Environmental Responsiveness of Polygalacturonic Acid-Based

Biomacromolecules 2011, 12, 359–369

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Environmental Responsiveness of Polygalacturonic Acid-Based Multilayers to Variation of pH Marta Westwood, Timothy R. Noel, and Roger Parker* Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Received September 20, 2010; Revised Manuscript Received November 26, 2010

The effect of pH on the stability of layer-by-layer deposited polygalacturonic acid (PGalA)-based multilayer films prepared with the polycations poly-L-lysine, chitosan, and lysozyme is studied. The response was characterized using a quartz crystal microbalance, dual polarization interferometry, and Fourier transform infrared spectroscopy which probe multilayer thickness, density, polymer mass (composition and speciation), and hydration. All multilayers showed irreversible changes in response to pH change becoming thinner due to the partial disassembly. Preferential loss of the polycation (50-80% w/w) and relative small losses of PGaLA (10-35% w/w) occurred. The charge density on the polycation has a strong influence on the response to the acid cycle. Most of the disassembly takes place at the pH lower that pKa of PGaLA, indicating that this factor was crucial in determining the stability of the films. The pH challenge also revealed a polycation-dependent shift to acid pH in the PGaLA pKa.

Introduction The layer by layer deposition of polyelectrolyte multilayers (PEM) has the potential to precisely structure and confer useful and novel properties to surfaces and interfaces. Initial studies employed synthetic polymers1 and, more recently, biopolymers have been studied including those suitable for biomedical2 and food applications.3,4 In addition to solid substrates layers can be applied to fluid substrates as in multilayer emulsions.5-7 Through suitable choice of the constituent polyelectrolytes the multilayers are expected to have properties which will be responsive to pH and salt concentration.8 Engineered into an encapsulation system this can confer multiple functionalities for enhanced delivery.9 A range of biomacromolecules, typically polysaccharides and proteins, can be used for the creation of films for biomedical and food applications.10 In these applications the component biomacromolecules have characteristic conformational properties depending upon factors such as their constituent monomers, chain sequence, intramolecular bonding, and solution conditions. In this study the polyanion chosen was polygalacturonic acid (PGaLA), a cell wall polysaccharide,11 more specifically a deesterified pectin that is typically extracted from the peel of citrus fruit. PGaLA is a weak polyelectrolyte based on a backbone of (1-4)-R-galacturonysyl units. In pectin, PGaLA backbone is interrupted in places by (1-2)-R-L-rhamnopyranosyl units.12 The pKa of anhydrogalacturonic acid residues in PGaLA are in the range 3.0-4.5,13 and the polymer has a persistence length of 10-12 nm, so it is classified as semiflexible.12 The aim of the study was to investigate the effect of varying the polycation on the properties of PGaLA-based multilayers. As compared with polyanions, relatively few polycations occur in nature. Poly-L-lysine (PLL), a polyamino acid, was chosen because it is highly charged and, because it is commonly used it allowed comparison with other studies including our own previous work.4,14,15 Its cationic charge at acidic pHs derives from its side chains each carrying a weakly basic ε-amino group with pKa about 9.16 The second polycation chosen was the * To whom correspondence should be addressed. Tel.: +44-1603-255000. Fax: +44-1603-507723. E-mail: [email protected].

polysaccharide chitosan (Chi), more specifically a glycosylaminoglycan,17 a β-(1-4)-linked polysaccharide derived from chitin. It is partially acetylated and the degree of acetylation (DA) determines the proportion of 2-acetamido groups present and the number of weakly basic 2-amino groups (pKa, 6.7), which are able to bind protons.18 With a persistence length of 6-8 nm chitosan is also classified as semiflexible.17 Finally, the basic globular protein lysozyme (Lys) was chosen. It has a high isoelectric point (IEP) at pH 11.1 and so it carries a positive charge over a large range of pH.19 Multilayers have a range of pH responsive behavior from simple swelling and shrinking20-23 to partial and complete dissolution.24 The aim is to understand these behaviors in terms of the basic properties of the constituent polyelectrolytes and the multilayer assembly conditions. An early attempt to understand the rules governing the assembly of polyelectrolyte multilayers was based upon whether charge balance was achieved by the polyelectrolytes (“intrinsic charge balance”25) or whether it was balanced by the counterions (“extrinsic charge balance”). While intrinsic charge balance was observed for model synthetic polymer multilayers,25 recent evidence has shown that extrinsic charge balance is common and that other factors determine the ratio of polyanion to polycation concentrations observed in multilayers. Studying a series of polysaccharides cross-linked with PLL, Crouzier and Picart26 showed a multilayer stochiometry with two lysine residues per sugar residue irrespective of the number of anionic charges carried by the sugar residues. Other work has shown hydrogen-bonding interactions can affect assembly and release in addition to charge interactions.27-29 In multilayers comprising weak polyelectrolytes, it has been found that the strong polyanion-polycation interactions can modify the pH at which weakly acidic or basic groups are ionized.20,21,30,31 Burke and Barrett20,21 studied the pH behavior of both poly(acrylic acid)-poly(allylamine hydrochloride) and hyaluronic acid-PLL (analogous to PGaLA-PLL) multilayers characterized by ζ-potentials measured using electrophoretic mobility to probe the charge on the uppermost surface of the multilayer. pKa shifts of up to 4 pH units were observed. The pKa of weakly acidic groups can be more directly characterized

10.1021/bm1011213  2011 American Chemical Society Published on Web 12/20/2010

360 Biomacromolecules, Vol. 12, No. 2, 2011

using FTIR-ATR provided there are suitable absorbance bands for both ionized and un-ionized forms. Sukhishvili22,23 used FTIR to study the ionization and pH stability of multilayers comprising a series of weak synthetic polyelectrolytes. Highly charged polycations increased the ionization of the polyanions, and at low pH, multilayers dissolved when the polyanion lost charge due to protonation. The response of a polyelectrolyte multilayer might be expected to have some of the properties of polyampholyte gels,32 which are suitable models for examining the behavior of specific distributions of positive and negative charges. Synthetic covalently cross-linked gels show some novel behaviors, such as the “antipolyelectrolyte effect” (swelling in response to increased salt concentration). However, as these systems are held together by covalent cross-links whereas in a PEM the “crosslinks” themselves are modulated by pH and salt the analogy has limited utility and PEM behavior is generally more complex.14 In our previous work we have observed the pH response of pectin-PLL14 and pectin-protein15 multilayers for low partially esterified charge density pectins. When the pH of the surrounding solution was increased above the IEP of the protein, the multilayer dissolved.19 The present study builds on our earlier work on pectin-based multilayers. In the present study, to maximize the ionic effects of the fully de-esterified pectin, polygalacturonic acid was chosen. One potential application of multilayer films is their use in the modulation of release in the human gastrointestinal tract. While passing down the gastrointestinal tract, a food first experiences conditions in the mouth before experiencing acid conditions in the stomach and, subsequently, being neutralized by hydrogen carbonate in the duodenum and small intestine.33 To mimic these environments, the response of the multilayers to a stepped pH cycle ranging from pH 7.0 down to 1.6 and returning to 7.0 was studied. The responsiveness of the assembled multilayers was characterized using a dual polarization interferometer (DPI),34-36 FTIR-ATR,37 and QCMD38,39 on an oxidized silicon surface, which provided complementary information about multilayer assembly, composition, interpenetration, and interactions between the successive layers of polyelectrolytes,40 and also their response to pH change.

Materials and Methods Polymers and Polymer Solution Preparation. Poly-L-lysine hydrobromide with a mean degree of polymerization of 70, hens egg lysozyme, and D2O (99.9%) were obtained from Sigma, and polygalacturonic acid4 was supplied by Fluka, Biochemika. Reagents were analytical grade. Chitosan with the degree of acetylation of 50% was prepared by homogeneous deacetylation of crab chitin (Sigma) following the method of Sannan41 to obtain material soluble at pH 7.0.40 Solutions of 0.6 mg mL-1 were used for the multilayer assembly and prepared by dissolving the biopolymer in 10 mM pH 7.0 phosphate buffer, containing 30 mM NaCl, to both control pH and provide a background level of ionic strength. The NaCl concentration was chosen to enhance the complexation between the proteins and polysaccharides.42 The pH was adjusted by adding 0.1 M NaOH. For pH responsiveness studies, the following buffers were prepared: HCl/KCl (pH 1.6-2.9), acetate (pH 3.6-4.8), and phosphate buffers (pH 6.0 and 7.0), each containing 30 mM NaCl. The hydrodynamic radii of the polymers as characterized using size exclusion chromatography40 were 7.1, 2.6, 9.2, and 2.0 nm for the PGalA, PLL, Chi, and Lys, respectively. For the chitosan, this corresponds to an estimated molecular weight of 130 kDa. Multilayer Formation. The multilayer structures were fabricated on a silicon oxide substrate by layer-by-layer deposition1 at 20 °C,

Westwood et al. following the procedure, which has been described in detail elsewhere.4,40 Prior to deposition, the substrate surfaces were cleaned using a UVozone chamber (Bioforce Nanosciences, Inc., Iowa, U.S.A.), then rinsed thoroughly with water, and dried with N2. For all of the multilayer films, the base layer was formed by deposition of PLL. The multilayer assembly of PGalA-based films was then subsequently formed by alternating exposure of the PLL-coated substrate to a PGalA solution and then to a polycation solution. All multilayer assemblies studied consisted of a total of 10 layers. Responsiveness Study. To assess the multilayer response to pH, the previously formed 10-layer films were first exposed to solutions of decreasing pH, starting from 7.0 down to 1.6. To study the reversibility of the process, the pH was then increased in the same steps back to 7.0. At each step the buffer solutions were left in contact with the films for a sufficient time to collect data of suitable accuracy. The QCMD measurements were taken in real time over a 5 min period after the introduction of each buffer solution. The DPI measurements were made while flowing the buffer solutions over the samples for 5 min. For the FTIR-ATR, the buffer was injected into the measurement cell and the spectra were recorded by coadding 512 scans in the frequency range 1500-1800 cm-1 with a resolution of 2 cm-1 over an 8 min period and subtracting a buffer background. After responsiveness studies, surfaces were cleaned with 2% v/v Hellmanex, rinsed with water, exposed to UV-ozone treatment, and then rinsed with water again. Surfaces were dried with N2. Quartz Crystal Microbalance with Dissipation Monitoring (QCMD). The multilayer assembly and responsiveness processes were monitored using a D300 QCMD (Q-Sense AB, Va¨stra Fro¨lunda, Sweden) with a QAFC 302 axial flow measurement chamber, as described elsewhere.4,40 Silicon dioxide coated AT-cut quartz crystals sandwiched between gold electrodes (QSX-303, Q-Sense AB, Va¨stra Fro¨lunda, Sweden) were used as the substrate. This QCMD sensor was placed in the measurement chamber and an AC voltage was applied to excite the fundamental resonant frequency (∼5 MHz) of the crystal and its overtones (3, 5, and 7). Each layer was formed during a 4 min exposure of the sensor surface to a biopolymer solution, and each deposition step was followed by a buffer rinse. The assembly and environmental response studies were carried out in real-time by monitoring changes in the oscillation frequency of the crystal. The responsiveness measurements were made as described above. A decrease in oscillation frequency indicates the adsorption of mass onto the QCMD sensor. If the mass is deposited evenly and forms a rigid elastic layer, then the negative frequency changes are proportional to the hydrated mass deposited (∆m), as described by the Sauerbrey equation.43

∆m )

-C∆fn n

(1)

where C is the mass sensitivity constant (C ) 17.7 ng cm-2 Hz1- for a 5 MHz crystal)44 and ∆fn is the frequency shift of the nth overtone). For viscoelastic materials, the Sauerbrey relation leads to an underestimation of the deposited hydrated mass and, under these conditions, a more detailed analysis was performed using the Voigt model with the QTools modeling package (Q-Sense AB, Va¨stra Fro¨lunda, Sweden). Dual Polarization Interferometry (DPI). The measurements were carried out using an AnaLight Bio200 DPI (Farfield Scientific, Crewe, U.K.) with a silicon oxynitride sensor chip.35,45 The sensor chip consists of a sandwich of two horizontally stacked waveguides, a sensing waveguide on top of a reference waveguide separated by an opaque cladding material. Laser light (wavelength, 632.8 nm) illuminating one end of the chip is switched between two polarizations: transverse magnetic (TM) and transverse electric (TE); the light travels along the waveguides and on exiting the chip produces an interference fringe pattern, which is detected by a camera located at the far field. A gasket on top of the sensing waveguide forms two measurement chambers, each 1 mm wide, 17 mm long, and 1 mm deep (17 µL volume). The

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Table 1. FTIR-ATR Calibration Constant, Penetration Depth, Wavenumber of the Absorption Peak, Calibration pH, and Absorbing Group for the Polymers Studied material, absorbing group

pH

absorbance peak wavenumber (cm-1)

dp, penetration depth (nm)

K (cm2 ng-1)

PGalA (GalA), carboxylate PGalA (GalA), carboxyl PLL, amide I Chi, amide I Lys, amide I

7.0 1.6 7.0 7.0 7.0

1610 1725 1645 1634 1646

490 457 479 482 479

6.0 × 10-5 2.12 × 10-5 4.8 × 10-5 4.1 × 10-5 5.6 × 10-5

principle of DPI measurement is that changes in the refractive index and thickness of an adsorbed layer on the sensing waveguide surface cause a phase change in the light traveling down the waveguide and, ultimately, a shift in the interference patterns detected by the camera which differ for vertically and horizontally polarized light. To extract structural information from these two phase changes, the simplest assumption made is that the adsorbed film behaves as a single homogeneous layer of uniform refractive index (RI), nf, and thickness, df. By solving Maxwell’s equations simultaneously for the two polarizations, unique values for the mean refractive index and optical thickness of the adsorbed film are obtained. At the start of each experiment, the sensing chip was calibrated with 40% w/w ethanol and water. Multilayers were assembled by injecting 100 µL of biopolymer solution into a continuously flowing buffer (flow rate 20 µL min-1) using an HPLC sampling valve. After each injection there was a rinsing step of 5 min. Once the multilayers were assembled the responsiveness measurements were made as described above. The de Feijter equation46 was used to quantify the adsorbed mass, Γ, from the mean optical adsorbed film thickness, df, the refractive index of the film, nf, and dn/dc, the refractive index increment of solutions of the constituent polymers.

Γ ) df

nf - nbruffer dn/dc

(2)

where nbuffer is the RI of the buffer. Fourier Transform Infrared Spectroscopy with Attenuated Total Reflection Mode (FTIR-ATR). Spectra were collected on a Nicolet Magna 860 FTIR spectrometer (Thermo Electron Corporation, Madison, U.S.A.) fitted with the MicroCircle liquid ATR cell (SpectraTech, Warrington, U.K.) with a silicon rod ATR crystal (Thermo Electron Corporation, Madison, U.S.A.), which presents a silicon oxide surface. All solutions were prepared in D2O-based phosphate buffer to eliminate the stretching mode associated with OH groups, which overlap with the amide I band. This also led to the exchange of H with D in the NH bond, which resulted in a shift of the amide II band (usually observed at 1500-1600 cm-1 in an aqueous solution) to lower wavenumbers. The multilayer deposition has been fully described elsewhere.40 In brief, 1.0 mL of each biopolymer solution was injected into the measurement cell and was left for 4 min prior to rinsing with 2.0 mL of deuterated buffer. To characterize assembly, spectra were collected over a 16 min period prior to the injection of the next biopolymer solution. Once assembled, the responsiveness measurements were made as described above. The spectra analysis and the determination of the mass adsorbed, based on the characteristic absorbance of PGalA (carboxylate and carboxylic groups)47 and the amide I bands of PLL,48 Chi, and Lys were carried out using the calibration method described by Sperline et al.49 The more elaborate methods using peak deconvolution and integrated peak areas were examined, but for our system, the overlap between the PGalA and PLL or lysozyme prevented the use of this approach. The calibration constants specific to each polymer were obtained based on the absorptivity of polymers in bulk solution that were measured independently. The calibration for the PGalA was performed with the monomer galacturonic acid (GalA) due to the limited solubility of the polymer at pH 1.6 and 7.0.

For the quantification, the penetration depth, dp, and the calibration constant K are required.49,50 The penetration depth of the evanescent wave is given by

dp )

λ 2 1/2 2πn1(sin2 θ - n12 )

(3)

where λ is the wavelength of the light in vacuum and n12 ) n2/n1. The refractive index of the crystal, n1, is 3.42 and that of the dilute polymer in solution, n2, is 1.33. The angle of incidence, θ is 45°. If it is assumed that the amount of adsorbed material is independent of concentration in the range studied, and that the amplitude of the evanescent wave remains constant within the thin layer, then the mass per unit area (areal mass) of polymer adsorbed, Γ, can be calculated from

(

A)K Γ+

cdp 2

)

(4)

where A is the peak absorbance for nonpolarised light, K is a constant, and c is the bulk solution concentration. All the absorbance-concentration curves exhibit essentially linear behavior above a certain threshold which corresponds to the concentration at which the surface becomes saturated to further adsorption. This threshold was at concentrations below 1.0 mg mL-1 for PLL, Chi, and GalA but the Lys adsorbed more strongly and so the threshold was about 2.0 mg mL-1. The calibration data is summarized in Table 1. The absorbance peaks of the polymers exhibited some overlap as shown in Figure 1, which shows the absorbance measured in solution scaled (using eq 4) to be equivalent to an absorbed layer with a concentration of 100 ng cm-2. In calculating concentrations of the polycation, there is a maximum overlap with the 1610 cm-1 carboxylate peak for chitosan (23.7% of the PGalA peak absorbance) and relatively less for PLL and lysozyme (8.4 and 6.8%, respectively). In calculating concentrations of polyanion in the presence of PGalA, the potential corrections are more similar, with lysozyme overlap the highest (28.1% of the lysozyme peak absorbance), followed by chitosan (26.2% of the peak chitosan absorbance) and PLL (21.3% of the peak PLL absorbance). The

Figure 1. FTIR-ATR spectra of the polymers scaled using eq 4 and the calibration constant, K, to correspond to a Γ of 100 ng cm-2. Polymers: PGalA, (solid line); PLL (dotted line); Chi (dashed line); and Lys (dash-dot line).

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quantitative importance of these potential corrections depends upon the particular concentrations occurring in the multilayers and will be discussed further in the Results and Discussion. In the case of proteins and polyamino acids it is well-known that the shape of amide I bands depend upon conformation and so care must be taken in relating absorbance data to polymer concentration.48

Results and Discussion Assembly of PGaLA-Based Multilayers at pH 7.0. PGaLAbased multilayers were assembled on oxidized silicon substrates to study their responsiveness to pH change. These studies were performed using QCMD, DPI, and FTIR techniques, which provided complementary information about the multilayer assembly, composition, interpenetration, and interactions between the successive layers of polyelectrolytes, and also their response to pH change. As polycationic cross-linkers PLL, chitosan, and globular lysozyme were chosen, and a detailed analysis of their multilayer growth with PGaLA has been reported.40 In this section a brief summary of the structural parameters of the constituent polymers and the resulting films that were deposited onto the sensor surfaces is presented. Sequential deposition of PGaLA and the polycations indicates that multilayer growth depends upon the strength of electrostatic interactions between successive layers, which in turn strongly depends on the polyelectrolyte structure, charge density and charge distribution. Chitosan with a degree of acetylation of 50% and amino groups with pKa of 6.7 was only partially charged18 at the assembly pH of 7.0, and the low charge density on chitosan resulted in the formation of highly hydrated films. From the conditions for multilayer growth1 it follows that during the deposition on PGaLA-topped surface, the chitosan adopts a conformation which compensates for the negative charge present at the surface, and also creates a positively charge surface for the following PGaLA adsorption. This was reflected by formation of the thickest but least dense structures. Pairing PLL (pKa of 9.0)16 with PGaLA (pKa of 3.5),51 which at the pH 7.0 were both fully charged, resulted in significantly stronger electrostatic interaction between consecutive layers. The PGaLA-PLL film was thinner and less hydrated. When the globular protein lysozyme is used as the polycation with a “patchy” distribution of positive and negative charge, multilayer growth did not result. Although lysozyme with IEP of 11.0 carried overall positive charge, it did not interact sufficiently strongly with PGaLA for multilayer growth. Due to the weak interactions between successive layers and greater affinity of lysozyme for PGaLA in solution, compared to the PGaLA present within the film, a stripping effect was observed that consequently resulted in a limited growth. This thin film of thickness 3.2 nm was, however, the densest structure, due to the packing ability of globular lysozyme. Whereas, PGaLA-Lys multilayers showed a limited growth, the PGaLA-PLL and PGaLA-Chi multilayers showed exponential growth, which is linked with the polycations ability to diffuse within the film during deposition.52 Composition data from FTIR-ATR showed that PGaLA-PLL and PGaLA-Chi multilayers were richer in the polyanion (PGaLA) with polycation/polyanion (PC/PA) mass ratios of 0.54 and 0.55, respectively, while the PGaLA-lysozme deposition was richer in polycation. None of the multilayer exhibited intrinsic charge balance. In each there was an excess of anionic charge carried by the PGaLA. The PGaLA-PLL system was closest to charge balance with a PC/PA charge ratio of 0.74, the PGaLA-Lys was intermediate, and the PGaLA-Chi system was furthest from intrinsic charge balance with a charge ratio of 0.28. A summary of the quantitative data describing the state

Figure 2. Multilayer film pH-response measured in situ with DPI. Optical thickness (-9-) and RI (-0-) as a function of pH (17 steps) for (A) PGaLA-PLL, (B) PGaLA-Chi, and (C) PGaLA-Lys multilayers. The arrows indicate direction of the pH cycle, starting from pH 7.0, reducing to 1.6 and increasing to 7.0. Horizontal bar denotes buffer RI of 1.3336.

of the multilayers as initially prepared is shown in Table 2 toward the end of this section. For further details of the assembly, see Westwood et al.40 DPI and FTIR-ATR Characterization of Multilayer pH Response. The environmental response of the three types of multilayer films to a pH cycle ranging from pH 7.0 down to 1.6 and returning to 7.0 was followed using DPI. Figure 2 shows changes that occur to the refractive index (RI) and optical thickness of the films as a function of pH. The pH challenge leads to an overall reduction in optical thickness in the range of 35-50%, depending on the system studied. The PGaLAChi multilayers (Figure 2B) show the largest reduction in optical thickness by reducing from 32 to 16 nm, whereas the PGaLAPLL multilayer (Figure 2A) reduces from 19 to 12 nm, and the PGaLA-Lys film thickness is reduced from 3.6 to 2.0 nm (Figure 2C). In terms of the overall changes in RI, the PGaLA-PLL multilayer typically shows a 15% decrease of RI, the PGaLAChi multilayers show a 30% decrease, and the PGaLA-Lys multilayers typically shows a 10% increase of RI value in response to the pH challenge. At this stage, it should be noted that the RI measured by DPI represented an effective RI of the whole structure, and it is proportional to the density of the film


(eq 2). Thus, the changes in RI observed during the pH responsiveness study directly corresponded to the changes in the density of the film. For the PGaLA-PLL multilayer (Figure 2A) there are minimal changes in thickness as the pH is reduced from 7.0 to 1.6. However, as the pH is returned to neutral, a significant reduction in thickness was observed in the pH range 1.6 to 4.2. In contrast there is a significant reduction of film RI as the pH is reduced below pH 2.9 corresponding to a reduction of the film density. Further decrease of pH to 1.6 leads to a RI decrease to 1.391, and consequently the lowest density of the film. As the pH is increased back to 7.0, RI increases slightly. Overall changes in RI and thickness reported for PGaLA-PLL clearly indicate the partial disassembly of the PGaLA-PLL film, and the polymer mass loss was estimated to be approximately 50%. The thickness of the PGaLA-chitosan multilayer varies throughout the whole pH range (Figure 2B). In contrast to the PGaLA-PLL system the charge on both the polyanion and the polycation are varying within the range of the pH cycle. PGaLAchitosan multilayers initially shrink as the pH is reduced from 7.0 to 3.6, and then swell as pH is lowered to 1.6. Initial shrinking of the PGaLA-chitosan film results in a pronounced reduction of the thickness from 32 to 24 nm, with a simultaneous RI increase to 1.41 suggesting the formation of a denser structure at pH 3.6. On increasing the pH, the multilayer shrinks in two stages, first in the pH range of 2.0 to 3.6 and then in the range of 6.0 to 7.0. It is worth pointing out that on the completion of the pH cycle the PGaLA-chitosan film is characterized by the same RI and, thus, the same film density, as prior to the pH challenge. Whereas there is no change in RI of the PGaLAchitosan film on pH challenge completion, the reduction of the film thickness by 50% indicates substantial dissolution of the PGaLA-chitosan film. In the case of PGaLA-lysozyme adsorbed film, major changes to thickness, and RI occur within the decreasing pH range from 6.0 to 2.9 (Figure 2C). While the thickness gradually decreases from 3.2 to 2 nm, the RI initially increases reaching a maximum value at pH 3.6 and then decreases, and reaches a final value of 1.462 at pH 1.6. Although the loss of thickness is partially offset by an increase in refractive index, a total polymer mass loss of 40% in response to pH changes is observed. Overall, DPI measurements show that the pH range over which the optical thickness and refractive index of the multilayers change are distinctly different, as might be expected from the ionizable groups of the constituent polymers. While all the multilayers become thinner, due to the partial disassembly, the density (polymer concentration) of the remaining fractions of PGaLA-PLL and PGaLA-chitosan multilayers decrease, while the density of the PGaLA-lysozyme remaining film increases on completion of pH cycle. To provide information on composition and speciation of weakly acidic and basic groups that underlie the thickness and density changes of the PGaLA-based films shown by DPI, FTIRATR measurements were performed. Figure 3 show changes in the spectra of the PGaLA-based assembled multilayers at pH 7.0, after the downward pH sweep at pH 1.6 and on returning to pH 7.0. Absorbance bands at 1610 and 1725 cm-1 correspond to the asymmetric stretching band of the carboxylate and carboxylic group of PGaLA, respectively. Absorbance bands at 1635, 1645, and 1646 cm-1 correspond to the amide I band of the polycation (chitosan, PLL, and lysozyme, respectively). No significant changes in the position of the amide I peak maxima were observed during the pH challenge, and so they remained at the wavenumbers at which the calibration measure-


363

Figure 3. FTIR spectra for (A) PGaLA-PLL, (B) PGaLA-Chi, and (C) PGaLA-Lys response to pH changes.

ments were performed. The absorption peaks for the PGaLA and the amide I bands of the polycations partially overlap in the region 1550-1700 cm-1 leading to a broad absorption peak dominated by the absorption due to PGaLA with a shoulder in the region of the amide I band for the PGaLA-PLL (Figure 3A) and PGaLA-Chi (Figure 3B) multilayers. Absorbance in the amide I region of lysozyme dominates the spectrum of the PGaLA-Lys multilayers (Figure 3C) at pH 7.0 with an overlapping PGaLA absorbance peak giving rise to a second less strong peak at 1610 cm-1. It should be noted that the amide I bands of the PGaLA-Chi and PGaLA-Lys spectra include contributions from the PLL base layer. The variation of the FTIR-ATR absorbance at 1610 cm-1, 1635-1646 cm-1 and 1725 cm-1 with pH are shown in Figure 4 provide more detailed information on the composition and speciation throughout the acid cycle. The changes observed in the spectra on the stepwise reduction of the pH to 1.6 are similar for PGaLA-PLL and PGaLA-Chi. As the pH is lowered, protons start binding to the galacturonate residues of the PGaLA, and progressively lose their charge and the corresponding electrostatic interactions within the multilayer. Consequently, we observe a decrease of the intensity of the peak at 1610 cm-1, while a new absorbance band assigned to the

364


Figure 4. Peak intensity of carboxylate COO- (-0-), amide I (-O-), and carboxylic COOH (-∆-) as a function of pH for (A) PGaLA-PLL, (B) PGaLA-Chi, and (C) PGaLA-Lys.

COOH group occurs at 1725 cm-1, as shown in Figure 3. As the pH is lowered, the significant changes in the 1610 cm-1 band of PGaLA-PLL and PGaLA-Chi occur when the pH reaches value of 4.2. Figure 4A,B shows that an initially constant value of the absorbance at 1610 cm-1 progressively decreases as the pH falls below 4.2. In the case of PGaLA-Chi, these changes are initially more rapid than for the PGaLA-PLL multilayers, indicating a weaker, more easily disassembled structure. For the PGaLA-Lys deposit (Figure 4C), the decrease in the 1610 cm-1 absorbance peak commences at pH 4.8 and falls rapidly as the pH approaches 1.6. The 1646 cm-1 amide I band of the lysozyme also decreases over the pH range of 4.8-1.6. As the pH is decreased from 7.0 to 1.6, the amide I band spectra of the PGaLA-Lys multilayer (Figure 3C) falls to 30% of its original value, indicating a large loss of lysozyme. Whereas the absorbance due to carboxylate (1610 cm-1) varies due to the binding of protons yielding carboxylic acid groups, as well as the loss of PGaLA from the multilayer, the decrease of intensity of amide I band indicates the loss of polycation by desorption. On pH increase to neutral, as expected, in all three multilayer systems the intensity of 1725 cm-1 decreases while the intensity of 1610 cm-1 increases due to the

Westwood et al.

ionization of COOH. Overall, net losses of PGaLA throughout the pH cycles are apparent in all three systems (Figure 4) at pHs above 5.0 at which point the ionization appears to be complete. The peak assigned to the carboxylic acid residues shows some hysteresis that is due to the loss of PGaLA from the multilayer and changes in the ionization behavior of the carboxylate groups due to irreversible changes in their environment,21 which will be described in more detail later. Overall, PGaLA-PLL system changes associated with amide I band are more extensive, showing a 60% mass loss of PLL as compared with 20% mass loss for PGaLA. For PGaLA-Chi multilayers, the changes observed during the pH decrease correspond to a 50% mass loss of chitosan and less than 10% of the PGaLA. The spectrum of the PGaLA-Lys multilayers (Figure 3C) is transformed due to a large preferential loss of the lysozyme (at least 80%) and a 15% loss of PGaLA over the pH cycle. Because changes in the infrared absorption are largely dependent upon loss of polymer or, in the case of the PGaLA, through changing speciation further analysis is required to interpret these spectra. Due to a limited overlap between the absorbance due to the PGaLA carboxylate groups and the amide I bands of the polycations (Figure 3), an apparent increase of the amide I band was observed on increasing the pH from 1.6 to 7.0 as shown in Figure 4. As noted in Materials and Methods, the magnitude of this effect follows the order that would be expected from the separation of the peak positions, that is, PGaLA-Chi (difference in wavenumber of peaks, 25 cm-1) > PGaLA-PLL (35 cm-1) ∼ PGaLA-Lys (36 cm-1). Thus, the overlap is most marked in the PGaLA-Chi results (Figure 2B). For this system a correction was made to the absorbance at 1635 cm-1 prior to calculating the polycation (Chi) concentration. Potential corrections to the PGaLA-PLL and PGaLA-Lys data were relatively small (less than 10%) and of a similar magnitude to the uncertainties in the calibration, and so these corrections were not applied. These calculations allow the constituent polymer mass, total polymer masses, and fractional ionization of the PGaLA to be calculated from the absorbance data (Figure 4). The total polymer mass was measured both by FTIR-ATR and, more directly, by DPI. To compare the pH-response on the polymer mass, each has been expressed as a percentage of its initial value and the polymer mass profiles are displayed in Figure 5. When the results are compared, the overall trends and magnitudes are similar. However, the DPI reports a reduction in polymer mass for the PGaLA-PLL and PGaLA-Chi multilayers as the pH is increased from 1.6 to 7.0, and this is not apparent in FTIR, due to the partial overlap of amide I band and COO- and calibration constant uncertainties. The fraction of PGaLA ionized, R,30 as a function of pH is shown in Figure 6. This shows that at the lowest pH there is still a significant proportion of ionized carboxylate (0.15-0.21) despite being more than 2 pH units below its solution pKa13. The main feature of Figure 6 is the hysteresis of ionization over the pH cycle, which gives an indication of the different strengths of interaction between polyelectrolytes incorporated into the multilayer films. In solution, the ionic interactions within the PGaLA chain mean that its ionization stretches over a greater range of pH than the monomeric acid.13,53 Due to this, polyelectrolyte effects the pKa of PGaLA over the range 3.0-4.5 and so the ionization might be expected to decrease to 50% at about pH 3.7. However, this is not observed experimentally and the pKa appears to be shifted to an apparent value, pKa(app). This phenomenon has been observed previously.20,21,30,31 The charge present on the multilayer film surface has a strong effect on the


Figure 5. Polymer mass remaining as a function of pH for (A) PGaLAPLL, (B) PGaLA-chitosan, and (C) PGaLA-lysozyme multilayers obtained with DPI (-∆-), and FTIR-ATR (-0-). The arrows indicate direction of the pH cycle.

Figure 6. pH response of the fractional ionization of the anyhydrogalacturonic residues in PGaLA-based multilayers measured using FTIR-ATR. Compositions: PGaLA-PLL, 9; PGaLA-chitosan, b; PGaLAlysozyme, 2.

acid-base equilibrium of adsorbing polyelectrolyte chains resulting in the shift in the pKa(app) toward lower pH value for polyacids.20 pKa(app) of PGaLA shifted toward more acidic value,


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due to the strong interactions with polycation, which suppress the protonation process. Whereas in our study the effect was probed using FTIR-ATR, in the work of Barrett and Burke, electrokinetic potentials were measured.20 This means that, whereas in our study we observed the effect averaged through the multilayer, in the study of Barrett and Burke, the effect was in the surface of the multilayer at the so-called electrokinetic shear plane.54 For the PGaLA-PLL multilayer, the pH at 50% PGaLA ionization, pKa(app), is about 2.0, whereas on the upward sweep it is about 3.9, a difference of 1.9 pH units (Figure 6A). This is because the strong polyanion-polycation interactions initially present in the multilayer have been broken, the polycation has been preferentially lost from the multilayer and on reionisation the PGaLA is closer to its bulk solution ionization behavior. For the PGaLA-Chi multilayer, pKa(app) is 2.4 on the downward sweep and 3.8 on the upward sweep, showing a shift of 1.4 pH units (Figure 6B). This is a smaller effect than is observed for the PGaLA-PLL multilayer, which is further evidence for the weaker interactions within the PGaLA-chitosan multilayer. On the upward pH sweep the apparent pKa(app) of the PGaLA is similar to that in the PGaLA-PLL multilayer. The ionization behavior of PGaLA in the PGaLA-Lys film is similar to that in the PGaLA-Chi film although at the lowest pH it does show the lowest degree of ionization at 0.16. Given that the PGaLALys film lost its polycations most readily suggests that it is not PGaLA-Lys interactions that are wholly responsible for inhibiting the PGaLA’s protonation. In this thin layer, the PLL, which forms a base layer, can be expected to continue to play a significant role in inhibiting protonation. Charge Balance within the Multilayers during pH Change. In addition to composition the FTIR-ATR gives access to charge balance within the multilayer films. The films are being adsorbed on an oxidized silicon surface which carries a negative charge. In comparison with the highest charge density polyelectrolyte, PLL, the charge spacing is relatively large and the charge density is low. The silanol groups on the surface are weakly acidic resulting in a pH dependent surface charge density. The titrations of Dove and Craven55 on colloidal silica showed a surface charge density of 2.7, 0.8, and 0.2 µC cm-2 at pH 7.0, 6.0, and 5.0, respectively. The PLL base layer has a concentration of about 60 ng cm-2 which, fully ionized, would be expected to carry a charge of 50 µC cm-2, which means that the underlying charge on the silica surface is small in comparison to the charges within the adsorbed multilayers. Using the FTIR-ATR measurements for the assembled multilayers allows a calculation of intrinsic charge balance, that is, the balance between those charges carried on the polymers (and surface; Table 2). The charge ratios show that there is an excess of negative charge within the multilayers i.e. the charge on the PGaLA exceeds that on the PLL, chitosan, and lysozyme. This is expressed as a ratio (positive charge/negative charge) and is reported in Table 2. For the purpose of these calculations, the net charge on lysozyme was used. During the pH cycle, the charge balance is reversed. The largest effect is for the PGaLA-PLL and PGaLA-lysozyme layers, the two higher polymer concentration multilayers, for which the intrinsic cationic charge exceeds the anionic charge by a factor greater than 2.0 at pH 1.6. The reversal of charge balance in the PGaLA-chitosan multilayer is relatively modest, with the positive/negative ratio reaching 1.11, close to intrinsic charge balance. On returning the pH to 7.0 at the end of the pH cycle the excess of negative charge in the multilayer returns. At the final pH there is over a 4-fold excess for the PGaLA-

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Table 2. pH Response of PGaLA-Based 10-Layer Multilayers Characterized Using DPI and FTIR-ATR DPI multilayer PGaLA-PLL PGaLA-chitosan PGaLA-lysozyme

a

FTIR-ATR

pHa

optical thickness (nm)

polymer concn (g mL-1)

polymer mass (ng cm-2)

polymer mass (ng cm-2)

polymer mass ratio (PC/PA)

charge ratio (()

7.0i 1.6 7.0f 7.0i 1.6 7.0f 7.0i 1.6 7.0f

18.3 ( 1 17.4 ( 1 11.9 ( 1 32 ( 1 28 ( 2 20 ( 5 3.2 ( 0.4 1.90 ( 0.01 1.97 ( 0.01

0.51 ( 0.05 0.39 ( 0.01 0.41 ( 0.02 0.31 ( 0.02 0.26 ( 0.02 0.25 ( 0.06 0.79 ( 0.07 0.84 ( 0.09 0.81 ( 0.08

920 ( 100 690 ( 100 500 ( 100 970 ( 150 715 ( 150 470 ( 20 250 ( 40 160 ( 20 160 ( 20

1310 ( 40 950 ( 30 910 ( 30 870 ( 90 120 ( 10 65 ( 10 335 ( 40 160 ( 5 160 ( 4

0.54 0.32 0.33 0.55 0.36 0.26 1.6 0.50 0.49

0.74 2.04 0.45 0.28 1.11 0.24 0.71 2.14 0.64

i, initial; f, final.

chitosan multilayer, a 2-fold excess for the PGaLA-PLL multilayer, and a positive/negative ratio of 0.64 for the lysozyme. This shows that the multilayers do not maintain constant intrinsic charge balance during environmental response and can sustain large charge imbalances of either sign characterized by positive/ negative charge ratios varying between 0.24 and 2.14. The recent work of Crouzier and Picart26 indicates that factors other than charge balance may control the stoichiometry of layerby-layer deposited polysaccharide-based multilayer films. In a comparative study of the assembly of multilayer films from a series of polysaccharides of increasing charge density (hyaluronan, chondroitin sulfate (CSA), heparin) with poly(L-lysine) they found that, after the deposition of 10 layers, the ratio of the number of polyanion monomers over the number of polycation monomers tended to a constant value of 0.5 irrespective of polysaccharide charge. This indicates that monomer ratio rather than charge ratio can determine stoichiometry. Initially our PGalA-PLL multilayer has a cation/anion charge ratio of 0.74 (Table 2), which corresponds to a polyanion/polycation ratio of 1.35 that contrasts with the value of 0.5 for the multilayer containing CSA, which possesses a similar charge density to PGalA. There could be several reasons for this difference. The charge on the CSA is carried by both sulfate and carboxyl groups and the underlying polysaccharide backbone with its mixed linkages is different, which gives rise to contrasting interactions and conformational properties. The larger monomeric groups of polysaccharides as compared with polyamino acids and their diverse monomer units and linkages means they are not behaving as model polyelectrolytes. QCMD Measurements, Hydrated Mass, and a Consolidated Description of Multilayer pH Response. The pH response of the films were also characterized using QCMD, which yields acoustic measurements of the hydrated mass of the multilayers in real time and also, through the dissipation, information on the changing material properties of the multilayers.14,15,40 In terms of the pH responsiveness, DPI (Figures 2 and 5) and FTIR-ATR (Figures 3-6) measurements showed that PGaLA-PLL films underwent irreversible changes in response to the acid cycle. In the QCMD, the response of the hydrated PGaLA-PLL film to the acid cycle can be described in four sections (Figure 7A): (i) pH 7.0-2.5, a small reduction of both ∆f3/3 and ∆D3 corresponding to a minor shrinkage of the multilayer, (ii) pH 2.5-1.6-2.9, a more extensive reduction of both ∆f3/3 and the ∆D3, corresponding to further shrinkage, (iii) pH 2.9-4.1, a sharp increase in both ∆f3/3 and ∆D3, which corresponds to swelling of the multilayer, (iv) pH 4.1-7.0, finally, as the pH is increased to 7.0 further decreases in ∆f3/3 and shrinkage. To give a consolidated description of all the measurements, each of the above sections of the QCMD response will be

described in the context of the DPI (Figure 2 and 5) and FTIRATR measurements (Figures 3-6), which we have presented previously. In the first section, pH 7.0-2.5, the QCMD shows that the multilayer undergoes a small shrinkage, which is consistent with the small reduction in optical thickness sensed by the DPI (Figure 2A). Although in the region of the bulk solution pKa of the polygalacturonic acid (3.0-4.5), the protonation has been largely suppressed by the strong polyanion-polycation interac-

Figure 7. QCMD frequency (s) and dissipation shifts (----) in response to pH changes for (A) PGaLA-PLL, (B) PGaLA-Chi, and (C) PGaLALys multilayers.


tions. Over this pH range, FTIR-ATR shows that the ionization of the PGaLA decreases to 0.8 (Figure 6). The origin of this shrinkage is likely to be due to a reduction of the Donnan pressure56,57 as the charge within the multilayer reduces. In the second section, pH 2.5-1.6-2.9, as the pH reduces to its minimum value the multilayer is in a highly dynamic state. QCMD shows a net loss of hydrated mass over this pH range. The RI (DPI, Figure 2A) shows a reduction corresponding to a decrease in density or, equivalently, an increase in hydration. This has been assigned to both loss of polymer mass and also the preferential loss of PLL which reduces the “crosslinking” of the multilayer. During multilayer assembly40 the DPI showed that the deposition of PLL resulted in an increase in density whereas PGaLA deposition led to a density decrease. This leads to the conclusion that during deposition the relatively small PLL polymer diffuses among the outermost PGaLA chains without causing a corresponding volume increase hence resulting in a density increase. The PGaLA-PLL multilayer showed exponential growth on assembly which is associated with the polymers being diffusively mobile52 within the multilayer. In the pH response experiment once the PGaLA loses its charge, the diffusive mobility within the multilayer means that the PLL is likely to be readily desorbed from the multilayer. In its acid form, PGaLA self-associates58 and so, at the lowest pHs, the PGaLA-rich multilayer (Table 2) may also be cross-linked through these hydrogen-bonded structures. At pH 1.6, the DPI (Figure 2A) shows a reduced thickness and hydrated mass, this contrasts with the behavior in the QCMD, and the conclusion is that conditions in the DPI experiments must be slightly different, perhaps due to the different geometries and hydrodynamic conditions in the two instruments. In the third section, pH 2.9-4.1, the QCMD shows a swelling feature in this pH range, which corresponds with the pH at which FTIR-ATR shows the PGaLA is reionizing (Figure 6). It might be expected that as the PGaLA ionises the polyanion-polycation interactions which developed during assembly would be re-established. However, the shift in pKa(app) indicates that this is not the case. The preferential loss of PLL during the acid cycle means there is insufficient material to reform the original structure of the multilayer and so the PGaLA ionization results in swelling. Finally, over the pH range 4.1-7.0, the QCMD shows the resumption of shrinkage in this pH range. The reionization of PGaLA is complete as the pH approaches 6.0 (Figure 6) and the internal structure of the multilayer readjusts to its final state of charge balance. The estimated charge ratio (Table 2) indicates there is a 2-fold excess of anionic charge that, combined with the shift in apparent pKa, indicates the multilayer has a distinctly different structure as compared with the one originally deposited. The pH-response of the PGaLA-chitosan multilayer characterized using QCMD is shown in Figure 7B. The frequency shifts show a stronger response for the PGaLA-chitosan multilayer as compared with the PGaLA-PLL multilayer, indicating that the polyanion-polycation interactions within the PGaLAchitosan multilayer are more pH sensitive due to the lower charge density and weakly basic behavior of the chitosan. The QCMD response can be described in three sections: (i) pH 7.0-4.1, the magnitude of ∆f3/3 decreases indicative of a reduction of hydrated mass or multilayer shrinkage, (ii) pH 4.1-1.6-2.5, ∆f 3/3 shows a small increase corresponding to multilayer swelling and, finally, (iii) pH 2.5-5.5, ∆f 3/3 decreases, culminating in a collapse of the signal at pH 5.5, which we interpret as a shrinkage of the multilayer followed by its detachment from the sensor surface.


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In the first section, pH 7.0-4.0, the reduction of hydrated mass is confirmed by the DPI (Figure 2B), the multilayer is shrinking and its density is increasing with no loss of polymer mass (Figure 5B). This is driven by the changing ionization of the PGaLA and the chitosan. Though the protonation of the PGaLA is shifted to lower pH by the polyanion-polycation interactions as the pH reaches 4.0 the absorbance due to the carboxylic acid groups is starting to appear in the FTIR-ATR spectrum (Figure 3B) and so the charge on the PGaLA is starting to decrease. However, the amine groups of the chitosan have a solution pKa of about 6.7,18 and, so, in this pH range they will be binding protons and increasing their cationic charge. Thus, we conclude that the shrinkage is due to an increase in the number of polyanion-polycation interactions due to the increasing polycation charge. By analogy with a gel, as the number of “crosslinks” increases, the network exerts a lower osmotic pressure and it will shrink.57 In the second section, pH 4.0-1.6-2.5, as the pH is further lowered from pH 4.0 to 1.6, and then, back to 2.5 there is a net swelling of the multilayer. The DPI (Figure 2B) confirms that over this pH range the optical thickness is increasing and the density is decreasing. It is over this pH range that FTIR-ATR shows the PGaLA becomes protonated (Figure 3B and 6) and, as with the PGaLA-PLL multilayer, the multilayer is in a highly dynamic state. Simultaneous with the protonation of the PGaLA (Figures 3B and 6) is a preferential loss of chitosan from the multilayer as quantified by the reduction in the PC/PA mass ratio (Table 2). Over the whole acid cycle there are net losses of PGaLA (Figure 4B), but whether it occurs prior to reaching pH 1.6, as suggested by FTIR-ATR (Figure 5B and Table 2) or on the upward pH sweep, as suggested by DPI (Figure 5B), is unclear. Charge balance calculations show that the PGaLA-chitosan multilayer is close to intrinsic charge balance at pH 1.6 (Table 2). At this pH, the levels of charge on the constituent polymers have reversed in relative magnitude. Whereas Figure 6 shows that the fractional ionization of the PGaLA is 0.20, the chitosan with a degree of acetylation 0.50 is expected to be fully ionised with 0.5 of its monomer units carrying positive charges. In the final section, pH 2.5-5.5, the QCMD ∆f 3/3 decreases (Figure 7B), indicating that the multilayer initially shrinks and then, at pH 5.5, ultimately the frequency drops to close to zero, which corresponds to desorption of the remaining multilayer from the QCMD chip. While the shrinkage of the multilayer is detected by the DPI, it does not show the same complete desorption phenomenon. Ono and Decher24 describe a multilayer system in which the layer close to the surface is sacrificial allowing a free-standing film to be prepared. While the end result is similar this is not the situation in the present PGaLA-Chi system. The initial PLL-PGaLA layer would be expected to remain in place as is the case for the PGaLA-PLL multilayer. In our situation, the shrinkage of the multilayer could generate stresses parallel to the QCMD chip surface, which would result in the film fracturing from the surface. The pH-response of the PGaLA-Lys adsorbed layer characterized using QCMD is shown in Figure 7C. In contrast to the PGaLA-PLL and PGaLA-Chi multilayers, there are a continuous series of changes occurring in the PGaLA-Lys adsorbed layer as the pH is changed as might be predicted from the polyampholyte character of this protein.19 As soon as the pH is reduced from 7.0, the negative charges on the protein reduce in number, increasing the net positive charge on the macromolecule. Over the pH range 4.8-3.6 ∆f3/3 decreases indicating loss of hydrated mass. The FTIR-ATR indicates there are simultaneous losses of lysozyme and PGaLA, a stripping process, similar to what

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was observed during the deposition of this layer.40 Once the pH has reached 1.6, the amide I absorbance is equivalent to that of the PLL precursor layer and results on the upward pH sweep cannot be linked with the behavior of lysozyme. Overall, as a result of the pH cycle, the PGaLA-Lys deposit partially dissolved, a process that was largely complete on the downward pH sweep.

Conclusions This study presents a detailed characterization of the effect of the polycation type on the pH response of PGaLA-based multilayers. The type and number of charges on the polycation affect both multilayer growth and its subsequent pH response. All the PGaLA-based multilayer films undergo irreversible changes in response to changes in pH, and in Table 2 the parameters describing the pH response of these multilayers is summarized. DPI measurements show that, while all the multilayers become thinner, due to the partial disassembly, the density (polymer concentration) of the residual PGaLA-PLL and PGaLA-chitosan multilayers decrease, while the density of the residual PGaLA-lysozyme film increases on completion of the pH cycle. The FTIR-ATR technique enables a quantitative analysis of multilayer composition and the investigation of the chemistry and interactions within the multilayer that relate directly to the stability of the multilayers to environmental changes. In particular, it allowed for the monitoring of the charge density changes at each step as the assemblies are exposed to different pH solutions. As shown in this study, lowering pH leads to a progressive decrease of the negative charge of PGaLA due to proton binding to COO- groups. This consequently results in weaker electrostatic interactions, which result in partial dissolution of the PGaLA-based films. Depending on the polycation used, we observed that for PGaLA-PLL system pH changes leads to a 60% mass loss of PLL as compared with 20% loss for PGaLA. For PGaLA-chitosan multilayers, the changes observed during the pH decrease correspond to a 50% reduction of chitosan and 10% of the PGaLA. For PGaLAlysozyme multilayers, we observed preferential loss of lysozyme by 80% and 35% loss of PGaLA over the pH cycle. In all cases, most of the disassembly took place at a pH lower that pKa of PGaLA, an indication that this factor was crucial for the stability of the films. Depending on the pH range of interest, the ionization properties of both the polycation and the polyanion determine the overall properties of the assembled multilayer. The layer by layer assembly results in a nonequilibrium structure and a charge state in which the pKa are shifted from their dilute solution values. Intrinsic charge balance is neither observed in the initial assembly nor during pH response. Intrinsic cationic charge/anionic charge ratios vary widely from 0.28-0.74 on assembly at pH 7.0 to 1.11-2.14 at pH 1.6 and 0.24-0.64 at pH 7.0, indicating a dynamic extrinsic charge balance. The self-association of the PGaLA in the acid form stabilizes the film structure at low pH. This provides insight into the behavior of PGaLA in multilayer assemblies and the relationship between structure, composition, and functionality, which will underpin the directed assembly of PGaLA-based, pH-responsive biomaterials. Acknowledgment. The UK Biotechnology and Biological Sciences Research Council supported this research from the Institute Strategic Programme Grant to the Institute of Food Research, Norwich, and through the award of the responsive mode grants (BBE0131711 and BBE0110041). The authors would also like to thank Dr. Nikolaus Wellner for his support.

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Environmental Responsiveness of Polygalacturonic Acid-Based

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