Nanospheres utilized in targeted drug delivery systems have seen much attention, however it is difficult to detect the nanospheres in an in-vivo test due to their nanoscale in size. This is a crucial step in targeted drug delivery to show the nanosphere being concentrated at the spot of interest. Nanospheres developed by oil in oil (o/o) emulsion technique have the advantage of encapsulating molecules, such as 1,6-Diphenyl-1,3,5-hexatriene (DPH), without damages and chemical alterations. In current study, DPH was encapsulated into a nanosphere as a fluorescing tracer to visualize the nanospheres trafficking in a mouse model of squamous cell carcinoma (SCC). The SCC tumors were established on nude mice. 0.5 ml of a 0.3 mg/ml solution of fluorcescent nanospheres were subcutaneously injected around the tumor. The injections of the drug carrier system were repeated at 2-day intervals till the sacrifice of the tumor-bearing animals on day 10. The tumors were retrieved for frozen and paraffin-embedded histological preparation. Fluorsescent microscopy was used to image the frozen sections, and compared with H&E stained sections. The fluorescence nanoparticles were easily identifiable under fluorescent microscopy, while typical histology images were unable to detect the nanospheres. The data suggest that fluorescent nanoparticles can be used to identify the location or localization of the nanospheres in an in-vivo environment in a simple and straightforward method that aids in characterization of targeted drug delivery.

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