Journal of Microencapsulation, 2009; 26(7): 649–658

RESEARCH ARTICLE

Anti-inflammatory effects of locally appliedenzyme-loaded ultradeformable vesicles onan acute cutaneous model

Sandra Simoes, Claudia Marques, Maria Eugenia Cruz and Maria Barbara Figueira Martins

Research Institute for Medicines and Pharmaceutical Sciences, Unit New Forms of Bioactive Agents (UNFAB)/INETIand Nanomedicine & Drug Delivery Systems Group of iMed.UL, Lisboa, Portugal

AbstractSuperoxide dismutase (SOD) and catalase (CAT) are active scavengers of reactive oxygen species andwere incorporated into ultradeformable vesicles with the aim of increasing enzyme bioavailability (skindelivery). These special very adaptable vesicles have been formulated and optimized for enzyme transportin order to penetrate into or across the intact skin barrier. Anti-inflammatory activity of SOD-loaded, CAT-loaded and of SOD- and CAT-loaded ultradeformable vesicles applied epicutaneously was measured usingdifferent protein doses on the skin, on an arachidonic acid-induced mouse ear oedema. The biologicalanti-oedema activity is a measurement of drug-targeting potentiation in the organ. Delivery by meansof deformable vesicles was compared to conventional vesicles or the absence of an enzyme carriermediated transport. This was done at various times following prophylactic application of the test for-mulations. Positive reference groups were treated epicutaneously with several low molecular weightnon-steroidal anti-inflammatory drugs (NSAIDs). The latter included indomethacin (3 mg kg�1), etofena-mate (30 mg kg�1) and piroxicam (1 mg kg�1) and reduced the oedema by 94� 4%, 81� 4% and 42� 5%,respectively, if measured 30 min after ear treatment with a NSAID. Of the enzyme-loaded carriers tested,only the enzyme-loaded ultradeformable vesicles reduced the swelling of ears significantly: SOD(90 mg kg�1), CAT (250 mg kg�1) and SOD (90 mg kg�1) plus CAT (250 mg kg�1) reduced the oedema by70� 12%, 65� 10% and 61� 19%, respectively, at t¼ 30 min. Aqueous enzyme solutions and emptycarriers had no such effect. The combination of two enzymes resulted in no increased therapeuticeffect, but the results are inconclusive since only two dose combinations were tested. The results pre-sented in this study suggest that antioxidant enzymes delivered by means of ultradeformable lipidvesicles can serve as a novel region-specific treatment of inflammation.

Key words: Antioxidant enzymes; superoxide dismutase; catalase; arachidonic-acid mouse ear inhibitiontest; skin delivery

Introduction

The anti-inflammatory actions of SOD and CAT were

extensively studied in terms of their ability to protect

cells against the superoxide radical and hydrogen peroxide

challenge. Both enzymes were also proposed for human

therapy. It is fair to say that such enzymes offer a better

protection than small anti-oxidants against acute massive

oxidative aggression, as is the case of inflammation

(Muzykantov 2001). Especially the enzymes involved in

superoxide anion dismutation (SOD), destruction of

H2O2 (CAT) and the reduction of lipoperoxides (glu-

tathione peroxidase) are highly efficient in this respect.

Non-enzymatic antioxidants are small weight molecules,

some of which can be administered orally. Although very

useful, such superoxide scavengers are consumed by reac-

tive oxygen species (ROS), which makes it necessary to use

high concentrations and/or frequent administrations.

Address for Correspondence: Sandra Simoes, Unit New Forms of Bioactive Agents (UNFAB)/INETI and Nanomedicine & Drug Delivery Systems Group ofiMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Estrada do Paco do Lumiar, 22, Edifıcio F, 1649-038 Lisboa, Portugal. Tel:þ 351210924732. Fax: þ 351 217163636. E-mail: sandra.simoes@ineti.pt

(Received 11 Nov 2008; accepted 14 Nov 2008)

ISSN 0265-2048 print/ISSN 1464-5246 online � 2009 Informa UK LtdDOI: 10.3109/02652040802630403 http://www.informahealthcare.com/mnc

(Received 11 Nov 2008; accepted 14 Nov 2008)

ISSN 0265-2048 print/ISSN 1464-5246 online � 2009 Informa UK LtdDOI: 10.3109/02652040802630403 http://www.informahealthcare.com/mnc

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Antioxidant enzymes, in contrast, are not consumed by

ROS and have a high affinity and rate of reaction for the

substrate.

SOD belongs to a group of metalloenzymes that cata-

lyse the superoxide anion into hydrogen peroxide. CAT

converts hydrogen peroxide into the non-harmful water

and oxygen. Conjugation of both enzymatic activities can

therefore increase the efficacy of antioxidative therapy

(Muzykantov 2001). The chief indications for such treat-

ment are chronic inflammatory diseases and the injuries

mediated by ROS (Corvo et al. 2000, Vorauer-Uhl et al.

2001, Jubeh et al. 2005, Jubeh et al. 2006). A large

number of studies have shown the advantages of antiox-

idant enzymes in the treatment of numerous pathologies

(Cuzzocrea et al. 2001). Enzymes, which can exhibit their

pharmacological activity in plasma or at tissue surface,

benefit from prolongation of plasma half-life. The thera-

peutic potential of these anti-inflammatory and anti-

arthritis agents is limited, however, by the difficulty in

reaching the target sites. Many proteins rapidly disappear

from the organism, and manifest short half-lives in the

blood system. Proteins with a low molecular weight are

cleared from the body through the kidneys and are

excreted in the urine. Therapeutic proteins may also be

degraded by proteases. Chemical modification with large,

inert groups (polyethylene glycols are an example) and

fusion to a second protein are two methods to extend

a protein’s serum half-life. These modifications increase

the effective molecular weight of a therapeutic protein

over the renal threshold and in some genetically engi-

neered fusion proteins, the fusion partner can actively

prevent degradation by proteases (Way 2002).

Chemically modified CAT delivery to hepatic non-par-

enchymal cells is a possible approach to prevent liver inju-

ries caused by ROS (Yabe et al. 1999). The combination of

CAT and SOD derivatives was also demonstrated to be

potentially useful. SOD does not penetrate the cellular

membrane, however, a large amount of exogenously

added SOD penetrated the cellular membrane and

increased total SOD activity (Edeas et al. 1996).

Intravenous administration of SOD and CAT incorporated

in liposomes increased the activity of the enzymes both in

circulation and in the lung (Turrens et al. 1984).

In premature rats exposed to hyperoxia, intratracheal

instillation of surfactant liposomes incorporating SOD

and CAT increases their lung antioxidant capacity

(Walther et al. 1995). Liposomal SOD treatment reversed

the radiation-induced fibrotic process in experimental

animals (Lefaix et al. 1996). Muzykantov (2001) reviewed

the targeting of SOD and CAT to vascular endothelium:

coupling of polyethylene glycol to the enzymes as well as

encapsulating the enzymes in liposomes increase their

bioavailability and protective effect. Intravenous adminis-

tration of SOD encapsulated in small sized long circulating

liposomes enhances localization of SOD at arthritic sites

(Corvo et al. 1999). Subcutaneous administration of the

same SOD liposomes showed equal activity to intravenous

administration (Corvo et al. 2000). SOD and CAT were

incorporated in negatively charged liposomes (Jubeh

et al. 2005) for local treatment of experimental colitis

and the results showed antioxidant targeting to the

inflamed epithelium of the colon (Jubeh et al. 2006).

Using an in vivo skin inflammation model, induced by

ROS generated by glucose oxidase as a producer of

H2O2, the inflammatory response was inhibited predomi-

nantly by CAT and to a lower extent by SOD, both attached

to polyethylene glycol (Trenam et al. 1991). Mentioned

strategies can improve the drug pharmacokinetics and

biodistribution, but do not eliminate another fundamental

problem: the need for a drug injection. The consequences

include oscillating drug levels and poor patient compli-

ance, particularly in the case of chronic diseases and fre-

quent administrations. On the other hand, especially

proteins would be transported through the skin directly

into the target tissue.

Numerous attempts have been made to find acceptable

alternatives to an injection. This includes drug adminis-

tration via the skin for local therapeutic effect, both on

diseased skin (topical delivery) and for systemic effect

(transdermal delivery) (Brown et al. 2006). However, due

to its structure and composition, the skin is an excellent

permeability barrier that protects the body against envir-

onmental aggression. Many attempts have thus been

made to overcome the skin permeability barrier

(El Maghraby et al. 2005, Choi and Maibach 2005, Fang

et al. 2006). Early studies have shown that skin fluidizers

can facilitate permeation of small drugs across the intact

skin but it remained practically impossible to deliver sig-

nificant amounts of large drugs, such as proteins, through

the skin without major organ perturbation (Cevc 1997,

Prausnitz 1997).

Topical application of SOD was proposed and investi-

gated for the treatment of skin and mucous membrane

lesions (Mizushima et al. 1991), with differing and even

contradictory results. Topically applied SOD encapsulated

in liposomes reduced the size of post-burn wound and the

formation of oedema in rabbits, compared to pure and

intralesionally injected SOD (Vorauer-Uhl et al. 2001).

Lipid peroxidation study in burned patients suggested

that polyethylene glycol-conjugated SOD can prevent the

conjugated dienes formation by suppressing oxygen radi-

cal production (Thomson et al. 1990). Using an acute,

localized gamma irradiation pig model it was shown that

a successful treatment of fibrosis, involving skin and ske-

letal muscle, with liposomal SOD may be possible (Lefaix

et al. 1996). In most studies dealing with the topical appli-

cation of SOD, however, the skin was not intact and

650 S. Simoes et al.

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enzyme delivery through the skin was by diffusion, per-

meation or using shunts such as hair follicules and glands.

Special mixed lipid carriers in the form of ultradeform-

able vesicles arguably deliver drugs transcutaneously via

the lymph into the blood circulation (Cevc et al. 1996, Cevc

et al. 1997, Cevc et al. 2002, Simoes et al. 2005). If desired,

the carriers can also be applied so as to mediate preferred

drug delivery into the deep subcutaneous tissue (Cevc and

Blume 2001, Rother et al. 2007). The resulting efficacy of

transcutaneous delivery can be rather high and the biodis-

tribution similar to that of a subcutaneous injection.

This study has shown for the first time that SOD incor-

porated in ultradeformable vesicles and applied onto

a skin area, not necessarily close to the inflamed tissue,

is able to promote non-invasive treatment of induced

arthritis (Simoes et al. 2005). In this work, SOD and CAT

were incorporated in ultradeformable vesicles and tested

for anti-inflammatory action in a murine ear oedema

model. Non-steroidal anti-inflammatory drugs (NSAIDs)

action served as positive control. As negative control, lipo-

somal SOD was used.

The objectives of this study were to examine the dermal

delivery of topically administered carrier-mediated antiox-

idant enzymes; to study the improvement of therapeutic

anti-inflammatory effect of enzyme-loaded ultradeform-

able vesicles; and to test whether the combination of

SOD and CAT could synergistically improve the anti-

oedema action in an acute inflammation model. The

results confirm that ultradeformable mixed lipid vesicles

mediate protein delivery into deep tissues, as evidenced by

the performance of epicutaneous application of SOD and

CAT incorporated into deformable vesicles to inhibit the

oedema formation.

Materials and methods

Enzyme-loaded ultradeformable vesicles

Based on previous formulation results, the ultradeform-

able systems used in this work comprised a mixture

of soybean phosphatidylcholine (SPC) and a surfactant,

in a ratio that ensured adequate vesicle adaptability, pro-

tein loading and stability (Simoes 2005). In brief, to pre-

pare highly adaptable vesicles suspension, SPC (S100,

Lipoid KG, Ludwigshafen, Germany) was mixed with the

bio-surfactant sodium cholate (p.a., Sigma, St. Louis, MO),

in molar ratio 3.75 : 1, and taken up in sodium phosphate

buffer solution (50 mM, pH¼ 7.4) to yield 10 w-% total lipid

suspension. This suspension was initially brought to uni-

formity by sequential filtration through decreasing pore

size filters under pressure filtration. The average vesicle

diameter (100� 50 nm) used was such that arguably

mainly unilamellar vesicles existed in each suspension.

To enlarge such vesicles via fusion, vesicles were frozen

and thawed five times and the vesicles in the resulting

paste were again broken down to the size of 150� 50 nm

by sequential filtration. The enzymes were added to the

intermediate blend of heterogeneous vesicles, which were

brought to the final size of �150� 50 nm by sequential

filtration, using track-etched polycarbonate membranes

(Poretics Corporation, Livermore, USA). Vesicles were pre-

pared at room temperature. Cu, Zn-superoxide dismutase

(SOD; from bovine erythrocytes, 98% protein, 2500–7000

units/mg protein) and catalase (CAT; from bovine liver,

2� crystallized, 40 000–60 000 units/mg protein) were pur-

chased from Sigma. All other chemicals were reagent

grade (Merck, Darmstad, Germany).

SOD-liposomes

Liposomes were made from a 1:1 mol/mol mixture of

SPC/cholesterol (cholesterol was obtained from Sigma)

dissolved and mixed in chloroform, which was then eva-

porated with a rotative evaporator. The resulting film was

hydrated with an enzyme solution in 50 mM phosphate

buffer (pH¼ 7.4) to make a 10 w-% lipid suspension. The

latter was sequentially sized by pressure filtration, through

a polycarbonate filter of decreasing pore size, to obtain

150 nm vesicles (Szoka et al. 1980).

Suspension characterization

The mean particle size and the particle size distribution in

terms of polydispersity index were measured by photon

correlation spectroscopy (PCS) at 90� with a Malvern

Zetasizer 3 (Malvern, Malvern, UK). SPC concentration

was determined with an enzymatic-colourimetric test

(Spinreact, Girona, Spain). Total protein quantification

was based on the method of Lowry et al. (1951), after pre-

vious vesicle disruption with 2% Triton X-100 and 20%

sodium dodecyl sulphate.

BIOXYTECH� ‘SOD-525TM kit’ (Oxys, Portland, USA),

a spectrophotometric assay for SOD, was used to measure

enzyme activity. CAT activity was assessed according to

Beers and Sizer (1952).

The separation of non-incorporated SOD and CAT was

performed by using a procedure involving a gel filtration

(sample volume: 800mL) on a Sephacryl S-400 HR column

(30 cm� 1.6 cm), eluted with sodium phosphate buffer

(50 mM, pH¼ 7.4, containing 5 mM NaChol) followed

by ultracentrifugation (Beckman L8-60M ultracentrifuge;

180 000 g for 2 h at 15�C) to concentrate the sample.

Incorporation efficiency (IE) was calculated as the

ratio [(final protein/final lipid)/(initial protein/initial

lipid)]� 100%.

Anti-inflammatory effects of locally applied enzyme-loaded ultradeformable vesicles 651

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Pressure-driven transport: measurement of vesicles

deformability

The flux of vesicles suspension (diluted with sodium phos-

phate buffer, 50 mM, pH¼ 7.4, 5 mM NaChol, to the final

concentration of 2 wt-% total lipid) through a micro-

porous filter with 30 nm pore diameter was driven by

different external pressures (0.3–1 MPa), created by

a nitrogen stream and measured as a function of time.

A 20 mL-filtration unit was employed, thus miniaturizing

the commercially available pressure filtration device of

Sartorius. The suspension was collected into a container

on a Sartorius LA620P scale (Sartorius, Gottingen,

Germany) to determine automatically the weight of fil-

tered suspension. The data were collected with the

Wedge software for Windows (TAL Technologies Inc.

Philadelphia) as a function of time and finally converted

into a flux or penetrability (flux/pressure value).

The average particle size was determined by PCS,

before and after the filtration experiment.

Formulation stability

Enzyme-loaded ultradeformable vesicles suspensions

were prepared, filtered through sterile filters, divided in

small aliquots (10 mL) placed in plastic sterile vials

under aseptic conditions and kept at 4�C. Samples were

analysed at given time points for assessment of retention

of catalytic activity, vesicle diameter and polydispersity

index, macroscopic appearance and sterility.

Non-steroidal anti-inflammatory drugs (NSAIDs)

Indomethacin (Elmetacin Spray, a product of Luitpold),

etofenamate (Feldene Gel, a product of Pfizer) and pirox-

icam (Reumon-Locao, a product of Bial) were purchased

from the local pharmacy. Elmetacin spray contained 1%

indomethacin together with a hydroethanolic-based exci-

pient composition; Feldene Gel contained etofenamate

5 mg g�1 gel within a base of propylenoglycol/ethanol;

Reumon-Locao contained 100 mg mL�1 piroxicam with

an undisclosed amount of isopropanol.

Arachidonic acid-mouse oedema inhibition test

(AA-MEIT)

This model is described as the most commonly used

in vivo test for assessing the anti-inflammatory effects of

drugs in the first stage trials (Young et al. 1989) and was

tested in female NMRI mice (28–32 g), aged 8–12 weeks

(Charles River, Santa Perpetua de la Mogoda, Spain).

All animals were used and kept under standard laboratory

conditions under the guidelines of the Institutional Animal

Care and Use Committee. Various formulations were

applied on the inside of an animal ear and left to dry

out. After the test drug administration, each animal was

kept individually in a separate cage. Two hours later, the

treated side was challenged with arachidonic acid (AA,

Sigma, St. Louis, MO), dissolved in ethanol (10 mL/2 mg

per ear) and the resulting oedema was determined 0.5 h,

1 h, 2 h and 24 h later. The tested compounds were applied

2 h before AA challenge to allow for an adequate absorp-

tion of the lipidic carrier suspension applied on the ear.

For the tested formulations, the volumes applied ranged

between 3.8–20.2 mL, in order to obtain the adequate dose

on the application site. The ear thickness was measured

with a Mitutoya micrometer (Ascona Tools, Redwood City,

USA) with three readings per ear. The degree of oedema

inhibition by a substance was calculated as the percentage

inhibition determined by comparing the drug treated

group with untreated controls. Internal control was

made by the application of the test formulations on the

right ear and the respective vehicle on the left ear of

the same mouse. Five mice were used per group.

Statistical treatment

Results are given as the mean of the measured values�

standard deviation, except otherwise specified. For animal

experiments, the data are represented as the mean�

standard deviation and were tested for significance using

one-way ANOVA test.

Results

The characteristics of the different type of vesicles pre-

pared and tested in this study are presented in Table 1.

The results show that incorporation of enzymes in differ-

ent type of vesicles with different preparation methods

did not affect the retention of enzymatic activity.

Enzyme-loaded deformable vesicles suffer the effect of

two modifiers on lipid vesicle structure: the surfactant

Table 1. Characteristics of the lipid vesicles used in this study.

Type of vesicle Enzyme Mean diameter

(nm)

IE (%) Ret. Act.

(%)

Deformable SOD 142� 4 32� 4 90� 2

Deformable CAT 142� 6 26� 6 95� 1

Liposome SOD 149� 9 34� 2 95� 1

IE¼ Incorporation efficiency; Ret. Act.¼Retention of enzymatic activity;

Enzyme concentration: 1 mg mL�1.

652 S. Simoes et al.

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and the protein. They can both increase membrane flex-

ibility. Transbarrier stress difference can be generated, for

example when a external pressure, produced by a gas

stream, is applied on a suspension and this is filtered

through narrow pores or naturally when a vesicle suspen-

sion is applied onto the skin, under non-occlusive condi-

tions and are left to dry out. In the latter, a transbarrier

hydration gradient is responsible for transbarrier stress

difference. The external pressure is then responsible for

the vesicle’s motion. Figure 1 presents the results of

penetrability measurement and shows qualitative similar

transport for different formulations. Enzyme-loaded ultra-

deformable vesicles show identical flow compared with

empty ultradeformable vesicles. Two different types of

enzyme-loaded formulations were tested: one with total

enzyme (encapsulated plus non-encapsulated); one with

only encapsulated enzyme (non-encapsulated enzyme

was removed by filtration and ultracentrifugation).

In both cases and for both enzymes, similar variation of

enzyme-vesicles mass flow as a function of time was

observed. The results reveal a non-linear dependence for

the mixed lipid vesicles, identifying unusual rheological

characteristics of ultradeformable vesicles, which can be

diagnostic of bilayer component demixing under stress.

SOD-liposomes exhibit no measurable flux for applied

pressure and for the conditions used, confirming no

deformability of lipid bilayer. Protein carrier penetration

of the mentioned artificial membrane results in no enzyme

loss. The lipid loss after barrier penetration is negligible

and the catalytic activity of the enzymes is also preserved

(data not shown). Protein-loaded carriers therefore can

pass through narrow pores even when the vesicle size

exceeds the pore diameter by a factor of �4. Thus, the

presence of protein does not have a major influence on

transport characteristics of the tested carriers. Aggregate

size was controlled before and after barrier crossing. After

passage through narrow pores, the aggregates with typical

initial size of 150� 50 nm, filtered through 30 nm pores,

reduced �30% in size. The results were independent of

the enzyme presence and driving pressure. It is possible

to conclude that when vesicle-radius/pore-radius is

�4 a typical vesicle size reduction of 30% can give final

vesicle-radius/pore-radius ratio equal to 2.8, which is still

greater than exclusion criteria based on pore size. With

this study it was also possible to conclude that the process

for removal of non-encapsulated enzyme, that involves gel

filtration and ultracentrifugation steps, does not affect the

adaptability characteristics of enzyme-loaded ultrade-

formable vesicles to cross membranes with known pores

size, driven by controlled external pressure.

Enzyme-loaded ultradeformable vesicles were stored at

4�C for up to 5 months. The stability evaluation included

vesicle size determination, enzymatic activity measure-

ment and sterility test. The time course of aggregates

size with and without incorporated enzymes is presented

in Figure 2(a) and shows that all the suspensions exhibited

similar behaviour. Vesicles do not show fusion or

0 20 40 60 80 100 120 140 1600

20

40

60

80

100

120

140

160

Mea

n di

amet

er (

nm)

Time after preparation (day)

Empty UDVSOD-UDV CAT-UDV

4 °C(a)

−20 20 40 60 80 100 120 140 1600

20

40

60

80

100

1204 °C

Act

ivity

ret

entio

n (%

)

Time after preparation (day)

SOD-UDV CAT-UDV

(b)

0

Figure 2. (a) Size evolution for empty ultradeformable vesicles (UDV) and SOD-loaded and CAT-loaded UDV during storage at 4�C. (b) Retention of

enzymatic activity of SOD-loaded and CAT-loaded UDV, during storage at 4�C. Enzyme concentration in the enzyme loaded carrier suspensions:

1 mg mL�1. Results are the median of three independent experiments.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.00

0.04

0.08

0.12

0.16

0.20

2rpore= 30 nm

Empty-UDV SOD-UDV SOD-UDV (filt+UC) CAT-UDV CAT-UDV (filt+UC)

Pen

etra

bilit

y (g

/s. M

Pa)

Driven Pressure (MPa)

Figure 1. Penetrability of nanoporous barrier to ultradeformable vesi-

cles (UDV) as a function of different formulations, for known pressure.

Comparison with empty-UDV is shown. Penetrability of systems without

non-incorporated enzyme is also presented (SOD-UDV (filtþUC); CAT-

UDV (filtþUC)). Enzyme concentration in the enzyme loaded carrier

suspensions: 1 mg mL�1 (n¼ 3).

Anti-inflammatory effects of locally applied enzyme-loaded ultradeformable vesicles 653

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aggregation after 150 days at 4�C. The polydispersity was

50.1. Therefore, the presence of sodium cholate, a polar

hydrophilic molecule, affords some advantages to the phy-

sical stability: an enhancement of the structure resistance

to undergo size variation and probably morphological var-

iation. The corresponding enzyme activity is presented in

Figure 2(b). Enzyme stability for the tested concentrations

is observed. Total retention of activity is observed for both

enzymes up to 1 month. For CAT, the activity remained

unchanged during the studied time period. In the case

of SOD, the activity decreased to 60% of the initial value.

When stored, both SOD-loaded and CAT-loaded ultrade-

formable vesicles suspensions were sterile. Keeping the

specimen under aseptic conditions for at least 5 months

show no contamination, i.e. no material growth was

detected on agar plates at 37�C. This composition was

found very suitable for enzyme incorporation and for

proceeding with animal experiments.

Oedema formation, determined at up to different time

points, 0.5 h, 1 h, 2 h and 24 h after topical application of

AA, was inhibited by epicutaneous application of SOD-

loaded ultradeformable vesicles formulations containing

45, 66, 90 and 120mg SOD/kg body weight (BW) but not

by 30 mg SOD/kg BW by means of the same type of carriers,

or by 120mg SOD/kg BW in an aqueous solution or by

SOD-loaded liposomes at 66 mg SOD/kg BW (Figure 3).

The differences observed between 30 mg SOD/kg BW and

45, 66, 90 mg SOD/kg BW are statistically significant

( p5 0.01). The difference between the lower and the

highest dose for this enzyme is also statistically significant

but in another level of significance ( p5 0.05). Results

measured after 24 h, in light of the large error bars, must

be considered as non-reliable. A non-linear dose-depen-

dence response was observed for the SOD incorporated in

membrane adaptable carriers (Figure 3, inset), whereas no

activity was seen for the negative controls.

Oedema formation was also inhibited by an epicuta-

neous application of CAT in ultradeformable carriers sus-

pension using doses of 100, 250 and 500mg CAT/kg BW.

No such effect was seen for CAT in an aqueous solution

500mg CAT/kg BW (Figure 4). The dose dependence

is approximately linear at 0.5 h after AA challenge

(Figure 4, inset).

The combination of SOD-loaded and CAT-loaded in

ultradeformable vesicles suspensions was no better than

single use of either of these two active ingredients, keeping

the SOD dose constant and testing two different CAT con-

centrations (90mg SOD/kg BWþ 100mg CAT/kg BW; 90 mg

SOD/kg BWþ 250mg CAT/kg BW) (Figure 5). This can be

due to the lack of synergism or simply are indication of the

saturation level, suggested in Figure 3.

Oedema inhibition by different formulations of the

anti-inflammatory enzymes and NSAIDs is compared in

Table 2. The positive reference groups, treated epicuta-

neously with the low molecular weight non-steroidal

anti-inflammatory agents, are believed to cross the skin

0.5 1 2 24

−20

0

20

40

60

80

100

120

140

160

180

200

[**]

Oed

ema

Inhi

biti

on (

%)

Time after challenge with AA (hours)

SODaq 120µg/kgUDV-SOD 30µg/kgUDV-SOD 45µg/kgUDV-SOD 66µg/kgUDV-SOD 90µg/kgUDV-SOD 120µg/kgLip-SOD 66µg/kg

[*]

Figure 3. Anti-inflammatory action of SOD administered epicuta-

neously in various formulations measured in AA-MEIT model. The tem-

poral evolution of oedema suppression is given as a function of the

locally applied enzyme dose. SOD aqueous solution (SODaq): 120mg

SOD/kg BW; SOD-loaded ultradeformable vesicles (UDV-SOD): 30 mg

SOD/kg BW, 45 mg SOD/kg BW, 66mg SOD/kg BW, 90 mg SOD/kg BW

and 120 mg SOD/kg BW; SOD-loaded liposomes (Lip-SOD): 60 mg SOD/

kg BW. Columns represent mean� SD for a group of five animals. Inset:

Oedema inhibition as a function of SOD associated with ultradeform-

able vesicles (UDV) applied on ear skin, 0.5 h after challenge with AA.

p5 0.01 vs UDV-SOD 30mg SOD/kg BW 0.5 h after challenge; p5 0.05 vs

UDV-SOD 30 mg SOD/kg BW 0.5 h after challenge.

00.5 1 2 24

20

40

60

80

100

120

140

160

180

200

Oed

ema

Inhi

biti

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%)

Time after challenge with AA (hours)

CAT 500 µg/kgUDV-CAT 100 µg/kgUDV-CAT 250 µg/kgUDV-CAT 500 µg/kg

Figure 4. Anti-inflammatory action of CAT administered epicuta-

neously in various formulations measured in AA-MEIT model. The tem-

poral evolution of oedema suppression is given as a function of the

locally applied enzyme dose. CAT aqueous solution (CATaq): 500mg

CAT/kg BW; CAT-loaded ultradeformable vesicles (UDV-CAT): 100mg

CAT/kg BW, 250mg CAT/kg BW and 500 mg CAT/kg BW. Columns repre-

sent mean� SD for a group of five animals. Inset: Oedema inhibition as

a function of CAT associated with vesicles (UDV) applied on ear skin,

0.5 h after challenge with AA.

654 S. Simoes et al.

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barrier rather efficiently (Heyneman et al. 2000). It is

therefore not surprising that indomethacin (3 mg kg�1

BW), etofenamate (30 mg kg�1 BW) and piroxicam

(1 mg kg�1 BW), used at fairly high absolute doses and

molar concentrations, exert comparably strong or better

anti-oedema effects than antioxidant enzymes.

Discussion

This study attempted to study the suppression of local

inflammation by topically applied SOD and CAT asso-

ciated with ultradeformable carriers using the acute

murine ear oedema model. Among numerous models of

inflammation developed to investigate the efficacy of ther-

apeutic drugs, those that involve the skin offer immediate

results, continuous monitoring and are easier to conduct

with non-invasive or non-terminating techniques (Young

et al. 1989). Like in other AA-MEIT studies (Muller-

Peddinghaus et al. 1993, Lloret and Moreno 1995, Hamer

et al. 1996, Burchardt and Muller-Peddinghaus 1997,

Puignero and Queralt 1997, Horizoe et al. 1998, Kim

et al. 1998), this study used indomethacin as a positive

reference. For comparison, it also applied other low mole-

cular weight non-steroidal anti-inflammatory agents. All of

these, when applied epicutaneously, change the AA-chal-

lenged ear thickness and consequently inhibit the ear

oedema formation (37–98%). A previous study obtained

indirect measurement of epicutaneously applied SOD

ultradeformable vesicles transdermal delivery on an

animal model of chronic inflammation. The therapeutic

efficacy (evaluated by physical and biochemical para-

meters) was obtained and the ability of SOD to interfere

with the establishment of the disease was also observed

(Simoes et al. 2005). In the present study, the indirect

measurement of deep skin targeting was examined.

Conventional and deformable carriers were compared in

terms of enhancement of enzyme skin bioavailability.

Enzyme-loaded liposomes failed to pass artificial mem-

branes and failed also the deposition of SOD in deep

skin tissues, as observed by anti-oedema effect.

Specially designed and optimized ultradeformable drug

carriers have been shown to deliver small and large mole-

cules through the skin (Cevc and Blume 1992, Cevc et al.

1996, Cevc et al. 1997, Cevc 1997, Simoes et al. 2005) fol-

lowing non-occlusive percutaneous administration and

reach the blood circulation via the lymph.

Ultradeformable carriers can be loaded with various mole-

cules with a vast range of sizes and lipophilicities. For each

drug, the composition must be optimized to obtain the

highest possible drug-carrier association, sufficient drug

stability and maximum carrier deformability (Simoes

et al. 2004, 2005). Ultradeformable carriers penetrate the

skin and thus differ from permeation enhancers, present

Table 2. Inhibition of oedema formation.

Tested compound and formulation Dose (mg/ear) Volume (mL/ear) Oedema inhibition

Mean� SD (%)a

SOD aqueous solution (120 mg kg�1) 3.8–4.4 3.8–4.4 3� 19

CAT aqueous solution (500 mg kg�1) 16.0–16.5 5.3–5.5 18� 9

SOD-loaded ultradeformable vesicles (90mg kg�1) 2.9–3.2 13.7–15.4 70� 12

CAT-loaded ultradeformable vesicles (250mg kg�1) 8.5–8.8 11.3–11.6 65� 10

(SODþCAT)-loaded ultradeformable vesicles

(90 mg kg�1þ 250 mg kg�1)

11.6–12.9 18.1–20.2 61� 19

Etofenamate (30 mg kg�1) 900.0–920.0 9.0–9.2 81� 4

Piroxicam (1 mg kg�1) 30.0–31.4 9.1–9.5 42� 5

Indomethacin (3 mg kg�1) 91.0–95.0 9.1–9.5 94� 4

a Measured 0.5 h after challenge with AA.

0.5 1 2 240

20

40

60

80

100

120

140

Oed

ema

Inhi

biti

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%)

Time after challenge (h)

UDV-(SOD90+CAT100) µg/kg UDV-(SOD90+CAT250) µg/kg

Figure 5. Anti-inflammatory action of SOD and CAT administered

epicutaneously, simultaneously, in various formulations measured in

AA-MEIT model. The temporal evolution of oedema suppression is

given as a function of the locally applied enzyme dose. SOD and CAT-

loaded ultradeformable vesicles (UDV-(CATþ SOD)): 100 mg CAT/kg BW

plus 90 mg SOD/kg BW and 250mg CAT/kg BW plus 90mg SOD/kg BW.

Results are the mean� SD. Columns represent mean� SD for a group

of five animals.

Anti-inflammatory effects of locally applied enzyme-loaded ultradeformable vesicles 655

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in commercially available compounds, that merely

improve drug permeation through the barrier.

For the NSAIDs tested, the transport through the skin is

typically driven by the drug’s concentration at the skin

surface. Such drugs thus diffuse through the stratum cor-

neum and then in the viable epidermis finally to be cleared

by the cutaneous blood capillary system (Cevc and Vierl

2007). In contrast, hydrophilic deformable carriers motion

is driven by hydrotaxis, i.e. by transcutaneous hydration

gradient maintained by non-occlusive formulation appli-

cation. Other formulations regarding the deformability of

the vesicles were tested (Simoes 2005). This study used

only one ultradeformable vesicles composition on

a basis of simplicity of preparation, ability to be handled

without temperature, organic solvents and mechanical

drastic conditions (that might be detrimental for enzyme

stability during the preparation process), proved physical

stability of the carrier, adequate and proved deformability,

easy to prepare in a scale-up situation and excellent repro-

ducibility from batch to batch. When dealing with enzyme

formulation it is important to keep the enzymatic activity

throughout the preparation process. The enzyme formula-

tion characterization is mandatory prior to any animal

experiment. Vesicle size is an important feature of deform-

able formulations. They are typically 150� 50 nm in dia-

meter, to ensure a vesicle diameter adequate for

deformation work. Micelles are small in diameter but

do not deform. Vesicles bigger than 200 nm can not, prob-

ably, complete the deformation work and consequent

delivery of the enzyme into skin to exert its anti-inflam-

matory action.

Proteins have a complex structure susceptible to

physical instability as denaturation, aggregation and pre-

cipitation and chemical instabilities such as oxidation,

deamidation, hydrolysis, etc. In general, lipid

vesicles formulations offer a good approach for protein

stabilization (Corvo et al. 1999). It has previously been

demonstrated that the presence of SOD in sodium cho-

late-phosphatidylcholine mixed systems does not affect

the state of lipid aggregation in these mixed systems

(Simoes et al. 2004). The high flexibility of such vesicles

membrane allows the carriers to squeeze through the

pores several times smaller than the vesicle’s diameter

(Cevc et al. 2002). Aggregate carriers cross the stratum

corneum as large entities, too big to enter the blood capil-

laries. Differential drug delivery into the living skin and

beyond thus becomes possible (Cevc et al. 2002). It is

reasonable to assume that SOD or CAT applied epicuta-

neously with ultradeformable vesicles experience a similar

fate. It has, indeed, previously been confirmed that such

proteins can cross artificial membranes with narrow pores

with a flux and permeability similar to that of water

(Simoes et al. 1998, Marques et al. 2002).

Biological characterization of topically applied

enzymes, in the dissolved form or associated with lipidic

carriers, has revealed that different formulations yield

different enzyme bioactivity. SOD or CAT applied in

a solution do not cross the skin. The transport is prevented

by the large molecular size of these enzymes and pro-

bably by protein self-aggregation on the skin surface.

Incorporation of enzymes into a suspension of conven-

tional liposomes is not advantageous in this respect:

SOD-loaded liposomes failed to inhibit the local inflam-

matory action of AA in the skin treated with such vesicles.

Maximum therapeutic success is achieved by using highly

deformable carriers, able to incorporate enzymes with full

catalytic activity and capable of crossing the stratum cor-

neum. This suggests that such carriers can transport both

tested enzymes into and across the skin.

The results obtained with SOD and CAT in ultradeform-

able carriers have shown that suppression of the local

oedema is enzyme-dose dependent. For SOD, in particu-

lar, a bell-shape response is obtained, which is typical for

this enzyme (Jadot et al. 1995) although not yet shown for

this inflammation model. The anti-inflammatory activity is

a function of the route of administration and by implica-

tion of the bioavailability. However, for doses ranging from

30–200 mg kg�1 dose-proportional anti-inflammatory

activity exists (Jadot et al. 1995).

It was previously determined for deformable vesicles

based on lipid-surfactant mixed composition that increas-

ing the total applied drug dose and the dose per area, the

systemic drug availability is promoted (Cevc et al. 1997)

and that the lag time for SOD to reach the blood is 4–6 h

(Simoes et al. 2002). A previous study found that SOD-

ultradeformable vesicles epicutaneously applied on mice

back skin can reduce AA induced ear oedema. The anti-

inflammatory effect had a maximum when the formula-

tion was applied 8 h prior to arachidonic acid challenge

(Simoes et al. 2003). The results herein presented show the

therapeutic efficacy of skin locally deposited enzymes. The

results showed a great reduction of AA-induced ear

oedema observed for the groups treated with SOD or

CAT in highly deformable carriers �70%. For the tested

doses, the combination of these enzymes did not bring any

improvement. The maximum enzyme-dependent reduc-

tion of oedema formation is thus below the value achieved

with highly established NSAIDs, which can exceed 90%

(see Table 2). One should not forget, however, that this

observation only relates to a macroscopic parameter (ear

thickness). The biochemical scene was not investigated to

date and could be the next focus of research. However, the

histopathologic assessment of the exposed side of the

challenged ear, done with light microscopy, revealed

lower level of inflammatory cells infiltration into the

tissue treated with SOD-loaded ultradeformable vesicles

when compared with non-treated controls. No such

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effect was observed when the less deformable liposomes

or mixed micelles were combined with the enzyme

(Simoes et al. 1998). It is also well known, that NSAIDs

are not just antioxidants, but rather exert a variety of

other biological actions, such as cycloxygenase-1 and -2,

and sometimes even lipoxygenase inhibition (Cash 1997,

Kimura 1997). The fact that NSAIDs yield stronger max-

imum effect than antioxidant enzymes is therefore not

surprising.

These results compare well with those measured

for various corticosteroid-loaded lipid carriers in

AA-MEIT (Cevc et al. 1997). In either case, this ensured

best performance amongst all tested formulations in terms

of corticosteroid delivery into or across the skin of murine

ears (Cevc et al. 1997).

In conclusion, this study has confirmed intradermal

delivery of antioxidative enzymes is possible and results

in good anti-inflammatory action. The proviso is that car-

riers are designed so as to overcome the skin barrier and

deliver the macromolecules to the sites of biological

action. Specifically enzyme incorporation into ultrade-

formable mixed lipid vesicles and their epicutaneous

application ensures good efficacy of local protein delivery

through the skin and the resulting therapeutic effects.

It was not possible to confirm the assumption that SOD

and CAT concomitant administration would be synergistic

in the animal model for the tested doses.

In further biological studies, the biological action of

similar enzymes delivered by means of ultradeformable

carriers deep below the skin will be explored.

Acknowledgements

Professor Gregor Cevc is acknowledged for the discussion

of the results. The work was financially supported by

Fundacao para a Ciencia e para a Tecnologia, project

POCTI/1999/FCB/35787.

References

Beers Jr RF, Sizer IW. 1952. A spectrophotometric method for measuringthe breakdown of hydrogen peroxide by catalase. J Biol Chem195:133–140.

Brown MC, Martin GP, Jones SA, Akomeah FK. 2006. Dermal and trans-dermal drug delivery systems: Current and future prospects. Drug Del13:175–187.

Burchardt ER, Muller-Peddinghaus R. 1997. Antiedematous effects ofcombination therapies with the leukotriene synthesis inhibitor BAYX1005 in the arachidonic acid induced mouse ear inflammation test.Prostaglandins Leukotrienes Essential Fatty Acids 56:301–306.

Cash CD. 1997. Are the reactive oxygen-derived species (ROS) interactiveporperties of the many therapeutic drugs from various categories perti-nent to their beneficial effects? Gen Pharmac 28:169–175.

Cevc G. 1997. Drug delivery across the skin. Exp Opin Invest Drugs6:1887–1937.

Cevc G, Blume G. 1992. Lipid vesicles penetrate into intact skin owing tothe transdermal osmotic gradients and hydration force. BiochimBiophys Acta 1104:226–232.

Cevc G, Blume G. 2001. New, highly efficient formulation of diclofenac forthe topical, transdermal administration in ultradeformable drug car-riers, Transfersomes. Biochim Biophys Acta 1514:191–205.

Cevc G, Blume G, Schatzlein A. 1997. Transfersomes mediated transepi-dermal delivery improves the regio-specificity and biological activity ofcorticosteroids in vivo. J Contr Rel 45:211–226.

Cevc G, Blume G, Schatzlein A, Gebauer D. 1996. The skin: A pathway forsystemic treatment with patches and lipid-based agent carriers. AdvDrug Deliv Rev 18:349–378.

Cevc G, Schatzlein A, Richardsen H. 2002. Ultradeformable lipid vesiclescan penetrate the skin and other semi-permeable barriers intact.Evidence from double label CLSM experiments and direct size mea-surements. Biochim Biophys Acta 1564:21–30.

Cevc G, Vierl U. 2007. Spatial distribution of cutaneous microvasculatureand local drug clearance after drug application on the skin. J Contr Rel118:18–26.

Choi MJ, Maibach HI. 2005. Elastic vesicles as topical/transdermal drugdelivery systems. Int J Cosmet Sci 27:211–221.

Corvo ML, Boerman OC, Oyen WJ, Jorge JC, Cruz ME, Crommelin DJ, et al.2000. Subcutaneous administration of superoxide dismutase entrappedin long circulating liposomes: In vivo fate and therapeutic activity in aninflammation model. Pharm Res 17:600–606.

Corvo ML, Boerman OC, Oyen WJG, Van Bloois L, Cruz MEM,Crommelin DJA, et al. 1999. Intravenous administration of superoxidedismutase entrapped in long circulating liposomes. II. In vivo fate ina rat model of adjuvant arthritis. Biochim Biophys Acta 1419:325–334.

Cuzzocrea S, Riley DP, Caputi AP, Salvemini D. 2001. Antioxidant therapy:A new pharmacological approach in shock, inflammation, and ische-mia/reperfusion injury. Pharmacol Rev 53:135–159.

Edeas MA, Peltier E, Claise C, Khalfoun Y, Lindenbaum A. 1996.Immunocytochemical study of uptake of exogenous carrier-freecopper-zinc superoxide dismutase by peripheral blood lymphocytes.Cell Mol Biol 42:1137–1143.

El Maghraby GMM, Campbell M, Finnin BC. 2005. Mechanisms of actionof novel skin penetration enhancers: Phospholipid versus skin lipidliposomes. Int J Pharm 305:90–104.

Fang J-Y, Hwang T-L, Huang Y-L. 2006. Liposomes as vehicles for enhan-cing drug delivery via skin routes. Curr Nanosci 2:55–70.

Hamer RR, Tegeler JJ, Kurtz ES, Allen RC, Bailey SC, Elliott ME, et al. 1996.Dibenzoxepinone hydroxylamines and hydroxamic acids: Dual inhibi-tors of cyclooxygenase and 5-lipoxygenase with potent topical anti-inflammatory activity. J Med Chem 39:246–252.

Heyneman CA, Lawless-Liday C, Wall GC. 2000. Oral versus topicalNSAIDs in rheumatic diseases: A comparison. Drugs 60:555–574.

Horizoe T, Nagakura N, Chiba K, Shirota H, Shinoda M, Kobayashi N, et al.1998. ER-34122, a novel dual 5-lipoxygenase/cyclooxygenase inhibitorwith potent anti-inflammatory activity in an arachidonic acid-inducedear inflammation model. Inflamm Res 47:375–383.

Jadot G, Vaille A, Maldonado J, Vanelle P. 1995. Clinical pharmacokineticsand delivery of bovine superoxide dismutase. Clin Pharmacokinet28:17–25.

Jubeh TT, Antler S, Haupt S, Barenholz Y, Rubinstein A. 2005. Local pre-vention of oxidative stress in the intestinal epithelium of the rat byadhesive liposomes of superoxide dismutase and tempamine. MolecPharm 2:2–11.

Jubeh TT, Nadler-Milbauer M, Barenholz Y, Rubinstein A. 2006. Localtreatment of experimental colitis in the rat by negatively charged lipo-somes of catalase, TMN and SOD. J Drug Target 14:155–163.

Kim B-J, Kim J-H, Heo M-Y, Kim H-P. 1998. Antioxidant and anti-inflam-matory activities of the mung bean. Cosmetics Toiletries 113:71–74.

Kimura K. 1997. Mechanisms of active oxygen species reduction by non-steroidal anti-inflammatory drugs. Int J Biochem Cell Biol 29:437–446.

Lefaix J-L, Delanian S, Leplate J-J, Tricaud Y, Martin M, Nimrod A, et al.1996. Successful treatment of radiation-induced fibrosis using Cu,Zn-SOD and Mn-SOD: An experimental study. Int J Radiat Oncol Biol Phys35:305–312.

Lloret S, Moreno JJ. 1995. Effects of an anti-inflammatory peptide (anti-flammin 2) on cell influx, eicosanoid biosynthesis and oedema

Anti-inflammatory effects of locally applied enzyme-loaded ultradeformable vesicles 657

Jour

nal o

f M

icro

enca

psul

atio

n D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 10

/28/

14Fo

r pe

rson

al u

se o

nly.

formation by arachidonic acid and tetradecanoyl phorbol dermal appli-

cation. Biochem Pharmacol 50:347–353.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measure-

ment with the folin phenol reagent. J Biol Chem 193:265–275.Muller-Peddinghaus R, Kohlsdorfer C, Theisen-Popp P, Fruchtmann R,

Perzborn E, Beckermann B, et al. 1993. BAY X1005, a new inhibitor of

leukotriene synthesis: In vivo inflammation pharmacology and pharma-cokinetics. J Pharmacol Exp Therap 267:51–57.

Marques CM, Simoes SI, Cruz ME, Cevc G, Martins MB. Formulation ofcatalase-loaded ultraflexible vesicles for transdermal delivery,

Proceedings of the 8th Liposome Research Days Conference; 2002.p 105.

Mizushima Y, Hoshi K, Yanagawa A, Takano K. 1991. Topical application

of superoxide dismutase cream. Drugs Exp Clin Res 17:127–131.Muzykantov VR. 2001. Targeting of superoxide dismutase and catalase to

vascular endothelium. J Contr Rel 71:1–21.Prausnitz MR. 1997. Reversible skin permeabilization for transdermal

delivery of macromolecules. Crit Rev Ther Drug Carrier Syst 14:455–483.Puignero V, Queralt J. 1997. Effect of topically applied cyclosporin A on

arachidonic acid (AA)- and tetradecanoylphorbol acetate (TPA)-

induced dermal inflammation in mouse ear. Inflammation 21:357–369.Rother M, Lavins BJ, Kneer W, Lehnhardt K, Seidel EJ, Mazgareanu S.

2007. Efficacy and safety of epicutaneous ketoprofen in Transfersome

(IDEA-033) versus oral celecoxib and placebo in osteoarthritis of theknee: Multicentre randomised controlled trial. Ann Rheum Dis

66:1178–1183.Simoes SI, Marques CM, Cruz MEM, Cevc G, Martins MB. Study of the

tissue distribution of SOD-loaded transfersomes. Proceedings of the 8thLiposome Research Days Conference; 2002. p 67.

Simoes SI, Marques CM, Cruz MEM, Cevc G, Martins MBF. Novel carriers

for antioxidant enzymes: Anti-inflammatory effects on arachidonic acid

induced ear edema in mice. Barrier Function of Mammalian Skin,Bristol-Rhode Island, USA; 2003.

Simoes SI, Martins MBF, Cruz MEM, Cevc G. Effects of transfersomal Cu/

Zn-superoxide dismutase on arachidonic acid-induced ear edema inmice. Proceedings of Cosm’ing; 1998. p 266.

Simoes SI, Richardsen HC, Martins MB, Cruz ME, Cevc G. Superoxide

dismutase delivery through the narrow pores in skin models by flexible

lipid aggregates, Proceedings of The III Spanish-Portuguese Conferenceon Controlled Drug Delivery; 1998. p 49.

Simoes SID. Preparation and characterization of enzyme-loaded

Transfersomes. PhD thesis. 2005. Chapter 4.

Simoes SI, Delgado TC, Lopes RM, Jesus S, Ferreira AA, Morais JA, et al.2005. Developments in the rat adjuvant arthritis model and its use intherapeutic evaluation of novel non-invasive treatment by SOD inTransfersomes. J Contr Rel 103:419–434.

Simoes SI, Marques CM, Cruz MEM, Cevc G, Martins MBF. 2004. Theeffect of cholate on solubilisation and permeability of simple and pro-tein loaded phosphatidylcholine/sodium cholate mixed aggregatesdesigned to mediate transdermal delivery of macromolecules. Eur JPharm Biopharm 58:509–519.

Simoes SI, Tapadas JM, Marques CM, Cruz MEM, Martins MBF, Cevc G.2005. Permeabilisation and solubilisation of soybean phosphatidylcho-line bilayer vesicles, as membrane models, by polysorbate, Tween 80.Eur J Pharmaceut Sci 26:307–317.

Szoka F, Olson F, Heath T, Vail W, Mayhew E, Papahadjopoulos D. 1980.Preparation of unilamellar liposomes of intermediate size (0.1–0.2mumol) by a combination of reverse phase evaporation and extrusionthrough polycarbonate membranes. Biochim Biophys Acta 60:559–571.

Thomson PD, Till GO, Woolliscroft JO, Smith DJ, Presad J. 1990.Superoxide dismutase prevents lipid peroxidation in burned patients.Burns 16:406–408.

Trenam CW, Dabbagh AJ, Morris CJ, Blake DR. 1991. Skin inflammationinduced by reactive oxygen species (ROS): An in vivo model. Br JDermatol 125:325–329.

Turrens JF, Crapo JD, Freeman BA. 1984. Protection against oxygen toxi-city by intravenous injection of liposome-entrapped catalase andsuperoxide dismutase. J Clin Invest 73:87–95.

Vorauer-Uhl K, Furnschlief E, Wagner A, Ferko B, Katinger H. 2001.Topically applied liposome encapsulated superoxide dismutasereduces postburn wound size and edema formation. Eur J Pharm Sci17:63–67.

Walther FJ, David-Cu R, Lopez SL. 1995. Anti-oxidant-surfactant lipo-somes mitigate hyperoxic lung injury in premature rabbits. Am JPhysiol 269:L613–L617.

Way JC. 2002. Second-generation protein drugs: Children of the revolu-tion. Drug Discov World 4:62–68.

Yabe Y, Nishikava M, Tamada A, Takakura Y, Hashida M. 1999. Targeteddelivery and improved therapeutic potential of catalase by chemicalmodification: Combination with superoxide dismutase derivatives. JPharmacol Exp Therap 289:1176–1184.

Young JM, De Young LM, 1989. Cutaneous models of inflammation for theevaluation of topical and systemic pharmacological agents.In: Spector J, Back N, editors. Pharmacological methods in the controlof inflammation. New York: Alan R Liss. pp 215–231.

658 S. Simoes et al.

Jour

nal o

f M

icro

enca

psul

atio

n D

ownl

oade

d fr

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ahea

lthca

re.c

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vers

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/28/

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r pe

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