GM-CSF-MA脂质体对急性白血病小鼠的治疗作用

目的:观察GM-CSF-MA(粒-巨噬细胞集落刺激因子-米托蒽醌及阿糖胞苷)脂质体对急性髓细胞白血病(AML)小鼠的治疗作用。方法:将AML小鼠随机分为5组,分别为GM-CSF-MA脂质体组、MA脂质体组、MA组、GM-CSF组和生理盐水对照组。观察各组小鼠的生存时间及外周血象、肝功(AST)、肾功(Cr)、磷酸肌酸激酶(CK)的变化。结果:GM-CSF-MA脂质体组小鼠的生存时间为(53.2±22.25)d,生命延长率为254.7%;外周血及骨髓中白血病细胞迅速减少,血象恢复快,与其它治疗组及对照组相比,有显著差异;治疗后GM-CSF-MA脂质体组AST、Cr恢复快,CK无明显变化,而MA组各项生化指标均明显升高,两组比较有显著差异。结论:GM-CSF-MA脂质体是一种高效、低毒的靶向治疗急性髓细胞白血病的新方法。     

Multifunctional and stimuli-sensitive pharmaceutical nanocarriers

Currently used pharmaceutical nanocarriers, such as liposomes, micelles, and polymeric nanoparticles, demonstrate a broad variety of useful properties, such as longevity in the body; specific targeting to certain disease sites; enhanced intracellular penetration; contrast properties allowing for direct carrier visualization in vivo; stimuli-sensitivity, and others. Some of those pharmaceutical carriers have already made their way into clinic, while others are still under preclinical development. In certain cases, the pharmaceutical nanocarriers combine several of the listed properties. Long-circulating immunoliposomes capable of prolonged residence in the blood and specific target recognition represent one of the examples of this kind. The engineering of multifunctional pharmaceutical nanocarriers combining several useful properties in one particle can significantly enhance the efficacy of many therapeutic and diagnostic protocols. This paper considers the current status and possible future directions in the emerging area of multifunctional nanocarriers with primary attention on the combination of such properties as longevity, targetability, intracellular penetration, contrast loading, and stimuli-sensitivity.     

超氧物歧化酶冻干制剂及脂质体制剂的研究

设计了十一种冻干制剂的配方,通过外观检查和稳定性考查,从中筛选出最佳配方,即以乳糖为赋形剂且冻干前浓度为2.5％的制剂。探讨了各种条件对制备SOD脂质体的影响,选出了提高包嵌率的较好条件。改进了测定SOD活性的极谱法,测定了SOD水溶液和两种SOD脂质体在兔血内SOD活性的变化,初步证实所制得的脂质体可延长SOD在体内的停留时间。     

载芬维A铵脂质体抑制A375黑素瘤细胞裸鼠皮下移植瘤的初步研究

目的 探讨载芬维A铵脂质体（4-HPR-L）对裸鼠皮下人恶性黑素瘤的抑制作用。方法 采用薄膜-超声分散法制备4-HPR-L。通过皮下接种黑素瘤A375细胞至BALB/c裸鼠右侧腋窝建立黑素瘤荷瘤裸鼠模型。取10只荷瘤裸鼠模型,随机等分为两组,分别给予尾静脉注射同浓度细胞膜近红外荧光探针（DiR）溶液和DiR脂质体（DiR-L）,应用小动物活体成像仪观察给药后6、12、24 h药物在体内分布情况。取30只荷瘤裸鼠,随机等分为3组,即对照组、4-HPR组和4-HPR-L组,分别经尾静脉每次注射5%（质量分数）葡萄糖溶液0.2 ml、25 mg/kg 4-HPR和4-HPR-L溶液,于接种A375细胞后第8、10、12、14、16、18、20、22天给药,动态监测给药后各组裸鼠的体重和肿瘤体积,观察生存情况。于末次给药后第2天处死裸鼠,取心、肝、脾、肺、肾及肿瘤组织,HE染色和免疫组化染色观察黑素瘤体内转移情况,并用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测肿瘤细胞凋亡情况。计量资料采用单因素方差分析和独立样本t检验进行分析。结果 小动物活体成像仪显示,DiR-L能较长时间滞留于肿瘤组织,给药24 h后在肿瘤部位仍可观察到较强荧光;定量分析显示,肿瘤组织中DiR-L荧光强度（22.85 ± 1.66）显著高于DiR（8.45 ± 0.97,t = 12.957,P < 0.01）。与对照组和4-HPR组相比,4-HPR-L组末次给药后第2天离体瘤重明显降低（F = 27.055,t值分别为4.768、6.640,均P < 0.05）。HE染色显示,4-HPR-L组2只裸鼠发生肝脏转移,而对照组、4-HPR组全部裸鼠发生肝脏转移。荷瘤鼠的生存期观察显示,4-HPR-L组裸鼠于接种后76 d内全部死亡,对照组和4-HPR组分别于接种后56 d和59 d内全部死亡。对照组凋亡指数为（12.14 ± 1.33）‰,4-HPR组为（67.17 ± 15.18）‰,4-HPR-L组为（152.73 ± 11.27）‰,3组间差异有统计学意义（F = 167.588,P < 0.05）,4-HPR-L组与对照组和4-HPR组相比,t值分别为18.162、11.075,均P < 0.05。结论 4-HPR-L能够有效抑制裸鼠皮下黑素瘤体积增长和黑素瘤细胞转移,并延长裸鼠生存期。     

Alkylphosphocholine-induced production of nitric oxide and tumor necrosis factor α by U 937 cells

The human histiocytic cell line U937, which expresses a number of monocyte markers and properties, was investigated with regard to its ability to be activated for NO and tumor necrosis factor (TNF) release after treatment with alkylphosphocholines (APC) and APC liposomes. Using APC multilamellar vesicles (MLV) a clear dose-dependent increase of NO production could be demonstrated for U 937 cells, whereas the corresponding soluble substances had no effect. The time course of NO release was characterised by a peak between 2 h and 12 h and a strong decrease after 24 h. LPS caused no NO release nor the production of TNF in U 937 cells. The simultaneous incubation of the cells with lipopolysaccharide and APC or APC-MLV, led to a strong increase in TNF production. Closer investigation of the time sequence of this synergistic effect demonstrated that cells, that had first been treated with hexadecylphosphocholine (HPC)-MLV and 4 h later with lipopolysaccharride secreted significantly more TNF into the supernatants than in the experiment where both substances were added simultaneously. From these results it was concluded that APC-MLV are possibly able to act as a primer in the process of lipopolysaccharide mediated TNF induction. Furthermore, a positive influence of phorbol 12-myristate 13-acetate (PMA) on the ability of U 937 cells to produce TNF following a treatment with HPC or HPC-MLV could be observed. PMA-pretreated cells were shown to release much more TNF compared to control cells, which led to the supposition that the immunomodifying activity of APC becomes effective only in more highly differentiated cell types.Abbreviations APC alkylphosphocholines - DPC dodecylphosphocholine - HPC hexadecylphosphocholine - TPC tetradecylphosphocholine - MLV multilamellar vesicles - PMA phorbol 12-myristate 13-acetate - TNF tumour necrosis factor Partly supported by the Bundesministerium für Forschung und Technologie (9319564A)     

Application of liposomes to sonoporation

A method to prepare liposomes is presented. Liposomes made in our laboratory were characterized acoustically and optically. The phase velocity and attenuation of liposomes in suspension (concentration = 10(9)/mL) were measured, ranging from 2 to 14 MHz, using ultrasound spectroscopy. Anti-rabbit IgG conjugated with Alexafluor 647 was delivered into Jurkat cells in suspension, using the liposomes, by 10 % duty cycle ultrasound tonebursts of 2.2 MHz (the in situ spatial peak-pressure amplitude = 80 W/cm2) with an efficiency of 13 %. It has been experimentally shown that liposomes may be an alternative stable agent to Optison for delivering macromolecules into cells.     

RGD-targeted paramagnetic liposomes for early detection of tumor: in vitro and in vivo studies

Magnetic resonance molecular imaging has emerged as a potential approach for tumor diagnosis in the last few decades. This approach consists of the delivery of MR contrast agents to the tumor by specific targeted carriers. For this purpose, a lipopeptide was constructed by using a cyclic RGD peptide headgroup coupled to palmitic acid anchors via a KGG tripeptide spacer. Targeted paramagnetic liposomes were then prepared by the incorporation of RGD-coupled-lipopeptides into lipid bilayers for specific bounding to tumor. In vitro, study demonstrated that RGD-targeted liposomes exhibited a better binding affinity to targeted cells than non-targeted liposomes. MR imaging of mice bearing A549 tumors with the RGD-targeted paramagnetic liposomes also resulted in a greater signal enhancement of tumor compared to non-targeted liposomes and pure contrast agents groups. In addition, biodistribution study also showed specific tumor targeting of RGD-targeted paramagnetic liposomes in vivo. Therefore, RGD-targeted paramagnetic liposomes prepared in the present study may be a more promising method for early tumor diagnosis.     

Chloride channel-mediated brain glioma targeting of chlorotoxin-modified doxorubicine-loaded liposomes

The chlorotoxin (ClTx), a scorpion-derived peptide, binding to gliomas with high specificity, was firstly applied to establish the ClTx-modified doxorubicin (DOX)-loaded liposome delivery system for targeting the brain glioma and improving the anticancer efficacy. In vitro physicochemical characterization of the novel liposome system presented satisfactory size of 100 nm with uniform distribution, high encapsulation efficiency and adequate loading capacity of both fluorescent probe and anticancer drug. It was demonstrated quantitatively by the spectrophotofluorometry and flow cytometry and qualitatively by the confocal microscopy that ClTx highly facilitated the uptake of liposomes by three glioma cell lines and one endothelial cell line. In vitro cytotoxicity studies proved that the presence of ClTx increased the cytotoxicity against glioma cells and endothelial cells with various levels for different cell lines. In BALB/c mice bearing U87 tumor xenografts, biodistribution of DiR-loaded liposomes by body imaging and anti-glioma pharmacodynamics of DOX-loaded liposomes were investigated. The ClTx-modified liposomes showed more accumulation in the subcutaneous and intracranial tumors, higher tumor growth inhibition and lower blood toxicity in the armpit tumor model. The in vitro and in vivo results exhibited good correlation of glioma targeting of the ClTx-modified liposomes. Significantly, with the ClTx as the targeting ligand, the liposomes might serve as an applicable delivery system for brain glioma therapy or imaging.     

阿苯达唑脂质体治疗66例人体包虫病的疗效观察

目的综合评价阿苯达唑脂质体(L-ABZ)治疗人体包虫病的疗效,并进行安全性考察,为该药在临床的应用提供客观依据.方法临床治疗了66例包虫病患者,其中囊型包虫病56例、泡型包虫病10例,口服阿苯达唑脂质体10mg*kg-1*d-1,一天两次,连续服用,疗程3～12个月,通过影像学指标观察,结合临床症状和体征及生化指标进行综合评价,动态随访3～24个月.按治愈率、有效率、部分有效率、无效率以及总有效率判断疗效,并对单囊型及与多子囊型以及原发、复发病人进行对照分析.结果共治愈20例(20/66),治愈率为30.3%;有效29例(29/66),有效率43.9%;部分有效10例(10/66),部分有效率15.1%;无效7例(7/66),无效率10.6%;总有效率为89.3%(59/66).其中,单囊型的治愈率明显高于多子囊型,但二者的有效率无显著性差异(P<0.1);而原发、复发病人的治疗阿苯达唑脂质体的疗效无明显差异(P＞0.1);泡型包虫病患者,约50%病例临床疗效明显.在安全性观察中,主观症状阳性率为13.6%(9/66),生化指标阳性率为12.1%(8/66).结论阿苯达唑脂质体口服液是目前有效的一种抗包虫病新药剂型,具有疗效较高,低毒、副作用小、安全性高的特点.     

载药脂质微气泡的制备及其应用

目的制备载紫杉醇脂质微气泡并测定其包封率、载药量、药物释放及观察其体内超声显像效果。方法制备载紫杉醇脂质微气泡,测定其包封率,载药量,粒径大小、分布和Zeta电位;观察苏丹黑对脂质微气泡的染色,以及超声辐照后苏丹黑的释放情况;观察辐照后载药脂质微气泡各层溶液药物的释放;并观察其在小鼠肝内的显像效果。结果载药脂质微气泡的浓度为2.3×109~3.5×109/ml,粒径范围为90%以上2~6μm,平均粒径为2.95μm。脂质微气泡紫杉醇的包封率大于90%,载药量为(26.5±0.7)%;Zeta电位为-(21.8±1.1)mV;苏丹黑染色后光镜下可见微气泡壁被染成黑色,超声辐照后微气泡稀释液变黑,并且超声辐照载药脂质微泡溶液后,上层和中间层的药物释放明显增加;静脉注射此载药微气泡后,小鼠肝可见良好、持续的增强显像效果。结论采用机械振荡法制备的紫杉醇脂质微气泡,包封率和载药量均较高,粒径分布好,体内显像效果好,超声辐照能促使微泡中的药物释放,有望实现实时监控下的体内定点靶向给药。