Adaptive Immune Responses of Legumin Nanoparticles

Legumin is one of the main storage proteins in the pea seeds (Pisum sativum L.) and the molecules of this protein have the capacity of binding together to form nanoparticles after aggregation and chemical cross-linkage with glutaraldehyde. The aim of this work was to study the adaptive immune response of legumin nanoparticles in rats. Following intradermal immunisation with the native protein legumin and legumin nanoparticles of about 250 nm, the humoral and cell-mediated immune responses were analysed in rats. The humoral responses against legumin and legumin nanoparticles were examined by western blot and ELISA analysis. Both techniques clearly showed that sera from rats immunised with legumin strongly expressed antibodies against this protein. On the contrary, serum samples from rats inoculated with legumin nanoparticles did not contain detectable amounts of antibodies. These results may be explained by a reduction on the antigenic epitopes of the protein induced by the glutaraldehyde used during the cross-linking step. Concerning the cell-mediated response, neither legumin nor legumin nanoparticles stimulated an immunogenic response. This absence of response of spleen lymphocytes for legumin and legumin nanoparticles may be explained by a cytostatic effect of legumin which was corroborated by the evaluation of the middle phase of cell apoptose. In fact, both legumin and legumin nanoparticles are potent inductors of a cytostatic phenomenon and showed a significant increase of the chromatin condensation (p <0.05) as compared with control.     

Enhancement of Nasal Absorption of Insulin Using Chitosan Nanoparticles

Purpose. To investigate the potential of chitosan nanoparticles as a system for improving the systemic absorption of insulin following nasal instillation. Methods. Insulin-loaded chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate anions. They were characterized for their size and zeta potential by photon correlation spectroscopy and laser Doppler anemometry, respectively. Insulin loading and release was determined by the microBCA protein assay. The ability of chitosan nanoparticles to enhance the nasal absorption of insulin was investigated in a conscious rabbit model by monitoring the plasma glucose levels. Results. Chitosan nanoparticles had a size in the range of 300–400 nm, a positive surface charge and their insulin loading can be modulated reaching values up to 55% [insulin/nanoparticles (w/w): 55/100]. Insulin association was found to be highly mediated by an ionic interaction mechanism and its release in vitro occurred rapidly in sink conditions. Chitosan nanoparticles enhanced the nasal absorption of insulin to a greater extent than an aqueous solution of chitosan. The amount and molecular weight of chitosan did not have a significant effect on insulin response. Conclusions. Chitosan nanoparticles are efficient vehicles for the transport of insulin through the nasal mucosa.     

Stealth PLA-PEG Nanoparticles as Protein Carriers for Nasal Administration

Purpose. The aim of the study was to encapsulate a model protein antigen, tetanus toxoid (TT), within hydrophobic (PLA) and surface hydrophilic (PLA-PEG) nanoparticles and to evaluate the potential of these colloidal carriers for the transport of proteins through the nasal mucosa. Methods. TT-loaded nanoparticles, prepared by a modified water-in-oil-in-water solvent evaporation technique, were characterized in their size, zeta potential and hydrophobicity. Nanoparticles were also assayed in vitro for their ability to deliver active antigen for extended periods of time. Finally, ¹²⁵I-TT-loaded nanoparticles were administered intranasally to rats and the amount of radioactivity recovered in the blood compartment, lymph nodes and other relevant tissues was monitored for up to 48 h. Results. PLA and PLA-PEG nanoparticles had a similar particle size (137-156 nm) and negative surface charge, but differed in their surface hydrophobicity: PLA were more hydrophobic than PLA-PEG nanoparticles. PLA-PEG nanoparticles, especially those containing gelatine as an stabilizer, provided extended delivery of the active protein. The transport of the radiolabeled protein through the rat nasal mucosa was highly affected by the surface properties of the nanoparticles: PLA-PEG nanoparticles led to a much greater penetration of TT into the blood circulation and the lymph nodes than PLA nanoparticles. Furthermore, after administration of ¹²⁵I-TT-loaded PLA-PEG nanoparticles, it was found that a high amount of radioactivity persisted in the blood compartment for at least 48 h. Conclusions. A novel nanoparticulate system has been developed with excellent characteristics for the transport of proteins through the nasal mucosa.     

Direct Nose-to-Brain Transfer of Morphine After Nasal Administration to Rats

Purpose The aim of this study was to quantify the olfactory transfer of morphine to the brain hemispheres by comparing brain tissue and plasma morphine levels after nasal administration with those after intravenous administration. Methods Morphine (1.0 mg/kg body weight) was administered via the right nostril or intravenously as a 15-min constant-rate infusion to male rats. The content of morphine and its metabolite morphine-3-glucuronide in samples of the olfactory bulbs, brain hemispheres, and plasma was assessed using high-performance liquid chromatography, and the areas under the concentration–time curves (AUC) were calculated. Results At both 5 and 15 min after administration, brain hemisphere morphine concentrations after nasal administration were similar to those after i.v. administration of the same dose, despite lower plasma concentrations after nasal administration. The brain hemispheres/plasma morphine AUC ratios for the 0–5 min period were thus approximately 3 and 0.1 after nasal and i.v. administration, respectively, demonstrating a statistically significant early distribution advantage of morphine to the brain hemispheres via the nasal route. Conclusion Morphine is transferred via olfactory pathways to the brain hemispheres, and drug transfer via this route significantly contributes to the early high brain concentrations after nasal administration to rats.     

Lymphatic Targeting of Polymeric Nanoparticles After Intraperitoneal Administration in Rats

Following intraperitoneal administration, the lymphatic targeting of polyacrylic nanoparticles has been evaluated in thoracic duct cannulated rats. The dosage forms administered consisted of carbon-14 polyhexylcyanoacrylate nanoparticles (PHCA) and polymethylmethacrylate (PMMA) nanoparticles. The carbon-14 concentrations were much higher in the excreted thoracic lymph than in the blood for both types of particles. The most dramatic results were found in the mediastinal nodes since the carbon-14 concentrations of rats receiving PHCA and PMMA nanoparticles by the ip route were 70-to more than 2000-fold higher than in the corresponding nodes of animals treated by the intravenous route. This potential lymphatic targeting could prove valuable in cancerology to treat tumors that metastasize in the peritoneal cavity or via lymphatic pathways such as colon carcinomas.     

Body Distribution of Camptothecin Solid Lipid Nanoparticles After Oral Administration

Purpose. The aim of this study was to investigate the specific changes in body distribution of camptothecin (CA) through incorporation into solid lipid nanoparticles (SLN) by peroral route. Methods. Camptothecin loaded solid lipid nanoparticles (CA-SLN) coated with poloxamer 188 were produced by high pressure homogenization. The CA-SLN were characterized by transmission electron microscopy and electrophoretic mobility measurement. In vitro release characteristics of camptothecin from CA-SLN were studied at different pH media. The concentration of camptothecin in organs was determined using reversed-phase high-performance liquid chromatography with a fluorescence detector after oral administration of CA-SLN and a camptothecin control solution (CA-SOL). Results. Our results showed that CA-SLN had an average diameter 196.8 nm with Zeta potential of –69.3 mV. The encapsulation efficiency of camptothecin was 99.6%, and in vitro drug release was achieved up to a week. There were two peaks in the camptothecin concentration-time curves in plasma and tested organs after oral administration of CA-SLN. The first peak was the result of free drug and the second peak was indicative of gut uptake of CA-SLN after 3 hours. In tested organs, the area under curve (AUC) and mean residence time (MRT) of CA-SLN increased significantly as compared with CA-SOL, and the increase of brain AUC was the highest among all tested organs. Conclusions. The results indicate SLN could be a promising sustained release and targeting system for camptothecin or other lipophilic antitumor drugs after oral administration.     

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.     

鼻腔给药NMFGF1纳米粒改善血管性痴呆小鼠认知功能障碍

目的 考察鼻腔给药包载非促分裂活性酸性成纤维细胞生长因子（non-mitogenic acid fibroblast growth factor,NMFGF1）的纳米粒（NMFGF1-NPs）对于血管性痴呆（vascular dementia,VD）小鼠认知功能改善作用及其机制。方法 应用水包水型乳化技术制备NMFGF1-NPs并进行质量表征。采用反复脑缺血再灌注法建立VD试验模型后分成假手术组、VD模型组、空白纳米粒组、NMFGF1溶液组及NMFGF1-NPs组,分别鼻腔给予相应形式药物干预。干预结束后,应用Morris水迷宫评价试验动物的学习及记忆功能,同时应用HE染色、FJB染色及Tunel凋亡染色等病理学方法评价试验动物海马神经元的形态、排列及凋亡指数（apoptosis index,AI）,另外应用ELISA及Western blotting等分子生物学方法探讨鼻腔给药NMFGF1-NPs改善VD的分子机制。结果 NMFGF1-NPs形态圆整,包封率（87.76±5.89）%。Morris水迷宫结果显示VD模型组小鼠的各项行为学指标均较假手术组有显著差异（P<0.01）,同时病理结果显示,VD模型组小鼠海马神经元CA1区域神经元排列紊乱、细胞形态结构缺失且AI较假手术组显著增加（P<0.01）,而经过鼻腔给药NMFGF1-NPs治疗组小鼠的各项行为学指标较VD模型组有显著改善,同时海马神经元细胞形态完整,排列整齐,AI指数较VD模型组及其他各个治疗干预组显著降低（P<0.01）。ELISA及Western blotting分析结果显示,鼻腔给药NMFGF1-NPs治疗组小鼠脑内MDA含量较VD模型组及其他各个治疗干预组显著下降（P<0.01）,同时SOD,NO含量及Nrf2,SOD-1,GSTO1/2表达显著增加（P<0.01）。结论 鼻腔给药NMFGF1-NPs给够通过激活Nrf2/ARE信号通路,发挥抗氧化应激损伤的作用,最终改善VD小鼠的学习及认知功能。     

Solid Lipid Nanoparticles in Lymph and Plasma After Duodenal Administration to Rats

Purpose. To evaluate the uptake and transport of solid lipid nanoparticles (SLN), which have been proposed as alternative drug carriers, into the lymph and blood after duodenal administration in rats. Methods. Single doses of two different concentrations of aqueous dispersions of unlabelled and labelled SLN (average diameter 80 nm) were administered intraduodenally to rats. At different times, samples of lymph were withdrawn by cannulating the thoracic duct and blood was sampled from the jugular vein. Monitoring continued for 45 and 180 minutes, for unlabelled and labelled SLN respectively. The biological samples were analysed by photon correlation spectroscopy (PCS), transmission electron microscopy (TEM) and gamma-counting. Results. TEM analysis evidenced SLN in lymph and blood after duodenal administration to rats; the size of SLN in lymph did not change markedly compared to that before administration. The labelled SLN confirmed the presence of SLN in lymph and blood. Conclusions. The uptake and transport of SLN in the lymph, and to a lesser extent in the blood, were evidenced. The in vivo physical stability of SLN may have important implications in designing drug-carrying SLN.     

Formulation Optimization and Bioavailability After Oral and Nasal Administration in Rabbits of Puerarin-Loaded Microemulsion

The main objective of the study was to investigate the efficacy of microemulsion (ME) to facilitate bioavailability of puerarin (PUE) after oral and nasal administration. The pseudo-ternary phase diagrams were constructed to screen the ME components and optimize the ME formulation. The optimized formulation for bioavailability assessment consisted of 20% Tween 80, 20% glycerin, and 1.6% ethyl oleate. The solubility (27.8 mg/mL) of PUE in ME was significantly improved compared to that (4.58 mg/mL) of crude PUE in water. The ME droplets were spherical with a mean particle diameter of 23.4 nm. After nasal (5 mg/kg) and oral (20 mg/kg) administration of a single dose of PUE as ME to fasted rabbits, the absolute bioavailability was 34.58% and 13.54%, respectively. It showed a shorter T_max (0.75 h) for nasal administration than that (1.0 h) for oral administration of PUE-loaded ME. The C_max of PUE-loaded ME was 0.55 μg/mL after nasal administration and 0.64 μg/mL after oral administration, respectively. The results showed that nasal administration might be a promising route to enhance the absorption of PUE in the form of ME.     

关于诱导广谱和持久免疫的流感疫苗开发的第二次会议报告

能防御不同流感病毒和诱导广谱和持久免疫的新型流感疫苗可以克服现有流感疫苗需每年接种的难题,特别适用于发展中国家,可用来控制流感流行和大流行。WHO于2005年12月在日内瓦召开了咨询会议,回顾了这种流感疫苗的研究现状,并确定了疫苗的研发日程。本文介绍会议的主要结论和建议。     

CpG ODN纳米粒增强免疫抑制小鼠对重组乙肝疫苗的免疫应答

目的：探讨CpG ODN壳聚糖纳米粒联合重组乙肝疫苗对免疫抑制小鼠的免疫增强作用。方法：选月环磷酰胺建立免疫抑制模型小鼠。将乙肝疫苗单独或和CpG ODN或CpG ODN纳米粒经后腿胫骨前肌注射到小鼠体内，ELISA方法检测抗HBsAg IgG抗体、IL-12水平；流式细胞仪检测外周血CD4^＋、CD8^＋T淋巴细胞亚群。结果：CpG ODN纳米粒联合疫苗组的CD4^＋细胞百分数，IL-12水平及抗HBsAg IgG抗体的含量显著高于CpG ODN联合疫苗组（P〈0．05）。结论：包裹在壳聚糖纳米粒中的CpG ODN较相同剂量的CpG ODN更能增强免疫抑制小鼠对乙肝疫苗的免疫应答。     

壳聚糖载药纳米粒研究进展

目的介绍壳聚糖载药纳米粒近年来的研究进展。方法总结壳聚糖纳米粒的制备方法、释药特性、生物摄取及其应用。结果不同的制备方法可得到不同粒径和表面特性的壳聚糖纳米粒。壳聚糖纳米粒改变了壳聚糖的摄取机制，广泛应用于药物的器官靶向、DNA转染效率提高、药物的非注射途释给药等方面。结论壳聚糖纳米粒作为一种新型的药物载体，具有重要的研究开发价值。     

Diesel Exhaust Particulates Induce Nasal Mucosal Hyperresponsiveness to Inhaled Histamine Aerosol

The prevalence of allergic rhinitis is increasing in many countries.It has been reported that the prevalence rate of allergic rhinitiscaused by pollens in air-polluted areas are higher than thatin nonpolluted areas. Therefore, it is important to determinewhether air pollutants are related to the increase in the prevalencerate of allergic rhinitis. In this respect, it is necessaryto elucidate whether exposure to air pollutants affects thenasal mucosa and causes nasal mucosal hyperresponsiveness tochemical mediators released by antigen-antibody reactions. Inthe present study using, guinea pigs, we investigated effectsof diesel exhaust particulates on (1) nasal airway resistance,(2) increases in nasal airway resistance and secretion inducedby histamine aerosol, and (3) vascular permeability and theincrease in vascular permeability induced by histamine in dorsalskin, since vascular permeability is an important factor involvedin increased nasal airway resistance and secretion. Intranasalpressure and nasal secretion from the nostril were measuredas markers of nasal airway resistance and exocrine activityof the nasal mucosa, respectively. A 30-min administration ofa suspension of diesel exhaust particulates into the nasal cavitiescaused a significant increase in intranasal pressure. The administrationalso augmented an increase in intranasal pressure and nasalsecretion induced by histamine aerosol. In dorsal skin, dieselexhaust particulates increased vascular permeability. Dieselexhaust particulates also augmented vascular permeability inducedby histamine. In conclusion, diesel exhaust particulates arepotent in increasing nasal airway resistance and augmentingincreases in nasal airway resistance and nasal secretion inducedby histamine. These properties of diesel exhaust particulatesare likely derived in part from the augmentation vascular permeabilityby these particulates.     

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。     

壳聚糖修饰的托氟啶固体脂质纳米粒的制备

摘 要 目的： 研究托氟啶固体脂质纳米粒及壳聚糖修饰的托氟啶固体脂质纳米粒的制备方法。方法: 采用薄膜 超声分散法制备托氟啶固体纳米脂质粒（TFu-SLNs）及壳聚糖修饰的TFu-SLNs,并对纳米粒的形态、粒径和表面电位进行测定,通过单因素考察及正交设计优化制备方法,同时考察处方稳定性。结果: 薄膜 超声分散法制备的TFu-SLNs平均粒径为160.2 nm,Zeta电位为－33.12 mV,壳聚糖修饰TFu-SLNs平均粒径为400.3 nm,Zeta电位为+12.87 mV。经壳聚糖修饰后,随着壳聚糖浓度的增加,电位逐渐增大。优化后的处方重复性、稳定性良好。结论:通过采用正交设计法对TFu固体脂质纳米粒处方进行优化,得到TFu固体脂质纳米粒及壳聚糖修饰的TFu固体脂质纳米粒的优化处方。     

粒细胞-巨噬细胞集落刺激因子壳聚糖缓释纳米粒的制备

目的:研究以生物可降解壳聚糖纳米粒作为粒细胞-巨噬细胞集落刺激因子(GM-CSF)新型缓释系统的可行性。方法:以三聚磷酸钠为交联剂,采用离子交联法制备负载GM-CSF和牛血清白蛋白(BSA)的纳米粒。用透射电镜观测纳米粒径和形态;用紫外分光光度计、荧光分光光度计分别测定BSA和GM-CSF包封率,并考察制剂体外药物释放情况。结果:纳米粒形态多呈球形,平均粒径为201nm,GM-CSF和BSA包封率分别为62.1%、58.5% ,3d时体外累积释放率分别为69%、82%。结论:应用离子交联法可制备负载GM-CSF的壳聚糖缓释纳米粒。     

Pharmacokinetics and Biodistribution of Oligonucleotide Adsorbed onto Poly(isobutylcyanoacrylate) Nanoparticles After Intravenous Administration in Mice

Purpose. The goal of this study was to evaluate the ability of nanoparticles to be used as a targeted delivery system for oligonucleotides. Methods. Pharmacokinetic and tissue distribution were carried out in mice by measuring the radioactivity associated to the model oligothymidylate ³³P-pdT₁₆ loaded to poly(isobutylcyanoacryrate) (PIBCA) nanoparticles. In addition, we have used a TLC linear analyzer to measure quantitatively on a polyacrylamide gel electrophoresis, the amount of non degraded pdT₁₆ Results. Organ distribution study has shown that nanoparticles deliver ³³P-pdT₁₆ specifically to the liver reducing its distribution in the kidney and in the bone marrow. Nanoparticles could partially protect pdT₁₆ against degradation in the plasma and in the liver 5 min after administration, whereas free oligonucleotide was totally degraded at the same time. Conclusions. Nanoparticles protect oligonucleotides in vivo against degradation and deliver them to the liver.     

甲磺酸帕珠沙星壳聚糖滴鼻剂的制备与质量控制

目的研究甲磺酸帕珠沙星壳聚糖滴鼻剂的制备方法并建立其质量控制方法。方法以壳聚糖为增稠剂和增效剂、甲磺酸帕珠沙星为主药制备滴鼻剂,采用高效液相色谱法测定主药含量,并对其稳定性进行考察。结果该产品的处方和制备工艺简单,质量稳定,甲磺酸帕珠沙星质量浓度在20.0～80.0μg/mL范围内与峰面积线性关系良好,r=0.9999(n=6);平均回收率为98.62%,RSD为0.53%(n=9)。结论滴鼻剂可作为甲磺酸帕珠沙星制剂新品种的开发研究对象。     

乳酸环丙沙星壳聚糖滴鼻剂的制备及质量控制

目的研制乳酸环丙沙星壳聚糖滴鼻剂并建立其质量控制方法。方法以壳聚糖为增稠剂、增效剂,乳酸环丙沙星为主药制备滴鼻剂。采用反相高效液相色谱法测定主药含量,并对其稳定性进行考察。结果所制备的制剂为无色、微粘、澄明液体,其鉴别符合中国药典2005版中的相关规定;乳酸环丙沙星在10～200μg·mL^-1内线性关系良好（r=0.9999）。平均回收率为99.19%,RSD为0.94%。日内RSD为0.53%,日间RSD为1.14%。结论本制剂制备工艺简单可行,质量稳定、可靠,控制方法灵敏、准确。