Abstract
Hepatic ischemia‐reperfusion injury (HIRI) is a prevalent issue during
liver resection and transplantation, with currently no cure or
FDA‐approved therapy. A promising drug, Cyclosporin A (CsA),
ameliorates HIRI by maintaining mitochondrial homeostasis but has
systemic side effects due to its low bioavailability and high dosage
requirements. This study introduces a biomimetic CsA delivery system
that directly targets hepatic lesions using mesenchymal stem cell (MSC)
membrane‐camouflaged liposomes. These hybrid nanovesicles (NVs),
leveraging MSC‐derived proteins, demonstrate efficient inflammatory
chemotaxis, transendothelial migration, and drug‐loading capacity. In a
HIRI mouse model, the biomimetic NVs accumulated at liver injury sites
entered hepatocytes, and significantly reduced liver damage and restore
function using only one‐tenth of the CsA dose typically required.
Proteomic analysis verifies the protection mechanism, which includes
reactive oxygen species inhibition, preservation of mitochondrial
integrity, and reduced cellular apoptosis, suggesting potential for
this biomimetic strategy in HIRI intervention.
Keywords: biomimetic delivery, cyclosporine A, ischemia‐reperfusion
injury, mesenchymal stem cell
__________________________________________________________________
This work develops a biomimetic cyclosporin A (CsA) delivery system
targeting hepatic lesion sites by constructing mesenchymal stem cell
membrane‐camouflaged liposomes. The constructed hybrid nanovesicles
effectively reduce liver damage and restore liver function at a
one‐tenth dose of free CsA, demonstrating the potential of the
nanovesicles to address the current challenges of hepatic
ischemia‐reperfusion injury intervention.
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1. Introduction
Hepatic ischemia‐reperfusion injury (HIRI) is a gradually exacerbated
phenomenon that arises during liver resection and transplantation when
blood flow is reinstated following a period of interruption.^[ [68]^1
^] This event leads to increased hepatocyte death due to adenosine
triphosphate consumption and oxygen depletion, and triggers an
inflammatory cascade and excessive production of reactive oxygen
species (ROS), causing significant hepatocyte damage.^[ [69]^2 ^] These
effects result in a cycle that increases the risk of early dysfunction
and limits the availability of organs for transplantation.^[ [70]^2 ,
[71]^3 ^] Hence, identifying effective protection strategies to
mitigate HIRI is a significant challenge in medical and public health
fields.
Mitochondria, which are critical in maintaining cellular homeostasis,
are implicated in the onset and progression of ischemic‐reperfusion
injury (IRI).^[ [72]^4 ^] Initial mechanical damage disrupts cell
membranes and cytoskeletal components, leading to increased
intracellular calcium levels and activation of the mitochondrial
permeability transition pore (mPTP). This results in mitochondrial
swelling and rupture.^[ [73]^5 ^] Later, damaged mitochondria release
cytochrome c and ROS, initiating cascade reactions that culminate in
widespread apoptosis and inflammation in the liver.^[ [74]^6 ^] Given
the crucial role of mitochondria, preserving or restoring mitochondrial
homeostasis is a key objective in HIRI prevention.
Cyclosporine A (CsA), an immunosuppressant isolated from fungi, has
been reported to pharmacologically inhibit the opening of mPTP.^[
[75]^7 ^] By binding with cyclophilin D (CypD) located in the inner
mitochondrial membrane, CsA can reduce mitochondrial calcium uptake and
ROS production, thereby alleviating mitochondrial dysfunction.^[ [76]^8
^] Previous research has highlighted the potential of CsA in HIRI
prevention.^[ [77]^9 ^] However, its poor aqueous solubility and high
binding affinity to plasma proteins lead to low bioavailability and
high‐dose requirements to achieve the desired protection effects.^[
[78]^10 ^] This, in turn, causes severe side effects such as
immunosuppression, cytotoxicity, and oncogenesis, and significantly
restricts its clinical application.^[ [79]^11 ^] In summary, the main
challenge in CsA‐mediated HIRI prevention lies in achieving targeted
delivery to the hepatic lesion sites while minimizing its adverse
effects on healthy cells.
Nanovehicles like liposomes offer a promising drug delivery strategy
due to their high drug‐loading capacity, sustained release profile, and
biocompatibility.^[ [80]^12 ^] However, they face challenges such as
rapid clearance by the mononuclear phagocyte system (MPS) and lack of
selectivity between healthy and injured liver cells^[ [81]^13 ^]
Natural cell membrane‐based nanomaterials, particularly the mesenchymal
stem cells (MSCs), offer an exciting approach to overcome these
challenges. Their low expression of major histocompatibility complex
allows them to evade rapid clearance by immune cells.^[ [82]^14 ^]
Meanwhile, their chemokine and adhesion molecule expression on their
membrane enables targeted migration to injured tissues.^[ [83]^15 ^]
These unique biological properties offer tremendous potential in
addressing the challenges associated with drug delivery by liposomes,
making them an attractive option for achieving biomimetic delivery of
CsA in the field of HIRI prevention.
In this study, we developed MSC membrane‐camouflaged CsA liposomes
(MMCLs) for targeted HIRI intervention. By combining human umbilical
cord‐derived MSC membrane with liposomal membrane, MMCLs were designed
to retain the inflammatory chemotaxis and immune privilege of MSCs,
along with efficient drug loading of liposomes (Scheme [84]1 ). The
fusion of membranes had minimal impact on the encapsulation capacity of
CsA. In vitro and in vivo experiments demonstrated the superior ability
of MMCLs to mitigate mitochondrial dysfunction, reduce ROS production,
and decrease cell apoptosis. In a HIRI mouse model, the inflammatory
microenvironment characterized by overexpression of pro‐inflammatory
cytokines and chemokines at the hepatic lesion sites provided a
suitable target. The biomimetic vesicles, MMCLs, exhibited efficient
accumulation in the injured liver and successfully evaded clearance by
the MPS in the liver and spleen. Benefiting from targeted delivery,
MMCLs significantly reduced liver damage and restored liver function at
a one‐tenth dose‐free CsA. Importantly, MMCLs mitigated the side
effects of the drugs on healthy organs and cells, presenting a
promising protection option for HIRI.
Scheme 1.
Scheme 1
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Schematic illustration of using biomimetic nanovesicles to prevent HIRI
by MMs with CLs. a) Construction of MMCLs by the extrusion method. b)
MMCLs exhibited inflammation chemotaxis to the hepatic lesion site and
escaped from macrophage clearance after tail intravenous injection.
Hepatocyte uptake of MMCLs can release CsA to bind with CypD on the
mitochondrial membrane, close the mPTP, alleviate mitochondrial
dysfunction, and reduce cell apoptosis.
2. Results and Discussion
2.1. Construction and Characterization of the Hybridized NVs
The preparation of MMCLs involved three steps: synthesis of CsA
liposomes (CLs), isolation of MSC membranes (MMs), and camouflaging CLs
with MSC membranes (Scheme [86]1a). CLs were first synthesized using a
thin‐film hydration method. The encapsulation efficiency and loading
capacity of CsA in CLs were determined to be 98.0% ± 2.8% and
4.8 ± 0.1%, respectively by high‐performance liquid chromatography
(HPLC). MMs were obtained following a previously reported method.^[
[87]^16 ^] The collected MMs did not exist nuclei, as verified by
prestaining the cell nuclei using DAPI and inverted fluorescence
microscopic images (Figure [88]S1, Supporting Information). The protein
content in the purified MMs obtained from 3 × 10^7 cells was determined
to be 330 µg. Subsequently, the purified MMs were sonicated, mixed with
CLs, and extruded through a series of polycarbonate membranes with pore
sizes of 400 and 200 nm to produce MMCLs. Various physicochemical
characterizations were performed as follows. HPLC detected only 2% of
CsA leakage in MMCLs, indicating that the membrane fusion step had no
significant effect on the loading capacity of CsA. Transmission
electron microscopy (TEM) images revealed that the CLs presented smooth
and spherical surfaces with a particle size of 100–200 nm, which was
consistent with the morphology of large unilocular liposomes reported
before.^[ [89]^17 ^] After co‐extrusion with wrinkled‐surface MMs, the
resulting MMCLs maintained a smooth spherical structure on their
surface with no multilamellar vesicles inside (Figure [90]1a), proving
that CLs and MMs were fused through the outer membrane rather than
encapsulated. Therefore, CsA as a hydrophobic drug should be located
within the phospholipid bilayer of the CLs and MMCLs, rather than in
the aqueous core. Dynamic lighting scattering (DLS) measurements showed
that the hydrodynamic diameter of MMCLs (152.8 ± 1.5 nm) was smaller
than that of MMs (217.1 ± 4.0 nm) and similar to that of CLs
(158.4 ± 1.4 nm), indicating the successful reconstruction of the two
types of NVs through co‐extrusion. The zeta potentials of CLs, MMs, and
MMCLs were all ≈−20 mV (Figure [91]1b).
Figure 1.
Figure 1
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Characterization of CLs, MMs, and MMCLs. a) Morphology of CLs, MMs, and
MMCLs under TEM. b) DLS size distribution and zeta potentials of CLs,
MMs, and MMCLs. c) Time‐dependent colloidal stability and
dispersibility of CLs and MMCLs in PBS or DMEM at 4 °C for 30 days. d)
Time‐dependent stability and dispersibility of CLs and MMCLs in 10% FBS
at 37 °C for 7 days. e) Drug release profile of CLs and MMCLs in PBS
buffer containing 0.5% Tween 80 under 37 °C for 48 h. f) Colocalization
analysis of CLs and MMs after co‐extruded at phospholipid‐to‐membrane
protein ratio of 1:0.2 and incubated with AML12 cells for 2 h. CLs were
labeled by DiO and MMs were labeled by DiI, as visualized using a CLSM.
Scale bar: 50 µm. g) The FITC‐positive ratio of CLs‐FITC, MMs, and
MMCLs after co‐extruded at phospholipid‐to‐membrane protein ratio of
1:0.2 as detected by nano‐flow cytometry. h) The protein profile of
liposomes, MMs, and MMLs as measured by SDS‐PAGE.
To assess the stability of the NVs, CLs, and MMCLs were dispersed in
different solutions and monitored for prolonged periods. As shown in
Figure [93]1c, the particle size of CLs and MMCLs slightly decreased
from 156 to 140 nm after 30 days of storage at 4 °C in phosphate buffer
solution (PBS) or Dulbecco's modified Eagle's medium (DMEM), while the
polydispersity index (PDI) remained consistently below 0.3,
demonstrating their good stability and dispersibility. Also, the
suspensions of CLs and MMCLs exhibited a strong Tyndall effect^[
[94]^18 ^] and showed no visible precipitation after 30 days of storage
(Figure [95]S2, Supporting Information), confirming the colloidal
stability of the NVs. Furthermore, the physiological temperature might
impact the structure of NVs, and serum proteins that nonspecifically
adhere to the surface of nanoparticles might lead to tacrophage
clearance. After storage in 10% fetal bovine serum (FBS) at 37 °C for
7 days, CLs and MMCLs both maintained stable particle size and low PDI
(Figure [96]1d), providing the potential for a prolonged blood
circulation half‐life. The release pattern was also investigated in PBS
buffer (pH 7.4 containing 0.5% Tween 80) at 37 °C. Both CLs and MMCLs
showed 12% drug release within 6 h. Then 42% of CsA was released from
CLs at 48 h, whereas 21% of CsA was released from MMCLs, indicating
that cell membrane hybridizing delayed the drug release of MMCLs
(Figure [97]1e).
To determine the decoration degree of MMs onto CLs, the colocalization
between CLs and MMs was measured at different phospholipid‐to‐membrane
protein weight ratios of 1:0.025, 1:0.05, and 1:0.2, respectively.
Initially, CLs and MMs were labeled with two lipophilic dyes,
3,3′‐dioctadecyloxacarbocyanine perchlorate (DiO) and
1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate
(DiI), respectively, defined as CLs‐DiO and MMs‐DiI. After
co‐extrusion, all groups were incubated with alpha mouse liver 12
(AML12) for 2 h and observed using a confocal laser scanning microscope
(CLSM). Obviously, as the proportion of MMs increased, there was
stronger DiI fluorescence in cells (Figure [98]1f; Figure [99]S3,
Supporting Information). Analysis of Pearson's correlation coefficient
(R) demonstrated that CLs‐DiO exhibited good colocalization with
MMs‐DiI at a phospholipid‐to‐membrane protein weight ratio of 1:0.2,
with an R‐value of 0.90, which was significantly higher than at ratios
of 1:0.05 (R = 0.71) and 1:0.025 (R = 0.55). Furthermore, to determine
the efficiency of membrane fusion, CLs labeled with DSPE‐PEG‐FITC
(CLs‐FITC) were co‐extruded with non‐labeled MMs at a ratio of 1:0.2 to
produce MMCLs. Nano‐flow cytometry analysis revealed that the
FITC‐positive ratio of CLs‐FITC was 100%. After fusion with
non‐fluorescent MMs, the FITC‐positive ratio of MMCLs decreased to
88.6% (Figure [100]1g). These results indicated that ≈90% MMs were
fully fused with CLs in the presence of a phospholipid‐to‐membrane
protein weight ratio of 1:0.2, which was determined as the appropriate
ratio for MMCLs preparation. Considering the crucial role of proteins
derived from MSCs, the protein remaining on the hybrid NVs was further
confirmed using sodium dodecyl sulfate‐polyacrylamide gel
electrophoresis (SDS‐PAGE). Compared with empty liposomes without
protein expression, the protein profiles of MSC membrane‐camouflaged
liposomes (MMLs) were completely consistent with that of MMs,
demonstrating that the total protein profiles of MMs were preserved
after co‐extrusion (Figure [101]1h). These findings provided strong
evidence for the successful decoration of CLs with the phospholipid
structure and protein profile of MMs, which is expected to impart
biomimetic properties to MMCLs.
2.2. Inflammatory Chemotaxis and Endocytosis Pathway of NVs
The innate immune plays a crucial role in response to cellular injury
during HIRI. Among these, pro‐inflammatory cytokines such as tumor
necrosis factor (TNF‐α) contribute to the activation of endothelial
cells, leading to increased permeability of the endothelium and the
recruitment and activation of leukocytes.^[ [102]^19 ^] It has been
observed that MSCs can also migrate through gaps and pores in the
activated endothelial cells during inflammation.^[ [103]^20 ^] To
explore the role of MMs in MSCs migration, we used liquid
chromatography‐tandem mass spectrometry (LC‐MS/MS) to perform protein
profiling of MMs and identified a total of 4456 high‐quality proteins.
Gene Set Enrichment Analysis (GSEA) showed the most highly enriched
categories in MMs included “Cell Adhesion,” “Cell Migration,” “Response
to Wounding,” and “Response to Cytokine” (Figure [104]2a; Figure
[105]S4, Supporting Information), indicating that a series of membrane
proteins were involved in the inflammatory chemotaxis process of MSCs
to the injury site. To confirm whether MMCLs decorated with MMs possess
similar properties to MSCs, human umbilical vein endothelial cells
(HUVEC) were cultivated in the upper chamber of a Transwell to form an
in vitro model of transendothelial migration. TNF‐α was added in the
bottom chamber to simulate inflammation‐activated HUVEC, and the mRNA
expression was assessed before and after TNF‐α treatment. The addition
of TNF‐α significantly increased the levels of adhesion molecules, such
as intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell
adhesion protein 1 (VCAM‐1), as well as the pro‐inflammatory cytokine
interleukin‐1β (IL‐1β), while decreasing the level of the
anti‐inflammatory cytokine interleukin‐10 (IL‐10) (Figure [106]S5,
Supporting Information), confirming the successful activation of
HUVEC.^[ [107]^20 , [108]^21 ^] Whereafter, the culture medium in the
upper chamber was replaced with fresh medium containing PKH26‐labeled
CLs or MMCLs. As demonstrated in Figure [109]2b, the transmigration
efficiency of CLs, without MMs modification, showed no significant
difference in the presence or absence of TNF‐α, indicating that
cytokines did not affect their penetration behavior. In contrast, the
number of MMCLs that crossed the endothelial layer in the TNF‐α‐treated
(TNF‐α +) group was significantly higher compared to the
TNF‐α‐untreated (TNF‐α −) group and the CLs groups. Similarly, the
uptake of MMCLs was significantly more than that of CLs by hepatocytes
cultured in the bottom chamber, 1.8 and 2.9 times higher at 2 and 4 h,
respectively (Figure [110]S6, Supporting Information). These
observations verified the inflammatory chemotaxis and transendothelial
migration properties of MMCLs, which were primarily attributed to the
decorated with MMs containing chemokine receptors and adhesion
molecules.
Figure 2.
Figure 2
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Inflammatory chemotaxis and endocytosis pathway of CLs and MMCLs. a)
Gene Set Enrichment Analysis (GSEA) of MMs. Enrichment plots for
significant datasets enriched in GSEA analysis show the profile of the
normalized ES and false discovery rate (FDR) ratio. b) The amount of
NVs in the bottom chamber at 2, 4, and 8 h after the addition of
PKH26‐labeled CLs or MMCLs (n = 3). c) CXCR4 and VLA‐4 expression of
CLs, MMCLs, and MMs detected by western blot assay. Inhibition by
AMD3100 and BIO5192 of the suppression of transendothelial migration
capability of PKH26‐labeled d) CLs and e) MMCLs (n = 5). Flow cytometry
analysis and quantification of mean fluorescence intensity of
PKH26‐labeled f) CLs and g) MMCLs internalized by AML12 cells under
different endocytosis inhibitors (n = 3). Data are presented as
mean ± SD. The statistical significance was analyzed using one‐way
ANOVA following Tukey's multiple comparisons test (*p < 0.05;
**p < 0.01; ***p < 0.001, ns = no significance).
Among the membrane proteins, previous studies have shown that C‐X‐C
chemokine receptor type 4 (CXCR4) and very late antigen‐4 (VLA‐4) are
classical chemokine receptors and adhesion molecules expressed on MMs.
The onset of inflammation in injured tissue causes the release of
cytokines which upregulate VCAM‐1 and activate VLA‐4, leading to the
initial arrest of MSCs on the endothelium surface.^[ [112]^22 ^]
Thereafter, MSCs traverse through the endothelial cells and migrate
toward injured sites following chemokine gradients, such as stromal
cell‐derived factor 1α (SDF‐1α), mediated by the CXCR4/SDF‐1α
pathway.^[ [113]^23 ^] Western blot images showed that the expression
of VLA‐4 and CXCR4 in MMs was largely preserved during the preparation
of MMCLs, but negative in CLs (Figure [114]2c). AMD3100, an inhibitor
of CXCR4,^[ [115]^24 ^] and BIO5192, an inhibitor of VLA‐4,^[ [116]^25
^] were applied in the in vitro model of transendothelial migration to
verify their effective role in inflammatory chemotaxis. As demonstrated
in Figure [117]2d, the addition of TNF‐α and inhibitors AMD3100 and
BIO5192 had no significant effect on the transendothelial ability of
CLs, which was ≈20%. Conversely, within the inflammatory environment,
AMD3100 and BIO5192 independently decreased the transendothelial
migration of MMCLs by 35% and 11%, respectively, and collectively
reduced migration by 41% when combined (Figure [118]2e). These results
proved that chemokine receptors represented by CXCR4, and adhesion
molecules represented by VLA‐4 play an important role in inflammatory
chemotaxis and transendothelial migration properties of MMCLs, which
can enable them to target injured liver cells within the bloodstream
more rapidly.
Furthermore, various chemical inhibitors were applied to investigate
the endocytic pathways of CLs and MMCLs by flow cytometry analysis. As
shown in Figure [119]2f,g, the cellular uptake of both NVs was
significantly inhibited by dynasore, which is a dynamin‐dependent
endocytosis inhibitor related to caveolin‐dependent and
clathrin‐mediated pathways.^[ [120]^26 ^] To separately elaborate the
function of clathrin and caveolin in the endocytosis, chloropromazine
(CPZ, clathrin‐mediated endocytosis inhibitor) and genistein
(caveolae‐dependent endocytosis inhibitor) were used, and the
quantification of fluorescence intensity hinted that the inhibition
rate of CPZ was basically the same as dynasore, while genistein had no
significant inhibition on cell uptake. The results revealed that the
clathrin‐mediated pathway could be a domain route of CLs and MMCLs that
entry into the cells whereas caveolin‐dependent mechanisms were not
favored. In addition, there was also no significant inhibition of cell
uptake by amiloride, which is a Na^+/H^+ pump‐related macropinocytosis
inhibitor.^[ [121]^27 ^] Taken together, these results indicated that
CLs and MMCLs followed the same internalization pathway, and
clathrin‐mediated endocytosis played the most significant role in
hepatocyte uptake.
2.3. Biodistribution and Pharmacokinetic Behavior of CsA‐loaded NVs in a HIRI
Mouse Model
A HIRI mouse model was established to evaluate the feasibility of a
bionic membrane‐assisted delivery to the injured liver.^[ [122]^28 ^]
To track the biodistribution of the NVs, the commercially available
tracer 1,1′‐dioctadecyl‐3,3,3,3′‐tetramethyl‐indotricarbocyaine iodide
(DiR) was used for labeling. It was determined that the supernatant
after DiR labeling had no fluorescence, both in vivo and ex vivo,
indicating that the free dye did not interfere with the imaging process
(Figure [123]S7, Supporting Information). Thereafter, 200 µL of
DiR‐labeled CLs or MMCLs were injected into HIRI mice via the tail
vein, and fluorescence imaging was conducted at various time points (2,
6, 24, and 48 h) following reperfusion. The results showed that MMCLs
were initially enriched in the liver within 2 h after reperfusion and
then decreased over time (Figure [124]3a). Quantitative analysis of the
ex vivo tissue fluorescence demonstrated a higher accumulation of MMCLs
in the liver than the CLs group, with 2.7‐ and 2.8‐fold higher
accumulation at 2 and 6 h, respectively (Figure [125]3b,c). Similarly,
immunofluorescence images of liver sections taken 2 h after reperfusion
revealed an increased number of MMCLs entering hepatocytes labeled with
cytokeratin‐18 (CK18) antibody, in comparison to CLs (Figure [126]S8,
Supporting Information). These observations confirmed that MMCLs
possessed a strong ability of chemotaxis toward the injured liver.
Interestingly, as the fluorescence of MMCLs decreased in the liver over
time, it gradually increased in the lung. After 24 and 48 h, the lung
distribution of MMCLs was 2.8‐ and 1.8‐fold higher, respectively,
compared to CLs (Figure [127]3d,e). Previous studies have revealed that
the lung is the first organ to encounter reperfusion from the
bloodstream following IRI in the liver. The increased oxidative stress
and release of inflammatory cytokines play a critical role in mediating
acute lung injury, which is a common postoperative complication after
liver transplantation.^[ [128]^29 ^] Therefore, we hypothesized that
the significantly higher distribution of MMCLs in the lung, in
comparison to CLs, could be attributed to the inflammatory chemotaxis
induced by MMs, which facilitated the NVs to penetrate the endothelium
readily under inflammatory conditions.^[ [129]^20 ^] This hypothesis,
however, warrants further investigation.
Figure 3.
Figure 3
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Biodistribution and pharmacokinetic behavior of CsA, CLs, and MMCLs in
a HIRI mice model through tail vein injection. a) Ex vivo tissue
distribution of DiR‐labeled CLs and MMCLs in the main organs at
different time points and the corresponding fluorescence signals of
liver, lung, and spleen at b) 2 h, c) 6 h, d) 24 h, and e) 48 h after
reperfusion (data are analyzed using two‐tailed t‐test, n = 5,
*p < 0.05; **p < 0.01; ***p < 0.001; and ns, no significant
difference). f) Immunofluorescence images of liver and spleen sections
after intravenous administration of PKH26‐labeled CLs or MMCLs, with
cell nuclei stained blue (DAPI) and macrophages stained green (F4/80).
Scale bar: 50 µm. Quantification of the concentration of CsA in g)
blood, h) liver, i) lung, and j) spleen after injection of free CsA,
CLs, or MMCLs (at a CsA dose of 0.1 mg kg^−1) into HIRI mice or
sham‐operated mice (n = 3). The samples were homogenized and subjected
to quantification of CsA concentration through LC‐MS analysis.
Conversely, CLs showed an abundant uptake by the spleen 2 h after
reperfusion, which can be directly observed by in vivo fluorescence
imaging (red arrows; Figure [131]S9, Supporting Information). This
tendency became even more pronounced at 24 and 48 h, reaching 3.1‐ and
4.3‐fold higher levels, respectively to MMCLs (Figure [132]3d,e). As
reported previously, the liver and spleen both play important roles in
immune surveillance and act as the blood‐cleansing device in the MPS.^[
[133]^30 ^] Opsonins present in the blood serum quickly bind to foreign
materials, enabling macrophages to easily recognize and remove these
nanoparticles.^[ [134]^14 , [135]^31 ^] Consequently, the accumulation
of NVs in the macrophages of the spleen and liver (Kupffer cells)
hinders the delivery of a sufficient dose of NVs to the destination.^[
[136]^13 ^] Therefore, the macrophages were stained with F4/80 antibody
to investigate the detailed distribution of CLs and MMCLs in the liver
and spleen sections. In the immunofluorescence images of liver and
spleen sections (Figure [137]3f), the PKH26‐labeled CLs were completely
colocalized with F4/80‐labeled cells, suggesting that the CLs were
phagocytosed by the hepatic and splenic macrophages. Whereas MMCLs were
hardly phagocytosed by Kupffer cells and splenic macrophages but
instead entered the hepatocytes, and the overall fluorescence intensity
of MMCLs in liver sections was much higher than that of CLs, which was
consistent with ex vivo imaging. These results were attributed to the
biomimetic functionalization of MMCLs with MMs, which disguised the
MMCLs as stem cells, preventing recognition by macrophages, and thereby
exhibiting a stronger ability to escape from the splenic and hepatic
MPS.
To determine if the pharmacokinetic behavior and distribution of CsA
were consistent with fluorescently labeled NVs, equivalent CsA dosage
(0.1 mg kg^−1) of free CsA, CLs, and MMCLs were injected into HIRI
mice, and the samples of blood and major organs were collected at 2, 6,
12, 24, and 48 h after reperfusion. The samples were homogenized and
subjected to quantification of CsA concentration through LC‐MS
analysis. It was found that the higher and more constant CsA
accumulation (120.8 ng mL^−1 at 2 h and 88.6 ng mL^−1 at 6 h) was
achieved in the blood of the MMCLs‐treated HIRI mice compared to free
CsA (31.5 ng mL^−1 at 2 h) and CLs (75.7 ng mL^−1 at 2 h and 24.1 ng
mL^−1 at 6 h). The elimination half‐life (t [1/2]) for CsA, CLs, and
MMCLs in HIRI mice were 4.0, 4.1, and 7.6 h, respectively
(Figure [138]3g). The prolonged blood circulation time could be
attributed to the biomimetic functionalization of MMCLs with MMs,
enhancing sustained drug release and reducing the clearance by MPS. In
the IR‐injured liver, CsA accumulation in the MMCLs‐treated mice was
2.8‐ and 2.7‐fold higher than that of the CLs‐treated mice
(Figure [139]3h), consistent with the fluorescence distribution of NVs.
In contrast, the concentration of CsA in the liver of sham‐operated
animals was negligible following the MMCLs treatment (Figure [140]3h).
Moreover, CsA was specifically enriched in the lung at 6 h
(Figure [141]3i). Together, these results demonstrated the inflammatory
chemotaxis of MMCLs induced by MMs. In addition, the content of CsA in
the spleen of MMCLs‐treated mice was lower than that of CLs‐treated
mice (Figure [142]3j), which was related to the low immunogenicity of
MMs. The dual capabilities of inflammation chemotaxis and macrophage
escape dramatically improved the biodistribution of MMCLs in the liver
of HIRI mice, concentrating them in the injured lesions. This enhanced
biodistribution is expected to improve the bioavailability of drugs
delivered by administrating MMCLs.
2.4. MMCLs Restored Mitochondrial Dysfunction in Hypoxia/Reoxygenation
(H/R)‐Injured Hepatocytes
After confirming that injured hepatocytes efficiently take up MMCLs
both in vitro and in vivo, we proceeded to study the protection
potential of MMCLs in H/R‐injured AML12 cells. To assess the
dose‐dependent response of drugs, we evaluated the viability of
H/R‐pretreated AML12 cells using a cell counting kit‐8 (CCK‐8) after
incubating with free CsA, CLs, or MMCLs at CsA concentrations ranging
from 0 to 500 ng mL^−1. As a control, AML12 cells cultured under normal
conditions were used. Subjecting these cells to 8 h of hypoxia followed
by 6 h of reoxygenation resulted in a significant decrease in cell
viability to 42.9% (Figure [143]4a). However, when treated with MMCLs
at a CsA concentration of 0.1 ng mL^−1, H/R‐induced cell death was
rescued, and cell viability increased to 88.5%. This protective effect
was superior to that of CsA or CLs at the same CsA concentration. The
enhanced efficacy of MMCLs may be attributed to their efficient uptake
by hepatocytes, which triggered the protection action of CsA during the
early stages of mitochondrial damage. Furthermore, we observed that
higher concentrations of CsA treatment led to a decrease in cell
viability and exhibited minimal protective effects beyond 50 ng mL^−1.
This finding indicated that CsA not only possesses protection benefits
but also exhibits significant cytotoxicity.^[ [144]^32 ^] This
dose‐dependent response underscores the importance of controlling the
CsA dosage to achieve the desired curative effect. Consequently, a CsA
concentration of 0.1 ng mL^−1 was selected for subsequent in vitro
experiments to validate its efficacy.
Figure 4.
Figure 4
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Verification of the protection effect of MMCLs on H/R‐injured AML12
cells in vitro. a) Cell viability of H/R‐injured cells after being
treated with free CsA, CLs, and MMCLs at CsA concentrations of 0 −
500 ng mL^−1 as evaluated by CCK‐8 assay. b) JC‐1 mitochondrial
membrane potential in normal cells and H/R‐injured cells following the
treatment with CsA, CLs, and MMCLs as imaged by CLSM (scale bar:
50 µm), and c) the corresponding fluorescence intensity ratio of JC‐1
polymeride and monomer (n = 5). d) Flow cytometry analysis of the
superoxide levels using MitoSOX Red staining, and e) the corresponding
positive ratio of MitoSOX Red (n = 5). f) CLSM images of AML12 cells
stained by DHE (scale bar: 50 µm), and g) the corresponding
fluorescence intensity of intracellular ROS (n = 5). h) HCI analysis of
cell death using PI staining (scale bar: 200 µm) and i) the
corresponding positive ratio of PI (n = 5). j) Flow cytometry analysis
of early and late apoptosis by annexin V‐FITC/PI dual staining assay,
and k) the corresponding rates of early and late apoptosis (n = 5).
Data are presented as mean ± SD. The statistical significance was
analyzed using one‐way ANOVA following Tukey's multiple comparisons
test (*p < 0.05; **p < 0.01; ***p < 0.001, ns = no significance).
First, we evaluated the mitochondrial function using JC‐1 mitochondrial
membrane potential assay. An increase in the JC‐1 monomer signal
indicates a decrease in mitochondrial membrane potential and early
apoptosis of cells. AML12 cells were subjected to H/R treatment and
then incubated with free CsA, CLs, or MMCLs for 4 h. The mitochondrial
membrane potential after drug treatments was significantly higher than
that in the H/R group, with MMCLs demonstrating a more pronounced
effect in maintaining mitochondrial homeostasis compared to CsA and CLs
(Figure [146]4b,c). Mitochondria are known as the primary site of
superoxide generation, where various antioxidant systems maintain a
balance under normal conditions but become insufficient during
ischemia/reperfusion insult.^[ [147]^33 ^] Considering that excessive
superoxide formation can lead to damage to mitochondrial DNA, proteins,
and lipids, inhibition of superoxide overproduction is crucial for the
suppression of IRI.^[ [148]^34 ^] Therefore, the levels of superoxide
in the H/R‐injured cells were assessed using a mitochondrial superoxide
indicator (MitoSOX Red) after treatment with free CsA, CLs, or MMCLs.
Compared to the control group with a MitoSOX Red positive ratio of
8.63%, H/R treatment induced the production of large amounts of
superoxide, resulting in a ratio of 16.38%. However, the addition of
MMCLs significantly downregulated the superoxide level in H/R‐injured
cells, reaching a MitoSOX Red level (9.08%) similar to that of the
control group. This effect was distinct from the treatments with free
CsA and CLs treatments (Figure [149]4d,e). Furthermore, the total
levels of ROS in the different groups were also investigated using a
dihydroethidium (DHE) assay kit. CLSM observations revealed that MMCLs
significantly reduced the ROS level in the H/R‐injured cells compared
to CsA and CLs (Figure [150]4f,g), effectively mitigating the damage to
cellular activity caused by by‐products of mitochondrial dysfunction.
These results demonstrated that MMCLs can efficiently maintain
mitochondrial membrane potential and reduce the production of
superoxide and ROS.
Building upon the aforementioned results, we further evaluated the
reparative effect of MMCLs on injured cells using propidium iodide (PI)
staining, visualized through a high‐content imaging (HCI) system. As
shown in Figure [151]4h,i, with the dual regulation of mitochondrial
function and ROS production, MMCLs reduced the positive rate of PI
staining from 34.1% to 5.5% in H/R‐injured AML12 cells. This reduction
was lower compared to the CsA (15.8%) and CLs (10.1%) treatment groups.
Similarly, the annexin V‐FITC/PI dual staining assay demonstrated that
the percentage of early and late apoptosis was the lowest in the MMCLs
group, confirming its superior protection effect on H/R injury
(Figure [152]4j,k). However, in the group treated with an equivalent
protein dose of MMs, the cell viability and total apoptosis rate of the
injured hepatocytes did not significantly change, indicating that the
independent effect of MMs on H/R injury can be considered negligible
(Figure [153]S10, Supporting Information). Overall, MMCLs effectively
mitigated mitochondrial dysfunction and ROS production caused by H/R
treatment, leading to a significant reduction in apoptotic hepatocytes
and contributing to the improved recovery of hepatic function in HIRI.
2.5. MMCLs Effectively Alleviated the Hepatic Injury In Vivo
Subsequently, the protection efficacy of free CsA, CLs, and MMCLs was
compared in a HIRI mouse model. The experiment involved different
groups, including a sham‐operated group of healthy mice (Sham) as a
control. As depicted in Figure [154]5a, the experimental groups
involved intravenous injection of PBS, CsA, CLs, or MMCLs into the
mice. An atraumatic clip was used to interrupt the blood supply of
liver lobes for 90 min to simulate an ischemic period, and then blood
and liver samples were collected after 8 h of reperfusion to evaluate
liver function. The levels of aspartate aminotransferase (AST), alanine
aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured.
Increased levels of these indicators are direct indications of liver
injury.^[ [155]^35 ^] The results showed that the HIRI mice injected
with PBS (HIRI group) exhibited significant elevations in AST, ALT, and
LDH levels compared to the Sham group (Figure [156]5b), confirming
liver damage caused by the ischemia/reperfusion surgery. Then, the
protective dose of CsA and MMCLs was optimized (Figure [157]S11,
Supporting Information), showing that treated with CsA or CLs at a
dosage of 1 mg kg^−1 (CsA‐1 or CLs‐1) most effectively reduced AST,
ALT, and LDH levels, but lower dosage of 0.1 mg kg^−1 of CsA (CsA‐0.1)
and CLs (CLs‐0.1) did not show any effect on these indicator levels. In
comparison, treatment with MMCLs at a low CsA dosage of 0.1 mg kg^−1
(MMCLs‐0.1) significantly down‐regulated the three hepatic enzymes,
despite the injection amount being only 1/10 of the effective dosage
used for CsA and CLs (1 mg kg^−1). To exclude the influence of MMs on
liver injury, liver function indicators were measured in HIRI mice that
were solely injected with MMs at a protein dosage equivalent to
MMCLs‐0.1. It was found that AST and LDH levels of the MMs‐treated
group did not change significantly, while liver sections injected with
MMs exhibited large areas of hepatocyte necrosis (Figure [158]S12,
Supporting Information), indicating that the independent effect of MMs
on liver injury can be considered negligible. Previous studies have
shown that hepatocyte membrane injury, such as lipid peroxidation
induced by ROS, leads to increased membrane permeability and the
release of hepatic enzymes like ALT into the bloodstream.^[ [159]^36 ^]
Prolonged hepatic injury may result in necrosis and subsequent
liberation of mitochondrial AST into the blood.^[ [160]^37 ^] Moreover,
LDH production was increased under hypoxic conditions, enabling cells
to generate adenosine triphosphate and maintain viability in a
low‐oxygen environment.^[ [161]^38 ^] This evidence collectively
demonstrated the potential of MMCLs administration at a relatively low
dosage of CsA in effectively restoring liver function, including
improving cell membrane permeability, maintaining mitochondrial
homeostasis, and enhancing energy metabolism in mice with HIRI.
Figure 5.
Figure 5
[162]Open in a new tab
Alleviation of the hepatic injury by CsA, CLs, and MMCLs in a HIRI
mouse model. a) Schematic illustration for the drug treatments in HIRI
mice. C57BL/6 mice were intravenously injected with PBS, CsA, CLs, or
MMCLs, followed by an I/R procedure, and then the blood and liver
samples were collected 10 h after injection. A sham‐operated group of
healthy mice (Sham) was set as a control. b) Evaluation of liver
functions of AST, ALT, and LDH after administration of PBS, CsA, CLs,
or MMCLs at a CsA dosage of 0.1 or 1 mg kg^−1 (n = 5). c) H&E staining
and immunohistochemical staining of liver sections in different
treatment groups. NA, necrotic area. Macrophages were stained using
F4/80 and neutrophils using Ly6G. d) Suzuki scores of the H&E staining
(n = 5). e) TUNEL images visualized by a panoramic scanning microscope
and the enlarged images of the red boxes. f) The mRNA expression of
inflammatory cytokines and chemokines in hepatic tissues was measured
by RT‐qPCR (n = 5). Percentage of g) macrophages (F4/80+) and h)
neutrophils (Ly6G+) in total cells of liver sections after the
indicated treatments (n = 5). Data are presented as mean ± SD. The
statistical significance was analyzed using one‐way ANOVA following
Tukey's multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001,
ns = no significance).
Due to the significant impact of HIRI on liver dysfunction and
subsequent cell death, the extent of hepatocyte necrosis in the
vicinity of liver lesions was assessed using hematoxylin−eosin (H&E)
staining. Necrotic areas (NA), characterized by apoptotic and dead
cells with crumbled nuclei and reddish cytoplasm, were visually
distinguishable from healthy cells by a distinct boundary (black dashed
line). To ensure a fair comparison of protection effects, all
subsequent experiments were conducted using a consistent CsA dosage of
0.1 mg kg^−1 across CsA, CLs, and MMCLs groups. Liver sections from
HIRI mice injected with PBS, CsA, and CLs exhibited significant areas
of hepatocyte necrosis (Figure [163]5c). However, the administration of
MMCLs markedly ameliorated liver damage with fewer areas of hepatocyte
necrosis and a clear reduction in the Suzuki score (1.8 ± 0.8) compared
to the HIRI group (4.8 ± 1.1) (Figure [164]5d). Also, a terminal
deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end labeling
(TUNEL) assay was employed to demonstrate the overall impact of
different drug treatments on the proportion of apoptosis cells labeled
green (Figure [165]5e), indicating that MMCLs most effectively mitigate
necrosis and apoptosis of hepatocytes in HIRI at a low dosage of 0.1 mg
kg^−1.
In fact, HIRI is a local sterile inflammatory response driven by innate
immunity.^[ [166]^39 ^] Assessing the inflammatory immune response of
the liver before and after drug intervention is critical to
understanding the occurrence and progression of liver damage.
Therefore, the expression of inflammatory cytokines and chemokines, as
well as activation and recruitment of macrophages and neutrophils in
liver tissue were evaluated. As shown in Figure [167]5f, the mRNA
levels of pro‐inflammatory cytokines (TNF‐α, tumor necrosis factor‐α;
IL‐1β, interleukin‐1β; IL‐6, interleukin‐6; IL‐18, interleukin‐18) and
chemokines (CCL12, C‐C chemokine ligand 12; CCL25, C‐C chemokine ligand
25) were strikingly upregulated after IR injury. The macrophage
(F4/80+) and neutrophil (Ly6G+) infiltration were investigated by
immunohistochemical staining, showing a marked increase in macrophages
and neutrophils in liver sections taken from the HIRI group compared to
the Sham group (Figure [168]5c). According to previous research,
injured hepatic cells release damage‐associated molecular patterns
(DAMPs), which can activate macrophages (Kupffer cells) that in turn
secrete inflammatory cytokines. This contributes to further hepatocyte
injury and the release of chemokines, which promote the recruitment of
C–C motif chemokine receptor 2+ (CCR2+) neutrophils into the
IR‐stressed liver and trigger parenchymal cell damage.^[ [169]^40 ^] In
this work, treatment of MMCLs significantly lowered TNF‐α, IL‐1β, IL‐6,
and IL‐18 expression, revealing the most effective anti‐inflammatory
function compared to CsA and CLs (Figure [170]5f). Concomitantly, MMCLs
strikingly decreased the expression of chemokines and inflammatory cell
infiltration, where the number of macrophages and neutrophils in liver
sections decreased by 49% and 90%, respectively (Figure [171]5g,h).
These results demonstrated that MMCLs can alleviate IR‐induced liver
damage by attenuating the innate inflammatory response.
Considering the importance of the biosafety of drugs in clinical
application, histomorphological observations and functional assessments
of major organs were performed in healthy mice after injection with
CsA, CLs, or MMCLs at their effective protective dosage. Notably, no
significant changes in cellular morphology were observed in the liver,
kidney, heart, and lung following treatment with different drugs
(Figure [172]S13, Supporting Information). However, AST and ALT of
liver function indicators, creatinine of kidney function indicators, as
well as creatine kinase (CK) of heart function indicators were
significantly higher than that of healthy mice with PBS after
administration of CsA (Figure [173]S14, Supporting Information). Also,
AST, ALT, CK, and CK‐MB values were increased in the CLs‐treated group,
indicating that the CsA dose of 1 mg kg^−1 presented liver, kidney, and
cardiac toxicity to some extent. In comparison, there was no
significant change in all the above indicators in the MMCLs‐treated
group, proving the superior biosafety at a protective dose of 0.1 mg
kg^−1 of MMCLs. Consequently, MMCLs hold significant potential as a
highly effective delivery system, capable of minimizing side effects,
widening the treatment window, and enhancing the clinical applicability
of CsA in HIRI.
2.6. Protection Mechanism of MMCLs on HIRI Mice
Proteomic analysis of mouse liver tissues was performed to investigate
the molecular mechanisms of MMCLs treatment on HIRI. Liver tissues from
the Sham group, HIRI group, and MMCLs treatment group were analyzed
using LC‐MS/MS, identifying a total of 3215 high‐quality proteins.
Among these 2776 proteins were detectable in all three groups (Figure
[174]6a). Principal component analysis revealed significant differences
in protein expression between the HIRI group and the other groups,
while the protein expression profile of the MMCLs treatment group
closely resembled that of the Sham group (Figure [175]6b). These
findings suggested a notable influence of IRI on hepatic protein
expression, which was effectively attenuated by the MMCLs treatment.
Figure 6.
Figure 6
[176]Open in a new tab
Protein profile analysis of liver samples from the Sham group, HIRI
group, and MMCLs treatment group. a) Venn diagram showing the overlap
of high‐quality proteins. b) Principal components analysis of the
enriched proteins. c) Volcano diagram showing the differently expressed
proteins between the HIRI and the MMCLs treatment group, fold change >
1.2 or < 0.8, p < 0.05. Functional enrichment analysis of the
differently expressed proteins d) upregulated or e) downregulated in
the MMCLs treatment group compared to the HIRI group. f) Heatmap
generated by clustering of the differentially expressed proteins in the
three groups. Red: up‐regulation; blue: downregulation. The expression
quantity of the corresponding proteins enriched in positive regulation
of g) mitochondrial fission and h) cellular apoptosis. Data are
analyzed using a two‐tailed t‐test, *p < 0.05; **p < 0.01;
***p < 0.001.
Differential protein expression analysis identified 126 differentially
expressed proteins between the HIRI and Sham groups, encompassing 70
upregulated and 56 downregulated proteins (Figure [177]S15, Supporting
Information). Through pathway enrichment analyses, it was found that in
the context of HIRI, an upregulation occurred in pathways such as
“respiratory electron transport,” “ROS generation,” “mitochondrial
organization,” “ferroptosis,” “apoptotic mitochondrial changes,” and
“regulation of myeloid leukocyte mediated immunity” (Figure [178]S16a,
Supporting Information). In contrast, pathways related to “autophagy,”
“steroid metabolic process,” “mRNA splicing,” “biosynthesis of
nucleotides sugars,” “negative regulation of MAPK pathway,” and “IL‐6
signaling pathway” exhibited significant downregulation (Figure
[179]S16b, Supporting Information). These results confirmed that during
HIRI, oxidative stress, cell apoptosis, and inflammatory responses were
activated, while cellular metabolism and biosynthesis were suppressed,
which was consistent with previous research findings.^[ [180]^41 ^]
Comparing the MMCLs treatment group with the HIRI group, 548
differentially expressed proteins were identified, of which 272 were
upregulated and 276 were downregulated (Figure [181]6c). Pathway
enrichment analysis revealed that MMCLs treatment group upregulated
pathways related to “carbohydrate catabolic processes,” “protein
catabolic processes,” “RNA metabolism,” “mRNA splicing,” “small
molecule biosynthetic processes,” “sister chromatid separation,” and
“mitotic anaphase” (Figure [182]6d). Conversely, pathways related to
“ROS,” “respiratory electron transport,” “assembly of mitochondrial
respiratory chain complex I,” “formation of neutrophil extracellular
traps,” “neutrophil degranulation,” “PPAR signaling pathways,” and
“ferroptosis” were found to be downregulated (Figure [183]6e). Among
these, the generation of ROS was observed to be the most prominently
downregulated pathway.
Elevated levels of proteins associated with the ROS signaling pathway
in the HIRI group were mitigated following MMCLs treatment
(Figure [184]6f). The majority of these proteins constitute the
mitochondrial electron transport chain,^[ [185]^42 ^] the primary locus
for ROS generation in IRI.^[ [186]^34 , [187]^43 ^] Elevated ROS levels
can induce mitochondrial oxidative stress leading to mitochondrial
damage characterized by excessive fission and abnormal function.^[
[188]^44 ^] In the HIRI group, proteins like Drp1 and Fis1, which
regulate mitochondrial fission,^[ [189]^45 ^] exhibited increased
expression, but this was attenuated following MMCLs treatment
(Figure [190]6g). Likewise, the expression of Bax and Bid, proteins
implicated in apoptosis,^[ [191]^46 ^] was diminished in the MMCLs
treatment group (Figure [192]6h). These results convincingly
demonstrated that MMCLs treatment substantially inhibited the
generation of mitochondrial ROS subsequent to HIRI, thereby preserving
mitochondrial integrity by reducing mitochondrial fission and
alleviating subsequent cellular apoptosis. In summary, these findings
provided invaluable insights into the molecular mechanisms underlying
HIRI and underscored the protection potential of MMCLs.
3. Conclusion
Here, we developed biomimetic nanovesicles by hybridizing liposomes
with MSC membranes for targeted CsA delivery to achieve early
intervention in HIRI. By membrane fusion, MMCLs exhibited inflammatory
chemotaxis and immune privilege due to the inherited CXCR4 and VLA‐4
expression from MSC membranes, along with efficient drug loading of
liposomes. Upon in vivo study, MMCLs accumulated in the injured liver
at a level 2.7 times higher than that of liposomes, and conversely 2.5
times less engulfed by splenic macrophages than liposomes. This
biomimetic delivery system remarkably reduced liver damage and restore
function at a 1/10 dose of free CsA in a HIRI mouse model, with no
significant side effects. Proteomic analysis revealed the protection
mechanism of MMCLs, including the downregulation of the proteins
associated with ROS production, reducing mitochondrial fission to
maintain mitochondrial integrity, and ultimately alleviating cellular
apoptosis. These findings provide strong evidence supporting the
potential of MMCLs as an efficient protection option for HIRI
prevention and highlight the versatility of MSC‐mediated biomimetic
nanovesicles as a flexible nanoplatform for injury‐targeted drug
delivery in vivo.
4. Experimental Section
Culture of Cell Lines
AML12 cells and HUVEC were purchased from Guangzhou Jennio Biotech
Co.,Ltd (Guangzhou, China). AML12 cells were cultured in DMEM: F12
medium supplemented with 10% FBS, 10 µg mL^−1 insulin, 5.5 µg mL^−1
transferrin, 5 ng mL^−1 selenium, and 40 ng mL^−1 dexamethasone. HUVEC
were incubated in a commercial endothelial cell medium (ScienCell
Research Laboratories, Inc., USA). All cells were cultured at 37 °C in
a humidified 5% CO[2] incubator.
Isolation and Culture of UC‐MSCs
The procedures for isolating UC‐MSCs have been proved by the Human
Research Ethics Committee of the Third Affiliated Hospital of Sun
Yat‐sen University. UC‐MSCs were isolated and cultured under aseptic
maintenance. Umbilical cords were collected after the donor agreed to
sign the consent form and washed twice using PBS to wipe out the
remnant blood. The umbilical cords were cut into 10 mm^3 piece^−1 and
placed in type I collagenase containing hyaluronidase (0.1%) and
CaCl[2](3 mm). After 4 h digestion at 37 °C, the umbilical cords were
transferred into low‐glucose (1 g L^−1) DMEM basic medium with 10% FBS
and cultured in the humidified atmosphere with 5% CO[2] at 37 °C.
Medium was refreshed every 3 days to remove nonadherent cells.
Membrane Derivation
Briefly, umbilical cord MSCs were grown to 80% confluence in multiple
T‐225 culture flasks and harvested, followed by washing in 1× PBS
thrice by centrifuging at 500 × g for 5 min. The purified cells
underwent freezing and thawing repeatedly from −20 to 37 °C three
times, followed by getting enucleated in a hand‐held Dounce homogenizer
(20 passes while on ice). After centrifugation at 3200 × g for 5 min at
4 °C, the supernatant was gathered and centrifuged at 15 000 × g for
30 min for membrane collection. The membrane fragments were
ultrasonicated (100 W) for 2 min and stored in 1× PBS at −80 °C for
subsequent experiments. The protein content in the purified MSC
membranes was determined by a bicinchoninic acid (BCA) protein
quantification kit (Beyotime, China) for further preparation of MMCLs.
Preparation and Characterization of CLs
CLs were prepared by a thin‐film hydration method. Phosphatidylcholine
(Ruixi, Xi'an, China), cholesterol (Sigma–Aldrich, USA), and CsA
(MedChemExpress, USA) (20:2.5:1, w/w) were dissolved in chloroform and
evaporated to form a thin film in a round flask. Subsequently, the film
was hydrated with 1× PBS at 40 °C for 30 min with a CsA concentration
of 0.5 mg mL^−1. Then, they were extruded with a Mini‐Extruder (Avanti
Polar Lipids, Inc., USA) using a polycarbonate membrane with a pore
size of 200 nm for ten cycles to develop CLs. To remove the free CsA,
CLs were placed in a dialysis bag (MWCO = 3500), submerged in 1× PBS,
and stirred at 100 rpm at 4 °C for 48 h.
Preparation and Characterization of MMCLs
To synthesize MMCLs, a modified extrusion approach was used according
to a previous report.^[ [193]^47 ^] Briefly, CLs were mixed with the
purified MSC membranes at the phospholipid‐to‐membrane protein weight
ratios of 1:0.025, 1:0.05, or 1:0.2, and then extruded through a
polycarbonate membrane with pore sizes of 400 and 200 nm. To remove the
free CsA, MMCLs were placed in a dialysis bag (MWCO = 3500), submerged
in 1× PBS, and stirred at 100 rpm at 4 °C for 48 h. Particle size and
zeta‐potential of CLs, MSC membranes, and MMCLs were measured by DLS
using an EliteSizer (Brookhaven, USA), and the morphology was observed
under a TEM (Talos L120C, ThermoFisher, USA) following uranyl acetate
(UA) staining.
Drug Loading and Release
To determine the encapsulation efficiency (EE) and loading capacity
(LC) of CsA in CLs, free CsA was separated by centrifugation at 8 000 ×
g for 15 min with an ultrafiltration tube (100 000 Da, Millipore). The
drug amount was quantified using an HPLC system (Waters 1525, USA)
under the following conditions: column, ZORBAX SB‐C18 column
(250 × 4.6 mm, 5 µm, Agilent); mobile phase, acetonitrile−methanol
(80:20, v/v); flow rate, 1.0 mL min^−1; detection wavelength, 210 nm;
column temperature, 70 °C. The EE and LC were calculated by the
following equations:
[MATH: EE%=1−C×n×VT×100% :MATH]
(1)
[MATH: LC%=T−C×n×VW+T−C×n×V×100% :MATH]
(2)
where C is the concentration measured of free CsA, n is dilution
multiple, V is the total volume, T is the quantity of reagent for CsA,
and W is the total weight of NVs.
To detect the releasing kinetic, CLs and MMCLs in PBS (1 mL) were
placed in a dialysis bag (MWCO = 3500) and submerged in a 300 mL
dissolution medium (PBS containing 0.5% Tween 80). All samples were
placed in a 37 °C orbital shaker and stirred at 100 rpm for 48 h. At
each time point, 1 mL dissolution medium was removed for HPLC analysis
and another 1 mL fresh medium was replenished.
Long‐Time Stability Measurement
1 mg mL^−1 CLs and MMCLs were suspended in 1× PBS, DMEM, and 10% FBS,
respectively, and stored at low temperature (4 °C) or physiological
temperature (37 °C) conditions. Their hydrodynamic size and PDI were
measured and monitored for a prolonged time by DLS analysis.
Evaluation of Membrane Fusion Between CLs and MSC Membranes
For DiO labeling of CLs, 20 µL of 100 µm DiO (Meilun, Dalian, China)
was dissolved in phosphatidylcholine‐containing chloroform to form the
thin film. For DiI labeling of MSC membranes, 1 µm DiI (Meilun, Dalian,
China) was used before the co‐extrusion process. A total of 5 × 10^4
AML12 cells were cultured in a 48‐well plate and treated with MMCLs
labeled with DiO and DiI. Following incubation for 2 h, cells were
washed with PBS and the colocalization between CLs and MSC membranes
was observed under the CLSM (LSM‐780, Carl Zeiss, Germany). The
Pearson's correlation coefficient was analyzed and measured with ImageJ
software.
In order to quantitatively evaluate the fusion efficiency, 10% of
DSPE‐PEG‐FITC (Ruixi, Xi'an, China) was dissolved in
phosphatidylcholine contained chloroform to form FITC‐labeled CLs
(CLs‐FITC). The free DSPE‐PEG‐FITC was separated by ultracentrifugation
in a SW41 Ti rotor (Beckman Coulter, USA) at 100 000 × g for 1 h. After
the co‐extrusion process, the fluorescence signal and particle count of
fused hybrid MMCLs were detected by a Flow NanoAnalyzer model (NanoFCM
Inc., Xiamen, China). According to the accurate count results of CLs,
MSC membranes, and MMCLs, the efficiency of membrane fusion was
evaluated.
Protein Analysis
The whole protein analysis of liposomes, MMs, and MMLs was performed by
SDS‐PAGE. Total proteins were extracted using RIPA lysates (Beyotime,
China) containing 1% protease inhibitor cocktail (APExBIO, USA) and
equivalent micrograms of proteins were mixed with 4× loading buffer
(Invitrogen, USA). All samples were heated at 100 °C for 10 min and
loaded onto 12% bis‐tris protein gels. Following electrophoresis at
100 V (Bio‐Rad, USA), staining was performed with Coomassie brilliant
blue (Biosharp, China). For western blot analysis, the protein was
transferred onto PVDF membrane (0.22 µm, Millipore) and treated with
primary antibodies against CXCR4 (60042‐1‐Ig, Proteintech, USA), and
VLA‐4 subunit alpha (19676‐1‐AP, Proteintech, USA), and HRP conjugated
anti‐rabbit IgG secondary antibody (ab205718, abcam, USA). Protein
bands were detected by a digital chemiluminescence system (Bio‐Rad,
USA).
Inducing the Inflammation of Vascular Endothelium In Vitro
HUVEC was treated with 100 ng mL^−1 TNF‐α (MedChemExpress, USA) for 4 h
to establish the in vitro model of inflamed vascular endothelium
according to the previous report.^[ [194]^48 ^] The mRNA expression of
ICAM‐1, VCAM‐1, IL‐1β, and IL‐10 were assessed by quantitative
real‐time PCR (qRT‐PCR). Before and after the TNF‐α treatment, HUVEC
was washed three times with PBS. Total RNA was extracted from the cells
by Trizol reagent and transcribed into cDNA for qRT‐PCR amplification.
Table [195]S1 (Supporting Information) lists the primers used in this
study, and the primers were synthesized by Sangon Biotech (Shanghai)
Co., Ltd.
Chemotactic Migration of MMCLs In Vitro
A Transwell assay was applied to evaluate the migration ability of CLs
and MMCLs. In brief, HUVEC was seeded on the upper chamber of the
transwell (0.4 µm, Corning, USA) at the cell density of 2 × 10^4 cells
per well and incubated overnight. Then, the serum‐free medium
containing 100 ng mL^−1 TNF‐α was substituted for the medium in the
bottom chambers for 4 h of activation. Meanwhile, CLs and MMCLs were
incubated with 1 µm of fluorescent lipophilic tracer PKH26
(Sigma–Aldrich, USA) with the provided buffer at room temperature prior
to ultracentrifugation at 100 000 × g for 1 h. Fluorescence was
quantified using the SpectraMax i3X multi‐mode microplate reader
(excitation at 560 nm, emission at 595 nm, cutoff at 590 nm).
Subsequently, HUVEC in the upper chamber were washed thrice with PBS
and incubated with PKH26‐labeled CLs or MMCLs with the same fluorescent
intensity. At time points of 2, 4, and 8 h, medium in the bottom
chamber was collected for the analysis of fluorescence intensity. The
ratio of the fluorescence intensity of the medium in the bottom chamber
to the initial fluorescence intensity of the medium in the upper
chamber after the addition of NVs was defined as the percentage of NVs
that crossed the endodermis. To investigate the abilities of hepatic
delivery of CLs and MMCLs after passing through the HUVEC monolayer,
HUVEC (2 × 10^4 cells per well) and AML12 cells (1 × 10^5 cells per
well) were seeded into the upper chamber and the bottom chamber of
Transwell plate, respectively. PKH26‐labeled CLs or MMCLs were added
into the upper chamber and incubated with HUVEC for 2 or 4 h. Then,
AML12 cells were washed three times using PBS and stained with Hoechst
33342. The fluorescence signal of cells was detected through CLSM
(LSM‐780, Carl Zeiss, Germany).
Inhibition Experiment
To investigate the roles of CXCR4 and VLA‐4 in chemotactic migration,
specific inhibitors AMD3100 (HY‐10046, MCE, USA) for CXCR4 and BIO5192
(HY‐107589, MCE, USA) for VLA‐4 were utilized. The experimental design
included control groups, AMD3100‐treated group, BIO5192‐treated group,
and a combination treatment group with both AMD3100 and BIO5192. An
inhibition experiment was conducted using an in vitro model of
transendothelial migration. PKH26‐labeled CLs and MMCLs were
pre‐incubated with AMD3100 at 10 µm or BIO5192 at 2 µm at 37 °C for
30 min. Subsequently, the NVs/inhibitor mixtures were loaded in the
upper chambers. 4 h later, the medium from the lower chamber was
collected and analyzed for fluorescence intensity to assess migration.
In Vitro Hepatocyte H/R Model
To induce in vitro ischemia injury, AML12 cells were incubated with
HBSS in an atmosphere containing 95% N[2] and 5% CO[2] at 37 °C to
mimic ischemia for 8 h. Reoxygenation was subsequently achieved by
replacing HBSS with a complete medium and placing cells in an incubator
containing 95% air and 5% CO[2] at 37 °C for 6 h.
Cell Viability Detection
AML12 cells were seeded in 96‐well plates at a density of 5 × 10^3 per
well. To evaluate the protective effect of free CsA, CLs, and MMCLs on
the H/R injured cells, drug treatment was carried out at the beginning
of reoxygenation. After incubation with a range of CsA concentrations
(0.1, 1, 10, 25, 50, 100, and 500 ng mL^−1) of free CsA, CLs, and
MMCLs, cell viability of the injured cells was assessed by a CCK‐8 kit
(BD Pharmingen, USA). Additionally, to clearly determine whether the
MMs offer protection against H/R injury, a separate group designated as
the MMs group was established. The quantity of MSC membrane proteins
used in this group is equivalent to that used in the corresponding
MMCLs group. The blank group was AML12 cells undergoing the same H/R
manipulation and treated with PBS instead of drugs. The absorbance at
450 nm was measured using a microplate reader (SpectraMax i3X,
Molecular Devices, USA). Cell viability was expressed as a percentage
relative to control cells without H/R treatment.
Measurement of Mitochondrial Integrity and Intracellular ROS Levels
The H/R injured AML12 cells were treated with PBS, free CsA, CLs, and
MMCLs with CsA concentration of 0.1 ng mL^−1 at the beginning of
reoxygenation. The control group was normal AML12 cells without H/R
manipulation and treated with PBS instead of drugs. JC‐1 fluorescence
probe (Beyotime, China) was performed to detect the mitochondrial
membrane potential according to the manufacturer's protocol. For
intracellular ROS analysis, a DHE fluorescence probe (MedChemExpress,
USA) was used according to the manufacturer's protocol, and the cell
nucleus was stained by Hoechst 33342. All samples were washed with PBS
and observed by CLSM (LSM‐780, Carl Zeiss, Germany). Also, the levels
of superoxide generated by mitochondria were assessed using MitoSOX Red
(ThermoFisher, USA), and the fluorescence signals in all samples were
analyzed by the flow cytometry (CytoFLEXLX, Beckman Coulter, USA).
Cell Apoptosis Detection
The H/R injured AML12 cells were treated with PBS, free CsA, CLs, MMs,
and MMCLs with CsA concentration of 0.1 ng mL^−1 at the beginning of
reoxygenation.
The quantity of the membrane proteins in MMs group was comparable to
that of the MMCLs group. The control group was normal AML12 cells
without H/R manipulation and treated with PBS instead of drugs. To
detect cell apoptosis after drug treatment, all cells were stained
using the Calcein‐AM/PI double staining kit BestBio (Shanghai, China).
Then all cells were analyzed through a high‐content analysis (HCA)
system (PerkinElmer, USA). Furthermore, the annexin V‐FITC/PI dual
staining kit (BD Pharmingen, USA) was used to detect the early and late
apoptosis of the above samples according to the manufacturer's
protocol. The fluorescence signals of cells were analyzed by flow
cytometry (CytoFLEXLX, Beckman Coulter, USA). The excitation/emission
wavelengths were 488 nm/525 nm for FITC and 535 nm/615 nm for PI.
Animals
C57BL/6 mice (male, 6−8 weeks old, weighting ≈20 g) were purchased from
Guangdong Yaokang Biotechnology Co., Ltd. (Guangdong, China). All mice
were housed in the Experimental Animal Center of Ruiye Model Animal
(Guangzhou) Biotechnology Co., Ltd (China) under specific pathogen‐free
(SPF) conditions and were given care according to the Guideline of Sun
Yat‐sen University for Animal Experimentation. Animals received free
access standard laboratory diet and water, which were maintained in a
constant environment, 50% humidity and 20 °C temperature, 12 h dark and
light cycle.
HIRI Mouse Model
All experimental processes of animals abided by the National Institutes
of Health Guide for the Care and Use of Laboratory Animals and were
approved by the Experimental Animal Ethics Committee of Ruiye Model
Animal (Guangzhou) Biotechnology Co., Ltd, China, approval no.
RYEth‐20230607256. The standard protocol of a mouse HIRI model was
conducted as previously described.^[ [196]^28 ^] Briefly, mice were
anesthetized with intraperitoneally 1% pentobarbital sodium at a dose
of 30 mg kg^−1. An atraumatic clip was used to interrupt the
artery/portal vein blood supply to the left and middle liver lobes for
an ischemic period of 90 min. Then, the atraumatic vascular clamp was
removed for reperfusion. Sham‐operated mice underwent the identical
procedure except for artery/portal vein occlusion.
In Vivo Biodistribution of CLs and MMCLs
C57BL/6 mice conducted with HIRI operation were averagely dived into
CLs and MMCLs groups (n = 5 per group). CLs and MMCLs were both labeled
with 1 µm DiR fluorescence dye (Meilun, Dalian, China) and then
injected into mice via the inferior vena cava immediately after the
initiation of reperfusion. After different time intervals (2, 6, 24,
and 48 h), in vivo, fluorescence signals were examined using the In
Vivo Xtreme II small animal imaging system (Bruker, Germany). After
that, the mice were euthanatized, and major organs including the heart,
lung, liver, spleen, and kidney were separated for ex vivo imaging of
the nanoparticles using the same imaging system. For immunofluorescence
imaging, CLs and MMCLs were both labeled with 1 µm PKH26
(Sigma–Aldrich, USA) and then injected into mice via inferior vena cava
immediately after the initiation of reperfusion. After 2 h, parts of
the liver and spleen of the two groups were isolated from mice and
embedded in an optimal cutting temperature compound (Servicebio, China)
and stored at −80 °C. The frozen liver and spleen slices were obtained
by a frozen slicer (Leica CM1950). After washing with PBS, sections
were stained with DAPI for 15 min. Images of sections were acquired
with CLSM (LSM 510, Carl Zeiss).
Determination of CsA Using LC‐MS
Sample preparation: Blood (100 µL for each) and fresh tissues (50 mg
for each) of major organs (lung, liver, and spleen) were collected from
mice post‐euthanasia. After washing with ice‐cold PBS to remove blood
and debris, tissues were homogenized in 1 mL of 0.05 m ZnSO[4]
extraction solution with methanol: water ratio of 1:1. The homogenate
was chilled at −20 °C for 1 h followed by centrifuge at 14 000 rpm for
10 min at 4 °C. Collected 500 µL of the supernatant for concentration.
The concentrated extract was re‐dissolved in 100 µL methanol and
centrifuged at 14 000 rpm for 10 min at 4 °C to remove any particles.
Standard Preparation: 1 mg CsA standard sample was accurately weighed
into a 1 mL volumetric flask, dissolved in methanol, ensuring thorough
mixing to achieve a 1 mg mL^−1 stock solution, and stored at −80 °C.
This stock was further diluted with methanol to create working
solutions with varying concentrations. LC‐MS Analysis: The system used
a Thermo Hypersil GOLD C18 column (2.1 × 100 × 1.9 µm, Thermo
Scientific, USA). Mobile phase A was 0.1% formic acid in water and
mobile phase B was a 1:1 mix of acetonitrile and isopropanol. The
gradient started at 5% B, increased to 80% B over 4 min, held for
4 min, and then returned to 5% B within 2 min. The flow rate was 0.2 mL
min^−1, and the column temperature was 60 °C. Mass spectrometry was
performed using an electrospray ionization (ESI) source in positive ion
mode. The transitions monitored for CsA were m/z 1220 → 1202.839.
Cytokines Measurement and Liver Function Evaluation
The blood of mice was collected into a glass container and allowed to
clot at room temperature for 2 h. Serum was obtained by centrifugation
at 150 × g for 5 min to sediment erythrocytes and then at 350 × g for
15 min. An automatic biochemical analyzer (7180‐ISE, Hitachi, Japan)
was applied to examine the serum levels of AST, ALT, and LDH.
H&E and Immunohistochemical Staining
Hepatic tissues were fixed in 4% paraformaldehyde, followed by
dehydration in an ethanol gradient. After paraffin embedding, the
samples were sectioned into 5 µm thickness slices and were processed
for H&E staining. Histopathological evaluation was conducted using the
Suzuki Score System^[ [197]^49 ^] and examined in a blinded fashion by
an experienced pathologist (Table [198]S2, Supporting Information).
Immunostaining for F4/80 and Ly6G was carried out on paraffin sections
using F4/80 antibody (Abcam, ab16911) and Ly6G antibody (Abcam,
ab25377). Then they were developed using a biotinylated alkaline
phosphatase‐conjugated secondary antibody and DAB substrate kits.
Images of sections were acquired with an optical microscope (E100,
Nikon, Japan). Immunohistochemical evaluation was conducted using
ImageJ software to calculate the proportion of positive cells in total
cells.
TUNEL Staining
The apoptotic liver tissue cells were assessed using the DeadEnd
Fluorometric TUNEL System (Promage, USA) according to the
manufacturer's protocol. After washing with PBS, sections were stained
with DAPI for 15 min. The apoptotic cells were visualized using a
panoramic scanning microscope (TissudFAXS SL Spectra, TissueGnostics,
Austria).
Label‐Free LC‐MS/MS Experiment
In the label‐free experiment, MMs and liver tissue samples were lysed,
and their proteins were extracted using a total protein extraction kit
(KeyGENE, Jiangsu, China). Protein digestion was performed using the
filter‐aided sample preparation (FASP) method as previously
described.^[ [199]^50 ^] In simple terms, the protein supernatant was
mixed with four volumes of ice‐cold acetone and incubated at −20 °C
overnight. Precipitated proteins were pelleted by centrifugation at
16 000 × g for 10 min at 4 °C and then washed with 80% acetone. The
proteins were suspended in 200 µL of UA buffer (8 m urea, 150 mm
Tris‐HCl, pH 8.0; Sigma) and incubated at room temperature for 1 h.
Next, 100 µL of 10 mm iodoacetamide (Sigma) was added and incubated in
the dark for 30 min. The sample was washed twice with 200 µL UA buffer
at 14 000 × g centrifugation for 10 min at room temperature. Then,
50 µL of trypsin working solution (5 µg of sequencing‐grade modified
trypsin (1:50 w/w, Promega, USA) dissolved in 50 µL of ultrapure water)
was added for digestion and incubated at 37 °C overnight. The digested
samples were desalted on C18 Cartridges (EmporeTM SPE Cartridges C18,
bed I.D. 7 mm, volume 3 mL; Sigma), concentrated by centrifugation at
14000 × g for 10 min, and reconstituted in 40 µL of 0.1% formic acid.
For LC‐MS/MS analysis, an Easy‐NLC 1000 Liquid Chromatograph coupled to
a Q Exactive mass spectrometer (ThermoFisher, USA) was used for a
120‐min run. Peptides were separated on an RP‐C18 analytical column at
a flow rate of 300 nL min^−1 over 120 min. After capillary separation,
digested samples were analyzed using a Q Exactive mass spectrometer.
Proteome Data Calculation and Analysis
The raw data were processed using Proteome Discoverer software (version
2.4.1.15, ThermoFisher, USA) and searched against the protein sequence
database downloaded from UniProt (Swissprot). The Comet search
parameters were set as follows: enzyme: trypsin, maximum missed
cleavages: 2, instrument: ESI‐TRAP, precursor mass tolerance: ± 10 ppm,
fragment mass tolerance: 0.05 Da, use average precursor mass: false,
modification groups from Quan method: TMT 6 plex, dynamic
modifications: oxidation; acetyl, static modifications, database
pattern: decoy, peptide FDR: ≤ 0.01. The expressed proteins from each
sample in each group were grouped, and the differentially expressed
proteins were used for bioinformatics analysis. The quality control of
the protein data and differential expression analysis were performed
using the DEP package (version 1.22.0) in R software (version 4.3.1).
Differentially expressed proteins were defined as those with a log2
fold‐change (logFC) greater than or equal to ± 0.26 (a 20% change in
expression) and a p‐value less than 0.05. Gene annotation and
enrichment analysis were conducted using Metascape
([200]https://metascape.org/gp/index.html#/main/step1). Gene set
enrichment analysis (GSEA) was conducted on the MM proteins using the
clusterProfiler package (version 4.8.3). This analysis specifically
employed Gene Ontology Biological Process (GO BP) gene sets to
determine enriched biological processes.
Statistical Analysis
GraphPad Prism was used for statistical analysis. All data were
represented as mean ± standard deviation (SD). Two‐tailed t‐test
analysis was used for a comparison between two groups, and the
differences among multiple groups were analyzed by one‐way ANOVA
following Tukey's multiple comparisons tests. p values < 0.05 were
considered significant.
Conflict of Interest
The authors declare no conflict of interest.
Supporting information
Supporting Information
[201]ADVS-11-2404171-s001.pdf^ (1.8MB, pdf)
Acknowledgements