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Agarose-polymeric microsphere beads: Specific new adsorbents for hemoperfusion
Reactive Polymers, 1 (1983) 241- 250 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
241
STATE-OF-THE-ART REPORT
AGAROSE-POLYMERIC MICROSPHERE BEADS: SPECIFIC NEW ADSORBENTS FOR HEMOPERFUSION SHLOMO MARGEL
The Plastics Research Department, The Weizmann Institute of Science, Rehot:ot (Israel) (Received March 18, 1983: accepted in revised form June 16, 1983)
Uniform agarose-polymeric microsphere beads of 1 mm diameter were synthesi:ed by encapsulating appropriate polymeric microspheres in an agarose matrix. The potential use of these beads for hemoperfusion was demonstrated. Agarose provides the high porosity and the biocompatibility of the beads," the encapsulaled microspheres are used for the removal of harmful substances from the circulatory system. Two types of polymeric microspheres were prepared." (a) polymercaptal microspheres of 0.8 micrometer average diameter," (b) polyacrolein microspheres of 0.2 micrometer average diameter. Agarose-polymercaptal microsphere beads were designed for the removal of heacv metals by hemoperfusion. As a model, the detoxification of mercury compounds was studied. Agarose-polyacrolein microsphere beads were designed for the removal of antibodie's by hemoperfusion, especially in autoimmune diseases. As a model, the removal of anti-BSA from immunized animals was studied.
INTRODUCTION
Hemoperfusion is a relatively new approach for the removal of harmful substances from the bloodstream. This goal is achieved by circulating the blood through an appropriate adsorbent which adsorbs the harmful material. The "cleaned" blood is then returned to the patient. The ideal adsorbent for hemoperfusion has to fulfill many requirements, of which the major ones are the following: (a) blood compatibility, which ensures minimum loss of red blood cells (RBC), white blood cells (WBC) and thrombocytes; (b) high specificity, so that only the harmful material is removed from the blood while all the other 0167-6989,/83/$03.00
important components remain in the blood; and (c) high affinity and capacity toward the substance that has to be removed from the blood, so that the adsorbent size and the time required for the hemoperfusion will be minimal. The hemoperfusion process was initiated in 1948 by Muirhead and Reid [1] who studied the use of ion-exchange resins as adsorbents for the removal of uremic toxins from poisoned animals. Following this work, Schreiner (1958) [2] employed unsuccessfully a lactated anion-exchange resin column to remove pentobarbital from a poisoned patient. These studies with ion-exchange resins, as well as others [3], were complicated by pyrogenic re-
~ 1983 Elsevier Science Publishers B.V.
242 actions, electrolyte disturbances and hemolysis. These complications could be reduced by pretreating the ionic resins [4,5]. However, the early resins for hemoperfusion were considered too dangerous for widespread clinical practice. In 1964 Yatzidis [6] presented his in vitro research with activated charcoal particles, showing their efficiency for removing barbital, phenobarbital, pentobarbital and salicylic acid. Later, Yatzidis et al. [7] reported on the use of charcoal hemoperfusion on two patients with barbiturate poisoning who both recovered. However, many side effects such as platelet depletion, a reduction in fibrinogen concentration and particle embolization sensations were noted. The hemoperfusion problems with activated charcoal were partially solved by the pioneering work of Kolff [8] and Chang [9-11]. Chang and his group rendered the sorbent biocompatible by coating, sometimes combined with encapsulation. Subsequently, charcoal has been coated with albumin collodion [11], nylon, agarose and poly(hydroxyethylmethacrylate) [12- 14]. Since then hemoperfusion with either charcoal, ionic or non-ionic resins [15-18] has become a more common clinical tool. These adsorbents have been used for the removal of a wide variety of poisons, such as barbiturates, nonbarbiturate hypnotics, sedatives, tranquilizers, antidepressants, alcohols, analgesics, antimicrobials, anticancer agents, herbicides, insecticides, cardiovascular agents, bilirubin, and others. Problems involving biocompatibility, especially disruption of the formed elements of the blood, have not yet been overcome completely. Although obviously the patient risk-benefit ratio must be considered, under extreme conditions greater risks are warranted. Unfortunately, charcoal and ionic resins are non-specific adsorbents and they remove from the blood many classes of biocompounds in addition to the toxic material. We envisualise the synthesis of novel ad-
sorbents for hemoperfusion, based on the encapsulation of appropriate polymeric microspheres in an agarose matrix. We synthesized two types of uniform, encapsulated microsphere beads, both 1 mm in size: (a) A g a r o s e - p o l y m e r c a p t a l microsphere beads, designed for the removal of heavy metals by hemoperfusion. As a model the detoxification of mercury compounds was studied. (b) Agarose-polyacrolein microsphere beads, designed for the removal of antibodies from the circulatory system. As a model we studied the removal of anti-BSA from immunized animals.
EXPERIMENTAL
Synthesis of polymercaptal microspheres Polymercaptal microspheres with 0.8 micrometer average diameter were formed by the reaction of glutaraldehyde and pentaerythritol tetrathioglycolate in the presence of Tween 20 as surfactant, as previously described [ 19].
Synthesis of polyacrolein microspheres Polyacrolein microspheres of 0,2 micrometer average diameter were prepared by polymerizing acrolein in an aqueous solution containing polyethylene oxide as surfactant with a cobalt radiation source, as previously described [20].
Synthesis of agarose-polymeric microsphere beads Uniform beads with a diameter of 1 mm were prepared by a procedure similar to that described by Losgen et al. [21]. A molten solution of agarose (0.8 g) containing appropriate polymeric microspheres (4-6% w/v) was drawn into the glass syringe apparatus
243
Binding a spacer to the agarose-polyacrolein microsphere beads
[
The spacer polylysine-glutaraldehyde was bound to the beads as previously described [22]. Summarily, the procedure was as follows. The beads were shaken in H20 with polylysine for 24 hours at room temperature. The polylysine-conjugated beads formed were washed free of unbound polylysine by repeated decantation. The conjugated beads in H20 were then shaken for 12 hours at room temperature with glutaraldehyde and the resultant polylysine glutaraldehyde conjugated beads were filtered and then washed with large amounts of distilled water.
Motor driven I
Piston
Thermal jacket
Coupling of proteins to the agarose-polyaldehyde microsphere beads [22]
Syringe
[
J
Fig. 1. Glass syringe apparatus for the preparation of uniform agarose-polymeric microsphere beads.
shown in Fig. 1. The apparatus was kept at 70°C while stirring the agarose-microspheres aqueous suspension at 300 rpm. Thereafter, the gel was forced out by a motor-driven piston and was injected dropwise into a tall vessel containing an ice-cold solvent mixture of toluene, chloroform and hexane in the ratio of 10 : 4 : 2, respectively. The beads which were formed were separated from the solvent mixture in a crude sieve, washed several times with dioxane and finally cleared of solvents by exhaustive washing with distilled water. The beads were stored in the cold in the presence of 0.05% w / v sodium azide until use.
Beads (1 ml) with or without polylysine glutaraldehyde spacer were shaken at 4°C for 24 hours with the desired proteins (10 mg protein in 5 ml phosphate-buffered saline solution (PBS) at pH 7.2). The remaining aldehyde groups were then quenched by shaking the beads for 12 hours with 0.05 ml ethanolamine solution, brought to pH 7.2 with HCI. The immuno-beads formed were then washed successively with PBS. Rabbit anti-bovine serum albumin (antiBSA) bound to BSA conjugated beads was eluted from the beads with 0.2 M glycine-HC1 buffer solution at pH 2.4.
Determination of proteins Quantitities of protein bound to the beads were determined by subtracting unbound protein from total protein measured according to the method of Lowry et al. [23]. Quantities of antibodies bound and eluted from the inamuno-beads were determined by the quantitative precipitation reaction developed by Heidelberger and Kendall [24].
244
~~
GLASS COLUMN BLOODOR SERUM ,I AGAROSEENCAPSULATED BLOOD PUMP ~] ~MICROSPHERES //I ~ G L A S S DISCWITH /~1 0.Sin m PORES
II~
RESERVOIR
Fig. 2. Schematic diagram of the apparatus employed for the in vitro hemoperfusion experiments.
In vitro experiments A Travenol roller-type blood pump cycled the citrated blood (containing either mercury compounds or anti-BSA) from the reservoir
Fig. 3. Photograph showing the
through the column of agarose-polymeric microsphere beads at a controlled flow rate (Fig. 2). Samples were drawn from the reservoir and checked for the concentration of mercury or anti-BSA as well as for blood compatibility. In vivo experiments' Rabbits were anesthetized with ether via a nose cone. Oxygen was administered via a face mask. The carotid artery and jugular vein were cannulated with medical grade PVC tubing. The carotid artery cannula was connected to an arterial outlet set via a three-way stopcock. A blood pump cycled the arterial blood from the carotid artery through the column of beads. The outlet of the column was attached to a venous inflow set consisting of a bubble trap. The blood was returned to the rabbit via a three-way stopcock to the cannula in the jugular vein.
in vivo
hemoperfusion system.
245
The inline tee stopcock in the venous inflow set permitted intravenous infusion of saline or cysteine solution. The complete system was equilibrated with sterile heparinized saline (10 units per milliliter) before use. The complete set as well as the extracorporeal blood were temperature-controlled in a water bath at 38°C (Fig. 3). The rabbits were fully heparinized by intravenous injection of 300 units heparin per kilogram body weight and maintained in the heparinized state by constant heparin addition.
Blood counting Formed elements of the blood were counted in a hemocytometer and with a Coulter Counter.
Mercury analysis Mercury was determined by flameless atomic absorption spectrophotometry with a mercury analyzer of Perkin-Elmer and an additional kit developed in our group for the direct determination of mercury [25,26].
R E S U L T S AND D I S C U S S I O N Agarose was chosen as the encapsulator polymer since it fullfils certain essential requirements for hemoperfusion applications, such as bio- and blood compatibility and high porosity. The encapsulation by agarose of the water-insoluble polyacrolein or polymercaptal polymer is difficult to carry out. However, by substituting the polymer powder by a suspension of microspheres, the encapsulation becomes easier to accomplish. Figure 4 shows Fig. 4. (a) and (b): Light microscopy photomicrographs of agarose beads and agarose-polymeric microsphere beads, respectively. (c) and (d): Cross-section pictures of agarose polymercaptal microsphere beads (obtained
peripher
periphery
by light microscopy) and of agarose polyacrolein microsphere beads (obtained by transmission electron microscopy), respectively.
246 TABLE 1 Intake of mercury compounds by polymercaptal microspheres in the presence of alkali and alkaline earth metallic compounds a [Hg] (ppm)
Metallic compounds (M)
Microspheres (rag)
Hg intake (%)
Ca, Na and Mg intake
(%) 20 (Mercuric chloride) 20 (Mercuric chloride) 1 (Mercuric chloride)
20 (Methyl mercury chloride) 1 (Methyl mercury chloride)
0.001 CaCI2 0.15 N aCl 0.1 NaC1 + CaC12 + MgC12 0.15 NaC1 0.1 NaC1 + CaC12 + MgCI 2
15 15 10
100 100 - 95
0 0 0
15 10
100 - 95
0 0
a The mercury compounds were stirred with polymercaptal microspheres for 15 min in 20 ml of aqueous solution.
photomicrographs of the uniform agarose beads (a) and the agarose-polymeric microsphere beads (b). Cross-section pictures of the agarose-polymercaptal microsphere beads and of the agarose-polyacrolein microsphere beads, showing the encapsulated microspheres, are given in Figs. 4(c) and 4(d), respectively.
Detoxification of mercury compounds with agarose-polymercaptal microsphere beads The polymercaptal microspheres bind mercury compounds specifically and do not interact with alkali and alkaline earth metallic compounds, as shown in Table 1. The microspheres have high affinity towards mercury compounds and can easily bind mercury, which was previously bound to the biological mercury binder cysteine (Table 2). In vitro hemoperfusion experiments, including a kinetic study (Table 3), illustrate the potential of these beads for extracorporeal use in cases of severe mercury poisoning. Cysteine accelerates the removal of mercury from blood, especially in cases of organic mercury poisoning (Table 3). These findings are in agreement with previous publications [27,28]. The distri-
bution percentage of mercury compounds between the plasma and the red blood cells is approximately 85 : 15 for HgCI 2 and 8 : 92 for CH3HgC1 [29]. The accelerating effect of cysteine is probably owing to its diffusion into the red blood cells, binding the mercury and diffusing back to the plasma. The beads then bind the mercury from the cysteine-mercury complex. The in vivo kinetic studies describing the removal of CH3HgC1 from poisoned rabbits are shown in Fig. 5. The rabbits were dosed TABLE 2 Rate of intake of mercury compounds from cysteine a Mercury compound
Time (min)
Reaction (%)
Mercuric chloride Mercuric chloride Mercuric chloride Mercuric chloride Methyl mercury chloride Methyl mercury chloride Methyl mercury chloride
10 60 240 1320 10 60 240
95 97 99 100 91 91 91
a Mercury compounds (20 ppm Hg) were stirred for 2 h with 20 ml 10 -2 M cysteine in PBS. Polymercaptal microspheres were then added (20 mg in the reaction with methyl mercury chloride and 50 mg in the reaction with mercuric chloride).
247 TABLE 3 Mercury intake from poisoned blood by agarose-polymercaptal microsphere b e a d s - - e f f e c t of cysteine Time
Mercury removal (%) a
(h)
Without cysteine
With cysteine
CH3HgCI
HgC12
CH3HgCI
HgCI 2
18 27 63 89
l0 25 44 90
30 63 64 88
13 45 64 88
0.5 2 4 12
3 g beads were shaken with 20 ml blood poisoned with 2 ppm mercury in the absence or presence of 10 2 M cysteine; control experiments (agarose beads) indicate negligible intake of mercury.
with 8 mg CH3HgC1 (2 mg/kg). In the chronic poisoning case the hemoperfusion procedure was started two days after the intoxication. In the acute poisoning case the treatment was
7O---
T--
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.o
5C
~O9
40
I
I
I--
I
I
Arterial outflow
~"Venus inflow
0 cO
o
30
o
2Q
N
I.© ~
'
q[
©
40
~
~
i Venous inflow
80
t Chronic intoxicatJor l
[
120 160 200 2 4 0 - Time (rain)
Fig. 5. In vivo kinetics describing the removal of methyl mercury chloride from rabbit blood. O and e : arterial and venous mercury concentration, respectively, in a chronic intoxication case: a and i : arterial and venous mercury concentration, respectively, in an acute intoxication case. 8 mg methyl mercury chloride (2 m g / k g ) was injected intravenously: the hemoperfusion procedure started two days after intoxication in the chronic poisoning case and 70 rain after intoxication in the acute poisoning case.
started 70 minutes after the intoxication. The differences between the arterial lines and the venous lines indicate the intake of mercury by the agarose p o l y m e r c a p t a l microsphere beads. Mercury is equilibrated between the blood and the tissues; therefore the mercury concentration in the blood did not change significantly. The clearance of the blood by the beads is defined by the following equation: c l e a r a n c e = Q v ( C ~ - C , ) , where Q~ is the flow rate ( ~ g / m i n ) and C~ and C,. are the artery and venous mercury concentration (/xg/ml), respectively. Integration of the clearance over the hemoperfusion time, of' clearance dt, will show the amount of mercury removed by the hemoperfusion procedure. Calculations show that 330 yg Hg was removed after 3 hours of hemoperfusion in the chronic poisoning case and 1000 yg Hg after 130 minutes in the acute intoxication case (approximately 4.5% and 13%, respectively, of the body mercury burden). The hemoperfusion treatment has to start as early as possible. However, it is also reasonable to expect that repeated hemoperfusion treatments will remove the body mercury burden significantly. The blood compatibility of the agarosepolymercaptal microsphere beads was checked by counting the formed elements during the in ~ivo hemoperfusion experiment (Table 4). The RBC decreased by 25%, the WBC decreased by 33% and the platelet count decreased by 13%, during 3 hours of hemoperfusion treatment. TABLE 4 Cell counts of the formed elements of rabbit blood pumped (10 m l / m i n ) through agarose-polymercaptal microsphere beads (30 g) Time(min)
RBC
WBC
Platelets
0 60 120 180
4,000,000 3,870,000 3,350,000 3,000,000
1,500 1,150 1,000 1,000
173,000 162,000 1:51.000
248
The potential use of the agarose-polymercaptal microsphere beads for the removal of mercury by hemoperfusion was demonstrated. A further investigation will have to be carried out in order to evaluate the potential use of these beads for the removal by hemoperfusion of other heavy metallic compounds such as arsenic, cadmium, lead, copper and others. A model of the removal of antibodies by hemoperfusion with agarose-polyacrolein microsphere beads: removal of anti-BSA The polyacrolein microspheres encapsulated by the agarose are provided with aldehyde groups through which various ligands containing primary amino groups, e.g., proteins, can bind covalently to form the Schiff base products. Leaching of proteins bound to the beads is not detected owing to the formation of polyvalent Schiff base bonds. The binding capacity of the agarose-polyacrolein microsphere beads towards various proteins is illustrated in Table 5. Steric requirements may explain the significant increase (3-6 fold) in the binding capacity of the beads bound to the spacer polylysine-glutaraldehyde. In a hemoperfusion model system, circulating rabbit anti-BSA was adsorbed to BSA conjugated beads. In practice, any antigen may be covalently bound to the beads and the appropriate circulating antibody (especially in autoimmune diseases) may be removed. A
I
~0.4
I
I
I
I
I
1 I
l
I
I
I
|
I
I
I
I
-
~)0.3 m
~
0.2
O0
30 60
120
180
MINUTES Fig. 6. Kinetics pooled i m m u n e culated through sphere beads at
of adsorption of r a b b i t anti-BSA from r a b b i t serum. 100 ml serum was cir17 g BSA agarose-polyacrolein microa flow rate of 62 m l / m i n .
kinetic study of the adsorption in vitro of rabbit anti-BSA from immunized rabbit serum is shown in Fig. 6; 62% was removed in 30 min, 78% in 60 min, 91% in 120 min and 95% in 180 min. The same beads were reused several times following the elution of the rabbit anti-BSA, with similar results. The kinetic results of a similar in vivo hemoperfusion
TABLE 5 Binding capacity of a g a r o s e - p o l y a c r o l e i n microsphere beads to various a m i n o ligands a Ligand
Spacer
Binding capacity (mg)
BSA BSA Rabbit Rabbit Bovine Bovine
Polylysine-glutaraldehyde Polylysine-glutaraldehyde Polylysine-glutaraldehyde
2.2 14.8 1.0 10.0 7.2 22.0
immunoglobulin immunoglobulin insulin insulin
a 1 ml beads was shaken with an excess quantity of the various ligands in 5 ml PBS for 12 hours at room temperature.
249
0.6
E
]
1
l
0.5
~3n
E 0.4 <~
oo 0.:5 nq
F-
z<
0.2
F-
O.Im
<1[ rr
0'00
:50 6 0 9 0 120 150 180 MINUTES
Fig. 7. Kinetics of removal of rabbit anti-BSA from immunized rabbits. Circulation of immunized rabbits blood at a flow rate of 12 m l / m i n through a column containing 30 g BSA agarose-polyacrolein microsphere beads.
experinaent are illustrated in Fig. 7; 62% of the anti-BSA was removed in 30 min, 80% in 60 min and 95% in 120 rain. The blood compatibility of the agarose-polyacrolein microsphere beads is summarized in Table 6; after 3 hours, the RBC count decreased by 2%, the WBC count by 7% and the thrombocytes by 14%,.
TABLE 6 Cell counts of the formed elements of rabbit blood pumped (12 ml/min) through agarose-polyacrolein microsphere beads (30 g) Time (rain)
RBC
WBC
Platelets
0 60 120 180
4,800,000 4,500,000 4,500,000 4,700,000
6,000 5,700 5,000 5,600
245,000 210,000 220,000 210,000
CONCLUSION The potential use of the agarose polymeric microsphere beads for hemoperfusion purposes was demonstrated. However, further experiments are needed before the beads will be ready for clinical trials. The blood compatibility of the beads, especially of the agarose polymercaptal microsphere beads has to be improved. This may be accomplished by a further coating of the beads. A study dealing with the influence of the beads on various blood constitutents such as enzymes, proteins and electrolytes has to be completed. Also, conditions have to be established for sterilizing the beads without damaging their specific activity.
REFERENCES 1 E.E. Muirhead and A.F. Reid, A resin artificial kidney, J. Lab. Clin. Med., 33 (1948) 896. 2 G.E. Schreiner, Role of hemodialysis artificial kidney in acute poisoning, Arch. Intern. Med., 102 (1958) 896. 3 J.L. Rosenbaum, G. Onesti and A.N. Brest, The rapid removal of cations from dogs with an ion exchange column, J. Amer. Med. Assoc., 180 (1962) 762. 4 A.S. Kissack, Jr., M.L. Geidman and K.E. Karlson, Studies with ion exchange resins, Trans. Amer. Soc. Artif. Intern. Organs, 8 (1962) 219. 5 T.F. Nealon, Jr., H. Sugerman. E. Shea and E. Fleegler, An extracorporeal device to treat barbiturate poisoning, J. Amer. Med. Assoc., 197 (1966) 158. 6 H. Yatzidis, A convenient hemoperfusion micro-apparatus over charcoal, Proc. Eur. Dial. Transplant Assoc., 1 (1964) 83. 7 H. Yatzidis, S. Voudiclari, D. Oreopoulos, N. Tsaparas, O. Triantaphyllidis, C. Gavaras and A. Stravroulaki, Treatment of severe barbiturate poisoning, Lancet, 2 (1965) 216. 8 C. Dunea and W.J. Kolff, Clinical experiences with the Yatzidis kidney, Trans. Amer. Soc. Artif. Intern. Organs, 11 (1965) 179. 9 T.M.S. Chang, Semipermiable microcapsules, Science, 146 (1964) 524. 10 T.M.S. Chang, F.C. Macintosh and S.C. Mason,
250
11 12
13
14
15
16
17
18
19
20
Semipermeable aqueous microcapsules, Canad. J. Physiol. Pharmacol., 44 (1966) 115. T.M.S. Chang, Artificial Cells, C.C. Thomas, Springfield, Ill., 1972. J.F. Winchester, R.O. Edwards, W.J. Tilstone and B.G. Woodcock, Activated charcoal hemoperfusion and experimental acetaminophen poisoning, Toxicol. Appl. Pharmacol., 31 (1975) 120. J.L. Rosembaum, M.S. Kramer and R. Roja, Resin hemoperfusion for acute drug intoxication, Arch. Intern. Med., 136 (1976) 262. J.D. Andrade, K. Kunitomo, R. Van Wagenen, D. Kastigir, D. Gough and W.J. Kolff, Coated adsorbents for direct blood perfusion: H E M A / activated charcoal, Trans. Amer. Soc. Artif. Intern. Organs., 17 (1971) 222. J.F. Winchester, M.C. Gelfand and W.J. Tilstone, Hemoperfusion in drug intoxication: clinical and laboratory aspects, Drug Metab. Rev., 8(1) (1978) 69. T. Barakat and I.W. MacPhee, Bilirubin and alkaline phosphatase clearance from blood-plasma by perfusion through activated carbon, Br. J. Surg., 58 (1971) 355. S. Sideman, L. Mor, D. Mordohovich, M. Mihich, O. Zinder and J.M. Brandes, In vivo hemoperfusion studies of unconjugated bilirubin removal by ion exchange resin, Trans. Amer. Soc. Artif. Intern. Organs, 27 (1981) 434. J.J.G. Perez, M.R. Rodriguez, A.T. Romirez, M.L.M. Perez, B.M. Cruz and J.B. Gomez, Hemoperfusion a traves de adsorbentes, Rev. Clin. Esp., 152 (4) (1979) 331. S. Margel, A novel approach for heavy metal poisoning treatment, a model. Mercury poisoning by means of chelating microspheres: hemoperfusion and oral administration, J. Med. Chem., 24 (1981) 1263. S. Margel, U. Beitler and M. Offarim, Polyacrolein
21
22
23
24
25 26
27
28
29
microspheres as a new tool in cell biology, J. Cell Sci., 56 (1982) 157. H. Losgen, G. Brunner, C.J. Holloway, B. Buttelmann, S. Husmann, P. Scharff and A. Siehoff, Large agarose beads for extracorporeal detoxification systems. I. Preparation and some properties and applications of the large agarose beads in haemoperfusion, Biomater., Med. Devices, Artif. Organs, 6 (1978) 151. S. Margel, Agarose-polyacrolein microsphere beads, new effective immunoadsorbents, FEBS Lett., 145 (1982) 341. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265. M. Heidelberger and F.E. Kendall, A quantitative study of the precipitin reaction between type III pneumococcus polysaccharide and purified homologous antibody, J. Exp. Med., 50 (1929) 809. S. Margel, A novel analysis of total inorganic and organic mercury, Israel patent pending, 1982. S. Margel, J. Hirsh and I. Harari, Hemoperfusion with chelating microspheres as a new treatment for severe mercury poisoning, Arch. Toxicol., in press. P.J. Kostyniak, T.W. Clarkson and A.H. Abbase, An extracorporeal complexing hemodialysis system for the treatment of methylmercury poisoning. II. In vivo applications in the dog, J. Pharmacol. Exp. Ther., 203(2) (1977) 253. S. Noy, J. Metanis, U. Taitlman, S. Bursztein, S. Yannai and K. Sachs, Chronic methylmercury poisoning, Harefuah, C(II) (1981) 518. F. Bakir, S.F. Damiuju, L. Amin-Zaki, M. Murtadha, A. Khalidi, M.Y. A1-Rawi, S. Tikriti, J.I. Dhabir, T.V. Clarkson, J.C. Smith and R.A. Doherty, Methylmercury poisoning in Iraq, Science, 181 (1973) 230.
241
STATE-OF-THE-ART REPORT
AGAROSE-POLYMERIC MICROSPHERE BEADS: SPECIFIC NEW ADSORBENTS FOR HEMOPERFUSION SHLOMO MARGEL
The Plastics Research Department, The Weizmann Institute of Science, Rehot:ot (Israel) (Received March 18, 1983: accepted in revised form June 16, 1983)
Uniform agarose-polymeric microsphere beads of 1 mm diameter were synthesi:ed by encapsulating appropriate polymeric microspheres in an agarose matrix. The potential use of these beads for hemoperfusion was demonstrated. Agarose provides the high porosity and the biocompatibility of the beads," the encapsulaled microspheres are used for the removal of harmful substances from the circulatory system. Two types of polymeric microspheres were prepared." (a) polymercaptal microspheres of 0.8 micrometer average diameter," (b) polyacrolein microspheres of 0.2 micrometer average diameter. Agarose-polymercaptal microsphere beads were designed for the removal of heacv metals by hemoperfusion. As a model, the detoxification of mercury compounds was studied. Agarose-polyacrolein microsphere beads were designed for the removal of antibodie's by hemoperfusion, especially in autoimmune diseases. As a model, the removal of anti-BSA from immunized animals was studied.
INTRODUCTION
Hemoperfusion is a relatively new approach for the removal of harmful substances from the bloodstream. This goal is achieved by circulating the blood through an appropriate adsorbent which adsorbs the harmful material. The "cleaned" blood is then returned to the patient. The ideal adsorbent for hemoperfusion has to fulfill many requirements, of which the major ones are the following: (a) blood compatibility, which ensures minimum loss of red blood cells (RBC), white blood cells (WBC) and thrombocytes; (b) high specificity, so that only the harmful material is removed from the blood while all the other 0167-6989,/83/$03.00
important components remain in the blood; and (c) high affinity and capacity toward the substance that has to be removed from the blood, so that the adsorbent size and the time required for the hemoperfusion will be minimal. The hemoperfusion process was initiated in 1948 by Muirhead and Reid [1] who studied the use of ion-exchange resins as adsorbents for the removal of uremic toxins from poisoned animals. Following this work, Schreiner (1958) [2] employed unsuccessfully a lactated anion-exchange resin column to remove pentobarbital from a poisoned patient. These studies with ion-exchange resins, as well as others [3], were complicated by pyrogenic re-
~ 1983 Elsevier Science Publishers B.V.
242 actions, electrolyte disturbances and hemolysis. These complications could be reduced by pretreating the ionic resins [4,5]. However, the early resins for hemoperfusion were considered too dangerous for widespread clinical practice. In 1964 Yatzidis [6] presented his in vitro research with activated charcoal particles, showing their efficiency for removing barbital, phenobarbital, pentobarbital and salicylic acid. Later, Yatzidis et al. [7] reported on the use of charcoal hemoperfusion on two patients with barbiturate poisoning who both recovered. However, many side effects such as platelet depletion, a reduction in fibrinogen concentration and particle embolization sensations were noted. The hemoperfusion problems with activated charcoal were partially solved by the pioneering work of Kolff [8] and Chang [9-11]. Chang and his group rendered the sorbent biocompatible by coating, sometimes combined with encapsulation. Subsequently, charcoal has been coated with albumin collodion [11], nylon, agarose and poly(hydroxyethylmethacrylate) [12- 14]. Since then hemoperfusion with either charcoal, ionic or non-ionic resins [15-18] has become a more common clinical tool. These adsorbents have been used for the removal of a wide variety of poisons, such as barbiturates, nonbarbiturate hypnotics, sedatives, tranquilizers, antidepressants, alcohols, analgesics, antimicrobials, anticancer agents, herbicides, insecticides, cardiovascular agents, bilirubin, and others. Problems involving biocompatibility, especially disruption of the formed elements of the blood, have not yet been overcome completely. Although obviously the patient risk-benefit ratio must be considered, under extreme conditions greater risks are warranted. Unfortunately, charcoal and ionic resins are non-specific adsorbents and they remove from the blood many classes of biocompounds in addition to the toxic material. We envisualise the synthesis of novel ad-
sorbents for hemoperfusion, based on the encapsulation of appropriate polymeric microspheres in an agarose matrix. We synthesized two types of uniform, encapsulated microsphere beads, both 1 mm in size: (a) A g a r o s e - p o l y m e r c a p t a l microsphere beads, designed for the removal of heavy metals by hemoperfusion. As a model the detoxification of mercury compounds was studied. (b) Agarose-polyacrolein microsphere beads, designed for the removal of antibodies from the circulatory system. As a model we studied the removal of anti-BSA from immunized animals.
EXPERIMENTAL
Synthesis of polymercaptal microspheres Polymercaptal microspheres with 0.8 micrometer average diameter were formed by the reaction of glutaraldehyde and pentaerythritol tetrathioglycolate in the presence of Tween 20 as surfactant, as previously described [ 19].
Synthesis of polyacrolein microspheres Polyacrolein microspheres of 0,2 micrometer average diameter were prepared by polymerizing acrolein in an aqueous solution containing polyethylene oxide as surfactant with a cobalt radiation source, as previously described [20].
Synthesis of agarose-polymeric microsphere beads Uniform beads with a diameter of 1 mm were prepared by a procedure similar to that described by Losgen et al. [21]. A molten solution of agarose (0.8 g) containing appropriate polymeric microspheres (4-6% w/v) was drawn into the glass syringe apparatus
243
Binding a spacer to the agarose-polyacrolein microsphere beads
[
The spacer polylysine-glutaraldehyde was bound to the beads as previously described [22]. Summarily, the procedure was as follows. The beads were shaken in H20 with polylysine for 24 hours at room temperature. The polylysine-conjugated beads formed were washed free of unbound polylysine by repeated decantation. The conjugated beads in H20 were then shaken for 12 hours at room temperature with glutaraldehyde and the resultant polylysine glutaraldehyde conjugated beads were filtered and then washed with large amounts of distilled water.
Motor driven I
Piston
Thermal jacket
Coupling of proteins to the agarose-polyaldehyde microsphere beads [22]
Syringe
[
J
Fig. 1. Glass syringe apparatus for the preparation of uniform agarose-polymeric microsphere beads.
shown in Fig. 1. The apparatus was kept at 70°C while stirring the agarose-microspheres aqueous suspension at 300 rpm. Thereafter, the gel was forced out by a motor-driven piston and was injected dropwise into a tall vessel containing an ice-cold solvent mixture of toluene, chloroform and hexane in the ratio of 10 : 4 : 2, respectively. The beads which were formed were separated from the solvent mixture in a crude sieve, washed several times with dioxane and finally cleared of solvents by exhaustive washing with distilled water. The beads were stored in the cold in the presence of 0.05% w / v sodium azide until use.
Beads (1 ml) with or without polylysine glutaraldehyde spacer were shaken at 4°C for 24 hours with the desired proteins (10 mg protein in 5 ml phosphate-buffered saline solution (PBS) at pH 7.2). The remaining aldehyde groups were then quenched by shaking the beads for 12 hours with 0.05 ml ethanolamine solution, brought to pH 7.2 with HCI. The immuno-beads formed were then washed successively with PBS. Rabbit anti-bovine serum albumin (antiBSA) bound to BSA conjugated beads was eluted from the beads with 0.2 M glycine-HC1 buffer solution at pH 2.4.
Determination of proteins Quantitities of protein bound to the beads were determined by subtracting unbound protein from total protein measured according to the method of Lowry et al. [23]. Quantities of antibodies bound and eluted from the inamuno-beads were determined by the quantitative precipitation reaction developed by Heidelberger and Kendall [24].
244
~~
GLASS COLUMN BLOODOR SERUM ,I AGAROSEENCAPSULATED BLOOD PUMP ~] ~MICROSPHERES //I ~ G L A S S DISCWITH /~1 0.Sin m PORES
II~
RESERVOIR
Fig. 2. Schematic diagram of the apparatus employed for the in vitro hemoperfusion experiments.
In vitro experiments A Travenol roller-type blood pump cycled the citrated blood (containing either mercury compounds or anti-BSA) from the reservoir
Fig. 3. Photograph showing the
through the column of agarose-polymeric microsphere beads at a controlled flow rate (Fig. 2). Samples were drawn from the reservoir and checked for the concentration of mercury or anti-BSA as well as for blood compatibility. In vivo experiments' Rabbits were anesthetized with ether via a nose cone. Oxygen was administered via a face mask. The carotid artery and jugular vein were cannulated with medical grade PVC tubing. The carotid artery cannula was connected to an arterial outlet set via a three-way stopcock. A blood pump cycled the arterial blood from the carotid artery through the column of beads. The outlet of the column was attached to a venous inflow set consisting of a bubble trap. The blood was returned to the rabbit via a three-way stopcock to the cannula in the jugular vein.
in vivo
hemoperfusion system.
245
The inline tee stopcock in the venous inflow set permitted intravenous infusion of saline or cysteine solution. The complete system was equilibrated with sterile heparinized saline (10 units per milliliter) before use. The complete set as well as the extracorporeal blood were temperature-controlled in a water bath at 38°C (Fig. 3). The rabbits were fully heparinized by intravenous injection of 300 units heparin per kilogram body weight and maintained in the heparinized state by constant heparin addition.
Blood counting Formed elements of the blood were counted in a hemocytometer and with a Coulter Counter.
Mercury analysis Mercury was determined by flameless atomic absorption spectrophotometry with a mercury analyzer of Perkin-Elmer and an additional kit developed in our group for the direct determination of mercury [25,26].
R E S U L T S AND D I S C U S S I O N Agarose was chosen as the encapsulator polymer since it fullfils certain essential requirements for hemoperfusion applications, such as bio- and blood compatibility and high porosity. The encapsulation by agarose of the water-insoluble polyacrolein or polymercaptal polymer is difficult to carry out. However, by substituting the polymer powder by a suspension of microspheres, the encapsulation becomes easier to accomplish. Figure 4 shows Fig. 4. (a) and (b): Light microscopy photomicrographs of agarose beads and agarose-polymeric microsphere beads, respectively. (c) and (d): Cross-section pictures of agarose polymercaptal microsphere beads (obtained
peripher
periphery
by light microscopy) and of agarose polyacrolein microsphere beads (obtained by transmission electron microscopy), respectively.
246 TABLE 1 Intake of mercury compounds by polymercaptal microspheres in the presence of alkali and alkaline earth metallic compounds a [Hg] (ppm)
Metallic compounds (M)
Microspheres (rag)
Hg intake (%)
Ca, Na and Mg intake
(%) 20 (Mercuric chloride) 20 (Mercuric chloride) 1 (Mercuric chloride)
20 (Methyl mercury chloride) 1 (Methyl mercury chloride)
0.001 CaCI2 0.15 N aCl 0.1 NaC1 + CaC12 + MgC12 0.15 NaC1 0.1 NaC1 + CaC12 + MgCI 2
15 15 10
100 100 - 95
0 0 0
15 10
100 - 95
0 0
a The mercury compounds were stirred with polymercaptal microspheres for 15 min in 20 ml of aqueous solution.
photomicrographs of the uniform agarose beads (a) and the agarose-polymeric microsphere beads (b). Cross-section pictures of the agarose-polymercaptal microsphere beads and of the agarose-polyacrolein microsphere beads, showing the encapsulated microspheres, are given in Figs. 4(c) and 4(d), respectively.
Detoxification of mercury compounds with agarose-polymercaptal microsphere beads The polymercaptal microspheres bind mercury compounds specifically and do not interact with alkali and alkaline earth metallic compounds, as shown in Table 1. The microspheres have high affinity towards mercury compounds and can easily bind mercury, which was previously bound to the biological mercury binder cysteine (Table 2). In vitro hemoperfusion experiments, including a kinetic study (Table 3), illustrate the potential of these beads for extracorporeal use in cases of severe mercury poisoning. Cysteine accelerates the removal of mercury from blood, especially in cases of organic mercury poisoning (Table 3). These findings are in agreement with previous publications [27,28]. The distri-
bution percentage of mercury compounds between the plasma and the red blood cells is approximately 85 : 15 for HgCI 2 and 8 : 92 for CH3HgC1 [29]. The accelerating effect of cysteine is probably owing to its diffusion into the red blood cells, binding the mercury and diffusing back to the plasma. The beads then bind the mercury from the cysteine-mercury complex. The in vivo kinetic studies describing the removal of CH3HgC1 from poisoned rabbits are shown in Fig. 5. The rabbits were dosed TABLE 2 Rate of intake of mercury compounds from cysteine a Mercury compound
Time (min)
Reaction (%)
Mercuric chloride Mercuric chloride Mercuric chloride Mercuric chloride Methyl mercury chloride Methyl mercury chloride Methyl mercury chloride
10 60 240 1320 10 60 240
95 97 99 100 91 91 91
a Mercury compounds (20 ppm Hg) were stirred for 2 h with 20 ml 10 -2 M cysteine in PBS. Polymercaptal microspheres were then added (20 mg in the reaction with methyl mercury chloride and 50 mg in the reaction with mercuric chloride).
247 TABLE 3 Mercury intake from poisoned blood by agarose-polymercaptal microsphere b e a d s - - e f f e c t of cysteine Time
Mercury removal (%) a
(h)
Without cysteine
With cysteine
CH3HgCI
HgC12
CH3HgCI
HgCI 2
18 27 63 89
l0 25 44 90
30 63 64 88
13 45 64 88
0.5 2 4 12
3 g beads were shaken with 20 ml blood poisoned with 2 ppm mercury in the absence or presence of 10 2 M cysteine; control experiments (agarose beads) indicate negligible intake of mercury.
with 8 mg CH3HgC1 (2 mg/kg). In the chronic poisoning case the hemoperfusion procedure was started two days after the intoxication. In the acute poisoning case the treatment was
7O---
T--
E ,~, 6.0 t-
.o
5C
~O9
40
I
I
I--
I
I
Arterial outflow
~"Venus inflow
0 cO
o
30
o
2Q
N
I.© ~
'
q[
©
40
~
~
i Venous inflow
80
t Chronic intoxicatJor l
[
120 160 200 2 4 0 - Time (rain)
Fig. 5. In vivo kinetics describing the removal of methyl mercury chloride from rabbit blood. O and e : arterial and venous mercury concentration, respectively, in a chronic intoxication case: a and i : arterial and venous mercury concentration, respectively, in an acute intoxication case. 8 mg methyl mercury chloride (2 m g / k g ) was injected intravenously: the hemoperfusion procedure started two days after intoxication in the chronic poisoning case and 70 rain after intoxication in the acute poisoning case.
started 70 minutes after the intoxication. The differences between the arterial lines and the venous lines indicate the intake of mercury by the agarose p o l y m e r c a p t a l microsphere beads. Mercury is equilibrated between the blood and the tissues; therefore the mercury concentration in the blood did not change significantly. The clearance of the blood by the beads is defined by the following equation: c l e a r a n c e = Q v ( C ~ - C , ) , where Q~ is the flow rate ( ~ g / m i n ) and C~ and C,. are the artery and venous mercury concentration (/xg/ml), respectively. Integration of the clearance over the hemoperfusion time, of' clearance dt, will show the amount of mercury removed by the hemoperfusion procedure. Calculations show that 330 yg Hg was removed after 3 hours of hemoperfusion in the chronic poisoning case and 1000 yg Hg after 130 minutes in the acute intoxication case (approximately 4.5% and 13%, respectively, of the body mercury burden). The hemoperfusion treatment has to start as early as possible. However, it is also reasonable to expect that repeated hemoperfusion treatments will remove the body mercury burden significantly. The blood compatibility of the agarosepolymercaptal microsphere beads was checked by counting the formed elements during the in ~ivo hemoperfusion experiment (Table 4). The RBC decreased by 25%, the WBC decreased by 33% and the platelet count decreased by 13%, during 3 hours of hemoperfusion treatment. TABLE 4 Cell counts of the formed elements of rabbit blood pumped (10 m l / m i n ) through agarose-polymercaptal microsphere beads (30 g) Time(min)
RBC
WBC
Platelets
0 60 120 180
4,000,000 3,870,000 3,350,000 3,000,000
1,500 1,150 1,000 1,000
173,000 162,000 1:51.000
248
The potential use of the agarose-polymercaptal microsphere beads for the removal of mercury by hemoperfusion was demonstrated. A further investigation will have to be carried out in order to evaluate the potential use of these beads for the removal by hemoperfusion of other heavy metallic compounds such as arsenic, cadmium, lead, copper and others. A model of the removal of antibodies by hemoperfusion with agarose-polyacrolein microsphere beads: removal of anti-BSA The polyacrolein microspheres encapsulated by the agarose are provided with aldehyde groups through which various ligands containing primary amino groups, e.g., proteins, can bind covalently to form the Schiff base products. Leaching of proteins bound to the beads is not detected owing to the formation of polyvalent Schiff base bonds. The binding capacity of the agarose-polyacrolein microsphere beads towards various proteins is illustrated in Table 5. Steric requirements may explain the significant increase (3-6 fold) in the binding capacity of the beads bound to the spacer polylysine-glutaraldehyde. In a hemoperfusion model system, circulating rabbit anti-BSA was adsorbed to BSA conjugated beads. In practice, any antigen may be covalently bound to the beads and the appropriate circulating antibody (especially in autoimmune diseases) may be removed. A
I
~0.4
I
I
I
I
I
1 I
l
I
I
I
|
I
I
I
I
-
~)0.3 m
~
0.2
O0
30 60
120
180
MINUTES Fig. 6. Kinetics pooled i m m u n e culated through sphere beads at
of adsorption of r a b b i t anti-BSA from r a b b i t serum. 100 ml serum was cir17 g BSA agarose-polyacrolein microa flow rate of 62 m l / m i n .
kinetic study of the adsorption in vitro of rabbit anti-BSA from immunized rabbit serum is shown in Fig. 6; 62% was removed in 30 min, 78% in 60 min, 91% in 120 min and 95% in 180 min. The same beads were reused several times following the elution of the rabbit anti-BSA, with similar results. The kinetic results of a similar in vivo hemoperfusion
TABLE 5 Binding capacity of a g a r o s e - p o l y a c r o l e i n microsphere beads to various a m i n o ligands a Ligand
Spacer
Binding capacity (mg)
BSA BSA Rabbit Rabbit Bovine Bovine
Polylysine-glutaraldehyde Polylysine-glutaraldehyde Polylysine-glutaraldehyde
2.2 14.8 1.0 10.0 7.2 22.0
immunoglobulin immunoglobulin insulin insulin
a 1 ml beads was shaken with an excess quantity of the various ligands in 5 ml PBS for 12 hours at room temperature.
249
0.6
E
]
1
l
0.5
~3n
E 0.4 <~
oo 0.:5 nq
F-
z<
0.2
F-
O.Im
<1[ rr
0'00
:50 6 0 9 0 120 150 180 MINUTES
Fig. 7. Kinetics of removal of rabbit anti-BSA from immunized rabbits. Circulation of immunized rabbits blood at a flow rate of 12 m l / m i n through a column containing 30 g BSA agarose-polyacrolein microsphere beads.
experinaent are illustrated in Fig. 7; 62% of the anti-BSA was removed in 30 min, 80% in 60 min and 95% in 120 rain. The blood compatibility of the agarose-polyacrolein microsphere beads is summarized in Table 6; after 3 hours, the RBC count decreased by 2%, the WBC count by 7% and the thrombocytes by 14%,.
TABLE 6 Cell counts of the formed elements of rabbit blood pumped (12 ml/min) through agarose-polyacrolein microsphere beads (30 g) Time (rain)
RBC
WBC
Platelets
0 60 120 180
4,800,000 4,500,000 4,500,000 4,700,000
6,000 5,700 5,000 5,600
245,000 210,000 220,000 210,000
CONCLUSION The potential use of the agarose polymeric microsphere beads for hemoperfusion purposes was demonstrated. However, further experiments are needed before the beads will be ready for clinical trials. The blood compatibility of the beads, especially of the agarose polymercaptal microsphere beads has to be improved. This may be accomplished by a further coating of the beads. A study dealing with the influence of the beads on various blood constitutents such as enzymes, proteins and electrolytes has to be completed. Also, conditions have to be established for sterilizing the beads without damaging their specific activity.
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21
22
23
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25 26
27
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29
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