Additive-containing gel structure and method for producing same, precursor of additive-containing gel structure and method for producing same, and kit for preparing additive-containing gel structure

Abstract

This method for producing an additive-containing gel structure includes: preparing a porous structure; introducing a solution containing a gel raw material into the internal space of the porous structure; preparing a freeze-dried product of the porous structure in which the solution containing the gel raw material is held in the internal space; and gelling the gel raw material by supplying a gelling liquid to the freeze-dried product. At least one of the solution containing the gel raw material and the gelling liquid contains one or more additives.

Description

Additive-containing gel structure and its manufacturing method, precursor of additive-containing gel structure and its manufacturing method, and kit for preparing additive-containing gel structure

[0001] The present invention relates to the entire field of technology that utilizes gels containing chemical substances and nanomaterials as a means of delivery and administration thereof, and more particularly to additive-containing gel structures and methods for producing the same, precursors of additive-containing gel structures and methods for producing the same, and kits for producing additive-containing gel structures, which are suitably usable in the medical field.

[0002] In the medical field, gels containing drugs and / or nanomaterials are frequently used as a means of delivering and administering drugs and nanomaterials.

[0003] Conventionally, gels containing drugs or nanomaterials were manufactured by first preparing a liquid by dissolving or dispersing the drug or nanomaterial and gel raw materials in a solvent such as water, and then gelling the mixture by changing the conditions to induce gelling or by adding a gelling agent. In other words, conventionally, the manufacturing process for gels containing drugs or nanomaterials consisted of a step of preparing a solution by dissolving the drug or nanomaterial and gel raw materials in a solvent, and a step of gelling. In the gelling step, it is necessary to mix the gel raw material solution with the gelling agent or to permeate the gelling agent into the gel raw material solution, and this process involves complicated work. On the other hand, it is preferable that drug and / or nanomaterial-containing gels used in medical settings can be manufactured simply. In addition, it is preferable that the drug and / or nanomaterial-containing gel raw materials can be stored in a dry state and can be immediately gelled and used when needed. Furthermore, while radiation sterilization using gamma rays or electron beams is mainly used for sterilizing gels, this sterilization process is extremely difficult to perform in a medical setting. In a medical setting, it is highly desirable to be able to easily manufacture sterile gels.

[0004] Hydrogels containing pharmaceuticals are disclosed, for example, in Non-Patent Documents 1 to 3. Non-Patent Document 3, in particular, states that multiple steps are required for the synthesis of hydrogels. When hydrogels are used for medical purposes, a sterilization step is added to the above multiple steps.

[0005] Given these circumstances, there was a strong demand for a method of delivering and administering drugs and nanomaterials that could be easily manufactured in medical settings and delivered under sterile conditions. Furthermore, this situation was not limited to medicine; in fields such as agriculture, horticulture, cosmetics, hygiene products, and fragrances and deodorizers, gels containing additives were used as a method of delivering and administering substances. For similar reasons, there was a need for a method that could deliver and administer the desired substance quickly, easily, and with stable quality in the required quantity, whenever needed.

[0006] Here, a gel is defined as "a substance in which polymers crosslink to form a three-dimensional network structure, or in which amphiphilic low-molecular-weight molecules self-assemble in oil, organic solvents, or water to form a three-dimensional network structure, and in which it takes up solvent molecules and swells but does not dissolve, thus taking on a state intermediate between solid and liquid." The three-dimensional network structure can be formed by physical crosslinking through physical interactions, chemical crosslinking through chemical reactions, or by a combination of both methods.

[0007] Thang, N. H., Chien, T. B., & Cuong, D. X., Polymer-Based Hydrogels Applied in Drug Delivery: An Overview. Gels, 9, 523 (2023)Ho, T.C., Chang, C.C., Chan, H.P., Chung, T.W., Shu, C.W., Chuang, K.P., Duh, T.H., Yang, M.H., & Tyan, Y.C., Hydrogels: Properties and Applications in Biomedicine. Molecules, 27, 2902 (2022)Hameed H., Faheem S., Paiva-Santos A.C., Sarwar H.S., Jamshaid M., A Comprehensive Review of Hydrogel-Based Drug Delivery Systems: Classification, Properties, Recent Trends, and Applications. AAPS PharmSciTech. 25, 64. (2024)

[0008] The present invention aims to solve the problems associated with the delivery and administration of drugs and nanomaterials using conventional gels, and to provide an additive-containing gel structure that enables simple, short-time delivery and administration of a desired drug or nanomaterial in a required amount with stable quality when needed, as well as a method for producing the same. Another object of the present invention is to provide a precursor that can produce the gel structure of the present invention simply in a short time with stable quality in a required amount when needed, a method for producing the precursor, and a kit for producing the gel structure.

[0009] The configurations of the present invention are shown below. (Configuration 1) A method for producing an additive-containing gel structure, comprising: preparing a porous structure; introducing a solution containing gel raw materials into the internal space of the porous structure; producing a freeze-dried product of the porous structure in which the solution containing gel raw materials is held in the internal space; and supplying a gelling liquid to the freeze-dried product to gel the gel raw materials, wherein at least one of the solution containing gel raw materials and the gelling liquid contains one or more additives. (Configuration 2) The method for producing an additive-containing gel structure according to Configuration 1, wherein the form of the porous structure is one of sponge-like, mesh-like, and nonwoven fabric-like. (Configuration 3) The method for producing an additive-containing gel structure according to Configuration 1 or 2, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials. (Configuration 4) The method for producing an additive-containing gel structure according to any one of Configurations 1 to 3, wherein the additive is one type. (Configuration 5) The method for producing an additive-containing gel structure according to any one of Configurations 1 to 3, wherein the additive consists of two or more types. (Configuration 6) A method for producing an additive-containing gel structure according to Configuration 5, wherein the solution containing the gel raw material contains a first additive, and the gelling solution contains a second additive different from the first additive. (Configuration 7) A method for producing an additive-containing gel structure according to any one of Configurations 1 to 6, wherein the gel raw material is a polymer. (Configuration 8) A method for producing an additive-containing gel structure according to Configuration 7, wherein the polymer is one or more selected from the group consisting of alginic acid and its sodium salt, collagen, gelatin, fibronectin, laminin, elastin, fibrin, polyamino acids, dextrin, cellulose, cellulose acetate, cellulose diacetate, cellulose nitrate, agarose, amylose, starch, hyaluronic acid, chondroitin sulfate, DNA, RNA, chitin, chitosan, natural rubber, regenerated fiber rayon, celluloid, polyurethane, polyvinyl alcohol, polyethylene glycol, and sulfur nitride polymer. (Configuration 9) A method for producing an additive-containing gel structure according to any one of Configurations 1 to 8, wherein the additive is an anticancer agent.(Configuration 10) The anticancer agent is a drug used to treat one or more cancers selected from the group consisting of colorectal cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, tongue cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, spleen cancer, cervical cancer, chronic myeloid leukemia, malignant melanoma, brain tumor, osteosarcoma, chondrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma, and is a method for producing the additive-containing gel structure according to Configuration 9. (Configuration 11) An additive-containing gel structure in which a gel containing an additive is formed in the internal space of a porous structure. (Configuration 12) The additive-containing gel structure according to Configuration 11, wherein the additive consists of one or more selected from the group consisting of drugs and nanomaterials. (Configuration 13) The additive-containing gel structure according to Configuration 11, wherein the additive is an anticancer agent. (Configuration 14) The additive-containing gel structure according to Configuration 13, wherein the anticancer drug is a drug used to treat one or more cancers selected from the group consisting of colorectal cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, tongue cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, spleen cancer, cervical cancer, chronic myeloid leukemia, malignant melanoma, brain tumor, osteosarcoma, chondrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma. (Configuration 15) A precursor for obtaining the additive-containing gel structure according to Configuration 11, which is a freeze-dried porous structure in which gel raw materials and additives are supported in the internal space. (Configuration 16) The additive is a precursor for the additive-containing gel structure according to Configuration 15, wherein the additive consists of one or more selected from the group consisting of drugs and nanomaterials. (Configuration 17) A method for producing a precursor of an additive-containing gel structure, comprising: preparing a porous structure; introducing a solution containing a gel raw material and an additive into the internal space of the porous structure; and producing a freeze-dried product of the porous structure in which the solution is retained in the internal space. (Configuration 18) The method for producing a precursor of an additive-containing gel structure according to Configuration 17, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.(Configuration 19) A gel structure preparation kit containing an additive, comprising a main component containing a precursor of a gel structure and an auxiliary component containing a gelling liquid, wherein the precursor is a freeze-dried product of a porous structure on which a gel raw material is supported, the gelling liquid is capable of gelling the gel raw material, and at least one of the freeze-dried porous structure and the gelling liquid contains an additive. (Configuration 20) The gel structure preparation kit containing an additive according to Configuration 19, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.

[0010] According to the present invention, the problems related to the delivery and administration of drugs and nanomaterials using conventional gels are solved, and it becomes possible to provide an additive-containing gel structure and a method for producing the same that can deliver and administer desired drugs and nanomaterials in the required quantity, with stable quality, at the required time, in a short time and with ease.

[0011] This is a flowchart illustrating the manufacturing process of the Convenience Gel of the present invention. This is a structural diagram schematically showing the structure at each step in the manufacturing process of the Convenience Gel of the present invention. This is a photograph illustrating the state of the porous structure at each step in Example 1. This is a photograph illustrating the state of the porous structure at each step in Example 1. This is a photograph illustrating the state of the porous structure at each step in Example 2. This is a photograph illustrating the state of the porous structure at each step in Example 2. This is a photograph illustrating the state of the porous structure at each step in Example 3. This is a photograph illustrating the state of the porous structure at each step in Example 4. This is a photograph illustrating the state of the porous structure at each step in Example 5. This is a photograph illustrating the state of the porous structure at each step in Example 6. This is a photograph illustrating the state of the porous structure at each step in Example 7. This is a photograph illustrating the state of the porous structure at each step in Example 8. This is a photograph illustrating the state of the porous structure at each step in Example 9. This is a photograph illustrating the state of the porous structure at each step in Example 9. This is a photograph illustrating the state of the porous structure at each step in Example 10. This is a photograph illustrating the state of the porous structure at each step in Example 10. This is a photograph illustrating the state of the porous structure at each step in Example 11. These are photographs illustrating the state of the porous structure at each step in Example 12. These are photographs illustrating the state of the porous structure at each step in Example 13. These are photographs illustrating the state of the porous structure at each step in Example 14. These are photographs illustrating the state of the porous structure at each step in Example 15. These are photographs illustrating the state of the porous structure at each step in Example 16. These are photographs illustrating the state of the porous structure at each step in Example 17. These are photographs illustrating the state of the porous structure at each step in Example 18. These are photographs illustrating the state of the porous structure at each step in Example 19. These are photographs illustrating the state of the porous structure at each step in Example 20. These are photographs illustrating the state of the porous structure at each step in Example 21. These are photographs illustrating the state of the porous structure at each step in Example 22. These are photographs illustrating the state of the porous structure at each step in Example 23.This is a photograph illustrating the state of the porous structure at each step in Example 24. This is a photograph illustrating the state of the porous structure at each step in Example 25. This is a photograph illustrating the state of the porous structure at each step in Example 26. This is a photograph illustrating the state of the porous structure at each step in Example 27. This is a photograph illustrating the state of the porous structure at each step in Example 28. This is a photograph illustrating the state of the porous structure at each step in Example 29. This is a diagram showing the sustained release behavior of the drug from the drug-containing convenience gel prepared in Examples 1 to 6. This is a diagram showing the sustained release behavior of the drug from the drug-containing convenience gel prepared in Examples 7 to 10. This is a diagram showing the sustained release behavior of the drug from the drug-containing convenience gel prepared in Examples 15 to 20. This is a diagram showing the sustained release behavior of the drug from the drug-containing convenience gel prepared in Examples 21 to 26. This is a diagram showing the sustained release behavior of the drug from the drug-containing convenience gel prepared in Examples 27 to 29. This is a photograph illustrating the state of the porous structure at each step in Example 30. This is a diagram showing the sustained release behavior of DTIC from the DTIC-containing convenience gel 30 prepared in Example 30. This figure shows the results of in vitro cell culture in Example 30, specifically the survival rate of human malignant melanoma cells cultured in the presence of DTIC-containing Convenience Gel 30 and the gel of Comparative Example 1. This figure shows the results of in vivo animal experiments in Example 30, specifically the in vivo chemiluminescence image of human malignant melanoma cells in mice implanted with DTIC-containing Convenience Gel 30 and the gel of Comparative Example 1. This is a photograph illustrating the state of the porous structure at each step in Example 31. This figure shows the sustained release behavior of IM from the IM-containing Convenience Gel 31 prepared in Example 31. This figure shows the results of in vitro cell culture in Example 31, specifically the survival rate of human chronic myeloid leukemia cells cultured in the presence of IM-containing Convenience Gel 31 and the gel of Comparative Example 2. This is a photograph illustrating the state of the porous structure at each step in Example 32. This figure shows the sustained release behavior of CBDCA from the CBDCA-containing Convenience Gel 32 in Example 32. This figure shows the results of in vitro cell culture in Example 32, specifically the survival rate of human cervical cancer cells cultured in the presence of CBDCA-containing Convenience Gel 32 and the gel of Comparative Example 3.These are photographs illustrating the state of the porous structure at each step in Example 33. These are the results of an in vivo animal experiment in Example 33, showing in vivo chemiluminescence images of human glioblastoma cells in mice implanted with Ara-C-containing Convenience Gel 33 and Comparative Example 4 gel, respectively. Images enclosed in rectangular frames represent the complete disappearance of the chemiluminescence signal from human glioblastoma cells (the same applies hereafter). These are photographs illustrating the state of the porous structure at each step in Example 34. These are the results of an in vivo animal experiment in Example 34, showing in vivo chemiluminescence images of human mammary gland cancer cells in mice implanted with CPA-containing Convenience Gel 34 and Comparative Example 5 gel, respectively. These are photographs illustrating the state of the porous structure at each step in Example 35. These are the results of an in vivo animal experiment in Example 35, showing in vivo chemiluminescence images of human pancreatic cancer cells in mice implanted with GEM-containing Convenience Gel 35 and Comparative Example 6 gel, respectively. These are photographs illustrating the state of the porous structure at each step in Example 36. This figure shows the results of an in vivo animal experiment in Example 36, illustrating the in vivo chemiluminescence images of human gastric cancer cells in mice implanted with DTX-containing Convenience Gel 36 and Comparative Example 7, respectively. This is a photograph illustrating the state of the porous structure at each step in Example 37. This figure shows the results of an in vivo animal experiment in Example 37, illustrating the in vivo chemiluminescence images of human liver cancer cells in mice implanted with 5-FU-containing Convenience Gel 37 and Comparative Example 8, respectively. This is a photograph illustrating the state of the porous structure at each step in Example 38. This figure shows the results of an in vivo animal experiment in Example 38, illustrating the in vivo chemiluminescence images of human lung cancer cells in mice implanted with PEM-containing Convenience Gel 38 and Comparative Example 9, respectively. This is a photograph illustrating the state of the porous structure at each step in Example 39. This figure shows the results of an in vivo animal experiment in Example 39, and depicts in vivo chemiluminescence images of human prostate cancer cells in mice implanted with DTX-containing convenience gel 39 and the gel of Comparative Example 10, respectively.This is a photograph for explaining the state of the porous structure in each step of Example 40. It is a diagram showing the in vivo chemiluminescence images of human colorectal adenocarcinoma cells in mice implanted with the CAP-containing ConveniGel 40 and the gel of Comparative Example 11, respectively, which are the results of the in vivo animal experiment in Example 40.

[0012] (Embodiment 1) Hereinafter, modes for carrying out the present invention will be described with reference to the drawings. The range A to B appearing in the text means A or more and B or less including the upper limit and the lower limit.

[0013] <Concept> Hereinafter, to make the content of the present invention easier to understand, descriptions will be made on the assumption of medical applications, but the present invention is not particularly limited to medical applications unless otherwise specified. In non-medical fields, the present invention is also similarly applicable when a gel containing an additive is used as a substance delivery method.

[0014] The gel structure of the present invention (hereinafter sometimes referred to as "ConveniGel") is a porous structure in which a gel is supported in internal spaces such as pores and voids, and has gel-like properties (properties possessed by a desired gel). The gel structure for delivering and administering a drug and / or nanomaterial provided by the present invention is obtained by allowing the gel portion to contain the drug and / or nanomaterial.

[0015] Since the gel raw material is supported on the porous structure in the precursor (lyophilized product) of this gel structure, the dissolution of the gel raw material and the gelation of the solution thereof are not performed in separate steps, and the dissolution and gelation of the gel raw material can be performed in the same step, that is, in the internal space of the porous structure. Thereby, the step of dissolving the gel raw material in a solvent and the step of mixing the gel raw material solution with a gelling agent, which were necessary in the conventional gel production method, can be simplified. That is, by supplying a liquid that causes the gel raw material to gel (hereinafter referred to as "gelling liquid") to the lyophilized product of the porous structure holding the solution containing the gel raw material, the gel raw material is immediately dissolved in the internal space of the porous structure and a gel is formed.

[0016] This is possible because the internal space of the porous structure promotes the penetration of the gelling solution, and the freeze-dried gel raw material supported within the internal space of the porous structure has a fine porous structure, making it easier to solvate. Therefore, if freeze-dried porous structures containing a solution of gel raw materials are prepared in advance, users can quickly produce gel-like materials in a simple process of adding the gelling solution when needed.

[0017] Furthermore, in this invention, additives such as drugs or nanomaterials are incorporated into the gel portion of the gel structure. This incorporation method involves adding the desired drug or nanomaterial to at least one of either the gel raw material solution or the gelation solution. Details will be explained in the section on the manufacturing method. Hereinafter, in the case where additives are added to both the gel raw material solution and the gelation solution, the additive added to the gel raw material solution will be referred to as the first additive, and the additive added to the gelation solution will be referred to as the second additive. The gel structure supporting the desired additive of this invention has the property of delivering and administering drugs or nanomaterials, similar to conventional gels containing drugs or nanomaterials.

[0018] Therefore, the gel structure of the present invention can be supplied quickly and easily in the required amount with stable quality when needed (it can be transformed from a precursor into a gel structure), and can deliver and administer desired drugs or nanomaterials quickly and easily in the required amount with stable quality when needed. Here, the porous structure may take various forms, including, for example, a sponge-like porous structure, a mesh-like porous structure, and a nonwoven porous structure.

[0019] One of the features of the present invention is that a porous structure is used to support the gel raw material and also to promote the diffusion of the gelling agent, thereby dissolving the gel raw material and forming a gel as is, and that the gel contains drugs or nanomaterials.

[0020] The method for manufacturing convenience gel involves forming a gel by permeating a freeze-dried gel raw material supported in the internal space of a porous structure with a gelling solution, and includes, for example, the following steps 1 to 4 as shown in Figure 1. For reference, Figure 2 shows explanatory diagrams illustrating the manufacturing process for a sponge-like porous structure, a mesh-like porous structure, and a nonwoven porous structure. Step 1: Step to prepare the porous structure (Step S11) Step 2: Step to introduce a gel raw material solution into the porous structure and fill the internal space of the porous structure with the gel raw material solution (Step S12) Step 3: Step to freeze-dry or freeze the porous structure with the internal space filled with the gel raw material solution, and freeze-dry the frozen material to produce a freeze-dried product (Step S13). Step 4: Step to obtain a structure in which gel is formed in the internal space of the porous structure by adding a gelling solution to the freeze-dried product (Step S14).

[0021] Here, at least one of the desired drug or nanomaterial is added to either the solution containing the gel raw materials or the gelling solution. The added drug and / or nanomaterial may be one type or two or more types. The destination to which the additive consisting of the drug and / or nanomaterial is added is not limited, but the following forms can be preferably exemplified. (1) If the additive is poorly water-soluble or poorly water-dispersible, the additive is added to the solution containing the gel raw materials. As a method of addition, first the additive is dissolved or dispersed in a non-aqueous solvent that is miscible with water, and then the solution or dispersion is added to the gel raw materials. This makes it possible to adjust the content of the additive. (2) If the additive is water-soluble or water-dispersible, the additive may be added to the solution containing the gel raw materials, or the additive may be added to the gelling solution. Alternatively, the additive may be added to both solutions. If it is desired to adjust the content of the additive in advance, the additive is added to the solution containing the gel raw materials. If it is desired to adjust the delivery and dosage of the additive depending on the situation, the additive is added to at least the gelling solution. The additive added to the solution containing the gel raw materials is the first additive, and the additive added to the gelation solution is the second additive. (3) If a water-soluble or water-dispersible additive is to be slow-released, the additive is added to the solution of the gel raw materials. (4) If a water-soluble or water-dispersible additive is to be fast-released, the additive is added to the gelation solution. (5) Regardless of the degree of water solubility or water dispersibility, if an additive is to be slow-released, the additive is added to the solution containing the gel raw materials, and if a water-soluble or water-dispersible additive is to be fast-released, the additive is added to the gelation solution. This allows for a combination of modalities for the delivery and administration of the additive. (6) If there are multiple types of additives, at least one of them may be added to the solution containing the gel raw materials, or at least one of them may be added to the gelation solution. However, if there is a concern that the additives may chemically react with each other or form precipitates in the solution containing the gel raw materials or the gelation solution, they should be added separately to the solution containing the gel raw materials and the gelation solution. (7) When nanomaterials are included in the gel as additives, at least one of the following is selected as the additive destination: a porous structure, a gel raw material solution, or a gelling liquid.

[0022] Examples of additives that are preferable to add to the solution containing the gel raw materials include various cancer drugs, itraconazole (antifungal), griseofulvin (antifungal), cyclosporine (immunosuppressant), phenytoin (antiepileptic), theophylline (bronchodilator), and indomethacin (nonsteroidal anti-inflammatory drug). Examples of additives that are preferable to add to the gelling solution include dexamethasone (corticosteroid), clemastine fumarate (sustained-release antihistamine), hydrocortisone sodium phosphate (corticosteroid), and prednisolone sodium succinate (synthetic corticosteroid).

[0023] In one embodiment, when the drug as an additive is a cancer treatment drug, the types of cancers and tumors targeted by the present invention are not particularly limited. The cancer treatment drug is released sustainedly from the convenience gel to produce a therapeutic effect. Examples of target cancers and tumors include colorectal cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, tongue cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, splenic cancer, cervical cancer, chronic myeloid leukemia, malignant melanoma, brain tumor, osteosarcoma, chondrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma. Furthermore, the present invention covers all stages (disease stages) from stage 0 to stage IV, which are indicators of the progression of cancer and tumors.

[0024] (Brain Tumors) Brain tumors include neuroepithelial tumors (such as astrocytoma, oligodendronoma, glioblastoma and other gliomas, ependymoma, choroid plexus tumor, other neuroepithelial tumors, neurocellular tumors, pineal gland tumors, and embryonic tumors), schwannoma (such as schwannomas and neurofibromas), meningeal tumors (such as meningiomas, other mesenchymal tumors, and malignant melanomas), lymphomas and hematopoietic neoplasms (such as malignant lymphomas and plasmacytomas), germ cell tumors (such as germ cell tumors, yolk sac tumors, choriocarcinomas, and teratomas), and sella turcica tumors (such as craniopharyngiomas and pituitary cell tumors).

[0025] In the drug treatment of brain tumors, cytotoxic anticancer drugs (hereinafter simply referred to as "anticancer drugs") are used as standard. Anticancer drugs attack cancer cells by inhibiting part of the mechanism by which cells proliferate. Examples of anticancer drugs include cytarabine, methotrexate, nimustine, vincristine, procarbazine, carboplatin, etoposide, temozolomide, carmustine, lomustine, ifosfamide, cisplatin, dacarbazine, doxorubicin, paclitaxel, fluorouracil, epirubicin, docetaxel, docetaxel hydrate, tegafur, tegafur-uracil, carmofur, doxifluridine, capecitabine, tegafur-gimeracil-oteracil potassium, mitomycin C, captothecin, giotrif, tarceva, ileza, taxol, irinotecan, oxaliplatin, erbitux, portraza, herceptin, perjeta, indomethacin, cyclophosphamide, gemcitabine, and vinorelbine. One or more types are selected from this group, but the present invention is not limited to the above-mentioned anticancer agents.

[0026] (Breast Cancer) There are no limitations on the stage or subtype of breast cancer. Breast cancer includes all stages, namely non-invasive cancer (stage 0) and invasive cancer (stages I to IV). Breast cancer also includes all subtypes, namely hormone receptor-positive / HER2-negative, hormone receptor-positive / HER2-positive, hormone receptor-negative / HER2-positive, and triple-negative.

[0027] Examples of anticancer drugs used in the drug treatment of breast cancer include cyclophosphamide, irinotecan, epirubicin hydrochloride, eriprine mesylate, gemcitabine, capecitabine, carboplatin, gemcitabine hydrochloride, cisplatin, tegafur-uracil combination, tegafur-gimeracil-oteracil potassium combination, doxifluridine, doxorubicin hydrochloride, docetaxel hydrate, paclitaxel, paclitaxel injection (albumin-suspended), vinorelbine, fluorouracil, folinate calcium, mitomycin C, mitoxantrone hydrochloride, and methotrexate. However, the present invention is not limited to the above anticancer drugs. When a large dose of one type of anticancer drug is administered (monotherapy), there is a concern that side effects may be severe and that cancer cells may acquire resistance to the anticancer drug. In such cases, polytherapy using multiple types of anticancer drugs simultaneously or sequentially is applied. In combination therapy, several anticancer drugs with different mechanisms of action are combined to enhance the effect on cancer while reducing side effects. Furthermore, it is believed that cancer cells are less likely to acquire drug resistance compared to monotherapy. Representative examples of combination therapy for breast cancer include AC therapy (combination of doxorubicin, trade name Adriamycin ADM, and cyclophosphamide CPA), EC therapy (combination of epirubicin EPI and cyclophosphamide CPA), and TC therapy (combination of docetaxel DTX and cyclophosphamide CPA; paclitaxel or nab-paclitaxel may be used instead of docetaxel). However, this invention is not limited to the above combinations. Examples of drugs used in hormone therapy include tamoxifen, toremifene, goserelin, leuprorelin, anastrozole, letrozole, exemestane, fulvestrant, medroxyprogesterone acetate, and ethinylestradiol. Furthermore, molecularly targeted drugs, such as anti-HER2 drugs, include trastuzumab, pertuzumab, trastuzumab emtansine, and lapatinib. Drugs that target the protein mTOR include everolimus, and drugs that target vascular endothelial growth factor (VEGF) include bevacizumab.Palbociclib and abemaciclib, which target the proteins CDK4 and CDK6, are used in combination with hormone therapy for metastatic or recurrent breast cancer. Olaparib, which targets the protein PARP, is used to treat hereditary breast and ovarian cancer syndrome. Opdivo targets the protein PD-1, and atezolizumab targets the protein PD-L1. These drugs bind to PD-1 or PD-L1, which puts the brakes on the immune response that attacks cancer cells, and release that brake. The above drugs may also be encapsulated in (broadly defined) lipid nanoparticles such as temperature-responsive liposomes.

[0028] (Pancreatic Cancer) The types of pancreatic cancer covered by this invention are not particularly limited. Examples of pancreatic cancers include pancreatic ductal carcinoma, pancreatic endocrine carcinoma (islet cell carcinoma), and intraductal papillary mucinous neoplasm. Furthermore, the cancer stages covered by this invention include all stages from stage 0 to stage IV.

[0029] Examples of anticancer drugs used in the drug treatment of pancreatic cancer include gemcitabine, 5-fluorouracil, irinotecan, oxaliplatin, nab-paclitaxel, tegafur, gimeracil, oteracil potassium, cytarabine, methotrexate, nimustine, vincristine, procarbazine, carboplatin, etoposide, temozolomide, carmustine, lomustine, ifosfamide, cisplatin, dacarbazine, doxorubicin, paclitaxel, epirubicin, docetaxel, docetaxel hydrate, carmofur, doxifluridine, capecitabine, mitomycin C, captothecin, giotrif, tarceva, ilesa, taxol, erbitux, portraza, herceptin, perjeta, indomethacin, cyclophosphamide, and vinorelbine. One or more types can be selected from the group consisting of these, but the present invention is not limited to the above anticancer drugs. Furthermore, as multi-drug therapies, FOLFIRINOX therapy, which combines three anticancer drugs, 5-fluorouracil, irinotecan, and oxaliplatin, with leucovorin (or leucovorin), an enhancer for 5-fluorouracil; GnP therapy, which combines gemcitabine and nab-paclitaxel; and S-1 therapy, which combines three components, tegafur, gimeracil, and oteracil potassium, are currently commonly used, but the present invention is not limited to these combinations.

[0030] (Gastric Cancer) The types of gastric cancer covered by this invention are not particularly limited. Gastric cancer is classified into general and special types. General types (adenocarcinoma) are classified into differentiated and undifferentiated types, and differentiated types are further subdivided into papillary adenocarcinoma and tubular adenocarcinoma, while undifferentiated types are subdivided into poorly differentiated adenocarcinoma, signet ring cell carcinoma, and mucinous carcinoma. Special types include carcinoid tumors, endocrine cell carcinomas, adenosquamous cell carcinomas, and squamous cell carcinomas. Furthermore, the cancer stages covered by this invention are all stages from stage 0 to stage IV.

[0031] Anticancer drugs used in the drug treatment of gastric cancer include, for example, docetaxel, oxaliplatin, 5-fluorouracil (often used in combination with levofolinate, a type of activated folic acid preparation, to enhance the anticancer effect), irinotecan, cisplatin, paclitaxel, tegafur, gimeracil, oteracil potassium, capecitabine, nab-paclitaxel, gemcitabine, cytarabine, methotrexate, nimustine, and bink. Listine, procarbazine, carboplatin, etoposide, temozolomide, carmustine, lomustine, ifosfamide, dacarbazine, doxorubicin, epirubicin, carmofur, doxifluridine, mitomycin C, captothecin, giotrif, tarceva, ilesa, taxol, erbitux, portraza, herceptin, perjeta, indomethacin, cyclophosphamide, and vinorelbine are examples. One or more can be selected from this group, but the present invention is not limited to the above anticancer drugs. In addition, tegafur / gimeracil / oteracil potassium + cisplatin (S-1 + CDDP therapy) and capecitabine + cisplatin (Cape + CDDP therapy) are currently commonly used as multidrug therapies, but the present invention is not limited to these combinations.

[0032] (Liver Cancer) The types of liver cancer covered by this invention are not particularly limited. Liver cancers include, for example, hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Furthermore, the cancer stages covered by this invention are all stages from stage 0 to stage IV.

[0033] Currently, drug therapy for liver cancer primarily involves molecularly targeted drugs, hormone therapy drugs, and immune checkpoint inhibitors. Molecularly targeted drugs target specific proteins or genes involved in the proliferation and survival of cancer cells, inhibiting their function by binding to them. Examples of molecularly targeted drugs include sorafenib, lenvatinib, regorafenib, ramucirumab, cabozantinib, lapatinib, imatinib, afatinib, aflibercept beta, ibritumomab tiuxetan, everolimus, erlotinib, osimertinib, gefitinib, gemtuzumab ozogamicin, sunitinib, sirolimus, cetuximab, dasatinib, dacomitinib, tamibarotene, temsirolimus, trastuzumab, tretinoin, necitumumab, panitumumab, bevacizumab, pertuzumab, bortezomib, and rituximab. Hormone therapy drugs inhibit the secretion or function of hormones and attack cancers that utilize hormones for growth. Examples of hormone therapy drugs include anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, phosfestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin, and letrozole. Examples of immunotherapy drugs include atezolizumab, bevacizumab, durvalumab, tremelimumab, nivolumab, pembrolizumab, ipilimumab, and avelumab. However, the present invention is not limited to the above drugs or combinations thereof.

[0034] In the drug treatment of liver cancer, anticancer drugs are sometimes used. Examples of anticancer drugs include fluorouracil, epirubicin, gemcitabine, cisplatin, paclitaxel, cytarabine, methotrexate, nimustine, vincristine, procarbazine, carboplatin, etoposide, temozolomide, carmustine, lomustine, ifosfamide, dacarbazine, doxorubicin, docetaxel, docetaxel hydrate, tegafur, tegafur-uracil, carmofur, doxifluridine, capecitabine, tegafur-gimeracil-oteracil potassium, mitomycin C, captothecin, giotrif, tarceva, ileza, taxol, irinotecan, oxaliplatin, erbitux, portraza, herceptin, perjeta, indomethacin, cyclophosphamide, and vinorelbine. One or more types can be selected from this group, but the present invention is not limited to the above-mentioned anticancer agents.

[0035] (Lung Cancer) The type of lung cancer targeted by this invention is not particularly limited. Lung cancer is classified into more than 10 types, among which the most frequent are adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Furthermore, the cancer stages targeted by this invention are all stages from stage 0 to stage IV.

[0036] Examples of anticancer drugs used in the drug treatment of lung cancer include pemetrexed, gemcitabine, docetaxel, vinorelbine, cisplatin, tegafur, irinotecan, carboplatin, paclitaxel, etoposide, oxaliplatin, 5-fluorouracil (often used in combination with levofolinate, a type of activated folic acid preparation, to enhance its anticancer effect), gimeracil, oteracil potassium, capecitabine, and nab-paclitaxel. Examples include cytarabine, methotrexate, nimustine, vincristine, procarbazine, temozolomide, carmustine, lomustine, ifosfamide, dacarbazine, doxorubicin, epirubicin, carmofur, doxifluridine, mitomycin C, captothecin, giotrif, tarceva, ilesa, taxol, erbitux, portraza, herceptin, perjeta, indomethacin, and cyclophosphamide. One or more can be selected from this group, but the present invention is not limited to the above anticancer agents.

[0037] (Prostate Cancer) The types of prostate cancer covered by this invention are not limited. Prostate cancer is classified into well-differentiated adenocarcinoma, moderately differentiated adenocarcinoma, and poorly differentiated adenocarcinoma according to its malignancy, and this invention covers all of them. Furthermore, the cancer stages covered by this invention are all stages from stage 0 to stage IV.

[0038] In the drug treatment of prostate cancer, hormone therapy drugs and anticancer drugs are currently used as standard. Examples of hormone therapy drugs include chlormadinone, bicalutamide, flutamide, leuprorelin, degarelix, estramustine, estradiol, enzalutamide, abiraterone, bisphosphonates, and dexamethasone, and one or more drugs from this group may be selected and used. As for anticancer drugs, docetaxel and cabazitaxel (often used in combination with prednisolone to enhance the therapeutic effect) are currently used as standard. However, in the present invention, one or more of the following can be selected and used: fluorouracil, epirubicin, gemcitabine, cisplatin, paclitaxel, vinblastine, cytarabine, methotrexate, nimustine, vincristine, procarbazine, carboplatin, etoposide, temozolomide, carmustine, lomustine, ifosfamide, dacarbazine, doxorubicin, docetaxel, tegafur, tegafur-uracil, carmofur, doxifluridine, capecitabine, tegafur-gimeracil-oteracil potassium, mitomycin C, captothecin, giotrif, tarceva, ileza, taxol, irinotecan, oxaliplatin, erbitux, portraza, herceptin, perjeta, indomethacin, cyclophosphamide, and vinorelbine. Depending on the patient's condition and treatment status, treatment with molecularly targeted drugs (some of which are classified as hormone therapy drugs) or immune checkpoint inhibitors may also be used. Examples of molecularly targeted drugs include olaparib, niraparib, talazoparib, enzalutamide, darolutamide, and apalutamide. In addition, the therapeutic effect of pembrolizumab, a type of immunotherapy drug, on prostate cancer is being studied, but in addition to pembrolizumab, one or more drugs from the group consisting of atezolizumab, bevacizumab, durvalumab, tremelimumab, nivolumab, ipilimumab, and avelumab may also be selected and used. However, the present invention is not limited to the drugs listed above.

[0039] (Colorectal Cancer) The type of colorectal cancer targeted by this invention is not particularly limited. Examples of colorectal cancer types include adenocarcinoma, squamous cell carcinoma, and adenosquamous cell carcinoma. Furthermore, the cancer stages targeted by this invention include all stages from stage 0 to stage IV.

[0040] In the drug treatment of colorectal cancer, anticancer drugs and molecularly targeted drugs are currently used as standard. Commonly used anticancer drugs include fluorouracil, tegafur-uracil, tegafur-gimerauracil-oteracil potassium combination preparations, capecitabine, irinotecan, oxaliplatin, and trifluridine-tipiracil hydrochloride. However, this invention is not limited to the anticancer drugs listed above. Examples of anticancer drugs include one or more selected from the group consisting of epirubicin, gemcitabine, cisplatin, paclitaxel, cytarabine, methotrexate, nimustine, vincristine, procarbazine, carboplatin, etoposide, temozolomide, carmustine, lomustine, ifosfamide, dacarbazine, doxorubicin, docetaxel, docetaxel hydrate, carmofur, mitomycin C, captothecin, giotrif, tarceva, ilesa, taxol, erbitux, portraza, herceptin, perjeta, indomethacin, cyclophosphamide, and vinorelbine. Currently, bevacizumab, aflibercept, and ramucirumab (all angiogenesis inhibitors), and cetuximab and panitumumab (both anti-EGFR antibody drugs) are commonly used as molecularly targeted drugs. However, the present invention is not limited to the above molecularly targeted drugs. Examples of molecularly targeted drugs include sorafenib, lenvatinib, regorafenib, cabozantinib, lapatinib, imatinib, afatinib, ibritumomab tiuxetan, everolimus, erlotinib, osimertinib, gefitinib, gemtuzumab ozogamicin, sunitinib, sirolimus, dasatinib, dacomitinib, tamibarotene, temsirolimus, trastuzumab, tretinoin, necitumumab, panitumumab, pertuzumab, bortezomib, and rituximab. Depending on the results of genetic testing, treatment with the BRAF inhibitor encorafenib, the MEK inhibitor binimetinib, and the immune checkpoint inhibitors pembrolizumab, nivolumab, and ipilimumab may also be effective. __

[0041] In addition to those listed above for each disease, examples of cancer therapeutic agents that are preferably used in the present invention include nedaplatin, nitrogen mustard, melphalan (Alkeran), and vinblastine.

[0042] Furthermore, when using poorly water-soluble anticancer agents (e.g., dacarbazine, paclitaxel, docetaxel, fluorouracil, temozolomide, irinotecan, ifosfamide, etoposide, carmustine, lomustine, lapatinib, etc.), they can first be dissolved in anhydrous ethanol, polyoxyethylene castor oil, dimethyl sulfoxide, or a mixture thereof. The resulting solution can then be added directly, or diluted with water or an aqueous solution, to the gel raw material solution, or both. On the other hand, when using water-soluble anticancer drugs (for example, doxorubicin, imatinib, carboplatin, cisplatin, cyclophosphamide, oxaliplatin, nedaplatin, nitrogen mustard, melphalan, nimustine, epirubicin, methotrexate, gemcitabine, cytarabine, vincristine, vinorelbine, procarbazine, etc.), they can be added directly, or diluted with water or an aqueous solution, to the gel raw material solution, the gelling solution, or both.

[0043] Although the above-mentioned additives are intended for medical use, the present invention is not limited to medical use, as stated above, and can also be applied to agriculture, horticulture, cosmetics, hygiene products, and fragrance / deodorizing applications. Examples of additives for agriculture and horticulture include ammonium sulfate (fertilizer), potassium phosphite (fertilizer), simeconazole (pesticides), probenazole (pesticides), mancozeb (pesticides), and sodium carbam salt (soil disinfectant). Examples of additives for cosmetics include hydroquinone (whitening agent), tretinoin (whitening agent), and EGF (epidermal growth factor). Examples of additives for hygiene products include silver nanoparticles (bactericidal substance). Examples of additives for fragrance / deodorizing applications include ethyl maltol (fragrance), heliotropin (fragrance), vanillin (fragrance), chlorine dioxide (deodorant), α-pinene (deodorant / bactericidal agent), and limonene (deodorant / bactericidal agent).

[0044] The freeze-dried porous structure, which is a precursor to the gel structure (convenience gel) of the present invention, and whose internal space is filled with a solution containing the gel raw material obtained in step 3 (hereinafter sometimes referred to as "gel structure precursor" or "convenience gel precursor"), exhibits minimal change over time and excellent storage stability, allowing for quick supply of gel when needed after storage. Furthermore, the gel raw material supported in the internal space forms a fine porous structure through freeze-drying, making it easily solvable. As a result, a synergistic effect occurs in which the porous structure surrounding the gelling agent promotes the penetration and diffusion of the gelling solution, shortening the time required in step 4 (time required for dissolution and gelation). Therefore, convenience gel can be produced in a short time simply by storing the convenience gel precursor and adding the gelling solution to it. The convenience gel precursor has stable quality and undergoes very little deterioration during storage. Similarly, the gelling solution also has stable quality and undergoes very little deterioration during storage. Convenience Gel is a gel structure that can be supplied easily, instantly, and stably in the required quantity, when needed, and can be used for the same purposes as gel in many cases.

[0045] The gel structure preparation kit (convenience gel preparation kit), which uses the freeze-dried convenience gel precursor produced in step 3 as the main component and the gelling solution as an auxiliary component, has high storage stability and features the ability to immediately obtain the gel structure of the present invention (convenience gel) from the kit state by adding the auxiliary component to the main component or immersing the main component in the auxiliary component. In other words, the gel structure preparation kit (convenience gel preparation kit) makes it possible to supply a structure with gel properties immediately, in the required amount, at the required time and place. Specifically, the gel structure preparation kit (convenience gel preparation kit) of the present invention comprises a main component containing a gel structure precursor and an auxiliary component containing a gelling solution, wherein the precursor is a freeze-dried porous structure on which gel raw materials are supported, and the gelling solution gels the gel raw materials.

[0046] <Details> In one embodiment, the additive-containing gel structure of the present invention can be easily obtained by introducing an additive (e.g., a drug or nanomaterial) and a gel raw material into a porous structure, then freeze-drying it to obtain a freeze-dried body, and then allowing a gelling liquid to penetrate the fine porous structure of the freeze-dried body and gel it. Alternatively, in another embodiment, the gel can be easily obtained by introducing a gel raw material into a porous structure, then freeze-drying it to obtain a freeze-dried body, and then allowing a gelling liquid containing an additive (a drug or nanomaterial) to penetrate the fine porous structure of the freeze-dried body and gel it.

[0047] Furthermore, the method for producing the additive-containing gel structure of the present invention may include, for example, a step of preparing a porous structure (step 1), followed by the following three types of steps.

[0048] <First Form> (Step 2A) A step of supplying a gel raw material solution or a gel raw material solution containing a drug to a porous structure to fill the internal space of the porous structure with the gel raw material solution or the gel raw material solution containing a drug. (Step 3A) A step of freezing the porous structure that has gone through Step 2A, freeze-drying the frozen material, and producing a freeze-dried material. (Step 4A) A step of forming a drug-containing gel structure by supplying a gelling liquid that does not contain a drug (when a gel raw material solution containing a drug is supplied in Step 2A), or a gelling liquid that contains a drug, to the freeze-dried material. If a gel raw material solution that does not contain a drug is supplied in Step 2A, the type of drug added to the gelling liquid in Step 4A is one or more types. If a gel raw material solution containing a drug is supplied in Step 2A, the type of drug added to the gelling liquid in Step 4A may be the same as the type of drug in Step 2A, or some or all types may be different.

[0049] <Second Form> (Step 2B) A step of introducing a gel raw material solution, or a gel raw material solution containing nanomaterials, into a porous structure, and filling the internal space of the porous structure with the gel raw material solution, or the gel raw material solution containing nanomaterials. (Step 3B) A step of freezing the porous structure that has gone through Step 2B, freeze-drying the frozen material, and producing a freeze-dried product. (Step 4B) A step of forming a nanomaterial-containing gel structure by supplying a gelling liquid that does not contain nanomaterials (when a gel raw material solution containing nanomaterials was used in Step 2B), or a gelling liquid that contains nanomaterials, to the freeze-dried product. If a gel raw material solution that does not contain nanomaterials is supplied in Step 2B, then one or more types of nanomaterials are added to the gelling liquid in Step 4B. If a gel raw material solution containing nanomaterials is supplied in Step 2B, then the types of nanomaterials added to the gelling liquid in Step 4B may be the same as the types of nanomaterials in Step 2B, or some or all of the types may be different.

[0050] <Third Form> (Step 2C) A step in which a solution of gel raw material containing both a drug and nanomaterials, or a solution of gel raw material containing a drug, or a solution of gel raw material containing nanomaterials, or a solution of gel raw material not containing a drug or nanomaterials, is introduced into a porous structure, and the internal space of the porous structure is filled with the solution of gel raw material not containing a drug or nanomaterials, or a solution of gel raw material containing nanomaterials, or a solution of gel raw material containing a drug, or a solution of gel raw material containing both a drug and nanomaterials. (Step 3C) A step in which the porous structure that has gone through Step 2C is frozen, the frozen material is freeze-dried, and a freeze-dried product is produced. (Step 4C) A step to form a drug- and nanomaterial-containing gel structure (∧ is a symbol meaning "and". In this specification, "drug- and nanomaterial-containing gel structure" is synonymous with "gel structure containing drug and nanomaterial". The same applies hereinafter.) by adding a gel solution that contains neither drug nor nanomaterial in Step 2C, or a gel solution that contains both drug and nanomaterial in Step 4C. In this embodiment, if the gelling solution further contains a drug, the type of drug may be the same as the drug in step 2C, or some or all of the types may be different. If a gelling solution containing nanomaterials but no drug is supplied in step 2C, then one or more types of drugs are added to the gelling solution in step 4C. In this embodiment, if the gelling solution further contains nanomaterials, the type of nanomaterials may be the same as the nanomaterials in step 2C, or some or all of the types may be different.If a gel raw material solution containing both the drug and the nanomaterial is supplied in step 2C, then in step 4C, a gelling solution containing neither the drug nor the nanomaterial can be used. On the other hand, in the above case, a gelling solution containing at least one of the drug and the nanomaterial can also be used in step 4C. In this case, the types of the drug and the nanomaterial may be the same as those in step 2C, or some or all of the types may be different.

[0051] (Step 1) The porous structure prepared in Step 1 is not particularly limited as long as it has an internal space, and may be, for example, a sponge, porous foam, mesh, woven fabric, nonwoven fabric, or a combination thereof. In this specification, the "internal space" of the porous structure is a space formed inside the porous structure, and includes pores, gaps, openings in the mesh, interfiber spaces, etc. However, in Step 4, the internal space must have an area that is open to the outside so that the gelling liquid (liquid that induces gelation) can enter from the outside.

[0052] The dimensions and shape of the porous structure are not limited and can be selected according to the application. Furthermore, the porous structure or freeze-dried material can be cut and used as needed. In one embodiment, the porous structure has numerous internal spaces such as voids, and the opening dimensions of each internal space are preferably in the range of 0.1 μm to 5000 μm, more preferably in the range of 0.5 μm to 3000 μm. The porosity is preferably in the range of 0.5% to 99.9%, more preferably in the range of 1.0% to 99.0%. In this specification, "opening dimensions of internal spaces" refers to dimensions representing the size of each internal space in the porous structure, and includes, for example, average values ​​of void diameter, mesh opening diameter (mesh opening), interfiber distance, or equivalent dimensions.

[0053] The material of the porous structure is not particularly limited, but examples include one or more selected from the group consisting of polymers, metals, and ceramics. That is, the porous structure may take the form of, for example, polymer only, metal only, ceramics only, polymer / polymer composite, polymer / metal composite, polymer / ceramic composite, metal / ceramic composite, polymer / metal / ceramic composite, etc. In the above forms, one or more types of polymers, metals, and ceramics can each be used.

[0054] In one embodiment, the polymer constituting the porous structure may be bioabsorbable or non-bioabsorbable. Examples of bioabsorbable polymers include poly-DL-lactic acid, poly-L-lactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, poly-ε-caprolactone, polybutylene succinate, polyethylene succinate, trimethylene carbonate, polydioxanone, collagen, gelatin, fibronectin, elastin, laminin, fibrinogen, peptides, hyaluronic acid, glycosaminoglycans, chondroitin sulfate, carboxymethylcellulose, polyalginic acid, chitosan, DNA, and RNA. Examples of non-absorbent polymers include sodium polyacrylate, polyurethane, polystyrene, polypropylene, polyethylene, polyamine, polyvinyl chloride, phenolic resin, urea resin, melamine resin, epoxy resin, fluororesin, nylon, vinylon, polyethylene terephthalate, cellulose, cellulose acetate, cellulose diacetate, cellulose nitrate, celluloid, recycled rayon, recycled cellulose, agarose, amylose, starch, chitin, natural rubber, polysiloxane, polyphosphazene, polysilane, sulfur nitride polymer, and synthetic ruby.

[0055] In one embodiment, all metals and alloys can be used as the metal constituting the porous structure. Typical examples include iron, carbon steel, chromium steel, nickel steel, stainless steel, aluminum, aluminum alloys, titanium, titanium alloys, nickel, nickel alloys, precious metals, magnesium alloys, CoCr alloys, Co-Cr-Al-Si alloys, copper, tantalum, niobium, and zirconium.

[0056] In one embodiment, all ceramics can be used to constitute the porous structure. Typical examples include alumina, zirconia, barium titanate, hydroxyapatite, silicon carbide, carbonate, silicon nitride, phosphate, tricalcium triphosphate, octacalcium phosphate, carbonate apatite, strontium apatite, silicate-substituted apatite, octacalcium phosphate, calcium silicate, dicalcium silicate, tricalcium silicate, magnesium silicate, zinc silicate, strontium silicate, zeolite, akermanite, bridigite, zinc oxide, aluminum nitride, boron nitride, ferrite, and Na. ２ O-CaO-SiO ２ -P ２ Bioglass is one example.

[0057] Porous structures can be commercially available or those fabricated using porous formation methods. For example, methods for fabricating porous polymer structures include particle elution, freeze-drying, sacrificial molding, phase separation, foam molding, fiber fusion, electrospinning, three-dimensional fabrication, and composite formation. When fabricating using porous formation methods, a mold for injecting the porous structure material may or may not be used.

[0058] The following is an example of a procedure for producing collagen sponges and gelatin sponges using freeze-drying as a method for creating porous structures. An aqueous collagen solution or an aqueous gelatin solution is placed in a freezer and frozen. After freeze-drying the frozen material, a cross-linking treatment is performed. The concentration of the aqueous collagen solution is 0.5 mg to 50 mg / mL, with a desirable concentration of 1 mg to 30 mg / mL. The concentration of the aqueous gelatin solution is 5 mg to 300 mg / mL, with a desirable concentration of 10 mg to 200 mg / mL. The freezer temperature is -100°C to -1°C, with a desirable temperature of -80°C to -1°C. The freezing time is 0.5 hours to 24 hours, with a desirable freezing time of 1 hour to 12 hours. The frozen material is freeze-dried under reduced pressure (1 Pa to 10 Pa) for 12 to 120 hours. After that, the freeze-dried material is cross-linked.

[0059] Any conventionally known method can be used for crosslinking porous structures. For example, there are crosslinking methods using crosslinking reaction solutions, crosslinking methods using heating, crosslinking methods using gamma rays, crosslinking methods using ultraviolet irradiation, and crosslinking methods using gases.

[0060] In the solution crosslinking method, crosslinking is performed using crosslinking agents such as carbodiimide, aldehydes, or epoxy compounds, and activators such as N-hydroxysuccinimide. However, since uncrosslinked collagen sponges or gelatin sponges dissolve in water, these crosslinking agents are dissolved in a mixed solvent of ethanol and water, and crosslinking is performed in several stages. The ratio of ethanol to water in the mixed solvent differs in each stage, changing from a high ratio to a low ratio from the first stage to the final stage. The ratio of ethanol to water ranges from 99 / 1 to 1 / 99. The crosslinking temperature is 4°C to 40°C, preferably room temperature. The concentration of the crosslinking agent is 5 mM to 500 mM, preferably 10 mM to 100 mM. The concentration of the activator is 5 mM to 500 mM, preferably 10 mM to 100 mM. After the final crosslinking reaction, the sponge is washed by immersing it in pure water at room temperature, and this is repeated at least four times as one washing cycle. After washing, to deactivate any unreacted active functional groups, the cross-linked collagen sponge or cross-linked gelatin sponge is immersed in an aqueous glycine solution at room temperature for several hours. The concentration of the aqueous glycine solution is 0.01 M to 10 M, preferably 0.05 M to 1.0 M. The temperature is 4°C to 37°C, preferably 4°C to 30°C. The immersion time is 1 hour to 24 hours, preferably 4 hours to 12 hours.

[0061] In the heating-based crosslinking method, the porous structure is crosslinked by heating it under vacuum. The vacuum level and temperature should be such that moisture is removed without denatured the porous structure. If the vacuum level is too low, the crosslinking efficiency will be low and the porous structure will be more prone to denature. If the temperature is too low, the crosslinking effect will be low and the crosslinking time will be longer. On the other hand, if the temperature is too high, the porous structure will be denatured. The vacuum level should be 100 Pa to 5000 Pa, preferably 100 Pa to 1000 Pa. The temperature should be 60°C to 200°C, preferably 100°C to 180°C. The crosslinking time should be 1 hour to 168 hours, preferably 3 hours to 120 hours.

[0062] The crosslinking method using gamma rays and ultraviolet light involves crosslinking a porous structure by irradiating it with gamma rays or ultraviolet light, respectively.

[0063] Preferably used crosslinking agents in the gas-assisted crosslinking method are aldehydes such as glutaraldehyde, formaldehyde, and paraformaldehyde, particularly glutaraldehyde. After the gas-assisted crosslinking treatment, the crosslinked material is further subjected to a 24-hour blocking treatment for unreacted aldehyde groups with a 0.1 M aqueous glycine solution, followed by 20 washes with distilled water.

[0064] The following describes the procedure for producing a collagen sponge using freeze-drying with ice microparticles as a method for creating a porous structure. First, pure water is sprayed into liquid nitrogen or another ultra-low temperature liquid filling a container to produce ice microparticles. The container containing the formed ice microparticles is transferred to a low-temperature chamber at 0°C or below, and the container is left to stand until the liquid nitrogen inside the container vaporizes and disappears. Then, in the low-temperature chamber at 0°C or below, ice of a predetermined particle size is sieved using a sieve with a large mesh opening and a sieve with a small mesh opening. The mesh opening of each sieve is nominally 20 μm to 2000 μm, preferably nominally 50 μm to 1000 μm. The sieved ice microparticles are left to stand in a low-temperature chamber at -8°C to -1°C for 0.5 to 24 hours to equilibrate the temperature of the ice microparticles at -8°C to -1°C. Then, an ethanol aqueous solution of collagen is prepared by mixing an acidic aqueous solution of collagen with ethanol. The concentration of the collagen acidic aqueous solution is 0.5 mg / mL to 50 mg / mL, with a preferred concentration of 1 mg / mL to 30 mg / mL. The pH of the collagen acidic aqueous solution is 1 to 6, with a preferred pH of 3 to 4. The prepared collagen ethanol aqueous solution is left to stand in a low-temperature chamber at -8°C to -1°C to equilibrate its temperature to -8°C to -1°C. The collagen ethanol aqueous solution, equilibrated to -8°C to -1°C, and the sieved ice particles, also equilibrated to -8°C to -1°C, are mixed in a low-temperature chamber at -8°C to -1°C in a predetermined volume mL to weight g ratio. Here, the ratio of collagen ethanol aqueous solution to ice particles can be 99:1 to 5:95 in volume mL to weight g, but a preferred ratio is 80:20 to 10:90. The mixture is stirred well so that the ice particles are uniformly dispersed in the collagen ethanol aqueous solution. The mixture is left to stand in a freezer at a temperature of -100°C to -20°C for 0.5 to 24 hours to freeze. The frozen material is freeze-dried under reduced pressure (1 Pa to 10 Pa) for 12 to 120 hours. After that, the freeze-dried material is cross-linked using the cross-linking method described above.

[0065] The following describes the procedure for producing a gelatin sponge using freeze-drying with ice microparticles as a method for creating a porous structure. First, ice microparticles are prepared using the method described above, and the sieved ice microparticles are left to stand in a low-temperature chamber at -10°C to -1°C for 0.5 to 24 hours to equilibrate the temperature of the ice microparticles to -10°C to -1°C. Then, gelatin is dissolved in an aqueous acetic acid solution to prepare an aqueous gelatin solution. The ratio of acetic acid to water (volume mL to volume mL) of the aqueous acetic acid solution is 10 (v / v)% to 50 (v / v)%, with a desirable ratio of 15 (v / v)% to 40 (v / v)%. The concentration of gelatin is 5 mg / mL to 400 mg / mL, with a desirable concentration of 10 mg / mL to 300 mg / mL. The aforementioned aqueous gelatin solution is left to stand in a low-temperature chamber at -10°C to -1°C to equilibrate its temperature to -10°C to -1°C. A gelatin acetic acid aqueous solution, equilibrated to a temperature of -10°C to -1°C, and the sieved ice particles, also equilibrated to a temperature of -10°C to -1°C, are mixed in a low-temperature chamber at -10°C to -1°C in a predetermined volume mL to weight g ratio. The ratio of gelatin acetic acid aqueous solution to ice particles may be 99:1 to 5:95 in volume mL to weight g, but a desirable ratio (volume mL to weight g) is 80:20 to 10:90. The mixture is stirred well so that the ice particles are uniformly dispersed in the gelatin acetic acid aqueous solution. This mixture is left to stand in a freezer at a temperature of -100°C to -20°C for 0.5 to 24 hours to freeze. The frozen material is freeze-dried under reduced pressure (1 Pa to 10 Pa) for 12 to 120 hours. After that, the freeze-dried material is crosslinked using the crosslinking method described above.

[0066] The method for fabricating a composite porous structure of polymers and nanomaterials is the same as the method for fabricating a polymer porous structure described above. In this case, instead of a polymer solution, a mixed solution prepared by dispersing nanomaterials in a polymer solution is used. Other fabrication conditions are the same as those for fabricating a polymer porous structure described above.

[0067] As the gel raw material solution, a solution of a polymer capable of gelling is used. The concentration of the polymer in the solution (mg / mL) is preferably 0.1 mg / mL to 600 mg / mL, and a desirable concentration is 0.5 mg / mL to 500 mg / mL. The solvent can be any liquid capable of dissolving the polymer. Typical solvents include pure water, cell culture medium, physiological saline, HEPES buffer, Tris buffer, MES buffer, phosphate buffer, ethanol, methanol, dimethyl sulfoxide, acetone, and t-butyl alcohol and mixtures thereof.

[0068] Examples of polymers that can gel include collagen, gelatin, polyamino acids, hyaluronic acid, chondroitin sulfate, fibronectin, laminin, elastin, fibrin, dextrin, cellulose, alginic acid and its sodium salts, chitosan, DNA, RNA, cellulose acetate, cellulose diacetate, cellulose nitrate, celluloid, regenerated fiber rayon, agarose, amylose, starch, chitin, natural rubber, polyurethane, protein, polysaccharides, polyvinyl alcohol, and polyethylene glycol. One or more of these polymers may be used.

[0069] Examples of drugs used in the present invention include drugs for treating common diseases, anticancer drugs, proteins, antibacterial agents, functional nanoparticles, imaging agents, cell growth factors, immunomodulators, nucleic acid drugs, plasmids, vaccines, and radiotherapy drugs.

[0070] In the present invention, drugs used to treat common diseases can be used as preferred agents. Examples include, but are not limited to, hypnotics and sedatives, anxiolytics, anticonvulsants, antipyretics, analgesics and anti-inflammatory drugs, antiparkinsonians, psychotropic agents, cold remedies, skeletal muscle relaxants, cardiac stimulants, antiarrhythmics, diuretics, antihypertensives, vasoconstrictors, vasodilators, antihyperlipidemia agents, antitussives, expectorants, bronchodilators, antidiarrheal agents, intestinal regulators, peptic ulcer agents, antacids, laxatives, enemas, choleretics, adrenal hormones, estrogen and progesterone agents, hemorrhoid agents, topical antiseptics and disinfectants, suppurative disease agents, analgesics, antipruritics, astringents, anti-inflammatorys, emollients, vitamins, calcium agents, hemostatic agents, anticoagulants, detoxification agents, gout treatments, diabetes medications, antimetabolites, antihistamines, and stimulant therapies.

[0071] In addition to the anticancer agents mentioned above, preferred agents used in the present invention include hormone therapy drugs such as anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, phosfestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin, and letrozole. Molecularly targeted drugs include, for example, afatinib, aflibercept beta, ibritumomab tiuxetan, imatinib, everolimus, erlotinib, osimertinib, gefitinib, gemtuzumab ozogamicin, sunitinib, sirolimus, cetuximab, sorafenib, dasatinib, dacomitinib, tamibarotene, temsirolimus, trastuzumab, tretinoin, necitumumab, panitumumab, bevacizumab, pertuzumab, bortezomib, lapatinib, ramucirumab, and rituximab. Immunotherapeutic drugs include, for example, nivolumab, pembrolizumab, ipilimumab, durvalumab, atezolizumab, and avelumab. Radiotherapy drugs, for example, N-isopropyl-4-iodoamphetamine hydrochloride ( １２３ I) Flucyclovin ( １８ F), Diethylenetriaminepentaacetate indium ( １１１ In), indium chloride ( １１１In), and iodophenylmethylpentadecanoic acid ( １２３ I) is one example.

[0072] Other medications used to treat this disease include insulin, epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, hepatocyte growth factor, vascular endothelial growth factor, β-transformation growth factor, bone morphogenetic factors, dexamethasone, and pilocarpine. Nucleic acid drugs include, for example, avacincaptad pegol, casimersen, eplontersen, etepliersen, fomivirsen, givosiran, goldirsen, imetelstat, inclisiran, inotersen, lumasirsen, mipomersen, nedosiran, nusinersen, olezarsen, patisiran, pegaptanib, tofersen, viltolarsen, and volanesorsen and vutrisiran.

[0073] In addition to disease-treating drugs, antibacterial agents can also be used. Examples include β-lactam (penicillin / cephalosporin), aminoglycoside, quinolone, fluoroquinolone, fosfomycin, colistin, and vicozamycin. Vaccines are also used, such as measles vaccine, rubella vaccine, varicella vaccine, BCG (tuberculosis) vaccine, mumps vaccine, coronavirus vaccine, influenza vaccine, and pneumococcal vaccine.

[0074] The nanomaterials used in this invention refer to materials manufactured using elements, compounds, etc., as raw materials, and whose dimension indicating size is 1 nm to several hundred nm. Examples of nanomaterials include iron tetroxide nanoparticles, iron dioxide nanoparticles, gold nanoparticles, silver nanoparticles, hydroxyapatite nanoparticles, carbon nanotubes, single-walled carbon nanotubes, multi-walled carbon nanotubes, graphene, fullerenes, nanodiamonds, PLGA nanoparticles, cobalt oxide nanoparticles, silica nanoparticles, zinc oxide nanoparticles, black phosphorus nanosheets, liposomes, dendrimers, nanogels, quantum dots, and composite nanoparticles thereof.

[0075] The nanomaterials used in this invention may take the form of spheres, ellipsoids, sea urchins (nanorods radiating outwards), petals, stars, dots, rods, fibers, wires, discs, films, sheets, cages, flakes, or clusters. These may be dense, porous (like nanocages), or hollow (like nanotubes).

[0076] (Step 2) In one embodiment, the porous structure of Step 1 is immersed in a solution containing gel raw materials in order to fill the internal space of the porous structure with the solution containing gel raw materials. In another embodiment, the solution containing gel raw materials can be added to the porous structure. In yet another embodiment, if the solution containing gel raw materials does not easily penetrate into the internal space of the porous structure, the air in the internal space can be replaced with the gel raw material solution by degassing under reduced pressure.

[0077] In the process of introducing a gel raw material solution into a porous structure, a gel raw material solution, a mixed solution of a drug and gel raw material, a mixed solution of a nanomaterial and gel raw material, or a mixed solution of a drug, a nanomaterial and gel raw material may be used. One or more of the above-mentioned drugs and nanomaterials may be used in combination.

[0078] To prepare a mixed solution of a drug and gel raw material, or a mixed solution of a nanomaterial and gel raw material, or a mixed solution of a drug, a nanomaterial and a gel raw material, for introduction into a porous structure, if the drug and / or nanomaterial is water-soluble and / or dispersible in an aqueous solution, the drug and / or nanomaterial may be directly added to the aqueous solution of the gel raw material and mixed until homogeneous. Alternatively, an aqueous solution of a water-soluble drug and / or dispersible nanomaterial may be prepared in advance and mixed with the aqueous solution of the gel raw material.

[0079] To prepare a mixed solution of a drug and gel raw material to be introduced into a porous structure, in the case of poorly water-soluble drugs, it is also possible to use a mixed solution prepared by first dissolving the drug in a non-aqueous solvent that can dissolve the drug, or in a mixed solvent containing a non-aqueous solvent, and then adding this drug solution to an aqueous solution of gel raw material and mixing until homogeneous. When mixing the drug solution and the aqueous solution of gel raw material, it is desirable to mix them in a ratio such that the drug does not precipitate from the mixed solution.

[0080] To prepare a mixture of nanomaterials and gel raw materials for introduction into a porous structure, if the nanomaterials do not disperse in aqueous solution, it is also possible to pre-prepare a dispersion of the nanomaterials by dispersing them in a non-aqueous solvent or a mixed solvent containing a non-aqueous solvent that can disperse the nanomaterials, and then add this dispersion to an aqueous solution of gel raw materials and mix until uniform. When mixing the dispersion of nanomaterials and the aqueous solution of gel raw materials, it is desirable to mix them in a ratio such that the nanomaterials do not precipitate from the mixture.

[0081] (Step 3) In Step 3, the porous structure (the porous structure after Step 2), in which the internal space is filled with a solution containing gel raw materials, is freeze-dried.

[0082] In one embodiment, freeze-drying can be exemplified by placing a porous structure, whose internal space is filled with a solution of gel raw materials, in a freezer. The freezing temperature should be the temperature at which the gel raw material solution freezes. The freezing temperature is between -196°C and -1°C, with a preferred temperature of -80°C to -1°C. Freezing can also be achieved by placing the porous structure filled with the gel raw material solution in a low-temperature liquid. Examples of low-temperature liquids include liquid cooled in a freezer, liquid cooled with dry ice, liquid nitrogen, and liquid air.

[0083] As a freeze-drying step, the frozen material is transferred to a freeze-dryer and freeze-dried. Freeze-drying is performed for 24 to 168 hours at a vacuum of 100 Pa or less, preferably 20 Pa or less, and more preferably 5 Pa or less.

[0084] Step 3 yields a freeze-dried porous structure (a precursor of the gel structure) that holds a solution of gel raw materials in its internal space. Therefore, steps 1 to 3 can also be described as a method for producing the precursor of the gel structure of the present invention. That is, the method for producing the precursor of the gel structure of the present invention includes preparing a porous structure (step 1), introducing a solution containing gel raw materials into the internal space of the porous structure (step 2), and producing a freeze-dried porous structure that holds a solution of gel raw materials in its internal space (step 3).

[0085] (Step 4) In Step 4, the gelling solution is supplied to the inside of the freeze-dried product prepared in Step 3, and the gelling solution is allowed to permeate the freeze-dried product. The amount of gelling solution supplied to the freeze-dried product may be the same as the volume of the freeze-dried product, or slightly more. Preferably, 80% to 120% of the external volume of the freeze-dried product is added as gelling solution. The prepared freeze-dried product can be made by dropping the gelling solution around the freeze-dried product, allowing the gelling solution to permeate the freeze-dried product. The amount of gelling solution dropped around the freeze-dried product may be the same as the volume of the freeze-dried product, or less, or slightly more. Preferably, 80% to 120% of the external volume of the freeze-dried product is added as gelling solution.

[0086] Any solvent capable of dissolving the gel raw materials can be used as the solvent in the gelling solution. Typical solvents include pure water, cell culture medium, physiological saline, HEPES buffer, Tris buffer, 2-morpholinoethanesulfonic acid (MES) buffer, phosphate buffer, ethanol, methanol, dimethyl sulfoxide, acetone, and t-butyl alcohol, as well as mixtures thereof.

[0087] As the gelling solution, a solution of a gelling agent that gels the gel raw materials is used. Typical gelling agents include polyvalent ions (divalent ions such as Ca, Sr, and Ba (e.g., calcium chloride, calcium sulfate, etc.), trivalent ions such as Fe(III) and Al, etc.), xanthan gum, carrageenan, guar gum, aldehydes, ketones, quinones, polyfunctional monomers, and radical reaction initiators.

[0088] In step 4, to induce gelation, the structure in which the gelling liquid has been immersed in the pores of the sponge (porous structure) may be heated or cooled, or incubated in an incubator at 25°C to 50°C. Light may also be irradiated onto it.

[0089] The sponge (porous structure) plays a role in supporting the gel raw material and, in step 4, plays a role in promoting the diffusion of the gelling agent. In step 3, the gel raw material held in the internal space of the porous structure is freeze-dried, thereby supporting the gel raw material in the pores (internal space) of the sponge. The gel raw material covers the pore walls of the sponge (walls forming the internal space of the porous structure) and exists within the pores, forming a three-dimensional network-like fine porous structure. Furthermore, in step 4, the dissolution and gelation of the gel raw material occur in the internal space, shortening the time required for the dissolution and gelation of the gel raw material. This is because the freeze-dried product of the gel raw material supported in the internal space of the porous structure has a fine porous structure and becomes easier to solvate. Therefore, if a freeze-dried product of a porous structure with the gel raw material supported in advance (precursor of the gel structure of the present invention) is prepared, the user can quickly and easily produce the additive-containing gel structure (convenience gel) of the present invention in a simple process of supplying the gelling liquid to this freeze-dried product (precursor of the gel structure of the present invention) when needed. Furthermore, in frozen gel raw materials, areas where the solvent was present before freeze-drying become voids or cavities after freeze-drying. When a gelling solution is added, the gelling solution quickly penetrates these voids and cavities. As a result, freeze-dried gel raw materials dissolve and gel in a shorter time than gel raw materials in other states.

[0090] As described above, the method for producing an additive-containing gel structure of the present invention comprises: preparing a porous structure (step 1); introducing a solution containing gel raw materials into the internal space of the porous structure (step 2); producing a freeze-dried product of the porous structure in which the solution containing the gel raw materials is held in the internal space (step 3); and supplying a gelling liquid to the freeze-dried product to gel the gel raw materials (step 4). At least one of the solution containing the gel raw materials or the gelling liquid contains an additive.

[0091] The additive-containing gel structure of the present invention has a gel containing the additive formed in the internal space of a porous structure. The precursor of the gel structure of the present invention is a freeze-dried product of a porous structure on which gel raw materials and additives are supported.

[0092] The additive-containing gel structure and its manufacturing method, the precursor of the additive-containing gel structure and its manufacturing method and usage method, and the additive-containing gel structure preparation kit of the present invention are not limited to the embodiments described above.

[0093] The present invention will be described in more detail below with reference to examples, but these examples are provided solely to aid in understanding the present invention and do not limit the present invention to them.

[0094] [Example 1] In Example 1, a mixed aqueous solution of doxorubicin (Dox) and sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare Dox-containing sodium alginate convenience gel 1 (hereinafter simply referred to as Dox-containing convenience gel 1).

[0095] First, a gelatin sponge was prepared using ice microparticles with a particle size of 250 μm to 355 μm as a pore-forming agent. Pure water was sprayed into liquid nitrogen in a container to produce ice microparticles. Next, these ice microparticles were transferred to a low-temperature chamber maintained at -15°C. The liquid nitrogen was evaporated by leaving the mixture in the low-temperature chamber for 2 to 2.5 hours. After that, the ice microparticles with a particle size of 250 μm to 355 μm were sieved out using a sieve with a mesh size of 355 μm and a sieve with a mesh size of 250 μm within the low-temperature chamber. The temperature inside the low-temperature chamber was changed to -5°C, and the ice microparticles were left to stand for 3 hours.

[0096] On the other hand, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing a 40.0 mg / mL gelatin-acetic acid aqueous solution. This gelatin-acetic acid aqueous solution was placed in a low-temperature chamber (-5°C) and stirred for 30 minutes. In the low-temperature chamber (-5°C), the ice particles were added to the gelatin-acetic acid aqueous solution to a ratio of 30 / 70 (mL / g) of gelatin-acetic acid aqueous solution / ice particles, and the mixture was slowly stirred for 10 minutes to prepare a gelatin-acetic acid aqueous solution / ice particle mixture. This gelatin-acetic acid aqueous solution / ice particle mixture was placed in a container whose temperature was equilibriumized in the low-temperature chamber (-5°C).

[0097] The mixture of gelatin acetic acid aqueous solution and ice microparticles was left in a low-temperature chamber (-5°C) for 30 minutes in its container. Then, it was transferred to a deep freezer (MDF-U72V, manufactured by Sanyo Electric Co., Ltd.) at -80°C and left to stand for 12 hours. The container was removed from the frozen material, and the frozen material was transferred to a freeze-drying container that had been left in the deep freezer at -80°C for 1 hour. Then, freeze-drying was performed in a freeze-dryer (FDU-2200, manufactured by Tokyo Rikakikai Co., Ltd.) at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain a gelatin sponge. This gelatin sponge was subjected to a cross-linking reaction sequentially in the following three steps. In the first cross-linking reaction step, 2-morpholinoethanesulfonic acid (MES, Sigma Corporation) was dissolved in ethanol / water (95 / 5, v / v) to prepare a final concentration 0.1 M MES solution. To this MES solution, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl, Fujifilm Wako Pure Chemical Industries, Ltd.) and N-hydroxysuccinimide (NHS, Fujifilm Wako Pure Chemical Industries, Ltd.) were added and dissolved while stirring to prepare a first-stage crosslinking reaction solution containing a final concentration of 50 mM WSCD·HCl and a final concentration of 20 mM NHS. The first-stage crosslinking reaction was carried out by immersing the freeze-dried gelatin in this first-stage crosslinking reaction solution at room temperature for 12 hours.

[0098] Next, a second crosslinking reaction solution was prepared by mixing 30 mL of ethanol / water (90 / 10, v / v) with a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS, similar to the first crosslinking reaction solution. The gelatin sponge, after the first crosslinking reaction, was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0099] Finally, a third-stage crosslinking reaction solution was prepared by mixing 30 mL of ethanol / water (85 / 15, v / v) with a 0.1 mmEs solution containing 50 mM WSCD·HCl and 20 mM NHS, similar to the first-stage crosslinking reaction solution. The gelatin sponge after the second-stage crosslinking reaction was immersed in the third-stage crosslinking reaction solution at room temperature for 12 hours. This carried out the third-stage crosslinking reaction. The gelatin sponge after the third-stage crosslinking reaction was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. Through these steps, a crosslinked gelatin sponge with a gelatin concentration of 40 mg / mL was obtained.

[0100] On the other hand, 10 mg of Dox was dissolved in 1 mL of ultrapure water to prepare a 10 mg / mL Dox aqueous solution. 250 mg of sodium alginate was dissolved in 9 mL of ultrapure water to prepare a sodium alginate aqueous solution. 1 mL of the 10 mg / mL Dox aqueous solution was added to 9 mL of this sodium alginate aqueous solution and stirred to prepare a Dox / sodium alginate mixed aqueous solution consisting of 1 mg / mL of Dox and 25 mg / mL of sodium alginate. The cross-linked gelatin sponge was cut into cylindrical shapes and immersed in the Dox / sodium alginate mixed aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin sponge were filled with the Dox / sodium alginate mixed aqueous solution. Subsequently, it was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours.

[0101] By adding a 3 mg / mL aqueous calcium chloride solution to the obtained freeze-dried product, sodium alginate was dissolved and immediately gelled to prepare Dox-containing convenience gel 1. The gelling time was less than 1 minute.

[0102] One of the effects of the present invention is the time it takes to supply the Convenience Gel from the freeze-dried Convenience Gel precursor. This effect was confirmed by the following comparative experiment. In the comparative experiment, a Dox-containing sodium alginate gel was prepared using a conventional method. Specifically, the same volume as in Example 1 was taken of a Dox / sodium alginate mixed aqueous solution consisting of the above-mentioned concentrations of 1 mg / mL Dox and 25 mg / mL sodium alginate, and gelled by immersion in the above-mentioned 0.3 mg / mL calcium chloride aqueous solution. As a result, the time required for the Dox / sodium alginate mixed aqueous solution to completely gel was more than 40 minutes, which was more than 40 times longer than the supply time of less than 1 minute in Example 1. If we consider the case where a drug-releasing gel is molded and the release amount is adjusted in a medical setting and implanted in the body during surgery, with the conventional method, the incision site cannot be closed with a scalpel for more than 40 minutes for gel implantation, whereas with the Convenience Gel of the present invention, the work can be completed in a short time of 1 minute. This difference is very large and will bring about a qualitative change and revolution in surgery.

[0103] Figure 3A shows the appearance of the cross-linked gelatin sponge cylinder, the cross-linked gelatin sponge cylinder / sodium alginate / Dox freeze-dried product, and Dox-containing convenience gel 1. The scale in the figure represents 1 mm. Figure 3B shows scanning electron microscope images of (a) the cross-linked gelatin sponge cylinder and (b) the cross-linked gelatin sponge cylinder / sodium alginate / Dox freeze-dried product. The cross-linked gelatin sponge cylinder / sodium alginate / Dox freeze-dried product and Dox-containing convenience gel 1 exhibit a red color, which is due to the color of the additive, Dox. Furthermore, the scanning electron microscope images revealed that the cross-linked gelatin sponge cylinder is porous and has interconnected pores. In addition, the pore diameter of the cross-linked gelatin sponge cylinder / sodium alginate / Dox freeze-dried product was smaller than that of the cross-linked gelatin sponge cylinder, indicating that sodium alginate / Dox is present in the pores and gaps of the cross-linked gelatin sponge cylinder. Furthermore, when Dox-containing convenience gel 1 was pressurized with a spatula, no water came out, indicating that the water was retained. This revealed that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0104] To investigate the sustained release behavior of Dox from Dox-containing convenience gel 1, one Dox-containing convenience gel 1 was immersed in 1 mL of HEPES buffer (pH 7.4) and incubated in a 37°C constant temperature bath while gently shaking. A total of three Dox-containing convenience gels 1 were used for the experiment, with N=3. After incubation for 1, 2, 3, 4, 5, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, and 480 hours, 500 μL of HEPES buffer (pH 7.4) was collected and replaced with another 500 μL of HEPES buffer (pH 7.4). The amount of Dox contained in the recovered 500 μL of HEPES buffer (pH 7.4) was quantified using a spectrofluorometer (FP-8500, manufactured by JASCO Corporation). Specifically, with an excitation wavelength of 480 nm, the fluorescence intensity at a wavelength of 590 nm was measured to determine the cumulative amount of Dox released from Dox-containing convenience gel 1, and the percentage (%) of Dox introduced was calculated. Figure 32 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 32, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0105] [Example 2] In Example 2, a mixed aqueous solution of paclitaxel (PTX) and sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare PTX-containing sodium alginate convenience gel 2 (hereinafter simply referred to as PTX-containing convenience gel 2).

[0106] Since PTX is a poorly water-soluble drug, it was first dissolved in ethanol, and then gradually diluted with ethanol aqueous solutions. Specifically, PTX was dissolved in 99.9% ethanol to prepare a 59.0 mg / mL PTX ethanol solution. Next, 100 μL each of ethanol aqueous solutions with concentrations of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 50%, 40%, 30%, 20%, and 10% (13 types in total) was added to 50 μL of the 59.0 mg / mL PTX ethanol solution to make a total volume of 1.35 mL (50 μL + 100 μL × 13 types) and then diluted. To this diluted solution, 100 μL each of 50%, 40%, 30%, 20%, and 10% ethanol aqueous solutions (a total of five types) were added to make a total volume of 1.85 mL (1350 μL + 100 μL x 5 types), and the solution was further diluted. To this diluted solution, 100 μL each of 40%, 30%, 20%, and 10% ethanol aqueous solutions were added to further dilute it, and then 100 μL each of 30%, 20%, and 10% ethanol aqueous solutions were added again (total volume 2.55 mL). To this solution, 100 μL each of 20% and 10% ethanol aqueous solutions were added, and then 10% ethanol aqueous solution was added 30 times, 100 μL each time (total volume 5.75 mL). Finally, 100 μL of 1% ethanol aqueous solution was added to this diluted solution 90 times to prepare an ethanol aqueous solution of PTX with a PTX concentration of 0.2 mg / mL and an ethanol concentration of 10.1% (total volume 14.75 mL).

[0107] To prepare the PTX-containing convenience gel, sodium alginate was added to an ethanol aqueous solution of PTX with a concentration of 0.2 mg / mL and an ethanol concentration of 10.1%, and the mixture was stirred to prepare a PTX / sodium alginate mixed aqueous solution with a concentration of 0.2 mg / mL of PTX and a concentration of 25 mg / mL of sodium alginate. The cross-linked gelatin sponge from Example 1 was cut into a cylindrical shape and immersed in the above PTX / sodium alginate mixed aqueous solution. By applying reduced pressure, the pores and gaps of the sponge were filled with the above PTX / sodium alginate mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a freeze-dried cross-linked gelatin sponge cylinder / sodium alginate / PTX product. By adding a 3 mg / mL calcium chloride aqueous solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby preparing the PTX-containing convenience gel 2.

[0108] Figure 4A shows the appearance of the cross-linked gelatin sponge cylinder, the cross-linked gelatin sponge cylinder / sodium alginate / PTX freeze-dried product, and PTX-containing convenience gel 2. The scale in the figure represents 1 mm. Figure 4B shows scanning electron microscope images of (a) the cross-linked gelatin sponge cylinder and (b) the cross-linked gelatin sponge cylinder / sodium alginate / PTX freeze-dried product. From the scanning electron microscope images, it was found that the cross-linked gelatin sponge cylinder has a porous structure in which pores are interconnected. The pore diameter of the cross-linked gelatin sponge cylinder / sodium alginate / PTX freeze-dried product was smaller than that of the cross-linked gelatin sponge cylinder, indicating that sodium alginate / PTX is contained in the pores and gaps of the cross-linked gelatin sponge cylinder. Furthermore, when the PTX-containing convenience gel 2 was pressurized with a spatula, no water came out, indicating that the water was retained. This revealed that the gel had formed in the pores and gaps of the cross-linked gelatin sponge.

[0109] To investigate the sustained release behavior of PTX from PTX-containing convenience gel 2, PTX-containing convenience gel 2 was incubated in the same manner as in Example 1. Since the experiment was conducted with N=3, a total of three PTX-containing convenience gels 2 were used. After incubation for 1, 2, 3, 4, 5, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, and 480 hours, 500 μL of HEPES buffer (pH 7.4) was collected, and a fresh 500 μL of HEPES buffer (pH 7.4) was added. The concentration of PTX in the recovered 500 μL of HEPES buffer (pH 7.4) was measured using a spectrophotometer (V-660, JASCO Corporation) to determine the absorbance at a wavelength of 230 nm. The cumulative amount of PTX released from PTX-containing convenience gel 2 was then calculated, and its percentage relative to the introduced amount was determined. Figure 32 shows the relationship between the mean ± standard deviation of the cumulative PTX release rate (%) relative to the introduced amount of PTX and the incubation time. Figure 32 shows that, similar to the case of conventional gels containing PTX, PTX was released gradually as the incubation time progressed.

[0110] [Example 3] In Example 3, a mixed aqueous solution of Dox / vincristine (VCR) / sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare Dox∧VCR-containing sodium alginate convenience gel 3 (hereinafter simply referred to as Dox∧VCR-containing convenience gel 3).

[0111] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared using ice microparticles with a particle size of 355 μm to 500 μm as a pore-forming agent and a 40.0 mg / mL gelatin acetic acid aqueous solution. The manufacturing method was the same as in Example 1, except that the particle size of the ice microparticles was changed to 355 μm to 500 μm.

[0112] Next, Dox, VCR, and sodium alginate were dissolved in ultrapure water to prepare aqueous solutions of Dox, VCR, and sodium alginate. The aqueous solutions of Dox and VCR were added to the aqueous solution of sodium alginate and stirred to prepare a mixed aqueous solution of Dox / VCR / sodium alginate with a VCR concentration of 0.2 mg / mL, a Dox concentration of 0.5 mg / mL, and a sodium alginate concentration of 25 mg / mL. Furthermore, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the mixed aqueous solution of Dox / VCR / sodium alginate, and under reduced pressure conditions, the pores and gaps of the cross-linked gelatin sponge were filled with the mixed aqueous solution of Dox / VCR / sodium alginate. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a 3 mg / mL aqueous calcium chloride solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby producing Dox∧VCR-containing convenience gel 3.

[0113] Figure 5 shows the appearance of (a) a cross-linked gelatin sponge prismatic body, (b) a lyophilized cross-linked gelatin sponge prismatic body / sodium alginate / Dox / VCR product, and (c) Dox∧VCR-containing convenience gel 3. The scale in the figure represents 1 mm. The cross-linked gelatin sponge prismatic body / sodium alginate / Dox / VCR product and the Dox∧VCR-containing convenience gel 3 exhibit a red color, which is due to the color of the additive Dox. Furthermore, when Dox∧VCR-containing convenience gel 3 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0114] To investigate the sustained release behavior of Dox from Dox∧VCR-containing convenience gel 3, one Dox∧VCR-containing convenience gel 3 was immersed in 500 μL of HEPES buffer (pH 7.4) and incubated in a 37°C constant temperature bath with slow shaking. A total of three Dox∧VCR-containing convenience gels 3 were used to conduct the experiment with N=3. After incubation for 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, 250 μL of HEPES buffer (pH 7.4) was collected and a fresh 250 μL of HEPES buffer (pH 7.4) was added. The concentration of Dox in the recovered 250 μL of HEPES buffer (pH 7.4) was measured using a spectrofluorometer (FP-8500) with an excitation wavelength of 480 nm, and the fluorescence intensity at a wavelength of 590 nm was measured. The cumulative amount of Dox released from the Dox∧VCR-containing convenience gel 3 was determined, and its percentage relative to the introduced amount was calculated. Figure 32 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the introduced amount of Dox and the incubation time. From Figure 32, it can be seen that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0115] [Example 4] In Example 4, a mixed aqueous solution of dexamethasone (Dex) and sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare Dex-containing sodium alginate convenience gel 4 (hereinafter simply referred to as Dex-containing convenience gel 4).

[0116] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared in the same manner as in Example 3. On the other hand, Dex and sodium alginate were dissolved in ultrapure water, respectively, to prepare an aqueous solution of Dex and an aqueous solution of sodium alginate. The aqueous solution of Dex was added to the aqueous solution of sodium alginate and stirred to prepare a mixed solution of Dex and sodium alginate with a concentration of Dex of 25 mg / mL and a concentration of sodium alginate of 25 mg / mL. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the mixed solution of Dex and sodium alginate. By applying reduced pressure, the pores and gaps of the cross-linked gelatin sponge were filled with the mixed solution of Dex and sodium alginate. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a 3 mg / mL aqueous calcium chloride solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby producing Dex-containing convenience gel 4.

[0117] Figure 6 shows the appearance of (a) a cross-linked gelatin sponge prism, (b) a cross-linked gelatin sponge prism / sodium alginate / Dex freeze-dried product, and (c) Dex-containing convenience gel 4. The scale in the figure represents 1 mm. When Dex-containing convenience gel 4 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that gel had formed in the pores and gaps of the cross-linked gelatin sponge.

[0118] To investigate the sustained release behavior of Dex from Dex-containing convenience gel 4, Dex-containing convenience gel 4 was incubated in the same manner as in Example 3. Since the experiment was conducted with N=3, a total of three Dex-containing convenience gels 4 were used. After incubation for 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, 250 μL of HEPES buffer (pH 7.4) was collected, and a fresh 250 μL of HEPES buffer (pH 7.4) was added. The concentration of Dex in the collected 250 μL of HEPES buffer (pH 7.4) was measured using a spectrophotometer (V-660) to determine the absorbance at a wavelength of 240 nm. The cumulative amount of Dex released from Dex-containing convenience gel 4 was then calculated, and its percentage relative to the introduced amount was determined. Figure 32 shows the relationship between the mean ± standard deviation of the cumulative Dex release rate (%) relative to the introduced amount of Dex and the incubation time. Figure 32 shows that, similar to the case of conventional gels containing Dex, Dex was gradually released as the incubation time progressed.

[0119] [Example 5] In Example 5, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a lyophilized product was obtained. Dox-containing collagen convenience gel 5 (hereinafter simply referred to as Dox-containing convenience gel 5) was then prepared by adding 37°C neutral phosphate buffer to the lyophilized product.

[0120] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared in the same manner as in Example 1. On the other hand, porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare an aqueous collagen solution. Dox aqueous solution, prepared in the same manner as in Example 1, was added to the prepared collagen solution and stirred at 4°C to prepare a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20.0 mg / mL. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the Dox / collagen mixed aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin sponge were filled with the Dox / collagen mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. Neutral phosphate buffer at 37°C was added to the freeze-dried product and incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel, thereby producing Dox-containing convenience gel 5. The time required for gelation was less than 5 minutes.

[0121] One of the effects of the present invention is the time it takes to supply the convenience gel from the freeze-dried convenience gel precursor. This effect was confirmed by the following comparative experiment. In the comparative experiment, Dox-containing collagen gel was prepared using a conventional method. Specifically, the same volume as in Example 5 was taken of a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20.0 mg / mL at 4°C, and mixed with a 10-fold concentrated neutral phosphate buffer at 4°C (the volume ratio of the mixed solution was Dox / collagen mixed aqueous solution: 10-fold concentrated neutral phosphate buffer = 9:1). The mixture was then incubated in a constant temperature bath at 37°C to induce gelation. As a result, the time required for the Dox-containing collagen aqueous solution to completely gel was 30 minutes or more, which was more than six times longer than the time of 5 minutes or less.

[0122] Figure 7 shows the appearance of (a) cross-linked gelatin sponge / collagen / Dox freeze-dried material and (b) Dox-containing convenience gel 5. The scale in the figure represents 1 mm. The cross-linked gelatin sponge / collagen / Dox freeze-dried material and Dox-containing convenience gel 5 are red in color, which is due to the color of the additive Dox. Furthermore, when Dox-containing convenience gel 5 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0123] To investigate the sustained release behavior of Dox from Dox-containing convenience gel 5, Dox-containing convenience gel 5 was incubated in the same manner as in Example 3. Since the experiment was conducted with N=3, a total of three Dox-containing convenience gels 5 were used. After incubation for 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, 250 μL of neutral phosphate buffer was collected and a fresh 250 μL of neutral phosphate buffer was added. The Dox concentration in the collected 250 μL of neutral phosphate buffer (pH 7.4) was measured using a spectrofluorometer (FP-8500) with an excitation wavelength of 480 nm, and the fluorescence intensity at 590 nm was measured. The cumulative amount of Dox released from Dox-containing convenience gel 5 was determined, and its percentage relative to the introduced amount was calculated. Figure 32 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the introduced Dox amount and the incubation time. Figure 32 shows that, similar to the case of conventional gels containing Dox, Dox was gradually released as the incubation time progressed.

[0124] [Example 6] In Example 6, an insulin / sodium alginate mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, an aqueous calcium chloride solution was added to the freeze-dried product to prepare insulin-containing sodium alginate convenience gel 6 (hereinafter simply referred to as insulin-containing convenience gel 6).

[0125] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared using ice microparticles with a particle size of 150 μm to 250 μm as a pore-forming agent and an 80.0 mg / mL gelatin acetic acid aqueous solution. The manufacturing method was the same as in Example 1, except that the particle size of the ice microparticles was changed to 150 μm to 250 μm and the concentration of the gelatin aqueous solution was changed to 80.0 mg / mL. Next, insulin and sodium alginate were dissolved in ultrapure water, respectively, to prepare insulin aqueous solution and sodium alginate aqueous solution. The insulin aqueous solution was added to the sodium alginate aqueous solution and stirred to prepare a mixed solution of insulin and sodium alginate with an insulin concentration of 5 mg / mL and a sodium alginate concentration of 10 mg / mL. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the mixed solution of insulin and sodium alginate, and the pores and gaps of the cross-linked gelatin sponge were filled with the mixed solution of insulin and sodium alginate by applying reduced pressure. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a 1.5 mg / mL aqueous calcium chloride solution to this freeze-dried material, sodium alginate was dissolved and immediately gelled, thereby preparing insulin-containing convenience gel 6.

[0126] Figure 8 shows the appearance of (a) a cross-linked gelatin sponge prism, (b) a cross-linked gelatin sponge prism / sodium alginate / insulin freeze-dried product, and (c) insulin-containing convenience gel 6. The scale in the figure represents 1 mm. When insulin-containing convenience gel 6 was pressed with a spatula and lifted, no water came out, indicating that the water was retained, which showed that gel had formed in the pores and gaps of the cross-linked gelatin sponge.

[0127] To investigate the sustained release behavior of insulin from insulin-containing convenience gel 6, insulin-containing convenience gel 6 was incubated in the same manner as in Example 3. Since the experiment was conducted with N=3, a total of three insulin-containing convenience gels 6 were used. After incubation for 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, 250 μL of HEPES buffer (pH 7.4) was collected, and a fresh 250 μL of HEPES buffer (pH 7.4) was added. The concentration of insulin in the collected 250 μL of HEPES buffer (pH 7.4) was measured using a spectrophotometer (V-660) to determine the absorbance at a wavelength of 273 nm. The cumulative amount of insulin released from insulin-containing convenience gel 6 was then calculated as a percentage of the total insulin introduced. Figure 32 shows the relationship between the mean ± standard deviation of the cumulative insulin release rate (%) relative to the total insulin introduced and the incubation time. Figure 32 shows that, similar to the case of conventional gels containing insulin, insulin was released gradually over time during incubation.

[0128] [Example 7] In Example 7, a PTX / sodium alginate mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a Dox / calcium chloride mixed aqueous solution was added to the freeze-dried product to prepare PTX∧Dox-containing sodium alginate convenience gel 7 (hereinafter simply referred to as PTX∧Dox-containing convenience gel 7). Here, PTX is the first additive and Dox is the second additive.

[0129] The manufacturing method is described in detail. First, a cross-linked gelatin sponge / sodium alginate / PTX freeze-dried product was prepared in the same manner as in Example 2. However, a prismatic cross-linked gelatin sponge was used here. Dox and calcium chloride were dissolved in ultrapure water, respectively, to prepare an aqueous solution of Dox and an aqueous solution of calcium chloride. The aqueous Dox solution was added to the aqueous calcium chloride solution and stirred to prepare a mixed aqueous solution of Dox with a concentration of 0.5 mg / mL of Dox and a concentration of 3 mg / mL of calcium chloride. Next, the mixed aqueous Dox / calcium chloride solution was added to the prismatic cross-linked gelatin sponge / sodium alginate / PTX freeze-dried product to dissolve the sodium alginate and immediately gel, thereby preparing PTX∧Dox-containing convenience gel 7.

[0130] Figure 9 shows the appearance of (a) a cross-linked gelatin sponge prismatic body / sodium alginate / PTX freeze-dried product and (b) PTX∧Dox-containing convenience gel 7. The scale in the figure represents 1 mm. The PTX∧Dox-containing convenience gel 7 is red in color, which is due to the color of Dox, one of the additives. When the PTX∧Dox-containing convenience gel 7 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0131] The sustained release behavior of Dox from PTX∧Dox-containing convenience gel 7 was investigated. The method was the same as in Example 3, except that the sample was changed to PTX∧Dox-containing convenience gel 7. Figure 33 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 33, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0132] [Example 8] In Example 8, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a Dox-Dex-containing neutral phosphate buffer was added to the freeze-dried product to prepare Dox∧Dex-containing collagen convenience gel 8 (hereinafter simply referred to as Dox∧Dex-containing convenience gel 8).

[0133] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared in the same manner as in Example 3. On the other hand, porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a collagen aqueous solution. Dox was dissolved in pure water to prepare a Dox aqueous solution. The Dox aqueous solution was added to the collagen solution and stirred at 4°C to prepare a mixed aqueous solution of Dox / collagen with a Dox concentration of 0.4 mg / mL and a collagen concentration of 4 mg / mL. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the Dox / collagen mixed aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin sponge were filled with the Dox / collagen mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a freeze-dried cross-linked gelatin sponge prismatic body / collagen / Dox product. Next, Dex was dissolved in neutral phosphate buffer to prepare a Dex-containing neutral phosphate buffer with a Dex concentration of 10.0 mg / mL. This was incubated in a 37°C constant temperature bath to bring the temperature of the Dex-containing neutral phosphate buffer to 37°C. The above 37°C Dex-containing neutral phosphate buffer was added to the above cross-linked gelatin sponge prismatic material / collagen / Dox freeze-dried product and incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby preparing Dox∧Dex-containing convenience gel 8.

[0134] Figure 10 shows the appearance of (a) a cross-linked gelatin sponge prismatic body, (b) a cross-linked gelatin sponge prismatic body / collagen / Dox freeze-dried product, and (c) a Dox∧Dex-containing convenience gel 8. The scale in the figure represents 1 mm. The cross-linked gelatin sponge prismatic body / collagen / Dox freeze-dried product and the Dox∧Dex-containing convenience gel 8 exhibit a red color, which is due to the color of Dox, one of the additives. Furthermore, when the Dox∧Dex-containing convenience gel 8 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0135] The sustained release behavior of Dox from Dox∧Dex-containing convenience gel 8 was investigated. The method was the same as in Example 5, except that the sample was changed to Dox∧Dex-containing convenience gel 8. Figure 33 shows the relationship between the mean ± standard deviation of the cumulative release rate (%) of Dox relative to the amount of Dox introduced and the incubation time. From Figure 33, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0136] [Example 9] In Example 9, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a Dox-Dex-containing neutral phosphate buffer was added to the freeze-dried product to prepare Dox∧Dex-containing collagen convenience gel 9 (hereinafter simply referred to as Dox∧Dex-containing convenience gel 9).

[0137] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared in the same manner as in Example 1. On the other hand, a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20.0 mg / mL was prepared in the same manner as in Example 5. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the Dox / collagen mixed aqueous solution. By applying reduced pressure, the pores and gaps of the sponge were filled with the Dox / collagen mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a freeze-dried cross-linked gelatin sponge prismatic body / collagen / Dox product. Next, Dex was dissolved in neutral phosphate buffer to prepare a Dex-containing neutral phosphate buffer with a Dex concentration of 10.0 mg / mL, and the temperature of the Dex-containing neutral phosphate buffer was raised to 37°C by incubation in a 37°C constant temperature bath. The above cross-linked gelatin sponge prismatic material / collagen / Dox freeze-dried product was mixed with the above-mentioned 37°C Dex-containing neutral phosphate buffer solution and incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby producing Dox∧Dex-containing convenience gel 9.

[0138] Figure 11A shows the appearance of (a) a cross-linked gelatin sponge prismatic body, (b) a cross-linked gelatin sponge prismatic body / collagen / Dox freeze-dried product, and (c) a Dox∧Dex-containing convenience gel 9. The scale in the figure represents 1 mm. Figure 11B shows scanning electron microscope images of (a) a cross-linked gelatin sponge prismatic body and (b) a cross-linked gelatin sponge prismatic body / collagen / Dox freeze-dried product. The cross-linked gelatin sponge prismatic body / collagen / Dox freeze-dried product and the Dox∧Dex-containing convenience gel 9 exhibit a red color, which is due to the color of Dox, one of the additives. Furthermore, scanning electron microscope images revealed that the cross-linked gelatin sponge prismatic body has a porous structure in which pores are interconnected. The pore diameter of the cross-linked gelatin sponge prismatic body / collagen / Dox freeze-dried material was smaller than that of the cross-linked gelatin sponge prismatic body, indicating that collagen / Dox was present in the pores and voids of the cross-linked gelatin sponge prismatic body. Furthermore, when the Dox / Dex-containing convenience gel 9 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, suggesting that gel was formed in the pores and voids of the aforementioned cross-linked gelatin sponge.

[0139] The sustained release behavior of Dox from Dox∧Dex-containing convenience gel 9 was investigated. The method was the same as in Example 5, except that the sample was changed to Dox∧Dex-containing convenience gel 9. Figure 33 shows the relationship between the mean ± standard deviation of the cumulative release rate (%) of Dox relative to the amount of Dox introduced and the incubation time. From Figure 33, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0140] [Example 10] In Example 10, an aqueous sodium alginate solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a Dox / calcium chloride mixed aqueous solution was added to the freeze-dried product to prepare Dox-containing sodium alginate convenience gel 10 (hereinafter simply referred to as Dox-containing convenience gel 10).

[0141] The manufacturing method is described in detail. First, a cross-linked gelatin sponge was prepared in the same manner as in Example 1. On the other hand, sodium alginate was dissolved in pure water to prepare a 25 mg / mL sodium alginate aqueous solution. The cross-linked gelatin sponge was cut into cylindrical shapes and immersed in the 25 mg / mL sodium alginate aqueous solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the sodium alginate aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product. Furthermore, Dox and calcium chloride were dissolved in ultrapure water, respectively, to prepare a Dox aqueous solution and a calcium chloride aqueous solution. The Dox aqueous solution was added to the calcium chloride aqueous solution and stirred to prepare a Dox / calcium chloride mixed aqueous solution with a Dox concentration of 1.0 mg / mL and a calcium chloride concentration of 3 mg / mL. Next, the Dox / calcium chloride mixed aqueous solution was added to the cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product to dissolve the sodium alginate and immediately gel it, thereby producing Dox-containing convenience gel 10.

[0142] Figure 12A shows the appearance of a cross-linked gelatin sponge cylinder, a cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product, and Dox-containing convenience gel 10. The scale in the figure represents 1 mm. Figure 12B shows scanning electron microscope images of (a) the cross-linked gelatin sponge cylinder and (b) the cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product. The Dox-containing convenience gel 10 exhibits a red color, which is due to the color of the additive, Dox. Furthermore, the scanning electron microscope image revealed that the cross-linked gelatin sponge cylinder has a porous structure with interconnected pores. The pore diameter of the cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product was smaller than that of the cross-linked gelatin sponge cylinder, indicating that sodium alginate is present in the pores and gaps of the cross-linked gelatin sponge cylinder. Furthermore, when the Dox-containing convenience gel 10 was pressurized with a spatula, no water came out, indicating that the water was retained. This revealed that the gel had formed in the pores and gaps of the cross-linked gelatin sponge.

[0143] The sustained release behavior of Dox from Dox-containing composite gel 10 was investigated. The method was the same as that in Example 1, except that the sample was changed to Dox-containing composite gel 10. The relationship between the incubation time and the mean value ± standard deviation of the cumulative Dox release rate (%) relative to the introduced Dox amount is shown in Figure 33. From Figure 33, it was found that, similar to the case of conventional gels containing Dox, Dox was sustained-released over the course of incubation time.

[0144] [Example 11] In Example 11, an aqueous mixed solution of magnetic nanoparticles / sodium alginate was introduced into the pores and voids of a crosslinked gelatin sponge, followed by freezing and freeze-drying. An aqueous calcium chloride solution was added to the resulting freeze-dried product, to thereby produce magnetic nanoparticle-containing sodium alginate composite gel 11 (hereinafter simply referred to as magnetic nanoparticle-containing composite gel 11).

[0145] The production method is specifically described below. First, a crosslinked gelatin sponge was produced in the same manner as in Example 3. Meanwhile, as nanomaterials, citric acid surface-modified Fe ３ O ４ magnetic nanoparticles were synthesized by the following procedure. Diethylene glycol (DEG) and N-methyldiethanolamine (NMDEA) were mixed at a volume ratio of 1:1 to prepare a DEG / NMDEA mixed solvent. Under a nitrogen atmosphere, 4 mmol of FeCl ２ ·4H ２ O and 2 mmol of FeCl ３ ·6H ２ O were added to 80 mL of the DEG / NMDEA mixed solvent, and the mixture was stirred for dissolution, to prepare a DEG / NMDEA mixed solvent of FeCl ２ ·4H ２ O / FeCl ３ ·6H ２ O. Separately, NaOH was added to the DEG / NMDEA mixed solvent, followed by stirring for dissolution, to prepare a 400 mM NaOH DEG / NMDEA mixed solvent. 40 mL of this 400 mM NaOH DEG / NMDEA mixed solvent was added to the aforementioned FeCl ２ ·4H ２ O / FeCl ３ ·6H ２The solution was added dropwise to a mixed solvent of O, DEG / NMDEA, and stirred at room temperature for 3 hours. Subsequently, under a nitrogen atmosphere, the reaction solution was heated to 220°C and stirred at 500 rpm for 12 hours. After that, the black precipitate was collected and washed three times with an ethanol / ethyl acetate mixed solvent prepared by mixing ethanol and ethyl acetate in a 1:1 volume ratio, and Fe was added. ３ O ４ Magnetic nanoparticles were obtained. This Fe ３ O ４ Magnetic nanoparticles are dispersed in 100 mL of 18 mM sodium citrate aqueous solution, and this dispersion is stirred at 60°C for 24 hours, thereby generating Fe ３ O ４ Magnetic nanoparticles were modified with sodium citrate. This reaction solution was centrifuged at 12,000 rpm for 15 minutes, and then washed three times with pure water. ３ O ４ Magnetic nanoparticles were obtained. These citrate-modified Fe ３ O ４ Magnetic nanoparticles were observed using a transmission electron microscope (JEOL JEM-ARM200F, manufactured by JEOL Ltd.).

[0146] Next, the citrate-modified Fe is added to the aqueous sodium alginate solution. ３ O ４ Disperse magnetic nanoparticles and citric acid-modified Fe ３ O ４ A citrate-modified Fe with a magnetic nanoparticle concentration of 10 mg / mL and a sodium alginate concentration of 15 mg / mL ３ O ４ A magnetic nanoparticle / sodium alginate dispersion was prepared. The cross-linked gelatin sponge was cut into prismatic shapes and used with the citrate-modified Fe ３ O ４ By immersing the sponge in a magnetic nanoparticle / sodium alginate dispersion and applying reduced pressure, the pores and gaps of the sponge are modified with citrate. ３ O ４ The container was filled with magnetic nanoparticles / sodium alginate dispersion. Subsequently, freeze-drying was performed in the same manner as in Example 1, and the cross-linked gelatin sponge prismatic body / sodium alginate / citric acid modified Fe ３ O ４A freeze-dried magnetic nanoparticle was obtained. This cross-linked gelatin sponge prismatic / sodium alginate / citric acid modified Fe ３ O ４ By adding a 2 mg / mL aqueous calcium chloride solution to freeze-dried magnetic nanoparticles, sodium alginate was dissolved and immediately gelled, thereby producing a magnetic nanoparticle-containing convenience gel 11.

[0147] Figure 13 shows (a) cross-linked gelatin sponge prism and (b) cross-linked gelatin sponge prism / sodium alginate / citric acid modified Fe ３ O ４ (c) The appearance of the magnetic nanoparticle freeze-dried material and the magnetic nanoparticle-containing convenience gel 11 is shown. The scale in the figure represents 1 mm. The magnetic nanoparticles had a petal-like morphology and a size of 30.7 ± 4.1 nm. Cross-linked gelatin sponge prismatic body / sodium alginate / citrate-modified Fe ３ O ４ The freeze-dried magnetic nanoparticle material and the magnetic nanoparticle-containing convenience gel 11 exhibit a black color, which is due to the color of the magnetic nanoparticles used as an additive. Furthermore, when the magnetic nanoparticle-containing convenience gel 11 was pressed with a spatula or lifted, no water was released, indicating that the water was retained. This suggests that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0148] The magnetothermal effect of a magnetic nanoparticle-containing convenience gel 11 was investigated. An alternating magnetic field (frequency 373.6 kHz, magnetic flux density 130 Gauss) was applied to the magnetic nanoparticle-containing convenience gel 11 for 10 minutes using a magnetic nanoparticle induction heating analyzer (NANOSCALE BIOMAGNETICS D5 SERIES G2, nB nanoScale Biomagnetics, Zaragoza, Spain). The temperature before and after the application of the alternating magnetic field was measured using an IR1 thermal imaging system (nB nanoScale Biomagnetics, Zaragoza, Spain). The temperature difference of the magnetic nanoparticle-containing convenience gel 11 before and after the application of the alternating magnetic field was calculated to investigate the temperature change after the application of the alternating magnetic field. Since the experiment was conducted with N=3, a total of three magnetic nanoparticle-containing convenience gels 11 were used, and the average value of the temperature change was calculated. As a result, after applying an alternating magnetic field for 10 minutes, the temperature of the magnetic nanoparticle-containing convenience gel 11 increased by 3.7 ± 0.2°C. This was due to the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle-containing convenience gel 11, indicating that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle-containing convenience gel 11 was maintained.

[0149] [Example 12] In Example 12, a gold nanorod / collagen aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing, the freeze-dried product was obtained. A neutral phosphate buffer was then added to the freeze-dried product to prepare a gold nanorod-containing collagen convenience gel 12 (hereinafter simply referred to as gold nanorod-containing convenience gel 12).

[0150] The specific manufacturing method is described below. First, a cross-linked gelatin sponge was prepared in the same manner as in Example 3. On the other hand, gold nanorods were synthesized as nanomaterials using the following procedure: In the presence of cetyltrimethylammonium bromide (CTAB), tetrachloroauric acid (HAuCl) was used. ４ ) is sodium borohydride (NaBH ４ Au seeds were prepared by reducing them with ) and then dispersion stabilizing them with CTAB. 10 mL of 0.01 MHA AuCl ４ 4 mL of 1 M HCl, 1.6 mL of 0.1 M ascorbic acid, and 2.20 mL of 10 mM AGNO. ３A 0.1 MCTAB solution was mixed with 150 mL of the Au seed solution to prepare a growth solution. While slowly stirring this growth solution, 0.48 mL of the Au seed solution was added, and the mixture was incubated for 12 hours to synthesize gold nanorods. Subsequently, the gold nanorods were recovered by centrifugation and washed with pure water to obtain gold nanorods. These gold nanorods were observed using a transmission electron microscope (JEOL JEM-ARM200F). The gold nanorods were also dispersed in a 5 mg / mL aqueous collagen solution to prepare a gold nanorod / collagen dispersion with a gold nanorod concentration of 2 mM and a collagen aqueous solution concentration of 5 mg / mL. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in the gold nanorod / collagen dispersion. By applying reduced pressure, the pores and gaps in the sponge were filled with the gold nanorod / collagen dispersion. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a freeze-dried product of cross-linked gelatin sponge prismatics / gold nanorods and collagen dispersion. A neutral phosphate buffer at 37°C was added to the freeze-dried dispersion of cross-linked gelatin sponge prismatics / gold nanorods and collagen, and incubated in a 37°C constant temperature bath for 10 minutes. This dissolved the collagen and immediately caused gelation, thereby producing a gold nanorod-containing convenience gel 12.

[0151] Figure 14 shows the appearance of (a) a cross-linked gelatin sponge prismatic body, (b) a freeze-dried dispersion of the cross-linked gelatin sponge prismatic body and gold nanorod-containing collagen, and (c) the gold nanorod-containing convenience gel 12. The scale in the figure represents 1 mm. The gold nanorods had a rod-like shape, with a major axis length of 64.3 ± 4.2 nm, a minor axis length of 15.6 ± 2.1 nm, and an aspect ratio of 4.1. The freeze-dried dispersion of the cross-linked gelatin sponge prismatic body and gold nanorod-containing collagen and the gold nanorod-containing convenience gel 12 were slightly brown in color, which is due to the color of the gold nanorods, which are an additive. Furthermore, when the gold nanorod-containing convenience gel 12 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0152] The heat generation effect of the gold nanorod-containing convenience gel 12 when irradiated with near-infrared light was investigated. The gold nanorod-containing convenience gel 12 was irradiated with near-infrared light at a wavelength of 805 nm and an intensity of 1.6 W / cm². ２ Near-infrared light (near-infrared laser light) was irradiated for 10 minutes. The temperature of the gold nanorod-containing convenience gel 12 was measured with a digital thermometer before and after 10 minutes of near-infrared light irradiation to investigate the heat generation effect. Since the experiment was conducted with N=3, a total of three gold nanorod-containing convenience gels 12 were used, and the average value of the temperature change was calculated. As a result, after 10 minutes of near-infrared light irradiation, the temperature of the gold nanorod-containing convenience gel 12 increased by 25.2 ± 0.9°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod-containing convenience gel 12, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod-containing convenience gel 12 was maintained.

[0153] [Example 13] In Example 13, a sodium alginate aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a dispersion of gold nanorod / calcium chloride aqueous solution was added to the freeze-dried product to prepare a gold nanorod-containing sodium alginate convenience gel 13 (hereinafter simply referred to as gold nanorod-containing convenience gel 13).

[0154] The manufacturing method is described in detail. First, in the same manner as in Example 3, ice nanoparticles with a particle size of 355 μm to 500 μm were used as a pore-forming agent, and a cross-linked gelatin sponge was prepared using a 40.0 mg / mL gelatin acetic acid aqueous solution. On the other hand, gold nanorods were synthesized in the same manner as in Example 12. These gold nanorods were dispersed in a 1.5 mg / mL calcium chloride aqueous solution to prepare a dispersion of gold nanorod-containing calcium chloride aqueous solution with a gold nanorod concentration of 1 mM and a calcium chloride aqueous solution concentration of 1.5 mg / mL. Next, the cross-linked gelatin sponge was cut into prismatic shapes and immersed in a 10 mg / mL sodium alginate aqueous solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the sodium alginate aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a cross-linked gelatin sponge prismatic body / sodium alginate freeze-dried product. A dispersion of the aforementioned calcium chloride aqueous solution containing gold nanorods was added to the cross-linked gelatin sponge prismatic body / freeze-dried sodium alginate to dissolve the sodium alginate and immediately induce gelation, thereby producing a gold nanorod-containing convenience gel 13.

[0155] Figure 15 shows the appearance of (a) a cross-linked gelatin sponge prismatic body, (b) a cross-linked gelatin sponge prismatic body / sodium alginate freeze-dried product, and (c) a gold nanorod-containing convenience gel 13. The scale in the figure represents 1 mm. The gold nanorod-containing convenience gel 13 has a slightly brownish color, which is due to the color of the gold nanorods, which are an additive. Furthermore, when the gold nanorod-containing convenience gel 13 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0156] In the same manner as in Example 12, the heat generation effect of the gold nanorod-containing convenience gel 13 when irradiated with near-infrared light was investigated. When the gold nanorod-containing convenience gel 13 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod-containing convenience gel 13 rose by 10.2 ± 1.0°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod-containing convenience gel 13, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod-containing convenience gel 13 was maintained.

[0157] [Example 14] In Example 14, a collagen aqueous solution was introduced into the pores and gaps of a cross-linked collagen sponge, and a lyophilized product was obtained by freezing and freeze-drying. A magnetic nanoparticle-containing neutral phosphate buffer was then added to the lyophilized product to prepare a magnetic nanoparticle-containing collagen convenience gel 14 (hereinafter simply referred to as magnetic nanoparticle-containing convenience gel 14).

[0158] The manufacturing method is described in detail. First, a cross-linked collagen sponge was prepared using ice microparticles with a particle size of 150 μm to 250 μm as a pore-forming agent, with a ratio (v / w) of 1:1 between 30 mg / mL of collagen aqueous solution and ice microparticles. (Preparation of ice microparticles) 300 mL of pure water was sprayed into 10 L of liquid nitrogen filled in a container. Next, the container was transferred to a low-temperature chamber maintained at -15°C. The liquid nitrogen was evaporated by leaving it standing in the low-temperature chamber for 2 to 2.5 hours. After that, the ice microparticles with a particle size of 150 μm to 250 μm were sieved out in the low-temperature chamber using sieves with a mesh size of 150 μm and sieves with a mesh size of 250 μm. After sieving, the temperature in the low-temperature chamber was changed to -5°C, and the ice microparticles were left standing for 3 hours.

[0159] (Preparation of Collagen Solution) Porcine type I atelocollagen was dissolved at 4°C in a mixed aqueous solution of dilute hydrochloric acid (pH 3.0) and ethanol (volume ratio of dilute hydrochloric acid to ethanol: 90 / 10) to prepare an ethanol aqueous solution of collagen at a concentration of 30 mg / mL. The prepared collagen solution was left at -5°C for 2 hours to maintain the solution temperature at -5°C.

[0160] (Preparation of the mold) A mold was prepared by placing a silicone rubber mold frame (internal dimensions: 5 cm long x 5 cm wide x 5 mm thick) on a copper plate covered with a perfluoroalkoxyalkane (PFA) film in a low-temperature chamber (-5°C). This mold was left to stand in the low-temperature chamber (-5°C) for 2 hours.

[0161] (Preparation of Collagen Sponge) In a low-temperature chamber at -5°C, 10 mL of the collagen solution and 10 g of the previously prepared ice microparticles were mixed and slowly stirred with a spatula for 10 minutes to prepare a collagen solution / ice microparticle mixture. The collagen solution / ice microparticle mixture was cast into the inside of the silicone rubber frame portion of the mold. Excess collagen solution / ice microparticle mixture was removed and the surface of the mixture was smoothed with a spatula. Next, a PFA film-coated glass plate cooled at -5°C was placed on top, and then the mixture was transferred to a deep freezer at -80°C and left to stand for 6 hours. Next, the PFA film-coated glass plate and PFA film-coated copper plate were removed from the frozen material and transferred to a freeze-drying container that had been left to stand in a deep freezer at -80°C for 1 hour. Next, freeze-drying was performed at room temperature under reduced pressure of 5 Pa or less for 72 hours to obtain a collagen sponge.

[0162] (Cross-linking reaction of collagen sponge) The cross-linking reaction of the collagen sponge described above was carried out in three steps. The cross-linking method was the same as in Example 1. After the third step of the cross-linking reaction, the collagen sponge was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed 10 times while shaking for 30 minutes.

[0163] (Preparation of magnetic nanoparticle-containing collagen convenience gel 14) The cross-linked collagen sponge described above was cut into prismatic shapes and immersed in a 5 mg / mL collagen aqueous solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the 5 mg / mL collagen aqueous solution. Subsequently, it was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain a collagen sponge / collagen freeze-dried product. Next, magnetic nanoparticles were synthesized in the same manner as in Example 11. These magnetic nanoparticles were dispersed in a neutral phosphate buffer to prepare a neutral phosphate buffer with a magnetic nanoparticle concentration of 20 mg / mL. This was incubated in a 37°C constant temperature bath to bring the temperature of the magnetic nanoparticle-containing neutral phosphate buffer to 37°C. The 37°C magnetic nanoparticle-containing neutral phosphate buffer was added to the collagen sponge / collagen freeze-dried product and incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby producing a magnetic nanoparticle-containing convenience gel 14.

[0164] Figure 16 shows the appearance of (a) a cross-linked collagen sponge prismatic body, (b) a cross-linked collagen sponge prismatic body / collagen freeze-dried product, and (c) a magnetic nanoparticle-containing convenience gel 14. The scale in the figure represents 1 mm. The magnetic nanoparticle-containing convenience gel 14 is black in color, which is due to the color of the magnetic nanoparticles used as an additive. Furthermore, when the magnetic nanoparticle-containing convenience gel 14 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked collagen sponge.

[0165] In the same manner as in Example 11, the magnetothermal effect of the magnetic nanoparticle-containing convenience gel 14 when an alternating magnetic field was applied was investigated. When an alternating magnetic field was applied to the magnetic nanoparticle-containing convenience gel 14 for 10 minutes, the temperature of the magnetic nanoparticle-containing convenience gel 14 rose by 3.4 ± 0.2°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle-containing convenience gel 14 was maintained.

[0166] [Example 15] In Example 15, a mixed aqueous solution of magnetic nanoparticles / Dox / VCR / sodium alginate was introduced into the pores and gaps of a cross-linked collagen sponge, and after freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare a magnetic nanoparticle ∧Dox∧VCR-containing sodium alginate convenience gel 15 (hereinafter simply referred to as magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 15).

[0167] The manufacturing method is described in detail. First, a cross-linked collagen sponge was prepared in the same manner as in Example 14. Magnetic nanoparticles were synthesized in the same manner as in Example 11. A mixed aqueous solution of Dox / VCR / sodium alginate was prepared in the same manner as in Example 3, with a concentration of VCR of 0.2 mg / mL, a concentration of Dox of 0.5 mg / mL, and a concentration of sodium alginate of 25 mg / mL. Next, the magnetic nanoparticles were dispersed in the mixed aqueous solution of Dox / VCR / sodium alginate to prepare a mixed aqueous dispersion of magnetic nanoparticles / Dox / VCR / sodium alginate with a concentration of magnetic nanoparticles of 10 mg / mL, a concentration of VCR of 0.2 mg / mL, a concentration of Dox of 0.5 mg / mL, and a concentration of sodium alginate of 25 mg / mL. Next, the cross-linked collagen sponge was cut into prismatic shapes and immersed in the aforementioned aqueous dispersion of magnetic nanoparticles / Dox / VCR / sodium alginate. By applying reduced pressure, the pores and gaps in the sponge were filled with the aqueous dispersion of magnetic nanoparticles / Dox / VCR / sodium alginate. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a 3 mg / mL aqueous calcium chloride solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby producing a convenience gel 15 containing magnetic nanoparticles ∧Dox∧VCR.

[0168] Figure 17 shows the appearance of (a) a cross-linked collagen sponge prismatic body, (b) a lyophilized cross-linked collagen sponge prismatic body / sodium alginate / magnetic nanoparticles / Dox / VCR, and (c) a convenience gel 15 containing magnetic nanoparticles ∧Dox∧VCR. The scale in the figure represents 1 mm. The lyophilized cross-linked collagen sponge prismatic body / sodium alginate / magnetic nanoparticles / Dox / VCR and the convenience gel 15 containing magnetic nanoparticles ∧Dox∧VCR are dark red in color, which is due to the black color of the magnetic nanoparticles and the red color of Dox. Furthermore, when the convenience gel 15 containing magnetic nanoparticles ∧Dox∧VCR is pressed with a spatula or lifted, no water comes out, indicating that the water is retained, which shows that the gel was formed in the pores and gaps of the cross-linked collagen sponge.

[0169] The magnetothermal effect of the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 15 was investigated in the same manner as in Example 11. When an alternating magnetic field was applied to the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 15 for 10 minutes, the temperature of the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 15 rose by 5.3 ± 0.1°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 15 is maintained.

[0170] The sustained release behavior of Dox from a convenience gel 15 containing magnetic nanoparticles ∧Dox∧VCR was investigated. The method was the same as in Example 3, except that the sample was changed to a convenience gel 15 containing magnetic nanoparticles ∧Dox∧VCR. Figure 34 shows the relationship between the mean ± standard deviation of the cumulative release rate (%) of Dox relative to the amount of Dox introduced and the incubation time. From Figure 34, it was found that, similar to the case of a conventional gel containing Dox, Dox was released gradually as the incubation time progressed.

[0171] [Example 16] In Example 16, a mixed aqueous dispersion of gold nanorods / Dox / collagen was introduced into the pores and gaps of a lactic acid / glycolic acid copolymer (PLGA)-crosslinked collagen composite mesh. After freezing and freeze-drying, a neutral phosphate buffer was added to the freeze-dried product to prepare a gold nanorod / Dox-containing collagen convenience gel 16 (hereinafter simply referred to as gold nanorod / Dox-containing convenience gel 16).

[0172] The manufacturing method is described in detail. First, a PLGA-crosslinked collagen composite mesh was prepared by compounding a commercially available PLGA knit mesh with collagen. Porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a 10 mg / mL collagen aqueous solution. In a low-temperature chamber at 4°C, the surface of a glass plate was covered with plastic wrap, and the aforementioned PLGA knit mesh (dimensions 80 mm × 55 mm × thickness 0.2 mm) was laid on top of it, and the 10 mg / mL collagen aqueous solution was dropped onto it. The droplet was spread with a glass rod to ensure that the solution evenly filled the voids in the PLGA knit mesh. Excess liquid was removed by rolling the glass rod. Next, it was frozen by standing at -80°C for 6 hours. A porous structure was formed by freeze-drying at room temperature under reduced pressure of 5 Pa or less for 3 days. Furthermore, a crosslinking reaction and treatment with a glycine aqueous solution were performed using the method shown in Example 1 to prepare a PLGA-crosslinked collagen composite mesh.

[0173] On the other hand, gold nanorods were synthesized in the same manner as in Example 12. Porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare an aqueous collagen solution. Dox was dissolved in pure water to prepare an aqueous Dox solution. The aqueous Dox solution was added to the collagen solution and stirred at 4°C to prepare a mixed Dox / collagen aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20 mg / mL. Next, the gold nanorods were dispersed in this mixed Dox / collagen aqueous solution to prepare a mixed gold nanorod / Dox / collagen aqueous dispersion with a gold nanorod concentration of 2 mM, a Dox concentration of 0.5 mg / mL, and a collagen concentration of 20 mg / mL.

[0174] Next, the PLGA-crosslinked collagen composite mesh was cut into a square shape and immersed in the gold nanorod / Dox / collagen mixed aqueous dispersion. After removing excess liquid, the pores and gaps of the PLGA-crosslinked collagen composite mesh were filled with the gold nanorod / Dox / collagen mixed aqueous dispersion by applying reduced pressure. Subsequently, freeze-drying was performed in the same manner as in Example 1. A neutral phosphate buffer at 37°C was added to the freeze-dried material, and it was incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby producing a gold nanorod / Dox-containing convenience gel 16.

[0175] Figure 18 shows the appearance of (a) a square PLGA-crosslinked collagen composite mesh, (c) a PLGA-crosslinked collagen composite mesh / collagen / gold nanorod / Dox freeze-dried product, and (d) a gold nanorod / Dox-containing convenience gel 16. The scale in the figure represents 1 mm. Figure 18 also shows (b) a scanning electron microscope image of the PLGA-crosslinked collagen composite mesh. From the scanning electron microscope image, it was found that a collagen sponge was formed in the voids of the PLGA knit mesh. In addition, the PLGA-crosslinked collagen composite mesh / collagen / gold nanorod / Dox freeze-dried product and the gold nanorod / Dox-containing convenience gel 16 exhibited a red color, which is due to the color of Dox, one of the additives. Furthermore, when the gold nanorod / Dox-containing convenience gel 16 was pressed with a spatula and lifted, no water came out, indicating that water was retained, which showed that gel was formed in the pores and voids of the PLGA-crosslinked collagen composite mesh.

[0176] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dox-containing convenience gel 16 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dox-containing convenience gel 16 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dox-containing convenience gel 16 rose by 23.2 ± 2.8°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 16, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 16 was maintained.

[0177] The sustained release behavior of Dox from gold nanorods∧Dox-containing convenience gel 16 was investigated. The method was the same as in Example 5, except that the sample was changed to gold nanorods∧Dox-containing convenience gel 16. Figure 34 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 34, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0178] [Example 17] In Example 17, a mixed aqueous solution of Dox / sodium alginate was introduced into the pores and gaps of a cross-linked collagen sponge, and after freezing and freeze-drying, an aqueous solution of calcium chloride containing magnetic nanoparticles was added to the freeze-dried product to prepare a magnetic nanoparticle-containing sodium alginate convenience gel 17 (hereinafter simply referred to as magnetic nanoparticle-containing Dox convenience gel 17).

[0179] The manufacturing method is described in detail. First, in the same manner as in Example 15, a cross-linked collagen sponge was prepared using ice nanoparticles with a particle size of 150 μm to 250 μm as a pore-forming agent, with a ratio (v / w) of 1 / 1 between 30 mg / mL collagen aqueous solution and ice nanoparticles. On the other hand, magnetic nanoparticles were synthesized in the same manner as in Example 11. These magnetic nanoparticles were dispersed in a 3 mg / mL calcium chloride aqueous solution, and Fe ３ O ４A dispersion of magnetic nanoparticles / calcium chloride aqueous solution with a magnetic nanoparticle concentration of 10 mg / mL was prepared. Next, Dox and sodium alginate were dissolved in ultrapure water to prepare aqueous solutions of Dox and sodium alginate, respectively. The aqueous Dox solution was added to the aqueous sodium alginate solution and stirred to prepare a mixed solution of Dox / sodium alginate with a concentration of 0.5 mg / mL of Dox and a concentration of 25 mg / mL of sodium alginate. Next, the cross-linked collagen sponge was cut into prismatic shapes and immersed in the mixed solution of Dox / sodium alginate. By applying reduced pressure, the pores and gaps in the sponge were filled with the mixed solution of Dox / sodium alginate. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding the dispersion of magnetic nanoparticles / calcium chloride aqueous solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby producing a magnetic nanoparticle / Dox-containing convenience gel 17.

[0180] Figure 19 shows the appearance of (a) a cross-linked collagen sponge prismatic body, (b) a cross-linked collagen sponge prismatic body / sodium alginate / Dox freeze-dried product, and (c) a convenience gel 17 containing magnetic nanoparticles ∧Dox. The scale in the figure represents 1 mm. The collagen sponge prismatic body / sodium alginate / Dox freeze-dried product is red, which is due to the color of Dox, one of the additives. The convenience gel 17 containing magnetic nanoparticles ∧Dox is black, and also slightly red. This is due to the black color of the magnetic nanoparticles and the red color of Dox, which are additives. Furthermore, when the convenience gel 17 containing magnetic nanoparticles ∧Dox was pressed with a spatula and lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked collagen sponge.

[0181] In the same manner as in Example 11, the magnetothermal effect of the magnetic nanoparticle ∧Dox-containing convenience gel 17 when an alternating magnetic field was applied was investigated. When an alternating magnetic field was applied to the magnetic nanoparticle ∧Dox-containing convenience gel 17 for 10 minutes, the temperature of the magnetic nanoparticle ∧Dox-containing convenience gel 17 rose by 4.8 ± 0.4°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle ∧Dox-containing convenience gel 17 was maintained.

[0182] The sustained release behavior of Dox from magnetic nanoparticle ∧Dox-containing convenience gel 17 was investigated. The method was the same as in Example 3, except that the sample was changed to magnetic nanoparticle ∧Dox-containing convenience gel 17. Figure 34 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 34, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0183] [Example 18] In Example 18, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a PLGA-crosslinked collagen composite mesh, and a gold nanorod-containing neutral phosphate buffer was added to the freeze-dried product obtained by freezing and freeze-drying to prepare a gold nanorod-containing Dox-containing collagen convenience gel 18 (hereinafter simply referred to as gold nanorod-containing Dox-containing convenience gel 18).

[0184] The manufacturing method is described in detail. First, a PLGA-crosslinked collagen composite mesh was prepared in the same manner as in Example 16. Next, a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20.0 mg / mL was prepared in the same manner as in Example 5. Gold nanorods were also synthesized in the same manner as in Example 12. These gold nanorods were dispersed in a neutral phosphate buffer to prepare a neutral phosphate buffer containing gold nanoparticles with a gold nanorod concentration of 2 mM, and the solution temperature was maintained at 37°C in a 37°C constant temperature bath. Subsequently, the PLGA-crosslinked collagen composite mesh was cut into squares and immersed in the Dox / collagen mixed aqueous solution. After removing excess liquid, the pores and gaps of the PLGA-crosslinked collagen composite mesh were filled with the Dox / collagen mixed aqueous solution by applying reduced pressure. Subsequently, freeze-drying was performed in the same manner as in Example 1. The aforementioned 37°C gold nanoparticle-containing neutral phosphate buffer was added to this freeze-dried material, and the mixture was incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby producing a gold nanorod ∧Dox-containing convenience gel 18.

[0185] Figure 20 shows the appearance of (a) a rectangular PLGA-crosslinked collagen composite mesh, (b) a PLGA-crosslinked collagen composite mesh / collagen / Dox freeze-dried product, and (c) a gold nanorod ∧Dox-containing convenience gel 18. The scale in the figure represents 1 mm. The PLGA-crosslinked collagen composite mesh / collagen / Dox freeze-dried product and the gold nanorod ∧Dox-containing convenience gel 18 exhibit a red color, which is due to the color of Dox, one of the additives. Furthermore, when the gold nanorod ∧Dox-containing convenience gel 18 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the PLGA-crosslinked collagen composite mesh.

[0186] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dox-containing convenience gel 18 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dox-containing convenience gel 18 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dox-containing convenience gel 18 rose by 18.7 ± 0.8°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 18, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 18 was maintained.

[0187] The sustained release behavior of Dox from gold nanorods∧Dox-containing convenience gel 18 was investigated. The method was the same as in Example 5, except that the sample was changed to gold nanorods∧Dox-containing convenience gel 18. Figure 34 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 34, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0188] [Example 19] In Example 19, a magnetic nanoparticle / sodium alginate mixed aqueous solution was introduced into the pores and gaps of a cross-linked collagen sponge, and after freezing and freeze-drying, a Dox-containing calcium chloride aqueous solution was added to the freeze-dried product to prepare a magnetic nanoparticle / Dox-containing sodium alginate convenience gel 19 (hereinafter simply referred to as magnetic nanoparticle / Dox-containing convenience gel 19).

[0189] The manufacturing method is described in detail. First, a cross-linked collagen sponge was prepared in the same manner as in Example 14. On the other hand, magnetic nanoparticles were synthesized in the same manner as in Example 11. These magnetic nanoparticles were dispersed in a 15 mg / mL aqueous sodium alginate solution to prepare a magnetic nanoparticle / sodium alginate mixed aqueous dispersion with a magnetic nanoparticle concentration of 10 mg / mL and a sodium alginate concentration of 15 mg / mL. Next, Dox and calcium chloride were dissolved in ultrapure water, respectively, to prepare aqueous Dox solutions and aqueous calcium chloride solutions. The aqueous Dox solution was added to the aqueous calcium chloride solution and stirred to prepare a Dox / calcium chloride mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a calcium chloride concentration of 2 mg / mL. Next, the cross-linked collagen sponge was cut into prismatic shapes and immersed in the aqueous dispersion of magnetic nanoparticles and sodium alginate. By applying reduced pressure, the pores and gaps in the sponge were filled with the aqueous dispersion of magnetic nanoparticles and sodium alginate. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding the aforementioned Dox / calcium chloride mixed aqueous solution to this freeze-dried material, sodium alginate was dissolved and immediately gelled, thereby producing magnetic nanoparticles ∧ Dox-containing convenience gel 19.

[0190] Figure 21 shows the appearance of (a) a cross-linked collagen sponge prismatic body, (b) a cross-linked collagen sponge prismatic body / sodium alginate / freeze-dried magnetic nanoparticle material, and (c) a convenience gel 19 containing magnetic nanoparticles ∧Dox. The scale in the figure represents 1 mm. The cross-linked collagen sponge prismatic body / sodium alginate / freeze-dried magnetic nanoparticle material is black, which is due to the color of the magnetic nanoparticles, one of the additives. The convenience gel 19 containing magnetic nanoparticles ∧Dox is dark red, which is due to the black color of the magnetic nanoparticles and the red color of Dox, which are additives. Furthermore, when the convenience gel 19 containing magnetic nanoparticles ∧Dox was pressed with a spatula and lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked collagen sponge.

[0191] The magnetothermal effect of the magnetic nanoparticle ∧Dox-containing convenience gel 19 was investigated when an alternating magnetic field was applied, in the same manner as in Example 11. When an alternating magnetic field was applied to the magnetic nanoparticle ∧Dox-containing convenience gel 19 for 10 minutes, the temperature of the magnetic nanoparticle ∧Dox-containing convenience gel 19 rose by 5.1 ± 0.1°C. It was found that the magnetic nanoparticles introduced into the magnetic nanoparticle ∧Dox-containing convenience gel 19 retained their magnetothermal effect.

[0192] The sustained release behavior of Dox from magnetic nanoparticle ∧Dox-containing convenience gel 19 was investigated. The method was the same as in Example 3, except that the sample was changed to magnetic nanoparticle ∧Dox-containing convenience gel 19. Figure 34 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 34, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0193] [Example 20] In Example 20, a mixed aqueous solution of gold nanorods and collagen was introduced into the pores and gaps of a PLGA-crosslinked collagen composite mesh, and a lyophilized product was obtained by freezing and freeze-drying. A neutral phosphate buffer containing VCR was then added to the freeze-dried product to prepare a gold nanorod / VCR-containing collagen convenience gel 20 (hereinafter simply referred to as gold nanorod / VCR-containing convenience gel 20).

[0194] The manufacturing method is described in detail. First, a PLGA-crosslinked collagen composite mesh was prepared in the same manner as in Example 16. On the other hand, gold nanorods were synthesized in the same manner as in Example 12. These gold nanorods were dispersed in a 0.5 mg / mL collagen aqueous solution to prepare a mixed aqueous dispersion of gold nanorod-containing collagen with a gold nanorod concentration of 2 mM and a collagen concentration of 5 mg / mL. In addition, VCR was dissolved in a neutral phosphate buffer to prepare a VCR-containing neutral phosphate buffer with a VCR concentration of 0.3 mg / mL, and incubated in a 37°C constant temperature bath to bring the temperature of the VCR-containing neutral phosphate buffer to 37°C. Next, the PLGA-crosslinked collagen composite mesh was cut into squares and immersed in the mixed aqueous dispersion of gold nanorod-containing collagen. After removing excess liquid, the pores and gaps of the PLGA-crosslinked collagen composite mesh were filled with the mixed aqueous dispersion of gold nanorod-containing collagen by applying reduced pressure. Next, freeze-drying was performed in the same manner as in Example 1. A VCR-containing neutral phosphate buffer at 37°C was added to the freeze-dried material, and the mixture was incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby producing a gold nanorod-VCR-containing convenience gel 20.

[0195] Figure 22 shows the appearance of (a) a rectangular PLGA-crosslinked collagen composite mesh, (b) a freeze-dried PLGA-crosslinked collagen composite mesh / collagen / gold nanorod, and (c) a gold nanorod / VCR-containing convenience gel 20. The scale in the figure represents 1 mm. The PLGA-crosslinked collagen composite mesh / collagen / gold nanorod freeze-dried product and the gold nanorod / VCR-containing convenience gel 20 are slightly brown in color, which is due to the color of the gold nanorod, one of the additives. Furthermore, when the gold nanorod / VCR-containing convenience gel 20 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the PLGA-crosslinked collagen composite mesh.

[0196] In the same manner as in Example 12, the heat generation effect of the gold nanorods∧VCR-containing convenience gel 20 when irradiated with near-infrared light was investigated. When the gold nanorods∧VCR-containing convenience gel 20 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorods∧VCR-containing convenience gel 20 rose by 22.2 ± 0.3°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorods∧VCR-containing convenience gel 20, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorods∧VCR-containing convenience gel 20 was maintained.

[0197] To investigate the sustained release behavior of VCR from gold nanorods∧VCR-containing convenience gel 20, gold nanorods∧VCR-containing convenience gel 20 was incubated in the same manner as in Example 3. Since the experiment was conducted with N=3, a total of three gold nanorods∧VCR-containing convenience gels 20 were used. After incubation for 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, 250 μL of neutral phosphate buffer was collected and a fresh 250 μL of neutral phosphate buffer was added. The concentration of VCR in the collected 250 μL of neutral phosphate buffer (pH 7.4) was measured using a spectrophotometer (V-660) to determine the absorbance at a wavelength of 297 nm. The cumulative amount of VCR released from the gold nanorods∧VCR-containing convenience gel 20 was then calculated as a percentage of the introduced amount. Figure 34 shows the relationship between the mean ± standard deviation of the cumulative VCR release rate (%) relative to the amount of VCR introduced and the incubation time. From Figure 34, it can be seen that, similar to the case of conventional gels containing VCR, VCR was released gradually as the incubation time progressed.

[0198] [Example 21] In Example 21, an aqueous sodium alginate solution was introduced into the pores and gaps of a β-TCP sponge, and after freezing and freeze-drying, a mixed aqueous dispersion of magnetic nanoparticles / VCR / calcium chloride was added to the freeze-dried product to prepare a magnetic nanoparticle / VCR-containing sodium alginate convenience gel 21 (hereinafter simply referred to as magnetic nanoparticle / VCR-containing convenience gel 21).

[0199] The manufacturing method is described in detail below. First, magnetic nanoparticles were synthesized in the same manner as in Example 11. These magnetic nanoparticles were dispersed in a mixed aqueous solution of VCR / calcium chloride to prepare a mixed aqueous dispersion of magnetic nanoparticles / VCR / calcium chloride with a concentration of magnetic nanoparticles of 10 mg / mL, a concentration of VCR of 0.2 mg / mL, and a concentration of calcium chloride of 3 mg / mL. In addition, an aqueous solution of sodium alginate with a concentration of 25 mg / mL was prepared. Next, a commercially available β-TCP sponge prismatic body was immersed in the aforementioned aqueous solution of sodium alginate and the pores and gaps of the sponge were filled with the aqueous solution of sodium alginate by applying reduced pressure. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding the mixed aqueous dispersion of magnetic nanoparticles / VCR / calcium chloride to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby producing a magnetic nanoparticle / VCR-containing convenience gel 21.

[0200] Figure 23 shows the appearance of (a) a β-TCP sponge prismatic body, (b) a β-TCP sponge prismatic body / sodium alginate freeze-dried product, and (c) a magnetic nanoparticle ∧VCR-containing convenience gel 21. The scale in the figure represents 1 mm. The magnetic nanoparticle ∧VCR-containing convenience gel 21 is black, which is due to the color of the magnetic nanoparticles, one of the additives. Furthermore, when the magnetic nanoparticle ∧VCR-containing convenience gel 21 was lifted, no water came out, indicating that water was retained, which showed that the gel was formed in the pores and gaps of the β-TCP sponge.

[0201] The magnetothermal effect of the magnetic nanoparticle-VCR-containing convenience gel 21 was investigated in the same manner as in Example 11. When an alternating magnetic field was applied to the magnetic nanoparticle-VCR-containing convenience gel 21 for 10 minutes, the temperature of the magnetic nanoparticle-VCR-containing convenience gel 21 rose by 5.1 ± 0.6°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle-VCR-containing convenience gel 21 was maintained. The sustained release behavior of VCR from the magnetic nanoparticle-VCR-containing convenience gel 21 was investigated. The method was the same as in Example 20, except that the sample was changed to the magnetic nanoparticle-VCR-containing convenience gel 21. Figure 35 shows the relationship between the mean ± standard deviation of the cumulative VCR release rate (%) relative to the amount of VCR introduced and the incubation time. From Figure 35, it was found that, similar to the case of a conventional gel containing VCR, VCR was sustained release as the incubation time progressed.

[0202] [Example 22] In Example 22, a collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked collagen sponge, and after freezing and freeze-drying, a gold nanorod / Dox-containing neutral phosphate buffer solution was added to the freeze-dried product to prepare a gold nanorod / Dox-containing collagen convenience gel 22 (hereinafter simply referred to as gold nanorod / Dox-containing convenience gel 22).

[0203] The manufacturing method is described in detail. First, a cross-linked collagen sponge was prepared in the same manner as in Example 14. Next, gold nanorods were synthesized in the same manner as in Example 12. These gold nanorods were dispersed in a neutral phosphate buffer containing Dox, and a neutral phosphate buffer containing gold nanorods and Dox was prepared with a concentration of 2 mM gold nanorods and a concentration of 0.5 mg / mL Dox. This was incubated in a 37°C constant temperature bath to bring the temperature of the neutral phosphate buffer containing gold nanorods and Dox to 37°C. Next, the cross-linked collagen sponge was cut into prismatic shapes and immersed in a 5 mg / mL collagen aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked collagen sponge were filled with the 5 mg / mL collagen aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1 to obtain a cross-linked collagen sponge / freeze-dried collagen product. A neutral phosphate buffer containing the aforementioned 37°C gold nanorods and Dox was added to this freeze-dried material, and the mixture was incubated in a 37°C constant temperature bath for 10 minutes to dissolve the collagen and immediately gel it, thereby producing a gold nanorod / Dox-containing convenience gel 22.

[0204] Figure 24 shows the appearance of (a) a cross-linked collagen sponge prismatic body, (b) a cross-linked collagen sponge prismatic body / collagen freeze-dried product, and (c) a gold nanorod ∧Dox-containing convenience gel 22. The scale in the figure represents 1 mm. The gold nanorod ∧Dox-containing convenience gel 22 is red in color, which is due to the color of the additives, gold nanorods and Dox. Furthermore, when the gold nanorod ∧Dox-containing convenience gel 22 was pressed with a spatula or lifted, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked collagen sponge.

[0205] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dox-containing convenience gel 22 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dox-containing convenience gel 22 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dox-containing convenience gel 22 rose by 22.1 ± 0.5°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 22, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 22 was maintained.

[0206] The sustained release behavior of Dox from gold nanorods ∧ Dox-containing convenience gel 22 was investigated. The method was the same as in Example 5, except that the sample was changed to gold nanorods ∧ Dox-containing convenience gel 22. Figure 35 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 35, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0207] [Example 23] In Example 23, a mixed aqueous solution of Dox / sodium alginate was introduced into the pores and gaps of a cross-linked gelatin-magnetic nanoparticle composite sponge, and a calcium chloride aqueous solution was added to the freeze-dried product obtained by freezing and freeze-drying to produce a magnetic nanoparticle ∧Dox-containing sodium alginate convenience gel 23 (hereinafter simply referred to as magnetic nanoparticle ∧Dox-containing convenience gel 23).

[0208] The manufacturing method is described in detail. First, magnetic nanoparticles were synthesized in the same manner as in Example 11. These magnetic nanoparticles were dispersed in ultrapure water to prepare an aqueous dispersion of magnetic nanoparticles with a concentration of 20.0 mg / mL. Next, 70 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing an 80.0 mg / mL gelatin-acetic acid aqueous solution. Then, the aforementioned aqueous dispersion of magnetic nanoparticles with a concentration of 20.0 mg / mL and the aforementioned 80.0 mg / mL gelatin-acetic acid aqueous solution were mixed in a 1:1 volume ratio to prepare a mixed dispersion aqueous solution of magnetic nanoparticles / gelatin in 35 (v / v)% acetic acid with a magnetic nanoparticle concentration of 10.0 mg / mL and a gelatin concentration of 40.0 mg / mL. Subsequently, a cross-linked gelatin-magnetic nanoparticle composite sponge was manufactured. The preparation method was the same as in Example 1, except that the 40.0 mg / mL gelatin acetic acid aqueous solution was replaced with a mixed dispersion aqueous solution of magnetic nanoparticles / gelatin with a concentration of 10.0 mg / mL of magnetic nanoparticles and a concentration of 40.0 mg / mL of gelatin in 35 (v / v) acetic acid. This cross-linked gelatin-magnetic nanoparticle composite sponge was observed with a scanning electron microscope. On the other hand, a mixed aqueous solution of Dox and sodium alginate was prepared with a concentration of Dox of 0.5 mg / mL and a concentration of sodium alginate of 25 mg / mL. Next, the cross-linked gelatin-magnetic nanoparticle composite sponge was cut into cylindrical shapes and immersed in the Dox / sodium alginate mixed aqueous solution. By applying reduced pressure, the pores and gaps of the composite sponge were filled with the Dox / sodium alginate mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a 3 mg / mL aqueous calcium chloride solution to this freeze-dried material, sodium alginate was dissolved and immediately gelled, thereby producing a convenience gel 23 containing magnetic nanoparticles ∧Dox.

[0209] Figure 25 shows the appearance of (a) a cross-linked gelatin-magnetic nanoparticle composite sponge cylinder, (c) a cross-linked gelatin-magnetic nanoparticle composite sponge cylinder / sodium alginate / Dox freeze-dried product, and (d) a convenience gel 23 containing magnetic nanoparticles ∧Dox. The scale in the figure represents 1 mm. Furthermore, Figure 25 shows (b) a scanning electron microscope image of the cross-linked gelatin-magnetic nanoparticle composite sponge cylinder. The cross-linked gelatin-magnetic nanoparticle composite sponge cylinder is black, which is due to the color of the magnetic nanoparticles, one of the additives. Furthermore, the scanning electron microscope image revealed that the cross-linked gelatin-magnetic nanoparticle composite sponge has a porous structure in which pores are interconnected. In addition, when the convenience gel 23 containing magnetic nanoparticles ∧Dox was pressurized with a spatula, no water came out, indicating that water was retained, which showed that gel was formed in the pores and gaps of the cross-linked gelatin-magnetic nanoparticle composite sponge.

[0210] The magnetothermal effect of the magnetic nanoparticle ∧Dox-containing convenience gel 23 when an alternating magnetic field is applied was investigated in the same manner as in Example 11. When an alternating magnetic field was applied to the magnetic nanoparticle ∧Dox-containing convenience gel 23 for 10 minutes, the temperature of the magnetic nanoparticle ∧Dox-containing convenience gel 23 rose by 5.3 ± 0.3°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle ∧Dox-containing convenience gel 23 is maintained.

[0211] The sustained release behavior of Dox from magnetic nanoparticle ∧Dox-containing convenience gel 23 was investigated. The method was the same as in Example 3, except that the sample was changed to magnetic nanoparticle ∧Dox-containing convenience gel 23. Figure 35 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 35, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0212] [Example 24] In Example 24, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin-gold nanorod composite sponge, and after freezing and freeze-drying, a neutral phosphate buffer was added to the freeze-dried product to prepare a gold nanorod∧Dox-containing collagen convenience gel 24 (hereinafter simply referred to as gold nanorod∧Dox-containing convenience gel 24).

[0213] The preparation method is described in detail. First, gold nanorods were synthesized in the same manner as in Example 12. Next, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing a 40.0 mg / mL gelatin-acetic acid aqueous solution. Glutamic acid was added to this 40.0 mg / mL gelatin-acetic acid aqueous solution and stirred to prepare a gelatin / glutamic acid mixed solution with a gelatin concentration of 40.0 mg / mL and a glutamic acid concentration of 4.0 mg / mL. Furthermore, the gold nanorods were dispersed in this gelatin / glutamic acid mixed solution to prepare a mixed aqueous dispersion of gold nanorods / gelatin / glutamic acid with a gold nanorod concentration of 2 mM, a gelatin concentration of 40.0 mg / mL, and a glutamic acid concentration of 4.0 mg / mL. A cross-linked gelatin-gold nanorod composite sponge was prepared using this mixed aqueous dispersion of gold nanorods, gelatin, and glutamic acid. The preparation method was the same as in Example 1, except that the 40.0 mg / mL gelatin-acetic acid aqueous solution was replaced with the above-mentioned mixed aqueous dispersion of gold nanorods, gelatin, and glutamic acid. This cross-linked gelatin-gold nanorod composite sponge was observed with a scanning electron microscope. Next, a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20 mg / mL was prepared. Subsequently, cylindrical pieces of the cross-linked gelatin-gold nanorod composite sponge were immersed in the Dox / collagen mixed aqueous solution and subjected to reduced pressure conditions to fill the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge with the Dox / collagen mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a neutral phosphate buffer at 37°C to this freeze-dried material and incubating it in a constant temperature bath at 37°C for 10 minutes, the collagen was dissolved and immediately gelled, thereby producing gold nanorod ∧Dox-containing convenience gel 24.

[0214] Figure 26 shows the appearance of (a) a cross-linked gelatin-gold nanorod composite sponge cylinder, (c) a cross-linked gelatin-gold nanorod composite sponge cylinder / collagen / Dox freeze-dried product, and (d) a gold nanorod∧Dox-containing convenience gel 24. The scale in the figure represents 1 mm. Figure 26 shows a scanning electron microscope image of (b) the cross-linked gelatin-gold nanorod composite sponge cylinder. The cross-linked gelatin-gold nanorod composite sponge cylinder is brown in color, which is due to the color of the gold nanorods, one of the additives. Furthermore, the scanning electron microscope image revealed that the cross-linked gelatin-gold nanorod composite sponge has a porous structure in which pores are interconnected. In addition, when the gold nanorod∧Dox-containing convenience gel 24 was pressurized with a spatula, no water came out, indicating that water was retained, which showed that gel was formed in the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge.

[0215] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dox-containing convenience gel 24 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dox-containing convenience gel 24 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dox-containing convenience gel 24 rose by 32.1 ± 0.5°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 24, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox-containing convenience gel 24 was maintained.

[0216] The sustained release behavior of Dox from gold nanorods ∧ Dox-containing convenience gel 24 was investigated. The method was the same as in Example 5, except that the sample was changed to gold nanorods ∧ Dox-containing convenience gel 24. Figure 35 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 35, it was found that, similar to the case of conventional gels containing Dox, Dox was released gradually as the incubation time progressed.

[0217] [Example 25] In Example 25, a Dex / sodium alginate mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin-gold nanorod composite sponge, and after freezing and freeze-drying, a Dox-containing calcium chloride aqueous solution was added to the freeze-dried product to produce a gold nanorod∧Dex∧Dox-containing sodium alginate convenience gel 25 (hereinafter simply referred to as gold nanorod∧Dex∧Dox-containing convenience gel 25).

[0218] The manufacturing method is described in detail. First, a cross-linked gelatin-gold nanorod composite sponge was prepared in the same manner as in Example 24. Next, a mixed solution of Dex and sodium alginate was prepared with a Dex concentration of 0.5 mg / mL and a sodium alginate concentration of 25 mg / mL. In addition, a mixed aqueous solution of Dox and calcium chloride was prepared with a Dox concentration of 0.5 mg / mL and a calcium chloride concentration of 3 mg / mL. Subsequently, cylindrical pieces of the cross-linked gelatin-gold nanorod composite sponge were immersed in the mixed solution of Dex and sodium alginate, and the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge were filled with the mixed solution of Dex and sodium alginate by applying reduced pressure. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding the aforementioned Dox / calcium chloride mixed aqueous solution to this freeze-dried material, sodium alginate was dissolved and immediately gelled, thereby producing a gold nanorod ∧Dex∧Dox-containing convenience gel 25.

[0219] Figure 27 shows the appearance of (a) a cross-linked gelatin-gold nanorod composite sponge cylinder, (b) a cross-linked gelatin-gold nanorod composite sponge cylinder / sodium alginate / Dex freeze-dried product, and (c) a gold nanorod ∧Dex∧Dox-containing convenience gel 25. The scale in the figure represents 1 mm. When the gold nanorod ∧Dex∧Dox-containing convenience gel 25 was pressed with a spatula and lifted, no water came out, indicating that the water was retained. This indicates that the gel was formed in the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge.

[0220] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dex∧Dox-containing convenience gel 25 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dex∧Dox-containing convenience gel 25 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dex∧Dox-containing convenience gel 25 rose by 30.8 ± 0.3°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dex∧Dox-containing convenience gel 25, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dex∧Dox-containing convenience gel 25 was maintained.

[0221] The sustained release behavior of Dox from a gold nanorod ∧Dex∧Dox-containing convenience gel 25 was investigated. The method was the same as in Example 3, except that the sample was changed to a gold nanorod ∧Dex∧Dox-containing convenience gel 25. Figure 35 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 35, it was found that, similar to the case of a conventional gel containing Dox, Dox was released gradually as the incubation time progressed.

[0222] [Example 26] In Example 26, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin-magnetic nanoparticle composite sponge, and after freezing and freeze-drying, a VCR-containing neutral phosphate buffer was added to the freeze-dried product to prepare magnetic nanoparticles∧Dox∧VCR-containing collagen convenience gel 26 (hereinafter simply referred to as magnetic nanoparticles∧Dox∧VCR-containing convenience gel 26).

[0223] The manufacturing method is described in detail. First, a cross-linked gelatin-magnetic nanoparticle composite sponge was prepared in the same manner as in Example 23. On the other hand, a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20 mg / mL was prepared. Also, a neutral phosphate buffer containing 0.3 mg / mL VCR was prepared, and the temperature of the solution was maintained at 37°C in a 37°C constant temperature bath. Next, the cross-linked gelatin-magnetic nanoparticle composite sponge was cut into cylindrical shapes and immersed in the Dox / collagen mixed aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin-magnetic nanoparticle composite sponge were filled with the Dox / collagen mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a VCR-containing neutral phosphate buffer at 37°C to this freeze-dried material and incubating it in a 37°C constant temperature bath for 10 minutes, the collagen was dissolved and immediately gelled, thereby producing magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 26.

[0224] Figure 28 shows the appearance of (a) a cross-linked gelatin-magnetic nanoparticle composite sponge cylinder, (b) a cross-linked gelatin-magnetic nanoparticle composite sponge cylinder / collagen / Dox freeze-dried product, and (c) a convenience gel 26 containing magnetic nanoparticles ∧Dox∧VCR. The scale in the figure represents 1 mm. The cross-linked gelatin-magnetic nanoparticle composite sponge cylinder is black, which is due to the color of the magnetic nanoparticles, one of the additives. The cross-linked gelatin-magnetic nanoparticle composite sponge cylinder / collagen / Dox freeze-dried product is slightly red, which is due to the color of Dox, one of the additives. Furthermore, when the convenience gel 26 containing magnetic nanoparticles ∧Dox∧VCR was pressed with a spatula, no water came out, indicating that water was retained, which showed that gel was formed in the pores and gaps of the cross-linked gelatin-magnetic nanoparticle composite sponge.

[0225] In the same manner as in Example 11, the magnetothermal effect of the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 26 when an alternating magnetic field was applied was investigated. When an alternating magnetic field was applied to the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 26 for 10 minutes, the temperature of the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 26 rose by 6.9 ± 0.4°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 26 was maintained.

[0226] The sustained release behavior of Dox from a convenience gel 26 containing magnetic nanoparticles ∧Dox∧VCR was investigated. The method was the same as in Example 5, except that the sample was changed to a convenience gel 26 containing magnetic nanoparticles ∧Dox∧VCR. Figure 35 shows the relationship between the mean ± standard deviation of the cumulative release rate (%) of Dox relative to the amount of Dox introduced and the incubation time. From Figure 35, it was found that, similar to the case of a conventional gel containing Dox, Dox was released gradually as the incubation time progressed.

[0227] [Example 27] In Example 27, a Dox / collagen mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin-gold nanorod composite sponge, and after freezing and freeze-drying, a Dex-containing neutral phosphate buffer was added to the freeze-dried product to prepare a gold nanorod∧Dox∧Dex-containing collagen convenience gel 27 (hereinafter simply referred to as gold nanorod∧Dox∧Dex-containing convenience gel 27).

[0228] The manufacturing method is described in detail. First, a cross-linked gelatin-gold nanorod composite sponge was prepared in the same manner as in Example 24. On the other hand, a Dox / collagen mixed aqueous solution with a Dox concentration of 0.5 mg / mL and a collagen concentration of 20 mg / mL was prepared. Also, a 10 mg / mL Dex-containing neutral phosphate buffer solution was prepared, and the temperature of the solution was maintained at 37°C in a 37°C constant temperature bath. Next, the cross-linked gelatin-gold nanorod composite sponge was cut into cylindrical shapes and immersed in the Dox / collagen mixed aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge were filled with the Dox / collagen mixed aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding a 37°C Dex-containing neutral phosphate buffer to this freeze-dried material and incubating it in a 37°C constant temperature bath for 10 minutes, the collagen was dissolved and immediately gelled, thereby producing a gold nanorod∧Dox∧Dex-containing convenience gel 27.

[0229] Figure 29 shows the appearance of (a) a cross-linked gelatin-gold nanorod composite sponge cylinder, (b) a cross-linked gelatin-gold nanorod composite sponge cylinder / collagen / Dox freeze-dried product, and (c) a gold nanorod ∧Dox ∧Dex-containing convenience gel 27. The scale in the figure represents 1 mm. The cross-linked gelatin-gold nanorod composite sponge cylinder is brown in color, which is due to the color of gold nanorods, one of the additives. The color of the gold nanorod ∧Dox ∧Dex-containing convenience gel 27 is even darker, which is due to the color of Dox, one of the additives. Furthermore, when the gold nanorod ∧Dox ∧Dex-containing convenience gel 27 was pressurized with a spatula, no water came out, indicating that water was retained, which showed that gel was formed in the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge.

[0230] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dox∧Dex-containing convenience gel 27 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dox∧Dex-containing convenience gel 27 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dox∧Dex-containing convenience gel 27 rose by 31.8 ± 0.8°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox∧Dex-containing convenience gel 27, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dox∧Dex-containing convenience gel 27 was maintained.

[0231] The sustained release behavior of Dox from a gold nanorod-containing Dox-Dex gel 27 was investigated. The method was the same as in Example 5, except that the sample was changed to a gold nanorod-containing Dox-Dex gel 27. Figure 36 shows the relationship between the mean ± standard deviation of the cumulative Dox release rate (%) relative to the amount of Dox introduced and the incubation time. From Figure 36, it was found that, similar to the case of a conventional gel containing Dox, Dox was released gradually as the incubation time progressed.

[0232] [Example 28] In Example 28, an aqueous sodium alginate solution was introduced into the pores and gaps of a cross-linked gelatin-magnetic nanoparticle composite sponge, and after freezing and freeze-drying, an aqueous calcium chloride solution containing Dox / VCR was added to the freeze-dried product to prepare magnetic nanoparticles ∧Dox∧VCR-containing convenience gel 28 (hereinafter simply referred to as magnetic nanoparticles ∧Dox∧VCR-containing convenience gel 28).

[0233] The manufacturing method is described in detail. First, a cross-linked gelatin-magnetic nanoparticle composite sponge was prepared in the same manner as in Example 23. On the other hand, a 20 mg / mL sodium alginate aqueous solution was prepared. In addition, a mixed aqueous solution consisting of 0.5 mg / mL Dox, 0.2 mg / mL VCR, and 2.5 mg / mL calcium chloride was prepared. Next, the cross-linked gelatin-magnetic nanoparticle composite sponge was cut into cylindrical shapes and immersed in the 20 mg / mL sodium alginate aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin-magnetic nanoparticle composite sponge were filled with the 20 mg / mL sodium alginate aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. By adding the mixed aqueous solution of Dox, VCR, and calcium chloride to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby producing a magnetic nanoparticle-Dox-VCR containing convenience gel 28.

[0234] Figure 30 shows the appearance of (a) a cross-linked gelatin-magnetic nanoparticle composite sponge cylinder, (b) a cross-linked gelatin-magnetic nanoparticle composite sponge cylinder / sodium alginate freeze-dried product, and (c) a convenience gel 28 containing magnetic nanoparticles ∧Dox∧VCR. The cross-linked gelatin-magnetic nanoparticle composite sponge cylinder, the cross-linked gelatin-magnetic nanoparticle composite sponge cylinder / sodium alginate freeze-dried product, and the convenience gel 28 containing magnetic nanoparticles ∧Dox∧VCR are all black in color, which is due to the color of the magnetic nanoparticles, one of the additives. Furthermore, when the convenience gel 28 containing magnetic nanoparticles ∧Dox∧VCR was pressurized with a spatula, no water came out, indicating that the water was retained, which showed that the gel was formed in the pores and gaps of the cross-linked gelatin-magnetic nanoparticle composite sponge.

[0235] In the same manner as in Example 11, the magnetothermal effect of the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 28 when an alternating magnetic field was applied was investigated. When an alternating magnetic field was applied to the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 28 for 10 minutes, the temperature of the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 28 rose by 5.1 ± 0.4°C. This indicates that the magnetothermal effect of the magnetic nanoparticles introduced into the magnetic nanoparticle ∧Dox∧VCR-containing convenience gel 28 was maintained.

[0236] The sustained release behavior of Dox from a convenience gel 28 containing magnetic nanoparticles ∧Dox∧VCR was investigated. The method was the same as in Example 3, except that the convenience gel 28 containing magnetic nanoparticles ∧Dox∧VCR was used. Figure 36 shows the relationship between the mean ± standard deviation of the cumulative release rate (%) of Dox relative to the amount of Dox introduced and the incubation time. From Figure 36, it was found that, similar to the case of a conventional gel containing Dox, Dox was released gradually as the incubation time progressed.

[0237] [Example 29] In Example 29, a collagen aqueous solution was introduced into the pores and gaps of a cross-linked gelatin-gold nanorod composite sponge, and after freezing, the freeze-dried product was obtained. Dex-containing neutral phosphate buffer was then added to the freeze-dried product to prepare a gold nanorod∧Dex-containing collagen convenience gel 29 (hereinafter simply referred to as gold nanorod∧Dex-containing convenience gel 29).

[0238] The manufacturing method is described in detail. First, a cross-linked gelatin-gold nanorod composite sponge was prepared in the same manner as in Example 24. On the other hand, a 5 mg / mL collagen aqueous solution was prepared. Also, a 10 mg / mL Dex-containing neutral phosphate buffer solution was prepared, and the temperature of the solution was maintained at 37°C in a 37°C constant temperature bath. Next, the cross-linked gelatin-gold nanorod composite sponge was cut into cylindrical shapes and immersed in the collagen aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin-gold nanorod composite sponge were filled with the collagen aqueous solution. Subsequently, freeze-drying was performed in the same manner as in Example 1. The Dex-containing neutral phosphate buffer solution was added to this freeze-dried product, and by incubation in a 37°C constant temperature bath for 10 minutes, the collagen was dissolved and immediately gelled, thereby producing a gold nanorod-Dex-containing convenience gel 29.

[0239] Figure 31 shows the appearance of (a) a cross-linked gelatin-gold nanorod composite sponge cylinder, (b) a cross-linked gelatin-gold nanorod composite sponge cylinder / collagen freeze-dried product, and a gold nanorod ∧Dex-containing convenience gel 29. The scale in the figure represents 1 mm. The cross-linked gelatin-gold nanorod composite sponge cylinder, the cross-linked gelatin-gold nanorod composite sponge cylinder / collagen freeze-dried product, and the gold nanorod ∧Dex-containing convenience gel 29 are brown in color, which is due to the color of the gold nanorods, one of the additives. Since water was retained, it was found that gel was formed in the pores and gaps of the gelatin-gold nanorod composite sponge.

[0240] In the same manner as in Example 12, the heat generation effect of the gold nanorod ∧Dex-containing convenience gel 29 when irradiated with near-infrared light was investigated. When the gold nanorod ∧Dex-containing convenience gel 29 was irradiated with near-infrared light for 10 minutes, the temperature of the gold nanorod ∧Dex-containing convenience gel 29 rose by 28.6 ± 0.6°C. This was due to the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dex-containing convenience gel 29, and it was found that the heat generation effect of the gold nanorods introduced into the gold nanorod ∧Dex-containing convenience gel 29 was maintained.

[0241] The sustained release behavior of Dex from gold nanorods∧Dex-containing convenience gel 29 was investigated. The method was the same as in Example 5, except that the sample was changed to gold nanorods∧Dex-containing convenience gel 29. Figure 36 shows the relationship between the mean ± standard deviation of the cumulative Dex release rate (%) relative to the amount of Dex introduced and the incubation time. From Figure 36, it was found that, similar to the case of conventional gels containing Dex, Dex was released gradually as the incubation time progressed.

[0242] [Example 30] In Example 30, a mixed solution of dacarbazine (DTIC) and sodium alginate was introduced into the pores and gaps of a PLGA-crosslinked collagen composite mesh. After freezing and freeze-drying, an aqueous calcium chloride solution was added to the freeze-dried product to prepare DTIC-containing sodium alginate convenience gel 30 (hereinafter simply referred to as DTIC-containing convenience gel 30). The killing effect of DTIC-containing convenience gel 30 on human malignant melanoma cells was confirmed by in vitro cell culture experiments and in vivo animal experiments.

[0243] The manufacturing method is described in detail. In the same manner as in Example 16, first, a PLGA-crosslinked collagen composite mesh was prepared by compounding a commercially available PLGA knit mesh with collagen. Porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a 10 mg / mL collagen aqueous solution. In a low-temperature chamber at 4°C, the surface of a glass plate was covered with plastic wrap, and a PLGA knit mesh (dimensions 80 mm x 55 mm x thickness 0.2 mm) was placed on top of it, and the 10 mg / mL collagen aqueous solution was dropped onto it. The droplet was spread with a glass rod to ensure that the mixed solution evenly filled the voids of the PLGA knit mesh. Excess liquid was removed by rolling the glass rod. Subsequently, it was frozen by standing at -80°C for 6 hours. A PLGA-uncrosslinked collagen composite mesh was prepared by freeze-drying at room temperature under reduced pressure of 5 Pa or less for 3 days.

[0244] Furthermore, the PLGA-uncrosslinked collagen composite mesh was subjected to a sequential crosslinking reaction and treatment with an aqueous glycine solution in the following three steps to produce a PLGA-crosslinked collagen composite mesh. In the first crosslinking reaction step, 2-morpholinoethanesulfonic acid (MES) was dissolved in ethanol / water (95 / 5, v / v) to prepare a 0.1 M MES solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added to this MES solution and dissolved while stirring to prepare a first-stage crosslinking reaction solution containing 50 mM WSCD·HCl and 20 mM NHS. The first-stage crosslinking reaction was carried out by immersing the freeze-dried PLGA-uncrosslinked collagen composite mesh in this first-stage crosslinking reaction solution at room temperature for 12 hours.

[0245] Next, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the second crosslinking reaction solution in 30 mL of ethanol / water (90 / 10, v / v), similar to the first crosslinking reaction solution. The PLGA-uncrosslinked collagen composite mesh, after the first crosslinking reaction, was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0246] Finally, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the third crosslinking reaction solution in 30 mL of ethanol / water (85 / 15, v / v), similar to the first crosslinking reaction solution. The PLGA-uncrosslinked collagen composite mesh, after the second crosslinking reaction, was immersed in the third crosslinking reaction solution at room temperature for 12 hours. This carried out the third crosslinking reaction. The PLGA-collagen composite mesh, after the third crosslinking reaction, was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. By these steps, a PLGA-crosslinked collagen composite mesh was obtained.

[0247] On the other hand, DTIC was dissolved in dimethyl sulfoxide (DMSO) to prepare a DMSO solution with a DTIC concentration of 30 mg / mL. 250 mg of sodium alginate was dissolved in 9 mL of ultrapure water to prepare an aqueous sodium alginate solution. 1 mL of the aforementioned DTIC DMSO solution was added to this aqueous sodium alginate solution and stirred to prepare a sodium alginate / DTIC mixed solution consisting of DTIC at a concentration of 3 mg / mL and sodium alginate at a concentration of 25 mg / mL. Subsequently, the PLGA-crosslinked collagen composite mesh was cut into 10 mm diameter discs and immersed in the sodium alginate / DTIC mixed solution. By applying reduced pressure, the pores and gaps of the PLGA-crosslinked collagen composite mesh were filled with the sodium alginate / DTIC mixed solution. Next, the material was frozen in a deep freezer at -80°C for 12 hours, followed by freeze-drying at room temperature under reduced pressure of 5 Pa or less for 72 hours to produce a freeze-dried product of PLGA-crosslinked collagen composite mesh / sodium alginate / DTIC. By adding a 3 mg / mL aqueous calcium chloride solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, producing DTIC-containing convenience gel 30. The gelling time was less than 1 minute. Because the supported gel is supported on a porous structure, it becomes a gel that can be easily formed in a short time when needed.

[0248] Furthermore, as a comparative example of the DTIC-containing convenience gel 30, a gel without DTIC (referred to as the gel of Comparative Example 1) was prepared. The preparation method was the same as that for the DTIC-containing convenience gel 30, except that the DTIC DMSO solution was replaced with DMSO.

[0249] Figure 37A shows the appearance of the PLGA-crosslinked collagen composite mesh / sodium alginate / DTIC freeze-dried product, DTIC-containing convenience gel 30, the pressurized DTIC-containing convenience gel 30, and the lifted DTIC-containing convenience gel 30. Since the DTIC-containing convenience gel 30 remained intact without releasing water even when pressurized with a spatula or when lifted, it was determined that the gel formed in the pores and gaps of the PLGA-crosslinked collagen composite mesh. Furthermore, to investigate the sustained release behavior of DTIC from the DTIC-containing convenience gel 30, one DTIC-containing convenience gel 30 was immersed in 500 μL of HEPES buffer (pH 7.4) and incubated in a 37°C constant temperature bath while slowly shaking. At incubation times of 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, 250 μL of liquid was sampled, and the remaining solution was replenished with 250 μL of HEPES buffer (pH 7.4). The absorbance of the sampled liquid (wavelength 330 nm) was measured using a UV-Vis spectrophotometer (V-660, manufactured by JASCO Corporation), the concentration of DTIC in the sampled liquid was calculated, the cumulative amount of DTIC released from the DTIC-containing convenience gel 30 was determined, and the percentage relative to the introduced amount was calculated. Figure 37B shows the relationship between the mean ± standard deviation of the cumulative DTIC release rate (%) relative to the introduced amount of DTIC and the incubation time. From the figure, it can be seen that, similar to conventional gels containing DTIC, DTIC was released gradually as the incubation time progressed.

[0250] To evaluate the cytotoxic effect of DTIC released from DTIC-containing convenience gel 30, in vitro cell culture experiments and in vivo animal experiments were performed. In these experiments, sterile DTIC-containing convenience gel 30 and sterile comparative example 1 gel were used. First, the lyophilized PLGA-crosslinked collagen composite mesh / sodium alginate / DTIC and the lyophilized PLGA-crosslinked collagen composite mesh / sodium alginate were sterilized by a known ethylene oxide gas sterilization method (EOG sterilization method) to obtain sterile lyophilized PLGA-crosslinked collagen composite mesh / sodium alginate / DTIC and sterile lyophilized PLGA-crosslinked collagen composite mesh / sodium alginate. The 3 mg / mL calcium chloride aqueous solution was sterilized by passing it through a 0.22 μm filter (a known filtration sterilization method) to prepare a sterile 3 mg / mL calcium chloride aqueous solution. Next, a sterile DTIC-containing convenience gel 30 was prepared by adding the sterile 3 mg / mL calcium chloride aqueous solution to the lyophilized lyophilized PLGA-crosslinked collagen composite mesh / sodium alginate / DTIC. Furthermore, a sterile comparative example 1 gel was prepared by adding the sterile 3 mg / mL calcium chloride aqueous solution to the lyophilized lyophilized PLGA-crosslinked collagen composite mesh / sodium alginate.

[0251] Then, to evaluate the in vitro cell-killing effect of DTIC released from the DTIC-containing convenience gel 30, cell viability was determined using the known WST-1 method. 20,000 human malignant melanoma cell lines (SK-Mel-28-Luc) were seeded in each well of a 24-well multi-well cell culture plate and incubated in Eagle's minimal essential medium (EMEM) containing 10% fetal bovine serum at 37°C and 5% CO2. ２Cell culture was performed under atmospheric conditions. After 12 hours of culture, one sterile DTIC-containing convenience gel 30 was added per well, and the cells were cultured for 24, 48, and 72 hours. The viability of cells in the wells after each culture time was determined by the WST-1 method. For comparison, the viability of cells in the presence of the sterile gel of Comparative Example 1 was determined using the sterile gel of Comparative Example 1.

[0252] Figure 37C shows the viability of SK-Mel-28-Luc cells cultured in the presence of DTIC-containing Convenience Gel 30 and in the presence of Comparative Example 1. The viability of SK-Mel-28-Luc cells cultured in the presence of Comparative Example 1 increased with culture time. Therefore, it was found that the gel of Comparative Example 1 did not exhibit cytotoxicity and did not affect the proliferation of SK-Mel-28-Luc cells. In contrast, the viability of SK-Mel-28-Luc cells cultured in the presence of DTIC-containing Convenience Gel 30 decreased with culture time. This is thought to be because DTIC released from DTIC-containing Convenience Gel 30 killed the SK-Mel-28-Luc cells. Therefore, it was found that DTIC-containing Convenience Gel 30 has the effect of killing human malignant melanoma cell lines.

[0253] Furthermore, animal experiments were conducted to evaluate the in vivo cell-killing effect of DTIC released from the DTIC-containing convenience gel 30. First, the aforementioned PLGA-crosslinked collagen composite mesh (disk-shaped with a diameter of 10 mm) was sterilized with a known 70 (v / v)% ethanol aqueous solution to prepare a sterilized PLGA-crosslinked collagen composite mesh. 100,000 SK-Mel-28-Luc cells were seeded on the upper surface of this sterilized PLGA-crosslinked collagen composite mesh and cultured in vitro for 6 hours. Then, the disc-shaped PLGA-crosslinked collagen composite mesh was turned over, and another 100,000 SK-Mel-28-Luc cells were seeded and cultured in vitro for 6 hours. After that, it was cultured in vitro for a further 12 hours. Next, one sterile DTIC-containing convenience gel 30 was sandwiched between two PLGA-crosslinked collagen composite meshes on which SK-Mel-28-Luc cells had been cultured, creating one set of implantation samples. One set of these implantation samples was implanted subcutaneously on the back of each nude mouse under general anesthesia. D-luciferin was injected subcutaneously at the implantation site 4 hours after implantation (day 0), and 1, 3, 5, 7, 9, and 11 days later, and in vivo chemiluminescence imaging was performed 5 minutes later. For comparison, a similar in vivo animal experiment was also performed using the sterile gel of Comparative Example 1. The results of the in vivo chemiluminescence imaging are shown in Figure 37D. For each experimental condition of DTIC-containing convenience gel 30 and Comparative Example 1, one nude mouse was used for each experiment.

[0254] In Figure 37D, the emission intensity in in vivo chemiluminescence imaging is converted to grayscale, but in reality, it is displayed in color. That is, the color changes in the order of red, yellow, green, and blue from areas with a high concentration of SK-Mel-28-Luc cells to areas with a low concentration. Therefore, areas that are shown particularly darkly in the grayscale image can be judged to be areas where SK-Mel-28-Luc cells are present. According to Figure 37D, immediately after implantation (day 0) of the DTIC-containing convenience gel 30 and the gel of Comparative Example 1, luminescent regions indicating the presence of SK-Mel-28-Luc cells were observed at the implantation site of the mice in both cases. In chemiluminescence imaging of SK-Mel-28-Luc cells in mice implanted with the gel of Comparative Example 1, a strong chemiluminescence signal was observed even on day 11, indicating that the SK-Mel-28-Luc cells were still alive. In contrast, the intensity of chemiluminescence imaging of SK-Mel-28-Luc cells in mice implanted with DTIC-containing Convenience Gel 30 decreased over time and disappeared by day 11. This suggests that the human malignant melanoma cells present within the PLGA-crosslinked collagen composite mesh surrounding the DTIC-containing Convenience Gel 30 died, causing the luminescence signal to disappear. In other words, it is thought that the DTIC released from the DTIC-containing Convenience Gel 30 killed human malignant melanoma cells located near the DTIC-containing Convenience Gel 30. These results demonstrate that DTIC released from the DTIC-containing Convenience Gel 30 can effectively kill human malignant melanoma cells.

[0255] [Example 31] In Example 31, an imatinib (IM) / sodium alginate mixed aqueous solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, an aqueous calcium chloride solution was added to the freeze-dried product to prepare IM-containing sodium alginate convenience gel 31 (hereinafter simply referred to as IM-containing convenience gel 31). The killing effect of IM-containing convenience gel 31 on human chronic myeloid leukemia cells was confirmed by in vitro cell culture experiments.

[0256] As a porous structure, a cross-linked gelatin sponge was prepared using a gelatin acetate aqueous solution with a particle size of 250-350 μm and a concentration of 80 mg / mL. On the other hand, IM and sodium alginate were dissolved in ultrapure water, respectively, to prepare IM aqueous solution and sodium alginate aqueous solution. The IM aqueous solution was added to the sodium alginate aqueous solution and stirred to prepare an IM / sodium alginate mixed aqueous solution consisting of IM at a concentration of 0.4 mg / mL and sodium alginate at a concentration of 15 mg / mL. The cross-linked gelatin sponge was cut into a cylindrical shape (6 mm in diameter, 6 mm in height), immersed in the IM / sodium alginate mixed aqueous solution, and subjected to reduced pressure conditions to fill the pores and voids of the cross-linked gelatin sponge with the IM / sodium alginate mixed aqueous solution. Subsequently, it was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain a freeze-dried cross-linked gelatin sponge cylinder / sodium alginate / IM product. By adding a 2 mg / mL aqueous calcium chloride solution to the obtained freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby preparing IM-containing convenience gel 31. As a comparative example of IM-containing convenience gel 31, the gel of Comparative Example 2 was prepared. The preparation method was the same as for IM-containing convenience gel 31, except that the IM / sodium alginate mixed aqueous solution was replaced with an aqueous sodium alginate solution.

[0257] Figure 38A shows the appearance of the cross-linked gelatin sponge cylinder / sodium alginate / IM freeze-dried product, IM-containing convenience gel, pressurized IM-containing convenience gel 31, and lifted IM-containing convenience gel 31. Since the IM-containing convenience gel 31 remained intact without releasing water even when pressurized with a spatula or lifted, it was determined that gel had formed in the pores and gaps of the cross-linked gelatin sponge. To investigate the sustained release behavior of IM from the IM-containing convenience gel 31, one IM-containing convenience gel 31 was immersed in 500 μL of HEPES buffer (pH 7.4) and incubated in a 37°C constant temperature bath while slowly shaking. 250 μL of liquid was sampled at each incubation time of 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours, and 250 μL of HEPES buffer was added to the remaining solution. The absorbance of the sampled liquid (wavelength 285 nm) was measured using a UV-Vis spectrophotometer to determine the concentration of IM contained in the liquid. From this concentration, the cumulative amount of IM released from the IM-containing convenience gel was determined, and its percentage relative to the introduced amount was calculated. Figure 38B shows the relationship between the mean ± standard deviation of the cumulative IM release rate (%) relative to the introduced amount and the incubation time. From the figure, it was found that, similar to conventional gels containing IM, IM was released gradually over time.

[0258] To evaluate the in vitro cell-killing effect of IM released from IM-containing convenience gel 31, cell viability was determined by the WST-1 method. In this experiment, sterile IM-containing convenience gel 31 and sterile comparative example 2 gel were used. Sterilized IM-containing convenience gel 31 was prepared by adding the aforementioned 2 mg / mL calcium chloride aqueous solution, sterilized by filtration sterilization, to the aforementioned cross-linked gelatin sponge cylinder / sodium alginate / IM freeze-dried product, sterilized by EOG sterilization. Sterilized comparative example 2 gel was prepared by adding the aforementioned 2 mg / mL calcium chloride aqueous solution, sterilized by filtration sterilization, to the aforementioned cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product, sterilized by EOG sterilization.

[0259] 40,000 K562 cells, a human chronic myeloid leukemia-derived cell line, were seeded in each well of a 24-well cell culture plate and incubated in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. ２ Cell culture was performed under atmospheric conditions. After 12 hours of culture, one sterile IM-containing convenience gel 31 was added per well, and the cells were cultured for 24, 48, and 72 hours. The cell viability after each culture time was determined by the WST-1 method. For comparison, the cell viability in the presence of the sterile comparative example 2 gel was determined.

[0260] Figure 38C shows the viability of K562 cells cultured in the presence of IM-containing convenience gel 31 and in the presence of the gel of Comparative Example 2. The viability of K562 cells cultured in the presence of the gel of Comparative Example 2 increased with culture time. This indicates that the gel of Comparative Example 2 did not exhibit cytotoxicity and did not affect the proliferation of K562 cells. In contrast, the viability of K562 cells cultured in the presence of IM-containing convenience gel 31 decreased with culture time. This is thought to be due to the sustained release of IM from the IM-containing convenience gel 31 killing the K562 cells. Therefore, it was found that IM-containing convenience gel 31 may kill human chronic myeloid leukemia-derived cells.

[0261] [Example 32] In Example 32, a mixed aqueous solution of carboplatin (CBDCA) and sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge. After freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare CBDCA-containing sodium alginate convenience gel 32 (hereinafter simply referred to as CBDCA-containing convenience gel 32). The killing effect of CBDCA-containing convenience gel 32 on human cervical cancer cells was confirmed by in vitro cell culture experiments.

[0262] As a porous structure, a cross-linked gelatin sponge was prepared using ice microparticles with a particle size of 250 μm to 350 μm and an 80 mg / mL gelatin acetic acid aqueous solution. On the other hand, CBDCA and sodium alginate were dissolved in pure water, respectively, to prepare CBDCA aqueous solution and sodium alginate aqueous solution. The CBDCA aqueous solution was added to the sodium alginate aqueous solution and stirred to prepare a CBDCA / sodium alginate mixed aqueous solution consisting of CBDCA at a concentration of 12 mg / mL and sodium alginate at a concentration of 10 mg / mL. The cross-linked gelatin sponge was cut into a cylindrical shape (6 mm in diameter, 6 mm in height), immersed in the CBDCA / sodium alginate mixed aqueous solution, and under reduced pressure conditions, the pores and gaps of the cross-linked gelatin sponge were filled with the CBDCA / sodium alginate mixed aqueous solution. Next, the mixture was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to prepare a cross-linked gelatin sponge cylinder / sodium alginate / CBDCA freeze-dried product. By adding a 1.5 mg / mL aqueous calcium chloride solution to this freeze-dried product, the sodium alginate was dissolved and immediately gelled, preparing CBDCA-containing convenience gel 32. As a comparative example of CBDCA-containing convenience gel 32, the gel of Comparative Example 3 was prepared. The preparation method was the same as that for CBDCA-containing convenience gel 32, except that the CBDCA / sodium alginate mixed aqueous solution was replaced with an aqueous sodium alginate solution.

[0263] Figure 39A shows the appearance of the cross-linked gelatin sponge cylinder / sodium alginate / CBDCA freeze-dried product, CBDCA-containing convenience gel 32, the pressurized CBDCA-containing convenience gel 32, and the lifted CBDCA-containing convenience gel 32. Since the CBDCA-containing convenience gel 32 remained intact without releasing water even when pressurized with a spatula or lifted, it was determined that the gel formed in the pores and gaps of the cross-linked gelatin sponge. To investigate the sustained release behavior of CBDCA from the CBDCA-containing convenience gel 32, one CBDCA-containing convenience gel 32 was immersed in 500 μL of HEPES buffer (pH 7.4) and incubated in a 37°C constant temperature bath while slowly shaking. A total of four CBDCA-containing convenience gels 32 were used to conduct the experiment with N=4. Incubation times were 1, 2, 4, 6, 12, 24, 48, 72, 96, and 120 hours. 250 μL of liquid was sampled at each incubation time point, and 250 μL of HEPES buffer was added to the remaining solution. The absorbance of the sampled liquid (wavelength 510 nm) was measured using a UV-Vis spectrophotometer to determine the concentration of CBDCA in the liquid. From this concentration, the cumulative amount of CBDCA released from the CBDCA-containing convenience gel 32 was determined, and its percentage relative to the introduced amount was calculated. Figure 39B shows the relationship between the mean ± standard deviation of the cumulative CBDCA release rate (%) relative to the introduced amount of CBDCA and the incubation time. From the figure, it can be seen that, similar to conventional gels containing CBDCA, CBDCA was released gradually as the incubation time progressed.

[0264] To evaluate the in vitro cell-killing effect of CBDCA released from CBDCA-containing convenience gel 32, cell viability was determined by the WST-1 method. In the in vitro cell culture experiment, sterile CBDCA-containing convenience gel 32 and sterile comparative example 3 gel were used. Sterilized CBDCA-containing convenience gel 32 was prepared by adding the aforementioned 1.5 mg / mL calcium chloride aqueous solution, sterilized by filtration sterilization, to the aforementioned cross-linked gelatin sponge cylinder / sodium alginate / CBDCA freeze-dried product, sterilized by EOG sterilization. Sterilized comparative example 3 gel was prepared using a sodium alginate aqueous solution without added CBDCA, in the same manner as the preparation method for sterile CBDCA-containing convenience gel 32.

[0265] Human cervical cancer cells (Hela cells) were seeded at 40,000 cells in each well of a 24-well cell culture plate and incubated in EMEM containing 10% fetal bovine serum at 37°C and 5% CO2. ２ Cell culture was performed under atmospheric conditions. After 12 hours of culture, one CBDCA-containing convenience gel 32 was added per well, and the cells were cultured for 24, 48, and 72 hours. The viability of the cells after each culture time was determined by the WST-1 method. For comparison, the cell viability in the presence of the sterile gel of Comparative Example 3 was used. Figure 39C shows the viability of Hela cells cultured in the presence of CBDCA-containing convenience gel 32 and in the presence of the gel of Comparative Example 3. The viability of Hela cells cultured in the presence of the gel of Comparative Example 3 increased with culture time. This indicates that the gel of Comparative Example 3 did not exhibit cytotoxicity and did not affect the proliferation of Hela cells. In contrast, the viability of Hela cells cultured in the presence of CBDCA-containing convenience gel 32 decreased with culture time. This is thought to be due to the CBDCA released from the CBDCA-containing convenience gel 32 killing the Hela cells. Therefore, it was found that CBDCA-containing Convenience Gel 32 can kill human cervical cancer cells.

[0266] [Example 33] In Example 33, an aqueous sodium alginate solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a cytarabine (Ara-C) / calcium chloride mixed aqueous solution was added to the freeze-dried product to prepare Ara-C-containing sodium alginate convenience gel 33 (hereinafter simply referred to as Ara-C-containing convenience gel 33). The killing effect of Ara-C-containing convenience gel 33 on human glioblastoma cells was confirmed by in vivo animal experiments.

[0267] First, a gelatin sponge was prepared using ice particles with a particle size of 350 μm to 500 μm as a pore-forming agent. Pure water was sprayed into liquid nitrogen in a container, and the mist-like water was frozen. Next, this frozen material was transferred to a low-temperature chamber at -15°C and left to stand for 2 to 2.5 hours to evaporate the liquid nitrogen. After that, the ice particles with a particle size of 350 μm to 500 μm were sieved out using a sieve with a mesh size of 500 μm and a sieve with a mesh size of 350 μm within the low-temperature chamber. The temperature inside the low-temperature chamber was changed to -5°C, and these ice particles were left to stand for 3 hours.

[0268] On the other hand, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing an 80 mg / mL gelatin-acetic acid aqueous solution. This gelatin-acetic acid aqueous solution was placed in a low-temperature chamber (-5°C) and stirred for 30 minutes. Subsequently, in the low-temperature chamber (-5°C), the ice particles were added to the gelatin-acetic acid aqueous solution to a ratio of 30 / 70 (mL / g) of gelatin-acetic acid aqueous solution / ice particles, and the mixture was slowly stirred for 10 minutes to prepare a gelatin-acetic acid aqueous solution / ice particle mixture. This gelatin-acetic acid aqueous solution / ice particle mixture was placed in a container whose temperature had been equilibriumized in the low-temperature chamber (-5°C). After the container containing the gelatin-acetic acid aqueous solution / ice particle mixture was left to stand in the low-temperature chamber (-5°C) for 30 minutes, it was transferred to a deep freezer at -80°C and left to stand for 12 hours. The container was removed from the frozen material and transferred to a freeze-drying container that had been left to stand in a deep freezer at -80°C for 1 hour. Then, freeze-drying was performed in a freeze-dryer at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain freeze-dried gelatin sponge.

[0269] A cross-linked gelatin sponge was prepared by sequentially performing a cross-linking reaction and treatment with an aqueous glycine solution in the following three steps. In the first cross-linking reaction step, 2-morpholinoethanesulfonic acid (MES) was dissolved in ethanol / water (95 / 5, v / v) to prepare a 0.1 M MES solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added to this MES solution and dissolved while stirring to prepare a first-stage cross-linking reaction solution containing 50 mM WSCD·HCl and 20 mM NHS. The first-stage cross-linking reaction was carried out by immersing the freeze-dried gelatin sponge in this first-stage cross-linking reaction solution at room temperature for 12 hours.

[0270] Next, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the second crosslinking reaction solution in 30 mL of ethanol / water (90 / 10, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the first crosslinking reaction was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0271] Finally, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the third crosslinking reaction solution in 30 mL of ethanol / water (85 / 15, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the second crosslinking reaction was immersed in the third crosslinking reaction solution at room temperature for 12 hours. This carried out the third crosslinking reaction. The gelatin sponge after the third crosslinking reaction was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. A crosslinked gelatin sponge was obtained by the above steps.

[0272] On the other hand, sodium alginate was dissolved in ultrapure water to prepare a 25 mg / mL sodium alginate aqueous solution. The cross-linked gelatin sponge was cut into cylindrical shapes (8 mm in diameter, 3 mm in height) and immersed in the 25 mg / mL sodium alginate aqueous solution. By applying reduced pressure, the pores and gaps of the cross-linked gelatin sponge were filled with the sodium alginate aqueous solution. Subsequently, the mixture was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain a cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product. Furthermore, Ara-C and calcium chloride were dissolved in ultrapure water, respectively, to prepare an Ara-C aqueous solution and a calcium chloride aqueous solution. The Ara-C aqueous solution was added to the calcium chloride aqueous solution and stirred to prepare an Ara-C / calcium chloride mixed aqueous solution with an Ara-C concentration of 0.12 mg / mL and a calcium chloride concentration of 3 mg / mL. Next, the Ara-C / calcium chloride mixed aqueous solution was added to the cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product to dissolve the sodium alginate and immediately gel it, thereby producing the Ara-C-containing convenience gel 33.

[0273] Furthermore, as a comparative example of the Ara-C-containing convenience gel 33, a gel without Ara-C (referred to as the gel of Comparative Example 4) was prepared. The preparation method was the same as that for the Ara-C-containing convenience gel 33, except that the Ara-C / calcium chloride mixed aqueous solution was replaced with a calcium chloride aqueous solution. Specifically, the gel of Comparative Example 4 was prepared by adding a 3 mg / mL calcium chloride aqueous solution to the cross-linked gelatin sponge cylinder / freeze-dried sodium alginate product, thereby dissolving the sodium alginate and immediately causing gelation.

[0274] Figure 40A shows photographs of the cross-linked gelatin sponge cylinder, the cross-linked gelatin sponge cylinder / freeze-dried sodium alginate, the Ara-C-containing convenience gel 33, the pressurized Ara-C-containing convenience gel 33, and the lifted Ara-C-containing convenience gel 33. Since the Ara-C-containing convenience gel 33 remained intact without releasing water even when pressurized with a spatula or when lifted, it was found that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0275] The killing effect of Ara-C-containing convenience gel 33 on human glioblastoma cells was evaluated in animal experiments. In the animal experiments, sterile Ara-C-containing convenience gel 33 and sterile comparative example 4 gel were used. First, the cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product was sterilized by the known ethylene oxide gas sterilization method (EOG sterilization method) to obtain sterile cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product. Subsequently, the Ara-C / calcium chloride mixed aqueous solution with an Ara-C concentration of 0.12 mg / mL and a calcium chloride concentration of 3 mg / mL was sterilized by passing it through a 0.22 μm filter (known filtration sterilization method) to prepare a sterile Ara-C / calcium chloride mixed aqueous solution. Finally, a sterile Ara-C-containing convenience gel 33 was prepared by adding the sterile Ara-C / calcium chloride mixed aqueous solution to the sterile cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product. In addition, a sterile comparative example 4 gel was prepared by adding a filtration-sterilized 3 mg / mL calcium chloride aqueous solution to the sterile cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product.

[0276] On the other hand, in order to mimic human glioblastoma cells present near Convenience Gel 33 in animal experiments, it was decided to implant Convenience Gel 33 subcutaneously into nude mice using a PLGA-crosslinked collagen composite mesh disc in which human glioblastoma cells had been cultured. First, the PLGA-crosslinked collagen composite mesh was prepared. As a preparation method, porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a 10 mg / mL collagen aqueous solution. In a low-temperature chamber at 4°C, the surface of a glass plate was covered with plastic wrap, and a commercially available PLGA knit mesh cut into a rectangle (dimensions 80 mm x 55 mm) was placed on top of it, and the 10 mg / mL collagen aqueous solution was dropped onto it. The droplet was spread with a glass rod so that the mixed solution evenly filled the voids of the PLGA knit mesh. Excess liquid was removed by rolling the glass rod. Subsequently, it was frozen by standing at -80°C for 6 hours. A PLGA-uncrosslinked collagen composite mesh was prepared by freeze-drying at room temperature under reduced pressure of 5 Pa or less for 3 days. This PLGA-uncrosslinked collagen composite mesh was then subjected to the three crosslinking steps described above and treatment with a glycine aqueous solution to produce a PLGA-crosslinked collagen composite mesh.

[0277] Next, the PLGA-crosslinked collagen composite mesh was cut into 8 mm diameter discs and sterilized with a known 70 (v / v) ethanol aqueous solution to prepare sterile PLGA-crosslinked collagen composite mesh discs. As human glioblastoma cells, commercially available luciferase-expressing human glioblastoma cell line U-251 MG-Luc cells were used. U-251 MG-Luc cells were seeded in a cell culture flask and cultured in EMEM containing 10% fetal bovine serum and 4 μg / mL puromycin at 37°C and 5% CO2. ２ Subculture was performed under atmospheric conditions. The subcultured U-251 MG-Luc cells were detached from the cell culture flask by trypsin treatment and suspended in the above EMEM medium, 1 × 10⁶ ６A suspension of U-251 MG-Luc cells at a concentration of cells / mL was prepared. 0.1 mL of the U-251 MG-Luc cell suspension was dropped onto the top surface of the sterile PLGA-crosslinked collagen composite mesh disc, seeded with 100,000 cells, and cultured in vitro for 6 hours. Then, the PLGA-crosslinked collagen composite mesh disc was inverted, another 0.1 mL of the U-251 MG-Luc cell suspension was dropped onto it, seeded with 100,000 cells, and cultured in vitro for 6 hours (200,000 cells were seeded on one PLGA-crosslinked collagen composite mesh disc). Subsequently, the cells were cultured in vitro for a further 60 hours.

[0278] Next, one sterile Ara-C-containing convenience gel 33 was sandwiched between two PLGA-crosslinked collagen composite mesh discs on which U-251 MG-Luc cells had been cultured, creating one set of implantation samples. One set of these implantation samples was implanted subcutaneously on the back of each nude mouse under general anesthesia. D-luciferin was injected subcutaneously at the implantation site 4 hours after implantation (day 0), and 1, 3, 5, 7, 9, and 11 days later, and in vivo chemiluminescence imaging was performed 5 minutes later. For comparison, a similar implantation experiment was performed using the sterile gel of Comparative Example 4. The results of in vivo chemiluminescence imaging are shown in Figure 40B. For each experimental condition of Ara-C-containing convenience gel 33 and Comparative Example 4, one nude mouse was used for each experiment.

[0279] Figure 40B shows the emission intensity in in vivo chemiluminescence imaging on a grayscale. The emission intensity changes from dark to light as the number of U-251 MG-Luc cells increases from areas with a high number to areas with a low number. Areas that appear particularly dark in the grayscale image can be judged as areas where many U-251 MG-Luc cells are present. According to Figure 40B, immediately after implantation (day 0) of Ara-C-containing convenience gel 33 and the gel of Comparative Example 4, luminescent regions indicating the presence of U-251 MG-Luc cells were observed at the implantation site of the mice in both cases. In chemiluminescence imaging of U-251 MG-Luc cells in mice implanted with the gel of Comparative Example 4, a strong chemiluminescence signal was observed even on day 11, indicating that the U-251 MG-Luc cells were still alive. In contrast, the chemiluminescence intensity of U-251 MG-Luc cells in mice implanted with Ara-C-containing convenience gel 33 decreased over time and disappeared by day 9. This suggests that the chemiluminescence signal disappeared because the U-251 MG-Luc cells present in the PLGA-crosslinked collagen composite mesh disc sandwiched with the Ara-C-containing convenience gel 33 died. In other words, it is thought that human glioblastoma cells present in the vicinity of the Ara-C-containing convenience gel 33 were killed by Ara-C released from the Ara-C-containing convenience gel 33. These results demonstrate that human glioblastoma cells can be effectively killed by Ara-C released from the Ara-C-containing convenience gel 33.

[0280] [Example 34] In Example 34, an aqueous sodium alginate solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a cyclophosphamide (CPA) / calcium chloride mixed aqueous solution was added to the freeze-dried product to prepare CPA-containing sodium alginate convenience gel 34 (hereinafter simply referred to as CPA-containing convenience gel 34). The killing effect of CPA-containing convenience gel 34 on human mammary cancer cells was confirmed by in vivo animal experiments.

[0281] First, a gelatin sponge was prepared using ice microparticles with a particle size of 250 μm to 350 μm as a pore-forming agent, and an 80 mg / mL gelatin acetic acid aqueous solution. Pure water was sprayed into liquid nitrogen in a container, and the mist-like water was frozen. Next, this frozen material was transferred to a low-temperature chamber at -15°C and left to stand for 2 to 2.5 hours to vaporize the liquid nitrogen. After that, the ice microparticles with a particle size of 250 μm to 350 μm were sieved out in the low-temperature chamber using a sieve with a mesh size of 350 μm and a sieve with a mesh size of 250 μm. The temperature in the low-temperature chamber was changed to -5°C, and these ice microparticles were left to stand for 3 hours.

[0282] On the other hand, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing an 80 mg / mL gelatin-acetic acid aqueous solution. This gelatin-acetic acid aqueous solution was placed in a low-temperature chamber (-5°C) and stirred for 30 minutes. Subsequently, in the low-temperature chamber (-5°C), the ice particles were added to the gelatin-acetic acid aqueous solution so that the ratio of gelatin-acetic acid aqueous solution to ice particles was 30 / 70 (mL / g), and the mixture was slowly stirred for 10 minutes to prepare a gelatin-acetic acid aqueous solution / ice particle mixture. This gelatin-acetic acid aqueous solution / ice particle mixture was placed in a container whose temperature had been equilibriumized in the low-temperature chamber (-5°C). After the container containing the gelatin-acetic acid aqueous solution / ice particle mixture was left to stand in the low-temperature chamber (-5°C) for 30 minutes, it was transferred to a deep freezer at -80°C and left to stand for 12 hours. The container was removed from the frozen material and transferred to a freeze-drying container that had been left to stand in a deep freezer at -80°C for 1 hour. Then, freeze-drying was performed in a freeze-dryer at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain freeze-dried gelatin sponge.

[0283] A cross-linked gelatin sponge was prepared by subjecting this freeze-dried gelatin sponge to the following three-step cross-linking reaction and treatment with an aqueous glycine solution. In the first cross-linking reaction step, 2-morpholinoethanesulfonic acid (MES) was dissolved in ethanol / water (95 / 5, v / v) to prepare a 0.1 M MES solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added to this MES solution and dissolved while stirring to prepare a first-step cross-linking reaction solution containing 50 mM WSCD·HCl and 20 mM NHS. The first-step cross-linking reaction was carried out by immersing the freeze-dried gelatin sponge in this first-step cross-linking reaction solution at room temperature for 12 hours.

[0284] Next, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the second crosslinking reaction solution in 30 mL of ethanol / water (90 / 10, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the first crosslinking reaction was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0285] Finally, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the third crosslinking reaction solution in 30 mL of ethanol / water (85 / 15, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the second crosslinking reaction was immersed in the third crosslinking reaction solution at room temperature for 12 hours. This carried out the third crosslinking reaction.

[0286] The gelatin sponge after the third cross-linking reaction was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. After that, it was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours. A cross-linked gelatin sponge was obtained by the above process.

[0287] On the other hand, sodium alginate was dissolved in ultrapure water to prepare a 25 mg / mL sodium alginate aqueous solution. The cross-linked gelatin sponge was cut into cylindrical shapes (8 mm in diameter, 3 mm in height), which were then immersed in the 25 mg / mL sodium alginate aqueous solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the sodium alginate aqueous solution. Subsequently, the sponges were frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to produce a cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product. This cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product was sterilized by ethylene oxide gas sterilization (EOG sterilization) to obtain a sterilized cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product.

[0288] Furthermore, CPA and calcium chloride were dissolved in ultrapure water, respectively, to prepare aqueous solutions of CPA and calcium chloride. The aqueous CPA solution was added to the aqueous calcium chloride solution and stirred to prepare a CPA / calcium chloride mixed aqueous solution with a CPA concentration of 4.1 mg / mL and a calcium chloride concentration of 3 mg / mL. This CPA / calcium chloride mixed aqueous solution was sterilized by passing it through a 0.22 μm filter (filtration sterilization method) to prepare a sterilized CPA / calcium chloride mixed aqueous solution. Next, the sterilized CPA / calcium chloride mixed aqueous solution was added to the sterilized cross-linked gelatin sponge cylinder / sodium alginate freeze-dried material to dissolve the sodium alginate and immediately gel, thereby producing a CPA-containing convenience gel 34. Because the supported gel was supported on a porous structure, it could be easily obtained in a short time when needed.

[0289] Furthermore, as a comparative example of the CPA-containing convenience gel 34, a gel without CPA (referred to as the gel of Comparative Example 5) was prepared. The preparation method was the same as that for the CPA-containing convenience gel 34, except that the CPA / calcium chloride mixed aqueous solution was replaced with a calcium chloride aqueous solution. Specifically, a 3 mg / mL calcium chloride aqueous solution that had been filtered and sterilized was added to the aforementioned sterilized cross-linked gelatin sponge cylinder / freeze-dried sodium alginate to dissolve the sodium alginate and immediately gel the mixture, thereby preparing the sterilized gel of Comparative Example 5.

[0290] Figure 41A shows photographs of the cross-linked gelatin sponge cylinder / freeze-dried sodium alginate, CPA-containing convenience gel 34, the pressurized CPA-containing convenience gel 34, and the lifted CPA-containing convenience gel 34. Since the CPA-containing convenience gel 34 remained intact without releasing water even when pressurized with a spatula or when lifted, it was found that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0291] The killing effect of CPA-containing Convenience Gel 34 on human mammary cancer cells was evaluated in animal experiments. To mimic the state in which human mammary cancer cells are present near Convenience Gel 34, Convenience Gel 34 was placed between PLGA-crosslinked collagen composite mesh discs in which human mammary cancer cells had been cultured and implanted subcutaneously into nude mice. First, the PLGA-crosslinked collagen composite mesh was prepared. As a preparation method, porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a 10 mg / mL collagen aqueous solution. In a low-temperature chamber at 4°C, the surface of a glass plate was covered with plastic wrap, and a rectangular piece of commercially available PLGA knit mesh (dimensions 80 mm x 55 mm) was placed on top, and the 10 mg / mL collagen aqueous solution was dropped onto it. The droplet was spread with a glass rod to ensure that the mixed solution evenly filled the voids of the PLGA knit mesh. Excess liquid was removed by rolling the glass rod. Next, the material was frozen by standing it at -80°C for 6 hours. The frozen material was freeze-dried at room temperature under reduced pressure of 5 Pa or less for 3 days to produce a PLGA-uncrosslinked collagen composite mesh. This PLGA-uncrosslinked collagen composite mesh was then subjected to the above three-step crosslinking reaction process and treatment with an aqueous glycine solution to produce a PLGA-crosslinked collagen composite mesh.

[0292] Next, the PLGA-crosslinked collagen composite mesh was cut into 8 mm diameter discs and sterilized with a 70 (v / v) ethanol aqueous solution to prepare sterile PLGA-crosslinked collagen composite mesh discs. As human mammary gland cancer cells, MDA-MB-231-Luc cells, a commercially available human mammary gland cancer cell line expressing luciferase, were used. First, MDA-MB-231-Luc cells were seeded in a cell culture flask and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum at 37°C and 5% CO2. ２ Subculture was performed under atmospheric conditions. The subcultured MDA-MB-231-Luc cells were detached from the cell culture flask by trypsin treatment and suspended in the above DMEM medium, 2 × 10⁻⁶ cells. ６A suspension of MDA-MB-231-Luc cells was prepared at a concentration of cells / mL. 0.1 mL of the MDA-MB-231-Luc cell suspension was dropped onto the top surface of the sterile PLGA-crosslinked collagen composite mesh disc, seeded with 200,000 cells, and cultured in vitro for 6 hours. Then, the PLGA-crosslinked collagen composite mesh disc was turned over, another 0.1 mL of the MDA-MB-231-Luc cell suspension was dropped onto it, seeded with another 200,000 cells, and cultured in vitro for 6 hours (i.e., a total of 400,000 cells were seeded on one PLGA-crosslinked collagen composite mesh disc). Subsequently, the cells were cultured in vitro for a further 12 hours.

[0293] Next, one sterile CPA-containing Convenience Gel 34 was sandwiched between two PLGA-crosslinked collagen composite mesh discs on which MDA-MB-231-Luc cells had been cultured, creating one set of implantation samples. One set of these implantation samples was implanted subcutaneously on the back of each nude mouse under general anesthesia. D-luciferin was injected subcutaneously at the implantation site 4 hours after implantation (day 0), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 days later, and in vivo chemiluminescence imaging was performed 5 minutes later. For comparison, a similar implantation experiment was performed using the sterile gel of Comparative Example 5. The results of in vivo chemiluminescence imaging are shown in Figure 41B. For each experimental condition of CPA-containing Convenience Gel 34 and Comparative Example 5, one nude mouse was used for each experiment.

[0294] Figure 41B shows the luminescence intensity in in vivo chemiluminescence imaging on a grayscale. The color changes from dark to light as the luminescence intensity decreases from high to low. Darker areas can be considered to be areas where many viable MDA-MB-231-Luc cells are present. According to Figure 41B, immediately after implantation (day 0) of the CPA-containing convenience gel 34 and the gel of Comparative Example 5, luminescence regions indicating the presence of MDA-MB-231-Luc cells were observed at the implantation site of the mice in both cases. In chemiluminescence imaging of MDA-MB-231-Luc cells in mice implanted with the gel of Comparative Example 5, a strong chemiluminescence signal was observed even on day 11, indicating that the MDA-MB-231-Luc cells were still viable. In contrast, the chemiluminescence signal of MDA-MB-231-Luc cells in mice implanted with CPA-containing convenience gel 34 decreased over time and disappeared by day 11. This is thought to be because the MDA-MB-231-Luc cells present in the PLGA-crosslinked collagen composite mesh disc sandwiched with the CPA-containing convenience gel 34 died, causing the chemiluminescence signal to disappear. In other words, it is thought that the CPA released from the CPA-containing convenience gel 34 killed human mammary cancer cells present in the vicinity of the CPA-containing convenience gel 34. These results demonstrate that CPA released from the CPA-containing convenience gel 34 can effectively kill human mammary cancer cells.

[0295] [Example 35] In Example 35, an aqueous sodium alginate solution was introduced into the pores and gaps of a cross-linked gelatin sponge, and after freezing and freeze-drying, a gemcitabine (GEM) / calcium chloride mixed aqueous solution was added to the freeze-dried product to prepare GEM-containing sodium alginate convenience gel 35 (hereinafter simply referred to as GEM-containing convenience gel 35). The killing effect of GEM-containing convenience gel 35 on human pancreatic cancer cells was confirmed by in vivo animal experiments.

[0296] First, a cross-linked gelatin sponge was prepared as a porous structure using ice microparticles with a particle size of 250 μm to 350 μm and a 60 mg / mL gelatin acetic acid aqueous solution. To control the pore structure of the gelatin sponge, ice microparticles with a particle size of 250 μm to 350 μm were used as a pore-forming agent. Pure water was sprayed into liquid nitrogen in a container, and the mist-like water was frozen. Next, this frozen material was transferred to a low-temperature chamber at -15°C and left to stand for 2 hours to evaporate the liquid nitrogen. After that, the ice microparticles with a particle size of 250 μm to 350 μm were sieved out in the low-temperature chamber using a sieve with a mesh size of 350 μm and a sieve with a mesh size of 250 μm. The temperature in the low-temperature chamber was changed to -5°C, and these ice microparticles were left to stand for 3 hours.

[0297] On the other hand, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing a 60 mg / mL gelatin-acetic acid aqueous solution. This gelatin-acetic acid aqueous solution was placed in a low-temperature chamber (-5°C) and stirred for 30 minutes. Subsequently, in the low-temperature chamber (-5°C), the ice particles were added to the gelatin-acetic acid aqueous solution to a ratio of 30 / 70 (mL / g) of gelatin-acetic acid aqueous solution / ice particles, and the mixture was slowly stirred for 10 minutes to prepare a gelatin-acetic acid aqueous solution / ice particle mixture. This gelatin-acetic acid aqueous solution / ice particle mixture was placed in a container whose temperature had been equilibriumized in the low-temperature chamber (-5°C). After the container containing the gelatin-acetic acid aqueous solution / ice particle mixture was left to stand in the low-temperature chamber (-5°C) for 30 minutes, it was transferred to a deep freezer at -80°C and left to stand for 12 hours. The container was removed from the frozen material and transferred to a freeze-drying container that had been left to stand in a deep freezer at -80°C for 1 hour. Then, freeze-drying was performed in a freeze-dryer at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain freeze-dried gelatin sponge.

[0298] A cross-linked gelatin sponge was prepared by sequentially performing a cross-linking reaction and treatment with an aqueous glycine solution in the following three steps. In the first cross-linking reaction step, 2-morpholinoethanesulfonic acid (MES) was dissolved in ethanol / water (95 / 5, v / v) to prepare a 0.1 M MES solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added to this MES solution and dissolved while stirring to prepare a first-stage cross-linking reaction solution containing 50 mM WSCD·HCl and 20 mM NHS. The first-stage cross-linking reaction was carried out by immersing the freeze-dried gelatin sponge in this first-stage cross-linking reaction solution at room temperature for 12 hours.

[0299] Next, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the second crosslinking reaction solution in 30 mL of ethanol / water (90 / 10, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the first crosslinking reaction was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0300] Finally, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the third crosslinking reaction solution in 30 mL of ethanol / water (85 / 15, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the second crosslinking reaction was immersed in the third crosslinking reaction solution at room temperature for 12 hours. This carried out the third crosslinking reaction.

[0301] The gelatin sponge after the third cross-linking reaction was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. A cross-linked gelatin sponge was obtained through the above process.

[0302] On the other hand, sodium alginate was dissolved in ultrapure water to prepare a 20 mg / mL sodium alginate aqueous solution. The cross-linked gelatin sponge was cut into cylindrical shapes (8 mm in diameter, 3 mm in height), which were then immersed in the 20 mg / mL sodium alginate aqueous solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the sodium alginate aqueous solution. Subsequently, the sponges were frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to produce a cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product. This cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product was sterilized by ethylene oxide gas sterilization (EOG sterilization) to obtain a sterilized cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product.

[0303] Next, GEM and calcium chloride were dissolved in ultrapure water to prepare GEM aqueous solution and calcium chloride aqueous solution. The GEM aqueous solution was added to the calcium chloride aqueous solution and stirred to prepare a GEM / calcium chloride mixed aqueous solution with a GEM concentration of 3.4 mg / mL and a calcium chloride concentration of 2.5 mg / mL. This GEM / calcium chloride mixed aqueous solution was sterilized by passing it through a 0.22 μm filter (filtration sterilization method) to prepare a sterilized GEM / calcium chloride mixed aqueous solution. Furthermore, the sterilized GEM / calcium chloride mixed aqueous solution was added to the sterilized cross-linked gelatin sponge cylinder / sodium alginate freeze-dried material to dissolve the sodium alginate and immediately gel it, thereby producing a sterilized GEM-containing convenience gel 35. The formed convenience gel 35, supported on a porous structure, can be easily formed in a short time when needed.

[0304] Furthermore, as a comparative example of GEM-containing convenience gel 35, a gel without GEM (referred to as the gel of Comparative Example 6) was prepared. The preparation method was the same as that for GEM-containing convenience gel 35, except that the GEM / calcium chloride mixed aqueous solution was replaced with a calcium chloride aqueous solution. Specifically, the cross-linked gelatin sponge was cut into cylindrical shapes (8 mm in diameter, 3 mm in height), immersed in a 20 mg / mL sodium alginate aqueous solution, and the pores and gaps of the sponge were filled with the sodium alginate aqueous solution by applying reduced pressure. Subsequently, it was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to prepare a cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product. This cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product was sterilized by EOG sterilization to obtain a sterilized cross-linked gelatin sponge cylindrical / sodium alginate freeze-dried product. Furthermore, calcium chloride was dissolved in ultrapure water to prepare a 2.5 mg / mL aqueous calcium chloride solution. This aqueous calcium chloride solution was filtered and sterilized to prepare a sterile aqueous calcium chloride solution. In addition, the sterile aqueous calcium chloride solution was added to the sterile cross-linked gelatin sponge cylinder / freeze-dried sodium alginate to dissolve the sodium alginate and immediately gel it, thereby producing a sterile gel of Comparative Example 6.

[0305] Figure 42A shows photographs of the cross-linked gelatin sponge cylinder / freeze-dried sodium alginate, GEM-containing convenience gel 35, the pressurized GEM-containing convenience gel 35, and the lifted GEM-containing convenience gel 35. Since the GEM-containing convenience gel 35 remained intact without releasing water even when pressurized with a spatula or when lifted, it was found that gel had formed in the pores and gaps of the cross-linked gelatin sponge.

[0306] The killing effect of GEM-containing Convenience Gel 35 on human pancreatic cancer cells was evaluated in animal experiments. To mimic the state in which human pancreatic cancer cells are present near Convenience Gel 35, Convenience Gel 35 was placed between PLGA-crosslinked collagen composite mesh discs in which human pancreatic cancer cells had been cultured and implanted subcutaneously into nude mice. First, the PLGA-crosslinked collagen composite mesh was prepared. As a preparation method, porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a 10 mg / mL collagen aqueous solution. In a low-temperature chamber at 4°C, the surface of a glass plate was covered with plastic wrap, and a rectangular piece of commercially available PLGA knit mesh (dimensions 80 mm x 55 mm) was placed on top of it, and the 10 mg / mL collagen aqueous solution was dropped onto it. The droplet was spread with a glass rod to evenly fill the voids of the PLGA knit mesh. Excess liquid was removed by rolling the glass rod. Next, the material was frozen by standing at -80°C for 6 hours. A PLGA-uncrosslinked collagen composite mesh was prepared by freeze-drying at room temperature under reduced pressure of 5 Pa or less for 72 hours. This PLGA-uncrosslinked collagen composite mesh was then subjected to the above three-step crosslinking process and treatment with a glycine aqueous solution to prepare a PLGA-crosslinked collagen composite mesh.

[0307] Next, the PLGA-crosslinked collagen composite mesh was cut into 8 mm diameter discs and sterilized with a 70 (v / v) ethanol aqueous solution to prepare sterile PLGA-crosslinked collagen composite mesh discs. As human pancreatic cancer cells, PaCa-2 / CMV-Luc cells, a commercially available human pancreatic cancer cell line expressing luciferase, were used. PaCa-2 / CMV-Luc cells were seeded in a cell culture flask and cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum at 37°C and 5% CO2. ２ Subculture was performed under atmospheric conditions. The subcultured PaCa-2 / CMV-Luc cells were detached from the cell culture flask by trypsin treatment and suspended in the above EMEM medium, 2 × 10⁻⁶ cells. ６A suspension of PaCa-2 / CMV-Luc cells was prepared at a concentration of cells / mL. 200,000 cells were seeded by dropping 0.1 mL of the PaCa-2 / CMV-Luc cell suspension onto the top surface of the sterile PLGA-crosslinked collagen composite mesh disc, and the cells were cultured in vitro for 6 hours. Subsequently, the PLGA-crosslinked collagen composite mesh disc was inverted, and another 200,000 cells were seeded by dropping 0.1 mL of the PaCa-2 / CMV-Luc cell suspension onto the top surface, and the cells were cultured in vitro for 6 hours (400,000 cells were seeded on one PLGA-crosslinked collagen composite mesh disc). The cells were then cultured in vitro for a further 12 hours.

[0308] Next, one sterile GEM-containing Convenience Gel 35 was sandwiched between two PLGA-crosslinked collagen composite mesh discs on which PaCa-2 / CMV-Luc cells had been cultured, creating one set of implantation samples. One set of these implantation samples was implanted subcutaneously on the back of each nude mouse under general anesthesia. Four hours after implantation (day 0), and at 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, and 14 days later, D-luciferin, a reagent that causes luciferase gene-expressing cancer cells to emit light, was injected subcutaneously at the implantation site, and in vivo chemiluminescence imaging was performed 5 minutes later. For comparison, a similar implantation experiment was performed using the sterile gel of Comparative Example 6. The results of in vivo chemiluminescence imaging are shown in Figure 42B. For each experimental condition of GEM-containing Convenience Gel 35 and Comparative Example 6, one nude mouse was used for each experiment.

[0309] Figure 42B shows the luminescence intensity in in vivo chemiluminescence imaging on a grayscale. That is, in the chemiluminescence image, the area changes from a dark gray region to a light gray region, corresponding to a region with a high number of PaCa-2 / CMV-Luc cells and a region with a low number of PaCa-2 / CMV-Luc cells. As shown in Figure 42B, immediately after implantation (day 0) of the GEM-containing convenience gel 35 and the gel of Comparative Example 6, luminescence regions indicating the presence of PaCa-2 / CMV-Luc cells were observed at the implantation site in both cases. Fourteen days after implantation, a strong chemiluminescence signal was observed in the chemiluminescence imaging of mice implanted with the gel of Comparative Example 6, indicating that the PaCa-2 / CMV-Luc cells were still alive. In contrast, in the chemiluminescence imaging of mice implanted with the GEM-containing convenience gel 35, the intensity of the chemiluminescence signal decreased with implantation time and disappeared by day 14. This suggests that PaCa-2 / CMV-Luc cells present in the PLGA-crosslinked collagen composite mesh disc sandwiched between the GEM-containing convenience gel 35 were killed. In other words, it is thought that the GEM released from the GEM-containing convenience gel 35 killed human pancreatic cancer cells present in the vicinity of the GEM-containing convenience gel 35. These results demonstrate that the GEM released from the GEM-containing convenience gel 35 can effectively kill human pancreatic cancer cells.

[0310] [Example 36] In Example 36, a mixed aqueous solution of docetaxel (DTX) and sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge. After freezing and freeze-drying, an aqueous solution of calcium chloride was added to the freeze-dried product to prepare DTX-containing sodium alginate 36 (hereinafter simply referred to as DTX-containing convenience gel 36). The killing effect of DTX-containing convenience gel 36 on human gastric cancer cells was confirmed by in vivo animal experiments.

[0311] First, a gelatin sponge was prepared using ice microparticles with a particle size of 250 μm to 350 μm as a pore-forming agent, and an 80 mg / mL gelatin acetic acid aqueous solution. Pure water was sprayed into liquid nitrogen in a container, and the mist-like water was frozen. Next, this frozen material was transferred to a low-temperature chamber at -15°C and left to stand for 2 to 2.5 hours to vaporize the liquid nitrogen. After that, the ice microparticles with a particle size of 250 μm to 350 μm were sieved out in the low-temperature chamber using a sieve with a mesh size of 350 μm and a sieve with a mesh size of 250 μm. The temperature in the low-temperature chamber was changed to -5°C, and these ice microparticles were left to stand for 3 hours.

[0312] On the other hand, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing an 80 mg / mL gelatin-acetic acid aqueous solution. This gelatin-acetic acid aqueous solution was placed in a low-temperature chamber (-5°C) and stirred for 30 minutes. Subsequently, in the low-temperature chamber (-5°C), the ice particles were added to the gelatin-acetic acid aqueous solution so that the ratio was 30 / 70 (volume mL to weight g), and the mixture was slowly stirred for 10 minutes to prepare a gelatin-acetic acid aqueous solution / ice particle mixture. This gelatin-acetic acid aqueous solution / ice particle mixture was placed in a container whose temperature had been equilibriumized in the low-temperature chamber (-5°C). After the container containing the gelatin-acetic acid aqueous solution / ice particle mixture was left to stand in the low-temperature chamber (-5°C) for 30 minutes, it was transferred to a deep freezer at -80°C and left to stand for 12 hours. The container was removed from the frozen material and transferred to a freeze-drying container that had been left to stand in a deep freezer at -80°C for 1 hour. Then, freeze-drying was performed in a freeze-dryer at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain freeze-dried gelatin sponge.

[0313] A cross-linked gelatin sponge was prepared by subjecting this freeze-dried gelatin sponge to the following three-step cross-linking reaction and treatment with an aqueous glycine solution. In the first cross-linking reaction step, 2-morpholinoethanesulfonic acid (MES) was dissolved in ethanol / water (95 / 5, v / v) to prepare a 0.1 M MES solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added to this MES solution and dissolved while stirring to prepare a first-step cross-linking reaction solution containing 50 mM WSCD·HCl and 20 mM NHS. The first-step cross-linking reaction was carried out by immersing the freeze-dried gelatin sponge in this first-step cross-linking reaction solution at room temperature for 12 hours.

[0314] Next, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the second crosslinking reaction solution in 30 mL of ethanol / water (90 / 10, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the first crosslinking reaction was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0315] Finally, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the third crosslinking reaction solution in 30 mL of ethanol / water (85 / 15, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the second crosslinking reaction was immersed in the third crosslinking reaction solution at room temperature for 12 hours. This carried out the third crosslinking reaction.

[0316] The gelatin sponge after the third cross-linking reaction was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. After that, it was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours. A cross-linked gelatin sponge was obtained by the above process.

[0317] On the other hand, DTX was dissolved in ethanol to prepare a DTX ethanol solution. Sodium alginate was dissolved in ultrapure water to prepare an aqueous sodium alginate solution. The DTX ethanol solution was added to this aqueous sodium alginate solution and stirred to prepare a DTX / sodium alginate mixed solution consisting of 0.002 mg / mL of DTX and 25 mg / mL of sodium alginate. Subsequently, the cross-linked gelatin sponge was cut into cylindrical shapes (8 mm in diameter, 3 mm in height) and immersed in the DTX / sodium alginate mixed solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the DTX / sodium alginate mixed solution. Subsequently, the sponge was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to produce a cross-linked gelatin sponge cylindrical / sodium alginate / DTX freeze-dried product. This cross-linked gelatin sponge cylinder / sodium alginate / DTX freeze-dried material was sterilized by ethylene oxide gas sterilization (EOG sterilization) to obtain sterilized cross-linked gelatin sponge cylinder / sodium alginate / DTX freeze-dried material.

[0318] Furthermore, calcium chloride was dissolved in ultrapure water to prepare a 3 mg / mL aqueous calcium chloride solution. This aqueous calcium chloride solution was sterilized by passing it through a 0.22 μm filter (filtration sterilization method) to prepare a sterile aqueous calcium chloride solution. In addition, by adding the sterile aqueous calcium chloride solution to the sterile cross-linked gelatin sponge cylinder / sodium alginate / DTX freeze-dried material, the sodium alginate was dissolved and immediately gelled, thereby producing a sterile DTX-containing convenience gel 36. Because the supported gel was supported on a porous structure, it could be easily obtained in a short time when needed.

[0319] Furthermore, as a comparative example of DTX-containing convenience gel 36, a DTX-free gel (referred to as comparative example 7 gel) was prepared. The preparation method was the same as that for DTX-containing convenience gel 36, except that the DTX / sodium alginate mixed solution was replaced with an aqueous sodium alginate solution. Specifically, a cross-linked gelatin sponge cylinder / sodium alginate freeze-dried product was prepared using a 25 mg / mL aqueous sodium alginate solution and sterilized with ethylene oxide gas. By adding the aforementioned sterilized 3 mg / mL aqueous calcium chloride solution to this sterilized freeze-dried product, the sodium alginate was dissolved and immediately gelled, thereby preparing the sterilized comparative example 7 gel.

[0320] Figure 43A shows photographs of the cross-linked gelatin sponge cylinder / sodium alginate / DTX freeze-dried product, DTX-containing convenience gel 36, the pressurized DTX-containing convenience gel 36, and the lifted DTX-containing convenience gel 36. Since the DTX-containing convenience gel 36 remained intact without releasing water even when pressurized with a spatula or when lifted, it was found that the gel was formed in the pores and gaps of the cross-linked gelatin sponge.

[0321] The killing effect of DTX-containing Convenience Gel 36 on human gastric cancer cells was evaluated in animal experiments. To mimic the state in which human gastric cancer cells are present near Convenience Gel 36, Convenience Gel 36 was placed on a PLGA-crosslinked collagen composite mesh disc in which human gastric cancer cells had been cultured, and implanted subcutaneously into nude mice. First, the PLGA-crosslinked collagen composite mesh was prepared. As a preparation method, porcine type I atelocollagen was dissolved in dilute hydrochloric acid (pH 3.0) at 4°C to prepare a 10 mg / mL collagen aqueous solution. In a low-temperature chamber at 4°C, the surface of a glass plate was covered with plastic wrap, and a rectangular piece of commercially available PLGA knit mesh (dimensions 80 mm x 55 mm) was placed on top, and the 10 mg / mL collagen aqueous solution was dropped onto it. The droplet was spread with a glass rod to ensure that the mixed solution evenly filled the voids of the PLGA knit mesh. Excess liquid was removed by rolling the glass rod. Next, the material was frozen by standing it at -80°C for 6 hours. The frozen material was freeze-dried at room temperature under reduced pressure of 5 Pa or less for 3 days to produce a PLGA-uncrosslinked collagen composite mesh. This PLGA-uncrosslinked collagen composite mesh was then subjected to the above three-step crosslinking reaction process and treatment with an aqueous glycine solution to produce a PLGA-crosslinked collagen composite mesh.

[0322] Next, the aforementioned PLGA-crosslinked collagen composite mesh was cut into 8 mm diameter discs and sterilized with a 70 (v / v) ethanol aqueous solution to prepare sterile PLGA-crosslinked collagen composite mesh discs. As human gastric cancer cells, MKN-7 / CMV-Luc cells, a commercially available human gastric cancer cell line expressing luciferase, were used. First, MKN-7 / CMV-Luc cells were seeded in a cell culture flask and cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. ２ Subculture was performed under atmospheric conditions. The subcultured MKN-7 / CMV-Luc cells were detached from the cell culture flask by trypsin treatment and suspended in RPMI1640 medium containing 10% fetal bovine serum as described above, in a 2 × 10⁶ solution. ６A suspension of MKN-7 / CMV-Luc cells was prepared at a concentration of cells / mL. 0.1 mL of the MKN-7 / CMV-Luc cell suspension was dropped onto the top surface of the sterile PLGA-crosslinked collagen composite mesh disc, seeded with 200,000 cells, and cultured in vitro for 6 hours. Then, the PLGA-crosslinked collagen composite mesh disc was inverted, and another 0.1 mL of the MKN-7 / CMV-Luc cell suspension was dropped onto it, seeded with another 200,000 cells, and cultured in vitro for 6 hours (i.e., a total of 400,000 cells were seeded on one PLGA-crosslinked collagen composite mesh disc). Subsequently, the cells were cultured in vitro for a further 12 hours.

[0323] Next, one sterile DTX-containing Convenience Gel 36 was sandwiched between two PLGA-crosslinked collagen composite mesh discs on which MKN-7 / CMV-Luc cells had been cultured, creating one set of implantation samples. One set of these implantation samples was implanted subcutaneously on the back of each nude mouse under general anesthesia. D-luciferin was injected subcutaneously at the implantation site at 4 hours (day 0), and at 1, 3, 5, 7, 8, 9, 10, and 11 days after implantation, and in vivo chemiluminescence imaging was performed 5 minutes later. For comparison, a similar implantation experiment was performed using the sterile gel of Comparative Example 7. The results of in vivo chemiluminescence imaging are shown in Figure 43B. For each experimental condition of DTX-containing Convenience Gel 36 and Comparative Example 7, one nude mouse was used for each experiment.

[0324] Figure 43B shows the luminescence intensity in in vivo chemiluminescence imaging on a grayscale. The color changes from dark to light as the luminescence intensity decreases from high to low. Darker areas can be considered to be areas where many viable MKN-7 / CMV-Luc cells are present. According to Figure 43B, immediately after implantation (day 0) of the DTX-containing Convenience Gel 36 and the gel of Comparative Example 7, luminescence regions indicating the presence of MKN-7 / CMV-Luc cells were observed at the implantation site of the mice in both cases. In chemiluminescence imaging of MKN-7 / CMV-Luc cells in mice implanted with the gel of Comparative Example 7, a strong chemiluminescence signal was observed even on day 11, indicating that the MKN-7 / CMV-Luc cells were still viable. In contrast, the chemiluminescence signal of MKN-7 / CMV-Luc cells in mice implanted with DTX-containing Convenience Gel 36 decreased over time and disappeared by day 11. This suggests that the chemiluminescence signal disappeared because the MKN-7 / CMV-Luc cells present in the PLGA-crosslinked collagen composite mesh disc sandwiched between the DTX-containing convenience gel 36 were killed. In other words, it is thought that the DTX released from the DTX-containing convenience gel 36 killed human gastric cancer cells in the vicinity of the DTX-containing convenience gel 36. These results demonstrate that DTX released from the DTX-containing convenience gel 36 can effectively kill human gastric cancer cells.

[0325] [Example 37] In Example 37, a mixed solution of fluorouracil (5-FU) and sodium alginate was introduced into the pores and gaps of a cross-linked gelatin sponge. After freezing and freeze-drying, an aqueous calcium chloride solution was added to the freeze-dried product to prepare 5-FU-containing sodium alginate convenience gel 37 (hereinafter simply referred to as 5-FU-containing convenience gel 37). The killing effect of 5-FU-containing convenience gel 37 on human liver cancer cells was confirmed by in vivo animal experiments.

[0326] First, a cross-linked gelatin sponge was prepared as a porous structure using ice microparticles with a particle size of 150 μm to 250 μm and a 40 mg / mL gelatin acetic acid aqueous solution. To control the pore structure of the gelatin sponge, ice microparticles with a particle size of 150 μm to 250 μm were used as a pore-forming agent. Pure water was sprayed into liquid nitrogen in a container, and the mist-like water was frozen. Next, this frozen material was transferred to a low-temperature chamber at -15°C and left to stand for 2 hours to evaporate the liquid nitrogen. After that, the ice microparticles with a particle size of 150 μm to 250 μm were sieved out in the low-temperature chamber using a sieve with a mesh size of 250 μm and a sieve with a mesh size of 150 μm. The temperature in the low-temperature chamber was changed to -5°C, and these ice microparticles were left to stand for 3 hours.

[0327] On the other hand, 35 (v / v)% acetic acid was added to porcine-derived gelatin powder, stirred at 45°C for 1 hour, and then stirred at room temperature for 4 hours to dissolve the gelatin, preparing a 40 mg / mL gelatin-acetic acid aqueous solution. This gelatin-acetic acid aqueous solution was placed in a low-temperature chamber (-5°C) and stirred for 30 minutes. Subsequently, in the low-temperature chamber (-5°C), the ice particles with a particle size of 150 μm to 250 μm were added to the gelatin-acetic acid aqueous solution so that the ratio of ice particles to the gelatin-acetic acid aqueous solution was 30 / 70 (v / w) in volume mL to weight g, and the mixture was slowly stirred for 10 minutes to prepare a gelatin-acetic acid aqueous solution / ice particle mixture. This gelatin-acetic acid aqueous solution / ice particle mixture was placed in a container whose temperature was equilibriumized in the low-temperature chamber (-5°C). After the container containing the gelatin-acetic acid aqueous solution / ice particle mixture was left to stand in the low-temperature chamber (-5°C) for 30 minutes, it was transferred to a deep freezer at -80°C and left to stand for 12 hours. The container was removed from the frozen material and transferred to a freeze-drying container that had been left to stand in a deep freezer at -80°C for 1 hour. Then, freeze-drying was performed in a freeze-dryer at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain freeze-dried gelatin sponge.

[0328] A cross-linked gelatin sponge was prepared by sequentially performing a cross-linking reaction and treatment with an aqueous glycine solution in the following three steps. In the first cross-linking reaction step, 2-morpholinoethanesulfonic acid (MES) was dissolved in ethanol / water (95 / 5, v / v) to prepare a 0.1 M MES solution. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSCD·HCl) and N-hydroxysuccinimide (NHS) were added to this MES solution and dissolved while stirring to prepare a first-stage cross-linking reaction solution containing 50 mM WSCD·HCl and 20 mM NHS. The first-stage cross-linking reaction was carried out by immersing the freeze-dried gelatin sponge in this first-stage cross-linking reaction solution at room temperature for 12 hours.

[0329] Next, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the second crosslinking reaction solution in 30 mL of ethanol / water (90 / 10, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the first crosslinking reaction was immersed in this second crosslinking reaction solution at room temperature for 12 hours. This carried out the second crosslinking reaction.

[0330] Finally, a 0.1 M MES solution containing 50 mM WSCD·HCl and 20 mM NHS was prepared as the third crosslinking reaction solution in 30 mL of ethanol / water (85 / 15, v / v), similar to the first crosslinking reaction solution. The gelatin sponge after the second crosslinking reaction was immersed in the third crosslinking reaction solution at room temperature for 12 hours. This carried out the third crosslinking reaction.

[0331] The gelatin sponge after the third cross-linking reaction was immersed in ultrapure water at room temperature and washed while shaking for 30 minutes. This washing was repeated a total of three times. Subsequently, it was immersed in a 0.1 M glycine aqueous solution at room temperature for 12 hours, and then immersed in ultrapure water at room temperature and washed three times while shaking for 30 minutes. A cross-linked gelatin sponge was obtained by the above process. This cross-linked gelatin sponge was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to obtain a freeze-dried cross-linked gelatin sponge.

[0332] On the other hand, sodium alginate was dissolved in ultrapure water to prepare an aqueous sodium alginate solution. 5-FU was dissolved in DMSO to prepare a DMSO solution of 5-FU. The DMSO solution of 5-FU was added to this aqueous sodium alginate solution and stirred to prepare a 5-FU / sodium alginate mixed solution with a 5-FU concentration of 5 mg / mL and a sodium alginate concentration of 20 mg / mL. The lyophilized cross-linked gelatin sponge was cut into cylindrical shapes (8 mm in diameter, 3 mm in height) and immersed in the 5-FU / sodium alginate mixed solution. By applying reduced pressure, the pores and gaps in the sponge were filled with the 5-FU / sodium alginate mixed solution. Subsequently, the sponge was frozen in a deep freezer at -80°C for 12 hours, and then freeze-dried at room temperature under reduced pressure of 5 Pa or less for 48 hours to produce a lyophilized cross-linked gelatin sponge cylindrical / sodium alginate / 5-FU lyophilized product. This cross-linked gelatin sponge cylinder / sodium alginate / 5-FU freeze-dried material was sterilized by ethylene oxide gas sterilization (EOG sterilization) to obtain sterilized cross-linked gelatin sponge cylinder / sodium alginate / 5-FU freeze-dried material.

[0333] Furthermore, calcium chloride was dissolved in ultrapure water to prepare a 2.5 mg / mL calcium chloride aqueous solution. This calcium chloride aqueous solution was sterilized by passing it through a filter with a pore size of 0.22 μm (filtration sterilization method) to prepare a sterilized calcium chloride aqueous solution. In addition, by adding the sterilized calcium chloride aqueous solution to the sterilized cross-linked gelatin sponge cylinder / sodium alginate / 5-FU freeze-dried material, the sodium alginate was dissolved and immediately gelled, thereby producing a sterilized 5-FU-containing convenience gel 37. The formed convenience gel 37, being supported on a porous structure, can be easily formed in a short time when needed.

[0334] Furthermore, as a comparative example of the 5-FU-containing convenience gel 37, a gel without 5-FU (referred to as the gel of Comparative Example 8) ...

Claims

1. A method for producing an additive-containing gel structure, comprising: preparing a porous structure; introducing a solution containing gel raw materials into the internal space of the porous structure; producing a freeze-dried product of the porous structure in which the solution containing the gel raw materials is held in the internal space; and supplying a gelling liquid to the freeze-dried product to gel the gel raw materials, wherein at least one of the solution containing the gel raw materials and the gelling liquid contains one or more additives.

2. The method for producing an additive-containing gel structure according to claim 1, wherein the form of the porous structure is one of a sponge, a mesh, or a nonwoven fabric.

3. The method for producing an additive-containing gel structure according to claim 1 or 2, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.

4. A method for producing an additive-containing gel structure according to any one of claims 1 to 3, wherein the additive is of one type.

5. A method for producing an additive-containing gel structure according to any one of claims 1 to 3, wherein the additive is of two or more types.

6. The method for producing an additive-containing gel structure according to claim 5, wherein the solution containing the gel raw material contains a first additive, and the gelling solution contains a second additive different from the first additive.

7. A method for producing an additive-containing gel structure according to any one of claims 1 to 6, wherein the gel raw material is a polymer.

8. The method for producing an additive-containing gel structure according to claim 7, wherein the polymer is one or more selected from the group consisting of alginic acid and its sodium salt, collagen, gelatin, fibronectin, laminin, elastin, fibrin, polyamino acids, dextrin, cellulose, cellulose acetate, cellulose diacetate, cellulose nitrate, agarose, amylose, starch, hyaluronic acid, chondroitin sulfate, DNA, RNA, chitin, chitosan, natural rubber, regenerated fiber rayon, celluloid, polyurethane, polyvinyl alcohol, polyethylene glycol, and sulfur nitride polymer.

9. A method for producing an additive-containing gel structure according to any one of claims 1 to 8, wherein the additive is an anticancer agent.

10. The method for producing an additive-containing gel structure according to claim 9, wherein the anticancer agent is a drug used to treat one or more cancers selected from the group consisting of colorectal cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, tongue cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, spleen cancer, cervical cancer, chronic myeloid leukemia, malignant melanoma, brain tumor, osteosarcoma, chondrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma.

11. An additive-containing gel structure in which a gel containing an additive is formed in the internal space of a porous structure.

12. The additive-containing gel structure according to claim 11, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.

13. The additive-containing gel structure according to claim 11, wherein the additive is an anticancer agent.

14. The additive-containing gel structure according to claim 13, wherein the anticancer agent is a drug used to treat one or more cancers selected from the group consisting of colorectal cancer, rectal cancer, gastric cancer, esophageal cancer, oral cancer, tongue cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, kidney cancer, spleen cancer, cervical cancer, chronic myeloid leukemia, malignant melanoma, brain tumor, osteosarcoma, chondrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, myxofibrosarcoma, and leiomyosarcoma.

15. A precursor for obtaining the additive-containing gel structure described in claim 11, which is a freeze-dried product of a porous structure in which gel raw materials and additives are supported in the internal space, the precursor for the additive-containing gel structure.

16. The precursor of the additive-containing gel structure according to claim 15, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.

17. A method for producing a precursor of an additive-containing gel structure, comprising: preparing a porous structure; introducing a solution containing gel raw materials and an additive into the internal space of the porous structure; and producing a freeze-dried product of the porous structure in which the solution is retained in the internal space.

18. A method for producing a precursor of an additive-containing gel structure according to claim 17, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.

19. A kit for preparing an additive-containing gel structure, comprising a main component containing a precursor of a gel structure and an auxiliary component containing a gelling liquid, wherein the precursor is a freeze-dried product of a porous structure on which a gel raw material is supported, the gelling liquid is capable of gelling the gel raw material, and at least one of the freeze-dried porous structure and the gelling liquid contains an additive.

20. The additive-containing gel structure preparation kit according to claim 19, wherein the additive consists of one or more selected from the group consisting of pharmaceuticals and nanomaterials.

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