主动载药与被动载药制备盐酸左氧氟沙星脂质体

建立了阳离子交换树脂－荧光分光光度法测定盐酸左氧氟沙星脂质体包封率的方法。该法具有简单、方便、省时、经济等优点。用所建立的方法考察了主动载药与被动载药制备的脂质体包封率,结果表明,主动载药中采用硫酸铵梯度法包封率最高,可达71．5％。     

Targeted and sustained drug delivery using PEGylated galactosylated liposomes

To achieve a sustained and targeted delivery of liposomes to liver parenchymal cells (PC), we modified distearoyl-L-phosphatidylcholine (DSPC)/cholesterol (Chol) (60:40) (DSPC/Chol) liposomes with a galactosylated cholesterol derivative (Gal-C4-Chol), and polysorbate (Tween) 20 or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (PEG(x)-DSPE). After intravenous injection, DSPC/Chol/Gal-C4-Chol (60:35:5) (Gal) liposomes were rapidly eliminated from the blood circulation and mostly recovered in the liver. The blood elimination of DSPC/Chol/Gal-C4-Chol/Tween 20 (55:35:5:5) (Tween 20-Gal) liposomes was slightly reduced as compared to Gal-liposomes. In contrast, a significant reduction in the blood elimination was observed with DSPC/Chol/Gal-C4-Chol/PEG(2000)-DSPE (59:35:5:1) (PEG(2000)-Gal) liposomes. Hepatic uptake of DSPC/Chol/Gal-C4-Chol/PEG(350)-DSPE (59:35:5:1) (PEG(350)-Gal) liposomes was intermediate between PEG(2000)-Gal-liposomes and Tween 20-Gal-liposomes. The uptake of PEG(350)-Gal-liposomes by liver PC was 7.7-fold higher than that by non-parenchymal cells (NPC). These results suggest that PEG(350)-DSPE can control the delivery rate of Gal-liposomes to liver PC without losing its targeting capability.     

PEGylated liposomes loading palmitoyl prednisolone for prolonged blood concentration of prednisolone

We investigated the pharmacokinetic behavior of palmitoyl prednisolone (Pal-PLS) and its liposomes with L-alpha-distearoylphosphatidylcholine (DSPC) and cholesterol (Chol) with or without L-alpha-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) after their intravenous administration in rats. Pal-PLS rapidly disappeared from the systemic circulation and prednisolone (PLS) was regenerated after the administration of DSPC/Chol liposomes. PEGylated liposomes including DSPE-PEG 2000, however, successfully maintained high blood concentrations of Pal-PLS and PLS. The blood profiles of drugs after the administration of liposomal Pal-PLS were analyzed according to a two-compartment model. The larger content of DSPE-PEG 2000 in DSPC/Chol liposomes showed a lower first order elimination rate constant from the central compartment (K(el)) and clearance (CL). The area under the concentration-time curve (AUC) of Pal-PLS and PLS in PEGylated liposomes was larger than DSPC/Chol liposomes. The mean resident time (MRT) of Pal-PLS and PLS was also prolonged by PEGylated liposomes. Although DSPC/Chol liposomes showed a high distribution of Pal-PLS in the liver and spleen, PEGylated liposomes significantly decreased the liver distribution of Pal-PLS. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations. In conclusion, PEGylated liposomes, including Pal-PLS, are useful for maintain the PLS concentration in the blood after intravenous administration.     

重组人生长激素脂质体的制备及载药研究

目的采用乙醇注入法结合反复冻融技术制备重组人生长激素脂质体,并考察影响包封率的主要因素。方法采用乙醇注入法制备空白脂质体,并通过反复冻融载药,采用葡聚糖凝胶柱分离结合CBB G-250染色法测定游离药物含量,计算包封率;考察孵化时间、孵化温度、冻融次数对包封率的影响;对冻干制品的外观、复溶速度、粒径及分布进行综合评分,优选冻干支持剂。结果孵化时间为40 min、孵化温度为10℃、冻融次数为3次时,能够获得较高包封率的脂质体,包封率为63.59%。海藻糖在脂质体冷冻干燥过程中具有最好的保护作用。结论乙醇注入法结合反复冻融可用于大分子蛋白质类脂质体的制备。     

聚乙二醇化尿酸酶脂质体的稳定性研究

目的 制备聚乙二醇化尿酸酶脂质体(PULPs)并考察其活性与稳定性.方法 采用逆向蒸发法制备PULPs,并考察其最适温度、最适pH、热稳定性、贮存稳定性及其抗胰蛋白酶水解的能力、抗部分金属离子及有机化合物影响的能力.结果 PULPs的最适温度为40℃,最适pH为8.5;PULPs的热稳定性、贮存稳定性、抗胰蛋白酶水解能力、抗部分金属离子及有机化合物影响的能力均优于尿酸酶.结论 PULPs在增强聚乙二醇化尿酸酶活性的同时增强了稳定性.     

主动载药法制备硫酸卷曲霉素脂质体及其质量评价

目的制备硫酸卷曲霉素脂质体,建立含量和包封率的测定方法,初步考察其体外释放规律。方法采用pH梯度法制备硫酸卷曲霉素脂质体,超滤法分离脂质体与游离药物,RP-HPLC测定脂质体的含量和包封率,透析法考察脂质体的体外释放行为。结果超滤法能很好地将脂质体与游离药物分离,测定硫酸卷曲霉素脂质体的含量为10.27mg/ml,包封率为47.8%,脂质体的体外释放规律符合一级动力学过程。结论pH梯度法适于制备硫酸卷曲霉素脂质体,超滤法可用于硫酸卷曲霉素脂质体包封率的测定,制备的脂质体具有一定的缓释效果。     

盐酸米托蒽醌聚乙二醇化脂质体的制备及对A549细胞体外抗肿瘤作用的研究

目的 研究盐酸米托蒽醌聚乙二醇化脂质体(DHAD-PEG-L)的制备及其抗肿瘤细胞增殖作用,比较DHAD-PEG-L与常规盐酸米托蒽醌(DHAD)制剂的抗肿瘤活性差异.方法 以主动载药法制备获得DHAD-PEG-L;以人非小细胞肺癌A549细胞为模型,采用MTT法考察DHAD-PEG-L和DHAD对肿瘤细胞的抑制效果,测定制剂作用肿瘤细胞48 h后的IC50值;以方差分析法比较DHAD-PEG-L与DHAD抗肿瘤活性的统计学差异.结果 DHAD-PEG-L和DHAD对A549细胞的IC50分别是(1.561±0.09)mg·L-1和(0.862±0.02)mg·L-1;药物经聚乙二醇(PEG)脂质体包封后,与常规制剂相比,高浓度时肿瘤抑制作用略有下降,低浓度时抑制作用增强.结论 PEG化技术可运用于脂质体,对DHAD进行减毒增效改造,预期可降低药物不良反应.     

Ampicillin-loaded liposomes and nanoparticles: comparison of drug loading, drug release and in vitro antimicrobial activity

In this paper, we report the physico-chemical properties of negatively charged liposomes and of polyisohexylcyanoacrylate nanoparticles loaded with ampicillin. Although the carriers were of the same size (200 nm), drug-loading capacity was 20 times higher for nanoparticles than for liposomes. After freeze-drying or storage at +4 degrees C, no drug escaped from polymeric nanoparticles. On the other hand, in the same conditions, ampicillin leaked rapidly from liposomes. Drug release in foetal calf serum was gradual (of zero order) with nanoparticles, whereas it was rapid with liposomes. Finally, the antimicrobial activity of ampicillin-entrapped liposomes or nanoparticles was studied in vitro.     

5-氟尿嘧啶半乳糖神经酰胺脂质体的抗耐药作用及部分机制研究

目的研究5氟尿嘧啶半乳糖神经酰胺脂质体(5FuGCL)体外对耐5FuHepG2细胞株的抑制作用并对其抗耐药机制进行探讨。方法在建立耐5Fu的HepG2细胞株模型的基础上,采用MTT法检测5FuGCL对其的抑制作用。另外,通过高效液相法(HPLC)检测细胞内液的药物含量、免疫组化法检测胸苷酸合酶(TS)的表达、化学法检测NO含量等研究其抗耐药作用机制。结果5FuGCL(75,150,300,600,1200μmol·L-1)对耐5Fu的HepG2细胞株有明显的抑制作用,IC50为158.6μmol·L-1,远小于游离5Fu(400.9μmol·L-1);5FuGCL(300μmol·L-1)对耐5Fu的HepG2细胞的抑制作用随时间的延长而增强,其中(24～48h)的抑制作用明显强于相同浓度的游离5Fu。5FuGCL(300μmol·L-1)与相同浓度的游离5Fu比较,能明显增加药物进入HepG2细胞内液的程度;5FuGCL(75,300,1200μmol·L-1)既可明显降低TS的表达,又可显著增加NO的含量,且5FuGCL(300,1200μmol·L-1)与相同浓度的游离5Fu相比,其作用明显增强。结论5FuGCL有明显的抗5Fu耐药作用,这可能是通过增加细胞内液5Fu的含量,抑制TS的表达和增加NO含量实现的。     

Improved anti-tumor efficacy and pharmacokinetics of bufalin via PEGylated liposomes

OBJECTIVE To determine the characterization,anti-tumor efficacy and pharmacokinetics of bufalin-loaded PEGylated liposomes compared with bufalin entity.METHODS Bufalin-loaded PEGylated liposomes and bufalin-loaded liposomes were prepared reproducibly with homogeneous particle size by the combination of thin film evaporation method and high pressure homogenization method.The particle size and zeta potential of the liposomes were determined by dynamic light scattering technique.The direct imaging of morphology of liposomes was charactered by transmission electron microscope.The content of bufalin in liposomes was analysed by HPLC method.The entrapment efficiency and the particle size was applied to assess the stability profile,after storage at 4℃ on day 0,7,15,30 and 90.The in-vitro release behaviours of bufalin from liposomes were conducted using dialysis bag technique at 37℃.In-vitro cytotoxicity studies were carried out using MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay on several kinds of tumor cel lines including SW620,PC-3,MDA-MB-231,A549,U251,U87 and HepG2.In-vivo pharmacokinetic study of bufalin liposomes was evaluated by HPLC method.RESULTS Their mean particle sizes were 127.6 nm and 155.0 nm,mean zeta potentials were 2.24 m V and-18.5 m V,entrapment efficiencies were 76.31%and 78.40%,respectively.In-vitro release profile revealed that the release of bufalin in bufalin-loaded PEGylated liposomes was slower than that of bufalin-loaded liposomes.The cytotoxicity of blank liposomes has been found within acceptable range,whereas bufalin-loaded PEGylated liposomes showed enhanced cytotoxicity to U251 cells compared with bufalin entity.In-vivo pharmacokinetics indicated that bufalinloaded PEGylated liposomes could extend eliminate half-life time of bufalin in plasma in rats.CONCLUSION The results suggested that bufalin-loaded PEGylated liposomes improved the solubility and increased the drug concentration in plasma.     

不同载药方法对介孔二氧化钛纳米粒载药及药物溶出速率的影响

目的考察不同载药方法对介孔二氧化钛纳米粒载药系统载药和溶出速率的影响。方法通过溶剂挥干法、熔融法、吸附平衡法分别将卡维地洛包载于介孔二氧化钛纳米粒中,用扫描电镜和透射电镜观测样品形貌,氮气吸附-解吸法测量载体的比表面积和孔体积,差示扫描量热法分析药物粒子的存在状态,X-射线衍射法进行物相分析,热失重法分析载药量,最后测定载药系统的溶出速率并进行长期稳定性试验。结果三种载药方法均能通过改变药物的存在状态使药物以非晶型存在于载体中而达到提高溶出速率的目的。其中溶剂挥干法所得的载药系统溶出速率提高显著,并且更具有长期稳定性。结论溶剂挥干法为介孔二氧化钛纳米粒载药的首选。     

超顺磁性四氧化三铁长循环脂质体的制备及表征

目的：构建磁共振对比剂超顺磁性四氧化三铁长循环脂质体（PS）,并对其理化性质与稳定性进行初步评价。方法：采用化学共沉淀法合成柠檬酸修饰的超顺磁性四氧化三铁纳米粒,并用脂质材料将纳米粒外层包被,制备得PS。采用透射电镜、动态光散射、凝胶色谱及磁共振成像等,对PS的形态、粒径、Zeta电位、包封率及弛豫率进行评价,并考察PS在4 ℃条件下的稳定性。结果：制备的PS为圆形或类圆形粒子,粒径为（141±1）nm,PDI为0.242±0.006,Zeta电位为（-17.8±1.0）mV,包封率为（91.73±5.18）%。体外MR成像结果表明,PS的弛豫率为355.20 mM^-1·s^-1。稳定性试验结果表明,PS在4 ℃条件下贮藏10 d,其粒径 [（151±3.2）nm]、PDI（0.273±0.012）、Zeta电位[（-19.9±0.3）mV]和包封率[（93.05±6.52）%],均无显著性变化。结论：本研究成功制备了超顺磁性四氧化三铁长循环脂质体MR对比剂,为进一步构建MR可视化肿瘤靶向药物传递系统提供了有效的物质基础。     

A calorimetric study on diflunisal release from poly(lactide-co-glycolide) microspheres by monitoring the drug effect on dipalmitoylphosphatidylcholine liposomes: temperature and drug loading influence

Diflunisal release from poly-Lactide-co-Glycolide (50:50, 34,000 MW) microspheres loaded with two different amounts of drug (2.5 +/- 0.5% and 10 +/- 0.5% w/w) was monitored by following the effects exerted by the drug on the thermotropic behavior of dipalmitoylphosphatidylcholine unilamellar vesicles at different temperatures. The effects of the drug released from the microspheres on the thermotropic behavior of lipid aqueous dispersion containing different molar ratios of drug was detected by differential scanning calorimetry and was compared with the effects exerted by the free Diflunisal. Diflunisal affects mainly the temperature (T_m) of the transition characteristic of phospholipid vesicles as model biomembrane, causing a shift toward lower values. This shift was modulated by the drug molar fraction with respect to the lipid concentration in the aqueous dispersion. Afterward, calorimetric measurements were performed on suspensions of blank liposomes added to weighed amounts of unloaded and differently Diflunisal-loaded microspheres as well as free powdered Diflunisal after incubation for increasing times at three different temperatures (25, 37, and 50°C). The T_m shifts of the lipid bilayer, caused by the drug released from polymeric system as well as by the free drug during incubation periods, were compared with that caused by free drug increasing molar fractions dispersed directly on the membrane, employed as a calibration curve to obtain the fraction of drug released. This in vitro study suggests that the kinetic process involved in drug release is influenced by the amount of drug loaded in the microspheres as well as by the temperature acting on drug solubility and membrane disorder. This drug release model, monitored by the calorimetric technique shows that a) the poly-Lactide-co-Glycolide microspheres are a good delivery system able to sustain the drug release; b) the differential scanning calorimetry technique applied on the drug interaction with biomembranes constitutes a good tool to follow the drug release; 3) this model, representing an innovative alternative in vitro model, should be used to determine the different kinetics involved in the drug transfer from a drug delivery system to a membrane as uptake site.     

Active methods of drug loading into liposomes: recent strategies for stable drug entrapment and increased in vivo activity

INTRODUCTION: The use of liposomes increases the therapeutic index of many drugs, and also offers drug targeting and controlled release. The commercial impact of liposomes is strengthened by the invention of several active drug encapsulation methods, allowing the encapsulation of several weak base or weak acid drugs with very high drug-to-lipid ratios. AREAS COVERED: In recent years, there have been reports on several new approaches to retain more hydrophobic drugs inside liposomes, in the circulation. Most of these methods apply drug precipitation inside preformed liposomes, as low soluble complexes with ions or chemicals. In some cases, drug derivatization was applied to enable active encapsulation of hydrophobic drugs, previously not reported to encapsulate, by active or remote loading. This review presents and compares most of the existing methods of active drug encapsulation and outlines recent strategies to achieve stable drug encapsulation in vivo. EXPERT OPINION: At present, there is no single universal encapsulation method that offers stable encapsulation of most drugs; each drug requires a different approach to manage all of its properties. Now is the time to combine all these strategies to achieve the goal of a complex, but successful, anticancer therapy.     

The impact of cholesterol and its metabolites on drug metabolism

INTRODUCTION: Global prevalence of Western-type diet has increased in the last decades resulting in occurrence of certain chronic diseases. This type of diet is also linked to high-cholesterol intake and increase in blood cholesterol. Many of the molecular mechanisms of dealing with increased levels of cholesterol and its metabolites have been elucidated in animal models and humans. It is also evident that cholesterol metabolism is closely connected to drug metabolism. Cholesterol/bile acids and drugs share many transporters, enzymes and regulatory proteins which are key points in the crosstalk. AREAS COVERED: This review presents an overview of the effect of cholesterol and its metabolites on drug metabolism with special emphasis on species-specific differences. The article focuses on the role of nuclear receptors farnesoid X receptor, vitamin D receptor and liver X receptor in the regulation of drug metabolism genes and the role of cholesterol biosynthesis intermediates, oxysterols and bile acids in the induction of drug metabolism through pregnane X receptor. EXPERT OPINION: Studies show that the regulation of drug metabolism by sterols is multileveled. Many species-dependent differences were observed which hinder the transfer of findings from model animals to humans. As of now, there is little evidence available for cholesterol impact on drug metabolism in vivo in humans. There is also the need to confirm the results obtained in animal models and in vitro analyses in human cells but this is very difficult given the current lack of tools.     

Pharmacogenetics of drug transporters and its impact on the pharmacotherapy

Most drug responses are determined by the interplay of several gene products that influence pharmacokinetics and pharmacodynamics, i.e., drug metabolizing enzymes, drug transporters, and drug targets. With the sequencing of the human genome, it has been estimated that approximately 500-1200 genes code for drug transporters. Concerning the effects of genetic polymorphisms on pharmacotherapy, the best characterized drug transporter is the multidrug resistant transporter P-glycoprotein/MDR1, the gene product of MDR1. Little such information is available on other drug transporters. MDR1 is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily, and is expressed mainly in intestines, liver, kidneys and brain. A number of various types of structurally unrelated drugs are substrates for MDR1, and their intestinal absorption, hepatobiliary secretion, renal secretion and brain transport are regulated by MDR1. The first investigation on the effects of MDR1 genotypes on pharmacotherapy was reported in 2000: a silent single nucleotide polymorphism (SNP), C3435T in exon 26, was found to be associated with the duodenal expression of MDR1, and thereby the plasma concentration of digoxin after oral administration. At present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical investigations on the association of MDR1 genotypes with the expression and function of MDR1 in tissues, and with pharmacokinetics and pharmacodynamics have mainly focused on C3435T; however, there are still discrepancies in the results, suggesting that the haplotype of the gene should be analyzed instead of a SNP. C3435T is also reported to be a risk factor for a certain class of diseases including the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this also might be explained by the effects on MDR1 expression and function. In this review, the latest reports on the effects of genetic polymorphisms of MDR1 on pharmacotherapy are summarized, and the pharmacogenetics of other transporters is briefly introduced.     

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.     

Pharmacogenomics and its potential impact on drug and formulation development

Recent advances in genomic research have provided the basis for new insights into the importance of genetic and genomic markers during the different stages of drug development. A new field of research, pharmacogenomics, which studies the relationship between drug effects and the genome, has emerged. Structural pharmacogenomics maps the complete DNA sequences of whole genomes (genotypes) including individual variations, and functional pharmacogenomics assesses the expression levels of thousands of genes in one single experiment. Together, these two areas of pharmacogenomics have generated massive databases, which have become a challenge for the research field of informatics and have fostered a new branch of research, bioinformatics. If skillfully used, the databases generated by pharmacogenomics together with data mining on the Web promise to improve the drug development process in a variety of areas: identification of drug targets, evaluation of toxicity, classification of diseases, evaluation of formulations, assessment of drug response and treatment, post-marketing applications, and development of personalized medicines.     

Ocular drug targeting by liposomes and their corneal interactions

Topical drug delivery is preferred in the eye to avoid under or over medication and undesired side effects of systemic administration. In order to maintain adequate drug levels in ocular tissues, frequent drug installation is necessary in vision threatening conditions like glaucoma, corneal ulcers due to microbial infections, etc. Only a part of the installed drug reaches the aqueous humor and the rest of it is drained by the nasolacrimal duct. Positively charged liposomes have been found to enhance the penetration of drugs into the cornea. The present study was conducted to visualize the interaction of liposomes with the cornea. Briefly, positively charged liposomes entrapped with Carboxyfluorescein (CF), Propidium iodide (PI), Horseradish peroxidase (HRP), Biotin, Hydroxy benzimide (Hoechst No: 33258), and Ethoxy benzimide (Hoechst No: 33342) were prepared by sonication. Their size and shape were analysed by laser dynamic light spectra and electron microscopy, respectively. The liposome encapsulated and free materials in buffer were instilled, in a volume of 20 microliters, into eyes of anesthetized albino rats. After 30 min and 1 h, the eyes were enucleated and quickly processed for cryosections of 3-5 microns thickness. The interaction of liposome entrapped propidium iodide and HRP was visualized on the outer epithelium of the cornea after specific processing.     

Ocular drug targeting by liposomes and their corneal interactions

Topical drug delivery is preferred in the eye to avoid under or over medication and undesired side effects of systemic administraion. In order to maintain adequate drug levels in ocular tissues, frequent drug installation is necessary in vision threatening conditions like glaucoma, corneal ulcers due to microbial infections, etc. Only apart of the installed drug reaches the aqueous humor and the rest of it is drained by the nasolacrimal duct. Positively charged liposomes have been found to enhance the penetration of drugs into the cornea. The present study was conducted to visualize the interaction of liposomes with the cornea. Briefly, positively charged liposomes entrapped with Carboxyfluorescein (CF), Propidium iodide (PI), Horseradish peroxidase (HRP), Biotin, Hydroxy benzimide (Hoechst No: 33258), and Ethoxy benzimide (Hoechst No: 33342) were prepared by sonication. Their size and shape were analysed by laser dynamic lightspectraand electron microscopy, respectively. The liposome encapsulated and free materials in buffer were instilled, in a volume of 20mul, into eyes of anesthetized albino rats. After 30min and 1h, the eyes were enucleated and quickly processed for cryosections of 3-5mum thickness. The interaction of liposome entrapped propidium iodide and HRPwas visualized on the outer epithelium of the cornea after specific processing.