Interactions between Solid Surfaces with Preadsorbed Poly

Poly(ethylenimine) (PEI) polyelectrolytes have been widely used to tune the stability, rheology, or adhesion properties of colloidal suspensions due t...
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Interactions between Solid Surfaces with Preadsorbed Poly(ethylenimine) (PEI) Layers: Effect of Unadsorbed Free PEI Chains Xiangjun Gong and To Ngai* Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

ABSTRACT: Poly(ethylenimine) (PEI) polyelectrolytes have been widely used to tune the stability, rheology, or adhesion properties of colloidal suspensions due to their strong tendency to adsorb to solid surfaces. They have also gained importance as gene carriers in biomedical applications, in which the anionic DNA chains are complexed and condensed to form PEI/DNA polyplexes. Some reported literatures have recently shown that the overdosed PEI chains, which are free in the solution mixture, also play a vital role in promoting the gene transfection, but the reason is unclear. In this work, we present the results of using total internal reflection microscopy (TIRM) to measure the interaction forces between a Brownian colloidal sphere and a flat glass plate in the presence of overdosed free PEI cationic chains, when both surfaces were saturated adsorbed with the PEI chains. The colloidal sphere preadsorbed with PEI chains was chosen to mimic the PEI/DNA polyplex. Results for the potential energy of interaction measured for model polyplex (e.g., PEI-coated sphere) interacting with a PEI-coated glass surface in the presence of overdosed free PEI chains at various pH values and salt concentrations were presented. As can be shown by direct force measurements, the interaction potentials in NaCl salt solution are dominated by repulsive forces originating from diffuse layer overlap and gravitational attraction. However, the presence of free PEI chains in the solution mixture produces a long-ranged (>60 nm) attractive force between two PEI-coated surfaces with the range and magnitude tunable by pH value, PEI, and salt concentrations. The possible mechanisms of this long-ranged attractive force are discussed. A better understanding of this free PEI-induced attractive force will be useful in the development of improved PEI/DNA polyplexes systems for biomedical applications.

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INTRODUCTION Poly(ethylenimine) (PEI) is a cationic branched weak polyelectrolyte, which has been used in a wide variety of industrial applications, including as stabilizer of colloidal particles,1 as flocculant in water purification,2−4 or as retention aid in papermaking.5 In all these applications, PEI polyelectrolytes strongly adsorb onto colloidal particles or macroscopic surfaces and change the interaction between the particles and between surfaces and their environment. Because of the importance of these processes in industry, numerous researchers have attempted to understand the driving forces for polyelectrolyte adsorption,6,7 the structure and properties of the adsorbed polyelectrolyte layer,6,8,9 and interaction forces acting between solid surfaces coated with such polyelectrolyte films.10,11 The adsorption of PEI to large surfaces and colloidal particles has been studied by adsorption experiments,6,12 electrokinetic methods,7,9 or dynamic light scattering (DLS) techniques.8,13,14 More recently, it became possible to study polyelectrolyte © 2013 American Chemical Society

adsorption to macroscopic planar substrates, especially by ellipsometry, streaming potential measurements, and quartz crystal microbalance (QCM).15,16 The effect of the PEI charge density on adsorption isotherms and hydrodynamic layer thickness has been well demonstrated by these classical approaches. However, the information related to the interaction forces between polyelectrolyte bearing surfaces only reveal indirectly. Therefore, quite a few studies have employed, for example, surface force apparatus (SFA),10,17,18 atomic force microscopy (AFM),19,20 or optical tweezers,21,22 to directly measure the interactions between adsorbed polyelectrolyte films. Interaction forces between mica or glass surfaces in PEI solution have been systematically investigated with the SFA.12 Generally, these studies conclude that PEI adsorbs to the negatively charged mica or glass surfaces, causing charge Received: February 11, 2013 Revised: April 18, 2013 Published: April 29, 2013 5974

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model polyplex (e.g., PEI-coated PS) can be varied by changing the amount of PEI, salt concentration, and pH value. Results for the potential energy of interaction measured for model polyplex interacting with a PEI-coated glass surface in the presence of overdosed free PEI chains at various pH values and salt concentrations were presented. We demonstrated that the presence of free PEI chains in the solution mixture produce a long-ranged (>60 nm) attractive force between two PEI-coated surfaces that is originated from neither van der Waals interactions nor the polymer bridging.

neutralization at the isoelectric point (IEP), subsequent charge reversal, and saturation. Therefore, at the starting, the force profiles between the charged solid surfaces are dominated by repulsion due to diffuse layer overlap. With increasing polymer dose, additional short-range attraction is observed, particularly at the IEP. The following charge reversal leads again to repulsive force due to diffuse layer overlap. The interaction forces acting between the negatively charged surfaces in the presence of PEI have also been investigated with the colloidal probe technique based on the AFM.23 Similarly, these studies have demonstrated that as the PEI dose is increased, the initially observed double-layer repulsive forces decrease in strength and a short-ranged attraction sets in. At intermediate dose, the long-ranged repulsive forces disappear, and only the attractive part can be seen. As the PEI dose is further increased, the long-ranged repulsive forces become dominant again.10 This dependence of the interaction forces on the polyelectrolyte dose is analogous to the SFA experiments. However, this study argued that the additional attractive forces are not likely originated from polymer strands bridging the two surfaces but attributed to the attractive electrostatic interaction of positive (covered) and negative (uncovered) patches of adjacent particles, so-called the patch-charge heterogeneities.23 While the effects of adsorbed PEI films on colloidal and surface interactions have been well studied both theoretically and experimentally, the effects of unadsorbed polyelectrolyte on surface interaction, particularly on preadsorbed PEI surfaces, are not as well understood. In addition to the industrial applications, PEI has gained importance as drug or gene carriers in pharmaceutical and biomedical applications.24,25 Anionic DNA chains are complexed and condensed by cationic PEI chains through the electrostatic interaction to form PEI/DNA polyplexes. The bounded PEI chains inside the polyplexes are known to provide a charge compensation so that DNA is condensed and protected from degradation. Interestingly, Yue and co-workers26,27 have recently shown that the unadsorbed PEI chains, which are free in the solution mixture, also play a vital role in promoting the gene transfection. The presence of free PEI chains in the solution leads to a significant faster and more efficient cellular uptake of polyplexes. In addition, DLS results revealed that at high free PEI dose the size distribution of the fully condensed playplexes would become broad, indicating the aggregation between the polyplexes, likely induced by the presence of free PEI chains.26 The question of interest here is: how do the unadsorbed, free PEI chains affect the stability of PEI/DNA polyplexes in solution mixture and subsequently promote the intracellular trafficking of the resultant polyplexes? To answer the above question, we used the total internal reflection microscopy (TIRM) to measure the interaction forces between a Brownian colloidal sphere and a flat glass plate in the presence of unadsorbed free PEI cationic chains, when both surfaces were saturated with physisorbed PEI chains. The TIRM technique is based on the use of evanescent wave light scattering by a single colloidal sphere near a flat surface to determine the equilibrium distribution of sphere−surface separations and the associated interaction energy. It has proven to be very successful in quantifying the mean potential energy of interaction between a polystyrene (PS) sphere and a glass surface in the presence of different polymers28−30 and nanoparticles.31,32 In this work, the PS sphere preadsorbed with PEI chains was chosen to mimic the PEI/DNA polyplex. Zeta potential data demonstrate that the surface potential of the

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EXPERIMENTAL SECTION

Materials and Sample Preparation. Micrometer-sized polystyrene (PS) sulfate latex spheres with a diameter of 6 μm (CV 4.1%, Interfacial Dynamics Co.) were used in both TIRM and zeta potential measurements. Silica particles with a diameter of 5 μm (CV 10.0%) were purchased from Polysciences Inc. and used for zeta potential measurement without further treatment. Branched PEI chains with a weight-averaged molar mass of about 25 000 g/mol were obtained from Sigma-Aldrich and used as received. The sodium chloride (NaCl, GR from BDH) was sealed at ∼200 °C to keep dry and pure. Fresh deionized water with the resistivity of 18.2 MΩ·cm at room temperature was used to prepare all aqueous samples. Hydrochloric acid (HCl, Merck) was used to adjust the pH of the solutions. The silica glass slides (BK-7, Fischer Scientific Co.) were initially dipped in 5% hydrofluoric acid (HF) solution, subsequently rinsed extensively with DI water, and finally dried with pure nitrogen gas. The treated slides were further cleaned by an ultraviolet (UV)-ozone plasma cleaner (Harrick Sci. Corp.) before assembling the TIRM sample cell.33 Prior to measuring the interaction forces by means of TIRM, the PS spheres and the glass slides were subjected to 72 h of contact with a 1 wt % of the PEI solution at pH = 7.6 (adjusted by HCl) and room temperature. After that, the glass slides were thoroughly rinsed with DI water, while the PS microspheres with the preadsorbed PEI layers (PEI-coated PS) were collected by centrifugation at a low rotation speed (3000 rpm) and then redispersed in the corresponding sample solutions. Total Internal Reflection Microscopy (TIRM). The principles of TIRM have been described in many previous literatures.34 In a TIRM measurement, an evanescent wave, which decays exponentially with the distance from the interface, is generated at the glass−water surface by a total internal reflection within the glass slide. When a micrometersized probe sphere approaches the surface close enough to enter the evanescent field, frustrated total reflection will occur and the sphere will scatter the evanescent field. The scattered intensity (I) of the colloidal sphere has been shown to be proportional to that of the evanescent wave and thus can be written as34

I(h) = Ih → 0 exp(− βh)

(1)

where h is the separation distance between the colloidal sphere and the glass surface, β−1 is the characteristic penetration length, and Ih→0 is the immobilized particle intensity which can be obtained by depositing the colloidal sphere on the bottom surface with a high concentration of salt solution (∼100 mM). Measuring I, which fluctuates due to Brownian motion, of the sphere perpendicular to the surface as a function of time thus provides a sensitive and nonintrusive method to determine the distance h between the sphere and the wall. In thermal equilibrium the probability distribution of finding the particle a certain distance to the wall, p(h), is given by the Boltzmann distribution

⎡ Φ(h) ⎤ p(h) = A exp⎢− ⎥ ⎣ kBT ⎦

(2)

(where A is a constant normalizing the integrated distribution to unity), which allows us to calculate the overall interaction potential Φ(h) from the measured scattering signal. In the TIRM measurements, a highly diluted PEI-coated PS dispersion was initially injected 5975

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into a sample cell, which composed a soft plastic frame with thickness 1 mm sandwiched between two glass slides, and the lower one was preadsorbed with the PEI layers. A PEI-coated PS sphere of average brightness was selected and held in place with optical tweezers by a solid-state Nd:YAG laser (output = 300 mW at λ = 532 nm), while the rest of the particles were washed away from the cell with the diluted NaCl (0.1 mM) solution. After that, the trapped particle was released and the interaction potentials between the PEI-coated PS sphere and PEI-coated glass slide in NaCl solution was recorded. In the next step, the PS sphere was trapped again, and the sample cell was gradually replaced with PEI solutions with different doses. It is noteworthy that this approach allows us to exchange the solution inside the sample cell between the pure NaCl solution and PEI solution repeatedly but keep using the same PEI-coated PS spheres and glass slide, which can significantly reduce the influence of sphere and surface variations. The conductivity of the sample solutions was recorded by the conductivity meter (Cond. Meter 470, Jenway). All TIRM experiments were performed at 25 °C. Zeta Potential. The electrophoretic mobility (μE) of the PEIcoated PS spheres and the PEI-coated silica particles in aqueous solutions were measured with a commercial zeta-potential spectrometer (ZetaPlus, Brookhaven). Similar to the samples used for TIRM measurements, the adsorption of PEI layers onto surfaces was prepared by immersing the PS sphere and silica particles in a PEI solution with a concentration of 1 wt % at pH 7.6 for 72 h at room temperature, respectively. Note that silica particles have an average diameter around 5 μm; the adsorption of PEI chain (∼4 nm) to the spherical particles is thus analogous to the adsorption to flat glass slide used in TIRM measurements. Generally, the mobility of the PS and silica particles coated with the PEI layers in the suspension was obtained by taking the average of 10 individual runs. Following the Smoluchowski equation (κRb ≫ 1), the zeta potential (ξpotential) can be calculated from μE as

ξpotential =

Figure 1. Zeta potential (ξpotential) of negatively charged PS latex particles with coating of branched PEI polyelectrolyte at various pH values. Note that repeatedly washing and redipsersion (from 2 to 8 times) shows a weak influence on the ξpotential of the resultant PEIcoated PS latex particles.

the ξpotential after the 4 and 8 times centrifugation steps (Figure 1). This is understandable because in the real situation adsorption of polyelectrolytes is basically irreversible. Once a polyelectrolyte chain has absorbed, it will not desorb except under extreme conditions, and any rearrangement or migration on the surface is highly unlikely.35 Therefore, we can assume that during our TIRM experiments the repeated rinsing of the sample cell between the salt and the PEI solution would not cause a significant effect on the properties of the adsorbed PEI layers at the probe particle and glass surfaces. A very similar absorption behavior was observed with the silica particles (Figure 2). The starting negatively charged silica surface (−70

μE η ε

(3)

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RESULTS AND DISCUSSION ξ potential of the PEI-Coated Solid Surfaces. The absorption properties of PEI layers onto the PS and silica particles were investigated by electrophoretic mobility measurements. The PS and silica particles were immersed in a highly concentrated PEI solution (1 wt %) for 72 h at room temperature to ensure that the particle surfaces were saturated with the PEI chains. Afterward, the suspensions were first centrifuged to remove the unbounded PEI chains. The collected particles were then resuspended in deionized water. After that, this washing procedure including centrifugation and resuspension steps were followed several times, and the particles after each washing were divided into different aqueous solutions at specific pH tuned with either HCl or NaOH. As a result, ξpotential of such a series of solutions were monitored and marked with the washing times. For the initial bare PS sphere, the ξpotential is ∼−60 mV, attributing to the deprotonation of the group on the surfaces. However, Figure 1 shows that after the spheres were subjected to 72 h of contact with PEI solution, the average ξpotential of the resultant PS spheres measured at different pH (ranging from 7.5 to 11.2) was approximately +80 mV, indicating that the adsorption of positively charged PEI onto the PS sphere surface is significant, which eventually leads to charge reversal or overcharging and that the surfaces are positively charged. The fact that such charge reversals is characteristic for polyelectrolytes adsorbing to oppositely charge particles.7,9 It is worthy to note that the purification or washing has only a weak influence on the ξpotential of the resultant PEI-coated PS spheres, as we see similar magnitude of

Figure 2. Zeta potential (ξpotential) of negatively charged silica particles with coating of branched PEI polyelectrolyte at various pH. Comparison between washing at 2 and 4 times shows that it has only a weak influence on the ξpotential of the resultant PEI-coated silica particles.

mV) became positively charged, suggesting that the PEI chains strongly adsorbed to the surface of silica spheres. The result of this adsorption leads to charge reversal at high polyelectrolyte dose and that the surfaces are positively charged. Also, the adsorbed PEI layers on the surface of silica particles show a weak influence by the washing steps. It should be mentioned that we have used silica spheres to mimic the PEI adsorption onto glass slide used in the TIRM measurements. Since the surface groups of pure silica spheres are SiOH which is the same with the glass slide after the above clean procedure, the absorption of PEI to these two substrates was considered to be quite similar.33,36 Effect of Unadsorbed PEI Chains on the Interaction between PEI-Coated Surfaces. Direct force measurements between the PEI-coated sphere and the PEI-coated glass surface were carried in NaCl and PEI solutions with the TIRM technique. At the beginning, the potential energy profile for the 5976

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PEI-coated sphere interacting with the PEI-coated surface in 0.15 mM NaCl, which corresponds to the solution conductivity of 12 μS/cm, was measured. After that, a 10 ppm PEI solution with the same conductivity of 12 μS/cm (adjusted by adding NaCl) was introduced into the sample cell to replace the 0.15 mM NaCl solution, while the PEI-coated PS sphere was held in place using optical tweezers. After complete replacement, the sphere was released and the interaction potential between the free-moving PEI-coated sphere and the PEI-coated glass surface in the presence of PEI solution was measured. Note that holding the probe sphere in an optical trap between these two consecutive experiments allows us to measure interactions between the same particle and substrate area before and after the introduction of PEI chains. Besides, the ionic strength of the PEI solution is adjusted the same as 0.15 mM NaCl solution; we thus can isolate the effect of the presence of PEI chains on the interaction between the PEI-coated sphere and substrate area. This procedure is repeated several times by alternatively rinsing the sample cell with 0.15 mM NaCl and then PEI solution with different concentrations, where the conductivity of all solutions is kept as 12 μS/cm. Figure 3

Figure 4. Potential energy profiles measured between the PEI-coated sphere and the PEI-coated glass surfaces in the presence of a higher ionic strength NaCl and PEI solutions at the same conductivity of 24 μS/cm. The six consecutive measurements were taken in the following sequence: 0.30 mM NaCl (○), 10 ppm PEI (●), 0.30 mM NaCl (△), 50 ppm PEI (▲), 0.30 mM NaCl (□), 100 ppm PEI (■). The solid line is the fitted line based on eq 6.

Φ(h) − Φ(hm) G = Be−κh + h kBT kBT

(4)

−1

where κ is the Debye length of the solvent, G is the net weight of the PS sphere, hm is the most probable height determined by the measured intensity histogram, and B indicates the amplitude of the electrostatic interaction which depends both on the Stern potential of the PS sphere and the glass surface. In addition, Φ(hm) was assumed to be 0 in Figures 3 and 4. Since eq 4 has a single minimum at h = hm, B is no longer an independent parameter and as a result

B=

G κhm e κ

(5)

Consequently, eq 4 can be replaced as

Figure 3. Potential energy profiles measured between the PEI-coated sphere and the PEI-coated glass surfaces in the presence of low ionic strength NaCl and PEI solutions at the same conductivity of 12 μS/ cm. The six consecutive measurements were taken in the following sequence: 0.15 mM NaCl (○), 10 ppm PEI (●), 0.15 mM NaCl (△), 50 ppm PEI (▲), 0.15 mM NaCl (□), and 100 ppm PEI (■). The solid line is the fitted line based on eq 6.

Φ(h) − Φ(hm) G G = e−κ(h − hm) + h κ kBT kBT

(6)

In this way, the shape of the potential energy profiles is not affected by B. Increasing the charge on either the probe sphere or the glass slide will shift the minimum to larger separation distance hm according to eq 6, but it does not affect the shape. Equation 6 was used to fit the measured interaction potentials in NaCl solutions, and the resulting solid curves are shown in Figures 3 and 4. The measured potential profile in the lower ionic strength (0.15 mM NaCl) solution has a decay length of about ∼25.7 nm. This value is in good agreement with the Debye length κ−1 ∼ 24.8 nm calculated from the solution composition. Besides, the fitting results in G ∼ 62.6 pN, which agrees well with the calculated value 60.9 fN of a 6 μm PS particle having an apparent density of ρ = 1.05 g/cm3. This type of agreement is typical for TIRM measurement.19,37,38 Obviously, increasing the electrolyte concentration leads to a shift of the separation distance, h, from ∼250 nm (0.15 mM NaCl with the conductivity of 12.0 μS/cm in Figure 3) to ∼150 nm (0.3 mM NaCl with the conductivity of 24.0 μS/cm in Figure 4) because the double-layer repulsion is weakened by the addition of salt. The solid curve shown in Figure 4 also fits the experimental data quite well; in particular, the measured decay length of about 12.4 nm, which is in fair agreement with the calculated Debye length κ−1 ∼ 17.6 nm. When PEI solution is added to the system, Figure 3 shows that the potential profiles shift closer to the PEI-coated glass surface and the separation distance decreases with increasing

summarizes the first six resulting profiles at this low ionic strength solution. For comparison, the direct force measurements were also conducted in a higher salt condition, where 0.3 mM NaCl was added, and PEI solutions were adjusted to a higher conductivity of ∼24 μS/cm. The resulting six profiles at the higher ionic strength are summarized in Figure 4. Therefore, a total of 12 consecutive measurements of the interaction potential profiles between the PEI bearing surfaces were obtained in the presence or absence of PEI polyelectrolyte, under both salt conditions. Figures 3 and 4 respectively show that either at 0.15 mM or at 0.3 mM NaCl solutions the potentials of the positively charged PEI-coated PS sphere above the PEI-coated glass slide composes of two parts: toward smaller distance the potential increases exponentially due to the electrostatic double layer repulsion between the adsorbed PEI layers on the sphere and glass surface. For larger distances the potential is completely dominated by the weight of the PS sphere. Since the separation distance is several Debye lengths, the van der Waals attraction is expected to be severely retarded, so as to be negligible. Then, for a 1:1 electrolyte, the total potential energy profile is followed as34 5977

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compared to the separation distance typically encountered (>60 nm), the presence of steric forces, direct bridging forces, or midplane crossing bridging forces is highly unlikely. We suspect that another force is acting. This attraction is always found by repeated experiments on over five different PEI-coated PS beads and silica surfaces with similar range and strength. To explore the origin of this attraction, ξpotential of the PEI-coated PS spheres and the PEIcoated silica particles in the presence of different concentrations of PEI polymers were investigated. Typically, the PEI-coated PS spheres and the PEI-coated silica particles were separately mixed with the PEI solutions at concentrations of 10, 50, and 100 ppm, and the conductivity of the solutions was adjusted by NaCl to 12 and 24 μS/cm. Figure 6 shows that for PEI-coated

bulk PEI concentration. This change in the separation distance is not expected to arise solely from any change in the ionic strength associated with the addition of the PEI polyelectrolyte since the conductivity of the three introduced PEI solutions was kept at 12 μS/cm. On the other hand, after rinsing the sample cell again with the 0.15 mM NaCl solution, the measured potential profiles are restored to the original position and are in good agreement with eq 6 represented by the solid curve shown in Figure 3. This result suggests that the replacement of the sample solution in the whole experiment has a weak effect to the PEI layers adsorbed onto the sphere and glass surfaces. For comparison, Figure 4 shows the potential energy profiles obtained for the PEI-coated PS sphere interacting with the PEIcoated glass slide in the presenting of three same amounts of free PEI solutions as those in Figure 3 but at a higher ionic strength (0.3 mM NaCl with conductivity of 24.0 μS/cm). The dependence of the interaction potentials on the PEI polyelectrolyte concentration is analogous to the results shown in Figure 3; namely, upon increasing the concentration of unadsorbed PEI, the profiles shift to smaller separation distances. Figure 4 shows that at a 100 ppm PEI solution the interaction potential is narrower and steeper, and hm is lowered to ∼60 nm. It seems that there is an attraction between the two PEI-coated surfaces in the presence of absorbed free PEI chains. This attractive force increases with increasing PEI concentrations, while the separation distance decreases. However, flushing the sample cell with pure 0.3 mM NaCl again can restore the original interaction potentials as shown in Figure 4. To isolate the effect of unadsorbed PEI on the interaction potentials, Figure 5 shows the potential files in the presence of

Figure 6. Zeta potential (ξpotential) of the PEI-coated PS sphere and the PEI-coated silica particles as a function of the PEI concentration at both low (12 μS/cm) and high (24 μS/cm) ionic strengths.

PS spheres, with increasing PEI concentration, ξpotential of the PEI-coated PS spheres in both low and high ionic strengths decreases. The dependence of the ξpotential on the PEI concentration is analogous to the PEI-coated silica particles, except that there has a large deviation of the ξpotential measured at 100 ppm PEI solution with conductivity of 12 μS/cm. It seems that the surface charge of the adsorbed PEI layers could be pronouncedly quenched by the overdosed PEI chains, which probably originates from the binding of protons and anions by the polyelectrolytes. In this manner, the excess PEI chains free in the solution not only remain dissolved in solution but also cause a reduction of the double-layer repulsion between the PEI-coated sphere and the PEI-coated surface via charge regulation, leading to a decrease in the particle−surface separation distance. However, this still could not account for why an additional weak attraction sets in, and the strength is enhanced with increasing the solution ionic strength as shown in Figure 5. Note that the attraction exists with a measurable distance larger than 60 nm; the range seems too large to be compatible with the van der Waals attraction. More importantly, van der Waals attraction between the two surfaces can be excluded since this attraction can be generated only by the addition of the free PEI chains, while it has not been seen in the same ionic strength NaCl solution. Moreover, the force measurements are carried out between the two surfaces which have been saturated adsorbed with PEI chains, and further adsorption is thus highly unlikely due to the strong repulsive electrostatic forces between the adsorbed PEI chains. Therefore, is it intuitive to ask whether the origin of the attraction is related to depletion forces induced by the excess PEI chains free in solution?39 First, independent studies have shown that the saturated adsorbed PEI layers on the PS and glass surface

Figure 5. Net potential energy profiles measured between the PEIcoated sphere and PEI-coated glass surfaces in the presence of different concentration of PEI at 12 and 24 μS/cm ionic strength solutions. The gravity part of the potential profiles measured in PEI solutions was removed from Figures 3 and 4.

unadsorbed PEI in both 0.15 and 0.3 mM ionic strength solutions, where the gravity parts shown in Figures 3 and 4 have been removed. It is clear to see that besides the double-layer repulsion a long-ranged attractive force has been found, particularly at higher PEI concentration and higher ionic strength. The attractive minimum increases up to ∼1.8 kBT at 100 ppm PEI with the conductivity of 24 μS/cm, pushing the PEI-coated sphere closer to the glass surface of about 60 nm. This behavior is in sharp contrast with the presence of the same ionic strength NaCl solution, where no long-ranged attractive force has been seen. Moreover, at the same PEI concentration, this attractive force is enhanced upon increasing the ionic strength of the solution. Since the adsorbed PEI layers on sphere and glass slide surfaces are very thin (∼4−6 nm) 5978

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are very thin, typically of a few nanometers.11 Therefore, the steric forces or the polymer bridging contributions also seem unlikely.29,40 Moreover, although it is hard to imagine a single PEI free chain with a size around 4 nm to induce such a longranged (>60 nm) depletion force, polyelectrolytes were found to have a much larger effective radius which might lead to an enhanced depletion attraction.41−43 One may argue that in Figure 5, with increasing ionic strength, the attractive force increases, which seems in sharp contrast to the polyelectrolyteinduced depletion attraction,42,44 where adding salt can weaken the depletion attraction caused by a reduction in osmotic pressure. However, it should be noted that the net energy profiles in Figure 5 also contain the electrostatic repulsion. Further addition of the salt also induces a weakening of the electrostatic repulsion due to the enhanced charge screening effect. This brings a “faked” increase to the observed attraction as the salt increases. Consequently, both the repulsive and attraction decreased, although there may in different amounts. However, it is rather difficult to dissolve the respective contribution weights of the two components in this case. In this way, the attraction caused by the depletion attraction force cannot be totally ruled out in this system. Finally, another possible origin of the attraction might be responsible for the inhomogeneous charge redistribution of the adsorbed polyelectrolyte layers due to the strong correlation between the polymer layers and the nearby free PEI chains, especially at low ionic strength. Because of branched nature of the PEI polyelectrolytes, one can suspect that the adsorbed films are strongly heterogeneous, consisting of individual adsorbed polyelectrolytes that are unevenly distributed on the oppositely charged PS sphere with sulfate groups and glass surfaces.13 When these two surfaces with the inhomogeneous charge distribution expose in the free PEI bulk and approach to each other, the PEI chains adsorbed on the surfaces keep contact with the newly induced counterions OH−, which might lead to the rearrangements of their charges on the chains and the corresponding confirmations. In such a way, a positively PEI charged patch might face a negatively charged uncovered PS surface, resulting in a net electrostatic attraction. However, the range and order of magnitude of such patch−charge interaction is still a matter of debate.23 To further study the effects of the presence of the free PEI chains, pH dependence of the interactions between the PEIcoated sphere and the PEI-coated glass surface at 100 ppm PEI solution was investigated. The conductivity of the PEI solutions at pH 9.3, 8.5, and 7.6 are around 12, 24, and 48 μS/cm, respectively, which corresponds to the NaCl solutions at the concentration of 0.15, 0.30, and 0.60 mM. In other words, ionic strength of the solutions increases as pH value decreases. On the other hand, with decreasing the pH value, the adsorbed PEI films should get more charges due to the protonation. Therefore, there is a completion between the charging and screening when the pH value of the PEI solutions is lowered. Figure 7 compares the measured energy potentials between the PEI-coated sphere and the PEI-coated glass surface in the presence of same ionic strengths of NaCl and PEI solutions at various pH values. We assume that at the same ionic strength the screening effect on the adsorbed PEI films interactions is the same, and thus the only difference in the aqueous environment is the existence of PEI polyelectrolytes or not. Figure 7 clearly shows that the potentials are strongly altered in the presence of PEI solutions due to the occurrence of attraction. With decreasing pH values we observe that the

Figure 7. Measured potential energy profiles between the PEI-coated PS sphere and the PEI-coated glass surfaces in the presence of NaCl and 100 ppm PEI electrolyte solutions at different pH values with the same ionic strength. The conductivity of the PEI solutions at pH 9.3, 8.5, and 7.6 is around 12, 24, and 48 μS/cm, respectively, which corresponds to the NaCl solutions at the concentration of 0.15, 0.30, and 0.60 mM.

potential minimum is shifted toward smaller distances and becomes deeper. Such an attraction disappeared when the aqueous environment was replaced with pure NaCl solution at the same ionic strength, indicating that the attractive forces are induced by the presence of the free PEI polyelectrolytes in the solution. At pH = 7.6, the PEI-coated sphere seems “stuck” at an equilibrium distance of ∼39.3 nm. A possible reason for this is that with increasing the ionic strength the repulsive forces between the two PEI-coated surfaces are weaken such that two heterogeneously charged surfaces approach closer, leading to the a stronger net electrostatic attraction between the charge patches on two surface. On the other hand, bridging forces could be also important between these two surfaces coated with adsorbed PEI polyelectrolytes at such short separation distance and high ionic strength. However, distinguishing these two mechanisms experimentally has proved difficult.23 For the separation distance greater than 60 nm, we argue that the entanglement between the PEI chains of the two surfaces is unimportant because the thickness of the adsorbed PEI films is typically within several nanometers. Figure 8 provides the information on ξpotential of the PEIcoated PS and the PEI-coated silica particles as a function of pH in the 100 ppm PEI solution. It can be seen that pH shows a pronounced influence of the PEI-coated silica surface, while the effect on the PEI-coated PS surface is not significant. With pH decreasing, ξpotential of the PEI-coated sphere first increases slightly from +38 mV at pH = 9.7 to +60 mV at pH = 9.2 and

Figure 8. Zeta potential (ξpotential) of the PEI-coated PS sphere and the PEI-coated silica particles as a function of pH value, where the PEIcoated PS and the PEI-coated silica particles are mixed with 100 ppm PEI solution at room temperature. 5979

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attraction. Direct force measurements show that this attractive force increases with increasing polyelectrolyte concentration and decreasing pH value. In addition, with increasing background electrolyte ionic strength, the attractive force increases. Our results show that the presence of steric forces, direct bridging forces, or midplane crossing bridging forces is highly unlikely. We argue that this attractive force mainly originates from two mechanisms: the free polyelectrolytes induced depletion attractive forces and patch−charge interaction due to the charge redistribution of the inhomogeneous adsorbed polyelectrolyte layers in the presence of free PEI chains. Increasing the free PEI polyelectrolytes in solution may likely charged the heterogeneous structures of the adsorbed polyelectrolyte layers in different degrees due to the ion−ion correlation in a limited geometry, leading to differently stretched polyelectrolyte chains. As a result, a complex interplay of electrostatic interactions between the two adsorbed polyelectrolyte films leads to the net attractive force. This finding might provide a new insight into the control of colloidal stability in the existence of large dosed cationic polyelectrolytes. Besides, a better understanding of this free PEI-induced attractive force will be useful in the development of improved PEI/DNA polyplexes systems for biomedical applications.

then levels off at +50 mV at pH 7.2, revealing a competition balance between the protonation of the adsorbed PEI layers and the screening effect of the added anions. On the other hand, ξpotential of the PEI-coated silica particles increase from +40 to around +90 mV with decreasing pH. This result indicates that the adsorbed PEI layers on the silica particles are charged as the pH value decreases. Thereby, one may expect that the strength of the double-layer repulsion should be enhanced and the separation distance between the particle− surface increases, but this is not the case as shown in Figure 7. The above ξpotential measurements also cannot provide an insight into the origin of the measured attraction. In the presence of free or better large dose PEI chains, our results have clearly demonstrated that there exists a long-ranged attraction with the range and magnitude depending on the pH value, polyelectrolyte, and salt concentration. From a certain perspective, these factors are all related to the ionic strength of the bulk solution. According to the definition of ionic strength, in the bulk of free PEI chains, several to tens of NH3+ charges on single protonized PEI chain contribute to the ionic strength much significantly than the corresponding free counterions OH−.45 As a result, increasing the PEI amounts will lead to a pronounced increase of the ionic strength in solution compared with adding 1:1 electrolyte like NaCl salt at the same molar concentration. This conclusion has been confirmed in the ξpotential measurements shown in Figure 6. From previous discussion, we speculate two possible reasons for the longranged attraction: the free PEI chain induced depletion and the patch−charge interaction. However, results shown in Figure 7 still cannot rule out or confirm any one of them. Namely, for the depletion interaction, decreasing pH will increase the protonation of the PEI chains and eventually enhance the attraction, while for patch−charge interaction, the adsorbed PEI films at the sphere and glass surfaces are strongly heterogeneous, consisting of unevenly distributed PEI chains. Under such conditions, increasing the free PEI chains or decreasing pH in solution may likely enhance and further charge the heterogeneous structures of the adsorbed polyelectrolyte layers in different degrees due to the ion−ion correlation in a limited geometry,46 leading to differently stretched polyelectrolyte chains. As a result, a complex interplay of electrostatic interactions between the two adsorbed polyelectrolyte films leads to the net attractive force. A similar behavior was found by Drechsler et al.,20 when they recently measured the interaction forces between two surfaces bearing with cationic polyelectrolyte brushes made of weak polybase poly(2-vinylpyridine). An additional strong attractive force at distance ∼30−50 nm is observed particularly when the polyelectrolytes are partly protonated and the salt concentration is low. However, due to the complexity of polyelectrolyte systems, a whole description of the complex interplay between the attraction and repulsion is difficult and will the subject of further work.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], Tel +852-3943 1222 (T.N.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of this work by the Hong Kong Special Administration Region (HKSAR) General Research Fund (CUHK403210, 2130237) is gratefully acknowledged. We thank Dr. Jin Fan (Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China) for illuminating and valuable suggestions.

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REFERENCES

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CONCLUSIONS In this work, we have investigated the effect of overdosed free branched cationic PEI polyelectrolytes on the interaction potentials between a colloidal and a glass surface when both surfaces are saturated adsorbed with physisorbed cationic PEI. The results show a strong reproducible long-ranged attraction (appeared at h > ∼60 nm) fully induced by the addition of free PEI chains at PEI concentrations as low as 10 ppm, and this range is too large to be compatible with van der Waals 5980

dx.doi.org/10.1021/la400543c | Langmuir 2013, 29, 5974−5981

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