Synthesis and Surface Chemistry Study of a New Amphiphilic PAMAM

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Synthesis and Surface Chemistry Study of a New Amphiphilic PAMAM Dendrimer Guodong Sui, Miodrag Micic, Qun Huo, and Roger M. Leblanc* Center for Supramolecular Science and Center for Advanced Microscopy, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33124 Received April 18, 2000. In Final Form: July 18, 2000 A new disk-shaped amphiphilic dendrimer has been synthesized by attaching sixty-four 12-hydroxydodecanoic acid chains to a fourth generation poly(amido amine) (PAMAM) dendrimer core. Surface pressure and surface potential-area isotherm measurements have shown that the dendrimer forms a stable monolayer at the air/water interface with limiting molecular area of 160 Å2/molecule. This small area relative to the huge size of the dendrimer suggests that the dendrimer molecules form an edge-on disk-shaped structure at the air/water interface. The topography of the dendrimer monolayer was observed by Brewster angle microscopy (BAM) at air/water interface as well as by environmental scanning electron microscopy (ESEM) as a Langmuir-Blodgett film. The striplike monolayer domains observed from BAM images correspond to the monolayer topography as observed from ESEM images.

Introduction Langmuir and Langmuir-Blodgett films with novel molecular architectures have always been a focal point in surface chemistry research area. In the late 1980s and early 1990s, disklike molecules1,2 have triggered the interest of surface chemists due to their unique anisotropic physical properties. Ringsdorf and Wendoff as well as other groups have investigated systematically the molecular structures and properties of various discotic molecules in Langmuir or Langmuir-Blodgett films and in bulk state.3-8 Face-on and edge-on models were used to characterize the arrangement of the discotic molecules at the air-water interface (Figure 1).3-5 The edge-on configuration was of more interest compared to the face-on configured monolayer, since the edge-on configured monolayers are columnar Langmuir films which may display anisotropic transport properties related to electron transport, energy transport, etc.9-12 Dendrimers, representing a new class of macromolecules and characterized by their treelike structure, have wide potential applications in medical science and material research.13,14 Recently, the design, synthesis, and surface * To whom correspondence may be addressed: Tel 305-284-2282; Fax 305-284-4571; E-mail [email protected]. (1) Chandrasekar, S.; Ranganath, G. S. Rep. Prog. Phys. 1990, 53, 57. (2) Gregg, B. A.; Fox, M. A.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 3024. (3) Albrecht, O.; Cumming, W.; Kreuder, W.; Laschewsky, A.; Ringsdorf, H. Colloid Polym. Sci. 1986, 264, 659. (4) Ortmann, E.; Wegner, G. Angew. Chem., Int. Ed. Engl. 1986, 25, 1105. (5) Laschewsky, A. Angew. Chem. Adv. Mater. 1989, 101, 1606. (6) Karthaus, O.; Ringsdorf, H.; Tsukruk, V. V.; Wendorff, J. H. Langmuir 1992, 8, 2279. (7) Tsukruk, V. V.; Wendorff, J. H.; Karthaus, O.; Ringsdorf, H. Langmuir 1993, 9, 614. (8) Dahn, U.; Erdelen, C.; Ringsdorf, H.; Festag, R.; Wendorff, J. H.; Heiney, P. A.; Maliszewskyj, N. C. Liq. Cryst. 1995, 19, 759. (9) Boden, N.; Bushby, R. J.; Clements, J.; Jesudason, M. V.; Knowles, P. P.; Willliams, G. Chem. Phys. Lett. 1988, 152, 94. (10) Karthaus, O.; Ringsdorf, H.; Urban, C. Makromol. Chem. Macromol. Symp. 1991, 46, 347. (11) Praefcke, K.; Singer, D.; Kohne, B.; Ebert, M.; Liebmann, A.; Wendorff, J. H. Liq. Cryst. 1991, 10, 147. (12) Moller, M.; Tsukruk, V. V.; Wendorff, J. H.; Bengs, H.; Ringsdorf, H. Liq. Cryst. 1992, 12, 17. (13) Fre´chet, J. M. Science 1994, 263, 1710.

Figure 1. Face-on and edge-on configurations of disklike molecules in Langmuir and Langmuir-Blodgett films.

chemistry studies of amphiphilic dendrimers in Langmuir or Langmuir-Blodgett films are attracting increased attention.15-20 One of the interesting features of amphiphilic dendrimers is that most of them are also disklike molecules, with a flat hydrophilic dendrimer core surrounded by flexible hydrophobic chains. Face-on and edgeon configurations were also found in the Langmuir monolayers of these macromolecules.19,20 In the face-on configuration, the dendrimer core lies flat on the water surface, while the hydrocarbon chains extend away from the interface. The molecules with relatively strong corewater interactions and weak core-core interactions would generally show the face-on arrangement. In the edge-on configuration, the dendrimer core sits perpendicular to the interface with part of the flexible chains submerged into the water and with other chains extended into the air. Recently, we reported the synthesis and surface chemistry study of an amphiphilic dendrimer (PDA-PAMAM (14) Zeng, F.; Zimmerman, S. C. Chem. Rev. 1997, 97, 1681. (15) Sayed-Sweet, Y.; Hedstrand, D. M.; Spinder, R.; Tomalia, D. A. J. Mater. Chem. 1997, 7, 199. (16) Schenning, A. P. H. J.; Elissen-Roman, C.; Weener, J.-W.; Barrs, M. W. P. L.; van der Gaast, S. J.; Meijer, E. W. J. Am. Chem. Soc. 1998, 120, 8199. (17) Sheiko, S. S.; Buzin, A. I.; Muzafarov, A. M.; Rebrov, E. A.; Getmanova, E. V. Langmuir 1998, 14, 7468. (18) Kampf, J. P.; Frank, C. W.; Malmstro¨m, E. E.; Hawker, C. J. Langmuir 1999, 15, 227. (19) Josefowicz, J. Y.; Maliszewskyj, N. C.; Idzik, S. H. J.; Heiney, P. A.; Mccauley, J. P. Jr.; Smith III, A. B. Science 1993, 260, 323. (20) Mindyuk, O. Y.; Heiney, P. A. Adv. Mater. 1999, 11, 341.

10.1021/la0005762 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/06/2000

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Experimental Section

Figure 2. Structure of the HA-PAMAM dendrimer.

dendrimer) by attaching sixteen 10,12-pentacosadiynoic acid (PDA) to a second generation poly(amido amine) (PAMAM) dendrimer core.21 From the surface pressure and surface dipole moment area isotherm studies, it was found that in the Langmuir monolayer PDA-PAMAM dendrimer molecules arrange themselves in a face-on configuration in which the hydrophilic core of the dendrimer is in contact with water and all the alkyl chains point to the air. This face-on arrangement is attributed to the relatively weak interactions between the small PAMAM dendrimer cores (second generation). From this previous study, we proposed that if the size of the PAMAM dendrimer core is increased, the corecore interactions should increase. With the increased corecore interactions, the amphiphilic dendrimer may adopt the edge-on configuration at the air/water interface. We herein report the design, synthesis, and study of a new amphiphilic dendrimer by attaching sixty-four 12-hydroxydodecanoic acid chains to a fourth generation PAMAM dendrimer core (HA-PAMAM, Figure 2). It needs to be pointed out that, other than the increased core size (from second generation to fourth generation), a total of 64 hydrophilic OH groups are presented in the out shell of this new dendrimer. Hydroxyl groups are the most commonly encountered functional groups to form intraor intermolecular hydrogen bonding in aqueous and organic solvent media.22,23 It is expected that the possible hydrogen bonding between the hydroxyl groups on the dendrimer will lead to a flat geometry of this dendrimer and therefore to adopt an edge-on configuration in Langmuir films. (21) Sui, G.; Micic, M.; Huo, Q.; Leblanc, R. M. Colloids Surf. A: Physicochem. Eng. Aspects 2000, 171, 185. (22) Lehn, J.-M. Supramolecular Chemistry; VCH: New York, 1995. (23) Atwood, J. L.; Davies, J. E. D.; MacNicol, D. D.; Vo¨gtle, F.; Lehn, J.-M. Comprehensive Supramolecular Chemistry; Pergamon: New York, 1996.

Materials. Starburst PAMAM dendrimers and other chemicals were purchased from Aldrich Chemical Co. and were used without further purification. All organic solvents for the synthesis were purchased from Fisher Scientific Co. as reagent grade and were also used without further purification. The 1H NMR spectra were recorded on a Bruker 300 MHz spectrometer. The IR spectrum was obtained from a Perkin-Elmer PE-GRAMS/2000 FT-IR spectrometer. The MALDI-TOF mass spectrum was conducted by the University of Illinois, School of Chemical Sciences. Synthesis. 12-Hydroxydodecanoic acid-N-hydroxysuccinimide Ester. A mixture of 12-hydroxydodecanoic acid (216.31 MW, 216.3 mg), N-hydroxysuccinimide (115.0 MW, 184.0 mg), and EDC (1(3-(dimethylamino)propy)-3-ethylcarbodiimide hydrochloride, 192 MW, 230.4 mg) were dissolved in 15 mL of CH2Cl2 and then was stirred for 24 h. The solution was washed with 20 mL of water and 20 mL of NaHCO3 saturated solution, followed by 20 mL of brine. Then the solution was dried over anhydrous Na2SO4. The solvent was then evaporated in vacuo to give white solids; 218 mg, yield 71%. FAB-MS (m/z): calcd for C16H27O5N 313; found 314 (M + 1)+. 1H NMR (CDCl3): δ 1.34 (m, 18 H), 1.73 (t, 2 H), 2.59 (t, 2 H), 2.82 (t, 4 H), 3.63 (t, 2 H), 4.09 (s, 1 H). 12-Hydroxydodecanoic Acid Functionalized Poly(amido amine) Dendrimer (Generation 4) (HA-PAMAM Dendrimer). To the 7 mL CH2Cl2 solution, 12-hydroxydodecanoic acid N-hydroxysuccinimide ester (297 MW, 90.0 mg) and 600 µL of starburst dendrimer (generation 4, 10 wt % in methyl alcohol) were added within 20 min, and the solution was stirred vigorously for 24 h. The solution was concentrated under vacuum. To the left solids, 30 mL of 1 N NaOH solution was added, and the suspension was stirred for 3 h. The suspension was filtered, and the solids were washed with 1 N NaOH solution, followed by distilled water and demineralized water. After being vacuum-dried, the product was obtained as white solids (34.1 mg, yield 37%). MALDI-TOF MS, m/z calcd for C1390H2656O252N250, 26868.0; found, 23155.1. IR (cm-1) (KBr): 3302 s, 2850 s, 1645 s, 1558 s, 1244 w, 721 w. 1H NMR (d-DMSO): δ 1.1-1.5 (m, 1152 H), 2.03 (s, 188 H), 2.18 (s, 750 H), 2.63 (s, 252 H), 3.07 (s, 128 H). General Methods for Surface Chemistry Study. All the surface chemistry studies were conducted in a clean room of class 1000 where temperature (20 ( 1 °C) and humidity (50 ( 1%) were controlled. The spreading solvent was chloroform and was purchased from Fisher Scientific Co. as HPLC grade. The water used as subphase for the monolayer film study was purified by a Modulab 2020 water purification system (Continental Water System Corp., San Antonio, TX) with a specific resistivity of 18 MΩ‚cm and a surface tension of 72.6 mN/m at 20 (1 °C. The injection volume of the dendrimer solution (0.1 mg/mL, CHCl3/ CF3COOH mixture solvent (v:v, 60:1)) was 30 µL for the surface pressure measurements, 70 µL for the surface potential measurements, and 40 µL for the Brewster angle experiments. After the solution was spread, a 15 min period was allowed to pass by for the complete evaporation of solvent before compression. The compression rate was set up at 5 Å2 molecule-1 min-1 for all the three different Langmuir troughs. Surface pressure measurements were made with a KSV minitrough (KSV Instrument Ltd., Helsinki, Finland). Two computer-controlled symmetrically movable barriers were used to regulate the surface area. The trough dimensions are 0.6 × 7.5 × 30.0 cm3. The surface pressure was measured by the Wilhelmy method with a sensitivity of (0.01 mN/m. Surface potential measurements were recorded using a homemade trough. Two symmetrically movable barriers controlled by the computer were used to regulate the surface area. The dimensions of this trough are 0.6 × 12.0 × 100.0 cm3. The surface potential was measured using the ionizing electrode method. A reference platinum electrode was immersed in the reference trough compartment, and an americium electrode (241Am) was placed at about 1-2 mm above the monolayer under study. Topographical Studies. For the purpose of Langmuir film topological studies at the air/water interface, we used a Brewster angle microscope (BAM). The system consists of a Nippon trough (5.0 × 47.0 cm2) equipped with a moving wall system (NL-LB

A New Amphiphilic PAMAM Dendrimer

Figure 3. Surface pressure-area isotherm (left) and surface dipole moment-area isotherm (right) of the HA-PAMAM dendrimer at air/water interface (pH ) 5.8 and T ) 20 ( 1 °C). 140SMWC, Nippon Laser & Electronics Lab., Nagoya, Japan), a helium-neon laser (wavelength 632.8 nm and power 10 mW), and a CCD camera. The images from the CCD were captured and digitized using a digital video capture mode (Snappy video Snapshot, Rancho Cordova, CA) for further analysis. The threedimensional representations of the BAM images were also presented by using a software NIH 1.62 image that allows noise reduction by using a low pass filter. The dark areas of the image represent the valleys, whereas the bright areas represent high peaks in the 3-dimensional figures. All the images presented in this work have dimensions of 400 µm × 400 µm. We also used a Philips/Electroscan environmental scanning electron microscope, XL 30-FEG, equipped with a field emission electron gun, to study the topography of a Langmuir-Blodgett film deposited on the surface of a mica plate. All imaging has been performed in a “wet mode”, using water vapor pressure in range of 0.6 Torr as the imaging gas and protective atmosphere. Images presented herein are gaseous secondary electron images (GSE). A wide range gaseous secondary electron detector was used without an external aperture. It has been previously demonstrated that a low-vacuum field emission gun ESEM could be successfully applied in Langmuir-Blodgett film studies.

Results and Discussion Surface Pressure and Surface Dipole MomentArea Isotherms. The surface pressure-area isotherm shown in Figure 3 suggests that the HA-PAMAM dendrimer forms a monolayer at air/water interface which evolves from a liquid expanded phase between surface pressure nil and 6 mN/m to a liquid condensed phase at surface pressure between 6 and 30 mN/m. The limiting molecular area was obtained by the extrapolation of the liquid condensed part of the isotherm at surface pressure nil with a value of 160 Å2/molecule. The surface dipole moment for nonionized monolayers, µ⊥, is used to describe the molecular orientation in a film. The surface dipole moment (mD) is obtained from surface potential measurement (∆V, mV) by the equation µ⊥ ) A∆V/12π, where A is the molecular area (Å2/molecule).24 Before compression, the total dipole moment is zero, due to the random distribution of the amphiphile dendrimer molecules. With compression, the amphiphiles start to orient themselves at about 500 Å2/molecule to form an organized structure, and the dipole moment of the monolayer starts to increase. The monolayer reaches a liquid condensed phase at about 250 Å2/molecule, and the (24) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interface, P73; Interscience Publisher: New York, 1966.

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surface dipole moment reaches its maximum value at 130 Å2/molecule. The surface dipole moment-area isotherm (Figure 3) shows that there is a steady increase from 500 to 130 Å2/molecule. Corresponding to the surface pressure-area isotherm, the surface dipole moment-area isotherm confirms that this dendrimer has a relatively small molecular area compared to its size. The molecular areas obtained from the isotherms can be used to determine the molecular configuration of the monolayer.20 Compared to the face-on configuration, the molecules with an edge-on configuration are in a more dense arrangement, so the monolayer with edge-on configuration will show a lower limiting molecular area. The limiting molecular area is generally of the order of 200-600 Å2 with the face-on configuration, while the edgeon arrangement was characterized by molecular area of 70-100 Å2. The PDA-PAMAM dendrimer21 we reported previously has a second-generation PAMAM core and sixteen 10,12pentacosadiynoic acid chains with a molecular area of 460 Å2/molecule. In this case, the face-on configuration applies, and the area of 460 Å2 is estimated to be the size of the dendrimer core. Compared with the PDA-PAMAM dendrimer, the HA-PAMAM dendrimer has a fourth generation PAMAM dendrimer core with 64 hydroxydodecane chains. However, its molecular area is only 160 Å2/ molecule. The molecular area would be much larger than that of the PDA-PAMAM dendrimer if the HA-PAMAM dendrimer molecules arranged themselves in a face-on configuration. Obviously, the molecular area of 160 Å2/ molecule suggests that the edge-on configuration applies to HA-PAMAM dendrimer in the Langmuir films, though the molecular area (160 Å2/molecule) is slightly larger than the characteristic molecular area (70-100 Å2/ molecule) of edge-on configured monolayers. Other than the increased core size, the number of hydrocarbon chains around the HA-PAMAM dendrimer is also increased compared to the case of PDA-PAMAM. This increased number of hydrocarbon chains leads to increased van der Waals interactions. Furthermore, as stated previously, the possible hydrogen bonding between the hydroxyl groups on the out shell of the dendrimer will also help the molecule to adopt a flat geometry. Except for the surface pressure and surface potential isotherm measurements, indirect experimental evidences also support the flat geometry and the edge-on configuration of HA-PAMAM dendrimer in free solution and at the air/water interface. The first evidence is the solubility of the dendrimer. Originally, it was thought that with 64 hydrophilic OH groups on the surface and medium long hydrocarbon chains (C12), the dendrimer should be quite soluble in aqueous or polar organic solvent. However, our test shows that this dendrimer is very difficult to dissolve in water and most of organic solvent. A very small amount of trifluoroacetic acid was obliged to mix with chloroform as spreading solvent for monolayer study. This is because the HA-PAMAM dendrimer molecules are stacked one by one with their flat geometry to form a filament-like or cylindrical assembly as suggested in our previous study.21 Because of the formation of these large stable assemblies, the solubility of the dendrimer is very poor. Furthermore, we have noticed that when using vertical-dipping method to prepare the Langmuir-Blodgett film onto hydrophilic mica (upward), the surface of one-layer film is still hydrophilic although the deposition ratio is near 0.8. This fact indirectly suggests that edge-on is the most likely configuration of HA-PAMAM molecules in the Langmuir

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Figure 4. Brewster angle microscopy images of Langmuir films on pure water at different surface pressures: A, 12 mN/m; B, 30 mN/m (pH ) 5.8 and T ) 20 ( 1 °C).

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We also used the ESEM microscopic technique to study the topological features of the HA-PAMAM dendrimer in LB films. The dendrimer film with well-expressed and preserved topography could be deposited on the hydrophilic mica substrate. It was found that the L-B film of HA-PAMAM molecules deposited onto mica is resistant to the electron beam irradiation up to intensity of 10 keV and that interaction of electron beam with sample does not disturb the fine structure of the film. Figure 5 represents an ESEM image of the deposited LangmuirBlodgett film at the surface pressure of 5 mN/m. It is possible to observe basically two types of different domains: rectangular and elliptical. Predominant domain patterns are elongated rectangular, with width in the range 5-15 µm and length between 60 and 200 µm. Such rectangular domains are packed together in larger, superdomains, which possess leaflike fractal forms. This fractal superdomain form is created by alignment of rectangular domains along their longer side. Individual rectangular domains are separated from each other in such a superdomain by 2-8 µm. It is interesting that the length of the rectangular domain expresses decreasing trend from center toward the end of superdomain. However, this decrease from center to the end of superdomain is not uniform, but more “saw-tooth”-like, which at the end provides a shape with a fractal dimensionality that closely resembles a palm leaf. The size of such superdomains is in the range of 100 µm in width and 100250 µm in length. Rectangular domains, arranged in superdomains, cover approximately 85-95% of the surface area. Compact circular or slightly ellipsoidal domains cover the remaining 5-15% of the total film area, with a diameter between 30 and 60 µm. With the increased surface pressure, redundancy of the deposited L-B film is decreasing, and the deposited film becomes quite uniform, with a single macrodomain covering an area millimeters in size. In general, topological features observed from BAM images of Langmuir films at the air/ water interface are similar to those expressed in the deposited L-B film as observed from ESEM images. In both cases, we were able to observe circular and rectangular regions, within the same dimension range. Conclusion

monolayer with part of hydroxyl groups on the dendrimer oriented toward the air. Topological Studies. Brewster angle microscopy has been used extensively to study the topography of monolayers at the air/water interface.25,26 In the edge-on configuration, disk-shaped dendrimer molecules are stacked upon each other and sitting perpendicular to the water surface. This configuration should lead to more compact packing of molecules than that of the face-on configuration, and we should observe more regular sized domains from the BAM images than that of the face-on configured dendrimer Langmuir films. The topography of the HA-PAMAM dendrimer monolayer was first observed by Brewster angle microscopy, as shown in Figure 4. One can see that, with increased surface pressure, the monolayer becomes more uniform and the regular sized striplike topography becomes more and more clear. This kind of domain was not observed in BAM images of the face-on configured PDA-PAMAM dendrimer Langmuir films.21 (25) Saville, P. M.; White, J. W.; Hawker, C. J.; Wooley, K. L.; Fre´chet, J. M. J. J. Phys. Chem. 1993, 97, 293. (26) Patino, J. M. R.; Sa´nchez, C. C.; Nin˜o, M. R. R. Langmuir 1999, 15, 4777.

In summary, the present study as well as our previous investigation on amphiphilic PAMAM dendrimers has shown that these disklike macromolecular species are able to form stable Langmuir monolayers at the air/water interface with different molecular configurations. In contrast to the study of small organic molecule-functionized lipid molecules, the use of branched dendrimers in monolayer studies opens a new door in the preparation of thin films with novel functions. It also should be mentioned that the design of the HAPAMAM dendrimer has other potential applications. One can see that, compared to conventional vesicles, this dendrimer also contains a very similar three-layer structure: the hydrophilic dendrimer inner core, the hydrophobic hydrocarbon middle layer, and the hydrophilic OH groups in the outer shell. Despite the flat geometry of the molecule, it might be regarded as a “minivesicle”. Considering the important application of micelles, vesicles, and liposomes in drug delivery,27 we may use this dendrimer as a molecular vehicle for drug delivery as well. In contrast to vesicles, which are molecular assemblies (27) Gregoriadis, G.; Allison, A. C. Liposomes in Biological Systems; John Wiley & Sons: New York, 1980.

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Figure 5. Environmental scanning electron microscopy image of Langmuir-Blodgett film deposited on mica at surface pressure 5 mN/m (imaging conditions: wet mode, 0.6 Torr, magnification 204× at 10 kV).

through noncovalent intermolecular interactions, the dendrimer is a single macromolecule which is built up completely by covalent bonding. The stability of these macromolecular carriers might be largely improved compared to vesicles with similar size. More importantly, the hydroxyl groups on the surface of the dendrimer can

be further modified by various functional groups such as carbohydrates, peptides, proteins, and fluorescence probes for molecular recognition studies toward a specific target in biological systems. LA0005762