Characterization of TK and PTK
The mass spectrum of 5,5-dimethyl-4,6-dithio-azelaic acid (TK) is showing in Figure.S1A, which was consistent with the theoretic molecular weight of TK. In addition, 1H NMR spectroscopy of TK showed peaks at 1.59, 2.90 and 2.68 ppm, it was assigned to Ha, Hb and Hc of TK, respectively (Figure.S1B). Compared with TK, there was two new peak appeared at 4.18 and 1.98 ppm in 1H NMR spectroscopy of poly(5,5-dimethyl-4,6-dithio-propylene glycol azelate) (PTK), which were assigned to Hd and He of propanediol (Figure.S2). The molecular weight of PTK was 20505 Da determined by gel penetration chromatography (Figure.S3).
Characterization of CsA@PPTK
Transmission electron microscopy showed that the appearance of CsA@PTK and CsA@PPTK was spherical with good dispersion (Figure.1A). Besides, CsA@PTK displayed a uniform structure from inside to outside, while CsA@PPTK showed obvious core-shell structure. This indicated that platelet membrane was coated on the surface of CsA@PTK. As compared to CsA@PTK, the size of CsA@PPTK did not show significant change. The zeta potential of CsA@PPTK obviously decreased to -25 Mv, which was similar to zeta potential of platelet (Figure.1B). The co-localization of the DiR-labeled platelet membrane (red) and coumarin 6 labeled CsA@PTK (green) further substantiated the successful coating of platelet membrane on the surface of CsA@PTK (Figure.1C). SDS-PAGE electrophoresis revealed that the majority protein of platelet membrane was retained in CsA@PPTK (Figure.1D). All above results indicated that the platelet membrane was coated on PTK nanoparticles successfully. The drug loading of CsA in CsA@PPTK was 4.97%. As shown in Figure.1E, the size and PDI of CsA@PPTK kept stable over 23 days, indicating platelet membrane coating increased the stability of CsA@PPTK. Moreover, CsA@PTK and CsA@PPTK did not cause significant hemolysis at concentration of 1 mg/mL.
ROS scavenging ability and in vitro drug release of CsA@PPTK
When CsA@PTK and CsA@PPTK was suspended in PBS, the suspension solution remained opalescence for 24 h. However, after CsA@PTK and CsA@PPTK was suspended in PBS solution containing 1 mM sodium hypochlorite (NaClO, an important ROS generated in ischemic myocardium under oxidative stress), CsA@PTK and CsA@PPTK suspension solutions became clear within 12 h. In PBS solution containing 10 mM NaClO, CsA@PTK and CsA@PPTK suspension solutions became clear within 3 h (Figure.2A, Figure.S4A). These results indicated that degradation rate of CsA@PTK and CsA@PPTK was accelerated with the increase of NaClO concentration. The degradation of CsA@PTK and CsA@PPTK exhibited ROS-dependent manner. The drug release tests indicated that after incubation with PBS solution containing 1 mM NaClO at 37 ℃ for 4 h, about 70% CsA was released from CsA@PTK and CsA@PPTK. When concentration of NaClO in PBS solution increased to 10 mM, 95% CsA was released from CsA@PTK and CsA@PPTK within 4 h. Nevertheless, when CsA@PTK or CsA@PPTK was incubated with PBS, less than 70% CsA was released within 24 h (Figure.2B, Figure.S4B). These results demonstrated that platelet membrane coating did not affect the ROS-responsive characteristic of CsA@PPTK. CsA@PTK and CsA@PPTK released CsA in ROS-dependent manner. The high ROS level microenvironment induced by ischemia reperfusion would facilitate the release of CsA from CsA@PPTK.
The in vitro ROS scavengingproperty of CsA@PPTK was assessed by DPPH assay[34]. When @PPTK, CsA@PTK and CsA@PPTK was incubated with DPPH solution for 15 min, the dark purple color of DPPH solution apparently faded as compared with DPPH or DPPH/CsA ethanol solution (Figure.2C). Quantitative analysis showed that the absorbance value of DPPH solution containing @PPTK, CsA@PTK and CsA@PPTK significantly decreased with the prolongation of incubation time (Figure.2D), revealing the clearance effect of @PPTK, CsA@PTK and CsA@PPTK on DPPH free radical.
The protective effectof CsA@PPTK on H/R injured H9c2 cells
At first, the cytotoxicity of PPTK on cardiomyocytes was assessed by MTT method. As shown in Figure.S5A, 0.2~1 mg/mL @PTK increased the activity of normal H9c2 cells after incubation for 24 h. In addition, 0.5 mg/mL, 1 mg/mL and 2 mg/mL @PTK improved the activity of hypoxia for 3 h and reoxygenation for 4 h (H/R) injured H9c2 cells (Figure.S5B). It was reported that the optimized concentration of CsA to protect H/R injured H9c2 cells was 15 and 30 μg/mL [16]. The drug loading of CsA@PPTK was 4.97%. Therefore, 1 mg/mL @PTK was much higher than the maximum dose of @PTK that needed in the experiment, indicating that under the therapeutic doses, CsA@PTK exhibited no cytotoxicity on cardiomyocytes.
As shown in Figure.2E, CsA, CsA@PTK and CsA@PPTK improved the activity of H/R injured H9c2 cells. There was no significant difference in activity of H/R injured H9c2 cells between 15 μg/mL and 30 μg/mL free CsA. The protective effect of CsA@PTK and CsA@PPTK on H/R injured H9c2 cells was similar to CsA when concentration of CsA was 15 μg/mL. However, when the concentration of CsA was 30 μg/mL, the protective effect of CsA@PTK and CsA@PPTK on H/R injured H9c2 cells was significantly stronger than that of CsA. This resulted from two factors. On the one hand, CsA@PPTK and CsA@PTK increased solubility of CsA. On the other hand, CsA@PPTK and CsA@PTK could scavenge ROS in H9c2 cells induced by H/R.
The protective effect of CsA@PPTK on mitochondria of H/R injured H9c2 cells
DCFH-DA staining demonstrated that there were a large amount of ROS in no-treated H/R injured H9c2 cells, indicating acute oxidative stress occurred after H/R (Figure.S6A). However, compared with no-treated H9c2 cells, ROS-positive cells and ROS level were significantly reduced in @PTK, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells. Besides, there were no significant difference in ROS level between @PTK treated and CsA@PTK treated H/R injured H9c2 cells (Figure.S6B). The above results suggested that ROS level were significantly inhibited by @PTK, CsA@PTK and CsA@PPTK in H/R injured H9c2 cells, and PTK exerted an important role in protection H9c2 cells from H/R injury by reducing ROS level.
The effect of CsA@PPTK on mitochondrial ROS is showing in Figure.3A-B, compared with control group, red fluorescence intensity was much stronger in H/R injured H9c2 cells, while red fluorescence intensity was much weaker in CsA, @PPTK, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells. The statistical results of red fluorescence intensity in H/R injured H9c2 cells are showing in Figure.3C. The ratio of red to blue fluorescence intensity in H/R model group was significantly higher than that in control group, indicating that mitochondria produced a large amount of ROS after H9c2 cells was injured by H/R. Compared with H/R model group, the ratio of red to blue fluorescence intensity in CsA, @PPTK, CsA@PTK and CsA@PPTK treated group was significantly reduced. Meanwhile, CsA@PPTK treated H/R injured H9c2 cells showed the lowest ratio of red to blue fluorescence intensity, indicating CsA, @PPTK, CsA@PTK and CsA@PPTK reduced mitochondrial ROS level in H/R injured H9c2 cells, and CsA@PPTK exhibited the strongest effect on reducing mitochondrial ROS level in H/R injured H9c2 cells.
The effect of CsA@PPTK on the opening of mPTP in H/R injured H9c2 cells was shown in Figure.3D-E. Compared with the normal group, the calcein fluorescence intensity in mitochondria in H/R injured H9c2 cells was significantly decreased, indicating that H/R could significantly increase the opening of mPTP in H9c2 cells. Compared with the H/R model group, the calcein fluorescence intensity in mitochondria was significantly increased in CsA, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells, indicating that CsA, CsA@PTK and CsA@PPTK played a protective role against H/R injury by reducing the opening of mPTP in H/R injured H9c2 cells.
Next, JC-1 staining was used to detect the mitochondrial membrane potential, and the results are showing in Figure.3F. Compared with normal cultured H9c2 cells, the red fluorescence was obviously decreased and green fluorescence was obviously increased in H/R injured H9c2 cells, suggesting mitochondrial membrane potential was damaged during H/R in H9c2 cells. However, compared with H/R model group, the ratio of red to green fluorescence was significantly increased in CsA, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells, indicating that CsA, CsA@PTK and CsA@PPTK recovered the damaged mitochondrial membrane potential.
Cellular uptake of CsA@PPTK by H/R injured H9c2 cells
The specific cellular uptake behavior of H9c2 cells towards CsA@PPTKwas studied by HPLC. The uptake of CsA@PPTK by H9c2 cells is shown in Figure.S7. The uptake of CsA@PTK and CsA@PPTK by normal and H/R injured H9c2 cells exhibited time-dependent manner. The uptake of CsA@PPTK in H/R injured H9c2 cells was much higher than that in normal H9c2 cells at 2 h. Compared with CsA@PTK, a large amount of CsA@PPTK was taken up by H/R injured H9c2 cells.
The effects of different uptake pathway inhibitors on the uptake of CsA@PPTK in H/R injured H9c2 cells were observed. As shown in Figure.S8, sucrose, colchicine, 2-deoxy-D-glucose and methyl-β-cyclodextrin showed no significant effect on the uptake of CsA@PPTK by H/R injured H9c2 cells. This suggested that the receptor-mediated endocytosis pathway did not involve in the uptake of CsA@PPTK by H/R injured H9c2 cells. Compared with the control group, the uptake of CsA@PPTK in H/R injured H9c2 cells significantly reduced at 4 ℃. In theory, adsorptive endocytosis is temperature dependent. The platelet membrane coated on the surface of CsA@PPTK is very similar with membrane of H/R injured H9c2 cells, which resulted in the strong adsorptive effect between CsA@PPTK and H/R injured H9c2 cells. Therefore, CsA@PPTK was taken up by H/R injured H9c2 cells mainly through adsorption endocytosis[35].
The ex-vivo targeted of CsA@PPTK
The specific binding of Coumarin 6@PPTK on aortic vessels with endothelia injured is showing in Figure.4A-B. The green fluorescence intensity of Coumarin 6@PPTK in normal aortic vessels was very weak, indicating little amount of Coumarin 6@PPTK was bound with normal aortic vessels. Compared with Coumarin 6@PTK, the green fluorescence intensity of Coumarin 6@PPTK in aortic vessels with endothelia injured was much stronger, suggesting a large amount of Coumarin 6@PPTK was bound with endothelia injured aortic vessels. The above data implied that CsA@PPTK could specifically bind with endothelia injured vessels in ischemic area.
In vivo targeting of CsA@PPTK
As shown in Figure.S9, after acute myocardial ischemic mice were intravenously injected a single dose of Cy7.5 labeled CsA@PPTK (Cy7.5@PPTK) 5 min before reperfusion, Cy7.5 labeled CsA@PTK (Cy7.5@PTK) was mainly distributed in the liver and spleen, and little amount of Cy7.5@PTK was distributed in heart. However, compared with Cy7.5@PTK, a large amount of Cy7.5@PPTK was distributed in the heart, while little amount of Cy7.5@PPTK was distributed in the liver of MI/RI mice. As shown in Figure.4C, Cy7.5@PPTK was distributed in the whole heart when it was intravenously injected to sham mice. However, Cy7.5@PPTK was mainly distributed in downstream areas of the occluded coronary artery heart when it was intravenously injected to MI/RI mice. Cy7.5@PTK distributed in the whole heart of MI/RI mice. Next, the heart was cut into six or seven transverse sections from apex to atrium, and the distribution of Cy7.5@PPTK in transverse sections is showing in Figure.4D. Cy7.5@PPTK distributed in all heart transverse sections of sham mice. Cy7.5@PTK also appeared in all heart transverse sections of MI/RI mice. However, Cy7.5@PPTK was mainly distributed in transverse sections of ischemic area especial in transverse section of apex of MI/RI mice heart. This indicated that Cy7.5@PPTK exhibited obvious ischemic myocardium targeting characteristic.
In vivo therapeutic effect of CsA@PPTK on MI/RI mice
On 28th day after reperfusion, echocardiography was applied to reveal the recovery of overall cardiac functions of MI/RI mice, and results are showing in Figure.5. The mice in sham group showed regular and stable heart beats along with a large amplitude. The amplitude of heart beats was reduced and ventricular cavity volume was increased in normal saline treated MI/RI mice (Figure.5A), suggesting an abnormal ventricular remodeling occurred. CsA and its preparations increased the amplitude of heart beats and decrease the ventricular volume of MI/RI mice as compared to normal saline. The left ventricular ejection fraction (LVEF) and fractional shortening (FS) reveal systolic function of the heart. Quantitative analysis revealed that EF and FS increased along the sequence of normal saline, CsA, @PPTK, CsA@PTK and CsA@PPTK in MI/RI mice (Figure.5B-C), manifesting the improvement of systolic function and inhibition of abnormal ventricular remodeling by using CsA, @PPTK, CsA@PTK and CsA@PPTK. Compared with normal saline, @PPTK obviously increased the heart function of MI/RI mice, indicating that ROS scavenging was an effective and necessary treatment method for MI/RI mice. CsA@PPTK treated mice showed the highest EF and FS in all group, signifying that ROS scavenging and anti-apoptosis could improve heart function significantly. The echocardiography was also applied to reveal the recovery of overall cardiac functions on 70th day post MI/RI, and results showed that CsA@PPTK performed the strongest effect on the recovery of heart function at dose of 2.5mg/kg (Figure.S10).This suggested that early reduction of reperfusion injury increased cardiac function for a longer period of time.
Figure.S11 shows the ROS level in infarcted heart area 1 day after reperfusion, red fluorescence represents ROS. The results indicated that compared with sham group, the ROS level in ischemic myocardium in normal saline treated group (MI/RI model group) was significantly increased. CsA, @PPTK, CsA@PTK and CsA@PPTK markedly decreased ROS level in ischemic myocardium. CsA@PPTK treated group showed the lowest ROS level in ischemic myocardium among 4 groups, which resulted from the following two reasons. Firstly, CsA inhibited the production of ROS in mitochondria. Secondly, PTK strongly scavenged ROS in ischemic cardiomyocytes and ischemic myocardium.
The phenotype of macrophage in the infarcted heart tissue was detected on 4th day post reperfusion. The M1 and M2 type macrophages were stained by their surface markers of CD86 (red) and CD206 (green), respectively. As shown in Figure.6A-B, the heart tissue exhibited strong green fluorescence in CsA@PPTK treated group, while heart tissue displayed weak green fluorescence in normal saline treated group. As compared with normal saline treated group, the ratio of M2 type macrophages to M1 type macrophages obviously increased in heart tissue in CsA, CsA@PTK, @PPTK and CsA@PPTK treated groups. CsA@PPTK treated group exhibited the highest ratio of M2 type macrophages to M1 type macrophages in heart tissue among 4 groups. Besides, as compared with normal saline treated group, the number of M2 type macrophages and M1 type macrophages significantly reduced in heart tissue in CsA, CsA@PTK, @PPTK and CsA@PPTK treated groups. CsA@PPTK treated groups showed a minimum of M2 type macrophages and M1 type macrophages in all groups. In addition, acute inflammatory reaction usually occurs after reperfusion, which is reflected by the secretion of inflammatory factors such as TNF-α from the activated immune cells [36]. The experimental results showed that compared with normal saline treated group, IL-1β and TGF-β concentration in serum was significantly decreased in CsA@PPTK treated MI/RI mice on 4th day after reperfusion (Figure.6C-D). Immunofluorescence staining results showed that compared with the sham group, Tregs in myocardial tissue significantly increased in the model group, and Tregs in myocardial tissue decreased after the treatment of CsA. Compared with model group, CsA@PPTK significantly increased the number of Tregs in the infarct area (Figure.6E-F). The above data demonstrated that CsA@PPTK alleviated MI/RI and recovered the heart function through regulating the phenotype of macrophage and reducing systemic inflammatory reaction.
TUNEL staining images of infarcted heart area on 7th day after reperfusion is showing in Figure.7A-B. The positive staining represents the apoptotic cardiomyocytes. Compared with normal saline treatment, CsA, @PPTK, CsA@PTK and CsA@PPTK significantly reduced the apoptosis of cardiomyocytes in ischemic area. CsA@PPTK treated group displayed a minimum number of apoptotic cardiomyocytes in ischemic area among all groups, which resulted from inhibiting the production of mitochondrial ROS and the recovery of damaged mitochondrial membrane potential of cardiomyocytes.
Masson trichrome staining and H&E staining of heart tissue are showing in Figure.7C. In masson trichrome staining section, normal heart muscle is represented in red and fibrotic areas are represented in blue. Compared with sham group, the blue areas were increased in normal saline treatment group. Meanwhile, the ventricles were significantly dilated, and the walls of the ventricles were significantly thinner in the model group than that in sham group. The percentage of myocardial fibrosis area in myocardial section area of mice is showing in Figure.7D. The area of myocardial fibrosis in normal saline group was significant greater than that in sham group. CsA, @PPTK, CsA@PTK and CsA@PPTK significantly reduced myocardial fibrosis as compared with normal saline. As compared with the same dose of CsA, CsA@PPTK significantly reduced myocardial fibrosis at the dose of 2.5 mg/kg. The percentage of left ventricular area to myocardial section area is showing in Figure.7E. The percentage of left ventricular area to myocardial section area in normal saline group was marked smaller than that in sham group. CsA@PPTK significantly increased the percentage of left ventricular area to myocardial section area as compared with normal saline. As compared with the same dose of CsA, CsA@PPTK significantly increased the percentage of left ventricular area to myocardial section area at the dose of 2.5 mg/kg. The above results demonstrated that CsA@PPTK strongly inhibited the cardiac remodeling and fibrosis, subsequently markedly improved the cardiac function of MI/RI mice. These results were also consistent with the echocardiography data. In addition, a large number of inflammatory cells were infiltrated, and the structure of cardiomyocytes was unclear in normal saline group. The cardiomyocytes in sham group were orderly arranged and the cell structure was intact. In 2.5 mg/kg CsA@PPTK treatment group, the arrangement of cardiomyocytes was regular, and the inflammatory cell infiltration was decreased. These results indicated that CsA@PPTK exhibited anti-inflammatory effect and protected myocardium through anti-inflammatory effect.
The CX43 is the main protein that constitutes the intercellular gap junction of ventricular myocytes[37]. The effect of CsA@PPTK on the expression of CX43 in myocardium of MI/RI mice is showing in Figure.8A-B. Compared with sham group, the expression of CX43 in the anterior wall of the left ventricle was significantly decreased in normal saline group. Compared with normal saline and same dose of CsA@PTK, CsA@PPTK significantly increased the expression of CX43 in the anterior wall of the left ventricle. These results indicated that CsA@PPTK significantly protected the activity of cardiomyocytes after MI/RI, and subsequently improved the cardiac function of MI/RI mice.
The effect of CsA@PPTK on the expression of MMP-9 in myocardium of MI/RI mice is showing in Figure.8C-D. Compared with sham group, the expression of MMP-9 in the left ventricle anterior wall was significantly increased in normal saline group. CsA, @PPTK, CsA@PTK and CsA@PPTK significantly reduced the expression of MMP-9 in the left ventricle anterior wall of MI/RI mice as compared with normal saline. CsA@PPTK markedly reduced the expression of MMP-9 in the left ventricle anterior wall as compared with the same dose of CsA@PTK, suggesting CsA@PPTK effectively protected heart from ventricular remodeling.
Interestingly, the arteriole density in the infarct area was also increased (Figure.8E) in CsA@PPTK treatment group, indicating that blood vessels were preserved in infarct area by using CsA@PPTK. This demonstrated that CsA@PPTK reduced apoptosis of endothelial cells of heart arteries, and subsequently improved blood supply in infarct area and cardiac function of MI/RI mice.