钆喷酸葡胺长循环脂质体的制备及其肿瘤细胞摄取能力研究

目的:制备用聚乙烯100硬脂基醚(Brij700)修饰的长循环钆喷酸葡胺脂质体,研究其被肿瘤细胞摄取的情况。方法:利用逆向蒸发法制备钆喷酸葡胺普通脂质体和长循环脂质体并进行质量检测;用磁共振造影仪(MRI)检测前列腺癌细胞(PC3)中摄取的钆喷酸葡胺的浓度,以SE-T1W信号值为指标比较肿瘤细胞对2种脂质体的摄取能力。结果:制备的钆喷酸葡胺普通脂质体和长循环脂质体平均粒径分别为824、363nm,包封率分别为63.16%、61.15%;PC3对2种脂质体摄取后的SE-T1W信号值分别为362、299;平均每个细胞的摄取数分别为2.12×10-4、2.70×10-4。结论:与钆喷酸葡胺普通脂质体比较,其长循环脂质体更利于肿瘤细胞的摄取。     

荧光法测定钆喷酸双葡胺脂质体的含量

本文建立了荧光法测定钆喷酸双葡胺脂质体含量的新方法,考察了荧光法测定钆喷酸双葡胺脂质体含量的最佳条件：激发波长275 nm,发射波长313 nm,pH为6.0~8.0,线性范围9.4 (g(ml-1 ~ 9.4 mg(ml-1,回收率100.2%,结果令人满意。     

钆喷酸葡胺致不良反应误吸二例

目的探讨钆喷酸葡胺在头部伽玛刀放疗患者行磁共振增强定位时的不良反应和安全性。方法报道2例钆喷酸葡胺不良反应致误吸及抢救经过。结果钆喷酸葡胺存在不良反应,在某种特殊情况下,会导致误吸。结论钆喷酸葡胺的不良反应发生率虽低,但其在头部伽玛刀放疗患者行磁共振增强定位时有误吸现象发生,应引起足够的重视,且应做好用药前的告知、预防误吸训练及用药中的急救准备工作。     

钆喷酸葡胺注射液致尿失禁1例

通过报道静脉注射钆喷酸葡胺(Gd-DTPA)致尿失禁1例,为临床使用提供参考信息,提示临床使用该药时应密切观察患者用药后情况。     

钆喷酸葡胺注射液引起过敏性休克死亡1例

1临床资料 患者,男,57岁,缘于15d前无明显诱因觉尿黄,如浓茶水样,伴有皮肤轻度瘙痒,给予保肝、降酶治疗,效果不佳,于2009年6月16日门诊以黄疸原因待查：1）病毒性肝炎;2）药物性肝损害收入我院。既往无药物、食物过敏史。入院后给予还原型谷胱甘肽、复方甘草酸苷、丁二磺酸腺苷蛋氨酸以及赤丹退黄颗粒等保肝降酶治疗,但患者淤胆现象较为明显,于是行核磁检查。     

钆喷酸葡胺注射液致过敏性休克1例

患者,男。58岁,于2012年7月18日来我院就诊,既往患者头颅CT、MRI等检查已明确诊断为蛛网膜下腔出血,小脑海绵状血管瘤。为进一步行伽马刀治疗小脑血管动静脉畸形来我院,门诊以“海绵状血管瘤”收入院。体检：P78次／min、BP120／80mmHg,心肺听诊无异常,无食物、药物过敏史,无近期用药史。     

钆喷酸葡胺注射液致过敏性休克1例

钆喷酸葡胺（ Gd-DTPA）临床主要作为核磁共振成像时顺磁性对比剂,可以增强显影效果,且少有过敏等不良反应,因而在临床检查中使用较为普遍。2013年8月11日,我科首次发生1例行磁共振强化检查时使用钆喷酸葡胺出现过敏反应的患者,经治疗患者基本痊愈,现将抢救经过报道如下。     

钆喷酸葡胺注射液致过敏性休克死亡1例

患者,男,53岁,体质量75 kg,因下肢静脉曲张伴溃疡入院. 20余年前无明显诱因下出现双下肢浅血管迂曲扩张,症状逐渐加重,1年前开始出现双下肢足内踝溃烂,为进一步诊治入院. 2010年6月3日上午行双下肢静脉磁共振检查,检查前一般情况尚可,神志清,两下肢静脉曲张双侧踝处可见溃疡,检查中经留置针通道注入钆喷酸葡胺针30 mL,约4 min后患者出现动弹状,脸色通红,口吐白沫,立即退出检查,患者呼吸困难,脉搏无法触及,立即静脉注射肾上腺素1 mg,再次检查,心脏停搏,立即给予心外按压,送急诊科抢救,给予地塞米松针10 mg、多巴胺针20 mg、胺碘酮150 mg、阿托品1.0 mg、甲泼尼龙40 mg等药物,行高频吸氧、心脏电除颤术、心肺复苏等抢救无效,宣告死亡.     

钆喷酸葡胺注射液不良反应的文献分析

目的分析钆喷酸葡胺注射液发生不良反应特点及相关因素,为临床合理用药提供参考。方法对万方数据库2002～2012年发表的文献进行检索,对涉及钆喷酸葡胺注射液不良反应病例的信息进行分类统计分析。结果发生不良反应的男女比例为1：1．2,多数不良反应发生在用药后30min内。非过敏反应有59例（44％）;轻度过敏性反应有47例（35％）;中度过敏性反应10例（8％）;重度过敏性反应18例（13％）,其中死亡有6例（4％）。结论虽然钆喷酸葡胺注射液不良反应发生率较低,但仍可引起严重的过敏反应,甚至休克死亡,由于其使用多在缺少临床医护监护的影像诊断科,所以更应加强其监测。     

钆喷酸葡胺对比剂的临床安全性分析

目的 探讨钆喷酸葡胺对比剂的不良反应,以利其安全使用.方法 对医院1500例静脉注射钆喷酸葡胺对比剂发生不良反应的病例进行分类分析,观察不良反应发生及处置过程.结果 发生钆喷酸葡胺不良反应12例,不良反应发生率为0.80%;其中轻度不良反应11例,重度不良反应1例.结论 静脉注射钆喷酸葡胺对比剂的不良反应发生率低,但严重不良反应一旦发生可危急生命,应引起临床重视.     

Long-circulating liposomes of indomethacin in arthritic rats--a biodisposition study

To improve the targeting efficiency of liposomes of indomethacin to the arthritic joints, circulation half-life of the liposomes was increased by grafting amphipathic polyethylene glycol-2000 to the bilayer surface. A comparative biodistribution study was performed between the conventional liposomes (PC:CH:PE--1:0.5:0.16) and long-circulating liposomes (PC:CH:PE-PEG--1:0.5:0.16) in arthritic rats. Pharmacokinetics of the drug changed significantly when administered in liposomal form. Pharmacokinetic parameters of the drug such as AUC0-t (trapezoidal), clearance and t1/2 (elimination half-life) changed significantly (p < 0.05) when encapsulated in liposomes. Significant difference in pharmacokinetics was observed in AUC0-t and clearance between the conventional liposomes and long-circulating liposomes. The increased AUC0-t and reduced clearance of the drug with long-circulating liposomes, increased the availability of the drug by reducing RES uptake, in turn localization in arthritic paw tissue was also increased. A concentration of 0.33 microgram of indomethacin/g of the tissue was achieved with S-liposomes after 24 h whereas it was only 0.26 microgram of drug/g of the tissue with conventional liposomes. From the study, in may be concluded that the targeting efficiency of the long-circulating liposomes was about four times more than the conventional liposomes.     

A detachable coating of cholesterol-anchored PEG improves tumor targeting of cell-penetrating peptide-modified liposomes

Cell-penetrating peptides (CPPs) have been widely used to enhance the membrane translocation of various carriers for many years, but the non-specificity of CPPs seriously limits their utility in vivo. In this study, cholesterol-anchored, reduction-sensitive PEG (first synthesized by our laboratory) was applied to develop a co-modified liposome with improved tumor targeting. Following optimization of the formulation, the in vitro and in vivo properties of the co-modified liposome were evaluated. The co-modified liposome had a much lower cellular uptake and tumor spheroid uptake, but a much higher tumor accumulation compared to CPP-modified liposome, indicating the non-specific penetration of CPPs could be attenuated by the outer PEG coating. With the addition of exogenous reducing agent, both the in vitro and in vivo cellular uptake was markedly increased, demonstrating that the reduction-sensitive PEG coating achieved a controllable detachment from the surface of liposomes and did not affect the penetrating abilities of CPPs. The present results demonstrate that the combination of cholestervsitive PEG and CPPs is an ideal alternative for the application of CPP-modified carriers in vivo.KEY WORDS: Cell penetrating peptide, Reduction-sensitive PEG, Tumor targeting, Cholesterol, Liposome     

Tumor targeting of doxorubicin by anti-MT1-MMP antibody-modified PEG liposomes

Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase (MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor.     

Immunospecific targeting of liposomes to erythrocytes

Immunoliposomes were made by covalently linking Fab' fragments (from rabbit antimouse erythrocyte IgG) to reverse-phase evaporation vesicles (REV) via maleimido-4-(p-phenylbutyrate) phosphatidylethanolamine (MPB-PE) as anchor molecule. These immunoliposomes were characterized in terms of size, charge, stability and antigen binding capacity. The effect of the liposomal Fab' density on the interaction with the target cell was studied. Two isolation methods were tested to separate free Fab' from liposomally bound Fab'. The necessity of deactivation of remaining reactive sites with dithiothreitol preincubation to increase the specificity of immunoliposome target cell interactions was demonstrated.     

钆喷酸葡胺脂质体在小鼠体内的分布与药代动力学

目的研究钆喷酸葡胺脂质体在小鼠体内的分布与药代动力学。方法应用ICP发射光谱分析法定量测定了钆喷酸葡胺及其脂质体在小鼠心、肝、脾、肺、肾及血中的钆浓度。结果钆喷酸葡胺及其脂质体在小鼠体内的血药时程变化符合二室模型。钆喷酸葡胺脂质体保持了相对较高的血药浓度 ,并且在肝脾中的分布明显增加。结论钆喷酸葡胺脂质体具有肝脾靶向性     

Long-circulating gadolinium-encapsulated liposomes for potential application in tumor neutron capture therapy

Gadolinium neutron capture therapy (Gd-NCT) is a promising cancer therapy modality. One of the key factors for a successful Gd-NCT is to deliver and maintain a sufficient amount of Gd in tumor tissues during neutron irradiation. We proposed to prepare a Gd delivery system by complexing a Gd-containing compound, diethylenetriaminepentaacetic acid (Gd-DTPA), with a polycationic peptide, poly-L-lysine (pLL), and then encapsulate the complexed Gd-DTPA into PEGylated liposomes. Complexation of Gd-DTPA with pLL not only enhanced the encapsulation efficiency of Gd-DTPA in liposomes, but also significantly limited the release of Gd-DTPA from the liposomes. A Gd-DTPA-encapsulated liposome formulation that contained 6.8+/-0.3 mg/mL of pure encapsulated Gd was prepared. The blood half-life of the Gd encapsulated into the liposome formulation was estimated to be about 24 h in healthy tumor-free mice. About 12 h after the Gd-encapsulated liposomes were intravenously injected into mice with pre-established model tumors, the Gd content in the tumors reached an average of 159 microg/g of wet tumor tissue. This Gd-DTPA encapsulated liposome may be used to deliver Gd into solid tumors for NCT and tumor imaging.     

表面带活性羧基的新型长循环脂质体的制备和组织分布研究

合成和纯化了一个带末端羧基的新的磷脂酰乙醇胺的聚乙二醇衍生物－DPPE－PEG3000－COOH。将这个化合物掺入脂质体后，脂质体在网状内皮系统（肝，脾）的聚集减少，在血中的循环时间明显延长，并表现为一级对数线性消除。脂质体表面的活性羧基可以和其它活性分子反应，这可能为脂质体带来新的功能。     

Enhanced Tumor Targeting of Doxorubicin by Ganglioside GMl-bearing Long-circulating Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating liposomes, composed of ganglioside GM1 (GM1)/distearoylphosphatidylcholine (DSPC)/cholesterol (CH) (0.13:1:1 in molar ratio) and sized to approximately 100 nm in mean diameter, with 98% entrapping efficiency by the transmembrane pH gradient method. Free DXR, DXR-DSPC/CH and DXR-GM1/DSPC/CH liposomes were injected intravenously into Colon 26 tumor-bearing Balb/c mice via the tail vein at a dose of 5.0 mg DXR/kg. DXR-GM1/DSPC/CH liposomes gave a higher blood level of the drug than did DXR-DSPC/CH liposomes or free DXR up to 24 hours after injection, and the area under the blood concentration-time curve (AUC) for DXR-GM1/DSPC/CH liposomes was 1.5 or 526 times higher than that for DXR-DSPC/CH liposomes or free DXR, respectively. DXR-GM1/DSPC/CH liposomes gave a decreased DXR concentration in the reticuloendothelial system (RES) of the liver and the spleen. Both liposomal formulations effectively reduced the DXR concentration in the heart as compared with that in the case of free DXR. At 6 hours after i.v. injection, DXR-GM1/DSPC/CH liposomes provided an approximately 3.3- or 9-fold higher peak DXR level in the tumor as compared with DXR-DSPC/CH liposomes or the free drug, respectively. These high tumor levels of DXR appear to reflect the prolonged residence time of the liposomes. The results suggest that encapsulation of DXR in GM1-bearing long-circulating liposomes will be useful for cancer chemotherapy.     

High-affinity targeting of biotin-labeled liposomes to streptavidin-conjugated ligands

Cell-specific delivery of drug-loaded liposomal carrier systems can be achieved through the use of liposomes with covalently attached proteins. For such targeting strategies to be successful a number of potential difficulties, related to the preparation of the liposomes as well as optimization of properties that maximize in vivo access and binding to a defined target cell population, must be overcome. The studies summarized here have attempted to identify specific factors that will promote binding of targeted liposomes to defined target surfaces. Liposomes containing biotinylated phospha-tidylethanolamine were used to demonstrate that the avidity of a targeted liposome for streptavidin-coated ELISA plates and cells is influenced by liposome lipid composition, the amount of targeting molecule present per liposome, the nature of the targeting ligand, and the target surface. Specifically, it is demonstrated that the three most important factors (in order of importance) controlling the apparent affinity of targeted liposomes are (1) target ligand concentration in the liposomal membrane; (2) the presence of a spacer grout between the biotin and the phospholipid headgroup; and (3) the addition of cholesterol. Other less important factors that influence target liposome binding include whether the target ligand is attached to a saturated phospholipid compared to an unsaturated lipid and whether the bulk phospholipid species in the liposome is unsaturated versus saturated. These studies suggest that targeted liposomes exhibiting a broad range of binding avidities, as estimated by the concentration of liposomes required to achieve saturation of a target surface, can be prepared by selective design of the liposomal carrier. Advantages of the biotinylated liposome for targeting include the relative ease of preparation the possibility of preparation of large-scale batches suitable for clinical development), the ease of incorporation of the targeting ligand, and, importantly, the ability to alter the apparent affinity of the liposome for the target cell through choice of the biotin-labeled lipid and targeting molecule concentration. The potential for developing a two-step targeting strategy based on the use of biotinylated liposomes is discussed.     

High-affinity targeting of biotin-labeled liposomes to streptavidin-conjugated ligands

Abstract

Cell-specific delivery of drug-loaded liposomal carrier systems can be achieved through the use of liposomes with covalently attached proteins. For such targeting strategies to be successful a number of potential difficulties, related to the preparation of the liposomes as well as optimization of properties that maximize in vivo access and binding to a defined target cell population, must be overcome. The studies summarized here have attempted to identify specific factors that will promote binding of targeted liposomes to defined target surfaces. Liposomes containing biotinylated phospha-tidylethanolamine were used to demonstrate that the avidity of a targeted liposome for streptavidin-coated ELISA plates and cells is influenced by liposome lipid composition, the amount of targeting molecule present per liposome, the nature of the targeting ligand, and the target surface. Specifically, it is demonstrated that the three most important factors (in order of importance) controlling the apparent affinity of targeted liposomes are (1) target ligand concentration in the liposomal membrane; (2) the presence of a spacer grout between the biotin and the phospholipid headgroup; and (3) the addition of cholesterol. Other less important factors that influence target liposome binding include whether the target ligand is attached to a saturated phospholipid compared to an unsaturated lipid and whether the bulk phospholipid species in the liposome is unsaturated versus saturated. These studies suggest that targeted liposomes exhibiting a broad range of binding avidities, as estimated by the concentration of liposomes required to achieve saturation of a target surface, can be prepared by selective design of the liposomal carrier. Advantages of the biotinylated liposome for targeting include the relative ease of preparation the possibility of preparation of large-scale batches suitable for clinical development), the ease of incorporation of the targeting ligand, and, importantly, the ability to alter the apparent affinity of the liposome for the target cell through choice of the biotin-labeled lipid and targeting molecule concentration. The potential for developing a two-step targeting strategy based on the use of biotinylated liposomes is discussed.