Microencapsulation of erythrocytes

Biochimica et Biophysica Acta, 717 (1982) 473-477

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Elsevier Biomedical Press BBA 21203

MICROENCAPSULATION OF ERYTHROCYTES MICHAEL V. SEFFON and RICHARD L. BROUGHTON

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, MSS 1.44 (Canada) (Received March 9th, 1982)

Key words: Encapsulation; Erythrocyte; Polyacrylate membrane

Human erythrocytes have been encapsulated in a polyacrylate membrane by a simple precipitation process. The encapsulated cells appeared to remain functional after encapsulation: the consumption of glucose and the ability to reversibly bind oxygen was unimpaired. Furthermore, storage at 4°C for almost 6 months had no effect on the Ps0 and nso values. This is the first time to our knowledge that live mammalian cells have been encapsulated in a polymer other than alginate.

Lim and Sun have recently reported [1] the microencapsulation of functioning pancreatic islets in a calcium alginate gel, which is subsequently treated with aqueous solutions of polylysine and poly(ethyleneimine). Intact erythrocytes [2] and plant chloroplasts [3] have also been entrapped in calcium alginate using methods adapted from the immobilizaton of microbial cells [4,5]. Immobilized animals cells would be useful for their potential ability to act as transplanted tissue without danger of immune rejection. Provided the surrounding membrane was impermeable to the higher molecular weight antibodies but permeable to oxygen, glucose, other substrates and/or the internally generated hormones, the immobilized cells would act in the host as they had in the donor. Alginate gels, even when treated with polylysine and poly(ethyleneimine), however, are not sufficiently biocompatible to be useful as long term implants; this proved to be a serious limitation of the microencapsulated islets [1]. It is presumed that the water solubilitiy of these polymers is responsible for this limited in vivo stability and biocompatibility. Hence it would be desirable to devise a microencapsulation process using a water-insoluble polymer. On the other hand, ceils can be relatively easily removed from alginate gels 0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

unlike water insoluble capsules. This can be an advantage of alginate gels in various applications (blood storage, monoclonal antibody production) where the cells need to be recovered intact after encapsulation. As a model system, human erythrocytes have been encapsulated in a water insoluble polyacrylate, EUDRAGIT RL, (an acrylic acid ester/methacrylic acid ester copolymer containing a low content of quaternary ammonium groups), to develop an appropriate immobilization system for such implants. Polyacrylates as a class (e.g., poly(methylmethacrylate), poly(hydroxyethylmethacrylate)) are generally accepted for their tissue compatibility. Of the various whole cell immobilization techniques available [4,5,7] microencapsulation methods are, in our experience, the simplest means of avoiding osmotic stresses. However, the solvents used to dissolve the polymer for microencapsulation should have very low water solubilities to minimize both the associated osmotic stress and the apparent toxicity. On the other hand, the permeability requirements of the surrounding microcapsule necessitates the use of polar polymers which are best dissolved in polar, water miscible solvents. Diethyl phthalate was the only corn-

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pound out of a great many examined that managed to reasonably satisfy both of these constraints. 0.5 ml of human erythrocytes collected in CPD, (Canadian Red Cross, Toronto) were washed, suspended in phosphate buffered saline, (0.143M NaC1/0.01 M phosphate) (50% hematocrit) and emulsified in 5 ml of an 8% (w/v) solution of Eudragit RL 100, (Robin Pharma GmbH, Darmstadt, F.R.G.) in diethyl phthalate. 4ml of this emulsion was further suspended in 20 ml mineral oil, in which the phthalate was not miscible to create an emulsion within an emulsion. Dropwise addition of 10 ml corn oil caused the phthalate to be drawn into the oil phase, leaving the polymer behind as a membrane encapsulating the droplets of cells. More corn oil was added to enhance the miscibility of diethyl phthalate in the oil phase, followed by centrifugation and two washes with fresh corn oil. A final wash with 25 ml mineral oil was used to remove the corn oil, folowed by the addition of 20 ml phoshate-buffered saline and centrifugation at 3000 rpm to separate the capsules from the mineral oil. The capsules were washed 2-3 times with phosphate buffered saline until the washes were hemoglobin-free and stored at 4°C in plasma. From the amount of hemoglobin in the washes (by s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of cyanomethemoglobin at 540 nm), an estimated 15% of the cells lyzed during encapsulation presumably due to mechanical damage during stirring. Because the polymer membrane was not freely permeable to hemoglobin there may have been hemoglobin from lyzed cells trapped inside the capsules so that the percentage of actually viable, intact, encapsulated cells may be somewhat lower than the remaining 85%. The microcapsules prepared in this manner are shown in Fig. 1. The actual capsules ('unit spheres') were the individual droplets of cells in RL100/diethyl phthalate prior to precipitation and are approx. 75-100/xm in diameter. However, these unit spheres were stuck together by excess polymer to form dumps that may be more than 1000 tam long and 200-300 #m wide. Improvements in the corn oil addition step have reduced this clumping. Assessing the viability or erythrocytes has been a source of controvery [8]. Erythrocytes, lacking nuclei, are in many ways senescent and do not

respond to viability assays used for most nucleated cell lines. The clinical assessment of post transfusion survival is the best assay [8] but was of course impractical in this case. Instead the presence of normal glucose consumption was used to indicate that most of the metabolic machinery of the cells was intact and functioning and hence that the cells could be considered viable. Measurement of the N a / K ratio could also be used as a similar measure of erythrocyte function. The encapsulated cells continued to consume glucose after encapsulation. Cells were suspended in CMRL 1969 tissue culture medium [9] containing 360 mg/dl glucose and 20 # g / m l gentamycin at pH 7.27 and maintained at room temperature for 5-6 days. The disappearance of glucose was measured using a glucose analyser (Yellow Springs International Model 27). The nutrients present in CMRL 1969 (or in plasma) were found to be essential to keeping the cells alive during the experimental period. The glucose concentrations were corrected to account for the volume of medium (0.1 ml) removed for concentration measurement. Constant average consumption rates were estimated by a linear least-squares method. The consumption rate was 32.9 -+- 6.0 /xg/h per ml erythrocytes averaged over 4 runs (-+S.D.) for free, unencapsulated cells and 48.8 -+ 9.3/~g/h per ml erythrocytes averaged over 3 runs (-+S.D.) for the encapsulated cells. Fig. 2 shows the results of two such runs. The reasons for the higher consumption rate for the encapsulated cells may be related to a higher pH locally within the capsules, to the experimental difficulty of maintaining a high pO 2 in the encapsulated cell suspension [10], or simply to an underestimate of the total number of viable cells encapsulated, it conceivably also could reflect subtle differences between encapsulated and free erythrocytes. That insignificant glucose was lost from solution by partitioning into the membrane, or by bacterial contamination, was shown by the absence of glucose consumption by microencapsulated dead cells (cells previously exposed to DMSO); the glucose concentration remained constant at 213 --+ 13 mg/dl over 5 days. Another meaure of erythrocyte function is the maintenance of its ability to reversibly bind oxygen. Bubbling air or nitrogen through the capsules caused a visible change in colour to red or

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Fig. I. A. Scanning electron micrograph of EUDRAGIT RL encapsulated erythrocytes showing a clump of 'unit spheres.' Scale mark is 10 ~am. B. Micrograph of encapsulated erythrocytes, prepared by rapid addition of corn oil to precipitate the polymer. Capsule diameter is approx. 20-30 ~am. Intact erythrocyte shown with arrow.

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purple, respectively, although it was more difficult to deoxygenate the oxygenated capsules than the reverse. The hemoglobin-oxygen saturation curves were measured using a Hem-O-Scan (American Instrument Co., Savage, Md) by Dr. J. Bonaventura, Duke University Marine Laboratories. The Hem-O-Scan uses dual wavelength spectroscopy coupled with oxygen monitoring by a Clark electrode to generate the saturation curve. This technique overcame the problem of the heterogeneous distribution of hemoglobin within the capsules, which made conventional spectroscopy difficult. The Ps0 and ns0 values of encapsulated erythrocytes are listed in Table I, showing the absence of an effect of storage time. (Ps0 is the oxygen partial pressure at 50% saturation and ns0 is the Hill equation constant [11] a measure of cooperativity between heine groups at 50% saturation.) The capsules remained red while stored at 4°C between measurements. To compare these values with literature values for normal erythrocytes [12], these results have been corrected (i.e., lowered) to pH 7.4 using the Severinghaus nomogram (Alog Po2/ApH----0.43) [13]. Values for ns0 were not adjusted. While these Ps0 values were lower than those found in fresh blood, they were similar to the values measured in stored blood. After about 7 days at 4°C [12] or 12-14h at 37°C [14], as ATP

and 2,3 diphosphoglycerate are consumed, the "°50 values fall to about 15-18 mmHg. The nso values were similar to those of normal cells, somewhat lower in the first series and higher in the second. Although the day-to-day variations are large, neither series shows the characteristic increase known to occur with storage of free cells [12]. This may be a benefit of encapsulation. Hem-O-Scan measurements, however, do not indicate the fraction of the encapsulated cells which retained normal saturation curves. Early experiments with larger capsules showed qualitative differences among capsules stored in phosphatebuffered saline without glucose, in phosphatebuffered saline with glucose and those stored in plasma. In the absence of glucose, the encapsulated cells lost their red colour and turned brown within 2 weeks. Those with glucose stayed red a week longer and those with a full complement of TABLE I CHARACTERISTIC PARAMETERS OF HEMOGLOBINOXYGEN SATURATION CURVE OF MICROENCAPSULATED ERYTHROCYTES Measured at pH 7.1, 37°C and adjusted to p H 7.4. Cells suspended in 0,9% NaCI/0.1 M phosphate with 5.6% CO 2 during measurement and stored in the same medium without C O 2 at 4°C between measurements. Series A and B differ in the rate of corn oil addition to the mineral oil suspension of cells and polymer in preparing the capsules. Effect of storage at 4°C

350

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-

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i

r

~

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325 3o0

Series A

E ._~ 275 250 225 =~ 200 (.9 175

Series B 15(

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110 210 310 40

S;

i

60

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7JO 80 90 100 110 120 1;0 1,~0 150

Time (hours)

Fig. 2. Consumption of glucose by free and encapsulated erythrocytes at p H 7.27, room temperature, 0.2 ml free erythrocytes ( 0 0 ) in 1 ml medium, 0.4 ml intact erythrocytes ((9 (9) encapsulated in E U D R A G I T in 1 ml medium.

Days after encapsulation

Ps0 (mmHg) n 5o

10 32 61 89 97 123 167 m e a n (-+ S.D.)

19.1 14.9 15.7 13.8 11.3 17.2 16.9 15.5--+2.5

2.3 2.2 1.6 2.4 1.8 2.2 1.4 2.0-+0.4

18.5 14.2 16.3 19.8 17.4 17.2-----2.1

3.8 2.9 2.8 2.9 3.0 3.1 -+0.4

25.7

2.4

12 39 48 73 117

mean (-+ S.D.) fresh whole blood [13] (pH 7.4)

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nutrients a s supplied by plasma remained red or purple, as opposed to brown, for more than 12 weeks. This observation indicates that the cellular apparatus for preventing the hemoglobin from converting to methemoglobin (the brown colour) must be in operation which is also a sign of cellular viability [15]. The reason for the difference in behaviour of encapsulated erythrocytes stored in phosphate-buffered saline for these experiments and for the Hem-O-Scan measurements remains to be clarified. Unfortunately, the capsules were left with trace amounts of diethyl phthalate and enough of the oils to make them slightly tacky. Although the oils and phthaiate appear to have little effect on the permeability of the capsules or the viability of the cells inside, these residues might cause an inflammatory response when implanted in animals. Refinements are being devised to remove the residual organic material. This process is now being extended to larger nucleated tissue cells (pancreatic islets and Chinese hamster ovary fibroblasts) and early results are encouraging. Although biocompatibility remains to be assessed, the ability to microencapsulate live animal cells in a non water-soluble polymer is a significant step in the development of immobilized animal cells for transplantation.

Acknowledgements We acknowledge the financial support of the N a t u r a l Sciences and Engineering Research

Council of Canada, the technical assistance of T.K. Kita, F.V. Lamberti and D.G. Phillips and the encouragement of Dr. A.M. Sun of Connaught Research Institute, Toronto.

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1-36 7 Chang, T.M.S. (1972) Artificial Cells, C.C. Thomas Springfield, Ill. 8 Beutler, E. (1973) in The Human Red Cell in Vitro (Greenwalt, T.J. and Jamieson, G.A., eds.), pp. 189-216, Grune and Stratton, New York 9 Healy, G.M., Teleki, S., Seefried, A.V., Walton, M.J. and Macmorine, H.G. (1971) Appl. Microbiol. 21, 1-5 10 Murphy,J.R. (1960) J. Lab. Clin. Med. 55, 282-293 11 White, A. et al (1978) Principles of Biochem, 6th. Edn., pp. 959-962, McGraw-Hill, New York 12 Bunn, H.F., May, M.H., Kocholaty, W.F. and Shields, C.E. (1969) J. Clin. Invest. 48, 311-321 13 Severinghaus,J.W. (1958) J. Appl. Physiol. 12, 485-486 14 Horvath, S.M., Malenfant, A., Rossi, F. and Rossi-Bernardi, L. (1977) Am. J. Hematology 2, 343-354 15 Harris, J.W. (1970) The Red Cell, revised edition, pp. 463-464, Harvard University Press, Cambridge, MA