Preparation Of Resveratrol Cubic Liquid Crystalline Nanoparticles And Its Anti-tumor Effects Study

Objective: Resveratrol cubic liquid crystalline nanoparticles(RES-LCNPs) was prepared,and its preparation process, formulation were screened and optimized. The morphology traits,partical size, zeta potential and stability were evaluated. In vitro drug release property of RES-LCNPs was analyzed. Furthermore, Hep G2 and A549 were served as models to investigate its anti-tumour activities.Methods: 1. RES-LCNPs was prepared by Bottom-up method on the basis of the preliminary experiment. Preparation process(stirring speed, dispersion time, ultrasonic power) and formulation factors(stabilizer types, stable dose, glyceryl monooleate amount, aqueous phase volume) of RES-LCNPs were screened by single factor analysis method. Then the formulation was further optimized by response surface methodology based on central composite design.2. The morphology traits of RES-LCNPs was observed through transmission electron microscopy(TEM), and its average size and zeta potential were measured by laser particle sizer.After that, differential scanning calorimetry(DSC) method was used to analyze whether RES was encapsulated in RES-LCNPs. The storage stability of RES-LCNPs and its lyophilized powders were investigated. Moreover, in vitro drug release properties of RES-LCNPs was also analyzed by equilibrium dialysis using 1.5% SDS solution as the release medium.3. Proliferation effects of RES-LCNPs on tumour cell lines HepG2 and A549 were investigated by MTT method, and the light microscopy and fluorescence microscope were used to observe the cell morphological changes. The effects on cell cycle and apoptosis were studied with flow cytometry(FCM). Moreover, the expression effects of tumor cell apoptosis related proteins(Bax、Bcl-2) were evaluated by Western blotting method.Results: 1. Preparation technology was as follows, the stirring speed was 1000 rpm, the dispersing time was 2 h and the ultrasonic power was 150 W. The optimal formulation was as follows, the ratio of GMO to RES concentration was 21.7:1, while RES concentration was 0.4 g/L and PVA concentration was 0.91 g/L. The predicted D value, which the dependent variable was0.592 while the measured value was 0.584 ± 0.007. The deviation between them was 1.35 %.2. Optimized RES-LCNPs showed good dispersity, morphological intactness and noconglutination. The average size of RES-LCNPs was(50.77 ± 5.14) nm, PDI was 0.249 ± 0.063 and zeta potential was(-22.6 ± 1.9) mV. DSC analysis suggested that RES was encapsulated in nanoparticles. When the temperature was 25℃, RES-LCNPs appeared flocculent precipitate on the 21 st day, small proportion of precipitation emerged on the 50 th day at 4℃, which indicated that RES-LCNPs showed good stability at 4℃. DL, EE and mean paricle size of RES-LCNPs lyophilized powder showed slight change during 90 days, which indicated ideal stability.Moreover, the accumulated release amount of RES were 66.62% and 80.76% when the RES-LCNPs’ release time were 8 h and 36 h, respectively, which demonstrated that RES-LCNPs had obvious sustained property, and the release mechanism reveled good correspondence to Weibull equation, whose regression equation was lnln[1/(1-Q)] = 0.609lnt- 1.4202(r=0.9816).3. RES-LCNPs of 10 μM, 20 μM, 40 μM, 80 μM acted on Hep G2 and A549 cells for 24 h.The cyto-inhibition rate increased with increasing drug concentration. Then, 40 μM RES-LCNPs acted on HepG2 and A549 cells for 6 h, 12 h, 24 h, 48 h, respectively. Cyto-inhibition rate went up as well with extended action time. Hep G2 and A549 cells processed by RES-LCNPs exhibited features, such as less cell amount, lower diopter and looser distribution, compared with those treated by the RES solution. The phenomenons of bright blue fluorescence, chromatin condensation and nuclei gathering were observed under an fluorescence microscope. The FCM detection presented that cell block appeared in G2/M phase and apoptosis happened significantly at the early and late stage. Furthermore, WB test demonstrated that RES-LCNPs could promote the expression of Bax whereas inhibit the expression of Bcl-2.Conclusion: Feasible preparation craft and formulation of RES-LCNPs had been assured.Characteristics of RES-LCNPs, such as roundness, no adhesion, small size as well as good stability were exhibited. Apart from these, it complied with a rather slow release pattern compared to the RES solution. Also, the inhibition effect of RES-LCNPs showed dependence to time and dose, and its cell apoptosis function was stronger than RES solution. What’s more, the cell block tended to happen in G/M phase and how the function of inducing apoptosis happened was possibly promoting the expression of Bax, inhibiting the expression of Bcl-2.