A Renal-Clearable Macromolecular Reporter for Near-Infrared Fluorescence Imaging of Bladder Cancer

Article abstract of DOI:10.1002/anie.201911859

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer-specific optical agents with high renal clearance. Herein, a macromolecular reporter (Cy

Full text of DOI:10.1002/anie.201911859

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Renal-clearable Macromolecular Reporter for Near-infrared
Fluorescence Imaging of Bladder Cancer
Authors: Kanyi Pu, Jiaguo Huang, Yuyan Jiang, Jingchao Li, Shasha
He, and Jingsheng Huang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201911859
Angew. Chem. 10.1002/ange.201911859
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10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
Molecularly Engineered Macrophage-Derived Exosomes with
Inflammation Tropism and Intrinsic Biosynthesis for
Atherosclerosis Treatment
Guanghao Wu[a], Qianru Zhao[a], Wanru Zhuang[a], Jingjing Ding[a], Chi Zhang[c], Haijun Gao[a], Dai-Wen
Pang[b], Jinfeng Zhang[a], Kanyi Pu* [c], Hai-Yan Xie*[a]
drugs are lack of specific targeting capability toward the
Abstract: Atherosclerosis is a major contributor to cardiovascular
inflammatory sites and usually show poor half-life, which not
diseases worldwide, and anti-inflammation is a promising strategy
merely compromise their practical therapeutic efficacy but also
for atherosclerosis treatment. Despite the progress made with
result in numerous side effects, even lead to death. Fortunately,
recent use of nanocarriers, development of effective
inflammation generally accompanies with leaky vasculature, thus
atherosclerosis targeting and inflammation-alleviation strategy is
nanoparticles can passively accumulate in the pathological areas.
still challenging. Here, we report the molecularly engineered M2
Various nanocarriers have been designed to load and deliver
macrophage-derived exosomes (M2 Exo) with inflammation-
drugs to atherosclerotic sites with improved permeability and
tropism and anti-inflammatory capabilities for atherosclerosis
retention.[3] However, the nanoparticle-based drug-delivery
imaging and therapy. These engineered M2 Exo are derived from
systems still suffer from some inherent disadvantages, such as
M2 macrophages and further electroporated with an FDA-
the immunogenicity and toxicity of the carrier itself, fast immune
approved hexyl 5-aminolevulinate hydrochloride (HAL). After
recognition and blood clearance, and poor biodistribution.[4]
systematic administration, the engineered M2 Exo exhibit
Consequently, developing more reliable and powerful approaches
excellent inflammation-tropism and anti-inflammation effects via
for the atherosclerosis treatment are highly desired but
the surface-bonded chemokine receptors and released anti-
challenging.
inflammatory cytokines from the anti-inflammatory M2
macrophages. Moreover, the encapsulated HAL can undergo
intrinsic biosynthesis and metabolism of heme to generate anti-
inflammatory carbon monoxide and bilirubin, which further
enhance the anti-inflammation effects and finally alleviate
atherosclerosis. Meanwhile, the intermediate protoporphyrin IX
(PpIX) of heme biosynthesis pathway permits the fluorescence
imaging and tracking of atherosclerosis. These M2 Exo
engineered with inherent inflammation tropism and intrinsic
biosynthesis of anti-inflammatory molecules can open a new
avenue towards the treatment of atherosclerosis.
Very recently, considerable progresses have been made in the
field of exosomes which are secreted nanovesicles (30–150 nm)
from various cell types, featuring with fascinating natural
properties, including outstanding biocompatibility, low cytotoxicity,
immunological inertness, specific targeting and long-term
circulating capability, making them effective drug delivery
vehicles.[5] Exosomes derived from tumor cells, immune cells and
mesenchymal stem cells are successfully applied in
encapsulating and delivering chemotherapeutic drugs, nucleic
acid drugs, neurotransmitters and even nanoparticles for the
treatment of different diseases, such as cancer and neurological
diseases, but have been rarely used in cardiovascular diseases
up to now.[6] Moreover, the specific accumulation of exosome-
based drug delivery systems is still limited even though they are
functionalized with targeting biomolecules, therefore, inevitably
leading to some adverse effects. It is worth noting that besides
the unique properties, exosomes enrich specific contents (e.g.
RNA, DNA, proteins and small molecules) derived from the
parental cells, thus grafting the native biological functions of
original cells that may be directly used for disease treatment. For
example, exosomes derived from different stem cells and cardiac
progenitor cells can enhance the cardiac function following
myocardial infarction owing to the abundant miRNA in the
exosomes.[7] Interestingly, it has been recently reported that
exosomes isolated from macrophages are of the intrinsic
inflammation-tropism capability owing to the various chemokine
receptors on their surface.[8] Moreover, M2 phenotype
macrophages can secrete anti-inflammatory cytokines such as
interleukin-10 (IL-10), interleukin-1Ra (IL-1Ra) and transforming
growth factor (TGF-β), implying the potential anti-inflammation
therapy effect of M2 macrophages-derived exosomes (M2 Exo).
[9] However, both the inflammation targeting and anti-inflammation
capabilities of M2 Exo have not been revealed yet to the best of
our knowledge.
Introduction
Atherosclerosis is a major contributor to cardiovascular diseases
which is one of the leading causes of morbidity and mortality
worldwide. Inflammation is not only the key indication of
atherosclerosis but also drives the whole disease progression.[1]
Therefore, anti-inflammation has been regarded as a promising
strategy for atherosclerosis treatment.[2] Despite some
remarkable successes, the existing clinical anti-atherosclerotic
[a]
G. Wu, Q. Zhao, W. Zhuang, J. Ding, H. Gao, Prof. J. Zhang, Prof.
H. Xie
School of Life Science
Beijing Institute of Technology
No.5 South Zhong Guan Cun Street, Beijing, 100081 (China)
E-mail: hyanxie@bit.edu.cn
[b]
[c]
D. Pang
Research Center for Analytical Sciences, College of Chemistry
Nankai University
No.94 Weijin Road, Nankai District, Tianjin, 300071 (China)
C. Zhang, Prof. K. Pu
School of Chemical and Biomedical Engineering, Nanyang
Technological University, Singapore, 637457, Singapore
E-mail: kypu@ntu.edu.sg
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under: https://doi.org/
This article is protected by copyright. All rights reserved.

10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
Herein, we molecularly engineer the M2 Exo to endow them with
inherent inflammation tropism and intrinsic biosynthesis
functionality for effective targeting and potent treatment of
atherosclerosis. The M2 Exo are collected from M2 macrophages
are further electroporated with an FDA-approved hexyl 5-
aminolevulinate hydrochloride (HAL) to obtain HAL-containing M2
exosomes (HAL@M2 Exo).[10] After systemic administration, the
as-prepared HAL@M2 Exo can bind to the inflammatory
endothelial cell in atherosclerosis sites via the chemokine
receptors presented on M2 Exo surface, and subsequently
undergo tethering, rolling, adhering and transmigrating across the
endothelial cell layers, eventually accumulating in the
atherosclerosis lesions.[11] Then, the uptake of HAL@M2 Exo by
inflammatory cells leads to the release of endogenous anti-
inflammatory cytokines and exogenous HAL. HAL initiates the
subsequent biosynthesis and metabolism of heme to produce two
extra anti-inflammatory compounds, carbon monoxide (CO) and
bilirubin.[12] The anti-inflammatory cytokines together with CO and
bilirubin significantly alleviate the inflammation-induced
atherosclerosis in vivo. Meanwhile, the intermediate
protoporphyrin IX (PpIX) of heme biosynthesis pathway can be
further used for fluorescence imaging and tracking of
into M2 Exo through electroporation. The successful HAL loading
was confirmed by high-performance liquid chromatography
(HPLC) analysis, and the loading capacity as well as
encapsulation efficiency of HAL were determined to be 25.14 %
and 19.34 %, respectively (Figure 2e and Figure S2). The average
size and zeta potential of the as-prepared HAL@M2 Exo
exhibited similar results as those of M2 Exo alone (Figure 2f),
indicating that the surface properties of M2 Exo would not be
changed greatly after drug loading. Furthermore, both M2 Exo
and HAL@M2 Exo showed excellent colloidal stability in
phosphate buffered saline (PBS) in a week (Figure 2g).
Generally, macrophages are defined as two phenotypes
including pro-inflammatory M1 phenotype and anti-inflammatory
M2 phenotype.[14] We then comparatively evaluated the secretion
levels of anti-inflammatory cytokines (e.g., IL-10, IL-1Ra and
TGF-β) and pro-inflammatory cytokines (e.g., interleukin-6 (IL-6),
tumor necrosis factor (TNF-α) and metalloproteinase-10 (MMP-
10)) in exosomes derived from pristine macrophages (M0 Exo),
exosomes derived from M1-polarized macrophages (M1 Exo)
(Figure S3), M2 Exo and HAL@M2 Exo. As presented in Figure
2h, both M2 Exo and HAL@M2 Exo showed much higher level of
anti-inflammatory factors but much lower quantity of pro-
inflammatory factors when compared with M0 Exo and M1 Exo,
suggesting the potential anti-inflammatory responses of M2 Exo.
For further verification, the inflammatory M1 cells were treated
with M2 Exo or HAL@M2 Exo for 24 h, and then the phenotype
transition of cells was evaluated by flow cytometry. We were glad
to find that M2 Exo or HAL@M2 Exo treatment could significantly
induce the reprogramming of inflammatory M1 cells as illustrated
by the clearly increased expression of M2 biomarkers: CD206 and
CD163; meanwhile the dramatically decrease of M1 biomarkers:
CD80 and CD86, indicating the effective anti-inflammatory
performance of M2 Exo and HAL@M2 Exo (Figure 2i).
atherosclerosis.[13]
The
smart
engineered
and
self-
biosynthesizable exosomes show great potential as a reliable
candidate for atherosclerosis treatment.
Figure 1. Scheme illustration of the anti-atherosclerosis treatment
by using HAL@M2 Exo. a) The preparation of HAL@M2 Exo. b)
The inflammation-tropism and anti-inflammation effect of
HAL@M2 Exo. c) The simplified biosynthesis and metabolism
pathway of heme induced by HAL.
Results and Discussion
Preparation and characterization of the HAL loading M2 Exo.
The pristine macrophages (M0) were firstly polarized to M2
phenotype by culturing RAW264.7 cells with interleukin-4 (IL-4)
(Figure S1). The corresponding M2 Exo collected by differential
ultracentrifugation were well dispersed with cup-shaped
nanostructure of approximate 180 nm in diameter (Figure 2a).
They were rich in M2 biomarkers (CD206 and CD163) as
characterized by different methods including flow cytometry,
western blotting and confocal laser scanning microscopy (CLSM),
in which CD63 is a representative exosome protein (Figure 2b-d).
Afterwards, the hydrophilic HAL molecules were encapsulated
Figure 2. Characterization of M2 Exo. a) TEM image of M2 Exo.
Scale bar: 200 nm. b, c) Flow cytometry and western blotting
analysis of CD63, CD206 and CD163 expressed on M2 Exo. d)
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10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
Fluorescence colocalization of CD63, CD206 and CD163 on M2
Exo. Scale bar: 1µm. e) HPLC analysis of HAL in M2 Exo. f) Sizes
and zeta potentials of M2 Exo and HAL@M2 Exo. g) The stability
of M2 Exo and HAL@M2 Exo during 7 days incubation monitored
according to the size and zeta potential in PBS. h) The content of
anti-inflammatory and pro-inflammatory cytokines in different
exosomes. i) Flow cytometry analysis of CD206, CD163, CD80 or
CD86 positive inflammatory cells before and after different
treatments. The results represent the mean ± standard deviation
(n = 3).
Efficient biosynthesis and metabolism of heme induced by
HAL in cells. It is reported that HAL can be enzymatically
synthesized to the intermediate protoporphyrin IX (PpIX) in Figure
3a. In the downstream biosynthesis, the PpIX is converted into
heme, which then efficiently converts into CO and bilirubin as the
final catabolic metabolites in cells.[15] Firstly, to verify the formation
of PpIX, characteristic red fluorescence of PpIX was visualized by
CLSM. As shown in Figure 3b, both free HAL or HAL@M2 Exo
treated inflammatory cells exhibited clearly bright red signals,
whereas M2 Exo alone or PBS treated groups showed negligible
fluorescence, indicating that diagnostic molecule PpIX could be
successfully biosynthesized from HAL substrate in the cells.
Notably, red signals of PpIX substantially increased in cells upon
addition of heme oxygenase-1 (HO-1) inhibitor, which were
attributed to the blocking of downstream pathway and thus the
enhanced accumulation of PpIX. To further confirm the successful
generation of the final metabolites, CO was detected trough
CLSM by using an allyl chloroformate functionalized fluorescein
(FL-CO-1) as the CO sensor and bilirubin was detected by ELISA
assay.[16] As expected, HAL-containing groups showed the
obvious CO and bilirubin production (Figure 3c and d). In contrast,
these two metabolites were hard to find in the absence of HAL or
in the presence of HO-1 inhibitor because lacking of essential
substrate or requisite enzyme. Interestingly, it was also found that
more metabolic products could be formed in HAL@M2 Exo
treated cells compared with free HAL treated group, probably
because that the high hydrophilicity of HAL molecules would
reduce its transmembrane transport while M2 Exo could improve
the uptake through endocytosis and membrane fusion. To further
support the above results, we detected the expression levels of
three key catalytic enzymes including uroporphyrinogen
decarboxylase (UROD) and protoporphyrinogen oxidase (PPOX)
that related to the PpIX biosynthesis as well as HO-1 that
associated with the bilirubin generation. It could be found that all
the three enzyme levels were remarkably up-regulated in HAL or
HAL@M2 Exo treated inflammatory cells (Figure 3e-g), which
were well in accordance with the above results. All together, these
results strongly suggested that exogenous HAL molecules could
prominently augment the enzyme-catalyzed biosynthesis and
Figure 3. The biosynthesis and metabolism of heme induced by
HAL in inflammatory cells. a) Simplified pathway. b, c)
Fluorescence imaging and corresponding mean fluorescence
intensity (MFI) statistics of PpIX or CO in cells after different
treatments. Scale bar: 5 µm. d) ELISA assays for bilirubin in cells
after different treatments. e, f) RT-PCR analysis for mRNA level
of UROD or PPOX in cells after different treatments. g) Western
blotting analysis of HO-1 in cells after different treatments. The
results represent the mean ± standard deviation (n = 3).
receptors associated to M2 Exo recruitment (Figure 4a). Firstly,
the CLSM imaging, western blotting and flow cytometry analyses
of both M2 Exo and HAL@M2 Exo verified the presence of three
chemokine receptors including CC chemokine receptor type 2
(CCR2), CXC chemokine receptor
2 (CXCR2) and CXC
chemokine ligand 2 (CXCL2) (Figure 4b, c and Figure S4), which
would respond to the inflammatory stimuli thus lead to the slowing
down and recruitment of M2 Exo based nanovehicles at
inflammatory sites. Then, the expression of adhesion molecules
including E-selectin, vascular cell adhesion molecule 1 (VCAM1)
and endothelial intercellular adhesion molecule 1 (ICAM1) on
inflammatory endothelial cells as well as their corresponding
chemokine receptors including CD44, monocyte very late antigen
4 (VLA4), and monocyte lymphocyte function-associated antigen
1 (LFA1) presented on M2 Exo membranes were respectively
examined by flow cytometry and western blotting. As depicted in
Figure 4d-g, the adhesion molecules were up-regulated on
activated human umbilical vein endothelial cells (HUVECs)
compared with the normal HUVECs; meanwhile, both M2 Exo and
HAL@M2 Exo showed high levels of the three receptors,
suggesting the strong molecular recognition between the
HAL@M2 Exo and activated HUVECs. Therefore, the initially
recruited M2 Exo or HAL@M2 Exo would firmly adhere to the
activated inflammatory endothelial cells via the E-selectin-CD44,
VCAM1-VLA4, and ICAM1-LFA1 interactions, which would further
facilitate their following transmigration. Moreover, the platelet
endothelial cell adhesion molecule (PECAM), one of the
endothelial transmembrane proteins playing important roles in
regulating transmigration,[17] was concurrently highly expressed
on M2 Exo based nanovehicles and activated HUVECs (Figure
4d-g), which would ultimately lead to the superior transmigration
metabolism of
heme,
synchronously
generating
the
corresponding functional intermediate and metabolites.
Excellent Inflammation-tropism capability of HAL@M2 Exo.
To evaluate the potential inflammation-tropism capability of the
as-prepared HAL@M2 Exo and clarify its relevant mechanisms,
we conducted a series of in vitro experiments to investigate the
presence of representative adhesion molecules and chemokine
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10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
and accumulation of M2 Exo based nanovehicles at inflammation
sites.
oxidized low-density lipoprotein (oxLDL) and LPS. Firstly, the
status of oxidative stress including generation of reactive oxygen
species (ROS) and inducible nitric oxide synthase (iNOS) as well
as expression of typical pro-inflammatory cytokines including
MMP-10, IL-6, TNF-α and interleukine-1β (IL-1β) were evaluated
in common inflammatory cells. As depicted in Figure 5a-c,
benefiting from the satisfactory anti-inflammation effect of HAL
and M2 Exo, both oxidative species and inflammatory cytokines
were obviously reduced in free HAL or M2 Exo treated groups
compared with the PBS control group (CN). Importantly, these
decreases were further significantly enhanced in the cells treated
with HAL@M2 Exo.
To further confirm the transmigration capability and uptake
efficiency of the HAL@M2 Exo, in vitro Transwell assays were
subsequently carried out as illustrated in Figure 4g. A monolayer
of activated HUVECs and inflammatory cells were respectively
incubated on the top chamber and bottom chamber for 24 h. Then,
four groups including (1): M2 Exo, (2): M2 Exo + LPS, (3):
HAL@M2 Exo, and (4): HAL@M2 Exo + LPS were individually
added to the upper chamber of the Transwell models. In LPS
induced inflammatory microenvironment, both M2 Exo and
HAL@M2 Exo exhibited dramatically higher efficiency of
transmigrating into basolateral chamber supernatant and
enhanced accumulation in inflammatory cells. In contrast, much
lower transmigration and uptake efficiency of both M2 Exo or
HAL@M2 Exo without LPS stimulation. All these results together
proved that HAL@M2 Exo were of excellent inflammation-tropism
capability owing to the intrinsic inflammatory affinities originating
from polarized M2 macrophages, which would be beneficial for
the following in vitro and in vivo anti-inflammation treatment.
Figure 5. Anti-inflammation and promoting cholesterol efflux
effects of HAL@M2 Exo. a) Fluorescence imaging of ROS
(Green: DCFH-DA labeled ROS) in inflammatory cells after
different treatments. Scale bar: 20 µm. b) Immunofluorescence
analysis of iNOS in inflammatory cells (Red: iNOS; Blue: cell
nuclei). Scale bar: 20 µm. c) ELISA assays of typical pro-
inflammatory cytokines in inflammatory cells after different
treatments. d) Fluorescence imaging of oxLDL in foam cells after
different treatments (Red: DiI-labeled oxLDL; Blue: cell nuclei;
Green: cell membrane). Scale bar: 20 µm. e) Western blotting
analyses of ABCA-1 and SR-BI in the foam cells after different
treatments. The results represent the mean ± standard deviation
(n = 3).
Subsequently, we investigated the anti-inflammation effect of
HAL@M2 Exo on foam cells which are crucial underpinnings to
early atherosclerosis establishment.[18] Notably, treatment of the
foam cells with free HAL, M2 Exo or HAL@M2 Exo could greatly
decrease the intracellular oxLDL content (Figure 5d and Figure
S5), which is a fundamental element involved in the progression
of foam cells. In addition, the expression levels of ATP-binding
cassette subfamily-A transporters-1 (ABCA-1) and scavenger
receptor class B type I (SR-BI) on foam cells after treatment were
also greatly up-regulated compared with the PBS control group
(Figure 5e), revealing that either HAL or M2 Exo containing
samples could facilitate the cholesterol efflux in foam cells
because these two receptors are responsible for the reverse
cholesterol transport (RCT) pathway. Notably, both the levels of
Figure 4. Inflammation-tropism of M2 Exo. a) Schematic model of
Exo adhesion cascade in the inflammatory sites. b) Fluorescence
imaging of chemokine receptors CCR2, CXCR2 and CXCL2 on
M2 Exo by using CD63 as the marker of exosomes. Scale bar:
1µm. c) Western blotting analysis of chemokine receptors CCR2,
CXCR2 and CXCL2 on M2 Exo or HAL@M2 Exo. d) Flow
cytometry analysis of cell-adhesion molecules E-selectin, VCAM1,
ICAM1 or PECAM positive HUVECs and activated HUVECs. e)
Flow cytometry analysis of receptors CD44, VLA4, LFA1 or
PECAM positive M2 Exo and HAL@M2 Exo. f) Western blotting
analysis of corresponding adhesion molecules and receptors in d,
e. g) The established Transwell model and time-dependent
quantification of M2 Exo or HAL@M2 Exo in the supernatant or
inflammatory cells of bottom chamber.The results represent the
mean ± standard deviation (n = 3).
Enhanced In vitro anti-inflammation effect of HAL@M2 Exo.
After validating the favorable tropism capability of HAL@M2 Exo,
we next comparatively examined the anti-inflammation effect of
HAL, M2 Exo and HAL@M2 Exo in common inflammatory cells
stimulated by LPS or typical foam cells simultaneously induced by
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10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
number of neutrophils in peritoneal exudates as well as the
enzymatic activity of myeloperoxidase (MPO) which is a typical
marker for neutrophils were determined 24 h after treatments. As
shown in Figure 6b and c, a large number of neutrophils infiltrated
and sequestrated in abdominal cavity and high MPO activity
exhibited in the control group. By contrast, administration of HAL
or M2 Exo based formulations could effectively diminish the
retention of infiltrated neutrophils in abdominal cavities,
meanwhile decrease the MPO activity. Particularly, the HAL@M2
Exo treated group displayed the lowest proportion of neutrophils
in peritoneal exudates, reflecting that HAL@M2 Exo could
significantly ameliorate acute peritonitis. Besides, the expression
levels of pro-inflammatory cytokines including MMP-10, IL-6,
TNF-α and IL-1β were correspondingly downregulated (Figure 6d
and Figure S7), further confirming that HAL@M2 Exo can
substantially alleviate inflammation in vivo.
ABCA-1 and SR-BI in HAL@M2 Exo treated group were clearly
higher than the other groups. Such RCT upregulation would be
further in favor of the ultimate anti-atherosclerotic outcome of
HAL@M2 Exo. Collectively, all these data implicated that the
combination of HAL and M2 Exo could effectively enhance the in
vitro anti-inflammation capability and HAL@M2 Exo is potentially
applied to the following in vivo therapy.
Superior in vivo anti-inflammation therapy in acute
peritonitis. Encouraged by the great inflammation-tropism and
inflammation-alleviation capabilities of HAL@M2 Exo in vitro, we
next investigated their in vivo performance by using an acute
peritonitis mouse model. The BALB/c mice were firstly
intraperitoneally injected with sterile zymosan solution (0.5 mL, 2
mg/mL).[19] After 12 h, different formulations including free HAL,
M2 Exo and HAL@M2 Exo were intravenously injected into the
mice respectively, and PBS was used as the control group. Time-
dependent in vivo accumulations of HAL or HAL@M2 Exo were
tracked by using the intrinsic fluorescence of HAL-converted PpIX.
It was observed that most HAL@M2 Exo could target and
accumulate in inflammatory abdominal cavity (indicated by the
red circles) while free HAL molecules were mainly located in other
sites, especially in liver (Figure 6a and Figure S6), demonstrating
the as-designed HAL@M2 Exo can effectively target to
inflammation sites and generate diagnostic signals in situ for in
vivo imaging.
Efficient In vivo treatment of early atherosclerosis. Finally,
we were in the position to evaluate the therapeutic effect of
HAL@M2 Exo on atherosclerosis. Firstly, an early atherosclerosis
mouse model was established by fed the ApoE deficient (ApoE-/-)
mice (male, 6-8 weeks) with cholesterol-rich diet for 12 weeks,[21]
and then randomly divided into four groups and intravenously
injected with PBS, HAL, M2 Exo or HAL@M2 Exo (5.5 mg/kg
HAL-equivalent and 9 mg/kg M2-equivalent), respectively (Figure
7a). After 24 h, the aortas and major organs including liver, spleen,
lung and kidney were excised from the mice treated with free HAL
or HAL@M2 Exo. It was found that the fluorescence originating
from endogenously biosynthesized PpIX in aortas of mice injected
with HAL@M2 Exo was much higher than that in free HAL treated
mice. More encouragingly, the HAL@M2 Exo treated group
exhibited the highest PpIX fluorescence signal in aortas among
all collected major organs (Figure 7b and Figure S8), again
demonstrating the specific inflammation-tropism of HAL@M2 Exo,
which was of great importance to boost its ultimate therapeutic
performance.
After 5 weeks treatments, the mice were all sacrificed. Then their
entire aortas as well as aortic valves were excised for Oil Red O
(ORO) staining which was applied for detecting lipid deposits in
atherosclerotic plaques (Figure 7c and d). Comparatively,
treatment with HAL@M2 Exo performed the optimal anti-
atherosclerotic effect where 75.2 % reduction of inflammation-
induced aortas lesion area as well as 73.9 % reduction of aortic
valves lesions were achieved in comparison with the PBS control
group. Then histological examination on aortic valves stained with
hematoxylin and eosin (H&E) was further carried out to directly
assess the anti-inflammatory outcome. As expected, necrosis
areas in the aortic valves were obviously diminished after
treatment with HAL@M2 Exo, which was also consistent with the
corresponding quantitative analyses (Figure 7e). After that, we
accordingly evaluated the expression levels of ABCA-1 and SR-
BI receptors in aortas by western blotting. In contrast to the control
group, these two RCT-pathway-modulated protein receptors were
greatly up-regulated in other three groups due to the improved
cholesterol-efflux capability of HAL or M2 Exo based formulations
(Figure 7f), which agreed well with the above-mentioned in vitro
results. These results together revealed that HAL@M2 Exo could
Figure 6. In vivo imaging and treatment of acute peritonitis using
HAL@M2 Exo. a) Fluorescence imaging of mice with acute
peritonitis at different time intervals after treatment with HAL or
HAL@M2 Exo. b, c) Flow cytometric analysis of neutrophils and
MPO activity in neutrophils in acute peritoneal exudates 24 h after
different treatments. d) ELISA assay of the expression levels of
typical pro-inflammatory cytokines in acute peritoneal exudates
24 h after different treatments. The results represent the mean ±
standard deviation (n = 6).
As is well-documented that dysregulated neutrophils play
considerable roles in inducing or even aggravating acute
inflammation-related diseases by releasing proteases and ROS
to the impaired tissues or organs.[20] Hence, to evaluate the
therapeutic effect of HAL@M2 Exo on this acute model, total
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10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
effectively relieve chronic inflammation-induced atherosclerosis in
vivo. Moreover, treatment with HAL@M2 Exo resulted in few
abnormalities in the organ histology and blood biochemical
markers (Figure S9), suggesting its good biocompatibility and
safe use.
HAL@M2 Exo. These HAL-engineered M2 macrophage-derived
exosomes would be promising for the treatment of atherosclerosis
and even many inflammation-associated diseases in future.
Experimental Section
See the Supporting Information for experimental section.
Acknowledgements
We thank Biological & Medical Engineering Core Facilities
(Beijing Institute of Technology) for providing advanced
equipment. This work was supported by the National Natural
Science Foundation of China (No. 91859123, and 21874011),
National Science and Technology Major Project (No. 2018ZX
10301405-001), and China Postdoctoral Science Foundation (No.
2018M630076).
Conflict of interest
The authors declare no conflict of interest
Keywords: exosomes • inflammation • membrane proteins •
biosynthesis
Figure 7. Treatment of early atherosclerotic plaques with
HAL@M2 Exo. a) Schematic diagram of the treatment protocol.
b) Fluorescence imaging of the aortas excised from mice. c)
Photographs of the excised aortas stained by ORO and the
corresponding quantitative analyses of plaque areas. d)
Cryosection photographs of the aortic valves stained by ORO and
the corresponding quantitative analyses of plaque areas. Scale
bar: 300 µm. e) H&E staining images and the necrosis area
statistics of aortic valves after different treatments. Scale bar: 200
µm. f) Western blotting analyses of ABCA-1 and SR-BI. The
results represent the mean ± standard deviation (n = 6).
[1] C. Weber, H. Noels, Nat Med 2011, 17, 1410–1422.
[2] a) M. Bäck, G. K. Hansson, Nat Rev Cardiol 2015, 12, 199–
211; b) I. Tabas, C. K. Glass, Science 2013, 316, 166–172;
c) H. Zhu, Y. Fang, Q. Miao, X. Qi, D. Ding, P. Chen, K. Pu,
ACS Nano 2017, 11, 8998–9009..
[3] a) J. H. Park, D. Dehaini, J. Zhou, M. Holay, R. H. Fang, L.
Zhang, Nanoscale Horiz. 2019, 10.1039.C9NH00291J; b) X.
Wei, M. Ying, D. Dehaini, Y. Su, A. V. Kroll, J. Zhou, W. Gao,
R. H. Fang, S. Chien, L. Zhang, ACS Nano 2018, 12, 109–
116; c) M. E. Lobatto, V. Fuster, Z. A. Fayad, W. J. M.
Mulder, Nat Rev Drug Discov 2011, 10, 835–852; d) J. Li, K.
Pu, Chem. Soc. Rev. 2019, 48, 38-71.
[4] a) J. Zhang, Y. Zu, C. S. Dhanasekara, J. Li, D. Wu, Z. Fan,
S. Wang, WIREs Nanomed Nanobiotechnol 2017, 9, e1412;
b) C. K. W. Chan, L. Zhang, C. K. Cheng, H. Yang, Y. Huang,
X. Y. Tian, C. H. J. Choi, Small 2018, 14, 1702793; c) W. J.
M. Mulder, F. A. Jaffer, Z. A. Fayad, M. Nahrendorf, Science
Translational Medicine 2014, 6, 239sr1-239sr1.
Conclusion
In this study, we have pioneered the deveolpment of HAL-
engineered M2 macrophage-derived exosomes for effective
treatment of atherosclerosis. The HAL@M2 Exo can not only
exert the superiority of anti-inflammatory M2 macrophages for
simultaneously targeting and alleviating inflammation in the
atherosclerosis lesions by surface-bonded chemokine receptors
and secreted anti-inflammatory cytokines, but also utilize the
FDA-approved natural precursor HAL for heme biosynthesis via a
series of enzyme-catalyzed reactions to generate extra anti-
inflammatory CO and bilirubin with boosting therapeutic effects.
Moreover, the red-fluorescent intermediate PpIX during HAL-
mediated heme biosynthesis processes exhibited fluorescence
imaging and tracking of atherosclerosis. Both in vitro and in vivo
experimental results demonstrated the remarkable inflammation-
tropism and inflammation-alleviation effects of the as-prepared
[5] a) F. Fernandez-Trillo, L. M. Grover, A. Stephenson-Brown,
P. Harrison, P. M. Mendes, Angew. Chem. Int. Ed. 2017, 56,
3142–3160; b) Y. Jiang, M. Shi, Y. Liu, S. Wan, C. Cui, L.
Zhang, W. Tan, Angew. Chem. Int. Ed. 2017, 56, 11916–
11920; c) K. Mori, M. Hirase, T. Morishige, E. Takano, H.
Sunayama, Y. Kitayama, S. Inubushi, R. Sasaki, M. Yashiro,
T. Takeuchi, Angew. Chem. Int. Ed. 2019, 58, 1612–1615.
[6] a) P. H. L. Tran, D. Xiang, T. T. D. Tran, W. Yin, Y. Zhang,
L. Kong, K. Chen, M. Sun, Y. Li, Y. Hou, et al., Adv. Mater.
2019, 1904040; b) Y. Lyu, D. Cui, J. Huang, W. Fan, Y. Miao,
K. Pu, Angew. Chem. Int. Ed. 2019, 58, 4983–4987; c) X.
Gao, S. Li, F. Ding, H. Fan, L. Shi, L. Zhu, J. Li, J. Feng, X.
Zhu, C. Zhang, Angew. Chem. Int. Ed. 2019, 58, 8719–8723.
[7] M. Adamiak, S. Sahoo, Molecular Therapy 2018, 26, 1635
[8] a) H. Kim, S. Y. Wang, G. Kwak, Y. Yang, I. C. Kwon, S. H.
Kim, Adv. Sci. 2019, 1900513; b) Y. W. Choo, M. Kang, H.
Y. Kim, J. Han, S. Kang, J.-R. Lee, G.-J. Jeong, S. P. Kwon,
S. Y. Song, S. Go, et al., ACS Nano 2018, 12, 8977–8993;
This article is protected by copyright. All rights reserved.

10.1002/anie.201911859
Angewandte Chemie International Edition
RESEARCH ARTICLE
c) Q. Hu, C. Qian, W. Sun, J. Wang, Z. Chen, H. N. Bomba,
H. Xin, Q. Shen, Z. Gu, Adv. Mater. 2016, 28, 9573–9580;
d) Q. Zhang, D. Dehaini, Y. Zhang, J. Zhou, X. Chen, L.
Zhang, R. H. Fang, W. Gao, L. Zhang, Nature Nanotech
2018, 13, 1182–1190; e) S. Thamphiwatana, P. Angsantikul,
T. Escajadillo, Q. Zhang, J. Olson, B. T. Luk, S. Zhang, R.
H. Fang, W. Gao, V. Nizet, et al., Proc Natl Acad Sci USA
2017, 114, 11488–11493;
[9] a) T. M. Raimondo, D. J. Mooney, Proc Natl Acad Sci USA
2018, 115, 10648–10653; b) J. Kim, H. Y. Kim, S. Y. Song,
S. Go, H. S. Sohn, S. Baik, M. Soh, K. Kim, D. Kim, H.-C.
Kim, et al., ACS Nano 2019, 13, 3206–3217; c) P. J. Murray,
J. E. Allen, S. K. Biswas, E. A. Fisher, D. W. Gilroy, S.
Goerdt, S. Gordon, J. A. Hamilton, L. B. Ivashkiv, T.
Lawrence, et al., Immunity 2014, 41, 339–340; d) A.
Aalipour, H.-Y. Chuang, S. Murty, A. L. D’Souza, S. Park, G.
S. Gulati, C. B. Patel, C. Beinat, F. Simonetta, I. Martinić, et
al., Nat Biotechnol 2019, 37, 531–539.
[10] a) E. L. P. Larsen, L. L. Randeberg, O. A. Gederaas, C.-J.
Arum, A. Hjelde, C.-M. Zhao, D. Chen, H. E. Krokan, L. O.
Svaasand, J. Biomed. Opt. 2008, 13, 044031; b) L. P. H.
Yang, Mol Diagn Ther 2014, 18, 105–116.
[11] Y. Bi, J. Chen, F. Hu, J. Liu, M. Li, L. Zhao, Neural Plasticity
2019, 2019, 1–21.
[12] a) H. J. Kim, M.-Y. Jeong, T. J. Parnell, M. Babst, J. D.
Phillips, D. R. Winge, J. Biol. Chem. 2016, 291, 17417–
17426; b) D.-W. Zheng, J.-X. Fan, X.-H. Liu, X. Dong, P.
Pan, L. Xu, X.-Z. Zhang, Adv. Mater. 2018, 30, 1801622; c)
Y. Lee, H. Kim, S. Kang, J. Lee, J. Park, S. Jon, Angew.
Chem. Int. Ed. 2016, 55, 7460–7463; c) R. Stocker, Y.
Yamamoto, A. McDonagh, A. Glazer, B. Ames, Science
1987,235, 1043–1046; d) S.-B. Wang, C. Zhang, Z.-X. Chen,
J.-J. Ye, S.-Y. Peng, L. Rong, C.-J. Liu, X.-Z. Zhang, ACS
Nano 2019, 13, 5523–5532.
[13] A.-N. Zhang, W. Wu, C. Zhang, Q.-Y. Wang, Z.-N. Zhuang,
H. Cheng, X.-Z. Zhang, J. Mater. Chem. B 2019, 7, 1087–
1095.
[14] a) P. M.-K. Tang, D. J. Nikolic-Paterson, H.-Y. Lan, Nat Rev
Nephrol 2019, 15, 144–158; b) J. Li, H. Duan, K. Pu, Adv.
Mater. 2019, 31, 1901607.
[15] a) M. Fujino, Y. Nishio, H. Ito, T. Tanaka, X.-K. Li,
International Immunopharmacology 2016, 37, 71–78; b) M.
Wachowska, A. Muchowicz, M. Firczuk, M. Gabrysiak, M.
Winiarska, M. Wańczyk, K. Bojarczuk, J. Golab, Molecules
2011, 16, 4140–4164.
[16] a) D.-W. Zheng, B. Li, C.-X. Li, L. Xu, J.-X. Fan, Q. Lei, X.-
Z. Zhang, Adv. Mater. 2017, 29, 1703822; b) W. Feng, D.
Liu, S. Feng, G. Feng, Anal. Chem. 2016, 88, 10648–10653.
[17] a) E. P. C. van der Vorst, Y. Döring, C. Weber, J Mol Med
2015, 93, 963–971; b) D. Tousoulis, C. Antoniades, C.
Stefanadis, Heart 2007, 93, 1001–1007; c) M. R. Williams,
V. Azcutia, G. Newton, P. Alcaide, F. W. Luscinskas, Trends
in Immunology 2011, 32, 461–469; d) Kolaczkowska, P.
Kubes, Nat Rev Immunol 2013, 13, 159–175.
[18] G. Chinetti-Gbaguidi, S. Colin, B. Staels, Nat Rev Cardiol
2015, 12, 10–17.
[19] a) J. M. Schwab, N. Chiang, M. Arita, C. N. Serhan, Nature
2007, 447, 869–874; b) X. Xu, H. An, D. Zhang, H. Tao, Y.
Dou, X. Li, J. Huang, J. Zhang, Sci. Adv. 2019, 5, 2953.
[20] a) M. Bäck, A. Yurdagul, I. Tabas, K. Öörni, P. T. Kovanen,
Nat Rev Cardiol 2019, 16, 389–406; b) H. Zhao, Z. Zeng, L.
Liu, J. Chen, H. Zhou, L. Huang, J. Huang, H. Xu, Y. Xu, Z.
Chen, et al., Nanoscale 2018, 10, 6981–6991; c) Y. Lyu, J.
Zeng, Y. Jiang, X. Zhen, T. Wang, S. Qiu, X. Lou, M. Gao,
K. Pu, ACS Nano 2018, 12, 1801–1810.
[21] S. C. Whitman, Clin Biochem Rev 2004, 25, 81–93.
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10.1002/anie.201911859
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RESEARCH ARTICLE
Entry for the Table of Contents (Please choose one layout)
Layout 2:
RESEARCH ARTICLE
Guanghao Wu, Qianru Zhao, Wanru
Zhuang, Jingjing Ding, Chi Zhang,
Haijun Gao, Dai-Wen Pang, Jinfeng
Zhang, Kanyi Pu*, Hai-Yan Xie*
Page No. – Page No.
Molecularly Engineered Macrophage-
Derived Exosomes with Inflammation
Tropism and Intrinsic Biosynthesis for
Atherosclerosis Treatment
M2 macrophage-derived exosomes molecularly engineered with HAL can actively
target and transmigrate to atherosclerotic lesions, wherein the biosynthesis and
metabolism of heme induced by HAL produced CO and bilirubin. Together with anti-
inflammatory cytokines in exosomes, the atherosclerosis is significantly alleviated.
Meanwhile, the intermediate PpIX can be used for imaging and tracking of
atherosclerosis.
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