Biol. Chem., Vol. 380, pp. 647– 652, June 1999 · Copyright © by Walter de Gruyter · Berlin · New York Dequalinium TM Vesicles Form Stable Complexes with Plasmid DNA which Are Protected from DNase Attack Jürgen Lasch 1, *, Axel Meye 2 , Helge Taubert 2 , Regine Koelsch 1 , Jasadee Mansa-ard 1 and Volkmar Weissig 3 1 Institute of Physiological Chemistry, 2 Institute of Pathology, Martin-Luther-University Halle, D-06097 Halle/Saale, Germany 3 Northeastern University, Bouve College of Pharmacy, Boston, MA 02115, USA * Corresponding author Upon sonication, the antimicrobial and antineoplastic compound dequalinium TM forms vesicles (DQAsomes, Weissig et al., 1998). Dequalinium TM (1,1-(1,10-de- camethylene-bis-[aminoquinaldinium])-chloride) was shown to be a fluorophore with an emission maximum at 366 nm. Addition of DNA results in a characteristic quenching of its intrinsic fluorescence. After density gradient centrifugation a band of dequalinium TM (DQA) tightly associated with DNA is located between the DNA and DQA bands. DQA/DNA-complexes contain- ing plasmid DNA at a molar ratio of DQA/DNA 6:1 are completely protected against DNase activity. Addition of negatively-charged lipids release intact DNA in the same manner as from cationic lipid/DNA complexes. As regards biological effects, DQAsomes show a dif- ferential cytotoxicity for normal and sarcoma cell lines. In vitro incubation with fluorescein-labeled oligodeoxynucleotides (5 -fluorescein-[GATC] 5 ) showed an increased uptake of the tagged oligodeoxynu- cleotide if complexed with dequalinium. We hypothesize that the DQA/DNA complexes are well-suited for ‘DQAsomal gene transfer’ in vitro and in vivo. Noteworthy, they display an intrinsic antitumor activity manifested by differential cytotoxicity for nor- mal and sarcoma cells. Key words: Antineoplastic DNA vehicle / Bolaamphiphile / Dequalinium / DNA / DNase-attack protection / DQA-complexes / DQAsomal gene transfer. Introduction The divalent cation dequalinium TM (DQA) is a potent and selective blocker of Ca 2+ -activated, small conductance K + -channels (Castle et al., 1993), an anti-microbial agent and has remarkable cancerostatic activities (Weiss et al., 1987). DQA is a bolaform molecule with two delocalized posi- tive charges separated by a lipophilic spacer (Figure 1), i.e.a single chain synthetic bolaamphiphile. It accumulates in mitochondria (Bodden et al., 1986, Weiss et al., 1987) like other lipophilic cations as a result of the relatively high electrical potential (–160 to –180 mV) across the mitochondrial membrane and strongly inhibits mitochondrial F1-ATPase and protein kinase C (Roten- berg et al., 1990; Moriyama et al., 1995). There is, however, still no consensus about the molecu- lar targets of this compound. Its anticarcinoma activity is based on increased accumulation and retention in malig- nant epithelial cells (Christman et al., 1990). DQA affects proliferation in highly metastatic melanoma cells and their motility and invasion as well (Helige et al., 1992). In addi- tion, it was shown to be more effective and less toxic in inhibiting growth of intraperitoneally implanted mouse carcinomas than other antineoplastic drugs like cisplatin, doxorubicin, 5-fluorouracil (Sweet and Singh, 1995). In line with these pharmacological effects it has some in vitro and in vivo cytotoxicity. Recently, we discovered that liposomal structures can be prepared from DQA, dubbed DQAsomes (Weissig et al., 1998). Here we describe more characteristics of these bolaform molecule vesicles and show that DQAsomes can efficiently bind plasmid DNA and protect it from de- oxyribonuclease-I digestion in vitro. The DQA/DNA com- plexes, which have much in common with the lipoplexes of DNA, were studied as potential DNA transport vehicles. Results Freeze-fracture electron microscopy showed unambigu- ously that dequalinium can form spherical vesicles (Weis- sig et al., 1998). Fig. 1 Chemical Structure of Dequalinium TM . Brought to you by | The University of Auckland Library Authenticated Download Date | 5/17/15 3:12 AM