Graphite Oxide Nanoparticles with Diameter Greater than 20 nm Are Biocompatible with Mouse Embryonic Stem Cells and Can Be Used in a Tissue Engineering System

The treatment of many degenerative diseases or tissue injuries mostly requires restoration or re-establishment of normal functions of the tissues. Tissue regeneration is achieved by replacing damaged tissue and/or stimulating the body's own repair mechanisms to heal previously irreplaceable tissues or organs. Regenerative medicine holds a future promise of providing functional and vital tissues or organs for the diseases that inherently have limited regenerative potential. For example, myocardial infarction, the current leading cause of death in the US, leads to significant cardiomyocyte loss, myocardial tissue damage, and impairment of myocardial function.[1] Current treatments of myocardial injury only attenuate the disease progression without facilitating myocardial repair. The natural regenerative potential of myocardial tissue remains suboptimal due to the limited mitotic capacity of cardiac cells and the decreased cellularity after injury. The only definitive treatment for advanced heart failure following a heart attack is heart organ transplant, but it is greatly limited by the availability of the organs.[2] Therefore, multiple efforts in pre-clinical research have specifically employed stem cells to restore the function and regenerate the myocardial tissue.[3-12] The damaged tissue can be replaced by a combination of living cells and biomaterials that aid in the cell survival, growth, mechanical support, and functions in vivo. In many cases, the viable stem cells can be transplanted into the biomaterials for targeted therapeutic applications. Embryonic stem cells (ESCs) derived from the inner cell mass of the blastocysts possess the highest pluripotency and the ability to regenerate functional tissues. The ESCs are capable of differentiating into cardiomyocytes to generate a large reservoir of ESC-derived cardiomyocytes (ESC-CMs) for the regeneration of cardiac tissue.[13, 14] However, transplantation of the ESC-CMs in vivo is hindered by suboptimal cell survival post transplantation, compromising their restorative potential. In addition, transplantation of the ESC-CMs into a beating heart encounters significant challenges in cell engraftment post-transplantation.[15] It is, therefore, of great clinical motivation to increase post-transplantation cell survival and engraftment. Currently, there are multiple biomaterial strategies to be utilized in conjunction with ESC therapy to address the survival and engraftment problems, but most types of biomaterials exhibit different challenges. It is difficult for the biomaterials to be delivered along with the ESCs through an endovascular catheter directly into the injured myocardium. Therefore, our selection criteria for a biomaterial candidate include the following: 1) biocompatibility with ESCs without affecting their pluripotency, survival, or proliferation; 2) nano-scale to be deliverable with the cells; and 3) cross-link or bind to hydrophobic biomolecules such as pro-survival molecules or extracellular matrix molecules to facilitate engraftment. Graphite Oxide (GO), a two-dimensional, single-layered sheet with both sp2 and sp3 carbon, has in recent years shown promise for biomedical use.[16-19] GO is naturally water-soluble but lacks solubility in buffered solutions due to a charge-screening effect.[20] This is a barrier for biomedical applications, which take place in buffered solutions or serum; however, covalent attachment of branched polyethylene glycol (PEG) allows excellent solubility for GO in buffered solutions with minimum toxicity both in vitro[17] and in vivo.[21] Importantly, GO possesses intrinsic fluorescence in the visible and near-infrared (NIR) regions,[17] which has helped to trigger the research into its biomedical potential. By attaching targeting peptides such as RGD to GO, selective cancer cell uptake was shown by NIR imaging.[17] The NIR region was selected for imaging because of the low endogenous absorption, scattering, and autofluorescence.[22] The high NIR absorption of GO has made it attractive as a photosensitizing agent for in vivo photothermal therapy.[23] Of note, a wide range of biomolecules can be conjugated to GO through a PEG linker to track GO in vivo through PET imaging,[24] attach targeting peptides for selective cell uptake,[17] and link chemotherapeutics to GO for drug delivery in vitro.[20] Manufactured GO has a range of sheet diameters from several hundred nanometers up to 10 microns. The PEGylation process of GO previously used long periods of bath sonication, resulting in small sheets ranging in size from 5-50 nm.[16] By tailoring the length of the bath sonication and the harshness of the subsequent centrifugation (used to remove multi-layered GO sheets), we are able to design GO with varying average sizes. Previous studies have shown that by using a graphene oxide (chemically reduced GO) surface, mesenchymal stem cell growth and differentiation were enhanced.[25] Our long term research goal is to develop an optimal ESC therapy strategy to treat the injured myocardium. For the forgoing reasons, the two objectives of this study are: 1) to examine the effects of GO particles on mouse ESC viability, proliferation, and gene expressions in vitro and 2) to identify the optimal size and concentration of GO particles with mouse ESCs. We hypothesize that GO particles will be biocompatible with mouse ESCs and will not alter or hinder the ESC survival, growth and pluripotency. This study identified an optimal range of GO size and concentration to be used in conjunction with mouse ESCs.