64Cu-labeled folate-conjugated shell cross-linked nanoparticles for tumor imaging and radiotherapy: synthesis, radiolabeling, and biologic evaluation.

Unlabelled: Long-circulating nanoparticles functionalized with ligands for receptors overexpressed by tumor cells have promising applications for active and passive tumor targeting. The purpose of this study was to evaluate 64Cu-radiolabeled folate-conjugated shell cross-linked nanoparticles (SCKs) as candidate agents to shuttle radionuclides and drugs into tumors overexpressing the folate receptor (FR).

Methods: SCKs were obtained by cross-linking the shell of micelles obtained from amphiphilic diblock copolymers. SCKs were then functionalized with folate, fluorescein thiosemicarbazide (FTSC), and 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA). The specific interaction of SCK-folate with the FR was investigated on KB cells. The biodistributions of 64Cu-TETA-SCK and 64Cu-TETA-SCK-folate were evaluated in athymic mice bearing small-size KB cell xenografts (10-100 mg), whereas the intratumor distributions were investigated by autoradiography in 0.3- to 0.6-g KB cell xenografts.

Results: A global solution-state functionalization strategy has been introduced for attaching optimum numbers of targeting and imaging agents onto the SCKs for increasing the efficiency of interaction with cell-surface receptors. Epifluorescence microscopy confirmed the specific interaction of FTSC-SCK-folate with the FR in vitro. 64Cu labeling of TETA-SCKs led to the radiolabeled compounds with 15%-20% yield and >95% radiochemical purity. The biodistribution results demonstrated high accumulation of 64Cu-labeled SCKs in organs of the reticuloendothelial system (RES) (56.0 +/- 7.1 %ID/g and 45.7 +/- 3.5 %ID/g [percentage injected dose per gram] in liver at 10 min after injection for folated and nonfolated SCKs, respectively) and a prolonged blood circulation. No increase of SCK tumor uptake deriving from folate conjugation was observed (5.9 +/- 2.8 %ID/g and 6.0 +/- 1.9 %ID/g at 4 h after injection for folated and nonfolated SCKs, respectively). However, tumor accumulation was higher in small-size tumors, where competitive block of SCK-folate uptake with excess folate was observed. Autoradiography results confirmed the extravasation of radiolabeled SCKs in vascularized areas of the tumor, whereas no diffusion was observed in necrotic regions.

Conclusion: Despite high RES uptake, the evaluated 64Cu-labeled SCKs exhibited long circulation in blood and were able to passively accumulate in tumors. Furthermore, SCK-folate uptake was competitively blocked by excess folate in small-size solid tumors, suggesting interaction with the FR. For these reasons, functionalized SCKs are promising drug-delivery agents for imaging and therapy of early-stage solid tumors.