Inclusion complex of biotinylated podophyllotoxin with hydroxypropyl-β-cyclodextrin: enhanced solubility, tumor selectivity and efficacy

Podophyllotoxin (PPT) is a natural lignan antitumor compound, but its clinical application is significantly limited by poor water solubility and lack of selectivity. To address these issues, PPT was conjugated with a tumor-targeting ligand, biotin (Bio), through different linkers to prepare three PPT-Bio conjugates: podophyllotoxin-biotin (PB), podophyllotoxin-6-aminohexanoic acid-biotin (PACAB), and podophyllotoxin-3,3’-dithiobispropionic acid-ethanolamine-biotin (PSSB). These conjugates were subsequently complexed with HP-β-CD to form the corresponding inclusion complexes, aiming to improve both tumor-targeting capability and water solubility of PPT. Among them, the PSSB/HP-β-CD complex exhibited 5.68-fold increase in solubility compared to free PPT. In vitro release studies demonstrated that the PSSB/HP-β-CD inclusion complex exhibited an accelerated release under both acidic and reductive environment. Cellular uptake studies revealed significantly higher internalization of all three inclusion complexes in Hela cells compared to free PPT. The targeting specificity of biotin was confirmed by competitive inhibition of the biotin receptor (BR). Further endocytosis pathway studies showed that the internalization process was energy-dependent and involved multiple endocytic pathways. Cytotoxicity assays indicated that all compounds and complexes exhibited concentration-dependent cytotoxicity, with the three inclusion complexes showing significantly stronger antitumor activity than free PPT. Notably, the PSSB/HP-β-CD complex demonstrated the highest potency (IC50 =17.03μM). Additionally, these complexes exhibited low toxicity on normal cells and good hemocompatibility. Preliminary pharmacokinetic evaluation indicated favorable tissue distribution of the biotinylated prodrug conjugates. Therefore, the biotinylated prodrug inclusion complexes, particularly PSSB/HP‑β‑CD, demonstrate great potential as targeted antitumor agents with improved solubility and efficacy.