Squaric Ester-Based, pH-Degradable Nanogels: Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators

Article abstract of DOI:10.1021/jacs.1c03772

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.

Full text of DOI:10.1021/jacs.1c03772

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Article
Squaric Ester-Based, pH-Degradable Nanogels: Modular
Nanocarriers for Safe, Systemic Administration of Toll-like Receptor
7/8 Agonistic Immune Modulators
Anne Huppertsberg, Leonard Kaps, Zifu Zhong, Sascha Schmitt, Judith Stickdorn, Kim Deswarte,
Francis Combes, Christian Czysch, Jana De Vrieze, Sabah Kasmi, Niklas Choteschovsky, Adrian Klefenz,
Carolina Medina-Montano, Pia Winterwerber, Chaojian Chen, Matthias Bros, Stefan Lienenklaus,
Niek N. Sanders, Kaloian Koynov, Detlef Schuppan, Bart N. Lambrecht, Sunil A. David,
Bruno G. De Geest, and Lutz Nuhn*
Cite This: J. Am. Chem. Soc. 2021, 143, 9872−9883
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ABSTRACT: Small-molecular Toll-like receptor 7/8 (TLR7/8)
agonists hold promise as immune modulators for a variety of immune
therapeutic purposes including cancer therapy or vaccination. However,
due to their rapid systemic distribution causing difficult-to-control
inflammatory off-target effects, their application is still problematic, in
particular systemically. To address this problem, we designed and
robustly fabricated pH-responsive nanogels serving as versatile
immunodrug nanocarriers for safe delivery of TLR7/8-stimulating
imidazoquinolines after intravenous administration. To this aim, a
primary amine-reactive methacrylamide monomer bearing a pendant
squaric ester amide is introduced, which is polymerized under
controlled RAFT polymerization conditions. Corresponding PEG-
derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-
reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester
amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive
degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or
conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the
nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic
off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline
the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of
small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer
immunotherapy purposes.
kinetic profile can pave the way to safer systemic applications in
cancer immunotherapy or as adjuvants during vaccination.9−11
One promising class of immunomodulators are agonists of
Toll-like receptor 7/8 (TLR7/8) that usually sense viral single-
stranded RNA and raise an immediate cellular innate immune
response.12 Imidazoquinolines, a class of synthetic TLR7/8
agonists, are particularly powerful in stimulating these receptors.
INTRODUCTION
■
Nanosized drug carriers can improve the pharmacokinetics of
potent but systemically toxic small-molecular drugs; however,
key challenges remain to establish robust fabrication processes,
prevent premature drug release, and guarantee sufficient carrier
integrity under physiological conditions. Moreover, opportu-
nities toward on-demand drug release and carrier degradation
would be desired.1−4 Polymer-based nanocarriers can be
designed to address these challenges chemically, especially for
applications in immunotherapy.5,6 A spatiotemporal control
over recently identified, highly potent immunomodulators is
crucial, since their drug leakage can cause uncontrolled systemic
immune responses combined with immense off-target tox-
icity.7,8 Covalent conjugation of immunomodulators to
polymer-based nanocarriers that control the drug’s pharmaco-
Received: April 9, 2021
Published: June 24, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Squaric ester-based nanogels derived from polymeric precursor micelles formed by self-assembly of squaric ester amide-containing
amphiphilic block copolymers. Subsequent transformation for in vivo application is achieved by amidation of pendant squaric ester amide groups inside
the amine-reactive, hydrophobic core including pH-responsive cross-linking, covalent drug or dye loading, and hydrophilization, affording fully
hydrophilic drug-loaded nanogels.
They can induce the maturation of antigen-presenting cells and
the release of cytokines that promote cellular immune responses,
especially via activation of T helper 1 (Th1) and cytotoxic T
cells.13,14 The latter play a key role in combating cancer and
intracellular pathogens.15,16 As TLR7/8 are located at the
endosomal membrane of phagocytosing innate immune cells,
they can be preferentially targeted by nanocarriers equipped
with imidazoquinoline-derived TLR7/8 agonists.17
nanogels. However, for this approach the hydrophilic triethylene
glycol monomethyl ether methacrylate block renders a lower
critical solution temperature in water,34 which can impair the
required shielding properties after exposure to biological media.
Additionally, the applied pentafluorophenyl ester exhibits only
limited hydrolytic resistance. Already slight traces of water can
risk its hydrolysis into methacrylic acid35 and, thus, impede
access to the nanogel formation process. In addition, during
amidation toxic pentafluorophenol is released,36 which requires
careful purification prior to applications in vitro or in vivo.
Consequently, alternative amine-reactive groups with higher
hydrolytic stability and nontoxic byproducts are needed. Tietze
et al. reported on squaric alkyl esters for selective amine coupling
reactions with high functional group tolerance as well as
excellent yields, even under mild conditions in aqueous media.37
Due to their homo-bifunctionality squaric alkyl esters can
consecutively be aminolyzed by two different amines, whereby
an enhanced aromatic stability after first amidation is obtained,
providing reduced reactivity for another amidation. Thus, it
assures a controlled sequential squaric bisamide formation.38
Compared to other reactive esters (including pentafluorophen-
yl) this alternative linking chemistry allows conjugation in
biocompatible solvents (e.g., water, ethanol) while releasing
alcohols as nontoxic byproducts.39
In this study, we therefore introduce polymerizable squaric
ester amides for a more robust and hydrolysis-resistant
postpolymerization modification of pH-degradable, immuno-
drug-loaded nanogels. Methacrylamides with pendant squaric
ester amides are grafted under controlled radical polymerization
conditions onto linear hydrophilic PEG chains and serve as
solvophobic groups for spontaneous micellar self-assembly now
also in polar protic solvents (Figure 1).
By sequential functionalization with primary amines, they can
be converted into a modular pH-responsive nanogel platform.
This process supports covalent conjugation of both hydro-
phobic and hydrophilic drugs into the core of these nanogels,
e.g., the amine-functionalized TLR7/8 agonist IMDQ. Due to
its high degree of PEGylation, this system features excellent
stability in blood that permits intravenous administration and
local maturation of antigen-presenting immune cells inside the
spleen, while circumventing systemic inflammatory responses
Previous studies demonstrated that undesired systemic
inflammation by the highly potent small-molecular TLR7/8
agonist 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-
c]quinolin-4-amine (IMDQ)18 can be circumvented through
its covalent conjugation to different types of nanocarriers.
Thereby, its activity upon subcutaneous administration can be
restricted to the site of injection and the draining lymph nodes. A
variety of nanocarriers has been investigated for this objective
including lipids,19,20 polysaccharides,21 synthetic polymers,22
micelles,23 and nanogels.24,25 These nanocarrier formulations
have generally been injected subcutaneously and provided a
local TLR7/8 activation by lymph node trafficking. However, a
few findings revealed that intravenous injections would result in
even improved immune responses.26−29 Especially during
intravenous nanoparticle vaccination, more stem-like antigen-
specific CD8+ T cells are generated that lead to superior
antitumor responses, especially during immune checkpoint
inhibition therapy.30 Yet, systemic treatments set new require-
ments for safety and allow for new targeting strategies as
compared to local administration. Thus, new efforts have to be
made in the design of novel blood-stable nanocarriers enabling
intravenous and organ-targeted TLR7/8 agonist administration.
A straightforward way to covalently load IMDQ into
nanocarriers is self-assembled reactive ester block copolymers
that are converted by amidation reactions into pH-degradable
nanogels.24,25,31 Via controlled radical polymerization processes
we have previously generated poly(triethylene glycol mono-
methyl ether methacrylate)-block-poly(pentafluorophenyl
methacrylate) as precursor polymers for which the pentafluor-
ophenyl esters trigger both micellar self-assembly in polar
aprotic solvents such as DMSO and selective reactivity toward
primary amines.32,33 This could also be applied for conjugation
of IMDQ and subsequent transformation into fully hydrophilic
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Figure 2. Synthesis of polymerizable squaric ester amides. (A) General chemical structure of synthesized monomers with a pendant squaric ester amide
group. (B) Synthesis route toward the most suitable monomer squaric ester amide methacrylamide (MA-SQ). (C) Schematic illustrations and (D)
synthesis schemes of RAFT homopolymerization of MA-SQ with a small molecular trithiocarbonate chain transfer agent (TTC-CTA) (C1 and D1)
and block copolymerization with macro-chain-transfer agent PEG-TTC-CTA (C2 and D2) (reaction conditions: (i) DMF, 70 °C, 0.2 equiv AIBN per
1.0 equiv TTC-CTA, and (ii) DMF, 70 °C, 50 equiv AIBN (to remove the TTC end group)). (E) Molecular weight distributions of polymers obtained
by radical polymerization: (E1) RAFT-derived homopolymer compared to the homopolymer obtained by FRP; (E2) RAFT-derived block copolymer
compared to PEG-TTC-CTA as macro-chain-transfer agent.
caused by the unbound TLR7/8 agonist. Altogether, these
observations demonstrate the broad versatility of amine-reactive
squaric ester-based precursor polymers and how they provide
access to a modular pH-degradable nanogel platform for safe
systemic administration of immune modulators.
Monomer and Polymer Syntheses. Initially, polymer-
izable squaric ester amide monomers with acrylate, acrylamide,
or methacrylamide groups were synthesized (Figure 2A). Direct
(meth-)acryloylation of 2-aminoethanol-functionalized squaric
ester amides failed due to occurring bisacryloyl derivatives and
autopolymerization. For instance, we carefully treated 3-ethoxy-
4-((2-hydroxyethyl)amino)cyclobut-3-ene-1,2-dione with
acryloyl chloride but obtained besides the desired squaric ester
amide acrylate product 2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-
yl)amino) ethyl acrylate (At-SQ) also a bisacrylated species (At-
SQ-At) at similar yields, as documented in the Supporting
Information (Figures S14−S23). We therefore opted for (meth-
)acrylamides by controlled monoamidation of squaric acid
diethyl ester with amine-functionalized (meth-) acrylamides.
Unfortunately, due to their aza-Michael reactivity, amino-
functionalized (meth-)acrylamides are only stable as protonated
salt species. While the corresponding methacrylamide-based
monomer was commercially available, we synthesized the TFA
salt of N-(3-aminopropyl)acrylamide as corresponding acryl-
amide derivative (Supporting Information Figure S7). It was
converted with squaric acid diethyl ester catalyzed by triethyl-
amine in ethanol and water and provided the desired acrylamide
monomer at acceptable yields (64%, A-SQ, NMR character-
ization in Supporting Information Figures S10−S13). However,
several attempts to polymerize it, even under free radical
polymerization (FRP) conditions, failed or yielded only
conversions below 30%. Fortunately, synthesis and polymer-
ization of the methacrylamide analogue was more promising.
RESULTS AND DISCUSSION
■
We first aimed to synthesize a polymerizable squaric ester amide
monomer that can be polymerized under controlled conditions
into well-defined homo- and block copolymers using reversible
addition−fragmentation chain transfer (RAFT) agents. While
squaric ester amides have already been studied as amine-reactive
groups for atom transfer radical polymerization (ATRP)40 or
RAFT41 chain transfer agents, to the best of our knowledge, the
use of squaric esters as functional side groups of monomers and
corresponding polymers has not been introduced before. They
provide access to a new class of hydrolysis-resistant, amine-
reactive precursor polymers for multiple postpolymerization
purposes.39 We investigated the polymer’s reactivity toward
different types of amines and observed a self-assembly behavior
of corresponding block copolymers in water or ethanol. The
core of the resulting micelles can be used to ligate fluorescent
dyes or IMDQ followed by core-cross-linking and hydro-
philization affording pH-degradable nanogels. Subsequent in
vitro and in vivo characterization revealed long blood circulation
and maturation of antigen-presenting cells in the spleen while
omitting inflammatory off-target effects.
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Figure 3. Amine selectivity of pendant squaric amide groups of p(MA-SQ)40. (A) Scheme for the conversion of polymeric squaric ester amides to
squaric bisamides by amidation with aminoethanol, morpholine, or triethylamine. UV/vis spectra during conversion with (B1) aminoethanol, (C1)
morpholine, or (D1) triethylamine and the corresponding molecular weight distributions of p(MA-SQ)40 before and after conversion with (B2)
aminoethanol, (C2) morpholine, or (D2) triethylamine. (E) Corresponding conversions estimated by UV absorbance over time.
Monoamidation of squaric acid diethyl ester with the
commercially available N-(3-aminopropyl)methacrylamide hy-
drochloride was again conducted in ethanol and water catalyzed
by triethylamine. It afforded a squaric ester amide methacryla-
mide monomer (MA-SQ) with a high yield of 90% (Figure 2B,
NMR and MS characterization in Supporting Information,
Figures S1−S6).
MA-SQ could afterward be polymerized under FRP
conditions at 70 °C using AIBN as radical source with
quantitative conversion. Consequently, it was an ideal candidate
for RAFT polymerization. We chose a trithiocarbonate as chain
transfer agent (TTC-CTA) and polymerized MA-SQ with either
a small-molecular TTC-CTA or a PEG-functionalized macro-
TTC-CTA (Supporting Information Figure S24−S26; note that
instead of commercially available macro-TTC-CTAs bearing an
ester group between PEG and the CTA group, we preferred to
apply an amide derivative to avoid possible ester aminolysis and
cleavage during amidation). At a monomer to CTA ratio of 50:1,
conversions of 75−80% were found affording p(MA-SQ)40 as
homopolymer and PEG113-b-p(MA-SQ)38 as block copolymer
(Figure 2C/D).
Consistent with earlier reports, NMR analysis validated that
squaric ester amide moieties withstood radical polymerization
conditions and remained intact (Figures S28 and S32).40,41
Molecular weight distributions of p(MA-SQ)40 obtained by size-
exclusion chromatography (SEC) revealed well-defined homo-
polymers with a number-average molar mass (Mn) of 13 800 g/
mol and polydispersity index (PDI) of 1.26 (Figure 2E1). In
stark contrast to the FRP, the RAFT homopolymers of MA-SQ
confirmed controlled polymerization conditions. Via NMR
spectroscopy, monomer conversion over time demonstrated
first-order kinetics for the polymer formation (Figures S34 and
S35). Similar conditions were applied during block copoly-
merization and provided polymers with a narrow PDI of 1.16
and Mn of 22 200 g/mol (Figure 2E2). The molecular weight
shift of the block copolymer (PEG113-b-p(MA-SQ)38) compared
to PEG-TTC-CTA clearly attested full MA-SQ monomer
grafting onto the macro-CTA. Additionally, DOSY NMR
analysis confirmed successful block copolymer formation by
providing identical diffusion coefficients of both blocks (Figure
S29). To avoid interference with the trithiocarbonate (TTC)
end group during subsequent amidation of the squaric ester
amides, both homo- and block copolymer were further treated
with an excess of AIBN for end group removal. This additional
step did not impair the composition or distribution of the
polymers (compare Supporting Information Figures S30 and
S33).
Amine-Selective Conversion. In order to verify amine
reactivity, the homopolymer p(MA-SQ)40 was first reacted with
an excess of primary, secondary, or tertiary amine. As model
compounds aminoethanol, morpholine, and triethylamine were
selected (Figure 3A, note that these reactions were conducted in
DMSO to ensure complete solubility of the homopolymer).
Interestingly, the formed squaric bisamides provide an
absorption maximum around 300 nm compared to the squaric
ester amide educts. Therefore, their aminolysis can easily be
monitored over time by UV/vis absorbance spectroscopy.39
A
strong and fast increase in absorbance at 300 nm revealed rapid
and quantitative conversion of the squaric ester amides by the
primary amine aminoethanol. The secondary amine morpholine
could only convert about 25% after 2 h, while no conversion was
observed with triethylamine as tertiary amine (Figure 3B1−D1,
Figure 3E, and Supporting Information Figure S37). The
respective homopolymers were isolated by precipitation in
diethyl ether. Recorded molecular weight distributions by SEC
demonstrated no significant change in Mn and PDI for p(MA-
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Figure 4. Nanogel fabrication process. (A) Scheme of sequential nanogel formation from squaric ester amide-derived, self-assembling precursor
polymers. Based on the cross-linking strategy, degradable nanogels (D-NG) as well as nondegradable or non-cross-linked control samples (ND-NG,
NCL-NG) can be obtained. (B) DLS size distribution of self-assembled micelles in ethanol and (C) fully fabricated nanogels D-NG, ND-NG, and
NCL-NG in PBS. (D) TEM image of D-NG. (E) AFM image of D-NG. (F1 and F2) Scheme of ketal-hydrolysis in acidic media resulting in D-NGs'
disassembly into soluble unimers (NCL-NG). (G) DLS study of D-NG at neutral pH in PBS compared to mildly acidic pH in HAc/Ac buffer by (G1)
size distribution and (G2) DLS count rate over time.
SQ)40 treated with triethylamine, while a complete peak shift to
smaller molecular weights was found after conversion with
aminoethanol (Figure 3B2−D2). The squaric ester amide
polymers treated with morpholine showed only a minimal peak
shift reflecting their incomplete conversion. These observations
were confirmed by NMR analysis (Figures S38 and S39).
Further studies indicated that full conversion by secondary
amine morpholine can still be achieved at elevated temperature
and extended reaction time (Figure S40, e.g., after 22 h at 70 °C
complete squaric ester amide aminolysis was demonstrated;
compare Figures S41 and S42). These results highlight the
suitability of squaric ester amides as reactive polymer side groups
for quantitative postpolymerization modification with preferen-
tially fast conversion of primary amines.
functionalities by amidation reactions. Following the design
concept of Figure 4A, we were interested in whether this
postpolymerization modification can also be combined with self-
assembly to sequentially access nanogels (by micelle formation,
covalent core-cross-linking, and transformation of the core from
hydrophobic to hydrophilic). The well-defined amphiphilic
block copolymer PEG113-b-p(MA-SQ)38 was suspended in
ethanol and treated with ultrasound, yielding narrowly dispersed
micelles. By dynamic light scattering (DLS) their sizes were
analyzed, revealing a z-average diameter (DZ) of 44 nm and a
narrow PDI of 0.13 (Figure 4B). Next, the polarity of the core
was switched chemically by exploiting its reactivity toward
hydrophilic amines. After addition of 5.0 equiv of PEG11-amine
the micelles disassembled within 30 min and provided fully
soluble polymers (Supporting Information Figures S43 and S44;
note that amines like aminoethanol did not increase the polarity
Nanogel Fabrication and Characterization. Polymeric
squaric ester amides can be used as precursors to introduce new
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Figure 5. Fluorescently labeled nanogels provide sufficient particle integrity for safe in vivo administration. (A) Scheme of sequential nanogel
fabrication with in situ installed fluorescent dye-labeling. (B) Oregon Green cadaverine (OG)-labeled nanogels (D-NG) investigated by fluorescence
correlation spectroscopy toward stability in plasma and the ability to disassemble upon exposure to endolysosomal pH values: (B1) normalized
autocorrelation curves (dots) and corresponding fits (solid line) of OG-labeled D-NG incubated in human blood plasma for 0, 6, and 24 h; (B2)
normalized autocorrelation curves (dots) and corresponding fits (solid line) of OG-labeled D-NG incubated in acidic buffer (pH 5.2) over time. (C)
Tetramethylrhodamine cadaverine (TMR)-labeled nanogels (D-NG) investigated for the in vitro uptake behavior in RAW murine macrophages using
flow cytometry and confocal microscopy imaging (n = 3): (C1) cellular mean fluorescence intensities (MFI) of RAW macrophages incubated with
TMR-labeled D-NG at 100, 50, and 10 μg/mL for 24 h; (C2) corresponding histograms of RAW macrophages incubated with TMR-labeled D-NG at
100, 50, and 10 μg/mL for 24 h (n = 3) or PBS (control). (C3) Confocal microscopy images of RAW macrophages incubated with TMR-labeled D-NG
at 100 μg/mL for 4 and 24 h (blue: nuclei stained with Hoechst 33258; green: cell membrane stained with cholera toxin B (CTB)-AF488; red: TMR-
labeled D-NG, scale bar 10 μm). (D) Near infrared dye 800RS cadaverine (NIR)-labeled nanogels (NIR-labeled D-NG) investigated for their
biodistribution after systemic application to BALB/c mice via tail vein injection using an in vivo NIR-imaging system (IVIS): (D1) whole body
fluorescence imaging 24 h after intravenous injection of 100 μL of NIR-labeled D-NG dispersion (2 mg/mL); (D2) ex vivo organ distribution imaging
and (D3) semiquantitative analysis (n = 4).
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to unfold the micelles). Thus, core hydrophilization is possible
to fabricate fully hydrophilic nanogels.
This was also the case for ND-NG and NCL-NG at both pH
levels over time (Supporting Information (Figure S54). Thus,
ketal-cross-linked nanogels represent ideal candidates for drug
delivery due to their transient supramolecular architecture that
unfolds into secretable smaller entities upon exposure to
endolysosomal pH values.
Next, polymeric micelles were modified by pH-responsive
cross-links prior to hydrophilization (note that the following
process is possible in both pure ethanol and buffered water;
compare Supporting Information Figure S50). In relation to 1.0
equiv of pendant squaric ester amide moieties, 0.15 equiv of the
ketal-containing bisamine 2,2′-bis(aminoethoxy)propane was
added. Its ketal unit renders the nanocarriers’ sensitivity to
endosomal acidic pH. As shown in previous reports, it can
effectively hydrolyze at pH 5, resulting in a disassembly of
nanogels into soluble polymer chains.33,42,43 In order to provide
a nondegradable nanogel version, the core of the micelles was
conjugated with 0.15 equiv of an ether-containing bisamine
(1,2-(bis(aminoethoxy)ethane). Additionally, a non-cross-
linked soluble polymer representing the ketal-cross-linked
nanogel after hydrolysis was obtained by aminolysis with 0.3
equiv of 2-aminoethanol. For each species, all remaining squaric
ester amide moieties were finally converted with 3.0 equiv of
PEG11-amine, resulting in fully hydrophilic nanogel or soluble
polymer species (Figure 4A). All samples were purified by
dialysis against water containing 0.1 wt % ammonia to prevent
premature ketal-hydrolysis. Subsequent lyophilization afforded
the nanogels as dry powders, which could be easily redispersed
in PBS buffer.
UV/vis absorbance spectroscopy could again be used to
monitor the conversion of the squaric esters inside the self-
assembled micelles/core-cross-linked nanogels by following the
increase of the absorption maximum around 300 nm related to
the formed squaric bisamides (compare Supporting Information
Figures S46−S48). Sequential addition of primary amines for
functionalization, cross-linking, and final conversion of the
remaining squaric ester amide moieties with PEG11-amine
yielded a stepwise increase of the absorbance. Note that no
difference between the absorption after addition of 3.0 equiv of
PEG11-amine and the purified nanogel after extensive dialysis
against water with 0.1 wt % ammonia could be found, confirming
quantitative squaric ester conversion inside the nanogels during
the fabrication process (compare Supporting Information
Figures S46−S48).
As shown by DLS analysis, the ketal-cross-linked, degradable
nanogel (D-NG) showed a very narrow monomodal distribu-
tion and a Dz of 44 nm, which was similar to the size of the
precursor micelle. The same was found for the nondegradable
nanogel (ND-NG, Dz = 46 nm), while for the non-cross-linked
version (NCL-NG) a significantly lower count rate was detected
indicating the absence of particular scattering. The average
volume diameter of the NCL-NG was 8 nm (Figure 4C). The
ketal-cross-linked nanogels were imaged by transmission
electron microscopy (TEM) and atomic force microscopy
(AFM). The dried particles could be recorded as spherical and
some slightly elongated morphologies with narrow dispersities
(Figure 4D and E; Supporting Information Figure S49). To
prove that the ketal-cross-linked nanogels fully disassemble into
soluble unimers under endolysosomal pH conditions (Figure
4F1,F2), several DLS stability studies at pH 7.4 or 5.2 were
performed. D-NG was exposed to acidic pH in acetate buffer at
10 mg/mL and subsequently monitored over time by DLS. A
gradual decline in count rate within 600 min was observed, while
a decrease of the particles’ size distribution demonstrated
complete nanogel disassembly into single polymer chains
(Figure 4G1,G2). In contrast, no change in count rate or size
was observed at neutral pH for D-NG in PBS (Figure 4G1,G2).
Nanogel Functionalization. To further investigate the
nanogels’ covalent drug delivery function, their squaric ester
amides were first used to introduce fluorescent labels. Prior to
the described cross-linking and core-hydrophilization, 0.01
equiv of amine-bearing fluorescent dyes (e.g., Oregon Green
cadaverine (OG), tetramethylrhodamine cadaverine (TMR), or
the near-infrared dye 800RS cadaverine (NIR)) was conjugated
to the core (Figure 5A). Covalent dye conjugation did not affect
the nanogel fabrication process. To ensure the absence of free
dye, nanogels were extensively spin-filtrated (MWCO: 10 000
g/mol) using a mixture of 0.1 wt % ammonia in water/EtOH (v/
v, 1:2) in addition to the described purification methods. Dye
labeling enables analyses of the nanocarriers’ behavior in
complex biological environment and, thus, provides key features
to evaluate their applicability for immunomodulating drug
delivery.
We first investigated the dye-labeled nanogel’s stability in
human blood plasma as complex biological medium relevant for
in vitro and in vivo application. Selective monitoring of nanogel
integrity over time was facilitated by fluorescence correlation
spectroscopy (FCS). It provides information about the
nanocarrier’s size in solution by autocorrelation analysis of
fluorescence intensity fluctuations that result from diffusional
motions of fluorescent species (e.g., labeled nanocarriers)
through a confocal volume.44 After incubation of pH-degradable
nanogels (OG-labeled D-NG) in human blood plasma for 0, 6,
and 24 h at 37 °C, FCS analysis confirmed that the nanogel’s
autocorrelation curve and the corresponding hydrodynamic
radius remained identical (Figure 5B1). Consequently, the
carriers are profoundly stable under these biologically relevant
conditions: Neither an increase of the nanogel’s size, which
would reflect aggregation with serum components nor its
decrease was observed, which would imply nanogel degradation.
The latter could only be observed by FCS when the ketal-cross-
linked OG-labeled D-NG was again exposed to pH 5.2 using
acetate buffer. As evident from the shift of the autocorrelation
curves, one can perfectly observe a sequential disassembly of the
nanogels into single unimers (Figure 5B2).
Next, cellular nanogel interaction was studied for TMR-
labeled D-NG using flow cytometry as well as confocal
microscopy. RAW macrophages were selected as model for
phagocytosing cells of the reticuloendothelial system that
usually rapidly engulf nanocarriers after systemic administration.
However, only a minor uptake of D-NG was observed after
dosing from 10 to 100 μg/mL. A weak increase in mean
fluorescence intensity from 500 (basal autofluorescence) to 700
could be recorded by flow cytometry (Figure 5C1), and the
resulting histogram provided only a minor but homogeneous
shift of all cells to increased fluorescence (Figure 5C2). These
observations were independent from the type of nanogel, as
similar low mean fluorescence intensities and low TMR-positive
cells were found for all species (D-NG, ND-NG, and NCL-NG;
compare Supporting Information Figure S56). These results
were further supported by confocal microscopy images of RAW
macrophages incubated at 100 μg/mL for both 4 and 24 h
(Figure 5C3 and Supporting Information Figure S55). All cells
provided a homogeneous intracellular localization of some
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Figure 6. Squaric ester amide-based nanogels with covalently attached TLR7/8 agonist IMDQ. (A) Scheme of sequential nanogel fabrication with
integral in situ conjugation of IMDQ. (B) TLR agonistic activity of soluble IMDQ (sIMDQ) and covalently attached IMDQ to degradable nanogels
(IMDQ-D-NG) measured by NF-κB activation of RAW Blue cells after 24 and 48 h via a Quanti-Blue reporter assay (n = 4). (C) Bioluminescence
images of heterozygous IFN-β (IFN-β+/Δβ‑luc) reporter mice intravenously injected with sIMDQ and covalently attached IMDQ-D-NG (5 μg of
IMDQ soluble or bound to nanogel in 100 μL of PBS); images were recorded before (0 h) as well as 4, 24, and 48 h after tail vein injection (n = 3). (D−
H) Results of flow cytometric analysis of isolated splenocytes (compare Figure S60 for the applied gating procedure) after systemic administration of
soluble IMDQ vs nanogel-conjugated IMDQ (1: fold increase of cells in the spleen in relation to PBS treated mice; 2: maturation plots by CD86 and
CD80 or CD69 for T cells; 3: corresponding MFI values). (D) Dendritic cells (DCs), (E) macrophages, (F) neutrophils, (G) B lymphocytes, and (H)
T lymphocytes.
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nanogels presumably inside vesicular compartments. Due to the
fact that particle uptake was only observed upon high particle
dosing at 100 μg/mL, these in vitro studies suggested the
potentially useful property of these nanogel carriers to withstand
rapid phagocytosis, making them applicable for systemic in vivo
applications.
nanogel samples in the tested experimental window (Figure
S57).
We then investigated the nanogel’s in vivo immune
stimulatory activity with a special focus on the activation of
antigen-presenting cells in the spleen while avoiding systemic
off-target effects. The latter can be visualized by bio-
luminescence imaging of heterozygous BALB/c IFN-β (IFN-
We therefore intravenously injected NIR-labeled D-NG into
mice’s tail veins and observed after 24 h still a strong fluorescent
signal distributed all over the body by the in vivo NIR imaging
system (IVIS) (Figure 5D1). These findings confirmed that the
nanogels remained in the blood circulation long enough to
address multiple organs. Subsequently, major organs were
harvested and imaged ex vivo to better quantitate the NIR signals
(Figure 5D2). Semiquantitative analysis confirmed that nano-
gels were sequestrated not only in the liver, a typical nonspecific
sink for systemically administered nanocarriers, and kidneys
(probably due to the renal clearance properties of the soluble
polymers after hydrolysis of ketal-cross-linked nanogels), but
also in the spleen. The spleen is a central organ of the lymphatic
system, being highly relevant for immunodrug delivery because
it harbors many antigen-presenting cells (Figure 5D3). We,
therefore, opted for a strategy to systemically deliver a TLR7/8
agonist into this organ and induce a local immune modulation.
Effective and Safe TLR7/8 Agonist Delivery for
Intravenous Routes of Administration. The highly potent
small-molecular TLR7/8 agonist IMDQ18 was selected as an
immune modulatory compound to trigger maturation of splenic
antigen-presenting cells. This compound has been demon-
strated to activate TLR7/8 even when it is bound to
macromolecular nanocarriers.24,45,46 Due to its primary benzylic
amine, IMDQ can straightforwardly be conjugated to pendant
squaric ester amide groups and, therefore, directly be
incorporated into the nanogel fabrication process (Figure 6A).
IMDQ conjugation did not affect the nanogel fabrication, as
cross-linked nanogels (IMDQ-D-NG and IMDQ-ND-NG)
were of similar size and the non-cross-linked species (IMDQ-
NCL-NG) was again completely soluble (compare Supporting
Information Figure S51). Quantification of the covalently
β
+/Δβ‑luc) reporter mice,47 in which luciferase as reporter gene is
genetically linked to the expression of type I interferon IFN-β, a
cytokine that is induced upon TLR7/8 stimulation. Mice were
intravenously injected into the tail vein, and bioluminescence
was monitored after 4, 24, and 48 h (Figure 6C and Supporting
Information Figure S59). For sIMDQ, a strong signal was
observed all over the body, reflecting a systemic inflammatory
response in those mice. A prominent IFN-β expression occurred
in the neck and abdomen and remained high up to 48 h
postinjection. The nanogel-conjugated IMDQ, however, did not
cause such a severe global immune activation all over the body.
After 4 h no severe systemic inflammation was recorded. Only
after 24 h a mild activity was observed with some expression in
the abdomen and neck region, but with reduced gain compared
to sIMDQ (Figure 6C and Supporting Information Figure S59).
These observations clearly underline that severe systemic
inflammatory responses are absent for the nanogel carrier
system.
To further corroborate the absence of soluble IMDQ’s acute
toxicity, we also monitored the systemic inflammatory cytokines
in sera of mice treated with soluble IMDQ vs nanogel-
conjugated IMDQ. The results are provided in the Supporting
Information Figure S62−S64 and confirm the observations of
the IFN-β reporter mice: After 4 h, increasing serum cytokine
levels of TNF-α, IL-6, and INF-γ are found in mice treated with
soluble IMDQ. They are significantly elevated compared to
mice treated with PBS, while mice treated with nanogel-bound
IMDQ only provide slightly increased but not significant TNF-
α, IL-6, and INF-γ levels (Supporting Information Figure S62).
After 24 h, those cytokines are again decreased to levels of
PBS-treated mice. The elevated cytokine levels for soluble
IMDQ-treated mice caused further liver toxicities by increased
ALT, AST, and GLDH enzyme serum activities (Supporting
Information Figure S63). Additionally, further influence on
pancreas toxicity by high lipase serum activity as well as kidney
toxicity by elevated blood urea nitrogen (BUN) can be observed
(Supporting Information Figure S64). These data underline
again the improved safety profile for the IMDQ-loaded nanogels
to circumvent these acute toxicities but focus the immune
stimulatory potential of the TLR7/8 agonist to the spleen.
One may speculate that such reduced systemic off-target
effects for the nanogel-conjugated IMDQ might simply be
caused by the minor receptor activation potency or reduced
bioavailability. However, when it comes to immune intervention
strategies (e.g., vaccination or cancer immunotherapy), site-
specific immune activation (i.e., spleen specific, rather than
systemic) is of important relevance and the major goal of this
study.
1
attached IMDQ was conducted by H NMR spectroscopy.
For the soluble NCL-NG species, a drug load of 2.7 wt % was
found (compare Supporting Information Figure S52) being
covalently attached to the polymer (confirmed by DOSY NMR,
Figure S53).
For demonstrating TLR7/8 stimulation activity, we used a
RAW-Blue macrophage reporter cell line that is genetically
engineered to secrete embryonic alkaline phosphatase (SEAP)
in response to TLR activation and downstream signaling via the
NF-κB pathway. SEAP expression itself can readily be detected
from the cell culture supernatant via UV/vis spectrophotometry
after addition of a coloring substrate (Quanti-Blue assay). RAW
Blue macrophages were pulsed with increasing doses of either
soluble IMDQ (sIMDQ) or IMDQ-D-NG (empty nanogel D-
NG served as a control). A strong TLR activation was found for
sIMDQ already at sub-micromolar concentrations, while
nonligated control nanogels did not induce any significant
activation (Figure 6B). IMDQ-D-NG led to a dose-dependent
activation, albeit less potent than sIMDQ, but still active in the
micromolar concentration range. Similar activities were found
after 24 as well as 48 h and could be confirmed for both IMDQ-
ND-NG and IMDQ-NCL-NG (Figure S58), which may
corroborate the weak cellular internalization. In parallel, RAW
Blue cellular viability was monitored by an MTT assay. No
significant influence on metabolic activity was found for all
Previous biodistribution analysis confirmed that a sufficient
amount of nanogels was found inside the spleen after systemic
activation (Figure 5D). To further validate that IMDQ-loaded
nanogels still activate spleen-resident immune cells, we isolated
splenocytes from the mice 48 h after injection and performed
quantitative flow cytometric analyses (for further details on the
gating procedure compare Supporting Information Figure S60).
Both sIMDQ and IMDQ-D-NG comparably increased the
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number of myeloid cells with no influence on B and T
lymphocytes (Figure 6D−H1). Dendritic cells (DCs), macro-
phages, and neutrophils are known to express and respond to
TLR7/8 activation effectively. Their numbers are therefore
increased to a similar extent for both sIMDQ and IMDQ-D-NG
(Figure 6D1−F1).
We then checked for maturation markers on those cells and
found again that IMDQ-D-NG triggered their expression to a
similar level as sIMDQ while omitting systemic inflammations
(Figure 6D2−H2 and Supporting Information Figure S61). For
DCs and neutrophils (Figure 6D3/4 and F3/4) the expression
levels of CD80 and CD86 were significantly enhanced by
IMDQ-D-NG as well as sIMDQ. They were also upregulated on
B lymphocytes that in contrast to T lymphocytes have antigen
presenting ability (Figure 6G3/4). Also, the T lymphocyte
activation marker CD69 was induced, again comparably, by
IMDQ-D-NG and sIMDQ treatment, indicating further down-
stream T lymphocyte activation (Figure 6H3).
Most interestingly, for macrophages no difference in the
expression of CD86 was observed (Figure 6E4), as CD86
upregulation is more typical for DCs. However, CD80
expression was more significantly triggered by IMDQ-D-NG
and even exceeded the expression levels of sIMDQ (Figure
6E3). Apparently, nanogel-bound IMDQ seems to have a
preferential stimulation potential in those highly phagocytotic
immune cells inside the spleen. These properties outperform the
treatment with sIMDQ and make the nanogels highly interesting
for further therapeutic immune interventions.
Altogether, these observations confirm the hypothesis that
our nanocarrier can deliver the TLR7/8 agonist into the spleen
and maintain its immune stimulatory activity inside that organ. It
prevents massive systemic immune activations and subsequent
severe inflammatory off-target effects but conserves organ-
specific immune stimulation inside the spleen, especially to
resident macrophages. Since this organ is highly relevant for
further immune intervention strategies including cancer
immunotherapy or vaccination,48,49 we consider squaric ester-
based, pH-degradable nanogels as a relevant platform to exploit
further systemic immunologic adjuvant strategies.
administrations routes for small-molecular TLR7/8 agonists
(and other relevant immune modulators) and, thus, provide
novel opportunities to explore their superior adjuvant potency
for systemic vaccination or cancer immunotherapy purposes.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03772.
■
sı
Experimental details including materials, instrumentation,
monomer and polymer syntheses, and characterization,
nanogel fabrication and characterization, IMDQ ligation
and quantification, in vitro and in vivo evaluation (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Lutz Nuhn − Max Planck Institute for Polymer Research, 55128
Mainz, Germany; orcid.org/0000-0003-0761-1106;
Phone: +49 (0) 6131-379-311; Email: lutz.nuhn@mpip-
mainz.mpg.de
Authors
Anne Huppertsberg − Max Planck Institute for Polymer
Research, 55128 Mainz, Germany
Leonard Kaps − Institute for Translational Immunology and
Research Center for Immune Therapy, University Medical
Center, Johannes Gutenberg-University Mainz, 55131 Mainz,
Germany; Department of Internal Medicine I, University
Medical Center of the Johannes Gutenberg-University Mainz,
55131 Mainz, Germany
Zifu Zhong − Department of Pharmaceutics and Cancer
Research Institute Ghent (CRIG), Ghent University, Ghent
9000, Belgium
Sascha Schmitt − Max Planck Institute for Polymer Research,
55128 Mainz, Germany
Judith Stickdorn − Max Planck Institute for Polymer Research,
55128 Mainz, Germany
Kim Deswarte − Department of Internal Medicine and
Pediatrics, Ghent University, VIB Center for Inflammation
Research, Ghent 9052, Belgium
CONCLUSIONS
■
In this study, we have introduced polymerizable squaric ester
amides as novel functionable tool to robustly fabricate pH-
responsive nanogels serving as a versatile nanocarrier platform
for safe delivery of the highly potent immunomodulator IMDQ
after intravenous administration. To the best of our knowledge,
the synthesis of methacrylamide monomers with a functional
pendant squaric ester amide moiety has not been reported
before. Its subsequent controlled RAFT block copolymerization
as well as its self-assembling behavior in polar protic solvents
provides access to precursor micelles with amine-reactive cores.
Through their sequential amidation, covalent dye or drug
loading, acid-sensitive cross-linking, and core-transformation
from hydrophobic to hydrophilic polarity can be performed,
affording fully hydrophilic nanogels with profound stability in
plasma and the bloodstream, with modest uptake by phagocytic
cells and with an inherently triggered degradation upon
exposure to mildly endolysosomal pH conditions. The carriers’
potential to covalently conjugate IMDQ as a highly potent
immunomodulator enables the drug’s safe and efficient delivery
by preventing systemic off-target inflammation but retaining its
spatial activity in the spleen. These findings underline the
potential of this carrier platform to access intravenous
Francis Combes − Laboratory of Gene Therapy, Department of
Nutrition, Genetics and Ethology, Ghent University, Merelbeke
9820, Belgium
Christian Czysch − Max Planck Institute for Polymer Research,
55128 Mainz, Germany
Jana De Vrieze − Department of Pharmaceutics and Cancer
Research Institute Ghent (CRIG), Ghent University, Ghent
9000, Belgium
Sabah Kasmi − Department of Pharmaceutics and Cancer
Research Institute Ghent (CRIG), Ghent University, Ghent
9000, Belgium
Niklas Choteschovsky − Institute for Translational
Immunology and Research Center for Immune Therapy,
University Medical Center, Johannes Gutenberg-University
Mainz, 55131 Mainz, Germany
Adrian Klefenz − Institute for Translational Immunology and
Research Center for Immune Therapy, University Medical
Center, Johannes Gutenberg-University Mainz, 55131 Mainz,
Germany
Carolina Medina-Montano − Department of Dermatology,
University Medical Center of Johannes Gutenberg-University
Mainz, 55131 Mainz, Germany
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Pia Winterwerber − Max Planck Institute for Polymer
Research, 55128 Mainz, Germany
Chaojian Chen − Max Planck Institute for Polymer Research,
55128 Mainz, Germany; orcid.org/0000-0002-2588-
2447
Matthias Bros − Department of Dermatology, University
Medical Center of Johannes Gutenberg-University Mainz,
55131 Mainz, Germany
Stefan Lienenklaus − Institute for Laboratory Animal Science
and Institute of Immunology, Hannover Medical School,
30625 Hannover, Germany
Niek N. Sanders − Laboratory of Gene Therapy, Department of
Nutrition, Genetics and Ethology, Ghent University, Merelbeke
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Kaloian Koynov − Max Planck Institute for Polymer Research,
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Detlef Schuppan − Institute for Translational Immunology and
Research Center for Immune Therapy, University Medical
Center, Johannes Gutenberg-University Mainz, 55131 Mainz,
Germany; Division of Gastroenterology, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston,
Massachusetts 02215, United States
Bart N. Lambrecht − Department of Internal Medicine and
Pediatrics, Ghent University, VIB Center for Inflammation
Research, Ghent 9052, Belgium; Department of Pulmonary
Medicine, Erasmus University Medical Center, Rotterdam
3015, Netherlands
Sunil A. David − ViroVax, LLC, Lawrence, Kansas 66047,
United States; orcid.org/0000-0003-1655-4641
Bruno G. De Geest − Department of Pharmaceutics and Cancer
Research Institute Ghent (CRIG), Ghent University, Ghent
9000, Belgium; orcid.org/0000-0001-9826-6170
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03772
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(18) Beesu, M.; Salyer, A. C. D.; Brush, M. J. H.; Trautman, K. L.; Hill,
J. K.; David, S. A. Identification of High-Potency Human TLR8 and
Dual TLR7/TLR8 Agonists in Pyrimidine-2,4-Diamines. J. Med. Chem.
2017, 60 (5), 2084−2098.
Notes
The authors declare no competing financial interest.
(19) Van Hoeven, N.; Fox, C. B.; Granger, B.; Evers, T.; Joshi, S. W.;
Nana, G. I.; Evans, S. C.; Lin, S.; Liang, H.; Liang, L.; Nakajima, R.;
Felgner, P. L.; Bowen, R. A.; Marlenee, N.; Hartwig, A.; Baldwin, S. L.;
Coler, R. N.; Tomai, M.; Elvecrog, J.; Reed, S. G.; Carter, D. A
Formulated TLR7/8 Agonist Is a Flexible, Highly Potent and Effective
Adjuvant for Pandemic Influenza Vaccines. Sci. Rep. 2017, 7 (1), 1−15.
(20) De Vrieze, J.; Louage, B.; Deswarte, K.; Zhong, Z.; De Coen, R.;
Van Herck, S.; Nuhn, L.; Kaas Frich, C.; Zelikin, A. N.; Lienenklaus, S.;
Sanders, N. N.; Lambrecht, B. N.; David, S. A.; De Geest, B. G. Potent
Lymphatic Translocation and Spatial Control Over Innate Immune
Activation by Polymer−Lipid Amphiphile Conjugates of Small-
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15390−15395.
(21) Yoo, E.; Salyer, A. C. D.; Brush, M. J. H.; Li, Y.; Trautman, K. L.;
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(22) Lynn, G. M.; Chytil, P.; Francica, J. R.; Lagová, A.; Kueberuwa,
G.; Ishizuka, A. S.; Zaidi, N.; Ramirez-Valdez, R. A.; Blobel, N. J.;
Baharom, F.; Leal, J.; Wang, A. Q.; Gerner, M. Y.; Etrych, T.; Ulbrich,
ACKNOWLEDGMENTS
■
A.H. and L.N. acknowledge support by the Max Planck
Graduate Center with the Johannes Gutenberg-Universität
Mainz (MPGC). Moreover, L.N. kindly acknowledges financial
support by the DFG through the Emmy Noether program as
well as the SFB 1066 projects Q02, B03, and B04. Both A.H. and
L.N. would also like to thank Tanja Weil for providing access to
excellent laboratory facilities.
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