Micromechanical Properties of Newly Developed Polyelectrolyte Microcapsules (PEMC)

Micromechanical Properties of Newly Developed Polyelectrolyte Microcapsules (PEMC)

Hans Bäumler

, Christina Kelemen

, Rita Mitlöhner

, Radostina Georgieva

, Astrid Krabi

, Silke Schäling

, Gerhard Artmann

, and Holger Kiesewetter

Summary.

Microcapsules fabricated from polyelectrolytes offer advantages in that they are permeable to polar molecules and are extremely stable against chemical and physical influences as compared with liposomes. Polyelectrolyte microcapsules (PEMC) were prepared by consecutive multiple adsorption of different polyanions and polycations on decomposable/dissolvable biological templates like red blood cells. Poly(styrene sulfonate) [PSS], dextransulfate and human serum albumin were adsorbed alternately with poly(allylamine hydrochloride) [PAH] onto glutardialdehyde treated red blood cells as tem- plate, which was decomposed after completing the coating by a hypochlorite solution. Deformability properties of PEMC were studied by means of micropipette technique and confocal laser scanning imaging. The morpho- logical properties of the PEMC were characterised by atomic force microscopy, confocal laser scanning microscopy and electrophoretic mobil- ity. Rheological properties were investigated by viscosimetry as well aggre- gometry (light backscattering). The detectable surface charge was always negative. The wall thickness as well as the bending modulus (BM) of the PEMC was dependent on the number of layers and the used polyelectrolytes.

The thicknesses were in the range between 7 and 28 nm and the BM was 4 to 5 magnitudes larger than the BM of red blood cells, which results in a slightly increased apparent blood viscosity and an increased aggregation time of a mixture of PEMC and blood (1 : 1).

Key words.

Microcapsules, Polyelectrolytes, Deformability, Electrophoresis

205

1Institute of Transfusion Medicine, Charité, Humboldt University of Berlin, D-10098 Berlin, Germany

2Department of Applied Biophysics, TFH Aachen, D-52428 Jülich, Germany

3Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam-Golm, Germany

Introduction

Recently, micro- and nano-sized polyelectrolyte capsules have been fabricated applying the layer-by-layer adsorption technique on charged colloidal parti- cles with subsequent decomposition and removal of the core [1–3]. The size of the capsules can be varied from 0.1 to tens of microns depending on the size of the template. The thickness of the capsule wall depends on the number of assembled polyelectrolyte layers and can be adjusted in the nanometer range [4]. A variety of colloidal particles, like melamine resin latexes (MRL) [1–3,5–7] or biological cells [3,8] have already been used as templates for the capsule preparation.

In the fabrication of the red blood cells (RBC) capsules a deproteinizer has been used resulting in an oxidation of both the biological template and the assembled polyelectrolyte layers. This causes a loss of amino groups in the film as well as a significant mass reduction. In addition, the oxidation and release of biological material lead to a transient osmotic expansion [7].

The reversibility of the expansion was explained by the elasticity of the shell wall representing a polymer net held together by bonds formed during oxidation.

The novel capsules may have applications in biology and medicine as micro-containers and micro-reactors for drugs, enzymes, DNA and other bioactive substances. Their size and shape, and their mechanical and chemi- cal stability mimic that of the cells. Especially, microcapsules fabricated on RBC are very interesting for this purpose because their elastic properties and their shape largely match the original biological template [8].

A new polyelectrolyte combination of the biocompatible dextran sulphate and poly(allylamine hydrochloride) was used for capsule preparation. The micromechanical properties of these new capsules were compared with poly(styrene sulphonate)/poly(allyl amine hydrochloride) capsules.

Materials and Methods

Materials

The sources of chemicals were the following:

Polyelectrolytes: poly (styrene sulphonate, sodium salt) (PSS), Mw 70000, Aldrich (Steinheim, Germany); poly (allyl amine hydrochloride), (PAH), Mw 70000 , Aldrich; dextran sulphate, (DxSO

), Mw 500000, Pharmacia Fine Chem- icals (Uppsala, Sweden), human serum albumin, (HSA), Mw ca. 65000, Sigma (St. Louis, MO, USA).

Labelled substances: fluorescein isothiocyanate labelled human serum

albumin, (FITC-HSA), fluorescein isothiocyanate labelled dextran, (FITC-

Dextran), Mw 77000; Sigma.

Other chemicals: glutaraldehyde (Grade I), Sigma-Aldrich (Schnelldorf, Germany); sodium hypochlorite (NaOCl), sodium chloride (NaCl) and phos- phate buffer solution (PBS).

Capsule Preparation

Erythrocyte Fixation

Erythrocytes were obtained from fresh human blood anti-coagulated with ethylene diamine tetra-acetate (EDTA) by means of centrifugation and sub- sequently washed twice in buffered NaCl solution (140 mM NaCl, 5.6 mM KCl, 5 .8 mM sodium phosphate buffer, pH 7.4). The cells were then stabilized with glutaraldehyde [9] at a final concentration of 2% for 60 min at 20°C. After fix- ation the cells were washed at least four times with distilled water and then resuspended in 154 mM NaCl solution.

Polyelectrolyte Film Assembly and Core Dissolution

The stepwise adsorption of oppositely charged polymers was performed using a filtration technique [3] and is schematically illustrated in Fig. 1. The polyelectrolyte assembly was performed either with PSS or DxSO

as the neg- atively charged polyelectrolytes followed by PAH as the positive polyelec- trolyte. The coating was always started with the negative polymer. Fixed erythrocytes were suspended in buffer-free 1 mg/ml polyelectrolyte and 0 .5 M NaCl with a final cell concentration of 10%(v/v). The pH values were 5.0 ± 0.1, 5.5 ± 0.1 and 6.0 ± 0.2 for the PAH, PSS and DxSO

solutions,

Fig. 1. Scheme of the layer-by-layer adsorption (LbL) of polyelectrolytes on a red blood cell template. PEMC, polyelectrolyte microcapsules

respectively. The cells were incubated under slight stirring for 15 min at room temperature. Washing the samples twice in a 100 mM NaCl (pH 6.5 ∏ 7.0) solu- tion finished each step.

For the preparation of the microcapsules erythrocytes with five, seven or fifteen layers (PSS/PAH)

PSS, (PSS/PAH)

PSS, (PSS/PAH)

PSS, or (DxSO

/PAH)

DxSO

, (DxSO

/PAH)

DxSO

, (DxSO

/PAH)

DxSO

, respec- tively, were suspended in a solution of 140 mM NaCl and 1.2% NaOCl. Within 20 min of incubation at 20°C the cellular template was dissolved obtaining hollow polyelectrolyte capsules [8]. Afterwards the sample was washed three times with a 154 mM NaCl solution and additionally with distilled water until a supernatant conductivity of 0.5 mS/m was reached. Since the capsules are permeable for small ions rinsing with water does not induce any osmotic response.

Confocal Laser Scanning Microscopy

Confocal images were taken with a confocal laser-scanning system CLSM 510 META attached to an inverse microscope from Zeiss (Jena, Germany), equipped with a 100¥ oil immersion objective with a numerical aperture of 1 .4.

Scanning Force Microscopy

Scanning Force Microscopy SFM images have been recorded in air at room temperature using a Nanoscope III Multimode SFM (Digital Instrument, Santa Barbara, CA, USA) in contact mode. Microlithographed tips on silicon nitride (Si

N

) cantilevers with a force constant of 0.58 N/m (Digital Instru- ment) have been used. Scanning Force Microscopy (SFM) images were processed by using the Nanoscope software. Samples have been prepared by applying a drop of the capsule solution onto a freshly cleaved mica substrate.

After allowing the capsules to settle the substrate was extensively rinsed in water and then dried under a gentle stream of nitrogen [10].

Electrophoretic Mobility Measurements

The electrophoretic mobility of the polyelectrolyte covered erythrocytes as well as of the capsules was measured by means of an electrophoresis cell/par- ticle analyser [11] (Electrophor, Hasotec, Rostock, Germany). The procedure has been described in detail elsewhere [12].

Micropipette Aspiration Technique

The deformability measurements were performed in distilled water under

video-microscopic control by means of a micropipette aspiration technique

[13]. Cylindrical glass capillaries with an internal diameter of 2.5 mm and 4 mm and a negative (suction) pressure in the range between 0 and 450 Pa were applied.

Viscosimetric and Aggregation Measurements

The viscosities of whole blood (number of RBC 5 ¥ 10

/l) as well as of blood PEMC mixtures (1 : 1; PEMC and RBC concentration 5 ¥ 10

/l) were deter- mined by means of a capillary viscometer (Fresenius, Bad Homburg, Germany) at a shear rate of 600 s

. The aggregation measurements of the same samples were performed by means of a light back scattering technique (Regulest, Nancy, France). The experimental set-up uses a rotational vis- cometer, which consists of two transparent coaxial cylinders with an annular gap between them of h = 1 mm (inner radius 10 mm and outer radius 11 mm) that can hold about 2 ml of red cell suspension. A Couette flow was established between the two cylinders. The shear rate g is generated across the gap by rotating the outer cylinder 5 s

£ g £ 800 s

. The inner cylinder remains sta- tionary to prevent the formation of Taylor vortices. The cell suspension was probed with a normally incident laser light (wavelength l = 780 nm, beam diameter d = 1 mm, power 3 mW) that enters perpendicularly to the flow direction. The laser light flux multiply scattered either from red cells or aggre- gates was detected in the backward direction at small angles 160° < f < 170°

by a photometric device. The analogue signal was digitized and processed by the computer [14–17].

Results and Discussion

After completing the adsorption cycles the template (i.e., the RBC core) was removed by digestion with the NaCl + NaOCl solution. This procedure dis- solves the proteins and lipid membrane, thereby causing an increase of the capsule’s internal osmotic pressure and a swelling of the microcapsule [8].

However, these osmotically active agents can diffuse through the polyelec- trolyte layer; after equilibration of the osmotic forces, the elastic recoil prop- erties of the microcapsule returns it to approximately the original RBC size.

Figure 2 presents atomic force microscopy (AFM) images of polyelectrolyte

shells prepared using discocytic and an echinocytic RBC as templates. Note

that while the more ellipsoidal discocytic-based shells show only relatively

few creases and folds, coating of crenated echinocytic RBC results in struc-

tured shells clearly showing the spikes of the original template. Subsequent

washings of the microcapsules in NaCl solution permits separation of the

remaining cellular components from the microcapsules. Figure 3 presents

CLSM images of PEMCs on discocyte as well as echinocyte templates in water.

Fig. 2. Atomic force microscopy (AFM) images of PEMC using discocytic red blood cells (a) and echinocytes (b) as templates show the original shape of the used RBC templates

Fig. 3. 3-D confocal laser scanning images of PEMC using discocytic red blood cells (a) and echinocytes (b) as templates. The original shape of the RBC template is mimicked by the PEMC

b a

The shells were labelled with FITC-HSA. The structure of the PEMC is clearly visible.

Subsequent washings of the microcapsules in NaCl solution permit sepa- ration of the remaining cellular components from the microcapsules.

The effects of the NaCl + NaOCl treatment on the surface charge of the polyelectrolyte layer were investigated by measuring the EPM of the micro- capsules before and after digesting the template. The EPM of the PE covered RBC is about twice as high as for the control RBC. After the digestion of the template the EPM of the PE coated RBC is not significantly different com- pared to the PEMC (Fig. 4), which is independent of the used PE. The depro- teinizer modifies the chemistry of the PE layer. The positive charges from the amino groups in PAH are lost, and PSS is partly released. This results in a structure that contains only negative charges.

The wall thickness of PEMC was determined by means of atomic force microscopic imaging. PEMC with 5, 9 and 15 polyelectrolyte layers using PAH/PSS, PAH/DxSO

, and PAH/(PSS + HSA), respectively were analyzed. The wall thickness increases in dependence on the number of layers linearly in the investigated range. Additionally, the wall thickness depends on the polymer

Fig. 4. Mean values and S.D. of the negative electrophoretic mobility of red blood cells (RBC), PAH/dextran sulphate coated RBC (RBC Dx), PAH/dextran sulphate PEMC (Dx PEMC), PAH/PSS coated RBC (RBC PSS), and PAH/PSS PEMC (PEMC PSS). The differences between the PE coated RBC and after dissolution of the template-PEMC-are not significant

used (Table 1). The polyelectrolyte combination of PAH and PSS shows the thickest wall compared to that of PAH and DxSO

. Since the diameter of the PEMC depends on the number of layers the density of the PEMC wall is highest for 5 PE layer and decreases with increasing number of PE-layer [18], which has consequences for the permeability of macromolecules.

The buckling of PEMC as a measure of bsending properties of PEMC was determined by means of a micropipette aspiration technique. The buckling pressure was dependent on the number of PE layers (wall thickness) as well as on the used PE. The buckling pressure of the investigated PEMC is much larger than the pressure necessary to buckle the membrane of RBC, which results in a bending modulus of 4 magnitudes larger than the bending modulus of RBC [19] (Table 2). But it has to be considered that the PEMC wall is very permeable for small molecules as well as macromolecules [12]. Con- sequently, water flows rapidly through the PEMC wall and in reality a much weaker pressure difference is applied to the PEMC wall than in the case of the lipid membrane of the RBC. Therefore the measured pressure, which has to

Number Wall thickness (nm)

of layers PSS/PAH DxSO4/PAH PSS + HSA/PAH

Mean SD Mean SD Mean SD

5 14,66 0,81 6,64 0,58 9,28 1,06

9 16,88 1,93 9,82 1,03 12,09 1,11

15 24,24 2,22 14,71 0,62 19,68 0,80

Wall thickness of PEMC of PPS poly (styrene sulphonate, sodium salt) (PSS), Mw 70000 and poly (allyl amine hydrochloride), (PAH), Mw 70000: PSS/PAH; dextran sulphate, (DxSO4), Mw 500000 and PAH: DxSO4/PAH and a mixture of human serum albumin, (HSA), Mw ca. 65000 and PSS (1 : 1) and PAH: (PSS + HSA)/PAH, respectively in depend- ence on the number of polyelectrolyte layers in the dry state determined by means of AFM imaging.

Table 2. Buckling pressure and calculated bending modulus using the model of Evans [19] for RBC and PEMC of different PE

Type of particle Buckling pressure Bending modulus

[cm H2O] [Nm]

RBC [19] 1.8 ¥ 10^-19

(PSS/PAH)2/PSS 320± 45 1.8 ¥ 10^-14

(DxSO4/PAH)2/DxSO4 120± 18

[(PSS + HSA)/PAH]2/(PSS + HSA) 80± 12 3.5 ¥ 10^-15 The buckling pressure was determined applying a suction pressure on a capillary of a diameter of 2.5 mm. (T = 22°C).

be applied on the PEMC can only be used as an rough estimate for the cal- culation of the bending modulus. Without doubt it can be stated that the investigated PEMC are deformable in contrast to the PEMC fabricated on melamin particles [7].

The influence of the PEMC on flow properties of blood suspensions was determined by means of viscometric measurements as well as by means of a light back scattering technique, which allows to determine the aggregation behaviour of blood. Figure 5 presents a CLSM image of a PEMC-blood mixture. It is obvious that the RBC do not interact with the yellow-green flu- orescent PEMC, but form rouleaux with each other. The primary aggregation time t

, which characterises the time of rouleau formation, is shorter for the

Fig. 5. Color laser scanning microscopy (CLSM) image of a mixture (1 : 1) of blood and fluorescein isothiocyanate (FITC) labelled PEMC in salt solution show that the RBC do not interact with the PEMC. ¥1000

PEMC-blood mixture than for the pure blood with the same particle con- centration (Fig. 6). The final aggregation time t

representative of the 3 D time rouleau formation is in case of the PEMC-blood mixture only half of the values of blood. This clearly confirms the result of the CLSM image that the presence of PEMC reduces the number of RBC aggregates as well as the number of RBC per aggregate.

The PEMC also influence the overall rheological properties of blood characterised by the apparent viscosity at high shear rates. The apparent vis- cosity is significantly higher for the PEMC-blood mixture than for blood (Fig. 7). The reason seems to be that the PEMC do not completely disaggre- gate like the RBC. This assumption is consistent with the finding of a higher shear stress necessary to disaggregate the RBC-PEMC mixture compared to blood.

Fig. 6. The primary aggregation time tA, which characterises the time of rouleau forma- tion for the PEMC blood mixture and the pure blood with the same particle concentra- tion (T = 37°C, PEMC and RBC concentration 5 ¥ 10¹²/l, 1 : 1 mixture) and the final aggregation time tFrepresentative of the 3-D time rouleau formation show that the pres- ence of PEMC reduces the RBC aggregation

Conclusion

The newly developed PEMC with the shape of red blood cells are negatively charged, which is advantageous in respect of their interaction with the endothelium. The deformability of the PEMC is not as high as RBC, but exceeds that of other particles. The PEMC are permeable for charged as well as non charged macromolecules. This enables the filling of PEMC with oxygen transporting molecules.

Acknowledgments.

The authors acknowledge the gift of the aggregometer by Jacques Dufaux (University Paris 7). This work was supported by grant 01K0- 31 P2813 of the Federal Ministry of Research and Technology of Germany.

References

1. Donath E, Sukhorukov GB, Caruso F, Davis S, Möwald H (1998) Neuartige Polymer- hohlkörper durch Selbstorganisation von Polyelektrolyten auf kolloidalen Templaten.

Angew Chemie 110:2323–2327

2. Sukhorukov GB, Brumen M, Donath E, Möhwald H (1999) Hollow polyelectrolyte shells: exclusion of polymers and donnan equilibrium. J Phys Chem B 103:6464–6440 Fig. 7. Rheological properties of blood with and without PEMC characterised by the apparent viscosity at high shear rates and the disaggregation shear stress (T = 37°C, PEMC and RBC concentration 5 ¥ 10¹²/l, 1 : 1 mixture)

3. Voigt A, Lichtenfeld H, Sukhorukov GB, Zastrow H, Donath E, Bäumler H, Möhwald H (1999) Membrane filtration for microencapsulation and microcapsules fabrication by layer–by–layer polyelectrolyte adsorption. Ind Eng Chem Res 38:4037–4043

4. Caruso F, Lichtenfeld H, Donath E, Möhwald H (1999) Investigation of electrostatic interactions in polyelectrolyte multilayer films: binding of anionic fluorescent probes to layers assembled onto colloids. Macromolecules 32:2317–2328

5. Radtchenko IL, Sukhorukov GB, Leporatti S, Khomutov GB, Donath E, Möhwald H (2000) Assembly of alternated multivalent ion/polyelectrolyte layers on colloidal par- ticles. Stability of the multilayers and encapsulation of macromolecules into polyelec- trolyte capsules. J Colloid Interface Sci 230:272–280

6. Sukhorukov GB, Antipov AA, Voigt A, Donath E, Möhwald H (2001) pH-controlled macromolecule encapsulation in and release from polyelectrolyte multilayer nanocap- sules. Macromol Rapid Commun 22:44–46

7. Bäumler H, Artmann G, Voigt A, Mitlöhner R, Neu B, Kiesewetter H (2000) Plastic behavoir of polyelectrolyte microcapsules derived from colloid templates. J Micro- encapsulation 17:651–655

8. Neu B, Voigt A, Mitlöhner R, Leporatti S, Gao CY, Donath E, Kiesewetter H, Möhwald H, Meiselman HJ, Bäumler H (2001) Biological cells as templates for hollow micro- capsules. J Microencapsul 18:385–395

9. Bäumler H, Djenev I, Iovchev S, Petrova R, Lerche D (1988) Polarizability of human red blood cells and conformational state of glycolcalyx. Stud Biophys 125:45–51 10. Georgieva R, Moya S, Hin M, Mitlöhner R, Donath E, Kiesewetter H, Möhwald H,

Bäumler H (2002) Permeation of macromolecules into polyelectrolyte microcapsules.

Biomacromolecules 3:517–524

11. Grümmer G, Knippel E, Budde A, Brockmann H, Treichler J (1995) An electrophoretic instrumentation for the multi-parameter analysis of cells and particles. Instr Sci Techn 23:265–276

12. Bäumler H, Donath E, Krabi A, Knippel W, Budde A, Kiesewetter H (1996) Elec- trophoresis of human red blood cells and platelets. Evidence for depletion of dextran.

Biorheology 33:333–351

13. Artmann G, Sung K-LP, Horn T,Whittemore D, Norwich G, Chien S (1997) Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation. Biophysical J 72:1434–1441

14. Snabre P (1988) Rhéologie des suspensions concentrées et agrégées de particles déformables. Thèse d’état (université Paris VII)

15. Snabre P, Mills P (1996) Rheology of weakly flocculated suspension of rigid particles.

J Phys III France 6:1811–1834

16. Snabre P, Bitbol M, Mills P (1987) Cell disaggregation behavior in shear flow. Biophys J 51:95–807

17. Siadat M (1989) Conception et réalisation d’un agrégamètre érythrocytaire automatisé et informatisé, thèse de doctorat, (université Paris VII)

18. Mitlöhner R (2003) Design und Charakterisierung von Polyelektrolytkapseln. Diplo- marbeit, TU Berlin

19. Evans EA (1989) Structure and deformation properties of red blood cells: concepts and quantitative methods. Methods Enzymol 173:3–35