Covalent Functionalization of Graphene Oxide with Poly-Lysine for the Use as mRNA Carriers

Graphene Oxide (GO) is an emerging carrier for a multitude of biomedical applications including messenger RNA (mRNA). GO is the oxidized version of graphene a two-dimensional planar structure consisting of sp2 hybridized carbons in repeating six-membered rings creating high potential for adsorption of drugs and macro molecules. The hope with GO based mRNA carriers is to serve as a thermodynamically stable alternative to lipid nanoparticles (LNPs).This master thesis presents an investigation into the applicability of covalent functionalization of GO with poly-lysine (PLL) and increased degree of functionalization through GO carboxylation with chloroacetic acid. With the goal to serve as a Proof-of-concept for the future development of mRNA carriers.Two types of reactions were investigated, peptide coupling and epoxide opening. The final carriers were assed with optical microscopy to determine size and morphology, 2,4,6-Trinitrobenzenesulfonic acid (TNBS) assay for the degree of PLL functionalization and differential scanning calorimetry (DSC) for qualitative mRNA binding data. The reactions and workup were validated using simplified molecular models such as NH3 instead of PLL as to distinguish between covalent bond formation and electrostatic adsorption with TGA-FTIR. Alkylation with chloroacetic acid was validated with a model system utilizing phenol as a model for GO which was assessed with LC-MS/MS, and corroborated with TGA-FTIR data. Workups removing leftover reactants or byproducts were either validated by 1H-NMR or TGA-FTIR.The final mRNA carriers displayed agglomeration of the GO sheets indicating that there will be difficulties achieving size and morphology requirements for a final medical product. Four out of seven investigated carriers had statistically significant concentrations of PLL and three out of seven gave positive results for mRNA binding. Both carriers from the epoxide opening reaction gave active mRNA carriers while one out of five carriers from the peptide coupling reaction gave an active mRNA carrier. Validation of the two N-C bond forming reactions failed likely due to high limit of detection of the method used (TGA-FTIR). Validation of alkylation with chloroacetic acid succeeded highlighting the applicability of TGA coupled analysis methods for proving covalent bond formation on GO. Validation of the workups proved the presence of leftover reactants and byproducts, and the need for further cleaning steps.