MTO vant in a ratio Added ratio of 1:1. After Kinesin Spindle Protein brief vortex, the suspension for 1030 minutes at 60 was incubated, followed by gentle shaking at room temperature for 12 h non-encapsulated MTO was removed from liposomes by dialysis using a dialysis membrane Spectra / Por. Message technology insertion into the peptide 1 Wed 19 on the surface chemical Of liposomes after step 2, the PIT resulting conjugate has recently been described. The PIT method was prior to performing vesicles LRP ligands were as follows optimized prepared: Cholesterol fluorescently labeled three anchor molecule was dissolved in distilled water st and was mixed with the liposome suspension to obtain mixtures of 1 to 5 mol% of 3, based on the total lipids. This mixture was incubated at 25 to 70 15, 30, 60 min or overnight with stirring at 700 rpm. 3 was carried out free liposomes Gr Enausschlusschromatographie using Antimetabolites Sepharose Centri spin separated 10 columns.
The fraction of liposomes in the void volume was collected for the analysis of fluorescence and lipids. Liposomes, the ligand for in vitro and in vivo in a similar way, using the following optimum: 2 mol% Chol, 19 Wed 1-peptide with 713 mol teaspoon mixed with liposomes and overnight 28th RAF Signaling Pathway Liposomes for in vivo experiments and by filter of 0.4 m extrusion prior to application. Determination of Liposomengr E characterization of the vesicles was carried out by a dynamic measurement of light scattering analyzer with a Gr E of submicron particles N5. The samples were immersed in a frozen glow discharged carbon lattice with L Chern in liquid ethane with a Vitrobot. The temperature was set to 22, relative humidity of 100%. The grids were vitrified in a Gatan cryo transmission medium and mounted in an electron microscope Tecnai F20 at 200 kV. The samples were imaged at 170 using conditions of low dose imaging. The pictures were taken with a mag AREA 25,000 and recorded a negative focus of 4 or 6 m on a CCD camera 2kx2k. Cell culture Madin Darby Canine Kidney cells were grown in Dulbecco’s modified Eagle’s medium cultured , erg Complements with 1% L glutamine, 7 g / l NaHCO 3, 1 g / L glucose and 10% heat-inactivated f Fetal K calf serum. The cells in the brain of the mouse endothelial cells were cultured in DMEM with 4.5 g / L glucose and 10% FCS. Glioblastoma cells and Doxorubicin human breast cancer cells were grown in RPMI 1640 with 10% FCS. The absorption cell uptake of liposomes was determined as described recently.
In brief: 3105 cells / well, grown in 24-well microtiter plate were treated with 600 l of calcein-loaded liposomes in serum-free media for different ZEITR trees at 37th Nonspecific uptake was incubated under Similar conditions after incubation for 15 determined at 4. Cellular Re calcein maximum concentration was determined by measuring the fluorescence after cell lysis with a buffer Ripa. All experiments were performed in triplicate in three independent Ngigen performed experiments. Transcytosis transcytosis as recently described as follows: 1105 MDCK cells, grown on durchl ssigen cell culture insert of a Transwell system were used after obtain a dense monolayer. The cells in the apical chamber of the transwell system were incubated with 400 l deficientDMEM nutritious containing liposomes at 37 for 24 h. Concentration in the calcein.