| Myocardial infarction（MI）is recognised as one of the leading causes of cardiovascular disease deaths worldwide.Although traditional clinical treatments for MI,including interventional and pharmacological therapies,have shown to improve myocardial function and extended life expectancy,there remains the issue of irreversible myocardial necrosis or progressive decline of cardiac function.After MI,the myocardial microenvironment is significantly altered by complex pathological changes during the inflammatory,proliferative and remodelling phases,including disruption of the dynamic balance of oxygen/blood,reactive oxygen species（ROS）,inflammatory factors and matrix metalloproteinases（MMPs）,resulting in massive cardiomyocyte death,inflammatory responses,extracellular matrix degradation and myocardial fibrosis.To reverse this phase of malignant bio-change and re-establish the function of the infarcted myocardium,the development of a novel therapeutic strategy that promotes myocardial repair to achieve effective treatment of MI is urgently needed.Injectable hydrogels are considered to be a minimally invasive technology that can overcome the limitations of traditional clinical procedures for stent implantation,achieved through the modulation of the sol-gel transition as a carrier for transporting various cells and drugs,providing passive targeting of therapeutic substances,improved retention/survival of transplanted cells and reduced toxic effects of therapeutic substances.However,current research on myocardial repair-based hydrogels remains in the initial stages,and few MI therapeutic hydrogels have the ability to both modulate the myocardial microenvironment and improve myocardial wall structure,which would greatly affect the ultimate myocardial therapeutic effect.To address these issues,several injectable hydrogels responsive to the microenvironment of infarcted myocardium were designed based on the changes in the development of different stages after MI,and loaded with corresponding therapeutic molecules in combination with the characteristics of specific lesion stages of MI,constituting a multi-functional therapeutic substance-loaded hydrogel system.By optimising the properties of the hydrogels,investigating the effects of the therapeutic substances loaded with hydrogels on the relevant cell functions,and their effects on improving the structure and function of the infarcted myocardium in SD rats,and ultimately comparing the effects of therapeutic strategies on the repair of the infarcted myocardium,with the aim of selecting the most appropriate treatment strategy for MI.PartⅠ:A dual crosslinked hydrogel-mediated integrated peptides andBMSC therapy for myocardial regenerationAims:Based on the early excess of ROS and the massive apoptosis of cardiomyocytes（CMs）in the myocardial microenvironment,and the design of a 4-arm-PEG-SG/PAMB-G-TK hydrogel system for ROS-response release tailored to liposome delivery for MI therapy.Methods:Chemical synthesis,microstructure and drug release of liposomes and hydrogels are examined using FT-IR,1H NMR,transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,rheometry and UV.Live-dead cell staining,tube formation,ROS assay and Western blot were used to verify the functional effects of liposome preparations loaded with hydrogels on relevant cell types and mitochondria.After 4 weeks,the infarcted myocardium was examined by ultrasound and electrocardiogram,and the infarcted heart was removed for H&E and Masson to examine the structure of the infarcted myocardium,and Western blot,immunofluorescence and immunohistochemistry to examine the expression of relevant factors and proteins.Results:The study showed that the PAMB-G-TK/4-arm-PEG-SG hydrogels exhibited myocardium-like mechanical strength and electrical conductivity with a response to ROS promoting liposome release.More interestingly,SS-31 released from the liposome system targeted cytochrome c（Cyt c）in the mitochondrial inner membrane in damaged CM and inhibited pathological ROS production to improve mitochondrial function,and sphingosine-1-phosphate（S1P）promoted endothelial cell angiogenesis by activating the PI3K/Akt pathway.In the rat MI model,this hydrogel scavenged excess ROS,improved mitochondrial ATP metabolism,promoted angiogenesis,and improved cardiac function.Summary:We designed a liposomal reagent to improve mitochondrial damage and loaded it in ROS-responsive PAMB-G-TK/4-arm-PEG-SG hydrogels for targeted improved CM activity and promoted angiogenesis in the early stages of MI treatment.However,the heart is a tissue with a continuous systolic-diastolic cycle and the rigid network of the hydrogel injected into the myocardium may have the disadvantage of breaking down after a continuous mechanical cycle,which is not conducive to long-term support of the collapsed myocardial wall and control release of the drug formulation.PartⅡ:A p H-responsive hydrogel promotes M1-type macrophagepolarization by releasing EGCG-PDA for myocardial repairAims:To address the disadvantages of the hydrogel’s tendency to rupture in the chapter 1,and to incorporate the characteristics of the microenvironment of the lesion after CM death.Chapter 2 was based onthe acidic microenvironment and the harsh local inflammatory at the site of MI,and we designed a p H-responsive release of epigallocatechin gallate（EGCG）-modified dopamine nanoparticles（EGCG-PDA NPs）composite hydrogel system for MI therapy.Methods:Chemical synthesis and microstructure of EGCG-PDA NPs and hydrogels were examined using FT-IR,1H NMR,TEM,particle size measurement and rheometry.Live-dead cell staining,immunofluorescence,ROS detection and Western blot were used to verify the effects of EGCG-PDA-loaded hydrogels on macrophage phenotype and CM function.An SD rat MI model was constructed and the hydrogel was injected into the infarcted myocardial site.The function of the infarcted myocardium was examined by small animal ultrasound,the structure of the infarcted myocardium was examined by H&E and Masson,and the expression of relevant factors and proteins was examined by Western blot,immunofluorescence and immunohistochemistry.Results:The hydrogel formed by complexation of phenylboronic acid with catechol at p H 9.0 from 4-arm-PEG-DA and 4-arm-PEG-PBA precursors solution exhibited excellent adhesion and self-healing properties.The hydrogel was injected into the infarcted myocardium and slowly released encapsulated EGCG-PDA NPs in response to acidic p H,showed antioxidant,anti-inflammatory effects and induced polarization of M1 macrophages into M2 macrophages,further enhancing cytokine secretion for cardiac repair.In the rat MI model,the hydrogel system reduced inflammation,promoted early polarization of M2 macrophages,promoted angiogenesis,reduced fibrosis,and improved cardiac function.Summary:We designed an EGCG-PDA NPs-loaded self-healing PAMB-G-TK/4-arm-PEG-PBA hydrogel system for MI treatment that responds to p H and is accompanied by regulation to promote the transformation of M1 to M2 macrophages.However,the hydrogel is composed of pure synthetic materials,which may be less biocompatible within the infarcted myocardium,and the short retention period of the hydrogel at the myocardial site due to p H responsiveness affects the long-term support of the infarct wall.PartⅢ:A dual crosslinked hydrogel-mediated integrated peptides andBMSC therapy for myocardial regenerationAims:To address the disadvantages of biocompatibility and mechanical properties present in chapter 1 and 2 and to incorporate the characteristics of the full MI phase.Chapter 3 was based on the excessive degradation of the extracellular matrix（ECM）by matrix metalloproteinases（MMPs）and the inability of CMs to regenerate after MI.Methods:Chemical synthesis,microstructure and drug release fromhydrogels were examined using FT-IR,1H NMR,SEM,rheometry and UV.Live-dead cell staining,tube formation and Western blot were used to verify the activity and functional effects of peptide-loaded hydrogels on relevant cell types.We constructed an SD rat MI model,small animal ultrasound and ECG to examine the function of infarcted myocardium,H&E,Masson and TTC to examine the structure of infarcted myocardium,and live imaging,Western blot,immunofluorescence and immunohistochemistry to examine the degradation of hydrogels and the expression of related factors.Results:We developed an integrated single"all-in-one"in situ dual crosslinking conductive hydrogel with favorable treatment properties termed as Ma HA/B-G-SH/Fe3+by ionic interactions and chemical covalency based on modified hyaluronic acid（HA）,gelatin（G）,and Fe3+.The resulting dual crosslinking dynamic hydrogel not only provides self-healing and mechanical properties adapted to the myocardial systolic-diastolic cycle with simultaneous electrical signal transmission to fibrous islands and normal tissue,but also leads to significant increase of the myocardial wall thickness very close to that of normal myocardium upon one single injection with complete degradation within28 days.Notably,the hydrogel covalently conjugated with a tailored peptide sequence of GGR-KLT and encapsulated with bone mesenchymal stem cells（BMSCs）was further used for in situ injection in a rat MI model,which exhibited（ⅰ）efficient inhibition of excessive matrix degradation dependent on early MMP-2 expression,（ⅱ）triggered on-demand release of KLT for at least 14 days and significant promotion of angiogenesis,and（ⅲ）synergistic BMSCs considerably enhanced myocardial regeneration within 28 days.Taken together,the dual cross-linked conductive hydrogel-mediated synergistic peptide and stem cell therapy can potentially restore and remodel the structure and function of injured myocardium throughout the stages of MI,thus demonstrating significant clinical translational potential.Summary:We have designed a dual cross-linked conductive hydrogel loaded with BMSCs in response to the on-demand release of pro-angiogenic peptides that can synergistically restore and remodel the structure and function of damaged myocardium throughout the MI process for long-term treatment after MI,overcoming the drawbacks of the above hydrogels due to the microenvironmental response that tends to lead to short myocardial retention time and low mechanical properties. |
