USOO64951.61B1 (12) United States Patent (10) Patent No.: US 6,495,161 B1 Soon-Shiong et al. (45) Date of Patent: Dec. 17, 2002

(54) CYTOPROTECTIVE BIOCOMPATIBLE 5,545,423 A * 8/1996 Soon-Shiong et al...... 424/484 CONTAINMENT SYSTEMS FOR 5,700,848 A 12/1997 Soon-Shiong et al...... 522/7 BIOLOGICALLY ACTIVE MATERIALS AND 5,759,578 A * 6/1998 Soon-Shiong et al...... 424/484 METHODS OF MAKING SAME 5,788.988 A * 8/1998 Soon-Shiong et al. ------424/484 OTHER PUBLICATIONS (75) Inventors: Patrick Soon-Shiong, Malibu; Neil Desai, Los Angeles; Nilesh Ron, Culver Lim and Sun, “Microencapsulated Islets as Bioartificial City; Andrew Sojomihardjo S., West Endocrine Pancreas,” Science, 210:908-910 (1980). Covina; Roswitha Heintz, Los Angeles; * cited b Francesco Curcio, Westlake Village, all cited by examiner of CA (US) Primary Examiner Dameron L. Jones (74) Attorney, Agent, or Firm-Stephen E. Reiter; Foley & (73) Assignee: VivoRx, Inc., Santa Monica, CA (US) Lardner (*) Notice: Subject to any disclaimer, the term of this (57) ABSTRACT past l5SSh i. adjusted under 35 In accordance with the invention, there are provided a -- y yS. methods, capsules, and delivery Systems useful in preparing biological containment Systems with properties (e.g., (21) Appl. No.: 09/264,187 mechanical Strength, capsule permeability and porosity, (22) Filed: Mar. 9, 1999 desired controlled release rates of the biologic or compo nents Secreted by the biologic, and immunoreactivity) that (51) Int. CI.7 ------A61 K 9/48 Ca be varied to adapt to a broader range of physiological (52) U.S. Cl...... 424/451; 424/450 conditions than known biological containment Systems. (58) Field of Search ...... 424/450, 451, There are also provided methods of making capsules con 424/489, 455, 456, 459, 460, 463, 461, taining cell aggregates therein, as well as the capsules 462, 484 formed thereby, which are useful as a quantitatively plentiful and low cost alternative to usage of freshly harvested cell (56) References Cited aggregates (e.g., islets from pancreas), Since the latter are U.S. PATENT DOCUMENTS usually available only in limited numbers. 4,352,883 A * 10/1982 Lim ...... 435/178 26 Claims, No Drawings US 6,495,161 B1 1 2 CYTOPROTECTIVE BOCOMPATIBLE employed, imperfections in the microcapsule membrane CONTAINMENT SYSTEMS FOR (allowing exposure of poly-L-lysine to the in vivo BIOLOGICALLY ACTIVE MATERIALS AND environment), failure of the microcapsule membrane to METHODS OF MAKING SAME completely cover the cells being encapsulated (thereby allowing exposure of the cells to the in Vivo environment), and the like. FIELD OF THE INVENTION Accordingly, there is a need in the art for new and better The present invention relates to new forms of biocom capsules for the encapsulation of biologically active mate patible containment Systems that envelop encapsulated or rials. In addition, there is a need for new methods of making free cells or other biologically active materials. In a particu capsules that encapsulate biologically active materials while lar aspect, the present invention relates to a System that permitting variation of certain properties (e.g., mechanical provides an immune barrier for the cells or other biologi Strength, capsule permeability and porosity, desired con cally active materials. In another aspect, the present inven trolled release rates of the biologic or components Secreted tion relates to a System that provides enhanced migration by the biologic, and immunoreactivity) across broad perfor and aggregation of the cells or other biologically active 15 mance ranges to address variable physiological conditions. materials within the containment System. In a further aspect, Further, there is a need for new methods of facilitating the present invention relates to a System that provides formation of and delivery Systems for cell aggregates. enhanced transfer of the Secretions of cells or other biologi cally active materials out of the containment System. BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, capsules (e.g., BACKGROUND OF THE INVENTION microcapsules and macrocapsules) have been developed for Microencapsulation of cells (e.g., pancreatic islets) by an the encapsulation of biologically active materials therein. alginate-PLL-alginate membrane (i.e., an alginate-poly-L- Invention capsules comprise at least one biocompatible lysine-alginate membrane) is a potential method for preven gellable material, wherein at least the outer layer of the tion of rejection of foreign cells by the host's immune 25 capsule is covalently crosslinked and optionally polyioni System. By this technique, researchers are able to encapsu cally crosslinked (or, in the case of macrocapsules compris late living islets in a protective membrane that allows insulin ing microcapsules therein, either polyionically crosslinked, to be Secreted, yet prevents antibodies from reaching the covalently crosslinked, or both polyionically croSSlinked islets, causing rejection of the cells. This membrane (or and covalently crosslinked), but not ionically crosslinked. microcapsule) protects the islet from rejection and allows Surprisingly, invention capsules permit enhanced migration insulin to be Secreted through its "pores' to maintain the and aggregation of the biologically active material within diabetic in normal glucose control. the capsule and enhanced control over the release rates of the Successful transplants of microencapsulated cells have biologically active material or components Secreted by the not been clinically feasible to date due to fundamental 35 biologically active material, while decreasing the risk of problems of transplant rejection and/or fibrotic reaction to biomineralization due to ions required for ionic crosslinking the microcapsule. In the treatment of diabetes, Lim and Sun, and enabling the biologically active material contained 1980; Science 210:908, reported the first successful implan within the capsule to retain a significant proportion of the tation of microencapsulated islets and described normaliza functionality of the unencapsulated biologically active mate tion of blood Sugar in diabetic rats. 40 rial. However, for microencapsulated cells to be clinically In a further aspect of the present invention, there also have useful and applicable in humans, it is important that the been developed methods of making invention capsules. One capsule be biocompatible, allow adequate diffusion for the of the invention methods comprises Subjecting a capsule encapsulated cells to respond appropriately to a Stimulatory whose outer layer is ionically crosslinked and covalently Signal and to provide the encapsulated cells with necessary 45 crosslinked, and optionally polyionically crosslinked (or, in nutrients, and optionally be retrievable. Retrievability is the case of macrocapsules comprising microcapsules desirable for a variety of reasons, e.g., So that accumulation therein, ionically crosslinked and either polyionically of the implanted materials can be avoided, So that encapsu crosslinked, covalently crosslinked, or both polyionically lated cells can be removed from the recipient when no longer crosslinked and covalently crosslinked), to conditions Suf needed or desired (e.g., when the product(s) of the encap 50 ficient to disruptionic crosslinking in at least the outer layer Sulated cells are no longer needed, if the encapsulated cells thereof. Surprisingly, invention methods facilitate the rela fail to perform as desired, etc.), So that encapsulated cells tively rapid formation of invention capsules under condi can be removed if/when they become non-viable, and the tions which are not cytotoxic, while decreasing the risk of like. biomineralization caused by the presence of ions required Biocompatibility of encapsulated islets remains a funda 55 for ionic crosslinking and enabling the biologically active mental problem. The term “biocompatible” is used herein in material contained within the capsule to retain a significant its broad Sense, and relates to the ability of the material to proportion of the functionality of the unencapsulated bio result in long-term in Vivo function of transplanted biologi logically active material. cal material, as well as its ability to avoid a foreign body, Additional methods of making invention capsules com fibrotic response. A major problem with microencapsulation 60 prise Simultaneously Subjecting a droplet comprising a Sus technology has been the occurrence of fibrous overgrowth of pension of biologically active materials in a covalently the epicapsular Surface, resulting in cell death and early graft crosslinkable carrier to conditions Sufficient to prevent Sub failure. Despite extensive Studies, the pathological basis of Stantial dissociation thereof and Subjecting the droplet to this phenomenon in alginate based capsules remains poorly conditions Sufficient to induce Substantial covalent understood. However, several factors have recently been 65 crosslinking thereof. Surprisingly, these invention methods identified as being involved in graft failure, e.g., the gulu facilitate the relatively rapid formation of invention capsules ronic acid/mannuronic acid content of the alginate under conditions which are not cytotoxic, while reducing to US 6,495,161 B1 3 4 Substantially Zero the risk of biomineralization caused by the tion of the detailed description and appended claims pro presence of ions required for ionic crosslinking and while vided herewith. enabling the biologically active material contained within the capsule to retain a significant proportion of the func DETAILED DESCRIPTION OF THE tionality of the unencapsulated biologically active material. INVENTION In a further aspect of the present invention, there also have been developed capsules containing cell aggregates therein, In accordance with the present invention, there are pro and methods for the production thereof. Invention capsules Vided microcapsules containing biologically active materials comprise a biocompatible gellable material, and have a core therein. Invention microcapsules comprise an ionically which is not ionically crosslinked, and at least an outer layer crosslinkable biocompatible gellable material, wherein at thereof which is covalently crosslinked, polyionically least the outer layer of Said microcapsule is covalently crosslinked, or both covalently crosslinked and polyioni crosslinked and optionally polyionically crosslinked, but not cally crosslinked. Surprisingly, invention capsules permit ionically crosslinked. enhanced migration and aggregation of the cell aggregates AS utilized herein, the term “microcapsule’ includes and constituent cells within the capsule and enhanced con 15 capsules of biocompatible gellable material directly Sur trol over the release rates of the components Secreted by the rounding biologically active material. Although the actual cell aggregates, while decreasing the risk of biomineraliza dimensions of the invention microcapsules are not critical, tion due to ions required for ionic crosslinking and enabling the term “microcapsules' includes capsules of biocompat the cell aggregates contained within the capsule to retain a ible gellable material the largest dimensions of which typi Significant proportion of the functionality of the unencap cally falls in the range of about 1 um up to about 1000 um, Sulated cell aggregates. with a preferable largest dimension falling in the range of Invention methods of making capsules containing cell about 100 um up to about 800 um. Commonly, all dimen aggregates therein described herein comprise Subjecting a Sions of the microcapsule exceed 20 nm. Invention micro capsule, comprising a biocompatible gellable material and capsules can be produced in a variety of shapes, i.e., in the having a core which is ionically crosslinked, to conditions 25 shape of a cylinder (i.e., a geometrical Solid generated by the Sufficient to disrupt ionic crosslinking therein. Surprisingly, revolution of a rectangle about one of its sides), a sphere invention methods facilitate enhanced migration and aggre (i.e., a Solid geometrical figure generated by the revolution gation of the cell aggregates and their constituent compo of a semicircle around its diameter), a disc (i.e., a generally nents within the capsule and enhanced control over the flat, circular form), a flat sheet (i.e., a generally flat polygo release rates of the biologically active material or of the nal form, preferably Square or rectangular), a wafer (i.e., an components Secreted by the biologically active material, irregular flat sheet), a dog-bone (i.e., a shape that has a while decreasing the risk of biomineralization due to ions central Stem and two ends which are larger in diameter than required for ionic crosslinking and facilitating the ability of the central stem, Such as a dumbbell), or the like. Invention the cell aggregates contained within the capsule to retain a microcapsules are generally formed So that the pore size of Significant proportion of the functionality of the unencap 35 at least the outer layer of the microcapsule is Sufficiently Sulated cell aggregates. large to allow unhindered diffusion of: In an additional aspect of the present invention, there also the biologically active material contained therein, or have been developed delivery Systems comprising the inven the compound Secreted by the biologically active material tion capsules. Surprisingly, invention delivery Systems per contained therein, or mit enhanced diffusion, acroSS and throughout the capsule, 40 the compound to be catalyzed and/or reacted by the of the biologically active material contained therein, or of biologically active material contained therein. the compound Secreted by the biologically active material Invention microcapsules are generally formed So that the contained therein, or of the compound to be catalyzed and/or pore size of at leat the outer layer of the microcapsule is reacted by the biologically active material contained therein, sufficiently small to block inward diffusion of molecules while decreasing the risk of biomineralization due to ions 45 which are capable of initiating an immune response to the required for ionic crosslinking and enabling the biologically biologically active material (e.g., IgG, complement proteins, active material contained within the capsule to retain a and the like), at least when the microcapsule is formed to not Significant proportion of the functionality of the unencap be contained within a larger macrocapsule. Sulated biologically active material. Biologically active materials contemplated for contain The present invention provides many advantages over the 50 ment and/or delivery in accordance with the present inven art. For example, invention methods, capsules, and delivery tion include individual living cells or groups of living cells Systems are useful in preparing biological containment Sys (e.g., cell aggregates), biological materials (for diagnostic tems with properties (e.g., mechanical strength, capsule purposes, e.g., for in Vivo evaluation of the effects of Such permeability and porosity, desired controlled release rates of biological materials on an organism, and conversely, the the biologic or components Secreted by the biologic, and 55 effects of the organism on Such biological materials), phar immunoreactivity) that can be varied to adapt to a broader macologically active drugs, diagnostic agents, agents of range of physiological conditions. This variation and adapt nutritional value, hemoglobin (to create artificial blood), and ability are due to the broader range of ratioS of ionic to the like. covalent linkages in the biocompatible gellable material AS utilized herein, the term “living cells' includes any permitted by the present invention. Further, invention meth 60 viable cellular material, regardless of the Source thereof. ods of making capsules containing cell aggregates therein, Thus, Virus cells, prokaryotic cells, eukaryotic cells, plant as well as the capsules formed thereby, are useful as a cells, and the like, are contemplated. Specifically contem quantitatively plentiful and low cost alternative to usage of plated living cells include islets of Langerhans (for the freshly harvested cell aggregates (e.g., islets from pancreas), treatment of diabetes) (including individual pancreatic islet Since the latter are usually available only in limited numbers. 65 cells (e.g., C., f, and ö cells of pancreatic islets), tumor cells Other advantages of the present invention can be readily (for evaluation of chemotherapeutic agents), human recognized by those of ordinary skill in the art upon inspec T-lymphoblastoid cells sensitive to the cytopathic effects of US 6,495,161 B1 S 6 HIV, dopamine secreting cells (for the treatment of Parkin tives thereof, taxotere and derivatives thereof, Son's disease), nerve growth factor cells (for the treatment of Vinblastine, Vincristine, tamoxifen, etoposide, Alzheimer's disease), hepatocytes (for treatment of liver pipOSulfan, and the like), dysfunction), adrenalin/angiotensin Secreting cells (for regu antianxiety agents (e.g., lorazepam, buspirone lation of hypo/hypertension), parathyroid cells (for replacing hydrochloride, prazepam, chlordiazepoxide thyroid function), norepinephrine/metencephalin Secreting hydrochloride, oxazepam, cloraZepate dipotassium, cells (for the control of pain), and the like. These living cells diazepam, hydroxy Zine pamoate, hydroxy Zine can be individual cells, or aggregates of cells held together hydrochloride, alprazolam, droperidol, halazepam, via intercellular adhesion mechanisms characteristic of the chlormezanone, dantrolene, and the like), individual cells (e.g., islets, and the like). immunosuppressive agents (e.g., cyclosporine, Examples of pharmacologically active agents include: azathioprine, mizoribine, FK506 (tacrolimus), and the analgesics/antipyretics (e.g., aspirin, acetaminophen, like), ibuprofen, naproxen Sodium, bupre norphine antimigraine agents (e.g., ergotamine tartrate, propanolol hydrochloride, propoxyphene hydrochloride, pro hydrochloride, iSome the pte ne mu cate, poxyphene napSylate, meperidine hydrochloride, 15 dichloralphenazone, and the like), hydromorphone hydrochloride, morphine Sulfate, Oxy Sedatives/hypnotics (e.g., barbiturates (e.g., pentobarbital, codone hydrochloride, codeine phosphate, dihydroco pentobarbital Sodium, Secobarbital Sodium, and the deine bitartrate, pentazocine hydrochloride, hydroc like), benzodiazapines (e.g., flurazepam hydrochloride, odone bit artrate, levorphanol tartrate, diflunisal, trolamine Salicylate, nalbuphine hydrochloride, mefe triazolam, tomazeparm, midazolam hydrochloride, and namic acid, butorphanol tartrate, choline Salicylate, the like), and the like), but albital, phenyltoloxamine citrate, diphenhydramine antianginal agents (e.g., beta-adrenergic blockers, cal citrate, methotrime praZine, cinname drine cium channel blockers (e.g., nifedipine, diltiazem hydrochloride, meprobamate, and the like), hydrochloride, and the like), nitrates (e.g., 25 nitroglycerin, isosorbide dinitrate, pentaerythritol anesthetics (e.g., cyclopropane, enflurane, halothane, tetranitrate, erythrityl tetranitrate, and the like), and the isoflurane, methoxyflurane, nitrous oxide, propofol, like), and the like), antipsychotic agents (e.g., haloperidol, loxapine antiasthmatics (e.g., AZelastine, Ketotifen, Traxanox, and Succinate, loxapine hydrochloride, thioridazine, thior the like), idazine hydrochloride, thiothixene, flu phenazine antibiotics (e.g., neomycin, Streptomycin, hydrochloride, fluphenazine decanoate, fluphenazine chloramphenicol, cephalosporin, amplicillin, penicillin, enanthate, trifluoperazine hydrochloride, chlorprom tetracycline, and the like), azine hydrochloride, perphenazine, lithium citrate, antidepressants (e.g., nefopam, oxypertine, doxepin prochlorperazine, and the like), hydrochloride, amoxapine, traZodone hydrochloride, 35 antimanic agents (e.g., lithium carbonate and the like), amitriptyline hydrochloride, map roti line antiarrhythmics (e.g., bretylium tosylate, esmolol hydrochloride, phenelZine Sulfate, desipramine hydrochloride, Verapamil hydrochloride, amiodarone, hydrochloride, nortriptyline hydrochloride, tranyl encainide hydrochloride, digoxin, digitoxin, mexiletine cypromine Sulfate, fluoxetine hydrochloride, doxepin hydrochloride, disopyramide phosphate, procainamide hydrochloride, imipramine hydrochloride, imipramine 40 hydrochloride, quinidine Sulfate, quinidine gluconate, pamoate, nortriptyline, amitriptyline hydrochloride, quinidine polygalacturonate, flecainide acetate, tocain isocarboxazid, desipramine hydrochloride, trimi ide hydrochloride, lidocaine hydrochloride, and the pramine maleate, protriptyline hydrochloride, and the like), like), antiarthritic agents (e.g., phenylbutazone, Sulindac, antidiabetics (e.g., biguanides, hormones, Sulfonylurea 45 penicillamine, Salsalate, piroXicam, azathioprine, derivatives, and the like), indomethacin, meclofenamate Sodium, gold Sodium antifungal agents (e.g., griseofulvin, keloconazole, thiomalate, ketoprofen, auranofin, aurothioglucose, tol amphotericin B, NyStatin, candicidin, and the like), metin Sodium, and the like), antihypertensive agents (e.g., propanolol, propafenone, antigout agents (e.g., colchicine, allopurinol, and the like), oxyprenolol, Nifedipine, reserpine, trimethaphan 50 anticoagulants (e.g., heparin, heparin Sodium, warfarin camsylate, phenoxybenzamine hydrochloride, par Sodium, and the like), gyline hydrochloride, deserpidine, diaZOxide, guanethi thrombolytic agents (e.g., urokinase, Streptokinase, dine monosulfate, minoxidil, rescinnamine, Sodium altoplase, and the like), nitroprusside, rauwolfia Serpentina, alseroxylon, phen 55 antifibrinolytic agents (e.g., aminocaproic acid and the tolamine meSylate, reserpine, and the like), like), anti-inflammatories (e.g., (non-steroidal) indomethacin, hemorheologic agents (e.g., pentoxifylline and the like), naproxen, ibuprofen, ramifenaZone, piroXicam, antiplatelet agents (e.g., aspirin, empirin, ascriptin, and (Steroidal) cortisone, dexamethasone, fluazacort, the like), hydrocortisone, prednisolone, prednisone, and the 60 anticonvulsants (e.g., valproic acid, divalproate Sodium, like), phenytoin, phenytoin Sodium, clonazepam, primidone, antineoplastics (e.g., adriamycin, cyclophosphamide, phenobarbitol, phenobarbitol Sodium, carbamazepine, actinomycin, bleomycin, duanorubicin, doxorubicin, amobarbital Sodium, methSuximide, metharbital, epirubicin, mitomycin, methotrexate, fluorouracil, mephobarbital, mephenytoin, phen Su Ximide, carboplatin, carnustine (BCNU), methyl-CCNU, 65 paramethadione, ethotoin, phenacemide, Secobarbitol cisplatin, etoposide, interferons, camptothecin and Sodium, cloraZepate dipotassium, trimethadione, and derivatives thereof, phenesterine, paclitaxel and deriva the like), US 6,495,161 B1 7 8 antiparkinson agents (e.g., ethoSuximide, and the like), hormones (e.g., androgens (e.g., danazol, testosterone antihistamines/antipruritics (e.g., hydroxy Zine cypionate, fluoxymesterone, ethyltostosterone, test hydrochloride, diphenhydramine hydrochloride, chlor oster one e nani hate, methylte Sto Sterone, pheniramine maleate, brompheniramine maleate, fluoxymesterone, testosterone cypionate), estrogens cyproheptadine hydrochloride, terfenadine, clemastine (e.g., estradiol, estropipate, conjugated estrogens), fumarate, triprolidine hydrochloride, carbinoxamine progestins (e.g., methoxyprogesterone acetate, nore maleate, diphenylpyraline hydrochloride, phenin thindrone acetate), corticosteroids (e.g., triamcinolone, damine tartrate, azatadine maleate, tripelennamine betamethasone, betamethasone Sodium phosphate, hydrochloride, dexchlorphenirarnine maleate, methdi dexamethasone, dexamethasone Sodium phosphate, lazine hydrochloride, trimprazine tartrate and the like), dexamethasone acetate, prednisone, methylpredniso agents useful for calcium regulation (e.g., calcitonin, lone acetate Suspension, triamcinolone acetonide, parathyroid hormone, and the like), methylprednisolone, prednisolone Sodium phosphate antibacterial agents (e.g., amikacin Sulfate, aztreonam, methylprednisolone Sodium Succinate, hydrocortisone chloramphenicol, chloramphenicol palmitate, chloram Sodium Succinate, methylprednisolone Sodium phe nicol Sodium Succinate, ciprofloxacin 15 Succinate, triamcinolone heXacatonide, hydrocortisone, hydrochloride, clindamycin hydrochloride, clindamy hydrocortisone cypionate, prednisolone, fluorocorti Sone acetate, paramethasone acetate, prednisolone cin palmitate, clindamycin phosphate, metronidazole, tebulate, prednisolone acetate, prednisolone Sodium metronidazole hydrochloride, gentamicin Sulfate, lin phosphate, hydrocortisone Sodium Succinate, and the comycin hydrochloride, tobramycin Sulfate, Vancomy like), thyroid hormones (e.g., levothyroxine Sodium cin hydrochloride, polymyxin B Sulfate, colistimethate and the like), and the like, Sodium, colistin Sulfate, and the like), hypoglycemic agents (e.g., human insulin, purified beef antiviral agents (e.g., interferon gamma, Zidovudine, insulin, purified pork in Sulin, gly buride, amantadine hydrochloride, ribavirin, acyclovir, and the chlorpropamide, glipizide, tolbutamide, tolaZamide, like), 25 antimicrobials (e.g., cephalosporins (e.g., cefazolin and the like), Sodium, cephradine, cefaclor, cephapirin Sodium, cefti hypolipidemic agents (e.g., clofibrate, dextrothyroxine ZOXime Sodium, cefoperaZone Sodium, cefotetan Sodium, probucol, lovastatin, niacin, and the like), disodium, cefutoxime azotil, cefotaXime Sodium, proteins (e.g., DNase, alginase, Superoxide dismutase, cefadroXil monohydrate, ceftazidime, cephalexin, lipase, and the like), nucleic acids (e.g., Sense or cephalothin Sodium, cephalexin hydrochloride anti-Sense nucleic acids encoding any therapeutically monohydrate, cefamandole nafate, cefoxitin Sodium, useful protein, including any of the proteins described cefonicid Sodium, ceforanide, ceftriaxone Sodium, herein, and the like), ceftazidime, cefadroxil, cephradine, cefuroxime agents useful for erythropoiesis stimulation (e.g., eryth Sodium, and the like), penicillins (e.g., amplicillin, 35 ropoietin and the like), amoxicillin, penicillin G benzathine, cyclacillin, ampi antiulcer/antireflux agents (e.g., famotidine, cimetidine, cillin Sodium, penicillin G potassium, penicillin V ranitidine hydrochloride, and the like), potassium, piperacillin Sodium, oxacillin Sodium, antinauseants/antiemetics (e.g., meclizine hydrochloride, bacampicillin hydrochloride, cloxacillin Sodium, ticar nabilone, prochlorperazine, dimenhydrinate, promet cillin disodium, azlocillin Sodium, carbenicillin indanyl 40 hazine hydrochloride, thiethylperazine, Scopolamine, Sodium, penicillin G potassium, penicillin G procaine, and the like), methicillin Sodium, nafcillin Sodium, and the like), oil-soluble Vitamins (e.g., vitamins A, D, E, K, and the erythromycins (e.g., erythromycin ethylsuccinate, like), erythromycin, erythromycin eStolate, erythromycin as well as other drugS Such as mitotane, Visadine, lactobionate, erythromycin Siearate, erythromycin 45 halonitroSoureas, anthrocyclines, ellipticine, and the ethylsuccinate, and the like), tetracyclines (e.g., tetra like, cycline hydrochloride, doxycycline hyclate, minocy and the like. cline hydrochloride, and the like), and the like), Examples of diagnostic agents contemplated for use in the anti-infectives (e.g., GM-CSF and the like), practice of the present invention include ultrasound contrast bronchodialators (e.g., Sympathomimetics (e.g., epineph 50 agents, radio contrast agents (e.g., iodo-octanes, rine hydrochloride, metaproterenol Sulfate, terbutaline halocarbons, renografin, and the like), magnetic contrast Sulfate, isoetharine, isoetharine meSylate, isoetharine agents (e.g., fluorocarbons, lipid soluble paramagnetic hydrochloride, albuterol sulfate, albuterol, bitolterol, compounds, and the like), as well as other diagnostic agents meSylate isoproterenol hydrochloride, terbutaline which cannot readily be delivered without some physical Sulfate, epinephrine bitartrate, metaproterenol Sulfate, 55 and/or chemical modification to accommodate the Substan epinephrine, epinephrine bit artrate), anticholinergic tially water insoluble nature thereof. agents (e.g., ipratropium bromide), Xanthines (e.g., Examples of agents of nutritional value contemplated for aminophylline, dyphylline, metaproterenol Sulfate, use in the practice of the present invention include amino aminophylline), mast cell Stabilizers (e.g., cromolyn acids, Sugars, proteins, carbohydrates, fat-Soluble Vitamins Sodium), inhalant corticosteroids (e.g., flurisolidebe 60 (e.g., vitamins A, D, E, K, and the like) or fat, or combina clomethasone dipropionate, beclomethasone dipropi tions of any two or more thereof. onate monohydrate), Salbutamol, beclomethasone As utilized herein, the term “ionically crosslinkable” dipropionate (BDP), ipratropium brornide, budesonide, means the ability of a biocompatible gellable material to ketotifen, Salmeterol, Xinafoate, terbutaline Sulfate, form ionically crosslinked networks in the presence of triamcinolone, theophylline, nedocromil Sodium, 65 multivalent cation(s) Such as calcium, zinc, barium, metaproterenol Sulfate, albuterol, flunisolide, and the Strontium, aluminum, iron, manganese, nickel, cobalt, like), copper, cadmium, lead, and the like, or mixtures of any two US 6,495,161 B1 9 10 or more thereof. This ability is due to the interaction of as polyethyleneimine, polyallylamine, polyetheramine, anions of the biocompatible gellable material (e.g., carboxy polyvinylpyridine, and the like), covalently crosslinkable groups on alginate) to ionically bond with the multivalent derivatives thereof, and the like. Polycationic material cations. Preferred multivalent cations include calcium, molecular weight can vary, depending on the degree of barium, and Strontium, with calcium being presently pre permeability desired. Polycationic material molecular ferred for ionically crosslinking a biocompatible gellable weights will typically fall within a range of about 1,000 to material comprising alginate. about 100,000 or higher, with a presently preferred molecu The characterization of an ionically crosslinkable bio lar weight in the range of about 10,000 up to about 50,000. compatible gellable material (or any portion thereof) as Presently, preferred polycationic materials for use in the being “not ionically croSSlinked' indicates that an insuffi practice of the present invention include polylysine (i.e., cient amount of multivalent cation(s) required to Substan poly-D-lysine (PDL), poly-DL-lysine, poly-L-lysine (PLL), tially crosslink the biocompatible gellable material is present poly-e-CBZ-D-lysine, poly-e-CBZ-DL-lysine, poly-e-CBZ in the biocompatible gellable material, either because an L-lysine), polyornithine (i.e., poly-DL-ornithine, poly-L- insufficient amount was always present or because an ornithine, or poly-8-CBZ-DL-omithine), and the like, and amount of Such multivalent cation(s) was removed by Sub 15 mixtures of any two or more thereof. jecting the biocompatible gellable material to conditions Covalently crosslinkable materials contemplated for use Sufficient to Substantially disrupt ionic crosslinking in the in the practice of the present invention include covalently biocompatible gellable material. crosslinkable polysaccharides (e.g., covalently crosslinkable Biocompatible gellable materials contemplated for use in alginates), covalently crosslinkable polyethylene glycols the practice of the present invention include ionically (i.e., covalently crosslinkable PEGs), covalently crosslink crosslinkable materials, covalently crosslinkable materials, able polycationic materials, covalently crosslinkable polyionically crosslinkable materials, and the like, and mix proteins, covalently crosslinkable peptides, other covalently tures of any two or more thereof. crosslinkable Synthetic polymers, and the like, and mixtures Ionically crosslinkable materials contemplated for use in of any two or more thereof. the practice of the present invention include anionic mate 25 Covalently crosslinkable alginates contemplated for use rials which are ionically crosslinkable (e.g., alginates and in the practice of the present invention include alginates other polysaccharides, chitosan, gellan gum, Xanthan gum, modified with a substituent X which is capable of undergo hyaluronic acid, heparin, pectin, carrageenan, and the like), ing free radical polymerization (X is a moiety containing a covalently crosslinkable derivatives thereof, and the like, carbon-carbon double bond or triple bond capable of free and mixtures of any two or more thereof. Alginates con radical polymerization; and X is linked covalently to the templated for use in the present invention include high alginate through linkages Selected from ester, ether, G-content alginate, high-M content alginate, Sodium thioether, disulfide, amide, imide, Secondary amines, tertiary alginate, and the like, and mixtures of any two or more amines, direct carbon-carbon (C-C) linkages, Sulfate thereof. esters, Sulfonate esters, phosphate esters, urethanes, Capsule properties like mechanical Strength, pore size, 35 carbonates, and the like). Examples of covalently crosslink and biocompatibility can be varied with the type and con able alginates include allyl and Vinyl ethers of alginate, centration of the alginate employed. For example, alginates acrylate and methacrylate esters of alginate, and the like. with differing C-L-guluronic acid (G blocks) to B-D- Covalently crosslinkable PEGs contemplated for use in mannuronic acid (M blocks) ratios are capable of yielding the practice of the present invention include linear or capsules with Significantly differing properties. G blockS 40 branched chain PEGs (including STAR PEGs) modified have a higher multivalent cation binding capacity than M with a Substituent X which is capable of undergoing free blocks. In addition, alginates having higher fractions of G radical polymerization (as described above); wherein X is blocks are more biocompatible than those containing a linked covalently to the PEG through linkages selected from larger fraction of M blocks since high M block alginates ester, ether, thioether, disulfide, amide, imide, Secondary have been found to induce fibrotic Overgrowth. Accordingly, 45 amines, tertiary amines, direct carbon-carbon (C-C) capsules Synthesized from alginates with high G/M ratioS linkages, Sulfate esters, Sulfonate esters, phosphate esters, are generally Stronger and more biocompatible than those urethanes, carbonates, and the like. Examples of Such capsules Synthesized from alginates with lower G/M ratioS. covalently crosslinkable PEGs include vinyl and allyl ethers Thus, the use in accordance with the present invention of of PEG, acrylate, diacrylate and methacrylate esters of PEG, alginates having at least 60% or greater G blockS is 50 and the like; and mixtures of any two or more thereof. preferred, with alginates having at least 70% or greater G PEGs having a wide range of molecular weights can be blocks being presently preferred. employed in the practice of the present invention. Thus, AS a further example, alginates with differing molecular mixtures of different molecular weights for covalently weights (MW) or alginate concentrations are capable of crosslinkable PEGs contemplated for use in the practice of yielding capsules with Significantly differing properties 55 the present invention include PEGs having a MW in the relating to mechanical Strength, pore size, and biocompat range of about 200 up to about 1,000,000 (with PEGs having ibility of the capsule. Thus, it is possible to further modify molecular weights in the range of about 500 up to about the end properties of the capsule by choosing alginates of 100,000 being preferred, and PEGs having molecular Specific types. weights in the range of about 1000 up to about 50,000 being Polyionically crosslinkable materials contemplated for 60 presently preferred). Such PEGs can be linear or branched use in the practice of the present invention include mixtures chain (including STAR PEGs). STAR PEGs are molecules of ionically crosslinkable materials and polycationic mate having a central core (such as divinyl benzene) which is rials and the like. Polycationic materials contemplated for anionically polymerizable under controlled conditions to use in the present invention include polyamino acids (e.g., form living nuclei having a predetermined number of active polyhistidine, polylysine, polyornithine, and the like), poly 65 Sites. Ethylene oxide is added to the living nuclei and mers containing primary amine groups, Secondary amine polymerized to produce a known number of PEG “arms,” groups, tertiary amine groups, or pyridinyl nitrogen(s) (Such which are quenched with water when the desired molecular US 6,495,161 B1 11 12 weight is achieved. Alternatively, the central core can be an Thus, increasing the contact time of the capsule with the ethoxylated oligomeric glycerol that is used to initiate photopolymerizing Solution gives more time for the photo polymerization of ethylene oxide to produce a STAR PEG of components to diffuse inwards into the capsule. Such desired molecular weight. enhanced penetration distance, upon photopolymerization, Covalently crosslinkable polycationic materials contem results in a capsule with a greater extent of covalent bonding plated for use in the practice of the present invention include therethrough. Hence, contact times of the capsule with the polycationic materials modified with a substituent X which photopolymerizing Solution determine the depth of photo is capable of undergoing free radical polymerization (as polymerization relative to the size of the capsule. A capsule described above); wherein X is linked covalently to the polycationic material through linkages Selected from ester, that has been uniformly photopolymerized throughout its ether, thioether, disulfide, amide, imide, Secondary amines, Volume would be expected to result in a homogeneous tertiary amines, direct carbon-carbon (C-C) linkages, Sul alginate matrix held together coherently by a uniformly fate esters, Sulfonate esters, phosphate esters, urethanes, distributed network of covalent bonds, in the absence of carbonates, and the like. Examples of covalently crosslink interspersed ionic bonds. Such a situation arises when the able polycationic materials include allyl and Vinyl ethers of contact time is Sufficiently long that photocomponents are polycations, acrylate and methacrylate esters of polycations, 15 allowed to diffuse throughout the entire volume of the and the like. capsule. On the other hand, a capsule that has been photo Free radical polymerization of the above-described polymerized only on the Surface would be expected to result covalently crosslinkable materials can be carried out in a in a composite capsule with a liquified (e.g., Substantially variety of ways, for example, initiated by irradiation with noncrosslinked) core Surrounded by a photocrosslinked Suitable wavelength electromagnetic radiation (e.g., visible modified-alginate gel layer on the Surface, in the absence of or ultraViolet radiation) in the presence of a Suitable interspersed ionic bonds. Such a situation arises when the photoinitiator, and optionally, cocatalyst and/or comonomer. contact time is So short that the photocomponents do not Alternatively, free radical polymerization can be initiated by diffuse throughout the entire Volume of the capsule, but are thermal initiation by a Suitable free radical catalyst. instead localized in a layer close to the outer Surface of the A variety of free radical initiators, as readily recognized 25 capsule. Upon photopolymerization, the capsule therefore by those of Skill in the art, can be employed in the practice possesses an ionically crosslinked alginate core Surrounded of the present invention. Thus, photoinitiators, thermal by a skin of covalently crosslinked and ionically croSSlinked initiators, and the like can be employed. For example, alginate. Subsequent disruption of ionic crosslinking via suitable UV initiators include 2,2-dimethoxy-2-phenyl invention methods yields a composite capsule with a liqui acetophenone and its water Soluble derivatives, benzoin fied core Surrounded by a covalently crosslinked layer. ethyl ether, 2,2-dimethyl phenoxyacetophenone, benzophe Capsules (e.g., microcapsules and macrocapsules) con none and its water Soluble derivatives, benzil and its water templated for use in the practice of the present invention Soluble derivatives, thioxanthone and its Water Soluble may be further characterized as comprising an Outer layer derivatives, and the like. For visible light polymerization, a and a core. Typically, the outer layer of a capsule comprises System of dye (also known as initiator or photosensitizer) 35 that portion of the biocompatible gellable material which is and cocatalyst (also known as coSynergist, activator, initi on the Outer Surface of the capsule, while the core of a ating intermediate, quenching partner, or free radical capsule is that portion of the biocompatible gellable material generator) are used. Examples of Suitable dyes are ethyl which is not the outer layer. eosin, eosin, eosin Y, erythrosin, riboflavin, fluorscein, rose Generally, the outer layer of a capsule has a thickness of bengal, methylene blue, thionine, and the like; examples of 40 at least about 1/500" of the largest dimension of the capsule Suitable cocatalysts are triethanolamine, arginine, methyl (e.g., 1 micron for 500 micron macrocapsule), with a thick diethanolamine, tiethylamine, and the like. ness of at least about 1/2.5" to about 2%s" of the largest A Small amount of a comonomer can optionally be added dimension of the capsule (e.g., 20–40 microns for 500 to the crosslinking reaction to increase the polymerization micron macrocapsule) being preferred, and a thickness of at rates. Examples of Suitable comonomers include Vinyl 45 least about /10" of the largest dimension of the capsule (e.g., pyrrolidinone, acrylamide, methacrylamide, acrylic acid, 50 microns for 500 micron macrocapsule) being presently ethacrylic acid, Sodium acrylate, Sodium methacrylate, preferred. When the outer layer of a capsule has been hydroxyethyl acrylate, hydroxyethyl methacrylate (HEMA), covalently crosslinked and optionally polyionically ethylene glycol diacrylate, ethylene glycol dimethacrylate, crosslinked, this enhanced thickness of the outer layer pentaerythritol triacrylate, pentaerythritol trimethacrylate, 50 provides enhanced immunogenic protection (e.g., enhanced trimethylol propane triacrylate, trimethylol propane prevention of direct exposure of any immunogenic agents at trimethacrylate, tripropylene glycol diacrylate, tripropylene the capsule Surface (e.g., polycations, unencapsulated bio glycol dimethacrylate, glyceryl acrylate, glyceryl logically active materials, and the like)) and enhanced methacrylate, and the like. Stability (e.g., Stability to long-term exposure to physiologi Photoinitiators, cocatalysts, and comonomers are collec 55 cal conditions), when compared to prior art microcapsules. tively referred to as photocomponents, and comprise the The core of a capsule contemplated for use in the practice active components of the photopolymerizing Solution. of the present invention can optionally be covalently Varying the concentrations and proportions of these com crosslinked and/or ionically crosslinked. Thus, in one ponents in the photopolymerizing Solution can be used to aspect, invention microcapsules can comprise a biocompat yield capsules with different mechanical Strengths and dif 60 ible gellable material whose core is ionically crosslinked. In fering permeabilities. These factors influence the in vivo a further aspect, invention microcapsules can comprise a Shelf life and performance of the capsule after transplanta biocompatible gellable material whose core is both tion into the body. In accordance with the present invention, covalently crosslinked and ionically crosslinked. In an addi varying the type and amount of the components in the tional aspect, invention microcapsules can comprise a bio photopolymerizing Solution provides a means of controlling 65 compatible gellable material whose core is covalently capsule properties like mechanical Strength, porosity or crosslinked, but not ionically crosslinked. In another aspect, permeability, and biocompatibility. invention microcapsules can comprise a biocompatible gel US 6,495,161 B1 13 14 lable material whose core is neither ionically crosslinked nor Similar to the outer layer of the invention microcapsules, covalently crosslinked. the outer layer of the optional microcapsule(s) contemplated In accordance with the present invention, there are further for use as part of the invention macrocapsules commonly is provided macrocapsules containing biologically active ionically crosslinked, covalently crosslinked, polyionically materials therein, optionally contained in at least one micro crosslinked, or any Suitable combination of any two or more capsule therein. Invention macrocapsules comprise a first thereof. Thus, in one aspect, invention macrocapsules can biocompatible gellable material which is ionically crosslink comprise at least one microcapsule, wherein at least the able and which contains the biologically active materials outer layer of the microcapsule(s) is covalently crosslinked. (and optionally present microcapsules) therein. When the In an additional aspect, invention macrocapsules can com microcapsules are not present within the macrocapsule, prise at least one microcapsule, wherein the outer layer of invention macrocapsules are further characterized in that at the microcapsule(s) is polyionically crosslinked. In another least the Outer layer of the macrocapsule is covalently aspect, invention macrocapsules can comprise at least one crosslinked and optionally polyionically crosslinked, but not microcapsule, wherein at least the outer layer of the ionically crosslinked. When the microcapsules are present microcapsule(s) is ionically crosslinked. within the macrocapsule, invention macrocapsules are fur Typically, the core of the optional microcapsule(s) con ther characterized in that at least the outer layer of the 15 templated for use as part of the invention macrocapsules is macrocapsule is covalently crosslinked, polyionically covalently crosslinked and/or ionically crosslinked. Thus, in crosslinked, or both covalently crosslinked and polyioni one aspect, the core of the microcapsule(s) contemplated for cally crosslinked, but not ionically crosslinked, and each of use as part of the invention macrocapsules is ionically the microcapsules contained within invention macrocap crosslinked. In a further aspect, the core of the microcapsule Sules comprises a Second biocompatible gellable material (s) contemplated for use as part of the invention macrocap containing the biologically active materials therein. Sules is covalently crosslinked and ionically crosslinked. In AS utilized herein, "macrocapsule’ includes capsules of another aspect, the core of the microcapsule(s) contemplated gel material Surrounding biologically active material, for use as part of the invention macrocapsules is covalently optionally contained within at least one microcapsule. The crosslinked, but not ionically crosslinked. In an additional term "macrocapsule' can include "macro-membranes,” 25 aspect, the core of the microcapsule(s) contemplated for use "macrogels,” “gel entrapped microcapsules,” “lace, as part of the invention macrocapsules is neither ionically “noodles,” “teabags,” “threads,” “worms,” and the like. crosslinked nor covalently crosslinked. Although the actual dimensions of the invention macrocap Capsules (e.g., microcapsules and macrocapsules) can be Sules are not critical, the term "macrocapsules' includes manufactured by various techniques known to those of Skill capsules of biocompatible gellable material the largest in the art, including but not limited to interfacial dimensions of which typically fall in the range of about 1000 polycondensation, emulsion polymerization, Simple and lum up to about 50000 um. Commonly, all dimensions of the complex coacervation, thermal and ionic gelation, phase invention macrocapsules are greater than 20 nm. Invention Separation, electroStatic precipitation, Solvent evaporation, macrocapsules can be produced in a variety of Shapes, i.e., and mechanical agitation. The Specific manufacturing tech in the shape of a cylinder (i.e., a geometrical Solid generated 35 nique employed is dictated by various factors, including the by the revolution of a rectangle about one of its sides), a chemistry of the biocompatible gellable material (i.e., the sphere (i.e., a Solid geometrical figure generated by the capsule shell material), the properties desired of the capsule revolution of a semicircle around its diameter), a disc (i.e., manufactured thereby, and the like. a generally flat, circular form), a flat sheet (i.e., a generally Biocompatible gellable material (e.g., alginate)- flat polygonal form, preferably Square or rectangular), a 40 containing microcapsules (and biocompatible gellable wafer (i.e., an irregular flat sheet), a dog-bone (i.e., a shape material-containing macrocapsules which comprise that has a central Stem and two ends which are larger in microcapsules) are generally produced employing a co-axial diameter than the central stem, Such as a dumbbell), or the pneumatic nozzle. The biocompatible gellable material Solu like. The macrocapsule is generally formed So that the pore tion (which contains the encapsulant (e.g., the biologically Size of at least the outer layer of the macrocapsule is 45 active material (for microcapsules and/or macrocapsules), or sufficiently large to allow unhindered diffusion of: the microcapsules containing the biologically active mate the biologically active material contained therein, or rial (for macrocapsules)) is extruded through the central the biologically active compound (e.g., insulin) Secreted bore, with air flowing around the Solution. The air preSSure by the biologically active material (e.g., pancreatic islet provides the force necessary to break up the extruded cells) contained therein, or 50 biocompatible gellable material Solution into droplets. In the compound to be catalyzed and/or reacted by the Such a System, the droplet Size can be altered by varying the biologically active material contained therein, ratio of the Solution flow rate to the air flow rate. Increasing while being sufficiently small to block inward diffusion of the latter relative to the former yields smaller droplets. molecules which are capable of initiating an immune Macrocapsules can also be Synthesized by extruding the response to the biologically active material (e.g., IgG, 55 biocompatible gellable material Solution manually through a complement proteins, and the like). Syringe attached with a needle. The droplets detach from the Like the core of the invention microcapsules, the core of needle when the drop size becomes big enough that the the invention macrocapsules can typically be covalently gravitational force tending to dislodge the droplet from the crosslinked and/or ionically crosslinked. Thus, in one needle exceeds the forces of Surface tension tending to keep aspect, invention macrocapsules comprise a core that is 60 the droplet attached to the needle. The size of the droplets ionically crosslinked. In a further aspect, invention macro can be controlled by choosing needles with an appropriate capsules comprise a core that is both covalently crosslinked gauge. and ionically crosslinked. In an additional aspect, invention Once the droplets of biocompatible gellable material macrocapsules can comprise a core that is covalently Solution have been formed, they can be Subjected to a variety crosslinked, but not ionically crosslinked. In another aspect, 65 of crosslinking conditions. invention microcapsules can comprise a core that is neither In one variety of crosslinking conditions, the droplets of ionically crosslinked nor covalently crosslinked. biocompatible gellable material Solution are ionically US 6,495,161 B1 15 16 crosslinked and covalently crosslinked to form capsules, and Subsequent to ionic crosslinking and covalent then Subjected to conditions Sufficient to disrupt ionic crosslinking, the capsules can optionally be rinsed thor crosslinking in at least the outer layer of the capsule. Under oughly with Saline in order to remove exceSS multivalent this aspect, the droplets are first Subjected to conditions cation(s) (whose removal helps reduce the chance of Sufficient to ionically crosslink the ionically crosslinkable biomineralization) and unreacted photocomponents (whose material Solution. Typically, these conditions comprise con removal helps reduce potential toxicity effects of these tacting the droplets with an ionic crosslinking medium photocomponents on the biologically active materials). The containing at least one multivalent cation(s) (e.g., calcium) capsules can optionally be incubated at about 37 C. in a to yield ionically crosslinked capsules (e.g., ionically Suitable culture medium. crosslinked microcapsules and ionically crosslinked Additional alternative treatments that can follow the cova macrocapsules). These ionically crosslinked capsules are lent crosslinking Step include Subjecting the capsule to Subsequently (or, optionally, Simultaneously) contacted with conditions Sufficient to disrupt ionic crosslinking in at least the photopolymerizing Solution for a predetermined amount the Outer layer of the capsule. This disruption of ionic of time. During this time, the components of the photopo crosslinking can promote migration and aggregation of the lymerizing Solution (that is, photoinitiators, cocatalysts, 15 biologically active material, as well as transport of the and/or comonomers) diffuse inwards into the ionically biologically active material or components Secreted by the crosslinked capsule. biologically active material out of the capsule. AS readily recognized by those of skill in the art, the Thus, in accordance with the present invention, there are predetermined time can be varied as a function of the size of additionally provided methods of making a microcapsule the capsule (i.e., Smaller capsules have larger ratios of having Substantially no ionic crosslinking in at least the Surface area to Volume, and thus require less time for outer layer thereof and containing biologically active mate equivalent diffusion of photocomponents), the concentration rials therein. Invention methods for making Such microcap of the individual components in the photopolymerizing Sules comprise: Solution and their concentrations relative to each other (i.e., Subjecting a microcapsule, wherein at least the outer layer different concentrations yield different properties of 25 thereof is ionically crosslinked, and wherein at least the capsule), and the like. In addition, the predetermined time Outer layer thereof is covalently crosslinked and option can be altered in order to vary the extent of the covalent ally polyionically crosslinked, and which contains bio crosslinkability relative to the ionic crosslinkability of the logically active materials therein, to conditions Suffi capsule. cient to disrupt ionic crosslinking in at least the outer The ionically crosslinked capsule containing the photo layer thereof, components can then optionally be Subsequently transferred thereby forming a microcapsule having Substantially no to another Solution containing a concentration of the multi ionic crosslinking in at least the outer layer thereof. valent cation(s) (e.g., Ca") which is Sufficiently high to In accordance with the present invention, there are further maintain an intact ionically crosslinked droplet, yet Suffi provided methods of making a macrocapsule having Sub ciently low (and definitely lower than the concentration of 35 Stantially no ionic crosslinking in at least the Outer layer the ionically crosslink initiating first multivalent cation(s) thereof and containing biologically active materials therein, Solution) to prevent mineralization due to possible local optionally contained within at least one microcapsule. Supersaturation of the multivalent cation (e.g., Ca") within Invention methods for making Such macrocapsules com the droplet. prise: The ionically crosslinked capsules are then Subsequently 40 Subjecting a macrocapsule, wherein at least the Outer layer (or, optionally, Simultaneously) Subjected to covalent thereof is ionically crosslinked, and which contains crosslinking conditions (e.g., photopolymerization (Such as biologically active materials therein, to conditions Suf under visible light from high pressure 100W mercury lamps ficient to disrupt ionic crosslinking in at least the outer (strong emission at wavelength of about 500 nm to about layer thereof, 550 nm) or argon ion laser light (wavelength of 514 nm at 45 thereby forming a macrocapsule having Substantially no powers between about 10 mW to about 2W))). The covalent ionic crosslinking in at least the outer layer thereof. When crosslinking time is generally rapid (on the order of milli microcapsules are not present within the macrocapsule, the Seconds (for photopolymerization via an argon ion laser) to macrocapsule can be further characterized in that at least the Seconds (for photopolymerization via a mercury lamp)), and outer layer thereof is covalently crosslinked and optionally varies with the concentrations of biocompatible gellable 50 polyionically crosslinked. When microcapsules are present material, initiator, cocatalyst, and comonomers in the ioni within the macrocapsule, the macrocapsule can be further cally crosslinked capsule. characterized in that at least the outer layer thereof is The time interval between the ionic crosslinking and the covalently crosslinked, polyionically crosslinked, or both covalent crosslinking of the droplets can be varied. This time covalently crosslinked and polyionically crosslinked. interval can vary from 0 Seconds (e.g., simultaneously 55 Conditions Sufficient to disrupt ionic crosslinking, either Subjecting the droplets to ionic crosslinking conditions and in at least the outer layer or the core of a microcapsule, a covalent crosslinking conditions) to about 5 minutes. The macrocapsule or their constituent biocompatible gellable shorter the time interval, the smaller the possibility exists materials, include contacting the relevant microcapsule, that the photocomponents will diffuse out of the droplets and macrocapsule or their constituent biocompatible gellable weaken the covalent crosslinking process, and the greater 60 materials with a Solution of Sodium citrate, ethylenediamine the probability that a stable covalently crosslinked capsule tetraacetic acid (EDTA), diethylenetriaminepentaacetic acid will be formed. This is especially applicable to smaller sized (DTPA), and other biocompatible chelators of multivalent droplets (e.g., microcapsules or Smaller macrocapsules), as cations, and the like, and mixtures of any two or more their larger ratios of Surface area (e.g., diffusion Surface) to thereof, in a concentration Sufficient to chelate Sufficient Volume increase the potential for loSS of photocomponents, 65 cations to Substantially disrupt ionic crosslinking in the and decrease the probability that a stable covalently relevant microcapsule, macrocapsule or their constituent crosslinked capsule will be formed. biocompatible gellable materials. In a preferred US 6,495,161 B1 17 18 embodiment, conditions Sufficient to disruptionic crosslink In accordance with another aspect of the present ing in at least the outer layer of a capsule or constituent invention, there are provided alternative methods of making biocompatible gellable material comprise contacting the a macrocapsule containing biologically active materials capsule or constituent biocompatible gellable material with therein, wherein droplets, comprising a Suspension of the a Solution of Sodium citrate having a concentration in the biologically active materials, optionally contained within at range of about 15 mM to about 1M. least one microcapsule, in a covalently crosslinkable carrier, In an alternative embodiment of the invention, capsules are simultaneously Subjected to: which are covalently crosslinked but not ionically conditions Sufficient to prevent Substantial dissociation crosslinked, are prepared by directly covalently thereof, and crosslinking, without first ionically crosslinking, the drop conditions Sufficient to induce Substantial covalent lets of biocompatible gellable solution formed as described above. crosslinking thereof, Thus, in accordance with this aspect of the present thereby forming the macrocapsule. Invention methods of invention, there are provided alternative methods of making making macrocapsules are optionally characterized, in one a microcapsule containing biologically active materials aspect, in that no ionic crosslinking is required (although therein, wherein droplets comprising a Suspension of bio 15 Such ionic crosslinking may be present) to Stabilize the logically active materials in a covalently crosslinkable car droplet prior to covalent crosslinking thereof, as the condi rier are simultaneously Subjected to: tions Sufficient to prevent Substantial dissociation of the conditions Sufficient to prevent Substantial dissociation droplet perform the requisite Stabilizing function of ionic thereof, and crosslinking. conditions Sufficient to induce Substantial covalent Capsules which comprise individual cells capable of crosslinking thereof, forming cell aggregates and which have been formed in thereby forming the microcapsule. Invention methods of accordance with the foregoing invention methods can be making microcapsules are optionally characterized, in one further characterized in that they are capable of facilitating aspect, in that no ionic crosslinking is required (although migration of the cells within the core of the capsules, and Such ionic crosslinking may be present) to Stabilize the 25 aggregation of the cells to form cell aggregates. droplet prior to covalent crosslinking thereof, as the condi Thus, in accordance with the present invention, there are tions Sufficient to prevent Substantial dissociation of the additionally provided capsules (e.g., microcapsules and droplet perform the requisite Stabilizing function of ionic macrocapsules) containing at least one cell aggregate crosslinking. therein. Invention capsules comprise an ionically crosslink As utilized herein, the term “covalently crosslinkable able biocompatible gellable material, and have a core and an carrier' includes all covalently crosslinkable materials as outer layer, wherein at least the outer layer of the capsule is described herein. covalently crosslinked or polyionically crosslinked or both Conditions Sufficient to prevent Substantial dissociation of covalently crosslinked and polyionically crosslinked, but not the droplet include contacting the droplet with a medium ionically crosslinked, and wherein Said at least one cell which is substantially immiscible with the droplet and which 35 aggregate is contained within the core which is not ionically does not substantially inhibit the induction of covalent crosslinked. crosslinking. Media which are substantially immiscible with When the capsule is a macrocapsule containing the droplet include those media which are capable of Sol microcapsules, there are at least two possible embodiments vating less than 10% of the volume of the droplet during the of this aspect of the invention. In a first embodiment, at least time period in which the droplet is in contact with the media. 40 the core of the microcapsule(s) of the macrocapsule is not Media which do not substantially inhibit the induction of ionically crosslinked, and the cell aggregate(s) is formed and covalent crosslinking include media which conduct Suffi contained within the core of the microcapsule(s) of the cient electromagnetic energy from an energy Source to the macrocapsule. In a Second embodiment, at least the core of droplet to enable initiation of covalent crosslinking in the the macrocapsule is not ionically crosslinked, and the cell covalently crosslinkable carrier, without destroying the 45 aggregate(s) is formed and contained within the core of the functionality of the biologically active material. macrocapsule. Media which are substantially immiscible with the droplet AS utilized herein, "cell aggregate' includes an aggrega and which do not substantially inhibit the induction of tion of individual living cells. Presently preferred cell aggre Substantial covalent crosslinking also comprise, for aqueous gates include pseudo islets, which are aggregates of indi droplets comprising biocompatible gellable materials con 50 vidual pancreatic islet cells (including a, f, or 6 pancreatic templated for use in accordance with the present invention, islet cells). The cell aggregates are formed within the Soybean oil, coconut oil, Safflower oil, Sunflower oil, Sesame microenvironment created by a capsule. Such a microenvi oil, other vegetable oils, and the like. In a preferred ronment as is present within the capsule provides a low embodiment, Such a Solution comprises Soybean oil. StreSS medium for the aggregation of Single cells into clumps Conditions Sufficient to induce Substantial covalent 55 of cells, or cell aggregates. The cell aggregates can, under at crosslinking of the droplet include irradiating the droplet least Some conditions, optionally be further characterized as with Sufficient energy to induce photocrosslinking of the exhibiting properties and functionality Substantially identi covalently crosslinkable carrier. This energy is generally in cal to those of naturally occurring islets of corresponding the form of electromagnetic radiation, Such as visible light, cells in Vivo. Accurate control over the average number and ultraViolet (UV) radiation, or lasers, although this energy 60 Size of cell aggregates encapsulated in each capsule can be can also include thermal energy. Two preferred embodi achieved by controlling the number of cells present per ments of Such conditions include contacting the droplet with capsule. Thus, the number of cells present per capsule could an argon ion laser at a wavelength of about 514 nm and at be varied by: a power level in the range of about 10 mW to about 2W for varying the culturing conditions for the unencapsulated no more than about 50 milliseconds, and contacting the 65 individual cells (i.e., varying the number of cell divi droplet with a high pressure (e.g., about 100 W) mercury Sion cycles experienced by each unencapsulated indi lamp for no more than about 5 minutes. vidual cell), and/or US 6,495,161 B1 19 20 varying the encapsulating conditions for the unencapsu The invention will now be described in greater detail with lated individual cells (i.e., varying the number of indi reference to the following non-limiting examples. Those of vidual cells per microcapsule), and/or ordinary skill in the art, when guided by the teachings of the varying the culturing conditions for the encapsulated Specification, may discover during the term of this patent individual cells (i.e., varying the number of cell divi 5 other embodiments of this invention which fall within the Sion cycles experienced by each encapsulated indi Scope of the appended claims. vidual cell). In accordance with the present invention, there are addi EXAMPLE 1. tionally provided methods of making a capsule containing at least one cell aggregate therein. Invention methods comprise Method of Testing Strength of Microcapsules Subjecting a capsule comprising an ionically crosslinked biocompatible gellable material wherein at least the outer The effect of varying the concentrations of the compo layer of the capsule is covalently crosslinked or polyioni nents comprising the photopolymerizing Solution (i.e., cally crosslinked or both covalently crosslinked and poly photoinitiators, cocatalysts, and comonomers) both indi ionically crosslinked, wherein Said capsule encapsulates a vidually and relative to each other has been characterized plurality of individual cells, to conditions Sufficient to dis 15 based on evaluation of bead strength. Trends in bead rupt ionic crosslinking within the core of the capsule. Strengths provide a window into understanding the effi Invention methods of making a capsule containing at least ciency of ionic and covalent crosslinking. In general, a one cell aggregate therein can be optionally characterized, in higher bead Strength indicates a stronger or closely one aspect, in that the ionic interactions within the capsule crosslinked matrix. Such a matrix is expected to have a are Sufficiently reduced to facilitate migration of the indi Smaller pore size due to the increased density of covalent vidual cells within the capsule, thereby facilitating aggre crosslinking between the polymer molecules. Based on Such gation and formation of at least one cell aggregate within the analysis, evaluating bead strengths provides an insight into capsule. mechanical properties, diffusional properties (porosity and When the capsule is a macrocapsule containing permeability), and in Vivo end performance of Such capsules microcapsules, there are at least two possible embodiments 25 of this aspect of the invention method. In a first embodiment, used for Xenotransplantation. Such experiments help con at least the core of the microcapsule(s) of the macrocapsule Struct an experimental database to intelligently manipulate is Subjected to conditions Sufficient to disrupt ionic capsule production conditions So as to yield capsules with crosslinking within the core of the microcapsule(s) of the desired end properties. macrocapsule, thereby rendering the core not ionically Modified alginate (acrylate derivatized alginate or AA) crosslinked, and the cell aggregate(s) is formed and con utilized in this Example 1 was prepared by chemically tained within the core of the microcapsule(s) of the macro modifying alginate by the incorporation of acrylate groups. capsule. In a second embodiment, at least the core of the The method for modification is included in U.S. Pat. No. macrocapsule is Subjected to conditions Sufficient to disrupt 5,700,848, Issue Date: Dec. 23, 1997, the entire contents of ionic crosslinking within the core of the microcapsule(s) of 35 which are hereby incorporated by reference herein. the macrocapsule, thereby rendering the core not ionically Modified-alginate, ionically crosslinked beads crosslinked, and the cell aggregate(s) is formed and con (comprising 2% AA) of 700 um average diameter were tained within the core of the macrocapsule. Synthesized by the conventional coaxial pneumatic nozzle Proliferation of the individual cells within the capsule can technique. Three photopolymerizing Solutions were used: be desirable because the number, Size and rate of formation 40 1xeosin (EE)/triethylamine (TEA)/vinyl pyrrolidinone (VP) of the cell aggregates can be directly proportional to the (each at a concentration of 0.025g/L, 2.5 ml/L, and 5 ml/L number of individual cells present within the capsule. Where respectively), 2xEE/TEA/VP (each at a concentration of proliferation of the individual pancreatic islet cells is 0.05 g/L, 5 ml/L, and 10 ml/L respectively), and 1xeosin Y desired, the invention methods of making a capsule con (EY)/TEA/VP (each at a concentration of 0.025 g/L, 2.5 taining at least one cell aggregate therein can optionally 45 ml/L, and 5 ml/L respectively). The time of exposure of the further include the step of: beads to the photopolymerizing Solution (also referred to as Subjecting the capsule to conditions Sufficient to promote Soaking time) was Systematically varied, while the photo proliferation of the at least one individual cell. polymerization time was kept constant at 5 minutes. The This step can take place either before or after the Step of mechanical integrity of the covalently crosslinked beads Subjecting the capsule to conditions Sufficient to disrupt 50 thereby Synthesized was tested by Subsequent immersion in ionic crosslinking within the core of the capsule, as 1M sodium citrate, followed by immersion in deionized described above. water. Strength analysis of the beads was performed using Conditions sufficient to promote proliferation of the indi Texture Analyzer (Stable Micro Systems, UK). The results vidual pancreatic cells include contacting the individual are presented in the following table: pancreatic cells with a Suitable culture medium. 55 In accordance with the present invention, there are also provided delivery Systems for biologically active materials. Invention delivery Systems comprise the invention micro Soaking Average strength of six microbeads, g capsules and/or the invention macrocapsules. The biologi time, min 1 x EE/TEA/VP 2 x EE/TEA/VP 1 x EY/TEA/VP cally active materials contemplated for use with the inven 60 tion delivery Systems include the biologically active 1. 157.2 215.1 12.55 materials contemplated for use with the invention micro 5 2012 403.9 136.6 capsules and the invention macrocapsules. All references cited in this application are hereby incor It is seen from the above table that as the concentrations porated herein by reference in their entirety, including the 65 of the photocomponents are doubled, the bead Strength entire contents of U.S. patent application Ser. No. 09/076, increases significantly, when the rest of the processing 339, Filed: May 11, 1998. conditions are identical. This is because higher concentra US 6,495,161 B1 21 22 tions of photocomponents can result in a greater extent of propidium iodide (AO/PI) staining. Function of the encap covalent croSSlinking contributing to the increased bead Sulated islets was examined and quantified by Static glucose Strength. For the same photopolymerizing Solution, increas stimulation (SGS). Unencapsulated (or free) islet viability ing the Soaking time of the beads in the photopolymerizing and function was assessed similarly. Briefly, the SGS tech Solution results in beads with increased Strength upon poly nique involves stimulation of islets with a high level of merization. This is because increased Soaking time allows glucose and measurement of the Secreted insulin (by RIA) in longer time for the photocomponents to diffuse in the bead response to the glucose level. During SGS, either encapsu resulting in a greater penetration distance. Subsequent pho lated or unencapsulated islets were incubated in RPMI topolymerization results in a stronger bead due to a greater culture medium containing a basal level of 60 mg% glucose extent of covalent crosslinking. It is also seen from the for 60 minutes, then transferred to a medium containing a above table that under identical experimental conditions and stimulatory level of 450 mg% glucose for 60 minutes, and at identical concentrations, EE results in the formation of a returned to basal medium (60 mg% glucose) for a further 60 stronger bead than EY. This can be attributed to the addi minutes. The Supernatant was collected at the end of each 60 tional hydrophobic interactions EE is capable of participat minute period. Insulin Secretion was assayed using RIA by ing in as compared to EY, due to the more hydrophobic 15 measuring insulin concentration (uu/ml per islet equivalent nature of EE compared to EY. count) in the Supernatant. Additional experiments were done by Systematically An increase in the Secreted insulin level above the basal varying the concentration of each of the components com Secretion during the Stimulation phase, followed by a return prising the photopolymerizing Solution, while keeping the in Secreted insulin to basal levels is a requisite for good islet concentrations of the rest of the components identical. In a function. The viability of the islets in this group of capsules certain range of concentrations it was found that each of the and in the free islets was quite high (70–85%), indicating photocomponents contribute to bead Strength. That is, in a that the encapsulation environment was not toxic to the cells. certain range of concentrations, increasing the concentration The encapsulated islets were also found to be functional of the photoinitiator, cocatalyst, and comonomer either in the group of capsules, yielding a SGS index of 5.14, as individually or in combination relative to the other compo 25 compared to an index of 9.59 for free islets. In such tests, an nents results in beads with increased Strengths after photo index >3 is indicative of healthy islets. The encapsulated polymerization. islets compared well to encapsulated islets where there was Thus, varying the concentrations of components in the no treatment with sodium citrate. The SGS index for these photopolymerizing Solution in addition to varying the Soak untreated, encapsulated islets was 6.01. ing time of the beads in Such Solutions provides a convenient These tests show that the encapsulation System employed means of controlling capsule properties like mechanical in this example (i.e., encapsulation followed by treatment Strength, porosity, and consequently in Vivo end perfor with Sodium citrate) is non-toxic to the cells, and the mance of the capsule after xenotransplantation. encapsulated islets remain healthy and retain normal func EXAMPLE 2 35 tion in Such a microenvironment. Method of Forming Macrocapsules Having at Least EXAMPLE 3 their Outer Layer not Ionically Crosslinked Method of Making Covalently Crosslinked Modified alginate (acrylate derivatized alginate or AA) Capsules not Requiring Prior Ionic Crosslinking utilized in this Example 2 was prepared in accordance with 40 the method described in Example 1. Modified alginate (acrylate derivatized alginate or AA) Freshly harvested human islet cells were encapsulated in utilized in this Example 3 was prepared in accordance with AA macrocapsules by extruding a mixture of the AA (at a the method described in Example 1. In addition, modified PEG (PEG diacrylate or PEGDA) was prepared by chemi concentration of 2% in Saline) and islet cells through a cally modifying PEG by the incorporation of acrylate syringe attached with a 23 G or 21 G needle into a Ca"-rich 45 solution (36 mM CaCl). The calcium ionically crosslinked groups. The method for modification is included in U.S. the AA matrix, resulting in Spherical ionically crosslinked patent application Ser. No. 09/076,339, Filed: May 11, 1998, beads approximately 2 mm in diameter. These beads were the entire contents of which are hereby incorporated by then immersed in a photopolymerizing Solution consisting reference herein. of eosin Y (0.025 g/l) (EY), triethylamine (2.5 ml/l) (TEA), 50 Photopolymerized AA and PEGDA coated capsules (e.g., and vinyl pyrrolidinone (5 ml/l) (VP). The photocomponents microcapsules and macrocapsules), which directly con were allowed to diffuse into the macrocapsules for 5 min. tained cells or contained cells further encapsulated in algi The macrocapsules were Subsequently transferred to a Solu nate microcapsules, were prepared in accordance with this tion with a lower level of calcium (10 mM CaCl), and then Example 3. immediately photopolymerized using 100 W high pressure 55 The apparatus for Synthesizing these capsules consisted of mercury lamps. The photopolymerization was carried out a System of coaxial nozzles Surrounded by an airjacket. The for 5 min. The resulting covalently crosslinked macrocap inner nozzle had a 22 G bore, and the outer nozzle had a 16 Sules were washed thoroughly with Saline to remove any G bore. The encapsulant (or the biologic to be encapsulated) unbound calcium or unreacted components of the photopo was to be extruded through the inner nozzle, while the lymerizing Solution. 60 biocompatible gellable material was to be simultaneously The covalently crosslinked, ionically crosslinked macro extruded through the outer nozzle. Air/nitrogen was to be capsules were treated by immersion for 5 min in a Sodium pumped through the outer jacket. The air flow rate was to be citrate solution (55 mM). Such treatment yielded a macro adjusted to yield capsules of differing Sizes. For example, capsule Solely crosslinked by covalent linkages without the increasing the air flow rate relative to the liquid flow rate ionic linkages. 65 would result in Synthesis of Smaller capsules. In vitro viability of the encapsulated human islets was A Suspension of cells (for microcapsule formation) or a examined in this group of capsules by acridine orange/ Suspension of the alginate microcapsules (for macrocapsule US 6,495,161 B1 23 24 formation) was prepared. The alginate microcapsules with Sion in accordance with the techniques described herein. The the cells encapsulated in them were produced using the average Size of the microcapsule in the current application conventional coaxial pneumatic nozzle System. was 800 umi-70 um. AA and PEGDA were dissolved in a solution comprising The initial loading of human pancreatic Single cells was in deionized water, the photoinitiator (EY), cocatalyst (TEA), the range of 5x10 to 15x10 cells/ml of the alginate Solution. In this particular application, the aforementioned and comonomer (VP). The suspension (i.e., of cells, or of the cell loading translates to approximately 1300-4000 single alginate microcapsules) was extruded through the inner cells/microcapsule. Upon aggregation of the cells within the nozzle, while the solution containing AA, PEGDA, and the microcapsule after degelling the microcapsule Via Sodium photocomponents was simultaneously extruded through the citrate treatment and after contacting the microcapsule with outer nozzle. The extruded droplets were allowed to fall into a Suitable culture medium, it was observed that a loading of Soybean oil. This resulted in a water-in-oil (w/o) emulsion, 1-15 pancreatic cell aggregates/microcapsule was achieved. in which oil prevented the dissociation of the hydrophilic The “cell aggregates' formed as described above were droplets. These droplets in the W/o emulsion were Simulta morphologically Similar to that of native, freshly isolated neously exposed to light from high preSSure mercury lamps human islets, Suggesting that the microcapsule indeed pro (100 watts). The photocomponents promoted covalent 15 Vided a low-StreSS environment for cell aggregation. These crosslinking of the AA and PEGDA biocompatible gellable cell aggregates were both viable and functional as estab material in the presence of the mercury lamp light. This lished through viability and function tests. resulted in the formation of a capsule coated by a mixture of Cell viability was assessed by acridine orange/propidium biocompatible materials (AA and PEGDA), in which each of iodide (AO/PI) staining, while function was assessed by the polymers is linked together (and possibly to each other) Static Glucose Stimulation (SGS) tests. The cell aggregates by covalent crosslinking, and in which the capsule is further had a viability 275% (usually, 70-90%), indicating that the characterized by an absence of ionic crosslinking. The core cell aggregates generated as described above were healthy of Such a capsule is either the cell Suspension or a Suspension islets. SGS indicated a stimulation index (SI) 22.0 (usually, of alginate microSpheres containing the cells. 2.0SSIs 40), Suggesting that the aforementioned cell aggre The capsules can be isolated from the w/o emulsion by 25 gates are capable of normal insulin Secretion function. filtration through a Sieve with a Suitable mesh rating, fol Successful reversal of diabetes was achieved in STZ lowed by repeated washings of the capsules with water. induced diabetic rats after transplantation of the encapsu Alternatively, the capsules can be recovered by the addition lated cell aggregates into these rats. These tests indicate that of excess water in a separatory funnel, thereby allowing the the cell aggregates are healthy and viable, and are capable of hydrophilic capsules to migrate to the water phase. This can both in vitro and in vivo function. Details of the AO/PI stain be done either in the presence or absence of a biocompatible and SGS test referenced above are described in Example #2 phase transfer agent. Repeated washings should be done to of U.S. patent application Ser. No. 09/076,339, Filed: May ensure Satisfactory removal of the oil phase. 11, 1998, the entire contents of which have already been incorporated herein by reference. EXAMPLE 4 35 This Example 5 demonstrates that viable, functional cell Preparation of Microcapsules Containing Cell aggregates can readily form in a capsule. Aggregates therein While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be Microcapsules, comprising unmodified alginate, a bio understood that modifications and variations are within the compatible gellable material which is ionically 40 Spirit and Scope of that which is described and claimed. crosslinkable, that encapsulates individual cells which are a That which is claimed is: coculture of C, B, and ö cells of pancreatic islets, were 1. A macrocapsule containing biologically active materi Synthesized by the conventional coaxial pneumatic nozzle als therein, wherein: technique. These microcapsules were then immersed in a 45 Said macrocapsule comprises a core and an outer layer, Solution of polylysine (PL), thereby resulting in a coating of wherein: PL around the alginate capsules to form an Outer layer of the the core comprises a first biocompatible gellable material biocompatible gellable material which was polyionically which is ionically crosslinkable and which optionally crosslinked. The resulting microcapsules were unmodified contains at least one microcapsule therein, wherein, alginate-polylysine (APL) microcapsules. After coating the 50 when at least one microcapsule is present, each micro microcapsules with PL, the core of these microcapsules was capsule comprises a Second biocompatible gellable liquified by degelling them through immersion of the micro material containing the biologically active materials capsules in sodium citrate (55 mM). therein, wherein: The microcapsules were then left Standing. Upon at least the Outer layer of Said macrocapsule is covalently Standing, the individual pancreatic islets cells within the 55 crosslinked or polyionically crosslinked or both poly microcapsule tended to aggregate in the microcapsule, ionically croSSlinked and covalently croSSlinked, but resulting in the formation of cell aggregates. not ionically crosslinked, and wherein: This Example 4 demonstrates that cell aggregates can when microcapsules are not present, at least the outer more easily form in a capsule when the core of the capsule layer of Said first biocompatible gellable material is is not ionically crosslinked. 60 covalently crosslinked and optionally polyionically crosslinked, but not ionically crosslinked. EXAMPLE 5 2. The macrocapsule according to claim 1, wherein the Preparation of Microcapsules for Aggregation of core of Said macrocapsule is ionically crosslinked. Proliferated Cells 3. The macrocapsule according to claim 2, wherein the 65 core of Said macrocapsule is covalently crosslinked. Alginate-PLL microcapsules, ranging from 300-1000 um 4. The macrocapsule according to claim 1, wherein the in diameter, were Synthesized by pneumatic coaxial eXtru core of Said macrocapsule is covalently crosslinked. US 6,495,161 B1 25 26 5. The macrocapsule according to claim 4, wherein the 21. The delivery system according to claim 20, wherein core of Said macrocapsule is not ionically crosslinked. Said living cells are Selected from the group consisting of 6. The macrocapsule according to claim 1, wherein the pancreatic islet cells, tumor cells, human T-lymphoblastoid core of Said macrocapsule is not ionically crosslinked. cells, islet of Langerhans cells, dopamine Secreting cells, 7. The macrocapsule according to claim 1, wherein at nerve growth factor cells, hepatocytes, adrenalin/ least the outer layer of each of Said microcapsules is angiotensin Secreting cells, parathyroid cells, and covalently crosslinked. norepinephrine/metencephalin Secreting cells. 8. The macrocapsule according to claim 7, wherein the 22. The delivery system according to claim 19, wherein core of each of Said microcapsules is covalently crosslinked. Said biologically active material comprises biological mate 9. The macrocapsule according to claim 2, wherein at rials. least the outer layer of each of Said microcapsules is 23. The delivery system according to claim 19, wherein covalently crosslinked. Said biologically active material comprises pharmacologi 10. The macrocapsule according to claim 9, wherein the cally active drugs. core of each of Said microcapsules is covalently crosslinked. 24. The delivery system according to claim 19, wherein 11. The macrocapsule according to claim 3, wherein at 15 Said biologically active material comprises diagnostic least the outer layer of each of Said microcapsules is agents. covalently crosslinked. 25. The delivery system according to claim 19, wherein 12. The macrocapsule according to claim 11, wherein the Said biologically active material comprises pancreatic islet core of each of Said microcapsules is covalently crosslinked. cells. 13. The macrocapsule according to claim 4, wherein at 26. A method of making a macrocapsule containing least the outer layer of each of Said microcapsules is biologically active materials therein and having Substan covalently crosslinked. tially no ionic crosslinking in at least the outer layer thereof, 14. The macrocapsule according to claim 13, wherein the Said method comprising Subjecting an ionically croSSlinked core of each of Said microcapsules is covalently crosslinked. macrocapsule which contains biologically active materials 15. The macrocapsule according to claim 5, wherein at 25 least the outer layer of each of Said microcapsules is therein to conditions Sufficient to disrupt the ionic crosslink covalently crosslinked. ing in at least the outer layer thereof, wherein: 16. The macrocapsule according to claim 15, wherein the a macrocapsule is formed having Substantially no ionic core of each of Said microcapsules is covalently crosslinked. crosslinking in at least the outer layer thereof, wherein: 17. The macrocapsule according to claim 6, wherein at Said biologically active materials are optionally contained least the outer layer of each of Said microcapsules is within at least one optionally present microcapsule, covalently crosslinked. wherein: 18. The macrocapsule according to claim 17, wherein the When microcapsules are not present, at least the Outer core of each of Said microcapsules is covalently crosslinked. layer of the macrocapsule is covalently crosslinked and 19. A delivery system for biologically active materials 35 optionally polyionically crosslinked, and when at least comprising a macrocapsule according to claim 1, wherein one microcapsule is present, at least the outer layer of Said biologically active material is Selected from the group the macrocapsule is covalently crosslinked or polyioni consisting of living cells, biological materials, pharmaco cally crosslinked or both covalently crosslinked and logically active drugs, and diagnostic agents. polyionically crosslinked. 20. The delivery system according to claim 19, wherein 40 Said biologically active material comprises living cells. k k k k k