Abstract
Senescence is a cellular response characterized by cells irreversibly
stopping dividing and entering a state of permanent growth arrest. One
of the underlying pathophysiological causes of senescence is the
oxidative stress-induced damage, indicating that eliminating the
reactive oxygen and nitrogen species (RONS) may be beneficial to
prevent and/or alleviate senescence. Herein, we developed ultra-small
polydopamine nanoparticles (UPDA NPs) with superoxide dismutase
(SOD)/catalase (CAT) enzyme-mimic activities, featuring broad-spectrum
RONS-scavenging capability for inducing cytoprotective effects against
RONS-mediated damage. The engineered UPDA NPs can restore
senescence-related renal function, tissue homeostasis, fur density, and
motor ability in mice, potentially associated with the regulation of
multiple genes involved in lipid metabolism, mitochondrial function,
energy homeostasis, telomerase activity, neuroprotection, and
inflammatory responses. Importantly, the dietary UPDA NPs can enhance
the antioxidant capacity, improve the climbing ability, and prolong the
lifespan of Drosophila. Notably, UPDA NPs possess excellent
biocompatibility stemming from the ultra-small size, ensuring quick
clearance out of the body. These findings reveal that UPDA NPs can
delay aging through reducing oxidative stress and provide a paradigm
and practical strategy for treating senescence and senescence-related
diseases.
Keywords: Nanomedicine, Polydopamine, Antioxidation, Anti-aging,
Oxidative stress
Graphical abstract
[45]Image 1
[46]Open in a new tab
1. Introduction
Senescence or biological aging is a state of irreversible cell growth
arrest accompanied with the halt of cell division before cells reach
the end of their lifespan [[47]1]. Senescence can lead to systemic
changes, which are associated with various chronic diseases such as
Alzheimer's disease [[48]2], diabetes [[49]3], atherosclerosis [[50]4]
and cancer [[51]5], posing a heavy burden on society, both medically
and economically. In addition to natural aging, senescence occurs in
response to various noxious stimuli, including DNA damage, oncogenic
stress, telomere dysfunction, inflammation, mitochondrial dysfunction,
epigenetic alterations, proteostasis imbalance, dysregulated
intracellular signaling pathways, and oxidative stress [[52]6,[53]7].
The free radical theory of aging posits that aging-associated
functional losses result from the accumulation of oxidative damage
caused by reactive oxygen and nitrogen species (RONS), referring to
reactive radical and non-radical derivatives of oxygen and nitrogen,
such as superoxide anion radical (O[2]^•-) [[54]8], hydrogen peroxide
(H[2]O[2]), hydroxyl radical (•OH) [[55]9], nitric oxide radical (•NO),
and peroxynitrite anion (ONOO^−), continuously produced in the process
of cell metabolism or upon the stimulation of environmental factors
[[56]10]. As one of the fundamental mechanisms driving aging, oxidative
stress has received continued and even increasing attention, which is a
phenomenon characterized by the imbalance between RONS-related oxidants
and antioxidants [[57]11]. RONS, whether endogenous or exogenous,
induce the oxidative modification of most biological macromolecules
involving proteins, lipids, carbohydrates, and nucleic acids, and
further change their stability and function [[58]11]. Therefore, there
is an urgent need to develop effective strategies to eliminate or
reduce excessive RONS for delaying the aging process.
Living organisms possess a series of highly effective enzymes including
catalase (CAT) [[59]12] and superoxide dismutase (SOD) [[60]13] with
the ability to remove RONS, protecting tissues or organs from oxidative
damage. These natural antioxidant enzymes are a class of biomolecules
(e.g., proteins and RNA), which are typically sensitive to
non-physiological or adverse environmental conditions (e.g., extreme
temperature and pH) [[61]14,[62]15] making them difficult to produce in
large quantities. Also, the activity of these enzymes relies on the
binding of mineral elements, such as copper, iron, manganese, and zinc,
the intake of which can be greatly compromised because of aging or
aging-related diseases [[63]16]. To address such challenges, a number
of artificial enzymes mimicking the activities of natural enzymes have
been developed [[64]17,[65]18], including nanozymes which have combined
advantages, such as ease of production, low cost, multifunction and
high stability. Among them, common inorganic nanozymes such as CeO[2]
[[66]19], platinum [[67]20], V[2]O[5] [[68]21] and 2D carbides and
nitrides (MXenes) [[69]22] with RONS-scavenging ability have been
potentially applied in various diseases including hepatic
ischemia-reperfusion injury [[70]23], neurodegenerative diseases (e.g.,
Alzheimer's [[71]24] and Parkinson's [[72]25] diseases), acute kidney
injury [[73]26] and so on. However, questions have been raised
regarding the biosafety and biocompatibility of inorganic materials, as
some have been reported to interact with biomolecules including DNAs,
proteins and lipids [[74]27]. To date, non-negligible toxicity issues
in biological systems (cells, tissues, organs, etc.) have restricted
inorganic nanozymes for further clinical translation [[75]28].
Compared to inorganic nanozymes, organic nanozymes exhibit satisfactory
biocompatibility and chemical flexibility [[76]29]. During the last
decade, an increasing number of organic nanozymes have been developed
for the detection of biomolecues or the treatment of cancer [[77]30],
while rarely proposed for combating oxidative stress-related diseases.
This is partly due to the fact that organic nanozymes are generally
designed to mimic the catalytic activity of specific enzymes, which
renders them insufficient to clear multiple types of RONS that can be
overproduced concomitantly in the progression of aging or aging-related
diseases. Therefore, developing nanoparticles with multiple-enzyme
properties is essential to overcome this limitation.
Polydopamine (PDA), which is widely used for synthesizing artificial
melanin particles, has significant advantages in terms of biosafety
[[78]31]. In previous reports, PDA is widely utilized as a theranostic
agent in the biomedical field ascribing to its binding affinity to
metal ions and response to various external stimuli such as light, pH,
and ultrasonic/magnetic field. A recent study showed that PDA is
redox-active and can either accept electrons from reducing agents or
donate electrons to oxidants with an effect of quenching free radicals
[[79]32]. Based on these two distinct properties, both the
antimicrobial activities and scavenging potentials for reactive oxygen
species (ROS) of PDA have been reported [[80]30,[81][32], [82][33],
[83][34]]. As PDA is enriched with the reductive groups of catechol
similar to natural polymer melanin and the size of melanin-like
nanoparticles can be easily adjusted with the alkali-based
self-polymerization technique, the antioxidative enzyme-like activities
of PDA with ROS scavenging performances are worthwhile to investigate
for further in vivo application. Particularly, downscaling the size of
polymers may not only make them bioexcretable but also expand their
contact area with the free radicals, which can maximize their
anti-aging effects by enhancing the electron-donating capacity. Herein,
we designed and developed ultra-small polydopamine nanoparticles (UPDA
NPs) via liquid-phase exfoliation technology, which are integrated with
CAT/SOD mimetic cascade activities and broad-spectrum ROS-scavenging
capabilities, capable of catalyzing O[2]^•- to generate H[2]O[2] and
O[2], decomposing H[2]O[2] into H[2]O and O[2], and removing •OH.
Additionally, UPDA NPs can serve as a scavenger for reactive nitrogen
species (RNS) such as •NO and ONOO^−. We verified that UPDA NPs inhibit
oxidative stress-induced senescence in vitro. Further evaluation
demonstrated that UPDA NPs possess excellent biocompatibility and
biosafety, and display anti-aging effects in vivo. Transcriptomics
analysis revealed that the anti-senescence effects of UPDA NPs are
likely related to but not limited to the regulation of the NF-κB
signaling pathway. Particularly, UPDA NPs can rescue drug-induced
senescent behaviors in mice and can effectively restore the lifespan of
Drosophila under oxidative stress ([84]Fig. 1). These results not only
demonstrate that UPDA NPs can be used as a paradigm of anti-aging
nanomedicine that exhibits desirable RONS-scavenging performance with
potential clinical application prospect, but also provide an
alternative and efficient strategy for the alleviation and treatment of
senescence.
Fig. 1.
[85]Fig. 1
[86]Open in a new tab
Schematic illustration of the underlying process and mechanism of UPDA
NPs in the efficient treatment for cellular senescence and aging of
model organisms. UPDA NPs can turn toxic ROS into non-toxic substances.
Specifically, O[2]^•- is converted to H[2]O[2] and O[2], and H[2]O[2]
is decomposed to O[2] and H[2]O, mimicking the activities of SOD and
CAT. Harmful RNS including •NO and ONOO^− can also be removed by UPDA
NPs. The RONS-scavenging capacities of UPDA NPs are translated to the
reversion of senescent phenotypes and behaviors in Drosophila and mice,
represented by the regulation of key senescence mediators at the
cellular and molecular levels.
2. Material and methods
2.1. Experimental design
This study was designed to develop an ultra-small organic nanomedicine
(UPDA NPs) with good biocompatibility and anti-senescence effects. For
the synthesis of UPDA NPs, we used techniques of self-polymerization
and liquid-phase exfoliation. A wide range of imaging and spectroscopic
analysis were performed for the characterization of UPDA NPs. For
evaluating the antioxidation effects of UPDA NPs, we determined their
RONS-scavenging efficacies and SOD/CAT activities. For confirming the
biocompatibility and biosafety of UPDA NPs in vivo, we examined the
major organs and hematological parameters of mice. To assess the
suppressive effects of UPDA NPs on oxidative stress-induced senescence,
different senescent models (doxorubicin (DOX)-treated human 293 T
cells, DOX-treated mice, and d-galactose (D-gal)-treated Drosophila)
were used for determining the expression levels of
senescence-associated secretory phenotype (SASP)-related molecules. For
understanding the anti-senescence mechanism of UPDA NPs,
transcriptomics analysis was performed to identify differentially
expressed genes (DEGs) and relevant pathways in the kidneys of
DOX-treated mice. To explore the possibility of applying UPDA NPs in
the treatment of senescence, we carried out phenotype evaluation,
physiological function assessment and behavioral studies in DOX-treated
mice and D-gal-treated Drosophila.
2.2. Chemical materials
Dopamine hydrochloride, sodium hydroxide (NaOH), sodium nitrite
(NaNO[2]), doxorubicin hydrochloride, 1,1-Diphenyl-2-picrylhydrazyl
radical (DPPH·), DETA NONOate (NOC18), 5,5-dimethyl-1-pyrrolineN-oxide
(DMPO),
4,5-Dihydro-4,4,5,5-tetramethyl-2-phenyl-1H-imidazol-1-yloxy-1-oxide
(PTIO), X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactoside), and
dihydroethidium (DHE) were purchased from Meryer (Shanghai) Chemical
Technology. Phosphate-buffered saline (PBS, pH 7.4,
Na[2]HPO[4]–NaH[2]PO[4], 10 mM) solution was prepared in the
laboratory. All chemicals and reagents were of analytical grade and
used as received without further purification. Ultrapure water
(18.2 MΩ cm^−1 at 25 °C) purified by a Milli-Q system was used
throughout the experiment.
2.3. Transmission electron microscope (TEM) imaging and spectroscopic
analyses
TEM imaging was conducted on a JEM-1400Flash electron microscope (JEOL,
Japan) at 120 kV. The sample was prepared by dispersing a small amount
of freeze-dried powder in PBS. Then, the suspension was dropped on 230
mesh copper TEM grids covered with thin amorphous carbon films. Fourier
transform infrared spectrum (FT-IR) spectra were measured by a VERTEX70
spectrometer (Bruker, Germany) in the range of 4000–400 cm^−1.
Ultraviolet visible (UV–vis) spectra were obtained using a VNANODROP
8000 spectrometer (Thermofisher scientific, U.S.A). A dynamic light
scattering (DLS) particle size analyzer (Malvern 2000, U.S.A) was used
to determine the hydrophilic diameters of the particles. X-ray
photoelectron spectroscopy (XPS) was performed with an ESCALAB 250Xi
(Thermofisher scientific, U.S.A) X-ray source. The crystal structure
and oxidation state of UPDA NPs were analyzed using a X-ray
diffractometer (Smartlab, China). The electron spin resonance (ESR)
spectroscopy signal was obtained on a Bruker A300 (X-band) spectrometer
(Bruker, Germany). All the measurements were performed at room
temperature if not specified otherwise.
2.4. Preparation of UPDA NPs
180 mg of dopamine hydrochloride was dissolved in 90 mL of deionized
water. 840 μL of 1 mol L^−1 (M) NaOH solution was added to the
dopamine hydrochloride solution at 60 °C and the mixture underwent
vigorous stir. The reaction lasted for 5 h. The solution color turned
to pale yellow as soon as NaOH was added and gradually changed to dark
brown. The product was collected by centrifugation at 16,000 rpm for
20 min and was then washed with deionized water three times. The
aqueous solvent was removed by freeze-drying to obtain black solids of
PDA. Under vigorous stir, 20 mg PDA nanoparticles was dissolved in
10 mL of 0.1 M NaOH. Then we swiftly dropped 0.1 M HCl into the
obtained solution to adjust the pH to 7.0 under sonication with an
output power of 600 W for 2 min. We obtained a bright black PDA
solution. The particles were retrieved by centrifugation with a
centrifugal filter (centrifugal filter device, MWCO = 30 kDa) at
8000 rpm for 8 min and was washed several times with deionized water
to remove the byproduct NaCl, followed by freeze-drying to obtain black
solids of UPDA NPs.
2.5. Determination of RONS-scavenging capability
The evaluation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) radical scavenging activity was performed according to a
previously reported method [[87]22]. Briefly, the ABTS radicals
(ABTS•^+) were generated by incubating 7 mM ABTS stock solution with
2.45 mM potassium persulfate in dark for 16 h. Then, the ABTS•^+
solution was diluted with PBS to reach an absorbance at 405 nm. 2 mL
UPDA NPs solutions (0, 20, 40, and 60 μg mL^−1, respectively) were
mixed with 2 mL ABTS solution and were placed in dark for 10 min.
Then the absorbance at 405 nm was monitored with a UV–vis
spectrophotometer. The ABTS•^+ scavenging abilities were calculated as
follows:
[MATH: ABTS•+scavengingratio(%)=((Acontrol−Asample)/Acontrol)×100, :MATH]
where A[control] is the absorbance of a standard solution without any
radical scavengers, and A[sample] is the absorbance after the reaction
with the radical scavengers, respectively.
The SOD-like activity was determined using an WST-1
(2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliu
m) assay kit (Nanjing Jiancheng Bioengineering Institute, China)
according to the manufacturer's instruction. Briefly, a tetrazolium
salt WST-1 reacts with O[2]^−, generating a formazan dye with a
characteristic absorption at 450 nm. This reaction is inhibited by SOD
due to the disproportionation of O[2]^−. Therefore, the SOD-like
activity is negatively correlated with the absorbance value of the
formazan dye, which was measured with the addition of different
concentrations of UPDA NPs (20, 40, 60, 90, 160, 190, 30, and
340 μg mL^−1) using a multiple plate reader (Molecular Devices,
U.S.A).
The CAT-like activity was determined based on the H[2]O[2]
decomposition reaction. A tube containing 10 mM H[2]O[2] and 0.1 mM
3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (CTPO) in PBS was
pumped with N[2] for 10 min. Then, UPDA NPs (1.25, 12.5, and
50 μg mL^−1) was added and incubated for 5 min. ESR spectra were
monitored at different time points. In the steady-state kinetic assay
for determining the CAT-like catalytic mechanism, a detection kit for
H[2]O[2] (Beyotime, China) was used. Briefly, H[2]O[2] of different
concentrations (5–60 μM) was added to 100 μg mL^−1 UPDA NPs in
separate test tubes. The detection solution was added and the reaction
mixture was incubated at room temperature for 30 min. Then, the
absorbance values were measured at 640 nm and the dynamics curve was
registered on a UV–vis spectrophotometer. The Michaelis-Menten constant
(K[m]) of UPDA NPs-catalyzed reaction was obtained by plotting the
initial velocity versus H[2]O[2] concentration. The maximum velocity
(V[max]) was calculated from the Lineweaver-Burk plot, where
1/V = 1/V[max]+ K[m] (V[max]C).
The amount of •OH was measured with the Fenton reaction, in which •OH
causes methylene blue (MB) to oxidize and its color fading degree is
proportional to the amount of •OH. Different concentrations of UPDA NPs
(0, 45, and 90 μg mL^−1) were added to the •OH-MB reaction solution,
and the absorbance at 664 nm was measured. The •OH-scavenging rate was
obtained with the following equation:
[MATH: Inhibition(%)=((Asample−Acontrol)/Asample)×100, :MATH]
where A[control] is the absorbance of the control group, and A[sample]
is the absorbance of the UPDA NPs-treated group.
DPPH• was used to evaluate the RNS scavenging activity of UPDA NPs.
Different concentrations of UPDA NPs (0, 5, 25, and 100 μg mL^−1)
were mixed with 40 μM DPPH• for 12 h and the absorbance spectra at
532 nm were recorded.
The ·NO-scavenging ability of UPDA NPs (50 μg mL^−1) was tested by
ESR spectroscopy using carboxy-PTIO (2-
(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) as the
trapper and the detection dye for ·NO. NOC18 was used as the source of
·NO. Carboxy-PTIO was dissolved in phosphate buffer (250 mM, pH 7.4),
and NOC18 was dissolved in NaOH (1 mM). In a test tube, 0.5%
methylcellulose was mixed with NOC18 (5 μM) for 30 min at room
temperature, and the mixture was then added into the carboxy-PTIO
solution (5 μM) in the absence or presence of UPDA NPs. The ESR
spectra were then recorded. The ESR signal of carboxy-PTIO is
characterized by five peaks. ·NO from NOC18 can reduce carboxy-PTIO to
carboxy-PTI and generate ·NO[2], which shows seven peaks of ESR
signals. The reduction of carboxy-PTIO by ·NO leads to a color change
from purple to yellow.
The ONOO^−-scavenging ability of UPDA NPs (0, 25, and 100 μg mL^−1)
was evaluated by UV–vis spectroscopy using pyrogallol red as the
indicator. The absorbance values were measured at 545 nm.
2.6. Cell culture
The 293 T cell line was acquired from ATCC, and cultured in dulbecco's
modified eagle medium (DMEM) (Hyclone, U.S.A) containing 10% Foetal
Bovine Serum (FBS) (Gibco, U.S.A) and 1% s7 penicillin/streptomycin
(Gibco, U.S.A) at 37 °C with 5% CO[2]. For DOX-induced senescence,
293 T cells were treated twice with 0.2 mM DOX with a 2-day interval
and were analyzed 7 days later. The expression of β-galactosidase was
detected by the cleavage of X-Gal. The levels of IL-6 and IL-1β were
determined using commercially available kits (Boster Bio, China) and
quantified from 3 replicates (n = 3).
2.7. Measurement of ROS scavenging activities in vitro
293 T cells were seeded into 96-well and 24-well plates at a density
of 1 × 10^4 cells per well and 1 × 10^5 cells per well,
respectively. After incubation for 24 h, cells were treated with
2 × 10^−7 M DOX, 8 g/L D-gal, or 250 μM H[2]O[2] and
100 μg mL^−1 UPDA NPs, were further incubated at 37 °C for a 2-day
interval, and were analyzed 7 days later. For determining cell
viability, cells seeded in 96-well plates were evaluated with cell
counting kit-8 (CCK-8, Beyotime, China). Cells without the addition of
UPDA NPs were regarded as the control. At least 50,000 cells were
analyzed in each sample. For determining the ROS-scavenging effect of
UPDA NPs (20 μg mL^−1), dichlorofluorescein (DCF), an oxidation
sensitive fluorescent dye, was used to detect the intracellular ROS
level [[88]35]. Briefly, DCFH-DA (2′,7′-dichlorodihydrofluorescein
diacetate) is a non-fluorescent chemical compound which can diffuse
through the cell membrane freely and can be hydrolyzed by an
intracellular esterase to generate DCFH
(2′,7′-dichlorodihydrofluorescein). The non-fluorescent DCFH can be
oxidized by the intracellular ROS to form fluorescent DCF. Therefore,
the quantity of intracellular ROS is correlated with the fluorescent
intensity of DCF. After the aforementioned incubation, cells were
gently rinsed three times with serum-free medium to remove the free
UPDA NPs. Then, 10 μM DCFH-DA in serum-free medium was added to the
cells, followed by incubation in dark at 37 °C for 30 min.
Afterwards, the cells were washed with serum-free medium three times to
remove unloaded DCFH-DA probe, imaged using a laser confocal microscope
(Zeiss, Germany), and subjected to flow cytometric analysis.
2.8. Immunocytochemistry
Normal and DOX-induced senescent 293 T cells were fixed with 4%
paraformaldehyde (PFA) for 15 min, permeabilized with 0.2% Triton
X-100 for 3 min and was then blocked with 10% (v/v) BSA (bovine serum
albumin) in PBS for 2 h at room temperature or at 4 °C overnight.
Subsequently, the cells were washed with PBS three times and were then
incubated with primary antibodies rabbit anti-Forkhead box O4 protein
(FOXO4) (1:150, Proteintech, U.S.A), rabbit anti-p16^ink4a (1:150,
Abclonal, China), or rabbit anti-lamin B1 (1:150, Abclonal, China) for
2 h. After the incubation, the cells were washed three times with PBS
and incubated with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:200,
Abcam, UK) for 2 h. Then, after three washes with PBS, the cells were
stained with DAPI for 5 min, followed by another three washes with
PBS. Finally, the cells were mounted and imaged on an inverted
fluorescence microscope (Zeiss, Germany). The fluorescence intensity
was obtained from 3 randomly selected regions of the cell culture to
determine the expression levels of FOXO4, p16^ink4a, and lamin B1 in
each group. Three independent batches of cells were analyzed to
determine the significance of difference between different groups.
2.9. Western blotting
293 T cells (1.0 × 10^6) were collected and total protein was
extracted with RIPA (Radioimmunoprecipitation) lysis buffer (Beyotime,
China). The protein lysates were then separated by SDS-PAGE and
transferred to nitrocellulose filter membranes. The membranes were
incubated with primary antibodies rabbit anti-p21 (1:1000, ABclonal,
China), rabbit anti-lamin B1(1:1000, Abcam, UK), rabbit anti-decoy
receptor 2 (DcR2) (1:1000, Proteintech, U.S.A), or mouse
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000,
ABclonal, China) and then with goat anti-rabbit IgG (H + L) DyLight™
800-Labeled or goat anti-mouse IgG (H + L) DyLight™ 680-Labeled
secondary antibodies (1:10,000, KPL, U.S.A). The immune-positive bands
were visualized using an Odyssey scanner (LI-COR Biosciences, USA). The
band densities of p21, lamin B1, and DcR2 were quantified in ImageJ and
normalized against those of GAPDH, representing their protein
expression levels respectively.
2.10. Animals
The animal care and experimental procedures were approved by the Animal
Ethics Committee of Shanghai University (ECSHU 2021–039). A maximum of
four mice per cage were kept on a 12-h light/dark cycle at a constant
temperature (22 °C) with food and water ad libitum. C57BL/6J mice of
26 weeks were used for DOX-induced senescent model. Mice were given two
intraperitoneal injections of DOX at a dose of 10 mg/kg. All mice were
kept in group housing until the start of the experiment and were then
randomly assigned to control and experimental groups.
2.11. Biocompatibility and biosafety evaluation
C57BL/6J mice (2 months old, 20–25 g) were intravenously administrated
with UPDA NPs at a single dose of 200 μL (100 μg mL^−1). The mice
injected with PBS were used as the control group. At day 30 post
injection, the mice were anesthetized with intraperitoneal injection of
chloral hydrate and sacrificed to harvest major organs (including
heart, liver, spleen, lung, and kidney) for hematoxylin and eosin
staining and histological analysis. Mouse blood was collected through
cardiac puncture. Routine blood parameters, including white blood cells
(WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA), mean
platelet volume (MPV), red blood cells (RBC), hemoglobin (HGB), mean
corpuscular hemoglobin (MCH), hematocrit (HCT), red blood cell
distribution width-standard deviation (RDWSD), mean corpuscular volume
(MCV), mean corpuscular-hemoglobin concentration (MCHC), platelet
(PLT), platelet distribution width (PDW), and red blood cell
distribution width-coefficient of variation (RDWCV), were analyzed on a
Sysmex XS-800i automated hematology analyzer (Sysmex, Japan).
2.12. Immunohistochemistry
Mice were anesthetized and perfused with PBS. The kidneys were
collected, fixed with 4% PFA, and cut to obtain 30 μm sections using a
vibratome (Invitrogen, U.S.A). After three rinses in PBS (5 min each),
the sections were blocked in PBST (PBS + 0.3% Triton X-100) with 3%
BSA for 2 h, and were then incubated in primary antibodies rabbit
anti-lamin B1 (Abclonal, China), mouse anti-p53 (Abcam, UK) or rabbit
anti-p21 (ABclonal, China) with a dilution of 1:1000 at 4 °C for
24 h, followed by three rinses in PBS. Then, the sections were
incubated with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:300, Abcam,
UK) or goat anti-mouse IgG H&L Alexa Fluor® 488 secondary antibodies
(1:300, Abcam, UK) for 2 h at room temperature. After the incubation,
the sections were washed with PBS for 5 min, and the nuclei were
counterstained with DAPI for 5 min. Finally, the sections were washed
in PBST and mounted in mounting medium. Fluorescence images were
acquired with a 20 × objective on a confocal microscope.
Flies were anesthetized with CO[2], dissected in PBS, and fixed with 4%
PFA at room temperature. Brain sections were prepared and incubated
with rabbit anti-caspase-3 primary antibody (1:500, Beyotime, China)
and then with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:200, Abcam,
UK), followed by DAPI staining and fluorescence imaging.
2.13. Determination of renal and hepatic function parameters
Blood samples were acquired from normal mice and DOX-induced senescent
mice with or without the treatment of UPDA NPs for plasma separation.
These samples were then centrifuged for 10 min at 1200 g. The
supernatants were transferred into 1.5 mL tubes and recentrifuged for
5 min at 1200 g. The supernatants were transferred again into 1.5 mL
tubes for determining the levels of creatinine (CRE), blood urea
nitrogen (BUN), alanine transaminase (ALT), and aspartate
aminotransferase (AST).
2.14. Assessment of swimming performance
We monitored the swimming behavior of different mouse groups by
continuously measuring their swimming speed. Swimming activity was
recorded for 30 days.
2.15. Transcriptomics analysis
DOX-induced senescent mice were randomly divided into two groups: the
control group (n = 5) and the group treated with 100 μg mL^−1 UPDA
NPs at a dosage of 200 μL (experimental group, n = 5). After 24 h
post injection, mice were sacrificed to collect the kidneys. Total RNA
was extracted using TRIzol® Reagent (Invitrogen, U.S.A) according to
the manufacturer's instructions and genomic DNA was removed using DNase
I (Takara Bio, U.S.A). Then RNA quality was determined by a 2100
Bioanalyser (Agilent, U.S.A) and quantified using an ND-2000 instrument
(NanoDrop Technologies, U.S.A). Only high-quality RNA samples
(OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN≥6.5, 28S:18S ≥ 1.0,
>1 μg) were used to construct sequencing library. RNA-seq
transcriptome library was prepared with a TruSeqTM RNA sample
preparation Kit (Illumina, U.S.A) using 1 μg of total RNA. Briefly,
messenger RNA with polyA tails was captured by oligo (dT) beads and was
then fragmented in fragmentation buffer. Next, double-stranded cDNA was
synthesized using a SuperScript double-stranded cDNA synthesis kit
(Invitrogen, U.S.A) with random hexamer primers (Illumina, U.S.A). The
synthesized cDNA was subjected to end-repair, phosphorylation, and
adenylation on the 3′ end. Libraries were enriched for cDNA fragments
of 300 bp on 2% Low Range Ultra Agarose gels followed by PCR
amplification using Phusion DNA polymerase (NEB, U.S.A) for 15 PCR
cycles. After the quantification with a TBS380 fluorometer (Turner
BioSystems, U.S.A), paired-end RNA-seq sequencing library was sequenced
with a HiSeq xten/NovaSeq 6000 sequencer (2 × 150bp read length,
Illumina, U.S.A). The raw paired end reads were trimmed and subjected
to quality control by SeqPrep ([89]https://github.com/jstjohn/SeqPrep)
and Sickle ([90]https://github.com/najoshi/sickle) with default
parameters. Then clean reads were separately aligned to reference
genome with orientation mode using the HISAT2 software
([91]http://ccb.jhu.edu/software/hisat2/index.shtml). The mapped reads
of each sample were assembled by StringTie
([92]https://ccb.jhu.edu/software/stringtie/index.shtml? t = example)
in a reference-based approach. To identify DEGs between two different
samples, the expression level of each transcript was calculated
according to the transcripts per million reads (TPM) method. RSEM
([93]http://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene
abundances. Differential expression analysis was performed using the
DESeq2 package with |log2FC|>1 and Q value ≤ 0.05 as the criteria of
DEGs. GO functional enrichment and KEGG pathway analyses were carried
out using Goatools ([94]https://github.com/tanghaibao/Goatools) and
KOBAS ([95]http://kobas.cbi.pku.edu.cn/home.do) with
Bonferroni-corrected p-value ≤0.05 considered to be the threshold of
enrichment. The protein-protein interaction (PPI) network of selected
DEGs was constructed using the STRING database [PMID: 30476243] and was
mapped with Cytoscape software (v 3.8.2). An interaction score >0.4 was
regarded as statistically significant.
2.16. Drosophila culture
Drosophila melanogaster (w1118) stocks were maintained and crossed
according to standard laboratory procedures. Flies were raised under a
12/12 h, light/dark cycle at 25 °C with 60% humidity.
2.17. Measurement of total antioxidant capacity (TAC) in drosophila
Different groups of Drosophila were sacrificed and washed with PBS.
20 mg of tissue was suspended in ice cold PBS or HBSS (Hanks’ Balanced
Salt Solution) and was thoroughly homogenized with an ultrasonic
homogenizer. The tissue homogenates were centrifuged at 12,000 g and
4 °C for 5 min. The supernatant was collected for further
experiments. The TAC in Drosophila after different treatments was
determined via the ABTS•^+ assay with a test kit (Beyotime, China).
Briefly, 20 μL peroxidase working solution, 10 μL supernatant of
tissue homogenates, and 170 μL ABTS working solution were added in
turn in a 96-well plate (one well per test) and were mixed gently. The
mixture was incubated at room temperature for 6 min and then the
absorbance (A[test]) was measured at 414 nm. The TAC in the Drosophila
group with the highest absorbance of ABTS•^+, which was the
D-gal-treated group, was set as 0. And the TAC in other groups was
calculated as (A[D-gal]-A[test])/A[D-gal] × 100%.
2.18. Determination of lifespan and climbing ability of drosophila
Newly eclosed wild-type female Drosophila were used for the lifespan
assay. 100 flies were used for each group. Flies were transferred for
fresh food and the death number was recorded every day. Data were
presented as survival curves. For the climbing assay, eight male and
eight female flies were placed in a plastic vial. The flies were gently
knocked to the bottom of the vial before timing. Climbing distances
within 4 s and 20 s were measured.
2.19. Statistical analysis
All quantitative data are shown as mean ± standard deviation in the
figures. For determining the statistical significance of differences in
the transcriptional levels of representative DEGs between the untreated
and UPDA NPs-treated mouse kidneys, the likelihood ratio test included
in the DEseq2 package of transcriptomics analysis was used. For
evaluating the lifespan difference of Drosophila with different
treatments, the log-rank test was used. For comparing the levels of
specific parameters in different treatment groups, such as the
expression of marker proteins, the biochemical indices and the
behavioral performances, the one-way ANOVA (analysis of variance) with
Tukey's post hoc test was used. The significance levels were set at
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
3. Results and discussion
3.1. Synthesis and characterization of UPDA NPs
The PDA nanoparticles (PDA NPs) were initially synthesized via
oxidative self-polymerization of dopamine in alkaline solution
([96]Fig. 2A), which showed very weak colloidal stability with the
particle size ranging from the scale of nanometer to that of micrometer
([97]Fig. 2B). The UPDA NPs can be facilely acquired from PDA NPs via
ultrasonication-mediated liquid-phase exfoliation technique
([98]Fig. 2A). Compared with PDA NPs, after exfoliation, the obtained
UPDA NPs exhibited benign dispersity and stability ([99]Fig. 2C). TEM
images revealed the uniform size and morphology of UPDA NPs
([100]Fig. 2C). The atomic force microscopy (AFM) image further
demonstrated the successful synthesis of UPDA NPs with high quality and
an average size of 3 nm ([101]Fig. S1, Supporting information), which
was consistent with what was observed in TEM images. The DLS analysis
showed that the hydrodynamic diameter of UPDA NPs was 2.7, 2.4, 5.6,
4.9, and 5.2 nm in deionized water, PBS, BSA, FBS, and DMEM,
respectively, and no obvious precipitation was observed after 72 h
([102]Fig. S2, Supporting information), which indicates that UPDA NPs
are relatively stable in various physiological environments and the
size falls below the glomerular filtration size threshold of 6–8 nm
([103]Fig. 2D) [[104]36]. Furthermore, the functional groups of UPDA
NPs were examined by utilizing an FT-IR spectrometer ([105]Fig. 2E)
with different adsorption bands indicating the presence of O–H, N–H,
and C–OH, in which the adsorption band at ∼3432 cm^−1 is assigned to
the stretching vibration of O–H and N–H, the peak located at
∼1677 cm^−1 is indexed to the stretching vibration of aromatic ring
and the bending ring vibration of N–H, the peaks at ∼1377 cm^−1 and
1498 cm^−1 are ascribed to the stretching and bending vibration of
C–OH, and the peak at ∼1590 cm^−1 is assigned to the stretching
vibration of the amide group, suggesting that UPDA NPs have been
successfully synthesized [[106]37]. UV–vis absorption spectra acquired
on UPDA NPs displayed a broad and intense absorption band ranging from
the ultraviolet region (100–400 nm) to the visible light region
(400–700 nm) and the absorption was enhanced with the increased
concentration of UPDA NPs, demonstrating excellent dispersity
([107]Fig. S3, Supporting information). The X-ray powder diffraction
(XRD) peak exhibited a bread-like pattern indicating the amorphous
nature of UPDA NPs with intermolecular stacking structure
([108]Fig. S4, Supporting information). Based on the XPS analysis, the
C, N, and O elements are present in the acquired UPDA NPs ([109]Fig. 2F
and [110]Fig. S5, Supporting information), further confirming the
successful synthesis of UPDA NPs without residual by-products.
Fig. 2.
[111]Fig. 2
[112]Open in a new tab
The synthesis diagram and characterization of UPDA NPs. (A) The
step-by-step production and key physical features of UPDA NPs. (B) A
representative TEM image of PDA NPs. The inset is a representative
photograph of PDA NPs dispersed in deionized water. (C) A
representative TEM image of UPDA NPs. The inset is a representative
photograph of UPDA NPs dispersed in deionized water. (D) DLS profiles
of UPDA NPs dispersed in various solutions including deionized water,
PBS, BSA, DMEM, and FBS. (E) The FT-IR spectrum of UPDA NPs. The
functional groups O–H, N–H, and C–OH were identified. (F) The
full-scale XPS spectrum of UPDA NPs. The C, N, and O elements were
identified.
3.2. RONS-scavenging capability and SOD/CAT-like activity of UPDA NPs
The classic ABTS•^+ assay was performed to assess the TAC of UPDA NPs,
in which ABTS•^+ is decolorized by antioxidants. The results showed
that UPDA NPs had high antioxidant activity in a
concentration-dependent manner ([113]Fig. 3A), and more than 80% of
total ROS could be eliminated by 40 μg mL^−1 UPDA NPs ([114]Fig. S6A,
Supporting information). Significantly, the TAC of UPDA NPs is twice as
much as that of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox), a water-soluble analog of vitamin E ([115]Fig. S6B,
Supporting information).
Fig. 3.
[116]Fig. 3
[117]Open in a new tab
The multiple RONS-scavenging effects of UPDA NPs and the underlying
mechanism. (A) TAC of UPDA NPs represented by the absorbance reduction
of radical cation ABTS•^+. The antioxidant activity of UPDA NPs
enhances with its concentration increasing. (B) SOD-like activity of
UPDA NPs represented by the percentage reduction of formazan formation.
O[2]^•- reacts with WST-1, generating formazan with an absorption peak
at 450 nm. (C) ESR spectra of CTPO demonstrating time-dependent O[2]
production from H[2]O[2] decomposition by UPDA NPs (50 μg mL^−1).
CTPO is an O[2]-sensitive probe. (D) CAT-like activity of UPDA NPs
represented by the absorbance reduction of Fe^3+-XO complex at 640 nm.
The Fe^3+ comes from the oxidation of Fe^2+ by H[2]O[2]. (E) Absorbance
of Fe^3+-XO complex upon the addition of 100 μg mL^−1 UPDA NPs to
varied concentrations of H[2]O[2]. (F) Michaelis-Menten curve and (G)
Lineweaver-Burk plot of CAT-like activity of UPDA NPs. (H)
•OH-scavenging ability of UPDA NPs represented by the absorbance
increase of MB. UPDA NPs prevents the degradation of MB by •OH. (I) ESR
spectra of DMPO demonstrating the concentration-dependent
•OH-scavenging effect of UPDA NPs. (J) Total RNS-scavenging ability of
UPDA NPs represented by the absorbance reduction of DPPH•. (K) ESR
spectra of PTIO demonstrating the •NO-scavenging ability of UPDA NPs.
The ESR signal of carboxy-PTIO is represented by 5 peaks. Carboxy-PTIO
reacts with ·NO, generating carboxy-PTI and •NO[2], which increases the
ESR signal to 7 peaks. UPDA UPs capture ·NO and reduce the ESR signal
to the original 5 peaks. (L) ONOO^−-scavenging ability of UPDA NPs
represented by the absorbance increase of pyrogallol red. (M) π
electronic delocalization of PDA particles. (N) Schematic diagram of
UPDA NPs donating hydrogen atoms with surface-active groups. (O) The
free radical-scavenging mechanism of UPDA NPs with catechol being the
core functional group. (For interpretation of the references to colour