基因壳聚糖纳米粒表面修饰和转染研究

目的:研究递送基因纳米粒表面修饰对体外基因转染的影响.方法:利用末端活化的聚乙二醇(PEG)制备PEG化基因壳聚糖纳米粒;通过两端活化的PEG将糖蛋白配基连接到纳米粒表面,完成肝靶向纳米粒的制备;用透射电镜观察表面修饰对纳米粒粒径大小、粒子形态的影响;使用蛋白质测定试剂盒测算纳米粒表面蛋白连接量;利用体外转染实验考察表面修饰对纳米粒转染活性的影响;用倒置荧光显微镜观察并用流式细胞仪测定转染结果.结果:纳米粒PEG化使转染效率大幅度升高,半乳糖基牛血清白蛋白(Galn-BSA)使体系的转染效率比PEG化纳米粒略有下降,但比不经修饰的纳米粒转染活性高.壳聚糖纳米粒的表面PEG化能提高纳米粒的体外稳定性,从而提高体外转染效率,并适合于进行冷冻干燥.结论:长循环壳聚糖基因递送纳米粒在基因治疗研究中可能会发挥重要作用.     

固体脂质纳米粒的研究进展

固体脂质纳米粒是新一代亚微粒给药系统，由于其生理相容性好，可控制药物释放以及良好的靶向性等优点，日益受到各国研究者的重视。本文综述了固体脂质纳米粒的制备方法，体外释药，给药途径及存在问题等方面的内容。     

壳聚糖纳米粒表面游离氨基与纳米粒特性研究

为研究对三聚磷酸钠(TPP)交联的壳聚糖纳米粒的表面游离氨基与纳米粒的性质之间的关联性,采用胶体滴定法测定壳聚糖纳米粒表面氨基游离率，考察表面游离氨基的数量及离解程度对纳米粒粒径、电位、形态及对药物包封率和体外释药特性的影响，并阐述这种变化机制。结果表明，随TPP浓度增加，表面游离氨基逐步减少，在一定TPP浓度范围内，纳米粒粒径减小，表面zeta电位降低，稳定性也随之下降，粒子易聚集，释药速度和程度也随之降低，但药物包封率未受到影响；随着pH升高，表面游离氨基离解程度降低，纳米粒粒径亦随之减小，表面zeta电位降低。酸性介质提高纳米粒的释药速度和程度，在中性和碱性介质中纳米粒的释药速度和程度明显降低。交联程度和pH影响表面游离氨基的数量或离解程度，进而影响纳米粒的体积相转变(溶胀/收缩过程)等重要性质。表面游离氨基与纳米粒性质间有密切的联系。     

壳聚糖纳米粒作为基因载体的研究:影响转染效率的因素

目的:以增强型绿色荧光蛋白质粒(pEGFP)为报告基因,探讨影响壳聚糖(CS)介导的基因转染率的因素.方法:复凝聚法制备pEGFP/CS纳米粒,选用HEK293细胞,考察CS相对分子质量、CS中的氨基与pEGFP中的磷酸基的比值(N/P比)、纳米粒粒径、介质pH值和血清对转染率的影响.结果:相对分子质量5×103的CS获得较高转染率;转染率随N/P比增加而提高;介质pH≤6.8有利于转染;在230～1 290nm范围,粒径对转染效率无影响;CS介导的转染不受血清影响.结论:CS相对分子质量、N/P比和介质pH值是影响转染率的因素,低相对分子质量水溶性CS有利于提高转染.     

壳聚糖纳米粒载体的应用研究进展

目的介绍壳聚糖纳米粒载体在药物、基因递送等方面的研究应用进展,为其在新领域的应用提供依据。方法广泛查阅中外文有关文献,整理分析归纳了其中27篇文献内容。结果壳聚糖纳米粒载体在药物和基因递送方面已经有诸多研究应用。结论壳聚糖纳米粒载体是一种有前途的非病毒递送载体,其特性和应用有待进一步探索。     

壳聚糖纳米粒的研究进展

壳聚糖是一类带正电的直链多糖，具有良好的生物相容性和生物可降解性，且具有多种生物活性，能有效增加药物通过眼部、鼻腔及胃肠道粘膜上皮的吸收，降低药物的吸收前代谢，提高药物的生物利用度，因此壳聚糖在缓控释给药系统中具有广阔的应用前景，但其溶解性能有待于进一步提高。本文就壳聚糖纳米粒的制备方法、作物特点及应用作一概述。     

以脂质体和纳米粒为载体的抗生素靶向给药

本文叙述了以脂质体或毫微粒为载体增加抗生素生物利用度的研究进展。胶体粒子的生物可降解性聚合物也被使用,而且显示了在未来的抗微生物化疗中使用的可能性,结果表明这些药物载体（脂质体,毫微粒）可增加感染部位的药物浓度而且降低了毒性,静脉注射抗生素的普通载体可以靶向单核吞噬细胞系统,隐性载体避免了这些细胞的大量捕获,扩大了这些载体的全身性作用。     

肝靶向氟尿嘧啶类脂纳米粒的研究

目的　为了提高氟尿嘧啶(5-Fu)的疗效,降低其毒副作用,制备具肝靶向的5-Fu类脂纳米粒。方法　利用氟尿嘧啶与硬脂酰氯进行反应,制备了5-Fu前体药物N₁-硬脂酰-5-Fu,通过红外光谱及核磁共振谱对合成的目标化合物进行结构确认。同时研究了前体药物的性质及稳定性。采用物理凝聚法制备类脂纳米粒,并研究其形态、粒径及粒径分布、载药量、体外释药特征、动物体内分布与药代动力学参数等。结果　平均粒径d_av=240.19 nm,载药量为20.53%。体外释药速率符合一级动力学模型。与5-Fu水针剂比较,类脂纳米粒组在肝脏中药物含量平均增加了一倍以上。家兔体内主要药动学参数为：V_c=0.04336 L.kg^-1,T_1/2β=1.2834 h,CL=0.1632 L.h^-1。结论　利用前体药物可提高药物的脂溶性,首次以物理凝聚法制备类脂纳米粒,小鼠体内分布研究表明类脂纳米粒有明显的肝靶向,有一定的优越性和参考价值。     

固体脂质纳米粒的研究进展

在新药的研发过程中,越来越多的数据显示,由于药物自身理化性质的限制,可能使一些很有希望的药物由于不符合药物动力学要求而遭淘汰.于是,脂质体、微乳、聚合物纳米粒等一系列药物载体被发展起来,并在选择性吸收和靶向给药等方面取得了显著的成就.然而以上几种给药系统又存在某些不足之处,如:生物可降解物质存在细胞毒性以及不易提高制备规模等[1].     

甘草酸表面修饰阿霉素壳聚糖纳米粒在小鼠体内的组织分布

目的：研究甘草酸表面修饰阿霉素壳聚糖纳米粒（GL-ADM-NPs）在小鼠体内的分布。方法：GL-ADM-NPs经小鼠尾静脉给药,采用液质联用（LC/MS/MS）法检测阿霉素在小鼠血浆、心、肝、脾、肺、肾中的浓度随时间的变化,并与游离阿霉素溶液（F-ADM）组和阿霉素壳聚糖纳米粒（ADM-NPs）组进行比较。以相对摄取率（Re）和峰浓度比（Ce）为指标评价其靶向性。结果：与F-ADM相比,ADM-NPs明显提高药物在肝脏中的浓度（Re和Ce分别为3.54和2.2） 经甘草酸修饰后GL-ADM-NPs对肝脏的靶向作用更强,Re和Ce分别达到5.83和3.42,在心和肾中药物分布显著降低,AUC分别为F-ADM的43.06%和62.58%。结论：GL-ADM-NPs改变了药物ADM的体内分布特征,具有明显的肝靶向性,并能显著降低心和肾毒性,有望成为肝癌治疗药物ADM的理想输送载体。     

叶酸和聚乙二醇双修饰的壳聚糖纳米粒的制备及其性能表征

目的 利用离子交联和化学交联相结合的方法制备壳聚糖纳米粒子(NPs),并对NPs分别进行了叶酸(FA)和聚乙二醇(PEG)的修饰。方法 通过红外光谱进行结构验证;用扫描电镜和粒度分析仪对粒子的微观形态、粒径、电位等进行了表征;通过与Hela细胞摄取实验对其靶向作用进行验证。结果 离子交联和化学交联相结合的方法制备壳聚糖纳米粒子粒径在200 nm左右并且粒径分布窄,修饰后的NPs(FA-NPs、PEG-NPs及FA+PEG-NPs)粒径不受功能基团修饰的影响。激光共聚焦试验证明FA-NPs及FA+PEG-NPs能显著提高细胞对粒子的摄取,而PEG-NPs则明显降低其对粒子的摄取。结论 FA+PEG-NPs有望成为一种新型的药物载体,用于抗癌药物对癌细胞的主动靶向。     

胰岛素壳聚糖纳米粒的制备及影响包封率因素的考察

采用离子感胶化法制备亲水性壳聚糖纳米粒。该法制备条件温和，是在下将两种水相混合而形成纳米粒。混合的两相中，其中一相含有壳聚糖和聚氧乙烯，另一相含有聚阴离子三聚磷酸钠。该纳米粒的粒度大小及ζ电位可以通过控制制剂处方组成及工艺进行调节。采用该法制备的胰岛素壳聚糖纳米粒，实验结果表明，胰岛素的包封率可高达90％，且制剂处方及工艺各因素对该纳米粒的包封率有不同程度的影响。该亲水性纳米粒可作为蛋白质或其它能与壳聚糖相互作用的大分子类药物给药系统的载体。     

Surface structured liposomes for site specific delivery of an antiviral agent-indinavir

The present investigation was aimed at targeting indinavir, a protease inhibitor to cells of mononuclear phagocyte system (MPS) via mannosylated liposomes. β-d-1-thiomannopyranoside residues were covalently coupled with dimyristoyl phosphatidylethanolamine (DMPE) to generate mannosylated-DMPE (Man-DMPE) conjugate which was further incorporated with disteroyl phosphatidyl choline and cholesterol to prepare mannosylated liposomes. The optimized mannosylated liposomes were nanometric in size (142?±?2.8?nm) with optimum entrapment efficiency (88.7?±?2.3%). Less than 20% cumulative drug release was observed from the prepared formulations in 24?h in phosphate buffer saline (pH 7.4). Cellular uptake studies performed on J774.A1 macrophage cell line via flow cytometric analysis depicted enhanced uptake of mannosylated liposomes as compared to plain liposomes. Annexin-V-fluorescein isothiocyanate/propidium iodide apoptosis assay delineated marginal cytotoxicity in macrophages from the developed formulation. Plasma and tissue distribution studies performed to assess the drug reach to macrophage rich regions depicted a significant level (P?<?0.05) of indinavir in macrophage rich tissues like liver, spleen, and lungs from mannosylated liposomes as compared to plain liposomes and free drug. The conducted studies suggest the potential of indinavir loaded mannosylated liposomes for anti-human immunodeficiency virus therapy.     

壳聚糖在新型给药系统中的应用

综述壳聚糖的物理化学和生物特性及在基因转染和不同给药系统中的应用研究近况。壳聚糖作为新型药用辅料,已受到越来越多的关注,对其应用的开发和研究已渗透到药剂学的多个领域。壳聚糖用作非病毒基因载体,也已成为近年来的研究热点之一。它也被广泛研究应用于眼部、鼻腔、口腔、胃内、小肠和结肠等靶向给药载体。     

Genuine DNA/polyethylenimine (PEI) Complexes Improve Transfection Properties and Cell Survival

Polyethylenimine (PEI) has been described as one of the most efficient cationic polymers for in vitro gene delivery. Systemic delivery of PEI/DNA polyplexes leads to a lung-expression tropism. Selective in vivo gene transfer would require targeting and stealth particles. Here, we describe two strategies for chemically coupling polyethylene glycol (PEG) to PEI, to form protected ligand-bearing particles. Pre-grafted PEG–PEI polymers lost their DNA condensing property, hence their poor performances. Coupling PEG to pre-formed PEI/DNA particles led to the expected physical properties. However, low transfection efficacies raised the question of the fate of excess free polymer in solution. We have developed a straightforward a purification assay, which uses centrifugation-based ultrafiltration. Crude polyplexes were purified, with up to 60% of the initial PEI dose being removed. The resulting purified and unshielded PEI/DNA polyplexes are more efficient for transfection and less toxic to cells in culture than the crude ones. Moreover, the in vivo toxicity of the polyplexes was greatly reduced, without affecting their efficacy.     

Chitosan-DNA Nanoparticles: The Effect of Cell Type and Hydrolysis of Chitosan on In Vitro DNA Transfection

Commercial chitosan (Ch) with low (LMWCh) and medium molecular weight (MMWCh) were hydrolyzed in diluted hydrochloric acid by heating at different temperatures. The viscosity average molecular weight of Chs was gradually decreased from 450 to 14 kDa as a function of temperature. Ch fractions were used for formation of Ch-DNA nanoparticles and tested for the ability to introduce DNA into HEK293, Swiss3T3, HeLa, and MDCK cells in vitro. The average diameter of nanoparticles was 200–220 nm. The surface charge of nanoparticles varied depending on the Ch/DNA ratio. The cell lines different response to DNA transfection with Ch fractions depended on molecular weight. HEK293 cells were efficiently transfected by nanoparticles prepared with Chs having a wide range of molecular weight (～14–195 kDa). Swiss3T3 cells were efficiently transfected by Ch polymers with about <17 kDa. In contrast, HeLa and MDCK cells were highly resistant to DNA transfection with Ch polymers. These results strongly suggest that Ch polymers may be widely used for DNA trasnfection of the mammalian cells under optimized conditions.     

载基因壳聚糖纳米粒的制备及其相关性质的研究

目的：制备壳聚糖载基因纳米粒，并对其体外相关性质进行初步研究。方法：采用复凝聚法制备载基因纳米粒；用纳米粒度仪测量粒度分布，分散性和Zeta电位；用透射电镜观察粒子的形态；用紫外分光光度法和比色法测定包封率和载药量，并对主要影响因素进行考察。用凝胶阻滞分析和电性结合分析对载药方式进行初步推测。结果：所制备的载基因纳米粒形态规则，大多呈球形，纳米粒平均粒径为263．2nm，粒径分布较窄，多分散度为0．213，Zeta电位为19．8mV；包封率大于90％，载药量约30％；凝胶阻滞和电性结合分析结果表明，非甲基化胞嘧啶鸟嘌呤的寡核苷酸链（CPG-ODN）与壳聚糖分子间可通过电性结合作用而完全结合。结论：采用复凝聚法可制备粒度分布均匀，形态规则，具有较高包封率和载药量的载基因壳聚糖纳米粒；电性结合作用是载基因壳聚糖纳米粒载药的主要方式。     

纳米羟喜树碱注射用灭菌粉末在家兔体内的药动学研究

目的考察纳米羟喜树碱注射用灭菌粉末经静脉注射给药后在家兔体内的药动学特征。方法建立测定家兔血浆中羟喜树碱含量的高效液相色谱法，标准曲线的回归分析采用加权最小二乘法；采用3p97药动学计算软件模拟房室模型并计算纳米羟喜树碱注射用灭菌粉末在家兔体内的药动学参数。结果纳米羟喜树碱注射用灭菌粉末在家兔体内的代谢符合1/C2权重的三室模型。相同剂量下普通羟喜树碱在家兔体内的半衰期较纳米制剂短。结论纳米羟喜树碱注射用灭菌粉末的药动学特性与普通羟喜树碱制剂的特性有明显区别。     

Development of Octreotide-Loaded Chitosan and Heparin Nanoparticles: Evaluation of Surface Modification Effect on Physicochemical Properties and Macrophage Uptake

Octreotide (OCT) is a therapeutic peptide which is administered for the treatment of acromegaly. The purpose of this study was to design a new polyethylene glycol (PEG)–conjugated nanoparticle (PEG-NP) to overcome the short half-life and poor stability of OCT. The developed PEG-NPs were compared with non-PEGylated NPs with respect to their size, morphological characteristics, loading efficiency, release profile, and macrophage uptake. The OCT-loaded NPs and PEG-NPs were prepared by ionic complexion of chitosan (Cs) with either heparin (Hp) or PEGylated heparin (PEG-Hp). The chemical structure of PEG-Hp was confirmed by IR and proton nuclear magnetic resonance. Morphological analyses by scanning electron microscopy showed that NPs and PEG-NPs have a uniform shape. Dynamic laser scattering measurements indicated that hydrodynamic diameter of NPs and PEG-NPs were 222.5 ± 10.0 nm and 334.9 ± 6.7 nm, respectively. NPs and PEG-NPs had a positive zeta potential of about 32.5 ± 1.1 mv and 20.6 ± 2.4 mv, respectively. Entrapment efficiency was 61.4 ± 1.0% and 55.7 ± 2.4% for NPs and PEG-NPs, respectively. Compared with the NPs, the PEG-NPs exhibited a slower release profile. Subsequently, fluorescein isothiocyanate–labeled chitosanCs was synthesized and used to evaluate the stealth characteristic of PEG-NPs. In vitro macrophage uptake of fluorescently labeled NPs was measured by flow cytometry.