α-Tocopherol liposome loaded chitosan hydrogel to suppress oxidative stress injury in cardiomyocytes

Abstract

Myocardial infarction (MI) continues to be a major contributor to the morbidity and mortality across the globe. Injectable hydrogel, a tissue-engineered scaffold, recently demonstrated very promising in myocardial repair. However, the undesirable retention and survival of transplanted cells has limited their applications due to the oxidative stress microenvironment of MI lesions. In this work, a thermosensitive α-tocopherol (AT) liposome loaded chitosan hydrogel was developed to suppress the oxidative stress injury in cardiomyocytes. AT was embedded in the liposomes to improve its solubility and stability. The innovative AT liposome loaded chitosan hydrogel (AT-LCH) system had an appropriate sol-to-gel transition temperature. Hydrogels possessed a highly porous structure with irregular pores interconnected throughout the construct as shown by SEM, and liposomes distributed uniformly in the porous structure. A sustained AT release was observed in AT-LCH. In addition, AT-LCH has shown an excellent biocompatibility to support the adhesion and survival of cardiomyocytes. Moreover, it can resist the oxidative stress environment and improve the survival of cardiomyocytes. In general, this work suggests that AT-LCH may present an ideal scaffold material for injectable cardiac tissue engineering.

Introduction

The morbidity and mortality of myocardial infarction (MI) is still on the rise year by year, cell transplantation is one of the promising therapy methods to repair the injured myocardium [1,2]. Cells transplanted with serum-free medium were carried out at the beginning of 2000s, however, a disadvantage of this approach is that there is limited cell retention and transplantation survival [3]. The impact of this may be related to the leakage of cells from the targeted site after intramyocardial delivery, and the hostile microenvironment of infarction lesion, such as inflammation, reactive oxygen species (ROS) accumulation or ischemia [4,5]. Tissue engineering has emerged as a promising solution for in situ cardiac tissue repair in infarcted heart after MI. In the field of injectable cardiac tissue engineering study, the desired seeded cells and/or therapeutic agents are delivered locally to the damaged area of the heart with some injectable nature or synthetic biomaterials as scaffold [6]. The scaffold provides a mimic extracellular matrix (ECM) of support for the cells, thereby improving the retention of transplanted cells. Scaffold biomaterials can be designed to stimulate cell growth and guide tissue regenerations.

Hydrogel was considered to be an appropriate injectable scaffold in delivering seeded cells in cardiac tissue engineering, due to their tunable properties and similarity with native myocardial ECM [7]. Fibrin [8], gelatin [9], chitosan [4], alginate [10], hyaluronan [11] and collagen [12] are the most commonly used natural hydrogel materials which can be applied in cardiac tissue engineering. Hydrogels possess great design flexibility, whose structure and composition can be tailored to achieve the required mechanical, rheological or degradation properties suitable for cardiac tissue engineering [2]. Recently, some functional hydrogels were used to improve the microenvironment of infarcted myocardium. It has been proved that hydrogels modified with QHREDGS peptide, ROY peptide and glutathione can support CMs survival [4,5,13]. Oxidative stress, which was characterized by the accumulation of free radicals, often occurred along with MI. Excessive production of free radicals can impair membrane lipids, proteins and DNA of transplanted cells, which would seriously affect the therapeutic effect of MI. Therefore, preparation of scaffold with antioxidant ability might improve the efficiency of cell transplanted.

Chitosan is widely used in the field of tissue engineering due to its unique antioxidant activity and good biocompatibility. As a promising biomaterial, chitosan has attracted considerable attentions for providing a new way of tissue repair and regeneration. It confirmed that chitosan based hydrogel can deliver seeded cells for myocardial repair as a carrier [4,[13], [14], [15], [16]]. Compared with normal saline control group, chitosan/β-glycerol phosphate disodium salt (β-GP) hydrogels was able to improve acute retention rate of the transplanted cells in MI significantly [15]. With the addition of β-GP, chitosan can form a thermo-sensitive hydrogel, which can keep flow state at low temperature and semisolid state at high temperature. β-GP can form a shield of water around chitosan chains in an acidic solution by hydrogen bonding and maintain the solubility of chitosan at higher pH values and lower temperatures. Hydrogen bonds between water molecules and chitosan chains would be released when the temperature raises, which makes the chitosan-chitosan hydrophobic interactions become dominant, resulting in phase transition from liquid to gel [17]. The formed semisolid hydrogel can support the cells transferred, simulate the ECM and provide an appropriate cell survival environment. However, chitosan hydrogels with high β-GP concentration produce ionic strengths that unsuitable for survival and proliferation of CMs. In order to decrease the cytotoxicity of chitosan based hydrogel and improve cell viability, less toxic cross linker hydroxyethyl cellulose (HEC) was added to reduce the concentration of β-GP. Nowadays, chitosan based hydrogels have been used to carry chondrocytes, osteoblasts, nerve cells, liver cells, skin cells and mesenchymal stem cells etc., which have been widely applied to the skin, liver, bone and cartilage tissue engineering research [[18], [19], [20]].

Intrinsic antioxidant ability of chitosan could not resist the oxidative stress microenvironment of MI sufficiently [4], the present study incorporated α-tocopherol (AT) into chitosan hydrogel. AT is the most active and effective form of vitamin E, which is widely distributed in nature and used as an antioxidant in clinic. Antioxidative properties of AT lies in the ability to scavenge free radicals generated from lipid biomolecules and ultimately interrupt free radical chain reaction [21]. It can minimize free radical-induced myocardial damage [22]. AT decreases lipid peroxidation and low-density lipoprotein oxidative susceptibility, thereby slowing the progression of atherosclerosis and occurrence of MI [23]. A recent meta-analysis research also suggested that tocopherol given alone would reduce the occurrence of MI in intervention trials [24].

However, poor solubility and easily oxidized properties of AT have limited its application and formulation. Liposomes was introduced to improve the solubility, thereby ensure the uniformly disperse of AT in the hydrogel. The use of liposome encapsulation also can protect the unstable drug and shield their function [[25], [26], [27]]. Additionally, incorporate liposomes into hydrogel might achieve the sustained release of drug, improve the retention time and extend the drug efficacy [28].

In general, this study intended to develop an AT contained chitosan hydrogel to increase the retention and survival rate of transplanted cells. Two main strategies were combined: firstly, AT was entrapped in liposomal carrier in an attempt to improve the solubility and stability, and secondly, the liposome was formulated into a chitosan based hydrogel to improve the antioxidant and biocompatibility. Then the injectable liposome loaded hydrogel was characterized in terms of temperature respo nse, in vitro drug release, degradation, biocompatibility and antioxidant properties, etc.