Preparation of SAA-loaded liposomes and IGF1R-targeted Lipo/SAA

The liposomes were prepared by ammonium sulfate gradients loading method. Briefly, HSPC 14.17 ​mg, CHEMS 4.87 ​mg, DSPE-PEG₂₀₀₀ 3.37 ​mg and DSPE-PEG₂₀₀₀-Mal 0.87 ​mg were dissolved and well mixed in 0.5 ​mL absolute ethanol. The mixture was evaporated to produce lipid film at 65 ​°C in a water bath and nitrogen atmosphere, then a 4 ​mL solution of 250 ​mM was added into the lipid film and stirred for 30 ​min. The liposome suspension was placed in a glass bottle in an ice bath and sonicated with a probe-type sonicator at 300 ​W power (sonic & Materials, Inc., 20 ​kHz) for 5 ​min. The liposomes were placed in 0.9% saline and then dialyzed overnight. After dialysis, the liposome suspension was incubated with SAA (1.80 ​mg) for 15 ​min and then was ultrafiltered and finally eluted three times with PBS, culminating in the production of SAA-loaded liposomes (Lipo/SAA). The obtained Lipo/SAA was stored at 4 ​°C.

Traut's reagent was used for the thiolation of IGF1R antibody. IGF1R antibody was diluted to a final concentration of 0.2 ​mg/mL with PBS. IGF1R antibody and 20-times molar excess of Traut's reagent were mixed, and the reaction proceeded at room temperature for 1 ​h under dark condition and in a nitrogen atmosphere. Residual reagent was removed by ultrafiltration centrifuge. Conjugations of the antibody to the liposomes were performed by incubation of thiolated IGF1R antibody and Lipo/SAA at room temperature for 24 ​h. The end product of IGF1R antibody-conjugated Lipo/SAA (IGF1R-targeted Lipo/SAA) was obtained. The product was then centrifuged at 3600 ​rpm for 30 ​min to remove unconjugated antibody.