| MOFs are new porous organic-inorganic hybrid functional materials. Compared with the classical inorganic microporous material, MOFs have rich spatial and topological structure, higher specific surface area and unique electromagnetic properties, which show important prospect in the H2and CO2gases adsorption. Recent studies suggest that MOFs also show a lots features as drug carriers, for example, high surface area are suitable for encapsulation of the drug. There are relatively unstable coordination bond between metal and ligand so that MOFs get biodegradability, MOFs also can carry multiple functional groups by modifying.MOFs usually comple metal ion with organic ligand by self-assembling by the mild conditions. In recent years, foreign scholars (Ferey; Wenbin Lin) study MOFs as a drug delivery system, the results show that the MOFs can achieve high drug loading rate and controlled release. For example, MOFs containing Cr3+and loading ibuprofen, the highest drug loading rate is0.220g/g carrier, at the same time they can release completely until18d, but the Cr3+limit their application in vivo. In view of human tissue contains metal element, MOFs synthesized with zinc which are more appropriate as drug carriers.Folic acid, an essential vitamin, joins nucleotide synthesis as cell growth and proliferation and passes into the cell mainly through the folic acid receptor. Folic acid receptor has high express level in vigorous tissues, especially in epithelial cancer, such as ovarian cancer, renal cancer, uterine cancer, testicular cancer, brain cancer, colon cancer, lung cancer and oral epithelial cancer and so on. Due to nontoxicity, low immunogenicity, stability, and binding force for folic acid receptor, folic acid has highly selection for the tumor. Many studies show that folic acid receptor as a therapeutic target in tumor treatment can enhance the anti-tumor effect, reduce the damage to normal tissue and overcome the drug resistance of tumor cells.5-fluorouracil (5-FU) is a commonly clinical antineoplastic drug for antimetabolite, mainly is used for stomach, intestines and liver and other cancer treatment, while it has strong toxicity and side effect. MOFs have high drug loading rate and controlled release, which can extend5-FU action time and improve short half-life toxicity and other shortcomings in vivo. IRMOF-3were synthesized, couple with folic acid and use5-FU as a model drug. The drug loading rate, release properties, cytotoxicity and target activity were evaluated in vitro, which provide prospect for IRMOF-3application when the domestic research have not been reported in this area.Purpose:1IRMOF-3were synthesized by solvent thermal process and characterized.2The surface of IRMOF-3were modified by folic acid and got Folic acid-IRMOF-3(FIR). The prescription process of coupling folic acid was optimized and FIR were characterized.3FIR were evaluated as drug carrier and drug release was studied in vitro.4Cytotoxicity of FIR and cell targeting characteristics were evaluated in vitro.Methods and results:1IRMOF-3were synthesized by solvent thermal process, and DMF was removed by the method of heating activation. IRMOF-3were confirmed by XRD, IR, SEM, TG. The results indicated that the structure of synthetic IRMOF-3was confirmed through XRD, IR spectrum. According to the results of TG, IRMOF-3displayed better thermo-stabilization and the most DMF was removed by heating activation. The average particle size about200-300nm, and IRMOF-3were round shape, smooth surface and uniform distribution.2Due to the principle that amide can be got by amino groups and carboxyl groups, IRMOF-3were modified by folic acid and obtain FIR. FIR were characterized by1H NMR, IR, SEM and MS to qualitative analysis. And the process of coupling folic acid was optimized and coupling rate as main indexes. According to L16(43) orthogonal design experiment, the study compared R value to determine sequence of the factors, and got an optimum prescription from various levels of K value. The results of1H NMR, IR and MS spectrum showed that the chemical shift and characteristic absorption peak of folic acid were shown in spectrum, which suggested that folic acid have been successfully coupled on IRMOF-3. The average particle size of FIR about200-300nm and uniformed distribution, the shape were circular or elliptic. Among the numerous influential factors, the sequence was carrier: folic acid (mass ratio)> reaction time> reaction temperature, the best condition was that mass ratio of carrier and folic acid was2:1, reaction time was2h, reaction temperature was40℃and the highest rate was0.709g/g carrier.3With loading time, carrier and drug quality ratio and loading rate as index, the study optimized the condition of loading drug to determine the best condition. Release experiment was observed in vitro. The result showed that drug loading of FIR was increased with the5-FU and FIR carrier mass ratio, and time lengthening also made FIR drug loading increase. At the same time, FIR loading rate was increased as the drug-loaded time. Factorial analysis showed that the FIR loading rate had a significant influence in different loading date (F=145.398, P=0) and different mass ratios (F=18.167, P=0), the drug loading date and the mass ratio had interactive effect (F=4.499, P=0.001).But the drug loading unchanged when5-FU and carrier quality ratio was1:2and the loading time was5d. So the optimal loading condition was that5-FU and FIR mass ratio was2:1and drug-loaded time was5d. According to diffusion method, the result showed that the accumulated release percentage of FIR containing5-FU was55.3%during84h and showed obvious sustained release effect.4A549cells (folic acid receptor deficient cells line), Hela cells (folic acid receptor expressed cells line) and KB cells (folic acid receptor over-expressed cells line) were cultured in10%fetal bovine serum without folic acid media, then continued incubated with media containing different concentration of5-fluorouracil (5-FU group, positive control group), IRMOF-3, IRMOF-3containing5-fluorouracil (5FU-IR, uncoupling folic acid), folic acid-conjugated IRMOF-3containing5-fluorouracil (5FU-FIR, connected with folic acid).Cell survival rate was determined by MTT method after48h.The results showed that the cell survival rate of IRMOF-3was much higher than5-FU in the three cells, IRMOF-3had better biocompatibility. The cell survival rate had no statistical significance between5FU-IR and5-FU in three kinds of cells (P values more than0.05), because5-FU and5FU-IR go into cells in the same way and the cell toxicity have no difference.The results also displayed that A549cells was folic acid receptor unexpressed cells,5FU-FIR group can not via folic acid receptor get into cells,and the cell survival rate had no significant difference between5FU-FIR and5-FU (P=0.379). Hela cells was folic acid receptor expressed cells,5FU-FIR had affinity with folic acid receptor and reached target cells by folic acid receptor, but may be5-FU entering hole had slow release process and can not completely release5-FU in48h, cell survival also had no significant difference in the two groups (P=0.441). KB cells was folic acid receptor high expressed cells, FIR can through the folic acid receptor into tumor cells and5-FU may be fast and accurate into the cell, and cell toxicity increased significantly. So the cell survival rate of the5FU-FIR group and5-FU group had significant difference (P=0.013).5In order to evaluate cell target of5FU-FIR,5FU-IR were used as control and three tumor cells of folic acid receptor different expressed (KB cells, Hela cells, A549cells) as the model in this experiment. The folic acid and the there cells were respectively cultured with5FU-FIR and5FU-IR for48h,then MTT method determined cell inhibition rate to study the effect of folic acid on cell growth and the tumor selectivity of5FU-FIR.The results showed that folic acid had no significant effect on cell growth in three kinds cells. Cell inhibition rate of5FU-IR were not subject to free folic acid in cells of folic acid receptor expressed and not expressed. The reason was that5FU-IR without coupling folic acid did not have the target activity and combined folic acid receptor.5FU-FIR can not bind to folic acid receptor and were swallowed through the passive way in A549cells. Cell inhibition rate was not affected by free folic acid at different concentration (F=0.495, P=0.690).But in Hela and KB cells, a small amount of folic acid receptor was blocked at low concentration of free acid and5FU-FIR can reach the cell interior. With exogenous folic acid concentration increased, folic acid receptor were slowly blocked and completely blocked at high concentration, then5FU-FIR were swallowed only through the passive mode and cell toxicity reduced greatly. So cell inhibition rate of different concentrations of folic acid had significant difference in the folic acid receptor expression cells for 5FU-FIR.(F=14.021,P=0.000in Hela cells,F=117.204,P=0.000in KB cells).In Hela cells and KB cells, the5FU-FIR had a strong targeting and targeted into the cell interior with folic acid receptor, whereas5FU-IR only through the endocytic means got into cells and cytotoxicity was less than5FU-FIR. A549cells were the cells folic acid receptor expressed negative,5FU-FIR and5FU-IR got into the cell in the same way without folic acid receptor, therefore there was no significant difference in cell inhibition rate for two groups. In summary,5FU-FIR were good antitumor medicine carrier for cell targeting and high affinity with folic acid receptor.Conclusion:Nanometer metal organic framework IRMOF-3own the properties of the right size, stability, repeatability, round or elliptic shape, smooth surface and uniform distribution. The folic acid was successfully coupled to IRMOF-3according to1H NMR, IR and MS spectrum. The folic acid-conjugated optimum condition was got by orthogonal experimental method. FIR loading5-FU have high loading rate by optimizing the loading condition and have better slow release in vitro, which can extend action time and improve short half-life for5-FU.The experiments of cell cytotoxicity and targeting effect show that toxicity of IRMOF-3is less than5-FU in the same drug concentration and IRMOF-3own good biocompatibility.5FU-FIR have targeting effect and are good carriers in cells of folic acid receptor expressed.The study of innovation:Metal organic frameworks IRMOF-3have many good properties, such as better compatibility, low toxicity, simple synthesis, tumor targeting with the coupling folic acid, large pore and surface area, high drug loading and controlled release. So IRMOF-3are good carriers of antitumor drugs. |
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