Preparation Of Folate Acid And Poly(Ethylene Imine) Functionalized Graphene Oxide Loaded With Coordination Complexes Nanomedicine For Targeted Therapy On Nasopharyngeal Carcinoma

Objective:To study on preparation of graphene oxide(GO) nanocarrier which coupling folate acid(FA) and poly(ethylene imine)(PEI), new complexes containing rigid ligand have been designed, synthesized and loaded on the prepared FA-PEI-GO nanocarrier and studied for their vitro anti-NPC targeting activities and their effect to induce the apoptosis, in order to provide scientific evidence for a new therapy towards NPC.Methods:1. GO was prepared by Hummers method firstly then PEI and FA was grafted onto GO as targeting molecule. By using XRD, RAMAN, AFM, SEM, FTIR and TGA to carry out characterization on its physical and chemical properties.2. Copper, Zinc and Ruthenium complexes containing 2,2-bpy, phen and pymtH had been synthesized by hydrothermal method and the normal temperature of volatile, and the structure was characterized by IR spectroscopy and single crystal X-ray diffraction analysis.3. Grafting the complexes on the FA-PEI-GO nanocarrier through ultrasonic concussion and exploring their micrograph, particle size, drug-loading rate and release pattern by using TEM, DLS and UV-Vis. Their antiproliferation activities were investigated towards nasopharyngeal carcinoma HNE-1 and CNE-2 cells through CCK-8 kit. The cell percentages of apoptosis and cell cycle distribution of complexes were measured with flow cytometry.Results:1. GO, FA-PEI-GO and complex 1-4 were synthesized succussfully. The molecular formula of complex 1 was C15H10CuN3, the molecular formula of complex 2 was C26H18CuN6O14, the molecular formula of complex 3 was C12H14N6O12Zn, the molecular formula of complex 4 was C44H36N4P2RuS2. Complex 1-4 were loaded on FA-PEI-GO with 323±11 μg/mL,203±27μg/mL,191±22μg/mL and 265±18μg/mL respectively.2. X-ray diffraction and Raman spectroscopy indicated the GO was synthesized successfully. The standard curve of GO concentration was observed at the absorption peak around 230 nm-1 in the UV-vis spectroscopy. FTIR spectrum demonstrated the absorption peaks at 2943 cm-1 and 2824 cm-1 of PEI and the absorption peaks at 1479 cm-1 of FA. The percentage of PEI was about 25% of PEI-GO via TGA result and the FA content of FA-PEI-GO was about 67.3±8.1μg/mL through the result of UV-Vis. The average particle size of X*FA-PEI-GO was about 100 nm.3. The IC50 values of complex 1 were 17.7±1.2μmol/L and 13.2±1.9 μmol/L, complex 2 were 6.7±0.8μmol/Lμmol/L and 2.9±0.7μmol/L, complex 3 were 30.6±2.2μmol/L and 26.1±2.7μmol/L, complex 4 were 35.0±1.5μ/L and 29.3±2.4 μmol/L, which measured through the MTT results towards HNE-1 and CNE-2 cells. CDDP*FA-PEI-GO to HNE-1 cells showed a stronger antiprolifercative activity than to CNE-2 cells at 0.5μg/mL,1 μg/mL and 2 μg/mL. Besides, complex 1-4*FA-PEI-GO showed anti-prolifercative activities towards HNE-1 cells through the results of CCK-8 kit. The results of flow cytometry analysis showed that complex 1*FA-PEI-GO, complex 2*FA-PEI-GO and complex 3*FA-PEI-GO inhibited the proliferation of HNE-1 mainly by inducing apoptosis, in the highest concentration, the apoptosis rate of them were 74.9±0.9%,94.6±1.3% and 41.2±1.1%, complex 4*FA-PEI-GO inhibited the proliferation of HNE-1 mainly by inducing necrosis, in 40μmol/L, the necrosis rate of complex 4*FA-PEI-GO was 36.9±0.8%. Further studies complex 1-4*FA-PEI-GO effected on the cell cycle distribution of HNE-1 cell, the results revealed that complex 1*FA-PEI-GO, complex 2*FA-PEI-GO and complex 4*FA-PEI-GO could arrest the cell cycle of HNE-1 cell at S phase, which may be one of mechanisms inducing cell apoptosis or necrosis, otherwise complex 3*FA-PEI-GO could arrest the cell cycle of HNE-1 cell at G2/M phase with dose-dependant manner.Conclusions:1. FA-PEI-GO nanocarrier shows good biocompatibility and can load coordination complexes efficiently and promise for targeted drug delivery and anti-prolifercation effect in folate receptor over-expressing NPC cells.2. The antiproliferation activities of complex 1-4 on HNE-1 and CNE-2 cells are stronger than all the ligands and metel. Complex 1-3 all ideally have a inhibition on the two nasophyaryngeal carcinomar cells and the complex 2 shows better anti-proliferation activities than CDDP. It shows that complexes containing rigid ligands can enhance the anti-nasophyaryngeal carcinomar activity.3. Complex 1-4*FA-PEI-GO all shows anti-prolifercative activities towards HNE-1 cells, and complex 2*FA-PEI-GO shows the most remarkable anti-prolifercative activities.4. Complex 1*FA-PEI-GO, complex 2*FA-PEI-GO and complex 3*FA-PEI-GO inhibits the proliferation of HNE-1 mainly by inducing apoptosis while complex 4*FA-PEI-GO mainly by inducing necrosis. Complex 1*FA-PEI-GO, complex 2*FA-PEI-GO and complex 4*FA-PEI-GO arrest the cell cycle of HNE-1 cell at S phase while complex 3*FA-PEI-GO mainly at G2/M phase. It shows that this series of nanomedicine have a well prospect in development and may be further studied for its mechanism.