www.elsevier.com/locate/jconrel

Journal of Controlled Releas

Liposomes as carriers for dermal delivery of tretinoin: in vitro

evaluation of drug permeation and vesicle–skin interaction

Chiara Sinicoa, Maria Manconia, Marcello Peppib, Francesco Laia,

Donatella Valentia, Anna Maria Faddaa,*

aDipartimento Farmaco Chimico Tecnologico, Universita di Cagliari, Via Ospedale 72, 09124 Cagliari, ItalybDipartimento di Citomorfologia, Laboratorio di Microscopia Elettronica e Microanalisi e Centro per lo studio dei metalli pesanti in medicina,

Universita di Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato, Cagliari, Italy

Received 27 July 2004; accepted 17 November 2004

Available online 16 December 2004

Abstract

The influence of liposome composition, size, lamellarity and charge on the (trans)dermal delivery of tretinoin (TRA) was

studied. For this purpose we studied both multilamellar (MLV) or unilamellar (UV) liposomes. Positively or negatively charged

liposomes were obtained using either hydrogenated (PhospholiponR90H) or non-hydrogenated soy phosphatidylcholine

(PhospholiponR90) and cholesterol, in combination with stearylamine or dicetylphosphate. Liposomal formulations were

characterized by transmission electron microscopy (TEM) and optical and light polarized microscopy for vesicle formation and

morphology, and by dynamic laser light scattering for size distribution. In order to obtain more information about the stability

and the thermodynamic activity of the liposomal tretinoin, TRA diffusion through a lipophilic membrane was investigated. The

effect of the vesicular incorporation of tretinoin on its accumulation into the newborn pig skin was also studied. The

experiments were performed in vitro using Franz cells in occlusive conditions and were compared to three different controls.

The tretinoin amount delivered through and accumulated in the several skin layers was detected by HPLC. Furthermore, TEM

in combination with osmium tetroxide was used to visualize the skin structure after the liposomal administration. Overall

obtained results showed that liposomes may be an interesting carrier for tretinoin in skin disease treatment, when appropriate

formulations are used. In particular, negatively charged liposomes strongly improved newborn pig skin hydration and TRA

retention, though no evidence of intact vesicle penetration was found.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Liposomes; Tretinoin; Pig skin; Topical application; Transmission electron microscopy

0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2004.11.020

* Corresponding author. Tel.: +39 70 6758565; fax: +39 70

6758553.

E-mail address: mfadda@unica.it (A.M. Fadda).

1. Introduction

During recent years trans retinoic acid or tretinoin

(TRA), a natural retinoid, has been the subject of

growing interest because of its ability to regulate

e 103 (2005) 123–136

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136124

epithelial cell growth and differentiation, sebum

production and collagen synthesis. These qualities

have led to its use in the treatment of various

proliferative and inflammatory skin diseases, such as

psoriasis, acne, epidermotropic T-cell lymphomas or

epithelial skin cancer [1]. Due to the severe side

effects when systemically administered [2], trans

retinoic acid is almost exclusively topically employed,

resulting in little if any TRA being absorbed systemi-

cally [2,3]. However, even its topical use is limited by

several disadvantages, such as skin irritation, very low

water solubility, and high instability in the presence of

air, light and heat. The low solubility may limit its

incorporation in a suitable vehicle, while its photol-

ability may render the topically applied drug ineffec-

tive [4]. Moreover, when topically applied trans

retinoid acid can lead to local irritation such as

erythema, peeling and burning at the application site

and increased susceptibility to sunlight. In order to

overcome all these drawbacks, tretinoin liposomal

formulations have been studied by several authors. In

particular, the incorporation of TRA in liposome

formulations has been selected in order to circumvent

the undesirable effects of the drug on the skin surface,

to maximize its accumulation into the skin, and to

prevent its fast degradation. Moreover, the use of

liposomes for targeting drugs into the pilosebaceous

structures has shown that liposomal incorporation

could be beneficial for treating hair follicle-associated

disorders, such as acne and alopecia. Different

investigators reported an increased skin accumulation

of TRA in vitro and a reduced irritancy in vivo after

treatment with these carriers. Foong et al. [5] reported

to find greater TRA concentration in the epidermis

and in the dermis after application of liposomal

formulations compared to conventional creams. In

one report, Masini et al. [6] found that the percentages

of tretinoin in the epidermis and dermis (in vitro

studies) were significantly higher with liposomes than

with gel formulations. Furthermore, Sch7fer-Kortinget al. [7] demonstrated that liposomal formulations

which were less concentrated in TRA than commer-

cial topical preparations had the same efficacy and

lower skin irritancy in man. Results of our previous

studies demonstrated that TRA can be incorporated in

high yields both in liposomes and niosomes, which

are also able to reduce the photodegradation rate of

this drug [8,9].

As liposomes have been reported to be able to give

slow drug release, cutaneous targeting and low trans-

dermal delivery of a drug, in this work we have studied

the influence of liposomal incorporation on the (trans)-

dermal delivery of tretinoin. The aim of this study is to

find new dermatological formulations suitable for

tretinoin. Therefore, we investigated the effect of

TRA incorporation on the deposition of the drug into

the different skin strata by using in vitro diffusion

experiments through newborn pig skin. In order to find

the optimal formulation for the topical delivery of

tretinoin, the influence of several parameters such as

vesicle size and composition, surface charge, and

vesicle stability were investigated. Moreover, in the

attempt to elucidate liposome–skin interactions, we

compared results of the permeation experiments with a

visualization study of the newborn pig skin using

transmission electron microscopy (TEM).

Many factors control the delivery of bioactive

molecules into the skin by using liposomal carriers.

They include drug physicochemical properties

(molecular weight, lipophilicity) as well as type of

liposomal formulations (lamellarity, lipid composi-

tion, surface charge, lipid concentration, size)

[10,11]. Several authors have studied the mecha-

nisms used by liposomes which can improve

(trans)dermal drug delivery. These studies were

carried out by using several techniques such as

diffusion experiments [12–14], visualization studies

using transmission electron microscopy (TEM)

[15,16], freeze fracture electron microscopy (FFEM)

[15–19] and/or fluoromicrography [20–22] as well as

confocal laser scanning microscopy (CLSM) [23–25],

X-ray diffraction and electron spin resonance (ESR)

[18,26,27]. Therefore, different results were obtained

which led to different interpretations on vesicle–skin

interactions. This can be summarized as follows: (1)

liposomes may penetrate intact into the skin

[11,28,29]; (2) vesicles do not penetrate intact but

they disintegrate on the skin surface and individual

lipid molecules can penetrate the stratum corneum

(SC) with consequent fluidization and modification of

the SC lipids [25]; (3) adsorption and fusion of

liposomes on the skin surface may occur with a

consequent mixing of liposomal bilayer with inter-

cellular skin lipids [18,23] and/or direct transfer of the

drug to the SC [30]; and (4) liposomes may exert an

occlusive effect [31].

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136 125

These different interpretations reported in literature

can be explained by the fact that vesicle–skin

interactions are powerfully dependent on the phys-

icochemical properties of the vesicular systems. In

fact, it has been shown that vesicle–skin interactions

are strongly affected by vesicle composition, and in

particular by their phase state and elasticity. Liquid-

state vesicles have demonstrated higher capability to

interact with human skin than gel-state vesicles

[16,19,30].

During this work we studied TRA delivery from

two different soy phosphatydilcholine-based lip-

osome formulations. In fact, in order to study the

influence of phase transition temperature (Tc) of

the main liposomal component, we prepared multi-

lamellar (MLV) or unilamellar (UV) liposomes

using two different commercial mixtures of hydro-

genated (PhospholiponR90H, P90H) or non-hydro-

genated soy phosphatydilcholine (PhospholiponR90,P90) as the main bilayer component. The head

groups of these phospholipids are consistent, but

the acyl chain components vary considerably. P90H

have fully saturated acyl chains and a high gel–

liquid Tc (80 8C, 52 8C in water). P90 is a

mixture of pure soy phosphatydilcholine, rich in

unsaturated and polyunsaturated fatty acids and

with a low Tc (b2 8C). Therefore, at the temper-

ature of the skin (i.e. 32 8C), P90H and P90

liposomal bilayers should be in a different thermo-

dynamic state.

Moreover, positively or negatively charged lip-

osomal formulations were obtained using either

P90H and P90 in combination with stearylamine

(SA) or dicetylphosphate (DCP), respectively. All

formulations also contained cholesterol. The effect of

the vesicular incorporation of tretinoin on its

accumulation into the newborn pig skin was also

investigated. The experiments were performed in

vitro using vertical diffusion Franz cells in occlusive

conditions and in comparison with three different

controls: a hydroalcoholic solution, an oil solution

and a commercial formulation of TRA (Retin-AR).At the end of the experiments, the tretinoin amount

accumulated into the several skin layers was detected

by HPLC. Furthermore, we used TEM in combina-

tion with osmium tetroxide (OsO4) to visualize the

newborn pig skin structure after the liposomal

administration.

2. Experimental methods

2.1. Materials

Soy phosphatidylcholine (PhospholiponR90, P90)and hydrogenated soy phosphatidylcholine (Phos-

pholiponR90H, P90H) were kindly obtained from

Natterman Phospholipids, Gmb. Cholesterol (Chol),

trans retinoic acid (TRA), dicetylphosphate (DCP),

stearylamine (SA) and all the other products were

analytical grade and were purchased from Aldrich,

Milan, Italy. Silicon membrane, PertheseR, was

purchased from Electromedics, Florence, Italy.

Retin-AR is a commercial preparation produced by

Janssen-Cilag, Milan, Italy.

2.2. Vesicle preparation

Liposomes were prepared from P90 or P90H,

cholesterol and TRA (5:0.6:2 molar ratio). All

vesicle formulations contained a constant amount

of DCP (2:1 TRA/DCP molar ratio) or SA (2:1

TRA/SA molar ratio) to obtain negatively or

positively charged vesicles. Multilamellar vesicles

(MLVs) were prepared according to the film

hydration method. The surfactant, Chol, TRA and

DCP or SA in chloroform solution were mixed in

the appropriate ratio. The lipid–drug mixture was

deposited as a thin film in a round-bottom flask by

roto-evaporating the chloroform under vacuum. The

vacuum was applied for 1 h to ensure total removal

of trace solvent. The film was hydrated at a

temperature above the gel–liquid transition temper-

ature of the amphiphiles (Tc) with phosphate-

buffered saline solution (PBS). Sonicated unilamel-

lar vesicles (UVs) were prepared starting from MLV

dispersion by sonication in a Soniprep 150 appara-

tus (MSE, Crowley), under a nitrogen stream, for 30

min (30 times for 1 min), at a temperature above

the Tc. Each vesicle suspension was purified from

non-incorporated TRA by gel chromatography on

Sephadex G50. Incorporation efficiencies (E%),

expressed as a percentage of the total amount of

TRA found in the studied formulations at the end of

the preparation procedure, were determined by

HPLC after disruption of vesicles with Triton X-

100. Tretinoin content of samples was analyzed at

350 nm using a liquid chromatograph Alliance 2690

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136126

(Waters), equipped with a photodiode array detector

and a computer integrating apparatus (Millennium

32). The column was a Nova-Pack C18 (60 A, 4

Am, Waters). The mobile phase was a mixture of

acetonitrile, water and acetic acid (84.5:15:0.5, v/v),

at a flow rate of 1.2 ml/min. All suspensions were

prepared under yellow light and kept in the dark at

all times. All formulations were diluted with PBS in

order to achieve the same TRA concentration (0.2

mg/ml). Diluted liposomal suspensions were stable

for at least 24 h without any appreciable vesicle

sedimentation.

2.3. Vesicle characterization

Vesicles were characterized by TEM and optical

and light polarized microscopy for vesicle formation

and morphology and by dynamic laser light scattering

(DLLS) for mean size and polydispersivity index (PI).

A drop of vesicle dispersion was applied to a carbon

film-covered copper grid and was stained with a 1%

phosphotungstic acid. Then samples were examined

and photographed with Zeiss EM 109 transmission

electron microscope at an accelerating voltage of 80

kV.

Optical and light polarized micrographs were

obtained using an optical microscope Zeiss Axioplan

2, at 25 8C. The temperature was kept constant F1 8Cby a hot stage device connected to a thermostatic bath.

Liposome size distribution was determined by

DLLS (N4 plus, Beckman Coulter) at 25 8C. Samples

were scattered (633 nm) at an angle of 908. Data werefitted by the method of inverse bLaplace trans-

formationQ and Contin.

2.4. Diffusion studies through silicone membrane

In vitro diffusion studies of TRA in liposomes

were performed through PertheseR using vertical

Franz diffusion cells (Rofarma, Milan). The receiver

compartment had a volume of 5.5 ml and an effective

diffusion area of 0.636 cm2. The receiver compart-

ment was filled with a hydroalcoholic solution

(ethanol/water 50:50) which was constantly stirred

with a small magnetic bar and thermostated at 37F1

8C throughout the experiments. 1 ml of each lip-

osomal suspension with or without (control) TRA

incorporated was placed on the silicone membrane

surface and then the diffusion cells were covered with

aluminum foil to prevent light exposure. A hydro-

alcoholic solution of TRA was also studied as a

reference. Samples of the receiver compartment were

extracted after elapsed times of 2, 4, 6, 8 and 24 h,

replaced with an equal volume of hydroalcoholic

solution to ensure sink conditions, and analyzed by

HPLC. At the end of the experiments, samples of the

donor phase were analyzed by HPLC for tretinoin

content. TRA recovery from the donor and receiver

compartments was always more than 96% of the

applied dose. Samples of the receiver compartment

were also analyzed for phospholipid content, using

thin-layer chromatography on high performance-TLC

silica gel uniplates (Analtech), a mixture of choro-

form/methanol/water (75:23:2, v/v) as eluant, and

iodine vapor for visualization.

2.5. Skin permeation studies

In vitro skin permeation studies were performed

using vertical diffusion Franz cells with an effective

diffusion area of 0.636 cm2. The experiments were

carried out using newborn pig skin. The skin,

previously frozen at �18 8C, was pre-equilibrated in

PBS solution at +25 8C 2 h before the experiments. A

circular piece of this skin was sandwiched securely

between the two halves of the cells with the SC side

facing the donor compartment. The receiver compart-

ment was filled with 5.5 ml of a hydroalcoholic

solution (ethanol/PBS 50:50) which was continuously

stirred and thermostated at 378F1 8C throughout the

experiments. 1 ml of TRA incorporated vesicle

suspensions (0.2 mg/ml) or control preparations were

placed on the membrane surface. As controls, a

hydroethanolic (W/Et) solution and an almond oil

solution of TRA (0.2 mg/ml) were used. To compare

the results we also tested a commercial cream, Retin-

AR (RetA, 0.025%). Before starting the experiments

the donor cell was sealed with parafilm and the cells

were covered with aluminum foil to prevent exposure

to light. At hourly intervals up to 9 h the receiver

compartment was removed and replaced with an equal

volume of pre-thermostated (37 8C) fresh hydro-

alcoholic solution. The complete substitution of the

receiver compartment was needed to ensure sink

conditions and quantitative determination of the small

amounts of TRA permeated.

Table 1

Diameter and entrapment efficiency (E%) of the prepared tretinoin

incorporating liposomes

Composition E% Average size (nm F S.D.

MLV UV MLV UV

P90/SA 71.80F5.1 93.03F1.7 598F67 297F74

P90H/SA 70.01F2.1 91.91F1.9 1163F84 205F53

P90/DCP 97.32F1.8 75.98F2.5 536F49 293F53

P90H/DCP 96.75F2.2 78.53F2.6 993F122 135F56

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136 127

The solutions were analyzed by HPLC. Studies

were performed in triplicate and the mean values were

used for the analysis of data.

2.6. Skin retention of tretinoin

At the end of permeation study the skin surface

was washed 3 times with PBS and then removed and

dried with filter paper. The SC was removed by

stripping with tapes of TesaR film (Germany) and

then extracted with 20 ml of methanol. The piece of

the skin was separated into the epidermis (EP) and

dermis (D) by pressing the skin surface against a hot

plate (60 8C) for 30 s and peeling off the epidermis

[32]. The separated skin samples were each soaked in

a flask with 20 ml of methanol for 24 h. Then the

methanolic samples were shaken in an ultrasound bath

for 4 times of 30 min each, in order to extract all the

drug accumulated in the skin pieces.

2.7. Skin analysis by transmission electron

microscopy

At the end of the permeation experiments the

pieces of skin treated with P90 or P90H/DCP

unilamellar vesicles were observed by TEM. The

skin was treated for 9 h with only PBS as control. The

skin samples incubated for different times with

liposomes were then cut into small ribbons approx-

imately of 2�4 mm in size. The ribbons were fixed

with Karnovski’s solution overnight, washed with

cacodylate solution buffer and then fixed with

osmium tetroxide in sodium cacodylate buffer (1:1)

for 1 h. Following post fixation, the samples were

dehydrated in 30%, 50%, 70%, 80%, 95% and 100%

acetone solutions and embedded in epon-araldite

resin.

Ultrathin sections were cut by an ultramicrotome

(Leica, Ultracut, UCT), collected on formvar-coated

grids and intensified with uranium acetate and lead

acetate. Finally samples were examined and photo-

graphed in a Joel JEM-1010 transmission electron

microscope.

2.8. Statistical analysis of data

Data analysis was carried out with the software

package Microsoft Excel, version 2001. Results are

expressed as meanFstandard error (3 independent

samples). Statistically significant difference was

determined using the Student’s t-test and analysis of

variance (ANOVA) with P=0.05 as a minimal level of

significance.

3. Results and discussion

3.1. Vesicle characterization

In the present work, in order to provide new topical

application forms suitable for tretinoin, we have

prepared and tested liposomes made with hydro-

genated or non-hydrogenated soy phosphatidylcho-

line. In particular we have prepared both negatively

and positively charged vesicles by using either DCP

(0.41 mg/ml final concentration) or stearylamine (SA,

0.13 mg/ml final concentration), respectively, as a

charge inducer. Vesicle size and tretinoin incorpora-

tion efficiencies are reported in Table 1. Polydisper-

sivity index (PI) of the prepared formulations ranged

from 0.2 to 0.3 for UVs and from 0.4 to 0.6 for MLVs.

Therefore, some of the samples were polydispersed

(PIN0.35), but analyses were always repeatable.

Dynamic light scattering analyses confirmed the

results obtained previously [8], that is, that vesicle

size is dependent on preparation method and bilayer

composition. As expected, vesicles prepared by the

film method (MLVs) were always larger than those

prepared by sonication (UVs). Moreover, the mean

size of UVs was large with respect to the preparation

method. In fact, sonicated liposome size ranged from

135 to 305 nm. This is due to the presence in the

bilayer of all studied vesicles, of the highly hydro-

philic amphiphiles DCP or SA that increase the

vesicle surface energy which causes vesicle enlarge-

ment, if compared with neutral liposomes [33]. For

-

)

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136128

the same reason, vesicle formulations containing SA

were always larger in size than those containing DCP.

As shown in Table 1, all studied formulations

presented a high entrapment efficiency (E%). It is

interesting to note that SA multilamellar liposomes

showed an incorporation efficiency that was lower

than that of the unilamellar vesicles. This could be due

to the higher energy required for TRA-incorporating

vesicle formation when SA was present in the lipid

phase. In fact, in the preparation of SA-containing

liposomes, sonication was more efficient than vortex-

ing, especially in comparison with the preparation of

negatively charged MLVs.

3.2. Diffusion studies through silicone membrane

In order to obtain more information about the

stability and the thermodynamic activity of tretinoin

in liposomal formulations, we studied the TRA

diffusion through a polymeric lipophilic membrane.

This silicone membrane, which offers a non-porous,

hydrophobic, inert and reproducible barrier, has been

used by several authors to evaluate factors such as

drug activity on permeation. The percentages of

Fig. 1. Cumulative amounts (%) of tretinoin permeated through a silicone

TRA-loaded liposomes. For each formulation, main component and cha

separately in triplicate in vertical Franz diffusion cells through a silicon m

samples of three separate experiments.

tretinoin permeated after 24 h from our liposomal

formulations and from a hydroalcoholic solution of

the free drug (control) are reported in Fig. 1. As may

be seen, the percentage of drug diffused from the

control solution was generally smaller than that from

the liposomal formulations. We had already obtained

similar results studying the diffusion of tretinoin and

8-methoxsalen (8-MOP) from liposomes and nio-

somes [8,34]. Our previous studies on the in vitro

diffusion of TRA and 8-MOP had shown that the

delivery of a lipophilic drug can be modulated by

varying the structure and/or bilayer composition of the

vesicular carriers. Moreover, these studies had shown

that vesicular formulations enhanced the lipophilic

drug diffusion if compared with a hydroalcoholic

solution of the free drug and that this result could be

the consequence of a higher thermodynamic activity

of the vesicle-incorporated drug with respect to the

control [34]. Results obtained during this study, using

liposomal formulations and a control with the same

drug concentration (i.e. 0.2 mg/ml), support this

explanation. In fact, in the control solution the drug

concentration was a lot less than the solute saturation

solubility, therefore the thermodynamic activity of the

membrane from a bfreeQ TRA hydroalcoholic solution (W/Et) and

rge inducer are reported. Diffusion experiments were carried out

embrane (PertheseR). Data represent the means for three replicate

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136 129

drug is most probably less than the activity in

liposomal formulations. Our results also show that

tretinoin diffusion is affected by the liposome

composition and structure. In fact, as previously

observed with negatively charged vesicular systems,

UVs always gave a higher drug release than MLVs

and P90 liposomes released greater TRA amounts

than P90H liposomes. Moreover, except for P90H

MLVs, negatively charged liposomes showed a higher

release rate than positively charged liposomes. This

result could be explained as a consequence of ion pair

formation between the drug (pKa=7.85) and stearyl-

amine counter ions. In fact, tretinoin is partially

ionized at pH=7 and therefore it could be more

strongly retained in the positively charged vesicles.

Finally, in our early work we suggested that

vesicles do not interact with Perthese membrane and

that the incorporated drug cannot diffuse through the

membrane. Indeed, we did not find phospholipids in

the receiver compartment at the end of the experi-

ments. However, we cannot exclude that the presence

of ethanol as a cosolvent in the donor compartment

could have produced a polarity of the membrane

resulting in a reduced partitioning and flux of the free

tretinoin.

Fig. 2. In vitro diffusion of liposomal and free tretinoin through newborn

are reported. Data represent the means for three replicate samples of three

3.3. In vitro skin permeation study

In order to evaluate the influence of composition,

structure and surface charge of the studied liposomes

on the TRA accumulation into and diffusion through

the skin, we carried out in vitro permeation studies,

using newborn pig skin and vertical Franz diffusion

cells. During this study we compared permeation data

obtained from liposomal tretinoin with those of

control formulations (oil and hydroalcoholic solution)

having the same drug concentration (i.e. 0.2 mg/ml).

Furthermore, as a control we also used a commercial

topical formulation of tretinoin (Retin-AR, 0.25 mg/

ml), because it was stated that liposomes less TRA

concentrated than commercial formulations had

shown the same efficacy [7].

The in vitro permeation study was carried out

through the whole newborn pig skin and in occlusive

conditions because, according to several authors ([16],

for instance) occlusive conditions are likely to

enhance drug accumulation into the skin layers. The

mean amount of TRA permeated per unit of surface

area from vesicles or control formulations was

determined during 9 h experiments. In Fig. 2,

permeation profiles (cumulative amounts of TRA

pig skin. For each formulation, main component and charge inducer

separate experiments.

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136130

permeated versus time) of tretinoin through the skin

from the different studied liposomes and controls are

shown. As can be seen, permeation curves do not

show a classic profile with a steady state phase.

Tretinoin flux is higher in the initial period of the

permeation experiments (0–3 h). Afterwards, for all

liposomal formulations, TRA permeation rate was

almost constant in all sampling times. Results seem to

indicate that TRA release and skin permeation

occurred rapidly especially when the drug was

delivered from positively charged liposomes. In fact,

after 1 h from the beginning of the experiments,

positively charged formulations delivered 0.463–

0.641 Ag/cm2 while negatively charged vesicles

delivered 0.098–0.180 Ag/cm2 of the drug. Then, a

plateau is obtained from the negatively charged P90

liposomes that showed the lowest TRA diffusion rate.

Table 2 shows the cumulative amounts of tretinoin

delivered in the receiver compartment 3 and 9 h after

the beginning of the experiments. It is evident that

there are some differences in the total amount of TRA

permeated.

In the first analysis, the dose permeated of

liposomal TRA was dependent on the main bilayer

component. In fact, this value increased from P90 to

P90H liposomes both in MLV and UV dispersions.

Results of our study show that vesicle size and

lamellarity did not affect TRA delivery through the pig

skin. In fact for each composition the permeation

profile was very similar for both multi- or unilamellar

vesicle dispersions. These results seem to confirm Du

Table 2

Results of in vitro permeation study from vesicular formulations and contro

the experiments (9 h); amount of TRA accumulated into the skin at th

Efficiency (LAC) value: TRA accumulated into SC/TRA delivered throug

Composition Structure TRA delivered (Ag/cm2FS.

3 h 9 h

P90/SA MLV 0.599F0.07 0.8

UV 0.544F0.04 0.7

P90H/SA MLV 0.897F0.06 1.5

UV 0.758F0.05 1.3

P90/DCP MLV 0.330F0.06 0.5

UV 0.263F0.04 0.4

P90H/DCP MLV 0.677F0.08 1.1

UV 0.565F0.05 1.0

W/Et 0.631F0.08 1.7

Oil 0.457F0.07 1.1

Retin-AR 0.404F0.08 0.9

Plessis et al. [13] suggesting that intact penetration of

liposomes does not occur. Some interesting data related

to vesicle composition can be pointed out. In fact,

surprisingly we have obtained higher drug diffusion

when TRA was delivered in vesicles made from

phospolipids with the highest Tc (P90H, Tc=52 8C)than in P90 vesicles (Tcb2 8C). TRA delivery through

the skin was enhanced by a factor of 2–2.35 in P90H

vesicles. It is well recognized that the thermodynamic

state of liposomal bilayers plays a fundamental role on

the drug transport rate through the skin in vitro.

Incorporation of drugs in gel-state liposomes is known

to result in a slower skin permeation rate than in fluid-

state vesicles [16]. It has also been suggested that the

fluid-state of the lipids is the main aspect involved in

the fluidity of the vesicle bilayers [19]. However, it is

also well known that cholesterol content may play a

crucial role for the effective delivery of liposomal

substance into the skin. The optimal amount of

cholesterol is between 30% and 50% of the mass of

the liposomal membrane [27]. Moreover, when lip-

osomal bilayer is heterogeneous (i.e. composed of

different phospholipids, cholesterol and other compo-

nents), regions of different ordering and dynamic of

phospholipid chains are formed in the liposomal

bilayer [27]. Results obtained during this work could

be explained as the consequence of the presence in the

liposomal bilayers of both cholesterol and the amphi-

patic drug tretinoin, which is present in high molar

ratio. Here, in the occlusive conditions, both choles-

terol and TRA could have affected the Tc of soy

ls: amount of TRA delivered through the skin at 3 h and at the end of

e end of the permeation experiments; and Locally Accumulation

h the skin ratio

D.) TRA accumulated (AgFS.D.) LAC

9 h 9 h

88F0.06 13.56F1.33 5.3

99F0.04 23.47F2.55 12.9

41F0.09 17.45F3.00 3.3

31F0.04 29.83F4.25 8.4

55F0.08 30.45F7.58 26.6

52F0.02 32.67F8.98 34.8

44F0.07 30.37F7.12 9.6

25F0.09 34.13F8.28 14.9

56F0.11 15.33F2.43 2.4

73F0.10 16.83F2.52 6.5

17F0.09 12.03F0.41 4.2

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136 131

hydrogenated phosphatidylcholine in such a way that

the fluidity of the P90H liposomal membrane increased

at the temperature of the experiments. A detailed study

of the thermal behavior of these formulations has just

started using Differential Scanning Calorimetry (DSC).

On the other hand, the differences in the TRA flux

could be simply related to a lower stability of the TRA-

incorporated P90 liposomes.

Obtained results also showed that positively

charged vesicles gave higher tretinoin transport rate

than negatively charged liposomes. In addition,

statistical comparison of the TRA amounts permeated

throughout 3–9 h showed that P90 liposomes pro-

vided drug permeation generally lower than the

controls. On the contrary, drug delivery from P90H/

SA liposomes was similar or slightly higher than

Retin-A, which was the control with the lowest TRA

diffusion through the pig skin. The highest drug

transport was obtained with the water/ethanol mixture

as a consequence of the enhancing property of ethyl

alcohol.

It is well known that the skin may act as a

negatively charged membrane [35,36] and it has been

generally reported that the presence of a charge on the

vesicle surface affects the drug diffusion through the

skin [32]. Negatively charged vesicles generally give

a higher flux than positively charged counterparts,

which in turn can improve drug accumulation in the

superficial skin strata. We also observed this behavior

studying the in vitro skin permeation of 8-MOP-

loaded vesicles [34]. However, in the present inves-

tigation, positively charged vesicles provided a

tretinoin permeation similar or even statistically

higher (Pb0.05, P90H formulations) than negatively

charged liposomes. As reported above for in vitro

TRA diffusion through the synthetic membrane, the

enhanced TRA diffusion through the pig skin

observed here with the SA-containing formulations

could be explained as the consequence of ion pair

formation between the organic acid tretinoin and SA

counter ions. The ion pair skin transport of TRA in

microemulsions, using various counter ions, has been

reported recently [37]. Here, the partially ionized TRA

(pKa=7.85; pH=7) and SA could behave as ion pairs

thus promoting tretinoin delivery through the skin. In

conclusion, overall results of the in vitro release and

skin permeation study indicated that TRA flux is

strongly affected by the liposome stability.

3.4. In vitro TRA regional distribution on the skin

The amounts of drug accumulated in the whole pig

skin are reported in Table 2. As can be seen, liposomal

formulations enhanced TRA accumulation into new-

born pig skin by a factor of 1.45–2.83, 1.14–2.22 and

1.04–20.03 over Retin-AR, hydroalcoholic and oil

solution, respectively. TRA oil solution gave the

highest accumulation compared the other controls,

while the lowest TRA accumulation was obtained

with Retin-AR. Results showed that the accumulation

of TRA provided by the liposomal formulations was

related both to vesicle structure and composition. In

fact, UVs always showed to be a better carrier than

MLVs for the delivery of TRA locally to the skin.

Moreover, this result was statistically important only

for SA-containing liposomes (Pb0.01). The highest

accumulation values were obtained from the nega-

tively charged vesicles and size and lamellarity of

negative liposomes did not greatly affect the TRA

delivery to the skin.

Fig. 3 shows the percentage of TRA delivered

through the skin and accumulated into the stratum

corneum (SC), epidermis (E) and dermis (D) for all

the studied liposomal formulations and controls at the

end of the experiments. As can be seen from Fig. 3,

the highest retention of tretinoin was always found in

the SC and in particular when negatively charged

liposomes were used. When carriers for TRA were

positively charged MLVs, the amount of drug

accumulated in the SC was very similar to that

obtained with the hydroalcoholic solution or Retin-

AR and even lower than that provided by the oil

solution.

To evaluate vesicular formulations as topical

carriers for TRA, it is interesting to compare the ratio

of TRA accumulated into SC versus TRA delivered

through the skin, which gives a dimensionless number

for the quantification of the Locally Accumulation

Efficiency (LAC) from the several formulations. LAC

values are listed in Table 2. The LAC value of

negatively charged liposomes was higher by a factor

of 2.3–8.3 than that of the commercially available

Retin-AR (LAC=4.2), suggesting that the main effect

of these systems was to accumulate the drug into the

skin. The only exceptions were positively charged

multilamellar liposomes whose LAC value was very

close to that of Retin-AR (P90/SA, LAC=5.3) or even

Fig. 3. Cumulative amounts of tretinoin permeated through the pig skin at the end of the experiments (R) and retained into stratum corneum

(SC), epidermis (E) and dermis (D) layers from the studied liposomal formulations and controls: hydroalcoholic solution (W/Et), oil solution (O)

and Retin-AR (RetA).

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136132

worse (P90H/SA, LAC=3.3). It is interesting to point

out that this last formulation had shown the highest

transdermal flux as compared to the other vesicular

formulations and the controls.

In addition, the comparison of LAC values

suggests that the accumulation of the tretinoin in the

skin may be improved by selecting proper vesicle

charge, structure and size as well as vesicle compo-

sition and therefore physico-chemical properties (Tc)

of the amphiphile used in the formulations. In

particular, it is promoted by small, negatively charged

vesicles and is favored when the main component of

liposomes has a Tc lower than skin temperature (P90,

Tcb2 8C).

3.5. Skin analysis by transmission electron

microscopy

In order to obtain more information on the

mechanism of interaction between TRA-vesicles and

Fig. 4. Transmission electron micrographs of pig skin treated with neg

conditions. (a) Overview of control pig skin incubated for 1 h with PBS. (

aggregates of vesicles (V) can be seen intact on the SC surface; corneocy

intercellular lipids (ILL) are larger, with an irregular appearance. (c) Overv

corneocytes are very swollen and not as dark as the control. Cellular corn

intercellular lamellar lipid regions (ILL1) larger than those of the upper fo

liposome is spread on the corneocyte surface and is fusing with it. (e) Ou

liposomes are still adhering to the pig skin surface. C=corneocytes; V=ve

skin, a few pig skin samples from the in vitro

permeation experiments were studied by transmission

electron microscopy (TEM). As reported above,

several studies, using several techniques, were carried

out in order to elucidate the mechanism of vesicle

transport and their interaction with the human skin in

vitro [16]. However, in spite of the evidence of

enhanced delivery through the SC of liposome-

entrapped drug, the relationship between vesicle and

skin interaction is still not clear. The study of the

interaction between vesicles and in particular SC, the

main barrier to the drug diffusion, is essential for an

appropriate evaluation of vesicles as drug carriers in

dermatological preparations. Therefore, for this pur-

pose and according to the results of the permeation

study, the UV/DCP formulations were chosen for the

visualization study using TEM. Skin samples were

removed at different times after the beginning of the

permeation experiments, fixated with Karnovski’s

solution, post-fixated with osmium tetroxide, and

atively charged liposomes or PBS solution (control) in occlusive

b) Pig skin after 1 h of treatment with P90H/DCP liposomes. Some

tes (C) are swollen and less electron dense than the control, while

iew of the pig skin treated with P90/DCP liposomes (1 h). Outermost

ified envelope (arrows) can also be observed. Inner SC layers show

ur SC layers. (d) Same sample of 4 c at higher magnifications. One

termost SC after 9 h of treatment with P90H/DCP liposomes. Two

sicles; ILL=intercellular lamellar lipid.

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136 133

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136134

finally examined by TEM. This analysis has provided

a reliable microscopic estimation of the skin mod-

ifications induced by TRA-incorporating vesicles as

compared with skin treated with PBS solution

(control). Representative electron micrographs of pig

skin 1 or 9 h after incubation in occlusive conditions

with PBS solution and P90/DCP or P90H/DCP

liposomes are depicted in Fig. 4. As can be seen

(Fig. 4a), the pig skin treated with PBS is mainly

organized in an alternating pattern of electron dense

and light bands. Intercellular spaces (light bands) are

very sharp and linear whereas corneocytes (black

bands) are flat and dark because of the presence of the

electron dense fibrous protein keratin. In Fig. 4b, an

overview of the ultrastructure of the pig skin after 1 h

of incubation with P90H/DCP liposomes is shown.

Aggregates of liposomes are still present on the skin

surface showing that vesicles close to the SC layers

were not rinsed off during the fixation procedure.

Some morphological alterations in the SC layers are

evident with respect to the control: corneocytes (C)

are very swollen, less electrondense; intercellular

spaces (ILL) are larger, wavy and more irregular than

PBS-treated skin. In fact, several folds, which increase

the intercellular pathway, are present.

Fig. 4c shows a micrograph of newborn pig skin

treated for 1 h with P90/DCP liposomes. As in Fig.

4b, the upper corneocytes appear swollen and not as

dark as the control. The four outermost layers of

corneocytes show quite clearly their boundary with

the intercellular lipids: the so-called cornified enve-

lope. Here, this cellular envelope has a highly

irregular appearance and it seems to create finger-like

structures within the intercellular lamellar lipids

(ILL).

The inner SC layers show a different organization

with intercellular lipid regions (ILL1) larger than in

the upper SC layers. No aggregates of vesicles are

present on the skin surface, but a single P90/DCP

vesicle tight to the external surface of the SC can be

seen. Fig. 4d, which shows the same sample at higher

magnifications, allows us to notice that the P90

liposome is spread on the corneocyte surface and is

fusing with it.

In Fig. 4e, a micrograph of the SC after 9 h of

treatment with P90H/DCP liposomes can be observed.

Two liposomal vesicles are still present intact on the

SC surface at the end of the experiment. They have a

spheroidal shape and one of them is close to the skin

surface while the other one is approaching the horny

layer. Moreover, in this micrograph the upper corneo-

cyte layer shows a swollen and flexible cytoplasm,

less electron dense than in the control: the SC treated

with liposomes appears very hydrated and elastic.

TEM analyses in this work have not shown any

evidence of vesicular structures in the pig skin.

Therefore, we can conclude that liposomes do not

seem to penetrate intact into deeper skin layers. On

the other hand, micrographs have given the evidence

of vesicle adsorption (both P90 and P90H liposomes)

and mixing or fusion (P90 vesicles) with the SC

surface. The main result of TEM analyses is that

liposomes may strongly affect the ultrastructure of the

intra- and intercellular regions of the horny layer.

On the basis of TEM images, it is evident that the

main advantage of liposomes is to improve the drug

residence time on the skin surface. This is particularly

true for P90H liposomes which are still present on the

pig skin surface at the end of the experiments (9 h;

Fig. 4e). Furthermore, liposomes enhance TRA

bioavailability because after their topical application,

they settle down on the skin surface close to the

outermost corneocyte layer. In these conditions,

material exchange between vesicles and intercellular

lipids may occur, allowing the diffusion of free

molecules as well as small membranal fragments into

the SC. Resulting perturbation of SC intercellular

lipids modifies the enthalpy of the SC lipid transition

giving rise to a new phase transition associated to the

applied lipids. The result is an increase of the

membrane permeability and a different partition of

lipophile molecules (TRA) in the intercellular spaces

[38,39]. In the end, liposomes form a lipid film on the

skin which retains water, both endogen and liposome-

entrapped, causing an improvement of SC intra- and

intercellular hydration. Vesicular components (i.e.

water and lipids) may join lipid bilayers in the SC

intercellular regions causing the swelling of the

intercellular lipids. Moreover, water also causes the

swelling of intracellular fibrous protein and hence of

corneocytes. Therefore, the compact structure of the

SC is opened and the barrier permeability is increased.

All these factors cause the retention of tretinoin in the

horny layer. However, they cannot improve TRA

diffusion through the inner more hydrophile skin

layers because of the lipophilicity of the drug which

C. Sinico et al. / Journal of Controlled Release 103 (2005) 123–136 135

shows higher affinity for the SC lipid matrix. Only

when TRA was incorporated into positively charged

liposomes a slightly improved diffusion through the

skin was obtained. This behavior can be explained as

a consequence of a better diffusion pattern of the more

hydrophile ionized TRA electrostatically linked to the

counter ion stearylamine.

4. Conclusions

Overall results obtained during this work have

shown that liposomes may be an interesting carrier for

tretinoin in skin disease treatment, when appropriate

formulations are used. In fact, tretinoin dermal

delivery was found to be affected by several factors

including vesicle composition, morphology and size.

In particular, it has been shown that negatively

charged liposomes strongly improved newborn pig

skin hydration and TRA retention, though no evi-

dence of intact vesicle penetration was found. Further

in vivo studies have to be carried out in order to

obtain more information on vesicle–skin interactions.

In addition, in order to find the best topical

application forms suitable for liposomal tretinoin, a

study on liposomes formulated in gels and poly-

ethylene glycol ointment is being carried out. These

studies are being performed both in occlusive and

non-occlusive conditions.

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