Research Of Preparation Of 5-Fluorouracil Loaded N-Succinyl-Chitosan As A Tumour Targeted Delivery

N-Succinyl-chitosan （Suc-Chi） was obtained by introduction of succinyl groups into chitosan N-terminal of the glucosarnine units. Suc-Chi, the amphiphilic copolymers is ideal as a novel carrier for stealthy nanoparticles and Tumor-targeted nanoparficles because of its good solubility, biocompatibility, low toxicity and long-term retention in the body and high affinity to tumor cells.In our study, 5-fluorouracil （5-FU） loaded N-Succinyl-chitosan （Suc-Chi） nanoparticles （5-FU-Suc-Chi/NPs） have been developed. The experimental results, which tumour targeted delivery system of 5FU-Suc-Chi/NPs were investigated, demonstrated that 5FU-Suc-Chi/NPs could be circulated in blood for a longer time and achieve effective tumor-specific drug delivery due to the enhanced permeability and retention （EPR） effect and its high affinity to tumor cells. Six main sections were included in this paper:1. Synthesis of N-succinyl-chitosan and its identification: A simple ring-opening reaction in dimethyl sulfoxide system was developed to synthesize successfully N-succinyl-chitosan. Suc-Chi was characterized by FTIR, ¹H NMR, ¹³C NMR. The degree of substitution （DS） was determined by cineration assay and testified further by ¹H NMR.2. Physical-chemical properties of N-succinyl-chitosan: Suc-Chi have a property as polyampholyte. Suc-Chi （DS=0.33） showed the good solubility in the acidic region below pH 4.5 and the basic region above pH 6.8. the N-acylated derivative couldn’t dissolved between pH. 4.5-6.8; The Isoelectric point （pI） of Suc-Chi （DS=0.33） is pH. 5.26; we concluded the glass transition temperatrure（Tg） at about 304.16℃; At pH 5.87, the octanol/water partition coefficient (P_app) achieves its maximum P_app, which is 0.16; The molecular weights （M_w） of the products were determined by gel permeation chromatography （GPC）.3. Preparation of 5-FU-Suc-Chi/NPs and optimization to the formulation: 5-FU-Suc-Chi-nanoparticles were prepared by a emulsification solvent diffusion method. A cellulase degradation method was developed to determine the entrapment efficiency （EE） of 5-FU-Suc-Chi/NPs. The formulation of 5-FU-Suc-Chi/NPs was optimized by orthogonal design on the basis of expcrimention of the effects of formulation and preparation factors. The ideal combination preparation and formulation is the initial 5-FU concentration: 1000μg/ml; ethanol: Span-80（v/v）=1:4; Suc-Chi concentration: 4mg/ml; ultrasonication time: 5min.4. Physieo-ehemieal characterization and in vitro evaluation of 5-FU-Sue-Chi/NPs: The morphological examination of the Suc-Chi nanopaxficles was performed using the transmission electron microscope and the scan electron microscope, The size （Z-average mean） and zeta potential of the nanopaxticles were analyzed by Zeta PotentiaL AnaLyzer. The obtained Suc-Chi nanoparticles was regular spherewith same particle size. The 5-FU entrapment efficiency of the nanoparticles and their loading capacity were 62.36±0.74% and 18.98±0.68% respectively. The Suc-Chi nanoparticles have a particle diameter （Z-average）approximately（236.6±64.7） nm and a negative zeta potential （-27.2±0.2） mV.A burst release was found in the initial 1hour followed by a continuous sustained release in vitro from 5-FU-Suc-Chi/NPs for 4 days, the drug of 5-FU injections was released completely within 1h. The drug release from 5-FU-Suc-Chi/NPs was sustained evidently compared with 5-FU injections. The drug release from 5-FU-Suc-Chi/NPs decreased with the increase of pH. The concentration-time curves of 5-FU and 5-FU-Suc-Chi/NPs were fitted to the first class kinetic equation and the bi-exponent and bi-phase kinetic equations respectively.5. In vivo biodistribution and evaluation for tumor targeting of 5-FU-Sue-Chi/NPs: The binding of Suc-Chi/NPs to human chronic myelogenous leukemia cell lines K562 cells was evaluated by using flow cytometry. The results of flow cytometry analysis demonstrated that the Suc-Chi/NPs and CS/NPs were able to bind to K562 cells effectively, the Suc-Chi/NPs displayed higher affinity to k562 cells than CS/NPs.The distributed amount of FITC-Suc-Chi/NPs in tissues and blood of Sarcoma180-bearing mice was estimated by the fluorescence measurement. These observations were further confirmed by our fluorescence microscopy results of the tissues sections. The rank order of tissue distribution was kidney＞tumor＞blood＞liver＞spleen＞lung. The results revealed that Suc-Chi/NPs were accumulated in the tumor tissue due to the EPR effect and its long systemic retention in blood circulation.A simple HPLC method was developed for the quantitation of 5-fluorouracil in vivo to evaluate the biodistribution and tumor selectivity of 5-FU-Suc-Chi/NPs in Sarcoma 180-beating mice. The 5-FU-Suc-Chi/NPs could be sustained at a high level in blood throughout a very long time, implying its long systemic retention in blood circulation. 5-FU-Suc-Chi/NPs were distributed mainly in tumor, liver and a small quantity of 5-FU-Suc-Chi-NPs was found in kidney and speen. 5-FU-Suc-Chi/NPs scarcely accumulated in the heart and lung, lowered the toxic effect of 5-FU to them. The results indicates that longevity in blood circulation and tumor targeting of 5-FU-Suc-Chi/NPs should be achieved. The relative tumor tissue exposures （Re）and the ratios of peak concentrations（Ce） were 9.43±1.86 and 2.75±0.26 respectively. This showed that the tumor affinity of 5-FU-Suc-Chi/NPs was increased Compared with 5-FU.6. In vivo pharmacokineties and pharmacodynamies of 5-FU-Sue-Chi/NPs: Pharmacokinetic analysis in plasma of showed that the T_1/2 of 5-FU-Suc-Chi/NPs was 11.98-fold of that of 5-FU, the AUC of serum of 5-FU-Sue-Chi/NPs was 3.5-fold of that of 5-FU, the MRT of 5-FU-Suc-Chi/NPs was 11.8-fold of that of 5-FU. The CL of 5-FU-Suc-Chi/NPs was 0.124-fold of that of 5-FU. The results further showed that the long-circulation characteristics of 5-FU-Suc-Chi/NPs was evident.The anti-tumor activity of 5-FU-Suc-Chi-NPs were evaluated by measuring the change in the tumor volume of Sarcoma180-bearing mice after intravenous injection of Suc-Chi/NPs, 5-FU-Suc-Chi/NPs, and 5-FU. The 5-FU-Suc-Chi/NPs showed good antitumour activities against Sarcoma 180 solid tumour and mild toxicity, compared with 5-FU injections.