The use of liposomes for the preparation of protein-free lipid emulsions models of chylomicron remnants.

Artificial chylomicron remnants were investigated as a new drug carrier system for the targeting of hepatic parenchymal cells. The emulsions presented here are similar in particle size and composition to natural lipoproteins. The preparations contained triolein, phospholipid, cholesterol and cholesteryl oleate. Egg yolk lecithin was either used to form multilamellar or unilamellar liposomes or it was incorporated into a lipid film prior to emulsification. Typically the lipid film contained triolein, cholesterol and cholesteryl oleate. When multilamellar liposomes were used however, cholesterol and cholesteryl oleate were incorporated into the vesicles. The emulsions were prepared by ultrasonication or by means of a microemulsifier. The unilamellar liposomes used with the microemulsifier yielded the best particle distribution, i.e. in the range of 40-60 as determined by quasi-elastic light scattering. The advantage of the method results from the complete emulsification of the components. The particle size remained unchanged during storage, although flocculation was observed. The results show that the synthesis of artificial chylomicron remnants in a microemulsifier is possible and reproducible.     

Liposomes as carriers of macrolides: preferential association of erythromycin A and azithromycin with liposomes of phosphatidylglycerol containing unsaturated fatty acid(s).

To assess the most favourable phospholipid composition of a liposomal carrier for antibiotics, small multilamellar liposomes were prepared from phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol of varying fatty acid composition in the presence of erythromycin A and azithromycin. Crude liposomes were subjected to Sepharose CL-4B column chromatography, and liposomes containing antibiotics were well separated from free antibiotics. These experiments established that the greatest association of antibiotics was achieved with liposomes prepared from phosphatidylglycerol rather than phosphatidylcholine or phosphatidylethanolamine. Furthermore, the composition of fatty acids in phosphatidylglycerol liposomes influenced the amount of antibiotics associated with liposomes; the highest amount was obtained with dioleoylphosphatidylglycerol followed by phosphatidylglycerol of fatty acid composition similar to that of egg yolk lecithin. It was established that purified liposomes, prepared from [3H]phosphatidylglycerol containing unsaturated fatty acid(s) bind about 25 per cent of originally present antibiotic. Both antibiotics, erythromycin A and azithromycin, were similar in respect to the amount of their association with liposomes. Determination of the size of phosphatidylglycerol/antibiotic liposomes established that the mean diameter of liposomes containing antibiotics was 200-350 nm, very close to that of liposomes without them.     

Effect of lecithins on BCG-alginate-PLL microcapsule particle size and stability upon storage

Alginate-PLL microcapsules containing BCG were prepared by emulsification/internal gelation of an alginate solution dispersed within a vegetable oil containing lecithins as emulsifiers. The lecithins studied were soy bean lecithin at 0.1, 0.5, 1 and 2% concentration; and dried egg yolk lecithin at 0.1, 0.25, 0.5 and 1%. The microcapsule particle size showed a dependence upon the amount and type of lecithin added to the oil. Increasing the emulsifying agent concentration was found to reduce particle size, from 50.9 microm obtained when lecithins were not used in the emulsification step, to 13.9 microm obtained when 1% dried egg yolk lecithin was employed. The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Once prepared, microcapsules were freeze-dried using 5% trehalose as cryoprotectant in order to preserve their stability upon storage. The stability of the microcapsules was assayed over 12 months at room temperature, finding that alginate-PLL microcapsules were stable up to 6 months. Moreover, in the case of microcapsules prepared with lecithins, a significant increase in particle size was observed, from 16.9 microm at the beginning of the study to 25.2 microm at 12 months storage.     

Effect of lecithins on BCG-alginate-PLL microcapsule particle size and stability upon storage

Alginate-PLL microcapsules containing BCG were prepared by emulsification/ internal gelation of an alginate solution dispersed within a vegetable oil containing lecithins as emulsifiers. The lecithins studied were soy bean lecithin at 0.1, 0.5, 1 and 2%concentration; and dried egg yolk lecithin at 0.1, 0.25, 0.5 and 1%. The microcapsule particle size showed a dependence upon the amount and type of lecithin added to the oil. Increasing the emulsifying agent concentration was found to reduce particle size, from 50.9mum obtained when lecithins were not used in the emulsification step, to 13.9mum obtained when 1% dried egg yolk lecithin was employed. The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Once prepared, microcapsules were freeze-fried using 5%trehalose as cryoprotectant in order to preserve their stability upon storage. The stability of the microcapsules was assayed over 12 months at room temperature, finding that alginate-PLL microcapsules were stable up to 6 months. Moreover, in the case of microcapsules prepared with lecithins, a significant increase in particle size was observed, from 16.9mum at the beginning of the study to 25.2mum at 12 months storage.     

Liposomes for Topical Use: A Physico-Chemical Comparison of Vesicles Prepared from Egg or Soy Lecithin

Developments in nanotechnology and in the formulation of liposomal systems provide the opportunity for cosmetic dermatology to design novel delivery systems. Determination of their physico-chemical parameters has importance when developing a nano-delivery system. The present study highlights some technological aspects/characteristics of liposomes formulated from egg or soy lecithins for topical use. Alterations in the pH, viscosity, surface tension, and microscopic/macroscopic appearance of these vesicular systems were investigated. The chemical composition of the two types of lecithin was checked by mass spectrometry. Caffeine, as a model molecule, was encapsulated into multilamellar vesicles prepared from the two types of lecithin: then zeta potential, membrane fluidity, and encapsulation efficiency were compared. According to our observations, samples prepared from the two lecithins altered the pH in opposite directions: egg lecithin increased it while soy lecithin decreased it with increased lipid concentration. Our EPR spectroscopic results showed that the binding of caffeine did not change the membrane fluidity in the temperature range of possible topical use (measured between 2 and 50 °C). Combining our results on encapsulation efficiency for caffeine (about 30% for both lecithins) with those on membrane fluidity data, we concluded that the interaction of caffeine with the liposomal membrane does not change the rotational motion of the lipid molecules close to the head group region. In conclusion, topical use of egg lecithin for liposomal formulations can be preferred if there are no differences in the physico-chemical properties due to the encapsulated drugs, because the physiological effects of egg lecithin vesicles on skin are significantly better than that of soy lecithin liposomes.     

The use of liposomes as a model for drug absorption: β-lactam antibiotics

Liposomes were prepared from egg phosphatidylcholine-cholesterol-diacetylphosphate (80: 20: 5) and total lipid extracts of rat intestinal mucosa, and the permeability of the liposomal membrane to eight β-lactam antibiotics was studied by using a dynamic dialysis method. Although all the antibiotics used here are ionized and poorly lipid-soluble at pH 6·5, some of them are orally active and efficiently absorbed from the small intestine. The release rate constants from the aqueous dispersion of drug-entrapped liposomes were approximately in the order of their absorbability. Intestinal lipid liposomes were more permeable to the antibiotics than egg lecithin liposomes and the release rate constants for the drugs from intestinal lipid liposomes were strongly correlative with their absorption rate constants, except for cephalothin and ampicillin, the deviations of which could be explained by their surface activity. It is suggested that lipid components of the intestinal mucosa and the bilayer structure may play an important role in the absorption process of the antibiotics. The validity of liposomes as a model for the intestinal absorption of drugs is also discussed.     

Liposomes from hydrogenated egg yolk lecithin

Egg yolk lecithin is a lipid, frequently been used for the liposome preparation. Such liposomes, however, are sensitive to oxidation and relatively permeable to encapsulated substances. The catalytic hydrogenation of egg yolk lecithin is one possibility to modify the properties mentioned. The authors deal with preparation and characterization of hydrogenated egg yolk lecithin. Liposomes from native and hydrogenated egg yolk lecithin--also in combination with cholesterol--are compared. Liposomes with hydrogenated egg yolk lecithin as phospholipid component exhibit a significantly increased encapsulation capacity and an essentially improved stability. The permeation of electrolytes, carboxyfluorescein and of the cytostatic drug daunorubicin is studied.     

Encapsulation efficiency of water-soluble and insoluble drugs in liposomes prepared by the microencapsulation vesicle method

The microencapsulation vesicle (MCV) method is a liposome preparation technique that reproducibly produces liposomes with homogeneous particle sizes with a high encapsulation efficiency. Liposomes encapsulating water-soluble drugs, lipophilic drugs and an amphiphilic drug were prepared by the MCV method and the encapsulation efficiency of the drugs was examined. Three kinds of egg yolk lecithin with different iodine values, i.e., purified egg yolk lecithin (PEL), partially hydrogenated purified egg yolk lecithin (R-20) and completely hydrogenated purified egg yolk lecithin (R-5), were used for membrane materials in order to explore the possible effects of membrane rigidity or surface area on the encapsulation efficiency of the drug. Water-soluble 5-fluorouracil showed 12-15% encapsulation efficiency, which was higher than those reported in the literature (less than 10%). With the MCV method, theoretically the initial drug-containing water phase was always separated from the dispersion medium by the lecithin-containing oil phase, which was advantageous to maintaining a higher encapsulation efficiency of the water-soluble drug. The encapsulation efficiency of lipophilic ibuprofen and flurbiprofen was around 90%. As for ketoprofen and liposomes were not formed when using hydrogenated egg yolk lecithin R-5, while the encapsulation efficiency using PEL or R-20 was around 80%. Amphiphilic amitriptyline hydrochloride resulted in a slightly higher encapsulation efficiency when dissolved in the water than the chloroform. Among the three kinds of lecithin, the most unsaturated PEL tended to show a higher encapsulation efficiency, probably due to differences in the packing geometry of the hydrophobic carbon chains in the membrane bilayer. The encapsulation efficiency of these drugs strongly correlated to the logP(octanol/water) and also tended to correlate to the logP(chloroform/water) for the order of the logP(chloroform/water) was almost the same as the order of the logP(octanol/water) in the drugs examined. As far as the results of this study, the logP(octanol/water) was considered to be a better indicator of the encapsulation efficiency of a drug in the MCV method.     

Antigen in chitosan coated liposomes enhances immune responses through parenteral immunization

To overcome the limitations of the use of conventional liposomes chitosan coated egg yolk lecithin based liposomes were used as antigen carrier through parenteral administration in two animal models such as fish and rabbit. Extracellular proteins (ECPs) antigen of Aeromonas hydrophila encapsulated in liposomes became more stable and induced better immune response. It enhanced both adaptive and innate immune responses than other preparations both at 21, 42 and 63days post-immunization and suggested to be a better antigen delivery system.     

Stability and drug release properties of liposomes containing cytarabine as a drug carrier

Liposomes were studied as a drug delivery system. Multilamellar vesicles, small unilamellar vesicles and large unilamellar vesicles containing cytarabine were prepared using egg yolk lecithin and cholesterol. Large unilamellar vesicles showed the highest encapsulation efficiency of all and their encapsulation efficiency increased as the buffer volume decreased. Cholesterol increased the stability of liposomal drug products as drug carriers and reduced the permeability of drug across the liposomal membrane. The release rate of cytarabine increased with incubation temperature and decreased with cholesterol incorporation in liposomal membrane. The release mechanism of cytarabine from large unilamellar vesicles in vitro was chiefly due to simple diffusion across the liposomal membrane rather than liposomal rupture.     

Liposomal gel with chloramphenicol: characterisation and in vitro release

The aim of our study was to develop a liposomal carrier system for the local treatment of bacterial vaginosis, capable to efficiently deliver entrapped drug during an extended period of time. Chloramphenicol was entrapped in liposomes composed of egg phosphatidylcholine/egg phosphatidylgycerol-sodium (9:1, molar ratio) and prepared by two different methods, the proliposome method and the polyol dilution method. Both liposome preparations were characterised and compared for particle size, polydispersity, entrapment efficiency and tested for in vitro stability in media that simulate human vaginal conditions (buffer pH 4.5 and vaginal fluid simulant). To achieve application viscosity of liposomes and to further improve their stability, liposomes prepared by the proliposome method were incorporated in the bioadhesive gel made of Carbopol 974P NF resin. In vitro release studies of liposomes incorporated in the gel have shown a prolonged release of entrapped chloramphenicol compared to control gel. Even after 24 hours of incubation in the vaginal fluid simulant, more than 40% of the originally entrapped drug was still retained in the gel. Storage stability studies have proven the ability of the Carbopol 974P NF gel to preserve the original size distribution of incorporated liposomes. All the performed experiments confirm the applicability of liposomes as a novel drug carrier system for the local treatment of bacterial vaginosis.     

Pharmacokinetics of small unilamellar liposomes and incorporated lonazolac after i.m. administration

The nonsteroidal anti-inflammatory drug lonazolac [3-(p-chlorophenyl)-1-phenylpyrazole-4]-acetic acid was incorporated into the bilayer of liposomes. The unilamellar and homogeneously sized liposomes with a diameter of 45 +/- 7 nm were prepared by controlled detergent dialysis. The maximal amount of lonazolac incorporation is 0.140 mg per mg egg phosphatidylcholine (egg PC). The liposomes were stable over a year. Pharmacokinetics of the 3H- and 14C-labelled liposomes and of the 14C-labelled lonazolac was investigated after i.v. and i.m. administration to rabbits. The i.m. injected liposomes showed a terminal half-life of 74 h and after 24 h 7 per cent of the administered lecithin was still in the plasma. Eight hours following the i.m. injection of the liposomes, the plasma levels of lonazolac were significantly higher compared to the reference. A portion of the liposomes was released from the muscle into the circulation. A great share of lonazolac was quickly interchanged between the liposomes and the surrounding tissue. In additional experiments small oligolamellar liposomes should be used to decrease the interchange of lonazolac and to enhance the plasma levels of liposomal incorporated lonazolac.     

Design of a polyvinyl alcohol hydrogen containing phospholipid as controlled-release vehicle for rectal administration of (+/-)-propranolol HCl

Polyvinyl alcohol hydrogels which contained phospholipid, egg yolk lecithin or hydrogenated soya lecithin were designed as a transrectal delivery system for propranolol hydrochloride. The hydrogel preparations containing phospholipid were prepared by a low-temperature crystallization method. The release profile of propranolol from hydrogel preparations containing phospholipid complied with Fickian diffusion (Higuchi model). The release of propranolol from the hydrogel preparation decreased with higher contents of phospholipid (approximately 2% w/w). In rats plasma concentrations of propranolol after rectal administration of hydrogel preparations containing phospholipid (1 and 2% w/w) were prolonged compared with those of rats receiving preparations without phospholipid.     

Development and evaluation of inhalational liposomal system of budesonide for better management of asthma

Budesonide is a corticosteroid used by inhalation in the prophylactic management of asthma. However, frequent dosing and adverse effects (local and systemic) remain a major concern in the use of budesonide. Liposomal systems for sustained pulmonary drug delivery have been particularly attractive because of their compatibility with lung surfactant components. In the present investigation, pulmonary liposomal delivery system of budesonide was prepared by film hydration method and evaluated for sustained release. Various parameters were optimized with respect to entrapment efficiency as well as particle size of budesonide liposomes. For better shelf life of budesonide liposomes, they were freeze dried using trehalose as cryoprotectant. The liposomes were characterized for entrapment efficiency, particle size, and surface topography; in vitro drug release was evaluated out in simulated lung fluid at 37° at pH 7.4. The respirable or fine particle fraction was determined by using twin stage impinger. The stability study of freeze dried as well as aqueous liposomal systems was carried out at 2-8° and at ambient temperature (28±40). The freeze dried liposomes showed better fine particle fraction and drug content over the period of six months at ambient as well as at 2-8° storage condition compared to aqueous dispersion of liposomes.     

A gamma scintigraphic evaluation of the precorneal residence of liposomal formulations in the rabbit

Multilamellar liposomes were prepared from dipalmitoyl phosphatidylcholine or egg lecithin in combination with cholesterol and either dicetyl phosphate or stearylamine. The size and charge of the colloidal preparations were characterized before labelling with [111In]8-hydroxyquinoline. Freshly labelled liposomes were instilled into the eyes of unanaesthetized NZW rabbits and their disposition and drainage followed using gamma scintigraphy. A positive surface charge was found to affect significantly liposomal drainage rate, whereas an increase in size restricted drainage from the inner canthal region. Drainage of the suspending medium was directly compared with liposomes by labelling the medium with [99mTc] sodium pertechnetate and following the simultaneous change in removal of 99mTc and 111In from the precorneal area. Slower drainage rates were obtained for the suspending medium compared with solutions of the isotopes suggesting that the liposomes restricted solution drainage.     

Stability of liposomes on long term storage

The effect of the lipid composition of liposomes on their storage for up to one year under different environmental conditions has been examined using 5,6-carboxyfluorescein as a model drug. When cholesterol and/or alpha-tocopherol were included in the liposomes, a significantly greater amount of dye was retained. The presence of alpha-tocopherol decreased the breakdown of phosphatidylcholine to lysophosphatidylcholine and also reduced the level of peroxidation. Carboxyfluorescein retention was further enhanced when liposomes were stored at 4 degrees C or at room temperature (20 degrees C) in an O2-free atmosphere. Lysophosphatidylcholine formation also slowed when the liposomes were kept at 4 degrees C, or in an O2-free atmosphere. It is concluded that egg yolk lecithin liposomes may be stored for long periods at low temperature in an O2-free atmosphere or with added stabilizers such as cholesterol and alpha-tocopherol.     

胸腺五肽脂质体的研制

目的:制备胸腺五肽脂质体并进行质量评价。方法:采用复乳法制备胸腺五肽脂质体,以聚乳酸-羟基乙酸共聚物(PLGA)及卵磷脂为成球材料、以胸腺五肽为主药制备脂质体。以明胶浓度、PLGA浓度和卵磷脂浓度为考察因素,以包封率和载药量为考察指标设计L(934)正交试验优化基质处方并进行验证试验。通过测定优化处方所制脂质体粒径、包封率、体外累积释放百分率等评价脂质体质量。结果:优化基质处方为明胶、PLGA和卵磷脂浓度分别为100、200、100mg.mL-1。所制脂质体形态完整,平均粒径为(9.03±0.83)μm,载药量与包封率分别为(1.81±0.03)与(74.4±1.4),20d的累积释药百分率达90以上。结论:所制胸腺五肽脂质体工艺简单、重现性好,包封率和载药量高,具有显著的缓释作用。     

Hydrophilic gels containing chlorophyllin-loaded liposomes: development and stability evaluation

The aim of this study was to develop and characterize hydrophilic gels containing chlorophyllin(CHL)-loaded liposomes as well as to evaluate their stability. Two different CHL-loaded liposome dispersions using non-hydrogenated and hydrogenated soybean lecithin were prepared, characterized for their particle size, polydispersity index and trapping efficiency and incorporated in Carbopol 940 NF hydrogel. The gels obtained were analyzed for flow properties, pH values and CHL content. The control liposome-free gel was obtained by incorporating the CHL solution in the hydrogel. The stability of the gels was evaluated in terms of rheological properties, pH values and CHL content during 6 months' storage at 20 +/- 2 degrees C. Suitable gel formulations for topical use were obtained revealing shear-thinning plastic flow behaviour without significant thixotropy during the whole period of examination. High yield values of the samples during the whole period indicated a long-term stability of the gel formulations. The gel formulations expressed a mild acid value acceptable for topical preparations. After 6 months' storage the CHL content was highest in the gel containing non-hydrogenated lecithin liposomes, followed by the gel containing hydrogenated lecithin liposomes and liposome-free gel, indicating that the encapsulation of CHL in liposomes led to a greater stability of CHL.     

Effect of positively and negatively charged liposomes on skin permeation of drugs

To clarify the effect of the surface charge of liposomes on percutaneous absorption, the permeation of liposomal drugs through rat skin was investigated in vitro and in vivo. Liposomes were prepared using egg yolk lecithin (EPC, phase transition temperature, -15 to -17 degrees C), cholesterol and dicetylphosphate (DP) or stearylamine (SA) (10:1:1, mol/mol). Also examined was the penetration behavior of positively and negatively charged liposomes, using a fluorescent probe (Nile Red). The in vitro penetration rate of melatonin (MT) entrapped in negatively charged liposomes was higher than that of positively charged ones (p<0.05). When the percutaneous absorption of ethosuximide (ES) encapsulated was estimated in vivo, the absorption of ES from negatively charged liposomes was slightly higher than that from positively charged liposomes. Additionally, the absorption of ES from both types of liposomes was superior to that from the lipid mixtures consisting of the same composition as the vesicles. The percutaneous absorption of betahistine (BH) from a gel formulation containing negatively charged liposomes of BH was much more than that from the formulation with positively charged ones, with 2-fold higher AUC (p<0.05). Histological studies revealed that the negatively charged liposomes diffused to the dermis and the lower portion of hair follicles through the stratum corneum and the follicles much faster than the positive vesicles at the initial time stage after application. Thus, the rapid penetration of negatively charged liposomes would contribute to the increased permeation of drugs through the skin.     

普罗布考亚微乳剂的制备

以中链甘油三酯为油相,蛋黄卵磷脂和泊洛沙姆188为乳化剂,采用高压乳匀法制备普罗布考亚微乳剂.以平均粒径、多分散系数(PDI)、ζ电位和稳定性等评价指标优化了处方.按优化条件制得的普罗布考亚微乳稳定性良好,平均粒径(168.8±1.5) nm,ζ电位(50.1±1.7) mV,载药量为(4.2±1.6)mg/ml.