Nanoparticles Of Liquid Crystalline Phase Enable Skin Delivery Biology Essay

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The ability of small-interfering RNA (siRNA) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for widespread use of this technology in skin is an effective delivery system. The aim of the present study was to evaluate nanodispersed systems based in liquid crystalline phases to deliver siRNA into the skin. The proposed systems present important properties to deliver macromolecules in biological medium, such as they are formed by substances that have absorption enhancing and fusogenic effects and also, the facilitated entrapment by cellular membranes due to their nanosized structure. Cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations and after dispersion in aqueous medium, nanoparticles of liquid crystalline phase were obtained and characterized by their physicochemical properties. Then, in vitro penetration study using diffusion cell and ear pig skin were carried out in order to evaluate the effect of the nanodispersions on the skin penetration of siRNA, and based on the results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo considering the parameters of skin penetration, skin irritation and ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the therapy of skin disorders once they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation.

Keywords: nanoparticles, liquid crystalline phase, small interfering RNA, topical delivery, skin penetration

1. Introduction

RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. RNAi takes advantage of the physiological gene silencing machinery unlike other mRNA targeting strategies [1]. The discovery of RNAi and the observation that siRNAs largely evade the immune response have opened up new therapeutic opportunities. The ability of siRNA to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. Clinical trials of siRNAs are currently underway, targeting the liver, kidney, lung, eye and skin [2, 3].

The skin is a uniquely attractive tissue for the investigation of RNAi therapeutic approaches due to its accessibility and the fact that there are large numbers of diseases amenable to cutaneous gene mediation [4]. Several in vitro and in vivo studies have used skin as a route to deliver siRNA for the treatment of melanoma [5], rheumatoid arthritis [4], wounds [6], allergic skin diseases (such as contact hypersensitivity and atopic dermatitis [7]) and dominant genetic skin conditions including pachyonychia congenita [8], alopecia areata [9] and psoriasis [10]. Therefore, topical delivery of siRNA can strategically modulate local gene expression in a variety of cutaneous disorders while avoiding systemic side effects [6]. Nevertheless, normal skin (especially the stratum corneum) represents a considerable barrier to topical nucleic acid delivery [11], so the clinical use of RNAi has been severely hampered by the lack of delivery systems for these molecules targeting cell populations in vivo due to their instability, inefficient cell entry, and poor pharmacokinetic profile [12]. Thus, carrier systems are required to overcome these barriers and the delivery is a key determinant as to whether or not RNAi-based therapeutics will have clinical relevance [13, 14].

In general, the ideal material for topical delivery of siRNA should be able to (i) bind and condense siRNA; (ii) overcome the barrier function of stratum corneum [15]; (iii) provide protection against degradation; (iv) direct siRNA to target cells; (v) facilitate its intracellular uptake; (vi) escape from endosome trafficking to the lysosome to reach the cell cytoplasm and avoid metabolism; (vii) promote efficient gene silencing [16, 17].

Carriers for siRNA delivery usually consist of cationic polymers, peptides or lipids that form complexes with the nucleic acid, protecting it from nuclease attack and facilitating cell uptake through electrostatic interactions with negatively-charged phospholipid bilayers or through specific targeting moieties [18].

Additionally, lyotropic liquid crystals, which can provide enhanced drug solubility, relative drug protection, and controlled release of drugs, while avoiding substantial side effects, seem to be promising candidates as alternative delivery means for various pharmaceuticals [19]. Liquid crystalline phases of monoolein were explored by Lopes et al. [20] to improve the skin penetration of a model peptide (cyclosporin A). The obtained results, which demonstrated that the developed system increased the skin penetration of cyclosporin A both in vitro and in vivo without causing skin irritation, suggested the potential applicability of liquid crystalline nanodispersions as a safe and promising strategy for topical delivery of several other macromolecules of dermatological interest [20].

Therefore, the present study aimed to evaluate nanoparticles of liquid crystalline phases as non-viral vehicles to improve the skin siRNA delivery. To this end, the cationic polymer polyethylenimine (PEI), commonly used in gene delivery applications, or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based liquid crystalline nanodispersions. The influence of PEI or OAM incorporation in the packing parameter of the lipid MO and consequently in the liquid crystalline phase formed, as well as, in its physicochemical properties was firstly assessed and optimized liquid crystalline nanodispersions were tested for in vitro skin penetration of siRNA. Then, the selected formulations were evaluated in vivo considering the parameters of skin penetration, skin irritation and ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models and compared by their effectively as siRNA delivery systems.

2. Materials and methods

2.1. Materials

Monoolein (Myverol 18-99) was supplied by Quest (Norwich, NY, USA), branched PEI (25 kDa), oleic acid (OA) and OAM were obtained from Sigma (St. Louis, MO, USA), poloxamer 407 (Pluronic F127ƒ’) was obtained from BASF (Florham Park, NJ, USA), the GAPDH antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the siRNAs used were Silencer Negative Control #1 siRNA (Catalogue #AM4635) and Silencer 6-carboxyfluorescein (FAM) GAPDH siRNA (Catalogue #AM4650), both purchased from Ambion (Austin, TX, USA).

2.2. Preparation of test nanodispersions

Systems containing MO or MO/OA in the oil phase and Tris-HCl 0.1 M, pH 6.5 containing 1.5% (w/w) of poloxamer 407 in the aqueous phase were incorporated with different percentages (0.25-5%, w/w) of the cationic polymer PEI or the cationic lipid OAM. For this preparation, MO was melted (42°C), and OA, PEI or OAM were added with stirring. Immediately thereafter, the aqueous phase was added and the resulting formulation was allowed to equilibrate at room temperature for 24 h. To obtain the dispersions, the gel with excess water was vortex-mixed and sonicated (22.5 kHz) in an ice-bath for 2 min. Then, the siRNA was incorporated into the dispersions to a final concentration of 2.5 µM and left for 30 min at room temperature.

2.3. Characterization of test nanodispersions

2.3.1. Polarized Light Microscopy

The developed systems were characterized under a polarized light microscope (Axioplan 2 Image Pol microscope, Carl Zeiss, Oberkochen, Germany) before and after the sonication process used to disperse the bulk gel.

2.3.2. Small Angle X-Ray Scattering (SAXS)

The liquid crystalline structure of the dispersed particles was analyzed by SAXS measurements, performed at the Crystallography Laboratory of the Physics Institute-USP, using a Nanostar equipment (Bruker). The dispersions were placed in cylindrical glass capillaries with internal diameter of 1.5 mm. The scattering curve of a single capillary was used as blank/parasite scattering, which has to be removed from the samples intensities, after transmission correction. The X-ray tube was operated at 40 kV and 30 mA, generating Cu Kα radiation, λ = 0.1540 nm, monochromatized by Göbel mirrors. A point focus beam at the sample holder, around 1mm diameter, was produced by the X-ray optics. The acquisition time of each scattering curve was 3 hours and a two-dimensional filament detector was utilized. With the equipment software the diffraction figure was integrated, in order to generate an intensity file as a function of 2θ, the scattering angle, or q, the scattering vector, q = (4°sin θ)/¬. The distance sample-detector was 650 mm, which provided a q range between 0.13 nm-1 and 3.13 nm-1. The liquid crystalline structures were determined by calculating the values ​​of the interplanar distances, d, from Bragg's law and these distances were associated to the Miller indices of the structure symmetry.

2.3.3. Light scattering

The mean diameter, particle size distribution and the zeta potential of the obtained dispersions were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK). The hydrodynamic diameter of the freshly prepared dispersions was measured at 25 °C with a scattering angle of 173° using a He-Ne laser, and the zeta potential was determined by the standard capillary electrophoresis cell of a Zetasizer Nano ZS at 25 °C. Data represent the average values from three separate measurements.

2.4. Screening of in vitro skin penetration of siRNA

After characterization of the nanodispersions the systems composed of MO/PEI/aqueous phase at 8:2:90 (w/w/w), MO/OAM/aqueous phase at 9.75:0.25:90 (w/w/w), MO/OA/PEI/aqueous phase at 8:2:5:85 (w/w/w) and MO/OA/OAM/aqueous phase at 8:2:2:88 (w/w/w) were selected and added to siRNA-FAM. For this, siRNA-FAM was incorporated into the nanodispersions to a final concentration of 10 µM and left for 30 min at room temperature. These systems were then evaluated with regards to their ability to carry the siRNA into the skin, to select the most promising in this aspect.

The penetration of siRNA into the skin was assessed using in vitro model of porcine ear skin, as previously described [20]. The skin from the outer surface of a freshly excised porcine ear was carefully dissected and dermatomized at 500 µm of thickness, stored at -20°C, and used within one month. On the day of the experiment, the skin was thawed and mounted on a Franz diffusion cell (diffusion area of 1.77 cm2; Hanson Instruments, Chatsworth, CA) with the stratum corneum facing the donor compartment (where the formulation was applied) and the dermis facing the receptor compartment. The latter compartment was filled with PBS solution (pH 7.4). The receptor phase was under constant stirring and maintained at 37 ± 0.5 °C during the experiments.

Two hundred microliters of the nanodispersions containing siRNA-FAM were applied to the surface of the stratum corneum, corresponding to a dose of 10 µM of siRNA-FAM. At 12 h post-application, skin surfaces were carefully cleaned and the diffusion area of the skin samples was frozen using acetone at - 30°C, embedded in Tissue-Tekƒ’ OCT compound (Pelco International, Redding, CA, USA), and sectioned using a cryostat microtome (Leica, Wetzlar, Germany). The skin sections (10 ­m) were mounted on glass slides. The slides were visualized without any additional staining or treatment through a 20X objective, using a Zeiss microscope (Axio Imager A.1, Carl Zeiss, Oberkochen, Germany) equipped with a filter for FAM and AxioVision software. Skin sections treated with 200 µL of PBS, nuclease-free water solution containing siRNA-FAM (dose of 10 µM) or the unloaded nanodispersions were used as controls.

2.5. In vivo efficacy

2.5.1. Preparation of nanodispersions and skin application in animal model

Based on the previous results of characterization and in vitro skin penetration, the systems selected for the in vivo studies were the nanodispersions composed of MO/OA/PEI/aqueous phase at 8:2:5:85 (w/w/w/w) and MO/OA/OAM/aqueous phase at 8:2:2:88 (w/w/w/w). For the following studies, each nanodispersion was combined with GAPDH siRNA-FAM at 10 ­M, mixed gently and incubated for 30 min at room temperature before use. Different control groups were included during the tests: the PBS-treated group, both nanodispersions with scrambled siRNA-FAM-treated groups and nuclease-free water solution containing GAPDH siRNA-FAM-treated group (referred to in the present study as naked GAPDH siRNA-FAM).

In vivo experiments were performed on 3-month-old sex-matched hairless mice of the HRS/J housed in a temperature-controlled room with access to water and food ad libitum until needed. All experiments were conducted in accordance with the National Institutes of Health guidelines for the welfare of experimental animals and with the approval of the Ethics Committee of the Faculty of Pharmaceutical Science of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil (Protocol n° Randomly chosen animals were divided into groups of 3-5 mice and were topically treated on the dorsal surface (area ¾2 cm2) with 100 ­L of the different nanodispersions described above. At 24 and 48 h post-application, the animals were sacrificed with an overdose of carbon dioxide, the surface of the skin was cleaned and the application area was dissected and analyzed concerning the skin penetration, the potential of irritation and the ability to knockdown GAPDH protein levels.

2.5.2. Microscopic study of skin penetration

Part of the hairless skin, treated as described above, was frozen with acetone at - 30°C, embedded in Tissue-Tekƒ’ OCT compound (Pelco International, Redding, CA, USA), and sectioned using a cryostat microtome (Leica, Wetzlar, Germany). The skin sections (8 ­m) were mounted on glass slides. The slides were visualized without any additional staining or treatment through a 20X objective using a Zeiss microscope (Axio Imager A.1, Carl Zeiss, Oberkochen, Germany) equipped with a filter for FAM (λexc = 492 nm and λem = 517 nm) and AxioVision software.

2.5.3. Skin irritation test

The skin sections obtained as described in section 2.5.1.were mounted on glass slides, stained with hematoxylin and eosin (H&E) and examined with a light microscope (Axioplan 2 Image Pol microscope, Carl Zeiss, Oberkochen, Germany). Skin irritation of the studied nanodispersions was evaluated according to the established endpoints of infiltration of inflammatory cells and epidermis thickening using the ImageJ Program (NIH, National Institutes of Health).

2.5.4. Ability to knockdown GAPDH protein levels - Western Blot analysis

Part of the hairless skin (1:4, w/w dilution), treated as described above, was homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2 and 1% protease inhibitor cocktail. Whole homogenates were centrifuged at 12,000 Ã- g for 10 min at 4 °C. Protein content was then determined (Bio-Rad Laboratories, CA) and equal amounts of protein (50 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [21], transferred to nitrocellulose membranes (GE Healthcare UK limited) and immunoblotted with 1:1000 antibody anti-GAPDH (FL-335, sc 25778, Santa Cruz Biotechnology). The membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (GE Healthcare, UK limited) and reactive proteins were visualized using ECL Western blotting detection reagents and analysis system (Amersham Biosciences). To ensure equal protein loading, the membranes were stripped and reprobed with anti-β-actin antibodies.

2.6. Statistical Analysis

Data were statistically analyzed by one-way ANOVA, followed by Bonferroni's multiple comparison t-test using GraphPad Prism® software. Results are expressed as the mean ± SEM and the level of significance was set at p < 0.05.

3. Results and Discussion

siRNA is a double-stranded molecule that can be designed to hybridize to a specific mRNA sequence. siRNA inhibits the translation of numerous genes both in vitro and in vivo. Therefore, topical introduction of siRNA targeted against genes involved in various cutaneous disorders represents a novel therapeutic approach to the treatment of inherited skin diseases, viral infections and skin cancer, among others. However, it is difficult to deliver siRNA into the skin by conventional methods based on passive diffusion because siRNA is a hydrophilic macromolecule [22].

Skin delivery techniques based on ballistic methods [23, 24], injection [25, 26], ultrasound and iontophoresis [27] have had success in the delivery of nucleic acids to skin cells. Direct topical application has also been used with mixed results [11]. Therefore, the development of a suitable delivery vehicle capable of increasing skin penetration of siRNA is of great interest so that RNAi-based therapeutics may have clinical relevance. In this report, we propose and compare the potential use of nanoparticles of liquid crystalline phase as a skin delivery system of siRNA.

3.1. Physicochemical properties of the test nanodispersions

Because parameters such as pH, temperature, and the presence of other compounds in the system can influence the packing parameter of the lipid and consequently the liquid crystalline phase formed [28], we first studied whether the cationic polymer PEI or the cationic lipid OAM affected the structure of the systems composed of MO/aqueous phase or MO/OA/aqueous phase.

For the MO/aqueous phase systems at 10:90 (w/w), the addition of PEI from 2 to 5% and OAM from 0.25 to 2% changed the liquid crystalline structure from cubic to hexagonal, both with excess of an aqueous phase. However, for the system composed of MO/OA/aqueous phase at 8:2:90 (w/w/w), the hexagonal structure of the gel containing excess of aqueous phase was maintained after the addition of different percentages (0.25-5 %) of both PEI and OAM. Figure 1 shows representative images of polarized light microscopy of the liquid crystalline structures obtained by the different systems after incorporation of PEI or OAM. The fan-like texture, typical of the hexagonal structure, can be observed in Figure 1E (hexagonal structure with excess of water, composed of MO/OA/OAM/aqueous phase at 8:2:2:88, w/w/w/w).

Lopes et al. [20] demonstrated that by dispersing the liquid crystalline system formed by MO and OA in excess of water in the presence of poloxamer 407, nanodispersion in aqueous medium could be obtained. It was also demonstrated that the dispersed nanoparticles retained the internal structure of the bulk phase and presented some advantages as a topical delivery system in comparison to bulk gel including increased skin uptake of drugs, less skin irritability, and higher fluidity [20].

In the present study, the sonication of the different liquid crystalline gels with excess of aqueous phase resulted in the formation of an anisotropic dispersion, as demonstrated by Figure 1F. The SAXS study (Table 1) revealed that the MO-based systems incorporated with PEI are not a very well organized single cubic phase, but contains hexagonal phase interstices. The internal structures of the bulk phase of the other systems were characterized as single hexagonal structure. In particular, for those containing PEI and OA, it was observed a more disordered hexagonal structure. The siRNA incorporation did not influence the liquid crystalline order.

Furthermore, the dispersed particles were nanometric (referred to as nanodispersions), as determined by light scattering, and maintained their diameter (around 200 nm) after the addition of siRNA (Table 2).

The values of the zeta potential ranged from -0.04 ± 0.35 to 25.10 ± 1.78 mV for the system described as (A) in Table 2, from 9.15 ± 1.83 to 23.10 ± 0.82 mV for system (B), from -4.87 ± 0.05 to 31.80 ± 0.60 mV for system (C) and from -4.79 ± 0.13 to 23.25 ± 0.75 mV for system (D), with increasing final concentration of PEI or OAM (from 0.25 to 5%, w/w).

Then, based on the characterization study and considering that our aimed was to evaluate the potential of nanodispersions of liquid crystalline phase as a skin delivery system of siRNA, we decided to choose two different nanodispersions incorporated with OAM in which the hexagonal phase was well-established and two different nanodispersions incorporated with PEI, bearing a positive surface charge. Table 3 describes the physicochemical properties of the selected systems after incorporation of siRNA at 10 ­M.

3.2. Screening of in vitro skin penetration of siRNA

By chosen the four different nanodispersions described in Table 3, we could observe the most relevant factors in the development of carrier systems able to overcome the different barriers found for topical delivery of siRNA.

The preliminary studies (data not shown) of in vitro skin penetration using porcine ear skin mounted in a Franz diffusion cell and visualization by fluorescence microscopy have demonstrated that all the nanodispersions showed increased in vitro penetration of siRNA-FAM when compared with the control formulation, a nuclease-free water solution containing siRNA-FAM. However, the presence of OA in the system, a known penetration enhancer [28], influenced the skin permeability, resulting in higher siRNA-FAM penetration into deeper skin layers and through tissue. Consequently, the nanodispersions containing OA were selected for further experiments.

3.3. In vivo efficacy of MO/OA/PEI/siRNA/aqueous phase and MO/OA/OAM/siRNA/aqueous phase nanodispersions

First, the in vivo skin penetration of FAM-labeled siRNA was assessed by fluorescence microscope visualization at 24 h and 48 h post-application of the selected nanodispersions (Figure 2). Because the skin presents autofluorescence, skin sections treated with only PBS were used as control and, as expected, untreated skin presented weak autofluorescence (Figure 2B). When naked siRNA (nuclease free water solution containing siRNA-FAM at 10 µM) was applied to the skin, FAM-labeled siRNA was observed only at specific points on the surface of the skin (Figure 2C). However, the incorporation of siRNA in MO/OA-based nanodispersions containing either OAM or PEI (Figures 2D and E, respectively) increased its skin penetration when compared to siRNA not complexed with a carrier (naked siRNA). There was also an increase in the penetration of siRNA-FAM with increased time for both nanodispersions.

Although not investigated, some speculation might be made about the mechanism by which the different nanodispersions influence siRNA skin penetration. Firstly, the nanosized of the studied systems might be determinant for the observed results, once it has been suggested that nanostructures are more likely to interact with the stratum corneum and enable siRNA to gain access to the living cells within the epidermis and dermis [29]. Additionally, the combined effects of the MO and OA, present in both tested nanodispersions and which have important characteristics to improve skin permeability, might have facilitated the siRNA penetration through the stratum corneum and deeper skin layers. Finally, the hexagonal phase, a potential intermediary in the membrane fusion process, may also have contributed to the fusion of the system particles with the intercellular lipids of the stratum corneum.

Considering that the potential of topical formulations to be used as a delivery system should be evaluated not only in terms of carrier capacity and percutaneous drug absorption but also in terms of its tolerability and toxicity [30], it is particularly important to consider potential skin irritation resulting from topical application of the proposed systems (the cationic polymer PEI and the cationic lipid OAM could induce adverse effects depending on the employed concentration). Photomicrographs illustrating skin tissues of hairless mice subjected to topical application of the nanodispersions or saline are shown in Figure 3. No histopathological alterations in the skin of animals treated with both nanodispersions incorporating GAPDH siRNA-FAM were seen by light microscopy, as compared to the PBS-treated control group. The same results were obtained for the animals treated with naked GADPH-siRNA-FAM, for the nanodispersions with scrambled siRNA-treated groups and for all groups examined 48 h post-application (data not shown). Furthermore, as demonstrated by Figure 4, no significant difference was observed in the epidermal thickness after treatment with the nanodispersions when compared to saline-treated animals. Therefore, by evaluating established endpoints of skin irritation (infiltration of inflammatory cells and epidermis thickening), it was demonstrated that the application of the two nanodispersions, under the conditions employed in the present study, did not cause significant skin irritation.

Finally, the ability of the carriers to effectively deliver siRNA was investigated in knockdown experiments of the model protein GAPDH. Western blot analysis of skin at 24 and 48 h post-application of the MO/OA/PEI/aqueous phase system as carrier of GAPDH siRNA demonstrated marked GAPDH protein reduction (Figure 5G) when compared with the naked GAPDH siRNA (Figure 5C). For the system composed of MO/OA/OAM/aqueous phase, a protein-specific suppression was only observed at 48 h post-treatment (Figure 5E). The results also demonstrated that for the PBS control group (Figure 5A) and for the negative control siRNA nanodispersions (Figures 5D and F), no knockdown effect was observed.

By comparing the in vivo efficacy of the developed nanodispersions important conclusions about the key determinant factors for a potential delivery system can be made:

(i) The use of the prototype for non-viral polymeric gene carriers PEI had a great influence on the observed in vivo efficacy. PEI holds a prominent position among the polycationic polymers used for gene delivery because of its well-established ability to condense nucleic acids via electrostatic interaction between the anionic phosphate in the nucleic acid backbone and the cationic primary, secondary, and tertiary amines of the polymer [31]; its relatively high cell uptake and its capability to escape from the endosomal pathway by the so-called "proton sponge effect", which consists in the protonation of amines in the PEI molecule, leading to osmotic swelling and subsequent burst of the endosomes without the need for an additional endosomolytic agent [32-34].

(ii) The different biological activities of the two nanodispersions studied might be partially determined by their physicochemical surface properties (zeta-potential). Considering that prior to cellular uptake, nanoparticles will interact with components of the cellular membrane and it is generally accepted that nanoparticles bearing a positive surface charge have uptake facilitated by electrostatic interactions with negatively charged cell membranes [18], the highest zeta potential observed for the PEI-based nanodispersion may have had great influence in ensuring carrier uptake. Thereby, to verify this statement in the proposed systems, future studies evaluating the efficacy and safety of the MO/OA-based systems incorporated with higher concentrations of OAM will be conducted.

(iii) Being a potential intermediary in the membrane fusion process, the well-organized hexagonal phase may have facilitated the fusion of the nanodispersed system composed of MO/OA/OAM/siRNA/aqueous with the stratum corneum and deeper skin layers. The hexagonal structure may also improve drug delivery to the skin by protecting the drug from physical and enzymatic degradation and by forming a depot in the skin surface and appendages, resulting in a prolonged release of the incorporated compound [20]. These characteristics, which may eventually translate into different kinetics of siRNA transfection, might explain the observed biological activity of the liquid crystalline nanodispersion only for the group evaluated 48 h post-application of the system and suggest the importance of evaluating this system for longer periods of time.


The present work established optimized nanoparticles of hexagonal phase dispersed in aqueous medium, presenting interesting properties, which enable their use as skin delivery system of siRNA. To our knowledge, this report is the first to demonstrate the potential of such systems as siRNA carriers into the skin and since the in vivo delivery of siRNA still represents a major hurdle for any therapeutic intervention, the nanodispersions studied may represent a promising new possibility for non-viral vehicle. Furthermore, they can be considered highly advantageous in the therapy of skin disorders, once the developed nanodispersions showed to be able to increase the siRNA skin penetration, cell uptake with further enhanced biological activity, without causing skin irritation. Additional studies will be pursued to verify if the changes in the proposed systems composition, dosing frequency, siRNA concentration, and other parameters can optimize the efficacy of siRNA in targeting skin disease-specific gene expression.


We thank Dr. Daniel de Paula (Unicentro, Brazil) for helpful discussions, José O. Del Ciampo for the light scattering analysis and Dimitrius L. Pitol and Nilce O. Wolga for technical assistance. We also thank Prof. Mamie Mizusaki Iyomasa for histological test. This work was supported by "Fundação de Amparo à Pesquisa do Estado de São Paulo" (FAPESP, Brazil, project # 04/09465-7) and "Conselho Nacional de Pesquisa" (CNPq, Brazil). F.T.M.C.Vicentini was the recipient of a FAPESP fellowship (process # 09/00332-8).