Langmuir 1998, 14, 6077-6081
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Interactions between Cationic Vesicles and Serum Proteins Lı´via A. Carvalho and Ana M. Carmona-Ribeiro* Departamento de Bioquı´mica, Instituto de Quı´mica, Caixa Postal 26077, S. Paulo, SP, Brazil Received March 27, 1998. In Final Form: April 22, 1998 Bovine serum albumin (BSA) and anti-bovine serum albumin (a-BSA) were separately incorporated in dioctadecyldimethylammonium bromide (DODAB) liposomes in water or in phosphate buffer saline (PBS). In water, 100% BSA or a-BSA incorporation in the liposomes contrasts with ca. 30% incorporation in PBS for both proteins, suggesting a major role for the electrostatic attraction between protein and liposome in determining the incorporation, though 30% incorporation can still be ascribed to the hydrophobic interaction. Neither of the two proteins, tested separately, can induce nonspecific liposome aggregation over an extensive range of combinations for DODAB and protein concentration as those possible in a microplate experiment. In water, electrophoretic mobility for the liposomes decreases slightly as a function of protein concentration but the liposome/protein complex remains positively charged even at the highest protein concentrations tested. Protein-induced rupture of liposomes containing [14C]sucrose was evaluated from dialysis of protein/ liposome mixtures. In water or in PBS, protein-induced leakage of radioactive liposomal contents was not observed, suggesting that the hydrophobic interaction between serum proteins and the DODAB bilayer is superficial. Finally, the absence of molecular recognition between BSA and a-BSA separately incorporated in the liposomes in water is consistent with preservation of the liposomal positive charge at full protein coverage on the cationic liposomes. These results shed new light onto the mechanisms by which cationic liposomes can deliver proteins or other important macromolecules of biological origin in vivo: opsonization possibly preserves liposome charge and integrity.
Introduction Synthetic quaternary ammonium compounds that form liposomes and vesicles in aqueous medium such as dioctadecyldimethylammonium chloride (DODAC) or bromide (DODAB)1 have outstanding properties as antigenspecific immunostimulators.2 In general, DODAB has proven to be effective in inducing humoral antibodies, resistance to challenge with virulent viruses, and delayedtype hypersensitivity (a marker for cell mediatedimmunity).3-6 On the other hand exogenous particles such as liposomes, emulsion droplets, nanoparticles, and other colloidal drug carriers have their clearance initialized in vivo by adsorbing serum proteins, the so-called opsonins. Serum proteins include albumin, immunoglobulins, complement components, and adhesins such as fibrinogen and fibronectin. An inverse correlation has been found between the amount of proteins adsorbed from serum and the circulation half-life of colloidal drug carriers.7-9 Despite the importance of opsonization, surprisingly little is known of the physicochemical factors determining adsorption of serum proteins on exogenous particles in general10-14 or on cationic DODAB liposomes, in particular. (1) Carmona-Ribeiro, A. M. Chem. Soc. Rev. 1992, 21, 209. (2) Katz, D.; Kraaijeveld, C. A.; Snippe, H. Synthetic lipoid compounds as antigen-specific immunostimulators for improving the efficacy of killed-virus vaccines. In The Theory and Practical Application of Adjuvants; Stewart-Tull, D. E. S., Ed.; John Wiley & Sons: Chichester, England, 1995. (3) Gall, D. Immunology 1966, 11, 369. (4) Prager, M. D.; Kanar, M. C.; Farmer, J. L.; Vanderzee, J. Cancer Lett. 1985, 27, 225. (5) Snippe, H.; Willers, J. M. N.; Inman, J. K.; Merchant, B. Immunology 1980, 39, 362. (6) Coon, J.; Hunter, R. J. Immunology 1973, 110, 183. (7) Chonn, A.; Semple, S. C.; Cullis, P. R. J. Biol. Chem. 1992, 267, 18759. (8) Gregoriadis, G. In Stealth Liposomes; Lasic, D., Martin, F., Eds. CRC Press: Boca Raton, FL, 1995; pp 7-12. (9) Storm, G.; Belliot, S. O.; Daemen, T.; Lasic, D. D. Adv. Drug Deliver. Rev. 1995, 17, 31. (10) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267.
In this work, major factors involved in opsonization of cationic liposomes are evaluated from a physicochemical study of the interaction between bovine serum albumin (BSA) or anti-bovine serum albumin (a-BSA) and cationic DODAB liposomes. These results are important to evaluate possible effects that serum proteins may have on stabilization of DODAB/biomolecule complexes in vivo. Materials and Methods Chemicals. Dioctadecyldimethylammonium bromide 99.9% pure (DODAB) was obtained from Fluka Chemie AG (Switzerland) and used as such without further purification. BSA and a-BSA were purchased from Sigma Immuno Chemicals. a-BSA (product no. B-7276) was developed in rabbit and fractionated to yield primarily the immunoglobulin fraction of antiserum. The antiserum was determined to be immunospecific for BSA versus normal bovine serum, BSA, by the supplier. The product came lyophilized from 0.01 M phosphate buffer saline, pH 7.2 and without addition of preservatives. BSA or a-BSA concentrations were determined from the Lowry method. The isoelectric point for BSA is at ca. pH 5.4 in water and at ca. pH 4.7 and 10 mM ionic strength. Because a-BSA is a polyclonal antibody, a range of isoelectric points is expected to occur. All other reagents were analytical grade and were used without further purification. Water was Milli-Q quality. Liposomes Preparation. A DODAB dispersion containing 10 mg/mL DODAB was prepared by heating the DODAB powder in water or in phosphate buffer saline (PBS) for 30 min at 56 °C, as previously described.2 The analytical DODAB concentration was determined by microtitration.15 Determination of BSA or a-BSA Incorporation in DODAB Liposomes. The interaction protein/liposome was promoted by mixing 0.5 mL of a 5 mg/mL DODAB dispersion with increasing (11) Haynes, C. A.; Norde, W. Colloids Surf. B 1994, 2, 517. (12) Malmsten, M.; Van Alstine, J. M. J. Colloid Interface Sci. 1996, 177, 502. (13) Malmsten, M. J. Colloid Interface Sci. 1995, 172, 106. (14) Malmsten, M.; Lassen, B.; Van Alstine, J. M.; Nilsson, U. R. J. Colloid Interface Sci. 1996 178, 123. (15) Schales, O.; Schales, S. S. J. Biol. Chem. 1941, 140, 879.
S0743-7463(98)00345-X CCC: $15.00 © 1998 American Chemical Society Published on Web 09/24/1998
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Table 1. Microplate Description in Terms of Protein/DODAB Concentrations per Well in the Microplate Experiment Used for Evaluation of Protein Induced-liposome Flocculationa A B C D E F G H
1
2
3
4
5
6
7
8
9
10
4 0.0078 2 0.0039 1 0.0019 0.5000 0.0009 0.2500 0.0005 0.1250 0.0003 0.0625 0.0001 0.0313 0.00006
2 0.0156 1 0.0078 0.5000 0.0039 0.2500 0.0019 0.1250 0.0009 0.0625 0.0005 0.0313 0.0003 0.0156 0.0001
1 0.0313 0.5000 0.0156 0.2500 0.0078 0.1250 0.0039 0.0625 0.0019 0.0313 0.0009 0.0156 0.0005 0.0078 0.0003
0.5000 0.0625 0.2500 0.0313 0.1250 0.0156 0.0625 0.0078 0.0313 0.0039 0.0156 0.0019 0.0078 0.0009 0.0039 0.0005
0.2500 0.1250 0.1250 0.0625 0.0625 0.0313 0.0313 0.0156 0.0156 0.0078 0.0078 0.0039 0.0039 0.0019 0.0019 0.0009
0.1250 0.2500 0.0625 0.1250 0.0313 0.0625 0.0156 0.0313 0.0078 0.0156 0.0039 0.0078 0.0019 0.0039 0.0009 0.0019
0.0625 0.5000 0.0313 0.2500 0.0156 0.1250 0.0078 0.0625 0.0039 0.0313 0.0019 0.0156 0.0009 0.0078 0.0005 0.0039
0.0313 1 0.0156 0.5000 0.0078 0.2500 0.0039 0.1250 0.0019 0.0625 0.0009 0.0313 0.0005 0.0156 0.0003 0.0078
0.0156 2 0.0078 1 0.0039 0.5000 0.0019 0.2500 0.0009 0.1250 0.0005 0.0625 0.0003 0.0313 0.0001 0.0156
0.0078 4 0.0039 2 0.0019 1 0.0009 0.5000 0.0005 0.2500 0.0003 0.1250 0.0001 0.0625 0.00006 0.0313
a Composition of mixtures in each cell is given in terms of BSA (or a-BSA) and DODAB concentrations (in mg/mL). Upper figures in each cell refer to protein concentration, whereas lower figures, to DODAB concentration.
volumes of a 5 mg/mL protein solution to a final volume of 1 mL in each mixture. Therefore, the total protein amount in the mixtures varied between 0 and 2.5 mg/mL, whereas the DODAB final concentration was fixed at 2.5 mg/mL. Thereafter, the mixture was incubated (l h, 40 °C) and centrifuged (15800g/l h/4 °C) to separate liposomes from free protein in solution. The supernatant was filtered through polycarbonate membranes (0.2 µm cutoff). Liposome retention by the filtering membranes was evaluated from determination of turbidity at 400 nm in the filtered solution. Protein incorporation into liposomes was obtained from protein determination16 in the filtered solutions where liposomes were completely absent. A similar procedure was used for BSA or a-BSA in water or in PBS. The percentage of protein incorporation was calculated from µg of protein adsorbed per mg DODAB. Assay for Protein-Induced Liposome Flocculation. Each well in a polystyrene microplate was precoated with 0.1 mL of BSA 1% in carbonate/bicarbonate buffer at pH 9.6 (overnight/4 °C), emptied, and washed three times with water before adding BSA solution over a range of concentrations (4 mg/mL up to tenths of (µg/mL). A 4 mg/mL BSA solution was successively diluted in the proportion of 1:2. Then 0.2 mL of these solutions was added to rows A to H/column 1 of the precoated microplate. Similarly, a 4 mg/mL DODAB dispersion prepared as previously described was sucessively diluted in the proportion of 1:2 and these solutions were added to rows A to H/column 10 of a second microplate, which was not precoated. For each of the two microplates, a multichannel pipet with eight channels was used for sucessive dilution of solutions from column 1 up to column 10 or from column 10 up to column 1 in the proportion of 1:2. Therefore, one microplate contained DODAB dispersions, whereas the other contained BSA solutions, both diluted over an extensive range of concentrations. Finally, contents of the two microplates were mixed by adding 0.1 mL of DODAB dispersions from column 1 in one plate to 0.1 mL of BSA solutions in column 1 of the other plate using a multichannel pipet. In this way, well A1 (row A and column 1) contained the most concentrated BSA solution (4 mg/mL) and the less concentrated DODAB dispersion (7.8 µg/ mL). Final BSA or DODAB concentrations are given in Table 1. Just after mixing liposomes and protein, the turbidity at 405 nm was recorded as a function of time for 30 min using an Easy Reader EAR 400AT (SLT-Labinstruments). A similar experiment was done for vesicles and BSA or a-BSA in PBS with the difference that the microplate to be used for a-BSA dilutions was precoated with Molico 10%. Determination of Liposome Rupture upon Interaction with Serum Proteins. The entrapment efficiency for DODAB liposomes was obtained from dialysis and radioactive labeling of the intraliposomal aqueous compartment.17 Equal volumes (16) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (17) Augusto, O.; Carmona-Ribeiro, A. M. Biochem. Educ. 1989, 17 (4), 209.
of liposomes prepared in water or in PBS solutions both containing [14C]sucrose (LS) and a dialysis control of water containing [14C]sucrose (S) were dialyzed in two separate bags against 2 L of water or PBS (changed three times), respectively, overnight with vigorous stirring. Before dialysis, aliquots of LS and S were reserved for the determination of [14C]sucrose entrapment efficiency of the liposomes (ENT). After dialysis, the radioactivity, in counts per minute (cpm), was determined for the two dialyzates and for the reserved aliquots. Entrapment can be taken as17
ENT ) (1/C)(cpm2/cpm1 - cpm2c/cpm1c) where 1 and 2 subscripts refer to counts of LS before and after dialysis, respectively; 1c and 2c subscripts are counts of S before and after dialysis, respectively; and C is the molar DODAB concentration. Thus, ENT is expressed in M-1. Protein-induced vesicle disruption was evaluated from two different assays: (1) the LS dialyzate containing a total radioactivity equal to cpmtotal was added to BSA or a-BSA solutions inside hemichamber a of an equilibrium dialysis chamber and leakage was inferred from the total radioactivity in hemichamber b (cpmb) once equilibrium was attained between the a and b compartments, which were separated by a cellulose dialysis membrane; (2) the LS dialyzate was added to BSA or a-BSA solutions and the mixture was placed inside a dialysis bag, submitted to 6 h of dialysis against water or PBS (2 L, 3X), radioactivity being counted before (cpm1) and after the dialysis procedure (cpm2). The percentiles of liposome rupture from the equilibrium and the conventional dialysis procedures described above were calculated respectively as
% Re ) 100[cpmb/(cpmtotal/2)] % Rd ) 100[(cpm1 - cpm2)/cpm1] Final DODAB and protein concentrations in the mixtures submitted to dialysis or to equilibrium dialysis were 2.5 and 0.34 mg/mL, respectively. Microelectrophoresis of the DODAB/BSA Complex. DODAB dispersions in water obtained as previously described were diluted to obtain 0.02 mM DODAB, mixed v/v with BSA solutions over a range of BSA concentrations and incubated (1 h/40 °C) before performing the electrophoretic mobility (EM) measurements as a function of DODAB concentration in the mixtures. Mobilities were measured using a Rank Brothers microelectrophoresis apparatus with a flat 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 vesicles over a given tracking distance were recorded, as was the direction of vesicle movement. Average velocities were calculated from data on at least 20 individual vesicles. EM was calculated according to the
Cationic Vesicle and Serum Protein Interactions
Figure 1. Protein incorporation expressed as mg of adsorbed protein per 2.5 mg of DODAB (A) or as a percentage of protein incorporation (B) as a function of total protein mass (mg) in the liposome/protein mixtures obtained in water or in PBS. There is 2.5 mg of DODAB per 1.0 mL of total volume in each mixture (4.0 mM DODAB). For these experiments, protein was added to previously prepared DODAB liposomes. equation EM ) cm(u/V)(1/t), where u is the distance over which the vesicle 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 vesicle a given distance u. Because of the large effect of DODAB concentration on EM,18 mobilities were determined as a function of DODAB concentration. The final DODAB concentration in the DODAB/ BSA mixtures was fixed at 0.01 mM so that effects of vesicle interaction on mobilities could be minimized.
Results and Discussion 1. Incorporation of BSA or a-BSA in DODAB Liposomes. To detect the occurrence of serum protein/ liposome interaction, its incorporation in the liposomes was measured over a range of experimental conditions, namely, for the interaction in water or in PBS for both BSA and a-BSA (Figure 1A, B). In Figure 1, at a fixed DODAB amount in each mixture, protein incorporation linearly increases as a function of added protein. One should notice that the DODAB concentration in the mixture is not a limiting factor toward protein adsorption. Excess DODAB in the mixtures causes the linear increase of adsorbed protein as a function of the total amount added. With increasing ionic strength, protein incorporation decreases, as depicted from the comparison between incorporation obtained in water (squares and circles) and in PBS (triangles). There is about 100% of protein incorporation in water against 25-40% incorporation in PBS. The larger figures (18) Carmona-Ribeiro, A. M.; Midmore, B. R. J. Phys. Chem. 1992, 96, 3542.
Langmuir, Vol. 14, No. 21, 1998 6079
for incorporation in water suggest that a substantial contribution of the electrostatic attraction between protein and liposome drives the incorporation in water. One should recall that the BSA isoelectric point is 5.4 so that BSA is negatively charged both in water (pH 6.4) and in PBS (pH 7.4). However, in PBS, the high ionic strength is screening the electrostatic attraction between BSA and DODAB though, in PBS, hydrophobically driven incorporation is still considerably large: 25-40%. It is important to recall that DODAB has often been used as an effective immunoadjuvant (2). This means that the liposome enhances antigen presentation to the immunological system. Recently, we have shown that DODAB can be remarkably effective for presentation of an antigenic protein in vivo.19 The main reason for this seems to be its ability to stretch the “lying-over” biomolecule so that more haptens are presented to the outside. Furthermore, in PBS, liposome integrity was maintained despite the interaction with the antigenic protein; i.e., no liposome rupture was detected.19 Similarly to the interaction between DODAB and serum proteins, percentages for protein incorporation in water were very high (ca. 75%), slightly decreasing to 55% in PBS.19 There is an interesting comparison to be done between the antigenic water soluble protein in ref 19 and BSA. Because BSA does not hold its native conformation in pure water as it does in PBS,20 it is likely that exposure of hydrophobic regions of the biomolecule are contributing to the 100% incorporation in the liposome. On the other hand, the antigenic protein in ref 19 that holds its native conformation in pure water yields more similar percentages of incorporation on the liposome in water or in PBS. Thus, there is possibly a very large component of hydrophobic interaction explaining the 100% BSA incorporation on the liposome in pure water. In vivo, opsonization occurs with BSA in its native conformation, which does not mean that the hydrophobic interaction driving BSA to the liposome is absent, as shown from the still large 35% BSA incorporation in PBS. As pointed out by one of the reviewers, coincidental adsorption for BSA and a-BSA is very surprising considering that the range of isoelectric points expected for polyclonal a-BSA is above pH 7. In this case, at pH 6.4 (water) or at pH 7.4 (PBS) a considerable fraction of antibody molecules could be positively charged, so that electrostatic repulsion between the cationic vesicle and the antibody would take place and one could not obtain 100% a-BSA incorporation (Figure 1). Because the result in Figure 1 is a fact, one is led to believe that cationic a-BSA also adsorbs on the cationic liposome either driven by the hydrophobic attraction or driven by the anionic a-BSA already deposited on the cationic vesicle. 2. Nonoccurrence of Protein-Induced Liposome Flocculation and Effect of BSA on Electrophoretic Mobility of DODAB Liposomes. Protein adsorption on DODAB bilayer vesicles in water could eventually lead to liposome flocculation since charge neutralization on the cationic liposome would possibly occur due to attachment of the negatively charged macromolecule. To evaluate the occurrence of protein induced-liposome flocculation in water over a range of DODAB/protein concentrations, a microplate experiment was performed. This allowed simultaneous evaluation of turbidity changes as a function of time for 80 combinations of DODAB/protein concentrations, as described in Table 1. Figure 2 shows turbidity as a function of time for most of these combina(19) Tsuruta, L. R.; Quintilio, W.; Costa, M. H. B.; Carmona-Ribeiro, A. M. J. Lipid Res. 1997, 38 (10), 2003. (20) Zimmerman, R. J.; Kanal, K. M.; Sanders, J.; Cameron, I. L.; Fullerton, G. D. J. Biochem. Biophys. Methods 1995, 30, 113.
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Carvalho and Carmona-Ribeiro
Figure 3. Turbidity at 405 nm as a function of time for DODAB/ BSA mixtures in a spectrophotometer experiment. Mixtures A9, B9, C9, D9, and E9 were equivalent to those in the microplate experiment in Figure 2, as described in Table 1. The procedure for precoating the polysterene cuvettes was analogous to the one used for microplate precoating.
Figure 2. Turbidity at 405 nm as a function of time for DODAB/ BSA mixtures in a microplate experiment to evaluate proteininduced liposome flocculation. Data for wells on rows A, B, C, D, E, and F and columns 1-10 are shown. Turbidities for wells G and H and columns 1-10 (not shown) were equal to zero due to the very low DODAB amount in each of these wells. These data indicate the absence of protein-induced liposome flocculation over the extensive range of BSA and DODAB concentrations tested (see Table 1).
tions with the exception of rows G1 to G10 and H1 to H10 which are not shown. Turbidity is not affected by time over the entire range of DODAB/protein concentrations tested, indicating the absence of protein-induced liposome flocculation (Figure 2). To confirm this result, a similar experiment was performed for each individual mixture in a precoated polystyrene cuvette (Figure 3). Again, no protein-induced flocculation was detected, indicating that the liposome/BSA interaction does not cause exposure of hydrophobic regions that could eventually cause liposome aggregation or cause any “bridging” between liposomes. In fact, opsonization by serum proteins in vivo coats a foreigner particle and this coating acts as a labeling to the start of processes that may lead to particle removal from the circulation. Similar experiments were performed for a-BSA yielding similar results (not shown). In agreement with the absence of protein-induced flocculation shown in Figures 2 and 3, though a decrease of electrophoretic mobilities for DODAB liposomes as a function of BSA concentration in BSA/DODAB mixtures was obtained, charge reversal or neutralization for the BSA/cationic vesicle complex did not take place (Figure 4). This shows that coating the vesicle with BSA does not cause charge neutralization of the liposome. Consistently, recognition between BSA and a-BSA incorporated in DODAB liposomes evaluated from appropriate microplate experiments (similar to those done for looking at non-
Figure 4. Electrophoretic mobilities (EM) for DODAB liposomes in water as a function of DODAB concentration in absence of protein (A) or as a function of BSA concentration at 0.01 mM DODAB (B).
specific protein-induced liposome flocculation) was absent (not shown). Specific flocculation due to recognition did not occur. Electrostatic repulsion between the positively charged DODAB/protein liposomes may well be accounting for the absence of biomolecular recognition between BSA and its antibody, which are separately incorporated in DODAB liposomes. Another possibility is hiding of recognition sites on the proteins as a consequence of
Cationic Vesicle and Serum Protein Interactions
Langmuir, Vol. 14, No. 21, 1998 6081
Table 2. Apparent Liposome Rupture (% R) As Determined from Equilibrium Dialysis (e) or Two Consecutive Dialysis (d) Procedures in the Absence or Presence of BSA or A-BSA % Re
% Rd
sample (1 mL)
water
PBS
water
PBS
2.5 mg of DODAB 2.5 mg of DODAB/ 0.34 mg of BSA 2.5 mg of DODAB/ 0.34 mg of a-BSA
25 26
52
22 21
88/50 43
25
40
25
37
protein adsorption on the liposome. Presently, we cannot discriminate between both possibilities. Although the albumin concentration in serum is 30-45 mg/mL, i.e., much higher than those we tested here, the present results could suggest that the positive charge on the liposome would be preserved upon formation of the albumin/liposome complex. This would explain how biomolecules can be delivered to cells by cationic liposomes. The complex cationic liposome/biomolecule would be coated by albumin in the first place so that the complex would remain positively charged and protected from the high ionic strength in the outside by the hydrophilic albumin coating. 3. Effect of BSA and a-BSA on Liposome Integrity. The effect of BSA on liposome integrity was evaluated from measurements of leakage of a radioactive marker contained in the vesicle aqueous compartment. The apparent percentages of rupture (% R) for DODAB liposomes alone or in the presence of BSA or a-BSA both in water and in PBS obtained from two different dialysis techniques are given in Table 2. Overall, measurements in water do not indicate any effect of the proteins on apparent liposomal rupture. In water, control, i.e., liposomes without protein, and experiment, i.e., liposomes with protein, yield similar percentages for % R obtained from both dialysis procedures. For the experiments in PBS, a significant decrease in apparent rupture is caused by the serum proteins (Table 2). (21) Carmona-Ribeiro, A. M.; Chaimovich, H. Biochim. Biophys. Acta 1983, 733, 172. (22) Carmona-Ribeiro, A. M.; Chaimovich, H. Biophys. J. 1986, 50, 621. (23) Carmona-Ribeiro, A. M.; Ortis, F.; Schumacher, R. I.; Armelin, M. C. S. Langmuir 1997, 13, 2215.
Before interpreting these results, one should have in mind that DODAB liposomes, besides containing radioactively labeled sucrose in their internal aqueous compartment, also adsorb this sugar at the bilayer surface.21 Thereby, upon an increase in the total DODAB bilayer surface offered to radioactive sucrose, there is an overestimation of the entrapment efficiency related to the increase in the total surface area available for sucrose adsorption.2 Upon dialysis, the apparent rupture of DODAB liposomes in water (Table 2) is probably due to desorption of the radioactive sucrose that was attached to the external liposome surface. In PBS, liposome rupture considerably increases due to vesicle aggregation and fusion induced by the increase in ionic strength.22 Curiously, serum proteins cause a small but significant decrease in the apparent liposome rupture, though apparent rupture remains still larger than corresponding figures obtained in water (Table 2). Snippe and co-workers indirectly offered several examples of a very high stability for DODAB-antigen complexes presented to the immunological system.2,5,23 The present results suggest that opsonization might act as a protective process toward DODAB-biomolecule stabilization in vivo. The concentration of albumin in serum is very large (30-50 mg/mL). In vivo stability for DODAB-biomolecule/albumin complexes appears as a strong possibility. Conclusions Major serum proteins, as are albumin and immunoglobulins, have a high affinity for cationic DODAB vesicles. Protein incorporation is 100% in water against ca. 3035% in PBS for both BSA and a-BSA, though neither of the two proteins can induce nonspecific liposome aggregation, liposome charge reversal, or vesicle rupture. The results suggest that preservation of the positive charge on the liposome after its coating by serum proteins is a possible mechanism by which cationic liposomes can deliver proteins or other important macromolecules of biological origin in vivo: opsonization possibly preserves liposome charge and integrity. Acknowledgment. Financial support from FAPESP and CNPq are gratefully acknowledged. L.A.C. thanks CNPq for an undergraduate fellowship. LA980345J
