PC or egg PC emulsifiers in combination with an inactivated influenza
vaccineor a recombinant malaria antigen, and these responseswere generally enhanced compared toantigen
without adjuvant. Therefore, we show that (1) some synthetic PCs (DMPC, POPC, and to a lesser extent 1,2-
dioleoyl-sn-glycero-3-phosphocholine) are effective stabilizers of squalene emulsion over a range of storage
temperatures while others are not (1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine,and 1,2-dilauroyl-sn-glycero-3-phosphocholine) and (2) the immunogenicity of stable
squalene emulsions is similar regardless of PC source.
KEY WORDS: oil-in-water emulsion; phosphatidylcholine; squalene; vaccine adjuvant.
Metabolizable oil-in-water emulsions have been demon-
strated to be safe and effective vaccine adjuvants, nutritional
supplements, and drug delivery vehicles. Various emulsifier
compositions exist, with squalene being the preferred metabo-
lizable oil for vaccine adjuvant applications (1–3). Commonly
used emulsifiers include Pluronics®, Tweens®, Spans®, and
phospholipids. Emulsifier selection is based on emulsion stabi-
lizing capacity and/or biological activity, since emulsifiers are
membrane active by definition and have been shown to have
various biological effects related to immune stimulation (4).
Lecithin and its main component, phosphatidylcholine
(PC), have been successfully utilized as emulsifiers in safe
and effective parenterally delivered emulsions. Perhaps the
widest use of phospholipid emulsifiers is in the application of
intravenous nutritional supplementation (5), such as in Intra-
lipid®, a soybean oil/egg lecithin emulsion. However, there
may be some disadvantages associated with egg lecithin (or
egg PC) as an emulsifier, and advantages to substituting egg-
derived phospholipids with synthetic phospholipids. First,
there is considerable heterogeneity of structure: egg PC may
contain at least 17 different PC species (6), whereas various
synthetic PCs are available commercially at 99% purity. Sec-
ond, egg lecithin consists of multiple monounsaturated and
polyunsaturated acyl chains, which are prone to oxidative
degradation, whereas various synthetic PCs are composed of
saturated acyl chains and are therefore more chemically stable
(7). Third, the egg phospholipids are derived from an animal
source instead of being synthetically produced.
We have previously published the physical stability profile
of squalene emulsions stabilized by egg PC (8,9). Moreover, we
showed that synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-
phocholine (POPC; a main component of egg PC) provided
equivalent stability to a squalene emulsion compared to egg
PC. However,noothersyntheticPCs wereevaluated. Synthetic,
homogeneous phospholipids have become widely available and
are now relatively inexpensive. By appropriate selection of
synthetic phospholipid, the chemical stability of the emulsifier
and the physical stability of the emulsion could be optimized.
Biological equivalence must also be addressed; changes in
source or purity of emulsifiers in vaccine adjuvant emulsions
Electronic supplementary material The online version of this article
(doi:10.1208/s12249-012-9771-x) contains supplementary material,
which is available to authorized users.
1Infectious Disease Research Institute, 1124 Columbia St, Ste 400,
Seattle, Washington 98104, USA.
2Protein Advances Inc., 1102 Columbia St, Ste 110, Seattle, Washington
3Immune Design Corp., 1124 Columbia St, Ste 700, Seattle, Washington
4To whom correspondence should be addressed. (e-mail: cfox@idri.
AAPS PharmSciTech, Vol. 13, No. 2, June 2012 (#2012)
1530-9932/12/0200-0498/0#2012 American Association of Pharmaceutical Scientists
have previously been shown to dramatically affect vaccine po-
tency (10,11). In the present manuscript, we seek to (1) build on
our previous work by evaluating the physical stability of squa-
lene emulsions containing other synthetic PCs (besides POPC),
and (2) compare the biological activity of the synthetic PC
emulsions as well as an egg PC emulsion in the context of
malaria and influenza vaccine formulations.
MATERIALS AND METHODS
Adjuvant Formulations. Shark liver squalene (≥98%
purity) was purchased from Sigma-Aldrich (St. Louis,
MO, USA). Egg phosphatidylcholine (egg PC), POPC,
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dis-
tearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmi-
toyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-
sn-glycero-3-phosphocholine (DMPC), and 1,2-dilauroyl-
sn-glycero-3-phosphocholine (DLPC) were obtained from
Avanti Polar Lipids, Inc. at 99% purity (Alabaster, AL, USA).
All emulsion formulations were prepared by high-speed mixing
an oil phase (containing squalene and phospholipid) and an
aqueous phase followed by high-pressure homogenization, as
described previously (8). The final concentration of phospholip-
id was 25 mM. Formulations were monitored for stability over
6 months at 5°C, room temperature (RT), 37°C, and 60°C.
Particle size, zeta potential, viscosity, hemolysis, and light scat-
tering optical profiling measurements were performed as de-
scribed previously (8). Briefly, particle size was determined at
the indicated timepoints by diluting an aliquot of each emulsion
size average (Z-avg) by dynamic light scattering (Malvern Zeta-
sizer APS). Zeta potential was measured shortly after emulsion
manufacture by diluting an aliquot of each emulsion 1:20-fold in
water and measuring zeta potential by microelectrophoresis
(Malvern Zetasizer Nano-ZS). A 20-mL sample of each
emulsion was removed from storage at 5°C and allowed to
equilibrate to room temperature before measuring viscosity
by a rotational viscometer (Brookfield DV-E). Emulsion he-
molytic activity was assayed following a modified version of a
previously published technique (12); a suspension of red
emulsion for ∼20 min, following which the sample was centri-
mixture, and the absorbance was measured at 398 nm. Laser
scattering optical profiling measurements were collected every
10 min for 4 h on undiluted emulsion samples at 60°C using a
870 nm laser and a charge-coupled detector (LUMiReader).
Immunization, Serum Collection, and Immunological
Assays. Female, 6–7 weeks old, BALB/c mice (Charles River
Laboratories, Wilmington, MA, USA) were immunized by in-
tramuscular injection in each hind quadricep. Two distinct anti-
gens were tested: (1) 2007–2008 Fluzone inactivated split-virus
vaccine, incorporating the representative influenza strains
A/Solomon Islands/2/2006 (H1N1), A/Wisconsin/67/2005
(H3N2) and B/Malaysia/2506/2004; and (2) recombinant Plas-
modium berghei circumsporozoite protein (PbCSP) produced
in-house using the codon-harmonized construct developed by
Walter Reed Army Institute of Research. Influenza and malaria
vaccine formulations, immunization regimens, and serum col-
lection were as described earlier (8), except that the influenza
antigen dose was 0.02 μg total HA. All procedures were per-
formed under specific pathogen-free conditions and in accor-
dance with the regulations and guidelines of the Infectious
Disease Research Institute animal care and use committee.
Antibody Responses. Micewereimmunizedtwice,3weeks
apart. Sera were analyzed for antigen-specific IgG, IgG1, IgG2a
antibodies, and hemagglutination inhibition (HI) antibody ac-
tivity, as described previously, with arbitrary anti-PbCSP units
titers were determined for influenza antibodies (8). A bone
marrow ELISPOT assay was used to determine the induction
of vaccine-specific long-lived antibody-secreting plasma cells in
samples collected 4 weeks following the Fluzone boost immuni-
zation with and without adjuvant as previously described with
minor modifications (13).
Antigen-Specific Cytokine Responses. MultiScreen 96-well
rat anti-mouse IL-5 capture Ab (eBioscience, San Diego, CA,
Table I. Composition of Emulsifiers
Structures and transition temperatures are taken from the manufacturer, except in the case of egg PC where the phase transition temperature
was obtained from (14)
Egg PC structure is representative of only one species; multiple structures comprise egg PC
499Immunomodulatory and Physical Effects of Phospholipid Composition
USA) and incubated overnight at 4°C. Plates were washed with
PBS, blocked with RPMI 1,640 and 10% FBS for at least 1 h at
the second Fluzone injection. Single cell suspensions were pre-
pared and seeded at 2×105cells per well in duplicate with either
media alone, concanavilin A (0.75 μg/ml), 5 hemagglutinating
units (HAU) inactivated A/Solomon Islands/2/2006(H1N1) or 2
HAU inactivated A/Wisconsin/67/2005(H3N2) for 48 h at 37°C.
The plates were then washed with 0.1% PBS-Tween 20 and
incubated overnight with a biotin-conjugated rat anti-mouse IL-
5 secondary Ab (eBioscience). The filters were developed using
the VectaStain ABC avidin peroxidase conjugate and Vectastain
AEC substrate kits according to the manufacturer’s protocol
(Vector Laboratories). The reaction was stopped by washing
the plates with deionized water, plates dried in the dark, and
spots counted using an automated ELISPOT reader (C.T.L.
Serie3A Analyzer, Cellular Technology Ltd.). Data were
analyzed using ImmunoSpot® software (CTL Analyzer LLC).
Statistical Analysis. All mouse experiments analyzed five
individual animals per group per timepoint. ELISPOT counts
and log10-transformed antibody titers were compared using
ANOVA with Tukey’s multiple comparison test. HI titers
were compared by ANOVAwith Tukey’s multiple comparison
test using the log2-transformed HI titers.
Physical Stability of Emulsions. Table I describes the
composition of the emulsifiers employed in this study. Egg
PC is a heterogeneous phosphatidylcholine mixture with
Table II. Emulsion Physical and Hemocompatibility Characterization
Phospholipid Dynamic viscosity (cP)Zeta potential (mV)Hemolysis (%)
Compared to (9) for corresponding data on egg PC and POPC emulsions. Inherent instrumental error of the viscometer is±0.12 cP. The positive
control for 100% hemolysis was deionized water
Fig. 1. Emulsion particle size stability at various storage temperatures. Shown are results following storage at a 5°C, b RT, c
37°C, and d 60°C. Particle size was measured using Malvern Instruments APS, with error bars representing standard size
deviation of three separate aliquots from one batch of each emulsion
500 Fox et al.
various acyl chain lengths and degrees of saturation, although
a major component has been identified as POPC (6,15). In
contrast, the synthetic phospholipids shown in Table I are
highly pure (≥99%) and have well-defined main phase tran-
sition temperatures (Tm) as lipid assemblies. DOPC and
POPC, which both contain monounsaturated acyl chains, have
low phase transition temperatures (<0°C) due to the packing
disorder imposed by the unsaturated chains. Egg PC, which
contains monounsaturated and polyunsaturated acyl chains,
also has a low Tm. DLPC consists of saturated acyl chains but
they are only 12 carbons in length, which results in a low Tm.
DMPC, DPPC, and DSPC have longer saturated acyl chains
(14, 16, and 18 carbons, respectively) and their phase transi-
tion temperatures increase according to chain length. For
example, DPPC configuration at room temperature is the
highly ordered gel phase, whereas above 41°C, the lipid forms
a liquid crystalline phase which is characterized by more pack-
ing disorder due to temperature-induced changes in acyl chain
Table II displays the physical and hemocompatibility
properties of the emulsions shortly after their manufacture.
The viscosity values are close to that of water (∼1 cP), indic-
ative of the low oil content in these emulsions (10% v/v when
viscosity is measured, but diluted to 2% v/v for immuniza-
tion). Zeta potential values are negative for emulsions
employing phospholipids with low Tmvalues (e.g., DOPC,
DLPC), whereas emulsions with higher Tmphospholipids
are positive (DSPC, DPPC). None of the emulsions displays
notable hemolytic activity when incubated with a suspension
of RBCs, although the DLPC emulsion appears slightly more
hemolytic than the others.
We recently reported that particle size stability of a syn-
thetic POPC–squalene emulsion stored at 5°C or room tem-
perature was equivalent or improved compared to an egg PC–
squalene emulsion (9). We sought to build on this work by
manufacturing squalene emulsions with various other synthet-
ic phospholipids besides POPC; Fig. 1 shows the particle size
stability of the emulsions stored at different temperatures. The
initial size of the POPC emulsion was reported earlier (9);
here, we have monitored the long-term stability of this same
lot for comparison to the other synthetic PC emulsions. Obvi-
ous anomalies in physical appearance such as phase separa-
tion qualified emulsions as visually unstable. Taking into
account data from all temperatures, the DMPC and POPC
emulsions demonstrated greater particle size stability than the
other emulsions studied. Minimal particle size change was
evident at 5°C, and gradual particle size change was apparent
with increasing temperature (Fig. 1). Comparatively, the
DOPC emulsion droplet size change was minimal at 5°C, but
noticeable at the higher storage temperatures. DPPC emul-
sion stability was highly dependent on temperature. When
stored above the DPPC Tm(41°C), the DPPC emulsion
showed good stability compared to the other emulsions. How-
ever, at the three storage temperatures below the Tm, the
DPPC emulsion was either visually unstable or showed more
particle size change than more stable emulsions. Interestingly,
the DLPC emulsion, containing the shortest saturated chain
emulsifier, was visually unstable at early timepoints at all four
storage temperatures. The DSPC emulsion also became visu-
ally unstable before the 6-month timepoint at all storage tem-
peratures. Size polydispersity values were similar among the
Fig. 2. Laser scattering optical profiling analysis of emulsion stability.
Integral transmission profiles of the 25–30 mm region of cuvettes
containing emulsion, measured over 4 h at 60°C
Fig. 3. Effect of emulsions on antibody responses induced by immu-
nization with recombinant malaria antigen. BALB/c mice were immu-
nized twice with PbCSP antigen formulated with emulsions containing
egg PC or synthetic PC, including POPC, DOPC, and DMPC. Anti-
gen-specific IgG a, IgG1 b, and IgG2a c were determined by ELISA.
n=5 per group, and data are shown as values from individual mice
(Log10) with the bar representing the mean. *p value<0.05 versus
immunization with protein alone
501 Immunomodulatory and Physical Effects of Phospholipid Composition
more stable emulsions (Electronic Supplementary Material
(ESM) Fig. 1).
Laser scattering optical profiling provides complementary
information regarding emulsion stability. Changes in the emul-
sion due to creaming, coalescence, etc. are detected as changes
in light transmission through the vertical profile of the emulsion
(8). The laser scattering optical profiles of the emulsions were
measured every 10 min over a period of 4 h at 60°C (ESM
Fig. 2). In order to compare data from different emulsions, the
integral transmission between a specific region (25–30 mm) in
in integral transmission is indicative of coalescence or particle
sizegrowth since larger particles willscatter morelight, allowing
less to be transmitted to the detector. An increase in integral
transmission is generally representative of creaming or phase
separation. The DMPC emulsion shows little change compared
this temperature. These optical profile data confirm the particle
size and visual stability observations described above. DLPC,
DPPC, and DSPC emulsions are not shown since these emul-
sions were already classified as visually unstable at 5°C before
the optical profiling measurements had been conducted.
Taken together, the physical stability data indicate
that among the synthetic lipids DMPC, POPC, and to a
lesser extent DOPC are the most effective emulsifiers of
squalene oil. Particle size and sodium dodecyl sulfate
polyacrylamide gel electrophoresis analysis following mix-
ing the emulsions with an inactivated influenza vaccine
indicated good compatibility (data not shown). Therefore,
the above stable synthetic PC emulsions, along with an
egg PC emulsion, were subsequently evaluated for biolog-
ical activity in a mouse model.
Emulsions Selectively Enhance Antibody Responses to a
Recombinant Malaria Protein. Antibody responses against a
recombinant malaria antigen, PbCSP, were measured in
individual mouse sera 2 weeks after a second immuniza-
tion with protein alone or with protein combined with
emulsion formulations. The anti-PbCSP IgG antibody lev-
els were significantly higher in mice immunized with
PbCSP injected in the presence of emulsions than mice
receiving protein alone (Fig. 3a, p values<0.05). IgG1
antibodies were also higher in mice immunized with
PbCSP with egg PC, POPC, and DMPC emulsions
Fig. 4. Effect of adjuvant formulation in humoral immune responses induced by immunization with inactivated split-virus
influenza vaccine (Fluzone). BALB/c mice were immunized with Fluzone formulated with emulsions containing egg PC or
synthetic PC, including POPC, DOPC, and DMPC. Antigen-specific IgG a, IgG1 b, and IgG2a c were determined by ELISA.
IgG-secreting bone marrow plasma cells against Fluzone were determined by ELISPOT d. Results for a–c are shown as the
endpoint titer (Log10), with each dot representing individual mice and the bar representing the mean. Results for d also
represent individual mice in each group, with the bar representing the mean ASPC/group. *p value<0.05 versus immuniza-
tion with vaccine alone and#p value<0.05 versus immunization with the vaccine containing the DMPC emulsion
502Fox et al.
compared to mice immunized with protein alone (Fig. 3b,
p values<0.05). In contrast, differences between the vari-
ous groups were not detected in the anti-PbCSP IgG2a
antibody levels (Fig. 3c, p values>0.05). These data indi-
cate that antigen-specific IgG1 antibodies are selectively
enhanced by including emulsions during exposure to
Emulsions Enhance Antibody Responses to Influenza
Proteins. We have previously observed elevated antibody
responses in animals injected with Fluzone formulated with
emulsions incorporating egg-derived PC (8,13), but have not
investigated if these effects are observed when synthetic lipids
are incorporated. To determine if emulsions elevated the an-
tibody response to native influenza proteins, we immunized
mice with a low dose of Fluzone vaccine in the presence or
absence of emulsion formulation incorporating natural or syn-
thetic phospholipids. Compared to vaccination with the Flu-
zone vaccine alone, total IgG, IgG1, and IgG2a antibody titers
were higher for all groups that received Fluzone with emulsion
formulations (Fig. 4).
We also assessed the effect of emulsion formulations on
the generation of antigen-specific antibody secreting plasma
cells (ASPC) within the bone marrow, as these long-lived cells
secrete antibody for extended periods of time after antigenic
exposure and can provide a basis for long-term protection.
Overall, our results indicate that similar numbers of ASPC
were generated among the various emulsion formulation
groups. Only the vaccine containing DOPC emulsion, however,
induced a significantly higher number of long-lived plasma cells
than Fluzone vaccine alone (Fig. 4d).
The HI assay is a meaningful predictive indicator of
influenza vaccine efficacy, with a titer of ≥40 generally con-
sidered enough to provide protection (17). To determine if
emulsion formulations could generate higher quality antibod-
ies capable of enhancing protection afforded by the Fluzone
vaccine, we compared HI titers of mice vaccinated with Flu-
zone alone or Fluzone with various emulsion formulations.
When measured 4 weeks after the final immunization, the
addition of emulsion formulation induces higher HI titers than
the Fluzone vaccine alone against both the A/Solomon
Islands/3/2006 (H1N1) and the A/Wisconsin/67/2005 (H3N2)
vaccine components (Fig. 5). Against the A/Solomon Islands/
3/2006 (H1N1) component, all vaccines containing emulsion
induced higher HI titers than Fluzone alone. Against the A/
Wisconsin/67/2005 (H3N2) component, egg PC and DOPC
emulsions induced higher HI titers than Fluzone alone.
Additional experiments indicated that, with the exception
of the DMPC emulsion, the number of IL-5-producing antigen-
specific splenocytes from mice immunized with Fluzone plus
emulsions was enhanced compared to cells from mice immu-
nized with Fluzone alone (Fig. 6, p values<0.05). Together, the
antibody and cytokine secretion data indicatean enhanced Th2-
type immune response is elicited by the vaccines containing
Antibody responses elicited with both the malaria and influ-
enza vaccines were enhanced in an equivalent manner regardless
of synthetic or egg-derived PC as emulsifier components. This
finding is meaningful for continued development of PC-emulsi-
fied emulsions based on synthetic instead of natural components.
The literature has shown why the biological equivalence of this
type of component substitution should not be taken for granted;
thereareseveralinstanceswhen substitution ofone phospholipid
emulsifier with another has resulted in differences in biological
activity. For instance, Yasuda et al. found that liposomes com-
posed of synthetic phosphatidylcholines showed a direct correla-
tion between immunogenicity (as indicated by the number of
antibody-secreting cells in the spleen) and lipid phase transition
temperature when used to immunize mice intraperitoneally with
a synthetic lipid-based antigen; higher phase transition was found
to directly correlate with increased immunogenicity (18). This
correlation between phase transition temperature and immuno-
genicity was not apparent in the current work, although the
phospholipids with the highest phase transition temperatures
(DPPC and DSPC) were not evaluated immunologically due to
poor emulsion stability. In another example of the importance of
emulsifier source, recent clinical trial results with a water-in-oil
Fig. 5. HI titers were enhanced by immunization with Fluzone for-
mulated with egg PC or synthetic PCs. BALB/c mice were immunized
with Fluzone and various emulsions, then serum HI titers determined
4 weeks after boosting. a HI titers against the A/Solomon Islands/3/
2006 (H1N1) component of Fluzone. b HI titers against the A/Wis-
consin/67/2005 (H3N2) component of Fluzone. Data are shown as the
(Log2) titer for each individual animal, with the bar representing the
mean. *p value<0.05 versus immunization with vaccine alone and
#p value<0.05 versus immunization with the vaccine containing
POPC emulsion or DMPC emulsion
503 Immunomodulatory and Physical Effects of Phospholipid Composition
vaccine adjuvant emulsion, Montanide® ISA 51, revealed that
the replacement of animal-source emulsifier with plant-source
material may have been the cause for significantly reduced in
vivo efficacy (11). The immunological data in the present work
demonstrate that influenza and malaria vaccines adjuvanted with
emulsions made with different natural or synthetic phosphatidyl-
cholines elicit similar antibody responses. Moreover, the
qualitative immunogenicity of the emulsions employed in this
work are in line with literature reports; for instance, emulsions
(without TLR agonists) appear to induce only modest increases
ences were evident between emulsions in experiments with the
influenza vaccine, illustrating that the lipid emulsifiers may differ
slightly in biological effects. However, in general, it appears that
Fig. 6. IL-5 producing cells are increased after immunization with Fluzone formulated with
emulsions. Splenocytes from BALB/c mice immunized with Fluzone and various emulsions
were stimulated with either a 5 HAU of inactivated A/Solomon Islands/3/2006(H1N1) or b
2 HAU-inactivated A/Wisconsin/67/2005(H3N2) and IL-5 producing cells determined by
ELISPOT. Data is represented as the number of spot forming units (SFU) per million
splenocytes for individual mice/group, with the bar representing the mean, n=5 per group.
*p value<0.05 versus immunization with vaccine alone and
immunization with the vaccine containing the DMPC emulsion
#p value<0.05 versus
504Fox et al.
egg PC can be replaced by synthetic phospholipids without a
detrimental effect on biological activity in the context of a simple
recombinant antigen malaria vaccine or a more complex inacti-
vated split-virus influenza vaccine.
Besides biological activity, it is important to consider
physical emulsion stability as it relates to emulsifier acyl chain
structure. It has occasionally been reported that purified PC
molecules do not effectively stabilize emulsions (20). For ex-
ample, a DOPC/DPPC/1,2-dipalmitoyl-sn-glycero-3-phos-
phoethanolamine (DPPE) synthetic mix did not produce
emulsions as stable as that observed with egg lecithin when
used to emulsify perfluorocarbons (21). However, the claim
that pure synthetic PCs do not emulsify effectively is easily
refuted by other reports (22), which make clear that stability is
dependent on the structure and properties of both oil and
emulsifier (23). The spontaneous curvature or packing struc-
ture of the emulsifiers determines their effectiveness in stabi-
lizing emulsions (22). Emulsifier spontaneous curvature or
packing is affected by (a) the phospholipid phase behavior,
(b) the degree of unsaturation of the lipid acyl chains, and (c)
the phospholipid miscibility in the oil (22). In the present
work, the importance of lipid phase transition is apparent
since emulsions containing the phospholipids with the highest
phase transition temperatures (DPPC and DSPC) were not
stable at most storage temperatures based on size and visual
appearance. Interestingly, DPPC produced a stable emulsion
only when stored above its main phase transition temperature.
Nevertheless, phase transition temperature was not the only
determining factor for physical stability; note that the DMPC
emulsion showed good stability (at all storage temperatures,
including temperatures above and below the phase transition)
compared to the DLPC emulsion even though DMPC has a
higher phase transition temperature. Thus, the complexity of
the emulsifier–oil interactions is evidenced by the fact that the
stability of the emulsions described in the present work could
not be predicted solely based on phospholipid phase transition
temperature, acyl chain length, or saturation. Likewise, the
effect of lipid phase transition temperature and acyl chain
structure on emulsion stability was difficult to predict in the
context of glyceryl trioctanoate emulsions: DLPC, DMPC, and
DPPC emulsions were more stable than DSPC, 1,2-dilinoleoyl-
sn-glycero-3-phosphocholine, and DOPC emulsions (24).
Emulsion dropletsize growth can occur by two mechanisms:
Ostwald ripening (Laplace pressure differences cause oil mole-
cules to diffuse from smaller droplets to larger droplets), or
coalescence (separate oil droplets merge and form a single drop-
let; 25). Since squalene is essentially insoluble in water, it is
unlikely that Ostwald ripening is responsible for the size growth
the likely mechanism for the size growth reported here. The rate
of coalescence is expected to increase with higher temperatures
due to increased rates of diffusion and droplet interaction events,
and the size data presented here are consistent with this expec-
tation. Emulsifiers that do not provide an effective steric or
electrostatic stabilization to the oil droplet via a well-packed
interfacial layer may be responsible for increased rates of coales-
cence. Our findings indicate that, overall, the unsaturated lipid
emulsifiers were more effective than the saturated lipids in stabi-
lizing squalene emulsions, with the exception of DMPC which
produced a highly stable emulsion even though it contains only
saturated acyl chains. This emphasizes again that it is difficult to
acyl chain structure of the phospholipid emulsifiers.
Finally, we note that various physical characterization data
shown in this manuscript correlate well with previous literature
reports. For example, the relationship of more negative zeta
lipids has been reported previously; it is attributed to increased
disorder and packing defects in liquid phase membranes which
causes exposure of the polar head groups and allows for more
available binding sites for solution anions (30,31). Furthermore,
the slightly increased hemolytic tendency of the DLPC emulsion
corresponds with another report from the literature that DLPC
and other short-chain phospholipids induce hemolysis via crea-
tion of nonspecific pores in lipid bilayers through lipid phase
separation, allowing permeation of ions (32).
In the present work, it was shown that vaccine-adjuvant
emulsions containing squalene oil and synthetic phospholipid
emulsifiers, namely DMPC or POPC, demonstrated long-term
particle size stability at various temperatures. In general, egg
PC and synthetic PC emulsions induced similar immune
responses in combination with a simple recombinant malaria
antigen or an inactivated split-virus influenza vaccine. Thus,
substitution of egg phosphatidylcholine with synthetic phos-
phatidylcholine did not result in loss of vaccine adjuvant bio-
logical activity. Ongoing work in our laboratory will compare
stability and immunogenicity of squalene–phospholipid emul-
sions with other classes of nonphospholipid surfactant struc-
tures, namely polysorbate 80 and poloxamer 188.
The authors wish to thank Susan Lin, Sandra Sivananthan,
Tim Dutill, Kristen Forseth, Tony Phan, Farah Mompoint, Tara
Evers, Alison Bernard, and Marah Hay for skilled technical
assistance and Dr. Martin Friede for helpful discussions. This
work was supported in part by National Institutes of Health
contract HHSN272200800045C and grant 42387 from the Bill
and Melinda Gates Foundation. The authors gratefully ac-
knowledge Dr. Evelina Angov for kindly providing the codon-
harmonized PbCSP construct developed by Walter Reed Army
Institute of Research.
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