三甲基壳聚糖分子量对载胰岛素纳米粒的影响

目的 研究不同分子量的三甲基壳聚糖(TMC)对载胰岛素口服纳米粒性质的影响,以寻找更适于口服的TMC分子量.方法 选用高、中、低分子量(400、200、50 kDa)的TMC为载体材料,采用离子交联法制备载胰岛素的聚电解质纳米粒;以不同分子量的TMC、三聚磷酸钠(TPP)和胰岛素(Ins)的浓度为考察因素,以纳米粒表面形态、粒径、Zeta电位、包封率和载药量为考察指标,进行单因素筛选试验,对比不同分子量的TMC对纳米粒性质的影响.结果与结论 中分子量TMC载胰岛素纳米粒的载药量较高,包封率合适,且释药较平稳,为相对较优的载体材料.     

用于改善吸收的甘草酸二铵/三甲基化壳聚糖纳米粒的制备和性质考察

目的制备甘草酸二铵(DG)/三甲基化壳聚糖(TMC)纳米粒并研究其基本性质。方法利用带正电荷的TMC与带负电荷的DG的静电相互作用制备DG/TMC纳米粒,以激光粒度测定仪和透射电镜(TEM)测定纳米粒的粒径和形态,以透析法测定纳米粒的释药度。结果 DG溶于p H 6.8~8.0的水中,浓度为50 g·L-1,TMC溶于p H 6.8~8.0的水中,浓度为30 g·L-1,室温下混合,温和搅拌,制得类球形DG/TMC纳米粒,平均粒径为72.8 nm,zeta电位为+29.81 m V。在p H=1盐酸、p H=7磷酸盐缓冲液、0.9%Na Cl溶液和蒸馏水中24 h累计释放度依次为99.2%、89.6%、85.8%和35.6%,口服相对生物利用度为220%,半衰期明显延长。结论 DG/TMC纳米粒制备方法简单可行,其释放行为与释放介质的离子强度及种类有关,具有缓释特点,能够改善DG的口服吸收。     

N-三甲基壳聚糖包衣PLGA纳米粒疫苗载药系统增加抗原交叉递呈的初步研究

摘 要 目的:探讨N-三甲基壳聚糖包衣聚乳酸 羟基乙酸共聚物［poly(lactic-co-glycolic acid),PLGA］纳米粒对诱导树突细胞交叉递呈的机制。方法: 复乳法制备PLGA纳米粒并用N-三甲基壳聚糖（N-trimethyl chitosan,TMC60）进行包衣,分别包载模型抗原卵清白蛋白、异硫氰酸荧光素（fluorescein isothiocyanate,FITC）标记的卵清白蛋白（FITC-OVA）。将TMC60包衣和未包衣的纳米粒分别作用于体外培养的小鼠骨髓系树突细胞（murine bone marrow-derived dendritic cell,BMDC）,用流式细胞仪检测BMDC对纳米粒子的吞噬动力学及BMDC表面分子CD83,MHCI和MHCII的表达;B3Z T细胞检测纳米粒被BMDC摄取后引起的交叉递呈反应。结果:TMC60包衣PLGA纳米粒可以促进BMDC吞噬作用;促进BMDC表达表面分子CD83和MHC I;增强BMDC对纳米粒包载抗原的交叉递呈作用。结论:TMC60包衣PLGA纳米粒可增强BMDC对外源性抗原的摄取及交叉递呈作用。     

载基因壳聚糖纳米粒的制备及其特性的研究

目的制备壳聚糖纳米粒,并连接上质粒,研究壳聚糖纳米粒的特性及其对DNA的结合及保护能力。方法采用离子交联法制备壳聚糖纳米粒,并用喷金扫描电子显微镜检测,了解粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基因);经琼脂糖凝胶电泳分析壳聚糖纳米载体与质粒DNA的结合能力,及不同pH值的壳聚糖纳米粒对质粒DNA的结合能力;并通过DnaseⅠ消化壳聚糖纳米-质粒结合物以观察壳聚糖纳米载体对质粒的保护作用。结果喷金扫描电镜检测证实壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5nm;琼脂糖凝胶电泳的结果显示壳聚糖纳米粒能有效地结合载体pGenesil-1质粒;不同pH值的壳聚糖纳米粒对质粒的保护作用不同,当pH值<7时壳聚糖纳米载体能100%结合质粒;DnaseⅠ消化试验证实壳聚糖纳米载体对质粒DNA有保护作用。结论采用离子交联法制备出粒径较小、均匀的壳聚糖纳米粒,并且壳聚糖纳米粒能有效地连接上质粒并对其有保护作用。     

杯状细胞靶向壳聚糖纳米粒的制备及吸收

目的将多肽CSKSSDYQC(CSK)化学结合到三甲基壳聚糖(trimethyl chitosan chloride,TMC)上,构建杯状细胞靶向口服给药系统,以期获得口服生物利用度较高的给药系统。方法通过化学合成的方法将CSK连接到TMC上,并用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)进行荧光标记。以粒径和成球率为指标,采用单因素实验得到纳米粒的制备处方。用乳酸脱氢酶细胞毒性测试(lactate dehydrogenase,LDH)法分别考察载体材料和纳米粒对Caco-2和HT29-MTX细胞存活率的影响。建立Caco-2/HT29-MTX共培养细胞单分子层,进行纳米粒的跨膜转运实验。利用免疫荧光法考察纳米粒在体内吸收情况。结果按照最优处方制得的FITC-TMC纳米粒和FITC-TMC-CSK纳米粒的粒径分别为214.5 nm和234.3 nm;电位分别为15.81 mV和8.96 mV;成球率分别为98.14%和91.58%。FITC-TMC-CSK的细胞毒性低于FITC-TMC,纳米粒的细胞毒性低于载体材料。FITC-TMC-CSK纳米粒的累积透过量高于FITC-TMC纳米粒,其表观渗透系数(P_(app))值是后者的2.17倍。FITC-TMC-CSK纳米粒在正常大鼠不同肠段吸收顺序为回肠>空肠>十二指肠。结论将CSK键合到TMC上构建杯状细胞靶向纳米粒,能有效提高纳米粒的吸收,在药物口服吸收的研究中具有广阔的应用前景。     

三甲基化壳聚糖卵清蛋白纳米粒的制备

目的：以N-三甲基壳聚糖盐酸盐（N—trimethyl chitosan chloride，TMC）为材料制备新型纳米粒（nanoparticles，NPs），包裹卵清蛋白（ovalbumin，OVA），以提高卵清蛋白的包封率。方法：利用TMC与三聚磷酸钠（tripolyphosphatesodium，TPP）之间的离子胶凝作用制备纳米粒；用纳米粒度及表面电位分析仪测定纳米粒的粒径及zeta电位；探讨OVA溶液的pH值及浓度，TMC溶液的浓度，TPP溶液的浓度等因素对OVA包封率的影响；用十二烷基硫酸钠一聚丙烯酰胺明胶电泳（Soldium Dodeoyl Sulfate—Polyacrylamide，SDS-PAGE）检验OVA在纳米粒制备及体外释放过程中有无降解。结果：本研究制备的TMC／OVA纳米粒为紧密球形，分布均匀，粒径约为135．4nm，zeta电位约为＋20mV；OVA的pH值及制备工艺是影响包封率的主要因素；SDS-PAGE电泳证实在纳米粒的制备及释放过程中OVA没有降解。结论：用离子胶凝法制备载蛋白多肽类疫苗的纳米粒，操作简便，采用合适的制备方法，调整处方可将包封率提高到90％以。     

壳聚糖纳米粒制备的研究进展

载药纳米粒作为药物、基因传递和控释的载体,是近年来出现的药物控释和缓释的新剂型。壳聚糖具有较好的生物黏附性、促吸收效应和酶抑制载体作用等特性。壳聚糖纳米粒作为一种新型药物载体,已成为目前国内外研究开发的热点。本文就壳聚糖纳米粒制备的研究进展情况进行了综述。     

载ACE-shRNA pGenesil质粒与壳聚糖纳米粒的结合及释放实验

目的制备适当粒径的壳聚糖纳米粒,并连接上质粒,研究壳聚糖纳米粒对质粒DNA的结合能力及在体外的释放。方法采用离子交联法制备壳聚糖纳米粒,通过喷金电镜观察其大小、形态及分布;经琼脂糖凝胶电泳分析纳米载体与质粒DNA的结合能力;在4种不同pH值的磷酸盐缓冲液(PBS)中观察壳聚糖质粒纳米粒的释放情况;通过紫外分光光度计检测其包埋率及释放率。结果喷金电镜证实壳聚糖纳米粒分布均匀,呈近似球形,平均粒径约5nm;琼脂糖凝胶电泳结果显示壳聚糖纳米粒能有效地结合质粒,当纳米粒与质粒的比例为10∶10时包埋率达98.7%;壳聚糖质粒纳米粒的性质较稳定,在pH值<7.5的PBS溶液能够平稳释放100h左右。结论制备出适当粒径且分布均匀的壳聚糖纳米粒,能有效地结合质粒,并且能够持续平稳地释放。     

多柔比星壳聚糖纳米粒的制备及其体外评价

目的 制备多柔比星壳聚糖纳米粒,并研究其包封情况和控释能力.方法 采用离子交联法制备多柔比星壳聚糖纳米粒,激光粒度仪测定粒径的分布,HPLC法测定纳米粒的包封率,透析法测定多柔比星壳聚糖纳米粒的体外释放情况.结果 壳聚糖的浓度增加,纳米粒的粒径增加、包封多柔比星的效率也增加;壳聚糖溶液的pH增加,纳米粒的粒径降低而包封多柔比星的效率增加;壳聚糖分子量增加对多柔比星的包封效率影响较小,但粒径增加,多柔比星突释减小,释放速率降低.结论 多柔比星可以通过离子交联法制备壳聚精纳米粒,其粒径、包封率可控,具有缓释效果.     

离子凝胶法制备水杨酸壳聚糖纳米粒

目的以壳聚糖为载体材料制备水杨酸壳聚糖纳米粒,并对其制备工艺及体系pH值对药物包封率的影响进行考察,初步探讨壳聚糖纳米粒的载药机制。方法以水杨酸为模型药物,采用离子凝胶法制备壳聚糖纳米粒,以包封率及粒径为指标,考察处方因素对纳米粒制备的影响。结果壳聚糖浓度、体系的pH值、药物质量浓度是影响制备工艺的主要因素;体系的pH值可显著提高壳聚糖纳米粒的包封率。结论药物与壳聚糖之间的离子相互作用较弱,并不是纳米粒载药的主要机制。     

N-三甲基壳聚糖对环孢素经皮渗透作用的试验研究

目的考察N-三甲基壳聚糖(N-trim ethyl ch itosan,TMC)作为透皮吸收促进剂的作用大小。方法用壳聚糖及碘甲烷为主要原料,合成季铵化程度为60%的TMC,即TMC60,通过1H-NMR确定其季铵化程度。以2%氮酮为阳性对照,以环孢素为模型药物,以离体小鼠皮肤为屏障,采用预处理法考察2%TMC60对环孢素透过离体皮肤的促进作用。结果经1H-NMR确定合成得到的TMC60季铵化程度为67.2%。体外透皮实验中,累积透过量及稳态透皮速率大小顺序均为:TMC60组>氮酮组>空白组,TMC60的促进作用强度大于同浓度的氮酮(P<0.05)。结论TMC对环孢素具有较好的经皮渗透作用。     

N-三甲基壳聚糖对双氯芬酸钠经皮渗透作用的研究

目的:考察N-三甲基壳聚糖(N-trimethyl chitosan,TMC)作为透皮吸收促进剂的作用。方法:用壳聚糖及碘甲烷为主要原料,合成季铵化程度为60%的TMC,即TMC60,通过1H-NMR确定其季铵化程度。以2%氮酮为阳性对照,以双氯芬酸钠为模型药物,以离体小鼠皮肤为屏障,采用预处理法考察2%TMC60对双氯芬酸钠透过离体皮肤的促渗作用。结果:经~1H- NMR确定合成得到的TMC60季铵化程度为67.2%。体外透皮实验中,累积透过量及稳态透皮速率大小顺序均为:TMC60组>氮酮组>空白组,TMC60的促进作用强度大于同浓度的氮酮(P<0.05)。结论:TMC对双氯芬酸钠具有较好的经皮促渗透作用。     

N-三甲基壳聚糖对环孢素经皮渗透作用的试验研究

OBJECTIVE To study the penetration effect of N-trimethyl chitosan(TMC) on Cyclosporin.METHODS TMC60 was synthesized by a two-step method using chitosan and methyl-iodide.The degree of quaternization(DQ) of TMC was determined by 1H-NMR.The excised mice ski     

TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283 ± 2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS–PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.     

N-三甲基壳聚糖包覆水飞蓟宾脂质体在小鼠体内的药动学及靶向性

目的:研究N-三甲基壳聚糖(TMC)包覆的水飞蓟宾脂质体(SLBL)在小鼠体内药动学,并对其肝靶向性进行研究。方法:小鼠尾静脉注射水飞蓟宾(SLB)溶液、SLBL、季铵化程度为20%的TMC包覆的SLBL(TMC20-SLBL),HPLC法检测不同时间血浆和各组织中SLB的浓度,采用DAS2.0软件统计学方法分析SLB在小鼠体内的药动学特征,采用相对摄取率re进行肝靶向性评价。结果:3种制剂中的SLB在小鼠体内的药动学特征符合二室模型,t_1/2β顺序依次为:SLBL>TMC20-SLBL>SLB溶液,AUC大小顺序为TMC20-SLBL>SLBL>SLB溶液,CL顺序为SLB溶液>SLBL>TMC20-SLBL。SLBL和TMC20-SLBL在肝组织的相对摄取率r_e分别为4.101和9.706。结论:与SLB溶液及SLBL相比,TMC20-SLBL具有明显的缓释作用及肝靶向性,生物利用度较高,有利于提高其治疗作用。     

Intestinal Absorption of Octreotide Using Trimethyl Chitosan Chloride: Studies in Pigs

Purpose. To investigate the enhancing effect of trimethyl chitosan chloride (TMC) on the enteral absorption of octreotide and to delineate the required doses of both TMC and peptide in vivo in juvenile pigs. Methods. Six female pigs (body weight, 25 kg) were operated to induce a stoma at the beginning of their jejunum and to insert an in-dwelling fistula for intrajejunal (IJ) administration of the formulations. A silicone cannula was inserted at the jugular vein for blood sampling. One week after surgery the pigs received IJ octreotide solution administrations with or without TMC at pH 7.4 or chitosan HCl at pH 5.5. For determining bioavailability (F) values, the pigs also received an octreotide solution intravenously (IV). Blood samples were taken from the cannulated jugular vein and subsequently analyzed by radioimmunoassay. Results. Intrajejunal administration of 10 mg octreotide without any polymer (control solution) resulted in F values of 1.7 ± 1.1% (mean ± SE). Chitosan HCl 1.5% (w/v) at pH 5.5 led to a 3-fold increase in F compared to the control (non-polymer containing) formulations. Co-administration of octreotide with 5 and 10% (w/v) TMC at pH 7.4 resulted in 7.7- and 14.5-fold increase of octreotide absorption, respectively (F of 13.9 ± 1.3% and 24.8 ± 1.8%). IJ administration of 5 mg octreotide solutions resulted in low F values of 0.5 ± 0.6%, whereas co-administration with 5% (w/v) TMC increased the intestinal octreotide bioavailability to 8.2 ± 1.5%. Conclusions. Cationic polymers of the chitosan type are able to enhance the intestinal absorption of the peptide drug octreotide in pigs. In this respect, TMC at neutral pH values of 7.4 appears to be more potent than chitosan HCl at a weak acidic pH of 5.5.     

N-三甲基壳聚糖包衣多柔比星脂质体肿瘤新生血管靶向性的体内评价

目的：考察N-三甲基壳聚糖（TMC）包衣的多柔比星（ADM）阳离子脂质体抗肿瘤活性及对肿瘤新生血管的靶向性。方法：采用动物移植性肿瘤实验法,建立小鼠H22肿瘤模型,比较TMC包衣脂质体组和其他各给药组的肿瘤抑制率并通过小鼠尾静脉注射异硫氰酸荧光素标记的葡聚糖（FITC—Dextran）,分析肿瘤组织中的在体荧光,定性观察肿瘤新生血管的形态排布情况,定量测定肿瘤组织中的血管相对密度。结果：TMC包衣的ADM脂质体组,其小鼠的瘤重抑制率迭53．47％,显著高于游离药物组和未包衣脂质体组（8．75％,34．88％）（P〈0．05）;给予TMC包衣ADM脂质体的小鼠,其肿瘤新生血管形态良好,排布均匀,血管分支少,而其他给药组的肿瘤新生血管多扭曲,粗细不均,一个节点有多个血管分支等;通过比较不同给药组肿瘤组织黏附的FITC—Dextran量,TMc包衣ADM脂质体组的肿瘤血管密度明显低于游离药物组和未包衣脂质体组（P〈0．05）。结论：TMC包衣的ADM阳离子脂质体不仅具有较强的抗肿瘤活性,而且具有很好的肿瘤新生血管靶向性。     

Influence of methylation process on the degree of quaternization of N-trimethyl chitosan chloride.

N-Trimethyl chitosan chloride (TMC) is a soluble chitosan derivative that shows effective enhancing properties for peptide and protein drug transport across mucosal membranes. TMC was synthesized by reductive methylation of chitosan in an alkaline environment at elevated temperature. The number of methylation process steps and the base used in the process was demonstrated to affect the degree of quaternization of the primary amino group and methylation of 3- and 6-hydroxyl groups. 1H-Nuclear magnetic resonance spectra showed that the degree of quaternization of TMC was higher when using sodium hydroxide as the base compared to using dimethyl amino pyridine. The degrees of quaternization as well as O-methylation of TMC increased with the number of reaction steps. O-Methylation resulted in decreased solubility of TMC. The high degree of quaternization of TMC with a low degree of O-methylation was prepared by employing one reaction step with two subsequent addition steps and a controlled alkaline environment of the mixture reaction.     

N-三甲基壳聚糖对尼美舒利脂质体凝胶大鼠体内药动学的影响

目的：研究N-三甲基壳聚糖（TMC）对尼关舒利（NIM）脂质体凝胶大鼠皮肤局部给药的药动学影响。方法：以壳聚糖及碘甲烷为主要原料,合成季铵化程度分别为40％和60％的TMC,即TMC40和TMC60。以泊洛沙姆407为基质制备5份NIM脂质体凝胶,1份不加TMC为NIM组,另4份凝胶分别用TMC40和TMC60（1．0％）作为包衣和混合材料,分别为TMC40C组,TMC40B组及TMC60C组,TMC60B组。经皮给药后采用RP—HPLC法测定大鼠皮肤和血浆中的药物浓度,计算药动学参数,并进行比较。结果：TMC40、TMC60季铵化程度分别为38％和59％。皮肤中的AUC0→t;NIM组〉TMC60C组〉TMC40C组〉TMC60B组〉TMC40B组（P〈0．05）;皮肤中的Cmax;NIM组、TMC60C组和TMC40C组均明显大于TMC60B组和TMC40B组（P〈0．05）;血浆中的AUC0→t;TMC60C组〉TMC60B组〉TMC40C组〉TMC40B组〉NIM组（P〈0．05）;血浆中的Cmax：TMC60C组〉TMC60B组〉TMC40C组〉TMC40B组和NIM组（P〈0．05）（其中C代表包衣组,B代表混合组）。结论：TMC的季铵化程度和不同作用方式对NIM脂质体的透皮吸收有较显著的影响,可通过合理选择,达到不同的制剂目的。     

Effect of chitosan structure properties and molecular weight on the intranasal absorption of tetramethylpyrazine phosphate in rats

The objective of this work was to assess and compare the absorption promoting effect of different molecular-weight chitosans, trimethyl chitosans and thiolated chitosans for intranasal absorption of 2,3,5,6-tetramethylpyrazine phosphate (TMPP). An in situ nasal perfusion technique in rats was utilized to test the rate and extent of TMPP absorption in situ. In vivo studies were carried out in rats and the pharmacokinetic parameters were calculated and compared with that of intravenous injection. All the chitosan derivatives investigated could enhance the intranasal absorption of TMPP significantly. However, thiolation could not improve the absorption-enhancing capacity of chitosan remarkably even when the thiolation ratio was as high as 152 μmol/g. In contrast, trimethylated chitosan exhibited stronger absorption-enhancing ability than the homopolymer chitosan. The permeation enhancing effect of chitosan increased with increasing molecular weight up to M_w 100 kDa. In vivo studies indicated that chitosan 100 kDa and TMC 50 kDa had comparable absorption-enhancing effect but chitosan 100 kDa functioned for more than 120 min versus 90 min for TMC. A good correlation was found between the in situ absorption data and plasma concentration in vivo for the polymers investigated. This study demonstrated that both chitosan structural features and chitosan molecular weight play a key role on promoting the intranasal absorption of TMPP. Taking safety reason into account, chitosan 100 kDa is the most promising as an intranasal absorption enhancer.