Biorelated Polyelectrolyte Coatings Studied by in Situ Attenuated Total

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Biorelated polyelectrolyte coatings studied by in-situ-ATR-FTIR spectroscopy: Deposition concepts, wet-adhesiveness, biomedical applications Martin Müller, Birgit Urban, and Simona Schwarz Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00897 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Biorelated polyelectrolyte coatings studied by in-situ-ATR-FTIR spectroscopy: Deposition concepts, wet-adhesiveness, biomedical applications Martin Müller1,2, Birgit Urban1, Simona Schwarz1 1. Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Str. 6, 01069 Dresden 2. Technische Universität Dresden, FR Chemie und Lebensmittelchemie, 01062 Dresden e-mail: [email protected]

Abstract In this conceptual contribution thin functional coatings consisting of either pure polyelectrolytes (PEL) or complexes between oppositely charged PEL at model and applied substrates are outlined. Latter PEL/PEL complexes were deposited by two concepts. In a first well known concept PEL multilayers (PEM) were consecutively deposited according to the Layer-by-Layer (LbL) technique. In a second less known concept PEL complex (PEC) nanoparticles (NP) preformed by mixing polycation (PC) and polyanion (PA) solutions were deposited in one step. Both concepts based on binary oppositely charged PEL are compared to one based on a single polycation system. Examples shall be given on adhesiveness, nanostructure, biomedical applications of PEM and PEC NP coatings. in-situ attenuated total reflection (ATR) infrared (IR), circular dichroism (CD), scanning force microscopy (SFM) were used for molecular, optical and microscopic characterization. At first, results on the adsorbed amount and wet-adhesiveness of pure (single component) PEL coatings as a function of charge density are given to motivate coatings of mixed oppositely charged PEL. Secondly, the wet-adhesiveness of PEM and PEC NP coatings of identical PEL compounds in aqueous media varying the molar charge ratio (n-/n+) and the deposition step z, respectively, is compared. Comparing the three PEL deposition concepts it is suggested, that the lack or absence of excess charge at the PEL/surface interface is one of the main factors for the wet-adhesiveness of all pure PEL, PEM and PEC NP coatings. Finally, the potential of PEM and PEC NP coatings for biomedical applications is outlined. Concerning biopassivation PEM coatings excessed or terminated by PA repel proteins with low isoelectric points. Concerning bioactivation PEM coatings loaded with antibiotics as well as PEC NP coatings loaded with therapeutic bisphosphonates showed retarded, optionally temperature responsive, drug release for applications in acute surgery and bone healing and immunoglobulin/PEL complex coatings might open theranostic applications.

Introduction Polyelectrolytes (PEL) are a fascinating polymer class, whose behavior in the bulk solution as well as at interfaces is determined in the first order by electrostatic interactions. Both intra- and intermolecular electrostatic self-repulsion on the segment and polymer (macro ion) level cause PEL in diluted regimes to adopt rather stretched conformations and show peculiar manifestations of selfassembly. Whereas, intermolecular electrostatic attraction cause PEL to be bound at oppositely charged surfaces and interfaces as well as to actively bind charged components like counterions of mineral or organic origin, oppositely charged PEL as well as proteins. However, in the second order all these repulsive and attractive electrostatic interactions can be screened or reduced by ionic strength due to dramatic Debye length reduction, by pH dependent proton uptake or release at weak polyacids or polybases, respectively or by complexation with an oppositely charged PEL. In these cases additional short range interaction forces come into play, which can turn general PEL properties like hydrophilic into hydrophobic. Moreover properties of PEL under such screened or reduced charge conditions assimilate again to uncharged hydrophilic polymers with respect to coiled conformation, hydrodynamics and molecular weight dependence of the hydrodynamic radius (RH versus M scaling). In this review PEL systems at interfaces are outlined, where they play a significant role to modify and functionalize substrates of different chemical origin, size and shape just using there inherent electrostatic and chemical properties not applying additional chemical bonding features.

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Numerous examples for industrial applications and developments concerning polyelectrolytes at interfaces prevail like in personal care (shampoo [1, 2, 3, 4], cosmetics [5, 6], contact lenses [7, 8]), laundry [9], textiles [10], paper making [11, 12], water treatment by flocculation [13, 14] and membrane based separation [15, 16, 17] to name only few. In all these PEL surface related applications, there are different concepts, ways, modalitities and options to bring PEL to surfaces in a sustained manner. Herein we like to focus on three concepts (i, ii, iii), which are given in the Fig. 1 and which are partly known from literature, partly reviewed herein and partly introduced under a new aspect herein.

(i)

(ii)

(iii)

Fig. 1. Three concepts of polyelectrolyte deposition at planar model substrate: Single component adsorption (i), consecutive adsorption (ii) and separate (or simultaneous) mixing and casting (drying) (iii). At first PEL layers can be deposited to technical and model substrates just by adsorption from a single component PEL solution (i), which is a classical concept treated numerously experimentally [18, 19] and theoretically in early [e.g. 20, 21, 22, 23] and more recent work [e.g. 24]. Secondly, PEL layers can be deposited at similar substrates by consecutive adsorption of oppositely charged PEL using the well-known Layer-by-Layer (LbL) approach [25, 26] (ii). Thirdly, PEL layers can be deposited at similar substrates mixing oppositely charged PEL in an initial step and casting and drying them afterwards [27] (iii) or by simultaneous mixing of oppositely charged PEL and adsorption in the presence of the substrate [28] (iii). Herein we present, review, summarize and compare examples and modalities of these three main PEL deposition concepts, which are based on findings of our own and of other groups. As main observables wet-adhesiveness (rinse stability), nanostructure and biomedically relevant properties and applications are outlined herein. As typical parameters PEL charge density, PEL composition and molar charge stoichiometry in pure and complexed PEL with partly synthetic but mostly biorelated PEL origin will be varied. Biorelated PEL are focused on, since they are either well available and low cost or are expected to behave beneficial in biomedical applications featured herein due to their structural similarity to compounds in cells and biofluids.

Experimental 1. Materials and Preparation Polyelectrolytes In the Fig. SI 1 (Supporting Informations SI) chemical structures of all used polyelectrolytes (PEL) are given. Mainly, biorelated polysaccharide based PEL were used. Cationic starches were synthesized and provided by the group of Prof. T. Heinze (University of Jena, Institute for Organic Chemistry and Macromolecular Chemistry, Center of Excellence for Polysaccharide Research). The used cationic starch system was based on an unmodified amylose polymer with molecular weight of 460.000 g/mol, which was modified with epoxy-propyl-trimethyl-ammonium chloride (EPTA) in a polymer-analogous reaction. Cationic starches with six different degrees of substitutions DS = 0.28, 0.37, 0.56, 0.62, 0.96 and 1.56 were used. Cationic ethylenediamino cellulose (EDAC, degree of substitution DS = 0.7, MW ≈ 100.000 g/mol (EDA-CELL-T17) was from TITK (Rudolstadt, Germany) and cellulose sulfate (CS, DS = 0.5) was from Euroferm (Erlangen, Germany). Poly(Llysine) (PLL, MW = 200.000 g/mol) was from Sigma-Aldrich (Germany). Typically, 0.01 – 0.001 M PEL solutions were used, whose pH values were adjusted by 0.1M HCl or 0.1M NaOH solutions.


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Substrates As model substrates grinded planar trapezoidal germanium (Ge) or silicon (Si) crystals with dimensions (52 (top) or 48 (bottom) x 20 x 2 mm3) were used, which are analytically applied as internal reflection elements (IRE). The outer surface of both materials Ge and Si plates are formed by thin layers of SiOxHy or GeOxHy, respectively. For both surfaces isoelectrical points of IEP = 2.3 (Si/SiOxHy) and IEP = 3.3 (Ge/GeOxHy) were determined [29]. Polyelectrolyte complexation (mixing) Typically 0.01 M polycation (PC) and 0.01 M polyanion (PA) solutions (related to monomer units) were mixed according to certain defined molar mixing ratios related to effectively charged monomer units and a more or less turbid (milky) PEL complex (PEC) dispersion was formed. Molar number or concentration of effectively charged monomer units were determined by colloid titration using the particle charge detector (PCD) provided by Mütek (Herrsching, Germany). These effective concentrations of charged monomer units (cPELEFF, typically smaller than 0.01M), and not (which are deviating from) the concentrations of monomer units (cPEL = 0.01 M), were the basis for the calculation of the molar mixing ratios ranging from n-/n+ = 0.25 to 4.0. Usually, individual volumes of polycation (VPC) and polyanion (VPA) solutions were calculated, so that the total volume was 2 mL. For PEC dispersions with n-/n+ < 1.0 the PC solution was presented and the PA solution was added, while for those with n-/n+ > 1.0 the PA solution was presented and the PC solution was added. This was done to prevent going beyond n-/n+ = 1.0 upon adding subsequently the respective oppositely charged PEL solution, which otherwise would cause flocculation. Cationic starch deposition Deposition of single component cationic starches of different substitution degrees (see above) was achieved in two ways. At first adsorption from cationic starch solutions with given DS and c = 0.01 M at Ge substrates for 1 hour, followed by rinsing with deionized water, was applied. The relative adsorbed amount of cationic starches with different DS was determined by FTIR spectroscopy (see below) based on the intensity (integral) of the diagnostic saccharide band around 1050 cm-1 due to ether (ν(C-O-C)) and hydroxyl (ν(C-OH)) linkages in polysaccharides. Direct comparison of the saccharide band intensities of cationic starch samples with different DS (correlated to adsorbed amount) was not straight forward, since higher DS result in lower intensities of the saccharide band. Thus a correction factor F was used, which was determined from ATR-FTIR spectra on dry casted films of all six starch samples from 0.01M solutions, where equally deposited amount was carefully taken into account. Thereby, the band at 1050 cm-1, diagnostic for saccharide moieties and the band at 1480 cm-1, attributed to the 2-hydroxy-propyl-trimethyl-ammounim-chloride modification, was ratioed according to F = A1050/A1480. This ratio was different for all six cationic starch samples but scaled not linear with DS. Therefore, the A1050 value (linear to adsorbed amount) of cationic starch with highest DS = 1.56 was arbitrarily corrected by a factor of F = 1.00 and the respective A1050 values of other starch samples were corrected by respectively calculated factors in the range F = 1.00 - 1.57. Secondly, cationic starch solutions for the six different DS values, respectively, were casted and dried onto Ge substrates (coating area A ≈ 4. 2 cm2) at T = 50 °C using a heating stage. An initial FTIR spectrum of the dry starch film was taken (see Fig. 3a). Thereafter, the starch coated Ge substrate was rinsed (desorption) and another FTIR spectrum was recorded (see Fig. 3a). A1050 values of intitial and after rinsing and drying were ratioed according to Q = AAFTER/ABEFORE x 100%. PEM deposition PEM deposition was followed by in-situ-ATR-FTIR spectroscopy as it was initially reported earlier [39]. Briefly trapezoidal Ge substrates (50 x 20 x 2 mm3) were housed on the front and backside by two sealing liquid compartments, respectively, allowing the consecutive injection or cycling of aqueous PEL solutions and pure water or buffer over the front side of the Ge model substrate (coating area A ≈ 4.2 cm2) within the flow cell. Typically for PEM of EDAC/CS 2 mL of a freshly prepared 0.01M EDAC solution at pH = 4.0 was injected into the flow cell and after 10 min


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redrawn, 2 mL of fresh pure water was three times injected and after 1 min redrawn and 2 mL of freshly prepared 0.01M CS solutions at pH = 4.0 was injected and redrawn after 10 min. Always fresh PEL solutions and fresh pure water were used in the respective steps. PEM deposition was monitored by in-situ-ATR-FTIR spectroscopy after each adsorption step z = 1 - 11 (PEM-z) recording ATR-FTIR spectra of the actual PEM coating in contact to the actual PEL solution and after rinsing. Dried PEM samples (PEM-11) showed thin homogeneous blueish coatings. Alternatively, Ge substrates were alternately dipped into beakers (50 ml) filled with PC solution (cPC = 0.01 M), millipore water, PA solution (cPA = 0.01 M) and millipore water using a dipping robot (Riegler-Kirstein, Berlin, Germany) and applying defined numbers of dipping cycles (PC/H2O/PA/H2O) at T = 25 °C. Dry PEM samples showed thin homogeneous blueish coatings within the total coating area A ≈ 10 cm2 on both sides of the Ge substrate and were characterized by transmission (TRANS) FTIR spectroscopy (see below). PEC deposition 50 microliters of freshly prepared PEC dispersions (see above) with defined molar mixing ratios n/n+ were solution casted and dried at T = 50 °C onto Ge model substrates (coating area A ≈ 4.2 cm2) using a heating stage. Optionally, the formed PEC films were rinsed by given aqueous media and again dried. PEC film samples were characterized by TRANS-FTIR spectroscopy (see below). Simultaneous mixing of oppositely charged PEL Either at first 10 mL of a 0.001M EDAC solution was continuously pumped through a reservoir over the Ge substrate surface (Fig. 2) and secondly 1 mL of 0.01 M CS solution was subsequently dosed into this circulating 0.001M EDAC solution (reservoir) in ten 100 uL portions or vice versa at first 10 mL of 0.001M CS solution was presented and 1 mL of 0.01M EDAC solution was dosed in ten 100 uL portions. The adsorbed amount was determined by in-situ-ATR-TIR spectroscopy (see below). 2. Analytical techniques Analytical techniques used herein are briefly described. More detailed descriptions of the used analytical techniques and protocols can be found in the related original literature. Ex-situ-Transmission (TRANS)-Fourier Transform Infrared (FTIR) spectroscopy was applied using Tensor II (BRUKER-Optics GmbH, Ettlingen, Germany) equipped with globar source and DTGS (Ditriglycinsulfat) recording FTIR spectra at 2 cm-1 spectral resolution. As standard substrates trapezoidal Ge plates (50 x 20 x 2 mm3), see above under substrates) housed in special sample holders (M. Ulrich, M.M. IPF Dresden) were used, which were coated by PEL or PEC as described above. Dry PEL or PEC coatings at Ge substrate were transmitted by the IR beam at the focus within the FTIR spectrometer. From single channel intensity spectra of polymer coated Ge plates (IS) and of uncoated Ge plates (IR) absorbance spectra were computed according to A = log IS/IR. For wet adhesion testing of PEL and PEC coatings at Ge IRE absorbance spectra for the initial dry state and the dry state after rinsing by water or buffer were recorded. In-situ-attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy was applied using Tensor II (BRUKER-Optics GmbH, Ettlingen, Germany) equipped with globar source and MCT (mercurium-cadmium-telluride) detector recording FTIR spectra at 2 cm-1 spectral resolution. As standard substrates trapezoidal Ge internal reflection elements (IRE) (50 x 20 x 2 mm3, 45° incident and leaving angle) housed in flow cells with liquid compartment (M. Ulrich, M.M. IPF Dresden) were used and applied either on precoated by PEL or PEC as described above or uncoated (adsorption measurements). A scheme of the in-situ-ATR-FTIR cell and the flow system driven by peristaltic pump (ISMATEC, München, Germany) is given in the Fig. 2. Coatings at Ge substrate were probed by evanescent IR beam at every reflection area out of 11 reflections. Similarly to TRANS-FTIR from single channel intensity spectra of polymer coated or adsorbed Ge IRE (IS) and of uncoated Ge IRE (IR) absorbance spectra were computed according to A = log(IS/IR).


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Fig. 2. Scheme of in-situ-ATR-FTIR cell housing trapezoidal internal reflection elements (IRE) based on silicon or germanium and flow system with stirrable reservoir driven by peristaltic pump for molecular characterization of PEL, PEM and PEC deposition. Scanning force microscopy (SFM) was applied using Nanostation II of Bruker Nano GmbH (Berlin, Germany). Tips (silicon) from Nanosensors (Darmstadt, Germany) with around 10 nm radii were used. The non-contact mode (topography, error and phase mode) was used to characterize PEL and PEC coatings under ambient conditions and scanning parameters (velocity, delay time, integration) were optimized by minimizing the amplitudes in the error mode image. Raw SFM data were further processed by either SISCANPro (BRUKER Nano GmbH, Karlsruhe, Germany) or SPIP software (Image Metrology, Horsholm, Denmark), respectively. Spectroscopic reflectometry was applied to determine thicknesses of dry polyelectrolyte coatings at Ge substrates using MProbeVis-MSP Thin Film Measurement system of Semiconsoft Inc. (Southborough, U.S.A.). As main components light source of tungsten-halogen (5 W), fibre optics probe and F4 Vis spectrometer with Si detector in the wavelength range 400-1100 nm were used.

Results and Discussion In the following three deposition concepts of polyelectrolytes (PEL) are described, which shall lead to wet-adhesive functional coatings at model and technical substrate materials close to applications in the life science and biomedical field. The first concept is based on the deposition of pure cationically modified polysaccharides with low charge density, the second on the consecutive deposition of oppositely charged polysaccharides using Layer-by-Layer (LbL) approach and the third on the deposition of preformed complexes of oppositely charged polysaccharides. As model substrate planar trapezoidal plates of either Ge or Si with thin GeOxHx and SiOxHy layers, respectively were used. Former zeta-potential analysis of those substrates revealed acidic surfaces with isoelectric points IEP = 3.3 and IEP = 2.3 for GeOxHx and SiOxHy, respectively [29]. Hence for values of pH > 4.0 predominantly used in the aqueous media of works of this report, a negative surface net charge prevailed. 1. Deposition of cationic starch For the deposition concept of cationically modified polysaccharides with low charge density a series of cationic starches with various degrees of cationic substitution and thus linear charge density was used. The effective number of cationic charges per saccharide unit was determined by colloid titration. Two deposition modes were applied, which were adsorption from cationic starch solution (1.1) as well as casting and drying a cationic starch solution at Ge model substrates followed by water rinsing (1.2), respectively.


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1.1. Cationic starch adsorption from solution In the Fig. 3a ATR-FTIR spectra on cationic starches (amylose) with varying degree of substitutions DS = 0.28 – 1.56, which have been adsorbed from 0.01M cationic starch solutions (pH = 7.3) at the negatively charged Ge model substrate and after one hour subsequently rinsed by pure water, are shown. a) DS = 1.56 DS = 0.96 DS = 0.62 DS = 0.56

integral A1050 / [cm-1]

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DS = 0.37 DS = 0.28

0.4 0.3 0.2 0.1 0.0 0

0.5

1

1.5

2

Degree of substitution DS b)

c) Fig. 3. Adsorption of cationic starch from solution at the Ge model substrate; a) in-situ ATR-FTIR spectra on adsorbed cationic starch from 0.01 M cationic starch solutions with degree of substitutions DS = 0.28, 0.37, 0.56, 0.62, 0.96 and 1.56. The spectra were recorded after adsorption time of 1 h and rinsing by pure water; b) Integral A of the diagnostic band around 1050 cm-1 as a measure for adsorbed cationic starch amount as a function of DS. The data points were fitted by an empirical reciprocal function A = B/DS (simplified eq. 1); c) Scheme on the effect of polycation charge density qm (scaling with degree of substitution DS) (left: high qm; right: low qm ) on the adsorbed amount at the oppositely charged surface. The molecular weight of the unmodified starch polymer was 460.000 g/mol. The bottom series of Fig. 3a represents in-situ ATR-FTIR spectra of adsorbed cationic starch with DS = 1.56, the next with DS = 0.96 up to top series with DS = 0.28. The band at the wavenumber position of around 1050 cm-1 is diagnostic for saccharide groups and its intensity scales with the adsorbed starch amount, when the thickness does not exceed some 300 nm [30]. Significantly, for lowest DS = 0.28 the overall adsorbed starch amount is higher compared to highest DS = 1.56, which suggests that the lower charge density caused a higher adsorbed starch amount. To check this further quantitatively, the integral values of the band at 1050 cm-1 (A1050), which is linear to the adsorbed amount, were


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plotted versus DS in the Fig. 3b. It has to be noted, that the applied starch concentration of c = 0.01 M (related to starch repeating units) was within the plateau region of adsorption isotherms (i.e. adsorbed amount versus c), which are not shown herein but for one exemplary series (DS = 0.62) in the Fig. SI 2 of the Supporting Informations (SI). In the Fig. 3b a reciprocal dependence between adsorbed amount (linear to A1050) and charge density (linear to DS) of starch could be obtained, which was already found by Fleer [31] for another cationic PEL system. The empirical relationship presented in this reference is given in a modified form in the following equation (1) in accordance to [20]: Γ= -K σ0 l2 / qm

(1)

Γ: surface concentration (i.e. adsorbed amount), Κ: constant, σ0: surface charge density, l: segment length, qm: PEL charge density Explanations for this experimental finding and empirical descriptions have been given [31, 20]. We like to emphasize self-repulsion of PEL on an intra- and intermolecular level as explanation motif, where on the one hand like charged segments under the applied salt free conditions should cause rather expanded and stretched polymer conformations leading to rather thin layers. On the other hand self-repulsion between already adsorbed cationic starch layers and further incoming cationic starch molecules makes the adsorption process self-limiting. Therefore highly charged starches form thinner layers of expanded starches and lower charged ones thicker layers of rather coiled starches based on both crucial factors conformation and sorption penalty. 1.2. Cationic starch desorption from dried films Related to these adsorption experiments further studies were performed, where cationic starches were brought onto Ge substrates by casting a defined volume of 0.01 M cationic starch solution and drying. Thereafter the dry cationic starch films were rinsed with pure water. FTIR spectra of the cationic starch layers were recorded for the initial dry state before and after rinsing with pure water, respectively, which are shown in the Fig. 4a for DS = 0.28 (bottom) and DS = 1.56 (top). a)

b)

Coverage / [%]

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50 DS = 0.28 DS = 1.56 FIT FIT

40 30 20 10 0 0

5

10

15 20 Time / [h]

Fig. 4. Desorption from dried films of cationic starches at the Ge model substrate; (a) in-situ ATRFTIR spectra on dried films from cationic starch solutions (0.01 M) for DS = 0.28 (bottom) and DS = 1.56 (top) before (black) and after (blue) rinsing for 1h with pure water. (b) Ratio Q of diagnostic IR band recorded after and before pure water rinsing of cationic starch films (DS = 0.28, 1.56) given in Fig. 4a versus rinsing time. The empirical equation Q = 100 ⋅ exp(-a ⋅ t) + b (in percentage) with empirical parameters a and b was used to fit the data assuming an exponential desorption process.


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Intensity ratios (Q) of the IR bands either at 1050 cm-1 (saccharide band) or 1480 cm-1, which can be assigned to the methyl groups of the modifying 2-hydroxy-propyl-trimethyl-ammonium group at the respective starch polymer, present after and before rinsing were calculated according to Q = AAFTER/ABEFORE x 100%. These values are empirical measures of sustained bound amount or adhesiveness, and plotted versus time for DS = 0.28 (black) and DS = 1.56 (red) in the Fig. 4b. Both dried cationic starch films (DS = 0.28 and 1.56) decrease in their bound amount upon water rinsing. However, significantly there is a larger relative residual amount of cationic starch of Q ≈ 10 % for DS = 0.28 compared to Q ≈ 2 % for DS = 1.56. Hence cationic starches with lower charge density better keep or sustain on the surface compared to higher charge densities. In the Fig. SI 2b of the Supporting Informations (SI) additional desorption data on cationic starch films deposited from lower concentrated 0.002 M solutions are given, which formed thinner starch layers. There the found desorption trend is even more pronounced than for the thicker films deposited from 0.01 M solution, since in thinner starch films less loosely bound starch amount prevails, so that upon rinsing the tight surface bound amount is higher relative to the total deposited amount. In contrast for thicker films deposited at 0.01 M starch solution the tight surface bound is smaller relative to the total deposited amount. This significant dependence of desorbability of cationic starch films on DS is in line with the adsorption results given in Fig. 3, where cationic starch with lower charge density revealed higher adsorbed amounts compared to cationic starches with higher charge density. 2. Consecutive deposition of oppositely charged PEL The second concept, which shall be described herein in the context of PEL deposition, is the wellknown and established LbL technique, where oppositely charged PEL are consecutively adsorbed from their solutions at various substrate materials resulting in PEL multilayers (PEM), followed by water rinsing, respectively [26, 32]. It is widely accepted, that formation of PEM assemblies is related to polyelectrolyte complexation and PEM get on the one hand their internal stability from electrostatic interaction between their polycation and polyanion components. However, on the other hand the driving force of the PEL complexation process is claimed to be entropically governed by counterion release, whenever a new like charged PEL with respect to counterion is approaching the outermost PEL layer of the PEM [33, 34]. In certain cases also weakly bound PEL within the PEM may diffuse in direction to the outermost PEM layer in order to contribute to compensate the charge of the newly bound PEL. In that case there is no linear relation between adsorbed PEM amount (deposition) and the adsorption step (z) [35, 36], since the first derivative (incremental change) of the adsorbed amount becomes dependent on z. 2.1. PEM deposition In the Fig. 5a in-situ ATR-FTIR spectra on the consecutive deposition of the two oppositely charged polysaccharides EDAC and CS at the Ge model substrate are given, where z = 12 deposition steps were applied. For z = 12 a thickness d ≈ 45 nm was reached evidenced by SFM cut depth and optical reflectometry at Ge/PEM, which is well below 200 nm and offers linear sensing of the deposition process by ATR-FTIR spectroscopy as it is critically outlined therein [30]. Generally, a subsequent increase of IR band intensities was obtained. In detail the IR band at around 1655 cm-1 can be assigned to the δ(NH3+) band of the EDAC component, the IR band (doublet) at around 1235 cm-1 to the ν(O=S=O) vibration of the CS component, while the composed band at around 1065 cm-1 to the ν(C-O) band of C-O-C and C-O-H groups of both polysaccharides CS and EDAC. Since quantitatively the intensities of these bands are related to their concentrations, plots of those versus deposition step z = 1 to 12, shown in the Fig. 5b, give insight to the individual polysaccharide deposition. First of all, the overall courses of intensities A of CS and EDAC related bands versus z are increasing due to LbL deposition following a weak exponential dependence according to A = A0 exp (a z)

(2)

The fitted curve (broken black line) of the composed EDAC/CS band (1065 cm-1) is given in the Fig. 5b and values of the empirical parameters were amplitude A0 = 0.117 + 0.015 cm-1 and growth


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2

5 0

1.5

-5 1655 1235 1065 FIT 3340 FIT

1

0.5

-10 -15

IR band integral / [cm-1]

factor a = 0.235 + 0.013. Equation was already given in [37] and the parameter a may be used to quantify the steepness or effectiveness of an exponential PEM growth. IR band integral / [cm-1]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-20 -25

0

-30 0

a)

2

4 6 8 10 Adsorption step z

12

b)

A B

c) d) Fig. 5. Consecutive adsorption of oppositely charged ethylenediamino-cellulose (EDAC) and cellulose sulfate (CS) according to the LbL concept at the Ge model substrate resulting in PEM of EDAC/CS; a) in-situ ATR-FTIR spectra of PEM-1 to PEM-12 (black) in contact to adsorbing EDAC or CS solution and spectral difference between PEM-11 and PEM-12 in contact to EDAC or CS solution, respectively, and after rinsing with pure water; b) Plot of the integrals (taken from Fig. 5a) of IR bands at 3340 (ν(OH)), 1655 (δ(OH)), 1235 (ν(SO2)) and 1065 cm-1 (ν(C-O)) diagnostic for water, EDAC and CS versus layer number z; c) SFM images (scale bar: 1 µm) of PEM-12 (left) and PEM-32 (right) of EDAC/CS obtained by LbL adsorption from 0.01M EDAC and CS solutions at pH = 4. d) Scheme of consecutive uptake (A) or pull-out (B) of oppositely charged PEL at the actual PEM/PEL solution interface. Furthermore significantly, the course of the CS component (1235 cm-1, black triangles) shows zig/zag-like behaviour, so that for the diagnostic CS band every odd step z = 1, 3, 5, 7, 9 the intensities are lower compared to the preceeding and succeeding steps z + 1 or z -1. Likewise for the diagnostic EDAC band (1655 cm-1, black cubes) most pronounced for the even steps at z = 6, 8, 10 the intensities are lower compared to preceeding and succeeding steps. This can be interpreted, that in every deposition step the actual polysaccharide (eg. EDAC) solution is able to partly pull out the oppositely charged polysaccharide (e.g. CS) from the outermost layer of the actual PEM and partly adsorb at the actual PEM, as it is shown in the scheme of Fig. 5d. The pull out can be explained by the huge volume concentration excess of the dissolved PEL in solution compared to that of the oppositely charged bound PEL in the outermost layer of the PEM. Under these conditions a part loss of the bound PEL is possible. Parallel to the loss of the oppositely charged PEL from the actual PEM under formation of a dispersed PEL complex there is uptake of the dissolved PEL at the outermost oppositely charged zone of the actual PEM. However, it can not be fully resolved, if the intensity loss of an individual PEL band, when oppositely charged PEL


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solution is added, could be also due to the migration of PEL from the interior (solid/substrate/PEM interface) in direction to the exterior zone of PEM (PEM/solution interface). Nevertheless, there is either loss or accumulation of PEL towards outermost PEM zone, when the oppositely charged PEL solution is present and in contact to an actual PEM assembly, which is in line with an earlier concise report of Kovacevic [38]. Moreover, in Fig. 5a negative IR bands of the ν(OH) due to water band can be observed, which we featured also in earlier references [39, 40]. As it was explained, the negative ν(OH) absorbance band is due to the spectral incompensation of Ge/GeOxHy/PEM in contact to water, where water is more distant from substrate and ν(OH) intensity is smaller compared to bare Ge/GeOxHy, where water is in direct contact to substrate, and ν(OH) intensity is larger. Plotting these negative difference intensities of ν(OH) versus the adsorption step z, decreasing courses were obtained, which are due to the increasing distance of the water front from the GeOxHy substrate caused by PEM formation. This course is approximately mirror-like to that of the band at 1065 cm-1 diagnostic to both EDAC and CS amount, which will be treated more quantitatively in a forthcoming paper. Fitting the course of ν(OH) by function (3), A = -A´0 exp (a´ z)

(3)

which is function (2) multiplied by -1, empirical parameters of A´0 = 1.099 + 0.244 cm-1 and a´= 0.261 + 0.021 were found. Interestingly, the value of this growth factor a´ is similar to a = 0.235 for the composed CS/EDAC course within the given error range, which confirms, that PEM deposition is correlated with expulsion of water from the interface. Additional evidence for the discussed erosive structure of PEM by mutual pull-out of bound PEL was obtained by SFM images on PEM-12 and PEM-32 of EDAC/CS given in the Fig. 5c. There granular structures of partly merged particles sizing around 100 nm can be obtained. Roughness values of RRMS = 6.94 nm for PEM-12 and R = 7.50 nm for PEM-32 manifest eroded structures, which are increasing with increasing consecutive PEL adsorption cycles. Interestingly, similar type of granular structures can be found in deposited preformed PEC NP of EDAC/CS shown in the Fig. 11c, which is commented on below in the respective section 3.2.2. 2.2. PEM rinse stability (wet-adhesiveness) To check for rinse stability in the Fig. 5a (blue difference spectra) spectral differences between PEM-11 and PEM-12 in contact to respective EDAC and CS solution and PEM-11 and PEM-12 in contact to pure water (rinsing) are given. No significant absorbance can be obtained and the ratios of respective band integrals from spectra of PEM before and after rinsing were 100 + 0.5 %, from which we conclude complete rinse stability and wet-adhesiveness of the EDAC/CS PEM system under water. As an explanation we consider, that charged segments in PEM assemblies close to the innermost (solid) substrate/PEM interface are globally stoichiometrically compensated, which results in a less hydrophilic phase and only low or no rinsability. Locally, at the outermost PEM/solution (liquid) interface charged segments might be not stoichiometrically compensated, but their (few) ion pairs with the underlying oppositely charged segments prevent them from rinsing out. Only incoming oppositely charged PEL are able to either pull out or bind at the outermost PEL segments as it is stated above. Hence it is the global charge compensation or the occurrence of only little excess charge amounts in PEM (with respect to all compensated charges), which cause wetadhesiveness of mixed PEL systems. This result is considerably in line with those for cationic starch deposition at Ge/GeOxHy substrates (section 1.), where it could be shown, that starches with lowest cationicity were adsorbed most or were desorbed (rinsed out) least. It will be also compared to that for films of PEL complexes, which are preformed by direct mixing of EDAC and CS solutions and thereafter casted and dried at Ge model substrates below (Section 3.). 2.3. Simultaneous mixing of PEL in the presence of substrate Closely related to the LbL concept of Decher [25, 26] there is another concept for an effective surface modification, which is also based on oppositely charged PEL. Likewise the LbL concept, polycation and polyanion solutions are contacted sequentially to a given substrate, but deviating from this the rinsing steps are omitted and only two and not many (“infinite”) processing steps are


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Langmuir

used. Concerning to this in-situ-ATR-FTIR spectra of adsorbed PEL material at Ge substrate are given in the Fig. 6.

1. CS / 2. EDAC

1. EDAC / 2. CS

Fig. 6. Bottom series (red): in-situ-ATR-FTIR spectra on adsorbed layers obtained by pumping 10 mL of 0.001M EDAC solution over Ge substrate (bottom), after addition of 1 mL 0.01M CS solution (middle) and rinsing in pure water (top). Top series (blue): in-situ-ATR-FTIR spectra on adsorbed layers obtained by pumping 10 mL of 0.001M EDAC solution over Ge substrate (bottom), after addition of 1 mL 0.01M CS solution (middle) and rinsing in pure water (top).

Fig. 7. Synergistic effect on cationic surface charge at silica ballotini upon mixing PDADMAC and PMA-X solutions (0.001M). Reproduced with permission from Ref. [28]. Copyright [1993, Elsevier].

In the lower red panel the bottom ATR-FTIR spectrum corresponds to an 0.001M EDAC solution (10 mL), which is pumped over the Ge substrate (see Fig. 1), while for the spectrum in the middle 0.01 M CS solution was given to 0.001M EDAC solution, so that still n-/n+ < 1, and third spectrum is due to rinsing. Evidently, no adsorbed EDAC amount was observed before mixing with CS solution, while after mixing with CS solution an EDA/CS complex film is deposited, which is wetadhesive. In the upper (blue) panel of Fig. 6 in-situ-ATR-FTIR spectra are given, where at first 0.001M CS solution (10 mL) was pumped over the Ge substrate (first blue spectrum), while the spectrum in the middle corresponds to addition of 0.01M EDAC solution, so that n-/n+ > 1 and third spectrum to rinsing with pure water. Similar to above almost no CS was adsorbed upon single CS addition (the very small signal is due to very low amounts of CS bound by nonelectrostatic interaction forces like hydrogen bonding), while significant adsorbed amounts were registered after mixing of EDAC solution in the presence of Ge substrate being still wet-adhesive after rinsing with pure water. This result can be seen related to findings on suspensions of glass beads or silica substrate particles similarly modified by the two oppositely charged synthetic PEL PDADMAC and copolymer of maleic acid using a similar “in-situ-mixing” concept in the presence of substrate [28]. Therein in a first step a polycation solution (0.001M) was dosed into the particle suspension (S) and at a certain amount a charge reversal from negative to positive was registered by zeta-Potential, which would not alter, if polycation excess was separated (e.g. by centrifugation). In a second step a defined volume of PA solution (0.001M), whose PA amount did not exceed the PC amount, was dosed into the particle suspension with the excess of unbound PC. Surprisingly, the positive zeta-potential of this ternary (substrate/PC/PA) suspension increased significantly with increasing PA amount and at a certain point reached a remarkable high maximum of positive zeta-potential, as it is shown in the Fig. 7. Obviously and seemingly counterintuitive, dosing negative polymer bound charges into the


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suspension containing cationically modified substrate particles and excessive of positive polymer bound charges results in an increase of positive surface charge at the substrate particles. As an explanation of such an “synergistic effect” it was suggested, that in the first step (S + PC) suspended particles (S) are irreversibly modified by a polycation shell and display a repulsive electrostatic barrier for further incoming excessive PC molecules (self-limiting). However, this PC shell is not homogeneous due to the self-repulsion of like charged PC. In the second step the excessive PC is subsequently complexed by the PA molecules and nonstoichiometric PEC particles with still cationic excess charge (PEC (+)) are formed. This cationic polymer bound excess charge is far lower compared to the cationic polymer bound charge of the uncomplexed PC and encounter a lower electrostatic barrier from the polycation shell at S. Therefore, additionally to the already bound smaller pure PC molecules larger PEC(+) particles are bound at free sites of the inhomogeneously modified S. Presumably free larger PEC(+) are also able to replace bound smaller PC molecules by entropic effects. Obviously, in the end the surface charge after PEC(+) modification is higher compared to after PC modification, although PC carry more charges compared to PEC(+). This situation is considerably related to that of cationic starch adsorption given in Section 1, where with decreasing charge density of cationic starches increasing adsorbed amount was obtained. The explanation was also, that self-repulsion of low charged cationic starch was lower compared to high charged cationic starch and therefore repulsive electrostatic barrier (PC + S-PC) could be overcome. Additionally, for both cases lower electrostatic barriers also allow additional attractive short range non-electrostatic forces (Van der Waals, hydrogen bonding). 3. Deposition of preformed PEC particles The third PEL deposition concept is based on either preforming polyelectrolyte complex (PEC) particles by directly mixing polycation and polyanion solution and then adding (adsorbing, casting) the resulted milky turbid dispersion to a substrate. Preforming (non-stoichiometric) colloidal PEC (“coacervate”) particles is a classical procedure initiated by the work of Kabanov and Zezin [41],Tsuchida [42], Dubin [43], Dautzenberg [44] and others [45]. The driving force of the PEC coacervation process is again claimed to be the increased entropy, when upon ion pairing of cationic and anionic groups bound (“condensed”) counterions are released (“evaporated”). Obviously, no or only low enthalpic contribution of the ion pairing process could be measured up to now [34, 46]. Furthermore, the obvious colloidal stability of the turbid PEC dispersions are provided by so called electrosteric contributions meaning that dangling like charged chains of the excess component in the periphery of PEC particles repel each other and thus PEC particles do not aggregate in their final state with hydrodynamic radii in the range of RH = 100 - 500 nm. However, it is claimed that PEC particles do aggregate in their very initial state, when upon ion pairing of few oppositely carged PEL so called primary PEC particles are formed, which then undergo controlled aggregation or growth process until the quasistable final state of secondary PEC particles is reached. The driving force for this controlled initial aggregation of primary PEC particles are attractive short range nonelectrostatic interactions, which are in the end opposed by repulsive electrosteric interactions (see above) [47, 48]. 3.1. Adsorption of PEC particles from dispersion In earlier work from our group preformed PEC particles were adsorbed from their freshly and separately prepared dispersions at model substrates [49, 50]. To monitor the PEC particle adsorption as a function of time or concentration in-situ-ATR-FTIR spectroscopy, which is sensitive for changes in the molecular composition at the solid/liquid interface of Si internal reflection elements, was used. 3.1.1. Adsorption kinetics and isotherm Typical ATR-FTIR spectra on the adsorption of PEC-0.66 particles consisting of a combination of the synthetic oppositely charged PELs poly(diallyldimethylammonium chloride) and poly(maleic acid-co-methylstyrene) (PDADMAC/PMA-MS) at an anionically modified silicon substrate, are given in the Fig. 8a [49]. PEC-0.66 particles obtained without and with centrifugation were used.


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Langmuir

Integrals A of diagnostic IR bands of PDADMAC or PMA-MS were determined and the corresponding adsorption kinetics of is given in the Fig. 8b. Moreover, an adsorption isotherm of PEC particles was recorded by in-situ-ATR-FTIR spectroscopy, which is given in the Fig. 8c and fitted by a Langmuir-type of function, where A and AMAX is the actual and maximum absorbance, respectively, cPEC is the PEC concentration, and K = k2/k1 equals the ratio of kinetic constants of back and forth reaction. Α/AMAX = cPEC/(cPEC + K) (4) Obviously, for this PEC system an adsorption isotherm with rounded shape was obtained, which is indicative for a low affinity adsorption isotherm. It was comparable to the adsorption isotherm of a standard carboxylated polystyrene (PS) latex system from the respective PS dispersion at the same anionically modified Si surface in the same concentration range (Fig. 8c) [49]. a)

b)

c)

Fig. 8. Adsorption of PEC NP (PDADMAC/PMA-M S) from dispersion at the anionically modified Si model substrate; a) ATR-FTIR spectra on the adsorption of uncentrifuged (open symbols) and centrifuged PEC NP; b) adsorption kinetics of PEC NP; c) adsorption isotherm of centrifuged PEC NP and carboxylated latex NP. Reproduced with permission from Ref. [49]. Copyright [2004, Elsevier]. 3.1.2. Rinse stability of adsorbed PEC particles (wet-adhesiveness) After rinsing adsorbed PEC particle layers with pure water the adsorbed amount did not vary significantly (data not shown). 3.1.3. Nanostructure SEM images of PEC particles of PDADMAC/PMAMS adsorbed from 0.002M dispersions after rinsing and drying are given in the Fig. 9a [49] and SFM images of refined (centrifuged) PEC particles consisting of PDADMAC and poly(styrenesulfonate sodium) (PDADMAC/PSS) spin coated from 0.002M dispersions, without rinsing after drying are given in the Fig. 9b [51]. Individually adsorbed PEC particles with relatively broadly distributed diameters of around 0.2 – 1 µm, which are larger compared to diameters of 0.1 – 0.2 µm [49, 50, 52] in the dispersed state obtained by dynamic light scattering, were obtained for this system. Obviously, adsorbed PEC particles have a tendency of merging and coalescing at this surface. Moreover PEC particles of PDADMAC/PSS spin coated from diluted dispersions onto Si substrate showed smaller diameters and more narrow size distributions, when the molecular weight was decreased and the concentration was reduced [51].


Langmuir

a) b) Fig. 9. Microscopic images of deposited PEC NP; a) SEM image of adsorbed PEC-0.66 particles (PDADMAC/PMA-MS) at the anionically modified Si/SiOxHy substrate (Reproduced with permission from Ref. [4949]. Copyright [2004, Elsevier]); b) SFM topography image of refined (centrifuged) PEC-0.66 particles of PDADMAC/PSS spin coated from 0.002M dispersions on silicon wafers. (Reproduced with permission from Ref. [Error! Bookmark not defined.Error! Bookmark not defined.]. Copyright [2006, Wiley-VCH]). 3.2. Casting and drying of PEC nanoparticles Another concept for PEC nanoparticle (NP) deposition is based on two steps, where in a first step polycation and polyanion solutions are mixed forming PEC NP and in a second step the formed colloidal PEC NP dispersion is casted onto the given substrate and dried. In the following results for the biorelated oppositely charged polysaccharide/polysaccharide system EDAC/CS and the oppositely charged polysaccharide/polypeptide system PLL/CS are presented. 3.2.1. PEC colloidal properties In the Fig. 10a the hydrodynamic radius (RH) of typical EDAC/CS particles with n-/n+ = 1.1 (EDAC/CS-1.1) is plotted versus aging time. RH / [nm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

200 EDAC/CS 0.5_1.1 pH 7 150 100 50 0 0

5

10 15 Time / [days]

b) a) Fig. 10. Casting and drying PEC NP at the GeOxHy model substrate; a) Hydrodynamic radius RH of EDAC/CS-1.1 particles as a function of aging time; b) ATR-FTIR spectra of casted and dried films of pure EDAC (bottom), pure CS (middle) and PEC (n-/n+ = 1.1) of EDAC/CS (top) before (black) and after (blue) water rinsing, respectively; c) SFM image (topography mode) of casted and dried EDAC/CS (n-/n+ = 1.1) particle film. RH of EDAC/CS particles were ranging between 100 – 150 nm. With increasing aging time there was only a slight decrease in size and this colloidal stability kept for several weeks, which is beneficial for providing samples for collaboration.


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Langmuir

3.2.2. Rinse stability and morphology of PEC NP coatings To check for the rinse stability of PEC particle coatings FTIR spectroscopy on the state before and after rinsing was applied. In the Fig. 10b FTIR spectra of dry EDAC/CS (n-/n+ = 1.1) coatings before and after rinsing with water in comparison to respective ones of the pure EDAC and CS compound are shown. While dry films of pure EDAC and CS films and EDAC/CS complexes casted from their pure solutions or dispersion show significant IR band intensities (e.g. saccharide band at 1100 cm-1) before rinsing, the band intensities disappear after rinsing for the pure EDAC and CS. However, the intensities of spectra from PEC films of EDAC/CS remain, so that wetadhesiveness can be only concluded for the PEC coating, while pure EDADC and CS films are readily dissolved in contact to water. Similarly to the analytical treatment of the cationic starch systems introduced above (3.1) a quantitative wet-adhesiveness parameter Q = AAFTER/ABEFORE can be defined based on FTIR spectroscopy [52], where AAFTER and ABEFORE denote the intensities of a diagnostic band of a coating before and after rinsing, respectively, with a given (aqueous) solvent. There are various explanations for the significant rinse stability of PEC coatings at mixing ratios close to one. Three aspects on their adhesiveness might be valid. At first, electrostatic attraction between negatively charged Ge/GeOxHy and the (complexed) polycation (EDAC) component might play a role. However, coatings of slightly cationic EDAD/CS PEC with n-/n+ = 0.9 and anionic PEC with n-/n+ = 1.1 seem to have similar rinse stabilities, so that electrostatic attraction seems to be a minor factor for wet-adhesiveness of PEC coatings. Secondly, PECs loose water upon drying and shrink to a considerable amount [51], so that they clamp at topographic features of surfaces with certain roughness. Thirdly, in coatings of densely packed adjacent PEC particles it is assumed, that dangling chains of one PEC particle might entangle with those of another PEC particle or might penetrate so that both (i.e. all) PEC particles are bridged. Hence, PEC particles also seem to adhere collectively, which is similar to latex spheres. Moreover in a recent report a preliminary result with respect to gluing two glass substrates manifesting also cohesive forces is given [53]. Such cohesive forces of certain PEC systems (e.g. carboxymethyl cellulose/poly(vinylamine)) between wet cellulose based sheets emphasizing the role of excessive cationic charge have been also described experimentally [54] and theoretically [55] by Pelton. In the following the immobilization effect is illustrated at the two different systems EDAC/CS and poly(L-lysine)/CS (PLL/CS). Starting with EDAC/CS the stoichiometry ratio of anionic (n-, CS units) and cationic charges (n+, EDAC units) was varied in a broad range n-/n+ = 0.25 - 2.0 and the influence on the rinse stability was studied. Representative spectra are given in Fig. 11a and in Fig. 11b the wet-adhesive parameter Q (see above) is plotted versus the mixing ratio. From such a master plot information on the wet-adhesiveness of individual PEL components can be obtained. Interestingly, the EDAC component was the more rinse stable the higher the mixing ratio n-/n+ (i.e. the more CS excess) was and the CS component was the more rinse stable the lower n-/n+ (i.e. the less EDAC excess) was. This is a clear indication, that individual PEL components in PEC coatings are more stable the more they are in shortage and less stable the more they are in excess, since charges of components in minority are fully compensated by opposite charges of excess components and thus kept by water rinse and charges of excess components are not and thus lost by water rinse (see Fig. 11d). Interestingly, the courses of the wet-adhesiveness parameter Q for EDAC and CS cross in their plots at mixing ratios close to one, so that obviously a certain charge symmetry prevails. Similar findings were found for PLL/CS system, where PLL was rinse stable for PEC samples with n-/n+ > 1 (CS in excess) and CS was rinse stable for PEC samples with n-/n+ < 1 (PLL in excess). Thereby the plateaus of complete integration of anionic CS for n-/n+ < 1 and of cationic PLL for n-/n+ > 1 were even more pronounced compared to the EDAC/CS system. Presumably, better matching of charge distances or of molecular weights for the PLL/CS compared to the EDAC/CS system might play a role. Conclusively, at the moment it seems, that PEC particle coatings with lowest excess charge feature highest rinse stability, which is seen to be related to the findings for cationic starch coatings (Results section 1.) and for PEM systems (Results section 2.). Obviously, it is the low (excess) charge, which is the most important factor for wet-adhesiveness or rinse stability. Otherwise, if high


Langmuir

(excess) charges are encountered at given substrates, the system looses excess charges as long as it gets neutral. Sometimes, this leads to the loss of the whole system, sometimes of part of the system and sometimes it stays stable. a)

b)

c)

d)

n-/n+ < 1 n-/n+ > 1

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Fig. 11. Wet-adhesiveness (rinse stability) of PEC NP coatings; a) TRANS-FTIR spectra on EDAC/CS (top) and PLL/CS (bottom) coatings for various molar mixing ratios n-/n+ = 0.25 – 2.0 before (black) and after rinsing with water; b) Plot of the wet-adhesiveness parameter Q (see text) versus the molar mixing ratio n-/n+ for EDAC/CS and PLL/CS coatings taken from TRANS-FTIR spectra given in Fig. 11a; c) SFM image (topography, scale bar: 1 µm) of deposited EDAC/CS-1.1 PEC particles at Ge substrate. PEC particles were preformed by mixing EDAC and CS solutions at the mixing ratio of n-/n+ = 1.1, then casted and dried at Ge, rinsed and again dried. d) Scheme on the rinsing effect of exccess PEL for n-/n+ < 1 and n-/n+ > 1 concerning PEC wet-adhesion. Finally for this section, as a qualitative proof of EDAC/CS adhesiveness the SFM image of a EDAC/CS PEC particle (n-/n+ = 1.1) film after casting from PEC dispersion, rinsing in pure water and drying is given in the Fig. 11c. Interestingly, a morphology of granular structures can be found in this PEC particle film of EDAC/CS, which was similar to that in PEM films of EDAC/CS given in Fig. 5c above. This suggests additionally to the correlation between PEM and PEC with respect to growth, composition and phase behavior claimed by e.g. Sukhishvili [56] also a correlation between PEM and PEC concerning structure and morphology on the nanoscopic and microscopic level. Such similarity between the morphology of single (one-off) deposited PEC films in comparison to manifold deposited PEM films might be explained by a readsorption process at PEM. As we suggested above, loosely bound or diffusive PELs within a PEM film can be pulled out by oppositely charged PELs of the added PEL solution. As a consequence phase separated PEL complex particles are formed (analogously to intermixing oppositely charged PELs) close to the


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PEM surface, which could readily (re)adsorb at the PEM surface. Hence, the granular morphology of PEM might be caused by such readsorbed PEC particles. 4. Biomedical applications 4.1. PEM coatings Polyelectrolyte complex related coatings can have several applications. One very promising application is their interaction to proteins. Proteins are polyampholytes, i.e. polyelectrolytes with both cationic and anionic units, whose net charge is regulated by the pH of the medium. Hence, basic proteins like e.g. lysozyme with a positive net charge and acidic proteins like e.g. human serum albumin with a negative net charge at neutral pH setting, respectively, interact primarily by electrostatic forces with polyelectrolyte bearing coatings like PEM. This can be used for both attractive binding under oppositely charged conditions as well as repulsive shielding under like charged conditions of protein and outermost PEL, respectively. 4.1.1. Passivation/protein inertness An example for PEM systems with either synthetic poly(ethyleneimine)/poly(acrylic acid) (PEI/PAC) or polysaccharide containing poly(ethyleneimine)/alginate (PEI/ALG) systems is given in the Fig. 12. Typical ATR-FTIR spectra of adsorbed HSA at PEI or ALG terminated PEMs are given for various adsorption times In the Fig. 12a [37]. Adsorbed amount / [%]

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100 80 60

HSA+PEI/ALG-9 HSA+PEI/PAC-5 HSA+CHT/ALG-9 HSA+CHT/ALG-8 HSA+PEI/ALG-8 HSA+PEI/PAC-4

40 20 0 0

15

30

45 60 Time / [min]

a) b) Fig. 12. Adsorption of human serum albumin (HSA, IEP = 4.7, MW = 66.000 g/mol, 1 mg/mL, PBS, pH=7.4) at PEM of PEI/ALG; a) ATR-FTIR spectra on HSA adsorption at PEM of PEI/ALG terminated either by ALG (bottom, blue) or PEI (top, red). Reproduced with permission from Ref. [37]. Copyright [2010, Wiley-VCH]. b) Adsorption kinetics of HSA at three different PEM systems terminating either by polycation or polyanion layer based on ATR-FTIR spectroscopy exemplarily shown in Fig. 12a. Reproduced with permission from Ref. [37, 57]. Copyright [2010, Wiley-VCH, 2001, Wiley-VCH]. An overview on the corresponding adsorption kinetics of HSA at three different PEMs is given in the Fig. 12b [37, 57]. Obviously, the result is unique. The acidic model protein HSA is strongly adsorbed at all studied polycation terminated PEMs due to electrostatic attraction and is only weakly bound at all studied polyanion terminated PEM due to electrostatic repulsion. There has been discussion, where proteins adsorbed at PEM are located in earlier classical reports [58, 59, 60]. This was experimentally addressed by us in an earlier report on the adsorption of acidic HSA at PEM with varying thickness (PEM-5, -7, -9, -11, -13) and oppositely charged outermost polycation layer by in-situ-ATR-FTIR spectroscopy [61]. It was found out, that the Amide I intensity diagnostic for bound protein amount was decreasing with increasing layer thickness. Since, intensities in ATR-FTIR spectra are decreasing with increasing distance from the internal reflection


Langmuir

element surface due to the exponentially decaying electrical field of the evanescent wave, it was suggested, that proteins are rather located at the outermost layer zone, than embedded in the interior of PEM. The passivation effect of PEM with like charged outermost PEL layer with respect to the target protein has been already industrially used for the modification of ophthalmic biomedical products like contact lenses [8] by consecutive spray coating of polycation and polyanion solutions onto the contact lens followed by drying. Passivation effect of PEM coating towards proteins of tear liquid under conservation of high oxygen permeability of the lens core was aimed at. 4.1.2. Sustained drug release Another interesting application of PEM coatings is their ability to deliver therapeutic drugs deposited at biomedical devices or implants. An example for the loading and releasing (delivery) of the antibiotic streptomycin (STRP) by PEM composed of synthetic and biorelated PEL is given in the Fig. 13 [62]. STRP content / [%]

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PEI/PAC-8-STRP-1:5 PEI/PAC-8-STRP-1:10 PEI/PAC-8_STRP-1:25 PEI/ALG-8-STRP-1:5 CHT/ALG-8-STRP-1:5

120 100 80 60 40 20 0 0

100

200

300

Release time / [min]

b) a) Fig. 13. Drug release of streptomycin (STRP) from PEM; a) ATR-FTIR spectra on PEM of PEI/ALG in the unloaded state (pink), after loading (black) and after releasing (green) of STRP; b) Release kinetic curves of STRP out of PEM of PEI/PAC, PEI/ALG and CHT/ALG. Reproduced with permission from Ref. [62]. Copyright [2013, Elsevier]. Three different PEM systems PEI/PAC, PEI/ALG, CHT/ALG were investigated. Fig. 13a shows insitu-ATR-FTIR spectra on PEM of PEI/ALG in the initial unloaded state, after STRP loading (black) and upon releasing of STRP (green). The STRP amounts were quantified by the diagnostic band at around 1100 cm-1 and plotted versus time in the Fig. 13b for various PEM systems. The release of STRP was found to be dependent on PEM composition and the STRP/PEM ratio, which was varied between 1:5 and 1:20 (STRP/PEM). The most delayed STRP release was obtained for the system PEI/ALG/STRP with STRP/PEM ratio of 1:5, showing 40% STRP release after 5 h and an extrapolated residual content of around 40% after 24h [62]. Drug eluting PEM coatings are relevant for the modification of implants or biomaterials, especially to reach antibacterial properties in the framework of nosocomial infections. Since various cationic polymer systems are known to have antibacterial properties per se [63, 64], PEMs terminating with a polycation layer and the additional option to elute antibiotic drugs like streptomycin, gentamycin, tetracycline should be interesting for providing dual septic systems. 4.2. PEC particle coatings As it is shown above adhesive polyelectrolyte layers can be fabricated even more simple, if preformed PEC particles are casted and dried onto materials. Moreover drugs can be incorporated


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by simple loading from a bulk drug solution or casting and drying a drug solution onto the PEC particle film. A couple of examples have been given recently with varying polyelectrolyte systems and drug types ranging from small charged molecules [65, 66, 67] up to amphoteric proteins [68, 52]. While in these works all colloid stability, wet-adhesiveness, cell compatibility [69], adaptation to bone substitute materials (BSM) and drug release was outlined, herein we like to focus on the drug release aspect.

120

T=25°C T=37°C T=50°C T=60°C

100 80 60

Absorbance Units

4.2.1. On demand drug release from PEC particle coatings In the last years we could show, that PEC particle coatings can be loaded by low molecular bone healing drugs such as the bisphosphonate derivative zoledronate (ZOL) [65, 66, 67] and proteinogenic growth factors like BMP-2 [68] and to release those in sustained way upon contact to buffer or cell media. However, the onset of release was ill defined and set in, as soon as the PEC coating encountered the release medium. Therefore, a recent development of our PEC concept has been the external switchability of drug release and out of several possible stimuli a thermal one was selected. PNIPAM based polymer systems are well known for their thermoresponsiveness in the interesting physiological range around 37 °C. Hence, based on our standard EDAC/CS system we replaced the anionic polysaccharide CS by the random copolymer poly(N-isopropylacrylamide-coacrylic acid) (PNIPAM-AA) and complexed it with EDAC [70]. Interestingly, complexation of EDAC/PNIPAM-AA revealed colloidally stable, adhesive and thermoresponsive PEC particles showing deswelling (compaction), when T = 37 °C was exceeded, which was associated to a volume phase transition (VPT) known for pure PNIPAM. Moreover, drugs like bisphosphonates could be loaded in coatings of EDAC/PNIPAM-AA particles, which were prepared at a mixing ratio of n-/n+ = 0.9. Concerning the thermoresponsive release performance in the Fig. 14 time courses of the relative ZOL content for the four different temperatures are shown. ZOL content / [%]

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0.06 0.05 0.04 0.03

40

0.02

20

0.01

0 0

300

600

900 1200 1500 Time / [min] Fig. 14. Elution of ZOL out of loaded EDAC/PNIPAM-AA (PEC-0.9, 0.01 M) particle coatings in HEPES buffer at T = 25 °C, 37 °C, 50 °C and 60 °C. Reproduced with permission from Ref. [70]. Copyright [2017, Springer].

Casted Rinsed

0 1800

1500

1200

900

Wavenumber [cm-1] Fig. 15. Wet-adhesiveness of immunoglobulin/CS complex particle films (top) in comparison to films of the single compounds CS (middle) and IGG (bottom).

For T = 25°C there was a slow decrease of the ZOL content to around 80% after 24h, for T = 37°C to around 60% and for T = 50°C to around 20%, while for 60°C ZOL was completely released after around 6h. Obviously, with increasing temperatures ZOL is eluted with increasing extent and rate from EDAC/PNIPAM-AA particle coatings. From the polymer scientific aspect this result was not expected on the first glance, since PNIPAM systems are known to swell at lower and compact at elevated temperatures and therefore a drug should be better integrated at higher T. However, in the


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case of EDAC/PNIPAMM-AA coatings it is suggested, that upon deswelling of the anionic PNIPAM-AA compound the conformation of the cationic EDAC compound, where most of the anionic drug ZOL is assumed to be bound by ion pairing, is disturbed as well. Hence, this perturbation might cause changes in the charge compensating features and structure within the PEC phase resulting in partial loss of the charged drug. From the medical application aspect a temperature T >> 37°C might be suspected to be physiologically no longer meaningful, since thermal necrosis and protein denaturation might occur. However, temperatures up to T = 60°C are not uncommon in hard tissue regeneration, where the application of PMMA bone cements cause also temperatures up to 60 - 70°C [71]. Presumably, short periods of heat at BSM like porous defect fillers (bone cements) and solid osteosynthetic plates might be still physiologically tolerable. Moreover, external local heat might be also achieved by e.g. magnetic fields upon iron oxide particles [72], which creates local hyperthermia and could locally trigger e.g. an embedding EDAC/PNIPAM-AA system. 4.2.2. Antibody/PEL complexes Protein/polyelectrolyte complex particles [73] is an interesting material for both immobilization or release of therapeutic proteins. In that framework antibody/PEL complexes are relevant for both diagnostic (e.g. “antigen fishing”) and therapeutic (e.g. “signal molecule elimination”) applications. Such theranostic applications will increase in the biomedical field in the next future. In the Fig. 15 FTIR spectra on films of the single compounds immunoglobulin G (bottom) and cellulose sulfate (middle) (CS) and particles of the complex of both (IGG/CS) are shown in the initial casted and dried state and after rinsing with HEPES buffer. In analogy to the wet-adhesiveness results for polycation/polyanion complex coatings above, there is also evidence, that only the complex IGG/CS is sufficiently rinse stable, which is a prerequisite for further biomedical applications. In the future we will apply this “complexing immobilization” strategy on antibodies against signal peptides of systemic bone diseases like osteoporosis and multiple myeloma, which are bound to commercial implants and BSM.

Summary and Outlook In this conceptual contribution concepts and modalities are described to deposit functional polyelectrolyte based coatings, which are wet-adhesive and serve as platforms for biomedical applications. At first, results on the adsorbed amount and wet adhesiveness of PEL layers, which were adsorbed from single component PEL solutions at Ge substrates, are given as a function of charge density. It could be shown, that the irreversibly adsorbed amount of cationic starches increases with decreasing charge density, so that lowest charge density resulted in the highest adsorbed amount. Profound wet adhesiveness of the PEL films with low charge density was proven. Secondly, results on the adsorbed amount and wet adhesiveness of PEL multilayer (PEM) coatings at Ge substrates, which were constructed by sequential adsorption from cationic PLL or EDAC and anionic CS solutions, are given as a function of adsorption cycles and outermost layer charge. With increasing adsorption cycle number exponentially increasing adsorbed amounts for the PEL pairs were found. No significant dependence of the wet adhesiveness of PEM coatings on outermost layer charge was found, since the net stoichiometry of the whole PEM assembly was not affected significantly by outermost layer variation. Thirdly, results on the adsorbed amount and wet-adhesiveness of PEL complex (PEC) nanoparticle (NP) coatings at Ge substrates, which were constructed by premixing solutions of polycations like EDAC or PLL with solutions of polyanions like CS at first and casting secondly the freshly formed PEC NP dispersions. Adsorption isotherms are given as a function of PEC concentration and wet- adhesiveness is characterized in dependence of the molar mixing ratio related to charged monomer units. With increasing PEC NP concentration increasing deposited amounts were obtained. With increasing deviation from the stoichiometric molar mixing ratio of n/n+ = 1 to n-/n+ > 1 or < 1 of the respective excess PEL component (polycation for n-/n+ < 1, polyanion for n-/n+ > 1) in PEC coatings can be rinsed off. This effect for PEC coatings is different to PEM coatings, since n-/n+ variations affect PEC coatings significantly and globally, while


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variations of the outermost layer from polycation to polyanion only have local charge effects and do not affect the global net charge of the PEM. Generally, all three PEL deposition concepts share the common effect of good wet adhesion, whenever interfacial excess charges are kept low either by low charge density related to single PEL component or by sufficient charge compensation related to PEM and PEC films. Vice versa all three concepts share the common effect of poor adhesion, whenever interfacial excess charges are high either by high charge density of single component PEL or by insufficient charge compensation within PEM and PEC films. Furthermore, especially for PEC particle films beside lack of excess charge also shrinking and water loss (expulsion) associated with the drying process might contribute to their wet-adhesiveness at planar model substrates, which could be even increased with increasing surface roughness due to mechanical anchoring or clamping on a nano- or microtopographical level. Additionally we suggest, that PEL deposition is effective and leads to sustainable wet adhesive coatings, whenever the deposition process at given solid substrates is associated with a volume phase separation in statu nascendi like complexation of two oppositely charged PEL (PEC, PEM) close to the solid/liquid interface. Concerning pure PEL (i.e. cationic starch) adsorption, where decreasing PEL linear charge density (qm) obviously leads to increasing adsorbed amount, we still see a lack of experimental and fundamental knowledge concerning the role of surface charge, where according to the empirical equation (1) given above increasing surface charge density (σ0) also leads to increasing adsorbed amount. Finally, the potential of PEC NP and PEM coatings for biomedical applications should be emphasized. On the one hand concerning biopassivation PEC NP and PEM coatings excessed or terminated by polyanions repel acidic proteins with low isoelectric points and vice versa. On the other hand concerning bioactivation both PEM and PEC coatings provide active loading and releasing of therapeutic antibiotics, bisphosphonates or growth factors to address implant based infections or local healing of systemically altered bone via the bone substitute material. Also PEM and PEC based coatings offer the immobilization and integration of specific and even bispecific antibodies [74] for theranostic applications. Moreover thermoresponsive PEC coatings were presented enabling the switchable (on-demand) release of drugs, which is relevant for spatiotemporal control of drug delivery during and after endoprosthetic surgery in the framework of hard tissue regeneration.

Supporting Information Additional material concerning chemical structures of the used polyelectrolytes (Fig. SI1), adsorption isotherm of cationic starch at Ge substrate (Fig. SI2), desorption of cationic starch casted from 0.002M solutions at Ge substrate (Fig. SI3) is available in the Supporting Informations (SI).

Acknowledgements Part of this work contributed to and was funded by the Transregio-Sonderforschungsbereich 79 (TRR 79, Materials for tissue regeneration within systemically altered bone, part project M7) involving universities and research institutes in Gießen, Heidelberg and Dresden.

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Brief Biographies Martin Müller leads the group ´Polyelectrolytes in Medicine´ at the Leibniz Institute of Polymer Research (IPF) Dresden, Germany and is private lecturer at the Technical University Dresden (TUD). Polyelectrolyte complexes are prepared as sizable, chargeable and shapable nanoparticles or Layer-byLayer films, in which a broad range of therapeutic drugs are loaded and released in a defined way. Also polymer brushes are studied. Main analytical tool is in-situ-ATR-FTIR spectroscopy among other microscopic and spectroscopic tools, which helps to detect ad- and desorption processes of drugs, catechols, lipids, polyelectrolytes and proteins at the solid/liquid interface as well as the conformation and orientation of polypeptides and proteins. Birgit Urban is chemical engineer in the group ´Polyelectrolytes in Medicine” at IPF Dresden and develops, performs and instructs on analytical techniques and protocols.

Simona Schwarz leads the group ´Polyelectrolytes in Environmental Applications´ at the Leibniz Institute of Polymer Research (IPF) Dresden, Germany. Polyelectrolyte (PEL) based solutions are provided for the treatment of water like the separation of heavy metal cations and low molecular anions and the flocculation of waste compounds in close cooperation with industry. Biorelated PEL like chitosan and ionically substituted starch as well as synthetic PEL based on acrylic compounds are applied. Main characterization tools are zeta-potential, colloid titration and various microscopic and light scattering based techniques sensitive to determine particle size and charge.

Cover Art Authors Sven Doering, Judith Nelke, Birgit Urban, Martin Müller


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Biorelated Polyelectrolyte Coatings Studied by in Situ Attenuated Total

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