High potency of lipid conjugated TLR7 agonist requires nanoparticulate or liposomal formulation

Article abstract of DOI:10.1016/j.ejps.2018.07.048

Conjugation of small molecule agonists of Toll-like receptor 7 (TLR7) to proteins, lipids, or polymers is known to modulate potency, and the physical form or formulation of these conjugates is likely to have a major effect on their immunostimulatory activity. Here, we studied the effect of formulation on potency of a 1,2?di?(9Z?octadecenoyl)?sn?glycero?3?phosphoethanolamine (DOPE) conjugated TLR7 agonist (DOPE-TLR7a) alongside assessing physical form using Dynamic Light Scattering (DLS), Nanosight Particle Tracking (NTA) analysis and Small Angle X-ray Scattering (SAXS). A very high potency of DOPE-TLR7a conjugate (EC₅₀ around 9 nM) was observed either when prepared by direct dilution from DMSO or when formulated into 400–700 nm large multilamella liposomes containing dimethyldioctadecylammonium bromide salt (DDA) and DOPE. When prepared by dissolution in DMSO followed by dilution in aqueous culture medium, 93 ± 5 nm nanoparticles were formed. Without dilution from solution in DMSO, no nanoparticles were observed and no immunostimulatory activity could be detected without this formulation step. SAXS analysis of the conjugate after DMSO dissolution/water dilution revealed a lamellar order with a layer spacing of 68.7 ? which correlates with arrangement in groups of 3 bilayers. The addition of another immunostimulatory glycolipid, trehalose?6,6?dibehenate (TDB), to DOPE:DDA liposomes gave no further increase in immunostimulatory activity beyond that provided by incorporating DOPE-TLR7a. Given the importance of nanoparticle or liposomal formulation for activity, we conclude that the major mechanism for increased potency when TLR7 agonists are conjugated to macromolecules is through alteration of physical form.

Full text of DOI:10.1016/j.ejps.2018.07.048

EuropeanJournalofPharmaceuticalSciences123(2018)268–276
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
High potency of lipid conjugated TLR7 agonist requires nanoparticulate or
liposomal formulation
Adam J.R. Gadda,1, Valeria Castellettoa, Elena Kabovaa, Kenneth Shanklanda, Yvonne Perrieb,
Ian Hamleya, Alexander J.A. Cobbc, F. Grecoa, Alexander D. Edwardsa,⁎
a
School of Chemistry, Food and Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G1 1XQ, United Kingdom
Department of Chemistry, 7 Trinity Street, King's College London, London SE1 1DB, United Kingdom
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Conjugation of small molecule agonists of Toll-like receptor 7 (TLR7) to proteins, lipids, or polymers is known to
TLR7 agonist
Lipid conjugate
Liposomes
Conjugation
Immunostimulatory
modulate potency, and the physical form or formulation of these conjugates is likely to have a major effect on
their immunostimulatory activity. Here, we studied the effect of formulation on potency of
a
1,2‑di‑(9Z‑octadecenoyl)‑sn‑glycero‑3‑phosphoethanolamine (DOPE) conjugated TLR7 agonist (DOPE-TLR7a)
alongside assessing physical form using Dynamic Light Scattering (DLS), Nanosight Particle Tracking (NTA)
analysis and Small Angle X-ray Scattering (SAXS). A very high potency of DOPE-TLR7a conjugate (EC50 around
9 nM) was observed either when prepared by direct dilution from DMSO or when formulated into 400–700 nm
large multilamella liposomes containing dimethyldioctadecylammonium bromide salt (DDA) and DOPE. When
prepared by dissolution in DMSO followed by dilution in aqueous culture medium, 93
5 nm nanoparticles
were formed. Without dilution from solution in DMSO, no nanoparticles were observed and no im-
munostimulatory activity could be detected without this formulation step. SAXS analysis of the conjugate after
DMSO dissolution/water dilution revealed a lamellar order with a layer spacing of 68.7 Å, which correlates with
arrangement in groups of 3 bilayers. The addition of another immunostimulatory glycolipid, trehalose‑6,6‑di-
behenate (TDB), to DOPE:DDA liposomes gave no further increase in immunostimulatory activity beyond that
provided by incorporating DOPE-TLR7a. Given the importance of nanoparticle or liposomal formulation for
activity, we conclude that the major mechanism for increased potency when TLR7 agonists are conjugated to
macromolecules is through alteration of physical form.
1. Introduction
administered with alum (Gupta et al., 1996) and the excision of the
injection site shortly after administration does not significantly impact
Recently the molecular pathways of immune activation both by
pathogen associated molecular patterns (PAMP) recognition via pattern
recognition receptors (PRR) such as TLR (Medzhitov and Janeway,
1998; Medzhitov et al., 1997; Takeda and Akira, 2004) and also by
particulate adjuvants for example by nucleotide-binding domain, leu-
cine-rich-containing family, pyrin domain-containing-3 (NLRP3)/in-
flammasome mediated pathways (Eisenbarth et al., 2008) have been
elucidated. These advances include insight into the immunomodulatory
properties of alum, which was thought to rely on a deposition effect to
enhance the persistence of presented antigens to antigen presenting
cells (APC) (Verdier et al., 2005). However this paradigm has been
challenged by studying the rapid release of 14C labelled antigens
on the immune response to the antigen (Lindblad, 2004). The increased
immunogenicity of alum adsorbed antigen may therefore lie in the ef-
ficiency of APC to phagocytise particulate antigens compared to the
macropinocytosis of antigen alone despite the lack of PRR agonist
(Morefield et al., 2005). The physical form of the antigen is therefore
clearly of equal importance to associated immunostimulatory activity
through PRR. Recent attempts at rational vaccine design therefore
manipulate both the innate molecular recognition pathways and the
physical form of the antigen. For example, when a small molecule
TLR7/8 agonist was conjugated to a temperature responsive polymer
support to form nanoparticles, the most significant increase in im-
munogenicity was seen from the formation of particles as opposed to
⁎
Corresponding author.
E-mail address: a.d.edwards@reading.ac.uk (A.D. Edwards).
Current address: School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom.
1
https://doi.org/10.1016/j.ejps.2018.07.048
Received 9 May 2018; Received in revised form 13 July 2018; Accepted 23 July 2018
Availableonline24July2018
0928-0987/©2018ElsevierB.V.Allrightsreserved.

A.J.R. Gadd et al.
EuropeanJournalofPharmaceuticalSciences123(2018)268–276
modulation of TLR7/8 agonist loading (Lynn et al., 2015).
example conjugated or unconjugated TLR7 agonist were both able to
induce dendritic cell (DC) maturation, but cross presentation was in-
creased when TLR7 agonist was conjugated directly to antigen (Oh
et al., 2011; Oh and Kedl, 2010).
This detailed molecular and cellular insight confirms multiple pre-
vious observations highlighting the importance of formulation and
physicochemical form of vaccine adjuvants for maximising im-
munostimulatory activity. For example, an increase in potency fol-
lowing liposomal formulation on muramyl dipeptide was observed as
far back as 1984 (Sone et al., 1984) the immunostimulatory activity of
which has been more recently attributed to nucleotide-binding oligo-
merization domain-like receptors (NOD) activation. Likewise, the in
vivo adjuvant activity of TLR2 stimulatory lipopeptides was found to be
strongly dependent on physical form, with particulate components
primarily responsible for vaccine activity in vivo (Tsunoda et al., 1999).
The physical form and self-assembly properties of TLR2 stimulatory
lipopeptides have recently been studied in much greater detail, re-
vealing that aggregation into micellular or fibrillar structures, has a
profound effect on biological activity (Castelletto et al., 2016; Hamley
et al., 2014).
The most intensely studied synthetic immunostimulatory small
molecule PRR agonists target the sensors of viral RNA TLR7 and TLR8
(Heil et al., 2003; Hemmi et al., 2002; Takeda and Akira, 2004). Indeed,
the imidazoquinoline Imiquimod was clinically approved in 1997,
5 years before its ability to trigger TLR7 was identified (Hemmi et al.,
2002). Many studies have reported the relative potency of different
families of small molecule agonists for TLR7 and TLR8, and while dif-
ferent cellular assays and endpoints are used, it is still informative to
compare their potency which varies widely. To date, the most potent
reported were in a library of 2‑substituted 8‑hydroxyadenines some of
which had minimum effective concentrations (MEC) as low as 1–10 nM
(Hirota et al., 2002). The majority of compounds in contrast have re-
ported activity in the 0.1–10 μM range including imidazoquinolines
with a ED50 or MEC in μM level (Kurimoto et al., 2004). Several
structure-activity relationship (SAR) studies have focused on identi-
fying a suitable location for conjugation to protein antigens or macro-
molecules without reducing potency following conjugation (Chan et al.,
2009; Wille-Reece et al., 2005a; Wu et al., 2007a). Interestingly, one of
the most studied examples, UC-1V150, is by no means the most potent
TLR7 molecule in its unconjugated form, with EC50 of around 500 nM
(Wu et al., 2007a). This contrasts with structurally similar 8‑ox-
oadenine derivatives reported with EC50 as low as 5 nM (Czarniecki,
2008) and rationally designed compounds based on pharmacophore
molecular docking studies as low as 0.5 nM have been reported (Yu
et al., 2013).
The potency of UC-1V150 is significantly increased following con-
jugation to proteins such as serum albumin (Wu et al., 2007b) even
taking into account that the conjugates typically have a drug substitu-
tion ratio of up to 5:1 meaning that stated molar concentrations un-
derestimate the total quantity of TLR7-agonist small molecule drug by
up to 5-fold. Similarly conjugation of UC1V150 to the anti-CD20 anti-
body rituximab demonstrates a similar increase in potency compared to
the free unconjugated TLR7 agonist (Gadd et al., 2015). Imidazoqui-
noline compounds such as 3M-012 conjugated to HIV Gag P41 protein
show a similar increase in potency over the unconjugated and the
protein mixed with TLR7/8 agonist (Wille-Reece et al., 2005b). As well
as protein/peptide conjugation of TLR agonists, other published ex-
amples of macromolecular conjugates include polymers such as PEG
(Chan et al., 2011), phospholipids (Chan et al., 2009) and glyco-
conjugates (Donadei et al., 2016; Shinchi et al., 2015). These examples
typically note an increase in potency compared to the unconjugated
agonist or agonist mixed with the antigen/scaffold. Other TLR agonists
have likewise been conjugated to macromolecules with modulation of
activity (Yu et al., 2017). Furthermore, conjugation alters in vivo re-
sponses including altered duration and location of cytokine induction,
and when humoral response was studied modification of the antibody
titre and ratio of immunoglobulin G (IgG) subtypes (Donadei et al.,
2016; Shinchi et al., 2015; Smirnov et al., 2011). Further benefits of
conjugation were also observed beyond simply increasing potency; for
A lipid modified member of the imidazoquinoline family TLR7
agonist was shown to be a potent vaccine adjuvant in vivo, but the in
vitro immunostimulatory activity of this compound was not reported
(Smirnov et al., 2011). However, the in vitro activity of the un-
conjugated agonist was reported in the low μM range similar for the
structurally related R848 compound. In contrast to the highly potent
UC-1V150 conjugated to the phospholipid DOPE that had EC50 of ap-
proximately 50 nM in vitro when analysing IL12p40 concentrations,
compared to the unconjugated agonist of aproximatly 1 μM (Chan et al.,
2009). Separately, immunostimulatory liposomal formulations have
been studied extensively for vaccine formulation as an antigen delivery
and/or depot system. While the majority of liposome-forming lipids
lack intrinsic immunostimulatory activity, addition of the glycolipid
trehalose‑6,6‑dibehenate (TDB) provides direct immunostimulatory
activity to liposomes by triggering the PRR minkle via Syk/CARD9
(Matsunaga and Moody, 2009; Vangala et al., 2006; Yamasaki et al.,
2008).
Here, we studied the influence of the physical form of lipid con-
jugated TLR7 agonist on immunostimulatory potency. We specifically
focussed on phospholipid-conjugated forms of 2-substituted 8‑hydro-
xyadenines which are potent TLR7 agonists (Chan et al., 2009). The
physical form of this conjugate and its influence on biological activity
has not previously been reported, and we therefore describe here how
either nanoparticles formed by dilution from solvent, or a liposomal
formulation, are required for this conjugate to be active in vitro and
provide further physical characterisation by SAXS.
2. Material and methods
2.1. Materials
RPMI 1640 supplemented with Glutamax & HEPES & Phenol Red,
performance plus heat inactivated foetal bovine serum, phosphate
buffered saline and 2‑mercaptoethanol were from Life Technologies
(Paisley, UK). ExtrAvidin alkaline phosphatase conjugate, Sigmafast
pNPP, PBS tablets, sodium azide and EDTA (0.5 M) were from Sigma
Aldrich (Gillingham, UK). The IL-12p40 capture antibody C 15.6 and
biotinylated IL-12p40/70 detection antibody C17.8 were from
eBioscience (Hatfield, UK). Nunc® Maxisorb 96 well ELISA plates were
from Thermo Fisher scientific (Paisley, UK). Reagents for chemical
synthesis were purchased from Sigma Aldrich (Gliiingham, UK) or
Fluorochem (Hadfield, UK) and used without further purification.
2.2. Synthesis
TLR7 agonist with carboxyl site on the para-benzyl group for con-
jugation (7, Fig. 1) and DOPE-TLR7 agonist conjugate (8, Fig. 1) were
synthesised according to previous methods (Wu et al., 2007b) and ob-
tained in good yields as white crystalline powders. A similar TLR7
agonist without the carboxyl conjugation site on the para-benzyl group
(9, Fig. 1) was also synthesised for activity comparison. Full synthetic
methodology and comprehensive characterisation data is provided in
Supplementary information, including a crystal structure for 9 (Sup-
plementary information).
4‑{[6‑Amino‑2‑(2‑methoxyethoxy)‑8‑oxo‑7H‑purin‑9(8H)‑yl]me-
thyl}benzoic Acid (7, Fig. 1)
1H NMR (DMSO‑d6) δ 12.94 (1H, s, ArC(O)OH), 10.03 (1H, s,
NHC(O)N), 7.89 (2H, d J = 8.2, 2x o-ArH), 7.39 (2H, d, J = 8.2, 2x m-
ArH), 6.51 (2H, s, NH2), 4.94 (2H, s, ArCH2N), 4.25 (2H, t, J = 4.2,
OCH2CH2OCH3), 3.57 (2H, t, J = 4.2, OCH2CH2OCH3), 3.26 (3H, s,
OCH2CH2OCH3). 13C NMR (DMSO‑d6) δ167.00, 159.83, 152.17,
149.08, 147.75, 141.98, 129.83, 129.55, 127.39, 98.34, 70.15, 65.26,
269

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EuropeanJournalofPharmaceuticalSciences123(2018)268–276
A
B
C
Fig. 1. Synthesis scheme of carboxyl modified TLR7 agonist (A). Synthesis of DOPE-TLR7a agonist conjugate by conjugation of carboxy modified TLR7 agonist to
phospholipid DOPE (B) and diagram of TLR7 agonist lacking a conjugation moiety (C).
+
58.01, 42.12. HRMS calculated for C16H18O5N5 (MH+) 360.1302
found 360.1302.
environment and only endotoxin free reagents used. To confirm the
absence of endotoxin contamination, a LAL assay was used to quantify
the amount of soluble endotoxin in all reagents. Endotoxin content of
sample was analysed by a Hycult biotech (Uden, Netherlands): LAL
Chromogenic Endpoint Assay Endotoxin quantification kit and was
performed according to manufacturer's instruction, unless otherwise
stated all compounds tested were below the limit of detection of
0.08 EU/mL.
IR (ν, cm−1): 3351, 3165, 2956 (Broad), 1703, 1630, 1603
(2R)‑3‑(((2‑(4‑((6‑amino‑2‑(2‑methoxyethoxy)‑8‑oxo‑7,8‑dihydro‑9H‑
purin‑9‑yl)methyl)benzamido)ethoxy)(hydroxy)phosphoryl)oxy)propa-
ne‑1,2‑diyl dioleate (8)
1H NMR (DMSO‑d6, 700 MHz, 45 °C): δ 10.35 (1H, s, NHC(O)N),
9.97 (1H, bs, ArC(O)NHCH2CH2), 9.37 (1H, bs, PO3OH), 7.86 (2H, d, J
8.2, 2x o-ArH), 7.34 (2H, d, J 8.2, 2x m-ArH), 6.60 (2H, bs, NH2), 5.31
(4H, m, 2x CH2CHCHCH2), 5.09 (1H, m, POCH2CHCH2O), 4.89 (2H, s,
NCH2Ar), 4.29 (1H, dd, J 3.1, 12.0, C(O)NHCH2CH2OPO3) 4.25 (2H, m,
OCH2CH2OCH3), 4.09 (1H, m, C(O)NHCH2CH2OPO3), 3.84 (2H, bs,
POCH2CHCH2O), 3.81 (2H, bs, POCH2CHCH2O) 3.57 (2H, m,
OCH2CH2OCH3), 3.26 (3H, s, OCH2CH2OCH3), 3.17, (1H, d, J 5.1), 2.24
(4H, m, 2x OC(O)CH2CH2), 1.95 (8H, m, 2x CH2CH2CHCHCH2CH2),
1.48 (4H, m, 2x OC(O)CH2CH2), 1.23 (40H, m, Lipid tail CH2), 0.85
(6H, m, 2x CH2CH2CH3).
2.4. Formulation of nanoparticles and liposomes
2.4.1. Nanoparticle formulation of DOPE-TLR7a agonist conjugate
Weighed amounts of DOPE-TLR7 agonist conjugates (typically 1 mg,
0.9 μmol) were pre-solubilized with DMSO (50 μL) before diluting with
H2O to form a stock solution at 150 μM. Samples were then diluted in
ether H2O or complete cell culture media (RPMI 1640, 10% FBS, 50 μM
2-ME) from the stock solution. Solutions of DOPE-TLR7 agonist con-
jugates were tested at concentrations relevant for in vitro stimulation of
RAW 264.7 macrophages (1–1000 nM). Unconjugated TLR7a or DOPE-
TLR7a conjugates stored as either dry powder or in complete media as
nanoparticle formulations showed no difference in activity when stored
at −20 °C over a period of 6 months. Although not formally evaluated,
we have no evidence to suggest any instability of these conjugates upon
storage.
13C NMR (DMSO‑d6, 175 MHz) δ 173.13, 172.88, 160.43, 152.74,
149.62, 148.45, 140.74, 134.04, 130.22, 128.01, 127.9, 99.00, 70.82,
62.93, 58.66, 46.04, 42.69, 40.59, 34.15, 33.97, 31.88, 29.70, 29.69,
29.44, 29.30, 29.23, 29.19, 29.12, 29.10, 29.03, 28.99, 19.16, 14.53.
HRMS calculated for C57H94N6O12P+ (MH+) 1085.6662 found
1085.6677
IR (ν, cm−1): 3438, 3352, 3193, 2927, 2856, 1742, 1708, 1674,
1638, 1617.
2.4.2. Production of liposomes containing TDB and DOPE-TLR7 agonist
conjugates
2.3. Endotoxin testing
Multilamellar vesicles (MLV) were synthesised by using the original
lipid-film hydration method (Bangham et al., 1965). MLV were selected
without further sonication or homogenization because previous studies
had shown that liposomal size had relatively little impact on im-
munostimulatory potency (Henriksen-Lacey et al., 2011). DDA, TDB
Endotoxin is a component of bacterial cell walls with potent in-
flammatory response through TLR4 and is also a common laboratory
contaminant. Synthesis and conjugation was completed in sterile
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EuropeanJournalofPharmaceuticalSciences123(2018)268–276
2.6. SAXS characterisation of DOPE-TLR7a conjugates
Table 1
Composition of MLVs containing DOPE-TLR7a conjugate, TDB and DOPE.
2.6.1. Sample preparation for Small Angle X-ray Scattering (SAXS)
An aliquot of DOPE-TLR7a conjugate was first dissolved in DMSO
then further diluted in TRIS buffered saline (TBS; 0.05 M Tris, 0.15 M
NaCl, pH 7.6) to give the corresponding concentrations used for SAXS
experiments. 1 wt% DOPE-TLR7a in 0.02 vol DMSO/vol TBS was di-
luted with TBS to give (for example) 0.25 wt% DOPE-TLR7a in 0.005
vol DMSO/vol TBS.
MLV composition
TDB w%/v
% (μM)
DOPE-TLR7a agonist
w%/v% (μM)
DOPE w%/v%
(μM)
0% DOPE-TLR7a − TDB
0% DOPE-TLR7a + TDB
1% DOPE-TLR7a − TDB
20% DOPE-TLR7a − TDB
20% DOPE-TLR7a + TDB
0 (0)
20 (250)
0 (0)
0 (0)
20 (250)
0 (0)
0 (0)
1 (11.5)
20 (230)
20 (230)
20 (336)
20 (336)
19 (320)
0 (0)
0 (0)
2.6.2. Small Angle X-ray Scattering (SAXS)
and DOPE-TLR7 agonist conjugate were dissolved in chloroform/me-
thanol (9:1 by volume) to give 10 mg/mL stock solutions. DDA was
used as the core constituent of MLVs and used at 60 w/v% when TDB
was present or 80 w/v% when no TDB was incorporated. DOPE was
used at 20 w/v% and substituted accordingly with DOPE-TLR7 agonist.
The organic solvent was removed by rotary evaporation, followed by
flushing with N2 to form a thin lipid film on the bottom of a round-
bottomed flask. The lipid film was hydrated in 10 mM Tris-buffer at
pH 7.4 by heating for 20 min at 60 °C. The phase transition temperature
(Tm) of DDA is 47 °C. This general method to formulate MLVs was used
to produce a variety of liposomal formulations as listed in Table 1. MLV
formulations were stable for a period of up to 2 weeks when stored at
4 °C, at which point aggregation and was evident and a reduction in
activity was observed.
Synchrotron SAXS experiments on solutions were performed using a
BioSAXS robots at beamline BM29 (ESRF, France). Solutions were
loaded into the 96 well plate of an EMBL BioSAXS robot, and then in-
jected via an automated sample exchanger into a quartz capillary
(1.8 mm internal diameter) in the X-ray beam. The quartz capillary was
enclosed in a vacuum chamber, in order to avoid parasitic scattering.
After the sample was injected in the capillary and reached the X-ray
beam, the flow was stopped during the SAXS data acquisition. BM29
operated with an X-ray wavelength λ = 1.03 Å (12 keV). The images
were captured using a PILATUS 1M detector, while data processing was
performed using dedicated beamline software ISPYB (BM29).
2.7. In vitro immunostimulatory activity with RAW264.7 macrophage cell
line
To clarify the final concentrations of immuno-modulatory compo-
nents in each preparation and the calculations involved to determine
the concentration of compounds used in vitro are described in more
detail as follows. To produce MLVs with 20 w%/v% of TLR7a-DOPE
conjugate: 125 μL of DDA (10 mg/mL) was transferred to a round
bottom flask with the addition of 25 μL of DOPE-TLR7a conjugate
(10 mg/mL). Following the above procedure, the lipid cake was then
resuspended in 1 mL of Tris-buffer to give a final following concentra-
tion of compounds: DDA = 1.25 mg/mL or 1.98 mM, DOPE-
TLR7a = 0.25 mg/mL or 230 μM, TDB = 0.25 mg/mL or 250 μM. MLV
were then diluted into complete culture medium to the desired con-
centration for in vitro stimulation assays. When both TLR7a-DOPE
conjugate and TDB were present in MLVs, the concentration of DOPE-
TLR7a was used to determine dosing during in vitro stimulations assays.
Raw 264.7 macrophages were subcultured according to ATCC
methods: Complete media contained RPMI 1640 Glutamax® (10% FBS,
2‑Mercaptoetanol 0.05 mM). Cells were cultured at 37 °C 5% CO2 in T-
75 flasks until approximately 80% confluency. Cells were then detached
using a solution of EDTA in PBS (1 mM, 10 mL) per T-75. Cell suspen-
sions were then centrifuged at 200 ×g for 5 min. The supernatant was
removed then the pellet re-suspended in complete media (5 mL). Cells
were then re-suspended in fresh culture media at 2 × 105/mL.
Stimulation assays were performed in tissue culture grade 96 well
plates. Test compounds were diluted serially 1:2 (100 μL final volume)
in complete media (RPMI 1640, 10% FBS, 0.05 mM 2-ME) then
2 × 106 cells/mL freshly passaged cells were added in 100 μL to give a
final volume of 200 μL. Plates were incubated for 24 h before the ana-
lysis of supernatants for pro-inflammatory cytokines. IL-12p40 con-
centrations in supernatants of RAW 264.7 stimulation assays were de-
termined by sandwich ELISA. Briefly, Nunc Maxisorb 96 well ELISA
plates were coated overnight with IL-12p40 capture antibody (C15.6) at
2 μg/mL in carbonate buffer (pH 9.6, 50 μL per well), followed by 2 h
with 200 μL per well block buffer (PBS, 2.5 v/v% FBS, 0.02 w/v%
NaN3). 100 μL per well biotinylated detection antibody (C17.8) at 1 μg/
mL in block buffer was added. Stimulated RAW 264.7 supernatants
were diluted 1:7.5 in block buffer, and compared to 1:2 dilution of
recombinant IL-12p40 standards with a top concentration of 10 ng/mL
in block buffer, with 150 μL sample per well. ExtrAvidin alkaline
phosphatase conjugate was prepared 3:10,000 dilution in PBS.
2.5. Particle size analysis using Nanosight Tracking Analysis (NTA) and
Dynamic Light Scattering (DLS)
Samples were prepared in 1 mL volume using milliQ ultrapure water
for NTA analysis, filtered through a 0.45 μm syringe filter immediately
prior to use. NTA was performed on a LM10 system with LM14 top plate
and 533 nm laser (Malvern, UK). Flow rate was determined by software
and set to 30 AU. Videos were recorded 5 time for each sample, 60 s
each, samples were repeated 3 times at a controlled temperature of
25 °C. A 200 nm particle standard was used as a calibration run diluted
1:1000 in miliQ water. For samples analysed in complete media, de-
tection threshold was increased to 6 to reduce autofluorescece noise. All
analysis was performed using NTA software v3.1 (Malvern, UK).
Liposomes were characterized using DLS measured used a Zetasizer
Nano-ZS (Malvern, UK). Samples were diluted into ultrapure water to
appropriate concentrations in low volume plastic cuvettes (Fisher
Scientific). A dispersant refractive index of 1.33 and material refractive
index of 1.45 was used for all samples. Individual measurements were
carried out for 10 s per run, with 12 runs per reading, repeated in tri-
plicate. This was repeated for three separate samples at 25 °C. ξ-
Potential values were also measured; using DTS-1061 folded capillary
tube cuvettes (Malvern, UK). Samples were diluted 1:100 in ultrapure
water to the same concentration used in sizing experiments. Samples
were measured using 20 sub-runs per reading, repeating 3 times for
each sample. Each sample was measured three times and the results
were processed using the Smoluchowski model (Fκa = 1.50).
3. Results
3.1. In vitro immunostimulatory activity of lipid conjugate nanoparticles
The TLR7 agonist UC-1V150 was conjugated to the phospholipid
DOPE as previously described and noted for their remarkable potency
compared to the unconjugated TLR7 agonist (Chan et al., 2009). We
discovered that pre-dissolving of the DOPE-TLR7a agonist conjugate in
DMSO was required to detect any activity with in vitro stimulation
(Table 2 and Fig. 2). We hypothesised that DOPE-TLR7a would be likely
to form micelles or nanoparticles in aqueous suspension following di-
lution from DMSO solution. To investigate the potency of alternative
formulations of the lipid-conjugated TLR7 agonist we added the con-
jugate to DOPE:DDA liposomes and found that this likewise increased
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EuropeanJournalofPharmaceuticalSciences123(2018)268–276
Table 3
Z-Average, PDI and Zeta potentials of MLVs determined by DLS.
Table 2
Immunomodulatory properties of TLR7 agonists, conjugates and liposomes.
Sample
EC50 nM
MEC nM
74
LIPOSOME composition
Z-average size (nm)
PDI
Zeta potential (mV)
Conjugates
Unconjugated TLR7 Agonist
(8)
Unmodified TLR7 Agonist
(9)
DOPE-TLR7a + DMSO
DOPE-TLR7a − DMSO
483
620
9
25
19
0% DOPE-TLR7a − TDB
0% DOPE-TLR7a + TDB
1% DOPE-TLR7a − TDB
20% DOPE-TLR7a − TDB
20% DOPE-TLR7a + TDB
636
679
424
526
616
0.45
0.18
0.80
0.34
0.37
59
76
67
65
60
9
20
12
11
20
190
1
< 1
No activity
No activity
(> 2000 nM)
(> 2000 nM)
Liposomes
MLV 1% DOPE-TLR7a
MLV 20% DOPE-TLR7a
MLV 20%
DOPE-TLR7a + TDB
MLV + TDB
45
34
33
23
20
19
6
< 2
< 2
with the Minkle/Syk/CARD9 activating TDB. This was in line with
previous studies where TDB was shown not to synergise with TLR3 and
TLR9 agonists for vaccine potency in MLVs (Milicic et al., 2012).
223
173
< 24
MLV no immunostimulatory
agent
> 200,000
> 200,000
3.2. High potency of lipid conjugated TLR7 agonist depends on nanoparticle
formation or liposomal formulation
The poor water solubility of the lipid conjugated TLR7 agonist
prevents direct addition of this compound to in vitro im-
munostimulatory assays, and it was therefore diluted in DMSO prior to
dilution into culture. We hypothesised that DOPE-TLR7a would be
likely to form micelles or nanoparticles in aqueous suspension following
dissolution in DMSO and dilution into water or culture medium. We
therefore used nanosight particle tracking analysis (NTA) to determine
the physical form of this preparation, and found nanoparticles with a
DOPE-TLR7 agonist conjugate (8)
TLR7 Agonist (7)
70
60
50
40
30
20
10
DOPE+TLR7 Agonist
(7) mixed (1:1)
DOPE
mean diameter of 93
1 nm when diluted into ultrapure water.
Complete cell culture medium showed background particulate matter
as expected given the high protein content and heterogenous compo-
sition of heat inactivated foetal bovine serum. A background fluores-
cence was also observed from phenol red in the culture medium, which
increased noise and reduced sensitivity with control 100 nm latex
particles. The increased viscosity of complete media over H2O was
taken into account by software correction viscosity values at 25 °C for
water = 0.89 cP and complete media = 0.96 cP. However, when di-
luted from DMSO into culture medium an increase in size of the DOPE-
TLR7 agonist conjugate was observed comparing to ultrapure water.
0
0.1
1
10
100
1000
10000
TLR7 Agonist or DOPE Concentration (nM)
TDB+DDA
50
45
40
35
30
25
20
15
10
5
1% TLR7 agonist (8) - TDB
20% TLR7 agonist (8) +TDB
20% TLR7 agonist (8) -TDB
TLR7 Agonist (7)
The increase from 93
1 nm in water compared to 155
5 nm in
culture medium could be attributed to agglomeration of particles, or to
incorporation of media components into micelles/nanoparticles. The
reduction of particle frequency in DOPE-TLR7a conjugates - not mir-
rored by the 100 nm latex standard - also supported the possibility of
agglomeration (Table 4).
DDA+DOPE
0
0.1
1
10
100
1000
10000
3.3. Structure analysis
TLR7 agonist, TDB or DOPE Concentration (nM)
To determine the physical form of the lipid conjugate in micellar/
nanoparticulate form, 0.15, 0.4 and 1 wt% DOPE-TLR7a was prepared
by dissolution/dilution and analysed by SAXS (Fig. 4). The SAXS scat-
tering shows two distinct peaks, at spacings d = 2π /qo = 68.7 and
33.9 Å (qo = position of the maxima in the scattering curve) and at a
positional ratio 1:2. These peaks suggest that DOPE-TLR7a is arranged
in a lamellar order with a layer spacing of 68.7 Å. The SAXS data for
1 wt% DOPE-TLR7a was modelled according to the form factor for a
bilayer structure, comprising three Gaussian functions to represent the
electron density variation across the two headgroups and the dense
lipid core (Pabst et al., 2000). The interaction between the bilayers was
Fig. 2. IL-12p40 concentration in supernantants of RAW 264.7 macrophages
cultures stimulated with Unconjugate TLR7 agonist, Unconjugated DOPE, TLR7
agonist mixed with DOPE 1:1 and DOPE-TLR7a conjugate (A). IL-12p40 con-
centration in supernatants of RAW 264.7 macrophages cultures after stimula-
tion with MLV containing DDA + TDB, DDA + DOPE, DDA + 1% (w/v) DOPE-
TLR7a conjugate, DDA + 20% (w/v) DOPE-TLR7a conjugate, DDA + TDB 20%
(w/v) + 20% (w/v)DOPE-TLR7a conjugate and TLR7 agonist (B). n = 3, error
bars show standard deviation.
potency by > 10-fold compared to unconjugated TLR7 agonist (Fig. 2B
and Tables 2 and 3).
Liposomes were analysed by DLS and found to have similar char-
acteristics to DDA:TDB MLVs. The incorporation of TLR7-agonist con-
jugated DOPE had little effect on size distribution (Table 3). Like
DMSO-dissolved/diluted nanoparticles, liposomes containing TLR7
agonist showed potent in vitro immunostimulatory activity with an
EC50 of around 9 nM (Table 2, Fig. 2B). Interestingly, when we changed
the liposomal composition by reducing the TLR7 content, a significant
reduction in activity was observed and found that there was no additive
or synergistic increase in activity when TLR7 agonist was combined
Table 4
Particle frequency of DOPE-TLR7a conjugate or 100 nm latex standards in
complete media or milliQ water determined by NTA.
Sample
Complete media counts per mL
H2O counts per mL
DOPE-TLR7a
100 nm latex
Blank
3.6 × 108
1.15 × 109
1 × 106
8 × 108
1.2 × 109
5 × 105
272

A.J.R. Gadd et al.
EuropeanJournalofPharmaceuticalSciences123(2018)268–276
Table 5
SAXS parameters extracted from the fittings of the experimental data shown in Fig. 4b.
2zH [Å]
Δ2zH [Å]
ρH [rel. units]
9.3e−4
σH [Å]
ρC [rel. units]
1.2e−4
σC [Å]
N
do [Å]
η
Ndiff
66
25.9
5.7
5.7
3.2
67
2.2
3.5
Key: Gaussian bilayer form factor: Gaussian half-width at half-maximum for polydispersity Δ2zH, inter-head group thicknesses 2zH, Gaussian half-width for outer layer
surface σH, electron density for headgroup ρH, Gaussian half-width for inner layer σC, relative electron density for inner layer ρC. Caillé structure factor: diffuse
background Ndiff; total number of layers N; layer thickness do; Caillé parameter η.
DOPE-TLR7 (8) + DMSO
DOPE-TLR7 (8) + DMSO
70
DOPE-TLR7 (8) - DMSO
1.E+09
DOPE-TLR7 (8) - DMSO
A
B
60
50
40
30
20
10
0
1.E+08
1.E+07
1.E+06
1.E+05
1
10
100
1000
1
10
100
Concentration (nM)
Concentration (nM)
DOPE-TLR7 agonist conjugate
100 nm Standard
C
5
10
8
4
3
Diluted from DMSO into H2O
H2O
6
4
2
0
2
1
0
0
50
100 150 200 250 300
0
50
100 150 200 250 300
Particle size (nm)
Particle size (nm)
5
10
8
4
3
Diluted from DMSO
into complete Media
Complete Media
6
4
2
1
0
2
0
0
50 100 150 200 250 300
0
50
100 150 200 250 300
Particle size (nm)
Particle size (nm)
Fig. 3. IL-12p40 concentration in supernatants of RAW 264.7 macrophages cultures after stimulation with DOPE-TLR7 agonist conjugate with or without pre-
dissolving in DMSO (A). Particle frequency determined by NTA tracking analysis of DOPE-TLR7 agonist conjugate with or without pre-dissolving in DMSO (B). The
effect of solvent on particle size of DOPE-TLR7 agonist conjugates pre-dissolved in DMSO, (top) H2O (bottom) complete media. In addition 100 nm latex standards
were also run in the same solvent systems (C). n = 3–5 error bars show standard deviation.
with the lamellar parameter 68.7 Å measured from the SAXS curve for
0.14–1 wt% DOPE-TLR7a (Fig. 4a). In contrast, SAXS experiments
measuring the structure of pure DOPE lamellar provide a layer spacing
of 74.6 Å in water (Koltover, 1998).
described by a modified Caillé structure factor (Caillé, 1972). The
model and its application to lipid-based bilayer structures have been
described in detail in our previous papers (Castelletto et al., 2013,
2012). The fitting was implemented using the software SASfit (Breßler
et al., 2015). Fig. 4b shows the fitting of the SAXS data for 1 wt% DOPE-
TLR7a using this model. The fitted parameters are listed in Table 5, and
the organization of DOPE-TLR7a, according to data in Fig. 4 and the
parameters in Table 5 is illustrated in Fig. 5.
4. Discussion
Conjugation of TLR7a to the phospholipid DOPE has previously
been shown to promote strong pro-inflammatory response in vitro, with
a similar increase in potency to other TLR7/8 agonist conjugates to
macromolecules such as polymers and peptides. However the inad-
vertent self-assembly of this compound by dissolving in DMSO prior to
The fitting of the SAXS data shows that DOPE-TLR7a molecules are
organized in bilayers with a repeat distance of 77
25 Å, such that the
parameters listed in Table 5 are correlated in groups over ~3 bilayers.
The repeat distance provided by the SAXS fitting is in good agreement
273

A.J.R. Gadd et al.
EuropeanJournalofPharmaceuticalSciences123(2018)268–276
into liposomes (Table 2 and Fig. 3A). Separately dissolving the TLR7a
agonist plus DOPE in DMSO before mixing and diluting in aqueous
media showed no increase in the in vitro pro-inflammatory response,
demonstrating incorporation via covalent coupling to lipid group is
essential to influence potency (Fig. 2A). Similar activity was observed
between TLR7 agonists without conjugation to the lipid group either
with a functional group for conjugation, such as a carboxylic acid (7),
and without functional group at the para-benzyl position (9), sug-
gesting the alteration of activity is due to lipid conjugation not simply
variation in groups on this para-benzyl position (Table 2). We were able
to diffract a crystal of compound 9 to confirm for the first time the solid
state structure of this important immunostimulatory compound (Sup-
plementary information).
Either using DMSO to dissolve the DOPE-TLR7a conjugate prior to
dispersion by dilution in aqueous solvents, or using well-established
lipid cake hydration method of forming MLVs showed a clear and large
increase in the number of particles formed and also a strong pro-in-
flammatory response in vitro (Fig. 3). Complete culture media typically
contains 10% FCS in addition to a large amount of amino acids, mi-
nerals and carbohydrates. Changes in ionic strength, counterions, pro-
tein concentration and pH can influence the formation, stability and
aggregation of particles (Maeda et al., 1997; Palladino and Ragone,
2011). Changing the solvent from water to complete media influenced
the average particle size and frequency of DOPE-TLR7a particles de-
tected by light scattering (Fig. 3c and Table 4). The increase in particle
size and reduction in particle frequency could be explained by the ag-
glomeration of DOPE-TLR7a particles. Control 100 nm latex particles
analysed in complete media displayed a broadening of particle size, the
mean increasing from 100 nm to 120 nm but particle frequency re-
mained constant (Table 4), suggesting that complete media may in-
teract directly with DOPE-TLR7a affecting particle size and frequency.
TDB is not only an immunostimulatory glycolipid, but has also been
reported to have a stabilising effect on cationic MLVs constructed from
DDA and DOPE (Christensen et al., 2007). Using DLS to analyse MLV
size distribution (Table 3) indicated that the inclusion of TDB did re-
duce the PDI of MLV formed from DDA, DOPE plus DOPE-TLR7a. The
resulting MLV all showed a strong pro-inflammatory induction in vitro.
Previous reports suggested that the immunostimulatory activity of TDB
( a )
0.15 ,
0.4 and
1 wt% DOPE-TLR7a
103
102
101
1
1: 68.7 Å
2: 33.9 Å
2
( b )
1 wt% DOPE-TLR7a
SAXS, fitting
103
102
101
0.01
0.1
q / Å-1
Fig. 4. (a) SAXS data measured for samples containing 0.15, 0.4 and 1 wt%
DOPE TLR7a. (b) SAXS data for 1 wt% DOPE-TLR7a fitted according to the form
factor for a lipid bilayer with a Gaussian electronic density profile, as described
in the text and fitted parameters in Table 5.
addition to culture, to overcome the poor aqueous solubility has not
previously been investigated. Likewise, the potency of liposomes in-
corporating DOPE-TLR7a conjugates has not been reported. We show
that attempts to directly disperse DOPE-TLR7a directly into aqueous
solution shows no sign of particulates when analysed by light scattering
techniques (Fig. 3B) and likewise no in vitro pro-inflammatory activity
can be detected without dilution from DMSO solution or incorporation
O
O
O
O
O
HO
O
O
O
OH
P
O
P
O
O
O
O
O
O
O
O
O
NH
N
HN
N
N
H
N
H
NH2
H2
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
HO
O
O
O
OH
P
O
P
O
O
O
O
O
O
O
NH
N
HN
N
N
H
N
H
NH2
H2N
N
N
N
N
O
O
O
O
O
O
O
O
O
HO
O
O
O
OH
P
O
P
O
O
O
O
O
O
O
O
O
NH
N
HN
N
N
N
NH2
H2
N
H
H
N
N
N
N
O
O
O
O
O
O
O
O
O
HO
O
O
O
OH
P
O
P
O
O
O
O
O
O
O
O
O
NH
N
HN
N
N
N
NH2
H2
N
H
H
N
N
N
N
O
O
O
O
(77 25)A
Fig. 5. Lamellar order of DOPE-TLR7a molecules and repeat distance of the bilayer, according to the fitting of the SAXS data measured for 1 wt% DOPE-TLR7a
(Fig. 4, Table 5).
274

A.J.R. Gadd et al.
EuropeanJournalofPharmaceuticalSciences123(2018)268–276
does not synergise with TLR 3 and 9 agonists,(Milicic et al., 2012) and
we similarly found no evidence of synergistic effect of combining
DOPE-TLR7a and TDB within liposomes in this simple in vitro system.
Adding TDB to MLV containing 20% DOPE-TLR7a showed no increased
pro-inflammatory activity (Fig. 3). Further study is now required to
evaluate the impact on formulation on different cell types expressing
TLR7, and molecular methods such as evaluation in knockout cells, or
reporter cell lines exclusively expressing a panel of TLRs and other PRR,
is now justified to understand if conjugation also affects receptor spe-
cificity, as well as potency, of these compounds. Initial experiments
using a GFP inducible Nf-κB cell line lacking TLR7 known as GTPT3
(Bloor et al., 2008) showed no induced GFP expression over back-
ground when stimulated with unconjugated TLR7 agonist, DOPE-TLR7a
conjugate or liposomes containing DOPE-TLR7a conjugate, but did
show a dose dependant response to LPS (data not shown). This pre-
liminary test suggests that DOPE-TLR7a conjugate in micellar or lipo-
somal formulation remains specific for TLR7.
produced in the synthesis of TLR7 agonist 7 and DOPE-TLR7a conjugate
8; 1H (400 MHz), 13C (100 MHz) NMR spectra, electrospray ionisation
(ESI) mass spectra of compounds 1–7, 9; 1H (700 MHz) 13C (175 MHz)
NMR spectrum, ESI mass spectrum of DOPE-TLR7a conjugate 8; single
crystal X-ray structure of unmodified TLR7 agonist 9; FTIR spectra of
compounds 6, 7, 8 and unconjugated DOPE. Supplementary data to this
article can be found online at https://doi.org/10.1016/j.ejps.2018.07.
048.
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Author information
No competing financial interests have been declared.
Acknowledgements
AG was supported by BBSRC doctoral training grant BB/F017189/1.
VC was supported by EPSRC grant EP/L020599/1 to IWH. We are also
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(Beamtime reference MX-1769) and Gabriele Giachin for assistance.
The University of Reading's Chemical Analysis Facility is acknowledged
for access to MS and NMR instrumentation and the Oxford Diffraction
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