MICROCAPSULE AND PRODUCTION METHOD THEREOF

Abstract

A microcapsule which is used in tissue regeneration, which may be specifically directed to the damaged tissues, and which forms an extracellular matrix-like structure at a certain point and thus allows cell proliferation.

Claims

1. A microcapsule which is used in tissue regeneration, which may be specifically directed to the damaged tissues, and which forms an extracellular matrix-like structure at a certain point, characterized in comprising: at least one living cell, at least one primary layer enclosing the living cell, at least one and at least one type of side group, which is present between the primary layer and the second layer, and allows the second layer to adhere to the primary layer and thus create a multi-layer structure, and to which another layer to enclose the primary layer is bound, at least one second layer comprising at least one functionalized polymer, at least one and at least one type of targeting factor, and at least one enhancing tissue formation/accumulation, a functionalized polymer which is coated onto the primary layer and which has the property of being targeted, a targeting factor, which is present on the functionalized polymer, allows targeting the damaged point, in which a scaffold is desired to be formed, and comprises a targeting agent and a targeting bridge factor, a tissue formation/accumulation factor which enhances the adhesion surface on the functionalized polymer, forms coupling points on the second layer, and comprises a tissue formation/accumulation bridge factor and a tissue formation/accumulation point, at least one tissue formation/accumulation bridge factor, at one end of which the tissue formation/accumulation point is located, and which, along with the bridging factor, forms a bridge between the functionalized polymer and the tissue formation/accumulation point.

2. The microcapsule of claim 1, characterized in comprising a primary layer, which forms a circular capsule by binding around the living cell, which is in the form of a polymer chain and is preferably alginate, and which is gelatin or elastin or cellulose or chitin or chitosan or carboxymethyl chitosan or chitosan derivatives, or alginate or poly methyl methacrylate, or all polyacrylamides, or polyethylene or polylactic acid or polyglycolic acid or PLGA (polylactic-co-glycolic acid) or polyethylene oxide or polyurethane or poly hydroxy methyl methacrylate or poly amino esters, or any type of crosslinkers thereof.

3. The microcapsule of claim 1, characterized in comprising side groups, which are identical or of different types, which may be coupled to one another via electrostatic or disulfide interactions, which have a positive or negative charge, which may be one or more of haloformyl or hydroxyl or aldehyde or alkyl or alkenyl or alkynyl or carboxamide, or primary amine or secondary amine or tertiary amine, or azide or azo or benzyl or carbonate ester or carboxylate or carboxyl or cyanate or thiocyanate or disulfide or ether or ester or halo or hydroperoxy, or primary ketimine or secondary ketimine, or primary amine or secondary amine, or imide or isocyanide or isocyanate or isothiocyanate or carbonyl or nitrate or nitryl or nitrosooxy or nitro or nitroso peroxy or phenyl or phosphino or phosphate or phosphono or pyridyl or sulfide or sulfo or sulfinyl or sulfhydryl.

4. The microcapsule of claim 1, characterized in comprising a functionalized polymer, which comprises a binding molecule and a bridging factor, which is gelatin or elastin or cellulose or chitin or chitosan or carboxymethyl chitosan or chitosan derivatives, or alginate or poly methyl methacrylate, or all polyacrylamides, or polyethylene or polylactic acid or polyglycolic acid or PLGA (polylactic-co-glycolic acid) or polyethylene oxide or polyurethane or poly hydroxy methyl methacrylate or poly amino esters, or any type of crosslinkers thereof.

5. The microcapsule of claim 3, characterized in comprising a bridging factor which allows the targeting factor and the tissue formation/accumulation factor to adhere to the functionalized polymer by means of a bridge.

6. The microcapsule of claim 1, characterized in comprising a targeting factor, which is present at one end of the targeting bridge factor, which may be a protein or an amino acid sequence or an aptamer or a DNA sequence or a receptor or an antibody, which comprises a targeting agent and a targeting bridge factor, wherein said targeting agent allows targeting to the point in which a scaffold is desired to be formed, is compatible with this point, and is preferably a cartilage-specific antibody, and thus allows targeting.

7. The microcapsule of claim 1, characterized in comprising a tissue formation/accumulation bridge factor one end of which comprises a tissue formation/accumulation point which may be a protein or an amino acid sequence or an aptamer or a DNA sequence or a receptor or rhodopsin-specific antibody.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0019] The microcapsule and the production method of this microcapsule, which have been developed for achieving the objects of the present invention, are illustrated in the accompanying drawings, in which:

[0020] FIG. 1. Schematic view of the cell coated with the primary layer.

[0021] FIG. 2. Schematic view of the side groups bound on the primary layer.

[0022] FIG. 3. Schematic view of the functionalized polymer bound to the primary layer and the side groups present on the primary layer.

[0023] FIG. 4. Schematic view of the second layer.

[0024] FIG. 5. Schematic view of the tissue formation/accumulation factor.

[0025] FIG. 6. Schematic view of the targeting factor.

[0026] FIG. 7. Schematic view of the microcapsule.

[0027] FIG. 8. Schematic view showing the steps of the microcapsule production method.

[0028] The parts in the drawings are enumerated individually and the reference numerals corresponding thereto are given below. [0029] 1. Microcapsule [0030] 2. Cell [0031] 3. Primary layer [0032] 4. Side group [0033] 5. Second layer [0034] 51. Functionalized polymer [0035] 511. Binding molecule (linker) [0036] 512. Bridging factor [0037] 52. Targeting factor [0038] 521. Targeting bridge factor [0039] 522. Targeting agent [0040] 53. Tissue formation/accumulation factor [0041] 531. Tissue formation/accumulation bridge factor [0042] 532. Tissue formation/accumulation point [0043] 100. Microcapsule production method

DETAILED DESCRIPTION OF THE INVENTION

[0044] A microcapsule which is used in tissue regeneration, which may be specifically directed to the damaged tissues, and which forms an extracellular matrix-like structure at a certain point basically comprises: [0045] at least one living cell (2), [0046] at least one primary layer (3) enclosing the living cell (2), [0047] at least one and at least one type of side group (4) to which another layer to enclose the primary layer (3) is bound, [0048] at least one second layer (5) comprising at least one functionalized polymer (51), at least one and at least one type of targeting factor (52), and at least one enhancing tissue formation/accumulation factor (53), [0049] a functionalized polymer (51) which is coated onto the primary layer (3) and which has the property of being targeted, [0050] a targeting factor (52) present on the functionalized polymer (51) and allows targeting the damaged point, and [0051] a tissue formation/accumulation factor (53) which enhances the adhesion surface on the functionalized polymer (51).

[0052] In an embodiment of the invention, the microcapsule (1) comprises living cell. The cell (2) is the innermost element. Once the living cell (2) within the microcapsule (1) reaches the damaged site, it creates an additional coupling surface on the damaged site for the formation of a scaffold. The microcapsule (1) moves along with the cell (2).

[0053] In an embodiment of the invention, the cell (2) is coated with a primary layer (3). The primary layer (3) is a polymer chain. The primary layer (3) may be gelatin, elastin, cellulose, chitin, chitosan, carboxymethyl chitosan and chitosan derivatives, alginate, poly methyl methacrylate, all polyacrylamides, polyethylene, polylactic acid, polyglycolic acid, PLGA (polylactic-co-glycolic acid), polyethylene oxide, polyurethane, poly hydroxymethyl methacrylate, poly amino esters, and any type of crosslinkers thereof. In the preferred embodiment of the invention, the primary layer (3) is alginate. A circular capsule is formed around the cell (2) by crosslinking the primary layer (3) and the crosslinker. The primary layer (3) constitutes the first layer of the multi-layer structure of the microcapsule (1).

[0054] In an embodiment of the invention, the primary layer (3) is a biodegradable polymer.

[0055] In an embodiment of the invention, the primary layer (3) is provided thereon with more than one side groups (4). The side groups (4) are the structures allowing the second layer (5) to attach to the surface. The side groups (4) on the primary layer (3) may be identical or more than one type. The side groups (4) may be coupled to one another via physical and/or chemical interactions. The side groups (4) may be one or more of haloformyl, hydroxyl, aldehyde, alkyl, alkenyl, alkynyl, carboxamide, primary amine, secondary amine, tertiary amine, azide, azo, benzyl, carbonate ester, carboxylate, carboxyl, cyanate, thiocyanate, disulfide, ether, ester, halo, hydroperoxy, primary ketimine, secondary ketimine, primary amine, secondary amine, imide, isocyanide, isocyanate, isothiocyanate, carbonyl, nitrate, nitryl, nitrosooxy, nitro, nitroso, peroxy, phenyl, phosphino, phosphate, phosphono, pyridyl, sulfide, sulfo, sulfinyl, and sulfhydryl. In the preferred embodiment of the invention, the side groups (4) are carboxyl and amine groups. The side groups (4) allow the formation of a multi-layer structure.

[0056] In an embodiment of the invention, there exists a second layer (5). The second layer (5) is the structure that enables the microcapsule (1) to be targeted to the preferred point. The second layer (5) is a layer in which targeting factors (52) and tissue formation/accumulation factors (53) are present on the functionalized polymer (51). First, the polymer is functionalized in order to obtain the second layer (5). The functionalized polymer (51) may be gelatin, elastin, cellulose, chitin, chitosan, carboxymethyl chitosan and chitosan derivatives, alginate, poly methyl methacrylate, all polyacrylamides, polyethylene, polylactic acid, polyglycolic acid, PLGA (polylactic-co-glycolic acid), polyethylene oxide, polyurethane, poly hydroxymethyl methacrylate, poly amino esters, and any type of crosslinkers thereof. In this embodiment of the invention, the functionalized polymer (51) is chitosan. The functionalized polymer (51) is coupled to the bridging factor (512) by way of a binding molecule (511). The bridging factor (512) allows the targeting factor (52) and the tissue formation/accumulation factor (53) to adhere to the functionalized polymer (51) by means of a bridge. The bridging factor (512) on the functionalized polymer (51) forms these bridges by means of the targeting bridge factor (521) and the tissue formation/accumulation factor (53). In this embodiment of the invention, the functionalized polymer (51) is chitosan.

[0057] In an embodiment of the invention, there exists a targeting factor (52). The targeting factor (52) allows the microcapsule (1) to be targeted to the site to which it is desired to reach. The targeting factor (52) comprises a targeting bridge factor (521) and a targeting agent (522). The targeting bridge factor (521), along with the bridging factor (512), forms a bridge between the functionalized polymer (51) and the targeting agent (522). At one end of the targeting bridge factor (521) is the targeting agent (522). The targeting agent (522) may be a protein, amino acid sequence, an aptamer, a DNA sequence, a receptor, or an antibody. In the preferred embodiment of the invention, the microcapsule (1) is used in cartilage tissue regeneration and the targeting agent (522) is cartilage-specific antibody.

[0058] In an embodiment of the invention, there exists a tissue formation/accumulation factor (53). The tissue formation/accumulation factor (53) forms the points of coupling on the microcapsule (1). The tissue formation/accumulation factor (53) comprises a tissue formation/accumulation bridge factor (531) and a tissue formation/accumulation point (532). The tissue formation/accumulation bridge factor (531), along with the bridging factor (512), forms a bridge between the functionalized polymer (51) and the tissue formation/accumulation point (532). At one end of the tissue formation/accumulation bridge factor (531) is the tissue formation/accumulation point (532). The tissue formation/accumulation point (532) may be a protein, amino acid sequence, an aptamer, a DNA sequence, a receptor, or an antibody. In the preferred embodiment of the invention, the tissue formation/accumulation point (532) is a rhodopsin-specific antibody.

[0059] With the microcapsule (1) according to the present invention, the need for open surgery in damaged tissue treatment is eliminated. The microcapsule (1) having living cell (2) therein may be directed/targeted to the damaged site of preference. The microcapsule (1) targeted to the site of preference and reaching that site forms a structure having extracellular (2) matrix properties at the damaged site. With the thus formed structure having extracellular (2) matrix properties, the cells (2) are proliferated and the tissue is regenerated.

[0060] The microcapsule production method (100) which is used in tissue regeneration, which may be specifically directed to the damaged tissues, and which forms an extracellular (2) matrix-like structure at a certain point and thus allows cell (2) proliferation comprises the process steps of: [0061] encapsulating the living cells (2) with a polymer, preferably alginate, and obtaining the primary layer (3) with side groups thereon (4) (101), [0062] functionalizing the polymer, preferably chitosan, and binding the bridging factor (512), preferably avidin, to the functionalized chitosan (102), [0063] coating the encapsulated cells (2) with the functionalized polymer (51) in order to form the second layer (5) (103), and [0064] obtaining the second layer (5) and the microcapsule (1) by binding, on the functionalized polymer (51), preferably the biotinylated targeting factor (52) and the tissue formation/accumulation factor (53) (104).

[0065] With the microcapsule production method (100) according to the invention, a microcapsule (1) which may be directed to a damaged site in the body and which carries living cell (2) is obtained.

[0066] In the microcapsule production method (100), first the step of encapsulating the living cells (2) with a polymer, preferably alginate, and obtaining the primary layer (3) with side groups thereon (4) (101) is performed. In this step, the CaCl2 dissolved in water and a low molecular weight sodium alginate polymer again dissolved in water are crosslinked. The chondrocytes proliferated in the cell (2) culture are added into the alginate solution and instilled into the CaCl2 solution. The cell (2) is thus coated with the primary layer (3).

[0067] Subsequent to the cell (2) encapsulation (101), the step of functionalizing the polymer, preferably chitosan, and binding the bridging factor (512), preferably avidin, to the functionalized chitosan (102) is performed. In this step, the chitosan is functionalized and avidinated. First of all, low molecular weight chitosan polymer is dissolved in acetic acid solution. And then, the ambient pH value is increased to the physiological pH range. Once the pH value is increased, chitosan is first treated with functionalizing agent, and then with avidin molecules. Avidin-bound chitosan polymer chains are washed with acetic acid solution at least once.

[0068] After the functionalization of the secondary polymer, preferably chitosan, and the binding of the bridge factor, preferably avidin (102), the encapsulated cells (2) are coated with a functionalized polymer (51) in order to form the second layer (5) thereof (103). Encapsulated cells (2) are washed with the preferred isotonic solution. In this embodiment of the invention, the cells (2) are washed with PBS (Phosphate Buffered Saline) buffer solution and transferred to the PBS solution. After washing, the avidin-bound chitosan polymers are brought to pH 4.5 and introduced into the PBS solution (pH 7.4) in which encapsulated cells (2) are present.

[0069] After the encapsulated cells (2) are coated with the functionalized polymer (51) in order to form the second layer (5) (103), the second layer (5) and the microcapsule (1) are obtained by binding, on the functionalized polymer (51), preferably the biotinylated targeting factor (52) and the tissue formation/accumulation factor (53) (104). The solution comprising therein biotinylated antibodies specific to the damaged cartilage, e.g. glycoprotein, 80 anti-GPNMB, anti-CD90/THY1, anti-GPCR, anti-S100A9, anti-CXCR4, and anti-periostin, is instilled into the solution in which the encapsulated cells (2) are present. Once the instillation is performed, it is kept for 0-24 hours at 30-45° C. The avidinated chitosan is also treated with biotinylated rhodopsin for 0-24 hours in a different environment.

[0070] In the production method (100) of a multi-layer microcapsule which can be targeted to the site of preference, first, the step of encapsulating the living cells (2) with a polymer, preferably alginate, and obtaining the primary layer (3) with side groups thereon (4) (101) is performed. After the primary layer (3) is obtained, the secondary polymer, preferably chitosan, is functionalized and the bridging factor (512), preferably avidin, is bound to the polymer, preferably chitosan (102). Later, the encapsulated cells (2) are coated with the functionalized polymer (51) in order to form the second layer (5) (103). Upon completion of the coating process, the second layer (5) and hence the microcapsule (1) are obtained by binding, to the functionalized polymer (51), the targeting factor (52) and the tissue formation/accumulation factor (53).