Polyelectrolyte Complexes and Layer-by-Layer Capsules from

Biomacromolecules 2002, 3, 579-590

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Polyelectrolyte Complexes and Layer-by-Layer Capsules from Chitosan/Chitosan Sulfate Gisela Berth,*,†,‡ Andreas Voigt,†,‡ Herbert Dautzenberg,† Edwin Donath,§ and Helmuth Mo¨hwald† Max Planck Institute for Colloids & Interface Research Golm, D-14424 Potsdam, Germany, Capsulution NanoScience AG, D-14476 Golm, Geiselbergstrasse 69, Germany, and University of Leipzig, Institute of Medical Physics & Biophysics, D-04103 Leipzig, Germany Received January 28, 2002

Polyelectrolyte complex formation of chitosans of varying average molecular weight and degree of acetylation with chitosan sulfate or poly(styrene sulfonate) was studied by static light scattering in dilute solution at various ionic strengths. Unlike the molecular weight, the degree of acetylation was found to have a significant effect on the resultant structural densities of the complexes. The same system was applied to the preparation of micrometer-sized hollow shells by means of a layer-by-layer technique (in total eight layers). Their behavior toward fluorescent probes such as fluorescein and rhodamin 6G or fluorescein isothiocyanate labeled dextrans at various ionic strengths and pH (observed by confocal laser light scanning microscopy) could be understood through a discussion of electrostatic forces between the highly charged shells and the probes to be dominant. At an ionic strength of 0.1 M and above, charge effects are largely suppressed (screening effect) and a size-dependent “cutoff” for the permeation of the macromolecular fluorophore was observed. Introduction Polyelectrolyte complexes (PEC) have been revealed to be useful in a broad field of applications. A sophisticated application is the formation of well-defined thin films consisting of only a few macromolecular layers by means of a layer-by-layer (LBL) technique which has been introduced by Decher and Hong.1 With suspended micrometer size or nanosized templates coated as a core instead of planar surfaces, the LBL technique could successfully be used to produce isolated hollow shells when the template was dissolved subsequently and the resultant low-molecular weight fragments were washed out. The remaining (solventfilled) shells reproduce precisely the size and shape of the template.2 Both the preparation and behavior of such shells primarily made of poly(styrene sulfonate) (PSS) as anionic component and poly(allylamine hydrochloride) (PAH) or poly(diallyl dimethylammonium chloride) (poly-DADMAC) as cationic components on several types of templates have been reported in detail3,4 and reviewed.5 For practical as well as academic reasons, the use of ionic polysaccharides in place of synthetic polyelectrolytes seems an interesting alternative. Polysaccharides are distinguished from many synthetic polyelectrolytes, for example, by their multifunctional monomers and their relatively high intrinsic chain stiffness. This applies also to the system chitosan and chitosan sulfate. Chitosans have long been found in cosmetics and pharmaceuticals. Sulfated chitosans have been positively valued with * To whom correspondence may be addressed at Capsulution Nanoscience AG, PF 600651, D-14406 Potsdam, Germany. E-mail: [email protected]. † Max Planck Institute for Colloids & Interface Research Golm. ‡ Capsulution NanoScience AG. § University of Leipzig.

respect to blood compatibility6 and influence on infections.7 Close relatives by chemical structure (and likely by conformation) have already been combined to PECs elsewhere when microcapsules by coacervation8 or double layers on metallic plates9 were made of 6-oxy-chitin (a poly(uronic acid)) and chitosan. Unfortunately, nothing specific was known about the PEC formation between chitosan and chitosan sulfate. Since “... multi-layer build-up has much in common with solution phase or coprecipitated polyelectrolyte complexes”,10 some basic studies beyond the molecular characterization of the polyelectrolyte components were necessary in order to develop a better understanding. Spanning molecular parameters of the components and solution phase PEC properties and multilayer assembly behavior would help to discuss effects which otherwise are difficult to address. To achieve these objectives, we have built on previous experience by our group: (i) Well-characterized chitosan samples of various average molecular weights and degree of acetylation (DA) were available from previous work.11-13 Molecularly dispersed solutions (free of any supramolecular assemblies) could be prepared with special care at sufficiently low polymer concentration which is of major importance for subsequent light scattering measurements with regard to (ii) and would certainly be useful for coating species on the nanoscale with regard to (iii). Only little was known about chitosan sulfate, so its characterization would have to be part of this study. (ii) When PECs from synthetic polyelectrolytes were studied, static light scattering (SLS) could successfully be used to investigate effects of various parameters on the course of the reaction14,15 as long as the PECs remained quasisoluble in nonstoichiometric systems at rather low polymer

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concentration. Thus obtainable information seemed to be worthwhile in the light of how to produce and manipulate shells made of the same materials under similar conditions. Colloid titration with toluidine blue as metachromatic indicator has been revealed to be useful13 to check the stoichiometry of PEC formation involving chitosan. (iii) The preparation of micrometer-sized hollow shells by consecutive coating of acid-soluble melamine formaldehyde (MF) or PSS latex templates according to the previously introduced LBL procedure2 would be adaptable to any pair of polyelectrolytes. Findings from (i) and (ii) would help to choose proper conditions for the coating procedure. The process of coating as well as the finally produced shells could then be studied using confocal laser scanning microscopy (CLSM) and ζ-potential measurements.2 In this paper we report on PEC formation between chitosans of various molecular weights or degree of acetylation and chitosan sulfate in dilute solution and the preparation and behavior of hollow capsules made of these materials. For reason of comparison, chitosan sulfate was occasionally replaced by poly(styrene sulfonate) in the PEC part. Some findings from chitosan sulfate characterization are also reported. Experimental Section Materials. The chitosans Chit B and Chit C were commercial products and the subjects of previous chemical and physicochemical characterization.11-13 Chitosan sulfate was a gift from Professor L. S. Galbraikh and Dr. L. A. Vikhoreva, Textile Institute, Department Chemical Fibers, Moscow, Russia. The elemental composition CHNOS was analyzed in a “varioEL” apparatus version F from “elementar Analysensysteme” GmbH, Germany. The sodium salt of poly(styrene sulfonate) used in the PEC experiments and colloid titration was prepared by anionic polymerization (humidity ∼ 11% was determined iodometrically). The weight average molecular weight was Mw ) 66 000 g/mol. Fluorescein and rhodamin 6B as well as the fluorescein isothiocyanate (FITC) labeled dextrans of known average molecular weight Mw (2 × 106 g/mol, 2.6 × 105 g/mol, 7.7 × 104 g/mol, 4.4 × 103 g/mol) and degree of substitution were commercial fine chemicals (supplier, Sigma, Germany). Methods. All SLS measurements were performed in a modified Sofica goniometer, model 42000, FICA, France, between 30 and 145° in steps of 5° at an operating wavelength of the laser light source of λo ) 632 nm. The Zimm procedure was used for the molecular weight determination of chitosan sulfate with δn/δc ) 0.170 mL/g. Clarification of the solutions by ultracentrifugation and membrane filtration was carried out as described in detail for chitosans.11-13 The specific refractive index increment of the (desalted) sample dissolved in acetate buffer (equilibrium dialysis) was determined at 25.0 °C in a ScanRef differential refractometer (Nanofilm Technology NFT, Germany); for details see ref 11. High-performance size exclusion chromatography (HPSEC) was performed on four serially connected commercial columns HEMA Bio 1000, 300, 100, 40 (each 300 mm long

Berth et al.

and 8 mm in diameter; stationary phase, hydrophilic modified hydroxy ethyl methacrylate; producer, MZ Analysentechnik, Germany; sample, 100 µL at ∼1 mg/mL) at 25 °C with acetate buffer, pH 4.5, as solvent and eluant. The concentration was monitored by means of a DRI detector (Shimadzu, Japan). Sedimentation velocity and sedimentation equilibrium experiments were carried out at 25 °C at 60 000 and 20 000 rpm, respectively, in a Beckman XL-I analytical ultracentrifuge applying an on-line Rayleigh interferometer as described in detail for chitosans.12 Colloid titration was performed in an UV/VIS spectrophotometer Lambda 2 (Perkin-Elmer, USA). Details are given elsewhere.13 Polyelectrolyte Complex Formation in Dilute Solution. A weighted amount of the polysaccharide (∼1 mg/mL chitosan, ∼2.5 mg/mL chitosan sulfate) was dissolved overnight in 0.02 M acetate buffer, pH 4.5, dialyzed twice against the 10-fold volume of fresh buffer, and ultracentrifuged (90 min at 40 000 rpm and 25 °C in a preparative Beckman ultracentrifuge model L-70, rotor Ti70.1). The supernatant was filtered through a 0.45 µm pore size membrane filter (minisart, Sartorius-Membranfilter GmbH, Germany). The precise concentration of the chitosan stock solutions was determined by colloid titration with poly(styrene sulfonate) and toluidine blue as indicator.13 Correspondingly, the chitosan sulfate stock solution was titrated with a chitosan solution of known concentration. For the subsequent PEC formation experiments, these stock solutions were diluted to 1 × 10-4 monomol/L as initial concentration of the chitosan solution (A) in the measuring cell and 2 × 10-4 monomol/L as concentration of the added counterpart solution (B) using the specified salt-containing buffers. Solution A was then filtered directly into the measuring cell through a membrane filter of 0.2 µm pore size. The starting volume was 10.0 mL. Complex formation was carried out under slight stirring by adding in steps of 0.5 mL (speed, 4 mL/h; Harvard apparatus 22 pump) a specified volume of the polymer solution B through a 0.2 µm pore size membrane filter to solution A directly in the Sofica goniometer cell at 25 °C. This experimental configuration has revealed to prevent local overreactions15 which would mess up systematic investigations. The PEC concentration was calculated according to cPEC ) c0

A

V0A V0A + VdB

(

)

mA + mB X MA

with c0 and V0 being the initial concentration and volume of solution A, Vd the added volume of solution B, m the masses of anion and cation per mole charges, respectively, and M the mass of the electrically neutral moiety. X means the molar mixing ratio. The effective molecular masses per charge unit were determined by colloid titration to be as follows: m+(ChitC) ) m+(ChitB) ) 228 g/mol; m+(ChitC50) ) m+(ChitB50) ) 341 g/mol; m-(chitosan sulfate) ) 242 g/mol; m-(PSS) ) 183 g/mol. The specific refractive index increments for the individual components were taken as δn/δc ) 0.203 mL/g uniquely for all chitosans, δn/δc ) 0.192 mL/g for PSS, and δn/δc ) 0.170 mL/g for chitosan sulfate. The weighted average

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value was taken for the specified PEC based on 1:1 stoichiometry. Experiments at a given polymer mixing ratio but varying ionic strengths were performed by adding salt-containing buffer accordingly. Preparation of Hollow Capsules. To coat the MF template, the polymer samples were dissolved under slight shaking at room temperature overnight in 0.02 M acetate buffer/0.1 M NaCl, pH 4.5. The solvent contained NaN3 (20 mg/L) for preservation. The polymer concentration was ∼1 mg/mL. To remove some remaining flakes, the solutions were filtered through a membrane filter of 8.0 µm pore size. The pH was checked and did not exceed a value of 4.75. About 4 mL of an aqueous suspension of the monodisperse sphere-like MF template of 3.7 µm diameter ((Microparticles GmbH, Germany; ∼10 vol % solid) was used, and the template was coated with eight layers in total (starting with chitosan sulfate) using the Amicon filtration unit as described previously.16 About 10 mL of polysaccharide solution was added at each step. After 5 min of settling time, the excess polymer was filtered off and residual polymer was removed by washing the suspension with ∼ 50 mL of acetate buffer first followed by 100 mL of water and so on until the number of eight layers was reached. Then the portion was poured into ∼300 mL of 0.1 N HCl (all at once) and washed with water until neutral (pH ∼ 6). The suspension of shells in water could be stored at 4 °C over at least 12 months. Variation of pH and Ionic Strength. To subsequently change the pH and/or ionic strength in the shell suspension, a small volume of the aqueous suspension was mixed with the 10-fold excess of solutions of various pH (0.02 M acetic acid, pH 3.2; 0.02 M acetate buffer, pH 4.5; 0.02 M sodium acetate, pH 6.7) but unique ionic strength (0.1 and 0.5 M NaCl). The mixture was equilibrated overnight and then used for microscopic studies and ζ-potential measurements, the latter were measured in the corresponding medium. MilliQ quality water and fresh-washed shells were used for the experiments without added salt. Incubation with Fluorophores. Added fluorescein and rhodamin 6G (5 µL at 0.5 mg/mL in water) and FITC-labeled dextrans (10 µL at 1 mg/mL in water) were allowed to react with 500 µL of the shell suspension for at least 2 h at room temperature before the samples were studied microscopically (CLSM). A further check was carried out after 2 weeks and 3 months, respectively. Atomic Force Microscopy (AFM). The AFM images were taken by means of a Digital Instruments Nanoscope IIIa instrument in tapping mode. A droplet of the aqueous shell suspension was air-dried on a mica plate at room temperature. Confocal Laser Scanning Microscopy (CLSM). The CLSM was carried out on a LEICA TCS system (Aristoplan, 100× oil immersion) using commercial software. ζ Potential. ζ potentials were measured using the Malvern Zetasizer 3000HS Instrument at 25 °C. Results and Discussion Characterization of the Polysaccharide Components. Characterization of Chitosan. Chitosans are linear copolymers built up of glucosamine (GlcN) and N-acetyl glu-

Table 1. Molecular Parameters of the Polymers Used

a

sample name

Mw,a g/mol

DA,b %

Chit C Chit B Chit C-50 Chit B-50 PSS chitosan sulfate

140 000 40 000 n.d. n.d. 66 000 660 000 (?)

25-31 22-27 50 50 n.d.

Mw, weight average molecular weight. b DA, degree of acetylation.

cosamine (GlcNAc). They are soluble in aqueous acidic solution. At a pH ∼ 4.5 well below the pKa ∼ 6.5, all the free amino groups bind protons and the macromolecules become cations whose charge density depends on the DA. In the cationic state, they are potential partners for PEC formation. For the molecular characterization,11-13 chitosans were dissolved in 0.02 M acetate buffer, pH 4.5, with 0.1 M NaCl. The weight-average molecular weights in Table 1 determined by SLS were found in good agreement with results from analytical ultracentrifugation as well as HPSEC on TSK columns coupled with molecular weight detection by multiangle laser light scattering. Gel chromatography experiments coupled with colloid titration revealed chemically largely homogeneous samples having a broad molecular weight distribution. Characterization of Chitosan Sulfate. The term “chitosan sulfate” may denote a mixture of mono-, di-, and trisubstituted chitosans. NMR studies18 have shown the 6- and 3,6O-sulfated sugar residues to occur in random distribution beside nonsubstituted GlcN or GlcNAc residues and Nsulfated residues. In our sample, the molar ratio of the elements N:C:S:H:O in the original matter was found to be 1:6.1:1.7:12:10.6 and turned to 1:6:1:12:7 after thorough dialysis against distilled water suggesting the removal of some inorganic salt impurities from the preparation. About 70% of the original matter was retrieved after dialysis. The 1:1 ratio for N:S is indicative of the monosubstituted derivative. On the basis of the assumption of 1:1 stoichiometry, colloid titration with Chit C (Table 1) in 0.02 M acetate buffer, pH 4.5, yielded an average molar mass of 242 g/mol charges, which seems quite reasonable. In view of the PEC formation with chitosan, chitosan sulfate was dissolved and analyzed in the same acetate buffer (pH 4.5; ionic strength ∼0.12 mol/L) that was used for chitosan. The elution line on HEMA-Bio columns is given in Figure 1 along with the elution profiles of four commercial PSS calibration standards. The polysaccharide elution line covers the whole range between void and total volume with a remarkable accumulation peak at the void volume suggesting a broad size distribution that exceeded the separation range of the medium. The weight-average molecular weight by SLS was found to be Mw ∼ 660 000 g/mol (Zimm plot in Figure 2). The radius of gyration (in the legend) plotted versus the degree of polymerization is close to the relationship for chitosans13 suggesting no discernible influence of the sulfate group on the chain conformation. The sedimentation coefficient measured in an analytical ultracentrifuge at a concentration of 1.5 mg/mL has shown a very broad distribution with a maximum near 2.5 S (see for comparison

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Figure 1. Elution line of chitosan sulfate on HEMA columns. The elution lines of four PSS calibration standards are added for comparison: O, Mw ) 388 000 g/mol; b, Mw ) 168 000 g/mol; 3, Mw ) 84 000 g/mol; 1, Mw ) 38 000 g/mol.

in ref 12). Parallel sedimentation equilibrium measurements led to an average molecular weight not above 33 000 g/mol (vbar: 0.506 mL/g taken from our chitosan studies). The HPSEC data would support the high molecular weight by SLS rather than the low one by ultracentrifugation. Somewhat contradictory findings by the different methods are a wide-spread phenomenon in the polysaccharide area17 often indicating the presence of some high-molecular weight particulate matter. However, the magnitude of the deviations as observed here appears more severe. The relatively low second virial coefficient B by SLS along with extremely saltdependent and, compared with chitosan, rather low intrinsic viscosities is likely to result from stable (soluble) selfaggregates formed when negatively charged sulfate groups interact with protonated amino groups. In general, zwitterionic polymers are known to cause big problems upon characterization. Superficially considered, it would make sense to use a higher pH upon polymer characterization. The

polyanion

solvent

Chit C

chitosan sulfate

Chit B Chit C-50 Chit B-50 Chit C Chit C-50 Chit C

chitosan sulfate chitosan sulfate chitosan sulfate PSS PSS chitosan sulfate

Chit C-50

chitosan sulfate

(a) 0.02 M acetate buffer pH 4.5 (b) ... plus 0.1 M NaCl (c) ... plus 0.5 M NaCl (d) ... plus 0.033 M CaCl2 (a) & (b) (a) & (b) & (c) & (d) (a) & (b) (a) & (b) (a) & (b) mixing ratio 0.8 ionic strength varied up to 0.5 M mixing ratio 0.8 ionic strength varied up to 0.5 M

same higher pH upon PEC formation would, however, produce other serious problems (pH gradient). So, unlike the chemical data, results of the macromolecular characterization are preliminary and are to be taken with reservation. Complex Formation in Dilute Solution Monitored by Static Light Scattering (SLS). Table 2 gives an overview of the systems studied. As long as the mixing ratio of the components was nonstoichiometric, the PECs remained quasi-soluble. Flocculation set in when charge stoichiometry was reached impeding then all further measurements. A typical data set plotted according to Zimm is shown in Figure 3. Most of the scattering curves could be fitted by a fit of first or second order thus providing the apparent molecular mass Mw,app and the z-average radius of gyration Rg,z from the scattering intensity R(q) at zero angle and the angular dependence of the scattered light, respectively. From these quantities, an apparent structural density can roughly be estimated14 assuming spheres of homogeneous density with the radius RG,z. Combining two different average values is not correct and leads in fact to somewhat underestimated density values. However, this approach seems to be justifiable

Figure 2. Zimm plot of chitosan sulfate in 0.02 M acetate buffer/0.1 M NaCl (ultracentrifuge supernatant filtered through 0.2 µm pore size membrane filter): Mw ) 660 000 g/mol; RG,z ) 114 nm; B ) 5.8 × 10-4 mL‚mol‚g-2.



Figure 3. Zimm plot for the reaction of Chit B with chitosan sulfate in 0.02 M acetate buffer (curves numbered according to increasing mixing ratios, see filled circles in Figure 5).

Figure 4. Complex formation between Chit C/chitosan sulfate under various conditions.

when only slightly different systems under similar conditions are to be compared. Figure 4 presents results obtained for the system Chit C/chitosan sulfate at varying ionic strengths. At the lowest ionic strength, the molecular weight starts at a rather low level and increases rapidly after 30% conversion of the present chitosan. Flocculation sets in exactly at the molar mixing ratio 1.0. In the presence of 0.1 or 0.5 M NaCl, the molecular weights are significantly higher from the beginning and increase further on in the course of titration until flocculation sets in. The highest molecular weight level is reached in the presence of 0.1 M NaCl. The same ionic strength realized by CaCl2 seems less effective in terms of the molecular weight increment. These findings are consistent with ideas developed from studies on synthetic poly-

electrolytes. Due to the screening effect of added salt, the presence of low amounts of salt reduces electrostatic interactions thus leading to higher chain flexibilities and enabling rearrangement processes. Both effects favor charge compensation via conformational adaptation so that structures on a low level of aggregation are formed where the complex particles bear the charge of the excess component. Both with increasing ionic strength and mixing ratio, the effective overcharge of the complex particles becomes reduced to such an extent that secondary aggregation is favored. The change of the respective radii of gyration corresponds to the pattern of molecular weights. The calculated structural densities do not depend on the ionic strength and are (almost) constant over the whole range of mixing ratios on an average level as low as 0.015 g/mL. Compared with often rather

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Figure 5. Complex formation between Chit B (circles) and Chit B-50 (triangles) with chitosan sulfate at various ionic strengths.

compact PECs from synthetic polymers,15 this means a high degree of swelling when only 1.5% of the volume of the complex particle is occupied by the polymer itself. However, compared with the molecularly dispersed components, the density value has increased by a factor of 25 or more. A somewhat different picture (Figure 5) is obtained when using the low-molecular weight Chit B instead of the highmolecular weight Chit C (closed and open circles). From the early stages on, the level of association is high even at low ionic strength. However, the structural densities are found on the same level as those in Figure 4. So, from the viewpoint of PEC formation, there is no reason to favor a particular molecular weight range for producing shells. The triangles in Figure 5 describe the PEC behavior when Chit B was replaced by the higher acetylated derivative Chit B-50. Figure 5 shows that the reduced charge density along the chain has only little effect on the association level, but the resultant structural densities increase remarkably. The effect is more pronounced with increasing mixing ratio, and the structural density value rises up to 0.35 g/mL shortly before flocculation. Figure 6 shows the corresponding plots for the reaction between Chit C-50 (Chit C re-acetylated to DA ) 50%) with chitosan sulfate. Compared with the data in Figure 4, the aggregation level is lower and less dependent on the ionic strength. Again, the most obvious difference is the structural density parameter which increases for all systems both with higher mixing ratio and increasing ionic strength in a similar manner as was shown in Figure 5. The low degree of swelling of these PECs would suggest using such highly acetylated chitosans at higher ionic strength as flocculants, and in fact, experiments on the flocculation19 of Escherichia

coli have shown chitosans with a DA ∼ 60% at higher ionic strength to be most effective. Figure 7 summarizes results with PSS as anionic component instead of chitosan sulfate. Combined with Chit C, the formed PECs show a strong tendency for association which is enhanced by added NaCl. The structural densities are found on the same low and constant level as those in Figure 4. Unlike that, Chit C-50 in the presence of 0.5 M NaCl exhibits a more compact structure on an almost constant level over the whole range thus differing from Figure 4 (lower level) and Figure 6 (strong increase). This comparison of data suggests significant effects of the DA of chitosan as well as the nature of the counterpart on the formed PECs. This specific behavior might be an effect of the charge distances on the components relative to each other (matching or mismatching). The findings correspond to results on PECs between NaPSS and cationic copolymers of various compositions,20,21 which revealed structural densities strongly dependent on the charge densities of the involved polyelectrolytes. Resuming the findings up to this point, one can say that the high structural density of the PECs formed by chitosans with a DA ≈ 50% (irrespective of the chain length) with chitosan sulfate distinguishes these compounds clearly from the rest. So the combination of these components in multilayer arrangements seems promising whenever compact membranes are desired. The higher degree of swelling in the other PEC compositions is supposed to lead to thicker and more “gel-like” membranes of good permeability. The results above have shown significant salt effects when the PEC preparation was carried out at varying ionic strength. So the question arises whether subsequently added salt would



Figure 6. Complex formation between Chit C-50 and chitosan sulfate under various conditions.

Figure 7. Complex formation between Chit C and Chit C-50 with PSS under various conditions.

affect the PEC performance (in the 0.02 M acetate buffer) as well. Figure 8 displays the effect of NaCl. The molar mixing ratio is 0.8 (cationic component in excess) and the

finally reached salt concentration is 0.6 M. Whereas there is only a slight response in the system Chit C/chitosan sulfate (which seems plausible in the context of the other findings

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Figure 8. Effect of subsequent increase of the ionic strength on a mixing ratio 0.8 for the complex formation between Chit C and Chit C-50, respectively, with chitosan sulfate.

on this system), the Chit C-50/chitosan sulfate system reacts by slight desegregation while the structural density goes through a flat maximum at I ∼ 0.15 M. This behavior could be of practical relevance in situations where changes of the degree of swelling are expected to have an effect. Up to here the pH of the systems was held near 4.5 ensuring full protonation of the available amino groups. Higher pH values would reduce the degree of protonation and hence the charge density along the chitosan backbone. Samples having a DA close to 50% are often called “watersoluble” because they remain dissolved in neutral or even slightly alkaline solutions. (The molecular basis of this phenomenon is not yet understood.) Such samples allow studying pH effects without being interfered by the limited solubility of low-acetylated chitosans. A 1:1 coprecipitate from Chit C-50/chitosan sulfate prepared under slightly acidic conditions dissolves suddenly at pH ∼ 7 and reprecipitates at decreasing pH. This process is reversible and can be followed by light scattering. Shifting the pH in small intervals from low to high values leaves the molecular weights (almost) unchanged on the high starting level (Figure 9) until they drop near pH 7 reaching nearly the level of the separate molecularly dispersed components by further addition of alkali. These findings indicate a complete decomposition of the PEC when all chains lose their positive charges. Mixing the components at higher pH and shifting the pH in small intervals from high to low values (data not shown) provides scattering curves which are indicative of bimodal

systems thus detecting immediate flocculation of tiny amounts probably due to a local reaction at the entry of acid. Such systems could be of practical interest since they allow (i) keeping the mixed components dissolved until their precipitation on a template is desired and (ii) producing relatively compact shells or coatings which readily redissolve near the neutral point. Preparation and Properties of Capsules. To check whether the process of LBL coating of small spherical particles would work under the chosen conditions, model experiments on a monodisperse PSS-latex (particle radius 478 nm by dynamic light scattering) were carried out using Chit C/chitosan sulfate. The ζ potential of the particles (suspended in the buffer) was measured after each coating, including a washing step for in total four layers. The values listed in Table 3 can be attributed to the excess charge of the polyelectrolyte in the uppermost layer as a first indication of a successful coating. Parallel efforts to directly measure the respective changes of the hydrodynamic radius by dynamic light scattering measurements (using an ALV goniometer) failed because the average diffusion coefficients were increasingly dominated by aggregates. Aggregation was, however, confined to a rather small mass proportion. The same conditions were used for the preparation of hollow capsules (“shells”) by coating monodisperse melamin formaldehyde latex (MF) particles (diameter of 3.7 µm) with in total eight layers of Chit C/chitosan sulfate and Chit B/chitosan sulfate, respectively (the first layer was made of chitosan sulfate owing to the positive charge of the MF



Figure 9. pH effect on the PEC made of Chit C-50 and chitosan sulfate (pH data taken from a parallel experiment without molecular weight detection by SLS).

Figure 10. Atomic force microscopy (AFM) image of an air-dried shell made of Chit C/chitosan sulfate (eight layers). Table 3. ζ Potential Detecting the Consecutive Coating of PSS Latex Particles (measured in acetate buffer, pH 4.5) ζ potential (mV) PSS latex “naked” 1. layer: Chit C 2. layer: chitosan sulfate 3. layer: Chit C 4. layer: chitosan sulfate

-50.2 +39.3 -48.1 +37.1 -42.5

particles). Figure 10 presents a representative AFM image of such a shell. These shells cannot be distinguished by AFM from those made of synthetic polymers.24 From the height profile of the flat and partly folded individual shell relative to the mica support, one can estimate the thickness per layer. The value of ∼ 2 nm agrees well with observations on capsules from synthetic materials. Since measured in the dried state, the value cannot be used to estimate the layer thickness of the swollen shells in suspensions.

The ζ potentials of the freshly prepared shells suspended in water were found to be positive (Table 4) as expected because of chitosan in the uppermost layer. After 2 weeks of storage they had turned to negative values (Table 4) whereas the pH ∼ 6 of the shell suspension was nearly unchanged. (No further charge reversal was noticed then over a period of 12 months.) This observation gave rise to check whether the ζ potential would respond to changes of the pH in the surrounding medium. Having equilibrated the shells in solutions of unique ionic strength but different pH, the respective ζ potentials as given in Table 4 were measured. One can see that at a pH as low as 3.4 the shells have recharged to a positive net charge. At higher pH values they retain their negative ζ potential (even at pH ∼ 4.5). Any variation of the ζ potential as function of the pH is indicative of amphoteric surfaces.22 The shell wall permeability toward species of different charge and size is of major interest and importance for practical purposes. It seems reasonable to anticipate a semipermeable membrane allowing exchange of matter when a concentration gradient between the shells interior and surrounding is built up. Both the surface charge of the shells and the degree of swelling of the network forming the shell wall are expected to have key functions in this context. An only qualitative approach in this direction is reported here using fluorophores of various molecular weight and confocal laser scanning microscopy (CLSM) for detection. Commonly used low-molecular weight fluorophores are fluorescein and rhodamin 6G. Being (weak) electrolytes themselves, their electrostatic and stoichiometric binding to polyelectrolyte molecules and assemblies of opposite charge could be used for quantitative conclusions.23,24 Their binding to different types of LBL shells has been revealed to be very useful in many microscopic studies when the size, shape, or the integrity of species3 were to judge. This includes the shells which are reported on here (Figure 11). While taking the images, one can often notice considerable amounts of

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Table 4. ζ Potentials of the Hollow Shells (average values over more or less broad distributions; measured in the given medium)

Chit C/ChitSulf Chit B/ChitSulf a

water, freshly prepared

water, after 2 weeks

pH ∼ 3.4a

pH ∼ 4.5b

pH ∼ 7.3c

+27.4 (pH 5.8) +29.3 (pH 5.9)

-27.0 (pH 6.0) -48.0 (pH 6.1)

+21.6 +21.4

-15.5 -31.3

-31.2 -35.3

pH ∼ 3.4: 0.02 M acetic acid/0.1 M NaCl. b pH ∼ 4.5: 0.02 M buffer/0.1 M NaCl. c pH ∼ 7.3: 0.02 M sodium acetate/0.1 M NaCl.

Table 5. Classification of Shells in Terms of the Scheme in Figure 12, Shells Made of Chit C/Chitosan Sulfate (eight layers in total)a,b water (pH ∼ 6.0) sodium chloride 0.1 N (pH ∼ 6.0) pH ∼ 3.3, I ∼ 0.1 M & 0.5 M pH ∼ 4.5, I ∼ 0.1 M & 0.5 M pH ∼ 7.3, I ∼ 0.1 M & 0.5 M

fluorescein

Mw ) 4400

Mw ) 77000

Mw ) 260000

Mw ) 2 mio

Rhodamin 6G

AEF C G H C

AE CB H H HTB

A E (F) CB H HG H

A (E) B H HG HI

A IH H G HI

G n.d. C H H

a M gives the average molecular weight in g/mol for the FITC-dextran used. b pH, measured directly in the suspension; ionic strength adjusted at two w levels (0.1 and 0.5 M).

Table 6. Classification of Shells in Terms of the Scheme in Figure 12, Shells Made of Chit B/Chitosan Sulfate (eight layers in total)a water (pH ∼ 6.0) sodium chloride 0.1 N (pH ∼ 6.0) pH ∼ 3.3, I ∼ 0.1 M & 0.5 M pH ∼ 4.5, I ∼ 0.1 M & ∼0.5 M pH ∼ 7.3, I ∼ 0.1 M & ∼0.5 M

fluorescein

Mw ) 4400

Mw ) 77000

Mw ) 260000

Mw ) 2 mio

Rhodamin 6F

AEF C G H C

AE C PEC debris only H CH

A IHB H H H

A IHB H G H HGI

A I GH G HGI

G n.d. n.d. n.d. n.d.

a M gives the average molecular weight in g/mol for the FITC-dextran used; pH measured directly in the suspension. Ionic strength adjusted at two w levels (0.1 and 0.5 M).

Figure 12. Scheme of principally obtainable CLSM images for a single suspended shell in the presence of fluorescent probes. The different green colors indicate regions of lower or higher fluorescent intensity according to the local concentration distribution of the probe (outside/shell wall/inside). Black regions mean the absence of fluorescent probes.

Figure 11. Confocal laser scanning microscopy (CLSM) image of fresh-prepared shells in water made of Chit C/chitosan sulfate (eight layers) and visualized with fluorescein.

smaller sized PEC precipitate beside the well-formed shells and it is especially this PEC “debris” which disturbs many global measurements on LBL shell suspensions. Microscopy and in particular CLSM offer an alternative to consider the behavior of individual shells. Fluorophores available in the high-molecular weight domain are, for example, dextrans labeled with fluorescein isothiocyanate (FITC-dextrans) covering the range from a few thousands up to a few million daltons (see Experimental Section). Corresponding to the extent of labeling, these fluorophores bear negative charges. This is worth mentioning at least for our probe with Mw ∼ 2 mio g/mol where on average 160 fluorescein units are fixed on a chain of almost 5 µm contour length. This makes the probe a potential partner for PEC formation.

Moreover, HPSEC experiments with on-line multiangle laser light scattering detection have shown that, except for the probe with Mw ) 4400 g/mol, the FITC-dextrans have broad and partly overlapping molecular weight distributions (personal communication with Dr. G. Rother, MPIKG), which requires consideration in discussing size effects below. When a fluorophore (FP) was added to a suspension of shells, a series of CLSM scenarios as schematically given (for a single shell) in Figure 12 was observed. Finding out the pattern according to which the images were dependent on the conditions in the suspension (pH, ionic strength) and the nature of the fluorescent probe itself was hoped to allow conclusions to be drawn with respect to the shell wall permeability. The different green colors indicate regions of lower or higher fluorescence intensity corresponding to the local concentration distribution of the probe. Black regions mean the absence of fluorescent probes. Parts A and C are extremes when all FP is totally rejected from the shell wall and interior (A) or can freely enter the


walls and interior so that the capsule is not detectable at all (C) unless one uses the transmission mode (window in C). Part B is an intermediate between A and C indicating an incomplete concentration equalization between inside/outside (see D). Part D would mean the capsule “sucks in” all FP from outside. This is unlikely for thermodynamic reasons (unless FP is bound inside somehow) but sometimes such images are obtained as a result of a strong radiance from the shell walls. Parts E and F describe a situation with FP on both sides of the membrane whereas the membrane itself seems to be a widely avoided zone for FP (see comments below). Part G is realized in cases of a high FP binding capacity (attraction by opposite charges) of the shell walls, and further addition of FP in excess can lead to situations as shown in parts H or I. In Tables 5 and 6 we have correlated the observed images with the designed pictures in Figure 12. Considering Table 5, one can see that in water without added salt the A type image dominates for all anionic FPs. In contrast, the cationic rhodamin 6G is largely bound to the shell wall according to type G. This is clear indication of mutual rejection in the case of like-charged species as well as attraction in the case of oppositely charged species. Finally, when small amounts of salt (NaCl) or alkali (NaOH) were added to the fluoresceincontaining shell suspension, the situation turned from type A to type C in a moment, indicating perfect concentration equalization. When acid (HCl) was added, the image turned immediately from type A to type G. Exactly the same situation was observed after 3 months of incubating the shells with fluorescein. This means a long-lasting exclusion of even small molecules from the membrane or shell interior as long as the electrostatic repulsive barrier is not reduced by the screening effects of salts. The size or molecular weight of the probe seems of minor importance. In the presence of salt, type C dominates the low molecular weight region. With increasing molecular weight of the probe, the picture turns to type I with minor amounts of H via type B. This means the shell walls are completely accessible and penetrable only for the low molecular weight probes. The high molecular weight probes become enriched onto/within the shell wall but cannot move into the shell interior. These results clearly show that when charge effects are suppressed by the screening effect of salt, the size of the permeating probe controls the process. In the acidic region (pH 3.3 and 4.5), type H images dominate. As long as there are free binding sites, the oppositely charged FPs are bound. Once attracted so closely, the molecules can move through the wall even if they are really long (reptation). At a pH slightly above the neutral point along with the adjusted ionic strengths one would expect images resembling those produced by the shells in a salt-containing solution at pH ∼ 6. This is found to be true in the case of fluorescein. For the FITC-dextrans, the actually observed images of type H and I show, however, enhanced enrichment of the probes in the shell wall thus detecting accessible positive charge sites in the shell region. Possibly this is a pH domain (right


above the pKa of the amino groups both in chitosan and in chitosan sulfate) where recharging and rearrangement processes take place which we have not yet understood. At the same time, permeation is markedly reduced at least for the two high molecular weight probes indicating again the size dependence of permeation. In principle the same tendencies can be seen in Table 6. Gradual differences due to the different chitosan molecular weights (comparison of Tables 5 and 6) or the two ionic strengths at each tested pH should not be overvalued because of the qualitative nature of our estimates. They are not suggested by the similar PEC structural densities either. In summary, the results illustrate the key role of charge effects for any transport of low molecular weight and high molecular weight substances through the shell wall. Oppositely charged species are bound as long as there are free binding sites. More interesting is the situation when species of the same sign of charge are brought into contact. In the absence of salt, repulsion between the highly charged shell surface and the fluorophore dominates even if the fluorophore is a small molecule such as fluorescein. As long as the species cannot approach each other, no permeation occurs. Our data have shown that as soon as charge effects between species of the same sign are suppressed (0.1 M NaCl seems sufficient), permeation takes place and is then controlled by the size of the test substance. There is no discernible barrier for small species, and the exchange is very fast. The size limit where exclusion starts to work in our model systems is not very clearsjust the tendencyswhich has to do with both the molecular weight distribution of the test substances and the varieties in the shell population. This is worthwhile information in order to understand the transport from the bulk of the suspension into the shells interior and reverse (see also ref 26). Our SLS results on the PECs rule out salt effects on the shell’s degree of swelling. So the width of meshes of the PEC network that builds up the shell wall is not supposed to change significantly. In addition to the shells described so far, we used the LBL procedure as well as coprecipitation by lowering the pH (onestep procedure) in order to prepare shells made of Chit C-50/ chitosan sulfate on MF templates. Their microscopic appearance differed from those in Figure 11 most of all by the fact that the shells were embedded in big amounts of coprecipitate. A separation for subsequent studies appeared to be difficult if not impossible. Conclusions Sophisticated microscopic experiments regarding the shell wall permeability seem desirable in order to get more substantial and quantitative data on a broader basis. The dominant charge effects upon permeation require a strict differentiation between studies in salt-free or salt-containing systems. Macromolecular fluorophores having a narrow molecular weight distribution would be helpful detectors for discussing size effects in terms of a cutoff limit. Acknowledgment. Ms. H. Zastrow and Dr. Stefano Leporatti are thanked for their help with the ζ potential and

590


AFM measurements, respectively. Ms. A. Vo¨lkel and Ms. M. Gra¨wert are thanked for the ultracentrifuge measurements and HPSEC, respectively. Ms. S. Pirok helped us with the elemental analyses. This study was partly supported by a grant from the BASF and BMBF 03C293A1. References and Notes (1) Decher, G.; Hong, J. D. Makromol. Chem. Makromol Symp. 1991, 46, 321. (2) Donath, E.; Sukhorukov, G.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2002. (3) Gao, Ch.; Leporatti, S.; Moya, S.; Donath, E.; Mo¨hwald, H. Langmuir 2001, 17, 3491. (4) Neu, B.; Voigt, A.; Mitlo¨hner, R.; Leporatti, S.; Gao, C. Y.; Donath, E.; Kiesewtter, H.; Mo¨hwald, H. Meiselmann, H. J.; Ba¨umler, H. J. Microencapsulation 2001, 18, 385. (5) Hammond, P. T. Curr. Opin. Colloid Interface Sci. 1999, 4, 430. (6) Amiji, M. M. Colloids Surf., B 1998, 10, 263. (7) Kochkina, Z. M.; Chirkov, S. N. Microbiology 2000, 69, 217. (8) Muzzarelli, R. A. A.; Miliani, M.; Cartolari, M.; Genta, I.; Perugini, P.; Modena, T.; Pavanetto, F.; Conti, B. S.T.P. Pharma Sci. 2000, 10, 51. (9) Muzzarelli, R. A. A.; Biagini, G.; DeBenedittis, A.; Mengucci, P.; Majni, G.; Tosi, G. Carbohydr. Polym. 2001, 45, 35. (10) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 8153. (11) Berth, G.; Dautzenberg, H.; Peter, M. G. Carbohydr. Polym. 1998, 36, 205-216. (12) Co¨lfen, H.; Berth, G.; Dautzenberg, H. Carbohydr. Polym. 2001, 45, 373.

Berth et al. (13) Berth, G.; Dautzenberg, H. Carbohydr. Polym. 2002, 47, 39-51. (14) Dautzenberg, H. Macromol. Chem. Phys. 2000, 201, 1765. (15) Dautzenberg, H. In Physical Chemistry of Polyelectrolytes; Radeva, T., Ed.; Marcel Dekker: New York, 2001. (16) Voigt, A.; Lichtenfeld, H.; Sukhorukov, G. B.; Zastrow, H.; Donath, E.; Ba¨umler, H.; Mo¨hwald, H. Ind. Eng. Chem. Res. 1999, 38, 4037. (17) Berth, G.; Dautzenberg, H. Recent Res. DeV. Macromol. Res. 1998, 3, 225. (18) Gamzazade, A.; Sklyar, A.; Nasibov, S.; Sushkov, I.; Shashkov, A.; Knirel, Yu. Carbohydr. Polym. 1997, 34, 113. (19) Strand, S. P.; Vandvik, M. S.; Vårum, K. M.; Ostgaard, K. Biomacromolecules 2001, 2, 126. (20) Philip, B.; Dautzenberg, H.; Linow, K. J.; Ko¨tz, J.; Dawidoff, W. Prog. Polym. Sci. 1989, 14, 91. (21) Dautzenberg, H.; Hartmann, J.; Grunewald, S.; Brand, F. Ber. BunsenGes. Conference Proceedings of “Polyelectrolytes Potsdam’ 95” 1996, 100, 1024. (22) Childress, A. E.; Elimelech, M. J. Membr. Sci. 1996, 119, 253. (23) Caruso, F.; Donath, E.; Mo¨hwald, H.; Georgieva, R. Macromolecules 1998, 31, 7365. (24) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald, H. Macromolecules 1999, 32, 2317. (25) Leporatti, S.; Voigt, A.; Mitlo¨hner, R.; Sukhorukov, G.; Donath, E.; Mo¨hwald, H. Langmuir 2000, 16, 4059. (26) Ibarz, G.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. AdV. Mater. 2001, 13,1324.

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