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Nasopharyngeal carcinoma is a poorly differentiated upper respiratory tract cancer that highly expresses human folate receptors (hFR). Binding of folate to hFR triggers endocytosis. The folate was conjugated into adenosine 5'-monophosphate (AMP) by 1,6-hexanediamine linkages. After reverse HPLC to reach 93% purity, the folate-AMP, which can only be used for transcription initiation but not for chain extension, was incorporated into the 5'-end of bacteriophage phi29 motor pRNA. A 16:1 ratio of folate-AMP to ATP in transcription resulted in more than 60% of the pRNA containing folate. A pRNA with a 5'-overhang is needed to enhance the accessibility of the 5' folate for specific receptor binding. Utilizing the engineered left/right interlocking loops, polyvalent dimeric pRNA nanoparticles were constructed using RNA nanotechnology to carry folate, a detection marker, and siRNA targeting at an antiapoptosis factor. The chimeric pRNAs were processed into ds-siRNA by Dicer. Incubation of nasopharyngeal epidermal carcinoma (KB) cells with the dimer resulted in its entry into cancer cells, and the subsequent silencing of the target gene. Such a protein-free RNA nanoparticle with undetectable antigenicity has a potential for repeated long-term administration for nasopharyngeal carcinoma as the effectiveness and specificity were confirmed by ex vivo delivery in the animal trial.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells