Synthesis Of Gd(?)-MOF And Application In 5-fu Delivery And Inhibiting Human Liver Cancer Cells

In this study,a novel metal organic framework material 1 was synthesized by solvothermal method,and its structure was characterized by different methods.Then the anticancer drug 5-FU was loaded into the metal organic framework material 1,and its in vitro release performance was observed.In addition,the toxicity of the metal organic framework material 1 was evaluated in the in vitro cell experiment,To analyze whether it has high drug loading,stability and biocompatibility,and whether it can become an ideal drug carrier in clinical treatment.Part ? Preparation and characterization of Gd(?)-MOFPurposes:In this study,5-fu drugs were loaded with metal-organic framework materials,and drug release tests and drug loading tests were carried out on them.In addition,the external release characteristics of metal-organic framework materials after drug loading were observed in vitro,so as to study the biocompatibility of the newly developed drug-loaded MOF.Materals and method:A new Gd(?)metal-organic framework(1)with the chemical formula of[Gd(BCB)(DMF)](H2O)2 has been successfully prepared using 4,4',4"-benzenetricarbonyltribenzoic acid(H3BCB)as organic linkers and 1D Gd(?)-based secondary building unit chain as metal nodes(47%based on Gd(NO3)36H2O).The products were characterized by elemental analysis,Fourier transform infrared spectroscopy,X-ray single crystal diffraction analysis and grand canonical Monte Carlo simulation(GCMC).To evaluate the biocompatibility of la,the in vitro cytotoxicity effects of la towards the cells line BEL-7404(liver cancer cells)and oral epidermal cells(normal cells)were determined using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)assays.Results:1,X-ray single crystal diffraction analysis results showed that the product belongs to Fdd2 space group.The crystal structure of the product was orthorhombic.The crystal structure unit was asymmetric.It contained two end-coordinated DMF ligands,two completely deprotonated BCB3-anions,two independent Gd3+ ions and two free water molecules..The Gd1 center was coordinated by eight oxygen atoms:six from six different BCB3+ ligands,and two from the terminally coordinated DMF molecules.Meanwhile,the Gd2 site was surrounded by eight oxygen atoms from six different BCB3-ligands.Elemental analysis(%)calcd for C33H26GdNO12:C,50.44;H,3.34;N,1.78.Found:C,50.16;H,3.13;N,1.92.IR(KBr pellet,cm-1):3123(br),1947(w),1773(w),1712(w),1610(vs),1549(s),1381(vs),1163(w),1109(m),1019(m),832(w),771(w),741(m),705(w),579(w).2,Complex 1 showed 1D channel of 13.7 × 6.4 A2 along the c axis,which was large enough for accommodating anticancer drug 5-Fu(5-Fluorouracil)molecules.To investigate the thermal stabilities,TGA measurements of complex 1 were performed in the temperature range of 30?800 ? under N2 gas flow.An initial weight loss of 14%was observed from 30 to 250?,indicating the loss of one coordinated DMF molecule and two lattice water molecules(calcd:13.9%).Their frameworks began to collapse and burn off with the decomposition of the H3BCB ligands when the temperature reached at approximately 500?,demonstrating their high thermal stability.Considering the following drug delivery experiments,the stability of 1 in the PBS solution had been checked by soaking the crystalline samples of 1 in PBS(pH=7.4)for one day at 37? in an oven,then the corresponding PXRD patterns were collected,which reveals that the PBS surroundings could not lead to the transformation of the framework of 1,confirming its good stability in the PBS.3,The N2 adsorption by at 77 K shows a reversible type-I adsorption behavior characteristic of microporous materials with a saturated uptake of 256 cm3/g,corresponding to a Brunauer-Emmett-Teifer(BET)surface area of 716 m2/g and a Langmuir surface area of 892 m2/g.A density functional theory based model fitted to the adsorption branch of the 77 K N2 isotherm showed the majority of the pores being 6.4 A in size.The drug loading experiments were carried out by impregnating of 1a.The 5-Fu storage capacity was 36.4 wt%based on the UV-Vis spectrum result.The delivery of 5-Fu occurred within 20 h(pH=7.4)without“burst effect”and further increasing the time could not lead to more 5-Fu released from the carrier.In contrast,when the pH value of the PBS solution was adjusted to 6.5,a faster release of 5-Fu from the 5-Fu loaded 1a was achieved compared to that at physiological pH 7.4.Meanwhile,it could be observed that 68%of loaded 5-Fu molecules were released into the solution within 35 h.The drug release profiles of 5-Fu loaded 1a indicated an enhanced release amount of the drug in acidic conditions(mimicking the acidic environment of the tumor cells)as compared to the slightly alkaline conditions(mimicking the pH of blood and normal tissues).4,To understand the interaction between the 5-Fu molecules and the framework,the GCMC simulation was carried out.The simulated favorable binding site of 5-Fu at zero loading indicates that the one 5-Fu molecule prefered to locate in the channel center with two H-bond interactions could be observed(C-O--H distance:2.614 A and 2.826 A).Furthermore,besides the H-bond interactions,there also existed electronic interaction between the F atom on the 5-Fu molecule and the open Gd(?)metal site with a distance of 3.370 A.All these interactions contribute to the strong binding energy of 5-Fu with the framework,resulting in the long-lasting drug release.5,In addition,the results from MTT assays revealed that the complex 1a was nontoxic(cell viability>80%)to the normal cells and liver cancer cells BEL-7404 in spite of concentrations up to 200 ?g/mL.These data indicated that this novel drug carrier possessed excellent biocompatibility.Conclusions:1,We have successfully designed a new metal-organic framework Gd(?)-MOF(1)by the thermal-flux reaction of(Gd(NO3)3](H2O)6 and H3BCB ligands.Studies have shown that this new drug carrier has high thermal stability and pH-dependent backbone stability,and has a large specific surface area,making it an ideal drug-load carrier.2,The novel metal-organic framework Gd(?)-MOF(1)is less toxic to normal cells and tumor cells,indicating good biocompatibility.Part ? Study on the inhibitory effect of Gd(?)-MOF loading with 5-Fu on the proliferation of human liver cancer cells in vitro and in vivo Ultrasound-guided Purposes:To evaluate the pharmacodynamics of the metal-organic framework loaded with the anticancer drug 5-Fu(5Fu@la),including in vitro cell experiments and ultrasound-guided animal experiments.Materals and method:MTT assays were used to evaluate the anticancer efficiency of 1a.At the same time,we injected a new metal-organic framework(5-Fu@1a)loaded with anticancer drug 5-Fu by intratumoral injection of human liver cancer cells BEL-7404 nude mice with intratumoral injection of antitumor drug 5-Fu and intratumoral injection of normal saline.In this study,ultrasound-guided intratumoral multi-point injection method was used to observe the needle insertion pathway and drug injection in real time,so that the drug could be accurately and evenly distributed throughout the tumor.Contrast-enhanced ultrasound was used to evaluate the experimental efficacy of intratumoral injection of 5-Fu@la.Results:1,5-Fu@1a exhibited the significant increase of anticancer activity against the human liver cancer cells BEL-7404 with the increase of the concentration.About 30%of BEL-7404 cells were killed under at 20 ?g/mL of 5-Fu@la in 24 h.It should be noted that further increase the time to 48 h could result in more cancer cell death,indicating the sustained and prolonged 5-Fu release from 1a.Furthermore,it could be observed that although the cancer cell viability of 5-Fu@1a was comparable with that of 5-Fu after 24 h,it was much lower than that of 5-Fu after 48 h,indicating the anticancer efficiency of 5-Fu was much improved by using 1a as the carrier.In comparison,it was found that the metal-organic framework of the same drug loading significantly inhibited tumor growth,while the 5-Fu@1a group did not show significant adverse reactions.2,At the same time,compared with the intratumoral injection of 5-Fu@1a in the human liver cancer cells BEL-7404 nude mice and the intratumoral injection of anti-tumor drug 5-Fu or physiological saline,it was found that 5-Fu@1a could inhibit growth of tumor significantly.And no significant adverse reactions adverse reactions were observed in 5-Fu@la group.In this study,the animals in the ultrasound-guied intratumoral injection of 5-Fu@1a group showed good mental activity,and did not show lethargy or emaciation.In the model group,the peripheral branching vessels of the tumors were enhanced obviously,and then most of the tumors were rapidly patchy enhanced and rapidly subsided.In 5-Fu group,the extent of parenchymal ultrasound enhancement was limited to peripheral enhancement and a little parenchymal patchy enhancement.After 3 weeks of treatment,the periphery of the tumors in group 5-Fu@la was slightly enhanced,and most of the parenchyma showed filling defect changes.PBD of group 5-Fu@1a was significantly lower than that in the other two groups.Quantitative analysis of contrast-enhanced ultrasound showed that 5-Fu@1a could inhibit angiogenesis of tumors.Conclusions:The study shows that the new metal-organic framework 1A loaded with 5-Fu has an effective anti-cancer ability,could significantly inhibit tumor growth and inhibit tumor angiogenesis,and is expected to be used in the treatment of hepatocellular carcinoma.