Ammonium bicarbonate (NH4HCO3) and dopamine hydrochloride were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Amino polyethylene glycol (mPEG-NH2, Mw = 5000) was supplied by Hunan Huateng Pharmaceutical Co., Ltd. (Hunan, China). Anhydrous calcium chloride (CaCl2), colchicine (Col) and sodium hydroxide (NaOH) were obtained from Aladdin (Shanghai, China). Evans blue (1%) and 2,3,5-triphenyltetrazolium chloride (TTC, 2%) were purchased from Solarbio, China. Anti-Rat IgG was purchased from Invitrogen (Carlsbad, CA). TUNEL Bright-Red Apoptosis Detection Kit was obtained from Yeasen biotech Co., Ltd. (Shanghai, China).
Synthesis of Colchicine-loaded Calcium Carbonate Nanoparticles (ColCaNPs)
The synthesis of ColCaNPs included 3 steps. Firstly, colchicine (8 mg) and CaCl2 (100 mg) were dissolved in anhydrous ethanol (120 mL). The mixture was transferred into a beaker and covered by parafilm with several pores. The beaker was left in a desiccator along with NH4HCO3 powder for 2 d at room temperature. Secondly, dopamine hydrochloride (600 mg) was added into the beaker with stirring under alkaline condition (pH~8.5) for 6 h, and then centrifuged. At last, mPEG-NH2 (20 mg) was added and stirred for 2 h. The mixture was purified by dialysis using 8000 D cutoff dialysis membrane against deionized water and freeze-dried to obtain ColCaNPs.
Nanoparticles without colchicine were designated as CaNPs, and nanoparticles without calcium carbonate were designated as ColNPs.
Characterization of Nanoparticles
The fourier transform infrared spectroscopy (FTIR) spectra were detected by a fourier transform infrared spectrometer (Nicolet iS50, USA). The powder X-ray diffraction (XRD) pattern was collected with an X ray diffractometer (Rigaku SmartLab, Rigaku, Japan). The hydrodynamic size was measured by dynamic light scattering (DLS) with a nano laser particle size analyzer (BT-90, Bettersize, China). The morphology was observed using a transmission electron microscope (TEM, HITACHI HT7700 Exalens, Japan).
Loading Capacity of Colchicine
The colchicine loading capacity was determined by UV-vis absorbance spectroscopy and calculated by the following equation:
Animal Models and Experiment Design
Male Wistar rats were purchased from the spifer Animal Center (SPF grade) and used for experiments when they were 12-14 weeks of age, weighing 280-320 g. The rats were fed adaptively for one week before experiments. Rats were divided into six groups according to different drug interventions: without drug intervention (MI), CaNPs, Col, ColCaNPs, ColNPs and sham operation (Sham). Each group consisted of 5 animals. The MI group was induced by ligating the left anterior descending (LAD) coronary artery of rats, but did not receive any treatment. The sham group underwent thoracotomy without coronary artery ligation. For drug interventions, different drugs were injected into the caudal vein at 30 min, 48 h or 96 h, respectively, after ligation of LAD. Blood was collected from the venous plexus of the anterior inner canthus before and 7 d after MI. One week later, the rats were sacrificed, and the heart, kidney and liver were taken out for relevant tests. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
Induction of MI Rat Model
All the rats were anesthetized with isoflurane, and after orotracheal intubation with a 22G venous catheter for controlled ventilation (Minivent, Harvard Apparatus) with controlled stroke (10 μL g-1) and frequency (150 min-1). After left thoracotomy and muscular dissection, ligation of the LAD was performed 2 mm below the left auricle with an 8–0 silk and a smooth catheter was applied on the artery to obtain an ischemia for 45 min. The ischemia was visually confirmed by the change in myocardial color turning into white and were assayed using a II lead electrocardiogram, then followed by reperfusion obtained by the catheter removal. Muscle and cutaneous plans were sutured with silk 6–0. Sham-operated animals were subjected to the same surgical procedure without LAD ligation.
The blood sample was centrifuged 10 min at 5000 rpm, and then the serum was stored at −80°C for the subsequent analyses. The LDH were measured based on the protocol. Inflammatory cytokines in this study included IL-1β, TNF-α, CRP, ALT and Cr were detected using ELISA kits (MDL Company) on the basis of the manufacturer's instructions
Measurement of Myocardial Infarct Size
Myocardial infarct size was determined by TTC method. All slices were incubated for 15 min in TTC (2%) at 37℃ in the dark for pathological examination. Images were captured with a microscope (ZEISS, Germany). TTC stained area (red, noninfarct area) and non-TTC stained area (white or pale, infarct area) were analyzed with an Image-Pro Plus image analysis software (Version 4.1, Media Cybernetics, LP, USA). The infarct size was calculated as the proportions of infarct myocardial to the whole myocardial tissues.
Histological Analysis and Immunohistochemistry
Briefly, fixed myocardial tissues were firstly dehydrated and embedded in paraffin. The kidney and liver transversely sectioned were performed with H&E staining for morphologic examination. Masson’s trichrome staining was also performed to detect myocardial fibrosis. Sections were observed using an OLYMPUS microscope (Japan).
We used the number of CD11c cells to document the M1 polarization and used the number of CD206 cells to document the M2 polarization. Paraffin-embedded heart tissue sections were stained with CD206 (1:100; ProteinTech Group) or CD11c (1:200; Abcam) to identify the CD206/CD11c antigen expressed by macrophages. Anti-Rat IgG was used as a secondary antibody to detect CD206/CD11c+ cells.
TUNEL staining was performed based on the standard protocol. Then cells were stained with TUNEL kit on ice according to the manufacturer's instructions. TUNEL-positive cells were counted under fluorescence microscopy (DM3000, Leica, Mannheim, Germany).
Rat tissues and cardiomyocytes were harvested and then split using RIPA protein extraction reagent (MDL, China). Proteins were transferred onto PVDF membranes, then incubated with the specific primary antibodies and the secondary antibodies. Protein lysates from cells were separated by 10% SDS-PAGE and then transferred onto NC membranes (Sigma). After blocking for 1 h, the membranes were incubated overnight at 4 °C with specific antibodies. Then, the secondary antibody (MD912577, MDL) were added and the membranes were maintained at room temperature for 1 h. The signals were determined by Immobilon Western chemiluminescent HRP Substrate (Millipore). Actin was acted as loading control. The primary antibodies were used as follows: anti-TLR4 (MDL, cat. no. MD2073-020), anti-NFκB (MDL, cat. no. MD7298), anti-NLRP3 antibody (MDL, cat. no. MD6251-020), anti-caspase-1 antibody (MDL, cat. no. ab2302), anti-IL-1β antibody (MDL, cat. No. MD 6444- 022), anti-TGF-β1 (MDL, cat. No. MD7237), anti-IL10 (MDL, cat. No. MD292- 020).
Results were expressed as the mean ± SD. Graph Pad Prism 6.0 and SPSS 20.0 software were used for statistical analysis. The significance of differences between two groups was assessed by Student's t-test or Mann-Whitney U test as appropriate, and ANOVA test or Kruskal-Wallis test used for comparisons among three or more groups. A P value < 0.05 was considered as statistically significant.