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A Novel Dendrimeric “Glue” for Adhesion of Phosphatidyl Choline-Based Liposomes Zili Sideratou,† John Foundis,† Dimitris Tsiourvas,† Ioannis P. Nezis,‡ Georgios Papadimas,‡ and Constantinos M. Paleos*,† Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece, and Faculty of Biology, Department of Cell Biology and Biophysics, University of Athens, Athens, Greece Received February 12, 2002. In Final Form: April 9, 2002 The interaction of phosphatidyl choline-cholesterol liposomes incorporating dihexadecyl phosphate as recognizable lipid with complementary guanidinylated diaminobutane poly(propylene imine) dendrimers of the fourth and fifth generation afforded liposome-dendrimer aggregates which were redispersed by the addition of an excess of a phosphate buffer. The higher generation dendrimeric derivative proved more effective when interacted with liposomes. This behavior was attributed to multivalent effects, which, as generally established, enhance the reactivity of multifunctional particles. Turbidimetry, atomic force microscopy (AFM) and optical microscopy were used for investigating the interaction of the complementary particles while the redispersion of the aggregates was studied by transmission electron microscopy (TEM). Liposomal membrane stability in the collapse and redispersion processes was assessed by the calcein fluorescence method, TEM, and AFM.
Introduction In recent years several reports have appeared dealing with the recognition of complementary liposomes.1-10 Both electrostatic and/or hydrogen bonding forces were employed for realizing these interactions, which lead either to tethered liposomes1,7 or to bigger aggregates through fusion.3,5,10 In certain cases multicompartmented aggregates were formed.6,9 The highlights of these investigations and analogous experiments involving interactions of liposomes with cells have recently been reviewed.11 The studies on the interaction of liposomes with dendrimers12-18 have only recently been initiated. Thus †
Institute of Physical Chemistry, NCSR “Demokritos”. Faculty of Biology, Department of Cell Biology and Biophysics, University of Athens. ‡
(1) Chiruvolou, S.; Walker, S.; Israelachvili, J.; Schmitt, F.-J.; Leckband, D.; Zasadzinski, J. A. Science 1994, 264, 1753-1756. (2) Noppi-Simson, D. A.; Needham, D. Biophys. J. 1996, 70, 13911401. (3) Paleos, C. M.; Sideratou, Z.; Tsiourvas, D. J. Phys. Chem. 1996, 100, 13898-13900. (4) Marchi-Artzner, V.; Jullien, L.; Belloni, L.; Raison, D.; Lacombe, L.; Lehn, J.-M. J. Phys. Chem. 1996, 100, 13844-13856. (5) Marchi-Artzner, V.; Jullien, L.; Gulik-Krzywicki, T.; Lehn, J.-M. Chem. Commun. 1997, 117-118. (6) Walker, S. A.; Kennedy, M. T.; Zasadzinski, J. A. Nature 1997, 387, 61-64. (7) Constable, E. C.; Meier, W.; Nardin, C.; Mundwiler, S. Chem. Commun. 1999, 1483-1484. (8) Paleos, C. M.; Kardassi, D.; Tsiourvas, D. Langmuir 1999, 15, 282-284. (9) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Tsortos, A.; Nounesis, G. Langmuir 2001, 16, 9186-9191. (10) Marchi-Artzner, V.; Cuedean-Bondeville, M.-A.; Gosse, C.; Sanderson, J. M.; Dedien, J.-C.; Gulik-Krzywicki, T.; Lehn, J.-M. ChemPhysChem 2001, 2, 367-376. (11) Paleos, C. M.; Sideratou, Z.; Tsiourvas D. ChemBioChem 2001, 2, 305-310. (12) Dvornic, P. R.; Tomalia, A. Macromol. Symp. 1994, 88, 123148. (13) Newkome, G. R.; Moorefield, C. N.; Vo¨gtle, F. Dendrimers and Dendrons: Concepts, Syntheses, Perspectives; Wiley-VCH: Weinheim, 2001. (14) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665-1688. (15) Schlu¨ter, A. D.; Rabe, J. P. Angew. Chem., Int. Ed. Engl. 2000, 39, 864-883. (16) Vo¨gtle, F.; Gestermann, S.; Hesse, R.; Schwierz, H.; Windisch, B. Prog. Polym. Sci. 2000, 25, 987-1041.
in the interaction of poly(amidoamine) dendrimers19,20 with dimyristoylphosphatidyl choline liposomes, several characterization methods were employed and the main conclusions are summarized in the following: (i) The dendrimers interacted with the membrane surface but did not permanently perturb the membrane properties. (ii) Measurements at low temperatures indicated that interaction is more effective for protonated high generation dendrimers. The latter have therefore potential as carriers for biomolecules to approach the cell membrane. Recently a cationic partial dendrimer21 was interacted with liposomes bearing positive, neutral, or negative charge. It was shown that there exists no preference, as expected, for anionic liposomes, a fact that suggests a predominantly hydrophobic interaction of cationic dendrimer with liposomes. It should be noted in this connection that due to the accumulation of guanidine moieties at the external surface of the dendrimers, multivalent effects may be exercised, affecting dendrimers’ interaction effectiveness with liposomes; the role of multivalency on chemical and biological processes was discussed in a seminal review by Whitesides et al.22 Multivalent reactivity is of significant biological interest and merits investigation employing such model interacting components. In the present study liposomes based on phosphatidyl choline-cholesterol and incorporating dihexadecyl phosphate as a lipid bearing a recognizable moiety were (17) Dendrimers. Topics in Current Chemistry; Springer: BerlinHeidelberg, 1998; Vol. 197. Dendrimers. Topics in Current Chemistry; Vo¨gtle, F., Ed.; Springer: Berlin-Heidelberg, 2000; Vol. 210. Dendrimers. Topics in Current Chemistry; Vo¨gtle, F., Ed.; Springer: BerlinHeidelberg, 2001; Vol. 212. Dendrimers. Topics in Current Chemistry; Vo¨gtle, F., Schalley, C. A., Eds.; Springer: Berlin-Heidelberg, 2001; Vol. 217. (18) Tully, D. C.; Fre´chet, J. M. J. Chem. Commun. 2001, 12291239. (19) Ottaviani, M. F.; Matteini, P.; Brustolon, M.; Turro, N. J.; Jockusch, S.; Tomalia, D. A. J. Phys. Chem. B 1998, 102, 6029-6039. (20) Ottaviani, M. F.; Daddi, R.; Brustolon, M.; Turro, N. J.; Tomalia, D. A. Langmuir 1999, 15, 1973-1980. (21) Purohit, G.; Sakthivel, T.; Florence, A. T. Int. J. Pharm. 2001, 214, 71-76. (22) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 2754-2794.
10.1021/la020150i CCC: $22.00 © 2002 American Chemical Society Published on Web 05/22/2002
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allowed to interact with guanidinylated poly(propylene imine) dendrimers of the fourth and fifth generation. Their interaction effectiveness was followed by turbidimetry and indirectly assessed by atomic force microscopy (AFM), transmission electron microscopy (TEM), and optical microscopy while the stability of the liposome membranes during their collapse with dendrimers and at the redispersion stage was assessed by the calcein fluorescence method and TEM. Materials and Methods Soybean hydrogenated phosphatidyl choline (Phospholipon 90H, Nattermann Phospholipid GMBH) and dihexadecyl phosphate (DHP, Sigma) were used for liposome preparation. Diaminobutane poly(propylene imine) dendrimers with 32 (DAB32) and 64 (DAB-64) primary amino groups, DSM Fine Chemicals, were used as received. 1H-Pyrazolo-1-carboxamidine hydrochloride was purchased from Fluka and purified by sublimation. Synthesis of Guanidinylated Dendrimers (GDAB). Partial guanidinylation of dendrimeric derivatives was obtained by applying a method analogous to the one previously reported23 for aliphatic primary amines. Thus to a concentrated dry DMF solution containing either 0.45 mmol of 1H-pyrazolo-1-carboxamidine hydrochloride (for reaction with DAB-32) or 0.85 mmol of 1H-pyrazolo-1-carboxamidine hydrochloride (for reaction with DAB-64 mmol) equimolar quantities of diisopropylethylamine were added. To this solution 0.1 mmol of DAB-32 or DAB-64 dissolved also in dry DMF was added. The reaction mixture was allowed to react for 24 h at room temperature under argon atmosphere. The product was precipitated with ether, centrifuged, dialyzed, and extensively dried. The introduction of guanidine group was approximately 12% as determined by proton NMR. 1H NMR: (250 MHz, DMSO-d6) δ ) 7.85 (broad s, NH), 7.20 (broad s, NH2+), 3.15 (m, NCH2CH2CH2NH), 2.95 (t, NCH2CH2CH2NH), 2.70 (t, CH2NH2), 2.50 (m, NCH2CH2CH2N, NCH2CH2CH2CH2N, NCH2CH2CH2NH2), 1.55 (m, NCH2CH2CH2N, NCH2CH2CH2CH2N, NCH2CH2CH2NH), 1.40 (broad s, NH2). Liposome Preparation. Unilamellar liposomes were prepared by the extrusion method24-26 employing a laboratory extruder (LiposoFast-Pneumatic, Avestin Inc.).27 In a typical experiment for preparing a 4 mL dispersion of liposomes, 0.038 mmol (9.5 × 10-3 M) of phosphatidyl choline (PC) and 0.002 mmol (5.0 × 10-4 M) dihexadecyl phosphate (DHP) (molar ratio 19:1) were mixed with cholesterol at varying concentrations and up to 50% molar with respect to PC. Liposomal dispersions were stable for several days; for characterization experiments, the dispersions were used following their preparation. Turbidimetry Redispersion of Aggregates. The interaction of the liposomes with their complementary dendrimers led to aggregates of various sizes. Their formation was monitored by the dispersion turbidity change at 400 nm using a Lamba-16 spectrophotometer (Perkin-Elmer) at 25 °C. Guanidinylated dendrimer solutions (2 × 10-4 M GDAB-32 or 1 × 10-4 M GDAB64) were progressively added to dispersions of PC-DHP liposomes with or without cholesterol (CHOL). The concentrations of PC and DHP were 9.5 × 10-5 and 5.0 × 10-6 M, respectively, while that of cholesterol varied from 10% up to 50% molar with respect to PC. Control experiments were performed using PC-CHOL liposomes (9.5 × 10-5 M PC, 4.75 × 10-5 M CHOL), which were interacted with guanidinylated dendrimer solutions (2 × 10-4 M GDAB-32 or 1 × 10-4 M GDAB-64) and their parent dendrimeric solutions (2 × 10-4 M DAB-32 or 1 × 10-4 M DAB-64). The aggregates obtained in the above-mentioned experiments were redispersed by an excess (50-fold) phosphate buffer (pH ) (23) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org. Chem. 1992, 57, 2497-2502. (24) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55-65. (25) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9-23. (26) Bangham, A. D.; Standish, M. M.; Watkins, J. C. J. Mol. Biol. 1965, 13, 238-252. (27) MacDonald, R. C.; MacDonald, R. I.; Menco, B. Ph. M.; Takeshita, K.; Subbarao, N. K.; Hu, L. Biochim. Biophys. Acta 1991, 1061, 297303.
Figure 1. Turbidity change during recognition experiments between PC-DHP liposomes with GDAB-32 (2 × 10-4 M) at various concentrations of cholesterol: 10% (2), 20% (O), 30% (b), 40% (0), 50% (9) molar with respect of PC and without cholesterol (4).
Figure 2. Turbidity change during recognition experiments between PC-CHOL-DHP (molar ratio 19:9.5:1) liposomes with GDAB-32 (2 × 10-4 M) or GDAB-64 (1 × 10-4 M) and redispersion experiments with phosphate buffer (GDAB-32, open circles; GDAB-64, filled circles). 7). The process was monitored by UV turbidimetric and microscopic studies. AFM and TEM and Optical Microscopy. AFM was employed for imaging the originally prepared small liposomes. A Multi-Mode Nanoscope III microscope (Digital Instruments) operating under the Tapping Mode was employed.3,8 The aggregates obtained after the interaction of liposomes with the dendrimers could be imaged by video-enhanced phase contrast optical microscopy. An Olympus BX-50 microscope coupled with a Kodak Megaplus model 1.4i camera (Imaging Technology Inc.) was used. For optical microscopy, to PC-CHOL-DHP (9.5 × 10-4 M PC, 4.75 × 10-4 M CHOL, and 5.0 × 10-5 M DHP) liposomal dispersions increasing quantities of guanidinylated dendrimers were gradually added; they were quickly agitated and used for microscopic observation. The redispersion of the aggregates was investigated by optical microscopy, AFM (Tapping Mode), and TEM. For TEM microscopy, liposomes were mounted on Formvar-coated grids and stained with 2% uranyl acetate employing a Philips EM300 microscope operating at 60 kV. Stability Assessment of Liposome Membranes. The evaluation of liposomal membrane stability at the stage of collapse with the recognizable dendrimeric particles was monitored by entrapping calcein in the liposome interior, allowing liposomes to interact with dendrimers and measuring the resulting fluorescence intensity. Encapsulation of calcein in liposomes was achieved by a method previously described.28 For these experiments guanidinylated dendrimeric solutions were added to
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Figure 3. AFM images of PC-CHOL-DHP (molar ratio 19:9.5:1) liposomes (A) and phase contrast optical microscopy image of a liposome aggregate after addition of 2 µL of GDAB-32 (2 × 10-4 M) to 2 mL of PC-CHOL-DHP (9.5 × 10-4 M PC, 4.75 × 10-4 M CHOL, and 5.0 × 10-5 M DHP) liposomal dispersion (B). Phase contrast optical microscopy (C) and TEM (D) images after mixing of 8 µL of GDAB-32 (2 × 10-4 M) with 2 mL of PC-CHOL-DHP (9.5 × 10-4 M PC, 4.75 × 10-4 M CHOL and 5.0 × 10-5 M DHP) liposomal dispersion. The bar in the lower right corner of the optical microscopy images corresponds to 5 µm while the bar in the TEM image corresponds to 250 nm. calcein-entrapped PH-CHOL-DHP liposomes and fluorescence intensity at 520 nm (excitation at 490 nm) was measured with a Perkin-Elmer LS-5B spectrophotometer.
Results and Discussion Interaction effectiveness between liposomes and dendrimers was assessed by turbidimetry as it has been extensively applied in previous studies.9,29-31 The increase of turbidity during titration was attributed to the formation of large particles due to the association of the liposomes with dendrimers acting as a “glue”. The change of turbidity of PC-DHP liposomes as a function of the added GDAB-32 solution (2 × 10-4 M) is shown in Figure 1. The plateau in the curves indicates that saturation of the recognizable groups of liposomes occurs after a certain concentration of the dendrimeric solution was added. The observed decrease in turbidity after the plateau was attributed to particle precipitation and not to dilution of the dispersion since very concentrated dendrimeric solutions were added in the recognition experiments. Analogous experiments were performed with liposomes, incorporating increasing quantities of cholesterol, usually up (28) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Tsortos, A.; Pyrpassopoulos S.; Nounesis, G. Langmuir 2002, 18, 829-835. (29) Stamatatos, L.; Leventis, R.; Zuckermann, M. J.; Silvius, J. R. Biochemistry 1988, 27, 3917-3925. (30) Hasegawa, M.; Kaku, T.; Kuroda, M.; Ise, N.; Kitano, H. Biotechnol. Appl. Biochem. 1992, 15, 40-47. (31) Stewart, R. J.; Boggs, J. M. Biochemistry 1993, 32, 10666-10674.
to the levels incorporated in biological cells or in liposomal drug delivery systems. With increasing cholesterol concentration, recognition was enhanced as evidenced by the turbidity increase (Figure 1) due to the formation of a greater number of large aggregates. These results are in line with our previous results on the enhancing role of cholesterol on the recognizability between liposomes bearing the guanidinium/phosphate complementary pair.9 A liquid-ordered phase32 was induced for the liposomal membrane, and under these conditions more effective recognition was achieved due to the enhanced lateral mobility of the recognizable molecules and the high degree of the alkyl-chain conformational order. Analogous experiments were performed when GDAB64 (1 × 10-4 M) was used. The concentration of the guanidinium groups was the same as in the previous experiments with GDAB-32 since the concentration of GDAB-64 was half of the one previously employed. In this last case interaction effectiveness was however higher compared to the analogous experiments with GDAB-32 (Figure 2). This may be attributed to multivalent effects,22 since for GDAB-64 dendrimers a greater number of guanidinium groups are located, on the average, at the external surface of the dendrimer. It should be noted that a minor turbidity increase was observed during the interaction between PC-CHOL (32) Mouritsen, O. G.; Jørgensen, K. Chem. Phys. Lipids 1994, 73, 3-25.
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liposomes with guanidinylated dendrimers. This may be due to the significantly less effective interaction of guanidinum groups with the sterically hindered phosphate group of PC. Also the same behavior was observed during the interaction between PC-CHOL-DHP with parent dendrimers. This was explained by the less effective interaction of the phosphate groups with the external amino groups of the parent dendrimers. It is interesting to note that addition of an excess of phosphate buffer, pH ) 7, leads to redispersion of the aggregates and precipitated particles as evidenced by the abrupt and significant decrease in turbidity studies, Figure 2. The high concentration of phosphate ions shifts the equilibrium toward the formation of dendrimeric phosphates, which are simultaneously detached from the liposomes leading to the formation of smaller particles. The size of the liposomes as determined by AFM microscopy (Tapping Mode) was found to range between 60 and 130 nm (Figure 3A). When liposomal dispersions were mixed with dendrimeric particles, interactions occurred spontaneously resulting in the formation of large aggregates, which finally precipitate. At low dendrimer concentrations it was possible to visualize aggregates, escaping precipitation, by phase contrast optical microscopy (Figure 3B). However at higher concentrations almost all of the aggregates do precipitate, which can be observed both by optical microscopy, Figure 3C, and by TEM, shown in Figure 3D. Redispersion of these aggregates with phosphate buffer leads to particles not visible with optical microscopy; for this purpose AFM and primarily TEM were employed. It becomes clear particularly with TEM that the particles (Figure 4) were elongated and had a size ranging between 80 and 100 nm; i.e., their length was almost equal to the diameter of the originally prepared liposomes. They were also dehydrated, a fact that may be attributed to osmotic phenomena due to the addition of an excess of a phosphate buffer. During the association of liposomes with dendrimers, calcein fluorescence intensity was only insignificantly increased. It is therefore deduced that collapse of the dendrimeric with liposomal particles does not lead to their disruption. During the redispersion experiments the fluorescence remained at a low level, which was explained by the fact that there was not a leakage to the bulk water (33) Komatsu, H.; Okada, S. Chem. Phys. Lipids 1997, 85, 67-74. (34) Osanai, S.; Nakamura, K. Biomaterials 2000, 21, 867-876.
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Figure 4. TEM images after redispersion of liposomedendrimeric aggregates with phosphate buffer. The bar in the lower right corner of the image corresponds to 100 nm.
phase. This property of liposomes was also proved by TEM according to which the liposomal membrane remained intact and the liposomes lost only water. It has been established33 that when calcein is remaining entrapped in the interior of the liposomes and consequently being at high concentration, it fluorescences only slightly because of self-quenching. On the contrary, if liposomes were disrupted or leakage occurred during fusion, calcein would have to be diluted in the bulk aqueous phase resulting in enhanced fluorescence since quenching would be reduced.34 This is actually occurring by the addition of alcohol, which disrupts liposomes, and fluorescence intensity is increased dramatically. It is therefore concluded that the interaction between complementary liposomes and dendrimers results in the precipitation of the liposomal dispersion, which was redispersed to normal and dehydrated liposomes by the addition of phosphate ions. On equal concentration of quanidinylated moieties, the higher generation dendrimeric derivative proved more effective when interacted with liposomes. This behavior was attributed to multivalent effects enhancing in general the reactivity of multifunctional particles. LA020150I
