N-羧烷基-6-O-壳聚糖硫酸酯的制备

以铜离子为模板定向合成了6-O-壳聚糖硫酯酯，然后分别用乙醛酸和丙酮酸在其N-位形成席夫碱，再经NaCNBH3还原制备了N-羧甲基-6-O-壳聚糖硫酸酯和N-（2-甲基-羧甲基）-6-O-壳聚糖硫酸酯，对制得的各化合物分别采用氧瓶燃烧法测定了硫含量（S%），采用HPGPC法测定了重均相对分子质量和相对分子质量分布宽度，并对各化合物的红外光谱（IR）和^13C-NMR核磁共振图谱进行了分析。     

几种O-羧甲基壳聚糖硫酸酯的制备

以甲壳质为原料制备了O-羧甲基壳聚糖，再经不同的硫酸酯化工艺分别制备了O-羧甲基-N-硫酸酯基壳聚糖（Ⅰ），O-羧甲基-O-硫酸酯基壳聚糖（Ⅱ），O-羧甲基-N，O-硫酸酯基壳聚糖(Ⅲ）等3种不同硫酸酯基取代位置的羧甲基壳聚糖衍生物，对各化合物分别采用氧瓶燃烧法测定了硫含量（S%），采用HPGPC法制定了重均相对分子质量和相对分子质量分布宽度。并对各化合物的红外光谱（IR）和^13C-NMR核磁共振图谱进行了分析。     

仿穿膜肽型壳聚糖衍生物N-辛基-N-精氨酸壳聚糖的合成和表征

以壳聚糖为母体,在其侧链氨基上引入亲水基精氨酸以及疏水基辛基,合成了一种新型的具有仿穿膜肽结构的壳聚糖衍生物——N-辛基-N-精氨酸壳聚糖(OACS)。同时通过FT-IR、1H NMR、元素分析和精氨酸显色法确证了OACS的化学结构以及其辛基和精氨酸的取代度。荧光光谱法测得系列OACS的临界胶束浓度为0.12～0.27 mg.mL/1;溶解度实验表明其在pH 1～12溶液中均易溶,并可自组装形成淡蓝色略带乳光的胶束溶液;马尔文粒径测定仪显示系列OACS形成的聚合物胶束平均粒径为158.4～224.6 nm,多分散系数为0.038～0.309,ζ电位为+19.16～+30.80 mV;原子力显微镜图谱显示所得胶束粒子分散均匀、大小规则圆整;MTT实验证实所得OACS在50～1 000μmol.L?1内安全性能良好。细胞实验结果表明,随着精氨酸取代度的升高,OACS胶束进入细胞的荧光量也随之增加,与壳聚糖相比,最大增加倍数可达40倍。因此,OACS有望作为一种兼具促吸收和载药功能的新型纳米载体。     

一种低分子壳聚糖硫酸酯铝的制备

本文采用正交试验设计方法对壳聚糖进行氧化降解,获得了一种低相对分子质量的水溶性壳聚糖,并在此基础上经硫酸化和盐交换制得了一种低相对分子质量壳聚糖硫酸酯式铝盐,壳聚糖氧化降解实验结果表明降解的最佳条件是温度为80度,过氧化氢浓度为5％,降解时间为1h,在此条件一产品收率可达52．68％,其粘均相对分子质量为3990,对制是的低相对分子质量壳聚糖硫酸酯铝进行了红外光谱分析和部分理化性质测定,经测定定样品中有机硫（S）含量为9．10％,铝（Al)含量为20．58％,样品制酸力为188．59mL.g^-1。     

N-正辛基-N''''-琥珀酰基壳聚糖的制备及其对4种肿瘤细胞的亲和性

目的以不同分子质量的壳聚糖为原料合成系列的N-正辛基-N'-琥珀酰基壳聚糖,考察其与肿瘤细胞的亲和性,为其作为抗肿瘤药物的靶向载体提供依据.方法用异硫氰酸荧光素(FITC)对N-正辛基-N'-琥珀酰基壳聚糖进行标记,再分别与人肝癌细胞(HepG2)、人白血病细胞(K562)、人非小细胞肺癌细胞(A549)及人胃癌细胞(BGC)共培养,通过流式细胞仪及酶联免疫检测仪测定N-正辛基-N'-琥珀酰基壳聚糖对肿瘤细胞的亲和性及抑制力.结果N-正辛基-N'-琥珀酰基壳聚糖对4种肿瘤细胞亲和性明显强于空白细胞株(P＜0.01),并具有一定的肿瘤抑制作用.结论N-正辛基-N'-琥珀酰基壳聚糖有望作为抗肿瘤药物的靶向载体.     

N-正辛基-N′-琥珀酰基壳聚糖胶束的制备及特征研究*

目的：制备两亲性的N-正辛基-N’-琥珀酰基壳聚糖（OSC）胶束，并研究其制剂学特征。方法：采用超声方法制备OSC胶束，并考察OSC的胶束化行为、胶束的形态、粒径及￡电位。结果：OSC胶束具有较低的临界胶束浓度，胶束粒子呈较规则的球形，粒径为200～250nm，￡电位在-40．0～-30．0mV，且疏水链段取代度增加，粒径变大，临界胶束浓度降低。结论：OSC胶束具有较好的物理稳定性。     

N-正辛基-N′-琥珀酰基壳聚糖胶束的制备及特征研究

目的:制备两亲性的N-正辛基-N′-琥珀酰基壳聚糖(OSC)胶束,并研究其制剂学特征。方法:采用超声方法制备OSC胶束,并考察OSC的胶束化行为、胶束的形态、粒径及ξ电位。结果:OSC胶束具有较低的临界胶束浓度,胶束粒子呈较规则的球形,粒径为200～250 nm,ξ电位在-40.0～-30.0 mV,且疏水链段取代度增加,粒径变大,临界胶束浓度降低。结论:OSC胶束具有较好的物理稳定性。     

N-正辛基-N′-琥珀酰基壳聚糖的制备及其对4种肿瘤细胞的亲和性

目的:以不同分子质量的壳聚糖为原料合成系列的N-正辛基-N′-琥珀酰基壳聚糖,考察其与肿瘤细胞的亲和性,为其作为抗肿瘤药物的靶向载体提供依据。方法:用异硫氰酸荧光素(FITC)对N-正辛基-N′-琥珀酰基壳聚糖进行标记,再分别与人肝癌细胞(HepG2)、人白血病细胞(K562)、人非小细胞肺癌细胞(A549)及人胃癌细胞(BGC)共培养,通过流式细胞仪及酶联免疫检测仪测定N-正辛基-N′-琥珀酰基壳聚糖对肿瘤细胞的亲和性及抑制力。结果:N-正辛基-N′-琥珀酰基壳聚糖对4种肿瘤细胞亲和性明显强于空白细胞株(P＜0．01),并具有一定的肿瘤抑制作用。结论:N-正辛基-N′-琥珀酰基壳聚糖有望作为抗肿瘤药物的靶向载体。     

壳聚糖-岩藻聚糖硫酸酯纳米粒的制备及性质研究

目的：本文以低分子量岩藻聚糖硫酸酯为交联剂,研究了聚电解质凝聚法制备壳聚糖-岩藻聚糖硫酸酯纳米微粒(Chitosan-fucoidan nanoparticles, CS-Fuc NPs)的制备工艺,并对纳米粒的胃肠道和贮藏稳定性进行研究。方法：采用聚电解质凝聚法制备壳聚糖-岩藻聚糖硫酸酯纳米微粒,采用体外模拟胃液和模拟肠液消化体系,检测纳米粒的胃肠稳定性,常温贮藏实验测定纳米粒的短期贮藏稳定性。结果：壳聚糖-岩藻聚糖硫酸酯纳米微粒的最优制备条件是：壳聚糖(1mg/ml)与岩藻聚糖硫酸酯(1mg/ml)体积比为1.1/1,pH 4.5,温度30℃。所得纳米粒粒径为227.8 nm,Zeta电位为38.4 mV,PDI为0.231,岩藻聚糖硫酸酯复合率(Fuc%)为94.92%。所制备的纳米颗粒在10周内没有显著变化,在模拟胃环境中稳定性良好,在模拟肠道环境中解聚性良好。结论： 壳聚糖-岩藻聚糖硫酸酯纳米粒制备工艺简单,性能良好,有望成为新型的口服药物运送载体。     

N-正辛基-N＇-琥珀酰基壳聚糖的制备及其对4种肿瘤细胞的亲和性

目的：以不同分子质量的壳聚糖为原料合成系列的N-正辛基-N’-琥珀酰基壳聚糖，考察其与肿瘤细胞的亲和性，为其作为抗肿瘤药物的靶向载体提供依据。方法：用异硫氰酸荧光素（FITC）对N-正辛基-N’-琥珀酰基壳聚糖进行标记，再分别与人肝癌细胞（HepG2）、人白血病细胞（K562）、人非小细胞肺癌细胞（A549）及人胃癌细胞（BGC）共培养，通过流式细胞仪及酶联免疫检测仪测定N-正辛基-N’-琥珀酰基壳聚糖对肿瘤细胞的亲和性及抑制力。结果：N-正辛基-N’-琥珀酰基壳聚糖对4种肿瘤细胞亲和性明显强于空白细胞株（P〈0．01），并具有一定的肿瘤抑制作用。结论：N-正辛基-N’-琥珀酰基壳聚糖有望作为抗肿瘤药物的靶向载体。     

青霉菌产壳聚糖酶制备低分子量壳聚糖的研究

利用Penicilliumsp.ZD-Z1发酵培养生产壳聚糖酶,研究其降解特性,并制备特定低分子量的壳聚糖。结果表明,在30℃、pH5、加内切酶ChA0.01u/ml的条件下降解4%壳聚糖,通过MHS方程η=1.02×10-4Mw1.27控制反应时间,可以得到低分子量的壳聚糖。     

水溶性O-羧甲基壳聚糖的制备及结构表征

目的:以甲壳素为原料制备水溶性O-羧甲基壳聚糖(O-CMC),并进行结构表征.方法:通过羧甲基化和脱乙酰化两步反应合成O-CMC,采用胶体滴定法测定产物的取代度和脱乙酰度,一点法测定黏均分子量,利用红外谱图确定羧甲基的取代位置.结果:甲壳素与氯乙酸在50℃进行羧甲基化反应4h,然后于50℃碱性条件下进行脱乙酰化反应30 min,即得产物.经超声纯化,制得水溶性O-CMC,产率为78.14％.红外光谱分析表明,合成产物的羧甲基仅局限于C6伯羟基取代,取代度为0.64,脱乙酰度为44.53％,黏均分子量为1.42×104.O-CMC在中性和碱性条件下水溶性好,等电点为4.46,0.5％水溶液pH为7.52.结论:本法制得的O-CMC结构表征明确,脱乙酰度和取代度符合设计要求,水溶性佳.     

Uptake and Cytotoxicity of Chitosan Molecules and Nanoparticles: Effects of Molecular Weight and Degree of Deacetylation

PURPOSE: To evaluate the effects of molecular weight (Mw) and degree of deacetylation (DD) on the cellular uptake and in vitro cytotoxicity of chitosan molecules and nanoparticles. METHODS: Chemical depolymerization and reacetylation produced chitosans of Mw 213,000 to 10,000 and DD 88-46%, respectively. Chitosan was labeled with FITC and transformed into nanoparticles by ionotropic gelation. Uptake of chitosan by confluent A549 cells was quantified by fluorometry, and in vitro cytotoxicity was evaluated by the MTT and neutral red uptakeassays. RESULTS: Nanoparticle uptake was a saturable event for all chitosan samples, with the binding affinity and uptake capacity decreasing with decreasing polymer Mw and DD. Uptake fell by 26% when Mw was decreased from 213,000 to 10,000, and by 41% when DD was lowered from 88% to 46%; the uptake data correlated with the zeta potential of the nanoparticles. Uptake of chitosan molecules did not exhibit saturation kinetics and was less dependent on Mw and DD. Postuptake quenching with trypan blue indicated that the cell-associated chitosan nanoparticles were internalized, but not the cell-associated chitosan molecules. Chitosan molecules and nanoparticles exhibited comparable cytotoxicity, yielding similar IC50 and IC20 values when evaluated against the A549 cells. Cytotoxicity of both chitosan entities was attenuated by decreasing polymer DD but was less affected by a lowering in Mw. CONCLUSIONS: Transforming chitosan into nanoparticles modified the mechanism of cellular uptake but did not change the cytotoxicity of the polymer toward A549 cells. Chitosan DD had a greater influence than Mw on the uptake and cytotoxicity of chitosan nanoparticles because of its effect on the zeta potential of the nanoparticles.     

低分子量半乳糖化脂肪酰壳聚糖的制备及其形成胶束的性质研究

目的制备具有主动肝靶向作用的低分子量半乳糖化脂肪酰壳聚糖聚合物胶束材料并进行结构表征,对其形成的胶束特性进行研究。方法以甲磺酸为溶剂及反应介质,利用脂肪酰氯(棕榈酰氯及月桂酰氯)与壳聚糖的OH进行酰化反应制备具有不同碳链长度及取代度的脂肪酰壳聚糖,再利用EDC.HCl活化乳糖酸,与所得的脂肪酰壳聚糖的2位氨基反应制备半乳糖化脂肪酰壳聚糖。用红外光谱、1H核磁共振谱对其结构进行表征及取代度的计算,并考察其溶解性能;制备其聚合物胶束,测定临界胶束浓度(CMC)和粒径。结果壳聚糖脂肪酰化的反应条件为20℃、1 h,半乳糖化反应的条件为30℃、24 h;壳聚糖棕榈酰化及月桂酰化反应中酰氯/壳聚糖的合适摩尔比分别为2∶1～8∶1、2∶1～10∶1。此条件下制得的脂肪酰壳聚糖及半乳糖化脂肪酰壳聚糖在二甲基亚砜(DMSO)中有较好的溶解性能;红外光谱、1H核磁共振光谱结果表明,成功合成了半乳糖化脂肪酰壳聚糖;脂肪酰基的取代度范围为0.28～1.13 mol,胶束的CMC约为0.39×10 2～2.82×10 2mg·mL-1,粒径大小119.8～546.0 nm。结论成功制备低分子量半乳糖化脂肪酰壳聚糖聚合物胶束材料。相同碳链长度条件下,脂肪酰氯与壳聚糖摩尔比越大,脂肪酰基的取代度越大,所得胶束的CMC及粒径越小;相同脂肪酰氯与壳聚糖摩尔比时,制得的半乳糖化脂肪酰壳聚糖的粒径小于脂肪酰壳聚糖的粒径。     

Optimization of Chitosan Succinate and Chitosan Phthalate Microspheres for Oral Delivery of Insulin using Response Surface Methodology

In the present study, a Box-Behnken experimental design was employed to statistically optimize the formulation parameters of chitosan phthalate and chitosan succinate microspheres preparation. These microspheres can be useful for oral insulin delivery system. The effects of three parameters namely polymer concentration, stirring speed and cross linking agent were studied. The fitted mathematical model allowed us to plot response surfaces curves and to determine optimal preparation conditions. Results clearly indicated that the crosslinking agent was the main factor influencing the insulin loading and releasing. The in vitro results indicated that chitosan succinate microspheres need high amount of crosslinking agent to control initial burst release compared to chitosan phthalate microspheres. The reason may be attributed that chitosan succinate is more hydrophilic than chitosan phthalate. The relative pharmacological efficacy for chitosan phthalate and chitosan succinate microspheres (18.66 ± 3.84%, 16.24 ± 4%) was almost three-fold higher than the efficacy of the oral insulin administration (4.68 ± 1.52%). These findings suggest that these microspheres are promising carrier for oral insulin delivery system.     

壳聚糖包覆脂质体递药系统的研究进展

脂质体在药物传递方面被广泛研究,但因结构稳定性差等因素使其应用受到了限制.壳聚糖是一种阳离子多糖,具有良好的生物相容性、生物降解性以及生物黏附性,并且可经化学改性成为性能更佳的壳聚糖衍生物.近年来,壳聚糖及其衍生物包覆脂质体在载药方面的研究得到了越来越多学者的关注.壳聚糖或其衍生物修饰脂质体,可提高其稳定性、黏附渗透性...     

Uptake of Chitosan and Associated Insulin in Caco-2 Cell Monolayers: A Comparison Between Chitosan Molecules and Chitosan Nanoparticles

PURPOSE: To evaluate the uptake of chitosan molecules (fCS) and nanoparticles (fNP), and their ability to mediate insulin transport in Caco-2 cell monolayers. METHODS: Cell-associated fCS and fNP were evaluated by fluorometry, trypan blue quenching, and confocal microscopy using FITC-labeled chitosan. Chitosan-mediated transport of FITC-labeled insulin was studied in Caco-2 cell monolayers cultured on permeable inserts. RESULTS: Caco-2 cells showed twofold higher association with fNP than fCS after 2-h incubation with 1 mg/ml samples. fNP uptake was a saturable (Km 1.04 mg/ml; Vmax 74.15 microg/mg/h), concentration- and temperature-dependent process that was inhibited by coadministered chlorpromazine. fCS uptake was temperature dependent, but was less sensitive to concentration and was inhibited by filipin. Postuptake quenching with 100 microg/ml of trypan blue suggests a significant amount of intracellular fNP, although the bulk of fCS was extracellular. Internalized fNP were located by confocal microscopy at 15 microm from the apical membrane, but there was no apparent breaching of the basal membrane. This might explain the failure of the nanoparticles to mediate significant insulin transport across the Caco-2 cell monolayer. CONCLUSIONS: Formulation of chitosan into nanoparticles transforms its extracellular interactions with the Caco-2 cells to one of cellular internalization via clathrin-mediated endocytosis.     

国产甲壳胺流变学性质及其影响因素的研究

用旋转粘度计测定甲壳胺的流变学性质，并考察了不同脱乙酰度、浓度、溶液pH值、离子强度、胃蛋白酶、温度以及不同性质的药物等因素对甲壳胺流变学性质的影响。结果表明，甲壳胺溶胶属假塑型流体且无触变性，上述因素对甲壳胺的流变学性质均有影响。其中甲壳胺浓度对其流变学性质影响最大，故以甲壳胺为缓控释辅料时应着重考虑其用量。     

Nasal Delivery of Insulin Using Novel Chitosan Based Formulations: A Comparative Study in Two Animal Models Between Simple Chitosan Formulations and Chitosan Nanoparticles

Purpose. To investigate whether the widely accepted advantages associated with the use of chitosan as a nasal drug delivery system, might be further improved by application of chitosan formulated as nanoparticles. Methods. Insulin-chitosan nanoparticles were prepared by the ionotropic gelation of chitosan glutamate and tripolyphosphate pentasodium and by simple complexation of insulin and chitosan. The nasal absorption of insulin after administration in chitosan nanoparticle formulations and in chitosan solution and powder formulations was evaluated in anaesthetised rats and/or in conscious sheep. Results. Insulin-chitosan nanoparticle formulations produced a pharmacological response in the two animal models, although in both cases the response in terms of lowering the blood glucose levels was less (to 52.9 or 59.7% of basal level in the rat, 72.6% in the sheep) than that of the nasal insulin chitosan solution formulation (40.1% in the rat, 53.0% in the sheep). The insulin-chitosan solution formulation was found to be significantly more effective than the complex and nanoparticle formulations. The hypoglycaemic response of the rat to the administration of post-loaded insulin-chitosan nanoparticles and insulin-loaded chitosan nanoparticles was comparable. As shown in the sheep model, the most effective chitosan formulation for nasal insulin absorption was a chitosan powder delivery system with a bioavailability of 17.0% as compared to 1.3% and 3.6% for the chitosan nanoparticles and chitosan solution formulations, respectively. Conclusion. It was shown conclusively that chitosan nanoparticles did not improve the absorption enhancing effect of chitosan in solution or powder form and that chitosan powder was the most effective formulation for nasal delivery of insulin in the sheep model.     

羧甲基壳聚糖在大鼠颅内组织相容性的初步观察

目的:探讨生物可降解缓释材料羧甲基壳聚糖(CMCS)在大鼠脑组织中的生物相容性,为开发其临床新用途奠定基础。方法:32只SD大鼠随机分为实验组和对照组,分别植入同面积的CMCS和明胶海绵。观察术后大鼠的行为改变和第3,7,14,30天的局部组织反应(HE染色)。结果:两组大鼠在实验期内均无明显行为改变,术后第3,7,14,30天的组织学观察表明CMCS为异物反应,与明胶海绵类似,无明显差异(P<0.05)。随时间推移,CMCS有不同程度降解。结论:CMCS具有良好的脑组织生物相容性,并可安全的降解,可作为颅内植入化疗缓释材料进一步开发。