Ion-Specific Responsiveness of Polyamidoamine (PAMAM

Apr 27, 2012 - By combining quartz crystal microbalance (QCM) and reflectivity, it was shown that poly(amidoamine) (PAMAM) dendrimers adsorbed at the ...
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Ion-Specific Responsiveness of Polyamidoamine (PAMAM) Dendrimers Adsorbed on Silica Substrates Maria Porus, Florent Clerc, Plinio Maroni, and Michal Borkovec* Department of Inorganic, Analytical Chemistry, University of Geneva, 30, Quai Ernest-Ansermet, 1205 Geneva, Switzerland ABSTRACT: By combining quartz crystal microbalance (QCM) and reflectivity, it was shown that poly(amidoamine) (PAMAM) dendrimers adsorbed at the silica−water interface undergo conformational changes when incubated in various electrolyte solutions. Adsorbed dendrimers swell in solutions of high ionic strengths and low pH, while they shrink in solutions of low ionic strengths and high pH. Dendrimer swelling is further ion specific, whereby the cations influence the swelling to a larger extent than anions. Among the cations studied, magnesium ions induce largest swelling, while the lithium ions the smallest. The responsiveness to stimulus by the electrolyte composition is mainly determined by attractive electrostatic interactions between the positively charged dendrimers and the negatively charged silica substrate. The dendrimer−substrate interactions are enhanced with increasing pH due to dissociation of the silanol groups at the silica surface, and they are strongly influenced by cations adsorbing on the water−silica interface. The electrostatic dendrimer−substrate interactions weaken in the presence of salt due to screening.

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INTRODUCTION Because of their compact and globular architecture, dendrimers were suggested to have substantial potential as delivery systems for genes or drugs.1−9 The approach pursued in gene delivery is to form complexes between dendrimers and nucleic acids capable to penetrate the cell membrane.1−5 In drug delivery, one rather attempts to bind the drug to the dendrimer and to trigger its release through a conformational transition.6−8 Because of the substantial promise in the latter approach, conformational transitions of dendrimers were studied in substantial detail theoretically and experimentally.10−16 Earlier computer simulations suggested substantial swelling with decreasing salt level, with increasing dendrimer charge, and with increasing quality of the solvent.9,17−19 With dynamic light scattering and small-angle neutron scattering experiments it was confirmed that the type of solvent modifies the size of dendrimers.11,12 Small-angle neutron and X-ray scattering experiments revealed that dendrimers are substantially swollen in aqueous solutions, but upon variations of the salt level or dendrimer charge any changes in size were hardly observable.13−16 The latter experiments were mostly carried out with poly(amidoamine) (PAMAM) dendrimers, as they are soluble in water and their charge can be conveniently tuned by adjusting the solution pH through the ionization of the amine groups present.20,21 Subsequent computer simulations were able to reproduce the absence of swelling in aqueous solution by including ion-pairing effects, and they further suggested the presence of a conformational transition in the dendrimer interior.8 In contrary to marginal swelling of PAMAM dendrimers dissolved in aqueous solutions, substantial swelling was reported when they were adsorbed to solid substrates.22 Atomic force microscopy (AFM) and quartz crystal microbalance (QCM) studies of adsorbed dendrimer films on silica− water interfaces revealed that dendrimers may swell by © 2012 American Chemical Society

adjusting the salt level and pH of the surrounding solution. Dendrimer−substrate interactions were suggested to be responsible for this behavior. The fact that such interactions might be relevant in controlling the shape of adsorbed dendrimers was proposed earlier based on computer simulation studies.23,24 In the present article, we explore conformational changes of adsorbed PAMAM dendrimers with QCM and optical reflectivity techniques. By combining these two techniques, it is possible to determine the water content of the adsorbed dendrimers unambiguously, and one obtains a more detailed picture of the underlying conformational transition. In particular, we clearly indentify attractive electrostatic interactions between the positively charged dendrimers and the negatively charged substrate to be principally responsible for the observed conformational changes. We further demonstrate that these conformational changes are ion specific, whereby cations play a more important role than anions. This observation can be again rationalized by the fact that the conformational changes are triggered by dendrimer−substrate interactions, as cations adsorb more strongly to the negatively charged water−silica interface than anions.25,26

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EXPERIMENTAL SECTION

Materials. Aqueous solutions of PAMAM dendrimers of generation 4, 7, and 10 were obtained from Dendritech (Midland, MI). Stock solutions of dendrimers were diluted by Milli-Q water down to a concentration of 5 mg/L. Solution pH was adjusted by KOH or HCl. Magnesium chloride (MgCl2, ≥99%) and potassium chloride (KCl, ≥99%) were purchased form Acros Organics (Fair Lawn, NJ). Lithium Received: March 1, 2012 Revised: April 19, 2012 Published: April 27, 2012 3919

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chloride (LiCl, ≥99%), rubidium chloride (RbCl, ≥99.8%), cesium chloride (CsCl, ≥99.9%), and barium chloride (BaCl2, ≥99.9%) were purchased from Sigma-Aldrich (Switzerland). Calcium chloride (CaCl2, ≥95%), sodium chloride (NaCl, ≥99.5%), and strontium chloride (SrCl2, ≥99%) were obtained from Fluka (Switzerland). Sodium perchlorate (NaClO4, ≥98%), sodium fluoride (NaF, ≥99%), sodium iodide (NaI, ≥99.5%), sodium nitrate (NaNO3, ≥99%), sodium bromide (NaBr, ≥99%), and sodium thiocyanate (NaSCN, ≥98%) were equally obtained from Sigma-Aldrich (Switzerland). These salts were all used without further purification. Silica-coated QCM sensor crystals were used as substrates (QSX 318, Q-Sense, Gotheborg, Sweden). The topmost coating consists of a sputtered silica layer of about 300 nm, which is attached to the gold surface of the QCM crystal through a titanium layer of about 50 nm as an adhesion promoter. Prior to use, they were cleaned in 2% Hellmanex solution (VWR, Switzerland) in an ultrasonic bath during 10 min, subsequently rinsed in Milli-Q water, dried in a flow of nitrogen, and treated in an UV-ozone cleaner for 20 min. Prior to every use, the precise thickness of the topmost sillica layer was measured by scanning angle null ellipsometry (Multiskop, Optrel, Berlin, Germany). The thickness was normally within 5% of the value given by the manufacturer. The root-mean-square roughness of the silica coatings of the crystals was measured to be around 5 nm with noncontact AFM imaging (Nanoscope II, Veeco). After each experiment, the crystals were cleaned as described above. The experiments were well reproducible, and the crystals could be reused many times. More details on the characterization of such crystals are given elsewhere.27 Reflectometry. The dry mass of the adsorbed dendrimers per unit area was measured by a home-built reflectometer. The crystal was installed into the stagnation point flow cell, which was covered by a capped optical prism with a central bore hole. Solutions were pumped into the cell with a peristaltic pump through the bore hole in the prism at a flow rate of 0.6 mL/min, which remains constant within about 10%. The cell was installed in the axis of a goniometer with two rotating arms holding the laser and the detector. The laser beam of 533 nm was modulated at a frequency of 229 Hz and focused at the stagnation point with an incident angle of 60°. As a result of light refraction within the prism, the actual angle of incidence on the surface is 71°, which is close to the Brewster angle of the silica−water interface. Upon reflection, the beam is separated in two polarization directions by a polarizing beam splitter. The respective intensities measured by photodiodes with a lock-in amplification detection scheme. The measured reflectivity signal R is proportional to the reflectances Rp and Rs parallel and perpendicular to the reflection plane, namely

R=A

increment of the dendrimers. The refractive indices of silica, titanium, and gold at 533 nm are 1.457, 1.846 + 2.535i, and 0.458 + 2.426i.28,29 The refractive index increment for the dendrimers is 0.194 mL/g, and the assumption of a laterally homogeneous film yields accurate values for the dry mass in spite of the island character of the dendrimer film.30,31 When adsorption processes in different electrolytes are studied within the same experimental run, it is essential to determine the respective baselines for each electrolyte separately since the reflectivity signal depends on the refractive index of the electrolyte solution. The detection limit for the dry mass is about 5 μg/m2. Further details on the reflectivity setup and data analysis are given elsewhere.32,33 Quartz Crystal Microbalance (QCM). The wet mass per unit area of the adsorbed dendrimers was measured with the Q-sense E4 (Gotherborg, Sweden) microbalance. This technique explores the piezolelectricity of quartz, whereby a resonant oscillation of the crystal is excited by applying an alternating electric voltage. The adsorbed wet mass per unit area Γwet is proportional to the shift in the resonance frequency according to

Δf = −

(1)

x=1−

where A is an unknown instrumental constant, which disappears when the reflectivity signal is normalized to its initial value as

S(t ) =

R(t ) − R(0) R(0)

(2)

(3)

where B is the sensitivity constant. A typical value for the sensitivity constant is 0.02 m2/mg. It is calculated from a homogeneous four-slab model, where the fourth topmost slab corresponds to the layers of adsorbed dendrimers. The refractive index n of this layer is obtained from the mixing law n = ns +

Γdry dn L dc

Γdry Γwet

(6)

where Γdry is the adsorbed mass per unit area obtained by reflectivity and Γwet is the wet mass per unit area from QCM. Note that this expression is equally valid for a laterally homogeneous as well as a heterogeneous adsorbed film and thus enables us to estimate the water content of the adsorbed dendrimers. Because of the roughness contribution,38 this estimate might be 10−20% too high, however. When discussing conformational changes of adsorbed dendrimers, we will always refer to the relative change in the wet mass

Adsorbed dry mass per unit area Γdry is then obtained from the relation S(t ) = B Γdry

(5)

where m is the overtone number and C = 0.177 mgHz−1 m−2 is the mass sensitivity constant.34 The frequency shift of the fifth overtone (m = 5) was used to estimate the wet mass as it features a lower noise with respect to the other overtones leading to a detection limit of about 20 μg/m2. The wet mass obtained from the other overtones was constant within the experimental error of about 10%. The shift in the dissipation signal ΔD was approximately proportional to Δf, and the magnitude of the ratio ΔD/Δf was in the range of (5−9) × 10−8. These two observations indicate relatively rigid films. The solutions are pumped through the circular parallel plate flow cell with the inlet and outlet situated at its rim with the same peristaltic pump as used for the reflectometry experiments at a flow rate of 0.6 mL/min. When different electrolytes during the same experimental run are used, the corresponding baselines must be determined separately for each electrolyte since the resonance frequency depends on the solution density and viscosity.35 Since one knows from AFM studies that adsorbed dendrimers films are laterally heterogeneous,30,31,36 one might expect that the wet mass includes contributions from water trapped in the neighborhood of the adsorbed dendrimers.37,38 The magnitude of this effect was estimated through the roughness correction to the frequency shift. Assuming Gaussian roughness, this contribution leads to a frequency shift of around 0.5 Hz. Water Content of the Adsorbed Dendrimers. Combination of the optical and piezoelectric data enables us to estimate the water content of the adsorbed dendrimers. The water mass fraction can be obtained from the ratio between the dry and wet adsorbed mass as

Rp Rs

m Γwet C

ΔΓwet Γ(0) wet

=

Γwet − Γ(0) wet Γ(0) wet

(7)

where Γwet is the wet mass in the presence of the simulating solution and Γ(0) wet the wet mass in the reference state. On the basis of eq 6, one can show that the resulting change in the water content is

(4)

Δx = x − x(0) = (1 − x(0))

where ns is the refractive index of the electrolyte solution, L is the thickness of the dendrimers layer, and dn/dc is the refractive index 3920

ΔΓwet Γ(0) wet

(8)

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where x(0) is the water mass fraction of dendrimers in the reference state.

by these variations. One observes that the adsorbed amount increases with increasing pH and that the wet mass obtained by QCM significantly exceeds the dry mass measured by reflectivity. The difference originates from the water trapped in the adsorbed dendrimers. No desorption of the dendrimers was observed after the rinse with the initial electrolyte solution. While the adsorbed films were found to be stable in all situations studied here, desorption phenomena were only observed for dendrimers of low generation and low pH and are discussed elsewhere.39 Figures 2a,b summarize the dependence of the dry and wet mass of PAMAM G10 dendrimers at the adsorption plateau on

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RESULTS AND DISCUSSION Adsorption of PAMAM dendrimers was studied at the water− silica interface with reflectivity and quartz crystal microbalance (QCM). Reflectivity demonstrates that the adsorbed films remain stable upon changes in the composition of the electrolyte solution. With QCM one can clearly demonstrate that changes in solution composition induce reversible conformational changes within the adsorbed dendrimers. Adsorption of PAMAM Dendrimers on Silica. Optical reflectivity and QCM were used to study adsorption of dendrimers on silica surfaces in situ. While the optical technique is sensitive to the dry adsorbed mass, QCM detects the total mass deposited on the crystal including the trapped water. The combination of these two techniques enables us to estimate the water content of the adsorbed dendrimers. Typical adsorption experiments are shown in Figure 1. The experiment

Figure 1. Probing adsorption of PAMAM G10 dendrimers on water− silica interface at different pH: (a) reflectivity signal, (b) QCM signal, (c) dry mass, and (d) wet mass. The substrate was equilibrated with an electrolyte solution of an ionic strength of 0.1 mM adjusted with KCl and pH indicated (solution I). The dendrimers are adsorbed from a solution of a concentration of 5 mg/L in the same electrolyte (solution II). The stability of the adsorbed layer is revealed by rinsing with the dendrimer-free electrolyte solution (solution I).

Figure 2. Properties of adsorbed layers of PAMAM G10 dendrimers on water−silica interface at saturation plateau. (a) Dry mass per unit area by reflectivity compared with the data from Cahill et al.31 (b) Wet mass mass per unit area by QCM as a function of pH for two different ionic strength adjusted with KCl. (c) Water mass fraction in the adsorbed dendrimers is obtained by combing the reflectivity and QCM results. Solid lines serve to guide the eye only.

was initiated by flushing the cell with an KCl electrolyte solution of an ionic strength of 0.1 mM at selected pH values to obtain a stable baseline (solution I). At time zero, a solution containing 5 mg/L PAMAM G10 dendrimers of an ionic strength of 0.1 mM and different pH values in the same electrolyte is injected (solution II). When the adsorption plateau has been reached, the stability of the adsorbed layer was checked by flushing the cell with pure electrolyte solution (solution I). Figures 1a,b show the reflectivity and QCM curves, while Figures 1c,d show the corresponding dry and wet adsorbed mass. While minor differences due to small variations in the flow rate can be observed in the increasing part of the curve, the adsorption plateau is reached in about 1 min and is not affected

pH at ionic strengths of 0.1 and 10 mM. Adsorbed mass increases with increasing pH and ionic strength. The measured dry mass can be directly compared with earlier reflectivity measurements by Cahill et al.31 One observes an excellent agreement in spite of the fact that the two sets of measurements were carried out on different types of silica surfaces. Cahill et al.31 used oxidized silicon wafers, while the present study uses a silica layer obtained by sputtering. Adsorption at low ionic strength ensured that dendrimers adsorbed individually and that they are well separated on the surface.31 The increase of 3921

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while the corresponding dry and wet adsorbed mass is displayed in the bottom. Initially, the response of the bare silica surface to a change in the ionic strength was investigated. The baselines are obtained with an electrolyte solution of 0.1 mM ionic strength (solution I) and then with a 100 mM KCl solution (solution II), and the cell is rinsed again (solution I). The reflectivity signal is substantially lower for the 100 mM solution due to the increase in the refractive index. The corresponding QCM signal remains approximately constant, since the corresponding changes in solution viscosity and density are minor. Subsequently, the substrate is flushed with a PAMAM G10 dendrimer solution of a concentration of 5 mg/L and an ionic strength of 0.1 mM (solution III). Both traces clearly reveal the formation of the dendrimer layer, and the increase of the wet mass is more important than in the dry mass since the adsorbed dendrimers contain substantial amounts of water. Comparison of these two values leads to a water mass fraction of about 60%. Flushing the substrate (solution I) does not lead to any changes in the adsorbed mass, which indicates that the adsorbed layer remains stable. Response to the stimulus is now investigated by pumping the 100 mM KCl solution through the cell (solution II). One observes substantial changes in the reflectivity and QCM signals. However, the adsorbed mass can only be obtained correctly with the respect to the baseline of the same solution. Moreover, the sensitivity constant B defined in eq 3 depends weakly on the ionic strength, and this variation must also be accounted for when calculating the adsorbed mass.32 When such an analysis is carried out, one finds that the dry mass remains constant within experimental error. This observation means that the amount of adsorbed dendrimers is constant upon changes in the ionic strength of the incubating solutions. Since the QCM signal of the bare crystal is insensitive to changes between solutions of 0.1 and 100 mM ionic strength, the QCM trace directly reveals the changes in the wet mass. Since the dry mass remains the same, the increase in the wet mass must originate from a swelling of the adsorbed dendrimers. Flushing the substrate with 0.1 and 100 mM pure electrolyte solutions indeed confirms that these changes are reversible and originate from conformational changes of the adsorbed dendrimers. This experiment thus demonstrates that dendrimers adsorbed in 0.1 mM solution swell reversibly when they are incubated in a KCl solution of 100 mM. The relative change in the wet mass is about 27%, which corresponds to an increase in the water mass fraction from about 60% to about 69%. Comparable changes in the water content were also observed by AFM imaging in solution.22 Figure 4 shows a similar stimulus experiment where CaCl2 is used instead of KCl as background electrolyte. The main difference to the previous experiment is that the QCM signal is significantly shifted even for the bare substrate when the electrolyte solution is changed from 0.1 to 100 mM. The reason for this shift is that the viscosity and density of these two solutions differ significantly. For this reason, the baselines for solutions I and II are not the same even in the QCM experiment, and this difference must be considered in the data analysis. When the respective correction is being made, the change in the wet mass is substantially smaller. In the case of CaCl2, we find a relative change in wet mass of 24%. Such corrections due to frequency shift due to changes of electrolyte concentrations were done throughout for all subsequent experiments.

the adsorbed dry mass with the ionic strength originates from the screening of the repulsive electrostatic forces between the adsorbing dendrimers. The higher the ionic strength, the weaker the electrostatic repulsion between the dendrimers, and the higher is the amount adsorbed. With increasing pH, the water−silica interface develops a negative charge due to dissociation of silanol groups40 and accumulates counterions in its diffuse layer. These counterions enhance the screening between the adsorbing dendrimers and promote their adsorption further.31 Similar trends in the adsorbed amount with the ionic strength and pH were also observed for other cationic dendrimers41,42 or polyelectrolytes.43−50 With the reflectivity and QCM data at hand, one can evaluate the water content of the adsorbed dendrimers from eq 6. Figure 2c shows that the water content lies in the range of 50−80% and decreases with increasing pH. These values can be compared to values between 30% and 80% measured for PAMAM dendrimers in solution by small-angle neutron scattering51 or from diffusion coefficients obtained by NMR.52 The present values probably overestimate the actual water content somewhat, as the QCM frequency shift of the adsorbed dendrimer layer is expected to be larger than for a homogeneous layer due to roughness effects.38 Indeed, somewhat smaller values for the water content were also found by comparing the volumes of individual dendrimers in wet and dry state by AFM.22 Studying the Response of Adsorbed Dendrimers to Electrolytes. Reflectivity and QCM experiments involving sequences of different solutions chosen to ensure appropriate preconditioning, layer formation, flushing, and stimulus were used to study the response of adsorbed dendrimer. A typical stimulus experiment carried out at pH 4 is illustrated in Figure 3. Reflectivity and QCM traces are shown in the upper row,

Figure 3. Probing swelling of PAMAM G10 dendrimers adsorbed on water−silica interface from KCl solutions of pH 4.0: (a) reflectivity signal, (b) QCM signal, (c) dry mass, and (d) wet mass. The surface is equilibrated with electrolyte solutions of an ionic strength of 0.1 mM (solution I), then flushed with a 100 mM KCl solution (solution II), and equilibrated again. Dendrimers are adsorbed from a solution of 5 mg/L in the electrolyte of 0.1 mM ionic strength (solution III). After rinsing (solution I), dendrimer swelling is induced with 100 mM KCl solution (solution II). The rinsing and swelling step is repeated a second time. 3922

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solution of an ionic strength of 10 mM, and then their swelling behavior is investigated when changing the ionic strength from 0.1 to 100 mM. The bare substrate is first flushed with solutions of ionic strength of 0.1 mM (solution I), 100 mM (solution II), and 10 mM (solution III). The reflectivity signal shows a decrease with increasing ionic strength, while the QCM signal remains constant. Subsequently, the substrate is flushed with a PAMAM G10 solution of a concentration of 5 mg/L in an electrolyte with an ionic strength of 10 mM (solution IV). One observes the resulting changes in the reflectivity and QCM signals and the corresponding increase in the dry and wet mass. Again, the increase in the wet mass is larger than in the dry mass due to the important water content. Comparison of these two values leads to a water mass fraction of 70%. The adsorbed dendrimer layer is subsequently flushed with 10 mM electrolyte (solution III). No changes in the reflectivity and QCM signal are observed, which indicates that the adsorbed layer remains unchanged. The dendrimer layer is further flushed with a 0.1 mM electrolyte solution (solution I), which leads to changes in the reflectivity and QCM signals. At this point, the QCM signal reveals a transient extending over few minutes, which probably indicates that the conformational kinetics is relatively slow. When the corresponding masses are calculated, one finds that the dry mass remains constant but that the wet mass is smaller than the one before, which indicates that the dendrimers did shrink. Subsequent pumping of 100 mM electrolyte solution leads again to changes in the reflectivity and QCM signals (solution II). When the corresponding adsorbed masses are calculated, one finds that the dry mass remains constant while the wet mass has increased which indicates that dendrimer have swollen. Changing back to solution I, the wet mass returns to its initial value. Flushing of the adsorbed dendrimer layer with 100 and 0.1 mM electrolyte solutions leads to analogous changes in the wet mass, indicating the reversibility of the process. By comparing the wet mass measured in 0.1 and 100 mM, we find that the relative change in the wet mass is 24%. This change is very similar to the value found for the experiment shown in Figure 3, where the dendrimers were adsorbed at an ionic strength of 0.1 mM. This finding indicates that swelling of the dendrimers is independent of the adsorption conditions. More evidence concerning this important point will be provided further below. A more efficient way to study the influence of stimuli was to incubate a preformed layer in different solutions. Figure 6 shows the frequency shifts of two typical sequences. Figure 6a shows a trace of an experiment testing the response of adsorbed dendrimers at pH 4.0 to changes in the ionic strength. The substrate is first flushed with a solution of an ionic strength of 0.1 mM (solution I), and the response of the bare substrate to solutions of different ionic strengths adjusted with KCl is tested. Dendrimers are adsorbed from a solution of a concentration of 5 mg/L at an ionic strength of 0.1 mM. After rinsing, the adsorbed layer is incubated in a series of solutions of different ionic strengths with subsequent rinsing. Figure 6b shows the trace where the response of adsorbed dendrimers to changes in pH was investigated. After testing the response of the substrate to 100 mM solution of pH 4.0 the dendrimers are adsorbed as shown in Figure 6a. Swelling is now induced with a 100 mM solution of given pH and flushed with the 0.1 mM solution. Such traces were converted to wet mass as described above, and the relative changes in the wet mass were evaluated.

Figure 4. Probing swelling of PAMAM G10 dendrimers adsorbed on water−silica interface from CaCl2 solutions at pH 4.0: (a) reflectivity signal, (b) QCM signal, (c) dry mass, and (d) wet mass. The surface is pre-equilibrated with a sequence of CaCl2 solutions of a concentration of 0.1 mM (solution I) and 100 mM (solution II). Dendrimers are adsorbed from a solution of 5 mg/L and an ionic strength of 0.1 mM (solution III). After rinsing with an electrolyte solution of a concentration 0.1 mM (solution I), dendrimer swelling is induced with an electrolyte of concentration 100 mM (solution II).

Experiments involving three different electrolyte solutions were used to address the question whether the adsorption conditions affect the response to stimulus. Figure 5 illustrates an experiment at pH 4 where dendrimers are adsorbed in a KCl

Figure 5. Probing swelling of PAMAM G10 dendrimers adsorbed on water−silica interface from 10 mM KCl solutions at pH 4.0: (a) reflectivity signal, (b) QCM signal, (c) dry mass, and (d) wet mass. The surface is equilibrated with a sequence of electrolyte solutions adjusted with KCl with ionic strengths of 0.1 mM (solution I), 100 mM (solution II), and 10 mM (solution III). Dendrimers are adsorbed from a solution of 5 mg/L in the electrolyte of 10 mM ionic strength (solution IV). After rinsing with electrolyte solutions (solutions III and I), dendrimer swelling is induced with an 100 mM electrolyte (solution II). 3923

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Figure 6. QCM swelling experiment with a series of various stimulating solutions. (a) Testing the ionic strength response at pH 4.0. The substrate is equilibrated in a solution of an ionic strength of 0.1 mM (solution I). The response of the bare substrate is tested with solutions of ionic strength of 100 mM (solution II) and 1000 mM (solution III) adjusted with KCl, and the substrate is rinsed again (solution I). Dendrimers are adsorbed at a concentration of 5 mg/L in a solution of pH 4.0 and an ionic strength of 0.1 mM (solution III) and flushed (solution I). Swelling of adsorbed dendrimers is induced by solutions of different ionic strengths as indicated (solutions V, VI, II, and III) subsequent rinsing (solution I). (b) Testing the pH response. The bare substrate is equilibrated in a solution of pH 4.0 and an ionic strength of 0.1 mM (solution I). The response of the bare substrate is tested in a solution of pH 4.0 and an ionic strength of 100 mM adjusted with KCl (solution II), and the substrate is rinsed (solution I). Dendrimers are adsorbed from the same solution as in (a, solution III), and the dendrimer layer is flushed (solution I). Swelling is induced with a solution an ionic strength of 100 mM and different pH values (solutions IV, II, V, VI, and VII) and flushed (solution I). These steps are repeated several times. Note that the labeling of solutions is different in (a) and (b).

Figure 7. Response of PAMAM G10 dendrimers adsorbed from an electrolyte solution of different ionic strengths and pH as indicated in the figure. The substrates were equilibrated in an electrolyte solution of an ionic strength 0.1 mM and pH 4.0. Relative change in wet mass is represented for different ionic strength and pH. Relative change in the wet mass (a) versus pH and (b) versus the ionic strength when adjusted with KCl electrolyte. (c) Water mass fraction versus the ionic strength including the plateau valued from Figure 2c. Solid lines serve to guide the eye only.

Ionic Strength and pH Dependence of Dendrimer Conformations. Variations of the wet mass of adsorbed dendrimer layers incubated in solutions of different pH and ionic strength were studied more systematically. Variations with pH are summarized in Figure 7a, while the changes with the ionic strengths are shown in Figure 7b. One observes that dendrimers swell with increasing pH and increasing salt concentration. Repeating the same experiments at lower ionic strength shows that the extent of swelling is smaller, even to the point that shrinking is observed at high pH. Additional stimulus experiments with dendrimers adsorbed under different conditions are summarized in Figure 7. Dendrimers adsorbed in a solution of an ionic strength of 0.1 mM and pH 4.0 were compared with dendrimers adsorbed at an ionic strength of 10 mM and pH 4.0 and 8.0. One finds that changes in wet mass are independent of the adsorption conditions used. The same data are represented in terms of the water mass fraction in Figure 7c. The water content found at the adsorption plateau shown in Figure 2c is also indicated. At pH 4, one observes that the water content increases with increasing salt concentration from about 60% to 70%, while at pH 8.0 the increase is from 50% to 60%. We confirm again that the water content is independent of the adsorption conditions

within experimental error, which suggests that the water content of adsorbed dendrimers depends only on the composition of the surrounding solution. The accuracy of the data shown in Figure 7 should be commented in more detail. The most accurate are measurements of the relative change of the wet mass, since these measurements refer to the same dendrimer film. When different substrates are involved, the errors become larger due to inevitable variations between different crystal surfaces. When these results are converted to the water content, the errors are augmented even further, since the QCM results are influenced by roughness effects and errors inherent to the reflectivity experiment. Therefore, the data shown in Figure 7c are much less accurate than the ones show in Figures 7a,b, especially for films of low adsorbed mass. We thus report only relative variation in the wet mass in the following. Figure 8 illustrates the extent of swelling for different dendrimer generations. The adsorbed layer was again equilibrated in 0.1 mM electrolyte solution of pH 4.0, and its swelling was investigated in a 100 mM KCl solution at the same pH. Since the adsorbed mass decreases with decreasing dendrimer generation, it becomes difficult to detect the 3924

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Figure 8. Response of PAMAM G10 dendrimers adsorbed from an electrolyte solution of pH 4.0 and an ionic strength of 0.1 mM equilibrated in the same electrolyte solution. Relative change in wet mass versus the dendrimer generation in a stimulating solution of pH 4.0 and an ionic strength 100 mM adjusted with KCl.

swelling with QCM for the small generations accurately. Nevertheless, one clearly observes that the extent of swelling decreases with decreasing generation. Even for the lowest generations investigated, the films remain stable under the conditions investigated, and no desorption was observed within experimental error. The trends in swelling and shrinking of adsorbed dendrimers can be qualitatively interpreted in terms of electrostatic attraction between positively charged PAMAM dendrimers and the negatively charged water−silica interface. These electrostatic forces are screened in the presence of salt. Therefore, they weaken when the salt concentration is increased, and the adsorbed dendrimers swell. The charge of the water−silica interface is strongly pH dependent. The interface is highly charged in basic conditions, while it is close to neutral in acidic ones.40,53 Therefore, the attractive electrostatic forces weaken with decreasing pH, and as a consequence the dendrimers swell progressively. With decreasing generation of the dendrimers, the number of charged groups within the dendrimer decreases. Therefore, the electrostatic forces weaken and the extent of swelling becomes less important, too. One should note that with decreasing pH the charge of the dendrimers increases, too,20 but this effect seems to play a less important role than the decrease of the charge of the water−silica interface. This picture is analogous to a previous interpretation of the stability of adsorbed dendrimer films on silica.39 Dendrimers of high generations adsorb irreversibly, especially at high pH and low ionic strengths, while reversible adsorption is observed for low generations, low pH, and high ionic strengths. The same attractive electrostatic forces between the positively charged dendrimers and the negatively charged silica were suggested to be responsible for this behavior. Ion Specificity of Dendrimer Conformations. The influence of the ion type on the swelling behavior of dendrimers was studied, too. Similar experiments as discussed above with KCl at pH 4.0 were carried out with solutions of other metal chloride salts for the same pH. The adsorbed dendrimer layer was equilibrated in a 0.1 mM salt solution, and the swelling was studied by incubating in a 100 mM salt solution. An example of such an experiment was already shown with CaCl2 in Figure 4. The swelling of adsorbed dendrimers shows substantial ion specificity, and the results are summarized in Figure 9. The relative changes in wet mass for different metal chloride salts

Figure 9. Ion-specific response of PAMAM G10 dendrimers adsorbed from an electrolyte solution of an ionic strength of 0.1 mM. They were equilibrated in electrolyte solution 0.1 mM of the chloride salts of the respective cation or potassium salts of the respective anion. Relative change in wet mass in a stimulating solution with a salt concentration of 100 mM. (a) Cations versus their crystallographic radius and (b) anions versus their position in the Hofmeister series. All solutions have pH 4.0.

are shown in Figure 9a as a function of the crystallographic radius.54 One observes that the extent of swelling strongly depends on the type of cation, whereby Li+ leads to the smallest swelling of 21%, while Mg2+ to the strongest swelling of 32%. One further observes the extent of swelling increases with increasing radius of the alkali metal cations, whereby K+ represents an exception. While the divalent earth alkali metal cations normally lead to larger swelling, they do not seem to follow any systematic trends. We suspect that the observed behavior is related to the affinity of the respective cations to the water−silica interface. Adsorption of different monovalent and divalent cations was studied to water−silica interfaces with reflectivity and computer simulations.25 It was also observed that Mg2+ and Sr2+ adsorb most strongly, which means that they reduce the surface charge most effectively, which explains the large swelling observed. The fact that swelling with divalent cations is comparable to the monovalent ones indicates that screening effects are complicated by additional interactions, such as ion−ion correlations or specific ion adsorption effects. Figure 9b represents the water mass uptake for sodium salts of different anions at pH 4.0. The adsorbed dendrimer layer was again pre-equilibrated in a 0.1 mM solution, and its swelling was studied by incubating in a 100 mM salt solution. One also observes that the type of anion leads to different extent of swelling, whereby ClO4− leads to smallest swelling of 10% while largest swelling of 24% is observes for Br−. The results are plotted as a function of the position in the Hofmeister series55 but no systematic correlation can be established. One observes that largest swelling is observed for the halide anions, while oxo-anions normally lead to smaller swelling. These effects probably depend on the unknown affinities of the respective anions to the positively charged dendrimers. 3925

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CONCLUSIONS PAMAM dendrimers adsorbed at the silica−water interface undergo conformational changes when incubated in different electrolyte solutions as revealed by combining QCM with reflectivity. Adsorbed dendrimers swell when incubated in solution of high ionic strengths and low pH, while they shrink in solutions of low ionic strengths and high pH. This behavior can be rationalized through attractive electrostatic interactions between the positively charged dendrimer and the negatively charge silica substrate. These interactions strengthen with increasing pH due progressive charging of the silica surface, but they weaken due to screening by salt. The extent of dendrimer swelling is ion specific. The type of cation plays a more important role, whereby divalent earth alkali metal cations lead to larger swelling than monovalent alkali metal cations. The influence of the type of anions is less important. This behavior can be qualitatively rationalized by attractive interactions between the dendrimer and the surface, which is strongly modified by the adsorption of the cations to the silica surface. Anions modify principally the interactions inside the dendrimers, and thus they influence the swelling behavior more weakly. The fact that swelling is dominated by the interactions between the dendrimers and the substrate is further corroborated by the following two observations. Preliminary experiments involving adsorbed PAMAM dendrimers on water−gold interfaces show that any swelling upon changes of the ionic strength is absent. This behavior involving the gold interface is entirely different from silica, which indicates that the substrate plays an essential role in the swelling process. Dendrimers in aqueous solutions were demonstrated to show little swelling upon changes in solution composition.13−16 This observation suggests that interactions between the dendrons within the dendrimer can be only marginally modified and cannot be easily exploited to control swelling or shrinking of dendrimers.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: +41 22 379 6405. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Swiss National Science Foundation and the University of Geneva.

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