Conjugation of hydrophobic drugs to motile pRNA 4WJ nanoparticles for spontaneous tumor targeting and undetectable toxicity

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Abstract
Chemotherapeutics are widely used in cancer treatments, but their toxicity, bioavailability and solubility present challenges. RNA nanotechnology has emerged as a promising modality for targeted delivery of chemotherapeutics. Structurally, RNA is thermostable, while conformationally it is dynamic and flexible. RNA's unique deformability and motility lead to rapid spontaneous tumor accumulation and glomerular excretion, thus fast body clearance, while its anionic charge and favorable small size prevent accumulation in vital organs, resulting in undetectable toxicity. We developed branched 4-way junction (4WJ) nanoparticles that were stable with a melting temperature >80 °C, even when conjugated with 24 drugs per 4WJ. Each 4WJ RNA component strand can conjugate six molecules of hydrophobic chemotherapeutic drugs, such as camptothecin, paclitaxel and SN-38. Thus, each 4WJ carries a total of 24 drug molecules spaced to prevent aggregation. RNA conjugation improved paclitaxel water solubility 32,000-fold. This protocol describes the construction of 4WJ RNA drug complexes for cancer therapy. Specific procedures include the modification of chemical drugs, conjugation of multiple prodrug molecules to each synthesized RNA component strand, assembly of RNA nanoparticles and their purification and characterization. Prodrugs are conjugated to RNA nanoparticles via efficient click chemistry, creating an ester linker that is cleaved by esterases in tumor tissues or cells, allowing the prodrugs to return back to their original structures and chemistry upon delivery and release, minimizing toxicity. Inclusion of tumor targeting ligands demonstrated specific delivery of high payload chemotherapeutics to tumors, controlled release of chemical drugs and strong tumor inhibition.

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