PRP-loaded pH-responsive hydrogel for the amelioration of ventricular remodeling following myocardial infarction

Scheme 1. Synthesis of PRP-PLL/PEG hydrogels (A) and their applications through multiple mechanisms to alleviate post-myocardial infarction ventricular remodeling (B). (Made By Figdraw).

Fig. 1. Characterization and mechanical property analysis of the hydrogels. A) PRP gel. B) Cell proliferation rate of HCFs (n = 3). C) Storage modulus G′ and loss modulus G″ of PLL/PEG, PRP-PLL/PEG hydrogels with a strain amplitude of 10 % and a frequency of 1Hz at 37 °C. D) Representative stress-strain curves of the hydrogels. E) SEM images of the hydrogels. ∗ indicates p < 0.05, n refers to biological replicates.

Fig. 2. Release assay of PRP in vitro. A) Release assay of PDGF, VEGF and EGF in pH7. B) Release assay of PDGF in different pH. C) PRP In Vitro Release Follows the Higuchi Model: (a) PDGF, (b) VEGF, (c) EGF.

Fig. 3. Biocompatibility of PRP-PLL/PEG hydrogels. A) Live/dead cell staining of H9C2. B) Cell viability of H9C2 (n = 3). C) H&E staining of (a) heart, (b) liver, and (c) kidney tissues from control and hydrogel groups at days 1, 7, and 14. NS indicates p ≥0.05, n refers to biological replicates.

Fig. 4. Western blot. A) Band of fibrotic protein. B-F) Protein expression quantification (n = 3); G) Transwell assay; H) Quantitative analysis of cell migration (n = 3). ∗/# indicates p < 0.05, ∗∗/## indicates p < 0.01, ∗∗∗∗/### indicates p < 0.001, ∗/# represent comparison with the control group and with the Ang II group, respectively; n refers to biological replicates.

Fig. 5. Cell apoptosis assay and ROS level. A) Flow cytometry; B) Apoptotic Quantitative analysis (n = 3). C) Detection of ROS level. ## indicates p < 0.01, ∗∗∗ indicates p < 0.001, ∗/# represent comparison with the control group and with the MI group, respectively, n refers to biological replicates.

Fig. 6. Tube formation assay. A) Fluorescence image of blood vessel formation (HUVEC); B-C) The nodes, total length Quantitative analysis of vascular (n = 3). ## indicates p < 0.01, ∗∗∗∗ indicates p < 0.001, ∗/# represent comparison with the control group and with the MI group, respectively, n refers to biological replicates.

Fig. 7. Protective effects of hydrogel on cardiac Function after myocardial infarction. A) General view of cardiac ligation; B) Electrocardiogram changes before and after ligation of the left anterior descending coronary artery (the paper speed: 25 mm/s; the gain: 10 mm/mV); C) Representative LVM-type echocardiographic images on postoperative days 14; Statistical analysis of LVEF (D) and LVFS (E) in SD rat cardiac function on postoperative days 14 (n = 3). NS indicates p ≥0.05, #/## indicates p < 0.05 and 0.01, respectively, ∗∗∗∗ indicates p < 0.001, ∗/# represent comparison with the control group and with the MI group, respectively, n refers to biological replicates.

Fig. 8. Evaluation of the in vivo anti-inflammatory and anti-fibrotic effects of hydrogels. A) Expression of fibrosis-associated proteins by WB; B-F) Fibrosis-related protein Quantitative analysis (n = 3); Representative H&E (G) and Masson staining (H) Results of Myocardial Sections; I) Statistical analysis of fibrotic area in SD rat cardiac function on postoperative day 14 (n = 3). NS indicates p ≥0.05, ## indicates p < 0.05, ∗∗∗∗ indicates p < 0.0001, ∗/# represent comparison with the control group and with the MI group, respectively, n refers to biological replicates.

Fig. 9. Immunohistochemical and fluorescence results of myocardial tissue after MI using hydrogel: A-D) Immunohistochemical staining results and statistical analysis of CD31, α-SMA, IL-1β, and casepase3 in myocardial tissue, respectively; E-H) Quantitative statistical analysis of CD31, α-SMA, IL-1β, and casepase3, respectively (n = 3). NS indicates p ≥0.05, ∗/# indicates p < 0.05, ## indicates p < 0.01, ∗∗∗/### indicates p < 0.001, ∗∗∗∗ indicates p < 0.0001, ∗/# represent comparison with the control group and with the MI group, respectively; n refers to biological replicates.

Fig. 10. Mechanism of myocardial tissue after MI using hydrogel. A) PCA analysis of MI and hydrogel group. B) Heat map of genes in different treatments. C) Volcano plots of all genes the MI and hydrogel group. The blue dots represented the upregulated differently expressed genes, the red dots were downregulated differently expressed genes, and the grey dots were non-differentially expressed genes. D) KEGG enrichment bubble diagram of differentially expressed genes; E) KEGG enrichment histogram of differentially expressed genes; F-H) The mRNA expression of genes (PI3K, AKT, mTOR) by qPCR (n = 3). ∗∗∗ indicates p < 0.001, ∗∗∗∗ indicates p < 0.0001, n refers to biological replicates.