Fabrication of gene loaded biotinylated microbubbles and gene-loaded BMBs
Biotinylated microbubbles (BMBs) carrying FITC-labeled SERCA2a/GFP-encoded Ad (named as S-BMBs), BMBs carrying rhodamine-labeled Cx43/RFP-encoded Ad (C-BMBs) and BMBs carrying both Ads (S/C-BMBs) as previously described [24]. Briefly, DSPC, DSPE-PEG2000 and DSPE-PEG2000-Biotin (molar ratios = 9:0.5:0.5) were dissolved in chloroform. The solvent was then evaporated under nitrogen flow at room temperature, producing a thin layer of phospholipid membrane. After two-hour vacuum treatment, the completely dried phospholipid membrane was hydrated at 60 °C with Tris (hydroxymethyl) aminomethane buffer saline, and transferred into vials (1 mL for each). After sealing the vials, perfluoropropane (Flura, Newport, TN, USA) was added. These vials with phospholipid suspension were mechanically vibrated for 30 s. The resulting BMBs were rinsed and incubated with streptavidin. After eliminating free streptavidin by PBS rinse, these BMBs (about 1×109) were further incubated with Ad to fabricate the gene-loaded BMBs.
Characterization of BMBs and gene-loaded BMBs
Particle size and size distribution of MBs were measured with Accusizer 780 Optical Particle Sizer (Particle Sizing Systems, Santa Barbara, CA, USA). A drop (about 20 μL) of each kind of BMBs suspension was applied to the microscope slide and observed under an optical microscope (Olympus, Tokyo, Japan). Furthermore, each kind of BMBs suspension was negatively stained with an aqueous solution of uranyl acetate and observed using a transmission electron microscope.
BMBs stability
The stability of BMBs was manifested by the change in the particle size and concentration over time. The particle size and concentration of 1ml of diluted BMBs were measured immediately after the completion of fabrication at room temperature, or after 15, 30, 45 and 60 mins by Accusizer 780 Optical Particle Sizer.
Gene loading capacity of BMBs
To determine the gene loading capacity of BMBs, FITC-labeled SERCA2a/GFP-encoded adenovirus (S-Ad) or rhodamine-labeled Cx43/RFP-encoded adenovirus (C-Ad) range from 1.25 μl to 20 μl (1×109 pfu/ml) was mixed with BMBs to fabricate gene loaded BMBs (S-BMBs or C-BMBs), respectively. Then, the gene loading capacity was determined by flow cytometry and qPCR. After determined the lowest amount of Ad to achieve the maximum gene loading efficiency. Furthermore, S-Ad and C-Ad was added into BMBs to making dual gene-loaded BMBs (S/C-BMBs) at the ratio of 1:1, 1:2 or 2:1, respectively. Next the dual gene loading capacity was determined by flow cytometry and qPCR.
Isolation culture and identification of BMSCs
Conforming to the Directive 2010/63/EU of the European Parliament, SD (Sprague Dawley) rats at 4 weeks of age were euthanatized with pentobarbital sodium (60 mg/kg body weight, Sigma-Aldrich Inc., USA) via intraperitoneal injection once. Then, bilateral tibial and femoral bones were dissected from body trunk and stored on ice in 75% alcohol. Subsequently, femurs and tibias were separated and both ends of each femur or tibia were cut, and a 22-gauge needle attached to a 10cc syringe containing complete medium was then inserted into the spongy bone exposed by removal of the growth plate. The marrow plug is then flushed from the bone with 5 ml of complete medium and collected in a 50 ml conical tube. Cells were then cultured in T‐75 cell culture flask, with a cell concentration of 1×105/mL, using Mouse Mesenchymal Stem Cell Growth Medium (Cyagen, China). BMSCs were then purified and passaged by attachment method. Cells were incubated under standard cell culture conditions with 5% CO2, at 37 °C and 95% relative humidity. The medium was changed every three days, and BMSCs were passaged when 80%-90% confluence was reached. BMSC identity was confirmed on the basis of morphological criteria, plastic adherence, and specific surface antigen expression: CD29(+), CD90(+), CD45(−).
Ultrasound-mediated gene transfection with BMBs in vitro
Ultrasound-mediated gene transfection was performed by using an ultrasound system including an arbitrary waveform generator (model AFG3102, Tektronix, USA), an RF power amplifier (model AR150A100B, AR, USA), and a single-element planar ultrasound (US) transducer (frequency = 1 MHz; Valpey Fisher Corp., MA, USA). Briefly, BMSCs were seeded in 12-well plates (1×105 cells per well) and transfection experiments were conducted when the cell confluence reached 70–80%. Then gene-loaded BMBs were added to the well, and to guarantee a close contact between transfection complexes and cells, the 12-well plate was sealed firmly and inverted for 15 min. Then ultrasound exposure was performed. The multiplicity of infection (MOI) was adjusted to 500 throughout the study. Ultrasound conditions were set as follows: frequency 1 MHz, power 2.0 W/cm2, duration 60 s, duty cycle 10%.
Detection of the in vitro gene transfection efficiency
To examine the ultrasound-mediated gene transfection efficiency, the following groups were included: (1) Control group; (2) Ad group, only Ad was used to transfect BMSCs; (3) Ad + US group, Ad was used to transfect BMSCs under the aid of ultrasound; (4) Ad + UTMD group, gene-loaded BMBs was use to transfect BMSCs under the aid of ultrasound. These BMSCs were cultured for another 48 h. Fluorescence microscopy was applied to detect GFP and RFP expression of each group, western blot and qPCR analysis to detect SERCA2a and Cx43 protein and mRNA level of each group.
Cell viability assay and in vivo biocompatibility
Cell viability was measured immediately after the gene transfection using a Cell Counting Kit-8 (CCK-8) according to the manufacturer’s protocol (Dojindo, Japan) to determine the possible cell damage caused by UTMD. Relative cell viability (RCV) was assessed using CCK-8 assay and then determined in a 96-well plate reader (BioTek Synergy 4) at 450 nm wavelength with the equation RCV (%) = At − Anc/Apc − Anc ×100%. Furthermore, to evaluate the in vivo biocompatibility of the BMBs, healthy SD rats were intravenously injected with gene-loaded BMBs. On the 15th day post-injection, fresh blood samples (1.0 mL) were obtained by cardiac puncture from the rats for serum biochemistry study. Subsequently, the major organs including lungs liver, spleen and kidneys of were carefully collected for H&E staining histology analysis. Healthy rats without any intervention were used as the control.
Animal model
The experimental protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval number of IACUC-20160218-057). Following the ARRIVE criteria [25], the experiment was carried out in strict accordance with the “3R” principle of substitution, reduction and optimization to minimize damage to animals. Male or female healthy SD rats (8~10 weeks old, weight 200-260 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental animals were well housed under standard conditions of room temperature and dark-light cycles with sufficient water and food.
On the first day of the experiment, rats were induced for anesthesia with Dexmedetomidine (0.3 mg/kg body weight, Sigma-Aldrich Inc., USA)/midazolam (4.0 mg/kg bw, Sigma-Aldrich Inc., USA)/butorphanol (5.0 mg/kg bw, Sigma-Aldrich Inc., USA) [26] via intraperitoneal injection once. Then, the rats were intubated with a 16G intravenous catheter (Introcan 16G, Braun Medical Co., Ltd., Germany) and ventilated with a rodent ventilator (HX-100E, Taimeng Software Co., Ltd., Chengdu, China). Left anterior descending artery (LAD) ligation was performed as previously described [27]. The rat’s heart was fully exposed by thoracotomy. Finding the coronary vein as a landmark, then permanently ligated the LAD approximately 2~3 mm distal from its origin with a depth of 0.5 mm by a 5/0 suture. Evidence of a MI was confirmed by pale and hypokinesia in the left ventricular anterior wall and ST-segment elevation on an electrocardiogram. Penicillin 160,000 u was intraperitoneal injected for 3 days after the surgery.
Experimental animal groups
Forty-eight successfully modeled SD rats were randomly divided into control and experimental groups: (1) Control group (MI + PBS, n = 8); (2) UTMD group (MI + PBS + US-BMBs, n = 8); (3) BMSCs group (MI + BMSCs, n = 8 ); (4) B+S+U group (MI + BMSCs + US-S-BMBs group, n = 8); (5) B+C+U group (MI + BMSCs + US-C-BMBs group, n = 8); (6) B+S/C+U group (MI + BMSCs + US-S/C-BMBs-1:1 group, n = 8). Another 8 randomly chosen healthy rats were operated without ligation and divided into SHAM groups.
BMSCs transplantation
Four weeks after the successful ligation, the rats underwent surgery again. The rats from SHAM, Control and UTMD groups were injected with PBS (100 μl). Rats from the rest groups were injected with BMSCs (5×106, 100 μl) at the myocardial infarct zone and the peri-infarct zone.
UTMD-mediated localized co-delivery of genes
Two days after BMSCs transplantation [20], UTMD-mediated gene localized co-delivery was performed. The treatment groups were infused with 100 μl of the S-BMBs, C-BMBs or S/C-BMBs respectively via the tail vein at a constant rate (15 ml/h), and then washed with PBS 100 μl. A Mindray Kunlun 7 ultrasound machine with a line array probe (L 11-3 U) was used at a setting of fundamental frequency 5.6 MHz-11.8 MHz, harmonic frequency 7 MHz ~ 9 MHz and mechanical index 0.53. Contrast mode was activated at the beginning of infusion. On seeing the filling of BMBs in the left ventricle cavity, the FLASH (mechanical index: 1[20]) function was triggered manually at an interval of 3 s ~ 5 s, lasting for 10 mins [28]. The rats of Control group were infused with PBS 100 μl and performed as above.
Left ventricular function analyzing
Echocardiography was performed 4 weeks after AMI and 2 weeks and 4 weeks post-treatment. A Vevo2100® ultrasound imaging system (High-Resolution Micro-Imaging System, VisualSonics, Canada) equipped with an 18 MHz transducer was used by an investigator blinded to group designation. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated by M-mode tracing. The dimension data are presented as the average of measurements of three selected beats.
Electrophysiological examination
Steady-state pacing was performed to test the ability of anti-arrhythmia after treatment. On the 28th day after BMSCs transplantation, Rats were induced for anesthesia by intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight, Sigma-Aldrich Inc., USA) once. ECG monitoring (BL-420F biological function experiment system, Taimeng Software Co., Ltd., Chengdu, China) was used to record the ECG activity in real time. The stimulation protocol was performed as described previously [29]. Briefly, the left chest was opened to fully expose the heart after anesthesia. A bipolar pacing electrode was placed in the left ventricular wall near the apex of the heart, with a diameter of 0.2 mm and a distance of 3 mm between the two electrodes. Continuous progressive stimulation (S1S1) was given to induce ventricular arrhythmias. Stimulus plan is that as below, pulse width of 0.1ms, step length at -2 ms, stimulus frequency starts at 5 Hz and stimulus voltage begins with 5 V, gradually increasing by 1 V. End point of stimulation is induction of ventricular tachycardia (VT) or ventricular fibrillation (VF) with more than 6 consecutive ventricular premature beats. After the Electrophysiological examination, the rats were euthanized by pentobarbital sodium (60 mg/kg body weight, Sigma-Aldrich Inc., USA) [26] via intraperitoneal injection once, and the hearts were taken for subsequent test. The procedure conformed to the Directive 2010/63/EU of the European Parliament.
Enzyme-linked immunosorbent assay (ELISA) analysis
ELISA was applied to determine the levels of SERCA2a and Cx43 in the infarcted myocardial tissue homogenates. commercial ELISA kits (R&D systems) were used per the manufacturer’s instructions. Samples and standards were prepared according to manufacturer's instructions.
Western blot assay
Protein expression levels of SERCA2a, Cx43, VEGF and cardiac-specific proteins (cTnT and α-actin) in the infarct zone were detected by Western blot according to the previous study. [30] Total proteins were extracted from treated myocardial infarct tissue using a RIPA buffer with protease and phosphatase inhibitors (G2002, Wuhan Servicebio Co., Ltd., China). The protein concentrations were determined by the Bicinchoninic acid (BCA) method as a protein standard. Proteins were separated by electrophoresis, transferred to a cellulose acetate membrane and blocked. The primary antibody (GB23303, Wuhan Servicebio Co., Ltd., China) was added followed by the secondary antibody (GB23302, Wuhan Servicebio Co., Ltd., China). The density of each band was quantitated by a densitometer with AlphaView Software for FluorChem Systems (ProteinSimpleTM).
Analysis of immunofluorescent protein expression
Immunofluorescence was performed to detect SERCA2a and Cx43 protein expression in the infarcted myocardium. Frozen sections of myocardial tissue were prepared as described previously [31]. Then, the prepared frozen sections were incubated with 5% (volume fraction) bovine serum albumin (Solarbio, China) for 30 mins. The primary antibody (at 1:100 /1:200 volume) was added, incubated overnight at 4 ℃, and then washed thrice for 5 mins with PBS. Next, the fluorescently labeled secondary antibody (at 1:300/1:400 volume) was added and incubated at 37 ℃ for 50 min. The slides were washed as above. Subsequently, the image was observed and photographed using a fluorescence microscope (Nikon Eclipse TI-SR, Nikon Inc., Japan). The fluorescence area ratio was quantitated with Image-Pro Plus version 6.0 software (Media Cybernetics, Bethesda, MD) by 2 observers blinded to the conditions.
Histological evaluation of neovascularization in the infarcted zone
To evaluate neovascularization, formaldehyde-fixed rat hearts were dehydrated and then embedded in paraffin. Paraffin-embedded sections (4 μm) were dewaxed and incubated with the primary antibody against factor VIII (ThermoFisher Scientific, America). After visualization with diaminobenzidine (DAB), we counted the positively stained micro-vessels under a Nikon eclipse E100 light microscope (Nikon Inc., Japan; 100× magnification). Microvascular endothelial cells showed a layer of brown ring-like precipitates with a diameter of less than 20 μm, which were calculated as previously described [32]. All histological analyses were independently performed by two experienced pathologists under double-blinded conditions.
Measurement of the infarct size
Twenty eighty days after treatment, the rats were euthanasized with overdose of pentobarbital sodium (60 mg/kg body weight, Sigma-Aldrich Inc., USA) via intraperitoneal injection once. Then, after cardiac perfusion with saline hearts of the rats were extracted and fixed in 4% paraformaldehyde (Wei Bio Technology Co., Ltd., Shanghai, China). Paraffin-embedded samples were sectioned at 4 μm, and Masson’s trichrome staining was performed. The infarcted zone was evaluated based on the percentage of blue staining, indicative of fibrosis, and quantitated with Image-Pro Plus version 6.0 software by two observers blinded to the conditions.
Statistical analysis
All data are summarized as mean ± standard deviation. The data were analyzed using SPSS 21.0. Parametric comparisons were tested by One-way analyses of variance (ANOVA) with subsequent post-hoc multiple comparisons using the least significant difference (LSD) test. Probability values P < 0.05 were considered statistically significant.