WO2016088384A1 - in vivoステルス性ナノ粒子 - Google Patents
in vivoステルス性ナノ粒子 Download PDFInfo
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- WO2016088384A1 WO2016088384A1 PCT/JP2015/006041 JP2015006041W WO2016088384A1 WO 2016088384 A1 WO2016088384 A1 WO 2016088384A1 JP 2015006041 W JP2015006041 W JP 2015006041W WO 2016088384 A1 WO2016088384 A1 WO 2016088384A1
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/44—Polymerisation in the presence of compounding ingredients, e.g. plasticisers, dyestuffs, fillers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Definitions
- the present invention relates to in-vivo stealth nanoparticles. More specifically, the present invention relates to nanoparticles for intra-blood delivery that can acquire stealth properties in blood vessels.
- Nanoparticle-based therapeutics and diagnostics have been developed for the treatment and diagnosis of various diseases including cancer.
- One of the properties that should be imparted in common to nanoparticles applied to drug delivery systems is stealth (the ability to avoid an immune response in blood, that is, retention in blood).
- stealth properties to the nanoparticles is surface modification.
- polymers such as polyethylene glycol
- opsonin for the purpose of inhibiting phagocytosis
- a molecular imprinting method is known as one of artificial receptor synthesis methods capable of specifically recognizing a target molecule.
- the MI method is a method for artificially constructing a binding site selective for a target molecule in a material using a molecule to be recognized (target molecule) as a template.
- Polymers synthesized using the MI method are called molecularly imprinted polymers (MIP).
- MIP radically polymerizes a template molecule (target molecule or derivative thereof) and a functional monomer (a molecule having a site that specifically interacts with the template molecule and a polymerizable functional group) together with a cross-linking agent. Is constructed by removing from the polymer (Non-patent Document 3).
- nanoparticles for drug delivery systems have taken a means of surface modification of the nanoparticles with molecules involved in the stealth performance. That is, it has been common knowledge that nanoparticles for drug delivery systems are manufactured in a manner to which desired stealth properties are imparted before being injected into the body. On the other hand, no molecularly imprinted polymer showing stealth property is known.
- an object of the present invention is to provide a molecularly imprinted polymer that can acquire stealth properties by a new mechanism.
- the present invention includes the following inventions in order to solve the above problems.
- the present invention is in vivo stealth nanoparticles.
- In vivo stealth nanoparticles are molecularly imprinted polymers that have plasma protein recognition sites on which plasma proteins are molecularly imprinted and contain components derived from biocompatible monomers.
- the in vivo stealth nanoparticles of the present invention are used for intravascular delivery.
- stealth in-vivo-stealth
- a plasma protein present in the blood vessel to the recognition site after being administered into the blood vessel.
- the plasma protein may be albumin.
- This configuration makes it possible to efficiently acquire stealth, because albumin occupying the most plasma protein is used to acquire stealth.
- the biocompatible monomer may be a zwitterionic compound.
- the in vivo stealth nanoparticle of any one of (1) to (3) above may have an average particle size of 10 nm or more and 100 nm or less.
- the average particle diameter in this invention means the arithmetic average diameter in the particle size distribution measured by a dynamic light scattering method.
- the in vivo stealth nanoparticle according to any one of (1) to (4) above may further contain a signal group.
- the nanoparticles can be traced from outside the living body to which the in vivo stealth nanoparticles are administered using a means for detecting the signal group.
- the in vivo stealth nanoparticle of any one of (1) to (5) above may carry a drug component.
- This configuration allows in vivo stealth nanoparticles to be used as drugs for drug delivery systems.
- the drug component may be contained in the molecularly imprinted polymer as a component derived from a drug monomer in which a polymerizable functional group is covalently bonded to the drug.
- This configuration facilitates the loading of drugs that ensure in-vivo stealth acquisition.
- a molecularly imprinted polymer that can acquire stealth property by a new mechanism of binding a plasma protein existing in a blood vessel to a recognition site after being administered into the blood vessel.
- purification are shown.
- the particle size distribution of the fluorescent HSA recognition nanoparticle [HSA] MIP-NGs of Example 1 obtained by DLS is shown.
- the change in the RU value and the particle concentration Show the relationship.
- mouth which injected the fluorescent HSA recognition nanoparticle [HSA] MIP-NGs of Example 1 after 10 minutes (a) and 14 hours (b) is shown.
- mouth which injected the fluorescent HSA recognition nanoparticle [HSA] MIP-NGs of Example 1 is shown.
- the confocal laser micrograph of the mouse liver which injected the fluorescent reference nanoparticle NIP-NGs of the comparative example 1 10 minutes after (a) and 15 hours after (b) is shown with the measurement point of the blood vessel in a liver.
- mouth which injected the fluorescent reference nanoparticle NIP-NGs of the comparative example 1 is shown. Measurement points of hepatocytes in the photograph of FIG. 10 are shown. The fluorescence intensity time-dependent change of the hepatocyte in the liver of the mouse
- the adsorption behavior (HSA vs Dox MIP) of the drug-supported HSA-recognized nanoparticle DOX1- [HSA] MIP-NGs of Example 2 to the HSA-immobilized gold substrate was compared with the HSA-recognized nanoparticle [HSA] that did not carry the drug of Example 1. It is shown together with the adsorption behavior of MIP-NGs (HSA vs Non-Dox MIP).
- the result of observing uptake into / 3T3 is shown.
- the particle size distribution of the fluorescent MSA recognition nanoparticle [MSA] MIP-NGs of Example 3 obtained by DLS is shown.
- substrates of the fluorescent MSA recognition nanoparticle [MSA] MIP-NGs of Example 3 is shown.
- support HSA recognition nanoparticle NF-DOX1- [HSA] MIP-NGs of Example 4 obtained by DLS is shown.
- support HSA recognition nanoparticle NF-DOX1- [HSA] MIP-NGs of Example 4 is shown.
- support HSA recognition nanoparticle NF-DOX2- [HSA] MIP-NGs of Example 5 obtained by DLS is shown.
- support HSA recognition nanoparticle NF-DOX2- [HSA] MIP-NGs of Example 5 is shown.
- support HSA recognition nanoparticle NF-DOX1- [HSA] MIP-NGs of Example 4, and a cell viability is shown.
- support HSA recognition nanoparticle NF-DOX2- [HSA] MIP-NGs of Example 5, and a cell viability is shown.
- the in vivo stealth nanoparticles of the present invention are molecularly imprinted polymers used for intravascular delivery.
- a molecularly imprinted polymer is a synthetic polymer having a binding site (target molecule recognition site) that is selective for a target molecule.
- the molecular imprint polymer is synthesized by a molecular imprint method which is one of template polymerization methods.
- the molecular imprint method is a method in which a target molecule is used as a template to artificially construct a binding site (target molecule recognition site) selective for the target molecule in a polymer.
- the target molecule to be recognized by the in vivo stealth nanoparticle of the present invention at the target molecule recognition site is a plasma protein.
- the plasma protein in the blood binds to the target molecule recognition site of the molecular imprint polymer, whereby stealth can be obtained.
- plasma proteins include albumin, ⁇ -globulin, fibrinogen, transferrin, ceruloplasmin, carcinoembryonic antigen, etc. Among them, albumin occupying the most is preferable. In this case, ensuring stealth in the blood of in vivo stealth nanoparticles is more efficiently performed.
- the plasma protein may be derived from human and non-human animals. Non-human animals include vertebrates including mammals such as mice, rats, monkeys, dogs, cats, cows, horses, pigs, hamsters, rabbits, and goats.
- the molecularly imprinted polymer constituting the in vivo stealth nanoparticle of the present invention is a crosslinked polymer containing at least components derived from a functional monomer and a biocompatible monomer.
- the functional monomer mentioned above refers to a molecule having both a functional group capable of binding to the target molecule, plasma protein, and a polymerizable functional group.
- the binding mode with plasma protein may be covalent or non-covalent.
- Non-covalent bonds include hydrogen bonds, ionic bonds, electrostatic interactions, van der Waals interactions, hydrophobic interactions, and the like.
- Examples of functional groups capable of binding to plasma proteins include acidic functional groups such as sulfonic acid groups and carboxyl groups, amino groups, cyclic secondary amino groups (for example, pyrrolidyl groups and piperidyl groups), pyridyl groups, imidazole groups, and guanidines. And a basic functional group such as a group, a carbamoyl group, a hydroxyl group, and an aldehyde group.
- Examples of the polymerizable functional group include a vinyl group and a (meth) acryl group (the same applies to other monomers).
- the functional monomer examples include pyrrolidyl acrylate, acrylic acid, methacrylic acid, acrylamide, 2- (dimethylamino) ethyl methacrylate, hydroxyethyl methacrylate, and the like.
- the functional monomer can be appropriately selected depending on the plasma protein used as a template.
- biocompatible monomer refers to a monomer that can constitute a biocompatible polymer.
- the biocompatible polymer is preferably a hydrophilic polymer and includes zwitterionic polymers and nonionic polymers.
- Biocompatibility refers to a property that does not induce adhesion of biological substances. By including a component derived from such a monomer, the molecular imprint polymer itself can be provided with preferable blood retention.
- the zwitterionic monomer capable of constituting the zwitterionic polymer includes an anionic group derived from an acidic functional group (for example, a phosphate group, a sulfuric acid group, and a carboxyl group) and a basic functional group (for example, a primary amino group). Both a cationic group derived from a secondary amino group, a tertiary amino group, a quaternary ammonium group and the like are included in one molecule. For example, phosphobetaine, sulfobetaine, carboxybetaine and the like can be mentioned.
- examples of the phosphobetaine include molecules having a phosphorylcholine group in the side chain, and preferably 2-methacryloyloxyethyl phosphorylcholine (MPC) and the like.
- examples of sulfobetaines include N, N-dimethyl-N- (3-sulfopropyl) -3′-methacryloylaminopropaneaminium inner salt (SPB), N, N-dimethyl-N- (4-sulfobutyl) -3 ′.
- SPB N-dimethyl-N- (4-sulfobutyl) -3 ′.
- SBB -Methacryloylaminopropaneaminium inner salt
- Carboxybetaines include N, N-dimethyl-N- (1-carboxymethyl) -2′-methacryloyloxyethanaminium inner salt (CMB), N, N-dimethyl-N- (2-carboxyethyl)- And 2'-methacryloyloxyethaneaminium inner salt (CEB).
- Having a component derived from a zwitterionic monomer is preferable from the viewpoint of drug release when a drug is supported on a molecular imprint polymer. Furthermore, it is preferable in that the particle diameter can be easily controlled to be smaller while maintaining blood dispersion stability.
- Nonionic polymers include polyether polymers such as poly (ethylene glycol) (PEG).
- PEG poly (ethylene glycol)
- the biocompatible monomer-derived component described above is, for example, more than 0% and 50% or less of the molecularly imprinted polymer (the amount expressed in% is based on moles), preferably 1% or more and 30% or less, more preferably 2% or more and 20 % Or less can be included.
- the content of the biocompatible monomer-derived component is not more than the above upper limit value, the molecularly imprinted plasma protein recognition site can be preferably maintained, and when it is not less than the above lower limit value, preferable retention in blood is obtained. be able to.
- the molecular imprint polymer may have a constituent derived from a water-soluble monomer.
- the water-soluble monomer in this case refers to a monomer capable of constituting an external stimulus responsive polymer such as a thermoresponsive polymer having a lower critical solution temperature (LCST) and a pH responsive polymer.
- LCST critical solution temperature
- Thermally responsive polymers with LCST include poly (N-isopropylacrylamide) (PNIPAM), poly (N, N-diethylacrylamide), poly (vinyl methyl ether), polyethylene oxide (PEO), polyethylene oxide side chains
- PNIPAM poly(N-isopropylacrylamide)
- PEO polyethylene oxide
- polymers for example, poly (diethylene glycol methacrylate), poly (triethylene glycol methacrylate), poly (oligoethylene glycol methacrylate)), saponified polyvinyl acetate, methyl cellulose, hydroxypropyl cellulose, and the like.
- the molecularly imprinted polymer exhibits hydrophilicity in a low temperature range, but exhibits hydrophobicity due to a temperature change to LCST or higher, and is easily taken up by cells.
- the pH-responsive polymer includes an anionic polymer (for example, poly (meth) acrylic acid) that exhibits hydrophilicity in the alkaline region and a cationic polymer (for example, polyacrylamide, methacryloxyethyltrimethylammonium chloride) that exhibits hydrophilicity in the acidic region.
- anionic polymer for example, poly (meth) acrylic acid
- a cationic polymer for example, polyacrylamide, methacryloxyethyltrimethylammonium chloride
- the molecular imprint polymer may have a component derived from a signal group-containing monomer.
- the signal group may be any functional group that can be detected and can be appropriately selected by those skilled in the art from, for example, a fluorescent group, a radioactive element-containing group, a magnetic group, and the like.
- the fluorescent group include groups derived from cyanine dyes such as fluorescein dyes and indocyanine dyes, rhodamine dyes, and quantum dots.
- the fluorescent group is preferably a near-infrared fluorescent group having high permeability to a living body.
- radioactive element-containing group examples include groups derived from sugars, amino acids, nucleic acids and the like labeled with a radioactive isotope such as 18 F.
- the magnetic group examples include those having a magnetic material such as ferrichrome, those found in ferrite nanoparticles, nanomagnetic particles, and the like.
- the in vivo stealth nanoparticles of the present invention can be used as a carrier for drugs and the like, and can carry drugs such as anticancer drugs, genes, contrast agents, fluorescent probes, and proteins such as enzymes.
- the anticancer agent may be any anticancer agent that is generally used for cancer treatment. Specifically, taxanes, platinum preparations, nitrosoureas, nitrogen mustards, triazines, anthracyclines, vinca alkaloids, epipodophyllotoxins, camptothecins, and fluorines. Pyrimidine chemicals can be exemplified. Taxanes include taxol, taxotere, paclitaxel, docetaxel and the like. Examples of platinum preparations include cisplatin and carboplatin. Examples of nitrosourea drugs include carmustine and lomustine. Examples of nitrogen mustard chemicals include cyclophosphamide. Examples of triazine drugs include dacarbazine.
- Anthracycline drugs include doxorubicin and the like.
- Vinca alkaloids include vincristine and vinblastine.
- epipodophyllotoxin drugs include etoposide.
- camptothecin drugs include irinotecan.
- fluoropyrimidine drugs include 5-fluorouracil and tegafur.
- the drug may be covalently supported on the stealth nanoparticles in vivo. Specifically, it can be supported by including a component derived from a drug monomer as a component of the molecular imprint polymer.
- the drug monomer has a structure in which the aforementioned drug is covalently bonded to a polymerizable functional group.
- the drug monomer can be obtained by reacting a desired drug with a monomer having a polymerizable functional group (polymerizable monomer).
- a reactive group on the drug side a group that does not interfere with the expression of the efficacy of the drug itself is appropriately selected by those skilled in the art.
- One reactive group can be appropriately determined by those skilled in the art.
- the reactive group on the polymerizable monomer side a group other than the polymerizable functional group and capable of reacting with the reactive group on the drug side is appropriately selected by those skilled in the art. Accordingly, the polymerizable monomer to be reacted with the drug is not particularly limited. For example, monomers having a carboxyl group such as acrylic acid and methacrylic acid; monomers having a hydroxyl group such as hydroxyalkyl acrylate and hydroxyalkyl methacrylate; methylol acrylate and methylol.
- Examples thereof include: methacrylates; monomers having vinyl groups such as allyl acrylate and allyl methacrylate; monomers having glycidyl groups such as glycidyl acrylate and glycidyl methacrylate; monomers having amino groups; monomers having sulfonic acid groups.
- the reactive group on the drug side and the reactive group on the polymerizable monomer side may be directly bonded by condensation or the like, and can be appropriately selected by those skilled in the art in the molecular design of the drug monomer. It may be bonded via a linking group.
- the average particle size of the in vivo stealth nanoparticles of the present invention may be 10 nm or more and 100 nm or less, preferably 40 nm or more and 60 nm or less, and more preferably 40 nm or more and 50 nm or less.
- the average particle diameter is less than or equal to the above upper limit value, the EPR (enhanced permeability and retention) effect can be easily expressed, and when it is greater than or equal to the above lower limit value, the specific surface area of the molecularly imprinted polymer can be secured and stealth It is easy to acquire sex.
- the average particle diameter means the arithmetic average diameter in the particle size distribution measured by the dynamic light scattering method.
- the dynamic light scattering method is based on the fluctuation of the scattered light intensity, which depends on the Brownian motion of the particle detected when the laser light is applied to the solution in which the particle is dispersed and the change in the scattered light is measured. This is a method for deriving the size (particle diameter).
- Particle size measuring devices based on the dynamic light scattering method are commercially available from various companies (for example, Otsuka Electronics, Sysmex, Beckman Coulter, etc.) and are suitably used for measuring the average particle size of the in vivo stealth nanoparticles of the present invention. be able to.
- the in vivo stealth nanoparticles of the present invention are synthesized by molecular imprinting methods.
- the target molecule is a plasma protein, its derivative or similar compound, and this template molecule is allowed to coexist during the radical polymerization reaction.
- a molecular recognition site that interacts complementarily with the template molecule is constructed together with the synthesis of the organic polymer.
- the polymerization reaction system in which the template molecule is allowed to coexist may contain at least a functional monomer, a biocompatible monomer, and a crosslinking agent.
- at least one of the above-described water-soluble monomer that is, a monomer capable of constituting an external stimulus-responsive polymer
- a signal group-containing monomer e.g., a drug monomer
- you may further contain at least any one of a polymerization initiator and a polymerization accelerator.
- oligomers and / or polymers of the monomers may be included.
- each monomer include the various compounds described in the above item 1-2 (constituent material of molecular imprint polymer) and item 1-3 (carrying drug).
- the crosslinking agent is preferably a molecule having at least two polymerizable functional groups (such as vinyl groups) in the molecule, and examples thereof include ethylene glycol dimethyl acrylate, N, N′-methylenebisacrylamide, and divinylbenzene. .
- polymerization initiator examples include azo polymerization initiators such as peroxides such as ammonium persulfate and potassium persulfate, azobisisobutyronitrile, 2,2′-azobis (2-methylpropionamidine) dihydrochloride, and the like. Is mentioned.
- polymerization accelerator examples include N, N, N ′, N′-tetramethylethylenediamine.
- an aqueous solvent such as a buffer solution is preferably used from the viewpoint of suppressing denaturation of the template molecule.
- the polymerization reaction can be initiated by simultaneously coexisting all the above components.
- a template molecule / functional monomer complex may be formed in advance, and then the template molecule / functional monomer complex may be subjected to a polymerization reaction together with a biocompatible monomer and a crosslinking agent.
- numerator and functional monomer origin component in the obtained organic polymer may be a covalent bond or a non-covalent bond.
- the binding mode is a non-covalent bond, it is not necessarily required to form a template molecule / functional monomer complex in advance, and this is preferable in that it can be easily cleaved.
- examples of the polymerization method for obtaining fine particles of the molecularly imprinted polymer include a non-emulsifier precipitation polymerization method, a dispersion polymerization method, an emulsion polymerization method, and a seed emulsion polymerization method.
- an organic polymer (molecular imprinted polymer) having a molecular recognition site that interacts with the template molecule in a substrate-specific manner can be obtained.
- the template molecule can be removed by cleaving the bond between the template molecule and the functional monomer-derived component and separating the released template molecule from the molecular imprint polymer.
- the cleavage of the template molecule can be appropriately determined by those skilled in the art based on the binding mode between the template molecule and the functional monomer-derived component.
- 1M NaCl solution polar solvent (eg alcohol such as methanol); surfactant (eg sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), tetradecyltrimethylammonium bromide (TTAB), cetyl bromide Trimethylammonium (CTAB), polyoxyethylene alkylphenyl ether (Triton), fatty acid ester (Span), polyoxyethylene ether fatty acid ester (Tween)); protein denaturants (eg, tris [2-carboxyethyl] phosphine hydrochloride ( (TCEP), urea, glycine salt, acid, alkali); an enzyme (eg, pepsin, trypsin, papain) or the like can be used for clea
- disconnecting a non-covalent bond may be performed simultaneously with the below-mentioned separation process.
- the mobile phase (buffer solution) used in the separation step can function as an eluent for the template molecule.
- the released template molecule is separated from the molecular imprint polymer.
- a separation method a person skilled in the art can appropriately select a separation method using a difference in physical properties between the two. Preferably, it can be separated by size exclusion chromatography.
- the in vivo stealth nanoparticle of the present invention can be used as a pharmaceutical composition together with a pharmaceutically acceptable component by supporting a drug.
- Pharmaceutically acceptable ingredients are solids and / or liquids that are non-toxic, inert and do not affect the target molecule recognition site of in vivo stealth nanoparticles, eg, sterile water, saline, stabilizers , Excipients, antioxidants, buffers, preservatives, pH adjusters, surfactants, binders and the like.
- the pharmaceutical composition can be prepared in a form that is administered into the body by methods such as injection and percutaneous absorption.
- Nanoparticle [HSA] Synthesis of MIP-NGs and NIP-NGs] [1-1.
- Example 1 Synthesis of fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs] N-isopropylacrylamide (NIPAm) 407 mg as a water-soluble monomer, N, N'-methylenebisacrylamide (MBAA) 30.8 mg as a cross-linking agent, pyrrolidyl acrylate (PyA) 70 mg as a functional monomer, fluorescent monomer Fluorescein acrylamide (FAm) 4mg, methacryloyloxyethyl phosphorylcholine (MPC) 59mg as biocompatible monomer, 2,2'-azobis (2-methylpropionamidine) dihydrochloride (V-50) 217mg as initiator, And 13.2 mg of human serum albumin (HSA, 50-60% in the blood) as the target protein was dissolved in 100 mL of 10 mM PBS (pH 7.
- pyrrolidyl acrylate is a monomer having the following structure, and is described in Inoue Y., Kuwahara A., Ohmori K., Sunayama H., Ooya T., Takeuchi T.
- the intermediate N-Boc-pyrrolidyl acrylate was synthesized from Boc-3-hydroxypyrrolidine and acryloyl chloride by the method described, and then obtained by deprotecting the Boc group.
- nanoparticles [HSA] MIP-NGs, NIP-NGs
- Purification was performed by separation using size exclusion chromatography. Specifically, Sephadex G-50 Medium was packed in a column with an inner diameter of 1.2 cm to a height of 33 cm, and 2 ml of the obtained nanogel emulsion ([HSA] MIP-NGs or NIP-NGs) was introduced. As an eluent, 10 mM PBS PBS buffer (pH 7.4) was used. Fractions were collected in 1.5-mL portions, and UV-vis measurement of each fraction was performed to confirm whether size exclusion chromatography separation was possible.
- FIG. 1 (a) shows the UV-vis spectrum of the polymer before purification
- FIG. 1 (b) shows the UV-vis spectrum of the sixth fraction after purification.
- the horizontal axis represents wavelength (nm)
- the vertical axis represents relative intensity.
- fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs can be completely separated from HSA by using a filler whose number of steps works on the polymer side.
- FIG. 2 shows the particle size distribution of fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs obtained by DLS.
- fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs when the excitation spectrum was measured at a fluorescence wavelength of 530 nm, it showed a maximum absorption around 500 nm.
- the fluorescence spectrum of fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs was measured at an excitation wavelength of 500 nm and 25 ° C., and showed a maximum absorption at 526 nm.
- the fluorescence reference nanoparticle NIP-NGs was measured in the same manner and showed the same maximum absorption.
- the fluorescence intensity (I 00 ) when the light source side is 0 ° ⁇ the detector side 0 °, the fluorescence intensity (I 09 ) when the light source side is 0 ° ⁇ the detector side 90 °, and the light source side 90 The fluorescence intensity (I 90 ) when the angle was ⁇ ° on the detector side (I 90 ) and the fluorescence intensity (I 99 ) when the angle was 90 ° on the light source side and 90 ° on the detector side were measured.
- the maximum wavelength was 526 nm.
- Fluorescence measurement was also performed on fluorescein acrylamide which is a fluorescent monomer before polymerization.
- the maximum wavelength was 510 nm.
- the anisotropy value A of fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs was calculated to be 0.205.
- the anisotropy value A of fluorescein acrylamide was calculated to be 0.0144.
- the anisotropy value A of fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs is clearly larger than that of fluorescein acrylamide. This result clearly shows that the depolarization due to Brownian motion was suppressed because the size was increased by the incorporation of fluorescent molecules into the nanogel particles.
- the obtained SAM film-formed substrate was washed with ethanol, and then N-ethyl- (dimethylaminopropyl) carbodiimide (EDC) (100 mg / mL) and N-hydroxysuccinimide (NHS) (100 mg / mL) was soaked in 0.3 mL of an aqueous solution in which was dissolved at room temperature for 30 minutes. As a result, the carboxylic acid was activated by NHS modification (activation step).
- EDC N-ethyl- (dimethylaminopropyl) carbodiimide
- NHS N-hydroxysuccinimide
- the substrate on which the carboxylic acid had been activated was incubated (25 ° C., 1.5 ⁇ h) in 10 ⁇ mM PBS buffer (pH 7.4) in which HSA® (1 mg / mL) was dissolved. In this way, an HSA-immobilized gold substrate was produced (HSA immobilization process).
- HSA-recognized nanoparticles [HSA] MIP-NGs were dispersed in 10 mM PBS buffer (pH 7.4), and the concentration was changed to 100, 200, 400, 800, 1600 ng / mL and used for measurement. Measurements were also performed on fluorescent reference nanoparticles NIP-NGs by the same operation.
- FIG. 5 shows changes with time in fluorescence intensity in veins and tissues.
- the horizontal axis represents time (minutes), and the vertical axis represents relative intensity (%).
- FIG. 6 shows changes in fluorescence intensity over time.
- FIG. 7 shows changes with time in fluorescence intensity in veins and tissues.
- the horizontal axis represents time (minutes), and the vertical axis represents relative intensity (%).
- mice Male, 4 weeks old were used to observe the liver.
- a hair removal cream was applied to the abdomen of the mouse to remove the hair.
- the skin was torn using an electronic knife and scissors so that the liver could be observed from the abdomen.
- a catheter was inserted into the tail vein for sample injection. It was confirmed that there was no leakage by introducing physiological saline with a 1 mL Terumo syringe.
- FIG. 8A shows a confocal laser micrograph of the liver of a mouse injected with the fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs of Example 1 after 10 minutes, and FIG. The confocal laser micrograph of the liver of the mouse
- FIG. 10 (a) shows a confocal laser micrograph of the mouse liver 10 minutes after injection of the fluorescent reference nanoparticle NIP-NGs of Comparative Example 1, and FIG. 10 (b) shows NIP-NGs injected. A confocal laser micrograph after 15 hours of mouse liver is shown.
- FIG. 10 (a) the blood vessels are clearly visible immediately after the injection (after 10 minutes), whereas, as shown in FIG. 10 (b), the blood vessels are almost visible after 15 hours from the injection. Instead, it was observed that many cells were taken up instead. This cell is considered a hepatocyte. Furthermore, it was observed that the number of hepatocytes that fluoresce increased with time.
- Adsorption behavior of drug-supported HSA-recognized nanoparticle DOX1- [HSA] MIP-NGs on HSA-immobilized gold substrate and adsorption amount (Absorption (RUmm 2 / g-protein) and particle concentration (NPs concentration (ng / nL)) 15 (n 2) is shown in Fig. 15.
- the amount of HSA adsorbed is defined by the amount of HSA immobilized on the SPR sensor chip.
- the drug-supported HSA-recognized nanoparticles DOX1- [HSA] MIP-NGs of this example are the same as the drug-unsupported HSA-recognized nanoparticles [HSA] MIP-NGs of Example 1. It was confirmed that the behavior was exhibited. Therefore, it is suggested that the drug-supported HSA-recognized nanoparticles DOX1- [HSA] MIP-NGs have the same HSA binding space as the drug-unsupported HSA-recognized nanoparticles [HSA] MIP-NGs of Example 1. It was done.
- the cells were observed using a confocal laser microscope.
- the observation conditions are as follows. Confocal laser microscope: Olympus IX81 Objective lens: 100 ⁇ (oil) Filter used: FITC
- FITC is a fluorescent image of nanoparticles
- Bright is a bright field image.
- MSA-recognized nanoparticles [MSA] MIP-NGs were purified in the same manner as in item 2 above except that Sephadex G-100 was used instead of Sephadex G-50, and the particle size was determined by DLS measurement. Asked.
- FIG. 18 shows the particle size distribution of fluorescent MSA-recognized nanoparticles [MSA] MIP-NGs obtained by DLS. From this, it is considered that nanosized MIP particles could be obtained in this example as well as [HSA] MIP-NGs in Example 1 and drug-supported [HSA] MIP-NGs in Example 2. .
- the amount of adsorption of the nanoparticles (NPs Absorption (RU x mm 2 / pmol-protein) and the particle concentration (NPs concentration (ng / mL)) is shown in Fig. 19.
- the fluorescent MSA-recognized nanoparticles [MSA] MIP-NGs are the same as the fluorescent HSA-recognized nanoparticles [HSA] MIP-NGs of Example 1.
- the binding capacity to IgG and Fibrinogen is low, while the binding amount to MSA is higher than the binding amount to HSA in the high concentration region, suggesting that a recognition space for MSA is formed. Therefore, it was shown that the nanoparticles of the present invention can be obtained for proteins other than HSA.
- Example 4 Synthesis of non-fluorescent drug-supported HSA-recognized nanoparticles NF-DOX1- [HSA] MIP-NGs] Except that the fluorescent monomer FAm was not used, the copolymerization was carried out in an emulsifier-free precipitation polymerization system in the same manner as in Example 2, and the non-fluorescent drug-supported HSA recognition nanoparticles NF-DOX1- [HSA] MIP-NGs were synthesized. Specific components and compositions for constructing the copolymerization reaction system are shown in Table 4 below. The polymerization was performed in a Schlenk flask under a nitrogen atmosphere at 70 ° C. for 12 hours.
- FIG. 20 shows the particle size distribution of non-fluorescent drug-supported HSA-recognized nanoparticles NF-DOX1- [HSA] MIP-NGs obtained by DLS.
- FIG. 21 shows the UV-Vis spectrum of non-fluorescent drug-supported HSA recognition nanoparticles NF-DOX1- [HSA] MIP-NGs. From these results, it was suggested that there is an absorption region derived from doxorubicin, and the anticancer agent is encapsulated in the particles.
- FIG. 22 shows the particle size distribution of non-fluorescent drug-supported HSA-recognized nanoparticles NF-DOX2- [HSA] MIP-NGs obtained by DLS.
- FIG. 23 shows the UV-Vis spectrum of non-fluorescent drug-supported HSA-recognized nanoparticles NF-DOX2- [HSA] MIP-NGs. From these results, it was suggested that there is an absorption region derived from doxorubicin, and the anticancer agent is encapsulated in the particles.
- cell viability was calculated based on the following formula.
- the cell viability decreased as the concentration of the nanoparticles of the present invention increased.
- the concentration of the non-fluorescent drug-supported HSA-recognized nanoparticles NF-DOX1- [HSA] MIP-NGs of Example 4 is 100 ⁇ g / mL
- the cell viability is 64%
- the non-fluorescent drug-supported Example 5 When the concentration of the HSA-recognized nanoparticle NF-DOX2- [HSA] MIP-NGs was used, the cell viability was 7%, and the cell viability was low.
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Abstract
Description
一方で、ステルス性を示す分子インプリントポリマーは知られていない。
本発明は、in vivoステルス性ナノ粒子である。in vivoステルス性ナノ粒子は、血漿タンパク質が分子インプリントされた血漿タンパク質認識部位を有し且つ生体適合性モノマーに由来する構成成分を含む分子インプリントポリマーである。本発明のin vivoステルス性ナノ粒子は、血管内搬送に用いられる。
上記(1)のin vivoステルス性ナノ粒子において、血漿タンパク質はアルブミンであってよい。
上記(1)または(2)のin vivoステルス性ナノ粒子において、生体適合性モノマーは双性イオン化合物であってよい。
上記(1)から(3)のいずれかのin vivoステルス性ナノ粒子は、平均粒子径が10nm以上100nm以下であってよい。
上記(1)から(4)のいずれかのin vivoステルス性ナノ粒子は、シグナル基をさらに含んでよい。
上記(1)から(5)のいずれかのin vivoステルス性ナノ粒子は、薬剤成分が担持されてよい。
上記(6)のin vivoステルス性ナノ粒子は、薬剤成分が、薬剤に重合性官能基が共有結合した薬剤モノマーに由来する構成成分として分子インプリントポリマーに含まれてよい。
本発明のin vivo ステルス性ナノ粒子は、血管内搬送に用いられる分子インプリントポリマーである。分子インプリントポリマーは、標的分子に選択性のある結合部位(標的分子認識部位)を有する合成ポリマーである。分子インプリントポリマーは、鋳型重合法の一つである分子インプリント法により合成されるものである。分子インプリント法は、標的分子を鋳型として、その標的分子に選択性のある結合部位(標的分子認識部位)を、ポリマー中に人工的に構築する方法である。
本発明のin vivo ステルス性ナノ粒子が標的分子認識部位で認識対象とする標的分子は、血漿タンパク質である。本発明のin vivo ステルス性ナノ粒子が血管内に投与された場合に、血液中の血漿タンパク質が分子インプリントポリマーの標的分子認識部位に結合することで、ステルス性を獲得することができる。血漿タンパク質としては、アルブミン、γ-グロブリン、フィブリノゲン、トランスフェリン、セルロプラスミン、および癌胎児性抗原などが挙げられるが、この中でも最も多くを占めるアルブミンであることが好ましい。この場合、in vivo ステルス性ナノ粒子の血液中でのステルス性確保がより効率的に行われる。なお、血漿タンパク質は、ヒト由来およびヒト以外の動物由来であってよい。ヒト以外の動物としては、マウス、ラット、サル、イヌ、ネコ、ウシ、ウマ、ブタ、ハムスタ-、ウサギ、およびヤギなどの哺乳動物を含む脊椎動物が挙げられる。
本発明のin vivo ステルス性ナノ粒子を構成する分子インプリントポリマーは、少なくとも機能性モノマーと生体適合性モノマーとにそれぞれ由来する構成成分を含む架橋ポリマーである。
スルホベタインとしては、N,N-ジメチル-N-(3-スルホプロピル)-3’-メタクリロイルアミノプロパンアミニウムインナーソルト(SPB)、N,N-ジメチル-N-(4-スルホブチル)-3’-メタクリロイルアミノプロパンアミニウムインナーソルト(SBB)などが挙げられる。
カルボキシベタインとしては、N,N-ジメチル-N-(1-カルボキシメチル)-2’-メタクリロイロキシエタンアミニウムインナーソルト(CMB)、N,N-ジメチル-N-(2-カルボキシエチル)-2’-メタクリロイロキシエタンアミニウムインナーソルト(CEB)などが挙げられる。
蛍光基としては、フルオレセイン系色素、インドシアニン色素などのシアニン系色素、ローダミン系色素、量子ドットなどに由来する基が挙げられる。蛍光基は、生体への透過性が高い近赤外蛍光基であることが好ましい。放射性元素含有基としては、18Fなどの放射性同位体でラベルした、糖、アミノ酸、核酸などに由来する基が挙げられる。磁性基としては、フェリクロームなどの磁性体を有するもの、フェライトナノ粒子、ナノ磁性粒子などにみられるものが挙げられる。
本発明のin vivoステルス性ナノ粒子は、薬剤等のキャリアとして用いることができ、抗がん剤、遺伝子、造影剤、蛍光プローブ、および、酵素などのタンパク質などの薬剤を担持することができる。
薬剤モノマーは、所望の薬剤と、重合性官能基を有するモノマー(重合性モノマー)とを反応させることによって得ることができる。
本発明のin vivoステルス性ナノ粒子の平均粒子径は、10nm以上100nm以下であってよく、好ましくは40nm以上60nm以下、さらには40nm以上50nm以下であってもよい。平均粒子径が上記の上限値以下であることにより、EPR(enhanced permeability and retention)効果の発現が容易であり、上記の下限値以上であることにより、分子インプリントポリマーの比表面積の確保およびステルス性の獲得が容易である。
本発明のin vivoステルス性ナノ粒子は、分子インプリンティング法によって合成する。具体的には、標的分子である血漿タンパク質、その誘導体または類似化合物を鋳型分子とし、この鋳型分子をラジカル重合反応時に共存させる。鋳型分子の共存により、鋳型分子に対して相補的に相互作用する分子認識部位が、有機ポリマーの合成とともに構築される。分子インプリントポリマーの合成方法の詳細は、例えば、参考文献「Komiyama, M., Takeuchi, T., Mukawa, T., Asanuma, H. "Molecular Imprinting", WILEY-VCH, Weinheim, 2002.」の記載などを参照して当業者が適宜決定することができる。
架橋剤は、分子中に重合性官能基(ビニル基など)を少なくとも2個持つ分子を用いることが好ましく、たとえば、エチレングリコールジメチルアクリレート、N,N'-メチレンビスアクリルアミド、ジビニルベンゼンなどが挙げられる。
本発明のin vivoステルス性ナノ粒子は、薬剤が担持されることで、薬学的に許容される成分とともに医薬組成物として利用可能である。薬学的に許容される成分は、非毒性、不活性かつin vivoステルス性ナノ粒子の標的分子認識部位に影響を与えない固体および/または液体であり、たとえば、滅菌水、生理食塩水、安定剤、賦形剤、酸化防止剤、緩衝剤、防腐剤、pH調整剤、界面活性剤、結合剤等などが挙げられる。
薬剤組成物は、注射および経皮吸収などの方法で体内投与される形態で調製されることができる。
[1-1.実施例1:蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの合成]
水溶性モノマーとしてのN-イソプロピルアクリルアミド(NIPAm)407mg、架橋剤としてのN,N’-メチレンビスアクリルアミド(MBAA)30.8mg、機能性モノマーとしてのピロリジルアクリレート(PyA)70mg、蛍光性モノマーとしてのフルオレセインアクリルアミド(FAm)4mg、生体適合性モノマーとしてのメタクリロイロキシエチルホスホリルコリン(MPC)59mg、開始剤としての2,2’-アゾビス(2-メチルプロピオンアミジン)2塩酸塩(V-50)217mg、および標的タンパク質としてのヒト血清アルブミン(HSA, 血中に50-60%存在)13.2mgを、シュレンクフラスコ内で100 mLの10 mM PBS (pH 7.4)に溶解させ、窒素雰囲気下70℃で12時間、無乳化剤沈殿重合を行った。これにより、ヒト血清アルブミンを鋳型とする分子インプリントポリマー([HSA]MIP-NGs)を合成した。
ヒト血清アルブミンを用いなかったことを除いて上記と同様の無乳化剤沈殿重合を行った。これにより、リファレンスナノ粒子(NIP-NGs)を合成した。
得られたナノ粒子[HSA]MIP-NGs,NIP-NGsのDLS測定を行った。DLS測定には動的光散乱光度計(DLS)(マルバーン株式会社製データサイザー)を用い、温度条件は25℃とした。
結果、蛍光性HSA認識ナノ粒子[HSA]MIP-NGsは、Z平均粒子径:44 nm、PDI: 0.38 nmであった。蛍光性リファレンスナノ粒子NIP-NGsは、Z平均粒子径:19 nm、PDI: 0.46 nmであった。いずれについても安定なナノ粒子を得ることに成功したことが示された。
[2-1.蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの精製]
重合後の蛍光性HSA認識ナノ粒子[HSA]MIP-NGsのナノゲルエマルションのUV-visスペクトルを測定したところ、フルオレセイン基に由来する510 nm程度の波長吸収とともに、モノマーおよびHSAに由来する200~300 nmの波長吸収も観測された。
そこで、不純物(HSAおよびモノマー)の吸収が観測されず、かつフルオレセイン基に由来する502nmの吸収が最も大きく観測された6番目のフラクションを[HSA]MIP-NGsの精製物として採用した。この際、蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの固形分濃度を測定すると、0.112 wt%であった。図1(a)に、精製前における重合物のUV-visスペクトルを示し、図1(b)に、精製後における6番目フラクションのUV-visスペクトルを示す。図1ではいずれも、横軸に波長(nm)、縦軸に相対強度を表す。
なお、より高分子側で段数が働く充填材を用いれば、蛍光性HSA認識ナノ粒子[HSA]MIP-NGsをHSAから完全分離することが可能と考えられる。
蛍光性リファレンスナノ粒子NIP-NGsについても同様に分取を行い、精製物を得た。この際、NIP-NGsの固形分濃度を測定すると、0.106 wt%であった。
精製された蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの平均粒子径を、精製前と同様に測定した。結果、蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの平均粒子径:23nm、PDI:0.45であった。図2に、DLSにより得られた蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの粒子径分布を示す。
精製された蛍光性HSA認識ナノ粒子[HSA]MIP-NGsを1000分の1に希釈(溶媒10 mM PBS buffer (pH7.4))し、蛍光測定を行った。
蛍光性リファレンスナノ粒子NIP-NGsについても同様に蛍光スペクトルを測定しところ、同様の極大吸収を示した。
精製された蛍光性HSA認識ナノ粒子[HSA]MIP-NGsを1000分の1希釈(溶媒10 mM PBS buffer (pH7.4))した。光源側に0°または90°、検出器側に0°または90°の偏光版を挿入し、蛍光測定を行った。具体的には、光源側0°-検出器側0°である場合の蛍光強度(I00)、光源側0°-検出器側90°である場合の蛍光強度(I09)、光源側90°-検出器側0°である場合の蛍光強度(I90)、および光源側90°-検出器側90°である場合の蛍光強度(I99)を測定した。極大波長は526nmであった。
[5-1.HSA固定化金基板の作製]
まず、金基板を水およびエタノールで洗浄した後、UV-O3処理した。その後すぐに、11-メルカプトウンデカン酸(1mM, エタノール) 5mL中に浸漬し、24 h 25℃でインキュベーションを行うことにより、金基板表面に11-メルカプトウンデカン酸の自己組織化単分子(SAM)膜を形成した(SAM膜形成工程)。
SAM膜形成工程によって得られた基板のXPS測定の結果、S2p軌道に由来する軌道が明確に確認された。したがって、基板の表面がカルボキシル基修飾されていることが判った。
表面プラズモン共鳴法(SPR)センサー装置(ビアコア社製Biacore Q)を用い、HSA固定化基板に対する蛍光性HSA認識ナノ粒子[HSA]MIP-NGsの吸着挙動について確認した。Running bufferに10 mM PBS (pH 7.4)を用い、温度25℃、Flow rate: 20μL/min、Injection volume: 20μLで測定を行った。蛍光性HSA認識ナノ粒子[HSA]MIP-NGsは、10 mM PBS buffer (pH 7.4)に分散させ,濃度を100, 200, 400, 800, 1600 ng/mLと変化させて測定に供した。
同様の操作により、蛍光性リファレンスナノ粒子NIP-NGsについても測定を行った。
精製した蛍光性HSA認識ナノ粒子[HSA]MIP-NGsを分散させた10 mM PBS (pH 7.4)を、ラットの尾の静脈にインジェクションした。ラットの耳の血管の蛍光動画を、共焦点レーザー顕微鏡(Nikon製)を用いて撮影した。10時間にわたり、動脈、静脈および組織中の蛍光強度を経時的に測定した。
積算時間1時間16分時の顕微鏡画像を図4に示す。図4中、四角で囲われた箇所(図中左から順に、組織、動脈、静脈の測定箇所を示す)で、蛍光強度の経時変化を調べた。静脈および組織中の蛍光強度の経時変化を図5に示す。図5は、横軸に時間(分)を表し、縦軸に相対強度(%)を表す。
以下のとおり、実施例1の蛍光性HSA認識ナノ粒子[HSA]MIP-NGsと、比較例1の蛍光性リファレンスナノ粒子NIP-NGsとについて、肝臓における血中滞留性を共焦点レーザー顕微鏡を用いて確認した。
Balb/cマウス(メス,生後4週間)を使用し、肝臓を観察した。まず、マウスの腹部に脱毛クリームを塗り、脱毛した。その後、腹部から肝臓を観察できるように、電子メスとはさみとを用いて皮膚を裂いた。また,サンプルをインジェクションするために、尾静脈へのカテーテル挿入を行った。生理食塩水を1mLテルモシリンジで導入することによって、漏れが無いことを確認した。
比較例1の蛍光性リファレンスナノ粒子NIP-NGsについても同様の実験操作を行った。
図8(a)に、実施例1の蛍光性HSA認識ナノ粒子[HSA]MIP-NGsをインジェクションしたマウスの肝臓の10分後の共焦点レーザー顕微鏡写真を示し、図8(b)に、[HSA]MIP-NGsをインジェクションしたマウスの肝臓の14時間後の共焦点レーザー顕微鏡写真を示す。
図10(a)に、比較例1の蛍光性リファレンスナノ粒子NIP-NGsをインジェクションしたマウス肝臓の10分後の共焦点レーザー顕微鏡写真を示し、図10(b)に、NIP-NGsをインジェクションしたマウス肝臓の15時間後の共焦点レーザー顕微鏡写真を示す。
[8-1.アミド結合を有するDoxorubicin methacrylate (DOXMA-1)の合成]
1H-NMR chart.1 (300 MHz, DMSO-d6)
δ=14.04 (br, 1H), δ=13.28 (br, 1H), δ=7.94, 7.67, 7.33 (m, 3H), δ=5.60, 5.47 (m, 2H), δ=5.27 (d, 2H), δ=4.95 (br, 1H), δ=4.83 (m, 2H), δ=4.56 (m, 2H), δ=4.16 (m, 1H), δ=3.97 (s, 3H), δ=3.43 (s, 1H), δ=2.97 (br, 2H), δ=2.18 (m, 1H), δ=1.97 (m, 1H), δ= 1.78 (s, 3H), δ=1.47 (m, 1H), δ=1.17 (d, 3H)
得られたDOXMA-1を無乳化剤沈殿重合系で共重合させることで、薬物を担持しつつHSA認識空間をもつ[HSA]MIP-NGs(DOX1-[HSA]MIP-NGs)の合成を行った。共重合反応系を構築する具体的な成分および組成を下記表2に示す。重合はシュレンクフラスコ中で窒素雰囲気下、70℃で12時間反応させた。
重合により得られたエマルションをSephadex G-100を用いてサイズ排除クロマトグラフィーを行って、薬剤担持HSA認識ナノ粒子DOX1-[HSA]MIP-NGsを精製した。その後、薬剤担持HSA認識ナノ粒子DOX1-[HSA]MIP-NGsを、動的光散乱法により粒子径を測定した。DLS測定の結果、Z平均粒子径は37 nm (PDI: 0.49)であった。図14に、DLSにより得られた薬剤担持HSA認識ナノ粒子DOX1-[HSA]MIP-NGsの粒子径分布を示す。このことから、薬剤担持型の[HSA]MIP-NGsにおいても、実施例1の薬剤非担持型の[HSA]MIP-NGsと同様にナノサイズMIP粒子を得ることが出来たと考えられる。
実施例1の項目5-1と同様にしてHSA固定化金基板を作成し、得られたHSA固定化金基板を用いて、項目5-2と同様にして薬剤担持HSA認識ナノ粒子DOX1-[HSA]MIP-NGsの吸着挙動を確認した。
本発明のナノ粒子の細胞内への取り込みを観察し、DDSにおける有用性を確認した。
共焦点観察用ガラスディッシュに血清D-MEM培地を用いて繊維芽細胞であるNIH/3T3を細胞数24万 cells/dishとなるように播種し、24時間CO2インキュベーター内で静置した。その後、精製した実施例1の薬剤非担持型のHSA認識ナノ粒子[HSA]MIP-NGsを、その濃度が100 μg/mLとなるように200 μL添加し、さらに24時間CO2インキュベーター内で静置した。なお、サンプルは観察前に血清D-MEM培地による洗浄を行っている。
共焦点レーザー顕微鏡:オリンパス社製IX81
対物レンズ: 100×(oil)
使用フィルター: FITC
図16の「With Dox」に示すように、薬剤担持型のHSA認識ナノ粒子DOX1-[HSA]MIP-NGsについても、細胞内に取り込まれていることが明らかになった。
ヒト乳がん細胞であるHelaについても上記項目11-1と同様に、薬剤非担持型のHSA認識ナノ粒子[HSA]MIP-NGsおよび薬剤担持型のHSA認識ナノ粒子DOX1-[HSA]MIP-NGsの取り込みを観察した。
標的タンパク質を、ヒト血清アルブミン(HSA)に替えてマウス血清アルブミン(MSA)としたことを除いて、項目1-1(実施例1)と同様に蛍光性MSA認識ナノ粒子[MSA]MIP-NGsを合成した。共重合反応系を構築した具体的な成分および組成を下記表3に示す。
実施例1の項目5-1と同様にしてHSA固定化金基板を作成し、HSAに替えてMSA(マウス血中アルブミン)を用いたことを除いて同様にしてMSA固定化金基板を作成し、HSAに替えてIgGを用いたことを除いて同様にしてIgG固定化金基板を作成した。
各種タンパク質固定化金基板のそれぞれに対し、項目5-2と同様にして蛍光性MSA認識ナノ粒子[MSA]MIP-NGsの吸着挙動を確認した。
蛍光性モノマーFAmを使用しなかったことを除いては基本的に実施例2と同様に無乳化剤沈殿重合系で共重合を行い、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsを合成した。共重合反応系を構築する具体的な成分および組成を下記表4に示す。重合はシュレンクフラスコ中で窒素雰囲気下、70℃で12時間反応させた。
重合により得られたエマルションをSephadex G-100を用いてサイズ排除クロマトグラフィーを行って、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsを精製した。その後、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsを、動的光散乱法により粒子径を測定した。DLS測定の結果、Z平均粒子径は17 nm (PDI: 0.46)であった。図20に、DLSにより得られた非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsの粒子径分布を示す。また、図21に、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsのUV-Visスペクトルを示す。これらの結果から、ドキソルビシン由来の吸収領域が存在し、抗がん剤が粒子内に封入されていることが示唆された。
[16-1.ヒドラゾン結合を有するDoxorubicin methacrylate (DOXMA-2)の合成]
Ethyl glycinate hydrochloride (5.0 g, 36 mmol)およびtriethylamine (10 mL, 72 mmol)をDCM(50 mL)に溶解した後、窒素雰囲気・氷冷下においてMethacryloyl chloride (3.78 g, 36 mmol)をDCM (30 mL)に溶解した溶液を滴化した。室温でover nightの反応を行い、その後、食塩水、クエン酸水溶液、および炭酸ナトリウム水溶液で三回ずつ洗浄し、再度食塩水で一回洗浄した。洗浄後、シリカゲルカラムクロマトグラフィー(ヘキサン:酢酸エチル= 100:00 - 50:50)によって精製を行った。減圧乾燥後、真空乾燥を行って、1H-NMRにて目的物の同定を行った。
1H-NMR (300 MHz, DMSO-d6) :
δ=8.35 (br, 1H), δ=5.71 (s, 1H), 5.39 (s, 1H), δ=4.07 (q, 2H), δ= 3.82 (m, 2H), δ= 1.85 (s, 3H), δ= 1.09 (t, 3H)
Ethyl glycinate methacrylate(0.5 g, 3.0 mmol)およびhydrazine hydrate (200 mg, 6.0 mmol)を無水メタノール(10 mL)中に混合し、室温でovernight反応させた。その後、溶媒を減圧除去後、シリカゲルクロマトグラフィー(EtOAc: MeOH= 100:00 - 50:50)によって精製を行った。減圧乾燥後、真空乾燥を行い、1H-NMRにて目的物の同定を行った。
1H-NMR (300 MHz, DMSO-d6):
δ=9.00 (br, 1H), δ=8.10 (br, 1H), δ=5.72 (s, 1H), δ=5.33 (s, 1H), δ=4.16 (b, 2H), δ= 3.65 (m, 2H), δ= 1.87 (s, 3H)
Methacryloyl glycine hydrazide(14.5 mg, 0.1 mmol)およびDOX hydrochloride(29 mg, 0.05 mmol)を無水メタノール(10 mL)中に混合し、室温でovernight反応させた。その後、溶媒を減圧除去後、MALDI-TOF-MSによって反応の進行を確認した。
MALDI-TOF-MS (matrix:CHCA): m/z=724.04 [M+Na].
薬剤モノマーとして上述のようにして得られたDOXMA-2を使用し、かつ、蛍光モノマーFAmを加えなかったことを除いては基本的に実施例と同様に無乳化剤沈殿重合系で共重合を行い、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsを合成した。共重合反応系を構築する具体的な成分および組成を下記表5に示す。重合はシュレンクフラスコ中で窒素雰囲気下、70℃で12時間反応させた。
重合により得られたエマルションをSephadex G-100を用いてサイズ排除クロマトグラフィーを行って、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsを精製した。その後、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsを、動的光散乱法により粒子径を測定した。DLS測定の結果、Z平均粒子径は81 nm (PDI: 0.45)であった。図22に、DLSにより得られた非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsの粒子径分布を示す。図23に、非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsのUV-Visスペクトルを示す。これらの結果から、ドキソルビシン由来の吸収領域が存在し、抗がん剤が粒子内に封入されていることが示唆された。
[18-1.sample]
96マイクロウェルプレートに無血清D-MEM培地を用いてNIH/3T3細胞を5000 cells/wellとなるように各ウェルに100 μLずつ播種し、24時間 CO2インキュベーター内で静置した。その後、カラムクロマトおよびフィルトレーションを施した実施例4の非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsおよび実施例5の非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsを、それぞれ、濃度が0-100μg/mLとなるように10 μLずつ添加し、さらに24時間CO2インキュベーター内で静置した。
NIH/3T3細胞を加えなかったことを除いて、上記の項目18-1と同様の操作を行って吸光度の測定を行った。
実施例4の非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsおよび実施例5の非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsを加えなかったことを除いて、上記の項目18-1と同様の操作を行って吸光度の測定を行った。
NIH/3T3細胞と、実施例4の非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX1-[HSA]MIP-NGsおよび実施例5の非蛍光性薬剤担持HSA認識ナノ粒子NF-DOX2-[HSA]MIP-NGsと、の両方を加えなかったことを除いて、上記の項目18-1と同様の操作を行って吸光度の測定を行った。
吸光度の測定を行った各試料の分類と内訳とを下記表6に示す。
以上の結果から、本発明の抗がん剤担持ナノ粒子が細胞に対して毒性を示すことが明らかになった。一方、抗がん剤を担持しないナノ粒子においては、細胞毒性がほとんど観察されなかったことから、抗がん剤を担持させることで、細胞毒性を生じさせることが可能になったと考えられる。したがって、本発明の抗がん剤担持ナノ粒子が、坑がん作用を持つナノキャリアとしての有用であることが示された。
Claims (7)
- 血漿タンパク質が分子インプリントされた血漿タンパク質認識部位を有し且つ生体適合性モノマーに由来する構成成分を含む分子インプリントポリマーであり、血管内搬送に用いられる、in vivoステルス性ナノ粒子。
- 前記血漿タンパク質がアルブミンである、請求項1に記載のin vivoステルス性ナノ粒子。
- 前記生体適合性モノマーが双性イオン化合物である、請求項1または2に記載のin vivoステルス性ナノ粒子。
- 平均粒子径が10nm以上100nm以下である、請求項1から3のいずれか1項に記載のin vivoステルス性ナノ粒子。
- シグナル基をさらに含む、請求項1から4のいずれか1項に記載のin vivoステルスナノ粒子。
- 薬剤成分が担持された、請求項1から5のいずれか1項に記載のin vivoステルスナノ粒子。
- 前記薬剤成分が、薬剤に重合性官能基が共有結合した薬剤モノマーに由来する構成成分として前記分子インプリントポリマーに含まれる、請求項6に記載のin vivoステルスナノ粒子。
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US10471011B2 (en) | 2019-11-12 |
EP3241563B1 (en) | 2020-03-04 |
JPWO2016088384A1 (ja) | 2017-10-12 |
JP6655845B2 (ja) | 2020-02-26 |
EP3241563A4 (en) | 2018-08-15 |
EP3241563A1 (en) | 2017-11-08 |
US20170319485A1 (en) | 2017-11-09 |
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