ENCAPSULATING AGENT WITH IMPROVED PROPERTIES ADAPTED FOR CELL ENCAPSULATION

Abstract

The invention is directed to a three-dimensional polymer network for encapsulating a pharmaceutical ingredient, the polymer network comprising (a) at least one first polymer; and (b) at least one cross-linking agent. The three-dimensional polymer network is remarkable in that the at least one first polymer comprises a first polyuronate derivative, the first polyuronate derivative being modified with a hydrophobic moiety; and in that the at least one cross-linking agent is calcium chloride and/or a cyclodextrin derivative.

Claims

1. A three-dimensional polymer network for encapsulating a pharmaceutical ingredient, said three-dimensional polymer network comprising a) at least one first polymer; and b) at least one cross-linking agent, wherein the at least one first polymer comprises a first polyuronate derivative, the first polyuronate derivative being modified with a hydrophobic moiety; and the at least one cross-linking agent is at least one of at least one calcium chloride and a cyclodextrin derivative.

2. The three-dimensional polymer network according to claim 1, wherein the three-dimensional polymer network further comprises a second polymer.

3. The three-dimensional polymer network according to claim 2, wherein the second polymer comprises a second polyuronate derivative.

4. The three-dimensional polymer network according to claim 2, wherein the second polymer comprises a second polyuronate derivative which is unmodified.

5. The three-dimensional polymer network according to claim 1, wherein the cyclodextrin derivative is selected from cyclodextrin, polymerized cyclodextrin, cyclodextrin modified with alginate with a degree of substitution equal to 1 or cyclodextrin modified with alginate with a degree of substitution inferior to 1.

6. The three-dimensional polymer network according to claim 1, wherein the first polyuronate derivative has a degree of substitution equal to 1 or a degree of substitution inferior to 1.

7. The three-dimensional polymer network according to claim 6, wherein the three-dimensional polymer network further comprises: a) a first polymer being a first polyuronate derivative modified with a hydrophobic moiety with a degree of substitution equal to 1, b) a second polymer being a second polyuronate derivative that is unmodified, and c) a cross-linking agent being calcium chloride and cyclodextrin, thereby forming an interpenetrating polymer network.

8. The three-dimensional polymer network according to claim 6, wherein the three-dimensional polymer network further comprises: a) a first polymer being a first polyuronate derivative modified with a hydrophobic moiety with a degree of substitution equal to 1, b) a second polymer being a second polyuronate derivative that is unmodified, and c) a cross-linking agent being calcium chloride and polymerized cyclodextrin, thereby forming an interpenetrating polymer network.

9. The three-dimensional polymer network according to claim 6, wherein the three-dimensional polymer network further comprises: a) a first polymer being a first polyuronate derivative modified with a hydrophobic moiety with a degree of substitution equal to 1, b) a second polymer being a second polyuronate derivative that is unmodified, and, c) a cross-linking agent being calcium chloride and cyclodextrin modified with alginate with a degree of substitution equal to 1, thereby forming an interpenetrating polymer network.

10. The three-dimensional polymer network according to claim 1, wherein the first polyuronate derivative and second polyuronate derivative are one of the derivatives selected from at least one of mannuronate derivatives, guluronate derivatives, alginate derivatives, pectin derivatives, iduronate derivatives, galacturonate derivatives, and lignin derivatives.

11. Three-dimensional polymer network according to claim 1 wherein the hydrophobic moiety modifying the first polyuronate derivative is selected from at least one of an alkyl moiety, a phenyl alkyl moiety, a fluoroalkane or any other hydrophobic derivative.

12. The three-dimensional polymer network according to claim 1, wherein the hydrophobic moiety modifying the first polyuronate derivative is covalently bounded to the first polyuronate derivative by at least one of an amide moiety, an ester moiety, a thioester moiety, a phosphonate moiety, an ether moiety, a thioether moiety, an imine moiety, or any other chemical group.

13. The three-dimensional polymer network according to claim 1, wherein the three-dimensional polymer network encapsulates at least one pharmaceutical ingredient.

14. The three-dimensional polymer network according to claim 13, wherein the pharmaceutical ingredient is cells.

15. The three-dimensional polymer network according to claim 13, wherein the pharmaceutical ingredient is Langerhans islets.

Description

DRAWINGS

[0033] FIG. 1 exemplarily illustrates a scheme of three-dimensional polymer network in accordance with the present invention.

[0034] FIG. 2 exemplarily illustrates a scheme of the crosslinking of two hydrophobic groups provided by a cyclodextrin derivative.

[0035] FIG. 3 exemplarily illustrates a scheme of the crosslinking of one hydrophobic group provided by a polymeric cyclodextrin derivative.

[0036] FIG. 4 exemplarily illustrates a generic chemical structure of the compounds used in the present invention.

DETAILED DESCRIPTION

[0037] In order to provide an encapsulating agent able to encapsulate a cell, it is interesting to achieve such encapsulating agent which is not only very stable but also very flexible, i.e. which can easily swell and shrink. These properties will confer protection to the encapsulated cell.

[0038] The three-dimensional polymer network according to various embodiments of the present invention, or the specific interpenetrating polymer network, comprises a first polymer network formed by an ionic cross-linked hydrogel formed by a polyuronate derivative. A polyuronate derivative is a water-soluble polyelectrolyte with a polysaccharide structure, and can be chosen among mannuronate derivatives, guluronate derivatives, alginate derivatives, pectin derivatives, iduronate derivatives, galacturonate derivatives, lignin derivatives and/or any combinations thereof.

[0039] Preferentially, alginate derivatives are used.

[0040] The polyuronate derivative, in particular the alginate derivative, is chemically modified with at least one hydrophobic moiety, which may be an alkyl moiety, a phenyl alkyl moiety, a fluoroalkane and/or any other hydrophobic derivatives.

[0041] The hydrophobic moiety is covalently bounded to the polyuronate derivatives by an amide moiety. Other chemical functionalities can also be employed, such as an ester moiety, a thioester moiety, a phosphonate moiety, an ether moiety, a thioether moiety, an imine moiety, or any other.

[0042] The polyuronate derivatives can also be chemically functionalized with 6-monodeoxy-6-monoamino-β-cyclodextrin hydrochloride.

[0043] The polyuronate derivatives are cross-linked with each other through a multivalent ion, preferentially Ca++. This is achieved by adding calcium chloride during the process of making such an interpenetrating polymer network.

[0044] The three-dimensional polymer network according to various embodiments of the present invention, or the specific interpenetrating polymer network, comprises a second polymer network formed by cyclodextrin moieties or by derivatives thereof such as polymerized cyclodextrins. Such polymerized cyclodextrin is formed through chemical connection of cyclodextrin moieties with epichlorhydrin.

[0045] FIG. 1 schematically represents a three-dimensional polymer network in accordance with the present invention.

[0046] The cyclodextrins are a class of compound which presents an outer hydrophilic surface and an inner hydrophobic cavity. Cyclodextrins are distinguished according to the number of saccharide rings which correspond to a specific cavity diameter. For instance, the cavity diameter of a-cyclodextrin is 4.7 Å-5.3 Å, the cavity diameter of β-cyclodextrin is 6.0 Å-6.5 Å, and the cavity diameter of γ-cyclodextrin is 7.5 Å-8.3 Å. This hollow hydrophobic cavity can interact with hydrophobic compounds through weak interaction, i.e., Van der Waals interactions.

[0047] The cyclodextrin derivatives can thus host the hydrophobic moiety which is present on the polyuronate derivatives.

[0048] These supramolecular interactions, in particular these Van der Waals interactions, which are showed either on FIG. 2 or on FIG. 3, allow a very high flexibility to the interpenetrating polymer network.

[0049] In FIG. 2, the cyclodextrin derivative cross-links two hydrophobic groups.

[0050] In FIG. 3, the cyclodextrin derivative (Le. a polymeric cyclodextrin) cross-links only one hydrophobic group.

[0051] This allows to the whole structure to be kept together, and therefore, to confer protection to any objects, like cell or cells, which are entrapped or encapsulated inside the three-dimensional polymer network schematically depicted in FIG. 1 (these objects are not shown).

[0052] Table 1 indicates an overview of the polymers and crosslinking agents which can be combined to furnish the three-dimensional polymer network:

TABLE-US-00001 TABLE 1 (Part 1) First polymer: hydrophobic- substituted polyuronate derivative Cross-linking Second polymer: n/a agents DS = 1 DS < 1 CaCl.sub.2 X X X CD X X polymerized CD X X CD-Alg (DS = 1) X X CD-Alg (DS < 1) X X IPN X Three-dimensional X X X X X X X polymer network

TABLE-US-00002 TABLE 1 (Part 2) First polymer: hydrophobic-substituted polyuronate derivative Second polymer: non-substituted Cross-linking polyuronate derivative agents DS = 1 DS < 1 CaCl.sub.2 X X X X X X X CD X X polymerized CD X X CD-Alg (DS = 1) X X CD-Alg (DS < 1) X X IPN X X X Three-dimensional X X X X X polymer network

[0053] The crosslinking agents can thus be chosen among calcium chloride (CaCl.sub.2), cyclodextrin derivatives (CD), polymerized cyclodextrin (p-CD) and/or any combination thereof. Among the cyclodextrin derivatives, a preferred derivative is a cyclodextrin modified with alginate. The degree of substitution, which corresponds to the (average) number of substituent groups attached per base unit or per monomeric unit can be equal to 1 or be inferior to 1.

[0054] The three-dimensional polymer network according to various embodiments of the present invention, or the specific interpenetrating polymer network, may comprise a first polymer and a second polymer, or only one polymer.

[0055] Preferentially, a hydrophobic substituted polyuronic acid salt (a polyuronate derivative modified with a hydrophobic moiety) is used as first polymer, or as the one polymer in case where only one polymer is used.

[0056] In case where two polymers are used, a hydrophobic substituted polyuronic acid salt (a polyuronate derivative modified with a hydrophobic moiety) is used as first polymer and a polyuronic acid salt which is unsubstituted (a polyuronate derivative which is unmodified) is used as second polymer.

[0057] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS=1) and cyclodextrin has been designed.

[0058] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS=1) and polymerized cyclodextrin has been designed.

[0059] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS=1) and a cyclodextrin modified with alginate (DS=1) has been designed.

[0060] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS=1) and a cyclodextrin modified with alginate (DS<1) has been designed.

[0061] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS<1), calcium chloride and cyclodextrin has been designed.

[0062] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS<1), calcium chloride and polymerized cyclodextrin has been designed.

[0063] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS<1) and cyclodextrin modified with alginate (DS=1) has been designed.

[0064] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt (DS<1), calcium chloride and a cyclodextrin modified with alginate (DS<1) has been designed.

[0065] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS=1), an unsubstituted polyuronic salt as second polymer, calcium chloride and cyclodextrin has been designed. This three-dimensional polymer network has been characterized as forming an interpenetrating polymer network.

[0066] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS=1), an unsubstituted polyuronic salt as second polymer, calcium chloride and polymerized cyclodextrin has been designed. This three-dimensional polymer network has been characterized as forming an interpenetrating polymer network.

[0067] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS=1), an unsubstituted polyuronic salt as second polymer, calcium chloride and a cyclodextrin modified with alginate (DS=1) has been designed. This three-dimensional polymer network has been characterized as forming an interpenetrating polymer network.

[0068] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS=1), an unsubstituted polyuronic salt as second polymer, calcium chloride and cyclodextrin modified with alginate (DS<1) has been designed.

[0069] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS<1), an unsubstituted polyuronic salt as second polymer, calcium chloride, and cyclodextrin has been designed.

[0070] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS<1), an unsubstituted polyuronic salt as second polymer, calcium chloride and polymerized cyclodextrin has been designed.

[0071] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS<1), an unsubstituted polyuronic salt as second polymer and cyclodextrin modified with alginate (DS=1) has been designed.

[0072] The three-dimensional polymer network containing hydrophobic substituted polyuronic salt as first polymer (DS<1), an unsubstituted polyuronic salt as second polymer, calcium chloride and cyclodextrin modified with alginate (DS<1) has been designed.

[0073] FIG. 4 indicates the generic structure of the compounds which have been used in the course of the present invention. The compound of formula 1 is the sodium salt of the polyuronic acids. Alternatively, the potassium salt (not shown) can be used. The compounds of formulas 2, 3, 4 and 5 are the hydrophobic modified polyuronic acids lacking of carboxylic acid moieties. The compounds of formulas 6 and 7 are the hydrophobic modified sodium salt of the polyuronic acids. The compounds of formulas 8, 9, 10 and 11 are the polyuronic acids modified with the cyclodextrin (CD). Finally, the compound 12 schematically represents a polymerized cyclodextrin.

[0074] In order to design the three-dimensional polymer network according to various embodiments of the present invention, or the specific interpenetrating polymer network, the following procedure has been applied.

[0075] The preparation of an aqueous solution of the first polymer is performed.

[0076] If a second polymer has to be incorporated to the polymer network, the preparation of an aqueous solution of the second polymer is performed.

[0077] A cyclodextrin derivative must be added to the aqueous solution of the polymer.

[0078] Both the aqueous solution are then mixed upon each other and added to a hardening aqueous solution of calcium chloride. After gentle stirring, the microcapsule becomes harder and can be filtered out from the solution.

[0079] The three-dimensional polymer network according to various embodiments of the present invention, or the specific interpenetrating polymer network, is particular in that its building blocks, i.e. the cross-linking moieties, confer an improved protection to any active ingredients which are entrapped or encapsulated in the inner core of the network.

[0080] Indeed, once the three-dimensional polymer network or the interpenetrating polymer network starts, for any reason(s), to disintegrate, the cyclodextrin moieties start to cross-link with other hydrophobic moieties present in their close surroundings. Therefore, the whole system can be maintained as such, and the entrapped or encapsulated objects, e.g. cells, can stay protected from the external environment.

[0081] It has been in particular shown that in such a system the cells are still alive at a level of pH up to 9.5, in presence of a high concentration of sodium ion.

[0082] Examples of cells which can be encapsulated are the Langerhans islets which comprise hormone-producing cells. The size of such islets is comprised between 200μm and 500μm.

[0083] The three-dimensional polymer network or the interpenetrating polymer network as described above is the main component of an encapsulating agent adapted for cell encapsulation or adapted for cells encapsulation. The encapsulating agent is either a microcapsule or a micro-sphere or a micro-bead.

[0084] The entrapped cells in the three-dimensional polymer network or in the interpenetrating polymer network (and subsequently in the encapsulating agent) is/are cell(s) able to be fed and able to secrete the proteins and/or the compounds used to treat disease, in particular burdensome diseases, such as cancer, diabetes, Parkinson's disease and/or any other.

[0085] These encapsulating agents can therefore act as a medicament and can further be implanted within the body of a living being, preferentially a human being. They can be grafted or simply inserted under the skin. They can also be swallowed by a subject.

[0086] Example of design of three-dimensional polymer network.

[0087] A microcapsule has been designed comprising alginic acid butyl amide sodium salt as first polymer (DS.sub.amide=0.41), alginic acid sodium salt (compound of formula 1 on FIG. 2) as second polymer, calcium chloride and β-cyclodextrin as cross-liking agents. 50 mL of a solution comprising 1.66% (w/v) aqueous alginic acid solution containing 0.82% (w/v) β-cyclodextrin are prepared (solution 1). 50 mL of a solution comprising 2% (w/v) aqueous alginic acid sodium salt solution are prepared (solution 2).

[0088] Both solutions 1 and 2 are mixed together, thereby forming a polymer aqueous solution. Then the desired active ingredient is added in a predetermined amount, e.g. 1000-5000 cells/mL. Polysorbate as surfactant can be added in order to improve the homogeneity of the dissolved compounds.1 L of a 0.1 M aqueous CaCl.sub.2 solution is prepared as a hardening bath. Then, the hardening bath is poured into a crystallizer dish (e.g. 2-3 cm filling height) and is stirred gently with a magnetic bar. The polymer solution is then dropped into the hardening bath with an encapsulator device or a microsyringe and the hardening of the microcapsules lasts at least 30 minutes under gentle stirring. The microcapsules are then filtered out from the solution.