In this thesis, functionalized graphene sheets were synthesized and their cellular uptake features, drug release properties, and anti-MDR ability were investigated and some significant conclusions were obtained. Hyperbranched polyglycerol (hPG) was successfully conjugated to the graphene backbone that was functionalized by nitrene through a [2+1] cycloaddition reaction. The resulting hPG-coating graphene sheets showed high polymer coverage, controllable size, good water dispersibility, excellent biocompatibility and can be easily post-functionalized. Amination and sulfation was applied to obtain positively charged and negatively charged graphene sheets, respectively. Furthermore, functionalized graphene sheets could be broken down into smaller sizes by horn sonication with corresponding time frames. In my first project, the cellular uptake characteristics of these graphene derivatives with similar polymer content, but different size and surface charges were investigated. It was found that large functionalized graphene sheets (1 μm) were preferable taken up via a phagocytic pathway, regardless of their surface charges. However, surface charge is a dominant factor for their small analogs (200 nm size). Small graphene sheets with positive charges mainly entered into the cells through clathrin-mediated endocytosis (CME), while this pathway did not play a significant role for the small ones with negative charge. Because of the surface charge, the negatively charged and positively charged graphene derivatives displayed size-independent and sizedependent uptake efficacy. Moreover, our results also revealed the cellular internalization of hPG-conjugated graphene sheets is negligible for all the sizes, which is attributed to the protein-resistant feature of hPG and low nonspecific interactions with biointerfaces. In the next project, we prepared graphene sheets with similar polymer content, size (around 150 nm), but different functionalities and surface charges according to our established protocol of the first project. The hydrophobic anticancer drug, DOX, was loaded onto these graphene derivatives and a pH-sensitive dye was connected onto their surface and employed as an antenna to receive strong signals from the acidic cell compartments. It was found that these functionalized graphene sheets with different functionalities underwent the same cellular uptake and acidification process, while their intracellular release properties were fundamentally different. The protonation of DOX in acidic conditions decreased their hydrophobic and π-π stacking interaction with graphene backbone and facilitated its release from both sheets with different surface charges. However, protonated DOX was positively charged and exhibited attractive and repulsive electrostatic interactions with negatively charged and positively charged hPG-conjugated graphene derivatives, respectively. While the release of DOX was accelerated by repulsive electrostatic force in the case of positive sheets, many of them were trapped on the surface of negative sheets via attractive electrostatic interactions. Therefore, the overall release rate and therapeutic effect was much higher in the first case. This study revealed that intracellular location and release features of the therapeutic agents are a function of their hydrophobic and electrostatic interactions with the graphene-based nanocarriers. In the third project, a graphene-based delivery nanoplatforms was introduced to overcome the newly-emerging MDR in tumor cells. Triphenylphosphonium and 2,3- dimethylmaleic anhydride were conjugated onto the hPG-covered nanographene sheets to achieve mitochondria targeting and charge-convention properties. The average size of these functionalized nanographene sheets were around 75 nm with a narrow sizedistribution, which was favorable for their accumulation in tumor site owning to the widely confirmed EPR effect. After internalization, these nanosheets targeted the mitochondria and finally disrupted them under laser irradiation, leading to the plunge of adenosine triphosphate (ATP) synthesis. Without enough “biological fuel,” the P-gP lost its function and the MDR was successfully reversed. Both of the in vitro and in vivo antitumor results confirmed these functionalized graphene sheets could effectively surmount the troublesome MDR tumors and remarkably promote the synergic antitumor theranostic efficacy. Moreover, serious side effects caused by chemotherapy agents could also be avoided with these graphene-based nanocarriers. During the doctoral studies, my work focused on the biological behavior of graphene derivatives and their potential application for antitumor therapy. Many promising results were obtained and several critical problems were addressed, including the cellular uptake properties, intracellular release features, and anti-MDR theranostic. These outcomes revealed that the biological behaviors of functionalized graphene sheets could be adjusted by their physiochemical characteristics and MDR reversal therapy could be achieved though the rational design of graphene-based nanoplatforms, which is of great significance for the future development of graphene-based nanomaterials for bioapplications.
