溴吡斯的明新型纳米乳体外释放和大鼠在体胃肠吸收

目的制备油包水型溴吡斯的明新型纳米乳(novel pyridostigmine bromide nanoemulsion,PPNE),研究其体外释放规律及各胃肠段吸收动力学特征。方法选用3种不同浓度pH 6.8、pH 7.4、pH 7.8的PBS以及pH 1.2的HCl作为释药介质,测定并绘制其体外释药曲线;并采用在体单向肠灌流实验模型,比较溴吡斯的明(pyridostigmine bromide,PB)和PPNE在胃肠道的吸收行为。结果在4种释放介质中,PB释放速率较快,而PPNE释放速率相对减缓;PPNE的吸收速率常数(K_a)和表观渗透系数(P_(app))较PB在十二指肠、空肠、回肠、结肠段中均增加。经t检验,PB和PPNE在4个肠段均有统计学意义(P<0.05)。结论 PPNE可提高药物的生物利用度,明显提高其药物在体肠段的吸收;体外释放中有良好的缓释效果。     

溴吡斯的明在大鼠离体肠的吸收动力学

目的观察溴吡斯的明在各肠段的吸收动力学特征。方法采用大鼠外翻肠囊模型,反相离子对色谱法测定不同浓度(25、50、100 mg.L-1)的溴吡斯的明在各肠段的吸收量,计算吸收速率常数(Ka)和表观渗透系数(Papp),并考察P-糖蛋白抑制剂(环孢素和维拉帕米)对药物吸收的影响。结果在25、50、100 mg.L-1溴吡斯的明条件下,Ka按十二指肠、空肠、回肠、结肠依次减小,不同浓度溴吡斯的明对同一肠段的Ka无显著影响(P>0.05),十二指肠、空肠、回肠间的Ka无显著差异(P>0.05),在50、100 mg.L-1溴吡斯的明条件下,结肠的Ka与十二指肠、空肠、回肠比较有显著差异(P<0.05,P<0.01)。随着溴吡斯的明浓度增加,各肠段Papp显著降低,不同肠段间的Papp有显著差异(P<0.05,P<0.01)。P-糖蛋白抑制剂对溴吡斯的明吸收无影响(P>0.05)。结论溴吡斯的明在十二指肠有较好吸收,在空肠和回肠有一定吸收,在结肠中吸收较少。     

溴新斯的明多囊脂质体的制备及其体外释放行为的初步研究

目的:制备溴新斯的明多囊脂质体(NB-MVL),并对其体外药物释放行为进行考察。方法:采用复乳法制备载药多囊脂质体,并用单因素试验优化处方,以动态透析技术与ƒ₂相似因子评价法对NB-MVL与溴新斯的明在体外不同释放介质中的释放行为进行研究,计算其药物释放度与相似因子。结果:NB-MVL的包封率受投料比、药物浓度等因素影响;NB-MVL在不同的释放介质中所获得的释放模型均符合Weibull模型,同时与溴新斯的明溶液在不同释放介质中所得累积释放曲线间的相似因子小于50。结论:本试验制备所得NB-MVL包封率较高,且体外药物释放行为与溴新斯的明存在显著性差异,表现出良好的缓释作用。     

溴吡斯的明磷脂复合物制备工艺

目的:星点设计-效应面法优化溴吡斯的明磷脂复合物制备工艺。方法:以溶剂挥发法制备溴吡斯的明磷脂复合物,以药物与磷脂的复合率为评价指标,采用单因素方法对溴吡斯的明磷脂复合物制备工艺影响因素的重要性进行考察,用星点设计对显著性因素进行优化,并且进行多元线性回归与二项式方程拟合,采用效应面法选取较佳工艺条件,并进行预测。结果:效应面法优选出的最佳工艺为:磷脂与溴吡斯的明以8∶1投料,主药质量浓度为4g.L-1,反应温度为40℃,以优化条件制备溴吡斯的明磷脂复合物,复合率为(84.02±1.68)%,与二项式拟合方程预测结果相差小于2%。结论:应用星点设计-效应面法优化溴吡斯的明磷脂复合物制备工艺,能够快速方便地得到最佳制备工艺,预测性良好。     

离子载体A23187介导pH梯度法制备重酒石酸长春瑞滨长循环脂质体

目的:采用A23187制备重酒石酸长春瑞滨长循环脂质体,优化了处方工艺,并考察了含量、包封率、药脂比和体外释放等检测指标。方法:采用A23187介导的pH梯度法制备了重酒石酸长春瑞滨脂质体;用HPLC法检测了脂质体中重酒石酸长春瑞滨的含量和脂质(HSPC)的含量,考察了药脂比;采用阳离子交换树脂分离脂质体和游离药物,HPLC法检测包封率;以4 mmol.L-1NH4Cl-PBS(pH 7.4)为体外释放介质考察了脂质体的体外释放行为。结果:重酒石酸长春瑞滨脂质体包封率为96.1%,药脂比为1∶5(w/w);高药脂比有利于延长药物体外释放的时间。结论:采用A23187介导的pH梯度法制备重酒石酸长春瑞滨脂质体工艺可行、载药量大、包封率高;所建立体外释放的检测方法快速、准确。     

一日1次溴新斯的明缓释片的制备及体内外释放行为研究

目的以溴新斯的明为模型药物制备日服1次缇释片,并对缓释片释放特征进行初步考察。方法采用亲水凝胶骨架与薄膜包衣控释技术制备溴新斯的明缓释片,以药物释放行为为评价指标,对处方及工艺进行单因素考察结果溴新斯的明缇释片体外药物释放可维持24 h,药物释放曲线符合一级动力学方程In(100-Q)=-0.147 4 t+4.775 4,体内药动学研究表明缓释片t_(1/2)(5.1±1.8)h,较普通片t_(1/2)(3.5±1.1)h延长,具有较为明显的缓释效果。结论亲水凝胶骨架与薄膜包衣控释技术联合应用可以成功制备易溶性药物溴新斯的明的缓释制剂。     

水溶性壳聚糖包覆多西他赛纳米脂质体的制备及性能研究

目的:制备羧甲基壳聚糖包衣多西他赛纳米脂质体,并考察其体外释放度。方法:采用薄膜分散法制备多西他赛阳离子脂质体,并用不同浓度的羧甲基壳聚糖包覆阳离子脂质体;用超滤法测定其包封率;用激光电位粒径测定仪分别测定其Zeta电位和粒径大小,并用透射电镜观察其形态;用透析法考察其体外释药性质。结果:所制的羧甲基壳聚糖包覆的脂质体包封率达99.98%;Zeta电位为-12.8 mV,平均粒径为(150±17)nm。结论:本实验制备的羧甲基壳聚糖包衣多西他赛纳米脂质体具有高包封率,粒径大小均匀,体外能显著延缓药物释放的性质。     

紫外分光光度法测定溴吡斯的明片中溴吡斯的明的含量

目的:建立测定溴吡斯的明片中溴吡斯的明含量的方法。方法:采用紫外分光光度法,以去离子水为空白,在269nm波长处测定溴吡斯的明的含量。结果:溴吡斯的明检测浓度在16.56～41.40μg·mL-(1r=0.9999)范围内与吸光度呈良好的线性关系;平均回收率为99.87%,RSD=0.19%(n=9)。结论:该法操作简便、精密度好、结果可靠,适用于溴吡斯的明片中溴吡斯的明含量的快速测定。     

肺靶向吡非尼酮脂质体的制备及体外释药性质研究

目的：研究肺靶向吡非尼酮脂质体的制备方法并考察其体外释药性质。方法：采用薄膜分散法制备吡非尼酮脂质体;用D-甘露糖修饰脂质体并添加适量十八胺调节脂质体表面电荷;用紫外分光光度法测定包封率;用正交实验优化处方,用透析法考察药物体外释放性质。结果：制得的脂质体平均粒径为581.1nm,表面电荷为-20.61mV,包封率为81.1%,稳定性好。药物体外释药符合Weibull方程。结论：采用薄膜分散法,用D-甘露糖修饰并添加十八胺可制得具有较高包封率及稳定性的吡非尼酮脂质体,有助于提高吡非尼酮的肺靶向性。     

溴吡斯的明磷脂复合物纳米粒的体外释放行为评价

目的:考察溴吡斯的明磷脂复合物纳米粒(PPNP)在不同释放介质中的体外释放行为。方法:采用动态透析技术研究PPNP在不同释放介质中的体外释放特性,紫外分光光度法测定药物释放度,并对其释放模型进行拟合,计算不同释放曲线间的相似因子。结果:PPNP与溴吡斯的明(PB)在0.1 mol.L-1盐酸溶液、pH6.8的PBS溶液、pH7.4的PBS溶液及0.1mol.L-1盐酸溶液(前2 h)和pH6.8的PBS溶液(2 h后)中的释药均符合Weibull模型。PPNP在各种释放介质中的释放曲线间的相似因子均大于50,但其与PB在各种释放介质中的释放曲线间的相似因子均小于50。结论:PPNP在不同释放介质中的释放行为相似,无显著性差别,PPNP在各种释放介质中的释放行为与PB相比存在显著性差别。PPNP改善了PB的溶出行为,具有一定的缓释效果。     

急进高原后肠道菌群介导的溴吡斯的明体内代谢研究

目的 探讨肠道菌群是否参与溴吡斯的明的代谢及急进高原后肠道菌群对其代谢产生的影响。方法 将大鼠随机分为平原给药组、平原空白组、高原给药组、高原空白组,共4组,每组6只大鼠。平原给药组大鼠禁食12 h后于上海（中国人民解放军海军军医大学）进行实验,分别将10.2 mg（约含溴吡斯的明2.3 mg）溴吡斯的明片剂粉末用水溶解后ig给药,于给药前（0 h）及给药后0.33、0.66、1.00、1.50、2.00、3.00、4.00、6.00、8.00、12.00、24.00 h由眼眶后静脉丛取血进行药动学分析。高原给药组大鼠于碌曲县海拔为4 300 m的高原地区,大鼠禁食12 h后开始药动学实验,过程同平原给药组。收集平原空白组和高原空白组大鼠的粪便,通过16S rRNA法分析急进高原后肠道菌群的变化情况;体外孵育实验结合LCMS/MS法分析肠道菌群与溴比斯的明的代谢关系。结果 急进高原后大鼠肠道菌群组成发生了变化,菌群种类和数量均减少;溴比斯的明与大鼠粪便体外孵育实验结果表明肠道微生物群参与溴吡斯的明吸收前的代谢,且在高原缺氧条件下,肠道微生物群对其代谢减慢;急进高原后溴吡斯的明药动学参数药时曲线下面积（AUC_0~t）增加了57.60%,清除率（CL）降低了35.94%。结论 肠道菌群参与溴吡斯的明吸收前代谢,可能与其生物利用度低有关;并且急进高原后肠道菌群介导的溴吡斯的明代谢减慢,从而增加其吸收。     

Liposomes containing cationic dimethyl dioctadecyl ammonium bromide: formulation, quality control, and lipofection efficiency

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.     

Liposomes Containing Cationic Dimethyl Dioctadecyl Ammonium Bromide: Formulation,Quality Control,and Lipofection Efficiency

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.     

Differential Adhesion of Normal and Inflamed Rat Colonic Mucosa by Charged Liposomes

PURPOSE: To study the adhesion properties of charged liposomes to the healthy and inflamed (colitis-induced) rat intestinal epithelium. METHODS: Neutral, positively charged, and negatively charged liposomes were prepared and tagged. The cationic or anionic liposomes contained increasing amounts (13, 22, or 36 mol%) of either the cationic lipid dimethyl-dioctadecylammoniumbromide (DODAB) or the anionic lipid 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG). Colitis was induced in rats by DNBS. Adhesion of the various types of liposomes was assessed in rat colon sacs. The effects of charge type, charge density (mol%), liposome size, and incubation time on the adhesion of the liposomes were compared in the inflamed and healthy epithelial tissues. RESULTS: Three times as many cationic liposomes adhered to the healthy colonic mucosa than neutral or anionic liposomes. However, anionic liposome adherence to the inflamed colonic mucosa was 2-fold that of either neutral or cationic liposomes (a finding that was verified by charged-dyes studies). Adherence was directly correlated with charge density. An inverse correlation was identified between cationic liposome size and healthy tissue adherence in short incubation periods. The adherence of cationic liposomes, which was also found to be time-dependent, decreased in healthy mucosa in the presence of high concentrations of aqueous Mg2+ rinse. CONCLUSIONS: Anionic liposomes could be useful for the topical delivery of anti-inflammatory drugs in inflammatory bowel disease therapy.     

阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响

目的制备包封荧光素钠（FS）的脂质体，考察阳离子脂质材料（DC-chol）和聚乙二醇（PEG）对脂质体包封率、细胞转染率及膜流动性的影响。方法以FS作为模型物质，制备并分离脂质体，测定脂质体包封率；通过观察荧光光谱的变化考察FS与脂质体膜之间的相互作用；以HepG₂ 2.2.15为细胞模型观察脂质体对FS细胞转染率的影响；通过荧光偏振技术考察阳离子脂质材料和PEG对脂质体膜流动性的影响。结果阳离子脂质材料和PEG能提高脂质体包封率（0.64％～86.57％）、细胞转染率（2.18％～48.46％）及脂质体膜流动性，PEG分子质量的增大有利于包封率、转染率的提高，并增加脂质体膜的流动性。结论在脂质体处方中加入阳离子脂质材料和高分子量的PEG有利于提高包封率、细胞转染率及增加脂质体膜的流动性。     

Effects of lipid composition and preparation conditions on physical-chemical properties, technological parameters and in vitro biological activity of gemcitabine-loaded liposomes

The effects of lipid composition and preparation conditions on the physicochemical and technological properties of gemcitabine-loaded liposomes, as well as the in vitro anti-tumoral activity of various liposome formulations were investigated. Three liposome formulations were investigated: DPPC/Chol/Oleic acid (8:3:1 molar ratio, liposomes A), DPPC/Chol/DPPS (6:3:1 molar ratio, liposomes B) and DPPC/Chol/DSPE-MPEG (6:3:1 molar ratio, liposomes C). Multilamellar liposomes were prepared by using the TLE, FAT and DRV methods, while small unilamellar liposomes were obtained by extrusion through polycarbonate filters. Light scattering techniques were used to characterize liposome formulations. Loading capacity and release profiles of gemcitabine from various liposome formulations were also investigated. Caco-2 cells were used to evaluate in vitro the antitumoral activity of gemcitabine-loaded liposomes with respect to the free drug and also the intracellular drug uptake. Preparation methods and liposome lipid composition influenced both physicochemical parameters and drug delivery features. Liposomes with a size ranging from 200 nm to 7 microm were obtained. The gemcitabine entrapment was higher than that expected probably due to an interaction with the liposome lipid components. The following decreasing loading capacity order was observed: liposome B>liposome C>liposome A. Gemcitabine release from various liposome formulations is modulated by two different processes, i.e. desorption from and permeation through liposomal bilayers. MTT assay showed a greater cytotoxic effect of gemcitabine-loaded liposomes with respect to the free drug. The following decreasing anticancer activity order was observed between the various liposome formulations: liposome C>liposome A>liposome B. The increased anticancer activity is correlated to the ability of the colloidal carrier to increase the intracellular drug uptake. Due to the encouraging results and to the high liposome modularity various applications of potential therapeutic relevance can be envisaged for liposomes.     

多西他赛他莫昔芬脂质体体外释放研究

目的考察多西他赛他莫昔芬复方脂质体体外释放。方法薄膜分散法制备单方和复方脂质体样品,通过透析法进行脂质体体外释放,采用高效液相色谱法测定他莫昔芬和多西他赛药物浓度,并对体外释放曲线进行数学模型拟合。结果单方和复方脂质体中,他莫昔芬和多西他赛均无突释,多西他赛较他莫昔芬释放快,他莫昔芬和多西他赛释放模型符合一级释放方程,他莫昔芬和多西他赛的释放曲线相似。结论他莫昔芬和多西他赛在单方和复方脂质体体外释放并无显著差异。     

Characterization of endotoxin and cationic liposome interaction

The objective of this study was to characterize the interaction of endotoxin with cationic liposomes used in nonviral gene delivery. Endotoxin-cationic liposome interaction was characterized using fluorescent anisotropy, and the Limulus amebocyte lysate (LAL) assay. Cellular toxicity of endotoxin-cationic liposome complex was examined using a dimethylthiazol diphenyltetrazolium bromide (MTT) assay. The effect of endotoxin on the lipid-DNA complex and subsequent transfection into COS-1 cells was also examined. A competitive interaction occurred between fluoroscein isothiocyanate (FITC)-labeled endotoxin and plasmid DNA for binding dioleoyl glycero trimethylammonium propane:dioleoyl glycero phosphoethanolamine (DOTAP:DOPE) liposomes using fluorescent anisotropy techniques. The LAL assay demonstrated no change in endotoxin activity upon interaction with liposomes. No loss of COS cell viability was detected via the MTT assay during a 5-hr exposure to endotoxin. Transient transfection studies indicate that increasing levels of endotoxin lowered activity more than 90% at 50,000 endotoxin units (EU)/ml. Endotoxin and cationic liposomes interact mainly by an electrostatic attraction. Endotoxin contamination can potentially impact transfection efficiency via competition with plasmid DNA for cationic liposome binding by increasing transfection variability at 50 EU/ml, a concentration of endotoxin contamination that can occur with small-scale plasmid preparations used for in vitro cell transfections, but would not be expected with typical GLP or GMP preparations used in clinical studies.     

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p<0.05), DOX uptake of these liposomes at 24h post-injection was similar to that of PEGylated liposomal DOX (p>0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p<0.01), neutral liposome control (p<0.01), as well as PEGylated liposomes (p<0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.