| Objectives:Modern pharmacology study has revealed that curcumol (Cur) has significant anti-hepatoma effect and plays an important role in liver protection. It also has anti-inflammatory and antibacterial capabilities. Cur compared with the traditional chemical drugs has the characteristic of low toxicity. Consequently, in the field of anti-hepatoma research, this compound has been drawing more and more attention in recent years. In order to make Cur rich in liver and improve its anti-hepatocarcinoma and liver protection effect, this study was aimed to make this compound possess liver-targeting property, through preparation of galactosylated liposome, which modified by galactose stearate. Meanwhile, this research also optimized the preparation methods, prescription and process of this liposome, as well as investigated its preliminary stability and safety, and finally valued its in vitro hepatic targeting and studied its anti-hepatoma effects in vitro and in vivo. In general, this research could provide a theoretical basis for the development of anti-hepatoma and liver protecting agents of Cur.Methods:1. The preparation of galactosylated fluorescent liposomes and its in vitro hepatic targetingThe D-galactose and vinyl stearate, which was dissolved in tetrahydrofuran, was used to synthesize galactose stearate under the catalysis of Novozym435immobilized lipase. The synthetic product was purified by silica gel column chromatography, and was analyzed by mass spectrometry and1H-NMR. Meanwhile, the content of target product was detected by high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), which could be used to calculate the yield of the target product.Then, by the thin film dispersion method, we used the obtained galactose stearate as a modification to prepare galactosylated liposome containing propidium iodide (PI), a kind of fluorescent dye. The particle diameter of galactosylated PI liposome (Gla-PI-L) was determinated by laser scattering method, and then its in vitro hepatic targeting was investigated in hepatoma cells (HepG2), gastric carcinoma cells (SGC7901) and lung carcinoma cells (A549).These three different types of cancer cells were incubated with different types of Gla-PI-L, in which the percentage of galactose stearate was different (0%,5%,10%,15%,20%,25%), and PI solution for a while. Cell fluorescence images were taken by a fluorescent inverted microscope and used for qualitative analysis. Meanwhile, the fluorescence intensity of these cells, after different treatments, was detected by fluorescence microplate for quantitative analysis.2. The preparation of galactosylated Cur liposomes and its stability and safety studyBy the second emulsification method, we used the obtained galactose stearate as a modification to prepare galactosylated liposome containing Cur (Gal-Cur-L). The content of Cur in Gal-Cur-L was detected by vanillin chromogenic method, the specificity, repeatability, stability and recovery rate of which were investigated respectively. Furthermore, the entrapment efficiency of the liposomes, obtained by petroleum ether extraction method, glucan gel column chromatography or dialysis method, were also investigated respectively; using the recovery percent of Cur and lipidosome as the detecting index, we ascertained the best method for determinating the entrapment efficiency of Gal-Cur-L.In order to better prepare Gal-Cur-L, the present study examined three preparation methods including ether injection method, film dispersion method and second emulsification method, and the encapsulation efficiency, particle diameter and stability factor were used as the indexes to elect the best preparation method. In this study, nine factors such as the drug-lipid ratio, phospholipid-cholesterol ratio, pH, ether dosage, triolein dosage, tween concentration, ultrasonic time, ultrasonic power and homogenization time, were selected for single factor test. Some factors that influence largely on the entrapment efficiency and particle diameter of Gal-Cur-L were further selected for orthogonal test. In this study, the morphology of Gal-Cur-L was analysed by optical microscopy and transmission electron microscopy respectively, and the encapsulation efficiency of it was measured by vanillin colorimetry-petroleum ether extraction method, while the particle and Zeta potential of this liposomes were detected by laser scattering method.Besides, in order to investigate the stability and security of Gal-Cur-L, the Gal-Cur-L was placed at4℃, regular tested its leakage rate, and assessed its reserved sample stability. Then the liposome after diluted to series of multiples, was also tested its leakage rate and observed its dilution stability. In addition, this study used rabbit red blood cell suspension to carry out the hemolytic test of the liposome, and the maximum tolerated dose of the liposome was also estimated in KM mice.3. The anti-hepatoma effects in vitro and in vivo of Gal-Cur-LHepatoma cells (HepG2) were selected to evaluate the in vitro antitumor activity of Gal-Cur-L. The experiment was divided into the following8groups:blank control group,5-FU positive control group (1.7mg·L-1), Gal-L control group, Cur control group (27mg·L-1), Cur-L control group, Gal-Cur-L13.5mg-L-1group, Gal-Cur-L27mg·L-1group and Gal-Cur-L54mg-L"1group. HepG2cells in different groups were incubated for24hours with each drug described above, and then MTT assay and lactate dehydrogenase (LDH) release were employed to evaluate cell viability and their damage degree.Furthermore, the transplanted hepatocarcinoma rat models were made by direct injcetion of Walker-256ascites to assess the anti-tumor effect of Gal-Cur-L in vivo. The experiment was also divided into the following8groups:normal control group, model control group,5-FU control group (52.3mg·kg-1), Cur control group (31.5mg·kg-1), Gur-L control group (31.5mg·kg-1), Gal-Cur-L15.75mg·kg-1group, Gal-Cur-L31.5mg·kg-1group and Gal-Cur-L63mg·kg-1group.The rats were treated with the above drugs by tail vein injection technique, and rats in the normal and model groups were injected with saline of the same volume with the same method. After the success of the model is determined, the therapy with the study drugs continued for20days. After that, the blood from the rat eye socket vEin was collected, and then the rats was executed by dislocation of cervical vertebra. Immediately, their livers were removed from the bodies, and tumor inhibitory rate was calculated, and the pathology of liver in every group was also observed by the method of hematoxylin-eosin (HE) staining. At the same time, by using the enzyme-linked immunosorbent assay (ELISA) kits, we also detected the levels of vascular endothelial growth factor (VEGF), endothelial inhibition (ES), aspertate aminotransferase (AST) and alanine aminotransferase (ALT) in serum of rats to comprehensively evaluate the therapeutical effect of these drugs on liver cancer.Results:1. The preparation of galactosylated fluorescent liposomes and its in vitro hepatictargetingMS analysis showed that the relative molecular weight of the target product is446, and1H-NMR analysis showed that the ester exchange reaction occurs between the the C6hydroxyl of galactos and ester bond of vinyl stearate. According to the results of HPLC-ELSD, we calculated that the galactose stearic acid ester yield was43.3%.Particle diameter test results showed that along with the increase of percentage of adding galactose stearic acid ester, the liposome size increased; and that the mean particle diameter of Gla-PI-L was less than200nm. Meanwhile, the qualitative and quantitative analysis demonstated that for the three kinds of cells treated with PI solution, they were scarcely stained, and the fluorescence intensity of them was all weak; Gla-PI-L with different ratios of galactose stearate could not make SGC-7901cells and A549be stained, but make HepG2be obviously stained, and the fluorescence intensity of HepG2cells could be expected to strengthen along with the increase of the percentage of galactose stearate, which was tend to balance when the percentage was20%. In general, these results suggest that galactosylated liposomes possess a good in vivo liver-targeting efficiency, which is intaked by liver-cell verged to balance when the percentage of galactose stearate was20%. Therefore, in the following experiments of this study the percentage of galactose stearate in galactosylated liposome was fixed in20%.2. The preparation of galactosylated Cur liposomes and its stability and safety studySpecificity study showed that other components of Gal-Cur-L had no interference in the determination of Cur. Reproducibility test revealed that the mean concentration of Cur in the tested samples of Gal-Cur-L was6.58mg-L-1(RSD1.73%), and the recovery experimental results indicated that the sample recovery was98%～101%, RSD<3%. The petroleum ether extraction method was the best method for determinating the entrapment efficiency of Gal-Cur-L.Preparation methods study results demonstated that the encapsulation efficiency, particle diameter and stability coefficient of Gal-Cur-L obtained by the second emulsification method were better than those of Gal-Cur-L obtained by ether injection method or film dispersion method. The results of single factor investigation showed pH, PBS and the amount of triolein had small effect on the encapsulation efficiency and particle diameter of Gal-Cur-L. From the results of orthogonal test for prescription, we got the best prescription:the ratio of lecithin-cholesterol was2:1, and that of drug-lipid was1:2, and the amount of ether and Twain were4mL and1.6g·L-1respectively. Meanwhile, from the results of orthogonal test for preparation technology, we found out that the optimum technology was15min for ultrasonic time, and20min for homogeneous time.Gal-Cur-L, obtained by the best prescription and preparation technology, was uniform milky emulsion with slight opalescence. There were no significant difference in appearance between Gal-Cur-L and Cur-L. The particle diameter of Gal-Cur-L showed a unimodal normal distribution, and the mean diameter of it was173.8±2.23nm. The diameter distributions of Cur-L and Gal-L were similar to that of Gal-Cur-L, the mean particle diameter of which was153.9±3.12nm and134.1±2.68nm respectively. Zeta potential of Gal-Cur-L was-40.86±1.69mV, and that of Cur-L was-34.13±1.26mV. the average packet of Gal-Cur-L closure rate was75.47±0.35%, stable coefficient of6.35±0.47%; Cur-L encapsulation efficiency was72.33±0.87%, the stability factor of5.68±0.31%. Furthermore, experimental results also indicated that the encapsulation efficiency of Gal-Cur-L and Cur-L was75.47±0.35%and72.33±0.87%respectively, while the stability coefficient of them was6.35±0.47%and5.68±0.31%, respectively.In addition, our preliminary stability test indicated that Gal-Cur-L was still steady at4℃for3months, and remained stable after10-fold dilution. The hemolysis test results revealed that there was no hemolytic phenomenon for Gal-Cur-L and Cur-L. Besides, the maximum tolerance test displayed that that the maximum tolerated dose of Gal-Cur-L is over240mL·kg-1.3. The anti-hepatoma effects in vitro and in vivo of Gal-Cur-LThe data of HepG2cell viability and LDH release rate indicated that, except Gal-L group, compared with blank control group, there were significant differences for the other groups (all p<0.05). Moreover, compared with Cur control group, different dosage groups of Gal-Cur-L and Cur-L control group all showed significant difference (all p<0.05); compared with Cur-L control group, different dosage groups of Gal-Cur-L all displayed significant difference (all p<0.05).Furthermore, the successful rate of transplanted hepatocarcinoma in rats was100%by direct injcetion of Walker-256ascites. The tumor inhibiting rates of drug therapy groups was higher than that of model control group. Compared with Cur control group, different dosage groups of Gal-Cur-L and Cur-L control group all showed significant difference (all p<0.05); compared with Cur-L control group, different dosage groups of Gal-Cur-L all displayed significant difference (all p<0.05).Compared with the model control group, the level of VEGF in each drug treated groups was significantly reduced, while the level of ES was significantly increased, and then the value of VEGF/ES was obviously decreased (all p<0.01). For the levels of VEGF, ES and VEGF/ES, compared with Cur control group, different dosage groups of Gal-Cur-L and Cur-L control group all showed significant difference (all p<0.05). Compared with Cur-L control group, different dosage groups of Gal-Cur-L all displayed significant difference (all p<0.05) for the levels of VEGF and ES.Compared with the model control group, the levels of AST and ALT value in each drug treated group were all significantly changed (all p<0.01), the levels of which also showed significant difference when compared to5-FU control group (p<0.05). For these two indexes mentioned above, compared with Cur control group, different dosage groups of Gal-Cur-L and Cur-L control group all showed significant difference (all p<0.05), except that there were no statistically significant difference between Cur control group and Gal-Cur-L low-dose group for the levels of ALT (p>0.05). Moreover, for these two indexes, compared with Cur-L control group, Gal-Cur-L middle-dose group and Gal-Cur-L high-dose group not displayed significant difference (p>0.05).In addition, the liver tissue HE staining showed that the hepatic lobule in the liver tissue of normal control group had the complete structure, while the structure of hepatic lobule, in model control group and different drug treated groups, was subjected to varying degrees of damage. In the liver tissue sections of model control group, there are a large number of cancer nodules, necrotic hepatocytes and a large number of inflammatory cells infiltration, while the cells of carcinoma tissues were dyed purple. Nevertheless, in the liver tissue sections of the drug therapy groups, especially the Gal-Cur-L middle-dose group and Gal-Cur-L high-dose group, the adverse changes mentioned above were all reversed and the area of cancer cell necrosis and foam cell hyperplasia was arised in defferent levels. Conclusion:The molecular weight and structure of galactose stearate obtained by enzymatic method are consistent with literature reports. The galactosylated PI liposome (Gla-PI-L) appears good targeting property, and presents highly distribution in hepatic cells (HepG2), while shows low distribution in gastric cancer cells (SGC7901)and lung cancer cells (A549). These data indicates galactosylated liposome has significant liver-targeting property, which is be a climb to the top when the ratio of galactose stearate in galactosylated liposome was20%.Meanwhile, the stability, repeatability and specificity tests revealed that vanillin colorimetric method works well for detecting the content of Cur in Gal-Cur-L. Furthermore, the results of the determination method for entrapment efficiency declare that the best way is petroleum ether extraction method, and the data of preparation technology show that the optimum technology is the second emulsification method. Besides, the particle diameter, Zeta potential, appearance, encapsulation efficiency and stability factor of Gal-Cur-L, obtained by the best prescription and preparation technology, are satisfied. Additionally, the preliminary investigation of the stability and safety of Gal-Cur-L exhibits that the liposomes prepared by this study can meet the requirements for animal experiment.In vitro anti-hepatoma results show that Gal-L, obtained by our prescription and preparation technology, has no adverse effect on cell growth. Besides that, in vivo anti-hepatoma experimental results, along with that of the experiment in vitro, indicate that Cur after modified by galactose stearate, can greatly increase its concentration in the liver, and its anti-hepatoma effect increases more significantly than Cur and Cur-L. The hepatoprotective effect of Gal-Cur-L and Cur-L was strengthend better than Cur. However, there was no statistically significant difference between Gal-Cur-L and Cur-L for hepatoprotective effect.In conclusion, Gal-Cur-L make the drug have good liver targeting property, significantly enhance its anti-hepatoma effect, and give full play to its anti-hepatoma and hepatoprotective effect. |
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