The brightness of Z sectioning images indicated that 5\CF\7CrpR permeated more deeply than the Rho\DOPE

The brightness of Z sectioning images indicated that 5\CF\7CrpR permeated more deeply than the Rho\DOPE. and hydrophobic bioactives. Hydrophilic T Combretastatin A4 cell epitope peptide was efficiently Combretastatin A4 delivered through mouse skin using the S/O nanodispersion system and lowered antigen\specific IgE levels in pollinosis model mice. Addition of the hydrophobic adju1vant R848 significantly lowered the antibody secretion and shifted the Th1/Th2\balance toward Th1\type immunity in the model mice, showing the potential to alleviate Japanese cedar pollinosis. and purified following our previously described method.25 A solution of the peptide in Milli\Q water was stored at 4?C until use. A water in oil (W/O) emulsion was prepared from an aqueous answer of 7CrpR (0.5 mg/ml, 2 ml) with or without R848 (0.25 mg/ml) and a cyclohexane solution of L\195 (12.5 mg/ml, 4 ml) using a polytron homogenizer PT2500E (Kinematica AG, Luzern, Switzerland). The W/O emulsion was flash\frozen in liquid nitrogen and the water and cyclohexane were removed by lyophilization for 24 h with a lyophilizer FDU\1200 (Eyela, Tokyo, Japan). The resulting solid paste was dispersed in IPM (1 ml) to yield a S/O nanodispersion made up of 1 mg/ml 7CrpR. Alternatively, the surfactant\protein complex was Tmem10 dispersed in IPM (1 ml) made up of R848 (0.5 mg/ml) to prepare a S/O nanodispersion containing R848 on the surface of the particles. Labeled S/O nanodispersions were prepare from 7CrpR labeled with Cy3 using a kit from GE healthcare (Buckinghamshire, UK). The size distributions of the S/O nanodispersions were analyzed using a Zetasizer Nano ZS light scattering instrument (Malvern, Worcestershire, UK). 3.2. Drug release test The drug release test was performed using custom\fabricated Franz\type diffusion cells with an effective diffusion area of 0.785 cm2 and a receptor volume of 5 ml. A polycarbonate film (Whatman Nuclepore Track\Etch Membrane, 0.1 m; GE healthcare) was set on a cell, and the receptor compartment filled with a phosphate buffered saline (PBS) answer made up of 1% sodium dodecyl sulfate. A S/O nanodispersion (200 l) was placed on the membrane and the cell was incubated for 48 h at 37?C. Samples (200 l) were extracted from the receptor compartment at 0, 3, 6, 24, and 48 h, and replaced with the same volume of fresh media. The peptide concentration was measured with a fluorescence spectrometer LS\55 (PerkinElmer, Waltham, MA) at 540 nm (ex)/570 nm (em). R848 concentration in the receptor chamber was analyzed by HPLC Combretastatin A4 and UV absorption (320 nm) on a Inertsil ODS\3 C18, 5 m, 4.6 250 mm column (GL Science, Tokyo, Japan), with a linear gradient from 95% water made up of 0.1% TFA to 90% acetonitrile containing 0.1% TFA over 40 min (flow rate: 1.0 ml/min). 3.3. Histology S/O nanodispersions made up of 5\CF\7crpR (1 mg/ml in IPM) and Rho\DOPE (50 g/ml in IPM) were prepared as previously described.29 Mouse ear auricles were collected from ddY mice (7\week\old, female, Kyudo) and stored at ?80?C until use. Tissue papers impregnated with S/O nanodispersions (25 l) were placed onto the dorsal skin of defrosted mouse auricles, tightly sealed in place with adhesive tape to model occlusive patches and incubated at 32?C for 24 h. After removal of patches, the ear pieces were washed thoroughly with 99% ethanol followed by Milli\Q water, and placed onto glass slides. Fluorescence images were obtained with a confocal laser scanning microscope LSM700 (Carl Zeiss, Oberkochen, Germany), by excitation at 488 nm (5\CF) and at 555 nm (Rhodamine). A series of Z sectioning images was obtained at 5 m intervals. The images showing 5\CF (green) and Rhodamine (red) were exported as individual jpeg files (8\bit RGB format, 512 pixels 512 pixels, each). The RGB pixel values of green and red images were converted to brightness values (G and R, respectively), using a software ImageJ without any image processing. 3.4. Sensitization and immunotherapy Mice were sensitized to Cj pollen according to our previous report.25 The Cj pollen extract was dissolved in PBS (100 g/ml). Cj pollen extract in PBS (100 l) and Imject Alum (100 l) were mixed for 30 min and administered to mice by s.c. injection once a week for 3 weeks. Six days after the final s.c. injection, histamine dihydrochloride in PBS answer (2 g/ml) was decreased into each nostril (5 l each). The Cj pollen extract dissolved in PBS was challenged into each nostril (5 l each) for 5 days from the day after the histamine administration. Blood samples were collected 3 days after the final.