| Adlay(Coix lachryma-jobi L. var. ma-yuen Stapf) refers to dry groats of coix, belonging to the family Gramineae. Domestic and international reaserches found that adlay possess many functional components, such as esters, free fatty acids, carbohydrates, lactam compounds, etc. Adlay has been reported to have antitumor, immunomodulatory, hypoglycemic activities. Coix seed oil is the main anti-tumor active component of coix seed, which has been used for clinical to treat liver tumor. Meanwhile, coixan is another anti-tumor active ingredient of coix seed, which has been confirmed to improve the immune system. However, they represent some drawbacks originating from their physicochemical property, leading to poor intestinal absorption. At present, it is still in its infancy for novel oral formulations beening devoted to enhance oral absorption of coix seed components. Amongst the various drug delivery systems, the microemulsion is defined as a dispersion consisting of oil, surfactants, cosurfactants and an aqueous phase, which is transparent, low viscosity and thermo dynamicallystable liquid solution. Additionally, microemulsion with small size and good stability can increase poorly soluble drugs for oral delivery. In previous study, we used coix seed oil as oil phase of microemulsion and prepared coix seed oil-triterpene components of Ganoderma microemulsion, which effectively increased absorption of coix seed oil and Ganoderma triterpenoids in vivo. However, the microemulsion delivery system mainly depended on the EPR effect leading to specificly accumulation on the tumor. In order to achieve further enhancing the recognition ability of tumor cells, surface modification with small molecule ligand is a feasible solution. In this study, coix seed oil and coixan used as two kinds of antitumor main components and coix seed components-based microemulsions(C-MEs) was prepared. In order to further improve hepatoma targeting and anti-hepatoma effect of C-MEs and then hydrophobic galactose esters with three different conformations were synthesized with exposed non-reducing D-galactose(Gal). The three kinds of galactose esters with small molecular weight were butyryl galactose ester(Gal(but)), octanoyl galactose ester(Gal(oct)) and acetyl galactose ester(Gal(ace)), which were able to be specific recognized by asialoglycoprotein receptor(ASGPR) on hepatocytes. C-MEs was modified by Gal(but) and Gal(oct) for obtaining butyryl galactose ester modified-coix components-based microemulsions(Gal(but)-C-MEs) and octanoyl galactose ester modified-coix components-based microemulsions(Gal(oct)-C-MEs). Meanwhile, physicochemical properties and hepatoma-targeting of Gal(but)-C-MEs and Gal(oct)-C-MEs were evaluated in vitro and in vivo, which provided theoretical basis and practical exploration for further improvement of the tissue targeting and anti-tumor effect of multi-components tumor targeting nano agents with Chinese medicine characteristics. We completed the following studies in experimental part: 1. Synthesis and characterization of galactose esters with different molecular conformationsUnder the catalysis of immobilized lipase Novozym 435, molar ratio of vinyl n-Octanoate, vinyl butyrate, vinyl acetate and galactose were both 1:1, and reacted in 60°C for 12-18 hours. The crude products were purified by silica gel column chromatography, which petroleum ether ratios to ethyl acetate(8:1, 12:1 and15:1) were respectively used as the elution agent. And then galactose esters were obtained. Meanwhile, the structures of products have been identified by FT-IR and 1H-NMR. Due to the higher difficulty of the purification of Gal(ace) and more by-products, so Gal(oct) and Gal(but) were selected to use for subsequent experiments. 2. Preparation and characterization of galactose ester modified-coix components-based microemulsionsIn the study, coix seed oil as the oil phase and coixan solution as water phase combined with pseudo ternary, Gal(but)-C-MEs and Gal(oct)-C-MEs were screened. The average size, PDI and zeta potential of Gal(but)-C-MEs and Gal(oct)-C-MEs were characterized. Meanwhile, the stability of microemulsions was evaluated.(1) Formulation screening of Gal(but)-C-MEs and Gal(oct)-C-MEsMicroemulsions were prepared by aqueous titration method and formulation prescription of microemulsion was to have been screened out combined with the pseudo ternary phase diagram. Gal(but)-C-MEs and Gal(oct)-C-MEs were prepared as follows: 150 mg of Cremophor? RH40, 50 mg of PEG400 and 32 mg of galactose ester were blended with vigorous magnetic stirring at room temperature for 2 h, 200 mg of coix seed oil was added to the mixture subsequently and stirred at room temperature for 2 h. Finally, 2 m L of coixan solution(120 mg/m L) was added to the resulting mixture drop by drop until the system became transparent and colloidal.(2) Characterization and quality evaluation of Gal(but)-C-MEs and Gal(oct)-C-MEsThe surface properties and morphology of Gal(but)-C-MEs and Gal(oct)-C-MEs were characterized by dynamic light scattering and transmission electron microscopy. The results show that the appearance of the two kinds of microemulsions was clear and transparent. The particle size, PDI and zeta potential of Gal(oct)-C-MEs and Gal(but)-C-MEs were 64.32 ± 5.63 nm, 0.094 ± 0.006,-0.715 ± 0.230 m V and 59.68 ± 6.65 nm, 0.070 ± 0.006,-2.950 ± 0.230 m V, respectively. Gal(oct)-C-MEs and Gal(but)-C-MEs displayed the spherical surface and evenly dispersed. And which were maintained strong stability at different dilution ratio, high speed centrifugation, storage time and at different p H values.3. Evaluation of Gal(oct)-C-MEs and Gal(but)-C-MEs at cellular level in vitro(1)The cytotoxicity of Gal(oct)-C-MEs and Gal(but)-C-MEsThe inhibitory effects of Gal(oct)-C-MEs and Gal(but)-C-MEs on Hep G2 cells were investigated by MTT experiment. The results showed that, compared to C-MEs, Gal(but)-C-MEs and Gal(oct)-C-MEs exhibited stronger cytotoxicity at coix seed oil concentration of 7.81-250.00 μg/m L in Hep G2 cells. IC50 values of C-MEs, Gal(but)-C-MEs and Gal(oct)-C-MEs were 71.23 ± 6.09, 62.55 ± 0.36, 47.98 ± 4.00 μg/m L, respectively. The inhibitory effect in Hep G2 cells of Gal(but)-C-MEs and Gal(oct)-C-MEs was stronger than that of C-MEs and IC50 values reduced 1.14 times and 1.48 times, respectively.(2)Uptake of Gal(oct)-C-MEs and Gal(but)-C-MEs in Hep G2 cellsThe uptake of Gal(oct)-C-MEs and Gal(but)-C-MEs was observed and determined by inverted fluorescence microscope and flow cytometry. Experimental results showed that fluorescence intensity of C-MEs, Gal(oct)-C-MEs and Gal(but)-C-MEs was significantly stronger than coix seed components mixture(Mixture) group, which displayed microemulsion enhancing cellular uptake of coix components. It should be noted that fluorescence intensity of Gal(oct)-C-MEs and Gal(but)-C-MEs was stronger than C-MEs, suggesting that C-MEs modified by Gal(oct) and Gal(but) was helpful to enhance the cellular uptake of coix seed components microemulison by tumor cells.(3)Study on the induced Hep G2 cell apoptotic rate of Gal(oct)-C-MEs and Gal(but)-C-MEsThe induced Hep G2 cell apoptosis effect of Gal(oct)-C-MEs and Gal(but)-C-MEs were determined by flow cytometry in vitro. Results showed that Hep G2 cells were treated with different formulation groups for 12 h, which found that C-MEs, Gal(but)-C-MEs and Gal(oct)-C-MEs significantly induced cell apoptosis. It was worth noting that apoptotic rate of Gal(oct)-C-MEs and Gal(but)-C-MEs groups were stronger than that of C-MEs group. The apoptotic rate was obtaind enhancement when concentration of coix seed oil increased.4. Hepatoma-targeting, antitumor efficacy and safety evaluation of Gal(oct)-C-MEs and Gal(but)-C-MEs in vivo(1)Evaluation of tumor targeting using Gal(oct)-C-MEs and Gal(but)-C-MEs in vivo in Hep G2 tumor-bearing nude miceSelection of Hep G2 nude mice model, we observed distribution of Gal(oct)-C-MEs and Gal(but)-C-MEs in vivo. Results showed that C-MEs, Gal(but)-C-MEs and Gal(oct)-C-MEs mainly accumulated in liver and tumor regions and biodistribution were significantly improve. Even still microemulsions gathered in tumor sites after 48 hours. Moreover, fluorescence intensity of Gal(oct)-C-MEs and Gal(but)-C-MEs groups were significantly more than C-MEs group. The results suggested that it might be the specific binding between the targeting ligand and asialoglycoprotein receptor. Gal(oct)-C-MEs group was most significant and used for subsequent experiments.(2)Anti-tumor effect of Gal(oct)-C-MEs in vivoSelection of Hep G2 nude mice model, we investigated anti-tumor effect of Gal(oct)-C-MEs in vivo. The results showed that tumor bearing nude mice after treatment, survival time of Gal(oct)-C-MEs group was longest, tumor inhibition rate of Gal(oct)-C-MEs was the highest and tumor weight was minimal. Furthermore, serum immune indices were determined by using ELISA kits for IL-6 and TNF-α. The results showed that TNF-α index of Gal(oct)-C-MEs, Kanglaite?, Mixture and C-MEs group increased compared with saline group. Moreover, they have significant difference(P < 0.05), which illustrated that body’s immune response leading to TNF-α index increased. The content of IL-6 among the groups had no obvious change, which the reasons still to be further discussed..(3)Safety evaluation of Gal(oct)-C-MEsSeveral indices representing the function of liver and kidney such as Aspartate aminotransferase(AST), Alanine aminotransfer- ase(ALT) and blood urea nitrogen(BUN) were detected by blood biochemical analyzer. The results showed that there were no significant differences about AST, ALT and BUN among all groups. At the same time, 100 μL of whole blood was used for blood routine analysis. Compared with saline group, WBC of Gal(oct)-C-MEs, Mixture, C-MEs increased significantly. Meanwhile, compared with Kanglaite injection group, WBC of Gal(oct)-C-MEs, Mixture, C-MEs also increased significantly. Moreover, they have significant difference(P < 0.05). HGB and PLT have similar results. It predicted that coix component enhanced immunity, enriched blood and prevented bleeding. In addition, the liver and kidney tissues of Gal(oct)-C-MEs was found that have no obvious abnormalities after H&E staining, indicating that there was no liver and kidney toxicity after treatment. |
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