Ind. Eng. Chem. Res. 1999, 38, 4037-4043
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Membrane Filtration for Microencapsulation and Microcapsules Fabrication by Layer-by-Layer Polyelectrolyte Adsorption Andreas Voigt,*,† Heinz Lichtenfeld,† Gleb B. Sukhorukov,† Heidemarie Zastrow,† Edwin Donath,† Hans Ba1 umler,‡ and Helmuth Mo1 hwald† Max-Planck-Institute of Colloids and Interfaces, D-14424 Potsdam, Germany, and Institute of Transfusion Medicine and Immunohematology, Medical Faculty Charite´ , Humboldt University of Berlin, D-10098 Berlin, Germany
Layer-by-layer polyelectrolyte adsorption at colloid particle surfaces as well as the removal of the core latex to obtain multilayer microcapsules is conducted by means of membrane filtration. As target particles for adsorption we use nonsoluble polystyrene sulfate latex, soluble melamine formaldehyde resin latex, and decomposable glutaraldehyde fixed human red blood cells. The materials adsorbed are poly(allylamine hydrochloride), poly(styrenesulfonate), poly(diallyldimethylammonium chloride), chitosan, and chitosansulfate. The coating process is carried out under different membrane filtration conditions with respect to the pressure regime, the filter materials, and the stirring conditions. We characterize the prepared multilayers at the particle surface or in the microcapsule (shell) form by atomic force microscopy, confocal laser scanning microscopy, transmission electron microscopy, single-particle light scattering, and electrophoresis. The quality, performance, and yield of the presented method are compared with the results obtained by centrifugation and sequential adsorption as alternative preparation strategies. Membrane filtration surpasses all other methods, so far established, with respect to the above criteria. Introduction Layer-by-layer polyelectrolyte/nanoparticle coating has been established quite recently as a method to fabricate designed complex multilayers.1 Selecting appropriate polymers and/or charged nanocolloids, one can build up multilayers as locally organized composite polysalt structures. The method of preparing them consists of a consecutive immersion of the multilayer carrier in the coating or washing solutions. This works for plane and macroscopic carriers only. If one is coating colloidal carriers such as poly(styrenesulfate), melamine formaldehyde resin latexes, or alumina powder, other preparation techniques are needed.2-4 Sukhorukov et al.2 have applied two different coating strategies. The more effective way was to separate the carrier particles from the coating and washing solutions by centrifugation. The other approach was to add the coating polymers consecutively to the particle suspension in amounts exactly corresponding to monolayer coverage (called sequential adsorption). Whereas the first strategy results in good quality, multilayer-coated nonaggregated particles, the second one creates a large fraction of flocculated particles. The first way is time-consuming and discontinuous and is always going through the state of particle aggregation; the second one is fast to carry out but needs precise determination of the components’ quantities added to the system in each step. The prepared complex particles are built up of a geometrically closed shell of the coating material and a core particle. There exists a class of core particles * To whom correspondence should be sent. Telephone: (+49) 0331 567 9235. Fax: (+49) 0331 567 9202. E-mail:
[email protected]. † Max-Planck-Institute of Colloids and Interfaces. ‡ Humboldt University of Berlin.
dissolvable under mild conditions, for example, acid pH in the case of special melamine formaldehyde resin. For a control of the multilayer quality, one dissolves the core and investigates the formed hollow microcapsules (shells).5 These polyelectrolyte and/or composite microcapsules have different applications. They can be used as microreaction vessels for synthesis (e.g., organic polymers), as templates for crystallization and precipitation (e.g., dyes, magnetic particles), or as carrier particles for oils or drugs. These are examples of systems which were prepared in our laboratories already. Further applications are under study now. We will present results of layer by layer coating of colloids by means of the membrane filtration method. We applied it successfully to a variety of coating substances at carrier particles of different origin and size. The method is very effective, and its advantages and shortcomings as compared with those of the abovementioned two strategies are discussed. The estimation of the quality of the prepared coatings and microcapsules obtained therefrom is done by atomic force microscopy (AFM), confocal laser scanning microscopy (CSLM), particle electrophoresis, and single-particle light scattering (SPLS). Experimental Section Materials. Polyelectrolytes. Sodium poly(styrenesulfonate), PSS, MW ∼ 70000, poly(allylamine hydrochloride), PAH, MW ∼ 8000-11000, poly(diallyldimethylammonium chloride), PDADMAC, MW ∼ 100000, and polyethylenimine, PEI, MW ∼ 50000 (PEI for AFM preparation) were obtained from Aldrich. Chitosan, MW ∼ 200000-300000, and chitosansulfate, MW ∼ 200000300000 we obtained from Prof. L. S. Gal’braikh and Dr. L. A. Vikhoreva from Textile Institute, Department of Chemical Fibers, Moscow, Russia.
10.1021/ie9900925 CCC: $18.00 © 1999 American Chemical Society Published on Web 08/21/1999
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Figure 1. Sketch of the membrane filtration procedure. The filtration can be performed continuously, controlling the sequential addition of the polyelectrolytes as well as the liquid level. The whole process from forming the first layer to harvesting the microcapsules was done in one and the same reaction vessel.
All polyelectrolytes were used without further purification. Solutions of 1 or 2 mg/mL PSS or PAH in 0.5 M NaCl (pro analysi, Merck, Darmstadt, Germany) were prepared. Chitosansulfate was prepared as a 1 mg/mL solution in 0.5 M NaCl. Chitosan was dissolved as 1 mg/ mL in 0.5 M NaCl and 0.3% v/v acetic acid.6,7 Complete dissolution was obtained after 40 min of ultrasound application in a Sonorex Super bath (level 10) from Bandelin, Berlin, Germany. Fluorescein isothiocyanate (FITC)-labeled PAH (FITCPAH) was prepared according to Sukhorukov et al.8 and was kindly provided by M. Auch, Max-Planck-Institute of Colloids and Interfaces, Berlin, Germany Magnetite Particles. Magnetite particles prepared by precipitation of Fe(II) and Fe(III) salts in ammonium hydroxide solution and stabilized by HCl 9 were provided by A. S. Susha. Other Chemicals. Water used for all experimental steps was obtained by reverse osmosis (Milli-RO 12 Plus; Millipore GmbH) followed by ion-exchange and filtration steps (Milli-QPlus 185, Millipore GmbH). Sodium hypochloride (12% chlorine) was purchased from Aug. Hedinger GmbH & Co., Stuttgart, Germany, and used as a deproteinizer (10% v/v solution) dissolving the fixed red blood cells. The So¨rensen citrate buffer was prepared from Merck products: citric acid monohydrate, NaOH, and HCl.10 Carrier Particles. Charged polystyrenesulfate latex (PS latex) of diameter 640 nm was prepared according to the method of Furusawa et al.11 Melamine formaldehyde resin particles (MF latex) with diameters of 3.7 and 5 µm were purchased from microparticles GmbH, Berlin, Germany. These particles were prepared under special conditions to keep them dissolvable in acid pH or sodium pyrosulfite solutions or different polar organic solvents. Human red blood cells fixed with glutaraldehyde were prepared according to the method of Donath et al.12 Membrane Filtration Equipment. The equipment was purchased from Sartorius AG, Go¨ttingen, Germany. We used a vacuum pump SM 166 92. It reaches a minimum of about 100 mbar. We used the same pump
also for pressure application up to a 3 bar maximum. For vacuum filtration we applied the 47 polycarbonate filtration unit SM 165 10, and for pressure filtration we used the SM 165 26. Membrane filters (47 mm) of the following types were used: Sartolon Polyamid SM 250 07-047 N (0.2 µm), Sartolon Polyamid SM 250 06-047 N (0.45 µm), Cellulose Acetate SM 111 04-047 N (0.8 µm), and Cellulose Nitrate SM 113 06-100 N (0.45 µm). Methods Adsorption. The polyelectrolyte adsorption was performed in PS and MF resin latex suspensions of 1-3% v/v. The concentration of the red blood cells was about 10% v/v. The volume of the particle and cell suspensions applied in the first adsorption step was between 10 and 50 mL. The adsorption time was always 5 min. Afterward the washing procedure with Millipore water was started. Filtration. In Figure 1 a sketch of the experimental procedure is shown. We used vacuum filtration, pressure filtration, and filtration without pressure manipulation. During the polyelectrolyte adsorption period one should apply a weak underpressure to the incubation chamber in relation to the lower filtrate chamber. This is done to prevent loss of the incubation medium with a simultaneous decrease of the polyelectrolyte to particle number ratio. Thus, the adsorption conditions are kept constant during the incubation. The preselection of the membrane filter depends on the charge sign of the polyelectrolyte to be adsorbed. In the case of polycations (PAH, Chitosan) we started with polyamide filters, and in the case of polyanions we started with cellulose acetate or nitrate. This was done to minimize the clogging of the filter by polyelectrolyte adsorption. Simultaneously, this should prevent strong adhesion of the coated particles after each adsorption cycle. However, the selection of the filters is not always governed by simple electrostatic considerations. One has to take into account other adhesion and aggregation mechanisms too. The velocity and strength of filter cake formation are important factors for successful filtration. They have to
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Figure 2. AFM picture (tapping mode) of PS latex of 1.28 µm diameter coated with six layers of PAH/PSS. The first layer at the negatively charged PS latex is PAH, and the last one is PSS. Axes are scaled in micrometers.
be minimized throughout the filtration process. The adhesion of PS and MF latex with an outermost layer of PSS or PAH to the filter surfaces is rather weak. For these particles the aggregation and/or flocculation is also weak. The filtration can be continued up to high particle concentrations (20%) without stopping the process or replacement of the lost suspension medium by the washing one. Usually we work with excess concentrations of the adsorbing polyelectrolytes. Therefore, it is of great advantage to concentrate the suspension as far as possible before the washing cycles start. A weak hairpencil is used to reverse weak aggregation after reexpansion of the suspension volume for washing. In the case of red blood cells great care has to be taken to inhibit aggregation during the formation of the first four or five layers. The uncoated and coated glutaraldehyde fixed cells have a high affinity for each other and form strong aggregates. The suspension should not be concentrated up to values exceeding about 5% to 10% (v/v), and filter cake formation has to be prevented as much as possible. There is also a very pronounced adhesion to the filter surface. After the first few adsorption layers were formed at the red blood cell surface, the situation is easier to cope with and comparable to that with the other carrier particle systems investigated by us. The chitosan/chitosansulfate system is a little bit more sticky than the PSS/PAH system but much easier to handle than the red blood cell system. Care must be taken not to destroy the coating layers during resuspension of rather weakly aggregated particles. Again, we had the best experiences applying the weak hairpencil and/or stirring the suspension from above. With permanent stirring, the aggregate formation is almost completely suppressible. Atomic Force Microscopy (AFM). The AFM images were obtained by means of a Digital Instruments Nanoscope IIIa in the tapping mode. Samples were prepared by applying a drop of the shell suspension to PEI-coated glass slides. After sedimentation of the microcapsules, the slides were rinsed in Millipore water and then dried under nitrogen atmosphere. Electrophoresis. The electrophoretic mobilities of the particles were measured by means of a Malvern Zetasizer 4. They were converted into ζ potentials using the Smoluchowski relation.2
Figure 3. Normalized light scattering intensity distributions (SPLS) of PAH/PSS-coated PS lattices of 640 nm. Particles with 11 and 21 layers are compared with uncoated ones.
Single-Particle Light Scattering (SPLS). The equipment and data analysis have been described in detail elsewhere.13 The dispersed particles flow is hydrodynamically focused by pressing the dispersion through a thin capillary. The particle concentration is adjusted as to make possible single-particle measurement in the scattering volume. The latter is 1.7 × 10-3 cm3. Forward scattered (5° to 10°) light pulses from particles flowing through the scattering volume were recorded. Confocal Laser Scanning Microscopy (CLSM). The images of the chitosan/chitosansulfate microcapsules labeled with FITC-PAH were obtained by means of a Leica confocal laser scanning microscope (Aristoplan, 100× oil immersion). Transmission Electron Microscopy (TEM). TEM measurements were obtained on a Phillips CM12 microscope operating at 120 kV. Results The AFM picture in Figure 2 illustrates PS latex surfaces after coating with three couples of PAH/PSS. They look smooth, and no polyelectrolyte aggregates are visible. The particles were coated by vacuum membrane filtration with a suction pressure of about 100 mbar. During the adsorption and washing processes the particle suspension is stirred permanently. Therefore, any tendency of aggregation and filter cake formation is inhibited. For the preparation we have used 450 nm cellulose nitrate membrane filters. Exchange for a polyamide filter or consecutive usage of negatively charged cellulose acetate and positively charged polyamide filters did not change the results. In Figure 3 the growth of the shell thickness at the surface of 640 nm PS latex is determined by SPLS. The scattering light intensity is increasing with particle size. There are shown the results of 11 and 21 PAH/PSS layers as compared with those of the uncoated particles. The preparation of the coated particles was identical to that used for the particles in Figure 2. A TEM picture of a 640 nm PS latex coated with a mixed layer of polyelectrolyte and magnetite is shown in Figure 4. Two couples of PAH/PSS were adsorbed as precursor layers. Thereafter, three couples of magnetite/ PSS were adsorbed. The filtrate solution was always uncolored and clear and obviously free of magnetite. This means that the magnetite added in excess is adsorbed completely. Magnetite aggregates are formed at the surface. They are clearly visible in via TEM.
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Figure 4. TEM picture of a PS latex particle of 640 nm coated with four layers of PAH/PSS followed by three pairs of layers each consisting of one layer of magnetite and one layer of PSS. The bar corresponds to 200 nm.
Figure 5. AFM picture (tapping mode) of a PSS/PAH 10-layer capsule prepared at a 3.7 µm melamine formaldehyde resin latex. The core latex was removed by addition of citrate buffer (So¨rensen) to adjust the pH to 1.4. In a few seconds at 22 °C the core latex was dissolved. Three washing cycles with Millipore water by means of membrane filtration cleaned up the preparation. The axis numbers are given in micrometers, and the height scale is in nanometers.
To obtain the formed adsorption shell in pure form, we coated dissolvable core particles (MF). The shell preparation and washing procedures were carried out here and in the following examples in the same membrane filtration equipment used for multilayer preparation. Any pressure or suspension stirring was reduced to minimal values so as not to damage the nonsupported shell structures. The resulting microcapsules of 10 layers of PAH/PSS at melamine formaldehyde resin particles of 3.7 µm are shown in the AFM picture of Figure 5. The core particles were dissolved in pH 1.4 citrate buffer. We see clearly the morphology of the flat shell as a folded thin skin. In Figure 6 we illustrate a failed preparation of a nine-layer PAH/PSS shell from a 5.0 µm MF core. The
Figure 6. AFM picture (tapping mode) of a PSS/PAH nine-layer capsule at a 5.0 µm melamine formaldehyde resin latex. The core latex was removed by pH 1.4 citrate buffer (So¨rensen). The capsule morphology was not intact because of excessive application of ultrasound and mechanical agitation during the filtration cycles. The formation of a dried filter cake was permitted.
Figure 7. AFM picture (tapping mode) of microcapsules obtained from glutaraldehyde fixed human red blood cells. The cells were coated with 10 layers of PSS/PAH, and the cell core was removed with a deproteinizer solution. The axis numbers are given in micrometers, and the height scale is in nanometers.
shell is broken, which was caused by strong mechanical and ultrasound influence during resuspension of a dried filter cake. Figure 7 shows an AFM picture of 10-layer PAH/PSS microcapsules grown at the surface of glutaraldehyde fixed human red blood cells. Despite the negative charge of the cells, one has to start with a PSS adsorption layer. This contraintuitive strategy results in stable coating conditions with respect to aggregation phenomena. The preparation has to be done under zero pressure or a mild excess of pressure or underpressure to prevent the formation of a filter cake of strongly aggregating cells. The same care has to be taken if one uses PDADMAC as a polycation (not shown here). High-quality results are always obtainable with these systems under zero pressure conditions (free filtration).
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Figure 8. AFM picture (tapping mode) of chitosan/chitosansulfate microcapsules consisting of 10 layers. The microcapsules were prepared at a 3.7 µm dissolvable MF latex, and the latex core was finally removed by So¨rensen citrate buffer of pH 1.4. The axis numbers are given in micrometers, and the height scale is in nanometers. Figure 10. CLSM picture of chitosan/chitosansulfate microcapsules consisting of 11 layers and adding one layer of FITC-labeled PAH as the outermost one. The width of the picture is 7.3 µm.
Figure 9. AFM picture (tapping mode) with high resolution in the center of one of the microcapsules shown in Figure 8. The phase representation is given. The axis numbers are given in micrometers, and the height scale is in degrees.
In Figures 8-11 we present results obtained with chitosan/chitosansulfate as shell-forming polyelectrolytes. Our motivation to study this combination was to reach a more biocompatible shell composition. In Figure 8 an AFM picture of 10-layer microcapsules and in Figure 9 a highly resolved detail of one of the shell surfaces are given. We cannot differentiate between real surface structures and AFM probe preparation artifacts. However, the roughness of the surface structure looks uniform and the shell formation conditions seem to be advantageous. For better estimation of the shell morphology FITC-labeld PAH was added as the 12th layer and the shell integrity was measured by CLSM, as shown in Figure 10. In Figure 11 the ζ potentials of the layer-by-layer growing microcapsules are given. Odd layer numbers correspond to chitosansulfate and are characterized by negative ζ potentials, and even layer numbers correspond to chitosan and provide positive ζ potentials, respectively. Discussion Our investigation was directed to find a strategy in polyelectrolyte multilayer and shell preparation by
Figure 11. ζ potential of uncoated and chitosan/chitosansulfatecoated 3.7 µm MF latex. Odd layer numbers correspond to chitosansulfate, and even layer numbers, to chitosan. The control corresponds to the pure particles (zero layers).
means of the membrane filtration method. We want to discuss the differences between this method and those established as centrifugation and sequential adsorption techniques. The main problem with all three methods is the control of the particle interaction. Usually one has to cope with the tendency of the particles to form more or less strong aggregates during different stages of the preparation cycle. The success in resuspension of the sediment formed in the centrifugation method is strongly dependent on the aggregation strength of the particles. It is rather difficult to resuspend particles of a strong aggregate without incalculable modifications or even damage of the prepared surface structures. In preparing 20-layer microcapsules on a PAH/PSS basis, we obtained an accumulated total loss of about 80% of the originally introduced material. This has to be compared with
