Injectable Nanocomposite Implants Reduce ROS Accumulation and Improve Heart Function after Infarction

Abstract In a myocardial infarction, blood supply to the left ventricle is abrogated due to blockage of one of the coronary arteries, leading to ischemia, which further triggers the generation of reactive oxygen species (ROS). These sequential processes eventually lead to the death of contractile cells and affect the integrity of blood vessels, resulting in the formation of scar tissue. A new heart therapy comprised of cardiac implants encapsulated within an injectable extracellular matrix‐gold nanoparticle composite hydrogel is reported. The particles on the collagenous fibers within the hydrogel promote fast transfer of electrical signal between cardiac cells, leading to the functional assembly of the cardiac implants. The composite hydrogel is shown to absorb reactive oxygen species in vitro and in vivo in mice ischemia reperfusion model. The reduction in ROS levels preserve cardiac tissue morphology and blood vessel integrity, reduce the scar size and the inflammatory response, and significantly prevent the deterioration of heart function.

buffer (10 × 10 −3 M Tris, 5 × 10 −3 M ethylenediaminete-traacetic acid (EDTA), and 1 × 10 −6 M phenylmethanesulfonyl-fluoride, pH 8.0) for 1 hour. Next, tissues were frozen and thawed three times in the hypotonic buffer. Tissues were washed gradually with 70% (v/v) ethanol and 100% ethanol for 30 minutes each. Lipids were extracted by 3x30 minutes washes of 100% acetone, followed by a 24-hour incubation in a 60/40 (v/v) hexane: acetone solution (solution was changed three times over 24 hours). The defatted tissue was washed in 100% ethanol for 30 min and incubated overnight at 4°C in 70% ethanol. Next, the tissue was washed four times with PBS (pH 7.4) and incubated in 0.25% Trypsin-EDTA solution (Biological Industries) overnight. The tissue was washed thoroughly with PBS and incubated in 1.5M NaCl (solution was changed three times in 24 hours), followed by washing in 50 × 10 −3 M Tris (pH 8.0), 1% triton-X100 (Sigma-Aldrich) solution for 1 hour. The decellularized tissue was washed in PBS followed by double distilled water and then frozen (-20°C) and lyophilized. The dry, decellularized omentum was ground into powder (Wiley Mini-Mill, Thomas Scientific, Swedesboro, NJ). The milled omentum was then enzymatically digested for 96 hours at room temperature under stirring, in a 1 mg mL −1 solution of pepsin (Sigma-Aldrich, 4000 U mg -1 ) in 0.1M HCl. Subsequently, the pH was adjusted to 7.4 using 5M NaOH and DMEM/F12 × 10 (Biological industries). The final concentration of decellularized omentum in the titrated solution was 1% (w/v).

Rheological properties.
Rheological measurements (n=6) were performed using a Discovery HR-3 hybrid Rheometer (TA Instruments, DE) with 8 mm diameter parallel plate geometry and a Peltier plate to maintain the sample temperature. Samples were prepared by encapsulation of AuNPs at different concentrations (50 and 0.5 µg mL -1 for high and low concentrations, respectively) or medium for control, to reach a final hydrogel percentage of 0.6% and 0.2%. In order to examine the solidification process, samples were loaded at a temperature of 4°C, which was then raised to 37°C, during which the oscillatory moduli of samples were monitored at a fixed frequency of 0.8 rad s −1 and a strain of 1%. U/mL Penicillin and 100 mg/mL streptomycin, and 0.5% (v/v) FBS. To enrich the cardiomyocytes population, cells were suspended in culture medium with 5% FBS and preplated twice (30 min). Next, cells were counted and seeded within the nanocomposite and pristine hydrogel at 2x10 7 cells in 100 µL pristine hydrogel (in order to achieve a final concentration of 0.6%). For ROS and bioluminescence experiments, cardiac cells were counted and seeded on white cell plates at a concentration of 5x10 5 cells per mL medium.
The cell-seeded implants or plates were cultivated at 37°C in a 5% carbon dioxide humidified incubator.
Cell viability. Implant viability was determined using a live/dead fluorescent staining assay with fluorescein diacetate (7 µg mL −1 , Sigma-Aldrich) and propidium Iodide (5 µg mL −1 , Sigma-Aldrich) for 20 min at 37°C. Live and dead cells within the different implants were visualized by an inverted fluorescence microscope.
Luminescence assay. All measurements were performed over time using Luminometer In order to identify the morphology of the heart tissue, Cine-FLASH (fast low angle shot) MRI was used to image the heart in a short-axis and long-axis view. Six short-axis slices covering the heart from the base to the apex and one long-axis slice were obtained to evaluate myocardial function and infarct size. The following sequence parameters were used: times. Samples were imaged using a confocal microscope (Nikon Eclipse NI-E). Images were processed and analyzed using NIS elements software (Nikon Instruments).
Heart immunohistochemistry. Heart sections were also stained for blood vessels detection.
Sections were fixed, permeabilized and blocked as specified above. Then, the sections were stained with α-CD-31 (100 µg, ab124432, Abcam) primary antibodies in blocking solution for