Targeted Delivery Of Ligustrazine By Folate Acid Modified Chitosan Nonoparticles Enhance The Inhibition Of Metastasis

Objective:To optimize the preparation of folate acid modified chitosan nonoparticles loaded with ligustrazine(active targeting nanoparticles)for tumor targeting and investigate its effect on the inhibition of tumor metastasis in vivo.Methods:1. To optimize the preparation of active targeting nanoparticles. Ionic gelation method was put to use in preparing the active targeting nanoparticles. Folic acid chitosan(FA-CS) concentration, sodium tripolyphosphate concentration, p H value as the main influence factors, encapsulation efficiency, drug loading, particle size as an evaluation index, Box-Behnken experimental design was used to optimize the preparation of nanoparticles. 2. Targeting property of active targeting nanoparticles in vitro. The expression levels of folate receptor were detected to screening cells by Western Blotting. Non-toxic dosage of active targeting nanoparticles was measured by MTT assay(Dosage for 95% cells viability). HPLC assay was used to determine intracellular concentration of ligustrazine in cells exposed to active targeting nanoparticles, passive nanoparticles and ligustrazine solution respectively for different hours.Concentration of ligustrazine in three kinds of cells with active targeting nanoparticles was determined by HPLC at 4 hours. 3. Targeting property of active targeting nanoparticles in vivo. Fluorescein isothiocyanate(FITC) reacts with the amino group of chitosan, synthesis of fluorescent labeled vector. Infrared spectroscopy and nuclear magnetic spectroscopy was used to analyze the structure characterization. In vivo imaging of H22 tumor bearingmice following intravenous administration of the same amount of fluorescence labeled different modified nanopaticles after 30 min, observe tumor tissue targeting of nanoparticles, and then observe the distribution of the nanoparticles in vitro tissue imaging. On the other hand,H22 tumor bearing mice were treated with active targeting nanoparticles, passive nanoparticles and ligustrazine solution respectively by tail vein injection. HPLC assay was used to determine concentration of ligustrazine in blood plasma and different organs at different times. 4. Active targeting nanoparticles of inhibiting tumor metastasis. H22 tumor metastasis model was established, and random Ly divided into 8 groups, respectively give: active targeting nanoparticles(40 mg/kg, high dose),(20 mg/kg, middle dose),(10 mg/kg, low dose), passive nanoparticles(20 mg/kg), ligustrazine solution(20 mg/kg), active empty nanoparticles, 5- FU(20 mg/kg), saline solution. Continuous administration of 21 days, Weight, the number of Pulmonary nodules in each group of mice was compared, the expressions of MMP-9, VEGF were detected by immunohistochemical SP method in xenografted tumor.Results:1. According to the optimized prescription(1mg/m L FA-CS, 0.89mg/m L TPP, p H4.61) preparing this FA-CS-LZ-NPs nanoparticle,the encapsulation efficiency, drug loading, particle size, PDI and Zeta potential of this nanoparticles were respectively(36.72±0.65)%,(9.44±0.17)%, 141.1±0.6 nm, 0.219±0.011, 20.8±1.4 mv, from the above all we can know that experimental observations and model prediction is more closer and model predictive is favorable. 2. Expression level of cell folic acid has significant difference in the Bel7402(High folate receptor expression) and L-02(Low folate receptor expression), can be used for further testing. The non-toxic dose of active targeting nanoparticles for Bel7402, L-02 cell is LZ 47.5μg/m L. Intracellular ligustrazine was 41.18±1.67μ g/m L for 4 hours’ incubation and the concentration was saturate for Bel7402 cells, which was significant higher than the CS-LZ-NPs group(19.10±2.02μ g/m L), LZ group(11.79±1.54μg/m L), and the negative control group of FA-CS-LZ-NPs for L-02 cells(21.24±2.22μg/m L). From this we can know that folic acid modification of nanoparticles is promoting high expression of cell folate receptors to intake of drugs. Hela, Bel7402, L-02 cells were respectively treated with FA-CS-LZ-NPs, LZ concentration of intracellular from high to low was: Hela > Bel7402 > L-02, proves that intracellular drug uptake and cell folate receptor expression levels were positively correlated. 3.In infrared spectrum,when compared with the FA-CS, FITC-FA-CS has an enhancement in 1575cm-1 bending vibration, is the results of the reaction for FITC isothiocyanate and chitosan amino. FITC-FA-CS had two 1H NMR peaks in δ6.715,δ6.949 compared with FA-CS. It supposed to be aromatic hydrocarbons of FITC. So it can be judged that FITC had coupled to the FA-CS molecules.In vivo imaging showed that the injection with FITC-FA-CS-NPs compared to the injection with FITC-CS-NPs and FITC solution, has the stronger fluorescence intensity on the tumor parts. In vitro organ imaging and in vivo LZ drug distribution results display, distribution of active nanoparticles group in tumor tissues was significantly higher than that of passive nanoparticles group and solution group. Nanoparticles after folic acid modified mainly concentrated in tumor, liver, kidney, and without folic acid nanoparticles mainly concentrated in the liver, lung, and less distribution of the tissue in the solution group. 4.The weight of 5-FU group of mice is decreased, other administration groups have different levels of growth, indicates that the toxic and side effects of ligustrazine nanoparticles is small. The expression level of MMP-9 and VEGF is in a dose-dependent downregulation, and the effect of active targeting nanoparticles is better. Compared with CS-LZ-NPs and LZ solution, FA-CS-LZ-NPs has a better effect in the inhibition of lung metastasis and its’ s in a dose-dependent manner.Conclusions:In this study, the preparation of Folate acid modified chitosan nonoparticles loaded with ligustrazine have suitable diameter, high encapsulation efficiency and loading capacity. In vitro experiments showed that the active targeting nanoparticles could successfully target Bel7402 cells(overexpressing the folate receptor). In vivo experiments showed that the active targeting nanoparticles could successfully target H22(overexpressing thefolate receptor) tumor model. Compared passive nanoparticles and LZ solution, active targeting nanoparticles has better inhibition of tumor metastasis on H22 transfer model by inhibit the expression of MMP-9 and VEGF protein.