Potentiometric Response Characteristics of Polycation-Sensitive

Stacey A. Nevins Buchanan, Lajos P. Balogh, and Mark E. Meyerhoff* ... University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-...
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Anal. Chem. 2004, 76, 1474-1482

Potentiometric Response Characteristics of Polycation-Sensitive Membrane Electrodes toward Poly(amidoamine) and Poly(propylenimine) Dendrimers Stacey A. Nevins Buchanan,† Lajos P. Balogh,‡ and Mark E. Meyerhoff*,†

Department of Chemistry, The University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055, and Department of Internal Medicine, The University of Michigan, 200 Zina Pitcher Place, Ann Arbor, Michigan 48109-0533

The potentiometric response characteristics of polycationsensitive membrane electrodes toward two classes of polycationic dendrimers are examined. Using appropriately formulated polymer membrane electrodes composed of a dinonylnaphthalenesulfonate (DNNS) salt in a plasticized polyurethane matrix, it is shown that poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI) dendrimers are readily detected at submicrogram per milliliter levels via a nonequilibrium response mechanism. The relationship between the total EMF response (at equilibrium) and the specific dendrimer structure is also examined. For both the PAMAM and PPI species, it is shown that the total EMF response does not change significantly with dendrimer generation number; however, the nonequilibrium analytically useful response curves are shifted to higher mass concentrations as the generation number is increased. The relative contributions of the terminal primary amines and the interior tertiary amines of the dendrimers to the observed EMF response are investigated by synthesis of various dendrimer derivatives (acetylated, quaternized, etc.). By comparing the total EMF responses for these derivatives as a function of sample pH, it is demonstrated that the lipophilic cation exchanger (DNNS) within the membrane phase can likely interact electrostatically with both protonated forms of the terminal primary amines and interior tertiary amines of the dendrimer structures. The practical application of the nonequilibrium potentiometric detection of dendrimers for monitoring their interaction with DNA is also demonstrated. Dendrimers are highly branched macromolecules, named for their dendritic or treelike form. These species are characterized by well-defined structures with relatively high monodispersity and end-group multivalency.1,2 During the past two decades, dendrimer chemistry has developed into a rapidly growing field, with * To whom correspondence should be addressed. E-mail: mmeyerho@ umich.edu. † Department of Chemistry. ‡ Department of Internal Medicine. (1) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III. Angew. Chem., Int. Ed. Engl. 1990, 29, 138-175. (2) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 16651688.

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numerous reports appearing regarding the synthesis and characterization of novel dendrimer structures. Furthermore, these materials have been proposed for use in a wide variety of biomedical applications. For example, several polycationic dendrimers have been shown to be effective gene therapy vectors,3-6 drug delivery platforms,7 or contrast agents for imaging.8-10 In addition, these species have been proposed for use in other fields, including lithography,11-13 light harvesting,14,15 and catalysis.16,17 With such applications growing, there is a need for better analytical methods to quantitate dendrimers and to examine their interactions with other macromolecules. Several types of dendrimers include acidic or basic moieties in their repeating branch units and terminal groups, resulting in a highly charged macromolecule (a polyion). Two examples of these are poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI) dendrimers, which gain polycationic character from the tertiary amines located at interior branch points and the primary amines at branch termini. The structures of a generation 3 PAMAM dendrimer and a generation 4 PPI dendrimer are shown in Figure 1. While the generation nomenclature differs for these two types of dendrimers, these are equivalent species, containing the same number of interior branch points and terminal groups (3) Haensler, J.; Szoka, F. C. Bioconjugate Chem. 1993, 4, 372-379. (4) Kukowska-Latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spindler, R.; Tomalia, D. A.; Baker, J. R., Jr. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4897-4902. (5) Bielinska, A. U.; Kukoswka-Latallo, J. F.; Baker, J. R., Jr. Biochim. Biophys. Acta 1997, 1353, 180-190. (6) Zinselmeyer, B. H.; Mackay, S. P.; Schatzlein, A. G.; Uchegbu, I. F. Pharm. Res. 2002, 19, 960-967. (7) Liu, M.; Fre´chet, J. M. J. Pharm. Sci. Technol. Today 1999, 2, 393-401. (8) Wiener, E. C.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow, O. A.; Tomalia, D. A.; Lauterbur, P. C. Magn. Reson. Med. 1994, 31, 1-8. (9) Wiener, E. C.; Konda, S.; Shadron, A.; Brechbiel, M.; Gansow, O. Invest. Radiol. 1997, 32, 748-754. (10) Yordanov, A. T.; Lodder, A. L.; Woller, E. K.; Cloninger, M. J.; Patronas, N.; Milenic, D.; Brechbiel, M. W. Nano Lett. 2002, 2, 595-599. (11) Tully, D. C.; Wilder, K.; Fre´chet, J. M. J.; Trimble, A. R.; Quate, C. F. Adv. Mater. 1999, 11, 314-348. (12) Tully, D. C.; Trimble, A. R.; Fre´chet, J. M. J.; Wilder, K.; Quate, C. F. Chem. Mater. 1999, 11, 2892-2898. (13) Li, H. W.; Kang, D. J.; Blamire, M. G.; Huck, W. T. S. Nano Lett. 2002, 2, 347-349. (14) Stewart, G. M.; Fox, M. A. J. Am. Chem. Soc. 1996, 118, 4354-4360. (15) Balzani, V.; Ceroni, P.; Juris, A.; Venturi, M. Campagna, S.; Puntoriero, F.; Serroni, S. Coord. Chem. Rev. 2001, 219, 545-572. (16) Zhao, M. Q.; Crooks, R. M. Angew. Chem., Int. Ed. 1999, 38, 364-366. (17) Niu, Y. H.; Yeung, L. K.; Crooks, R. M. J. Am. Chem. Soc. 2001, 123, 68406846. 10.1021/ac035265l CCC: $27.50

© 2004 American Chemical Society Published on Web 01/22/2004

Figure 1. Structures of (a) a poly(amidoamine) (PAMAM) generation 3 amine-form dendrimer (G3A) and (b) poly(propylenimine) (PPI) generation 4 amine-form dendrimer (DAB-32). Both dendrimers contain 30 interior tertiary amines and 32 primary amines at branch termini.

but differing in their core and linking group compositions. For clarity, the PPI dendrimers in this report are thus identified by the number of end groups (e.g., DAB-32) rather than generation number. These two classes of dendrimers, in particular the PAMAM dendrimers, have been employed in many of the applications cited above. Unfortunately, these species are not readily quantitated in solution, since they lack a strong chromophore group that absorbs above 230 nm. Potentiometric polyion sensors are sensitive analytical tools that are useful for detecting highly charged macromolecules in samples as complex as whole blood.18-20 These membrane electrodes exhibit large and reproducible potentiometric responses

toward polyions. Such response is attributed to a change in the nonequilibrium steady-state phase boundary potential at the sample/membrane interface. This results from the extraction of the polyion into the organic membrane, stabilized by cooperative ion-pairing interactions with lipophilic ion exchangers20 (e.g., dinonylnaphthalenesulfonate doped in a polymeric membrane for polycation sensing). It has recently been shown that fully (18) Ma, S. C.; Yang, V. C.; Fu, B.; Meyerhoff, M. E. Anal. Chem. 1993, 65, 2078-2084. (19) Fu, B.; Bakker, E.; Yun, J. H.; Yang, V. C.; Meyerhoff, M E. Anal. Chem. 1994, 66, 2250-2259. (20) Fu, B.; Bakker, E.; Yun, J. H.; Meyerhoff, M. E. Macromolecules 1995, 28, 5834-5840.

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reversible polyion response can be achieved by operating such sensors in a pulsed galvanostatic mode,21 and this advance could ultimately make these sensors even more useful. PAMAM and PPI dendrimers represent a new class of polyions for investigation with these sensors; prior to this work, only linear polyionic species (e.g., heparin, polyphosphates, protamine, etc.) have been studied. Thus, the primary goal of this fundamental analytical study was to examine the nature of polycation sensor response as a function of dendrimer structure and composition. To this end, various PAMAM and PPI derivatives were synthesized and characterized, and the equilibrium and nonequilibrium potentiometric responses of DNNS-based polycation sensors toward these species are examined in detail. MATERIALS AND METHODS Reagents. Tris(hydroxymethyl)aminomethane and 2-nitrophenyl octyl ether (NPOE) were purchased from Sigma Chemical Co. (St. Louis, MO). Tetrahydrofuran was a product of Fisher (Fair Lawn, NJ). Iodomethane, 1,2,2,6,6-pentamethylpiperidine, tetramethylammonium hydroxide pentahydrate, formic acid, and formaldehyde were purchased from Aldrich. Amine-form generation 3 (G3A) PAMAM dendrimers were obtained from Aldrich (Milwaukee, WI), and amine-form generations 5 and 7 (G5A and G7A) PAMAM dendrimers were purchased from Dendritech (Midland, MI). Carboxyl-terminated generation 2.5 PAMAM dendrimers (G2.5) and generations 3, 4, and 5 PPI dendrimers (DAB-16, DAB32, and DAB-64, respectively) were products of Aldrich. Linear double-stranded DNA (500 bp, 50% G-C content) was obtained from Gensura (San Diego, CA) and was stored at 4 °C prior to use. M48 polyurethane was provided by Medtronic Inc. (Minneapolis, MN). Poly(acetylenepyridinium) PAMAM copolymers were prepared as described previously.22 Calcium dinonylnaphthalenesulfonate (DNNS) was a gift from King Industries (Norwalk, CT). All other reagents were analytical grade or better. Preparation of Polyion-Sensitive Electrodes. Cylindrical polycation-sensitive electrodes were prepared as described previously.23 Briefly, membranes with a composition of 1% (w/w) DNNS, 49.5% (w/w) NPOE, and 49.5% (w/w) M48 were utilized. The electrodes were filled with an internal electrolyte composed of 20 mM phosphate buffer (containing 60 mM Na+ and 42 mM Cl-), and a Ag/AgCl wire was inserted to complete the assembly. Planar rotating polycation-sensitive electrodes were prepared as described recently,24 using the same membrane composition as the cylindrical electrodes. EMF Measurements. The EMF response of the polycationsensitive electrodes was measured against an external Ag/AgCl wire placed in the test solution (phosphate buffer, containing 60 mM Na+). Small volumes of dendrimer stock solutions (0.1-20 µg/µL) were added to the well-stirred 3-5 mL of buffer solution (identical to the internal filling solution of the electrode), and the potentiometric response was recorded at fixed time intervals (typically, 2 min) to obtain the nonequilibrium dendrimer dose responses. To obtain equilibrium responses, greater volumes of the most concentrated dendrimer stock solutions were added to the buffer solution (to yield final concentrations of 20 µg/mL) and the EMF was recorded after 5 min. The response of the sensors to high concentrations of polycation is rapid, while the reverse EMF responses are very slow, owing to the strong ionpairing interactions in the membrane phase, making the poten1476

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tiometric responses irreversible. Thus, sensors were used once and then discarded. Rotating electrode measurements were facilitated by a specially designed apparatus, as described previously.24 For both static and rotating measurements, the electrodes were connected to a VF-4 electrode interface module (World Precision Instruments, Sarasota, FL) coupled to a NB-MIO analog/ digital input/output board (National Instruments, Austin, TX) in a Macintosh IIcx computer. LabView 2 software was used to record all data. Gel Electrophoresis. Dendrimer samples were analyzed using poly(acrylamide) gel electrophoresis (PAGE). Precast gradient gels (4-20%) were purchased from ISC BioExpress (Kaysville, UT). Separations were performed using a Mini-Protean electrophoresis system (Bio-Rad, Hercules, CA) at 200 V (normal polarity, 25 V/cm) for 30-60 min using a Tris-borate-EDTA separation buffer, pH 8.3 (89 mM Tris, 89 mM boric acid, and 2.5 mM Na2EDTA). Gels were stained for 10 min in 0.025% (w/v) Coomassie Blue R-250 solution (7% (v/v) acetic acid, 40% (v/v) methanol) and then placed in destaining solution overnight (5% (v/v) methanol, 7% (v/v) acetic acid). Electrospray Ionization Mass Spectrometry (ESI-MS). Mass spectrometry data were obtained using a Waters/Micromass LCT instrument (Milford, MA). The instrument included a Z-spray ion source for electrospray ionization. Two hexapole rf lenses introduce ions into the orthogonal acceleration reflectron timeof-flight mass analyzer. Dendrimers were dissolved in solutions of 90% (v/v) methanol and 0.3% (v/v) acetic acid to concentrations of ∼1.0 µg/mL and introduced into the instrument by direct infusion at 100 µL/min. Spectra were deconvoluted using MassLynx software and the MaxEnt 1 algorithm. Synthesis of Acetylated Dendrimers. Generation 3 PAMAM dendrimers (G3A) and generation 4 PPI dendrimers (DAB-32) were acetylated at their terminal primary amine groups via reaction with acetic anhydride. The synthesis procedure is available in Supporting Information. Analysis via ESI-MS indicated complete conversion of all terminal amine sites to their corresponding acetylated forms. PAGE analysis (at pH 8.3) showed bands that migrated only slightly from the sample wells during the separation, which is consistent with the expected dramatic change in the charge state upon acetylation. Synthesis of Quaternary Ammonium Dendrimers. The quaternary ammonium derivative of the generation 4 PPI dendrimer (DAB-32-Me) was prepared following a procedure adapted from Krieder and Ford.25 The terminal primary and interior tertiary amines of the dendrimer were quaternized by the addition of methyl groups in two steps. A detailed description of this procedure can be found in Supporting Information. The quaternary ammonium derivative of the generation 3 PAMAM dendrimer (G3Me) was prepared following a procedure adapted from Gong and co-workers.26 The method of Krieder and Ford was not attempted for the PAMAM dendrimer, as it was anticipated that the high reaction temperature required would cause the dendrimer to decompose via a retro-Michael addition (21) Shvarev, A.; Bakker, E. J. Am. Chem. Soc. 2003, 125, 11192-11193. (22) Balogh, L.; de Leuze-Jallouli, A.; Dvornic, P.; Kunugi, Y.; Blumstein, A.; Tomalia, D. A. Macromolecules 1999, 32, 1036-1042. (23) Han, I. S.; Ramamurthy, N.; Yun, J. H.; Schaller, U.; Meyerhoff, M. E.; Yang, V. C. FASEB J. 1996, 10, 1621-1626. (24) Ye, Q.; Meyerhoff, M. E. Anal. Chem. 2001, 73, 332-335. (25) Krieder, J. L.; Ford, W. T. J. Polym. Sci. Poly. Chem. 2001, 39, 821-832.

Table 1. Theoretical Dendrimer Characteristicsa dendrimer

MW

primary amines

tertiary amines

mass-tocharge ratiob

diameter (Å)

G3A G5A G7A DAB-16 DAB-32 DAB-64

6909 28826 116493 1687 3514 7168

32 128 512 16 32 64

30 126 510 14 30 62

111.4 113.5 114.0 56.2 56.7 56.8

36 54 81 27 34 44

a As reported by Dvornic et al.28 and Tomalia et al.29 b The massto-charge values were calculated based on the fully protonated form of the dendrimer.

reaction.27 In this case, the procedure used by Gong et al. involved generating the quaternary species directly from the amine form. Briefly, the PAMAM dendrimer was alkylated with an excess of iodomethane in the presence of triethylamine in methanolic solution. A more detailed description of the synthesis procedure can be found in Supporting Information. RESULTS AND DISCUSSION Table 1 lists various features of the two classes of dendrimers examined in this study.28,29 For both, the theoretical values for the ideal dendrimer structures are tabulated. Actual dendrimer samples are expected to contain some small fraction of defective dendrimers (e.g., loops or missing branches). Mass spectrometry data were obtained for all generations of the PPI dendrimers examined. These data indicated that the desired dendrimer structures were present, along with some fraction of defective structures. For example, the mass spectra for the generation 4 PPI dendrimer (DAB-32) and the generation 3 PAMAM dendrimer (G3A) are shown in Figure 2. It can be seen that both samples contain the expected dendrimer structure, as well as some defective species, which are formed by the presence of branch and/or loop defects.30 All generations of the PPI dendrimers used in this study were readily analyzed in this manner. As has been reported in the literature,31 it was found that the higher generation PAMAM dendrimer materials tend to be more complex, which are not readily analyzed for discrete molecular weights using mass spectrometry (data not shown). However, analysis of all samples via PAGE indicated that the principal components of each dendrimer sample were the generation of interest but that lower generation PAMAM molecules and dimers of the principal components were also present to some degree. Absorbance measurements of the dye-processed gel indicated that the G3 PAMAM sample contained 87% G3A dendrimer, the G5 PAMAM sample contained 73% G5A, and the G7 PAMAM sample contained 57% G7A dendrimer (based on the integrated optical density of the Coomassie Blue-stained bands of the gel, average of two injections). (26) Gong, A.; Liu, C.; Chen, Y.; Zhang, X.; Chen, C.; Xi, F. Macromol. Rapid Commun. 1999, 20, 492-496. (27) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117-132. (28) Dvornic, P. R.; Tomalia, D. A. In Polymer Data Handbook; Mark, J. E., Ed.; Oxford University Press: New York, 1999; pp 853-856. (29) Tomalia, D. A.; Rookmaker, M. In Polymer Data Handbook; Mark, J. E., Ed.; Oxford University Press: New York, 1999; pp 857-860. (30) Bosman, A. W.; Bruining, M. J.; Kooijman, H.; Spek, A. L.; Janssen, R. A. J.; Meijer, E. W. J. Am. Chem. Soc. 1998, 120, 8547-8548. (31) Schwartz, B. L.; Rockwood, A. L.; Smith, R. D.; Tomalia, D. A.; Spindler, R. Rapid Commun. Mass Spectrom. 1995, 15, 1552-1555.

To explore the relationship between the dendrimer generation number and the total sensor equilibrium EMF response, a study of the EMF response toward each dendrimer species was performed. Equilibrium EMF response conditions20 were achieved by injecting a given volume of dendrimer solution into 3 mL of well-stirred buffer to yield a sample of 20 µg/mL dendrimer, and the sensor EMF response was recorded 5 min after sample addition. To examine the roles of the different amine groups in the dendrimer structures, the sample pH was controlled to fix the degree of protonation of the various amine sites. Three 20 mM phosphate buffers were prepared (pH 11.2, 7.0, and 3.0); these values were chosen with the dendrimer amine pKa values in mind.32,33 PAMAM dendrimers are reported to have macroscopic pKa values of 9-10 (primary amines) and 4-5 (tertiary amines),32 while PPI dendrimers are reported to have macroscopic pKa values of 10.0 (primary amines) and 6.7 (tertiary amines).33 It should be noted that a study of PPI dendrimers by Koper and co-workers34 has demonstrated the highly complex nature of protonation for these molecules, where a single macroscopic pKa value is not necessarily sufficient to describe the basicity of a given type of amine group (i.e., primary or tertiary). Thus, the pKa values reported by the respective manufacturers were used as approximate values for the purposes of this study. Table 2 summarizes the equilibrium EMF response data obtained for various generations of PAMAM and PPI dendrimers as a function of pH. Note that the largest response occurs at the lowest pH, where the vast majority of amine sites are fully protonated. At high pH, the EMF response is greatly reduced, as the dendrimer amine sites are nearly fully deprotonated. For comparison, the equilibrium EMF response of Polybrene (a linear quaternary ammonium polycation) was determined under the same conditions. This species should exhibit no pH dependence since it is fully quaternized, but the EMF response would be affected by slight differences in ionic strength of the three test buffers because the activity of the sodium ion (with a concentration of 60 mM in all three buffers), which poises the initial EMF of the membrane electrode, would be different in each of the three buffers (i.e., yielding a different baseline EMF before the addition of dendrimer). The data shown are therefore normalized to the Polybrene response at pH 11.2 to eliminate the contribution of the differences in ionic composition of the buffers to the EMF response. This is accomplished by scaling the net EMF responses toward the different dendrimers in proportion to the total EMF response of Polybrene in a given buffer. The differences in equilibrium EMF response at the two higher pH values (pH 7 and pH 11) between the two types of dendrimers are significant; these differences can be attributed to the larger pKa values reported for the PPI dendrimers.33 As for differences between generations, the number of primary amines doubles with each generation number, as shown in Table 1. Given the globular shape of the PAMAM and PPI dendrimers, it was expected that if the terminal amine groups are indeed located at the surface of the molecules, the charge density would therefore increase with (32) Brothers, H. M., II; Piehler, L. T.; Tomalia, D. A. J. Chromatogr., A 1998, 814, 233-246. (33) http://www.dsm.com/astramol/properties/∼en/ (accessed January 2003). (34) Koper, G. J. M.; van Genderen, M. H. P.; Elissen-Roma´n, C.; Baars, M. W. P. L.; Meijer, E. W.; Borkovec, M. J. Am. Chem. Soc. 1997, 119, 65126521.

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Figure 2. ESI-MS data for (a) a generation 3 PAMAM dendrimer (G3A) and (b) a generation 4 PPI dendrimer (DAB-32).

Table 2. Equilibrium EMF Response Data for Amine-Form PAMAM and PPI Dendrimers as a Function of Sample pH ∆EMF (mV)a sample

pH 3.0

pH 7.0

pH 11.2

Polybrene G3A G5A G7A DAB-16 DAB-32 DAB-64

120.1 ( 3.7 125.1 ( 1.5 115.5 ( 0.6 110.4 ( 3.4 120.0 ( 2.7 128.8 ( 1.1 123.7 ( 1.3

120.1 ( 1.3 87.1 ( 1.0 86.1 ( 0.9 85.8 ( 1.1 107.6 ( 0.8 105.9 ( 1.0 98.8 ( 1.1

120.1 ( 0.8 2.2 ( 0.4 4.0 ( 0.4 6.9 ( 2.1 60.9 ( 0.4 55.7 ( 0.7 57.1 ( 0.6

a The average net response of four sensors is shown ((SD), where the response was recorded 5 min after addition of a given dendrimer sample to 3 mL of well-stirred 20 mM phosphate buffer (to give a dendrimer concentration of 20 µg/mL). The data are normalized to Polybrene response at pH 11.2 to correct for ionic strength effects.

generation number. Prior studies with polyion sensors20,35,36 suggest a greater total equilibrium EMF response with increasing charge density of the polyion. However, this was not found to be 1478

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the case in this study. Table 2 illustrates that there is only a small difference in potentiometric response with generation number under all pH conditions examined, and there does not appear to be a consistent trend among the three generations of each dendrimer in terms of generation number and total EMF response. The fact that the sensor response does not differ significantly with generation number is consistent with a constant mass-to-charge ratio, which would be a more accurate description of the distribution of the cationic sites in the molecules if there is some degree of backfolding of the end groups. Indeed, there has been conflicting evidence in the literature as to the location of the dendrimer end groups.30,37-39 Clearly, the fact that potentiometric data indicate that there is no significant difference in the thermodynamics of extraction for higher generation PAMAM and PPI dendrimers strongly supports the notion that bending of the branches may occur to a significant degree when the dendritic structure is extracted as an ion pair-type complex (with DNNS) within the (35) Dai, S. Ph.D. Thesis, University of Michigan, 2000. (36) Esson, J. M.; Meyerhoff, M. E. Electroanalysis 1997, 9, 1325-1330. (37) de Gennes, P. G.; Hervet, H. Phys. Lett. 1983, 44, L351-L361.

Table 3. Equilibrium EMF Response Data for PAMAM and PPI Dendrimer Derivatives as a Function of Sample pH ∆EMF (mV)a sample

pH 3.0

pH 7.0

pH 11.2

Polybrene G3A G3Ac G2.5b G3Me DAB-32-Ac DAB-32 DAB-32-Me

118.6 ( 1.2 125.1 ( 1.5 82.0 ( 3.0 77.5 ( 0.4 124.7 ( 0.5 105.2 ( 0.3 121.3 ( 1.0 130.2 ( 1.9

118.6 ( 0.7 87.1 ( 1.0 31.2 ( 0.8 2.8 ( 0.8 87.0 ( 0.5 99.9 ( 5.8 106.7 ( 1.0 125.7 ( 1.7

118.6 ( 0.8 2.2 ( 0.4 1.2 ( 2.7 5.6 ( 5.7 13.6 ( 0.5 5.1 ( 0.7 55.7 ( 0.7 124.5 ( 1.7

a The average net response of four sensors is shown ((SD), where the response was recorded 5 min after addition of a given dendrimer sample to 3 mL of well-stirred 20 mM phosphate buffer (to give a dendrimer concentration of 20 µg/mL). The data are normalized to Polybrene response at pH 11.2 to correct for ionic strength effects. b G2.5 is the half-generation PAMAM species, with 30 interior tertiary amines and 32 terminal carboxyl groups.

organic membrane phase of the sensor. Alternately, DNNS could intercalate into the core region of such dendrimers; see below. Dendrimer Composition and Equilibrium Potentiometric Response. To investigate the roles of the dendrimer composition and charge in the overall EMF response, a series of PAMAM and PPI derivatives were prepared. To eliminate the contribution of the charge from the terminal primary amines, acetylated G3 PAMAM and G4 PPI dendrimers were synthesized and characterized (G3Ac and DAB-32Ac, respectively). In these derivatives, only the interior tertiary amines can be protonated and thus dominate the overall polycationic character of the molecule. To eliminate the pH dependence of the dendrimer charge, quaternary ammonium forms of the G3 PAMAM and G4 PPI dendrimers were also prepared and characterized (G3Me and DAB-32Me, respectively). A half-generation PAMAM dendrimer was also analyzed; this generation 2.5 (G2.5) PAMAM dendrimer has 32 terminal carboxyl groups with 30 interior tertiary amines, and is thus zwitterionic under certain conditions. The polyion sensor equilibrium EMF responses to these derivatives as a function of pH were found to be quite different from the amine form dendrimers. Table 3 summarizes the response data obtained for the derivatives (normalized to Polybrene response). For the acetylated species (G3Ac and DAB32Ac), it can be seen that these derivatives exhibit significant potentiometric response only at low pH (when the interior tertiary amines are predicted to be protonated). This was also true for the half-generation PAMAM species (G2.5), which is expected to have a net positive charge only at low pH, when the carboxyl groups could be neutral and the interior amines could be protonated. This suggests that the lipophilic ion exchanger DNNS can in fact interact favorably (electrostatically) with the interior protonated amines of the dendrimers, further supporting a model where significant bending of the terminal chains occurs38,40 to facilitate this interaction, or the possibility of the DNNS species being able to intercalate into the core of the dendrimer to enhance electrostatic binding. (38) Lesanec, R. L.; Muthukumar, M. Macromolecules 1990, 23, 2280-2288. (39) Zook, T. C.; Pickett, G. T. Phys. Rev. Lett. 2003, 90, art. 015502. (40) Zhang, Z.-Y.; Smith, B. D. Bioconjugate Chem. 2000, 11, 805-814.

Figure 3. Synthesis of poly(amidoamine)-poly(acetylene) copolymers.

The methylated G3 PAMAM dendrimer (G3Me) exhibits a significant response even at pH 11.2 relative to the amine-form starting material (G3A), indicating that quaternization was somewhat successful but not complete. Indeed, this species does exhibit a pH-dependent EMF response, further suggesting that it is not fully quaternized. Elemental analysis of this derivative confirmed that only partial conversion to a fully quaternized species had been achieved (see Experimental Section). In comparison, as shown in Table 3, the methylated G4 PPI dendrimer (DAB-32Me) exhibits little pH dependence in its potentiometric response. These data are supported by elemental analysis of this material, which indicated complete quaternization had been achieved (see Supporting Information). This sample produces the largest total EMF response of all dendrimers examined. The DNNS-based sensor’s equilibrium EMF response toward another class of PAMAM dendrimerssionic poly(acetylene) copolymers (PAMAM-PAc)swas also examined. These dendrimer derivatives were prepared as described by Balogh and coworkers,22 where the globular PAMAM dendrimer serves as the core, surrounded by a “crust” of the cationic, rigid poly(acetylene) chains. The chains are attached at the PAMAM primary amines by addition of the acetylene monomer, N-alkyl-2-ethynylpyridinium trifluoromethanesulfonate. The preparation of such species is illustrated in Figure 3. Given that the monomer contains a quaternary salt of pyridine, the resultant poly(acetylene) chains are polycationic with no pH-dependent charge. These derivatives are named by the core dendrimer (G3 or G4 PAMAM) followed by the mole ratio of monomer to primary amines. For example, the G3-PAc-0.5 copolymer consists of a G3 core with an average of one monomer unit for every two primary amines. It is expected that this mole ratio represents the average chain length; the G3-PAc-0.5 species, for example, may contain some fraction of longer chains attached at amine sites. The equilibrium response data for various PAMAM-PAc copolymers in 20 mM phosphate buffer (pH 7.0) are listed in Table 4. The pH dependence of the potentiometric response toward these species was also examined; as anticipated, no significant pH effects were observed (data not shown). This suggests that the lipophilic ion exchanger DNNS in the membrane phase is interacting primarily with the pyridinium surface groups of these copolymers, as opposed to the PAMAM dendrimers, where the data indicate the ion exchanger also interacts favorably with the interior tertiary amines when they are ionized. For comparison, the EMF response data for the poly(acetylene) homopolymer are shown; a similar equilibrium EMF response is observed for this sample relative to the dendrimer copolymers. Prior investigations of polyion sensor response toward various linear polyionic species have identified several characteristics that affect the overall ion-exchange constant. These include charge Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Table 4. Equilibrium EMF Response Data for PAMAM-PAc Copolymers samplea

∆EMF (mV), pH 7.0b

G4-PAc-0.5 G3-PAc-1 G4-PAc-1 G3-PAc-4 G4-PAc-4 G4-PAc-16 homopolymer

93.4 ( 1.5 104.2 ( 0.7 102.9 ( 0.7 105.7 ( 1.4 106.7 ( 1.7 113.8 ( 1.1 100.1 ( 0.9

a The sample is named by the generation number of the PAMAM starting material (G3 or G4) followed by the mole ratio of the pyridinium monomer added to the moles of primary amines. b The average net response of four sensors is shown ((SD), where the response was recorded 5 min after addition of a given dendrimer sample to 3 mL of well-stirred 20 mM phosphate buffer, pH 7.0 (to give a dendrimer concentration of 20 µg/mL).

density, with increasing charge density correlating to a more favorable extraction into the membrane phase.20,35,36 The equilibrium response data presented here for dendritic polyions are consistent with this model, provided that there is either some degree of backfolding of the dendrimer terminal groups or the DNNS ion exchanger can intercalate within the dendrimer structure to allow for electrostatic interactions with the protonated interior amines. Studies of various polyphosphates have shown an increase in potentiometric response with chain length (for a given repeating ionic unit);36 this effect was attributed to a greater degree of cooperative ion-pairing with increasing chain length. This trend, however, has not been observed for the dendrimers examined in this study, where only minor differences in EMF responses are observed with increasing generation number. Last, the lipophilicity of linear polyions has been shown to affect the response, where greater potentiometric responses were observed for peptides with more hydrophobic side chains.35 The larger potentiometric response generated by the alkylated quaternary dendrimers can, in part, be attributed to this effect as well. The effect of different anions on the EMF response has not been explicitly examined in this study, but it is possible that the overall magnitude of the EMF response could be impacted by solutionphase anions that interact favorably with the protonated amines. For example, anionic surfactants in the solution phase could bind with polycations, as demonstrated by Esson et al.,41 thus reducing the EMF response. Dendrimer Composition and Nonequilibrium, Analytically Useful Potentiometric Responses. A large and reproducible super-Nernstian response can be achieved when the polyion sensor is exposed to relatively low concentrations of polyion for short periods of time. Under these conditions, polyion sensors serve as a useful analytical tool in the quantitation of polyions. This nonequilibrium potentiometric response toward low concentrations of a polycation can be described by the following equation:19

∆EMF ) -

(

zDaδm RT ln 1 c F STDmδa poly

)

(1)

where R and F are the gas and Faraday constants, respectively, T is the temperature, z is the charge of the polyion, cpoly is the 1480 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

Figure 4. Nonequilibrium EMF response of different PAMAM generations (a) versus concentration (in µg/mL) and (b) versus concentration (in M). The average net response of four sensors is shown ((SD), where the response was recorded 120 s after each addition of small aliquots of a given dendrimer sample to 5 mL of well-stirred 20 mM phosphate buffer (pH 7.0). The solid lines shown are trend lines, not theoretical response curves.

polyion concentration in the sample, ST is the total ion-exchanger concentration in the sensor membrane, Da and Dm are the diffusion coefficients of the polyion in the aqueous and membrane phases, respectively, and δa and δm are the diffusion layer thicknesses in the aqueous and membrane phases, respectively. It should be noted that eq 1 only predicts the initial portion of the nonequilibrium response curve when a fraction of the original countercations in the membrane phase are replaced by the polycations.19 The nonequilibrium potentiometric response profiles of the DNNS-based sensors toward the different generations of amineform PAMAM and PPI dendrimers were explored and are shown in Figures 4 and 5, respectively. Figures 4a and 5a show the EMF response data plotted versus mass concentration (in µg/mL), and Figures 4b and 5b show the same data plotted as a function of molar concentration. Detection limits for all dendrimer generations examined are in the 5-100 nM range (submicrogram per liter), where the detection limit is defined as at least a 3-mV (3σ) change from baseline potential. In general, the EMF response curve is shifted to a higher mass concentration for the larger dendrimers. This trend is more apparent for the three PAMAM dendrimers (41) Esson, J. M.; Ramamurthy, N.; Meyerhoff, M. E. Anal. Chim. Acta 2000, 404, 83-94.

Figure 6. Nonequilibrium EMF response of static and rotation polycation sensors toward G3A PAMAM dendrimers. The average net response of three sensors is shown ((SD), where the response was recorded 120 s after each addition of small aliquots of a given dendrimer sample to 5 mL of well-stirred 20 mM phosphate buffer (pH 7.0). The static planar sensor response profile (with stirring) is shown in comparison to that of the rotated planar sensor (3000 rpm, no stirring). The solid lines shown are trend lines, not theoretical response curves.

Figure 5. Nonequilibrium EMF response of different PPI generations (a) versus concentration (in µg/mL) and (b) versus concentration (in M). The average net response of four sensors is shown ((SD), where the response was recorded 120 s after each addition of small aliquots of a given dendrimer sample to 5 mL of well-stirred 20 mM phosphate buffer (pH 7.0). The solid lines shown are trend lines, not theoretical response curves.

compared to the PPI dendrimers. It should be noted that a given mass of dendrimer, regardless of generation number, contains the same quantity of tertiary and primary amines. The generation number indicates how these identical repeating units are connected as molecules; thus, with increasing generation number, these groups are transported together as larger, more bulky molecules to the surface of the polymer membrane. As shown in eq 1, polyion sensor theory predicts that the nonequilibrium response is dependent on the diffusion coefficient and on the charge of the dendrimer. Both the charge and the diffusion coefficient are dependent on the generation number. With every generation, the number of amine sites doubles. The diameter of a PAMAM dendrimer increases by ∼10 Å with each generation number, with the generations examined here ranging from 36 to 81 Å (see Table 1).28,29 The Stokes-Einstein equation predicts an associated diffusion coefficient decreasing from 1.4 × 10-9 to 6.0 × 10-10 cm2 s-1 for these three PAMAM dendrimers (assuming a spherical geometry, with the viscosity of water being 0.891 × 10-2 kg cm-1 s-1 at 25 °C). Thus, if the diffusion coefficient were the principal factor governing the nonequilibrium EMF

response profiles, the detection limits on a molar basis should increase with increasing generation number. Instead, these data suggest that the increase in charge concentration with generation number is the dominant factor. For example, the G7A dendrimer contains ∼10 times more amine sites than the G3A dendrimer; a given molar concentration of G7A dendrimer will therefore displace a greater quantity of Na+ from the sensor membrane and generate a larger EMF response. In comparison, the diffusion coefficient changes by only a factor of 2. Thus, the dendrimer with a lower diffusion coefficient, but much greater charge, generates a larger EMF response. This is anticipated from eq 1. In comparison, the PPI dendrimers did not exhibit a significant difference in response profile (on a mass concentration basis) for the two smaller generations (DAB-16 and DAB-32, generations 4 and 5, respectively; see Figure 5a), but a shift was observed for the largest of the three dendrimers of this type examined (DAB-64, generation 6). However, it should be noted that the PAMAM dendrimers examined are generations 3, 5, and 7; the PPI dendrimers differ only by one generation: 4, 5, and 6. Based on reported diameters for these species29 the diffusion coefficients are predicted to range from 1.8 × 10-9 to 1.1 × 10-9 cm2 s-1 for these three PPI dendrimers. As with PAMAM dendrimers, the number of amine sites doubles with each generation of PPI dendrimers. The same trend is seen in Figure 5b, where the sensor shows the poorest sensitivity on a molar concentration basis toward the smaller, lower generation PPI dendrimer. It has recently been found that a rotating polyion sensor configuration offers a significant improvement in detection limits.24 This is attributed to a decrease in the aqueous-phase diffusion layer thickness (relative to a static sensor in a stirred solution); this decrease affords an order of magnitude decrease in detection limits, depending on the rotation speed of the sensor. Figure 6 shows a comparison of the mass concentration response profiles Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 7. Titration of 1.0 and 2.0 µg/mL 500-bp linear doublestranded DNA using generation 3 amine-form PAMAM dendrimers (G3A), as monitored using polycation-sensitive membrane electrodes. The average net EMF response of four sensors is shown ((SD), where the response was recorded 120 s after each addition of small aliquots of a given dendrimer sample to 5 mL of well-stirred 50 mM Tris buffer containing 120 mM NaCl (pH 7.4). The solid lines shown are trend lines, not theoretical response curves.

of DNNS-based polyion sensors toward G3A PAMAM dendrimers in phosphate buffer (pH 7) using both rotating and nonrotating sensors. The total EMF response observed for the two types of sensors differed to a small extent; this may be attributed to differences in the sensor preparation procedures, where attaching the sensor membrane to the Tygon tubing may introduce various contaminants. Nonetheless, rotation of the planar sensor at 3000 rpm results in a detection limit improvement by nearly 1 order of magnitude (0.045 vs 0.34 µg/mL) relative to a static planar sensor with stirring. Furthermore, this is an improvement of 50% with respect to the static cylindrical electrode with stirring (as used for all other studies presented here). Rotation at higher speeds affords the possibility of achieving even lower detection limits.24 One practical application for the potentiometric detection of dendrimers relates to studying their interaction with DNA. As stated previously, both PAMAM and PPI dendrimers have been shown to be effective vectors for gene therapy.3-6 It has been found that the dendrimers and DNA form a complex, where the condensed DNA is less vulnerable to endogenous nucleases, and can be transported across the cell membrane relatively intact.5 The mechanism by which the DNA is bound by the dendrimer and then subsequently available in the cell for transcription is not yet fully understood.40 Thus, techniques that allow monitoring dendrimer/DNA binding could offer insight into this process. Polyion sensors can be conveniently used to monitor such binding processes. In this case, the polyion sensor serves as the end point detector in the titration of a given quantity of DNA with the dendrimer of interest.42 Since the sensor responds only to the free (unbound) dendrimer concentration in solution, the titration progress can be readily monitored. Figure 7 shows the titration of 500-bp double-stranded linear DNA with generation 3 amineform PAMAM dendrimers (G3A) as monitored with a static polycation sensor. With increasing DNA concentration, the den(42) Yun, J. H.; Ma, S. C.; Fu, B.; Yang, V. C.; Meyerhoff, M. E. Electroanalysis 1993, 5, 719-724. (43) Chen, W.; Turro, N. J.; Tomalia, D. A. Langmuir 2000, 16, 15-19.

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drimer response curve is shifted to higher concentrations, as expected. By comparison with the dendrimer calibration curve, the amounts of bound and free dendrimer can be determined for each point along the titration curve. The binding stoichiometries for these titrations were determined from the titration end points (defined as half the total EMF change observed for the titration); mole ratios of 25.2:1 and 27.0:1 (G3A to 500-bp DNA) were found, which corresponds to an average of 1 molecule of dendrimer bound for every 19 base pairs. These end points correspond to charge ratios of ∼0.8:1 (assuming 32 positively charged amines per G3A molecule at this pH). If dendrimer/DNA binding is nonspecific, and the binding is considered as a noncooperative process, the binding sites on the DNA can treated as independent binding domains. The binding constant can then be estimated from the titration curve as the reciprocal of the molar concentration of dendrimer at which half the binding sites are occupied (halfway to the end point). This relatively simplistic method is commonly used for estimating binding constants of biological macromolecules with multiple binding sites (e.g., divalent antibodies). By this method, a binding constant value of 1.2((0.23) × 107 M-1 (an average of n ) 4) was found for both concentrations of DNA titrated. While apparent binding strengths have been estimated previously using ethidium bromide as a probe,43 to the best of the authors’ knowledge, this is the first report of a PAMAM/DNA binding constant. CONCLUSIONS We have demonstrated the rapid and sensitive potentiometric detection of two classes of polycationic dendrimers in aqueous solutions. The total (equilibrium) potentiometric response originates from interactions between the lipophilic ion-exchanger sites (DNNS) in the membrane phase of the sensor and both the primary amines at dendrimer branch termini and the tertiary amines at interior dendrimer branch sites. It has been further shown that the total potentiometric response is not affected by the generation number, presumably due to the fact that interior amine sites play a role in the thermodynamics of ion-pair formation in the membrane phase. Using nonequilibrium responses, both PAMAM and PPI dendrimers can be detected at submicrogram per milliliter levels (5-100 nM levels) with good reproducibility. This analytical response has been shown to be useful for obtaining fundamental information about the stoichiometry and binding strength of these polycationic dendrimers with DNA. It is anticipated that a similar potentiometric method can be used to study interactions between such dendrimers and other macromolecules of interest, including proteins. ACKNOWLEDGMENT We thank the National Institutes of Health (EB00784) for supporting this work. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 16, 2003. AC035265L

October

27,

2003.

Accepted