血栓靶向尿激酶脂质体的制备及其体内溶栓效果

目的制备血栓靶向的尿激酶脂质体,并在大鼠颈总动脉血栓模型上考察其溶栓情况。方法通过液相合成法合成出靶向于血栓的特异性配体H-Arg-Gly-Asp-Ser-OH (RGDS),并将其与monocarboxyl poly (ethylene glycol) 3 500 distearoyl phosphatidylethalnolamine (DSPE-PEG_{3 500}-COOH)偶联后插入到脂质体双层膜中得到血栓靶向尿激酶脂质体;通过制备方法的改进,以氢化豆磷脂在室温下制备尿激酶脂质体;在大鼠颈总动脉血栓模型上,考察了血栓靶向脂质体的体内溶栓效果。 结果所得的尿激酶脂质体包封率高、粒径小,稳定性好;与空白对照组的栓重相比,在相同剂量(60 kU·kg^-1,小剂量)下,游离尿激酶组几乎无任何改变,尿激酶脂质体组血栓重量稍有减轻但无显著性差异,血栓靶向尿激酶脂质体组血栓重量明显减轻(P<0.001);干重时的情况略有不同。与同剂量的普通脂质体相比,血栓靶向尿激酶脂质体溶栓效果显著改善(湿重时P<0.01,干重时P<0.05),表现出明显的靶向溶栓能力。 结论所制备的血栓靶向尿激酶脂质体具有靶向溶栓的效果。     

三七总皂苷口服脂质体的制备及大鼠体内药动学研究

目的制备三七总皂苷(TPNS)口服胆盐脂质体,考察其药剂学特征及在大鼠体内的药动学行为。方法薄膜分散法制备TPNS胆盐脂质体,对其外观形态、粒径分布、包封率进行评价,并以注射用血栓通和血栓通胶囊为参比制剂,将18只大鼠随机分成血栓通胶囊灌胃组、TPNS脂质体灌胃组和血栓通溶液尾静脉注射组进行药动学行为考察。结果制备的TPNS脂质体呈类球形结构,粒径为(163.0±5.28)nm,包封率为(80.6±2.14)%,3组实验的AUC_(0~t)分别为865.39、1442.79和2124.38h·μg·mL~(-1),自制TPNS脂质体相对于血栓通胶囊的生物利用度为166.72%。结论 TPNS胆盐脂质体外观良好,包封率较高,能显著提高TPNS口服给药的生物利用度。     

4′-甲基-7-(2-羟基-3-异丙胺基丙氧基)-黄酮盐酸盐对实验性血栓形成的影响

4′-甲基-7-(2-羟基-3-异丙胺基丙氧基)-黄酮盐酸盐(SIPI-549)对大鼠和家兔实验性血栓形成有剂量(或浓度)依赖性的抑制作用。ⅳ7.5～20.0 mg/kg,使大鼠颈动—静脉旁路血栓湿重减轻。家兔半体内试验(ⅳSIPI-549 10 mg/kg),可使Chandler管中的雪暴现象出现时间和血栓形成时间延长,血栓湿重和干重减轻。家兔体外实验与半体内试验的结果基本一致。     

4′-甲基-7-(2-羟基-3-异丙胺基丙氧基)-黄酮盐酸盐对实验性血栓形成的影响

4′-甲基-7-(2-羟基-3-异丙胺基丙氧基)-黄酮盐酸盐(SIPI-549)对大鼠和家兔实验性血栓形成有剂量(或浓度)依赖性的抑制作用。ⅳ7.5～20.0 mg/kg,使大鼠颈动—静脉旁路血栓湿重减轻。家兔半体内试验(ⅳSIPI-549 10 mg/kg),可使Chandler管中的雪暴现象出现时间和血栓形成时间延长,血栓湿重和干重减轻。家兔体外实验与半体内试验的结果基本一致。     

纳米脂质体促进柚皮素对弹性蛋白酶诱导的小鼠腹主动脉瘤的抑制作用（英文）

腹主动脉瘤(AAA)是一种严重的炎症性血管疾病,其特征是基质金属蛋白酶(MMPs)升高、细胞外基质(ECM)降解和血管炎症反应。近年来的研究表明,柚皮素(NGN)作为一种生物活性黄酮,有抑制AAA的作用,但是较低的水溶性及口服吸收特性限制了NGN抑制AAA的有效性。本研究开发了一种NGN纳米脂质体处方(NGN-NL),以增强NGN对弹性蛋白酶诱导的小鼠AAA的抑制作用。首先,我们优化了NGN-NL的处方,发现磷脂和NGN的比例为9:1时为最佳处方。此外,我们观察到,与脂质体处方中的药物及辅料的物理混合物相比, NGN制备成脂质体后具有更强的抑制LPS诱导的巨噬细胞M1极化的作用。在同等剂量下, NGN-NL可比游离NGN展现出更强的抑制巨噬细胞M1极化的作用。此外,脂质体载体本身也呈现一定程度的抑制M1极化的特性,可协同增强NGN抑制巨噬细胞M1极化的效果。体内研究表明,以含有25mg/kg NGN剂量的NGN-NL对小鼠隔日进行腹腔注射,与游离NGN在使用两倍剂量(50mg/kg)下产生的AAA抑制效率相近。具体表现为:改善腹主动脉直径扩张、保持主动脉结构完整性、减缓弹性蛋白降解、减...     

颌面血管瘤局部注射用平阳霉素脂质体的制备和优化

目的制备平阳霉素脂质体,建立包封率测定方法,考察处方影响因素,优化处方。方法采用硫酸铵梯度法制备平阳霉素脂质体;以Sephadex G-50微柱离心法分离脂质体和游离药物,应用HPLC法测定药物含量,计算包封率;通过单因素考察优化硫酸铵梯度法的处方工艺,考察不同处方因素对包封率的影响。结果 Sephadex G-50微柱可完全吸附平阳霉素游离药物,空白脂质体回收率为98.3%~101.2%;平阳霉素浓度在5~200μg·m L-1内与峰面积线性关系良好(r=0.9999),日内和日间精密度(RSD)均<2%,回收率为98.3%~100.1%。通过单因素考察优化平阳霉素脂质体的处方为:磷脂100 mg;胆固醇20 mg;平阳霉素15 mg。优化工艺为采用浓度为250 mmol·L-1硫酸铵溶液制备空白脂质体,加入平阳霉素溶液后在40℃水浴中载药40 min。结论硫酸铵梯度法适用于制备平阳霉素脂质体,微柱离心法测定包封率准确可靠,处方工艺优化后的平阳霉素脂质体的包封率可以达到63.7%。     

抑肽酶和氨甲环酸对脑肿瘤切除术患者的疗效分析

目的分析抑肽酶和氨甲环酸对脑肿瘤切除术患者血液保护作用的疗效。方法收集入我院行脑肿瘤切除术的患者90例,将其按照随机原则进行分组,即分为对照组、抑肽酶组和氨甲环酸组,其中抑肽酶组患者在术前和术中给予患者抑肽酶静脉滴注,剂量为8×103 k IU/kg,氨甲环酸组同法静脉滴注氨甲环酸,剂量为12 mg/kg,对照组患者同法给予生理盐水静脉滴注,记录患者术中出血量,记录术前、术后的血小板值以及溶栓二聚体的量。结果对照组患者的术中出血量为(1005.23±192.91)mL,血小板术前为(197.86±51.32)×10~9/L,术后为(139.53±58.13)×10~9/L,溶栓二聚体的量术前为(0.42±0.24)mg/L,术后1 d为(1.08±0.22)mg/L;抑肽酶组患者的术中出血量为(657.38±182.43)mL,血小板术前为(201.14±55.47)×10~9/L,术后为(167.72±53.16)×10~9/L,溶栓二聚体的量术前为(0.49±0.18)mg/L,术后1 d为(0.52±0.21)mg/L;氨甲环酸组患者的术中出血量为(835.46±200.61)mL,血小板术前为(194.74±56.55)×10~9/L,术后为(148.43±53.12)×10~9/L,溶栓二聚体的量术前为(0.46±0.21)mg/L,术后1 d为(0.81±0.17)mg/L。结论抑肽酶能显著减少脑肿瘤切除术患者的术中出血量,提高了手术的安全性,效果优于氨甲环酸,值得在临床的日后工作中大力推广应用。     

栀子提取物类脂质体在大鼠体内的分布及靶向性研究

目的:研究栀子提取物类脂质体在大鼠体内的药物分布特点及靶向性。方法:采用改良薄膜分散法制备栀子提取物类脂质体。将30只大鼠按体质量随机均分为空白组、栀子提取物(57 mg/kg)组和栀子提取物类脂质体(131 mg/kg)组,ig相应药物,空白组ig等体积蒸馏水,每天1次,连续7 d。末次给药1 h后,取大鼠心、肝、脾、肺、肾组织,采用高效液相色谱法检测其中栀子提取物有效成分栀子苷的含量,通过药物靶向指数(DTI)定量评价栀子提取物类脂质体的体内靶向性。结果:与栀子提取物组比较,栀子提取物类脂质体组大鼠心、脾、脑组织中栀子苷含量明显增加(P<0.01),DTI分别为1.718、1.972、13.071;肝、肾组织中栀子苷含量明显降低(P<0.01),DTI分别为0.431、0.467。结论:栀子提取物类脂质体改变了栀子苷在大鼠体内的组织分布,可靶向作用于脑组织,减少了肝的首关效应。     

Box-Behnken效应面法优化丁香挥发油脂质体处方

目的:采用Box-Behnken效应面法优化丁香挥发油脂质体处方。方法:乙醇注入法制备丁香挥发油脂质体,以磷脂与丁香挥发油质量比(X1)、磷脂与胆固醇质量比(X2)、磷脂浓度(X3)为研究对象,包封率为评价指标,通过Box-Behnken效应面法筛选丁香挥发油脂质体最佳处方。结果:X_1=5,X_2=3.9,X_3=11.72mg/mL;丁香挥发油脂质体包封率为(73.74±2.27)%,与预测值偏差为0.81%;载药量为(12.79±0.43)%,粒径为(73.67±3.58)nm,PDI为0.221±0.024,Zeta电位为(-24.3±6.8)mV。结论:BoxBehnken效应面法可以简单有效的运用于丁香挥发油脂质体的处方优化。优化得到的脂质体处方合理,包封率较高,理化性质考察合格。     

麦胚凝集素修饰的长春瑞滨阳离子脂质体的处方优选及细胞毒性试验

目的:制备麦胚凝集素(WGA)修饰的长春瑞滨(VRB)阳离子脂质体(WGA-VRB阳离子脂质体),优化处方并进行细胞毒性试验。方法:以磷脂、胆固醇为辅料,以3β-[N-(N′-N′-二甲基氨基乙烷)-氨基甲酰基]胆固醇盐酸盐(DC-Chol)为阳离子材料,以二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)为长循环链,采用薄膜分散法及硫酸铵梯度法制备WGA-VRB阳离子脂质体。以包封率为指标,采用星点设计-响应面法优化处方中DC-Chol、胆固醇和VRB用量。测定所制VRB脂质体和WGA-VRB阳离子脂质体中VRB含量,比较二者和空白阳离子脂质体作用于人乳腺癌细胞MCF-7和人非小细胞肺癌细胞A549后对细胞存活率的影响。结果:5 m L WGA-VRB阳离子脂质体的最优处方为磷脂22 mg、胆固醇12 mg、DC-Chol 8 mg、VRB 0.5 mg,其包封率为(92.24±1.21)%(n=3),与预测值的相对误差为5.3%。VRB脂质体和WGA-VRB阳离子脂质体中VRB含量分别为(96.01±3.26)、(93.39±1.59)μg/m L(n=3);与空白阳离子脂质体和VRB脂质体比较,WGA-VRB阳离子脂质体可明显降低MCF-7、A549细胞的存活率(P<0.05)。结论:成功制得WGA-VRB阳离子脂质体,其对MCF-7、A549细胞存活的抑制作用强于VRB脂质体。     

液相色谱-串联质谱法研究重组水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的一级结构

利用液质联用研究重组水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的一级结构。采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的相对分子质量；采用液质联用分别分析HV12p-rPA的胰蛋白酶(trypsin)和胰凝乳蛋白酶(chymotrypsin)酶解产物。MALDI-TOF-MS测得HV12p-rPA的相对分子质量为41 472 Da，与理论值相符；HV12p-rPA的胰蛋白酶(trypsin)酶解产物进行液质联用分析结果表明该融合蛋白中含有瑞替普酶序列；HV12p-rPA的胰凝乳蛋白酶(chymotrypsin)酶解产物进行液质联用分析，结果表明融合蛋白中含有水蛭素12肽，并检测出融合蛋白中的链接肽(DEGGGSY)，融合蛋白的N末端MASDF和C末端LDWIRDNMRP；采用液质联用进行肽图分析，识别出的多肽匹配度Xcorr均超过1.5，部分多肽的Xcorr超过3.0，表明本实验测定过程中多肽识别结果准确可靠；目标蛋白的氨基酸序列覆盖率超过85%。结果确定了HV12p-rPA融合蛋白的序列与理论值相符。     

Preparation of novel double liposomes using the glass-filter method

The glass-filter method, a newly developed preparative method for liposomes, was applied for preparation of novel double liposomes. Double liposomes were prepared by filtering a suspension of liposomes prepared using a G4 filter (pore size: 10-16 microm) into a G3 filter (pore size: 40-100 microm) coated with a similar lipid layer. The morphological structure of the double liposomes was confirmed using scanning electron microscopy by the freeze-fracture method to be multivesicular vesicles consisting of small liposomes enveloped in larger liposomes. The diameter of liposomes prepared using the G4 filter was 0.8-2 microm and that of liposomes prepared using the G3 filter or double liposomes was 5-10 microm. These results suggested that the particle size of liposomes is dependent on the pore size of the glass-filter. The encapsulation efficiencies of double liposomes for brilliant blue FCF (BB) and erythrosine (ER) were higher than those of liposomes prepared by the standard Bangham method. Double liposomes showed suppressed release of BB or ER compared with normal liposomes. In particular, no release of BB was observed from the double liposomes prepared with stearylamine. These findings implied that the outer lipid layer protects the inner liposomes. The glass-filter method is the only method that we can get the double liposomes in a short period, and double liposomes prepared by this novel method had adequate size and good stability in vitro.     

Polymerized phospholipid vesicles containing amphotericin B: evaluation of toxic and antifungal activities in vitro

We have prepared lipid vesicles (liposomes) composed of polymerized bis[12-(methacryloyloxy)dodecanoyl]-L-alpha-phosphatidylcholine (DPL) which contain the antifungal polyene antibiotic amphotericin B (AMB). It was necessary to devise a novel method for incorporating AMB into the liposomes subsequent to polymerization. The polymer liposome AMB was as effective as AMB in "conventional" liposomes in terms of inhibiting fungal growth in vitro. However, in contrast to "conventional" liposomes, the polymerized DPL vesicles did not protect mammalian cells against AMB induced toxicity.     

Encapsulation of lipopeptides within liposomes: effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C8, C12 and C16. The C12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers.     

Topical application of liposomal cobalamin hydrogel for atopic dermatitis therapy

Topical vitamin B12 was shown to be effective for atopic dermatitis. However, vitamin B12 itself is light sensitive and has low skin permeability, thus reducing its therapeutic effectiveness. In the present study, we prepared a liposomal hydrogel of adenosylcobalamin (AdCbl), a vitamin B12 derivative, and investigated possible beneficial effects of AdCbl on atopic dermatitis using an NC/Nga murine atopic dermatitis model. AdCbl was loaded into liposomes prepared by a thin film hydration method using a pH gradient method that employed citric acid buffer solution. This resulted in AdCbl-loaded liposomes that were 106.4 +/- 2.2 nm in size. The loading efficiency was 40% (of the initial AdCbl amount). Lipo-AdCbl had enhanced skin permeability, being about 17-fold compared with AdCbl-gel. Topical administration of Lipo-AdCbl-gel to 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like skin lesions in NC/Nga mice ameliorated lesion intensity scores, dorsal skin thickness, and total serum IgE in a concentration-dependent manner. Other preparations, including AdCbl solution, AdCbl cream, liposomes alone, and a mixture of AdCbl solution and liposomes had little effect. Taken together, our findings indicate that Lipo-AdCbl-gel has protective effects against atopic dermatitis symptoms, and suggest that it may be of benefit in the treatment of human inflammatory skin diseases.     

荆豆凝集素修饰脂质体对小鼠口服吸收胰岛素的促进作用

目的研究荆豆凝集素(UEA1)修饰的胰岛素脂质体在小鼠胃肠道的吸收作用。方法采用碳二亚胺偶联法制备荆豆凝集素修饰的磷脂酰乙醇胺(PE)，将PE-UEA1参入胰岛素脂质体中制成凝集素修饰脂质体，并证实UEA1凝集活性不受影响。分别对正常小鼠及糖尿病模型小鼠灌胃给予350 u·kg^-1胰岛素的修饰脂质体溶液，用葡萄糖酶试剂盒测定小鼠血糖，并与同剂量普通胰岛素脂质体比较。结果对于正常小鼠，荆豆凝集素修饰脂质体在4 h使血糖降至初始水平的(84±15)%，8 h降至(78±11)%，12 h为(90±12)%。胰岛素普通脂质体几乎没有降糖作用，与生理盐水对照组相当。对于糖尿病模型小鼠，荆豆凝集素修饰脂质体在4 h使血糖降至初始水平的(73±7)%，8 h降至(74±9)%，12 h为(86±9)%。结论荆豆凝集素修饰的脂质体可能通过与M细胞表面特异性受体的结合促进大分子药物的胃肠吸收。     

Preparation of a TK/GCV administration system mediated by transferrin modified pro-cationic liposomes

Transferrin modified pro-cationic liposomes were prepared and used to investigate the effect of targeting therapeutic genes to human hepatoma carcinoma cells in vitro. The main lipid CHETA, cholest-5-en-3beta-yl[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl]amino]ethyl] carbamate (C36H61N3O4S2), was synthesized and used to prepare pro-cationic liposomes. The thymidine kinase (TK) gene loaded pro-cationic liposomes were prepared by first mixing the plasmid DNA and protamine together, and then incubating the resulted polyplexes with blank pro-cationic liposomes preformed by the thin film dispersion-sonication method. Transferrin (Tf) was adsorbed on the surface of pro-cationic liposomes via electrostatic interactions to form transferrin modified pro-cationic liposomes. Cellular association was measured by fluorimetry at excitation and emission wavelengths of 490 and 520 nm, respectively. The viability of TK gene infected cells following administration of ganciclovir (GCV) was investigated by MTT assay. The transferrin modified TK gene pro-cationic liposomes had a mean diameter of 240 +/- 12 nm and zeta potential of -24.10 +/- 2.5 mV (n = 3). The transmission electron microscopy image indicated that most of the liposomes were relatively regular and spherical with a condensed core inside. Cell-associated fluorescence of Tf-liposomes and unmodified liposomes (without transferrin) was 7.8 x 10(6), and 3.2 x 10(6) per milligram protein, respectively. Compared to Lipofectamine 2000 (Invitrogen, USA) the pro-cationic liposomes and transferrin modified pro-cationic liposomes had less cytotoxicity to cells. The transduced TK gene HepG2 cells were more sensitive to GCV than the un-transduced TK gene ones and the human normal Chang liver cells were not affected by the TK/GCV system mediated by procationic liposomes.     

蔗糖八硫酸酯三乙胺梯度法制备重酒石酸长春瑞滨脂质体

 目的：制备重酒石酸长春瑞滨脂质体,并对制备工艺进行考察。方法：以蔗糖八硫酸酯三乙胺（triethylammonium sucrose octasulfate,TEA8SOS）梯度法制备重酒石酸长春瑞滨脂质体,以阳离子交换树脂分离脂质体与游离药物;并以紫外分光光度法和激光粒度仪分别测定重酒石酸长春瑞滨脂质体的包封率和粒径。结果：重酒石酸长春瑞滨脂质体包封率约为95%,粒径为129.5 nm。结论：以蔗糖八硫酸酯三乙胺梯度法制备的重酒石酸长春瑞滨脂质体包封率较高,方法可行。     

Synthesis of transferrin (Tf) conjugated liposomes via Staudinger ligation

Staudinger ligation was evaluated as a strategy for synthesizing receptor targeted liposomes. First, an activated lipid derivative was synthesized by reacting dioleoyl phosphatidylethanolamine (DOPE) and 2-(diphenylphosphino) terephthalic acid 1-methyl 4-penta-fluorophenyldiester. Second, transferrin (Tf) was activated with p-azidophenyl isothiocyanate. Third, liposomes containing the activated lipid were prepared and then coupled to the activated Tf via the Staudinger reaction. These liposomes were evaluated in KB cells for cellular uptake and cytotoxicity, and in mice for pharmacokinetic properties. Tf-derivatized liposomes encapsulating calcein prepared by this conjugation method effectively targeted Tf receptor expressing KB cells. In addition, the Tf-targeted liposomes entrapping doxorubicin showed greatly enhanced in vitro cytotoxicity relative to non-targeted control liposomes. Pharmacokinetic parameters indicated that these liposomes retained long circulating properties relative to the free drug. In summary, Staudinger ligation is an effective method for the synthesis of receptor targeted liposomes.     

Chemotherapy with hybrid liposomes for lymphoma without drugs in vivo

Hybrid liposomes composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (n) dodecyl ether (C(12)(EO)(n), n=21 and 25) were prepared with the method of sonication. Clear solution of hybrid liposomes having hydrodynamic diameter of 80-100 nm could be maintained over 3 weeks. Hybrid liposomes induced apoptosis for human lymphoma (MOLT-4 and RAJI) cells in vitro. No toxicity was observed in the rats after intravenously injecting hybrid liposomes in vivo. We clearly demonstrated that a mouse model of lymphoma was established and prolonged survival was obtained in mice models of lymphoma after the treatment with hybrid liposomes without drugs in vivo. The results in this study should support the prolonged survival for patients with lymphoma in clinical applications.