Charactcrization of Polyclcctrolytc Microcapsulcs by Confocal Lascr Scanning Microscopy and Atomic...

7/29/2019 Charactcrization of Polyclcctrolytc Microcapsulcs by Confocal Lascr Scanning Microscopy and Atomic Forcc Microscopy

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Laser Physics, Vol. 15, No. 4, 2005, pp. 545551.Original Text Copyright 2005 by Astro, Ltd.Copyright 2005 byMAIK Nauka /Interperiodica (Russia).

1. INTRODUCTIONBiotechnology and biomedicine fields utilize immo-bilization technologies to maintain the viability and/orcatalytic activity of biological material. The semiperme-able membrane, which acts as a protective barrier andprovides for the controlled transport of species to andfrom the encapsulated biological material in a desiredway, is a key factor in this approach. It has been gener-ally agreed that encapsulation in microcapsules is themost promising immobilization technology today [1].Various types of natural and synthetic polymers havebeen used as the capsule-making materials. Of them, thepolyelectrolyte-based microcapsules have been themost widely used, due to their simple preparation proto-cols and the mild conditions that are the prerequisitesfor the encapsulation of biological material [1].

In order to meet the goals of encapsulation, the cap-sule and membrane have to fulfill a number of differentcriteria. Depending on the type of application, informa-tion on the following properties is of high importance:mechanical and chemical stability, selective permeabil-ity, geometry (size, shape), surface chemistry, andtopology. For the immunoprotection of living cells,these parameters determine the biocompatibility of themicrocapsules used. These requirements have not beenmet so far, as was recently reasoned by, for example, deVos et al.

[2] and Orive et al.

[3], who showed that both

the quality of the cells and the encapsulation materialand 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] andatomic force microscopy (AFM) [6, 7]. Using thesetechniques, new features on the micron or submicronscale have been identified. The primary goal of thiscontribution is to further explore the potential of optical

and scanning force microscopies with respect to newapproaches in sample labeling and data quantification.

2. AIM

The aim of the present study was to select a set ofquantitative parameters derived from CLSM and AFMimaging methods to characterize microcapsule geome-try, surface quality, and microstructure. The workinghypothesis for CLSM imaging was that cationic andanionic fluorescent labels diffusing to the capsular inte-rior interact with the residual charge of polyanions andpolycations, respectively; thus, the imaging of the spa-tial distribution of both types of polyelectrolytes will be

possible without chemical modification of reactants bycovalent labeling. Atomic force microscopy was usedfor quantitative characterization of the surface qualityof microcapsules with different coating layers.

3. MATERIAL

High-viscosity sodium alginate (SA) (ISP Algi-nates, UK), sodium cellulose sulfate (CS) (AcrosOrganics, Belgium), and poly(methylene-co-guani-dine) hydrochloride (PMCG) (Scientific Polymer Prod-ucts Inc., Ontario, NY) were used for capsule forma-tion. The fluorescent labels (Rhodamine 123,Rhodamine 110, Eosin Y, and Fluorescein) were fromAcros Organics (Belgium). The chemical structure ofthe polymers and probes used is shown in Scheme 1.

4. FORMATION OF POLYELECTROLYTECAPSULES

Microcapsules based on the recipe developed in [8]were produced by dropping a polyanion solution con-sisting 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,

Characterization of Polyelectrolyte Microcapsulesby Confocal Laser Scanning Microscopy

and Atomic Force Microscopy

J. Podsko

ov

1

, D. Chorvt, Jr.

2, *, G. Kollrikov

3

, and I. Lack

3

1 Faculty of Mathematics, Physics, and Informatics, Comenius University, Bratislava, Slovakia

2International Laser Center, Bratislava, Slovakia

3 Polymer Institute of the Slovak Academy of Sciences, Bratislava, Slovakia

*e-mail: [email protected] October 20, 2004

Abstract

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

BIOPHOTONICS


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1.0 wt % CaCl

2

, and 0.9 wt % NaCl at pH 7.5. An air-stripping nozzle was used to form the final capsule ofaround 1 mm. The drop collection and reaction timeswere 5 and 40 s, respectively, in order to avoid the non-uniform capsules obtained when the reaction and col-lection times are similar [9]. In the next step, the cap-sules 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. Thefirst one was used as a control (C0), while the next threewere used to estimate the effect of the last coating layerobtained by capsule C0 treatment in 0.1 wt % coatingsolutions of CS with molecular weights of 760 kDa(C1) and 185 kDa (C2) and SA with a molecular weight

O

OOOC

O

OO

OH O

OO

OOH

OH

OHOH

OOC

HOOOC

OH

OH OOC

O

OH

OH

CH2

O

SO3

O

OCH2

O

OH

OH n

CH2 NH C

NH

NH

n

O

C ONa

O

Br

O

BrBr

NaO

Br

O

C OH

O

OHO

O

C OH

O

NH2H2N+

O

C OCH3

O

NH2H2N+

G G M M

(a)

(b) (c)

(d) (e)

(f) (g)

Na

+

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 fluorescent labels Eosin Y (d), Fluorescein (e), Rhodamine 110 (f), and Rhodamine

123 (g).

OH


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CHARACTERIZATION OF POLYELECTROLYTE MICROCAPSULES 547

of 300 kDa (C3), respectively, for 10 min. The capsuleswere stored in 0.9 wt % NaCl containing 200 ppm ofNaN

3

. All solutions were filtered via a 0.45-

m cellu-lose acetate syringe filter.

5 .METHODS

Optical Microscopy and ConfocalLaser Scanning Microscopy

For a semiautomatic measurement of the capsulewall thickness and capsule diameter, a system based onoptical microscopy and digital image processing wasdeveloped (www.prover.sk). To characterize micro-structure of the wall of microcapsules, confocal laserscanning microscopy was used. The transmission,reflection, and fluorescence emission were simulta-

neously measured by the confocal laser scanning micro-scope LSM510 META (Scheme 2) on Axiovert 200(Zeiss) using 40

/1.2 W C-Apochromat or 10

/0.05objectives, the 488-nm laser line, and emission bands at435485 nm (reflection) and 535590 nm (fluores-cence).

Microcapsules were labeled with cationic(Rhodamine 123, Rhodamine 110) and anionic (EosinY, Fluorescein) fluorescent labels of concentration10

7

mol/L for an incubation time of 1 h.

Atomic Force Microscopy

The AFM probe, a NT-MDT Solver P47 AFM appa-ratus, was mounted on a small spring silicon cantilever,which deflected a laser beam into a four-segmented

photodiode. The motion of the probe was sensedthrough the displacement of the laser. The sensor outputtriggered the control electronic to move the z

piezo-driver, so that the laser stayed at its original position.The voltage required for this movement was stored foreach point (

x

,y

) and plotted as a gray image. The dif-ferent voltage values are represented as a topographyimage with different heights for every pixel on theimage. Microcapsules were mechanically fixed bymetallic mesh, fixed to the AFM stand by a magneticfield, and observed in a liquid (physiological) medium.We achieved the reduction of lateral forces using thesemicontact (tapping) measuring mode with resonancefrequency 5080 kHz and scanning frequency 0.51.5 Hz/line. A 10

10

m area on the surface of eachsample was scanned with a resolution of 512

512 pix-els. The z

resolution of our measurements was in therange of 1 nm; thexy

resolution, in the range of tens ofnm. The measurements were made for six microcap-sules of each type and at two areas for each microcap-sule. To investigate the influence of the last coatinglayer of the microcapsules on their morphology andsurface properties, four types of microcapsules withdifferent coatings (C0C3) were investigated.

PMT 2

PMT 1

DM 3

CP 2

CP 3

CP 1DM 2

DM 1

SM

PD

S

O

Ar:ion laserMulti-channel PMT

(b)

(a)

Scheme 2.

Scheme of the confocal laser scanning microscope: (a) dual-channel filter-based confocal detection, (b) multispectralconfocal detection (Zeiss META). S, sample; O, objective; SM, scanning mirrors; PD, photodiode; PMT, photomultiplier; CP, con-focal pinhole; DM, dichroic mirror.


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6. RESULTS AND DISCUSSION

Optical Microscopy and ConfocalLaser Scanning Microscopy

The determination of the geometrical parameters ofthe microcapsule (type C0), expressed as capsule sizeand capsular membrane thickness, were investigatedusing three complementary techniquesopticalmicroscopy and confocal microscopy in transmission

(Fig. 1a) and reflection (Fig. 1b) modes. The averagediameter was 1225

60

m and the membrane thick-ness was 82

3

m for a given representative batch ofmicrocapsules. The important outcome from Fig. 1 isthat obtained values characterizing the membranethickness from transmission microscopy were in accor-dance with the confocal reflection measurements. Thepossibility of visualizing the membrane by the confocal

(a) (b)

Fig. 1.

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

20

m

(a)

20

m 20

m

20

m20

m20

m

(d)

(b)

(e)

(c)

(f)

Fig. 2.

Representative images of microcapsules labeled with Fluorescein ((a) transmission, (b) reflection, (c) fluorescence emission)and Rhodamine 123 ((d) transmission, (e) reflection, (f) fluorescence emission).


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reflectance image has, to our knowledge, not beenemphasized yet.

Figure 2 shows representative images of microcap-sules labeled with anionic Fluorescein and cationicRhodamine 123. This figure demonstrates the differ-ences in spatial patterns using different microscopymodes 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 mem-brane could not be identified from the transmissionimage regardless of the fluorescent label type (Figs. 2a,2d). The capsule reflection pattern (Figs. 2b, 2e) allowsone to visualize the membrane structure, whichinvolves not only the membrane outline visible in trans-mission mode (Fig. 1a), which is a common mode ofthe characterization of a capsular membrane, but alsothe membrane layers of different reflective properties,which are localized at the interface between the mem-

brane and the capsule core. This feature was commonto all capsules of the same batch.

The different charges of the fluorescent labelsresulted in their distinct spatial distributions. Fluores-cein (Fig. 2c) was predominantly localized to the outermembrane region, and its concentration sharply dropstowards the membrane interior. On the other hand, cat-ionic Rhodamine 123 (Fig. 2d) was observable in the

entire membrane volume, with higher intensity at theinner membrane region. The cross-section profiles offluorescence and reflection images, which quantify therespective intensities, are shown in Fig. 3 for all fluores-cent labels used. The results suggest that the outermembrane of a polyelectrolyte microcapsule has a rel-atively higher positive residual charge than the mem-brane interior. The residual negative charge exhibits amore complicated profile. Going from the membraneouter layer to the interior, the residual negative charge

0

20

Intensity, arb. units

Distance,

m

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

40 60 80 100 120 140 1600

Reflection

Fluorescence

0

20


Distance,

m

0.1

0.2

0.3

0.40.5

0.6

0.7

0.8

0.9

1.0

40 60 80 100 120 140 1600

Reflection

Fluorescence

180

0

20


Distance,

m

0.1

0.2

0.3

0.4

0.5

0.6

0.70.8

0.9

1.0

40 60 80 100 120 1400

Reflection

Fluorescence

0

20


Distance,

m

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.80.9

1.0

40 60 80 100 120 140 160

Reflection

Fluorescence

1.1

0200.1

Fig. 3.

Representative intensity profiles of fluorescence (normalized to 1.0) and reflection (normalized to 0.5) in microcapsuleslabeled with Fluorescein (a), Eosin Y (b), Rhodamine 123 (c), and Rhodamine 110 (d).

(a) (b)

(c) (d)


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first decreases but then sharply increases at the innermembrane wall, in agreement with the intensity ofreflection, indicating the possible presence of nonfluo-rescent precipitates with a spatial distribution similar tothe binding site of the Rhodamines.

We also tested the microcapsules for the presence ofan autofluorescence background. Both reflection andfaint fluorescence signals were present in unlabeledsamples, but intrinsic fluorescence was negligible com-pared to the signal level from the fluorescent labels.Preliminary results with other fluorescent labels pointout that the behavior shown in Figs. 2 and 3 maydepend on the chemical structure of the label and can-not be generalized. A further insight into the real distri-bution of membrane-forming polymers could be madepossible by applying covalently bound fluorescentlabels.

Atomic Force Microscopy

Typical images of microcapsule surfaces taken withthe AFM technique are shown in Fig. 4. For quantitativeanalysis, the characteristic surface roughness R

a

and

standard deviation for thez

direction on the sample sur-faceR

q

were employed. These parameters are in accor-dance with DIN and ISO evaluation and are defined asfollows [10].

The parameter R

a

(DIN 4768) defines the averagevalue of the surface roughness within the area beinganalyzed:

(1)

wherez

mean

= .

The parameterR

q

(ISO 4287/1) defines the value ofthe standard deviation for thez

coordinate on the sam-ple surface within the area being analyzed:

(2)

Ra1

NxNy------------- z i j,( ) zmean ,

j 1=

Ny

i 1=

Nx

=

1NxNy------------- zijj 1=

Nyi 1=Nx

Rq1

NxNy------------- z i j,( ) zmean( )

2

j 1=

Ny

i 1=

Nx

.=

00

2000

4000

6000

8000

2000

4000

6000

8000

10000nM

002000

4000

60008000

2000

4000

6000

8000

10000nM

00

2000

4000

6000

8000

2000

4000

6000

8000

nM

00

2000

4000

6000

8000

2000

4000

6000

8000

10000nM

500

400

300

200

100

0

nM

500

400

300

200

100

0

nM

nM

400

300

200

100

0

nM

400

300

200

100

0

500

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

C0 C1

C2 C3


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The resulting values ofRa andRq obtained from theinvestigated microcapsules are summarized in thetable. They show that microcapsules coated with high-molecular-weight CS exhibit higher roughness than theother types. However, taking into account the standarddeviation of the values, the differences in roughness forboth parametersRa andRq in all capsule types were notsignificant (ttest,p < 0.01).

7. CONCLUSIONS

The aim of this study was to demonstrate the possi-bility of using advanced imaging techniques to studyhydrogel polyelectrolyte microcapsules. Confocal laserscanning microscopy was used in an attempt to visual-ize the spatial distribution of the residual charge, whichmay correspond to the real composition of the polyelec-trolyte complex. Noncovalently bound fluorescentlabels with either positive or negative charge wereemployed to monitor the distribution of the residualopposite charge of the polyelectrolytes.

We observed that cationic and anionic fluorescentlabels were distributed in different regions of polyelec-trolyte capsules, creating specific spatial patterns in the

microcapsule wall. Anionic labels were located prefer-entially at the outer membrane surface, whereas cat-ionic ones were distributed within the entire volumewith distinct areas of high fluorescence that corre-sponded to the reflection signal. The discovery of resid-

ual negative charge at the inner membrane representsan important message for the encapsulation technology,because it may interfere with the diffusion properties ofthe capsule with respect to the permeating positivecharges.

The results from AFM measurements, expressed insurface roughness quantities, show that coating withsodium alginate or cellulose sulfate does not signifi-

cantly change the capsule surface topology.

ACKNOWLEDGMENTS

This work was supported by the Slovak Science andTechnology Assistance Agency (contract no. APVT-20-016002).

REFERENCES1. I. Lack, Polyelectrolyte Complexes for Microcapsule

Formation, in Fundamentals of Cell ImmobilisationBiotechnology, Focus on Biotechnology, Ed. by V. Nedo-vic 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. Skjak-

Braek, 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.

Mean values ofRa and Rq computed for the microcapsuleswith different coatings

Capsule type Rq [nm] Ra [nm]

C0 54 33 43 28

C1 97 39 77 31

C2 48 25 38 20

C3 48 18 38 14

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