MXPA97007492A - Microencapsulacion farmaceut - Google Patents

Abstract

A method for producing a microencapsulated pharmaceutical formulation comprising causing a dye to bind to the surface of pharmaceutical particles or a radiant energy source to the dye in the presence of liquid polymerizable or polymerizable material to cause the material to crosslink, produce a conformational crosslinked polymer on particular surfaces. Preferably, the polymer provides an immunoprotective layer around the particles, as long as the pharmaceutical components leave the microcapsules. Microencapsulated pharmaceutical formulations and their medical use are also described, especially for the treatment of diabetes by encapsulating insulin-secreting cells.

Description

PHARMACEUTICAL MICROENCAPSÜLACIQN FIELD OF THE INVENTION This invention relates to a microencapsulation method, especially for pharmaceutical purposes. This is particularly applicable to the preparation of pharmaceutical formulations which comprise immunoisolated cells, which produce and secrete therapeutic substances, for example insulin, and cut * the medical use of those formulations.

ANTgCBDEMTgS Dg LA INVEMCIOM The cellular immunoisolation is a procedure which involves the placement of cells or cell agglutinates within a semipermeable membrane barrier before transplantation to avoid rejection by the immune system. This can be applied for all cell types that secrete a bioactive substance either naturally or through genetic engineering means. In practice, the main work has been done with tissues that secrete REF: 25734 insulin. The molecular weight cut-off (Pm) of the encapsulation membrane can be controlled by the encapsulation procedure to exclude inward diffusion of inoglobulins and lytic factors from the complement system, but allow the passage of smaller molecules such as glucose and insulin. The barrier therefore allows ß cells to respond physiologically to changes in blood glucose but avoids any contact with the components of the immune system. Under these circumstances, xenogeneic tissue could be used, thus eliminating the source of the problem, and immunosuppression would not be required to prevent rejection or recurrence of the disease since the grafted islets could be isolated from the host's immune system. Studies, which explored the immunoisolation principle using diffusion chambers to isolate islet tissue or pancreatic fragments, were performed with little success (reviewed in 1, 2), although transient relief and hyperglycemia were achieved, the available membrane materials did not they allowed the experimental stimulation / secretion of insulin transport (3) More recently, the use of hollow capillary fibers in conjunction with isolated allogeneic or xenogeneic islets within a semipermeable chamber as an extracorporeal or intravascular insulin secretory device has been successful used for the short-term regression of diabetes in roedo res (4, 5), dogs (6,7) and monkeys (8). Exraporeal or intravascular methods, although essential for testing the validity of the encapsulation technique, are not suitable for human applications, especially in young children. The diffusion chambers - the method of choice for human applications - are, however, still hampered by problems of consistency (9). Several methods of manufacturing polymer capsules have been developed, based on different engineering techniques. The encapsulation procedures are more commonly distinguished by their geometrical appearance, i.e. micro or macrocapsules. In macroencapsulation, cells or cell agglutinates are encapsulated within hollow fibers of selective permeability or flat sheet membranes. Since they are made of thermoplastics, these capsules are mechanically stable and relatively easy to recover. Several researchers have reported the successful use of thermoplastic hollow fiber capsules to transplant islet cells in models of diabetes in rodents. We have previously reported that, given the appropriate surface microgeometry and chemical composition, the reaction of the tissue formed around implanted thermoplastic-based macrocapsules is minimal in both the brain (10) and the peritoneal cavity (11, 12). the mouses. We have also reported that the long-term brain survival of macroencapsulated PC12 cells in a dopaminergic cell lineage, when transplanted through the species (13) and that these * implants significantly improve behavior in experimental rat and primate Parkinsonian models (14) Using the same encapsulation systemLacy and colleagues have reported the correction of streptozotocin-induced hyperglycemia in rats implanted with macroencapsulated subcutaneous islet cells (15). More recently, Scharp and his colleagues have reported the survival of 2 weeks of encapsulated human islets in diabetic patients using the same acrylic-based macroencapsulation system (16). Using a similar acrylic system, we have recently reported the successful transplantation of bovine chromatin cells into the intrathecal space of humans suffering from terminal cancer pain. The explanted devices showed an absence of reaction of the host to the capsule as well as viable chromaffin cells. Recovered, the capsules released amounts of catecholamine comparable to those measured in vitro before transplantation. Although mechanically stable and biocompatible, hollow fiber based systems require a low packing density to allow the proper viability of the transplanted cells. The requirement to scale this system of material to correct * diabetes in a human may require a device of 50 m of impractical length. Another limitation of this technique is the thickness of the capsule wall and its potential influence on the diffusion kinetics of glucose. The diffusion barrier may incur short-term hypoglycemic episodes due to excessive insulin secretion. We have shown that macroencapsulation using semipermeable hollow fibers is a viable technology for the xenogeneic transplantation of endocrine tissue in humans. Although this technology has also been used experimentally for the encapsulation and transplantation of islets, it is not suitable for effective packaging. The wall thicknesses of the capsules are usually a minimum of 100 μm and in the hollow fiber the cells are immobilized within a hydrogel matrix core typically 500-600 μm in diameter. This creates diffusion distances of several hundred μm between host and transplanted cells and can adversely affect diffusion kinetics. This diffusion barrier can induce a "delay" of significant time in the detection of glucose levels within the blood that causes phase shifts in insulin secretion and thus "the erratic regulation of glucose levels in the blood." Also, the geometrical restrictions of fiber technology result in very poor packing densities and up to several meters of encapsulated fiber from transplanted islet may be required.A solution to these problems may be the use of the microencapsulation technique. microencapsulation, cell agglutinates are immobilized in hydrogel microspheres of 500-600 μm Typically the semipermeable membrane is formed on the surface of the microsphere.Several chemical systems have been used.In the most common form, the membrane of the capsules formed by ionic or hydrogen bonds between two weak polyelectrolytes; typically an acidic polysaccharide, such as alginic acid, and a cationic polyamino acid, such as polylysine. Practically, the capture of the cells is obtained by the gelation of a charged polyelectrolyte induced by exposure to a multivalent counter ion. A counterpolyelectrolyte is then absorbed interfacially onto the immobilization matrix of the cell. The microcapsules have an ideal shape for diffusion. In vitro tests showed that the release of insulin from microencapsulated islets was equivalent to that of nonencapsulated cells, but they are mechanically fragile, particularly when polyelectrolytes are used, and they are also chemically unstable since they depend only on the ionic bonds for their integrity, leading to the rupture of the microcapsules after several weeks of the implantation in the brain of non-human primates.The intraperitoneal implantation of such microcapsules has been reported to reverse the diabetes in experimental models of diabetes of rodent and more recently in humans.The poor biocompatibility of the system however gives rise to questions about its use in young diabetic patients.In an effort to correct stability and provide biocompatibility, Sefton and his collaborators are developing microcapsules based on the precipitation of a solution polymeric organic around islet agglutinates. The problems of toxicity and disparity of the permeability characteristics of the solvent still prevent this method. In general, the use of microcapsule systems in humans is limited by problems of long-term stability and process limitations to ensure a uniform thin coating on * a large volume of islets.

BRIEF DESCRIPTION OF THE INVENTION According to one aspect of the present invention there is provided here a method for producing a microencapsulated pharmaceutical formulation; The method comprises causing a dye to bind to the surface of particles or agglutinates of pharmaceutical particles (hereinafter generally referred to as "particles") and to apply radiant energy to the dye in the presence of a liquid (or polymerizable) polymeric material to make The material is crosslinked, producing a conformational layer of cross-linked polymer on the particulate surfaces. Desirably the dye binds specifically to the surface (particularly to islet surfaces or cell membranes). In general, the dye is a fluorescent dye. The mechanism of crosslinking may involve a less induced excitation of the dye to its triplet state, creating free radicals from a suitable electron donor. These free radicals initiate the crosslinking of the polymer resulting in the formation of the hydrogel. The "pharmaceutical" particles do not need to be directly pharmaceutical in effect, but they can be for example cells or agglutinates of cells that produce and secrete a pharmaceutically active substance. The polymer suitably provides an immunoprotective layer, i.e., one that prevents the body's immune system from mounting an immune response to the particles, and at the same time allows the therapeutic components of the particle to exit the microcapsules. This is particularly appropriate where the particles comprise cells which produce and secrete a therapeutic substance such as a protein, the polymer is permeable to the therapeutic substance and cellular nutrients, but of course not to the cells themselves. The polymer is preferably a hydrogel, which can be crosslinked by irradiating the dye with a suitable energy source such as a laser, or converted to a hydrogel after crosslinking. Typically the material before crosslinking contains polymer molecules, for example 400 g / mol-18500 g / mol. The dye can be applied to the surface of the particles, by dyeing or other means, before contacting the thus treated cells with the liquid polymeric material (or polymer former), and exciting the dye to crosslink the polymer. The use of specifically incorporated dyes in the membrane allows to restrict the diffusion phenomenon, thereby improving the thickness of the coating and reducing phototoxicity Alternatively, the material can be contacted with the particles simultaneously with the dye. Molecules of the material (for example chains or micelles) can be labeled with a dye, and be able to bind to the particles, for example where the particles are cells, the material can be able to bind to the cell membrane, such as by amphiphilic interactions, by protein binding, or by other chemical means, or by any receptor-ligand or antibody-antigen interactions. The polymer is crosslinked by exciting the dye with an appropriate energy source, for example laser light of an appropriate frequency. The dye with the polymer solution (or polymer former) and the particulate material are suitably placed in a laser integration chamber which ensures uniform polymerization through the uniform distribution of the laser light. The * energy is supplied until a conformational coating of the desired thickness is formed around the material.

"Conformational" coating means a thin coating that conforms to the shape of the material, for example cells or cell agglutinates. In a further aspect, the present invention provides a microencapsulated pharmaceutical formulation that can be obtained by the above method. In a further aspect of the present invention there is provided a pharmaceutical formulation in which a particulate pharmaceutical material is coated in a conformational manner with a polymer crosslinked covalently by the action of an irradiated dye. In "'one more aspect, the present invention provides the microencapsulated pharmaceutical formulations mentioned above for medical use. In a further aspect, the present invention provides the use of a microencapsulated pharmaceutical formulation comprising cells that secrete insulin in the preparation of a medicament for the treatment of diabetes, wherein the cells are coated in a conformational manner with a covalently crosslinked polymer. by the action of an irradiated dye In a further aspect, the present invention provides an implant for the therapeutic regulation of glucose comprising microencapsulated particles obtainable from the above method.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the principle of interfacial photopolymerization.

Figure 2 shows an installation for the microencapsulation of biological particles using an integrating sphere for homogeneous irradiation. Fiber feed allows use in a sterile environment. Figure 3 is a graph representing the polymerization time as a function of the concentration of photosensitizer (Eosin Y). Figure 4 is a graph representing the polymerization time as a function of the electron donor concentration (TEOA). Figure 5 is a graph showing the influence of the laser intensity on the polymerization time. Figure 6 is a bar graph showing the influence of the concentration of PEG on the polymerization time. Figure 7 is a graph representing the pore diameter of the PEG-based hydrogels calculated from hydraulic permeability measurements. Figure 8 is a graph showing the relative polymerization time of the PEG-based polymer for different dyes.

Figure 9 shows an encapsulation with eosin Y dye of (a) islets of rat Langerhans showing a "" coating of 50 μm thickness in oblique illumination (bright area below, dark zone above the islet); (b) agglutinated TC tet beta, with a coating of 20 μm; (c) encapsulated rat islet using eosin NCS dye (10 to 20 μm coating visible as a bright ridge above the islet, oblique illumination). The islets have an average diameter of 200 μm.

DETAILED DESCRIPTION OF THE INVENTION Our advance in encapsulation technology incorporates the advantages of both micro and macroencapsulation techniques previously described. The mechanical stability of a covalently crosslinked hydrogel is combined with the biocompatibility and size of a microencapsulating hydrogel. A customary laser polymerization process has been developed which individually coats or coats cell agglutinates in large volumes with a permanently crosslinked hydrogel skin of approximately 10 to 20 μm in thickness. The uniform, thin immunoisolating membrane is called "conformational coating". This process allows a distance of "minimum glucose diffusion to beta cells and a maximum packing coefficient for cell transplantation." Based on the assumption that 500,000 islet equivalents are required to reverse human diabetes, it can be assumed that require 2 ml of conformationally coated islets for the therapeutic regulation of glucose.The conformational coating encapsulation methodology is illustrated in Figure 1. The islets are first suspended in a solution composed of a fluorescent membrane staining dye. attached to the membrane is an energy donor for the subsequent polymerization reaction.After staining, the islets are washed and resuspended in a polymer solution which will polymerize easily in a hydrogel around the islets after excitation of the dye with a appropriate laser frequency, the islets are then placed in a camera Laser design as usual (Figure 2) that ensures uniform polymerization through a uniform distribution of laser light until a conformational coating of the desired thickness is formed around the suspended islets. In preliminary experiments, we have studied the following parameters of the conformational coating procedure: a) Selection ¿ß. a fciafcß. ajea Specific staining of membranes ealularaa Two types of dyes can be considered: (i) Free diffusion dyes, (for example, eosin Y, eosin B, fluorescein, Rhodamine). (ii) Dyes capable of specifically incorporating into the cell membrane (e.g., Dil, ElO, eosin isothiocyanate, fluorescein and phospholipids derived from eosin). (i) Free diffusion dyes: Homogeneous impregnations were obtained using 1 mM eosin Y solutions. Impregnation times of 1 minute to 10 minutes were used, followed by 1 or two washes. To visualize eosin uptake, a fluorescence microscope was used with focus on islets of impregnated and agglutinated Rat Langerhans of a beta cell lineage. It was shown that eosin Y was absorbed into the cell cytoplasm 5 minutes after staining in a 1 mM solution. (ii) Dyes for membrane staining: To reduce diffusion problems, we chose several dyes capable of binding to cell membranes. •• The first two are cationic membrane markers that belong to the family of dialkylcarbocyanines, Dil and DiO. Those amphiphilic markers interact with the double lipid layer. Eosin 5-isothiocyanate (Eosin NCS), which is known to bind to membrane proteins, was also tested. Also, a phospholipid derived from fluorescein (FLPE) appears to be a promising dye for microencapsulation. The islets of rat Langerhans and the agglutinates of the beta cell lineage have been impregnated with the dyes mentioned above. The fluorescence microscopy with focal showed a fluorescent layer that indicates a specific permanent absorption of the dyes on the surface of the membrane. The FLPE impregnation parameters were optimized. Impregnation times greater than 5 minutes and dye concentrations greater than 250 μM do not significantly increase dye incorporation. The best results were obtained at a temperature of 4 ° C. These encouraging results using specific staining open the way for new applications. An alternative way of controlling the thickness of the coating could be the use of polymeric chains or micelles, marked with a dye, and having a final group capable of binding to a cell membrane. This binding can be achieved by amphiphilic interactions, by protein binding (as with the isothiocyanate group), or by other chemical means. The absorption wavelength of the dye has to be adjusted to the laser-eosin source or fluorescein for an argon laser. The length of the chains will give rise to the control of the thickness of the coating, and the molecular weight of a high polymer could prohibit the permeation of the membrane, thereby eliminating a possible phototoxic action. For example, a dextran labeled with eosin, which possesses lipophilic or charged end groups, could satisfy the need for conformational polymer coatings with growth. Another way to achieve the specific binding of the dye could be the use of immunochemistry, coupling eosin to the specific antigens of an islet. The specificity and absence of membrane permeation could again allow the conformational type of the islet. (b) Development of the photopolymer The polymer system used to conformationally coat beta cells is a biocompatible polyethylene glycol (PEG) based on hydrogel. This consists of three elements: an aqueous solution of poly (ethylene glycol 400 diacrylate) (PEG-DA) or poly (ethylene glycol multiacrylate 18 500) (PEG-MA), the initiator of the reaction, triethanolamine (TEOA), and an appropriate dye that acts as a photosensitizer (eosin Y, eosin NCS, FLPE, etc.). Three parameters of the process need to be controlled simultaneously, to form the thin uniform coating and ensure both proper functioning as an immunoprotective membrane and its mechanical durability for processing and transplantation. These parameters are the concentration of dye, the amount of initiator of the reaction for the polymerization, and the intensity of the laser, all optimized as a function of the reaction time. The reaction time was determined experimentally using an optical holographic technique for the PEG17'18 system. A PEG photopolymer, contained in a quartz cuvette, was exposed to an interference pattern created by the intersection of 2 argon laser beams (wavelength of 514 nm), thereby recording a lattice in the photopolymer. A low power He-Ne laser is used to test the growth of this grid by measuring its diffraction efficiency. The polymerization time is defined as the time necessary to achieve 90% of the maximum diffracted intensity. First the concentration of dye necessary to give a fast and complete polymerization reaction at a maximum laser intensity of 1 W / cm2 was determined. The TEOA concentration of 90 mM was not used as a factor limiting the reaction rate. Figure 3 presents the polymerization time as a function of the eosin Y concentration for a 10 μm thick film. An optimum concentration was demonstrated, which corresponds to 70% of the absorption of light through the film. Concentrations of higher dyes 1 lead to inhomogeneous polymerization due to incomplete bleaching of the dye. From those measurements, 1 mM dye concentrations were used for cell microencapsulation. Using the optimized dye concentration and an irradiation intensity of 1 W / cm2, the TEOA concentration of minimum reaction initiator-necessary to complete the polymerization was determined. In this manner, a minimum amount of TEOA monomer not consumed will remain after the polymerization. Figure 4 shows the influence of the TEOA concentration on the polymerization time. A concentration of at least 90 mM was required to maximize the reaction rate. The irradiation time necessary for the photopolymerization was determined using the optimized reaction parameters. Figure 5 shows the relationship between polymerization time and laser intensity. For example, a laser intensity of 1 W / cm2 was required for 13 seconds to complete the polymerization.

The use of a low concentration of PEG-DA (concentration of 10% (weight / volume) in physiological medium) resulted in a very low reaction rate (Figure 6). In addition, the gels obtained were mechanically brittle, and showed a higher water permeability than a 5% (weight / volume) concentrated agarose gel (Figure 7). Based on these measurements, PEG concentrations of 20% to 30% were used. From a photochemical point of view, the dyes mentioned in section a) were very different. The photopolymerization speed measurements by the holographic technique gave irradiation times of 17 minutes for the DiO and 10 minutes for the Dil (see Figure 8). The eosin NCS was 1.4 times slower than the eosin Y. This behavior can be explained by the high conversion efficiency of the eosin triplet, which allows a high quantum yield of the reaction. The FLPE induces the crosslinking of the photopolymer twice the time required for eosin Y. Other dyes have also proven to be efficient photosensitizers, for example, eosin B, rose bengal and stilbene. cl Encapsulation gives biologic cellular agglutinates i) Encapsulation with free diffusion dyes: The microencapsulation of the islets of Langerhans has been previously reported. ' Encapsulation was evaluated using the eosin Y photosensitizer. The primary cells (islets of rat Langerhans) and a genetically engineered mouse beta cell line (TC tet beta) have been successfully encapsulated (see Figure 9 (a) and (b)). ). A 30% (w / v) PEG-DEA solution containing 90 mM TEOA was used, and the islets were impregnated in a physiological solution of 1 mM eosin Y for 5 minutes. The intensity of irradiation was 1 W / cm2 for 20 s. Coating thicknesses of 50 μm down to 20 μm were obtained. It has been shown that the coating thicknesses can be controlled both by the irradiation time and by the polymer concentration. Prolonged irradiation times results in a thicker coating due to diffusion of the dye, while a higher polymer concentration results in a thinner coating due to the increased viscosity. However, two main advantages arise; First, the absorption of the dye within the cells leads to toxicity due to the photogeneration of free radicals. Second, the sedimentation and convextion generate non-uniform flows around the islet, resulting in irregular coatings (tail formation) and can lead to polymerization of the polymer solution mass. To overcome these problems, specific membrane staining dyes have been demonstrated for microencapsulation. (li) Encapsulation with membrane staining dyes: Staining of the rat islets of Langerhans was carried out in a 1 mM NCS eosin solution. Due to the low kinetics of incorporation, prolonged impregnation times (up to 2 hours) were necessary. After the two washes in saline and resuspension in the polymer solution "of PEG-DA at 30% (weight / volume), the islets were irradiated with intensities that fluctuated from 100 mW lW / cm2 for 10 to 30 seconds. conformational coating with thicknesses of 10 to 20 microns In a single suspension, approximately 80% of the islets were encapsulated with a visible coating surrounding the whole islet (see Figure 9 (c).) Comparative encapsulation experiments gave better results with the eosin NCS dye attached to the membrane than with the eosin Y, giving a thinner conformational coating, without the "tail effect." The tendency to induce massive polymerization of the islet solution was also reduced. encapsulated in culture showed that they survive the photopolymerization process, additional studies are necessary to evaluate the complete functionality of the islet. s demonstrated that the use of membrane-bound dyes for the microencapsulation of biological particles allows to reduce the thickness of the coating, thus giving kinetics of faster release. New dyes are currently under investigation.

Fluorescein-derived phospholipids such as fluorescein DHPE (FLPE) have been shown to bind specifically to the cell membrane and induce polymerization with high efficiency. Eosin DHPE can also be used, its high efficiency in the triplet state leads to an efficient photopolymerization. di Toxicity of the process During the microencapsulation process, the biological particles were placed under conditions that are far from those of the biological media. Damages of a chemical, thermal, mechanical or photochemical nature can limit the viability or functionality of the cell. The viability of the cell was measured by means of a fluorescent vital staining method (fluorescein diacetate (FDA) or calcein AM assays), and the functionality was evaluated by the dynamic measurement of insulin secretion under the stimulation of the glucose The evaluation of chemical toxicity was achieved by incubating the islets of Langerhans in the prepolymer solution (PEG-DA at 30% (weight / volume) - and 90 mM TEOA). The islets showed a 100% viability until a 4 minute incubation. Since the microencapsulation process lasts approximately 30 s, it is expected that the prepolymer will not induce chemical damage. The toxicity of the dye impregnation has also been verified. The chemical toxicity for eosin Y and FLPE has not been measured under the staining conditions mentioned above. Without 2 * 7, however, the eosin NCS has been shown to inhibit insulin secretion after 10 minutes of impregnation in a 1 mM solution. The cell agglutinates are extremely sensitive to mechanical stress. The disintegration of cell agglutinates has been reported after exposure to a shear stress of 5 N / m for 10 s. These types of shear stress are easily obtained21 in other microencapsulation processes such as extrusion of microdroplets through nozzles. In the interfacial depolymerization process, almost no shear stress occurs. No cell disintegration was observed during our encapsulation experiments. The heating of the cell agglutinates caused by the absorption of laser light or the heat of polymerization can damage cellular tissues. Computer calculations have shown that under the photopolymerization conditions used (eg intensity less than or equal to 1 W / cm2, concentration of 1 mM dye, 10 to 30 s of polymerization) a maximum temperature increase of 1.4 degrees is expected. As a consequence, damage from the laser-induced heating is not expected.

Phototoxic effects can result from the detoxification of the dyes via the generation of toxic free radicals. The evaluation of this effect on cell viability is shown in Figure 10. The islets of rat Langerhans, impregnated with a solution of eosin Y lmM, were exposed to various intensities and times of laser irradiation, and viability was measured at day after. 100% viability was measured for irradiation time of 10 s and laser intensities ranging from 50 mW / cm2 to lW / cm2. Longer irradiation time "leads to a decrease in viability, together with a reduced insulin secretion.Thus, irradiation time of less than 30 s has to be used for the microencapsulation process, the viability of the rat islets was measured exposed to the laser, stained with FLPE (see Figure 11) After staining in a 20 μM FLPE solution for 5 minutes at 4 ° C, 100% viability was obtained at an irradiation intensity of 50 mW / cm2 , and from 70% to 1 W / cm2, these results make the FLPE dye a promising photoinitiator for microencapsulation.

The viability of the encapsulated Rat Langerhans islets was also evaluated. After encapsulation in a 30% PEG-DA membrane using a 1 mM eosin Y concentration and an irradiation of 10 s at 1 W / cm2, a viability of 70% ± 10% was measured (mean of eight samples ± standard deviation) . This last result implies that minor damage occurs in the cell membrane during the entire microencapsulation process. e) Development of a photoactivated hydrogel based on the bifurcation of the b nzofßnone A second encapsulation technology material will be developed using heterobifunctional binders based on the benzophenone (BP) chemistry. The advantage of this chemical technology is its ability to effectively crosslink virtually any hydrogel material. The chemicals of BP are activated by light in the near UV range (350 nm) and will react easily with the C-H bonds. In preliminary experiments it was observed that islet cells could tolerate exposure to 100 mW per cm for several minutes. The BP chemicals can be designed as usual so that one end reacts thermochemically with a specific chemical functional group and the other end containing the BP chemical can then be photoactivated to initiate a crosslinking reaction. The chemical of BP can also be used effectively in an aqueous environment. In collaboration with Dr H Sigrist of the University of Bern, our laboratory developed a bifunctional binder that can be derived thermochemically on albumin with the BP unit on the other end available for crosslinking. Albumin-BP could prove to be an ideal system to conformally coat a polymeric skin around the ß cell agglutinates. This can be achieved by first adsorbing the albumin-BP on the β-cells and then photoactivating the β-cells in a hydrogel solution. In this way, albumin-BP adsorbed on the cell membrane will be fixed immediately while at the same time a hydrogel skin is reticulated around the ß cell agglutinates. The albumin-BP can be produced in large quantities and sealed on a lineage of β cells to be compared with the interfacial polymerization reaction described above. The derivation of a phospholipid, for example phosphatidiethanolamine (PE), with chemical products from BP should also be evaluated. If this is achieved, the lipid can be effectively incorporated into the membrane and the BP product can then be activated to polymerize any hydrogel system around the islet aggregates. The advantage of using a dye compared to the albumin derived is the proximity and immobilization-of the dye within the membrane compared to the adsorbed albumin system. The polymer "skin" formed by the activation of BP fixed to the membrane dye can form a more tightly bound hydrogel membrane. Several hydrogel-based systems must be tested experimentally to cross-link with BP chemical products. Those include pure PEG, polyvinyl alcohol and agarose. It is known that these hydrophilic systems are highly biocompatible since they show the lowest protein adsorption and therefore prevent any significant cellular adhesion. These crosslinked hydrogels may prove to be especially interesting since they could show improved mechanical and chemical stability compared to the polyelectrolyte systems currently used for transplant studies.

REFERENCES 1. Hegre, Ó. D. Transplant of islet cells in "The Diabetic Pancreas", eds. Volk and Arquilla, Plenum New York, 1985. 2. Tze, W. J. and Tai, J.: Manipulation of pancreatic islet cells in alotransplantation. Trans Proc. 14: 714, 19823. Theodoron, N. A., Vrbova, H., Tyhurst, M., and Howell, S.-L .: Problems with the use of polycarbonate diffusion chambers for the transplantation of syngeneic pancreatic islets in rats. Diabetology 18: 313, 1980. 4. Chick, W. L., Perna, J. J., Lauris, V., Low, D., et al: Artificial pancreas using living beta cells: effects of glucose homeostasis in diabetic rats, Science 197: 780, 1977.

. Tze, W. J., Wong, F.C. and Chen, L.M.: Implantable artificial capillary unit for halograft and pancreatic islet xenograft, Diabetology 16: 247, 1979. 6. Tze, W. J., Tai, J., Wong, F.C. Davis, H.R.: Studies with implantable artificial capillary units containing rat islets in diabetic dogs. Diabetology 19: 541, 1980. 7. Sullivan, S.J., Maki, T., Borland, K.M., Mahoney, M-- D., Solomon, B., et al Pancreas, artificial biohybrid; long-term implantation studies in diabetic, pancreatectomized dogs. Science 252: 718, 1991. 8. Syn, A.M., Parisus, W., Healy, G.M., Vacek, I., et al: The use in diabetic rats and monkeys of artificial capillary units containing islets of cultivated Langerhans. Diabetes 26: 1136, 1977. 9. Colton, C, Avgoustiniatos, E. S. Bioengineering in the development of artificial hybrid pancreas. J. Biochem. Eng. 113: 152, 1991.

Winn, S.R., Aebischer, P., Galletti, P.M. Tissue-brain reaction with selective permeability polymer capsules. Biomed. Mater. Res., 23: 31, 1989.

Christenson, L., Aebischer, P., McMillan, P., Galletti, P. M. Tissue reaction with intraperitoneal implants: species and effect differences of corticosteroids and doxorubicin. J. Biomed Mater. Res., 23: 705, 1989.

Christenson, L. Wahlberg, L., Aebischer, P. Contribution of mast cell cells to tissue reaction with polymer capsules implanted intraperitoneally and effect of local release of corticosteroids. J. Biomed. Mater. Res. 25: 1119, 1991.

Aebischer, P., Tresco, P.A., Winn, S.R., Greene, L.A., Jaeger, C. B. Long-term cross-species brain transplantation of a cell lineage that secretes dopamine encapsulated in polymer. Exp. Neurol. 111: 269, 1991.

Tresco, P.A., Winn, S.R., Tan, S., Jaeger, C.B., Greene, L.A., Aebischer, P. Transplantation of PC12 cells encapsulated with polymer reduces the rotational behavior induced by lesions. Cell. Transpl. , in press.

Lacy, P.E., Hegre, 0. D., Gerasimidi-vazeou, A., Gentile, F.T., Dionne, K. E .: Maintenance of normoglycemia in diabetic mice by xenograft-cutaneous islets encapsulated. Science 24: 1782, 1991.

Scharp, D.W., Lacy, P.E., Santiago, J.V., McCullough, C.S., et al. Results of our first intraportal islet allografts in insulin-dependent diabetic patients of type 1. Transpl. 51:76, 1991.

Jordán, 0. and Marquis Weible F. "Holographic control of the formation of hydrogen for biocompatible photopolymers", 2629, 46 (1995). 18. Jordán, 0. and Marquis Weible F., "Characterization of the photopolymerization by the holographic technique applied to a diffuse hydrogel", presented to Applied Optics (1995). 19. Sawhney, A.S., Parhak C. P. and Hubbell, J.A., "Modification of the surfaces of the islets of Langerhans with immunoreactive poly (ethylene glycol) coatings via interfacial polymerization", Biotech. Bioeng. 44: 383-386 (1994).

. Hubbell J. A., US Patent No. W093 / 16687 PCT / US93 / 01776, 1993. 21. Hua J. M., Erickson L. E. Ylin T. Y. and Glasgow L. A., "A Review of the Effects of Cutting and Interfacial Phenomena on Cell Viability." Crit. Rev. Biotech. 13: 305-32 (1993).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, it is Claims as property what is contained in the following:

Claims (22)

1. A method for producing a microencapsulated pharmaceutical formulation, characterized in that it comprises causing a dye to bind to the surface of pharmaceutical particles or agglutinated particles and apply a radiant energy source to the dye in the presence of a liquid polymerizable material to make the material reticulate, produce a conformational layer of cross-linked polymer on the particulate surfaces.

2. The method according to claim 1, characterized in that the polymer provides an immunoprotective layer, and at the same time allows the therapeutic components of the particles to leave the microcapsules.

3. The method according to claim 1 or claim 2, characterized in that the pharmaceutical formulation comprises cells or cell agglutinates which produce and secrete a pharmaceutically active substance.

4. The method according to claim 3, characterized in that the polymer is permeable to the nutrients of the cell

5. The method according to any of the preceding claims, characterized in that the dye is excited up to a triplet state when irradiated by the source of energy, and leads to the formation of free radicals which initiate crosslinking.

6. The method according to any of the preceding claims, characterized in that the dye is able to bind to the surface of the particles.

7. The method according to claim 6, characterized in that the dye is Dil, DiO or eosin NCS, eosin Y, fluorescein DHPE or eosin DHPE.

8. The method according to any of the preceding claims, characterized in that the polymerizable material is a hydrogel which can be crosslinked by irradiating the dye with a laser, which can produce light of an appropriate frequency.

9. The method of compliance with any of the claims. Prior art, characterized in that the dye is applied to the surface of the particles before contacting the particles with the liquid polymerizable material and energizing the dye to crosslink the material. The method according to any of claims 1 to 8, characterized in that the polymerizable material is contacted with the particles simultaneously with the dye.

11. The method according to any of the preceding claims, characterized in that the energy is supplied until a conformational coating of the desired thickness is formed around the material. Í2. A microencapsulated pharmaceutical formulation, characterized in that it is obtained by the method according to any of claims 1 to 11.

13. A microencapsulated pharmaceutical formulation, characterized in that a particulate pharmaceutical material is coated in a conformational manner with a polymer crosslinked covalently by the action of an irradiated dye.

14. The formulation according to claim 13, characterized in that the polymer coating is approximately 10 to 20 μm thick.

15. the formulation according to claim 13 or claim 14, characterized in that the polymer provides an immunoprotective layer, which * - while allowing the therapeutic components of the particles to leave the microcapsules.

16. the formulation according to any of claims 13 to 15, characterized in that the particulate material comprises cells or agglutinates or aggregates of cells, which produce and secrete a pharmaceutically active substance.

17. The formulation according to any of claims 13 to 16, characterized in that the polymer is permeable to the nutrients of the cell.

18. The formulation according to claim 16 or claim 17, characterized in that the particles are insulin-secreting cells.

19. The microencapsulated pharmaceutical formulation according to any of claims 12 to 18 for medical use.

20. The use of a pharmaceutical formulation * -microencapsulated comprising cells that secrete insulin in the preparation of a medicament for the treatment of diabetes, wherein the cells are coated in a conformational manner with a polymer crosslinked covalently by the action of an irradiated dye.

21. The use according to claim 20, wherein the cells secreting insulin are β-cells.

22. An implant for the therapeutic regulation of glucose, characterized in that it comprises particles that can be obtained by the method according to any of claims 1 to 11.