Novel Cationic Amphiphiles As Delivery Agents for Antisense Oligonucleotides R
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3334–3341 Nucleic Acids Research, 1999, Vol. 27, No. 16 © 1999 Oxford University Press Novel cationic amphiphiles as delivery agents for antisense oligonucleotides R. K. DeLong1, Hoon Yoo1,S.K.Alahari1,M.Fisher1,S.M.Short1,S.H.Kang2,R.Kole1,2, V. Janout3,S.L.Regan3 and R. L. Juliano1,* 1Department of Pharmacology and 2Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA and 3Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA Received as resubmission April 19, 1999; Revised and Accepted May 7, 1999 ABSTRACT INTRODUCTION There has been great interest recently in therapeutic Currently there is substantial interest in the use of antisense use of nucleic acids including genes, ribozymes and oligonucleotides, ribozymes, high molecular weight DNA and antisense oligonucleotides. Despite recent improve- other types of nucleic acids to modify gene expression for ments in delivering antisense oligonucleotides to therapeutic purposes (1–5). Antisense oligonucleotides are cells in culture, nucleic acid-based therapy is still usually administered in vitro or in vivo either in free form or often limited by the poor penetration of the nucleic complexed with various cationic lipid-based formulations (6–9). acid into the cytoplasm and nucleus of cells. In this Gene therapy has primarily relied on viral vectors (1), but there report we describe nucleic acid delivery to cells is increasing interest in use of non-viral gene delivery systems based on lipids, polymers or combinations of both (10–14). using a series of novel cationic amphiphiles containing The ability of a nucleic acid to achieve a desired function cholic acid moieties linked via alkylamino side within cells is influenced by several critical factors in addition chains. We term these agents ‘molecular umbrellas’ to its own innate biological activity. These include the hetero- since the cationic alkylamino chains provide a ‘handle’ geneity of uptake within the cell population; the degree to for binding of nucleic acids, while the cholic acid which the nucleic acid reaches appropriate cellular compart- moieties are likely to interact with the lipid bilayer ments (cytosol and nucleus); and, when a carrier is used, the allowing the highly charged nucleic acid backbone to extent to which the nucleic acid can dissociate from the carrier traverse across the cell membrane. Optimal gene and and attain its biologically active form in the target cell. The oligonucleotide delivery to cells was afforded by a commercially available ‘cytofectins’ (cationic liposomes) derivative (amphiphile 5) containing four cholic acid currently widely used as carriers for genes and oligonucleotides moieties. With this amphiphile used as a constituent typically form large, complex aggregates with nucleic acids in cationic liposomes, a 4–5 log increase in reporter (15). Despite some recent progress (16), there is still relatively gene delivery was measured. This amphiphile used little known about the ability of such complexes to enter key alone provided a 250-fold enhancement of oligo- cellular compartments or to release biologically active nucleic nucleotide association with cells as observed by flow acids. In addition, most cationic lipid complexes do not func- cytometry. A substantial fraction of cells exposed to tion well in the presence of plasma proteins, although there are complexes of amphiphile 5 and fluorescent oligo- some exceptions. While some studies with cationic lipids have nucleotide showed nuclear accumulation of the provided evidence for gene expression in vivo (17,18), there are still many problems with access of the complex to desired fluorophore. Enhanced pharmacological effectiveness tissue sites and with attaining and sustaining appropriate levels of antisense oligonucleotides complexed with of gene expression in the target issue. amphiphile 5 was observed using an antisense In vivo studies with therapeutic antisense oligonucleotides splicing correction assay that activates a Luciferase have primarily utilized administration of the oligonucleotide reporter. Intracellular delivery, nuclear localization alone, without complexation to carriers (2,19,20). However, a and pharmacological effectiveness of oligonucleo- substantial amount of in vitro data suggests that carrier tides using amphiphile 5 were similar to those systems, including cytofectins, can substantially increase the afforded by commercial cytofectins. However, in con- delivery of antisense compounds to cells, as well as the con- trast to most commercial cytofectins, the umbrella sequent pharmacological effects (6,7). Thus it is possible that amphiphile showed substantial delivery activity even suitable carrier systems could increase the in vivo efficacy of in the presence of high concentrations of serum. antisense oligonucleotides as well. *To whom correspondence should be addressed. Tel: +1 919 966 4383; Fax: +1 919 966 5640; Email: [email protected] Present address: R. K. DeLong, Megabios Corp., Burlingame, CA, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Nucleic Acids Research, 1999, Vol. 27, No. 16 3335 In the studies reported here, we have examined novel cationic Preparation of nucleic acid complexes amphiphiles as delivery agents for nucleic acids, particularly Amphiphile/liposome/DNA complexes. Amphiphiles 1–5 dis- for antisense oligonucleotides. These amphiphiles utilize one solved in methanol (1 mg/ml), were combined with an equal or more cholic acid moieties tethered to alkylamino side chains volume of dioleoylphosphatidyl ethanolamine (DOPE) (1 mg/ that are derivatives of spermine and/or spermidine (21). The ml) (Avanti Polar Lipids) in a solution of chloroform. After amphiphiles were designed to act as ‘molecular umbrellas’ evaporation of the solvent, the mixture was suspended in sterile where the hydrophobic portion of the cholic acid moiety water (1 mg/ml). Each lipid–amphiphile solution (1L–5L) was allows membrane interaction and penetration and the vortexed for 5 min, bath sonicated (30 min) and stored at 4°C µ hydrophilic face of the cholic acid moiety, along with the under N2. Complexes of plasmid (1 g/ml) or oligonucleotide alkylamino side chain, permits interaction with the negatively (10 nM–1 µM) were formed by combining the DNA stock charged nucleic acid. In studies with cultured cells, we have dissolved in 100 µlofOpti-Mem(GibcoBRL)withanequal tested several forms of ‘molecular umbrella’ amphiphiles for volume of the (1L–5L) amphiphile/liposomes (10 µg/ml). The their ability to enhance DNA transfection and for their ability complexes were incubated for 30 min at room temperature and to deliver antisense oligonucleotides. These amphiphiles then added directly to cells in 1 ml cell culture medium. proved to be only moderately effective as DNA transfection agents and were clearly less efficient than the commercially Amphiphile/oligonucleotide complexes. Stocks of oligonucleo- available cytofectins Lipofectin® and Lipofectamine®.How- tide (50–100 µM) were diluted to the appropriate concentration ever, the umbrella amphiphiles were quite effective in promoting for complexation (10 nM–10 µM) with sterile water. The oligo- delivery of antisense oligonucleotides to the nucleus, as nucleotide was then stirred within 0.5 ml glass V-vials (Wheaton) observed by fluorescence microscopy. Further, we found that in 40–100 µl sterile water. Amphiphile at 5–10 µg/ml in 1% the umbrella surfactants could enhance the pharmacological methanol/water was then added rapidly via a 0.5 cc U-100 effectiveness of antisense oligonucleotides to a significant Insulin Syringe (Becton Dickinson) and stirred for 15 min at extent. However, in contrast to many lipid cytofectins, the room temperature. The complexes were incubated for 30 min umbrella amphiphile/oligonucleotide complexes retained sub- at room temperature and then added directly to cells in 1 ml stantial activity in the presence of serum. This suggests that cell culture medium. these compounds may have some value for in vivo delivery of antisense oligonucleotides. Cytofectin complexes. Complexes of oligonucleotides with Lipofectin® or Lipofectamine® were prepared as previously described (6), essentially according to the manufacturer’s MATERIALS AND METHODS directions. Cholic acid–polyamine conjugates (molecular umbrellas) Plasmid transfection assay Specific procedures that were used to prepared the cholic acid– For transfections, ECV304 or MDR3T3 cells were seeded in 6- polyamine conjugates 1, 3, 4 and 5 were essentially those well dishes ~24 h prior to transfection and used at 60–80% previously described (21). Compound 2, which was prepared confluence. Cells were transfected for 4 h in serum-free by similar methods, exhibited the expected 1H NMR spectrum. medium with ~1 µgofpGL3plasmid(Promega)andeither 20 µg/ml of Lipofectamine® (Gibco BRL) or amphiphile (5–10 µg/ Cell culture ml); cells were then placed in serum-containing growth Handling and maintenance of multi-drug resistant NIH 3T3 medium and allowed to recover for 24 h. Transfected cells cells has been previously described (6). Human endothelial were lysed in a buffer containing 0.25 M Tris (pH 7.8), 1% Triton cells (ECV304; ATCC) were routinely grown in Medium 199 X-100 and 1 mM dithiothreitol (DTT). Lysate protein concen- (Gibco) supplemented with 10% heat inactivated fetal bovine trations were determined using the bicinchonic acid assay (Pierce Chemical Co.). Cell lysates were stored at –70°Cuntil