Reprogramming Exosomes to Escape from Immune Surveillance for Mitochondrial Protection in Hepatic Ischemia-Reperfusion Injury

Background: Therapeutic interventions such as synthetic drugs and microRNA (miR) modulators have created opportunities for mitigating hepatic ischemia/reperfusion injury (HIRI) by alleviating mitochondrial dysfunction. However, delivering multi-therapeutic ingredients with low toxicity to hepatocytes still lags behind its development. Methods: In this study, we endowed exosomes with delivery function to concentrate on hepatocytes for multidimensionally halting mitochondria dysfunction during HIRI. Concretely, exosomes were reprogrammed with a transmembrane protein CD47, which acted as a “camouflage cloak” to mimic the “don't eat me” mechanism to escape from immune surveillance. Besides, HuR was engineered bridging to the membrane by fusing with CD47 and located in the cytoplasm for miR loading. Results: This strategy successfully delivered dual payloads to hepatocytes and efficiently protected mitochondria by inhibiting the opening of mitochondrial permeability transition pore (mPTP) and upregulating mitochondrial transcription factor A (TFAM), respectively. Conclusions: The reprogramming of exosomes with CD47 and HuR for targeted delivery of CsA and miR inhibitors represents a promising therapeutic strategy for addressing HIRI. This approach shows potential for safe and effective clinical applications in the treatment of HIRI.


Lentivirus-mediated transfection of CD47-HuR fusion gene into LO2 cells
Lentiviral vectors GV367 for stable expression of CD47-HuR fusion gene were successfully synthesized by GeneChem Incorporation (Shanghai, China).They carry fragments to express green fluorescent protein (GFP) and puromycin resistance protein (PAC), which are shown in Figure S8.Then, vectors incorporated with CD47-HuR fusion protein were fabricated.Specific primers used for validating the fusion gene by GeneChem were listed in Table S1.LO2 cells were seeded in 24-well plates (5×10 4 cells/well) for lentiviral transfection following the previous methods [1], and were named LO2 CD47-HuR cells.
Transfection mixes A and B were prepared separately for each well.Mix A contained 250 µL Opti-MEM and 5 µL mimics/inhibitors, while mix B contained 250 µL Opti-MEM and 5 µL Lipofectamine TM 2000.Both mixtures were vortexed together for 10 sec and incubated for 5 min.The culture medium was removed from cells and the cells were rinsed with PBS.Then, 1 mL medium was added to the cells followed by an additional 500 µL transfection mixture (A+B) to the well.After transfection, cells should then be incubated in a fresh medium for another 48 h.The sequences of miR-590-3p and miR-7057-3p mimics/inhibitors are shown in Table S2.

Extraction of protein from cells, liver tissue, and Exos
To prepare the cellular lysate for western blot, 0.5 mL RIPA containing 1% PMSF was added to the cells after being rinsed with PBS thrice.Cells were then lysed for 30 min at 4 ℃.Besides, dissected liver tissues were precisely weighed and placed into a Precellys tissue grinding tube.Subsequently, the tissues were lysed with cell lysis buffer (20 μL/mg) and thoroughly ground until most of the tissues were dissolved.To extract proteins from Exos, the sample was added to Exo-specific lysis buffer at a volume ratio of 1:1, and the resulting mixture was incubated on ice for 10 min for efficient lysis.Afterward, these supernatants were collected by centrifugation at a speed of 12,000 rpm for 15 min.The extracted proteins were then subjected to protein quantification and western blot.

Bicinchoninic acid (BCA) protein assay and western blot
The BCA protein assay kit was used to determine the protein concentration according to the manufacturer 's instructions., and the expression levels of interest proteins were examined by western blot.Briefly, equal amounts (30 µg) of sample proteins were loaded onto a well of polyacrylamide gel electrophoresis (PAGE) and electrophoresis was run at 80 V for 30 min, followed by 110 V for 50 min.Afterward, the proteins were transferred to polyvinylidene difluoride (PVDF, Millipore, USA) membranes at a current of 250 mA for 60~120 min.Then, PVDF membranes were blocked with 5% skim milk powder for 60 min at room temperature, and rinsed with 1× Tris-buffered saline containing 1% Tween 20 (TBST) buffer.After that, the PVDF membranes were incubated with primary antibodies at 4 °C overnight and subsequently horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse secondary antibody in 1× TBST buffer at room temperature with shaking for 60 min.β-actin was referred to as control.Finally, the expression levels of proteins were monitored by an enhanced chemiluminescent (ECL) reagent (Life-iLab, China) and visualized with an ECL detection system (BIO-OI, China).The detailed information of primary and secondary antibodies used in this study and their concentrations are shown in Table S3.

RNA extraction
Enzyme-free EP tubes, reagents, and consumables were exposed to UV irradiation for sterilization.Cells were gently washed thrice for RNA extraction.Then, appropriate amounts of Trizol (Takara, Japan) were added (1 mL/well).After shaking, the cell suspension was transferred by pipette into an enzyme-free 1.5 mL EP tube, added with 200 μL chloroform, and centrifuged at 12,000 rpm for 15 min at 4 ℃.After that, the colorless aqueous phase layer was transferred into a new EP tube.700 μL isopropanol was added, and the mixture was centrifuged at 12,000 rpm for 10 min at 4 ℃.Next, the supernatant was discarded.Subsequently, the concentration of RNA was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).Finally, the RNA samples were stored at − 80 ℃ for further use.
The resulting cDNAs were diluted 5~10 times and stored at -20 ℃ for further use.For fluorescent quantitation, 12.5 μL TB Green Premix Ex TaqⅡ and 10.3 μL DEPC water were added to mixtures.The resulting mixtures were then added to an 8-tube strip with each tube containing 2 μL cDNA from different experimental groups.The PCR amplification was performed using a fluorescence quantitative PCR instrument.The process started with a pre-denaturation step at 95 ℃ for 30 sec, followed by 40 cycles of denaturation at 95 ℃ for 5 sec, annealing at 60 ℃ for 30 sec.The data analysis was performed using the 2 -△△ CT formula.The primer sequences for SybrGreen were designed by Sangon Biotech (Shanghai, China) and were listed in Table S4.

Cell viability assay
For cytocompatibility testing, LO2 cells were seeded in 96-well plates, 24-well plates, or 6-well plates.24 h after cell adhesion, the fresh cell culture medium containing Exo CD47-HuR , CsA@Exo CD47-HuR , miRi@Exo CD47-HuR , or CsA/miRi@Exo CD47-HuR were added for incubation (24 h).After OGD/R, the cells were stained using the LIVE/DEAD Fixable Dead Cell Stain kit (Beyotime, China) to differentiate between live and dead cells.The cell viability was assessed three times using a microplate reader (Thermo Fisher, USA) by measuring the absorbance at 450 nm with a cell counting kit-8 assay.Besides, the Annexin V-FITC/PI (AV/PI) apoptosis assay kit (Beyotime, China) was used to distinguish apoptotic cells.Then a flow cytometer (CytoFLEX, BeckmanCoulter, USA) was used to measure the apoptosis rates of LO2 cells.Flowjo software (version 10.8.1) was used to analyze the results.

Observation of mitochondrial morphology by CLSM
To observe the mitochondrial morphology, LO2 cells were incubated with different groups of Exos for 24 h on a 24-well glass slide and then suffered from OGD/R.After that, MitoTracker was used to stain active mitochondria for 30 min at 37 ℃, followed by rinsing 3 times by PBS and fixed with 4% paraformaldehyde in Hanks' balanced salt solution (HBSS) for 15 min.The cell nuclei were stained blue using DAPI for 30 min.After washing 3 times in PBS, CLSM equipped with a 63×/1.4NA oil objectives and laser/filter sets was used to observe DAPI (blue) and MitoTracker (red).

Characterization of the mitochondrial membrane potential (ΔΨm)
ΔΨm was assessed in live cells using JC-1.LO2 cells were seeded in 6-well plates or 24-well plates and treated with different groups of Exos for 24 h.After washing with PBS, each well was mixed with 0.1% JC-1 and incubated for 20 min at 37 ℃. 2 mL cell culture medium was added to each well and washed twice with JC-1 staining buffer.Images were scanned by CLSM, and the CytoFLEX flow cytometer was also used to determine ΔΨm.The ratio of JC-1 aggregate/JC-1 monomer fluorescence intensity was carried out to measure ΔΨm.

Assessment of mitochondrial permeability transition pore (mPTP) opening
Calcein-AM/CoCl2 assay (Beyotime, China) was performed to evaluate the opening condition of the mPTP as described previously [2].LO2 cells were seeded in 24-well plates, incubated with different groups of Exos for 24 h, and washed with PBS twice.Each well was incubated with Calcein-AM (2 μM) for 30 min at 37 °C.After washing in PBS twice, the cells were exposed to CoCl2 (2 mM) for 15 min to detect the distribution of cobalt inside mitochondria.The degree of mPTP opening was reflected by green (Calcein-AM) fluorescence assessed by CLSM and the CytoFLEX flow cytometer.

Measurement of intracellular reactive oxygen species (ROS) and mitochondrial ROS
After LO2 cells were treated as described above, the ROS assay kit (Beyotime, China) was used to assess the intracellular ROS levels in accordance with the manufacturer's instructions.In brief, LO2 cells were stained with 2',7'-dichlorofluorescein diacetate (DCFH-DA, green) probe for 30 min in the dark and the fluorescence images were captured by CLSM.MitoSox Red (Yeasen Biotechnol., China), a probe specifically targeting mitochondria, was used to selectively detect hyperoxide produced by mitochondria as previously described [3].LO2 cells were loaded with 5 μM MitoSox Red diluted in HBSS for 10 min at 37 °C.The fluorescence intensity was observed under CLSM.

Transaminase detection
Mouse blood samples were collected using the eye-ball bleeding method with EDTA-Na2 anticoagulant tubes.The samples were then centrifuged at 2,000 rpm for 15 min, and the plasma was tested for the activity of ALT and AST using an automated blood biochemical analyzer.

Hematoxylin & Eosin (H&E) staining
First, tissues were gently rinsed with PBS buffer or physiological saline, and then we fixed the tissues by soaking them in a 4% paraformaldehyde solution.Next, a defatting agent was applied to remove water and fat.The samples were then immersed and embedded in melted paraffin to fix and protect their morphology.Then, the embedded tissues were cut into thin slices of 5~8 μm using a microtome.H&E staining was performed as previously reported [4] and analyzed under a microscope (Nexcope NIB410, USA).The Suzuki score [5] was used to evaluate the degree of liver tissue damage after HIRI, and the details are described in Table S5.

Detection of ROS in liver tissues
First, frozen sections were thawed at room temperature and the moisture content was controlled.A circle was drawn around the tissue with a histological pen, and a self-quenching fluorescent reagent was added for 5 min, followed by rinsing with running water for 10 min.Then, the red fluorescence probe Dihydroethidium (DHE, Sigma-Aldrich, Shanghai, China) was added to the circle, and the samples were incubated at 37°C in the dark for 30 min.The slides were washed on a shaking platform in PBS 3 times, each time for 5 min.DAPI staining solution was added and the samples were incubated at room temperature in the dark for 10 min.The slides were washed again on the shaking platform in PBS 3 times, each time for 5 min.Finally, the anti-fluorescence quenching sealing agent was added for slide sealing.

Tissue mitochondrial purification
All solutions in the kit (Beyotime, China) were thawed at room temperature, mixed thoroughly, and immediately placed on ice.After completing the animal experiments, a small piece of liver left lobe tissue weighing approximately 50~100 mg was excised and weighed in a 1.5 mL centrifuge tube.The tissue was washed once with PBS and placed in a centrifuge tube on ice.Next, 10 volumes of pre-cooled mitochondrial separation reagent A were added, and the tissue was homogenized on ice approximately 10 times.Note: If the tissue weighs 80 mg, it can be roughly estimated that the tissue volume is close to 80 µL, and thus 800 µL of mitochondrial separation reagent A should be added.The homogenate was centrifuged at 1,000 g and 4 °C for 5 min to obtain higher purity mitochondria.The supernatant was carefully transferred to another centrifuge tube and centrifuged at 3,500 g and 4 °C for 10 min.
The supernatant was carefully removed, and the precipitate was the isolated mitochondria.Typically, 40 µL corresponding mitochondrial storage solution could be used to resuspend the mitochondria obtained from 100 mg tissue.The expected protein concentration of the isolated mitochondrial sample was 10~20 mg/mL.Then, the extracted mitochondria were subjected to subsequent detection within 6 h.

Detection of ΔΨm in liver tissues by JC-1
Dilute 0.9 mL JC-1 staining working solution and add 0.1 mL purified tissue mitochondria with a total protein content of 10~100 µg.
Mix the samples and measure them using a spectrofluorometer FS5 (Edinburgh Instruments, UK).The green fluorescence was detected at an excitation wavelength of 490 nm and an emission wavelength of 530 nm, while the red fluorescence was measured using an excitation wavelength of 525 nm and an emission wavelength of 590 nm.The ratio (red/green) was calculated to evaluate ΔΨ.A smaller ratio indicated lower ΔΨ and therefore decreased mitochondrial function, which could be used to evaluate cellular apoptosis and mitochondrial function.

Detection of mPTP opening in liver tissue
The purified mPTP colorimetric assay kit (GENMED, USA) was used to determine the degree of mPTP opening.Firstly, the tested purified mitochondrial samples were prepared and transferred into the corresponding wells of a 96-well enzyme-linked immunosorbent assay plate.Next, GENMED buffer solution (Reagent A) was added, mixed, and left to stand for 1 min at room temperature.Then, an induction solution (Reagent B) was added and the samples were immediately placed into a spectrophotometer (at a wavelength of 540 nm), and the initial absorbance (Initial A540) was recorded.Subsequently, the absorbance ratio (A540/Initial A540) was calculated at each time point by monitoring continuously every 30 s for 10 min, which represented the actual absorbance value at the designated time divided by the absorbance value at 0 min.If the absorbance ratio decreased, it indicated an increased activity of mPTP.The membrane channel pore induction opening curve was constructed, in which the absorbance ratio (A540/Initial A540) was used as the vertical axis (Y) and the detection times were used as the horizontal axis (X).DCFH-DA (green) together with its fluorescence intensity quantification (E).Scale bar: 100 μm.Groups were designed as: normal LO2 cells (G1); LO2 cells after OGD/R (G2); LO2 cells pretreated with Exos CD47-HuR before OGD/R (G3); LO2 cells pretreated with CsA@Exos CD47-HuR before OGD/R (G4); LO2 cells pretreated with miRi@Exos CD47-HuR before OGD/R (G5); LO2 cells pretreated with CsA/miRi@Exos CD47-HuR before OGD/R (G6).miRi refers to hsa-miR-590-3p inhibitor.Data are expressed as mean ± S.D. (n = 3), NS means no difference (non-repeated ANOVA followed by Tukey's test).

Figure
Figure S2 Identification and validation of miRNA (miR) modulators regulating TFAM.The putative seedmatching sites between hsa-miR-590-3p (A) or mmu-miR-7057-3p (E) and TFAM predicted by the TargetScan database.Protein expression of TFAM detected by western blot was significantly high after transfected with hsa-miR-590-3p inhibitor in LO2 cells (B, C) as well as mRNA expression of TFAM by qRT-PCR (D).Protein expression of TFAM detected by western blot was significantly high after transfected with mmu-miR-7057-3p inhibitor in NCTC-1469 cells (F, G) and mRNA expression of Tfam by qRT-PCR (H).miR-NC represents negative control of miR inhibitor, and anti-miR represents miR inhibitor.Data are mean ± S.D. (n = 3), (Student's t-test).

Figure
Figure S5 Selection of the optimal time of "Oxygen-Glucose Deprivation/Reoxygenation" (OGD/R).(A) Western blot of apoptosis-related protein and TFAM expressions in LO2 cells under OGD for 2, 4, 6, 8, and 10 h, followed by 24 h reoxygenation for the applied time together with quantification (B).(C) Expression of TFAM mRNA tested by qRT-PCR.(D) Western blot of apoptosis-related protein and TFAM expressions in LO2 cells under OGD for 6 h, followed by 3, 6, 12, and 24 h reoxygenation for the applied time together with quantification (E).(F) Expression of TFAM mRNA tested by qRT-PCR.Data are expressed as mean ± S.D. (n = 3), (non-repeated ANOVA followed by Tukey's test).

Figure
Figure S7 Protection properties in elevating mitochondrial membrane potential (Δψm) and scavenging ROS of CsA/miRi@Exos CD47-HuR in vitro.(A) Δψm analysis of LO2 cells after different treatments, using JC-1 stain.Green fluorescence exhibited JC-1 monomer in cytoplasm and red fluorescence exhibited JC-1 aggregates in core mitochondria.Scale bar: 100 μm.(B) Quantitative analysis of fluorescence intensity of JC-1 monomer and JC-1 aggregates, respectively.(C) Quantitative analysis of the mPTP opening stained with Calcein AM (green).(D) Representative confocal images showing reactive oxygen species (ROS) stained with

Figure
Figure S8 Schematic map of the GV367 lentiviral vector.The vector contains the Ubi promoter (Ubi), multiple cloning site (MCS), SV40 poly(A) signal (SV40), enhanced green fluorescent protein (EGFP) reporter, and puromycin resistance marker (puromycin).The fusion gene was inserted between the AgeI and NheI cloning sites.The construct was verified by sequencing and assigned the control number CON238.