Covalent Conjugation of Small-Molecule Adjuvants to Nanoparticles Induces Robust Cytotoxic T Cell Responses via DC Activation

Article abstract of DOI:10.1021/acs.bioconjchem.6b00277

Specific recognitions of pathogen associated molecular patterns by Toll-like receptors (TLRs) initiate dendritic cell (DC) activation, which is critical for coordinating innate and adaptive immune responses. Imidazoquinolines as small-molecule TLR7 agonists often suffer from prompt dissemination and short half-life in the bloodstream, preventing their localization to the corresponding receptors and effective DC activation. We postulated that covalent incorporation of imidazoquinoline moieties onto the surface of biocompatible nanoparticles (～30 nm size) would enhance their chemical stability, cellular uptake efficiency, and adjuvanticity. The fully synthetic adjuvant-nanocomplexes led to successful DC activation at lower nanomolar doses compared with free small-molecule agonists. Once a model antigen such as ovalbumin was used for immunization, we found that the nanocomplexes promoted an unusually strong cytotoxic T lymphocyte response, revealing their unique immunostimulatory capacity benefiting from multivalency and efficient transport to endosomal TLR7.

Full text of DOI:10.1021/acs.bioconjchem.6b00277

Subscriber access provided by Northern Illinois University
Article
Covalent Conjugation of Small-Molecule Adjuvants to Nanoparticles
Induces Robust Cytotoxic T Cell Responses via DC Activation
Woo Gyum Kim, Bongseo Choi, Hyun-Ji Yang, Jae-A Han, Hoesu
Jung, HyungJoon Cho, Sebyung Kang, and Sung You Hong
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00277 • Publication Date (Web): 09 Aug 2016
Downloaded from http://pubs.acs.org on August 10, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Bioconjugate Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
Covalent Conjugation of Small-Molecule Adjuvants to Nanoparticles
Induces Robust Cytotoxic T Cell Responses via DC Activation
7
8
,
,
Woo Gyum Kim,† § Bongseo Choi,‡ § HyunꢀJi Yang,† JaeꢀA Han,‡ Hoesu Jung,‡
,
,
HyungJoon Cho,‡ Sebyung Kang,‡ * and Sung You Hong† *
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
‡
†School of Energy and Chemical Engineering and School of Life Sciences, Ulsan National Institute of Science
and Technology (UNIST), UNISTꢀgil 50, Ulsan 689ꢀ798, Republic of Korea
ABSTRACT: Specific recognitions of pathogen associated molecular patterns by Tollꢀlike receptors
(TLRs) initiate dendritic cell (DC) activation, which are critical for coordinating innate and adaptive
immune responses. Imidazoquinolines as smallꢀmolecule TLR7 agonists often suffer from their prompt
dissemination and short halfꢀlife in the bloodstream, preventing their localization to the corresponding
receptors and effective DC activation. We postulated that covalent incorporation of imidazoquinoline
moieties onto the surface of biocompatible nanoparticles (~30 nm size) would enhance their chemical
stability, cellular uptake efficiency, and adjuvanticity. The fully synthetic adjuvantꢀnanocomplexes led
to successful DC activation at lower nanomolar doses compared with free smallꢀmolecule agonists.
Once a model antigen such as ovalbumin was used for immunization, we found that the nanocomplexes
promoted an unusually strong cytotoxic
T lymphocyte response, revealing their unique
immunostimulatory capacity benefiting from multivalency and efficient transport to endosomal TLR7.
ꢀ
INTRODUCTION
Dendritic cells (DCs) are the most potent antigenꢀpresenting cells (APCs), which coordinate between
the innate and adaptive immune systems.1 They are specialized to engulf and process antigens and
subsequently present epitopes to elicit robust immune responses.2ꢀ4 APCs express various types of
pattern recognition receptors including lectins or TLRs to distinguish between selfꢀ and nonꢀselfꢀ
structures. Recognition of pathogen associated molecular patterns (PAMPs) by TLRs generally induces
DC activation.5ꢀ7 Activated DCs present foreign epitopes of antigens onto major histocompatibility
complexes (MHCs), and increase the expression of coꢀstimulatory molecules (CD80, CD86) to help
cognate interaction with T cell receptor (TCR). The expression of chemokine receptor CCR7 leads DCs
to migrate into lymph nodes, where naïve T cells are transformed into functional T lymphocytes
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 19
1
2
3
including cytotoxic T lymphocytes (CTLs).8,9
4
5
6
7
8
TLR7, located within endosomal compartment, is a promising adjuvant targetꢀsite for DCꢀ
mediated immunization. It recognizes nucleotideꢀderived compounds, including singleꢀstranded RNA
or lowꢀmolecularꢀweight imidazoquinoline derivatives, such as R837 (imiquimod) and R848
(resiquimod).5,8,10 Yet, promotion of robust CTL responses by smallꢀmolecule adjuvants is highly
challenging due to their prompt dissemination through diffusion.8,11ꢀ13 To overcome these hurdles,
polymeric or inorganic nanoparticles (NPs) encapsulating imidazoquinolines have been introduced to
enhance stability and biodistribution of TLR7 agonists, consequently improving DC activation
efficiency.14ꢀ17 Here, we describe the first synthetic approach for preparing covalently linked
imidazoquinolineꢀnanoconjugates for inducing robust CTL responses (Figure 1). Our design can
entirely avoid the potential timeꢀbased release of smallꢀmolecule agonists from the nonꢀcovalently
functionalized nanocarriers through the interactions between cell membranes and engineered NPs.
However, the challenges associated with our approach are twoꢀfold. First, the design of nanocomplexes
requires multiꢀstep reactions to achieve a molecularly wellꢀdefined structure. Second, the synthetic
nanocomplexes should effectively initiate TLRꢀmediated DC activation and subsequently induce T cell
immunity. To validate our working hypothesis, we designed alkyneꢀfunctionalized imidazoquinoline
derivatives and covalently conjugated them with biocompatible NPs to examine their role in DC
activation and generation of CTL response.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. General attributes of AdjuvantꢀNPs in inducing DC activation and a robust CTL response.
2
ACS Paragon Plus Environment

Page 3 of 19
Bioconjugate Chemistry
1
2
3
ꢀ
RESULTS AND DISCUSSION
4
5
6
7
8
Although liveꢀattenuated vaccines can elicit longꢀterm immunity, they have a potential risk of
infection, and practically not suitable vaccine candidates against pathogens such as influenza, HIV, or
Ebola virus.18 In contrast, subunit vaccines provide superior safety profiles and allow tunable design at
the molecularꢀlevel to elicit predictable immune responses. However, they are shortꢀlived and poorly
immunogenic. Thus, immunostimulatory adjuvants are required to generate potent T cell immunity.8,18
The advent of engineered nanocomplexes loaded with imidazoquinoline analogues opens up new
opportunities to effectively target TLR7, yet investigations have been established on the basis of nonꢀ
covalent encapsulation chemistry. Although CpG oligodeoxynucleotideꢀNP complexes have been
previously demonstrated,19,20 NPs covalently incorporating the smallꢀmolecule cognate ligands without
repeating monomer units have not been reported so far.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To synthesize wellꢀdefined molecular adjuvantꢀnanocomplexes, we designed and prepared an
imidazoquinoline analogue (Adjuvant 1) with a terminal alkyne moiety to couple with azide coated
iron oxide NPs (see Figure 2a). Adjuvant 2 was synthesized from 2,4ꢀquinolinediol as previously
described by the David group.21ꢀ24 Based on the previous structureꢀactivity relationship studies,21,25ꢀ28 nꢀ
butyl group was introduced at Cꢀ2 position to increase TLR7 agonistic potency. Further, an alkyne
functionality as a versatile anchor was placed at Nꢀ1 position for nextꢀstage chemical reactions, since
the site modification does not significantly compromise agonistic potency. Molecular structure of
1
TLR7 agonist, Adjuvant 1, was confirmed by H and 13C nuclear magnetic resonance (NMR) spectra
(Figures 2b and 2c). As a conjugation platform displaying multivalency, waterꢀsoluble and surfaceꢀ
engineered iron oxide NPs were selected because of their biocompatibility, facile surface modification,
monodisperse size, biomoleculeꢀfree structure, and enhanced stability. In addition, they can be
potentially applied as multifunctional agents for diagnostic/therapeutic purposes (e.g. magnetic
resonance imaging (MRI) or photothermal therapy).29ꢀ34 Biocompatible NPs with monodisperse size
ranges of ~30 nm can be used as nanocarriers in vivo, which are optimal for internalization by
immature DCs by facilitating endolysosomal pathway, and can be trafficked into the draining lymph
nodes, thereby enhancing their adjuvanticity.11,20,35ꢀ38 Amineꢀsurfaceꢀmodified iron oxide NPs (Amineꢀ
NPs) were then reacted with Spacer 3 with an activated ester moiety, to afford AzideꢀNPs, since azido
functionality can be readily installed and is highly orthogonal and versatile for further transformations.
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 19
1
2
3
4
5
6
7
Finally, Adjuvant 1 was conjugated by CuIꢀcatalyzed Huisgen 1,3ꢀdipolar cycloaddition reaction, and
treated with 0.1 M Tris buffer (pH 6) to form AdjuvantꢀNPs (for the details, see the Experimental
Section and the Supporting Information).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. (a) Synthetic scheme of AdjuvantꢀNPs (i) HBTU, TEA, DCM, (ii) CuSO4 ⋅ 5H2O, sodium ascorbate,
DMF, (iii) 0.1 M Tris buffer (pH 6), (iv) diluted hydrogen chloride solution. (b, c) 1H and 13C NMR spectra
(MeOD) of Adjuvant 1.
Coreꢀ and hydrodynamic sizes of the synthetic nanocomplexes were determined by
transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses, respectively
(Figures 3a and 3b). TEM image revealed spherical and monodisperse particles with ~11 nm core
diameters of AdjuvantꢀNPs without any signs of particle aggregation, even after multiꢀstep chemical
modifications. DLS data analysis showed an effective diameter of 31.6 nm and a narrow size
distribution with polydispersity index (PDI) = 0.258. Previously NPs having ~30 nm size have been
demonstrated to be efficiently uptaken by DCs.19,20 Moreover, we carried out spectroscopic studies to
examine the effectiveness of imidazoquinoline conjugation. UVꢀVis spectrum of AdjuvantꢀNPs showed
distinct imidazoquinoline peaks at about 225, 246, and 321 nm with slight peak shifts (Figure 3c). To
quantify the loading level, a fluorescence assay was conducted (Figure 3d), since iron oxide NPs are
weakly fluorescent. NHSꢀfluorescein is an amine reactive fluorescent probe bearing an activated ester
moiety, thus fluorophore can be appended to AdjuvantꢀNPs to generate FluoresceinꢀAdjuvantꢀNPs.
4
ACS Paragon Plus Environment

Page 5 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
Based on the standard curve of NHSꢀfluorescein and iron concentration of NPs, the loading amount of
imidazoquinolines in AdjuvantꢀNPs was estimated to be 0.139 ꢁmol/[mg Fe] (see Supporting
Information, Figures S1 and S2).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Characterization of AdjuvantꢀNPs. (a) TEM image of AdjuvantꢀNPs, (b) DLS analysis, (c) UVꢀVis
spectra, and (d) fluorescence spectra.
Further, we evaluated DC activation efficacies by using synthetic TLR7 agonists. Adjuvantꢀ
NPs or free Adjuvant 1' were intraperitoneally injected into mice, and their DCs were harvested 18
hours later. DC activation markers including CD80, CD86, MHC I, and CCR7 were stained with
phycoerythrin (PE)ꢀconjugated antibodies and analyzed by flow cytometry. R848,10 AdjuvantꢀNPs, or
Adjuvant 1' effectively increased the expression levels of the markers (Figure 4 and Figures S3ꢀS5).
Highly waterꢀsoluble Adjuvant 1' acted as an effective stimulant of DC activation at 115.6 nmol or
even at a concentration as low as 69.4 nmol (Figure S3). AmineꢀNPs showed weak selfꢀadjuvant effect
(Figure S5). Remarkably, 13.9 nmol of AdjuvantꢀNPs (concentration in loading levels of cognate
ligands) and 115.6 nmol of free Adjuvant 1' induced comparable immunostimulatory activities. This is
attributed to the enhanced avidity as well as effective internalization of the nanocomplexes to the
endosomal TLR7 of DCs. ILꢀ12p40 is known as one of important proꢀinflammatory cytokines secreted
from activated DCs to educate naïve CD8+ T cells.39 The secreted ILꢀ12p40 levels of the AdjuvantꢀNP
treated group were significantly higher than those of groups that were treated with PBS or AmineꢀNPs
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 19
1
2
3
4
5
6
7
8
9
(Figure S6a). Adjuvant 1’ also induced the production of the secreted ILꢀ12p40 with an almost
identical level. To directly detect the antigenꢀspecific CD8+ T cell response, we carried out MHC I
tetramer assays and observed increased populations of OTꢀ1 peptideꢀspecific CD8+ T cells in the
groups treated with Adjuvant 1’ or AdjuvantꢀNPs compared to those of AmineꢀNPs (Figure S6b).
These data suggested that adjuvantꢀNPs induce efficient DC activation leading to the effective
presentation of antigenic peptides on MHC I and subsequent antigenꢀspecific CTL effect.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Adjuvant effects on in vivo DC activation. (a) flow cytometry analyses, and (b and c) the mean
fluorescence intensity (MFI) levels of DC activation markers (CD80, CD86, CCR7, and MHC I). The P values <
0.05(*) <0.01(**) <0.001(***) were considered significant.
Since TLR7 is expressed inside endosomal compartments of DCs, effective delivery of
antigens and adjuvants into DCs is indispensable for their proper maturation and subsequent immune
6
ACS Paragon Plus Environment

Page 7 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
response. However, free small molecules hardly localize to TLRs; thus, they require effective delivery
vehicles. To examine cellular internalization of the nanocomplexes and their appropriate localization in
DCs, we prepared FluoresceinꢀAdjuvantꢀNPs as probes (see Supporting Information) and studied their
uptake using confocal fluorescence microscopy (Figure 5a). The complexes were cultured with
immature DCs in vitro in the presence of ovalbumin (OVA) as a model antigen. After 18 hours, the
cells were fixed, and the nuclei and low pH endosomes were stained with DAPI and LysoTracker,
respectively. Fluorescent confocal cell images clearly demonstrated that FluoresceinꢀAdjuvantꢀNPs
were localized within endosomes thanks to their suitable particle size (~30 nm) (Figure 5b and Figure
S7),19,20 which can assist imidazoquinoline cognate agonists effectively interact with TLR7 within DC
endosomes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. (a and b) Fluorescence imaging studies to examine the internalization of FluoresceinꢀAdjuvantꢀNPs in
DCs. Samples were characterized by confocal fluorescent microscopy.
The efficient DC activation and nanocomplex internalization prompted us to test whether
these mature DCs can elicit sufficient cytotoxic CD8+ T cell responses. We performed an in vivo CTL
assay based on the carboxyfluorescein diacetate succinimidyl ester (CFSE) assay to monitor OVAꢀ
specific T cell proliferation.40,41 Mice were intraperitoneally immunized with 25 ꢁg of OVA protein as
an antigen in the presence of PBS, R848, AmineꢀNPs, or AdjuvantꢀNPs as TLR7 agonists. Groups of
mice were primarily immunized for 2 weeks, and additionally boosted for 1 week. After immunization,
mice were intravenously injected with 50:50 mixtures of OTꢀ1 peptideꢀpulsed (CFSEhi) and unpulsed
(CFSElow) syngeneic splenocytes to evaluate OVAꢀspecific CTL activity. The population of OTꢀ1
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 19
1
2
3
4
5
6
7
8
9
peptide pulsed target cells was analyzed by flow cytometry. It is speculated that if OTꢀ1 specific T cells
are effectively stimulated by matured DCs with OVA protein and adjuvants, the percentage of OTꢀ1
peptideꢀpulsed (CFSEhi) syngeneic splenocytes would be lysed and their population decreased because
of T cell cytotoxicity (Figure 6a). Remarkably, injection of AdjuvantꢀNPs with 27.8 nmol of adjuvant
together with OVA protein caused 84% target cell lysis. In contrast, smallꢀmolecule R848 (28.5 or
142.5 nmol) or AmineꢀNPs showed negligible to poor (0ꢀ25%) cytotoxic responses (Figure 6b). It is
speculated that superior CD8+ T cell efficacy of AdjuvantꢀNPs at low doses of imidazoquinoline
moiety is associated with the enhanced avidity of fully synthesized multivalent adjuvantꢀNPs and
effective DC internalization.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. In vivo CTL assay on splenocytes. (a) Percentages of OTꢀ1 peptide unpulsed CFSElow (left) and that of
pulsed CFSEhigh (right) were analyzed by flow cytometry. Each group was stimulated with indicated adjuvants;
Sample A: R848 10 ꢁg, 28.5 nmol; sample B: R 848 50 ꢁg, 142.5 nmol; sample C: amineꢀNPs 100 ꢁg Fe; sample
D: amine NPs 200 ꢁg Fe; sample E: AdjuvantꢀNPs (100 ꢁg Fe, 13.9 nmol of imidazoquinoline); sample F:
AdjuvantꢀNPs (200 ꢁg Fe, 27.8 nmol of imidazoquinoline) along with OVA protein. (b) The conversion of the
percentages of CFSEhigh based on the negative control of PBS treated group. The P values < 0.01(**) were
considered significant.
ꢀ
CONCLUSION
We chemically synthesized structurally wellꢀdefined molecular adjuvantꢀnanoparticle conjugates
through multiꢀstep reactions and investigated their potency of their immunostimulatory activity. The
nanocomplexes displaying multiple lowꢀmolecularꢀweight ligands were efficiently internalized by
8
ACS Paragon Plus Environment

Page 9 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
immature DCs, and they subsequently enhanced in vivo DC activation by facilitating multivalent
interactions between imidazoquinoline moieties and endosomal TLR7. In addition, they induced
increased expression levels of activation markers in the low nanomolar range. Their cellular
localization was validated by fluorescent labeling of the nanocomplexes. Coꢀadministration of the
synthetic adjuvantꢀnanocomplexes and OVA protein elicited unusually robust antigenꢀspecific
cytotoxic T cell responses. Considering the significant challenges generating cellꢀmediated immunity
via smallꢀmolecule based adjuvant systems, we believe that our synthetic approach can provide a
versatile platform for the rational designing of nextꢀgeneration vaccines.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
EXPERIMENTAL SECTION
Synthesis of Adjuvant 1: Adjuvant 221 (400 mg, 1.11 mmol) and TEA (547 ꢀL, 3.89 mmol, 3.5 equiv)
were dissolved in DCM (80 mL). 4ꢀpentynoic acid (142 mg, 1.45 mmol, 1.3 equiv) and HBTU (549
mg, 1.45 mmol, 1.3 equiv) were added at 0 °C, and the solution left to stir overnight at room
temperature. The reaction mixture was concentrated in vacuo and the residue was purified by flash
column chromatography (DCM:MeOH:NH4OH = 9.5:0.5:0.1) to yield the title compound as a clear oil
1
(261 mg, 53%). H NMR (400 MHz, MeOD) δH 0.92 (t, J 7.4 Hz, 3H), 1.42 (dt, J 14.7, 7.4 Hz, 2H),
1.77 (dt, J 15.4, 7.6 Hz, 2H), 2.20 (t, J 2.6 Hz, 1H), 2.34ꢀ2.49 (m, 4H), 4.33 (s, 2H), 5.82 (s, 2H), 7.00
(d, J 8.1 Hz, 2H), 7.09 (m, 1H), 7.27 (d, J 8.1 Hz, 2H), 7.41 (m, 1H), 7.65 (dd, J 8.3, 0.4 Hz, 1H), 7.78
(d, J 7.8 Hz, 1H); 13C NMR (150 MHz, MeOD) δC 14.1, 15.7, 23.4, 27.8, 30.7, 35.9, 43.6, 49.5, 70.3,
83.5, 115.7, 121.5, 123.4, 126.2, 126.7, 126.9 128.5 129.4 135.4, 136.0, 139.8, 144.8, 152.5, 156.0,
173.8.; HRMS (ESI): Calcd for C27H30N5O+ [M+H]+: 440.2445, found 440.2445.
Synthesis of AzidoꢀNPs: Spacer 342 (30 mg, 116 ꢀmol) dissolved in DMF was added to the AmineꢀNPs
(1.5 mg Fe). Mixture was stirred at room temperature for a day, then dialyzed in DI water was
conducted for 3 times to remove nonꢀconjugated molecules in excess.
Synthesis of AdjuvantꢀNPs: Adjuvant 1 (2.55 mg, 5.80 ꢀmol, 15 equiv) dissolved in DMF,
CuSO4⋅6H2O (1.45 mg, 5.80 ꢀmol, 15 equiv) and (+)ꢀsodium Lꢀascorbate (1.15 mg, 5.80 ꢀmol, 15
equiv) was added to AzideꢀNPs and stirred at room temperature for a day. The reaction mixture was
9
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 19
1
2
3
4
5
6
7
8
9
dialyzed in DI water for two times then, treated with 0.1 M Tris buffer (pH 6) for 2 times to form
ammonium salt of imidazoquinoline moiety. Solution was filtered through 0.2 ꢀm pore size filter and
concentrated to 3 mg/mL Fe dissolved in autoclaved PBS buffer by using centrifugal filter (3000 rpm,
12 min).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
10.1021/acs.bioconjchem.xxxxxxx. Details of synthetic and immunological experimental procedures
and NMR spectra are provided (PDF)
ꢀ
AUTHOR INFORMATION
Corresponding Authors
*Email: sabsab7@unist.ac.kr
*Email: syhong@unist.ac.kr
Authors Contributions
§W.G.K. and B.C. contributed equally to this work. W.G.K., B.C., S.K. and S.Y.H. designed the
research project. W.G.K., B.C., H.ꢀJ.Y., J.ꢀA.H., H.J. performed the experiments, W.G.K., B.C., H.ꢀ
J.Y., J.ꢀA.H., H.J., H.C., S.K., and S.Y.H. analyzed the data, and W.G.K., B.C., S.K., and S.Y.H. wrote
the paper.
Notes
The authors declare no competing financial interests.
ꢀ
ACKNOWLEDGMENTS
This work was supported by National Research Foundation of Korea (NRFꢀ2016K1A3A1A25003511
and NRFꢀ2010ꢀ0028684). Authors are grateful to Prof. Francesco Peri for many helpful discussions.
ꢀ
REFERENCES
10
ACS Paragon Plus Environment

Page 11 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
(1) Tacken, P. J., de Vries, I. J. M., Torensma, R. and Figdor, C. G. (2007) Dendriticꢀcell immunotherapy: from ex
vivo loading to in vivo targeting. Nat. Rev. Immunol. 7, 790ꢀ802.
(2) Palucka, K. and Banchereau, J. (2012) Cancer immunotherapy via dendritic cells. Nat. Rev. Cancer 12, 265ꢀ
277.
(3) Mandal, S., Hammink, R., Tel, J., EksteenꢀAkeroyd, Z. H., Rowan, A. E., Blank, K. and Figdor, C. G. (2015)
Polymerꢀbased synthetic dendritic cells for tailoring robust and multifunctional T cell responses. ACS Chem. Biol.
10, 485ꢀ492.
(4) Mandal, S., EksteenꢀAkeroyd, Z. H., Jacobs, M. J., Hammink, R., Koepf, M., Lambeck, A. J. A., van Hest, J.
C. M., Wilson, C. J., Blank, K., Figdor, C. G. et al. (2013) Therapeutic nanoworms: towards novel synthetic
dendritic cells for immunotherapy. Chem. Sci. 4, 4168ꢀ4174.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5) O'Neill, L. A. J., Golenbock, D. and Bowie, A. G. (2013) The history of Tollꢀlike receptorsꢀredefining innate
immunity. Nat. Rev. Immunol. 13, 453ꢀ460.
(6) Iwasaki, A. and Medzhitov, R. (2010) Regulation of adaptive immunity by the innate immune system. Science
327, 291ꢀ295.
(7) Tom, J. K., Dotsey, E. Y., Wong, H. Y., Stutts, L., Moore, T., Davies, D. H., Felgner, P. L. and EsserꢀKahn, A. P.
(2015) Modulation of innate immune responses via covalently linked TLR agonists. ACS Cent. Sci. 1, 439ꢀ448.
(8) Moyle, P. M. and Toth, I. (2013) Modern subunit vaccines: development, components, and research
opportunities. ChemMedChem 8, 360ꢀ376.
(9) Palucka, K., Banchereau, J. and Mellman, I. (2010) Designing vaccines based on biology of human dendritic
cell subsets. Immunity 33, 464ꢀ478.
(10) Hemmi, H., Kaisho, T., Takeuchi, O., Sato, S., Sanjo, H., Hoshino, K., Horiuchi, T., Tomizawa, H., Takeda, K.
and Akira, S. (2002) Small antiꢀviral compounds activate immune cells via the TLR7 MyD88ꢀdependent signaling
pathway. Nat. Immunol. 3, 196ꢀ200.
(11) Moon, J. J., Huang, B. and Irvine, D. J. (2012) Engineering nanoꢀ and microparticles to tune immunity. Adv.
Mater. 24, 3724ꢀ3746.
(12) Rajagopal, D., Paturel, C., Morel, Y., Uematsu, S., Akira, S. and Diebold, S. S. (2010) Plasmacytoid dendritic
cellꢀderived type I interferon is crucial for the adjuvant activity of Tollꢀlike receptor 7 agonists. Blood 115, 1949ꢀ
1957.
(13) Warshakoon, H. J., Hood, J. D., Kimbrell, M. R., Malladi, S., Wu, W. Y., Shukla, N. M., Agnihotri, G., Sil, D.
and David, S. A. (2009) Potential adjuvantic properties of innate immune stimuli. Hum. Vaccin. 5, 381ꢀ394.
(14) Kasturi, S. P., Skountzou, I., Albrecht, R. A., Koutsonanos, D., Hua, T., Kakaya, H. I., Ravindran, R., Stewart,
S., Alam, M., Kwissa, M. et al. (2011) Programming the magnitude and persistence of antibody responses with
innate immunity. Nature 470, 543ꢀ550.
(15) Heo, M. B. and Lim, Y. T. (2014) Programmed nanoparticles for combined immunomodulation, antigen
presentation and tracking of immunotherapeutic cells. Biomaterials 35, 590ꢀ600.
(16) Ilyinskii, P. O., Roy, C. J., O'Neil, C. P., Browning, E. A., Pittet, L. A., Altreuter, D. H., Alexis, F., Tonti, E.,
Shi, J., Basto, P. A. et al. (2014) Adjuvantꢀcarrying synthetic vaccine particles augment the immune response to
encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release.
Vaccine 32, 2882ꢀ2895.
(17) Tacken, P. J., Zeelenberg, I. S., Cruz, L. J., van HoutꢀKuijer, M. A., van de Glind, G., Fokkink, R. G.,
Lambeck, A. J. A. and Figdor, C. G. (2011) Targeted delivery of TLR ligands to human and mouse dendritic cells
strongly enhances adjuvanticity. Blood 118, 6836ꢀ6844.
(18) Coffman, R. L., Sher, A. and Seder, R. A. (2010) Vaccine adjuvants: putting innate immunity to work.
Immunity 33, 492ꢀ503.
(19) de Titta, A., Ballester, M., Julier, Z., Nembrini, C., Jeanbart, L., van der Vlies, A. J., Swartz, M. A. and
Hubbell, J. A. (2013) Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory
recall at low dose. Proc. Natl. Acad. Sci. U.S.A. 110, 19902ꢀ19907.
(20) Molino, N. M., Anderson, A. K. L., Nelson, E. L. and Wang, S.ꢀW. (2013) Biomimetic protein nanoparticles
facilitate enhanced dendritic cell activation and crossꢀpresentation. ACS Nano 7, 9743ꢀ9752.
(21) Shukla, N. M., Malladi, S. S., Mutz, C. A., Balakrishna, R. and David, S. A. (2010) Structureꢀactivity
relationships in human tollꢀlike receptor 7ꢀactive imidazoquinoline analogues. J. Med. Chem. 53, 4450ꢀ4465.
(22) Shukla, N. M., Mutz, C. A., Ukani, R., Warshakoon, H. J., Moore, D. S. and David, S. A. (2010) Syntheses of
fluorescent imidazoquinoline conjugates as probes of Tollꢀlike receptor 7. Bioorg. Med. Chem. Lett. 20, 6384ꢀ
6386.
(23) Shukla, N. M., Lewis, T. C., Day, T. P., Mutz, C. A., Ukani, R., Hamilton, C. D., Balakrishna, R. and David,
S. A. (2011) Toward selfꢀadjuvanting subunit vaccines: model peptide and protein antigens incorporating
covalently bound tollꢀlike receptorꢀ7 agonistic imidazoquinolines. Bioorg. Med. Chem. Lett. 21, 3232ꢀ3236.
(24) Shukla, N. M., Mutz, C. A., Malladi, S. S., Warshakoon, H. J., Balakrishna, R. and David, S. A. (2012) Tollꢀ
like receptor (TLR)ꢀ7 and ꢀ8 modulatory activities of dimeric imidazoquinolines. J. Med. Chem. 55, 1106ꢀ1116.
(25) Schiaffo, C. E., Shi, C., Xiong, Z., Olin, M., Ohlfest, J. R., Aldrich, C. C. and Ferguson, D. M. (2014)
Structureꢀactivity relationship analysis of imidazoquinolines with Tollꢀlike receptors 7 and 8 selectivity and
enhanced cytokine induction. J. Med. Chem. 57, 339ꢀ347.
(26) Shi, C., Xiong, Z., Chittepu, P., Aldrich, C. C., Ohlfest, J. R. and Ferguson, D. M. (2012) Discovery of
imidazoquinolines with Tollꢀlike receptor 7/8 independent cytokine induction. ACS Med. Chem. Lett. 3, 501ꢀ504.
(27) Ryu, K. A., Stutts, L., Tom, J. K., Mancini, R. J. and EsserꢀKahn, A. P. J. (2014) Stimulation of innate
immune cells by lightꢀactivated TLR7/8 agonists. J. Am. Chem. Soc. 136, 10823ꢀ10825.
11
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 12 of 19
1
2
3
4
5
6
7
8
9
(28) Yoo, E., Salunke, D. B., Sil, D., Guo, X., Salyer, A. C. D., Hermanson, A. R., Kumar, M., Malladi, S. S.,
Balakrishna, R., Thompson, W. H. et al. (2014) Determinants of activity at human Tollꢀlike receptors 7 and 8:
quantitative structureꢀactivity relationship (QSAR) of diverse heterocyclic scaffolds. J. Med. Chem. 57, 7955ꢀ
7970.
(29) Dobrovolskaia, M. A. and McNeil, S. E. (2007) Immunological properties of engineered nanomaterials. Nat.
Nanotech. 2, 469ꢀ478.
(30) Gupta, A. K. and Gupta, M. (2005) Synthesis and surface engineering of iron oxide nanoparticles for
biomedical applications. Biomaterials 26, 3995ꢀ4021.
(31) Na, H. B., Song, I. C. and Hyeon, T. (2009) Inorganic nanoparticles for MRI contrast agents. Adv. Mater. 21
,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2133ꢀ2148.
(32) de Vries, I. J. M., Lesterhuis, W. J., Barentsz, J. O., Verdijk, P., van Krieken, J. H., Boerman, O. C., Oyen, W.
J., Bonenkamp, J. J., Boezeman, J. B., Adema, G. J. et al. (2005) Magnetic resonance tracking of dendritic cells in
melanoma patients for monitoring of cellular therapy. Nat. Biotechnol. 23, 1407ꢀ1413.
(33) Song, X., Gong, H., Yin, S., Cheng, L., Wang, C., Li, Z., Li, Y., Wang, X., Liu, G. and Liu, Z. (2014) Ultraꢀ
small iron oxide doped polypyrrole nanoparticles for in vivo multimodal imaging guided photothermal therapy.
Adv. Funct. Mater. 24, 1194ꢀ1201.
(34) Yang, K., Hu, L., Ma, X., Ye, S., Cheng, L., Shi, X., Li, C., Li, Y. and Liu, Z. (2012) Multimodal imaging
guided photothermal therapy using functionalized graphene nanosheets anchored with magnetic nanoparticles.
Adv. Mater. 24, 1868ꢀ1872.
(35) Irvine, D. J., Hanson, M. C., Rakhra, K. and Tokatlian, T. (2015) Synthetic nanoparticles for vaccines and
immunotherapy. Chem. Rev. 115, 11109ꢀ11146.
(36) Leleux, J. and Roy, K. (2013) Micro and nanoparticleꢀbased delivery systems for vaccine immunotherapy: an
immunological and materials perspective. Adv. Healthcare Mater. 2, 72ꢀ94.
(37) Mintern, J. D., Percival, C., Kamphuis, M. M. J., Chin, W. J., Caruso, F. and Johnston, A. P. R. (2013)
Targeting dendritic cells: the role of specific receptors in the internalization of polymer capsules. Adv. Healthcare
Mater. 2, 940ꢀ944.
(38) Smith, D. M., Simon, J. K. and Baker, J. R. (2013) Applications of nanotechnology for immunology. Nat. Rev.
Immunol. 13, 592ꢀ605.
(39) Koch, F., Stanzl, U., Jennewein, P., Janke, K., Heufler, C., Kampgen, E., Romani, N. and Schuler, G. (1996)
High level ILꢀ12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and
downregulation by ILꢀ4 and ILꢀ10. J. Exp. Med. 184, 741ꢀ746.
(40) Han, J.ꢀA., Kang, Y. J., Shin, C., Ra, J.ꢀS., Shin, H.ꢀH., Hong, S. Y., Do, Y. and Kang, S. (2014) Ferritin
protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell (DC)ꢀbased vaccine
development. Nanomedicine 10, 561ꢀ569.
(41) Quah, B. J. C., Warren, H. S. and Parish, C. R. (2007) Monitoring lymphocyte proliferation in vitro and in
vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2, 2049ꢀ
2056.
(42) Lampkins, A. J., O'Neil, E. J. and Smith, B. D. (2008) Bioꢀorthogonal phosphatidylserine conjugates for
delivery and imaging applications. J. Org. Chem. 73, 6053ꢀ6058.
TOC Image
12
ACS Paragon Plus Environment

Page 13 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. General attributes of Adjuvant-NPs in inducing DC activation and a robust CTL response.
308x120mm (150 x 150 DPI)
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 14 of 19
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. (a) Synthetic scheme of Adjuvant-NPs (i) HBTU, TEA, DCM, (ii) CuSO4 5H2O, sodium ascorbate,
DMF, (iii) 0.1 M Tris buffer (pH 6), (iv) diluted hydrogen chloride solution. (b, c) 1H and 13C NMR spectra
(MeOD) of Adjuvant 1.
207x152mm (150 x 150 DPI)
ACS Paragon Plus Environment

Page 15 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Characterization of Adjuvant-NPs. (a) TEM image of Adjuvant-NPs, (b) DLS analysis, (c) UV-Vis
spectra, and (d) fluorescence spectra.
246x160mm (150 x 150 DPI)
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 16 of 19
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Adjuvant effects on in vivo DC activation. (a) flow cytometry analyses, and (b and c) the mean
fluorescence intensity (MFI) levels of DC activation markers (CD80, CD86, CCR7, and MHC I). The P values
< 0.05(*) <0.01(**) <0.001(***) were considered significant.
262x278mm (150 x 150 DPI)
ACS Paragon Plus Environment

Page 17 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. (a and b) Fluorescence imaging studies to examine the internalization of Fluorescein-Adjuvant-NPs
in DCs. Samples were characterized by confocal fluorescent microscopy.
327x169mm (150 x 150 DPI)
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 18 of 19
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. In vivo CTL assay on splenocytes. (a) Percentages of OT-1 peptide unpulsed CFSElow (left) and
that of pulsed CFSEhigh (right) were analyzed by flow cytometry. Each group was stimulated with indicated
adjuvants; Sample A: R848 10 µg, 28.5 nmol; sample B: R 848 50 µg, 142.5 nmol; sample C: amine-NPs
100 µg Fe; sample D: amine NPs 200 µg Fe; sample E: Adjuvant-NPs (100 µg Fe, 13.9 nmol of
imidazoquinoline); sample F: Adjuvant-NPs (200 µg Fe, 27.8 nmol of imidazoquinoline) along with OVA
protein. (b) The conversion of the percentages of CFSEhigh based on the negative control of PBS treated
group. The P values < 0.01(**) were considered significant.
303x169mm (150 x 150 DPI)
ACS Paragon Plus Environment

Page 19 of 19
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
TOC image
250x157mm (150 x 150 DPI)
ACS Paragon Plus Environment