Lipophilic prodrug conjugates allow facile and rapid synthesis of high-loading capacity liposomes without the need for post-assembly purification.

Dihydropyridopyrazoles are simplified synthetic analogues of podophyllotoxin that can effectively mimic its molecular scaffold and act as potent mitotic spindle poisons in dividing cancer cells. However, despite nanomolar potencies and ease of synthetic preparation, further clinical development of these promising anticancer agents is hampered due to their poor aqueous solubility. In this article, we developed a prodrug strategy that enables incorporation of dihydropyridopyrazoles into liposome bilayers to overcome the solubility issues. The active drug was covalently connected to either myristic or palmitic acid anchor via carboxylesterase hydrolyzable linkage. The resulting prodrugs were self-assembled into liposome bilayers from hydrated lipid films using ultrasound without the need for post-assembly purification. The average particle size of the prodrug-loaded liposomes was about 90 nm. The prodrug incorporation was verified by differential scanning calorimetry, spectrophotometry and gel filtration reaching maximum at 0.3 and 0.35 prodrug/lipid molar ratios for myristic and palmitic conjugates, respectively. However, the ratio of 0.2 was used in the particle size and biological activity experiments to maintain long-term stability of the prodrug-loaded liposomes against phase separation during storage. Antiproliferative activity was tested against HeLa and Jurkat cancer cell lines in vitro showing that the liposomal prodrug retained antitubulin activity of the parent drug and induced apoptosis-mediated cancer cell death. Overall, the established data provide a powerful platform for further clinical development of dihydropyridopyrazoles using liposomes as the drug delivery system.