Preparation,Characterization, and Pharmacodynamics of Thermosensitive Liposomes Containing Docetaxel

A novel thermosensitive liposome (TL) containing docetaxel (DTX) was designed to enhance DTX-targeted delivery and antitumor effect. TL loading DTX (DTX-TL) were prepared by thin film hydration. The mean particle size of the liposomes was about 100 nm, and the drug entrapment efficiency was more than 95%. The phase transition temperature of liposomes was about 42°C. In vitro drug release showed that drug released at 37°C was obviously less than that at 42°C. For in vivo experiments, the human breast tumor model was established by subcutaneous xenotransplantation on nude mice; liposomes and injection containing DTX were injected i.v. in nude mice, followed by exposure of the tumors to hyperthermia (HT) for 30 min after administration. The tumor inhibition ratio of DTX-TL group was significantly higher than the normal injection group. Combining TL with HT enhanced the delivery of DTX and thereby its antitumor effects. The liposomes reported in this paper could potentially produce viable clinical strategies for improved targeting and delivery of DTX for the treatment of breast cancer.     

In vitro and in vivo antitumor effects of lupeol-loaded galactosylated liposomes

Lupeol liposomes, modified with Gal-PEG-DSPE, were developed following a thin-film dispersion method. Then, the morphology, physicochemical properties, and in vitro release properties of those liposomes were investigated. The scanning electron microscopic images showed that most of the liposomes were spherical particles; they were similar in size and uniformly dispersed. Both lupeol liposomes and Gal-lupeol liposomes exhibited an average particle size of about 100 nm. The encapsulation efficiency was greater than 85%. The encapsulation efficiency of lupeol liposome and Gal-lupeol liposome, stored with 15% sucrose as glycoprotein for 6 months, was higher than 80%; although the particle size increased, they remained within 200 nm. The cell-uptake study demonstrated that the Gal-lupeol-liposome uptake efficiency was the highest in HepG2 cells. The HepG2 cells treated with the Gal-lupeol liposomes had higher apoptotic efficiency than the lupeol liposome and free lupeol. After HepG2 cells were treated with Gal-lupeol liposome, the expressions of AKT/mTOR-related proteins (p-AKT308 and p-AKT473) were also significantly reduced than the lupeol-liposome and free lupeol group. The in vivo targeting studies showed that Gal-NR-L exhibited liver-targeting effects on FVB mice. The pharmacodynamic study was performed by transfecting AKT and c-MET via the high-pressure tail vein of FVB mice. After Gal-lupeol-L administration, the liver index and liver weight of mice were less than those non-targeted group. The histopathological study showed that the lobular structure in the mice liver was clearer, the vacuoles were more obvious, and the cytoplasm was more abundant after Gal-lupeol-L administration. Also, the qRT-PCR study showed that AFP, GPC3, and EpCAM mRNA expression levels were significantly lower than those non-targeted lupeol-liposomes.     

多西他赛他莫昔芬脂质体体外释放研究

目的考察多西他赛他莫昔芬复方脂质体体外释放。方法薄膜分散法制备单方和复方脂质体样品,通过透析法进行脂质体体外释放,采用高效液相色谱法测定他莫昔芬和多西他赛药物浓度,并对体外释放曲线进行数学模型拟合。结果单方和复方脂质体中,他莫昔芬和多西他赛均无突释,多西他赛较他莫昔芬释放快,他莫昔芬和多西他赛释放模型符合一级释放方程,他莫昔芬和多西他赛的释放曲线相似。结论他莫昔芬和多西他赛在单方和复方脂质体体外释放并无显著差异。     

Preparation,Optimization and Characterization of Bovine Lactoferrin‐loaded Liposomes and Solid Lipid Particles Modified by Hydrophilic Polymers Using Factorial Design

Bioadhesive liposomes and solid lipid particles (SLPs) modified by pectin and chitosan for oral administration of bovine lactoferrin (bLf) were prepared using a 2⁴ full‐factorial design to identify the key formulation variables influencing particle size and drug entrapment efficiency (EE). Netlike structures of the polymer–particle mixture consisting of a polymeric network in which multiple particles were imbedded were observed by scanning electron microscopy (SEM). Chemical stability of bLf after encapsulation into pectin‐ and chitosan‐modified liposomes and SLPs was confirmed by Fourier transform infrared spectra (FTIR). Bovine lactoferrin was located within phospholipid bilayer, whereas in SLPs bLf was within the matrix. The crystalline nature of bLf after encapsulation was investigated by differential scanning calorimetry (DSC) of drug‐loaded particles, indicating amorphous dispersion of bLf in the polymer–lipid matrix of pectin‐ and chitosan‐modified liposomes and SLPs. In vivo pharmacokinetic investigation of bLf in pectin‐ and chitosan‐modified liposomes and SLPs showed prolonged mean residence time (MRT) of bLf in rat blood and increased the relative bioavailability (F_bio%) by 1.95‐ to 2.69‐fold compared with free bLf. The developed carrier systems are considered to be promising vehicles for oral delivery.     

PEG修饰鳖甲肽HGRFG脂质体的药动学研究

目的 制备鳖甲肽HGRFG脂质体以及聚乙二醇（polyethylene glycol,PEG）修饰后的长循环脂质体,考察经修饰的脂质体较未修饰脂质体在动物体内分布及滞留情况。方法 采用BLB/c裸鼠荧光活体成像实验,进行脂质体体内分布及代谢研究;采用药动学实验,初步探究2种载药脂质体在血浆内的滞留时间。结果 2种载药脂质体均能广泛分布于实验动物体内。与未经修饰的脂质体载药组相比,经PEG修饰的长循环脂质体载药组中裸鼠荧光全部消失的时间明显延长,药物在血浆滞留时间也明显延长。结论 经PEG修饰后的长循环脂质体可以延长药物在实验动物体内的滞留时间,延长药物半衰期。     

Design and synthesis of novel galactosylated polymers for liposomes as gene drug carriers targeting the hepatic asialoglycoprotein receptor

The 18-mer oligodeoxynucleotides (ODNs) that can inhibit survivin gene expression were selected as a model gene drug to study hepatic-targeting drug delivery system. Novel galactosylated polymers (cholesteryloxycarbonylamino) ethylamine-α,β-polyasparthydrazied (CHE-PAHy-Lacs), which target asialoglycoprotein receptor on hepatic parenchymal cells (PC), were designed and synthesized as non-toxic, non-antigenic and non-teratogenic ligands for liposomes. The liposomes incorporating different CHE-PAHy-Lacs were prepared and characterized by zeta potential and particle size analyzer. The drug encapsulation efficiency was measured by gel filtration method. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate was used as a marker for all the liposome preparations in the in vivo experiments. The CHE-PAHy-Lac liposomes produced a significant improvement in the encapsulation efficiency of ODNs (28.73–51.37%) compared with conventional liposomes (9.88%). The in vivo results showed that the liposomes incorporating CHE-PAHy-Lac, which contained about 30% (w/w) galactosyl residues, exhibited marked accumulation in the liver and hepatic PC. These results suggest that the novel galactosylated polymers used for liposomes have a great potential as a gene delivery system for hepatic targeting.     

川陈皮素自组装前体脂质体的制备及其大鼠体内药代动力学研究

本文研制川陈皮素自组装前体脂质体, 并以混悬剂为对照考察其经大鼠灌胃给药后的药代动力学行为。采用一种新型前体脂质体法制备川陈皮素自组装前体脂质体, 考察其水合后粒径、包封率和稳定性等理化性质; 大鼠分别灌胃给予川陈皮素混悬剂和水合后的脂质体, 以尼莫地平为内标, 采用HPLC法测定血浆中药物浓度, 用Kinatica 4.4程序计算药代动力学参数。制得的川陈皮素前体脂质体经水合后包封率可达80%以上,平均粒径为212.1 nm, 稳定性好; 药代动力学研究显示,与混悬剂相比川陈皮素脂质体在体内吸收较快, 相对生物利用度为264.3%, MRT增加。结果表明, 川陈皮素自组装前体脂质体制备工艺简单可行; 川陈皮素制成自组装前体脂质体后, 大鼠口服吸收显著增加。     

替诺福韦阳离子脂质体的制备及其对人体肝细胞SMMC-7721摄取和细胞毒性研究

目的:研究替诺福韦阳离子脂质体的制备及其促进肝实质细胞摄取的情况和细胞毒性作用。方法:采用叔丁醇冻干法制备替诺福韦阳离子脂质体,测定其包封率及理化性质;以SMMC-7721细胞为模型,研究脂质体对肝实质细胞摄取替诺福韦的促进作用,MTT法检测不同条件下载药脂质体对细胞的毒性情况。结果:制备的脂质体包封率为(88.3±1.6)%,粒径为(278.4±67.6)nm,Zeta电势为(31±5)mV;经半乳糖基及PEG修饰的脂质体较游离药物进入肝实质细胞的浓度明显升高,且时间延长;当替诺福韦脂质体、脂质浓度分别为7.5、30μg·mL-1时,细胞存活率在80%以上,毒性较小。结论:所制备的阳离子脂质体具有显著增加细胞摄取替诺福韦和保护替诺福韦的作用,有望成为抗病毒药物如替诺福韦等的高效传递系统。     

两亲性壳聚糖包覆紫杉醇脂质体的制备及体外释放研究

目的:制备两亲性壳聚糖N-辛基-N,O-羧甲基壳聚糖包覆紫杉醇脂质体(PTX-LP-OCC),并考察其理化性质及体外释放行为。方法:采用基于乙醇的前体脂质体法制备紫杉醇脂质体并以OCC包覆,并以普通脂质体(PTX-LP)为对照,测定其包封率、粒径大小、电位,观测其形态及稳定性,然后采用全体液平衡反向透析法研究体外释放行为。结果:紫杉醇脂质体包封率为89.5%,粒径为236.5 nm,Zeta电位为-31.4 mV,多糖包覆修饰后药物包封率无显著变化,粒径及Zeta电位显著增加,脂质体稳定性显著提高,药物释放呈缓释特征,且突释显著降低。结论:两亲性壳聚糖包覆脂质体是一个有前景的抗肿瘤药物递送载体     

多肽GRGDS修饰的紫杉醇长循环靶向脂质体的体外评价

目的:本研究以甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(glycine-arginine-glycine-aspartic acid-serine,GRGDS)五肽修饰的脂质体作为抗癌药物.紫杉醇的载体,对其体外理化性质和细胞毒作用进行评览价.方法:采用化学偶联合成DSPE-PEG-GRGDS,以此作为导向性材料,采用薄膜分散法制备载紫杉醇的PEG修饰长循环脂质体(GRGDS-SSL-PTX),并对脂质体的包封率、粒径和体外释放率等性质进行了考察,同时采用人卵巢癌SKOV-3细胞和人乳腺癌MCF-7细胞进行了体外细胞生长抑制的评价.结果:与普通紫杉醇长循环脂质体(SSL-PTX)相比,本研究制备的紫杉醇主动靶向脂质体(GRGDS-SSL-PTX)的粒径、包封率、载药量、体外释放及稳定性等理化性质无显著差异,包封率约为95%,平均粒径为(115.5±2.2)和(117.5±1.3)nm.冰冻蚀刻透射电镜观察结果表明,脂质体外观基本圆整且均匀分散.体外释放结果表明,12 h内分别有67.9%和72.3%的PTX从SSL-PTX和GRGDS-SSL-PTX中释放.体外细胞毒实验结果表明,GRGDS-SSL-PTX对人卵巢癌SKOV-3细胞和人乳腺癌MCF-7细胞的生长抑制作用均有增强,分别为SSL-PTX的1.42倍和2.12倍.结论:GRGDS五肽修饰的紫杉醇靶向脂质体成功制备,将有利于体内肿瘤的靶向治疗效果.     

水飞蓟素前体脂质体的制备和大鼠药代动力学的研究水飞蓟素前体脂质体的制备和大鼠药代动力学的研究

目的为了提高水飞蓟素的口服生物利用度，研制水飞蓟素前体脂质体并对其理化性质进行考察；研究水飞蓟素前体脂质体的大鼠体内生物利用度。方法采用薄膜载体沉积法制备水飞蓟素前体脂质体，通过研究水合后脂质体的包封率、粒径、稳定性来考察其理化性质；将水飞蓟素前体脂质体在体外进行水合，再给予大鼠灌胃，用RP-HPLC法测定不同时间血浆中总的和游离的水飞蓟素的浓度，通过3P97程序计算药代动力学参数。结果用该法制得的前体脂质体包封率可达90%以上，平均粒径为238.8 nm，稳定性较好；药代动力学研究表明水飞蓟素脂质体在体内吸收较快，生物利用度较高。结论采用薄膜载体沉积法制备水飞蓟素前体脂质体，制备工艺简单，易于工业化生产；将水飞蓟素制备成前体脂质体提高了水飞蓟素的生物利用度。     

Preparation of paraoxonase‐1 liposomes and studies on their in vivo pharmacokinetics in rats

A liposome formulation of the enzyme paraoxonase‐1 (PON1) was prepared for purposes of prolonging and maintaining its activity in vivo. Following purification of PON1 from rabbit serum, liposomes containing PON1 (L‐PON1) were prepared using a film‐dispersion method with a soybean phospholipid–cholesterol mixture (5 : 1, w/w). The pharmacokinetic behaviour of conventional injectable PON1 and L‐PON1 was compared following a single intravenous injection in rats. The enzyme activity of PON1 and its pharmacokinetic parameters were calculated based on a two‐compartment model following conventional injection. The level of PON1 encapsulation in L‐PON1 was 86.20 ± 3.12%. The particle size distribution of L‐PON1 was a narrow unimodal form, with an average diameter of 126 nm. The results suggest that compared with conventional injectable PON1, L‐PON1 has an improved half‐life and enhanced enzyme activity in rats. In conclusion, PON1 can be encapsulated into a lipid bilayer for enhanced stability.     

Preparation and evaluation of N(3)-O-toluyl-fluorouracil-loaded liposomes

This study was aimed at developing a liposome delivery system for a new and potential antitumor lipophilic prodrug of 5-fluorouracil (5-Fu)-N(3)-O-toluyl-fluorouracil (TFu), intended to improve the bioavailability and therapeutic efficacy of 5-Fu by oral and intravenous administration. TFu-loaded liposomes were prepared by a modified film dispersion-homogenization technique, the formulation and manufacture parameters were optimized concerning the drug encapsulation efficiency. TFu-loaded liposomes were characterized according to particle size, size distribution, zeta potential, drug entrapment efficiency, drug loading and physical stability, respectively. In vitro release characteristics, in vivo pharmacokinetic properties and bioavailabilities were also investigated. The formulated liposomes were found to be relatively uniform in size (400.5 +/- 9.6 nm) with a negative zeta potential (-6.4 +/- 0.8 mV). The drug entrapment efficiency and loading were (88.87 +/- 3.25%) and (8.89 +/- 0.19%), respectively. The physical stability experiments results indicated that lyophilized TFu-loaded liposomes were stable for at least 9 months at 4 degrees C. In vitro drug release profile of TFu-loaded liposomes followed the bi-exponential equation. The results of the pharmacokinetic studies in mice indicated that the bioavailability of TFu-loaded liposomes was higher than the suspension after oral administration, and was bioequivalent comparing with TFu 50% alcohol solution after intravenous (i.v.) administration. These results indicated that TFu-loaded liposomes were valued to develop as a practical preparation for oral or i.v. administration.     

Melittin liposomes surface modified with poloxamer 188: in vitro characterization and in vivo evaluation

Melittin liposomes surface modified with poloxamer 188 were developed, and the effect of poloxamer 188 was investigated with regard to anti-cancer effect and vascular stimulation. Melittin liposomes surface modified with poloxamer 188 at different concentrations (0%, 2%, and 5%) were prepared using the adsorption method, followed by in vitro characterization, including entrapment efficiency, zeta potential, particle size, and morphology. Subsequently, the influence of repeated freeze-thawing on the liposomes was investigated, and the effect of poloxamer 188 on the repeated freeze-thawing process was explored. Vascular stimulation effects of MLT, and MLT liposome that surface coated with or without poloxamer were all studied. Pharmacokinetics of the different MLT preparations were determined and the anticancer activity of the MLT formulations was investigated. The particle size of the liposomes gradually increased with increasing poloxamer 188 content, while the entrapment efficiency did not change significantly. After the first freeze-thaw cycle, size and PDI were both markedly reduced, entrapment efficiency rose, and there was no significant change of zeta potential. The vascular irritation caused by MLT could be reduced to an extent by encapsulation in liposome, but not completely eliminated, while liposomes coated with poloxamer 188 can effectively abolish the phenomenon. Melittin liposomes with surface modified by poloxamer exhibit enhanced bioavailability, effective anticancer activity, and reduced side effects compared with melittin solution. Poloxamer plays an important role in melittin liposomes.     

A Scalable,Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

No HeadingPurpose. A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.Methods. Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.Results. Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.Conclusions. This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.     

甘草次酸阳离子脂质体的制备及稳定性考察

目的:制备甘草次酸阳离子脂质体,并研究其稳定性。方法:用乙醇注入法制备甘草次酸阳离子脂质体。考察其粒径、包封率、过氧化值、在血浆中的稳定性和放样稳定性等性质。结果:所得脂质体的粒径小而均匀,呈球形和类球形,包封率为(91.6±1.2)%;离心加速试验结果显示脂质体的稳定性参数KE值较小,脂质体在血浆中释放缓慢,在4℃下放置6个月,其外观、包封率、粒径等各项指标无明显改变。结论:制得的甘草次酸脂质体包封率较高,稳定性良好。     

Pharmacokinetics of oligodeoxynucleotides encapsulated in liposomes: effect of lipid composition and preparation method

1. The effect of the method employed to prepare liposomes and their lipid composition were evaluated in terms of the encapsulation efficiency and pharmacokinetic features of two oligodeoxynucleotides of a 21 mer: the normal (N-Odn) and the phosphorothioate (S-Odn) oligodeoxynucleotide. 2. Liposomes were prepared by the classical method of multilamellar vesicles (MV) and by the dehydration-rehydration method (DR). Two lipid mixtures were used to prepare liposomes - the predominant lipid being phosphatidylcholine (PC) and sphingomyelin (SM) respectively. 3. The DR method for liposome preparation provided the highest encapsulation e ciency, regardless of liposome lipid composition and the type of oligodeoxynucleotide involved (N-Odn or S-Odn). 4. The pharmacokinetics of free and liposome encapsulated oligodeoxynucleotides was studied in mouse following i.v. administration. Liposome encapsulated oligodeoxynucleotides exhibited a significantly lower plasma clearance and longer half-life and residence time than free oligodeoxynucleotides. The method used to obtain the liposomes affected plasma clearance, which was lower for liposomes elaborated by the DR method than for liposomes prepared with the MV method. The use of S-Odn in place of N-Odn decreased the plasma clearance of oligodeoxynucleotide when administered encapsulated in liposomes, regardless of the lipid composition and method used to obtain the liposomes.     

Preparation,characterization and evaluation of multi-component-loaded liposomes containing Tat-TOS,phospholipase D inhibitor and doxorubicin

In the present study, we aimed to co-load the α-TOS conjugate Tat-TOS with the phospholipase D inhibitor FIPI and the antitumor drug doxorubicin (DOX) in a liposome delivery system for antitumor metastasis. Firstly, Tat-TOS was synthesized by solid-phase synthesis, and its structure was confirmed. The ability of free and liposomal Tat-TOS to induce apoptosis in vitro was evaluated by flow cytometry. Biodistribution of Tat-TOS-loaded liposomes was investigated by a molecular imaging system. Multi-component-loaded liposomes modified with Tat-TOS containing FIPI and DOX was prepared by thin film dispersion method in combination with pH gradient method and post-insertion method. Physicochemical properties were determined, and the in vitro uptake ability of the formulations was evaluated. The results showed that the prepared liposomes were characterized by a uniform particle size distribution and small particle size. The encapsulation efficiency of FIPI and DOX exceeded 85%. Both free and liposomal Tat-TOS significantly improved the activity of inducing apoptosis of tumor cells. The liposomes modified with Tat-TOS were apparently accumulated in normal lung tissue and tumor metastasized lung. Multi-component-loaded liposomes exhibited the strongest cell uptake capacity, suggesting a stronger anti-metastatic effect and anti-tumor activity in vivo.     

雷替曲塞pH敏感脂质体的制备研究

目的 制备雷替曲塞pH敏感脂质体，并优化其处方和制备工艺，对所优化的pH敏感脂质体进行评价。方法 采用单因素考察和正交设计试验优化处方，考察最优处方制得的雷替曲塞pH敏感脂质体的粒径、Zeta电位、外观形态、包封率、体外释放度和细胞毒性作用。结果 按最优处方制备的雷替曲塞pH敏感脂质体，平均粒径为(227.0±21.4)nm，PDI为0.223±0.061，Zeta电位为(-44.2±3.6)mv，包封率为(58.3±2.1)%；体外释放结果表明载药脂质体在pH5.0和pH6.0的释放介质中释放快速，在pH7.4的释放介质中释放缓慢，pH敏感作用明显；细胞毒性试验证实脂质体载体本身安全性较高，而载药脂质体制剂有较高的细胞毒性。结论 本研究表明，优化得到的雷替曲塞pH敏感脂质体具有简便的制备方法、适宜的理化性质和较高的安全性，具有广阔的临床应用前景。     

The effect of ileal brake activators on the oral bioavailability of atenolol in man

The aims of this study were to develop novel liposome formulations for tranexamic acid (TA) from various lipid compositions [neutral (hydrogenated soya phosphatidylcholine and cholesterol), positive (stearylamine) or negative (dicetyl phosphate) charged lipid], and to investigate the effects of concentrations of TA (5 and 10% in DI water) and charges on the physicochemical properties of liposomes. Liposomes were prepared by chloroform film method with sonication. The physical (appearance, pH, size, morphology) and chemical (drug encapsulation efficiency, transition temperature, enthalpy of transition) properties of liposomes were characterized. The TA contents were determined spectrophotometrically at 415 nm, following derivatization with 2,4,6-trinitrobenzosulfonic acid. The charged liposomes demonstrated better physical stability than the neutral liposomes. The percentages of TA entrapped in all liposome formulations varied between 13.2 and 15.6%, and were independent of TA concentrations and charges of liposomes. Charges affected the physical stability, pH and size of liposomes. The particle sizes of negative blank and positive liposomes (with and without the entrapped drug) were approximately 10 times larger than the negative liposome with the entrapped TA. The multilamellar 7:2:1 molar ratio of hydrogenated soy phosphatidylcholine/cholesterol/dicetyl phosphate entrapped with 10% TA liposome (10%TA,-) was selected for further release study, due to its high physical stability, small particle size and relatively high drug encapsulation efficiency.