Laser Physics, Vol. 15, No. 4, 2005, pp. 545551.

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. Podskoov1, D. Chorvt, Jr.2, *, G. Kollrikov3, and I. Lack3
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

AbstractPolymer 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 uorescent labels were employed.

1. INTRODUCTION Biotechnology and biomedicine elds 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 fulll 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, specically, 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 identied. 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 quantication. 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 uorescent 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 modication 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) (Scientic Polymer Products Inc., Ontario, NY) were used for capsule formation. The uorescent 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,

545

546

PODSKOOV et al. (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 (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

H2N

NH2

H2N

NH2

Scheme 1. Structure of used polymers: sodium alginate with guluronic G and mannuronic M acid residues (a); cellulose sulfate (b); poly(methylene-co-guanidine) (c); and the uorescent labels Eosin Y (d), Fluorescein (e), Rhodamine 110 (f), and Rhodamine 123 (g).

1.0 wt % CaCl2, and 0.9 wt % NaCl at pH 7.5. An airstripping nozzle was used to form the nal capsule of around 1 mm. The drop collection and reaction times were 5 and 40 s, respectively, in order to avoid the nonuniform capsules obtained when the reaction and collection times are similar [9]. In the next step, the capsules were immersed in 50-mM sodium citrate at pH 7

for 10 min to equilibrate the polyelectrolyte complex. The batch of capsules was divided into four parts. The rst one was used as a control (C0), while the next three were used to estimate the effect of the last coating layer obtained by capsule C0 treatment in 0.1 wt % coating solutions of CS with molecular weights of 760 kDa (C1) and 185 kDa (C2) and SA with a molecular weight
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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 lter-based confocal detection, (b) multispectral confocal detection (Zeiss META). S, sample; O, objective; SM, scanning mirrors; PD, photodiode; PMT, photomultiplier; CP, confocal pinhole; DM, dichroic mirror.

of 300 kDa (C3), respectively, for 10 min. The capsules were stored in 0.9 wt % NaCl containing 200 ppm of NaN3. All solutions were ltered via a 0.45-m cellulose acetate syringe lter. 5 .METHODS Optical Microscopy and Confocal Laser Scanning Microscopy For a semiautomatic measurement of the capsule wall thickness and capsule diameter, a system based on optical microscopy and digital image processing was developed (www.prover.sk). To characterize microstructure of the wall of microcapsules, confocal laser scanning microscopy was used. The transmission, reection, and uorescence emission were simultaneously measured by the confocal laser scanning microscope LSM510 META (Scheme 2) on Axiovert 200 (Zeiss) using 40/1.2 W C-Apochromat or 10/0.05 objectives, the 488-nm laser line, and emission bands at 435485 nm (reection) and 535590 nm (uorescence). Microcapsules were labeled with cationic (Rhodamine 123, Rhodamine 110) and anionic (Eosin Y, Fluorescein) uorescent labels of concentration 107 mol/L for an incubation time of 1 h.
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Atomic Force Microscopy The AFM probe, a NT-MDT Solver P47 AFM apparatus, was mounted on a small spring silicon cantilever, which deected a laser beam into a four-segmented photodiode. The motion of the probe was sensed through the displacement of the laser. The sensor output triggered the control electronic to move the z piezodriver, so that the laser stayed at its original position. The voltage required for this movement was stored for each point (x, y) and plotted as a gray image. The different voltage values are represented as a topography image with different heights for every pixel on the image. Microcapsules were mechanically xed by metallic mesh, xed to the AFM stand by a magnetic eld, and observed in a liquid (physiological) medium. We achieved the reduction of lateral forces using the semicontact (tapping) measuring mode with resonance frequency 5080 kHz and scanning frequency 0.5 1.5 Hz/line. A 10 10 m area on the surface of each sample was scanned with a resolution of 512 512 pixels. The z resolution of our measurements was in the range of 1 nm; the xy resolution, in the range of tens of nm. The measurements were made for six microcapsules of each type and at two areas for each microcapsule. To investigate the inuence of the last coating layer of the microcapsules on their morphology and surface properties, four types of microcapsules with different coatings (C0C3) were investigated.

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PODSKOOV et al.

(a)

(b)

Fig. 1. Measuring of capsule diameter and membrane thickness using CLSM in transmission (a) and reection (b) modes.

(a)

20 m

(b)

20 m

(c)

20 m

(d)

20 m

(e)

20 m

(f)

20 m

Fig. 2. Representative images of microcapsules labeled with Fluorescein ((a) transmission, (b) reection, (c) uorescence emission) and Rhodamine 123 ((d) transmission, (e) reection, (f) uorescence emission).

6. RESULTS AND DISCUSSION Optical Microscopy and Confocal Laser Scanning Microscopy The determination of the geometrical parameters of the microcapsule (type C0), expressed as capsule size and capsular membrane thickness, were investigated using three complementary techniquesoptical microscopy and confocal microscopy in transmission

(Fig. 1a) and reection (Fig. 1b) modes. The average diameter was 1225 60 m and the membrane thickness was 82 3 m for a given representative batch of microcapsules. The important outcome from Fig. 1 is that obtained values characterizing the membrane thickness from transmission microscopy were in accordance with the confocal reection measurements. The possibility of visualizing the membrane by the confocal
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CHARACTERIZATION OF POLYELECTROLYTE MICROCAPSULES Intensity, arb. units (a) Intensity, arb. units 1.0 1.0 Reflection 0.9 0.9 Fluorescence 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0 20 40 60 80 100 120 140 160 0 20 40 60 Distance, m (c) Intensity, arb. units Intensity, arb. units 1.0 1.1 Reflection 0.9 1.0 Fluorescence 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 0.1 0 20 40 60 80 100 120 140 20 0 20 40 Distance, m (b) Reflection Fluorescence

549

80 100 120 140 160 180 Distance, m (d) Reflection Fluorescence

60 80 100 120 140 160 Distance, m

Fig. 3. Representative intensity proles of uorescence (normalized to 1.0) and reection (normalized to 0.5) in microcapsules labeled with Fluorescein (a), Eosin Y (b), Rhodamine 123 (c), and Rhodamine 110 (d).

reectance image has, to our knowledge, not been emphasized yet. Figure 2 shows representative images of microcapsules labeled with anionic Fluorescein and cationic Rhodamine 123. This gure demonstrates the differences in spatial patterns using different microscopy modes in the presence of differently charged labels. When using a high-aperture objective (40/1.2 W), the working range of the objective is limited and the membrane could not be identied from the transmission image regardless of the uorescent label type (Figs. 2a, 2d). The capsule reection pattern (Figs. 2b, 2e) allows one to visualize the membrane structure, which involves not only the membrane outline visible in transmission mode (Fig. 1a), which is a common mode of the characterization of a capsular membrane, but also the membrane layers of different reective properties, which are localized at the interface between the memLASER PHYSICS Vol. 15 No. 4 2005

brane and the capsule core. This feature was common to all capsules of the same batch. The different charges of the uorescent labels resulted in their distinct spatial distributions. Fluorescein (Fig. 2c) was predominantly localized to the outer membrane region, and its concentration sharply drops towards the membrane interior. On the other hand, cationic Rhodamine 123 (Fig. 2d) was observable in the entire membrane volume, with higher intensity at the inner membrane region. The cross-section proles of uorescence and reection images, which quantify the respective intensities, are shown in Fig. 3 for all uorescent labels used. The results suggest that the outer membrane of a polyelectrolyte microcapsule has a relatively higher positive residual charge than the membrane interior. The residual negative charge exhibits a more complicated prole. Going from the membrane outer layer to the interior, the residual negative charge

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PODSKOOV et al. 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. 4. Typical images of the surface of polyelectrolyte microcapsules using AFM microscopy in a liquid environment.

rst decreases but then sharply increases at the inner membrane wall, in agreement with the intensity of reection, indicating the possible presence of nonuorescent precipitates with a spatial distribution similar to the binding site of the Rhodamines. We also tested the microcapsules for the presence of an autouorescence background. Both reection and faint uorescence signals were present in unlabeled samples, but intrinsic uorescence was negligible compared to the signal level from the uorescent labels. Preliminary results with other uorescent labels point out that the behavior shown in Figs. 2 and 3 may depend on the chemical structure of the label and cannot be generalized. A further insight into the real distribution of membrane-forming polymers could be made possible by applying covalently bound uorescent labels. Atomic Force Microscopy Typical images of microcapsule surfaces taken with the AFM technique are shown in Fig. 4. For quantitative analysis, the characteristic surface roughness Ra and

standard deviation for the z direction on the sample surface Rq were employed. These parameters are in accordance with DIN and ISO evaluation and are dened as follows [10]. The parameter Ra (DIN 4768) denes the average value of the surface roughness within the area being analyzed: 1 R a = -----------NxNy 1 where zmean = -----------NxNy

z ( i, j ) z
i=1j=1 Nx Ny i=1

Nx

Ny

mean

(1)

z . j = 1 ij

The parameter Rq (ISO 4287/1) denes the value of the standard deviation for the z coordinate on the sample surface within the area being analyzed: Rq = 1 -----------NxNy

( z ( i, j ) z
i=1j=1

Nx

Ny

mean )

(2)

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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

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ual negative charge at the inner membrane represents an important message for the encapsulation technology, because it may interfere with the diffusion properties of the capsule with respect to the permeating positive charges. The results from AFM measurements, expressed in surface roughness quantities, show that coating with sodium alginate or cellulose sulfate does not signicantly change the capsule surface topology. ACKNOWLEDGMENTS This work was supported by the Slovak Science and Technology Assistance Agency (contract no. APVT-20016002). REFERENCES
1. I. Lack, Polyelectrolyte Complexes for Microcapsule Formation, in Fundamentals of Cell Immobilisation Biotechnology, Focus on Biotechnology, Ed. by V. Nedovic and T. Willaert (Kluwer Academic, Dordrecht, 2004), ISBN 1-4020-1887-8, Vol. 8A, pp. 103120. 2. P. deVos, A. F. Hamel, and K. Tatarkiewicz, Diabetologia 45, 159 (2002). 3. G. Orive, R. M. Hernndez, A. R. Gascn, et al., Nature Med. 9, 104 (2003). 4. A. Lamprecht, U. F. Schaefer, and C.-M. Lehr, Eur. J. Pharm. Biopharm. 49, 1 (2000). 5. B. L. Strand, Y. A. Morch, T. Espevik, and G. SkjakBraek, Biotechnol. Bioeng. 84, 386 (2002). 6. K. Xu, D. Hercules, I. Lack, and T. G. Wang, J. Biomed. Mater. Res. 41, 461 (1998). 7. Ch. Gao, S. Leporatti, E. Donath, and H. Moehwald, J. Phys. Chem. 104, 7144 (2000). 8. I. Lack, M. Briov, A. V. Anilkumar, et al., J. Biomed. Mater. Res. 39, 52 (1998). 9. A. V. Anilkumar, I. Lack, and T. G. Wang, Biotechnol. Bioeng. 75, 581 (2001). 10. NT MDT Solver P47 User Manual, www.ntmdt.com.

The resulting values of Ra and Rq obtained from the investigated microcapsules are summarized in the table. They show that microcapsules coated with highmolecular-weight CS exhibit higher roughness than the other types. However, taking into account the standard deviation of the values, the differences in roughness for both parameters Ra and Rq in all capsule types were not signicant (t test, p < 0.01). 7. CONCLUSIONS The aim of this study was to demonstrate the possibility of using advanced imaging techniques to study hydrogel polyelectrolyte microcapsules. Confocal laser scanning microscopy was used in an attempt to visualize the spatial distribution of the residual charge, which may correspond to the real composition of the polyelectrolyte complex. Noncovalently bound uorescent labels with either positive or negative charge were employed to monitor the distribution of the residual opposite charge of the polyelectrolytes. We observed that cationic and anionic uorescent labels were distributed in different regions of polyelectrolyte capsules, creating specic spatial patterns in the microcapsule wall. Anionic labels were located preferentially at the outer membrane surface, whereas cationic ones were distributed within the entire volume with distinct areas of high uorescence that corresponded to the reection signal. The discovery of resid-

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2005