用作基因载体的阳离子脂质体及其相关材料与制备技术

综述近年来阳离子脂质体在基因转染中的应用及其相关材料和制备技术。基因疗法面临的技术问题之一是寻找合适的基因载体,阳离子脂质体作为一种非病毒载体,具有无免疫原性、可自然降解等优点,在肺部、眼部疾病以及癌症的治疗中极具应用价值。随着研究的不断深入,新型脂质体材料相继出现、制备工艺也不断更新,阳离子脂质体的理化性质和转染效率均得到了极大的改善,推动了基因疗法和基因转染研究的发展。     

星点设计-效应面法优化平阳霉素脂质体的处方及工艺

目的：优化载平阳霉素脂质体的处方及其制备工艺。方法：以其包封率为考察指标,以平阳霉素与所用脂质的比、所用磷脂与胆固醇的比、制备温度为考察项,采用星点设计-效应面法优化盐酸平阳霉素脂质体的处方及工艺。结果：确定其最优处方为平阳霉素与脂质的比为0.15（W/W）,所用磷脂与胆固醇的比为4.5（W/W）,制备温度为42.3℃,实测值与预测值的最终差异显示无统计学意义。该脂质体的平均粒径为136 nm,Zeta电位为（-28.55±6.81）mV。结论：所选制备工艺较为合理,星点设计-效应面法可用于该制剂的处方优化。     

星点设计-效应面法优化吴茱萸碱脂质体的处方

摘 要 目的：优化吴茱萸碱脂质体的处方。 方法: 采用薄膜分散法制备脂质体,并采用星点设计 效应面法以包封率为考察指标,对大豆卵磷脂与药物质量比、大豆卵磷脂与胆固醇质量比、大豆卵磷脂浓度进行二项式拟合,通过三维效应面图和等高线选择最佳处方,对预测结果进行验证,并观察其外观、粒径及pH。结果: 最佳处方为大豆卵磷脂与药物质量比30.58∶1,大豆卵磷脂与胆固醇的质量比15.22∶1,大豆卵磷脂浓度为42.26 mg·mL^-1,制得吴茱萸碱脂质体的平均包封率为92.89%。吴茱萸碱脂质体外观为乳白色半透明,呈圆球形或椭圆形的球粒,粒径为126 nm,pH为6.94±0.17。结论:采用该处方工艺制备吴茱萸碱脂质体稳定、可行。     

星点设计-效应面法优化α-细辛脑脂质体喷鼻剂的制备工艺

目的:采用星点设计-效应面法优化α-细辛脑脂质体喷鼻剂的制备工艺。方法:以乙醇注入法制备α-细辛脑脂质体喷鼻剂,采用星点设计-效应面法优化制备工艺,在单因素试验的基础上以磷脂含量、磷脂与胆固醇的质量比和磷脂与药物的质量比为自变量,以包封率为因变量,优化处方工艺,进行验证试验并在电镜下观察脂质体形态。结果:二项式方程拟合度高(R2=0.9074),预测性好;星点设计-效应面法优选出的最佳工艺条件为磷脂含量为4.5%、磷脂与胆固醇的质量比为7.5、磷脂与药物的质量比为45,测得包封率为68.1%;最佳工艺验证结果与二项式拟合方程预测值偏差为1.73%;制备的脂质体形态完整,双分子膜结构清晰,均属于单室脂质体。结论:应用星点设计-效应面法能够精确有效地优化α-细辛脑脂质体喷鼻剂的制备工艺,稳定可行。     

星点设计-效应面法优化溴新斯的明多囊脂质体的制备工艺

目的采用星点设计-效应面法优化溴新斯的明多囊脂质体的制备工艺。方法采用复乳法制备溴新斯的明多囊脂质体,以包封率为评价指标,用星点设计优化处方,并进行多元线性回归与二项式方程拟合,采用效应面法选取较佳工艺条件,并进行预测。结果以磷脂与胆固醇为1.41∶1投料、主药浓度为22.98 g·L-1、三油酸甘油酯为24.93 mmol·L-1的优化条件制备溴新斯的明多囊脂质体,包封率为80.34%±1.53%,与二项式拟合方程预测结果相差小于2.5%。结论应用星点设计-效应面法优化溴新斯的明多囊脂质体的制备工艺,能快速方便地得到最佳制备工艺,预测性良好。     

星点设计-效应面法优化醋酸地塞米松脂质纳米粒的制备工艺

目的 优化醋酸地塞米松脂质纳米粒(DXMA-NLC)的制备工艺.方法 采用薄膜超声法制备DXMA-NLC.以投药量、表面活性剂的浓度和大豆油的量为考察因素,以粒径和包封率为考察指标,根据星点设计原理安排实验,并用二项式拟合建立指标与因素之间的数学关系,经效应面法预测最佳工艺条件.结果 采用15 mg DXMA、0.04...     

星点设计-效应面法优化羟基喜树碱/mPEG-S-S-C18纳米粒的制备工艺

目的 制备载羟基喜树碱（hydroxycamptothecin,HCPT）还原响应mPEG-S-S-C18纳米粒,采用星点设计-效应面法筛选优化制备工艺。方法 采用乳化-溶剂挥发法制备HCPT/mPEG-S-S-C18纳米粒,应用单因素法考察投药量、水相/油相体积比、超声功率以及超声时间对载药纳米粒包封率和载药量的影响。在此基础上,以包封率和载药量作为评价指标,采用Design-Expert V8.0.6软件进行星点设计,优化载药纳米粒的制备工艺。结果 优化获得的HCPT/mPEG-S-S-C18纳米粒制备工艺投药量为1.0 mg,水相/油相体积比为4.56∶1,超声功率为562.5 W。该工艺制备的载药纳米粒包封率为（58.14±1.04）%,载药量为（3.46±0.22）%,平均粒径为（322.9±9.52） nm,多分散性指数为0.195±0.05,Zeta电位为（-17.5±2.11） mV。结论 乳化-溶剂挥发法适用于制备HCPT/mPEG-S-S-C18纳米粒,星点设计-效应面法可优化获得载药纳米粒的最佳制备工艺,所得的载药纳米粒包封率和载药量较高,所建立的数学模型预测性良好。     

Preparation and gene delivery of alkaline amino acids-based cationic liposomes

Cationic lipids 1, 2, and 3, based on hydrophobic cholesterol linked to L-lysine, L-histidine or L-arginine, respectively, were designed and tested as gene delivery vectors. Physicochemical and biological properties of all liposomes and lipoplexes were evaluated, including lipid-DNA interactions, size, morphology, zeta potential, acid-base buffering capability, protection of DNA from DNase I digestion, and cytotoxity. The efficiency of luciferase gene transfection of lipoplexes 1-3 was compared with that of commercial dioleoyl-trimethylammonium propane (DOTAP) and polyethyleneimine (PEI) in 293T cells and HepG2 cells with or without poly(ethylene glycol) PEG stabilizer. The complexation and protection of DNA of liposome 3 was the strongest among the three liposomes. The efficiency of gene transfection of liposomes 1-3 was two-to threefold higher than that of PEI and/or DOTAP in 293T cells. Liposomes 1 and 3 in PEG as stabilizer showed sixfold higher transfection efficiency than that of PEI and/or DOTAP, whereas liposome 2 showed very low transfection efficiency. In HepG2 cells, the transfection efficiency of all the cationic liposomes was much lower than that of DOTAP. In conclusion, lipids 1-3 were efficient and non-toxic gene vectors; the headgroup of cationic lipids and the stabilizer of liposome formulation had an important influence on gene transfection.     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

DC-Chol/DOPE cationic liposomes: A comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

Cationic liposomes (CLs) composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposomes) have been classified as one of the most efficient gene delivery systems. Our study aims to examine the effect of the molar ratio of DC-Chol/DOPE, PEGylation and serum on the pDNA (plasmid pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol/DOPE, respectively. The transfection efficiency of DC-Chol/DOPE liposomes increased along with increased weight ratio of DC-Chol/siRNA. However, the pDNA transfection efficiency decreased along with increased weight ratio of DC-Chol/pDNA from 3/1. As expected, PEGylation decreased siRNA and pDNA transfection efficiency of DC-Chol/DOPE liposomes. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-Chol/pDNA from 3/1 did not lead to higher pDNA transfection efficiency, whereas increased weight ratio of DC-Chol/siRNA resulted in increased siRNA transfection efficiency. Furthermore, the serum did not significantly inhibit the pDNA and siRNA transfection efficiency of DC-Chol/DOPE liposomes. In conclusion, our results elucidated the influence factors of DC-Chol/DOPE liposome transfection and would reveal that siRNA and pDNA transfection mechanisms were different in DC-Chol/DOPE liposomes.     

Formulation optimization of itraconazole loaded PEGylated liposomes for parenteral administration by using design of experiments

The aim of this study was to systematically characterize and optimize the encapsulation process of itraconazole (ITZ) into the PEGylated liposomes. ITZ was used as a model compound for poorly soluble drugs. PEGylated liposomes were prepared using the film hydration method combined with sonication in order to produce small unilamellar vesicles (SUV). The concentration of encapsulated ITZ was measured by a reversed phase-high-performance liquid chromatography (RP-HPLC) method. Systematic characterization of encapsulation process was performed using the design of experiments (DoE) approach. The systematic screening of process parameters and well designed settings of parameter levels in optimization phase improved the ITZ encapsulation process. Optimization demonstrated that only minimal range of design space could be used to achieve the highest encapsulation efficiency (EE (%)), more precisely the EE of 90% was gained using 25 mg/ml of lipid and drug loading of 0.3% (w/w). However, the desirable drug loading could be predicted and adjusted by using mathematical modeling behind the DoE approach. The entire encapsulation process was shown to be repeatable with high significance (p < 0.05). It could be demonstrated that DoE plays an important role in optimization experiments leading to robust results supporting high quality.     

Ethosomes and ultradeformable liposomes for transdermal delivery of clotrimazole: A comparative assessment

The objective of work was to formulate, evaluate and compare the transdermal potential of novel vesicular nanocarriers: ethosomes and ultradeformable liposomes, containing clotrimazole (CLT), an anti-fungal bioactive. The ethosomal formulation (ET₄) and ultradeformable liposomal (UL) formulation (TT₃) showed highest entrapment 68.73 ± 1.4% and 55.51 ± 1.7%, optimal nanometric size range 132 ± 9.5 nm and 121 ± 9.7 nm, and smallest polydispersity index 0.027 ± 0.011 and 0.067 ± 0.009, respectively. The formulation ET₄ provided enhanced transdermal flux 56.25 ± 5.49 μg/cm²/h and decreased the lag time of 0.9 h in comparison to TT₃ formulation (50.16 ± 3.84 μg/cm²/h; 1.0 h). Skin interaction and FT-IR studies revealed greater penetration enhancing effect of ET₄ than TT₃ formulation. ET₄ formulation also had the highest zone of inhibition (34.6 ± 0.57 mm), in contrast to TT₃ formulation (29.6 ± 0.57 mm) and marketed cream formulation (19.0 ± 1.00 mm) against candidal species. Results suggested ethosomes to be the most proficient carrier system for dermal and transdermal delivery of clotrimazole.     

遗传算法在经皮给药微乳载体处方优化中的应用

以萘普生为模型药物，用遗传算法优化经皮给药微乳载体的处方。用伪三元相图法确定由Tween 80、IPM、乙醇和水组成的微乳区域。用3因素3水平的中心设计法制备载药量为1.12%的萘普生模型微乳，并进行离体兔皮的体外渗透实验。以稳态渗透速率的二次回归模型为目标函数，用遗传算法对中心设计结果进行优化，筛选出具有最大透皮速率的萘普生微乳载体处方。所得优化处方的组成为：21.41% Tween 80、15.17%乙醇、4.14% IPM和59.28%水，预计的稳态渗透速率为183.57 μg·cm^-2·h^-1。回代试验表明，以优化处方制备的萘普生微乳，其稳态渗透速率的平均值为189.43 μg·cm^-2·h^-1，高于预测值。结果表明，用遗传算法筛选微乳经皮给药载体处方，方法可行，结果合理、可靠。     

Encapsulation of ATP into liposomes by different methods: optimization of the procedure

Different methods and conditions for ATP incorporation into PEGylated liposomes were compared in order to obtain a preparation with a maximized ATP content. Such a preparation may find the application for the in vivo treatment of ischemic tissues suffering from an insufficient ATP supply. Several different methods of liposome preparation and purification were used and HPLC was employed to determine the concentration of ATP in the liposomes. Thin lipid film hydration produced vesicles with the lowest ATP encapsulation (ca. 5?mol%). A pH gradient method yielded liposomes with ca. 10?mol% of ATP. Reverse phase evaporation and freezing-thawing methods resulted in a maximum entrapment of ATP on the level of 36–38?mol%. The freezing-thawing method was chosen for further investigation because of its simplicity and absence of a need to use organic solvents. The separation of the non-entrapped ATP by gel-filtration, centrifugation or dialysis yielded virtually identical liposomal preparations. The incorporation of PEG (as PEG-distearoyl phosphatidylethanolamine, PEG-DSPE) into the liposomal membrane decreases the quantity of the entrapped ATP (from 38?mol% for liposomes with 0.5?mol% of PEG-DSPE to only 17?mol% for liposomes with 5?mol% of PEG-DSPE).     

Design and characterization of liposomes containing long-chain N-acylPEs for brain delivery: penetration of liposomes incorporating GM1 into the rat brain

Purpose. To develop a suitable liposomal carrier to encapsulate neu- roactive compounds that are stable enough to carry them to the brain across the blood-brain barrier with the appropriate surface characteristics for an effective targeting and for an active membrane transport. Methods. Liposomes containing glycosides and a fusogenic lipid were prepared by extrusion. Photon correlation spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry were used to characterize liposomal preparations. Tissue distribution was determined by using ³H-cholesterylhexadecylether as a marker. Results. The incorporation of glycoside determinants and N-palmitoylphosphatidylethanolamine gives liposomes with similar initial size, trapped volume, negative surface charge, bilayer fluidity, and melting temperature, except for monosialoganglioside-containing liposomes, which showed less negative surface charge and the highest size, trapped volume and melting temperature. All glycosilated formulations gave liposomes able to retain up to the 95% of encapsulated carboxyfluorescein after 90 min at physiologic temperature even in the presence of serum. Monosialoganglioside liposomes were recovered in the cortex, basal ganglia, and mesencephalon of both brain hemispheres. The liver uptake was higher for sulfatide- and glucose-liposomes, whereas the higher blood levels were observed for glucose- and mannose-liposomes. Conclusions. These results show the suitability of such liposomal formulations to hold encapsulated drugs. Moreover, the brain uptake of monosialoganglioside liposomes makes them good candidates as drug delivery systems to the brain.     

Application of Box-Behnken design in understanding the quality of genistein self-nanoemulsified drug delivery systems and optimizing its formulation

The purpose of the present study was to understand the effect of formulation variables of self- nanoemulsified drug delivery systems (SNEDDS) on the rapid dissolution of a model drug, genistein (GN). A three-factor, three-level Box-Behnken design was used to explore the main and interaction effect of several independent formulation variables including the amount of Maisine 35-1 and Labrafac Lipophile WL 1349 (1:1, w/w) (X₁), Cremophor EL and Labrasol (3:1, w/w) (X₂), and Transcutol P (X₃). Droplet size (Y₁), turbidity (Y₂), and dissolution percentage of GN after 5 (Y₃) and 30 (Y₄) min were the dependent variables. A mathematical relationship, Y₃?=???89.3447?+?5.9524X₁?+?1.0683X₂?+?0.462X₃???0.0825X₁²???0.0075X₂²???0.0009X₃²?+?0.0104X₁X₂ ??0.0113X₁X₃?+?0.0009X₂X₃ (r2?=?0.9604), was obtained to explain the effect of all factors and their co-linearities on the dissolution of GN at 5?min. Formulation optimization was then performed to maximize dissolution percentage of GN at 5?min (Y₃). The optimized formulation was predicted to dissolution 93.34% of GN at 5?min, when X₁, X₂ and X₃ values were 37.1, 101.7 and 77.3?mg, respectively. A new batch was prepared according to the optimized formulation, and the observed and predicted values of Y₃ were in close agreement. In conclusion, the Box-Behnken experimental design allowed us to understand the effect of formulation variables on the rapid dissolution of GN from SNEDDS, and optimize the formulation to obtain a rapid drug dissolution at 5?min.