Bioscience Reports, Vol. 15, No. 1, 1995 Cholesterol Enhances Cationic Liposome- Mediated DNA Transfection of Human Respiratory Epithelial Cells Michael J. Bennett, 1 Michael H. Nantz, 1 Rajiv P. Balasubramaniam, 1 Dieter C. Gruenert, 3 and Robert W. Malone 2,4 Received December 14, 1994; accepted January 27, 1995 Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1- (2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphos- phatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy. KEY WORDS: cationic lipdsome; cholestergl; transfection; human respiratory epithelial cells. ABBREVIATIONS: N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI); dioleoylphosphatidylethanolamine (DOPE); N-[1-(2,3-dioleoyloxy) propyl]-N,N,N- trimethyl ammonium chloride (DOTMA); Eagle's modified essential medium (Mem); Dulbecco's Modified Eagle's Medium (DMEM). INTRODUCTION Methods for polynucleotide delivery are essential for the correlation of gene expression with phenotype, investigation of the regulation of gene expression, and gene therapy. Commonly used gene delivery methods include the use of i Gene Therapy Program, Department of Chemistry, University of California, Davis, California 95616. z Medical Pathology, University of California, Davis, California 95616. 3 Gene Therapy Core Center, Cystic Fibrosis Research Center and Cardiovascular Research Institute, Department of Laboratory Medicine, University of California, San Francisco, California 94143. 4 To Whom correspondence should be addressed. 47 0144-8463/95/0200-0047507.50/0 (cid:14)9 1995 Plenum Publishing Corporation
`
`Moderna Ex 1009-p. 1
`Moderna v Protiva
`
`

`

`48 Bennett, Nantz, Balasubramaniam, Gruenert and Malone recombinant viral vectors, physical methods (such as direct DNA injection), or pharmaceutical reagents. One pharmaceutical gene delivery technique involves the use of DNA packaged with lipids (liposomes). Originally, neutral or weakly charged lipids were utilized for liposomal gene delivery (1, 2, 3). The discovery that cationic liposomes spontaneously associate with DNA, fuse with cell membranes, and deliver the DNA into cytoplasm (4) has greatly advanced the utility of liposomal polynucleotide transfection. Synthetic cationic transfection lipid preparations spontaneously interact with DNA in solution to form lipid-DNA complexes. When placed onto tissue culture cells, these lipid-DNA complexes interact with the plasma membrane (4). As a consequence of this process, a portion of the exogenous DNA becomes localized in the nucleus and is subsequently transcribed. There are two leading hypotheses defining the mechanism of cationic lipid-mediated transfection: 1) plasma membrane fusion and subsequent cytoplasmic delivery (4) or 2) a pathway involving endocytic uptake (5, 7, 8). These hypotheses are not mutually exclusive, and may be active to a greater or lesser extent in different cell types. Either transfection pathway may be facilitated by alterations in liposome formulation which effect the fluidity of lipid/polynucleotide/cell membrane complexes. Sterols are commonly used for modulating the fluidity of both natural and artificial membranes (9). Cationic cholesterol derivatives have also been shown to mediate effective DNA transfection of cells and tissues when formulated into sonicated vesicles with DOPE (10). Therefore, these experiments have been designed to investigate the effect of cholesterol, the predominant mammalian sterol, on the efficacy of cationic liposome transfection formulations. Since respiratory epithelial cells are a potential therapeutic target for cationic lipid-mediated gene therapy, experiments focusing on the transfection of the apical surface of polarized respiratory epithelial cells were performed. Human surface airway epithelial cells have been isolated and cultured in vitro, but even under optimal conditions such cells have a limited life span and senesce or terminally differentiate (11, 12). To circumvent this problem, cultures of polarized immortal human bronchial epithelial cells have recently been developed (13), and this cell line (16HBE140-) was chosen as the principal target for the analysis of the effects of cholesterol on cationic lipid-mediated DNA transfection. MATERIAL AND METHODS Chemicals Dioleoylphosphatidylethanolamine was purchased from Avanti Polar Lipids (Inc. (Birmingham, A1). Cholesterol was purchased from Sigma Chemical Company (St. Louis, MO). The cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N-[1- (2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) was prepared using a procedure developed in our laboratories (14).
`
`Moderna Ex 1009-p. 2
`Moderna v Protiva
`
`

`

`Liposome-Mediated DNA Transfection 49 Cell Culture Human respiratory epithelial cells (16HBE14o-) were cultured by plating on 24 well tissue culture plastic ware coated with fibronectin, vitrogen (collagen), and bovine serum albumen as previously described (11, 12). Growth media consisted of Eagle's modified essential medium (MEM) supplemented with 10% fetal bovine serum, and cells were transfected as subconfluent monolayers. As a control for cell specific effects, NIH 3T3 cells were also tested for DNA transfection using the various liposome formulations. NIH 3T3 were obtained from ATCC (CRL 1658). 3T3 cells were cultured on standard 24 well tissue culture plates using Dulbecco's Modified Eagle's Medium and 10% calf serum. Liposome Formulation An appropriate mass of the cationic lipid DORI and a neutral lipid (DOPE and/or cholesterol), as solutions in chloroform, were added to 1.9 ml sample vials to yield a 50:50 mole ratio of DORI:neutral lipid. The chloroform was removed via rotary evaporation at 37~ The resulting thin lipid films were placed under high vacuum overnight to insure that all traces of solvent have been removed. The lipid mixture was resuspended with vortex mixing using 1 ml sterile water for injection (American Reagent Laboratories Inc.), resulting in a total lipid concentration of 2/xmole lipid/1 ml water. This solution was sonicated until clear using a Branson sonifer 450 sonicator equipped with a cup horn (35~ 15 minutes). Piasmid DNA The plasmid pCMVL consists of the P. pyralis luciferase cDNA subcloned into the plasmid pRc/CMV (Invitrogen). This plasmid was transformed into competent E. coli DH5-a cells, amplified in terrific broth, and prepared by alkaline lysis with the isolation of covalently closed circular plasmid DNA using two rounds of CsC1-EtBr gradient ultracentrifugation. The plasmid DNA was subsequently treated with DNAse-free RNAse, phenol/chloroform extracted, and purified by precipitation from an ethanol/sodium acetate solution. DNA purity was determined by agarose gel electrophoresis and optical density (OD 260/280 greater than or equal to 1.8). Transfection of Cultured Cells Tissue culture well plates (24 well) were plated 24 hours prior to transfection at approximately 80% confluency with either NIH 3T3 cells or 16HBE14o- cells. The growth media was removed via aspiration and the cells were washed once with 0.5 ml PBS/well. The liposome/DNA complexes were formed through sequential addition of an appropriate amounts of DMEM (serum-free), plasmid pCMVL (4 micrograms), and liposome formulation into a 2 ml Eppendoff tube
`
`Moderna Ex 1009-p. 3
`Moderna v Protiva
`
`

`

`50 Bennett, Nantz, Balasubramaniam, Gruenert and Malone gave a total volume of 800 ml. The addition of these substances was followed by thorough vortex mixing and incubation for 15 minutes at room temperature. A 200 ml aliquot of the resultant transfection complex was added to each well and the cells were incubated for 4 hours at 37~ At this time, 500 microliters of the appropriate growth media + 10% calf serum/well was added and the cells were incubated for an additional 48 hours. Luciferase Assay Relative luciferase activity was determined by using the Enhanced Luciferase Assay Kit and Monolight 2010 luminometer (both from Analytical Luminescence Laboratories, San Diego, CA). This was accomplished by directly applying 233.3 mL of concentrated luciferase lysis buffer to each well and placing the cells on ice for 15 minutes. Removal of growth media was not necessary prior to the application of the lysis buffer. This technique proved to be more efficient and avoids the possibility of cell loss during media removal. An analogous experiment where the growth media was removed afforded similar results. Luciferase light emissions from 31.1 microliters of the lysate were measured over a 10 second period, and results expressed as a function of an assumed total lysate volume of 933.3/xl. RESULTS To investigate the possibility that added cholesterol could enhance liposomal transfection ability, liposomes containing mixtures of the cationic lipid DORI and a neutral lipid component, DOPE and/or cholesterol, were formulated and screened for their ability to transfect pCMVL DNA into human airway epithelial cells and NIH 3T3 murine fibroblasts. Optimal experimental conditions were obtained by varying the ratio of DOPE to cholesterol while keeping the amount of DORI (50:50 mole ratio of DORI:neutral lipid mixture) and the DORI:DNA phosphate charge ratio (2:1) constant. These experimental conditions were specifically selected to allow direct comparison between the tri-component liposomes and 50:50 DOPE:DORI liposomes. Previous work by ourselves and others (6) has shown that a 50:50 DOPE:DORI formulation is optimal for bi-component transfection preparations composed of DOPE and DORI. The addition of cholesterol to the cationic lipid formulations enhances the transfection of differentiated human respiratory epithelial cells (Figure 1). This observation was consistent for all formulations, including those composed of only DORI and cholesterol as well as the tri-component liposomes. Furthermore, variable transfection activity was observed among the tri-component formula- tions. The most active DNA transfection preparation was formulated using a molar ratio of 30: 50: 20 DOPE: DORI: cholesterol. To ascertain whether the enhancement of transfection which was observed with added cholesterol was limited to respiratory epithelial cells, NIH 3T3 cells
`
`Moderna Ex 1009-p. 4
`Moderna v Protiva
`
`

`

`Liposome-Mediated DNA Transfection 51 2.1E+07 "~ 1.8E+07 (cid:12)9 t~ 1.5E+07. "" 1.2E+07 .e 9.0E+06. r~ 6.0E+06 3.0E+06 "~ 0.0E+00 ] 50:50 DORI:Cholesterol [] 10:50:40 DOPE:DORI:Cholesterol [] 20:50:30 DOPE:DORI:Cholesterol [] 30:50:20 DOPE:DORI:Cholesterol [] 40:50:10 DOPE:DORI:Cholesterol [] 50:50 DOPE:DORI Fig. 1. Formulations containing 50 mole % of the cationic lipid DORI and varying amounts of DOPE and cholesterol were screened for their ability to functionally deliver the plasmid pCMVL (1/xg) into human respiratory epithelial cells. Cell lysates obtained 48 hours after transfection were analyzed for luciferase specific activity. Each data point reflects the mean value of total light units derived from four transfections and the standard deviation from this mean. were transfected using the same formulations as were used for respiratory epithelial cell transfection (Figure 2). Again, it was determined that the tri-component formulation consisting of 30:50: 20 DOPE: DORI: cholesterol was optimal for transfection of NIH 3T3 cells with DNA. 4.0E+07 ,~ 3SE+07 3.0E +07 - r~ 2_5E +07 2.0E +07 o "~ 1.5E+07 ,~ 1.0E +07 ~.~ 5.0E +06 0.0E +00 T [] 51150 DORI:Cholesterol [] 111 50:4 0 DOPE ".D ORI: Clao le slerol 1~ 21150:3 0 DOPE :D ORI: Cho le sle rol [] 311 50 ~20 DOPE :D ORI: Cho b sle rol [] 41150:10 DOPE :D ORI: Cho b sle rol [] 51150 DOPE:DORI Fig. 2. Optimization of NIH 3T3 fibroblasi transfection mediated by liposomes containing the cationic lipid DORI (50 mole %) and variable amounts of DOPE and cholesterol. Analysis for luciferase specific activity 48 hours after transfection indicates that liposomes comprised of a 30:50:20 mole ratio of D OPE:DORI:cholesterol are the most effective in mediating pCMVL delivery. These results compliment those obtained with the human respiratory epithelial cells.
`
`Moderna Ex 1009-p. 5
`Moderna v Protiva
`
`

`

`52 Bennett, Nantz, Balasubramaniam, Gruenert and Malone DISCUSSION The inclusion of cholesterol in cationic lipid transfection preparations enhances DNA transfection of differentiated human respiratory epithelial cells. This finding is not limited to epithelial cells, but is also applicable to a commonly used murine fibroblastic cell line. Respiratory epithelial cell transfection is important for investigations of gene expression, transport and secretion, and for the development of human gene therapies. Furthermore, methods for DNA transfection of cultured mammalian cells are critical to a wide range of experimentation concerning the biochemistry and molecular biology of nucleic acids. Therefore, this observation may be of interest to investigators who do not routinely transfect respiratory epithelial cells. The theoretical basis for the observation that the addition of cholesterol to cationic lipid preparations enhances DNA transfection of mammalian cells is speculative. It is known that cholesterol and some of it's derivatives modulate the physical state of both natural and artificial membranes. In general, at tempera- tures above the phase transition temperature of phospholipid-containing membr- anes (e.g. 37~ cholesterol decreases membrane fluidity by restricting the mobility of fatty acyl chains (15). Thus the optimized tri-component formulation which is described (30:50:20 DOPE:DORI:cholesterol) is likely to be more rigid than the corresponding formulations which lack cholesterol. If this is the case, one explanation for cholesterol enhancement of transfection would be that a more rigid lipid component of a cationic lipid:DNA complex facilitates endocytic uptake rather than direct membrane fusion. Therefore, these results may support the hypothesis that endocytosis is the principal mechanism for cationic liposome- mediated polynucleotide transfection. ACKNOWLEDGEMENTS We thank Jill G. Malone and Amy Sawyer for assistance with cell culture, Jill G. Malone and Karl Bassler for assistance with manuscript preparation and Phillip Montbriand for plasmid DNA preparation. This work was supported by grants from the Cystic Fibrosis Foundation ($884) and the California Tobacco Related Disease Research Program (4KT-0205) (RWM), and NIH grants DK47766 and DK46002 (DCG). REFERENCES 1. Fraley, R. and Papahadjopoulos, D. (1982) Curr. Top. Microbiol. Immunol. 96:171-91. 2. Uchida, T. (1988) Exp. Cell Res. 178:1-17. 3. Gould-Fogerite, S., Mazurkiewicz, J. E., Raska Jr., K., Voelkerding, K., Lehman, J. M. and Mannino, R. J. (1989) Gene 84:429-38. 4. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. and Danielsen, M. (1987) Proc. Natl. Acad. Sci USA 84:7413-7.
`
`Moderna Ex 1009-p. 6
`Moderna v Protiva
`
`

`

`Liposome-Mediated DNA Transfection 53 5. Farhood, H., Bottega, R., Epand, R. M. and Huang, L. (1992). Biochim. Biophys. Acta 1111:239-46. 6. Felgner, J. N., Kummar, R., Sridhar, C. N., Wheeler, C., Tsai, Y. J., Border, R., Ramsay, P., Martin, M. and Felgner, P. (1994) J. Biol. Chem. 269:2550-2561. 7. Pinnaduwage, P., Schmitt, L. and Huang, L. (1989) Biochim. Biophys. Acta 985:33-8. 8. Debs, R., Pian, M., Gaensler, K., Clements, J., Friend, D. S. and Dobbs, L. (1992) Am. J. Respir. Cell Mol. BioL 7"-406-13. 9. New, R. R. C. (1990) In: Liposomes--A Practical Approach. (New, R. R. C. ed), IRL Press, Oxford, UK, pp. 19-22. 10. Gao, X. and Huang, L. (1991). Biochem. Biophys. Res. Commun. 179:280-5. 11. Gruenert, D. C., Basbaum, C. B. and Widdicombe, J. H. (1990) In: Vitro Cell Dev. Biol. 26:411-8. 12. Lechner, J. LaVeck (1985) J. Tissue Culture Methods 9:43-48. 13. Cozens, A. L., Yezzi, M. J., Kunzelmann, K., Ohrui, T., Chin, L., Eng, K., Finkbeiner, W. E., Widdicombe, J. H. and Gruenert, D. C. (1994) Am. J. Respir. Cell Mol. Biol. 10:38-47. 14. Bennett, M. J., Malone, R. W. and Nantz, M. H. (Submitted). Tetrahedron Lett. 15. Bloch, K. (1991) In: Biochemistry of Lipids, Lipoproteins and Membranes. (Vance, D. E. and Vance, J. eds), Elsevier, New York, NY. pp. 368-369.
`
`Moderna Ex 1009-p. 7
`Moderna v Protiva
`
`