药用豆磷脂的HPLC法测定

本文报告了用HPLC法对药用豆磷脂进行定性定量分析。作者选用硅胶为固定相,以正己烷-异芮醇-醋酸盐缓冲液(pH5.8)(7.5:8:1)为流动相,紫外检测波长206nm,使得磷脂酰胆碱(PC),磷脂酰乙醇胺(PE),溶血磷脂酰胆碱(LPC),磷脂酰肌醇(PI)和磷脂酸(PA)获得满意分离,并对主成分PC进行了含量测定,平均回收率为99.8%,方法的变异系数为0.8%.     

丙泊酚中/长链脂肪乳注射液中溶血磷脂酰胆碱和溶血磷脂酰乙醇胺的含量测定

目的建立HPLC法测定丙泊酚中/长链脂肪乳注射液中溶血磷脂酰胆碱(LPC)和溶血磷脂酰乙醇胺(LPE)的含量。方法采用HPLC法,色谱柱:Lichrospher 100 DIOL(250 mm×4 mm,5μm);流动相:乙腈-水-冰醋酸-三乙胺(85∶15∶0.45∶0.05)为流动相A,正己烷-异丙醇(18∶30)溶液为流动相B;梯度洗脱;柱温:35℃。结果溶血磷脂酰胆碱和溶血磷脂酰乙醇胺的线性范围分别为0.03~0.25(r1=0.9997)和0.01~0.10(r2=0.9962)mg·mL^-1,定量限分别为0.72和0.31μg。结论该方法准确、可行,精密度高,可用于溶血磷脂酰胆碱和溶血磷脂酰乙醇胺的含量测定。     

欧芹素乙对人脐静脉内皮细胞的保护作用及对血管内皮生长因子表达的影响

目的:研究欧芹素乙对内皮细胞的保护作用;溶血磷脂酰胆碱(LPC)对人脐静脉内皮细胞株HUVEC细胞中血管内皮生长因子(VEGF)表达的影响以及欧芹素乙的影响.方法:应用四唑盐(MTT)法检测溶血磷脂酰胆碱对HUVEC细胞的毒性作用及欧芹素乙的保护作用;应用基础酶联免疫吸附试验(ELISA)检测各组条件培养基中VEGF蛋白含量;采用RT-PCR法及Reahime PCR方法检测溶血磷脂酰胆碱对VEGF mRNA的表达及欧芹素乙的影响.结果:MTT检测结果显示,溶血磷脂酰胆碱对HUVEC细胞具有较强的生长抑制作用,而欧芹素乙对溶血磷脂酰胆碱所致的细胞增殖抑制具有较好的保护作用.ELISA结果显示,HUVEC细胞暴露于溶血磷脂酰胆碱后,VEGF蛋白含量明显升高;加入欧芹素乙后剂量依赖性地降低VEGF蛋白的表达.RT-PCR结果显示,溶血磷脂酰胆碱可以增加3种VEGF异构体的转录水平,其中VEGF165的表达显著增加,欧芹素乙可剂量依赖性地抑制溶血磷脂酰胆碱引起的VEGF mRNA的高表达.结论:欧芹素乙对溶血磷脂酰胆碱引起的细胞损伤有明显的保护作用;欧芹素乙可抑制溶血磷脂酰胆碱所诱导的HUVEC细胞中VEGF蛋白及VEGF mRNA的高表达,对内皮细胞起到保护作用.     

羟基喜树碱脂质体制备工艺的研究

目的 确定羟基喜树碱(HCPT)脂质体(HCPT-lipo)的处方和制备工艺.方法 筛选乙醇、丙酮、甲醇、乙腈等有机溶剂,进行色谱条件专属性试验并在色谱条件下进样分析;测定HCPT-lipo中药物包封率(ER),以ER(％)=WE/Wz×100％求算HCPT-lipo的包封率.对比干膜法、pH梯度/真空-注入法、逆相蒸发法、高压乳匀法、乳化蒸发法5种不同方法制得脂质体的包封率,确定制备HCPT-lipo的技术.结果 每1mL脂质体溶液(含HCPT0.5 mg)加入3mL甲醇超声能够得到完全澄清透明的溶液.在与样品峰相应的位置上,空白脂质体对HCPT-lipo样品中HCPT测定无干扰;HCPT原料在0.824～692 ng具有良好的线性关系.乳化蒸发法可制得包封率高,稳定性好的脂质体.药脂比宜＞12.5∶1.结论 制得的脂质体在粒径和包封率等关键指标上都比较满意,基本达到了试验设计的目的.     

5-氟尿嘧啶核苷前药脂质体中磷脂含量的测定

目的:建立测定5-氟尿嘧啶核苷(5-FUR)前药脂质体中磷脂含量的方法。方法:以乙醇裂解5-FUR前药脂质体,用氯仿萃取后,在488nm波长处用硫氰铁铵显色法测定脂质体中磷脂的含量。结果:磷脂检测浓度的线性范围为0.01~0.10mg·mL-1(r=0.9996,n=3);平均回收率为99.66%(RSD=3.17%)。结论:本方法简便、快速、灵敏,可用于5-FUR前药脂质体的质量控制。     

海参皂苷nobiliside A脂质体及其溶血行为的初步研究

本文旨在制备海参皂苷nobiliside A(Nob A)脂质体，建立脂质体中Nob A的含量和包封率的测定方法，考察该脂质体的体内外溶血行为。以高效液相色谱-蒸发光散射检测法(HPLC-ELSD)测定脂质体中Nob A含量，微柱离心法测定其包封率。以溶血率和包封率、粒径为指标，单因素考察了制备方法、磷脂用量、胆固醇用量和药脂比。根据单因素的考察结果，以薄膜超声法制备3批脂质体，与同浓度的药物溶液进行体内外溶血行为的比较。结果显示：用于含量测定和包封率测定的HPLC-ELSD和微柱离心法准确、快速，采用薄膜超声法制备的脂质体形态圆整，粒径较为均匀。当磷脂与胆固醇比例为2∶1，药脂比为1∶40时，Nob A脂质体的平均包封率为95.7%，平均粒径为87.6 nm。Nob A以脂质体作为给药载体，其体内外溶血行为大大降低，有望成为静脉注射用脂质体。     

正交试验优化硫酸长春新碱脂质体的制备工艺

目的制备性质稳定的硫酸长春新碱脂质体。方法采用单因素试验考察磷脂与胆固醇比例、药脂比、脂质浓度、载药温度、载药时间、外水相p H值对硫酸长春新碱脂质体包封率的影响。以包封率为指标,分别以氢化磷脂(SPC-3)和二硬脂酰磷脂酰胆碱(DSPC)为磷脂材料,通过正交试验考察载药温度、药脂比和载药时间对制备工艺的影响,优化出硫酸长春新碱脂质体的最佳制备工艺。结果硫酸长春新碱脂质体的最佳制备工艺为:将药物溶液(按照药物含量计)和空白脂质体溶液(按照脂质含量计)按照1∶20的比例混合,用Na2HPO4直接调节外水相p H值至7.2。SPC-3脂质体在65℃条件下载药,载药时间30 min。DSPC脂质体在60℃条件下载药,载药时间10 min。结论优选出的硫酸长春新碱脂质体的处方工艺稳定可行。     

HPLC-ELSD法同时测定氟比洛芬酯注射液中溶血磷脂酰胆碱和溶血磷脂酰乙醇胺的含量

摘要：目的:建立同时测定氟比洛芬酯注射液中溶血磷脂酰胆碱（LPC）和溶血磷脂酰乙醇胺（LPE）含量的方法。方法:采用HPLC法,蒸发光散射检测器,色谱柱为Zorbax RX-SIL（250 mm×4.6 mm,5μm）柱,以流动相A:甲醇-水-冰醋酸-三乙胺（85∶15∶0.45∶0.05）,流动相B:正己烷-异丙醇-流动相A（20∶48∶22）梯度洗脱,流速为1.0 ml·min-1,柱温为40℃,漂移管温度为75℃,雾化气流速为1.5 L·min-1,进样量为20μl。结果:LPC和LPE分别在82.72～517.03μg·ml-1（r=0.999 9）、19.19～119.93μg·ml-1（r=0.999 0）范围内,浓度的对数值与其峰面积的对数值呈现良好线性关系; LPC和LPE的回收率分别为95.44%（RSD=1.10%,n=9）,97.54%（RSD=1.52%,n=9）。36批制剂中均检出LPC和LPE,并且溶血磷脂含量随时间和温度增加而增加。结论:建议贮藏条件为25℃以下密闭储存,不可冷冻,有效期为12个月。该方法操作简便,可用于氟比洛芬酯注射液中有害杂质溶血磷脂的含量测定。     

新型阿霉素热敏脂质体的研制

目的:探讨以二棕榈酰磷脂酰胆碱(DPPC)和单棕榈酰磷脂酰胆碱(MPPC)为原料制备的温度敏感阿霉素脂质体的制备工艺和理化性质.方法:采用膜过滤挤压法和pH梯度法制备阿霉素脂质体,以紫外分光光度法检测样本中阿霉素含量,计算阿霉素热敏脂质体在不同温度下的药物释放特性,并对其包裹率、粒径,pH值进行研究.结果:阿霉素热敏脂质体在39℃下迅速释放,前20 s内释放药物50%,42℃下药物释放达到60%以上.包封率99.5%,平均粒径90.8 nm,pH值为6.5.结论:制备的热敏感阿霉素脂质体优于常规脂质体,具有良好的温度控释特性.     

HPLC-ELSD测定尼莫地平脂肪乳注射液中溶血磷脂酰胆碱的含量

目的：建立HPLC-ELSD测定尼莫地平脂肪乳注射液中溶血磷脂酰胆碱（LPC）的含量.方法：采用Kromasil 100-5SIL（250 mm×215;4.6 mm,5 μm）色谱柱,以甲醇-冰醋酸（500：10,三乙胺调节pH至6.0） 为流动相,流速为1.0 ml·min^-1,柱温为30℃,使用蒸发光散射检测器（雾化气：氮气,漂移管温度：60℃）.结果：样品中磷脂酰胆碱（PC）及LPC与相邻杂质峰分离效果良好;LPC定量限为218.2 ng,检测限为65.46 ng;平均回收率为100.9%,RSD为3.3%（n=9）.结论：本法简便、专属性强、准确可靠,可用于尼莫地平脂肪乳注射液中溶血磷脂酰胆碱的含量测定.     

Modified hydrolysis kinetics of the active lactone moiety of 10-hydroxycamptothecin by liposomal encapsulation

The key structural requirement for the antitumor activity of 10-hydroxycamptothecin (HCPT) is the intact lactone moiety which is always instability and suffered from pH-dependent hydrolysis. The aim of this study was to evaluate the protection effects of liposomal encapsulation on the labile lactone ring. Mono-modal dispersed quasi-spherical liposomes with mean diameter of 145?nm and high drug entrapment efficiency of 90% were obtained under optimal conditions. The in vitro hydrolysis kinetics behaviors of lactone were studied in varied pH buffers. Compared to that of free HCPT in solution formulation, both the hydrolysis half-life and observed equilibrium constant of liposomal HCPT were increased significantly along with the decreased apparent hydrolysis rate constant. The plasma pharmacokinetics was studied by assessing the lactone stability versus time profiles in vivo following intravenous administration of free and liposomal HCPT. The liposomal encapsulation led to a twofold increase in the AUC values and significant decrease in the plasma clearance of lactone (P?<?0.05). There was a good correlation between in vitro and in vivo stability of HCPT-lactone. These results suggested a potential application of the novel liposome formulation for the stable delivery system of HCPT.     

Camptothecin-catalyzed phospholipid hydrolysis in liposomes

Hydrolysis of phospholipid (PL) within camptothecin (CPT)-containing liposomes was studied systematically, after elevated lyso-phosphatidylcholine (LPC)-concentrations in pH 5, CPT-containing liposomes (22.1+/-0.9 mol%) relative to control-liposomes (7.3+/-0.5 mol%) occasionally had been observed after four months storage in fridge. Liposomes were prepared by dispersing freeze-dried PL/CPT mixtures in 25 mM phosphate buffered saline (PBS) of varying pH (5.0-7.8) and CPT concentrations (0, 3 and 6 mM). PL-hydrolysis was monitored by HPTLC, quantifying LPC. In an accelerated stability study (60 degrees C), a catalytic effect of CPT on PL-hydrolysis was observed after 40 h, but not up to 30 h of incubation. The pH profile of the hydrolysis indicated a stability optimum at pH 6.0 for the liposomes independent of CPT. The equilibrium point between the more active lactone- and the carboxylate-form of CPT was found to be pH 6.8. As a compromise, pH 6.0 was chosen, assuring >85% CPT to be present in the lactone form. At this pH, both control- and CPT-liposomes showed only minor hydrolysis after autoclaving (121 degrees C, 15 min). Storage at room temperature and in fridge (2 months), as well as accelerated ageing (70 degrees C, 25 h), gave a significant elevation of LPC content in CPT-liposomes relative to control-liposomes. This study demonstrates a catalytic effect of CPT on PL-hydrolysis, the onset of which seems to require a certain threshold level of hydrolytic degradation.     

PCM and TAT co-modified liposome with improved myocardium delivery: in vitro and in vivo evaluations

In this study, PCM and TAT co-modified liposome was developed as a novel drug carrier for myocardium delivery with evaluation of its in vitro and in vivo properties. Liposomes containing fluorescent probe coumarin-6 were prepared by thin-film hydration. The PCM ligands specifically bind to the PCM receptors in the extracellular connective tissue of primary myocardium cells (MCs), while the TAT ligands functioned as a classical cell penetrating peptide to make liposomes internalized by MCs. The unmodified liposome (L), PCM-modified liposome (PL), TAT-modified liposome (TL) and PCM and TAT co-modified liposome (PTL) were prepared and characterized. The cellular uptake and intracellular distribution of various liposomes by MCs demonstrated that PTL had the best delivery capability. Peptide inhibition assay indicated that the uptake of PL could be inhibited by PCM. However, TAT could almost not suppress the uptake of TL. In addition, the CCK-8 experiments showed that liposomes had low cytotoxicity. In vivo fluorescent images of frozen sections and HPLC-fluorescence analysis further demonstrated that PTL had highest myocardium distribution. The results of this study demonstrated that PCM and TAT co-modifying could improve the myocardial targeting ability of liposome.     

羟基喜树碱脂质体的制备及质量评价研究

目的:制备羟基喜树碱(HCPT)脂质体,并对其质量进行评价.方法:采用薄膜分散-高压乳匀法制备羟基喜树碱脂质体;用激光粒度分析仪测定其Zeta电位、粒径大小;考察其在0.9%NaCl溶液、水、5%葡萄糖溶液中8 h的稳定性;用凝胶柱层析法考察包封率;采用薄膜透析法考察体外释药性质.结果:羟基喜树碱脂质体Zeta电位为(-33.1±1.3) mV,平均粒径(182.5±5.6) nm,8 h内在水、5%葡萄糖溶液中稳定性良好;包封率(91.2±1.2)%;体外释药曲线符合Higuchi方程Q=1.291 6t1/2 0.309 8,r=0.980 3.结论:本试验制备的羟基喜树碱脂质体稳定性好,大小均匀, 包封率高,并具有延缓药物释放的性质.     

Hydroxycamptothecin liposomes based on thermal and magnetic dual-responsive system: preparation,in vitro and in vivo antitumor activity,microdialysis-based tumor pharmacokinetics

Due to the absence of lactone form of hydroxycamptothecin, the commercially available hydroxycamptothecin injection exhibits inefficient therapeutic effects. In this study, we constructed a novel delivery system (thermosensitive magnetic liposomes) that protects lactone form of hydroxycamptothecin from blood or water. After hydroxycamptothecin was loaded into the thermosensitive magnetic liposome (HCPT/TML), its in vitro and in vivo antitumor activity and microdialysis-based tumour pharmacokinetics were determined. The results demonstrated that HCPT/TMLs possessed favourable physicochemical features and significant cytotoxicity against the Huh-7 cells in vitro. In the in vivo antitumor study and tumour pharmacokinetics, HCPT/TMLs displayed effective targeting delivery and antitumor effects, which corresponded to the determined hydroxycamptothecin concentration in tumour tissue. In conclusion, this thermal and magnetic dual-responsive system can efficiently deliver hydroxycamptothecin to tumour tissue and has great potential application in cancer treatment.     

RGD环肽介导的靶向脂质体体内药动学及荷瘤动物活体成像研究

本文测定了大鼠单剂量(5 mg.kg1)尾静脉注射RGD环肽介导的羟基喜树碱(hydroxycamptothecin,HCPT)靶向脂质体(HCPT-RGD-LP)和HCPT长循环脂质体(HCPT-LP)的血药浓度,比较两组的药动学行为;研究了HCPT-LP及HCPT注射剂在正常小鼠血浆和心、肝、脾、肺、肾中的分布情况;采用人肝癌HepG2细胞移植裸鼠,以DiR为荧光探针,通过活体成像比较RGD环肽修饰的DiR靶向脂质体(DiR-RGD-LP)和DiR长循环脂质体(DiR-LP)在荷瘤裸鼠体内的分布。结果显示,HCPT-RGD-LP组和HCPT-LP组的主要药动学参数t1/2β、CL、Vc、AUC048 h、AUC0∞、MRT048 h和MRT0∞均无显著性差异(P>0.05);HCPT-LP在小鼠体内的循环时间明显长于HCPT注射剂,且药物在肝脏中的分布浓度较高;荷瘤裸鼠中,DiR-RGD-LP组肿瘤部位的荧光强度显著高于DiR-LP组,提示RGD环肽用于脂质体修饰能明显提高给药系统的肿瘤靶向性。     

Detection of bacterial endotoxin in paclitaxel liposome

Based on current research, there are three technologies during the test of bacterial endotoxin of liposomes: (1) extraction of bacterial endotoxin from liposomes; (2) addition of bacterial endotoxin in the process of recovery test; and (3) elimination of the interference factors from drugs and excipients. In the present study, we pointed out that the key technologies to test bacterial endotoxin from paclitaxel liposome included following steps: extraction of bacterial endotoxins from ethanol-dissolved liposomes; preparation of positive control of recovery solution by adding 0.01 mL standard endotoxins in 1 mL liposome ethanol solution; and the use of 0.5% human albumin to eliminate the interference from detection, and accurate detection of the bacterial endotoxin of liposomes.     

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.     

Development of vitamin loaded topical liposomal formulation using factorial design approach: drug deposition and stability

Long-term exposure of the skin to UV light causes degenerative effects, which can be minimized by using antioxidant formulations. The major challenge in this regard is that a significant amount of antioxidant should reach at the site for effective photoprotection. However, barrier properties of the skin limit their use. In the present study, Vitamin E acetate was encapsulated into liposome for improving its topical delivery. However preparation of liposomes is very difficult due to number of formulation variables involved therein. In the present work systematic statistical study for the formulation of liposomes for topical delivery of Vitamin E using the factorial design approach was undertaken. Amount of phospholipid (PL) and cholesterol (CH) were taken at three different levels and liposomes were prepared using ethanol injection method. Liposomes were characterized for encapsulation efficiency, vesicle size, zeta potential, and drug deposition in the rat skin. Gels containing liposomal dispersion (batch with higher skin deposition of VE) were prepared in Carbopol^® 980 NF and were characterized for gel strength, viscosity and drug deposition in the rat skin. Stability of liposome dispersion and gel formulation was studied at 30 °C/65% RH for 3 months. Results of regression analysis revealed that vesicle size and drug deposition in the rat skin were dependant on the lipid concentration and lipid:drug ratio. Drug deposition in rat skin had an inverse relationship with respect to PL and CH concentration. Prepared liposomal dispersion (50 mg PL:6 mg CH) showed seven-fold increase in drug deposition compared to control (plain drug dispersion). Gel formulation demonstrated six-fold and four-fold increase in drug deposition compared to control gel and marketed cream, respectively. Liposome dispersion and gel formulation were found to be stable for 3 months. Factorial design was found to be well suited to identify the key variables affecting drug deposition. Improved drug deposition from liposomal preparations demonstrates its potential for dermal delivery.