A detachable coating of cholesterol-anchored PEG improves tumor targeting of cell-penetrating peptide-modified liposomes

Cell-penetrating peptides (CPPs) have been widely used to enhance the membrane translocation of various carriers for many years, but the non-specificity of CPPs seriously limits their utility in vivo. In this study, cholesterol-anchored, reduction-sensitive PEG (first synthesized by our laboratory) was applied to develop a co-modified liposome with improved tumor targeting. Following optimization of the formulation, the in vitro and in vivo properties of the co-modified liposome were evaluated. The co-modified liposome had a much lower cellular uptake and tumor spheroid uptake, but a much higher tumor accumulation compared to CPP-modified liposome, indicating the non-specific penetration of CPPs could be attenuated by the outer PEG coating. With the addition of exogenous reducing agent, both the in vitro and in vivo cellular uptake was markedly increased, demonstrating that the reduction-sensitive PEG coating achieved a controllable detachment from the surface of liposomes and did not affect the penetrating abilities of CPPs. The present results demonstrate that the combination of cholestervsitive PEG and CPPs is an ideal alternative for the application of CPP-modified carriers in vivo.KEY WORDS: Cell penetrating peptide, Reduction-sensitive PEG, Tumor targeting, Cholesterol, Liposome     

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.     

Enhancing effect of cetylmannoside on targeting of liposomes to Kupffer cells in rats

In order to evaluate whether surface modification of liposomes by cetylmannoside (Man) could be useful for targeting to Kupffer cells, the effect of Man on disposition of liposomes was examined after intravenous administration to rats. In the case of small unilamellar vesicles (SUV), no difference in disposition was observed between control liposomes (PC-SUV) and modified liposomes (Man-SUV). On the other hand, in the case of multilamellar vesicles (MLV), modified liposomes (Man-MLV) were rapidly eliminated from the circulation, and showed higher accumulation (51.4% of dose) in the liver as compared with control liposomes (PC-MLV, 25.7% of dose). In the spleen, splenic clearance of Man-MLV (0.068 ml/min) was comparable to that of PC-MLV (0.068 ml/min), although Man-MLV showed lower accumulation (5.7% of dose) than PC-MLV (14.7% of dose). This lower accumulation in the spleen of Man-MLV might be due to the low blood concentration caused by the high accumulation in the liver. Thus, it is considered that liposomal size is important in revealing the effects of Man, and Man-MLV is able to enhance only the affinity for the liver. The cellular distribution in the liver of Man-MLV 2 h after intravenous administration to rats gave encouraging evidence that Kupffer cells might be involved in the enhanced hepatic uptake of the liposomes. These results suggest the usefulness of Man-MLV for targeting to Kupffer cells. Furthermore, the involvement of plasma protein(s) in the uptake of Man-MLV is suspected.     

N-三甲基壳聚糖包覆水飞蓟宾脂质体在小鼠体内的药动学及靶向性

目的:研究N-三甲基壳聚糖(TMC)包覆的水飞蓟宾脂质体(SLBL)在小鼠体内药动学,并对其肝靶向性进行研究。方法:小鼠尾静脉注射水飞蓟宾(SLB)溶液、SLBL、季铵化程度为20%的TMC包覆的SLBL(TMC20-SLBL),HPLC法检测不同时间血浆和各组织中SLB的浓度,采用DAS2.0软件统计学方法分析SLB在小鼠体内的药动学特征,采用相对摄取率re进行肝靶向性评价。结果:3种制剂中的SLB在小鼠体内的药动学特征符合二室模型,t_1/2β顺序依次为:SLBL>TMC20-SLBL>SLB溶液,AUC大小顺序为TMC20-SLBL>SLBL>SLB溶液,CL顺序为SLB溶液>SLBL>TMC20-SLBL。SLBL和TMC20-SLBL在肝组织的相对摄取率r_e分别为4.101和9.706。结论:与SLB溶液及SLBL相比,TMC20-SLBL具有明显的缓释作用及肝靶向性,生物利用度较高,有利于提高其治疗作用。     

Silymarin causes caspases activation and apoptosis in K562 leukemia cells through inactivation of Akt pathway

Cancer is one of the largest causes of death in both men and women. Akt, overexpressed in a number of human malignancies including leukemia, is an important target in cancer prevention and/or therapy. Silymarin, a flavonoid antioxidant, has high human acceptance being used clinically for the treatment of liver diseases. In this study, Akt activity was inhibited by silymarin without changes in total Akt level associated with a prominent caspases-9 and -3 activation as well as PARP cleavage, accompanied by a strong apoptotic death and growth inhibition of K562 cells. These findings suggest that silymarin could serve as a candidate for anti-leukemia drug.     

Silymarin improves the behavioural, biochemical and histoarchitecture alterations in focal ischemic rats: a comparative evaluation with piracetam and protocatachuic acid

Comparative neuroprotective potential of silymarin, piracetam and protocatechuic acid ethyl ester (PCA) was evaluated in focal ischemic rats. Various pharmacological, biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite content, brain water content) and behavioural (memory impairment, motor control, neurological score) including infarct size and histopathological alterations were evaluated. Silymarin (200 mg/kg) and PCA treatment significantly improved behavioural, biochemical and histopathological changes, and reduced water content and infarct size. However, piracetam only improved behavioural and histopathological changes, reduced water content and infarct size. The findings indicate that silymarin exhibits neuroprotective activity better than PCA and piracetam in focal ischemia/reperfusion reflected by its better restoration of behavioural and antioxidant profile.     

具有肾靶向性的三巯基葡萄糖-胆固醇偶联物脂质体配体的合成

目的设计并合成一种新型的具有肾靶向性的三巯基葡萄糖-胆固醇偶联物脂质体配体。方法以胆固醇与癸二醇的缩合产物为原料,与分支试剂四(ω-甲磺酰氧丙氧甲基)甲烷缩合成醚,再与1,2,3,4-四-O-乙酰基-1-巯基-吡喃葡萄糖在碘化钾和DIPEA的作用下成醚,最后经甲醇钠溶液脱保护后得到目标配体。结果成功制备了一个胆甾三巯基葡萄糖偶联物。结论目标化合物经IR、1HNMR、MS确证。     

扑热息痛滴剂、酏剂和片剂在正常人中的生物利用度比较

本文报道7名健康受试者分别口服1g单剂量扑热息痛滴剂(A)、酏剂(B)和片剂(C)后的生物利用度和药代动力学比较。血药浓度用紫外分光光度法测定。结果表明:A和B比C吸收迅速、达峰早,Cp和AUC较C犬(P均<0.05)。与C相比,A和B能维持在有效治疗血浓的时间更长。A和B更适合于儿科应用。     

水飞蓟素胶丸的制备及其含量测定

目的　制备水飞蓟素胶丸 ,建立测定胶丸中水飞蓟宾含量的方法。方法　水飞蓟素原料制成微粉 ,研磨乳化后制丸 ,并用HPLC测定主要成分水飞蓟宾的含量。结果　胶丸符合中国药典标准 ;胶丸中水飞蓟宾的含量为 6 2 .7mg·g-1,平均回收率为 97.5 % ,RSD =0 .80 % (n =6 )。结论　所制制剂工艺简单 ,有效成分的检测方法准确可靠。     

Specific incorporation of asialofetuin-labeled liposomes into hepatocytes through the action of galactose-binding protein

Asialofetuin-labelled liposomes (AF-liposomes) having mean diameters of 0.13 micron ([S]) and 0.35 micron ([L]) were obtained by the subsequent extrusion method in combination with dialysis. Intravenously administered AF-liposomes [S] were rapidly cleared from the systemic circulation. By pre- and post-treatment of the rats with free asialofetuin (AF), the rate of elimination decreased to that of non-labelled liposomes (N-liposomes) [S]. The liver uptake of AF-liposomes [S] (60 per cent of dose in 30 min) was about twice that of N-liposomes [S]. Forty-seven per cent of the AF-liposomes [S] incorporated into the liver were found in the parenchymal cell fraction as against 20 per cent in the non-parenchymal cell fraction. In contrast, N-liposomes [S] were taken up primarily by non-parenchymal cells as was also the case for AF- or N-liposomes [L]. Both the lipid and aqueous markers of AF-liposomes [S] were detected in subcellular fractions of the liver, but their distribution was noted to change differently with the lapse of time. Intravenously administered AF-liposomes [S] were specifically recognized by galactose-binding protein and underwent disruption in the cell after being taken up by hepatocytes. AF-liposomes [S] may possibly be utilized to deliver drugs into hepatocytes.     

The bioavailability of oral nifedipine formulations: a statistical and simulation approach

The bioequivalence of three oral forms of nifedipine was assessed in a triple crossover study on 12 healthy volunteers. Single 10 mg dose was given and ten blood samples were drawn during the first 8 h after administration. Highly sensitive gas chromatographic method was used for the nifedipine assay. Pharmacokinetic parameters which describe bioavailability and general kinetic behaviour of the drug (AUC, Cmax, tmax, beta, MRT) were calculated from individual plasma profiles. They were subjected to statistical analysis (paired t-test, Hauck's inverted t-test, and Westlake's method of confidence intervals). Analogue-hybrid simulation and identification was used to generate plasma profiles of nifedipine after single and multiple dosing. Averaged plasma concentrations were used for this purpose. The three formulations studied were bioequivalent in terms of the rate of absorption. The simulation proved to be an efficient tool to substitute in vivo multiple dosing studies for assessment of bioavailability. The specific statistical methods should be preferred in bioequivalence data evaluation due to their greater power and inclusion of extraneous bioequivalence limits interval. Despite the differences among the formulations studied, each one of them should be viewed according to its intended clinical use.     

Analysis of silibinin in rat plasma and bile for hepatobiliary excretion and oral bioavailability application

Silibinin is an herbal ingredient isolated from milk thistle. The aim of this study was to develop a simple liquid chromatographic system to assay silibinin in plasma and bile for pharmacokinetic study. Silibinin was given oral and intravenously. The plasma sample (25 microL) was vortex-mixed with 50 microL of internal standard solution (naringenin 10 microg/mL in acetonitrile) to achieve protein precipitation. Silibinin in the rat plasma and bile was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile -10 mM monosodium phosphate (pH 5.45 adjusted with orthophosphoric acid) (50:50, v/v) and the flow-rate of 1 mL/min. The UV detection wavelength was 288 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-100 microg/mL. Intra- and inter-assay precision and accuracy of silibinin fell well within the predefined limits of acceptability (<15%). An ultrafiltration method was used in this experiment and the protein binding of silibinin was 70.3+/-4.6%. After silibinin administration in rats, the disposition of silibinin in the plasma and bile fluid was due to rapid distribution and equilibration between the blood and hepatobiliary system, and the bile levels of unconjugated silibinin and total silibinin were greater than those in the plasma. The oral bioavailability of silibinin in rats was estimated to be 0.73%.     

水飞蓟素缓释滴丸的研制及其体外释放特性

目的:研究水飞蓟素缓释滴丸的处方和工艺,并对其体外释放特性进行了评价.方法:采用联合载体材料即聚乙二醇6000和泊洛沙姆188为速释性固体分散体载体材料,硬脂酸为缓释性骨架材料;熔融法制备水飞蓟素缓释滴丸,考察了滴丸成型的影响因素;并与市售片剂进行了释放度比较.结果:选择处方组成为水飞蓟素:聚乙二醇6000:泊洛沙姆188:硬脂酸(1:2:1:1.5);滴头直径为2.3 mm/4.1 mm,滴距为6 cm,滴速为40滴·min-1;该缓释滴丸10 h的最大累积溶出百分率可达92.5%.结论:所制得水飞蓟素缓释滴丸具有良好的缓释效果,为研发水飞蓟素新制剂提供参考.     

Silymarin enhanced cytotoxic effect of anti-Fas agonistic antibody CH11 on A375-S2 cells

Silymarin is a polyphenolic flavonoid from milk thistle (Silybum marianum), which has anti-inflammatory, cytoprotective as well as antioxidant effects. Our previous study demonstrated that silymarin has anti-apoptotic effect against UV irradiation. In this study, we assessed the effect of silymarin on anti-Fas agonistic antibody CH11-treated human malignant melanoma, A375-S2 cells. Pretreatment with silymarin (3 × 10^- 4 mol/L) significantly induced cell apoptosis in CH11-treated A375-S2 cells. Mitochondrial transmembrane potential (ΔΨ_m) was also down-regulated by silymarin pretreatment. Caspase-8, -9, -3 and pan-caspase inhibitors partially reversed silymarin-induced apoptosis of CH11-treated cells. The expression of Fas-associated proteins with death domain (FADD), a downstream molecule of the death receptor pathway, was increased by silymarin pretreatment, followed by cleavage of procaspase-8, whose activation induced cell apoptosis. Moreover, cleavage of procaspase-3 and digestion of its substrate, the inhibitor of caspase-activated DNase (ICAD), were also increased by silymarin pretreatment. These results suggested that silymarin could also exaggerate the apoptotic effect of anti-Fas agonistic antibody CH11 on A375-S2 cells.     

Selective liposome targeting of folate receptor positive immune cells in inflammatory diseases

Activated macrophages play a key role in the development and maintenance of inflammatory diseases such as atherosclerosis, lupus, psoriasis, rheumatoid arthritis, ulcerative colitis, and many others. These activated macrophages, but not resting or quiescent macrophages highly up-regulate folate receptor beta (FR-β). This differential expression of FR-β provides a mechanism to selectively deliver imaging and therapeutic agents utilizing folate as a targeting molecule. In an effort to determine whether inflammatory diseases can be targeted utilizing a folate-linked nanosize carrier, a PEG-coated liposome was prepared that incorporated a folate conjugated PEG that also could transport imaging or therapeutic cargo. We demonstrate that these folate-liposomes specifically bind to folate receptor positive cells and accumulate at sites of inflammation in mouse models of colitis and atherosclerosis. These two animal models show that folate-targeted liposomes could be successfully utilized to deliver fluorescent molecules and an anti-inflammatory drug (betamethasone) for diagnostic and therapeutic applications.     

Silymarin,the antioxidant component and Silybum marianum extracts prevent liver damage

Liver disorders are one of the common recent problems affects on the human health. These disorders due to many environmental polluted sources. Many herbal, medicinal and pharmaceutical plants and their extracts are widely studied by many researchers. Silybum marianum got a bright reputation in relieve the liver diseases, and that might be for the potent silymarin mixture. Mechanism of action for silymarin conducted mainly to the antiradical and anticarcinogenic roles. Ethyl acetate (100 mg/kg bw) and ethanol seed extracts for S. marianum (100 mg/kg bw) were tested against the injection (i.p.) by carbon tetrachloride (2 ml/kg bw) the inducer of liver damage. Their activity were compared with standard hepatic drug hepaticum (100 mg/kg bw) for 10 days. Ethanolic extract showed the most significantly decrease in the liver enzymes. For the oxidative experiments, ethyl acetate showed the most increase for glutathione level and the risk factor HDL/LDL significantly. Hepaticum was the most powerful group for the significant decreasing for malondialdehyde and fucosidase activity. Some equal improvements were noticed in the histopathological studies for the protective groups.     

HPLC法测定人血浆中舒必利浓度及其人体药动学和相对生物利用度研究

目的:建立HPLC法测定人血浆中舒必利浓度,并研究其药动学及相对生物利用度。方法:采用固相萃取HPLC法,以甲氧氯普胺为内标,乙腈∶5 mmol/L磷酸二氢钾溶液(45∶55,V/V)为流动相,检测波长:λex 244 nm,λem 280 nm。18名受试者随机分成两组,采用双周期交叉试验设计,以HPLC法测定血药浓度,计算药动学参数并进行方差分析,以双单侧t检验进行生物等效性判定。结果:舒必利血浆浓度在6.76~1 689.6 ng/mL范围内样品浓度与峰面积比之间线性关系良好,回归方程为Y=0.00289079X+0.00247349(r=0.9998,n=9),方法回收率90%~110%,日内日间RSD<5%。18名男性健康志愿者单剂量口服100 mg受试制剂或参比制剂后AUC0→36分别为(3 996.0±579.5)ng.h.mL-1和(3 939.6±469.7)ng.h.mL-1,AUC0→∞分别为(4 715.5±753.2)ng.h.mL-1和(4 629.7±773.2)ng.h.mL-1,Tmax分别为(3.3±0.8)h和(2.7±1.0)h,Cmax分别为(362.9±118.7)ng.mL-1和(358.9±98.9)ng.mL-1,t1/2分别为(10.3±3.8)h和(9.7±4.1)h。受试制剂对参比制剂的相对生物利用度为101.9%±13.7%。结论:HPLC法能快速、准确的测定人血浆中的舒必利浓度;受试制剂与参比制剂具有生物等效性。     

The effectiveness of N-acetylcysteine in isolated hepatocytes,against the toxicity of paracetamol,acrolein, and paraquat

The protective effect of N-acetylcysteine against the toxicity of paracetamol, acrolein, and paraquat was investigated using isolated hepatocytes as the experimental system. N-acetylcysteine protects against paracetamol toxicity by acting as a precursor for intracellular glutathione. N-acetylcysteine protects against acrolein toxicity by providing a source of sulfhydryl groups, and is effective without prior conversion. Paraquat toxicity can be decreased by coincubating the cells with N-acetylcysteine, but the mechanism for the protective effect is not as clear in this instance. It is probable that N-acetylcysteine protects against paraquat toxicity by helping to maintain intracellular glutathinone levels.     

Long circulating liposomes of 2',3'-dideoxyinosine: Formulation and stability

2',3'-Dideoxyinosine (ddI), an anti-human immunodeficiency virus (HIV) agent, was encapsulated in liposomes. The influence of the phospholipid/cholesterol ratio, concentration of phospholipid (PL), and chain length of PL on the encapsulation of ddI in multilamellar vesicles (MLVs), frozen and thawed multilamellar vesicles (FAT MLVs), and large unilamellar vesicles (LUVs) was studied. An optimum formulation was then selected to prepare long circulating liposomes. Stability studies at 4, 25, and 37°C and under certain stress conditions were performed. Release characteristics in phosphate buffer (pH 7.4) at 37°C were studied. Results show an increase in encapsulation efficiency (EE) with increasing amounts of cholesterol, a decrease in EE and increase in encapsulation yield (EY) with increasing concentrations of PL, and an increase in EE with increases in PL chain length, in both MLVs and LUVs. Freezing and thawing of MLVs had no influence on EE at a PL concentration of 10 mg/mL but increased EE at higher concentrations of PL. Various stability tests showed the formulation to be stable to leakage of entrapped drug when stored at 4, 25, and 37°C for 6 months, when subjected to mechanical stress, and on exposure to human serum. The release studies indicated that 70% of ddI was released over a period of 72 h.     

Receptor-mediated gene delivery to HepG2 cells by ternary assemblies containing cationic liposomes and cationized asialoorosomucoid

Unilamellar cationic liposomes have been prepared from an equimolar mixture of 3beta[N',N'-dimethylaminopropane)-carbomoyl] cholesterol (Chol-T), a higher homologue of 3beta[N',N'-dimethylaminoethane)-carbomoyl] cholesterol (DC-Chol), and dioleoylphosphatidyl-ethanolamine. The DNA binding capabilities of Chol-T and Chol-T/DOPE liposomes have been demonstrated in lipid impregnated paper-DNA binding assays and gel retardation experiments, respectively. These liposomes have been combined with pRSVL plasmid DNA and N-ethyl-N'-(3-trimethylpropylammonium) carbodiimide iodide modified asialoorosomucoid (Me+ CDI urea-AOM) to generate ternary electrostatic assemblies intended for selective entry into cells displaying the galactose-specific lectin. This effect has been evaluated in the human hepatocellular carcinoma cell line HepG2 in which high levels of luciferase activity were achieved (up to 1.84 x 10(7) relative light units/mg protein) after transfection with complexes containing liposomes (1-3 microg), Me+CDI urea-AOM (2 microg), and DNA (0.5 microg) in 0.5 mL culture medium. Transfections conducted in the presence of free asialoorosomucoid afforded much lower luciferase activity (up to 1.5 x 10(5) relative light units/mg protein) confirming that DNA uptake was predominantly via asialoorosomucoid receptor-mediated endocytosis. We concluded therefore that modular complexes used in our study display the carbohydrate moiety of the glycoprotein component prominently, thus permitting interaction of terminal galactose units with their cognate receptors on the cell membrane.