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Guetal. Journal of Nanobiotechnology (2022) 20:268
https://doi.org/10.1186/s12951-022-01485-8
RESEARCH
An enhanced antioxidant strategy
ofastaxanthin encapsulated inROS-responsive
nanoparticles forcombating cisplatin-induced
ototoxicity
Jiayi Gu1,2,3†, Xueling Wang1,2,3†, Yuming Chen1,2,3, Ke Xu1,2,3, Dehong Yu1,2,3,4* and Hao Wu1,2,3*
Abstract
Background: Excessive accumulation of reactive oxygen species (ROS) has been documented as the crucial cellular
mechanism of cisplatin-induced ototoxicity. However, numerous antioxidants have failed in clinical studies partly due
to inefficient drug delivery to the cochlea. A drug delivery system is an attractive strategy to overcome this drawback.
Methods and results: In the present study, we proposed the combination of antioxidant astaxanthin (ATX) and ROS-
responsive/consuming nanoparticles (PPS-NP) to combat cisplatin-induced ototoxicity. ATX-PPS-NP were constructed
by the self-assembly of an amphiphilic hyperbranched polyphosphoester containing thioketal units, which scavenged
ROS and disintegrate to release the encapsulated ATX. The ROS-sensitivity was confirmed by 1H nuclear magnetic
resonance spectroscopy, transmission electron microscopy and an H2O2 ON/OFF stimulated model. Enhanced release
profiles stimulated by H2O2 were verified in artificial perilymph, the HEI-OC1 cell line and guinea pigs. In addition, ATX-
PPS-NP efficiently inhibited cisplatin-induced HEI-OC1 cell cytotoxicity and apoptosis compared with ATX or PPS-NP
alone, suggesting an enhanced effect of the combination of the natural active compound ATX and ROS-consuming
PPS-NP. Moreover, ATX-PPS-NP attenuated outer hair cell losses in cultured organ of Corti. In guinea pigs, NiRe-PPS-NP
verified a quick penetration across the round window membrane and ATX-PPS-NP showed protective effect on spiral
ganglion neurons, which further attenuated cisplatin-induced moderate hearing loss. Further studies revealed that
the protective mechanisms involved decreasing excessive ROS generation, reducing inflammatory chemokine (inter-
leukin-6) release, increasing antioxidant glutathione expression and inhibiting the mitochondrial apoptotic pathway.
Conclusions: Thus, this ROS-responsive nanoparticle encapsulating ATX has favorable potential in the prevention of
cisplatin-induced hearing loss.
Keywords: ROS-responsive, Poly(propylene sulfide), Astaxanthin, Cisplatin-induced ototoxicity
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Open Access
Journal of Nanobiotechnology
†Jiayi Gu and Xueling Wang are First authors
*Correspondence: dehongyu@126.com; wuhao@shsmu.edu.cn
1 Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth
People’s Hospital, Shanghai Jiao Tong University School of Medicine,
Shanghai, China
Full list of author information is available at the end of the article
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Page 2 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
Introduction
To date, the precise mechanism underlying cisplatin-
induced ototoxicity is still unclear. However, accu-
mulation of reactive oxygen species (ROS) has been
documented as a critical mediator in cisplatin-induced
ototoxicity, involved in many pathological processes.
In the cochlea, cisplatin upregulates the expression of
NADPH oxidase 3 and xanthine oxidase, two major
sources of ROS [1–3]. ROS then facilitate the imbalance
between ROS and the antioxidant system by deplet-
ing antioxidant molecules, including superoxide dis-
mutase, glutathione peroxidase, and catalase [4–6],
which is capable of inducing cochlear lipid peroxidation
by increasing concentrations of toxic chemicals, such as
malondialdehyde or 4-hydroxynonenal (4-HNE) [7–9].
Besides, excessive ROS opens calcium-permeable chan-
nels in sarcoplasmic/endoplasmic reticulum membranes
and plasma membranes [10], leading to an increase in
cytosolic calcium levels and eventual apoptotic and
autophagic cell death [11].
A wide range of therapeutic molecules including anti-
oxidants (apocynin, vitamin C), anti-inflammatory
(curcumin, epigallocatechin-3-gallate), anti-apoptotic
(pifithrin‐α) compounds, have been applied in preclinical
studies due to their preventative and restorative effects.
Of these compounds, antioxidants exhibit application
prospects. Astaxanthin (ATX), a xanthophyll carot-
enoid, has strong antioxidant capacity by scavenging
free radicals, quenching singlet oxygen, enhancing anti-
oxidant enzymes and inhibiting lipid peroxidation [12].
Meanwhile, it is associated with maintaining the mito-
chondrial redox state and functional integrity against
oxidative stress [13]. However, due to severe restriction
of the blood-labyrinth barrier, lack of sufficient drug con-
centrations in the inner ear often exist after intravenous
injection or oral intake (systemic administration) [14].
Even after intratympanic injection (local administration),
ATX can hardly penetrate the round window membrane
(RWM) and enter the inner ear due to its hydrophobicity
and instability. In our previous study, ATX first demon-
strated its otoprotective effect against cisplatin-induced
ototoxicity with the aid of lipid-polymer hybrid nano-
particles (LPN) [15]. erefore, inner ear drug delivery
strategies have attracted much attention due to their
contribution in allowing drug access to the inner ear,
improving cochlear drug concentrations and ultimately
strengthening drug efficacy [16, 17].
Recently, increasing attention has been paid to the
development of stimuli-responsive nanoparticles,
which realize the accurate spatiotemporal control of
drug release and minimize toxicity by avoiding dam-
age to non-target sites [18]. e stimuli that activate the
Graphical Abstract
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Page 3 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
responsiveness of nanomaterials can either be endog-
enous (redox, pH, enzyme) or exogenous (light, heat,
magnetic field and ultrasound) [19]. Notably, ROS-
responsive drug delivery systems in endogenous patho-
logical conditions have been extensively studied [20–23].
According to the literatures, ROS-responsive systems
include polysulfides, polyselenides, polythioketals, poly-
oxalates, oligoproline- and catechol-based materials [24].
Poly(propylene sulfide) (PPS), as a type of polysulfide,
can be successively oxidized to sulfoxide by hydrogen
peroxide (H2O2) and to sulfone by hypochlorite [25].
e two-step reaction results in a large change in polar-
ity and increases water solubility, consequently lead-
ing to an extensive disintegration of PPS and control
of drug release. As reported, Poly(propylene sulfide)-
poly(ethylene glycol) (PPS-PEG) has been used for drug
delivery in a broad spectrum of diseases, including can-
cers, diabetes and inflammation-related injuries [26–28].
Recently, ROS-responsive nanoparticles encapsulating
berberine have been developed and applied in noise-
induced hearing loss [29].
In this study, we fabricated a novel ROS-responsive/
consuming nanoparticle system loaded with the antioxi-
dant ATX (ATX-PPS-NP) and confirmed its ‘on demand’
spatiotemporal release invitro and in vivo. is system
combined the ROS-consuming effect of PPS-PEG and
the antioxidant efficacy of ATX. e protection against
cisplatin-induced ototoxicity was verified in HEI-OC1
cells, cochlear explants and guinea pigs, where antioxi-
dant/anti-inflammatory/anti-apoptotic processes were
involved. ATX-PPS-NP eventually reduced the loss of
spiral ganglion neuron (SGNs) and prevented hearing
loss in animal models of cisplatin-induced hearing loss.
us, providing a novel strategy for the prevention of cis-
platin-induced hearing loss.
Materials andmethods
Materials
PPS-PEG (molecular weight [MW] 54 kDa) and FITC-
conjugated PPS-PEG (FITC-PPS-PEG, MW 54 kDa)
were purchased from Xi’an Ruixi Biological Technol-
ogy Co., Ltd (Xi’an, China), and successful synthesis
was confirmed by 1H nuclear magnetic resonance (1H-
NMR) spectra. As shown in Additional file 1: Fig. S1,
FTIR spectra illustrated an absorption peak at 1024 cm−1
after treating PPS-PEG with 30% H2O2 solution, indicat-
ing the chemical bond change into S = O according to
the literature. Astaxanthin and Nile red were purchased
from Sigma (St Louis, MO, USA). Cisplatin was provided
by Aladdin (Shanghai, China). Chlorpromazine (CPZ),
filipin, nystatin and nocodazole were purchased from
Sigma (St Louis, MO, USA), and EIPA was from Med-
ChemExpress (Shanghai, China). ROS probes, Amplex
UltraRed Reagent and 2ʹ, 7ʹ-dichlorofluorescein diacetate
(DCFHDA), were obtained from Invitrogen (Carlsbad,
CA, USA) and Sigma.Texas Red conjugated cisplatin was
supplied by Ursa Bioscience (Abingdon, MD, USA).e
FITC-Annexin V/PI double staining kit was obtained
from BD Biosciences (San Diego, CA, USA).e GSH-
Glo™ Glutathione Assay kit was obtained from Promega
(Madison, WI, USA).e IL-6 ELISA kit was provided
by R&D Systems (Minneapolis, MN, USA). Antibod-
ies, includingcleaved-caspase 3, cleaved-caspase 9, P53,
cytochrome-C andα-tubulin, were purchased from CST
(Danvers, MA, USA), and Myosin VIIa was from Pro-
teus Biosciences (Ramona, CA, USA).4-hydroxynonenal
(4-HNE) was purchased from Abcam (Cambridge, MA,
USA).
Cell culture
HEI-OC1 (generated at House Ear Institute, Los Angeles,
CA, USA), an inner ear cell line derived from the audi-
tory organ of the transgenic mouse Immortomouse™, is
a potential tool for studying the mechanisms of ototoxic
drugs invitro [30]. HEI-OC1 cells were cultured in Dul-
becco’s Modified Eagle’s Medium (DMEM) and supple-
mented with 10% (v/v) fetal bovine serum (FBS) in a 10%
CO2 incubator at 33°C.
Animals
Healthy female adult guinea pigs (weighing 200–250g,
with no otitis media) and mouse pups (C57BL/6J, P0-3)
were used. roughout the study, all animals were main-
tained under standard laboratory conditions, housed
under standard animal facility conditions with adequate
food and water. All animal studies were approved and
conducted in accordance with the guidelines of the Ethics
Committee of the Shanghai Jiaotong University, School
of Medicine (Shanghai, China).
Preparation ofastaxanthin‑loaded ROS‑responsive
nanoparticles (ATX‑PPS‑NP)
ATX-PPS-NP was fabricated using a single emulsion and
solvent evaporation method as described previously [15].
irty milligrams of PPS-PEG and 2mg ATXwere dis-
solved in 1ml of dichloromethane solution, followed by
pouring 3ml of sodium cholate solution (3%, w/v) into
the organic solvent. e mixture was sonicated at 260W
for 4min (Scientz Biotechnology Co., Ltd., Ningbo City,
China). e resulting oil/water emulsion was further
diluted in 36 ml of 0.5% sodium cholate solution and
stirred overnight to remove the organic solvent. ATX-
PPS-NP were collected by centrifugation (11,000×g,
30min) and washed twice to remove excessive emulsifier
and unloaded ATX. FITC-PPS-NP were prepared in the
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Guetal. Journal of Nanobiotechnology (2022) 20:268
same way except that PPS-PEG was replaced with FITC-
PPS-PEG. NiRe-PPS-NP were loaded with the fluores-
cent probe Nile red. e NP were stored at 4°C.
Characterization
Particle size, polydispersity index (PDI) and z-potential
values were measured by zeta potential measurement
(Nano ZS90 Zetasizer, Malvern Instruments Co., Ltd.,
UK) equipped with dynamic light scattering (DLS).
Encapsulation efficiency (EE %) and drug loading (DL
%) of the ATX-PPS-NP were determined using liquid
chromatography combined with mass spectrometry
(LC–MS) as described previously [16].
ATX stability
Absorbance measurements were performed to assess
the stability of ATX loaded in NP [31]. Natural ATX
in EtOH/DCM (% v/v; 60/40) was used as the stand-
ard for the ATX calibration curve (absorbance-con-
centration curve) based on Beer–Lambert law, under
the maximum absorption (λmax) at 490 nm. At vari-
ous time points (0, 7, 14 and 21 days), ATX-PPS-NP
or ATX were diluted and dissolved in EtOH/DCM for
the ATX concentrations measurements by microplate
reader.e remaining ATX was normalized by dividing
the concentration of ATX at day 0.
ROS‑responsive release prole invitro
e release kinetics of PPS-NP invitro was simulated
by the fluorescence quenching of loaded Nile red, a
special fluorescent probe which fluoresces strongly in a
hydrophobic environment, but quenches when released
into an aqueous phase where it is poorly soluble. Ten
microliters of NiRe-PPS-NP was added to H2O2 solu-
tion diluted in artificial perilymph (0, 5 and 500mM)
to each well of a 96-well plate which was stirred and
placed in a gas bath at 37°C. e Nile red fluorescence
was measured using a microplate reader (Tecan Spark,
Tecan Group Ltd.) at an excitation/emission wave-
length of 535nm/612nm at specific time points from
0 to 72h.
To evaluate ROS-responsive drug release, H2O2 solu-
tion was used to stimulate ROS release. Briefly, NiRe-
PPS-NPwere subjected to 0 or 500mM H2O2 solution at
each ON phase for 24h, and replaced byPBS for another
24h for the OFF phase,circulating for a maximum dura-
tion of 7days. At the end of each interval,Nile red fluo-
rescence was measured by a microplate reader (Tecan
Spark, Tecan Group Ltd.) at an excitation/emission wave-
length of 535nm/612nm.
To further evaluate disintegration of ATX-PPS-NP in
the ROS environment, transmission electron microscopy
(TEM) was used to observe the morphological changes in
PPS-NP. Briefly, ATX-PPS-NP were treated with 0.3% or
3% H2O2 solution for 24 or 48h.
ROS‑responsive release prole—in HEI‑OC1 cells
HEI-OC1 cells were seeded and treated with 60 μM
cisplatin for 24 h, followed by 24 h-culture in NiRe-
PPS-NP. After washing twice, nuclei were stained with
Hoechst 33,342. H2O2 solution (200μM) was used as a
positive control. Fluorescent images were captured with
a laser confocal microscope (LSM880, Zeiss, Germany).
Cellular uptake assay
HEI-OC1 cells (5 × 104 cells per well) were seeded in a
24-well plate and allowed to adhere overnight. e cells
were then incubated with FITC-PPS-NP for 1, 3, 6, 12
and 24h. e cells were washed with PBS, fixed with
4% paraformaldehyde (PFA) for 15 min and stained
with Hoechst 33,342. e FITC fluorescence was exam-
ined using a laser confocal microscope (LSM-880,
Zeiss, Germany). To quantify cellular uptake, cells were
prepared as previously described in a 6-well plate and
collected for flow cytometry (BD Biosciences, Fortessa,
CA, USA).
To examine the endocytosis mechanism of PPS-NP,
HEI-OC1 cells were pre-incubated with the following
inhibitors based on previous studies [32] at nontoxic
concentrations: 15 µg/ml of chlorpromazine (CPZ), to
inhibit clathrin-mediated endocytosis; 100μM of EIPA
(MCE), to block macropinocytosis and phagocytosis;
6µg/ml filipin and 50µg/ml of nystatin, to inhibit cav-
eolae-mediated endocytosis; and 2µg/ml nocodazole, to
disrupt microtubules. e inhibitors were added to the
cell culture medium or incubated for 45min at 4°C prior
to addition of the FITC-PPS-NP suspensions for another
3h. Subsequently, the cells were washed and collected for
flow cytometry (BD Biosciences, Fortessa, CA, USA).
ROS‑responsive accumulation ofPPS‑NP
In HEI‑OC1 cells
HEI-OC1 cells were cultured in dishes for confocal
images. e cells were pretreated with CDDP (60 μM)
or free medium for 24h, and additionally co-incubated
with Amplex UltraRed Reagent and FITC-PPS-NP.
Immediately, the cells wereimaged with a laser confocal
microscope (LSM880, Zeiss) in a time series, at 10min
intervals for up to 30min. H2O2 solution (200μM) was
used as a positive control.
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Guetal. Journal of Nanobiotechnology (2022) 20:268
In cultured Organ ofCorti
e organs of Corti were dissected from neonatal mice
(postnatal days 1–3) and cultured in the medium as
described previously [17]. Explants were co-incubated
with Texas Red conjugated cisplatin and FITC-PPS-NP
for 24h. e tissues were then stained with Myosin VIIa
and Hoechst 33,342. Fluorescent images were captured
with a laser confocal microscope (LSM880, Zeiss).
Protective eect inHEI‑OC1 cells
Cell viability assay
Cell viability in different patterns of cisplatin and drug
was examined using the CCK-8 assay (Dojindo, Japan). In
brief, HEI-OC1 cells (1 × 105 cells per well) were seeded
in 96-well plates and incubated overnight. e cells were
divided into six groups (ATX, PPS-NP, ATX-PPS-NP,
administrated at the same ATX concentration of 1μg/ml)
as follows: (1) Control, (2) drug alone for 24h, (3) cispl-
atin (60μM) for 24h, (4) pretreatment with drug for 4h
and withdrawal of the drug followed by treatment with
CDDP for 24 h (pre-4 h), (5) pretreatment with drug
for 4h followed by a co-treatment with CDDP for 24h
(pre-4h + co-24h) and (6) direct co-treatment with drug
and CDDP for 24h (co-24h). After washing three times,
100μl of dissolved CCK-8 solution was added to each
well and incubated for 2h at 33°C. e absorbance was
measured using a microplate reader (Tecan Spark, Tecan
Group Ltd.) at 450nm. All data were normalized based
on background intensity. Blank PPS-NP with the same
concentrations were also examined in all cases with nan-
oparticles involved.
Cell apoptosis study
Cellular apoptosis was quantified by flow cytometry
using an FITC-Annexin V/PI double staining kit (BD
Biosciences, San Diego, CA, USA). HEI-OC1 cells were
pretreated with ATX, PPS-NP and ATX-PPS-NP for
4h followed by co-treatment with CDDP for 24 h. e
cells were then rinsed, trypsinized and collected after
centrifugation. e cells were resuspended in 100μl of
1 × binding buffer containing 5μl FITC-Annexin V and
PI, incubated for 15min at room temperature in the dark
and then analyzed by flow cytometry (BD Biosciences,
Fortessa, CA, USA). Both Annexin V + /PI − cells repre-
senting early-apoptotic cells and Annexin V + /PI + cells
representing late-apoptotic cells were considered apop-
totic. Blank PPS-NP at the same concentrations were also
examined in all cases with nanoparticles involved.
Molecular mechanism oftheprotective eect inHEI‑OC1
cells
HEI-OC1 cells were divided into six groups: (1) Con-
trol, (2) CDDP, 60μM, 24h, (3–5) pretreated with ATX,
PPS-NP and ATX-PPS-NP, respectively, for 4h followed
by co-treatment with CDDP for 24 h. Each group was
included in the following experiments.
Intracellular ROS level
e intracellular ROS level was detected using a fluo-
rescent dye, 2’,7’-dichlorofluorescein diacetate (DCF-
HDA). Non-fluorescent DCFH is converted into the
highly fluorescent DCF by an intracellular oxida-
tion. For the assay, cells were seeded in a 24-well
plate overnight and treated with drugs as above. After
rinsing twice, 10 μM DCFHDA was added and cul-
tured for 30 min at room temperature in the dark.
Images were acquired by laser confocal microscopy
(488nm/530nm) and fluorescence intensities of ran-
domly chosen cells were quantified.
Glutathione (GSH) andinterleukin‑6 (IL‑6) assay
HEI-OC1 cells (1 × 105 cells per well) were seeded in
96-well plates and incubated overnight, followed by
drug administration as above. After 24h, the medium
was removed and the content of GSH was calculated
using aGSH-Glo™ Glutathione Assay kit. e removed
medium was collected for IL-6 detection using an
ELISA kit,according to the manufacturer’s protocol.
Apoptosis‑associated proteins
e levels of apoptosis-associated proteins were evalu-
ated by western blot. HEI-OC1 cells were cultured
and treated with drugs as above. After washing twice
with ice cold PBS on ice and lysed in RIPA, the pro-
tein extracts were subjected to 12% SDS-PAGE and
electrotransferred to PVDF membranes. is was fol-
lowed by blocking and incubation with specific anti-
bodies to cleaved-caspase 3, cleaved-caspase 9, P53 and
cytochrome-C (α-tubulin served as the control stand-
ard). e relative intensity was quantified by ImageJ
software.
Protective eect incultured Organ ofCorti
e organs of Corti were cultured as above and divided
into five groups: (1) Control, (2) CDDP, 60 μM, 24 h,
(3–5) pretreated with ATX, PPS-NP and ATX-PPS-NP,
respectively, at the same ATX concentration of 1 μg/
ml, for 4h and followed by co-treatment with CDDP for
24h. After fixation, hair cells were labelled with Myosin
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Page 6 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
VIIa and quantified in the apex, middle and basal turns.
Fluorescent images were captured with a laser confocal
microscope (LSM880, Zeiss). e numbers of surviv-
ing hair cells in 1μm tissue of Organ of Corti were cal-
culated, and the percentages of those were compared in
groups.
Protective eect inguinea pigs
Evaluation ofRWM penetration
NiRe-PPS-NP were used to investigate whether PPS-
NP could penetrate the RWM. Briefly, guinea pigs were
anaesthetized with ketamine (60mg/kg, IP) and xyla-
zine (10 mg/kg, IP). NiRe-PPS-NP in gelatin sponge
were placed on the RWM as previously reported [15],
for 0.5, 2 and 6h, respectively (n = 3). After fixation,
the RWM was microdissected and prepared for confo-
cal imaging according to theprocedures described in
previous studies [33].A series of images of the RWM
by the z-axis scanning model at 1-µm vertical intervals
from the middle ear side to the scala tympani side were
collected and mean fluorescence intensities (MFI) were
calculated. e outer epithelium layer (OE), connective
tissue layer (CT), and inner epithelium layer (IE) of the
RWM z-stack series images were differentiated by cell
morphology.
In vivo release prole ofATX
Guinea pigs were divided into the control and cisplatin-
treated groups and intraperitoneally injected with PBS
and cisplatin (12mg/kg), respectively. At designed time
points of 0.5, 2, 6 and 12 h after RWM application of
ATX-PPS-NP in the control group and 2h in the cispl-
atin group, the cochleae were dissected after euthanasia
and perilymph was collected by aspiration using a sharp
glass pipette inserted into the scala tympani from the
RWM. Samples were stored at −80°C. e concentra-
tions of ATX were quantified using LC–MS. After dilut-
ing the samples, a 30µL aliquot of the sample was added
with 150µL IS (diclofenac, 100ng/ml) in ACN. e mix-
ture was vortexed for 10min and centrifuged at 3200g
for 10min and then vortexed for 10min and centrifuged
at 5800rpm for 10min. A 5µL aliquot of the supernatant
was injected for LC–MS analysis. e minimum concen-
tration detected was 1ng/ml.
Protective eect—Auditory Brainstem Responses (ABR) tests
To evaluate auditory function, ABR tests were recorded
in response to pure tone stimuli (4, 5.6, 8, 11.2, 16, 22.6,
32 and 45kHz) between 0 and 90dB in 5dB increments
using a closed-field microphone. e acoustic signals
were generated with a RZ6 signal processor and BioSig
software (TDT, Alachua FL, USA). resholds were
defined as the lowest stimulus level at which a response
was observed.
Guinea pigs were administrated with ATX-PPS-NP
(0.36 mg/ml, 7μl), ATX (0.36 mg/ml,7μl) or normal
saline (N.S.) on the RWM. One hour later, the animals
were intraperitoneally injected with cisplatin (12mg/kg).
ABR tests were conducted before the operation and one
or three days after CDDP injection.
Mechanism oftheprotective eect inanimals
Guinea pig cochlea samples were prepared as reported
previously [34]. Briefly, mouse cochleae were quickly dis-
sected from the temporal bone following rapid decapi-
tation. After fixation, decalcification and progressive
dehydration, the samples were embedded in paraffin.
Mid-modiolar section samples were then cut at 5–7μm
thickness and mounted on glass slides. Paraffin sections
were deparaffinized in xylene, rehydrated through an
ethanol series, and stained with H&E.For immunostain-
ing of 4-HNE in SGNs,cochlear sections were prepared
as previously described [35] and imaged with an optical
microscope. R2, R3, R4 was defined as cochlear turns
from apex to base.ImageJ software was used to measure
the area of SGN regions. SGN density was defined by the
number of SGNs/area of the SGN region. e integrated
optical density (IOD) of 4-HNE staining in the SGN area
was measured by ImageJ software.
Statistics
All data are reported as mean ± standard deviation (SD).
Analysis of Variance (ANOVA) with Bonferroni correc-
tion for multiple comparisons was used to determine
treatment effects. p < 0.05 was considered significant.
Results
Characterization ofATX‑PPS‑NP
e physicochemical characteristics of nanoparticles
were validated (Fig. 1) by measuring particle size, PDI
and z-potential values. As shown in Fig.1B and Table1,
the particle size of PPS-NP and ATX-PPS-NP were
169.57 ± 7.87 nm and 182.27 ± 5.05 nm in diameter,
respectively, and fitted the Gaussian shape line. A low
PDI value (0.38 ± 0.02) also indicated a relatively uniform
distribution of nanoparticles. Encapsulation efficiency
(EE %) and drug loading (DL %) were 68.07% ± 3.02%
and 4.31% ± 0.22%, respectively. Moreover, PPS-NP and
ATX-PPS-NP were positively charged at 10.86 ± 3.27mV
and 14.73 ± 0.87mV, respectively.
We investigated the stability of ATX-PPS-NP by meas-
uring the remaining concentrations of ATX at different
time points (day 0, 7, 14, 21) under light or heat stimu-
lation. As shown in Fig. 1C, ATX-PPS-NP significantly
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Page 7 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
improved the stability of ATX on days 7, 14, and 21
under 37°C with or without light (dark), characterized
by the remaining concentration of ATX which was nota-
bly higher in the ATX-PPS-NP group than in the ATX
group. Furthermore, on day 21, ATX-PPS-NP preserved
a greater amount of ATX, 78% and 71%, respectively, in
dark and light environments, compared with 46% and
32% in free ATX groups (p < 0.01, p < 0.001). Temperature
had no impact on ATX stability when encapsulated in
PPS-NP under light (Fig.1D).
ROS‑responsive release prole invitro
e ROS-responsive release from PPS-NP was assessed
by the loss of fluorescence of encapsulated Nile red
(NiRe) (Fig.1E, F). Specifically, at each time point, the
fluorescence value which was subtracted from that of
Fig. 1 Physicochemical characterization of ATX-PPS-NP and in vitro ROS-responsive release. A The preparation of ATX-PPS-NP. B Gaussian
distribution of the ATX-PPS-NP. C–D In 4 °C or 37 °C environments, the remaining percentages of ATX alone or encapsulated in PPS-NP in dark
or light conditions within 21 days. *p < 0.05, **p < 0.01, ***p < 0.001 vs ATX dark, ###p < 0.001 vs ATX light. E In vitro Nile red (NiRe) release from
NiRe-PPS-NP in various concentrations of H2O2 solutions. F In vitro release of NiRe with intermittent exposure (OFF/ON cycles every other day) to
500 mM H2O2 solution. G Representative TEM images of ATX-PPS-NP in control and various oxidation groups
Table 1 Particle size, zeta potential, PDI values, drug loading (DL%) and encapsulation efficiency (EE%) of PPS-NP and ATX-PPS-NP
Nanoparticles (NP) Particle Size (nm) Zeta potential (mV) PDI EE % DL %
PPS-NP 169.57 ± 7.87 10.86 ± 3.27 0.29 ± 0.03 / /
ATX-PPS-NP 182.27 ± 5.05 14.73 ± 0.87 0.38 ± 0.02 68.07 ± 3.02 4.31 ± 0.22
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Page 8 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
the sample prior to H2O2 addition was normalized by
the control. Under 5 mM H2O2 oxidation, approxi-
mately 45% of NiRe was released over 48h, double higher
than 20% released from the control group, while NiRe
released from NiRe-PPS-NP oxidized by 500mM H2O2
was approximately 100%. Moreover, NiRe release at each
time point was higher in the oxidation group than in the
control. ese results indicated that ROS could quickly
trigger the disintegration of NiRe-PPS-NP at an early
stage and the encapsulated drug was released in a dose-
dependent manner upon exposure to H2O2. To illustrate
the smart release of encapsulated drugs, NiRe-PPS-NP
wereintermittently exposed to 0 or 500mM H2O2 solu-
tion OFF/ON cycles every other day. e slope of the
release curve was steeper during the ON phases than
during the OFF phases, indicating that “on demand”
release could be achieved. Together, the release pro-
files demonstrated ROS concentration-dependent, on
demand release of the loaded drugs from PPS-NP.
To further evaluate disintegration of ATX-PPS-NP
in the ROS environment, TEM was used to observe the
morphological change in PPS-NP. As shown in Fig.1G,
PPS-NP exhibited a spherical structure with an average
diameter between 100–200 nm, in agreement with the
size measurement performed by DLS. After oxidation
with 0.3%-3% H2O2 solution for 24–48h mimicking ROS
stimulation, NP progressively disintegrated and fused,
suggesting ROS-responsiveness of PPS-NP.
Cellular uptake andendocytosis mechanism
To investigate the endocytosis of PPS-NP and the
underlying mechanism, we fabricated FITC-PPS-NP as
mentioned above, where FITC was used as a tracer flu-
orescein in the formulation of PPS-NP. Confocal laser
scanning microscopy and flow cytometry were per-
formed to observe and quantify the uptake profile in
HEI-OC1 cells, respectively. As shown in Fig.2A–C, cel-
lular uptake showed a time-dependent increase between
1–12h. After reaching the peak at 12h, the uptake activ-
ity decreased at 24h.
To elucidate the endocytic pathways of PPS-NP, we
studied the effects of selective inhibitors and low temper-
ature (4°C) on cellular uptake. Of the inhibitors studied,
treatment with CPZ led to significantly reduced fluores-
cence, suggesting that clathrin-mediated endocytosis
may contribute to the uptake of PPS-NP in HEI-OC1
cells (Fig.2D), in line with the size of particles (about
100nm in diameter) internalized inthe clathrin-medi-
ated pathway [36]. Furthermore, low temperature (4°C)
also decreased the internalization of FITC-PPS-NP, sug-
gesting energy-dependent endocytosis.
ROS‑responsive accumulation ofPPS‑NP inHEI‑OC1 cells
andcultured Organ ofCorti
Firstly, we screened the optimal dose and time for the
CDDP-treated HEI-OC1 cell model. e results showed
that 60 μM CDDP for 24 h induced approximately
70% cell viability and a significant generation of ROS
Fig. 2 In vitro cellular uptake of FITC-PPS-NP. A Confocal images demonstrated cellular uptake of FITC-PPS-NP for various periods of time (1, 3, 6, 12
and 24 h) in HEI-OC1. B, C Flow cytometry assay of cellular uptake and quantifications of MFI of FITC. D Endocytosis mechanism of PPS-NP. **p < 0.01
vs control, ***p < 0.001 vs control
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Guetal. Journal of Nanobiotechnology (2022) 20:268
(Additional file1: Fig. S2). en we evaluated the ROS-
responsive accumulation of FITC-PPS-NP. In Fig.3A–C,
HEI-OC1 cells treated with CDDP (60μM, 24h) were co-
cultured with FITC-PPS-NP and Amplex Ultrared, which
respectively trafficked intracellular locations of NP and
labeled ROS generated, and H2O2 (200μM, 3h) was used
as a positive control. Serial images of live cells demon-
strated thatFITC-PPS-NP progressively entered cells and
approached where ROS were located, suggesting passive
ROS-responsive accumulation of FITC-PPS-NP invitro.
A stronger green fluorescence was observed in the CDDP
group compared with the control group, indicating that
ROS promoted the uptake ofFITC-PPS-NP.
Figure 3D showed the intracellular ROS-responsive-
ness of NiRe-PPS-NP. In HEI-OC1 cells, NiRe-PPS-
NP were quickly internalized. After exposure to CDDP
(60μM), the red fluorescence was quenched, indicating
ROS-triggered release of NiRe into the aqueous phase in
response to oxidative stress, while H2O2 (200μM) was
used as a positive control.
In Fig. 3E, Texas Red and FITC fluorescence traf-
ficked CDDP and NP, respectively, while Myosin VIIa
labeled hair cells. Compared with a fairly weak FITC sig-
nal in the control group,the whole and detailed images
of organotypic culture inthe cisplatin group showed a
stronger green fluorescence, mainly inthe spiral limbus
and hair cells, suggesting that cisplatin treatment pro-
moted the uptake of NP. e merging signal of Texas Red
and FITC indicated that FITC-PPS-NP targeted the spe-
cific sites damaged by cisplatin, where ROS seemed to be
generated.
Cytoprotective andanti‑apoptotic activity ofATX‑PPS‑NP
As illustrated in Fig. 4A, HEI-OC1 cells were treated
with CDDP and different drugs, including ATX (1μg/
ml), PPS-NP (drug-free) and ATX-PPS-NP (1 μg/
ml). Cell viability was significantly increased in the
ATX pre-4 h group (p < 0.001) and drug-free PPS-NP
pre-4h + co-24h group (p < 0.001), which was inhibited
by CDDP. ATX-PPS-NP resulted in higher viability after
Fig. 3 ROS-responsive accumulation and release profile in HEI-OC1 cells and cultured Organ of Corti. A–C Live cell imaging of cellular uptake of
FITC-PPS-NP within 30 min under CDDP (60 μM, 24 h) stimulation, H2O2 (200 μM, 3 h) as a positive control. D Confocal images showed the Nile Red
fluorescence changed at various time points in control or CDDP (60 μM) treated HEI-OC1 cells, H2O2 (200 μM) as a positive control. E Representative
confocal images of cochlear explants. Texas Red (TR) and FITC fluorescence respectively labeled CDDP and NP, while Myosin VIIa labeled hair cells
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Guetal. Journal of Nanobiotechnology (2022) 20:268
pre-4h + co-24h of administration, up to 1.5-fold com-
pared with that of the CDDP group. ese results suggest
that ATX-PPS-NP, combining ROS-consuming PPS-NP
and antioxidant ATX, demonstrated an enhanced protec-
tive effect against cisplatin-induced cytotoxicity.
Annexin V-FITC/PI double staining was used to label
apoptotic cells and the percentage of these cells was
analyzed by flow cytometry (Fig.4B, C). CDDP induced
18.43% of apoptosis, which was reduced by ATX-PPS-NP
(p < 0.01), while no significant difference occurred in the
ATX and drug-free PPS-NP group, confirming that ATX-
PPS-NP exhibited notable efficiency in attenuating cell
apoptosis.
As shown in Fig.4D, E, the intact arrangement of hair
cells was destroyed following CDDP administration and
was partially restored by ATX-PPS-NP. To evaluate the
protective effect of ATX-PPS-NP, hair cell counts were
calculated. e results revealed that the CDDP group
exhibited severe loss of hair cells and ATX-PPS-NP pre-
served hair cells in the basal turn ofthe organ of Corti
explants.
The mechanisms oftheantioxidant, anti‑inammatory
andanti‑apoptotic eects ofATX‑PPS‑NP
e underlying mechanisms of CDDP-induced ototox-
icity are complicated, which resulted in the multifacto-
rial protective effects of ATX-PPS-NP. We studied these
effects from three aspects. Firstly, we explored the anti-
oxidant effect. After screening the optimal drug treat-
ment to reduce ROS production, pre 4h + co 24 h was
Fig. 4 Cytoprotective and anti-apoptotic activity of ATX-PPS-NP against CDDP-induced toxicity in vitro. A A parallel comparison of cell viability
in CDDP-treated HEI-OC1 cells with administrations of ATX (1 μg/ml), PPS-NP (drug free) and ATX-PPS-NP (1 μg/ml) in three types. *** p < 0.001 vs
control, ### p < 0.001 vs CDDP, &&& p < 0.001 vs ATX-PPS-NP. B Flow cytometry showing the percentage of apoptotic cells labeled by Annexin V-FITC/PI
double staining in ATX (1 μg/ml), PPS-NP (drug free) and ATX-PPS-NP (1 μg/ml) pretreated HEI-OC1 cells, followed by CDDP (60 μM, 24 h) exposure.
C Quantifications of apoptotic cells. ** p < 0.01, *** p < 0.001 vs control, # p < 0.05, ## p < 0.01, ### p < 0.001 vs CDDP, &&& p < 0.001 vs ATX-PPS-NP. D
Immunostaining of Myosin VIIa labeling hair cells in cochlea explants in control, CDDP (60 μM), ATX (1 μg/ml), PPS-NP (drug free) and ATX-PPS-NP
(1 μg/ml) group. E Quantifications of hair cells in apex, middle and basal turn of cochlea explants. *, #, & p < 0.05 vs control, **, ##, && p < 0.01 vs control,
***, ##, &&& p < 0.001 vs control, $ p < 0.05 vs CDDP, $$ p < 0.01 vs CDDP
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Guetal. Journal of Nanobiotechnology (2022) 20:268
selected for the following studies (Additional file1: Fig.
S3). In Fig. 5A–B, as demonstrated by a reduction in
MFI of DCFHDA (green fluorescence), the level of ROS
induced by CDDP decreased following treatment with
ATX-PPS-NP (p < 0.05), suggesting the highest antioxi-
dant efficacy of ATX-PPS-NP against CDDP-induced
accumulation of ROS among the three drugs stud-
ied. On the other hand, neither the ATX nor drug-free
PPS-NP groups displayed significant reduction in ROS,
confirming that a combination of ROS-consuming PPS-
NP and the ROS-scavenging compound ATX led to an
enhanced antioxidant effect.
It is acknowledged that the excessive generation of ROS
overwhelms the redox balance [5]. We then detected
the GSH level to investigate the antioxidant efficacy of
ATX-PPS-NP. As shown in Fig.5C, CDDPsignificantly
decreased GSH level, while ATX-PPS-NP restored GSH
level to approximately that of the control group when
Fig. 5 The mechanisms of antioxidant, anti-inflammatory and anti-apoptotic effects of ATX-PPS-NP.A Confocal images of intracellular ROS level
stained by DCFHDA (green fluorescence) in ATX (1 μg/ml), PPS-NP (drug free) and ATX-PPS-NP (1 μg/ml) pretreated HEI-OC1 cells, followed by
CDDP (60 μM, 24 h) administration. B Quantifications of MFI of DCFHDA. C GSH levels. D IL-6 levels. E–F The expression levels and qualifications of
apoptosis-associated proteins evaluated by Western blot analyses. * p < 0.05, ** p < 0.01, *** p < 0.001 vs control, # p < 0.05, ## p < 0.01, ### p < 0.001 vs
CDDP, &&& p < 0.001 vs ATX-PPS-NP
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Guetal. Journal of Nanobiotechnology (2022) 20:268
compared to the ATX or PPS-NP group. is indicated
that ATX-PPS-NP strengthened theantioxidant defense
system by reducing the depletion of GSH contents.
Secondly, the accumulation of ROS has also been iden-
tified as an important mediator of the inflammatory
response. To clarify the anti-inflammatory effect of ATX-
PPS-NP, a cytokine assay (IL-6) was performed. Com-
pared to the control group, a significantly higher level of
IL-6 was detected in the CDDP group. Treatment with
ATX-PPS-NP was effective in decreasing cochlear pro-
inflammatory cytokine levels (Fig.5D). By contrast, free
ATX alone or blank PPS-NP showed no significant sup-
pression of IL-6.
Lastly, in terms of protein expression involved in
the process of apoptosis, we evaluated the protein
level of cleaved-caspase 3, cleaved-caspase 9, P53 and
cytochrome-C (Fig.5E, F). e results demonstrated that
the expression of cytochrome-C and cleaved-caspase 3
was significantly elevated by CDDP treatment and sub-
sequently reduced by ATX-PPS-NP. However, cleaved-
caspase 9 and P53 were unchanged. Cytochrome-C is
an initial factor in the mitochondrial apoptotic pathway
and caspase-3 serves as a converging point in intrinsic
and extrinsic pathways. ese results suggest that exces-
sive ROS induced by CDDP triggered mitochondrial
depolarization and the cytochrome-C release, activating
caspase-3 and ultimately leading to intrinsic apoptosis,
which was suppressed by ATX-PPS-NP.
Overall, ATX-PPS-NP had potent antioxidant, anti-
inflammatory and anti-apoptotic effects on CDDP-
treated cells in vitro, eventually leading to increased
cellular viability and attenuation of cell apoptosis.
Evaluation ofRWM penetration andinvivo release prole
Previous studies on inner ear drug delivery systems
mostly focused on their pharmacokinetics or cochlear
distributions, but seldom on the interaction with the
RWM. Here, we adapted a novel method reported by
Chen etal. [33] to observe the RWM penetration pro-
cess by NP. In detail, fluorescent images were captured
in the three layers of the RWM (OE, outer epithelium
layer; CT, connective tissue; IE, inner epithelium layer) at
Fig. 6 Evaluation of RWM penetration and in vivo release profile. A Fluorescent images of NiRe-PPS-NP penetrating three layers (OE, CT and IE) of
round window membrane in control, 0.5, 2 and 6 h time points. B MFI of Nile Red along the RWM from middle ear side to scala tympani side. C ATX
concentrations in control cochlear perilymph in various time points (0.5, 2, 6 and 12 h) and CDDP-treated ones (2 h). *** p < 0.001
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Guetal. Journal of Nanobiotechnology (2022) 20:268
various time points after administration of NiRe-PPS-NP
(Fig.6A). As illustrated in Fig.6B, the MFI of NiRe-PPS-
NP increased quickly in the 0.5h group, indicating the
successful and rapid migration of NP from the middle ear
side to the scala tympani side of the RWM. A significant
increase in MFI was observed in the 2h group, suggest-
ing stable transport of NP into the inner ear. MFI in the
6h group was reduced to a level lower than that in the
0.5h group, which indicated that amajority of NP had
penetrated theRWM into the cochlea.
To assess the penetrating capacity of ATX-PPS-NP
through the RWM, we examined the ATX concentration
in perilymph following the administration of a single dose
of ATX-PPS-NP into the RWM. As shown in Fig. 6C,
at 0.5h after administration, the concentration of ATX
which had diffused into the inner ear was 76.23 ± 9.74ng/
ml. is was sustained for almost 12h. In contrast, ATX
concentrations in the perilymph of CDDP-treated ani-
mals were much higher than those without CDDP dam-
age, 272.97 ± 58.18 ng/ml vs. 46.00 ± 24.77 ng/ml (***
p < 0.001), suggesting the ROS-triggered drug release of
ATX-PPS-NP in guinea pigs, consistent with the ROS-
responsive drug release invitro (Fig.1E–G, 3D).
Protective eects onauditory function inguinea pigs
andunderlying mechanisms
Encouraged by the efficient cytoprotection of ATX-
PPS-NP in vitro, the protective effect was then
studiedin vivo.Firstly, H&E stainingverified thatATX-
PPS-NP were well-tolerated,no inflammatory response
was observed in the RWM (Additional file1: Fig. S4). e
surgical procedures used for RWM administration were
further confirmed to be safe, based on no elevation in
ABR thresholds (Fig.7A). We then classified cisplatin-
induced hearing loss into moderate and severe types
according to the duration of CDDP treatment. It can be
seen in Fig. 7A that ATX-PPS-NP administration pro-
tected against CDDP-induced moderate hearing loss by
13.57, 15.00 and12.86dB SPL at 4.0, 5.6 and 8.0 kHz,
respectively, while no significant changes appeared in the
unencapsulated ATX group. Taken together, these results
verified that ATX-PPS-NP showed superior protection
in HEI-OC1 cells, cochlear explants and animal mod-
els compared with equivalent ATX alone, revealing the
enhanced antioxidant effect of ATX-PPS-NP. Addition-
ally, no improvement in hearing was observed in animals
with severe hearing loss, indicating that ATX-PPS-NP
was only effective in protection againstmoderate hearing
loss. We found that pretreatment ofATX-PPS-NP atten-
uated cisplatin-induced moderate hearing loss at low fre-
quencies, with a negligible effectat high frequencies.
e present studyrevealed thatfocal vacuolization and
nuclear degeneration were observed in cisplatin-treated
SGNs, which was alleviated in the ATX-PPS-NP group
(Fig.7D).e SGN density was determined as the num-
ber of SGNs per unit area in the Rosenthal canal. As
shown in Fig. 7B, ATX-PPS-NP preserved SGNs from
destruction by cisplatin in the apical turn of the coch-
lea,consistent with the structural site where the protec-
tion of auditory function occurred. Animals treated with
CDDP (Day 1) is an appropriate model for studying the
mechanisms of ATX-PPS-NP in SGNs alone.
However, the number or morphology of hair cells and
supporting cells were not influenced in CDDP-induced
moderate hearing loss, and the thickness of the stria
vascularis was not changed (Additional file1: Fig. S5).
e mismatching of protection targets between coch-
lear explants and guinea pigs could be the result of the
specific damaging regions by different administration
types. Direct exposure to culture medium containing
cisplatin led to outer hair cell (OHC) loss, while systemi-
cally administrated cisplatin firstly caused SGN loss. In
addition, functional impairments or subcellular changes
which could not be obtained without performing ultra-
structural level morphological analyses in OHCs may
occur, but OHC count was unchanged on day 1 after
CDDP administration.
To evaluate the in vivo antioxidant efficacy of ATX-
PPS-NP, 4-HNE (a cytotoxic end product of lipid per-
oxidation, one of the markers for oxidative stress) was
stained with 3, 3’-diaminobenzidine (DAB) for assess-
ment of ROS generation. Figure 7C, E showed higher
expression of 4-HNE in the cytoplasm of SGNs in the
CDDP (Day 1) group compared to the control. Weaker
expression of 4-HNE was observed in the ATX-PPS-NP
group, with no changes in the ATX group. e quantifi-
cations of IOD/area demonstrated a reduction in 4-HNE
in the apical and middle turns, which verified the strong
ROS-scavenging ability of ATX-PPS-NP.
Discussion
Up to now, cisplatin is an indispensable chemothera-
peutic compound for a broad spectrum of solid tumors.
However, its high therapeutic efficacy is often coupled
with a potential ototoxicity with an average incidence
over 60% [1]. With no pharmaceutical drugs approved by
FDA, astaxanthin is considered as a promising candidate
against cisplatin-induced ototoxicity due to its power-
ful antioxidant property, with ROS-scavenging capacity
6000 times higher than vitamin C, 550 times higher than
vitamin E, 75 times higher than a-lipoic acid [37]. How-
ever, its bioavailability in inner ear is limited by low coch-
lear ATX concentration on account of its hydrophobicity,
short half-life and susceptibility to chemical degrada-
tion under certainconditions (light, temperature, alkali,
oxidation and isomerization) [38]. To overcome these
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Page 14 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
Fig. 7 Protective effects on auditory function in guinea pigs and underlying mechanisms. A ABR thresholds of various groups. *** p < 0.001 control
vs CDDP (Day 1) and CDDP (Day 3), # p < 0.05 ATX-PPS-NP + CDDP (Day 1) vs CDDP (Day 1), ## p < 0.01 ATX-PPS-NP + CDDP (Day 1) vs CDDP (Day 1), &
p < 0.05 ATX-PPS-NP + CDDP (Day 1) vs ATX + CDDP (Day 1) B & D. H&E staining of SGNs and calculations of SGN density in 2–4 turns of guinea pig
cochleae. * p < 0.05 vs control, ** p < 0.01 vs control, *** p < 0.001 vs control, # p < 0.05 vs CDDP. C & E. Immunohistochemistry of 4-HNE in SGNs and
quantifications of integrated optical density (IOD) of 4-HNE staining. *** p < 0.001 vs control, ## p < 0.01 vs CDDP
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Page 15 of 17
Guetal. Journal of Nanobiotechnology (2022) 20:268
limitations, PEGylation was applied in the fabrication
of nanoparticles due to its effect in prolonging circula-
tion half-life in serum bydecreasing protein absorption
and avoiding the formation of aggregates [39]. Here, we
verified that the encapsulation of PPS-NP preserved the
stability of ATX (Fig.1C, D),in consonance with meth-
ods using microencapsulation with chitosan, polymeric
nanospheres, emulsions and β-cyclodextrin in previous
studies [40]. Previous studiesindicated that size between
150 and 300nm with a positivesurface charge has advan-
tages in permeating the RWM andentering the cochlea
faster [41], which conferred ATX-PPS-NP advantages in
providing sufficient ATX concentration in inner ear.
As previous studies have shown that ROS-responsive
systems have the potential to achieve oxidation-specific
drug release in a series of disease models [26–29], here,
ATX-PPS-NP demonstrated ROS-responsive accumula-
tion and enhanced drug release either in artificial peri-
lymph, HEI-OC1 cell line or guinea pigs. Besides, the
process of ROS-triggering disintegration of PPS-NP was
also ROS-consuming, which facilitated an enhanced anti-
oxidant effect when combining with ATX. Moreover,
evidences indicatedoxidative stress initiated the inflam-
matory process, resulting in the synthesis and secretion
of proinflammatory cytokines [42], and activated mito-
chondrial apoptotic machinery. e reduction of IL-6
levels and the protein expressions of cytochrome-C and
cleaved-caspase 3 in ATX-PPS-NP group suggested that
an enhanced antioxidant efficacy further led to enhanced
anti-inflammatory and anti-apoptotic effects on CDDP-
treated cells. Eventually, it was verified that ATX-PPS-NP
showed superior cellular protection in HEI-OC1 cell line,
cochlear explants and guinea pigs compared with equiva-
lent ATX. us, ATX-PPS-NP could reduce ATX doses
andpotentially minimize the side-effects caused by off-
target effects [43], with protective efficacy guaranteed.
Outer hair cells of the organ of Corti, SGNs and stria
vascularis are three major targets of cisplatin-induced
ototoxicity. Time sequence studies [44, 45] explor-
ing impairment processes at these 3 sites have verified
that theyrun in parallel, other than secondary injuries
dependent on another ototoxic process. A recent study
found thata delay in ABR wave 1 latency which probably
implied the damage to SGN mitochondria and myelina-
tion was the earliestfunctional and cellular changes after
cisplatin treatment,indicating SGN as an early and direct
damaging targetin cisplatin-induced ototoxicity [46]. In
the present study, the damaging target was determined
by the analysis of histological and immunohistochemi-
cal staining of the basilar membrane and mid-modiolar
sections. Notably, ATX-PPS-NPs increased the SGNs
survival and preserved the greater morphology of SGNs
in apical turns (Fig.7B, D), with nonoticeable changes of
OHCs and SCs in Organ of Corti (Fig. S5). However, the
lack of SGN cell lines and technological difficulty in the
culture of Organ of Corti containing intact SGNs con-
stricts the evaluation of the protection of ATX-PPS-NP
on SGNs invitro.
4-HNE is a cytotoxic end product of lipid peroxida-
tion and can be eliminated by conjugating to GSH [35].
Here, we found that ATX-PPS-NP facilitated a reduced
4-HNE generation (Fig.7C, E) and elevated level of GSH
(Fig.5C), suggesting the involvement of ATX-PPS-NP in
the mitigation of lipid peroxidation and the further cell
death induced by CDDP. As to the functional analysis,
the pretreatment ofATX-PPS-NPs attenuated cisplatin-
induced moderate hearing loss at low frequencies, with
negligible effectsat high frequencies (Fig.7A). e rea-
son why auditory function protection occurred in low
frequencies is that, on one hand, cisplatin induces more
severe hearing loss at high frequencies. On the other
hand, drug efficacy is likely to depend on the intrinsic
properties of the cochlea. For example,the capacity of
spiral ganglion cells to respond to ROS challenges may
vary along the tonotopic axis, with a higher SOD2immu-
nopositivity in SGNs located at the apex than those at the
base [47].
Conclusion
In the present study, we developed a novel drug delivery
system combining antioxidant ATX and ROS-responsive/
consuming nanoparticles (PPS-NP) to combat cisplatin-
induced ototoxicity. ATX-PPS-NP were proven to be ROS-
responsive in artificial perilymph, the HEI-OC1 cell line and
guinea pigs. In addition, ATX-PPS-NP accumulated in ROS-
specific sites and subsequently disintegrated for smart drug
release. In HEI-OC1 cells, ATX-PPS-NP mitigated ROS
level (DCFHDA) and inflammatory reaction (IL-6). Moreo-
ver,ATX-PPS-NP enhanced cell viability andalleviated cispl-
atin-induced mitochondrial apoptotic pathwayby reducing
the protein expression of cytochrome-C and cleaved cas-
pase-3. In guinea pigs, RWM evaluation and LC–MS analy-
sis verified the feasible penetration of ATX-PPS-NP into the
inner ear. Based on these findings, we administrated ATX-
PPS-NP on the RWM before CDDP injection and observed
partial recovery of auditory function from moderate hearing
loss. ATX-PPS-NPs increased the SGNs survival and miti-
gated the lipid peroxidation induced by CDDP.erefore,
this work provides an enhanced antioxidant therapy by com-
bining ROS-responsive/consuming nanoparticles and ATX
for cisplatin-induced ototoxicity.
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Guetal. Journal of Nanobiotechnology (2022) 20:268
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12951- 022- 01485-8.
Additional le1: Figure S1. Nuclear magnetic resonance 1H NMR
spectra of PPS-PEG and FITC-PPS-PEG. Figure S2. Screening the optimal
dose and time for CDDP administration in HEI-OC1. Figure S3. Screening
the optimal application of drugs (ATX, PPS-NP, ATX-PPS-NP) in cell models.
Figure S4. H&E staining of round window membrane (RWM). Figure S5.
Structural changes of cochlea in CDDP (day1)-treated mice.
Author contributions
JG executed experiments and drafted the manuscript. XW designed
experiments and analyzed data. YC and KX collected data. DY conceived the
study. HW revised the manuscript. All authors read and approved the final
manuscript.
Funding
The present study was supported by the National Natural Science Foun-
dation of China (No. 81970874), Natural Science Foundation of Shang-
hai (No. 19ZR1429400), Natural Science Foundation of Shanghai (No.
21ZR1437600), Shanghai Municipal Science and Technology Major Project (No.
2018SHZDZX05), Shanghai Key Laboratory of Translational Medicine on Ear
and Nose Diseases (No. 14DZ2260300), and Innovative research team of high-
level local universities in Shanghai.
Availability of data and materials
Date and material are available for any research.
Declarations
Ethics approval and consent to participate
All animal studies were approved and conducted in accordance with the
guidelines of the Ethics Committee of the Shanghai Jiaotong University,
School of Medicine (Shanghai, China).
Consent for publication
We agree for publication.
Competing interests
The authors declare that they have no known competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.
Author details
1 Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth
People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai,
China. 2 Ear Institute, Shanghai Jiao Tong University School of Medicine,
Shanghai, China. 3 Shanghai Key Laboratory of Translational Medicine on Ear
and Nose Diseases (14DZ2260300), Shanghai, China. 4 Materdicine Lab, School
of Life Sciences, Shanghai University, Shanghai, China.
Received: 14 March 2022 Accepted: 31 May 2022
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