JP2005154514A - Functional biodegradable material and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a functional biodegradable material wherein various hydrophilic functional sites or water-soluble polymers are supported on a surface of a biodegradable material such as polylactic acid and a lactic acid-glycolic acid copolymer. <P>SOLUTION: The functional biodegradable material contains the biodegradable material and a surface modifying molecule which modifies the surface of the biodegradable material. The functional surface modifying molecule is represented by the formula: [moiety having affinity for the biodegradable material]-[hydrophilic spacer moiety]<SB>n</SB>-[linker moiety]-[hydrophilic functional moiety] (wherein n is 1 or 0). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biodegradable material whose surface is modified with a functional molecule and a method for producing the same.

Biodegradable materials such as polylactic acid and lactic acid / glycolic acid copolymers are reduced in molecular weight by hydrolysis in vivo, and the degradation products are non-toxic, and thus are widely used as medical materials. For example, biodegradable polymers are used for sutures, carriers for drug delivery systems (drug delivery systems), matrices or support materials for cell culture for regenerative medicine, and the like. When used as a carrier for drug delivery, since it does not require mechanical strength, a relatively low molecular weight (approximately 10 ³ to 10 ⁴ Da) polylactic acid or a copolymer of lactic acid / glycolic acid is used. It is mainly used as a carrier for fat-soluble drugs and physiologically active substances, and side effects of drugs can be reduced by sustained release in vivo.

  In the preparation method, the drug and polylactic acid or lactic acid / glycolic acid copolymer are dissolved in an organic solvent such as dichloromethane, chloroform, acetone or ethyl acetate, and an aqueous solution in which a surfactant such as polyvinyl alcohol (PVA) or lecithin is dissolved. The emulsion is formed in the method, and the organic solvent is evaporated by raising the temperature or reducing the pressure to prepare fine spherical particles (oil-in-water (O / W) method). In the case of a water-soluble drug, a technique in which the drug is dissolved in an aqueous solution such as gelatin in advance and prepared by a water-in-oil-in-water (W / O / W) method is used (Non-patent Document 1). ~ 3).

  In addition, when microparticles made of polylactic acid or a lactic acid / glycolic acid copolymer are administered into the blood, the retention time in the blood is extremely short because it is rapidly trapped in the reticuloendothelial system as in the case of the liposome preparation. The residence time in blood greatly depends on the particle size, and is preferably about 100 to 300 nm. Therefore, a technique is used in which surface modification is performed with a polyoxyethylene chain or the like, which is a water-soluble polymer, so that the reticuloendothelial system is not easily recognized. Specifically, using a diblock copolymer of lactic acid and polyoxyethylene as a surface modifier, polylactic acid microparticles surface-modified with polyoxyethylene chains were prepared by the W / O method, and retained in blood in mice. The time has been successfully extended (Non-Patent Documents 4 to 5).

  Most of the previous studies on microparticles made of polylactic acid or lactic acid / glycolic acid copolymer have extended the residence time in blood by surface modification with polyoxyethylene chains, etc., or sustained release time when used as a drug sustained-release agent The purpose is to control. The sustained release of the drug carried by hydrolysis in the body can extend the effective time and reduce the side effects of the drug. However, as a more advanced function, the drug is accumulated at a pathological site such as a specific organ or tissue. Thus, side effects on other organs or tissues can be greatly reduced. However, in order to recognize and accumulate a specific site on the fine particles, it is necessary to bind the recognition site to the surface. Examples of recognition sites include low molecular weight to high molecular weight compounds such as antibodies, proteins, oligopeptides, polysaccharides, oligosaccharides, and low molecular ligands. However, polylactic acid or lactic acid / glycolic acid copolymer does not have a functional group in the molecule except for the main chain end groups (carboxyl group and hydroxyl group), and cannot recognize the recognition site.

  Actually, a technique of modifying a polylactic acid end group carboxylic acid in order to introduce a thiol group into the surface of a fine particle made of polylactic acid has been disclosed (Non-patent Document 6). For example, after preparing polylactic acid microparticles prepared by the O / W method, the terminal carboxyl group is activated by the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) method to react with cysteine or cysteamine. This introduces a thiol group. Although introduction of about 1000 thiol groups per polylactic acid fine particle has been reported, a method for binding an antibody or a recognition site using this thiol group is not disclosed. The terminal carboxyl groups are not all present on the surface of the fine particles when forming the fine particles by the O / W method, and the surface density is extremely low. Furthermore, the carboxyl groups that are activated by EDC and react with cysteine or cysteamine are a part of them, and as a result, only about 1000 thiol groups can be introduced per particle of polylactic acid fine particles. When a recognition site is further bound to this, the number of particles supported on the particle surface is further reduced, and it is considered that a high molecular weight protein can hardly be supported, and the particles have sufficient recognition ability during flow. Cannot be granted. Furthermore, it is considered that the surface density of the carboxyl group varies greatly depending on the molecular weight or composition ratio of the polylactic acid or lactic acid / glycolic acid copolymer or the preparation method such as the O / W method, and the functional group should be introduced quantitatively. Is considered difficult.

In addition, adsorption of proteins such as antibodies and surface modifiers on the surface of polylactic acid microparticles has been performed, but desorption due to dilution is seen in blood, and desorption / adsorption competitively with plasma proteins. Since the reaction proceeds, it is not realistic to carry the recognition site by physical adsorption.
Quellec, P. et al., J. Biomed. Mater. Res. 42, 45-54, 1998 Murakami, H., et al., Int. J. Pharm. 187, 143-152, 1999 Allemann, E. et al., Int. J. Pharm. 87, 247-253, 1992 Mosqueira VCF et al., Pharm. Res. 18, 1411-1419, 2001 Mosqueira VCF et al., J. Drug Targeting, 7, 65-78, 1999 Nobs, L., et al., Int. J. Pharm., 250, 327-337, 2003

  As described above, a method for stably introducing a sufficient amount of recognition sites on the surface of fine particles using a biodegradable material such as polylactic acid or a lactic acid / glycolic acid copolymer has not been established. To specifically realize a system with a mechanism that recognizes and accumulates specific organs and tissues and gradually releases the drug in a drug carrier using fine particles of polylactic acid or lactic acid / glycolic acid copolymer No effective functional biodegradable material is known. In addition, as a cell culture matrix for regenerative medicine, one that carries a cell adhesion factor is not yet known.

  Therefore, the present invention can be applied to the surface of a biodegradable material such as polylactic acid, lactic acid / glycolic acid copolymer, etc., with hydrophilic functional sites such as antibodies, proteins, polysaccharides or oligosaccharides, peptides, or polyoxyethylene chains. It is an object of the present invention to provide a functional biodegradable material carrying a water-soluble polymer and a method for producing the same.

  The present inventors have modified the surface by co-assembling amphiphilic molecules consisting of a portion having a high affinity for a biodegradable material typified by polylactic acid or a lactic acid / glycolic acid copolymer and a hydrophilic portion. It has been found that a functional biodegradable material can be produced by forming a biodegradable material and attaching a hydrophilic functional moiety via a linker. More specifically, it succeeded in binding a water-soluble polymer such as a polyoxyethylene chain as a recognition site or hydrophilic part such as an antibody, receptor protein, oligosaccharide, polysaccharide or oligopeptide as a functional part, The present invention has been achieved.

That is, according to the present invention, a biodegradable material and a surface modifying molecule that modifies the surface of the biodegradable material, wherein the functional surface modifying molecule has the general formula:
[Part having affinity with biodegradable material]-[Hydrophilic spacer part] _n- [Linker part]-[Hydrophilic functional part]
Provided is a functional biodegradable material, wherein n is 1 or 0.
Further, according to the present invention, the biodegradable material and the functional surface modifying molecule are dissolved in a common solvent, and this is dispersed in a polyvinyl alcohol aqueous solution to form an O / W emulsion, and the solvent is removed. A method for producing a functional biodegradable material in the form of spherical particles is provided.
Furthermore, according to the present invention, the general formula:
[Parts that have affinity with biodegradable materials]-[Hydrophilic spacer parts] _n
(Wherein n is 1 or 0), the amphiphilic compound and the biodegradable material are dissolved in their common solvent and dispersed in an aqueous polyvinyl alcohol solution to form an O / W emulsion. The solvent is removed to obtain spherical particles composed of the biodegradable material surface-modified with the amphiphilic compound, and a hydrophilic functional part is directly or linker on the amphiphilic compound on the spherical particle surface. There is provided a method for producing a spherical functional degradable material, characterized in that bonding is performed via

  According to the present invention, the surface of a biodegradable material such as polylactic acid, lactic acid / glycolic acid copolymer, etc., hydrophilic functional sites such as antibodies, proteins, polysaccharides or oligosaccharides, peptides, polyoxyethylene chains, etc. A functional biodegradable material carrying a water-soluble polymer and a method for producing the same are provided.

  The present invention is described in more detail below.

The functional biodegradable material of the present invention includes a biodegradable material and a functional surface modifying molecule that modifies the surface of the biodegradable material. Functional surface modifying molecules have the general formula:
[Part having affinity with biodegradable material]-[Hydrophilic spacer part] _n- [Linker part]-[Hydrophilic functional part]
It is represented by In the above formula, n is 1 or 0.

The biodegradable material is a material that can be decomposed in a living body by hydrolysis or the like, and includes materials composed of aliphatic polyesters, polysaccharides, polyols, esters thereof, and the like. Such biodegradable materials include, for example, polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymers, polycaprolactone, lactic acid / caprolactone copolymers, polyester compounds such as glycolic acid / caprolactone copolymers, and acetylcellulose. , Chitin, chitosan, polyvinyl alcohol, polyvinyl acetate and the like. Biodegradable materials differ in mechanical strength, hydrolysis rate, moldability, crystallinity, viscoelasticity, etc. depending on the chemical properties, molecular weight, copolymer composition, etc. of the polymer constituting the biodegradable material. Any degradable material can be used. The molecular weight of the polymer such as polylactic acid and lactic acid / glycolic acid copolymer constituting the biodegradable material is desirably 10 ³ Da to 10 ⁵ Da, but is not particularly limited thereto. The copolymer composition of the lactic acid / glycolic acid copolymer may be 50/50 to 90/10 in molar ratio, for example, 70/30, 85/15, 90/10, etc., but is not limited thereto. It is not a thing.

The form of the biodegradable material varies depending on the use, for example, spherical fine particles when used as a carrier for drug delivery, and a tube or plate shape for an implantable sustained-release material. When used as a tissue culture matrix for regenerative medicine, it is a porous columnar or pellet, cast membrane, or a shape suitable for each tissue or organ. When used as a suture, it is fibrous. When used as a joining material used for joining a fractured part or an osteotomy part, it has a screw shape or a pin shape. Thus, although the shape of a biodegradable material changes with uses, the biodegradable material used for this invention should just be a shape suitable for the use. The following description is a spherical biodegradable material suitable for drug delivery, but is not particularly limited to this shape or this biodegradable material. In the case of spherical fine particles, the biodegradable material preferably has a diameter of about 50 nm to 10 μm.
The polylactic acid or lactic acid / glycolic acid copolymer has a hydrophobic structure because it does not have a water-soluble functional group in the main chain or side chain. For example, the surface of fine particles prepared from polylactic acid or a lactic acid / glycolic acid copolymer by an O / W method, an oil-in-oil (O / O) method, or a W / O / W method is coated with polylactic acid or lactic acid • It is considered that the carboxyl group present at the end of the glycolic acid copolymer is slightly present. In order to obtain fine particles of polylactic acid or lactic acid / glycolic acid copolymer, polylactic acid or lactic acid / glycolic acid copolymer dissolved in an organic solvent such as dichloromethane, chloroform or ethyl acetate is dispersed in an aqueous solution of polyvinyl alcohol. After the emulsion is formed, the solvent is removed under reduced pressure. Polyvinyl alcohol is adsorbed on the surface of the obtained fine particles, and this improves the dispersion stability. When polyvinyl alcohol is not present, the dispersion stability of the fine particles is remarkably lowered and tends to aggregate rapidly. When the terminal carboxyl group of polylactic acid or lactic acid / glycolic acid copolymer is used for the reaction, the density of the carboxyl group present on the surface of the fine particle changes depending on the conditions at the time of emulsion formation. Since it is covered with polyvinyl alcohol, it is considered difficult to introduce a high molecular weight recognition site even if a low molecule such as a thiol group can be introduced.

  Therefore, in one aspect of the present invention, a molecule having both a hydrophilic part and a part having affinity with the biodegradable material, that is, a formula [part having affinity with the biodegradable material]-[hydrophilic spacer. The molecule represented by [Part] is oriented on the surface of fine particles made of a biodegradable material, a linker is introduced at the end of the hydrophilic spacer part of this molecule, and then a hydrophilic functional part is bonded. As an example of such a molecule, 1,2-dipalmitoyl 3-sn-glycerophosphatidylethanolamine (DPPE) is used as an amphipathic molecule having a part having affinity for a biodegradable material as a hydrophobic part. 1,2-distearyl 3-sn-glycerophosphatidylethanolamine (DSPE), 1,4-dipalmitoyl glutamate (DPE), 1,4-distearyl glutamate (DSE), L-glutamic acid, lysine, or A compound having a dendritic branched structure described in Japanese Patent No. 3181276, or having a maleimide at one end of a polyoxyethylene chain and the other end having an amide bond or urethane bond with the amino group of the molecule Polyoxyethylene-binding lipid, polyoxyethylene having polyamide and terminal maleimide As a hydrophobic part of a compound having a hydrophilic part and a hydrophobic part in the molecule, such as a diblock copolymer with a chain, a diblock copolymer of a polyester and a polyethylene oxide chain having a maleimide at the terminal, or the compounds shown above , An alkyl chain having 12 or more carbon atoms, or an alkenyl chain having 1 to 4 unsaturated bonds and having 12 or more carbon atoms. Also included is a case where a water-soluble molecule such as polyoxyethylene is bonded to the third hydroxyl group of glycerol in which two alkyl chains or alkenyl groups are bonded to the 1,3-position or 1,2-position of glycerin. It is. Illustrative of these hydrophobic groups are primary alcohol and secondary alcohol residues having 12 to 22 alkyl carbon atoms. That is, examples of the linear primary saturated alcohol include lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol and the like, and other linear primary unsaturated compounds such as 1,1-dodecenol, 1-oleyl alcohol, and linoleinyl alcohol. Alcohol residues of alcohol, branched primary saturated alcohol, branched primary unsaturated alcohol, secondary saturated alcohol or secondary unsaturated alcohol are listed. Moreover, the dialkylglycerol residue couple | bonded with the said 1st saturated alcohol and the 1st unsaturated alcohol at the 1,3 position or the 1,2 position of glycerol is mention | raise | lifted. Further, alcohol residues such as sterols such as cholesterol, cholestanol, sitosterol and ergosterol are also included in the range of hydrophobic groups.

The hydrophilic spacer portion is not particularly limited as long as it is a water-soluble polymer and biocompatible. Examples include polyoxyethylene, polysaccharide, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, chitosan or polyol, polyacrylic acid, polymethacrylic acid, poly L-lysine, poly L-glutamic acid, chitosan, alginic acid, polyvinyl. Alcohol or derivatives thereof. The molecular weight of these hydrophilic spacer portions may be any molecular weight as long as the sufficient spacer effect can be maintained without inhibiting the recognition ability of the functional molecule, but is preferably 400 Da to 10,000 Da, and preferably 1000 Da to 5000 Da. The compounds having a dendritic branch structure described in Japanese Patent No. 3181276 include compounds represented by the following structural formulas 1 and 2.

In Structural Formula 1 and Structural Formula 2, q is 1 to 4, R ₀ is a hydrogen atom, and R ₁ and R ₂ each independently have an alkyl group, particularly 1 to 30 carbon atoms. It is an alkyl group.
Specifically, after surface modifying molecules are dissolved in an organic solvent such as polylactic acid or lactic acid / glycolic acid copolymer in chloroform, dichloromethane, ethyl acetate or the like, fine particles are formed by the O / W method. The particle size of the fine particles can be controlled by the stirring speed at the time of forming the emulsion. For example, the particle size is 150 ± 50 nm when stirred at 3000 rpm, the particle size is 350 ± 150 nm when stirred at 1000 rpm, and the particle size is 1500 when stirred at 800 rpm. ± 750 nm. In this case, various forms of functional materials can be prepared by forming into pellets, plates, tubes, fibers, or the like.

  In addition to the O / W method, the emulsion method is widely applied to the fine particle preparation method such as the O / O method or the O / W / O method. Further, the surface modifying molecule is not limited to the above compound as long as the molecular structure is similar. The particle diameter of the fine particles thus prepared is 10 nm or more and 10 μm or less, preferably about 100 nm to 300 nm.

  The surface modification molecules are oriented at the fine particle interface, and the hydrophobic part faces the fine particle surface inside and the hydrophilic part faces the water phase side of the fine particle surface, so that the interface modification is possible. Accordingly, the form of the biodegradable material is not limited to the particles as described above. Even if the shape and size of the biodegradable material, the particle size of the fine particles, or the copolymer composition of the biodegradable polymer is changed, any surface modification is possible.

  By the above-described method, for example, various hydrophilic functional parts can be bonded to fine particles comprising a surface modifying molecule and polylactic acid or lactic acid / glycolic acid via a linker. Examples of hydrophilic functional molecules include antibodies (subclasses such as IgG, IgM, IgE, IgA, IgD and their degradation parts), receptor proteins, oligopeptides as their ligands, enzymes, polysaccharides, etc. Examples include functional molecules such as hepatocyte recognition sites such as galactose, N-acetylgalactosamine, mannose, N-acetylglucosamine, fucose, and sialic acid, and RGD which is a collagen recognition site and platelet membrane protein GPIa / GPIbα which is a receptor for IIa complex or von Willebrand factor, GPIIb / IIIa which is a receptor for fibrinogen, and an oligopeptide containing fibrinogen which is a receptor for GPIIb / IIIa or a dodecapeptide which is a recognition site thereof, or , Sugar recognition Chin family and the selectin family, and adhesion molecules such as integrin family and the cadherin family, the receptor for the extracellular matrix.

The linker is not particularly limited as long as it is capable of crosslinking with the SH group introduced into the fine particle and the functional site and has a known biotolerability. Preferably, one end reacts with the SH group and the other end is the OH group. , A compound that reacts with either a COOH group or an NH ₂ group. Examples of such a linker include dicarboxylic acid, aminocarboxylic acid, bismaleimide compound, bishalocarbonyl compound, halocarbonylmaleimide compound, dithiomaleimide, dithiocarboxylic acid and maleimide carboxylic acid. It preferably has a carbon chain of C ₂ -C _10.

  In addition, the surface modification molecule to which a functional molecule such as oligosaccharide or oligopeptide is bonded in advance is similarly dissolved in an organic solvent such as polylactic acid or lactic acid / glycolic acid copolymer and chloroform, followed by the O / W method. The fine particle functional biodegradable material of the present invention can also be prepared by forming fine particles by the above method. However, in that case, it is necessary that the amphiphilic molecule including the functional molecule is dissolved in the organic solvent.

  Furthermore, in the case of a cast film, sponge, or pellet, a polylactic acid or a lactic acid / glycolic acid copolymer and an amphiphilic molecule are dissolved in an organic solvent such as chloroform in the same manner, and then molded. Remove the solvent and bind the water-soluble functional molecules.

  It is possible to encapsulate a fat-soluble drug in the O / W method and a water-soluble drug in the biodegradable material by the W / O / W method, and realize a sustained release at a specific site recognized by the functional molecule. Can be greatly reduced. Drugs included in biodegradable materials include anticancer agents, antibiotics, enzyme agents, enzyme inhibitors, antioxidants, lipid uptake inhibitors, hormone agents, anti-inflammatory agents, steroid agents, vasodilators, angiotensin converting enzyme inhibitors Agent, angiotensin receptor antagonist, smooth muscle cell proliferation / migration inhibitor, platelet aggregation inhibitor, anticoagulant, chemical mediator release inhibitor, vascular endothelial cell proliferation or inhibitor, aldose reductase inhibitor, mesangial Cell growth inhibitor, lipoxygenase inhibitor, immunosuppressant, immunostimulator, antiviral agent, Maillard reaction inhibitor, amyloidosis inhibitor, NOS inhibitor, AGEs inhibitor or radical scavenger, protein, peptide, nucleic acid, poly Examples include, but are not limited to, nucleotides, genes and their analogs No.

  For example, examples of anticancer agents include cyclophosphamide, ifosfamide, nitrogen mustard-N-oxide, thiotepa, brusphan, carbocon, nimustine hydrochloride, ranimustine, melphalan, inprosulfan tosylate, dacarbazine, procarbazine hydrochloride, Cytarabine, cytarabine oxfert, enositabine, mercaptopurine, thioinosine, fluorouracil, doxyfluridine, tegafur, methotrexate, carmofur, hydroxycarbamide, vincristine sulfate, vinblastine sulfate, vindesine sulfate, etoposide, chromomycin A3, daunorubicin hydrochloride, doxorubicin hydrochloride hydrochloride Pirarubicin, epirubicin hydrochloride, dactinomycin, mitoxantrone hydrochloride, bleo Isine, pepromycin sulfate, mitomycin C, neocarnostatin, L asparaginase, acegraton mitopronitol, sodium dextran sulfate, octreotide acetate, cisplatin, carboplatin, tamoxifen citrate, medroxyprogesterone acetate, estramustine phosphate sodium, acetic acid Examples include goserelin, leuprorelin acetate, and irinotecan hydrochloride.

  Examples of antibiotics include benzylpenicillin potassium, benzylpenicillin benzathine, phenoxymethyl penicillin potassium, pheneticillin potassium, cloxacillin sodium, flucloxacillin sodium, ampicillin, sultamicillin tosylate, bacampicillin hydrochloride, tarampicillin hydrochloride, lenampicillin , Hetacillin potassium, cyclacillin, amoxicillin, pibmesilinum hydrochloride, aspoxicillin, carbenicillin sodium, calindacillin sodium, sulbenicillin sodium, ticarcillin sodium, piperacillin sodium, cephalolidine, cephalothin sodium, cefazolin sodium, cefapirin sodium, cefradine, cephalexin , Cephatrizine propylene glycol, cefloxa , Cefaclor, cefadroxyl, cefotiam hydrochloride, cefotiam hexetyl, cefuroxime sodium, cefuroxime axetil, cefamandol sodium, cefdinir, cefetamethopoxyl hydrochloride, ceftipten, cefmetazole sodium, cefoxitin sodium, Cefotetan sodium, cefminox sodium, cefpuperazone sodium, cefpyramide sodium, cefthrodine sodium, cefotaxime sodium, cefoperazone sodium, ceftizoxime sodium, cefmenoxime hydrochloride, ceftriaxone sodium, ceftazidime, cefpimi Zole sodium, cefixime, cefterampipoxil, cefzonam sodium, cefpodoxyproxetyl, cefodizime, cefpirom sulfate Ratamoxef sodium, flomoxef sodium, imipenem, cilastatin sodium, aztreonam, carmonam sodium, strepeptomycin sulfate, kanamycin sulfate, fradiomycin sulfate, amikacin sulfate, gentamicin sulfate, paromomycin sulfate, pecanamicin sulfate, liposta sulfate Mycin, dibekacin sulfate, tobramycin, sisomycin sulfate, mythromycin sulfate, astronomycin sulfate, netilmycin sulfate, isepamicin sulfate, arbekacin sulfate, erythromycin, kitasamycin, acetyl kitasamycin, oleandomycin phosphate, josamycin, acetyl spiramycin, midecamycin , Midecamycin acetate, rokitamycin, roxithromycin, clarithromycin, tetrasai hydrochloride Culin, oxytetracycline hydrochloride, tetracycline metaphosphate, demethylchlortetracycline hydrochloride, loritetracycline, doxycycline hydrochloride, minocycline hydrochloride, chloramphenicol, chloramphenicol sodium succinate, chloramphenicol palmitate, thianphenicol, hydrochloric acid Examples include aminoacetic acid thianphenicol, colistin sulfate, colistin sodium methanesulfonate, polymyxin B sulfate, bacitracin, vancomycin hydrochloride, lincomycin hydrochloride, clindamycin, spectinomycin hydrochloride, fosfomycin sodium, and fosfomycin calcium.

  Examples of enzyme agents include chymotrypsin, crystalline trypsin, streptokinase / streptodolase, hyaluronidase, urokinase, nasarplase, alteplase, lysozyme chloride, semi-alkaline proteinase, serrapeptase, tisokinase, duteprase, batroxobin, pronase, promeline, etc. .

  Examples of antioxidants include tocopherol, ascorbic acid, uric acid and the like.

  Examples of anti-inflammatory agents include choline salicylate, sazapyrine, sodium salicylate, aspirin, diflunisal, flufenamic acid, mefenamic acid, fructaphenine, tolfenamic acid, diclofenacnatrim, tolmetin sodium, sulindac, fenbufen, ferbinacethyl, indomethacin, indomethacin farnesyl, Acemetacin, Progouritacin maleate, Ampenac sodium, Nabumetone, Ibuprofen, Flurbiprofen, Flurbiprofen axetil, Ketoprofen, Naproxen, Protidic acid, Planoprofen, Fenoprofen calcium, Thiaprofenic acid, Oxaprozin, Loxoprofen Sodium, aluminoprofen, zaltoprofen, phenylbutazone, clofezone Keto phenylbutazone, piroxicam, tenoxicam, ampiroxicam, tiaramide hydrochloride, hydrochloric tinoridine, benzydamine hydrochloride, epirizole, Erumofazon and the like.

  Examples of steroids include cortisone acetate, hydrocortisone (phosphate ester, acetate salt), hydrocortisone butyrate, hydrocortisone sodium succinate, prednisolone (acetate, succinate, tertiary butyl acetate ester, phosphate ester), methylprednisolone (acetate) ), Methylprednisolone sodium succinate, triamcinolone, triamcinolone acetonide (triamcinolone acetate), dexamethasone (phosphate ester, acetate salt, sodium phosphate salt, sulfate ester), dexamethasone palmitate, betamethasone (phosphate salt, sodium salt) , Acetic acid parameterzone, fludrocortisone acetate, halopredone acetate, clobetasol propionate, halcinonide, beclomethasone propionate, betametharate valerate Emissions, betamethasone acetate, cortisone acetate, and the like.

  Examples of angiotensin converting enzyme inhibitors include alacepril, imidapril hydrochloride, temocapril hydrochloride, delapril hydrochloride, benazepril hydrochloride, captopril, cilazapril, enalapril maleate, lisinopril and the like. Examples of vasodilators include theophylline, diprofylline, proxyphylline, aminophylline, choline theophylline, prostaglandin, prostaglandin derivatives, alprostadil alphadex, alprostadil, limaprost alphadex, papaverine, cyclandelate, cinnarizine, fumarate Bencic acid, cinepazide maleate, dirazep hydrochloride, trapidil, diphenidol hydrochloride, nicotinic acid, inositol hexanicotinate, nicamethate citrate, nicotinic alcohol tartrate, tocopherol nicotinate, hepronicart, isoxpurine hydrochloride, bamethane sulfate, tolarizone hydrochloride, dihydromesylate Ergotoxin, ifenprodil tartrate, moxysilito hydrochloride, nicergoline, nicardipine hydrochloride, nilva Pin, nifedipine, benidipine hydrochloride, diltiazem hydrochloride, nisoldipine, nitrendipine, manidipine hydrochloride, balnidipine hydrochloride, efonidipine hydrochloride, verapamil hydrochloride, trimetazidine hydrochloride, captopril, enalapril maleate, alacepril, delapril hydrochloride, cilazapril, ricinopril hydrochloride, benzapril hydrochloride hydrochloride Examples include todralazine hydrochloride, budralazine, cadralazine, indapamide, carbochromene hydrochloride, efloxate, etaphenone hydrochloride, oxyfedrine hydrochloride, nicorandil, amyl nitrite, and isosorbide nitrate.

  Examples of the smooth muscle cell migration / proliferation inhibitor include heparin sodium, dalteparin sodium (low molecular weight heparin), heparin calcium, dextran sulfate and the like.

  Examples of the platelet aggregation inhibitor include ticlopidine hydrochloride, cilostazol, ethyl icosapentate, beraprost sodium, sarpgrelate hydrochloride, batroxobin, dipyridamole and the like.

  Examples of the anticoagulant include heparin sodium, dalteparin sodium (low molecular heparin), heparin calcium, dextran sulfate, warfarin potassium, argatroban and the like.

  Examples of the chemical mediator release inhibitor include tranilast, ketofetin fumarate, azelastine hydrochloride, oxatomide, amlexanox, and repirinast.

  Examples of immunosuppressive agents include cyclosporine and the like.

  Examples of the antiviral agent include acyclovir, ganciclovir, didanosine, zidovudine, sorivudine, vidarabine and the like.

  Moreover, the kind of the drug for diagnosis to be enclosed is not particularly limited as long as the formation of the drug carrier is not impaired. Specific examples include X-ray contrast agents, radioisotope-labeled nuclear medicine diagnostic agents, diagnostic agents for nuclear magnetic resonance diagnosis, and the like. For example, X-ray contrast agents include meglumine amidotrizoate, sodium iotalamate, meglumine iotalamate, gastrografin, iodamide meglumine, lipiodol ultrafluid, adipiodon meglumine, oxagrate, meglumine iotroxate, iotrolan, iopanoic acid Iopamidol, iohexol, ioversol, iopodate sodium, iomeprol, isopaque, iodoxamic acid and the like.

  As an application example, for example, when a recombinant GPIbα or GPIa / IIa of platelet membrane protein is supported on microparticles made of a biodegradable material having a particle size of about 50 nm to 10 μm by the above method, it is used as an intravenous hemostatic agent. it can. In addition, when a biodegradable material into which a cell adhesion factor such as RGD is introduced by the same method is used, cells can be effectively immobilized as a matrix for various tissue formation as a regenerative medical material. It can also be used as a release agent from a biodegradable material placed at a specific site in the body by intramuscular injection or the like.

Hereinafter, a method for binding IgG (molecular weight 160 kDa) is exemplified, but the present invention is not limited to this. In a chloroform solution, a compound represented by the structural formula 1 (provided that q = 4, R ₁ and R ₂ = an alkyl group having 18 carbon atoms) and a crosslinking agent N-succinimidyl 3- (2-pyridyldithio) propionate ( SPDP) was reacted to introduce pyridyldithio (PD) groups and mixed with polylactic acid to prepare polylactic acid microparticles having PD groups introduced by the O / W method. In addition, a PD group can be introduced into IgG by SPDP, reduced to a thiol group by dithiothreitol, and then mixed with PD group-introduced polylactic acid fine particles to bond them together. Since the molecular modification to IgG can be minimized, it is possible to suppress functional degradation.

  Further, polyoxyethylene chain-bonded glutamic acid type lipid having a maleimide group at the terminal was mixed with polylactic acid to prepare polylactic acid microparticles having a maleimide group introduced by the O / W method. In addition, a PD group can be introduced into IgG by SPDP, reduced to a thiol group by dithiothreitol, and then mixed with maleimide group-introduced polylactic acid fine particles to bond both. Since IgG is bonded to the terminal via a polyoxyethylene chain, a decrease in IgG recognition ability can be suppressed by a so-called spacer effect. In addition, the surface modification of polylactic acid with a polyoxyethylene chain is also possible.

  Further, a polyblock having a hydrophobic part of polyamide or polyester and a hydrophilic part with a polyoxyethylene chain having a terminal maleimide group is mixed with polylactic acid as a surface modifying molecule, and a polyimide having a maleimide group introduced by the O / W method. Lactic acid fine particles were prepared. In addition, a PD group can be introduced into IgG by SPDP, reduced to a thiol group by dithiothreitol, and then mixed with maleimide group-introduced polylactic acid fine particles to bond both. As in the previous section, since IgG is bound to the end via a polyoxyethylene chain, a decrease in IgG recognition ability can be suppressed by a so-called spacer effect, and surface modification of polylactic acid with a polyoxyethylene chain is also possible. . In addition, polyamide or polyester, which is a hydrophobic part, has good compatibility with polylactic acid. In particular, in the case of polyamide, hydrogen bonds are also involved, so that a compound having a higher molecular weight than IgG can be stably supported. It becomes possible.

[Example]
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1
Lactic acid / glycolic acid copolymer (lactic acid / glycolic acid molar ratio = 85/15, hereinafter PLGA) (20 mg) was dissolved in chloroform (10 mL) and gradually dissolved in a 1 wt% aqueous solution of polyvinyl alcohol (PVA) (100 mL). The mixture was stirred for 30 minutes and the solvent was removed with an evaporator to obtain PLGA fine particles. Unbound PVA was removed by centrifugation (10000 rpm, 30 minutes). The particle diameter of the obtained PLGA fine particles correlated with the stirring speed. Specifically, the particle size was 150 ± 50 nm when stirred at 3000 rpm, the particle size was 350 ± 150 nm when stirred at 1000 rpm, and the particle size was 1500 ± 750 nm when stirred at 800 rpm.

Example 2
PLGA (20 mg) and DSPE (2.0 mg) were dissolved in chloroform (10 mL) and dropped into a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in phosphate buffer (PBS) for 30 minutes. After stirring (stirring speed: 850 rpm), the solvent was removed with an evaporator. Unbound phospholipid and PVA were removed by centrifugation (2500 rpm, 30 minutes) to obtain PLGA fine particles having a particle size of 950 ± 420 nm in which lipid DSPE was oriented on the surface.

N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (6.2 mg) was dissolved in 99.5% ethanol solution (1 mL) to obtain an SPDP solution having a concentration of 20.0 × 10 ⁻³ mol / L. . The SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to the above-mentioned dispersion (4 mL) of PLGA fine particles adjusted to a concentration of 0.55 wt%, and stirred at 23 ° C. for 2 hours. PLGA microparticles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the SPDP-coupled PLGA microparticle dispersion was made up to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLGA fine particles. The binding ratio between the PLGA fine particles and IgG was measured by a fluorescence measurement method using fluorescein isothiocyanate (FITC), and was PLGA fine particles / IgG = 70/1 (weight ratio).

Example 3
PLGA (20 mg) and 1,4-dipalmitoyl-glutamate (DPE) (1.8 mg) were dissolved in chloroform (10 mL) and added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS. Then, after stirring for 30 minutes (stirring speed: 750 rpm), the solvent was removed with an evaporator. Unbound lipid and PVA were removed by centrifugation (2500 rpm, 30 minutes) to obtain PLGA microparticles having a particle size of 2600 ± 1200 nm with the lipid oriented on the surface.

An SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to the dispersion (4 mL) of the PLGA fine particles adjusted to a concentration of 0.55 wt%, and stirred at 23 ° C. for 2 hours. PLGA microparticles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the SPDP-coupled PLGA microparticle dispersion was made up to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLGA fine particles. The binding ratio between the PLGA fine particles and IgG was measured by a fluorescence measurement method using FITC, and was PLGA fine particles / IgG = 66/1 (weight ratio).

Example 4
Polylactic acid (PLA) (20 mg) and DPPE (1.9 mg) were dissolved in chloroform (10 mL), and the resulting solution was dropped into a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS. Then, after stirring for 30 minutes (stirring speed: 850 rpm), the solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 950 ± 400 nm with DPPE oriented on the surface were obtained.

An SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to 4 mL of the PLA fine particle dispersion adjusted to a concentration of 0.55 wt%, and the mixture was stirred at 23 ° C. for 2 hours. PLGA microparticles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the volume of the SPDP-bound PLA microparticle dispersion was adjusted to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLA microparticles. The binding ratio between PLA fine particles and IgG was measured by a fluorescence measurement method using FITC, and was PLA fine particles / IgG = 68/1 (weight ratio).

Example 5
PLA (20 mg) and 1,4-distearoyl-glutamate (DSE) (1.8 mg) were dissolved in 10 mL of chloroform, and the obtained solution was dissolved in 1 wt% PVA aqueous solution (pH 7.4; 100 mL) dissolved in PBS. The solution was dropped into the solution, stirred for 30 minutes, and then the solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 2500 ± 1200 nm with DSE oriented on the surface were obtained.

An SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to 4 mL of the PLA fine particle dispersion adjusted to a concentration of 0.55 wt%, and the mixture was stirred at 23 ° C. for 2 hours. PLA fine particles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the volume of the SPDP-bound PLA fine particle dispersion was adjusted to 4 mL. Next, a 20 mM SPDP solution (2.3 μL) was reacted with 7.5 wt% IgG (60 μL) (room temperature, 30 minutes), and 6 mM of 200 mM DTT was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLA microparticles. The binding ratio between PLA fine particles and IgG was measured by a fluorescence measurement method using FITC and was PLA fine particles / IgG = 62/1 (weight ratio).

Example 6
PLA (20 mg) and a compound represented by the structural formula 1 (where q = 4, R ₁ and R ₂ = 18 carbon atoms alkyl group) (4.0 mg) were dissolved in chloroform (10 mL), and PVA dissolved in PBS Was added dropwise to a 1% by weight aqueous solution (pH 7.4; 100 mL), stirred for 30 minutes (stirring speed: 800 rpm), and then the solvent was removed by an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 1400 ± 800 nm with lipids oriented on the surface were obtained.

An SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to 4 mL of the PLA fine particle dispersion adjusted to a concentration of 0.55 wt%, and the mixture was stirred at 23 ° C. for 2 hours. PLA fine particles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the volume of the SPDP-bound PLA fine particle dispersion was adjusted to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLA microparticles. The binding ratio between PLA fine particles and IgG was measured by a fluorescence measurement method using FITC, and was PLA fine particles / IgG = 78/1 (weight ratio).

Example 7
DPGA (Mal-PEG-DPPE) (8.2 mg) having PLGA (20 mg) and a polyethylene oxide chain having a maleimide at the end bound thereto was dissolved in 10 mL of chloroform, and the resulting solution was added to 1 weight of PVA dissolved in PBS. % Aqueous solution (pH 7.4; 100 mL), and after stirring for 30 minutes (stirring speed: 850 rpm), the solvent was removed with an evaporator. Unreacted PEG lipids and PVA were removed by centrifugation (2500 rpm, 30 minutes) to obtain PLGA fine particles with a particle size of 1000 ± 50 nm in which Mal-PEG-DPPE was oriented on the surface.

  The PLGA fine particle dispersion was made up to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLGA fine particles. The binding ratio between PLGA microparticles and IgG was measured by the micro BCA method and was PLGA microparticles / IgG = 52/1 (weight ratio).

Example 8
DPE (Mal-PEG-DPE) (7.2 mg) having PLGA (20 mg) and a polyethylene oxide chain having maleimide at the end bound thereto was dissolved in chloroform (10 mL), and a 1 wt% aqueous solution of PVA (pH 7) dissolved in PBS. .4; 100 mL) and stirred for 30 minutes (stirring speed: 750 rpm), and then the solvent was removed by an evaporator. By removing free PEG lipids and PVA by centrifugation (2500 rpm, 30 minutes), PLGA fine particles having a particle size of 2400 ± 1100 nm with Mal-PEG-DPE oriented on the surface were obtained.

  The PLGA fine particle dispersion was made up to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLGA fine particles. The binding ratio between the PLGA fine particles and IgG was measured by the micro BCA method, and was PLGA fine particles / IgG = 45/1 (weight ratio).

Example 9
PLA (20 mg) and Mal-PEG-DSPE (8.5 mg) were dissolved in 10 mL of chloroform and added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS and stirred for 30 minutes (stirring speed). 850 rpm), the solvent was removed by an evaporator. By removing unbound Mal-PEG-DSPE and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle diameter of 1100 ± 550 nm in which Mal-PEG-DSPE was oriented on the surface were obtained.

  The volume of the PLA fine particle dispersion was adjusted to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLGA fine particles. The binding ratio between PLA fine particles and IgG was measured by the micro BCA method, and was PLGA fine particles / IgG = 52/1 (weight ratio).

Example 10
PLA (20 mg) and Mal-PEG-DSE (7.8 mg) were dissolved in 10 mL of chloroform and added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred for 30 minutes (stirring speed). 750 rpm), the solvent was removed with an evaporator. By removing unbound Mal-PEG-DSE and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 2400 ± 1000 nm with Mal-PEG-DSE oriented on the surface were obtained.

  The volume of the PLA fine particle dispersion was adjusted to 4 mL. Next, 7.5% by weight IgG (60 μL) was reacted with 20 mM SPDP solution (2.3 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-IgG obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted IgG was separated by centrifugation (2500 rpm, 30 minutes) to obtain IgG-PLA microparticles. The binding ratio between PLA fine particles and IgG was measured by the micro BCA method, and was PLA fine particles / IgG = 55/1 (weight ratio).

Example 11
PLA (20 mg) and DPPE (1.9 mg) were dissolved in chloroform (10 mL) and added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS. After stirring for 30 minutes (stirring speed: 750 rpm), The solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 2500 ± 1100 nm with DPPE oriented on the surface were obtained.

20.0 × 10 ⁻³ mol / L SPDP solution (600 μL) was added to the PLA fine particle dispersion (4 mL) adjusted to a concentration of 0.55 wt% and stirred at 23 ° C. for 2 hours. PLA fine particles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the volume of the SPDP-bound PLA fine particle dispersion was adjusted to 4 mL. Next, 60 μL of tridecane peptide (CHHLGGAKQAGGDV, 7.5% by weight) was added to this PLA dispersion, followed by stirring at 4 ° C. for 30 hours. Unreacted substances were separated by centrifugation (2500 rpm, 30 minutes) to obtain tridecane peptide-PLA fine particles. The binding ratio between the PLA fine particles and the tridecane peptide was measured by a fluorescence measurement method using FITC, and was PLA fine particle tridecane peptide = 44/1 (weight ratio).

Example 12
PLA (20 mg) and Mal-PEG-DSE (7.8 mg) were dissolved in chloroform (10 mL), added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred for 30 minutes ( After stirring (750 rpm), the solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 2500 ± 1200 nm with Mal-PEG-DSE oriented on the surface were obtained.

20.0 × 10 ⁻³ mol / L SPDP solution (600 μL) was added to the PLA fine particle dispersion (4 mL) adjusted to a concentration of 0.55 wt% and stirred at 23 ° C. for 2 hours. PLA fine particles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the volume of the SPDP-bound PLA fine particle dispersion was adjusted to 4 mL. Next, 70 μL of tridecane peptide (CHHLGGAKQAGGDV, 7.5% by weight) was added to this PLA dispersion, followed by stirring at 4 ° C. for 30 hours. Unreacted substances were separated by centrifugation (2500 rpm, 30 minutes) to obtain tridecane peptide-PLA fine particles. The binding ratio between the PLA fine particles and the tridecane peptide was measured by a fluorescence measurement method using FITC, and was PLA fine particles / tridecane peptide = 42/1 (weight ratio).

Example 13
After DSPE (10 mg) and SPDP (30 mg) were reacted in a chlorophore solution, PD-DSPE (8 mg) was obtained with a silica gel column (developing solvent: chloroform / methanol = 8/1). Under a nitrogen atmosphere, PD-DSPE (8 mg) and tridecane peptide (10 mg) were reacted in methanol and washed with pure water to obtain tridecane peptide-DSPE (8 mg).

  PLGA (20 mg) and tridecane peptide-DSPE (1.5 mg) were dissolved in chloroform (10 mL), dropped into 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred for 30 minutes. The solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLGA fine particles having a particle size of 2549 ± 1000 nm in which tridecane peptide-DSPE or tridecane peptide-DSPE was oriented on the surface were obtained. The binding ratio between the PLGA fine particles and the tridecane peptide was measured by a fluorescence measurement method using FITC, and was PLGA fine particles / tridecane peptide = 52/1 (weight ratio).

Example 14
Mal-PEG-DSE (10 mg) and tridecane peptide (8 mg) were reacted in methanol and washed with pure water to obtain tridecane peptide-PEG-DSE (7 mg).

  PLA (20 mg) and tridecane peptide-PEG-DSE (2.5 mg) were dissolved in 10 mL of chloroform, dropped into 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred for 30 minutes. The solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 2549 ± 1000 nm in which tridecane peptide-PEG-DPE or tridecane peptide-PEG-DSE is oriented on the surface are obtained. Obtained. The binding ratio between the PLA fine particles and the tridecane peptide was measured by a fluorescence measurement method using FITC, and was PLA fine particles / tridecane peptide = 53/1 (weight ratio).

Example 15
PLA (20 mg) and tridecane peptide-PEG-DPE (2.5 mg) prepared in the same manner as in Example 13 were dissolved in chloroform (10 mL), and a 1 wt% aqueous solution of PVA (pH 7.4) dissolved in PBS. ; 100 mL), stirred for 30 minutes, poured into a pellet molding machine, and the solvent was removed under reduced pressure. The binding ratio between the pellet-like PLA and the tridecane peptide was measured by a fluorescence measurement method using FITC, and was PLA / tridecane peptide = 80/1 (weight ratio).

Example 16
RGDSK (100 mg) is dissolved in DPPE (10 mg) in a dimethylformamide solution, mixed with glutaraldehyde (3 mg) to react (room temperature, 10 hours), added dropwise to pure water to remove unreacted substances, RGDSK -DPPE (8 mg) was obtained. PLA (20 mg) and RGDSK-DPPE (1.5 mg) were dissolved in 10 mL of chloroform, dropped into a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, stirred for 30 minutes, and then on an evaporator. The solvent was removed. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 454 ± 100 nm with RGDSK-DPPE oriented on the surface were obtained. The binding ratio between PLGA fine particles and RGDSK was measured by a fluorescence measurement method using FITC, and was PLA fine particles / RGDSK = 100/1 (weight ratio).

Example 17
PLGA (20 mg) and Mal-PEG-DSE (10.0 mg) were dissolved in chloroform (10 mL), added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred for 30 minutes ( After stirring (750 rpm), the solvent was removed with an evaporator. Unbound Mal-PEG-DSE and PVA were removed by centrifugation (2500 rpm, 30 minutes) to obtain PLGA microparticles having a particle diameter of 2600 ± 800 nm with Mal-PEG-DSE oriented on the surface.

  The PLGA fine particle dispersion was brought to a constant volume of 4 mL, and then 20 mM SPDP solution (8 μL) was reacted with 5.0 wt% rGPIbα (90 μL) (room temperature, 30 minutes), followed by addition of 200 mM DTT (10 μL). (Room temperature, 30 minutes). SH-rGPIbα obtained by separation with Sephadex G25 was added to the PLGA dispersion and stirred at 4 ° C. for 30 hours. Unreacted rGPIbα was separated by centrifugation (2500 rpm, 30 minutes) to obtain rGPIbα-PLGA fine particles. The binding ratio between PLGA microparticles and rGPIbα was measured by the micro BCA method and was PLGA microparticles / IgG = 45/1 (weight ratio).

Example 18
PLA (20 mg) and DPE (1.8 mg) were dissolved in chloroform (10 mL), dropped into 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred for 30 minutes (stirring speed 850 rpm). Thereafter, the solvent was removed with an evaporator. By removing unbound lipid and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles having a particle size of 950 ± 300 nm with DPE oriented on the surface were obtained.

An SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to the dispersion (4 mL) of the PLA fine particles adjusted to a concentration of 0.55 wt%, and stirred at 23 ° C. for 2 hours. PLA fine particles and unreacted SPDP were separated by centrifugation (2500 rpm, 30 minutes), and the volume of the SPDP-bound PLA fine particle dispersion was adjusted to 4 mL. Next, 10% by weight fibrinogen (45 μL) was reacted with 20 mM SPDP solution (2.5 μL) (room temperature, 30 minutes), and 200 mM DTT (6 μL) was added (room temperature, 30 minutes). SH-fibrinogen obtained by separation with Sephadex G25 was added to the PLA dispersion and stirred at 4 ° C. for 30 hours. Unreacted fibrinogen was separated by centrifugation (2500 rpm, 30 minutes) to obtain fibrinogen-PLA fine particles. The binding ratio between the PLA fine particles and fibrinogen was measured by a fluorescence measurement method using FITC, and was PLA fine particles / fibrinogen = 77/1 (weight ratio).

Example 19
Polycaprolactone (20 mg) and polylysine-DSE (2.5 mg) were dissolved in chloroform (10 mL), added dropwise to a 1 wt% aqueous solution of PVA (pH 7.4; 100 mL) dissolved in PBS, and stirred (stirred) for 30 minutes. After 1000 rpm), it was poured into a rectangular parallelepiped molding box, and the solvent was removed with an aspirator. Furthermore, it was dried in a vacuum dryer for 12 hours to obtain a sponge-like straight type polycaprolactone.

An SPDP solution (600 μL) having a concentration of 20.0 × 10 ⁻³ mol / L was added to the polycaprolactone immersion liquid (4 mL) adjusted to a concentration of 0.55 wt%, and the mixture was stirred at 23 ° C. for 2 hours. Polycaprolactone and unreacted SPDP were separated by filtration to obtain a PD group conjugate. Next, amino sugar (7.5% by weight; 70 μL) into which cysteine was previously introduced was added, and the mixture was invaded at 4 ° C. for 30 hours. Unreacted substances were separated by filtration to obtain amino sugar-polycaprolactone. The binding ratio was measured by a fluorescence measurement method using FITC, and was polycaprolactone / amino sugar = 115/1 (weight ratio).

Example 20
DPGA (PEG-DPPE) (4.2 mg) and busulfan (10 mg) in which PLGA (20 mg) and a polyethylene oxide chain are bonded are dissolved in chloroform (10 mL), and a 1% by weight aqueous solution of PVA (pH 7.4) dissolved in PBS. ; 100 mL), and after stirring for 30 minutes, the solvent was removed with an evaporator. Free PEG lipids and PVA were removed by centrifugation (2500 rpm, 30 minutes) to obtain PLGA fine particles having a particle size of 250 ± 100 nm in which PEG-DPPE encapsulating busulfan was oriented on the surface.

Example 21
PLA (20 mg) and DPE (PEG-DPE) (4.8 mg) and busulfan (10 mg) combined with polyethylene oxide chains were dissolved in chloroform (10 mL), and a 1% by weight aqueous solution of PVA (pH 7.4) dissolved in PBS. ; 100 mL), and after stirring for 30 minutes, the solvent was removed with an evaporator. By removing free PEG lipids and PVA by centrifugation (2500 rpm, 30 minutes), PLA fine particles with a particle size of 210 ± 60 nm in which PEG-DPE encapsulating busulfan was oriented on the surface were obtained.

  By orienting the surface modifying molecule on the surface of the biodegradable material according to the present invention, adsorption of blood cell components such as plasma proteins, platelets and leukocytes can be suppressed, and an antithrombotic material can be obtained. Moreover, if a cell adhesion factor is utilized as a functional molecule, it functions as a matrix for various cells and tissue culture. Furthermore, when the shape of the biodegradable material is spherical, it can be used as a drug carrier of an intravenous formulation, and the functional part at the end of the surface modifier can be recognized and accumulated at a specific site. If used as a carrier, it can be used as a drug carrier or an implantable drug sustained-release material. In addition, the functional biodegradable material of the present invention can be used in various fields.

Claims (12)

A biodegradable material and a surface modifying molecule that modifies the surface of the biodegradable material, wherein the functional surface modifying molecule has the general formula:
[Part having affinity with biodegradable material]-[Hydrophilic spacer part] _n- [Linker part]-[Hydrophilic functional part]
A functional biodegradable material represented by the formula (where n is 1 or 0).   The functional biodegradable material according to claim 1, comprising a medicinal component.   The functional biodegradable material according to claim 1, wherein the biodegradable material includes polylactic acid or a lactic acid / glycolic acid copolymer.   The functional biodegradable material according to any one of claims 1 to 3, which is in the form of spherical particles having a diameter of 10 nm or more and 10 µm or less.   The functional biodegradable material according to any one of claims 1 to 4, wherein the [part having affinity with the biodegradable material] is formed from a hydrocarbon chain, an aliphatic polyester, or an aliphatic polyamide. . The [hydrophilic functional part] contains a thiol group, and the [part having affinity with the biodegradable material] or the [hydrophilic spacer part] consists of an OH group, a COOH group, and an NH ₂ group at the terminal. 6. The method according to claim 1, comprising at least one functional group, wherein the linker moiety binds to the thiol group at one end and binds to the at least one functional group at the other end. Functional biodegradable material as described.   The functional biodegradable material according to any one of claims 1 to 6, wherein the [hydrophilic spacer portion] contains polyoxyethylene, a polypeptide, a polyol, or a polysaccharide.   The functional biodegradable material according to claim 1, 2, 3, 4, or 6, wherein the surface modifying molecule comprises a phospholipid or a polyoxyethylene-linked phospholipid structure.   The functional biodegradable material according to any one of claims 1 to 7, wherein the surface modifying molecule includes L-glutamic acid, lysine, or a dendritic branched structure.   The functional biodegradable material according to any one of claims 1 to 9, wherein the [hydrophilic functional moiety] is one or more proteins, peptides, or saccharides.   Spherical particles characterized in that a biodegradable material and a functional surface modifying molecule are dissolved in a common solvent and dispersed in a polyvinyl alcohol aqueous solution to form an O / W emulsion, and the solvent is removed. The manufacturing method of the functional biodegradable material in the form of this. General formula:
[Parts that have affinity with biodegradable materials]-[Hydrophilic spacer parts] _n
(Wherein n is 1 or 0), the amphiphilic compound and the biodegradable material are dissolved in their common solvent and dispersed in an aqueous polyvinyl alcohol solution to form an O / W emulsion. The solvent is removed to obtain spherical particles made of the biodegradable material surface-modified with the amphiphilic compound, and a hydrophilic functional part is directly linked to the amphiphilic compound on the spherical particle surface. A method for producing a spherical functional degradable material, characterized in that they are bonded together.