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To establish the biodistribution profile of the PLGA nanoparticles with dual surface modifications of PEG and folic acid (FA) in mice xenografted with MDA-MB-231 human breast cancer cells with high expression of folate receptor (FR); and to illustrate that the modified nanoparticles can target the loaded indocyanine green (ICG) to the tumor with high FR expression.ICG-loaded nanoparticles were prepared with PLGA (non-modified nanoparticles, NM-NP) or mPEG-PLGA and FA-PLGA (dual modified nanoparticles, DM-NP). Biodistribution of the ICG-loaded nanoparticles (1.25 mg/kg) after i.v. injection was investigated on athymic mice transplanted with MDA-MB-231 tumor.ICG concentration in plasma from the DM-NP group was significantly (p < 0.05) higher than the NM-NP group from 90 min to the end of the study (12 h). After 4 h, the drug concentration in the tumor tissue from the DM-NP started to be significantly (p < 0.05) higher than the NM-NP until 12 h. Compared to the NM-NP, the DM-NP increased the AUC0–12 h in plasma by 245% and the AUC0–12 h in tumor by 194%, while decreased the AUC0–12 h in liver by 13%.The accumulation of DM-NP into the tumor was significantly higher than NM-NP due to the long circulation and FR-mediated uptake.