Dual-ligand supramolecular nanofibers inspired by the renin-angiotensin system for the targeting and synergistic therapy of myocardial infarction

Rationale: The compensatory activation of the renin-angiotensin system (RAS) after myocardial infarction (MI) plays a crucial role in the pathogenesis of heart failure. Most existing studies on this subject focus on mono- or dual-therapy of blocking the RAS, which exhibit limited efficacy and often causes serious adverse reactions. Few studies have been conducted on targeted therapy based on the activated RAS post-MI. Thus, the development of multiple-functional nanomedicine with concurrent targeting ability and synergistic therapeutic effect against RAS may show great promise in improving cardiac function post-MI. Methods: We utilized a cooperative self-assembly strategy constructing supramolecular nanofibers— telmisartan-doped co-assembly nanofibers (TDCNfs) to counter-regulate RAS through targeted delivery and combined therapy. TDCNfs were prepared through serial steps of solvent exchange, heating incubation, gelation, centrifugation, and lyophilization, in which the telmisartan was doped in the self-assembly process of Ang1-7 to obtain the co-assembly nanofibers wherein they act as both therapeutic agents and target-guide agents. Results: TDCNfs exhibited the desired binding affinity to the two different receptors, AT1R and MasR. Through the dual ligand-receptor interactions to mediate the coincident downstream pathways, TDCNfs not only displayed favorably targeted properties to hypoxic cardiomyocytes, but also exerted synergistic therapeutic effects in apoptosis reduction, inflammatory response alleviation, and fibrosis inhibition in vitro and in vivo, significantly protecting cardiac function and mitigating post-MI adverse outcomes. Conclusion: A dual-ligand nanoplatform was successfully developed to achieve targeted and synergistic therapy against cardiac deterioration post-MI. We envision that the integration of multiple therapeutic agents through supramolecular self-assembly would offer new insight for the systematic and targeted treatment of cardiovascular diseases.


S1.1 Synthesis of NBD-β-Alanine and peptides:
980 mg of Alanine (1.1 equiv.), 4.14 g of K2CO3 (3 equiv.), and 2 g of NBD-Cl (1 equiv., 10 mmol) were added in 15 mL H2O. 20 mL of MeOH was then added dropwise under N2 protect (low yields if without nitrogen protection) and stirred for further 3 h at room temperature (LC-MS detection). After that, MeOH was removed by a rotary evaporator. The obtained solution was acidified to pH 3 to obtain precipitate (NBD-β-Alanine, yield: 87%). (HBTU) as the coupling reagent. The growth of the peptide chain was allowed to proceed according to the standard Fmoc solid-phase peptide synthesis protocol. After the last coupling step, the peptide derivatives were cleaved from the resin with a mixture of 1% trifluoroacetic acid and 99% dichloromethane for 10 minutes, then dried via rotary evaporation followed by diethylether precipitation.

S1. 2 Synthesis of peptides
Cy5.5-G-DRVYIHP ( Cy5.5 Ang1-7) were prepared through liquid-phase synthesis with the ratio of Cy5.5-NHS easter and peptides at 1: 1.5, respectively. Briefly, the peptides were dissolved in DMSO and adjusted pH to 8-9, then Cy5.5-NHS Ester was added to solution and stirred at room temperature for 24 h in the dark.
The products were purified by High Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry.
DL and EE were calculated using the following equation.
In vitro Drug Release of Tel and The Stability of TDCNfs. TDCNfs (25 mg) was dissolved in PBS (5 mL) and the pH was adjusted to 7.4. Proteinase K was added at a concentration of 0.138 mg/mL and the reaction mixtures were incubated at 37 °C for 24 h. Next, 500 μL of the sample was removed at each time point and the release of Tel was analyzed, while the remained SAA1-7 in the sample was analyzed using LC-MS simultaneously.

S1.4 Transmission electron microscopy
The SAA1-7 with 50% molar ratio of Tel was incubated at 40 ℃ for 0 min, 30 min, 60 min, and 120 min, respectively. TEM samples in different time points were prepared.
Besides, the samples of TDCNfs diluted in PBS (pH=7.4) for 1 h, 1 month, 2 months were prepared. The sample preparation procedures: 10 µL samples of each were placed on a carbon-coated copper grid and incubated for 30 seconds to allow the peptide nanostructures to adhere to the substrate, then rinsed twice with ultrapure water. The samples were then stained with a saturated uranyl acetate solution and placed in a desiccator overnight before analysis.

S1.8 Rheology
The rheology test was done on an AR 1500 ex (TA Instrument) system; 25 mm parallel plate at a gap of 500 μm was used during the experiment. The hydrogel of SAA1-7, TDCNfs, CY5.5 TDCNfs were characterized by the mode of dynamic frequency sweep in the region of 0.1-100 rad/s at a strain of 1%.
Primary neonatal rat cardiomyocytes (NRCMs) and neonatal rat cardiac fibroblasts (NRCFs) were isolated from the hearts of neonatal SD rats.
Briefly, after the digestion of hearts, the cells were collected and suspended in DMEM medium supplemented with 10% FBS and incubated with 95% O2, 5% CO2.

S2.4 Intracellular TUNEL and ROS Measurements
NRCMs were incubated in OGD condition for 1 h, and then cultured with PBS or different compounds (diluted in PBS) at 10 μM under the same conditions for 2-3 h.
The cells were then fixed in 4% paraformaldehyde and apoptotic cells were dyed by TUNEL staining using a one-step TUNEL apoptosis assay kit according to the subjected to permanent left anterior descending (LAD) ligation as described previously [1]. In brief, the animals were anesthetized using 1.5% sodium pentobarbital (50 mg/kg) through intraperitoneal injection, and mice were ventilated with a rodent ventilator subsequently. We exposed mice hearts by the fourth intercostal space incision and a subsequent pericardium removal. The LAD was ligated using an 8-0-prolene suture permanently to perform MI generation. Sham group mice underwent the same surgical procedure without LAD ligation.

S3.2 Live animal imaging experiments
To investigate their biodistribution, Cy5.5 TDCNfs was intravenously injected into the mice with or without myocardial infarction at a dose of 0.2 mg and Cy5.5 SAA1-7, Cy5.5 Ang1-7 was intravenously injected into the mice with myocardial infarction at the same dose. At 1 h, 12 h and 24 h post-injection, the images were captured. After 24 h, mice were sacrificed, hearts and the major organs were excised. Images were captured at an excitation wavelength of 630 nm and an emission wavelength of 700 nm using an in vivo imaging system. Images were analyzed using the BRUKER Molecular Imaging Software.

S3.3 In vivo co-localization
The hearts of mice were then dissected after intravenous injection of compounds at 24 h. Immunofluorescence staining on frozen section of heart samples as reported previously [2]. Anti-AT1R primary antibody (1:200 dilution) was used as the primary antibodies. Staining signals were visualized with FITC-labeled Goat anti-Rabbit IgG (1:200 dilution). The sections were counterstained with DAPI and examined using a laser confocal scanning microscopy.