Preparation, characterization and biological evaluation of β-cyclodextrin-biotin conjugate based podophyllotoxin complex

Abstract

Podophyllotoxin is a natural occurring aryltetralin lignin with pronounced cytotoxic activity. However, its clinical application for cancer treatment has been blocked due to its poor water solubility and selectivity. In this work, biotin as a tumor specific ligand was coupled with β-cyclodextrin and the resulting biotin modified β-cyclodextrin was used to complex with podophyllotoxin to improve its aqueous solubility and tumor selectivity. The solubility of β-cyclodextrin was greatly enhanced(>16 times) by conjugating with biotin. podophyllotoxin/ mono-6-biotin-amino-6-deoxy-β-cyclodextrin inclusion complex was prepared by freeze-drying method and the complex behavior between mono-6-biotin-amino-6-deoxy-β-cyclodextrin and podophyllotoxin was studied by water solubility, phase solubility, Job's plot, UV spectroscopy, Proton Nuclear Magnetic Resonance, Rotating-frame Overhauser Effect Spectroscopy, Powder X-ray diffraction and Scanning electron microscopy. The solubility of podophyllotoxin/ mono-6-biotin-amino-6-deoxy-β-cyclodextrin complex was greatly improved(9 times) compared with Podophyllotoxin. The stability constant of podophyllotoxin/ mono-6-biotin-amino-6-deoxy-β-cyclodextrin complex (K_s= 415.29 M^-1) was 3.2 times that of podophyllotoxin/β-cyclodextrin complex. The possible inclusion mode of podophyllotoxin/mono-6-biotin-amino-6-deoxy-β-cyclodextrin complex was inferred from the Proton Nuclear Magnetic Resonance and Rotating-frame Overhauser Effect Spectroscopy. The cellular uptake study showed that the introduction of biotin increased the cellular uptake of rhodamine-B/mono-6-biotin-amino-6-deoxy-β-cyclodextrin complex. Moreover, cell cytotoxicity study showed that the antitumor activity of podophyllotoxin/ mono-6-biotin-amino-6-deoxy-β-cyclodextrin complex was more potent than podophyllotoxin/β-cyclodextrin complex and free podophyllotoxin. The superior water solubility and enhanced cytotoxicity suggested that the mono-6-biotin-amino-6-deoxy-β-cyclodextrin associated inclusion complex might be a potential and promising delivery system for hydrophobic chemotherapeutics such as podophyllotoxin.

Keywords: Biotin; Complex; Podophyllotoxin; Solubility; β-cyclodextrins.