Structural Transformative Antioxidants for Dual-Responsive Anti-Inflammatory Delivery and Photoacoustic Inflammation Imaging

Article abstract of DOI:10.1002/anie.202100873

We have synthesized a PEGylated, phenylboronic acid modified L-DOPA pro-antioxidant (pPAD) that can self-assemble into nanoparticles (pPADN) for the loading of a model glucocorticoid dexamethasone (Dex) through 1,3-diol/phenylboronic acid chemistry and hydrophobic interactions for more effective treatment of inflammation. Upon exposure to ROS, pPADN convert into the active form of L-DOPA, and a cascade of oxidative reactions transform it into antioxidative melanin-like materials. Concomitantly, the structural transformation of pPADN triggers the specific release of Dex, along with the acidic pH of inflammatory tissue. In a rat model of osteoarthritis, Dex-loaded pPADN markedly mitigate synovial inflammation, suppress joint destruction and cartilage matrix degradation, with negligible in vivo toxicity. Moreover, in situ structural transformation makes pPADN suitable for noninvasive monitoring of therapeutic effects as a photoacoustic imaging contrast agent.

Full text of DOI:10.1002/anie.202100873

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How to cite:
International Edition: doi.org/10.1002/anie.202100873
German Edition: doi.org/10.1002/ange.202100873
Drug Delivery
Structural Transformative Antioxidants for Dual-Responsive Anti-
Inflammatory Delivery and Photoacoustic Inflammation Imaging
Caiyan Zhao+, Jingxiao Chen+, Jiamin Ye+, Zhi Li, Lichao Su, Junqing Wang, Ye Zhang,
Jinghua Chen, Huanghao Yang, Jinjun Shi,* and Jibin Song*
Abstract: We have synthesized a PEGylated, phenylboronic
acid modified L-DOPA pro-antioxidant (pPAD) that can self-
assemble into nanoparticles (pPADN) for the loading of
a model glucocorticoid dexamethasone (Dex) through 1,3-
diol/phenylboronic acid chemistry and hydrophobic interac-
tions for more effective treatment of inflammation. Upon
exposure to ROS, pPADN convert into the active form of L-
DOPA, and a cascade of oxidative reactions transform it into
antioxidative melanin-like materials. Concomitantly, the struc-
tural transformation of pPADN triggers the specific release of
Dex, along with the acidic pH of inflammatory tissue. In a rat
model of osteoarthritis, Dex-loaded pPADN markedly miti-
gate synovial inflammation, suppress joint destruction and
cartilage matrix degradation, with negligible in vivo toxicity.
Moreover, in situ structural transformation makes pPADN
suitable for noninvasive monitoring of therapeutic effects as
a photoacoustic imaging contrast agent.
debris and byproducts.[2] However, when this process is out of
control, these pro-inflammatory factors would lead to hyper-
inflammation, resulting in chronic diseases such as auto-
immune disease, degenerative disease, cancer, and others.[3] It
is worth noting that inflammatory responses are often
associated with overproduction of reactive oxygen species
(ROS).[4] Higher levels of toxic ROS not only induce
oxidative injury, cell apoptosis and necrosis, but also activate
inflammatory signaling through NF-kB, amplify pro-inflam-
matory pathways, and exacerbate immune responses.[5] Of
note, the secondary species (e.g., COH, COCl, and OONOÀ) are
more chemically reactive and less controllable than primary
species (e.g., O2CÀ and H2O2), due to the lack of specific
enzyme control systems in the body.[6] The persistent presence
of these uncontrolled ROS inevitably potentiates inflamma-
tion in the lesions. In addition, the infiltration and activation
of inflammatory cells induce accelerated glycolysis and
increased lactic acid secretion, acidifying the environment
of inflamed tissues.[7]
Introduction
Glucocorticoids such as dexamethasone (Dex) play an
important role in the treatment of inflammation, as they can
quickly slow the progress of inflammation and relieve pain.[8]
However, glucocorticoids injected into inflamed tissues such
as the joint in osteoarthritis (OA) are cleared rapidly.[9] In
addition, there is always the risk of untoward side-effects
through nonspecific targeting, such as suppression of immune
function and the risk of inducing diabetes, muscular atrophy,
osteoporosis, and other conditions.[10] Thus, the use of
glucocorticoids is often limited. To achieve better therapeutic
efficacy and minimize adverse effects, new approaches that
can prolong the retention time of glucocorticoids at the site of
inflammation and control drug release locally in lesions with
minimal exposure of normal tissues are sought.
Inflammation plays a pivotal role in response to danger
signals from infection and tissue damage.[1] The activated
immune system triggered by pathogen-associated molecular
patterns or damage-associated molecular patterns then re-
cruits monocytes, polarizes macrophages, and secretes in-
flammatory mediators such as interleukin-1 and cyclooxyge-
nase-2 (COX-2), promoting the clearance of injury tissue
[*] Dr. C. Zhao,[+] J. Ye,[+] Dr. Z. Li, L. Su, Prof. H. Yang, Prof. J. Song
MOE Key Laboratory for Analytical Science of Food Safety and
Biology, College of Chemistry, Fuzhou University, Fujian Science &
Technology Innovation Laboratory for Optoelectronic Information of
China
Herein, we report a transformative antioxidative bioma-
terial-based platform for ROS and pH dual-responsive Dex
delivery to effectively treat OA. Biomaterials with intrinsic
ROS scavenging activity have recently gained significant
attention, among which nano-antioxidants composed of
natural biomolecules represent a promising paradigm for
treatment of ROS-related diseases.[4c,11] Particularly, dopa-
mine or L-DOPA with catechol structure has been widely
suggested as an effective antioxidant; it can be readily
oxidized to form cross-linked polydopamine (a major pigment
of melanin) in an oxidative environment.[12] Melanin, a natural
antioxidative metabolite, has also been applied in antioxida-
tive treatment.[13] However, these melanin-based/like bioma-
terials are associated with several drawbacks, such as limited
stability due to autoxidation of catechol in aqueous solution
Fuzhou 350108 (P. R. China)
E-mail: jibinsong@fzu.edu.cn
Dr. C. Zhao,[+] Dr. J. Chen,[+] Dr. J. Wang, Dr. Y. Zhang, Prof. J. Shi
Center for Nanomedicine, Brigham and Women’s Hospital
Harvard Medical School
Boston, Massachusetts 02115 (USA)
E-mail: jshi@bwh.harvard.edu
Dr. J. Chen,[+] Prof. J. Chen
Key Laboratory of Carbohydrate Chemistry and Biotechnology
Ministry of Education, School of Pharmaceutical Sciences
Jiangnan University
Wuxi 214122 (P. R. China)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202100873.
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and their complicated structure and variable perfor-
pPAD, enabling self-assembly into stable pPADN. In a rat
mance.[14,15] Moreover, the use of antioxidative biomaterials
for delivery of anti-inflammatory drugs to treat inflammation
remains largely unexplored.
model of OA, the therapeutic effect of Dex-loaded pPADN
was evaluated. Additionally, based on the photoacoustic (PA)
imaging feature of melanin nanomaterials,[18] and the widely
exploration of PA imaging in measuring oxidative stress,[19]
the use of pPADN for ROS-specific PA imaging performance
was explored.
In this study, we synthesized a PEGylated, phenylboronic
acid modified L-DOPA pro-antioxidant (pPAD) that can self-
assemble into nanoparticles (pPADN) for specific loading
and dual-responsive delivery of Dex (Scheme 1). Phenyl-
boronic acid is an efficient ROS-responsive group,[16] that
Results and Discussion
To endow the unstable antioxidant L-DOPA with control-
lable, intelligent and stable performance, its phenol and
amine groups were sequentially protected by self-immolative
and ROS-responsive phenylboronic acid groups, which can
also reversibly bind with the 1,3-diols present in glucocorti-
coids. To make it amphiphilic, mPEG2k-NH2 was then coupled
to the carboxyl group of L-DOPA to form PEGylated
phenylboronic acid-conjugated L-DOPA (pPAD) polymer
(Scheme S1, Figure S1–S11).
The pPAD polymer with hydrophobic block of phenyl-
boronic acid-conjugated L-DOPA derivative and hydrophilic
block of PEG was self-assembled into nanoparticles
(pPADN) in aqueous solution. In theory, pPADN provided
a stable reservoir for Dex on account of the specific and
reversible covalent bonding of phenylboronic acid and 1,3-
diol, as well as the hydrophobic interaction. To explore the
self-assembly process of Dex-pPADN, the interaction of Dex
and pPAD was analyzed by isothermal titration calorimetry
(ITC). Figure S12 shows the enthalpy change DH =
À1386 calmolÀ1 and the entropy change DS = 25.5 calm-
oLÀ1 degÀ1. The titrations were exothermic (DH < 0) and
spontaneous (DG < 0; DG = DHÀTDS). This suggests that
Dex can self-assemble with pPAD to form Dex-pPADN, in
which both the phenylboronic acid/1,3-diol chemistry and
hydrophobic interaction may play a role. Given the impor-
tance of encapsulation efficiency and particle size in nano-
particle drug delivery, these two parameters were then
evaluated. As shown in Figure 1a,b, keeping the concentra-
tion of Dex constant at 1 mgmLÀ1, the drug encapsulation
efficiency gradually increased while the particle size first
decreased and then increased as the ratio of copolymer/drug
increased. Dex-pPADN showed an ultrahigh encapsulation
efficiency of ꢀ 92% and lowest particle size at the 10:1 mass
ratio of pPADN to Dex, which was selected for subsequent
investigation. As a ROS non-sensitive control, the PEGy-
lated, phenyl-protected L-DOPA (pBD) was constructed by
replacing the phenylboronic acid groups of pPAD with phenyl
structures (Scheme S2, Figure S13–S17). Drug encapsulation
efficiency and particle size of Dex-pBDN were also analyzed.
As shown in Figure S18, the Dex encapsulation efficiency of
pBD was significantly lower than that of pPAD at the same
polymer/Dex ratios. In addition, the particle size of Dex-
pBDN did not decrease as the ratio of polymer/Dex increased
(Figure S19). These results may have been caused by the fact
that Dex molecules can be specifically bound to pPAD
through 1,3-diol/phenylboronic acid chemistry and hydro-
phobic interaction, but pBD can load Dex only through
hydrophobic interaction. The unique interactions between
Scheme 1. Schematic illustration of the preparation of Dex-pPADN for
treatment of osteoarthritis. pPAD is first synthesized via protecting the
amine and phenol groups of L-DOPA with phenylboronic acid groups
and then PEGylating. Dex-pPADN is formulated by loading Dex with
pPAD through 1,3-diol/phenylboronic acid chemistry and hydrophobic
interactions. Dex-pPADN dissociates and is transformed into a mela-
nin-like compound in response to excess ROS, along with the release
of Dex in acidic inflammatory tissue, which synergistically induces the
inhibition of pro-inflammatory factors, the scavenging of ROS, and the
activation of a near-infrared (NIR) photoacoustic (PA) signal.
allows the transformation of pPAD to active L-DOPA upon
oxidant trigger and through further oxidation to form
melanin-like structure. Therefore, the pPAD scaffold is
expected to play an antioxidant role in the presence of excess
ROS. Moreover, the boronic ester formed from phenylbor-
onic acid and diol is known to be one of the strongest single-
pair reversible functional group interactions in aqueous
solution. It has been widely used to construct glucose and
pH sensors, among which 1,2- or 1,3-diols bind particularly
easily with phenylboronic acid due to the formation of stable
five- or six-membered ring structures.[17] Thus, the 1,3-diol
present on glucocorticoids provides an ideal site for inter-
action with phenylboronic acid, which along with the hydro-
phobic interaction, leads to specific and high Dex loading and
controllable Dex release. In addition, mPEG2k-NH2 becomes
linked to the carboxyl group of L-DOPA to form amphiphilic
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pPADN, which would defi-
nitely be beneficial for im-
proving drug availability
and minimizing undesired
side effects.
The boronic acid/esters
in Dex-pPADN are readily
detached after attack by
OONOÀ and H2O2, produc-
ing the antioxidative form
of L-DOPA. We went on to
assess the anti-oxidative
performance
of
Dex-
pPADN by studying its
OONOÀ and H2O2 scav-
enging activity. As shown
in Figure 1e, Dex-pPADN
significantly
scavenged
H2O2 in a concentration-de-
pendent manner, but Dex-
pBDN and pBDN failed to
scavenge H2O2. In addition,
consistent with the results
of the H2O2-scavenging as-
say, Dex-pPADN displayed
preferable concentration-
dependent ONOOÀ scav-
enging ability (Figure 1 f,g).
Almost 89% of ONOOÀ
was quenched when it was
co-incubated with 10 mM of
Dex-pPADN. Whereas nei-
ther pBDN nor pDex-BDN
scavenged ONOOÀ (Fig-
ure S20). These findings im-
ply that the protection of
the amine and phenolic
groups of L-DOPA is an
Figure 1. Preparation and characterization of Dex-pPADN and related materials. The effects of pPAD/Dex
ratios on a) encapsulation efficiency and b) particle size. c) TEM images and hydrodynamic diameter
distribution of Dex-pPADN at the pPAD/Dex ratio of 10:1. d) ROS- and pH-triggered release of Dex from Dex-
pPADN in vitro. e) H2O2 scavenging effect of various nanoformulations at the same conditions (concentration
of pPADN: 20 mM; incubation time: 4 h); inset plot shows the H2O2 scavenging effect of Dex-pPADN with
different concentrations (incubation time: 4 h). f) ONOOÀ scavenging effect of Dex-pPADN with different
concentrations. g) The quantification of the ONOOÀ scavenging effect of various nanoformulations. h) UV-
vis-NIR absorption spectra; i) PA signal intensity and PA images of Dex-pPADN after incubation with different
concentrations of ONOOÀ. (G1: 0 mM ONOOÀ, G2: 5 mM ONOOÀ, G3: 10 mM ONOOÀ, G4: 20 mM
ONOOÀ, G5: 40 mM ONOOÀ).
pPAD and Dex improved encapsulation capacity of pPAD for
Dex and a certain amount of Dex could condense pPADN
into smaller nanostructures.
effective way to render it stable and inactive. Meanwhile,
protection using ROS-responsive boronic acid groups not
only preserves its naturally antioxidative activity but also
endows it with the unique ability to bind with glucocorticoids.
Antioxidative stability and colloidal structure stability
were further evaluated. As shown in Figure S21,S22, the
scavenging activity of the Dex-pPADN against both H2O2 and
ONOOÀ showed no obvious change after 6-month storage. In
addition, the hydrodynamic diameter of Dex-pPADN in
saline solution can be maintained at ꢀ 90 nm for at least 6
months (Figure S23). Since glucose could form covalent
adducts with boronic acids, we then evaluated the stability
of Dex-pPADN in saline solution with the presence of glucose
(0.90 mgmLÀ1, similar to normal blood glucose content).
Results showed that nanoparticle size of Dex-pPADN was not
significantly changed and the Dex leakage was less than 1%
even after incubation for 7 days (Figure S24). Such good
antioxidative stability and structural stability are predictive of
its successful advancement to in vivo studies.
The physicochemical characteristics of Dex-pPADN were
further investigated at the polymer/drug ratio of 10/1. As
shown in Figure 1c, dynamic light scattering (DLS) measure-
ments revealed that the hydrodynamic diameter of Dex-
pPADN was 89.6 Æ 9.5 nm. Transmission electron microscopy
showed that the morphology of Dex-pPADN is a homoge-
neous spherical structure. Subsequently, we investigated the
drug release kinetics of Dex-pPADN under inflammatory or
physiological environments simulated in vitro. As shown in
Figure 1d, Dex was released from Dex-pPADN much more
quickly at pH 6.5 and/or in the buffer with H2O2, whereas Dex
release was slower in the buffer without H2O2 at pH 7.4.
Nearly 79% of encapsulated Dex was released after 48 h
under the oxidative and acid condition. In contrast, only
approximately 26% of Dex was released after 48 h under
conditions similar to the normal physiological environment.
These findings suggest the selective release of Dex from Dex-
L-DOPA monomer is instable and readily undergoes self-
polymerization to form a melanin-like structure in an
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oxidative environment, coupled with a color change from
shown in Figure S29, ROS was overproduced after LPS
colorless to dark brown. Polydopamine has a broad absorp-
tion band in the near infrared (NIR) region and shows good
PA effect. By exploiting these features, we further inves-
tigated the ROS-induced variation of Dex-pPADN. UV-vis-
NIR absorption and PA imaging performance of Dex-
pPADN were studied before and after co-incubation with
ONOOÀ. ONOOÀ concentrations ranging from 5 to 40 mM
were applied to oxidize Dex-pPADN. As shown in Figure 1h,
the UV-vis-NIR absorption spectra revealed that Dex-
pPADN showed no absorption in the NIR region, but
obviously enhanced absorption signals were observed after
the ONOOÀ trigger. Meanwhile, the absorption signals
showed an increase in ONOOÀ concentration-dependent
manner. In addition, digital picture visually confirmed that
the color of Dex-pPADN solution gradually changed from
colorless to brown as the increase of ONOOÀ concentrations
(Figure S25). PA imaging results were agreed with UV-vis-
NIR absorption assay (Figure 1i). As the concentration of
ONOOÀ increased, PA signal of Dex-pPADN is significantly
enhanced. Next, we used another oxidant, ClOÀ to further
study the ROS-mediated oxidation of Dex-pPADN (Fig-
ure S26). The color change of the solution from colorless to
brown was observed as the concentration of ClOÀ increased,
consistent with the ONOOÀ assay. Moreover, the chemical
structure change of pPAD in oxidation atmosphere was
investigated via 1H NMR (Figure S27). The reaction with
ONOOÀ resulted in a significant change of peaks at d 8.0–
6.5 ppm from benzene ring, which do not correspond to the
protons from monomer L-DOPA. Based on these results, we
speculated that not only was pPAD transformed into L-
DOPA in the presence of ONOOÀ, but also further oxidized,
leading to the formation of melanin-like product.
COX-2 plays an essential role in the development and
progression of inflammation. Therefore, COX-2 inhibition
activity of Dex-pPADN were evaluated by western blot (WB)
in activated macrophage Raw 264.7 cells. LPS is a component
of cell walls from Gram-negative bacteria and a toll-like
receptor (TLR4) agonist that is widely used as an activator of
macrophages to create inflammation models.[20] After 15 h of
incubation with 1.0 mgmLÀ1 of LPS, significant macrophage
activation was observed, as evidenced by upregulated ex-
pression of COX-2 (Figure S28). In contrast, when the
activated Raw 264.7 cells were treated with Dex-pPADN,
the expression of COX-2 was significantly reduced, with
a concentration-dependent inhibition effect. Furthermore,
the inhibition effect of Dex-pPADN, pPADN, and dexame-
thasone sodium phosphate (Dex-p, a water-soluble form)
against activation of macrophages was compared under the
same conditions. As shown in Figure 2a, expression of COX-2
was down-regulated by these drugs. pPADN treatment only
moderately suppressed the activation of macrophages with
92% expression of COX-2, whereas treatment with Dex-p or
Dex-pPADN dramatically lowed this value to 66% or 30%,
respectively.
stimulation, with bright green fluorescence in Raw 264.7 cells.
As expected, Dex-pPADN effectively decreased ROS levels
in activated Raw 264.7 cells in a dose-dependent manner; the
effective concentration of Dex-pPADN against overproduced
ROS was 50 mgmLÀ1. Furthermore, the ROS scavenging
effect of Dex-pPADN, pPADN, and Dex-p was compared
(Figure 2b). Both pPADN and Dex-pPADN dramatically
reduced levels of overproduced ROS relative to the LPS
treatment group. However, Dex-p only slightly decreased
ROS levels. Quantitative results from flow cytometry were
consistent with the above results obtained with a confocal
laser scanning microscope (CLSM) (Figure 2c). Dex-pPADN
showed the best effect in inhibiting the generation of both
cytotoxic ROS and inflammatory COX-2.
It should be noted that the inflammatory mediators
released by activated macrophage can induce different levels
of activation, apoptosis, and necrosis of chondrocytes and
synoviocytes in OA. Hence the ability of Dex-pPADN to
inhibit inflammation-induced death of ATCD5 cells was
evaluated by CCK-8. Neither pPADN nor Dex-pPADN
elicited obvious cytotoxicity even at high pPADN concen-
trations such as 400 mgmLÀ1 (Figure S30). However, the
viability of ATCD5 cells showed dose-dependent decrease
after incubation with the cell supernatant harvested from LPS
activated macrophages (Figure S31). When the concentration
of cell supernatant was 20%, the death rate of ATCD 5 cells
reached ꢀ 45%. In comparison, treatment with Dex-pPADN
(100 mgmLÀ1 of pPADN) reduced this value to ꢀ 5% (Fig-
ure 2d). In addition, annexin V-FITC/PI co-staining assay
verified that the supernatant harvested from activated macro-
phages induced apoptosis in ATCD5 cells, which was
significantly reversed by pre-treatment or post-treatment
with Dex-pPADN. As shown in Figure 2e–f, the presence of
cellular supernatant harvested from LPS-activated macro-
phages caused apoptosis in 51.73% of ATCD5 cells; pre/post-
treatment with Dex-pPADN reduced this value to 18.91 or
27.23%, respectively. Further study using TUNEL assay also
revealed that the apoptosis status of ATCD5 cells induced by
inflammatory mediators from activated macrophage was
effectively reversed via pre/post-treatment with Dex-pPADN
(Figure 2g), suggesting that Dex-pPADN protected ATCD5
cells from damage by pro-inflammatory factors.
Given the promising anti-inflammatory activity of Dex-
pPADN in vitro, its efficacy in treating OA was evaluated in
a rat model. Following arthritis induction, Dex-pPADN were
delivered into arthritic knees via intra-articular injection
every 4 days (Figure 3a).
Healthy rats injected with PBS and OA rats injected with
PBS, pPADN, or Dex-p served as controls. The degree of
swelling in knee joints was assessed by measuring knee width.
As shown in Figure S32, OA mice developed severe swelling
of knee joints. After Dex-pPADN treatment, knee joint
swelling was dramatically ameliorated, showing the strongest
therapeutic efficacy (Figure 3b). Treatment with either
pPADN or Dex-p only moderately suppressed knee joint
swelling. At the study endpoint, the knee joints in different
treatment groups were photographed and compared. As
shown in Figure 3c, arthroncus was clearly observed in the
Excess ROS contributed to inflammation and amplified
pro-inflammatory pathways. We went on to investigate ROS
levels in LPS-activated Raw 264.7 cells using a commercial kit
and 2’,7’-dichlorofluorescein diacetate (DCFH-DA). As
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Figure 2. Anti-inflammatory, anti-oxidative, and cellular protective activities of Dex-pPADN. a) COX-2 expression in Raw 264.7 cells detected by
Western blot. (Lane 1: COX-2; Lane 2: GAPDH). b) ROS levels in Raw 264.7 cells visualized with a confocal laser scanning microscope (CLSM).
G1: PBS; G2: LPS; G3: LPS+pPADN; G4: LPS+Dex-p; and G5: LPS+Dex-pPADN. c) ROS levels in Raw 264.7 cells analyzed by flow cytometry.
d) Cell viabilities of ATCD5 cells under different treatment conditions (G1: untreated control; G2–G5: treated with supernatant collected from
activated Raw264.7. G2: PBS; G3: 25 mgmLÀ1 of Dex-pPADN; G4: 50 mgmLÀ1 of Dex-pPADN; G5: 100 mgmLÀ1 of Dex-pPADN). e) Flow cytometry
analysis of ATCD5 cells. f) Quantitative analysis of apoptotic and necrotic ATCD5 cells. g) Merged CLSM images of ATCD5 cells under different
treatment conditions. (e–g: G1: untreated control; G2–G4: treated with supernatant collected from activated Raw264.7. G2: PBS; G3: pre-treated
with Dex-pPADN; G4: post-treated with Dex-pPADN). Statistical significance was calculated by t-test (n=3): ** p<0.01, *** p<0.005, and ****
p<0.001.
OA model group, but the symptoms in the Dex-pPADN
treatment group were significantly alleviated to a level
comparable to that of healthy mice. It has been reported that
inflammation in OA is often accompanied by hyperthermia in
knee joints due to the increased synovitis and/or subchondral
bone activity.[21] Thus thermography of knee joints was
recorded using a digital infrared thermal imaging camera at
the study endpoint to evaluate the therapeutic efficacy of
different treatments. As shown in Figure 3d–e, the temper-
ature of arthritic knees was ꢀ 48C higher that of healthy knee
joints. In comparation, the temperature of knee joints was
dramatically lowered for OA rats after treatment with
pPADN or Dex-p; Dex-pPADN treatment further decreased
this value to the lowest level seen. These findings suggest that
Dex-pPADN has robust efficacy for OA treatment.
To further assess the therapeutic effect of Dex-pPADN,
micro-CT imaging was applied to analyze bone morphology
and bone microstructure. As shown in Figure 3 f, severe bone
and cartilage erosion occurred around inflamed knee joints in
the OA model group, but only slight articular cartilage
degradation was observed after pPADN or Dex-p treatment.
In particularly, there were nearly no pathological changes in
inflamed knee joints after treatment with Dex-pPADN. The
relevant indicators of bone structure were further calculated
and compared through histomorphometric analysis of CT
images. BV/TV (Bone Volume/Total Volume) is generally
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massive synovial villous hy-
perplasia, which are typical
symptoms of OA. In com-
parison, treatment with
Dex-pPADN showed al-
most no synovial hyperpla-
sia or cartilage erosion. Sa-
franin O staining was then
performed to assess carti-
lage degradation and extra-
cellular cartilage matrix
(ECM) changes. Articular
cartilage in healthy rats
clearly exhibited strong ex-
pression of proteoglycans.
However, in the OA model
group, articular cartilage
was severely damaged, ac-
companied by weak distri-
bution of proteoglycans in
ECM. After treatment with
Dex-pPADN, these symp-
toms were efficiently alle-
viated, as evidenced by the
almost-intact cartilage sur-
face and evenly distributed
proteoglycans in ECM. The
therapeutic effect of Dex-
pPADN in inhibiting the
degradation of cartilage
ECM was further con-
firmed by immunofluores-
cence staining analysis of
aggrecan levels. As shown
in Figure 4a, aggrecan was
severely depleted in carti-
lage ECM of OA rats, but
uniformly distributed in the
Figure 3. Therapeutic efficacy of Dex-pPADN in OA rat model. a) Schematic illustration of the establishment
and treatment schedule of the OA model. b) Knee width change of OA rats at various time points after
different treatments. c) Representative digital images of knee joints in different groups. d) Quantification of
knee joint temperature in different groups. e) Representative thermographic images of healthy knees or
arthritic knees with different treatments. f) Representative CT images of healthy knees and arthritic knees
with different treatments. g–i) Quantitative analysis of Bone Volume/Total Volume (BV/TV), Trabecular
Number (Tb.N), and Trabecular Separation (Tb.Sp) of knee joints in different groups. G1: healthy rats; G2–
G5: OA rats (G2: saline treatment; G3: pPADN treatment; G4: Dex-p treatment; G5: Dex-pPADN treatment).
Statistical significance was calculated by t-test (n=4): * p<0.05 and **** p<0.001.
Dex-pPADN
treatment
group. L-DOPA with cate-
chol structure has been sug-
gested as an effective anti-
used to reflect bone mass. Tb.N (trabecular number) and
Tb.Sp (trabecular separation) are main factors referring to the
spatial structure of trabecular bone. The lowered Tb.N value
and increased Tb.Sp value indicate that bone catabolism was
greater than bone anabolism.[22] As shown in Figure 3g–i, the
BV/TV value was dramatically lower in the OA model group
compared to healthy rats. In addition, significant lower Tb.N
and higher Tb.Sp were also observed in OA model group,
indicating osteoporosis. In comparison, Dex-pPADN treat-
ment remarkably restored these indicators to near-normal
levels. Taken together, these results suggest that Dex-pPADN
significantly prevented OA-mediated cartilage erosion and
bone destruction.
oxidant. Therefore, we also used L-DOPA as a ROS scav-
enging control to study if it has the same ability to reduce
symptoms (Figure S33). Results showed that L-DOPA mod-
erately alleviated the cartilage erosion and a small amount of
aggrecan was present in ECM.
To evaluate the systemic response of Dex-pPADN, serum
levels of TNF-a, IL-12, and IL-1b in rats subjected to various
treatments were assessed (Figure 4e–g). These pro-inflam-
matory cytokines were significantly upregulated accompany-
ing the establishment of OA. Conversely, these cytokines
were decreased in Dex-p, pPADN, and Dex-pPADN treat-
ment groups. In particular, the Dex-pPADN treatment group
showed the lowest levels of these pro-inflammatory cytokines.
These results suggest that Dex-pPADN not only effectively
alleviated synovitis and degradation of cartilage and its
extracellular matrix, but also suppressed the over-expression
of pro-inflammatory factors.
Furthermore, the therapeutic efficacy of Dex-pPADN was
analyzed by histological examination (Figure 4a–d). H&E
stained images of knee joints from the OA model group
showed severe bone destruction, neutrophil infiltration, and
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displayed continuous low
PA signals at all investigated
time points. However, knee
joints in the OA model
group showed an initial low
PA signal, whose intensity
increased with time, reach-
ing a maximum 2 h post
injection. In contrast, only
a small enhancement in PA
signal was observed over
time after treatment with
pPADN or Dex-p. In the
case of Dex-pPADN, the
PA signal in knee joint re-
gion was still quite low 2 h
post injection, close to that
in the healthy group. Over-
all, these results indicate
that Dex-pPADN can act as
a PA contrast agent to ach-
ieve effective diagnosis and
treatment guidance for OA.
The feasibility of moni-
toring OA progression via
assessing inflammation-as-
sociated ROS was further
verified using a conventional
ROS-sensitive luminescent
probe L-012. At the end of
treatment, L-012 was deliv-
ered to the knee joint region
of all rats via intra-articular
injection. Luminescent sig-
nals were noninvasively
Figure 4. Histopathology analysis and inflammation evaluation of OA rats. a) H&E staining images, safranin
O staining images, and immunofluorescence staining images of knee joints in different groups. Serum levels
of b) TNF-a, c) IL-12, and d) IL-1b in rats with different treatments. Statistical significance was calculated by
t-test (n=4): * p<0.05, ** p<0.01, *** p<0.005, and **** p<0.001.
To assess the potential side-effects of Dex-pPADN, the
main organs in all groups were harvested and subjected to
H&E staining at the end of treatment. As shown in Fig-
ure S34, no obvious tissue damage was found in Dex-pPADN
or pPADN treatment groups, whereas slight hemorrhage and
necrosis were detected in liver and kidney in the Dex-p
treatment group. Furthermore, Dex-pPADN and Dex-p were
intravenously injected into rats every fourth day for 5 times.
After 20 days, the major organs were collected for H&E
staining (Figure S35). Damaged renal tubules were found in
kidney tissues from the Dex-p treated rats, and vacuolar
degeneration could be clearly observed in liver sections. In
comparison, much fewer damaged structures were noticed in
monitored via an in vivo imaging system. As shown in
Figure 5e,f, the luminescent signal in inflamed knee joints was
stronger than that in healthy knee joints. After treatment with
various drug formulations, the decrease in the intensity of
luminescent signals in inflamed regions was observed. Con-
sistent with the PA imaging results, the luminescent signal in
the Dex-pPADN treatment group was weakest, indicating not
only effective mitigation of oxidative stress and inflammatory
responses of Dex-pPADN in OA rats, but also the potential to
identify inflammatory regions and monitor treatment out-
comes.
the saline or Dex-pPADN group. These results suggest that Conclusion
the specific encapsulation with pPADN effectively reduced
the adverse effects of Dex.
In conclusion, we have developed a Dex-pPADN platform
On the basis of the structural transformation of Dex-
pPADN under an oxidative microenvironment, we explored
the additional ability of Dex-pPADN to monitor OA
progression by imaging the inflammation-associated ROS
using an in vivo PA imaging system. At the study endpoint, all
rats received intra-articular injection of Dex-pPADN; PA
imaging in the articular cavity region was carried out. As
shown in Figure 5a–d, the knee joints of healthy rats
and demonstrated its potential as an anti-inflammatory
strategy in OA management. Unlike existing anti-inflamma-
tory drug carriers, pPADN (prepared from an amphiphilic
phenylboronic acid-decorated L-DOPA derivative) provided
a scaffold for reversible coupling with glucocorticoids to
facilitate ROS- and pH-responsive self-assemblies of gluco-
corticoids based on 1,3-diol/phenylboronic acid chemistry and
hydrophobic interaction. This is the first demonstration of
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addressing inflammation-re-
lated diseases and improv-
ing glucocorticoid-based
treatment of inflammation.
Acknowledgements
This work was supported by
the Prostate Cancer Foun-
dation Young Investigator
Award, the National Natural
Science Foundation of Chi-
na
(Nos.
21874024;
51973084), and the joint re-
search projects of Health
and Education Commission
of Fujian Province (2019-
WJ-20), and the Natural
Science Foundation of Fu-
jian
Province
(No.
2020J02012).
Conflict of interest
The authors declare no con-
flict of interest.
Keywords: antioxidants ·
drug delivery · inflammation ·
L-DOPA ·
Figure 5. In vivo therapeutic monitoring via evaluation of ROS levels in OA rats with different treatments.
a) Representative PA images of a healthy articular cavity or an arthritic articular cavity with different
treatments. b) PA signal intensity of Dex-pPADN (excited at 710 nm) at various time points in different
groups. c) PA signal intensity at 710 nm in different groups after injection with Dex-pPADN for 2 h. d) PA
spectra of Dex-pPADN in different groups after injection with Dex-pPADN for 2 h. (b–d: G1: healthy rats; G2:
OA rats treated with saline; G3: OA rats treated with pPADN; G4: OA rats treated with Dex-p; G5: OA rats
treated with Dex-pPADN). e) In vivo luminescence images of healthy knees and arthritic knees in rats with
different treatments. f) Corresponding L-012 luminescence intensity in different groups. (G1: healthy rats; G2:
OA rats treated with saline; G3: OA rats treated with pPADN; G4: OA rats treated with Dex-p; G5: OA rats
treated with Dex-pPADN). Statistical significance was calculated by t-test (n=3): * p<0.05, ** p<0.01, ***
p<0.005, and **** p<0.001.
photoacoustic imaging
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Version of record online: && &&
,
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Research Articles
Drug Delivery
PEGylated, phenylboronic acid modified
L-DOPA-derived nanoparticles (pPADN)
specifically integrated reactive oxygen
species (ROS) scavenging and anti-
inflammatory drug delivery to treat
osteoarthritis. The pPADN sensed and
eliminated elevated ROS via cascade
oxidative reactions and delivered gluco-
corticoids to the site of inflammation.
C. Zhao, J. Chen, J. Ye, Z. Li, L. Su,
J. Wang, Y. Zhang, J. Chen, H. Yang,
J. Shi,* J. Song*
&&&& — &&&&
Structural Transformative Antioxidants
for Dual-Responsive Anti-Inflammatory
Delivery and Photoacoustic Inflammation
Imaging
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