MMP 9-instructed assembly of bFGF nanofibers in ischemic myocardium to promote heart repair

Background: The only effective treatment for myocardial infarction (MI) is the timely restoration of coronary blood flow in the infarcted area, but further reperfusion exacerbates myocardial injury and leads to distal coronary no-reflow, which affects patient prognosis. Angiogenesis could be an important therapeutic strategy for re-establishing the blood supply to save the ischemic myocardium after MI. Basic fibroblast growth factor (bFGF) has been shown to promote angiogenesis. However, direct intravenous administration of bFGF is not a viable option given its poor half-life in vivo. Methods: Herein, we developed a peptide Lys-Lys-Pro-Leu-Gly-Leu-Ala-Gly-Phe-Phe (K2) to encapsulate bFGF to form bFGF@K2 micelle and proposed an enzyme-instructed self-assembly (EISA) strategy to deliver and slowly release bFGF in the ischemic myocardium. Results: The bFGF@K2 micelle exerted a stronger cardioprotective effect than free bFGF in a rat model of myocardial ischemia-reperfusion (MI/R). In vitro results revealed that the bFGF@K2 micelle could be cleaved by matrix metallopeptidase 9 (MMP-9) to yield bFGF@Nanofiber through amphipathic changes. In vivo experiments indicated that intravenous administration of bFGF@K2 micelle could lead to their restructuring into bFGF@Nanofiber and long term retention of bFGF in the ischemic myocardium of rat due to high expression of MMP-9 and assembly-induced retention (AIR) effect, respectively. Twenty-eight days after MI/R model establishment, bFGF@K2 micelle treatment significantly reduced fibrosis and improved cardiac function of the rats. Conclusion: We predict that our strategy could be applied in clinic for MI treatment in the future.


Reagents and equipment
All chemicals are reagent grade and have not been further purified for use. 5(6)-Carboxy-tetramethylrhodamine N-succinimidyl ester (TMR-NHS) was purchased from Shanghai Yuanye Co.Ltd. Recombinant MMP-9 protein standard was purchased from Sino Biological (Beijing, China). Recombinant basic fibroblast growth factor (bFGF), MMP-9, alpha-smooth muscle actin (α-SMA) antibodies, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining kit were purchased from Beyotime (China). MMP-9 ELISA kit was purchased from MultiSciences (China). bFGF antibodies and MTT kit were purchased from Abcam (UK). CD31 and alpha-sarcomeric actin (α-SA) antibodies were purchased from Santa Cruz (USA). Electrospray ionization (ESI) mass spectra were obtained on a Finnigan LCQ (ThermoFisher Corporation). 1 H NMR and 13 C NMR spectra were obtained on a Bruker AV 400. The rheology test were obtained on MCR302 (Austria). Zeta and dynamic light scattering (DLS) were performed on a Malvern Nano-ZS ZEN3600 (UK). The circular dichroism (CD) spectra were obtained on J-1700 (JASCO, Japan).

Myocardial ischemia reperfusion animal model
Male Sprague Dawley rats aged 8-10 weeks, weighing 200-220 g, were obtained from the Experimental Animal Center of Anhui Medical University. All experimental procedures and protocols used in this study have been reviewed and approved by the Ethics Committee of Experimental Animals, Anhui Medical University (LLSC20190476).
The myocardial ischemia-reperfusion (MI/R) model was established via the left anterior descending branch ligated for 30 min and then reperfused. Briefly, all rats were anesthetized with S3 pentobarbital (40 mg/kg), then intubated and connected to a ventilator for mechanical ventilation, equipped with a Powerlab system to observe the electrocardiogram (ECG). The successful signs of ischemia were a pale ischemic appearance on the surface of the local myocardium, ST-segment elevation, and Q wave in lead II. The sham group underwent a sham operation without coronary artery ligation.

MTT assay
Cytotoxicity of peptide K2 and LAGFF was evaluated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded into 96-well cell culture plates at 5 × 10 4 /well and incubated in a 5% CO2 atmosphere at 37°C. The solutions of peptide diluted by DMEM (100 μL/well) at concentrations of 5, 10, 20, and 40 μg/mL in 100 μL medium were added to the wells, respectively. The cells were incubated with the peptides for 24 or 48 h. A solution of 5 mg/mL MTT dissolved in phosphate-buffered saline (PBS) (pH 7.4) (10 μL/well) was added to each well. A solution of DMSO (100 μL/well) was added to dissolve the formazan. The data were obtained using a 96-well plate reader to detect its absorption at 490 nm.

In vivo pharmacokinetic studies and tissue biodistribution
TMR-NHS was used to label the bFGF. 24h, 48h, and 72h after injection, hearts and major organs of rats were removed, fresh frozen in OCT, and cryosectioned. Heart cryosections were fixed with 4% paraformaldehyde in PBS and incubated with the antibody against α-SA to show the myocardium and then the TMR fluorescence was observed to identify the presence of bFGF in the ischemic myocardium. In addition, cryosections of liver, lung, spleen and kidney were directly used to observe the fluorescence of TMR. All pictures were captured using Zeiss microscopy and analyzed by NIH Image J software.

Western blot
The myocardial tissue including the ischemic area and border area was collected and lysates were prepared by freezing in radioimmunoprecipitation assay (RIPA) for 20 min. The cell debris was removed by centrifugation (12000 rpm, 4 ℃, 15 min), and then the cell lysates were mixed with SDS sample buffer at 100 °C for 10 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Immun-Blot polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membranes were blocked in 5% skim milk for 4 h, then incubated with primary antibody (anti-bFGF or anti-MMP-9), and the β-Actin and Gapdh were used as internal control, respectively. Membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase. Immunoreactive bands were visualized with an ECL system. Protein bands were quantified by densitometric analysis using ImageJ software.

TUNEL staining
After treatment with saline, K2 micelle, bFGF, and bFGF@K2 micelle for 2 days, MI/R rats were sacrificed and the hearts were removed. Apoptotic cells in the border zone of the ischemic myocardium were analyzed using TUNEL kit according to the instructions, and DAPI counterstain was performed to visualize the nuclei. The TUNEL positive cells were observed under a fluorescent microscope and counted using NIH Image J software.

Immunohistochemistry and histopathology
After euthanasia, hearts of rats were taken out, fixed with 4% paraformaldehyde overnight at 4℃, then, fresh frozen in OCT and cryosectioned for immunohistochemistry. Blood vessels were stained with antibody against CD31 or α-SMA (1:500) mounted with fluoromount for fluorescent imaging under microscopy. For histopathological examination, hearts and other major organs were S5 formalin-fixed, paraffin-embedded, and sectioned. Masson's trichrome-staining was used to assess the fibrosis area and the left ventricular wall thickness of the heart slices. Sections of other major organs were stained with hematoxylin and eosin (H&E) to evaluate the biocompatibility of micelles using light microscopy. All images were analyzed using NIH Image J software.

Time (min)
Flow ( Table S2. HPLC condition for Figure S5 Time (