<Emphasis Type="Italic">In vitro</Emphasis> anti-inflammatory efficacies of liposomal suspensions of acetylsalicylic acid

Egg phosphatidylcholine (EPC) liposome containing acetylsalicylic acid (ASA) (EPC/ASA liposome) was prepared by a film hydration and sonication method, and the effect of liposome on the in vitro anti-inflammatory efficacy and the in vitro skin permeation of ASA was investigated. The mean diameter of EPC liposome and EPC/ASA liposome was about 271 and 175 nm, respectively. Both of liposomes were multi-lamellar vesicles on transmission electron microscopy photos. The in vitro viability of cell (Raw264.7) treated with EPC/ASA liposome suspension was significantly lower than the viability of cell treated with ASA solution. The amount of nitrite and prostaglandin E2 produced by cell treated with EPC/ASA liposome suspension was significantly lower than the amount produced by cells treated with ASA solution, indicating EPC liposome boosted the anti-inflammatory efficacy of ASA. The amount of ASA permeated through hairless mouse skin was inappreciable for 24 h when ASA solution was applied, whereas the permeation amount markedly increased to about 185 μg/cm² for 24 h when EPC/ASA liposome suspension was applied. EPC liposome also enhanced the permeation into stratum corneum and epidermis/dermis.     

Effects of frequency and power of ultrasound on the size reduction of liposome

The solutions of liposome made of l-α-dilauroyl phosphatidylcholine are sonicated at various powers and frequencies (43-480 kHz), and the resultant change in the size of liposome is measured by the dynamic light scattering method. The ultrasonic power dissipated into the solution is determined by the calorimetric method in order to compare the effects of ultrasound of different frequencies. The faster reduction of the mean size of liposome is achieved at the lower frequency. Comparing at the same frequency and total energy, short-time irradiation of strong ultrasound is more efficient than long-time irradiation of weak ultrasound. These results indicate that the small number of cavitation events with stronger physical disturbance on liposome can reduce the size of the liposome more efficiently than the large number of cavitation events with weaker disturbance.     

Fluorimetric study of phospholipase A-induced efflux of Ca2+ from liposomes]

Artificial vesicles, liposomes, were prepared from the total fraction of phospholipids of rat liver mitochondria. Electron microscopy showed that the structure of liposomes depended on cation composition of the medium in which they were formed. Fluorescence of chlorotetracycline increased in the suspension of liposomes loaded with Ca+2 due to the formation of Ca+2-chlorotetracycline-phospholipid membrane complex. Incubation of liposome suspension with phospholipase A in the presence of EDTA resulted in a decrease of chlorotetracycline fluorescence indicating a break of the integrity of liposome membranes and Ca+2 efflux.     

阿霉素脂质体的制备及其体外抗肿瘤活性的初步研究

目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。     

Effect of tryptophan oligopeptides on the size distribution of POPC liposomes: a dynamic light scattering and turbidimetric study

A chemical regulation of POPC liposome size distribution was investigated, based on the affinity of indole-containing compounds for phosphocholine membranes. In particular, tryptophan oligopeptides have shown interesting properties of size regulation, both when liposomes were formed in their presence and when the peptides were added to a preformed liposome suspension. Combining dynamic light scattering (DLS) and turbidimetric data, it was possible to show how such peptides had an influence on the size distribution of spontaneously formed liposomes prepared by the thin film hydration, reverse-phase evaporation and ethanol (or methanol) injection methods. In the presence of Trp-Trp or Trp-Trp-Trp, a disappearance of large vesicle aggregates was observed, as suggested also by light microscopy analysis. On the contrary, no effect was detected using extruded vesicles. Turbidimetric titration allowed the determination of the relative efficacy of the size regulators, Trp-Trp-Trp being about 20 times more powerful than the dimer, while the monomer had no effect. In addition, other indole-containing compounds and the antimicrobial peptide indolicidin were tested, showing similar behaviours. Discussing the results according to the current knowledge about the preference of Trp residues for interfacial regions in lecithin bilayers, this study confirms the relevant role of tryptophan in the biomembrane binding properties of many peptides and introduces a new behavior in the field of liposomes-peptides interactions.     

Liposome filtration. Dependence on transition temperature

Liposomes formed by vortexing and passed through polycarbonate surface retention membranes showed appreciable differences in filtration behavior depending on the temperature of filtration relative to the liposome gel-liquid crystal transition temperature. Below transition, liposomes were filterable and size distributions could be determined; the cumulative volume distributions were log-normal. Above transition, liposomes were not filterable: smaller liposomes were formed until a limiting size was reached. These results suggest that liquid crystal liposome size distributions cannot be determined by filtration. This filtration behavior is a physical property of liposomes, related to the gel-liquid crystal transition, not previously reported. This property could be exploited as a new method for controlling liposome size distributions, but the implications for lipid membranes, including biological membranes, are general.     

Photometric assays of cell shrinkage-the resolution of a conflict

Two groups have induced the shrinkage of yeast cells and measured its influence on visible light transmittance. Interestingly, they observed opposite effects; Bryant, Latimer and Seiber found shrinkage to increase suspension transmittance, Bussey found it to decrease transmittance. Each group corroborated its findings with light scattering theory. Bryant et al. used their own theoretical method while Bussey used an equation of Koch. We now find that the opposite effects of shrinkage on suspension transmittance were probably caused by differences in yeast cell size and in the designs of the photometer optical systems. All observed effects are found to be predicted by our method in terms of particle size and photometer geometry. The cited agreement of Bussey's findings with Koch's equation is fortuitous since the experiments were outside the proper domain of that approximate equation—the yeast cells were too large as was the acceptance angle of the photometer.     

Free will and evolution

The circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of a suspension of arbitrarily shaped optically active particles may be calculated by using the Rayleigh-Debye theory. Subject to restrictions on the size of the particles and the refractive index of the suspending medium the CD and ORD spectra of the suspension are the same as the intrinsic molecular spectra. The size of the particles can be comparable to or larger than the wavelength of the incident light provided that the optical properties of particles and medium are sufficiently similar. Therefore, it should be possible to experimentally reduce scattering artefacts in CD and ORD spectra of suspensions by suitably choosing a medium in which the particles are suspended.     

Electronic determination of size and number in isolated unfixed adipocyte populations

Adipose tissue cellularity and metabolism are traditionally expressed in terms of mean cell size and number. The need for a simple method allowing rapid determination of cell size and number of freshly isolated, unfixed adipocyte preparations led us to compare estimates of cell size determined by the established method of optical sizing to a proposed method of electronic cell sizing and counting. In collagenase-isolated, unfixed adipocytes whose mean diameters ranged from approximately 40 to 65 microns (obtained from healthy rats weighing 100-360 g) the electronic method provided estimates of the mean cell diameter and size distribution that did not differ from the optical sizing technique. Estimates of mean cell diameter and cell number by the electronic method were rapid and reproducible (coefficients of variation 0.5 and 3.8%, respectively) and a less than 20 sec delay until sample analysis, after mixing of the adipocyte suspension, did not alter these estimates. Electronic determination of cell size and number, using freshly isolated, unfixed rat adipocyte populations (mean cell diameter less than or equal to 60 microns), is rapid and reliable. It will be particularly useful for studies of hormone binding and transport processes where it may be necessary to tightly control cell density.     

Electric birefringence as a means of studying the effect of anaesthetics on liposomes

Birefringence can be induced in liposome suspensions using electric fields. The fields interact predominantly with anisotropic electrical polarisabilities which give rise to induced dipole moments. Using pulsed electric fields, the optical and electrical polarisabilities and the geometrical size of the liposomes can be measured simultaneously. These parameters have been found to be very sensitive to the presence of small amounts of fluidising additives of polar and ionic nature. Illustrative data are presented for the influence of the amines ammonium chloride, methyl ammonium chloride and lignocaine and of benzyl alcohol on phosphatidylcholine/serine liposomes. Structural changes in the vesicle membranes were detected, which appeared to correlate with the biological functions, thus indicating that electric birefringence is a rapid and useful method for studying interactive phenomena in lipid membrane systems.     

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.     

Preparation and characterization of galactose-modified liposomes by a nonaqueous enzymatic reaction

In this study, NOH (NOH?=?N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide) was enzymatically synthesized as a targeting molecule and incorporated into liposomes to prepare a liposome surface modified with galactose. Glycyrrhetinic-acid-loaded liposome (GA-LP) and glycyrrhetinic-acid-loaded liposome surface modified with galactose (NOH-GA-LP) were prepared by the ethanol-injection method. NOH-GA-LP was characterized by morphology, particle size, zeta potential, encapsulation efficiency, release in vitro, and stability. The size of spherical particles was in the range of 179-211?nm. Spherical particles exhibit a positive electrical charge (38.7 mV) and possess high encapsulation efficiency (91.3%) and show sustained release (72% over 48 hours) in vitro. This novel approach for the liposome surface modified with galactose by enzymatic synthesis is expected to provide potential application as a drug carrier for active targeted delivery to hepatocytes.     

A simple method for mass-production of liposomes, in particular large liposomes, suitable for delivery of free amino acids to filter feeding zooplankton

In marine fish larviculture the live feed organisms are often enriched in order to enhance their nutritional value. One of the challenges is to enhance the phospholipids (PL) content, and another is to enhance the content of specific water soluble nutrients, like free amino acids (FAA). There are a few studies where this has been achieved by the use of liposomes. The aim of this study was to develop a simple method for mass-production of liposomes within a size range of 1-5 mum a size range suitable to feed live food organisms. Furthermore, the liposomes should have a high FAA concentration and be stable under conditions typical for short-time enrichment of live feed organisms. The method used in the present study is based on a combination of a reverse-phase evaporation method for preparing liposomes and re-hydration of freeze-dried, empty liposomes. The liposomal membrane was made of soy phosphatydilcholine and was loaded with a highly concentrated free amino acids solution. Most of the liposomes produced were 2-8 mum in diameter and the FAA encapsulation efficiency was 42.6%. Two experiments simulating 2 hr of live food enrichment were used to evaluate the liposomes. The results showed the liposome did not disintegrate or aggregate when suspended in seawater and that only 9% of the FAA content of the liposomes was lost after 2 hr suspension. The developed method was easy and reliable, producing tens of grams of liposomes per batch.     

Spray-freezing of liposomes

In freeze-etch studies it was found that liposomes of some lecithins exhibited wrinkled structures on fracture faces, when quenched from above the transition temperature. The formation of this artifact can be prevented by spray-freezing the liposome suspension.     

Effect of diglucosamine on the entrapment of protein into liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Effect of Diglucosamine on the Entrapment of Protein into Liposomes

Liposomes, which had entrapped bovine serum albumin (BSA), were modified with diglucosamine by two methods. The liposome was prepared by a freeze-thawing method in the presence of the disaccharide, or the disaccharide was added to the liposome prepared in advance without it. To examine the effects of diglucosamine, the morphology, mean particle size, and zeta potential of both liposomes were compared with those of BSA-entrapping liposome prepared without the disaccharide. Diglucosamine caused no remarkable change in shape and no aggregation of the liposome. The presence of the disaccharide was confirmed on the surfaces of modified liposomes, and the entrapment of BSA into the liposomes was increased by the disaccharide. The entrapment behavior was affected by the way the disaccharide was added, and the difference in the way the BSA was entrapped was also indicated.     

Preparation of dry reconstituted liposomal powder by freeze-drying at room temperature

The aim of this study was to develop a novel, one-step method of liposome preparation by freeze-drying at room temperature as well as to investigate the physicochemical properties of dry reconstituted liposomal powder that was prepared. The method was based on utilizing sublimation of a volatile solid inert carrier, that is, chlorobutanol hemihydrate (CBN), instead of ice, which was less sophisticated and simpler than the conventional freeze-drying process. The optimum conditions used in the sublimation process of CBN were a temperature of 25–30°C and a pressure of 1.5–2.0 mBar for 8 hours. The influence of various parameters, such as type, particle size, and ratio of sugar lyoprotectant (i.e., mannitol or sucrose) and CBN to lipid on reconstitution time, liposome size, zeta potential, vesicle type, and lamella structure of reconstituted liposomes, were studied. The results revealed that the obtained liposomes were oligolamellar vesicles with particle sizes ranging from 400 to 1,000?nm. Type and ratio of sugar and CBN to lipid were found to significantly affect the reconstitution time. On the other hand, liposome size was independent of type of sugar and ratio of CBN to lipid, yet became smaller at higher sugar-to-lipid ratio and smaller sugar and CBN size. In all cases, traces of residual solvents were definitely below the acceptable limit.     

细胞表面电荷的光镊测量方法及应用研究

分析了细胞表面电荷测量的意义、方法和现状,提出采用光镊技术测量细胞表面电荷的方法,介绍了实现该方法的系统构成和检测原理。在电场力的作用下,处于悬浮液中的带电细胞产生电泳。在外加电场力为零时利用激光的陷阱力捕获该细胞,然后施加并逐渐加大外加电场力,直到细胞刚好逃脱光阱力的束缚时的瞬间光阱力,理论上就是细胞所带电荷在电场中产生的库仑力与粘滞力之和。     

Preparation and characterization of galactose-modified liposomes by a nonaqueous enzymatic reaction

In this study, NOH (NOH?=?N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide) was enzymatically synthesized as a targeting molecule and incorporated into liposomes to prepare a liposome surface modified with galactose. Glycyrrhetinic-acid–loaded liposome (GA-LP) and glycyrrhetinic-acid–loaded liposome surface modified with galactose (NOH-GA-LP) were prepared by the ethanol-injection method. NOH-GA-LP was characterized by morphology, particle size, zeta potential, encapsulation efficiency, release in vitro, and stability. The size of spherical particles was in the range of 179–211?nm. Spherical particles exhibit a positive electrical charge (38.7 mV) and possess high encapsulation efficiency (91.3%) and show sustained release (72% over 48 hours) in vitro. This novel approach for the liposome surface modified with galactose by enzymatic synthesis is expected to provide potential application as a drug carrier for active targeted delivery to hepatocytes.     

Characterization of plant suspension cultures using the focused beam reflectance technique

Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R ²>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.