Effect of capsule diameter on the permeability to horseradish peroxidase of individual HEMA-MMA microcapsules

Journal of Controlled Release 49 (1997) 217–227

Effect of capsule diameter on the permeability to horseradish peroxidase of individual HEMA-MMA microcapsules Jeong Rim Hwang, Michael V. Sefton* Department of Chemical Engineering and Applied Chemistry, Center for Biomaterials, University of Toronto, Toronto, Ontario M5 S 1 A4, Canada Received 24 March 1995; received in revised form 28 April 1997; accepted 28 April 1997

Abstract Poly (2-hydroxyethyl methacrylate-methyl methacrylate), (HEMA-MMA) microcapsules to be used for the transplantation of live mammalian cells were prepared by an interfacial precipitation process. A submerged jet coextrusion technique was used to make various sizes of capsules as small as 450 mm in outside diameter. A novel method based on an enzyme-chromogenic substrate assay was used to measure the capsule permeability at the individual capsule level. Capsule size, shearing frequency (number of capsules sheared per unit time), capsule permeability and the capsule-to-capsule variation in permeability were dependent, directly or indirectly, upon the shearing force (i.e. hexadecane flow-rate) applied to the nozzle during preparation. The average permeability coefficients of those capsules to horseradish peroxidase, (HRP, MW 40kDa, a model protein), were in the range of 2310 210 to 9310 210 cm 2 / sec; the permeability varied from 1310 210 to 3310 210 cm 2 / s for 500 mm capsules (the best) and 0.1310 210 to 6310 210 cm 2 / s for 660 mm capsules (the poorest). Scanning electron microscopy illustrated that finger-like macrovoids were formed under the external skin layer of the capsule membrane, but that an interconnected open-cell structure was formed under the internal skin. Modelling the precipitation process in two dimensions (i.e. between two glass slides) suggested that the faster precipitation of HEMA-MMA at the external surface dominated the formation of the membrane structure, compared with the slower precipitation at the internal surface.  1997 Elsevier Science B.V. Keywords: Microcapsule; Interfacial precipitation; Phase-inversion membrane; HEMA-MMA hydrogel; Permeability

1. Introduction Membranes prepared by phase-inversion have been widely used in various biomedical applications, frequently configured as hollow fibers. We have been interested in the formation of similar asymmetric membranes but as microcapsules to be used for transplanting live mammalian cells or tissues. These cells isolated from the immune system by the *Corresponding author.

permselective capsule wall are to be used as drug delivery systems to secrete biologically active agents in response to physiological stimuli in vivo. Microcapsules, not necessarily with asymmetric membranes, have been studied for a wide range of applications: bioartificial pancreas [1], dopamine delivery for Parkinson’s disease [2], and bioartificial liver [3]. To be effective, the capsule wall must have a molecular weight cut-off of |100 kDa to prevent rejection induced by antibodies which are .150 kDa

0168-3659 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved. PII S0168-3659( 97 )00091-6

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in size. However, to maintain the normal physiological state of the encapsulated cells, the biocompatible membrane must be permeable to essential nutrients and growth factors and must allow for the diffusion of specific therapeutic agents and metabolites out of the capsule. Our specific interest is the use of hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer as a hydrogel material for the capsule membrane because of its biocompatibility, water insolubility and processibility [4]. Capsules with HEMA-MMA are made by interfacial precipitation, which results in an asymmetric membrane with high permeability yet have a suitable thickness to maintain mechanical integrity. The basic principles of interfacial precipitation are not significantly different from those involved in the phase inversion preparation of traditional flat or hollow fiber membranes [5–7]. Hence, it was expected that the several parameters, well established as affecting the preparation of traditional phase-inversion membranes, could be directly introduced to the microencapsulation process to control capsule structure and permeability. Changing the nonsolvent composition, for example, has already been found to alter the structure of earlier HEMA-MMA microcapsules [8]. Microcapsules were prepared by shearing droplets from a stationary nozzle using a rapidly flowing fluid stream [9] and triethylene glycol (TEG, MW150) was used instead of poly(ethylene glycol) (PEG, MW200), as the solvent for the polymer. The lower molecular weight of TEG was expected to increase the exchange rate of solvent and coagulant at the membrane solution / coagulant interface at the initial moment of immersion-precipitation. This exchange rate has a key role in determining the surface porosity [10]. A previous study with protein producing HepG2 cells in capsules made by an earlier process [11] demonstrated that there was considerable capsule-tocapsule variability in protein release from the encapsulated cells and a measureable release of fibrinogen (340 kDa) from a defective subpopulation (|20– 30% of the total) of the capsules. In this study the permeability of individual capsules was measured so as to define the capsule-to-capsule variation and the effects of process alterations on permeability. The very small volume of the microcapsule (,0.05 ml) made it difficult to determine the permeability differ-

ences among the microcapsules since the amount of material released was far below the detection limit of most convenient assay systems [8]. Previously aliquots of |100 capsules were used for sufficient sensitivity. Since the enzyme activity assay for horse-radish peroxidase (HRP) was sensitive enough to detect the extremely small amounts of HRP released from the small individual capsules, HRP was used as a model protein. In addition to the external coagulation bath there is also polymer precipitation on the inside due to mixing between the membrane solvent and the aqueous core (normally containing cell suspension). Hence, the microcapsule membranes were formed through coagulation from both external and internal surfaces. Because of the differences in the composition of the two aqueous phases inside and outside of the capsules, differences in membrane structure are expected. The kinetic basis of this difference was measured.

2. Experimental

2.1. Preparation of microcapsules Poly (2-hydroxyethyl methacrylate-methyl methacrylate) hydrogel copolymer (HEMA-MMA), nominally containing 75 mol% HEMA was synthesized by solution polymerization as described before [12], using azobisisobutyronitrile as initiator (0.1 mol%). The polymer was dissolved (10% w / v) in triethylene glycol, TEG (MW 150, Aldrich), and stored overnight to degas it. HEMA-MMA capsules were prepared by submerged jet coextrusion (Fig. 1) following a previously reported procedure [9] with some variation. The triple-barrelled extrusion nozzle (Fig. 2) was composed of a polypropylene (PP) barrel for extruding polymer solution surrounded by a glass barrel for guiding shearing liquid to the exterior of the PP barrel. A 22G stainless steel needle was located at the center of the PP barrel for extruding core material into a polymer solution droplet at the tip of the PP barrel. Both tips of the PP barrel and stainless steel needle were tapered to facilitate droplet formation. The polymer solution annular space at the

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Fig. 1. A schematic diagram of the submerged jet coextrusion apparatus for making small diameter capsules.

nozzle tip was 125 mm thick. Other key dimensions of the nozzle are given in Fig. 2. The polymer solution was pumped to the nozzle at 0.78 ml / h by syringe pump (model A-99, Razel Scientific Instruments Inc.). The core solution, 20% w / v Ficoll-PBS solution (Ficoll 400, MW 400 kDa,

Fig. 2. Triple-barreled spinneret used to make small diameter capsules. Extruded polymer solution droplet was sheared-off from the nozzle tip by the flowing hexadecane.

Pharmacia; PBS: phosphate buffered saline, pH 7.4, in g / l: 8.7 NaCl, 6.1 Na 2 HPO 4 , 1.1 NaH 2 PO 4 ) was coextruded at 0.33 ml / h by a second syringe pump. Ficoll  was used as a viscosity enhancer to improve capsule centering based on its use in making larger capsules [13]. Hexadecane (Aldrich), the hydrophobic shearing fluid, was pumped through the glass barrel (Fig. 2) at 65–165 ml / min using a peristaltic pump (Master Flex, Cole-Parmer). After being extruded from the nozzle, the nascent microcapsules (produced at a rate termed the shearing frequency) passed through a layer of hexadecane and entered a coagulation bath of Pluronic L101 nonionic surfactant (100 ppm, BASF Chemicals) dissolved in PBS. L101 is used to lower the hexadecane / PBS interfacial tension as before (8). The microcapsules were kept in the L101-PBS coagulant bath for 30 min after the last capsule was prepared while suspended by an overhead magnetic stirrer. A typical batch consisted of 2000 capsules and took an extrusion time of |30 min. The microcapsules were transferred into fresh PBS, washed, and then collected into 100 mm petri dishes (Falcon, Becton Dickinson). After further washing with fresh PBS, the microcapsules were incubated in PBS (the

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PBS was replaced each day) for 4 days before incubating with the test protein solution. Approximately 40 cured microcapsules were incubated in 2 ml of 5 mg / ml HRP solution (molecular weight: 40 kDa, 1100 units / mg, Sigma) in a glass scintillation vial for 3 days at room temperature. Afterwards, the microcapsules were transferred into fresh PBS and washed for 5 min.

2.2. Determination of microcapsule permeability Into each well of a 96-well flat-bottomed plate (Linbro, Mclean, VA), one capsule was transferred along with PBS and the transferred PBS was removed. Fresh PBS (300 ml) was added to each well as the release medium. The plate was covered and shaken gently (30 cycles / min) on an orbital shaker (Labquake Shaker, Lab Industries) at room temperature. Aliquots (30 ml) of release medium were removed at each time point (0, 1, 2, 4, and 8 hour) and analyzed for HRP concentration by enzyme activity as described below. Fresh PBS (30 ml) was added to the release medium after each aliquot was removed so that the volume of release medium was kept constant. After the last aliquot was removed, each capsule was carefully broken apart within the release medium using sharp 22G needles to release completely the remainder of the HRP. The amount of HRP released after crushing was also measured (30 ml aliquot), and added to the cumulative amount of released HRP to estimate, by mass balance, the total initial loading of HRP in the capsules.

2.3. Enzyme activity The enzyme assay was calibrated using solutions of HRP in PBS in the range of 1 to 100 ng / ml. Aliquots (30 ml) in triplicate were added to a 96well flat-bottomed plate. 3,39,5,59-tetramethylbenzidine (TMB) in dimethyl formamide / phosphate buffer containing hydrogen peroxide (TMB Peroxidase EIA Substrate Kit, Bio-Rad Laboratories) was added (70 ml) and after 5 min at room temperature the reaction was stopped with 100 ml of 0.5M H 2 SO 4 . The absorbances at 450 nm were determined with a MR700 microplate reader (Dynatech Laboratories Inc). Calibration curves were linear from 1

to 100 ng / ml. Aliquots of release medium were analyzed in the same way after diluting the 30 ml aliquot into the 1 to 100 ng / ml measurement range. Storage for as long as 12 days at room temperature had no effect on the calibration curve, indicating that microcapsules could be incubated for a few days in HRP solution without losing activity.

2.4. Evaluation of release kinetics The release kinetics were analyzed as before [11] according to the following equation [14] for drug delivery through a spherical membrane with a changing core concentration assuming there was no HRP in the capsule wall at time t50: Mt /M` 5 1 2 e 2 b P m t (1)

b 5 3b /a 2 (b 2 a). Mt /M` is the fractional release of model protein, b is the geometry factor of capsule membrane, Pm is the permeability coefficient, and t is release time. ‘b‘ and ‘a‘ are respectively the outside and inside radii of the microcapsule averaged over .20 capsules and determined from SEM micrographs. The geometry factors could not be determined for the same individual capsules as were used for the permeability measurements but were assumed to be the same for all capsules of a particular batch. Also no correction is made for boundary layer effects outside (or inside) the capsules; hence Pm should be considered an overall mass transfer coefficient, rather than an intrinsic membrane property. Since the permeabilities were so low, it was presumed that the limiting resistance was indeed the membrane.

2.5. Morphological studies Microcapsules were observed under a Zeiss Axiovert 135 optical microscope, and a scanning electron microscope (SEM) was used to examine the membrane morphology. Microcapsules were cryofractured by immersing them in liquid nitrogen and ‘cutting‘ them in half with a sharp scalpel, sputter coated with gold, and examined with a Hitachi S-520 SEM. Since the capsule centre was determined by

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eye, this is part of the experimental error in mean diameter.

2.6. Phase separation kinetics Difference in phase separation kinetics of the external and internal surfaces was modelled as follows [7]: A drop of polymer solution was placed on a microscope slide, and gently pressed by another microscope slide to form a thin liquid film. Subsequently, a drop of nonsolvent coagulant was placed at the edge of the slides. Precipitation of polymer occurred as the coagulant contacted the polymer solution, and the precipitation front moved inward as the coagulant penetrated the polymer solution. Optical micrographs were recorded at regular intervals to estimate the precipitation rate of HEMA-MMA. Two different coagulants were examined, L101-PBS and Ficoll-PBS since these are involved in the precipitation of the polymer solution at the external and internal surfaces of the capsule membrane, respectively, during membrane formation.

3. Results

3.1. Preparation of HEMA-MMA microcapsules

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hydrophobic shearing liquid, was applied to the nozzle tip. Correspondingly, the rate of production (‘shearing frequency‘) also increased since the polymer and core flow-rates were held constant. The capsule size gradually decreased (Fig. 4) during the curing step in the PBS / L101 as the solvent was extracted and thereafter as the wall became thinner. Residual solvent in the capsule membrane was assumed to be removed within the first hours after preparation but that the macroporous capsule membrane slowly densified through a process of creep (presumably surface energy driven) over the first 2|3 days. Capsule dimensions are given in Table 1 for the particular batches studied further. HEMA-MMA capsules had a macroscopically smooth and homogeneous external surface as shown in Fig. 6(a) at least at low magnification. Many pits or surface depressions that may be surface pores were found on the external surface in Fig. 6(b) at a higher magnification. The capsule membrane is shown in cross section in Fig. 5. The spherical lumen of small capsules (OD: 450 mm) was located near the center of the microcapsule (Fig. 5a), while that of larger capsules (OD: 660 mm) was significantly off-centered (Fig. 5b); note also the high eccentricity value in Table 1. Presumably gravity pulled the polymer solution down to cause the eccentricity and had more of an

Fig. 3 shows that smaller capsules were formed as expected, when a higher flow-rate of hexadecane, the

Fig. 3. Effect of hexadecane flow-rate on the shearing frequency (j, defined as the number of capsules produced per minute) and the final (day 7) average outside diameter (h) of the resulting capsules.

Fig. 4. Decrease in the outside diameter of HEMA-MMA microcapsule due to the shrinkage of HEMA-MMA while cured in PBS after preparation. The decrease in outside diameter was insignificant after 4 days. The flow-rates of hexadecane were: 65(j), 90 (h), 120 (♦), and 165 (x) ml / min. Final diameters are as reported in Fig. 3 and Table 1.

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Table 1 Dimensions of microcapsules from SEM of fully cured microcapsules Hexadecane flow-rate (ml / min)

Outside diameter (mm)

Inside diameter (mm)

d(mm)

Eccentricity

Geometry factor, b

165 120 90 65

45069 500615 580625 660634

34066 410610 490620 440620

6.0 5.0 1.0 70.5

0.027 0.020 0.003 0.213

4.2310 4 61.9310 3 4.0310 4 63.1310 3 3.3310 4 63.6310 3 1.8310 4 61.9310 3

d 5 the average distance between the centers of the core and the whole capsule. Eccentricity 5 d / bb 5 3b /a 2 (b 2 a), a: internal radius b: external radius average 6 sd; n.20

effect with the larger capsule since these were produced more slowly. The larger capsules also had thicker walls, suggesting that the fluid mechanics controlling the formation of these capsules was somehow substantially different than for the others. An interconnected open-cell substructure was located beneath the internal skin layer, as shown in Fig. 6 (c). The substructure appeared to be composed of nodules of polymer connected to each other. This

open-cell substructure, presumably offers low resistance to mass transfer through the membrane [15]. On the contrary, finger-like macrovoids were produced beneath the external skin layer (Fig. 6d). These macrovoids extended a reasonably uniform 25 mm into the membrane as shown in Fig. 5 (a) and (b), indicating that the wet phase-inversion appeared to occur uniformly around the capsule shell. The depressions seen in Fig. 6 (b) were not visible in cross-

Fig. 5. Scanning electron micrographs of HEMA-MMA microcapsules: (a) Small diameter capsule (OD: 450 mm) (b) Large diameter capsule (OD: 660 mm).

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Fig. 6. Scanning electron micrographs of cross-sectional structures of small diameter (OD; 450 mm) HEMA-MMA microcapsules:(a) 3120 (b) external surface 312 000 (c) Internal skin structure of small diameter capsule (d) External skin structure of small diameter capsule

section suggesting that the depressions may not be holes in the outer skin. This is discussed further below.

3.2. Capsule permeability HEMA-MMA microcapsules prepared here had a narrow distribution of HRP loading (Fig. 7), indicating that the core lumen volumes and walls were not significantly different among microcapsules of same outside diameter. Larger capsules had more HRP, as expected. No attempt was made to distinguish the

fraction of HRP that was present in the lumen and that present in the wall. The individual capsule permeability to HRP, on the other hand, was broadly distributed. Different distributions in HRP release were obtained for the different capsules (Fig. 8) with the 500 mm capsules being the most uniform of all. An even broader distribution was observed for large diameter capsules prepared by the earlier submerged jet method [11]. An apparent permeability coefficient was estimated from each cumulative release curve using the average b value given in Table 1, ignoring that some HRP was initially present in the wall, that b varied

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J. Rim Hwang, M.V. Sefton / Journal of Controlled Release 49 (1997) 217 – 227 Table 2 Average HRP permeabilities of HEMA-MMA microcapsules: effect of diameter

Fig. 7. HRP loadings of microcapsules of different sizes. Narrow distribution in the loading of HRP in each case indicates that there was no significant variation of same size capsules in their core lumen volumes. HRP loadings were 197 (622), 400 (674), 502 (6102), and 776 (661) ng / ml for OD 450(x), 500(♦), 580(h), and 660 mm(j) microcapsules, respectively.

from capsule-to-capsule and that no correction was made for boundary layer effects. These permeability coefficients (Fig. 8, Table 2) varied from a mean of 2.2310 210 cm 2 / s to 9.0310 210 cm 2 / s for small capsules (OD: 450 mm) and large capsules (OD: 660

Capsule diameter(mm)

450

500

580

660

Average Standard deviation Coefficient of variation Number of capsules

2.2 1.7 0.80 24

2.0 0.5 0.25 26

3.2 2.0 0.62 23

9.0 4.4 0.48 26

mm), respectively. The higher permeability coefficients of the larger capsules may be due to the higher eccentricity made by the improper shearing-off of capsule droplet from the nozzle tip causing locally thinner walls (Fig. 5b) and presumably more defects. However there was no correlation between the coefficient of variation of the permeability and the capsule eccentricity (Table 1). The 500 mm capsules had the smallest degree of variation, suggesting that these were the optimum sized capsules to be made by this nozzle configuration. The variation for the 660 mm capsule appears to be the biggest only because of the scale used in Fig. 8.

3.3. Phase separation kinetics

Fig. 8. Distributions in the estimated permeability coefficient of HEMA-MMA microcapsules with different outside diameters. Each point was calculated from the release curve of a single capsule. Outside diameters of microcapsules are 450 mm(x), 500 mm(♦), 580 mm(h), and 660 mm (j). b was assumed to be the same for each capsule of the same batch; b given in Table 1. Means and standard deviations are given in Table 2.

Inspection of the morphologies at the inside and outside of the capsule (Fig. 6c, d) suggested that there were two different coagulation modes occurring at the two sides of the capsule membrane. To understand the difference, the precipitation rate of HEMA-MMA was monitored in a two dimensional model of capsule wall formation. The penetration distance of the precipitation front in this model system plotted against the square root of time is shown in Fig. 9. Penetration distance was linearly related to the square root of time in agreement with the results of others [7,16]. Interestingly the penetration of L101-PBS coagulant was faster than that of Ficoll  -PBS suggesting that precipitation occurs faster on the outside of the capsule membrane than on the inside. The slower coagulation process at the internal surface is a consequence of the viscosity of the 20% Ficoll  solution and the altered chemical potential of the water relative to that in the external solution.

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these depressions / pores, suggesting that these depressions are more pore-like than the SEMs suggest.

4.2. Capsule-to-capsule variation in permeability

Fig. 9. Difference in penetration distances (6 sd, n54) of the precipitation fronts of HEMA-MMA copolymer with different nonsolvent coagulants in two dimensional membrane formation model. Nonsolvent coagulants were L101-PBS(j) and Ficoll  PBS(h).

4. Discussion

4.1. Mechanism of membrane formation Through contact with two different coagulants L101-PBS, and Ficoll  -PBS at the external and internal surfaces, respectively, the capsule membrane forms at both interfaces by instantaneous demixing [17] but at two different rates. The results in Fig. 9 indicate that coagulation at the external surface of capsules proceeds more rapidly than that at the internal surface. The fast phase separation from the external surface dominates the formation of the capsule membrane except for the skin layer at the internal surface so that the microporous open-cell substructure underlying the inner skin (Fig. 6c) likely reflects the penetration of the precipitation front from the outside in rather than from the inside out. It is not known whether there are morphological or permeability differences between inner and outer skins or which one is rate limiting. It is not even clear whether the depressions seen in the outer skin are holes or not. From the cross-sectional views (Fig. 6d), no pores that traverse the outer skin are apparent. On the other hand, in another paper [18] a correlation was demonstrated between the permeability of the capsule and the size distribution of

The results presented in Fig. 8 illustrate the capsule-to-capsule variation in permeability to HRP. The loading of HRP (Fig. 7) was fairly uniform since the three day incubation period was sufficient to fully saturate the capsules regardless of permeability; the residual variation in loading was fully attributable to variation in capsule diameter. The variation in permeability likely arose from two sources: eccentricity in the capsule and an intrinsic variation even in centered capsules due to variation in the rate of instantaneous demixing leading to a variation in membrane characteristics. As the demixing process proceeds very fast, small differences in the preparation process, even within a single batch, can make a significant difference in the resulting capsules. The capsule membranes in this work were prepared capsule-by-capsule from the extrusion nozzle through continuous flow of hexadecane shearing liquid. The hexadecane flow is slightly periodic (due to the peristaltic pump that is used) and hence each nascent capsule may stay at the nozzle for slightly different times. Similarly each capsule may penetrate the hexadecane / PBS interface at slightly different rates and be exposed to the two fluids for different durations. Thus the exposure of capsules to hexadecane and the initial exposure to coagulant may be different for each capsule. It is suspected that these very small time differences can account for the unavoidable variation in permeability seen in the well-centered smaller diameter capsules. It also remains to be seen if the exposure to hexadecane has an effect on the rate of demixing. For the larger diameter capsules, their eccentricity may also be important. Such eccentric capsules are more likely to have flaws or defects leading to pinholes than the better centered, smaller capsules. Their thin spots are also more vulnerable to cracks and defects, through subsequent handling. To compensate for the eccentricity the capsules are made with thicker walls than would otherwise be necessary. The 660 mm capsules had a wall thickness of 110 mm, while the better centered 500 mm capsules had a 45 mm thick wall. This difference in wall

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thickness further exacerbates the subtle differences associated with the rates of demixing that was described above. It is also possible that thicker walls are less dense and hence more permeable. It is worth noting that there was no correlation between eccentricity and the variability in permeability (compare Table 1 and Table 2), suggesting that the effect of variable demixing rates is more important. Eccentricity also plays a role in the calculation of the estimated permeability. The formula for b assumes a perfectly centered spherical shell. A similar formula for eccentric spheres (like the ones in the heat transfer literature for eccentric cylinders) is not available. This would introduce a constant error if the eccentricity were constant from capsule-to-capsule and so would not change the coefficient of variation. On the other hand, since the eccentricity varies from capsule to capsule, the value of b would vary and this also contributes to the variability in permeability. Whether it makes the permeability variation appear worse than it really is, is unknown, since the values of eccentricity, a, b or b cannot be determined for the identical capsules for which the permeability was measured. To translate the slopes into reasonable units a constant b was assumed; the calculated permeabilities are then just estimates based on this assumption.

4.3. Conclusions Small capsules (OD: |450 mm) were prepared by interfacial precipitation wet phase-inversion from a HEMA-MMA solution in triethylene glycol (TEG) using a submerged jet coextrusion technique. The low molecular weight of TEG was expected to contribute to a faster exchange of solvent and coagulant at their interface. Smaller microcapsules were prepared when the flow-rate of hexadecane was increased leading to a higher shearing force on the nascent capsule at extrusion. A narrower distribution in HRP permeability coefficient was obtained for some of the smaller microcapsules which had a better centered capsule structure. However the smallest diameter capsules studied here had the broadest permeability distribution although the average permeability was much less than that for the biggest, poorly centered, capsules.

The skins formed at the external and internal surfaces of the capsule membrane had different morphologies consistent with the faster exchange of solvent and coagulant at the external surface.

References [1] F. Lim, A.M. Sun, Microencapsulated islets as bioartificial endocrine pancreas, Science 210 (1980) 908–910. [2] P.A. Tresco, S.R. Winn, P. Aebischer, Polymer encapsulated neurotransmitter secreting cells: Potential treatment for Parkinson’s disease, ASAIO J. 38 (1992) 17–23. [3] V. Dixit, M. Arthre, G. Gitnick, Repeated transplantation of microencapsulated Hepatocytes for sustained correction of hyperbilirubinemia in Gunn rats, Cell Transplant. 1 (1992) 275–279. [4] M.V. Sefton, L. Kharlip, V. Horvath, T. Roberts, Controlled release using microencapsulated mammalian cells, J. Control. Rel. 19 (1992) 289–298. [5] I. Cabasso, E. Klein, J.K. Smith, Polysulfone hollow fibers. I. Spinning and properties, J. Appl. Polymer Sci. 20 (1976) 2377–2394. [6] I. Cabasso, E. Klein, J.K. Smith, Polysulfone hollow fibers. II. Morphology, J, J. Appl. Polymer Sci. 21 (1977) 165–180. [7] H. Strathmann, Production of microporous media by phase inversion process, in: D.R. Lloyd (Ed.), Materials Science of Synthetic Membranes, ACS Symp. Ser., No 269, American Chemical Society, Washington DC, 1985, pp. 165–195. [8] C.A. Crooks, J.A. Douglas, R.L. Broughton, M.V. Sefton, Microencapsulation of mammalian cells in a HEMA-MMA copolymer: Effects on capsule morphology and permeability, J. Biomed. Mater. Res. 24 (1990) 1241–1262. [9] H. Uludag, V. Horvath, J.P. Black, M.V. Sefton, Viability and protein secretion from human hepatoma (HepG2) cells encapsulated in 400 mm polyacrylate microcapsules by submerged nozzle–liquid jet extrusion, Biotechnol. Bioeng. 44 (1994) 1199–1204. [10] R.M. Boom, T. Boomgaard, C.A. Smolders, Mass transfer and thermodynamics during immersion precipitation for a two-polymer system. Evaluation with the system PES-PVPNMP-water, J. Memb. Sci. 90 (1994) 231–249. [11] H. Uludag, J.R. Hwang, M.V. Sefton, Microencapsulated human hepatoma (HepG2) cells: Capsule-to-capsule variations in protein secretion and permeability, J. Contr. Rel. 33 (1995) 273–283. [12] W.T.K. Stevenson, R.A. Evangelista, M.E. Sugamori, M.V. Sefton, Preparation and characterization of thermoplastic polymers from hydroxy alkyl methacrylates, J. Appl. Polymer Sci. 34 (1987) 65–83. [13] H. Uludag, M.V. Sefton, Metabolic activity and proliferation of CHO cells in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules, Cell Transplant. 2 (1993) 175–182. [14] W.R. Good and P.I. Lee, Membrane controlled reservoir drug

J. Rim Hwang, M.V. Sefton / Journal of Controlled Release 49 (1997) 217 – 227 delivery systems, in: R.S. Langer, D.L. Wise (Eds.), Medical Applications of Controlled Release, Volume I, CRC Press, Boca Raton, 1984, pp. 1–39. [15] H. Borhorst, F.W. Altena, C.A. Smolders, Formation of asymmetric cellulose acetate membranes, Desalination 38 (1981) 349–360. [16] Y.S. Kang, H.J. Kim, U.Y. Kim, Asymmetric membrane formation via immersion precipitation method. I. Kinetic effect, J. Memb. Sci. 60 (1991) 219–232.

227

[17] J. van’t Hof, Wet spinning of asymmetric hollow fiber membranes for gas separation, Ph.D. thesis, University of Twente, The Netherlands, 1988. [18] J.R. Hwang, M.V. Sefton, Effect of polymer concentration and a pore forming agent (PVP) on HEMA-MMA microcapsule permeability, J. Memb. Sci. 108 (1995) 257–268.