Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA

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Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA
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  ELSEVIER International Journal of Pharmaceutics 139 (1996) 69 78 international journal of pharmaceutics Effect of cationic liposome composition on in vitro cytotoxicity and protective effect on carried DNA Rita Cortesi c, Elisabetta Esposito a, Enea Menegatti a, Roberto Gambari b, Claudio Nastruzzi a,* aDepartment of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy bBiotechnology Center, University of Ferrara, Ferrara, Italy CDepartment of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK Received 29 January 1996; revised 18 April 1996; accepted 29 April 1996 Abstract Positively charged liposomes were prepared by using three different cationic surfactants, namely cetyl-trimethyl-am- monium bromide (CTAB), didodecyl-dimethyl-ammonium bromide (DDABI2) and dioctadecyl-dimethyl-ammonium bromide (DDAB~8). A study of the parameters influencing the in vitro toxicity of cationic liposomes on cultured cell lines was performed, demonstrating the lower cytotoxicity of DDABls-containing liposomes. In addition, the stability of 2DNA complexed to cationic liposomes after exposure to serum exo- and endonucleases was evaluated. Our results indicated that DDAB~2 and DDAB18 liposomes are able to efficiently protect DNA from degradation, thus representing a potential approach to deliver nucleic acid in vivo. Keywords: Cationic liposomes; DNA stability; Cytotoxicity 1. Introduction Abbreviations: PC, phosphatidylcholine; CH, cholesterol; CD, cationic detergent; DDAB12, didodecyl-dimethyl-ammo- nium bromide; DDAB~s, dioctadecyl-dimethyl-ammonium bromide; CTAB, cetyl-trimethyl-ammonium bromide; 2DNA, lambda phage DNA. * Corresponding author, Dipartimento di Scienze Farma- ceutiche, Universita degli Studi di Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy. Tel: + 39-532-291259; Fax: + 39-532-291296: E-mail: nas@ifeuniv.unife.it. With the advent of molecular biology and bio- technology techniques, the use of antisense and gene transfection protocols has assumed a very important role as experimental therapy (Uhlmann and Peyman, 1990). Gene therapy is indeed emerging as a clinically viable pharmacological regimen for genetic, neoplastic and infectious dis- eases. Gene transfection, consisting of the intro- 0378-5173/'96/$15.00 ~ 1996 Elsevier Science B.V. All rights reserved PH S0378-5173(96)045 74-7  70 R. Cortesi et al. / International Journal of Pharrnaceutics 139 1996) 69-78 duction of normal exogenous genes into target cells, allows the replacement of the defective gene product, hence restoring normal cell function (Feigner et al., 1987). In addition, antisense oligonucleotides or chemically modified ana- logues, designed to be complementary to viral or eukaryotic mRNAs, can be used to inhibit both in vitro and in vivo protein synthesis (To and Nei- man, 1992; Neckers et al., 1992). More generally, different nucleic acid molecules, including (a) mR- NAs or complete genes (Mitchell and Tjian, 1989; Faisst and Meyer, 1992), (b) antisense oligonucle- otides (Uhlmann and Peyman, 1990), (c) triplex- forming oligonucleotides (Alunni-Fabbroni et al., 1995) and (d) polymerase chain reaction (PCR)- generated DNA fragments (Gambari and Nastruzzi, 1994), have been considered for modu- lating gene transcription. The above mentioned nucleic acid molecules provide opportunities to either replace the missing/defective gene or arrest the expression of specific genes. Nevertheless, after administration, nucleic acid molecules should remain stable in extracellular environment in order to exert a pharmacological effect. In this view, both antisense and transfec- tion technologies require reliable and efficient sys- tems for their delivery into target cells. On the basis of this consideration, the design of an effi- cient nucleic acid delivery system represents one of the key steps for these therapeutic agents (Langer, 1990). Various approaches have been proposed, such as neutral or cationic liposomes and polymeric microparticles (Janoff, 1992; Gre- goriadis, 1988; Thierry et al., 1992; Cortesi et al., 1994b). Particulate systems could indeed reduce the metabolization of oligonucleotides and en- hance specific cellular uptake (Feigner, 1990; Hug and Sleight, 1991). Moreover, liposomes are known to preserve the entrapped compound from enzymatic degradations and to allow targeting strategies. For the preparation of liposomes in- tended for nucleic acid delivery, two approaches appear particularly suitable: (a) the use of a neu- tral lipid composition by 'minimal volume entrap- ment' technique (Thierry et al., 1992) and (b) the use of cationic liposomes prepared by extrusion technique (Cortesi et al., 1994a). It should be pointed out that, in contrast with 'conventional' ones, cationic liposomes do not entrap DNA molecules within their interior. The nucleic acid molecules are bounded by ionic interactions on the surface of preformed cationic liposomes. The surfaces of these vesicles are positively charged due to the presence of quaternary ammonium detergents in the liposome composition, such as DOTMA N- 1- 2,3-dioleyloxy)propyl)-N,N,N- trimethyl-ammonium chloride) (Feigner et al., 1987; Thierry et al., 1992), DEBDA (diisobutyl- cresoxy-ethoxy-ethyl-dimethyl-benzyl-ammonium hydroxyde) (Ballas et al., 1988) or CTAB (cetyltrimethyl-ammonium bromide) (Pin- naduwage et al., 1989). In this way, negatively charged nucleic acids complex the surface of pre- formed cationic liposomes. For this reason, the term 'liposome-associated DNA' should be more correctly used to describe this type of liposome. In spite of their simple preparation protocol, cationic liposomes display some disadvantages, such as cytolitic and cytotoxic activity. Yoshihara and Nakae (1986) have demonstrated that cationic liposomes containing stearylamine showed an in vivo toxicity in rabbit. This effect was attributed to hemolysis of the erythrocytes and was directly related to the amount of steary- lamine present in the liposome composition. Moreover, Feigner et al. (1987) reported that vesicles containing DOTMA, used to facilitate the fusion of liposome/DNA complex with plasma membrane, could result in a cytotoxic effect. This phenomenon is attributable to the presence of the cationic detergent, causing a disruption-solubiliza- tion process of cell membranes (Lappalainen et al., 1994). The aims of this work were the evaluation of parameters influencing the in vitro toxicity of cationic liposomes on cultured cell lines. In addi- tion, the study investigated the stability of DNA molecules complexed to cationic liposomes after exposure to exo- and endonucleases present in serum. In particular this report describes: (a) the preparation of positively charged liposomes by using three different cationic surfactants, namely didodecyl-dimethyl-ammonium bromide (DDAB~2), dioctadecyl-dimethyl-ammonium bro- mide (DDAB18) and cetyl-trimethyl-ammonium bromide (CTAB); (b) the cytotoxic activity of the  R. Cortesi et al. / International Journal of Pharmaceutics 139 1996) 69-78 71 obtained cationic liposomes on in vitro cultured human K562 erythroleukemic cells; and finally (c) the protective effect of cationic liposome complex- ation on lambda phage DNA (2DNA) degrada- tion mediated by serum nucleases. 2. Experimental procedures 2.1. Materials Egg phosphatidyl choline was purchased from Lipid Products (Surrey, England). 2 phage DNA (2DNA) was obtained from Pharmacia (Uppsala, Sweden). Cholesterol was obtained from Fluka (Bucs, Switzerland). Quater- nary ammonium detergents didodecyl-dimethyl- ammonium bromide (DDAB~2) and dioctadecyl-dimethyl-ammonium bromide (DDABI8) were purchased from Fluka, cetyl- trimethyl-ammonium bromide (CTAB) was pur- chased from Sigma Chemical Co. 2.2. Liposome preparation Cationic liposomes were prepared by the re- verse-phase evaporation (REV) method (Szoka and Papahadjopoulos, 1978). The aqueous phase consisted of 1 ml of water, the organic phase was a solution of egg phosphatidylcholine (PC), cholesterol (CH) and the cationic detergent (CD) in 4 ml of diethyl ether. The molar ratio of the liposome constituents was PC:CH:CD 8:2:1, mol/ mol/mol. The biphasic system was vortexed and soni- cared at 0°C for 5 rain in a bath-type sonicator. The ether present in the obtained stable emulsion was removed by rotary evaporation under re- duced pressure at room temperature, resulting in a turbid, white liposome dispersion. In order to obtain homogeneously sized vesicles, the REV liposomes were then extruded through polycar- bonate filters with different pore sizes. The forma- tion of the liposome-DNA complex was carried on by mixing the unilamellar vesicles composed of PC, CH and CD (1 mg of total lipid in 1 ml of water) with a solution containing 0.3 /zg of 2DNA. 2.3. Liposome morphology Shape and surface characteristics of the ob- tained liposomes were studied by freeze fracture electron microscopy. Briefly, after freezing the sample by propane jet technique, the cryofixed preparation was fractured at 108°K in a Balzen BAF 300 at 10 -s Pa. Photomicrographs were taken on Agfa Scientia 23D56 cut films and devel- oped in Geratone G5C for 3.5 min at 293°K. The dimensional analysis of liposomal suspensions was performed by a photon correlation granu- lometer SEM F60 (SEMATech, Nice, France) equipped with a computer-driving RD goniometer and an argon ion laser, operating at a few mW of power of 2 = 514nm. 2.4. Analysis of the electrophoretic mobility of liposome-DNA complexes VET~o0 containing increasing concentrations of cationic detergent (8:2:0.5, 8:2:1, 8:2:1.5, 8:2:2, mol/mol/mol) were mixed with 2DNA. Lipo- some/2DNA complexes (9:1, w/w ratio) were in- cubated at 37°C for 5 min, then each sample was subjected to electrophoresis. Electrophoresis was carried on in a 0.8% agarose gel at constant voltage (100 mV) for 2 h in the absence or in the presence of increasing concentrations of CD. The relative band migration was determined, after staining the gels with ethidium bromide. 2.5. DNA stability studies The stability of liposome/2DNA complexes to- wards fetal calf serum (FCS) containing nucleases was studied following the above protocol. 2DNA (0.3 mg) (Pharrnacia, Uppsala, Sweden) were complexed to different amounts of cationic lipo- somes, resulting in final liposomes/DNA ratios of 3:1, 9:1 and 12:1, w/w. The liposome/DNA com- plexes were then incubated at 37°C in a thermo- static bath. At different time intervals, comprising between 0 and 240 min, samples were withdrawn and stored at -20°C until electrophoretic analy- sis was performed. Electrophoresis was performed on 0.8% agarose gels containing 0.5 mg/ml ethid- ium bromide for 2 h at 25 mV constant current.  72 R. Cortesi et al. / International Journal of Pharmaceuties 139 1996) 69-78 After electrophoresis, the 2DNA bands were visu- alized by ultraviolet (UV) shadowing (Maniatis et al., 1982). The quantitative analysis of band inten- sity was performed by a computerized scanning analysis of digitalized images. For this analysis, the 'NIH Image', a public domain image process- ing and analysis program for the Macintosh, was used. 2.6. Cytotoxicity studies The cytotoxicity of cationic liposomes was de- termined on in vitro cultured human leukemic K562 cells (Lozzio and Lozzio, 1975). Standard conditions for cell growth were a-medium (Gibco, Grand Island, NY), 50 mg/1 streptomycin, 300 mg/1 penicillin, supplemented with 10% FCS (Irvine Scientific, Santa Ana, CA) in 5% CO2 at 90% humidity. Cell growth was determined by counting with a Model ZF Coulter Counter (Coulter Electronics Inc., Hielah, FL). Counts of viable cells were performed after 0.1% Trypan blue exclusion test. 3. Results and discussion As previously stated in the introduction, the use of cationic liposomes as a delivery system for nucleic acids could result in a series of advantages over 'conventional' liposomes. Nucleic acids are not exposed to denaturing conditions possibly present in other liposome preparation protocols (i.e., sonication, organic solvents) (Felgner et al., 1987; Bennet et al., 1992; Thierry et al., 1992). The liposome/DNA complexation can be per- formed just before use, by simply mixing nucleic acid to 'preformed' vesicles. Finally, both lipo- somes and DNA can be stored in lyophilized form and rehydrated and mixed just prior the use, resulting in a quantitative 'association' yield. 3.1. Liposome preparation Positively charged liposomes were produced by employing a protocol based on reverse phase evaporation followed by extrusion of the liposome suspension through polycarbonate filters with ho- mogeneous pore size. Liposomes were subjected to one extrusion cycle through two stacked 200 nm pore size filters, followed by three extrusion cycles through two stacked 100 nm pore size membranes. This choice was made on the basis of many papers that describe vesicles with an aver- age diameter around 100 nm as good candidates for both in vitro and in vivo studies using differ- ent administration routes (i.e., intramuscular, en- dovenous, subcutaneous) (Janoff, 1992). The extruded liposomes were named VET~00, accord- ing to Mayer et al. (1986), where VET indicates vesicles produced by extrusion techniques and the subscript number the pore size of the membrane used for the extrusion. Three different cationic detergents were alterna- tively used for the production of positively charged liposomes, namely CTAB, DDAB12 and DDAB18. 3.2. Morphological analysis The morphological and dimensional analysis of the produced liposomes was performed by freeze- fracture electron microscopy technique and dy- namic light scattering. Electron microscopy demonstrated that the liposomal suspensions were constituted of unilamellar vesicles with an average diameter reflecting the pore size of the utilized membrane. Dynamic light scattering studies demonstrated that the extruded vesicles present a narrow size distribution. In Table 1 are reported the volume-weighted and intensity-weighted parti- cle size analyses of the three cationic liposome suspensions as determined by dynamic light scat- tering. 3.3. Cytotoxicity studies In order to obtain information about the cyto- toxicity of cationic liposomes, in vitro assays were performed by cultivating human erythroleukemic K562 cells in the presence of liposomes. Increas- ing concentrations of cationic liposomes, having a PC:CH:CD 8:2:1, mol/mol/mol composition, were added to the cell. Concentrations comprised be- tween 0 and 40 mg/ml of liposome corresponding to 0.1-10 mM of CD were tested. The results,  R. Cortesi et al. / International Journal of Pharmaceutics 139 1996) 69-78 Table 1 Particle size distribution of cationic liposomes produced by extrusion technique* 73 Cationic surfactant Mean diameter (S.D.) (nm) Dispersion (S.D.) (nm) Polydispersity (S.D.) Volume-weighted particle size analysis DDABls 114.8 (10.8) 19.4 (3.2) 0.215 (0.062) DDABI2 123.9 (33.0) 60.0 (9.8) 0.235 (0.069) CTAB 104.5 (32.5) 49.5 (10.5) 0.224 (0.079) Intensity-weighted particle size analysis DDAB~8 120.1 (31.0) 55.7 (9.3) 0,215 (0.062) DDABI2 140.0 (37.3) 67.8 (11.1) 0.235 (0.068) CTAB 121.3 (37.7) 57.4 (12.2) 0.224 (0.079) *Data refer to VETioo constituted of PC:CH:CD 8:2:1 (mol/mol/mol). Data were obtained by using a photon correlation granulometer SEM F60 (SEMATech, Nice, France) computer-driving RD goniometer and an argon ion laser. equipped with a reported in Fig. 1, indicate that CTAB- and DDAB~2-1iposomes showed a rather pronounced cytotoxicity, with ICso values of 0.88/~M and 0.85 ItM, respectively (where IC50 is the compound concentration inhibiting 50% of the cell growth). On the contrary, DDAB~8-1iposomes displayed only a low cytotoxic effect (ICs0 > 25 mM). Liposomes (lag/ml) 0 10 20 30 40 50 L , I i L i L I [ I O e- 2 100~ E, [] [] ~ ~ ~ [] E~ 80 ~ 0 60 40 20 t 0 o • 0 O ~ J I 1111111 I I~111 11~ J i i iiiill 0.1 1 10 100 Cationic detergent (p.M) Fig. 1. In vitro cytotoxic effect of different VETlo o cationic liposomes, containing CTAB (O), DDABt2 (O) or DDAB18 (D), on human erythroleukemic K562 cells. The PC:CH:CD molar ratio was 8:2:1, mol/mol/mol. Determinations were performed after 5 days of cell culture. Data represent the % of cell number/ml compared to untreated control K562 cells. The reported results are the average of 3 independent experiments, SD _< 7'7,,. Moreover, we compared the cytotoxicity of the CTAB-, DDAB~2-, and DDABls-liposomes with the respective cationic detergent solutions (micel- lar solutions). As shown in Fig. 2 (panels A and B), the curve trends of CTAB- and DDABlz-lipo- some suspensions are almost superimposable with those of the respective micellar solution, whilst in the case of DDAB~s (Fig. 2C) the two curves diverge one from the other. As clearly evident, the cytotoxicity of DDABzs is lower when this cationic detergent is in liposome form. Table 2 summarizes the ICs0 values found for the three cationic detergents used as liposome suspensions or micellar solutions. Furthermore, the influence of liposome/CD molar ratio on liposome cytotoxicity was evalu- ated. Four liposome compositions characterized by different PC:CH:CD molar ratios were tested, namely 8:2:0.5, 8:2:1, 8:2:1.5 and 8:2:2 (mol/mol/ mol). The obtained results demonstrated that within the molar ratio interval used, no apprecia- ble difference in the cytotoxic activity was ob- served. Nevertheless, in the case of CTAB and DDABIs, one could observe a slightly increased antiproliferative effect for liposomes containing a higher proportion of cationic detergent (data not shown). 3.4. Binding migration studies In order to evaluate the strength of the interac- tion occurring between DNA and liposomes and
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