Langmuir 2005, 21, 7483-7490
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Interaction of Functional Dendrimers with Multilamellar Liposomes: Design of a Model System for Studying Drug Delivery Alexandros Pantos,† Dimitris Tsiourvas,† George Nounesis,‡ and Constantinos M. Paleos*,† Institutes of Physical Chemistry and of Radioisotopes and Radiodiagnostic Products, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece Received April 18, 2005. In Final Form: May 31, 2005 Multilamellar liposomes consisting of phosphatidylcholine-cholesterol-dihexadecyl phosphate (19:9.5:1 molar ratio) and dispersed in aqueous or phosphate buffer solutions were interacted with poly(propylene imine) dendrimers which were partially functionalized with guanidinium groups. The remaining toxic external primary amino groups of the dendrimers were reacted with propylene oxide, affording the corresponding hydroxylated derivatives. Microscopic, ζ-potential, and dynamic light scattering techniques have shown that liposomal-dendrimeric molecular recognition occurs due to the interaction between the complementary phosphate and guanidinium groups. Calcein liposomal entrapment experiments demonstrate a limited leakage, i.e., less than 13%, following liposomes interaction with the modified dendrimers. Calorimetric studies indicate that the enthalpy of the interaction is dependent on the number of guanidinium groups present at the dendrimeric surface and the medium. The process is reversible, and redispersion of the aggregates occurs by adding concentrated phosphate buffer. Two corticosteroid drugs, i.e., betamethasone dipropionate and betamethasone valerate, were encapsulated into the functionalized dendrimers. Drug transport from guanidinylated dendrimers to multilamellar liposomes ranges from 40% to 85%, and it is also dependent on the medium and the degree of dendrimer guanidinylation.
Introduction Surface-functionalized dendrimers1-10 have been recently employed as systems for drug delivery11-15 and gene transfer.16-20 Specificity toward particular cells is an indispensable property that a successful drug delivery * To whom correspondence should be addressed. Phone: +30-210-6503666. Fax: +30-210-6529792. E-mail: paleos@ chem.demokritos.gr. † Institute of Physical Chemistry. ‡ Institute of Radioisotopes and Radiodiagnostic Products. (1) Newkome, G. R.; Moorefield, C. N.; Vo¨gtle, F. Dendrimers and Dendrons. Concepts, Syntheses, Perspectives; Wiley-VCH: Weinheim, Germany, 2001; see also references therein. (2) Fre´chet, J. M. J.; Tomalia, D. A. Dendrimers and Other Dendritic Polymers; Wiley Series in Polymer Science; John Wiley & Sons Ltd.: Chichester and New York, 2001; see also references therein. (3) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665. (4) Schlu¨ter, A. D.; Rabe, J. P. Angew. Chem., Int. Ed. 2000, 39, 864. (5) Vo¨gtle, F.; Schalley, C. A. In Dendrimers; Vo¨gtle, F., Ed.; Topics in Current Chemistry; Springer: Berlin-Heidelberg, 1998; Vol. 197; 2000; Vol. 210; 2001; Vol. 212; 2001; Vol. 217. (6) Ardoin, N.; Asrtuc, D. Bull. Soc. Chim. Fr. 1995, 132, 875. (7) Zeng, F. W.; Zimmerman, S. C. Chem. Rev. 1997, 97, 1681. (8) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M. Langmuir 2000, 16, 1766. (9) Paleos, C. M.; Tsiourvas, D.; Sideratou, Z.; Tziveleka, L. Biomacromolecules 2004, 5, 524. (10) Vo¨gtle, F.; Gestermann, S.; Hesse, R.; Schwierz, H.; Windisch, B. Prog. Polym. Sci. 2000, 25, 987. (11) Gillies, E. R.; Fre´chet, J. M. J. Drug Discovery Today 2005, 10, 35. (12) Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey, H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. J. Controlled Release 2000, 65, 133. (13) Stiriba, S. E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. 2002, 41, 1329. (14) Padillo De Jesus, O. L.; Ihre, H. R.; Cagne, L.; Fre´chet, J. M. J.; Szoka, F. C., Jr. Bioconjugate Chem. 2002, 13, 453. (15) Boas, U.; Heegaard, P. M. H. Chem. Soc. Rev. 2004, 33, 43. (16) Bielinska, A. U.; Chunling, C.; Johnson, J.; Baker, J. R., Jr. Bioconjugate Chem. 1999, 10, 843. (17) Eichman, J. D.; Bielinska, A. U.; Kukowska-Latallo, J. F.; Baker, J. R., Jr. Pharm. Sci. Technol. Today 2000, 3, 232.
system must fulfill. This has been achieved by the introduction of targeting ligands at the surface of the carriers, in this case of dendrimers, which must be complementary to a specific cell receptor. On the other hand, since liposomes are considered the closest analogues to biological cells, it would be useful and preferable before investigating dendrimer and cell interaction to resort to a simpler system, i.e., that of the interaction of a functional dendrimer with multilamellar liposomes. Processes and phenomena encountered during this interaction are associated with the application of dendrimers as drug delivery systems. In this regard it should be noted that multilamellar liposomes have already been applied as cell models for investigating drug transport from unilamellar liposomes when the latter were employed as drug delivery systems.21,22 Specifically, it has been established22 that fast and effective transport of the drugs occurs from unilamellar to multilamellar complementary liposomes due to molecular recognition. In a recent study the interaction of phosphatidylcholine-cholesterol liposomes incorporating dihexadecyl phosphate with complementary guanidinylated poly(propylene imine) dendrimers of the fourth and fifth generations was investigated.23 It was found that dendrimer-liposome aggregates were obtained which were redispersed by the addition of a high concentration of phosphate buffer, suggesting that this process is revers(18) Luo, D.; Haverstick, K.; Belcheva, N.; Han, E.; Saltzman, W. M. Macromolecules 2002, 35, 3456. (19) Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem. 2002, 13, 510. (20) Zinselmeyer, B. H.; Mackay, S. P.; Schatzlein, A. G.; Uchegbu, I. F. Pharm. Res. 2002, 19, 960. (21) Shabbits, J. A.; Chiu, G. N. C.; Mayer, L. D. J. Controlled Release 2002, 84, 161. (22) Pantos, A.; Tsiourvas, D.; Paleos, C. M.; Nounesis, G. Langmuir, in press. (23) Sideratou, Z.; Foundis, J.; Tsiourvas, D.; Nezis, I. P.; Papadimas, G.; Paleos, C. M. Langmuir 2002, 18, 5036.
10.1021/la0510331 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/29/2005
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ible. This study is now extended by employing drug-loaded poly(propylene imine) dendrimers of the fourth generation (DAB) which have been partially functionalized at the surface with guanidinium groups. In addition, for applying this functional dendrimer to cells in future experiments, the toxic external primary groups12 have been reacted with propylene oxide, resulting in the hydroxylation of the dendrimer. The so-prepared dendrimers were allowed to interact at room temperature with multilamellar liposomes prepared from hydrogenated phosphatidylcholine (PC), cholesterol (CHOL), and dihexadecyl phosphate (DHP) at a 19:9.5:1 molar ratio. A PC:CHOL molar ratio of 2:1 was used to approximate biological membranes and because at this ratio the organization of the recognizable moieties within the liquidordered phase of the bilayer membrane enhances molecular recognition24 between the guanidinium groups of the dendrimer and the phosphate groups of DHP at the liposomal interface. The strong binding between the guanidinium and the phosphate group is attributed to the combined electrostatic and hydrogen bond interactions.25 Binding is also affected by organizational26 and polyvalent27,28 effects exercised in liposomal and dendrimeric surfaces. In addition, the low concentration of DHP in the bilayer employed, i.e., DHP:PC ) 1:19 molar ratio, does not perturb the liquid-ordered phase, while, due to the high binding efficiency of the recognizable moieties, reproducible and accurate results can be obtained. Drug transport from dendrimers to multilamellar liposomes during their interaction is also investigated employing two lipophilic corticoid derivatives. The processes taking place during the interaction of either empty or drug-loaded dendrimers with the multilamellar acceptor liposomes were investigated with optical microscopy, dynamic light scattering, ζ-potential, and calcein entrapment experiments as well as by isothermal titration calorimetry. Thus, the structural features of the resulting (24) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Tsortos, A.; Nounesis, G. Langmuir 2000, 16, 9186. (25) Hirst, S. C.; Geib, S. J.; Fan, E.; Hamilton, A. D. Isr. J. Chem. 1992, 32, 105. (26) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1996, 118, 8524. (27) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2755. (28) Kitov, P. I.; Bundle, D. R. J. Am. Chem. Soc. 2003, 125, 16271.
particles and the thermodynamic parameters of dendrimer-liposome interactions were investigated. Experimental Section Materials. Soybean hydrogenated phosphatidylcholine (Phospholipon 90H, Nattermann Phospholipid GmbH) and dihexadecyl phosphate (Sigma) were used. Nucleopore filters of 400 nm pore size (Whatman) were employed for liposome extrusion. Betamethasone dipropionate (BTP) and betamethasone valerate (BTV) were purchased from Sigma-Aldrich. Poly(propylene imine) dendrimer of the fourth generation with 32 primary amino end groups (DAB) was purchased from DSM. Propylene oxide (99%), 1H-pyrazole-1-carboxamidine hydrochloride (99%), and N,Ndiisopropylethylamine (99%) were purchased from Aldrich. Calcein (97%) was purchased from Merck. Functionalization of Dendrimers. The functionalization of DAB dendrimer was achieved in two steps, as shown in Scheme 1. The first step involves complete or partial hydroxylation of DAB dendrimer, while in the second step guanidinylation of the remaining primary amino groups occurs. Complete or Partial Hydroxylation of DAB. To 0.10 mmol of DAB-32 dissolved in water was added dropwise 3.25, 2.65, or 2.05 mmol of propylene oxide also dissolved in water for obtaining either completely hydroxylated dendrimers (DAB-G0) or partially hydroxylated dendrimers with 6 or 12 primary amino groups (DAB-N6 and DAB-N12). The mixture was allowed to react for 12 h at room temperature and dialyzed using a 1.200 cutoff membrane for removing low molecular weight compounds. Water was removed under reduced pressure, and the product was dried under vacuum for 24 h. Hydroxylation was established by 1H NMR (500 MHz, D2O): δ 3.85 (m, NHCH2CH(OH)CH3), 2.70 (m, NHCH2CH(OH)CH3, CH2NH2), 2.60 (t, CH2CH2NHCH2), 2.50 (NCH2CH2CH2NH, NCH2CH2CH2NH2), 2.35 (t, NCH2CH2CH2CH2N), 1.60-1.65 (NCH2CH2CH2NH2, NCH2CH2CH2NH), 1.50 (NCH2CH2CH2N), 1.30 (t, NCH2CH2CH2CH2N), 1.15 (d, NHCH2CH(OH)CH3. The number of unreacted primary amino groups was determined by both NMR and fluorescence spectroscopy employing the fluorescamine method.29 Guanidinylation of Partially Hydroxylated DAB Derivatives. The unreacted primary amino groups of hydroxylated derivatives were guanidinylated with 1H-pyrazole-1-carboxamidine hydrochloride employing a method analogous to the one reported in the literature,30 affording dendrimeric derivatives bearing 6 (DAB-G6) or 12 (DAB-G12) guanidinium groups. Thus, to 0.05 mmol of DAB-N12 or DAB-N6 dissolved in water was added dropwise an aqueous solution containing 0.66 or 0.33 mmol (29) Uderfriend, S.; Stein, S.; Bohlen P.; Leimgruger, W.; Weigele, M. Science 1972, 178, 871. (30) Bernatotovicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org. Chem. 1992, 57, 2497.
Interaction of Dendrimers with Liposomes of 1H-pyrazole-1-carboxamidine hydrochloride and 0.66 or 0.33 mmol of N,N-diisopropylethylamine. The mixture was allowed to react for 20 h at room temperature and dialyzed using a 1.200 cutoff membrane for removing low molecular weight compounds. Water was removed under reduced pressure, and the product was dried under vacuum for 24 h. The introduction of guanidinium groups was established by 1H and 13C NMR. 1H NMR (500 MHz, D2O): δ 3.85 (m, NHCH2CH(OH)CH3), 3.15 (t, CH2CH2NHC(NH)NH2+), 2.70 (t, NHCH2CH(OH)CH3), 2.60 (t, CH2CH2NHCH2), 2.50 (NCH2CH2CH2NH, NCH2CH2CH2N), 2.35 (t, NCH2CH2CH2CH2N), 1.65 (NCH2CH2CH2NH), 1.50 (NCH2CH2CH2N), 1.30 (t, NCH2CH2CH2CH2N), 1.15 (d, NHCH2CH(OH)CH3. 1H NMR (500 MHz, DMSO-d6): δ 7.85 (br s, NH), 7.20 (br s, NH2+ ). 13C NMR (500 MHz, D2O): δ 158.5 (CH2NHC(NH)NH2+), 67.0 (NHCH2CH(OH)CH3), 55.4 (NHCH2CH(OH)CH3), 51.0-52.5 (NCH2CH2CH2N, NCH2CH2CH2CH2N), 47.8 (CH2NHCH2CH(OH)), 40.0 CH2NHC(NH)NH2+), 25.5 (NCH2CH2CH2NH), 24.0 (NCH2CH2CH2N), 23.5 (NCH2CH2CH2CH2N), 20.5 (NHCH2CH(OH)CH3). Preparation of Liposomes. Large multilamellar liposomes with a diameter of 400 nm were prepared by the extrusion method. In a typical experiment for preparing a 2 mL dispersion of liposomes, 0.038 mmol (1.9 × 10-2 M) of PC, 0.019 mmol of CHOL (9.5 × 10-3 M) (molar ratio PC:CHOL ) 2:1), and 0.002 mmol (1.0 × 10-3 M) of DHP (molar ratio PC:DHP ) 19:1) were dissolved in chloroform/methanol solution (2:1, v/v) for the formation, in the usual manner, of a lipid film. The film was hydrated (2 mL) with either water or phosphate buffer, 10 mM (pH 7.4), and the sample was vortexed for 10 min at temperatures above 65 °C. The suspension obtained was extruded through two stacked polycarbonate filters of 400 nm pore size, and only three cycles were applied at 65 °C to obtain multilamellar liposomes.22,31 The liposomal dispersion was then centrifuged at 16.000g for 30 min. Following centrifugation, the multilamellar liposomes precipitated as a pellet while the unilamellar liposomes remained in the supernatant layer.22 The pellet was washed two times with water or phosphate buffer and finally redispersed in 2 mL of water or phosphate buffer, respectively. Incorporation of Drugs in Functional Dendrimers. The functional dendrimeric derivatives and BTP or BTV were dissolved in chloroform. Subsequently, the solvent was distilled off, forming a film. Following dispersion of the film in water or phosphate buffer, 10 mM (pH 7.4), a dendrimeric solution incorporating the drug was obtained which was centrifuged at 16.000g for 40 min. The nonincorporated drug was precipitated, while the drug-loaded dendrimeric derivative remained in the supernatant layer. Dynamic light scattering experiments proved that dendrimeric aggregates or drug particles were not present in solution. The concentration of the incorporated drug inside the dendrimer was determined spectroscopically. Interaction Experiments. Multilamellar liposomes were allowed to interact with functionalized dendrimers DAB-G0, DAB-G6, and DAB-G12 by adding a dendrimeric aqueous or 10 mM (pH 7.4) phosphate buffer solution to multilamellar liposomal dispersions at several dendrimer:DHP molar ratios. The resulting precipitates were redispersed by the addition of concentrated phosphate buffer (0.4 M, pH 7.4), yielding the original multilamellar liposomes. For investigating drug transport from functional dendrimers to multilamellar liposomes during their interaction, a drug-loaded dendrimeric aqueous or 10 mM phosphate buffer solution was added to a multilamellar liposomal dispersion in water or 10 mM phosphate buffer, respectively, up to a final dendrimer:DHP molar ratio of 1:25. The mixture was incubated for 10 min at 25 °C and centrifuged at 16.000g for 40 min. The resulting pellet was washed two times with water or 10 mM phosphate buffer. For the spectroscopic determination of the drugs present in the obtained aggregates, the pellet was dried under vacuum for 24 h and dissolved in 2.5 mL of methanol (solution I). For the determination of the drug content in the multilamellar liposomes obtained after redispersion of the aggregates, concentrated phosphate buffer (0.4 M, pH 7.4) was added to the pellet and the redispersed liposomes obtained were centrifuged (31) New, R. R. C. In Liposomes: A practical approach; Rickwood, D., Hames, B. D., Eds.; IRL Press: Oxford, U.K., 1990; pp 55-56.
Langmuir, Vol. 21, No. 16, 2005 7485 at 16.000g for 40 min. The precipitated multilamellar liposomes were washed two times with phosphate buffer, dried under vacuum for 24 h, and dissolved in 2.5 mL of methanol (solution II) for the determination of the drug present in the liposomes, as described below. Spectroscopic Determination of BTP and BTV. For determining the incorporation of BTP or BTV into the dendrimers, solutions (100-200 µL) of drug-loaded dendrimers in water or 10 mM phosphate buffer (pH 7.4) were diluted to 3 mL by the addition of methanol and water to a final 50% v/v water-methanol mixture. The concentration of BTP or BTV in these solutions was determined, employing a Perkin-Elmer LS-5B spectrophotometer, by the use of the first-order derivative of their UV spectra and measuring the value of the peak at 271 nm (for BTP) or 274 nm (for BTV). At this wavelength the value of the first-order derivative spectra of the functional dendrimers is zero. The concentration of the dendrimers was also determined by measuring the absorbance of the solutions at 210 nm and subtracting the absorbance of the drug at this wavelength. For the determination of BTP or BTV in the liposomes, the first-order derivative of the UV spectrum of the methanolic solution I or II was registered by measuring the value of the peak at 269 nm (for BTP or BTV). Standard spectroscopic curves of BTP, BTV, DAB-G0, DAB-G6, and DAB-G12 in a water-methanol mixture (50% v/v) and in methanol were used. All the experiments were repeated three times at least. Characterization Techniques. The initially obtained multilamellar liposomes were investigated by dynamic light scattering, ζ-potential experiments, and atomic force and optical microscopy. Interaction between functional dendrimers and multilamellar liposomes was investigated by optical microscopy, isothermal titration calorimetry, dynamic light scattering, and ζ-potential measurements. The stability of the liposomes during interaction was studied by calcein-entrapped experiments. The redispersion of the obtained aggregates by the addition of concentrated phosphate buffer was followed by dynamic light scattering, ζ-potential experiments, and atomic force and optical microscopy. Dynamic light scattering studies were performed employing an AXIOS-150/EX (Triton Hellas) apparatus with a 30 mW laser source, and an Avalanche photodiode detector at an angle of 90° was employed. For these experiments 8 µL dispersions of multilamellar liposomes in water or phosphate buffer as well the resulting dispersions following their titration with various dendrimeric solutions, before and after the addition of the concentrated phosphate buffer, were diluted with 0.9 mL of water or phosphate buffer, respectively. Ten scattering measurements were collected for each dispersion, and the results were averaged. AFM images (tapping mode) were obtained employing a MultiMode Nanoscope III microscope (Digital Instruments) employing tapping mode operation. Samples were observed by placing droplets of liposome dispersions on a freshly cleaved mica surface. Video-enhanced phase contrast optical microscopy images were obtained employing an Olympus BX-50 microscope coupled with a Kodak Megaplus model 1.4i camera using an IC-PCI image board (Imaging Technology Inc.). The ζ-potential measurements were conducted using ZetaPlus of Brookhaven Instruments Corp. In a typical experiment, 40 µL of the dispersion of multilamellar liposomes in water or phosphate buffer was diluted to 1.5 mL, and a dendrimeric solution was progressively added. Following each addition the mixtures were quickly agitated and introduced into the instrument cell. Ten ζ-potential measurements were collected for each dispersion, and the results were averaged. Isothermal titration calorimetric experiments were carried out on an MCS-ITC calorimeter (Microcal Inc., Northampton, MA). Thus, a solution of dendrimers (0.4 mM) was placed in a 250 µL titration syringe, while the multilamellar liposomal dispersion in water or in phosphate buffer (1.9 mM PC) was placed in a 1.334 mL reaction cell. A dendrimeric solution in water or in phosphate buffer, respectively, was titrated into the liposomal dispersion via 40 injection sequences of 4.0 µL per injection. The injections were preprogrammed at 300 s intervals and were performed automatically at 26 °C under stirring at 400 rpm. The exothermic heat flow (dQ/dt) data were collected every second for the first 100 s after each injection and every 5 s for
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the remaining time interval and were analyzed using the inbuilt Origin software. Liposome stability at the stage of their interaction with the modified dendrimers was evaluated by allowing multilamellar liposomes, in which calcein was entrapped, to interact with guanidinylated or nonguanidinylated dendrimers and measuring the resulting fluorescence intensity. For these experiments, a lipid film consisting of PC, CHOL, and DHP was now hydrated with 2 mL of solution containing calcein. It consisted of 70 mM calcein and 10 mM phosphate buffer. The nonencapsulated calcein was removed by gel filtration through a Sephadex G-50 column (15 mm × 28 cm) using the phosphate buffer solution as an eluent. The fluorescence intensity of calcein-entrapped multilamellar liposomal dispersions, during the addition of dendrimeric solutions, was monitored at 520 nm (excitation at 390 nm) using a Perkin-Elmer LS-5B spectrophotometer. The percentage of calcein leakage was determined by the following equation:
% calcein leakage ) [(F - Fo) × 100]/(Fmax - Fo) where F is the fluorescence intensity of the resulting mixture, Fo is the fluorescence intensity of the initially prepared multilamellar liposomal dispersion before the addition of the complementary functional dendrimer, and Fmax is the maximum fluorescence intensity corresponding to 100% calcein leakage after the addition of 100 µL, 1% w/w, of Triton X-100 solution.
Results and Discussion A two-step process was used for the preparation of the functional dendrimers as shown in Scheme 1. In the first step complete or partial hydroxylation (62.5% or 81.25%, molar) of poly(propylene imine) dendrimer of the fourth generation was performed by reacting the primary amino groups of the dendrimer with propylene oxide. The degree of hydroxylation was determined by 1H NMR as well as by determining the number of unreacted primary amino groups of the partially hydroxylated dendrimers with fluorescamine.29 Specifically, by integrating the peak at 1.10 ppm attributed to the terminal CH3 group or the peak at 3.85 ppm attributed to the CH group and the peak at 1.30 ppm attributed to the β-CH2 of the tertiary amino groups of the dendrimeric core, it was found that 100%, 80%, and 61%, molar, of the surface amino groups reacted for DAB-G0, DAB-N6, and DAB-N12, respectively. Furthermore, the number of unreacted primary amino groups per dendrimeric molecule was determined with a higher accuracy employing the fluorescamine method. It was found that 0.2, 6.2, and 12.3 primary amino groups were present in DAB-G0, DAB-N6, and DAB-N12, respectively. These results are in good agreement with the 1H NMR results. In the second step, unreacted primary amino groups of the hydroxylated dendrimer reacted with 1Hpyrazole-1-carboxamidine hydrochloride and N,N-diisopropylethylamine, affording the guanidinylated derivatives as established by 1H and 13C NMR spectroscopy. Specifically, a new triplet appearing at 3.15 ppm is attributed to the R-CH2 relative to the guanidinium group, and the peaks at 7.15 and 7.75 ppm are attributed to the guanidinium moieties. Furthermore, in the 13C NMR spectra the guanidinylation of primary amines is demonstrated by the appearance of a new peak at 158.5 ppm corresponding to the C atom of the guanidinium moiety. Liposomal Characterization. The size of the initially prepared multilamellar liposomes was large enough, and therefore, it was possible to observe them by videoenhanced phase contrast optical microscopy. Furthermore, AFM (tapping mode) was employed for visualizing the liposomes (Figure 1A). In addition, dynamic light scattering experiments were employed for quantifying their size, showing a monomodal size distribution with an
Figure 1. AFM images of multilamellar PC-CHOL-DHP liposomes before interaction (A) and of the particles observed following the addition of concentrated phosphate buffer (200 mM) into the dispersion of aggregates resulting from the interaction of the multilamellar liposomes with the guanidinylated dendrimeric derivatives (B). Table 1. ζ-Potential Values (mV) of Multilamellar Liposomes in Water and in Phosphate Buffer Solutions
multilamellar liposome
water
phosphate buffer (10 mM, pH 7.4)
PC-CHOL PC-CHOL-DHP
-2.9 ( 0.9 -27.5 ( 1.3
-2.4 ( 0.7 -20.8 ( 1.0
phosphate buffer (400 mM, pH 7.4) -1.9 ( 1.8 -14.2 ( 1.5
average diameter of about 400 nm and a polydispersity index of 0.10-0.15. Values of the ζ-potential reflect the surface charge of liposomes and can therefore be used to predict their interaction effectiveness as far as electrostatic forces are concerned. ζ-potential values of multilamellar liposomes PC-CHOL and PC-CHOL-DHP dispersed in water or in phosphate buffer are measured, and the results are summarized in Table 1. It is evident that for the PCCHOL multilamellar liposome the ζ-potential values in water or in buffer solutions are practically zero, while the incorporation of DHP into the PC-CHOL liposomal bilayer leads to liposomes with negative ζ-potential values. In the presence of increasing concentrations of phosphate buffer (10 and 400 mM) the ζ-potential values of PC-CHOL-DHP liposomes are significantly reduced. The increase of the total concentration of phosphate anions,
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Table 2. Drug Encapsulation into the Dendrimeric Derivatives in Water or in 10 mM Phosphate Buffer (pH 7.4) water
phosphate buffer
drug
dendrimer
[drug] (mM)
[dendrimer] (mM)
drug:dendrimer molar ratio
[drug] (mM)
[dendrimer] (mM)
drug:dendrimer molar ratio
BTP
DAB-G0 DAB-G6 DAB-G12 DAB-G0 DAB-G6 DAB-G12
4.39 ( 0.10 2.80 ( 0.09 2.60 ( 0.11 3.77 ( 0.05 15.11 ( 0.15 16.15 ( 0.11
3.08 ( 0.05 2.85 ( 0.10 3.35 ( 0.09 3.15 ( 0.08 3.12 ( 0.12 2.91 ( 0.09
1.43 0.98 0.78 1.19 4.84 5.54
3.40 ( 0.14 1.62 ( 0.05 1.50 ( 0.06 2.97 ( 0.10 13.35 ( 0.14 15.01 ( 0.11
2.96 ( 0.05 3.32 ( 0.05 3.29 ( 0.07 2.92 ( 0.09 3.28 ( 0.12 3.19 ( 0.15
1.15 0.49 0.46 1.01 4.07 4.73
BTV
at constant pH, leads to a smaller number of dissociated phosphate moieties of DHP in the liposomal surface. Drug Incorporation into Dendrimers. The encapsulation of BTP or BTV by the dendrimers was determined by measuring the concentration of the drug in the dendrimeric solutions employing UV spectroscopy after removal of the nonencapsulated drug. The results are summarized in Table 2. It can be deduced that the guanidinylation of the dendrimers reduces the solubilization of BTP possibly due to the more open structure of the dendrimeric derivatives due to the presence of the charged guanidinium surface groups. Furthermore, in phosphate buffer solutions, drug incorporation into the dendrimers was further reduced especially in the case of the guanidinylated dendrimers. This is attributed to the more open structure32,33 that the dendrimeric derivatives attain in lower pH due to the partial protonation of the tertiary amino groups in the interior of the dendrimers and of the secondary amino groups located near the surface which disfavors the incorporation of the drug. Apparently the guanidinylated dendrimeric derivatives have a more open structure than the hydroxylated ones, and this leads to a significant difference in drug solubilization compared to that of DAB-G0. The same effect of the phosphate buffer on the solubilization of BTV is also observed. However, the solubilization of BTV is significantly higher than that of BTP in the guanidinylated derivatives DAB-G6 and DAB-G12 either in water or in buffer solution, while their solubilizations are comparable in DAB-G0 solutions. This clearly indicates favorable interactions between BTV and the guanidinium groups of the dendrimers, leading to a major increase of the drug: dendrimer molar ratio. Interaction Experiments. Microscopic Studies. Molecular recognition between PC-CHOL-DHP multilamellar liposomes dispersed in water and guanidinylated or nonguanidinylated dendrimers occurred spontaneously, leading to the formation of large aggregates at dendrimer: DHP molar ratios higher than 1:30 as observed by phase contrast optical microscopy. As the dendrimer:DHP molar ratio increased, the interaction led to even larger aggregates. Analogous interaction experiments in phosphate buffer, 10 mM (pH 7.4), led to the formation of smaller aggregates as compared to the ones obtained in water. Interestingly, the process is reversible since the obtained aggregates were redispersed by the addition of concentrated buffer (200 mM). AFM images of the obtained dispersions reveal the presence of particles of about the same size as that of initial multilamellar liposomes (Figure 1B). However, they exhibit a prolate shape, while the initially obtained multilamellar liposomes were spherical. This change in morphology is attributed to osmotic effects occurring due to the addition of the concentrated phosphate buffer.23 (32) Lee, I.; Athey, B. D.; Wetzel, A. W.; Meixner, W.; Baker, J. R., Jr. Macromolecules 2002, 35, 4510. (33) Govorun, E. N.; Zeldovich. K. B.; Khokhlov, A. R. Macromol. Theory Simul. 2003, 12, 705.
Light Scattering Studies. The optical microscopy and AFM results are consistent with DLS measurements. The size increase obtained during the interaction of PC-CHOL-DHP multilamellar liposomes with guanidinylated or nonguanidinylated dendrimers in water is shown in Figure 2. When the dendrimer:DHP molar ratio was 1:25, a ∼10-fold or 7-fold increase in the size of the aggregates was observed when DAB-G12 or DAB-G6 was added, respectively. However, only a 3-fold increase was observed when DAB-G0 was added. At higher dendrimer: DHP molar ratios the size of the aggregates becomes even larger. On the other hand, when the dendrimer:DHP molar ratio is 1:30, an insignificant size increase is obtained for
Figure 2. Particle size distribution of multilamellar PCCHOL-DHP (a) and of the resulting aggregates following interaction in water with DAB-G0 (A), DAB-G6 (B), and DABG12 (C) at dendrimer:DHP molar ratios of 1:30 (b), 1:25 (c), or 1:20 (d).
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Figure 3. Particle size distribution of the aggregates obtained after the interaction of DAB-G6 dendrimers with PC-CHOLDHP multilamellar liposomes (d) and of the particles formed during their redispersion by the addition of phosphate buffer at various concentrations: 50 mM (c), 100 mM (b), and 200 mM (a).
DAB-G0. However, the addition of DAB-G6 and DAB-G12 at these molar ratios yields a bimodal size distribution, clearly suggesting the presence of the original multilamellar liposomes as well as of aggregates with approximately double radius. The observed difference between DAB-G6 or DAB-G12 and DAB-G0 is attributed to the presence of guanidinium groups in the dendrimeric molecule, which lead to a more efficient binding of the dendrimer with the phosphate groups of DHP in the liposomal bilayer. The size increase observed upon addition of the nonguanidinylated dendrimer is attributed to electrostatic interactions of the tertiary and secondary amino groups of DAB-G0 with the phosphate groups of DHP. It should be noted that in control experiments with multilamellar PC-CHOL liposomes, i.e., with liposomes not incorporating the recognizable DHP amphiphile, an insignificant size increase was observed during their interaction with guanidinylated or nonguanidinylated dendrimers. This can be attributed to the weak interaction of the dendrimeric guanidinium groups with the sterically hindered phosphate group of PC. In phosphate buffer, an analogous size increase was observed although the size of the particles was smaller. At 1:25 dendrimer:DHP molar ratios, the size of the aggregates increased by approximately 8-fold or 3-fold when DAB-G12 or when DAB-G6 was added and insignificantly when DAB-G0 was used. This is a result of the competition between the phosphate groups of the lipid bilayer and the phosphate groups dissolved in the bulk aqueous phase. The redispersion of the aggregates, following the addition of concentrated phosphate buffer solution, was also monitored by DLS experiments. When the concentration of the buffer becomes 50 mM, the particles have about 1/10 of the initial size, while at 200 mM their size becomes comparable to that of the initially obtained multilamellar liposomes (Figure 3), in line with optical microscopy and AFM experiments. ζ-Potential Measurements. The interaction between the negatively charged phosphate groups of the liposomal bilayer and the positively charged guanidinium or amino groups of dendrimers at low dendrimer:DHP molar ratios, i.e., before any precipitation occurs, was followed by ζ-potential measurements. In water, as the dendrimer: DHP molar ratio increases, the liposomal ζ-potential values progressively increase to more positive values (Figure 4A), indicating that, due to interactions between the phosphate and guanidinium groups, dendrimers are located in the surface of the particle. At a dendrimer:DHP
Figure 4. Variation of liposomal ζ-potential values following their interaction with DAB-G0 (open squares), DAB-G6 (filled circles), and DAB-G12 (open circles) dendrimers in water (A) or in 10 mM phosphate buffer (B).
molar ratio of about 1:60 for the nonguanidinylated dendrimer and 1:80 for the guanidinylated ones, the ζ-potential values become zero, and therefore, it is possible for the noncharged liposomal particles to further interact and aggregate. At even higher molar ratios the ζ-potential values increase further, and positive plateau values are reached when the dendrimer:DHP molar ratios become 1:30 and 1:40 for the nonguanidinylated and guanidinylated dendrimers, respectively. It is evident that at these molar ratios dendrimers are located in the surface of the liposomes. Aggregation can be induced not only due to the presence of guanidinium groups but also due to the presence of secondary and tertiary amino groups (DAB-G0) although in this case the interaction is smaller. The ζ-potential plateau value is significantly higher in the case of DAB-G6 compared to DAB-G0 but lower in the case of DAB-G6 compared to DAB-G12. Analogous results were observed when the same experiments were performed in 10 mM phosphate buffer (pH 7.4). In this case, the ζ-potential values were significantly lower (Figure 4B), while the obtained plateau value is always negative. It is evident that phosphate ions present in solution interact with the positively charged groups of the dendrimers, and therefore, dendrimerliposome interaction is less effective. Following the addition of concentrated phosphate buffer in aggregates resulting from the mixing of multilamellar liposomes with guanidinylated or nonguanidinylated dendrimers, the ζ-potential values of the obtained particles decreased as the buffer concentration increased (Figure 5). At 200 mM phosphate concentration the resulting particles have not only the dimensions of multilamellar liposomes, as observed by DLS experiments, but also the same ζ-potential values (cf. Table 1). Therefore, the addition of the concentrated buffer solution not only induces the disruption of the aggregates, but fully reverses the interaction between the dendrimeric molecules and the phosphate groups located at the external surface of the multilamellar liposomes. Isothermal Titration Calorimetry. The binding properties of the three dendrimeric derivatives DAB-G0, DAB-G6, and DAB-G12 with PC-CHOL-DHP multila-
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Figure 5. Variation of ζ-potential values of particles obtained following mixing of DAB-G0 (open symbols) or DAB-G12 (filled symbols) with multilamellar liposomes during their redispersion by the addition of concentrated phosphate buffer.
Figure 6. Isothermal titration data for the sharp exothermic peaks during the first four injections of DAB-G0 (dotted line), DAB-G6 (dashed line), and DAB-G12 (dashed-dotted line) with PC-CHOL-DHP multilamellar liposomes in water. The control experiment, that is, aqueous dispersion of PC-CHOL multilamellar liposomes titrated with DAB-G12, is also presented (solid line). Inset: Raw ITC data for the entire titration experiment (40 injections) of DAB-G12 with PC-CHOL-DHP multilamellar liposomes in water.
mellar liposomes in water or phosphate buffer solution were explored by employing isothermal titration calorimetry. Thus, multilamellar PC-CHOL-DHP liposomal dispersions have been titrated with guanidinylated or nonguanidinylated dendrimeric solutions. Exothermic enthalpy changes related to the interaction were observed during the first injections, reaching a saturation plateau quite fast (Figure 6). The observed exothermic enthalpy changes include contributions from the binding of the dendrimeric guanidinium groups with the PC and DHP phosphate groups as well as of the dendrimeric secondary and tertiary amino groups with the PC and DHP phosphate groups. The latter effect is clearly demonstrated by the titration experiments of the multilamellar liposomes with DAB-G0 (Figures 6 and 7). Control experiments for the interaction of dendrimers with multilamellar PC-CHOL liposomes have also been carried out. In this case significantly less binding enthalpy change was measured during the titration (Figure 6) corresponding to the weak binding of the dendrimers with the phosphate groups of the PC as well to dilution heat. Upon subtraction of the calorimetric contribution from the control experiment, a single-site binding model has been
Figure 7. Isothermal titration calorimetric data (symbols) and fitted curves (lines) for the interaction of DAB-G0 (filled circles), DAB-G6 (open squares), and DAB-G12 (filled squares) with multilamellar PC-CHOL-DHP liposomes in water (A, top) and in phosphate buffer (B, bottom). A one set of binding sites model with constant stoichiometry has been used to fit the experimental results. Table 3. Fitting Results of the ITC Data for the Interaction of Dendrimeric Derivatives with PC-CHOL-DHP Multilamellar Liposomes in Water dendrimer
∆H (kcal mol-1)
Kapp (M-1)
DAB-G0 DAB-G6 DAB-G12
-1.6 × -2.8 × 102 -3.8 × 102
1.7 × 105 4.7 × 105 4.1 × 105
102
applied for the interaction between guanidinium and amino groups of the dendrimers with DHP phosphate groups, leading to the ∆H and apparent Kapp values listed in Table 3. Since the interacting concentration of DHP cannot be accurately estimated, the so-obtained values are only approximate. The isothermal titration data and the corresponding curves are shown in Figure 7. The results from ITC experiments in phosphate buffers have also been carried out. A much weaker interaction picture emerges due to the competition between the liposomal and the buffer phosphate groups. The results are also shown in Figure 7. Not surprisingly, fitting attempts with the one-set-of-sites binding model are not successful. Calcein Entrapment Experiments. The stability of the multilamellar liposomes during their interaction with the dendrimers was investigated employing the calcein fluorescence method. Calcein entrapped in liposomes does not fluoresce due to self-quenching because of its high concentration in the aqueous core. However, it gives a
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Table 4. Drug Transfer (%) from Dendrimers to Multilamellar Liposomes (a) in the Aggregates Obtained after Their Interaction in Water or in 10 mM Phosphate Buffer (pH 7.4) and (b) in the Multilamellar Liposomes Obtained Following Redispersion of the Aggregates drug transfer (%) in aggregates
drug transfer (%) after redispersion
drug
dendrimer
water
phosphate buffer
water
phosphate buffer
BTP
DAB-G0 DAB-G6 DAB-G12 DAB-G0 DAB-G6 DAB-G12
24.4 ( 2.4 62.5 ( 1.9 84.5 ( 2.1 32.9 ( 2.0 59.0 ( 1.5 78.1 ( 2.3
19.8 ( 1.2 48.5 ( 1.6 68.4 ( 1.5 27.1 ( 1.0 39.5 ( 2.1 57.5 ( 2.0
15.8 ( 0.9 28.1 ( 1.7 45.1 ( 1.8 15.9 ( 1.2 29.0 ( 1.0 42.0 ( 1.5
12.1 ( 1.1 24.5 ( 1.3 40.0 ( 1.4 14.1 ( 0.9 26.1 ( 1.5 38.2 ( 1.2
BTV
strong signal once released to the bulk aqueous phase.34,35 According to this technique, calcein was entrapped in the multilamellar liposomes employing a procedure described in previous papers.22,36 Following their interaction with the modified dendrimers at a 1:25 dendrimer:DHP molar ratio, only a slight fluorescence intensity increase was observed, indicating less than 13% leakage of calcein as shown in Figure 8.
mM phosphate buffer the drug present in the aggregates decreases slightly. In this case, the decrease of drug transport can be rationalized by the competitive interaction of the phosphate groups in the bulk with the guanidinium dendrimeric groups, leading to less effective adhesion with the multilamellar liposomes. Upon the addition of concentrated phosphate buffer, the redispersion of the aggregates in the medium and the separation of the no-longer-interacting dendrimers, drugs are still present in the obtained multilamellar liposomes. Determination of BTP or BTV in the multilamellar liposomes indicates that, in all cases, ca. 50% (Table 4) of the amount of drugs found in the aggregates before redispersion is still present, suggesting that they are located in the lipid bilayer since their solubility in water is extremely low. Drug transport is induced by the use of guanidinylated dendrimers since drug transport values of about 40-45% could be obtained in the case of DAB-G12 while only 12-15% was observed in the case of the nonguanidinylated derivative. Conclusions
Figure 8. Emission fluorescence intensity as a function of time of calcein solution encapsulated in multilamellar PC-CHOL-DHP liposomes following their interaction with DAB-G0 (open symbols) and DAB-G12 (closed symbols) as well as the corresponding calculated calcein leakage (inset).
Drug Transport from Dendrimers to Multilamellar Liposomes. The interaction between drug-loaded dendrimers and multilamellar liposomes results in the transport of drugs from the dendrimeric derivatives to the “empty” liposomes as summarized in Table 4. The experiments demonstrate that about 25% of BTP or BTV is present in the precipitated aggregates when DAB-G0 is used. When the guanidinylated dendrimers DAB-G6 and DAB-G12 are used, the amount of drugs in the precipitate increases substantially, becoming about 60% and 80%, respectively. These significant differences, observed in the transport of drug using guanidinylated or nonguanidinylated dendrimers, can be attributed to the functionalization of the dendrimeric molecules. The presence of guanidinium groups at the external surface of the dendrimers results in a more effective adhesion with the multilamellar liposomes as the ITC and DLS experiments demonstrate. As expected, when the interaction is taking place in 10 (34) Komatsu, H.; Okada S. Chem. Phys. Lipids 1997, 85, 67. (35) Osanai, S.; Nakamura, K. Biomaterials 2000, 21, 867 (36) Pantos, A.; Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Giatrellis, S.; Nounesis, G. Langmuir 2004, 20, 6165.
Guanidinylation of poly(propylene imine) dendrimers led to derivatives which interact effectively with complementary multilamellar liposomes consisting of phosphatidylcholine-cholesterol-dihexadecyl phosphate (19:9.5:1) which were dispersed in aqueous or phosphate buffer solutions. Various methods including calorimetric studies have established that molecular recognition between these nanoparticles is induced by the degree of guanidinylation of the dendrimeric surface. This process is however reversible, and redispersion of the aggregates occurs by adding concentrated phosphate buffer. In addition, in an attempt to study the drug transport properties of these functional dendrimers, it has been shown that during this interaction effective transport of betamethasone dipropionate and betamethasone valerate had occurred from the guanidinylated dendrimers to the liposomal aggregates. Drug transport ranged from 40% to 85% and was dependent on the number of guanidinium moieties at the dendrimer surface and certainly on the interaction medium. Following their redispersion, up to 40-45% of the amount of the drugs used still remained in the isolated multilamellar liposomes. Acknowledgment. This work was partially supported by the “Excellence in the Research Institutes” Program, Action 3.3.1, cofunded by the Greek Ministry of Development and EU, and by COST Chemistry, Action D27. We thank Dr. Z. Sideratou and Dr. L. Tziveleka for their help in the NMR experiments. LA0510331
