Abstract
Pharmacological intervention represents the most prevalent strategy for
managing hyperhomocysteinemia-induced atherosclerosis (AS). However,
conventional drugs are often hampered by the liver first-pass effect
and limited targeted efficacy. To address these challenges, we
exploited the cholesterol efflux-promoting effects of Atorvastatin and
reactive oxygen species (ROS)-scavenging capacity of Astragaloside IV
in plaque macrophages to develop new biomimetic membrane-modified
nanomaterials with targeting capability. This biomimetic membrane
grants the nanodrug immune evasion capability, while hyaluronic acid
ensures precise targeting to plaque sites. In vitro studies revealed
that the nanomaterials can accurately target the CD44 receptor, which
is highly expressed on macrophages within plaques. More importantly,
the sustained and stable release of the two drugs could promote
cholesterol efflux and reduce lipid deposition by activating the
LXRα-mediated ABCG1, ABCA1/SR-B1 signaling pathway. Meanwhile,
these nanomaterials could activate Parkin-mediated autophagy,
ameliorate damaged mitochondria and inhibit the production of ROS. In
vivo studies demonstrated that the nanomaterials significantly enhanced
its half-life in the bloodstream and exhibited remarkable biological
safety in the ApoE^−/− mouse model. These findings indicate that the
biomimetic nanomaterials possess a potential capability to safeguard
against the advancement of AS.
Keywords: Hyperhomocystinemia, Atherosclerosis, Macrophage-derived foam
cells, Astragaloside IV, Atorvastatin
Graphical abstract
Image 1
[35]Open in a new tab
Highlights
* •
Uses [Mø+RBC]m to extend circulation, reduce phagocytosis via CD47,
overcoming conventional nanocarrier limits.
* •
Uses HA-PEG[2000]-DSPE to target CD44 on plaque macrophages,
enabling precise atherosclerotic lesion drug delivery.
* •
Combines Atorvastatin and Astragaloside IV to simultaneously
modulate lipid metabolism and mitochondrial dysfunction in AS.
1. Introduction
Atherosclerosis (AS) is a chronic illness that poses a serious threat
to human health and stands as the primary factor contributing to the
incidence and fatality rates of cardiovascular diseases [[36]1].
Epidemiological research has indicated that elevated homocysteine
levels (HHcy) constitute an independent factor of AS risk [[37]2]. In
HHcy-induced atherosclerotic lesions, the transformation of macrophages
into foam cells is a hallmark for this pathophysiology [[38]3]. Its
development is that the cholesterol-rich oxidized low-density
lipoprotein (oxLDL) causes endothelial damage and recruits monocytes to
differentiate into macrophages. These oxLDL internalized macrophages
become cholesterol-loaded foam cells [[39]4]. The metabolic disorder
between cholesterol influx and efflux results in a substantial increase
in oxLDL. In the context of AS, exposure to oxLDL results in
mitochondrial dysfunction by reducing the membrane potential [[40]5].
Impaired mitochondria then significantly increases reactive oxygen
species (ROS) production, leading to oxidative stress and tissue damage
[[41]6]. Autophagy, a physiological self-protective process, enables
organisms to eliminate damaged or unwanted intracellular substances,
such as lipid droplets and dysfunctional mitochondria [[42]7].
Mitochondrial ATP generation plays a pivotal role in maintaining cell
homeostasis and survival [[43]8]. Therefore, the elimination of damaged
mitochondria by autophagy (known as mitophagy) plays a key role in AS
caused by excessive oxLDL.
Atorvastatin (AT) is a class of clinical hypolipidemic drugs that can
reduce cholesterol biosynthesis by inhibiting
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [[44]9].
Inhibition of cholesterol synthesis leads to reduced cholesterol
production and upregulation of low-density lipoprotein receptors.
Moreover, evidences have shown that AT can promote cholesterol efflux
and reduce lipid deposition to treat AS by promoting LXRα-mediated
ABCA1, ABCG1/SR-B1 signaling [[45]10,[46]11]. In addition, more and
more natural compounds derived from Chinese herbal medicines have
become an important resource for the development of drugs for
cardiovascular diseases therapy [[47]12]. Astragaloside IV (AS-IV), the
main component of the traditional Chinese medicine astragalus [[48]13],
shows variouspharmacological effects including anti-inflammatory and
anti-oxidative stress [[49]14]. Moreover, AS-IV could inhibit ROS
production, reduce mitochondrial damage and maintain cell viability and
autophagic process by upregulating the cellular PINK1/Parkin signaling
pathway [[50]15,[51]16]. As an E3 ubiquitin ligase, Parkin selectively
targets malfunctioning mitochondria with diminished membrane potential
selectively. Subsequently, Parkin initiated mitophagy to selectively
eliminate damaged mitochondria [[52]17]. Considering the function of
AS-IV to ameliorate mitochondrial damage and mitigate the oxidative
stress response to treat AS, we combined AT and AS-IV for the
synergistic treatment of AS to address the limitations of single
medication.
Conventional oral drugs for AS therapy often showed a short half-life
and frequent administration. Moreover, high doses due to the hepatic
first-pass effect may cause adverse effects such as gastrointestinal
bleeding or liver injury. Thus, how to efficiently delivery drugs to
the targets is a serious challenge.
Recently, the development of nanotechnology provides an alternative for
simultaneously delivering two kinds of drugs to plaques [[53]18]. By
adopting this kind of strategy, we developed biomimetic liposome with
precise targeting to plaque sites. The liposome can effectively promote
the transfer of accumulated cholesterol from the atherosclerotic
arteries to the liver, and then achieve discharge [[54]19]. However,
changes in performance of nanomaterials once entered the body may be
recognized and eliminated by the immune system [[55]20]. To mitigate
the clearance of nanocarriers by the reticuloendothelial system (RES),
a biomimetic membrane was constructed to simulate the natural immune
evasion strategies of cells. Erythrocytes, characterized by their
extended circulation half-life, high abundance, and enucleated nature,
were selected as the ideal carrier to prolong circulation time and
enhance targeting efficiency [[56]21]. The core mechanism involves the
CD47 protein on the membrane surface transmitting a “don't eat me”
signal via the SIRPα signaling pathway, thereby inhibiting phagocytosis
by the RES [[57]22]. While mediating immune evasion [[58]23], due to
its “homing” capability, enables macrophages to actively target
atherosclerotic plaques [[59]24,[60]25]. Furthermore, M1-type
macrophages overexpress the CD44 receptor on the surface of
atherosclerotic plaques [[61]26], therefore, modification with
HA-PEG[2000]-DSPE (hyaluronic acid and DSPE-PEG[2000]-NH[2] complex)
can enhance the active targeting of nanomaterials to plaque sites. This
study aims to integrate the advantages of both membranes to construct a
nanodrug deliver system that combines long-circulating properties,
precise delivery, and low immunogenicity. A multi-membrane synergistic
strategy (e.g. erythrocyte-macrophage membrane composite modification)
combined with targeted molecular engineering (HA-PEG[2000]-DSPE
modification) provides a potential solution for the targeted treatment
of AS (see [62]Scheme 1).
Scheme. 1.
[63]Scheme. 1
[64]Open in a new tab
The preparation process of HA-M@LP@ST and the therapeutic strategy for
AS.
2. Materials and methods
2.1. Supplies and reagents
AS-IV (98 % purity) was sourced from Nanjing Chunqiu Bioengineering
Co., Ltd. (Nanjing, China), and AT (95 % purity) from Beijing Puxitang
Biotechnology Co., Ltd. (Beijing, China). L-α-Phosphatidylcholine
(SPC), cholesterol, and hyaluronic acid were obtained from Shanghai
Long Biotechnology Co., Ltd. (Shanghai, China), Dalian Meilun
Biotechnology Co., Ltd. (Dalian, China), and Solarbio (China),
respectively. N-Hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO,
cell culture grade), and DCFH-DA fluorescent probe were also supplied
by Solarbio. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC),
homocysteine (Hcy), and Oil Red O were procured from Sigma (USA).
DiO/DiI dyes, DSPE-PEG[2000]-NH[2], and the cell membrane protein
extraction kit were sourced from Yisheng Biotechnology Co., Ltd.
(China), Huaten Biotechnology Co., Ltd. (China), and Beyotime
Biotechnology Co., Ltd. (China). Chlorin e6 (Ce6) and oxLDL (including
DiI-labeled oxLDL) were obtained from Shanghai Yuanye Biotechnology
Co., Ltd. (Shanghai, China) and Guangzhou Yiyuan Biotechnology Co.,
Ltd. (China). Chloroform and methanol were purchased from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Preparation of nanoparticles
2.2.1. The preparation of biomimetic membrane [Mø+RBC]m
Initially, whole blood was obtained from C57BL/6 mice. After
centrifugation, plasma was removed, and erythrocytes were washed with
1 × PBS until the supernatants became clear. The erythrocyte pellet was
then resuspended in 0.25 × PBS and incubated overnight at 4 °C to
induce lysis. The resulting mixture was sonicated with an 80 W probe
for 5 min to ensure complete erythrocyte disruption, yielding membrane
fragments. Following centrifugation at 13,000 rpm for 30 min, the
erythrocyte membrane (RBCm) was collected from the intermediate pellet
layer and stored at −20 °C.
RAW 264.7 cells were cultured in 10 % FBS-supplemented medium until
reaching a density of 2.5x10^^7 cells/mL. Following cell detachment,
cells were centrifuged at 1000 rpm for 10 min at 4 °C to pellet.
Macrophage membranes were extracted using a cell membrane protein and
cytoplasmic protein extraction kit. The supernatants were collected and
centrifuged at 13,000 rpm for 30 min to isolate the macrophage membrane
(Møm) fragment pellet and store at −20 °C. RBCm and Møm pellets were
mixed at a 1:1 (mass ratio) and incubated at 37 °C for 1 h to obtain
the hybridized membrane [Mø+RBC]m [[65]27].
2.2.2. Synthesis of HA-PEG[2000]-DSPE (HA)
EDC (25 mg), NHS (50 mg), and hyaluronic acid (5 mg) were dissolved in
1 mL PBS buffer and stirred at room temperature for 30 min.
Subsequently, DSPE-PEG[2000]-NH[2] (25 mg) was added, and the reaction
mixture was incubated at 37 °C for 24 h with continuous stirring. The
products were dialyzed using a 2.5 kDa molecular weight cutoff dialysis
bag for 12 h. Following lyophilization, the products were stored at
−20 °C.
2.2.3. Preparation of HA-M@LP@ST
Liposomes were prepared using the thin-film dispersion method. The
lipid components were formulated at a mass ratio of
lecithin/cholesterol/AS-IV/AT = 20:5:1:0.12 (mass ratio). Specifically,
lecithin and cholesterol were dissolved in chloroform, while AS-IV and
AT were dissolved in methanol, and the volume was adjusted to 5 mL with
chloroform. The mixture was rotary evaporated at 45 °C to form a
uniform film, which was hydrated with distilled water for 1 h. Followed
by probe sonication at 80 W for 5 min. The resulting solution was
centrifuged at 13600 rpm for 30 min to collect the precipitate of
drug-loaded liposome naoparticleds (LP@ST). LP@ST were mixed with [Mø
+RBC]m at a 20:1 (mass ratio) and incubated in a 37 °C water bath for
1 h to obtain membrane-modified drug-loaded liposome nanoparticles
(M@LP@ST). Subsequently, M@LP@ST were mixed with HA-PEG[2000]-DSPE at a
10:1 (mass ratio) and incubated in a 37 °C water bath for 30 min to
prepare HA-PEG[2000]-DSPE modified M@LP@ST(HA-M@LP@ST) and stored at
4 °C.
The formula for estimating encapsulation efficiency (EE %) is as
follows:
[MATH: EE(%)=(Loadeddrug/Totaldrug)×100% :MATH]
2.3. Characterization of HA-M@LP@ST
2.3.1. Characterization of prepared nanoparticles
The morphology of the nanoparticles was assessed via Transmission
Electron Microscopy (TEM, JEM-2100 Plus). The dimensions and electrical
surface potential (zeta potential) of the nanoparticles
were determined using Light Scattering Zetasizer Nano Z Zetasizer (DLS,
NanoZS90).
2.3.2. Membrane localization characterization
Møm and RBCm were initially labeled with DiO (green, Ex/Em: 484/501 nm)
and DiI (red, Ex/Em: 550/567 nm), respectively. Following incubation at
37 °C for 1 h, DiO-Møm and DiI-RBCm were generated. These fluorescent
solutions were then mixed and incubated at 37 °C for an additional 1 h
to yield DiO/DiI-[Mø+RBC]m. Subsequently, DiO and DiI labeled [Mø+RBC]m
and liposomes were incubated separately at 37 °C for 1 h, resulting in
DiO-liposomes and DiI-[Mø+RBC]m. The two fluorescent solutions were
mixed and incubated at 37 °C for 1 h to obtain DiO/DiI-M@liposomes.
Membrane fusion was assessed via live-cell confocal imaging (Olympus
FV1200). UV–Vis (Shimadzu) absorption spectra of Møm, RBCm, and
[Mø+RBC]m were acquired using a UV–Vis spectrophotometer. Protein
samples of Møm, RBCm, [Mø+RBC]m, and HA-M@LP@ST were prepared according
to the membrane protein extraction kit protocol. These samples were
then separated on 10 % SDS-PAGE and stained with Coomassie brilliant
blue. Western blot analysis was performed to detect the membrane
markers of CD11b (Møm) and CD47 (RBCm) in the protein samples.
2.3.3. Stability and drug release experiments
HA-M@LP@ST were sequentially incubated in NaCl, DMEM, and 10 % FBS
solutions in reverse chronological order for a duration of 7 days. On
the final day, the size and degree of polydispersity (PDI) of the
nanoparticles were consistently evaluated by DLS.
Dialysis bags containing HA-M@LP@ST in PBS (pH 7.4) at 37 °C were
employed to investigate the release kinetics. The AT content was
measured using UV spectrophotometry, while the AS-IV concentration was
determined via fluorescence spectrophotometry (HITACHI). The formula
below was utilized to calculate the rate of drug release:
[MATH: Releaseamount(%)=(Mt/Mo)×100% :MATH]
where Mt represents the mass of the drug released at a specific time
point and Mo denotes the initial amount of drug incorporated into the
nanomaterials.
2.4. In vitro experiments
2.4.1. Cell culture
The following cell lines were cultivated in an incubator maintained at
37 °C with 5 % CO[2]: Mouse macrophage cell line (RAW 264.7), murine
hepatocytes (NCTC 1469), human umbilical vein endothelial cell line
(HUVEC), vascular smooth muscle cell line (VSMC), and rat cardiac
myoblast (H9C2) cells. These cells were grown in DMEM medium enriched
with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin
solution (Gibco/Invitrogen). The cells were acquired from the Cell Bank
of the Chinese Academy of Sciences.
2.4.2. Cellular uptake
In 24-well plates, RAW 264.7 cells were treated with Hcy (100 μM) for a
duration of 24 h. Initially, free AT and AS-IV were dissolved in DMSO
and diluted using a medium supplemented with 10 % fetal bovine serum to
final concentrations of 5 μM AT, 30 μM AS-IV and HA-M@LP@ST containing
30 μM AS-IV and 5 μM AT. Following a 2 h pre-incubation period, the
samples were exposed to DiI-oxLDL (40 μg/mL) and incubated for an
additional 24 h. After counterstaining with DAPI, the cells were
observed using two-photon confocal microscopy (CLSM Nikon).
Additionally, the cells were incubated with oxLDL (80 μg/mL) for 24 h.
Following fixation with 4 % paraformaldehyde, the cells were stained
with 0.3 % Oil Red O (ORO), washed with 60 % isopropanol, and
visualized under an inverted microscope (Olympus, IX-73).
Assays to evaluate cellular immune evasion and targeted cellular uptake
were conducted in the 12-well plates. Both RAW 264.7 cells and RAW
264.7 cells stimulated with Hcy (100 μM) were exposed to HA-M@LP@Ce6
(0, 2.5, 5, and 10 μg/mL Ce6) for a period of 4 h. In the targeted
experiment, one group was pre-treated with hyaluronic acid solution for
2 h before adding HA-M@LP@Ce6. Subsequently, the cells were stained
with DAPI for 5 min and visualized using a live-cell imaging system.
2.4.3. In vivo inhibition of oxidative stress assay
Following pretreatment of cells with AT, AS-IV, and HA-M@LP@ST for 2 h,
cells were stimulated with Hcy (100 μM) for 4 h. Subsequently, cells
were incubated with DCFH-DA (10 μM) and MitoSOX Red (3 μM) probes for
30 min. Mitochondrial membrane potential was detected in accordance
with the JC-1 Mitochondrial Membrane Potential Kit. Fluorescence
signals were observed and quantified using CLSM.
2.4.4. Cell immunofluorescence
RAW 264.7 cells were stimulated with Hcy (100 μM) for 24 h, followed by
a 24 h incubation with AT, AS-IV, and HA-M@LP@ST. Cells were
subsequently fixed in 4 % paraformaldehyde, blocked with goat serum,
and incubated overnight at 4 °C with LC3/ABCG1/ABCA1 antibody. After a
2 h incubation with a fluorescently conjugated secondary antibody and
5 min DAPI staining, fluorescence signals were visualized and
quantified via CLSM.
2.4.5. In vitro biocompatibility
An MTT assay was used to evaluate the cytotoxic effects of AS-IV
combined with AT, LP@ST, and HA-M@LP@ST on RAW 264.7 cells, HUVECs,
VSMCs, H9C2, and NCTC 1469 cells, with absorbance measured at 490 nm
using a microplate reader (PerkinElmer EnSpire). Zebrafish embryos at 1
day post-fertilization were incubated with AS-IV+AT, LP@ST, and
HA-M@LP@ST in a 96-well plate for 72 h. Embryo viability, heart rate
and representative images were recorded daily under an inverted
microscope. Hemolytic and blood coagulation experiments were performed
based on reference [[66]27].
2.4.6. Western blot
Protein expression quantity were evaluated across the experimental
groups. Total protein extraction was carried out using RIPA buffer,
followed by quantification via BCA assay. The proteins were resolved by
electrophoresis and transferred onto a PVDF membrane sourced from MA,
USA. To block nonspecific binding sites, the membrane was first
pretreated with 5 % non-fat milk in TBST. Following this, the membrane
was incubated with the primary antibody at 4 °C overnight. Afterward,
the PVDF membrane was exposed to an HRP-conjugated secondary antibody
at room temperature for a period of 2 h.
2.4.7. Transcriptomic analysis and TEM
The RAW 264.7 cells from the control, model (100 μM Hcy), and
HA-M@LP@ST (100 μM Hcy + HA-M@LP@ST) groups were collected and
flash-frozen in liquid nitrogen. High-throughput sequencing was
performed by GENE DENOVE Inc. Data analysis was performed online by
using the Omicsmart platform. Cells were harvested and placed in a
centrifuge tube containing a 2.5 % glutaraldehyde solution. Samples
were stored at 4 °C and sent to Scientific Compass Company for
processing.
2.5. In vivo research
2.5.1. Animals
C57BL/6 mice were purchased from Hunan Silek Jingda Experimental Animal
Co., LTD. ApoE^−/− mice were purchased from Beijing Weishang Lide
Biotechnology Co., LTD. 6-week-old C57BL/6 background male ApoE^−/−
mice were randomly assigned to five groups and subjected to a high-fat,
high-methionine diet (HFD + HMD) for 12 weeks. The mice in the model
group received normal saline, AS-IV group (8 mg/kg). AT group
(2 mg/kg). AS-IV+AT group (8 mg/kg AS-IV+2 mg/kg AT). HA-M@LP@ST group
(8 mg/kg AS-IV+2 mg/kg AT). To ensure the stability of the free drugs
during in vivo administration and minimize precipitation, it is
recommended to first dissolve the drugs in Tween 80, then dilute them
with 0.9 % sodium chloride solution, and perform ultrasonic treatment
to ensure complete dissolution. All treatments were administered via
tail-vein injection, twice weekly. All animal care and experimental
protocols adhered to the “Regulations on the Administration of
Laboratory Animals” of the Ministry of Health of the People's Republic
of China and were approved by the Animal Care Committee of Ningxia
Medical University (License number: 2023-G109).
2.5.2. In vivo pharmacokinetics and plaque targeting capability
Adult male C57BL/6 mice were injected with Ce6 (5 mg/kg) and
HA-M@LP@Ce6 via the tail vein. Subsequently, blood samples were
collected at various time points: 0h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h.
The blood circulation half-life (t[1/2]) was calculated using ICP-MS
analysis performed on a Perkin-Elmer 3300 XL instrument sourced from
the USA. 12h post-injection of Ce6, M@LP@Ce6 and HA-M@LP@Ce6 in
ApoE^−/− mice, the aorta and various organs were isolated and imaged
using a Kodak multimodal imaging system.
2.5.3. In vivo anti-AS study
After completing the animal experiments, the ApoE^−/− mice were
euthanized. The aorta was then stained with ORO to assess the size of
the lesion area. We examined frozen sections of the aortic root for
lipid accumulations by employing ORO staining. Additionally, paraffin
sections were stained using hematoxylin and eosin (H&E) staining,
Masson's trichrome and frozen section were stained using DHE staining.
An immunohistochemical assessment was carried out using antibodies for
F4/80 (Affinity Biosciences), CD31^+ (Affinity Biosciences), α-smooth
muscle actin (α-SMA, Affinity Biosciences), LXRα (Proteintech), ABCG1
(Proteintech), ABCA1 (Bioss) followed by scanning with a digital
imaging system (3DHISTECH, Hungary). For the immunofluorescence
staining of the aortic root sections, antibodies against Parkin
(Proteintech), LC3II (Abmart) and p62 (Proteintech) were utilized and
visualized using CLSM. The expression levels of LXR target genes
SREBP1C (Proteintech), SCD1 (ABclonal), and ACC1 (Proteintech) in the
liver were assessed via immunofluorescence. Blood samples were
collected, and the concentrations of MCP-1, IL-1β, IL-6, TNF-α, and
IL-10 in the serum were determined through ELISA. Finally, a
semi-quantitative analysis was carried out with the assistance of
Image-Pro Plus 6.0 software.
2.5.4. In vivo safety evaluation
Organs (heart, liver, spleen, lungs, kidneys) and Blood samples were
collected from ApoE^−/− mice for in vivo safety assessment. Biochemical
and hematological analyses were performed on blood, while organ
specimens were stained with H&E.
2.6. Statistical analysis
Statistical analyses were performed using one-way analysis of variance
(ANOVA). Data are expressed as mean ± standard deviation (SD). All
statistical tests were implemented in GraphPad Prism software (version
8.0; GraphPad Inc, USA). Significance levels were defined as follows:
∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
3. Results and discussion
3.1. AS-IV and AT exert their effects in scavenging ROS and inhibiting foam
cell formation
Inflammatory macrophages stimulated with Hcy play a pivotal role in the
progression of AS. Consequently, this cell model was used to assess the
therapeutic potential of AS-IV and AT in the context of AS. Initially,
we evaluated the effect of AS-IV, AT and the combination on the
viability of normal RAW 264.7 cells. MTT assay revealed that the
viability of normal RAW 264.7 cells remained above 80 % in the presence
of 30 μM AS-IV and 10 μM AT ([67]Fig. 1A), the result of which is
consistnent with our previous research [[68]10] indicating no
significant adverse effects. Thus, 30 μM AS-IV and 10 μM AT were used
for subsequent experiments. Meanwhile, the concentration of AT was
adjusted to 5 μM when combined with AS-IV. Considering the occurrence
of high ROS in the inflammatory macrophages of AS plaques [[69]28],
DCFH-DA fluorescent probe was used to evaluate the efficacy of AS-IV
and AT on scavenge. As shown in [70]Fig. 1B and C, RAW 264.7 cells with
Hcy treatment emitted intense green fluorescence, which reflected high
ROS levels. Conversely, green fluorescence was markedly reduced in
cells treated with AS-IV and AT. The relative mean fluorescence
intensity (MFI) decreased by 84.91 % and 90.49 %, respectively, after
treatment with AS-IV and AT. These results confirmed the protective
effects of AS-IV and AT against oxidative damage in RAW 264.7 cells,
attributed to their ROS-scavenging function.
Fig. 1.
[71]Fig. 1
[72]Open in a new tab
Evaluation of the effects of AS-IV and AT on ROS scavenging and
inhibition of foam cell formation. (A) The combined impact of AS-IV and
AT on the viability of normal RAW 264.7 cells, as measured by MTT
assay. (B&C) Fluorescence images and quantification of ROS levels in
inflammatory RAW 264.7 cells after treatment with AS-IV and AT. Scale
bar: 100 μm. (D&E) Light microscopy images and quantification of
oxLDL-induced macrophage-derived foam cells stained using ORO following
treatment with AS-IV and AT. Scale bar: 20 μm.
In addition, a key event in AS progression is the formation of foam
cells from macrophages, resulting in lipid deposition [[73]29]. Next,
we subjected inflammatory RAW 264.7 cells to treat with oxLDL for
subsequent ORO staining ([74]Fig. 1D and E). The model group exhibited
a substantial quantity of cellular lipid droplet, indicating the
formation of macrophage-derived foam cells. In contrast, the number of
intracellular lipid droplet were markedly reduced following treatment
with AS-IV and AT. Upon statistical analysis, the lipid droplets in the
AS-IV and AT groups decreased by 34.5 % and 44.8 %, respectively. These
results confirmed that AS-IV and AT could markedly diminish the number
of lipid droplet in macrophages and inhibit foam cell formation. Taken
together, these results demonstrated that AS-IV and AT can treat AS by
scavenging ROS and inhibiting foam cell formation.
3.2. Synthesis and characterization of HA-M@LP@ST
Compared with traditional drugs, nanomaterials exhibit advantages in
the precision and targeting capability for AS treatment [[75]30].
Building upon these advantages, a novel nanodrug, designated as
HA-M@LP@ST, was developed using the thin-film dispersion method, with
the synthetic procedures outlined in [76]Fig. 2A. TEM image of blank
liposomes revealed uniformly dispersed spherical nanostructure with an
average diameter of ∼200 nm ([77]Fig. 2B). To confer stealth property
and prolong systemic circulation duration, HA-PEG[2000]-DSPE (HA) was
prepared by incorporating DSPE-PEG[2000]-NH[2] into the hyaluronic acid
formulation. Subsequent modification with biomimetic membrane and HA
yielded HA-M@LP@ST with an average diameter of ∼100 nm ([78]Fig. 2C).
The incorporation of DSPE-PEG into liposome can significantly reduce
the mean particle size by inhibiting nanoparticle aggregation [[79]19].
Subsequent DLS measurementindicated an average diameter of 151.9 ± 2.6
nm for HA-M@LP@ST ([80]Fig. 2D). These results reflect the differences
in the measurement environment between TEM and DLS [[81]31]. The Zeta
potential of HA-M@LP@ST was determined to be approximately
-25.4 ± 2.2 mV ([82]Fig. 2E).
Fig. 2.
[83]Fig. 2
[84]Open in a new tab
Preparation and characterisation of HA-M@LP@ST. (A) HA-M@LP@ST
synthesis step. (B&C) TEM images of Liposome and HA-M@LP@ST. Liposome
Scale bar: 200 nm. HA-M@LP@ST Scale bar: 100 nm. (D) The particle size
of HA-M@LP@ST were analyzed by DLS. (E) The Zeta potential measurement
of HA-M@LP@ST. (F) Multidimensional live-cell fluorescence imaging maps
were created for Møm, RBC m, [Mø+RBC]m and Liposome. Scale bar: 20 μm.
(G) SDS-PAGE analysis of retention protein bands of Møm RBCm,
[Mø+RBC]m, and HA-M@LP@ST. (H) Western blot detection of CD44 level in
macrophages with Hcy intervention. (I&J) The results of size and PDI
measurements for HA-M@LP@ST stored in NaCl, DMEM, and 10 % FBS solution
for 7 days. (K) Release rates of AS-IV from LP@ST and HA-M@LP@ST in
PBS. (L) Release rates of AT from LP@ST and HA-M@LP@ST in PBS.
We subsequently investigated the fusion efficiency of Møm, RBCm, Møm
and RBCm were labeled with green (DiO) and red (DiI) fluorescent dyes,
respectively, to create DiO/DiI-[Møm+RBCm] vesicles. Multidimensional
live cell imaging revealed significant co-localization (yellow signal)
of the two kinds of vesicles ([85]Fig. 2F). UV spectrophotometry assay
also demonstrated overlapping absorbance peaks for [Møm+RBCm] ([86]Fig.
S1A). Next, liposome and the biomimetic membrane were labeled with DiO
and DiI, respectively, to form DiO/DiI-M@LP@ST. Efficient fusion
between the hybrid membrane and liposome was evidenced by significant
co-localization of the fluorescent signal (yellow signal) ([87]Fig.
2F). SDS-PAGE analysis revealed the retention of Møm and RBCm proteins
within the HA-M@LP@ST ([88]Fig. 2G). Western blot analysis further
confirmed the presence of cell membrane markers CD11b and CD47 in
HA-M@LP@ST ([89]Fig. S1B). To verify whether HA-M@LP@ST actively target
CD44-overexpressing inflammatory macrophages, we employed western blot
assay to detect the levels of CD44 on the surface of macrophages in the
Hcy-treated group and the control group ([90]Fig. 2H). The results
indicated that Hcy treatment significantly upregulated the expression
of CD44, which was consistent with previous report [[91]32].
In addition, stability test showed that the size of HA-M@LP@ST remained
unchanged after being stored at room temperature for 7 days ([92]Fig.
2I). All PDI values after 7 days were <1.0 ([93]Fig. 2J) and smaller
PDI values indicate a more uniform distribution of HA-M@LP@ST sizes.
Finally, we examined the release behavior of LP@ST and HA-M@LP@ST in
PBS. The release rates of AS-IV were approximately 71.8 % for LP@ST and
89.8 % for HA-M@LP@ST, while those of AT were 68.9 % and 83.9 %,
respectively ([94]Fig. 2K and L). These findings suggest that AS-IV and
AT in HA-M@LP@ST can be gradually released to accumulate at the lesion
site.
3.3. HA-M@LP@Ce6 can be efficiently uptaken by Hcy treated RAW 264.7 cells
and extend the blood circulation time
Using Ce6-labeled HA-M@LP (HA-M@LP@Ce6), we investigated the effect of
membrane coating on the macrophages uptake The red fluorescence
depicted in [95]Fig. 3A clearly indicated that LP@Ce6 were successfully
captured by normal RAW 264.7 cells. However, both M@LP@Ce6 and
HA-M@LP@Ce6 exhibited significantly reduced fluorescence intensity,
demonstrating their ability to evade normal macrophage uptake.
Furthermore, the evasion ability of HA-M@LP@Ce6 were correspondingly
enhanced with biomimetic membrane concentration increase ([96]Fig. 3C
and D). Given the overexpression of CD44 on inflammatory macrophages
located within atherosclerotic plaques [[97]33], we subsequently
explored the targeting efficacy of nanomedicine towards these cells.
Initially, we incubated the nanomaterials with inflammatory RAW
264.7 cells and observed a significantly higher fluorescence intensity
in M@LP@Ce6 and HA-M@LP@Ce6 compared to normal RAW 264.7 cells
([98]Fig. 3A). To further confirm the pivotal role of HA, we pretreated
the cells with an free HA solution for 2 h prior to the HA-M@LP@Ce6
treatment. Accordingly, a marked reduction in fluorescence intensity
was observed in this group ([99]Fig. 3B), confirming HA-CD44 mediated
targeting through competitive receptor blockade. These findings
systematically validate that HA-M@LP@Ce6 can escape from normal
macrophage clearance and actively target CD44-overexpressing
inflammatory macrophages, suggesting therapeutic potential for AS.
Fig. 3.
[100]Fig. 3
[101]Open in a new tab
Pharmacokinetic analysis and evaluation of targeting ability of
HA-M@LP@Ce6. (A&B) Confocal fluorescence imaging and quantification of
LP@Ce6, M@LP@Ce6 and HA-M@LP@Ce6 in RAW 264.7 cells. Scale bar: 20 μm.
(C&D) Confocal fluorescence imaging and quantification of
concentration-dependent immune escape by HA-M@LP@Ce6 in RAW
264.7 cells. Scale bar: 20 μm. (E&F) Pharmacokinetic images and curves
of Ce6 and HA-M@LP@Ce6 in C57BL/6 mice. (G&H) Evaluation of the
targeted ability and quantification of Ce6, M@LP@Ce6 and HA-M@LP@Ce6 in
ApoE^−/− mice. (I&J) Distribution and quantification of HA-M@LP@Ce6 in
major organs (heart, liver, spleen, lung, kidney and intestine) in
ApoE^−/− mice.
Liposomes, owing to the amphipathic nature of the phospholipids,
closely mimic natural cell membranes and effectively facilitate
cellular uptake [[102]34]. However, conventional liposomes suffer from
instability in plasma, resulting in a shortened half-life and rapid
clearance from the blood circulation [[103]35]. To overcome these
limitations, we have utilized the immune escape property and long
half-life of biomimetic membrane. Then, we administered HA-M@LP@Ce6 to
C57BL/6 mice and subsequently collected orbital blood samples at
various time points for analysis. As illustrated in [104]Fig. 3E,
HA-M@LP@Ce6 markedly extended the blood circulation time, achieving an
approximately 45.25 % improvement compared to the Ce6 group ([105]Fig.
3F). These findings suggest that the incorporation of a biomimetic
membrane can significantly improve the pharmacokinetics of liposomal
formulation, thereby facilitating drug accumualtion in the plaque sites
of AS.
We further substantiated the targeted uptake capability of HA-M@LP@Ce6
in vivo. ApoE^−/− mice were subjected to high-fat, high-methionine diet
(HFD + HMD) for 12 weeks to establish an AS model. After performing the
tail vein injection of HA-M@LP@Ce6, we dissected the mice at the 12-h
time point and utilized in vivo imaging technology to track the
distribution of HA-M@LP@Ce6, while quantitatively assessing its
accumulation at plaque sites. Relatively intense fluorescence
signal was found in the aortic arch and abdominal aorta of the mice
([106]Fig. 3G and H) attributed to the formation of plaques in ApoE^−/−
mice [[107]36]. Ce6 served as a mimic for free drugs, exhibiting weaker
fluorescence intensity in the aortic region among the tested agents. In
contrast, the strongest signal was found in the plaque areas of
mice with HA-M@LP@Ce6 administration. This suggests that HA conjugation
enhances the accumulation of HA-M@LP@Ce6 at AS plaque sites.
Additionally, fluorescence imaging of these organs revealed that
HA-M@LP@Ce6 were predominantly concentrated in the liver, kidneys, and
intestine ([108]Fig. 3I and J). The variations in distribution pattern
suggest a favorable impact on the prolonged circulation and effective
accumulation of nanodrugs within atherosclerotic lesions [[109]36]. In
conclusion, the combination of biomimetic membrane and HA enables
HA-M@LP@Ce6 to actively target towards atherosclerotic plaques,
enhancing the potential for therapeutic applications in AS.
3.4. RNA-seq of HA-M@LP@ST
We conducted transcriptome analysis on the control group (normal RAW
264.7 cells), model group (RAW 264.7 cells stimulated with Hcy), and
HA-M@LP@ST group (RAW 264.7 cells with Hcy+HA-M@LP@ST treatment) to
explore the mechanisms of therapeutic action. Principal Component
Analysis (PCA) revealed unique transcriptomic features among RAW
264.7 cells across three groups ([110]Fig. 4A). Subsequently, the
overall gene expression profiles of the three groups were graphically
displayed using a Venn diagram ([111]Fig. 4B). Differentially Expressed
Genes (DEGs) analysis demonstrated significant differences between the
control, model, and HA-M@LP@ST group. To further explore the
therapeutic mechanisms of HA-M@LP@ST group, we examined the
relationship of gene expression among these groups. In comparison to
the model group, a significant number of DEGs were identified in the
HA-M@LP@ST group. The Venn diagram presented co-expression of 1255
genes in the model and HA-M@LP@ST group, while 424 genes were expressed
by HA-M@LP@ST group compared with model group. From the volcano plot,
it was found that there were 1737 DEGs between the control and model
groups, of which 80 genes were upregulated and 1657 genes were
downregulated, respectively ([112]Fig. 4C). Model and HA-M@LP@ST group
were 1255 DEGs, of which 1120 and 135 genes were upregulated and
downregulated, respectively ([113]Fig. 4D). KEGG pathway enrichment
analysis revealed the important role of HA-M@LP@ST group in regulating
lipid metabolism and AS, autophagy and ABC transporter signaling
pathways after optimal therapy ([114]Fig. 4E). Subsequently, we further
explore the role of HA-M@LP@ST in these signaling pathways.
Fig. 4.
[115]Fig. 4
[116]Open in a new tab
Transcriptomic analysis of HA-M@LP@ST. (A) PCA of DEGs based on control
group (normal RAW 264.7 cells), model group (RAW 264.7 cells stimulated
with Hcy), and HA-M@LP@ST group (RAW 264.7 cells stimulated with
Hcy + HA-M@LP@ST treatment). Each data point corresponds to the PCA
result of an individual sample. (B) Venn diagram depicting the number
of DEGs when comparing HA-M@LP@ST group with the Control group and the
Model group, respectively. (C&D) Volcano plots illustrating the up- and
down-regulated genes in the comparisons between HA-M@LP@ST group versus
Control and Model group, respectively. (E) KEGG pathway enrichment for
the identified major relevant differentially expressed genes.
3.5. HA-M@LP@ST inhibit foam cell formation by promoting cholesterol efflux
A key feature of AS is the entry of lipoproteins into the body, where
they are stored by macrophages in the arteries. The assimilation of
macrophages with modified lipids, such as oxLDL, leads to the formation
of foam cells [[117]37]. Consequently, Dil-labeled oxidized LDL
(Dil-oxLDL), which can emit a distinctive red fluorescence, functioned
as the lipid basis to monitor the effect of HA-M@LP@ST on the uptake
efficiacy of macrophages. Upon treating inflammatory RAW 264.7 cells
with Dil-oxLDL, a pronounced red fluorescence was discernible within
the cytoplasm ([118]Fig. 5A), the result of which directly indicated
the cellular uptake amount of oxLDL. In contrast, the red fluorescence
signal was notably diminished by 60.9 % in the HA-M@LP@ST-treated group
compared to the model group. However, the inhibition rate was only
34.6 % was found in the AS-IV+AT group ([119]Fig. 5B). This result
clearly demonstrated the strong inhibitory effect of HA-M@LP@ST
on oxLDL uptake.
Fig. 5.
[120]Fig. 5
[121]Open in a new tab
HA-M@LP@ST promotes cholesterol efflux and reduces foam cell formation.
(A&B) Confocal fluorescence images and quantification of DiI-oxLDL
uptake in inflammatory RAW 264.7 cells after HA-M@LP@ST treatment.
Scale bar: 20 μm. (C&D) Light microscopy images and quantification of
oxLDL-induced macrophage-derived foam cells were stained using ORO
following treatment with HA-M@LP@ST treatment. Scale bar: 20 μm. (E–H)
Confocal fluorescence images and quantitative analysis of ABCA1 and
ABCG1 on activated macrophages under different treatments. (I&J)
Western blot analysis and quantification of LXRα, ABCG1, and SR-B1
proteins in macrophages with different treatments.
Subsequently, we investigated the effect of HA-M@LP@ST on the foam cell
formation. ORO staining indicated that inflammatory RAW 264.7 cells
incubated with 80 μg/mL oxLDL for 24 h exhibited considerable
accumulation of lipid droplets, which reflected foam cell formation
([122]Fig. 5C). However, HA-M@LP@ST treatment showed a marked
inhibitory effect, which was higher than that of AS-IV+AT treatment.
Quantitative analysis revealed inhibit rates of 61.33 % and 31.82 % for
the HA-M@LP@ST and AS-IV+AT treatment, respectively ([123]Fig. 5D).
This result highlights the key role of HA-M@LP@ST for inhibiting foam
cell formation by reducing oxLDL uptake. In addition, for the purpose
of clearly distinguishing the specific contributions of various design
elements, such as liposomes, biomimetic membrane, or HA, in the overall
construction, we synthesized various nanoparticle formulations,
including sole liposomes, HA modified liposomes and membrane
coating liposomes as controls. By subsequently assessing the effect of
these nanoparticles on foam cell formation in vitro, we found
substantial foam cell formation following oxLDL exposure. Compared to
the model group, Liposomes, HA@LP, and M@LP exhibited ultra low
efficacy on foam cell formation, suggesting negligible effect of
them on AS therapy ([124]Fig. S2A and B).
Driving excess cholesterol efflux is considered to be a major
atheroprotective process in macrophages [[125]38]. The three primary
transporters facilitating cholesterol efflux are scavenger receptor B1
(SR-B1), ATP-binding cassette transporter G1 (ABCG1) and ATP-binding
cassette transporter A1 (ABCA1) [[126]39,[127]40]. Both ABCG1 and ABCA1
genes are under the direct regulation of liver X receptors (LXRs)
[[128]41]. Subsequently, in order to delve deeper into the mechanism
underlying the effect of HA-M@LP@ST on lipid accumulation, we measured
the levels of lipid efflux related proteins in cells.
Immunofluorescence images showed the upregulation of ABCG1 and ABCA1
caused by HA-M@LP@ST ([129]Fig. 5E–H). Western blot assay demonstrated
notable increase of LXRα in the HA-M@LP@ST group, accompanied by
elevated levels of ABCG1 and SR-B1 ([130]Fig. 5I and J). These results
suggest the activation of cholesterol reverse transport pathway,
thereby highlighting the role of HA-M@LP@ST's for facilitating
cholesterol efflux and mitigating lipid accumulation, which finally
inhibited the formation of foam cells.
3.6. HA-M@LP@ST can improve mitochondrial damage of macrophages by promoting
autophagy
Autophagy of macrophages is a protective mechanism for inhibiting AS
[[131]42] by clearing damaged organelles, especially damaged
mitochondria [[132]43]. As the autophagosome formation implies
successful induction of autophagy [[133]44], we firstly performed TEM
imaging and observed the accumulation of autophagosomes in inflammatory
RAW 264.7 cells treated with HA-M@LP@ST ([134]Fig. 6A).
Immuno-fluorescence staining also demonstrated the stronger green
fluorescence signal representing higher levels of the autophagosome
marker LC3II in HA-M@LP@ST treated cells compared to the model group.
Meanwhile, co-ocalization images showed increased production of
autophagosomes within macrophage ([135]Fig. 6B and C). Parkin is an E3
ubiquitin ligase that increases mitochondrial viability. Activated
Parkin can be linked with LC3 to form mitochondrial autophagosomes to
initiate the degradation program of mitochondria [[136]45]. Western
blot assay indicated significant upregulation of Parkin and LC3II
proteins, whereas the downregulation of p62 protein in the HA-M@LP@ST
group when compared to the model group ([137]Fig. 6D and E). These
observations suggested that HA-M@LP@ST treatment promoted autophagy in
macrophages and accelerated the degradation of p62. In conclusion,
HA-M@LP@ST is able to promote Parkin-mediated autophagy in macrophages.
Fig. 6.
[138]Fig. 6
[139]Open in a new tab
HA-M@LP@ST cleared mtROS and restores mitochondrial function. (A)
Autophagosomes were visualized by TEM. Scale bar: 500 nm. (B&C)
Confocal fluorescence images and quantification of LC3II in
inflammatory RAW 264.7 cells. Scale bar: 50 μm. (D&E) Western blot
analysis detected and quantified the protein expression of Parkin,
LC3II, and p62. (F&G) Confocal fluorescence images and quantification
of total ROS in HA-M@LP@ST cleared inflammatory RAW 264.7 cells. Scale
bar: 100 μm. (H&I) Images and quantification of mitochondrial
superoxide levels were obtained using MitoSOX red fluorescence
staining. Scale bar: 20 μm. (J) Mitochondrial membrane potential assay
using JC - 1 staining. Scale bar: 20 μm. (K) The ratio of red MFI
(aggregated JC - 1) to green MFI (monomer JC - 1) was calculated to
quantify the mitochondrial membrane potential. (L) Quantification of
mitochondrial ATP levels.
Considering the tight relationship between ROS and autophagy, we
accordingly investigated the effect of HA-M@LP@ST on the ROS levels of
macrophages. The results revealed significant reduction of total ROS
levels in cells treated with AS-IV+AT and HA-M@LP@ST, compared to the
model group ([140]Fig. 6F). Statistical analysis further emphasized
that the relative MFI in the AS-IV+AT group exhibited 86 % decrease
compared to the model group, and the relative MFI in the HA-M@LP@ST
group demonstrated 95.8 % reduction ([141]Fig. 6G). In addition, we
also evaluated the ability of nanoparticles to reduce ROS levels in
vitro. Our findings revealed significant elevation of ROS in
Hcy-stimulated RAW 264.7 cells. Compared to the model group, Liposome,
HA@LP, and M@LP exhibited limited efficacy in ROS scavenging ([142]Fig.
S2C and D). Using the MitoSOX Red superoxide indicator to evaluate the
effect of HA-M@LP@ST on mitochondrial ROS (mtROS) levels. it was found
that Hcy treated RAW 264.7 cells showed strong red fluorescence due to
the substantial mtROS accumulation. Conversely, HA-M@LP@ST
administration caused 55.68 % reduction in red fluorescence intensity
([143]Fig. 6H and I). Considering the reduction of membrane
potential (Δψm) is the hallmark of mitochondrial dysfunction [[144]46],
we utilized a mitochondrial membrane potential assay kit to evaluate
the effects of HA-M@LP@ST on the mitochondrial membrane potential in
macrophages. The control groupwith healthy mitochondria displayed red
fluorescence due to the formation of J-aggregates within the
mitochondrial matrix. In contrast, the model group showed green
fluorescence, indicating a reduction in Δψm values. Notably, RAW
264.7 cells treated with HA-M@LP@ST exhibited strong red fluorescence
alongside weak green fluorescence ([145]Fig. 6J and K), suggesting the
enhancement of Δψm in inflammatory RAW 264.7 cells. Mitochondria are
responsible for generating the majority of cellular adenosine
triphosphate (ATP). Based on this, we examined the intracellular ATP
levels of inflammatory RAW 264.7 and found the increase of ATP levels
in macrophages after HA-M@LP@ST treatment ([146]Fig. 6L). In
conclusion, HA-M@LP@ST promotes the partial recovery of mitochondrial
function by eliminating mtROS, regulating the mitochondrial membrane
potential and increasing ATP synthesis.
3.7. Therapeutic efficacy of HA-M@LP@ST against AS in vivo
We established an animal model specific to the plaque development. The
specific experimental design is illustrated in [147]Fig. 7A. Weekly
assessment revealed no notable weight loss in any of the groups, as
depicted in [148]Fig. 7B. Ultrasound imaging ([149]Fig. S3A) showed
that HA-M@LP@ST group exhibited the decrease of blood flow velocity and
the reduction of inner media thickness of the membrane compared to the
model group ([150]Fig. S3B and C). Then, we euthanized the ApoE^−/−
mice and harvested whole aorta along with major organs. After ORO
staining of the whole aorta and subsequent statistical analysis, it was
shown that the HA-M@LP@ST group exhibited the most significant
anti-atherosclerotic effect ([151]Fig. 7C and D).
Fig. 7.
[152]Fig. 7
[153]Open in a new tab
Anti-atherogenic efficacy of HA-M@LP@ST in vivo. (A) Schematic diagram
of the experiment design. (B) Statistical results of body weight change
in ApoE^−/− mice at 12 weeks. (C) Representative images of whole aortic
ORO stained plaques (red area) in ApoE^−/− mice in different groups.
(D) Gross morphology and quantification of the entire aortic plaque
area, n = 7. (E–H) Representative histochemical images of aortic root
cross-sections stained with H&E (E), F4/80 antibody (G).
Semi-quantitative analysis of the necrotic core area (F), macrophage
infiltration in the plaque area (H). Scale bar: 100 μm. n = 3. (I)
Diagram of the aortic arch in ApoE^−/− mice. White dashed lines
represent the plaque areas within the aortic arch. (J&K) ORO staining
and quantification of frozen cross-sections of aortic root in different
groups. Scale bar: 500 μm. n = 3.
It is well-established that the necrotic core area closely associated
with the progression of plaque formation and the severity of disease
[[154]47]. Therefore, we performed systematic histopathological
evaluations. H&E staining of aortic root sections revealed distinct
morphological differences. Notably, the aortic root plaques in the
model group displayed necrotic core rich in lipids, whereas the
decrease in the size of the necrotic core was noted in the HA-M@LP@ST
group. Compared to the model group, the HA-M@LP@ST group reduced the
necrotic cores by 50.38% ([155]Fig. 7E and F). Necrotic core in plaque
is closely related to the infiltrating macrophage number [[156]48].
Immunohistochemistry results showed that the number of macrophages
(represented by F4/80) was reduced by about 54.7 % compared to the
model group ([157]Fig. 7G and H). The above results showed that the
HA-M@LP@ST significantly reduced the necrotic core within the aortic
arch. Morphometric analysis of aortic arch region (indicated by the
dotted line) exhibited significant reduction in plaque area upon
dissection of the aorta ([158]Fig. 7I). Then, we prepared aortic root
ORO staining to substantiate the therapeutic efficacy of HA-M@LP@ST
([159]Fig. 7J and K). The findings indicated that the HA-M@LP@ST group
exhibited the most significant reduction in plaque area. Quantitative
analysis indicated that the ORO-positive area reduced by approximately
23.7 %, 33 %, and 55 % in the AS-IV, AT, and AS-IV+AT groups,
respectively, whereas the HA-M@LP@ST group reduced 67.3 %, in
comparison to the model group. These findings unequivocally indicated
that the HA-M@LP@ST possessed the most remarkable anti-lipid deposition
effect.
Subsequently, we assessed the impact of HA-M@LP@ST on the stability of
plaques. Masson Trichrome staining indicated that HA-M@LP@ST increased
the collagen content around the plaque by 50.6 %, compared to the model
group ([160]Fig. 8A and B). The upregulation of collagen content
contributed to theincrease in fibrous cap thickness, indicating
enhanced plaque stability following treatment. To further investigate
collagen synthesis in the fibrous cap, we monitored the quantitative
levels of VSMCs. As shown in [161]Fig. 8C, enrichment of VSMCs in the
plaques was observed after HA-M@LP@ST treatment, with VSMC levels
upregulated by approximately 41.8 %. This increase suggests that
HA-M@LP@ST promote collagen synthesis within the fibrous cap.
Additionally, neovessels in atherosclerotic plaques, formed by CD31^+
endothelial cells, are structurally deficient and permeable,
predisposing to intraplaque hemorrhage and plaque instability
[[162]49]. As illustrated in [163]Fig. 8D, the levels of perivascular
CD31^+ endothelial cells decreased by 63.1 % in the HA-M@LP@ST group
compared to the model group. These results clearly indicate that
HA-M@LP@ST effectively curbs the development of atherosclerotic plaques
and enhances the stability of vulnerable atherosclerotic plaques,
suggesting their potential for AS therapy.
Fig. 8.
[164]Fig. 8
[165]Open in a new tab
HA-M@LP@ST improved plaque stability in a mice model of AS. (A)
Illustrative histochemical images depicting cross-sections of the
aortic root stained with Masson's trichrome, an α-SMA antibody, and a
CD31^+ antibody. (B–D) Semi-quantitative analysis of collagen content,
VSMC expression, and CD31^+ staining in the plaque area. Scale bar:
100 μm. n = 3.
3.8. HA-M@LP@ST can regulate oxidative stress and cholesterol efflux in vivo
In order to further explore the mechanism of HA-M@LP@ST for in vivo AS
treatment, we firstly performed dihydroethidium (DHE) staining on
aortic root sections obtained from AS mice to assess the levels of ROS.
As we expected, HA-M@LP@ST treatment significantly reduced red
fluorescence intensity, reflecting the low levels of ROS ([166]Fig. 9A
and B). Immunofluorescence imaging indicated that HA-M@LP@ST
significantly promoted the levels of LC3II and Parkin while inhibiting
p62 ([167]Fig. 9C–H). Immunochemical assay showed the upregulation of
HA-M@LP@ST on the levels of LXRα, ABCG1 and ABCA1 in the aortic arch
([168]Fig. 9I–N). It was reported that lessening the lipid accumulation
in existing plaque involves boosting cholesterol efflux [[169]50].
These results suggest that HA-M@LP@ST can reduce oxidative damage,
promote autophagy and enhance cholesterol efflux to alleviate the AS.
Fig. 9.
[170]Fig. 9
[171]Open in a new tab
HA-M@LP@ST exhibit effects in fighting oxidative damage, enhance
cholesterol efflux. (A&B) ROS levels and quantification in ApoE^-/-
mice aortic roots. Scale bar: 200 μm. (C–H) Immunofluorescence staining
and quantification assay of the levels of Parkin, LC3II and p62
proteins in the aortic roots. Scale bar: 200 μm. n = 3. (I–N) LXRα,
ABCG1 and ABCA1 levels in the aortic roots detected by
immunohistochemistry and quantified. Scale bar: 100 μm. n = 3. (O–S)
Serum levels of the proinflammatory factors IL-6, MCP-1, IL-1 β, TNF-
α, and the anti-inflammatory factor IL-10 in ApoE^−/− mice are
presented.
Furthermore, LXR activation in hepatocytes was reported to stimulate
the expression of downstream genes including sterol regulatory
element-binding protein 1c (SREBP1C), stearoyl-CoA desaturase 1 (SCD1),
and acetyl-CoA carboxylase (ACC1), thereby promoting hepatic de novo
lipogenesis [[172]51]. Using immunofluorescence staining to assess the
levels of LXR target genes (SREBP1C, SCD1, and ACC1) in liver sections,
we found slight upregulation of SREBP1C, SCD1, and ACC1 proteins in the
model group, which suggested a potential activation of the LXR
signaling pathway. In contrast, HA-M@LP@ST NPs administration decreased
the levels of these proteins ([173]Fig. S4), which indicated the
inhibition of HA-M@LP@ST NPs on this signaling pathway.
Given the intimate link between AS and inflammatory factors, we
measured the levels of inflammatory cytokines in the serum of ApoE^−/−
mice. As illustrated in [174]Fig. 9O–S, compared with all other treated
groups, low levels of major cytokines (MCP-1, TNF-α, IL-1β, and IL-6)
were observed in the serum of ApoE^−/− mice with HA-M@LP@ST treatment.
Meanwhile, the levels of IL-10 were increased in the ApoE^−/− mice
treated with HA-M@LP@ST. These findings strongly indicate that
HA-M@LP@ST possess the ability to suppress the inflammatory response
during the development of AS.
3.9. Biosafety of HA-M@LP@ST
Biological safety constitutes the primary criterion for assessing the
clinical translation potential of nanomedicines. we performed a series
of biological safety experiments. At first, MTT assay was employed to
assess the cytotoxicity of HA-M@LP@ST on various cells. Notably, all
cell survival rates remained above 80 % ([175]Fig. S5A–E). Given that
nanoparticles can potentially activate human blood coagulation and lead
to thrombosis [[176]52], we performed rigorous blood compatibility
experiments, including coagulation assays and hemolysis test ([177]Fig.
S5F–I), These experiments indicated a minimal risk of HA-M@LP@ST for
inducing thrombosis and hemolysis. In addition, various concentrations
of HA-M@LP@ST were incubated with zebrafish eggs to evaluate its
biocompatibility ([178]Fig. S6A). The results showed minimal changes of
zebrafish in survival rates ([179]Fig. S6B) and heart rate ([180]Fig.
S6C) compared to the control group. Collectively, these findings
indicate high biocompatibility of HA-M@LP@ST.
The next step was to investigate the biosafety of HA-M@LP@ST in
ApoE^−/− mice vivo. Following a 12-week treatment regimen, we conducted
H&E staining on the primary organs of the mice and did not find notable
drug damage ([181]Fig. S7). Subsequently, the collected plasma
supernatants were subjected to hematological evaluation ([182]Fig.
S8A–M). All routine hematological parameters, encompassing RBC,
platelets (PLT), hemoglobin levels (HGB), and other indices, fell
within the normal physiological ranges. Furthermore, clinical
biochemical analyses demonstrated that biochemical markers such as
blood urea (UREA) and serum creatinine (CREA) also maintained normal
levels, suggesting negligible impact of HA-M@LP@ST on the liver and
kidney function. In contrary, the levels of total cholesterol (TC),
triglycerides (TG) and low-density lipoprotein (LDL) exhibited varying
degrees of decrease when compared to the model group, indicating that
HA-M@LP@ST possessed significant lipid-lowering efficacy. Additionally,
the reduction in homocysteine (Hcy) levels suggested that HA-M@LP@ST
could effectively decrease hyperhomocysteinemia. Considering that
clinical patients with hyperlipidemia often present with concurrent
hyperglycemia, we incorporated blood glucose monitoring into animal
experiment ([183]Fig. S8N). Blood glucose levels slightly increased
four weeks post-modeling. However, blood glucose levels decreased to
varying degrees after withHA-M@LP@ST treatment. These results
collectively indicate the favorable biosafety of HA-M@LP@ST and the
effectiveness in lowering blood glucose levels.
4. Conclusion
In this study, we designed and synthesized a versatile nanomedicine of
HA-M@LP@ST. This nanomedicine can evade phagocytosis by the immune
system, thereby enabling prolonged circulation and targeted delivery to
the lesion site of AS for drug release. Moreover, it can facilitate
cholesterol efflux and mitigate foam cell formation by activating the
LXRα/ABCG1 and ABCA1/SR-BI signaling pathway. Additionally, it can
regulate parkin-mediated autophagy, enhance impaired mitochondrial
function, and alleviate oxidative damage in macrophages. The
synergistic combination of these two treatment mechanisms improves the
efficacy of HA-M@LP@ST on AS.
CRediT authorship contribution statement
Hanshuang Ding: Writing – original draft, Visualization, Validation,
Software, Resources, Project administration, Methodology,
Investigation, Formal analysis, Data curation, Conceptualization. Yi
Liu: Project administration, Methodology. Tongtong Xia: Project
administration, Methodology, Investigation. Huiping Zhang: Supervision,
Resources, Funding acquisition, Conceptualization. Yinju Hao: Writing –
review & editing, Supervision, Resources, Investigation, Funding
acquisition, Conceptualization. Bin Liu: Writing – review & editing,
Supervision, Project administration, Methodology, Conceptualization.
Yideng Jiang: Writing – review & editing, Supervision, Resources,
Project administration, Methodology, Investigation, Funding
acquisition, Conceptualization.
Disclosure
The authors declare that they have no known competing interests.
Declaration of competing interest
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.
Acknowledgements