KR102144615B1 - Micelle comprising polydeoxyribonucleotide, drug delivery matrixs and process for the preparation thereof - Google Patents

Abstract

The present invention relates to a micelle comprising a polydeoxyribonucleotide (PDRN), a drug delivery system, and a method for preparing the same, comprising: a polymer polymer; drug; And a crosslinking agent for drug delivery, wherein the polymer is poly(ethylene oxide) and poly(glycidyl methacrylate) bonded poly(ethylene oxide)-b-poly(glycidyl methacrylate) Block copolymer, the drug is polydeoxyribonucleotide (polydeoxyribonucleotide, PDRN), the crosslinking agent of the present invention, characterized in that the dipropagyl 3,3'-dithiodipropionate containing a disulfide bond The micelles for drug delivery containing PDRN can be conveniently manufactured, and can stably protect the drug and obtain a drug effect continuously for a long time by sustained release of the drug. The micelles manufactured by the manufacturing method of the present invention can be stored for a long time and handled easily because PDRN is enclosed in micro micelles to ensure stability during storage. In addition, since the dispersibility is increased, it is better dispersed in the body to increase the absorption rate, and thus the efficacy of PDRN can be increased.

Description

Micelle comprising polydeoxyribonucleotide, drug delivery matrixs and process for the preparation thereof

The present invention relates to a micelle containing polydeoxyribonucleotide (PDRN), a drug delivery system, and a method for manufacturing the same, and more specifically, PDRN is enclosed in micro micelles to ensure stability during storage, thereby enabling long-term storage and , It relates to a PDRN-containing micelle, a drug delivery system, and a method for producing the same, which can be easily handled, as well as increased dispersibility, thereby increasing the effect of the PDRN drug.

In a drug delivery system that solubilizes most poorly soluble drugs that are insoluble in water and delivers them into the body, drug delivery technology using additives such as various surfactants or alcohols, drug delivery technology using polymers, PEG-PLGA Most are drug delivery technologies using a copolymer of two or more polymer blocks having a hydrophilic block and a hydrophobic block property.

This method of solubilizing poorly soluble drugs has been reported to cause side effects on the nervous and digestive systems because various types of surfactants or additives such as alcohol are added to inject into blood vessels in the body.

In addition, in the case of drug delivery technology using a copolymer, problems such as lack of biocompatibility of the synthetic polymer of the copolymer unit and a complicated process have been raised. In addition, the efficiency of excretion in vivo is reduced due to the high molecular weight.

Therefore, there is an urgent need to develop a new formulation that can use poorly soluble drugs as a drug delivery system with improved biocompatibility and biodegradability without the above problems.

Meanwhile, a polymersome is a sphere type in which molecules having different properties (hydrophobicity & hydrophilicity) in one molecule are self-assembly. The diblock copolymer is the simplest structure among the polymersomes and has a core-capsule type self-assembled spherical structure including hydrophilic and hydrophobic molecules in one molecule. In addition, the size of the spherical particles formed by self-assembly of the diblock copolymer may range from several tens of nanometers (nm) to several micrometers (㎛).

The amphiphilic molecule forming the polymersome is called an amphiphilic block copolymer, and the amphiphilic block copolymer forms a membrane in an aqueous or oil phase by a self-assembly method by a hydrophobic effect. , Polymersomes formed of diblock copolymers form polymersomes that exhibit unique properties depending on the properties of the blocks used. Such an amphiphilic diblock copolymer exhibits a structure similar to a typical low-molecular amphiphilic compound having a hydrophobic tail and a hydrophilic head, and this structure induces a micelle structure through a self-assembly process.

On the other hand, poloxamer is a polyoxyethylene-polyoxypropylene tertiary block copolymer (Poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblock copolymer) is hydrophobic due to its excellent surfactant properties due to its amphiphilic structure. And it is used to improve the solubility of the oil-soluble substance in water, or to improve the miscibility of two or more substances having different hydrophobicity.

The poloxamers exist in various types according to the molecular weight and content of polyoxypropylene, and are currently widely used in various industries such as solubilizing agents, emulsifying agents and wetting agents. In addition, the range of use is expanding day by day, such as continuous preparations, ophthalmic preparations, injections, and anticancer drugs for sustained release of drugs from the burn area.

In recent years, polymeric micelles based on a core-shell structure have been studied extensively because of their advantages such as hydrophobic drug solubility, long-term circulation time and specific tissue targeting. The core-shell structure of an amphiphilic block copolymer consists of an insoluble core and a water-soluble corona. Polymer micelles are typically in the range of tens of nanometers in size and are suitable for enhanced permeability and retention (EPR) effects. The EPR effect is characterized by the accumulation of molecules in the range of 40 to 200 nanometers in cancer cells larger than in normal cells. In other words, the side with normal cells has a tight boundary with blood vessels, whereas the side with fast-growing cancer cells has a loose boundary with blood vessels, so molecules or particles of this size cannot penetrate normal cells, while cancer The blood vessel wall on the tissue side passes well and is transferred to and accumulated in cancer cells, and is effectively used for drug delivery to cancer cells.

However, the inherent instability of micelles has been a significant problem prior to many applications. In other words, the polymer micelles can be separated from each other as a single substance at a very diluted concentration during blood circulation and escaped. It does not achieve targeted release and may cause toxicity to normal living tissues. Therefore, improving the stability of polymer micelles is an urgent problem that has been recently studied.

On the other hand, polydeoxyribonucleotide (hereinafter referred to as'PDRN') is known as a tissue regeneration material, and PDRN is manufactured from DNA extracted from salmon semen.

PDRN is used for the treatment of wounds caused by skin transplantation and tissue repair, but it is also used at the judgment of the medical staff for some tissue regeneration or inflammation treatment where there is no proper treatment, and also releases various growth factors by stimulating the A2 receptor, a signal transducer for skin regeneration. It is known as a component that promotes, produces capillaries by VEGF (vascular endothelial cell proliferation factor), improves blood circulation, has anti-inflammatory action, and prevents capillary leakage.

The present inventors believe that PDRN is encapsulated in micro micelles to ensure stability during storage, enabling long-term storage, easy handling, and increased dispersibility to increase the drug efficacy of PDRN by increasing the absorption rate by being better dispersed in the body. The present invention was completed by preparing PDRN-containing micelles.

Accordingly, a technical problem to be solved in the present invention is to provide a micelle for drug delivery including a polydeoxyribonucleotide (PDRN).

Another technical problem to be solved in the present invention is to provide a drug delivery system comprising the micelles.

Another technical problem to be solved in the present invention is to provide a method of manufacturing the micelles and drug delivery systems.

In order to solve the above technical problem, in the present invention, a polymer polymer; drug; And a crosslinking agent for drug delivery, wherein the polymer is poly(ethylene oxide) and poly(glycidyl methacrylate) bonded poly(ethylene oxide)-b-poly(glycidyl methacrylate) It is a block copolymer, the drug is a polydeoxyribonucleotide (PDRN), and the crosslinking agent is a dipropagyl 3,3'-dithiodipropionate containing a disulfide bond. Provide micelles.

Polydeoxyribonucleotide (PDRN), an active ingredient of the present invention, is a material extracted from the testis of salmon or herring and has a molecular weight ranging from 100Da to 1500kDa. Since the use is different depending on the molecular weight size, for example, when used for an external product such as a wound covering, PDRN having a molecular weight of about 200 to 500 kDa is used, so a process of purifying the PDRN and classifying it by molecular weight is required. In the purification process, the same column as GPC is used. In the case of GPC, a small amount of purification is possible, but in the case of large-scale purification, PDRN purification is performed using expensive equipment. If the PDRN purification process is performed with such expensive equipment, the process cost increases, and thus the purified PDRN by size must be supplied at a high price. If a product is developed using this purified PDRN, the problem of increasing the unit price for the product occurs. .

PDRN is mainly used for DNA injection, and DNA injection is a substance that stimulates growth factors such as fibroblasts and collagen to promote cell proliferation and healing. Compared to other treatments or injections, it is more than 30% effective faster and more powerful, and the effect lasts. The period is long. Not only does it surpass the efficacy and stability of existing injections, but also relieves discomfort due to no pain caused by inflammation, and has no side effects as it induces balanced regeneration through the human body's own mechanism rather than an artificial method. Unlike general pain and inflammation treatments, it is a treatment that helps regeneration of cells and tissues. It is not a temporary role to relieve symptoms in a short period of time, but a fundamental healing can be expected. The effect of tissue regeneration is also not supplying growth factors with a short half-life, but genes Continuous effect can be expected by inducing the production of growth factors by promoting expression.

According to one embodiment of the present invention, "micelles" formed by polymers are generally formed by self-assembly of amphiphilic polymers in an aqueous solution and have a unique core-shell structure thereof. And a poorly soluble drug may be encapsulated in the core. An amphiphilic polymer having both hydrophilicity and hydrophobicity is used, consisting of a hydrophilic moiety having hydrophilicity and a hydrophobic block having hydrophobicity. When these amphiphilic polymers are dispersed in an aqueous solution, they tend to clump themselves (ie, self-aggregation) in order to minimize contact with water and stabilize free energy due to hydrophobic interactions by hydrophobic blocks. A fine core is formed by the hydrophobic blocks thus agglomerated, and a shell is formed around the fine core by a hydrophilic block to form a polymer micelle having a physical bond between molecules. The solubility of the polymer micelles in an aqueous solution is increased by the hydrophilic block. "Mycel" in the present invention has an average particle size of 50 to 400 nm.

In the present invention, "biodegradable" means that the block copolymer can be chemically decomposed in the body to form non-toxic compounds. The rate of degradation is the same as or different from the rate of release of the drug (drug, etc.).

In the present invention, "biocompatibility" means a function of interacting with the human body without undesirable subsequent effects.

In the present invention, "continuous release" refers to the continuous release of a drug (drug, etc.) over a predetermined period.

In the present invention, "controlled release" refers to the control of the rate and/or amount of a drug delivered according to the drug (drug, etc.) delivery formulation of the present invention. Controlled release may be continuous or discontinuous and/or linear or nonlinear. This can be achieved using one or more types of polymeric compositions, drug (drug, etc.) loading, excipients or degradation enhancing agents, or the inclusion of other modifiers, administered alone, in combination or sequentially to provide the desired effect.

In the present invention, "drug" refers to any compound or composition that induces a desired pharmacological, immunogenic and/or physiological effect by local and/or systemic action when administered to an organism (human or non-human animal). Mention. Thus, the term includes compounds or chemicals traditionally regarded as drugs, vaccines, and biomedical agents including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like.

In the present invention, "therapeutic effect" refers to any improvement in the disease of a subject, human or animal treated according to the method, and to be detected by a preventive or preventive effect, or by physical investigation, laboratory or mechanical methods. And obtaining any relief in the severity of the signs and symptoms of a possible disease, disease or condition.

Unless otherwise defined in the context of a specific disease or condition in the present invention, the terms "treatment" or "treating" as used herein means (i) a disease, disease and/or disease prone to but not yet diagnosed as ill. Preventing the occurrence of a disease, disease, or disease in an unspecified animal or human; (ii) inhibiting a disease, disease or condition, ie inhibiting its progression; And/or (iii) alleviating the disease, disorder or condition, ie causing a regression of the disease, condition and/or condition.

In one embodiment of the present invention, the micelle is a hydrophilic shell; And a hydrophobic core.

In one embodiment of the present invention, the polymer polymer includes an azide group, and the crosslinking agent and the drug may include an alkyne group.

In one embodiment of the present invention, the drug may include a hydrazone linker.

In one embodiment of the present invention, the polymer polymer may include an alkyne group, and the crosslinking agent and the drug may include an azide group.

In one embodiment of the present invention, the hydrophilic portion of the polymer polymer is made of poly(ethylene oxide), poly(oligo(ethylene glycol)methyl ether methacrylate) and poly(2-dimethylamino)ethyl methacrylate). It may be one or more selected from the group.

In one embodiment of the present invention, the hydrophobic portion of the polymer polymer is poly(glycidyl methacrylate), poly(α-propargyl carboxylate-ε-caprolactone), poly(α-propargyl-ε- Caprolactone), poly(azido ε-caprolactone-co-ε-caprolactone), poly(azido ε-caprolactone), poly(γ-propargyl-glutamate), poly(5-( 4-(prop-2-yne-1-yloxy)benzyl)-1,3-dioxolane-2,4-dione (tyrosine (alkynyl)-OCA), poly(N-(prop-) 2-ain-1-yl)acrylamide), poly(N-(3-azidopropyl)acrylamide), poly(1-ethanyl-4-vinylbenzene), poly(γ-azido-propyl- It may be at least one selected from the group consisting of L-glutamate) and poly(propargyl acrylate).

In one embodiment of the present invention, the polymer polymer may be poly(ethylene oxide)-b-poly(glycidyl methacrylate) including an azide group or an alkyne group.

In one embodiment of the present invention, the drug is a polydeoxyribonucleotide (PDRN).

In one embodiment of the present invention, the crosslinking agent may be dipropargyl 3,3'-dithiodipropionate.

In another embodiment of the present invention, there is provided a PDRN drug delivery system having a wound treatment, tissue repair, skin regeneration, blood circulation improvement and anti-inflammatory action including the micelles.

In another embodiment of the present invention, in the method of manufacturing the micelle, 1) preparing an amphiphilic polymer polymer comprising a hydrophilic portion and a hydrophobic portion and containing an azide group; 2) introducing an alkyne group to the drug through a hydrazone linker; 3) preparing a crosslinking agent containing a disulfide bond and an alkyne group; And 4) mixing and reacting the polymer polymer, the drug, and the crosslinking agent. It provides a method of manufacturing a micelle for drug delivery comprising a.

In another embodiment of the present invention, in the method of manufacturing the micelle, 1) preparing an amphiphilic polymer polymer comprising a hydrophilic portion and a hydrophobic portion and containing an azide group; 2) introducing a drug into the polymer polymer; 3) preparing a crosslinking agent containing a disulfide bond and an alkyne group; And 4) reacting by mixing the polymer polymer into which the drug has been introduced and the crosslinking agent. It provides a method for producing a micelle for drug delivery.

In one embodiment of the present invention, step 1) may prepare a polymer polymer through RAFT polymerization or ATRP molecular controlled polymerization.

In one embodiment of the present invention, the hydrophilic moiety in step 1) is poly(ethylene oxide), poly(oligo(ethylene glycol)methyl ethermethacrylate) and poly(2-dimethylamino)ethyl methacrylate). It is at least one selected from the group consisting of, and the hydrophobic moiety is poly(glycidyl methacrylate), poly(α-propargyl carboxylate-ε-caprolactone), poly(α-propargyl-ε-caprolactone) , Poly(azido ε-caprolactone-co-ε-caprolactone), poly(azido ε-caprolactone), poly(γ-propargyl-glutamate), poly(5-(4-(pro P-2-yne-1-yloxy)benzyl)-1,3-dioxolane-2,4-dione (tyrosine (alkynyl)-OCA), poly(N-(prop-2-yne-) 1-yl)acrylamide), poly(N-(3-azidopropyl)acrylamide), poly(1-ethynyl-4-vinylbenzene), poly(γ-azido-propyl-L-glutamate) And it may be at least one selected from the group consisting of poly (propargyl acrylate).

In one embodiment of the present invention, the drug in step 2) is polydeoxyribonucleotide (PDRN).

In one embodiment of the present invention, the crosslinking agent in step 3) may be dipropargyl 3,3'-dithiodipropionate.

The drug contained in the drug delivery system of the present invention is delivered by injection or other method (e.g., implantation, placement in a body cavity or possible space, tissue of the body) as appropriate to a person or other mammal with a therapeutically effective disease state or symptom. Coating the surface or coating the surface of the implantable device), but it is particularly preferred that the composition is delivered parenterally.

The'parenteral' includes intramuscular, intraperitoneal, intra-abdominal, subcutaneous, intravenous and intraarterial.

The composition of the present invention may be typically formulated as an injection formulation. The injectable compositions of the present invention can be injected or inserted into the body of a human or other mammal by any suitable method, preferably by injection through a hypodermic needle. For example, it may be administered intraarterial, intravenous, genitourinary, subcutaneous, intramuscular, subcutaneous, intracranial, intraperical, intrapleural, or other body cavity or possible space by injection or other means. Or, via a catheter or syringe, for example during an arthroscopic procedure, into the joint, or into the genitourinary tract, into the vessel, into the palate or into the pleura, or into any body cavity or possible space in the body, surgery, surgery, diagnosis or It can be introduced during interventional procedures. In other applications, open surgical or traumatic wounds, burns, or topical application to the skin or other tissue surface.

In particular, the present invention is characterized in that it is a sustained-release composition that allows a drug to be slowly released in the body when injected into a specific site in the body.

The composition of the present invention is suitable for use as a sustained and controlled release matrix of drugs. When the matrix is coupled with one or more drugs homogeneously contained therein, a biodegradable sustained release drug delivery system is provided.

As used herein, the term “sustained release” (ie, sustained release or controlled release) refers to a human or other mammal being introduced, or to an open wound, burn or tissue surface, or to a body cavity or latent body space. Introduced within, means to continuously release one or more drug streams over a predetermined period of time at a therapeutic level sufficient to achieve the desired therapeutic effect over a predetermined period of time.

In one embodiment of the present invention, it was confirmed that most of the encapsulated drugs can be released continuously for about 21 to 25 days without initial rapid release.

In addition, since the composition for drug delivery of the present invention is decomposed into a substance harmless to the human body after a certain period of time and is discharged to the outside through the kidney, the composition according to the present invention is injected into a specific part of the body using a general syringe or catheter. In one case, since the drug is slowly released and the drug is maintained at a certain concentration in the circulating blood for a long period of time, the drug is not only excellent in expression of efficacy, but also does not require a separate surgical removal procedure for removing the drug carrier.

Thus, according to the sustained-release drug delivery system of the present invention, the drug can be released to the target site of the subject in a controlled manner.

In one embodiment, micelles are used to provide site-specific release of a drug to a subject. In another embodiment, the micelles comprise one or more drugs that can be administered to the subject, such that the drug is released by diffusion from the micelles and/or degradation of the micelles. At this time, the administration value of the composition will vary depending on the type and severity of the disease, disease or disease to be treated. It should be further understood that for any particular subject, a particular dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or directing the composition. In vivo administration can be based on in vitro release studies in cell culture or in vivo animal models.

As such, the compositions of the present invention release drugs in a controlled manner, thus providing optimal delivery of drugs. As a result of controlled delivery, the drug is delivered for the desired period. Slower and more constant rates of delivery can in turn lead to a decrease in the frequency with which the drug must be administered to the animal. In addition, in the case of the dosage, 0.5 ~ 100 mg/kg per day, can be administered once or several times, but appropriately varies depending on the degree of disease symptoms, age, weight, health status, sex, administration route, and treatment period. Can be.

In addition to PDRN, drug substances that may be included in the micelles of the present invention include, for example, proteins, polypeptides, carbohydrates, inorganic substances, antibiotics, anti-neoplastic agents, local anesthetics, anti-angiogenic agents, vasoactive agents, anticoagulants, immunomodulators, cells. Toxic agents, antiviral agents, antibodies, neurotransmitters, psychotropic agents, oligonucleotides, lipids, cells, tissues, tissues or cell aggregates, and combinations thereof. In addition, there are cancer chemotherapeutic agents such as cytokines, chemokines, lymphokines and indeed purified nucleic acids, and vaccines such as attenuated influenza viruses. Actually purified nucleic acids that can be incorporated include genomic nucleic acid sequences, cDNA encoding proteins, expression vectors, antisense molecules and ribozymes that bind to complementary nucleic acid sequences and inhibit translation or transcription. As such, there is no limitation on the types of drugs that can be used basically.

In addition, various techniques for encapsulating (loading) drugs into micelles are known. The drug is contained in the micelles in an amount of about 0.01 to about 100% by weight, preferably about 1 to about 95% by weight, and more preferably about 10 to about 70% by weight. The amount or concentration of drug contained in micelles will depend on the rate of absorption, inactivation and excretion of the drug.

In one embodiment, the micelles of the present invention, prepared by the above method, enter the body through an intravenous injection and circulate through the bloodstream, and have the property of loosening the cells constituting the cancer tissue by the enhanced permeability and retention (EPR) effect. It is delivered to the target site where the tumor is located, and over time, the drug contained in the micelles will be actively diffused, or the micelles will be degraded and released by controlled means to the target site.

All the contents described above with respect to the micelle particles and the drug delivery composition may be applied or applied mutatis mutandis to the drug delivery system.

Herein, a method of treating a disease, disease, or disease including introducing the drug to a subject (patient) in need thereof, and a method of manufacturing the delivery system of the present invention are also included.

All the contents described above with respect to the micelle particles and the drug delivery composition may be applied or applied mutatis mutandis to an external preparation for skin.

In the present specification, the term "skin external preparation" is a concept encompassing the entire composition applied to the skin, and includes, for example, various cosmetics such as basic cosmetics, makeup cosmetics, and hair cosmetics; It is a concept including ointments, creams, lotions, gels, patches, etc., including pharmaceutical compositions applied to external skin, including pharmaceuticals and quasi-drugs.

The micelles for drug delivery comprising the PDRN of the present invention can be conveniently prepared, and can stably protect the drug and obtain a drug effect continuously for a long time by sustained release of the drug. The micelles manufactured by the manufacturing method of the present invention can be stored for a long time and handled easily because PDRN is enclosed in micro micelles to ensure stability during storage. In addition, since the dispersibility is increased, it is better dispersed in the body to increase the absorption rate, and thus the efficacy of PDRN can be increased.

1 is a graph showing the in vitro PDRN release pattern from micelles.

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

Inclusion or inclusion in the entire specification refers to the inclusion of certain constituent elements without particular limitation, and cannot be interpreted as excluding or excluding the addition of other constituent elements.

Unless expressly stated throughout the specification, terminology is only intended to refer to specific embodiments and is not intended to limit the present invention. In addition, the singular forms used herein also include plural forms unless the phrases express an obvious opposite meaning.

In one embodiment of the present invention, an amphiphilic polymer comprising a hydrophilic moiety and a hydrophobic moiety; A drug bonded to the polymer by an alkyne-azide chemical bond; And a crosslinking agent including a disulfide bond and bonded to the polymer by an alkine-azide chemical bond. It provides a micelle for drug delivery comprising a PDRN.

The'alkine' means alkyne and generally refers to a compound having a triple bond between atoms in a large category, but is not limited thereto.

The alkyne-azide chemical bond is bonded by a click reaction, but is not limited thereto.

In one embodiment of the present invention, the micelle is a hydrophilic shell; And a hydrophobic core.

In one embodiment of the present invention, the polymer polymer includes an azide group, and the crosslinking agent and the drug may include an alkyne group.

In one embodiment of the present invention, the drug may include a hydrazone linker.

In one embodiment of the present invention, the polymer polymer may include an alkyne group, and the crosslinking agent and the drug may include an azide group.

In one embodiment of the present invention, the hydrophilic portion of the polymer polymer is made of poly(ethylene oxide), poly(oligo(ethylene glycol)methyl ether methacrylate) and poly(2-dimethylamino)ethyl methacrylate). It may be one or more selected from the group.

In one embodiment of the present invention, the hydrophobic portion of the polymer polymer is poly(glycidyl methacrylate), poly(α-propargyl carboxylate-ε-caprolactone), poly(α-propargyl-ε- Caprolactone), poly(azido ε-caprolactone-co-ε-caprolactone), poly(azido ε-caprolactone), poly(γ-propargyl-glutamate), poly(5-( 4-(prop-2-yne-1-yloxy)benzyl)-1,3-dioxolane-2,4-dione (tyrosine (alkynyl)-OCA), poly(N-(prop-) 2-ain-1-yl)acrylamide), poly(N-(3-azidopropyl)acrylamide), poly(1-ethanyl-4-vinylbenzene), poly(γ-azido-propyl- It may be at least one selected from the group consisting of L-glutamate) and poly(propargyl acrylate).

In one embodiment of the present invention, the polymer polymer may be poly(ethylene oxide)-b-poly(glycidyl methacrylate) including an azide group or an alkyne group.

In one embodiment of the present invention, the crosslinking agent may be dipropargyl 3,3'-dithiodipropionate.

In another embodiment of the present invention, a drug delivery system having PDRN efficacy including the micelles is provided.

The drug delivery system may be prepared by a conventional method and is not particularly limited as long as it is a configuration capable of delivering a drug to a specific location in the body.

In another embodiment of the present invention, in the method of manufacturing the micelle, 1) preparing an amphiphilic polymer polymer comprising a hydrophilic portion and a hydrophobic portion and containing an azide group; 2) introducing an alkyne group to the drug through a hydrazone linker; 3) preparing a crosslinking agent containing a disulfide bond and an alkyne group; And 4) reacting by mixing the high molecular polymer, the drug, and the crosslinking agent; it provides a method for producing a micelle for drug delivery comprising PDRN.

In another embodiment of the present invention, in the method of manufacturing the micelle, 1) preparing an amphiphilic polymer polymer comprising a hydrophilic portion and a hydrophobic portion and containing an azide group; 2) introducing a drug into the polymer polymer; 3) preparing a crosslinking agent containing a disulfide bond and an alkyne group; And 4) reacting by mixing the polymer polymer into which the drug has been introduced and the crosslinking agent. It provides a method for preparing a micelle for drug delivery comprising PDRN.

In one embodiment of the present invention, step 1) may prepare a polymer polymer through RAFT polymerization or ATRP molecular controlled polymerization.

The RAFT polymerization reaction means Reversible Addition-Fragmentation Chain Transfer polymerization, and the ATRP molecular control polymerization reaction means atom transfer radical polymerization.

In one embodiment of the present invention, the hydrophilic moiety in step 1) is poly(ethylene oxide), poly(oligo(ethylene glycol)methyl ethermethacrylate) and poly(2-dimethylamino)ethyl methacrylate). It is at least one selected from the group consisting of, and the hydrophobic moiety is poly(glycidyl methacrylate), poly(α-propargyl carboxylate-ε-caprolactone), poly(α-propargyl-ε-caprolactone) , Poly(azido ε-caprolactone-co-ε-caprolactone), poly(azido ε-caprolactone), poly(γ-propargyl-glutamate), poly(5-(4-(pro P-2-yne-1-yloxy)benzyl)-1,3-dioxolane-2,4-dione (tyrosine (alkynyl)-OCA), poly(N-(prop-2-ine- 1-yl)acrylamide), poly(N-(3-azidopropyl)acrylamide), poly(1-ethynyl-4-vinylbenzene), poly(γ-azido-propyl-L-glutamate) And it may be at least one selected from the group consisting of poly (propargyl acrylate).

In one embodiment of the present invention, the crosslinking agent in step 3) may be dipropargyl 3,3'-dithiodipropionate.

Hereinafter, preferred embodiments, etc. are presented to aid the understanding of the present invention. However, the following examples and the like are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the examples.

<Example 1> PEO (polyethylene oxide)-b-PGMA-N3 (block copolymer), dipropargyl 3,3'-dithiodipropionate (disulfide-containing crosslinking agent) and PDRN (drug) containing Preparation of micelles

1-1. Synthesis of PEO-b-PGMA block copolymer

A block copolymer of PEO-b-PGMA was synthesized through RAFT using a PEO-RAFT macro initiator.

The general procedure is to put PEO-RAFT (0.25 g, 0.11 mmol), GMA (0.711 g, 5 mmol), AIBN (0.008 g, 0.05 mmol), and DMF (3 mL) in a round flask, and then replace with nitrogen for 30 minutes and 75 The mixture was stirred in an oil bath at °C for 7 hours. Thereafter, the product was dissolved in THF and purified by precipitation in an excess of ethyl ether. The polymer was dried in a vacuum oven at 40° C. overnight (0.7 g, yield: 73%).

1-2. Introduction of an azide group into the PEO-b-PGMA-N3 block copolymer

PEO-b-PGMA-N3 block copolymer (Mn of PEO / Mn of PGMA = 1) was prepared through the epoxide ring-opening reaction of the PGMA segment (segments). A mixture of PEO-b-PGMA (0.40 g, 1.4 mmol of epoxide moieties), NaN3 (0.406 g, 6.3 mmol), NH4Cl (0.334 g, 6.3 mmol) and DMF (5 mL) was added to a round flask. Stirred at 50° C. for 24 hours. After removing the insoluble salt through the filter, the filtrate was dialyzed in purified water (MW cutoff, 1 kDa) for 24 hours. Purified water was changed about every 4 hours. The final product was obtained by freezedrying (0.39 g, 97%).

1-3. Synthesis of dipropargyl 3,3'-dithiodipropionate (crosslinking agent containing disulfide bonds)

To prepare dipropargyl 3,3'-dithiodipropionate, a crosslinking agent containing a disulfide bond, 3,3'-dithiodipropionic acid (3.55 g, 16.9 mmol), propargyl alcohol (3.3 g) , 59.2 mmol) and a benzene solution (80 mL) of p-toluenesulfonic acid monohydrate (1.6 g, 8.5 mmol) were refluxed for 2 hours. The reaction mixture was washed with saturated aqueous NaHCO ₃ , brine, and dried MgSO ₄ and then concentrated. The product was isolated (2.6 g, 13.3 mmol, 79%) by flash column chromatography on silica gel (hexane/AcOEt, 10/1, v/v).

1-4. Introduction of an alkyne group to PDRN (drug) through a hydrazone linker (preparation of propargylamidopropanohydrazide-prednisolone 21-acetate)

Propargyl amine was acrylated with tert-butyl acrylate through Michael addition reaction. At room temperature, a solution of propargyl amine (0.6 g, 10.9 mmol) was added to dry methanol (10 mL) containing tert-butyl acrylate (0.9997 g, 7.8 mmol). The mixture was stirred at 50° C. for 24 hours. Excess reagent and solvent were removed by applying vacuum.

The crude reaction mixture (1.36 g, 8 mmol) was dissolved in anhydrous methanol (4 mL), and hydrazine monohydrate (5 mL, 82 mmol) was slowly added. Then, the reaction mixture was gently stirred at 50° C. for 12 hours. Methanol and by-products were removed by distillation under reduced pressure, and the oily residue was dissolved in propan-2-ol. The solvent was removed by distillation under reduced pressure again, and this procedure was repeated twice to remove unreacted hydrazine hydrate to prepare propargylamidopropanohydrazide.

Thereafter, PDRN (100 mg, 0.248 mmol) was dissolved in 10 mL methanol at 30°C. The prepared propargylamidopropanohydrazide (175 mg, 1.24 mmol) was added to the solution, and the suspension was stirred vigorously in a dark room. After 30 minutes, 0.2 mL of acetic acid was added to the suspension, and the mixture was stirred at 30° C. for 64 hours. The solvent was removed under reduced pressure, and propargylamidopropanohydrazide-PDRN was obtained by recrystallization with chloroform-ether at 60°C.

1-5. Preparation of drug-carrying micelles of PEO-b-PGMA-N3 block copolymer

PEO-b-PGMA-N3 ( 5.0mg, azide group of 14.3X10 ^{- 3} mmol), dimethyl 3,3'-dimethyl propargyl thio di propionate (0.36mg, 1.2X10 ^{- 3} mmol) and propargyl amino ^{(- 3 mmol 3.0 mg, 5.7X10} ) was dissolved uniformly in dry (anhydrous) DMF (1 mL) doped Fano hydrazide -PDRN to. Thereafter, 30 mL of DI was adjusted to pH 8.5 in a borate buffer solution to avoid hydrolysis of hydrozone bonds. And DI was dripped while stirring vigorously for one day. Copper (II) sulfate pentahydrate (2.2 mg, 8.8 μmol) to water (100 μL) and sodium ascorbate (1.75 mg, 8.8 μmol) to water (100 μL) were sequentially added, and the mixture was copper. It turned pale yellow due to the formation of copper complexes. The solution was stirred at room temperature for 24 hours. Subsequently, the formed solution was transferred to a dialysis bag (MW cutoff, 1 kDa), placed in 2 L of purified water having a pH of 8.5, and the purified water was changed every 4 hours, followed by dialysis for two days. The micelle solution was passed through a 0.45 μm filter and freeze-dried.

<Test Example 1> DLS and TEM measurements for micelles with crosslinked cores (micelles of Example 1) and micelles without crosslinking cores

In order to further confirm the crosslinked hydrophobic core, changes in the mechanical diameter of the core-crosslinked micelles and the core-non-crosslinked micelles were measured through DLS.

It can be seen that the non-crosslinked micelles have a particle size distribution of 82 nm, and the crosslinked micelles have a particle size distribution of 78 nm. The particle size after crosslinking slightly decreased due to the reinforced miniaturization and densification of the crosslinked structure.

In addition, the size and shape of the core-crosslinked micelles and the core-crosslinked micelles were confirmed through TEM. As a result, both non-crosslinked and crosslinked micelles showed uniform spherical distribution. The average diameter of uncrosslinked micelles was approximately 75 nm and the average diameter of crosslinked micelles was approximately 65 nm. The size obtained from the TEM measurement was smaller than the value obtained from DLS due to the shrinkage due to drying of the micelles during the TEM sample preparation process.

<Test Example 2> Analysis of drug loading efficiency of micelles of Example 1

PDRN, which has very low solubility in water, was used as a demonstration drug mounted in a hydrophobic core. PA functionalized with a hydrazone bond and an alkyne functional group was covalently bonded to the micelle core by a click reaction at the same time as the core crosslinking reaction. As a result of injecting azide: crosslinking agent: PA-alkine at a molar ratio of 1: 0.14: 0.66, the PA loading efficiency was measured to be 83%. This result is much higher than that of a physically mounted system that typically shows 6-32% efficiency. That is, it can be seen that the excellent performance of drug loading to micelles can be improved through an alkine-azide click reaction.

<Test Example 3> Measurement of stability of micelles of Example 1

Oxidation stability was measured using a Metrohm 743 Rancimat (Metrohm 743 Rancimat) at 130 ℃, 20ℓ / min ventilation conditions (induction time). As a comparative example, an emulsion composition containing PDRN was used.

As shown in Table 1, it was confirmed that the micelles of Example 1 of the present invention had a longer oxidation induction period than that of the composition of Comparative Example and thus had high oxidation stability.

<Test Example 4> Analysis of drug release pattern of micelles of Example 1

In order to prove that the micelles of the present invention effectively prevent the early release of the drug and have a dual response of pH and reduction reaction, the PDRN release pattern was analyzed. Crosslinked micelles loaded with PDRN were incubated in PBS at 37°C with pH 7.4 or 5 with or without DTT. Figure 1 shows a graph of in vitro PDRN release from micelles of Example 1 according to different DTT concentrations and pH at 37°C.

1 is a graph showing the in vitro PDRN release pattern from micelles.

As shown in FIG. 1, at pH 7.4, PDRN had little initial release due to a covalent bond between PDRN known as a triazole ring and a PGMA-N3 core. When the concentration of DTT was increased to 10 mM, the amount of PA released after 72 hours was measured to be about 21% of the initial loading value. In contrast, PDRN release was significantly increased at pH 5. As mentioned above, the acid-responsive hydrazone bond is decomposed at pH 5, so that PDRN is easily diffused and released from the core of the micelle. At high DTT concentrations, a large amount of PA is released by the decomposition of a large number of disulfide bonds at pH 7.4 and 5, indicating a reduction-responsive PDRN release characteristic. The PA release value reached 80% after 72 hours in the presence of 10 mM DTT at pH 5.

Claims (7)

High molecular polymer; drug; And as a drug delivery micelle comprising a crosslinking agent,
The high molecular weight polymer is a poly(ethylene oxide)-b-poly(glycidyl methacrylate) block copolymer in which poly(ethylene oxide) and poly(glycidyl methacrylate) are bonded,
The drug is polydeoxyribonucleotide (PDRN),
The crosslinking agent is a micelle for drug delivery comprising PDRN, characterized in that dipropargyl 3,3'-dithiodipropionate containing a disulfide bond. The method of claim 1,
The high molecular weight polymer contains an azide group,
The drug and the crosslinking agent contain an alkyne group,
The drug and the cross-linking agent drug delivery micelles comprising PDRN, characterized in that the polymer and the alkine-azide (alkyne-azide) is bonded by a chemical bond. The method of claim 1,
The PDRN is a micelle for drug delivery comprising a PDRN, characterized in that contained in the micelle in an amount of 0.01 to 100% by weight. The method of claim 1,
The micelles for drug delivery comprising PDRN having an average particle size of 50 to 400 nm. A drug delivery system comprising micelles for drug delivery according to any one of claims 1 to 4. In the method for manufacturing a micelle according to any one of claims 1 to 4,
1) preparing an amphiphilic polymer comprising a hydrophilic portion and a hydrophobic portion and containing an azide group;
2) introducing an alkyne group to the drug through a hydrazone linker;
3) preparing a crosslinking agent containing a disulfide bond and an alkyne group; And
4) reacting by mixing the polymer, the drug, and the crosslinking agent;
Method for producing a micelle for drug delivery comprising a. In the method for manufacturing a micelle according to any one of claims 1 to 4,
1) preparing an amphiphilic polymer comprising a hydrophilic portion and a hydrophobic portion and containing an azide group;
2) introducing a drug into the polymer polymer;
3) preparing a crosslinking agent containing a disulfide bond and an alkyne group; And
4) reacting by mixing the polymer polymer into which the drug is introduced and the crosslinking agent;
Method for producing a micelle for drug delivery comprising a.

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