ELSEVIER Journal of Controlled Release 30 ( 1994) l-15

Review

Liposomes and niosomes as topical drug carriers: dermal and

transdermal drug delivery
Hans Schreief, Joke Bouwstrab
“Ctwtrrfor Lung Research, Vunderbilr Uni~~er.vi@School oj’h4edicine. B-1308 MCN, N~~shville. TN 37232-2650. USA
“DiGsion of Pharmctc~eutiwl Trc~hrmlog,v. Lriden/Amrterdom Center@ Drug Rrserrrch, _7300 RA Leidm, The Nethrr1rrld.v

(Received I I May 1993: accepted in revised form 20 September 1993)

Abstract

A critical analysis of (trans)dermal delivery of substances encapsulated within liposomes and niosomes is presented. Topical
liposomes or niosomes may serve as solubilization matrix, as a local depot for sustained release of dermally active compounds,
as penetration enhancers, or as rate-limiting membrane barrier for the modulation of systemic absorption of drugs. The mecha-
nism( s) of vesicle-skin interaction and drug delivery are being extensively investigated using radioactive- or fluorescence-
labeled marker molecules and drugs, and various electron and (laser) light microscopic visualization techniques, and different
models describing the interaction with and fate of vesicles in the skin have been proposed. With the current experimental data
base on hand, most investigators agree that direct contact between vesicles and skin is essential for efficient delivery. although
phospholipids per se apparently do not penetrate into deeper skin layers. Investigators have mostly focused on dermal cortico-
steroid liposome products. However, localized effects of liposome-associated proteins such as superoxide dismutase, tissue
growth factors and interferons appear also to be enhanced. The delivery of liposome-encapsulated proteins and enzymes into
deeper skin layers has been reported, although the mechanism of delivery remains to be elucidated. An objective assessment of
the performance of topical liposome formulations vs. conventional dosage forms is frequently obscured by investigators com-
paring equal concentrations, rather than equivalent thermodynamic activities of their respective formulations. We conclude that
liposomes and niosomes may become a useful dosage form for a variety of dermally active compounds, specifically due to their
ability to modulate drug transfer and serve as nontoxic penetration enhancers.

Key words: Liposome; Niosome: Topical drug delivery; Dermal drug delivery; Transdermal drug delivery; Corticosteroid

1. Introduction delivery is the low penetration rate of substances

through the skin. The diffusional barrier for most sub-
One of the major disadvantages in transdermal drug stances is localized in the upper layer of the skin, the
stratum corneum, which consists of comeocytes
Corresponding author. Tel. (615) 323 1775; fax: (615) 343 2684. embedded in a lipid matrix.
Abbreviations: DMPC, dimyristoylphosphatidylcholine; DOPC.
Several techniques have been explored to increase
dioleylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcho-
line: DSPC, distearoylphosphatidylcholine; EPC, egg phosphatidyl-
the drug penetration rate across skin including ionto-
choline; SPC; soy phosphatidylcholine; NDB-PE, N-( 7-nitro-2, I ,3- phoresis [ I] and penetration enhancement [ 21, partic-
benzoxadiazol-4-yl)-dipalmitoylphosphatidyle~hanolamine; non- ularly for the delivery of peptides and proteins [ 3-51.
ionic walkyl polyoxyethylene ether surfactants, (C,EO,); phos- Here we focus on a third alternative method, the encap-
phate-buffered saline (PBS).

016%3659/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved

SSDlOl68-3659(93)EOl42-3
sulation of drugs in lipid vesicles prepared from phos-
pholipids (liposomes) or nonionic surfactants
(niosomes) which have been shown to facilitate trans-
port of drugs into and across skin.
While liposomes have been investigated for many
years as parenteral drug carrier systems, particularly
for the selective delivery of anticancer, antibiotic and
antifungal agents [6], they have only for approxi-
mately one decade been considered for topical drug
delivery, including ophthalmic 17.81, pulmonary [ 9-
121 and dermal/ transdermal I 7,13- 161 delivery.
Dermal liposome products have since 1987 been
exploited by the cosmetics industry, with currently in
excess of 100 liposome and niosome products on the
market. However, the first therapeutic topical liposome
preparation, a ‘liposome gel’ of the antifungal drug
econazole ( PevarylR; Cilag AC) has been introduced
only recently in Switzerland.
The rationale for the use of lipid vesicles as topical
drug carriers is four-fold:
(i) they may serve as ‘organic’ solvent for the solu-
bilization of poorly soluble drugs, for instance corti-
costeroids; as a result, higher local drug concentrations
at the thermodynamic activity maximum can be
applied;
(ii) they may serve as a local depot for the sustained
release of dermally active compounds including anti-
biotics, corticosteroids or retinoic acid;
(iii) by virtue of penetration of individual phospho-
lipid molecules or nonionic ether surfactants into the
lipid layers of the stratum corneum and epidermis they
may serve as penetration enhancer and facilitate dermal
delivery leading to higher localized drug concentra-
tions;
(iv) they may serve as rate-limiting membrane bar-
rier for the modulation of systemic absorption, i.e., they
may serve as controlled transdermal delivery systems.
2. Mechanism of dermal and transdermal delivery
2.1. Skin penetration studies using radioactive or
jluorescent markers and drugs
Mezei and Gulasekharam [ 17,181 were the first to
demonstrate that liposomes loaded with triamcinolone
acetonide, in both a lotion [ 17 1 and gel [ 18 ] dosage
form. facilitated 3-5-fold accumulation of radiola-
belled drug within epidermis and dermis, while sys-
temic drug levels were very low. Urinary excretion and
organ accumulation, specifically in the thalamic region,
were greatly reduced relative to radioactive counts
found following application of a control ointment.
These results were encouraging and sparked much
interest in the use of liposomal drug preparations for
topical application.
It was unfortunate, however. that the authors con-
cluded that liposomes apparently penetrated the skin,
effectively acting as ‘transdermal carriers’. This
hypothesis has met with considerable skepticism by
many investigators [ 19-23 1. It was doubted that rela-
tively large particles such as liposomes would be phys-
ically able to traverse the stratum corneum and
penetrate the epidermal and dermal layer [ 19,20],
especially if one considers that the intercellular spaces
in the stratum corneum are completely tilled with lipids
which form lamellar phases with well-defined repeat
distances [ 24 ]
Mezei’s apparent findings have later been attributed
to a potential artifact of the experimental method
employed, i.e., the use of alcohol swabs to remove
nonabsorbed compound from the skin surface which
may have caused a transient penetration enhancement
and promoted transdermal absorption of radioactively
labelled triamcinolone acetonide [ 221.
In an elegant series of experiments, Ganesan et al.
[ 191 and Ho et al. [ 20 J demonstrated unequivocally
in an in vitro hairless mouse skin system (finite dose
diffusion cell) that neither liposomes nor phospholipid
molecules diffuse across intact skin: (i) radiolabelled
phospholipid could not be detected in the acceptor com-
partment; and (ii) lipophilic drugs such as hydrocorti-
sone and progesterone had practically identical skin
transfer coefficients, whether they were incorporated
within liposomes or not. From these findings. the
authors [ 191 developed a scheme as shown in Fig. 1
which indicates that skin permeation of water-soluble
compounds such as glucose depends entirely on their
intrinsic skin permeability constant (I’,), while skin
permeation of highly lipophilic compounds such as
progesterone depends entirely on a permeability con-
stant determined by the interaction of the liposome
carrier and the skin (P#), with a combination of both
P, and P# operative for substances that are partly
hydrophilic and partly lipophilic such as hydrocorti-
sane (although its permeation was found to be 99%
determined by the liposome-skin permeability coeffi-
cient P#).
The study was carried out with equal concentrations
in all formulations. The authors stated that the loading
capacity of liposome formulations is much higher
(lower thermodynamic activity at equal concentrations
in liposome compared to control formulation) which,
at saturation concentrations, would facilitate increased
total transport of drug through skin. Transport of pro-
gesterone was not affected by the acyl chain length of
the phospholipids, nor by the presence of charged phos-
pholipids in the vesicle membrane, supporting the con-
clusion that no fusion of vesicles with the skin took
place.
Similarly, Knepp et al. [ 2 1,221 immobilized lipo-
somes in an agarose matrix (in order to prevent direct
contact between vesicles and skin) in vitro to hairless
mouse skin and showed that progesterone was not
transported across skin together with the phospholipid
DONOR SKIN RECEIVER
GLUCOSE
I---!
I I
PROGESTERONE n;c;-Cr’=-
Fig. 1. Schematic description of skin permeation mechanisms of
liposome-entrapped solutes. Water-soluble non-membrane-interact-
ing solutes such as glucose are not absorbed when encapsulated in
liposomes; flux is determined by the bulk concentration (Cn) and
the intrinsic skin permeability constant P,. Flux of partially water-
soluble, lipophilic compounds such as hydrocortisone is a function
of both Cn and P, as well as the liposome-associated concentration
C, and the skin permeability constant resulting from the interaction
with the liposomes (P*). Flux of highly lipophilic substances such
as progesterone is entirely determined by C, and P#. (Modified
from Ref. 19; with permission.)
carrier, nor did (radiolabelled) phospholipid cross the
skin barrier over a 48-h observation period. The rate of
transport of progesterone across hairless mouse skin
was similar whether an aqueous liposome (DPPC) dis-
persion or the same dispersion immobilized within an
agarose matrix was used, indicating that the rate-limi-
ting step was phase transfer of progesterone from the
liposome bilayer into the aqueous environment prior to
skin transfer, rather than direct liposome-to-skin trans-
fer as proposed by Ganesan et al. [ 191. In a second
study [ 221, involving a total of seven formulations
which differed with respect to phospholipid acyl chain
length and degrees of saturation, or contained 1% free
fatty acid (oleic and stearic; two formulations), it was
shown that the cumulative amounts of progesterone
released into buffer were not significantly different with
any of the variables tested at both 4°C and 35X, again
pointing towards a release mechanism based on the
lipophilicity of the drug and not on the physical state
(i.e., gel vs. liquid-crystalline) of the formulation.
Interestingly, while the release of progesterone into
buffer was independent of the formulation, the trans-
dermal delivery of progesterone varied widely for the
different formulations: transdermal delivery from lipo-
somes made with saturated phospholipids (DMPC and
DPPC) was approximately ten-times smallercompared
to unencapsulated drug; delivery from liposomes made
with unsaturated phospholipids (EPC and DOPC) was
only reduced by about 50%.
In a very elegant set of experiments Knepp et al.
[ 221 traced the apparent penetration enhancement
effect to the presence of free fatty acids: transdermal
delivery of progesterone from liposomes made from
saturated DPPC (gel state) liposomes, and unsaturated
(liquid-crystalline) EPC or DOPC liposomes served
to define the lower and upper boundaries of transdermal
delivery (Figs. 2, 3).
When either 1% unsaturated (oleic) or I % saturated
(stearic) free fatty acid was added to DPPC liposomes
the resulting transdermal delivery of progesterone was
pointedly different: delivery from stearic acid-contain-
ing liposomes was practically identical to delivery from
DPPC liposomes, whereas delivery from oleic acid-
containing liposomes was almost identical to delivery
from EPC or DOPC liposomes and was saturable over
a range of 0. l-l 0% of oleic acid present (Fig. 4).
Evidently, presence of free unsaturated fatty acids in
the liposome formulations contributed to a ‘fluidiza-
 IN - VITRO SKIN DELIVERY IN - VITRO SKIN DELIVERY

---t
e FPEEPG

1
--o- FFZSEFG 10

--c PGiEPC PGiDOPC

T
+ PGiDMPC
IT T
- PGiDPPC

0 10 20 30 40

TIME (HOURS) TIME (HRS)

Fig. 2. Transdermal delivery rate ( mean k SD; II = 6) of progester- Fig. 3. Transdermal delivery ritte ( mean + SD: II = 6) of progester-
one (PG) across hairless mouse skin in vitro alone or encapsulated one ( PC) acrws hairless mouse skin in vitro alone or encapwlated
in EPC and DMPC lipwomeh. (From Ref. 22; with permission.) in DPPC and DOPC lipo\omes. ( From Ref. 21-i uith permission. )

tion’ of the lipid domains within the stratum comeum
which, in turn, facilitated transdermal flux of proges-
terone.
An interesting approach to the assessment of the
effect of phospholipids on human skin was taken by
Jacobs et al. [ 231, who pretreated skin on the arms of
human volunteers with egg phospholipid dispersions
for 7 days prior to the application of four commercial
corticosteroid preparations (creams of hydrocortisone
0. I%, clobetasone butyrate 0.05%, betamethasone
0.1% and clobetasol propionate 0.05%). Blanching of
the treated skin was then assessed and quantitated on
an arbitrary scale of O-4. Marginal increases in the
blanching response and reduction in tachyphylaxis
were found for all preparations, except clobetasone
butyrate, following treatment with liposomes. The
authors speculated that liposomes may either form a
thin lipid film on the surface of the skin, or increase the
total lipid content of the skin such that water loss is
retarded and liquid-crystalline matrices in the intersti-
tial spaces of the uppermost skin layer can form. In
either case, steroids would preferentially partition into
such lipid domains and form a depot which could
explain the observed higher dermal and epidermal cor-
ticosteroid concentrations. Although, as a conse-
quence, duration of drug activity should be prolonged
in phospholipid-pretreated skin, this could only be
demonstrated for clobetasol propionate cream.
Another explanation for the increased blanching
response might be that individual phospholipids (or
fractions of free fatty acids associated with the phos-
pholipids) act as penetration enhancers. This has also
been concluded from a more recent study [ 25 ] where
the penetration of various drugs dissolved in propylene
glycol and tetra glycol was monitored in the presence
of egg yolk lecithin and commercial soy bean lecithin.
 H. Schreier. J. Bouw~stra / Journal
Y- PG/EPC
--t PGA% ONDP
_ PG/l% SA/DP
----t PG/DPPC
TIME (HOURS)
Fig. 4. Transdermal delivery rate (mean + SD: n = 6) of progester-
one (PG) across hairless mouse skin in vitro encapsulated in EPC
and DPPC liposomes, and in DPPC liposomes containing 1% oleic
(OA) or I % stearic (SA) acid. (From Ref. 22; with permission.)
Komatsu et al. [ 26,271 studied the influence of lipo-
somes consisting of EPC, cholesterol and dicetylphos-
phate on the penetration of butyl paraben in vitro and
in vivo. In vitro, the radioactive trace ( [ “CIDPPC)
penetrated only to a very small extent through the skin
when incorporated in EPC liposomes [ 261, in agree-
ment with earlier studies [ 191. An increase in the phos-
pholipid content at a constant drug concentration
resulted in lower penetration rates of [ 14C]butyl para-
ben. This was explained by a lower thermodynamic
activity of the drug in the liposome bilayer. Pretreat-
ment of skin by liposomes did not result in higher
penetration rates of [ “C]butyl paraben which indi-
cated that neither the vesicles nor their molecular com-
ponents did act as penetration enhancers.
In vivo, butyl paraben encapsulated in liposomes
penetrated to deeper regions in the skin, while the radi-
oactively labelled phospholipid remained on the skin
surface [ 271.
ofControlledRelease 30 (1994) I-15 5
In a very recent study [28] it was also shown that
gel-state (C,,EO,) niosomes did not increase the pen-
etration of estradiol through human skin after pretreat-
ment, while pretreatment with liquid-crystalline
(C,,EOI or C9,9E0,0) vesicles resulted in a signifi-
cant increase in estradiol transport. Penetration of estra-
diol across human skin was studied using vesicle
formulations saturated with estradiol in order to ascer-
tain equivalent thermodynamic activity in all formu-
lations. Hence, the influence of the formulations on
drug transport was directly comparable which is an
essential parameter of the study design as has been
shown before [ 19,251. Estradiol encapsulated in liq-
uid-crystalline vesicles resulted in much higher estra-
diol fluxes than when applied in a buffer (PBS)
solution.
In additional experiments, Hofland et al. [28]
showed that, although pretreatment with vesicles
resulted in higher estradiol fluxes compared to
untreated stratum corneum, the fluxes were sign@-
cantly lower than when estradiol was encapsulated in
vesicles. Since these higher estradiol fluxes cannot be
explained by penetration enhancement of the surfactant
only, it was postulated that niosomes fuse at the inter-
face of the stratum comeum and that the high local
estradiol concentration in the vesicle bilayers generates
a high thermodynamic activity of estradiol in the upper
part of the stratumcomeum. Fusion of niosome vesicles
on the surface of skin has been demonstrated by elec-
tron microscopy [29] as illustrated in Fig. 5. If this
mechanism is valid, vesicles are a more promising car-
rier for lipophilic drugs than for hydrophilic drugs.
Egbaria et al. [ 301 investigated effects of the prep-
aration method and lipid composition on the disposition
of drugs in, and diffusion across skin. Liposomes pre-
pared by the dehydration-rehydration method [ 3 11
were observed to penetrate deeper into skin strata than
large unilamellar vesicles. In addition, liposomes pre-
pared from ceramides were more effective in penetrat-
ing into skin than liposomes prepared from
phospholipids. While it would be not unexpected that
liposomes of different composition penetrate into skin
more or less efficiently, it is unclear as to what role the
preparation technique would play to determine skin
penetration. One could speculate that the lamellarity,
homogeneity, size, or a combination thereof (all of
which determine the overall effective liposome surface
available for interaction with skin) of the different
Fig. 5. Niosome vesicles (decyloxyethyleneoleylether) forming hpid stack\ which are adsorbed onto the skm surface. f\. fu\ing vesicle: c.
corneocyte. ( From Ref. 29; with permiwon.)

preparations may have influenced the efficient inter- using double labelling. Mouse skin was compared with
calation in skin, although this has not been considered pig skin. Liposomes consisting of EPC.
in this study, and the underlying mechanism is not at [‘HI cholesterol and [ ‘“Cl cholesterol sulfate were pre-
all clear. pared by reverse-phase evaporation [ 331, manual
Du Plessis et al. [32] studied the influence of the shaking and dehydration-rehydration [ 3 1 1.The studies
nature and condition of skin on the penetration of lipids were carried out under nonocclusive conditions. After
24 h no differences were found in [ ‘“Clcholesterol
uptake with the three liposome formulations: the
receiver compartment contained no detectable amounts
of lipid, which was in agreement with the findings of
Ganesan et al. [ 191 and Komatsu et al. [ 26). Differ-
ences were found, however, in the uptake of radiola-
belled lipids in mouse vs. pig stratum corneum.
Surprisingly, the total amount of lipids found was less
in mouse skin than in pig skin. However. as the amount
of lipid applied to pig skin was twice the amount
applied to mouse skin, interpretation of these results is
difficult which is compounded by the nonocclusive
conditions, as differences in volume may lead to dif-
ferent states of dehydration over time.
In the same study [32], the ratio of choles-
terol : cholesterol sulfate at the surface, in the stratum
corneum, and in deeper skin layers was determined. It
appeared that in all layers the ratio between cholesterol
sulfate and cholesterol was almost equal to one which
is indicative for simultaneous transport, possibly in
bilayer fragments. However, again the conclusion from
this study is obscured by the fact that radioactive cho-
lesterol is known to exchange very rapidly with endog-
enous cholesterol.
Further evidence that liposomes penetrate no deeper
than the stratum corneum layer has been provided by
Lasch et al. [ 341 who labelled liposomes with both a
hydrophilic high molecular weight fluorescent marker
(FITC-dextran, 70 000 M,) and a fluorescent lipid
marker (NBD-DPPE). Fluorescence micrographs
taken after 0.5, 5 and 24 h showed unequivocally that,
despite a rapid dispersion within the stratum corneum,
no further penetration of either label into epidermis,
dermis or deeper layers of the skin took place. The
authors conclude that intact liposomes are confined to
the outermost layer of the skin and do not penetrate
through the skin.
While current experimental evidence would princi-
pally exclude absorption enhancement mechanisms for
hydrophilic drugs, other than perhaps enhancement via
complexation with individual phospholipids, Artman
et al. [ 35,361 performed a series of experiments with
vesicles prepared from a commercial soy phospholipid
product (NAT 106; mainly consisting of SPC), and
observed that liposome-encapsulated antibodies with
molecular weights between 20 000 and 50 000 Da dis-
tributed rapidly into deep cutaneous regions, whereas
antibodies did not penetrate into skin when applied as
aqueous solution. Similar results were obtained using
[ “S ] heparin and “““‘technetium as marker. This is an
unexpected and rather remarkable finding in the light
of most other studies where no skin penetration of ves-
icles, or phospholipids, was observed.
Bouwstra et al. [37] observed that the same lipo-
some formulation ( NAT 106) induced large structural
changes in the lamellar structure of the skin (Fig. 6)
which may to some extent corroborate the findings of
Artman et al. [ 35,361.
Similarly, Jarosh et al. [38] studied the effect of
DNA repair enzymes when applied to skin in lipo-
somes. After one hour of application, enzymes were
detected histochemically in the epidermal cells while
very few traversed into the systemic circulation. The
results strongly indicated that DNA repair enzymes
encapsulated in liposomes resulted in an enhanced
DNA repair in a dose-dependent, saturable manner.
Although, the authors’ conclusion that liposomes pen-
etrated mouse and human skin is faulty, since only the
localization of the enzyme, and not of the liposomes or
lipids was monitored.
In another remarkable, and rather controversial devi-
ation from the general dogma that liposomes cannot
penetrate skin, Cevc has put forward a hypothesis, and
presented some preliminary experimental support
[ 391, that under certain conditions, i.e., without occlu-
sion and with a specific formulation of lipids called
‘transfersomes’ whose exact structure and composition
has yet to be disclosed, the hydration driving force for
these vesicles into the skin is larger than the resistance
encountered when passing through the narrow lipid-
filled channels separating the corneocytes of the stra-
tum corneum. The driving force is supposedly mainly
generated by the large hydration gradient across the
skin, varying from 15 to 20% in stratum corneum to
70% in stratum granulosum. According to the hypoth-
esis, vesicles applied under occlusion should not pen-
etrate, since occlusion essentially eliminates the
hydration gradient. To follow the fate of the dermally
applied ‘transfersomes’, the distribution of ‘H-labelled
dipalmitoylphosphatidylcholine in the various tissue
compartments and blood was measured. With ‘stan-
dard’ liposomes and ‘transfersomes’ applied under
occlusion, only a few percent of the lipids were found
in the dermis after 8 h of application. However, when
transfersomes were applied nonocclusively high levels
of radioactivity were found in the deeper layers of the
Fig. 6. NAT 106 liposomes have a strong effect on the microstructure of the stratum corneum. Corneocytea (C) were swollen considerably; the
smooth ultrastructure of the intercellular skin architecture was disrupted, with flattened lipid islands ( L) which were not always spherical. (From
Ref. 37; with permission.)

skin which is in striking agreement with Cevc’s hypoth-
esis.
More recently, Cevc and co-investigators [ 401 have
disclosed the composition of anesthetic transfersomes
containing 2% lidocaine. The lipid composition was
described as a mixture of phosphatidylcholine with 20-
50 mol% ( IO-24% by weight) sodium cholate as well
as 3-7% ethanol. While the authors argue that a more
conventional explanation of the observed effect, i.e.,
stratum comeum fluidization, is unlikely because of
lack of a dose-effect relationship, the fact that the
human study with anesthetic transfersomes was done
“ between formulations, no conclusions can be drawn
..I under occlusion by a watertight wrapping for 25
min” [ 4 I ] contradicts the hypothesis which predicts
abolishment of the hydration driving force upon occlu-
sion and favors penetration enhancement as an alter-
native to the ‘hydration driving force’ hypothesis.
With respect to the follicular route, Lieb et al. [ 4 I ]
studied the localization of carboxyfluorescein in ham-
ster ear skin, which contains large amounts of pilose-
baceous units. The location of carboxyfluorescein
applied in Hepes buffer, in 5% propylene glycol, in
multilamellar vesicles and in 0.05% sodium lauryl sul-
fate in Hepes buffer were compared. Since the ther-
modynamic activity of carboxyfluorescein varied
with respect to the mechanism involved in the variation
of carboxyfluorescein localization. Surprisingly, it was
concluded that carboxyfluorescein was selectively
delivered into the pilosebaceous area of the ear, while
the results indicate that the majority of the marker was
found in the epidermis.
2.2. Physicochemical studies of the interaction oj
lipid vesicles with skin
The composition of skin lipids is unique and varies
greatly from stratum comeum to the basal layer (421.
Specifically, the very low phospholipid content of the
stratum comeum lipids which consist, to approximately
50%, of ceramides [42] is indicative for a particular
role of the latter in the protective properties of the
stratum corneum. Abraham et al. [43] have investi-
gated the formation of the lipid lamellae in the stratum
comeum. Small unilamellar liposomes prepared from
ceramides (40%)) cholesterol (25%), cholesterol sul-
fate ( 10%) and free fatty acids (25%) were shown to
transform to large unilamellar liposomes and finally to
lamellar lipid sheets when calcium chloride was added
to the dispersion. Golden et al. [ 441 characterized the
physical properties of these skin lipids and found a
transition temperature of the mixture around 60-80°C
accompanied by an abrupt change in permeability at
about 70°C. Hence, it was documented that the highly
structured lipid lamellae in skin are the main barrier
and controlling parameter for water flux across skin.
Freeze-fracture electron microscopic techniques for
the visualization of skin ultrastructure have been devel-
oped by Holman et al. [45] and BoddC et al. [ 461.
Following exposure of liquid state niosomes to skin,
Hofland et al. [ 471 have demonstrated the appearance
of structural changes deeper in the stratum corneum,
resembling multilamellar vesicular structures. The
authors speculate that either intact niosomes migrated
into the stratum corneum, or that molecularly dispersed
high local concentrations of nonionic surfactants could
form curved lamellar structures within the lipid inter-
stitial spaces of the stratum comeum. An example is
given in Fig. 7.
In a FTIR-AR study [ 481, the effect of a commercial
liposome formulation (NAT 50) prepared in D,O on
the inward flux of D,O and the outward flux of H,O
was studied and compared to D,O-treated skin in vivo
(under occlusion). Liposome treatment of skin
resulted in an increased DzO inward flux compared to
control, while the HZ0 outward flux was decreased.
These differences were attributed to a penetration
enhancing effect of the individual phospholipids. The
phospholipids could be detected in the lower layers of
the stratum corneum. However Bouwstra et al. [37]
could not find changes in the stratum comeum lipid
structure after application of the NAT 50 liposomes in
vitro. In their study, a fusion of the vesicles on the
surface of the skin was observed which would be
expected to facilitate accumulation of Hz0 in the stra-
tum comeum.
One-dimensional electron paramagnetic resonance
imaging has been employed to monitor the fate of a
liposome-associated spin probe following dermal
application of various types of liposomes [ 491. The
authors claim that they were able to observe and quan-
titate skin transfer of intact liposomes using this tech-
nique, although they are seemingly unaware of the prior
controversy as to the feasibility of intact liposomes
entering the skin, nor do they make an effort to discuss
their findings relative to the well-founded mechanistic
studies published earlier [ 19-2 1I. From the data pre-
sented it remains unclear whether the differences
found, e.g., between small unilamellar, reverse-phase
evaporation and multilamellar vesicles, in both liquid-
crystalline or gel state, are significant and meaningful.
Although the technique might provide valuable infor-
mation, an independent validation is mandatory before
it can be accepted as a valid experimental method.
2.3. Comparative studies of liposomes und
conventional dermal dosage forms
Investigators have routinely compared liposome
preparations with conventional creams of various com-
pounds, a summary of which is given below. Although
these studies may be important for the application of
liposomes for topical uses from a practical point of
view, it should be pointed out that no mechanistic infor-
mation can be obtained from them for two reasons: (i)
When comparing a liposome preparation with a con-
ventional cream, both the physical structure of the drug
carrier as well as the nature of the components from
which it has been prepared change. Therefore, one can-
not conclude whether the obtained differences are due
to the often seemingly ‘magic’ action mechanism of
the vesicle structures or to the interaction of individual
Fig. i ‘. Freeze-fracture electron micrograph of trioxyethylenedodecylether niuwmw. Between corneocytes (C ). clusters of vehicular htructl
(VS) are found in the intercellular lipid lamellae ( ILL). ( From Ref. 17; with permission. )

lipid I molecules with the compound in question, or with
the skin components, or both (e.g., penetration
enhz mcement) ; (ii) the principal problem with most
lipo! tome studies comparing drug transport through
skin with different formulations is the fact that most
inve stigators (with the exception of [ 19,25-271 ) use
ryu~rl concentrations of the drug in the various forn lU-
lations under study. Hence, the thermodynamic activ ity
of the drug in the various formulations is differ ent
which greatly affects the partitioning of the drug
between formulation and skin. In other words, withcJut
using equi~~~lrn~ concentrations of drug to ensure ol Xi-
mal thermodynamic activity, differences in drug trans-
port are observed due to a difference in solubility of
the drug in the formulation.
In the first one of these publications, Mezei et al.
[ 17,181 applied triamcinolone in liposomes and com-
pared it to triamcinolone in DermabaseR. As mentioned
before the liposome formulation favorably altered drug
distribution. In a more recent publication from the same
group tetracaine was incorporated in multilamellar
phospholipids and the anesthetic effects of the liposo-
mal cream and a PontocainR cream were compared
[ 501. It appeared that tetracaine encapsulated in lipo-
somes was more active.
Michel et al. [ 511 encapsulated 1% MRZ 3199 (a
pyridine carboxylic acid phenyl ester) in soybean lec-
ithin and incorporated the liposomes in a 1o/cCarbopol
934 gel matrix. Penetration of 1% MRZ 3199 in the
liposome preparation was compared with that of a
cream on human skin of six volunteers. The study
showed improved penetration into skin with liposomes
compared to the cream. In a related study [ 521, DL-a-
tocopherol nicotinate and 2-( r-butyl)4-cyclohexyl-
phenylnicotinate N-oxide (L440) were incorporated in
liposomes and compared with o/w as well as w/o
emulsions. Again drug applied in liposomes resulted in
higher concentrations in the stratum comeum as deter-
mined by stripping. In a mouse edema test, the anti-
inflammatory activity of L440 was found equal when
applied as liposomal or o/w emulsion formulation,
while the w/o emulsion performed less satisfactory.
Egbaria et al. (K. Egbaria, C. Ramachandran and N.
Weiner, personal communication) investigated the
topical delivery of cyclosporin and compared the accu-
mulation in hairless mouse stratum corneum when
applied in skin lipid liposomes, phospholipid lipo-
somes, emulsions and a hydroalcoholic solution in
vitro. The accumulation in stratum corneum increased
in the order skin lipid liposomes > phospholipid lipo-
somes > emulsions > hydroalcoholic solution. The
total amount of drug in deeper layers of the skin and
the acceptor compartment was less with liposomes
compared to the emulsion and hydroalcoholic solution.
Krowczynski and Storek [.53] compared triamcin-
olone absorption in skin following application in lipo-
somes prepared from lecithin and cholesterol and in a
cream. It appeared that triamcinolone applied in lipo-
somes resulted in increased absorption compared to the
cream.
Lasch and Wohlrab [ 54,55 ] studied the distribution
of cortisol and hydrocortisone in the skin after appli-
cation in a cream and in liposomes. In both studies an
improved concentration-time profile was observed in
different layers of the skin when using liposomes.
Korting et al. [56] compared the efficacy of beta-
methasone diproprionate encapsulated in liposomes
and in a cream. The liposomes were prepared from egg
lecithin and incorporated in a polyacrylate gel. The
studies were carried out in vivo in ten patients with
atopic eczema and ten patients with psoriasis vulgaris.
It was concluded that betamethasone encapsulated in
liposomes improved the antiinflammatory action. but
not the antiproliferative effect.
3. Potential drug products
Most investigators [ I7-22,53-561 have concen-
trated on the potential use of liposomes for the dermal
delivery of steroid compounds. including triamcino-
lone acetonide [ 17, I8 J and a lipophilic prodrug thereof
(triamcinolone acetonide-2 I -palmitate) [ 521, hydro-
cortisone [ 18.561, betamethasone dipropionate [ 561,
cortisol [ 551, progesterone [ 19-221 and dihydrotes-
tosterone [ 57 ] Interestingly, there is a significant body
of literature on both the formulation, physicochemical
interaction and the therapeutic efficacy of liposome-
corticosteroid compounds, largely generated by inves-
tigators interested in the intra-articular corticosteroid
therapy, e.g., of rheumatoid arthritis [ 58-611. which
seems to be mostly ignored by current investigators.
In addition to corticosteroids, the transdermal deliv-
ery of the a,-blocker bunazosin HCI [62], the non-
steroidal antiinflammatory agent flufenamic acid [ 631,
and the CAMP phosophodiesterase inhibitor dyphylline
(for the treatment of psoriasis) [ 161 via liposome have
been reported. Liposomes loaded with clindamycin
hydrochloride [ 641 were reported to show a better effi-
cacy than non-liposome lotions in therapy of acne vul-
garis.
The application of liposomes for the topical delivery
of proteins has emerged only recently. Superoxide dis-
mutase (SOD) activity on skin has been shown to be
retained better following UV radiation when SOD was
applied incorporated within liposomes [ 651, Brown et
al. [ 661 showed that liposome incorporation prolonged
the exposure of incisions to epidermal growth factor
and to transforming growth factor-p (TGF-B), result-
ing in increased tensile strength, while application of
the factors in solution failed to improve treatment over
controls.
Margalit et al. 1671 have prepared ‘bioadhesive’
liposomes by anchoring epidermal growth factor, gel-
atin or collagen in the liposome surface using a cross-
linking agent (glutaraldehyde). In preliminary cell cul-
ture studies (A431 cell line) they could demonstrate
significant binding of these bioadhesive liposomes to
cell surfaces while untreated liposomes did not bind.
They could also show that diffusion of encapsulated
material was essentially unhindered by the presence of
the anchor, although gelatin appeared to provide an
additional ‘compartment’ from which drugs (vinblas-
tine. progesterone, fluconazole) were released with a
different diffusion rate.
As a means to improve treatment of cutaneous virus
infections, specifically herpes simplex virus infections,
Egbaria et al. 13 1] evaluated the deposition of inter-
feron-a (IFN-a) formulated with ‘skin lipids’ and
showed that liposome-associated IFN-LU was delivered
to deep skin layers. Similarly. 70-80s of a dose of
liposomally encapsulated gamma-interferon was found
associated with skin over a 24-h period, although
approximately one-third of the dose was only deposited
onto the stratum corneum [ 681. Since the order of
deposition in deeper skin layers decreased with
decreasing number of hair follicles (ham-
ster >> human > hairless mouse), the authors specu-
lated that the transfollicular route may be an important
pathway for the deposition of drugs in deeper strata of
the skin.
Ho et al. [69 1 succeeded in treating herpes simplex
genitalis infection in guinea pigs with liposomes pre-
senting the recombinant glycoprotein D antigen of her-
pes simplex virus (HSV- I ). This is a unique use of
liposomes as adjuvants in a topical application which
will likely be exploited more to treat a variety of local-
ized infectious states in the future.
4. Safety of topical liposomes and niosomes
One important aspect of penetration enhancement
(if this mode of action is accepted as a major route of
interaction of liposomal lipids and skin lipids) via lipo-
somes and niosomes which has until very recently been
essentially ignored is the potential toxicity of lipid
mixtures when applied repeatedly or chronically to
skin. Hofland et al. [ 701 have employed inhibition of
cell proliferation of SV-40-transformed human keratin-
ocytes to study dermal toxicity of nonionic surfactant
vesicles ( niosomes) A 1O-fold stronger inhibition of
proliferation was found with polyoxyethylene alkyl
chains linked with ester bonds, compared to those
linked with ether bonds, while the presence of choles-
terol appeared to have no effect on cell proliferation.
Freeze-fracture electron micrographs revealed clusters
of vesicular lipid lamellae which were clearly different
from endogenous lipids found within the intercellular
lipid lamellae. The authors speculate that indeed intact
surfactant vesicles migrate into the skin lipid layers, or
that such vesicular structures form following molecular
dispersion and diffusion of surfactants into the skin
lipid lamellae.
5. Conclusions
The field of dermal/transdermal liposomes and nio-
somes as it presents itself today is characterized by both
significant differences in the results obtained by an
increasing number of investigators, but also by some
strikingly similar experimental findings. suggesting
some common mechanisms of (inter)action.
With respect to phospholipid transport, it appears
that the majority of investigators [ 19-22,27,32,33 1 has
found no accumulation of radioactively or fluores-
cence-labelled phospholipid in deeper skin layers or in
the acceptor compartment. Hence, most investigators
would agree that no transport of lipids is taking place
across whole skin. An exception is the hypothesis put
forward by Cevc [ 391, who maintains that so called
transfersomes can transfer large amounts of lipid ( radi-
oactivity) in form of ‘lipid aggregates’ to deeper layers
of the skin when applied nonocclusively.
Another mechanism which has been reported for
niosomes [ 28 ] is fusion of vesicles on the surface of
the skin which might lead to the establishment of large
concentration gradients across the skin for niosome-
intercalated lipophilic drugs.
Secondly, most studies are in agreement that direct
contact between vesicles and skin is essential for pen-
etration and drug delivery. If direct contact is
obstructed, drug transport is not increased as shown by
Hofland et al. [ 28 1. Komatsu et al. [ 271 and Hofland j2 1 K. Knutson. S.L. Krill and J. Zhang, Solvent-medicated alter-
ations of the stratum comeum. J. Controlled Release, II
et al. [ 281 both showed that a penetration enhancement
( 1990) 93-103.
of the molecular components cannot fully account for {3] P.G. Green. R.S. Hinz, A. Kim, C. Cullander, G. Yamane. F.C.
the increase in drug transport observed when drugs are Szoka, Jr. and R.H. Guy, Transdermal iontophoresis of amino
encapsulated in vesicles. acids and peptides in vitro. J. Controlled Release. 21 ( 1992)

It appears that vesicles have a more pronounced 187-190.

effect on transport of Iipophilic drugs than on that of I-I P. Green. B. Shroot. F. Bcrnerd. W.R. Pilgrim and R.H. Guy,
In vitro and in vivo ionrophoresis of a tripeptide across nude
hydrophilic drugs. The exceptions are studies by Art- rat skin. J. Controlled Release, 20 ( 1992) 209-2 17.
man et al. [ 35,361 and Cevc et al. (personal commu- IS V.H.L. Lee. Enzymatic barriers to peptide and protein absorp-
nication) who reported enhanced transport of large tion and the use of penelration enhancers to modify absorption,

hydrophilic drugs through skin. in: S.S. Davis. L. Ilium and E. Tomlinson (Eds.), Delivery
systems for Peptide Drugs. NATO ASI Series A, Life Sciences.
‘4 critical flaw of many studies that attempt to com-
Vol. 125. Plenum Press, New York, NY. 1986. pp. 87-104.
pare (and demonstrate the inferiority of) currently 161 G. Lopez-Berectein and I.J. Fidler (Eds.), Liposomes in the
employed topical dosage forms such as creams, gels, Therapy of InfectiousDiseases and Cancer. Alan R. Liss. New
lotions, etc., with liposomal formulations is the lack of York. 1989.
identical t~erF~~(~~~~3~~i~~~c~~i~d~t~~~ns, i.e., saturation M. Mezei, Liposomes in the topical appiic~ltioll of drugs: a
review. in: Liposomes as Drug Carriers, G. Gre~oriadis (Ed.}.
acrir+v of the drug, rather than equivalent drug con-
J. Wiley & Sons. Chichesrer. 1988. pp. 663-677.
centmtions. If Iiposomal formuiations were to replace M.K. Niesman. The use of liposomea as drug carriers in oph-
conventional dosage forms superior performance under thalmology, Crit. Rev. Ther. Drug Carrier Syst.. 9 ( 1992) I-
identical thermodynamic conditions must be demon- 38.
strated. P.J. Mihalko, H. Schreier and R.M. Abra, Liposomes: a pul-
monary perspective. in: Liposomei; as Drug Carriers, G. Gre-
In order to better understand the mechaIlisn1
goriadis (Ed. 1, J. Wiley & Sons. Chichester. 1988. pp. 67Y-
involved in {trans)de~al transport of drugs when 694.
applied in vesicles to skin, more emphasis should be I.W. Kellaway and S.J. Farr. Liposomes as drug delivery sys-
placed on comparative studies employing double-label- tems to the lung. Adv. Drug Del. Rev., 5 ( IWO) 149-161.

Iing (both fluorescence and radioactivity) and visual- H. Schreier. Liposome aerosols, J. Liposome Res.. 2 ( 1992)
14s- 1%.
ization techniques including freeze-fracture electron
H. Schrcier, R.J. Gonzalez-Rot& and A.A. Stecenko. Pulmo-
rnicros~opy [ 7 I 1 and confocal laser light microsc~)py nary delivery of liposornes. J. Controlled Release, 24 ( 1993 i
1721. This should afford better correlations of fluxes 20%2.1.
of carrier vs. active agent molecules. Observation and M. Schifer-Korting. H.C. Korting and 0. Braun-Falco, Lipo-
some preparations: a step forward in topical drug therapy for
identification of the nature of vesicular structures in the
skin disease’? J. Am. Acad. Dermatol.. 21 ( 1989) 1271-1275.
skin will further elucidate the mechanisms involved in
[ 141 K. Egbariaand N. Weiner. Liposomes asa topical drug delivery
enhancement or inhibition of dermal and transderr~lal system. Adv. Drug Del. Rev.. 5 ( 1990) 287-300.
drug transport when applied in vesicular carrier sys- [ 1S/ H.C.Korting. P. Blecher, M. Schafer-K~~rtin~ and A. Wendel.
tems. Liposomes and niosomes for delivery of agents Topical liposome drugs to come: what the patent literature tells
us, J. Am. Acad. Dermatol., 25 ( I99 I ) IO68-1071.
to and through the skin continue to be an area of
[ Ih] E. Touitou. N. Shaco-Ezra, N. Dayan. M. Jushynski. R. Rafae-
research to be further explored for a better understand- ioff and R. Azoury. Dyphylline liposomes for delivery to the
ing and characterization of the transport path and inter- skin, J. Pharm. Sci., 81 ( 1992) 131-134.
action with the skin. [I7 J M. Mezei and V. Gcllasekharam. Liposomes: a selective drug
delivery system for the topical routeof admillistration. 1. Lotion
dosage form. Life Sci., 26 ( 1980) 1473-1477.
[18 ] M. Mezei and V. Gulasekharam. Liposomes: a selective drug
delivery system for the topical route of administration: gel
References dosage form, J. Pharm. Pharmacol.. 34 C1982) 473-474.
I 19 1M.G. Ganesan, N.D. Weiner. G.L. Flynn and N.F.H. Ho, Infu-
ence of lipos~~mal drug entrapment on ~rcutaneous abso~tion.
[ 1j R.R. Bumette. lontophoresis. in: J. Hadgrafr and R.H. Guy Inr. J. Pharm.. 20 ( 1984) 139-1.54.
( Eds. ), Transdermal Drug Delivery: Developmenral Issues and [ZO] N.F.H. Ho. M.G. Ganesan. N.D. WeinerandGL. Flynn.Mech-
Research Initiatives, Marcel Dekker. New York. NY, 1989. pp. onisms of topical delivery of liposomally entrapped drugs. J.
247-29 I. Controlled Release, 2 ( 1985) 61-65.
 V.M. Knepp. R.S. Hinn. F.C. %oka, Jr. and R.H. Guy. Con- 1371 J.A. Bouwstra. H.E.J. HoHand. F. Spies and H.E. Junpinger.
trolled drug release from a novel liposomal delivcry system. I. Changes in the structure of human stratum corncum mduccd
Investlgatlon oftransdermal potential. J. Controlled Rcle:rw. 5 by lipowmes. in: Liposome Dermatics, Griwbach Confcrencc.
( 198X)71 l-221. 0. Bran-Falco. H.C. Kortmg and H.I. Maihach ( Ed\.1.
V.M. Knepp. F.C. Sztrkn. Jr. and R.H. Guy, Controlled drug Springer-Vcrlaf. Berlin, IYY2. pp. 111-I 36.
release from a novel lipowmal delivery aystcm. II. Tran\dermnl [3X I D.B. Jvro\h. Lipo\omc-zncap~ulated cwyme\ for DNA repair.
delivery characteristvx.. I Controlled Releaw. I2 ( IYYO) 1% in: Liptrsome Dcrmatio. Grieshach Conference. 0. Braun-
30. Falco. H.C. Kortingand H.I. Maihach ( Ed\ 1.Sprtnger-Verlq.
(7-3 I M. Jacob\, G.P. Martm and C. Marriott.. Effect or phwphatib Berlin. lYY7. pp. 3X-360.
dylcholine on the topical hmavailability of corticosteroid\
I39 G. Cevc and G. Blume. Lipid Veh~clc\ penetrate tnto intact \hln
aseased by the hutnun \kin blanching assay. J. Pharm. Phar-
owing to the tran\dermal osmotic gradient\ and hqdratwn
Illmll.. 30 ( 198X) x2’)-833.
I’orcc, Blochim. Bwphy\. Acta. I IO3 ( I YY I ) 726-232.
J.A. Bouwstra, G.S. Coot%. J.A. van der Spek and W. Bras.
I10 M.E. Planas. P. Gonxder. L. Rodrigtw. S. Sawhe/ and G
Structural investigations of human stratum corneum by small
Ce\c. Noninva\ive percutaneous induction oftopical ;ulalgc\ia
angle X-ray wattering. J. Invst. Dermatol.. 97 ( IYYI ) lOO5-
by a new type of drug carrier. and prolongation trf locd pain
1012.
Insensitivity hq anc\thetic lipowmrr. Anesth. Anslg.. 75
M. Mah_jour. B. Mawr, Z. Rahidbaigi and M.B. Fawri. Effect
(1993) 6lS321
of egg yolk lecithins and commerc~ul w) bean lecithins on 1n
31 L.M. Llcb. Ch. Ramachandran. K. Egharia and N. Weiner.
vitro skin permeation of drug\. J. Controlled Releuw. II
Topical dellwry cnhnncemcnt with tnultilatnellar I~povrme\
( I YYO) 133-152.
into pilo\ehaceou\ unit\. I. In \ itro evaluatton uvng Huorcvxnt
H. Komathu. H. Okamoto. K. Miyagawa. M. Hahida and H.
Sc/aki. Percutaneous ahsorption of butyl parahen from Itpo- technlquc\ \\ith hamster ear model. J. In\c\t. Dermatol., YO

wmes in vitro, Chem. Phnrm. Bull., 34 ( IYX6) 3123-3320. ( 1002) 10X-l 13.

H. Komatw. K. Htgahi. H. Ohamoto. K. M~yagaw. M. Hash- I 1’ H.J. Yardlcy and R. Summerly. Lipid compo4tmn and metah-
ida and H. Sc/.aka, Preservative activity and In viva percuta- oliwl in norm;d and divxwd cpidcl-nils. Phat-m. Ther., Ii
ncous penetration of butyl par&en entrapped in lipo~on~c~. ( 14x I ) 357-3x3.
Chcm. Pharm. Bull.. 33 ( IYXh) 3415-3431. 143 I W. Abraham. P.W. Wertf, L. Landmann and I1.T. Downing,
[2X 1 H.E.J. HoHand. H.E. Junginger and J.A. Bouw\tra. Ehtradiol Stratum cornt‘um lipid liposome\: calcium-induced tran\lol--
permeation from non-ionic wfnctant \e\icle\ through hutnan mation Into lamellar \hect\. J. Inve\t. Dermntol.. XX ( IYX7 I
skin in vitro. Pharm. Res., in prcsh. 712-714
H.E.J. Holland. J.A. Bouww-a. F. Spie\. H.E. Bodde and H.E. G.M. Golden. D.B. Guach. A.H. Kennedy. J.F. McKlc and
Junginger. Interaction\ herwren non-ionic suri’actant bcsicles R.O. Potts. Stratum corneum lipid phase transitions and wtcr
and human \kin in vitro. wbmitted to Br. J. Dermatol. barrtcr propertlcs. Biochemistry. 26 ( I987 ) 23X2-13Xx.
K. Egharia. C. Ramachandran. D. Kittayanond and N. Weiner. B.P. Holman. F. Spies and H.E. BoddC. An optimized l’rt‘cw
Topical deli\erq of I~posomally encqxulated intcrl’eron ew- l’racture replicatwn procedure i’or human \kin. J. In\c\t. Dw
uated by in vitro dil’l‘usion \tudw. Antinucroh. Agents Chen- n13tol.. 01 ( IYYO) 337-335.
other.. 34 ( IYYO) 107-I IO.
I H.E. BoddC. B. Holman. F. Spia. A. Weerhrim. J. Kcmpcnaal-,
C.J. Kirby and G. Gregoriadia. Dehydration-rehydration w\t-
M. Momma\ and M. Ponec. Freeze-fracture cltxtron micro\-
cle\: a simple tnethod for high-yield drug entraptncnt in lipo-
copy on in vitro reconwuctcd human epidcrmi\. J. Invc\t
wmeh. Biotechnology, 2 t lYX1) Y79-Y81.
Dcrmatol.. 95 ( IYYO) 10X-I 16.
J. du PIeshis. K. Egharia. N. Weiner. Influence of formulatton
1471 H.E.J. Holland. J.A. Bouustra, H.E. Bodd2. F. Spies and H.E.
lactor\ on the deposition of lipowmal component\ into the
Junginger. Non-iomc wfxtnnt \e<icles in tran\dermal l’w
different stmta or the \ktn, J. Sot. Cosmet. Chem.. 43 ( 1993
mulatiow controlled release and In vitro cl’fect on human \kin.
Y3- 100.
Pharm. Rcs., 6 ( Suppl. ) C19X9) S 17X.
F. SzokaJr. and D. Papahadjopoulo\. Procedure forprcparation
ol’liposomes with large internal aqueous space and high capture IJX I H.E. Bodde. L A.R.M. Pechtold. M.T.A. Subncl and F.H.N. de
Hnan. Monitoring in viw \hin hydration by lipowtnr\ using
hy reverse-phase evaporation, Proc. Natl. Acad. Sci. USA, 75
Int’mrzd apcctroxopy m conjugation with tape stripping. in.
(I’)781 4191~4198.
Llposomc Dermatic\. Griesbach Conference, 0. Braun-Falco.
J. Lasch, R. Lauh and W. Wohlrah. How deep do intact lipo-
somea penetrate into human qkin? J. Controlled Release. IX H.C. Korting and H.I. Mnihach ( Ed\. 1.Springer-Vcrlag. Bw

( 1991) 55-58. lin. 1992, pp. 137-149.

[ 35 I C. Artman, J. Roding. M. Ghycq and H.G. Prazel, Lipowmes 1391 V. Gahrijelcic. M. Sentjurc and J. Kristl, Evaluation of Ilpo-
frotn soya phospholipids a\ percutaneou\ drug carriers. Ar/- wtnv ah drug carriers into the \kin hy Otlr-dtllleli\iOn;II EPR
neim. Forsch. Drug Re\., 30 ( 1990) 1363-1365. imaging. Int. J. Pharm.. 63 ( 1990) 75-7’).
[X5] C. Artman. J. Roding. M. Ghycry and H.G. Prarel. Liposomes [SOI A. Geazteh and M. Mexi, Topical anesthesia of the shm hy
from wya phospholipids as percutaneous drug carrier<. At-T- liposomc encapsulated tetracaine, Anesth. Analg.. 67 ( IYXX)
ncim. Forsch. Drug Re\.. 30 ( 1990) 136551368. IO7Y- I oxI
 H. Schreier, .I. Bouwstrn / Journal
[ 5 I ] C. Michel. Th. Purmann, E. Mentrup, G. Michel and J. Kreuter,
Topical application of an antiphlogistic drug in liposomes,
Proc. Int. Symp. ControlledRelease Bioact. Mater., 18 ( 1991)
485-486.
[52] C. Michel, T. Purman, E. Mentrup, E. Seiller and J. Kreuter,
Effect of liposomes on percutaneous penetration of lipophilic
materials, Int. J. Pharmaceut., 84 ( 1992) 93-105.
[ 531 L. Krowczynski and T. Stozek, Liposomen als Wirkstofftrlger
in der percutanen Therapie, Pharmazie, 39 ( 1984) 627-628.
[54] J. Lasch and W. Wohlrab, Liposome-bound cortisol: a new
approach to cutaneous therapy, Biomed. Biochim. Acta, IO
(1986) 1295-1299.
1551 W. Wohlrab and J. Lasch, Penetration kinetics of liposomal
hydrocortisone in human skin, Dermatologica, 174 ( 1987) I8-
22.
[ 561 H.C. Korting. H. Zienicki, M. Schafer-Korting and 0. Braun-
Falco, Liposome encapsulation improves efficacy of betame-
thasone dipropionate in atopic eczema but not in psoriasis vul-
garis, Eur. J. Clin. Pharmacol., 39 ( 1990) 349-35 I.
[57] A.J.M. Vermorken, M.W.A.C. Hukkelhoven, A.M.G. Ver-
meesch-Markslag, C.M.A.A. Goes, P. Wirtz and J. Ziegen-
meyer, The use of liposomes in the topical application of
steroids. J. Pharm. Pharmacol., 36 ( 1984) 334-336.
[58] U.H. Shaw, C.G. Knight and J.T. Dingle, Liposomal retention
of a modified anti-inflammatory steroid, Biochem. J., 158
( 1976) 473476.
[59] F.J.T. Fildes and J.E. Oliver, Interaction of cortisol-21.palmi-
tate with liposomes examined by differential scanning calorim-
etry, J. Pharm. Pharmacol., 30 (1978) 337-342.
[ 601 M. Arrowsmith, J. Hadgraft and I.W. Kellaway, The interaction
of cortisone esters with liposomes as studied by differential
scanning calorimetry, Int. J. Pharm., 16 ( 1983) 305-3 18.
1611 M. Arrowsmith. J. Hadgraft and I.W. Kellaway. The in vitro
release of steroids from Iiposomes, Int. J. Pharm., 14 ( 1983)
191-208.
[ 621 A. Kato, Y. lshibashi and Y. Miyake, Effect ofegg yolk lecithin
on transdermal delivery of bunazosin hydrochloride. J. Pharm.
Pharmacol., 39 ( 1987) 399-400.
c?fControlledRelease 30 (1994) I-15 15
[ 631 T. Kimura. N. Nagahara, K. Hirabayashi, Y. Kurosaki and T.
Nakayama, Enhanced percutaneous penetration of flufenamic
acid using lipid disperse systems containing glycosylceram-
ides, Chem. Pharm. Bull., 37 (1989) 454-457.
[64] N. Skalko. M. Cajokav and I. Jalsenjak, Liposomes with clin-
damycin hydrochloride in therapy of acne vulgaris, Int. J.
Pharm., 85 (1992) 97-101.
[ 651 Y. Miyachi, S. Imamura and Y. Niwa, Decreased skin supe-
roxide dismutase activity by a single exposure of ultraviolet
radiation is reduced by liposomal superoxide dismutase pre-
treatment. J. Invest. Dermatol., 89 ( 1987) I I l-l 12.
[66] G.L. Brown, L.J. Curtsinger. M. White, R.O. Mitchell, J.
Pietsch, R. Nordquist, A. von Fraunhofer and G.S. Schultz,
Acceleration of tensile strength of incisions treated with EGF
and TGF-P, Ann. Surg., 208 ( 1988) 788-794.
[67] R. Margalit. M. Okon. N. Yerushalmi and E. Avidor, Bioad-
hesive liposomes as topical drug delivery systems: molecular
and cellular studies, J. Controlled Release, I9 ( 1992) 275-
288.
[68] J. du Plessis, K. Egbaria, C. Ramachandran and N. Weiner,
Topical delivery of liposomally encapsulated gamma-inter-
feron, Antiviral Res., 18 ( 1992) 259-265.
[69] R.J.Y. Ho, R.L. Burke and T.C. Merigan, Antigen-presenting
liposomes are effective in treatment of recurrent herpes simplex
virus genitalis in guinea pigs, J. Viral., 63 ( 1989) 2951-2958.
[70] H.E.J. Holland, J.A. B0uwstra.M. Ponec,H.E.BoddC,F. Spies,
J. Coos Verhoef and H.E. Junginger, Interactions of non-ionic
surfactant vesicles with cultured keratinocytes and human skin
in vitro: a survey of toxicological aspects and ultrastructural
changes in stratum comeum, J. Controlled Release, I6 ( I99 I )
155-168.
17 I ] J.A. Bouwstra, B.A.I. van den Bergh. M.A. Salomons-de Vries
and F. Spies, Visualization of interactions between liposomes
and skin. Proc. Int. Symp. Controlled Release Bioact. Mater.,
20 (1993) 440-441.
[72] A.J. Hoogstraate, C. Cullander. J.F. Nagelkerke, J. Verhoef,
H.E. Junginger and H.E. BoddC. Diffusion rates and transport
pathways of FITC-labelled model compounds through buccal
epithelium, Proc. Int. Symp. Controlled Release Bioact. Mater.,
20 ( 1993) 234-235.