ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 348, No. 1, December 1, pp. 199–206, 1997 Article No. BB970385 Maintenance of Transfection Rates and Physical Characterization of Lipid/DNA Complexes after Freeze-Drying and Rehydration Thomas J. Anchordoquy, 1 John F. Carpenter, and David J. Kroll Department of Pharmaceutical Sciences and Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received August 1, 1997, and in revised form September 8, 1997 based diseases (e.g., AIDS) 2 and treat hereditary dis- It is well established that cationic liposomes form eases and cancer (1 – 4). Although viruses are capable of complexes with DNA and effectively transfect cells in extremely efficient gene delivery, problems associated vivo and ex vivo. Lipid/DNA complexes have proven with their immunogenicity and concerns about poten- safe and nonimmunogenic in clinical trials; however, tial infection have led to the development of synthetic they are known to aggregate readily in liquid formula- delivery vehicles, e.g., cationic liposomes (5 – 7). Lipo- tions. This physical instability requires clinicians to some-based delivery systems offer significant advan- prepare lipid/DNA complexes immediately prior to in- tages over viral vectors, and have proven safe and effec- jection. In order to eliminate problems associated with tive in clinical trials (8–10). However, the use of these this temporal requirement, we investigated the feasi- synthetic vectors is severely limited by their instability bility of preserving complexes as a dried preparation in liquid formulations (10, 11). Therefore, it would be that could be tested, stored, and rehydrated as needed. advantageous to develop dried formulations of lipid/ To this end, our study evaluated the ability of different DNA complexes that, upon rehydration, possessed pre- stabilizers to preserve transfection rates of complexes dictable physical characteristics and transfection effi- during acute freeze-drying stress. Our data show that ciencies comparable to fresh preparations. The primary complexes lyophilized in 0.5 M sucrose or trehalose goal of this study was to investigate the ability of vari- possessed transfection rates similar to those of fresh ous stabilizers to protect lipid/DNA complexes during preparations. In addition, dried complexes that exhib- freeze-drying (lyophilization) stress. ited full transfection activity upon rehydration had The preparation of lipid/DNA complexes for transfec- sizes comparable to nonlyophilized controls. Our work tion is typically accomplished by mixing separate solu- demonstrates that lipid/DNA complexes can be stabi- tions of DNA and cationic liposomes. Because these lized as dried powders that offer significant advan- complexes readily form aggregates possessing reduced tages over current liquid formulations. Furthermore, transfection efficiency, current clinical protocols re- the correlation of transfection rates with maintenance of complex diameter suggests that size plays a critical quire that preparations of lipid/DNA complexes be in- role in lipid-based DNA delivery.  1997 Academic Press jected immediately after mixing (8, 10). This instability Key Words: lyophilization; freeze-drying; cationic li- not only is inconvenient, but it introduces variability posomes; stability; lipid/DNA complexes; gene deliv- in mixing regimes and does not allow the effectiveness ery; transfection. of lipid/DNA complexes to be evaluated prior to use. In contrast, stable formulations would allow samples to be prepared under controlled laboratory conditions that result in complexes possessing consistent physical properties and maximal transfection efficiencies. To The ability to deliver heterologous DNA to cells offers the potential to develop potent vaccines against viral- 2 Abbreviations used: AIDS, acquired immunodeficiency syndrome; SDS, sodium dodecyl sulfate; DOTAP, 1,2-dioleoyl-3-trimethylam- 1 To whom correspondence should be addressed at School of Phar- monium-propane; DOPE, L-a dioleoyl phosphatidylethanolamine; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buf- macy, C238, University of Colorado, Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Fax: (303) 315-6281. fered saline; BCA, bicinchonic acid. 199 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.