CA2034641A1 - Homologous guluronic acid alginate coating composition for in-vivo application and implatation and method of using same - Google Patents

Abstract

Homoloqous Guluronic Acid Alginate Coating Composition for In-Vivo Application and Implantation and Method of Using Same Abstract Disclosed is a transplantation or implantation composition which provokes a reduced immune response comprising material encapsulated within a physical semi-permeable barrier. At least the outermost layer of the barrier contains alginate comprised substantially of .alpha.-l-guluronic acid with minor amounts .beta.-D-mannuronic acid.
The methods of making such a composition and use of the same are also disclosed.

Description

2~3~
DESCRIP~I_N

HomQl~o ous Guluronic Acid Alqinate Co~
Composition fo~ In-YlYo A~plication and Impl~nta 1~ Meth~d o~ ~s$n~ Same This is a continu~tion o~ co-pending application U.S.
Serial No. 07/446,462l ~iled December 5t 1989.
.
Backqround of the Invention Field of the Invention This invention relates to the ~ields of polymer chemistry, immunology and transplantation, and more particul~rly to the ~ield of materials for use in conjunction with transplantation and lmplantation of foreign cells and biological materlals.

A~t ~àckarou~
Evidence exists that transplantation of insulin-producing cells (islets) can cure the di~betic animal of the need ~or insulin therapy. The major obstacle prev~nting clinlcal success in islet transplantation 2s a therapy for diabetes to date has been immunogenicity of the cell and re~ection of the transplanted graf~.
Survival of islet allografts and or xenografts has been ~chieved by various methods o~ immunosuppression and/or related immunologiaal techniques. However, such tech-niques have had only limited ~uc~ess in that thetransplanted islet cell~ ~urvive only a ~hort while before re~ection occurs. In addition, the extended u3e of immunosuppressive agents Orten re~ults ln ~e~ere complications, ~uch as, renal damage and even a~ncer in the transpl~nt recipient.
One ~olution to this problem of gra~t re~ection is the introduction o~ ~ physical, s~ml-p~rmeable barrier between tha transplanted islets and th~ host' l~une system by the method of mi~roencap~lation.

~icroencapsulation is a proc~s in which small, discrete materials, viable biological tissue or cells, liquid droplets, or gases are completely enveloped by an intact membrane which is preferably compatible with the biological system in which it is placed. The function of the microcapsule ~embrane is to protect th~ ma~erial within from immunological recognition by the ~ost and to control the ~low of materials inside and outside the microcapsule acro s the membrane.
A large body of literature on microencapsulation has been produced including Darquy, S. and Reach, G.
~iabetologi~ (19 5) 28:776-780; Lim, F~ and Sun, A.
S~çnc~, (1980) 210:908-910; Lim, F. and ~OS5, R. J~uLnal f Eh~m~ceutiçaL Sc~enc~s, (April, 1981) 351- 354;
O'Shea, et al. ~iQ5hçmig~ e~ B~gih~ d~Q~ ~04 (1984) 133- 136; Leung, et al. ~r~ifici~l ~q~n~, (1983) 7(2):208-212; Araki, et al. iabetes, Vol. 34, September 1985, 250-85~; and U.S. Patent Nos. 4,690,682; 4,409,331;
4,391,90g, among others.
In addition to islet cells, other materials such as tissue, charcoal, microbial cells, yeasts, chloroplasts, plant protoplasts, mitochondria and enzymes have been immobilized and entrapped u~ing microencapsulation technique~.
Attempts have been made to tran~plant ~uch encapsulated material into a patient to perform th~
~pecific ~unction oS that material inside the r~clpi~nt patient. For example, activated charcoal could be used to detoxify blood, while pancr~atic ti sue could 6uppl~ment the patient's supply of in~ulin. ~QÇ~_~ g" Lim ~nd Sun ~1980) Saienc~ ~lQ, 908; ~'Sh~a, ot ~ 1984) Biochim.
Biophys. Acta ~Q~, 133.
While ~uch attempts have be~n partially success~ul, the patient's body o~ten reacts in ways that impair the activity of the microcapsules by fibroblast overg~owth o~
this substance by the body. ~ potential ~echanism ~or the induction of fibrobla~ts iB the ac~ivation Or macrophages, 6 4 ~

and the resultant ~timulation of cytokines by the capsule substance. Cytokines are molecule~ secrQted by the body in response to a new ~et of antigens, and ~re often toxic to the encapsulated cQlls. Some cytokin~s in turn ~timulate the immune system o~ the patient. Thus, immune response can still be a limiting ~actor in the Qff~ctive life o~ the encapsulated material.
In addition, ~ibroblast C~118 tend to ov~rgrow the microcapsules, also apparently in response to the newly released cytokines. Dinerallo, in LYMPHOKINES AND ~HE
IMMUNE RESPONSE (Cohen, ed. 1990) CRC Press, p. 156: Pi~la and Korn, in LYMPHOKINES AND THE IMMUNE RESPONSE ~Coh~n, ed. 1990) CRC Press, pp. 255-273. ~his growth of fibroblasts cause~ the microcapsules to lose their porosity. As a result, the cellular material inside the microcapsules cannot receive nutrients and the product of the cellular material cannot permeate the mic:rocap~ule wall. This can cause the encap~ulated living material ~o die, and can impair the e~fectiven~ss of tha microcapsule~
as a delivery sys~em.
Among the materials used. ~or encapsulation are calcium alginate gels. Lim ~nd Sun, in l9RO, ~;ucc~;sPully microencapsulated is}ets using al~inate gel, poly-L-ly ine and polyethyl~nimine. The encapsulated is12ts were injected intraperitoneally int4 diabetic rats. The animals' blood g}ucose levels dropped to normal for tWQ to three weeks, ~uggesting that the encapsul~tion procass had protQcted th~ islets from invasion by th~ r~cipionts' immune ~y~t~m. However, these ~tudies ahowed that the microcapsules were e~entually rej~cted ~s a r~uIt of fibrosis, or ~ibroblast ~ormation around ~h~ ~icrocapsule, which ~v~ntually r~stricts th0 rlow o~ nutrients to the cells contained in the microcapsul~ and the outflow o~
material from the microcapsulss created by the i61et cells disposed th~rein.
Alginat~, the principal material o~ the microcapsule~, is a heterogeneous group o~ linaar binary co~olymers o~ 1-4 linked ~-D-mannuronic ~cQd3~ and its C-5 epimer ~-L-guluronic acid (G). The monomers are arranged in a blockwise pattern along the polymexic rhain where homopolymeric regions (M blocks ~nd G blocks) are interspaced with seguences containing both ~onomers (MG
blocks). The proportion and sequential arrangem~nt o~ the uronic acid~ in alginate depend upon the 6pecies o~ algae and the kind of algal tissue ~rom ~hich the ~aterial is prepared. Various properties of diXferent types of alginates are based upon ~he guluronic acid makeup o~ the particular alginate. For example, viscosity depends mainly upon the molecular size, whereas the affinity for divalent ions essential for the gel-forming properties are related to the guluronic acid content. Specifically, two consecutive di-axially linked G residues provide binding sites for calcium ions and long sequence~ of such sites form cross-links with similar sequences in other alginate molecules, giving rise to gel networks.
Commercial alginates are produced mainly ~rom ~m~ari~ ~u~erbQ~ea, ~açrocystis pyri~e~a, minaria diqitata, Ascophyllum nodosum, minaria_i~p~nica, clonia maxima, ~sC~i3~n~L~e~s ~nd ~3ga~m_SD~
Additionally, alginates may be obtained from certain bacteria. ~o~bacter vinelandii produces Q-acetylated algin~te with a content of L-guluronlc ~cid ranging ~rom 15 to 90%. P~eudomonas aer~g~nQse under certain qrowth conditions produces poly-~nnuronic ~cid and such bacteria as well as cert~in other alginate producing Pseudomonades are not able to produce polymers containing G-hlocks.
Also, alginat~s having high or low conten~ o~ G or M residues may be obtained fro~ specl~ic portions of algal ti~sue. For ~xample, alginate having a high contant of gU1UrOniC aC1d maY be Obtained PrOm the OUter COrteX Of 01d 3~iPe~ Of L. hYP~r~O~. A1ginate haVing a high COntent Of ~ulUronic aC~d Can ~1BO be PrePared bY Ch~miCa1 fraCtiOnat~On ~r bY enZYma~iC mOdi~iCatiO~ US~ng mannUrOnan C-5 epimerase. ~his enZYme 1S able to ~46~

introduce G-blocks into an existing ~lginate polymer, producing polymers with high G-block content.
It is believed that alginate it~elf is one of the materials of the microcapsules which cau~es fibrosis, ~uch that attempted implanta~ion or tran~plantation af ~lginate encapsulated ~aterial i~ viable only ~or ~ ~hort term.
A measure of the poten~ial to caus~ ~ibro~is can be obtained ~rom the ability of certain subs~ancQs to induce cytokine produc~ion, including tumor necrosi~ factor-~
(TNF-~), interleukin~ l) and int~rleukin-6 ~IL-6).
These cytokines play an important role in immune responses and in in~lammatory reactions. These macrophag~derived mediators are Xnown to regulate fibroblast proli~eration (Libby et al., J. Clin. Invest. ~1986) 7~:1432; Vilcek et al., J. Exp. ~ed~ (1986) ~ :632). A possible mechanism for the fibrotic reaction to implanted microcapsules is the activation of macrophages, either by a contaminant within commercial alginate (e.g., polyphenols or lipopolysaccharides (LPS)), or by alginate monomers directly, with su~sequent release o~ cykokines responsible for ~ibroblast migration ~nd proliferation. LPS are known to stimulate ~he immune response~ Additionally, polysaccharides other than LPS have been reported to have an immunostimulating effect, including antitumor activity and stimulation of ~onocyte ~unction However, littl~ is known about the e~cts o~ polysaccharid~s on cytokine production ~rom monocytas.

~ he present invention provides a success~ul approach to microencapsulation and implantation which has not heretofoxe been discovered.
It i~ on~ object of thq pr~nt inv~ntion to provide a material which may b~ i~planted or tran~planted in YiYQ
which is non-i~munogenic and non-fibroblast inducing.

~ t is yet another ob~ect of the present invention to provide a microencapsulation system utilizing purified alglnate having a high guiuron~c acid content.
It is another ob~ect of the invention to provide a S microencapsulation system which does not include poly-L-lysine in its outer ooating and which eliminates the growth o~ fibroblasts around the microcapsules.
It is yet another object of the present invention to provide a microcapsule which does no~ induce tumor lo necrosis factor (TNF) or interleukin release and thereby prevents fibroblast ~ormation therearound.
It is another object of the present invention to pro~ide a microencapsulation ~ystem which i5 gubstantially nontoxic to the cells by minimizing the am~unt of poly-phenol in the alginate.
It is an object of the present invention to provide a composition and means for reducing production of cytokines such as TNF, IL-1 and I~;6 production in y1yQ.
It is anoth~r object of this inventicn to provide a composition ~nd means for preventing or trea~ing sepsis caused ~y TNF, IL-l and/or IL-6 production.
The present inven~ion pro~ides a material comprised of alginate, and particularly, alginate comprising primarily guluronic ~ci~, with minimal ~mounts of ~annuronic ~cid, the material being useful ~n ~ for implnnt~tion ~nd transplantation in mammali~n bodies. The materlal ~y take ~any ~orms, ~uch a~ ~heets, organ capsulation ~nd the like, but is pr~ferably used for ~ icroencapsul~tion o~ living ~ell~ and ti~sue which are foreign to the host in which th~y are i~plhnted. The present invantion ~l~o protects i~le~ cells or other transplanted tis~ue ~rom i~munologic~l cell re~ection.
The present invention also provides ~ ~icroencapsulation system w~ich.limits fibroblast overgrowth.
The present invantion al80 ~cts to depress production YiYQ cf cytokine~ 8u~h ~ TNF, I~-1 and IL-6. As such, it is useful as a pharmaceutical to inhibit the pro~uction --.

.

7 ~3~
o~ these cytokines ~or such purpo~e~ a~ the treatment of ~epæis, immune rejection, and inflammatory re~ponee.
Specifically, the present lnvention relate~ to enc~psulation of cell~ or other biological material with an outer coating of alginate which is completely or substantially ~omprised of guluronic acid and is completely or substantially fre~ of mannursnic acid.
Additionally, the same alginate can be used ~n various ~orms to inhibit cytokine production.

Brie~ ~es~crlption o~ ~he Drawinas FIGURE 1 is a ~raph ~howing the induction o~ TNF by Poly M, heterologous GMGM polymeric and Poly G alginates.
FIGURE 2 is a graph showing the dampening ePfect of induction of TNF by Poly G when combined with Poly M.
1~ FIGURE 3 is a graph showing the induction o~ IL-1 by Poly M, heterologous GMGM polymeric and Poly G alginates.
FIGURE ~ is a graph showing the dampening ef~ect o~
induction of IL-l by Poly G when co~bined with Poly M.
FIGURE 5 is a graph showing the induction of IL-6 by Poly M, heterologous GMGM polymeric and Poly G alginates.
FIGURE 6 is a graph showing thQ dampening effect of induction o~ IL-6 by Poly G when comblned with Poly M.

Det~iled Description of_the:~I~vention The present invention compri~es material which can be implanted or tran~pl~nted i~ iYP into mammal~ without induclng any substanti~l immunoganic r~action or fibroblast ~ormation. One important use for this ma~erial i8 ~or encap~ul~tion of biological ~ateri~ls, ~uch ~s in microcapsule~. The pre~ent lnvention i8 also a process for mlcroencapsulating biolog~cal cell~ and other material~ for u~e in implantation or transplantation RS a drug or biological mater~al delivery ~y~tsm. ~5 used herein, the term biologlcal material~ includes prokaryotic and cukaryotic ~ell~ which are either naturally occurring or genetically enyineered, drugs or pharmaceutical6, en2ym2s, parts o~ cells such as mltoc~Q~r~ ~nd protoplasts or any other naturally occurrlng sr synthesized material which may be implanted.
The material used ln the present invention is S alginate compri.~ed 6ubstantially o~ ~-L-guluronic acid (G) which may be referr~d to herein as guluronic acid. Small amounts of ~annuronic acid t~-D-mannuronic acid) (M) are also present. There are at le~t 65% G re~idues or ~ore, and pref~rably about 85% G residues and 15% or l~s M
residues. Algina~e so comprised ellc$t~ a ~ery low response ~rom monocytes in ~he production of tumor necrosis factor (TNF) and o~ IL-l and IL-6, which, as a result, does not elicit fibrosis.
~his high-G alginate is used in thP formation of microcapsules for the transplantation or implantation of for~ign ~at2rial into a mammal. The high-G alginate is used in place of at lea~t one layer of polyanionic polymer, such as low-G alginate, ln the microcapsule membrane. The high-G alginate can be used as the inn~r or first layer #urrounding the encapsulated ~aterial. In some microcapsules this may be the only layer surrounding the ~ncapsulated material. Additionally a gecond layer of polyanionic polymer can be used, oither immediately surrounding the gelled ~irst layer, or ~urrounding a second layer ~ade of a polycationic polymer.
Alternatively, tho polyanionic polymer layer can be u~ed as the second layer ~urrounding a first l~yer made of either a polycationic polymer or a base~en~ membrane.
Preferably, the outermost layer of polyanionic polymer is high-G alginate. Any other layer o~ polyanionic poly~er can al~o be composed of high-G alginater. Procedures ~or ~aking microcapsules are w~ll known in ~he ~rt and example6 are described in, ~or example, Lim and Sun, "Microenc~psula~ed islets as bio~rtl~iclal endocrine pancreas" ~lgDE~L 1980; 21:908: O'Sh~a et al, "Prolonged ~urvival of transplanted i~lets o~ Langerhsn~ ~ncapsul~ted in ~ biocompatible ~e~brane". Biochim ~iophys ~ 1984:

.. . . . . .. .-, 9 ~3~64~
804: 133; Goosen et al, ~'Optimization of microancapsulation parameters: Semipermeable~icrocapsules as a bioartificial pancreas": Bio~echnol ~ioena 1985; 27:
146; Sun et al "Transplantation of microencapsulated i~les S of Langerhans as a in~ulin deliver system" ~$~s~L_in Phar~aceutical $ciçnces 1985; 93; Tze et al, "Biocompatibility and immunological ~tudles of ~icroencapsulation with cross-linked alginate capsul~
rans~Lantatign 1982; 33; 563; Sun and Lim (1980) Science, ~lQ:908-9lo, Tsang et al. U.S. ~a~ent No. 4,663,286, and Rha et al. U.S. Patent No~ 4,744,933 tMay 17, 1988), whlch are incorporated herein by reference.
Matarial to be implanted or transplanted is first coated with a negatively charged material such as algina~e to form a gel coating therearound, as described above.
Alternatively, the cellular material is coated with a tissue basement membrane such as Matrigel (Collaborative Rese~rch, Inc., Bed~ord, Massachusetts). T~e ~olubilized tissue basem~nt membrane contains pro~eoglycans, collagen, and laminin and/or intactin. By use of the Matrigel, the proteoglycan ~orm5 a matrix which permits transfer of selected materials inside and out of the cell~. Further, the use o~ the tissue ba~ement membrane eliminates the need for a gelling and then reliquification of the gel material ~urrounding the cell. Additionally, it is believad that the ti~sue basement me~brane provides both struc~ural elements supporting the cell~ and al50 mediates variou~ ~ignificant ~ellular events and cellular runctions in~luding prolifera~ion and di~erentiation. Thus, by incorporation of the ~tructural element~ of the extracellular matrix of the ti~sue bagement membranQ or ~n equiv~lent thereof into a micro~ncap~ulated bioenvironment, the viabllity and functionality o~ the immuno-isolated islet cells is enhanced. This environment not only ~nhances ~slet recovery but the microencapsulation process also contributes to the long term viability and function o~ the islet cells. ln the .. , .... _, . _ . . _ _ . .. , . _ _ . . .

203~

preferred embodiment, the molecular weight of the proteoglycans used as the ti~sue basement ~embrane have a molecular weight of 200,000 to 300,000 Da~tons.
Following coating o~ the transplantable material with S the tissue basement membrane matrix or an equivalent thereof, ~he transplantable material ls next coated with a positively charged material such as poly-L-lysine. The use of poly-L-ly~ine ~s a coating material i8 well-~nown in the art and is described in various re~erences ref~rred to above. lt has been Pound, however, that the use of poly-L-lysine having a molecular weight of less than 20,000 is particularly advantageous in that it mi~imlzes the induction of fibroblast formation. Alternatively, a positively charged polysaccharide ~uch as chitosan may be used as the second layer o~ the coating.
As a third and outer ~oating layer, the positively charged ~econd layer is coated with a layer of alginate of the present invention comprising high G content, greater than 65% and preferably greater than 85% G residues, as described above. However, it should be noted that any minimization of mannuron~c :~cid re~idue~, with the eguivalent increase in G resldues in the alginate, is contemplated by the pre~ent invention and will enhance the decrease o~ ~lbro~is.
For ~n vivo applications of the present invention, the composition comprising ~lginate havln~ a high G
content may be u~ed in the ~orm of organ capsulat~on, sheets of algin~te ~or i~pl~nt~tiont hollow ~iber~, microcapsule ~nd membranes ~ormed of the ~ub~ect compositio~.
In accordance with these ~indin~s, the pre~ent inve~tion compri~es the use of Poly G alginate as a coating material ~or any biological materials which may be lmplanted or tran~planted. The materi~l to be implanted or transplantad can include livin~ ti~ue, llvlng cell~, actlvated charcoal, or any other material o~ the approprlate ~ize whl~h is u~ful when transplanted within 11 2~ 4~
immunoisolation systems. Material to be transplanted is pref~rably chosen ~or its ability t~ ~unction in ViYo when transplanted into a recipient body. Living cells can include, but are not limit~d to, islets of Langerhans cell~ which can produce insulln, hepatic cell3 or liver tissue, and red blood c~lls. ln particul~r, cell ~uch as islet cells ~rom the ~slet~ of L~ngerhans may be puri~ied in accorddnce with co pendinq application Seri~l No.
232,328 or by other methods known in the ~rt.
Experim~nts were run to test the efficacy of high-G
alginate in microcapsul~s. High-G alginate was used to make microcapsul~s that were tested ~or cellular re~ponse.
~hese responses were compared to responses elicitçd by alginates with a vsriety of compositi~ns: high-M
alginate, alginate with continuous blocks predominantly composed of M (M blocks), and alginate with continuous blocks predominantly composed of ~ (G blocks). Induction of various cytokines were tested, including TNF, IL-l and IL-6, as a means of monitoring the immune respon~e to the various alginates.
Microcapsules with at least one outer layer composed of high-G alginak0 werQ found to ~licit the lowest amounts of the Yarious cytokines. See Figures 1-6. As a result, . such microcapsules would b~ the most ef~ective for implantation or tr~nsplantation of ~aterial into a ma~malian body, becau~e they would ~inimize the i~mune responsa by the body to the ~icrocapsulss.
~ or use in ~his invention, alginate can be prepared according to ~ethod~ well known ~n ~he art. For ~xample, alginate can be commercialy obtAined fro~ num~rous ou~lets including Sigma ~St. Louis, M0) and Protan ~/S (Drammen, Norway). Poly G alginate ~ay be obt~ined ~rom Protan (Norway or Sea~tle), or may be obtained by isolation of ~he material ~rom natural sources or by chemical 35 conversion by method~ reported ~ n the litera~cure. Some alginate i~ relatively high in M re~idues and must be converted to low M ~or u~ in thi~ inv~ntion. An ~xz~mple .

_. , of a procedure which can be used for reduclng ~h~ lçve~ of M in alginate follows.
Another use ~or the composition of this invsntion is to inhibit ln ViyQ production of cytokines such a~ TNF, S IL-l and IL-6. ~s Fl~ures 2, ~, and 6 show, the high-G
alginate depresses production of both of these cytokines.
Thes~ cy~o~ines play a role ln a number of di ea e ~tates, including but not limited to inflam~atory response, kransplant rejection, and sepsi~. There~ore, this hlgh-G alginate composition can be adminlstered t~ treat suchdisease state~. The concentration of high-G alginate useful for such treatment is shown in Figures 2, 4 and 6, Table 1, and described in Examples 5 and 6, below.

Example 1 Alqinate P~epara~lon Commercial alginate from the algae Laminaria hyperborea ~LF 10/60, batch nr. BL 5417368) containing 64%
guluronic acid residues was obtained from Protan A/S, Drammen, Norway. LPS contamination in the algina~e was removed by the method described by Karplus et al. ("A new method for reduction of endotoxin cont~mination ~rom protein solut~ons"s ~A~ Y~ ~9~ (1987) ~Q~ 211) u~ing a combination of 2Olymyxin-B-sepharose 4B (PB-seph 4B) tPharmacia, Uppsala, Sweden) affinlty binding and endotoxin-protein dis~ociation with the dialyzable sur~actant octyl-0-D-glucopyrano~ide OBDG, slgma, St.
Louis, MO, USA).
Briefly, 1% (w/v) OBDG was added ~o 1% tw/v) LF 10/60 ~olution (dis~olved in elution buf~r con~s~ing of Na~CO3 pH R.5), and mixed for 30 min. 2t xoom temp¢r ture. Equal volume6 of the PB-Seph 4B gel and OBDG/alginatQ ~olution were mixed and trans~erred to a dialysis bag (MW
12-14000). The bag was then placed in a container with phosphat~ buffered saline (P~S) and dialyzed for 48 hours at room temperature. Subsequently, the PB-Seph 4B-gel was removed by centrifugation at 2750 r.p.m., ~or 10 min. at ......... , ~, ... . .. . .. .. .
. .

13 ~ 6~
4 C. 0.2% NaCl (w/v) was added to the alginate solution and the alginats w~ precipitated with 96% ethanol. The alginate was then washed twice with 96~ ethanol and finally once with 96% ethanol and once wi~h diethylether before it was dried. This alginate i3 rererred to herein as poly-G alginate or G-block 41ginat~.
For studies comparing the e~fe~t of hlgh M and high G alginate, as well as ~lginates having M blocks and G
blocks, on cellular respon~es, variou~ ~ype~ o~ alginate were obtained as follows:
M-block alginate (95~ M ~nd degree Or polymerization ~DPn) = 35) was obtained ~rom an ~lginate enriched mannuronic acid isolated from the intracellular substance of ~scophyllum nodosum (~.nodosum) fruiting bodies as des~ribed by Haug et al. ("Correl~tion between chemical structur~ and physical properties of alginates" ~çt~.~hem ~~n~ 1967:21:768).
Alginate fraqments containing ~ore than 85% of ~
units and DPn-40 (G-blocks) were prepared from min~ria ~lgl~3~ Alginate ~ragments with predominantly an alternating structure, MG-blocks (63% M and DPn~2~) were isolated from & nodosum by the ~-thod described by Haug et al. ~"Studies on the sekvens of uronic acid re~idues in alginic ~cid 1967:21:691).
An alglnate ~ample w~th a lower content o~ guluronic ~cid reslduec (46~) was i~olated ~rom ~i~sues: o~
n~do~um ~ de~cri~ed by Haug ~t ul.
~he monomer co~position and ~equential ~rrangement as well a~ the DPn were analyzed by~H-n.~.r. ~pectroscopy on a Bruker 400 WM ~pectrometer e~ deRcrib~d pr~viously by Gra dalen ~t ~1. tNA p.m.r. ~tudi~ o~ composition and sequence of uronat~ re~idues in alginate~; Carbohydr Res 1979: 68:23).
Endotoxin content in the puri~ied and unpurified algin~t~s wa~ quanti~ied by the LA~-assay (Coatest Endotoxin from Kabi Vitrum, Sto~khol~, Sw~den).

~4 Exam~le 2 20 3~641 M~nocyte Cultivation Monocytes were isolated from human A~ blood buffy coat ~The Bloodbank, UniVersity of Trondheim, Norway) as describe~ by Boyum ("Separation of ~onocytes and lymfocytes~" Soan ~ lmmunol 1976 5:9). ~onocytes in 24 well culture pla~es (Costar, Cambridge~ MA, USA) Were cultured in complete medium sonsist$ng of RPMI 1640 (Gibco, Paisley, U.K.) with 1% glutamine, 40 mg/ml garamyc~n and 25% A- serum. (The sloOdbank, u~iversity o~
Trondheim)~
The alginate, the M blocks, the G-blocks and the MG
bloc~s were dissolved in PBS and sterile filtered through 0.2 ~m filter (Nuclepore, Pleasanton, CA, USA). Commercial unfiltered alginate was sterilized by autoclaving. The polysaccharide solutions were diluted in complete medium and added to the monocytes for 16-24 hours before the supernatan~s were harvested. El__gQ~ rain 026:06) derived ~PS tSigma), or b/1-3 ~ polyglucose (obtained from Professor R. Seljelid, University of Troms0, Norway) were added to some monocyte cultures.
Alginate g~ls were made in 24-well ~ulture plates (Costar) by adding 0.5 ml 10 mg/ml ~terile filtered al~inate solution ln the wells. Then 1 ~l of 0.1 M CaCl2 was added for lO min. before the supernatant over the gel was removed. The qels were then washed twice with 1.5 ml saline, and fin~lly twice with complete ~edium. Human monocytes at a concentration o 0.5 x lOh cells/well were added to the alginate gel~, and the plate was incubated for 16-24 hours be~ore the supernatants were harvested.

xol}PL~
As~ay fo~ Dete~tion ~o~. T~F~ S.upernatants F~om Mon~cvtes Amount of TNF-~ was determined by its cytotoxic ef~ect on the fibrosar~oma cell line WEHI 164 clone 13, as described in Espevik et al (IlA highly ~ensitive cell line, 2~3~41 WE~II 163 clon~ 13, for measuring cy~otoxic factor/tumor necrosis factor from human monocytes~" I~mun~l Methods 1986; 95:gg.) Dilutions of r~combinant TN~ r-TNF-~, Genentech, Xnc. South San Francisco) were included a6 a standard. Th~ TNF-~ specificity o~ the a~say was veri~ied by a monoclonal ~ntibody aga~nst rTNF-~ which compl~tely neutrallzed the re~ord~d activity (data not ~hown).

Assay for Detection of I~-1 in Supernatants From Monocytes Amount of IL-1 was deterMined by a two stage assay.
The first stage involves the mousP thymocyte EL-4 NO~ I
cell line which produces high concentrations of IL-2 (interleukin-2) in response to human IL-1, a~ described by Gaering et al. Dilutions o~ r-IL-1 (Glaxo, Geneva, Switzerland) were included as standard. A~t~r incubation in CO2 for 24 hour~, 100 ml of each o~ the upernatants were tran~ferred into replicate 96-well microplates. The second sta~e in this assay involves the IL-2 dependent mouse T cell line HT-2 as described by Mosmann, T. ("Rapid colorimetric assay for cellular growth and ~urvival:
Application to proliferation and ~ytotoxicity assays.l' J.
lmmunol 1987; 139:4~16). One hundred ml of ~T-2 suspension (1.2 x 105 cells/ml were added to each well and incubated for an additional 24 hours. The IL-l activity was compIetely neutralized by two polyclonal antibodies again~t rIL-lb. R~sult~ ars present~d as pg~ S.E.
of triplicat~d d@termination~.

ExamplQ 5 Viability_Usinq ~TT-a~say L~(4.5-dim~thyl~h~zQl-2~yl~-?.5-dlphenvltetrazolium bro~id.e) Vlability in the assays for TNF-~, IL-1 ~nd ~L-6 was meaQured in a colorimetric assay for growth ~nd ~urvival by uslng a tetrazolium ~alt a~ deqcribed by Mosmann, u~r~.

. . . .... . . .... .. .... . .. ... . . ..... . . ......... ............. . .
~.. ` ,.

~3~

As shown in Figur~ 1, three separate alginate compositions were tested for their ability to induce monocytes to rela~se TNF. The algin2t~ compositions included Poly G alginate, heterologous GMGM alginata compri6ing linear binary copolymer~ of 1-4 linked ~-D-mannuronic acid (~) and its C-5 epimer ~-L-guluronic acid (G), and Poly ~ D-mannuronic ~cid) alginate. The ~oregoing ~hree types o~ alginate material are re~renced in Figures 1 through 6 ~s Poly G, GMGM and Poly M. The lo algina~es were di~solved in tis~ue culture mediu~ in varyinq concentrations set forth in Figure 1, 3 and 5 in which equal concenkrations of monocytes were placed.
Figure l shows that Poly M and GMGM alginate induced substantial TNF production by the monocytes on the order of 7000 to 10,000 picograms of TNF per ~illiliter, whereas Poly ~ alginate induced ~F production ~wo orders of magnitude less, or at ~pproximately 200 pg/ml of TNF. ~NF
is known as an induoer of fibrobl~3t growth. Figure 3 shows the equivalent result~ with r~spect to I~-l produc~ion by the monocytes. Figure 5 ~hows the equivalent results with respect to IL-6 produ~tion.
As shown in Figure 2, Poly ~ apparently lnhibits the production of TNF by monocytes. Flgure 2 shows the results of an experiment in which Poly M and Poly M pIus 2s l mg/ml of Poly G w~s added to a culture of monocytes and the TNF production was measured. As can be ~een from the graph, the Poly M pluc Poly G sample lnduced ~ubstanti~lly lower TNF production than Poly M alone. Thus it appears that Poly G not only has very ll~ited TNF induction capability, it also inhibitc ~oly M alginate~ abili~y to induce TNF production o~:~onocytes, and nccordlngly, would inhibit Poly M alglnate lnduction o~ ribrosis. Figure 4 shows the equivalent results with respect to IL-I
production by ~he monocytes. Figure 6 shows the equivalent results wIth r~ pect to I~-6 production.

, 17 2~3~6~
Exam~ 6 GrQwth o~ Monocytes on Alg~a~
Table 1 ~ows the result~ o~ ~n experiment which demonstrates cytokine relea~e from ~onocytes cultured on alginate gels. Monocytes on ~issue culture pl~tes were detached by a rubber police~an, washed once in Han~s Balanced Salt Solution, and added to cultur~ wells with alginate g21, or ~ulture wells w~th LPS or grow~h ~edia.
Algina~e gels w~re made as describçd above. Supernatants were harvested a~ter 16-24 hour~ and as~ayed for TNF, IL-6 and IL-1. As can be seen ~rom the table, the monocytes culturad on L~ 10/60, which has a 64% G residue content, induced substantially less production of eac~ of TNF, IL-l and IL-6 compared with A. nodosum algina~e gel, which has a G residue content of 4~%. LPS also showed a great capacity to induce cytokine production.

Table l CYTOKINE RELEASE FROM MONOCY~ES CUL~URED ON ALGINATE GELS
20 ¦Treatment ¦TNF-~(Pg/m1) ¦IL-6 (pg/ml) ¦IL-1 (pg/ml)¦

¦LF 10/60 ¦¦7000~1-1100 ¦10900~1-1600¦6400+1-100 alginate gal¦¦
25 ¦ A. nOdOBUm ~ 15600+1-5300 ¦ 15200~1-2000 ¦ 16300~1-800 ¦
¦alginate gal ll ¦1 ~g/~l LPS ¦¦12400+1-2600¦22200~1-5100¦9600+1-900 ¦Growth ¦¦50+1-10 ¦70+1-20 ¦90+1-10 ¦Medium ll .. . . _ ~, . . ... , . ____. . .. _ ._ ~ ,_ . ~, _.. _ ._ . ... __, _ _ .
,:

: ' , 18 2~3~41 ExamP1~-7 M~croencapsulati,on Q ~ sle~s o~_ ~ nqÇ~a~s Cultured rat islets o~ ~angerh~ns (2 X 103 islets i~
O.2 ml medium) w~re suspended uniformly in 2 ml of a 1.5%
(w/w) sodium alginate solution (~iscosity 51 op ) in phy6iological salins. Spherical droplets containing i~lets were produced by syringe pump/air ~e~ Qxtrusion through a 22-gauge needle and coll~cted in 1.5% (w/w~ ~alcium chloride solution. ~he ~upexnatant w~s d~canted a~d the gelled spheric~l alginate drople~, containing isl~t~, were washed with dilute CHES (2-cyclohexylamino-ethane sulfonic acid) solution and 1.1% calcium chloride solution.
After a~pir~ting off the supernatant, the gelled droplets were incubated ~or 6 minutes in 0.05~ (w/w) polylysine having a molecular weight of 17,000.
The supernatant was decanted and the polylysine capsules were washed with dilute CHES, 1.1% calcium chloride solution and physiological saline. The washed polylysine capsules were incubated ~or 4 ~inutes ~n 30 ml of 0.03% ~odium alginate to p~rmit the ~ormation of ~n outer alginate membr~ne on the initial polylysine membr~ne, by ionic interaction between the negatively charged alginate and the positively charged polylysine.
The alginate used in the outer and inner coating is poly G alginate produced ~8 described above.
The result~ng ~icrocapGules were wa~hed with fialine, 0.05M citrate buffer ~or 6 ~inutes ~o reliqui~y the ~nner caleium slginat~, ~nd washed again wlth ~aline. The mi~rocapsules were ~ound ~o be per~ec~ly spherical and each to contain fro~ 1 ~o 2 viable isletæ. ~he microcapsules had diameters of 700 + 50 ~ and wall thicknecses of about 5 ~m. The microcapsules were suspended in nutrient ~edium at 37~C.
It will be obvious to a person of ordinary ~kill in th~ art that the pre~ent invention is not.limited in i~s application to ~pecific biological ~ater1als to be .

.

19 ~ 6~
encapsulated, 6uch as the islet cells described in detail ~bove, or by the speclfically described other inner layers of ~icrocapsule discussed herein. It will also be appreciated that the 6ubject invention i8 not limited to only three layer microcapsules, but that two layer capsules may also be employed using the high G content alginate of the present invention, and if desired, the low molecular weight poly-~-lysine (below 20,000 dal~ons).
The only limit~cion~ of the pre~en~c invention are set forth in the claims appended hareto and any equivalents thereo~.

': .

Claims (37)

1. A transplantation or implantation composition which provokes a reduced immune response comprising material encapsulated within a physical semi-permeable barrier comprised of alginate that is greater than 50% .alpha.-1-guluronic acid.

2. The barrier of claim 1 wherein the alginate is at least 65% .alpha.-1-guluronic acid.

3. The barrier of claim 2 wherein the alginate is at least 85% .alpha.-1-guluronic acid.

4. The transplantation or implantation material of claim 1 comprised of purified islet capable of producing insulin.

5. The barrier of claim 1 selected from the group consisting of microcapsules, hollow fibers, and vascular prostheses.

6. The alginate of claim 1 containing minor amounts of .beta.-D-mannuronic acid.

7. The alginate of claim 1 containing minor amounts of hyaluronic acid.

8. The barrier of claim 1 comprising alternating layers of polyanionic polymer and polycationic polymer, wherein the outermost layer is composed of polyanionic polymer.

9. The barrier of claim 8 wherein the polyanionic polymer is alginate.

10. The barrier of claim 8 wherein the polycationic polymer is selected from the group consisting of poly-L-lysine and chitosan.

11. The barrier of claim 10 wherein the poly-L-lysine has a molecular weight of less than 20,000 daltons per molecule.

12. A method of encapsulating material to be transplanted or implanted in a manner that provokes a reduced immune response comprising coating said material with at least one layer of alginate, said alginate being more than 50% .alpha.-1-guluronic acid.

13. The method of claim 12 wherein the alginate is at least 65% .alpha.-1-guluronic acid.

14. The method of claim 13 wherein the alginate is at least 85% .alpha.-1-guluronic acid.

15. The method of claim 12 wherein the transplantation or implantation material is comprised of purified islets capable of producing insulin.

16. The method of claim 1 wherein the barrier is selected from the group consisting of microcapsules, hollow fibers, and vascular prostheses.

17. The method of claim 1 wherein the alginate contains minor amounts of .beta.-D-mannuronic acid.

18. The method of claim 1 wherein the alginate contains minor amounts of hyaluronic acid.

19. The method of claim 12 wherein the barrier is comprised of alternating layers of polyanionio polymer and polycationic polymer, wherein the outermost layer is composed of polyanionic polymer.

20. The method of claim 19 wherein the polyanionic polymer is alginate.

21. The method of claim 19 wherein the polycationic polymer is selected from the group consisting of poly-L-lysine and chitosan.

22. The method of claim 21 wherein the poly-L-lysine has a molecular weight of less than 20,000 daltons per molecule.

23. An alginate composition for in vivo application in mammals comprising at least 65% .alpha.-L-guluronic acid.

24. The alginate composition of claim 23 comprising at least 85% .alpha.-L-guluronic acid.

25. A method of treatment of diseases caused or exacerbated by cytokines comprising administering an alginate composition comprising at least 65% .alpha.-L-guluronic acid.

26. A method of treatment of diseases caused or exacerbated by a cytokine selected from the group consisting of TNF, IL-1 and IL-6, comprising administering An alginate composition comprising at least 65% .alpha.-L-guluronic acid.

27. The method of claim 25 or 26 wherein the alginate composition comprises at least 85% .alpha.-L-guluronic acid.

28. The method of claim 25 or 26 where the disease state is selected from septic shock, transplant rejection, and inflammatory response.

29. A method of inhibiting in vivo production of cytokines in mammals comprising administering an alginate composition comprising at least 65% .alpha.-L-guluronic acid.

30. A method of inhibiting in vivo production in mammals of cytokines selected from the group consisting of TNF, IL-1 and IL-6 comprising administering an alginate composition comprising at least 65% .alpha.-L-guluronic acid.

31. The method of claim 29 or 30 wherein the alginate composition comprises at least 85% .alpha.-L-guluronic acid.

32. A composition which inhibits in vivo production of cytokines in mammals comprising an alginate composition comprising at least 65% .alpha.-L-guluronic acid.

33. A composition which inhibits in vivo production of cytokines selected from the group consisting of TNF, IL-1 and IL-6 in mammals comprising an alginate composition comprising at least 65% .alpha.-L-guluronic acid.

34. The composition of claim 32 or 33 wherein the alignate composition comprises at least 85% .alpha.-L-guluronic acid.

35. A method of using an alginate composition comprising at least 65% .alpha.-L-guluronic acid to suppress inflammation caused by production of cytokines in mammals.

36. A method of using an alginate composition comprising at least 65% .alpha.-L-guluronic acid to suppress inflammation caused by production in mammals of a cytokine selected from the group consisting of TNF, IL-1 and IL-6.

37. The method of claim 35 or 36 wherein the alginate composition comprises at least 85% .alpha.-L-guluronic acid.