Langmuir 2004, 20, 6575-6579
6575
Doxorubicin-Poly(acrylic acid) Complexes: Interaction with Liposomes M. V. Kitaeva,† N. S. Melik-Nubarov,*,† F. M. Menger,‡ and A. A.Yaroslavov† School of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119899, Leninskie Gory, Russian Federation, and Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received February 2, 2004. In Final Form: May 21, 2004 Complexation of antitumor drug, doxorubicin (DOX), with poly(acrylic acid) (PAA) in buffer solutions was examined. The DOX-to-PAA binding was governed by electrostatic and stacking interactions resulting in a complex of characteristic composition with a PAA/DOX ) 1.6 molar ratio. Sizes of the complex particles were found to lie in 600-900-nm range. However, the particles were able to interact with small neutral egg yolk lecithin liposomes (80-100 nm in diameter), a ternary DOX/PAA/liposome complex being formed. The observations and conclusions we made may be useful for interpreting biological effects of polymerbased bioactive constructs.
1. Introduction Complexation of polyelectrolytes with amphiphilic lowmolecular-weight ligands, surfactants, and dyes has been studied for a long time. It has been found that the stability of such complexes in aqueous media derives from electrostatic interactions between ionic groups of the ligand and macromolecule and from hydrophobic interactions between the components.1-4 Binding of ligands is accompanied by charge neutralization of the macromolecule and by increases in particle size5,6 resulting finally in phase separation and particle sedimentation.5-8 Since the early 1980s, polycomplexes have been used for delivery of drugs into living cells.9-11 In particular, anionic carriers such as polyaspartate;9-10 polyglutamate;11 and block ionomers of aspartate, benzyl aspartate,12-14 and benzyl glutamate15 with poly(ethylene oxide) dem* To whom correspondence should be addressed. E-mail:
[email protected]. † M.V. Lomonosov Moscow State University. ‡ Emory University. (1) Vitagliano, V.; Costantino, L.; Zagari, A. J. Phys. Chem. 1973, 77, 204-210. (2) Peyratout, C.; Donath, E.; Dehne, L. J. Photochem. Photobiol., A 2001, 142, 51-57. (3) Lindman, B.; Thalberg, K. In Interactions of surfactants with polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; pp 205-276. (4) Goddard, E. D. J. Colloid Interface Sci. 2002, 256, 228-235. (5) Dautzenberg, H. Macromolecules 1997, 30, 7810-7815. (6) Berret, J.-F.; Herve, P.; Aguerre-Chariol, O.; Oberdisse, J. J. Phys. Chem. B 2003, 107, 8111-8118. (7) Svensson, A.; Piculell, L.; Karlsson, L.; Cabane, B.; Jo¨nsson, B. J. Phys. Chem. B 2003, 107, 8119-8130. (8) Chen, J.; Heitmann, J. A.; Hubbe, V. Colloids Surf., A 2003, 223, 215-230. (9) Pratesi, G.; Savi, G.; Pezzoni, G.; Bellini, O.; Penco, S.; Tinelli, S.; Zunino, F. Br. J. Cancer 1985, 52, 841-848. (10) Bogush, T.; Smirnova, G.; Shubina, I.; Syrkin, A.; Robert, J. Cancer Chemother. Pharmacol. 1995, 35, 501-505. (11) Ehtezazi, T.; Govender, T.; Stolnik, S. Pharm. Res. 2000, 17, 871-878. (12) Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Pharm. Res. 1993, 10, 970-974. (13) Jeong, Y. I.; Nah, J. W.; Lee, H. C.; Kim, S. H.; Cho, C. S. Int. J. Pharm. 1999, 188, 49-58. (14) Kataoka, K.; Matsumoto, T.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Fukushima, S.; Okamoto, K.; Kwon, G. S. J. Controlled Release 2000, 64, 143-153. (15) Oh, I.; Lee, K.; Kwon, H. Y.; Lee, Y. B.; Shin, S. C.; Cho, C. S.; Kim, C. K. Int. J. Pharm. 1999, 181, 107-115.
onstrated good potential for targeting doxorubicin (DOX), an antitumor drug, into cells and tissues. The therapeutic effect of DOX was significantly improved upon binding to polyanions.12,16-18 Developing useful biomedical applications requires studying the behavior of drug-containing polycomplexes in an actual biological environment. Up to now, contributors have concentrated on drug release from the polycomplexes while using animal blood and organs as well as model semipermeable membranes.15-18 Much less attention was paid to the study of polycomplex-cell interactions. Binding of the polycomplex to the cell membrane is no doubt a key step initiating a cascade of biochemical reactions in cells. For this reason, we focused on complexes of DOX with poly(acrylic acid) (PAA), interacting with neutral bilayer lipid vesicles as cellmimetic objects. 2. Experimental Section 2.1. Materials. DOX from the Russian Institute of Antibiotics (Russia), egg yolk lecithin (EL) from Bioleck (Ukraine), Ndansylphosphatidylethanol amine (dansyl-PE), PAA with Mw ) 5.000, and buffer components tris(hydroxymethyl) aminomethane (Tris) and N-2-hydroxyethylpiperazine-N′-ethanesulfonate free acid (Hepes) were purchased from Sigma-Aldrich (U.S.A.) and used as received. 2.2. Methods. To prepare small unilamellar EL vesicles with diameter 80-100 nm, we followed the sonication procedure described previously.19 The same procedure was used for preparing EL vesicles with dansyl-PE incorporated into the bilayer; 0.5 wt % of dansyl-PE was added to EL. Absorbance spectra of DOX were recorded with a Specord M-40 spectrophotometer (Carl Zeiss, Germany). Fluorescence measurements were done using a F-4000 fluorescence spectrophotometer (Hitachi, Japan). Mean hydrodynamic diameters of the DOX-PAA complex particles and products of their interaction with vesicles were measured by photon correlation spectroscopy using an Autosizer IIc (Malvern, U.K.) equipped with a He-Ne laser. (16) Janes, K. A.; Fresneau, M. P.; Marazuela, A.; Fabra, A.; Alonso, M. J. J. Controlled Release 2001, 73, 255-267. (17) Yokoyama, M.; Okano, T.; Sakurai, Y.; Ekimoto, H.; Shibazaki, C.; Kataoka, K. Cancer Res. 1991, 51, 3229-3236. (18) Nakanishi, T.; Fukushima, S.; Okamoto, K.; Suzuki, M.; Matsumura, Y.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. J. Controlled Release 2001, 74, 295-302. (19) Yaroslavov, A. A.; Kul’kov, V. E.; Polinsky, A. S.; Baibakov, B. A.; Kabanov, V. A. FEBS Lett. 1994, 28, 121-123.
10.1021/la0497144 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/02/2004
6576
Langmuir, Vol. 20, No. 16, 2004
Kitaeva et al.
Figure 2. Dependence of relative DOX fluorescence intensity on the PAA/DOX ratio (λex ) 490 nm). [DOX] ) 50 µM.
Figure 1. Absorbance (A) and fluorescence (λex ) 490 nm, B) spectra of DOX (1) and the DOX-PAA mixture (2). [DOX] ) 50 µM; [PAA] ) 150 µM. To estimate the amounts of DOX and PAA uninvolved in a DOX-PAA complex, the following procedure was used. The complex particles were separated from a supernatant by passing suspension samples through the microcentrifuge PTMK filters with a pore diameter of 5 nm (Millipore, U.S.A.). To avoid nonspecific binding of unbound DOX on the filters, the latter was blocked by prepassing 50 µM DOX solutions until equalizing DOX concentrations over and under the filters. The thus-treated filters were additionally blocked with 50 µM PAA solutions to avoid nonspecific PAA binding. DOX concentrations in the filtrates were measured spectrophotometrically at λ ) 490 nm taking ) 10 500 cm-1 M-1.20 PAA concentrations in the filtrates were determined on the basis of PAA’s ability to quench DOX fluorescence. The corresponding results and calibration curve are represented in Results and Discussion. The microcentrifugal filtration technique was also used for controlling a possible dissociation of the DOX-PAA complex when interacting with EL vesicles. The experiments were performed in a 20 mM Hepes-Tris buffer solution, pH 7.0, at 20 °C. To prepare solutions, doubledistilled water was used after additionally passing it through a Milli-Q system (Millipore, U.S.A.) equipped with ion-exchange and adsorption columns as well as a filter to remove large particles.
3. Results and Discussion 3.1. Complexation of DOX with PAA. The DOX molecule contains an amino group with a pK of 8.6;21 meanwhile, the pK0 for PAA carboxylic groups is equal to 4.8. 22 Therefore, complexation of DOX to PAA was studied at pH 7 to realize electrostatic interactions between both components. In Figure 1A is represented an absorbance spectrum for DOX in a pH 7 buffer with a maximum at 480 nm and two shoulders at 500 and 535 nm (curve 1). Addition of an aqueous solution of PAA to an equimolar buffer solution of DOX was accompanied by a 10-nm red (20) Soderlund, T.; Jutila, A.; Kinnunen, P. K. J. Biophys. J. 1999, 76, 896-907. (21) Harrigan, P. R.; Wong, K. F.; Redelmeier, T. E.; Wheeler, J. J.; Cullis, P. R. Biochim. Biophys. Acta 1993, 1149, 329-338. (22) Nagasawa, M.; Murase, T.; Kondo, K. J. Phys. Chem. 1965, 69, 4005-4012.
Figure 3. Dependence of relative PAA-DOX complex fluorescence intensity (λex ) 490 nm) on NaCl (A) and ethanol (B) concentrations. [DOX] ) 50 µM; [PAA] ) 150 µM. The fluorescence intensity equal to 1 corresponds to that of the 50 µM DOX solution in 20 mM Hepes-Tris buffer, pH 7.0.
shift of λmax in the absorbance spectrum of DOX, a decrease in optical density of the solution being observed (curve 2). The red shift apparently resulted from stacking interactions of DOX molecules immobilized along PAA chains. This indicated, in turn, a formation of the DOX-PAA complex stabilized by multipointed ionic bonds between the DOX ensemble and the PAA chain. A similar effect has been observed for dyes bound to oppositely charged polyelectrolytes.1,2,23,24 Being excited at 490 nm, DOX emits bright fluorescence with a maximum at 555 nm and a shoulder at 588 nm (Figure 1B, curve 1), which is quenched in the presence of PAA (curve 2). A dependence of the relative fluorescence intensity, measured at λem ) 555 nm, on the [PAA]/[DOX] ratio is given in Figure 2. Because the (poly)acrylate anion is not a fluorescence quencher, the observed significant decrease in DOX fluorescence intensity is obviously due to the stacking interactions of PAA-bound DOX molecules. As reported earlier, DOX molecules have a strong tendency to self-association and formation of regular structures in aqueous solutions at [DOX] g 130 µM. 25 Our results show a similar effect, but at lower concentrations of DOX, when it is binding to PAA. Addition of either NaCl or ethanol to a solution of the DOX-PAA complex resulted in an increase in the DOX fluorescence intensity (Figure 3A,B, respectively), that
Doxorubicin-Poly(acrylic acid) Complexes
Langmuir, Vol. 20, No. 16, 2004 6577
Figure 4. Calibration curve for finding PAA concentration based on PAA-induced quenching DOX fluorescence (see eq 1).
is, a complex dissociation, thus corroborating contributions of electrostatic and stacking interactions in the stabilization of the DOX-PAA complex. DOX-to-PAA complexation was accompanied by appearance of large particles. Their size was found to fluctuate within the 600-900-nm range when changing the [PAA]/[DOX] ratio from 0.3 up to 5. Binding of DOX to PAA was also studied by measuring concentrations of both components in the filtrates after passing mixed DOX-PAA solutions through the microcentrifuge filters with 5-nm pores. Filters pretreated with DOX and PAA were used. Appearance of DOX in the filtrates was controlled spectrophotometrically. For PAA, its ability to quench DOX fluorescence was used for analysis. The curve from Figure 2 was satisfactorily described by a linear equation
I/I0 ) 1 - B[PAA] at [PAA] < [DOX] (here, B is the proportional coefficient equal to 6 × 10-3 M-1) which could be also written as
[PAA] )
1 - I/I0 B
(1)
Rearrangement of the dependence from Figure 2 as the [(I/I0)/B] - [PAA] plot gave the calibration curve for finding PAA concentration on the basis of PAA-induced quenching DOX fluorescence (Figure 4). It should be emphasized that the calibration is applicable for [PAA] < 80 µM. For higher PAA concentrations, solutions to be analyzed were initially diluted with the pH 7 buffer to satisfy the above PAAto-DOX ratio. Before the actual DOX-to-PAA binding measurements, permeability of the filter toward DOX and PAA was tested. Thus, two series of DOX and PAA solutions with different concentrations were passed through the pretreated filters and the concentrations of each component in the filtrates were measured. It was found that the concentrations above and under the filter were the same, indicating no loss of DOX and PAA when passing them through the filter. Subsequently, the microcentrifugal filtration experiment was repeated with samples, containing a constant DOX concentration ([DOX] ) 50 µM) but increasing PAA concentrationes, and DOX concentrations under the filter were measured. It was expected that DOX-PAA aggregates of microscale size would remain on the filter, allowing the estimation of a concentration of DOX uncomplexed with PAA. Actually, absorbance spectra of all passed solutions had a maximum at 480 nm and two (23) Rabinowitch, E.; Epstein, L. F. J. Am. Chem. Soc. 1941, 63, 69-78. (24) Michaelis, L.; Granick, S. J. Am. Chem. Soc. 1945, 67, 12121219. (25) Menozzi, M.; Valentini, L.; Vannini, E.; Arcamone, F. J. Pharm. Sci. 1984, 73, 766-770.
Figure 5. Dependence of concentration of DOX (1) and PAA (2) uninvolved in the DOX-PAA complex on the PAA/DOX ratio. [DOX] ) 50 µM.
Figure 6. Dependence of the degree of PAA carboxylic groups ionization (R) on pH value.
shoulders at 500 and 535 nm, that is, corresponded to the spectrum of free DOX solution represented by curve 1 from Figure 1B. In other words, in all these solutions only DOX noncomplexed with PAA could be found. The results are represented in Figure 5 as a dependence of [DOX]unbound on φ ) [PAA]/[DOX] (curve 1). It is seen that free DOX concentrations linearly decreased when raising the φ value and then achieved an ultimate low level. Binding of DOX developed until φ0 ) [PAA]0/[DOX] was achieved. The microcentrifugal filtration technique was used also for controlling concentrations of PAA uninvolved in the complex with DOX (Figure 5, curve 2). It is seen that free (unbound to DOX) PAA could be found in solution only at φ > φ0, that is, at [PAA] > [PAA]0. Two main conclusions follow from the above findings. First, in the excess of DOX all added PAA was involved in complex with PAA. Second, under these conditions DOX is distributed between added PAA and the bulk solution so that a part of the DOX molecules form maximum amounts of ionic bonds within each macromolecule, DOXPAA complexes of ultimate (characteristic) composition being formed. This is accompanied by loss of stability of DOX-PAA complexes and their aggregation. The other part of DOX remains in solution. From the data of Figure 5, a PAA/DOX molar ratio in the characteristic complex was estimated: φ0 ) [PAA]0/[DOX] ) 1.6. Thus, only (1/1.6) × 100% ∼ 60% of PAA carboxylic groups were able to form an ionic bond with amino groups of DOX. At the same time, a degree of ionization of PAA carboxylic groups at pH 7 is equal to 70% (Figure 6). This indicates a low negative charge of DOX-PAA complex particles. A reduced portion of carboxylic groups involved in complexation with DOX (only 60% against 70% available) apparently resulted from strong steric restrictions hindering PAA carboxylic groups from interacting with bulky DOX molecules. It has been shown earlier that cationic amphiphilic compounds, for example, one-tailed surfactants, induce additional ionization of carboxylic groups when interacting with PAA, the portion of elec-
6578
Langmuir, Vol. 20, No. 16, 2004
Kitaeva et al.
Figure 7. pH dependence of the relative amount of DOX complexed with PAA. [DOX] ) 50 µM; [PAA] ) 150 µM.
trostatically complexed carboxylic groups achieving 100%.26 As follows, binding of DOX did not influence the ionization state of PAA carboxylic groups. In Figure 7 is represented an effect of the pH on efficiency of DOX binding to PAA with a maximum at pH 6.5. Both the decrease and the increase in pH values is accompanied by deteriorating DOX binding due to the increase in the degree of ionization of PAA and DOX, respectively. This allows the conclusion that interaction of DOX with PAA is mainly governed by the degree of ionization of both components. Additional stabilization of the DOX-PAA complex is achieved due to intermolecular DOX stacking interactions. 3.2. Interaction of DOX-PAA Complexes with Liposomes. Effective binding of DOX to PAA and the formation of DOX-PAA complex particles of microscale size was, thus, proved. Hence, a principal question arises of whether DOX incorporated into these particles is able to reach the cell membrane. To answer the question, small unilamellar lipid vesicles (liposomes) were used as cellmimetic species. The interaction of the DOX-PAA complex with the liposomal membrane was controlled by the fluorescence resonance energy transfer technique.27,28 For this, EL liposomes containing dansyl-PE were prepared. Being excited at 350 nm, the label is characterized by a broad emission band with the maximum at 520 nm, which overlaps the excitation band of DOX (λmax ) 490 nm). As expected, addition of increasing amounts of DOX to suspensions of such liposomes resulted in quenching of the dansyl fluorescence (Figure 8A). At the same time, a peak at 555 nm and a shoulder at 588 nm, which are characteristic of the emission spectrum of DOX, appeared (cf. Figure 8A and Figure 1B). The same kind but diminished fluorescence changes were observed as a solution containing the DOX-PAA complex was added to a suspension of dansyl-labeled liposomes (data are not shown). The dependencies of fluorescence intensities at 520 and 555 nm on concentrations of free and PAA-coupled DOX are given in Figure 8B. The fluorescence changes described above proved that an energy transfer from dansyl-PE to DOX resulted from mutual approach of both components. It means that not only free DOX molecules but also DOX involved in complexation with PAA were capable of interacting with liposomes. It has been shown earlier that free DOX was able to incorporate into the liposomal membrane so that a part of the drug, represented by condensed aromatic rings, are biting deeper into the hydrophobic part of the lipid bilayer; meanwhile the amino group of the drug is exposed to the surrounding water solution.29 The same (26) Kogej, K. J. Phys. Chem. 2003, 107, 8003-8010. (27) Griffin, E. A.; Vanderkooi, J. M.; Maniara, G.; Erecinska, M. Biochemistry 1986, 25, 7875-7880. (28) Dupou-Cezanne, L.; Sautereau, A. M.; Tocanne, J. F. Eur. J. Biochem. 1989, 181, 695-702. (29) Heywang, C.; Chazalet, M. S.-P.; Masson, M. C.; Bolard, J. Biophys. J. 1998, 75, 2368-2381.
Figure 8. (A) Fluorescence spectra of binary (dansyl-PE/EL vesicles + DOX) mixtures (λex ) 350 nm). Total lipid concentration 1 mg/mL ([dansyl-PE] ) 7 µM). [DOX] ) 0 (1), 1.1 (2), 2.2 (3), 4.3 (4), 8.5 (5), 12.3 (6), 16.3 (7), 20 (8), 23.7 (9), and 27.4 µM (10). (B) Changes in fluorescence intensity of dansyl-PE (1, 2) and DOX (3, 4) on the concentration of DOX (1, 3) and DOX complexed with PAA (2, 4). [PAA]/[DOX] ) 3 (2, 4).
Figure 9. Time dependence of changes in fluorescence intensity of DOX after addition of DOX-PAA complex to 4 mg/mL EL vesicle suspension (A) and ultimate levels of DOX fluorescence recovery for different vesicle concentrations (B). [DOX] ) 50 µM; [PAA] ) 150 µM.
mechanism could be suggested for DOX involved in complex with PAA. If so, interaction of a DOX-PAA complex with liposomes should be accompanied by a disruption of DOX stacking interactions and, consequently, by an increase in DOX fluorescence. To check this prediction, an EL liposome suspension was added to the DOX-PAA complex solution and the kinetics of changes in fluorescence intensity at λem ) 555 nm was recorded (Figure 9A). It is seen that after component mixing the fluorescence intensity began sharply increasing and reaching a maximum value within a few minutes. The ultimate levels of DOX fluorescence that were reached for different liposome concentrations are represented in Figure 9B. These results corroborate the hypothesis about
Doxorubicin-Poly(acrylic acid) Complexes
Langmuir, Vol. 20, No. 16, 2004 6579
Figure 11. Scheme of formation of DOX-PAA complexes and their interaction with liposomes. Figure 10. Dependence of relative DOX concentration in the filtrate after passing an equilibrated (DOX-PAA complex + EL vesicles) mixture through a 200-nm microcentrifugal filter on vesicle concentration. [DOX] ) 50 µM; [PAA]/[DOX] ) 3.
incorporation of DOX, precomplexed with PAA, into the liposomal membrane. Such incorporation could develop though dissociation of the DOX-PAA complex or with retention of the DOXPAA ionic contacts. To distinguish between them, a series of samples with equal concentrations of DOX-PAA complex but with different concentrations of liposomes were prepared. After equilibration, the mixtures were passed through the microcentrifugal filters with 5-nm pores. PAA and DOX concentrations in the filtrates were then measured. Neither PAA nor DOX in the filtrates was found. This indicates no dissociation of the DOXPAA complex when contacting liposomes. Thus, addition of the DOX-PAA complex to EL liposomes was accompanied by incorporation of DOX molecules into the liposomal membrane, with the ionic contacts between amino groups of DOX and the carboxylic groups of PAA being retained. By using photon correlation spectroscopy, a mean particle size in the system after mixing of the DOX-PAA complex with liposomes was estimated. The mean size was found to decrease from 800 down to 200 nm when elevating the liposome concentration from 0 up to 8 mg/ mL. The decrease in the particle size after complex/ liposome mixing was also demonstrated as follows. Samples with equal complex concentration and increasing liposome concentrations were prepared and passed after equilibration through the filters with 200-nm pores. A dependence of relative DOX concentration found under the filter on liposome concentration is given in Figure 10.
This dependence obviously reflects an increase in the portion of particles whose size was below 200 nm. As follows from the figure, in the original DOX-PAA solution this portion was about 20% and points to a rather broad distribution in DOX-PAA complex particles. Addition of liposomes increased a portion of the 200-nm particles up to 70%. 4. Concluding Remarks On the basis of the above results, a mechanistic presentation for complexation of DOX with PAA and interaction of the resulting complex with liposomes can be suggested (Figure 11). With DOX-to-PAA binding, a DOX-PAA complex of characteristic composition (PAA/ DOX ) 1.6 molar ratio), stabilized by electrostatic and stacking interactions, is formed. The process is accompanied by an appearance of particles whose sizes lie within the 600-900-nm range. Nevertheless, the particles are able to interact with small neutral liposomes such that DOX molecules incorporated into the lipid bilayer and ionic contacts between DOX amino groups and PAA carboxylic groups are retained. The interaction results in a decrease of the DOX-PAA complex particle size. The established phenomena may be important for understanding the mechanism of how polymer-based bioactive constructs interact with cells. Acknowledgment. The authors highly appreciate the support of some parts of this research by the Russian Foundation for Fundamental Research (Grant 02-0333185) and the Fogarty International Research Cooperation Award (Grant TW05555). LA0497144
