Trifluralin liposomal formulations active against Leishmaniadonovani infections

The purpose of this study was to increase the therapeutic index of the antiparasitic drug, trifluralin (TFL), to allow its parenteral administration without the need of toxic solvents. This was achieved by incorporating TFL in liposomes with high loading capacity. These formulations were stable in freeze-dried form during at least one year and in frozen form during at least three months. Therapeutic activity, assessed on a visceral model of infection, showed that TFL liposomes reduced the number of parasites by up to one third or one half as compared to negative control and to free TFL, respectively.     

Liposome-Mediated calcium phosphate formation in metastable solutions

Summary The present study examined the effect of anionic liposomes on precipitate formation in supersaturated calcium phosphate solutions. The liposomes were prepared by dispersing 7∶2∶1 molar mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol in buffered aqueous solutions containing 0 or 50 mM inorganic phosphate (P₁). Unencapsulated P_I was removed by gel filtration. The liposomes were then suspended in reaction solutions containing 2.25 mM Ca²⁺ and either 0, 1.0, or 1.5 mM P₁. All experiments were carred out at 22°C, pH 7.4, and 240 mOsm. External solution Ca²⁺ and P_I losses were found to be appreciable only when the membranes of liposomes containing entrapped P_I were made permeable to Ca²⁺ with the addition to the suspension of the ionophore X-537A. The Ca²⁺ losses, moreover, were up to 3 times as great (1.5 vs 0.5 mM) when accompained by external P₁ losses than in P_I-free external solutions where Ca²⁺ alone was involved. Previous studies showed that in this latter situation, decline in external Ca²⁺ concentration was the result of precipitate formation in the aqueous interiors of the liposomes. The present findings suggest that when the external solution phase was metastable, the apparent coupling of large additional Ca²⁺ losses with intraliposomal precipitation was due to secondary precipitation brought about by the seeding action of intraliposomal crystals penetrating into the external solution. The results may explain in part the origin of extravesicular mineral deposits in matrix vesicle calcification.     

Liposomes with nifedipine and nifedipine-cyclodextrin complex: calorimetrical and plasma stability comparison

Inclusion complexes of nifedipine with 2-hydroxypropyl-ß-cyclodextrin (HPβCD) were formed by the spray- and freeze-drying methods. Nifedipine or its inclusion complexes (Nifedipine-CD complex I and II) were incorporated into liposomes prepared by the ethanol injection method. The highest entrapment value (77.7% of the starting material) was achieved for liposomes with N-CD complex II. The interaction of nifedipine with lipid bilayers was measured calorimetrically. DPPC liposomes mixed with nifedipine or N-CD complex II showed a slight shift of the transition temperature of DPPC towards lower temperatures compared to DPPC liposomes alone or mixed with HPßCD. However, with nifedipine, an additional transition peak was seen at lower temperatures in the second and all subsequent scans which could not be detected for the N-CD complex. Plasma stability studies showed that liposomes containing N-CD complex II are more stable than liposomes containing nifedipine. Encapsulation of drug-cyclodextrin complexes into liposomes can increase the entrapment of the lipophilic drug and reduce its release from the carrier.     

Effect of membrane cholesterol on calcium phosphate formation in aqueous suspensions of anionic liposomes

Summary The present study examined the effect of membrane cholesterol on liposome-mediated calcium phosphate precipitation in metastable aqueous solutions (2.25 mM Ca²⁺ and 1.5 mM inorganic phosphate) at 22°C, pH 7.4 and 240 mOsm. The liposomes were prepared from 7:2:X molar mixtures of phosphatidylcholine, dicetylphosphate, and cholesterol (x=0, 1, 5, or 9) and contained either 0 or 50 mM encapsulated phosphate. The membranes were made permeable to Ca²⁺ by addition of the cationophore, X-537A. Changes in external Ca²⁺ concentration were used as the principal monitor of the course of precipitation. Without encapsulated phosphate, 7:2:X liposomes (with or without ionophore) induced no precipitation. With 50 mM encapsulated phosphate and in the presence of ionophore, precipitation significantly depended on the cholesterol level in the membrane. At 0 and 10 mole% cholesterol, precipitate developed rapidly both within and outside the liposomes. At 35 and 50 mole% cholesterol, no observable intraliposomal precipitation occurred, and extraliposomal precipitation started only after an induction period of 24 hours. Delayed extraliposomal precipitation also took place in PO₄-containing liposomes without added ionophore. In this latter case, however, cholesterol was essential for this precipitation to occur with the optimum level being around 10 mole%. Suppression of ionophore-mediated intraliposomal precipitation at higher cholesterol levels could be related to the inflexible cholesterol molecules making the membrane more rigid, thereby restricting Ca-ionophore transport. This restriction could be reversed with ethanol. Delayed extraliposomal precipitation in the absence of added ionophore (or at higher cholesterol levels in its presence) could be explained by seeding from low, unobserved levels of intraliposomal precipitate formed during slow, unfacilitate Ca²⁺ leakage into the liposomal interior.     

两性霉素B脂质体无菌检查法研究

在一般无菌检查方法的基础上，结合两性霉素Ｂ与卵磷脂的理化特点，确立了两性霉素Ｂ脂质体的无菌检查方法。用无菌甲醇４０ｍｌ溶解样品（含两性霉素Ｂ４ｍｇ），通过０．４５μｍ薄膜（ＴＹＰＥＦＨＭＩＬＬＩＰＯＲＥ），用ｐＨ７．３磷酸盐缓冲液（含０．１％吐温８０，０．００２％甘牛胆酸钠）和生理盐水各２００ｍｌ先后冲洗，按一般无菌方法进行检验。实验结果表明，本方法简单、易行、可靠。     

Studies on the intracerebral injection of vincristine free and entrapped within liposomes in the rat

The cerebral tissue response and behaviour of rats injected with vincristine of increasing concentration, free and entrapped within liposomes was studied. In separate experiments, blood, urine and brain levels of vincristine were measured after intracerebral injection of free and liposome-entrapped vincristine. When entrapped within liposomes, vincristine clearance from the brain was significantly reduced and the amount of tissue damage was directly related to the amount of drug given, being slightly greater at the same dose when entrapped. These results indicate a potential application for drugs entrapped within liposomes acting as a depot preparation in the treatment of cerebral gliomas.     

Enhancement of the transdermal delivery of catechins by liposomes incorporating anionic surfactants and ethanol

The aim of this study was to develop and evaluate liposomal formulations encapsulating tea catechins, which possess antioxidant and chemopreventive activities. Liposomes were characterized for size, zeta potential, and entrapment efficiency. Both in vitro and in vivo skin permeation were examined using nude mouse skin as a model. The results suggested that the liposomal composition plays an important role in affecting the efficiency of transdermal catechin delivery. Incorporation of anionic surfactants such as deoxycholic acid (DA) and dicetyl phosphate (DP) in the liposomes in the presence of 15% ethanol increased the (+)-catechin permeation by five to seven-fold as compared to the control. The flexibility of bilayers is suggested as an important factor governing the enhancing effect of liposomes. Intercellular spaces within the stratum corneum but not shunt routes are the major pathways for catechin delivery from liposomes. (+)-Catechin and (−)-epicatechin are isomers which showed similar encapsulation efficiencies and skin permeation in liposomes. (−)-Epigallocatechin-3-gallate showed the highest encapsulation rate and in vivo skin deposition level in liposomes among all catechins tested. The stability and in vitro tranepidermal water loss test indicated the safety of the practical use of liposomes developed in this study.     

甲氨喋呤脂质体药物释放动力学研究

本文应用荧光分光光度法研究了甲氨喋呤脂质体冻干粉针在几种输液中药物释放动力学。结果表明:甲氨喋呤脂质体的药物释放速率符合一级动力学方程。甲氨喋呤脂质体在不同输液中药物释放速率常数不同。温度升高、药物释放速率常数增大。     

An effect of antibiotic amphotericin B on ion transport across model lipid membranes and tonoplast membranes

A pH sensitive fluorescence probe piranine trisulfonate, entrapped inside small unilamellar liposomes formed with egg yolk phosphatidylcholine, was applied to investigate effect of polyene antibiotic amphotericin B (AmB) on proton transport across lipid membranes. Time dependencies of fluorescence-monitored pH changes inside lipid vesicles, upon sudden acidification of the liposome suspension, were analyzed in terms of two-exponential kinetics. It appears that addition of AmB at 3 mol%, with respect to lipid, considerably increases the rate constant of the fast component of proton transport (a change from (60 to 149) x 10(-3)s(-1)) and decreases the rate constant of the slow component (a change from (11 to 5) x 10(-3)s(-1)). Incorporation of 0.1 mol% AmB results in the decrease of both parameters (to (33 and 2) x 10(-3)s(-1), respectively). The increase in the rate of proton transfer across the lipid membrane is interpreted as related to the formation of membrane channels by AmB, at higher concentration of the drug or nonspecific destabilization of the membrane structure. At low concentrations, at which formation of molecular structures of AmB is not possible, the antibiotic molecules are oriented horizontally with respect to the plane of the membrane and act in making the membrane more compact and less permeable to ions. The presence of sterols (cholesterol, ergosterol and cholesterol dimer) in the lipid phase, in the concentration 3 mol% and lower, decreased the rate constants of proton transfer across the membranes but did not influence significantly the effect of AmB on the ion transport. The presence of AmB in the bathing solutions of tonoplast membranes isolated from Conocephalum conicum at the concentrations range 1 x 10(-7) to 3.6 x 10(-5) does not influence considerably the ion current, as monitored by means of the patch-clamp technique.     

9-硝基喜树碱及其脂质体对HepG2、L02细胞周期和凋亡作用的研究

目的 研究9-硝基喜树碱及其脂质体对人肝癌细胞株HepG2和人正常肝细胞L02细胞周期、凋亡的影响及其可能的作用机制.方法 HepG2、L02细胞用含不同浓度9-硝基喜树碱及其脂质体的培养液孵育后,利用MTT法测定细胞活性,流式细胞术检测细胞周期及细胞凋亡,WesternBlot验证周期相关蛋白和凋亡相关蛋白的表达变化.结果 9-硝基喜树碱及其脂质体对两种细胞生长呈时间和剂量依赖性抑制.药物处理后S期和G2/M期细胞明显增多,浓度大于0.1 μmol/L时24 h后HepG2细胞完全阻滞于S期;0.1 μmol/L孵育72 h后,超过95%HepG2细胞阻滞于G2/M期.药物对细胞凋亡的诱导作用也呈明显的剂量和时间依赖关系.Western Blot显示:Bax、Caspase3表达增高,Cyclin A、Cdk2、Cyclin E、Bcl-2表达减低.与L02相比,HepG2细胞对药物更加敏感.结论 9-硝基喜树碱及其脂质体可以通过调控细胞周期和诱导凋亡有效抑制细胞生长,其对肿瘤细胞的抑制作用强于正常肝细胞.9-硝基喜树碱脂质体体外抗肿瘤效果略强于9-硝基喜树碱单体.

Abstract：

Objective To investigate the modulating effects and explore their mechanism of 9-nitrocamptothecin and its liposomes to induce apoptosis and inhibit cell cycle in HepG2 and L02 cell lines. Methods Cells were incubated with 9-nitrocamptothecin(9NC) or with 9-nitrocamptothecin liposomes for 24 h, 48 h and 72 h, then, the cell viability was measured via MTT assay; cell cycle and apoptosis was evaluated by flow cytometry after stained by PI and Annexin V-PE/7AAD. Additional, Western Blot was used to evaluate the expression of cell cycle and apoptosis related protein. Results Both cells viability were apparently inhibited by the 9-nitrocamptothecin and 9-nitrocamptothecin liposomes, the inhibitory effect showed a time-dependent and dose-dependent manner. Both S and G2/M phases arrest were observed after incubated with drugs. HepG2 cell was completely arrested in S phase when 9NC concentration over than 0. 1 μmol/L after incubation for 24 h, while more than 95% cells arrested in G2/M phase when 9NC concentration is 0. 1 μmol/L after incubation for 72 h. Apoptosis induction effect also showed a time-dependent and dose-dependent manner. Western Blot results showed the expression of Bax and Caspase-3 were upregulated while Cyclin A, Cdk2, Cyclin E and Bcl-2 were downregulated. More importantly, the compounds were more cytotoxic to the cancer cell lines than to the normal liver cell. Conclusions 9-nitrocamptothecin and 9-nitrocamptothecin liposomes can potently inhibit cell growth via regulation of cell cycle and induction of apoptosis, and this effect was preferentially in cancer cell. Inhibitory of 9-nitrocamptothecin liposomes was slightly better than the 9-nitrocamptothecin.