Langmuir 1997, 13, 2215-2218
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Interactions between Cationic Vesicles and Cultured Mammalian Cells A. M. Carmona-Ribeiro,* F. Ortis, R. I. Schumacher, and M. C. S. Armelin Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, Caixa Postal 26077, Sa˜ o Paulo 05599-970, SP, Brazil Received July 31, 1996. In Final Form: December 10, 1996 The interaction of small cationic vesicles composed of dioctadecyldimethylammonium bromide (DODAB) with normal versus transformed mouse fibroblasts is described using cell microelectrophoresis, turbidimetry, and cell viability assays over a wide range of DODAB concentrations (10-7-10-3 M). Normal and transformed cells (104 cells/mL) attain a point of zero charge at, respectively, 18.0 and 1.6 µM DODAB. Further increasing DODAB concentration (C) generates positively charged cells. At 105 cells/mL and C g 50 µM, DODAB induces cell-cell adhesion. For transformed and normal cells, peak adhesion occurs at 100 and 1000 µM DODAB, respectively. Upon 0.5 h interaction time with 100 µM DODAB, at 104 cells/mL, 20% of cell death is obtained for normal cells whereas transformed cells remain unaffected. Transformed cells have a higher affinity for DODAB vesicles than their normal counterparts but are more resistant to DODAB-induced cell death. The results indicate that DODAB vesicles interact with cells with very high affinity at low ionic strength and are not toxic below 1 mM, suggesting that they might successfully deliver oppositely charged proteins or DNA strands to cells. These results may be of importance for liposome-mediated processes currently being used for drug or gene delivery to cells.
1. Introduction Phospholipids in water form closed and concentric multilamellar vesicles that are widely used as model systems for cell membranes.1 The medical utility of liposomes made up of phospholipids or synthetic amphiphiles as drug, gene, or antigen carriers is irrefutable.2-4 However, a few gaps of knowledge still exist concerning basic physicochemical aspects of the interaction between liposomes or vesicles and biological surfaces. These gaps are particularly deep for interactions between synthetic amphiphile vesicles5 made up of the cationic amphiphile dioctadecyldimethylammonium bromide (DODAB) and biological surfaces. We recently characterized, from a physicochemical point of view, the interaction between small cationic vesicles and bacteria.6,7 There is a high affinity between cationic vesicles and bacteria (nonreversible adsorption) with limiting adsorption values for the vesicle-forming amphiphile pointing to vesicle adhesion to bacteria without vesicle rupture.6 The fact that vesicles in the rigid gel state do not disintegrate upon adsorption to the bacterial cell surface might be of considerable significance to deliverance by endocytosis of vesicles containing drugs inside the vesicle aqueous compartment. Bacteria flocculation and death are induced by cationic vesicles, although cell death is not related to aggregation since it also takes place in nonaggregated cells at very low bacteria concentrations.7 Here we characterize the interaction of cationic liposomes composed of DODAB with both normal * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, March 15, 1997. (1) Bangham, A. D. Liposome Letters; Academic Press: London, 1983. (2) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (3) Gregoriadis, G., Ed. Liposome Technology; CRC: Boca Raton, FL, 1993. (4) Katz, D.; Kraaijeveld, C. A.; Snippe, H. Synthetic Lipoid Compounds as Antigen-specific Immunostimulators for Improving the Efficacy of Killed-virus Va¨ccines. In Theory and Practical Application of Adjuvants; Stewart-Tull, D. E. S., Ed.; John Wiley & Sons: Chichester, England, 1995. (5) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 1992, 21, 209. (6) Ta´pias, G. N.; Sicchierolli, S. M.; Mamizuka, E. M.; CarmonaRibeiro, A. M. Langmuir 1994, 10, 3461. (7) Sicchierolli, S. M.; Mamizuka, E. M.; Carmona-Ribeiro, A. M. Langmuir 1995, 11, 2991.
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and transformed mammalian cells. The experimental strategy adopted consists of determining electrophoretic mobility, turbidity of cell suspensions, and cell viability for normal and transformed cells over a wide range of DODAB concentrations. Although SVT2 transformed cells present a higher affinity for DODAB, they are more resistant to DODAB-induced cell death than their normal 3T3 counterparts. The moderate DODAB cytotoxicity obtained below 1 mM DODAB suggests a good potential for DODAB as a system to deliver drugs, antigenic proteins, or genes to mammalian cells at low ionic strength. 2. Material and Methods 2.1. Cells and Culture Conditions. Normal Balb-c 3T3 (clone A31) mouse fibroblasts (17) and its SV40-transformed SVT2 counterpart8 were obtained from the American Type Culture Collection (Rockville, MD). Cell lines were cultured under a 5% CO2 atmosphere, at 37 °C, in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Cell suspensions were obtained from monolayer cultures upon incubation with 0.264 M D-glucose for 30 min. Cell concentration (cells/mL) was determined using a Neubauer chamber. Further dilutions of cell suspensions were always made in 0.264 M D-glucose solution. 2.2. Vesicle Preparation. DODAB (99.9% pure from Sigma Chemical Co.) was used without further purification. Small unilamellar DODAB vesicles (SV) with 86 nm mean zeta-average diameter9 were prepared by sonication in a 0.264 M D-glucose solution with tip.5 SV were centrifuged at 10000g for 1 h at 15 °C to precipitate multilamellar liposomes and titanium particles ejected from the titanium probe during sonication. DODAB concentration was determined by microtitration.10 2.3. Interaction between Vesicles and Cells. Due to the low stability of DODAB vesicles in the presence of electrolyte at room temperature,5 cells were suspended in 0.264 M D-glucose before vesicle addition. Thereafter, all desired dilutions of vesicles or cells were made in 0.264 M D-glucose solution so that cells and vesicles were kept in a perfectly isotonic environment throughout and the vesicles presented high colloidal stability. Interaction between vesicles and cells was induced by adding the vesicles to the cells. 2.4. Cell Microelectrophoresis. Equal volumes of the cell suspensions (5 × 104 cells/mL) and SV, both in 0.264 M D-glucose, (8) Aaronson, S. A; Todaro, G. Science, 1968, 162, 1024. (9) Carmona-Ribeiro, A. M.; Midmore, B. R. Langmuir 1992, 8, 801. (10) Schales, O.; Schales, S. S. J. Biol. Chem. 1941, 140, 879.
© 1997 American Chemical Society
2216 Langmuir, Vol. 13, No. 8, 1997 were mixed and allowed to interact at 25 °C for 1 h before measuring electrophoretic mobilities (EM) using a Rank Brothers microelectrophoresis apparatus with a flat quartz cell at 25 °C. The sample to be measured was placed into the electrophoresis cell, electrodes were connected, and a voltage of (60 V was applied across the cell. Velocities of individual cells over a given tracking distance and direction of cell movement were recorded. Average velocities were calculated from data obtained with at least 20 individual cells. EM was calculated according to the equation EM ) cm(u/V)(1/t) where u is the distance over which the cell is tracked (micrometers), cm is the interelectrodes distance (7.27 cm), V is the voltage applied ((60 V), and t is the average time, in seconds, required to track one cell in a given distance u. 2.5. Cell Adhesion Experiments. Turbidity at 500 nm was continuously recorded as a function of time upon vesicle addition to the cells using a Hitachi U-2000 spectrophotometer in the single-beam mode. The time lag between mixing and recording was usually smaller than 10 s. 2.6. Viability Assays. 2.6.1. Fluorescence Microscopy. Adherent cells were cultured on sterile glass coverslips as a subconfluent monolayer. Thereafter, cells were washed with Dulbecco’s phosphate-buffered saline (PBS) to remove the culture media, incubated with DODAB SV (0.01, 0.1, or 1 mM) for 30 min, washed again with PBS, and finally incubated with 0.15 mL of the combined LIVE/DEAD assay reagents for 30 min. The LIVE/DEAD EukoLight viability/citotoxicity kit L-3224 (Molecular Probes, Inc., Eugene, OR) is a two-color fluorescence cell viability assay based on the simultaneous determination of live and dead cells with two probes that measure two recognized cell viability parameters, namely, intracellular esterase activity and plasma membrane integrity. Live cells present esterase activity that converts the nonfluorescent cell-permeant calcein acetoxymethyl ester (calcein AM) to the intensely fluorescent calcein, producing an intense uniform green; whereas ethydium D-1 enters cells with damaged membranes and undergoes a 40× enhancement of fluorescence upon binding nucleic acids, thereby producing a bright red fluorescence in dead cells. Fluorescence microscopy was performed using a Nikon Fluophot with a 200 W Hg lamp. 2.6.2. Flow Cytometry. Cell suspensions were treated with increasing DODAB concentrations (0-1 mM) for 30 min, centrifuged at 3500g for 5 min and incubated in calcein-AM (1 µg/mL) for 15 min. The suspensions were centrifuged again, resuspended in PBS, and analyzed in a FACSTAR-PLUS cytofluorimeter-sorter (Beckton & Dickinson, Heidelberg, FRG). Routinely, 20 000 cells were acquired; photomultiplier for fluorescence, forward scatter, and sideward scatter were adjusted to the log mode. Data were analyzed by the Lysis II program (Beckton & Dickinson). Individual measurements of forward scatter and green fluorescence (calcein) signals were plotted as a linear-log countour-plot. The dashed region in this plot (see Figure 4) represents affected cells.
3. Results and Discussion 3.1. DODAB Effect on the Cell Surface Charge. The electrophoretic mobility of normal A31 and its SV40transformed SVT2 counterpart increases as a function of DODAB concentration (C) (Figure 1). At 104 cells/mL, there is a DODAB concentration where EM is equal to zero: 1.6 and 18.0 µM DODAB for A31 and SVT2, respectively. Mobilities measured in the absence of DODAB were -2.92 and -2.27 for SVT2 and A31, respectively. A direct correlation between surface sialic acids and surface charge on the cell is likely to occur. DODAB readily neutralizes the negative surface charge on SVT2. Neuraminidase susceptible sialic acid,11 alkaline phosphatase susceptible phosphate groups, and unidentified anionic, amino, and sulfydryl groups have been detected on the cell surface.12,13 Overall, the observation that transformed cells have a higher number of charged groups and hence a larger EM in the absence of DODAB (11) Patinkin, D. Cancer Res. 1970, 30, 489. (12) Mehrishi, J. N. Nature 1970, 226, 452. (13) Mehrishi, J. N.; Grassetti, N. R. Nature 1969, 224, 563.
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Figure 1. Effect of DODAB concentration (C) on electrophoretic mobility (EM) of mammalian fibroblasts in suspension containing 0.264 M D-glucose. Normal A31 and transformed SVT2 cells are represented by the symbols b and O, respectively. Cell concentration is 104 cells/mL.
Figure 2. Effect of DODAB concentration on turbidity at 500 nm for a suspension of mammalian fibroblasts in 0.264 M D-glucose. Cell concentration is 105 cells/mL. DODAB molar concentrations are 10-6 (a), 5 × 10-6 (b), 10-5 (c), 5 × 10-5 (d), 10-4 (e), 5 × 10-4 (f), and 10-3 (g).
is in acordance with the literature.14 This explains, in part, the higher affinity for cationic vesicles displayed by SVT2 cells (Figure 1). 3.2. DODAB Effect on Cell-Cell Adhesion. Cell adhesion was monitored upon DODAB addition as turbidity at 500 nm as a function of time (Figure 2). SVT2 and A31 initial adhesion rates attain a maximum at 0.1 and 1 mM DODAB, respectively. Adhesion rate is expected to be related not only to charge neutralization but also to accessibility of anionic groups on the cell surface to the cationic SV. An intricate network of cell adhesion molecules and receptors involved in cell-cell and cellsubstratum interactions like adhesins and integrins are expressed by normal cells.15 They create a considerable steric hinderance to be overcome by the vesicles which slows down DODAB-induced adhesion of normal cells. Although it is more difficult for the vesicles to reach anionic sites on the normal cell surface, once neutralization is achieved, the extent of adhesion observed for the normal cells is greater than that for SVT2 (Figure 2). A decreased sialic acid content on cell adhesion molecules correlates (14) James, A. M. Chem. Rev. 1979, 79, 389. (15) Cunningham, B. A. Trends. Biochem. Sci. 1986, 11, 423.
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Figure 4. Effect of DODAB concentration on incorporation of calcein by A31 cells as determined using flow cytometry. DODAB concentrations are 0 (A), 0.01 (B), 0.1 (C), and 1.0 mM (D).
Figure 3. Fluorescence microscopy of A31 (on the left) and SVT2 cells (on the right) after 0.5 h interaction with small cationic DODAB vesicles. DODAB concentrations are 0.01 (A, D), 0.1 (B, E), and 1 mM (C, F).
with enhanced adhesion properties.16 Therefore, the more extensive adhesion obtained for A31 cells is consistent with its comparatively lower sialic acid content relative to SVT2. Consistently, DODAB neutralization of sialic acid groups enhances cell adhesion (Figure 2). Because of the defective nature of the extracellular matrix and its adhesion proteins displayed by SVT2 cells, the maximal extent of adhesion attained by these cells is much smaller than that displayed by A31 cells (Figure 2). However, the adhesion rate attains its maximum at smaller DODAB concentrations since anionic groups on the SVT2 cell are readily accessible to neutralization. In fact, the extracellular matrix on normal A31 cells represents a protective network that is likely to sterically prevent the close approach of cationic vesicles. It is important to emphasize that cell-cell adhesion was observed for cells in suspension only from 105 cells/mL. At smaller cell concentrations, rapid adhesion could not be induced by DODAB. A similar result was obtained for the interaction between DODAB SV and bacteria with rapid flocculation being induced from 107 bacteria/mL.7 It is interesting to notice that mammalian cells are still inside the range of colloidal particles and could therefore have their colloidal stability eventually described under the light of current formalisms available for colloids. 3.3. DODAB Cytotoxicity. Interaction between a subconfluent cell monolayer and DODAB for 30 min causes cell death in both cell types (Figure 3C,B,E,F). However, in the subconfluent monolayer, cell concentration is low and quantification of cell death as a percentile of the total number of cells attached to the coverslip is technically difficult. Quantification was achieved using flow cytometry for cells in suspension. Increasing DODAB concentration causes loss of the calcein content of the cell (Figure 4). This was observed for A31 and for SVT2 cell lines with similar fluorescence distributions (not shown). The (16) Sunshine, J.; Balak, K.; Rutishauser, U.; Jacobson, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5986.
Figure 5. Resistance of A31 and SVT2 cell lines to treatment with DODAB. The relative cytotoxicity is expressed as percentage of dead cells as a function of DODAB concentration in a logarithmic scale. Final cell concentration in the mixtures is 104 cells/mL.
forward signal (a measure of cell size) also drops significantly in DODAB-treated populations, indicating changes in the membrane structure. Figure 5 shows that increasing DODAB concentration decreases viability of both cell lines. At 1 mM DODAB, there is 50% cell death (Figure 5). Although SVT2 presents a higher affinity for DODAB (Figure 1), it is also more resistant to DODAB-induced death as depicted from its higher viability at 0.1 mM DODAB (Figure 5). Cytotoxicity of DODAB vesicles does not seem to be a problem below 1 mM DODAB. Furthermore, cytotoxicity is greater for normal cells indicating that drug or gene delivery could possibly be achieved where it is necessary, namely, in the defective transformed cells (more resistant to DODAB than normal cells). Although many synthetic cationic lipids have already been synthesized with the specific purpose of testing their capabilities for gene transfer,17 only a few are effective. DODAB is among the effective ones, being as effective as synthetic lipospermine (known as Transfectam) over 0.5 h interaction time.18 Since natural lipid headgroups are either zwitterionic or anionic, masked “cationic” plasmids should associate spontaneously and rapidly with the cell membrane through a cooperative ionic interaction and (17) Remy, J. S.; Sirlin, C.; Vierling, P.; Behr, J. P. Bioconjugate Chem. 1994, 5, 647. (18) Behr, J. P.; Demeneix, B.; Loeffler, J-P.; Perez-Mutul, J. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6982.
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subsequently be internalized by endocytosis. To our knowledge, there is only one report on the capability of dioctadecyldimethylammonium chloride, which is different from DODAB only by its counterion, to promote transfection.18 Melanotrophs, which are mammalian primary endocrine cells, were incubated for various time periods with DNA-DODAC complexes, and expression of the bacterial chloramphenicol acetyltransferase gene was evaluated.18 After 30 min incubation, incorporation of radioactively-labeled uridine, used to evaluate cell viability, was about the same as that obtained for the DNA-lipospermine complex. Unfortunately, the next measurement was obtained only at 18 h of incubation time,18 when the relatively high DODAB concentration used (1 mM) is indeed expected to be cytotoxic (Figure 5). At 18 h, uridine incorporation significantly fell18 as a consequence of cell death (Figure 5). A systematic evaluation of the full potential of DODAB to transfect genes is still lacking in the literature as is a complete and systematic determination of the effects of interaction time and DODAB concentration on cytotoxicity. These issues are currently being dealt with in our laboratories for both mammalian cells and bacteria. Another important open issue regards the possibility of using DODAB at physiological ionic strength. In the present experiments the interaction between DODAB vesicles and cells was carried out in isotonic D-glucose solution. The fact that these synthetic amphiphile vesicles are unstable in a colloidal sense, i.e. they flocculate upon addition of monovalent salt,19-24 does not mean that they (19) Carmona-Ribeiro, A. M.; Yoshida, L. S.; Chaimovich, H. J. Phys.Chem. 1985, 89, 2928. (20) Carmona-Ribeiro, A. M. J. Phys. Chem. 1989, 93, 2630.
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cannot be stabilized “n vivo”. Snippe and co-workers indirectly offered several examples of a very high stability for DODAB-antigen complexes presented to the immunological system.4 DODAB is well-established as an efficient immunoadjuvant able to improve humoral and cellular response to several antigens in experimental animals and also in humans.4 Therefore, it is premature to discard DODAB vesicles as carriers in vivo only on basis of their low colloid stability in the presence of salt. We are presently investigating the interaction between DODAB and albumin in order to evaluate possible effects that serum proteins may have on DODAB stabilization in vivo. Conclusions At low ionic strength, DODAB small vesicles bind with high affinity to normal and transformed mammalian cells in culture, cause cell-cell adhesion at high cell concentration in suspension, and are moderately cytotoxic below 1 mM DODAB. Transformed cells are more resistant than normal cells to DODAB-induced cell death. The full potential of DODAB as a drug, protein, or DNA delivery system remains to be explored. Acknowledgment. This work was supported by FAPESP, CNPq-PADCT, ICGEB-UNIDO, Fundac¸ a˜o Banco do Brasil, and CABBIO. F.O. holds an undergraduate training fellowship from FAPESP. LA960759H (21) Carmona-Ribeiro, A. M. J. Phys. Chem. 1992, 96, 9555. (22) Carmona-Ribeiro, A. M. J. Phys. Chem. 1993, 97, 4247. (23) Carmona-Ribeiro, A. M. J. Phys. Chem. 1993, 97, 11843. (24) Tsuruta, L. R.; Carmona-Ribeiro, A. M. J. Phys. Chem. 1996, 100, 7130.
