Preparation And Characterization Of Chitosan Based Hydrogel Modulating The Microenvironment After Myocardial Infarction

In China, the incidence of myocardial infarction (MI) has increased steadily. Thecurrent clinical treatments, such as drug therapy and intervention therapy, canalleviate the symptoms and improve the quality of life of patients. However, thesemethods cannot repair damaged/infarcted myocardium, leading to the high mortality.Rencently, cell therapy has been used to repaire the injured myocardium, but thehostile microenvironment after MI (e.g. ischemia, hypoxia and inflammation) candecrease the retention and survival of the implanted cells, resulting in the long-termeffect of cell therapy is not significant. Injectable cardiac tissue engineering mayovercome the limitation of cell therapy, in which cells are delivered within aninjectable scaffold, and achieve better cardiac repair and reconstruction.In injectable cardiac tissue engineering, the injectable scaffold plays a crucialrole to improve the retention and survival of the transplanted cells in MI region.However, the current injectable biomaterials are designed to improve their chemical,physical and processing properties, and the ischemia and hypoxia microenvironmentafter MI are not sufficiently considered when the biomaterials are designed.On the one hand, a large number of reactive oxygen species (ROS) are producedin ischemic surroundings after MI and impair membrane lipids, proteins, and DNA oftransplanted/host cells, which is one of the main obstacles to influence the treatmenteffect of MI. Therefore, it is necessary to design the injectable hydrogel with excellentantioxidant capabilities to remove excessive ROS and thus suppress oxidative stressdamage. Recently, some antioxidant hydrogels have been investigated to repair thedamaged skin and nucleus pulposus, but not yet used in the myocardial tissueengineering. On the other hand, revascularization of the infarcted myocardium is veryimportant for cardiac repair, affecting the survival, proliferation and maturation oftransplanted/host cells. To induce angiogenesis in MI region, hematopoietic stem cellsor angiogenic growth factors can be injected in vivo. However, the direct injection ofthe cells is limited due to the low retention and survival rate of transplanted cells. Theactivity of growth factors is affected by the harsh microenvironment, which furtherinfluences the therapeutic effect, and meanwhile the high price also limits the application of growth factors. Therefore, it’s very important to develop the injectablebiomaterial with angiogenic activity under hypoxic microenvironment.Chitosan with unique bioactive and antioxidative properties is widely used asinjectable hydrogel. It has a certain antioxidant capacity and angiogenic activityowing to the active groups on the molecular chain. Unfortunately, the antioxidantactivity of chitosan decreases with the increase of the molecular weight (MW) due tothe complex intermolecular and intramolecular hydrogen bonds, and the ability ofchitosan to form hydrogels decreases along with the decrease of MW. On the otherhand, angiogenic activity of chitosan is insufficient under the microenvironment afterMI. Thus, there is a need to design chitosan-based hydrogels with high MW while stillretaining the good antioxidative and angiogenic activity under MI condition.Glutathione (GSH) is well known to be a major barrier against ROS. However,as one of antioxidants with low molecular weight, it’s less effective owing to the poorthermal stability. To overcome this limitation, GSH has been covalently linked on apolymeric matrix to enhance its stability and physicochemical properties. But how toconnect GSH to a polymeric matrix to improve antioxidation has not been reported.RoY, a12amino-acid synthetic peptide, induces angiogenesis under hypoxiccondition and alleviates hind limb ischemia of mice by activating the stress proteinGRP78receptor on endothelial cells. Therefore, there is a potential application tograft RoY with angiogenic activity on chitosan chain, which is expected to improveangiogenic activity under hypoxic environment.In this study, with the purpose of improving the microenvironment after MI, thenew injectable chitosan based hydrogel with good antioxidative and angiogenicactivity under MI condition have been developed. And this study was carried out asfollows:Part l: Preparation and characterization of chitosan-glutathioneantioxidative hydrogelExperiment One： Preparation and characterization of chitosan-glutathioneconjugate. GSH was introduced onto the chitosan chloride (CSCl) chain via EDCchemistry. The formation of the CSCl-GSH conjugates was monitored by1H NMRand Fourier transform infrartd (FTIR) spectroscopy, and the thermal stability wasinvestigated by differential scanning calorimeter (DSC). The amount of GSH inCSCl-GSH conjugates was spectrophotometrically determined by quantifying thiolgroups using Ellman’s reagent. By studying the ability to remove different types of ROS, the antioxidative properties of the conjugates were evaluated. And the intrinsicrelationship between the grafted degree (GD) or concentration and the antioxidantcapacity of CSCl-GSH conjugates was also analyzed.Results found that GSH has been grafted on the CSCl chiain via the formation ofamide bonds, and GD can be controlled via changing the feed ratio of CSCl and GSHand EDC amount. CSCl-GSH conjugates have good thermal stability, and the thermaldegradation temperature increases with the increase of GD. CSCl-GSH conjugatescould effectively scavenge the superoxide anion, hydroxyl radical and DPPH radicalseven at high concentration. The antioxidative activity increases with the increase ofconjugate concentration; CSCl-GSH conjugates with high GD display much higherantioxidative activity than that of CSCl for superoxide anion and DPPH radicals,while the conjugate with GD10%showed the highest antioxidative capacity.Experiment Two：Preparation and characterization of CSCl-GSH hydrogel.Thermosensitive CSCl-GSH hydrogels were developed via mixing CSCl-GSH, GPand HEC solutions. The viscosity, degradation performance and toxicity of thehydrogels were evaluated. Then the adhesion and survival of cardiomyocytes (CMs)on/in the hydrogels were also studied.Results suggested that CSCl-GSH hydrogels were liquid at room temperature,and rapidly formed gels when the temperature was higher than36℃. And theintroduction of GSH was beneficial for the formation of thermosensitive hydrogel.Besides, CSCl-GSH hydrogels have shown a good biocompatibility to support theadhesion and survival of CMs, compared with CSCl hydrogel.Experiment Three：Modulation of CSCl-GSH hydrogel on biological behavior ofcardiomyocytes under ROS microenviroment. To induce the oxidative stress, CMscultured in CSCl-GSH hydrogels were treated by H2O2. Intracellular ROS weremeasured using DHE and DCFH-DA staining, and NO level of CMs was determinedby the Griess method. Then the activities of catalase and superoxide dismutase ofCMs were measured. And MDA levels were determined to evaluate intracellular lipidperoxidation. In addition, by quantitative analysis of cleaved caspase3, apoptosis ofCMs in CSCl-GSH hydrogels was studied.Under ROS surrounding, CMs grew well in CSCl-GSH hydrogels. Compared toCSCl hydrogels, CSCl-GSH hydrogels could remove the excessive intracellular ROSeven when they were exposed to high ROS microenvironment. And the activities ofantioxidative enzyme of CMs were decreased. Moreover, the hydrogels were able to reduce intracellular MDA levels and lipid peroxidation, suppressing oxidative stressdamage. In addition, CSCl-GSH hydrogels could inhibit apoptosis of CMs byinactivation of cleaved caspase3.Part2: Preparation and characterization of chitosan-RoY hydrogel withangiogenic activityExperiment One：Preparation and characterization of CSCl-RoY hydrogel. RoYpeptide was grafted onto the CSCl chain via EDC chemistry. The formation of theCSCl-RoY conjugates was monitored by1H NMR and FTIR spectroscopy, and thethermal stability was investigated by DSC. CSCl-RoY hydrogels were prepared andthe degradation performance was evaluated.Results suggested that CSCl-RoY conjugates with different GD and goodthermal stability were synthesized via the formation of amide bonds. Meanwhile,CSCl-RoY hydrogels owned good thermosensitive property and suitable degradationrate, compared with CSCl hydrogels.Experiment Two: Modulation and related mechanisms of CSCl-RoY hydrogel onbiological behavior of human umbilical vein endothelial cells (HUVECs). HUVECswere cultured on/in CSCl and CSCl-GSH hydrogels under normal (21%O2) andhypoxia (3%O2) conditions, respectively. The survival, adhesion, proliferation,migration and tube formation of HUVECs were studied. Moreover, the expression ofheat shock protein GRP78, Akt and ERK1/2phosphorylation levels were analyzed.Results show that comparing with CSCl hydrogels, CSCl-RoY hydrogels werebeneficial for the survival, proliferation, migration and tube formation of HUVECsunder hypoxic condition. And the effect was related with GD of RoY. CSCl-RoYhydrogels with high/moderate GD displayed higher activity to improve cellproliferation and tube formation than the other hydrogels; CSCl-RoY hydrogels withhigh GD were superior in promoting cell migration. Moreover, membrane GRP78ofHUVECs in CSCl-RoY hydrogels was expressed lower while phosphated Akt andERK1/2related to cell survival and proliferation was expressed higher than that inCSCl hydrogels under hypoxic condition.Experiment Three: Effect of CSCl-RoY hydrogel on angiogenesis and injuredmyocardium of rats in vivo. PBS, CSCl and CSCl-RoY hydrogels were injected intoMI zone of rats, respectively. Infarct size, ventricular wall thickness, density anddiameter of small blood vessels were analyzed using Masson’s trichrome staining andimmunofluorescence staining of vascular-specific antibodies. Compared with PBS and CSCl hydrogels, CSCl-RoY hydrogels couldsignificantly increase vessel density, and increase vessel diameter to some extent,showing higher angiogenic activity. Moreover, CSCl-RoY hydrogels couldsignificantly reduce infarct size, and decrease wall thickness to some extent.In summary, in order to improve the antioxidative activity of chitosan with highMW, GSH was covalently linked onto the CSCl chain to form CSCl-GSH conjugates,and then CSCl-GSH hydrogels with good thermal sensitivity and biocompatibilitywere prepared. Under ROS microenvironment, CSCl-GSH hydrogels could not onlysupport the adhesion and survival of CMs, but also remove the excessive intracellularROS and thus suppress the oxidative stress damage and apoptosis. Meanwhile，RoYpeptide with specific angiogenic activity was also grafted onto the CSCl chain，andthermosensitive CSCl-RoY hydrogels were prepared. CSCl-RoY hydrogels couldmodulate membrane surface GRP78receptor of HUVECs, activate Akt and ERK1/2signaling pathway, and finally promote cell survival, proliferation, migration and tubeformation. Moreover, CSCl-RoY hydrogels could induce angiogenesis in infarct zone,attenuate myocardial injury and promote tissue repair. Therefore, it suggests that thetwo chitosan-based hydrogels could modulate microenvironment after MI and havethe potential for the treatment of MI.