Laser Physics, Vol. 15, No. 4, 2005, pp. 545–551.

Original Text Copyright © 2005 by Astro, Ltd. Copyright © 2005 by MAIK “Nauka /Interperiodica” (Russia).

BIOPHOTONICS

Characterization of Polyelectrolyte Microcapsules by Confocal Laser Scanning Microscopy and Atomic Force Microscopy
J. Podskoçová1, D. Chorvát, Jr.2, *, G. Kolláriková3, and I. Lacík3
1 Faculty

of Mathematics, Physics, and Informatics, Comenius University, Bratislava, Slovakia 2 International Laser Center, Bratislava, Slovakia 3 Polymer Institute of the Slovak Academy of Sciences, Bratislava, Slovakia
*e-mail: dusan@ilc.sk Received October 20, 2004

Abstract—Polymer microcapsules based on sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) were characterized with respect to their geometry, surface quality, and microstructure. To quantitatively describe these properties, atomic force microscopy in a liquid environment, optical microscopy, and confocal laser scanning microscopy using noncovalently bound fluorescent labels were employed.

1. INTRODUCTION Biotechnology and biomedicine fields utilize immobilization technologies to maintain the viability and/or catalytic activity of biological material. The semipermeable membrane, which acts as a protective barrier and provides for the controlled transport of species to and from the encapsulated biological material in a desired way, is a key factor in this approach. It has been generally agreed that encapsulation in microcapsules is the most promising immobilization technology today [1]. Various types of natural and synthetic polymers have been used as the capsule-making materials. Of them, the polyelectrolyte-based microcapsules have been the most widely used, due to their simple preparation protocols and the mild conditions that are the prerequisites for the encapsulation of biological material [1]. In order to meet the goals of encapsulation, the capsule and membrane have to fulfill a number of different criteria. Depending on the type of application, information on the following properties is of high importance: mechanical and chemical stability, selective permeability, geometry (size, shape), surface chemistry, and topology. For the immunoprotection of living cells, these parameters determine the biocompatibility of the microcapsules used. These requirements have not been met so far, as was recently reasoned by, for example, de Vos et al. [2] and Orive et al. [3], who showed that both the quality of the cells and the encapsulation material and technology should be optimized. In recent years, new experimental techniques for microcapsule characterization have been introduced, specifically, confocal laser scanning microscopy (CLSM) using covalently bound labels [4, 5] and atomic force microscopy (AFM) [6, 7]. Using these techniques, new features on the micron or submicron scale have been identified. The primary goal of this contribution is to further explore the potential of optical

and scanning force microscopies with respect to new approaches in sample labeling and data quantification. 2. AIM The aim of the present study was to select a set of quantitative parameters derived from CLSM and AFM imaging methods to characterize microcapsule geometry, surface quality, and microstructure. The working hypothesis for CLSM imaging was that cationic and anionic fluorescent labels diffusing to the capsular interior interact with the residual charge of polyanions and polycations, respectively; thus, the imaging of the spatial distribution of both types of polyelectrolytes will be possible without chemical modification of reactants by covalent labeling. Atomic force microscopy was used for quantitative characterization of the surface quality of microcapsules with different coating layers. 3. MATERIAL High-viscosity sodium alginate (SA) (ISP Alginates, UK), sodium cellulose sulfate (CS) (Acros Organics, Belgium), and poly(methylene-co-guanidine) hydrochloride (PMCG) (Scientific Polymer Products Inc., Ontario, NY) were used for capsule formation. The fluorescent labels (Rhodamine 123, Rhodamine 110, Eosin Y, and Fluorescein) were from Acros Organics (Belgium). The chemical structure of the polymers and probes used is shown in Scheme 1. 4. FORMATION OF POLYELECTROLYTE CAPSULES Microcapsules based on the recipe developed in [8] were produced by dropping a polyanion solution consisting of 0.9 wt % SA and 0.9 wt % CS (in 0.9 wt % NaCl at pH 7.0) into a solution of 1.2 wt % PMCG,

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The first one was used as a control (C0).0 wt % CaCl2. (a) OOC O OH O OH G O OH O OH OOC G (b) – SO3 OOC O HO O OH OH O OOC O OH O M M (c) Na+ CH2 NH C NH NH O OH CH2 OH (d) O C ONa Br O O Br (f) O C OH (g) O C OCH3 + n n O CH2 O OH O OH O OH (e) O C OH Br NaO Br HO O O H2N O NH2 + H2N O NH2 Scheme 1. Rhodamine 110 (f). poly(methylene-co-guanidine) (c). and 0.5.1 wt % coating solutions of CS with molecular weights of 760 kDa (C1) and 185 kDa (C2) and SA with a molecular weight LASER PHYSICS Vol. Structure of used polymers: sodium alginate with guluronic G and mannuronic M acid residues (a). The drop collection and reaction times were 5 and 40 s. The batch of capsules was divided into four parts. cellulose sulfate (b). in order to avoid the nonuniform capsules obtained when the reaction and collection times are similar [9]. An airstripping nozzle was used to form the final capsule of around 1 mm. Fluorescein (e). and the fluorescent labels Eosin Y (d). the capsules were immersed in 50-mM sodium citrate at pH 7 for 10 min to equilibrate the polyelectrolyte complex. 15 No. and Rhodamine 123 (g). respectively.546 PODSKOÇOVÁ et al. In the next step.9 wt % NaCl at pH 7. while the next three were used to estimate the effect of the last coating layer obtained by capsule C0 treatment in 0. 1. 4 2005 .

To characterize microstructure of the wall of microcapsules. 15 No. was mounted on a small spring silicon cantilever. To investigate the influence of the last coating layer of the microcapsules on their morphology and surface properties. and observed in a liquid (physiological) medium. 5 . PMT. CP. objective. (b) multispectral confocal detection (Zeiss META). Rhodamine 110) and anionic (Eosin Y. The voltage required for this movement was stored for each point (x. four types of microcapsules with different coatings (C0–C3) were investigated. The measurements were made for six microcapsules of each type and at two areas for each microcapsule. DM. The z resolution of our measurements was in the range of 1 nm.METHODS Optical Microscopy and Confocal Laser Scanning Microscopy For a semiautomatic measurement of the capsule wall thickness and capsule diameter. for 10 min. y) and plotted as a gray image. The sensor output triggered the control electronic to move the z piezodriver. Fluorescein) fluorescent labels of concentration 10−7 mol/L for an incubation time of 1 h. A 10 × 10 μm area on the surface of each sample was scanned with a resolution of 512 × 512 pixels. dichroic mirror. sample. We achieved the reduction of lateral forces using the semicontact (tapping) measuring mode with resonance frequency 50–80 kHz and scanning frequency 0. The capsules were stored in 0. LASER PHYSICS Vol.9 wt % NaCl containing 200 ppm of NaN3. PD. which deflected a laser beam into a four-segmented photodiode.05 objectives. scanning mirrors.5– 1. SM.sk). Microcapsules were mechanically fixed by metallic mesh. in the range of tens of nm. confocal pinhole. so that the laser stayed at its original position. the 488-nm laser line. O. photodiode. a NT-MDT Solver P47 AFM apparatus. of 300 kDa (C3). The transmission. The different voltage values are represented as a topography image with different heights for every pixel on the image.CHARACTERIZATION OF POLYELECTROLYTE MICROCAPSULES PD (a) PMT 1 S CP 2 DM 3 CP 3 O 547 PMT 2 (b) DM 1 SM CP 1 DM 2 Multi-channel PMT Ar:ion laser Scheme 2. Scheme of the confocal laser scanning microscope: (a) dual-channel filter-based confocal detection. Microcapsules were labeled with cationic (Rhodamine 123. The motion of the probe was sensed through the displacement of the laser.5 Hz/line. respectively. . All solutions were filtered via a 0. photomultiplier.2 W C-Apochromat or 10×/0. 4 2005 Atomic Force Microscopy The AFM probe. reflection. and fluorescence emission were simultaneously measured by the confocal laser scanning microscope LSM510 META (Scheme 2) on Axiovert 200 (Zeiss) using 40×/1. fixed to the AFM stand by a magnetic field. a system based on optical microscopy and digital image processing was developed (www. the xy resolution. S. and emission bands at 435–485 nm (reflection) and 535–590 nm (fluorescence).prover.45-μm cellulose acetate syringe filter. confocal laser scanning microscopy was used.

15 No. The important outcome from Fig. (a) 20 μm (b) 20 μm (c) 20 μm (d) 20 μm (e) 20 μm (f) 20 μm Fig. expressed as capsule size and capsular membrane thickness. were investigated using three complementary techniques—optical microscopy and confocal microscopy in transmission (Fig. (e) reflection. The possibility of visualizing the membrane by the confocal LASER PHYSICS Vol. 1 is that obtained values characterizing the membrane thickness from transmission microscopy were in accordance with the confocal reflection measurements. Representative images of microcapsules labeled with Fluorescein ((a) transmission. (b) reflection. (f) fluorescence emission). 6.548 PODSKOÇOVÁ et al. 4 2005 . (a) (b) Fig. (c) fluorescence emission) and Rhodamine 123 ((d) transmission. The average diameter was 1225 ± 60 μm and the membrane thickness was 82 ± 3 μm for a given representative batch of microcapsules. 1. 1b) modes. Measuring of capsule diameter and membrane thickness using CLSM in transmission (a) and reflection (b) modes. 1a) and reflection (Fig. 2. RESULTS AND DISCUSSION Optical Microscopy and Confocal Laser Scanning Microscopy The determination of the geometrical parameters of the microcapsule (type C0).

The results suggest that the outer membrane of a polyelectrolyte microcapsule has a relatively higher positive residual charge than the membrane interior.1 0 20 40 60 80 100 120 140 –20 0 20 40 Distance.2 0.5 0. μm (d) Reflection Fluorescence 60 80 100 120 140 160 Distance.1 0 0 –0. This figure demonstrates the differences in spatial patterns using different microscopy modes in the presence of differently charged labels. 2c) was predominantly localized to the outer membrane region. which is a common mode of the characterization of a capsular membrane. The capsule reflection pattern (Figs. μm (b) Reflection Fluorescence 549 80 100 120 140 160 180 Distance.1 0 0 0 20 40 60 80 100 120 140 160 0 20 40 60 Distance. 2a.2 0.4 0.6 0. not been emphasized yet. arb. which are localized at the interface between the memLASER PHYSICS Vol.2 0. μm (c) Intensity.9 0.9 Fluorescence 0.0 Fluorescence 0.8 0. which quantify the respective intensities. 1a). arb. Going from the membrane outer layer to the interior. 2e) allows one to visualize the membrane structure. arb. and its concentration sharply drops towards the membrane interior. This feature was common to all capsules of the same batch. 3 for all fluorescent labels used.CHARACTERIZATION OF POLYELECTROLYTE MICROCAPSULES Intensity.9 0. Rhodamine 123 (c). 15 No. Eosin Y (b).0 1. 3. units (a) Intensity. units 1.1 0.7 0.6 0.4 0. 2d) was observable in the entire membrane volume.0) and reflection (normalized to 0.0 Reflection 0.1 0. with higher intensity at the inner membrane region.6 0. μm Fig. The cross-section profiles of fluorescence and reflection images. units Intensity.8 0.1 Reflection 0. are shown in Fig. Figure 2 shows representative images of microcapsules labeled with anionic Fluorescein and cationic Rhodamine 123.0 1.2 W).3 0. 2b.9 1.8 0.5 0.3 0.4 0.5) in microcapsules labeled with Fluorescein (a).5 0.7 0.4 0. The different charges of the fluorescent labels resulted in their distinct spatial distributions. the residual negative charge .3 0. and Rhodamine 110 (d).7 0.8 0.5 0. units 1. On the other hand. cationic Rhodamine 123 (Fig. The residual negative charge exhibits a more complicated profile.3 0. 2d). which involves not only the membrane outline visible in transmission mode (Fig.2 0. Representative intensity profiles of fluorescence (normalized to 1.7 0. Fluorescein (Fig. When using a high-aperture objective (40×/1.6 0. reflectance image has. 4 2005 brane and the capsule core. to our knowledge. but also the membrane layers of different reflective properties. the working range of the objective is limited and the membrane could not be identified from the transmission image regardless of the fluorescent label type (Figs. arb.

15 No. These parameters are in accordance with DIN and ISO evaluation and are defined as follows [10]. j ) – z i=1j=1 Nx Ny i=1 Nx Ny mean . 4 2005 .550 C0 PODSKOÇOVÁ et al. but intrinsic fluorescence was negligible compared to the signal level from the fluorescent labels. j = 1 ij The parameter Rq (ISO 4287/1) defines the value of the standard deviation for the z coordinate on the sample surface within the area being analyzed: Rq = 1 -----------NxNy ∑ ∑ ( z ( i. We also tested the microcapsules for the presence of an autofluorescence background. indicating the possible presence of nonfluorescent precipitates with a spatial distribution similar to the binding site of the Rhodamines. A further insight into the real distribution of membrane-forming polymers could be made possible by applying covalently bound fluorescent labels. in agreement with the intensity of reflection. Atomic Force Microscopy Typical images of microcapsule surfaces taken with the AFM technique are shown in Fig. For quantitative analysis. the characteristic surface roughness Ra and standard deviation for the z direction on the sample surface Rq were employed. 2 and 3 may depend on the chemical structure of the label and cannot be generalized. The parameter Ra (DIN 4768) defines the average value of the surface roughness within the area being analyzed: 1 R a = -----------NxNy 1 where zmean = -----------NxNy ∑ ∑ z ( i. (2) LASER PHYSICS Vol. first decreases but then sharply increases at the inner membrane wall. Preliminary results with other fluorescent labels point out that the behavior shown in Figs. j ) – z i=1j=1 Nx Ny mean ) 2 . C1 nM 500 400 nM 10000 8000 6000 4000 2000 0 0 C2 nM 500 400 nM 10000 8000 6000 4000 2000 0 0 8000 6000 4000 2000 300 200 100 0 nM 8000 6000 4000 2000 0 0 2000 6000 4000 8000 200 100 0 C3 nM 400 300 nM 10000 200 8000 100 6000 4000 0 2000 300 8000 6000 4000 2000 0 0 nM 500 400 300 200 8000 6000 4000 2000 100 0 Fig. Typical images of the surface of polyelectrolyte microcapsules using AFM microscopy in a liquid environment. 4. 4. (1) ∑ ∑ z . Both reflection and faint fluorescence signals were present in unlabeled samples.

49. Hercules. A. Moehwald. Vol. Lacík. D. J. 84. NT MDT Solver P47 User Manual. Wang. Hamel. Diabetologia 45. 2. ISBN 1-4020-1887-8. 386 (2002). 7. Xu. J. Strand. Res. J. whereas cationic ones were distributed within the entire volume with distinct areas of high fluorescence that corresponded to the reflection signal. P.01). Mater. Biotechnol. and T. and C. The discovery of resid- LASER PHYSICS Vol. T. Morch. Focus on Biotechnology. R. Donath. 9. Espevik. 4. 75. Leporatti. I. I. B. Lacík. Ed. The results from AFM measurements. Bioeng. A. the differences in roughness for both parameters Ra and Rq in all capsule types were not significant (t test. U. Tatarkiewicz. K. G. Hernández. 10. 8. Lacík. Biotechnol. F. Biomed. 7144 (2000). 4 2005 . L. 5. www. M. 104 (2003). “Polyelectrolyte Complexes for Microcapsule Formation.-M. G. R. 103–120. Anionic labels were located preferentially at the outer membrane surface. show that coating with sodium alginate or cellulose sulfate does not significantly change the capsule surface topology. Ch.com.. I. 7. and G. because it may interfere with the diffusion properties of the capsule with respect to the permeating positive charges. et al.ntmdt. Chem. Lehr. Pharm. Confocal laser scanning microscopy was used in an attempt to visualize the spatial distribution of the residual charge. 581 (2001). and K. Orive. pp. Bioeng. V. Nature Med. REFERENCES 1. Eur. A. 3. taking into account the standard deviation of the values.” in Fundamentals of Cell Immobilisation Biotechnology. S. 52 (1998). They show that microcapsules coated with highmolecular-weight CS exhibit higher roughness than the other types. 39.. 6. APVT-20016002). and H. Gao. deVos. Phys. which may correspond to the real composition of the polyelectrolyte complex. 2004). SkjakBraek. p < 0. A. Wang. 9. 8A. Lacík. et al.CHARACTERIZATION OF POLYELECTROLYTE MICROCAPSULES Mean values of Ra and Rq computed for the microcapsules with different coatings Capsule type C0 C1 C2 C3 Rq [nm] 54 ± 33 97 ± 39 48 ± 25 48 ± 18 Ra [nm] 43 ± 28 77 ± 31 38 ± 20 38 ± 14 551 ual negative charge at the inner membrane represents an important message for the encapsulation technology. Mater. and T. G. CONCLUSIONS The aim of this study was to demonstrate the possibility of using advanced imaging techniques to study hydrogel polyelectrolyte microcapsules. 15 No. Res. E. However. Y. 461 (1998). Willaert (Kluwer Academic. The resulting values of Ra and Rq obtained from the investigated microcapsules are summarized in the table. Biopharm. Schaefer. Bri¡¡ová. A. Anilkumar. A. M. Gascón. Lamprecht. 1 (2000). ACKNOWLEDGMENTS This work was supported by the Slovak Science and Technology Assistance Agency (contract no. Dordrecht. Anilkumar. 41. I. Noncovalently bound fluorescent labels with either positive or negative charge were employed to monitor the distribution of the residual opposite charge of the polyelectrolytes. 104. V. by V. expressed in surface roughness quantities. We observed that cationic and anionic fluorescent labels were distributed in different regions of polyelectrolyte capsules. F. Biomed. Nedovic and T. creating specific spatial patterns in the microcapsule wall. J. 159 (2002).

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