奥沙利铂长循环热敏脂质体的包封率测定及体外释放考察

目的:建立奥沙利铂脂质体的包封率和含量测定方法,并对其体外释放进行考察.方法:采用逆相蒸发法制备奥沙利铂脂质体,用葡聚糖凝胶G-50柱分离载药脂质体和游离药物,以水为洗脱液,RP-HPLC法测定包封率,采用透析法考察脂质体在37℃和41℃的体外释放规律.结果:柱层析分离方法的药物回收率为99.25%,柱加样回收率为100.37%;药物含量测定方法的工作曲线为A =3E 06c-3290.8(n=5),r=0.9999,线性范围0.0201～2.0100 mg/ml,方法回收率为100.70%;脂质体在41℃条件下的释放规律符合一级动力学过程.结论:本方法简便、灵敏、准确,可以用于测定奥沙利铂脂质体的含量和包封率,所制得的脂质体具有明显的热敏释放特征.     

盐酸表柔比星长循环热敏冻干脂质体的处方工艺研究与体外释药机制探讨

目的研究盐酸表柔比星长循环热敏冻干脂质体（EPI-LTSL）的处方工艺,并探讨其体外释药机制。方法pH梯度法制备EPI-LTSL并进行冷冻干燥,正交试验优化处方,单因素试验优化工艺。通过观察不同温度下EPI在不同介质中形成沉淀的性状,探索EPI-LTSL的体外释药机制。结果最佳处方及工艺为药脂比1∶10,DPPC/MSPC为82∶8,水化介质为250mmol/L柠檬酸盐（pH4.0）。空白脂质体经15000psi（103MPa）高压均质循环5～6次,100nm聚碳酸酯膜挤压过膜3次;调节脂质体外相pH后,35℃水浴20min载药。冻干脂质体内外相乳糖浓度为9%,采用中速预冻（-65℃）及缓慢的解吸干燥可以制备较好的冻干品。EPI与柠檬酸盐形成的沉淀在42℃可以完全溶解,而在25℃成絮状或片状沉淀。结论制备的EPI-LTSL载药量和包封率较高,粒径较小。冻干脂质体外观平整,内部疏松多孔,色泽鲜亮,复水迅速且粒径变化较小。以柠檬酸盐为水化介质制备的EPI-LTSL,在25℃时包载于脂质体内相的EPI以沉淀稳定存在,而在42℃时可完全溶解释放出来。     

99Tcm-MIBI脂质体的制备及特征分析

目的 研究99Tcm-甲氧基异丁基异腈（MIBI）脂质体的制备方法,并对其特征进行分析。方法 采用乙醇注入-超声法制备99Tcm-MIBI脂质体,测定脂质体粒径大小和包封率,对超声时间、大豆卵磷脂与胆固醇的质量比等条件进行优化,观察99Tcm-MIBI脂质体的稳定性。结果 当制备的99Tcm-MIBI质量浓度为0.01 mg/mL时,平均粒径大小为（171.4±25.2）nm,包封率的平均值为（8.5±1.3）%。大豆卵磷脂与胆固醇的质量比为4:1和超声时间为5 min为最佳条件。电镜图显示,制备第1天后粒径较小,形态较为规则、均匀,呈完整球形或椭圆形;30 d后粒径大小及形态未见明显改变。结论 采用乙醇注入-超声法制备的99Tcm-MIBI脂质体粒径较小、包封率高,且较稳定。     

交错融合脂质体的制备

目的研究交错融合脂质体（IFVs）的制备。方法采用改良方法制备交错融合脂质体（IFVs）,应用激光散射粒度分析仪（PCS）对其粒径大小及分布情况进行测定,利用高压液相色谱仪测定其包封率。结果 IFVs脂质体粒径为100 nm左右。包封率为（69.0±5.4）%,远高于其他类型脂质体,并且性质稳定。结论 IFVs是一种大单层脂质体,其特点是粒径均匀（100 nm）,包封容积大,可达20~25μl/μmol脂质,可以携带足够量药物。     

马尼地平脂质体的制备及其质量研究

目的优选马尼地平脂质体的处方和制备工艺,并对其进行质量研究。方法采用乙醇注入法制备马尼地平脂质体,并对其形态学、粒径、包封率、初步稳定性等性质进行了研究。结果在透射电子显微镜下观察,制备的马尼地平脂质体为均匀的球状或近球状,平均粒径为233．9nm,包封率为89．6％,脂质体4℃放置稳定。结论选择乙醇注入法优化工艺制备马尼地平脂质体稳定可行,为进一步开发马尼地平新制剂提供了实验依据。     

促黄体激素释放激素a(LHRHa)靶向鸦胆子油脂质体的制备及质量评价

目的 制备促黄体激素释放激素a(LHRHa)靶向鸦胆子油脂质体并评价其质量.方法 采用薄膜分散法结合生物素-链霉亲和素桥接法制备LHRHa靶向鸦胆子油脂质体,正交设计优选处方,透射电镜下观察脂质体形态,采用Zetasizer Nano ZS分析仪测定脂质体粒径、Zeta电位,葡聚糖凝胶柱层析结合分光光度法测定包封率,通过离心加速实验及渗漏率考察脂质体稳定性,体外细胞实验鉴定脂质体靶向性.结果 所得优化处方为磷脂与胆固醇比4∶1,药物与脂质比3∶10,DSPE-PEG-Biotin与卵磷脂质量比为3％,超声乳化时间为8min.按此处方制备的脂质体形态圆整,粒径为155.1±14.5nm,Zeta电位为-24.1±0.54mV,平均包封率达92.2％,对卵巢癌A2780/DDP细胞的亲和力约为普通脂质体的2.7倍.结论 LHRHa靶向鸦胆子油脂质体制备工艺可行,同时具有包封率高、稳定性及靶向性良好等优点.     

HER-2靶向硼脂质体的制备与影响因素研究

目的制备具有良好靶向性的硼脂质体，为硼中子俘获治疗的研究与应用建立一个有效的靶向给药途仆方法采用新型的主动载药方法-pH梯度法制备硼脂质体，分析不同药脂比例对包封率的影响，优化Trastuzumab连接到DSPE—PEG-NHS分子微团的时间因素，采用微团转移法将Trastuzumab-PEG-DSPE掺入预先制备的硼脂质体，比较不同时间和温度对掺入量的影响，测试脂质体的稳定性、温度对Trastuzumab与受体结合能力的影响以及硼脂质体的受体靶向特异性。结果制备的硼脂质体大小均匀，直径约为100nm，圆形小囊状，药物包封率高。Trastuzumab与DSPE-PEG-NHS室温下孵育1h可达到足够高的连接率。60℃的水浴中作用4h，90％的Trastuzumab-PEG-DSPE掺入了脂质体脂膜。脂质体稳定，37℃保存2周仍有95％的抗体与脂质体相连。实验所用温度对Trastuzumab与HER-2结合的能力影响不大。制备的硼脂质体与HER-2受体的结合能被浓度不断增高的Trastuzumab所置换。结论本法制备的硼脂质体大小均匀，外观圆整，药物包封率高，有良好稳定性。实验过程对抗体特性无明显影响，硼脂质体具备与HER-2受体特异性结合的能力。     

鬼臼毒素固体脂质纳米粒的制备及对人表皮细胞体外增殖的影响

目的研究鬼臼毒素固体脂质纳米粒(POD-SLN)对人表皮细胞增殖的影响。方法采用超声乳化法制备POD-SLN混悬液,采用扫描电镜观察其粒径大小和形态,粒径分析仪测定其粒径和电位,高效液相法测定其包封率,并观察其稳定性。用POD-SLN作用于体外培养的人表皮细胞,并于6、12、24、48h检测细胞的生长情况。实验设POD-SLN组、鬼臼毒素普通脂质体组、鬼臼毒素(POD)组、空白固体脂质纳米粒(SLN)组、空白对照组,MTT法测定各组对人表皮细胞增殖的抑制作用。结果制备的POD-SLN呈圆球形或椭圆形,稳定性好,粒径为87.2±10.3nm,电位25.3±0.8mV,包封率为83.2%±2.5%。POD-SLN对人表皮细胞增殖的抑制作用呈浓度和时间依赖性。POD-SLN、POD普通脂质体及POD作用48h对人表皮细胞的抑制率最高分别为91.05%、77.02h.46%,IC50分别为2.11、16.65、101.42μg/L。空白SLN对人表皮细胞增殖无影响。结论该制备工艺可行,所制备的POD-SLN在体外能有效抑制人表皮细胞增殖,且作用强于POD及普通POD脂质体。     

具有肝脏靶向性的白藜芦醇脂质体

目的制备白藜芦醇脂质体(RES-LIP)和半乳糖苷修饰的白藜芦醇脂质体(RES-GLIP),探讨并比较二者的肝靶向作用。方法采用薄膜超声分散法制备RES-LIP和RES-GLIP;透射电镜观察其形态及粒径分布;RP-HPLC测定载药量、包封率。小鼠尾静脉给药后,RP-HPLC测定白藜芦醇(Resveratrol,RES)在血清及肝、脾、肾、肺中的浓度。结果RES-LIP和RES-GLIP的粒径分别为(100.5±40.2)nm和(80.4±42.3)nm,载药量分别为19.81%和19.11%,包封率分别为96.52%和95.87%。RES-sol、RES-LIP和RES-GLIP的靶向效率分别为0.29、0.46和3.87。RES-GLIP的靶向作用明显强于RES-LIP和RES-sol。结论RES-GLIP在体内具有良好的肝靶向性,制备半乳糖苷修饰的白藜芦醇脂质体对提高药物疗效,减少用药剂量,降低药物毒副作用具有显著意义。     

正交试验优选冬凌草甲素脂质体制备工艺

目的优选冬凌草甲素脂质体的较优制备工艺。方法以胆固醇、大豆磷脂为载体,采用乙醇注入法制备冬凌草甲素脂质体。采用包封率作为为评价指标,以正交实验设计选出制备脂质体的最佳处方。结果通过正交设计优选出冬凌草甲素脂质体的制备工艺条件为:冬凌草甲素∶卵磷脂为1∶20,胆固醇与卵磷脂质量比为5∶1,超声时间为20 min,温度为50℃。结论该制备工艺得到的脂质体包封率较高,形态较好,方法可行。     

优化积雪草苷非离子表面活性剂囊泡的处方和工艺

 目的 筛选积雪草苷非离子表面活性剂囊泡的最优处方和最佳制备工艺。方法 采用薄膜蒸发法制备积雪草苷囊泡,以包封率和载药量为指标对非离子型表面活性剂的种类和用量、投药量、胆固醇用量、孵化时间、孵化温度等处方因素和工艺条件进行单因素考察,正交设计筛选处方、优化制备工艺,并对积雪草苷囊泡的最优处方和工艺进行验证。结果 因素考察试验中,对积雪草苷囊泡包封率和载药量影响的顺序依次为:投药量>孵化温度>孵化时间。优化处方及工艺为:积雪草苷36 mg,孵化温度30 ℃,孵化时间30 min,其包封率和载药量分别为89.56%和25.26%,镜下观察成典型囊形。结论 采用薄膜蒸发法所制备的积雪草苷囊泡呈圆形,分散性好,囊泡的包封率和载药量均较高。     

Gadolinium-DTPA liposomes as a potential MRI contrast agent. Work in progress

To evaluate the use of liposomes containing Gadolinium-DTPA (Gd-DTPA) as potential intravascular contrast agents, we synthesized and tested Gd-DTPA liposomes. A freeze-thaw extrusion process was used to synthesize neutral unilamellar vesicles. Using this technique, we prepared 0.4 micron vesicles with encapsulation efficiency as high as 39% for Gd-DTPA. In vitro dialysis showed that essentially 100% of the Gd-DTPA was retained with the liposomes after 72 hours of dialysis. MR imaging of in vitro samples showed concentration-dependent increase in signal intensity with Gd-DTPA liposomes. Imaging of rats after intravenous injection of Gd-DTPA liposomes showed sustained intravascular contrast enhancement of vascular structures and liver greater than free Gd-DTPA. There was no evidence of acute toxicity in rats during the imaging experiments or on follow-up of two months. Paramagnetic liposomes may be useful to enhance the vasculature, liver, and spleen.     

Evaluation of the dehydration-rehydration method for production of contrast-carrying liposomes

We measured the amounts of three types of radiographic contrast media (RCM) that could be entrapped in liposomes prepared by the dehydration-rehydration vesicle (DRV) technique. To make DRVs, one initially makes water-containing, small unilamellar vesicles, adds contrast media and lyophilizes the mixture. Upon rehydration, the DRVs re-form, passively entrapping RCM. Diatrizoate, iohexol and iotrolan proved to be entrappable in similar amounts (diatrizoate was best), but all of these amounts were less than for other small molecules, such as carboxyfluorescein (P less than 0.05). Entrapment was directly proportional to lipid concentration (r = 0.76; P less than 0.002), and inversely related to iodine concentration (r = 0.86; P less than 0.002). Under ideal conditions with neutral lipids, 19.45 +/- 9.9% of diatrizoate was entrapped, corresponding to 1.05 +/- 0.50 g I per g lipid. These values are close to those achievable for large unilamellar vesicles. Use of an automated mixing device (the Microfluidizer) in place of sonication, facilitated production of large liposome batches and improved entrapment (P less than 0.05). Computed tomography (CT) scans of rats showed 30 and 218 HU of liver and spleen enhancement, respectively, per g I/kg injected DRVs. These studies showed this method (possible augmented by the Microfluidizer) allows efficient production of contrast-carrying liposomes.     

Selective MR imaging of labeled human peripheral blood mononuclear cells by liposome mediated incorporation of dextran-magnetite particles

Human peripheral blood mononuclear cells (PBMCs) were incubated with large unilamellar vesicles (LUV) containing encapsulated dextran-magnetite particles (DMP). This resulted in an efficient incorporation of DMP. Electron microscopy revealed the presence of DMP in cells mainly in phagosomes and secondary lysosomes. DMP-labeled PBMCs showed a strong increase of the transverse relaxation rate (up to 16.6 s^?1 for 5 x 10⁷ cells/ml) and, accordingly, a great loss of signal intensity in MR imaging. The fraction of DMP containing PBMCs could be enriched by magnetic cell separation. The major population of the DMP containing cells proved to be monocytes. When PBMCs depleted of monocytes were used for labeling, DMP uptake was observed also in the peripheral blood lymphocytes. The labeling of PBMCs presented here may be used in future studies of selective MR imaging of in vivo cell migration in a variety of immunologically compromised tissue states, e.g., tumors, transplantations, and abscesses.     

A phospholipid spin label used as a liposome-associated MRI contrast agent

Given current clinical use of phospholipid bilayer structures (liposomes/vesicles) as nontoxic drug delivery vehicles, we have addressed the possibility of employing the phospholipids themselves as MRI contrast agents. To this end we have synthesized phosphatidylcholine with a nitroxide spin label replacing one methyl residue of the choline headgroup. This material was mixed with natural phosphatidylcholine in mole ratios from 1:50 to 1:1 and used to prepare sonicated unilamellar vesicles in saline. Expected structural features of these vesicles were verified by freeze-fracture electron microscopy. Proton T1 values of saline were readily decreased to less than 0.3 s by such preparations, yielding a net relaxivity of 0.6 M-1 s-1. The approach seems to be a realistic way of firmly associating a contrast agent of minimal toxicity with ordinary liposomes/vesicles in a manner that is not subject to leakage.     

An improved method for the preparation of liposomal gadolinium-DTPA. Ionophore-mediated active entrapment of gadolinium

We have developed an improved method for the production of liposomal gadolinium-DTPA (Gd-DTPA). The ionophore A23187 facilitates the uptake of externally added Gd into the interior aqueous space of a unilamellar lipid vesicle, where it is chelated by passively entrapped DTPA to form the Gd-DTPA chelate in situ. The presence of a pH gradient across the vesicle membrane is not essential for Gd uptake, the extent of which apparently is limited only by the interior concentration of the chelator. Once formed internally, the Gd-DTPA complex is retained within the vesicles for at least several days at room temperature. Biodistribution studies in mice indicate that liposomal Gd-DTPA prepared by this ionophoretic loading procedure exhibits biodistribution and clearance characteristics similar to 153Gd-DTPA-labeled liposomes prepared by means of passive entrapment of the preformed chelate.     

高效液相色谱法测定奥扎格雷钠脂质体的包封率

 目的 建立高效液相色谱法(high performance liquid chromatography,HPLC)测定奥扎格雷钠脂质体包封率的检测技术。方法 采用HPLC,以C₁₈色谱柱分离,以0.05 mol/L磷酸二氢钾溶液(pH=7.0)-甲醇-乙腈(80∶15∶5)为流动相,检测波长为274 nm测定。结果 奥扎格雷在10～100 μg/ml浓度范围内线性关系良好,r=0.9997。日内、日间RSD均小于0.5%,方法回收率为(100.2±0.42)%,样品的平均包封率为(94.89±0.44)%。结论 该测定方法简单,准确度高,灵敏度好,适合奥扎格雷钠脂质体的包封率测定。     

Tumor uptake of 67Ga-carrying liposomes

The in vivo distribution, excretion, and tumor localization of liposome-encapsulated 67Ga in normal and Ehrlich tumor (solid form)-bearing mice were studied. In normal mice, multilamellar vesicles (MLVs) were taken up mainly by the liver and spleen, whereas small unilamellar vesicles (SUVs) exhibited a broader tissue distribution. When 67Ga was encapsulated in MLVs or SUVs, the excretion of the radiotracer in the urine and feces was less than that observed for free tracer at 72 h after i.v. administration. In tumor-bearing mice, SUVs were found to accumulate preferentially in tumors. The tumor uptake of neutral, positive, and negative SUVs was 10%-13% of the administered dose per gram of tumor tissue at 24 h after their injection. These values were about three times higher than those found for free 67Ga-nitrilotriacetic acid (67Ga-NTA) or 67Ga-citrate. Significant differences in tumor uptake due to different surface charges of liposomes were not observed. Enhanced tumor-to-blood and tumor-to-muscle ratios were also observed at 24 h after injection. These results suggest that 67Ga-carrying liposomes may be a useful for tumor imaging.