Asymmetric flow field‐flow fractionation of liposomes: 2. Concentration detection and adsorptive loss phenomena

The applicability of different concentration detection methods for online quantification of liposomes upon asymmetric flow field‐flow fractionation was investigated. Filter‐extruded egg phosphatidylcholine liposomes of different size were used. Online quantification using a differential refractive index (dRI) detector was found feasible for relatively high sample loads in the magnitude of 100 μg lipid (under the chosen fractionation conditions). UV–Vis detection of the turbidity of liposomes was ruled out as online detection method because turbidity increases with particle size and the signal is not only concentration but also particle‐size dependent. Staining of liposomes by Rhodamine phosphatidylethanolamine or Sudan Red and subsequent online UV–Vis detection at the absorption maximum of the dye enabled quantification with much higher sensitivity than dRI detection. Furthermore analyte loss and carry‐over phenomena upon repeated injection of varying liposome sample loads were studied using regenerated cellulose (RC) membranes as accumulation wall. It could be shown that RC membranes are prone to adsorption in case of very small sample loads (0.5 μg). This effect may be overcome by pre‐saturation of the membrane with sample loads of at least 2 μg. For higher sample loads adsorptive losses play a minor role. Recovery from pre‐saturated membranes reached approximately 100% and carry‐over was found negligible.     

Asymmetric flow field‐flow fractionation of liposomes: optimization of fractionation variables

The purpose of this study was to investigate the influence of ionic strength of the carrier liquid, cross flow rate, focus flow rate, and sample load on the retention behavior of liposomes in asymmetric flow field‐flow fractionation (AF4). Two differently prepared samples of large unilamellar vesicles (LUV) were used. Experiments were performed varying the factors systematically and evaluating their effect on both retention behavior of the liposomes and on particle size as obtained from online coupled multi‐angle light scattering (MALS) analysis. The results showed that the focus flow rate had the least influence on the elution of liposomes. Elution of LUV is mainly governed by the chosen cross flow condition and ionic strength of the carrier liquid as well as its sample load. Optimal fractionation and size analysis were achieved using a sample load of about 10 μg, a cross flow gradient from 1.0 to 0.1 mL/min over 35 min and a carrier solution of NaNO₃ with a concentration of 10 mM.     

纳米白藜芦醇脂质体的制备及分配系数测定

采用薄膜旋转蒸发-超声法制备了纳米白藜芦醇脂质体(RES-Lip),并用透射电子显微镜(TEM)和动态光散射技术(DLS)对产物进行表征;测定了膜材比(卵磷脂与胆固醇质量比m PC:m Chol=5:1,8:1,10:1,12:1)和药脂比(药物与卵磷脂质量比m RES:m PC=1:25,1:40,1:50,1:60)对RES-Lip脂质体-水分配系数(Plip/w)的影响,以及油-水分配系数(lg P_(o/w))和脂质体-水分配系数(lgP_(lip/w))随p H值的变化趋势,计算了RES-Lip中药物与磷脂双分子膜之间的吉布斯自由能。结果表明,实验中所制备的RES-Lip呈球形囊泡结构,粒径约为100 nm;当膜材比和药脂比分别为10:1和1:40时,lgP_(lip/w)最大,说明此时RES与磷脂膜间的综合作用力最大;RES-Lip的分配系数(lg P_(o/w)和lgP_(lip/w))随体系p H的变化趋势相同,说明RES与磷脂膜的作用力中以疏水作用为主,氢键、静电作用为辅;RES-Lip中RES与脂质体膜之间的吉布斯自由能为-17.07 k J·mol~(-1)。     

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69+/-8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.     

Amitriptyline overdose treatment by pegylated anionic liposomes

Liposomes composed of DOPG, 50:50 DMPC:DOPG, 95:5 and 85:15 DOPG:DPPE-mPEG-2000, and 55:15:30 DMPC:DPPE-mPEG-2000:CH were studied for their ability to sequester amitriptyline in human serum. The effects of lipid type and loading, liposome size, PEG inclusion, protein interaction and storage were considered. Liposome size had no effect on drug uptake, as 40, 100, and 284 nm liposomes bound similar amounts of drug in buffer. The optimal amount of PEG-modified lipid incorporated into liposomes was found to be 5%. 95:5 DOPG:DPPE-mPEG-2000 liposomes loaded at 1.44 mg lipid/mL were most effective at shielding protein interactions while still allowing amitriptyline to diffuse to the bilayer surface and bind. Absolute reductions of 99% in buffer and human serum samples were observed, while the free drug concentration reduction relative to binding in serum without liposomes was nearly 90% across a drug concentration range of 1-20 muM. With such reductions, serum drug concentrations could be rapidly reduced from toxic to therapeutic levels. Furthermore, storage tests revealed that such liposomes may be stored for at least one month without a change in drug binding ability. These findings strongly suggest that predominantly anionic liposomes incorporated with PEG are excellent candidates for amitriptyline overdose treatment.     

Liposomes Assembled from a Dual Drug‐tailed Phospholipid for Cancer Therapy

We report a novel dual drug‐tailed phospholipid which can form liposomes as a combination of prodrug and drug carrier. An amphiphilic dual chlorambucil‐tailed phospholipid (DCTP) was synthesized by a straightforward esterification. With two chlorambucil molecules as hydrophobic tails and one glycerophosphatidylcholine molecule as a hydrophilic head, the DCTP, a phospholipid prodrug, undergoes assembly to form a liposome without any additives by the thin lipid film technique. The DCTP liposomes, as an effective carrier of chlorambucil, exhibited a very high loading capacity and excellent stability. The liposomes had higher cytotoxic effects to cancer cell lines than free DCTP and chlorambucil. The in vivo antitumor activity assessment indicated that the DCTP liposomes could inhibit the tumor growth effectively. This novel strategy of dual drug‐tailed phospholipid liposomes may be also applicable to other hydrophobic anticancer drugs which have great potential in cancer therapy.     

Possibility of Targeting Treatment for Ischemic Heart Disease With Liposome (Ⅰ)

This paper reports the basic research on the possibility of using targeting treatment for ischemic heart disease with liposome as drug carrier. Studies have been performed on isolated rat cardiomyocytes, or isolated perfused rat and rabbit hearts. Results show that cardiomyocytes may interact with liposome through fusion, endocytosis, adsorption and molecular exchange of phospholipid. Forms of cellular uptake of liposome depend chiefly on the physicochemical properties of liposomes. Anoxia changes the pattern of liposome uptake by cardiomyocytes and increases uptake of liposomes. Uptake of liposomes, especially of positively charged liposomes by ischemic myocardium is significantly increased. The quantity of increase of liposome uptake is in the following order: ischemia-reperfusion area>peripheral area of the infarct>non-ischemic area>infarcted area. The above results indicate that liposome as drug carrier might promote the delivery of drug into ischemic myocardium and cardiomyocytes.     

Effects of diclofenac on EPC liposome membrane properties

In this work the interaction of a non-steroidal anti-inflammatory drug (NSAID), diclofenac, with egg yolk phosphatidylcoline (EPC) liposomes, used as cell-membrane models, was quantified by determination of the partition coefficient. The liposome/aqueous phase partition coefficient was determined by derivative spectrophotometry, fluorescence quenching, and measurement of zeta-potential. Theoretical models based on simple partition of the diclofenac between two different media, were used to fit the experimental data, enabling the determination of K_p. The three techniques used yielded similar results. The effects of the interaction on the membranes characteristics were further evaluated, either by studying membrane potential changes or by effects on membrane fluidity. The liposome membrane potential and the size and size-homogeneity of liposomes were measured by light scattering. The effects of diclofenac on the internal viscosity or fluidity of the membrane were determined by use of spectroscopic probes—a series of n-(9-anthroyloxy) fatty acids in which the carboxyl terminal group is located at the interfacial region of the membrane and the fluorescent anthracene group is attached at different positions along the fatty acid chain. The location of the diclofenac on the membrane was also evaluated, by fluorescence quenching using the same series of fluorescent probes. Because the fluorescent anthracene group is attached at different positions along the fatty acid chain, it is possible to label at a graded series of depths in the bilayer. The interactions between the drug and the probe are a means of predicting the location of the drug on the membrane.     

Thick-target PIGE analysis of plant materials preconcentrated by dry ashing

An analytical methodology has been developed for the UV-spectrophotometric determination of carbaryl in waters after its preconcentration onto a polyether type polyurethane foam followed by on-line elution. The aforementioned strategy offers an easy way for in-field sampling and to improve the analytical sensitivity. Several chemical and flow variables (mass of sorbent, sample flow rate, sample volume and carrier flow rate) were studied to ensure the best performance of the system. Recovery studies, carried out on natural water samples spiked with known amounts of carbaryl at concentration levels between 250 and 500 mug l(-1), provided recovery percentages between 94 and 105%. A detection limit of 12 mug l(-1) was achieved and a variation coefficient of 3.4% was obtained at 0.50 mug ml(-1).     

Determination of liposome–buffer distribution coefficients of charged drugs by capillary electrophoresis frontal analysis

The potential of using CE frontal analysis (CE‐FA) to study the interactions between a range of charged low molecular weight drug substances and liposomes was evaluated. The liposomes used were net negatively charged and consisted of 2‐oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phosphocholine and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphate monosodium salt in a ratio of 80/20 mol%. Apparent distribution coefficients (D_mem), defined as the molar concentration of drug substance in the membrane phase divided by the molar concentration of drug substance in the aqueous phase, were successfully determined for six positively and eight negatively charged drug substances with log D_mem ranging from 1.35 to 3.63. The extent of liposome–buffer distribution was found to be dependent on the drug concentration. The results obtained with the developed CE‐FA method were in good agreement with results obtained by equilibrium dialysis. Furthermore, the CE‐FA method was faster, less labor intensive and required smaller sample volumes (～50 μL) compared with equilibrium dialysis. Thus, CE‐FA is an efficient and useful tool for the characterization of interactions between liposomes and low molecular weight drug substances.     

Liposome solubilization induced by surfactant molecules in a microchip

The dynamics of liposome solubilization was monitored by dynamic light scattering and optical microscopy. A newly designed Y-shape microchannel connected to a room was incorporated into a microchip and the reaction processes of the liposome suspension and surfactant solution were observed in the room after mixing the two fluids and stopping the flow. By using this microchip, we succeeded in real-time monitoring of liposome solubilization and the following dynamic processes of solubilization were proposed: 1) Deformed liposomes become spherical. 2) The liposome size increases until the surfactant/liposome ratio in the liposome membrane reaches a threshold value. 3) Mixed micelles of surfactants and phospholipids are released and the liposomes collapse.     

Partition and location of nimesulide in EPC liposomes: a spectrophotometric and fluorescence study

Study of the mechanism of action of anti-inflammatory drugs (NSAIDs) and their side-effects may fall in the domain of membranology. In this work the extent of the interaction between an NSAID (nimesulide) and membrane phospholipids was quantified by the partition coefficient, K_p , using egg phosphatidylcholine (EPC) liposomes as cell membrane models. The liposome/aqueous phase partition coefficients were determined under physiological conditions, by derivative spectrophotometry and fluorescence quenching. Derivative spectrophotometry allows elimination of background signal effects (light scattering) due to the presence of liposomes. Theoretical models, accounting for simple partition of the NSAID between two different media, were used to fit the experimental data, allowing the determination of K_p in multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs). The location of nimesulide in MLVs and LUVs was evaluated by fluorescence quenching using spectroscopic probes located at different sites on the membrane. All n-AS probes were quenched and the relative quenching efficiencies were ordered as 2-AS<6-AS9-AS<12-AS; this suggests the drug is deeply buried in the membrane. Fluorescence quenching using the 12-AS probe was also used to determine the partition coefficient of the drug in MLVs and LUVs. The two techniques yield similar results. Finally, measurement of zeta-potential in the presence of different concentrations of nimesulide was performed to investigate possible changes in the zwitterionic phosphatidylcholine membranes. The membrane surface potential was not altered, which seems to be an indication that nimesulide binds to lipid bilayer mostly by hydrophobic interactions.     

A versatile liposome/cyclodextrin supramolecular carrier for drug delivery through the blood-brain barrier

For many commercial drugs, reaching the central nervous system in large amount without damaging the blood-brain-barrier (BBB) remains a challenging task. We present here a supramolecular strategy aiming at using a well-defined cyclodextrin-coated liposomes as drug carrier and adamantoyl saccharides as BBB-interacting ligands. In this study, the liposome is constituted of n-alkyldimethylammoniumcyclodextrins incorporated in the lipid bilayer of a 3/7 cholesterol/dipalmitoylphosphatidylcholine mixture and the ligand is constituted of an adamantoylglucose molecule whose adamantoyl moiety can be included in the CD cavity. The whole supramolecular assembly has been characterized by light-scattering and ³¹P NMR measurements. Toxicity and permeability studies on an in vitro model of the BBB clearly demonstrated a 5-fold improved ability of the modified liposome to enter the BBB-endothelial cells compared to the non-coated liposome. Fluorescence labelling of these liposomes is also displayed with DiI as a fluorescent probe.     

Investigation of Single‐Drug‐Encapsulating Liposomes using the Nano‐Impact Method

Encapsulating liposomes are widely used for controlled drug delivery. We report the use of nano‐impact experiments for the electrochemical attomolar quantification of the liposome load, uniquely at the single liposome level, using vitamin C encapsulated liposomes as a model. The size of the liposomes and their picomolar concentration are also determined in biological buffer in real time.     

基于非对称场流分离技术分离表征小米淀粉

基于非对称场流分离技术耦合多角度激光光散射检测器和示差折光检测器,建立了分离表征小米淀粉的方法。研究了进样量、交叉流流速、半衰期（t_1/2）、载液离子强度和pH值对小米淀粉分离效果的影响;考察了该方法的重现性;探究了小米淀粉分子结构。结果表明,在进样体积为50 μL、进样质量浓度为0.50 g/L、交叉流流速为1.2 mL/min、t_1/2=3 min、载液为10 mmol/L pH 7.00 NaNO₃（含3 mmol/L NaN₃）的条件下,小米淀粉分离效果最佳。该方法具有良好的重现性,得到的小米淀粉的回转半径相对标准偏差为3.4%、摩尔质量相对标准偏差为7.0%。     

Formulation and evaluation of celastrol-loaded liposomes

The main purpose of this study was to evaluate the intestinal absorption and the antineoplastic effect of the poorly water-soluble drug celastrol when liposomes were used as oral drug delivery system. Liposomes were prepared by the ethanol-injection method. An optimized liposome formulation composed of phospholipid, cholesterol and Tween-80 resulted in favorable encapsulation efficiency at 98.06 ± 0.94%. Homogeneous and stable particle size of 89.6 ± 7.3 nm and zeta potential of -(87.7 ± 5.8) mV were determined by laser particle size analyzer. Subsequently, the four-site perfusion rat intestinal model revealed that celastrol-loaded liposomes had improved effective permeability compared to the free drug in four intestinal segments (p < 0.05). Moreover, celastrol-loaded liposomes could also inhibit the tumor growth in C57BL/6 mice. These results suggest that liposomes could be a promising perioral carrier for celastrol.     

A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating

To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as L-cysteine, and thereby internalization of the liposomes was significantly facilitated. When L-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells.     

Effect of Different Pretreatment Methods on the Measurement of the Entrapment Efficiency of Liposomes and Countermeasures in Free Drug Analysis

Further investigation into the methods for the analysis of the entrapment efficiency of liposomes has been prompted by the urgent need to improve traditional methods which offer more risk to liposome leakage. The aim of this study was to investigate the suitability of hollow fiber centrifugal ultrafiltration (HF-CF-UF) coupled with HPLC as an alternative method for the routine determination of the entrapment efficiency of liposomes and to compare it with previously developed nonequilibrium procedures based on size-exclusion chromatography (SEC). By comparison, evaluation of the entrapped fraction with SEC resulted in 3 or 4 % lower entrapment values. More importantly, the free drug concentrations were three- to sixfold higher than with HF-CF-UF. In addition, we investigated the reasons for the different values obtained with SEC and a dynamic equilibrium theory was put forward based on this. In addition, we have developed and characterized an equilibrium method of separating free and liposomal drugs for liposomal formulations based on HF-CF-UF. The method was validated over the concentration range of 7.9–235 μg mL^?1 for indomethacin and 0.258–8.24 μg mL^?1 for vitamin A. Inter- and intra-day precision (RSD %) were ≤1.2 % for indomethacin and ≤1.8 % for vitamin A, respectively. The recoveries of both free drug and total drug were higher than 96.0 % with RSD ≤1.3 % (n = 5). Taken together, our improved method differs from those previously reported with regard to higher recovery, small sample volume, less laborious, and most importantly without damaging or disturbing liposomes. Validation results suggested that our method was sufficiently accurate and sensitive to be used to evaluate the entrapment efficiency of a liposome formulation without complicated pretreatment.     

Performance of hollow-fiber flow field-flow fractionation in protein separation

Since hollow-fiber flow field-flow fractionation (HF FIFFF) utilizes a cylindrical channel made of a hollow-fiber membrane, which is inexpensive and simple in channel assembly and thus disposable, interests are increasing as a potential separation device in cells, proteins, and macromolecules. In this study, performance of HF FIFFF of proteins is described by examining the influence of flow rate conditions and length of fiber (polyacrylonitrile or PAN in this work) on sample recovery as well as experimental plate heights. The interfiber reproducibility in terms of separation time and recovery was also studied. Experiments showed that sample recovery was consistent regardless of the length of fiber when the effective field strength (equivalent to the mean flow velocity at the fiber wall) and the channel void time were adjusted to be equivalent for channels of various fiber lengths. This supported that the majority of sample loss in HF FIFFF separation of apoferritin and their aggregates may occur before the migration process. It is finally demonstrated that HF FIFFF can be applied for characterizing the reduction in Stokes' size of low density lipoproteins from blood plasma samples obtained from patients having coronary artery disease and from healthy donors.     

氢化物发生-原子吸收光谱法测定食品中的汞

研究了采用氢化物发生-原子吸收光谱法测定食品中痕量汞的方法,通过采用氢化物发生器,选择合适浓度的载流和硼氢化钾,获得了较为满意的分析结果。在测定汞含量5～20μg/kg的食品标准样品时,测试结果相对标准偏差RSD为2.2%～3.7%,回收率为84.9%～97.5%,检出限为0.2μg/L,完全满足食品行业汞元素痕量检测要求,操作简便、快速。