Development of a Poly(2-ethyl-2-oxazoline)-Based Fluorescence Imaging Probe Targeting the Folate Receptor in Tumor Tissues

Poly(2-ethyl-2-oxazoline) (PEtOx) is a water-soluble and biocompatible polymer. In this study, we developed a cancer-targeted fluorescence imaging probe based on PEtOx by conjugating both cancer-targeting moieties and fluorescent dyes to secondary amino groups introduced by hydrolyzing the acyl groups of PEtOx. Due to the overexpression of folate receptors in tumors such as ovarian cancers, we selected folic acid (FA) as the cancer-targeting moiety in this study. After partial hydrolysis of propionyl groups in PEtOx (50 kDa) by refluxing with hydrochloric acid, the FA derivative and fluorescence dye (indocyanine green [ICG] derivative) were sequentially reacted to obtain FA-PEtOx-ICG. The hydrolysis ratio of PEtOx was approximately 8%, and the number of FA and ICG derivatives introduced into PEtOx was approximately 11 and 5, respectively. The cellular uptake of FA-PEtOx-ICG by SKOV3 (human ovarian cancer cells) overexpressing folate receptors was evaluated by fluorescence microscopy. FA-PEtOx-ICG was highly internalized by SKOV3 cells compared to control PEtOx-ICG. Moreover, the cellular uptake was significantly suppressed by the co-addition of excess FA, suggesting the specific binding of FA-PEtOx-ICG to folate receptors. For in vivo evaluation, an animal model of peritoneal metastases was prepared by intraperitoneal injection of green fluorescent protein (GFP)-transfected SKOV3 cells into female nude mice, and the mice were fed a folate-deficient diet. The submillimeter peritoneal metastasis foci (GFP-positive) were detected by FA-PEtOx-ICG with spectral fluorescence imaging. Furthermore, the fluorescence signals of FA-PEtOx-ICG in SKOV3 tumors were significantly reduced when mice were fed a folate-containing diet. These results indicate the specific binding of FA-PEtOx-ICG to the folate receptors in vivo.