Unusual pH-Dependent Polarity Changes in PAMAM Dendrimers: Evidence for pH-Responsive Conformational Changes Wei Chen, ² Donald A. Tomalia, ‡ and James L. Thomas* ,² Department of Chemical Engineering and Applied Chemistry, Columbia University,New York, New York 10027, and Center for Biologic Nanotechnology, University of Michigan, Ann Arbor, Michigan 48109 Received May 8, 2000 Revised Manuscript Received October 20, 2000 Poly(amidoamine) (PAMAM) dendrimers have been the subject of many studies because of their novel structural properties and attractive potential applica- tions in a variety of fields, such as drug delivery, gene therapy, and chemical separations. 1-6 PAMAM den- drimers are polyelectrolytes, and in aqueous solution can be protonated at termini (primary amines) and at branch points (tertiary amines), with the extent of protonation depending on the solution pH. In general, the protonation of polyelectrolytes strongly affects con- formation; greater molecular charge results in a more expanded and more hydrated polymer conformation, as demonstrated by viscometry, light scattering, and X-ray scattering and indirectly by means of environmentally responsive fluorescence probes. 7 The pH-dependent conformational behavior of dendrimers is a subject of considerable debate, however. We have used the polarity-responsive probe 5-(di- methylamino)-1-naphthalenesulfonic acid (DNS, 1) to explore changes in dendrimer structure that may occur with pH titration. The fluorescence quantum yield and Stokes shift of DNS vary with solvent polarity and to a lesser extent with solvent microviscosity. Polar solvents stabilize a twisted intramolecular charge transfer (TICT) species which has a long wavelength emission; high microviscosity can slow the molecular rotation needed to form this twisted conformation. 8 The anionic sul- fonate moiety of DNS can ionically bind to cationic amines, such as the terminal amines of PAMAM den- drimers, as shown below. Although the binding is weak, it is saturable; consequently, the average environment of the charged amines can be assayed through the fluorescence spectroscopy of the associated DNS probe. Experimental Section. Polyamidoamine dendrim- ers were synthesized as previously described. 9 Briefly, to an ethylenediamine core, Michael addition of methyl acrylate followed by exhaustive amidation with ethyl- enediamine results in a four-armed “dendrimer” termed generation 0, 2. Repetition of the sequence is used to make successively higher generations. (The dendrimer generation is equal to the number of branch points between the ethylenediamine “core” and the termini and is consequently a logarithmic measure of the molecular size.) 5-Dimethylamino-1-naphthalenesulfonic acid, 1 (99+% purity), was purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. In all experiments, distilled deionized water was used (Millipore filtration system). Results. The binding of DNS to PAMAM generation 6 (G6) was investigated by adding increasing amounts of dendrimers into 2 μM aqueous DNS solution at fixed pH. At pH 8 (Figure 1, squares), DNS showed an increase in the fluorescence intensity and a blue shift ² Columbia University. ‡ University of Michigan. * Corresponding author. Figure 1. Emission peak intensity (A) and emission maxi- mum wavelength (B) of 2 μM DNS with increasing amount of dendrimer G6 at different pH: pH 10 (4), pH 9 (0), and pH 8 (O). Excitation was at 310 nm. The smooth curves are fits to a simple binding equilibrium. 9169 Macromolecules 2000, 33, 9169-9172 10.1021/ma000791p CCC: $19.00 © 2000 American Chemical Society Published on Web 11/22/2000