The Assembly and Disassembly of Biopolyelectrolyte Multilayers and

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 19, 2016 | http://pubs.acs.org Publication Date: March 3, 2009 | doi: 10.1021/bk-2009-1007.ch002

Chapter 2

The Assembly and Disassembly of Biopolyelectrolyte Multilayers and Their Potential in the Encapsulation and Controlled Release of Active Ingredients from Foods J. Moffat, R. Parker, T. R. Noel, D. Duta, and S. G. Ring Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom

The assembly of multilayers using layer-by-layer (LbL) deposition of oppositely charged biopolyelectrolytes has the potential to form novel barriers with applications in controlled delivery in the food and pharmaceutical industries. Multilayer assembly was studied using FTIR-ATR and a quartz crystal microbalance to give information on the mass of the deposited layers, their chemical characteristics and hydration. The polyanions examined were pectins of varying degrees of esterification, while chitosan, poly-L-lysine and, at a pH below its pI, β-lactoglobulin were the polycations. Multilayer formation occurred at pH's when both anionic and cationic polyelectrolytes carried a charge. The hydration of the structures was sensitive to variations in pH and ionic strength, resulting in environmentally responsive behavior. Dis assembly could be triggered by change in pH, or pH history, resulting in an initial weakening of attractive interactions and subsequent solubilisation of the components. A centrifugation technique for applying a chitosan/pectin multilayer coating to emulsion droplets is also described.

© 2009 American Chemical Society

In Micro/Nanoencapsulation of Active Food Ingredients; Huang, Qingrong, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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Introduction The structure and composition of foods affect the site and rate of release of their components and, i f manipulated in a rational way, offers the opportunity for foods with controlled or modified release functionalities. Multiple phases are present in foods (/); both in foods themselves and when they are mixed with digestive fluids and so interfacial processes assume a key role in the science of the release process. In pharmaceutical technology there are many types of functional coatings and structures for achieving controlled and modified release of bioactive compounds (2). Designing foods as delivery systems, however, presents a number of distinctly different challenges. Polyelectrolyte multilayer approaches offer a means of structuring interfaces with potentially useful functionalities (3). The majority of research has been on synthetic polyelectrolyte multilayers (3, 4) but more recent studies have reported results for biopolyelectrolytes (5, 6) including food biopolymers (7, 8). In previous work we have studied the assembly of biopolyelectrolyte multilayers comprising anionic polysaccharides with cationic polysaccharides, polyamino acids or globular proteins using a range of complementary techniques (9, 10, 11). Surface plasmon resonance was used to follow the sequential layer by layer deposition of pectin and chitosan in real-time under conditions of continuous flow (9). The stoichiometry of pectin/poly-L-lysine multilayers was such that the polymers carry a net positive charge contrary to the common assumption of intrinsic charge balance (//). In the present work the aim was to identify the factors underlying the disassembly of multilayers in response to changing environmental conditions. In order to test whether multilayer environmental responsiveness investigated upon plane surfaces can be related to release characteristics in three dimensional systems, the multilayers need to be deposited on disperse systems. A centrifugation method for achieving this is also described.

Materials and Methods Materials Poly-L-lysine hydrobromide (PLL) with a mean degree of polymerization of 70 and β-lactoglobulin ( B L G ) were obtained from Sigma, low molecular weight chitosan (nominal degree of acetylation 75-85%) was obtained from Aldrich, citrus pectins with degrees of esterification 51% and 71% were obtained from CP Kelco (Denmark) and citrus polygalacturonic acid (PGA) was obtained from Fluka. D 0 (99.9%), hexadecane, L-a-phosphatidylcholine, sucrose and buffer salts were also obtained from Sigma. Chemical and physicochemical 2


37 characterisation of the pectins has been reported previously (//, 12). For multilayer deposition polymers were dissolved at a concentration of 0.6 mg mL" in 10 m M buffer solution with 30 m M NaCl. For base layers P L L was dissolved at a concentration of 0.08 mg mL" . 1

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Multilayer-coated emulsion preparation Five g of hexadecane was blended for 3 min with 95 g of 1% w/w L-aphosphatidylcholine in 100 m M pH 3.0 acetate buffer using an ultra Turrax T25 fitted with an S-25N head. 1 mL of the emulsion was then dispersed in 40 mL of 25% w/w sucrose. A gradient was formed in a 15 mL centrifuge tube in the order: emulsion/25% w/w sucrose; chitosan/20% w/w sucrose; pectin (or PGA)/15% w/w sucrose; chitosan/10% w/w sucrose; pectin (or PGA)/5% w/w sucrose. The polymer concentration was 0.08 mg/mL in pH 3.6 acetate buffer. Each layer was separated by a layer of buffer containing sucrose of the appropriate concentration. After centrifugation at 2000 g for 30 min, emulsion droplets were harvested from each of the buffer layers.

Fourier Transform Spectroscopy

Infrared-Attenuated Total Reflection (FTIR-ATR)

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Infrared spectra were collected, over the range 2000-1500 cm" , on a Nicolet 860 FTIR spectrometer (Thermo Electron Corporation, Madison, U S A ) fitted with a silicon cylindrical A T R crystal in a MicroCircle liquid A T R cell (SpectraTech, Warrington, U K ) . Methods used were as detailed in Krzeminski et al. (11). The silicon crystal presented a silica surface for multilayer deposition. The multilayer was deposited by a layer-by-layer (LbL) procedure (3). A base layer of the cationic polyamino acid P L L is first deposited on the silica surface of the A T R crystal of the FTIR by injecting P L L solution into the sample cell. After an adsorption time of 3 min, the excess solution is washed off by injecting buffer solution (as used to dissolve the PLL). This is followed by injecting a polyanion solution, followed by a buffer wash, a polycation solution, a buffer wash and so on until the required number of layers has been deposited. Absorbances were measured relative to a baseline at 1800 cm" and quantitative analysis was based on methods of Sperline et al. (13). 1

Quartz Crystal Microbalance Measurements were made with a D300 quartz crystal microbalance with dissipation monitoring ( Q C M D , Q-Sense A B , Vâstra Frôlunda, Sweden) with a


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Q A F C 302 axial flow measurements chamber as described in Krzeminski et al., (11). The sensing element was a disc-shaped piezoelectric quartz crystal sandwiched between two gold electrodes which presented a gold surface for multilayer deposition. A n approximate analysis of the frequency shift of the microbalance can be made using the Sauerbrey approach (14) to give the hydrated mass of the adsorbed layer. A more refined analysis takes the viscoelasticity of the multilayer into account (15) and was performed using QTools software (Q-Sense A B , as above).

Microelectrophoresis Electrophoretic mobility of the emulsion droplets in pH 5.6 acetate buffer was determined using a Zetasizer 3 (Malvern Instruments) fitted with a A Z 4 electrophoresis cell calibrated using a latex electrophoretic mobility standard.

Results The disassembly of multilayers was studied by first assembling multilayers directly on the surface of the sensing element of the spectrometer or microbalance and then following their behavior as the solvent conditions were changed. Pectin/protein multilayers can be assembled at pH's below the isoelectric point of the protein under conditions under which the pectin carries a negative charge due to its ionized anhydrogalacturonic acid groups and the protein carries a net positive charge because its positively charged residues outnumber its negatively charged residues. Figure 1 shows the result of a pH titration on a 8 layer pectin/p-lactoglobulin multilayer probed using FTIR-ATR. 1

The spectrum of the multilayer in the frequency range 1500 - 1800 cm" is dominated by the amide I band (1635 cm" ) of the B L G with small absorption bands arising from the - C O O D stretch vibrations of the anhydrogalacturonic acid groups (1730 cm" ), -C=0 uronic acid methyl ester and the asymmetric stretch o f - C O O " (1612 cm" ) of the pectin (16). The p K of the uronic acid of pectin vary in the range 3.5 - 4.5 (17) and so both ionised and unionised species can be expected at pD 3.6. The carboxyl and carboxylate groups of the acidic amino acid residues (aspartic and glutamic acid) of B L G absorb in the region of 1725 cm" and 1572 cm", respectively. Figure 1 shows spectra for the multilayer as it is rinsed with buffers of increasing pH over the range 3.6 - 5.4. Initially, over the range 3.6 - 4.8, the absorbance associated with carboxyl groups of pectin and/or the protein (1725-1730 cm" ) shows a small decrease. Simultaneously there is an increase in the absorbance associated with the aspartate and glutamate residues of the protein (1572 cm" ). This indicates that while there is an increasing number of negative charges on the protein and a 1

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The Assembly and Disassembly of Biopolyelectrolyte Multilayers and

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