Liposomes and Niosomes as Topical Drug Delivery Systems

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Review

Skin Pharmacol Physiol 2005;18:209–219 DOI: 10.1159/000086666

Liposomes and Niosomes as Topical Drug Delivery Systems

M.J. Choi H.I. Maibach

Department of Dermatology, University of California, School of Medicine, San Francisco, Calif. , USA

has fewer side effects and improves patient compliance [1] . The skin is a major target for TT drug delivery. The stratum corneum (SC) provides a main barrier against drug transport, and SC intercellular lipids help to regulate penetration. This lipid matrix, composed of ceramides, free fatty acids, cholesterol (CHOL), CHOL sulfate, and minor lipids, plays a major role in barrier function [2, 3] . Despite the advantages of TT drug delivery, low SC per-meability may limit its usefulness. To increase permeabil-ity, chemical and physical approaches have been exam-ined to lower SC barrier properties. These approaches include tape stripping [4–7] , iontophoresis [8–10] , elec-troporation [8, 9, 11–14] and vesicular systems, such as liposomes and niosomes [15–18] .

Among these approaches, liposomes (phospholipid-based artifi cial vesicles) and niosomes (non-ionic surfac-tant vesicles) are widely used to enhance drug permeation across the skin in cosmetic and dermatologic fi elds. In addition to conventional liposomes and niosomes, proli-posomes and proniosomes are also used to enhance TT drug delivery. They are converted into liposomes and nio-somes upon simple hydration [19, 20] . Mezei and Gu-lasekharam [15, 16] fi rst reported the potential use of li-posomes in topical skin applications. Subsequently, vari-ous vesicular systems have been developed [21–24] . Vesicular delivery systems have attracted considerable attention in TT drug delivery for many reasons. These penetration enhancers are biodegradable, non-toxic, am-phiphilic in nature, and effective in the modulation of

Key Words Proliposomes � Topical/transdermal delivery � Follicular transport � Liposomes � Niosomes � Proniosomes

Abstract The skin acts as a major target as well as a principle bar-rier for topical/transdermal (TT) drug delivery. The stra-tum corneum plays a crucial role in barrier function for TT drug delivery. Despite major research and develop-ment efforts in TT systems and the advantages of these routes, low stratum corneum permeability limits the use-fulness of topical drug delivery. To overcome this, meth-ods have been assessed to increase permeation. One controversial method is the use of vesicular systems, such as liposomes and niosomes, whose effectiveness depends on their physicochemical properties. This re-view focuses on the effect of liposomes and niosomes on enhancing drug penetration, and defi nes the effect of composition, size and type of the vesicular system on TT delivery.

Copyright © 2005 S. Karger AG, Basel

Introduction

The topical/transdermal (TT) route for drug adminis-tration has advantages over other pathways. It avoids he-patic fi rst pass effect, provides continuous drug delivery,

Received: May 18, 2004 Accepted after revision: February 7, 2005 Published online: July 5, 2005

Howard I. Maibach Department of Dermatology, School of Medicine University of California, Box 0989, Surge 110 90 Medical Center Way, San Francisco, CA 94143-0989 (USA) Tel. +1 415 476 2468, Fax +1 415 753 5304, E-Mail [email protected]

© 2005 S. Karger AG, Basel 1660–5527/05/0185–0209$22.00/0

Accessible online at: www.karger.com/spp

Choi/Maibach Skin Pharmacol Physiol 2005;18:209–219 210

drug release properties [25] . Their effectiveness is strong-ly dependent on their physiological properties, such as composition, size, charge, lamellarity and application conditions.

Most drugs permeate via the intercellular lipid matrix, such as the intercellular and transcellular routes. How-ever, vesicular systems use three potential pathways of drug permeation into the viable tissue – through hair fol-licles with associated sebaceous glands, via eccrine sweat ducts or across continuous SC between these appendages. Follicular pathway is important for small or macromol-ecule delivery with vesicular systems [26–28] .

This review focuses on the common liposomes and niosomes and does not discuss elastic liposomes, such as transfersomes and ethosomes. We shall review elastic li-posomes in our next study (in preparation). Here, we de-fi ne the effect of composition, size and type of the vesicu-lar system, i.e. liposomes, niosomes, proliposomes and proniosomes, on TT drug delivery.

Skin Interaction of the Vesicular System

Vesicle–skin interactions were visualized with isolated human SC incubated for 48 h and vesicles prepared from CHOL and polyoxyethylenealkylether surfactants. Hof-land et al. [18] reported that, after this incubation time, liquid as well as gel-state vesicles fused at the superfi cial layer of the SC, but that only liquid-state vesicles induced perturbations in lipid organization and formation of wa-ter pools within the SC. Abraham and Downing [29] showed fusion and adsorption of vesicles onto the SC surface, forming stacks of lamellae and irregular struc-tures on top of the skin depending on vesicle composition. Hence, vesicle–skin interactions are strongly dependent on the physiological properties of the vesicular systems.

To further investigate interaction between liposomes and human SC in vitro, Hofl and et al. [30] examined li-posomal formulations from commercially available phos-pholipid mixtures (Nattermann): NAT106, NAT89 and NAT50. The main difference between these phospholip-id formulations was the hydrophilicity of the head groups. NAT50 liposomes only fused on the SC surface and did not perturb SC lipid organization. With NAT89, rough structures were formed in the outermost four SC layers, indicating either intrusion of rough ultrastructures formed by fusion of vesicles or alteration of the SC lipid lamellae. NAT106, containing a high fraction of phosphatidylcho-line (PC), induced marked changes in SC ultrastructure. The corneocytes were considerably swollen and the ultra-

structure of intercellular lipid lamellae showed fl attened spherical structures. From the above results, they ob-served two types of interaction: fi rst, interactions at the skin–formulation interface involving adsorption and fu-sion of the liposomes onto the SC surface, resulting in new structure formation. Secondly, liposome–skin interac-tions are found in the deeper layers of the SC and involve alteration of the bilayer ultrastructure.

To explore vesicular system mechanisms, soybean PC liposomes and Span 60 niosomes were selected as model vesicles to investigate the reasons for permeation en-hancement [31] . Pretreatment of skin with phospholipid and non-ionic surfactant was performed to clarify wheth-er phospholipid or surfactant affected skin structure. Af-ter a 12-hour pretreatment, permeation across soybean PC and Span 60-treated skin was signifi cantly higher (p ! 0.05) than that across non-treated skin. Surfactant-treated formulations were superior to phospholipid-treated and non-treated formulations in facilitating the permeation of enoxacin, as well as drug deposition into the skin. Total amount of enoxacin permeated from sur-factant-treated, liposome-treated and non-treated con-trol group was 178.4 8 19.7, 59.3 8 9.4 and 31.5 8 5.9 � g/cm 2 , respectively. Skin residual of enoxacin perme-ated from the same group was 177.1 8 35.7, 135.3 8 26.3 and 38.8 8 7.1 � g/cm 2 , respectively. These results indicated that soybean PC and Span 60 can serve as per-meation enhancers for enoxacin delivery. The action of liposomes and niosomes as permeation enhancers might predominantly be on the intercellular lipid SC, raising SC fl uidity.

Follicular Pathway

Traditionally, the main pathway for the TT delivery of active agents across the skin was thought to be through SC intercellular routes and transcellular routes. However, alternative means, such as via appendage transport, i.e., follicular transport, is gaining more acceptance; this path-way is important for TT drug delivery with vesicular sys-tems [28] . The pilosebaceous unit (hair follicle, hair shaft and sebaceous gland) provides a route that bypasses in-tact SC; it also represents a drug target. Sebaceous gland cells are more permeable than corneocytes; thus, drugs can reach the dermis by entering the follicle (bypassing the invaginated SC), and passing through the sebaceous gland [24] .

Experiments with the Syrian hamster ear model dem-onstrated that carboxyfl uorescein (CF)-loaded liposomes


Skin Pharmacol Physiol 2005;18:209–219 211

delivered much higher drug concentrations into the seba-ceous glands compared with conventional CF formula-tion [32] . Li et al. [33–35] found that liposomal entrapped calcein, melanin and DNA resulted in specifi c delivery into the hair follicles of histocultured mouse skin, while aqueous control solution of these molecules showed no follicular localization. These results indicate that the fol-licular pathway plays a major role in TT drug delivery with vesicular systems. Hoffman [36] and Ciotti and Weiner [28] developed novel liposomal formulations (non-ionic liposomes; niosomes) for enabling delivery into hair follicles and sebaceous glands. Niosomal formu-lations had higher effi cacy in skin delivery than naked molecules and conventional formulations. Naked mole-cules are trapped in the SC and cannot enter the follicle. However, niosomal delivery of these molecules is time-dependent and able to selectively give them access to hair follicles.

Using a combination of liposomes and DNA, Cotsa-relis [37] and Raghavachari and Fahl [38] demonstrated that liposome composition, hair follicle structure and hair cycle stage act as important parameters infl uencing trans-fection of human hair follicles. As far as liposome com-position was concerned, non-ionic liposomes were their most effi cient vehicles for transdermal systems, and the traditional PC-based liposome carriers were not effective as delivery vehicles for DNA when compared with non-ionic liposomes. Fan et al. [39] reported that the presence of normal hair follicles was required to elicit immune re-sponse to expressed antigen after topical DNA applica-tion. Wu et al. [40] demonstrated that the level of gene expression that could be achieved with normal follicular structure was higher than that observed in the abnormal follicles. Hoffman et al. [41] reported that early anagen follicular cells were the primary cell population trans-formed during dermal gene transduction. From these re-sults, normal follicles and hair cycle were important for inducing immune response to protein and DNA in the follicle.

How do liposomes permeate into follicles and hair shafts? Liposomes may permeate follicles via a lipid-rich channel that coats the follicular duct with subsequent en-try into the follicular cells. Weiner at al. [42] suggested two ways by which liposome-entrapped molecules could be incorporated into hair shafts; one is by liposome de-livery of the molecules fi rst into the follicular matrix cells, and then into the hair shaft as the matrix cells grow and differentiate into new hair shafts. The other is by direct permeation of liposomes-entrapped molecules into hair shafts. Vesicular systems can effi ciently convey small and

large molecules into skin through a follicular pathway. As the latter is increased upon drug delivery, vesicular sys-tems have the potential of selectively targeting hair fol-licle with large and small molecules, including genes, opening the fi eld of gene therapy and other molecular therapy of the hair process to restore hair growth, physi-ologically restore or alter hair pigment, and prevent or accelerate hair loss.

Topical Delivery of Liposomes

Various vesicular carriers have been suggested as ve-hicles for TT delivery of drugs. Mezei and Gulasekharam [15, 16] fi rst reported that liposomes loaded with triam-cinolone acetonide (TRMA) facilitated a 3- to 5-fold ac-cumulation within the epidermis and dermis. The effect of liposomes on drug penetration is controversial. Knepp et al. [43] compared progesterone delivery with free and liposomal formulation in hairless mouse skin. The asso-ciation of progesterone with egg PC liposome halves the transdermal delivery of the drug when compared with the control system. Progesterone incorporation into the di-palmitoyl PC liposomes lowers penetration by more than one order of magnitude. With caffeine, the fl ux from aqueous solution was higher (25.5 � g/h/cm 2 ) than from a liposome system containing PC/CHOL (50: 1, molar ra-tio) with 40 nm average size (6.9 � g/h/cm 2 ). However, the quantity of caffeine accumulated in the skin after 24 h was greatest for small liposome vesicles (2,260 � g/cm 2 ). This value is 12 times greater than that for aqueous solu-tion [44] .

Liposomes have several functions in TT delivery: one is a retention effect into the SC and another is an enhanc-ing effect. In case of high-penetrating drugs, such as pro-gesterone, trihexyphenidyl HCl and caffeine [43–45] , li-posome formulation penetrates less than control formula-tion but has higher retention in the SC. For some drugs, including minimally permeable ones and macromole-cules, liposome formulations greatly enhance penetra-tion. In case of unfractionated heparin, heparin solution could not penetrate but liposomes loaded with heparin permitted 8.2-fold accumulation of the drug within the skin [46] . Table 1 summarizes the effect of liposomes on TT delivery of various drugs.

Liposomes have another function on percutaneous penetration: with enoxacin, permeation after liposome pretreatment was higher than in non-treated skin. Hence, liposomes interacted with the SC and then perturbed its structure [31] . To investigate the function of liposomes


in penetration, Verma et al. [47] compared the penetra-tion of liposomes with entrapped and non-entrapped drugs utilizing CF-entrapped liposomes (CF inside lipo-somes), simple mixtures of liposomes and CF (CF outside liposomes), and mixtures of entrapped and non-entrapped liposomes (CF in and outside liposomes). The effect of separation of the nonentrapped from liposomally en-trapped CF was investigated by measuring the penetra-tion of CF across human skin under non-occlusive condi-tions in vitro using diffusion cells. The liposomal formu-lation containing CF both inside and outside the vesicles showed statistically enhanced penetration of CF into the human SC as compared with the formulations containing CF only outside of the liposomes and CF in buffer. Con-focal laser scanning microscopy revealed that the formu-lation in which CF was present outside the liposomes showed bright fl uorescence intensity in the SC and weak fl uorescence in the viable epidermis. However, the CF in buffer failed to show any fl uorescence in the viable epi-dermis. Taken together, the results indicate that phospho-lipid vesicles not only carry the entrapped hydrophilic

substance, but also the nonentrapped hydrophilic sub-stance into the SC and possibly into the deeper skin lay-ers.

Topical Delivery of Niosomes

Niosomes, non-ionic surfactant vesicles, are widely studied as an alternative to liposomes. These vesicles ap-pear to be similar to liposomes in terms of their physical properties. They are also prepared in the same way and, under a variety of conditions, form unilamellar or multi-lamellar structures. Niosomes alleviate the disadvantages associated with liposomes, such as chemical instability, variable purity of phospholipids and high cost [48] . They have the potential for controlled and targeted drug deliv-ery [49] .

Niosomes enhanced the penetration of chemicals and drugs through the SC [28] . Table 2 summarizes the effect of niosomes on the TT delivery of various drugs. With minoxidil (2%), niosomal formulation results in a direc-

Table 1. Effect of liposomal system on the skin penetration of drugs and macromolecules

Compositionsa Experimental animals Drug <Enhancing factor References

PC:CHOL mouse tacrolimus <9.0 70PC/CHOL (1:1) mouse dithranol <6.0 50Skin lipids hairless mouse 5-aminolevulinic acid <0.1/2.5b 71PC/CHOL (50:1) hairless mouse caffeine <0.25/12.0b 44PC mouse cyclosporin A <6.5 72PC:CHOL:PS (1:0.5:0.1) hairless mouse peptide <1.4–2.0 55DPPC/CHOL (1.1:0.5) rabbit triamcinolone acetonide <3–5 16Soybean PC nude mouse enoxacin <1.4 31Soybean PC nude mouse trihexyphenidyl HCl <0.6/3.3b 45Skin lipids human corticosteroid 13 63DMPC/CHOL:PS (7:5:3) human corticosteroid <8 63PC:SM (2:1) human low-molecular heparin <0.8 46PC:SM (2:1) human unfractionated heparin <8.2 46Egg PC hairless mouse progesterone <0.5 43DPPC hairless mouse progesterone <0.1 43Soybean PC hairless mouse paromomycin <3.0 73DOPE/CHOH/DSPC rat pDNA <3.0 38DMPC/DMPG rat adriamycin <2.4 74Egg PC rat triamcinolone acetonide <4.0 59DPPC rat triamcinolone acetonide <2.4 59Skin lipids rat triamcinolone acetonide 17.0 59

a Skin lipid is composed of ceramides/cholesterol (CHOL)/palmitic acid/CHOL sulfate. PC = Phosphatidyl-choline; DPPC = dipalmitoyl PC; DMPC = dimyristoyl PC; DSPC = distearyl PC; DMPG = dimyristoyl phos-phatidylglycerol; PS = phosphatidylserine; SM = sphingomyelin.

b Skin retention.



tionally superior enhancement of hair growth compared with common formulation [28] . Transdermal permeation and skin partitioning of enoxacin from Span niosomes (85.8 8 19.2 � g/cm 2 ) were much higher than from the free form (31.5 8 5.91 � g/cm 2 ) and from liposome (45.0 8 8.3 � g/cm 2 ) [31] . The effect of niosomes was not al-ways signifi cantly higher than that of liposomes. With dithranol delivery, transdermal permeation of liposomes is much higher than that from the conventional creams and from niosomes. The permeation rate constants, 23.1 � g/cm 2 /h with a liposomal system and 7.8 � g/cm 2 /h with a niosomal system in comparison with 4.1 � g/cm 2 /h obtained for the cream base clearly indicate the enhanc-ing effect of vesicles on permeation and penetration [50] . Depending on the drugs and on vesicle composition, the

effect of liposomes and niosomes differs. Table 3 sum-marizes enoxacin and dithranol permeation using lipo-some and niosome formulations.

Niosomes can also aid penetration of peptides, pro-teins and DNA. Span and Tween are widely used in nio-somes for chemical drugs, whereas polyoxyethyleneether and diacyl glycerides are used for protein and DNA as components. Ciotti and Weiner [28] reported the expres-sion level of human IL-1 pDNA by using different formu-lations. Expression of pDNA from niosomes were sig-nifi cantly greater than the PC liposomes and control for-mulation tested. The expression level of niosomes, PC and control was 163 8 15 pg/cm 2 , 43 8 5 pg/cm 2 and 30 8 5 pg/cm 2 , respectively. Raghavachari and Fahl [38] also reported that nonionic liposomes (niosomes) were

Table 2. Effect of niosomal system on the skin penetration of drugs and macromolecules

Type of vesicles Experimentalanimals

Drug Enhancing factor

References

GDL:CHOL:POE (57:15:28) hairless mouse peptide 3.0–4.0 55GDL:CHOL:POE:DOTAP (50/15/23/12) hamster pDNA 5.4 28GDL:CHOL:POE (58:15:27) rat pDNA 6.0 38GDL:CHOL:POE (58:15:27) rat adriamycin 1.8 74GDL:CHOL:POE:DOTAP (50/15/23/12) rat adriamycin 2.5 74Span60:CHOL (2:1) mouse dithranol 2.0 50Span60:CHOL (1:1) nude mouse enoxacin 2.7 31Span60 rat estradiol 2.1 52Span40:SoyPC:CHOH (4.5:4.5:1) rat levonorgestrel 1.3–3.7 48

GDL = Glyceryl dilaurate; POE = polyoxyethylene-10-stearylether; DOTAP = 1,2-dioleaoyloxy-3-(trimethyl-ammonio)-propane.

Table 3. Effect of liposome and niosome formulations on enoxacin and dithranol permeation across intact skin

Drug Vehicle Composition Fluxa Enhancingfactorb

Enoxacin liposomes soybean PC/CHOH (9:1, weight ratio) 44.9788.25 1.4liposomes DMPC/CHOH (7:3, molar ratio) 19.21810.88 0.6niosomes Span 60/CHOH (1:1, molar ratio) 85.79819.20 2.7

Dithranol cream O/W cream 186820 1liposomes PC/CHOH (1:1, molar ratio) 1,050850 5.7niosomes SPAN/CHOH (2:1, molar ratio) 400830 2.15

a Total amount of enoxacin and dithranol permeated across intact nude mouse and hairless mouse skin dur-ing 48 h (�g/cm2).

b Based on the buffer and cream system in case of enoxacin and dithranol, respectively. Free drug fl ux of enoxacin was 31.50 8 5.88 �g/cm2.


most effi cient for TT delivery systems; rat pups treated with niosome formulations showed much higher reporter gene expression than cationic and phospholipid lipo-somes. Jayaraman et al. [51] ascribed the high effi ciency of niosome formulations as facilitators of drug delivery to the unique lipid composition, suggesting that niosome components, such as polyoxyethyleneether and glyceryl dilaurate, act as penetration enhancers similar to non-ionic detergents, such as Span and Tween.

Similar to liposomal systems, the types of non-ionic surfactant and CHOL contents are important factors af-fecting the effi ciency of transdermal delivery. Fang et al. [52] reported the effect of non-ionic surfactant on estra-diol delivery. The trend of estradiol fl ux from niosome formulations was ordered: Tween 20 1 Span 60 1 Span 85 1 Span 40. Vanhal et al. [53] reported the effect of CHOL content on estradiol delivery. They demonstrated that reduced CHOL content of niosomes increased trans-dermal estradiol delivery.

How can niosomes increase permeation? Several mechanisms may explain the ability of niosomes to mod-ulate drug delivery [25, 31, 48, 54] . The mechanism of drug permeation with niosomes may resemble that of li-posomes. One possible reason for niosomes to enhance permeability is their ability to modify SC structure; the intercellular lipid barrier in the SC may become looser and more permeable by niosome treatment [31, 54] . An-other reason is altering adsorption and fusion of niosomes with the skin’s surface, which leads to a high thermody-namic activity gradient of drug at the interface. Schreier and Bouwstra [54] demonstrated the fusion of niosome vesicles of estradiol on the surface of skin by electron mi-croscopy. In niosome formulation, non-ionic surfactant itself, acts as a permeation enhancer, which might partly contribute to the enhancement of drug permeation from niosomes.

Fleisher et al. [55] reported that nonionic emulsions are potent enhancers of niosomal systems. Nonionic emulsions are composed of niosomes and isopropyl my-ristate at a 80: 20 ratio by weight followed by sonication for 2 min. With growth hormone releasing peptide, non-ionic emulsions enhanced a 4- to 6-fold peptide level in living skin and in the receiver phase. They suggested that isopropyl myristate might facilitate fl uidization of the SC lipid domains allowing a greater degree of drug perme-ation. In addition to peptides, the absorption of mannitol (an uncharged polar hydrophilic marker) into and across hairless mouse skin was 10-fold higher following topical in vivo application of nonionic emulsions compared to that from niosomes.

Liposomal Size

Pores in the skin are normally 0.3 nm narrow and can be opened without major skin damage merely to 20–40 nm at the most. Hence, it is diffi cult to contemplate how an entire liposome could cross the skin and partici-pate in transdermal transport [56–58] . Because drug car-riers must pass through fi ne pores, liposome’s size may infl uence TT delivery of drugs through microroutes, such as the intercellular and the transcellular route. It was as-sumed that a decrease in particle size of the liposomes would result in an increase of the amount of drug found in the deeper skin [59] . However, if liposomes mainly permeate through follicles, liposome size may not infl u-ence TT drug delivery. Vesicular systems can transport drugs into the skin through the transcellular and follicle route; it is not clear which pathway is more important.

To compare the effects of different vesicles, Yu and Liao [59] prepared small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) with negative surface charge. The negative SUV (J value, 1.4 8 0.2) with a di-ameter of 35 nm gave a signifi cantly higher permeation than the corresponding MLVs (J value, 0.68 8 0.09) of the same charge. Egbaria et al. [60] also compared cyclo-sporin A (CsA) penetration in MLV and large unilamellar vesicles (LUV) containing skin lipids and phospholipids. Regardless of lipid compositions, the total amount of CsA in deeper SC and deeper skin strata at 24 h was MLV 1 LUV. Generally, the size of MLV was higher than that of LUV. Because they did not provide the accurate size for MLV and LUV, the effect of vesicle type on drug penetra-tion could not be defi nitively compared.

Yu and Liao [59] reported the effect of MLV size on the TRMA permeation. There was no difference in MLV with different size, from 1 to 0.2 � m. The size of MLV may not infl uence TT drug delivery. In addition to lipo-somal size, other factors may be involved in TT delivery in negatively charged liposomes. To further investigate the effect of liposomal size, du Plessis et al. [61] prepared various sizes of liposomes and compared the effect of size with CsA-entrapped liposomes containing PC/CHOL/ cholesteryl sulphate (1: 0.5: 0.1, molar ratio). The smaller liposomal particle sizes (60 nm) did not result in higher CsA levels in the deeper skin strata of hairless mouse, hamster and pig skin. The intermediate particle size of 300 nm resulted in both the highest reservoirs in the deep-er skin strata as well as the highest drug concentration in the receiver. Taken together, these results indicate that an optimum particle size for optimal drug delivery ex-ists.



To further investigate the infl uence of vesicle size on drug penetration, Verma et al. [62] used hydrophilic and lipophilic fl uorescent compounds entrapped in lipo-somes. In a lipophilic fl uorescent substance, maximum fl uorescence in the skin was observed with smaller lipo-somes of 71 nm in diameter. In case of CF, the liposomes with a size of 120 nm in diameter showed statistically enhanced penetration of CF into the skin as compared with larger and smaller ones. The results indicated that CF penetration was inversely related to the size of the li-posomes ( table 4 ). These results indicate that larger vesi-cles may not penetrate well into the deeper layers of the skin and stay in/on the SC forming a lipid layer after dry-ing. They demonstrated that the large vesicles with a size 1 600 nm are not able to deliver their contents into deep-er layers of the skin and these liposomes stay in/on the SC. The liposomes with size ! 300 nm delivered their con-tents to some extent into deeper layers of the skin. How-ever, the liposomes with size ! 70 nm seem to be promis-ing for dermal delivery. Taken together, these results also indicate that the size of liposomes infl uences TT drug de-livery into the skin, and a size of 100 8 30 nm may show the greatest penetration of hydrophilic and lipophilic drugs into the skin. However, a more extensive study should be undertaken to defi ne the exact optimal particle size for each formulation.

Composition

Depending on composition, bilayers of vesicles are in either a liquid crystalline or a rigid gel state. Lipid com-positions infl uenced the TT delivery activities of various drugs, such as progestrone and TRMA [43, 59] . Knepp et

al. [43] observed that incorporation of progesterone in liquid-state liposomes resulted in a higher skin perme-ation rate than when incorporated in gel-state liposomes. Yu and Liao [59] reported that TRMA in the PC liposome (J value, 1.0 8 0.1) showed a signifi cantly higher perme-ated amount of TRMA than that in the dipalmitoyl PC liposomes (J value, 0.64 8 0.05) and ointment (J value, 0.25 8 0.02). Also, they reported that liposomes consist-ing of skin lipid composition (J value, 4.7 8 0.2) more signifi cantly enhanced the skin permeation of TRMA than other liposomes.

Fresta and Puglisi [63] reported that skin-lipid lipo-somes provided higher drug disposition within the deep-er skin layers than control ointment and a phospholipid-based liposomes formulation. There was no signifi cant difference in mean corticosteroid concentration within the SC layer for the various formulations. Drug concen-tration in the epidermis provided by phospholipid-based liposomes and skin-lipid liposomes was, respectively, 8 and 13 times higher than that obtained by the conven-tional ointment formulation. In the case of dermis, the drug concentration was increased by a factor of 11 and 19 times in phospholipid-based and skin-lipid liposome formulations, respectively. Therapeutic effectiveness of skin-lipid liposomes was 6 and 1.3 times higher than that obtained with an ointment and phospholipid-based for-mulation, respectively. Liu et al. [64] also reported that the skin lipid liposomes provided the most effective TT delivery of acyclovir palmitate. Taken together, skin-lip-id liposomes appeared to be a suitable TRMA, acyclo - vir palmitate and corticosteroid delivery system, increas-ing penetration and pharmacological effectiveness. How-ever, Egbaria et al. [60, 65] compared CsA penetration in hairless mouse and human skin with skin lipid and phos-pholipid liposome formulations. Unlike TRMA and cor-ticosteroid, phospholipid liposomes showed higher pen-etrated amount of CsA than skin lipid liposomes. The accumulation of CsA in the SC at 24 h was in the follow-ing order: skin lipid MLV 1 phospholipid MLV 1 skin lipid LUV 1 phospholipid LUV. The total amount of drug in the deeper SC and deeper skin at 24 h was in the following order: phospholipid MLV 1 skin lipid MLV 1 phospholipid LUV 1 skin lipid LUV. Drug accumulation of CsA differs, depending on the species. Whereas skin lipid liposomes were more effective than phospholipid-based liposomes in depositing drug in deeper skin strata in rodent species (mouse and guinea pig), the opposite effect was observed in human cadaver skin. Liposomal lipid composition was an essential factor affecting the ef-fi ciency of TT drug delivery. Hence, a more extensive

Table 4. Amount of CF delivered by liposomes with different par-ticle size into the different strata of human abdominal skin using Franz cell in vitro after a 14-hour non-occlusive application

Average size ofLiposomes, nm

Stratumcorneum

Deeperskin

Receptorcompartment

Buffer 31.8782.57 0.0780.01 0.0080.0073a 74.0780.47 0.2080.01 0.1180.01

120 72.8881.69 0.7780.08 0.2780.03191 65.2881.74 0.1680.03 0.1880.03377 61.1982.62 0.1180.01 0.1180.01810 39.9582.64 0.0280.00 0.0480.00

a Data from a 12-hour non-occlusive application.


study is needed to defi ne optimal compositions for TT drug delivery.

To further investigate the effect of composition, Fang et al. [31] compared skin permeation of enoxacin com-bined with saturated (egg) PC with skin permeation of the same drug combined with natural (soybean) PC. PC with saturated long alkyl chain (dimyristoyl PC; DMPC) pro-vided rigid bilayers with low permeability for encapsu-lated molecules. DMPC liposomes (20.6 8 2.9 � g/cm 2 ) reduced enoxacin permeation (p ! 0.05) based on the to-tal amount of enoxacin permeated during a 48-hour in vitro permeation experiment. The amount of enoxacin permeated (45.0 8 8.3 � g/cm 2 ) was signifi cantly in-creased in soybean or egg PC liposomes as compared with that in the free form (31.5 8 5.9 � g/cm 2 ) or in DMPC liposomes (20.6 8 2.9 � g/cm 2 ). These data indicated that the lipid composition of liposomes infl uenced the trans-dermal activity of entrapped enoxacin. The presence of unsaturated fatty acids in the phospholipids may be re-sponsible for enhancer effects. The packing nature of un-saturated fatty acids changed the fl uidity of the SC lipid structure and facilitated drug skin permeation.

Surface Charge

To investigate the effect of liposome surface charge on drug permeation, Yu and Liao [59] incorporated nega-tively and positively charged lipids into liposomes. The effect of liposome size on TT delivery differs, depending on surface charge. The negative SUV showed a signifi -cantly higher permeated amount than the corresponding MLV. However, there was no signifi cant difference in other charged vesicles. The positive MLV (J value, 1.4 8 0.5) were slightly higher than SUV (J value, 1.2 8 0.1) of the same charge, and the neutral SUV (J value, 0.9 8 0.1) showed to be signifi cantly lower than MLV (1.2 8 0.2) of the same charge.

To enhance the topical delivery of lipophilic drug (rho-damine B), Katahira et al. [24] investigated the electro-static interaction between the positive and negative com-ponents incorporated in the liposome bilayer. The higher in vitro rat skin permeability of lipophilic drug was ob-served in positive and neutral multilamellar liposomal preparations; negative liposome composed of PC and dicetyl phosphate (DCP) showed lower skin permeability to lipophilic drug. However, Manosroi et al. [66] reported that rat skin permeability of amphotericin B entrapped in charged liposomes (40–50 ng/cm 2 /h) was 10-fold high-er than that in neutral liposomes (4 ng/cm 2 /h) in the SC

and deeper skin layers. The respective permeability of amphotericin B in various liposome formulations through the SC was ordered: positively charged 1 negatively charged 1 neutral liposome. Similar results were obtained from acyclovir palmitate liposome formulations [64] . Skin retention of acyclovir palmitate from positive lipo-somes was signifi cantly higher than that from other for-mulations. This may be due to the fact that the cell sur - face of the skin is negatively charged [59] . The positive charges on the surface of liposome formulation can bind to the negative charges of the skin, thereby enhancing the penetration of liposomal formulation into the skin.

To further investigate the effect of negatively charged lipid on enoxacin permeation, Fang et al. [31] incorpo-rated DCP into liposome and niosome formulation. DCP reduced the permeation of enoxacin across the skin with-out affecting enoxacin partitioning into the SC. Since the absorption of liposomes or niosomes into the skin is due to physical or electrostatic forces, it was expected that charged liposomes or niosomes might alter this interac-tion. Lower enoxacin skin permeation of negatively charged niosomes could be attributed to repulsion within the skin surface under physiological conditions.

Proliposomes

Proliposomes, free-fl owing particles that immediately form a liposomal system upon hydration [19, 67] are com-posed of drug, phospholipids and a water-soluble porous powder. Proliposomes can be stored sterilized in a dried state. By controlling the size of the porous powder in pro-liposomes a relatively narrow range of reconstituted lipo-some size can be obtained. Because of these properties, proliposomes appear to be a potential alternative to lipo-somes in design and fabrication of liposomal dosage forms. Proliposomes (proniosomes) offer a versatile lipo-some (niosome) delivery concept with a potential for use with a wide range of active compounds.

When proliposomes are applied to mucosal mem-branes, they are expected to form liposomes upon hydra-tion by mucosal fl uids. The resulting liposomes may act as a sustained release dosage form of the loaded drugs. Microscopic observation revealed that proliposomes are converted to liposomes almost completely within min-utes following contact with water. Proliposomes may form liposomes with sweat when applied under occlusive conditions in vivo [68] . If drug can be delivered transder-mally from proliposomes in the form of patches, sus-tained drug absorption would be achieved without using



sophisticated devices and/or artifi cial membranes since proliposomes themselves may control the release and dosage rate of a drug.

Hwang et al. [69] reported in vitro skin permeation of nicotine from proliposomes. The nicotine fl ux from pro-liposomes was initially retarded compared with that of nicotine powder. The initial fl ux of nicotine from the powder was more than twice that of proliposome prepa-rations (172 � g/cm 2 for powder vs. 73 � g/cm 2 for proli-posomes). These results indicate that sustained transder-mal delivery of nicotine is possible using proliposome formulations if the latter are topically applied under oc-clusive condition.

Proniosomes

Proniosomes can be converted into niosomes upon simple hydration of the skin. Hence, proniosomes offer a versatile vesicle delivery concept with the potential for delivering drugs via the TT route. They are mainly com-posed of non-ionic surfactants (Span and Tween), phos-pholipids, alcohols and water with or without CHOL. The method of proniosome preparation is based on the simple idea that a mixture of surfactant/alcohol/aqueous phase can be used to form a concentrated proniosome gel, which can spontaneously be converted to stable niosomal dis-persion by dilution with excess aqueous phase [20] .

The types of non-ionic surfactants, alcohols, CHOL, and PC in proniosomes are important factors affecting the effi ciency of transdermal drug delivery. Unlike nio-somes, presence or absence of CHOL in the lipid bilayers of vesicles did not make any difference in encapsulation and permeation of the associated drugs [52] . Vora et al. [48] and Fang et al. [52] studied the effect of formulation composition, surfactant and alcohol type and preparation method on transdermal permeation profi le from various formulations. Different effects of non-ionic surfactants on levonorgestrel permeation profi le showed that the fl ux value was the highest for Span 80 and the lowest for Span 60. No signifi cant difference was observed in the skin permeation profi le of formulations containing Span 40 and 60 due to their higher phase transition temperature that is responsible for their lower permeability. However, Fang et al. [52] reported different effects of non-ionic sur-factants in the case of estradiol: Span 60 showed the high-est fl ux on estradiol permeation profi le. Proniosome for estradiol and levonorgestrel formulations differed in their content of CHOL and type and content of alcohol.

The effect of different alcohols on drug permeation profi le was isopropanol 1 butanol 1 propanol 1 ethanol. The formulation containing isopropanol gave the highest drug permeation with a fl ux value of 3.2 � g/cm 2 /h among the different alcohols. The fl ux values for ethanol, propa-nol and butanol were 2.3, 2.6 and 2.9 � g/cm 2 /h, respec-tively [48] . Different effect of alcohols was involved in proniosome size. Vesicles with ethanol are the largest due to the slowest phase separation with this alcohol because of its greater solubility in water. Isopropanol results in the smallest vesicles, which may be due to the branched chain present in it. Similar to the effect of liposome size, the size of proniosomes also infl uenced TT drug delivery.

Vora et al. [48] reported that soya lipids showed better penetration-enhancing properties than egg lecithin for-mulations. Soya lecithin contains unsaturated fatty acids, oleic acid and linoleic acids, which have penetration-en-hancing properties of their own as compared with egg lecithin, which contains saturated fatty acids.

Proniosomes have similar mechanism to niosomes to modulate drug delivery across skin. Proniosomes consist of nonionic surfactants, phospholipids and alcohols as a component. Both phospholipids and nonionic surfac-tants in proniosomes can act as penetration enhancers, which are useful for increasing the permeation of many drugs. Also, proniosomes utilize high alcohol content, which itself acts as penetration enhancer. However, there are different effects of nonionic surfactants and CHOL in proniosomes and niosomes. Reducing CHOL content in niosomes increases estradiol delivery into the skin [53] . But this phenomenon did not appear in proniosomes. There was no signifi cant difference between the estradiol fl ux with/without CHOL in proniosomes. The fl ux of es-tradiol from Span 60 was signifi cantly higher than other nonionic surfactants in proniosomes, whereas Tween 20 showed the highest fl ux in the niosome formulation [52] . These data indicate that niosomes and proniosomes infl u-ence estradiol permeation differently. Either direct trans-fer of drug from proniosomes to the skin, or the penetra-tion enhancer effect by non-ionic surfactant may contrib-ute to the mechanism of estradiol permeation from proniosome formulations. Proniosomes may become a useful dosage form for drug permeation into the skin, es-pecially due to their simple, scaling-up production proce-dure and ability to modulate drug delivery across the skin. Hence, a more extensive study should be undertaken to fi nd out the optimal proniosome formulation for drug permeation into the skin.


Conclusions

Phospholipids as components of liposomes and nio-somes have an affi nity for biological membranes. Mixing of liposomes with skin lipids in the intercellular layers and phospholipids could be one mechanism contributing to the enhancement of drug permeation into the skin by lipid vesicles. The same phenomenon is observed in nio-somes. Liposomes and niosomes disrupt the membrane properties of the SC and directly fuse with the upper lay-

er of the skin, thereby enhancing the skin permeation of drugs. As far as TT drug delivery is concerned, a vesicular dosage form is superior to an ointment form or a buffer solution. Drug concentration in the various skin layers was increased when vesicular systems were used. System-ic absorption and, as a consequence, the possible side ef-fects were signifi cantly reduced. The advantages of ve-sicular systems have the potential of strengthening the effi cacy and safety of the drug. Hence, many topical drugs may be developed using vesicular systems.

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Liposomes and Niosomes as Topical Drug Delivery Systems