WO2010149785A1

WO2010149785A1 - Cationic liposomes for the delivery of high molecular weight compounds

Info

Publication number: WO2010149785A1
Application number: PCT/EP2010/059102
Authority: WO
Inventors: Barbara Geusens; Jo Lambert; Niek Sanders
Original assignee: Universiteit Gent; Pierre Fabre S.A.
Priority date: 2009-06-26
Filing date: 2010-06-25
Publication date: 2010-12-29

Abstract

The present invention relates to a specific type of liposomes, denominated as SECosomes, made from a cationic lipid such as 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP), a surfactant such as sodium cholate (NaChol), a stabilizer such as cholesterol or a derivate thereof, and, an alcohol such as ethanol. Said liposomes are capable of delivering high molecular weight compounds, such as siRNAs, efficiently across tissues and into cells of tissues, such as skin.

Description

CATIONIC LIPOSOMES FOR THE DELIVERY OF HIGH MOLECULAR WEIGHT COMPOUNDS

Technical field of invention

Background art

The use of lipid vesicles or liposomes or non-viral carriers as drug delivery systems for e.g. skin treatment has attracted increasing attention in recent years. Conventional liposomes were the first to be described for the delivery of drugs to the outer layers of the skin [I]. Because they are generally characterized by a lack of penetration, their accumulation in the stratum corneum (SC) makes them of little value for disorders that require treatment in the deeper skin layers. Especially designed vesicles such as ultradeformable liposomes, flexible liposomes, and ethosomes have been described to deliver drugs into the deeper layers of the skin [2, 3]. Flexible liposomes were the first generation of elastic vesicles introduced by Cevc and were reported to penetrate intact skin while carrying therapeutic concentrations of drugs when applied under non-occlusive conditions [4]. Flexible liposomes consist of phospholipids and an edge activator or surfactant, which destabilizes the lipid bilayers of the vesicles but increases their deformability. Several in vivo studies have reported that ultradeformable liposomes are able to transfer therapeutic amounts of drugs, including macro molecules through the SC [2, 4-11].

Ethosomes are a type of lipid vesicular system composed mainly of phosho lipids, ethanol at relatively high concentrations and water [3]. Like flexible liposomes, ethosomes were found to penetrate the skin and allow enhanced delivery of various compounds, even to the systemic circulation.

We previously developed and investigated the physicochemical characteristics and stability of a still another type of ultradeformable liposome composed of DOTAP/NaChol 6:1 w:w. Moreover, its in vitro transfection efficiency, where siRNA against a target gene is used as a delivery parameter, is evaluated in human primary melanocytes [12]. However, none of the existing ultradeformable liposomes (i.e. flexible liposomes , ethosomes and ultradeformable liposomes composed of DOTAP/NaChol 6:1 w:w.) were highly efficient in carrying high molecular weight compounds, such siRNA, into or across the skin. Hence, there is a need to synthesize a new type of liposome which is superior, compared to the existing ones, to vehicle compounds, such as high molecular weight compounds, into and across tissues such as skin.

Brief description of Figures

Figure 1 : Diameter (grey bars) and zeta potential (squares) of different types of liposomes (A) and their corresponding complexes (B). All complexes were small and positively charged. Figure 2: Cryo-transmission electron microscopy images of SECosomes (A) and their corresponding complexes The complexes suggest a bilamillar and flexible character. Figure 3: Diameter of the SECoplexes as a function of their storage time at 4 ⁰C. Each point represents the mean ± SEM of three (n=3). Figure 4: Penetration studies through human skin. The same concentration of transferplexes, ethoplexes and SECoplexes was applied for 1 hr at room temperature. Multiphoton microscopy images show autofluorescence of the skin in red, and the FAM- labeled complexes in green (Fig 4A). FLIM analysis was done by means of the lifetime distribution decay curves of free and bound NAD(P)H (Fig 4B). No difference in distribution could be observed for the transferplexes and ethoplexes, whereas a clear shift to the left was seen for the SECoplexes.

Description of invention

The present invention provides for a new, specific type of liposome which is surprisingly superior compared to the existing ones, to efficiently vehicle compounds, such as high molecular weight compounds, into and across tissues such as skin.

The new type of liposomes, sometimes also denominated as particles, lipid vesicles or non- viral carriers, contain a cationic lipid such as 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP), cholesterol or a derivate thereof as a stabilizer, and, a surfactant, such as sodium cholate (NaChol) or Tween 20, at a relatively specific weight ratio, as well as a high percentage of an alcohol, such as 15-35% ethanol or propanol. The new type of cationic liposomes is given the name 'SECosomes' (Surfactant-Ethanol-Cholesterol). They differ from the existing skin-penetrating lipid-based formulations, like surfactant-containing flexible liposomes or ethanol-containing ethosomes, not only in their composition but they also show surprisingly superior properties such as delivery and skin penetration of compounds, such as high molecular weight compounds such as siRNAs, miRNA's (micro- RNAs) or anti-miRNA's (antagomirs). Therefore, physicochemical properties, biological effect and penetration capacity is compared to that of 'traditional' flexible liposomes and ethosomes.

Hence, the present invention relates to cationic liposomes comprising a 6:1 :1 w:w:w ratio of 1) a cationic lipid such as l,2-dioleoyl-3-trimethylammonium propane (DOTAP), 2) a surfactant such as sodium cholate (NaChol) and 3) cholesterol or a derivate thereof, respectively, and, a high percentage of an alcohol, such as 15-35% ethanol or propanol, preferably 30 % ethanol. The term 'cationic' refers, for example, to the positive electrical charge of DOTAP which is e.g. due to the positive charge in the trimethylammonium propane (TAP) head group of DOTAP. DOTAP is a well known synthetic phospholipid [13]. The term 'a 6:1 :1 w:w:w ratio' indicates that the weight of the total amount of DOTAP within a liposome composed of DOTAP, cholesterol and a surfactant such as NaChol is about 6 times higher than the weight of the total amount of cholesterol or the surfactant such as NaChol, the latter two compounds having about the same weight within a liposome composed of DOTAP, cholesterol and the surfactant such as NaChol. A skilled person will appreciate that a 6:1 :1 w:w:w ratio can also be equal to 'about a 6:1 :1 w:w:w ratio' which corresponds to a 5-7: 1-2: 1-2 w:w:w ratio range. Hence, and for example, a ratio of 7,0: 1,5: 2,1 w:w:w or 5.1 : 1,8: 1,0 w:w:w corresponds to 'about a 6:1 :1 w:w:w ratio'. Similarly, 15-35% alcohol, preferably 30% alcohol such as ethanol, corresponds to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 % alcohol, preferably 30% alcohol such as 30% ethanol. Similar to DOTAP, NaChol, cholesterol and ethanol are compounds well known in the art [14-16]. It should be understood that other compounds than DOTAP, NaChol, cholesterol or ethanol, which have similar physicochemical properties than the latter 4 compounds, can be used to synthesize the liposomes of the present invention. For example, alternatives for DOTAP are bis[2-(l l- phenoxyundecanoate)ethyl]-dimethylammonium bromide, JV-hexadecyl-iV- { 10-[O-(4- acetoxy)-phenylundecanoate]ethyl}dimethylammonium bromide or bis[2-(l l- butyloxyundecanoate)ethyl]dimethylammonium bromide. Cholesterol derivatives may be readily substituted for the cholesterol element of the present liposome invention. Many cholesterol derivatives are known to the skilled artisan. Examples include but are not limited to cholesteryl acetate and cholesteryl oleate. Other surfactants (detergents) include but are not limited to, α-tocopherol polyethylene glycol succinate (TPGS), PS-80, sodium dodecyl-sulfate, sodium salts of N-lauroylsarcosine, lauryldimethylamine oxide, cetryltrimethylammonium bromide, sodium salt of bis (2-ethylhexyl-sulfosuccinate), sodium deoxycholate, Span 20 (sorbitan monolaurate), Span 40 (sorbitan monopalmitate), Span 60 (sorbitan stearate), Span 80 (sorbitan monooleate), polysorbate 80 (T ween 80) or Tween 20. Ethanol can be substituted by other alcohols such as methanol, propanol, butanol and others. The present invention further relates to the usage of the cationic liposomes of the present invention to vehicle any compound (including low molecular weight compounds), but especially high molecular weight compounds across or into tissues. The term 'high molecular weight compound' refers to any chemical entity or molecule, such as nucleic acids, peptides, proteins, natural and synthetic polymers, drugs such as antibiotics and the like, having a molecular weight greater than 1 kDa, preferably greater than 5kDa and more preferably greater than 1OkDa. Said high molecular weight compounds have preferably a negative charge. More specifically, the present invention relates to the usage as indicated above, wherein said high molecular weigh compounds are negatively charged nucleic acids and more specifically wherein said nucleic acid is a siRNA, miRNA, dsRNA, mRNA or an antagomer (anti-miRNA). Preferred targets of said siRNA's are tyrosinase, myosin Va Exon F, melanophilin and protease activated receptor-2 (PAR-2) as are exemplified further. As such, the present invention relates to a new therapeutic for hyperpigmentation (13). Other preferred targets of siRNA molecules of the present invention are IL12B, DEFB4, TNF-alpha and mir-203 as are also exemplified further. As such, the present invention further relates to a new therapeutic for psoriasis. The present invention provides liposomes which are, compared to the existing ones, surprisingly superior to efficiently vehicle said compounds, especially high molecular weight compounds into and across tissues. A preferred tissue is human or animal skin and said high molecular weight compounds are preferably delivered into epidermal keratinocytes, melanocytes, or epithial cells of the skin and migrate even into the deeper layers, such as the dermis. With the terms 'efficiently vehicle said high molecular weight compounds into and across tissues' is meant that the liposomes of the present invention are capable to bring (or transfer or deliver or vehicle) said compounds across specific tissues or tissue layers and/or to bring them into a specific tissue or into cells of a specific tissue wherein said compounds can fulfill their (biological) function, and this in a manner which is significantly better compared to already known liposomes.

The present invention further relates to a process to produce a cationic liposome as indicated above comprising dissolving a cationic lipid such as DOTAP and cholesterol (or a cholesterol derivate) in an organic solvent such as chloroform at e.g. a concentration of lOmg/ml; dissolving sodiumcholate or any other surfactant in an organic solvent such as ethanol at e.g. a concentration of 10mg/ml; synthesizing cationic lipid/cholesterol or cholesterol derivates/surfactant, such DOTAP/Chol/surfactant (NaChol), cationic liposomes in a 5-7:1-2:1-2 ratio, preferably a 6:1 : 1 w:w:w ratio, using dissolved cationic lipid (such as DOTAP) and cholesterol or cholesterol derivate, and, dissolved sodiumcholate or other surfactant obtained in the preceding steps using e.g. rotary vacuum evaporation; removing traces of solvent; and, hydrating said cationic lipid/cholesterol or cholesterol derivates/surfactant (preferably DOTAP/Chol/NaChol) cationic liposomes with a mixture of 20-35% alcohol, preferably with 30 % ethanol in buffer, or, hydrating said liposomes with a higher than 35 % alcohol and subsequent dilution/dialysis in order to finally obtain 20-35% alcohol.

The present invention is hereby following illustrated by specific working examples.

Examples

Example 1: Characterization and use of Secosomes Materials l,2-dioleoyl-3-trimethylamonium propane chloride (DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL, USA), cholesterol (Choi) and sodium cholate (NaChol; cholic acid, sodium salt) were purchased from Sigma (Bornem, Belgium). Particle preparation and complex formation SECosomes, ethosomes and flexible liposomes were prepared using the solvent evaporation method. DOTAP and cholesterol were dissolved in chloroform in a concentration of 10 mg/ml (w/v). Then, sodium cholate (NaChol) was dissolved in >99% ethanol by means of sonication in a concentration of 10 mg/ml. In clean, dry round bottom flasks, DOTAP, cholesterol and NaChol were mixed to obtain a weightweightweight (w:w:w) ratio of 6:1 :1 DOTAP/Chol/NaChol for both SECosomes and flexible liposomes . A 6:1 (w:w) ratio of DOTAP/Chol was used to synthesize the ethosomes. The solvent was later removed by rotary vacuum evaporation above the lipid transition temperature and traces of solvent were removed by N₂. The resulting film was then hydrated. A hydro alcoholic solution of 70% Hepes buffer (20 mM, pH 7.4) and 30% ethanol was used for both the SECosomes and ethosomes [17], while a 100% solution of Hepes Buffer was used to rehydrate the flexible liposomes-fϊlm. After overnight incubation at 4°C in the absence of light, all particles were manually extruded 31 times through a 100 nm polycarbonate membrane filter (Whatman, Brentfort, UK). The corresponding siRNA-complexes of the different types of formulations are prepared by diluting the siRNA targeted against tyrosinase with 2OmM HEPES buffer (pH 7.4) to a final volume of lOOμl. The liposomes are then added under vortex mixing to give a liposome :siRNA ratio of 16:1 (w:w). The mixture is left to stand for 20 minutes at room temperature before being used.

Name Composition (w:w:w) % HB : % EtOH

SECosomes DOTAP/Chol/NaChol 6:1:1 70:30

Ethosomes DOTAP/Chol 6:1 70:30

Flexible liposomes DOTAP/Chol/NaChol 6:1:1 100:0

Table 1 : Description of the composition of the different formulations

Size and Zeta-potential measurements

The average particle size and zeta potential of the three liposomal formulations and their corresponding complexes, were determined using the Zetasizer Nano series (Malvern, Worcestershire, UK). Prior to the measurements, they were diluted in 2OmM Hepes Buffer (pH 7.4) and measurements were carried out at 25 ⁰C. Each sample was measured three times and the mean value is calculated.

Deformability measurements Deformability was determined as previously reported [18]. Briefly, we pushed the liposomes and their complexes three times through two polycarbonate membrane filters with a pore diameter of 30 nm (Whatman, Brentfort, UK). By comparing the size of the particles before and after this experiment we were able to evaluate the deformability. Besides DOTAP/Chol/NaChol SECosomes, we also tested DOTAP/Chol 6:1 (w:w) ethosomes (30% EtOH), DOTAP/Chol 6:1 (w:w) liposomes and DOTAP/Chol/NaChol 6:1 :1 (w:w:w) flexible liposomes together with their siRNA complexes. The latter were prepared by the rotary evaporation method as described previously.

Cryo- transmission electron microscopy 1-2 μl of SECosomes and SECoplexes (SECPX, i.e SECosomes comprising a siRNA) was placed on a glow discharged holey carbon film. The excess of liquid was blotted with a filter paper prior to vitrification in liquid ethane. The grids were examined in a Philips CMlO, CM 120 or CM 200 (Philips, Eindhoven, The Netherlands) cryo-electron microscope equipped with a Gatan cryo-stage (model 626, Gatan, Pleasanton, CA). Images were recorded on a slow-scan CCD camera under low-dose conditions (model 794 MSC, Gatan, Pleasanton, CA).

Polyacrylamide gel electrophoresis

The presence of free siRNA in the SECPX was assessed by gel electrophoresis on a 20% non-denaturing polyacrylamide gel (PAGE). SECPX were prepared as previously described at a final siRNA concentration of 5 μM. Subsequently, 5 μl of loading buffer

(30% glycerol solution) was added to each sample and this mixture was loaded onto a 20% polyacrylamide gel prepared in TBE buffer (10.8 g/1 Tris base, 5.5 g/1 boric acid and

0.58 g/1 EDTA). Electrophoresis was performed at 100 V during 2 h. Afterwards the siRNA was visualized by incubation of the gel in 1/10000 SYBR Green staining solution

(Molecular Probes, Eugene, OR) followed by UV transillumination and gel photography. Cell culture

Primary epidermal melanocytes were established as described previously [19]. Briefly, foreskins from neonatals were incubated overnight at 4°C in 10% Dispase II (Roche Diagnostics GmbH, Mannheim, Germany) to separate the epidermal layer, with melanocytes anchored to the basal membrane, from the underlying dermis. Melanocytes were cultured in Ham's FlO medium (Invitrogen Ltd, Paisley, United Kingdom) supplemented with 2.5% fetal calf serum (FCS), 1% Ultroser G, 5 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml endothelin-1 (ET-I), 0.33 nM cholera toxin (CT), 0.033 nM isobutyl-methyl-xanthine (IBMX), 5.3 nM 12-0-tetradecanol phorbol- 13 -acetate (TPA) and antibiotics/antimycotics. The cells were housed in an incubator at 37°C, 99% humidity, and 10% CO₂.

Transfection procedures

Cells are seeded at 6 x 10⁵ cells/well in P60 dishes 24h prior to transfection. Culture medium is removed from each well and cells are washed with Ix PBS. The complexes are added dropwise onto the cells and incubated for 4h with the cells in serum-free minimal medium (Ham FlO medium + L-glutamine + 5 ng/ml bFGF + 1% P/S + 0,2% Fungizone) at 37°C under 10% CO₂. Lipoplexes are then removed and replaced with 4 ml of the complete culture medium. After 48h incubation, the cells are washed with Ix PBS and harvested by addition of Trypsin-EDTA solution. All transfections are done in triplicate. A scrambled siRNA (Eurogentec, Seraing, Belgium), that shows no homology to any known gene, is used as a control.

RNA extraction from the harvested melanocytes, cDNA synthesis, real-time quantitative

PCR and western blotting ire all done as described previously by Van GeIe et al. [20]

Storage stability of SECPX

SECosome/siRNA complexes, SECoplexes (SECPX), are stored at 4°C in the absence of light. Subsequently, the particle size and silencing efficiency of the SECPX is determined at 1, 7, 14, 21 and 28 days after storage.

Skin penetration studies

Human excised skin was obtained from patients who had undergone plastic surgery.

Subcutaneous fat was removed and the full thickness skin was stored for 1 day at 4° C in the presence of DMEM (+10% P/S) medium prior to use. The next day, the skin was taken out of the medium and allowed to adjust to room temperature. The skin was cleaned with PBS and excess fluid was removed with filter paper. Complexes were made according to the procedure described above, using a fluorescently labeled siRNA-FAM to monitor the particles. The siRNA has a scrambled nucleotide composition and shows no complementarity/homology to any known mRNA. The same concentration of complexes as we used for transfection procedures, was applied onto the full thickness skin under non- occlusive circumstances for 1 hr. Tissue paper was used to remove excess fluid. Untreated skin was used as a negative control. For microscope observations, the skin pieces were mounted on a glass slide with the epidermal side facing up.

Microscopy

In the multiphoton microscopy (MPM) imaging system (JenLab GmbH SchillerstraBe 107745 Jena Germany), ultrashortpulse excitation laser light was tightly focused onto a sample by using a high-NA oil- immersion microscope objective (4OX, NAl.3). The excitation light represented a pulsed femtosecond laser, which was a tunable, computer- controlled titanium-sapphire laser (MaiTai, Spectra Physics) whose wavelength was set to 740 nm for endogenuos autofluorescence and 900 nm for monitoring the nanoparticle complexes containing 5-carboxyfluorescein (5 -FAM). A narrow-bandpass interference filter (FDIG) centered at 500-580 nm was used to selectively detect FAM signal. A broad- bandpass filter (BG39), which a long wavelength cutoff of 700 nm was employed to block the excitation laser light so that the skin autofluorescence emission was captured. The two images from the two channels were overlaid at a postprocessing stage. A TCSPC (time correlated single photon counting) 830 detector (Becker & Hickl, Berlin, Germany) was integrated into the tomography system to enable FLIM measurements. FLIM analysis of skin penetration of the complexes was undertaken using SPC 830 2.9 on a dual core computer using a Windows XP platform. The module builds up a photon distribution over the scan coordinates, x and y, and the time in the fluorescence decay, t [21]. Signals from four photon counting detectors working in different wavelength intervals are processed simultaneously. The four path detection channels were equipped with pre-selected spectral filters: with band pass of 350 nm-450 nm (Channel 1); 450 nm- 515 nm (Channel 2); 515 nm-620 nm (Channel 3); 620 nm-670 nm (Channel 4). Fluorescence lifetime analysis was done by the SPCImage data analysis package of Becker & Hickl. The software uses iterative convolution with a single, double, or triple- exponential model to determine the decay parameters in the individual pixels of the scan. A description of the data analysis can be found in [22].

Results

Characterization of SECosomes and SECPX

New liposomes consisting of the cationic lipid, DOTAP, an edge activator, sodium cholate, and a stabilisator, cholesterol were prepared with 30% of ethanol. Inclusion of cholesterol is needed to increase the stability of the particles, because particles could not be formed without.

Also DOTAP/Chol ethosomes with 30% of EtOH and DOTAP/Chol/NaChol transfersomes, were prepared. By comparing the physicochemical and biological properties of all three formulations, the beneficial effect of combining sodium cholate and ethanol will be examinated (see also Table 1). Laser diffraction studies showed the differences in size between all three formulations, as shown by the black bars. For flexible liposomes, the diameters ranged from 97-98 nm, while for SECosomes, the diameters ranged from 57-59 nm. Diameters of ethosomes ranged in between, around 65-66 nm. Zetapotential measurements showed positive surface charges for all the three formulations, without any significant differences. Fig. IB depicts the size and zeta potential of the corresponding siRNA complex-formulations. Obviously, the size of the complexes was increased after mixing them with siRNA. A decrease in zeta potential was seen for the flexible liposome/siRNA complexes (= transferplexes) and indicates that at least a fraction of the siRNAs is bound to the surface. In contrast, no decrease was observed for the ethosome or the SECosome complexes.

Deformability test

In order to test whether the SECosomes are flexible and deformable, they were filtrated 3 times through 2 30 nm membranes and there diameters before and after filtration were compared. Again, flexible liposomes and ethosomes were tested in parallel. The same procedure was done for the corresponding complexes.

Table 2 shows the mean diameter of the particles and their respective complexes before and after filtration through 2 30 nm membranes. Non-elastic, rigid DOTAP/Chol particles, not containing EtOH or NaChol, were prepared as a reference control. Observations before and after filtration showed that they were mainly retained by the membrane or broken up into smaller fragments as the count rate measured by the DLS instrument sharply dropped to almost the background signal. Addition of either NaChol or EtOH (30%), forming flexible liposomes and ethosomes respectively, enables the particles to change their shape and deform under stress (Table 2). Deformability was even more pronounced for SECosomes, as their particle size before and after filtration did not change. The same trend was also observed for the corresponding complexes, although for the flexible liposome complexes no accurate DLS measurements could be obtained after filtration. The kcps increased significantly from 227 to 459, indicating that the particles were broken up into smaller fragments that started to cluster together into aggregates of different sizes, as suggested by the software program. As a consequence, no accurate size could be detected. On the other hand, ethoplexes and SECoplexes suggested a deformable character. Although a decrease in size could be observed after filtration, the particle sizes still exceeded the pore diameter by 3 times. The size difference before and after filtration was the least pronounced for the SECoplexes.

Before filtration (nm) After filtration (nm) Relative decrease (%)

DOTAP/Chol 98 ± 2 - -

Flexible liposomes 98 ± 1 75 ± 1* 30%*

Ethosomes 79 ± 3 70 ± 2 11%

SECosomes 58 + 1 56 + 1 3%

DOTAP/Chol LPX 120 ± 6 - -

Transferplexes 112 + 1 - -

Ethoplexes 123,0 +/- 3,6 98 ± 3 20%

SECoplexes 101 + 2 86 15%

Table 2: Mean diameter (±S.D.) of different liposome and complex formulations, before and after filtration through a microporous filter with pore diameter of 30 nm

Cryo-transmission electron microscopy

To have an idea about the morphology of our new liposomal formulation cryo-transmission electron microscopy (Cryo-TEM) was performed. Cryo-TEM images of SECosomes revealed the presence of spherical vesicular structures, at first sight not indicative for their deformable character (Fig. 2A). Their round shape simply results from the lowest energy requirement state they are in. The corresponding SECPX show more bilamillar rather than typical multilamillar structures of which their morphology suggests deformability (Fig. 2B, 2C). The reason for this observed morphology is the increased concentrations (factor 100) that need to be addressed in order to acquire cryo-TEM images. The increased concentrations cause aggregation and induce pressure, causing the complexes to deform, thereby indicating their elastic properties.

Transfection experiments SiRNA targeted against tyrosinase is used to evaluate the ability of the SECosomes to deliver siRNA to primary epidermal melanocytes. The knockdown percentage of tyrosinase mRNA levels is established as a delivery parameter. In parallel, a gene knockdown assay is performed for the ethosome and flexible liposome formulations. Cells transfected with the scrambled (non-silencing) siRNA are used as positive control and are considered as 100%. Particles containing scrambled siRNA do, when compared to untreated cells, not reduce the tyrosinase mRNA levels. As the commonly used transfection reagent HiPerFect (Qiagen, Hilden, Germany) is recommended for transfection of primary cells with siRNA, we decide to compare the transfection activity of our 3 formulations to that of HiPerFect. Real-time quantitative PCR (QPCR) reveals that there is a difference between the three formulations in their ability to deliver siRNA against tyrosinase.

Physicochemical and biological stability

Carriers for in vivo siRNA delivery should fulfill a number of requirements including the ability to be stored without losing their biological stability. This is especially important when repeated administration is needed, since the effects of siRNA are only transient. Therefore, we investigated the effect of storage time at 4°C on the maintenance of the gene silencing capacity and physicochemical alterations of the SECoplexes. Freshly made complexes, prepared immediately before the transfection experiments, were used as controls. We found that the complexes retained their biological activity up to 28 days of storage at 4°C without any decrease in gene silencing capacity. In addition, the SECoplexes could be defined as stable since their size does not change significantly and no large aggregates occur (Figure 3) [23]. Skin penetration studies through human skin

Multiphoton images were taken, parallel to the surface, at increasing skin depths, ranging from 20 μm until 100-150 μm. Figure 4 A shows optical sections of excised human skin treated with transferplexes, ethoplexes and SECoplexes, from top to bottom, respectively. In all cases the same concentration was used and analysis was done 1 hr after application. Due to the endogenous fluorophores that are stimulated by two-photon excitation (740 nm), we can clearly distinguish different cellular patterns that refer to subsequent cell layers. Epidermal skin fissures appear as clefts or grooves, and gradually disappear with increasing dephts. Red is auto fluorescence signal, green is the signal from the fluorophore (FAM) and yellow is overlap of both signals. Untreated skin was used as a control. When excited at 900 nm, using the narrow-bandpass filter, no fluorescence signal could be observed from control skin. For all three conditions a clear migration of the complexes was observed in the skin fissures. Where transferplexes and ethoplexes were clearly visible at a depth of 80 μm and 100 μm, respectively, the SECoplexes were even detected in the dermis at a depth of 150 μm. In addition, no uptake by individual cells was observed for the transferplexes and ethoplexes, since they are not able to cross the stratum corneum surrounding the keratinocyte clusters. For the SECoplexes, however, yellow fluorescent signal could be detected inside these clusters, in individual cells, at different depths (Figure 4A, white arrows). To confirm our MPM observations, FLIM images were taken in parallel, as it is an excellent way to distinguish overlapping autofluorescence from complexes [24]. Moreover, it gives you an idea about the redox state of the cells and how uptake of the complexes may influence this [25]. The distribution of the lifetime values for free and bound NAD(P)H are given in Figure 4B. No difference in fluorescence decay curves of free and bound NAD(P)H between control skin and skin treated with transferplexes or ethoplexes was observed, as both curves nicely overlap. A clear shift to the left was observed for SECoplex treated skin, both for free and bound NAD(P)H decay curves, suggesting that this difference in redox state of the cell is due to the uptake of the SECoplexes.

Biological effect in human skin.

Human excised skin is obtained from patients who had undergone plastic surgery. Subcutaneous fat is removed and the full thickness skin is used immediately. SECoplexes were made according to the procedure described above, using a siRNA targeted against the protease-activated receptor 2 (PAR-2) expressed in keratinocytes. Different concentration of complexes are tested onto the full thickness skin under non-occlusive circumstances. Different time points are analyzed, ranging from 1 hr until 8 hrs. Tissue paper is used to remove excess fluid. Untreated skin is used as a negative control. Total RNA extraction is performed on the skin tissue and specific PAR-2 primers are used to perform qPCR analysis. In addition, in order to observe a possible difference in phenotypic effect between untreated and siRNA-treated skin, both skin pieces are irradiated by means of a solar simulator.

Conclusions:

1) The size and the zeta potential of the different formulations are shown in Fig. Ia. Diameters of ethosomes ranged in between, around 65-66 nm and were smaller than flexible liposomes. In addition, SECosomes even show a smaller diameter compared to the ethosomes. All formulations showed a narrow size distribution as estimated by their low polydispersity index .

2) All siRNA-complex formulations were increased in size and positively charged (Figure IB). Since there was no decrease observed for the ethosome nor the SECosome complexes, compared to their non-complexed vesicles, we suggested that the siRNA molecules are not just bound to the outer surface of the vesicle but are rather 'sandwiched' in between several vesicles, protecting them from shear stress and degradation. The positive surface charges are high enough to inhibit, via electrostatic repulsion, the aggregation of the complexes and to improve their solubility in hydro-aqueous solutions as well as their interaction with the negatively charged cell membrane and thus their cellular uptake.

3) The differences in particle size before and after 30 nm filtration were observed to be the smallest for the SEComes as well as for the SECoplexes, indicating their ultradeformable character.

4) After filtration, the SECoplexes retain their transfection effiency, suggesting that the siRNA molecules are fully protected by the SECosomes from shear forces and RNases. This however was not observed for the other 2 formulations. 5) SECosomes show typical round- shaped unilamillar structures (Figure 2A). The complexes, on the other hand, present a deformed bilamillar.morphology (Figure 2B, 2C). A 16:1 ratio SECosome: siRNA allows siRNA molecules to be 'sandwiched' in between vesicles layers without forming multilamillar aggregations [12]. Moreover, their morphology suggests flexible vesicle membranes, confirming the results of the deformability test.

6) Transfection experiments using hard-to-transfect human primary melanocytes show the excellent properties of SECoplexes as non- viral siRNA delivery systems. 7) High-resolution multiphoton analysis in combination with fluorescence lifetime imaging is used to determine the distribution and detection of the different formulations. The complexes were applied onto freshly excised human skin and after 1 hour incubation period the skin was imaged. Owing to the excitation of endogenous fluorophores of the skin by multiphoton excitation and the correlation of the resulting auto fluorescence image with the nanoparticle fluorescence pattern, the identification of accumulative spots and penetration pathways is possible with subcellular resolution. Figure 4A showes the migration pathways of the different formulations in the skin fissures. The transferplexes and ethoplexes migrate until a depth of 80μm and lOOμm respectively. The SECoplexes, however, migrated even into the dermis, at a depth of 150 μm. Moreover, the SECoplexes seem to have been taken up by individual keratinocytes at different depths. Such penetration through the stratum corneum was not observed for the other two types of complexes.

In order to confirm uptake by individual keratinocytes, FLIM analysis was performed. Figure 4B shows the graphs of the fluorescence decay curve of free and bound NAD(P)H, which is a marker for the redox state of the cell. No difference in patterns between control and treated skin could be observed for the transferplexes and ethoplexes, The fluorescence lifetime decay curves of both free and bound NAD(P)H showed the same trend. In contrast, a clear shift in lifetime was observed when treated with SECoplexes. This difference in pattern confirms uptake of the complexes by the cells, thereby changing their redox state.

Example 2: Topical application of Secosome encapsulated siRNAs reduces and prevents

UV-induced skin pigmentation Introduction

Human skin color results from the presence of melanin that is produced by melanocytes located in the basal layer of the epidermis¹. In response to the ultraviolet (UV) wavelengths of sunlight melanocytes become activated, divide, and synthesize melanin. Melanin plays an important role in preventing UV-induced skin damage ² and is synthesized in unique membrane-bound organelles, melanosomes, whose phenotype usually varies according to the type of melanin they contain. The rate-limiting enzyme for the synthesis of melanin is tyrosinase (TYR), an enzyme responsible for the hydroxylation of the amino acid tyrosine to DOPA (3,4-dihydroxyphenylalanine). Following maturation, melanosomes are transported from the cell centre to the cell periphery down the melanocytes' dendrites. Retention of melanosomes at the dendrite periphery results from the combined action of Myosin Va exon F (MyoVa exF), melanophilin (Mlph) and Rab27a, that together form the tripartite complex. The next step involves the extrusion of the melanosomes and their incorporation into the neighboring keratinocytes. This transfer process is not fully understood but apparently involves the phagocytosis of released melanosomes by keratinocytes³. An important regulator of melanosome transfer is the protease-activated receptor-2 that is present on the membrane of keratinocytes. Activation of PAR-2 results in increased phagocytic activity of cultured keratinocytes towards isolated melanosomes ⁴'⁵. Pigmentary disorders occur for various reasons such as loss of melanocytes, increase or decrease in melanocyte activity. Epidermal hyperpigmentation results either from an increased number of active melanocytes in combination with increased melanin production or from a normal number of melanocytes that produce higher than normal amounts of melanin pigment ⁶. Overexposure to UV radiation can lead to a pathological increase in melanin production, characterized by a predominant lesion called lentigo. For medical and cosmetic reasons, it is often desired to alter hyperpigmented skin areas. Various whitening substances are used to deplete skin colour for cosmetic purposes ⁷. Currently available topical agents used to treat hyperpigmentation include tyrosinase inhibitors, retinoids, hydroquinones, and melanocyte-cytotoxic agents. Unfortunately, the results of these treatments are sometimes disappointing and cause unwanted side-effects ⁷'⁸. This denotes that there is a need for more effective, more specific, and safer depigmentation therapies. RNA interference has emerged as a potent tool for post-transcriptional gene silencing ⁹. Short interfering RNA (siRNA) assembles with proteins to form an RNA-induced silencing complex that targets complementary mRNA for degradation ¹⁰. Because this tool allows the specific downregulation of the given gene and observation of the responses, it has been widely used in studies of cellular function of a specific gene. RNA interference has also been successfully used for therapeutic purposes in cutaneous therapy ⁿ. In melanogenic cells, for example, siRNA has been used to target and downregulate microphthalmia-associated transcription factor (MITF) and its subsequent transcriptional target, TYR 12. In another study, siRNA-mediated specific down-regulation of TYR has shown to inhibit melanin synthesis in fish embryos ¹³. Also in human cultured melanocytes, siRNA 214 was investigated to target tyrosinase to inhibit melanogenesis in melanocytes for the control of skin pigmentation ¹⁴. Van GeIe et al. ¹⁵ developed an siRNA against Myosin Va exon F, a motor protein that is part of the tripartite complex involved in intracellular melanosome transport in the melanocytes. Silencing this protein in cultured human primary melanocytes resulted in a perinuclear clustering of the melanosomes, consistent with the phenotypic effect seen in melanocytes of Griscelli patients. This approach could therefore lead to a treatment for hyperpigmentation disorders. The present example evaluates inhibition of pigmentation by means of topically applied siRNAs. The SECosomes of the present invention were used as a delivery vehicle. Four different siRNAs targeted against tyrosinase, PAR-2, Myosin Va exon F and melanophilin were used individually and synergistically. In order to study local pigmentary responses to cutaneous siRNA therapy, we first needed to establish a hyperpigmentation model that was kept viable throughout the entire experiment and that served as a representative model of the in vivo environment of human skin. Although animal models have often been used to study topical delivery purposes, the observations arising from their use can be confounded by interspecies variations in morphology and permeability. In this light, human skin explants provide perhaps the closest laboratory model attainable to the in vivo environment in terms of biological complexity and fidelity to human physiology. In contrast to skin equivalents, all cellular elements and their interactions are included in human full-skin culture explants. Therefore, this approach offers improved translation between the investigative laboratory and the clinical setting. Materials and methods

Source of skin

Human abdominal skin from healthy patients was collected after plastic surgery. Subcutaneous fat was removed by blunt sectioning. The Caucasian skin tissue explants were thoroughly washed in PBS and tissue pieces measuring 25 mm in diameter were deposited on grids in 6-well petri dishes and were maintained in Bio-EC BEM medium (BIO-EC laboratory, Longjumeau,France), placed under 10% CO2 in an incubator at 37°C.

Experimental procedures

In a first test experiment, skin specimens were merely incubated in Bio-EC BEM medium to evaluate skin viability and possible tissue degradation as a function of time. The culture medium was changed every other day. The same skin specimens were also subjected to UV-irradiation using a Oriel solar simulator with Xenon lamp (900 W/cm2). Irradiation was done using 1.78 J/cm2 as the estimated UV dose at which a visual establishment of reaction to UV started. Before solar simulated radiation (SSR), culture medium was replaced by a thin layer of sterile PBS, in which only a part of the dermis was incubated, to prevent side effects of possible presence of photoactive compounds in the medium. After irradiation the PBS was replaced by the standard Bio-EC BEM base medium. A second group of tissue specimens from another donor received 1 MED SSR on day 0, after which topical treatment started twice daily for 4 days using a combination of siRNA-SECosome formulations against PAR-2, Tyr, Mlph and MyoVa exF in order to deplete the skin colour of the established (hyper)pigmentation model. Hydroquinone was used as a positive control. Tissues were harvested 96 hours after the start of the experiment. Finally, a third group of explant skin samples received topical application of siRNA-SECosome formulations twice daily during the first three days in culture prior to SSR on day 4. Five different formulations were tested; SECosome/siRNA complexes against PAR-2, Tyr, Mlph and MyoVa exF were applied individually at a dose of 6 μg/cm2. A combination of all 4 formulations was also tested to evaluate the synergistic effect. Total sun block (SPF 50+, Bioderma) was used as a positive control. Morphological analysis

Skin biopsies of 4 mm diameter were taken every day for routine histology to observe skin viability and SSR-induced tanning. For the SECosome/siRNA treated specimens, biopsies were only taken at the beginning (DO) and end (D4) of the experiment. In short, the biopsies were immediately fixed in 10% phosphate-buffered formalin, embedded in paraffin, cut in 5 μm-thin sections and stained with 1% hematoxylin and eosin (H&E). Skin biopsies were examined on tissue changes in the epidermis and dermis. Characteristics indicating degeneration of the tissue are, among others, loosening of the epidermis from the dermis, a decrease in number of epithelial cell layers and occurrence of vacuoles in epidermal cells. On a cellular level the occurrence of necrotic cells is recognized by cell swelling and dark shrunken nuclei, whereas apoptotic epithelial cells, also called sunburn cells, are recognized by their shrunken appearance and irregular shrunken nuclei. Melanin pigments were visualized using Fontana-Masson (F&M) stain. F&M staining identifies silver nitrate reducing molecules. In skin, this non-specific stain identifies primarily melanin. F&M stained sections were used for image analysis. All images were obtained and analyzed with Image J software. Parameters measured were surface area of silver deposits within melanocytes and keratinocytes and the total surface area of the tissue, and the relative pigmented area was calculated. A value of 100% was assigned to untreated (non- irradiated) controls, and values of treatment groups were normalized to their relevant controls.

UV-induced tanning - Visual assessment

Throughout the entire experiment, all tissue explants are photographed using a digital camera for visual assessment of depigmentation. The degree of pigmentation was assessed using a colormeter (DSM II ColorMeter; Cortex Technology); the outcome measure of this device is the Lvalue- the higher the Z-value, the lighter the skin.

RNA extraction and gene amplifications From 8 mm biopsy skin tissues, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) according to manufacturer's recommendations. Tissue biopsies were stored in KNAlater at 4°C prior to use. Before disruption, they were snap frozen in liquid nitrogen followed by motar and pestle grinding. Next, homogenization was done using QIAshredder homogenizers. RNA was quantified using a ND- 1000 spectrophotometer (Isogen, LifeScience, St-Pietersleeuw, Belgium). Total RNA (2 μg) was treated with the RQl RNase-free DNase from Promega (Leiden, The Netherlands). In addition, treated RNA samples were desalted prior to cDNA synthesis using the iScript cDNA Synthesis Kit (BioRad, Eke, Belgium) according to the manufacturer's recommendations and subsequently diluted with nuclease-free water (Sigma) to 12.5 ng/μl cDNA.

In order to assess the RNA purity and integrity, we performed a SPUD assay for the detection of enzymatic inhibitors (Nolan 2006) and a capillary gel electrophoresis analysis (Experion; Bio-Rad) to establish an RNA quality index (RQI). Quantification of gene expression levels of PAR-2, Tyr and Mlph transcripts was performed using primer sequences that are commercially available (Eurogentec, Seraing, Belgium). For MyoVa exF, primer sequences were designed using Primer Express software (Applied Biosystems, Foster City, CA). Relative gene expression levels were determined using a SYBR Green I RT-PCR assay as described by Vandesompele et al. ¹⁷ and the comparative Ct method was used for quantification. PCR reagents (SYBR Green I Master Mixes) were obtained from Eurogentec (Seraing, Belgium) and used according to the manufacturer's instructions. PCR reactions were run on a BioRad MyiQ Real-Time PCR detection system (BioRad, Eke, Belgium). To correct for differences in RNA quantities and cDNA synthesis efficiency, relative gene expression levels were normalized using the geometric mean of three housekeeping genes (GAPDH, UBC and SDHA for skin tissue) according to Vandesompele et al. ¹⁸.

Results and discussion Skin viability in culture conditions

In order to exploit and study the siRNA knockdown effects, it is imperative to ensure that the skin model remains genetically viable during the entire experimental procedure. According to Ng et al. ¹⁹ who addressed the viability of ex vivo skin for DNA vaccine expression in function of time, we kept our skin explants in culture for a maximum of 4 days or 96 hours. On gross inspection, the specimens appear viable and in good general condition at all time points. No dryness, wrinkles or fluffiness was observed. H&E staining of the explants showed normal skin architecture with no damage or necrotic centers. However, 72 h after being put in culture, slight morphological changes were observed. On a cellular level some epidermal cells show first signs of degeneration, such as the appearance of a halo around the nuclei, which became more prominent during time. Overall, the tissue structure stayed well preserved; the stratum corneum, epidermis and dermis are well recognizable.

UV-induced pigmentation

In order to use siRNA therapy for the depletion and prevention of UV-induced pigmentation, a reliable pigmentation model needed to be established first. Caucasian skin explants were used to examine the effects of UV-irradiation (solar simulated radiation; SSR) on pigmentation in ex vivo conditions. A UV dose of 1.78 J/cm2 was used as the estimated UV dose at which visible colouration for this skin type started. Although a change in skin colour was noticeable immediately after SSR, tanning became more pronounced at later timepoints. Freshly excised (Day 0) skin explants showed an obvious increase in pigment granules 24 h after solar simulator radiation was applied at a dose of 1.78 J/cm2, compared to non- irradiated control skin. This subsequently caused a visible tanning of the epidermis. This phenotypic effect could be observed macroscopically and was confirmed by the colorimeter L-values, being 64.92 and 44.49, for non-irradiated and irradiated skin, respectively. Fontana-Masson staining revealed a 5.9 increase in intracellular melanin granules in the pigmented, irradiated skin sample compared to the non-irradiated control sample.

Skin morphology and viability after SSR

Besides the beneficial effects of sunlight, it is known that UV-irradiation during excessive and prolonged sun exposure can cause severe acute damage. One of the first signs of UV or SSR-induced effects on skins morphology is the formation of sunburn cells, a hallmark of UV-induced damage ²¹. These effects have been described previously in human volunteers , human skin explants ' , reconstructed skin models and animal models . Histologically, sunburn cells show a pyknotic nucleus and a shrunken glassy, eosinophilic cytoplasm. Inspection of fresh skin histology immediately after SSR revealed minimal damage to the cells and architecture of the skin, compared to non-irradiated control skin. When the irradiated explant sample was kept in culture for another 96 h, more degenerative effects could be recognized. Nuclei become darker (cfr. sunburn cells), the majority of epithelial cells showed white halos around their nuclei and vacuoles were present in the epidermal layers.

Differential gene expression after UV-irradiation In order to evaluate the knockdown effects of the different SECosome/siRNA formulations on human skin explants, a relevant differential gene expression profile should be realized as a result of SSR. We especially focus on the mRNA levels of four genes of interest, as described above. Biopsies (8 mm) were taken from freshly isolated skin; before SSR, immediately after SSR and 24 h after SSR. Similarly, biopsies were obtained from skin that was incubated for 96 h in culture medium. Again, conditions before, after and 24 h after SSR were tested. RNA quality and integrity were inspected before qPCR analysis was performed. All 6 samples displayed a RNA Quality Indicator (RQI) > 8, reflecting almost entirely intact RNA without major degradation products. Immediately after SSR, areduction in mRNA expression levels is revealed. Particularly, genes involved in transport mechanisms of the melanosomes (MyoVa exF, Mlph, PAR-2) are downregulated. However, a slight increase could be observed for tyrosinase, which is involved in the early steps of melanogenesis. 24 h after SSR, basal levels were restored and an upregulation of Mlph and Tyr was noted. Since mRNA levels precede protein expression levels, the visual tanning of the skin - 24 h after SSR - and the increase in melanin granules results from the rise in tyrosinase mRNA expression levels (19%) immediately after SSR. Consequently, the further increase in tyrosinase expression levels (27%) results in a more pronounced tanning effect at longer time points.

Topical application of SECosome/siRNA formulations Our primary goal was to develop a 'gene cream' for topical skin application in order to successfully reduce hyperpigmentation. SECosome/siRNA formulations were topically applied on an established hyperpigmented skin explant in order to deplete the skin color. Hyperpigmentation was induced as a result of UV-irradiation (cfr. solar lentigines) on day 0 and subsequently treated with a mixture of SECosome/siRNA complexes for 4 days in order to obtain a macroscopic effect. The mixture contained siRNAs directed against MyoVa exF, Mlph, Tyr and PAR-2. We used hydroquinone in Neostrata pigment lightning gel as a positive control. The results of a 4 day treatment of hydroquinone (HQ) and SECosome/siRNA mix versus control indicate that after SSR and before treatment started, all three specimens had a comparable skin color with L-values ranging from 38 to 41. Although visual assessment and colorimeter measurements suggest HQ induces the most pronounced depigmenting effects, microscopic analysis (FM) and quantification of melanin granules suspect differently. According to software analysis of the FM histological section, 15% less pigment granules were observed after treatment with SECosome/siRNA mix, whereas for HQ this was only 4%. In another set-up, we used the same SECosome/siRNA mix to evaluate whether it could also prevent SSR- induced tanning. Skin specimens were treated for 4 days, twice daily with a mix of SECosome/siRNA complexes. In parallel, individual formulations of MyoVa exF, Mlph, Tyr and PAR-2 were also tested. On day 4, the specimens were irradiated at 1.78J/cm2. Sun block (SPF 50+) and a mixture of naked siRNA (without the SECosomes) were used as positive and negative control, respectively. Skin explants were thus treated for 4 consecutive days (twice daily) with sun block (SPF 50+) and naked siRNA mix as a positive and negative control, respectively, after which they were subjected to SSR. Evaluation was done on a macroscopic and histological level, before, after and 24 h after SSR. The results indicate that sun block efficiently prevents UV-induced stimulation of pigmentation and the amount of melanin granules remains unaffected throughout the entire experiment. The highly increased L- value (83,28) is due to the sticky white opaque sun cream that was not entirely removed. Naked siRNA molecules that are topically applied do not have any effect on prevention of UV-induced pigmentation. This could be expected since these negatively charged high molecular weight molecules do not readily penetrate intact skin. As a consequence, a 2-fold upregulation of melanin granules was calculated. The tanning effect that was seen after SSR when naked siRNAs were applied confirms earlier findings that explant skin maintains the ability to respond to UV-irradiation (SSR). SECosome/siRNA formulations were applied for each siRNA individually, and a combination of all 4 in a SECosome/siRNA mix. First macroscopic observations show a 23% decrease in L-value which matches the 20% increase in pigment granules immediately after UV. Evaluation at a later time point (24h after UV) showed a status quo in pigment granule quantity, but an increase in L-value. Where software analysis displayed a status quo in melanin content immediately after SSR compared to the situation before UV, we found that 24 h after SSR, a 40% decrease in melanin granules was noticed. At first sight, this even surpasses the UV-prevention effect of sun block, and shows an effective knockdown. Again, the degree of pigmentation on a macroscopic scale (L- value) does not entirely correlate with the software quantification that was carried out on a histological level. Skin explants were treated for 4 consecutive days (twice daily) with SECosome/siRNA formulations targeted against MyoVa exF, Mlph, Tyr or PAR-2 individually. In addition, a combination of all 4 was also tested. Evaluation was done on a macroscopic and histological level, before, after and 24 h after SSR. The fact that a decrease in melanin granules was observed 24 h after irradiation, whereas immediate observations displayed unchanged levels, suggests that UV-irradiation induces an improved biological effect of the siRNA mix formulation that could only be visualized at later time points. It is a well- known aspect of UV-irradiation to enhance percutaneous penetration in vitro ³¹, since it induces alterations in epidermal barrier function ³²'³³. It could therefore be suggested that enhanced penetration occured as a result of SSR, and that the siRNA effects were more pronounced when higher quantities of the complexes reached deeper layers. In addition, the mixed formulation targets multiple key molecules at the same time, causing a cumulative and hence more pronounced effect.

General conclusion Although hyperpigmentation or abnormal accumulation of melanin is an issue of cosmetic concern, it has a huge market potential, especially in the Asian countries. Cutaneous siRNA therapy for specific knock-down of unwanted or aberrant genes, without affecting other targets, has gained a lot of interest for a variety of skin disorders ⁿ. Interfering in the pigmentation pathway by means of RNAi is a valuable way to reversibly inhibit protein translation of key molecules involved in this process. SiRNA treatment in order to deplete hyperpigmentation besides prevention of UV-induced tanning revealed positive results for the formulation containing SECosomes and a mix of different siRNAs. Indeed, these results indicate that different siRNA molecules work synergistically to obtain the best effect, both in reducing pigmentation as well as in preventing SSR tanning.

References indicated in example 2

(1) Nordlund JJ, Boissy RE, Hearing VJ, King RA, Ortonne JP: The Pigmentary System:

Physiology and Pathophysiology. New York, Oxford University Press, 1998. (2) Raposo G, Marks MS: Melanosomes— dark organelles enlighten endosomal membrane transport. Nat Rev MoI Cell Biol 2007;8:786-797.

(3) Van Den Bossche K, Naeyaert JM, Lambert J: The quest for the mechanism of melanin transfer. Traffic 2006;7:769-778. (4) Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M, Shapiro SS: Inhibition of melanosome transfer results in skin lightening. J Invest Dermatol 2000;115:162-167.

(5) Seiberg M, Paine C, Sharlow E, Andrade-Gordon P, Costanzo M, Eisinger M, Shapiro SS: The proteaseactivated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions. Exp Cell Res 2000;254:25-32.

(6) Demis DJ: Clinical Dermatology-Melasma. Philadelphia, Lippincott Co, 1989.

(7) Solano F, Briganti S, Picardo M, Ghanem G: Hypopigmenting agents: an updated review on biological, chemical and clinical aspects. Pigment Cell Res 2006;19:550-571.

(8) Hacker SM: Common disorders of pigmentation: when are more than cosmetic cover- ups required? Postgrad Med 1996;99:177-186.

(9) Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-811.

(10) Hammond SM, Bernstein E, Beach D, Hannon GJ: An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells. Nature 2000;404:293-296.

(11) Geusens B, Sanders N, Prow T, Van GeIe M, Lambert J: Cutaneous short-interfering RNA therapy. Expert Opin Drug Deliv 2009;6:1333-1349.

(12) Nakai N, Kishida T, Shin- Ya M, Imanishi J, Ueda Y, Kishimoto S, Mazda O: Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf. Gene Ther 2007; 14:357-365.

(13) Boonanuntanasarn S, Yoshizaki G, Takeuchi T: Specific gene silencing using small interfering RNAs in fish embryos. Biochem Biophys Res Commun 2003;310:1089-1095.

(14) An SM, Koh JS, Boo YC: Inhibition of melanogenesis by tyrosinase siRNA in human melanocytes. BMB Rep 2009;42:178-183. (15) Van GeIe M, Geusens B, Schmitt AM, Aguilar L, Lambert J: Knockdown of myosin Va iso forms by RNAi as a tool to block melanosome transport in primary human melanocytes. J Invest Dermatol 2008;128:2474-2484. (17) Vandesompele J, De Paepe A, Speleman F: Elimination of primer-dimer artifacts and genomic coamplifϊcation using a two-step SYBR green I real-time RT-PCR. Anal Biochem 2002;303:95-98.

(18) Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3.

(19) Ng KW, Pearton M, Coulman S, Anstey A, Gateley C, Morrissey A, Allender C, Birchall J: Development of an ex vivo human skin model for intradermal vaccination: tissue viability and Langerhans cell behaviour. Vaccine 2009;27:5948-5955. (20) Ravnbak MH, Philipsen PA, Wiegell SR, WuIf HC: Skin pigmentation kinetics after UVB exposure. Acta Derm Venereol 2008;88:223-228.

(21) Kulms D, Schwarz T: Molecular mechanisms of UV-induced apoptosis. Photodermatol Photoimmunol Photomed 2000; 16:195-201.

(22) Camouse MM, Domingo DS, Swain FR, Conrad EP, Matsui MS, Maes D, Declercq L, Cooper KD, Stevens SR, Baron ED: Topical application of green and white tea extracts provides protection from solar simulated ultraviolet light in human skin. Exp Dermatol 2009;18:522-526.

(23) Hofmann-Wellenhof R, Smolle J, Roschger A, Strunk D, Hubmer M, Hoffmann C, Quehenberger F, Horn M, Kerl H, Wolf P: Sunburn cell formation, dendritic cell migration, and immunomodulatory factor production after solar- simulated irradiation of sunscreen-treated human skin explants in vitro. J Invest Dermatol 2004;123:781-787.

(24) Duval C, Schmidt R, Regnier M, Facy V, Asselineau D, Bernerd F: The use of reconstructed human skin to evaluate UV-induced modifications and sunscreen efficacy. Exp Dermatol 2003; 12 Suppl 2:64-70. (25) Rijnkels JM, Whiteley LO, Beijersbergen van Henegouwen GM: Time and dose- related ultraviolet B damage in viable pig skin explants held in a newly developed organ culture system. Photochem Photobiol 2001;73:499-504.

(26) Daniels F, Jr., van der Leun JC, Johnson BE: Sunburn. Sd Am 1968;219:38-46.

(27) Sheehan JM, Young AR: The sunburn cell revisited: an update on mechanistic aspects. Photochem Photobiol Sci 2002; 1 :365-377.

(28) Abdel-Malek ZA, Kadekaro AL, Swope VB: Stepping up melanocytes to the challenge of UV exposure. Pigment Cell Melanoma Res;23: 171-186. (29) Cario-Andre M, Pain C, Gall Y, Ginestar J, Nikaido O, Taieb A: Studies on epidermis reconstructed with and without melanocytes: melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair- skinned Caucasians. J Invest Dermatol

2000;115:193-199. (30) Gilchrest BA, Park HY, Eller MS, Yaar M: Mechanisms of ultraviolet light-induced pigmentation. Photochem Photobiol 1996;63:l-10.

(31) Gelis C, Mavon A, Delverdier M, Paillous N, Vicendo P: Modifications of in vitro skin penetration under solar irradiation: evaluation on flow-through diffusion cells.

Photochem Photobiol 2002;75:598-604. (32) Haratake A, Uchida Y, Schmuth M, Tanno O, Yasuda R, Epstein JH, Elias PM,

Holleran WM: UVBinduced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response. J Invest Dermatol

1997;108:769-775.

(33) Holleran WM, Uchida Y, Halkier-Sorensen L, Haratake A, Hara M, Epstein JH, Elias PM: Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function. Photodermatol Photoimmunol Photomed 1997;13:117-128.

Example 3: Secosome encapsulated siRNAs for use to treat psoriasis.

The present example relates to the development of a topical liposomal vector (Secosomes) for the protection, transport and delivery of siRNA's and antagomirs (anti-miRNAs) to target genes important in the pathogenesis of psoriasis. By intervening at genetic level by means of a topical applied therapy, we create a specific, better tolerated and patient- friendly therapy for psoriasis. SiRNA's and antagomirs against genes linked to psoriasis are developed and their silencing capacity is examined in psoriasis cell cultures. The most potent siRNAs and antagomirs are complexed with cationic liposomes to form lipoplexes. In a next phase these lipoplexes are tested on psoriasis 3D skin models and mouse models for their capacity to induce regression of the psoriasis phenotype. An analysis of the most potent combinations of siRNA's and liposomal formulations is performed. An optimalisation of the liposomal vector is sometimes achieved to allow deeper penetration and/or cell specific delivery by means of a ligand. Introduction and objectives

Psoriasis is a common chronic immune-mediated inflammatory papulosquamous skin disorder, with a strong genetic basis. Despite the availability of many treatments for psoriasis, their adverse effects or inadequate efficacy have created the need for more effective, better tolerated and more cost-effective therapies¹. The role of multiple genes in the pathogenesis of psoriasis and the recent discovery of microRNAs (miRNA) offer new perspectives for antisense therapies as an alternative therapeutic strategy. Silencing of gene activity by RNA interference (RNAi) has been an exciting new avenue of research and its broad applicability has led to the introduction of small interfering RNAs (siRNA) and anti- miRNAs (antagomirs) as new therapeutic agents². Two types of small RNA molecules - miRNA and small interfering RNA (siRNA) - are central to RNA interference. The behavior of siRNA and miRNA is similar, but their origin is different, with siRNA an exogenous synthetic molecule and miRNA an endogenous genome-encoded molecule. The genetic make-up of psoriasis is multifactorial: multiple genes and the environment conspire to increase one's risk of developing psoriasis. Recently knowledge of the genetic basis of this condition has advanced considerably with the advent of genome-wide association studies. These scans show significant associations between psoriasis and single nucleotide polymorphisms (SNPs) in genes encoding IL12, IL23R, IL23A,TNFAIP3 (TNF-α induced protein 3) and TNIPl (TNFAIP3 interacting protein 1), IL4 en IL 13, and the 'late cornifϊed envelop (LCE) gene cluster ^3"6. Polymorphisms in the promoter region of TNF-alpha are also associated with psoriasis, and are responsible for the elevated expression level of TNF-α in psoriasis lesions ^7"9. In addition, gene dosage analyses illustrate that an amplification of copy number in the β-defensin gene clusteron chromosome 8 is associated with an increased risk for developing psoriasis. DEFB4, encoding human β-defensin 2 (hBD-2), shows the strongest association with psoriasis¹⁰. Recently, much attention has focused on the impact of miRNAs in disease states, and deregulation of miRNAs seems to play also a role in the pathogenesis of psoriasis. MiR- 203 is a psoriasis-specific miRNA and its expression is upregulated in psoriasis affected skin¹¹. Consequently, both silencing of deregulated genes by siRNAs and inhibition of disease-specific miRNA's by means of antagomirs are promising RNAi-based therapeutic approaches for psoriasis.

Diseases of the skin are amenable to RNAi-based therapies and there is a variety of potential applications for RNAi therapy in the skin¹². The skin is an easy target organ for topical siRNA delivery as it allows direct access to target cells in the skin. Application of topical or local siRNA therapy is easily confined to the affected skin and systemic absorption and toxicity is minimal. Topical siRNA application, on the other hand, requires penetration-enhancing techniques as the large negatively charged hydrophilic molecules cannot passively diffuse into the skin¹².

Since there is an urgent need for specific, painless and patient-friendly therapeutics, we want to stress the importance of RNAi-based liposome-mediated topical applications. Hence, the present example relates to the development of a liposome-based delivery system for siRNAs and antagomirs to target psoriasis-related genes. The liposome or vector offers protection against degradation and mediates transports and delivery of the encapsulated molecules into the target cell. We choose IL12B (encoding p40 subunit of IL23), DEFB4 (encoding hBD-2), TNF-α and miR-203 as targets for RNAi therapy because of their strong and consistent association with psoriasis (IL12B ³'⁶'²¹'²² ₅ DEFB4 ¹⁰'²³, TNF-α ²⁴'²⁵ , miR-203¹¹ ), their overexpression in psoriasis-lesions (IL12B²⁶, DEFB4 ²⁷'²⁸ ,TNF-α ²⁴'²⁹ , miR-203¹¹) and their expression by keratinocytes (IL12B³⁰, DEFB4 ²⁷'³¹ , TNF-α ²⁵, miR-203¹¹).

Methodology

1) Testing of siRNAs and antagomirs directed towards 4 target genes: IL12B, DEFB4, TNF- α en mir-203. In order to test the silencing capacity of the antisense molecules, we develop keratinocyte cell cultures with psoriasis phenotype that display the 4 genes of interest.

The cell cultures are evaluated using ELISA assays. The differential expression of the target genes in the cell culture are investigated using qPCR and western blotting. Next, siRNAs against IL12B, DEFB4, TNF-α and antagomirs against mir-203 are developed and transfected in keratinocytes cultured in vitro using lipofectamine. Their ability to knockdown the targeted genes are tested by means of qPCR and a luciferase reporter construct for siRNA and antagomir evaluation, respectively. The antisense molecules that provide a knockdown percentage of at least 70% are considered efficient and are used in further experiments.

2) Development and characterization of cationic liposomes (Secosomes) as non- viral drug carriers. We synthesize cationic liposomes as non-viral vector for the selected antisense molecules using the rotation-evaporation method. Lipoplexes are prepared by complexation of the cationic liposomes with the negatively charged antisense molecules. These lipoplexes are characterized by zeta potential and dynamic light scattering (DLS) measurements. The transfection efficiency of the different lipoplexes (siRNA/antagomir+liposome) and their cytotoxicity are tested in the psoriasis cell cultures. 3) Penetration studies and biological evaluation of siRNA- and antagomir- liposomal complexes. The complexes are evaluated biologically by topical administration on reconstructed 3D skin models and on psoriasis mouse models. For the latter we use human non-lesional psoriasis-skin and transplant it onto immunodefϊcient beige/nude/Xid (BNX) mice. Induction of psoriasis lesions is accomplished by intradermal injection of autologous peripheral blood mononuclear cells (PBMCs). Evaluation of the penetration capacity of the liposomes and the place of delivery are investigated by multiphoton microscopy (MPM).

References indicated in example 3

1 Naldi L, Raho G. Emerging drugs for psoriasis. Expert Opin Emerg Drugs 2009; 14: 145-63.

2 Grimm D. Small silencing RNAs: state-of-the-art. Adv Drug Deliv Rev 2009; 61: 672- 703.

3 Nair RP, Duffϊn KC, Helms C et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet 2009; 41: 199-204. 4 Zhang XJ, Huang W, Yang S et al. Psoriasis genome-wide association study identifies susceptibility variants within LCE gene cluster at Iq21. Nat Genet 2009; 41: 205-10.

5 Capon F, Di Meglio P, Szaub J et al. Sequence variants in the genes for the interleukin-

23 receptor (IL23R) and its ligand (IL12B) confer protection against psoriasis. Hum Genet

2007; 122: 201-6. 6 Cargill M, Schrodi SJ, Chang M et al. A large-scale genetic association study confirms

IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet

2007; 80: 273-90.

7 Arias AI, Giles B, Eiermann TH et al. Tumor necrosis factor-alpha gene polymorphism in psoriasis. Exp Clin Immunogenet 1997; 14: 118-22. 8 Li C, Wang G, Gao Y et al. TNF-alpha gene promoter -238OA and -308OA polymorphisms alter risk of psoriasis vulgaris: a meta-analysis. J Invest Dermatol 2007;

127: 1886-92. 9 Mossner R, Kingo K, Kleensang A et al. Association of TNF -238 and -308 promoter polymorphisms with psoriasis vulgaris and psoriatic arthritis but not with pustulosis palmoplantaris. J Invest Dermatol

2005; 124: 282-4. 10 Hollox EJ, Huffmeier U, Zeeuwen PL et al. Psoriasis is associated with increased beta-defensin genomic copy number. Nat Genet 2008; 40: 23-5.

11 Sonkoly E, Wei T, Janson PC et al. MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis? PLoS One 2007; 2: e610.

12 Geusens B, Sanders N, Prow T et al. Cutaneous short-interfering RNA therapy. Expert Opin Drug Deliv 2009; 6: 1333-49. 20 Gonzalez-Gonzalez E, Ra H, Hickerson RP et al. siRNA silencing of keratinocyte- specific GFP expression in a transgenic mouse skin model. Gene Ther 2009; 16: 963-72.

21 Scanlon JV, Exter BP, Steinberg M et al. Ustekinumab: Treatment of Adult Moderate- to-Severe Chronic Plaque Psoriasis (September) (CE). Ann Pharmacother 2009.

22 Capon F, Bijlmakers MJ, WoIf N et al. Identification of ZNF313/RNF114 as a novel psoriasis susceptibility gene. Hum MoI Genet 2008; 17: 1938-45.

23 Peric M, Koglin S, Dombrowski Y et al. Vitamin D analogs differentially control antimicrobial peptide/" alarmin" expression in psoriasis. PLoS One 2009; 4: e6340.

24 Johansen C, Funding AT, Otkjaer K et al. Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2. J Immunol 2006; 176: 1431-8.

25 Jakobsen M, Stenderup K, Rosada C et al. Amelioration of Psoriasis by Anti-TNF-alpha RNAi in the Xenograft Transplantation Model. MoI Ther 2009.

26 Lee E, Trepicchio WL, Oestreicher JL et al. Increased expression of interleukin 23 pl9 and p40 in lesional skin of patients with psoriasis vulgaris. J Exp Med 2004; 199: 125-30. 27 Van Ruissen F, de Jongh GJ, Zeeuwen PL et al. Induction of normal and psoriatic phenotypes in submerged keratinocyte cultures. J Cell Physiol 1996; 168: 442-52. 28 Gambichler T, Skrygan M, Tomi NS et al. Differential mRNA expression of antimicrobial peptides and proteins in atopic dermatitis as compared to psoriasis vulgaris and healthy skin. Int Arch Allergy Immunol 2008; 147: 17-24. 29 Ettehadi P, Greaves MW, Wallach D et al. Elevated tumour necrosis factor-alpha (TNF- alpha) biological activity in psoriatic skin lesions. Clin Exp Immunol 1994; 96: 146-51. 30 Piskin G, Sylva-Steenland RM, Bos JD et al. In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin. J Immunol 2006; 176: 1908-15.

31 Harder J, Meyer-Hoffert U, Wehkamp K et al. Differential gene induction of human beta-defensins (hBD-1, -2, -3, and -4) in keratinocytes is inhibited by retinoic acid. J Invest

Dermatol 2004; 123: 522-9.

Example 4: Testing different components for synthesis of SECosomes and investigating the physicochemical properties of the SECosomes.

SECosomes of the present invention were produced according to the present invention using the following well-known compounds: the cationic lipids DOTAP and DODAP, the surfactants sodium cholate and Tween 20, cholesterol (stabilizer) and the alcohols ethanol and propanol.

The following SECosomes were made and were given the following number: 1) DOTAP/NaChol/cholesterol (w:w:w = 6:1 :1) and 40% ethanol, 2) DOTAP/NaChol/cholesterol (w:w:w = 6:1 :1) and 20% ethanol, 3) DOTAP/NaChol(w:w = 6:1) and 20% ethanol, 4) DOTAP/NaChol(w:w = 6:1) and 30% ethanol, 5) DOTAP/NaChol(w:w = 6:1) and 40% ethanol, 6) DOTAP/NaChol/cholesterol (w:w:w = 6:1 :1) and 20% propanol, 7) DOT AP/T ween 20/cholesterol (w:w:w = 6:1 :1) and 30% ethanol, 8) DODAP/NaChol/cholesterol (w:w:w = 6:1 :1) and 30% ethanol, 9) DODAP/Tween/cholesterol (w:w:w = 6:1 :1) and 30% ethanol, and 10) DOD AP/T ween/cholesterol (w:w:w = 6:1 :1) and 30% propanol. The diameter (nm) and zeta potential (mV) of each SECosome was determined according to the invention.

The results:

SECosome N° diameter zeta potential

1 - -

2 81.5 56.2

3 72.7 57.7

4 45 50.5 5 _ _

6 73.3 53.9

7 43.2 50.4

8 56.8 -15.3

9 39.5 2.6

10 30.5 -2.3

Conclusions:

1. no liposomes were formed when 40% ethanol was used (SECosomes N° 1 and 4).

The physicochemical properties of the liposomes are positive when 20% or 30% ethanol or propanol are used (SECosomes N° 2 and 6).

2. when no cholesterol is used (SECosomes N° 3-5), micelles are formed due to combination of ethanol and surfactant which sometimes results in very small diameters.

3. Tween 20 (SECosomes N° 7) might be used instead of nacholate but the diameter is rather small.

4. DODAP is a cationic lipid that is pH sensitive. At pH 7.4 this lipid is not positively charged ( SECosomes 8-10).

References used in application (except in Examples 2 and 3)

1. Mezei M, Gulasekharam V: Liposomes--a selective drug delivery system for the topical route of administration. Lotion dosage form. Life Sci 1980, 26(18): 1473- 1477.

2. Cevc G: Transfersomes, liposomes and other lipid suspensions on the skin: permeation enhancement, vesicle penetration, and transdermal drug delivery. Crit Rev Ther Drug Carrier Syst 1996, 13(3-4):257-388.

3. Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M: Ethosomes - novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J Control Release 2000, 65(3):403-418.

4. Cevc G, Blume G: Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force. Biochim Biophys Acta 1992, 1104(l):226-232. 5. Cevc G: Transdermal drug delivery of insulin with ultradeformable carriers.

CHn Pharmacokinet 2003, 42(5):461-474.

6. Cevc G: Lipid vesicles and other colloids as drug carriers on the skin. Adv Drug Deliv Rev 2004, 56(5):675-711. 7. Cevc G, Blume G: New, highly efficient formulation of diclofenac for the topical, transdermal administration in ultradeformable drug carriers, Transfersomes. Biochim Biophys Acta 2001, 1514(2):191-205.

8. Cevc G, Blume G: Biological activity and characteristics of triamcinolone- acetonide formulated with the self-regulating drug carriers, Transfersomes.

Biochim Biophys Acta 2003, 1614(2):156-164.

9. Cevc G, Blume G: Hydrocortisone and dexamethasone in very deformable drug carriers have increased biological potency, prolonged effect, and reduced therapeutic dosage. Biochim Biophys Acta 2004, 1663(l-2):61-73. 10. Cevc G, Gebauer D, Stieber J, Schatzlein A, Blume G: Ultraflexible vesicles, Transfersomes, have an extremely low pore penetration resistance and transport therapeutic amounts of insulin across the intact mammalian skin. Biochim Biophys Acta 1998, 1368(2):201-215.

11. Cevc G, Schatzlein A, Richardsen H: Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements. Biochim Biophys Acta 2002, 1564(l):21-30.

12. Geusens B, Lambert J, De Smedt SC, Buyens K, Sanders NN, GeIe MV: Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes. J Control Release 2009, 133(3) :214-

220.

13. Sioud M, Sorensen DR: Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 2003, 312(4): 1220-1225.

14. Kirby C, Clarke J, Gregoriadis G: Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro. Biochem J 1980,

186(2):591-598.

15. Sloan KB, Beall HD, Taylor HE, Getz JJ, Villaneuva R, Nipper R, Smith K: Skin penetration enhancement of mefenamic acid by ethanol and 1,8-cineole can be explained by the 'pull' effect. IntJPharm 1998, 171:185-193. 16. Kim A, Lee EH, Choi SH, Kim CK: In vitro and in vivo transfection efficiency of a novel ultradeformable cationic liposome. Biomaterials 2004, 25(2):305-313. 17. Lopez-Pinto JM, Gonzalez-Rodriguez ML, Rabasco AM: Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes. Int J Pharm 2005,

298(1):1-12. 18. Trotta M, Peira E, Debernardi F, Gallarate M: Elastic liposomes for skin delivery of dipotassium glycyrrhizinate. IntJPharm 2002, 241(2):319-327. 19. Naeyaert JM, Eller M, Gordon PR, Park HY, Gilchrest BA: Pigment content of cultured human melanocytes does not correlate with tyrosinase message level.

Br J Dermatol 1991, 125(4):297-303. 20. Van GeIe M, Geusens B, Schmitt AM, Aguilar L, Lambert J: Knockdown of

Myosin Va isoforms by RNAi as a Tool to Block Melanosome Transport in

Primary Human Melanocytes. J Invest Dermatol 2008, doi:10.1038/jid.2008.100.

21. Becker W: Advanced Time-Correlated Single Photon Counting Techniques. Springer 2005.

22. Becker, Hickl: DCS-120 Confocal Scanning FLIM Systems. Available on wwwbecker-hicklcom .

23. Vorauer-Uhl K, Wagner A, Katinger H: Long term stability of rh-Cu/Zn- superoxide dismutase (SOD)-liposomes prepared by the cross-flow injection technique following International Conference on Harmonisation (ICH)- guidelines. Eur J Pharm Biopharm 2002, 54(l):83-87.

24. Konig K, Riemann I: High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed 0/tf 2003, 8(3):432-439.

25. Niesner RA, Andresen V, Gunzer M: Intravital two-photon microscopy: focus on speed and time resolved imaging modalities. Immunol Rev 2008, 221:7-25.

Claims

1. A cationic liposome comprising:

-a 5-7:1-2:1-2 w:w:w ratio of: 1) a cationic lipid, 2) a surfactant and 3) cholesterol or a derivate thereof, respectively, and -20-35 % alcohol.

2. A cationic liposome according to claim 1, wherein said cationic lipid is l,2-dioleoyl-3- trimethylammonium propane (DOTAP).

3. A cationic liposome according to any of claims 1-2, wherein said surfactant is sodium cholate (NaChol).

4. A cationic liposome according to any of claims 1-3, wherein said alcohol is ethanol.

5. A cationic liposome according to any of claims 1-4, comprising:

-a 6:1 :1 w:w:w ratio of: cationic lipid l,2-dioleoyl-3-trimethylammonium propane (DOTAP), sodium cholate (NaChol) and cholesterol, respectively, and -30 % ethanol.

6. Use of a cationic liposome according to any of claims 1-5 to vehicle high molecular weight compounds into tissues.

7. Use according to claim 6, wherein said high molecular weight compound is a siRNA, a miRNA, an anti-miRNA, dsRNA or mRNA.

8. Use according to claim 7, wherein said siRNA is targeted against tyrosinase, myosin Va Exon F, melanophilin, protease-activated receptor-2 (PAR-2), IL12B, DEFB4, TNF-alpha or mir-203.

9. Use according to any of claims 6-8, wherein said tissue is skin.

10. Use according to claim 9, wherein said high molecular weight compounds are delivered into epidermal keratinocytes, melanocytes or epithial cells of the skin.

11. A process to produce a cationic liposome according to any of claims 1-5 comprising: -dissolving a cationic lipid such as DOTAP and cholesterol or a derivate thereof in an organic solvent,

-dissolving a surfactant such as sodiumcholate in an organic solvent, -synthesizing 'cationic lipid/cholesterol or a derivate thereof/surfactant' cationic liposomes in a 5-7:1-2:1-2 w:w:w ratio, preferably a 6:1 : 1 w:w:w ratio, using dissolved cationic lipid and cholesterol or a derivate thereof, and, dissolved surfactant such a sodiumcholate obtained in the preceding steps, -removing traces of solvent, and

-hydrate said 'cationic lipid/ cholesterol or a derivate thereof /surfactant' cationic liposomes with a mixture of 20-35 % alcohol, or, hydrate said liposomes with a higher than 35 % alcohol and subsequent dilution/dialysis in order to finally obtain 20-35% alcohol.