Atomic force microscopy observation of highly arrayed phospholipid bilayer vesicle on a gold surface

Tapping mode atomic force microscopy (TM-AFM) imaging of a phospholipid bilayer vesicle (liposome) immobilized on a gold surface was performed to investigate morphologies of the electrode surfaces produced through application of three different sample preparation methods. We compared both methods from a morphological viewpoint using TM-AFM images. Liposomes, composed of zwitterionic and anionic phospholipids, were prepared by extrusion. Results indicate that the surface with immobilized L1-liposome, which was fabricated by the amino coupling method, seemed to form large amounts of aggregated or fused liposomes. In contrast, L2-liposome-containing 1-octadecanthiol that was directly attached on the gold surface using thiol-gold binding force was immobilized as a uniform surface topology without liposome aggregation. Finally, we attempted to arrange individual L3-liposome, prepared by mixing zwitterionic and anionic phospholipids, onto the gold layer by electron-beam (e-beam) lithography technique. A third method, L3-liposome formation on the sensor surface, is greatly anticipated for biosensor applications.     

脂质体水凝胶结构变化与形成机理

通过明胶和壳聚糖为原料制备互穿聚合物网络结构（interpenetrating polymer network,IPN）水凝胶,采用质构考察不同比例配方和交联剂转谷氨酰胺酶（microbial transglutaminase,mTG）质量浓度对IPN水凝胶的影响,同时研究将脂质体包埋进IPN水凝胶后形成的脂质体水凝胶结构及胶联机理。结果表明：明胶与壳聚糖按质量比4∶1混合、mTG质量浓度10 mg/mL条件下,制备的脂质体水凝胶可成胶且质构性质表现最优,硬度可达到18.68 g;通过膨胀系数、傅里叶变换红外光谱、差热分析及交联率分析等可知,脂质体与水凝胶通过化学方式进行联接,同时存在氢键的作用,且脂质体被包裹入凝胶内部网状结构。脂质体水凝胶的研究多以包埋缓释为主,较少有针对其结构和交联方式的报道。本研究结果对开发新型脂质体和脂质体水凝胶等运载体系并将其应用于食品营养物的包埋、缓释提供新思路。     

Detection of a heat stress-mediated interaction between protein and phospholipid membrane using dielectric measurement

The dielectric response of lipid bilayer membrane vesicles (liposomes) prepared using either phosphatidylcholine from egg (EPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was analyzed at a frequency range of 0.1 to 100 MHz. A marked dielectric dispersion for EPC and POPC liposome suspensions was observed above 1 MHz. An appropriate analysis of the dielectric dispersion curve was performed using the Cole-Cole equation and the Debye equation and was found to provide a method for the determination of dielectric parameters. Among the dielectric parameters, the characteristic frequency of a second dispersion around 50 MHz varied corresponding with changes in the test conditions. Of particular note is that an anomalous change in the characteristic frequency in the presence of protein corresponded to the degree of hydrophobic interaction between proteins and liposomes. The value of the frequency around 50 MHz, as well as the decrease in permittivity over the frequency range tested, are indicators of the interaction between proteins and liposomes.     

Evaluation of interaction between liposome membranes induced by stimuli responsive polymer and protein

Immobilized liposome chromatography was utilized as a novel method for the quantitative evaluation of the interaction between liposome membranes. The capacity factors evaluated from the elution profile showed that interaction between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposome membranes occurred in the presence of a stimuli responsive polymer and protein under specific stimulus conditions. The occurrence of such interaction was supported by experimental results for POPC liposome membrane fusion under corresponding stimuli conditions.     

Comparing the stability of retinol in liposomes with cholesterol, β-sitosterol,and stigmasterol

In this study, cholesterol (CH), β-sitosterol (SI), and stigmasterol (ST) were explored to improve the stability of retinol in the liposome bilayer. Retinol was incorporated into liposomes composed of soybean-derived L-α-phosphatidylcholine (PC) and 10% sterol (w/w), which were prepared as multilamellar vesicles. Under all conditions, the efficiency of retinol incorporation into liposomes was higher than 99%, and the average particle size of liposomes was similar to that of PC alone. Liposomes were stored at 4 and 25 °C, with and without light, respectively, for 10 days. It was found that during the storage, CH and SI were effective in stabilizing the retinol in liposomes. These results indicate that an appropriate phytosterol could improve the stability of retinol in liposomes.     

Antimicrobial peptide isolated from ovalbumin hydrolysate by immobilized liposome-binding extraction

Antimicrobial peptides are of greatest potential as a new group of antibiotics. Enzymatic hydrolysis was performed using flavourzyme, pepsin, trypsin, neutrase, papain and alcalase to obtain antimicrobial peptides from ovalbumin. Pepsin hydrolysate exhibited the highest antimicrobial activity compared with other fractions. To purify and characterize antimicrobial peptide, an immobilized liposome-binding extraction method was developed, in which the liposome was regarded as a mimic biomembrane system. The immobilized liposome stationary phase was prepared, and the maximum adsorption capacity and Langmuir constant were 81.30 μM/g and 8.19 mL/mg, respectively. Four fragments were simultaneously predicted by the comparison between reverse-phase high-performance liquid chromatography chromatograms of pepsin hydrolysate before and after interaction with immobilized liposome membrane. A novel cationic antimicrobial peptide named Opep2 was sequenced as RVASMASEKMKI. In addition, antimicrobial mode experiments showed that Opep2 interacted with cell wall membrane and caused potassium efflux, nucleotide leakage and ultimately cell death. Because of the high efficiency and procedural simplicity, the immobilized liposome-binding extraction method could be more efficient for the screening of antimicrobial peptides than conventional methods, which provides scientific references for antimicrobial peptide production.     

Formation of biopolymer-coated liposomes by electrostatic deposition of chitosan

ABSTRACT:  The purpose of this study was to prepare stable biopolymer-coated liposome suspensions using an electrostatic deposition method. Liposome suspensions were produced by homogenizing 1% soy lecithin in acetate buffer (0.1 M, pH 3). Cationic chitosan (Mw approximately 200 kDa) solutions were mixed with anionic liposome suspensions ( d approximately 100 and 200 nm), and the effect of phospholipid concentration, chitosan concentration, and liposome size on the properties of the particles formed was determined. The particle size and charge (ζ-potential) were measured using dynamic light scattering and particle electrophoresis. The particle charge changed from –38 mV in the absence of chitosan to +60 mV in the presence of chitosan, indicating complex formation between the anionic liposomes and cationic chitosan molecules. Below a minimum critical chitosan concentration ( c _min), large aggregates were formed that phase separated within minutes, whose origin was attributed to formation of coacervates. On the other hand, above a maximum critical chitosan concentration ( c _max), large flocs were formed that sedimented within hours, whose formation was attributed to depletion flocculation. Minimum and maximum critical chitosan concentrations depended on liposomal concentration and size. At c _min < c < c _max, chitosan-coated liposomes were formed that did not aggregate and were stable to sedimentation. Coated liposomes had better stability to aggregation than uncoated liposomes when stored at ambient temperatures for 45 d. This study indicates that chitosan can be used to form biopolymer-coated liposomes with enhanced stability over uncoated liposomes.     

Biophysical studies on collagen-lipid interaction

The potential use of liposomes as a delivery system is still limited by the poor understanding of the interaction mechanisms of liposomes underlying with biological media. Interaction between liposomes and protein is important for the structure and function of cells. In the present work, the interaction between collagen and dipalmitoyl phosphatidylcholine (DPPC) liposomes was studied by solubilization using a nonionic detergent, octylglucoside (OG), as well as a monolayer technique. The solubilization of the liposomal membrane was found to proceed in three stages of transition from the vesicular form to the mixed micellar form. Moreover, the amount of detergent needed to completely solubilize the liposomal membrane was increased after the incubation of liposomes with collagen, indicating an increased membrane resistance to the detergent and hence, a change in the natural membrane permeation properties. The addition of collagen in the subphase of different monolayer films induced a considerable shift towards a larger area/molecule in the compression-isotherm curves. This is either due to the insertion of collagen into the monolayer via its hydrophobic residues or to adsorption causing a protein layer to be located parallel to the lipid monolayer. It was concluded that collagen significantly altered the physical state of the liposome membrane, which may be attributed to collagen interaction with the liposomal surface and/or to its incorporation within the bilayer membrane.     

TG-DHA脂质体对高脂饮食小鼠食欲的调节作用

目的:制备TG-DHA脂质体并探究其对高脂饮食小鼠食欲调节因子表达的影响。方法:首先用薄膜分散法制备TG-DHA脂质体。然后将C57BL/6J小鼠随机分为对照组（C）、模型组（M）、TG-DHA脂质体组（L-DHA）。灌胃剂量1000 mg/kg.bw,灌胃8周后,用酶联免疫法检测血清瘦素、胰岛素、阿黑皮素原（Proopiomelanocortin,POMC）水平;用RT-PCR法检测下丘脑神经肽Y（Neuropeptide Y, NPY）、α-黑色素细胞刺激激素（α-melanocyte stimulating hormone,α-MSH）,胆囊收缩素（Cholecystokinin,CCK）、肽YY（Peptide YY,PYY）、胰高血糖素样肽-1（Glucagon like peptide-1, GLP-1）的mRNA表达。结果:TG-DHA脂质体的包封率高达85.83%。与M组相比,L-DHA组显著上调了血清瘦素（23.03%）、POMC（1.30倍）含量（P<0.01）以及α-MSH（1.60倍）、PYY（1.32倍）、GLP-1（78.10%）mRNA的表达量（P<0.05）。结论:TG-DHA脂质体可通过改善实验小鼠血清食欲激素,下丘脑神经肽及脑-肠肽等食欲调节因子表达异常,抑制高脂饮食小鼠的食欲,从而改善高脂饮食诱导的肥胖。     

Oxidative refolding of lysozyme assisted by negatively charged liposomes: relationship with lysozyme-mediated fusion of liposomes

Oxidative refolding of denatured/reduced lysozyme was examined in the presence of charged liposomes composed of neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Surface charge density of liposomes had a marked effect on the interaction between liposomes and reduced lysozyme which is observed in the early stage of the refolding. It was found that there was a critical level of surface charge density of liposomes (-0.15 C/nm2) at which the interaction between liposomes and lysozyme drastically changed. At the surface charge density of liposomes ranging from -0.15 to -1.4 C/nm2, the interaction between liposomes and lysozyme resulted in aggregate formation. In contrast, at the surface charge density ranging from 0 to -0.15 C/nm2, no aggregate formation was observed if the lysozyme/liposome molar ratio was less than 600. On the basis of the experimental results, a model for the interaction between charged liposomes and lysozyme was proposed, focusing on the mechanism of protein-induced fusion of charged liposomes as well as protein refolding on liposomes. Then, the optimal condition for oxidative refolding of lysozyme was examined in the presence of charged liposomes by controlling the lysozyme-liposome interaction. The reactivation yield of lysozyme was improved up to 85% in the presence of liposomes with a surface charge density of -0.14 C/nm2.     

Electrochemical glucose biosensing by pyranose oxidase immobilized in gold nanoparticle-polyaniline/AgCl/gelatin nanocomposite matrix

A novel pyranose oxidase (PyOx) biosensor based on gold nanoparticles (AuNPs)–polyaniline(PANI)/AgCl/gelatin nanocomposite has been developed for the glucose detection. PyOx was immobilized on the surface of glassy carbon electrode (GCE) via the nanocomposite matrix. The electrode surface was imaged by scanning electron microscopy (SEM). Amperometric detection of the consumed oxygen during the enzymatic reaction was monitored at −0.7 V. After optimization studies, analytical characterization of the biosensor was carried out. The linear response of the AuNPs–AgCl/PANI/gelatin modified PyOx biosensor is found to be from 0.05 to 0.75 mM glucose with the equation of y = 2.043x + 0.253; R² = 0.993. Finally, proposed biosensor was used to analyze glucose content in real samples. Obtained data from the biosensing system was compared with a commercial enzyme assay kit based on spectrophotometric Trinder reaction as a reference method.     

Aβ/Cu-catalyzed oxidation of cholesterol in 1,2-dipalmitoyl phosphatidylcholine liposome membrane

The amyloid β protein with 42 amino acid residues (Aβ), which is a causative protein of Alzheimer's disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by Aβ/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water.     

Functionality of porcine skin hydrolysates produced by hydrothermal processing for liposomal delivery system

Porcine skin proteins were hydrolyzed using hydrothermal processing (HTP) and fractionated by membrane ultrafiltration (UF) into peptides with different molecular weights (1–10 kDa). The porcine skin hydrolysates (PSHs) were analyzed for their antioxidant, antiaging, and skin permeation properties. Additionally, the PSHs were incorporated into liposomes with different charges to maintain and/or enhance their beneficial effect. The results showed that both HTP and UF‐II (1–3 kDa) had significantly high antioxidant and antiaging effects and good skin permeability. Based on the results for PSH‐loaded liposomes, the best carrier for incorporating PSHs was a positively charged liposome that had been prepared using positively charged lipid. PSH loaded with positively charged liposomes (PSH/LIP‐P) had the following droplet properties: 71.11 nm, 0.208 PDI (polydispersity index), and 38.37 mV. PSH/LIP‐P also had an incorporation efficiency of 70.72%, and the liposomes maintained their physical droplet stability during storage.

Practical applications

The findings of this study highlighted porcine skin hydrolysates (PSH) produced by hydrothermal processing and membrane ultrafiltration with high antioxidant and antiaging effects and good skin permeability. PSH‐loaded liposome capsules maintained a small size and high incorporation efficiency for PSH. The PSHs and PSH‐loaded liposomes could be tested further for their in vivo functionality as nutraceutical or cosmeceutical ingredients. Hence, the finding could be useful information for developing potential commercial products in the food, cosmetics, and related industries.     

Catalase-based thin-layer enzyme cell used in organic-phase FIA system for determination of moisture in oily foods

The aim of our present work was to study the possibility of constructing a biosensor based on immobilized catalase enzyme (EC 1.11.1.6.) in organic-phase solutions. The catalase enzyme was immobilized by glutaraldehyde on a natural protein membrane in a thin-layer enzyme cell, connected to a stopped-flow injection analyser (SFIA) system with an amperometric detector. Adding FMCA to acetonitrile, the optimal concentration was 7.5 mg l^–1, while with TBATS it was 2.7 mg l^–1. The optimal pH value of the immobilized enzyme in buffer was about 6.0. On studying the role of the buffer solution used in the carrier solvent, the activity of the enzyme changed dramatically. The signal was highest when there was no buffer added to the carrier solution and decreased rapidly when the content increased (0–1.5%). Utilizing these results, a quick analytical method was developed to monitor indirectly the water content (activator) in various butter and margarine samples by maintaining a fixed substrate concentration. The water content of samples was compared with the results obtained by the gravimetric reference method (AOAC Method 920.116); the correlation coefficient (r) was 0.993.     

Heat-enhanced production of chitosanase from Streptomyces griseus in the presence of liposome

The effects of heat stress and liposome treatment on the growth of Streptomyces griseus cells and chitosanase production were investigated on the basis of using the designed strategy of a stress-mediated bioprocess. The effective conditions for increasing the interaction between chitosanase and the 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) liposome under heat stress condition were determined on the basis of the results of circular dichroism (CD) and dielectric dispersion analysis (DDA). Under these effective conditions, S. griseus cells were cultivated for the effective production of chitosanase. The results obtained from both CD spectra and DDA showed that heat stress enhances the interaction of the POPC liposomes and chitosanase. The strongest interaction between them could be obtained in the specific temperature range of 40-45 degrees C. The enhancement of the target chitosanase production was conducted under heat stress at 41 degrees C in the presence and absence of the POPC liposomes. The growth rates of S. griseus cells in the cases of heat (41 degrees C) and heat (41 degrees C)/POPC treatments were respectively 1.2 and 1.4 times higher than that obtained under the control condition. In the heat (41 degrees C) and heat (41 degrees C)/POPC treatments, chitosanase activity increased to 1.8 and 2 times, respectively, higher than that obtained under the control condition. Heat stress and the addition of the POPC liposomes could therefore be utilized to induce the potential functions of bacterial cells for the enhancement of the final target production.     

Determination of isoflavones in commercial soy products by HPLC and coulometric electrode array detection

The concentrations of the isoflavones daidzein, genistein and glycitein were determined in a large number of commercially-available soy products by high-performance liquid chromatography with coulometric electrode array detection using estriol as internal standard.During extraction, the naturally occurring glycosides were converted into their respective aglycones by acid hydrolysis. The analytes were separated on a C-18 reversed phase column, eluted with methanol/acetonitrile/50 mM sodium acetate pH 4.8 (40/5/55, v/v/v), and detected by a coulometric electrode array detector using twelve electrodes set to potentials between +250 and +800 mV (in increments of 50 mV) against palladium reference electrodes.The isoflavones could be determined over a wide concentration range (0.8–1135.0 mg/kg for daidzein, 1.9–1442.5 mg/kg for genistein and 0.5–154.6 mg/kg for glycitein). The recovery of the isoflavones in the different matrices was determined by the standard addition method and varied between 40.9–94.4%. The detection limits (S/N=3) depend on the matrix of the soy product and were found for daidzein between 0.3–1.6 mg/kg, for genistein between 0.3–1.7 mg/kg and for glycitein between 0.5–2.3 mg/kg.     

Dielectric response of cells and liposomes and its utilization for evaluation of cell membrane-protein interaction

The dielectric measurements of an Escherichia coli (E. coli) cell suspension and liposome suspensions were carried out in the frequency range between 0.1 and 100 MHz to detect the heat stress-mediated interaction between proteins and cell membranes. The dielectric relaxation dispersion was observed to be above 1 MHz. The dielectric parameter (amplitude of dispersion, deltaepsilon) based on the Cole-Cole equation was anomalously changed with increasing temperature. The value of deltaepsilon of liposomes was varied at various temperatures depending on the type of protein present. The change in deltaepsilon of liposomes correlated with the amount of protein translocated across the phospholipid membrane. The changes in the value of deltaepsilon of E. coli cells with temperature variation was similar to those of liposomes in the presence of proteins, suggesting that the variation in dielectric parameters reflected the interaction between the phospholipid membrane and proteins. It was found that the dielectric measurement could be utilized for the detection of the interaction between proteins and liposomes.     

Quantification of structural properties of cell-sized individual liposomes by flow cytometry

We describe a new high-throughput method of quantifying the structural properties of individual cell-sized liposomes. An internal aqueous solution of liposomes was labeled with a green fluorescent marker and the membrane with a red marker. The double-labeled liposomes were analyzed using flow cytometry, and the internal aqueous volume and lipid membrane volume of each liposome were measured. The experimental results indicate that both the internal aqueous and lipid membrane volumes positively correlate with the intensity of forward-scatter (FS) and side-scatter (SS) signals in a logarithmic scale. In addition, liposomes in 18 small areas gated by log(FS) and log(SS) were sorted by fluorescence-activated cell sorting (FACS), and observed by optical microscopy. Structural characteristics observed in the microscopy images of heterogeneous liposomes correlated with FACS data. Because this method does not employ any particular assumption about the shape and structure of liposomes, flow cytometry is a powerful tool for estimating the internal and membrane volumes of individual cell-sized liposomes with heterogeneous shapes and structures.     

维生素E脂质体的制备工艺筛选、优化及其性质研究

目的优化维生素E（V_E）脂质体的制备工艺并考察其性质。方法采用乙醇注入法、乙醚注入法、逆相蒸发法、薄膜水化法和复乳法分别制备V_E脂质体,以包封率和保留率为考察指标,选择最优制备方法 :经L₉（3⁴）正交试验设计优化选择,确定脂质体的最佳配方。结果薄膜水化法制备所得的V_E脂质体包封率最高,V_E保留率较高;用薄膜水化法制备脂质体的最佳配方为磷脂:V_E:胆固醇=20:0.8:1.5;用透射电镜观察最佳实验组V_E脂质体发现其具有指纹状结构,Zeta电位为-30.9±0.9 mV,平均粒径为33.7 nm。结论薄膜水化法制得的V_E脂质体具有包封率高,V_E保留率高,粒径均匀等特点。     

Comparative study of the oxidative and physical stability of liposomal and nanoliposomal polyunsaturated fatty acids prepared with conventional and Mozafari methods

The relative oxidative stability of freshly prepared and stored liposomal and nanoliposomal systems of docosahexaenoic acid (DHA, 22:6 n−3) and eicosapentaenoic acid (EPA, 20:5 n−3) were investigated. The effects of organic solvents on the oxidative stability of liposomal polyunsaturated fatty acids (PUFAs) produced by two methods, the Bangham thin-film hydration (conventional rotary evaporation method and using organic solvents) and Mozafari (direct hydration and without using organic solvents) methods, were compared. The highest physicochemical stability was observed in PUFA liposomes prepared by the Mozafari method, followed by conventional liposomes and bulk PUFAs. There was no significant change in physicochemical stability during 10 months of cold storage (4 °C) in the dark. Moreover, the comparison between liposomes (>200 nm) and nanoliposomes (50–200 nm) revealed that the surface charge, physical stability and oxidative stability of liposomal PUFAs increased as the size of the liposomes decreased. The differences in the oxidative stability of PUFAs may be due to the protective effects of aqueous systems, which indicate the advantage of using non-organic solvent (water and CO₂) techniques in liposome manufacturing.