Accumulation of free or complexed IGF-1 in selected
organs was measured at three time points. The aim of this study was to compare accumulation and pharmacokinetics of free and complexed IGF-1 to the brain in order to evaluate the therapeutic potential for INCL. 2. Materials and Methods 2.1. Radiolabeling IGF-1 and IGFPB-3 were offered by Insmed Incorporation (Richmond, VA, USA). IGF-1 and IGFBP-3 were radiolabeled with 125I with Iodo-Gen method. selleck Briefly, precoated iodination tubes (Pierce) were rinsed with 1 mL of phosphate-buffered saline, pH 7.4, (PBS), and 125I (22 MBq, Map Medicals, Finland) was incubated at room temperature for 10 minutes. After incubation IGF-1 (200μg) or IGFBP-3 (200μg) Inhibitors,research,lifescience,medical was added and the reaction mixture was further incubated for 15 minutes at RT. The solution was purified using HiTrap Sephadex column (GE Healthcare) using PBS as a mobile phase at flow rate 1 mL/min. Labeling efficiency was 29–43% with specific activity of 0.22MBq/nmol Inhibitors,research,lifescience,medical and 0.37MBq/nmol for the IGF-1 and 11–17% for the IGFBP-3 with specific activity of 0.29MBq/nmol and 0.33MBq/nmol, respectively. 2.2. Nanoparticles
Thermally hydrocarbonized Inhibitors,research,lifescience,medical mesoporous silicon nanoparticles (THCPSi) were prepared as described earlier . Nanoparticles (800μg) were mixed with radiolabelled IGF-1 (200μg) in 2mL of 10mM HEPES pH 7.4. The suspension was mixed at RT for two hours sonicating every 30 minutes. 94% of IGF-1 was incorporated in the particles and the loading degree was 23.5% (w/w). The in vitro release was studied using fresh mouse plasma diluted 1:2 in PBS. Nanoparticles were mixed with diluted plasma and incubated at +37°C.
Inhibitors,research,lifescience,medical A sample of the particles was centrifuged immediately and at 20, 60, 120, and 240 minutes time points (n = 3/time point). Radioactivity of the supernatant was measured by Gamma Counter (1277 Gammamaster automatic Gamma Counter, LKB Wallac, Finland). 2.3. Animals A homozygous knockout mouse model Cln1-/-, showing overall neurologic features highly similar to the clinical Inhibitors,research,lifescience,medical symptoms of INCL, was used in this study . The Cln1-/- mice were backcrossed to C57BL/6 for more than 10 generations, and the congeneity was confirmed with the Mouse Medium below Density SNP Panel (Illumina). The genotypes of the mice were determined by PCR of DNA from tail biopsies. Total of 36 nine-week-old (n = 3/group) female mice were used for the biodistribution studies. The mice received chow and water ad libitum. All animal procedures were performed according to protocols approved by the ethical boards for animal experimentation of the National Public Health Institute and University of Helsinki, as well as National Animal Experiment Board of Regional State Administrative Agencies of Southern Finland (Agreement number 09-06737), and all experiments were done in accordance with good practice of handling laboratory animals and genetically modified organisms. 2.4.