Cell culture. HeLa cells were purchased from the Chinese Academy of Sciences Shanghai Institute of Cell Bank (Shanghai, China). HeLa cells with mitochondrial complex I damaged were generated using 0.1 μM Rot and previously described38. The HeLa cells with or without Rot treatments were cultured in DMEM with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin under a humidified 37 °C 5% CO2 atmosphere.
Animal model. C57BL/6J male and Balb/c female mice were purchased from the East China Normal University Laboratory Animal Technology Co., Ltd. (Shanghai, China). The guidelines for human and responsible use of animals in research approved by the regional ethics committee of China Pharmaceutical University (2021-11-009) were employed to guide all the animal protocols and procedures.
For Rot-treated LHON mouse model, Balb/c female mice were anesthetized and treated with 3 μL 1, 2.5 or 5 mM Rot (solvent: trilaurin) by intravitreal injection. And the vehicle group was treated with 3 μL trilaurin.
IPF mouse model was established using 40 μL BLM (1, 2 or 4 U/kg) by endotracheal intubation. And the PBS group was treated with 40 μL PBS.
Preparation and characterization of MLPers. MLPers were prepared by adhering Lipo 2000 NPs to extracted exogenous mitochondria. In brief, exogenous mitochondria with 40 μg mitochondrial proteins were extracted from HeLa cells, heart tissues of Balb/c female mice or C57BL/6J male mice according to the protocol of Mitochondrial Isolation Kits (MP-007, Invent Biotechnologies). 2 μL Lipo 2000 was mixed with 1 μg mRNA for 0.5 h at 4 °C and the prepared Lipo 2000 NPs were further mixed with exogenous mitochondria for 30 min. The MLPers were obtained after centrifugation at 16000 × g for 20 min to remove free Lipo 2000 NPs. The average hydrodynamic diameters and zeta potentials of the Mito and MLPers were analyzed by dynamic light scattering (DLS) and a Nano ZS Zetasizer DLS analyzer (Brookhaven, Germany).
For determination of the mRNA contents in MLPers, mRNA labeled with Cy3 fluorophore (R1999, APExBIO) was loaded in ML@Cy3-mRNA. After 30 min of incubation and 20 min of centrifugation, the supernatant and sediment were collected and were resuspended in Heprin solution for 30 min. The fluorescence intensity (FI) of Cy3-mRNA were analyzed by multifunctional microplate reader (Molecular Devices ID5, USA).
For visualization of the adhesion of mRNA-loaded Lipo 2000 NPs and free mitochondria, mRNA (1 μg) was marked with Cy5 fluorophore (R1010, APExBIO) and exogenous mitochondria (40 μg mitochondrial proteins) were labeled with MitoTracker Green. MLPers and free mitochondria were respectively seeded into the 96-well plate and the images were recorded by inverted fluorescence microscopy (Nikon, Japan). In addition, SEM (Hitachi, Japan) was also carried out to determine the morphology of MLPers.
Functionality and activity of mitochondria after binding with Lipo 2000 NPs in MLPers were examined by Janus-green-b staining. The images of extracted mitochondria with or without Lipo 2000 NPs were collected by inverted fluorescence microscopy. For the activity of mitochondria, the free Mito group and MLPers were stained with JC-1 Kit after 0, 1 and 3 days of storage. The FI was detected by multifunctional microplate reader.
The stability of Lipo 2000 NPs on the surface of extracted mitochondria, Cy3-mRNA loaded Lipo 2000 NPs were adhered to mitochondria for 0 or 1 day in free DMEM media. Then, the solutions were centrifugated for 20 min at 16000 × g. The supernates were detected by multifunctional microplate reader and the sediments were resuspended in Heprin solution for 30 min to replace the Lipo 2000 NPs on the surface of mitochondria. After 16000 × g centrifugation for 20 min, the supernate and sediment were analyzed by multifunctional microplate reader to calculate the contents of mRNA shedding from MLPers and the contents of mRNA internalized into extracted mitochondria after different storage times.
Mitochondrial heterogeneity detection. To evaluate mitochondrial heterogeneity, we performed mitochondrial protein content, mtDNA copy number, immunofluorescence of mitochondrial complex I, MMP, mitochondrial morphology and mitochondrial mass detections.
Mitochondrial protein content assay. Mitochondria and nucleus were extracted from HeLa cells, eye tissues from Balb/c or lung tissues from C57BL/6J by Mitochondria Isolation Kit after different treatments. The mitochondrial and nuclear protein contents were tested by BCA Detection Kit, and the mitochondrial heterogeneity was calculated according to the ratio of mitochondrial/nuclear protein contents.
mtDNA copy number. Genomic DNA of frozen eye or lung tissues was extracted using the Blood, Cell, and Tissue Genomic DNA Extraction Kit (TIANGEN Biotech Co., Ltd.) according to the manufacturer’s protocol. Quantification of mtDNA copy number was assessed in triplicates using 2 × SYBR Green PCR Master Mix from TIANGEN Biotech Co., Ltd.. The mtDNA levels were calculated by Double standard curve method.
For mitochondrial complex I contents, HeLa cells, eye or lung tissues were collected after treated with various groups and the localization of complex I proteins by immunofluorescence in fixed HeLa cells, eye or lung tissues was carried out as described previously52. The antibodies were used as following: Anti-Complex I Antibody (1:1000, ab109798, Abcam), Cy3 Goat Anti-Mouse IgG (H+L) (1:100, AS008, ABclonal). The images were recorded using Pannoramic MIDI/250 (3D HISTECH, Hungary) or CLSM (Carl ZEISS 700, Germany).
To evaluate the change of MMP in Rot-treated HeLa cells, 0.1 μM Rot-treated HeLa cells were cultured in the 35 mm glass bottom dishes (5×104 cells/well) or 24-well plate (5×104 cells/well). After 24 h, the cells from different treatment groups were stained by JC-1 dye (1×) at 37 °C for 15 min. Then, the images were observed by CLSM at 543 nm (JC-1 aggregates) and 488 nm (JC-1 monomers) excitation. In addition, multifunctional microplate reader was used to record the FI at 543 nm.
Mitochondrial morphology was monitored by TEM (Hitachi, Japan). In brief, the cells, eye or lung tissues were fixed with electron microscopic fixative solution, dehydrated and infiltrated in the resin. The prepared samples were detected by TEM.
Mitochondrial mass detections. MitoTracker Green was carried out to report the mitochondrial mass in different treatment groups. After various treatments, the cells, eye or lung tissues were covered with MitoTracker Green (500 nM). The samples were observed by CLSM or Pannoramic MIDI/250. The mitochondria from HeLa cells of different groups were isolated and the MFI of MitoTracker Green was detected by flow cytometry (BD FACSCalibur, USA).
Live/dead cells double stain.After HeLa cells were treated with 0.1 μM Rot for 12 h, various formulations were added into Rot-treated HeLa cells for 8 h. Then the medium was removed and cells were treated with Calcein-AM/PI Double Stain Kit (Yeasen Biotechnology (Shanghai) Co., Ltd., China). The images were recorded by inverted fluorescence microscopy and showed by ImageJ software.
Complex I activity. HeLa cells were cultured with 0.1 μM Rot for 24 h, and different treatment groups were added into 6-well plate for 24 h. The cells were collected and the complex I activity was detected by Complex I Activity Detection Kit (Solarbio Life Science, China).
ATP contents assay. HeLa cells were plated in 6-well plate overnight, and treated with 0.1 μM Rot for 24 h. The Rot-treated HeLa cells were covered with Lipo@mParkin (0.4 and 0.6 μg mParkin/well), free Mito and MLPers including 80 μg mitochondrial proteins and 0.4 μg mParkin/well. After 24 h for incubation, the ATP contents were estimated by ATP Bioluminescent Assay Kit and the chemiluminometer (Luminoskan Ascent, ThermoFisher Scientific, USA) was used to detect the luminescence in various groups.
ROS level detection. HeLa cells were seeded in 24-well plate for 14-16 h and treated with 0.1 μM Rot for 24 h. The Rot-treated HeLa cells were cultured with Lipo@mParkin (0.2 and 0.3 μg mParkin/well), free Mito and MLPers including 40 μg mitochondrial proteins and 0.2 μg mParkin/well for 24 h. DCFH-DA (5 μM) as a ROS probe was carried out to assess the ROS level by flow cytometry.
Cellular uptake and the mechanism study. The Rot-treated HeLa cells were treated with Lipo@Cy5-mRNA (0.2 and 0.3 μg Cy5-mRNA/well), free Mito and ML@Cy5-mRNA including 40 μg mitochondrial proteins and 0.2 μg Cy5-mRNA/well (exogenous mitochondria was labeled with MitoTracker Green or MitoTracker Red). After 1, 4 or 24 h, the cells were collected to analysis the MFI of MitoTracker Green/Red and Cy5-mRNA by flow cytometry. And the images were recorded by CLSM.
For the mechanism of cellular uptake, Rot-treated cells were covered with different cellular uptake inhibitors for 1 h including chlorpromazine (8.5 μg/mL), genistein (56.75 μg/mL) and amiloride (0.133 mg/mL). Then, Lipo@Cy5-mRNA (0.2 μg Cy5-mRNA/well) and ML@Cy5-mRNA were added into 24-well plate for 4 h. Flow cytometry was used to evaluate the MFI of Cy5-mRNA.
To explore the location of exogenous mitochondria and total cellular mitochondria in vivo and in vitro, exogenous mitochondria were marked with MitoTracker Green and the total cellular mitochondria after different treatments for 24 h were labeled by MitoTracker Red. Then, CLSM was used to record the images in various treatment groups.
Western blot. For evaluation of Parkin protein expression, the Rot-treated HeLa cells were cultured in the 6-plate and added Lipo@mParkin (0.4 and 0.6 μg mParkin/well), free Mito and MLPers were incubated for 24 h. The cellular proteins were extracted through Whole cell lysis assay and protein concentration was ascertained by BCA Protein Assay Kit (KeyGEN Biotechnology, China). Parkin Rabbit pAb (1:1000, A11172, ABclonal) and β-Actin Mouse Monoclonal Antibody (1:1000, AF2811, Beyotime Biotechnology) were used as antibodies and the images were detected by the chemiluminescence imaging system (Tanon, China). The band intensity of Parkin and β-actin proteins was quantified using integrated density value by ImageJ software.
The expression of Parkin protein in LHON and IPF mouse models was referred to Parkin protein expression in Rot-treated HeLa cells. And the immunofluorescence images were shown using Pannoramic MIDI/250.
Colocalization between lysosomes and mitochondria.HeLa cells were cultured in 35 mm glass bottom dishes for 14-16 h. After Rot treatment for 12 h, cells were covered with different treatments for 8 h. The Rot-treated HeLa cells were stained by MitoTracker Green probe at 37 °C for 30 min and were rinsed by PBS. LysoTracker Deep Red probe was used to label lysosomes. The colocalization between lysosomes and mitochondria was recorded by CLSM.
The exogenous mitochondrial uptake and mGFP transfection of MLPers in vivo. To evaluate the exogenous mitochondrial uptake in vivo, MitoTracker Red probe was performed to label exogenous mitochondria. The PBS, free Mito and MLPers were injected into normal Balb/c and C57BL/6J mice. After 24 h, the eyes from Balb/c mice and lung tissues from C57BL/6J were analyzed by fluorescent photos.
For mGFP transfection in vivo, normal Balb/c and C57BL/6J mice were treated with ML@mGFP for 3, 7, 14 days, and the eye or lung tissues were collected for further florescence analysis.
In vivo therapeutic effect in LHON mouse model. To establish LHON mouse model, Balb/c female mice were anesthetized and 3 μL 2.5 mM Rot were injected into vitreous chamber. After 7 days, the mice were randomly assigned to different formulations, including PBS, Lipo@mParkin (0.05 and 0.075 μg mParkin/eye), Mito (10 μg allogeneic mitochondrial proteins from Balb/c mice’ heart tissues) and MLPers (10 μg allogeneic mitochondrial proteins and 0.05 μg mParkin/eye).
After 1 week, eyes from various treatments were gathered in eye fixation fluid and longitudinal retinal sections were prepared for H&E staining. The thickness of retina in H&E sections was analyzed by the CaseViewer software. For ROS level detection, mice were injected intravitreally with DCFH-DA (20 μM) in 3 μL PBS and eye tissues were collected for fluorescence analysis. In addition, eye tissues were rapidly grinded in lysis buffer and the ATP contents in different groups were assessed by ATP Bioluminescent Assay Kit (Beyotime Biotechnology, China). The optomotor test was performed as previously described53. In brief, each mouse was placed in a circular platform and encompassed in black-and-white striped roller for 5 min. The roller was turned clockwise and counterclockwise for 2 min each and the movement times of head were recorded.
In vivo therapeutic effect in IPF mouse model. C57BL/6J male mice were subjected to BLM at a dose of 2 U/kg in 30 μL PBS and were treated with various formulations, including PBS, Lipo@mParkin (0.2 and 0.3 μg mParkin), Mito (40 μg allogeneic mitochondrial proteins from C57BL/6J mice’ heart tissues) and MLPers (40 μg allogeneic mitochondrial proteins and 0.2 μg mParkin). Mice were inhalation of these treatments through endotracheal intubation at 7th day.
After 21 days, lung tissues from different groups were harvested for H&E staining and Masson staining. The fibrosis areas were analyzed by Image J. Hydroxyproline contents in lungs were assessed by Hydroxyproline Detection Kit (Nanjing Jiancheng Bioengineering Institute, China). For the contents of IL-4 and TGF-β in lungs, Enzyme-linked Immunosorbent Assay (ELISA) was carried out by Servicebio (Wuhan, China).
Safety evaluation of allogeneic mitochondria-mediated MLPers in vivo. According to the dose and frequency for intravitreal injection or endotracheal intubation of MLPers, the normal Balb/c female mice and C57BL/6J male mice were injected with 1-fold, 2-fold, and 4-fold the dose of MLPers. The body weights were recorded during dosing. After two or three weeks, the serum samples from Balb/c female mice were prepared for the ALT, AST, BUN and inflammatory factors detections. mtDNA copy number in the whole blood was also assessed. In addition, the heart, liver, spleen, lung, kidneys and eyes tissues were collected and investigated by H&E staining and organ index analysis. For C57BL/6J male mice, the whole blood, serum samples and organ samples were harvested and evaluated referred to Balb/c female mice after the third dose during 3 weeks.
Statistics. Mitochondrial heterogeneity (%) = 100% – (Normal mitochondrial number detected by different indicators/Total mitochondrial number in the PBS or vehicle group × 100). Quantitative data in these experiments were presented as mean ± standard deviation (s.d.) from sample numbers (n). And the comparison of the mean values between multiple groups adopted the one-way analysis of variance (ANOVA) test, the pairwise comparisons adopted Tukey’s honest significant difference (HSD) post hoc test, LSD or Scheffe test when the data satisfied the homogeneity of variance, and the Games-Howell test was used for the comparison between multiple groups if the above conditions were not met the homogeneity of variance. All tests were two-sided. Comparisons of all groups were analyzed using the SPSS 19.0. The Image J software (National Institutes of Health, USA) was applied for quantitative analysis in fluorescence intensity for confocal images.