Preparation Of Polymer Gene Vectors-graphene Oxide Complex And Studies On Its Biocompatibility

As the discovery of graphene, research about2-D materials encounters newchallenge. As the same time, graphite oxide–after it was discovered for more than150years-has attract great concerns because it can be used as the raw materials forlow cost and large scale produce graphene. After exfoliated, one can get grapheneoxide in solution from graphite oxide. Graphene oxide has lots of oxygen-containinggroups which make the properties of graphene oxide great different from graphene.Recently, a lot of literatures about application of graphene oxide have been published.Among these reports, there are some attempts of using graphene oxide as bio-ormedicine materials. Some reports shows that GO is nontoxic both in vitro and in vivo.GO has impressively drug loading ratio benefits from its large specific surface area.Massive oxygen-containing groups make GO easy to be modified. All these propertiesmake GO a great potential to be a powerful drug carrier.In other hand, as the development of medical and molecular biology, theknowledge about disease comes into genome era. After the accomplishment of HumanGenome Project and progress in detection method, causing genes of many diseasesincluding cancers have been identified. As a result, a new technology named genetherapy appears. For design gene vectors using in gene therapy, many new criterianeed to be satisfied. Comparing with traditional drugs, genetic materials have theirspecial natures and need different delivery pathways to make therapeutic gene work.Up to now, there are mainly two types successful gene vectors which arebiology-based vectors and synthesis vectors. For viral or bacteria vectors, its mainbottleneck is their safety concerns. These biology-base vetors might revert towild-type virion and they are inherently immunogenic. So development of novelvectors with high efficient and low toxicity is one of the research focuses in genetherapy.In this thesis, we choose GO as starting material. After chemical modification,we get GO-based gene delivery vectors. Both cell transfection and protein absorption experiments were carried out to evaluate its ability as gene vectors.At first, GO was gotten using an improved Hummers method. Carboxyl was thenintroduced into GO using NaOH and chloroacetic acid. For endowing GO the abilityof loading gene, two widely used polymer poly-L-lysine(PLL) andpolyethyleneimine(PEI) were conjugated onto GO. PEG molecules was introduce intothe complex of GO and low molecular weight PEI. After characterizing all ofsynthesized complexes, their stabilities in different solution environment wereobserved. Results showed that both high molecular weight PEI-GO conjugation andPEGylated PEI-GO were stable in both high concentration of NaCl solution andDMEM solution.Because there was report about high molecular weight PEI-GO complex used astransfection vectors and it was highly toxicity, we did not observe the transfectionefficient of high molecular weight PEI-GO complex in this thesis. Instead, GO-PLL,low molecular weight PEI-GO and PEGylated low molecular weight PEI-GO wasinvestigated. All of the observed sample showed low cytotoxicity but had highly DNAplasmid binding ability. In the cell transfection experiment, GO-PLL showed poorlytransfection efficient while GO-PEI-PEG gave an excellent stability and transfectionefficient.At last, we investigated the interaction between our vectors and protein. Bovineserum albumin (BSA) was chosen as model protein. Both Bradford assay and proteininherent absorbance was used for protein quantitative analysis method and the resultswere compared. Bradford assay was chosen as our protein quantitative analysismethod because of its versatility. Because of its low transfection efficient, interactionbetween GO-PLL and protein was not test. Results of relationship between proteinconcentration and vector stability in solution showed that there were obviouslydifferent among our vectors. As BSA concentration increased, GO appeared topartially aggregation at first and became stable in BSA solution of high concentration.High molecular weight PEI-GO complex showed a reversed stability which was stablein BSA solution of low concentration and became aggregation in BSA solution of highconcentration. Low molecular weight PEI-GO complex aggregated in all tested BSAsolution. After PEGylation, GO-PEI-PEG was stable in all tested BSA solution.Relationship between protein amount absorption on vectors and pH value of solutionshowed that protein amount absorption on GO decreased as pH value increased. These results suggested that the interaction between BAS and GO were dominated byelectrostatic interaction. Through Langmuir and Freundlich adsorption isothermformulas, BSA absorbed on GO and GO-PEI-PEG should follow the single layerabsorption model. Both GO-PEI samples did not follow the Langmuir a adsorptionisotherm formula. Especially, high molecular weight PEI-GO complex showed atypical multiple layers absorption behavior. The adsorption isotherms also showedthat the binding ability between protein and PEGylated samples were weaker thanother samples which made less protein absorption on GO-PEI-PEG after adsorptionequilibrium. Because most of biological interaction started from protein interaction,vectors with less protein absorption should more safety in practical usage.