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13.1 13.2 13.3 13.4 13.5

Vesicular Carriers for Enhanced Delivery through the Skin
Elka Touitou and Biana Godin

Introduction ............................................................................................................. 255 Liposomes—Phospholipid Vesicles for Topical Administration of Drugs............... 255 Niosomes and Elastic Niosomes—Nonionic Surfactant Vesicles............................. 259 Tranfersomes—Ultradeformable Liposomes ........................................................... 263 Ethosomes—Soft Phospholipid Vesicles for Enhanced Dermal and Transdermal Delivery........................................................................... 264 13.6 Concluding Remarks................................................................................................ 273 References .......................................................................................................................... 273

Dermal and transdermal delivery requires efficient penetration of compounds through the skin barrier, the bilayer domains of intercellular lipid matrices, and keratin bundles in the stratum corneum (SC). Lipid vesicular systems are a recognized mode of enhanced delivery of drugs into and through the skin. However, it is noteworthy that not every lipid vesicular system has the adequate characteristics to enhance skin membrane permeation. Specially designed lipid vesicles in contrast to classic liposomal compositions could achieve this goal. This chapter describes the structure, main physicochemical characteristics, and mechanism of action of prominent vesicular carriers in this field and reviews reported data on their enhanced delivery performance.

Liposomes were first proposed for drug topical administration to the skin more than 25 years ago by Mezei and Gulusekharam [1,2]. The basic components of liposomes are phospholipids (phosphatidylcholine, phophatidylethanolamine, phophatidylserine, dipalmitoyl phosphatidylcholine, and others), cholesterol, and water. Liposomes may vary significantly in terms of size (from tens of nm to microns) and structure. In liposomes, one or more concentric bilayers surround an aqueous core generating small or large unilamellar vesicles (SUV, LUV) or multilamellar vesicles (MLV), respectively [3]. In the early 1980s Mezei and Gulusekharam published the results of their investigation on topical administration of triamcinolone acetonide (TA) delivered from liposomes, showing



Enhancement in Drug Delivery

higher drug concentrations in the upper skin strata and reduced drug percutaneous transport, as compared to a conventional formulation [1,2]. This local delivery behavior of liposomes was further confirmed by many studies where various drugs were tested in vitro or in vivo [4–16]. Investigators have mostly focused on dermal corticosteroid and retinoid liposome products. However, localized effects of liposome-encapsulated proteins, enzymes, tissue growth factors, interferons, and other immunomodulators have also been reported [17–20]. Another study showed that drug delivery to or across the skin could be modulated either by the use of liposomes or permeation enhancers, respectively. In this work, a high flux of caffeine through the skin was achieved from a system containing a combination of the enhancers diethylglycol and oleic acid, whereas the application of caffeine from small unilamellar liposomes resulted in accumulation of large drug quantities within the skin (Figure 13.1). Quantitative skin autoradiography results confirmed these findings, showing that the highest concentration of caffeine was accumulated in epidermis [10].
(a) Flux, µg/h ϫ cm2 1200 1000 800 600 400 200 0 PEG SV Aq.soln. PEG + enhanc. Aq.soln. + enhanc.


Qs24, µg/cm2 2500 2000 1500 1000 500 0 PEG SV Aq.soln. PEG + enhanc. Aq.soln. + enhanc.

FIGURE 13.1 Modulation of caffeine skin delivery by carrier design. Effect of different formulations on the flux (a) and the skin accumulation (Qs24) (b) of caffeine in in vitro permeation experiments through the hairless mouse skin for 24 h. Each system contained 3% of the drug. SV-small vesicle liposomes (mean diameter: 40 nm); PEG, polyethylene glycol base; Aq. soln., aqueous solution; PEG þ enhanc., PEG base containing 10% oleic acid and 20% transcutol; Aq. soln. þ enhanc., aqueous system containing 10% oleic acid and 20% transcutol.

have a retarding effect on the incorporated drug release. Hoffman’s group observed liposomal delivery of the active Lac-Z gene and its expression mostly in the hair follicles [26. In a further study. A number of clinical implications of drug reservoir formation in the upper skin layers by delivery from liposomes have been reported [4–6. The findings of this work show that after 24 h structural changes characterizing toxic dermatitis in the epidermis visualized by light and electron microscopies were more pronounced for the conventional formulation tested [22]. and 0.05% of the drug on the other. Tolerability is another aspect that should be considered during the design of topical preparations. 5% propylene glycol. These findings are in agreement with the results obtained by Egbaria and Weiner who tested the penetration of radiolabeled lipids (14C-dipalmitoylphosphatidylcholine and 3H-cholesterol) from liposomes into various strata of excised hairless mouse using the striping technique [16]. morphological changes following in vitro application of a liposomal (0. SUV structures were visualized on the stratum corneum surface at the interface of the liposomal dispersion and the stratum corneum (Figure 13. the liposomes appear to fuse and adsorb on the stratum corneum. From these studies it appears that the efficiency of liposomal-incorporated drugs was superior to other formulations in the treatment of disorders.Vesicular Carriers for Enhanced Delivery through the Skin 257 It is commonly agreed that direct contact between liposomes and skin is necessary for efficient delivery. evaluated efficacy and local tolerability [13]. In a double-blind study on 20 acne vulgaris patients. The in vitro skin penetration behavior of carboxyfluorescein incorporated in multilamellar liposomes (phosphatidylcholine: cholesterol: phosphatidylserine) and in another four nonliposomal systems (HEPES pH 7.05% and 0. Near the interface.025% or 0. This retarding release behavior from liposomes was further confirmed by a lower drug transport rate as compared to the gel measured across hairless mouse skin [30].13]. a liposomal betamethasone dipropionate was more efficient than a nonliposomal preparation in eczematous but not in psoriatic patients [6]. forming clusters of bilayers and rough structures on the top of the outermost corneocytes. 10% ethanol. due to their structure. [8] examined the . [25]. The work carried out by Hofland et al. Knepp et al.05% sodium lauryl sulfate) was studied by Lieb et al. Schafer-Korting et al.01%) on one side of the body and a commercial gel preparation with either 0. in an interesting setup of in vitro and in vivo experiments in mice.4 buffer. Liposomes applied on the skin were also investigated for their delivery proprieties to the pilosebaceous units [15. scaling. It was shown that liposomes. by using freeze fracture electron microscopy. No changes in the ultrastructure of the intercellular lipid regions were detected within the stratum corneum. in a doubleblind. The results of this study confirmed the work hypothesis that liposomes remain adsorbed on the skin surface thus producing drug reservoir in the upper cutaneous strata.025%) or conventional (0. In early studies.2).05%) tretinoin were investigated on reconstructed epidermis. Results indicate that despite the lower tretinoin content. randomized paired study on patients suffering from atopic eczema or psoriasis vulgaris. and rating redness. and burning on to a 4-point scale. who received for 10 weeks liposomal tretinoin (0. reported that progesterone release from agarose gel was faster than from liposomes embedded in the gel [29]. resulted in an important contribution to the understanding of the interaction between liposomes and human stratum corneum [21]. Further. Using two fluorescent techniques the authors found a higher accumulation of the probe within skin follicles when delivered from liposomes [25]. Another study by Foldvari et al. In this work the highest quantities of lipid probes were detected on the stratum corneum surface and no radioactivity was measured in the receiver compartment. the liposomal preparation was equipotent to the reference gels. For example. but significantly superior in terms of skin tolerability. which do not affect the deep layers of the skin. In this study.28].23–28]. not even in the lipid regions between the first and second layers of corneocytes. The measured clinical parameters included: quantity of comedones=papules=pustules.

258 Enhancement in Drug Delivery FIGURE 13. No changes in ultrastructure could be found deeper in the stratum corneum. the direction of platinum shadow scale bar (0. Pt. The liposomes are fusing edge to edge. (Reproduced from Hofland.1 mm). J.) . and patches of liposomal clusters (P) are formed on the top of the outermost corneocytes (C). et al. The liposomes appear to adsorb (A) on to the surface of the stratum corneum. 132. Br. Dermatol.. H. With permission from Blackwell Publishing.E. 1995. 853..2 Stratum corneum incubation with liposome suspension (S).

suggesting that this base favored dyphylline accumulation within the skin. Working under nitrogen or argon also minimizes the oxidation of lipids during preparation [32. freeze–thaw processes.3 NIOSOMES AND ELASTIC NIOSOMES—NONIONIC SURFACTANT VESICLES Nonionic surfactant vesicles (niosomes) were first proposed by Handjani-Vila et al. it is important to take into consideration that the base in which liposomes are incorporated could also affect the drug delivery profile as well as the interactions between the liposomes and skin structures. Some of these oxidation by-products tend to be rather toxic in biological systems. From a formulative point of view. gel filtration. [36] as systems to improve accumulation of the active molecule within the skin and thus benefit cosmetic products. These techniques suffer from one or more drawbacks such as the use of solvents (sometimes pharmaceutically unacceptable). the lipid vesicles may be subject to a series of adverse effects including aggregation. and hydrolysis [31– 35]. Results of this work show that the presence of additives decreased both the total amount released and the amount of drug in peak sample.4]. It is important to keep in mind that the delivery properties of the final formulation could be altered by the vehicle used and should be tested to confirm any performance characteristics claimed. With these bases. temperature. 7a. A number of liposomes are currently marketed by cosmetic raw material companies for incorporation in different bases to obtain final products. the lowest skin permeation flux and a superior skin partitioning of liposomal dyphylline were reported for the PEG base. a PEG-enhancer base. veegum. The stability of liposomes is of great concern during the design of topical formulations. Liposomes for topical delivery are prepared by the same classic methods widely described in the literature for preparation of these vesicles. buffer species. reverse phase evaporation. and the state of aggregation. Storage at low temperatures and protection from light and oxygen reduces the chance of oxidation. [9]. and solvent injection. cholesterol present in the liposomal vesicles is readily oxidized. extrusion). an additional sizing process to control the size distribution of final products (sonication. phospholipid oxidation. 7-keto-cholesterol. and methylcellulose. emulsification. Furthermore. The appearance of peak was also delayed in the presence of most additives. and the need for special equipment. Oxidation of phospholipids in liposomes mainly takes place via a free radical chain mechanism in unsaturated fatty acyl chain-carrying phospholipids. Further protection can be achieved with the addition of antioxidants. The hydrolysis is dependent on several factors such as pH.5a.and 7-hydroperoxides. The effect of formulation bases on dyphylline skin permeation from liposomes was examined by Touitou et al. and the 5. carbopol gel. The oxidation products 25-hydroxycholesterol. and water) was studied. As liposomal preparations contain an aqueous phase.6b-triol. One of the major drawbacks of liposomes is related to their preparation methods [3. cholestane-3b. 13. with the exception of methylcellulose when used at low concentrations. These methods include hydration of a dry lipid film. The majority of the liposome preparation methods are complicated multistep processes. or dialysis. acyl chain length and headgroup. In this work the effect of four bases for dyphylline liposomes (polyethylene glycol (PEG). fusion. multiple-step entrapment procedure for preparing drug-containing liposomes. . which had activity causing toxic effects on aortic smooth muscle cells [35].33]. were found in a concentrate.and 7b-hydroxycholesterol.Vesicular Carriers for Enhanced Delivery through the Skin 259 release and the pharmacodynamic effect of tetracaine delivered from liposomes in the presence of gel-forming polymers including carbopol. ionic strength. creating a stability problem for lipid-based drug products. Liposome preparation is followed by homogenization and separation of unentrapped drug by centrifugation. These reports opened the way for an intensive investigation of these vesicles as carriers for skin administration of drugs [37–47].

40. these vesicles are analogous to liposomes. and others. and mechanism of action of niosomes was published by Uchegbu and Vyas [41].5. respectively) [40]. crown ether. vesicular structures of about 100 nm size have been observed between the first and second layers of human corneocytes 48 h after incubation as well as in the deeper strata of the skin [37]. It is commonly accepted that the hydrophilic lipophilic balance (HLB) is a parameter that could indicate the vesicleforming potential of surfactants. low HLB values could predict vesicle formation [52.) to obtain a homogeneous colloidal dispersion and separation of the unentrapped drug [36. perfluoroalkyl surfactants.5. homogenization. alkyl glycerol ethers. For amphiphils such as sorbitan esters and alkyl ethers. chemical composition. Wasag-7. polysorbate–cholesterol mixtures. Occlusion is a condition that could affect drug transport from niosomes and through the stratum corneum. and 42+2 ng=cm2=h. when cholesterol at an appropriate concentration was added to the amphiphil [44]. Such an effect was reported for saturated estradiol niosomal formulations composed of polyoxyethylene alkyl ether surfactants and sucrose ester surfactants with cholesterol and dicetyl phosphate. Niosomes. A number of works investigated the interaction between niosomes and human skin. for which occlusion enhanced the drug human stratum corneum transport [43]. The investigated systems resulted in a relatively low encapsulation efficiency of the drug.48–55]. ester. In this case it could be assumed that a kind of amphiphilic complex with a lower HLB was responsible for the vesicle formation. Among the hydrophilic headgroups found in these amphiphils are ethylene oxides.55]. In certain cases.55]. sorbitan esters. m ¼ 3 or 7. etc. characteristics. all being saturated systems containing 1. small unilamellar niosomes and a micellar solution. glycerols. With niosomes prepared from C12 alcohol polyoxyethylene ether and cholesterol.75 mM estradiol. The hydrophilic and the hydrophobic moieties are generally linked by ether. prepared from polyoxyethylene alkyl ethers (CnEOm. were tested for skin delivery of protonated and unprotonated lidocaine. An excellent review on the structure. No significant differences in the flux through human stratum corneum membrane were obtained when the drug was applied from vesicles or a control solution through an occlusive or nonocclusive mode [45]. Niosome formation requires the presence of a particular class of amphiphile and aqueous solvent. extrusion. Among the surfactants we can enumerate polyoxyethylene alkyl ethers. However. Hofland et al. In another study.41. a highly hydrophilic molecule. The methods for preparation of niosomes are similar and as complicated as those used for liposomes. crown ether derivatives. The hydrophobic moiety of the surfactant may contain one or more alkyl (C12–C18) or perfluoroalkyl (C10) groups or a steroidal group. two-photon fluorescence microscopy confirmed that fluorescent probe encapsulated in niosomes was confined to the intercellular spaces within the apical stratum corneum layers [56]. One of the most frequently utilized techniques consists of the hydration of a mixture of the surfactant–lipid at elevated temperature followed by optional size reduction (by sonication. Basically.7). The authors concluded that the structures visualized in the deeper regions could be vesicles reorganized from individual molecules that penetrated the skin. In this study examining drug delivery from multilamellar niosomes. or amide bonds. electron micrographs illustrated that niosomes containing surfactants and cholesterol affected only the most superficial corneocytes. Moreover. polyhydroxyls. niosomes were obtained from polysorbate 20 (HLB 16. cholesterol is required for vesicle formation. and sugars. investigated the in vitro permeation behavior of estradiol from niosomes (n-alkyl polyoxyethylenes=cholesterol) through human stratum corneum. and 0.260 Enhancement in Drug Delivery Niosomes are bilayer structures formed from amphiphiles in aqueous media. very low permeation fluxes were detected (64+17. 45+15. Many types of surfactants have been used for formulation of niosomes [41.52. 1. n ¼ 12 or 18) or stearate=palmitate sucrose ester. .

Thin layer chromatography measurements showed that after 1 h application of elastic and rigid vesicles. in contrast to rigid vesicle material.5) could be encapsulated within the vesicles (~30%). various L-595=PEG-8-L=sulfosuccinate vesicles. [56–58] introduced a new type of niosomes. or aqueous solutions of these drugs. following 12 h in vivo application. the quantity of PEG-8-L vesicle constituent in stratum corneum significantly decreased as compared to the rigid vesicle material. addition of polymerized surfactants. only one niosomal system comprised of glyceryl dilaurate= cholesterol=polyoxyethylene-10-stearyl ether improved drug delivery into skin shafts as compared to other niosomal systems. the surfactants. skin occlusion enhanced drug permeation from elastic vesicles as well as from the buffer solution. were investigated in comparison to a saturated buffer solution. In one of these works. steric and electrostatic stabilizers to the formulation [41. In this work. the encapsulated drug.3). interferon-a (INF-a) and cyclosporin into the pilosebaceous units of the hamster ear [46]. In a further study. Data obtained by tape-stripping suggested that the lipid probe and the drug from elastic vesicles can enter the deeper layers of the stratum corneum. the elastic surfactant vesicles containing octaoxyethylene laurate-ester (PEG-8-L) and sucrose laurate-ester (L-595). Elastic vesicles also improved ketorolac permeation across the skin (Figure 13. storage temperature. Niemiec et al. Van den Bergh et al. For this purpose. the interfacial polymerization of surfactant monomers in situ. vesicle aggregation may be prevented by inclusion of . from very rigid to very elastic. fatty acid spin labels were incorporated into the systems [58]. This behavior was explained by the entrapment ability of the hydrophilic moiety within the aqueous core of the vesicles.6) remained unattached. These authors investigated the delivery of lidocaine HCl and lidocaine base from vesicles through silicone membrane and nude mice skin [44]. To study the elasticity of the vesicles. hydroethanolic. ketorolac-loaded elastic (PEG-8L: L-595) and rigid vesicles (L-595 only) applied nonocclusively to the skin were used to quantify skin distribution profiles of a deuterium-labeled phospholipid (as a marker for the vesicle constituent) and ketorolac [59]. However. Niosomes were also investigated for their ability to deliver drugs to hair follicles. In this study. The amount of lidocaine permeated through nude mice skin from these niosomes was similar to liposomes and only about twofold greater than from a micellar system. The in vivo interactions of these elastic vesicles with hairless mouse skin were evaluated following nonocclusive application in comparison to rigid vesicles composed of sucrose stearate-ester (Wasag-7) [57].52]. In general. Mechanistic studies on elastic vesicles were further conducted by Honeywell-Nguyen et al. showed that niosomes could be obtained from polyoxyethylene sorbitan monolaurate–cholesterol in aqueous environment. Carafa et al. It was found that only the charged molecule (loading pH 5. after a longer application time.Vesicular Carriers for Enhanced Delivery through the Skin 261 More recently. cholesterol. It may be possible to stabilize niosomes by a variety of methods such as the use of membrane-spanning lipids. the effect of elastic and rigid vesicles on the penetration of pergolide across human skin was further assessed in vitro using flow-through Franz diffusion cells [60]. investigated the deposition of two polypeptide drugs.64. The results of these studies indicate that the molar content of PEG-8-L in vesicles directly affects the bilayers elasticity. It is noteworthy that the highest pergolide skin permeation was obtained when a simple saturated buffer solution not containing vesicles was applied under occlusion. liposomes. Disrupted organization of skin bilayers and increased skin permeability were observed following application of elastic vesicles but not the rigid ones. The stability of various niosomal formulations depends on factors such as preparation methods. The lipophilic unionized form of lidocaine (loading pH 8.65]. and additive mixture [41. a sixfold increase in the amount of elastic vesicle constituents was present within the stratum corneum compared to Wasag-7 rigid vesicles.52. [59–63].

The cumulative amounts found after 1 and 4 h of elastic vesicle treatment (corresponding to the time periods chosen for this study). (Reproduced from Honeywell-Nguyen.0 Cumulative amount (ng/cm2) 50. Rigid ves. As an example. J.0 0. Elastic vesicles were clearly more effective as compared with rigid vesicles in the enhancement of ketorolac transport across the skin. The concentrations of ether surfactants required to inhibit cell proliferation by 50% were 10-fold lower than for ester surfactants. 902.0 150. 123. et al.0 1 2 3 4 2000.55].. However. FIGURE 13. Neither the HLB nor the critical micelle concentration values or cholesterol content affected keratinocyte proliferation..L.0 Enhancement in Drug Delivery 8000.) molecules that stabilize the system. Dermatol.0 0 5 10 Hours (h) 15 20 Elastic ves. the toxicity of polyoxyethylene alkyl ether vesicles containing C12–18 alkyl chains and 3 and 7 oxyethylene units was assessed by measuring the effect on proliferation of cultured human keratinocytes [47].0 100. however. It was found that the length of either polyoxyethylene headgroup or alkyl chain had only a minor influence on keratinocyte proliferation.000.0 6000. Steric and electrostatic stabilization of sorbitan monostearate niosomes could be achieved by inclusion of cholesteryl polyoxyethylene ethers or dicetyl phosphate [54. Invest. 2004.262 10.0 0. were still very low.3 The cumulative amount of ketorolac as a function of time from elastic and rigid vesicle formulations across human skin in vitro. Skin safety of niosomes was tested in a number of studies. With permission from Blackwell Publishing.0 0 4000. P. the ether surfactants were much more toxic than esters tested in this study. .

water and sometimes very low concentrations of ethanol ( 7%) [66]. in the early 1990s [66– 69]. reported that the vesicles failed to transfer detectable quantities of cyclosporin A through the hydrated abdominal mice skin [71]. . The preparation process is usually followed by homogenization. Voltaren measured deep in the hind legs was close to 10. following epicutaneous application of insulin associated with transfersomes (Transferinsuline) the first signs of hypoglycemia were observed at 90–180 min and a maximum transfersome-mediated decrease in blood glucose levels was about 35% of the effect of the same insulin dose administrated through subcutaneous injection.Vesicular Carriers for Enhanced Delivery through the Skin 263 13.5 mg=g for Transfenac and Voltaren. the deformable vesicle is able to squeeze and forge through the small pores [66. deformable liposomes. It is suggested that these lipid vesicles could avoid the osmotic tension by dehydration. or other mechanical means to reduce the size of the lipid vesicles. Transfersomes dehydrate on the skin surface by evaporation. Similar results were obtained in pigs (Figure 13. or both. rats. An early study confirmed that occlusion is detrimental to transfersome penetration enhancement ability.4). The micelles may further delipidize the stratum corneum creating small pores through which the drug could penetrate. murine skin permeation of fluorescently labeled lipids from transfersomal suspensions and liposomes is comparable [67]. Thus.72. [74–76] designed and characterized transfersomes with immunomodulators interleukin-2 (IL-2) and INF-a and reported that both molecules retained their biological activity and could be efficiently encapsulated in these vesicles.4 TRANFERSOMES—ULTRADEFORMABLE LIPOSOMES Transfersomes. In a study carried out in normoglycemic human volunteers. are generated. and pigs [70]. under the occlusive conditions. concentrated micellar systems of cholates or cholates–phospholipids. Transfersomes are prepared by the same methods as liposomes.77–79] and these studies have been recently reviewed by Cevc [80]. respectively. Another possible explanation for the fact that transfersomes are able to deliver molecules only under nonoccluded conditions could be that as a result of water evaporation from the applied sodium cholate–phospholipid aggregates system. The mechanism of skin permeation by transfersomes as proposed by the authors involves a number of processes.6 mg drug=kg body weight the ratio of diclofenac quantities delivered from Transfenac vs.73]. and adjuvants [67. Guo et al. a surfactant edge activator (such as sodium cholate).7 and 3.68. vaccines. The cumulative pharmacodynamic response of at least 50% of the subcutaneous dose was obtained after skin application of insulin from transfersomes with systemic normoglycaemia that lasted for 16 h [67. A study on diclofenac-containing transfersomes (Transfenac) carried out in comparison to a commercial Voltaren emulgel formulation evaluated the distribution of radiolabeled drug and a number of pharmacokinetic parameters in mice. In rats. nonoccluded administration is vital to allow for enhanced delivery from transfersomes. The transfersome technology has been tested for delivery of a number of additional molecules including steroids. The authors claim that due to the presence of the polar surfactant molecule in the lipid phase of the aggregate. The relative drug quantities measured in the mice dorsal muscles epicutaneously treated with 9 mg diclofenac=kg were 6. following application of 0. were introduced by Cevc et al. Hofer et al. The main components of these systems are phospholipids. Furthermore.70]. which occur when the system is applied nonoccluded to the skin. The results of this study clearly demonstrated that. sonication. resulting in an osmotic pressure difference between the region of higher water concentration inside the skin and the nearly dry surface of the skin. thereby opening narrow intercellular pores in the stratum corneum and penetrating the barrier.

Biophys. Acta.25 mg=kg body weight (right-dashed). 1514. (Reproduced from Cevc. and water) in a hydroethanolic milieu. 13. In further work these authors replaced sodium cholate with Span 80 and Tween 80 for preparation of ultradeformable vesicles of estradiol [82]. on the hind thigh of mice. and delivery properties.50 mg/kg 9. the authors compared the potential use of ultradeformable and standard liposomes as skin drug delivery systems and found that the ultradeformable formulation was superior to standard liposomes for in vitro skin delivery of 5-FU [81]. and 9 mg=kg body weight (left-dashed) of the drug in ultradeformable vesicles. ethanol. were so named to emphasize the . 16-.5 ETHOSOMES—SOFT PHOSPHOLIPID VESICLES FOR ENHANCED DERMAL AND TRANSDERMAL DELIVERY Although frequently referred to as a kind of liposomes. In one work.25 mg/kg 4. Interestingly.) Various aspects of skin drug delivery from transfersomes were also studied by Barry’s group [81–85].264 Enhancement in Drug Delivery 2. Ethosomes. mechanism of action. transfersomes. and Tween 80 as compared with aqueous control.Remote-site Application.. G. 4. Ethosomal carriers contain soft lipid vesicles (mainly composed of phospholipids. They have appropriate features.25 mg=kg body weight (right-dashed). invented by Touitou [86–88].Remote-site FIGURE 13. 191. Span 80.4 Left: Tissue concentration of diclofenac-derived 3H-radioactivity at t ¼ 12 h after an epicutaneous application of 2. 4. in these systems the entrapment efficiency was less than 10%. The maximum fluxes of estradiol through the human epidermal membrane increased by 18-. t = 12 h. designed to allow for enhanced delivery by passive transport to the deep skin strata and through the skin [86–90]. Their delivery enhancing properties can be modulated by changes in the composition and structure. With permission from Elsevier. ethosomes are very different from other lipid vesicles by their composition.5 mg=kg body weight (hatched).00 mg/kg On the hind-legs skin. 2001. Right: Biodistribution of diclofenac-derived 3H-radioactivity 12 h after an application of 2. 3 NMRI mice Transfenac 102 Voltaren-Emulgel (µg/g tissue) 3H-diclofenac 101 100 10Ϫ1 in usc Sk M le Sk in M us cle Bl oo d d er ad Liv Bl er Sk in M us cle Sk in M us cle Bl oo d d er ad Liv Bl er Application. The authors concluded that these nonionic surfactants are as efficient edge activators as sodium cholate in the preparation of transfersomes. and 15-fold for vesicles containing sodium cholate.5 mg=kg body weight (hatched). structure. G. and 9 mg=kg body weight (left-dashed) in the commercial hydrogel. Biochim. and Blume.

This suggestion was further strengthened by fluorescent anisotropy FIGURE 13.Vesicular Carriers for Enhanced Delivery through the Skin 265 presence of ethanol in a vesicular structure. 65. d).94] or multilamellar [86.. with their sizes ranging from 30 nm to microns [96]. In contrast to this belief. the authors were able to visualize the ethosomal vesicles. e).95]. lipophilic. and water by TEM: (b) Entrapment of fluorescent probes by phopholipid vesicles as visualized by CSLM. f ). Ethosomal systems may contain unilamellar [93. transmission electron microscopy (TEM). the existence of vesicles and the structure of ethosomes were evidenced by various methods including 31P-NMR. To gain insights into the characteristics of ethosomes that allow them to efficiently promote drug delivery into and through the skin the transition temperature (Tm) of vesicular lipids and free energy measurements of the vesicle bilayers were assessed by differential scanning calorimetry (DSC) and fluorescence anisotropy [86. Phosphorous NMR spectra of ethosomal systems exhibited a solid-state lineshape. D-289 (b. or calceine (c.93–96] (Figure 13. the phospholipid bilayer configuration typically observed in phosphatidylcholine vesicles in the aqueous environment. J. For example. (Reproduced from Touitou.) . White represents the highest concentration of probe.95] lipid vesicles.94. the paramagnetic-ion NMR spectra pointed toward a greater ethosomal phospholipid membrane fluidity and permeability to cations.92]. Liposomes (a–c) or ethosomes (d–f ) were prepared with one of three fluorescent probes: rhodamine red (a. The presence of ethanol. whereas negatively stained transmission electron micrographs showed that multilamellar ethosomes are characterized by a structure with phospholipid bilayers throughout the vesicle [86. One of the distinguishing characteristics of ethosomes is their ability to efficiently entrap molecules of various lipophilicities (Figure 13. allows for efficient entrapment of hydrophilic. respectively. 2000. Moreover. Due to the interdigitation effect of alcohol on lipid bilayers. and scanning electron microscopy (SEM) [86]. ultracentrifugation studies demonstrated that the encapsulation efficiency of ethosomes could be as high as 90% and 83% in the case of the lipophilic drugs testosterone and minoxidil.5a). 30% ethanol. With permission from Elsevier. et al.95]. in comparison to liposomes [86]. Control. it was previously thought that the ethanol milieu is destructive to vesicular structures and that vesicles could not coexist with high concentrations of alcohol [91.5 (a) Visualization of a typical multilamellar ethosome containing 2% PL. Scanning electron micrographs confirmed a three-dimensional nature of ethosomal vesicles. Release. and amphiphilic molecules [86]. and are explained by the fluidity of phospholipid bilayers in ethosomes [86.5b). Using the electron microscopy techniques. E. The Tm values were reported to be 208C–358C lower for ethosomes as compared to liposomes composed from the same phospholipids but without ethanol. together with high-vesicle lamellarity. This is different from the problematic entrapment of lipophilic compounds in liposomes restricted by a limited number of phospholipid bilayers surrounding the aqueous core. 403.

These results evidenced that ethosomal vesicles possess a soft malleable structure.5% of initial) of phosphatidylcholine (PL) permeated the skin during a 24 h experiment.) . Control. which could be related to the fluidizing effect of ethanol on the phospholipid bilayers [91. For example. which shed more light on understanding this sequence of synergistic processes. et al. were obtained by researchers from the skin permeation studies.92]. Based on the results obtained in fluorescent anisotropy and DSC experiments as well as in skin permeation studies. suggesting that the vesicles might have traversed the skin strata.266 Enhancement in Drug Delivery measurements of AVPC (9-Antrylvinyl labeled analog of phosphatidylcholine) where a 20% lower value was measured in comparison to liposomes [86]. 65. E. fluorescently labeled bacitracin Intercellular pathway Keratinocyte Stratum corneum Stratum corneum lipid multilayers Drug Ethanol Ethosome (I) Action of ethanol (II) Action of ethosomes a b c FIGURE 13. Further. Release. The authors suggest that the fluidizing effect of ethanol on the lipid bilayers of stratum corneum together with the characteristics of ethosomes contribute to the skin permeation enhancement of drug from the ethosomal carrier.6 Proposed mechanism for permeation of molecules from ethosomal system through stratum corneum (SC) lipids. In a further confocal laser scanning microscopy (CLSM) study by Godin and Touitou. the alcohol interferes with the lipid organization in stratum corneum.6).. the soft vesicle penetrates the fluidized stratum corneum bilayers forging a pathway through the skin by virtue of its particulate nature and later on fuses with cell membranes in the deeper skin layers releasing the active agent there. ethosomes are much less rigid. The softness of ethosomal vesicles imparts to them the ability to penetrate the disturbed stratum corneum lipid bilayers and promote delivery of active agents into the deep layers of the skin and through the skin. 403. a mechanism for enhanced delivery by the ethosomal system was proposed [86] (Figure 13. it was found that an important amount (10. (Reproduced from Touitou. With permission from Elsevier. In comparison to liposomes. Further data. Besides this important fluidizing effect of ethanol on the phospholipid vesicle bilayers. J. 2000.

B. Charged and hydrophilic large molecules (polypeptides. penetrated the rat skin through the intercorneocyte pathways. which typically exist along the lipid domain of the stratum corneum [95] (Figure 13.95. liposomes and hydroethanolic solution..1% FITCBac following an 8 h skin exposure: ethosomes vs. With permission from Elsevier. when calceine was applied from liposomes or from a hydroethanolic solution. J. which does not usually cross lipid bilayers. In contrast.Vesicular Carriers for Enhanced Delivery through the Skin 267 FIGURE 13. no deep penetration of RR from liposomal dispersion was visualized. In contrast. the ability of a rhodamine red dihexadecanoyl glycerophosphoethanolamine (RR). When testing the enhanced skin permeation properties of ethosomes. which was delivered from trihexyphenidyl HCl ethosomes deeply into the skin (170 mm) with a significantly higher intensity than the two control systems [93] (Figure 13. respectively [97].8). proteins) were challenging compounds tested with ethosomal carriers [93. it is important to compare them to hydroethanolic solutions.97].98–100]. to penetrate deep into the skin was evaluated by CLSM [86. lower MaxFI values were obtained followed by a sharp decrease of fluorescent intensity to zero at the depths of 60 and 80 mm. Touitou. The authors reported that for calceine. hydroalcoholic solution. a maximum fluorescence intensity (MaxFI) value of 150 arbitrary units (AU) was obtained when delivered from ethosomes. liposomes.) (FITC-Bac) delivered in vivo from ethosomes. 365. Control. delivery of two additional fluorescent probes possessing distinct characteristics. which contain the system ingredients in various combinations. and dropped to zero only at 160 mm. The obtained CLS micrographs of nude mice skin after 8 h application of RR from ethosomes.7). which is a phopholipid probe.or intracorneocyte fluorescence were observed with FITC-Bac hydroethanolic solution and liposomes. a hydrophilic probe calceine and an amphiphilic cationic probe D-289 (4-(4-diethylamino) styryl-N-methylpyridinium iodide) from ethosomes and control systems was assessed [93. E. The ability of trihexyphenidyl hydrochloride . in contrast to liposomes that remained on the skin surface. An important number of agents from various pharmacological groups and with a variety of physicochemical characteristics were formulated with ethosomes and tested in vitro. This value was reached at the skin depth of 20 mm. In agreement with previously described works on liposomes. 2004. In further CLSM studies. 94.7 Reconstituted CLSM optical slices of the stratum corneum of the skin following skin delivery of FITC-Bac in vivo in SD rats. As RR is used as an indicator of lipid fusion. remained constant throughout approximately 50 mm. or phospholipid ethanolic solutions.97]. In research using fluorescent probes. and liposomes illustrate that delivery from ethosomes resulted in the highest intensity of fluorescence up to a depth of 150 mm. the results obtained in these experiments suggest that ethosomes traversed the skin strata to a high depth. (Reproduced from Godin. respectively. significantly lower fluorescence staining of the intercellular penetration pathway and no inter. Similar data were measured for the amphiphilic cationic probe D-289. in animals and clinical studies. Comparison of skin permeation routes from systems containing 0.96. Release.

268 Enhancement in Drug Delivery FIGURE 13. In this work the anti-inflammatory effect of CBD ethosomal systems was also evaluated. following 8 h application from systems containing 0.537 mg CBD=g muscle) [96. Lodzki et al. N. THP fluxes were 87. Testosterone. phosphate buffer. insulin application from a control nonethosomal formulation was not able to reduce the blood levels of glucose . 1879. a molecule with log P ~ 8. Significant differences in the pharmacodynamic profiles between CBD-treated and -untreated animals at all times during the experiment were observed.9) [96. and hydroethanolic drug solution. respectively. to be delivered across the skin from ethosomes was investigated [93].03% D-289 and 1% THP: (a) liposomes (containing 2% PL) (b) 30% hydroethanolic solution and (c) ethosomes (containing 2% PL and 30% ethanol). These compounds accumulate within stratum corneum layers and encounter problems in their clearance into the viable epidermis interface where partitioning into a predominantly aqueous environment is required. Furthermore.) (THP). and Touitou. significantly greater quantities of this cationic molecule were detected in the skin following delivery from ethosomes than from any other control system tested. CBD’s plasma concentrations reach steady-state levels of 0. (Reproduced from Dayan. THP ethosomes designed in this work were found to efficiently entrap THP (75%) and be stable for at least 2 years at room temperature.15 mg=cm2) and in the underlying muscle (11. The possibility to tailor the desirable hypoglycemic effect by modulating system composition presents another advantage reported for ethosomal insulin [99].99. It is noteworthy that the ethosomal insulin system resulted in a favorable pharmacodynamic profile with the plateau effect lasting for at least 8 h. The application of 100 mg ethosomal composition containing 3% CBD on nude mice skin for 24 h resulted in a significant drug amount transported through the skin (559 mg=cm2) and formation of an important CBD skin reservoir (845 mg=cm2). As known. from an ethosomal carrier [101–103]. 21. Pharmacokinetic study in ICR mice revealed that following system application to the animal abdomen for 72 h. examined the transdermal delivery of cannabidiol (CBD). 51.101]. In further work with ethosomal insulin. an anti-Parkinsonian agent.07 + 24.100].21 mg=cm2=h) than for liposomes. With permission from Elsevier.8 Visualization of penetration of fluorescent probe D-289 to full-thickness nude mice skin by CLSM. and 4. Biomaterials.67 mg=mL in 24 h and last at least until the end of the experiment. E. highly lipophilic molecules (log P > 5) exhibit insufficient transdermal absorption.. On the other hand. The obtained data show that the ethosomal carrier enabled percutaneous absorption of insulin by passive diffusion. . a significant decrease (up to 60%) in blood glucose levels (BGL) in both normal and diabetic rats was measured (Figure 13. In this study. The authors further reported that in vivo application of CBD ethosomes to the abdominal skin of CD1 nude mice resulted in significant localization of the molecule within the skin (110. indicating that the inflammation was prevented by transdermal delivery of ethosomal CBD [102]. 2000.5 times higher for ethosomes (0.

control) 50% 40% 30% 20% 10% 0% 0 1 2 3 4 Time (h) 5 7 6 8 FIGURE 13. no treatment in (a) diabetic and (b) normal SD-1 rats. control) 40% 30% 20% 10% 0% 0 1 2 3 4 Time (h) 5 7 6 8 (b) Normal rats 70% 60% Delta of % glucose concentration decrease (Ethosomes vs. A 30-fold higher quantity of testosterone was found to permeate in vitro through rabbit pinna skin from an ethosomal patch. is an additional lipophilic molecule tested with ethosomes. USA) for systemic absorption in vivo in rats and skin permeation through human skin in vitro [104]. each containing 0. prescribed as a hormone replacement therapy in primary and secondary hypogonadism. AUC and Cmax values measured in rabbits treated with Testosome were 2. In a more recent study. AndroGel (Unimed. a nonpatch ethosomal testosterone system was designed and tested vs. (848. the estimated application area needed for obtaining physiological testosterone human plasma levels is only 40 cm2.25 mg hormone=cm2. For this ethosomal testosterone formulation.38 mg) as compared to Testoderm (Alza).4 times higher than in animals treated with Testoderm [86]. respectively. Testosome.Vesicular Carriers for Enhanced Delivery through the Skin 269 (a) Diabetic rats 50% Delta of % glucose concentration decrease (Ethosomes vs.2 and 2. A single dermal application of 400 mg ethosomal formulation containing 1% testosterone resulted in Cmax and AUC values of 1970 + 251 ng=dL and 9313 + 385 and ng=dL*h.9 Delta blood glucose levels measured following in vivo application to the abdominal area of ethosomal insulin vs. These works demonstrated that ethosomes possess the ability not only to enhance the drug partitioning .16 + 158. which is approximately 10-fold lesser than for the currently marketed products.

27 Â 107 cfu=g tissue on days 7 and 10. In vivo experiments with ethosomal bacitracin demonstrated that the antibiotic was efficiently delivered into deep skin layers from ethosomes but not from liposomes or a hydroethanolic solution [95]. double-blind. ZC (1. respectively.9 days. 3.2 vs. It is worth mentioning that in in vitro experiments occlusion had no effect on drug skin transport and similar flux values were obtained for occlusive and nonocclusive applications. Optimization of drug delivery to the target tissue is the important goal in modern therapy. but also to permit its clearance into the hydrophilic environment. In these studies. In the parallel arm 80% of lesions crusted on the third day from the initiation of treatment with EA compared to only 10% in the ZC group. 5. In this work ACV ethosomal cream was compared to a commercial product (Zovirax. The assessed clinical parameters were the time to crust formation. Of particular interest are the results of the in vivo study on Staphylococcus aureus infected mice. effective bacterial kill with ethosomal erythromycin could ultimately result in minimizing bacterial resistance. both the time to crust formation and the healing period were significantly reduced with EA vs. thereby potentially increasing patient compliance. bacterial counts of the infected tissues were 1.96. aureus. and was nontoxic to dermal 3T3 cultured fibroblasts in live=dead viability=cytotoxicity assay [94].105–107]. A very efficient healing of S. In contrast. In the crossover arm. In this work the authors compared the efficiency of ethosomal erythromycin applied to the skin-infected site with parenteral or topical administration of the drug in a hydroethanolic solution. only 10% with ZC (Figure 13.5 days and 4. . In these animals. The results of this clinical trial show that treatment with EA formulation significantly improved all evaluated clinical parameters. a new formulation containing ethosomal ACV (EA) was designed and evaluated in a two-armed.270 Enhancement in Drug Delivery into the lipophilic layers of the skin. Insufficient drug delivery into the basal epidermis where virus replication occurs is at the origin of the low efficiency of acyclovir (ACV) dermal products for Herpes simplex topical treatment [108–110]. Keeping this in mind. The fraction of abortive herpetic lesions was ~30% in the case of EA vs. time to loss of crust (the healing period). The results clearly showed that EA significantly improved all evaluated clinical parameters in both parallel and crossover arms. and the percentage of abortive lesions (lesions not progressive beyond the papular stage). animals treated with topical hydroethanolic erythromycin solution developed deep dermal abscesses with destroyed dermal structures colonized by S.95. It was found that the therapy with ethosomal erythromycin was as effective as the systemically administered erythromycin. Recently. a novel approach to treat these problematic infections by local application of antibiotic in ethosomal carrier was investigated [94. aureus-induced deep dermal infections and zero bacterial skin counts were measured when the mice were treated with ethosomal erythromycin.06 Â 107 and 0. ethosomes enabled the delivery of the polypeptide antibiotic bacitracin in vitro and in vivo through human cadaver and rat skin. Histological evaluation of the skin treated with ethosomal antibiotic revealed normal skin structure with no bacterial colonies. respectively. This new therapeutical mode could result in decreased drug exposure and associated side effects. Further work shows that the ethosomal system significantly improved antibacterial action of erythromycin as compared to free drug in in vitro susceptibility tests. bacterial infections localized within deep dermal and subdermal tissues are cured only by systemic antibiotic administration. leading to transdermal delivery.8 vs. respectively). randomized study. Currently. which could be explained by the efficient delivery of the drug to its target tissue. Data discussed above suggest the possibility to substitute systemic antibiotic administration with topical ethosomal drug application in clinical use. designed to test the hypothesis that ethosomal erythromycin is able to eradicate deep dermal infection [107].10). In addition to these therapeutic benefits. ZC) in 40 subjects (61 herpetic episodes) [111].

4 3. 50.9 4.8 5.) Ethosomal carriers were found to be very efficient in promoting delivery of molecules to pilosebaceous and hair follicular units.5 1. of abortive lesions.11). randomized. With permission from John Wiley & Sons.10 Parameters assessed in a two-armed.3 6. Zovirax cream (ZC). Quantitative skin autoradiography [113–115] was further utilized to assess the levels of H3-minoxidil within pilosebaceous elements of the skin. double-blind clinical study in RHL patients with two formulations containing 5% acyclovir: ethosomal acyclovir (EA) vs.5% minoxidil and 50 mCi H3-minoxidil to the dorsal region of hairless rats for up to 24 h resulted in the accumulation of the tritiated drug within the skin follicles (Figure 13. (c) number of abortive lesions (%).Vesicular Carriers for Enhanced Delivery through the Skin (a) 271 Parallel arm 12 10 t. (b) data obtained in crossover arm. days 6 4 2 0 Days to crust formation ZC EA Days to loss of crust 3.112]. Drug Dev. E. days 8 6 4 2 0 Days to crust formation ZC EA Days to loss of crust 1. Tailored minoxidil ethosomes were investigated in vivo for drug localization into the pilosebaceous units as a potential way to improve its therapeutic effect in hair loss disorders [96.5 (b) Crossover arm 10 8 t. et al. In vivo application of ethosomes and liposomes containing 0. % 30 20 10 0 Parallel arm ZC EA Crossover arm 10 7 33 27 FIGURE 13. The obtained results demonstrated that the ethosomal system was 5 times more efficient in . 2000.6 4. (Reproduced from Touitou. 406. (a) Data obtained in parallel arm.2 (c) Abortive lesions 40 No.. The designed minoxidil ethosomes appeared as multilayered vesicles with an encapsulation capacity of 83+6%. Res..

In animal studies on rabbits no acute skin irritation was observed following a single-dose 48 h occlusive application of patches containing the ethosomal systems. the produced vesicles are of uniform size and no homogenization=size-reducing steps are required.88]. respectively.005) [90].5 nmol=g tissue. The mean size of empty and cationic trihexyphenidyl .272 Enhancement in Drug Delivery (a) (b) (c) FIGURE 13.5% minoxidil and 50 mCi tritiated drug to the dorsal region of hairless rat (6 cm2) in vivo. (a) skin autoradiogram.11 Localization of H3-minoxidil within the pilosebaceous units. methods used for preparation of ethosomes do not require special equipment and can be scaled up without difficulty.93. Moreover. cumulative 14 d repeated ethosomal patch application also did not generate any significant erythema [86]. In this study no significant difference in erythema index (DEI) was measured between skin areas treated with ethosomes and saline [116]. Furthermore. or 48 h application of ethosomes containing 2% PL and 45% ethanol on healthy human volunteers. In another study following 12. The stability of ethosomal systems incorporating various drugs was assessed in a number of studies by comparing the average diameter and the structure of the vesicles during a 2-year period at room temperature [86. The specific methods for ethosomes preparation are given in patents published on ethosomes [87. In contrast to liposomes and other vesicular carriers described in this chapter. targeting minoxidil to the skin shaft than liposomes (22 vs. which are used to prepare stable ethosomal formulations depending on drug and the target of drug delivery. 24. The tolerability and safety of ethosomes were tested in cultured skin cells. on animals and in humans. An in vitro live=dead viability=cytotoxicity viability test carried out with various vesicular systems and controls indicated that ethosomal carriers were not toxic to 3T3 fibroblasts and cultured cells kept their viability [97]. (c) superimposition of a and b. (b) histological image. p < 0. the formulation was very well tolerated and no signs of erythema were detected. Skin images were obtained following 12 h application of ethosomal system containing 0.94]. 4. There are a number of methods.

Mezei. open vast potential therapeutic uses. Oxford: Oxford University Press. O. ethosomes.I. The high tolerability and efficiency of vesicular systems. and the elastic vesicles. and elastic niosomes.. a selective drug delivery system for the topical route of administration.Vesicular Carriers for Enhanced Delivery through the Skin 273 loaded vesicles remained unaffected during the storage interval. Crit Rev Ther Drug Carrier Syst 11:97. H. Moreover. or follicular delivery. whereas the diameter of ethosomes following a 1-year interval was 117 + 18 nm. 1990. 1992. Gulusekharam.C. . 13. REFERENCES 1. Data on SupraVir cream (Trima. Wohlrab. a classic liposomes remain confined to the upper skin layers. Gulusekharam. Liposomes. transdermal. Furthermore.. 1982. Maibach. Liposome-bound cortisol: A new approach to cutaneous therapy. Schmid. Lipid vesicles for enhancing drug skin permeation have been specially designed using various approaches. A number of pharmaceutical and cosmetic products covered by patents on ethosomes are marketed. their common feature is their ability to improve the delivery of drugs across the skin barrier. Eur J Clin Pharmacol 39:349. Studies conducted with ethosomal systems in vitro. and V. 4. Lasch. Israel). J. 1990. In another study with ethosomes containing 1% erythromycin no significant variations in the dimensions of ethosomes throughout the storage at room temperature were measured [94]. The above mentioned drugs that have been tested with ethosomal carriers are clearly not exhaustive but intended to illustrate the wide range of molecules that could be tailored with ethosomes for facilitating dermal. Thus. 5. transfersomes. skin permeation experiments showed that the cream after 3 years retains its initial penetration enhancing capacity [117].R. M. Heidelberg: Springer-Verlag. M. For this purpose. and in humans confirm that these soft vesicles enable efficient transport of active agents into the deep strata and across the skin. 6. New. Korting. 1980.C.6 CONCLUDING REMARKS In summary.C. Life Sci 26:1473. and W. Korting. et al. Acyclovir in SupraVir cream has been shown by HPLC assay to be stable for at least 3 years at 258C. and V. Mezei. Although each vesicle type has its own characteristics. TEM micrographs confirmed that erythromycin unilamellar ethosomes kept their configuration during the stability evaluation experiments. Liposomes. 1986.C. M.. the liposomal carriers could be efficient in local treatment of skin disorders and for cosmetic uses. 3. have been invented. a selective drug delivery system for the topical route of administration: Gel dosage form. The initial mean size of the vesicles was 123 + 15 nm. These carriers might offer advanced local and systemic new therapies with agents that are unable to efficiently penetrate the stratum corneum via passive diffusion. H. indicate that the formulation and the drug has a long shelf life with no stability problems. Liposomes: A drug carrier system for topical treatment in dermatology. 7. Liposome encapsulation improves efficacy of betamethasone dipropionate in atopic eczema but not in psoriasis vulgaris. and H.. visualization by negative stain TEM confirmed that the vesicular structure of the ethosomes persisted after 2 years of storage and no significant structural changes occurred over that time in both systems [86. soft vesicles with fluid bilayers.. and H. R. J Pharm Pharmacol 34:473. an ethosomal formulation of acyclovir. Korting. such as ethosomes. Liposomes—a practical approach. Braun-Falco. Gries Conference: Liposome Dermatics.. Biomed Biochim Acta 45:1295.H. resulting in the formation of drug reservoir mainly in the horny strata and generally do not penetrate into the deeper layers of the skin.93]. 1994. in vivo. 2.

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