Powder containing particles of gelatin derivative and application of same in medical treatment

Abstract

The present invention provides a powder which is capable of forming a tissue covering material that is capable of exhibiting a further function while having high adhesive strength to living tissues.　The powder includes first particles that each contain a crosslinked gelatin derivative, and second particles that are different from the first particles and can provide a further function. The gelatin derivative has a structure represented by formula (1). In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, R1 represents a hydrocarbon group having 1 to 20 carbon atoms, and R2 represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.

Description

Powder containing particles of gelatin derivatives and its medical applications

[0001] The present invention relates to powders containing particles of gelatin derivatives and their medical applications.

[0002] Gelatin is used in various medical applications due to its excellent biocompatibility and biodegradability. In Patent Document 1, the present inventors have reported particles of a crosslinked gelatin derivative suitable for wound dressings, adhesion barriers, hemostatic agents, etc. The particles can be applied to biological tissues, for example, by spraying, where they gel and adhere to the tissue, functioning as a tissue dressing.

[0003] WO2020 / 137903 publication

[0004] The crosslinked gelatin derivative particles of Patent Document 1 exhibit excellent adhesiveness to biological tissues as tissue dressings. If additional functions can be imparted to the tissue dressing while maintaining this adhesiveness, it is expected that the tissue dressing will be applicable to a wider range of medical uses. Furthermore, it would be even more desirable if the adhesive strength to biological tissues could be further increased.

[0005] The present invention solves the above-mentioned problems. That is, in one embodiment, the present invention provides a powder capable of forming a tissue dressing that has high adhesive strength to biological tissue and can also exhibit additional functions. In another embodiment, the present invention provides a tissue dressing that has even higher adhesive strength to biological tissue.

[0006] As a result of extensive research into achieving the above object, the present inventors provide the following powder and its medical application.

[0007] [1] A powder comprising first particles containing a crosslinked gelatin derivative and second particles that are separate entities from the first particles and can provide an additional function, wherein the gelatin derivative has a structure represented by formula (1) described below. [2] The powder according to [1], wherein the second particles contain or are a particulate drug. [3] The powder according to [2], wherein the drug is hydrophobic or positively or negatively charged. [4] The powder according to any one of [1] to [3], wherein the ratio (W2 / W1) of the mass of the second particles (W2) to the mass of the first particles (W1) is 0.1 / 50 to 60 / 50. [5] The powder according to [4], wherein the ratio (W2 / W1) of the mass of the second particles (W2) to the mass of the first particles (W1) is 0.1 / 50 to 40 / 50. [6] The powder according to [4], wherein the ratio (W2 / W1) of the mass of the second particles (W2) to the mass of the first particles (W1) is 1 / 50 to 60 / 50. [7] The powder according to any one of [1] to [6], wherein the average particle size of the first particles is 0.1 μm to 100 μm. [8] The powder according to any one of [1] to [7], wherein the average particle size of the second particles is 5 nm to 100 μm. [9] The powder according to [8], wherein the average particle size of the second particles is 10 nm to 100 nm.

[10] The powder according to any one of [1] to [9], wherein the ratio (D2 / D1) of the average particle size of the second particles to the average particle size of the first particles is 0.001 to 0.1.

[11] The powder according to any one of [1] to

[10] , wherein the water contact angle is 50 degrees or more when measured 1 second after water is dropped onto the powder.

[12] The powder according to any one of [2] to

[11] , wherein the drug is at least one selected from the group consisting of an anticancer drug, an immunosuppressant, a growth factor, a cell differentiation inducer, an antimetabolite, a growth factor inhibitor, an immune checkpoint inhibitor, an antibiotic, an anti-inflammatory agent, and a protein preparation.

[13] The powder according to

[12] , wherein the drug includes at least one selected from the group consisting of a hydrophobic anticancer drug, a hydrophobic immunosuppressant, and a hydrophobic cell differentiation inducer.

[14] The powder according to

[13] , wherein the crystallinity of the hydrophobic anticancer drug is 30 to 50% as determined by crystal structure analysis by XRD.

[15] The powder according to

[13] or

[14] , wherein the hydrophobic anticancer drug is selected from the group consisting of cabazitaxel, paclitaxel, docetaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, tamoxifen, anastrozole, Gleevec, 5-fluorouracil (5-FU), floxuridine, leuprolide, flutamide, zoledronate, doxorubicin, vincristine, gemcitabine, streptozocin, vinorelbine, retinoic acid series drugs, adriamycin, mitomycin, daunomycin, prednisone, testosterone, mitoxantrone, aspirin, salicylic acid, ibuprofen, naproxen, fenoprofen, indomethacin, phenylalanine, cyclophosphamide, celecoxib, valdecoxib, and nimesulide.

[16] The powder according to

[14] , wherein the hydrophobic immunosuppressant is selected from the group consisting of steroids (e.g., hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, and betamethasone), cyclosporine, mizoribine (Bredinin), cyclophosphamide (Endoxan), azathioprine (Azanin), tacrolimus (FK506), sirolimus (rapamycin), deoxyspergualin (gusperimus hydrochloride), everolimus, ABT-578 (zotarolimus), basiliximab (anti-IL-2 receptor monoclonal antibody), muromonab CD3 (anti-CD3 monoclonal antibody), and albulin (antilymphocyte globulin).

[17] The powder according to

[14] , wherein the hydrophobic cell differentiation inducer is tamibarotene.

[18] The powder according to

[12] , wherein the antimetabolite is pemetrexed.

[19] The powder according to

[12] , wherein the growth factor inhibitor is bevacizumab.

[20] The powder according to

[12] , wherein the immune checkpoint inhibitor is nivolumab.

[21] The powder according to any one of [2] to

[12] , wherein the drug includes a growth factor and the powder further includes a cationic polymer.

[22] The powder according to

[21] , wherein the growth factor is selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), and Wnt7b.

[23] The powder according to any one of [1] to

[22] , wherein the second particles comprise inorganic fine particles.

[24] The powder according to

[23] , wherein the second particles comprise magnetic particles and an anticancer drug, preferably a hydrophobic anticancer drug.

[25] The powder according to

[23] , wherein the second particles comprise calcium phosphate and a growth factor.

[26] A tissue dressing material comprising the powder according to any one of [1] to

[25] .

[27] A cancer-killing material comprising the powder according to

[24] .

[28] The cancer-killing material according to

[27] , wherein the magnetic particles are iron oxide.

[29] A bone substitute material comprising the powder according to

[25] .

[30] The powder according to [1], wherein the second particles are inorganic fine particles.

[31] The powder according to

[30] , wherein the second particles contain a metal.

[32] The powder according to

[30] or

[31] , wherein the ratio (W2 / W1) of the mass of the second particles (W2) to the mass of the first particles (W1) is 20 / 50 to 60 / 50.

[33] The powder according to

[32] , wherein the ratio (W2 / W1) of the mass of the second particles (W2) to the mass of the first particles (W1) is 20 / 50 to 40 / 50.

[34] The powder according to

[32] , wherein the ratio (W2 / W1) of the mass of the second particles (W2) to the mass of the first particles (W1) is 40 / 50 to 60 / 50.

[35] The powder according to any one of

[30] to

[34] , wherein the average particle size of the first particles is 0.1 μm to 100 μm.

[36] The powder according to any one of

[30] to

[35] , wherein the average particle size of the second particles is 5 nm to 100 μm.

[37] The powder according to

[36] , wherein the average particle size of the second particles is 10 nm to 100 nm.

[38] The powder according to any one of

[30] to

[37] , wherein the ratio of the average particle size of the second particles to the average particle size of the first particles (D2 / D1) is 0.001 to 0.1.

[39] The powder according to any one of

[30] to

[38] , wherein the second particles are magnetic particles or calcium phosphate.

[40] The powder according to any one of

[30] to

[39] , which has a water contact angle of 50 degrees or more when measured 1 second after dropping water.

[41] A tissue-dressing material containing the powder according to any one of

[30] to

[40] .

[42] A cancer-killing material comprising the powder according to

[39] or

[40] , wherein the second particles are magnetic particles.

[43] The cancer-killing material according to

[42] , wherein the magnetic particles are iron oxide.

[44] A bone filler comprising the powder according to

[39] or

[40] , wherein the second particles are calcium phosphate.

[45] A method for covering or filling tissue, comprising the step of locally administering an effective amount of the powder according to any one of [1] to

[25] and

[30] to

[40] to a predetermined site of the tissue to form a gel.

[46] A method for covering or filling a tissue excision site, comprising the step of locally administering an effective amount of the powder according to any one of [1] to

[25] and

[30] to

[40] to the excision site or its vicinity in a patient after surgery involving tissue excision, thereby forming a gel.

[47] The method according to

[45] or

[46] , wherein the second particles comprise an anticancer drug, and the tissue is tumor tissue.

[48] A method for treating cancer in a patient, comprising the step of locally administering a therapeutically effective amount of the powder according to any one of

[12] to

[15] ,

[23] , and

[24] , wherein the second particles comprise an anticancer drug, to tumor tissue or its vicinity in a patient having a tumor, thereby forming a gel.

[49] The method according to

[48] , wherein the second particles further comprise magnetic particles.

[51] A method for treating or alleviating inflammation in a patient suffering from ulcerative colitis, comprising the step of locally administering a therapeutically effective amount of the powder according to any one of

[12] ,

[13] , and

[16] , wherein the second particles comprise an immunosuppressant, to the inflamed site or its vicinity in the patient, thereby forming a gel.

[52] A method for treating polycystic kidney disease in a patient, comprising the step of locally administering a therapeutically effective amount of the powder according to any one of

[12] ,

[13] , and

[17] , wherein the second particles comprise a cell differentiation inducer, to the renal tissue or its vicinity of the patient, thereby forming a gel.

[53] A method for promoting tissue regeneration in a patient, comprising the step of locally administering a therapeutically effective amount of the powder according to any one of

[12] ,

[13] , and

[18] , wherein the second particles comprise a growth factor, to the site where tissue has been removed or its vicinity of the patient, after tissue removal, thereby forming a gel.

[54] A method for killing residual malignant pleural mesothelioma in a patient after removal of malignant pleural mesothelioma, comprising the step of locally administering a therapeutically effective amount of the powder according to

[12] , wherein the second particles comprise an antimetabolite, to the site where the cancer has been removed or its vicinity in the patient, thereby forming a gel.

[55] A method for treating malignant pleural mesothelioma in a patient, comprising the step of locally administering a therapeutically effective amount of the powder according to

[12] , wherein the second particles comprise a growth factor inhibitor, to the cancer tissue or the tissue where the cancer has been removed or its vicinity in the patient, thereby forming a gel.

[56] A method for treating kidney cancer, non-small cell lung cancer, head and neck cancer, melanoma, Hodgkin's disease, or liver cancer in a patient, comprising the step of locally administering a therapeutically effective amount of the powder according to

[12] , wherein the second particles comprise an immune checkpoint inhibitor, to the cancer tissue or the site where the cancer tissue has been removed or its vicinity in the patient, thereby forming a gel.

[57] The method according to any one of

[45] to

[56] , wherein the gel has a water content of 50 to 70%.

[58] The method according to any one of

[45] to

[57] , wherein the gel has a pressure resistance of 70 mmHg or more.

[0008] The present invention provides a powder suitable for forming a tissue dressing. The tissue dressing formed with the powder of one embodiment has high adhesive strength to biological tissue and can exhibit additional functions. Furthermore, the tissue dressing formed with the powder of another embodiment can exhibit even higher adhesive strength to biological tissue.

[0009] 1 is a schematic diagram illustrating the configuration of a powder according to one embodiment, comprising gelatin derivative particles (C10MP) and inorganic particles (SPION).

[0023] FIG. 1 is a schematic diagram illustrating an example of a usage form in which a gel formed by hydration of a powder according to one embodiment is used as a tissue dressing (cancer killing material).

[0024] FIG. 2 is an enlarged view (conceptual diagram) of region 2B shown in FIG. 2.

[0025] FIG. 2 is an enlarged view (conceptual diagram) of region 2C shown in FIG. 2.

[0026] FIG. 1 is a schematic diagram illustrating a method for producing a powder comprising gelatin derivative particles (C10MP) and inorganic particles (SPIOS) in an example.

[0027] FIG. 1 is an SEM photograph of first particles (C10MP), second particles (SPION), and raw gelatin particles (OrgMP) produced in an example.

[0028] FIG. 2 is a graph showing the particle size distribution of first particles (C10MP) and raw gelatin particles (OrgMP) produced in an example.

[0029] FIG. 1 is a graph showing the particle size distribution of second particles (SPION) produced in an example. 1 is a micrograph showing the hydration of powder (SPION / C10MP) and powder (SPION / OrgMP). 2 is an IR spectrum of gel (SPION / C10MP) and gel (SPION / OrgMP) formed after powder hydration. 3 is a graph showing the storage modulus of gel (OrgMP), gel (SPION / C10MP), and gel (SPION / OrgMP) formed after powder hydration. 4 is a schematic diagram illustrating the interaction between each component in gel (SPION / C10MP) formed after powder hydration. 5 is a schematic diagram illustrating an adhesive strength test of gel formed after powder hydration in the examples. 6 is a graph showing the results of an adhesive strength test of gel (OrgMP), gel (SPION / OrgMP), gel (C10MP), and gel (SPION / C10MP) formed after powder hydration.

[0023] Figure 1 is a schematic diagram illustrating a pressure strength test of a gel formed after powder hydration in an example. Figure 2 is a graph showing the results of a pressure strength test of gels (OrgMP), (SPION / OrgMP), (C10MP), and (SPION / C10MP) formed after powder hydration. Figure 3 is a graph showing the results of a heat generation test of gels (OrgMP), (SPION / OrgMP), (C10MP), and (SPION / C10MP) formed after powder hydration. Figure 4 is a graph showing the survival rate of cancer cells in an in vitro test using a gel formed by hydration of powder (SPION / C10MP) as a cancer killing material.1 is a graph showing the survival rate of normal cells in an in vitro test in which a gel formed by hydration of a powder (SPION / C10MP) was used as a cancer-killing material. 2 is a graph showing the therapeutic effect (change in cancer size) in an in vivo test in which a gel formed by hydration of a powder (SPION / C10MP) was used as a cancer-killing material. 3 is a graph showing the therapeutic effect (mouse survival rate) in an in vivo test in which a gel formed by hydration of a powder was used as a cancer-killing material. 4 is a schematic diagram illustrating the preparation method, configuration, and medical application example of a powder according to another embodiment in which second particles contain a drug. 5 is a diagram showing X-ray diffraction patterns of commercially available PTX and reprecipitated PTX. Figure 24(a) shows photographs of the shape of a water droplet taken one minute after dropping it onto a powder mixture of PTX particles and C10MP particles (PTX / C10MP), OrgMP, C10MP, and a powder mixture of PTX particles and OrgMP particles (PTX / OrgMP). Figure 24(b) shows the water contact angle of each particle or powder. The results of measuring the water content of colloidal gels formed with OrgMP, PTX / OrgMP, C10MP, and PTX / C10MP are shown. This is a schematic diagram illustrating a pressure strength test of gels formed after powder hydration. Figure 27(a) shows the results of measuring the pressure strength of colloidal gels formed with OrgMP, PTX / OrgMP, C10MP, and PTX / C10MP. Figure 27(b) shows a schematic diagram of the internal structure of the formed colloidal gel. 1 is a graph showing the percentage of PTX released from colloidal gels formed with PTX / OrgMP and PTX / C10MP when the colloidal gels were immersed in PBS. 2 is a schematic diagram showing the protocol for a test in which PTX / C10MP was injected into the peritoneum of a cancer-bearing mouse model. 3 is a graph showing the change in tumor volume over time in each group in which PTX, OrgMP, C10MP, PTX / OrgMP, or PTX / C10MP was applied to the peritoneum of a cancer-bearing mouse model.

[0010] The following description of the constituent elements may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0011] In the description of groups (atomic groups) in this specification, when a notation does not specify whether the group is substituted or unsubstituted, it encompasses both unsubstituted and substituted groups, as long as it does not impair the effects of the present invention. For example, the term "alkyl group" encompasses not only alkyl groups without a substituent (unsubstituted alkyl groups) but also alkyl groups with a substituent (substituted alkyl groups). This also applies to each compound.

[0012] The present invention relates to a powder comprising first particles containing a cross-linked gelatin derivative and second particles that are separate from the first particles and may provide an additional function, as well as therapeutic applications using the same.

[0013] The powder can be produced by a very simple method in which the first particles and the second particles are separately prepared and then mixed together. Powders according to various embodiments of the present invention will be described in detail below.

[0014] <First Particles> Examples of the first particles include the powder disclosed in Patent Document 1 (WO 2020 / 137903), the contents of which are incorporated herein by reference. Specifically, the first particles contain a crosslinked gelatin derivative (hydrophobized gelatin), and the gelatin derivative has a structure represented by the following formula (1):

[0015]

[0016] In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, and R １ represents a hydrocarbon group having 1 to 20 carbon atoms, and R ２ represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.

[0017] The divalent linking group for L is not particularly limited, and examples thereof include -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -N(R)- (wherein R represents a hydrogen atom or a monovalent organic group (preferably a hydrocarbon group having 1 to 20 carbon atoms)), an alkylene group (preferably an alkylene group having 2 to 10 carbon atoms), an alkenylene group (preferably an alkenylene group having 2 to 10 carbon atoms), and combinations thereof, with -C(O)- being preferred. Accordingly, L is preferably a single bond or -C(O)-.

[0018] *-CHR １ R ２ (* indicates the binding position) is preferably bound to the ε-amino group of the raw material gelatin (raw material gelatin), and more preferably bound to the ε-amino group of lysine (Lys) in the gelatin. *-CHR is bonded to an amino group, preferably the amino group of lysine, with or without a linking group (in other words, directly). １ R ２ Examples of the method for bonding include a method utilizing the so-called reductive amination reaction (a method using an aldehyde or a ketone) and a method utilizing the Schotten-Baumann reaction (a method using an acid chloride).

[0019] The —NH— structure of formula (1) is, for example, expressed as a peak at 3300 cm in an FT-IR (Fourier transform infrared) spectrum. －１ It can be detected by nearby bands.

[0020] The hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, and examples thereof include a chain hydrocarbon group having 1 to 20 carbon atoms, an alicyclic hydrocarbon group having 3 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 14 carbon atoms, and groups formed by combining these.

[0021] R ２ is 1 to 20 hydrocarbon groups, R ２ is R １ may be the same as or different from R １ , and R ２ The alkyl group may be linear or branched.

[0022] The chain hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, but examples thereof include methyl, ethyl, propyl, butyl, hexyl, octyl (or capryl), nonyl (or pelargornyl), decyl, dodecyl (or lauryl), and tetradecyl (or myristyl) groups. Among these, R １ is preferably an alkyl group having 1 to 13 carbon atoms, more preferably an alkyl group having 7 to 12 carbon atoms, still more preferably an alkyl group having 8 to 11 carbon atoms, and particularly preferably an alkyl group having 9 to 11 carbon atoms. ２ Although there are no particular limitations on the group, it is preferably a hydrogen atom.

[0023] Examples of the alicyclic hydrocarbon group having 3 to 20 carbon atoms include a cyclopropyl group, a cyclopentyl group, a cyclohexyl group, an adamantyl group, and a norbornyl group.

[0024] The aromatic hydrocarbon group having 6 to 14 carbon atoms is not particularly limited, but examples thereof include a phenyl group, a tolyl group, and a naphthyl group.

[0025] The group obtained by combining the above is not particularly limited, but examples thereof include aralkyl groups having 6 to 12 carbon atoms, such as a benzyl group, a phenethyl group, a naphthylmethyl group, and a naphthylethyl group.

[0026] Furthermore, as reported by the present inventor in Japanese Patent No. 7132465, a crosslinked gelatin derivative having a structure represented by formula (1) exhibits angiogenesis promoting action in surrounding cells when injected into a living body. The contents of Japanese Patent No. 7132465 are incorporated herein by reference. From the viewpoint of promoting angiogenesis and adhesion to tissues, in formula (1), -CHR １ R ２ The total number of carbon atoms is preferably 9 to 20, and more preferably 12 to 14. When the total number of carbon atoms is within this range, a gel formed by applying the powder to a living body exhibits excellent angiogenic ability, and when the gel is used as a bone filling material that enables blood vessel induction, bone regeneration can be promoted.

[0027] The gelatin derivative represented by formula (1) is preferably at least one gelatin derivative selected from the group consisting of the following formulas (2) and (3), and more preferably the gelatin derivative represented by formula (2).

[0028]

[0029]

[0030] In formulas (2) and (3), the meanings of the symbols are the same as those in formula (1) already explained, and the preferred embodiments are also the same.

[0031] In this specification, the "derivatization rate" is defined as the molar ratio of the content of imino groups bonded to hydrophobic groups (hydrocarbon groups) in the gelatin derivative to the content of amino groups in the starting gelatin. The derivatization rate of the gelatin derivative is not particularly limited, but is generally preferably 20 to 80 mol%, more preferably 30 to 70 mol%. In other words, the imino group / amino group (molar ratio) in the obtained gelatin derivative is preferably 20 / 80 to 80 / 20, more preferably 30 / 70 to 70 / 30. In this specification, the derivatization rate is calculated by quantifying the number of amino groups in the starting gelatin and the number of amino groups in the gelatin derivative using the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method), and using the values ​​obtained, using the following formula: Derivatization rate (mol %) = [Number of amino groups in starting gelatin - Number of amino groups in gelatin derivative] / [Number of amino groups in starting gelatin] x 100

[0032] Gelatin used as a raw material for gelatin derivatives (hereinafter also referred to as "raw material gelatin") may be naturally derived, synthesized (including fermented and genetically modified), or may be naturally derived or synthesized gelatin that has been subjected to some kind of treatment. More specifically, examples include naturally derived gelatin obtained from the skin, bones, tendons, etc. of mammals, birds, fish, etc., and treated gelatin obtained by treating naturally derived gelatin with an acid or alkali (and optionally heat-extracting). Among these, alkali-treated gelatin is preferred because it can produce a powder that has a more excellent effect of the present invention.

[0033] Furthermore, when the powder is administered to a living body, for example, when used as a wound dressing, it is preferable to use endotoxin-reduced gelatin with a reduced endotoxin content. There are no particular limitations on such endotoxin-reduced gelatin, and known gelatins can be used, including, for example, the gelatin described in JP 2007-231225 A, the contents of which are incorporated herein by reference.

[0034] Examples of mammal-derived gelatin include porcine and bovine gelatin. Fish-derived gelatin is not particularly limited, but gelatin derived from cold-water fish (cold-water fish) such as salmon, trout, Pacific cod, Alaska pollock, sea bream, tilapia, and tuna (hereinafter also referred to as "cold-water fish-derived gelatin") is preferred.

[0035] Cold-water fish-derived gelatin is a polymer in which two or more amino acids are linked in a linear chain, and contains 190 or less imino acids, more specifically, 80 or less hydroxyprolines and 110 or less prolines per 1000 constituent amino acids. The room temperature fluidity of cold-water fish-derived gelatin is thought to be due to the number of hydroxyprolines being 80 or less or the number of prolines being 110 or less. If either of these conditions is met, the denaturation temperature is thought to be below room temperature, resulting in room temperature fluidity.

[0036] Thai gelatin has a hydroxyproline number of 73, a proline number of 108, and a denaturation temperature of 302.5 K. Tilapia gelatin has a hydroxyproline number of 82, a proline number of 110, and a denaturation temperature of 309 K. In comparison, porcine gelatin has a hydroxyproline number of 95, a proline number of 121, and a denaturation temperature of 316 K.

[0037] Furthermore, gelatin derived from cold-water fish has an amino acid sequence similar to that of gelatin derived from animals, is easily decomposed by enzymes, and is highly biocompatible.

[0038] The molecular weight of the starting gelatin is not particularly limited, but the weight average molecular weight (Mw) is preferably 5,000 to 100,000, more preferably 10,000 to 50,000, and even more preferably 20,000 to 40,000. In this specification, the weight average molecular weight means the weight average molecular weight determined by gel permeation chromatography (GPC).

[0039] The first particle contains the above-mentioned crosslinked gelatin derivative. In this specification, "crosslinked" does not include a reversible physical crosslinked structure, but means a crosslinked structure obtained by an irreversible crosslinking reaction. Therefore, a "crosslinked gelatin derivative" is a gelatin derivative having an irreversible crosslinked structure obtained by a crosslinking reaction caused by applying energy to the gelatin derivative with heat, light, energy rays, etc., and / or by a crosslinking agent. Typically, the functional group (-NH ２ , -OH, -SH, -COOH, etc.

[0040] The first particles may contain a crosslinked gelatin derivative and may contain other components as long as the effects of the present invention are achieved. The content of the crosslinked gelatin derivative in the first particles 10 is not particularly limited. However, in order to more easily obtain a powder having the effects of the present invention, the crosslinked gelatin derivative is preferably contained in an amount of 90% by mass or more, and more preferably 99% by mass or more, relative to the total mass of the first particles 10. Examples of crosslinked gelatin derivatives include non-crosslinked gelatin derivatives, solvents, buffering agents, colorants, preservatives, excipients, and pharmaceuticals (antithrombotic drugs, antibacterial agents, anticancer drugs, immunosuppressants, growth factors, etc.). However, since the particles are typically produced by thermally crosslinking a gelatin derivative, other components contained within the particles may aggregate, deform, deteriorate, or denature during thermal crosslinking. For example, if inorganic fine particles or pharmaceuticals (second particles) were incorporated into the first particles, they would aggregate during thermal crosslinking, increasing the particle size and potentially reducing the adhesiveness of the gel to tissue. Furthermore, if the inorganic fine particles or the drug (second particles) aggregate, deform, change in quality, or denature during thermal crosslinking, the intended function may not be fully exerted. For example, as described below, if magnetic particles are used as the second particles, the gel 40 can be heated by an external magnetic field, and this function can be used to locally kill cancer cells (see Figure 4). However, if the magnetic powder in the gel 40 aggregates, it becomes difficult to heat it by an external magnetic field, and there is a risk that a sufficient cancer cell killing effect will not be achieved.

[0041] The average particle size of the first particles 10 may be, for example, 0.1 μm to 100 μm, 0.5 μm to 50 μm, 1 μm to 30 μm, or 1 μm to 10 μm. In this specification, the "average particle size" can be measured by the method described in the examples below.

[0042] The method for producing the first particles is not particularly limited, and the first particles can be produced, for example, by the method described in Patent Document 1 (WO2020 / 137903).

[0043] <Second particle>

[0044] As shown in Figure 1, powder 30 of one embodiment contains particles (first particles) 10 containing a crosslinked gelatin derivative, as well as inorganic fine particles (second particles) 20. The first particles 10 and the second particles 20 are separate entities, and are different from a form in which one is contained within the other. Powder 30 of this embodiment contains a mixture of two types of particles (first particles 10 and second particles 20).

[0045] The second particles 20 contained in the powder 30 are not particularly limited as long as they are inorganic fine particles, and inorganic fine particles having various functions can be used, and they are expected to be used for various purposes, particularly medical purposes, depending on their functions.

[0046] For example, magnetic particles may be used as the second particles 20. When a powder 30 containing first particles 10 and magnetic particles (second particles) 20 is applied to biological tissue, a gel 40 containing the magnetic particles (second particles) 20 can be formed, and the gel 40 can be heated by applying an external magnetic field. It is known that cancer cells are more susceptible to heat than normal cells. The powder 30 can be used as a cancer killing material that can kill only cancer cells while minimizing damage to normal cells (see Figure 4).

[0047] The magnetic particles are not particularly limited, but preferably have low biotoxicity, for example, iron oxide (e.g., Fe ３ O ４ , Fe ２ O ３ ), ferrite, chromium oxide, gold, cobalt, etc., and among these, iron oxide, which is composed of metals that are contained in large amounts in the body when ionized, is preferred.

[0048] Furthermore, for example, calcium phosphate may be used as the second particles 20. Calcium phosphate such as hydroxyapatite has osteoconductivity (the property of attracting bone to the surrounding area) and is therefore used as a main component of artificial bone. For this reason, powder 30 containing first particles 10 and calcium phosphate (second particles) 20 can be applied only to the affected area (such as a fractured area) by spraying or the like and used as a bone filling material.

[0049] Other inorganic fine particles include calcium phosphate having osteoinductive properties, such as β-tricalcium phosphate (β-TCP), α-tricalcium phosphate (α-TCP), and octacalcium phosphate (OCP).

[0050] From the viewpoint of further improving the adhesive strength of the gel 40 to tissue, it is preferable that the second particles 20 contain a metal. The strength of the gel 40 is improved by the interaction between the metal-containing fine particles and the functional groups (e.g., carboxy groups) of the gelatin derivative, thereby further increasing the adhesive strength of the gel 40. The metal contained in the second particles 20 may be determined based on the function of the second particles 20, but metals with low biotoxicity are preferred, such as sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), aluminum (Al), vanadium (V), chromium (Cr), cobalt (Co), nickel (Ni), copper (Cu), and zinc (Zn). Other inorganic particles include, for example, titanium oxide and cerium oxide.

[0051] As illustrated in Figure 22, powder 60 in another embodiment includes first particles 10 containing a cross-linked gelatin derivative and second particles 50 containing or consisting of a particulate drug. Again, second particles 50 are separate entities from first particles 10, and are different from the form in which one is contained within the other. Therefore, powder 60 in this embodiment is also a mixture containing first particles 10 and second particles 50. First particles 10 in this embodiment are similar to the first particles in the previous embodiment.

[0052] The gelatin derivative constituting the first particle 10 is hydrophobic and has functional groups such as carboxyl groups. Therefore, in this embodiment, when the first particle hydrates to form a colloidal gel, the drug interacts with the gelatin derivative through hydrophobic or electrostatic interaction, is incorporated into the gel, and is locally released from the gel that adheres to the tissue.

[0053] Therefore, in this embodiment, the drug is not particularly limited, and the drug may be a low-molecular-weight compound or a high-molecular-weight bioactive substance. In a preferred embodiment, the drug is selected from hydrophobic, negatively, or positively charged drugs. The hydrophobic drug interacts hydrophobically with the hydrophobic first particles 10, improving the tissue adhesiveness and pressure resistance of the gel formed from the powder 60. Because the colloidal gel formed from gelatin is negatively charged, a positively charged drug can electrostatically bind to the colloidal gel. Furthermore, when used together with a cationic additive, a negatively charged drug can electrostatically bind to the colloidal gel via the cationic additive.

[0054] Examples of drugs include anticancer drugs, immunosuppressants, growth factors, cell differentiation inducers, metabolic antagonists, growth factor inhibitors, immune checkpoint inhibitors, antibiotics, anti-inflammatory agents, and protein preparations, and these may be used alone or in combination of two or more.

[0055] The anticancer agent is not particularly limited, but hydrophobic anticancer agents are preferred, and examples thereof include cabazitaxel, paclitaxel, docetaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, tamoxifen, anastrozole, Gleevec, 5-fluorouracil (5-FU), floxuridine, leuprolide, flutamide, zoledronate, doxorubicin, vincristine, gemcitabine, streptozocin, vinorelbine, retinoic acid series, adriamycin, mitomycin, daunomycin, prednisone, testosterone, mitoxantrone, aspirin, salicylic acid, ibuprofen, naproxen, fenoprofen, indomethacin, phenyltazone, cyclophosphamide, celecoxib, valdecoxib, and nimesulide.

[0056] The immunosuppressant is not particularly limited, but hydrophobic immunosuppressants are preferred, and examples thereof include low molecular weight compounds such as steroids (e.g., hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone), cyclosporine, mizoribine (bredinin), cyclophosphamide (endoxan), azathioprine (azanin), tacrolimus (FK506), sirolimus (rapamycin), deoxyspergualin (gusperimus hydrochloride), everolimus, and ABT-578 (zotarolimus), as well as high molecular weight active substances such as basiliximab (anti-IL-2 receptor monoclonal antibody), muromonab CD3 (anti-CD3 monoclonal antibody), and arbulin (antilymphocyte globulin).

[0057] The cell differentiation inducer is not particularly limited, but a hydrophobic cell differentiation inducer is preferred, for example, tamibarotene. The metabolic antagonist is not particularly limited, but pemetrexed is an example. The growth factor inhibitor is not particularly limited, but bevacizumab is an example. The immune checkpoint inhibitor is not particularly limited, but nivolumab is an example.

[0058] Examples of growth factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), and Wnt7b.

[0059] Since the polymeric biologically active substance, such as a growth factor, is positively or negatively charged, and the colloidal gel formed by the gelatin constituting the first particles is negatively charged, the positively charged active substance can be electrostatically bound to the colloidal gel. Furthermore, when a negatively charged polymeric biologically active substance, such as a growth factor, is used together with a cationic additive, it can be electrostatically bound to the colloidal gel via the cationic additive. Examples of cationic additives include cationic polymers such as polylysine, chitosan, and polyallylamine.

[0060] When the drug is a low-molecular-weight compound, the release rate of the drug can be adjusted by its degree of crystallinity. Specifically, the higher the degree of crystallinity, the slower the release rate, and the lower the degree of crystallinity, the faster the release rate. Therefore, it is possible to set an appropriate release rate by combining this with the hydrophobicity of the drug. For example, when the drug is a hydrophobic anticancer drug, immunosuppressant, or cell differentiation inducer, a crystallinity of 30-50% can be set to achieve sustained release over a period of several months to six months. The degree of crystallinity can be adjusted by changing the temperature and drug concentration during reprecipitation of the drug. Specifically, a drug solution is prepared, and a poor solvent is added dropwise to the drug to reprecipitate it. The solution temperature is set to 10-40°C, the drug concentration is set to 0.1-10 w / v%, and the poor solvent is slowly added dropwise. This allows the degree of crystallinity to be 30-50%. To lower the degree of crystallinity, precipitation can be carried out at a lower temperature for a shorter time, while to increase the degree of crystallinity, precipitation can be carried out at a higher temperature for a longer time.

[0061] The average particle diameter of the second particles 20, 50 may be adjusted appropriately depending on their type (function), but is preferably, for example, 5 nm to 100 μm or 10 nm to 10 μm. If the average particle diameter of the second particles 20, 50 is within the above range, the interaction between the hydrophobized gelatins is not significantly affected, and the gel 40 can be imparted with additional functions while maintaining the excellent adhesive properties of the gel 40. Furthermore, when the second particles are magnetic particles, from the viewpoint of improving heating efficiency, the average particle diameter is more preferably 5 nm to 100 nm, 10 nm to 100 nm, or 10 nm to 20 nm. Furthermore, when the second particles are particulate drugs, from the viewpoint of inclusion in the gel, the average particle diameter is more preferably 10 nm to 100 nm, 20 nm to 100 nm, or 50 nm to 100 nm. In this specification, the term "average particle diameter" can be measured by the method described in the Examples below.

[0062] The second particles 20 contained in the powder 30 may be of one type alone or may be a mixture of two or more types as long as the effects of the present invention are achieved. The second particles 20 may be produced by a known method, or may be commercially available products.

[0063] The second particles may contain, for example, both a drug and inorganic fine particles. For example, the second particles may contain an anticancer drug as a drug and further contain magnetic particles. In this case, the anticancer drug and the magnetic particles may be integrated into a particle, or may be separate particles. In this embodiment, the latter is preferred. Furthermore, the second particles may contain a growth factor such as VEGF or Wnt7b as a drug, and may further contain calcium phosphate. In this case, too, the growth factor and calcium phosphate may be integrated into a particle, or may be separate particles. In this case, too, the latter is preferred in this embodiment.

[0064] <Powder> The powders 30 and 60 contain a mixture of particles (first particles) 10 containing the above-described crosslinked gelatin derivative and functional particles (second particles) 20 and 50. The ratio (W2 / W1) of the mass (W2) of the second particles 20 and 50 to the mass (W1) of the first particles 10 is not particularly limited, but it is preferable to select an appropriate ratio depending on the type of functional particles (second particles). For example, when the second particles are or contain functional inorganic particles 20, the ratio (W2 / W1) is preferably 20 / 50 to 60 / 50, 20 / 50 to 50 / 50, or 20 / 50 to 40 / 50 from the viewpoint of enhancing the adhesive strength of the gel 40 formed from the powder 30. Furthermore, when the second particles are or contain magnetic particles 20, the ratio (W2 / W1) is preferably 40 / 50 to 60 / 50 from the viewpoint of enhancing heat generation efficiency. When the second particles are particulate drug 50 or contain a drug, the ratio (W2 / W1) is preferably 0.1 / 50 to 60 / 50, 0.1 / 50 to 50 / 50, or 0.1 / 50 to 40 / 50 from the viewpoint of increasing the adhesive strength of gel 70 formed from powder 60. In particular, when second particles 50 are or contain a growth factor, a physiologically active protein, or an antibody, the ratio (W2 / W1) is preferably 0.1 / 50 to 60 / 50.

[0065] The powders 30, 60 may be composed only of the first particles 10 and the second particles 20, 50, or may contain other components different from the first particles 10 and the second particles 20, 50, as long as the effects of the present invention are achieved. The proportion of the total mass of the first particles 10 and the second particles 20, 50 in the powders 30, 60 may be, for example, 90 mass% or more, 99 mass% or more, or 100 mass%. Other components that the powder 30 may contain are not particularly limited, and examples thereof include non-crosslinked gelatin derivatives, solvents, buffering agents, colorants, preservatives, excipients, and pharmaceuticals (antithrombotic drugs, antibacterial agents, anticancer drugs, immunosuppressants, growth factors, etc.).

[0066] The ratio (D2) / (D1) of the average particle diameter (D2) of the second particles 20, 50 to the average particle diameter (D1) of the first particles 10 is not particularly limited, and may be, for example, 0.001 to 100, 0.001 to 1, or 0.001 to 0.1. Furthermore, from the viewpoint of increasing the adhesive strength of the gels 40, 70 to tissue, it is preferable that the particle diameter of the second particles 20, 50 be smaller than the particle diameter of the first particles (i.e., the ratio (D2) / (D1) is less than 1), and may be, for example, 0.001 to 0.1. Furthermore, when the second particles 20 are magnetic particles, from the viewpoint of increasing heat generation efficiency, the ratio (D2) / (D1)=0.001 to 0.1, or 0.001 to 0.01 is preferable. Furthermore, when the second particles are particulate drugs, the ratio (D2) / (D1) is preferably 0.001 to 0.1, or 0.01 to 0.1, from the viewpoint of adhesiveness and deliverability.

[0067] There is no particular limitation on the method for producing the powders 30 and 60 of this embodiment. For example, the powders 30 and 60 may be produced by preparing the first particles and the second particles, respectively, and mixing them in a desired ratio by a known method.

[0068] The resulting powders 30 and 60 contain the first particles and the second particles in the aforementioned ratios (W2 / W1) and (D2) / (D1), for example, and the gel formed thereby is hydrophobic overall. Therefore, depending on the structure of the gelatin derivative and the values ​​of the ratios (W2 / W1) and (D2) / (D1), the powders 30 and 60 may have a water contact angle of 50 degrees or more, preferably 60 degrees or more, and more preferably 70 degrees or more. Furthermore, the water contact angle is typically 90 degrees or less, and in many cases 80 degrees or less. Here, the "water contact angle" in this specification is measured by the method described in the Examples below. The water contact angle is an indicator of the hydrophobicity of the gelatin derivative and, by extension, the gel formed therefrom. Within the aforementioned water contact angle range, the adhesive strength to tissue and the pressure resistance are increased, and if the first particles contain a hydrophobic compound as a drug, these strengths are further improved.

[0069] Furthermore, due to its hydrophobicity, the colloidal gel formed from powders 30 and 60 has a lower water content of gelatin than a colloidal gel formed from a powder in which the first particles are made of unhydrophobized gelatin, e.g., 50-70%, preferably 55-65%. Herein, "water content" is determined by the method described in the Examples below. Such a decrease in water content indicates that the gel is denser due to hydrophobic interactions, which is understood to improve the pressure resistance of the gel. In fact, colloidal gels 40 and 70 formed from powders 30 and 60 have a pressure resistance of 70 mmHg or more, preferably 80 mmHg or more, in a pressure resistance test. Furthermore, colloidal gels 70 formed from powder 60 in which the second particles contain a hydrophobic drug have improved pressure resistance due to hydrophobic interactions with the hydrophobic drug. Specifically, the powder 60 has a pressure resistance of 90 mmHg or more, and preferably 100 mmHg or more. Furthermore, powder 60 in which the second particles do not contain a hydrophobic drug typically has a pressure resistance of 100 mmHg or less, and powder 60 in which the second particles contain a hydrophobic drug typically has a pressure resistance of 120 mmHg or less. Here, in this specification, "pressure resistance" is determined by the method described in the Examples below.

[0070] <Powder Uses> When the powders 30 and 60 according to the present embodiment are applied (e.g., sprayed) in a dry state to biological tissue, the first particles 10 absorb blood and moisture and gel, exhibiting excellent adhesive properties to biological tissue while retaining the functional second particles 20 and 50. By utilizing this property, the powders 30 and 60 according to the present embodiment can be used as tissue dressings or tissue fillers with various functions. For example, an effective amount of the powders 30 and 60 can be locally administered to the excision site or its vicinity after surgery involving tissue resection to form a gel that coats or fills the tissue resection site. In addition to being usable as a tissue dressing, certain embodiments of the powders can also be used as cancer killing agents and bone fillers. Similar to the powder disclosed in Patent Document 1 (WO 2020 / 137903), the powders can also be used as wound dressings, adhesion inhibitors, hemostatic agents, and the like. The application amount can be adjusted appropriately depending on the purpose of use and the application site.

[0071] When magnetic particles are used as the second particles 20, the gel formed from the powder 30 can be used not only as a wound dressing but also as a cancer killing agent. Furthermore, after the gel adheres to tissue, the hydrophobic groups exposed on the surface of the first particles sink into the gel in an aqueous environment, gradually making the exposed surface hydrophilic, making it less likely to adhere to tissue. It can also function as an adhesion inhibitor. Furthermore, since it can be applied by spraying, it can be used not only in open surgery but also in wounds in endoscopic surgery. For example, when the powder 30 of this embodiment is applied (sprayed) to an affected area of ​​biological tissue (e.g., a damaged area after endoscopic resection of a cancer tumor) ( FIG. 2( a) ), the powder 30 applied to the tissue hydrates and forms a gel 40 ( FIG. 2( b) ). At this time, hydrophobic interactions occur between the first particles 10 ( FIG. 3( a) ), between the first particles 10 and tissues (cell membranes, cell matrix) ( FIG. 3( b) ), and further between the second particles and the first particles (e.g., coordinate bonds between metal-containing microparticles and carboxy groups) ( FIG. 3( c) ). It is presumed that the gel 40 of this embodiment has improved bulk strength and adhesiveness (adhesive force) to tissues, and is also more stable in water ( FIG. 3( d) ). In this way, by applying the powder 30 to the affected area (wound) during endoscopic cancer tumor resection surgery, the wound can be covered and postoperative adhesions can be suppressed. Furthermore, by applying an external magnetic field, any remaining cancer cells can be killed ( FIG. 4 ).

[0072] Furthermore, as described above, when calcium phosphate is used as the second particles, the powder can be applied only to the affected area (such as a fracture site) using a spray, syringe, etc., and used as a bone filler for a jawbone defect during oral surgery or when applying an implant.

[0073] As shown in FIG. 22 , when the second particles 50 contain an effective amount of a drug, they can be used not only as wound dressings but also for the treatment of various diseases, depending on the type of drug. They can also function as adhesion barriers. For example, when the second particles 50 contain an effective amount of an anticancer drug, a therapeutically effective amount of powder 60 can be locally administered to the tumor tissue or its surrounding area of ​​a patient with a tumor to form a gel, thereby treating the patient's cancer. Furthermore, by locally administering powder 60 to the affected area (wound) during endoscopic cancer tumor resection surgery, wound coverage can be achieved, postoperative adhesions can be suppressed, and any remaining cancer cells can be killed by the sustained release of the anticancer drug. In these embodiments, the anticancer drug is preferably a hydrophobic anticancer drug. Furthermore, the second particles may contain magnetic particles in addition to the anticancer drug. In this case, applying an external magnetic field to heat the tumor tissue can be expected to have an additive or synergistic effect with the anticancer drug in killing cancer cells.

[0074] When the second particles comprise an effective amount of an immunosuppressant, a therapeutically effective amount of the powder can be administered topically to a patient suffering from ulcerative colitis at or near the site of inflammation to form a gel, thereby treating or alleviating inflammation in the patient. In this embodiment, the immunosuppressant is preferably a hydrophobic immunosuppressant.

[0075] When the second particles include a cell differentiation inducer, a therapeutically effective amount of the powder can be administered locally to or near the renal tissue of a patient with autosomal dominant polycystic kidney disease to form a gel, thereby treating polycystic kidney disease in the patient. In this embodiment, the cell differentiation inducer is preferably a hydrophobic cell differentiation inducer.

[0076] When the second particles contain a growth factor, a therapeutically effective amount of the powder can be locally administered to the patient at or near the excised tissue site after tissue resection to form a gel, thereby promoting tissue regeneration in the patient. As described above, the growth factor is positively or negatively charged. When a negatively charged growth factor is included, the second particles preferably further contain a cationic polymer. The tissue to be resected may include tumor tissue. Furthermore, when the second particles contain a growth factor such as VEGF or Wnt7b as a drug and the gel formed from the powder is used as a bone substitute, calcium phosphate may be further included. This embodiment is expected to further promote bone formation.

[0077] When the second particles include an antimetabolite, a therapeutically effective amount of the powder can be administered locally to a patient after removal of the malignant pleural mesothelioma at or near the site of removal to form a gel, thereby treating residual malignant pleural mesothelioma in the patient. In this embodiment, the antimetabolite is preferably a hydrophobic antimetabolite.

[0078] When the second particles include a growth factor inhibitor, a therapeutically effective amount of the powder can be administered locally to a patient after removal of the malignant pleural mesothelioma at or near the site of removal to form a gel, thereby treating residual malignant pleural mesothelioma in the patient. In this embodiment, the growth factor inhibitor is preferably a growth factor inhibitor.

[0079] When the second particles comprise an immune checkpoint inhibitor, a therapeutically effective amount of the powder can be administered locally to a patient suffering from kidney cancer, non-small cell lung cancer, head and neck cancer, melanoma, Hodgkin's disease, or liver cancer at or near the site of cancer removal to form a gel, thereby treating the patient's kidney cancer, non-small cell lung cancer, head and neck cancer, melanoma, Hodgkin's disease, or liver cancer. In this embodiment, the immune checkpoint inhibitor is preferably an immune checkpoint inhibitor.

[0080] The present invention will be described in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the examples shown below.

[0081] 1. First Particles <Synthesis of Gelatin Derivative (Hydrophobic Gelatin)> The amino group of raw material gelatin (hereinafter referred to as "Org-ApGltn") was reacted with decanal to form a Schiff base, and the resulting Schiff base was then reduced to a stable secondary amine with a reducing agent to synthesize hydrophobic gelatin (hereinafter referred to as "C10-ApGltn") (see FIG. 5). Details are described below.

[0082] 100 g of Alaska pollack-derived gelatin (Org-ApGltn) (Nitta Gelatin Co., Ltd., weight-average molecular weight (Mw): 38,552 Da, amino group content: 339 μmol / g) was dissolved in 105 mL of ultrapure water, and while stirring at 50°C, decanal (Tokyo Chemical Industry Co., Ltd., 67.8 mmol) in an amount twice the amino group content of Org-ApGltn (339 μmol / g) was added to the solution together with ethanol (Junsei Kagaku Co., Ltd.) to form an imine bond between the decanal and the amino group of Org-ApGltn. After stirring at the same temperature for 1 hour, 2-picoline borane (Junsei Kagaku Co., Ltd., 50.85 mmol) was added to the mixture together with ethanol to reduce the imine. The resulting mixed solution (Org-ApGltn concentration: 20% by mass / volume, water:ethanol = 105:45 mL) was stirred at 50°C for 17 hours to allow the reaction to proceed. The reaction solution (150 mL) was then added dropwise to 1500 mL of cold ethanol (-7 to 4°C) to purify C10-ApGltn. The resulting reprecipitate was washed with 1500 mL of ethanol (1 hour x 3 times) to remove unreacted decanal and 2-picoline borane. The resulting mixture was then vacuum dried for 3 days to obtain C10-ApGltn in a yield of 92.9% by mass.

[0083] The introduction of a decyl group into the obtained C10-ApGltn was confirmed by Fourier transform infrared spectroscopy and １ This could be confirmed by H-NMR. The derivatization rate of C10-ApGltn (hereinafter referred to as "DS") was calculated from the values ​​obtained by quantifying the number of amino groups in the raw gelatin and the number of amino groups in the hydrophobized gelatin using the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method). The DS was 47 mol%.

[0084] <Production of first particles> 0.5 g of the gelatin derivative obtained by the above method was weighed into a graduated vial (50 mL), and 7.5 mL of MilliQ (registered trademark) water was added. Next, after dissolving in a water bath at 50°C, MilliQ water was added to make up to 10 mL. At this time, the content (final concentration) of the gelatin derivative in the gelatin solution was 5 mass / vol%.

[0085] Next, while stirring with a stirrer bar at room temperature, ethanol (EtOH) was added dropwise until the gelatin solution became cloudy, yielding a gelatin solution containing intermediate particles. The gelatin solution was then left to stand in a freezer (-30°C) for at least 2 hours. The vial was then removed from the freezer, its opening covered with Kimwipes (registered trademark), and freeze-dried to yield an intermediate powder containing intermediate particles. The resulting intermediate powder was heated at 150°C for 6 hours to crosslink it, yielding first particles. The synthesized first particles are referred to as "C10MP" or "C10" as appropriate.

[0086] <Production of raw material gelatin particles> Raw material gelatin particles were produced in the same manner as the first particles described above, using raw material gelatin (Org-ApGltn) instead of the synthesized gelatin derivative (C10-ApGltn). The synthesized raw material gelatin particles will be referred to as "OrgMP" or "Org" as appropriate.

[0087] 2. Second Particles <Production of Second Particles> The second particles are produced under high pH conditions by the method described below. ２＋ and Fe ３＋ The second particles were synthesized by co-precipitation of the above (see FIG. 5). The synthesized second particles are referred to as "SPION" or "SP" as appropriate.

[0088] (1) FeCl ２ (2 g) and FeCl ３ (5.4 g) to N ２ The compound was dissolved in ultrapure water (5 mL) under flow conditions by stirring with a stirrer (in a draft, at room temperature, at a rotation speed of 300 rpm). (2) A 1.8 M NaOH aqueous solution (40 mL) was placed in a three-neck flask, and N ２The mixture was stirred with a stirrer (1200 rpm) under flow conditions while heated to 50°C in an oil bath. (3) The solution prepared in (1) above was added dropwise to the NaOH aqueous solution in (2). A black precipitate formed upon addition. (4) After the addition was completed, stirring was continued for another hour under the same conditions to obtain a suspension. (5) The resulting suspension was transferred to a centrifuge tube, centrifuged at 13,000 rpm, and the supernatant was removed. (6) 50 mL of ultrapure water was added to resuspend the precipitate, obtaining a resuspension. Half of the resuspension was ultrasonically cleaned, and then centrifuged at 13,000 rpm for 60 minutes to remove the supernatant, obtaining a precipitate. The precipitate was then washed nine times by adding ultrapure water, centrifuging, and removing the supernatant. The remaining half of the resuspension was adjusted to pH 7.4, ultrasonically washed, and centrifuged at 13,000 rpm for 60 minutes to remove the supernatant and obtain a precipitate. Furthermore, the precipitate was washed six times by adding ultrapure water, centrifuging, and removing the supernatant. If a sufficient amount of precipitate could not be obtained by centrifugation, the resuspension was placed on a stirrer overnight and precipitated by a magnetic field. (7) The precipitate obtained in (6) above was dried under reduced pressure to obtain syn-Fe ３ O ４ As a result, powder (second particles) of 10 ...

[0089] 3. Evaluation of Particles The prepared first particles (C10MP), raw gelatin particles (OrgMP), and second particles (SPION) were observed using an SEM. The particle size distribution and average particle diameter of each particle were also determined. SEM photographs of each particle are shown in Figure 6, and the particle size distributions of each particle are shown in Figures 7 and 8. The average particle diameters of each particle were C10MP: 2.3 µm, OrgMP: 3.5 µm, and SPION: 11.4 nm. The conditions for calculating the average particle diameter of each particle were as follows:

[0090] <First particles (C10MP), raw gelatin particles (OrgMP)> Each particle was observed using a scanning electron microscope (SEM, JCM7000, manufactured by JEOL Ltd., Tokyo, Japan), and the particle size was counted using Image J. Specifically, the particle size of 100 particles was calculated from the scale length on the SEM image using Image J. The average particle size was determined by calculating the average of the particle sizes of the 100 particles. A frequency distribution table (a table showing the distribution by dividing the data into classes) was prepared for the particle size distribution, and this distribution was summarized in a graph. <Second particles (SPION)> The average particle size of SPION was counted using Image J using images obtained with a high-resolution transmission electron microscope (HRTEM; JEM2100F, manufactured by JEOL Ltd., Tokyo, Japan). Specifically, Image J was used to calculate the particle diameters of 100 randomly selected particles from the scale length on the SEM image. The average particle diameter was calculated by averaging the diameters of the 100 particles. A frequency distribution table (a table showing the distribution by dividing the data into classes) was created to show the particle size distribution, and this distribution was summarized in a graph.

[0091] 4. Powder (1) <Production of Powder> The produced particles were mixed in the mass ratios shown in Table 1 to produce Samples 1-1 to 1-4, 2-1 to 2-4, and 3.

[0092]

[0093] 5. Evaluation of Powder (1) <Observation of Powder Hydration> The aggregation behavior of particles in a humid environment was observed. Sample 1-3 (SP / C10 = 40 mg / 50 mg) and Sample 2-3 (SP / Org = 40 mg / 50 mg) were each added to 200 μL of physiological saline to prepare a suspension. After culturing this suspension for a maximum of 2 hours, the aggregation behavior of the particles was observed using a bright-field microscope (Keyence Corporation, BZ-X710). A bright-field micrograph is shown in Figure 9.

[0094] Sample 2-3 (SP / Org) remained isolated from each other at all hydration times, and no particle fusion was observed. On the other hand, in Sample 1-3 (SP / C10), the particles fused (aggregated) over the hydration time, forming a colloidal gel through hydrophobic interactions. Sample 1-3 (SP / C10) exhibited a densely packed structure after 30 minutes of hydration, and after 60 minutes of hydration, almost all of the C10MP particles fused together to form large aggregates. This result also suggests that hydrophobic interactions between C10MP particles are the driving force behind colloidal gel formation under humid conditions.

[0095] In Figure 9, the black areas indicated by white arrows (triangles) are the secondary particles (SP). This observation confirmed that in Sample 1-3 (SP / C10), even when the secondary particles were present, the hydrophobic interactions between the C10MP particles were not inhibited, leading to gelation and particle fusion.

[0096] <IR Spectra of Hydrated Powders (Gels)> The IR spectra of the hydrated gels of Sample 2-1 (Org), Sample 1-1 (C10), Sample 2-3 (SP / Org), and Sample 1-3 (SP / C10) were measured. The results are shown in Figure 10. For reference, the FT-IR spectrum of Sample 3 is also shown in Figure 10.

[0097] The samples for FT-IR spectrum measurement were prepared as follows: First, a predetermined amount of each sample was weighed out: Sample 2-1 (Org = 50 mg), Sample 1-1 (C10 = 50 mg), Sample 2-3 (SP / Org = 40 mg / 50 mg), Sample 1-3 (SP / C10 = 40 mg / 50 mg). Each of these samples was hydrated with 300 μL of PBS in a silicone mold with a diameter of 10 mm and a thickness of 1.5 mm, and the resulting colloidal gel was freeze-dried. The dried colloidal gel was used as the measurement sample.

[0098] In FIG. 10(a), when attention is paid to the peaks derived from the carboxyl groups of gelatin, the peaks of the carboxyl groups of Samples 2-1 and 1-1, which do not contain second particles, are shifted to the lower wavenumber side in Samples 2-3 and 1-3, which contain second particles (1638 cm －１ →1629cm －１From this peak shift, it was confirmed that in Samples 2-3 and 1-3, the carboxyl groups contained in the gelatin were coordinately bonded to the metal (Fe) contained in the secondary particles (see FIG. 10(b)).

[0099] <Storage modulus of powder (gel) after hydration> Samples 2-1 to 2-4 (using raw material gelatin Org) and samples 1-1 to 1-4 (using gelatin derivative C10) were evaluated as follows. First, each sample (powder) was weighed out to achieve the specified mass ratio shown in Table 1, with 50 mg of gelatin particles (Org or C10). Each of these samples was placed in a silicone mold with a diameter of 10 mm and a thickness of 1.5 mm, and the surface of the powder was smoothed using a spatula. Then, 300 μL of PBS was placed on each sample (powder), and the sample was hydrated at 37° C. for 30 minutes to prepare a measurement sample. The prepared measurement sample was placed on the stage of a viscoelasticity measuring device (Rheoplus, Anton Paar) and clamped with a jig having a diameter of 10 mm. The storage modulus G' of the sample was measured under the conditions of a stage temperature of 37°C, a shear strain of 1%, and an angular frequency of 10 rad / s. The results are shown in Figure 11.

[0100] As shown in Figure 11, compared to Samples 2-1 to 2-4 (using raw gelatin Org), Samples 1-1 to 1-4 (using gelatin derivative C10) had a higher storage modulus G' and formed stronger gels. This is presumably due to the hydrophobic interaction between the hydrophobic groups (decyl groups) introduced into the gelatin derivative of the first particles, which caused the particles to fuse (aggregate) and increase gel strength, as confirmed in the <Observation of Powder Hydration> (see Figure 9) (see Figure 12). Furthermore, comparing Samples 1-1 to 1-4 in Figure 11, the higher the proportion of second particles (SP), the higher the gel strength. This is presumably due to the interaction between the second particles and the carboxyl groups of the gelatin derivative, as confirmed in the <IR spectrum after powder hydration> (see Figure 10) (see Figure 12). As such, in Samples 1-1 to 1-4, as shown in FIG. 12, it is presumed that two types of interactions contribute to the improvement of gel strength: interactions between first particles (C10MP-C10MP interaction) and interactions between first particles and second particles (SPION-C10MP interaction).

[0101] <Adhesive Strength> Samples 2-1 to 2-4 (using raw material gelatin Org) and samples 1-1 to 1-4 (using gelatin derivative C10) were evaluated as follows. The test method was performed in accordance with the standard of the American Society for Testing and Materials (ASTM F-2258-05) (see FIG. 13).

[0102] First, a fresh porcine stomach (purchased from Tokyo Shibaura Organs) was opened, and the mucosal layer was removed to expose the submucosal serosal tissue. The resulting tissue was cut into 2.5 cm square tissue pieces and fixed to the upper and lower stages of a testing device (Texture Analyzer TA-XT2i, manufactured by Stable Micro Systems) using cyanoacrylate (manufactured by Henkel Japan Co., Ltd.). The temperature of the porcine stomach lining tissue during measurement was maintained at 37°C using a hot plate. To remove excess moisture from the tissue surface, an industrial paper cloth (trade name "Kimwipe") was pressed against the tissue at 80 kPa for 3 minutes (Figure 13(a)). Each sample was sprayed so as to cover the tissue on the stage of the lower jig (Figure 13(b)). The upper jig was then placed on top of the sample in the lower jig and pressed against the lower jig at 80 kPa for 3 minutes (Figure 13(c)). The upper jig was then raised at 10 mm / min, and the adhesive strength of the sample was measured ( FIG. 13( d) ). The results are shown in FIG. 14 . As a control, FIG. 14 also shows the adhesive strength between two tissues on which no particles were sprayed (No materials). The amount of each sample used in the test (amount sprayed on the tissue) was an amount that achieved the specified mass ratio shown in Table 1, with 50 mg of gelatin particles (Org or C10). For comparison, the results of similar tests using 20 mg, 40 mg, and 60 mg of SP (second particles) instead of each sample are also shown in FIG. 14 .

[0103] As shown in Fig. 14, Samples 1-1 to 1-4 (using gelatin derivative C10) had higher adhesive strength than Samples 2-1 to 2-4 (using raw material gelatin Org). This is presumably due to the hydrophobic interactions between the first particles 10, between the first particles 10 and tissues (cell membranes, cell matrix), and further between the second particles and the first particles (for example, coordinate bonds between the metal-containing second particles and carboxy groups in the first particles), as shown in Fig. 3.

[0104] Next, samples 1-1 to 1-4 are compared in Figure 14. As the proportion of second particles (SP) increased, adhesive strength increased, peaking at SP / C10 = 40 / 50. Further increases in the proportion of second particles resulted in a slight decrease in adhesive strength. The increase in adhesive strength as the proportion of second particles (SP) increased is presumed to be due to an increase in gel strength caused by the interaction between the first particles and the second particles. Furthermore, the slight decrease in gel strength in sample 1-4 (SP / C10 = 60 / 50) is presumed to be due to the mass proportion of second particles (SP) exceeding that of the first particles, thereby affecting (weakening) the interaction between the first particles and the tissue.

[0105] <Compression Strength> The compression strength of colloidal gels was measured using Samples 2-1 and 2-3 (using raw gelatin Org) and Samples 1-1 and 1-3 (using gelatin derivative C10) according to ASTM-F2392-04R, with minor modifications (see Figure 15). First, a 10 mm diameter submucosal layer of a 35 mm diameter circular duodenum was removed with surgical scissors to expose the submucosal tissue. A 1 mm pinhole was created in the center of the exposed submucosal tissue to create a perforation model after ex-vivo endoscopic submucosal dissection (ESD) (Figure 15(a)). Each sample (powder) was applied to a 10 mm diameter, 1.5 mm thick area of ​​the perforation model using a silicone mold to close the pinhole (Figure 15(b)). 300 μL of PBS was then added dropwise to hydrate (Figure 15(c)). The tissue was then left to stand for 30 minutes to form a colloidal gel. The tissue on which the colloidal gel had formed was fixed to a pressure tester, and air pressure was applied from the gel side at a rate of 2 mL / min to measure the pressure resistance of the colloidal gel (FIG. 15(d)). The amounts of each sample used in the test were as follows: Sample 2-1 (Org = 50 mg), Sample 2-3 (SP / Org = 40 mg / 50 mg), Sample 1-1 (C10 = 50 mg), and Sample 1-3 (SP / C10 = 40 mg / 50 mg).

[0106] As shown in FIG. 16 , compared to samples 2-1 and 2-3 (using raw gelatin Org), samples 1-1 and 1-3 (using gelatin derivative C10) had higher pressure resistance. Similar to the results of the <Adhesion Strength Test>, this is presumably due to hydrophobic interactions between the first particles 10, between the first particles 10 and tissues (cell membranes, cell matrix), and between the second particles and the first particles (e.g., coordinate bonds between the metal-containing second particles and the carboxy groups in the first particles) (see FIG. 3 ). Furthermore, when comparing samples 1-1 and 1-3 in FIG. 16 , sample 1-3, which contains a higher proportion of second particles (SP), tended to have slightly higher pressure resistance. This is presumably due to increased gel strength resulting from interactions between the first particles and the second particles.

[0107] <Heat generation test of powders (gels) after hydration> The heat generation characteristics of gels obtained from Samples 1-1 to 1-4 (powders) in response to an alternating magnetic field (AMF) were measured using a magnetic field generator (G2 D5, Nanoscale Biomagnetics, Zaragoza, Spain). First, each sample (powders) was placed in a silicone mold with a diameter of 10 mm and a thickness of 1.5 mm and hydrated with 300 μL of PBS to prepare a colloidal gel. The resulting colloidal gel was placed on the stage of the magnetic field generator, and an AMF of 130 G and 373.35 kHz was applied to the colloidal gel for up to 600 seconds. The temperature change on the colloidal gel was measured using a thermal camera (Xi 400, Optris, Berlin, Germany). The results are shown in Figure 17. The amount of each sample used in the test was determined to be the predetermined mass ratio shown in Table 1, based on 50 mg of gelatin particles (C10).

[0108] As shown in Figure 17, the heat generation properties improved as the proportion of SP in the sample increased. In particular, Sample 1-3 (SP / C10 = 40 / 50) and Sample 1-4 (SP / C10 = 60 / 50) showed temperatures of 40°C or higher after the application of a magnetic field, which was sufficient to kill cancer cells.

[0109] <Tests (in vitro) using powder as a cancer killing agent> In order to confirm the effect of Sample 1-3 (SP / C10 = 40 / 50) as a cancer killing agent, the following tests (A-1) to (A-4) and tests (B-1) to (B-4) were conducted.

[0110] Test (A-1): First, KM12-Luc cells, which are cancer cells, were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. 3.0 × 10 KM12-Luc cells were placed on an 8-well glass plate. ５ The cells were seeded at a density of 100 cells / well and incubated in 5% CO ２ The cells were incubated at 37°C under ambient atmosphere for 24 hours. Additionally, a gel (colloid gel) was prepared by hydrating Sample 1-3 (SP / C10 = 40 mg / 50 mg) in an 8 mm x 8 mm x 1 mm silicone mold with 300 μL of PBS. The resulting colloid gel was added to incubated KM12-Luc cells, and AMF (130 G, 373.35 kHz) was applied to the colloid gel for 10 minutes. After removing the colloid gel from the medium, the medium was replaced with 10% WST-8 / DMEM and incubated for 2 hours. Finally, the absorbance at 450 nm was measured using the WST-8 assay, and the cell number was counted using a calibration curve. The results are shown in Figure 18.

[0111] Test (A-2): A test was carried out in the same manner as Test (A-1), except that Sample 1-3 (SP / C10) was not used. Test (A-3): A test was carried out in the same manner as Test (A-1), except that AMF was not applied. Test (A-4): A test was carried out in the same manner as Test (A-1), except that Sample 1-3 (SP / C10) was not used and AMF was not applied. The results of Tests (A-2) to (A-4) are shown in Figure 18.

[0112] Tests (B-1) to (B-4): Tests were conducted in the same manner as Tests (A-1) to (A-4), except that normal cells, human mesenchymal stem cells (hMSCs), were used instead of cancer cells. The results are shown in Figure 19.

[0113] As shown in Figure 18, it was confirmed that applying an AFM to Sample 1-3 (SP / C10) (Test A-1) resulted in a high cancer cell killing effect. This is presumably because Sample 1-3 was heated to 40°C or higher by the AFM application (see Figure 17). On the other hand, as shown in Figure 19, when applying an AFM to Sample 1-3 (SP / C10) to normal cells (hMSCs) (Test B-1), cell killing was observed, but the reduction in cell number was not as significant as that observed with cancer cells. This is because normal cells are more resistant to high-temperature environments than cancer cells. These results confirm that applying an AFM to Sample 1-3 (SP / C10) functions as a cancer killing agent with minimal damage to normal cells.

[0114] <Test (in vivo) using powder as cancer killing agent (1)> In order to confirm the effect of Sample 1-3 (SP / C10=40 / 50) as a cancer killing agent, the test described below was carried out.

[0115] First, 2.5 × 10 cultured cancer cells (KM12-Luc cells) were ６ A PBS solution containing 0.1 mL of cancer cells was injected into the subcutaneous tissue of the back of nude mice. After the cancer cell injection, the cancer cells proliferated and the tumor size (tumor volume) increased to 100 mm. ３ Mice that had grown to 100 μL were used as cancer-bearing mouse models in the following tests. The size of the mouse tumor (tumor volume) was determined using the following method. First, 30 mg / mL luciferin (100 μL) was injected into the abdomen of the mouse, and 10 minutes later, luminescence from the tumor was confirmed using an IVIS imaging system (Lumina 2, PerkinElmer Inc., Waltham, Massachusetts, USA). From the luminescence, a: length of the tumor, b: width of the tumor were calculated, and the size of the tumor was calculated according to the following formula: Cancer size (tumor volume) (mm ３ ) = (a b ２ ) / 2 (In the above formula, a: length of the cancer (length of the tumor at its longest point), b: width of the cancer (length of the tumor at its shortest point).)

[0116] Mice were divided into two groups, group (I) (n=5) and group (II) (n=5). After anesthesia, a subcutaneous space was created between the tumor and abdominal tissue in both groups. A gel (10 mm diameter, 1.5 mm thickness) made from sample 1-3 (SP / C10) was implanted only into the subcutaneous space of mice in group (I). The implanted gel was made by hydrating sample 1-3 (SP / C10 = 40 mg / 50 mg) with 300 μL of PBS.

[0117] Mice in groups (I) and (II) were exposed to AMF (130 G and 373.35 kHz) daily for days 1 to 3, and every other day from day 4 onwards, and the skin surface temperature was monitored with a thermal camera. Similarly, tumor size was determined from the luminescence of the tumor using the IVIS observation described above, every day for days 1 to 3, and every other day from day 4 onwards. These AMF exposures and IVIS observations were repeated for 12 days. The changes in tumor size (tumor volume) in mice in groups (I) and (II) are shown in Figure 20.

[0118] After day 12, AMF challenge and IVIS observation were performed every two days to calculate the tumor size (tumor volume) of mice in groups (I) and (II). When the tumor length (a) reached 20 mm, the mice were sacrificed as a humane endpoint and the survival rate was calculated. After 18 days, all mice were sacrificed. The survival rates of mice in groups (I) and (II) are shown in Figure 21.

[0119] As shown in Figure 20, in group (II) (without gel implantation), cancer continued to grow for 12 days from the start of the study. In contrast, in group (I) (with SP / C10 gel implantation), cancer growth was significantly suppressed. Furthermore, as shown in Figure 21, on day 16 from the start of the study, the mouse survival rate in group (II) (without gel implantation) was 20%. In contrast, in group (I) (with SP / C10 gel implantation), the mouse survival rate on day 16 was 100%.

[0120] 6. Powder (2) (Powder Forming Colloidal Gel with Tissue Adhesion and Anticancer Drug Sustained-Release Capability) <Powder Production> C10MP was used as the first particles. Details of the production method are as described above. The average particle diameter of the first particles was 2.3 μm, and the conditions for calculating the average particle diameter of the first particles were also as described above. Paclitaxel (PTX) obtained by reprecipitation of commercially available paclitaxel (PTX) (manufactured by Nacalai Tesque) was used as the second particles. Reprecipitation was performed using the following procedure. (1) Paclitaxel (PTX; 20 mg) was dissolved in 2 mL of ethanol at 25°C to prepare a 1 w / v% PTX solution. (2) While stirring at 400 rpm, 2 mL of ultrapure water at 25°C, a poor solvent, was added dropwise to the solution, allowing the PTX to recrystallize within the solution. (3) The solution was temporarily frozen at -30°C for 24 hours. (4) PTX crystals were obtained by freeze-drying for 24 hours.

[0121] The crystal structures of the commercially available and reprecipitated paclitaxel (PTX) were analyzed by XRD. XRD analysis was performed as follows: the commercially available PTX and the reprecipitated PTX were fixed on a silicon sample holder. The sample holder was set in an XRD device (Powder XRD / SmartLab3, Rigaku) ​​and measurements were performed under the following conditions. Figure 23 shows the X-ray diffraction patterns of commercially available PTX and reprecipitated PTX. As shown in Figure 23, the peak positions of the reprecipitated PTX coincided with those observed for the commercially available PTX, confirming that the reprecipitated PTX had a crystalline structure. Meanwhile, the crystallinity of the commercially available PTX was 74±3%, while that of the reprecipitated PTX was 40±3%. Therefore, it is predicted that the crystallinity can be reduced by reprecipitation, and that the reduction in crystallinity increases the dissolution rate of PTX from the gel. The average particle size of the second particles was 2.8 μm. The calculation conditions for the average particle size were the same as those for the second particles described above.

[0122] As shown in Figure 22, first particles 10 and second particles 50 were mixed in a mass ratio of 50 / 1 (first particles / second particles) to produce powder 60 consisting of C10MP and paclitaxel (PTX) (hereinafter referred to as PTX / C10MP as appropriate) as sample 4.

[0123] <Water Contact Angle of Powder (2)> The powder (PTX: 0.4 mg / C10MP: 20 mg) prepared by the above procedure was weighed into a 2 mL tube and stirred with a vortex mixer for 10 seconds. The mixed particles were attached to a slide glass with double-sided tape (area 1 × 2 cm ２ ) and fixed. A 10 μL water droplet was dropped onto the particle using a contact angle measuring device (DM700, Kyowa Interface Science), and the water contact angle was measured 1 second later. For comparison, the water contact angles of OrgMP, C10MP, and PTX / OrgMP were also measured in the same manner. The measurement results are shown in Figures 24(a) and (b). Figure 24(a) shows a photograph of the shape of a water droplet on each particle or powder, and Figure 24(b) shows the water contact angle of each particle or powder. The water contact angle increased for hydrophobized gelatin particles or powders containing them, while there was no change in the water contact angle with or without PTX.

[0124] <Water Content of Colloidal Gel Formed from PTX / C10MP> The powder (PTX: 1 mg / C10MP: 250 mg) prepared by the above procedure was weighed into a 2 mL tube and vortexed for 10 seconds. The powder was added to a silicone mold (diameter (D): 10 mm x depth: 1 mm) and hydrated with 500 μl of PBS for 30 minutes. After removing excess PBS with a Kimwipe, the mass of the gel (WW) was measured. After freeze-drying the sample, the dry gel mass (WD) was measured. The water content was calculated using the following formula: Water content (%) = 100 × (WW - WD) / WW, where WW is the weight of the colloidal gel in the wet state and WD is the weight of the colloidal gel in the dry state. For comparison, the water content was calculated similarly for gels formed from OrgMP, C10MP, and PTX / OrgMP (PTX: 1 mg / OrgMP: 250 mg). The measurement results are shown in Figure 25. As shown in Figure 25, the water content of the gel formed from OrgMP was lower than that of the gel formed from C10MP. This is thought to be due to an increased crosslink density within the gel due to hydrophobic interactions. This structure is expected to improve pressure resistance.

[0125] <Compression Strength of PTX / C10 Colloid Gel> The compression strength of the colloid gel was measured using a method based on ASTM-F2392-04R with slight modifications. As shown in Figure 26, porcine gastric mucosa was washed, saline was injected into the submucosal tissue with a needle, and the mucosal tissue was then excised with scissors. The mucosal tissue was then cut into 35 mm diameter circles, and a 3 mm diameter pinhole was created in the center of each circle to create a perforation model after ex-vivo ESD (endoscopic submucosal dissection). PTX / OrgMP (PTX: 1 mg / OrgMP: 50 mg) or PTX / C10MP (PTX: 1 mg / C10MP: 50 mg) was sprayed to a thickness of 1 mm over a 15 mm diameter area using a silicone mold, so as to fill the pinhole. After allowing the particles to stand on the tissue for 10 minutes, 500 μL of PBS was added to the particles and left for 30 minutes to form a colloidal gel. The tissue with the gel formed was fixed to a pressure tester, and air pressure was applied from the gel side. The pressure at which the gel ruptured was measured to evaluate the pressure resistance. Figure 27(a) shows the measurement results, and Figure 27(b) shows a schematic diagram of the internal structure of the formed colloidal gel. As shown in Figure 27(a), hydrophobization significantly increased the pressure resistance. Furthermore, mixing PTX powder with C10MPs further increased the pressure resistance. This is thought to be due to the hydrophobic PTX interacting with the hydrophobic groups of gelatin, allowing PTX to act as a composite material. Similar effects are expected for other hydrophobic compounds.

[0126] <PTX sustained-release characteristics of PTX / C10MP colloidal gels> 50 mg of OrgMP or C10-MP and 1.0 mg of paclitaxel were weighed into a 2 mL tube and vortexed for 10 seconds to obtain a powder. For comparison, 1.0 mg of paclitaxel alone was used. PTX / OrgMP, PTXC10MP, or PTX was placed in a silicone mold (diameter (D) = 10 mm), and 500 μL of PBS was added dropwise and allowed to hydrate for 30 minutes to form a gel. The formed colloidal gel was immersed in 50 mL of PBS in a 50 mL centrifuge tube and incubated at 37°C. After 3, 6, 9, 12, 15, and 18 days, the tube was inverted three times to mix. 10 mL of the supernatant was collected for measurement and stored at -30°C. After completely removing the solution on the gel, fresh PBS (50 mL) was added and the mixture was again incubated at 37°C. The collected sample was lyophilized and then dissolved in the eluent described below. The concentration of PTX eluted by HPLC was calculated using the obtained sample. The measurement conditions were as follows: Eluent: acetonitrile / water / methanol = 48 / 41 / 11 (v / v); Flow rate: 1 mL / min; UV wavelength: 242 nm; Column: C18 Nova-Pak column (Waters).

[0127] The measurement results are shown in Figure 28. PTX and PTX / Org were not stable in PBS, and most of the PTX diffused into the PBS within 9 days. On the other hand, PTX / C10 continued to release PTX for 18 days, revealing that 37% of the total PTX was released in 18 days.

[0128] <Test using powder as a cancer killing agent (in vivo) (2)> As shown in Figure 29, human colon cancer-derived cells (KM12-Luc) were suspended in PBS (2.5 x 10 ７ 100 μL of the 100 μL IVF solution (100 μL / mL) was injected into the back of BALB / c nude mice, and after 8 days, the average tumor volume was 75 mm ３It was confirmed that the tumor volume was 0.01 mg / mL. The following gels or suspensions were then implanted or injected into the tissue between the tumor and peritoneum of the mice. Group (I) (n=5): Control (untreated) Group (II) (n=3): PTX (1 mg / mL PTX / PBS suspension) Group (III) (n=3): OrgMP (50 mg) gel Group (IV) (n=3): C10MP (50 mg) gel Group (V) (n=3): PTX / OrgMP (1 mg / 50 mg) gel Group (VI) (n=3): PTX / C10MP (1 mg / 50 mg) gel The tumor volume was determined every 3 days using the following formula: Tumor volume (mm ３ ) = (a b ２ ) / 2 where a: tumor length (length at the longest point of the tumor), b: tumor width (length at the shortest point of the tumor). Tumor imaging was also performed using IVIS every 6 days. IVIS observations were performed 10 minutes after 100 μL of 30 mg / mL luciferin / PBS was injected into the peritoneum of the mice. Figure 30 shows the change in tumor volume over time for each group, and Figure 31 shows the change in survival rate over time for each group. As shown in Figure 30, the PTX / C10MP group showed significantly suppressed tumor growth compared to the other groups on day 12.

[0129] The powder of this embodiment can be used as tissue dressing materials with various functions, such as cancer killing materials and bone filling materials, as well as wound dressing materials, adhesion barriers, and hemostatic materials.

[0130] 10 First particle 20, 50 Second particle 30, 60 Powder 40, 70 Gel

Claims

1. first particles containing a crosslinked gelatin derivative; a second particle that is separate from the first particle and can provide an additional function; The gelatin derivative is a powder having a structure represented by the following formula (1): 【Chemistry 1】 In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, and R １ represents a hydrocarbon group having 1 to 20 carbon atoms, and R ２ represents a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.

2. The powder of claim 1 , wherein the second particles comprise a particulate drug.

3. 3. The powder of claim 2, wherein the agent is hydrophobic or positively or negatively charged.

4. 2. The powder according to claim 1, wherein the ratio (W2 / W1) of the mass (W2) of the second particles to the mass (W1) of the first particles is 20 / 50 to 60 / 50.

5. 5. The powder according to claim 4, wherein the ratio (W2 / W1) of the mass (W2) of the second particles to the mass (W1) of the first particles is 0.1 / 50 to 40 / 50.

6. 5. The powder according to claim 4, wherein the ratio (W2 / W1) of the mass (W2) of the second particles to the mass (W1) of the first particles is 1 / 50 to 60 / 50.

7. The powder according to claim 1, wherein the first particles have an average particle size of 0.1 μm to 100 μm.

8. The powder according to claim 1, wherein the second particles have an average particle size of 5 nm to 100 μm.

9. The powder according to claim 8, wherein the average particle size of the second particles is 10 nm to 100 nm.

10. 2. The powder according to claim 1, wherein a ratio (D2 / D1) of an average particle size of the second particles to an average particle size of the first particles is 0.001 to 0.

1.

11. 2. The powder according to claim 1, which has a water contact angle of 50 degrees or more when measured 1 second after water is dropped onto it.

12. 3. The powder according to claim 2, wherein the drug is at least one selected from the group consisting of anticancer drugs, immunosuppressants, growth factors, cell differentiation inducers, metabolic antagonists, growth factor inhibitors, immune checkpoint inhibitors, antibiotics, anti-inflammatory drugs, and protein preparations.

13. The powder according to claim 12 , wherein the drug comprises at least one selected from the group consisting of a hydrophobic anticancer drug, a hydrophobic immunosuppressant, and a hydrophobic cell differentiation inducer.

14. The powder according to claim 13, wherein the hydrophobic anticancer drug has a crystallinity of 30 to 50% as determined by crystal structure analysis by XRD.

15. 14. The powder of claim 13, wherein the hydrophobic anticancer drug is selected from the group consisting of cabazitaxel, paclitaxel, docetaxel, cisplatin, carboplatin, nedaplatin, oxaliplatin, tamoxifen, anastrozole, Gleevec, 5-fluorouracil (5-FU), floxuridine, leuprolide, flutamide, zoledronate, doxorubicin, vincristine, gemcitabine, streptozocin, vinorelbine, retinoic acid series drugs, adriamycin, mitomycin, daunomycin, prednisone, testosterone, mitoxantrone, aspirin, salicylic acid, ibuprofen, naproxen, fenoprofen, indomethacin, phenylalanine, cyclophosphamide, celecoxib, valdecoxib, and nimesulide.

16. 15. The powder of claim 14, wherein the hydrophobic immunosuppressant is selected from the group consisting of steroids (e.g., hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone), cyclosporine, mizoribine (Bredinin), cyclophosphamide (Endoxan), azathioprine (Azanin), tacrolimus (FK506), sirolimus (rapamycin), deoxyspergualin (gusperimus hydrochloride), everolimus, ABT-578 (zotarolimus), basiliximab (anti-IL-2 receptor monoclonal antibody), muromonab CD3 (anti-CD3 monoclonal antibody), and albulin (antilymphocyte globulin).

17. 15. The powder of claim 14, wherein the hydrophobic cell differentiation inducer is tamibarotene.

18. The powder of claim 2 , wherein the agent comprises a growth factor and the powder further comprises a cationic polymer.

19. 19. The powder of claim 18, wherein the growth factor is selected from the group consisting of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), and Wnt7b.

20. The powder of claim 1 , wherein the second particles comprise inorganic fine particles.

21. 21. The powder of claim 20, wherein the second particles comprise magnetic particles and an anti-cancer drug, preferably a hydrophobic anti-cancer drug.

22. 21. The powder of claim 20, wherein the second particles comprise calcium phosphate and a growth factor.

23. A tissue dressing containing the powder according to any one of claims 1 to 22.

24. A cancer-killing material comprising the powder of claim 21.

25. 25. The cancer killing material of claim 24, wherein the magnetic particles are iron oxide.

26. A bone substitute comprising the powder according to claim 22.

27. The powder according to claim 1 , wherein the second particles are inorganic fine particles.

28. 28. The powder of claim 27, wherein the second particles comprise a metal.

29. The powder according to claim 27, wherein the ratio (W2 / W1) of the mass (W2) of the second particles to the mass (W1) of the first particles is 20 / 50 to 60 / 50.

30. The powder according to claim 29, wherein the ratio (W2 / W1) of the mass (W2) of the second particles to the mass (W1) of the first particles is 20 / 50 to 40 / 50.

31. The powder according to claim 29, wherein the ratio (W2 / W1) of the mass (W2) of the second particles to the mass (W1) of the first particles is 40 / 50 to 60 / 50.

32. The powder according to claim 27, wherein the average particle size of the first particles is 0.1 μm to 100 μm.

33. The powder according to claim 27, wherein the average particle size of the second particles is 5 nm to 100 μm.

34. The powder according to claim 33, wherein the average particle diameter of the second particles is 10 nm to 100 nm.

35. The powder according to claim 27, wherein the ratio (D2 / D1) of the average particle diameter of the second particles to the average particle diameter of the first particles is 0.001 to 0.

1.

36. 28. The powder of claim 27, wherein the second particles are magnetic particles or calcium phosphate.

37. 28. The powder of claim 27, having a water contact angle of 50 degrees or greater when measured 1 second after water is dropped on the powder.

38. A tissue dressing containing the powder according to any one of claims 27 to 37.

39. 37. A cancer-killing material comprising the powder of claim 36, wherein the second particles are magnetic particles.

40. 40. The cancer killing material of claim 39, wherein the magnetic particles are iron oxide.

41. A bone substitute comprising the powder described in claim 36, wherein the second particles are calcium phosphate.

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