全反式维甲酸前体脂质体的制备及磷脂过氧化值的测定

目的：制备全反式维甲酸（all-trans retinoic acid,ATRA）前体脂质体,并测定前体脂质体中磷脂的过氧化值（POV）。方法：采用山梨醇为固化载体,改良旋转蒸发-载体吸附法制备ATRA前体脂质体。水化后,采用微柱离心法测定全反式维甲酸脂质体的包封率。以丙二醛（MDA）法测定磷脂的过氧化值来表示在制备全反式维甲酸前体脂质体过程中磷脂的过氧化程度。结果：ATRA前体脂质体的包封率为84.98%。制备前后磷脂的过氧化值分别为0.080和0.089。结论：该方法操作简单易行,适合实验室范围制备前体脂质体。且在制备前后,磷脂的过氧化值差为0.009,说明磷脂在制备过程中比较稳定。     

全反式维甲酸增强依维莫司对小细胞肺癌的体内外抗肿瘤作用

目的 探讨雷帕霉素靶蛋白(mTOR)抑制剂依维莫司与全反式维甲酸(ATRA)联用对小细胞肺癌细胞株NCI-H446增殖及迁移能力的影响.方法 将不同浓度ATRA分别和依维莫司联合作用于NCI-H446细胞,采用MTT试验检测药物作用24h、48h、72h后的细胞存活率;细胞划痕实验观测药物处理24h后的细胞迁移能力.不同药物浓度作用于荷瘤小鼠,通过裸鼠移植瘤模型观察全反式维甲酸与依维莫司的体内协同抑瘤作用.结果 MTT实验表明ATRA联合依维莫司抑制NCI-H446细胞增殖呈时间及浓度依赖性,高浓度联合用药组72h细胞存活率最低;划痕实验结果表明联合用药组抑制细胞迁移作用显著,ARTA药物浓度越高迁移抑制作用越强;裸鼠皮下抑制瘤模型中各组在给药3周后发现联合用药组抑瘤作用高于依维莫司组和对照组.结论 依维莫司与ATRA联合应用在抑制人小细胞肺癌细胞增殖、迁移方面具有协同作用,在小细胞肺癌治疗可能具有良好的临床应用前景.     

高压液相色谱法检测全反式维甲酸微囊在体内的缓慢释放

目的 将全反式维甲酸与全反式维甲酸微囊在体内的释放过程进行比较.方法 应用高压液相色谱(HPLC)技术,测定了全反式维甲酸(ATRA)的标准曲线.结果 5只家兔在1次口服20mg的裸氏全反式维甲酸后,药物在血浆中达到高峰浓度时间(Tpeak)约为1 h,峰浓度(Cpmax)为0.081 μg/mL,血浆清除半衰期(t...     

米托蒽醌长循环脂质体的制备及药代动力学的研究

目的研究米托蒽醌长循环脂质体的制备方法并考察其在家兔体内的驻留行为。方法用乙醇注入合并硫酸铵梯度法制备米托蒽醌长循环脂质体。用两亲性聚乙二醇2000-二硬脂酰磷脂乙醇胺(PEG-DSPE)修饰脂质体膜。以RP-HLPC柱切换方法测定家兔血浆中米托蒽醌药物浓度。结果长循环脂质体平均粒径为60 nm,药物包封率为93.6%。以相同剂量(2 mg·kg^-1)经iv分别给予家兔长循环脂质体和普通脂质体,前者平均驻留时间(MRT)为9.8 h,后者为3.6 h,长循环脂质体药时曲线下面积(AUC)提高了6.4倍。证明米托蒽醌长循环脂质体延长了在血液循环中的时间。结论用乙醇注入合并硫酸铵梯度法可制得包封率高、粒径小的脂质体,以PEG修饰磷脂膜可以增加长循环脂质体的AUC,并延长其在血液循环中的时间。     

替加氟磁性长循环脂质体在大鼠体内的药代动力学和靶向性

目的研究替加氟磁性长循环脂质体经肝动脉给药的药代动力学和组织靶向性。方法采用高效液相色谱仪检测大鼠生物样品中替加氟的浓度。结果磁控并加热的替加氟磁性长循环脂质体组的肝内8h药时曲线下面积(AUC)是游离替加氟组的17.4倍,是替加氟长循环脂质体组的3.9倍;其肝外组织血浆和肾的AUC比游离替加氟组低。其肝靶向效率达到73.9%。结论替加氟磁性长循环脂质体经肝动脉给药能显著增加药物的肝脏靶向性,可能降低其肾毒性。     

全反式维甲酸联合索拉菲尼对人SMMC-7721肝癌细胞株的作用

目的观察全反式维甲酸联合索拉菲尼对SMMC-7721细胞增殖及凋亡的影响。方法选用ATRA(10-5mol/L)及不同浓度的索拉菲尼(6μmol/L、9μmol/L、12μmol/L),并选用10-5mol/L的ATRA分别联合索拉菲尼(6μmol/L、9μmol/L、12μmol/L)作用于SMMC-7721肝癌细胞24h、48h、72h;期间药物对SMMC-7721肝癌细胞生长的抑制阻滞作用通过MTT比色法进行观测,同时细胞生长过程中利用倒置显微镜观察形态变化,其后药物作用48h时SMMC-7721细胞的周期分布和凋亡的情况利用流式细胞术分析。结果不同浓度的索拉菲尼单药、全反式维甲酸单药及联合用药均对SMMC-7721肝癌细胞的生长明显抑制并改变细胞的形态,促进肝癌细胞的凋亡比例增加。其凋亡作用有随剂量-时间改变的依赖性。两药联合应用相比单用全反式维甲酸或索拉菲尼其细胞凋亡作用更为显著。12μmol/L索拉菲尼、9μmol/L索拉菲尼分别与ATRA联合作用于细胞48h及72h相比较,其抑制作用无明显差异。两药联合作用时可见细胞周期阻滞在S期的肝癌细胞较单药应用时显著增多(P<0.01),凋亡率增加。结论 ATRA、索拉菲尼均有阻滞肝癌SMMC-7721细胞增殖及促凋亡的作用,联合索拉菲尼作用增强并具协同作用。     

熊果酸磷脂纳米注射剂在小鼠体内的分布研究

目的:考察熊果酸磷脂纳米(UA-PL-NP)注射剂在小鼠体内的组织分布。方法:取昆明小鼠尾静脉注射UA-PL-NP后,分别于给药后5min、1h和4h收集血浆和各组织,采用反相高效液相(RP-HPLC)法检测各组织中熊果酸(UA)药物浓度。结果:(1)UA-PL-NP给药后UA主要分布于肝和胃肠组织(。2)主要分布组织的药物浓度呈现随给药剂量增加而线性增加的趋势(。3)胃、肠组织药物浓度于停药后迅速下降,至停药4h后浓度下降至极低水平;而肝组织药物浓度于停药后不断升高,停药后4h仍维持较高浓度([52.15±18.61)mg·kg-1]。结论:UA-PL-NP给药初期迅速分布于肝、胃、肠等组织,停药后胃肠组织药物释放入血后经再分布达平衡,使UA较长时间高度浓聚于肝组织,具有肝靶向分布特征。     

拓扑替康脂质体的制备及体内分布研究

目的：制备由不同磷脂组成的盐酸拓扑替康脂质体,并比较其在荷瘤小鼠体内的分布行为.方法：采用非饱和的大豆卵磷脂（SPC）与饱和的氢化大豆卵磷脂（HSPC）作为包封材料,利用硫酸铵梯度法分别制备盐酸拓扑替康脂质体SPC-L与HSPC-L,静脉注射后测定荷瘤小鼠血浆及各组织中的药物浓度,研究其体内分布过程.结果：与游离药物相比,脂质体包封后拓扑替康的血浆浓度明显增加,SPC-L与HSPC-L组的血浆AUC（0～∞）分别是游离药物组的7.0倍和18.8倍,同时其内酯/羧酸盐相对比例增加.肝、脾、肺以及肿瘤组织中药物浓度也明显增加,按每1.0 g组织中药物的量计算,HSPC-L组脾中的药物分布增加最多,是游离药物组的137.5倍;与SPC-L组比较,HSPC-L组肿瘤中的药物分布增加较多,相对于游离药物组的肿瘤摄取率为4.1.结论：脂质体包封改变了拓扑替康在体内的分布行为,与SPC-L比较,HSPC-L更为有效地提高了拓扑替康的体内稳定性,增加了其在血浆及各组织中的分布.     

不同磷脂组成莪术油包合物脂质体的抗肿瘤作用比较

目的:考察不同磷脂组成莪术油包合物脂质体的抗肿瘤药效。方法:采用乙醇注入法制备了莪术油包合物脂质体。KM小鼠接种H22瘤株造成移植性荷瘤小鼠模型,比较莪术油不同制剂[包括莪术油溶液,莪术油包合物(ZTO-HPCD)、莪术油包合物SPC脂质体(ZTO-HPCD-SL)及莪术油包合物复合磷脂脂质体(ZTO-HPCD-SHL)]给药后荷瘤小鼠的抑瘤效果,比较给药后各组荷瘤小鼠的体质量和免疫器官(脾和胸腺)指数。并且还考察了对腹水癌模型小鼠生命延长率的影响。结果:磷脂组成对莪术油包合物脂质体的抗肿瘤效果具有显著影响。相同剂量下的莪术油溶液、ZTO-HPCD、ZTO-HPCD-SL、ZTO-HPCD-SHL的抑瘤率分别为17.90%、17.49%、39.77%、59.42%,与莪术油溶液相比,莪术油包合物脂质体制剂给药后未发现对免疫器官存在明显的抑制作用,具有一定的生命延长作用。结论:采用包合物脂质体与复合磷脂脂质体技术可以显著提高莪术油的抗肝癌效果。     

表达谱基因芯片筛选肝癌细胞中ATRA下游基因的应用研究

目的：全反式维甲酸( All Trans-Retinoic acid, ATRA)处理肝癌细胞系HepG2细胞后进行表达谱基因芯片技术分析,探索其对肝癌细胞基因表达谱的影响。方法80μmol/L的全反式维甲酸处理HepG2细胞24 h后,提取细胞的mRNA,应用基因表达谱芯片技术对差异表达的mRNA进行检测和分析,以等体积无水乙醇处理作为对照组。结果全反式维甲酸处理HepG2细胞后,共筛选出661个差异表达基因,这些差异基因与肿瘤细胞的信号转导、增殖分化等都有密切的关系。结论成功的应用表达谱芯片技术筛选出ATRA处理细胞后的差异表达基因,为进一步探讨ATRA对肝癌细胞的作用机制奠定了基础,也为临床应用ATRA诱导肝癌分化提供理论依据。     

Preparation and ocular pharmacokinetics of ganciclovir liposomes

Ophthalmic liposomes of ganciclovir (GCV) were prepared by the reverse phase evaporation method, and their ocular pharmacokinetics in albino rabbits were compared with those obtained after dosing with GCV solution. The in vitro transcorneal permeability of GCV liposomes was found to be 3.9-fold higher than that of the solution. After in vivo instillation in albino rabbits, no difference was found in the precorneal elimination rate of GCV from liposome vs solution dosing. The aqueous humor concentration-time profiles of both liposomes and solution were well described by 2-compartmental pharmacokinetics with first-order absorption. The area under the curve of the aqueous humor concentration-time profiles of GCV liposomes was found to be 1.7-fold higher than that of GCV solution. Ocular tissue distribution of GCV from liposomes was 2 to 10 times higher in the sclera, cornea, iris, lens, and vitreous humor when compared with those observed after solution dosing. These results suggested that liposomes may hold some promise in ocular GCV delivery.     

黄芩苷脂质体的制备及在小鼠体内的分布

目的制备黄芩苷脂质体并考察其在小鼠体内的分布 ,为黄芩苷脂质体治疗乙型肝炎提供依据。方法用逆相蒸发法制备黄芩苷脂质体 ,测定其粒径及包封率 ,观察静脉注射黄芩苷脂质体及黄芩苷水溶液后 ,不同时间在小鼠体内的分布。结果 5批黄芩苷脂质体平均粒径为 (36 0± 4 2 )nm ,平均包封率为 (5 6 .0± 5 .3) %。黄芩苷脂质体主要分布于肝、脾 ,以肝脏中分布最多 ,而在血中清除加快。结论逆相蒸发法制备黄芩苷脂质体条件易控制 ,重复性好 ,可获得较高包封率和符合肝靶向要求的脂质体 ;黄芩苷脂质体具有一定的肝靶向性 ,可延缓黄芩苷在体内的代谢为乙肝治疗提供新途径。     

Body distributioin of RGD-mediated liposome in brain-targeting drug delivery

RGD conjugation liposomes (RGD-liposomes) were evaluated for brain-targeting drug delivery. The flow cytometric in vitro study demonstrated that RGD-liposomes could bind to monocytes and neutrophils effectively. Ferulic acid (4-hydroxy-3-methoxycinnamic, FA) was loaded into liposomes. Rats were subjected to intrastriatal microinjections of 100 units of human recombinant IL-1beta to produce brain inflammation and caudal vein injection of three formulations (FA solution, FA liposome and RGD-coated FA liposome). Animals were sacrificed 15, 30, 60 and 120 min after administration to study the body distribution of the FA in the three formulations. HPLC was used to determine the concentration of FA in vivo with salicylic acid as internal standard. The results of body distribution indicated that RGD-coated liposomes could be mediated into the brain with a 6-fold FA concentration compared to FA solution and 3-fold in comparison to uncoated liposome. Brain targeted delivery was achieved and a reduction in dosage might be allowed.     

Liposomal encapsulation enhances antiviral efficacy of SPC3 against human immunodeficiency virus type-1 infection in human lymphocytes

Because encapsulation of antiviral drugs in liposomes resulted generally in improved activity against retroviral replication in vivo, the antiviral effects of free-SPC3 and liposome-associated SPC3 were compared in cultured human lymphocytes infected with HIV-1. SPC3 was entrapped in various liposomal formulations, either different in size (mean diameter of 100 and 250 nm), SPC3 concentration or cholesterol content. Liposome-associated SPC3 were tested for both inhibition of cell-cell fusion and infection with HIV-1 clones. SPC3 inhibited HIV-1-induced fusion at a micromolar concentration range. When associated with liposomes, SPC3 was found to be about 10-fold more potent than free SPC3 in inhibiting syncytium formation. Continuous treatment with free SPC3 also inhibited virus production in a dose-dependent manner, with inhibition of HIV infection of C8166 T-cells or human peripheral blood lymphocytes (PBLs) at micromolar concentrations. Liposomal entrapment was found to increase the antiviral efficacy of SPC3 by more than 10- and 5-fold in C8166 and PBLs, respectively. These data suggest that the liposome approach may be used to improve SPC3 antiviral efficacy.     

In Vitro andIn Vivo Studies of Different Liposomes Containing Topotecan

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistribution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S180 tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19-fold increase in the area under the curve (AUC(0-->infinity)), respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in AUC(0-->infinity) compared with H-Lip, but there was no significant increase in t(1/2) and AUC(0-->infinity) for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bone marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.     

Uptake of liposomes by cultured cardiomyocytes

Small unilamellar liposomes (SUV) of different phospholipid/polymer composition were labeled with NBD-PC, which served as a bilayersituated fluorescence marker. Neonatal cardiomyocytes were incubated with liposomes and then the cell-associated fluorescence was measured. The factors influencing the liposome uptake by cardiomyocytes such as concentration of lipid, time of incubation, membrane fluidity of liposomes, charge lipid/polymer modification of liposomes and anoxia of cultured cardiomyocytes were investigated. The liposome uptake by cardiomyocytes increased dose-dependently and time-dependently. Liposome uptake was strongly influenced by the electrical charge and modified polymer. After 2 h incubation, the uptake of positively charged liposomes was 1.7-fold higher than that of negatively charged one and both higher than that of the neutral one. The presence of PE-PEG2000 distinctly reduced the liposome uptake and the difference between the uptake of charged and neutral liposome. Anoxia increased the uptake of liposome at the first hour (increased 20%), but after 2 h incubation the liposome uptake by hypoxia cellswas less than that of normoxia cells (decreased 18%). Mechanisms involved are also discussed.     

Polyethylene glycol-coated liposomes for oral delivery of recombinant human epidermal growth factor

The present study was to investigate the feasibility of oral delivery of recombinant human epidermal growth factor (rhEGF). Polyethylene glycol (PEG)-coated liposomes containing rhEGF was prepared and evaluated for their stability and permeability in Caco-2 cells. In the animal study, we also determined plasma concentration and gastric ulcer healing effect after oral administration of rhEGF liposomes or the solution. Encapsulation of rhEGF into liposomes, suppressed the degradation in Caco-2 cell homogenate compared with the solution. The flux of rhEGF from dipalmitoylphosphatidylcholine (DPPC) liposome across Caco-2 cell monolayer from the apical to basolateral side was three times greater than that from phosphatidylcholine (PC) liposome or the solution. After oral administration of rhEGF liposomes or the solution in rats, the area under the concentration-time curve (AUC) of rhEGF increased 1.7- and 2.5-fold for PC and DPPC liposomes, respectively. The gastric ulcer healing effect was significantly increased in DPPC liposome compared with PC liposome and the solution. The enhanced curative ratio of rhEGF encapsulated into DPPC liposome may be due to the resistance to enzyme degradation, higher permeability and increased plasma AUC. Therefore, PEG-coated liposomes containing rhEGF could be used as an oral delivery formulation with enhanced encapsulation efficiency.     

Inhibition of metastatic lung cancer in C57BL/6 mice by liposome encapsulated all trans retinoic acid (ATRA)

The purpose of this study was to investigate whether all trans retinoic acid (ATRA) incorporated in liposome composed of distearoylphosphatidylcholine (DSPC/cholesterol) could inhibit the metastatic lung cancer in mice more efficiently than free ATRA. Metastatic lung cancer model was developed by intravenous injection of B16F10 cells and it is also referred as melanoma model. In this present study, C57BL/6 mice were divided into several groups as per experimental design and the free ATRA and liposome encapsulated ATRA were given for 21 days at a dose of 0.60 mg/kg body weight/day after cell line implantation. After 21 days, mice were sacrificed at different time interval for ATRA level analysis in serum and lung tissue by HPLC method and the remaining mice were kept for anticancer study. The ATRA level increased significantly in serum and lung tissue in liposome encapsulated ATRA treated mice. In cancer bearing mice, tumor nodule formation decreased and life span increased after receiving liposome encapsulated ATRA treatment than free ATRA treated mice. This result implies that the liposome encapsulated ATRA has maintained more ATRA concentration in lung tissue and showed more inhibition on the lung tumor nodule formation. The results indicate a possible use of liposome encapsulated ATRA in prevention of lung metastasis.     

Galactosylated liposomes as oligodeoxynucleotides carrier for hepatocyte-selective targeting

18mer oligodeoxynucleotides (ODNs) which can inhibit survivin gene expression were selected as a model gene drug. The glycolipid (5-cholestan-3beta-yl)-1-[2-(lactobionyl amido) ethylamido] formate (CHE-LA) which specific target to the cells expressing galactose receptors was synthesized through the reaction of lactone of lactobiono-1,5-lactone (LA) and the amino-group of 2-(cholesteryloxycarbonylamino) ethylamine (CHE). The galactosylated liposome incorporated with CHE-LA containing oligodeoxynucleotides was prepared with SPC, cholesterol, CHE-LA and oligodeoxynucleotides by the thin-film hydration method. 1,1'-Dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate (Dil) was used as a marker for all the liposome preparations. Compared with conventional liposomes (CL), the galactosylated liposomes (GL) exhibited a drastically increased distribution to the liver in vivo and the galactosylated liposomes containing oligodeoxynucleotides (GLO) can also more efficiently induced an apoptosis of HepG2 cells in vitro than the conventional liposome containing oligodeoxynucleotides (CLO). In addition, the GLO represented an improving of the ODNs entrapment efficiency.     

碱性成纤维细胞生长因子脂质体对异丙肾上腺素引起的大鼠心肌损伤的保护作用

目的考察碱性成纤维细胞生长因子(bFGF)脂质体对异丙肾上腺素(isoproterenol,ISO)引起的大鼠心肌损伤的保护作用。方法大鼠腹腔注射ISO(80 mg.kg-1)建立大鼠急性心肌损伤模型后,尾静脉注射相同剂量的bFGF水溶液、bFGF普通脂质体和bFGF长循环脂质体,48 h后取心室部分常规切片HE染色,并对血清中乳酸脱氢酶(LDH)、心肌组织中胶原(collagen)、丙二醛(MDA)及ATP酶的含量进行测定。结果bFGF水溶液给药组没有明显减轻ISO对大鼠心肌组织的损伤程度,而bFGF脂质体给药组则显著减轻了ISO引起的心肌损伤程度。bFGF普通脂质体组的LDHc、ollagen、MDA较ISO组分别降低了31.5%、24.3%、37.7%,ATP酶增加了27.9%;bFGF长循环脂质体组的LDH、collagen、MDA较ISO组分别降低了47.1%、29.7%、44.0%,ATP酶增加了32.7%。结论与相同剂量的bFGF水溶液相比bFGF脂质体对ISO引起的大鼠心肌损伤具有明显的保护作用,且长循环脂质体的效果优于普通脂质体,其原因为脂质体作为药物载体能够保护bFGF在体内的稳定性并增加其在心肌组织中的分布。