![Average particle size (z-average) of DDA/TDX liposomes stored at 4 °C (A) and 25 °C (B) as an indication of the colloidal stability upon storage [DDA/TDB (black dots), DDA/TDA (gray dots), DDA/TDS (black triangles), DDA/TDP (gray triangles), DDA/TDMyr (black squares) and DDA/TDL (gray squares)]. Results denote mean values + SD (n = 3).](https://www.researchgate.net/profile/Camilla-Foged/publication/268157235/figure/fig2/AS:392212803276804@1470522227599/Average-particle-size-z-average-of-DDA-TDX-liposomes-stored-at-4-C-A-and-25-C-B_Q320.jpg)
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Research paper
Influence of trehalose 6,60-diester (TDX) chain length on the
physicochemical and immunopotentiating properties of DDA/TDX
liposomes
Rie Selchau Kallerup
a,b,c,
⇑
, Cecilie Maria Madsen
a,c
, Mikkel Lohmann Schiøth
b
, Henrik Franzyk
b
,
Fabrice Rose
a
, Dennis Christensen
c
, Karen Smith Korsholm
c,1
, Camilla Foged
a,
⇑
,1
a
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
b
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
c
Department of Infectious Disease Immunology, Vaccine Adjuvant Research, Statens Serum Institut, Copenhagen, Denmark
article info
Article history:
Received 28 June 2014
Accepted in revised form 29 October 2014
Available online xxxx
Keywords:
Adjuvant
Liposome
Vaccine delivery
Dimethyldioctadecylammonium
Trehalose-6,6
0
-dibehenate
Nanomedicine
Protein antigen
abstract
Linking physicochemical characterization to functional properties is crucial for defining critical quality
attributes during development of subunit vaccines toward optimal safety and efficacy profiles. We
investigated how the trehalose 6,6
0
-diester (TDX) chain length influenced the physicochemical and
immunopotentiating properties of the clinically tested liposomal adjuvant composed of dimethyldioctad-
ecylammonium (DDA) bromide and analogues of trehalose-6,6
0
-dibehenate (TDB). TDB analogues with
symmetrically shortened acyl chains [denoted X: arachidate (A), stearate (S), palmitate (P), myristate
(Myr) and laurate (L)] were incorporated into DDA liposomes and characterized with respect to size,
polydispersity index, charge, thermotropic phase behavior and lipid–lipid interactions. Incorporation of
11 mol% TDX into DDA liposomes significantly decreased the polydispersity index when TDA, TDS, TDP
and TDMyr were incorporated, whereas both the initial size and the charge of the liposomes were
unaffected. The long-term colloidal stability was only decreased when including TDL in DDA liposomes.
The fatty acid length of TDX affected the phase transition of the liposomes, and for the DDA/TDP and
DDA/TDS liposomes a homogeneous distribution of the lipids in the bilayer was indicated. The membrane
packing was studied further by using the Langmuir monolayer technique. Incorporation of TDS improved
the packing of the lipid monolayer, as compared to the other analogues, suggesting the most favorable
stability. Finally, immunization of mice with the recombinant tuberculosis fusion antigen Ag85B-ESAT-
6-Rv2660c (H56) and the physicochemically most optimal formulations (DDA/TDB, DDA/TDS and DDA/
TDP) induced comparable T-cell responses. In conclusion, of the investigated TDB analogues, incorporation
of 11 mol% TDS or TDP into DDA liposomes resulted in an adjuvant system with the most favorable
physicochemical properties and an immunological profile comparable to that of DDA/TDB.
Ó2014 Elsevier B.V. All rights reserved.
1. Introduction
Subunit vaccines have in recent years become attractive vaccine
candidates due to superior safety profiles; subunit vaccines are
based on the antigen(s) from pathogenic microorganisms that best
stimulate the immune system, often highly purified recombinant
antigens, which reduces the risk of adverse reactions. However, in
comparison to traditional vaccines based on live attenuated and
whole inactivated pathogens, recombinant antigens are commonly
associated with reduced immunogenicity due to their lack of intrin-
sic immunostimulatory capacity. Hence adjuvants are required in
the vaccine formulation to potentiate the immune response against
co-administered antigen(s) thereby enhancing vaccine efficacy [1,2].
http://dx.doi.org/10.1016/j.ejpb.2014.10.015
0939-6411/Ó2014 Elsevier B.V. All rights reserved.
Abbreviations: APC, antigen-presenting cell; DDA, dimethyldioctadecylammo-
nium; DLS, dynamic light scattering; DSC, differential scanning calorimetry; IFN-
c
,
interferon
c
; IL-5, interleukin 5; IL-17a, interleukin 17a; PAMP, pathogen-associated
molecular pattern; PDI, polydispersity index; PRR, pattern-recognition receptor;
TEM, transmission electron microscopy; TDA, trehalose 6,6
0
-diarachidate; TDB,
trehalose 6,6
0
-dibehenate; TDL, trehalose 6,6
0
-dilaurate; TDMyr, trehalose 6,
6
0
-dimyristate; TDP, trehalose 6,6
0
-dipalmitate; TDS, trehalose 6,6
0
-distearate.
⇑
Corresponding authors. Department of Pharmacy, Faculty of Health and Medical
Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø,
Denmark. Tel./fax: +45 35 33 64 02.
E-mail addresses: rie.kallerup@sund.ku.dk (R.S. Kallerup), camilla.foged@sund.
ku.dk (C. Foged).
1
These authors contributed equally.
European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
Substantial progress has been made in discovery of new effi-
cient adjuvants for subunit vaccines, but only a few of these are
utilized in licensed vaccine products. Adjuvants constitute a large
group of structurally heterogeneous compounds, but can generally
be classified as delivery systems or immunopotentiating com-
pounds, with some compounds possessing both properties [2,3].
Most immunopotentiators are ligands for pattern-recognition
receptors (PRRs) on antigen-presenting cells (APCs) that recognize
so-called pathogen-associated molecular patterns (PAMPs)
expressed by pathogens [4]. The combination of delivery systems
and immunopotentiators appears to represent extraordinarily
effective adjuvants due to concurrent enhanced antigen delivery
and potent stimulation of innate immunity. Especially adjuvants
of a particulate nature are attractive due to the particulate nature
of pathogens and the passive targeting of particulates to the APCs
[5]. Subunit vaccine formulations are therefore highly complex
formulations consisting of one or more antigens, often covalently
linked into larger fusion proteins, in combination with adjuvants.
This poses a demand for extensive physicochemical and functional
characterization of such vaccine formulations in order to define
and understand critical quality attributes important for optimal
safety and efficacy [6,7].
Cationic liposomes have been shown to interact efficiently with
APCs [8], and in vivo studies have demonstrated an improved and
prolonged adjuvant effect of cationic liposomes as compared to
both neutral and negatively charged liposomal formulations [9],
which renders cationic liposomes promising adjuvants. The cat-
ionic liposomal adjuvant CAF01 (Statens Serum Institut, Denmark)
based on the surfactant dimethyldioctadecylammonium (DDA)
bromide and the glycolipid trehalose-6,6
0
-dibehenate (TDB) is
particularly interesting as it, in addition to inducing an antibody-
mediated immune response, also stimulates a strong CD4
+
T-cell
response characterized by a mixed Th1/Th17 profile. This is in con-
trast to most adjuvants included in licensed vaccines such as the
aluminum-based adjuvants and the MF59 emulsion, which only
stimulate antibody-mediated responses [10–14]. Induction of
cell-mediated immunity is a prerequisite for preventing diseases
such as tuberculosis, malaria and AIDS. CAF01 has been tested in
phase 1 clinical trials in combination with the recombinant tuber-
culosis fusion antigen Ag85B-ESAT6 (NCT ID: NCT00922363) and
an HIV-1 peptide mix (NCT ID: NCT01141205; NCT01009762),
and has been shown to be safe and well-tolerated [15].
The immunopotentiating compound TDB is a simplified synthetic
analogue of trehalose-6,6
0
-dimycolate (TDM), also known as the cord
factor, which is an immunostimulatory glycolipid present in the
outer membrane of the mycobacterial cell wall [16]. The enhanced
adjuvant activity of CAF01, as compared to DDA liposomes, can be
assigned to the presence of TDB, which has been shown tobe a ligand
for the C-type lectin receptor Mincle [macrophage-inducible Ca
2+
-
dependent (C-type) lectin; CLEC4E]. The Mincle receptor is a simple
type II transmembrane PRR expressed on the surface of macrophages
that functions in the capture and internalization of mycobacteria
[17]. The extracellular C-type carbohydrate recognition domain is
responsible for ligand binding and is attached to the cell surface by
a short stalk and a transmembrane anchor. Mincle forms a heteroo-
ligomer with the
c
-subunit of the Fc receptor and signals through the
FcR
c
-Syk-CARD9 signaling pathway [13,18]. The crystal structure
and binding mechanism of Mincle have recently been reported,
suggesting the simultaneous recognition of the sugar headgroup
and the lipid acyl chains [19–21]. Furthermore, binding studies have
shown that the sugar headgroup interaction is Ca
2+
dependent, and
that the receptor possesses a hydrophobic groove adjacent to the
two putative sugar binding sites that can accommodate a fatty acid
tail of glycolipids.
For immunopotentiators such as TDB incorporated into particu-
late delivery systems, it is not only the bulk molecular properties
that affect the adjuvanticity of the formulation; the colligative
and physicochemical properties of the adjuvant formulation may
also have a tremendous impact [22–24]. We therefore hypothe-
sized that the chain length of TDB analogues is important for the
adjuvant activity upon incorporation into DDA liposomes. The aims
of the present study were therefore (i) to synthesize a library of
TDB analogues (generally denoted TDX) with symmetrically short-
ened acyl chains, (ii) to incorporate them into DDA liposomes, (iii)
to evaluate how the acyl chain length affected the physicochemical
properties of the liposomes, and (iv) to evaluate the adjuvant
activity of the DDA/TDX liposomes.
2. Materials and methods
2.1. Materials
DDA was obtained from Avanti Polar Lipids (Alabaster, AL, USA).
TDB was synthesized by Clausen-Kaas A/S (Farum, Denmark). All
other chemicals and reagents were obtained commercially at
analytical grade.
2.2. Synthesis of TDX glycolipids
TDX was synthesized and purified by previously reported
pathways as described below and in Fig. 1. The final identity and
purity of the resulting compounds were confirmed by NMR (see
supporting information).
2.2.1. Synthesis of 2,3,4,2
0
,3
0
,4
0
-hexa-O-(trimethylsilyl)-
a
,
a
-trehalose
The procedure reported by [25] was followed, which enables
the silylation of carbohydrates and sterically hindered alcohols
under very mild conditions. Briefly, trehalose was treated with
N,O-bis(trimethylsilyl)acetamide and a catalytic amount of tetra-
butylammonium fluoride (TBAF) in an aprotic solvent i.e. N,N-
dimethylformamide (DMF), and subsequently the intermediate
fully silylated trehalose was selectively deprotected to afford the
desired hexasilyl ether in good yield. The structure was confirmed
by NMR (see supporting information for details).
2.2.2. Synthesis of TDB 6,6
0
-diester analogues (TDX)
Trehalose 6,6
0
-diesters were synthesized according to [26] by
coupling fatty acids to the selectively protected
a
,
a
0
-trehalose,
i.e. 2,3,4,2
0
,3
0
,4
0
-hexa-O-(trimethylsilyl)-
a
,
a
-trehalose, in the pres-
ence of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyr-
idine (DMAP). The deprotection procedure of the resulting
trimethylsilyl ether-protected trehalose 6,6
0
-diesters was per-
formed by a protocol similar to a recently reported protocol [27].
Five 6,6
0
-diesters with symmetrically shortened acyl chains
were obtained in good yield (40–60%). The diesters synthesized
were trehalose 6,6
0
-diarachidate (TDA), trehalose 6,6
0
-distearate
(TDS), trehalose 6,6
0
-dipalmitate (TDP), trehalose 6,6
0
-dimyristate
(TDMyr) and trehalose 6,6
0
-dilaurate (TDL) with acyl chains of
20, 18, 16, 14 and 12 carbon atoms, respectively. The complete
diester analogue series is denoted TDX. The structures of the com-
pounds, their purity and their chemical stability (after 1.5 years)
were confirmed by NMR (see supporting information for details).
2.3. Preparation of liposomes
The DDA/TDX (89:11) liposomes were prepared by using the
thin film method essentially as described previously [10], com-
bined with a probe sonication step to ensure a narrow size distri-
bution. Briefly, weighed amounts of DDA and TDX were dissolved
in CHCl
3
–MeOH (9:1, v/v) in a round-bottomed flask. The organic
solvent was evaporated under vacuum resulting in a thin lipid film.
2R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
The film was stripped twice with EtOH and dried overnight to
remove trace amounts of the organic solvents. The lipid film was
rehydrated by adding Tris buffer (10 mM, pH 7.4), and sonicated
for 5 min using a Sonifier
Ò
cell disruptor (Branson, Danbury, CT,
USA), followed by heating to 60 °C for 1 h with 2 min of vigorous
vortex mixing every tenth min. After 20 min of rehydration, the
liposomes were tip-sonicated for 20 s with a 150 W Branson
tip-sonicator (50% of the duty cycle) to reduce the size of the
liposomes. The final concentrations of DDA and TDB/TDA/TDS/
TDP/TDMyr/TDL were 2.5 mg/mL and 0.50/0.47/0.44/0.41/0.39/
0.36 mg/mL, respectively, corresponding to a DDA:TDX molar ratio
of 89:11. There was no loss of lipid during the preparation proce-
dure (confirmed by HPLC, results not shown). The liposomes were
stored at 4 °C until further use.
2.4. Size, polydispersity and zeta-potential
The average particle size distribution and polydispersity index
(PDI) were determined by dynamic light scattering using the photon
correlation spectroscopy technique. The surface charge of the parti-
cles was estimated by analysis of the zeta-potential (laser-Doppler
electrophoresis). For determination of the size distribution and
PDI, the samples were diluted 10-fold in 10 mM Tris buffer (n= 3).
For determination of the zeta-potential, the samples were diluted
300 times in 10 mM Tris buffer (n= 3). The measurements were
done in triplicate at 25 °C using a Zetasizer Nano ZS (Malvern Instru-
ments, Worcestershire, UK) equipped with a 633 nm laser and 173°
detection optics. For viscosity and refractive index, the values of
pure water were used. Malvern DTS v.6.20 software was used for
data acquisition and analysis. A Nanosphere™ Size Standard
(220 ± 6 nm, Duke Scientific Corporation, Palo Alto, CA, USA) and a
zeta-potential transfer standard (50 ± 5 mV, Malvern Instru-
ments) were used to verify the performance of the instrument.
2.5. Cryo-TEM
Morphological analysis was carried out by cryo-transmission
electron microscopy (cryo-TEM) using a Philips CM120 BioTWIN
transmission electron microscope (Philips, Eindhoven, The Nether-
lands). Samples for cryo-TEM were prepared under controlled tem-
perature and humidity conditions within an environmental
vitrification system. A small droplet (5
l
L) was deposited onto a
Pelco Lacey carbon-filmed grid and spread carefully. Excess liquid
was removed resulting in the formation of a thin (10–500 nm)
sample film. The samples were immediately plunged into liquid
ethane and kept at 180 °C. The vitrified samples were subse-
quently transferred from liquid nitrogen to an Oxford CT3500 cryo
holder connected to the electron microscope. The sample temper-
ature was continuously kept below 180 °C. All observations were
made in the bright field mode at an acceleration voltage of 120 kV.
Digital images were recorded with a Gatan Imaging Filter 100 CCD
camera (Gatan, Pleasanton, CA, USA).
2.6. Liposome colloidal stability
The colloidal stability of the liposomal formulations was stud-
ied by storing the undiluted liposome dispersions (n= 3) for
4 months in 10 mM Tris buffer at 4 °Cor25°C, respectively. The
particle size distribution was measured at regular intervals as
described above.
2.7. Differential scanning calorimetry
The gel-to-liquid crystalline phase transition temperature (T
m
)
and the width of the phase transition peak at half height (T
½
)of
the undiluted liposomal dispersions were determined by using dif-
ferential scanning calorimetry (DSC). All samples and buffers were
degassed prior to the measurements. The thermograms were
recorded with a Nano DSC (TA instruments, New Castle, DE, USA)
at a scanning rate of 0.5 °C/min in the temperature range of
20–60 °C for analysis. Three scans were recorded for each sample
(n= 3), and an average of the three scans was used for data analy-
sis. Origin
Ò
7 scientific plotting software (Origin Lab Corporation,
Northampton, MA, USA) was used for baseline correction and data
analysis. The change in enthalpy (
D
H) was determined by integrat-
ing the area under the baseline-corrected excess heat capacity, C
p
,
curve obtained for each sample.
2.8. Langmuir isotherms
Monolayers consisting of DDA/TDX (50:50 mol%) were formed
at room temperature by spreading a total amount of 27.7 nmol lipid
in CHCl
3
–MeOH (9:1) on an aqueous subphase in a KSV Minitrough
1 (KSV Instruments Ltd., Helsinki, Finland) with a surface area of
243 cm
2
using a Hamilton microsyringe. The aqueous subphase
consisted of 10 mM Tris buffer (pH = 7.4). In order to allow total
evaporation of the organic solvent, compression of the monolayer
was initiated 10 min after spreading the lipid monolayer. The
monolayer was compressed with a barrier speed of 10 mm/min,
and the surface pressure was measured by using a Wilhelmy plati-
num plate (KSV Instruments Ltd.). Each sample was compressed
once in three independent experiments (n= 3). KSV software (KSV
Instruments Ltd.) was used for data analysis.
2.9. Immunization of mice
Female CB6F1 (F1 of BALB/c C57BL/6) mice were obtained
from Harlan Laboratories (Boxmeer, The Netherlands). The mice
were allowed free access to water and food. All animal experiments
were approved by the Danish Council for Animal Experiments and
O
OR
O
RO
RO
RO
OH
OR
RO b
1R = H
2R = TMS
3a-e R = TMS; n = 10, 12, 14, 16 and 18
4a-e R = H; n = 10, 12, 14, 16 and 18
O
OH
a
O
OR
O
RO
RO
RO
OR
RO
O
c
O
O
n
O
O
n
Fig. 1. Reagents and conditions: (a) N,O-bis(trimethylsilyl)acetamide, TBAF (cat.), DMF, rt, 30 min, followed by propan-2-ol, 0 °C, K
2
CO
3
in MeOH, 2 h (yield 70%); (b)
CH
3
(CH)
n
COOH, DCC, DMAP, CH
2
Cl
2
, rt, 16 h; (c) DOWEX-H
+
,CH
2
Cl
2
–MeOH (1:1), rt, 30–60 min; yields: n= 10 (TDL = 4a, 31%), n= 12 (TDMyr = 4b, 57%), n= 14 (TDP = 4c,
67%), n= 16 (TDS = 4d, 66%), and n= 18 (TDA = 4e, 71%).
R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx 3
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
done in accordance with the EU directive 2010/63EU for animal
experiments. The vaccines were prepared by mixing the liposomal
dispersions with 5
l
gMycobacterium tuberculosis antigen H56
(Ag85B-ESAT-6-Rv2660c fusion protein) [28] in 10 mM sterile Tris
buffer (pH = 7.4). The vaccines were left at room temperature with
intermittent mixing to allow for full adsorption of antigen to the
liposomes [12].
The adjuvants selected for the animal experiments were DDA/
TDP, DDA/TDS and DDA/TDB liposomes. As controls were used mice
receiving antigen only and naïve mice. The DDA dose was 250
l
g,
and the TDX dose was as follows: 50
l
g TDB, 44
l
g TDS and 41
l
g
TDP (corresponding to a content of 11 mol%). Mice were immunized
subcutaneously at the base of the tail with 200
l
L of vaccine three
times with a two-week interval. The mice were euthanized three
weeks after the final immunization. Suspensions of spleen cells
were obtained by passage of spleens through a metal mesh followed
by centrifugation at 700gfor 5 min. The cells were washed once
with RPMI 1640 (Gibco Invitrogen, Carlsbad, CA, USA) and once with
RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal
bovine serum, 5 10
6
Mb-mercaptoethanol, 1 % (v/v) penicillin–
streptomycin, 1% (v/v) sodium pyruvate, 1 mM
L
-glutamine, and
10 mM HEPES.
2.10. Detection of interferon (IFN)-
c
, interleukin (IL)-17a and IL-5
cytokines from activated T-cells by enzyme-linked immunosorbent
assay (ELISA)
Isolated splenocytes were cultured in duplicate in round-
bottomed 96-well micro-titer plates at a density of 2 10
6
cells/
well in a volume of 200
l
L RPMI supplemented as described above.
The cells were re-stimulated with 5
l
g/mL H56 and allowed to
incubate for 72 h at 37 °C. Wells containing medium alone or
3
l
g/mL concanavalin A (Sigma–Aldrich, St. Louis, MO, USA) were
included as negative and positive controls, respectively. After
incubation, supernatants were collected and IFN-
c
, IL-17a and
IL-5 production was quantified by using a standard enzyme-linked
immunosorbent assay (ELISA) protocol. Briefly, purified rat
a
-mouse IFN-
c
or IL-17a (BD Biosciences, San Jose, CA, USA) was
used as capture antibodies, and biotin-conjugated rat
a
-mouse
IFN-
c
or IL-17a (BD Biosciences) was used as detection antibodies,
followed by horse-radish peroxidase (HRP)-conjugated streptavi-
din (BD Biosciences) and TMB (3,3
0
,5,5
0
-tetramethylbenzidine) Plus
Ready-to-use substrate (Kem-En-Tec, Taastrup, Denmark). The
reaction was stopped at the optimal color development with
0.2 M H
2
SO
4
, and absorbance was read at 450 nm with wavelength
correction at 570 nm. The IL-5 production was measured by using
the OptEIA Mouse IL-5 set according to the manufacturer’s instruc-
tions (BD Biosciences).
2.11. Intracellular flow cytometry analysis
Isolated splenocytes (1 10
7
cells/well) were stimulated for 1 h
with 5
l
g/mL H56 in the presence of 1
l
g/mL anti-CD28 and anti-
CD49d (BD Pharmingen, San Diego, CA, USA) in a total volume of
200
l
L supplemented RPMI 1640. Cells were subsequently incu-
bated for 5–6 h at 37 °C after addition of 10
l
g/mL brefeldin A
(Sigma Aldrich) and 0.7
l
L/mL monensin/Golgi-stop (BD Pharmin-
gen). Following overnight storage at 4 °C, the cells were washed in
FACS buffer and stained with anti-CD4 and anti-CD44 monoclonal
antibodies (BD Pharmingen) in FACS buffer for 30 min at 4 °C. The
cells were washed with FACS buffer, permeabilized by using the
Cyto-fix/Cyto-perm kit (BD Pharmingen) according to the manu-
facturer’s instructions, and stained intracellularly for 30 min at
4°C using anti-IFN-
c
and anti-tumor necrosis factor (TNF)-
a
mAbs
(eBiosciences, San Diego, CA, USA), respectively, in Perm wash buf-
fer. After washing, the cells were resuspended in FACS buffer and
analyzed by flow cytometry using a six-color BD FACS Canto flow
cytometer (BD Biosciences, San Jose, CA, USA). Responses were
analyzed with the FlowJo software V7.2.2 (Tree Star Inc., Ashland,
OR, USA).
2.12. Statistics
Statistical calculations were done by one-way ANOVA at a 0.05
significance level followed by Dunnet’s multiple comparison test,
using GraphPad Prism 6 (GraphPad Software La Jolla, CA, USA).
For the animal experiments three separate experiments were con-
ducted (n=6,n= 8 and n= 8) and the results were pooled.
3. Results
3.1. Synthesis of TDX and formulation of DDA/TDX liposomes
TDB analogues were synthesized essentially as described previ-
ously [25–27] and incorporated into the bilayer of DDA liposomes
by the thin-film method combined with probe sonication at a
molar ratio of 11 mol% and characterized in terms of initial average
particle size, PDI and zeta-potential (Table 1). Incorporation of TDX
did not significantly affect the average particle size of the resulting
liposomes, as compared to DDA/TDB liposomes (140.3 ± 14.7 nm),
which corresponds to previously reported values [29].
A slightly lower degree of polydispersity was seen for the DDA/
TDA, DDA/TDS, DDA/TDP and DDA/TDMyr liposomes, reflected by a
significantly decreased PDI, as compared to DDA/TDB liposomes.
This decrease is most likely due to a more compatible length of
the lipid tails in the bilayer, resulting in a better packing due to
more favorable interactions between the lipid tails. The volume-
based particle size distribution (Fig. 2A) appeared almost unimodal.
Importantly, Cryo-TEM images confirmed the formation of lipo-
somes with an approximate size of 130–150 nm as illustrated by
representative DDA/TDP liposomes (Fig. 2B). The surface charge of
the liposomes was not affected by the TDX component.
3.2. The colloidal stability is affected by the acyl chain length
Incorporation of 11 mol% TDB has previously been shown to
stabilize physically unstable DDA liposomes [10,30] via hydrogen
bond formation between the trehalose headgroups and the buffer
medium. The colloidal stability, measured as the change in average
particle size of the formulations as a function of time, was evalu-
ated during storage for 4 months at 4 °C and 25 °C, respectively.
The average particle size of the formulations was not affected by
the storage temperature, and the colloidal stability was maintained
for 4 months (Fig. 3), except for the DDA/TDL formulation, for
which a rapid increase in average particle size was observed upon
storage at 4 °C. The visible aggregates formed in DDA/TDL formula-
tions upon storage were confirmed by cryoTEM images (data not
shown). The zeta-potential of the formulations remained constant
during the stability study (data not shown).
Table 1
Composition and physical characteristics of DDA/TDX liposomes (89:11). Results
denote mean values ± SD (n= 3). Statistically significant differences from DDA/TDB
liposomes are indicated:
⁄
p< 0.05,
⁄⁄
p< 0.01, and
⁄⁄⁄
p< 0.001.
Formulation z-Average (nm) PDI Zeta-potential (mV)
DDA/TDB 140.3 ± 14.7 0.256 ± 0.02 62.5 ± 4.3
DDA/TDA 130.3 ± 8.7 0.223 ± 0.01
⁄⁄⁄
57.0 ± 10.1
DDA/TDS 137.9 ± 7.7 0.209 ± 0.001
⁄⁄⁄
62.9 ± 4.8
DDA/TDP 133.4 ± 8.8 0.213 ± 0.01
⁄⁄⁄
59.3 ± 11.1
DDA/TDMyr 131.8 ± 5.2 0.216 ± 0.02
⁄⁄⁄
60.6 ± 11.6
DDA/TDL 146.1 ± 13.4 0.265 ± 0.01 55.9 ± 8.2
4R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
3.3. The length of the acyl chain affects the thermotropic phase
behavior
In order to elucidate the effect of the acyl chain length on the
thermotropic phase behavior, the gel-to-liquid crystalline phase
transition of the lipid bilayers of DDA/TDX was characterized by
using DSC. Liposomes undergo a transition from a gel to a liquid
crystalline phase at the main T
m
. An obvious change in the appear-
ance of the thermograms clearly demonstrates an influence of the
TDX component on the thermodynamic behavior (Fig. 4 and
Table 2). When incorporating TDB or TDA into the DDA bilayer,
two interconnected transition peaks were observed (most pro-
nounced for the DDA/TDB formulation) at approximately 41 °C
and 46 °C, respectively, as reported previously for DDA/TDB lipo-
somes [10,29]. This suggests the presence of lipid microdomains
with different T
m
enriched in either DDA (T
m
of approximately
47 °C) or TDB (T
m
of approximately 42 °C) [10] and TDA (T
m
of
approximately 44 °C), respectively. In contrast, incorporation of
TDS or TDP into the DDA lipid bilayer resulted in a single and nar-
rower phase transition. This was also reflected in the T
½
values,
which were approximately 1.4 °C and 1.1 °C for DDA/TDS and
DDA/TDP, respectively, as compared to approximately 2.9 °C for
DDA/TDB. For these two compositions, the T
m
was also signifi-
cantly increased with approximately 4 °C(p< 0.001), as compared
to DDA/TDB liposomes. A broader phase transition was evident for
lipid bilayers consisting of DDA and TDMyr or TDL (T
½
approxi-
mately 3.5 °C and 4.9 °C, respectively).
Fig. 2. (A) A representative volume-based particle size distribution of DDA/TDP
liposomal suspension (black) and DDA/TDB liposomes (gray). (B) CryoTEM images
of DDA/TDP formulations.
Fig. 3. Average particle size (z-average) of DDA/TDX liposomes stored at 4 °C (A)
and 25 °C (B) as an indication of the colloidal stability upon storage [DDA/TDB
(black dots), DDA/TDA (gray dots), DDA/TDS (black triangles), DDA/TDP (gray
triangles), DDA/TDMyr (black squares) and DDA/TDL (gray squares)]. Results denote
mean values + SD (n= 3).
Fig. 4. Representative thermograms of DDA/TDX liposomes. The curves have been
normalized to the molar content.
Table 2
Thermodynamic properties of DDA/TDX (89:11) liposomes. Results denote mean ± SD
(n= 3). For each sample, three scans were performed, and values represent averages
of the three scans. Statistically significant differences between the T
m
for each
formulation, as compared to the T
m
of DDA/TDB, are indicated:
⁄
p< 0.05,
⁄⁄
p< 0.01
and
⁄⁄⁄
p< 0.001.
Formulation T
m
(°C)
D
H(kJ/mol) T
½
(°C)
DDA/TDB 41.42 ± 0.15 36.81 ± 1.81 2.94 ± 1.60
DDA/TDA 43.93 ± 0.38
⁄
32.52 ± 1.87 2.61 ± 0.69
DDA/TDS 45.54 ± 0.27
⁄⁄⁄
35.31 ± 2.23 1.38 ± 0.40
DDA/TDP 45.95 ± 0.13
⁄⁄⁄
37.54 ± 1.77 1.09 ± 0.04
DDA/TDMyr 44.63 ± 0.90
⁄⁄
31.04 ± 2.02 3.45 ± 0.38
DDA/TDL
a
43.93 ± 0.46
⁄
24.57 ± 2.73
⁄⁄⁄
4.92 ± 2.76
a
For DDA/TDL, values are based on an average of the second and the third scan
due to an irreversible first scan.
R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx 5
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
The change in enthalpy (
D
H) was generally not significantly
affected by the incorporation of TDB, TDA, TDS, TDP or TDMyr into
the DDA bilayer, whereas a large and significant decrease was
observed when TDL was incorporated (Table 2).
3.4. The TDX component affects the packing and hydration of the DDA
monolayer
The Langmuir technique is a widely used method to investigate
lipid–lipid interactions [31]. We applied the technique to investi-
gate further how the lipid–lipid interactions were affected by the
acyl chain length by incorporation of the different TDX compo-
nents into a DDA monolayer spread onto an aqueous subphase. A
molar ratio of 50 mol% was chosen in order to ensure a detectable
influence of the TDX component. The results are shown in Fig. 5,
and the mean values for the surface pressure (G) as well as the
mean molecular area (A) at the collapse point and at the phase
transition from the liquid-expanded to the liquid-condensed phase
are summarized in Table 3. The monolayers of DDA showed a
phase transition from the liquid-expanded phase to the liquid-con-
densed state at a surface pressure of 14.0 ± 0.4 mN/m with a mean
molecular area of 67.0 ± 0.1 Å
2
, and the monolayer collapsed at a
surface pressure of 46.6 ± 0.5 mN/m with a mean molecular area
of 43.1 ± 0.04 Å
2
, as reported previously [29,32,33]. Lipid monolay-
ers consisting of DDA/TDX (50:50 mol%) were compared. Incorpo-
ration of TDB, TDA or TDS into the DDA monolayer resulted in a
significant increase of the surface pressure at the collapse point
(48.9–58.4 mN/m), suggesting that TDB, TDA and TDS stabilize
the DDA monolayer. The surface pressure was not significantly
affected at the collapse point by incorporation of TDP, TDMyr or
TDL. The mean molecular area was significantly decreased at the
collapse point for the DDA/TDS monolayer (25.1 ± 0.2 Å
2
) in com-
parison to the DDA monolayer, suggesting that the presence of
TDS at this specific molar ratio results in a significantly denser
packing of the lipids. In contrast, the presence of TDB, TDA, TDP,
TDMyr and TDL resulted in significantly larger molecular areas at
the collapse point, showing that the DDA/TDX monolayers are
more loosely packed with these lipids.
For DDA/TDB, DDA/TDA and DDA/TDS, a significantly lower sur-
face pressure at the phase transition (3.2–11.5 mN/m) as compared
to that of DDA (14.0 mN/m) was observed. The opposite was observed
for the DDA/TDP monolayer (16.8 mN/m) while no phase transition
was detectable for the DDA/TDMyr and DDA/TDL monolayers. This
is probably a result of the low phase transition temperatures of
TDMyr and TDL, which apparently are in the fluid state at room tem-
perature, and the monolayers are thus in the liquid expanded phase
until the collapse point. The mean molecular area at the phase tran-
sition was also significantly affected by incorporation of TDB, TDA
or TDS into the DDA monolayer. Thus, incorporation of TDB or TDA
resulted in a significant increase in the mean molecular area
(78.9 ± 0.1 Å
2
and 88.9 ± 0.1 Å
2
, respectively) at the phase transition,
whereas the opposite was observed for incorporation of TDS into the
DDA monolayer (58.8 ± 0.2 Å
2
). For TDP there was only a small, but
significant difference in the surface pressure at the phase transition,
but there was no difference in the molecular area.
3.5. Incorporation of TDP or TDS into DDA liposomes ensures adjuvant
efficacy comparable to DDA/TDB liposomes
The DDA/TDS and DDA/TDP liposomes were chosen for further
evaluation in terms of immunological properties and compared
to DDA/TDB liposomes, as these liposomes had the most promising
physicochemical properties (stability and membrane packing). The
adjuvants were evaluated for their ability to induce Th1 and Th17
cells (based on INF-
c
, TNF-
a
, and IL-17a secretion), as DDA/TDB lip-
osomes have previously been reported to stimulate a CD4
+
T-cell
response characterized by a mixed Th1/Th17 response [13,14,34].
Thus, co-administration of the recombinant tuberculosis fusion
antigen Ag85B-ESAT-6-Rv2660c (H56) with DDA/TDB, DDA/TDS
or DDA/TDP liposomes resulted in a significantly increased release
of IFN-
c
compared to the groups immunized with antigen alone
(Fig. 6A). The same was observed for the cytokine IL-17a
(Fig. 6B). As expected, none of the formulations or the antigen itself
resulted in a significant increase in the production of the more
Th2-associated cytokine IL-5 (Fig. 6C).
Intracellular flow cytometry was used to further investigate the
relative effect of the DDA/TDS and DDA/TDP formulations com-
pared to the DDA/TDB formulations based on the T cell-mediated
immune response on a single-cell level. Co-administration of anti-
gen with DDA/TDB, DDA/TDS or DDA/TDP formulations resulted in
a significant increase in the frequency of antigen-specific IFN-
c
+
and TNF-
a
+
CD4
+
T-cells as compared to immunization with anti-
gen alone (Fig. 7A and B, respectively). We also found that all for-
mulations induced circulating antibodies of the IgG1 and IgG2a
isotypes with no differences between the groups (data not shown).
Three separate but similar experiments were performed and the
results were combined to increase the statistical power. Overall,
our results are consistent with previous findings that TDB
functions as a Th1/Th17 immunopotentiator [13,14,34], and the
adjuvant activity of DDA/TDS and DDA/TDP was comparable to
that of DDA/TDB as no statistically significant differences in the
immunopotentiating effects were observed.
Fig. 5. Pressure/area isotherms of Langmuir monolayers composed of DDA (black
dots), DDA/TDB (gray solid), DDA/TDA (gray dashed), DDA/TDS (black solid), DDA/
TDP (black dashed), DDA/TDMyr (black dash-dot) and DDA/TDL (gray dots) on a
10 mM Tris buffer subphase (pH = 7.4). The DDA/TDX binary monolayers had a
50:50 M composition. The total molar lipid concentrations are identical for all
experiments. The curves represent averages of three experiments.
Table 3
Surface pressures (
p
) and mean molecular areas (A) at the collapse point for DDA/TDX
(50 mol%) monolayers on 10 mM Tris subphases. Results denote mean ± SD from
three batches of each formulation. Statistically significant differences from the DDA
monolayer are indicated:
⁄
p< 0.05,
⁄⁄
p< 0.01, and
⁄⁄⁄
p< 0.001.
DDA/TDX (50/
50)
Collapse point Phase transition
G(mN/m) A(Å
2
)G(mN/m) A(Å
2
)
DDA 46.6 ± 0.5 43.1 ± 0.04 14.0 ± 0.4 67.0 ± 0.1
⁄⁄⁄
DDA/TDB 49.6 ± 0.6
⁄⁄⁄
46.5 ± 0.1
⁄⁄⁄
6.6 ± 0.4
⁄⁄⁄
78.9 ± 0.1
⁄⁄⁄
DDA/TDA 48.9 ± 0.2
⁄⁄⁄
46.5 ± 0.02
⁄⁄⁄
3.2 ± 0.2
⁄⁄⁄
88.9 ± 0.04
⁄⁄⁄
DDA/TDS
a
58.4 ± 0.4
⁄⁄⁄
25.1 ± 0.2
⁄⁄⁄
11.5 ± 0.3
⁄⁄⁄
58.8 ± 0.4
⁄⁄⁄
DDA/TDP 47.4 ± 0.5 45.5 ± 0.2
⁄⁄⁄
16.8 ± 0.4
⁄⁄⁄
66.6 ± 0.2
DDA/TDMyr 45.6 ± 0.1 50.7 ± 0.1
⁄⁄⁄
––
DDA/TDL 45.5 ± 0.9 44.7 ± 0.03
⁄⁄⁄
––
a
The mean surface pressure and the molecular area at collapse point and phase
transition for the DDA/TDS monolayer are significantly different from the other
combinations of DDA/TDX.
6R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
4. Discussion
A promising approach toward development of new and effec-
tive adjuvants is to combine delivery systems with immunopoten-
tiating compounds. By rational selection of immunopotentiators
one may tailor the adjuvanticity toward a desired immunological
profile. Nevertheless, the physicochemical properties of the formu-
lation should be taken into consideration, since they may have an
impact on the adjuvanticity of the formulation.
DDA liposomes are known to be physically unstable upon stor-
age, which can be explained by the small headgroup in the DDA
molecule that interacts only to a low degree with the surrounding
water molecules. Furthermore, the cationic headgroups of the DDA
molecules cause intermolecular repulsions in bilayers eventually
resulting in instability of DDA liposomes. It has previously been
shown that incorporation of 11 mol% TDB into DDA liposomes con-
fers stability to the liposomes and facilitates an increased immune
response as compared to formulations consisting of DDA or DDA in
combination with a higher or lower TDB content [10]. Generally,
incorporation of a TDX component in the present study provided
colloidal stability of the DDA liposomes due to a decreased electro-
static repulsion between the DDA molecules when neutral TDX
molecules were present. However, TDL was apparently unable to
stabilize DDA liposomes as aggregation was observed upon storage
at 4 °C.
Temperature affects the diffusivity of the lipids in the bilayer
that may undergo conformational changes, rotations around the
axis of the lipids, lateral diffusion, protrusion out of the bilayer
and flip-flop movements between each leaflet of the bilayer [35].
Since these movements are slowed down by a decrease in temper-
ature, it can be expected that protrusion out of the bilayer does not
necessary result in a subsequent insertion, leaving the bilayer in a
DDA-like state, which is known to be unstable in terms of size,
when the storage temperature is 4 °C. Also, the state of the lipid
and thereby the fluidity of the lipids are dependent on temperature.
If the phase transition from gel to liquid crystalline phase is
assumed to take place between 4 °C and 25 °C degrees for TDL, then
the DDA/TDL formulation will be gel-like for the DDA part and
liquid-like for the TDL part when stored at 25 °C. This could have
a positive effect on the colloidal stability of the formulation,
explaining the observed higher degree of colloidal stability of the
Fig. 6. Production of cytokines (A) INF-
c
, (B) IL-17a and (C) IL-5 upon restimulation
of splenocytes isolated from CB6F1 mice (BAlb/6 C57BL/6) immunized with 5
l
g
H56 co-administered with DDA/TDB, DDA/TDS and DDA/TDP liposomes. Cytokine
levels were measured by ELISA. The shown data are from three separate
experiments (n=6,n= 8 and n= 8) plotted as individual values (triangles represent
experiment 1, dots represent experiment 2 and squares represent experiment 3).
Statistically significant differences from control group (Ag) are indicated:
⁄
p< 0.05,
⁄⁄
p< 0.01 and
⁄⁄⁄
p< 0.001.
Fig. 7. Antigen-specific CD4
+
T-cell response after immunization with 5
l
g H56 co-
administered with DDA/TDB, DDA/TDS and DDA/TDP liposomal formulations or
5
l
g H56. The IFN-
c
and TNF-
a
response from CD4
+
T-cells was measured by
intracellular flow cytometry after restimulation of the isolated splenocytes with
H56. The shown data are from three separate experiments (n=6,n= 8 and n=8)
plotted as individual values (triangles represent experiment 1, dots represent
experiment 2, and squares represent experiment 3). Statistically significant
differences from control group (Ag) are indicated:
⁄⁄⁄
p< 0.001.
R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx 7
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
DDA/TDL formulation at 25 °C than at 4 °C. The TDL component
thus sets the limit for how short a chain length can be incorporated.
The results obtained by using the Langmuir technique support
that increased hydration properties are obtained via incorporation
of TDX since the monolayer could resist a higher surface pressure
at the collapse point when DDA was combined with the TDX com-
ponent at a 50:50 M ratio, as compared to DDA alone. This is due to
the nature of the glycolipid headgroup of the TDX component,
which allows for increased formation of hydrogen bonds by inter-
action with the surrounding water molecules. This hydration effect
is suggested to account for the stabilizing effect TDB in DDA/TDB
liposomes [10,33], which is in line with the findings on colloidal
stability in the present study. The increase in surface pressure is
most likely due to an interaction of the TDX headgroup with the
aqueous subphase, as well as the intercalation of uncharged TDX
headgroups between the cationic DDA headgroups reducing inter-
molecular repulsion. Nonetheless, other factors are also implicated
since the increase in surface pressure was only detected for the
DDA monolayers in which TDB, TDS and TDP were incorporated.
Furthermore, only insertion of TDS into the DDA monolayer
resulted in a decreased mean molecular area at the collapse point
as compared to the other DDA/TDX combinations. The decreased
mean molecular area observed for the DDA/TDS monolayer is
due to a more dense packing of the lipids. This can be explained
by the presence of equally long lipid tails in the DDA and TDS mol-
ecules which allows for favorable lipid–lipid interactions and thus
a tighter lipid packing. Incorporation of shorter or longer lipid tails
leads to larger differences between the chain lengths in the DDA/
TDX monolayer forcing it either to accommodate the excess carbon
atoms of the longer lipid chain lengths or to fill out the space left
when incorporating lipids with a shorter chain length.
The effect of the TDX component on the phase transition is not as
clear. The significantly decreased surface pressure observed for
monolayers consisting of DDA and TDB, TDA or TDS would be
expected only for the monolayer containing DDA and TDB or TDA,
due to the presence of longer lipid tails, which must be accommo-
dated in the monolayer. Other factors must be taken into account.
The incorporation of TDP into the DDA monolayer resulted in an
increased surface pressure at the phase transition. The mean molec-
ular area at the phase transition was also affected by the TDX com-
ponent. Only when incorporating TDS into the DDA monolayer a
decrease in the mean molecular area was observed. This is most
likely due to a favorable interaction between the lipid tails of
DDA and TDS due to their identical length. The increase in mean
molecular area at the phase transition observed when incorporat-
ing TDB or TDA into the DDA monolayer can be explained by the for-
mation of microdomains, which is also reflected in the
thermograms obtained by DSC. No phase transition of the DDA/
TDMyr or DDA/TDL monolayer was observed possibly caused by a
low T
m
for these systems as well as a more fluent monolayer at
room temperature (experimental temperature). From the DSC
results (Table 2) it is evident that the T
m
of the system is influenced
by incorporation of 11 mol% TDX. When the content of the TDX
component was increased to 40 mol% (data not shown) the T
m
of
the DDA/TDMyr and DDA/TDL systems was altered as compared
to results obtained with 11 mol% (Table 2), thus T
m
was shifted from
44.6 °C(Table 2) to 36.8 °C (data not shown) for DDA/TDMyr and
from 43.9 °C(Table 2) to 30.7 °C (data not shown) for DDA/TDL.
For the remaining systems no differences were observed (data not
shown). This illustrates that increasing the amount of the shorter
TDX analogues, for which an influence on T
m
is very pronounced,
also affects the packing of the monolayer in the Langmuir experi-
ments, and hence confirms the assumption that the absence of a
phase transition is a consequence of a low T
m
.
The appearance of the thermograms from the DSC measurements
also clearly reveals an effect of chain length, since interconnected
peaks, sharp peaks or broad peaks arise for the different DDA/TDX
formulations. The presence of interconnected peaks is most likely
due to the formation of local microdomains enriched in the TDX
component [36]. These local structures become more pronounced
as the difference in chain length between the DDA and TDX mole-
cules increases. The single sharp phase transition observed for the
DDA/TDS and DDA/TDP liposome dispersions is due to a more homo-
geneous phase consisting of lipids with similar tail lengths display-
ing more uniform thermodynamic behavior. A general decrease of
T
m
for the DDA/TDX formulations (Table 2) as compared to the T
m
(47 °C) of a DDA formulation [10] indicates that the TDX is incorpo-
rated since a decrease of T
m
is typically associated with a decreasing
chain length in a liposomal bilayer structure [37,38]. This is also evi-
dent from the sharpness of the transition peaks observed for both
the DDA/TDS and DDA/TDP liposome dispersions in which the
length of the acyl chains are C
18
and C
16
, respectively, and thus more
comparable to the C
18
lipid tails of the DDA molecule than the acyl
chains of the other TDX analogues.
The combined results of the physicochemical characterization
formed the basis for the selection of DDA/TDS and DDA/TDP formu-
lations to be tested in vivo for adjuvanticity. These formulations
were compared to the DDA/TDB formulation to investigate whether
the immunological properties were preserved. All three formula-
tions induced comparable Th1 responses, assessed by the level of
IFN-
c
production, the frequency of IFN-
c
and TNF-
a
expressing
CD4
+
T cells and the lack of IL-5 production in response to restimu-
lation with the vaccine antigen. It has previously been shown that
the use of DDA/TDB liposomes as adjuvant results in a mixed
Th1/Th17 profile [13,14,34]. In the present study, both the DDA/
TDP and DDA/TDS liposome-adjuvanted formulations induced a
significant increase in IL-17a as compared to antigen alone and
the variation was high within the different treatment groups.
Th17 responses are highly dependent on the mouse strain and
external factors, such as food and intestinal bacterial flora, which
may, at least partially, explain the variation. Altogether, there were
no significant differences between the formulations for any of the
evaluated immunological parameters and, thus, improving the
physicochemical properties of DDA/TDB by substitution of TDB
with TDS or TDP did not compromise its adjuvanticity.
Insufficient antigen adsorption to the liposomes during vaccine
preparation could affect the immunological response and the nat-
ure of the antigen must also be taken into account. However, H56
is a negatively charged antigen at physiological pH due to a pI
value of 4.9 [39], and our recent studies have shown that H56 is
almost completely adsorbed to DDA/TDB liposomes [39].
TDB is a well-known immunopotentiator exerting its effect
through binding to the Mincle receptor followed by signaling
through the FcR
c
-Syk-Card9 pathway [13]. The Mincle receptor
has been extensively studied in terms of immune activation mech-
anisms and structural characteristics of ligands [13,14,18,40,41].
When TDX is incorporated into DDA liposomes, the TDX molecules
are arranged in the lipid bilayer with their hydrophobic tails
inserted into the DDA bilayer and the hydrophilic headgroups fac-
ing the surrounding aqueous environment. Thus, only the head-
groups are presumably presented for interaction with Mincle. A
study by Khan et al. [40] concluded that an acyl chain length above
18 carbon atoms is necessary for immune activation. However,
these studies involved TDB or analogues thereof and not liposomes.
This illustrates the importance of addressing the colligative proper-
ties of the adjuvant formulations as such and not only single-mole-
cule properties, since the present study shows that incorporation of
TDS or TDP into a DDA bilayer gives rise to a comparable immune
activation as seen with a DDA/TDB formulation.
A systematic evaluation of the optimal molar content of the TDX
component, e.g. using the Langmuir monolayer technique, could
result in valuable information on both physicochemical and immu-
8R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
nological properties, since the DDA bilayer might be affected differ-
ently depending on the nature of the TDX component. In the study
by Davidsen et al. a clear concentration-dependent effect on the
thermodynamic behavior was observed. A more favorable formula-
tion in terms of immune activation could result from a higher mol%
of TDX, as it would allow for a possible increased interaction with
the Mincle receptor with ensuing higher immune activation. How-
ever, an increased amount of TDB in DDA liposomes (20 mol%) has
in fact previously been shown to have a negative effect on the
immune activation [10]. This could be a result of a less favorable
packing of the TDB component due to the longer lipid chain, as com-
pared to DDA, or a less homogeneous lipid phase. The lower
immune activation seen for DDA/TDB (80:20) could also be due to
an overstimulation of the immune system or saturation of interac-
tions with Mincle. A better packing is ensured when incorporating
TDS into the DDA bilayer and an investigation of the effect of differ-
ent mol% on the immunological response could provide knowledge
on whether or not the immune system is exhausted, and whether or
not an increase in Mincle interaction could be feasible, leading to an
increased immune activation.
With this study we have demonstrated that incorporation of TDS
into DDA liposomes results in the most favorable physicochemical
properties as compared to the other investigated TDX analogues,
with an adjuvant activity comparable to that of DDA/TDB liposomes.
We are currently investigating the effect of headgroup modifica-
tions and the degree of acylation on receptor binding and immuno-
stimulatory properties using the TDS molecule as a lead compound.
5. Conclusion
In conclusion, the present study provides a thorough character-
ization of how incorporation of TDB analogues with symmetrically
shortened acyl chains into a DDA liposomal formulation affects the
physicochemical properties of DDA/TDX liposomes. In addition,
these optimal formulations were compared immunologically. The
main phase transition temperature was affected leading to a T
m
value closer to that of DDA, when incorporating TDS or TDP ana-
logues with chain lengths comparable to that of DDA. Furthermore,
a more uniform lipid bilayer and sharp phase transition of the lipids
were observed for the DDA/TDS and DDA/TDP formulations as illus-
trated by a narrow phase transition temperature peak. The initial
liposome size and surface charge were not affected by the chain
length, and neither was the stability during storage of the lipo-
somes, except for the shortest analogue (TDL) that rendered the for-
mulation unstable at 4 °C. Only when incorporating TDS into a DDA
monolayer, an increased surface pressure and decreased mean
molecular area at the collapse point were found. This is due to
favorable lipid–lipid interactions between the DDA and TDS com-
ponent. Shortening the acyl chain length from C
22
(TDB) to a C
16
(TDP) or a C
18
(TDS) length did not compromise the immunopoten-
tiating effect, which leads to the conclusion that incorporation of
TDS or TDP into a DDA liposomal formulation confers favorable
physicochemical properties while maintaining the immunopotenti-
ating effect.
Role of the funding source
The work was funded by The Danish Council for Independent
Research | Medical Sciences (Grant No. 09-067412) and the Faculty
of Health and Medical Sciences, University of Copenhagen, Denmark
(RSK). The Danish National Advanced Technology Foundation
(Grant Nos. 007-2007-1 and 069-2011-1), the Danish Strategic
Research Council (Centre for Nano-Vaccine, Grant No. 09-067052),
the European Union’s Seventh Framework Programs NEWTBVAC
(Grant Agreement No. FP7-HEALTH-F3-2009-241745) and ADITEC
(Grant Agreement No. FP7-HEALTH-F2010-280873) provided addi-
tional funding. We acknowledge the Danish Agency for Science,
Technology and Innovation for funding the Zetasizer Nano ZS and
The Danish National Advanced Technology Foundation and the Dan-
ish Ministry of Science, Technology and Innovation for funding the
nano-DSC. The Drug Research Academy, University of Copenhagen,
is kindly acknowledged for co-funding the KSV Minitrough 1. The
NMR equipment used in this work was purchased via Grant No.
10-085264 from The Danish Research Council for Independent
Research | Nature and Universe. The funding sources had no
involvement in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; nor in the deci-
sion to submit the paper for publication.
Conflict of interest
KSK and DC are employed by Statens Serum Institut, a non-profit
government research facility, which holds a patent on the use of
TDB in adjuvant formulations and of which the CAF adjuvants are
proprietary products.
Acknowledgments
The authors wish to acknowledge Rune Fedelius Jensen (Statens
Serum Institut) and the Department of Biological Services (Statens
Serum Institut) for excellent technical assistance. We acknowledge
Gunnel Karlsson (Lund University) for performing the Cryo-TEM
analysis.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejpb.2014.10.015.
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10 R.S. Kallerup et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Please cite this article in press as: R.S. Kallerup et al., Influence of trehalose 6,60-diester (TDX) chain length on the physicochemical and immunopotenti-
ating properties of DDA/TDX liposomes, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.10.015
