BRAIN RESEARCH ELSEVIER Brain Research 695 (1995) 231-236 Short communication Interaction of cationic liposomes with cells of electrically active neuronal networks in culture Hassan M.E. Azzazy a,1, Keelung Hong b, Ming-Chi Wu c, Guenter W. Gross a,, a Department of Biological Sciences, PO Box 5218, Unit,ersity of North Texas, Denton, TX 76203, USA h Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA c Department of Biochemistry andMolecularBiology, UniL,ersity of North Texas Health Science Center, Fort Worth, TX 7610Z USA Accepted 31 May 1995 Abstract Incubation of rhodamine-labeled cationic liposomes with mature murine spinal cultures results in strong fluorescence that is evenly distributed on somata and neurites of neurons in 7 different cultures. Staining of the glial carpet is minimal. Rhodamine-labeled dextran, encapsulated in liposomes, also stains neurons. Electron microscope data show external attachment and intact internalization of liposomes. Spontaneous electrical bursting activity is altered but not lost after incubation. Keywords: Liposome endocytosis; Neuronal network; Multielectrode array; Cell culture Many important cellular mechanisms can be determined only by the manipulation of intracellular processes. This often requires probes or genetic vectors that normally do not cross the cell membrane. To traverse this barrier, several methods have been investigated. One of these utilizes liposomes (phospholipid vesicles) as vehicles for transfer of genetic materials, antibodies, or bioactive drugs [5,9,19]. Liposomes are biodegradable, hold the promise of limited vehicle toxicity, and have a long shelf life. Early experiments showed that negatively charged lipo- somes exhibited a higher transfer efficiency than neutral liposomes and were used in cell cultures as well as in vivo [24]. However difficulties, such as low nucleic acid encap- sulation efficiency and DNA degradation due to sonication during preparation, were encountered with these methods [17]. Further improvements were achieved by the introduc- tion of cationic liposomes [6,8,16,22]. These vehicles inter- act spontaneously with negatively charged nucleic acids, leading to entrapment of DNA molecules. This avoids problems associated with the generation of a high concen- tration of free liposomes in the transfection mixture. A1- * Corresponding author. Fax: (1) (817) 565-4135. E-mail: gross@nervous.cnns.unt.edu I Present address: Clinical Chemistry Lab., Univ. of Maryland Medi- cal Center, Baltimore, MD 21201, USA. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993( 95)0071 0-5 though cationic liposomes were considered sensitive to serum proteins and were used initially only for in vitro studies [8], Brigham et al., [4] and Stewart et al. [23] detected exogenous DNA in tissues of mice injected with DNA-containing cationic liposomes. Thus, it appears that this vehicle may be used to transfect cells in vivo as well as in vitro. At present, cationic liposomes are considered efficient non-viral transfection vehicles for animal cells [7,28,29]. Cationic liposomes may have several mechanisms of interaction with cells. Ultrastructural studies have shown endocytosis via smooth, 'coatless' vesicle pinocytosis [21] as well as coated pit endocytosis [24]. Zhou and Huang [28] also reported that complexes of DNA and cationic liposomes containing lipopolylysine were taken up by cells via endocytosis. However, other laboratories proposed that cationic liposomes fuse with cell surfaces [6,22]. Finally at least one report suggested 'hemifusion' of liposomes to cells and substantial mixing of lipids between vesicle and target membranes [22]. Straubinger [24] considered an understanding of the mechanism of interaction "critical to improving the efficiency by which liposomes deliver their contents to cells". However, the complexity of liposome/target tissue interactions makes each experimen- tal system somewhat unique and predictions of interaction mechanisms and transfer efficiency are still very difficult. It has been our goal to develop a gentle liposome