KR100612734B1 - Composition comprising magnetic nanoparticle encapsulating magnetic material and drug with biodegradable synthetic polymer - Google Patents

Abstract

The present invention provides a composition containing magnetic nanoparticles in which a magnetic material and a therapeutic agent are encapsulated into a biodegradable polymer, a method of preparing the magnetic nanoparticles, and a magnetic field to concentrate the magnetic nanoparticles on a target site. A method for delivering targeted therapy.

Magnetic materials, biodegradable polymers, magnetic nanoparticles, targeted drug delivery methods

Description

Composition consisting magnetic nanoparticles encapsulating magnetic material and drug with biodegradable synthetic polymer

1 is a method for producing magnetic nanoparticles using an emulsion diffusion method, wherein the ratio of oil phase to aqueous phase is 1: 1.5 (a), 1: 2 (b), 1: 3 (c), and 1: 4 (d), respectively. SEM image of the magnetic nanoparticles prepared as) is shown.

FIG. 2 is a SEM image photograph of magnetic nanoparticles prepared in the method of manufacturing magnetic nanoparticles using an emulsion diffusion method in which the ratio of magnetite to PCL is 1: 5 (a) and 2: 5 (b), respectively. It is shown.

Figure 3 shows the average diameter of the magnetic nanoparticles according to the ratio of magnetite: PCL in the manufacturing method of the magnetic nanoparticles using the emulsion diffusion method.

4 is a method for producing magnetic nanoparticles using an emulsion diffusion method, wherein the ratio of magnetite to PCL is 1: 0 (a), 1: 5 (b), 2: 5 (c), and 3: 5 (d), respectively. The TEM image of the magnetic nanoparticles prepared by) is shown.

5 is a method for producing magnetic nanoparticles using the multiple emulsification method, wherein the polyvinyl alcohol concentrations for the aqueous phase 2 are 0.25% (a), 0.5% (b), 1% (c) and 2% (d, respectively). SEM image of the magnetic nanoparticles prepared as) is shown.

FIG. 6 is a SEM image photograph of magnetic nanoparticles prepared by using magnetic emulsification method using a multiple emulsification method with a ratio of magnetite to PCL of 1: 5 (a) and 2: 5 (b), respectively. It is shown.

Figure 7 shows the average diameter of the magnetic nanoparticles according to the ratio of magnetite: PCL in the method of producing magnetic nanoparticles using the multiple emulsification method.

8 shows FTIR analysis results of magnetite (a), pure PCL particles (b), magnetic nanoparticles (c) prepared using emulsion diffusion method, and magnetic nanoparticles (d) prepared using multiple emulsification method. It is shown.

9 shows the results of VSM analysis of magnetite (a), magnetic nanoparticles (b) prepared using emulsion diffusion method, and magnetic nanoparticles (c) prepared using multiple emulsification method.

Figure 10 shows the release aspect of the therapeutic agent (gemcitabine) of the magnetic nanoparticles (a) prepared using the emulsion diffusion method and the magnetic nanoparticles (b) prepared using the multiple emulsification method.

Figure 11 shows the release pattern of the therapeutic agent (cisplatin) of the magnetic nanoparticles (a) prepared using the emulsion diffusion method and the magnetic nanoparticles (b) prepared using the multiple emulsification method.

Figure 12 (a) and (b) is a control mouse administered only the composition according to the present invention, (c) and (d) shows an experimental group mouse administered the composition according to the present invention and applied a magnetic field to the left tumor will be.

Figure 13 shows the results of blue iron staining on the tumor tissue without the magnetic field in the experimental group of mice.

Figure 14 shows the blue iron staining results for tumor tissue to which the magnetic field was applied in the experimental group of mice.

Figure 15 shows the results of blue iron staining for the spleen (a), pancreas (b), liver (c) tissues other than the tumor in the experimental group of mice.

Figure 16 shows the tumor suppression effect of magnetic nanoparticles containing gemcitabine in vivo.

The present invention relates to a composition for use in a targeting drug delivery system, and more particularly, a composition containing a magnetic material and magnetic nanoparticles encapsulating a therapeutic agent into a biodegradable polymer, the magnetic nano The present invention relates to a method for producing particles and a method for delivering a targeted therapeutic agent capable of concentrating the magnetic nanoparticles on a target site by using a magnetic field.

Targeted therapeutic agent delivery is one of therapeutic agent delivery methods to allow selective and quantitative accumulation of therapeutic agents in a desired organ or tissue, regardless of the mode or site of administration. This delivery method can maximize the therapeutic effect by increasing the concentration of the therapeutic agent at the target site, while minimizing side effects by reducing the concentration of the therapeutic agent in non-target tissues and organs. In addition, the amount of treatment required to achieve a therapeutic effect, as well as the cost of treatment, can be significantly reduced.

Basic strategies for targeted delivery of therapeutics, which are generally used in a variety of experimental and clinical uses, include: (a) direct application of therapeutics to affected areas (organs, tissues); (b) Passive accumulation of therapeutic agents through the leaky vasculature (eg, tumors, infarction, inflammation, etc.); (c) physical targeting (pH and temperature) using abnormal pH and / or temperature at target sites such as tumors or inflammation. (D) targeting by the action of an external magnetic field of the therapeutic agent attached to the magnetic carrier; And (e) the use of vector molecules having a high specific affinity for the affected site.

Among the above strategies, the targeted drug delivery method using the magnetic field is particularly useful because it allows the concentration of the therapeutic agent only at a limited target site away from the reticular endothelial system due to the action of the magnetic field. US Pat. No. 4,345,588 is a method for delivering a therapeutic agent to a target capillary bed of the body, which can be administered intravascularly, rearranged using a magnetic field, produces a biodegradable microsphere comprising the therapeutic agent, and target capillary bed Disclosed is a method of treatment, characterized in that it is administered so as to be permanently confined to the target capillary bed in order to be released therein. However, the particles disclosed in this patent are micro sized, which does not penetrate the gaps between intact cells, which leads to limitations in their application. In addition, the microparticles are encapsulated with natural materials such as lipids, proteins, and carbohydrates, which are disadvantageous in that it is difficult to separate and wash the prepared particles.

In addition, International Patent Publication No. 01/56546 discloses a magnetoliposome-containing composition for treating a target site of a biological tissue, a treatment method using the same, and a method of preparing a magnetoliposome-containing composition. However, the patent requires a step of applying an electric field to the target site to activate the prodrug as a therapeutic agent, which may cause damage to normal tissue or cells in the surrounding area due to heat generated. If the drug is released and remains in an undecomposed state, it may have a detrimental effect on the human body.

On the other hand, encapsulating a therapeutic agent with magnetic nanoparticles is known to be particularly useful for reducing the toxicity of the therapeutic agent. Korean Patent Laid-Open No. 2001-0086811 relates to a method for producing biodegradable microparticles using an emulsion diffusion method, and in particular, can control the release rate of a therapeutic agent and have a strong affinity to biological tissues to penetrate deeply into tissues. A method for producing biodegradable microparticles having a particle size of is disclosed. However, the target-oriented therapeutic agent delivery method using nanoparticles prepared by the above-mentioned manufacturing method and encapsulating the therapeutic agent alone has a problem that the therapeutic agent cannot be substantially concentrated only on the target target site.

Accordingly, an object of the present invention is to provide a composition comprising magnetic nanoparticles that can concentrate a therapeutic agent substantially on a target site and penetrate deeply in the site to maximize the action of the therapeutic agent while minimizing side effects. It is.

In one aspect, the present invention relates to a composition containing magnetic nanoparticles encapsulating a magnetic material and a therapeutic agent into a biodegradable polymer.

In another aspect, the present invention relates to a method for preparing a magnetic nanoparticle, comprising the following steps: (a) an organic phase in which a therapeutic agent, a degradable polymer and a magnetic substance are partially dissolved in water In an oil phase; (b) saturating the solution in an aqueous phase in which the stabilizer is dissolved to reach phase equilibrium; (c) emulsifying the saturated solution using a homogenizer; (d) adding water to the emulsified solution to diffuse the solvent partially dissolved in water into the aqueous phase and (e) subjecting the solution to filtration, serum replacement, centrifugation and vacuum drying to form the final magnetic nanoparticles. Obtaining.

In another aspect, the present invention relates to a method for producing a magnetic nanoparticle, comprising the following steps: (a) a therapeutic agent, Preparing a water phase 1 by dissolving the magnetic material and the emulsifier in distilled water; (b) dissolving the biodegradable polymer in a solvent partially dissolved in water to prepare an oil phase; (c) adding the aqueous phase 1 to the oil phase and stirring to prepare a primary W / O type emulsion; (d) adding distilled water (aqueous phase 2) in which an emulsifier is dissolved to the primary W / O-type emulsion and stirring to prepare a W / O / W-type emulsion; and (e) filtration, serum replacement, and centrifugation of the emulsion. Separation and vacuum drying to obtain the final magnetic nanoparticles.

As a still further aspect, the present invention relates to a method for delivering a targeted therapeutic agent, comprising the following steps: (a) magnetic material and magnetic nanoparticles encapsulating the therapeutic agent with a biodegradable polymer; Administering the composition to the subject and (b) subjecting the magnetic nanoparticles to the target site for a period of time by acting an external or internal magnetic field at the target site to release the therapeutic agent as the biodegradable polymer degrades. step.

In order to achieve the above object, the present invention provides a composition containing magnetic nanoparticles encapsulating a magnetic material and a therapeutic agent into a biodegradable polymer.

Biodegradable synthetic polymers usable for the production of magnetic nanoparticles according to the invention include polyphosphazenes, polylactides, polylactide-co-glycolides, polycaprolactones, polyanhydrides, polymaleic Acids and derivatives thereof, polyalkylcyanoacrylates, polyhydrooxybutylates, polycarbonates, polyorthoesters, and the like. Preferably, one is selected from polyesters including polylactide, polylactide-co-glycolide, polycaprolactone and polyanhydride which can be readily biodegraded by the action of esterases in serum. Can be.

Among the aforementioned biodegradable polymers, polycaprolactone (hereinafter referred to as PCL) is a kind of linear aliphatic polyester molecule, which has some unique properties due to its molecular structure composed of five nonpolar methylene groups and one relatively polar ester group. have. While the physical properties are similar to polyolefins due to the high olefin content, the presence of aliphatic ester bonds that are unstable to hydrolysis induces the polymer to degrade in vivo. The molecular structure also allows the PCL to be compatible with many other polymers, thus making it possible to prepare various PCL polymer blends with unique properties. In addition, PCL is slowly degraded and does not create an acidic environment, such as polylactide and polylactide-co-glycolide, while biodegrading releases therapeutic agents and produces low molecular weight byproducts without toxicity. Therefore, in preparing the magnetic nanoparticles according to the present invention, PCL is most preferred as a biodegradable polymer.

The magnetic material of the magnetic nanoparticles according to the present invention includes a Fe-based magnetic compound such as magnetic iron oxide and the like, and the most preferable magnetic material is magnetite (Fe ₃ O ₄ ). In the present invention, the magnetic material is cut into an ultrafine state, the size of which should be 100 nm or less, preferably 30 nm or less, and most preferably 10 nm or less. Techniques for producing such a very small size of magnetite are known in the art, for example, fine milling, vacuum deposition and chemical precipitation. Fine milling in a ball mill may also be used to prepare a colloidal suspension of magnetite. Commercially, magnetite in fine powder form or suspension can be purchased, for example, from Ferrofluidics Corporation, Burlington, Massachusetts, and the size of these particles ranges from 10 to 20 nm.

The kind and chemical properties of the therapeutic agent that can be loaded onto the magnetic nanoparticles of the present invention are not particularly limited and may be variously selected according to the disease to be treated. The therapeutic agent may be loaded onto the magnetic nanoparticles in the form of a powder, in the form of an aqueous solution if water-soluble, or in the form of a solution dissolved in an organic solvent if it is fat-soluble. Thus, the magnetic nanoparticles of the present invention can be used to deliver a variety of water soluble and hydrophobic therapeutic agents to a target site. In particular, the composition containing the magnetic nanoparticles of the present invention can concentrate the therapeutic agent in the desired position, and therefore, when used to deliver a therapeutic agent such as an anticancer agent, an immunosuppressive agent, an anti-inflammatory analgesic agent, etc., which have limited clinical use due to side effects. Will be useful.

Anticancer agents usable in the present invention include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, mel Melphalan, chlorambucil, busulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin, bleomycin, Plicomycin, etoposide, tamoxifen, taxol, taxotere, transplatinum, vincristin, vinblastin And irinotecan Etc. are included.

Therapeutic gemcitabine and cisplatin used as one embodiment of the present invention are in the form of an organic coordination complex, which is used to treat small-cell lung cancer, metastatic ovarian tumor, pancreatic cancer and advanced bladder cancer. However, it is known to cause serious side effects on the kidney and digestive system, and its clinical use is limited.

Immunosuppressants available in the present invention include cyclophosphamide, azathiopurine, 6-mercaptopurin (6-mercaptopurin, 6-MP), cytarabine, bromouracil (BUdR). , Fluorouracin (FUdR), methotrexate, mitomycin C, actinomycin D, cortisone, predonisolone, dexamethasone, and the like.

The anti-inflammatory analgesics available in the present invention include naproxen, diclofenac, diclofenac, indomethacin, sulindac, piroxicam, ibuprofen, azaprofen Azapropazon, nabumeton, tiaprofenic acid, indoprofen, fenoprofen, flurbiprofen, pyrazolac, zaltopro Zaltoprofen, nabumetone, bromfenac, ampiroxicam and lornoxicam.

Magnetic nanoparticles according to the present invention can be prepared according to methods known in the art. A preferred embodiment is the preparation of magnetic nanoparticles using emulsion diffusion or multiple emulsification. On the other hand, although the diameter of a magnetic nanoparticle is 1000 nm or less normally, in this invention, it is preferable that it is 500 nm or less. Therefore, the manufacturing method to be described below aims to produce magnetic nanoparticles having a uniform size distribution while having a diameter of 500 nm or less. Hereinafter, each manufacturing method will be described step by step to meet the above object.

The method for preparing the magnetic nanoparticles according to the present invention using an emulsion diffusion method includes the following steps: (a) an organic phase in which a therapeutic agent, a biodegradable polymer and a magnetic substance are partially dissolved in water. (B) saturating the solution in an aqueous phase (aqueous phase) in which the stabilizer is dissolved to reach phase equilibrium; (c) emulsifying the saturated solution using a homogenizer; ( d) adding water to the emulsified solution to diffuse a solvent partially dissolved in water in the water phase and (e) subjecting the solution to filtration, serum replacement, centrifugation and vacuum drying to obtain final magnetic nanoparticles. Steps.

In the above production method, propylene carbonate, ethyl acetate, benzyl alcohol, methyl ethyl ketone, and the like may be used as the solvent partially dissolved in water. Ethyl acetate is particularly preferred because of its low toxicity, suitable solubility and low boiling point.

In the above production method, sodium stabilizer sulfate, polyvinyl alcohol, dididodecyldimethylammonium bromide, etc., which are less harmful to the human body, may be used as the stabilizer of step (b), and the stabilizer is about 1 to 10 with respect to the aqueous phase. Dissolution at a concentration of% (w / v) is suitable.

In the above production method, particles of various shapes and sizes may be produced according to the ratio of oil phase to aqueous phase. In order to prepare the magnetic nanoparticles according to the present invention, the aqueous phase volume will preferably be 2 to 4 times the oil phase volume. If the aqueous phase volume is less than 2 times, there is no significant change in size but particles are aggregated whereas if the aqueous phase volume exceeds 4 times, particles having defects and irregularities on the surface are produced. Particularly, when the ratio of oil phase to aqueous phase is about 1: 2, particles having a relatively uniform size distribution are produced. Therefore, in the method of manufacturing magnetic nanoparticles according to the present invention, the ratio of oil phase to aqueous phase is Preferably about 1: 2.

In the above production method, particles of various shapes and sizes may be produced according to the ratio of magnetic material: biodegradable polymer. In order to prepare the magnetic nanoparticles according to the present invention, the weight of the magnetic material will be preferably 0.2 to 0.8 times the weight of the biodegradable polymer. As the amount of magnetic material increases, the size of the magnetic nanoparticles slightly increases, whereas the efficiency of encapsulating the magnetic material into the biodegradable polymer increases dramatically. However, when the amount of the magnetic material is equal to or greater than the amount of the biodegradable polymer, not only the magnetic nanoparticles are not formed well but also the size of the particles increases. Therefore, in the method for producing magnetic nanoparticles according to the present invention, the amount of the magnetic material is most preferably about 0.8 times the amount of the biodegradable polymer.

On the other hand, the present inventors confirmed that when the chemical properties of the therapeutic agent is hydrophobic, the emulsion encapsulation method is excellent in the encapsulation efficiency. Therefore, when preparing magnetic nanoparticles containing a hydrophobic therapeutic agent, it will be preferable to prepare by using the emulsion diffusion method. In addition, the inventors have confirmed that the release rate of the therapeutic agent is relatively high magnetic nanoparticles prepared using the emulsion diffusion method. Therefore, it would be desirable to use magnetic nanoparticles prepared by emulsion diffusion to treat diseases requiring rapid release of the therapeutic agent.

Another method of preparing magnetic nanoparticles according to the present invention, multiple emulsification, includes the following steps: Dissolving a magnetic material and an emulsifier in distilled water to prepare an aqueous phase 1; (b) dissolving a biodegradable polymer in a solvent partially dissolved in water; (c) preparing the oil phase in the oil phase; Adding and stirring to prepare a primary W / O type emulsion; (d) adding distilled water (aqueous phase 2) in which an emulsifier is dissolved to the primary W / O type emulsion, and stirring to prepare a W / O / W type emulsion Manufacturing step; And (e) subjecting the emulsion to filtration, serum replacement, centrifugation and vacuum drying to obtain final magnetic nanoparticles.

In the above production method, propylene carbonate, ethyl acetate, benzyl alcohol, methyl ethyl ketone, and the like may be used as the solvent partially dissolved in water. Ethyl acetate is particularly preferred because of its low toxicity, suitable solubility and low boiling point.

In the above production method, as the emulsifier to be dissolved in the aqueous phase 1 and the aqueous phase 2, sodium aryl sulfate, polyvinyl alcohol, dididodecyldimethylammonium bromide and the like which are less harmful to the human body may be used.

In the above production method, particles of various shapes and sizes may be produced according to the emulsifier concentration of the aqueous phase 2. In order to prepare the magnetic nanoparticles according to the present invention, the concentration of the emulsifier may be 0.25 to 2% (w / v). When the concentration of the emulsifier is 0.25% or less, not only does it stabilize the solution in emulsification but also produce aggregated particles. On the other hand, as the concentration of the emulsifier increases, the particle size becomes smaller and uniformly dispersed. When the concentration of the emulsifier is 2% or more, it produces magnetic nanoparticles of a similar shape and size as when the concentration of the emulsifier is 2%. do. Therefore, in the method for producing magnetic nanoparticles according to the present invention, the concentration of the emulsifier is most preferably about 2%.

In the above production method, particles of various shapes and sizes may be produced according to the ratio of magnetic material: biodegradable polymer. In order to prepare the magnetic nanoparticles according to the present invention, the weight of the magnetic material will be preferably 0.2 to 0.8 times the weight of the biodegradable polymer. As the amount of magnetic material increases, the size of the magnetic nanoparticles slightly increases, whereas the efficiency of encapsulating the magnetic material into the biodegradable polymer increases dramatically. However, when the amount of the magnetic material is equal to or greater than the amount of the biodegradable polymer, not only the magnetic nanoparticles are not formed well but also the size of the particles increases. Therefore, in the method for producing magnetic nanoparticles according to the present invention, the amount of the magnetic material is most preferably about 0.8 times the amount of the biodegradable polymer.

On the other hand, the present inventors have confirmed that the encapsulation efficiency is excellent when the chemical properties of the therapeutic agent is hydrophilic when using multiple emulsification method. Therefore, when preparing magnetic nanoparticles containing a hydrophilic therapeutic agent, it is preferable to prepare by using a multiple emulsification method. In addition, the inventors have confirmed that the magnetic nanoparticles prepared using the multiple emulsification method have a relatively slow release rate of the therapeutic agent. Therefore, it is desirable to use magnetic nanoparticles prepared by multiple emulsification methods to treat diseases requiring slow release of the therapeutic agent.

Compositions comprising magnetic nanoparticles according to the present invention may be administered via conventional routes known in the art and may be formulated accordingly. Available routes of administration of the compositions include, for example, intravascular, lymphatic, parenteral, subcutaneous, intramuscular, intranasal, intraperitoneal, interstitial, oral, intratumoral administration and the like. Administration in vitro. In the case of intravascular administration, it is usually injected into a vein, but may also be injected into an artery. The composition according to the invention can be injected into the gap or into the cavity of every body.

The present invention provides a target-oriented therapeutic agent delivery method capable of specifically concentrating magnetic nanoparticles encapsulating a magnetic substance and a therapeutic agent into a biodegradable polymer using a magnetic field at a target site. The composition is administered to a subject, followed by application of an external or internal magnetic field to the target site for a period of time to localize the magnetic nanoparticles to the target site (ie, disease site). Here, the application time of the magnetic field is the time taken to concentrate 70%, preferably 80%, and most preferably 90% or more of the dose at the target site, which is the distance between the administration site and the target site. And various conditions, including the route of administration. In addition, the strength of the magnetic field used to concentrate the magnetic nanoparticles of the present invention on the target site is preferably in the range of about 0.2 to 0.3 Tesla. The magnetic nanoparticles concentrated on the target site by this process may cause a therapeutic effect by releasing the therapeutic agent therein while being degraded by the action of enzymes in the serum.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, and according to the gist of the present invention to those skilled in the art that the scope of the present invention is not limited by these examples. Will be self explanatory.

Example 1

Preparation of ingredients and therapeutics

Magnetite (Fe ₃ O ₄ ) was purchased from Idaho University and poly ε-caprolactone (MW = 42,500 Da) was obtained from Aldrich Chemical Co., USA. Ethyl acetate and nonionic surfactant polyvinyl alcohol (PVA) were purchased from Aldrich Chemical Co., USA. The average molecular weight and saponification of the PVA are 15,000-20,000 Da and 88%, respectively. Ammonium thiocyanate, hydroperoxide, diphosphoric acid (KH ₂ PO ₄ ) and monophosphoric acid (K ₂ HPO ₄ ) were purchased from Duksan Pure Chemical Co., Korea. All of these materials were used as HPLC grade without further purification. The distilled water used in the present invention is ultra pure water (DDI) distilled water prepared by Elgastat's UHQ system (UK).

Gemcitabine hydrochloride (Gemza, Lilly co., Indianapolis, U.S.A.) and diaminedicloplatinum (cisplatin, Aldrich Chemical Co., U.S.A.) were used as hydrophilic and hydrophobic agents, respectively.

Example 2

Comparison of the size and shape of magnetic nanoparticles prepared by varying the ratio of oil phase to aqueous phase in the method of manufacturing magnetic nanoparticles by emulsion diffusion method

An organic phase was prepared by dissolving gemcitabine 50 mg, PCL 0.5 g (5% w / w) and magnetite 0.4 g in 10 ml of ethyl acetate. Meanwhile, an aqueous phase was prepared to have a final volume of 15, 20, 30 and 40 ml, respectively, in which each polyvinyl alcohol (PVA) was dissolved at 5% concentration in D.D.I distilled water. 10 ml of the oil phase was saturated in the aqueous phase to reach phase equilibrium, and the saturated solution was emulsified for 10 minutes using a 30% power probe sonicator (ULH700S, Ulssohitech, Korea). Under mild stirring conditions (150 rpm), 200 ml of water was added to the emulsified solution to diffuse ethyl acetate into the aqueous phase. The final magnetic nanoparticles were obtained by filtering the product after the reaction using a membrane filter paper (pore size: 0.45 μm), replacing the serum, and centrifuging three times at 10,000 rpm for 30 minutes to dry in a 35 ° C. vacuum oven. It was.

On the other hand, the size and morphology of the magnetic nanoparticles thus prepared were confirmed using SEM, and the results are shown in FIG.

According to the photograph of FIG. 1, the particle size also tends to increase as the volume of the aqueous phase increases. When the oil phase: water phase ratio was 1: 2 (b), it was possible to produce relatively small particles, and when the volume of water was reduced to 6.7 ml without changing the volume of ethyl acetate (10 ml), the aggregated particles It was prepared, when the oil phase: water phase ratio of 1: 4 (d) the produced particles showed surface defects and irregularities.

Example 3

Comparison of the size and shape of magnetic nanoparticles prepared by varying the ratio of magnetite to PCL in the method of manufacturing magnetic nanoparticles by emulsion diffusion method

The amount of magnetite was changed in the range of 0.1 to 0.5 g to prepare the same method as in Example 2 (provided that the oil phase: water phase ratio was 1: 2). The morphology, average size, and size distribution of the prepared magnetic nanoparticles were confirmed using SEM, TEM, and DLS, and the results are shown in FIGS. 2, 3, and 4, respectively.

It can be seen from the photograph of FIG. 2 that the magnetic nanoparticles prepared by the emulsion diffusion method are smooth, well-individualized and have a uniform size distribution. In addition, as shown in Figure 3, the average diameter of the magnetic nanoparticles produced by the emulsion diffusion method increased from 150 to 170nm as the amount of magnetite increased from 0.1g to 0.5g.

Example 4

Comparison of the size and shape of magnetic nanoparticles prepared by varying the concentration of the emulsifier for aqueous phase 2 in the method of manufacturing magnetic nanoparticles by multiple emulsification

Gemcitabine 50mg, Magnetite 0.4g And Aqueous phase 1 was prepared by dispersing PVA (0.1% w / w) in 2 ml of DDI distilled water, and an oil phase was prepared by dissolving PCL (5% w / w) in 10 ml of ethyl acetate. The aqueous phase 1 was added to the oil phase and emulsified for 4 minutes using a 30% power probe sonicator. The W / O emulsion was then added to 20 ml of aqueous phase 2 containing 0.05 mg to 0.4 mg of PVA and emulsified for 1 minute using a 15% power probe sonicator. The double emulsion (W / O / W) thus prepared was diluted with 100 ml of DDI distilled water and the system was kept under gentle stirring. The final magnetic nanoparticles were obtained by filtering the product after the reaction using a membrane filter paper (pore size: 0.45 μm), replacing the serum, and centrifuging three times at 10,000 rpm for 30 minutes to dry in a 35 ° C. vacuum oven. It was.

On the other hand, the size and morphology of the magnetic nanoparticles thus prepared were confirmed using SEM, and the results are shown in FIG.

In general, as the concentration of the emulsifier increases from the photograph of FIG. 5, the particle size was confirmed to be uniformly dispersed. When the concentration of the emulsifier is 0.25% or less (a), agglomerated particles were prepared, and magnetic nanoparticles were prepared when the concentration of the emulsifier was about 2% (d).

Example 5

Comparison of the size and shape of magnetic nanoparticles prepared by varying the ratio of magnetite to PCL in the method of manufacturing magnetic nanoparticles by multiple emulsification

The amount of magnetite was changed in the range of 0.1 g to 0.5 g to prepare the same method as in Example 4 except that the concentration of PVA in the aqueous phase 2 was 2%. The shape, average size, and size distribution of the prepared magnetic nanoparticles were confirmed using SEM and DLS, and the results are shown in FIGS. 6 and 7, respectively.

It can be seen from the photograph of FIG. 6 that the magnetic nanoparticles prepared by the multiple emulsion method have a larger size distribution and more aggregate than the magnetic nanoparticles prepared by the emulsion diffusion method. In addition, as shown in Figure 7, the average diameter of the magnetic nanoparticles produced by the multiple emulsification method was increased from 350 to 370nm as the amount of magnetite increased from 0.1g to 0.5g.

Example 6

Efficiency in which magnetite is encapsulated in the magnetic nanoparticles of the present invention

It was confirmed whether magnetite was loaded into the magnetic nanoparticles prepared in Example 2 (provided that the oil phase: water phase ratio was 1: 2) and Example 4 (however, the concentration of PVA in the water phase 2 was 2%). In order to perform the FTIR analysis, the results are shown in FIG.

As can be seen in the Fig. 8 (b), the spectrum of pure PCL particles 1726㎝ ^-1, carboxylic acid of the aliphatic group that corresponds to the band-specific band in 2943㎝ ^-1 corresponding to the band of the hydro group Was formed. The IR spectrum (a) of the magnetite was formed in the low frequency region (1000-300 cm ^-1 ) due to the structure of the iron oxide. On the other hand, as shown in (c) and (d) of Figure 8, the most characteristic band (570cm ^-1 ) of the magnetic nanoparticles can be confirmed in comparison with that of the magnetite (570cm ^-1 ), Through the spectrum of the magnetic nanoparticles prepared in the above embodiment it can be seen that all the bands of pure PCL particles are present. That is, magnetite was loaded into the magnetic nanoparticles.

The actual concentration of magnetite loaded in the magnetic nanoparticles can be measured by absorbance, inducing the oxidation of Fe ²⁺ to Fe ³⁺ by adding HCl / H ₂ O ₂ = 2: 3 v / v solution and 1 The absorbance of the thiocyanate composite formed by adding% ammonium thiocyanate was measured at a wavelength of λ = 480 nm.

The encapsulation efficiency of the magnetite calculated using the numerical values obtained from these measurement results is shown in Tables 1 (emulsification diffusion method) and 2 (multi-dispersion method) together with the size of the final magnetic nanoparticles. In the two manufacturing methods, the size of the magnetic nanoparticles increased consistently with the amount of magnetite, but the change was small. On the other hand, the encapsulation efficiency was different depending on the preparation method and the amount of magnetite, and the maximum encapsulation efficiency of the magnetite was about 7.84% and 15.8% of the theoretical loading, respectively.

Magnetite / PCL Ratio (w / w) Average size (nm) Magnetite Loading (mg) Encapsulation Efficiency (%) 1/5 150 ± 0.5 0.6 3.43 2/5 158 ± 0.5 1.3 4.4 3/5 164 ± 0.5 2.7 5.4 4/5 167 ± 0.5 3.5 7.84

Magnetite / PCL Ratio (w / w) Average size (nm) Magnetite Loading (mg) Encapsulation Efficiency (%) 1/5 322.4 ± 0.5 1.86 1.18 2/5 324 ± 0.5 3.81 13.32 3/5 329 ± 0.5 5.56 14.83 4/5 339.5 ± 0.5 6.7 15.80

(Here, magnetite loading: amount of magnetite encapsulated in 1 mg of magnetic nanoparticles, encapsulation efficiency: ratio of the magnetite loading to theoretical loading.)

Example 7

Magnetic Properties of Magnetic Nanoparticles According to the Present Invention

The magnetic properties of the magnetite and the magnetic nanoparticles prepared in Example 2 (wherein the ratio of oil phase to aqueous phase is 1: 2) and Example 4 (wherein the concentration of PVA in aqueous phase 2 is 2%) are It was confirmed using a vibrating sample magnetometer (VSM), and a magnetic hysteresis loop was shown in FIG. 9.

As shown in (a) of FIG. 9, the magnetization curve of the magnetite at room temperature showed no magnetic hysteresis, which is consistent with the typical superparamagnetic properties of the 100 nm magnetite magnetic nanoparticles. The magnetic field of 10koe or more basically saturated the magnetic powder at room temperature, and the value of 48emu was confirmed as saturation magnetization in the 6koe magnetic field. As shown in (b) and (c) of FIG. 9, the maximum magnetic properties of the magnetic nanoparticles were measured to be about 10.2 emu / g at a magnetic field strength of 6koe. Under this magnetic field, the curve did not show any hysteresis, but also residual electricity and coercitivity. Saturation magnetization of magnetic nanoparticles is smaller than that of giant magnetites, but these composite particles have paramagnetic properties.

Example 8

Experiment for release of therapeutic agent of magnetic nanoparticles according to the present invention

Magnetic properties were determined according to the numerical values shown in Table 3 using the production methods of Example 2 (wherein, the oil phase: water phase ratio is 1: 2) and Example 4 (wherein the concentration of PVA is 2% in the water phase 2). Nanoparticles were prepared.

The therapeutic release experiment of the magnetic nanoparticles was carried out in an aqueous release medium containing phosphate buffer at 37 ± 0.5 ℃. The dried magnetic nanoparticles were placed in a flask containing 30 ml of an aqueous release medium, which was placed in a shaking incubator (SI-900, JO Tech., Korea) and spun at a speed of 150 rpm. 24 hours At intervals, 3 ml of aqueous solution was withdrawn and supplemented with 3 ml of DDI distilled water. The amount of therapeutic agent released could be monitored by measuring absorbance using a UV spectrometer (UV16A, Shimadzu, Japan) with a standard calibration curve. Absorbance for gemcitabine and cisplatin was measured at wavelengths of 267.8 nm and 270.6 nm, respectively, and the results are shown in FIGS. 10 and 11.

According to this, the emission profile showed a big difference according to the method of manufacturing the magnetic nanoparticles. For particles produced by emulsion diffusion, very fast release could be observed because the therapeutic agent is located near the surface of the particles. Conversely, in the case of particles produced by multiple emulsification, very slow release profiles were observed, indicating that the therapeutic agent is located inside the polymer matrix and the release occurs due to the collapse of the polymer.

Example 9

Efficiency in which the therapeutic agent is encapsulated in the magnetic nanoparticles of the present invention

The therapeutic release experiment showed the efficiency (through total amount of gemcitabine or cisplatin released until day 30) of encapsulating the therapeutic agent in the magnetic nanoparticles according to the present invention, and the results are shown in Table 3.

According to this, the maximum encapsulation efficiencies of gemcitabine and cisplatin of magnetic nanoparticles prepared by emulsion diffusion and multiple emulsion methods are about 18.6%, 52.5% and 71.4% and 30.4% of theoretical loading, respectively. Cisplatin (hydrophobic) was produced by emulsion diffusion, while gemcitabine (hydrophilic) was produced by multiple emulsification. In conclusion, it was found that the multiple emulsification method is suitable for encapsulating the water-soluble therapeutic agent and the emulsion diffusion method in the magnetic nanoparticles.

Way remedy Therapeutics / PCL Ratios Therapeutic Loading (mg) Encapsulation Efficiency (%) Emulsification-Dispersion Gemcitabine 0.01 0.11 10.8 0.1 1.93 18.6 Cisplatin 0.01 0.39 39.2 0.1 4.74 52.2 Multiple emulsification Gemcitabine 0.01 0.49 49.4 0.1 6.58 71.4 Cisplatin 0.01 0.24 24.6 0.1 2.8 30.4

(Wherein therapeutic loading: amount of therapeutic agent encapsulated in 1 mg of magnetic nanoparticles,

Encapsulation Efficiency: The ratio of the therapeutic loading to the theoretical loading.)

Experimental Example

Anticancer Effect of Magnetic Nanoparticles According to the Present Invention

(1) Pancreatic Cancer Cell Line Culture

Human pancreatic cancer cell line (ATCC: CRL-2119) is a moderately differentiated human adenocarcinoma derived from pancreatic catheter and contains DMEM and Ham F-12 nutrient media (including NaHCO ₃ 1.2 g / l and 15 mM HEPES). Incubated in a 1: 1 mixture of DEEM: F-12. The culture medium was added 5% fetal calf serum and 1% antibiotic. Cell cultures were incubated at 37 ° C. in air moistened with 5% CO ₂ , and the medium was changed every 3 days.

(2) xenograft

HPAC cells 1 × 0 ⁷ were injected symmetrically into the subcutaneous tissue in the flanks of BALB / c nude mice. Tumor masses occupy most of the sides symmetrically, covering the costal ridge. All mice were maintained under the same conditions in terms of food and water.

(3) administration of therapeutic agents and application of magnetic fields;

At 14 days after tumor injection, mice developed grafts of HPAC tumor cells symmetrically. The mice were randomly assigned to one of two treatment groups: (a) control group (number = 7) and (b) experimental group (number = 8). The days on which mice were separated in two different treatment protocols were randomly named 0 days. On day 0, 7, 14 and 20, all 15 mice were dosed intravenously through the blood vessels of the tail using a 26 gauge needle with the magnetic nanoparticles prepared in Example 2 (per kg of mouse weight). At a dosage of 120 mg of magnetic nanoparticles). Upon administration, the mice belonging to the experimental group were fixed to minimize movement, and then fixed with 0.25 Tesla magnets on the skin on the left tumor mass by simple tape attachment to be affected by the magnetic field for 2 hours. Mice in the control group also received the same amount of therapeutic agent in the same manner but did not apply a magnetic field (FIG. 12).

The strength of the magnetic field applied in this step was chosen because temporary 0.25 in vivo studies showed that about 0.25 Tesla magnets were most effective in targeting the magnetic nanoparticles of the present invention to tumor masses.

(4) tumor cutting and blue iron staining

Mice surviving by day 21 were sacrificed by cervical dissection, these mice were dissected and recovered for transplanted tumors, fixed in para-formaldehyde and freeze-cut for iron analysis by Prussian blue staining.

Frozen fragments of the tumor tissue were fixed with acetone, treated with H ₂ O ₂ , and staining was followed by Gomori's Prussian blue iron reaction. Blue iron staining results are shown in FIGS. 13, 14 and 15.

13, 14, and 15, it was confirmed that the magnetic nanoparticles according to the present invention were locally concentrated at the site where the magnetic field was applied.

(5) data and statistical analysis

Tumor size was measured two-dimensionally for length and longest width using calipers. Tumor volume was calculated by the following equation (1), the results are shown graphically in FIG. The calculations were averaged for each of the experimental and control groups and compared using the Unpaired Welch's Correction test.

V = (a × 2b) / 2 [a = length (cm), b = width (cm)]

In the present invention, the inventors made two comparisons for tumor size. The first comparison is comparing the left (magnetic field applied) and right (non-magnetic field applied) tumors within the experimental group. A second comparison was made between the tumors of the control group and the left tumors of the experimental group.

In the first comparison, we found that tumors with magnetic fields were reduced in size compared to tumors without magnetic fields. In the second comparison, it was confirmed that the tumors to which the magnetic field was applied were reduced in size compared with the tumors of the control group. On the other hand, it can be seen that the difference in the first comparison is larger than the difference in the second comparison. In the experimental group, magnetic nanoparticles are relatively concentrated in the left tumor due to the action of the magnet on the left tumor, whereas in the control group, the magnetic nanoparticle concentration of the left tumor is similar to that of the right tumor. Because. In conclusion, the concentration of magnetic nanoparticles increased in the order of the right tumor of the experimental group, the left and right tumors of the control group, and the left tumor of the experimental group. In particular, in the left tumor of the experimental group it was confirmed that not only the growth of the tumor is suppressed but also degraded.

The composition comprising the magnetic nanoparticles according to the present invention may minimize the side effects and maximize the action of the drug on the lesion by allowing the therapeutic agent to be substantially concentrated only at the disease site and penetrate deep into the tissue.

Claims (16)

A composition comprising magnetic nanoparticles encapsulating a magnetic material and a therapeutic agent with polycaprolactone. delete delete The composition of claim 1, wherein the magnetic material is a Fe-based magnetic compound. 5. A composition according to claim 4, wherein said Fe-based magnetic compound is magnetite. The composition of claim 1, wherein the magnetic nanoparticles have a size of 500 nm or less. (a) dissolving a hydrophobic therapeutic agent, polycaprolactone and a magnetic material of 0.2 to 0.8 times the amount of polycaprolactone in an organic phase (oil phase) partially dissolved in water; (b) The solution is saturated in an aqueous phase two to four times the volume of the oil phase in which a stabilizer selected from the group consisting of sodium arylsulfate, polyvinyl alcohol and dododecyldimethylammonium bromide is dissolved. Reaching equilibrium; (c) emulsifying the saturated solution using a homogenizer; (d) adding water to the emulsified solution to diffuse the solvent partially dissolved in water into the aqueous phase and (e) subjecting the solution to filtration, serum replacement, centrifugation and vacuum drying to form the final magnetic nanoparticles. Method of producing a magnetic nanoparticle comprising the step of obtaining. delete delete delete (a) preparing aqueous phase 1 by dissolving a hydrophilic therapeutic agent, 0.2-0.8 times the magnetic material and the emulsifier in the amount of polycaprolactone below in distilled water; (b) dissolving polycaprolactone in a solvent partially dissolved in water to prepare an oil phase; (c) adding the aqueous phase 1 to the oil phase and stirring to prepare a primary W / O type emulsion; (d) adding distilled water (aqueous phase 2) in which an emulsifier is dissolved at a concentration of 0.25 to 2% to the primary W / O emulsion, and stirring to prepare a W / O / W emulsion; and (e) A method for producing magnetic nanoparticles comprising the step of obtaining the final magnetic nanoparticles by filtration, serum replacement, centrifugation and vacuum drying the emulsion. delete delete delete delete delete

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