Synthesis and Characterization of CdSe/ZnS quantum dots and Oil-soluble CdSe/ZnS core-shell quantum dots (QDs) were purchased from the Suzhou Xingshuo Nanotechnology Company. The QDs were phase transferred using glutathione (GSH) and cross-linked with carboxyl-PEG-carboxyl groups (Sigma-Aldrich CAT#14565, Mn = 2000) to obtain PEGylated polymer-encapsulated QDs. One mL of CdSe/ZnS QDs (10 mg) chloroform solution was added to 10 mL of 250 mg GSH plus 200 mg sodium hydroxide aqueous solution which were magnetically stirred. The chloroform in the mixture gradually evaporated at room temperature. The ligand exchange of QDs is also completed at the water-oil interface. The QDs aqueous solution was dialyzed against borate buffer (pH 8.0, 20 mM) for at least 72 hours to remove free glutathione in the solution. The glutathione molecules on the QD surface are cross-linked through carboxyl-PEG-carboxyl which helps to improve colloidal stability. The carboxyl group in the PEG crosslinker is activated by the activator 1-(3-dimethylaminopropyl) 3-ethyl carbodiimide hydrochloride (EDC) (10mg/mL). After 15 minutes of activation at room temperature, the active PEG crosslinker was added to the GSH-QDs aqueous solution (where the molar ratio of QD/PEG is 1/1000) under magnetic stirring at room temperature for 2 hours. The resulting PEGylated QDs solution was dialyzed in borate buffer for 72 hours to remove unbound PEG and small molecules. Finally, the QDs solution was concentrated by rotary evaporation to about 5 μM for later use.
Modification of peptides
Gly-Gly-Gly-Gly-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe is a peptide targeting ischemic myocardium with lysine residues at the end which was synthesized by GenScript. The purity of peptide detected using high-pressure liquid chromatography was >95%. The target peptide was covalently coupled to the carboxyl group on the surface of the QDs through the terminal amino group. In the general coupling process, the PEGylated QDs were dispersed in phosphate buffer (pH 6.0, 25mM) and EDC was added (Concentration: about 1mg/mL). The reaction was activated at room temperature for 0.5 hour. The excess activator was removed using ultrafiltration and the targeting peptide (concentration about 1mg/mL) was added and reacted at room temperature for 1 hour. The excess targeting peptide was removed using ultrafiltration to obtain the target. QDs was coupled to peptides (Peptide-PEG-QDs, PPQD).
Package of DNA Plasmids
Polyvinylimide (Mw 2000) was dispersed in PB buffer (1mg/mL). The pH was adjusted to neutral. The targeted peptide-coupled QDs (PPQDs) were mixed with different proportions of polyvinylimide and quickly Stirred. Moreover, 1% agarose gel electrophoresis was performed to analyze the electrostatic self-assembly process of QDs and PEI in order to get a better QD/PEI ratio. Under a better QD/PEI ratio, the plasmids with different DNA concentrations were mixed with QD/PEI. Agarose gel electrophoresis was used to analyze the plasmids better ratio loaded on the QDs (Agarose concentration 1%, voltage 80V, time 15 minutes).
Animals
Male SPF grade Sprague-Dwaly (SD) rats (weighing 250-300g) and c57BL6/J male mice (weighing 18–22 g) were purchased from the Second Military Medical University Experimental Animal Center. The Animal Research Ethics Committee approved the animal protocols of the Second Military Medical University in accordance with the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication vol. 25 no. 28, revised 1996). The experimental animals were kept under standard room conditions with a temperature of 25±1°C, the humidity of 60% and 6 a.m. to 6 p.m. natural lights. Standard rodent chow and water were given routinely.
The experimental animals were divided into the sham group, the IR group and the CSE group. The IR treatment was induced by ligating the left anterior descending artery (LAD) for 30 minutes and reperfusion for 24 hours as previously described(8). The sham group underwent thoracotomy without ligation. The IR group were subjected to IR treatment and intravenous injection with 20 μl vector nanocarriers before reperfusion. The CSE group were subjected to IR treatment and intravenous injection with 20 μl CSE nanocarrier before reperfusion.
In vivo fluorescence imaging system
The Bruker in-vivo F PRO system was applied in vivo whole-animal imaging analysis. Images of the post-injection of CSE nanocarriers were captured and analyzed. The excitation and emission filters were set to 410 and 700 nm (bandpass, ±15 nm) respectively.
Echocardiography
Echocardiography was performed blindly four days after the surgery (VisualSonics Vevo 2100, M-mode, and 30MHz probe). The conscious mice were examined at the mid-papillary level. LV dimensions in diastole and systole especially the ejection fraction (EF) were measured following the standard procedures and calculations.
Infarct size measurement
Evans blue dye (2%, Solarbio, China) was injected in the vein but the area at risk (AAR) remained non-dyed (17). The heart's infarct size was assessed using the TTC-staining technique and digital measurement with Image-Pro Plus software (Media Cybernetics). The area at risk/left ventricle ratio (AAR/LV) and scar area/left ventricle ratio was quantified and calculated.
Apoptosis
TUNEL staining was conducted as previously described(20). Image-Pro Plus software (Media Cybernetics) was used to detect the apoptosis rates of TUNEL sections.
Cell culture and treatment
The neonatal cardiomyocytes of rats were cultured and used to conduct the hypoxia-reoxygenation (H/R) experiments [9]. The cardiomyocytes were divided into four groups. For the N+Vector group, the cells were cultured in DMEM with 50 μl vector nanocarriers in 5%CO2 and 95% air for 24 hours. Moreover, in the N+CSE group the cells were cultured in DMEM with 50 μl CSE nanocarriers in 5%CO2 and 95% air for 24 hours. Additionally, for the HR+Vector group, the cells were cultured in DMEM with 50 μl vector nanocarriers with 1% O2, 5% CO2 and 94% N2 atmosphere for 24 h and 5% CO2 and 95% air for 6 hours. lastly, for the HR+CSE group, the cells were cultured in DMEM with 50 μl CSE nanocarriers with 1% O2, 5% CO2 and 94% N2 atmosphere for 24 hours and 5% CO2 and 95% air for 6 hours.
ATP and LDH test
The conditioned cell culture supernatant (200 μl) was collected after HR and was used to determine LDH levels using a spectrophotometric kit (Roche Diagnostics) and ATP concentrations using an ATP assay (Colorimetric/Fluorometric, Abcam, ab83355) according to the manufacturer's instructions.
Western blotting
Protein concentrations were determined using the BCA protein assay kit according to the manufacturer's protocol. Western blotting (WB) was conducted as previously described. Primary antibodies were listed as follows: CSE, Abcam, ab96755; CHOP, Cell Signaling Technology, 2895S; GRP78, Abcam, ab21685; eIF2a, Abcam, ab169528; ATF-6, Cell Signaling Technology, #65880; Parkin, Cell Signaling Technology, #2132; ATG7, Cell Signaling Technology, #2631 ; Nix, Abcam, ab109414; LC-3, Abcam, ab192890; GAPDH, ThermoFisher, MA5-15738-D680. The grayscale value of the bands was quantified using Image lab software (BIO-RAD, USA).
Statistical analysis
GraphPad Prism 7.0 was used to analyze the data in the study. The continuous data were expressed as the mean ± standard error (SEM). One-way ANOVA was used followed by Post-hoc t Turkey's test to compare the variables of different groups. A P-value <0.05 was considered to have statistical significance.