Size exclusion properties of polyelectrolyte complex microcapsules prepared from sodium cellulose sulphate and poly[diallyldimethylammonium chloride]

Journal of Membrane Science 162 (1999) 165±171

Size exclusion properties of polyelectrolyte complex microcapsules prepared from sodium cellulose sulphate and poly[diallyldimethylammonium chloride] Horst Dautzenberga,y, Ute Schuldta, Dietmar Lercheb, Holger Woehleckec, Rudolf Ehwaldc,* a

UniversitaÈt Potsdam, Forschungsgruppe Polyelektrolytkomplexe, Kantstr. 55, Teltow D-14513, Germany b L.U.M. GmbH, Rudower Chaussee 5, Berlin D-12489, Germany c Humboldt-UniversitaÈt zu Berlin, Institut fuÈr Biologie, Invalidenstr. 42, Berlin 10115, Germany Received 13 January 1999; received in revised form 1 April 1999; accepted 8 April 1999

Abstract Diffusion experiments aided by high performance size exclusion chromatography were carried out to describe the size exclusion properties of microcapsules prepared from cellulose sulphate and poly[diallyldimethylammonium chloride] (PDADMAC). The membrane cut-off was characterised by the dependence of the distribution ratio (luminal concentration/ external concentration) of dextran molecules on their Stokes' radius (1±9 nm) after diffusion periods of 72 and 240 h. When capsules were prepared from standard cellulose sulphate and different polydisperse PDADMAC preparations with high mass portions of size fractions >10 kDa, the cut-off range was narrow with an upper size limit (USL) of permeation close to 2 nm and independent of the residence time in the PDADMAC bath or the presence of salt (155 mM NaCl) in the reacting polymer solutions. Capsules prepared with PDADMAC rich in size fractions with lower molecular weight (<3 kDa) showed higher values of the USL. The cut-off was dependent on the residence time and salt concentration when capsules were produced using polydisperse PDADMAC with high mass portions of small molecules (<0.6 kDa). Prolonged residence of such capsules in a reaction bath containing 155 mM NaCl resulted in a strong increase of the USL. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Size exclusion chromatography; Dextran; Membrane cut-off; Pores; Diffusion

1. Introduction Microcapsules in the strict sense, i.e. membrane coated small spheres with an enclosed liquid phase, have potential applications in biotechnology, phar*Corresponding author. Tel.: +30-20938816; fax: +3020938635; e-mail: [email protected] y Deceased.

macy and medicine, which depend on the size exclusion and permeability properties of their membranes [1,2]. Polyelectrolyte complex (PEC) microcapsules may be produced by dropping a solution of sodium cellulose sulphate into a solution of poly[diallyldimethylammonium chloride] (PDADMAC). The rapid reaction of cellulose sulphate with the polycation (Fig. 1) at the interface between the two polymer

0376-7388/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 9 9 ) 0 0 1 3 5 - 0

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Fig. 1. Chemical structure of the polyelectrolytes used for preparation of PEC microcapsules.

solutions results in a mechanically stable membrane around the capsule core [3,4]. The core (lumen) usually contains a solution of nonreacted cellulose sulphate. Although polycations are known to rupture biomembranes and to precipitate proteins or nucleic acids, a completely biocompatible encapsulation process is possible. Materials to be included, e.g. cells or enzymes, can be dissolved in the nondenaturating cellulose sulphate solution under physiological conditions. At capsule formation these materials get trapped within the capsule lumen. They are well protected from polycations, which are not soluble in the luminal solution of nonreacted cellulose sulphate. Animal and human tissues or cells do not show detectable damage by PEC encapsulation. Due to the simplicity, reproducibility and biocompatibility of the encapsulation process, PEC capsules are of great interest for biotechnological and medical applications [5±9]. PEC membranes formed by the reaction of cellulose sulphate with PDADMAC have size exclusion properties in the ultra®lter range [7]. For several applications (e.g. long-term inclusion of enzymes and cells, access of substrates and release of products) it is essential to produce capsules with de®ned size exclusion properties. A sharp membrane cut-off, i.e. a narrow molecule

size range of transition from high permeability (below a critical size) to complete long-term exclusion (above a critical size) is desirable. The aim of this study is to characterise PEC capsule membranes with respect to their size exclusion properties. Attention was focused on the in¯uence of several important variables of the capsule formation process: (1) molecular weight dispersion of PDADMAC, (2) residence time of the capsules in the PDADMAC solution, (3) presence of a monovalent salt in the PDADMAC bath. We describe the size exclusion properties of PEC capsules by their diffusional exchange with polydisperse dextran. The applied method [10] gives the membrane cut-off as result of only one diffusion experiment. The membrane cut-off can be described by an upper size limit (USL), i.e. the Stokes' radius of a neutral polymer reaching 5% of the equilibrium concentration in the encapsulated liquid phase, and the mean size limit (MSL), i.e. the Stokes' radius of those molecules which reach half of the equilibrium concentration. 2. Experimental 2.1. Cellulose sulphate preparation Sodium cellulose sulphate was prepared in laboratory scale by heterogeneous sulphation of cotton cellulose [11]. It has a degree of substitution of 0.34 and a viscosity of 24 mPa s of the aqueous solution at standard conditions (10 g lÿ1, 258C). The mean molecular weight Mw is 416 kDa (measured with pullulan standards by size exclusion chromatography). 2.2. PDADMAC samples The ®ve types of PDADMAC used in this study were selected from a series of laboratory samples and technical batches supplied by Katpol, Wolfen, Germany. All samples were obtained by radical polymerisation initiated by peroxide [12]. With regard to the molecular size dispersion they represent two types (Fig. 2): one characterised by a relatively low mean molecular weight (MK10 and B40) and the other by medium mean molecular weight and relatively broad molecular weight dispersion (PDADMACs 13 200,

H. Dautzenberg et al. / Journal of Membrane Science 162 (1999) 165±171

167

2.4. Dextran diffusion experiments and size exclusion parameters

Fig. 2. Molecular weight distribution of applied PDADMAC types measured by size exclusion chromatography. Eluent: 0.5 M NaNO3; column: HEMA BIO linear (inner diameter: 8 mm, height: 300 mm); elution rate: 0.8 ml/min). The abscissa refers to calibration of the column with pullulan standards. The ordinate gives the increase in the eluted portion of PDADMAC (wt) per log of molecular weight (dwt/d log MW).

14 000 and 22 000). Samples of the latter type differed markedly in the relative amount of high-molecular weight fractions (above the size of a 100 kDa pullulan). One of the samples with lower mean molecular weight, PDADMAC B40, has a rather broad dispersion and contains high mass fractions of components with very low molecular size (below 500 Da), the other one (PDADMAC MK10) has a more narrow dispersion. 2.3. Capsule preparation The aqueous sodium cellulose sulphate solution (40 g lÿ1) was pressed through a steel injection needle with constant ¯ow. Droplets with a weight of about 15 mg (diameter about 3 mm) fell due to their own weight into the stirred aqueous PDADMAC solution. The concentration of the PDADMAC solutions was 20 or 40 g lÿ1. Part of the capsules was prepared without salt addition, another part in the presence of NaCl (9 g lÿ1, 155 mm). After different residence times they were separated from the precipitation bath. Independent of the presence or absence of NaCl in the reaction bath, the capsules were washed (six times) with 155 mM NaCl and stored in 155 mM NaCl before the investigation of their size exclusion properties.

A dextran probing solution (DPS) with broad continuous molecule size dispersion was prepared by mixing several commercial polydisperse dextrans as described previously [10]. This solution had dextran size fractions with Stokes' radii between 1 and 9 nm in similar concentrations. Its electrolyte composition (100 mM NaCl, 1 g lÿ1 NaN3, 10 mM Na phosphate pH 7) was the same as of the eluent used for the chromatographic fractionation. Investigated samples (10±20 capsules, fresh weight about 300 mg) were equilibrated in 100 ml of the eluent buffer at 68C for at least 24 h, ®ltered on a sieve, and incubated for diffusion periods of 3 or 10 days in a volume (V0) of 0.3 ml of the DPS at 68C. After the diffusion period, all dextran molecules were diluted by exchange with the external ®lm volume (Vf) at the capsules' surface, and those smaller than the exclusion limit were further diluted by diffusion into the internal liquid phase volume (Vi). Both the DPS thus modi®ed by diffusional exchange with the sample and the original DPS were chromatographed on a calibrated Superdex HR 200 column (Pharmacia, Sweden). The applied HPLC apparatus was the same as described previously [10]. In this study, the data analysis was improved to obtain distribution ratios (ratio between the mean luminal concentration Ci and the external equilibrium concentration C*). The latter were plotted on the Stokes' radius. This allows for a de®nition of the upper size limit and the mean size limit by coordinated values of

. The original and modi®ed dextran samples were fractionated on a Superdex HR 200 column (30 cm) and the concentration signals (angle of rotation) of elugrams were stored together with the coordinated elution times and Stokes' radii, to obtain a function of dilution quotients (q) on Stokes' radius [10]. These quotients give, for a given elution time or Stokes' radius, the ratio between the concentration of an eluted dextran fraction of the original DPS (C) and the concentration of the corresponding fraction of the modi®ed DPS (C*): q ˆ C=C  ;

(1) 0

where q is a constant (qˆq ) for all fractions with Stokes' radii above the upper size limit, since all excluded dextran molecules originally dissolved in

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the volume V0 equilibrate with the additional volume of the external liquid ®lm (Vf) only. V0 ‡ V f ˆ C=C …Ci ˆ 0†; V0

q0 ˆ

(2)

where also q is a constant, but of higher value q00 , for all dextran fractions with Stokes' radii small enough to allow for equilibration with the whole inner liquid phase (Vi). q00 ˆ

V 0 ‡ Vf ‡ Vi ˆ C=C …C ˆ Ci †: V0

(3)

In the cut-off range, q is size-dependent and its value is intermediate between q0 and q00 . qˆ

V 0 ‡ Vf ‡
Vi ˆ C=C  …
C ˆ Ci †: V0

(4)

The distribution ratio ˆCi/C* may be obtained as

ˆ

q ÿ q0 : q00 ÿ q0

(5)

By a suitable software the original elution diagrams (detector units versus elution time) were transformed to a plot of the distribution ratio on rs (Fig. 3). USL is the Stokes' radius of the dextran fraction characterised by a distribution ratio of 0.05, MSL the size of dextran molecules with a distribution ratio of 0.5.

3. Results The size dependence of dextran diffusion into PEC capsules is shown in Fig. 3. Curves A are representative for capsules prepared with the PDADMACs with higher molecular weight (PDADMAC 13 200, 14 000, and 22 000). Low molecular weight fractions of the dextran probing solution (<1.4 nm) entered the whole available luminal diffusion space of these capsules up to the equilibrium value. Dextran fractions with a Stokes' radius larger than 1.9±2.0 nm (equal to that of a globular protein with about 18 kDa) were completely excluded. Cut-off values were not markedly changed by prolongation of the diffusion time from 3 to 10 days. Capsules obtained with PDADMAC 13 200, 14 000 and 22 000 were similar in their size exclusion properties. When capsules were prepared using the above mentioned PDADMAC preparations, neither a variation of the residence time (5±60 min)

Fig. 3. Size exclusion curves of PEC capsules. Curves represent the dependence of the distribution ratio (luminal concentration/ external concentration) of dextran size fractions on their Stokes' radius. The second abscissa gives the molecular weights of standard proteins used for calibration of the size scale. (A) Capsules prepared with 40 g lÿ1 PDADMAC 13 200 without addition of NaCl; residence time 5 min; full line: diffusion time 3 d; dotted line: diffusion time 10 d. (B) Capsules prepared with 20 g lÿ1 PDADMAC MK10 without addition of NaCl; residence time 5 min; diffusion time 3 d. (C) Capsules prepared with 20 g lÿ1 PDADMAC B40 in the presence of 155 mM NaCl; residence time 30 min; diffusion time 3 d.

nor the addition of NaCl (155 mM) did effect the membrane cut-off (Table 1). When capsules were produced in a solution of PDADMAC B40 (signi®cant mass portion of fractions with very low-molecular weight), the permeationcontrolling pore size was higher than usual and showed a complex dependence on residence time and salt addition. Capsules prepared with B40 in the absence of additional salt had a fairly sharp cutoff, which shifted to higher values by shortening the residence time (Table 1). Capsules prepared with B40 in the presence of additional NaCl (155 mM) had a markedly increased upper size limit and a broad size cut-off of their membranes (Fig. 3C). The latter was broadened and increased, when the residence time was prolonged (Table 1). Also with PDADMAC MK10 (high portion of molecular weight fractions <10 kDa), USL and MSL were relatively high (Table 1). It is worth mentioning that molecules with a size of more than 2 nm

H. Dautzenberg et al. / Journal of Membrane Science 162 (1999) 165±171 Table 1 Influence of the residence time, PDADMAC type and NaCl on the cut-off limits of PEC capsules PDADMAC

PDADMAC (wt%)

NaCl

Reaction MSL time (min) (nm)

USL (nm)

13 200 13 200 13 200

4 4 4

ÿ ÿ ÿ

5 30 60

1.6 1.6 1.6

1.8 1.9 1.8

13 200 13 200 13 200

4 4 4

‡ ‡ ‡

5 30 60

1.6 1.6 1.6

1.8 1.8 1.9

14 000 14 000 14 000 14 000

2 2 2 2

‡ ‡ ‡ ‡

5 30 60 120

1.5 1.5 1.5 1.5

1.7 1.7 1.6 1.7

22 000 22 000 22 000

4 4 4

ÿ ÿ ÿ

5 30 60

1.5 1.6 1.6

1.8 1.8 1.8

22 000 22 000 22 000

4 4 4

‡ ‡ ‡

5 30 60

1.6 1.6 1.7

1.8 1.8 1.9

B40 B40 B40

2 2 2

ÿ ÿ ÿ

5 30 60

2.8 2.2 2.0

3.6 2.6 2.3

B40 B40 B40

2 2 2

‡ ‡ ‡

5 30 60

2.3 3.0 3.7

3.7 6.2 7.1

MK10

2

ÿ

5

2.6

3.6

All PEC capsules were prepared with 4 wt% cellulose sulphate. NaCl was added (‡) or not added (ÿ) to both reacting polyelectrolyte solutions in a concentration of 155 mM.

reached a distribution ratio of close to 1 in the capsules prepared with MK10 (Fig. 3B). It is known that concentrated solutions of large polymer molecules (e.g. dextran), when enclosed within a dialysis chamber, may have a similar size exclusion effect (size-dependent reduction of distribution ratio) on permeable polymers as cross-linked gels (e.g. Sephadex) of an equal water content [13]. In the case of the encapsulated nonreacted cellulose sulphate (about 40 g lÿ1 )

169

such a size exclusion effect was not signi®cant for dextran molecules with rs up to 2 nm. 4. Discussion The applied HPLC-aided dextran diffusion technique [10] is well suited to quantify the in¯uence of reaction parameters on size exclusion properties of microcapsules. As a main result of this study it may be stated that the PEC capsule membranes obtained with different types of PDADMAC characterised by a high portion of size fractions >10 kDa have a sharp membrane cut-off. The narrow size limits of the cut-off range point to a homogeneous pore structure of the membrane and homogeneous samples of individual capsules. As the cut-off range was practically not increased by prolongation of the diffusion time (Fig. 3A), large diffusion channels do not exist in the membranes of the investigated samples. Their presence should induce slow permeation of larger dextran species. The dispersion of PDADMAC in the range of high molecular weight does not seem to be very critical for the membrane pore structure as long as the samples contain larger portions of species with molecular weight >10 kDa. With suitable PDADMAC the pore size controlling structure of the capsule membrane is obtained in a fast reaction and does not change at prolongation of the residence time in the PDADMAC bath (Table 1). Presence of salt or buffer is necessary for encapsulation of most biomaterials. The PEC membrane is unpermeable for at least the larger part of the reacting polyelectrolytes. The Donnan-distribution of counterions may be osmotically unbalanced and therefore induce capsule swelling and shrinkage in the reaction bath. During washing and further applications, the medium of PEC capsules must contain a salt in suf®cient concentration (100±200 mM) to reduce the colloid-osmotic pressure difference across the capsule membrane [7]. The salt ions stabilise the capsule volume in the absence of a compensating external polyelectrolyte and prevent swellingmediated changes in the membrane structure. On the other hand, too high salt concentrations (0.5 M) might destabilize the PEC due to the electrical shielding effect of the ions [7].

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If the PEC complex is formed with PADMAC of higher molecular weight, addition of NaCl to the reacting polymer solutions in a concentration of 155 mM does not alter the pore structure of the capsule membrane (Table 1). We have found, however (Table 1), that the ®nal pore structure of capsule membranes was effected by NaCl of this concentration when the PEC had been formed with PDADMAC B40. The observed cut-off broadening by the combined effects of salt, high residence time and low-molecular weight PDADMAC was mainly due to a strong increase of the USL. Permeation of dextran species with a size much lower than the USL was not fast enough for equilibration within 3 days. Probably the additional large pores have a small fraction in the whole membrane area (Fig. 3C). PEC membranes obtained with PDADMAC B40 are highly asymmetric structures, even in a morphological sense. They have a dense outer primary layer that has been rapidly formed at the interface between the polymer phases and a more porous layer at the inner face. The inner layer thickens at increasing residence time by the reaction of permeating lowmolecular-weight PDADMAC with nonpermeating cellulose sulphate. The thickening process is absent in capsules prepared with high-molecular weight (nonpermeating) PDADMAC [4,7]. When capsules are prepared from PDADMAC B40, the percentage of nonreacted cellulose sulphate decreases with increasing residence time and the contemporary decrease in the concentration of osmotically active counter-ions (Na‡) within the capsules causes shrinkage. Salt addition accelerates both this secondary thickening process of the capsule membrane and the related shrinkage [4,7]. It is possible that these processes are connected with the observed formation of larger pores. We have shown in the study, that the PEC capsules prepared with different PDADMAC preparations of suf®ciently high molecular weight (PDADMAC 13 200, 14 000 and 22 000) have a sharp membrane cut-off in the range of 1.6±2 nm. Molecules with a size above this range do not diffuse through the capsule membrane within 10 days. The cut-off is low enough for stable inclusion of enzymes with a molecular weight >20 kDa. It may be interesting to note that different reaction parameters (residence time, salt

concentration and, in a certain range, also PDADMAC concentration) did not alter signi®cantly the membrane cut-off. PDADMAC with a high portion of low-molecular weight components (<3 kDa) are less suitable for the formation of capsules with de®ned and stable membrane cut-off. Acknowledgements The authors are grateful for ®nancial support of this study by the Gesellschaft fuÈr WirtschaftsfoÈrderung und Marktplanung m.b.H (program Marktorientierte Industrieforschung und wirtschaftlicher Strukturwandel, Project no. 764/98). The applied method of membrane pore size determination was developed with the support of the Deutsche Forschungsgemeinschaft, Project no. Eh 144-1-1.

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