Liquid Crystalline Nanodispersions Of Topical Delivery Of Sirna Biology Essay

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The ability of small-interfering RNA 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. Therefore, the present study was aimed to evaluate the potential application of nanodispersions of monoolein (MO) and oleic acid (OA) containing the cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) as topical carrier systems for the delivery of siRNA. Firstly, it was evaluated the influence of different concentrations of PEI or OAM in the liquid crystalline phase formed by MO and MO/OA-based systems as well as in the physicochemical properties of the dispersions obtained after sonication of such systems. Then, based on the characterization study as well as on the in vitro skin penetration study the systems composed of 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 and investigated with regards to their ability to in vivo deliver siRNA. The results showed that these nanodispersions were effective in optimizing the skin penetration of siRNA as well as the reduction in the levels of the model protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH), without causing skin irritation. Therefore, the present study showed the potential use of liquid crystalline nanodispersions as a carrier to deliver siRNA into skin, suggesting a new siRNA delivery system to be used in gene therapy of many skin disorders.

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

Introduction

RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. Unlike other mRNA targeting strategies, RNAi takes advantage of the physiological gene silencing machinery1.

The discovery of RNAi and the observation that siRNAs largely evade the immune response have opened up new therapeutic opportunities. The potency (IC50 in the picomolar range) and selectivity (single-nucleotide (nt) discrimination) of siRNAs make these inhibitors attractive drug candidates. Clinical trials of siRNAs are currently underway targeting the liver, kidney, lung, eye and skin2.

The skin is a uniquely attractive tissue for investigation of RNAi therapeutic approaches due to its accessibility as well as the fact that there are large numbers of diseases amenable to cutaneous gene mediation3. Many in vitro and in vivo studies have used skin as a route to delivery siRNA in the treatment of melanoma4, rheumatoid arthritis3, wound5, allergic skin diseases such as contact hypersensitivity and atopic dermatitis6, dominant genetic skin including pachyonychia congenital7, alopecia areata8 and psoriasis9.

Therefore, topical delivery of siRNA can strategically modulate local gene expression in a variety of cutaneous disorders while avoiding systemic side effects5. Nevertheless, normal skin (especially the stratum corneum) represents a considerable barrier to topical nucleic acid delivery10, so the clinical use of RNAi has been severely hampered by the lack of delivery of these molecules to target cell populations in vivo due to their instability, inefficient cell entry, and poor pharmacokinetic profile11, being the delivery a key determinant as to whether or not RNAi-based therapeutics will have clinical relevance12.

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 moieties13.

Furthermore, liquid crystalline phases of monoolein, which present interesting properties for topical delivery system, were explored by Lopes et al.14 about their ability 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, suggests the potential applicability of liquid crystalline nanodispersions as a safe and promising strategy for topical delivery of several other macromolecules of dermatological interest14.

Therefore, the present study was aimed to develop and evaluate the potential application of liquid crystalline nanodispersions as topical carrier systems for the delivery of siRNA. Firstly, we rationalized that introduction of polyethylenimine (PEI), a cationic polymer commonly used in gene delivery applications or the cationic lipid oleylamine (OAM) in the liquid crystalline phases could improve the retention of anionic siRNA molecules. Then, in vivo studies using animal model, were carried out aiming to verify the potentiality of liquid crystalline formulations containing PEI or OAM as topical carrier systems for the delivery of siRNA. The ability of the developed carriers to effectively deliver siRNA was investigated considering the parameters skin penetration, skin irritation and capability to knockdown GAPDH protein levels.

Experimental Section

Materials

Monoolein (MO) (Myverol 18-99) was supplied by Quest (Norwich, NY, USA), branched polyethylenimine (PEI) (25 kDa), oleic acid (OA) and oleylamine (OAM) were obtained from Sigma (St. Louis, MO, USA), poloxamer 407 (Pluronic F127) was obtained from BASF (Florham Park, NJ, USA), the glyceraldehyde-3-phosphate dehydrogenase (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).

Preparation of the formulations

Formulations 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 with the cationic lipid OAM. For that, MO was melted (42ï‚°C), and OA, PEI or OAM added under stirring. Immediately thereafter, the aqueous phase was added, and the resulting formulation was allowed to equilibrate at room temperature for 24 h.

For the obtainment of the dispersions, the gel with excess water was vortex-mixed and sonicated (22.5 kHz) in ice-bath for 2 min. Then, the siRNA was incorporated into the dispersions in the final concentration of 2.5 µM and left for 30 min at room temperature.

Characterization of the formulations

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 liquid crystalline gel. The mean diameter, particle size distribution and the zeta potential of the obtained dispersions were determined using a 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° with a He-Ne laser, and the zeta potential was determined by the standard capillary electrophoresis cell of Zetasizer Nano ZS at 25 °C. All the average values were performed with the data from three separate measurements.

Screening of in vitro skin penetration of siRNA

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 based on the previously characterization and added with siRNA-FAM. For that, siRNA-FAM was incorporated into the nanodispersions in the final concentration of 10 µM and left for 30 min at room temperature. These systems were evaluated with regards to their ability to penetrate the siRNA through the skin in order to select the most promising.

The penetration of siRNA in the skin was assessed in an in vitro model of porcine ear skin, as previously described14. The skin from the outer surface of a freshly excised porcine ear was carefully dissected and dermatomized, stored at -20C, and used within a 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.

Two hundred microlitres of the dispersions incorporated with siRNA-FAM were applied to the surface of the stratum corneum. At 12 h post-application, skin surfaces were carefully cleaned, and the diffusion area of skin samples was frozen by using acetone at -30C, embedded in Tissue-Tek 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 PBS or with nuclease-free water solution containing siRNA-FAM (at 10 µM) or with the unloaded dispersions were used as control.

In vivo nanodispersions functionality

Formulations

Based on the previous results of characterization and in vitro skin penetration, the systems selected for the in vivo functionality studies were 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 dispersion was added with GAPDH siRNA-FAM at 10 M, mixed gently and incubated for 30 min at room temperature before use.

Different control groups were included: PBS-treated group, both nanodispersions with scrambled siRNA-FAM-treated groups and nuclease-free water solution containing GAPDH siRNA-FAM-treated group (referred in the present study as naked GAPDH siRNA-FAM).

Animals and experimental protocol

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 use. All experiments were conducted in accordance with 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 nï‚° 09.1.118.53.2).

Randomly chosen animals were divided into groups of 3-5 and topically treated on the dorsal surface (area 2 cm2) with 100 L of the different formulations described above. At 24 and 48 h post-application, the animals were killed with an overdose of carbon dioxide, the surface of the skin cleaned and used for the following studies.

In vivo skin penetration

Part of the hairless skin, treated as described above, was frozen by using acetone at -30C, embedded in Tissue-Tek 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 and AxioVision software.

Skin irritation

The skin sections obtained as described in section "in vivo skin penetration" were mounted on glass slides, stained with haematoxylin and eosin (H&E) and examined with light microscopy (Axioplan 2 Image Pol microscope, Carl Zeiss, Oberkochen, Germany).

Skin irritation of the studied formulations was evaluated according to the established endpoints of infiltration of inflammatory cells and epidermis thickening using the ImageJ Program (NIH-National Institute of Health).

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 ï‚°C.

Protein content was determined (Bio-Rad Laboratories, CA) and equal amounts of protein (50 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)15, 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.

Statistical Analysis

Data were statistically analyzed by one-way ANOVA, followed by Bonferroni's multiple comparison t-test. The level of significance was set at p < 0.05.

Results and Discussion

siRNA is a double-stranded molecule which can be designed to hybridize with 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, skin cancer, etc. However, it is difficult to deliver siRNA into the skin by conventional methods based on passive diffusion because siRNA is a hydrophilic macromolecule16.

Skin delivery techniques based on ballistic methods17,18, injection19,20, ultrasound and iontophoresis21 successfully deliver nucleic acids to skin cells. Direct topical application has also been used with mixed results10.

Therefore, the development of suitable delivery vehicle capable of increasing skin penetration of siRNA is of great interest in order that RNAi-based therapeutics may have clinical relevance. In this report, we proposed the potential use of liquid crystalline nanodispersions as a topical carrier of siRNA.

Liquid crystalline phases of MO, such as reverse hexagonal and cubic phases, present interesting properties for a topical delivery system, and hence have been studied to deliver compounds of pharmaceutical interest to the skin and mucosa. These liquid crystalline phases are (i) bioadhesive, (ii) present a permeation enhancer as the structure forming lipid (MO), and (iii) present ability to incorporate compounds independently of their solubility, to protect them from physical and enzymatic degradation, and to sustain their delivery.14 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,22 we first studied whether the cationic polymer PEI or the cationic lipid OAM affects 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% have changed the liquid crystalline phase from cubic to hexagonal, both with excess aqueous phase. But for the formulation composed of MO/OA/Aqueous phase at 8:2:90 (w/w/w), the hexagonal phase gel containing excess aqueous phase was maintained after 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 phases obtained by the different systems after incorporation of PEI or OAM. The fan-like texture, typical of the hexagonal phase, can be observed in Figure 1E (hexagonal phase with excess water, composed of MO/OA/OAM/Aqueous phase at 8/2/2/88, w/w/w/w).

Lopes et al.14 have demonstrated that by dispersing the liquid crystalline phase formed by MO and OA in excess water in the presence of poloxamer 407, an nanodispersion in aqueous medium can be obtained. It was demonstrated that the dispersed nanoparticles retain the internal structure of the bulk phase and presents some advantages as a topical delivery system in comparison with the bulk gel: increase skin uptake of drugs, less skin irritability, higher fluidity, and others14.

In the present study the sonication of the different liquid crystalline gel with excess aqueous phase resulted in the obtainment of an anisotropic dispersion as demonstrated by Figure 1F. Previous studies using small-angle X-ray diffraction have demonstrated that the internal structure of the bulk phase was maintained after the sonication process. Besides this, the dispersed particles were nanometric (referred as nanodispersions) as determined by light scattering and maintained their diameter after the addition of siRNA as shown on Table 1.

The values of zeta potential ranged from -0.04  0.35 to 25.10  1.78 mV for system described as (A) in Table 1, 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 the final concentration of PEI or OAM from 0.25 to 5%, w/w. These alterations in surface charge is especially relevant design criteria for ensuring carrier uptake, e.g. it is generally accepted that nanoparticles bearing a positive surface charge have a facilitate uptake by electrostatic interactions with negatively charged cell membranes13.

Then, based on the characterization study four different liquid crystalline nanodispersions were selected to be evaluated with regards to their potential as topical carrier systems for the delivery of siRNA. Table 2 describes the physicochemical properties of the selected systems after incorporation of siRNA at 10 M.

The skin penetration of siRNA-FAM incorporated in the previously selected systems was assessed in vitro (using porcine ear skin mounted in a Franz diffusion cell) and visualized by fluorescence microscopy at 12 h post-application. The results (data not shown) demonstrated that all the studied systems increased the in vitro penetration of siRNA-FAM when compared with the control formulation, a nuclease-free water solution containing siRNA-FAM. However, the MO/OA-based systems were more effective than the MO-based one. The increased skin penetration of siRNA observed by application of both MO and MO/OA-based systems is probably due to the action of MO as penetration enhancer. MO, the main structural lipid of the nanodispersion, due to its ability to remove skin ceramides and increase lipid fluidity in the stratum corneum is a penetration enhancer that has already been demonstrated to increase skin penetration of peptides and other compounds23. Nevertheless, the differences observed in the in vitro skin penetration of siRNA-FAM might rely on the difference of constituents of both systems. The presence of OA in the system might influence the skin permeability, and thus, results in higher siRNA-FAM penetration in deeper skin layers and through this tissue. OA is also a known penetration enhancer, and it has already been shown to increase the skin absorption of several drugs, including peptides22.

Therefore, the nanodispersions composed of 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 for the further experiments. Firstly, the in vivo skin penetration of FAM labeled siRNA was assessed by fluorescence microscope visualization at 24 and 48 h post-application of the selected nanodispersions and the results are shown in Figure 2.

Because the skin presents autofluorescence, skin sections treated with only PBS were used as control and as expected untreated skin presented a very weak autofluorescence (Figures 2B). When naked siRNA (nuclease free water solution containing siRNA-FAM at 10 µM) was applied on the skin, FAM-labeled siRNA was observed only at specific points in the surface of the skin (Figure 2C). But the incorporation of siRNA on MO/OA-based systems containing both PEI or OAM increased its skin penetration when compared to the siRNA not complexed with a carrier (naked siRNA). Besides this, it can be noted that the siRNA-FAM following the treatment with nanodispersion containing OAM was predominantly confined to the superficial layers of epidermis and to hair follicles; on the other hand, treatment of the skin with the MO/OA/PEI nanodispersion resulted in fluorescent staining of stratum corneum and viable epidermis (Figures 2D and E, respectively). Finally, there was an increase in the penetration of siRNA-FAM with increased time for both nanodispersions.

Being a potential intermediary in the membrane fusion process, the hexagonal phase may facilitate the fusion of the nanodispersed system with 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 can form a depot in the skin surface and appendages, resulting in a prolonged release of the incorporated compound14.

Considering that the potentiality of this topical formulation to be used as delivery system should be evaluated not only in terms of carrier capacity and percutaneous drug absorption, but also in terms of its tolerability and toxicity24, together with the fact that the cationic polymer PEI as well as the cationic lipid OAM may induce adverse effects depending on the employed concentration, it is particularly important to consider potential skin irritation resulting from topical application of the proposed systems.

Photomicrographs illustrating skin tissues of animals 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 (Figure 3). The same results were obtained for the animals treated with naked GADPH-siRNA-FAM and also for the nanodispersions with scrambled siRNA-treated groups and for all the groups examined 48 h post-application of the investigated systems (data not shown). Furthermore, as demonstrated by Figure 4, no significant difference was observed in the epidermis thickness after the 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 both nanodispersions, in the conditions employed in the present study, did not cause skin irritation.

Finally, the ability of the carriers to effectively deliver siRNA was investigated in knockdown experiments of a model protein, namely glyceraldehyde 3-phosphate dehydrogenase (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 5F) when compared with the naked GAPDH siRNA (Figure 5B), which showed minimal protein silencing effect. For the system composed of MO/OA/OAM/Aqueous phase, only a weak protein-specific suppression can be observed (Figure 5D), which might be explained by the smaller skin penetration of this formulation. The results also demonstrated that for the PBS control group (Figure 5A), as well as for the negative control siRNA nanodispersions (Figures 5C and E) no knockdown effect was observed.

A variety of synthetically and biologically-derived polymers have been investigated for use as nucleic acid carriers including poly(dimethylaminoethyl methacrylate) (pDMAEMA), poly(L-lysine), polyethylenimine (PEI), and chitosan13. PEI has been one of the prototypes for non-viral polymeric gene carriers and has been the most efficient gene carrier with the highest cationic charge density potential25. It is an attractive carrier for intracellular 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 polymer26.

Lu et al.( 2009) suggested that although most of the cellular uptake of siRNA lipoplexes is via endocytic pathways, these pathways do not appear to play a significant role in the delivery of siRNA that mediate RNAi. Instead, a minor but rapid pathway, probably mediated by fusion between siRNA lipoplexes and the plasma membrane, is responsible for the functional siRNA delivery27.

Therefore, the promising results obtained, which suggest the effectiveness of the developed nanodispersions as a siRNA delivery system, might be due to: (i) The properties of the liquid crystalline phase formed. The hexagonal phase may facilitate the fusion of the systems particles with the stratum corneum and deeper skin layers; may protect the siRNA from physical and enzymatic degradation and may form a depot in the skin which results in a prolonged release of the incorporated compound; (ii) The combined effects of the different components of the formulation, that have important characteristics to improve skin penetration, cell uptake and consequently the functionality of siRNA.

Conclusion

The present work brings novelty in proposing a delivery system based in liquid crystalline phase for skin siRNA delivery. To our knowledge, this is the first report to demonstrate the potentiality of such systems as siRNA carriers on the skin. This approach can be considered highly advantageous in the therapy of skin disorders, especially those one where inflammatory processes mainly take place.

Liquid crystalline nanodispersions of MO and OA incorporated with the cationic polymer PEI or with the cationic lipid OAM showed to be non-irritant and a promising strategy to deliver siRNA to the skin. Further, additional studies will be addressed to verify if the changes in proposed systems composition, dosing frequency, siRNA concentration, and other parameters could optimize efficacy of siRNA in targeting skin disease-specific gene expression.

Acknowledgments

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. 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).

Figure Captions

Figure 1. Photomicrographs using polarized light microscopy. Hexagonal phase gel composed of: (A) MO/PEI/Aqueous phase at 7:3:90 (w/w/w), (B) MO/OAM/Aqueous phase at 9.5:0.5:90 (w/w/w), (C) MO/OA/PEI/Aqueous phase at 8:2:2:88 (w/w/w/w) and (D and E) MO/OA/OAM/Aqueous phase at 8:2:2:88 (w/w/w/w). (F) Dispersion obtained after sonication (composed of MO/PEI/Aqueous phase at 7:3:90, w/w/w) Panels (A-D) the objective used was 20X and for panel (E and F) 32X.

Figure 2. Microscopic evaluation of hairless mice skin after treatment for 24 and 48 h with different formulations. (A) Light microscopy of skin treated with PBS (H&E). Fluorescence microscopy of skin section treated with: (B) PBS solution; (C) naked siRNA-FAM (nuclease-free water solution containing siRNA-FAM at 10 M); (D) MO/OA/OAM/Aqueous phase at 8:2:2:88 w/w/w/w incorporating siRNA-FAM (10 M) and (E) MO/OA/PEI/Aqueous phase at 8:2:5:85 w/w/w/w incorporating siRNA-FAM (10 M). Sections were visualized using FAM filter through a 20X objective. Three batches of each formulation were tested, and representative pictures are shown.

Figure 3. Photomicrographs of hairless mice skin sections of animals treated with: (A) PBS; (B) MO/OA/OAM/Aqueous phase at 8:2:2:88 w/w/w/w incorporating GAPDH siRNA-FAM (10 M) and (C) MO/OA/PEI/Aqueous phase 8:2:5:85 w/w/w/w incorporating GAPDH siRNA-FAM (10 M) for 24 h. Sections were stained with H&E and visualized by conventional light microscope through a 100X objective.

Figure 4. Epidermis thickness of hairless mice skin sections at 24 h (A) and 48 h (B) post-application of the different formulations. Both negative control siRNA and GAPDH siRNA were added at a final concentration of 10 M. Results are represented by means  S.E.M. (n=3-5). No statistical significant difference was detected. Statistical analysis was performed by one-way ANOVA followed by Bonferroni's test of multiple comparisons (p < 0.05).

Figure 5. Silencing of GAPDH protein in hairless mice skin at 24 and 48 h post-application of the following systems: (A) PBS solution; (B) naked negative control siRNA (nuclease-free water solution containing negative control siRNA at 10 M); (C) naked GAPDH siRNA (nuclease-free water solution containing GAPDH siRNA at 10 M); (D), MO/OA/OAM/Aqueous phase at 8:2:2:88 w/w/w/w incorporating negative control siRNA (10 M), (E) MO/OA/OAM/Aqueous phase at 8:2:2:88 w/w/w/w incorporating GAPDH siRNA (10 M); (F) MO/OA/PEI/Aqueous phase at 8:2:5:85 w/w/w/w incorporating negative control siRNA (10 M) and (G) MO/OA/PEI/Aqueous phase at 8:2:5:85 w/w/w/w incorporating GAPDH siRNA (10 M). The data are representative of three independent experiments, 3-5 animals per group.

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