Preparation, Characterization and Applications of Liposomes: State of the Art Laouini Et Al

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

A. Laouini1, C. Jaafar-Maalej1, I. Limayem-Blouza2, S. Sfar2, C. Charcosset1 ∗, and H. Fessi1

1Laboratoire d’Automatique et de Génie des Procédés (LAGEP), UMR-CNRS 5007, Université Claude Bernard Lyon 1, CPE Lyon, Bat 308 G, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France 2Laboratoire de Pharmacie Galénique, Faculté 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 fields. This bibliographic paper offers a general review on the backgroundDelivered and development by Ingenta of liposomes to: with a focus on preparation methods including classic (thin film hydration, reverse-phaseGuest User evaporation, ethanol injection…) and novel scalable techniques. Furthermore, liposomeIP : 134.214.70.49 characterization techniques including mean size, zeta- potential, lamellarity, encapsulationTue, efficiency, 13 Novin 2012 vitro drug 12:28:57 release, vesicles stability and lipid analysis synthesized from different published works are reported. The current deepening and widening of liposome interest in many scientific 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 opti- mism 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

1. Introduction ...... 147 Liposomes, defined as microscopic spherical-shaped 2. Liposomes ...... 149 vesicles, consist of an internal aqueous compartment 2.1. Definition ...... 149 entrapped by one or multiple concentric lipidic bilayers. 2.2. Classification ...... 149 3. Liposomes Preparation Procedures ...... 151 Liposomes membrane is composed of natural and/or syn- 3.1. General Ingredients ...... 151 thetic lipids which are relatively biocompatible, biodegrad- 3.2. Preparation Method ...... 151 able and non-immunogenic material. Because of their 4. In-Vitro Liposomes Characterization ...... 153 unique bilayer-structure properties, liposomes are used as 4.1. Lamellarity Determination ...... 153 carriers for both lipophilic and water-soluble molecules. 4.2. Size Analysis ...... 156 4.3. Zeta Potential ...... 158 Hydrophilic substances are encapsulated in the inte- 4.4. Encapsulation Efficiency ...... 158 rior aqueous compartments. Lipophilic drugs are mainly 4.5. Lipid Analysis ...... 159 entrapped within lipid bilayers. 4.6. In-Vitro Drug Release ...... 159 As asserted by different authors, liposomes have attrac- 4.7. Liposomes Stability ...... 160 tive biological properties, including the biocompatibility 5. Liposomes Applications ...... 160 and biodegradability. They show promise as active vec- 5.1. Pharmaceutical Applications ...... 160 5.2. Cosmetic Applications ...... 165 tors due to their capacity to enhance the encapsulant 5.3. Food Applications ...... 165 performance by increasing drug solubility, and stability; 6. Conclusion ...... 166 delivering encapsulated drugs to specific target sites, and References and Notes ...... 166 providing sustained drug release.1 Their sub-cellular size allows relatively higher intracellular uptake than other par- ∗Author to whom correspondence should be addressed. ticulate systems; improving in vivo drug bioavailability.

J. Colloid Sci. Biotechnol. 2012, Vol. 1, No. 2 2164-9634/2012/1/147/022 doi:10.1166/jcsb.2012.1020 147 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

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, nano- emulsions) by the use of membranes which allows reproducible production process with less energy consumption. REVIEW

C. Jaafar-Maalej after obtaining a Master degree in Biomedical engineering at the Univer- sity 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 d’Automatique et de Génie des Procédés” (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. Delivered by Ingenta to: Guest User IP : 134.214.70.49 Tue, 13 Nov 2012 12:28:57 I. Limayem-Blouza after obtaining a Master degree in Biomedical engineering at the Uni- versity of Lyon 1, France, Iméne 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).

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 Compiègne (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, first in the “Laboratoire des Sciences du Génie Chimique” (LSGC, Nancy, France), from 1992 to 1994; then in “Laboratoire d’Automatique et de Génie des Procédés” (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, ultrafiltration and microfiltra- tion, preparation of emulsions and particles using membranes, and membrane crystallization for biotechnological, pharmaceutical and environmental applications.

148 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art

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 scientiﬁc committee of the French Medical Agency. Head of the joint research unit between the University of Lyon1 and the National Center of Sci-

entiﬁc Research (CNRS) (Chemical Engineering and Automated Process, LAGEP, UMR REVIEW 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 efficiency inspite of drug solubility, low toxicity due to effect which organizes amphiphilic molecules (phospho- phospholipids content, drug protection against degrada- lipids) so as to minimize entropically unfavorable inter- tion factors like pH and light and the reduction of tissue actions between hydrophobic acyl-chains and surrounding irritation. Delivered by Ingentaaqueous to: medium.13 14 This effect is further settled by vari- Liposomes have been extensively studied as drug carri-Guest Userous intermolecular forces such as electrostatic interactions, ers in the pharmaceutical and medical fields.1 2 IPResearch : 134.214.70.49hydrogen bonding, as well as Vanderwaals and dispersion has expanded considerably over the last 30Tue, years, 13 increas- Nov 2012forces. 12:28:5715 ing applications area from drug and gene delivery to diag- Liposomes were defined as an artificial microscopic nostics, cosmetics, long-lasting immune-contraception to vesicle consisting of a central aqueous compartment sur- 3 food and chemical industry. The superiority of liposomes rounded by one or more concentric phospholipid lay- as drug carriers has been widely recognized. Ten liposo- ers (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 4 preclinical development or in clinical trials. within these vesicles developing large potential applica- Several reviews about liposomes as drug delivery 5 6 7 8 tions. Numerous researchers have worked with these struc- systems and specific application via oral, topical, tures since Bangham’s discovery, making of liposomes the pulmonary,9 10 and ophthalmic11 route have been pub- most 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. Classification technology with respect to numerous multidisciplinary applications. As a contribution for updating the state of Liposomes can be classified in terms of composition and knowledge, a focuses on liposomes preparation method mechanism of intracellular delivery into five types17 and recent characterization techniques including mean (i) conventional liposomes; size, zeta-potential, lamellarity, encapsulating efficiency, in (ii) pH-sensitive liposomes; vitro active release, stability and lipid analysis have been (iii) cationic liposomes; described as well as the most significant achievements and (iv) immunoliposomes and applications. (v) long-circulating liposomes. Otherwise, vesicle size is a critical parameter in determin- 2. LIPOSOMES ing circulation half-life of liposomes, and both size and number of bilayers influence the extent of drug encapsu- 2.1. Definition lation within liposomes. Thus, liposomes were typically Liposomes were first produced in England in the 60’s, classified on the basis of their size and number of bilayers by Bangham who was studying phospholipids and blood into (Fig. 2): clotting.12 According to legend, he was experimenting with (i) Small unilamellar vesicles (SUV): 20–100 nm; new laboratory equipment, and he made a noted obser- (ii) Large unilamellar vesicles (LUV): > 100 nm; vation about phospholipids forming closed multilamellar (iii) Giant unilamellar vesicles (GUV): > 1000 nm; vesicle spontaneously in aqueous solution which took two (iv) Oligolamellar vesicle (OLV): 100–500 nm and years to be proved. The phospholipid reorganisation in (v) Multilamellar vesicles (MLV): > 500 nm.18

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 149 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al. REVIEW

Delivered by Ingenta to: Guest User IP : 134.214.70.49 Tue, 13 Nov 2012 12:28:57

Fig. 1. Schematic drawing of liposomes structure and lipophilic or hydrophilic drug entrapment models.

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

150 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art

New developed types of liposome, designated as dou- 3.2.1.2. Reverse-Phase Evaporation (REV) Technique. ble liposome (DL)19 and multivesicular vesicles (MVV),20 A lipidic film is prepared by evaporating organic solvent were recently reported. These liposomes, which could be under reduced pressure. The system is purged with nitro- prepared by novel preparative technique, are thought to gen and the lipids are re-dissolved in a second organic improve drug protection against several enzymes.21 phase which is usually constituted by diethyl ether and/or isopropyl ether. Large unilamellar and oligolamellar vesi- REVIEW cles are formed when an aqueous buffer is introduced into 3. LIPOSOMES PREPARATION this mixture. The organic solvent is subsequently removed PROCEDURES and the system is maintained under continuous nitrogen. These vesicles have aqueous volume to lipid ratios that are 3.1. General Ingredients 30 times higher than sonicated preparations and 4 times 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, Phos- low salt concentrations) is entrapped within the vesicles, phatidylglycerol, Phosphatidylcholline, Phosphatidylser- encapsulating even large macromolecular assemblies with ine, Phosphatidylinositol) Phosphatidylcholine (also high efficiency.27 known as lecithin) and phosphatidylethanolamine con- 3.2.1.3. 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 con- the lipid into an organic phase (ethanol or ether), followed tain other constituents such as cholesterol,Delivered hydrophilic by Ingentaby the injection to: of the lipid solution into aqueous media, polymer conjugated lipids and water. Guest Userforming liposomes.28 Cholesterol has been largely used to improve theIP bilayer: 134.214.70.49The ethanol injection method was first described in characteristics of the liposomes. It improvesTue, the membrane 13 Nov 20121973. 12:28:5729 The main relevance of the ethanol injection fluidity, bilayer stability and reduces the permeability of method resides in the observation that a narrow distribu- water soluble molecules through the membrane.22 tion of small liposomes (under 100 nm) can be obtained A clear advantage of liposomes is the fact that the by simply injecting an ethanolic lipid solution in water, in lipid membrane is made from physiological lipids which one step, without extrusion or sonication.30 decreases the danger of acute and chronic toxicity. The ether injection method differs from the ethanol injection method since the ether is immiscible with the aqueous phase, which is also heated so that the solvent is 3.2. Preparation Method removed from the liposomal product. The method involves 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 dis- There 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 per- the 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 efficiencies. Method. This is the original method which was initially 24 3.2.1.4. Detergent Dialysis. Liposomes, in the size used for liposomes production. A mixture of phospho- range of 40–180 nm, are formed when lipids are solubi- lipid and cholesterol were dispersed in organic solvent. lized with detergent, yielding defined mixed micelles.32 As Then, the organic solvent was removed by means of evap- the detergent is subsequently removed by controlled dialy- oration (using a Rotary Evaporator at reduced pressure). sis, phospholipids form homogeneous unilamellar vesicles Finally, the dry lipidic film deposited on the flask wall was with usefully large encapsulated volume. hydrated by adding an aqueous buffer solution under agita- Other methods have been already used for lipo- tion 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 popu- However, 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 (1–5 m diameter). Thus, liposome the environment and to human health, requiring complete size reduction techniques, such as sonication for SUVs for- removal of residual organic solvent. Furthermore, conven- mation or extrusion through polycarbonate filters forming tional methods consist of many steps for size homoge- LUVs25 26 were useful to produce smaller and more uni- nization and consume a large amount of energy which is formly sized population of vesicles. unsuitable for the mass production of liposomes.

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 151 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

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 filtration and filled 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 Heat- process was as follows: freezing at −40 C for 8 h; pri- ing 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 modi- 25 C for 10 h.39 The chamber pressure was maintained at fied ethanol injection techniques which are increasingly 20 Pascal during the drying process. On addition of water, attractive. the lyophilized product spontaneously forms homogenous REVIEW 3.2.2.1. Heating Method. A new method for fast pro- liposome 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 aque- the polydispersity of the liposome preparation.39 There- ous medium followed by the heating of these compo- fore, 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 final been developed for liposomes preparation using supercrit- liposomal product. Temperature and mechanical stirring ical carbon dioxide.41 This method allowed aqueous dis- provide adequate energy for the formation of stable lipo- Delivered by Ingentapersions to: of liposomes to be obtained through emulsion somes. Guest Userformation by introducing a given amount of water into a Reza Mozafari et al. confirmed by TLC that no degra- IP : 134.214.70.49homogeneous mixture of supercritical carbon dioxide/L- dation of the used lipids occurred at the aboveTue, mentioned 13 Nov 2012 12:28:57 temperatures.37 The particle size can be controlled by the R-dipalmitoylphosphatidylcholine/ethanol under sufficient phospholipid nature and charge, the speed of the stirring stirring and subsequent pressure reduction. and the shape of the reaction vessel. Otherwise, employ- Transmission electron microscopy observations revealed ment of heat abolishes the need to carry out any further that vesicles are large unilamellar with diameters of 0.1– 41 sterilisation procedure reducing the time and cost of lipo- 1.2 m. The trapping efficiency of these liposomes indi- some production. cated more than 5 times higher values for the water-soluble 3.2.2.2. Spray-Drying. Since spray-drying is a very solute compared to multilamellar vesicles prepared by simple and industrially applicable method, the direct the Bangham method. The trapping efficiency for an oil- spray-drying of a mixture of lipid and drug was applied in soluble substance, the cholesterol, was about 63%. Results the preparation of liposomes.38 The spray-drying process showed that the SCRPE is an excellent technique that per- is considered to be a fast single-step procedure applied in mits one-step preparation of large unilamellar liposomes the nanoparticles formulation. exhibiting a high trapping efficiency for both water-soluble 42 43 Hence, liposomes were prepared by suspending lecithin and oil-soluble compounds. and mannitol in chloroform. The mixture was sonicated 3.2.2.5. Modified Ethanol Injection Method. Novel for 8 min (bath sonicator) and subjected to spray-drying approaches based on the principle of the ethanol injection 44–46 on a Buchi 190 M Mini Spray Dryer. The spray-drying technique such as the microfluidic channel method, 47–50 conditions were as follows: inlet and outlet temperatures the crossflow-injection technique, and the membrane were 120 C and 80 C, respectively; airflow rate was contactor method51 were recently reported for liposome 700 NI/hr; and the flow rate was 1000 ml/hr. The dried production. product was hydrated with different volumes of phosphate 3.2.2.5.1. The Crossflow Injection Technique. The con- buffered saline (PBS; pH 7.4) by stirring for 45 min.38 The cept of continuous crossflow injection is a promising main factor influencing the liposomal size was the volume approach as a novel scalable liposome preparation tech- of aqueous medium used for hydration of the spray-dried nique for pharmaceutical application. Wagner et al. used product.38 However, mannitol plays an important role in a cross flow injection module made of two tubes welded increasing the surface area of the lipid mixture, enabling together forming a cross.47–50 At the connecting point, successful hydration of the spray-dried product. the modules were adapted with an injection hole. The 3.2.2.3. Freeze Drying. This new method was influencing parameters such as the lipid concentration, the described for the preparation of sterile and pyrogen-free injection hole diameter, the injection pressure, the buffer submicron narrow sized liposomes.39 40 It is based on flow rate, and system performance were investigated.47 the formation of a homogenous dispersion of lipids in A minimum of buffer flow rate is required to affect batch water-soluble carrier materials. Liposome-forming lipids homogeneity and strongly influencing parameters are lipid and water-soluble carrier materials such as sucrose were concentration in combination with increasing injection dissolved in tert-butyl alcohol/water cosolvent systems in pressures. After exceeding the upper pressure limit of the

152 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art linear range, where injection velocities remain constant, cell, the intracellular fate is affected by the lamellarity. The the vesicle batches are narrowly distributed, also when liposomes lamellarity made from different lipids or prepa- injecting higher lipid concentrations. Reproducibility and ration procedures varies widely. That is why, the analysis scalability data show similar results with respect to vesicle of liposomes lamellarity is an important parameter to be size and size distribution and demonstrate the stability and considered. robustness of the novel continuous liposome preparation Liposome lamellarity is often accomplished by methods REVIEW technique.49 that are based on the visible or fluorescence signal change 3.2.2.5.2. Microfluidization. By using a microfluidic of lipids marker upon reagents addition. This approach is hydrodynamic focusing (MHF) platform, Jahn et al. gen- reviewed in more detail, since it is a relatively simple pro- erated liposomes by injecting the lipid phase and the water cedure that can be easily carried out in a standard lab. Sev- phase into a microchannel.45 Microfluidic flow is gener- eral lipids can be used and results rely on the comparison ally laminar due to the small channel dimensions and rela- of the total signal to the signal achieved from the reaction tively low flow rates. Well-defined mixing is then obtained between the lipids marker and the specified reagents.58 by interfacial diffusion when multiple flow streams are For example the UV absorbance of 2,4,6- injected in a microchannel. The size of the liposomes was trinitrobenzensulfonic acid (TNBS) at 420 nm increases in mainly controlled by changing the flow rate.44 the mixture as a result of complex formation with primary 3.2.2.5.3. Membrane Contactor. Recently, Jaafar- amines. This property has been used for the detection of Maalej et al. applied the ethanol injection technique aminolipids at 420 nm. As the lipid bilayers are slightly while using a membrane contactor for large scale lipo- permeable to the TNBS reagent, an overestimate of the somes production. In this method, a lipid phaseDelivered (ethanol, by Ingentaexternal to: surface can be expected. To correct the reagent phospholipid and cholesterol) was pressed throughGuest the Userleakage through the bilayer, three incubation times were membrane with a specified pore size. NitrogenIP gas: 134.214.70.49 at used. The obtained external surface area at each incubation pressure below 5 bar was sufficient for passingTue, the 13 organic Nov 2012time 12:28:57 was plotted against incubation time and the graph phase through the membrane. At the same time, the was extrapolated to time zero. Under certain conditions, aqueous phase flew tangentially to the membrane surface the bilayer permeability of TNBS is minimized such as and swept away the formed liposomes within the mem- the only aminolipids on the exterior bilayer contribute brane device. The new process advantages are the design to the signal. Lysis of liposomes by a detergent such as simplicity, the control of the liposome size by tuning the Triton X-100 allows TNBS to interact with interior amino- process parameters and the scaling-up abilities.51 lipids and yields the total signal. TNBS has remained the As a result, these techniques lead from the conven- commonly used method for the estimation of the degree tional batch process to potential large scale continuous of lamallarity. However, this method has disadvantages procedures. which make it impotent in most cases; the TNBS assay requires large amount of material (milligrams) which makes the multiple sample application difficult and affect 4. IN-VITRO LIPOSOMES CHARACTERIZATION assay precision when the amount is limited.59 60 In another method, the addition of periodate to phos- In order to assess the liposome quality and to obtain quan- phatidylglycerol results in the diol oxidation and releas- titative measures that allow comparison between differ- ing of formaldehyde. The released formaldehyde reacted ent batches of liposomes, various parameters should be with chromotropic acid to yield a product which was monitored. For liposomes applications in analytical and subsequently detected at 570 nm. This method has been bioanalytical fields, the main characteristics include the used for the determination of external reactive groups on average mean diameter and polydispersity index; encapsu- liposomes.58 lation efficiency; the ratio of phospholipids to drug con- Otherwise, the quenching of N-(7-nitrobenz-2-oxa-1,3- centration and lamellarity determination. Other commonly diazol-4-yl) (NBD) fluorescence is obtained by sodium monitored parameters include surface charge through zeta dithionite. NBD-labeled lipids are highly fluorescent potential measurement, phase transitions through differen- at low concentration (<1 mol%) in membranes, but tial scanning calorimetry and quantification of residual sol- undergo self-quenching at increased concentrations. In this vents through gas chromatography. A detailed description approach, the initial NBD labelled lipids fluorescence is of today’s most commonly methods and novel techniques from all lipids in the sample. Under appropriate condi- of liposome characterization is presented in this report. tions, the addition of sodium dithionite quenches the fluorescence of only the NBD labelled existing on the outer 4.1. Lamellarity Determination bilayer. Fluorescence was monitored on spectrofluorometer with excitation and emission wavelengths of 450 nm and Lipid bilayers number of liposomes influences the encap- 530 nm respectively. The percentage of external lipid is sulation efficiency and the drugs release kinetics. Further- found by dividing the change in fluorescence upon dithion- more, when liposomes are taken up or processed in the ite addition by the total fluorescence.61–63

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 153 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

Table I. Preparation methods and applications through published papers.

Active Membrane Solvent/ Preparation method substance composition adjuvants Size Zeta EE% Study details References

Hydration lipidic film Ibuprofen Egg 9:1 solvent mixture 4.2 –5.2 17% *The cholesterol content (52) phosphatidylcho- of chloroform and 6 μm 54 mV 61% *Llipid alkyl chain length line (PC) methanol *Charged lipids Phosphatidylcho- Distilled water line (DMPC) Effects on liposome Distearoyl characteristics (size, zeta a,d phosphatidylcho- e,capsulation efficiency) line (DSPC) Dilignoceroyl phosphatidylcholine (C24PC) REVIEW Stearylamine (SA), dicetylphosphate (DCP) cholesterol (Chol)

Reverse -phase Acetazolamide Egg L- Chloroform: 5.8 —10% *Effect of the cholesterol (53) evaporation phosphatidylcho- methanol mixture 7.6 μm 45% content and charged lipids line, type X-E, and (2:1, v/v) on liposome characteristics cholesterol ether *Photomicroscopic Analysis Conventionnel stearylamine or *Differential Scanning techniques dicetyl phosphate Calorimetry measurements *Stability Study *In vivo Studies (male rabbits) Ethanol injection salidroside Egg yolk absolute ethanol 50 0 20% *The effect of salidroside- (54) phosphatidylcho- 100 nm –18 mV 40% loading capacity on EE lineDelivered by Ingenta to: *Effect of cholesterol on EE cholesterol and particle size Guest User *Turbidity measurements IP : 134.214.70.49 *In vitro release study Detergent dialysis —Egg-Tue, 13 NovTwo 2012 detergents 12:28:57 60 –7 — *Dynamic light scattering and (55) phosphatidylcho- sodium cholate 160 nm –51 mV zeta potential line (EPC, and octyl-_-d- Measurements egg- glucopyranoside *Cryo-transmission electron phosphatidylglyc- microscopy erol EPG) Custom-made Comparison of size and dialysis cell size distribution data (7.1cm2 exchange obtained from DLS and cryo- area) TEM Emulsion Bovine serum Egg Chloroform and 10 —43% *Factors impacting (56) (double emulsification albumin (BSA) phosphatidylcho- ether 30 μm 71% encapsulation process) line (PC), Triolein 4.0% glucose efficiency of multivesicular (TO), cholesterol aqueous solution liposomes (Chol) *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. Heating method Plasmid DNA Dipalmitoylphosp Sterile phosphate —160 — *Transfection efficiency and (57) from hatidylcholine buffered saline 2250 nm cytotoxicity were evaluated Escherichia coli (DPPC), glycerol (final using Chinese hamster dicetylphosphate concentration ovary-K1 (CHO-K1) cells. (DCP), 3%, v/v) cholesterol (70:20:10 molar ratio) Spray drying Metronidazole Lecithin Mannitol 268 —24 *Serum stability study (38) and verapamil chloroform 395 nm 47% hydrochloride Drug- Cyclodextrin Complex Freeze drying Ketoprofen (K), Soybean Sucrose 110 — — *Size measurements (39) phosphatidyl- Tert-butyl alcohol/ 410 nm *Entrapment studies ciprofloxacin choline, and lactate (CL) soybean Water *Electron microscopy and propranolol phosphatidyl- *Thermal analysis Novel hydrochloride serine *X-ray diffraction techniques (PH)

Calcein (Cal) — — (40) 100 nm *Size measurement Soybean Sucrose, *Entrapment efficiency phosphatidyl- cholesterol (4:1) *Thermal analysis choline, and Tert-butyl alcohol/ *Cryomicroscopy hydrogenated water *Mass loss analysis soybean *Residual solvent content phosphatidyl- serine

Continued.

154 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art

Table I. Continued.

Active Membrane Solvent/ Preparation method substance composition adjuvants Size Zeta EE% Study details References

Supercritical reverse- Glucose L-R- The cell <2000 nm 0 15% *The trapping efficiency (42) (43) phase evaporation Dipalmitoylphosp temperature was 30 mV 20% *Dynamic light scattering, method hatidylcholine raised to 60 °C, *zeta potential (DPPC and CO2 *freeze-fracture was introduced to transmission electron REVIEW achieve the microscopy experimental investigating conditions Physicochemical (typically properties of chitosan-coated 200 bar). 40 min liposomes with stirring at equilibration, the pressure was released to obtain the liposomal dispersion. Microfluidic — Egg Ethanol 50 (46) channel phosphatidyl- or ethanol/tert- 160 nm choline or butanol (1:1 v/v) hydrogenated soy phosphatidylcholine Cholesterol (2.5:1 w/w) Modified ethanol (44) injection Dimyristoylphosp — hatidylcholine Isopropyl alcohol 25 (DMPC) 50 nm CholesterolDelivered and by Ingenta to: dihexadecyl phosphate (5:4:1Guest User in dry chloroform)IP : 134.214.70.49 Crossflow — Dipalmitoyl- Ethanol 100 — — (48) injection Tue,phosphatidyl- 13 Nov 2012 12:28:57200 nm choline Cholesterol and stearylamine (7:2:1)

— (47) Recombinant Idem Idem 200 25 human 300 nm 27% superoxide dismutase (rh- Cu/Zn-SOD) from Escherichia coli Membrane Indomethacin Lipoid E80 Ethanol or 100 –35 IMC: 63% *Size measurement (51) contactor (IMC), Cholesterol ethanol/tert- 180 nm –6 (mV) *Zeta potential Beclomethason (20% w/w PL) butanol (1:1 v/v) BDP: 98% *Entrapment efficiency e dipropionate *Thermal analysis (BDP) *Electron transmission microscopy

These methods appear to close a gap in the method- Presuming that the shift reagent (Mn2+) only interacts ology to determine external surface structure of vesicles. with the phospholipids located in the outermost monolayer, However, these methods assume that the lipid of interest the degree of lamellarity can be calculated by the ratio of is distributed evenly over all lipid layers, and the reagents 31P-NMR signal before and after Mn2+ addition. Used used to elicit the signal change are impermeable to the for a long time in the field of liposome research, this tech- membrane over the time course of the measurements.64 nique has been found to be quite sensitive to experimental Other numerous methods for the lamellarity determi- conditions which can have distinct effect on the analy- nation such as magnetic resonance were mainly used sis. For example, Mn2+ is able to penetrate the liposomal to study the outside-inside distribution of phospholipids bilayer especially when used at high concentrations. At within bilayer and the characterization of model membrane low pH or in the presence of complexing agents (such as structures. A straightforward application of nuclear mag- HEPES or TRIS buffer at certain concentration), no pen- netic resonance in the quality control of liposomes is the etration of Mn2+ occurs. Therefore, under well-defined determination of size and lamellarity. Dispersions of MLVs conditions, the analysis of liposomes by 31P-NMR is the give rise to very broad powder 31P-NMR spectra due to presence of shift reagent in an elegant and accurate method the restricted anisotropic motion whereas SUVs are char- giving useful information about the outer to inner phos- acterized using a narrow line spectra. It is well known that pholipids ratio amount.65 66 the paramagnetic ion Mn2+ interacts with the negatively Other techniques for lamellarity determination include charged phospholipids phosphate causing perturbations small angle X-ray scattering (SAXS). For this pur- of the nuclear spin relaxation times which broaden the pose, liposome dispersions put into glass capillaries and 31P-NMR resonance and reduces the quantifiable signal. curves were recorded with a camera equipped with a

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 155 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

one-dimensional position sensitive detector. Blank scatter- without sample manipulation. The result is with a high ing curves were obtained from the same capillaries filled resolution three-dimensional profile 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 req- the electron distance distribution p(r) (the probability to uisite surface immobilization does not adversely affect the find 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 pro- To confirm the lamellarity results by an imaging vide information on morphology, size, as well as on the REVIEW method, freeze fracture technique with subsequent trans- possible aggregation processes of liposomes during their mission electron microscopy was used. For this purpose, storage. Imaging in aqueous medium allows the liposomes carbon film 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 min- a thin film stretched over the holes. The specimens were utes after deposition on mica support, after which they instantly shock-frozen in melting nitrogen or by plung- collapsed. For this reason, the liposomes images have to be ing them into liquid ethane or propane in a temperature- obtained 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 a cryo-electron microscope. To determine theDelivered mean lamel- by Ingentareplace to: the wide variety of microscopic techniques mea- larity, micrographs of three different areas of the specimenGuest Usersuring liposomal size.71–74 were investigated.66 69 70 IP : 134.214.70.49HPLC using SEC can be used to separate and quantify Whatever is the technique, the lamellarityTue, determination 13 Nov 2012liposome 12:28:57 populations according to a time-based resolu- is essential to define 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 4.2. Size Analysis the column. This mechanism leads to separation based on large particles elution before smaller particles. Con- The average size and size distribution of liposomes are ventional SEC is frequently used for liposomes separation important parameters especially when the liposomes are from unencapsulated materials as a final purification step, intended for therapeutic use by inhalation or parenteral but the use of HPLC-SEC for analysis offers increased res- route. Several techniques are available for assessing sub- olution of liposome populations and reduced sample size micrometer liposome size and size distribution which and enhances reproducibility. One recommended commer- include microscopy techniques, size-exclusion chromatog- cially available column is the ethylene glycol-metacrylate raphy (SEC), field-flow fractionation and static or dynamic gel which has a separation range from 20 to 500 nm, this light scattering. ‘hydroxylated poly-ether-based’ gel shows a larger exclu- Several variations on electron microscopy (EM) such sion limit than other gels. An osmotically balanced mobile as transmission EM using negative staining, freeze frac- phase flowing at relatively low pressures (1–1.5 mega- ture TEM, and cryo EM, provide valuable information on pascal) helps to prevent damages, swelling or shrinkage liposome preparations since they yield a view of morphol- of liposomes. HPLC-SEC can offer a powerful technique ogy and can resolve particles of varying size. However, for not only size distribution determination, but also sta- sample preparation is complicated as it requires removal bility in terms of aggregation and vesicle permeability. of liposomes from their native environment. These tech- Three methods have been described in literature: dynamic niques can also generate artefacts, induce shrinkage and light-scattering analysis of SEC fractions; rechromatogra- shape distortion, and are time consuming to obtain a rep- phy of SEC fractions on a calibrated column with tur- resentative size distribution of the population, thus are bidity measurements; and SEC with on-line turbidity and not amenable to being routine measurements. Some of refractive index detection. The rechromatography method these problems may be overcome yielding reproducible was judged to be the most reliable, although the sen- and accurate results by giving careful attention to sam- sitivity suffered from the dilution in the two chromato- ple preparation. A recently developed microscopic tech- graphic steps. Disadvantages of HPLC for liposomes size nique known as atomic force microscopy (AFM) has been determination mainly stem from recovery issues. These utilized to study liposome morphology, size and stability. include unwanted adsorption of lipids on the column pack- AFM, scanning probe microscopes with dimensional reso- ing and destruction of liposomes containing lipids with lution approaching 0.1 nm, provides unique possibility for higher affinity to the column material than the compos- visualizing small liposomes in natural environment even ite lipids. Both lipids necessitate a preliminary step of

156 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art presaturation of the LC column with lipids prior to anal- a single wavelength. The FFF mechanism for liposomes ysis. In addition, the lipid bilayer rigidity, which is a analysis differs in that FFF flow separates vesicles on a function of the lipid composition, plays a role in the lipo- hydrodynamic size basis, whereas sedimentation FFF sep- somes retention and recovery. The bilayer rigidity dic- arates them on a weight basis. Flow FFF enables rapid, tates whether liposomes of large diameter can be deformed convenient and non invasive measurement of vesicle size and thereby pass through relatively narrow pores or lipo- distribution without prior calibration using size standards. REVIEW somes of small diameter which may be excluded from Other advantage of the FFF technique is the wide range relatively large pores, dependent on pore’s shape and ori- of particle sizes that can be separated (1 nm – 100 m) entation. The net effect is therefore difficult to predict. with high resolution. The only limit of this technique is Thus, while hydrodynamic size and subsequent molecu- the complexity and expense of instrumentation.78–81 lar weight information can be obtained through this tech- Dynamic 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 com- in liposome size distribution analysis. DLS measures position) set of standards. Lastly, while suitable packing the time-dependant fluctuations of light scattered from materials are available for small to moderately sized lipo- particles experiencing Brownian motion, which results somes resolution, it is not the case for large liposomes from collisions between suspended particles and solvent (>800 nm).75–77 molecules. When a particle is suspended in a solution and Field-flow fractionation (FFF) is a technique which illuminated by light, it scatters light given that its index overcomes some of the limitations of HPLC in liposomes of refraction differs from that of suspending solvent. In analysis. It includes electrical, thermal, sedimentationDelivered and by Ingentaother words, to: its polarizability differs from that of the sol- flow FFF techniques that rely on a field application whichGuest Uservent. This means that the arriving electric field is oscil- is perpendicular to the direction of flow. FFF usesIP a : chan- 134.214.70.49lating and is able to displace the cloud of electrons and nel wall which consists of a semipermeableTue, membrane 13 Nov 2012thereby 12:28:57 cause atoms to oscillate. The strengths of the tech- chosen with a MWCO suitable for the liposomes under nique include the ability to make measurements in native study. This membrane allows only the carrier fluid to pass. environments; its sensitivity to small quantities of high In flow FFF, there are two liquids flows acting on the sam- molecular weight aggregate; ease of commercially avail- ple components. The channel flow that runs through the able operating instrument; minimal sample volume, con- channel and the crossflow that flowing perpendicular to centration and preparation requirements. It also covers a the channel passes through the inlet frit into this channel large size range of species spanning the low nanometer to and exits through the membrane and outlet frit. A com- low micrometer range. However, the technique does not mon procedure for sample injection is called ‘stop-flow yield particle shape information; it can yield a bias towards relaxation’, in which a small volume sample is injected reporting larger diameters when small quantities of high into the channel flow. After a short delay period that allows molecular weight or aggregates or impurities are present the sample to move into the channel from the injector, in the sample.82–84 the channel flow is stopped for a time, allowing only the Measurement of particle size distribution could also be crossflow to act on the sample. The laminar flow profile 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 fluid in channel walls, while the perpendicular flow propels all par- which they are suspended can be accurately analysed.85 86 ticles toward the membrane wall. Diffusion due to Brown- Centrifugal sedimentation of particles suspended in a fluid ian motion of particles in a size-based manner reduces the is a well-known method of measuring the size distribu- accumulation of smaller particles against the membrane tion of particles in the range of 0.015–30 m in diameter. wall. Retention times in this technique are proportional to The sedimentation velocity of any particle could be cal- the hydrodynamic diameter of the particles since smaller culated if the particle density, fluid density, fluid 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 first, in normal mode FFF, small liposomes elute first as a very thin layer at the surface of the fluid. A light beam due to their higher diffusion coefficient. 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 fluid 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 flows 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 fluid and the diam- used in FFF, but the most common is a UV/VIS spec- eter 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 fluid’s

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 157 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

surface.86 Sedimentation velocity increases as the square of suspension with zeta potentials > +30 mV or < −30 mV the particle diameter, so that particles which differ in size are normally considered stable.87 88 by only few percent settle at significantly different rates. The time needed to reach the detector is used to calculate 4.4. Encapsulation Efficiency the size of the particles. This method for size analysis has a high resolution compared to the other analysis method, The liposome preparations are a mixture of encapsulated it has also a high sensitivity which enables him to detect and un-encapsulated drug fractions. The first step for the small additional peaks and pick up small changes. More- determination of the encapsulation efficiency is the sepa- over, high accuracy is assured since all analyses are run ration between the encapsulated drug (within the carrier) REVIEW against a known calibration standard; the calibration can and the free drug. Several separation techniques have been be either external (standard injected before the sample) or reported in the literature. The mini-column centrifugation internal (standard mixed with the sample).85 is a method based on the difference of size between the Several other techniques, considered to be less conven- drug loaded liposomes and the free drug. Indeed, undi- tional, have been applied for liposome size distribution luted liposome suspension is applied dropwise to the top of analysis but are not discussed in this paper, such as NMR, sephadex gel column and the column is spun at 2000 rpm flow cytometry, capillary zone electrophoresis, etc. 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 Delivered by Ingentathe gel andto: were collected from the first and second stage Three of the fundamental states of matter are solids, Guest Userof centrifugation.89 liquids and gases. If one of these states is finely dis- IP : 134.214.70.49The separation between the free drug and the encapsu- persed in another then we have a ‘colloidal system’. Most Tue, 13 Nov 2012lated 12:28:57 drug could also be achieved by the use of a dialysis colloidal dispersions in aqueous media carry an electric membrane with an appropriate cut-off. The liposme sam- charge. 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 surround- The ultracentrifugation technique was reported as a sim- ing medium. The more important mechanisms are: ioniza- ple 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 specifically 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 quan- property which is exhibited by any particle in suspension. tified. Techniques used for this quantification depend on It has long been recognized that the zeta potential is a very the nature of the encapsulant and include spectrophotome- good index of the interaction magnitude between colloidal try, fluorescence 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 effi- zeta 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 concen- no force to prevent the particles flocculating. tration. This method can be applied if the liposomes do To measure the zeta potential, a laser is used to provide not undergo any purification (SEC, dialysis) following a light source illuminating particles within the samples. preparation. Either technique are applied to separate lipo- The 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 sta- 13 is detected. When an electric field is applied to the bility in terms of leakage or the effect of various dis- cell, any particles moving through the measurement vol- ruptive conditions on the retention of encapsulants. In ume will lead to fluctuation of the detected light with a some cases, size distribution and encapsulation efficiency frequency proportional to the particle speed. This infor- determinations 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 computer and hence potential zeta is calculated. Particles encapsulant.

158 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art

The terminology varies widely with respect to the abil- Enzymatic assays for phosphatidylcholine and choles- ity 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 efficiency’, ‘trap- phospholipids and release free choline. The free choline ping efficiency’ or ‘encapsulation efficiency (EE)’ which is then oxidized to form betaine aldehyde, betaine and is typically defined as the total amount of encapsulant hydrogen peroxide, by choline oxydase. The generated REVIEW 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 quantified at 505 nm.98 molecules (dependent on lipid/buffer composition, lipo- The latter method relies on hydrolysis of choles- some 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 define the encapsulation efficiency, 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 concentra- phenol in the presence of peroxidase to yield a blue- tion of encapsulant by the concentration of lipid. A similar coloured quinoneimine dye which shows strong absorption definition is suggested expressing EE on a weight (mg) at 505 nm.99 98 22 encapsulant per mM of lipid basis. Another commonly Chromatographic techniques such as HPLC, GC and used parameter is the captured volume, defined as Lof thin layer chromatography (TLC) can be used to separate entrapped volume/ mol of lipid. This numberDelivered ranges from by Ingentaand quantify to: the lipids composing lipid bilayers.100 Chro- 0.5 L/nmol for SUV and MLV to 30 l/nmol for LUV.Guest Usermatographic approaches are advantageous since they can Unlike the ‘percent encapsulation’ parameter citedIP : previ- 134.214.70.49separate and quantify each lipid in the mixture. TLC meth- ously, these representations require knowledgeTue, of the13 phos-Nov 2012ods 12:28:57 for phospholipid analysis often rely on lipid separa- 94 95 pholipids concentration. tion using a mixture of chloroform, methanol and water. Detection is frequently accomplished using molybdenum 4.5. Lipid Analysis blue in sulfuric acid and ninhydrin stains for the detection of phosphate and primary amino groups, respectively. Several chemistry techniques are commonly used for the For HPLC analysis, detection of lipids in the UV range is determination of phospholipid content. Most of these tech- limited to 200–210 nm due to their lack of chromophores. niques include the use of molybdate-containing reagents GC analysis of lipids typically requires a derivatization yielding a blue-colored product. One such method is the step to ensure sufficient volatility of the components, either Bartlett assay which relies on the digestion of organic through trimethyl silylation or methyl esterification prior materials in liposome samples by 160 C sulfuric acid, oxidation to inorganic phosphates by hydrogen peroxide, to detection by flame ionization or mass spectroscopy. In phosphomolybdate formation upon interaction with ammo- many cases, pre-treatment of liposomes to disrupt the lipid nium molybdate, followed by reduction through interaction bilayers is completed prior to chromatographic analysis with 1,2,6-aminonaphtolsulfonic acid at 100 C. A blue including dilution of the liposome suspension with alco- product is formed which can then be analysed at 830 nm hols such as 2-propanol, ethanol or methanol. The pro- for the quantitative assessment of the phospholipids in the cedure choice is dependent on the mobile phase and the preparation.96 degree of lipid solubility. In the ascorbic acid method, ammonium molybdate reacts with orthophosphates formed from acid digestion 4.6. In-Vitro Drug Release to yield phosphomolybdic acid. This compound is then reduced with ascorbic acid to yield a blue-colored solution, In vitro drug release can be performed using the dialy- analysed at 820 nm.96 sis tube diffusion technique. The dialysis bag membrane Phospholipids can also be analyzed through complex should be selected following screening of various mem- formation with ammonium ferrothiocyante, extraction into brane, no drug adsorption may occur and the membrane chloroform, and absorbance measurement at 488 nm.96 should be freely permeable to the active ingredient (the cut A convenient and sensitive fluorescence assay for off molecular weight shouldn’t be a limiting step in the dif- phospholipid vesicles has also been reported by:97 when fusion process). Some millilitres aliquot of liposome sus- phospholipid vesicles are added to an aqueous solu- pension is placed in the dialysis bag, hermetically tied and tion of 1,6-diphenyl-1,3,5-hexatriene (DPH) a fluorescence dropped in the receptor compartment containing the disso- enhancement of several hundred-fold is observed which lution medium. The entire system is kept at 37 C under can be used for a phospholipid concentration determina- continuous magnetic stirring and the receptor medium is tion. The fluorescence is a function of the type of phos- closed to avoid evaporation of the dissolution medium. pholipid, salt concentration, and time of incubation. The kinetic experiments are carried out respecting the sink

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 159 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

conditions in the receptor compartment. Samples of the 5. LIPOSOMES APPLICATIONS dialysate are taken at various time intervals and assayed for the drug by HPLC, spectrophotometer or any other 5.1. Pharmaceutical Applications convenient method. The sample volume is replaced with The use of liposomes as systemic and topical drug deliv- fresh dissolution medium so as the volume of the receptor ery systems has attracted increasing attention. Liposomes compartment remains constant. Every kinetic experiment can be formulated in liquid (suspension), solid (dry pow- is performed in triplicate and the average values are taken der) or semi-solid (gel, cream) forms. In vivo, they can be to establish the release proﬁle of the drug from the lipo- administered topically or via parenteral route. some suspension.101 102 REVIEW 5.1.1. Systemic Liposomal Drugs

4.7. Liposomes Stability After systemic (usually intravenous) administration, liposomes are typically recognized as foreign particles and The liposomes stability is a major consideration for lipo- consequently endocytosed by the mononuclear phagocytic some production and administration steps: from process to system cells (MPS), mostly fixed 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 sta- physical integrity and does not adversely influence the bilized liposome, which are not avidly taken up by MPS chemical integrity of the active ingredientDelivered during its by Ingentacells, have to: different biodistributions properties and have life. Researchers are attempting to deliver low andGuest high Usershown enhanced accumulation in sites of trauma, such as molecular weight drugs in a variety of polymerIP : matri- 134.214.70.49tumours, infections and inflammation. This accumulation ces and liposome suspensions. The successfulTue, 13 introduc- Nov 2012is simply12:28:57 due to their prolonged circulation and small size; tion of dosage forms depends upon a well-defined stability enabling them to extravasate.105 study. In designing a stability study, physical, chemical Based on the liposome properties introduced above, sev- and microbial parameters must be considered and evalu- eral techniques of drug delivery can be envisaged: ated. This wisdom is also required for the liposome dosage —Liposomes can be applied to protect the entrapped drug form. A stability study must include a section for product against enzymatic degradation whilst in circulation. The characterization and another section concerning the prod- lipids used in their formulation are not susceptible to enzy- uct stability during storage. matic degradation; the entrapped drug is thus protected All liposome preparations are heterogeneous in size, the while the lipid vesicles circulate within the extracellu- average size distribution of liposomes changes upon their lar fluid. As an example, -lactamase sensitive antibi- storage. Liposomes tend to fuse and grow into bigger vesi- otics such as the penicillins and cephalosporins have cles, which is a thermodynamically more favourable state. been encapsulated in order to be protected against the - 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 vesi- encapsulated drug and facilitate the gastrointestinal trans- cles. 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 lipo- oral 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 specific target site should allow efficacy increase at low cause permeability changes within liposome bilayer. In dose with attendant decrease of toxicity. Indeed, pump- addition, 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 profile of a but, more fundamentally, increase undesirable side effects. drug molecule may entirely be different from its liposome Hence, the benefits of drug targeting include reducing preparation stability profile. Thus, it is essential to develop drug waste, and it is possible to deliver a drug to a tis- stability 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. Effec- are 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 lipo- spatial and temporal distribution of the encapsulated drug somal preparations.103 104 molecules in the body, which may significantly reduce

160 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art

Table II. Liposome applications in the pharmaceutical ﬁeld.

The intention of encapsulation Molecule Therapeutic class Conclusion Reference

Protection Insulin Hypoglycaemic agent 1.3 units of insulin entrapped in dipalmitoyl-phosphatidylcholine/ (107) against cholesterol liposomes administered to normal rats decreased REVIEW enzymatic blood glucose level in4htoabout 77% of those before degradation 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 4h. Liposome-entrapped insulin significantly reduced glucose and (126) 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. Factor-VIII Coagulation factor Factor-VIII loaded liposomes were given orally to a patient with (127) 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. Cysteamine Treatment of acute Oral administration of liposome-entrapped cysteamine induces an (128) radiation syndromeDeliveredincrease by Ingenta in the concentration to: of exogenous sulphur compounds in “ARS” plasma, liver and spleen. This fact can be related to a protection Guestof cysteamine User in the digestive tract. Pencillins AntibioticsIP : The134.214.70.49 entrapped drugs are protected against -lactamase enzyme (109) cephalosporins Tue, 13 Novwhile 2012 they are 12:28:57 in circulation in the extracellular fluid. Drug targeting Cyclosporine Immunosuppressive The in vivo pharmacokinetics and renal toxicity of a liposomal (129) formulation and the commercially available cyclosporine are compared. The apparent volume of distribution was significantly 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 significantly prolonged biologic half-life (47.91 ± 13.15 vs 30.95 ± 8.89 h). Kidney function was assessed via the calculation of the glomerular filtration 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 (130) 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 significantly 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. Amikacin Antibiotic The clearance of liposomal amikacin was over 100-fold lower than (131) 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 filtered 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, significantly alters the disposition of amikacin within the body and therefore decreases the potentially toxic level of renal tubular exposure.

Continued. J. Colloid Sci. Biotechnol. 1, 147–168, 2012 161 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

Table II. Continued.

The intention of encapsulation Molecule Therapeutic class Conclusion Reference

Doxorubicin Antineoplastic Doxorubicin has been shown to have a 4.5-times-lower (110) 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 signiﬁcant extent Enhancement of Amphotericin B Treatment of fungal Owing to its aqueous insolubility, amphotericin B is (111)

REVIEW drug systemic infections typically formulated into detergent micelles. solubilisation 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 efficiently to macrophages and, additionally, toxicity can be significantly reduced. Enhancement of Polyvinylpyrrolidone Blood plasma substitute At an equal concentration of 125I-labelled (132) drug uptake polyvinylpyrrolidone, the rate of uptake of the liposome-entrapped macromolecule by the tissue was over 4-times that of the free macromolecule. Vitamin K Prevention or treatment The effect of liposomal -associated vitamin K , (133) 1 Delivered by Ingenta to: 1 of hemorrhage administered orally, was investigated using rabbits Guest Userwith warfarin-induced hypoprothrombinaemia, and IP : 134.214.70.49evaluated in comparison with other dosage forms of Tue, 13 Nov 2012the 12:28:57 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. desferrioxamine Chelating agent Liposomal desferrioxamine given intravenously to (134) iron-overloaded 59Fe labeled mice doubled the 59Fe excretion for a given dose of the drug. Vaccination Inactivated hepatitis Prevention of Hepatitis A Purified hepatitis-A-virion capsule, viral phospholipids (113) A virus and envelope glycoprotein from influenza 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.

unwanted toxic side effects and increase the efficacy of —In order to enhance solubilisation, the amphotericin B, the treatment. The first step, therefore, is to determine the which is the drug of choice in the treatment of systemic antigens that are produced by the tumour cells. Then to fungal infections, has been widely studied for liposome target the drug via specific receptor ligands, which may encapsulation. Owing to its aqueous insolubility, ampho- be specific antibodies for antigens produced by tumour tericin B is typically formulated into detergent micelles. cells.109 Two liposomal formulations have been approved But, micelles are unstable upon systemic administration, by the US food and drug administration (FDA) and are and several neuro- and nephrotoxicity limit the dose that commercially available in the USA, Europe and Japan for can be administered. However in a stable colloid par- the treatment of Kaposi’s sarcoma. Doxil® is a formu- ticle, such as liposomes, encapsulated drug is delivered lation of doxorubicin precipitated in sterically stabilized much more efficiently to macrophages and, additionally, liposomes (on the market since 1995) and DaunoXome® toxicity can be significantly reduced.111 Following this is daunorubicin encapsulated in small liposomes (on the rationale, a lipid-based amphotericin B formulation is market since 1990). Doxil® has been shown to have a actually commercially available in the Europe and US 4.5-times-lower medium-pathology score for doxorubicin- market (respectively since 1990 and 1997): AmBisome® induced cardiotoxicity than the free drug. In squamous including amphotericin B into small liposomes. cell lung carcinoma, the same drug is capable of reducing —Otherwise, liposomes can also be used to enhance the tumor burden to a significant extent.110 drug intracellular uptake. The lipid formulation promotes

162 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art the cellular penetration of the encapsulated drug especially Later, the liposomes ability of enclosing many differ- antibiotics, reducing the effective dose and incidence of ent biological materials and delivering them to the epider- toxicity. 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 promis- for varying skin diseases treatment. REVIEW 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 success- dependant manner across the stratum corneum. How- fully launched by the Swiss Serum Institute in 1994. Puri- ever, multilamellar liposomes can deliver drugs within fied hepatitis-A-virion capsule, viral phospholipids and 30 minutes to the stratum corneum, epidermidis, and envelope glycoprotein from influenza virus are mixed with dermis in significantly higher concentrations than conven- phosphatidylcholine 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 influenza, hepatitis B, diphtheria be considered: corticoids, retinoids and local anaesthetics. and tetanus.113 Mezei et al. was the first to report increased corti- —Cationic liposomes have been shown to complex (neg- costeroid concentrations in epidermis and corium com- atively charged) DNA, and such complexes were able to bined with a reduced percutaneous absorption in an animal transfect cells in vitro, resulting in the expressionDelivered of the by Ingentamodel.116 to:This is particularly important as far as adverse protein encoded in the DNA plasmid in the target cells,Guest Usereffects from extensive corticosteroid therapy are reduced. and making liposomes useful in direct gene transfer.IP : Obvi- 134.214.70.49These findings were the same with human skin; Lasch ously for gene therapy (the treatment of diseasesTue, 13 on Nov the 2012investigated 12:28:57 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 con- discovered that cationic lipid-based DNA complexes can centration profiles 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 insufficient in the therapy of acute Today, enthusiasm for the systemic use of liposomal dermatoses.117 For this reason, higher drug concentrations drugs is not as widespread as it was. While the long will mean an improved therapeutic effect. In a clinical trial, list of diseases considered candidate for systemic appli- Korting investigated the effect of betamethasone dipro- cation of liposomal drugs has been reduced to just a few pionate in a liposomal preparation and in a commercial indications, the topical application of liposomal prepara- conventional preparation in patient suffering from atopic tions has recently attracted more interest. eczema.118 In this double-blind, randomized, paired trial 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 inflammation compared to Skin treatment applications of liposomes are based on the the conventional preparation. similarity between the lipid vesicles bilayer structure and The retinoids is the second important group of drug natural membranes which includes the ability of lipid vesi- which seems to be a promising candidate for liposomal cles, with specific lipid composition, to alter cell mem- encapsulation. One main field for the topical administra- brane fluidity and to fuse with them. In the dermatological tion of retinoids is uncomplicated acne vulgaris. Commer- field, liposomes were initially used because of their mois- cial tretinoin gels shows local irritant effects and flare-up turizing and restoring action.114 reactions at the beginning of the treatment. These char- In Schmid’s work, stratum corneum liposomes have acteristics, often compromising patient compliance, can been used in the treatment of atopic dry skin in order to be overcome by the liposomal formulation of tretinoin. restore the barrier function and to vehicle an active sub- Masini et al. reported a reduced irritancy in animal experi- stance at the same time.115 The composition and properties ments after treatment with liposomal tretinoin, which may of liposomes play an important role in their interaction be explained by gradual drug release from the liposomal and possible penetration within the epidermis. In addition, preparation.119 Schafer-Korting et al. performed a double liposomes provide valuable raw material for the regener- blind study to evaluate the efficacy and tolerability of ation of skin by replenishing lipid molecules and mois- liposomal tretinoin in patients with uncomplicated acne ture. Lipids are well hydrated and, even in the absence of vulgaris.120 The results clearly showed that the less con- active ingredients, humidify the skin. Often this is enough centrated liposomal drug commands equal efficacy and to improve skin elasticity and barrier function, which are less skin irritation compared to its commercial conven- the main causes of skin aging. tional counterparts. This equal efficacy might be attributed

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 163 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

Table III. Liposome-based products on the market.

Molecule Treated disease Product Company Status

Doxorubicin Kaposi’s sarcoma and DOXIL Ben Venue Laboratories for On the market since 1995 AIDS-related cancers. Johnson and Johnson, (USA) and 1996 (Europe) Ovarian cancer and USA multiple myeloma. CAELYX Schering-Plough, Europe MYOCET Enzon Pharmaceuticals for Cephalon, Europe and for

REVIEW Sopherion Therapeutics, USA and Canada Amphotericin B Systemic fungal AMBISOME First, NeXstar On the market since 1990 infections Pharmaceuticals which (Europe) and 1997 (USA) 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, Delivered by Ingentaand Sumitomo to: Pharmaceuticals in Japan. FUNGISOMEGuest User Lifecare Innovations, India AMPHOTECIP : 134.214.70.49 Intermune, USA Tue,ABELCET 13 Nov 2012 Enzon 12:28:57 Pharmaceuticals, USA AMPHOLIP Bharat Serums & Vaccines, India AMPHOCIL Samaritan pharmaceuticals, USA ABLC The liposome company, USA Daunorubicin Speciﬁc types of DAUNOXOME First, NeXstar On the market since 1996 leukaemia (acute Pharmaceuticals. Then the (USA and Europe) myeloid leukemia and drug was sold to Diatos in acute lymphocytic 2006 leukemia). Inactivated hepatitis Hepatitis A EPAXAL Crucell Company who On the Swiss market since A virus merged with the Swiss 1994 and now in more Serum and Vaccine than 40 country. Institute in 2006 Lidocaine Anaesthesia for skin LMX4 LMX5 Ferndale Laboratories, USA On the US market since itching, burning or 1998 pain.

to an improved bioavailability combined with a slower ethanolic solutions of phospholipids much care is taken drug release. In conclusion, the liposomally encapsulated to remove the remaining traces of alcohol. Data pre- tretinoin seems to be superior to the conventional dosage sented by Touitou et al. indicated that the presence of form. ethanol with a relatively high concentration in systems of During the last few years, many attempts have been lipid vesicles, termed ethosomes, was reported to influ- made to provide adequate local anaesthesia of the skin. ence the stratum corneum penetration and permeation of For this, prolonged application time and high anaesthetic drugs.122 Encapsulation experiments showed that etho- concentration are required. Studies performed by Gesztes somes are able to entrap both hydrophilic and lipophilic et al. indicated that tetracaine encapsulated liposomes pro- drugs. These entrapment results were supported by in vitro vide better local anaesthesia (low drug concentration and studies on the delivery of drugs in and through the skin. long anaesthesia duration) than a conventional anaesthetic Again, the ethosomal system was shown to be far superior cream. Similar results were obtained using other local to the control systems, both in terms of the drug con- anaesthetics such as lidocaine which is commercialized in centration in the skin and the flux of the drug through the US market since 1998 (ELA-Max®).121 the skin. Patches containing testosterone in an ethoso- Furthermore, it has been commonly believed that high mal system were compared in vivo in rabbits with the concentrations of ethanol are detrimental to liposomal for- commercial patch Testoderm®. The results showed signifi- mulations. Therefore, when liposomes are prepared from cantly higher testosterone blood levels from the ethosomal

164 J. Colloid Sci. Biotechnol. 1, 147–168, 2012 Laouini et al. Preparation, Characterization and Applications of Liposomes: State of the Art system. Horwitz et al. tested clinically the ethosomal car- Table IV. Some liposomal cosmetic formulations currently on the rier for dermal delivery of the antiviral drug, acyclovir in a market. double-blind randomized study with Zovirax®.123 Results Product Manufacturer Key ingredients indicated that ethosomes performed significantly better Capture Christian Dior Liposomes in gel than the commercial drug form. For example, the average Efect du Soleil L’Oréal Tanning agents in liposomes REVIEW time to crusting of lesions was shorter for the ethosomal Future Perfect Estée Lauder Vitamin E, A, cerebroside, system. The permeation enhancement from ethosomes Skin Gel ceramide suggests a synergetic mechanism between ethanol, lipid Aquasome LA Nikko Chemical Co Liposomes with humectant vesicles and skin. Indeed, in comparison to liposomes, Eye Perfector Avon Soothing cream for eye 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 flexible characteristics which allow them to penetrate more easily into lasting perfumes, hair conditioners, aftershaves, lipsticks, deeper layers of skin. In another hand ethanol may disturbs make-up and similar products are also gaining large frac- 138 the organization of the stratum corneum lipid bilayer and tions of the market. Some of the liposomal cosmetic enhances its lipid fluidity. formulations currently available in the market are shown Liposomes based on a natural marine lipid extract con- in Table IV. taining a highly polyunsaturated fatty acid ratio were recently introduced as Marinosomes® by Moussaoui et al. 5.3. Food Applications for the prevention and treatment of skinDelivered diseases.124 by Ingenta to: The majority of microencapsulation techniques currently Cansell et al. have reported that Marinosomes® contributedGuest User used in the food industry are based on biopolymer matri- to reduce inflammation induced by croton oil by regulatingIP : 134.214.70.49 ces composed of sugars, starches, gums, proteins, syn- PGE and IL-8 production in keratinocyte cultures.Tue, 13125 Nov 2012 12:28:57 2 thetics, dextrin and alginates. Nevertheless, liposomes have recently begun to gain in importance in food 5.2. Cosmetic Applications products.139 140 Indeed, the ability of liposomes to solu- 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 indus- the 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 first liposo- liposomes for controlled delivery of functional compo- mal 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 flavours. The applications are for example dairy 135 in 1986. Liposomes have been also used in the treat- products preparation, stabilization of food components ment of hair loss; minoxidil, a vasodilator, is in the active against degradation, and delivery and enhanced efficiency ingredient in products like “Regaine” that claim to pre- of 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 encapsu- water 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 lipo- compared 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 eco- 1.5-fold increase in skin water content, whereas liposome nomic profit 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 con- require well controlled conditions.141 tent was measured daily and the results showed that skin In addition to improved fermentation, liposomes were humidity was increased significantly 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 bac- Since 1987, several cosmetic products have been com- teria is questioned due to health concerns and natural alter- mercially 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 encapsula- and 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

J. Colloid Sci. Biotechnol. 1, 147–168, 2012 165 Preparation, Characterization and Applications of Liposomes: State of the Art Laouini et al.

between the casein matrix and fat globules of curd and Finally, the major challenge for liposome is the large cheese.142 These applications of enhancing natural preser- scale production method. Pharmaceutically acceptable pro- vatives, including antioxidants such as vitamin E and C, cedures are those that can be easily scaled to larger will undoubtedly become very important due to recent batch sizes and economically feasible. However, unlike the dietary trends which tend to reduce the addition of artificial classical pharmaceutical dosage forms (tablets, capsules, preservatives and increase portion of unsaturated fats in suppository) which are produced in large batch sizes, the diet. liposome based drugs even those already in the market In other areas of the agro-food industry, biocides encap- are produced in small size batches and thus are costly for sulated into liposomes have shown superior action due to the manufacturers. Scale-up process to larger size batches REVIEW prolonged presence of fungicides, herbicides, or pesticides is often a monumental task for the process development at reduced damage to other life forms. Liposome surface scientists. can be made sticky so that they remain on the leafs for However the accumulation of many novel experiences longer times and they do not wash into the ground.143 studying the practical aspects of liposomes, added to new developments in basic research, will bring the field of lipo- 6. CONCLUSION some biotechnology to the place it deserves in the future. An encouraging sign is the increasing number of clinical Since they have been discovered in the 60’s by Bangham, trials involving liposomes. liposomes have drawn attention of researchers. Nowadays, they always remain a topical issue; new preparation meth- References and Notes ods have been developed as well as new characterizationDelivered by Ingenta to: techniques. Guest User1. T. Lian and R. J. Ho, J. Pharm. 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Received: 21 March 2012. Accepted: 30 June 2012.

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