吴茱萸碱与脂质体模拟生物膜的相互作用:FTIR和DSC研究

药物与生物膜相互作用的研究对于了解药物药效和改善其生物性能具有重要的意义。但生物膜的组成复杂,直接研究药物活性成分与生物膜的相互作用比较困难。以脂质体作为生物膜模型,研究了吴茱萸碱与脂质体的相互作用,分析了吴茱萸碱分子在脂质体中的包封位置,探讨了吴茱萸碱抗炎作用可能的作用机制。以二棕榈酰磷脂酰胆碱（DPPC）为膜材,应用薄膜分散法制备含有不同摩尔百分比（x）的吴茱萸碱脂质体,应用傅立叶变换红外光谱（FTIR）和差示扫描量热（DSC）技术分析随着脂质体中药物摩尔百分比的增大,DPPC分子各红外特征吸收峰频率、峰形及量热参数的变化情况,从而探讨药物在脂质体中的包封位置及吴茱萸碱分子对脂质体膜流动性的影响。实验数据表明,在0＜x＜10 mol%的浓度范围内,DPPC头部区域磷酸基团的不对称伸缩振动频率没有明显变化,脂质体相变温度和相变焓均随药物摩尔百分比的增大而减小。在0＜x＜5 mol%浓度范围内,DPPC界面区域的水化的羰基峰的吸收波数由1 726.0 cm-1增加到1 731.8 cm-1,当x=10 mol%时,该波数又减小到1 728.0 cm-1。在10 mol%≤x＜20 mol%浓度范围内,磷酸基团的不对称伸缩振动的波数由1 242.0 cm-1减小为1 236.3 cm-1,水化的羰基峰的吸收频率没有明显变化,脂质体相变温度和相变焓均随药物摩尔百分比的增大而增大。纯DPPC脂质体中亚甲基的对称伸缩振动波数为2 848.4 cm-1,载药后该波数都增大到2 850.3 cm-1。这些结果表明吴茱萸碱在脂质体中的包封位置具有浓度依赖性：在0＜x＜10 mol%浓度范围内,吴茱萸碱主要作用于DPPC分子的疏水尾链区域,少部分药物分子作用于DPPC分子的界面区域。在10 mol%≤x＜20 mol%浓度范围内,吴茱萸碱分子则主要作用于DPPC分子的头部区域,少部分药物分子作用于DPPC分子的疏水尾链区。所有载药脂质体的相变温度均低于纯DPPC脂质体的相变温度,即不同浓度的吴茱萸碱均可以使脂质体的膜流动性增加,并且,当药物摩尔百分比为10 mol%时,吴茱萸碱对生物膜流动性的增加效应最为明显。研究工作对于进一步揭示吴茱萸碱与生物膜的相互作用机制具有重要意义。     

Solubilization kinetics of phospholipid vesicles by sodium taurocholate

The solubilization kinetics of phospholipid vesicles, about 100 nm in diameter and composed of egg phosphatidylcholine (EPC) and EPC/cholesterol in molar ratio 7/3, by sodium taurocholate (TC) used as a model bile salt were investigated by monitoring the turbidity at 500 nm and by quasielastic light scattering (QELS). The solubilization process was found to be dependent on the rate of TC addition. Although the solubilization profiles were identical whatever the rate of TC addition, an increase in the amount of TC needed to solubilize phosphatidylcholine liposomes was observed at higher rates. These results suggest that at low TC concentrations the permeability of the membrane to taurocholate is the rate-limiting step of the solubilization. In the case of cholesterol-containing vesicles, the effect of the rate of addition of TC was observed only at the solubilization characteristic points, called B and C, corresponding to a sharp decrease in the turbidity. This suggests that cholesterol greatly reduces the permeability of the membrane. In addition, the kinetic process was found to be independent of the micellar concentration of the detergent added to the aqueous medium, indicating that the solubilization of liposomes by TC was independent of the initial state of aggregation of the detergent. The calculated values of lipid/TC aggregates and of the partition coefficient show that the kinetic effect observed at high TC concentrations prior to complete solubilization might also be due to the diffusion of the detergent into the membranes. This gives rise to the differences in composition of the aggregates as a consequence of the variation in the rate of TC addition. In addition, QELS scattered intensity variations confirm the presence of a kinetic process for the solubilization of liposomes by TC. In conclusion, our results suggest that solubilization of lipid vesicles by TC is governed by kinetic parameters that might be controlled by liposome membrane permeability at low TC concentrations and by the lateral diffusion of the detergent into aggregates at higher TC concentrations.     

Influence of cyclodextrins on the physical stability of DPPC-liposomes

This paper reports on the physical stability of DPPC-(dipalmitoyl phosphatidyl choline) liposomes in various aqueous dispersions and its control by uncharged polymers. The effect of natural (-, β-, γ-) cyclodextrins (CDs) on the stability of bare and polymer-bearing liposomes and also, the attachment of the CD molecules and the macromolecules, respectively, to the DPPC-bilayers of small unilamellar vesicles (SUV) were studied.

It was found that above a CD/DPPC ratio, each cyclodextrin caused a definite destruction in the phospholipid bilayers. The extent of membrane destabilization due to a cyclodextrin closely related to the amount of the CD molecules bound to the DPPC-bilayers.

The polymer-coated liposomes formulated by incorporating a dissolved homopolymer or copolymer into the phospholipid bilayer of the vesicles exhibited higher physical stability. Uncharged polymers effectively hindered the disintegration of the liposomal membranes brought about by the CD molecules. The polymer layers formed around the phospholipid bilayers ensured an enhanced steric stabilization for the DPPC-liposomes. Methylcellulose (MC) with high molecular mass and a polyvinyl alcohol-co-vinyl propional copolymer alike exhibited efficient stabilizing effect.     

Cyclodextrin-mediated entrapment of curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA in liposomes for treatment of xenograft lung tumor in rats

We recently reported a novel curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA as a potent anti-proliferative agent, and showed that it induces autophagic cell death in lung cancer cells. We are now reporting a drug-in-CD-in-liposome approach to formulate CLEFMA liposomes that could be labeled with Tc-99m radionuclide for non-invasive imaging of their biodistribution. CLEFMA encapsulation was enabled by hydroxypropyl-β-cyclodextrin. In vitro studies showed that CLEFMA possessed more potent anti-proliferative activity in lung adenocarcinoma H441 cells than naturally occurring curcumin. At the same time, it had no effect on the proliferative capacity of normal lung fibroblasts. CLEFMA liposomes retained the antiproliferative potency of free CLEFMA, while maintaining its non-toxic nature in normal lung fibroblasts. In nude rats bearing xenograft H441 tumors, the tumor volume significantly reduced after i.v. treatment with CLEFMA liposomes (p<0.05); the tumor inhibition was determined to be 94%. The anti-tumor activity of CLEFMA liposomes was confirmed by the observation that F-18-fluorodeoxyglucose uptake in tumors of treated rats was reduced as compared to those of control rats. Tc-99m-labeled CLEFMA liposomes accumulated in liver (33.7%); spleen showed the largest accumulation on per gram tissue basis (6.2%/g). Upon histopathological examination of liver, lung and kidney, we found no apparent toxicity from multiple CLEFMA liposome administrations. The results demonstrate the utility of liposomes to serve as a carrier for CLEFMA. This study is the first to demonstrate the efficacy of novel curcuminoid CLEFMA in a preclinical model.     

Interactions of amphiphilic block copolymers with lipid model membranes

Studies on interactions between amphiphilic block copolymers and lipid membranes have been focused traditionally on ABA triblock copolymers of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), widely due to their commercial availability. However, new architectures of amphiphilic block copolymer have been synthesized in recent years partially taking advantage of new polymerization techniques. This review focuses on amphiphilic block copolymers with potential biological activity and on model membrane systems used for studying interactions with such block copolymers. Experimental methods to study block copolymer–phospholipid interactions in Langmuir monolayers, liposomes, and planar bilayers are summarized. This work is intended to convey a better understanding of amphiphilic block copolymers used for in vitro and in vivo experiments in medicine and pharmacy. Recent developments and open questions are addressed.     

Polymer-associated liposomes as a novel delivery system for cyclodextrin-bound drugs

It is known that cyclodextrins (CDs) extract lipid components from bilayer of liposomes. This could undermine the potential benefits of liposomes as drug carriers. In this study, we demonstrated that PC-Chol liposomes with various CDs or rhapontin (Rh)-hydroxypropyl betaCD (HPbetaCD) complexes could be stabilized by association with the amphiphilic polyelectrolyte, poly(methacrylic acid-co-stearyl methacrylate). Based on the results of differential scanning calorimetry, photocorrelation spectroscopy and transmission electron microscopy, the polymer-associated liposomes had the same vesicular form as liposome with clear boundaries and retained structural integrity for at least 1 month. In addition, the polymer-associated structure was unaffected by the type of CD, the composition and concentration of lipid components, and the concentration of the Rh-HPbetaCD complex. This contrasted with PC-Chol liposomes, whose structure was dependent on these factors. Using structurally different polymer-associated liposomes and PC-Chol liposomes containing the Rh-HPbetaCD complex, we also showed that the stability of vesicles could influence the skin permeability of CD-drug complexes.     

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.     

Determination of carbofuran in water by homogeneous immunoassay using selectively conjugate mastoparan and terbium/dipicolinic acid fluorescent complex

Homogeneous immunoassay (LITRFIA) for carbofuran (CF) determination was performed using liposomes and mastoparan (Mast) conjugate as cytolitic agent. Mast was conjugated to the 5-(2-2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)-pentanoic acid (CPCF) both randomly and selectively to a single (V¹- or K⁴-) amino-group. The conjugated compounds have been tested for the cytolytic activity on liposomes trapping Tb/citrate complex. Dipicolinic acid (DPA) was used as fluorescent chelating agent. The CPCF–V¹–Mast derivative (retaining almost the same lytic activity as Mast) was used in the immunoassay in competition with standard CF. Liposome lysis was proportional to the standard concentrations in a dynamic range between 10 pg and 10 ng. Assay has been performed for tap water analysis and for 10 real samples taken from an agricultural area to the south of Milan. Recovery in samples spiked with two different CF concentrations was between 92.5 and 105%.     

The overall adhesion-spreading process of liposomes on a mercury electrode is controlled by a mixed diffusion and reaction kinetics mechanism

Using high-resolution chronoamperometric measurements, with sampling each 1.333 μs, the initial step of the adhesion-spreading of liposomes on a mercury electrode was studied. These measurements allow getting a deeper insight into the first interaction of the liposomes with the mercury electrode, and they show that the overall adhesion-spreading process at different potentials is partially controlled by a fast but weak interaction equilibrium resulting in a mixed diffusion- and reaction-kinetics-controlled mechanism of the overall reaction. The authors dedicate this contribution to Keith Oldham on the occasion of his 80th birthday. Since my (FS) first meeting with Keith Oldham in Alan Bond’s laboratory in Australia in 1987, I had the privilege to get Keith’s unerring advice and have stimulating discussions with him for which I like to cordially thank him.