Liposome State of Art-France - 2012 PDF

Copyright 2012 American Scientic Publishers All rights reserved Printed in the United States of America
Journal of Colloid Science and Biotechnology Vol. 1, 147168, 2012
Preparation, Characterization and Applications of Liposomes: State of the Art

A. Laouini1 , C. Jaafar-Maalej1 , I. Limayem-Blouza2 , S. Sfar2 , C. Charcosset1 , and H. Fessi1
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Laboratoire dAutomatique et de Gnie des Procds (LAGEP), UMR-CNRS 5007, Universit Claude Bernard Lyon 1, CPE Lyon, Bat 308 G, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France 2 Laboratoire de Pharmacie Galnique, Facult de Pharmacie, Rue Avicenne, 5000 Monastir, Tunisie Liposomes, spherical-shaped nanovesicles, were discovered in the 60ies by Bangham. Since that, they were extensively studied as potential drug carrier. Due to their composition variability and structural properties, liposomes are extremely versatile leading to a large number of applications including pharmaceutical, cosmetics and food industrial elds. This bibliographic paper offers a by Ingenta to: with a focus on preparation methgeneral review on the background Delivered and development of liposomes Guest User evaporation, ethanol injection) and novel ods including classic (thin lm hydration, reverse-phase IP : 134.214.70.49 scalable techniques. Furthermore, liposome characterization techniques including mean size, zetaTue, 13 Nov 12:28:57 potential, lamellarity, encapsulation efciency, in2012 vitro drug release, vesicles stability and lipid analysis synthesized from different published works are reported. The current deepening and widening of liposome interest in many scientic disciplines and their application in pharmaceutics, cosmetics and food industries as promising novel breakthroughs and products were also handled. Finally, an opinion on the usefulness of liposomes in various applications ranging from unsubstantiated optimism to undeserved pessimism is given. The obtained information allows establishing criteria for selecting liposomes as a drug carrier according to its advantages and limitations.
Keywords: Liposomes, Preparation Methods, Characterization, Phospholipids, Therapeutic

Application, Cosmetic, Food.
CONTENTS
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Denition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Classication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Liposomes Preparation Procedures . . . . . . . . . . . . . . . . . . . . . . 3.1. General Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Preparation Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. In-Vitro Liposomes Characterization . . . . . . . . . . . . . . . . . . . . . 4.1. Lamellarity Determination . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Size Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Zeta Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Encapsulation Efciency . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Lipid Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. In-Vitro Drug Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Liposomes Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Liposomes Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Pharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . . 5.2. Cosmetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. INTRODUCTION
147 149 149 149 151 151 151 153 153 156 158 158 159 159 160 160 160 165 165 166 166
Author to whom correspondence should be addressed.
Liposomes, dened as microscopic spherical-shaped vesicles, consist of an internal aqueous compartment entrapped by one or multiple concentric lipidic bilayers. Liposomes membrane is composed of natural and/or synthetic lipids which are relatively biocompatible, biodegradable and non-immunogenic material. Because of their unique bilayer-structure properties, liposomes are used as carriers for both lipophilic and water-soluble molecules. Hydrophilic substances are encapsulated in the interior aqueous compartments. Lipophilic drugs are mainly entrapped within lipid bilayers. As asserted by different authors, liposomes have attractive biological properties, including the biocompatibility and biodegradability. They show promise as active vectors due to their capacity to enhance the encapsulant performance by increasing drug solubility, and stability; delivering encapsulated drugs to specic target sites, and providing sustained drug release.1 Their sub-cellular size allows relatively higher intracellular uptake than other particulate systems; improving in vivo drug bioavailability.
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A. Laouini is a Ph.D. student at the University of Lyon (France). His thesis is dealing with the application of membrane contactors for the preparation of colloidal systems. Prior to joining the University of Lyon, he received an M.Sc. in pharmacotechnie in 2010 and a degree of pharmacist in 2007 from the University of Monastir (Tunisia). His Current research interests focus on developing advanced drug carriers (liposomes, micelles, nanoemulsions ) by the use of membranes which allows reproducible production process with less energy consumption.
C. Jaafar-Maalej after obtaining a Master degree in Biomedical engineering at the University of Lyon 1, France, Chiraz Jaafar Maalej earned his Ph.D. degree in pharmacotechnie, with his thesis addressing the development of beclomethasone-liposomes suitable for pulmonary route. She is currently a postdoc fellow at the Laboratoire dAutomatique et de Gnie des Procds (LAGEP). Current research is directed toward the development of novel drug delivery systems for pediatric application from preformulation to in vivo pharmacokinetic studies in rats.
I. Limayem-Blouza after obtaining a Master degree in Biomedical engineering at the University of Lyon 1, France, Imne Limayem Blouza obtained her Ph.D. degree (2006 at the University of Lyon 1) in pharmacotechnie, with her thesis addressing the development of drug carriers (nano-particles) by the use of membrane contactor. She is actually aggregate professor in galenic pharmacy at the Faculty of Pharmacy of Monastir (Tunisia).
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S. Sfar is Pharm.D., Ph.D. in pharmaceutical sciences. Currently she is a research professor of technological pharmacy and she is responsible of a research unit for medicines development. Professor Souad Sfar has published on various aspects of pharmaceutical technology such as nanotechnology, bioavailability, pharmaceutical dosage forms, etc.
C. Charcosset graduated from Ecole Centrale de Lyon (France) in 1987. She obtained a Ph.D. in Compigne (France) in 1990; she was then appointed as a postdoctoral fellow at MIT (Boston, USA) from 1990 to 1992. She obtained a Research Scientist position at CNRS, rst in the Laboratoire des Sciences du Gnie Chimique (LSGC, Nancy, France), from 1992 to 1994; then in Laboratoire dAutomatique et de Gnie des Procds (LAGEP, Villeurbanne, France). She has actually a Research Director position. Her past and current research deals with membrane and membrane processes, such as characterization of membranes by confocal microscopy, membrane chromatography, ultraltration and microltration, preparation of emulsions and particles using membranes, and membrane crystallization for biotechnological, pharmaceutical and environmental applications.
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H. Fessi Master of Sciences in Chemistry and Ph.D. in Pharmaceutical Sciences of the University of Paris XI. Professor of Pharmaceutical Technology at the University Lyon I and expert member of the scientic committee of the French Medical Agency. Head of the joint research unit between the University of Lyon1 and the National Center of Scientic Research (CNRS) (Chemical Engineering and Automated Process, LAGEP, UMR 5007). His research interests focus on: The elaboration of original processes leading to biocompatible, biodegradable nanocolloid dispersions such as: nanospheres, nanocapsules, polymer nanoparticles, proteic nanospheres, liposomes, emulsions, cyclodextrin based dispersions and injectable submicronic dispersions. Biological and pharmaceutical evaluation of the elaborated colloidal dispersion systems in terms of interaction with the mucosal membranes. Optimization of the bioavailability and local tolerance of the administrated drug via oral, intramuscular and ocular routes. Pharmaceutical engineering aspect: The study of the industrial extrapolation of the developed processes leading to the colloidal systems elaboration. Other advantages of liposomes include high encapsulation aqueous solution is mainly driven by the hydrophobic efciency inspite of drug solubility, low toxicity due to effect which organizes amphiphilic molecules (phosphophospholipids content, drug protection against degradalipids) so as to minimize entropically unfavorable intertion factors like pH and light and the reduction of tissue actions between hydrophobic acyl-chains and surrounding irritation. Delivered by Ingenta aqueous to: medium.13 14 This effect is further settled by variLiposomes have been extensively studied as drug carriGuest User ous intermolecular forces such as electrostatic interactions, Research ers in the pharmaceutical and medical elds.1 2 IP : 134.214.70.49 hydrogen bonding, as well as Vanderwaals and dispersion 15 has expanded considerably over the last 30 Tue, years,13 increasNov 2012 12:28:57 forces. ing applications area from drug and gene delivery to diagLiposomes were dened as an articial microscopic nostics, cosmetics, long-lasting immune-contraception to vesicle consisting of a central aqueous compartment surfood and chemical industry.3 The superiority of liposomes rounded by one or more concentric phospholipid layas drug carriers has been widely recognized. Ten liposoers (lamellas) (Fig. 1). Furthermore, hydrophilic (in the mal and lipid-based formulations have been approved by aqueous cavity), hydrophobic (within lipidic membrane) regulatory authorities and many liposomal drugs are in and amphiphilic substances are able to be incorporated preclinical development or in clinical trials.4 within these vesicles developing large potential applicaSeveral reviews about liposomes as drug delivery tions. Numerous researchers have worked with these strucsystems5 and specic application via oral,6 7 topical,8 tures since Banghams discovery, making of liposomes the pulmonary,9 10 and ophthalmic11 route have been pubmost popular nanocarrier systems.16 lished. Clearly, within the frame of a single review paper it is impossible to address all the pertinent issues, this bibliographic paper attempt to review liposomes current 2.2. Classication technology with respect to numerous multidisciplinary applications. As a contribution for updating the state of Liposomes can be classied in terms of composition and knowledge, a focuses on liposomes preparation method mechanism of intracellular delivery into ve types17 and recent characterization techniques including mean (i) conventional liposomes; size, zeta-potential, lamellarity, encapsulating efciency, in (ii) pH-sensitive liposomes; vitro active release, stability and lipid analysis have been (iii) cationic liposomes; described as well as the most signicant achievements and (iv) immunoliposomes and applications. (v) long-circulating liposomes.
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2. LIPOSOMES
2.1. Denition Liposomes were rst produced in England in the 60s, by Bangham who was studying phospholipids and blood clotting.12 According to legend, he was experimenting with new laboratory equipment, and he made a noted observation about phospholipids forming closed multilamellar vesicle spontaneously in aqueous solution which took two years to be proved. The phospholipid reorganisation in
Otherwise, vesicle size is a critical parameter in determining circulation half-life of liposomes, and both size and number of bilayers inuence the extent of drug encapsulation within liposomes. Thus, liposomes were typically classied on the basis of their size and number of bilayers into (Fig. 2): (i) Small unilamellar vesicles (SUV): 20100 nm; (ii) Large unilamellar vesicles (LUV): > 100 nm; (iii) Giant unilamellar vesicles (GUV): > 1000 nm; (iv) Oligolamellar vesicle (OLV): 100500 nm and (v) Multilamellar vesicles (MLV): > 500 nm.18 149
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Fig. 1. Schematic drawing of liposomes structure and lipophilic or hydrophilic drug entrapment models.
Fig. 2. Liposomes classication based on size and lamellarity.
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3.2.1.2. Reverse-Phase Evaporation (REV) Technique. A lipidic lm is prepared by evaporating organic solvent under reduced pressure. The system is purged with nitrogen and the lipids are re-dissolved in a second organic phase which is usually constituted by diethyl ether and/or isopropyl ether. Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into this mixture. The organic solvent is subsequently removed 3. LIPOSOMES PREPARATION and the system is maintained under continuous nitrogen. PROCEDURES These vesicles have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times 3.1. General Ingredients higher than multilamellar vesicles. Most importantly, a Generally, liposome composition includes natural and/or substantial fraction of the aqueous phase (up to 62% at synthetic phospholipids (Phosphatidylethanolamine, Phoslow salt concentrations) is entrapped within the vesicles, phatidylglycerol, Phosphatidylcholline, Phosphatidylserencapsulating even large macromolecular assemblies with ine, Phosphatidylinositol) Phosphatidylcholine (also high efciency.27 known as lecithin) and phosphatidylethanolamine con3.2.1.3. Solvent (Ether or Ethanol) Injection Technique. stitute the two major structural components of most The solvent injection methods involve the dissolution of biological membranes. Liposome bilayers may also conthe lipid into an organic phase (ethanol or ether), followed to: tain other constituents such as cholesterol,Delivered hydrophilicby Ingenta by the injection of the lipid solution into aqueous media, Guest User forming liposomes.28 polymer conjugated lipids and water. IPbilayer : 134.214.70.49 The ethanol injection method was rst described in Cholesterol has been largely used to improve the 29 13 Nov 2012 12:28:57 1973. The main relevance of the ethanol injection characteristics of the liposomes. It improvesTue, the membrane method resides in the observation that a narrow distribuuidity, bilayer stability and reduces the permeability of tion of small liposomes (under 100 nm) can be obtained water soluble molecules through the membrane.22 by simply injecting an ethanolic lipid solution in water, in A clear advantage of liposomes is the fact that the one step, without extrusion or sonication.30 lipid membrane is made from physiological lipids which The ether injection method differs from the ethanol decreases the danger of acute and chronic toxicity. injection method since the ether is immiscible with the aqueous phase, which is also heated so that the solvent is removed from the liposomal product. The method involves 3.2. Preparation Method injection of ether-lipid solutions into warmed aqueous 3.2.1. Classical Technique phases above the boiling point of the ether. The ether vaporizes upon contacting the aqueous phase, and the disThere are four classical methods of liposome manufacture. persed lipid forms primarily unilamellar liposomes.31 An The difference between the various methods is the way in advantage of the ether injection method compared to the which lipids are drying down from organic solvents and ethanol injection method is the removal of the solvent from then redispersed in aqueous media.23 These steps are perthe product, enabling the process to be run for extended formed individually or are mostly combined. periods forming a concentrated liposomal product with 3.2.1.1. Hydration of a Thin Lipid Film: Bangham high entrapment efciencies. Method. This is the original method which was initially 3.2.1.4. Detergent Dialysis. Liposomes, in the size used for liposomes production.24 A mixture of phosphorange of 40180 nm, are formed when lipids are solubilipid and cholesterol were dispersed in organic solvent. lized with detergent, yielding dened mixed micelles.32 As Then, the organic solvent was removed by means of evapthe detergent is subsequently removed by controlled dialyoration (using a Rotary Evaporator at reduced pressure). sis, phospholipids form homogeneous unilamellar vesicles Finally, the dry lipidic lm deposited on the ask wall was with usefully large encapsulated volume. hydrated by adding an aqueous buffer solution under agitaOther methods have been already used for lipotion at temperature above the lipid transition temperature. somes preparation such as: calcium induced fusion,33 This method is widespread and easy to handle, however, nanoprecipitation,34 and emulsion techniques.35 36 dispersed-phospholipids in aqueous buffer yields a popuHowever, these classical techniques require large lation of multilamellar liposomes (MLVs) heterogeneous amounts of organic solvent, which are harmful both to both in size and shape (15 m diameter). Thus, liposome the environment and to human health, requiring complete size reduction techniques, such as sonication for SUVs forremoval of residual organic solvent. Furthermore, convenmation or extrusion through polycarbonate lters forming tional methods consist of many steps for size homogeLUVs25 26 were useful to produce smaller and more uninization and consume a large amount of energy which is unsuitable for the mass production of liposomes. formly sized population of vesicles. New developed types of liposome, designated as double liposome (DL)19 and multivesicular vesicles (MVV),20 were recently reported. These liposomes, which could be prepared by novel preparative technique, are thought to improve drug protection against several enzymes.21
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3.2.2. New Large-Scale Liposome Technique
appropriate ratios to form a clear isotropic monophase solution. Then the monophase solution was sterilized Since industrial scale production of liposomes has become by ltration and lled into freeze-drying vials. In recent reality, the range of liposome preparation methods has study, a laboratory freeze drier was used and freeze-drying been extended by a number of techniques such as Heatprocess was as follows: freezing at 40 C for 8 h; priing Method, Spray drying, Freeze Drying, Super Critical mary drying at 40 C for 48 h and secondary drying at Reverse Phase Evaporation (SCRPE), and several modi25 C for 10 h.39 The chamber pressure was maintained at ed ethanol injection techniques which are increasingly 20 Pascal during the drying process. On addition of water, attractive. the lyophilized product spontaneously forms homogenous 3.2.2.1. Heating Method. A new method for fast proliposome preparation. After investigation of the various duction of liposomes without the use of any hazardous parameters associated with this method it is found that the chemical or process has been described.23 This method lipid/carrier ratio is the key factor affecting the size and involves the hydration of liposome components in an aquethe polydispersity of the liposome preparation.39 Thereous medium followed by the heating of these compofore, TBA/water cosolvent system was used for economy nents, in the presence of glycerol (3% v/v), up to 120 C. concerns. Glycerol is a water-soluble and physiologically acceptable 3.2.2.4. Super Critical Reverse Phase Evaporation chemical with the ability to increase the stability of lipid (SCRPE). The SCRPE is a one-step new method that has vesicles and does not need to be removed from the nal been developed for liposomes preparation using supercritliposomal product. Temperature and mechanical stirring ical carbon dioxide.41 This method allowed aqueous disprovide adequate energy for the formation of stable lipoDelivered by Ingenta persionsto: of liposomes to be obtained through emulsion somes. Guest User formation by introducing a given amount of water into a Reza Mozafari et al. conrmed by TLC that IP no : degra134.214.70.49 homogeneous mixture of supercritical carbon dioxide/Ldation of the used lipids occurred at the above mentioned Tue, 13 Nov 2012 12:28:57 R-dipalmitoylphosphatidylcholine/ethanol under sufcient temperatures.37 The particle size can be controlled by the stirring and subsequent pressure reduction. phospholipid nature and charge, the speed of the stirring Transmission electron microscopy observations revealed and the shape of the reaction vessel. Otherwise, employthat vesicles are large unilamellar with diameters of 0.1 ment of heat abolishes the need to carry out any further 1.2 m.41 The trapping efciency of these liposomes indisterilisation procedure reducing the time and cost of lipocated more than 5 times higher values for the water-soluble some production. solute compared to multilamellar vesicles prepared by 3.2.2.2. Spray-Drying. Since spray-drying is a very the Bangham method. The trapping efciency for an oilsimple and industrially applicable method, the direct soluble substance, the cholesterol, was about 63%. Results spray-drying of a mixture of lipid and drug was applied in 38 showed that the SCRPE is an excellent technique that perthe preparation of liposomes. The spray-drying process mits one-step preparation of large unilamellar liposomes is considered to be a fast single-step procedure applied in exhibiting a high trapping efciency for both water-soluble the nanoparticles formulation. and oil-soluble compounds.42 43 Hence, liposomes were prepared by suspending lecithin 3.2.2.5. Modied Ethanol Injection Method. Novel and mannitol in chloroform. The mixture was sonicated approaches based on the principle of the ethanol injection for 8 min (bath sonicator) and subjected to spray-drying technique such as the microuidic channel method,4446 on a Buchi 190 M Mini Spray Dryer. The spray-drying the crossow-injection technique,4750 and the membrane conditions were as follows: inlet and outlet temperatures contactor method51 were recently reported for liposome were 120 C and 80 C, respectively; airow rate was production. 700 NI/hr; and the ow rate was 1000 ml/hr. The dried 3.2.2.5.1. The Crossow Injection Technique. The conproduct was hydrated with different volumes of phosphate cept of continuous crossow injection is a promising buffered saline (PBS; pH 7.4) by stirring for 45 min.38 The approach as a novel scalable liposome preparation techmain factor inuencing the liposomal size was the volume nique for pharmaceutical application. Wagner et al. used of aqueous medium used for hydration of the spray-dried a cross ow injection module made of two tubes welded product.38 However, mannitol plays an important role in together forming a cross.4750 At the connecting point, increasing the surface area of the lipid mixture, enabling the modules were adapted with an injection hole. The successful hydration of the spray-dried product. inuencing parameters such as the lipid concentration, the 3.2.2.3. Freeze Drying. This new method was injection hole diameter, the injection pressure, the buffer described for the preparation of sterile and pyrogen-free ow rate, and system performance were investigated.47 submicron narrow sized liposomes.39 40 It is based on A minimum of buffer ow rate is required to affect batch the formation of a homogenous dispersion of lipids in homogeneity and strongly inuencing parameters are lipid water-soluble carrier materials. Liposome-forming lipids concentration in combination with increasing injection and water-soluble carrier materials such as sucrose were pressures. After exceeding the upper pressure limit of the dissolved in tert-butyl alcohol/water cosolvent systems in 152
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linear range, where injection velocities remain constant, cell, the intracellular fate is affected by the lamellarity. The liposomes lamellarity made from different lipids or prepathe vesicle batches are narrowly distributed, also when ration procedures varies widely. That is why, the analysis injecting higher lipid concentrations. Reproducibility and of liposomes lamellarity is an important parameter to be scalability data show similar results with respect to vesicle considered. size and size distribution and demonstrate the stability and Liposome lamellarity is often accomplished by methods robustness of the novel continuous liposome preparation that are based on the visible or uorescence signal change technique.49 of lipids marker upon reagents addition. This approach is 3.2.2.5.2. Microuidization. By using a microuidic reviewed in more detail, since it is a relatively simple prohydrodynamic focusing (MHF) platform, Jahn et al. gencedure that can be easily carried out in a standard lab. Severated liposomes by injecting the lipid phase and the water eral lipids can be used and results rely on the comparison phase into a microchannel.45 Microuidic ow is generof the total signal to the signal achieved from the reaction ally laminar due to the small channel dimensions and relabetween the lipids marker and the specied reagents.58 tively low ow rates. Well-dened mixing is then obtained For example the UV absorbance of 2,4,6by interfacial diffusion when multiple ow streams are trinitrobenzensulfonic acid (TNBS) at 420 nm increases in injected in a microchannel. The size of the liposomes was the mixture as a result of complex formation with primary mainly controlled by changing the ow rate.44 amines. This property has been used for the detection of 3.2.2.5.3. Membrane Contactor. Recently, Jaafaraminolipids at 420 nm. As the lipid bilayers are slightly Maalej et al. applied the ethanol injection technique permeable to the TNBS reagent, an overestimate of the while using a membrane contactor for large scale lipoDelivered external to: surface can be expected. To correct the reagent somes production. In this method, a lipid phase (ethanol,by Ingenta Guest leakage through the bilayer, three incubation times were phospholipid and cholesterol) was pressed through the User IP gas : 134.214.70.49 used. The obtained external surface area at each incubation membrane with a specied pore size. Nitrogen at Tue, 13 Nov 2012 12:28:57 time was plotted against incubation time and the graph pressure below 5 bar was sufcient for passing the organic was extrapolated to time zero. Under certain conditions, phase through the membrane. At the same time, the the bilayer permeability of TNBS is minimized such as aqueous phase ew tangentially to the membrane surface the only aminolipids on the exterior bilayer contribute and swept away the formed liposomes within the memto the signal. Lysis of liposomes by a detergent such as brane device. The new process advantages are the design Triton X-100 allows TNBS to interact with interior aminosimplicity, the control of the liposome size by tuning the lipids and yields the total signal. TNBS has remained the process parameters and the scaling-up abilities.51 commonly used method for the estimation of the degree As a result, these techniques lead from the convenof lamallarity. However, this method has disadvantages tional batch process to potential large scale continuous which make it impotent in most cases; the TNBS assay procedures. requires large amount of material (milligrams) which makes the multiple sample application difcult and affect 4. IN-VITRO LIPOSOMES assay precision when the amount is limited.59 60 CHARACTERIZATION In another method, the addition of periodate to phosphatidylglycerol results in the diol oxidation and releasIn order to assess the liposome quality and to obtain quaning of formaldehyde. The released formaldehyde reacted titative measures that allow comparison between differwith chromotropic acid to yield a product which was ent batches of liposomes, various parameters should be subsequently detected at 570 nm. This method has been monitored. For liposomes applications in analytical and used for the determination of external reactive groups on bioanalytical elds, the main characteristics include the 58 liposomes. average mean diameter and polydispersity index; encapsuOtherwise, the quenching of N-(7-nitrobenz-2-oxa-1,3lation efciency; the ratio of phospholipids to drug condiazol-4-yl) (NBD) uorescence is obtained by sodium centration and lamellarity determination. Other commonly dithionite. NBD-labeled lipids are highly uorescent monitored parameters include surface charge through zeta at low concentration ( < 1 mol%) in membranes, but potential measurement, phase transitions through differenundergo self-quenching at increased concentrations. In this tial scanning calorimetry and quantication of residual solapproach, the initial NBD labelled lipids uorescence is vents through gas chromatography. A detailed description from all lipids in the sample. Under appropriate condiof todays most commonly methods and novel techniques tions, the addition of sodium dithionite quenches the uof liposome characterization is presented in this report. orescence of only the NBD labelled existing on the outer bilayer. Fluorescence was monitored on spectrouorometer 4.1. Lamellarity Determination with excitation and emission wavelengths of 450 nm and Lipid bilayers number of liposomes inuences the encap530 nm respectively. The percentage of external lipid is sulation efciency and the drugs release kinetics. Furtherfound by dividing the change in uorescence upon dithionite addition by the total uorescence.6163 more, when liposomes are taken up or processed in the
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Table I. Preparation methods and applications through published papers.
Preparation method Hydration lipidic film Active substance Ibuprofen Membrane composition Egg phosphatidylcholine (PC) Phosphatidylcholine (DMPC) Distearoyl phosphatidylcholine (DSPC) Dilignoceroyl phosphatidylcholine (C24PC) Stearylamine (SA), dicetylphosphate (DCP) cholesterol (Chol) Solvent/ adjuvants 9:1 solvent mixture of chloroform and methanol Distilled water Size 4.2 6 m Zeta 5.2 54 mV EE% 17% 61% Study details *The cholesterol content *Llipid alkyl chain length *Charged lipids
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References
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Effects on liposome characteristics (size, zeta a,d e,capsulation efficiency)
Reverse -phase evaporation Conventionnel techniques
Acetazolamide
Egg LChloroform: phosphatidylchomethanol mixture line, type X-E, and (2:1, v/v) cholesterol ether stearylamine or dicetyl phosphate
5.8 7.6 m
10% 45%
Ethanol injection
salidroside
Detergent dialysis
nm Delivered by Ingenta100 to: Guest User IP : 134.214.70.49 EggTwo detergents 60 Tue, 13 Nov 2012 12:28:57 phosphatidylchosodium cholate 160 nm line (EPC, eggphosphatidylglycerol EPG) and octyl-_-dglucopyranoside Custom-made dialysis cell (7.1cm2 exchange area) Chloroform and 10 ether 30 m 4.0% glucose aqueous solution
Egg yolk phosphatidylcholine cholesterol
absolute ethanol
50
0 18 mV
20% 40%
*Effect of the cholesterol content and charged lipids on liposome characteristics *Photomicroscopic Analysis *Differential Scanning Calorimetry measurements *Stability Study *In vivo Studies (male rabbits) *The effect of salidrosideloading capacity on EE *Effect of cholesterol on EE and particle size *Turbidity measurements *In vitro release study *Dynamic light scattering and zeta potential Measurements *Cryo-transmission electron microscopy Comparison of size and size distribution data obtained from DLS and cryoTEM *Factors impacting encapsulation efficiency of multivesicular liposomes *The protein release from liposomes-in-alginate (LIA) in vitro *Analysis of bovine serum albumin in LIA also studied, Verify the improvement of entrapment efficiency and release time prologation. *Transfection efficiency and cytotoxicity were evaluated using Chinese hamster ovary-K1 (CHO-K1) cells.
(53)
(54)
7 51 mV
(55)
Emulsion (double emulsification process)
Bovine serum albumin (BSA)
Egg phosphatidylcholine (PC), Triolein (TO), cholesterol (Chol)
43% 71%
(56)
Heating method
Spray drying
Freeze drying
Novel techniques
Plasmid DNA Dipalmitoylphosp from hatidylcholine Escherichia coli (DPPC), dicetylphosphate (DCP), cholesterol (70:20:10 molar ratio) Metronidazole Lecithin and verapamil hydrochloride DrugCyclodextrin Complex Ketoprofen (K), Soybean phosphatidylciprofloxacin choline, and lactate (CL) soybean and propranolol phosphatidylhydrochloride serine (PH)
Sterile phosphate buffered saline glycerol (final concentration 3%, v/v)
160 2250 nm
(57)
Mannitol chloroform
268 395 nm
24 47%
*Serum stability study
(38)
Sucrose Tert-butyl alcohol/ Water
110 410 nm
*Size measurements *Entrapment studies *Electron microscopy *Thermal analysis *X-ray diffraction
(39)
Calcein (Cal) 100 nm Soybean phosphatidylcholine, and hydrogenated soybean phosphatidylserine Sucrose, cholesterol (4:1) Tert-butyl alcohol/ water
*Size measurement *Entrapment efficiency *Thermal analysis *Cryomicroscopy *Mass loss analysis *Residual solvent content
(40)
Continued.
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Table I. Continued.
Preparation method Active substance
Membrane composition
Solvent/ adjuvants
Size
Zeta
EE%
Study details
References
Supercritical reversephase evaporation method
Glucose
L-RDipalmitoylphosp hatidylcholine (DPPC
Microfluidic channel
Egg phosphatidylcholine or hydrogenated soy phosphatidylcholine Cholesterol (2.5:1 w/w)
< 2000 nm The cell temperature was raised to 60 C, and CO2 was introduced to achieve the experimental conditions (typically 200 bar). 40 min with stirring at equilibration, the pressure was released to obtain the liposomal dispersion. Ethanol 50 160 nm or ethanol/tertbutanol (1:1 v/v)
0 30 mV
15% 20%
*The trapping efficiency *Dynamic light scattering, *zeta potential *freeze-fracture transmission electron microscopy investigating Physicochemical properties of chitosan-coated liposomes
(42) (43)
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Modified ethanol injection 50 nm Delivered by Ingenta to: Guest User IP : 134.214.70.49 100 Tue, 13 Nov 2012 12:28:57 200 nm Dimyristoylphosp hatidylcholine Isopropyl alcohol (DMPC) Cholesterol and dihexadecyl phosphate (5:4:1 in dry chloroform) DipalmitoylEthanol phosphatidylcholine Cholesterol and stearylamine (7:2:1) 25
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Crossflow injection
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Recombinant Idem human superoxide dismutase (rhCu/Zn-SOD) from Escherichia coli Indomethacin Lipoid E80 (IMC), Cholesterol Beclomethason (20% w/w PL) e dipropionate (BDP) Idem 200 300 nm 25 27%
(47)
Membrane contactor
Ethanol or ethanol/tertbutanol (1:1 v/v)
100 180 nm
35 6 (mV)
IMC: 63% *Size measurement *Zeta potential BDP: 98% *Entrapment efficiency *Thermal analysis *Electron transmission microscopy
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These methods appear to close a gap in the methodology to determine external surface structure of vesicles. However, these methods assume that the lipid of interest is distributed evenly over all lipid layers, and the reagents used to elicit the signal change are impermeable to the membrane over the time course of the measurements.64 Other numerous methods for the lamellarity determination such as magnetic resonance were mainly used to study the outside-inside distribution of phospholipids within bilayer and the characterization of model membrane structures. A straightforward application of nuclear magnetic resonance in the quality control of liposomes is the determination of size and lamellarity. Dispersions of MLVs give rise to very broad powder 31P-NMR spectra due to the restricted anisotropic motion whereas SUVs are characterized using a narrow line spectra. It is well known that the paramagnetic ion Mn2+ interacts with the negatively charged phospholipids phosphate causing perturbations of the nuclear spin relaxation times which broaden the 31P-NMR resonance and reduces the quantiable signal.
Presuming that the shift reagent (Mn2+) only interacts with the phospholipids located in the outermost monolayer, the degree of lamellarity can be calculated by the ratio of 31P-NMR signal before and after Mn2+ addition. Used for a long time in the eld of liposome research, this technique has been found to be quite sensitive to experimental conditions which can have distinct effect on the analysis. For example, Mn2+ is able to penetrate the liposomal bilayer especially when used at high concentrations. At low pH or in the presence of complexing agents (such as HEPES or TRIS buffer at certain concentration), no penetration of Mn2+ occurs. Therefore, under well-dened conditions, the analysis of liposomes by 31P-NMR is the presence of shift reagent in an elegant and accurate method giving useful information about the outer to inner phospholipids ratio amount.65 66 Other techniques for lamellarity determination include small angle X-ray scattering (SAXS). For this purpose, liposome dispersions put into glass capillaries and curves were recorded with a camera equipped with a 155
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without sample manipulation. The result is with a high one-dimensional position sensitive detector. Blank scattering curves were obtained from the same capillaries lled resolution three-dimensional prole of the vesicle surface with the liposome suspension solvent. Data were evaluated under study. The technique permits liposomes visualization using the Indirect Fourier Transformation which provides without alteration of their native form; given that the reqthe electron distance distribution p(r) (the probability to uisite surface immobilization does not adversely affect the nd two electrons with distance r in the measured sample). sample and that the force of the probe itself does not have SAXS is considered as a good method evaluating vesicles deleterious effects on the vesicles. AFM analysis is rapid, lamellarity with high accuracy.67 68 powerful and relatively non invasive technique. It can proTo conrm the lamellarity results by an imaging vide information on morphology, size, as well as on the method, freeze fracture technique with subsequent transpossible aggregation processes of liposomes during their mission electron microscopy was used. For this purpose, storage. Imaging in aqueous medium allows the liposomes carbon lm grids were used for specimen preparation. observation under physiological condition. Using AFM A drop of the sample was put on the untreated coated grid. technology, experimental data indicate that liposomes in Most of the liquid was removed with blotting paper leaving water dispersion maintained their integrity only few mina thin lm stretched over the holes. The specimens were utes after deposition on mica support, after which they instantly shock-frozen in melting nitrogen or by plungcollapsed. For this reason, the liposomes images have to be ing them into liquid ethane or propane in a temperatureobtained within 10 min after deposition. Therefore, special controlled freezing unit. After freezing, the specimens attention has to be given to the experimental conditions were inserted into a cryo-transfer holder and transferred to and especially the analytical times, AFM technique can by Ingenta a cryo-electron microscope. To determine theDelivered mean lamelreplace to: the wide variety of microscopic techniques meaGuest User larity, micrographs of three different areas of the specimen suring liposomal size.7174 IP : 134.214.70.49 were investigated.66 69 70 HPLC using SEC can be used to separate and quantify Tue, 13 Nov 2012 12:28:57 Whatever is the technique, the lamellarity determination liposome populations according to a time-based resoluis essential to dene liposome structure as it is a very tion of hydrodynamic size. The porous packing material important prerequisite for liposomes success in therapy. used in this technique excludes larges species from the internal pore volume leading to their shorter retention on the column. This mechanism leads to separation based 4.2. Size Analysis on large particles elution before smaller particles. Conventional SEC is frequently used for liposomes separation The average size and size distribution of liposomes are from unencapsulated materials as a nal purication step, important parameters especially when the liposomes are but the use of HPLC-SEC for analysis offers increased resintended for therapeutic use by inhalation or parenteral olution of liposome populations and reduced sample size route. Several techniques are available for assessing suband enhances reproducibility. One recommended commermicrometer liposome size and size distribution which cially available column is the ethylene glycol-metacrylate include microscopy techniques, size-exclusion chromatoggel which has a separation range from 20 to 500 nm, this raphy (SEC), eld-ow fractionation and static or dynamic hydroxylated poly-ether-based gel shows a larger exclulight scattering. sion limit than other gels. An osmotically balanced mobile Several variations on electron microscopy (EM) such phase owing at relatively low pressures (11.5 megaas transmission EM using negative staining, freeze fracpascal) helps to prevent damages, swelling or shrinkage ture TEM, and cryo EM, provide valuable information on of liposomes. HPLC-SEC can offer a powerful technique liposome preparations since they yield a view of morpholfor not only size distribution determination, but also staogy and can resolve particles of varying size. However, bility in terms of aggregation and vesicle permeability. sample preparation is complicated as it requires removal Three methods have been described in literature: dynamic of liposomes from their native environment. These techlight-scattering analysis of SEC fractions; rechromatograniques can also generate artefacts, induce shrinkage and phy of SEC fractions on a calibrated column with turshape distortion, and are time consuming to obtain a repbidity measurements; and SEC with on-line turbidity and resentative size distribution of the population, thus are refractive index detection. The rechromatography method not amenable to being routine measurements. Some of was judged to be the most reliable, although the senthese problems may be overcome yielding reproducible sitivity suffered from the dilution in the two chromatoand accurate results by giving careful attention to samgraphic steps. Disadvantages of HPLC for liposomes size ple preparation. A recently developed microscopic techdetermination mainly stem from recovery issues. These nique known as atomic force microscopy (AFM) has been include unwanted adsorption of lipids on the column packutilized to study liposome morphology, size and stability. ing and destruction of liposomes containing lipids with AFM, scanning probe microscopes with dimensional resohigher afnity to the column material than the composlution approaching 0.1 nm, provides unique possibility for visualizing small liposomes in natural environment even ite lipids. Both lipids necessitate a preliminary step of 156
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a single wavelength. The FFF mechanism for liposomes presaturation of the LC column with lipids prior to analanalysis differs in that FFF ow separates vesicles on a ysis. In addition, the lipid bilayer rigidity, which is a hydrodynamic size basis, whereas sedimentation FFF sepfunction of the lipid composition, plays a role in the lipoarates them on a weight basis. Flow FFF enables rapid, somes retention and recovery. The bilayer rigidity dicconvenient and non invasive measurement of vesicle size tates whether liposomes of large diameter can be deformed distribution without prior calibration using size standards. and thereby pass through relatively narrow pores or lipoOther advantage of the FFF technique is the wide range somes of small diameter which may be excluded from of particle sizes that can be separated (1 nm 100 m) relatively large pores, dependent on pores shape and oriwith high resolution. The only limit of this technique is entation. The net effect is therefore difcult to predict. the complexity and expense of instrumentation.7881 Thus, while hydrodynamic size and subsequent molecular weight information can be obtained through this techDynamic light scattering (DLS), otherwise known as nique, the accuracy of this determination is based on the photon correlation spectroscopy (PCS), is extensively used use of well-matched (both by shape and chemical comin liposome size distribution analysis. DLS measures position) set of standards. Lastly, while suitable packing the time-dependant uctuations of light scattered from materials are available for small to moderately sized lipoparticles experiencing Brownian motion, which results somes resolution, it is not the case for large liposomes from collisions between suspended particles and solvent (>800 nm).7577 molecules. When a particle is suspended in a solution and illuminated by light, it scatters light given that its index Field-ow fractionation (FFF) is a technique which of refraction differs from that of suspending solvent. In overcomes some of the limitations of HPLC in liposomes Delivered to: its polarizability differs from that of the solother words, analysis. It includes electrical, thermal, sedimentation andby Ingenta Guest User vent. This means that the arriving electric eld is oscilow FFF techniques that rely on a eld application which IP : 134.214.70.49 lating and is able to displace the cloud of electrons and is perpendicular to the direction of ow. FFF uses a chanTue, 13 Nov 2012 12:28:57 thereby cause atoms to oscillate. The strengths of the technel wall which consists of a semipermeable membrane nique include the ability to make measurements in native chosen with a MWCO suitable for the liposomes under environments; its sensitivity to small quantities of high study. This membrane allows only the carrier uid to pass. molecular weight aggregate; ease of commercially availIn ow FFF, there are two liquids ows acting on the samable operating instrument; minimal sample volume, conple components. The channel ow that runs through the centration and preparation requirements. It also covers a channel and the crossow that owing perpendicular to large size range of species spanning the low nanometer to the channel passes through the inlet frit into this channel low micrometer range. However, the technique does not and exits through the membrane and outlet frit. A comyield particle shape information; it can yield a bias towards mon procedure for sample injection is called stop-ow reporting larger diameters when small quantities of high relaxation, in which a small volume sample is injected molecular weight or aggregates or impurities are present into the channel ow. After a short delay period that allows in the sample.8284 the sample to move into the channel from the injector, the channel ow is stopped for a time, allowing only the Measurement of particle size distribution could also be crossow to act on the sample. The laminar ow prole achieved using density gradient stabilized sedimentation slow down the movement of particles located closer to the whereby particles that are lower in density than the uid in channel walls, while the perpendicular ow propels all parwhich they are suspended can be accurately analysed.85 86 ticles toward the membrane wall. Diffusion due to BrownCentrifugal sedimentation of particles suspended in a uid ian motion of particles in a size-based manner reduces the is a well-known method of measuring the size distribuaccumulation of smaller particles against the membrane tion of particles in the range of 0.01530 m in diameter. wall. Retention times in this technique are proportional to The sedimentation velocity of any particle could be calthe hydrodynamic diameter of the particles since smaller culated if the particle density, uid density, uid viscosity particles reach an equilibrium position further from the and centrifugal acceleration are known. If the conditions of channel walls. Whereas in HPLC-SEC, large liposomes sedimentation are stable, the particles begin sedimentation elute rst, in normal mode FFF, small liposomes elute rst as a very thin layer at the surface of the uid. A light beam due to their higher diffusion coefcient. The carrier liquid or an X-ray beam passes through the centrifuge at some used in FFF needs to be chosen carefully so that there is distance below the surface of the uid and measures the no appreciable swelling of the membrane, as this can lead concentration of particle as they settle. The time required to non-uniform ows in the channel. Aqueous solutions for particles to reach the detecting beam depends upon the are usually used as carrier liquids, although non-aqueous speed and geometry of the centrifuge, the difference in solvents have also been used. Many detectors have been density between the particles and the uid and the diamused in FFF, but the most common is a UV/VIS speceter of the particles. The particles sediment at velocities, trophotometer. Photodiode arrays have been used to obtain depending upon their size until reaching the detector beam the entire spectra of eluting samples instead of monitoring which is positioned at a known distance below the uids
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surface.86 Sedimentation velocity increases as the square of the particle diameter, so that particles which differ in size by only few percent settle at signicantly different rates. The time needed to reach the detector is used to calculate the size of the particles. This method for size analysis has a high resolution compared to the other analysis method, it has also a high sensitivity which enables him to detect small additional peaks and pick up small changes. Moreover, high accuracy is assured since all analyses are run against a known calibration standard; the calibration can be either external (standard injected before the sample) or internal (standard mixed with the sample).85 Several other techniques, considered to be less conventional, have been applied for liposome size distribution analysis but are not discussed in this paper, such as NMR, ow cytometry, capillary zone electrophoresis, etc.
suspension with zeta potentials > +30 mV or < 30 mV are normally considered stable.87 88 4.4. Encapsulation Efciency
The liposome preparations are a mixture of encapsulated and un-encapsulated drug fractions. The rst step for the determination of the encapsulation efciency is the separation between the encapsulated drug (within the carrier) and the free drug. Several separation techniques have been reported in the literature. The mini-column centrifugation is a method based on the difference of size between the drug loaded liposomes and the free drug. Indeed, undiluted liposome suspension is applied dropwise to the top of sephadex gel column and the column is spun at 2000 rpm for 3 min to expel the void volume containing the liposomes into the centrifuge tube. Then 250 l of water was added and centrifugation was repeated. The non entrapped 4.3. Zeta Potential drug remained bound to the gel, while vesicles traversed the gel and Delivered by Ingenta to: were collected from the rst and second stage Three of the fundamental states of matter are solids, of centrifugation.89 Guest User liquids and gases. If one of these states is nely disThe separation between the free drug and the encapsuIP : 134.214.70.49 persed in another then we have a colloidal system. Most lated drug could also be achieved by the use of a dialysis Tue, 13 Nov 2012 12:28:57 colloidal dispersions in aqueous media carry an electric membrane with an appropriate cut-off. The liposme samcharge. There are many origins of this surface charge ple is dialysed against a buffer solution for 2 hours.84 depending upon the nature of the particle and its surroundThe ultracentrifugation technique was reported as a siming medium. The more important mechanisms are: ionizaple and fast method for the separation of drug-loaded tion of surface groups (dissociation of acidic groups on the liposomes from their medium. The sample is centrifuged surface of a particle giving a negatively charged surface, at 50000 rpm for 50 min at +4 C.90 Centrifugation at conversely a basic surface will take on a positive charge) 3000 rpm for 30 minutes can also be used. But prior to the and adsorption of charged species (surfactant ions may be centrifugation, liposomes should be aggregated in order to specically adsorbed on the particle surface leading in the enable their sedimentation by adding an equal volume of case of cationic surfactants to a positively charged surface protamine solution (10 mg/ml) to the sample.91 92 and in the case of anionic surfactants to a negatively one). Once drug-loaded liposomes are separated from their The zeta potential of a particle is the overall charge that medium, the lipidic bilayer is disrupted with methanol a particle acquires in a particular medium. It is a physical or Triton X-100 and the released material is then quanproperty which is exhibited by any particle in suspension. tied. Techniques used for this quantication depend on It has long been recognized that the zeta potential is a very the nature of the encapsulant and include spectrophotomegood index of the interaction magnitude between colloidal try, uorescence spectroscopy, enzyme-based methods and particles. Measurements of zeta potential are commonly electrochemical techniques. used to predict the stability of colloidal systems. If all the Other methods such as HPLC or FFF can also be particles in suspension have a large negative or positive applied for the determination of the encapsulation efzeta potential then they will tend to repel each other and ciency. In this case, the encapsulation percent can be there will be no tendency to aggregation. However, if the expressed as the ratio of the un-encapsulated peak area particles have low zeta potential values then there will be to that of a reference standard at the same initial concenno force to prevent the particles occulating. tration. This method can be applied if the liposomes do To measure the zeta potential, a laser is used to provide not undergo any purication (SEC, dialysis ) following a light source illuminating particles within the samples. preparation. Either technique are applied to separate lipoThe incident laser beam passes through the centre of the some encapsulating materials from un-encapsulated drug sample cell and the scattered light at an angle of about and hence can also be used to monitor the storage sta13 is detected. When an electric eld is applied to the bility in terms of leakage or the effect of various discell, any particles moving through the measurement volruptive conditions on the retention of encapsulants. In ume will lead to uctuation of the detected light with a some cases, size distribution and encapsulation efciency frequency proportional to the particle speed. This infordeterminations could be combined in one assay by using mation is passed to a digital signal processor, then to a FFF coupled to a concentration detector suitable for the encapsulant. computer and hence potential zeta is calculated. Particles 158
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The terminology varies widely with respect to the abilEnzymatic assays for phosphatidylcholine and cholesity of various liposome formulations to encapsulate the terol analysis are commercially available and widely used. target molecules. Many papers express results in term of The former method used phospholipase D to hydrolyze percent encapsulation, incorporation efciency, trapphospholipids and release free choline. The free choline ping efciency or encapsulation efciency (EE) which is then oxidized to form betaine aldehyde, betaine and is typically dened as the total amount of encapsulant hydrogen peroxide, by choline oxydase. The generated found in the liposome solution versus the total initial input hydrogen peroxide causes oxidative coupling of phenol of encapsulant solution. This value depends not only on and 4-aminoantipyrine mediated by peroxidase to yield the ability of the liposomes to capture the encapsulant quinoneimine dye which is quantied at 505 nm.98 molecules (dependent on lipid/buffer composition, lipoThe latter method relies on hydrolysis of cholessome lamellarity, preparation procedure ) but also on the terol esters with cholesterol ester hydrolase, followed initial molar amount of encapsulant.93 by oxidation of the cholesterol by cholesterol oxidase Other authors dene the encapsulation efciency, or and subsequent production of hydrogen peroxide. This encapsulation capacity, as the molar amount of marker per product also oxidatively couples 4-aminoantipyrine to mole of lipid which is obtained by dividing the concentraphenol in the presence of peroxidase to yield a bluetion of encapsulant by the concentration of lipid. A similar coloured quinoneimine dye which shows strong absorption denition is suggested expressing EE on a weight (mg) at 505 nm.99 98 encapsulant per mM of lipid basis.22 Another commonly Chromatographic techniques such as HPLC, GC and used parameter is the captured volume, dened as L of thin layer chromatography (TLC) can be used to separate to: the lipids composing lipid bilayers.100 Chroentrapped volume/ mol of lipid. This numberDelivered ranges fromby Ingenta and quantify Guest User 0.5 L/nmol for SUV and MLV to 30 l/nmol for LUV. matographic approaches are advantageous since they can IP : 134.214.70.49 Unlike the percent encapsulation parameter cited previseparate and quantify each lipid in the mixture. TLC methTue, 13 phosNov 2012 ously, these representations require knowledge of the ods12:28:57 for phospholipid analysis often rely on lipid separapholipids concentration.94 95 tion using a mixture of chloroform, methanol and water. 4.5. Lipid Analysis Several chemistry techniques are commonly used for the determination of phospholipid content. Most of these techniques include the use of molybdate-containing reagents yielding a blue-colored product. One such method is the Bartlett assay which relies on the digestion of organic materials in liposome samples by 160 C sulfuric acid, oxidation to inorganic phosphates by hydrogen peroxide, phosphomolybdate formation upon interaction with ammonium molybdate, followed by reduction through interaction with 1,2,6-aminonaphtolsulfonic acid at 100 C. A blue product is formed which can then be analysed at 830 nm for the quantitative assessment of the phospholipids in the preparation.96 In the ascorbic acid method, ammonium molybdate reacts with orthophosphates formed from acid digestion to yield phosphomolybdic acid. This compound is then reduced with ascorbic acid to yield a blue-colored solution, analysed at 820 nm.96 Phospholipids can also be analyzed through complex formation with ammonium ferrothiocyante, extraction into chloroform, and absorbance measurement at 488 nm.96 A convenient and sensitive uorescence assay for phospholipid vesicles has also been reported by:97 when phospholipid vesicles are added to an aqueous solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) a uorescence enhancement of several hundred-fold is observed which can be used for a phospholipid concentration determination. The uorescence is a function of the type of phospholipid, salt concentration, and time of incubation.
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Detection is frequently accomplished using molybdenum blue in sulfuric acid and ninhydrin stains for the detection of phosphate and primary amino groups, respectively. For HPLC analysis, detection of lipids in the UV range is limited to 200210 nm due to their lack of chromophores. GC analysis of lipids typically requires a derivatization step to ensure sufcient volatility of the components, either through trimethyl silylation or methyl esterication prior to detection by ame ionization or mass spectroscopy. In many cases, pre-treatment of liposomes to disrupt the lipid bilayers is completed prior to chromatographic analysis including dilution of the liposome suspension with alcohols such as 2-propanol, ethanol or methanol. The procedure choice is dependent on the mobile phase and the degree of lipid solubility. 4.6. In-Vitro Drug Release In vitro drug release can be performed using the dialysis tube diffusion technique. The dialysis bag membrane should be selected following screening of various membrane, no drug adsorption may occur and the membrane should be freely permeable to the active ingredient (the cut off molecular weight shouldnt be a limiting step in the diffusion process). Some millilitres aliquot of liposome suspension is placed in the dialysis bag, hermetically tied and dropped in the receptor compartment containing the dissolution medium. The entire system is kept at 37 C under continuous magnetic stirring and the receptor medium is closed to avoid evaporation of the dissolution medium. The kinetic experiments are carried out respecting the sink 159
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conditions in the receptor compartment. Samples of the dialysate are taken at various time intervals and assayed for the drug by HPLC, spectrophotometer or any other convenient method. The sample volume is replaced with fresh dissolution medium so as the volume of the receptor compartment remains constant. Every kinetic experiment is performed in triplicate and the average values are taken to establish the release prole of the drug from the liposome suspension.101 102
5. LIPOSOMES APPLICATIONS
5.1. Pharmaceutical Applications The use of liposomes as systemic and topical drug delivery systems has attracted increasing attention. Liposomes can be formulated in liquid (suspension), solid (dry powder) or semi-solid (gel, cream) forms. In vivo, they can be administered topically or via parenteral route. 5.1.1. Systemic Liposomal Drugs
After systemic (usually intravenous) administration, liposomes are typically recognized as foreign particles and The liposomes stability is a major consideration for lipoconsequently endocytosed by the mononuclear phagocytic some production and administration steps: from process to system cells (MPS), mostly xed Kuppfer cells, in the storage and delivery. liver and spleen. Liposomes can serve as an excellent A stable pharmaceutical dosage form maintains its drug-delivery vehicle to these cells. Thus, sterically staphysical integrity and does not adversely inuence the bilized liposome, which are not avidly taken up by MPS chemical integrity of the active ingredient during its cells, have Delivered by Ingenta to: different biodistributions properties and have shown enhanced accumulation in sites of trauma, such as life. Researchers are attempting to deliver low andGuest high User tumours, infections and inammation. This accumulation molecular weight drugs in a variety of polymer IP :matri134.214.70.49 is simply due to their prolonged circulation and small size; ces and liposome suspensions. The successful introducTue, 13 Nov 2012 12:28:57 enabling them to extravasate.105 tion of dosage forms depends upon a well-dened stability Based on the liposome properties introduced above, sevstudy. In designing a stability study, physical, chemical eral techniques of drug delivery can be envisaged: and microbial parameters must be considered and evaluLiposomes can be applied to protect the entrapped drug ated. This wisdom is also required for the liposome dosage against enzymatic degradation whilst in circulation. The form. A stability study must include a section for product lipids used in their formulation are not susceptible to enzycharacterization and another section concerning the prodmatic degradation; the entrapped drug is thus protected uct stability during storage. while the lipid vesicles circulate within the extracelluAll liposome preparations are heterogeneous in size, the lar uid. As an example, -lactamase sensitive antibiaverage size distribution of liposomes changes upon their otics such as the penicillins and cephalosporins have storage. Liposomes tend to fuse and grow into bigger vesibeen encapsulated in order to be protected against the cles, which is a thermodynamically more favourable state. lactamase enzyme. Rowland et al. reported that liposomes Fusion and breakage of liposomes on storage also poses offer protection in the gastrointestinal tract environment of a critical problem leading to drug leakage from the vesiencapsulated drug and facilitate the gastrointestinal transcles. Therefore, visual appearance and size distribution are port of a variety of compounds.106 As clearly evidenced by important parameters to evaluate physical stability. Dapergolas, liposomes are candidates to be explored for In the other hand, the major ingredient in the lipooral delivery of peptides (insulin) and proteins (vaccines), some formulations is the lipid. The liposomes lipids are which are orally degradable.107 derived from natural and/or synthetic phospholipid sources Liposomes can be used for drug targeting. It has been containing unsaturated fatty acids which are known to proved that restricting the distribution of the drug to the undergo oxidative reactions. These reactions products can specic target site should allow efcacy increase at low cause permeability changes within liposome bilayer. In dose with attendant decrease of toxicity. Indeed, pumpaddition, interactions of drug with the phospholipid also ing a drug through the whole body is not only wasteful alter the chemical stability; hence the stability prole of a but, more fundamentally, increase undesirable side effects. drug molecule may entirely be different from its liposome Hence, the benets of drug targeting include reducing preparation stability prole. Thus, it is essential to develop drug waste, and it is possible to deliver a drug to a tisstability protocols evaluating the chemical integrity of the sue or cell region not normally accessible to the free or drug over a period of time. untargeted drug.108 Liposomes have been widely applied Finally, majority of therapeutic liposome formulations in drug targeting especially in cancer treatment. Effecare parenteral products and therefore must be sterilized tive chemotherapy is severely limited by the toxic side to remove the microbial contamination from the product. effects of the drugs. Liposome encapsulation can alter the Thus, it is important to control microbial stability of lipospatial and temporal distribution of the encapsulated drug somal preparations.103 104 molecules in the body, which may signicantly reduce 4.7. Liposomes Stability 160
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Table II. Liposome applications in the pharmaceutical eld. The intention of encapsulation Protection against enzymatic degradation Molecule Insulin Therapeutic class Hypoglycaemic agent
Conclusion
Reference (107)
Factor-VIII
Cysteamine
Drug targeting
Pencillins cephalosporins Cyclosporine
Amikacin
1.3 units of insulin entrapped in dipalmitoyl-phosphatidylcholine/ cholesterol liposomes administered to normal rats decreased blood glucose level in 4 h to about 77% of those before treatment. Higher doses (4.2 and 8.4 units) extended this effect over 24 h. 1.0 units of insulin entrapped in the same liposomes had an even more pronounced effect in diabetic rats: levels of blood-glucose were reduced to 57% of pre-treatment values after 4 h. Liposome-entrapped insulin signicantly reduced glucose and raised insulin in 54% of rats and 67% of the rabbits. Among the rats that responded, blood glucose fell from a basal of 318 21 mg/dl to a nadir of 186 22 mg/dl at 2 h. while insulin rose from 30 7 U/ml to a peak of 399 75 U/ml at 1 h. Coagulation factor Factor-VIII loaded liposomes were given orally to a patient with severe haemophilia A. Plasma concentration of factor-VIII rose to therapeutically effective levels. Factor-VIII activity did not rise when the free drug is orally administered. Oral administration of liposome-entrapped cysteamine induces an Treatment of acute increase in the concentration radiation syndrome Delivered by Ingenta to: of exogenous sulphur compounds in plasma, liver and spleen. This fact can be related to a protection ARS Guest User of cysteamine in the digestive tract. IP :The 134.214.70.49 Antibiotics entrapped drugs are protected against -lactamase enzyme Tue, 13 Nov 12:28:57 while2012 they are in circulation in the extracellular uid. Immunosuppressive The in vivo pharmacokinetics and renal toxicity of a liposomal formulation and the commercially available cyclosporine are compared. The apparent volume of distribution was signicantly greater in liposomal formulation compared to the commercially form (13.82 2.9 vs 7.67 3.01 L/Kg), most likely due to the signicantly prolonged biologic half-life (47.91 13.15 vs 30.95 8.89 h). Kidney function was assessed via the calculation of the glomerular ltration rate GFR, no dose-limiting nephrotoxicity was found with the liposomal formulation, suggesting a potential alternative to the toxic commercial formulation. A change in the pharmacokinetic parameters of cyclosporine due to liposomal encapsulation was observed. A peak concentration was reached in 50 min in case of liposomes compared with 225 min in case of commercially available formulation. The rate of absorption was signicantly faster following the liposome administration. Generally, there was less inter-individual variation in pharmacokinetics parameters when cyclosporine was orally administered in the liposomal formulation. Thus, an oral liposomal formulation can be developed to offer the advantages of low variability and fast onset of action. Antibiotic The clearance of liposomal amikacin was over 100-fold lower than the conventional amikacin clearance. Half-life in plasma was longer than that reported for other amikacin formulations. The levels in plasma remained > 180 mg/ml for 6 days after the administration of the last dose and the free amikacin concentration in plasma never exceeded 17.4 6 1 mg/ml. The low volume of distribution (45 ml/kg) indicates that the amikacin in plasma largely remained sequestered in long circulating liposomes. Less than half the amikacin was recovered in the urine, suggesting that the level of renal exposure to ltered free amikacin was reduced, possibly as a result of intracellular uptake or the metabolism of liposomal amikacin. Thus, by sequestering this antibiotic in liposomes with long circulation times, this formulation not only maintained antibiotic levels in the body for over 1 week after treatment, but also, signicantly alters the disposition of amikacin within the body and therefore decreases the potentially toxic level of renal tubular exposure.
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(127)
(128)
(109) (129)
(130)
(131)
Continued.
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Table II. Continued. The intention of encapsulation Molecule Doxorubicin Therapeutic class Antineoplastic Conclusion
Laouini et al.
Reference (110)
Enhancement of drug solubilisation
Amphotericin B
Enhancement of drug uptake
Polyvinylpyrrolidone
Vitamin K1
desferrioxamine
Vaccination
Inactivated hepatitis A virus
Doxorubicin has been shown to have a 4.5-times-lower medium-pathology score for doxorubicin-induced cardiotoxicity than the free drug. In squamous cell lung carcinoma, the same drug is capable of reducing tumor burden to a signicant extent Treatment of fungal Owing to its aqueous insolubility, amphotericin B is systemic infections typically formulated into detergent micelles. However, micelles are unstable upon systemic administration, and several neuro- and nephrotoxicity limit the dose that can be administered. If the drug is formulated into liposomes, it is delivered much more efciently to macrophages and, additionally, toxicity can be signicantly reduced. Blood plasma substitute At an equal concentration of 125 I-labelled polyvinylpyrrolidone, the rate of uptake of the liposome-entrapped macromolecule by the tissue was over 4-times that of the free macromolecule. Prevention or treatment The effect of liposomal -associated vitamin K1 , Delivered by Ingenta to: of hemorrhage administered orally, was investigated using rabbits Guest User with warfarin-induced hypoprothrombinaemia, and IP : 134.214.70.49 evaluated in comparison with other dosage forms of Tue, 13 Nov 2012 the12:28:57 vitamin. The coagulation recovery time of the liposomal preparation was much faster than that of the other preparations: This time was 6.2 h for the oral liposomal preparation, 13.6 h for solubilised vitamin and 19.6 h for stabilized emulsion. Chelating agent Liposomal desferrioxamine given intravenously to iron-overloaded 59 Fe labeled mice doubled the 59 Fe excretion for a given dose of the drug. Prevention of Hepatitis A Puried hepatitis-A-virion capsule, viral phospholipids and envelope glycoprotein from inuenza virus are mixed with phosphatidylcholine and phosphatidylethanolamine in the presence of excess of detergent. Liposomes containing antigen and some viral lipids and proteins are formed. The liposomes potentiate the immune response to the vaccine antigen.
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unwanted toxic side effects and increase the efcacy of the treatment. The rst step, therefore, is to determine the antigens that are produced by the tumour cells. Then to target the drug via specic receptor ligands, which may be specic antibodies for antigens produced by tumour cells.109 Two liposomal formulations have been approved by the US food and drug administration (FDA) and are commercially available in the USA, Europe and Japan for the treatment of Kaposis sarcoma. Doxil is a formulation of doxorubicin precipitated in sterically stabilized liposomes (on the market since 1995) and DaunoXome is daunorubicin encapsulated in small liposomes (on the market since 1990). Doxil has been shown to have a 4.5-times-lower medium-pathology score for doxorubicininduced cardiotoxicity than the free drug. In squamous cell lung carcinoma, the same drug is capable of reducing tumor burden to a signicant extent.110 162
In order to enhance solubilisation, the amphotericin B, which is the drug of choice in the treatment of systemic fungal infections, has been widely studied for liposome encapsulation. Owing to its aqueous insolubility, amphotericin B is typically formulated into detergent micelles. But, micelles are unstable upon systemic administration, and several neuro- and nephrotoxicity limit the dose that can be administered. However in a stable colloid particle, such as liposomes, encapsulated drug is delivered much more efciently to macrophages and, additionally, toxicity can be signicantly reduced.111 Following this rationale, a lipid-based amphotericin B formulation is actually commercially available in the Europe and US market (respectively since 1990 and 1997): AmBisome including amphotericin B into small liposomes. Otherwise, liposomes can also be used to enhance the drug intracellular uptake. The lipid formulation promotes
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the cellular penetration of the encapsulated drug especially Later, the liposomes ability of enclosing many differantibiotics, reducing the effective dose and incidence of ent biological materials and delivering them to the epidertoxicity. mal cells or even deeper cell layers was investigated. This According to the studies performed by Sullivan et al., offered new perspectives and leads to the conclusion that liposomes may be useful as immunotherapeutic agents: liposomes may be useful vehicles for topical drug delivery the use of antigen-presenting liposomes may be a promisfor varying skin diseases treatment. ing approach in the therapy of infectious diseases like Typically, conventional dosage forms, such as solution, HIV infection or Herpes simplex virus genital infection.112 creams, and ointments, deliver drugs in a concentration A liposomal vaccine against hepatitis A has been successdependant manner across the stratum corneum. Howfully launched by the Swiss Serum Institute in 1994. Puriever, multilamellar liposomes can deliver drugs within ed hepatitis-A-virion capsule, viral phospholipids and 30 minutes to the stratum corneum, epidermidis, and envelope glycoprotein from inuenza virus are mixed with dermis in signicantly higher concentrations than convenphosphatidylcholine and phosphatidylethanolamine in the tional preparations. presence of excess detergent forming liposomes leading Among the great variety of candidates for liposome to potentiate the immune response. The same company is encapsulation, there are mainly three groups of drugs to developing vaccines for inuenza, hepatitis B, diphtheria be considered: corticoids, retinoids and local anaesthetics. and tetanus.113 Mezei et al. was the rst to report increased cortiCationic liposomes have been shown to complex (negcosteroid concentrations in epidermis and corium comatively charged) DNA, and such complexes were able to bined with a reduced percutaneous absorption in an animal Delivered to: transfect cells in vitro, resulting in the expression of theby Ingenta model.116 This is particularly important as far as adverse Guest User protein encoded in the DNA plasmid in the target cells, effects from extensive corticosteroid therapy are reduced. IP :Obvi134.214.70.49 and making liposomes useful in direct gene transfer. These ndings were the same with human skin; Lasch Tue, 13 Nov 12:28:57 ously for gene therapy (the treatment of diseases on the 2012 investigated the effect of liposomally entrapped cortisol on molecular level by switching genes on or off), it was human skin ex vivo; he revealed improved cortisol condiscovered that cationic lipid-based DNA complexes can centration proles which is of real importance because transfect certain cells in vivo upon localized or systemic cortisol is known to have no adverse effects in long-term administration.13 therapy but to be often insufcient in the therapy of acute dermatoses.117 For this reason, higher drug concentrations Today, enthusiasm for the systemic use of liposomal will mean an improved therapeutic effect. In a clinical trial, drugs is not as widespread as it was. While the long Korting investigated the effect of betamethasone diprolist of diseases considered candidate for systemic applipionate in a liposomal preparation and in a commercial cation of liposomal drugs has been reduced to just a few conventional preparation in patient suffering from atopic indications, the topical application of liposomal preparaeczema.118 In this double-blind, randomized, paired trial tions has recently attracted more interest. the liposomal preparation, containing markedly less active substance was slightly superior in patients with atopic 5.1.2. Topical Liposomal Drugs eczema reducing parameters of inammation compared to the conventional preparation. Skin treatment applications of liposomes are based on the The retinoids is the second important group of drug similarity between the lipid vesicles bilayer structure and which seems to be a promising candidate for liposomal natural membranes which includes the ability of lipid vesiencapsulation. One main eld for the topical administracles, with specic lipid composition, to alter cell memtion of retinoids is uncomplicated acne vulgaris. Commerbrane uidity and to fuse with them. In the dermatological cial tretinoin gels shows local irritant effects and are-up eld, liposomes were initially used because of their mois114 reactions at the beginning of the treatment. These charturizing and restoring action. acteristics, often compromising patient compliance, can In Schmids work, stratum corneum liposomes have be overcome by the liposomal formulation of tretinoin. been used in the treatment of atopic dry skin in order to Masini et al. reported a reduced irritancy in animal experirestore the barrier function and to vehicle an active subments after treatment with liposomal tretinoin, which may stance at the same time.115 The composition and properties be explained by gradual drug release from the liposomal of liposomes play an important role in their interaction preparation.119 Schafer-Korting et al. performed a double and possible penetration within the epidermis. In addition, blind study to evaluate the efcacy and tolerability of liposomes provide valuable raw material for the regenerliposomal tretinoin in patients with uncomplicated acne ation of skin by replenishing lipid molecules and moisvulgaris.120 The results clearly showed that the less conture. Lipids are well hydrated and, even in the absence of centrated liposomal drug commands equal efcacy and active ingredients, humidify the skin. Often this is enough less skin irritation compared to its commercial convento improve skin elasticity and barrier function, which are tional counterparts. This equal efcacy might be attributed the main causes of skin aging.
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Table III. Liposome-based products on the market. Molecule Doxorubicin Treated disease Kaposis sarcoma and AIDS-related cancers. Ovarian cancer and multiple myeloma. Product DOXIL Company Ben Venue Laboratories for Johnson and Johnson, USA Status
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On the market since 1995 (USA) and 1996 (Europe)
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Amphotericin B
Systemic fungal infections
Daunorubicin
Inactivated hepatitis A virus
Specic types of leukaemia (acute myeloid leukemia and acute lymphocytic leukemia). Hepatitis A
Schering-Plough, Europe Enzon Pharmaceuticals for Cephalon, Europe and for Sopherion Therapeutics, USA and Canada AMBISOME First, NeXstar Pharmaceuticals which was acquired by Gilead Sciences in 1999. Thus, the drug is marketed by Gilead in Europe and licensed to Astellas Pharma (formerly Fujisawa Pharmaceuticals) for marketing in the USA, and Sumitomo Delivered by Ingenta to: Pharmaceuticals in Japan. Guest User FUNGISOME Lifecare Innovations, India IP : 134.214.70.49 AMPHOTEC Intermune, USA Tue, 13 Nov 2012Enzon 12:28:57 ABELCET Pharmaceuticals, USA AMPHOLIP Bharat Serums & Vaccines, India AMPHOCIL Samaritan pharmaceuticals, USA ABLC The liposome company, USA DAUNOXOME First, NeXstar Pharmaceuticals. Then the drug was sold to Diatos in 2006 EPAXAL Crucell Company who merged with the Swiss Serum and Vaccine Institute in 2006 Ferndale Laboratories, USA
CAELYX MYOCET
On the market since 1990 (Europe) and 1997 (USA)
On the market since 1996 (USA and Europe)
On the Swiss market since 1994 and now in more than 40 country. On the US market since 1998
Lidocaine
Anaesthesia for skin itching, burning or pain.
LMX4 LMX5
to an improved bioavailability combined with a slower drug release. In conclusion, the liposomally encapsulated tretinoin seems to be superior to the conventional dosage form. During the last few years, many attempts have been made to provide adequate local anaesthesia of the skin. For this, prolonged application time and high anaesthetic concentration are required. Studies performed by Gesztes et al. indicated that tetracaine encapsulated liposomes provide better local anaesthesia (low drug concentration and long anaesthesia duration) than a conventional anaesthetic cream. Similar results were obtained using other local anaesthetics such as lidocaine which is commercialized in the US market since 1998 (ELA-Max ).121 Furthermore, it has been commonly believed that high concentrations of ethanol are detrimental to liposomal formulations. Therefore, when liposomes are prepared from 164
ethanolic solutions of phospholipids much care is taken to remove the remaining traces of alcohol. Data presented by Touitou et al. indicated that the presence of ethanol with a relatively high concentration in systems of lipid vesicles, termed ethosomes, was reported to inuence the stratum corneum penetration and permeation of drugs.122 Encapsulation experiments showed that ethosomes are able to entrap both hydrophilic and lipophilic drugs. These entrapment results were supported by in vitro studies on the delivery of drugs in and through the skin. Again, the ethosomal system was shown to be far superior to the control systems, both in terms of the drug concentration in the skin and the ux of the drug through the skin. Patches containing testosterone in an ethosomal system were compared in vivo in rabbits with the commercial patch Testoderm . The results showed signicantly higher testosterone blood levels from the ethosomal
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Table IV. Some liposomal cosmetic formulations currently on the system. Horwitz et al. tested clinically the ethosomal carmarket. rier for dermal delivery of the antiviral drug, acyclovir in a 123 double-blind randomized study with Zovirax . Results Product Manufacturer Key ingredients indicated that ethosomes performed signicantly better Capture Christian Dior Liposomes in gel than the commercial drug form. For example, the average Efect du Soleil LOral Tanning agents in liposomes time to crusting of lesions was shorter for the ethosomal Future Perfect Este Lauder Vitamin E, A, cerebroside, Skin Gel ceramide system. The permeation enhancement from ethosomes Aquasome LA Nikko Chemical Co Liposomes with humectant suggests a synergetic mechanism between ethanol, lipid Eye Perfector Avon Soothing cream for eye vesicles and skin. Indeed, in comparison to liposomes, irritation ethosomes are less rigid. Thus, the effects of ethanol which Flawless Finish Elisabeth Arden Liquid make-up were considered to be harmful to classic liposomal formulations may provide the vesicles with soft exible characlasting perfumes, hair conditioners, aftershaves, lipsticks, teristics which allow them to penetrate more easily into make-up and similar products are also gaining large fracdeeper layers of skin. In another hand ethanol may disturbs tions of the market.138 Some of the liposomal cosmetic the organization of the stratum corneum lipid bilayer and formulations currently available in the market are shown enhances its lipid uidity. in Table IV. Liposomes based on a natural marine lipid extract containing a highly polyunsaturated fatty acid ratio were 5.3. Food Applications recently introduced as Marinosomes by Moussaoui et al. Delivered 124by Ingenta to: for the prevention and treatment of skin diseases. The majority of microencapsulation techniques currently Guest User Cansell et al. have reported that Marinosomes contributed used in the food industry are based on biopolymer matriIP : 134.214.70.49 to reduce inammation induced by croton oil by regulating ces12:28:57 composed of sugars, starches, gums, proteins, synTue, 13 Nov 2012 125 PGE2 and IL-8 production in keratinocyte cultures. thetics, dextrin and alginates. Nevertheless, liposomes have recently begun to gain in importance in food products.139 140 Indeed, the ability of liposomes to solu5.2. Cosmetic Applications bilise compounds with demanding solubility properties, The properties of liposomes can be utilized also in the sequester compounds from potentially harmful medium, delivery of ingredients in cosmetics. Liposomes offer and release incorporated molecules in a sustained and advantages because lipids are well hydrated and can reduce predictable way can be used in food processing industhe dryness of the skin which is a primary cause for try. Based on studies on liposomes for pharmaceutical ageing. Also, liposomes can supply replenish lipids and and medical uses, food scientists have begun to utilize importantly linolenic acid to the skin. The rst liposoliposomes for controlled delivery of functional compomal cosmetic product to appear on the market was the nents such as proteins, enzymes, vitamins, antioxidants, anti-ageing cream Capture launched by Christian Dior and avours. The applications are for example dairy in 1986.135 Liposomes have been also used in the treatproducts preparation, stabilization of food components ment of hair loss; minoxidil, a vasodilator, is in the active against degradation, and delivery and enhanced efciency ingredient in products like Regaine that claim to preof antimicrobial peptides. vent or slow hair loss.136 The skin care preparations with The sustained release system concept can be used in empty or moisture loaded liposome reduce the transdermal various fermentation processes in which the encapsuwater loss and are suitable for the treatment of dry skin. lated enzymes can greatly shorten fermentation times and They also enhance the supply of lipids and water to the improve the quality of the product. A classical example stratum corneum.137 Various liposome formulations were is cheese-making; after preliminary studies in which lipocompared in vivo for cosmetics application;114 liposome some systems were optimized the cheese ripening times formulations prepared from egg phospholipids exhibited a were shorten by 30 to 50%. This means a substantial eco1.5-fold increase in skin water content, whereas liposome nomic prot knowing that ripening times of some cheeses, formulations prepared from soya phospholipids showed no such as Cheddar, are about one year during which they advantage compared to the references. Skin water conrequire well controlled conditions.141 tent was measured daily and the results showed that skin In addition to improved fermentation, liposomes were humidity was increased signicantly for the formulation tried in the preservation of cheeses. Addition of nitrates to containing 20% egg phospholipids during 6 days. cheese milk to suppress the growth of spore-forming bacSince 1987, several cosmetic products have been comteria is questioned due to health concerns and natural altermercially available; they range from simple liposome natives are under study. Lysozyme is effective but quickly pastes which are used as a replacement for creams, gels inactivated due to binding to casein. Liposome encapsulaand ointments to formulations containing various extracts, tion can both preserve potency and increase effectiveness moisturizers, antibiotics, etc. Unrinsable sunscreens, long because liposomes become localized in the water spaces
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between the casein matrix and fat globules of curd and cheese.142 These applications of enhancing natural preservatives, including antioxidants such as vitamin E and C, will undoubtedly become very important due to recent dietary trends which tend to reduce the addition of articial preservatives and increase portion of unsaturated fats in the diet. In other areas of the agro-food industry, biocides encapsulated into liposomes have shown superior action due to prolonged presence of fungicides, herbicides, or pesticides at reduced damage to other life forms. Liposome surface can be made sticky so that they remain on the leafs for longer times and they do not wash into the ground.143
6. CONCLUSION
Since they have been discovered in the 60s by Bangham, liposomes have drawn attention of researchers. Nowadays, they always remain a topical issue; new preparation methReferences and Notes Delivered by Ingenta to: ods have been developed as well as new characterization Guest User 1. T. Lian and R. J. Ho, J. Pharm. Sci. 90, 667 (2001). techniques. 2. R. Banerjee, J. Biomater. Appl. 16, 14 (2001). IP : 134.214.70.49 In the pharmaceutical eld, liposomes have long been B. Keller, Trends Food Sci. Technol. 12, 25 (2001). Tue, Nov 12:28:57 of great interest by offering a promising way for 13 both sys- 20123. 4. N. Maurer, D. B. Fenske, and P. R. Cullis, Expert Opin. Biol. Ther. temic and locally acting drugs used for therapeutic appli1, 923 (2001). cations in humans and animals. As a result of the great 5. A. Samad, Y. Sultana, and M. Aqil, Curr. Drug Deliv. 4, 297 (2007). potential of liposomes in the area of drug delivery, several 6. J. A. Rogers and K. E. Anderson, Crit. Rev. Ther. Drug Carrier companies have been actively engaged in expansion and Syst. 15, 421 (1998). evaluation of liposome products. Most of them concern 7. S. M. Michalek, N. K. Childers, J. Katz, F. R. Denys, A. K. Berry, anticancer and antifungal drugs that, administered in their J. H. Eldridge, J. R. McGhee, and R. Curtiss, Curr. Top Microbiol. free form, are toxic or exhibit serious side-effects and their Immunol. 146, 51 (1989). 8. K. Egbaria and N. Weiner, Adv. Drug Deliv. Rev. 5, 287 (1990). encapsulation into liposomal vesicles signicantly dimin9. I. Kellaway and S. Farr, Adv. Drug Deliv. Rev. 5, 149 (1990). ishes these unwanted properties. However, there are few 10. H. Schreier, R. Gonzalez-Rothib, and A. Stecenkoc, J. Control Rel. commercially available pharmaceutical products based on 24, 209 (1993). drug-in-liposome formulations. Liposome based formula11. S. Ebrahim, G. Peyman, and P. Lee, Surv. ophthalmol. 50, 167 tion have not entered the market in great numbers because (2005). 12. A. D. Bangham, J. C. Glover, S. Hollingshead, and B. A. Pethica, of some problems limiting their development. Biochem. J. 84, 513 (1962). Even that batch to batch reproducibility, low drug 13. D. D. Lasic, Trends Biotechnol. 16, 307 (1998). entrapment, particle size control, and short circulation 14. D. Lasic and D. Papadjopoulos, Science 267, 1275 (1995). half-life of vesicles seem to have been resolved, some 15. J. Israelachvili, S. Marcelja, and R. Horn, Rev. Biophys. 13, 121 other problems are still limiting the widespread use of (1980). 16. A. D. Bangham, M. M. Standish, and G. Weissmann, J. Mol. Biol. liposomes, among them the stability issues, sterilization 13, 253 (1965). method and production of large batch sizes. 17. A. Sharma and U. Sharma, Int. J. Pharm. 154, 123 (1997). Some of the stability problems may be overcome by 18. F. Bordi C. Cametti, and S. Sennato, Advances in Planar lyophilisation. The nal product is freeze-dried liposome Lipid Bilayers and Liposomes edited by Academic Press (2006), mixed with a suitable cryoprotectant that are particularly pp 281320. 19. Y. Ebato, Y. Kato, H. Onishi, T. Nagai, and Y. Machida, Drug stable and have to be reconstituted immediately prior to Develop. Res. 58, 253 (2003). administration. 20. G. J. Grant, Y. Barenholz, E. M. Bolotin, M. Bansinath, Another challenge is the identication of a suitable H. Turndorf, B. Piskoun, and E. M. Davidson, Anesthesiology method for sterilization of liposome formulations as phos101, 133 (2004). pholipids are thermolabile and sensitive substance to 21. K. Katayama, Y. Kato, H. Onishi, T. Nagai, and Y. Machida, Drug Dev. Ind. Pharm. 29, 725 (2003). procedures involving the use of heat, radiation and/or 22. S. Vemuri and C. T. Rhodes, Pharm. Acta Helv. 70, 95 (1995). chemical sterilizing agents. The alternative technique of 23. M. R. Mozafari, Cell Mol. Biol. Lett. 10, 711 (2005). liposome sterilization is ltration through sterile mem24. A. Bangham, J. De Gier, and G. Greville, Chem. Phys. Lipids 1, 225 branes (0.22 m). However, this method is limited by (1967). liposome size and is not suitable for large vesicles 25. F. Olson, C. A. Hunt, F. C. Szoka, W. J. Vail, and (>0 22 m). D. Papahadjopoulos, Biochim. Biophys. Acta 557, 9 (1979). 166
Finally, the major challenge for liposome is the large scale production method. Pharmaceutically acceptable procedures are those that can be easily scaled to larger batch sizes and economically feasible. However, unlike the classical pharmaceutical dosage forms (tablets, capsules, suppository ) which are produced in large batch sizes, liposome based drugs even those already in the market are produced in small size batches and thus are costly for the manufacturers. Scale-up process to larger size batches is often a monumental task for the process development scientists. However the accumulation of many novel experiences studying the practical aspects of liposomes, added to new developments in basic research, will bring the eld of liposome biotechnology to the place it deserves in the future. An encouraging sign is the increasing number of clinical trials involving liposomes.
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61. S. Mukherjee, H. Raghuraman, S. Dasgupta, and A. Chattopadhyay, 26. B. Mui, L. Chow, and M. J. Hope, Methods Enzymol. 367, 3 (2003). Chem. Phys. Lipids. 127, 91 (2004). 27. F. J. Szoka and D. Papahadjopoulos, Proc. Natl. Acad. Sci. USA 62. K. Matsuzaki, O. Murase, K. Sugishita, S. Yoneyama, K. Akada, 75, 4194 (1978). M. Ueha, A. Nakamura, and S. Kobayashi, Biochim. Biophys. Acta 28. J. Szebeni, J. H. Breuer, J. G. Szelenyi, G. Bathori, G. Lelkes, and 1467, 219 (2000). S, R. Hollan, Biochim. Biophys. Acta 798, 60 (1984). 63. A. Chattopadhyay, Chem. Phys. Lipids. 53, 1 (1990). 29. S. Batzri and E. D. Korn, Biochim. Biophys. Acta 298, 1015 (1973). 64. H. J. Gruber and H. Schindler, Biochim. Biophys. Acta 1189, 212 30. P. Stano, S. Bufali, C. Pisano, F. Bucci, M. Barbarino, (1994). M. Santaniello, P. Carminati, and P. L. Luisi, J. Liposome Res. 65. L. D. Mayer, M. J. Hope, and P. R. Cullis, Biochim. Biophys. Acta 14, 87 (2004). 858, 161 (1986). 31. D. W. Deamer, Ann. NY Acad. Sci. 308, 250 (1978). 66. M. Frhlich, V. Brecht, and R. Peschka-Sss, Chem. Phys. Lipids 32. O. Zumbuehl and H. G. Weder, Biochim. Biophys. Acta 640, 252 109, 103 (2001). (1981). 67. M. Mller, S. Mackeben, and C. C. Mller-Goymann, Int. J. Pharm. 33. D. Papahadjopoulos, S. Nir, and N. Dzgnes, J. Bioenerg. 274, 139 (2004). Biomembr. 22, 157 (1990). 68. H. Jousma, H. Talsma Spies, F. Joosten JGH, and Junginger, HE, 34. E. Cauchetier, H. Fessi, Y. Boulard, M. Deniau, A. Astier, and Crommelin, DJA, Int. J. Pharm. 35, 263 (1987). M. Paul, Drug Dev. Res. 47, 155 (1999). 69. M. Hope, M. Bally, L. Mayer, A. Janoff, and P. Cullis, Chem. Phys. 35. T. Nii and F. Ishii, Int. J. Pharm. 298, 198 (2005). Lipids. 40, 89 (1986). 36. H. Cheung Shum, D. Lee, I. Yoon, T. Kodger, and D. Weitz, Lang70. H. Hauser, Biochim. Biophys. Acta 772, 37 (1984). muir 24, 7651 (2008). 71. A. F. Palmer, P. Wingert, and J. Nickels, Biophys. J. 85, 1233 37. M. R. Mozafari and A. Omri, J. Pharm. Sci. 96, 1955 (2007). (2003). 38. N. Skalko-Basnet, Z. Pavelic, and M. Becirevic-Lacan, Drug Dev. 72. J. Jass, T. Tjrnhage, and G. Puu, Methods Enzymol. 367, 199 Ind. Pharm. 26, 1279 (2000). (2003) . 39. C. Li and Y. Deng, J. Pharm. Sci. 93, 1403 (2004)Delivered . by Ingenta to: 73. P. M. Frederik, and D. H. W. Hubert, Methods Enzymol. 391, 431 40. J. Cui, C. Li, Y. Deng, Y. Wang, and W. Wang, Int. J. Pharm. Guest User (2005). 312, 131 (2006). IP : 74. B. Ruozi, G. Tosi, F. Forni, M. Fresta, and M. A. Vandelli, Eur. J. 41. K. Otake, T. Imura, H. Sakai, and M. Abe, Langmuir 17, 134.214.70.49 3898 Tue, 13 Nov 2012 12:28:57 Pharm. Sci. 25, 81 (2005). (2001). 42. K. Otake, T. Shimomura, T. Goto, T. Imura, T. Furuya, S. Yoda, 75. P. Lundahl, C. M. Zeng, C. Lagerquist Hgglund, I. Gottschalk, and Y. Takebayashi, H. Sakai, and M. Abe, Langmuir 22, 2543 (2006). E. Greijer, J. Chromatogr. B Biomed. Sci. Appl. 722, 103 (1999). 43. T. Imura, K. Otake, S. Hashimoto, T. Gotoh, M. Yuasa, 76. S. Lessieur, C. Gabrielle-Madelmont, M. Paternostre, and S. Yokoyama, et al., Colloid Surface B 27, 133 (2002). M. Ollivon, Chem. Phys. Lipids. 64, 57 (1993). 44. A. Jahn, W. N. Vreeland, D. L. DeVoe, L. E. Locascio, and 77. S. Lessieur, C. Gabrielle-Madelmont, M. Paternostre, and M. Gaitan, Langmuir 23, 6289 (2007). M. Ollivon, Anal. Biochem. 192, 334 (1991). 45. A. Jahn, W. N. Vreeland, M. Gaitan, and L. E. Locascio, J. Am. 78. M. H. Moon, I. Park, and Y. Kim, J. Chromatogr. A. 813, 91 (1998). Chem. Soc. 126, 2674 (2004). 79. B. A. Korgel, J. H. Van Zanten, and H. G. Monbouquette, Biophys. 46. P. Pradhan, J. Guan, D. Lu, P. G. Wang, L. J. Lee, and R. J. Lee, J. 74, 3264 (1998). Anticancer Res. 28, 943 (2008). 80. L. Gimbert, K. Andrew, P. Haygarth, and P. Worsfold, Trends Anal. 47. A. Wagner, K. Vorauer-Uhl, and H. Katinger, Eur. J. Pharm. BioChem. 22, 615 (2003). pharm. 54, 213 (2002c). 81. F. Yang, K. Caldwell, and J. Giddings, J. Colloid Interf. Sci. 92, 81 48. A. Wagner, K. Vorauer-Uhl, G. Kreismayr, and H. Katinger, J. Lipo(1983). some Res. 12, 271 (2002b). 82. T. Provder, Prog. Org. Coat. 32, 143 (1997). 49. A. Wagner, K. Vorauer-Uhl, G. Kreismayr, and H. Katinger, 83. S. Klchens, V. Ramaswami, J. Birgenheier, L. Nett, and J. Liposome Res. 12, 259 (2002a). D. F. OBrien, Chem. Phys. Lipids. 65, 1 (1993). 50. A. Wagner, M. Platzgummer, G. Kreismayr, H. Quendler, 84. N. Berger, A. Sachse, J. Bender, R. Schubert, and M. Brandl, Int. G. Stiegler, B. Ferko, G. Vecera, K. Vorauer-Uhl, and H. Katinger, J. Pharm. 223, 55 (2001). J. Liposome Res. 16, 311 (2006). 85. P. Schuck, Biophys. J. 78, 1096 (2000). 51. C. Jaafar-Maalej, C. Charcosset, and H. Fessi, J. Lip. Res. 21, 1 86. S. Fitzpatrick, US patent 5786898 (1998). (2011). 87. J. Lyklema and G. Fleer, Colloid Surface 25, 357 (1987). 52. A. R. Mohammed, N. Weston, A. G. A. Coombes, M. Fitzgerald, 88. R. Hunter and H. Midmore, J. Colloid Interf. Sci. 237, 147 (2001). and Y. Perrie, Int. J. Pharm. 285, 23 (2004). 89. M. N. Padamwar and V. B. Pokharkar, Int. J. Pharm. 320, 37 (2006). 53. R. M. Hathout, S. Mansour, N. D. Mortada, and A. S. Guinedi, 90. A. Laouini, C. Jaafar-Maalej, S. Sfar, C. Charcosset, and H. Fessi, AAPS. Pharm. Sci. Technol. 8, 1 (2007). Int. J. Pharm. 415, 53 (2011). 54. M. Fan, S. Xu, S. Xia, and X. Zhang, J. Agric. Food Chem. 55, 167 91. W. Sun, N. Zhang, A. Li, W. Zou, and W. Xu, Int. J. Pharm. (2007). 353, 243 (2008). 55. M. Holzer, S. Barnert, J. Momm, and R. Schubert, J. Chromatogr. 92. X. Wang, L. Cai, X. Zhang, L. Deng, H. Zheng, C. Deng, J. A. 1216, 5838 (2009). Wen, X. Zhao, Y. Wei, and L. Chen, Int. J. Pharm. 410, 169 56. C. Dai, B. Wang, H. Zhao, B. Li, and J. Wang, Colloids Surf. B (2011). Biointer. 47, 205 (2006). 93. D. R. Arin and A. F. Palmer, Biotechnol. Prog. 19, 1798 (2003). 57. S. Moazam Mortazavia, M. Reza Mohammadabadib, K. Khosravi-Daranic, and M. Reza Mozafari, J. Biotechnol. 94. G. Ramaldes, J. Deverre, J. Grognet, F. Puisieux, and E. Fattal, Int. 129, 604 (2007). J. Pharm. 143, 1 (1996). 58. K. A. Edwards and A. J. Baeumner, Talanta 68, 1432 (2006). 95. W. Perkins, S. Minchey, and P. Ahl, Chem. Phys. Lipids. 64, 197 59. T. Morol, A. Subramanian, and W. Velander, J. Immunol. Methods (1993). 203, 45 (1997). 96. J. C. Stewart, Anal. Biochem. 104, 10 (1980). 60. E. Brekkan, L. Lu, and P. Lundahl, J. Chromatogr. A. 711, 33 97. E. London and G. W. Feligenson, Anal. Biochem. 88, 203 (1995). (1978).
REVIEW
167
Laouini et al.
123. E. Horwitz, S. Pisanty, R. Czerninski, M. Helser, E. Eliav, and 98. M. Takayama, S. Itoh, T. Nagasaki, and I. Tanimizu, Clin. Chim. E. Touitou, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. Acta 79, 93 (1977). 87, 700 (1999). 99. H. Kobayashi, M. Yoshida, and K. Miyashita, Chem. Phys. Lipids 124. N. Moussaoui, M. Cansell, and A. Denizot, Int. J. Pharm. 242, 361 126, 111 (2003). (2002). 100. R. Singh, M. Ajagbe, S. Bhamidipati, Z. Ahmad, and I. Ahmad, 125. M. S. Cansell, N. Moussaoui, and M. Mancini, Int. J. Pharm. J. Chromatogr. A 1073, 347 (2005). 343, 277 (2007). 101. L. H. Reddy, K. Vivek, N. Bakshi, and R. S. R. Murthy, Pharm. 126. J. F. Arrieta-Molero, K. Aleck, M. K. Sinha, C. M. Brownscheidle, Dev. Technol. 11, 167 (2006). L. J. Shapiro, and M. A. Sperling, Horm. Res. 16, 249 (1982). 102. N. Ammoury, H. Fessi, J. P. Devissaguet, F. Puisieux, and S. Benita, 127. H. C. Hemker, W. T. Hermens, A. D. Muller, and R. F. Zwaal, J. Pharm. Sci. 79, 763 (1990). Lancet 1, 70 (1980). 103. J. Plessis, C. Ramachandran, N. Weiner, and D. Mller, Int. J. 128. D. Jaskierowicz, F. Genissel, V. Roman, F. Berleur, and M. Fatome, Pharm. 127, 273 (1996). Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 47, 615 (1985). 104. E. Casals, A. M. Galn, G. Escolar, M. Gallardo, and J. Estelrich, 129. K. Vadiei, R. Perez-Soler, G. Lopez-Berestein, and D. Luke, Int. J. Chem. Phys. Lipids 125, 139 (2003). Pharm. 57, 125 (1989). 105. R. K. Jain, Adv. Drug Deliv. Rev. 46, 149 (2001). 106. R. N. Rowland and J. F. Woodley, Biochim. Biophys. Acta 620, 400 130. M. Al-Meshal, S. Khidr, M. Bayomi, and A. Al-Angary, Int. J. (1980). Pharm. 168, 163 (1998). 107. G. Dapergolas and G. Gregoriadis, Lancet 2, 824 (1976). 131. R. M. Fielding, L. Moon-McDermott, R. O. Lewis, and M. J. 108. R. M. Fielding, Clin. Pharmacokinet 21, 155 (1991). Horner, Antimicrob. Agents Chemother. 43, 503 (1999). 109. M. Uhumwangho and R. Okor, J. Med. Biomed. Res. 4, 9 (2005). 132. R. N. Rowland and J. F. Woodley, Biochim. Biophys. Acta 673, 217 110. D. D. Lasic, Nature 380, 561 (1996). (1981). 111. J. Adler-moore and R. Proft, J. lip. Res. 3, 429 (1993). 133. M. Nagata, T. Yotsuyanagi, M. Nonomura, and K. Ikeda, J. Pharm. 112. S. M. Sullivan, R. K. Gieseler, S. Lenzner, J. Ruppert, T. G. Pharmacol. 36, 527 (1984). Delivered by Ingenta to: Gabrysiak, J. H. Peters, G. Cox, L. Richer, W. J. Martin, and M. J. 134. S. P. Young, E. Baker, and E. R. Huehns, Br. J. Haematol. 41, 357 Guest User Scolaro, Antisense Res. Dev. 2, 187 (1992). (1979). IP : S. 134.214.70.49 113. R. Glck, R. Mischler, B. Finkel, J. U. Que, B. Scarpa, and J. J. 135. C. C. Miiller-Goymann, European Journal of Pharmaceutics and Cryz, Lancet 344, 160 (1994). Biopharmaceutics 58, 343 (2004). Tue, 13 Nov 2012 12:28:57 136. H. Lautenschlger, Liposomes Handbook of Cosmetic Science and 114. G. Betz, A. Aeppli, N. Menshutina, and H. Leuenberger, Int. J. Technology, Taylor and Francis Group, CRC Press, Boca Raton Pharm. 296, 44 (2005). (2006), pp. 155163. 115. M. Schmid and H. Korting, Adv. Drug Deliv. Rev. 6, 335 (199). 137. V. B. Patravale and S. D. Mandawgade, International Journal of 116. M. Mezei and V. Gulasekharam, Life Sci. 26, 1473 (1980). Cosmetic Science 30, 19 (2008). 117. J. Lasch and W. Wohlrab, Biomed. Biochim. Acta 45, 1295 (1986). 138. A. Sharma, S. Kumar, and M. N. Mahadevan, IJRAPR 2, 54 (2012). 118. H. C. Korting, H. Zienicke, M. Schfer-Korting, and O. Braun139. T. M. Taylor, P. M. Davidson, B. D. Bruce, and J. Weiss, Crit. Rev. Falco, Eur. J. Clin. Pharmacol. 39, 349 (1990). Food Sci. Nutr. 45, 587 (2005). 119. V. Masini, F. Bonte, A. Meybeck, and J. Wepierre, J. Pharm. Sci. 140. M. R. Mozafari, C. Johnson, S. Hatziantoniou, and C. Demetzos, 82, 17 (1993). J. Liposome Res. 18, 309 (2008). 120. M. Schfer-Korting, H. C. Korting, and E. Ponce-Pschl, Clin. 141. B. A. Law and J. S. King, J. Diary Res. 52, 183 (1991). Investig 72, 1086 (1994). 142. C. Kirby, Delivery Systems for Enzymes. Chem. Br. 26, 847 (1990). 121. A. Gesztes and M. Mezei, Anesth. Analg. 67, 1079 (1988). 143. A. Tahibi, J. D. Sakurai, R. Mathur, and D. F. H. Wallach, Proc. 122. E. Touitou, N. Dayan, L. Bergelson, B. Godin, and M. Eliaz, Symp. Contr. Rel. Bioact. Mat. 18, 231 (1991). J. Control Release 65, 403 (2000).
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Received: 21 March 2012. Accepted: 30 June 2012.
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Copyright 2012 American Scientic Publishers All rights reserved Printed in the United States of America

Journal of Colloid Science and Biotechnology Vol. 1, 147168, 2012

Preparation, Characterization and Applications of Liposomes: State of the Art

Keywords: Liposomes, Preparation Methods, Characterization, Phospholipids, Therapeutic

Author to whom correspondence should be addressed.

J. Colloid Sci. Biotechnol. 2012, Vol. 1, No. 2

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Fig. 2. Liposomes classication based on size and lamellarity.

J. Colloid Sci. Biotechnol. 1, 147168, 2012

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

3.2.2. New Large-Scale Liposome Technique

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Effects on liposome characteristics (size, zeta a,d e,capsulation efficiency)

Reverse -phase evaporation Conventionnel techniques

Egg yolk phosphatidylcholine cholesterol

Emulsion (double emulsification process)

Bovine serum albumin (BSA)

Egg phosphatidylcholine (PC), Triolein (TO), cholesterol (Chol)

Sterile phosphate buffered saline glycerol (final concentration 3%, v/v)

*Serum stability study

Sucrose Tert-butyl alcohol/ Water

J. Colloid Sci. Biotechnol. 1, 147168, 2012

Preparation, Characterization and Applications of Liposomes: State of the Art

Supercritical reversephase evaporation method

L-RDipalmitoylphosp hatidylcholine (DPPC

Egg phosphatidylcholine or hydrogenated soy phosphatidylcholine Cholesterol (2.5:1 w/w)

Ethanol or ethanol/tertbutanol (1:1 v/v)

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

Pencillins cephalosporins Cyclosporine

J. Colloid Sci. Biotechnol. 1, 147168, 2012

Preparation, Characterization and Applications of Liposomes: State of the Art

Enhancement of drug solubilisation

Enhancement of drug uptake

Inactivated hepatitis A virus

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

On the market since 1995 (USA) and 1996 (Europe)

Systemic fungal infections

Inactivated hepatitis A virus

On the market since 1990 (Europe) and 1997 (USA)

On the market since 1996 (USA and Europe)

Anaesthesia for skin itching, burning or pain.

Preparation, Characterization and Applications of Liposomes: State of the Art

J. Colloid Sci. Biotechnol. 1, 147168, 2012

Preparation, Characterization and Applications of Liposomes: State of the Art

Preparation, Characterization and Applications of Liposomes: State of the Art

J. Colloid Sci. Biotechnol. 1, 147168, 2012

Preparation, Characterization and Applications of Liposomes: State of the Art

Received: 21 March 2012. Accepted: 30 June 2012.

J. Colloid Sci. Biotechnol. 1, 147168, 2012

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