Materials and reagents
Materials and reagents were included in Additional file 1.
Extraction and purification of the PM
Whole blood samples were collected from rats by cardiac puncture with EDTA-treated anticoagulant vacutainer tubes. After that, the whole blood was centrifuged at 200 × g for 20 min at 20 ℃. The supernatant fraction was collected and centrifuged at 800 × g for 15 min. The white sediments were platelets, which were resuspended with PBS containing EDTA and PGE1 to avoid platelet activation. Then, platelets were lysed by repeated freeze–thaw at − 80 °C and room temperature for 3 times, followed by repeated washes with PBS. Subsequently, platelets were further broken by the ultrasonic cell disruptor (SCIENTZ-950E) for 5 min on ice (100 W, 53KHZ) [11, 15]. Platelet membrane (PM) vesicles were prepared by continuous extrusion through 0.2 and 0.1 μm polycarbonate filters (Avanti® Mini-Extruder set). Finally, total protein of PM was measured by BCA assay.
Synthesis of PTPN nanoparticles
pH-responsive tPA nanoparticles (TPN) were synthesized as described in our previous work [10]. Briefly, PEI–PBA was achieved by reacting 38.5 mg of PBA, 50 mg of PEI and acid-binding agent trimethylamine in 2 mL of DMSO at 70 °C for 24 h followed by dialysis and lyophilization. PC–tPA was formatted via a Schiff base reaction between the aldehyde group on the PC and the primary amino group on the tPA at 50:1 molar ratio. 29 μL of PC (1 mg/mL) was added into 100 μL of tPA solution (1 mg/mL) at room temperature followed by adjusting pH to 8.0. This reaction was stirred for 1 h and then removed excess PC by ultrafiltration. After that, obtained PC–tPA was stored at 4 ℃ for further use.
Prepared PEI–PBA and PC–tPA at mass ratio of 3:1 were mixed to construct a pH-responsive nanocomplex via boronate ester reaction. Briefly, PC–tPA solution (0.15 mL, 0.12 mg/mL) was added dropwise to PEI–PBA solution (0.15 mL, 0.36 mg/mL) under stirring for 10 min. Meanwhile, mPEG-Dopamine (0.5 mg/mL) was added and continued stirring to obtain the TPN.
Then, TPN and prepared PM were mixed at different protein mass ratios 1:10 for synthetizing PM coated TPN (PTPN) [16, 17]. In brief, TPN (0.5 mL, 0.05 mg/mL) was mixed with PM (0.5 mL, 0.5 mg/mL) and pre-warmed at 37 ℃ followed by continuous extrusion through 0.2 and 0.1 μm polycarbonate filters.
Characterization of nanoparticles
The particle size and zeta potential of nanoparticles suspended in different pH PBS solutions were determined by dynamic light scattering (DLS) zetasizer (Malvern, Zetasizer 3000). The stability of TPN and PTPN (suspended in PBS or DMEM containing 10% FBS) was evaluated by DLS at 0 h, 12 h, 24 h, 2 d, and 3 d. In addition, transmission electron microscope (TEM) was applied to visualize the morphology of TPN, PM and PTPN.
SDS–PAGE gel with silver staining and Western blot were used to analyze protein profiles of the PM, TPN and PTPN. Briefly, equivalent amounts of protein (4 μg) of PM, TPN and PTPN were separated on a 10% SDS–PAGE gel followed by fixation with ethanol and acetic acid. Subsequently, the gels were incubated with silver staining sensitizer for 2 min and then with silver nitrate for 10 min. Lastly, the gel was incubated with chromogen solution and terminated with stop solution. Similarly, SDS–PAGE gel after electrophoresis was transferred to the 0.22 μm polyvinylidene difluoride membrane (PVDF) and incubated with anti-CD34, anti-CD61, and anti-CD42b overnight. After that, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and detected by electro-chemiluminescence (GE, Amersham Imager600).
Cell model
Primary cardiomyocytes were isolated and cultured according to a previously established method [18]. Briefly, hearts harvested from the neonatal Sprague–Dawley (SD) rats were serially digested with 0.1% trypsin. After that, single-cell suspension was added into cultural dish to remove the fibroblasts by differential attachment for 1 h. The purity of primary cardiomyocytes was confirmed as > 80% by immunofluorescence staining of α-actinin (Additional file 1: Fig. S1) [19]. Human Umbilical Vein Endothelial cells (HUVEC) were purchased from ATCC.
The oxidative stress cell model induced by hypoxia and H2O2 was used in this work. Cell were cultured in low glucose serum-free DMEM in the hypoxic chamber with 1% O2 for 12 h [20]. To mimic the oxidative stress induced by ischemia/reperfusion, cells were treated with 500 μM H2O2 for 2 h [21].
Animal model
Male ICR mice and SD rats aged 6–8 weeks were purchased from Shanghai JSJ Laboratory Animal Co., Ltd (Shanghai, China). All experimental procedures and animal studies were approved by the Animal Ethics Committee of Shanghai Chest Hospital (Approval number: KS(Y)21,357).
Establishment of the MI rat model: Rats were anesthetized by intraperitoneal (i.p.) injection of 2% paraformaldehyde sodium, followed by endotracheal intubation with 16G cannula (tidal volume = 8 ml/kg, frequency = 80/min) [22]. Then, the chest was opened after blunt dissection of the pectoralis major muscle and pectoralis minor muscle. A 2.5 mm × 2.5 mm filter paper saturated with 15% FeCl3 solution was placed on the left anterior descending coronary artery (LAD) for 5 min to induce coronary thrombosis. Finally, the filter paper was removed, and the incision was sutured layer by layer.
Establishment of the femoral artery thrombosis rat model: After anesthesia, the left femoral artery of the rat was exposed and separated from the femoral vein and nerve. A 2.5 mm × 2.5 mm filter paper saturated with 15% FeCl3 solution was placed on the femoral artery [23].
Establishment of the carotid artery thrombosis model: ICR mice were used to establish carotid artery thrombosis for live imaging analysis. Briefly, after anesthesia with 0.5% paraformaldehyde sodium and removal of the cervical hair with depilatory cream, the carotid artery was exposed under stereomicroscope (SZX7, Olympus) and covered with a 1 mm × 1 mm filter paper containing 15% FeCl3 solution for 5 min [23].
Safety evaluation
Cell viability was measured by Cell Counting Kit-8 (CCK-8) [24]. In detail, HUVEC were seeded into 96-well plates at a density of 5000 cells per well for 12 h. After that, the medium was replaced with 100 μL of DMEM containing different concentrations (0, 100, 200, 500 and 1000 μg/ml) of tPA, TPN, PM and PTPN for 6, 12, 24 and 48 h. Then, 10 μL of CCK-8 regent was added into each well and incubated for 1 h. Absorbance at 450 nm was measured using the microplate reader (ThermoFihser, VarioscanLUX), and the cell viability was calculated using the following equation:
$${\text{Cell viability}}~(\% )~ = \frac{{{\text{Absorbance}}~({\text{experimental}}~{\text{group}}) - {\text{Absorbance}}~({\text{blank}})}}{{{\text{Absorbance}}({\text{ctrl}}~{\text{group}}) - {\text{Absorbance}}~({\text{blank}})}} \times 100$$
Blood routine examination and clotting function assay were applied to evaluate the biocompatibility of the nanocarrier components. Briefly, 500 μL of normal saline, tPA, PM, TPN and PTPN (20 μg/mL) were injected via the tail vein to the SD rats for acute toxicity test. After 24 h, blood was collected from the retro-orbital venous plexus and placed in sodium citrate anticoagulant tubes. Blood routine examination was performed with the automated blood analyzer (Mindray, BC-2800 Vet) and clotting function was detected with the automatic blood coagulation analyzer (Rayto, RAC-120).
Rats were sacrificed at 4 weeks post intravenous administration. Heart, liver, spleen, lung and kidney were fixed in 4% paraformaldehyde for 24 h, dehydrated, paraffin embedded, and Hematoxylin–Eosin (H&E) stained to observe tissue histomorphometry.
Binding efficiency of PTPN in vitro
To evaluate the PTPN binding efficiency to HUVEC in vitro [25, 26], HUVEC were seeded into a 24-well confocal plate at a density of 2 × 104 cells per well for 12 h. After that, FITC-labeled TPN, DiI-labeled PM or PTPN extracted with FITC-labeled TPN and DiI-labeled PM (500 ng/mL) was added into the medium. Then, cells were moved to the hypoxic chamber with 1% O2 for 1 h. Subsequently, HUVEC were counterstained with Hoechst. Finally, binding efficiency was detected by confocal microscopy (ZEISS, LSM 980 with Airyscan2) and flow cytometry (BD FACSCANTO II).
Thrombus-targeted ability in vivo
A carotid artery thrombosis model was established to assess the thrombus-targeted ability of PTPN. 100 μL of FITC-labeled PTPN or FITC-labeled TPN (20 μg/ml) was injected via the tail vein of the ICR mice. The distribution of nanoparticles was monitored continuously for 3 h using the In Vivo Imaging System (IVIS; PerkinElmer) [27].
Thrombolysis ability of PTPN in vitro
Rat whole blood was collected from the retro-orbital venous plexus and placed in 1.5 mL microcentrifuge tubes for coagulation at room temperature [8]. After that, thrombus clots were moved to 24-well plates and incubation with 200 μL of ddH2O, tPA (10 μg/mL), PM (10 μg/mL), TPN (10 μg/mL), PTPN (suspended in PBS, pH = 7.4, 10 μg/mL) or PTPN (suspended in PBS, pH = 7.4, 10 μg/mL) respectively. Thrombus clots were extracted and photographed at 1 h or 3 h. After that, solutions containing lysed red blood cells were moved to 96-well plate and absorbance were detected at 538 nm.
Drug release assay in vitro
Rhodamine labeled tPA was used to synthesize PTPN for release assay. PTPN was dissolved in PBS at pH 7.4 and pH 6.4 respectively, then the release of Rhodamine-tPA at different time points was obtained by ultrafiltration (100 kD, 3000 rpm, 10 min) and detected by fluorescence spectrophotometer.
Evaluation of tPA activity
As tPA mediates plasminogen conversion to plasmin, the activity evaluation of tPA released from PTPN was measured. PTPN was suspended in PBS at pH 7.4 or pH 6.4 (10 μg/mL), respectively. Subsequently, 2 μL of plasmin substrate-AMC, 20 μL lytic PTPN solution and 48 μL of plasmin assay buffer were added into the plate to detect the plasmin activity. Fluorescent signal was captured every 2 min for 20 min at 37 °C at Ex/Em = 360/450 nm by a fluorometry (ThermoFihser, VarioscanLUX).
Thrombolysis ability of PTPN in vivo
Nnormal saline, tPA, TPN or PTPN (0.1 mg/kg tPA) was administered via the tail vein in rats after establishment of femoral artery thrombosis. After 6 h, the femoral artery was removed, and H&E staining was performed to detect the recanalization of blood vessels. After FeCl3 induced AMI in rats, normal saline, TPN or PTPN (0.1 mg/kg tPA) was administered via the tail vein, and after 6 h the heart was harvested for staining.
Efficacy of PTPN in AMI treatment
CD31 immunohistochemical (IHC) staining and picrosirius red staining were performed to observe pathological changes of heart. In brief, rats in different groups were sacrificed and perfused with PBS, and then with 4% paraformaldehyde. 5 μm paraffin sections were used for histologic and IHC study. For Picro Sirus Red staining, the sections were immersed into picrosirius red solution for 8 min, dehydrated with anhydrous ethanol twice, and dewaxed twice with dimethylbenzene (5 min each).
For CD31 immunofluorescence, the sections were dewaxed and placed in EDTA buffer (pH = 6.0) to repair antigens subsequently washed with PBS for three times. After that, the sections were immersed in 3% H2O2 solution and protected from light for 25 min at room temperature. After washing with PBS, the sections were blocked with 5% rabbit serum and incubated with anti-CD31 (1:200) at 4 °C overnight. Then, the sections were incubated with Cy3-conjugated secondary antibodies for 1 h at room temperature.
Mitochondrial morphology of primary cardiomyocytes
Primary cardiomyocytes were seeded into the 24-well confocal plate at a density of 2 × 104/mL and cultured overnight. The medium was replaced with 500 μL of DMEM containing PBS, tPA, TPN or PTPN (500 ng/mL) for 1 h incubation. H2O2 was added into the cultural medium at a final concentration of 500 μM for 2 h at 37 °C. The medium was then replaced with DMEM containing MitoTracker™ Red CMXRos and Hoechst 38,450 for 30 min at 37 °C. Finally, primary cardiomyocytes were washed with PBS, and photographed with a confocal microscope.
Mitochondrial membrane potential assay
Primary cardiomyocytes were cultured and stimulated as described above. Then, the cells were incubated with warm DMEM containing a JC-1 probe for 30 min at 37 °C, washed with warm PBS, re-stained with Hoechst 38,450, and photographed.
ATP production assay
Primary cardiomyocytes were seeded into an opaque white 96-well plate at a density of 5000 cells per well and cultured overnight. The medium was replaced with 100 μL of DMEM containing PBS or PTPN (500 ng/ml) for 1 h, incubated with H2O2 (500 nM) for 2 h, and equilibrated for 10 min at room temperature. 100 μL of detection reagent was added into each, shaken well, and incubated at room temperature for signal stabilization [28]. Luminescence was recorded by a plate reader (ThermoFihser, VarioscanLUX) at an integration time of 0.5 s.
Mitochondrial OxPhos protein expression
Oxidative phosphorylation (OxPhos) supplies most energy for eukaryotic cells, which consists of five protein subunits. Protein expression of OxPhos complexes were detected by Western blot with OxPhos Rodent WB cocktail antibody. Other steps of Western blot were the same as described above.
RNA sequencing and analysis
Primary cardiomyocytes were collected using a scraper and counted. After washing with PBS, cells were lysed with Trizol (1 mL/106 cells) and stored at − 80 °C. The library preparation and sequencing were conducted by the Haplox (China). Differential expression genes (DEGs) were selected by ∣Fold Change∣ ≤ 1.5 with P value < 0.05. GO function annotations and KEGG pathway database were applied to analyze the potential function and pathway of DEGs. Statistical analysis and graphical plotting were performed using R software.
Statistical analysis
All numerical data are presented as the means ± SEM. Details and methods are included in the figure legends. Differences between two groups were compared with unpaired Student’s T test, and differences between multiple groups were compared by one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test. P values are noted in each figure and P value < 0.05 was considered statistically significant.