SATA was dissolved in N-N-dimethyl formamide in a ratio of 1:100

SATA was dissolved in N-N-dimethyl formamide in a ratio of 1:100. The SATA was employed for thiolation, which is necessary for antibodies to crosslink with the maleimide group of the DSPE-PEG2000-maleimide lipid. The SATA solution was mixed with the antibody solution in a molar ratio of 8:1 SATA:antibody and incubated for 45 minutes at room temperature during continuous rotation. Unbound SATA was removed, according to the manufacture protocol by using a 50kDa Vivaspin 6 ultrafiltration

device (GE-Healthcare, 28-9323-18). The protein concentration Inhibitors,research,lifescience,medical of the antibodies was determined by UV spectroscopy (Implen NanoPhotometer). In order for the SATA to crosslink with the maleimide groups, the sulfhydryl groups were deacetylated by mixing the SATA/antibody solution with Inhibitors,research,lifescience,medical hydroxylamine solution (0.5M hydroxylamine HCl; 0.5 HEPES M HEPES; 25mM EDTA) and incubated for one hour at room temperature before mixing the SATA:antibody solution with Inhibitors,research,lifescience,medical liposomes. Finally, the conjugation was performed by mixing the liposomes with the deacetylated SATA:antibody

solution in a molar ratio of ratio of 1:1000 for DSPE-PEG2000-maleimide:antibody and incubated for 2 hours at room temperature followed by incubation on a rotator at 4°C overnight. Unbound antibody and self-aggregated liposomes were separated from immunoliposomes by gel filtration chromatography

using a 4B sepharose gel. Inhibitors,research,lifescience,medical Mean particle size of the various liposomes was determined by dynamic light scattering and the zeta potential by laser Doppler electrophoresis using a Zetasizer Nano ZS (Malvern). Determination of particle size using the Zetasizer Nano ZS generates a Z-average value of mean Inhibitors,research,lifescience,medical particle size, polydispersity of the size distribution, and the mean size of individual peaks present in the particle suspension. All measurements were performed on four separate samples and data was analyzed using Malvern Zetasizer Software v.6.2. The concentrations of the conjugated antibodies were determined using the RC DC Protein Assay (BioRad, Cat. No. 500-0121). A standard curve was prepared consisting of five dilutions ranging from 0.2mg/mL mafosfamide to 1.5mg/mL nonimmune IgG from human serum in HEPES buffer. Liposome samples were diluted 1:2 in HEPES buffer to ensure that the samples were within range of the standard curve. All standards and samples were prepared in duplicate. Absorbance was read at 750nm using a spectrophotometer (Thermo Fischer Scientific, Genesys 10 UV-Vis Scanner) using disposable semimicropolystyrene cuvettes (Sarstedt, Germany). The antibody concentration of the various liposome samples was calculated from the plotted standard curve.

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