Influence of Protamine Functionalization on the Colloidal Stability of

Article pubs.acs.org/Langmuir

Influence of Protamine Functionalization on the Colloidal Stability of 1D and 2D Titanium Oxide Nanostructures Paul Rouster,† Marko Pavlovic,† Endre Horváth,‡ László Forró,‡ Sandwip K. Dey,§ and Istvan Szilagyi*,† †

School of Chemistry and Biochemistry, University of Geneva, CH-1205 Geneva, Switzerland School of Basic Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland § Materials Program, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, Arizona 85287, United States ‡

S Supporting Information *

ABSTRACT: The colloidal stability of titanium oxide nanosheets (TNS) and nanowires (TiONW) was studied in the presence of protamine (natural polyelectrolyte) in aqueous dispersions, where the nanostructures possessed negative net charge, and the protamine was positively charged. Regardless of their shape, similar charging and aggregation behaviors were observed for both TNS and TiONW. Electrophoretic experiments performed at different protamine loadings revealed that the adsorption of protamine led to charge neutralization and charge inversion depending on the polyelectrolyte dose applied. Light scattering measurements indicated unstable dispersions once the surface charge was close to zero or slow aggregation below and above the charge neutralization point with negatively or positively charged nanostructures, respectively. These stability regimes were confirmed by the electron microscopy images taken at different polyelectrolyte loadings. The protamine dose and salt-dependent colloidal stability confirmed the presence of DLVO-type interparticle forces, and no experimental evidence was found for additional interactions (e.g., patch-charge, hydrophobic, or steric forces), which are usually present in similar polyelectrolyte−particle systems. These findings indicate that the polyelectrolyte adsorbs on the TNS and TiONW surfaces in a flat and extended conformation giving rise to the absence of surface heterogeneities. Therefore, protamine is an excellent biocompatible candidate to form smooth surfaces, for instance in multilayers composed of polyelectrolytes and particles to be used in biomedical applications.

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reverse the anticoagulant effect of heparin.11 Since protamine gives rise to a pI value of 1212 and thus carries a net positive charge under physiological conditions and exhibits considerable affinity for oppositely charged surfaces, it has been used for the biomimetic mineralization of titania particles as well as a template for preparing titania−protamine composites with advantageous properties for enzyme immobilization.13,14 The latter study demonstrated that the embedded biocatalytic proteins usually kept their functional integrity and exhibited higher thermal and recycling stability than the bare enzymes. Protamine has been sometimes applied as a building block in polyelectrolyte or polyelectrolyte−particle multilayers in order to create novel structures of biomedical relevance. In one study, the core of the composite was dissolved to generate hollow structures, where bioactive molecules could be loaded and

INTRODUCTION

Titanium oxide and its derivatives are commonly used materials in catalysis, paint fabrication, water purification, novel device development and biomedical treatments.1−3 Nanostructures of various phases, chemical compositions, and shapes are obtained depending on the experimental synthesis conditions applied.4,5 Although spherical titanium oxide nanoparticles have attracted most of the interest to date, recent studies have indicated that 1D (i.e., nanowires or TiONW) and 2D (i.e., nanosheets or TNS) nanostructures may possess additional attributes with respect to photovoltaic, chemical, biochemical, and mechanical properties.1,4,6,7 Chief among them is the potential application of TiONW and TNS for biomedical processes.8−10 Despite their potential and importance for their use in living systems, the various interactions between functionalized TiONW and/or TNS and biomacromolecules have not been revealed yet. For example, biocompatible protamine, a weak polyelectrolyte mainly composed of amino acid arginine, is commonly used in postsurgery medicines due to its ability to © XXXX American Chemical Society

Received: May 31, 2017 Revised: July 28, 2017 Published: August 22, 2017 A

DOI: 10.1021/acs.langmuir.7b01815 Langmuir XXXX, XXX, XXX−XXX

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calculated to be 30 m2/g. The calculations of the outer surface area are detailed in the Supporting Information. The PZC values of TiONW and TNS are similar to the ones reported for other titanium oxide derivatives.25−27 Electrophoresis. Electrophoretic mobility experiments were performed using a Zetasizer Nano ZS (Malvern Instruments) device. During sample preparation, 0.5 mL of bare TNS or TiONW of 10 mg/ L and 36 mg/L concentration, respectively, was added to 4.5 mL of solution containing precise amounts of protamine and KCl. The concentration range was 0.1−100 mg/L for protamine and 1−2000 mM for KCl in the dispersions. The samples were then allowed to rest overnight before performing the mobility experiments. This time interval was long enough to complete the protamine adsorption processes, even in the case of saturated polyelectrolyte layers formed on the particles. Here, the average of five individual measurements on a sample is reported with an error of 5% for such an experimental procedure. For isolated particles with low zeta potential, as in the case of TiONW and TNS, the electrophoretic mobility (u) was converted to zeta potential (ζ) using the model developed by von Smoluchowski:28

released under specific conditions owing to their interactions with protamine.15 In another study,16 a similar layer-by-layer technique was used to prepare an alumina−titania composite in the presence of protamine. The dissolution of the alumina template and pyrolysis of the polyelectrolyte resulted in the formation of a continuous network of titania nanotube arrays. Also, multilayered ultrathin clay-protamine films have been demonstrated.17 Moreover, protamine-assisted titania coating of silicon nanopillars was achieved in a layer-by-layer process to show the advantages with respect to solar absorption for the nanocomposites.18 In the aforementioned studies, the high affinity of protamine to oppositely charged particle surfaces and subsequent charge inversion upon adsorption were always concluded on the basis of zeta potential measurements. Protamine has also been used in various other biomedical applications, where its role was mostly to form biocompatible surfaces with advantageous charging properties. For instance, selective membranes containing protamine were developed for biosensor applications.19 Drug nanoparticles were formulated with protamine-coated clay platelets to improve their applicability in living systems.20 It has been shown that saturated protamine layers on inorganic carriers enhance the efficiency of DNA delivery processes into cells.21 In addition, protamine−nucleic acid complexes have been prepared and adsorbed on functionalized gold nanoparticles in order to formulate a system for gene delivery applications.22 These examples clearly indicate the importance of protamine interactions with dispersed colloids or nanoparticles; however, the effects of protamine adsorption on charging and aggregation properties of such particles have not yet been comprehensively investigated. The colloidal stability of these systems is indeed the central question since aggregation of the nanostructures may prevent their in vivo applications. In the present study, the colloidal stability of protaminefunctionalized TNS and TiONW was investigated. Specifically, the charging properties were assessed by electrophoresis, while particle aggregation was followed using time-resolved light scattering experiments. Additionally, the structure of aggregated clusters was probed by electron microscopy. These studies allowed clarification of the mechanism of protamine adsorption and its effect on the stability of colloidal dispersions of protamine-functionalized TNS and TiONW.

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ζ=

uη ε0ε

(1)

where η is the viscosity of the medium, ε0 is the dielectric permittivity of the vacuum, and ε is the relative dielectric constant of water at the respective temperature. The low potential of the bare and protamine-functionalized TNS and TiONW also allowed the estimation of the surface charge density (σ) using the Debye−Hückel theory that relates the change of the double layer potential (ψDL) with the inverse Debye length (κ) as29 σ = εε0κψDL

(2)

Since a very thin hydrodynamically stagnant layer around the charged particles was assumed, the zeta potentials measured by electrophoresis were used in eq 2. Additionally, knowing the solution composition (and concentration of the background electrolyte), the magnitude of κ was calculated using 2NAe 2I εε0kBT

κ=

(3)

where NA is Avogadro’s number, e is the elementary charge, kB is Boltzmann’s constant, T is the absolute temperature, and I is the ionic strength defined as

I=

EXPERIMENTAL SECTION

Materials. High purity chemicals, protamine sulfate, and KOH from Sigma-Aldrich and KCl from Acros Organics were purchased and used for the experiments as received. Ultrapure water (Millipore) was used for sample preparation, and the measurements were performed at 25 °C and pH 9 adjusted by KOH. This pH value provides a slightly alkaline environment and also allows maintaining the acid−base processes in the samples. The TNS and TiONW were prepared hydrothermally, and detailed synthetic procedures and results of solid state characterizations are given elsewhere.7,23 The formation of the titanium oxide materials was unambiguously confirmed by recording and analyzing the X-ray diffraction patterns shown in Figures S1 and S2 for TNS and TiONW, respectively. Briefly, the main properties of the nanostructured particles can be summarized as follows. For TNS, the point of zero charge (PZC) and calculated geometrical surface area were 5.2 and 73 m2/g, respectively.23 For TiONW, the corresponding numbers were 4.1 and 186 m2/g.24 Note that the significant difference in surface areas is due to the layered structure (i.e., the inner porosity) of the latter material. However, since intercalation of larger molecules is not feasible in the interlayer space of TiONW, the effective outer surface area exposed to the adsorption processes of macromolecules was

1 2

∑ zi2ci i

(4)

where zi is the valence, and ci is the molar concentration of ion i. Light Scattering. Dynamic light scattering (DLS) measurements were performed using an ALV/CGS-3 goniometer (ALV GmbH). The sample preparation procedure in the DLS experiments was very similar to the one described above for electrophoresis. The only difference is that the final volume was 2 mL for DLS, and the measurements were started immediately after the addition of the particles to the solutions containing protamine and KCl of known concentrations. The scattering angle was set at 90°, and the correlation function was accumulated for 20 s. The translational diffusion coefficient (D) of the particles was determined by using a second order cumulant fit. The hydrodynamic radius (Rh) was then calculated with the Stokes− Einstein equation:30 Rh =

kBT 6πηD

(5)

Time-resolved DLS experiments were carried out to assess the stability of the dispersions and expressed in terms of the stability ratio (W) as30−34 B


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W=

1 dR h(t ) R h(0) dt

TiONW, respectively, the mobility values were similar to that exhibited by the bare particles when measured under identical experimental conditions.23,35 The mobilities increased with increasing the protamine dose, which can be explained by the adsorption of these macromolecules on the oppositely charged titanium oxide surfaces, followed by charge neutralization of the surfaces occurring at the isoelectric point (IEP) observed at 256 mg/g and 61 mg/g for TNS and TiONW, respectively. With further addition of protamine, beyond the dose corresponding to the IEPs, the charge on TNS and TiONW surfaces reversed to positive, and the electrophoretic mobility values increased. Such an overcharging phenomenon is typical in systems containing polyelectrolytes and oppositely charged surfaces36−42 and is driven by entropy gain upon adsorption43 and hydrophobic interactions between the polyelectrolyte chains44 and ion correlation effects.45 Moreover, on the basis of zeta potential measurements for silica surfaces,15 clay platelets20 as well as for titania nanotubes16 and spherical particles,14 similar charge reversal upon protamine adsorption has also been reported. Continued adsorption and increasing electrophoretic mobilities eventually led to the onset of the adsorption saturation plateau (ASP), which was located at 988 mg/g and 359 mg/g for TNS and TiONW, respectively. For TNS, the value is significantly higher than what was reported in the presence of PDADMAC polyelectrolytes of high linear charge density.23 This deviation is most likely due to the lower charge of protamine, which is due to the moderate repulsion between the polyelectrolyte chains on the surface, allowing for adsorption in a more compact conformation. In contrast, a lower loading value of 200 mg/g was reported to form a saturated protamine layer on a montmorillonite platelet.20 Given the fact that those experiments were carried out at lower pH, the possession of higher protamine charge and repulsion between the protamine macromolecules resulted in a lower amount of adsorption on the clay surface. Note, above the ASP, further adsorption of protamine is prohibited, and the excess remains dissolved in the bulk state. To make sure that the surface is saturated with the polyelectrolyte, the electrophoretic mobilities were measured at different time intervals at the onset of the ASPs. It was found that the changes in the values for both TNS and TiONW were within the experimental error after a few minutes of equilibration time. Therefore, the applied protocol that the dispersions were left resting overnight before the electrophoretic measurements indeed allowed enough time for the formation of the saturated protamine layer on the surfaces. If one converts the protamine dose from mg/g to mg/m2 (corresponding to the surface coverage of the nanostructures) units, trends in the mobilities with varying the protamine dose are similar in this alternate representation (Figure 1a). However, the location of the charge neutralization points and the adsorption saturation plateaus occur at significantly lower value when depicted with respect to the surface area. Indeed, the IEP appears to be between 2 and 4 mg/m2, and the onsets of the ASP are located in the range of 8−14 mg/m2 for both TNS and TiONW. Given the accuracy of the electrophoretic measurements, the values in these regimes can be considered as similar within experimental error. Such a collapse of the mobility curves upon conversion of the units can be explained by the fact that TNS possesses a higher outer surface area compared to the TiONW and that thus the adsorbed amount shifts toward lower values. These results clearly indicate that the shape (either 1D or 2D) of the titanium oxide

t → 0(fast)

1 dR h(t ) R h(0) dt

(6)

t→0

where “fast” indicates experimental conditions, under which aggregation is entirely controlled by the diffusion of TNS and TiONW. Such a condition was achieved in 1 M KCl solutions, where the electrostatic repulsion was screened by the salt and particle collisions resulting in the formation of aggregates. In essence, stability ratios close to unity indicate rapidly aggregating systems (i.e., unstable dispersions), while higher values refer to slower aggregation (i.e., more stable dispersions). The hydrodynamic radius of the primary particles (Rh(0)) was found to be 99 and 225 nm for TNS and TiONW, respectively (Table 1).

Table 1. Dispersion Characteristics of the Bare and Protamine-Coated TNS and TiONW

TNS TNS-protamine TiONW TiONWprotamine

Rh (nm)a

PDIa

CCC (mM)b

σ (mC/m2)c

99 98 225 232

0.38 0.38 0.34 0.31

8.5 25.6 7.3 12.4

−8.3 7.9 −8.2 8.0

ASP (mg/m2)d 13.6 11.9

a

Hydrodynamic radius and polydispersity index were measured by DLS in stable dispersions. The standard deviation of the size measurements is about ±5 nm. bThe CCC values were determined from the stability ratio versus ionic strength plots, and their accuracy is 5%. cThe surface charge densities were estimated with the Debye− Hückel model (eq 2) with an average error of 10%. dThe onsets of the adsorption saturation plateaus were obtained experimentally from the electrophoretic measurements performed at 1 mM ionic strength. Microscopy. Transmission electron microscopy (TEM) images were recorded using a Tecnai G2 Sphera device (FEI). The microscope was equipped with a LaB6 cathode and operated with an acceleration voltage of 120 kV. Prior to the imaging of the samples, CF200H-CU-UL carbon hexagonal meshes (Electron Microscopy Sciences) were treated with plasma for 20 s. Thereafter, about 5 μL of colloidal dispersion was drop-cast on the mesh and allowed to adsorb for 2 min followed by the removal of the excess of solution with a filter paper. The coated mesh was then placed on the holder, which was mounted on to the microscope for imaging.

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RESULTS AND DISCUSSION Charging and aggregation properties of bare and protaminefunctionalized TNS and TiONW were assessed in electrophoretic and time-resolved DLS experiments, respectively. In addition, the structure and orientation of nanostructured 1D and 2D particles forming aggregated clusters were determined by TEM. The effect of protamine dose, ionic strength, and dimensionality of the particles on the mechanism of protamine adsorption and colloidal stability was clarified. Colloidal Stability in the Presence of Protamine. The influence of protamine-functionalized TNS and TiONW on the stability of colloidal dispersions was studied at 1 mM ionic strength and pH 9. Under these experimental conditions, protamine is positively charged due to its high pI of about 12,12 while negative surface charges have been reported for titanium oxide nanoparticles due to their lower PZC.23,35 The electrophoretic mobilities were measured first, and they were negative at low protamine doses (Figure 1a). At very low protamine loadings, below 70 mg/g (i.e., 70 mg of protamine per gram of titanium oxide) and 17 mg/g for TNS and C


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Figure 1. Electrophoretic mobilities (a) and stability ratios (b) of TNS and TiONW at different protamine doses in mg/g (left column) and in mg/ m2 (right column) measured at 1 mM KCl concentration and pH 9. The lines are to guide the eyes.

away from this region (i.e., at low and high protamine loadings), the stability ratios were higher or not even measurable indicating stable dispersions. Although the tendency was the same in both mg/g and mg/m 2 representations of the applied loading, the shift in the fast aggregation regime disappeared, and the plots overlapped once the doses were converted from mg/g to mg/m2. This again is due to the higher surface area of TNS. Similar to protamine adsorption and charging behavior, no significant differences between the colloidal stability of TNS and TiONW in the presence of protamine were observed. In general, such a trend of polyelectrolyte concentrationdependent stability ratios correlates well with the results of electrophoretic measurements and can be interpreted in terms of the Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory.28,29,49 This model was developed to predict the colloidal stability of charged particles in electrolyte solutions and states that the overall interparticle force is the sum of the repulsive double layer and attractive van der Waals forces. The strength of the former depends on the electric potential of the particles, whereas the attractive component is always present, and its extent is the same for the same type of materials.28 For the aggregation of TNS and TiONW particles at different protamine doses, one can readily observe that the lack of surface charge around the IEP and vanishing of the double layer repulsion, coupled with the predominance of the attractive van der Waals forces, led to fast aggregation. At low protamine doses, however, the charge of the bare TNS and TiONW is only partially compensated by the adsorbed polyelectrolytes,

nanostructures has no significant influence on the amount of adsorbed protamine and on the corresponding charging behavior. In addition, the very similar adsorption behavior of the polyelectrolyte on both TNS and TiONW shows that the surface density of the oxygen atoms, which can enhance the adsorption of the protamine on the particles, is similar and that hence the facets of the different nanostructures are of similar composition and surface energy. The aggregation studies for TNS and TiONW were conducted under exactly the same experimental conditions (i.e., polyelectrolyte dose, pH, and ionic strength) used in the electrophoretic studies above. As explained in the Experimental Section, the colloidal stability was expressed in terms of the stability ratio. Note that in the case of reaction limited aggregation, the inverse of the stability ratio is equal to the fraction of collisions, which leads to dimer formation. On the other hand, when the aggregation is controlled solely by the diffusion of the particles, all collisions are successful, which leads to a stability ratio of unity. Typical U-shaped stability ratio versus protamine dose curves were obtained with both titanium oxide nanostructures (Figure 1b), which are similar to other dispersions containing polyelectrolytes and oppositely charged colloidal particles.24,35,36,46,47 However, the one measured for TNS appears to be narrower compared to the one for TiONW. For protamine doses around the IEPs, stability ratios close to unity were obtained indicating the instability of the systems due to rapid aggregation of the particles. Such protamine-induced destabilization was also reported for gold particles.48 Further D


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Figure 2. TEM images of TNS (top) and TiONW (bottom) in the presence of protamine below (a), around (b), and above (c) the isoelectric point. The images were taken with the same samples as those used in the DLS measurements.

nanostructures, stable dispersions were obtained below and above the IEP, whereas unstable dispersions and aggregation were observed in the vicinity of the IEP. These results are in good agreement with the colloidal stability studies discussed earlier. Effect of Ionic Strength. The influence of the background electrolyte concentration (adjusted by KCl) on the colloidal stability of the titanium oxide nanostructures at different protamine doses was also studied. Therefore, the data obtained from electrophoretic and time-resolved DLS measurements shown in Figure 1 were repeated at 10 mM KCl concentration. This ionic strength is slightly above the critical coagulation concentration (CCC) measured earlier for both TNS23 and TiONW35 at pH 9 (Table 1). The electrophoretic mobilities were first determined (Figure 3a). At low polyelectrolyte coverage below 0.7 mg/m2 and 0.6 mg/m2 for TNS and TiONW, respectively, negative mobility values were obtained. In this regime, the magnitude of the mobilities was lower for both types of dispersions than the ones for bare particles in 10 mM KCl solutions. This difference is due to the low amount of adsorbed polyelectrolyte on the surface, which neutralizes a small fraction of the surface charge of the titanium oxide nanostructures. By further increasing the protamine coverage, charge neutralization of the particles appeared around 3.9 mg/m2 and 1.8 mg/m2 for the TNS and TiONW, respectively. However, charge reversal was observed at higher protamine doses, and the onsets of the ASPs fell in a similar range for both particles around 20 mg/m2. Although the trend of the protamine dose-dependent mobilities was the same as that at 1 mM ionic strength, an important effect of KCl concentration on the surface charge properties was revealed. Specifically, the onset of the ASP values clearly increased with increasing salt concentration levels. This is attributed to the reduced repulsion between the adsorbed protamine chains at higher ionic strengths. Such a charge screening effect gives rise to a more compact conformation of the macromolecules on the surface and thus

and therefore, the repulsive forces induced by the overlapping electrical double layers overcome the attractive interactions, which led to an electrostatic stabilization of the system. A similar scenario prevails at high protamine doses above the IEP, where the charge of the particles is reversed. Here, a double layer forms around the positively charged protamine-functionalized TNS and TiONW giving rise to the restabilization of the dispersions due to electrostatic repulsion. The same types of interparticle forces were found irrespective of the shape or dimension of the titanium oxide nanostructures. Although the importance of additional forces of non-DLVO origin due to surface charge heterogeneities24,50,51 and interactions between adsorbed polyelectrolyte chains23,52,53 have been reported in other particle−polyelectrolyte systems, evidence of such forces was not found in the current study. To confirm the results of the aggregation measurements, TEM images of both TNS and TiONW were taken at different protamine doses, namely, below, above, and in the vicinity of the IEP. At low protamine doses, stable dispersions were observed in the DLS measurements, and indeed, separated particles could be seen in the images (Figure 2a). Note that the drying process during the sample preparation for the TEM experiments could cause the formation of some particle dimers or trimers. However, the images are significantly different for both TNS and TiONW at the IEP (Figure 2b). Aggregation clearly occurred leading to the formation of clusters of larger sizes, and no individual particles or lower ranked aggregates could be found at these intermediate doses indicating unstable dispersions. Although it was found earlier that the TNS and TiONW nanostructures tend to aggregate in a face-to-face orientation in the presence of polyelectrolytes,23 dendrimers,24 or simple salts,36 this conclusion cannot be unambiguously taken from the present results. Furthermore, stable dispersions were observed above the IEP, as indicated by the presence of individual nanostructures or slightly aggregated particles (Figure 2c). On the basis of the TEM analysis, one can conclude that irrespective of the shape of the titanium oxide E


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colloidal stability with varying KCl concentrations are consistent with the DLVO theory. Clearly, interparticle forces of electrostatic origin are assumed to be operative between the polyelectrolyte-modified nanostructures. Similar observations have been reported in other systems, where charged colloidal particles were dispersed together with oppositely charged polyelectrolytes in aqueous solutions.23,24,35,36 The protamine dose at the onset of the ASP results in the full coverage of the outer surface of the titanium oxide nanostructures. Therefore, comparisons of the hydrodynamic radii and polydispersity index (PDI) of bare and protaminefunctionalized TNS and TiONW (Table 1) showed very similar values (before and after the coating processes). This indicates that aggregation did not occur upon the formation of a saturated protamine layer. Colloidal Stability of Bare and Protamine-Coated TNS and TiONW. The charging behavior and resistance against saltinduced aggregation of the protamine-coated particles, in a wide range of ionic strength, were explored using electrophoretic and DLS measurements. The mobilities of the coated particles are shown in Figure 4a. The corresponding values

Figure 3. Electrophoretic mobility (a) and stability ratio (b) values of TNS and TiONW nanostructures measured at different protamine coverages at pH 9 and 10 mM KCl concentration. The lines are to guide the eyes.

to a higher adsorbed amount. Similar results have also been reported in other particle-oppositely charged polyelectrolyte systems.54 Also compared to the 1 mM KCl case, the IEPs were similar and independent of the ionic strengths and shape of the particles, the latter due to the very similar surface charge densities of the bare TNS and TiONW (Table 1). The increase of the electrolyte concentration, however, induced a more striking change in the aggregation behavior of both titanium oxide nanostructures. From the plot of the stability ratio versus protamine dose (Figure 3b), TNS were found to be unstable below the IEP; this is due to the fact that the salt concentration was higher than the CCC value of bare particles. The stability ratios being close to unity in this regime indicate that the aggregation was controlled solely by the diffusion of TNS. Above the IEP, however, a slight restabilization of the system occurred as indicated by an increase in the stability ratio values. Nevertheless, the colloidal stability of the functionalized TNS is rather limited, and the stability ratio values are lower than 10 in this regime. Note that a stability ratio of 10 means that 10% of particle collisions result in dimer formation.30 In contrast, irrespective of the protamine dose used, no stable dispersions were found at 10 mM ionic strength for the TiONW system. As a consequence, one can presume that the surface charges of the bare or functionalized nanowires are very similar and are screened by the salt at 10 mM ionic strength, which leads to fast particle aggregation. The observed trends in

Figure 4. Electrophoretic mobility (a) and stability ratio (b) of bare and protamine-coated TNS (circle) and TiONW (square) as a function of the ionic strength set by KCl at pH 9..

measured for the bare TNS and TiONW are also presented for comparison. Obviously, the originally negative nanostructures became positively charged after the protamine coating procedure due to the overcharging phenomenon discussed previously. F


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This fact also confirms that the stabilization forces are of electrostatic (DLVO) origin, since the very similar surface charge densities were accompanied by almost the same CCCs, as discussed before. The effect of protamine coating on the colloidal stability of titanium oxide is rather unusual since polyelectrolyte adsorption often leads to the rise of interparticle forces of non-DLVO origin such as attractive patch-charge, bridging interactions, and repulsive steric forces. The present results clearly suggest that protamine forms a flat and homogeneous layer on the TNS and TiONW surfaces and that thus the aggregation mechanism is driven solely by DLVO-type (double layer and van der Waals) forces, which are the major interactions in the case of homogeneous surface charge distribution. In addition, these observations are generic and irrespective of the shape of the nanostructures. On the basis of these results, wherever smooth polyelectrolyte layers are required on negatively charged substrates, protamine can be suggested as a coating agent. For instance, this information is especially important, when polyelectrolyte−particle multilayers are to be built.17,59,60 The protamine layer could therefore provide a smooth base for the stacking of building blocks. In addition, biomimetic mineralization of particle surfaces with protamine may be a promising route to obtain biocompatible delivery systems.

In general, the magnitudes of the electrophoretic mobilities of both bare and coated titanium oxide materials are the same within experimental error. However, a decrease in the magnitude of the mobilities is observed with increasing KCl concentration. Note that values are close to zero at the highest ionic strengths. This observation can be explained by the progressive screening of the surface charges of the particles by the salt constituents and the subsequent thinning of the electrical double layer with increasing KCl concentration. On the basis of these results, one can conclude that the mobility values for both the bare or protamine-coated particles are similar in magnitude for a given ionic strength, again indicating that the shape of the nanostructures does not play a significant role in the charging properties. This finding also suggests that the intercalation of K+ ions into the layered structure of the TiONW is negligible. The stability ratios determined under identical experimental conditions as applied in the mobility studies are presented in Figure 4b. The tendencies are the same for both bare and coated particles. Stable dispersions with high stability ratio values are observed at low ionic strengths, whereas unstable dispersions were found at high salt concentrations. The sharp transition between the stable and unstable regimes occurred at the CCC. Such an aggregation behavior is similar to other colloidal dispersions in electrolytic solutions30,33,55,56 and can be explained in terms of the DLVO theory.28,29,49 Indeed, as the ionic strength increases, more ions are available to screen the surface charges of the particles leading to a decrease in the repulsive strength of the electrical double layer, which vanishes around the CCC. After this point, the attractive van der Waals forces become predominant, which gives rise to diffusion controlled aggregation of the particles. For both bare TNS and TiONW, the CCC was around 8 mM. Protamine coating of the TNS led to a slightly higher CCC value, which indicates that the polyelectrolyte coating enhances the colloidal stability of the particles to a small extent. However, the presence of the protamine layer on the TiONW did not marginally influence the stability of the dispersions. Generally, polyelectrolyte coatings on inorganic particles often lead to an improved colloidal stability in comparison to the bare ones.23,24 However, in the present systems, a significant stabilization effect of the protamine layer could not be concluded. This behavior is most likely due to the flat conformation of the adsorbed protamine on the TNS or TiONW surfaces, which solely gives rise to electrostatic stabilization via overlapping double layers. The improved particle stability upon polyelectrolyte coating is usually due to the joint effect of repulsive electrostatic and steric forces.23,52,53 The latter interaction is especially important once the adsorbed polyelectrolytes form tails and loops on the surface, and hence, their overlap with other adsorbed polyelectrolytes on another particle leads to the rise of osmotic pressure, which induces repulsion between the colloids or nanostructures.57,58 In the current context, the flat structure of the protamine layer on the surfaces does not allow any steric interactions, and therefore, the TNS and TiONW possess only limited colloidal stability after covering them with the polyelectrolyte. The surface charge density of the protamine-coated particles was determined from the ionic strength dependence of the electrophoretic mobilities (eq 2). Data illustrated in Table 1 indicate that protamine-coated TNS and TiONW possess very similar surface charges and that their magnitude is identical to the ones measured for the bare titanium oxide nanostructures.

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CONCLUSIONS

A comparison of the colloidal behavior of titanium oxide nanosheets and nanowires was made in the presence of protamine on the basis of results from electrophoretic, light scattering, and electron microscopy studies. It was found that on both types of nanostructured surfaces, the oppositely charged polyelectrolyte adsorbed giving rise to charge neutralization and overcharging at appropriate protamine loadings. Aggregation studies revealed that the dispersions are stable at low and high protamine doses, while diffusion controlled aggregation resulted in unstable samples around the charge neutralization point. Such charging and aggregation behaviors were generic and irrespective of the shape of the titanium oxide nanostructures. Once the measured data are presented as a function of the protamine dose normalized to the surface area, the stability regimes were quantitatively identical. The major interparticle forces were interpreted in terms of the DLVO theory, i.e, repulsive double layer and attractive van der Waals forces are responsible for the aggregation processes in the presence of protamine of different concentrations. This finding is rather atypical since forces of non-DLVO origin have been reported earlier in several particle−polyelectrolyte systems. It was also observed that protamine adsorbed in a flat conformation on the nanostructures leading to the lack of surface heterogeneities and hence to conditions unfavorable for the emergence of patch-charge or steric interactions between the polyelectrolyte-functionalized TNS or TiONW. For scientists working on the development of particle−polyelectrolyte composites in dispersions or on thin films, these results are especially important since the homogeneous surface produced upon protamine adsorption provides an excellent basis for facile future steps during the preparation of such composite materials. G


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b01815. Powder XRD spectra of the materials and details of surface area calculation (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Endre Horváth: 0000-0001-7562-2267 Istvan Szilagyi: 0000-0001-7289-0979 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Swiss Secretariat for Education, Research and Innovation (C15.0024), Swiss National Science Foundation (150162), and COST Action CM1303. We thank Professor Michal Borkovec for the access to the light scattering instruments in the Laboratory of Colloid and Surface Chemistry. The technical help of Mr. Olivier Vassalli during the time-resolved measurements is gratefully acknowledged.

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REFERENCES

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