JP5614885B2 - Water-insoluble elastin microparticles - Google Patents

Description

The present invention relates to novel water-insoluble elastin microparticles, a method for producing the same, and uses thereof. More specifically, the present invention uses water-insoluble elastin microparticles that can be used as a sustained-release carrier for drugs, a method for producing the same, and the use of the microparticles. The present invention relates to a sustained drug release carrier.

The drug delivery system (hereinafter sometimes abbreviated as DDS) is intended to “activate drugs in the required amount at the required place when necessary”, and enables effective use and side effects of drugs. It is expected to play a very important role in reducing the patient's quality and improving the patient's quality of life (QOL). Currently, development of DDS technology in various administration forms such as oral, arteriovenous injection, transdermal, and transpulmonary is progressing. For example, an intra-arterial administration method of an anticancer drug solution is one of the local therapies that are frequently used in the clinic. However, in the case of this method, even if an anticancer agent solution is injected, the contact time of the drug with the tumor tissue is short, so that a large therapeutic effect cannot be expected.
In the drug delivery system, it is important to develop a drug carrier (DDS carrier) for carrying the drug to the target site. As an example of DDS using the DDS carrier, mitomycin C, which is an anticancer agent, is coated with ethyl cellulose. Chemoembolization is known in which the microcapsules are injected into a tumor artery. However, the number of known DDS carriers is still insufficient, and development of new DDS carriers is desired.

By the way, the pharmacokinetics of microparticles injected into the vascular system and the transition to each organ include the anatomical positional relationship between the administration site and the vascular system / organ, the structure of the capillary bed and vascular wall, and the particles in the organ. It is known that it is defined by the size and surface characteristics. Since the properties of the blood vessel wall vary greatly depending on the organ and lesion, the fine particles show different organ distributions depending on the particle diameter after intravascular administration, and the organs also depend on the anatomical positional relationship between the administration site and the organ. Distribution is different. For example, intravenously injected fine particles having a diameter of 12 μm or more are trapped in the circulating blood stream and mechanically embolized in the lung capillary bed. Injecting the same particles into the artery causes embolization in the capillaries of the organ under the basin.

On the other hand, micro-order microparticles with a diameter of about 0.2 to 3 μm are phagocytosed and accumulated by reticulo-endothelial system (RES) including Kupffer cells of the liver. Therefore, when there is a lesion such as liver cancer, when these microparticles are used as a carrier and an anticancer agent is carried on this fine particle and intravenously injected, it is specifically phagocytosed by reticuloendothelial tissues such as liver Kupffer cells. Therefore, it is effective for treating liver cancer and the like. Moreover, since it is phagocytosed by liver Kupffer cells more specifically than other tissues, side effects such as anticancer agents are expected to be small. In addition, it is said that microparticles with a diameter of 0.1 μm or less leak out of the blood vessel at the liver, cancer lesions, and inflamed sites, making it impossible to target the microparticles (see Non-Patent Document 1).

On the other hand, the present inventors have focused on elastin, a biopolymer, and have studied its application as a drug carrier. Elastin is a major constituent of animals such as the aorta, ligaments, and skin of animals, and usually exists as an insoluble protein having a three-dimensional network structure in vivo. It is widely known that water-soluble elastin can be obtained by hydrolyzing such elastin with an acid or alkali or treating with an enzyme. The solubilized elastin thus obtained causes a phenomenon called coacervation in an aqueous solution. Coacervation is a reversible effect in which an elastin aqueous solution becomes cloudy when heated to near body temperature, and when left as it is, it is separated into two layers of a transparent equilibrium solution and a pale yellow highly viscous coacervate, and when cooled, it returns to the original homogeneous solution. This is a series of phenomena (see Patent Documents 1, 2, and 3).

The present inventors paid attention to the coacervate droplets formed during the coacervation, and succeeded in producing nanoparticles of about 200 nm that are more stable and insoluble in water by irradiating them with γ rays. The patent application was filed first (Japanese Patent Application No. 2010-091952). These nanoparticles are expected to be used as a DDS carrier for transdermal administration from the viewpoint of particle size.

However, as described above, micro-order sized microparticles are necessary for the treatment of diseases of the liver and spleen, and the previously developed nanoparticles are insufficient in size. Conventionally, micro-order DDS carriers are known. However, since these materials are synthetic polymers, the synthesis steps such as chemical synthesis and modification become complicated, and it can be said that they are safe in terms of safety. There is no problem.

Yoshinobu Fukumori et al., Chemical Engineering, 63 (10), 567-571 (1999)

Japanese Patent No. 4078431 JP 2007-45722 A JP 2009-219422 A

It is an object of the present invention to provide water-insoluble elastin microparticles suitable as a drug sustained-release carrier, particularly a drug carrier used for the treatment of liver and spleen by intravenous injection. Another object of the present invention is to provide a method for efficiently producing such water-insoluble elastin microparticles by a simple operation. Another object of the present invention is to provide a sustained-release drug carrier suitable for drugs used for the treatment of liver and spleen by intravenous injection.

In the present invention, in order to solve the above problems, a first invention, in coacervate droplets of the water-soluble elastin from fish, obtained by irradiating the radiation, the particle size Ru 1~10μm der water Insoluble elastin microparticles are provided. As a second invention, there is provided a method for producing the above microparticles by irradiating a water-soluble elastin coacervate droplet derived from fish with radiation. Furthermore, as a third invention, a sustained-release drug carrier comprising the above microparticles as a constituent component is provided.

In the present invention, for example, a coacervate droplet is prepared by heating an aqueous solution of water-soluble elastin derived from fish such as bonito and tuna, and the droplet is irradiated with radiation such as γ-rays. Water-insoluble micro-order particles can be produced. Micro-order particles are larger in size than nano-order particles and are easily trapped in the liver and spleen. Therefore, DDS for transporting drugs to the lesion sites of the liver and spleen to treat liver and spleen diseases. Application as a carrier can be expected. Moreover, since the microparticle of the present invention is a polymer material derived from a living body, it is decomposed by an enzyme and does not remain in the living body. Furthermore, in the present invention, since microparticles are produced by irradiation with radiation, they can be produced as sterile microparticles.

It is a figure which shows the procedure which produces the water-soluble elastin derived from a bonito from a bonito arterial sphere. It is a figure which shows the particle size distribution of the water-insoluble elastin microparticles derived from skipjack. It is a transmission electron micrograph of water-insoluble elastin microparticles derived from skipjack.

  In the present invention, water-insoluble elastin microparticles are produced by irradiating water-soluble elastin coacervate droplets derived from animals with radiation. There are no particular restrictions on the animal biological tissue that is the raw material for water-soluble elastin. Specific examples thereof include sucking pigs, horses, cattle, sheep, and other mammals that have traditionally been awarded for their high content of elastin. Examples include ligaments and aortic blood vessels obtained from animals, and arterial spheres of fish such as skipjack, tuna, yellowtail, and salmon. Among these, fish-derived water-soluble elastin is preferable because it easily forms micro-order particles after irradiation with radiation. In particular, when water-soluble elastin-derived coacervate droplets derived from bonito are used, water-insoluble particles in the range of more than 400 nm to 2 μm can be easily obtained by adjusting the radiation dose.

Various methods for obtaining water-soluble elastin from animal biological tissues have been proposed in the past, and are not particularly limited, but the following method proposed by the present inventor is preferable (see Patent Documents 1 and 2). .

The first method is performed as follows. First, collagen and other unwanted proteins are removed from animal tissue to obtain insoluble elastin, and then this insoluble elastin is immersed in an acidic solubilizing solution such as oxalic acid or an alkaline solubilizing solution such as sodium hydroxide. To produce water-soluble elastin. Collagen and other unwanted proteins are usually removed using an aqueous solution of an alkaline substance such as sodium hydroxide, potassium hydroxide, calcium hydroxide, or barium hydroxide. The concentration of these alkaline substances is 0.005 to 0.5 mol per liter, and the animal tissue is immersed in an alkaline solution at a liquid temperature of 0 to 105 ° C. for about 5 to 60 minutes. It is preferable to repeat the immersion a predetermined number of times.

Also, when removing collagen and other unwanted proteins, soak the animal's biological tissue in a salt solution containing a salt such as sodium chloride, potassium chloride, calcium chloride or barium chloride before the treatment with an alkaline solution. Processing (preprocessing) can also be performed.

The animal body tissue is preferably homogenized using a homogenizer. The homogenization is not particularly limited as long as it can shred the living tissue, such as a mixer or a meat chopper, and it is preferable to use an instrument capable of shredding in a 3 mm square or less, more preferably a paste. The smaller the size of the shredded biological tissue, the higher the efficiency of removing collagen and other unnecessary proteins. The homogenized biological tissue may be degreased by boiling it with hot water or a hot dilute alkaline aqueous solution, or treating it with an organic solvent, for example.

Examples of the solubilized solution include oxalic acid, formic acid, acetic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, betaine, difluoroacetic acid, trifluoroacetic acid, phosphoric acid, sulfamic acid, perchloric acid, trichloroacetic acid. An acidic solution containing an acid component such as is used. The acid concentration of the acidic solution is preferably 0.05 to 5 mol per liter, and the liquid temperature is preferably 90 to 105 ° C.

The solubilizing solution may also be an alkaline solution containing an alkaline substance such as sodium hydroxide, potassium hydroxide, calcium hydroxide, or barium hydroxide. It is preferable that the concentration of the alkaline substance added to the alkaline solution is 0.01 to 5 mol per liter and the liquid temperature is 90 to 105 ° C.

The second method includes a pretreatment step including at least one of a removal process of an unnecessary part of an animal living tissue, a degreasing process of an animal living tissue, and a shredding process of an animal living tissue; An alkaline extraction step of immersing a living tissue in an alkaline solution and filtering out collagen and other unnecessary proteins, and an alkali or acid dissolution step of dissolving the residue after the alkali extraction step with an alkali or an acid are repeated a predetermined number of times by filtration. This is a method for producing water-soluble elastin by sequentially performing a filtrate recovery step for obtaining a filtrate containing water-soluble elastin and a water-soluble elastin production step for producing water-soluble elastin from the filtrate. The alkali used in the alkali dissolution step is preferably any one of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, or a mixture. The acid used in the acid dissolving step is oxalic acid, formic acid, acetic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, betaine, difluoroacetic acid, trifluoroacetic acid, phosphoric acid, sulfamic acid, perchlorine. An acid, at least one of trichloroacetic acid, or a mixture is preferable.

This operation differs from the above-described first method in which collagen and other unnecessary proteins are removed from biological tissue to obtain insoluble elastin, and then this insoluble elastin is solubilized to obtain water-soluble elastin. This is a method for obtaining water-soluble elastin directly without obtaining insoluble elastin from tissue. That is, the degreased and shredded biological tissue is immersed for about 5 to 60 minutes in an alkaline solution of 0.005 to 0.5 mol per liter and 0 to 105 ° C., and the immersion is repeated a predetermined number of times for elastin. To obtain a treated tissue from which other collagens and unnecessary proteins have been removed, and then repeatedly immerse the treated tissue in an alkaline solution of 0.01 to 5 mol per liter at 90 to 105 ° C. for about 20 to 360 minutes. (The alkaline solution has a higher concentration and longer immersion time than the unnecessary protein removal operation), or the treated tissue is 0.05 to 5 moles per liter at 90 to 105 ° C in an acidic solution. It is a method of obtaining water-soluble elastin by repeatedly dipping and dissolving for about 20 to 360 minutes.

As described above, the water-soluble elastin obtained by the first or second method is then subjected to, for example, dialysis treatment to remove low-molecular-weight elastin to obtain water-soluble elastin having a molecular weight of about 3,500 or more. be able to. Particularly preferably used is high molecular weight water-soluble elastin having an average molecular weight of about 100,000 to 300,000.

The water-soluble elastin thus obtained has a concentration of 0.1 to 60 mg / ml, preferably a concentration of 0.5 to 30 mg / ml, and a solvent of a broad buffer solution (pH 1.0 to 11.0). Coacervate droplets can be formed by adjusting each of them and heating at a temperature rising rate of 0.1 to 40 ° C./min. And the production | generation of a coacervate droplet can be confirmed by measuring the turbidity of the aqueous solution of elastin in the temperature range of 4-80 degreeC.

In the present invention, water-insoluble microparticles are obtained by irradiating coacervate droplets obtained as described above with radiation such as γ rays or electron beams, that is, ionizing radiation. Radiation can be easily irradiated by, for example, a Co-60 gamma irradiation apparatus. Irradiation conditions may be appropriately selected. The irradiation temperature is usually 10 to 80 ° C., preferably 30 to 70 ° C., and the irradiation amount is usually 5 to 50 kGy, preferably 10 to 30 kGy. What is necessary is just to adjust irradiation time suitably so that it may become a desired irradiation amount. Upon irradiation, the coacervate droplets crosslink and convert to water insoluble particles.

The generated water-insoluble particles can be easily separated from the reaction system by filtration or centrifugation. These particles are micro-order sized particles, and specifically have a particle size of more than 300 nm, 10 μm or less, preferably more than 400 nm, 10 μm or less, more preferably about 1 to 3 μm. The microparticles exist stably over a long period of time, and are stable for 2 months at 4 ° C. and 37 ° C., for example.
The water-insoluble microparticles thus obtained are novel microparticles using animal-derived elastin, and are used as sustained-release carriers for therapeutic drugs for the liver and spleen because of their particle sizes.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
(Production of bonito-derived water-soluble elastin)
In accordance with the procedure shown in FIG. 1, unnecessary proteins soluble in NaCl water and soluble in NaOH aqueous solution were removed from skipjack arterial spheres, and then solubilized to produce bonito-derived water-soluble elastin. In the figure, x6 and x2 represent the number of repetitions of the same process.

Bonito was used as an animal living tissue, and the low-elastin content portions such as attached fat and muscle were scraped off with a blade and then homogenized using a homogenizer. The homogenized bonito body tissue is immersed in a diluted alkaline solution (0.01 to 0.02N) at 4 ° C. for 10 days, and further heat treated at 80 to 90 ° C. for 1 hour to degrease to obtain an arterial defatted tissue. It was.

This arterial sphere degreased tissue was placed in a container, and 10-fold volume (10 ml per 1 g weight) of 1M sodium chloride aqueous solution was added to the weight of the arterial sphere degreased tissue and immersed therein, followed by stirring at 4 ° C. for 24 hours. This operation is for removing unnecessary proteins. After removing the solubilized components, the same operation was repeated, and a total of 6 immersion treatments were performed.

The arterial degreased tissue that had undergone the immersion treatment was placed in a 10-fold volume of 0.05 M aqueous sodium hydroxide solution and stirred at 50 ° C. for 30 minutes to perform collagen removal treatment. Next, after separating the arterial defatted tissue and the alkaline solution, treatment with 0.05 M aqueous sodium hydroxide solution at 50 ° C. for 15 minutes was performed twice, and with 0.01 M aqueous sodium hydroxide solution at 4 ° C. for 15 minutes. The treatment was repeated 3 times to obtain insoluble elastin.

Next, 0.25 M oxalic acid (solubilized solution) having a volume 10 times the weight of insoluble elastin is added and stirred at 100 ° C. for 30 minutes to fragment insoluble elastin and solubilize in water. After separating the sex component, the same operation was performed 4 times in total on the residue to obtain water-soluble elastin derived from skipjack.

  The isolated bonito-derived water-soluble elastin was subjected to SDS (sodium dodecyl sulfate) -PAGE (electrophoresis of polyacrylamide gel) under non-reducing conditions, and the molecular weight distribution was measured. The gel concentration was 15.0%, the staining method was CBB, and the molecular weight marker was protein molecular weight marker “Daiichi” II. The molecular weight distribution was widely distributed in the range of 3,500 to 300,000, particularly many components having a molecular weight of 43,000 or more, and the average molecular weight was about 200,000.

In order to examine the amino acid composition of the isolated bonito-derived water-soluble elastin, amino acid analysis was performed. About 1 mg of each powder was dissolved in 1 ml of 6N · HCl, placed in a tube for hydrolysis, replaced with nitrogen gas under reduced pressure, and the tube was sealed. Then, it hydrolyzed at 110 degreeC for 48 hours. The hydrolyzed sample was subjected to amino acid analysis, and the amino acid composition was calculated. The results are shown in Table 1.

(Production of water-soluble elastin microparticles)
Bonito-derived water-soluble elastin was taken in a 10 ml vial and dissolved in water to prepare a 2.0 mg / ml aqueous solution. This aqueous solution is heated to 45 ° C., and microparticles are produced by irradiating γ-rays at a dose rate of 8.8 kGy / hour using a Co-60 γ-ray irradiator (irradiation amount 10 kGy) and crosslinking between molecules. did. The obtained microparticles were separated and obtained by centrifugation. The yield of the particles was 1.1 mg, and the yield was 5.5%.

The particle size of the particles after γ-ray irradiation was measured by a dynamic light scattering method (DLS) using NICOMP (Imaging Technology Group, 380ZLS). Each sample was placed in a 3 ml cell and measured for 2 minutes × 3 times. The measurement result of the particle size distribution is shown in FIG. The horizontal axis in FIG. 2 is the particle size (unit is nm), and K represents 1,000. From this result, it was confirmed that 91.5% of the generated crosslinked particles were included in the particle size range of 1280 nm ± 209.3 nm.

The photograph which observed the bonito origin particle | grains produced by the gamma ray irradiation with the transmission electron microscope (TEM) was shown in FIG. From this result, it was found that the microparticles are almost spherical particles. In order to confirm the stability of the microparticles in the aqueous dispersion, the zeta potential was measured, and it was confirmed that the particles had a sufficient negative charge at -40.5 mV and were very stable particles. It was.

As described above, the crosslinked microparticles of the present invention are substantially spherical and stable particles, and when used as a DDS carrier, the drug is easily loaded in shape, and the DDS carrier loaded with a drug is in the blood. It is expected to be easy to move.

According to the present invention, micro-order stable crosslinked particles can be prepared by a simple operation of heating an aqueous solution of water-soluble elastin derived from animals to produce coacervate droplets and irradiating them with radiation. Particles of this size have the property of being trapped in the liver and spleen, so if injected intravenously, they are thought to accumulate in the liver and spleen. It is useful as a carrier for therapeutic drugs such as cancer, particularly as a sustained release carrier. Moreover, since this crosslinked particle is a living body-derived polymer material, it is decomposed by an enzyme and does not remain in the living body. Furthermore, since it can be produced by irradiation, aseptic microparticles can be obtained.

Claims (4)

The coacervate droplets of the water-soluble elastin from fish, obtained by irradiating the radiation, elastin microparticles having a particle diameter of Ru 1~10μm der water-insoluble. 2. The method for producing microparticles according to claim 1, wherein the coacervate droplets of water-soluble elastin derived from fish are irradiated with radiation. The method for producing microparticles according to claim 2 , wherein the irradiation temperature of radiation is 10 to 80 ° C. and the irradiation amount is 5 to 50 kGy. A sustained-release drug carrier comprising the microparticle according to claim 1 as a constituent.

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