Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer–Lipid Amphiphile Conjugates of Small-Molecule TLR7/8 Agonists

Article abstract of DOI:10.1002/anie.201905687

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid–polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid–polymer amphiphiles for optimized lymphoid targeting.

Full text of DOI:10.1002/anie.201905687

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Potent lymphatic translocation and spatial control over innate
immune activation by polymer-lipid amphiphile conjugates of
small molecule TLR7/8 agonists
Authors: Jana De Vrieze, Benoit Louage, Kim De Swarte, Zifu Zhong,
Ruben De Coen, Simon Van Herck, Lutz Nuhn, Camilla Kaas
Frich, Alexander Zelikin, Stefan Lienenklaus, Niek Sanders,
Sunil David, Bart Lambrecht, and Bruno de Geest
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201905687
Angew. Chem. 10.1002/ange.201905687
Link to VoR: http://dx.doi.org/10.1002/anie.201905687
http://dx.doi.org/10.1002/ange.201905687

10.1002/anie.201905687
Angewandte Chemie International Edition
COMMUNICATION
Potent lymphatic translocation and spatial control over innate
immune activation by polymer-lipid amphiphile conjugates of
small molecule TLR7/8 agonists
Jana De Vrieze,[a] Benoit Louage,[a] Kim De Swarte,[b] Zifu Zhong,[c] Ruben De Coen,[a] Simon Van
Herck,[a] Lutz Nuhn,[d] Camilla Kaas Frich,[e] Alexander N. Zelikin,[e] Stefan Lienenklaus,[f] Niek N.
Sanders,[c] Bart N. Lambrecht,[b] Sunil A. David,[g] Bruno G. De Geest*[a]
Abstract: Uncontrolled systemic inflammatory immune triggering has
hampered clinical translation of several classes of small molecule
immune-modulators such as imidazoquinoline TLR7/8 agonists for
vaccine design and cancer immunotherapy. Taking advantage of the
inherent serum protein-binding property of lipid motifs and their
tendency to accumulate in lymphoid tissue we design amphiphilic
lipid-polymer conjugates that confer suppression of systemic
inflammation but provoke potent lymph node immune activation. In
Scheme 1| Concept of lymph node targeting lipid-polymer amphiphile
conjugates. A hydrophilic polymer backbone provides good water-solubility of
our work we provide a rational base for the design of lipid-polymer
amphiphiles for optimized lymphoid targeting.
the amphiphile construct. The lipid moiety has high affinity for albumin which,
following local administration, mediates transportation via the interstitial flow to
lymphoid tissue. Small molecule immunostimulatory drugs (i.e. TLR7/8a) are
linked to the polymer backbone and upon arrival in lymphoid tissue provoke
potent innate immune activation.
Spatial control over the activity of small molecule immune-
stimulatory compounds is key in the successful design of potent
vaccines and cancer immune-therapeutics.[1–3] For example, Toll
like receptors (TLRs) 7 and 8, localized on the endosomal
membrane of innate immune cells, recognize viral RNA as natural
ligand and triggering causes robust type I interferon responses
that are strong mediators of anti-viral and anti-tumoral immune
responses.[4,5] Potent small molecule agonists of TLR7/8
(TLR7/8a) have been discovered, but like many other small
molecule drugs, are prone to rapid distribution throughout the
body, thereby losing site-specific activity and causing systemic
inflammation. [6–8]
The aim of this work was to devise a simple strategy that
offers spatial control of the immune-stimulatory activity of small
molecule TLR7/8a. Previously, we and others have demonstrated
that nanoparticle-conjugation is a potent method to restrict TLR
agonistic activity,[9–12] upon local subcutaneous administration, to
the site of administration and to draining lymphatic tissue where
TLR7/8 triggering provokes robust activation of innate immune
cell subsets. These papers emphasize the key contribution of the
colloidal nature of the resulting conjugate to achieve lymph node
focused immune-stimulation.[13] Indeed, fully hydrophilic TLR7/8a
conjugates that exist in unimeric single chain form in solution,
exhibit a dramatic reduction in lymphatic translocation. Systems
based on self-assembly of random co-polymers, block
copolymers or crosslinked nanogels have an inherent lack of
control over macromolecular and/or supramolecular structure.
Moreover, non-degradable by-products are prone to long-term
accumulation, which limit the potential for clinical translation.
These considerations foster new efforts towards the design of
more simple TLR7/8a carrier systems that are equally or more
potent in lymphatic translocation and innate immune activation.
Lipid-mediated non-covalent albumin binding has proven a
powerful strategy for lymphoid delivery of imaging agents[15] and
polymer-drug conjugates[17] and is also known to prologue drug
[16]
circulation in the blood stream
upon systemic administration.
[a]
Jana De Vrieze, Dr. Benoit Louage, Dr; Ruben De Coen, Dr. Simon
Van Herck, Prof.Dr. Bruno G. De Geest. Department of
Pharmaceutics, Ghent University, Ghent, Belgium
E-mail: br.degeest@ugent.be
Kim De Swarte, Prof.Dr. Bart N. Lambrecht. IRC VIB-UGent,
Zwijnaarde, Belgium
Zifu Zhong, Prof.Dr. Niek N. Sanders. Department of Nutrition,
Genetics and Ethology, Ghent University, Merelbeke, Belgium.
Dr. Lutz Nuhn. Max Planck Institute for Polymer Research, Mainz,
Germany
Dr. Camilla Kaas Frich, Prof.Dr. Alexander N. Zelikin. Department of
Chemistry, Aarhus University, Aarhus C, Denmark.
Dr. Stefan Lienenklaus. Institute for Laboratory Animal Science and
Institute of Immunology, Hanover Medical School, Hanover,
Germany
Recent work from the Irvine group reported that lipid amphiphiles
containing CpG as a macromolecular TLR9 agonist, show, upon
subcutaneous injection, robust lymphatic translocation by hitch
hiking onto albumin molecules in the interstitial flow from the
injection site to draining lymphoid tissue.[14] In addition,
[b]
[c]
[d]
[e]
[f]
cholesterol-conjugates have also been reported to bind to other
[18]
serum proteins, such as lipoproteins, too.
Translation of this
approach to small molecule TLR7/8a (i.e. design of lipid-TLR7/8a
conjugates) has proven successful to reduce unwanted systemic
immune-stimulation but fails to provoke immune-stimulation in
draining lymphoid tissue, due to inherent solubility issues.[19,20]
Here, we circumvent these issues by designing lipid-polymer
amphiphiles consisting of a lipid motif as hydrophobic part and a
hydrophilic polymer connected to the lipid moiety at one of its
chain ends. Onto the polymer backbone multiple TLR7/8a motifs
[g]
Prof.Dr. Sunil A. David. Department of Medicinal Chemistry,
University of Minnesota, Minneapolis, MN, USA.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201905687
Angewandte Chemie International Edition
COMMUNICATION
(1)
(A)
O
HO
O
S
S
S
S
(2)
+
OH
O
DIC/DMAP
S
S
(B)
rhodamine-cadaverine
H2
N
(1)
O
NC
IMDQ
N+
O
NC
OH
HO
O
OH
H2N
TEA
AIBN
70 °C
OH
O
F
F
O
ACVA
O
HO
O
O
TEA
N
N
O
F
O
F
O
NH
O
NH
F
F
H2
N
H
N
F
F
NH2
H2
N
N
N
F
OH
F
H2
N
(2)
O
O
NC
NC
OH
HO
O
H2
N
TEA
O
O
AIBN
70 °C
O
F
F
ACVA
O
TEA
O
F
O
F
O
NH
O
NH
F
F
F
F
F
OH
F
Scheme 2| Synthesis of lipid-polymer amphiphile conjugates. (A) A cholesterol-functionalized chain transfer agent (CTA) for RAFT polymerization is
synthesized by esterification. (B) RAFT polymerization of pentafluorophenyl acrylate yields a reactive ester backbone that is by post-polymerization modification
substituted with an amine-containing dye or with the imidazoquinoline TLR7/8 agonist IMDQ followed by reacting remaining PFP-esters with 2-aminoethanol to
generate a hydrophilic polymer backbone.
Table 1| Characterization of cholesterol-polyPFPA and polyPFPA.
Subsequently, these polymers were tested for their interaction
with cells in vitro, thereby probing for the influence of the
Theor
SEC
CTA
M/CTA/
AIBN
conv.
(%)
DP
Mn
Mn
D
cholesterol moiety. These experiments were performed on murine
RAW macrophages as model innate immune cells and conducted
at 4 °C to block endocytosis and at 37 °C to mimic normal
physiological conditions. Flow cytometry (Figure 1A) indicated a
clear dose-response binding of the cholesterol-polymer
amphiphiles at both temperatures (A1: 4 °C, A2: 37 °C), with
minimal binding of the non-amphiphile control polymer.
Interestingly, also at 4 °C where endocytosis is blocked,
significant cellular association of the cholesterol-polymer
amphiphiles was observed. To shed light on the underlying
interaction, confocal microscopy was performed (Figure 1B),
revealing that pulsing cells with cholesterol-polymer amphiphiles
at 4 °C results in the conjugates’ red fluorescence being
exclusively located at the cell membrane. These findings suggest
that cholesterol-polymer conjugates anchor to the phospholipid
cell membrane through hydrophobic interaction. These findings
are in line with previous reports on the ability of cholesterol
analogues to drive cell surface anchoring of macromolecular
species.[24–26] When cells are pulsed at 37 °C, confocal
microscopy depicts a punctuated pattern, indicating that the
cholesterol-polymer amphiphiles become endocytosed into
vesicular compartments (i.e. endosomes and lysosomes).
Interestingly, control polymers that lack the cholesterol moiety do
not show any interaction with the cell membrane and show very
low cell uptake at physiological temperature, at least within the
tested experimental window. To further explore the influence of
amphiphile design on cell membrane anchoring ability, we
synthesized two other cholesterol-polymer amphiphiles with a DP
of respectively 30 and 60. Flow cytometry analysis (Figure S14)
indicated that polymers with a shorter chain length did less
efficiently anchor to cell membranes at 4 °C. Hence cholesterol-
polymer amphiphiles with a DP of 100 were selected for further
evaluation.
(kDa)
(kDa)
cholesterol-
PABTC
120/1/0.2
120/1/0.2
90
89
108
107
26.3
12.9
1.22
1.15
PABTC
25.7
13.5
are conjugated. This concept is schematically shown in Scheme
1. In first instance, we investigated whether amphiphilic lipid-
polymer conjugates allow for
translocation compared to a non-amphiphilic hydrophilic polymer.
Hereto, we synthesized cholesterol-functionalized chain
a more efficient lymphatic
a
transfer agent (CTA) for reversible addition-fragmentation chain
transfer (RAFT) polymerization[21] by esterification of 2-propanoic
acid butyl trithiocarbonate (PABTC) and cholesterol according to
Scheme 2. The cholesterol-CTA was used together with
unmodified PABTC (as a control) for RAFT polymerization of
pentafluorophenyl acrylate (PFPA)[22] (degree of polymerization
(DP) of 100) to generate a reactive ester polymer backbone that
can later on be used for conjugation of TLR7/8 agonist molecules,
as well as for fluorescent labelling (Scheme 2). Polymer analysis
by NMR spectroscopy and size exclusion chromatography (SEC)
is summarized in Table 1. Overall, the measured dispersity was
relatively low, indicating good control over polymerization. The
trithiocarbonate RAFT end group was removed by treating the
polymer with an excess of 4,4'-azobis(4-cyanovaleric acid)
(ACVA) to avoid later on issues on disulfide formation.[23] In a next
step, the polymers were fluorescently labelled by nucleophilic
substitution with rhodamine cadaverine targeting a degree of
substitution (DS: number of modified repeating units per 100) of
1. Finally, a hydrophilic polymer backbone was generated by
reacting remaining PFP-ester motifs with an excess of 2-
aminoethanol (Scheme 2).
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10.1002/anie.201905687
Angewandte Chemie International Edition
COMMUNICATION
1000
800
600
400
200
0
cholesterol-polymer
cholesterol-polymer
amphiphile
non-amphiphile
polymer
****
A1
B
C1
100
80
60
40
20
0
polymer
*
4 °C
4 °C
**
cholesterol-polymer amphiphile
hydrophillic non-amphiphile polymer
0.002
0.01
0.02
1
10
100
concentration [µg/mL]
concentration [mg/mL]
****
25000
20000
15000
10000
5000
0
cholesterol-polymer
A2
cholesterol-polymer
amphiphile
hydrophillic non-amphiphile
polymer
C2
37 °C
100
80
60
40
20
0
37 °C
polymer
****
Triton X-100
****
0.002
0.01
0.02
0,02
0,1
0,2
concentration [mg/mL]
concentration [mg/ml]
Figure 1| In vitro cell interaction of lipid-polymer amphiphile conjugates. (A) Flow cytometry analysis of murine RAW macrophages pulsed at 4°C (A1) and
37 °C (A2) with amphiphile conjugates and control. t-test: *:p<0,1; **:p<0,01; ****:p<0,0001 (n=3, mean + SD). (B) Corresponding confocal microscopy images
depicting an overlay of the red fluorescence channel (note that conjugates were labeled with rhodamine) and the transmitted light channel. Scale bar represents 15
micron. Cytotoxic (C1) (based on MTT assay, n=3) and hemolytic activity (n=3) (C2) of amphiphile conjugates and controls.
100
80
60
40
20
0
***
***
A
B
800
600
400
200
0
Importantly, cytotoxicity measured by MTT assay (Figure 1C1)
and membrane-destabilizing activity measured by chicken red
blood cells (RBC) lysis assay (Figure 1C2), showed no significant
detrimental effect for both cholesterol-polymer amphiphiles and
hydrophilic non-amphiphile polymers. Taken together, these
findings highlight the translational potential of the cholesterol-
polymer amphiphiles having much more benign properties than
an e.g. an ionic amphiphile surfactant such as Triton-X.
cholesterol-
polymer
cholesterol-
polymer
polymer
polymer
cholesterol-polymer
polymer
**
****
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
**
***
*
****
***
****
****
Subsequently, we tested lymphatic translocation of the lipid-
polymer conjugates. Hereto mice were injected subcutaneously
into the footpad, followed by dissection of the draining popliteal
lymph node (a schematic overview of the mouse lymphoid system
is shown in Figure S15) at 4 h, 24 h and 96 h post injection. FACS
analysis shows a dramatic effect of the presence of the
cholesterol motif on lymphatic translocation (Figure 2A).
Whereas non-amphiphilic polymers show relatively low lymphatic
translocation above baseline (cfr. Figure S16 for a corresponding
histogram), amphiphilicity promotes highly efficient lymphatic
translocation with very high percentages of dendritic cells (DCs),
B cells and macrophages in the draining lymph node being
positive for the cholesterol-polymer conjugates (Figure 2B).
These data are confirmed by confocal microscopy (Figure 2C)
which shows massive accumulation of polymer in the subcapsular
sinus and underlying B cell follicles as soon as 4 h post injection
and lasting for at least up to 4 days. Based on the observation that
amphiphilic cholesterol-polymer conjugates reach draining
lymphoid tissue that early post-injection [9,14,27] it is reasonable to
conclude that these compounds exhibit highly efficient passive
translocation to lymphoid tissue and do not require uptake by
immune cells at the site of injection for subsequent well-mediated
lymphatic transportation.
96
**
h
96
*
h
96
ns
h
4
h
48
*
h
48
*
h
4
h
4
h
48
*
h
**
**
6000
4000
2000
0
12500
10000
7500
5000
2500
0
20000
15000
10000
5000
0
**
96
h
4
h
96
h
96
h
4
h
48 h
48
h
48 h
4
h
4 h
96 h
48 h
C
4 h
96 h
48 h
Figure 2| In vivo behavior of lipid-polymer amphiphile conjugates. (A) Flow
cytometry analysis on total lymphocyte population in the draining popliteal
lymph node of mice, 24h post-injection in the footpad. t-test; ***:p<0,001 (n=3,
mean + SD). Dotted line in panel A depicts the baseline value of the untreated
control. (B) Flow cytometry analysis of conjugate uptake by immune cell subsets
at different times post-injection. t-test; ****:p<0,0001; ***:p<0,001; **:p<0,01;
*:p<0,1 (n=3, mean + SD). (C) Corresponding confocal microscopy images of
popliteal lymph nodes (tissue sections) at different time points post-injection in
the footpad. Scale bar represents 200 micron.
Lipid amphiphiles have been reported to exhibit affinity to albumin,
and lipoproteins, and hence promote lymphatic translocation to
lymphoid tissue by hitch hiking the interstitial albumin flow.[14,16,17]
To verify whether this hypothesis also holds true for our
cholesterol-polymer conjugate design we conducted a series of
experiments to investigate albumin binding. Biolayer
interferometry (BLI) using albumin-coated sensors showed a high
affinity of the cholesterol-polymer amphiphiles whereas low
affinity was found for the non-amphiphile control (Figure 3A).
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10.1002/anie.201905687
Angewandte Chemie International Edition
COMMUNICATION
albumin
cholesterol-polymer
albumin 1:1
cholesterol-polymer
albumin 5:1
cholesterol-polymer
albumin 10:1
albumin
polymer
albumin1:1
polymer
albumin 5:1
polymer
B1
B2
Aqueous SEC on mixtures of albumin and polymer at different
ratio (Figure 3B) showed a pronounced decrease in retention
time with increasing ratio of cholesterol-polymer to albumin,
thereby indicating both species to interact with each other. By
contrast, hydrophilic non-amphiphilic polymers had no influence
on the retention time of albumin. Taken together, these data proof
that the cholesterol-polymer amphiphiles have very strong
albumin-binding properties, which supports the hypothesis that
albumin-binding is a potent mediator of lymphoid translocation
and is not only capable of translocating of relatively small
constructs such as amphiphile dyes, peptides and
oligonucleotides,[14] but also larger macromolecules such as those
used in this work.
A
cholesterol-polymer
polymer
albumin 10:1
3.6
3.4
3.2
3.0
1.0
0.5
0.0
1.0
0.5
0.0
2.8
500
750
1000
1250
1500
15
time [min]
20
15
20
time [min]
time [s]
Figure 3| Albumin binding assessment. (A) Bio-layer interferometry
sensorgrams of affinity towards albumin. Albumin-coated sensors were
immersed at time point 500s into cholesterol-polymer or polymer solutions to
measure adsorption and immersed into PBS solution at time point 1100s to
measure desorption. (B) SEC elugrams of albumin and mixture of albumin and
cholesterol-polymer (B1) or polymer (B2) at different ratios.
To further explore the role of the lipid motif on albumin binding
and lymphatic translocation, we synthesized polymers containing
a monoalkyl (i.e. steraryl) lipid and dialkyl (i.e. 1,2-distearoyl-sn-
glycero-3- phosphoethanolamine (DSPE)) lipid moiety at their
chain end respectively (Scheme S1 in Supporting Information). In
vitro cell uptake and in vivo lymphatic transportation experiments
indicated these amphiphiles to be sub-optimal relative to
cholesterol-based amphiphiles (Figure S17-18 in Supporting
Information). Albumin-binding studies showed that monoalkyl
amphiphiles did bind albumin, but to a lesser extent than
cholesterol amphiphiles. Confocal microscopy at 4 °C also did not
indicate cell membrane anchoring of the monoalkyl amphiphiles,
thereby highlighting the influence of cholesterol-mediated
membrane anchoring on increasing cellular uptake of the
amphiphile conjugates. Dialkyl amphiphiles did strongly bind to
albumin (Figure S19). However, due to the increased
hydrophobicity of the DSPE motif, relative to a monoalkyl or
cholesterol moiety, the resulting amphiphiles showed large
aggregates (Figure S19), which is likely responsible for reduced
cellular uptake and lymphatic translocation. These findings
highlight the subtle balance between albumin binding and
colloidal stability and its influence on tissue mobility upon
subcutaneous administration. Indeed, whereas we and others
have previously shown that colloidally stable nanoparticles with
sub 200 nm dimension and inert surface can exert high mobility
towards lymphoid tissue,[9,11,12,28] even for colloids with particularly
low albumin-binding properties,[13,28] more hydrophobic
compounds that are prone to aggregate formation. The latter
mainly form a depot at the site of injection,[12,20] which has been
associated with lower efficacy when used as delivery vehicle for
immunostimulatory drugs. Although we demonstrate for the
particular case in this paper that cholesterol-polymer amphiphiles
exhibit superior in vitro and in vivo performance, a window of
opportunity remains to further engineer a dialkyl amphiphile
system with improved aqueous solubility and likely also superior
lymphatic translocation capacity, especially in view of the findings
by the Irvine group regarding dialkyl-oligonucleotide
conjugates.[14]
upon extensive dialysis the final product did not contain any free
soluble IMDQ (Figure S20) and UV-vis spectrophotometry
analysis (Figure S21) was used to quantify the IMDQ load. We
also confirmed by BLI that IMDQ-conjugation did not impair the
albumin-binding ability of the conjugate (Figure S22).The ability
of these conjugates to trigger TLR activation was tested using the
RAW-Blue NF-kB activation in vitro reporter cell assay and
(Figure 4A) showed that although a significant loss in activity
occurred upon ligation of IMDQ onto the polymer backbone
(which is in line with previous findings by us and others),
cholesterol-polymer conjugates are still able to trigger innate
immune activation.
To assess the capacity of the conjugates to induce innate
immune activation in vivo, mice received
a subcutaneous
equivalent dose of IMDQ in the footpad, followed by dissection of
the draining popliteal lymph node (cfr. Figure S15 for a schematic
of the mouse lymphoid system) and flow cytometry analysis. The
latter demonstrated that both IMDQ in soluble form and when
conjugated to cholesterol-polymer are capable of inducing
activation of DCs (expressed by upregulation of the maturation
markers CD80 and CD86), with IMDQ conjugated to cholesterol-
polymer inducing a significantly higher extent of innate immune
activation (Figure 4B). Control cholesterol-polymer that did not
contain IMDQ, did not induce immune activation above the naive
control. To further assess innate immune activation with a spatio-
temporal resolution, we made use of an IFN-β-luciferase reporter
mouse model, with a firefly luciferase gene under the control of
the IFN-β promotor.[30] Type I interferons (IFN) are a major
cytokine family induced by TLR7/8 triggering and potent
promotors of anti-viral and anti-tumoral immune responses.[31]
Bioluminescence imaging of type I IFN induction allows for non-
invasive spatio-temporal evaluation of in vivo innate immune
activation. As shown in Figure 4C, subcutaneous injection of
IMDQ in soluble form in the footpad of mice prompts a rapid
systemic inflammatory response. By contrast, cholesterol-
polymer-IMDQ dramatically abrogates systemic inflammation, but
by contrast focuses immune activation to the site of administration
and the draining popliteal lymph node (marked by the black arrow
in panel C1 of Figure 4), thereby confirming the flow cytometry
data.
Having proven the ability of the cholesterol polymer system to
efficiently mediate cell uptake in vitro and in vivo, we used this
approach for the design of amphiphilic cholesterol-polymer
TLR7/8a conjugates. Hereto, the polyPFPA backbone was
substituted with the small molecule imidazoquinoline TLR7/8a
IMDQ,[7,29] according to Scheme 2. HPLC analysis showed that
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10.1002/anie.201905687
Angewandte Chemie International Edition
COMMUNICATION
cholesterol-polymer-IMDQ
****
****
0.8
A
B1
B2
IMDQ
80
60
40
20
0
ns
800
**
0.6
0.4
0.2
0.0
600
400
200
0
naive
cholesterol-polymer
IMDQ
cholesterol-polymer-IMDQ
0.0001 0.001 0.01
0.1
1
10
100
IMDQ concentration [µM]
cholesterol-polymer-
IMDQ
cholesterol-
polymer
C1
C2
IMDQ
cholesterol-polymer
IMDQ
cholesterol-polymer-IMDQ
*
15
10
5
0
Figure 4| In vivo innate immune activation by lipid-polymer amphiphile conjugates. (A) TLR agonistic activity measured as NF-kB activation by a RAW-Blue
reporter assay. (n =6, mean + SD). (B) FACS analysis of the draining popliteal lymph nodes post footpad injection (n=4, mean + SD). (B1) Analysis on DC count
and (B2) activated DCs (CD80+, CD86+ cells). One-way Anova; ****:p<0,0001; **:p<0,01; n=4, mean + SD. (C) Bioluminescence images (C1) and quantification of
local versus systemic response (C2) of luciferase reporter mice (IFNβ+/Δβ-luc), images taken 4h post footpad injection. One-way Anova; *:p<0,1; n=3, mean + SD.
[8]
[9]
A. Z. Dudek, C. Yunis, L. I. Harrison, S. Kumar, R. Hawkinson, S. Cooley,
J. P. Vasilakos, K. S. Gorski, J. S. Miller, Clin. Cancer Res. 2007, 13,
7119–7125.
In summary, we have reported on a simple yet efficient
amphiphile design to deliver potent immunostimulatory small
molecules to lymphoid tissue after subcutaneous administration.
The amphiphile approach strongly limits systemic toxicity and
J. J. Moon, H. Suh, A. Bershteyn, M. T. Stephan, H. Liu, B. Huang, M.
Sohail, S. Luo, S. Ho Um, H. Khant, et al., Nat Mater 2011, 10, 243–251.
could be applied to
a wide variety of small molecule
[10] M. Ballester, C. Nembrini, N. Dhar, A. de Titta, C. de Piano, M. Pasquier,
E. Simeoni, A. J. van der Vlies, J. D. McKinney, J. A. Hubbell, et al.,
Vaccine 2011, 29, 6959–6966.
immunostimulatory drugs. Further ongoing design optimization
focuses on positioning lipid and drug motifs at opposite polymer
chain end to further increase the translational potential of the
amphiphile approach.
[11] a) L. Nuhn, N. Vanparijs, A. De Beuckelaer, L. Lybaert, G. Verstraete, K.
Deswarte, S. Lienenklaus, N. M. Shukla, A. C. Salyer, B. N. Lambrecht,
et al., Proc. Natl. Acad. Sci. 2016, 113, 8098–8103; b) L. Nuhn, L. Van
Hoecke, K. Deswarte, B. Schepens, Y. Li, B. N. Lambrecht, S. De Koker,
S. David, X. Saelens, B. G. De Geest, Biomaterials 2018, 178, 643-651;
c) L. Nuhn, S. De Koker, S. Van Lint, Z. Zhong, J. P. Catani, F. Combes,
K. Deswarte, Y. Li, B. N. Lambrecht, S. Lienenklaus, N. N. Sanders, S.
A. David, J. Tavernier, B. G. De Geest, Adv. Mater. 2018, 30, e1803397.
[12] G. M. Lynn, R. Laga, P. A. Darrah, A. S. Ishizuka, A. J. Balaci, A. E.
Dulcey, M. Pechar, R. Pola, M. Y. Gerner, A. Yamamoto, et al., Nat.
Biotechnol. 2015, 33, 1201-1210.
Acknowledgements
BDG acknowledges the FWO Flanders, Kom Op Tegen Kanker
and Ghent University (BOF-GOA for funding). LN is grateful for
the support of the DFG (SFB 1066-B4 and Emmy Noether
Program) and the Fonds der Chemischen Industrie (Liebig
Program).
[13] S. Van Herck, K. Deswarte, L. Nuhn, Z. Zhong, J. P. Portela Catani, Y.
Li, N. N. Sanders, S. Lienenklaus, S. De Koker, B. N. Lambrecht, et al.,
J. Am. Chem. Soc. 2018, 140, 14300–14307.
[14] H. Liu, K. D. Moynihan, Y. Zheng, G. L. Szeto, A. V Li, B. Huang, D. S.
Van Egeren, C. Park, D. J. Irvine, Nature 2014, 507, 519–22.
[15] G. Liberale, A. Bohlok, A. Bormans, F. Bouazza, M. G. Galdon, I. El
Nakadi, P. Bourgeois, V. Donckier, Eur. J. Surg. Oncol. 2018, 44, 1301–
1306.
Keywords: lipid amphiphiles • innate immunity • immune-
modulators • lymph nodes • polymers
[1]
a) Q. Sun, M. Barz, B. G. De Geest, M. Diken, W. E. Hennink, F. Kiessling,
T. Lammers, Y. Shi, Chem. Soc. Rev. 2019, 48, 351–381; b) L. Lybaert,
K. Vermaelen, B. G. De Geest, L. Nuhn, J. Controlled Release 2018, 289,
125-124.
[16] C. K. Frich, F. Krüger, R. Walther, C. Domar, A. H. F. Andersen, A.
Tvilum, F. Dagnæs-Hansen, P. W. Denton, M. Tolstrup, S. R. Paludan,
et al., J. Control. Release 2019, 294, 298–310.
[2]
L. Milling, Y. Zhang, D. J. Irvine, Adv. Drug Deliv. Rev. 2017, 114, 79–
101.
[17] Y.-R. Zheng, K. Suntharalingam, T. C. Johnstone, H. Yoo, W. Lin, J. G.
Brooks, S. J. Lippard, J. Am. Chem. Soc. 2014, 136, 8790–8798.
[18] C. Wolfrum, S. Shi, K.N. Jayaprakash, M. Jayaraman, G. Wang, R.K.
Pandey, K.G. Rajeev, T. Nakayama, K. Charisse, E.M. Ndungo, et al.
Nat. Biotechnol. 2007, 25, 1149-1157.
[3]
[4]
[5]
J. Hubbell, S. N. Thomas, M. Swartz, Nature 2009, 462, 449–460.
A. Iwasaki, R. Medzhitov, Science 2010, 327, 291–5.
S. S. Diebold, T. Kaisho, H. Hemmi, S. Akira, C. Reis e Sousa, Science
2004, 303, 1529–31.
[19] M. Chan, T. Hayashi, C. S. Kuy, C. S. Gray, C. C. N. Wu, M. Corr, W.
Wrasidlo, H. B. Cottam, D. A. Carson, Bioconjug. Chem. 2009, 20, 1194-
1200.
[6]
[7]
B. J. Weigel, S. Cooley, T. DeFor, D. J. Weisdorf, A. Panoskaltsis-Mortari,
W. Chen, B. R. Blazar, J. S. Miller, Am. J. Hematol. 2012, 87, 953–956.
T. Y.-H. Wu, M. Singh, A. T. Miller, E. De Gregorio, F. Doro, U. D’Oro, D.
A. G. Skibinski, M. L. Mbow, S. Bufali, A. E. Herman, et al., Sci. Transl.
Med. 2014, 6, 263ra160.
[20] D. Smirnov, J. J. Schmidt, J. T. Capecchi, P. D. Wightman, Vaccine 2011,
29, 5434-5442.
This article is protected by copyright. All rights reserved.

10.1002/anie.201905687
Angewandte Chemie International Edition
COMMUNICATION
[21] C. Boyer, V. Bulmus, T. P. Davis, V. Ladmiral, J. Liu, S. Perrier, Chem.
Rev. 2009, 109, 5402–36.
[27] S.T. Reddy, A.J. van der Vlies, E. Simeoni, V. Angeli, G.J. Randolph,
C.P. O'Neil, L.K. Lee, M.A. Swartz, J.A. Hubbell, Nat. Biotechnol. 2007,
25, 1159-1164.
[22] A. Das, P. Theato, Chem. Rev. 2015, 116, 1434-1495.
[23] H. Willcock, R. K. O’Reilly, Polym. Chem. 2010, 1, 149–157.
[24] S. Boonyarattanakalin, S. E. Martin, S. A. Dykstra, B. R. Peterson, J. Am.
Chem. Soc. 2004, 126, 16379–86.
[28] S. De Koker, J. Cui, N. Vanparijs, L. Albertazzi, J. Grooten, F. Caruso, B.
G. De Geest, Angew. Chem. Int. Ed. Engl. 2016, 55, 1334-1339.
[29] N. M. Shukla, S. S. Malladi, C. a. Mutz, R. Balakrishna, S. a. David, J.
Med. Chem. 2010, 53, 4450–4465.
[25] E. C. Woods, N. A. Yee, J. Shen, C. R. Bertozzi, Angew. Chem. Int. Ed.
Engl. 2015, 54, 15782–8.
[30] S. Lienenklaus, M. Cornitescu, N. Zietara, M. Łyszkiewicz, N. Gekara, J.
Jabłónska, F. Edenhofer, K. Rajewsky, D. Bruder, M. Hafner, et al., J.
Immunol. 2009, 183, 3229–36.
[26] H. Liu, B. Kwong, D. J. Irvine, Angew. Chemie Int. Ed. 2011, 50, 7052–
7055.
[31] L. Zitvogel, L. Galluzzi, O. Kepp, M. J. Smyth, G. Kroemer, Nat. Rev.
Immunol. 2015, 15, 405–14.
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Entry for the Table of Contents (Please choose one layout)
Layout 2:
COMMUNICATION
Jana De Vrieze, Benoit Louage, Kim De
Swarte, Zifu Zhong, Ruben De Coen,
Simon Van Herck, Lutz Nuhn, Camilla
Kaas Frich, Alexander N. Zelikin, Stefan
Lienenklaus, Niek N. Sanders, Bart N.
Lambrecht, Sunil A. David, Bruno G. De
Geest*
lipidic motif
transporation via
interstitial fow
TLR7/8a
albumin
hydrophillic polymer
Lipid-polymer amphiphile conjugates of potent small molecule immune-modulators
restrict innate immune activation to lymphoid tissue
Page No. – Page No.
Potent lymphatic translocation and
spatial control over innate immune
activation by polymer-lipid
amphiphile conjugates of small
molecule TLR7/8 agonists
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