## 2 Materials and Methods
### Treatment Formulations  
The three treatments administered to the rats were naringenin dissolved in DMSO, naringenin encapsulated in PEG-PCL, and PEG-PCL micelle without any naringenin. Naringenin, a flavanone and MARK4 inhibitor with a molecular weight of 272 g/mol and poor water solubility, was obtained from Indofine Chemical Company Inc. (Hillsborough, NJ, USA). PEG-PCL was obtained from Polymer Source (Dorval, QC, Canada).  
In the first naringenin solution, naringenin was dissolved in 1% DMSO/99% phosphate-buffered saline (PBS), resulting in a 1.3 mg naringenin/mL solution. To prevent the precipitation of naringenin, this solution required being vortexed just prior to treatment administration. Vortexing entailed placing \(\sim\)10 mL of a solution in a vial, holding the vial against a vortex pad for 5-10 s, and then visually inspecting the vial to confirm that the solution appeared homogenous.  
In efforts to minimize the potential toxicity of DMSO, eliminate the need for pre-treatment vortexing, and develop a nanoparticle platform that could later be modified to target cardiac tissue, we developed an alternative naringenin loaded into polymeric micelle solution. In this solution, naringenin was encapsulated in a PEG-PCL micelle [PEG (4000)-PCL (3000); PDI 1.15). Stock solutions of naringenin (10 mg/mL) and PEG-PCL (50 mg/mL) were made in tetrahydrofuran (THF). An amount of 100 uL of naringenin solution was combined and sonicated with 100 uL of PEG-PCL solution. This mixture was introduced to 4 mL ultrapure water (Millipore Milli-Q water) and sonicated at 37 \({}^{\circ}\)C for 10 min to ensure homogenization. The vial was placed in a dark environment to evaporate THF overnight. The sample was purified and concentrated using spin filtration (molecular weight cutoff of approximately 50 kDa) at 4000 RPM for 25 min to remove the free drug (i.e., unloaded naringenin). After that, it was resuspended in a final volume of 2 mL ultrapure water and then filtered with a 0.22-micron syringe filter to remove large/aggregate particles.  
The naringenin-loaded micelles were characterized by UV-absorption spectroscopy in acetonitrile (ACN) to capture their absorbance spectra (Varian-Cary 100 Bio spectrophotometer, Agilent Technologies, Santa Clara, CA, USA). Micelles' hydrodynamic diameter and zeta potential were also examined using dynamic and electrophoretic light scattering, respectively (Zetasizer Nano ZS, Malvern Panalytical, Malvern, UK). Micelles' diameter was confirmed using transmission electron microscopy (JEM-1010 microscope, JEOL, Tokyo, Japan) at 80 kV. The integration of naringenin into micelles was demonstrated by Fourier transform infrared (FTIR) spectroscopy (VERTEX 70v FT-IR Spectrometer, Bruker Scientific LLC, Billerica, MA, USA). The samples naringenin-loaded micelle, naringenin alone, PEG-PCL micelle alone, and a physical mixture of naringenin and PEG-PCL-micelle were mixed with potassium bromide and compressed into a disk for FTIR analysis.  
The concentration of naringenin (Nar) was assessed by dissolving the micelles in ACN and measuring at a wavelength of 285 nm. The encapsulation efficiency and loading of naringenin were evaluated spectrometrically utilizing the subsequent equations:  
* Encapsulation efficiency = (Nar mass after loading)/(Nar mass before loading) \(\times\) 100%;
* Drug loading = (Nar mass after loading)/((Nar mass after loading + PEG-PCL mass after loading)) \(\times\) 100%.  
Encapsulation efficiency was used to quantify how little naringenin was was wasted during the naringenin encapsulation process. Drug loading was used to quantify the percentage of naringenin-encapsulated micelle mass contributed by naringenin itself.  
The third treatment solution, an empty PEG-PCL micelle, was created without naringenin (as described above).