The skin penetration model. Skin biopsies were placed in agar in cell inlets. The inlets were placed in supplemented DMEM medium and incubated at 37°C and 5% CO 2 

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... of elastic vesicles into both human and mice skin in vitro and to study interactions between vesicles and skin (11,13). The results of those studies showed the location of intact elastic vesicles in the upper three layers of the stratum corneum and that the intercellular lipid layer was disturbed by the application of elastic vesicles. TEM has also been used to demonstrate that metallic nanoparticles with a size of 10 nm were able to penetrate the skin (14) and to study changes in ultrastructure in the skin after drug delivery by iontophoresis (15,16). The objectives of the current study were to investigate the effect of Posintro TM nanoparticles on human skin penetration of model compounds and to elucidate the mechanism by which this takes place. The results can be used to evaluate their potential as a transcutaneous delivery system. Quillaja saponaria A (Quil A) is a saponin derivative and was obtained from Brenntag Biosector, Denmark, and mega-10 (decanoyl- N -methylglucamide) was from Bachem, Germany. Cholesterol (>98%), dioleoyl phosphatidylethanol- amine (>99%), and 1-palmitoyl-2-oleoyl- sn -glycero-3-phos- phocholine (POPC, >99%) were obtained from Avanti Polar Lipids, AL, USA. Methyl nicotinate, DC-cholesterol ( ∼ 95%), sucrose (>99.5%), 4 ′ -6-diamidino-2-phenylindole (DAPI), and agarose were purchased from Sigma-Aldrich, Denmark. Acridine (2,8-bis(dimethylamino)-10-dodecyl-acri- dinium bromide) was obtained from ACROS, Belgium. Dulbecco ’ s modi fi ed Eagle ’ s medium (DMEM) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 500 ng/ml amphotericin B, and 10% ( v / v ) fetal calf serum were all purchased from BioWhittaker, Cambrex. Fluorescent mounting medium was purchased from DakoCytomation, Denmark. Tissue-TEK OCT was purchased from Sakura Finetek, Denmark. Phosphate-buffered saline (PBS) pH 7.4 was prepared from distilled, deionized water (Milli-Q Water system, Millipore, Axeb, Denmark). Three percent ( w / v ) glutaraldehyde, glycide ether 100 (Epon), 1% ( w / v ) aqueous osmium tetroxide, methylene blue, and ethanol were all purchased from Roth GmbH, Germany, while copper grids and formvar solution were from Plano, Germany. Uranyl acetate was obtained from SERVA, Germany, and propylene oxide and cacodylate buffer pH 7.4 were from Polysciences Europe, Germany, and lead citrate was from Merck, Germany. Posintro TM nanoparticles were prepared by the standard dialysis method fi rst described by Höglund et al. in 1989 (17). The particles used for the CLSM studies were slightly modi fi ed, as the fl uorophore (acridine) was incorporated into the nanoparticles, which were subsequently named acridine – Posintro TM . Brie fl y, cholesterol, DC-cholesterol, and POPC (and acridine) were dissolved in 20% ( w / v ) mega-10 in Milli- Q water. The saponin, Quil A, was dissolved in Milli-Q water, and appropriate amounts of the components were mixed and stirred for 2 h at 37°C. The theoretical weight ratio before dialysis between Quil A/POPC/total cholesterol was 5:1:1, and the total lipid concentration was 0.2% ( w / v ). The amount of DC-cholesterol was either 25% of the total cholesterol or 50% of the total cholesterol. Where nothing else is men- tioned, the amount of DC-cholesterol is 25% of the total cholesterol. The mixture was dialyzed (Slide-A-Lyzer® cassette, 10,000 MW cutoff, Thermo Scienti fi c, Denmark) against PBS for 48 h at 26°C and 48 h at 5°C with buffer changes every 24 h in order to remove the detergent. Acridine – Posintro TM particles were ultracentrifuged (UC) in a continuous sucrose gradient prepared by the freeze – thaw method described previously (18). Brie fl y, 4.5 ml 25% ( w / v ) sucrose solution was placed in an ultraclear centrifuge tube (Beckman Coulter, CA, USA) and frozen at − 20°C. The day before centrifugation, the tube was placed at 4°C for slow thawing. Acridine – Posintro TM suspension of 0.7 ml was placed on top of the sucrose gradient and centrifuged for 4 h at 15°C and 50,000 rpm (112,000× g ). The visible band was collected by gradient harvesting (Auto-Densi Flow®, MO, USA) and dialyzed against PBS at room temperature for 48 h with two buffer changes in order to remove the sucrose. Size distribution was measured by dynamic light scattering (DLS), and zeta potential was measured by laser Doppler electrophoresis (LDE; Zetasizer Nano ZS, Malvern, UK) in small volume cuvettes before and after UC. TEM images were recorded of the prepared Posintro TM and acridine – Posintro TM by negative staining. Brie fl y, Posintro TM was placed on a formvar-coated copper grid and negatively stained using a fi ltered aqueous solution of 1% ( w / v ) uranyl acetate. Fresh human abdominal skin was obtained after cosmetic surgery from Frederiksborg Klinikken, Denmark (approved by the local ethical committee, no. H-Ø-2001-1-39G) and processed the same day. Subcutaneous fat was removed, and skin biopsies with a diameter of 8 mm were punched out. The skin penetration model (Fig. 1) was prepared by pouring heated liquid agar into the bottom of cell culture inserts (Becton Dickinson Biosciences, Denmark). The inserts were transferred to deep six-well plates (Becton Dickinson Biosciences, Denmark) and incubated at 4°C for 1 h. The skin biopsies were placed in the hardened agar with the stratum corneum facing upwards, and 11 ml of supplemented DMEM medium was added to each well. The skin biopsies were cultured and maintained at 37°C and 5% CO 2 in a humidi fi ed atmosphere. CLSM was used to visualize the penetration of acridine – Posintro TM nanoparticles by localization of the fl uorescent label (acridine) in human skin. Samples of 20 μ l were applied on the surface of the skin biopsies, which were subsequently incubated as described above for 24, 48, or 72 h. The applied samples were composed of 0.05 mg/ml acridine in 5% ( w / v ) mega-10 in PBS and acridine – Posintro TM nanoparticles in PBS, corresponding to 0.05 mg/ml acridine. After 24, 48, or 72 h of exposure, skin biopsies were removed from the penetration model setup, washed in PBS, and quick-frozen in Tissue-Tek. Subsequently, the frozen samples were cut in cryo-dermatome (Leica CM1100, Germany) into 4- μ m-thin sections, which were incubated at room temperature for 30 min with DAPI nuclear stain (1 μg/ml in PBS). The sections were mounted in fl uorescent mounting medium and visualized on a CLSM (Leica TCS SPE, Germany). The excitation wavelength was 405 nm for DAPI and 488 nm for acridine. The emission was visualized in the ranges 410 – 490 and 500 – 535 nm for DAPI and acridine, respectively. Each treatment was repeated three times, and representative images obtained with the same settings were recorded. The potential of the Posintro TM nanoparticles to penetrate into the stratum corneum upon cutaneous application was examined using TEM. Samples of 20 μl Posintro TM were applied to human skin in vitro , either in the absence or presence of a cross-linked polyvinyl-pyrrolidone-based hydrogel containing 65% water (Coloplast A/S, Denmark, patent no. WO/2004/031253) covering the skin surface. The hydrogel was applied in order to study the effect of hydration. After 24 and 72 h of incubation under the conditions described above, skin biopsies exposed to Posintro TM with or without hydration were cut in half and divided into seven small skin pieces of approximately 1 mm 3 . The small skin pieces were fi xed in 3% ( v / v ) glutaraldehyde in Milli-Q water overnight at 4°C, followed by fi xation in 1% ( w / v ) osmium tetroxide in 0.1 M cacodylate buffer pH 7.4 for 1 h at room temperature. After fi xation, the small pieces were dehydrated in a series of graded ethanol solutions (50%, 70%, 80%, 96%, and 100% ( v / v )) and in propylene oxide with gradually increasing amounts of Epon. The tissue samples were embedded in Epon overnight at 60°C for polymerization. Semithin sections (1 μm) were cut and stained with methylene blue for visualization with light microscopy to verify the integrity of the skin. Several ultrathin (approximately 90 nm) sections were cut on a microtome (Ultracut E, Reichert-Jung, Austria), collected on formvar-coated grids and counterstained with uranyl acetate and lead citrate. The sections were examined in a Philips TEM (EM400, Eindhoven, The Netherlands). Each treatment was repeated three times, and representative micrographs were collected. Human abdominal or mammary skin obtained from cosmetic surgery (same approval as described previously) was frozen at − 20°C immediately after receipt and removal of the subcutaneous fat. Prior to the experiment, the skin was thawed and hydrated overnight in PBS at 5°C. The skin was then placed in a 60°C water bath for 1 min, after which the epidermis was carefully separated from the dermis and placed on top of a cellulose membrane in Franz diffusion cells. The donor solution consisted of 10 mg/ml of the model substance, methyl nicotinate, in the absence or presence of Posintro TM or the different components of the nanoparticles in similar concentrations as theoretically present in the intact nanoparticles. Methyl-nicotinate-containing donor solutions com- prised: PBS, 10% ( w / v ) Tween 80 in PBS, Posintro TM with 25% ( w / w ) DC-cholesterol in PBS, Posintro TM with 50% ( w / w ) DC-cholesterol in PBS, Quil A in PBS, cholesterol in PBS with 10% ( w / v ) Tween 80, DC-cholesterol in PBS with 10% ( w / v ) Tween 80, POPC in PBS with 10% ( w / v ) Tween 80, and a mixture of the above-mentioned components in PBS with 10% ( w / v ) Tween 80. A test volume of 200 μl was applied to a skin diffusion area of 0.2 cm 2 , and the 3 ml stirred receptor phase (PBS) was kept at 37°C, maintaining a skin surface temperature of 32°C. Three-hundred-microliter samples were withdrawn from the receptor compartment after 1/2, 1, 3, 5, 8, 12, and 24 h and immediately replaced by 300 μ l PBS. For each test solution, eight replicates were performed on skin from eight different skin ...