Gene Therapy (1998) 5, 855–860  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt BRIEF COMMUNICATION Influence of the DNA complexation medium on the efficiency of lipospermine/DNA particles

A Kichler1, W Zauner1, M Ogris1 and E Wagner1,2 1Institute of Biochemistry, University of Vienna; and 2Boehringer Ingelheim R&D Vienna, Austria

Dioctadecylamidoglycylspermine (DOGS, Transfectam) is plex formation. The best transfection conditions consisted

a cationic lipid able to interact with DNA to form complexes of particles formed in RPMI medium, NaHCO3/Na2HPO4 or that mediate efficient gene transfer into various eukaryotic sodium citrate solutions. Mixing in a 150 mM sodium chlor- cells. The state of condensation of the plasmid changes ide solution (as recommended) resulted in lower gene with the medium composition. We therefore investigated to expression. When the helper lipid 1,2-dioleoyl-sn-glycero- what extent the DNA condensation buffer influences the 3-phosphoethanolamine (DOPE) was present in the transfection efficiency of Transfectam/DNA particles. Our DNA/cationic lipid formulation, the increase in reporter results show that in a variety of cell lines, a greater than activity was also observed, although to a lower extent. 100-fold difference in luciferase gene expression is Thus, choosing the optimal conditions for formulating observed with Transfectam/DNA complexes at a +/− DNA/lipid complexes considerably reduces the amount of charge ratio of 0.75 depending on the conditions of com- lipid and DNA needed to obtain maximum gene transfer.

Keywords: gene transfer; cationic lipids; lipospermine; DNA compaction

The development of highly efficient gene transfer mote binding and uptake of the complexes through non- methods is a prerequisite for gene therapy. One of the specific endocytosis.15,16 However, the low gene transfer approaches involves artificial self-assembling systems. efficiency observed in vivo with synthetic vectors may be Typically, (poly)cationic molecules interact with DNA to due to the interactions of the positively charged transfec- form particles that are able to deliver the DNA into the tion particles with blood components. In contrast, lipo- cells. Cationic lipids are some of the more promising vec- plexes (cationic lipid/DNA complexes6) with charge tors.1–4 However, it is estimated that only one out of 104 ratios close to neutrality should allow: (1) a reduced bind- plasmid molecules presented to the cell by cationic lipids, ing to the anionic extracellular matrix; and (2) a reduced reaches the nucleus and is expressed. In contrast, for activation of the complement system.17 In addition, there every 10 recombinant adenoviruses that reach a cell, at is a dose-dependent toxicity associated with the use of least one will successfully transfer and express its gene.5 lipoplexes. Thus, it is necessary to reduce the quantity of Efforts to improve the lipofection (gene transfer using material (ie the DNA and its carrier) required to achieve cationic lipids6) efficiency include direct modification of efficient in vivo. the cationic lipid,7,8 addition of helper molecules such as We report that the optimization of the the lipid DOPE (1,2-dioleoyl-sn-glycero-3-phospho- DNA/Transfectam condensation step using suboptimal ethanolamine),9 addition of replication-defective adeno- amounts of cationic lipid leads to highly efficient formu- viruses10 or fusigenic peptides.11,12 Despite encouraging lations. advances, the number of plasmid copies required to Complexes of DNA and 2, 1 and 0.75 charge equiva- obtain high gene expression is still high. lents of Transfectam were prepared (the term charge Cationic lipids with multivalent headgroups, such as equivalent indicates the amount of lipid used for the for- lipospermines, are usually more efficient for in vitro gene mation of the complexes; one charge equivalent corre- transfer than the monovalent cationic lipids.13 However, sponds to the amount required to neutralize all the nega- transfection with these lipids in vitro requires DNA com- tive charges carried by the phosphate groups of the plexes bearing a strong net positive charge, ie in previous plasmid; the charge ratio is calculated by taking into studies using Transfectam (dioctadecylamidoglycyl- account the fact that one molecule of Transfectam carries spermine, DOGS), it was found that the transfection three positive charges, as three ammonium groups are efficiency was optimal when the +/− charge ratio was protonated at physiological pH13). When formulated in about 3 to 6.14 These positive charges are thought to pro- Roswell Park Memorial Institute (RPMI; Gibco-BRL, Gai- thersburg, MD, USA) culture medium instead of 150 mm NaCl solution as recommended by the manufacturer, these lipoplexes gave a 600, 250 and 200 times higher Correspondence: A Kichler, at his present addresss: Genethon, Groupe de Vectorologie, 1, Rue de l’Internationale, BP 60, 91002 Evry Cedex 2, luciferase reporter gene expression (Figure 1a), thus France reaching the values obtained with highly positively Received 8 August 1997; accepted 13 January 1998 charged complexes (compare with Transfectam Optimization of lipospermine-based transfection formulations A Kichler et al 856

Figure 1 Influence of the buffer composition used for the DNA condensation step on reporter gene expression. The plasmid coding for the Photinus pyralis luciferase gene under control of the cytomegalovirus enhancer/promoter18 (pCMVL) was used. Complexes were prepared as follows: plasmid DNA (3 or 4 ␮g) and the desired amount of cationic compound were each diluted into 75 ␮l of buffer (as indicated in the Figures) and gently mixed. After about 15 min, the two solutions were mixed. After 10 more min, the mixture was diluted with serum-free medium to a final volume of 1.5 ml; 750 ␮l of the transfection mixture was put on each well of the duplicate. (a) Complexes of 0.75, 1, 2 and 4 charge equivalents of Transfectam/3 ␮g

pCMVL prepared in 150 mm NaCl, H2O or RPMI were used. The DNA complexes were finally mixed with RPMI medium and added to the human melanoma cells H225 (75 000 cells per well in 24-well plates; the cells were kindly provided by S Schreiber and G Stingl, University of Vienna, Austria). After 4 h, the transfection medium was replaced by fresh RPMI supplemented with 2 mml-glutamine, 1 mm sodium pyruvate and 10% fetal calf serum (FCS; Gibco-BRL). The cells were lysed after 24 h in 200 ␮l of 250 mm Tris pH 7.3, 0.5% Triton X-100. The cell lysate was centrifuged for 5 min at 10 000 g to pellet debris. Luciferase light units were recorded using a Clinilumat LB9502 instrument (Berthold, Bad Wildbad, Germany) from an aliquot of the supernatant with 10 s integration. Luciferase background (approximately 300 light units) was subtracted from each value and the transfection efficiency was expressed as total light units per well and represents the mean value of the duplicates (±s.d.). (b) Determination of the RPMI component(s) responsible for the increased expression. Transfectam used at 1 charge equivalent was complexed with 3 ␮g DNA (per duplicate) in either

150 mm NaCl, NaCl/Na citrate (pH 5.5), or NaHCO3/Na2HPO4. For the remaining formulations, the cationic lipid pre-incubated in NaHCO3/Na2HPO4 (or NaCl), was mixed with the DNA diluted in NaCl (or NaHCO3/Na2HPO4). The efficiencies of these formulations were compared to Transfectam 4 eq/DNA complexes prepared in NaCl 0.15 m.

4 eq/DNA prepared in 150 mm NaCl, which leads to the optimized amounts of cationic lipid, ie 4 charge equiva- highest luciferase activity on H225 cells). By replacing the lents, and DNA (2 ␮g per well for a 24-well plate) were RPMI medium with DMEM (Dulbecco’s modified Eagle’s mixed in RPMI, there was no enhancement in reporter medium; Gibco-BRL), the enhancement observed with gene expression. In fact, a decrease of the efficiency was suboptimal amounts of lipid (0.75, 1 and 2 eq) was less observed, which is consistent with the data obtained by significant, but the efficiency remained better than with Barthel and colleagues.14 water or 150 mm NaCl (not shown). In contrast, when In order to determine which components are respon- Optimization of lipospermine-based transfection formulations A Kichler et al 857

Figure 2 The enhancement of reporter gene expression can be observed on several cell types. The efficiencies of different lipoplexes, prepared in NaCl

(light bars) or in NaHCO3/Na2HPO4 (dark bars), were determined on (a) HepG2 cells (120 000 cells per well were plated on the day before transfection in 24 well-plates) and (b) B16 cells (70 000 cells per well); both cell types were cultured in DMEM supplemented with antibiotics, glutamine and 10% heat-inactivated FCS. The transfection experiments were carried out as indicated in Figure 1. Total luciferase activity of the cells is shown and represents the mean value of the duplicates (±s.d.). sible for the increased efficiency with suboptimal human hepatocarcinoma cell line HepG2, B16 murine amounts of lipid, we tested each salt, alone or in combi- melanoma cells or the murine fibroblasts NIH 3T3, com- nation with other components of the RPMI medium. parable results to those observed on H225 cells were

NaHCO3 (23.8 mm; pH 7.4) and Na2HPO4 (5.6 mm;pH obtained (Figure 2, and Otmane Boussif, personal 7.4) used alone (data not shown) or mixed together (pH communication), showing that the effect is not cell type- 8; Figure 1b) gave a similar enhancement to that of the dependent. An enhancement, although to a lower extent, complete culture medium. In addition, we tested other was observed using Lipofectamine20 (Gibco-BRL) under buffers for their ability to augment the efficiency of 1 suboptimal conditions (0.75 eq; see Figure 3). Lipofectam- charge equivalent Transfectam/DNA complexes; a buffer ine is a mixture of the lipospermine DOSPA (2,3-diole- containing 150 mm NaCl and 20 mm sodium citrate (pH oyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 5.5) was as efficient as NaHCO3/Na2HPO4 (Figure 1b). 1-propanaminium trifluoroacetate) and the helper lipid Thus, in contrast to the medium effect observed by DOPE (DOSPA/DOPE, 3/1, w/w). It was previously Caplen et al19 with the cationic formulation DC- shown that DOPE can increase the efficiency of Transfec- Chol/DOPE, the effect we observed is not pH-dependent tam when suboptimal amounts of lipid are used.9 No (ie it does not rely on a modified protonation state of the enhancement in reporter gene expression was obtained amines of the lipospermine). with Transfectam 2 eq/DOPE 1.5 eq/DNA particles. Using the lipoplexes prepared in the These results confirm the Lipofectamine data, demon-

NaHCO3/Na2HPO4 buffer on other cell lines, such as the strating that the presence of DOPE diminishes the Optimization of lipospermine-based transfection formulations A Kichler et al 858

Figure 3 Influence of the DNA complexation medium on the efficiency of different lipospermine-based formulations. Transfectam- and Lipofectamine-

based complexes were prepared (as described in Figure 1) either in 0.15 m NaCl (light bars) or in a NaHCO3/Na2HPO4 solution (dark bars). The Transfectam/DOPE complex was obtained by mixing the desired amounts of Transfectam and helper lipid DOPE (the amount of helper lipid used is given in equivalents (mol/mol) to Transfectam) before dilution with the DNA solution. To obtain the peptide formulation, the fusigenic peptide INF521 (1 mg/ml solution in HBS/glycerol (3:1)) was added to the preformed Transfectam/DNA complexes; after a 10 to 20 min period, the transfection volume was adjusted to 1.5 ml with culture medium, and 750 ␮l of this transfection mixture per well was pipetted on to H225 cells. Total luciferase activity of the cells is shown and represents the mean value of the duplicates (±s.d.).

enhancement obtained by the DNA complexation step is complexed with the cationic lipid to form transfection- performed in NaHCO3/Na2HPO4. competent particles. We have recently shown that fusigenic peptides Since high reporter gene expression was obtained with derived from the N-terminus of hemagglutinin from the Transfectam 0.75 eq/DNA complexes formulated in

influenza virus are able to increase the efficiency of NaHCO3/Na2HPO4 while only a small part of the DNA Transfectam/DNA complexes up to 1000 times, when is complexed, we asked what would happen if the Transfectam is used in suboptimal amounts.12 Preparing amount of DNA used for transfection were reduced. ␮ these ternary complexes in NaHCO3/Na2HPO4 further Figure 5 shows that using 0.5 or 1 g of DNA per well improved their efficiency by a factor of seven (Figure 3). instead of 2 ␮g reduces the efficiency of Transfectam 4 When the cationic lipid was diluted in eq/DNA (NaCl) and Transfectam 0.75 eq/DNA

NaHCO3/Na2HPO4 and the DNA in 150 mm NaCl for (NaHCO3/Na2HPO4 or NaCl) complexes. However, complexation, the complexes remained very efficient in reporter gene expression obtained with the Transfectam gene transfer. However, when the lipid was diluted in 4 eq (NaHCO3/Na2HPO4) complexes remained high. 150 mm NaCl and the DNA in NaHCO3/Na2HPO4, the In the presence of 20% serum (of both heat-inactivated resulting complexes lost their gene transfer efficacy serum and non-decomplemented serum) the transfection

(Figure 1b). These formulations were then used in a DNA efficiency of the NaHCO3/Na2HPO4 formulation was retardation assay (see Figure 4). The following obser- affected in a comparable manner to the NaCl preparation vations could be made: (1) Transfectam 1 eq complexed (data not shown). Finally, we asked whether the addition to DNA in a 150 mm NaCl solution was able to retard of the anionic ions to the lipoplexes has an influence on all the DNA. The same result was obtained when, before the size of the DNA complexes. The particle size mixing, Transfectam was diluted in NaCl and the plas- measurements obtained by laser light scattering showed

mid in NaHCO3/Na2HPO4; (2) however, with the com- that the complexes formulated in sodium chloride were plexes obtained when the lipid is diluted in smaller than those prepared in NaHCO3/Na2HPO4 (400– NaHCO3/Na2HPO4 and the DNA in NaCl or 750 nm versus 950–1050 nm). NaHCO3/Na2HPO4, only a small part of the DNA is Taken together, our data show that only a small part retarded. A similar pattern was observed when 4 charge of the Transfectam/DNA complexes prepared in 150 mm equivalents of Transfectam were used (Figure 4). Note sodium chloride is involved in gene transfer. Formulation that a sodium chloride dispersion containing 4 charge of complexes in NaHCO3/Na2HPO4 leads to the same equivalents of Transfectam was able to prevent intercal- expression levels as Transfectam 4 eq/DNA/NaCl com- ation of ethidium bromide in DNA (Figure 4, lane 2 and plexes but either less lipid needs to be used or fewer com- 5, lower panel). These results show that there is, plexes needs to be applied to the cells (see Figure 5). depending on the complexation buffer used, an even or Because there is a dose-dependent toxicity associated uneven distribution of the cationic lipid among the DNA with the use of lipoplexes22,23 (and AK unpublished molecules. Thus, a possible explanation for efficient results), it is advantageous to minimize the amount of transfection by NaHCO3/Na2HPO4 lipoplexes formed at complexes and lipid used. In addition, with complexes low ratios is that only a small portion of the total DNA prepared in sodium chloride, there is a narrow window Optimization of lipospermine-based transfection formulations A Kichler et al 859

Figure 5 Transfection levels obtained with decreasing amounts of plasmid DNA depend on the condensation buffer. HepG2 cells were transfected with decreasing amounts of lipoplexes. The complexes of 0.75 (-ᮀ-), and 4(-᭺-) charge equivalents were prepared either in 0.15 m sodium chloride

(open symbols) or in NaHCO3/Na2HPO4 (closed symbols). Total luciferase activities per well are shown and are the means of the duplicates (±s.d.).

Figure 4 Analysis of the interaction between plasmid DNA and Trans- DNA-transfection procedure. Proc Natl Acad Sci USA 1987; 84: fectam by gel retardation assay. One microgram plasmid DNA in 25 ␮l 7413–7417. buffer (150 mm NaCl or 23.8 mm NaHCO3/5.6 mm Na2HPO4) was mixed with aliquots of lipid (1 or 4 charge equivalents of Transfectam) also 3 Leventis R, Silvius JR. Interactions of mammalian cells with diluted in 25 ␮l of buffer. After a 10-min maturation period, 25 ␮l of the lipid dispersions containing novel metabolizable cationic complexes were then applied on an agarose gel. The gel was prepared with amphiphiles. Biochim Biophys Acta 1990; 1023: 124–132. 0.9% agarose in Tris-EDTA with 0.5 ␮g/ml ethidium bromide. Electro- 4 Gao X, Huang L. A novel cationic liposome reagent for efficient phoresis was carried out at 70 V and 35 mA for 80 min. Lane 1, plasmid; transfection of mammalian cells. Biochem Biophys Res Commun + lane 2, Transfectam/NaCl DNA/NaCl; lane 3, Transfectam/NaHCO3- 1991; 179: 280–285. + Na2HPO4 DNA/NaHCO3-Na2HPO4; lane 4, Transfectam/NaHCO3- 5 Crystal RG. The gene as the drug. Nature Med 1995; 1: 15–17. + + Na2HPO4 DNA/NaCl; lane 5, Transfectam/NaCl DNA/NaHCO3- 6 Felgner PL et al. Nomenclature for synthetic gene delivery sys- Na2HPO4. In the upper panel 1 eq of Transfectam was used; for the lower tems. Hum Gene Ther 1997; 8: 511–512. panel Transfectam was used at 4 charge equivalents. 7 Remy JS, Sirlin C, Vierling P, Behr JP. Gene transfer with a series of lipophilic DNA-binding molecules. Bioconj Chem 1994; 5: 647–654. 8 Felgner JH et al. Enhanced gene delivery and mechanism studies for efficient transfection – ie conditions outside those with a novel series of cationic lipid formulations. J Biol Chem optimal, result in a considerable decrease of reporter gene 1994; 269: 2550–2561. expression. In contrast, the window is much larger with 9 Remy JS et al. Targeted gene transfer into hepatoma cells with transfection particles prepared in RPMI medium or lipopolyamine-condensed DNA particles bearing multi- antennary galactosyl ligands: a stage towards artificial viruses. NaHCO3/Na2HPO4. This point could be particularly important for in vivo applications. The isolation and Proc Natl Acad Sci USA 1995; 92: 1744–1748. 10 Raja-Walia R et al. Enhancement of liposome-mediated gene characterization of these highly efficient transfer into vascular tissue by replication deficient adenovirus. Transfectam/DNA/NaHCO3-Na2HPO4 particles will be Gene Therapy 1995; 2: 521–530. the focus of future investigations. 11 Kamata H, Yagisawa H, Takahashi S, Hirata H. Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Res 1994; 22: 536–537. Acknowledgements 12 Kichler A, Mechtler K, Behr JP, Wagner E. 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