阅读说明：本技术 膜 (Film ) 是由 R·J·麦肯 T-M·塞斯蒂 B·罗伯 于 2019-10-01 设计创作，主要内容包括：本发明涉及一种治疗组合物(5,6),其包含内部部分(7)和完全或部分围绕所述内部部分的生物相容性膜(4)。所述生物相容性膜包含至少两个层：通过电纺丝形成的热塑性聚氨酯聚合物纤维的多孔非织造网络的第一层(1),并且具有大于或等于50％的孔隙率；小于5μm的平均孔径；并具有10至250μm的厚度；以及通过电纺丝形成的热塑性聚合物纤维的多孔非织造网络的第二层(2)。所述第二层(2)的均值平均纤维直径大于第一层(1)中的均值平均纤维直径,和/或其中第二层(2)的平均孔径大于第一层(1)的平均孔径。所述内部部分(7)包含治疗剂(3)。本发明还涉及膜和治疗组合物例如用于封装治疗细胞的用途。(The present invention relates to a therapeutic composition (5, 6) comprising an inner portion (7) and a biocompatible membrane (4) completely or partially surrounding the inner portion. The biocompatible film comprises at least two layers: a first layer (1) of a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning and having a porosity greater than or equal to 50%; an average pore diameter of less than 5 μm; and has a thickness of 10 to 250 μm; and a second layer (2) of a porous nonwoven network of thermoplastic polymer fibres formed by electrospinning. The mean average fiber diameter of the second layer (2) is larger than the mean average fiber diameter in the first layer (1), and/or wherein the average pore size of the second layer (2) is larger than the average pore size of the first layer (1). The inner portion (7) comprises a therapeutic agent (3). The invention also relates to the use of the membrane and therapeutic compositions, for example, for encapsulating therapeutic cells.)

1. A therapeutic composition (5, 6) comprising an inner portion (7) and a biocompatible membrane (4, 10) completely or partially surrounding the inner portion; wherein the biocompatible film comprises at least two layers:

a first layer (1) of a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning and having a porosity greater than or equal to 50%; an average pore diameter of less than 5 μm; and has a thickness of 10 to 250 μm; and

a second layer (2) of a porous non-woven network of thermoplastic polymer fibres formed by electrospinning, the porosity of the second layer (2) being substantially equal to or higher than the porosity of the first layer (1); and/or wherein the mean average fiber diameter of the second layer (2) is greater than the mean average fiber diameter of the first layer (1); and/or wherein the average pore size of the second layer (2) is larger than the average pore size of the first layer (1); and

wherein the inner portion (7) comprises a therapeutic agent (3).

2. The therapeutic composition of claim 1, wherein the biocompatible film (4, 10) is in the form of a pouch or bag (5, 6, 11) that partially or completely encapsulates an inner portion (7) comprising the therapeutic agent (3).

3. The therapeutic composition of claim 2, further comprising a carrier (8), the therapeutic agent (3) being disposed on or in the carrier (8), preferably wherein the therapeutic agent (3) is:

attached to the surface of a carrier (8);

arranged in a hole of the carrier (8); and/or

Encapsulated in a carrier (8).

4. The therapeutic composition according to claim 2 or 3, wherein the pouch or bag (6) is arranged such that the first layer (1) faces or is in contact with the encapsulated inner portion (7) and optionally the therapeutic agent (3); and the second layer (2) faces outwards.

5. The therapeutic composition according to claim 2, wherein the pouch or bag (5) is arranged such that the first layer (1) is facing outwards; and the second layer (2) faces or is in contact with the encapsulated inner portion (7) and optionally the therapeutic agent (3).

6. The therapeutic composition according to claim 3, wherein the pouch or bag (5) is arranged such that the first layer (1) is facing outwards; and the second layer (2) faces or is in contact with the encapsulated inner portion (7), the carrier (8) and optionally the therapeutic agent (3).

7. The therapeutic composition of claim 2 or 3, wherein the biocompatible film (10) comprises three layers: the first layer (1) is arranged between two second layers (2), wherein the inner portion (7) is arranged at an inwardly facing surface of one of the second layers (2) when the biocompatible film (4) is in the form of a bag or pouch (11); the other of the two second layers (2) providing an outward facing surface; and wherein the carrier is optionally arranged within the inner portion (7).

8. The therapeutic composition according to any one of the preceding claims, wherein the porosity of the first layer (1) is from 50% to 90%, optionally from 50% to 80%.

9. The therapeutic composition according to any one of the preceding claims, wherein the first layer (1) has an average pore size of less than 2 μ ι η.

10. The therapeutic composition according to any one of the preceding claims, wherein the thickness of the first layer (1) is from 10 to 150 μ ι η, preferably from 20 to 150 μ ι η, most preferably from 50 to 150 μ ι η or from 50 to 200 μ ι η.

11. The therapeutic composition according to any of the preceding claims, wherein the mean diameter of the polymer fibers of the first layer (1) is less than 700nm, preferably less than 600nm, preferably less than 500nm, and most preferably 100-500nm, even more preferably 50-500 nm.

12. The therapeutic composition according to any one of the preceding claims, wherein the second layer (2) is or comprises polyurethane or any other biocompatible thermoplastic polymer or polymer blend.

13. The therapeutic composition according to any one of the preceding claims, wherein the first layer (1) and/or the second layer (2) of the biocompatible film (4, 10) is non-biodegradable.

14. The therapeutic composition according to any one of the preceding claims, wherein the therapeutic agent (3) is selected from the group consisting of: a therapeutic cell, a drug, a nucleic acid, a polynucleotide, a protein, a polypeptide, an antibody, a particle such as a lipid nanoparticle, an extracellular vesicle, or an exosome, optionally wherein the polynucleotide comprises DNA, RNA, RNAi, saRNA, or siRNA.

15. The therapeutic composition of claim 3, wherein the biocompatible film (4) completely or partially surrounds the carrier.

16. The therapeutic composition of claim 3 or 15, wherein the carrier (8) comprises a porous non-woven network of polymer fibres, or a hydrogel, gelatin, collagen (optionally fibres or sponge) or acellular tissue.

17. The therapeutic composition according to any of the preceding claims, comprising cells (3), wherein the cells are preferably pancreatic beta cells or pancreatic islet cells.

18. The therapeutic composition of claim 4 or 7, wherein the second outward-facing layer further comprises a hydrogel, gelatin or collagen (optionally fiber or sponge) or acellular tissue.

19. The therapeutic composition according to any one of the preceding claims, wherein the second layer (2) is formed by electrospun fibres, and

(i) porosity from 70 to 98%, preferably from 80 to 95%; and/or

(ii) An average pore diameter of 5 to 80 μm, preferably 10 to 50 μm; and/or

(iii) The mean diameter of the polymer fibres is from 1 to 10 μm, preferably from 2 to 8 μm, most preferably from 3 to 7 μm.

20. The therapeutic composition according to any one of the preceding claims, wherein

(i) The porosity of the second layer (2) compared to the porosity of the first layer (1) is in the range of at least 120%, 110%, 100%, optionally 100% to 110%, 100% to 150%, 100% to 175%, or 100% to 190%, or 100% up to 199% or 200% of the porosity of the first layer (1); and/or

(ii) The average pore size/diameter of the second layer (2) is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times the pore size/diameter of the first layer (1); and/or

(iii) The polymer fibers of the second layer (2) have a mean diameter that is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times the diameter of the first layer (1).

21. The therapeutic composition of any one of the preceding claims, further comprising one or more additives, wherein the additives are preferably disposed within the carrier (8)Or in one or both of the first layer (1) or the second layer (2), wherein an additive is selected from: growth factors such as VEGF, cross-linking agents, growth factors, catalase and other enzymes; or oxygen-releasing materials, e.g. CaO₂Or haemoglobin, peroxides (e.g. H)₂O₂、CaO₂、MgO₂、Li₂O₂、Na₂O₂) Sodium percarbonate (Na)₂CO₃) Perfluorocarbons, hydroxyapatite, tricalcium phosphate (bone growth promoting material), most preferably CaO₂And/or MgO₂。

22. A film comprising at least two layers, wherein:

(i) the first layer (1) is a biocompatible film comprising a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning, wherein the biocompatible film has a porosity of greater than or equal to 50%; the average pore diameter is less than 5 mu m; and a thickness of 10 to 250 μm; and

(ii) the second layer (2) is disposed on the first layer, and wherein the second layer (2) is a porous non-woven network of thermoplastic polymer fibers formed by electrospinning, the fibers of the second layer (2) may or may not be polyurethane, the mean average fiber diameter of the second layer (2) being greater than the mean average fiber diameter of the first layer (1), and/or wherein the average pore size of the second layer (2) is greater than the average pore size of the first layer (1).

23. The film according to claim 22, wherein the first layer (1) and/or the second layer (2) are as defined in any one of claims 1 to 21.

24. A film or therapeutic composition according to any preceding claim for use in a method of treatment of the human or animal body by therapy.

25. The membrane or the therapeutic composition according to claim 24 for use in a method of immunoprotection of a therapeutic cell or for use in a method of treatment of (preferably type I) diabetes.

26. A method of treatment of the human or animal body in need thereof comprising administering to the human or animal body a therapeutically effective amount of a therapeutic composition or film according to any preceding claim.

27. A device comprising the therapeutic composition according to any one of claims 1 to 21, wherein the inner portion (7) comprises pancreatic β or islet cells, with or without carrier material (8).

28. A device comprising the therapeutic composition according to any one of claims 1 to 21, wherein the inner portion (7) comprises hepatocytes.

29. A device comprising a therapeutic composition according to any of claims 1-21, wherein the inner portion comprises red blood cells and/or white blood cells, preferably engineered white blood cells, such as CAR-T cells.

30. A method of preparing a therapeutic composition according to any one of claims 1-21, comprising:

(i) an electrospinning process to produce a biocompatible film (4) comprising a porous non-woven network of thermoplastic polyurethane polymer fibres; and

(ii) shaping the biocompatible film to produce a therapeutic composition in which the inner portion (7) is wholly or partially surrounded; optionally the shaping is performed by welding the edges.

Technical Field

The present invention relates to a biocompatible membrane that allows for the selective delivery of materials to target cells in vivo. In particular, the present invention relates to a therapeutic composition comprising an interior portion and a biocompatible film completely or partially surrounding the interior portion, wherein the biocompatible film comprises a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning. The invention also relates to a method for producing the membrane and to the use of the membrane in therapy.

Background

Diabetes is a condition in which the body fails to produce enough insulin or does not respond to insulin. According to the centers for disease control and prevention, 2900 million people in the united states have diabetes, and 8600 million people have pre-diabetes, which is a serious health condition that increases the risk of developing type 2 diabetes and other chronic diseases. Of all diabetic patients, approximately 5%, or nearly 150 million Americans, suffer from type 1 diabetes (T1D). It is estimated that the annual cost of T1D for the U.S. healthcare system in 2010 is approximately $ 144 billion. Type 1 diabetes is an autoimmune disease in which the immune system of the patient becomes abnormal, attacking and destroying the beta (β) cells of the pancreas. Beta cells are responsible for regulating blood glucose (glucose) levels by producing precise amounts of the essential hormone insulin. Patients with T1D and many type 2 patients require insulin to survive. Since β -cell loss is the major pathogenesis of T1D, the disease is an ideal candidate for cell replacement therapy.

Cell therapy has been proposed to treat diabetes. The cells must respond to blood/serum glucose levels by releasing or not releasing insulin. There are many companies developing cell therapies, whether pancreatic beta cells or stem cells can differentiate into beta cells. To be successful in vivo, it is necessary to implant cells into a membrane bag (or equivalent) having the following desired properties:

promoting (or at least allowing) angiogenesis;

allowing the glucose and insulin molecules to circulate;

preventing entry of host immune cells and destruction of introduced cells;

preventing the introduced cells from escaping into the host (they may lead to cancer);

support of living cells for 2 years (current commercial goal);

allow easy cleanup (once a problem occurs).

To date, no material has been found that meets all of these characteristics.

There remains a need to provide improved materials for encapsulating cellular material in vivo.

Disclosure of Invention

The present invention provides the desirable characteristics discussed above, in particular by a therapeutic composition comprising the film of claim 1.

The present invention utilizes biocompatible membranes that can act as size selective membranes or "molecular sieves" to control the delivery of foreign substances to specific target cells in vitro and in vivo. The membrane contains pores of a suitable size to allow the passage of solute molecules, such as glucose, but to prevent the passage of larger particles, such as cells. The membrane can be packaged with target cells of interest, optionally on a scaffold, to provide artificial tissues and organs for treatment of disease. Advantageously, the membrane may shield any encapsulated cells from the host's immune system.

The film of the present invention is shaped to form a therapeutic composition in the form of a "pouch" or "bag" (bag) containing a therapeutic agent. The therapeutic agent is encapsulated by the film. As noted above, the membrane is selectively permeable, allowing certain molecules to pass through. When the therapeutic agents are cells, they are usually permanently fixed inside the membrane (bag). However, when the therapeutic agent is a smaller entity (e.g., a drug molecule), it may diffuse out of the pouch through the membrane and into the patient.

Accordingly, in a first aspect, the present invention provides a therapeutic composition (5, 6) comprising an inner portion (7) and a biocompatible film (4, 10) completely or partially surrounding the inner portion, wherein the biocompatible film comprises at least two layers:

a first layer (1) of a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning and having a porosity greater than or equal to 50%; an average pore diameter of less than 5 μm; and a thickness of 10 to 250 μm; and

a second layer (2) of a porous nonwoven network of thermoplastic polymer fibres formed by electrospinning, wherein the mean average fibre diameter (mean average fibre diameter) of the second layer (2) is greater than the mean average fibre diameter of the first layer (1); and/or wherein the average pore size of the second layer (2) is larger than the average pore size of the first layer (1); and

wherein the inner portion (7) comprises a therapeutic agent (3).

In some embodiments, the second layer (2) is defined as having a porosity substantially equal to or higher than the porosity of the first layer (1).

In some embodiments, the second layer (2) is defined as having a mean average fiber diameter greater than the mean average fiber diameter in the first layer (1).

In some embodiments, the second layer (2) is defined as having an average pore size greater than the average pore size of the first layer (1).

In some embodiments, the second layer (2) is defined as having a porosity substantially equal to or higher than the porosity of the first layer (1), and the mean average fiber diameter of the second layer (2) is greater than the mean average fiber diameter in the first layer (1). In some embodiments, the second layer (2) is defined as having a porosity substantially equal to or higher than the porosity of the first layer (1), and wherein the average pore size of the second layer (2) is greater than the average pore size of the first layer (1). In some embodiments, the second layer (2) is defined as having a mean average fiber diameter greater than the mean average fiber diameter in the first layer (1), and wherein the average pore size of the second layer (2) is greater than the average pore size of the first layer (1).

In some embodiments, the fibers of the second layer (2) may be polyurethane, while in other embodiments they may be as defined herein, or may be any other biocompatible thermoplastic polymer or polymer blend. In some embodiments, the biocompatible film (4, 10) is in the form of a pouch or bag (5, 6, 11) that partially or completely encapsulates an interior portion (7) comprising the therapeutic agent (3). In some embodiments, the therapeutic composition further comprises a carrier (8) having disposed thereon or therein a therapeutic agent (3), preferably wherein the therapeutic agent (3) is:

attached to the surface of a carrier (8);

placed in the pores of a carrier (8); and/or

Encapsulated in a carrier (8).

In some embodiments, the pouch or bag (6) is arranged such that the first layer (1) faces or contacts the encapsulated interior portion (7) and optional therapeutic agent (3); and the second layer (2) faces outwards. In some embodiments, the pouch or bag (5) is arranged such that the first layer (1) faces outwardly; while the second layer (2) faces or contacts the inner part of the encapsulation (7) and optionally the therapeutic agent (3). In some embodiments, the pouch or bag (5) is arranged such that the first layer (1) faces outwardly; while the second layer (2) faces or contacts the encapsulated inner portion (7), the carrier (8) and optionally the therapeutic agent (3). In some embodiments, the biocompatible film (10) comprises three layers: -arranging a first layer (1) between two second layers (2), wherein an inner portion (7) is arranged within an inwardly facing surface of one of said second layers (2) when the biocompatible film (4) is in the form of a bag or pouch (11); the other of the two second layers (2) providing an outward facing surface; and wherein the carrier is optionally arranged in the inner portion (7).

In some embodiments, the porosity of the first layer (1) is from 50% to 90%, optionally from 50% to 80%. In some embodiments, the first layer (1) has an average pore size of less than 2 μm. In some embodiments, the thickness of the first layer (1) is from 10 to 150 μm, preferably from 20 to 150 μm, most preferably from 50 to 150 μm or from 50 to 200 μm. In some embodiments, the average diameter of the polymer fibers of the first layer (1) is less than 700nm, preferably less than 600nm, preferably less than 500nm, and most preferably 100-500nm, even more preferably 50-500 nm.

In some embodiments, the second layer (2) is or comprises polyurethane or any other biocompatible thermoplastic polymer or polymer blend and/or other thermoplastic polymers, optionally those described herein, including polyethylene.

In some embodiments, the first layer (1) and/or the second layer (2) of the biocompatible film (4, 10) is non-biodegradable. In some embodiments, the therapeutic agent (3) is selected from: a therapeutic cell, a drug, a nucleic acid, a polynucleotide, a protein, a polypeptide, an antibody, a particle such as a lipid nanoparticle, an extracellular vesicle, or an exosome, optionally wherein the polynucleotide comprises DNA, RNA, RNAi, saRNA, or siRNA. In some embodiments, the biocompatible film (4) completely or partially surrounds the carrier. In some embodiments, the carrier (8) comprises a porous non-woven network of polymer fibers, or a hydrogel, gelatin, collagen (optionally fibers or sponge), or acellular tissue. In some embodiments, the composition comprises a cell (3), wherein the cell is preferably a pancreatic beta cell or an islet cell.

In some embodiments having an outwardly facing second layer, the second layer (2) further comprises hydrogel, gelatin or collagen (optionally fibers or sponges) or acellular tissue.

In some embodiments, the second layer (2) is formed from electrospun fibers, and

(i) porosity from 70 to 98%, preferably from 80 to 95%; and/or

(ii) An average pore diameter of 5 to 80 μm, preferably 10 to 50 μm; and/or

(iii) The average diameter of the polymer fibers is 1 to 10 μm, preferably 2 to 8 μm, most preferably 3 to 7 μm.

In some embodiments:

(i) the porosity of the second layer (2) compared to the porosity of the first layer (1) is in the range of at least 120%, 110%, 100%, optionally 100% to 110%, 100% to 150%, 100% to 175%, or 100% to 190%, or 100% up to 199% or 200% of the porosity of the first layer (1); and/or

(ii) The average pore size/diameter of the second layer (2) is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times the pore size/diameter of the first layer (1); and/or

(iii) The polymer fibers of the second layer (2) have a mean diameter (mean diameter) that is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times the diameter of the first layer (1).

Any combination of the above is contemplated. In some embodiments, the term "second layer (2) having a porosity substantially equal to or higher than the porosity of the first layer (1)" means that the second layer has a porosity greater than the first layer. In some embodiments, the term means that the porosity is substantially equal to the porosity of the first layer, which may mean that the porosity of the second layer is exactly equal to the porosity of the first layer, e.g., to 2 significant digits. It is also contemplated that in some embodiments, the porosity of the second layer may be slightly less than the porosity of the first layer, and for example, a 5% variation is contemplated herein.

In some embodiments, the therapeutic composition further comprises one or more additives, wherein the additives are preferably disposed within the carrier (8) or within one or both of the first layer (1) or the second layer (2), wherein the additives are selected from the group consisting of: growth factors such as VEGF, cross-linking agents, growth factors, catalase and other enzymes; or oxygen-releasing materials such as CaO₂Or haemoglobin, peroxides (e.g. H)₂O₂、CaO₂、MgO₂、Li₂O₂、Na₂O₂) Sodium percarbonate (Na)₂CO₃) Perfluorocarbons, hydroxyapatite, tricalcium phosphate (bone growth promoting material), most preferably CaO₂And/or MgO₂。

In some embodiments, the porosity of the second layer (2) is within a range of at least 120%, 110%, 100%, 90%, or 80% of the porosity of the first layer (1).

In some embodiments, the average pore size/diameter of the second layer (2) is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times the pore size/diameter of the first layer (1). In some embodiments, the pore size/diameter of the second layer (2) is at most 100 times the pore size/diameter of the first layer (1). In some embodiments, the pore size/diameter of the second layer (2) is 2 to 5, 2 to 10, 2 to 20, 2 to 50, 2 to 100, 5 to 10, 5 to 20, 5 to 50, 5 to 100, 10 to 20, 10 to 50, 10 to 100, 20 to 50, 20 to 100, or 50 to 100 times the pore size/diameter of the first layer (1).

In some embodiments, the polymer fibers of the second layer (2) have a larger diameter than the polymer fibers of the first layer (1). In some embodiments, the mean diameter of the polymer fibers of the second layer (2) is at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times the diameter of the first layer (1). In some embodiments, the polymer fibers of the second layer (2) are at most 100 times the diameter of the first layer (1). In some embodiments, the polymer fibers of the second layer (2) are from 2 to 5, 2 to 10, 2 to 20, 2 to 50, 2 to 100, 5 to 10, 5 to 20, 5 to 50, 5 to 100, 10 to 20, 10 to 50, 10 to 100, 20 to 50, 20 to 100, or 50 to 100 times the diameter of the first layer (1).

An advantage of the arrangement where the second layer (2) or one of the second layers (2) has an inwardly facing surface (e.g. as shown in fig. 10 and 13) is that no carrier is required. Alternatively, the second layer (2) serves as a carrier.

In a second aspect, there is also provided a film comprising at least two layers, wherein

(i) The first layer (1) is a biocompatible film comprising a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning, wherein the biocompatible film has a porosity of greater than or equal to 50%; the average pore diameter is less than 5 mu m; a thickness of 10 to 250 μm; and

(ii) the second layer (2) is disposed on the first layer, and wherein the second layer (2) is a porous non-woven network of thermoplastic polymer fibers formed by electrospinning, the fibers of the second layer (2) may or may not be polyurethane, the mean average fiber diameter of the second layer (2) is greater than the mean average fiber diameter of the first layer (1), and/or the average pore size of the second layer (2) is greater than the average pore size of the first layer (1).

In some embodiments, the first layer (1) and/or the second layer (2) are as defined herein.

In any aspect, in some embodiments, the film or therapeutic composition may be used in a method of treatment of the human or animal body by therapy. In some embodiments, the film or therapeutic composition can be used in a method of treating (preferably type I) diabetes.

There is provided a method of treatment of the human or animal body in need thereof comprising administering to the human or animal body a therapeutically effective amount of a therapeutic composition, or a method for immunoprotection of a therapeutic cell, or a method for treatment of (preferably type I) diabetes. Preferably also provided is a device comprising a therapeutic composition, wherein the inner portion comprises pancreatic β or islet cells, with or without carrier material. Also provided is a device comprising the therapeutic composition, wherein the inner portion comprises hepatocytes. Also provided is a device comprising a therapeutic composition, wherein the inner portion comprises red blood cells and/or white blood cells (e.g., B cells or T cells), preferably engineered white blood cells, e.g., engineered T cells, including CAR-T cells (chimeric antigen receptor T cells).

In a third aspect, there is provided a method of producing a therapeutic composition comprising:

(i) an electrospinning process to produce a biocompatible membrane (4) comprising a porous non-woven network of thermoplastic polyurethane polymer fibres; and

(ii) shaping the biocompatible film to produce a therapeutic composition in which the inner portion (7) is wholly or partially surrounded; the shaping is optionally performed by welding (weld) edges.

Advantageously, according to any aspect of the invention, the membrane of the invention is permeable to many biomolecules (typically less than 100nm in size, e.g. glucose), but prevents molecules/particles larger than this size from passing. Advantageously, the membrane is impermeable to the passage of cells.

The membranes used in the present invention can be used to encapsulate target cells of interest, thereby controlling the microenvironment of the cells. The porous nature of the polymer fiber network allows solutes to pass through the membrane and thus reach the target cell of interest. Thus, the cells are maintained and viable. However, the pore size is controlled so that larger molecules, such as cells, cannot pass through the membrane. Thus preventing the introduced cells from escaping from the host (which is crucial from a regulatory point of view). In addition, host immune cells are prevented from entering the target cells and destroying them. Finally, the membrane can separate and remove the target cells from the host if this proves necessary.

Accordingly, the present invention satisfies many of the desirable features outlined above.

Drawings

Figure 1 is an SEM image showing the top layer of the therapeutic composition formed in example 1.

Fig. 2 is an SEM image showing the bottom layer of the therapeutic composition formed in example 1.

Fig. 3 is an SEM image, corresponding to the combination of images visible in both layers in fig. 1 and 2.

Fig. 4 is a scanning electron micrograph of the electrospun precursor prepared in example 2.

Fig. 5A shows a schematic of how an electrospun precursor is folded prior to welding with a decellularized collagen membrane.

FIG. 5B shows a liquid crystal display device havingA therapeutic composition of an enclosed acellular collagen membrane.

FIG. 5C shows a liquid crystal display device havingA therapeutic composition of an enclosed acellular collagen membrane.

Fig. 5D shows a therapeutic composition with an acellular collagen membrane 100% surrounded;

figure 6A shows a therapeutic composition made from an electrospun precursor, with the inlet left open. The syringe contains a hydrogel that is injected into the "bag".

Figure 6B shows a hydrogel-filled therapeutic composition in which the electrospun membrane completely surrounds the hydrogel (i.e., 100% coverage).

FIG. 7 shows II-8 release as a response to LPS concentration measured by ELISA.

FIG. 8 shows ATP detection in culture media. Samples marked with x are taken from the wells of the removal pack. The detected signal is too low and therefore does not appear on the graph. The other series represent ATP release from control wells with THP-1 cells in the wells.

Fig. 9 shows a flat two-layer biocompatible membrane (4) comprising a layer (1) with smaller diameter fibers and a second layer (2) with larger diameter fibers. The layers are not shown to scale and one layer may or may not be thicker than the other.

Fig. 10 shows the biocompatible film (4) of fig. 9 folded (in 2D form, but equally applicable to 3D) to a configuration in which the layer (1) with the smaller diameter fibres is facing outwards so as to be in contact with the subcutaneous environment of the patient into which the bag or pouch (5) is to be provided. The layer (2) with the larger diameter fibers faces inwards to come into contact with the therapeutic agent (in this case, the cells (3) loaded on the carrier (8)) which can be placed in the inner part (7) of the bag or pouch (5).

Fig. 11 shows the biocompatible membrane (4) of fig. 9 folded (in 2D form, but equally applicable to 3D), in fig. 11 a bag or pouch (6), so that the layer (1) with smaller diameter fibers faces inwards and can thus be brought into contact with a therapeutic agent (in this case, cells (3) loaded on a carrier (8)) that can be placed inside the inner part (7) of the bag or pouch (6). The layer (1) can also replace the need for a carrier here, or can be joined with a further carrier, for example a hydrogel. The layer (2) with the larger diameter fibres faces outwards and can thus be in contact with the subcutaneous environment of the patient into which the bag or pouch (6) is to be provided.

Fig. 12 shows the biocompatible film (4) of fig. 9 folded (in 2D form, but equally applicable to 3D) to a configuration in which the layer (1) with the smaller diameter fibres is facing outwards so as to be in contact with the subcutaneous environment of the patient into which the bag or pouch (5) is to be provided. In this embodiment, the biocompatible film is folded and prepared in such a way that the resulting bag or pouch (9) is a biconcave disc comprising an inner portion (7) which itself comprises the therapeutic agent (in this case, cells (3)) loaded on a carrier (8).

Fig. 13A shows a three-layer biocompatible film (10) comprising a first layer (1) of smaller diameter fibers between two second layers (2) of larger diameter fibers.

Fig. 13B shows the three-layer biocompatible film (10) from fig. 13A folded (in 2D form, but equally applicable to 3D) to form a bag or pouch (11), wherein the outer layer (2) with larger diameter fibers may be in contact with the subcutaneous environment of the patient into which the bag or pouch is to be provided, and the inner layer (2) with larger diameter fibers may be in contact with the inner portion (7) containing the therapeutic agent (in this case, the cells (3) are supported on a carrier (8)). The layer (1) with the smaller diameter fibres is located between the two layers (2) with the larger diameter fibres and does not directly contact the external subcutaneous environment of the patient nor the inner part (7) of the bag or pouch.

The advantage of the arrangements shown in figures 10 and 13, i.e. where the second layer or one of the second layers has an inwardly facing surface, is that no carrier is required. In contrast, the second layer (2) acts as a carrier.

Detailed Description

Schweicher et al, Front Biosci (Landmark Ed), 2014; 19: 49-76, "Membranes to achieve immunoprotection of transplanted islets" reviews the use of semipermeable Membranes for encapsulation and immunoprotection of transplanted islets or beta cells for the treatment of diabetes. This article summarizes that despite many promising encapsulation studies and the development of many devices, cell encapsulation has not yet had an impact in a clinical setting. Some factors that limit the widespread use of encapsulated islets include incomplete isolation of the islets from the immune system and insufficient accessibility of the cells within the device to nutrient nutrients.

Devices for cell encapsulation have used various organic and inorganic materials. Hydrogels have been most successful to date among organic (polymeric) materials, although thermoplastic polymers have also been used due to their mechanical and chemical stability. Zondervan et al, Design of polyurethane films for encapsulation of Langerhans islets (Design of a polyurethane membrane for the encapsulation of islets of Langerhans), Biomaterials, 1992; 13(3): 136-144, mention is made of the use of polyurethane. However, the use of electrospinning to produce polyurethanes is not disclosed.

Zhuo, etc.; j Appl Polym Sci, 2008 discloses a method of preparing polyurethane nanofibers by electrospinning. The nanofibers electrospun from DMF solution have ultrafine diameters of about 700 to 50 nm. In particular, it was found that the solution concentration plays a major role in influencing the conversion of the polymer solution into ultrafine fibers, and the diameter increases with increasing solution concentration.

WO 2008/112190 provides a bioartificial pancreas and a method for producing the same to produce insulin in diabetic animals. The bioartificial pancreas comprises a perforated midsection with a fill port for introducing insulin producing cells, along which a biocompatible polymer network is deposited, and an immune barrier membrane formed on the midsection.

WO 2008/039530 provides a tissue engineered intervertebral disc comprising an inner layer and an outer layer, wherein the outer layer is a nanofibrous polymer carrier comprising polymer nanofibres and the inner layer comprises a hydrogel composition into which therapeutic cells are placed and cultured.

US 2017/0325933 provides a vascular prosthesis comprising a layer of fibroblasts, a layer of smooth muscle, a layer of endothelial cells and a lumen, all surrounded by a cortical layer of synthetic polymer produced by a process including electrospinning.

CN 107596448 provides a biofilm scaffold material and a method for producing said membrane, wherein said membrane comprises an outer layer, an intermediate layer and an inner layer. The inner layer comprises an electrospun fibrous membrane consisting of polyurethane, the intermediate layer comprises a mixture of polyurethane and polycaprolactone comprising a Ca salt, and the outer layer comprises polycaprolactone comprising a Ca salt.

CN 103623410 provides an antibacterial composition and an implant using the same for producing artificial organs and tissues for human body. The antimicrobial composition is coated with polyurethane by high voltage electrospinning to form the antimicrobial agent contained within the boundaries of the thermoplastic polyurethane elastomer.

CN 101785875 provides a method for preparing a superfine nanofiber vascular prosthesis, which uses polyurethane electrospinning to produce a prosthesis with high porosity, promotes material exchange, and simultaneously inhibits the proliferation of cells to the subendothelial layer of adjacent blood vessels.

CN 101708344 provides a nanofiber vascular prosthesis and a production method thereof. The inner part of the vascular prosthesis is prepared by mixing a solution of gelatin and glacial acetic acid with a cross-linking agent and sodium heparin, and then adding polyurethane as the outer layer by electrospinning. The inner layer improves blood compatibility, while the outer layer is biostable and can improve physical and mechanical properties.

CN 108498857 provides a method for the preparation of an artificial fullerene-carrying nucleus pulposus, comprising an inner layer of xerogel, coated around it by electrospinning with a polyurethane film. It contains xerogels without affecting their hydration and dimensional characteristics and prevents unwanted migration of xerogel implants and extends the shelf life of such devices.

GB 2518800 provides a duodenal intestinal membrane made of electrospun biocompatible materials, useful for the treatment of diabetes and steatosis. The duodenal epithelial membrane is placed in the duodenum to inhibit food contact with the intestinal mucosa and prevent physiological effects on the intestinal mucosal cells. The material is obtained by electrospinning and provides beneficial medical device properties including greater adhesion, reduced damage and greater bounce suppression.

WO 2006/080009 a2 provides an implantable bioreactor wherein a first compartment is capable of being in fluid communication with the vasculature of a patient and a second compartment is configured for containing cells, wherein the compartments are separated by a membrane. The membrane separating the compartments or the entire device can be made of electrospun materials, including electrospun polyurethane. The device can be used for treating diabetes, and the cells in the cell compartment are insulin-secreting cells.

Luo et al (Biomaterials (102(2016)249- > 258) describe the use of two identical electrospun polyurethane membranes sandwiched between two PET meshes the objective is to produce an implantable immune barrier membrane in which therapeutic cells are contained and prevent fibrotic deposition after implantation, which may affect intracellular nutrient supply.

The present invention is now directed to the inner portion constituting the interior of the therapeutic composition. Which is completely or partially surrounded by a biocompatible membrane. By "surrounding" is meant that the biocompatible membrane surrounds or encloses the inner portion in three dimensions, which means that the inner portion is typically at least 50%, more preferably 60%, 70%, 80%, 90% or 95% surrounded by the biocompatible membrane.

The inner portion is typically not hollow, but is formed of a substantially continuous block. The inner portion can, for example, comprise a carrier (including a stent) on or into which the therapeutic agent is disposed. Thus, the therapeutic composition is typically a continuous block that is completely enclosed, with an interior portion thereof being encapsulated by the biocompatible film.

The therapeutic composition is generally not tubular, i.e., preferably it does not have a hollowed-out portion that is in contact with the external environment. The therapeutic composition may be in the form of a bag or packet.

In the context of the present invention, the terms "bag" and "bag" have their usual meaning, referring to a non-rigid container in which the material from which the bag or bag is constructed forms the outer boundary of the environment in which the targeted contents may be placed to prevent the entry and exit of the targeted contents from other than the intended inlet and outlet of the bag or bag. The inlet and outlet may be sealed with the material from which the bag or pouch is constructed to completely enclose the interior of the bag or pouch. In the present invention, the biocompatible film forming the pouch or bag is impermeable to the therapeutic agent, thus serving to confine the therapeutic agent while being permeable to the bag or pouch of the selected molecule that is smaller than the therapeutic agent. The pouch or bag of the present invention may take a variety of shapes and configurations, particularly based on circles, squares and other polygons to form, among other things, discs, cuboids and other polyhedrons.

The shape of the bag or pouch may be approximately spherical. The average maximum diameter may be from 0.5 to 10cm, preferably from 1 to 5 cm.

In the context of the present invention, a carrier is defined herein and may comprise a scaffold, as also mentioned herein. For example, the carrier may be a hydrogel or collagen fibers, but collagen may also be described as a scaffold.

In some embodiments, the therapeutic composition (5, 6) is formed in the shape of a biconcave disc, e.g., similar to a red blood cell (erythrocyte). One example is shown in (9) in fig. 12. For example, an O-ring (O-ring) of a suitable material may be used to help form and maintain the shape of the biconcave disk by providing structure around the perimeter of the biconcave disk.

By partially surrounding, it is meant that the biocompatible membrane does not completely surround the inner portion, but provides a degree of coverage. The inner portion is typically surrounded by a biocompatible film by at least 50%, more preferably by at least 60%, 70%, 80%, 90% or 95%.

By completely surrounding, it is meant that the biocompatible film completely covers, wraps, or encloses the interior portion such that the interior portion is completely encapsulated (i.e., 100% surrounded) by the biocompatible film.

The film according to the invention is a sheetlike polyurethane material produced by electrospinning. The electrospun polyurethane according to the present invention provides a material which differs structurally from the polyurethane material produced by the process disclosed by Zondervan et al, as described above, in that the polyurethane network is formed by cross-linking a mixture of linoleic acid and linear poly (ether urethane) with dicumyl peroxide. The electrospinning process produces a more uniform and adjustable network of polyurethane fibers than prior art methods.

Preferably, the fibers are nanofibers. The term "nanofiber" refers to a micro-fiber whose diameter is conveniently measured in nm or μm.

Thus, the mean diameter of the polymer fibres in the membrane is less than 1000nm, typically less than 900nm, 800nm, 700nm, 600nm or 500nm, and most preferably 100-500nm or 50-500 nm. The relative standard deviation of the fiber diameter distribution around the mean fiber diameter is typically less than or equal to 30%.

Typically, the mean diameter of the polymer fibers in the scaffold is measured by Scanning Electron Microscopy (SEM). Typically, the standard deviation of the mean is also measured by SEM.

A fiber network is a random distribution of fibers in space that forms an interconnected web with spaces between the fibers. The network has small spaces between the fibers that make up the network, forming pores or channels in the network that allow fluid to pass through.

The porous network of fibers is a nonwoven network, i.e., the fibers are generally randomly oriented within the porous network. Thus, the polymeric fibers in the porous nonwoven fibrous network are not oriented in any particular way-that is, the fibers in the porous nonwoven network are generally randomly oriented or at least nearly randomly oriented. Thus, the degree of alignment of the polymer fibers in the film is low, if not completely random.

The membrane, which may be referred to as a barrier membrane, is size selective, i.e., selectively permeable to molecules or particles of a certain size. The following discussion of the barrier film applies in particular to the first layer (1)

Thus, the membrane according to the invention acts as a size selective barrier. The porous membrane is impermeable to cells. The pores or channels in the membrane are large enough to allow diffusion of ions, metabolites, proteins and/or bioactive molecules (e.g., glucose), but prevent the cells from penetrating and permeating the nanofiber network. The flow of fluid and molecules through the membrane may be measured using techniques known in the art, one example of which is described herein.

The flux profile of therapeutic molecules and nutrients may be 1X 10-6cm²S to 1X 10-7cm²The value of/s is desirably not less than 1X 10-5cm²And s. The flux properties should be comparable to other types of barrier membranes described in the literature (Thanos, c.g., Gaglia, j.l.&Pagliuca，F.W.，Cell Therapy：Current Status and Future Directions19-52(Humana Press，Cham，2017))。

A barrier film according to the present invention may be defined as a thin, flexible, sheet-like layer of material comprising a network of fibers. It may act as a border or lining. For example, the membrane may act as a barrier in a living organism.

Typical barrier films of the invention have a thickness of 25 to 250 μm, for example 10 or 20 to 150 μm.

The barrier film typically has a porosity equal to or greater than 50%, such as greater than or equal to 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In other words, the membrane typically has greater than 50% air by volume, such as greater than or equal to 60%, 65%, 70%, 75%, 80%, 85%, or 90% air (when the membrane is not fluid-filled). In a preferred embodiment, the porosity is from 50 to 80%.

The porous network of polymer fibres in the barrier membrane may have an average pore size of, for example, 0.5 to 100 μm, for example 1 μm to 10 μm, preferably less than 20, 15, 10 or 5 μm. The Langerhans islets in the pancreas are typically 100-200 μm in diameter, and thus such pores will prevent the passage of such cells. However, the pore size may be difficult to measure accurately because the pore size depends on the distance between the measured scaffolds, and no two pores have the same shape due to the random orientation of the nanofibers. To address this problem, the pore size can be measured by using an SEM and embedding the largest inscribed circle into the irregular polygon of the pore. The mean average for a given film sample can then be calculated.

The pore size is adjusted to be smaller than typical cell diameters (approximately less than 20 microns). Such porosity is advantageous in preventing cell proliferation through the scaffold membrane. The pore diameter can be calculated from the average pore area using the following formula:

equation 1: a formula for converting the average pore area (a) to the average pore diameter (d).

In the present invention, an exemplary pore size may be defined as the diameter of the largest inscribed circle that may be embedded in an irregular polygon formed by the intersection of three or more fibers. The mathematical description of this method can be found in Martinez, O. An Efficient Algorithm (2012) to Calculate the Center of the largest Inscribed Circle in An Irregular Polygon.

The membrane may have a gradient in pore size, porosity, or average fiber diameter. In a preferred embodiment of the invention, the membrane has a two-layer structure, wherein the pore size, porosity and/or average fiber diameter in the two layers are different.

The porosity, average pore size and average fiber diameter of the nonwoven network are interrelated as explained, for example, in Greiner and Weddorff, angelw.chem.int.ed.2007, 46, 5670-.

The polymer used to form the membrane is a biocompatible polymer. The membrane is non-cytotoxic. Preferably, the polymer is not a bioerodible or biodegradable polymer. The polymer is a thermoplastic polyurethane polymer. Typically, polyurethanes are permanent (nonabsorbable) polymers. This allows the therapeutic composition to be easily removed from the body if desired.

In some embodiments, the thermoplastic polyurethane may be a polycarbonate-urethane. In some embodiments, the thermoplastic polyurethane may be a silicone-polycarbonate-urethane. In some embodiments, the thermoplastic polyurethane may be a polyether-urethane. In some embodiments, the thermoplastic polyurethane may be a silicone-polyether-urethane. In some embodiments, the thermoplastic polyurethane may be a polyester-urethane. In some embodiments, the thermoplastic polyurethane may be a polymeric polyol-urethane. In some embodiments, the thermoplastic polyurethane may be a polyester-ether-urethane.

The film according to the invention may comprise further components in addition to the network of polymer fibers.

The film-forming polymer network is generally homogeneous, extensible into different forms, and has a controlled structure and properties.

The therapeutic composition according to the invention comprises a membrane according to the first aspect of the invention and a therapeutic agent, such as one or more drugs or cells. The film may partially or completely encapsulate the therapeutic agent. The film typically forms the outer portion of the therapeutic composition, while the therapeutic agent forms the inner portion.

The cells microencapsulated by the membrane of the invention may be supported on or in a carrier, which is preferably a scaffold. The cell-loaded scaffold in the present invention may advantageously further comprise a component suitable to provide mechanical strength and maintain the open-cell structural integrity of the porous network of fibers. The second component may advantageously limit deformation or stretching of the porous network of fibers, thereby minimizing adverse changes in porosity and pore size that are detrimental to the porous network, thereby facilitating cell growth. A method of preparing a scaffold having cells disposed thereon is disclosed in WO 2013/117926.

The scaffold is suitable for supporting cell growth and typically comprises a porous network of fibres. The fibers are typically polymeric fibers. The stent of the present invention may be elongated or cylindrical.

Alternatively, the therapeutic agent may be mixed with a different carrier material. Suitable materials include hydrogels, polymer foams and acellular tissues, and/or alginates.

In some embodiments, the barrier film, i.e. the first layer (1), may be surrounded by an outer layer of material, e.g. the second layer (2). The outer layer (second layer) may completely or partially surround the barrier membrane (first layer). Typically, the outer layer of the material forms another layer on top of the biocompatible film. The outer layer may serve to promote adhesion and blood vessel formation in the body. The outer layer may be formed from electrospun fibers and/or other carrier scaffold materials (e.g., hydrogels, gelatin foams, decellularized tissue, etc.).

In the context of a cell encapsulation device, this outer layer will be discussed further below.

The therapeutic compositions of the present invention can be used to more effectively implant therapeutic cells into a target tissue, such as damaged or diseased tissue. For example, such therapeutic compositions can provide a pathway for cells to optimally position in the body. The therapeutic compositions of the present invention may serve as an organ substitute. The cells can be, for example, pancreatic beta cells, and the therapeutic composition can serve as an artificial treatment for the pancreas. Alternatively, the cells may be stem cells.

Thus, the therapeutic compositions are useful for treating diabetes, particularly type I diabetes.

In some embodiments, the cell type or treatment may be associated with any one or more of the following. In some embodiments, the therapeutic uses or cell types are those associated with diabetic cell therapy: not only pancreatic beta cells, but also those cells in the path of development to them, such as ipscs, hpscs, and/or hES, pancreatic progenitor cells, endocrine progenitor cells, and beta cells.

In general, in some embodiments, the cell type can be any stem cell or progenitor cell. An example may be a cell (including a stem cell or progenitor cell) that will differentiate into a desired phenotype.

In some embodiments, the therapy is for AMD (age-related macular degeneration). Thus, in some embodiments, the cell type may be a retinal pigment epithelial cell.

In some embodiments, the therapy is for hemophilia or cancer, particularly hematologic cancer, such as leukemia. Thus, in some embodiments, the cell type may be a factor ix (fix) and factor XIII producing hepatocyte.

In some embodiments, the cell may be or include one or more genetically modified cells. Such as white blood cells. In some embodiments, the leukocytes may have been extracted from the patient or tissue match and engineered ex vivo and then returned to the patient. In some embodiments, the therapy is for ALS. Thus, in some embodiments, the cell type may be an astrocyte.

The treatment may comprise surgical implantation of the composition of the invention into the human or animal body, for example in the vicinity of the liver.

The therapeutic compositions of the present invention can advantageously improve the survival of therapeutic cells in vivo.

The therapeutic compositions of the invention are generally elongate and may, for example, be cylindrical, as this may facilitate delivery to tissue by injection or catheter. However, as will be discussed further below, the therapeutic composition may in principle be in any shape. In some embodiments, it is not generally in the shape of a hollow tube.

Thus, the therapeutic composition may have the shape of a polygonal prism. The polygonal column may be, for example, a triangular, quadrangular, pentagonal, hexagonal, heptaprismatic, octagonal, nonaprismatic or decaprismatic column. The polygonal column may be, for example, a hexagonal column. Typically, the polygon prism is a straight prism, and may be, for example, a straight triangular prism, a straight quadrangular prism, a straight pentagonal prism, a straight hexagonal prism, a straight heptaprismatic, a straight octagonal prism, a straight nonaprismatic, or a straight decaprismatic. The polygonal column may be, for example, a right hexagonal column. The polygonal prism is typically a straight regular prism, and may be, for example, a straight regular triangular prism, a straight regular quadrangular prism, a straight regular pentagonal prism, a straight regular hexagonal prism, a straight regular hepta-prism, a straight regular octagonal prism, a straight regular nona-prism, or a straight regular deca-prism. The polygonal column may be, for example, a right regular hexagonal column.

The therapeutic composition may have the shape of a cylinder or a polygonal column. The polygonal column may, for example, be as further defined above, and may, for example, be a straight regular polygonal column.

When the therapeutic composition has the shape of a cylinder or a polygonal column, the height may be, for example, 5mm to 10 cm. The height of the cylinder or polygon is typically from 8mm to 8cm, or for example from 1cm to 6 cm.

When the therapeutic composition has the shape of a cylinder or a polygonal column, the diameter of the cylinder or polygonal column is typically 2mm to 5 cm. The meaning of cylinder diameter is well known. The diameter of the polygon is also well known, which is the maximum distance between any pair of vertices. Thus, the diameter of a polygon prism is the maximum distance between any pair of vertices on any polygon facet of the prism. Thus, in the case of a hexagonal prism, particularly a straight regular hexagonal prism, the diameter is the diameter of any one hexagonal facet of the prism measured from the apex (or the intersection of the two sides) of the hexagonal facet through the center of the facet to the opposite apex (the intersection of the two opposite sides of the facet) of the facet.

The diameter of the cylinder or polygon may be, for example, 4mm to 3cm, or, for example, 6mm to 2 cm.

Advantageously, therapeutic compositions having the dimensions, heights, and diameters described herein are generally large enough to support cell growth, extending in all three dimensions sufficient to provide the advantages of 3D cell culture over 2D cell layers.

Electrospinning typically produces flat sheets of porous, non-woven, fibrous polymer film. During industrial production of such films, they are typically collected on a rotating drum, a flat collector, or in a roll-to-roll manner. In order to use such membranes as immunoprotective cell encapsulation devices, they need to be appropriately shaped. This can be achieved by joining two panels or a portion of a panel together or folding a portion of a panel onto itself. Adhesive bonding may be achieved by various techniques known in the art, such as laser Welding (Weber, m., Hoheisel, a. & glasmancher, B. (2016.) automatic control of the laser Welding Process of heart valve stents (automatic control of the laser Welding Process of heart valve stents), Current direction in biological Engineering, 2(1), page 301), sonic Welding (wire, e. et al., Ultrasonic Welding as a Preliminary Study of the bonding Process of Electrospun nanofiber mats (Preliminary Study of Ultrasonic Welding a Joining Process for electrically spun nanofiber mats), nano-materials, 8, 2018), bonding (y 133, y. hot Welding, n. S., join, S., part of heat fibers, bonding of fibers (60. about. hot fibers, 746. about, solvent bonding (Rianjanu, A., Kusumaamaja, A., Suyono, EA & Triyana, K. Solvent steaming improves the mechanical strength of electrospun polyvinyl alcohol nanofibers (Solvent vapor treatment improved mechanical strength of electrospinning polyvinyl alcohol nanofibers), Heliyon 4, e00592(2018)) or gluing (Musiari, F. et al, Feasibility study of electrospinning nanofibers to enhance adhesion (Feasibility testing of adhesive bonding force by electrospinning nanofibers), Procedia Structure. Integ. 2, 112-jar (2016)).

In addition, the production of small-sized tubular structures of electrospun material with diameters of several mm to cm is a well-known technique in the art (Krishnan, l. et al, the vascularization and cell separation potential of novel electrospun cell delivery vehicles, j. biomed. mater. res., a 102, 2208-19 (2014)). The combination of the above-described bonding techniques and small tube production can be used to form a closed therapeutic device for use in the present invention.

After the electrospun material is produced, it can be cut to the desired size and shape. It can then be folded and glued onto itself (e.g., by sonic welding) to create the final 3-dimensional product. Alternatively, two separate pieces of electrospun material may be secured together by sonic welding to make a bag or pouch. Optionally, another layer may be added to the final product, for example, collagen fibers (e.g., in the form of a collagen film), which may surround the electrospun membrane to varying degrees (e.g., which may partially or completely surround the electrospun membrane, e.g., which may surround 60, 80, or 100%). The present invention further provides a therapeutic composition comprising: (i) cells, biomolecules, or other active agents; (ii) and (4) a bracket. The biomolecule or other active agent may be a drug, nucleic acid, nucleotide, protein, polypeptide, antibody or exosome. The nucleic acid may comprise DNA, RNA, RNAi, SaRNA or SiRNA. Optionally, the therapeutic composition comprises (i) cells, e.g., adhesion-treating cells, and (ii) a scaffold. The cells may be disposed within a porous network of fibers in the scaffold. The cells may be disposed in the pores of the scaffold. The cells may be disposed (e.g., may be adhered) on the surface of the scaffold. The cells may be disposed in the pores of the scaffold, or may be disposed (e.g., may be adhered) on the surface of the scaffold.

The polymer of the scaffold may be the same or different polymer than the polymer of the fibers in the membrane. Typically the same polymer. In one embodiment of the invention, the scaffold may be formed of collagen, collagen fibers or collagen sponge. Suitable polymers are discussed further below.

In the therapeutic composition of the present invention, a membrane may be disposed around at least a portion of the inner portion.

The present invention also advantageously provides a device for encapsulating therapeutic cells and then implanting and retrieving them from a subject when necessary. The device is a preferred embodiment of the therapeutic composition according to the first aspect of the invention. ****

The apparatus may preferably comprise:

an encapsulated inner portion comprising a therapeutic agent;

a barrier film layer (first layer) encapsulating an inner portion, as disclosed herein; and

an outer layer (second layer) surrounding the barrier film layer.

References herein to an outer layer may also be understood to refer to a second layer.

The outer layer of the device may comprise one or more different layers.

The barrier membrane prevents the implanted therapeutic cells from escaping into the patient and prevents the patient's immune cells from reaching the therapeutic cells.

The outer layer is typically a cell permeable material, such as a stent material, which allows blood vessels to form adjacent to the inner barrier membrane.

In a preferred embodiment, the outer layer comprises electrospun fibers of relatively large diameter (typically 1000-. The outer layer typically has a porosity, pore size, and average fiber diameter greater than the membrane layer.

Preferably, the outer layer is formed of electrospun polyurethane fibers, and

(i) porosity from 70 to 98%, preferably from 80 to 95%; and/or

(ii) An average pore diameter of 5 to 80 μm, preferably 10 to 50 μm; and/or

(iii) The mean diameter of the polymer fibers is from 1 to 10 μm, preferably from 2 to 8 μm, most preferably from 3 to 7 μm.

Preferably, the outer layer is formed of fibers, preferably electrospun polyurethane fibers. The outer layer may be absorbable or non-absorbable. Alternatively, the outer layer may be a hydrogel. In another aspect, it may comprise decellularized tissue.

The inner portion optionally comprises a carrier material and a therapeutic agent. The material may be a scaffold material as discussed above with respect to the therapeutic composition. The inner part material may be fibrous in nature, for example electrospun polyurethane fibres. Alternatively, it may be a hydrogel, foam or tissue alginate, gelatin or collagen (optionally fibres or sponges), or acellular tissue.

Preferably, the inner portion (including any carrier or scaffold) and/or the outer layer (i.e. second layer) may comprise a material formed from fibres. Such fibers typically comprise a biocompatible polymer. Any suitable biocompatible polymer may be used, and the biocompatible polymer may be, for example, a natural polymer or a synthetic polymer. In some embodiments, the polymer is a bioerodible or biodegradable polymer.

The fibers of the inner portion (including any carrier or scaffold) and/or the outer layer (i.e., second layer) in the upper section may, for example, comprise any of the following polymers: poly (L-lactide); polyglycolic acid; polyhydroxybutyrate; polystyrene; polyethylene; polypropylene; polyethylene oxide; a polyester urethane; polyvinyl alcohol; polyacrylonitrile; a polylactide; polyglycolide; a polyurethane; a polycarbonate; a polyimide; a polyamide; an aliphatic polyamide; an aromatic polyamide; a polybenzimidazole; polyethylene terephthalate; poly [ ethylene-co-vinyl acetate ]; polyvinyl chloride; polymethyl methacrylate; polyvinyl butyral; polyvinylidene fluoride; poly (vinylidene fluoride-co-hexafluoropropylene); cellulose acetate; polyvinyl acetate; polyacrylic acid; polymethacrylic acid; polyacrylamide; polyvinylpyrrolidone; polyphenylene sulfide; hydroxypropyl cellulose; polyvinylidene chloride, polytetrafluoroethylene, polyacrylate, polymethacrylate, polyester, polysulfone, polyolefin, polysilsesquioxane, siloxane, epoxy, cyanate ester, bismaleimide polymer; polyketones, polyethers, polyamines, polyphosphazenes, polysulfides, organic/inorganic hybrid polymers or copolymers thereof, such as poly (lactide-co-glycolide); polylactide-co-poly (epsilon-caprolactone) or poly (L-lactide) -co-poly (epsilon-caprolactone); or blends thereof, such as a blend of polyvinyl alcohol and polyacrylic acid.

The fibers may comprise a bioerodible or biodegradable polymer, for example selected from the group consisting of poly (L-lactide); polyglycolic acid; polyhydroxybutyrate and poly (ester-urethane) polymers.

The fibers may alternatively comprise, for example, a biopolymer, or a blend of a biopolymer and a synthetic polymer. For example, the following biopolymers and blends of biopolymers with synthetic polymers may be used:

collagen; collagen/polyethylene oxide; collagen/poly (epsilon-caprolactone); collagen/poly (lactide) -co-poly (epsilon-caprolactone); gelatin; gelatin/poly (epsilon-caprolactone); gelatin/polyethylene oxide; casein/polyvinyl alcohol; casein/polyethylene oxide; a lipase; cellulase/polyvinyl alcohol; bovine serum albumin/polyvinyl alcohol; luciferase/polyvinyl alcohol; alpha-chymotrypsin; fibrinogen; silk; regenerating silk; regenerating the Bombyx mori filaments; bomyx mori filaments/polyethylene oxide; silk fibroin; silk fibroin/chitosan; silk fibroin/chitin; silk/polyethylene oxide (coaxial); artificial spider silk; chitin; chitosan; chitosan/polyethylene oxide; chitosan/polyvinyl alcohol; quaternized chitosan/polyvinyl alcohol; hexanoyl chitosan/polylactide; cellulose or cellulose acetate.

The fibers may alternatively, for example, comprise a blend of two or more polymers, a copolymer (which may, for example, be a block copolymer) or a blend of a polymer and an inorganic material.

Non-limiting examples of such blend blends of two or more polymers include polyvinylpyrrolidone/polylactide blends; polyaniline/polystyrene blends; polyaniline/polyethylene oxide blends; polyvinyl chloride/polyurethane blends, poly [ (m-phenylenevinylene) -co- (2, 5-dioctyloxy-p-phenylenevinylene) ]/poly (ethylene oxide) blends; poly [ 2-methoxy-5- (2' -ethylhexyloxy) -1, 4-phenylenevinylene ] (MEH-PPV)/polystyrene blend, polyaniline/polystyrene blend; polyaniline/polycarbonate blends, polyethylene terephthalate/polyethylene terephthalate-co-polyethylene isophthalate blends, polysulfone/polyurethane blends; chitosan/polylactide blends, polyglycolide/chitin blends and polylactide/poly (lactide-co-glycolide) blends.

Non-limiting examples of such block copolymer systems include polylactide-b-poly (ethylene oxide) block copolymers; poly (lactide-co-glycolide) -b-poly (ethylene oxide) block copolymers; poly [ (trimethylene carbonate) -b- (epsilon-caprolactone) ] block copolymers; polystyrene-b-polydimethylsiloxane and polystyrene-b-polypropylene block copolymers; polystyrene-b-polybutadiene-b-polystyrene block copolymers and polystyrene-b-polyisoprene block copolymers.

Thus, the nanofibers may for example comprise any of the materials listed in the preceding paragraphs. Nanofiber scaffolds comprising the above polymers, copolymers, and blends of two or more polymers can be produced by electrospinning, as detailed in Greiner and Wendorff, angelw.chem.int.ed.2007, 46, 5670-.

The therapeutic compositions or devices of the present invention may further comprise additives, preferably mixed with the fibers of the electrospun material. Such additives may include growth factors, such as VEGF. Alternatively, the additive may be an oxygen-releasing material, such as CaO₂Or hemoglobin. Since one important problem of islet transplantation is premature islet death due to hypoxic conditions, the use of oxygen-releasing agents may solve the hypoxic problem. Alternative additives include cross-linking agents. For example, calcium ions are used for crosslinking of hydrogels. Suitable additives may be selected from: haemoglobin, peroxides (e.g. H)₂O₂、CaO₂、MgO₂、Li₂O₂、Na₂O₂) Sodium percarbonate (Na)₂CO₃) Perfluorocarbons, hydroxyapatite, tricalcium phosphate (bone growth promoting material), growth factors, catalase and other enzymes. In some embodiments, other additives may include antimicrobial agents, antiviral agents, antifungal agents, and/or silver nanoparticles.

Examples of growth factors may include any one or more of: colony stimulating factors (m-CSF, G-CSF, GM-CSF), Epidermal Growth Factor (EGF), Erythropoietin (EPO), Fibroblast Growth Factor (FGF), Hepatocyte Growth Factor (HGF), liver cancer-derived growth factor (HDGF), interleukins, Keratinocyte Growth Factor (KGF), Migration Stimulating Factor (MSF), Macrophage Stimulating Protein (MSP), also known as hepatocyte-like growth factor protein (HGFLP), myostatin (GDF-8), neuregulin (e.g., neuregulin 1, 2, 3 or 4), neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), Nerve Growth Factor (NGF), neurotrophin-3 or 4), Placental Growth Factor (PGF), platelet-derived growth factor (PDGF), nephropathies (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors, such as transforming growth factor alpha (TGF-alpha) or beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), Vascular Endothelial Growth Factor (VEGF), or factors involved in the Wnt signaling Pathway (Wnt signaling Pathway).

In particular, the growth factor may be insulin and/or an insulin-like growth factor. Also preferred are cytokines comprising the above interleukins.

Additives may be present in any of the component layers of the compositions or devices of the present invention. It is preferably present in the outer and/or inner part.

Typically, the cells in the therapeutic composition or device of the invention comprise adherent therapeutic cells. Adherent cells are cells that are capable of adhering to a culture vessel specifically treated for adherent cell culture. The concept of adherent cells is well known to those skilled in the art. One skilled in the art can recognize whether cells are adherent. The therapeutic cells are cells capable of having a therapeutic effect. The therapeutic cells are typically living cells. The therapeutic cells are typically cells capable of repairing damaged or diseased tissue. The therapeutic cells are preferably autologous. In other words, the cells are preferably from the following patients: the cells will be administered in vivo to repair damaged or diseased tissue. Alternatively, the cells are preferably allogeneic. In other words, the cells are preferably from a patient that is immunologically compatible with the patient to whom the cells will be administered in vivo to repair damaged or diseased tissue, as described above. The cells may be semi-allogeneic. Semi-allogeneic populations are typically generated from two or more patients that are immunologically compatible with the patient to whom the cells are to be administered in vivo. In other words, all cells are preferably genetically identical to, or sufficiently genetically identical to, the patient to whom they are to be administered in vivo, so that the cells are immunologically compatible with the patient to whom they are to be administered in vivo.

The composition typically comprises more than one cell, e.g., at least about 2, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 200, at least about 500, at least about 1000, at least about 2000, at least about 5000, at least about 10000, at least about 50000, at least about 100000, at least about 2 × 10⁵At least about 5 x 10⁵At least about 1 × 10⁶At least about 2 x 10⁶At least about 5 x 10⁶At least about 1 × 10⁷At least about 2 x 10⁷At least about 5 x 10⁷At least about 1 × 10⁸Or at least about 2 x 10⁸And (4) cells. In some cases, the composition may comprise at least 1.0 x 10⁷At least 1.0 x 10⁸At least 1.0 x 10⁹At least 1.0 x 10¹⁰At least 1.0 x 10¹¹Or at least 1.0 × 10¹²Individual cells or even more.

The number of cells in the composition generally depends on the size of the shape. The number of cells is usually about 0.5X 10⁵To about 3X 10⁵E.g. about 1X 10⁵To about 2X 10⁵Particularly in the cylindrical stents of the present invention, the length is from about 4mm to about 8mm and the diameter is from about 200 μm to about 500 μm.

The adhesion therapy cells can comprise pancreatic beta cells, such as beta cell aggregates. A beta cell aggregate is an aggregate of two or more cells, at least one of which is a beta cell. An example of a beta cell aggregate is islets of Langerhans. For example, islets of Langerhans can be isolated from cadaveric donor pancreases.

The present invention provides a method of making the barrier membrane, therapeutic composition or device of the present invention. In a preferred embodiment, the biocompatible membrane can be fabricated into a sheet-like membrane by electrospinning. Such a sheet-like film can then be readily shaped into the final therapeutic composition or device.

Alternatively, the films of the present invention can be incorporated into coatings of existing structures having any combination of these layers and subsequently sealed by sonic welding, heat sealing, solvent bonding, gluing, laser welding, and the like.

A suitable method for making nanofiber nonwoven is disclosed in US20120115386a 1.

The invention further provides a method of producing a therapeutic composition of the invention as defined herein, the method comprising combining in a culture vessel (i) a scaffold and (ii) a cell, biomolecule or other active agent. (i) Both the scaffold and (ii) the cells, biomolecules or other active agents may be as further defined anywhere herein.

In one embodiment, a method for producing a therapeutic composition of the invention comprises combining in a culture vessel (i) a scaffold and (ii) an adherent therapeutic cell, drug, nucleic acid, nucleotide, protein, polypeptide, or exosome.

The method of producing the therapeutic composition of the present invention may, for example, comprise: (i) binding the scaffold, and (ii) allowing the adherent therapeutic cells to penetrate and proliferate on the surface and inside the outer portion of the scaffold, thereby producing the therapeutic composition.

The number of cells added to the container generally corresponds to the number of cells that should be present in the composition of the invention. The proportion of added cells attached to the scaffold can be measured by removing the scaffold from the container and determining how many cells, if any, remain in the container. Techniques for culturing cells are well known to those skilled in the art.

The scaffold and cells may be combined in any suitable culture vessel. The container may be a flask or a plate well, such as a standard 6-, 24-or 96-well plate. Such flasks and plates are commercially available from the following sources: corning, Fisher scientific, VWR supplier, Nunc, Starstedt or Falcon.

The invention further provides a therapeutic composition of the invention for use in a method of treatment of the human or animal body by therapy.

In all cases, the therapeutic cells are preferably derived from the patient or an allogeneic donor. The extraction of cells from the patient should ensure that the cells themselves are not rejected by the patient's immune system. Any difference between the donor and recipient will eventually lead to cell clearance, but not before repair of at least a portion of the damaged or diseased tissue.

The therapeutic compositions of the present invention can be administered to any suitable patient. The patient is typically a human patient. The patient may be an infant, a juvenile or an adult. The patient may be known to have damaged or diseased tissue, or suspected of having damaged or diseased tissue. The patient may be predisposed to, or at risk of, the relevant disease or injury. The patient may have diabetes.

Transfection of cells is well known in the art. Cells are generally transfected with nucleic acids encoding the agents. For example, viral particles or other vectors encoding the agents may be used.

The nucleic acid causes expression of the agent in the cell. The nucleic acid molecule will preferably comprise a promoter operably linked to the sequence encoding the agent, which promoter is active or can be induced in the cell.

The composition may be administered by any route. Suitable routes include, but are not limited to, intravenous, intramuscular, intraperitoneal, or other suitable routes of administration. The composition is preferably applied directly to damaged or diseased tissue. Injection or insertion through a catheter is particularly preferred.

As described above, the film of the present invention is formed by electrospinning. Preferably, any scaffold used in the present invention can also be produced by electrospinning.

In a preferred embodiment of the present invention, there is provided a method of producing a biocompatible porous membrane, the method comprising electrospinning a fiber (preferably, nanofiber) precursor solution onto a collection substrate to produce a biocompatible membrane comprising a nonwoven network of thermoplastic polyurethane polymer fibers; wherein the nanofiber precursor solution comprises a polymer dissolved in a solvent.

The network of polymer fibers may comprise a single layer of fibers or multiple (two or more) layers. The porosity of each layer and the pore size within each layer may be the same or different.

The electrospinning process can be easily adapted to produce films with a multilayer structure. The production of the multilayer structure will be discussed further below.

Thus, the membrane according to the first aspect of the invention may have a gradient structure. This means that at least one characteristic of the membrane (e.g. density, stiffness, porosity, fibre size, pore size) changes across the body of the membrane from one side to the other. The properties are changed by changing the conditions during electrospinning.

As described herein, typical multilayer films comprise a bilayer structure. When the membrane is combined with a scaffold, in some embodiments it is preferred that the layer having the smaller average pore size is in contact with the scaffold. This then forms a three-layer structure together with the stent.

The polymer fibers in the film are produced by electrospinning, as described in further detail below. The fibers forming the scaffold (as discussed further below) may also be formed by electrospinning or by other suitable methods known to those skilled in the art, including but not limited to melt spinning, dry spinning, wet spinning, and extrusion. Electrospinning is preferred.

The use of electrospinning provides certain advantages. Such as repeatability from batch to batch and is compatible with current automated equipment. Furthermore, polymer (nano) fibers having a specific mean fiber diameter and a low standard deviation from the mean can be produced very stably. This provides control over porosity and pore size.

The polymer fibers of the membrane and the scaffold may comprise the same polymer or different polymers. If they are the same polymer, the polymer is a polyurethane.

The fibers of the scaffold may be the same or different from the fibers of the inner and/or outer portions. In some embodiments, the scaffold fibers are the same as the fibers of the inner portion. In some embodiments, the scaffold fibers are the same as the fibers of the outer portion (if those of the outer portion are different from the fibers of the inner portion). For example, stents typically comprise or consist of polymers, which are both bioabsorbable and biocompatible, such as polylactide, polyglycolide, poly (lactide-co-glycolide) (PLGA) or Polycaprolactone (PCL), for example, with polyhydroxybutyrate or polyester urethane also being useful.

More generally, the fibers of the scaffold may be selected from the following:

a polylactide; polyglycolide; poly (lactide-co-glycolide) (PLGA); polycaprolactone (PCL); polyhydroxybutyrate; poly (epsilon-caprolactone); polystyrene; polyethylene; polypropylene; polyethylene oxide; a polyester urethane; polyvinyl alcohol; polyacrylonitrile; a polylactide; polyglycolide; a polyurethane; a polycarbonate; a polyimide; a polyamide; an aliphatic polyamide; an aromatic polyamide; a polybenzimidazole; polyethylene terephthalate; poly [ ethylene-co- (vinyl acetate) ]; polyvinyl chloride; polymethyl methacrylate; polyvinyl butyral; polyvinylidene fluoride; poly (vinylidene fluoride-co-hexafluoropropylene); cellulose acetate; polyvinyl acetate; polyacrylic acid; polymethacrylic acid; polyacrylamide; polyvinylpyrrolidone; polyphenylene sulfide; hydroxypropyl cellulose; polyvinylidene chloride, polytetrafluoroethylene, polyacrylate, polymethacrylate, polyester, polysulfone, polyolefin, polysilsesquioxane, siloxane, epoxy, cyanate ester, bismaleimide polymer; polyketones, polyethers, polyamines, polyphosphazenes, polysulfides, organic/inorganic hybrid polymers or copolymers thereof, such as poly (lactide-co-glycolide); polylactide-co-poly (epsilon-caprolactone) or poly-L-lactide-co-poly (epsilon-caprolactone); or blends thereof, such as a blend of polyvinyl alcohol and polyacrylic acid.

In some embodiments, the scaffold may comprise collagen in the form of fibers or collagen sponge. Collagen scaffolds are useful due to the high biocompatibility of collagen. Alternatives to collagen that may be used in some embodiments include the polymers listed above and the bioerodible or biodegradable polymers listed below.

The fibers of the scaffold may independently comprise or consist of a bioerodible or biodegradable polymer, for example selected from the group consisting of polylactide, polyglycolide, poly (lactide-co-glycolide) (PLGA), Polycaprolactone (PCL), poly (epsilon-caprolactone) (PCL), polyhydroxybutyrate, and poly (ester-urethane).

The fibers of the scaffold may alternatively, for example, independently comprise or consist of a biopolymer or a blend of a biopolymer and a synthetic polymer.

The film according to the first aspect of the invention may comprise a plurality of layers. For example, the film may comprise 1, 2, 3, 4 or 5 different layers. The layers may include different levels of porosity.

Typically, the thickness (depth) of the layers in the films of the present invention is from about 30 μm to about 1000 μm. The layers in the film may, for example, have a thickness (depth) of from about 30 μm to about 800 μm, for example from about 40 μm to about 600 μm, or from about 50 μm to about 400 μm, or from 50 μm to 200 μm or from 50 μm to 150 μm. The layers in the film may have a thickness (depth) of, for example, about 50 μm to about 200 μm, or, for example, about 80 μm to about 120 μm.

The porosity and pore size of each layer in the membrane may be as defined above for the membrane according to the first aspect of the invention. Thus, each layer may independently have a porosity equal to or greater than 50%. Further, each layer of the membrane may independently have an average pore size of 10 μm to 20 μm.

The electrospinning process is well known per se and is described, for example, in the following review articles: z. M.Huang et al, compositions Science and Technology, 63(2003) 2223-. The skilled person will know how to apply this technique appropriately. Discussed briefly below.

The electrospinning process generally involves electrospinning a fiber precursor solution onto a collecting substrate or onto a previous layer on a collecting substrate while rotating the collecting substrate at a specific speed, wherein the fiber precursor solution comprises a specific desired polymer dissolved in a solvent.

Generally, in electrospinning, the polymer or polymer blend to be used to prepare the fiber network is dissolved in a suitable solvent until a homogeneous solution of the desired concentration is obtained. The concentration of the polymer solution must generally be high enough to achieve sufficient chain entanglement to form continuous fibers. The polymer solution is then typically loaded into a container (typically a syringe) connected to a conductive (typically metal) capillary. The capillary tube is connected to a high voltage (usually to the positive pole of a high voltage dc power supply) and is held at a fixed distance from the grounded collection device. The collecting means may be metallic and is typically covered in a collecting substrate on which the fibres are deposited. The collecting device is preferably rotatable to ensure uniform deposition of the material. Fibers are typically produced by passing a polymer solution through a metal capillary at a fixed flow rate while applying a high voltage to the capillary to establish an electric field between the capillary and a collection device. The applied voltage should be high enough to overcome the surface tension of the polymer droplet at the capillary tip. As charge accumulates on the surface of the droplet, the surface area must be increased to accommodate the additional charge, which is created by forming taylor cones from the droplet, from which the continuous fibers are then extracted. As the fibers travel toward the grounded collector, the solvent evaporates rapidly and the fibers are further elongated due to instability caused by the golomb repulsion of surface charges on the jets. Instability in the jet caused by high charge density leads to rapid whipping of the jet, producing solid (dry) filaments of nanometer/micrometer diameter. If it is desired to form a random layer of nonwoven fibers on the substrate, the collector is rotated slowly (e.g., at a speed of about 100 rpm). Alternatively, if a layer of aligned fibers is desired, the collector may be rotated at a higher speed (e.g., at a speed of about 2500 rpm). Multiple layers of different fiber arrangements, from a randomly arranged nonwoven layer to a highly arranged fibrous layer, can be deposited by varying the rotation speed during deposition. After a fixed amount of material has been deposited to produce one or more layers of a particular desired thickness, the layer or layers are dried to remove any residual solvent/moisture from the fibers. Typically, it is dried under vacuum, for example at room temperature (about 25 ℃) for 24-48 hours.

Any suitable polymer may be used in the fiber precursor solution or in the solution used in the electrospinning process. The polymer used may be any of the polymers listed above with respect to the membrane or scaffold. All of these polymers can be used in electrospinning processes to produce porous three-dimensional networks of nanofibers, for details see Greiner and Wendorff, angelw.chem.int.ed., 2007, 46, 5670-5703.

Any suitable solvent may be used in the nanofiber precursor solution. A variety of solvents may be used in electrospinning including, for example, water and polar, non-polar, protic and aprotic organic solvents. The solvent is chosen to be suitable for the polymer or blend used, in particular so that a homogeneous solution of the desired concentration of polymer can be obtained.

The concentration of polymer in the solution should be high enough to achieve sufficient chain entanglement to form continuous fibers. Typically, the concentration of polymer in the solvent is from about 1 wt% to about 20 wt%. The concentration of polymer in the solvent may be, for example, from about 2 wt% to about 10 wt%. For example, the concentration of polymer in the solvent can be from about 3 wt% to about 5 wt%.

Typically, the distribution capillaries of the fiber forming module have an inner diameter of about 0.5mm to about 1.0 mm.

To ensure uniform deposition on the collecting substrate, electrospinning typically also includes moving at least a portion of the fiber collecting device relative to the fiber forming module during the deposition. Thus, typically, electrospinning further comprises moving at least a portion of the fiber collection device during the deposition process.

Deposition of multiple layers on the collection substrate is continued until multiple layers of a particular desired thickness are obtained. The thickness of the multiple layers may be as further defined above for the films of the invention, and may for example be from about 30 μm to about 1000 μm, or for example from about 50 μm to about 200 μm or 150 μm, for example from about 80 μm to about 120 μm.

Thus, the step of feeding the fiber precursor solution through a dispensing capillary while applying the voltage is typically performed until the thickness of the layers of the scaffold precursor have the appropriate thickness.

Typically, the flow rate at which the fiber precursor solution is fed through the dispensing capillary is from 100 μ l/hr to 3000 μ l/hr. More typically, it is from 400 μ l/hr to 2500 μ l/hr, for example about 2000 μ l/hr.

The distance between the dispensing capillary and the collection substrate is typically 200mm to 400 mm. More typically, it is from 200mm to 300mm, for example about 250 mm.

The voltage applied between the dispensing capillary and the fiber collection device is typically 2kV to 15 kV. More typically it is from 4kV to 10kV, for example about 5-8 kV.

Typically, electrospinning is carried out at a temperature of 22 ℃ to 28 ℃. More typically, electrospinning is carried out at a temperature of 23 ℃ to 27 ℃, e.g., about 25 ℃.

Typically, electrospinning is carried out in air at a relative humidity of 20% to 45%. Electrospinning can be carried out, for example, in air at a relative humidity of 35% to 45%, e.g., about 40%.

The electrospinning process for producing a membrane or scaffold may further comprise: drying the multilayer polymeric fiber thus produced to remove residual solvent; cutting the plurality of layers of polymer fibers into elongated strips, thereby producing the stent precursor. Typically, the scaffold precursor is dried under vacuum. Typically, drying is performed under vacuum at room temperature.

Typically, the electrospinning process further comprises: the resulting membrane or scaffold is removed from the collection substrate. The collection substrate typically comprises a release paper sheet, aluminum foil, or silicone coated sheet.

The invention may also be defined by the following further aspects:

yet another aspect of the invention provides a therapeutic composition comprising an interior portion and a biocompatible membrane completely or partially surrounding the interior portion; wherein the biocompatible film comprises a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning. The membrane may be formulated to act as a cell encapsulation device, selectively allowing passage of agents such as nutrients, but not cells.

In yet another aspect of the invention there is provided a film comprising at least two layers, wherein

(i) The first layer is a biocompatible film comprising a porous nonwoven network of thermoplastic polyurethane polymer fibers formed by electrospinning; and

(ii) a second layer disposed on the first layer.

In yet another aspect of the invention, a biocompatible membrane is provided comprising a porous non-woven network of thermoplastic polyurethane polymer fibers formed by electrospinning for use in a method of immunoprotection of a therapeutic cell.

In a further aspect of the invention there is also provided a film or therapeutic composition as defined above for use in a method of treatment of the human or animal body by therapy.

Yet another aspect of the invention provides a method for the manufacture of a therapeutic composition according to the first aspect of the invention, the method comprising:

(i) an electrospinning process to produce a biocompatible membrane comprising a porous non-woven network of thermoplastic polyurethane polymer fibers; and

(ii) the biocompatible membrane is molded to produce a therapeutic composition in which the interior portion is completely or partially surrounded.

The following applies to any aspect of the invention unless otherwise indicated. In some embodiments, the biocompatible membrane has a porosity of greater than or equal to 50%, preferably from 50% to 80%.

In some embodiments, the biocompatible membrane has an average pore size of less than 5 μm, and preferably an average pore size of less than 2 μm.

In some embodiments, the biocompatible film has a thickness of 10 to 250 μm, preferably 10 to 150 μm or 20 to 150 μm, most preferably 50 to 150 μm or 50 to 200 μm.

In some embodiments, the mean diameter of the polymer fibers is less than 700nm, preferably less than 600nm, preferably less than 500nm, and most preferably 100-500nm, even more preferably 50-500 nm.

In some embodiments, the biocompatible membrane is non-biodegradable.

In some embodiments, the biocompatible membrane comprises a bilayer structure. In some embodiments, the bilayer is arranged such that the layer with the higher porosity faces inwardly, e.g. towards the wound site, while the lower porosity layer faces outwardly to prevent bacteria and e.g. particulates from entering the wound site, while preferably still allowing oxygen and/or water to enter the wound site. Such an arrangement may be useful, for example, in internal wound care, such as periodontitis, nerve sheaths, hernia repair patches, artificial periosteum, and/or fistulas. In this way, a corresponding internal wound care device is also provided. This may also include any of the additives described herein.

In some embodiments, the composition further comprises a therapeutic agent, wherein the therapeutic agent is preferably in the inner portion, and preferably wherein the therapeutic agent is selected from the group consisting of: a therapeutic cell, a drug, a nucleic acid, a nucleotide, a protein, a polypeptide, an antibody, a particle such as a lipid nanoparticle, an extracellular vesicle, or an exosome, optionally wherein the nucleic acid comprises DNA, RNA, RNAi, SaRNA, or SiRNA.

In some embodiments, the composition further comprises a carrier on or in which the therapeutic agent is disposed, preferably wherein the therapeutic agent is attached to a surface of the carrier, disposed in a well of the carrier, or both.

In some embodiments, the biocompatible membrane completely or partially surrounds the carrier.

In some embodiments, the carrier comprises a porous non-woven network of polymer fibers, or a hydrogel, gelatin, collagen sponge, or acellular tissue.

In some embodiments, the composition comprises cells, wherein the cells are preferably pancreatic beta cells or islet cells.

In some embodiments, the composition further comprises an outer layer disposed on the outer surface of the biocompatible membrane, preferably wherein the outer layer is formed of electrospun fibers, preferably polyurethane fibers, and/or comprises a hydrogel, gelatin or collagen sponge or acellular tissue.

In some embodiments, the porosity of the outer layer is higher than the porosity of the biocompatible membrane, and/or wherein the mean average fiber diameter of the outer layer is greater than the mean average fiber diameter in the biocompatible membrane, and/or wherein the average pore size of the outer layer is greater than the average pore size of the inner layer.

In some embodiments, the outer layer is formed of electrospun polyurethane fibers, and

(i) a porosity of 70 to 98%, preferably 80 to 95%; and/or

(ii) An average pore diameter of 5 to 80 μm, preferably 10 to 50 μm; and/or

(iii) The mean diameter of the polymer fibres is from 1 to 10 μm, preferably from 2 to 8 μm, most preferably from 3 to 7 μm.

In some embodiments, the composition further comprises one or more additives, wherein the additives are preferably disposed within the carrier or outer layer (if present), further wherein the additives are selected from growth factors such as VEGF, cross-linking agents, growth factors, catalase and other enzymes; or oxygen-releasing materials such as CaO₂Or haemoglobin, peroxides (e.g. H)₂O₂、CaO₂、MgO₂、Li₂O₂、Na₂O₂) Sodium percarbonate (Na)₂CO₃) Perfluorocarbons, hydroxyapatite, tricalcium phosphate (bone growth promoting material), most preferably CaO₂And/or MgO₂Which can be used as a cross-linking agent in alginate hydrogels; the catalase is also preferably combined with an oxygen-releasing material that advantageously scavenges any released toxic hydrogen peroxide.

Yet another aspect of the present invention provides a film comprising at least two layers, wherein (i) the first layer is a biocompatible film comprising a porous nonwoven network of thermoplastic polyurethane polymer fibers formed by electrospinning; (ii) a second layer disposed on the first layer. In some embodiments, the first layer is defined as having a porosity of greater than or equal to 50%, preferably from 50% to 80%; having an average pore diameter of less than 5 μm; has a thickness of 10 to 150 μm, preferably 20 to 150 μm, most preferably 50 to 150 μm or 50 to 200 μm; or a biocompatible membrane wherein the mean diameter of the polymer fibers is less than 700nm, preferably less than 600nm, preferably less than 500nm, and most preferably 100-500nm, even more preferably 50-500 nm. In some embodiments, the second layer further comprises an outer layer disposed on the outer surface of the biocompatible membrane, preferably wherein the outer layer is in the form of electrospun fibers, preferably polyurethane fibersAnd/or comprises a hydrogel, gelatin or collagen sponge, or acellular tissue; a porosity higher than that of the biocompatible membrane, and/or wherein the mean average fiber diameter of the outer layer is greater than that of the biocompatible membrane, and/or wherein the average pore diameter of the outer layer is greater than that of the inner layer; and wherein it is formed from electrospun polyurethane fibers, and (i) a porosity of from 70 to 98%, preferably from 80 to 95%; and/or (ii) an average pore diameter of 5 to 80 μm, preferably 10 to 50 μm; and/or (iii) the mean diameter of the polymer fibres is from 1 to 10 μm, preferably from 2 to 8 μm, most preferably from 3 to 7 μm. In some embodiments, the film further comprises one or more additives, wherein the additives are preferably selected from the group consisting of: growth factors such as VEGF, cross-linking agents, growth factors, catalase and other enzymes; or oxygen-releasing materials such as CaO₂Or haemoglobin, peroxides (e.g. H)₂O₂、CaO₂、MgO₂、Li₂O₂、Na₂O₂) Sodium percarbonate (Na)₂CO₃) Perfluorocarbons, hydroxyapatite, tricalcium phosphate (bone growth promoting material), most preferably CaO₂And/or MgO₂Which can be used as a cross-linking agent in alginate hydrogels; the catalase is also preferably combined with an oxygen-releasing material that advantageously scavenges any released toxic hydrogen peroxide.

Detailed Description

The invention is further illustrated in the following examples.

Example 1

Thermoplastic aromatic polycarbonate based Polyurethane (PU) (Chronoflex, Advansource, usa) is used to make device precursors/films by electrospinning. Solutions containing 5.0 wt% or 25 wt% PU in hexafluoro-2-propanol (HFIP) (Sigma Aldrich, UK) were prepared.

The device precursor comprised two distinct electrospun fiber layers, a nonwoven top layer and a bottom layer. Electrospinning scaffold precursors were prepared by delivering the polymer solution to the bottom and top layers by syringe pumps at a constant feed rate of 8.333X 10^ (-7) L/s and electrospinning was performed vertically at an accelerating voltage of +5kV DC- +8 kV. In a climate controlled electrospinning machine (LE-100, bionica, spain), the temperature and relative humidity were kept constant (25 ℃ and 40% RH, respectively). The fibers were collected on a release paper wrapped around a rotating collector 20cm from the needle tip. In preparing the top and bottom nonwoven layers, the collector was rotated at a speed of 200 rpm. Longitudinal translation was also applied using a programmable motorized stage at a translation (translation) speed of 40 mm/s. Electrospinning was carried out for 270 minutes to produce the desired sheet thickness, i.e., the desired thickness of the device precursor.

Fiber diameter and scaffold morphology characterization was performed by Scanning Electron Microscopy (SEM) (Phenom G2 Pro equipped with fibermetric software, Phenom World, the netherlands) using automated image characterization of multiple images to determine mean fiber diameter and relative standard deviation. The fibermetric software will automatically identify the position of the fibers in the captured SEM image and measure the diameter of each fiber 20 times at a specific position. Typically, about 100 such measurements are performed per image. The diameter of the fibers can also be obtained by manually measuring/analyzing multiple SEM images.

The fibers on the bottom layer had an average fiber diameter of 600nm with a tolerance of + -30%. The fibers on the top layer had an average fiber diameter of 5 μm with a tolerance of ± 40%. The thickness of the sheet was measured using a micrometer. The target average thickness of the material was 150 μm with a tolerance of ± 20%.

The fiber mat was dried in a vacuum oven at 25 ℃ for 24 hours at-10 mbar to reduce the amount of residual solvent remaining during the manufacturing process.

The figures illustrate these layers. The files ending with T (FIG. 1) are the top layer, and the files ending with B (FIG. 2) are the bottom layer. The last picture (fig. 3) is a combination where both layers are visible.

Example 2

Electrospinning

Thermoplastic aromatic polycarbonate based Polyurethane (PU) (Chronoflex, Advansource, usa) is used to make device precursors/films by electrospinning. Solutions containing 4 wt% PU in hexafluoro-2-propanol (HFIP) (Sigma Aldrich, UK) were prepared. An electrospinning precursor was prepared by delivering the polymer solution via a syringe pump at a constant feed rate of 15mL/h, and electrospinning was performed vertically at an accelerating voltage of +20kV DC- +8 kV. In a climate controlled electrospinning machine (LE-100, bionica, spain), the temperature and relative humidity were kept constant (at 25 ℃ and 40% RH, respectively). The fibers were collected on release paper wrapped around a rotating collector 25cm from the needle tip. The collector was rotated at 100 rpm. A longitudinal translation was also applied using a programmable motorized stage at a translation speed of 40 mm/s. Electrospinning was carried out for 250 minutes to produce the desired sheet thickness, i.e., the desired thickness of the device precursor.

Fiber diameter, pore size and scaffold morphology characterization was performed by Scanning Electron Microscopy (SEM) (Phenom G2 Pro equipped with fibermetric software, Phenom World, the netherlands) using automated image characterization of multiple images to determine mean fiber diameter and mean pore diameter. The fibermetric software will automatically identify the position of the fibers in the captured SEM image and measure the diameter of each fiber 20 times at a specific position. Typically, about 100 such measurements are performed per image. The diameter of the fibers can also be obtained by manually measuring/analyzing multiple SEM images. The fibermetric software also automatically measures the free space area between fibers as a measure of pore size. The resulting data was converted to mean pore size using equation 1, assuming circular pores is conventional practice.

Equation 1: a formula for converting the average pore area (a) to the average pore diameter (d).

The average fiber diameter of the fibers was 445nm with a tolerance of + -190 nm. The average pore diameter was 1.5 μm. + -. 0.8. mu.m. The thickness of the sheet was measured using a micrometer. The target average thickness of the material was 160 μm with a tolerance of ± 20%. Figure 1 shows a scanning electron micrograph of a fibre produced using the method described above.

The fiber mat was dried in a vacuum oven at 25 ℃ for over 24 hours at-10 mbar to reduce the amount of residual solvent remaining during the manufacturing process.

Post-treatment

Surrounding the solid component:

the scaffolds were fabricated into various proofs (proof) of concept (concept) therapeutic compositions using a sonic welder. The electrospun precursor was cut to a specific size and folded, and then sonically welded to itself (fig. 5 a-c). The commercial acellular collagen membrane (Chondro-Geistlich) to different extents, i.e. to wrap/surroundAnd 100%.

Surrounding liquid component:

the hydrogel was prepared by dispersing 1 wt% hyaluronic acid in 50/50 mixture of distilled water and isopropanol. The mixture was homogenized on a roller mixer for 24 hours. As described above, a sonic welder is used to create an empty "bag" in which a small inlet remains open. The hydrogel is filled into a syringe and injected into a "bag" and then the inlet is again closed using a sonic welder and a fully enclosed therapeutic composition is formed. See fig. 6A and 6B.

Cell working:

studies have shown that secreted proteins are released from cells contained in "bags" made of electrospun precursor material after the cells are stimulated with agents designed to induce protein secretion when added to the external environment.

Summary of experimental design:

THP-1 cells are an immune derived cell line that, when stimulated with the appropriate agent Lipopolysaccharide (LPS), secrete interleukin IL-8. The level of secreted IL-8 is directly proportional to the concentration of LPS administered to the cells. IL-8 can be detected using ELISA-based immunoassays. Thus, detection of IL-8 in the culture medium surrounding the bag demonstrates that electrospun material allows secreted proteins, such as IL-8, to cross the membrane. To check for "escape" of cells from the "bag", we measured the level of ATP in the culture medium at the end of the experiment. ATP is produced by the cells in a "just in time" manner and is not secreted, so detection of ATP in the external medium means contamination of the cells to the external environment.

Method

The electrospun material was folded and welded along the edges as described above, forming a bag with an opening along one short side. Each bag is designed to stand upright in a 24-well plate. The bags were placed into a 24-well plate, one bag per well, using forceps, such that the bags were supported by the walls of the well plate with the openings facing upward to allow the media containing the cells to be placed in the bags.

THP-1 cells, a non-adherent cell type, were used for the study. At T175cm²Stock solutions of THP-1 cells were cultured in flasks. Aliquots of these cells were counted using a Luna cell counter and diluted to a concentration of 200,000 cells per ml, and 500 μ l of this stock solution was placed into the opening of the bag (100,000 cells per t-bag per well). In addition, 1ml of the same medium (without cells) was put into the wells quickly, but outside the bag. Then, the 24-well plate containing these bags was heated at 37 ℃ with 5% CO₂And incubation at 95% humidity for 24 hours.

After 24 hours, various concentrations of lipopolysaccharide (LPS-bacterial cell wall extract) were added to the medium outside the packet by removing 500. mu.l of medium from each well and re-administering 500. mu.l of medium containing 2X concentration of LPS. The plates were then re-incubated for 24, 48, 72 and 96 hours, and at each time point an aliquot of medium (70 μ l) was removed from the external solution and frozen (-30 ℃) for later use.

Detection of IL-8 in culture Medium

The level of IL-8 released from the cells into the culture medium was detected using a plate-based ELISA system. Briefly, the immunoadsorbent plates were coated with a primary antibody to human IL-8 according to the manufacturer's instructions. Aliquots of medium (20 μ l) removed from the external environment at defined time points were added to the immunoassay plates and allowed to interact with the antibody-coated plates overnight at 4 ℃. The wells were then washed with buffer to remove media and unbound material, and a secondary antibody labeled with horseradish peroxidase (HRP) was added to all wells, washed to remove unbound secondary antibody, and then treated with HRP substrate. Thus, the HRP signal is proportional to the amount of IL-8 in the medium. FIG. 7 shows IL-8 signals measured under various experimental conditions. The data show that the amount of LPS added results in a dose response of THP-1 cells, and that the "pocket" allows both IL-8 and LPS to pass through the membrane. The interior of the bag provides a good environment for the cells and the cells remain viable during the experiment.

ATP detection

To detect cells released from the pack into the external environment, at the end of the experiment, the pack was physically removed from the well plate using forceps and placed into a separate plate. The media from each well was mixed with a 1ml pipette and transferred to a 1.5ml centrifuge tube, one tube per well. The tube was centrifuged at 800G for 5 minutes to pellet any cells in the tube. The medium was removed by careful aspiration, leaving a small amount (about 50. mu.l) in the tube. The ATP concentration in each tube was then measured using Promega ATP GLO detection reagent (following the manufacturer's protocol). ATP is present only when cells are present in the tube, and thus the reading is directly proportional to the number of cells in the tube. FIG. 8 shows the detection of ATP in wells with the removal of the packet (marked with an asterisk) and the detection of ATP in the presence of cells. The data clearly show that no cells were able to escape the bag throughout the experiment.

Post-analysis visual inspection of holes

In addition to detecting ATP, after removal of the media, the 24-well microplates were visually inspected with a microscope to identify any cells that adhered to the wells or remained in the plate after aspiration of the media. No cells were detected.

Example 3

Preparing bags or sacks having fibres of greater diameter on their outer surface

The flat sheet of biocompatible film (4) shown in fig. 9 is folded into the bag or pouch (5) in fig. 11 such that the layer (2) with the larger diameter fibers faces outwards and can be brought into contact with the subcutaneous environment of the patient into which the bag or pouch (5) is provided. The layer (1) with the smaller diameter fibers faces inwards so as to be accessible to the therapeutic agent, in this case the cells (3), placed in a bag or pouch (5).

Example 4

Preparing bags or sacks with larger diameter fibres on the inner surface

As in example 3 above, but the flat sheet of biocompatible film (4) shown in fig. 9 is folded into the bag or pouch (6) in fig. 10 so that the layer (2) with larger diameter fibers faces inward so as to be accessible to the therapeutic agent (in this case cells (3)) placed in the bag or pouch (6). The layer (1) with the smaller diameter fibres is facing outwards and can thus be in contact with the subcutaneous environment of the patient into which the bag or pouch (6) is provided.

Example 5

Preparing double-dished bags or pouches

Starting from example 2 or 3, the design of the bag or pouch can be enhanced to resemble a double-concave dish (9), as shown in fig. 12. In fig. 12, the biocompatible film (4) has been folded (in 2D, but equally applicable to 3D) into a configuration with the layer (1) with the smaller diameter fibers facing outwards so as to be accessible to the subcutaneous environment of the patient into which the bag or pouch (5) is provided. In this embodiment, the biocompatible film is folded and prepared in such a way that the resulting bag or pouch (9) is a biconcave disc comprising an inner portion (7) which itself comprises the therapeutic agent (in this case cells (3) loaded on a carrier (8)). FIG. 12 shows an embodiment wherein the outermost layer comprises fibers of smaller diameter; the opposite is that the layers are exchanged and the larger diameter fibers are on the outer surface, with a corresponding similar structure. For example, an O-ring of suitable material may be used to help form and maintain the shape of the double-concave disk by providing structure around the periphery of the double-concave disk.

Example 6

Preparation of three-layer films and related bags or pouches

As shown in fig. 13A, a three-layer biocompatible film (10) comprising a layer (1) of smaller diameter fibers between two layers (2) of larger diameter fibers may be formed by electrospinning. This three-layer film (10) can be used to form a pack or pouch in the same manner as the two-layer film described above, resulting in a three-layer pack or pouch as shown in fig. 13B, formed by folding the three-layer film (10) (in 2D, but equally applicable to 3D) to form a pack or pouch (11), wherein the outer layer (2) with the larger diameter fibers can be in contact with the subcutaneous environment of the patient into which the pack or pouch is provided, and the inner layer (2) with the larger diameter fibers can be in contact with the inner portion (7) containing the therapeutic agent, in this case the cells (3) supported on the carrier (8). The layer (1) of smaller diameter fibers is located between the two layers (2) of larger diameter fibers and ideally does not directly contact the patient's external subcutaneous environment nor the interior portion (7) of the bag or pouch.