Protamine Microsized

Aug 16, 2003 - With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 μm. The microcapsules were loaded with horseradish ...
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Biomacromolecules 2003, 4, 1191-1197

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Loading the Multilayer Dextran Sulfate/Protamine Microsized Capsules with Peroxidase Nadezda G. Balabushevich,†,‡ Olga P. Tiourina,† Dmitry V. Volodkin,†,‡ Natalia I. Larionova,‡ and Gleb B. Sukhorukov*,† Max-Planck Institute of Colloids and Interfaces, Golm/Potsdam, 14476, Germany, and Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia Received January 29, 2003; Revised Manuscript Received June 25, 2003

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 ( 0.2 µm, which is more than that diameter of the templates (5.12 ( 0.15 µm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 µm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 × 108 to 2.2 × 108 peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months. Introduction Design and biofunctionalization of microparticles are the challenging research fields in biotechnology, especially when the enzyme properties are to be imparted in these particles. Fabrication of micron- and submicron-sized polymer spheres, capsules or liposomes containing proteins is a substantial task for applications in biomedicine, cosmetology, ecology, and pharmaceutical and food industries.1 A novel concept of microencapsulation of different types of materials was recently developed.2 It is based on the layerby-layer (LbL) assembly of oppositely charged macromolecules onto the surface of colloid particles. The LbL method explores the electrostatic interaction at each step of adsorption and can involve many substances as layer constituents, such as synthetic polyelectrolytes, proteins, nucleic acids, lipids, inorganic nanoparticles, and multivalent dyes.3-5 Subsequently, the LbL technology was transferred from flat microscopic substrates to surfaces of submicron-and fewmicron-sized colloidal particles.6 Up to now, different colloidal cores were used to template the LbL polyelectrolyte assembly on their surfaces, for instance, organic latex particles, inorganic particles, dye and drug nanocrystals, compact form of DNA, protein aggregates, gel beads, and biological cells.6-12 The colloidal core can be decomposed in the conditions where the polyelectrolyte multilayers are stable leading to formation of hollow polyelectrolyte capsules.13,14 The most important features of polyelectrolyte multilayer shells, which make them promising for encapsula* To whom correspondence should be addressed. Fax: +49 331 567 9202. E-mail: [email protected]. † Max-Planck Institute of Colloids and Interfaces. ‡ Lomonosov Moscow State University.

tion of different materials, are the possibilities for a wide range of controlling the capsule wall properties, such as shell thickness tuneable in the nanometer range, compatibility, affinity, and degradation.7 Polyelectrolyte multilayers are sensitive to pH value or to solvent mixture. Therefore, the permeability of polyelectrolyte multilayer shells undergoes reversible changes when the pH value changes over pK value of polyelectrolyte used for shell assembly.15 The reversible changes in permeability make possible the encapsulation of enzymes. This was shown by chymotrypsin16 and urease17 encapsulation in polyelectrolyte capsules composed of poly(styrene sulfonate) (PSS) and poly(allylamine) (PAH). The proteins and enzymes might be also embedded into the melamine formaldehyde (MF)/PSS complex formed in the capsule interior by spontaneous deposition.18 For many applications, the capsules must be composed of biocompatible polymers. In this paper, the encapsulation of peroxidase was performed in capsules made of dextran sulfate and protamine. The encapsulation payload, enzyme activity, and release were studied. Experimental Section Materials. Dextran sulfate, Mw 500 000, protamine, horseradish peroxidase, peroxidase fluorescein isothiocynate labeled (FITC-peroxidase), 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), rhodamine 6G, and 6-carboxyfluorescein were purchased from Sigma. Amplex Red reagent was purchased from Molecular Probes, U.S.A. MF particles with a diameter of 5.12 ( 0.15 µm were purchased from Microparticles GmbH, Germany. Preparation of Polyelectrolyte Microcapsules. The polyelectrolyte multilayer assembly was fabricated on MF

10.1021/bm0340321 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/16/2003

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particles at pH 5.0 and 0.2 M NaCl by alternate adsorption of dextran sulfate and protamine. The polymer concentration was 5 mg/mL. Each adsorption cycle was completed with three centrifugation steps followed by re-suspension in pure water before the next polymer was added to the particles. Such a washing procedure was used to avoid polyelectrolyte complex formation outside the particle surface.13 The cores were hydrolyzed at pH 1.7 in HCl solution. Then microcapsules were washed with water until pH 3.0. The concentration of the microcapsule suspension was calculated directly from numerous confocal images by counting the amount of microcapsules in the defined volume and equalled (7 ( 3) × 1010 particles/L. The suspension of microcapsules at pH 3.0 could be stored at 4 °C over a month long period. Encapsulation of Peroxidase into the Dextran Sulfate/ Protamine Microcapsules. A 0.05-0.20 mL microcapsule suspension in water was centrifuged (2000 g, 2 min), and the supernatant was removed. Then the microcapsules were mixed with 0.95-0.80 mL of peroxidase solution (1-3 mg/ mL) in the universal buffer (0.02 M H3PO4, 0.02 M CH3COOH, 0.02 M H3BO3 + 0.1 M NaOH, pH 4-8). After incubation for 1 h at room temperature, this mixture was centrifuged (2000 g, 2 min) and washed 1-3 times with the buffer. The supernatants, washings, and microcapsules suspension were used to determine the protein content and the enzyme activity. Determination of Protein Concentration. The protein concentration in solution and in suspensions of microcapsules was determined according to the Lowry method.19 To determine the concentration of the encapsulated protein, microcapsules were ultrasonicated and centrifuged (5000 g, 3 min) before optical measurements. The loading capacity (L) was evaluated by the following equation: L ) [c]NA/Mw[N] where [c] is the concentration of the encapsulated protein, mg/mL; NA is the Avogadro constant; Mw is the molecular weight of peroxidase, g/mol; [N] is microcapsule concentration, number of microcapsules per L. Assay of the Peroxidase Activity. 0.905-0.925 mL of 0.1 M acetate buffer, pH 5.0, 0.005-0.025 mL of peroxidase solution (0.05-0.04 mg/mL) or microcapsule suspension, and 0.020 mL of ABTS solution (8 mg/mL) were placed in a quartz cell. Then 0.050 mL of a H2O2 solution (0.5%) was added.20 The absorbance was measured at 403 nm using a UV spectrometer (Cary UV-visible). The activity of peroxidase was 550 U/mg. 1 U oxidizes 1 µmole ABTS per min at 25 °C, pH 5.0. The activity of the encapsulated peroxidase vs the activity of an equal amount of the native protein was expressed in present. Effect of pH on Peroxidase Release from Microcapsules. Peroxidase was encapsulated at pH 8.0 as mentioned above, the suspension was centrifuged, and the supernatant was removed. The protein content in microcapsules was accepted as 100%. 1 mL of universal buffer solution (pH 4.0, 5.0 or 8.0) was added to the capsules, and after incubation for 10 min, the mixture was centrifuged and the protein content was determined in the supernatant. Precipi-

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tated microcapsules were resuspended in a new portion (1 mL) of the same buffer solution and the procedure was repeated. The cumulative release of protein was calculated as a ratio of total protein released, determined in supernatants after a certain time of incubation, to initial protein in microcapsules prepared at pH 8.0. Effect of a Number of Polyelectrolyte Layers on Enzyme Release from Microcapsules. Peroxidase was encapsulated at pH 8.0, and a part of the capsules loaded with protein was covered by 2 bilayers of dextran sulfate/ protamine to produce 6 bilayer microcapsules. Then the microcapsules were resuspended in the buffer (pH 7.0) to obtain the final concentration 0.2 mg protein/mL. The microcapsules suspension was incubated at 20 °C with constant agitation at 40 rpm. After 10, 30, 60, 120, 180, 240, and 420 min, the aliquots (0.05 mL) of the suspension were withdrawn and centrifuged. The supernatants were used to determine the enzyme activity. Confocal Laser Scanning Microscopy (CLSM). The CLSM was carried out on a LEICA TCS system (Aristoplan (Germany), 100 × oil immersion) using commercial software. Scanning Electron Microscopy (SEM). SEM was conducted using a Zeiss DSM 40 instrument operated at an acceleration voltage of 3 keV. Samples were prepared by applying a drop of the suspension onto glass wafers. The water was evaporated before the samples were covered with a thin gold film. Raman Spectroscopy. Raman spectra and images were made in water using a Confocal Raman Microscope (CRM200, Witec) with a piezo scanner (P-500, Physik Instrumente) and objectives (× 60, NA ) 0.80 or ×100 oil, NA ) 1.25, Nikon). In a typical experiment, a circularly polarized laser (CrystaLaser, λ ) 532 nm) was focused on the material with diffraction limited spot size (∼λ/2). An avalanche photodiode detector (APD) was used to record high resolution Raman images. To have a Raman spectroscopic analysis into microparticles, we focused the beam spot exactly inside the spherical particle, but for measuring onto the surface, of particles it was focused on the edge. Concentrated solutions of protamine (50 mg/mL) and dextran sulfate (100 mg/mL) were used for spectroscopic analysis of polyelectrolytes. Visualization of Active Peroxidase in Microcapsules. 0.01 mL of a microcapsule suspension was mixed with 0.08 mL of 0.25 M phosphate buffer (pH 7.4), 0.05 mL of 10 mM Amplex Red reagent in DMSO, and 0.05 mL of 20 mM H2O2. Resorufin production was followed by fluorescence CLSM at 587 nm. Results and Discussion To elaborate biocompatible capsules containing enzymes, the pair of oppositely charged polyelectrolytes should to be properly chosen according to the following prerequisites. First, these polymers should not affect the enzyme activity. Second, the capsules composed of these polymers should be fairly stable, which is not always the case because some core dissolution procedure might lead either to the shell rupture or even to disassembling of multilayer films.

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Table 1. Retention of the Enzymatic Activity of Free Peroxidase (in the Presence of Components for Microcapsule Preparation) and Encapsulated Enzyme in 0.1 M Acetate Buffer, pH 5.0, 20 °C relative activity of peroxidase, % free enzyme duration of storage

buffer

dextran sulfatea

protaminea

melamin formaldehyde hydrolysatea

encapsulated enzyme

1h 4 days 7 days

100 ( 4 85 ( 4 83 ( 4

101 ( 5 95 ( 4 91 ( 4

124 ( 6 102 ( 5 98 ( 5

124 ( 6 104 ( 5 89 ( 3

57 ( 3 55 ( 2 53 ( 2

a

Ratio in solution component: peroxidase ) 100:1 (w/w).

Dextran sulfate with a molecular weight of (Mw) 500 000 was chosen as the polyanion. As the polycation, we selected the strong basic protein protamine (salmin) with the Mw about 5000 and a high content of arginine, which is up to 70% of the total amount of amino acids.21 Such a polyelectrolyte combination has not been used so far for multilayer build up. Both polyelectrolytes revealed no influence on peroxidase activity in 0.1 M acetate buffer at pH 5.0 known as the optimal condition for enzyme function. The weight ratio of polyelectrolytes to enzyme in these experiments varied from 1:1 to 100:1. Table 1 shows that the chosen polyelectrolytes only slightly influenced the activity of the enzyme for 7 days. According to our results (Table 1), the addition of the solution obtained by dissolution of MF particles at acidifying up to pH 1.7 to peroxidase does not suppress the enzyme activity. It is worth mentioning that protamine and melamine formaldehyde hydrolysate gave some activation of peroxidase (24%) after a short incubation. This effect disappeared after incubation for a few days. So some quantity of MF and polyelectrolytes did not negatively influence peroxidase. The capsules were fabricated according to the conventional method14 by the sequential adsorption of 4 bilayers of dextran sulfate/protamine on MF cores in 0.2 M NaCl (pH 5.0). As it is stressed by Gao et al.,22 the process of core dissolution called for a very slow adjusting of pH to the required value and had to be tuned for MF cores from different lots. At fast pH adjustment to low pH values, the capsule wall dissolves in a minute time scale after core dissolution. In this study then, MF cores were decomposed at pH 1.7, and the capsules were washed thoroughly in these conditions. It should take into account that native MF cores from different lots were dissolved at the same pH at which a degradation of this cores coated by the polyelectrolyte shell was observed. Dextran sulfate/protamine microcapsules prepared were kept at pH 3.0 for prevention of microflora growth. The morphology of the capsules was studied by means of confocalmicroscopy (Figure 1a). The capsules became swollen compared to initial templates (5.12 µm). The average size of the microcapsules at pH 3-5 was about 8.0 ( 0.2 µm. With a pH increase up to 7-8, the microcapsules enlarged (swelled) up to a diameter of 9-10 µm. In basic solutions (pH 9-11), the microcapsules changed in form (Figure 1d), but this pH range is not used for working with enzymes. The results of SEM (Figure 2) showed that the capsules were porous visually as if they had a loose shell. The presence of a salient middle region testified that the capsules were not hollow. We can assume that there was a weakly

Figure 1. CLSM image of dextran sulfate/protamine microcapsules: a. at pH 5; b. loaded with 6-carboxyfluorescein at pH 5; c. loaded with rhodamine 6G at pH 5; d. loaded with rhodamine 6G at pH 9; e. (transmission), f. (fluorescence), loaded with FITC-peroxidase at pH 5.

Figure 2. SEM image of dextran sulfate/protaminemicrocapsules.

cross-linked gellike matrix inside the shell, which prevented the loss of the round form by microcapsules on drying. So, microcapsules prepared in this work differed from the hollow capsules fabricated using PSS and PAA.14,18,23

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Figure 3. Imaging of peroxidase activity inside dextran sulfate/ protamine microcapsules: A. transmission; B-E. fluorescence image of resorufin after 1, 11, 12, and 13 min, respectively.

To elucidate the capsule interior composition, we applied various dyes which permeated through the microcapsule wall and bound exclusively either to the positively or negatively charged groups.24 The low molecular weight, negatively charged 6-carboxyfluorescein (Mw 376) and the positively charged rhodamine 6G (Mw 479) penetrated and were homogeneously distributed inside the microcapsules in the pH range 5-8 (Figure 1b-d). Impregnation of differently charged dyes inside the capsule revealed the presence of a homogeneous matrix with both positively and negatively charged groups inside the microcapsules. Positively charged FITC-peroxidase (Mw 44000, isoelectric point pI 8.8) also concentrated inside the microcapsules at pH 3-8 compared to the solution surrounding the microspheres (Figure 1 e, f). To confirm the localization of the active enzyme into microcapsules, we used the reagent Amplex Red (10-acetyl3,7-dihydroxyphenoxazine). In the presence of peroxidase, the Amplex Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 563 and 587 nm. Figure 3 shows that an increase of the fluorescence emission of resorufin was observed inside microcapsules but not in a surrounding solution. Our recently results (ref 25) show that, in the range of pH 5-8, various proteins such as insulin (Mw 6500, pI 5.5), aprotinin (Mw 6500, pI 10.5), trypsin (Mw 24 000, pI 10.5), chymotrypsin (Mw 25 000, pI 8.8), glucose oxidase (Mw 160 000, pI 5.5), and catalase (Mw 250 000, pI 5.4) can penetrate into microcapsules such as peroxidase. In contrast, alginate/protamine microcapsules, prepared on MF cores dissolved at pH 1.7, adsorbed only positively charged substances.26 Therefore, the presence of the alginate gel in the interior of the microcapsules was suggested but not proved.26 Meanwhile, an existing negatively charged complex, formed by PSS and MF degradation products, was postulated for PSS/PAH or poly(diallyldimethylammonium chloride) (PDADMAC) microcapsules.18 The positively charged water-soluble substances (including proteins) were spontaneously deposited inside these microcapsules.18 The microcapsules prepared in this study regardless of their age adsorbed in the interior both positively and negatively charged species of various molecular masses. Apparently, the existence of other components can be suggested besides melamine formaldehyde resins inside the capsules. We used confocal Raman spectroscopy to detect

Figure 4. Raman spectroscopy analysis: Raman spectra of protamine, (A); dextran sulfate, (B); melamine formaldehyde cores (C); a shell (D) and interior (E) of coated MF cores; and a shell (F) and interior (G) of hollow microcapsules.

the presence of polyelectrolytes (protamine and dextran sulfate) inside as well as on the surface of microparticles prepared in this study (coated MF cores and microcapsules). Raman spectra of polyelectrolytes used for multilayer coating, native, and coated MF cores and also of fabricated hollow microcapsules are given in Figure 4. One can see the Raman spectrum of protamine (Figure 4A) as a line with no exact peaks for detection of this polyelectrolyte, wereas dextran sulfate showed a spectrum with an intense peak at about 1100 cm-1 (Figure 4B, peak 1). This peak, which can be assigned to the vibration of sulfonate groups in the polymer, was used as a fingerprint for dextran sulfate. The Raman spectrum of melamine formaldehyde microcores is shown in Figure 4C, peak 2. The pronounced peak with an experimental frequency 977 cm-1 is typical for melamine and melamine formaldehyde resins.27,28 We can see clearly the appearance of the Raman peak assigned to dextran sulfate on the spectrum obtained from surface signal of the multilayer polyelectrolyte coated MF core (Figure 4D). However, upon focusing inside the coated MF particle, a single peak corresponding to melamine formaldehyde resins (Figure 4E) was found, indicating a dense structure of the MF core because of which the polyelectrolytes cannot penetrate inside the particle. However, when the coated core was exposed to conditions at which it dissolves, the polyelectrolytes penetrated into the space contributed by the dissolving core, which was confirmed by spectra corresponding to the signal from the capsule shell (Figure 4F) and from the interior of the capsule (Figure 4G). The appearance of the peak of dextran sulfate was evident from the spectra in addition to the peak of the MF resin. Thus, we can conclude that dextran sulfate from the shell interacted with some of the positively charged MF degradation products. It could cause a special redistribution of the polymers, which resulted in the formation of a porous pHsensitive gellike matrix on the basis of MF-oligomers complexes with both polyelectrolytes used for the LbL assembly. It can be concluded that the properties of the matrix, which is crucial for the spontaneous adsorption of substances in the interior of the microcapsules prepared on

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MF cores, seemed to be mainly governed by the polyelectrolytes used for the shell fabrication. The process of gelation was rather quick when dextran sulfate and protamine, as well as alginate and protamine, were used for LbL assembly. However, it is well-known29 that equilibration in processes with PSS participation establishes rather slowly. It seems to be a cause of the finding that freshly prepared PSS/ PDADMAC microcapsules were not favorable for the spontaneous deposition of the enzyme.30 Further, we studied the principles of peroxidase encapsulation in multilayer dextran sulfate/protamine microcapsules such as pH and the number of microcapsules. The influence of the pH values on the process of peroxidase encapsulation and its keeping inside is shown in Figure 5. With the pH increased from 5 (according to conditions of capsule fabrication) to 8, the loading capacity increased from 0.2 × 108 to 2.2 × 108 peroxidase molecules per microcapsule, respectively. The microcapsule swelling at higher pH seemed to be a reason for an increase in the peroxidase encapsulation payload. Figure 5 demonstrates that the enzyme was partially released from the microcapsule after 3-fold washings of the microcapsules with the buffer used for protein encapsulation. On the other hand, when peroxidase-loaded microcapsules were incubated in buffer solution without its change and stirring, release of the enzyme for 24 h was negligible. Figure 5 shows that the pH of washing solutions in the range 5-8 did not influence on the relative activity of encapsulated peroxidase, which was found about 50% using ABTS. Table 2 shows that at pH 5.0 the amount of protein encapsulated at pH 5.0 and its activity increased proportionally with an increase in the number of microcapsules. The specific activity of encapsulated peroxidase was 57% of initial enzyme activity. At the storage of protein-loaded microcapsules, the specific activity of encapsulated peroxidase after 7 days decreased down to 4%, whereas the loss of activity of native peroxidase was 17% (Table 1). The effect of the pH of the medium on the release of the peroxidase encapsulated at pH 8.0 is shown in Figure 6. Microcapsules were not washed before the release test. One can see at pH 4.0 and 5.0 the protein was released very fast for 10 min (70-80%) but then the leakage was noticeably slow. We suppose that the reason of this change in release rate is the decreasing size of the microcapsules at low a pH value. However, when the pH at which the microparticles were fabricated and the pH at which the protein was released are the same (pH 8.0), the protein release for 10 min was calculated about 20%, which can be explained by the release of protein molecules that interacted weakly with the microcapsule surface followed by a very slow release of the peroxidase encapsulated inside. The microcapsules containing peroxidase can be then coated with additional layers of polyelectrolytes. But this process caused considerable (up to 70%) loss of enzyme because of multiple washing steps between adsorption steps. Nevertheless, the leakage of peroxidase from microcapsules with 6 polyelectrolyte bilayers at the first 4 h was slower than from to initial 4 bilayer microcapsules (Figure 7). Note that peroxidase released from the capsules recovered its activity to 90-95% of the native enzyme activity. So we

Figure 5. Influence of pH value on peroxidase encapsulation into dextran sulfate/protamine microcapsules and specific activity of the encapsulated enzyme. Number of microcapsules 1.4 × 107. Each bar represents the mean of at least three experiments ((S.D.). Table 2. Properties of Peroxidase Loaded Dextran Sulfate/ Protamine Microcapsules at pH 5.0, 20 °C number of microcapsules, ×107

protein into microcapsules, mg

peroxidase activity, U

relative activity of peroxidase, %

0.35 0.70 1.40

0.016 ( 0.001 0.035 ( 0.002 0.070 ( 0.003

5.0 ( 0.2 11.0 ( 0.5 22.0 ( 0.6

57 ( 3 57 ( 2 57 ( 3

presume that the decrease in the activity of the encapsulated peroxidase (57% to control values) could be the consequence of extensive interpolyelectrolyte gel formation inside the

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Figure 6. Influence of pH value on peroxidase release from dextran sulfate/protamine microcapsules. Expressed as % of the protein encapsulated at pH 8.0. Each bar represents the mean of at least three experiments ((S.D.).

Figure 7. Release profiles of peroxidase activity at pH 7.0 from dextran sulfate/protaminemicrocapsules with 4 and 6 polyelectrolyte bilayers. Expressed as % of peroxidase activity used for encapsulation at pH 8.0. Each bar represents the mean of at least three experiments ((S.D.).

microcapsules which created the diffusional limitations for negatively charged substrate ABTS. So positively charged peroxidase with a molecular mass Mw of 44 000 penetrated dextran sulfate/protamine microcapsules through rather large pores of the shells and concentrated inside the microcapsules compared to the surrounding solution. Peroxidase seemed to remain inside capsules because of the interaction with the gellike matrix composed of polyelectrolytes and MF oligomers. It is notewortly that this process did not depend on the age of the microcapsules in contrast with peroxidase deposition in the interior of PSS/PDADMAC microcapsules prepared on MF cores.30 Peroxidase content in dextran sulfate/protamine microcapsules was 10 times higher than chymotrypsin content in hollow microcapsules on the basis of PSS and PAH.16 In contrast to preferential peroxidase adsorption in PSS/PDADMAC microcapsule wall and bulk30 in this work peroxidase was found to distribute homogeneously within dextran sulfate/protamine microcapsules. Changes of pH, which resulted in microcapsules swelling lead to the protein release. Encapsulated peroxidase had a rather high activity, which was stable for 12 months. Conclusions Our results demonstrate that peroxidase can be successively entrapped in the polyelectrolyte multilayer microcapsules whose shells were made of biocompatible polymers, as those used in this work, dextran sulfate/and protamine.

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Apparently peroxidase was embedded in a gellike structure in the microcapsule interior formed by MF residues in the complex with polyelectrolytes, which penetrated inside the capsules after the core was decomposed. The amount of finally incorporated peroxidase depended on pH at which the enzyme was sucked in. This points to the electrostatic nature of protein binding to the interior. In all investigated cases, the peroxidase was homogeneously distributed and exhibited no preferential adsorption onto the microcapsule wall and bulk as it was for microcapsules formed by another polyelectrolyte combination of MF cores. The experiments on exploring this approach to prepare microcapsules loaded with other enzymes, enzyme combination, and to study influence of shell composition on protein uptake and subsequent release are under investigation in our laboratory. Such microcapsules containing proteins/enzymes appeared to be especially promising for the development of stable analytical systems and bioseparation. Acknowledgment. This work was supported by both Sofja Kovalevskaja Program of Alexander von Humboldt Foundation, German Ministry of Education and Research and by the Ministry of Industry, Science and Technologies of Russian Federation in the frame of the Russian-German scientific program. The authors are grateful to Prof. Dr. H. Mo¨hwald for continuous support, Prof. Dr. E. Donath for stimulating discussion, and Dr. D. Shenoy for reading the manuscript and corrections. We thank also Wenfei Dong for help with the Raman spectroscopy. References and Notes (1) Arshady, R., Ed.; Microspheres. Microcapsules & Liposomes; Citus Ltd: London, 1999; Vol. 1, pp 2. (2) Sukhorukov, G. B. In Series “Studies in Interface Science”: NoVel Methods to Study Interfacial Layers; Miller, R., Mo¨bius, D., Eds.; Elsevier Publishers: Amsterdam, The Netherlands, 2001; Vol. 13, p 383. (3) Decher, G.; Hong, J. D. Macromol. Chem. Macromol. Symp. 1991, 46, 321. (4) Decher, G. Science 1997, 277, 1232. (5) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319. (6) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Budde, A.; Mo¨hwald, H. Colloids Surf. A 1998, 137, 253. (7) Antipov, A. A.; Sukhorukov, G. B.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 2001, 105, 2281. (8) Trubetskoy, V. S.; Loomis, A.; Hagstrom, J. E.; Budker, V. G.; Wolff, J. A. Nucleic Acids Res. 1999, 27, 3090. (9) Balabushevitch, N. G.; Sukhorukov, G. B.; Moroz, N. A.; Volodkin, D. V.; Larionova, N. I.; Donath, E.; Mo¨wald, H. Biotechnol. Bioeng. 2001, 76, 207. (10) Bobreshova, M. E.; Sukhorukov, G. B.; Saburova E. A.; Elfimova L. I.; Sukhorukov, B. I.; Sharabchina, L. I. Biophysics 1999, 44, 813. (11) Pommersheim, R.; Schrezenmeir, J.; Vogt, W. Macromol. Chem. Phys. 1994, 195, 1557. (12) Neu, B.; Voigt, A.; Mitlo¨hner, R.; Leporatti, S.; Donath, E.; Gao, C. Y.; Kiesewetter, H.; Mo¨hwald, H.; Meiselman. H. J.; Ba¨umler, H. J. Microencapsulation 2001, 18, 385. (13) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Mo¨hwald, H. Polym. AdV. Technol. 1998, 9, 759. (14) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S.; Mo¨hwald, H. Angew. Chem. 1998, 37, 2201. (15) Sukhorukov, G. B.; Antipov, A. A.; Voigt, A.; Donath, E.; Mo¨hwald, H. Macromol. Rapid Commun. 2001, 22, 44. (16) Tiourina, O. P.; Antipov, A. A.; Sukhorukov, G. B.; Larionova, N. I.; Lvov, Y.; Mo¨hwald, H. Macromol. Biosci. 2001, 1, 209. (17) Lvov Y.; Antipov, A.; Mamedov, A.; Mo¨wald, H.; Sukhorukov, G. B. Nano Lett. 2001, 1, 125.

Dextran Sulfate/Protamine Microsized Capsules (18) Gao, C. Y.; Donath, E.; Mo¨hwald, H.; Shen, J. Angew. Chem., Int. Ed. 2002, 41 (20), 3789. (19) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (20) Childs, R.; Bardsley, W. Biochem. J. 1975, 145, 93. (21) Callanan, M. J.; Carroll, W. R.; Mitchell, E. R. J. Biol. Chem. 1957, 229, 279. (22) Gao, C.; Moya, S.; Lichtenfeld, H.; Casoli, A.; Fielder, H.; Donath, E.; Mo¨hwald, H. Macromol. Mater. Eng. 2001, 286, 355. (23) Sukhorukov, G. B.; Donath, E.; Moya, S.; Susha, A. S.; Voigt, A.; Hartmann, J.; Mo¨hwald. H. J. Microecapsulation 2000, 17, 177. (24) Caruso, F.; Lichtenfeld, H.; Donath, E.; Mo¨hwald. H. Macromolecules 1999, 32, 2317.

Biomacromolecules, Vol. 4, No. 5, 2003 1197 (25) Larionova, N. I.; Balabushevich, N. G.; Sukhorukov, G. B.; Tiourina, O. P.; Zimina, E. P. XI Intern. BRG Workshop on Bioencapsulation; Strasbourg, 2003, May 25-27, # 20. (26) Tiourina, O. P.; Sukhorukov, G. B. Int. J. Pharm. 2002, 242, 155. (27) Koglin, E.; Kip, B.; Meier, R. J. Phys. Chem. 1996, 100, 5078. (28) Scheepers, M. L.; Gelan, J. M.; Carleer, R. A.; Adriaensens, P. J.; Vanderzande, D. J.; Kip, B. J.; Brandts P. M. Vibr. Spectrosc. 1993, 6, 55. (29) Michaels, A. Encyclopedia of Polymer Science and Technology; Interscience Publ.: New York, 1969; Vol. 10, pp 765. (30) Gao, C. Y.; Liu, X. Y.; Shen, J. C.; Mo¨hwald, H. Chem. Commun. 2002, 17, 1928.

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