摘要
N-琥珀酰壳聚糖(N-succinyl-chitosans，Suc-Chi)是由壳聚糖和丁二酸酐反应合成的一种羧基酰化的水溶性壳聚糖衍生物。N—琥珀酰壳聚糖是一种两亲聚合物，具有较好的生物相溶性，较低的毒性、较长的体内半期及肿瘤亲和性。这些特性使其成为一种新型“隐形NPs”和“高肿瘤趋向性NPs”的较为理想载体材料。
本文设计以N-琥珀酰壳聚糖为载体，5-氟尿嘧啶为抗肿瘤模型药，制备纳米粒(5-FU-Suc-Chi／NPs)。5-FU-Suc-Chi／NPs肿瘤靶向传递系统的研究结果，证实了5-FU-Suc-Chi／NPs具有长循环特性，并可以通过EPR效应及其对肿瘤的亲和力实现肿瘤靶向。本文研究的主要方法、内容和结论包括以下几个方面：
1 N-肛琥珀酰壳聚糖的制备及结构表征：采用非均相法以二甲亚砜作为溶剂，合成N-琥珀酰壳聚糖，通过IR、~1H NMR、~(13)C NMR对产物进行结构确证。采用灰分分析法测定Suc-Chi样品取代度，并通过~1H NMR法验证。
2 N-琥珀酰壳聚糖理化性质及药用特性：N-琥珀酰壳聚糖是一种聚两性电解质，Suc-Chi(DS=0.33)在4.5～6.8 pH值范围内不溶，在pH 4.5以下和pH 6.8以上溶解性较好；Suc-Chi(DS=0.33)等电点(pI)为5.20，玻璃化温度(Tg)为304.16℃；Suc-Chi(DS=0.33)zata电位(ξ)随pH增大而降低，在pH约为5.0时zata电位等于零；Suc-Chi(DS=0.33)油／水分配系数(P_(app))在pH 5.87达到最大值，P_(app))0.16。采用凝胶渗透色谱法(GPC)法测定N-琥珀酰壳聚糖不同降解产物的分子量(Mw)。
3 5-FU-Suc-Chi／NPs制备及工艺处方优化：采用乳化溶剂扩散法制备5-FU-Suc-Chi／NPs。建立纤维素酶降解法测定5-FU-Suc-Chi／NPs包封率的分析方法。以包封率、载药量、粒径为评价指标，在单因素实验基础上，按正交设计L_9(3~4)表进行试验，优化处方。最优处方：5-FU初始浓度为1000μg／mL；乙醇与Span-80体积比为1∶4；Suc-Chi浓度为4mg／mL；超声时间为5 min。
4 5-FU-Suc-Chi／NPs理化特征及体外释放行为：采用透射电镜和扫描电镜观察5-FU-Suc-Chi／NPs外观形态，激光粒度仪测定其粒径，结果表明5-FU-Suc-Chi／NPs外观平滑呈球形或类球形，形态比较均匀完整；分散性好，粒径分布窄，呈正态分布，其平均粒径为236.6±64.7 nm；zeta电位为-27.2±0.2 mv；包封率和载药量分别为62.36±0.74％和18.98±0.68％。
采用透析袋法测定5-FU-Suc-Chi／NPs体外释药速度，在释药的初始阶段出现突释，1h药物释放达到45％，24小时达到67％，随后持续缓慢释放一直到第4d。5-FU注射液释药速度较快，1h基本释药完毕。两者体外释药存在显著差异(P＜0.05)，说明5-FU-Suc-Chi／NPs有明显的缓释作用。5-FU-Suc-Chi／NPs释放速度随介质pH值升高而减慢。5-FU初始浓度与5-FU-Suc-Chi／NPs释药量之间不存在线性关系。采用Origin7.5软件拟合药物体外释药模型的研究表明，5-FU注射液的释放符合一级动力学方程，5-FU-Suc-Chi／NPs的释放较符合双指数双相动力学模型。
5 5-FU-Suc-Chi／NPs的体内组织分布及靶向性：采用流式细胞仪检测荧光强度来评价FITC-Suc-Chi／NPs与K562白血病肿瘤细胞之间的亲和性。结果表明，FICT-Suc-Chi／NPs与FICT-CS／NPs对K562白血病肿瘤细胞都具有较高的亲和力，两者相比FICT-Suc-Chi／NPs的亲和力更强。建立FITC-Suc-Chi／NPs的体内荧光分析方法，检测空白Suc-Chi／NPs在Sarcoma180-荷瘤小鼠体内组织分布，并通过组织切片的荧光显微照片确证FICT-Suc-Chi／NPs的组织分布结果。研究发现，FICT-Suc-Chi／NPs能够长时间的滞留在血液中，在各组织及血浆中分布的数量次序依次为肾＞肿瘤＞血液＞肝＞脾＞肺。结果表明，FICT-Suc-Chi／NPs具有长循环特性和肿瘤靶向性。
建立5-FU体内分析方法，评价5-FU-Suc-Chi／NPs在荷瘤小鼠体内靶向性。研究发现，5-FU-Suc-Chi／NPs中的5-FU能够在血中长时间的滞留，与5-FU原形药相比较具有明显的缓释作用。5-FU-Suc-Chi／NPs中的5-FU在荷瘤小鼠体内分布趋势为：1 h时，肝＞肿瘤＞血＞肾＞脾＞心＞肺；1d时，肿瘤＞肝＞脾＞肾＞血＞肺＞心；3d时，肿瘤＞肝＞肾＞脾＞血＞肺＞心。表明5-FU-Suc-Chi／NPs具有肿瘤靶向作用，可降低心脏、肾脏的毒副作用。肿瘤相对摄取率Re和峰浓度比Ce分别为：9.43±1.86，2.75±0.26，说明5-FU-Suc-Chi／NPs对肿瘤的趋向性大于5-FU注射液。
6 5-FU-Suc-Chi／NPs的药动学及药效学：建立5-FU在大鼠血浆中分析方法，5-FU-Suc-Chi／NPs在大鼠体内动力学研究发现，5-FU-Suc-Chi／NPs与5-FU注射液的主要药物动力学参数之间存在显著性差异。5-FU-Suc-Chi／NPs的T_(1／2)显著延长11.98倍，AUC显著增大3.5倍，MRT显著延长11.8倍，CL显著减小0.124倍；表明5-FU-Suc-Chi／NPs具有长循环特性，实现了药物的缓释作用。
5-FU-Suc-Chi／NPs在Sarcoma 180-荷瘤小鼠体内药效学的研究表明，5-FU-Suc-Chi／NPs与5-FU注射液相比较，5-FU-Suc-Chi／NPs能够更有效地抑制Sarcoma 180肿瘤的生长，而且组织毒性较温和。
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, ~(13)C 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.
引文
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