JP2008001764A - Method for producing particulate shaped body made of protein, and particulate shaped body made of protein obtained by the method - Google Patents

Abstract

By using a radiation stronger than ultraviolet rays, a crosslinked structure is stably introduced into the protein, and a nano- and sub-micron size order biodegradable particulate shaped body made of the protein is produced.
A method for producing a particulate compact comprising a protein comprises mixing a raw material containing a protein powder and water or a buffer so as to be 0.00025 to 0.1% by weight. And the prepared raw material mixture is irradiated with ionizing radiation at an irradiation dose of 100 Gy or more and less than 200 kGy.
[Selection] Figure 1

Description

  The present invention relates to a method for producing a particulate shaped product comprising a protein by irradiation with radiation, and a particulate matter comprising a nano and submicron size order protein whose form does not change even at a high temperature above the solubilization temperature obtained by the method. The present invention relates to a molded body.

  Albumin is a general term for a group of soluble proteins contained in cells and body fluids of animals and plants, and is contained in serum, milk, egg white and the like. Examples include porcine albumin, bovine albumin (BSA), human serum albumin (HSA), whey albumin, ovalbumin and the like. Among them, HSA accounts for about 60% of plasma proteins, and is the second most simple protein in the blood after hemoglobin. Many studies have been conducted since ancient times, but Carter et al. Since then, it has attracted a lot of attention again in various fields. Particularly in the field of medical materials, an albumin-heme complex in which a porphyrin iron complex (FeP), which is uniquely designed and synthesized by utilizing the nonspecific molecular binding ability of HSA, is included in the hydrophobic domain of HSA. Has been synthesized and is expected to be applied to artificial red blood cells. Moreover, since biocompatibility is high, the application to the medical adhesive of the biopolymer-aldehyde type compound which uses albumin as a biopolymer is proposed.

  The above albumin is a water-soluble protein, and a molded product using these water-soluble proteins is difficult to stably maintain a certain form. It is necessary to introduce certain water resistance and physical strength by making the structure of the molded product a bridge structure. Currently, in order to make the structure of a molded product into a crosslinked structure, a crosslinking treatment such as glutaraldehyde or formaldehyde is used for crosslinking. However, when such a crosslinking agent is used, there is a concern about cytotoxicity due to the residual crosslinking agent in which an unreacted crosslinking agent remains (see, for example, Non-Patent Document 1 and Non-Patent Document 2). When the cross-linking agent is used, it is necessary to remove the residual cross-linking agent by washing treatment using water or the like, but it is difficult to completely remove the remaining cross-linking agent. Further, although it is bound to the protein, it does not react completely and it is necessary to inactivate the crosslinking agent having an unreacted group.

Thus, since the method of carrying out the crosslinking treatment using the crosslinking agent has a problem in safety, an insolubilization technique by irradiating the protein with ultraviolet rays and carrying out the crosslinking treatment is disclosed as a countermeasure (for example, patents). (See Literature 1 and Patent Literature 2.) Specifically, in Patent Document 1, a medical base material containing gelatin and collagen as essential base material components and cross-linked by irradiation with ultraviolet rays is disclosed in Patent Document 2, and gelatin is used in Patent Document 2. An anti-adhesive material that has been molded and then cross-linked by irradiation with ultraviolet rays is disclosed.
JP 11-47258 A (Claim 1) JP 2000-37450 A (Claim 1) J. Appl. Toxicol. 21, 131 (2001) Toxicology 175, 175 (2002)

  However, in the case of using ultraviolet irradiation as shown in Patent Document 1 and Patent Document 2, since ultraviolet light has a low transmission ability, there is a problem in that it is not suitable for a cross-linking treatment of a thick protein sample. In addition, tissue culture is performed at a temperature close to body temperature, but when gelatin cross-linked by irradiation with ultraviolet rays is used as a tissue culture carrier, since the gelatinization temperature of gelatin is lower than 37 ° C., the solution becomes a solution, Incubation was difficult. Therefore, improvement in water absorption, strength and heat resistance of the cell culture carrier has been essential for application. Fibronectin is most suitable as a molecule having both biocompatibility and cell adhesion required as a cell culture carrier, but the same modification is required for fibronectin or a culture carrier coated with fibronectin.

The object of the present invention is to produce a nano- and sub-micron size biodegradable particulate shaped body composed of protein by stably introducing a bridging structure into protein by using radiation stronger than ultraviolet rays. It is to provide a method.
Another object of the present invention is to provide a nano- and sub-micron size particulate shaped product comprising a protein having biocompatibility, water absorption and heat resistance in the use of medical materials such as tissue culture and drug delivery system. There is.

  As a result of intensive research on the above problems, the present inventors prepared a dilute raw material mixture by thoroughly mixing a raw material containing a water-soluble protein and water or a buffer solution, and the prepared dilute raw material mixture It has been found that by irradiating with ionizing radiation, a particulate shaped body composed of nano- and sub-micron-sized proteins whose form does not change even at a high temperature above the solation temperature can be obtained.

  In the method for producing a particulate molded body comprising a protein of the present invention, a raw material mixture is prepared by mixing a raw material containing protein and water or a buffer solution, and the prepared raw material mixture is irradiated with ionizing radiation. Thus, by using radiation that is stronger than ultraviolet rays, it is possible to produce nano- and sub-micron size order biodegradable particulate compacts made of protein, in which a crosslinked structure is stably introduced into the protein. The particulate molded body made of the protein obtained by the above production method does not change in shape even at a high temperature above the solation temperature, and has biocompatibility, water absorption and heat resistance. It can be used for medical materials.

Next, the best mode for carrying out the present invention will be described.
In the method for producing a particulate molded body comprising a protein of the present invention, a raw material mixture is prepared by mixing a raw material containing protein and water or a buffer solution, and this prepared raw material mixture is irradiated with ionizing radiation. It is characterized by that. Examples of the buffer include phosphate buffer, TE (tris-HCl-EDTA) buffer, HEPES buffer, and the like. By using the buffer, the effect of stabilizing the structure of the protein in the solution can be obtained. The raw material containing protein is preferably in the form of powder. As the protein contained in the raw material, a water-soluble protein is used. The raw material mixture is preferably prepared by adding water or a buffer to the raw material and mixing so that the concentration of the liquid mixture becomes 0.00025 to 0.1% by weight. Among these, a particularly preferable concentration is 0.00025 to 0.001% by weight. The concentration of the raw material mixture is within the above-mentioned concentration range because if the amount is less than the lower limit, a sufficient amount of the particulate formed body cannot be produced, and if the upper limit is exceeded, the average particle diameter of the formed particulate formed body This is because it is difficult to control.

  The protein contained in the raw material includes albumin, bird egg white, mammals, fish, insects, etc. contained in the serum of mammals such as pigs, cows or humans, egg whites of chickens, milk of mammals such as milk and breast milk, etc. For lysozyme contained in erythrocytes, hemoglobin contained in erythrocytes, etc., muscle contraction of animals, cell movement, intracellular transport of intracellular substances, fibrinogen, fibrin involved in blood coagulation, casein which is the main component of milk, cell adhesion Consists of fibronectin, a cell surface protein involved, elastin widely distributed in stretchable organs such as the dermis, ligaments, tendons, and vascular walls of the skin, and keratin and basement membranes that constitute animal skin, nails, and hair And laminin, which is a cell adhesion glycoprotein. Examples of albumin include mammalian albumin or ovalbumin selected from the group consisting of porcine albumin, bovine albumin or human albumin. Further, these proteins can be further purified and used, for example, in order to satisfy standards such as Japanese Pharmacopoeia or purified albumin. Furthermore, derivatives in which the side chains of these proteins are chemically modified can also be used. Chemical modifications include carboxymethylation, carboxyethylation, methylation, hydroxyethylation, acetylation, tosylation and the like.

  The raw material mixture thus prepared is irradiated with ionizing radiation. Since ionizing radiation having higher energy than ultraviolet rays can stably introduce a cross-linking structure into proteins and does not use a cross-linking agent or ultraviolet rays, a safe particulate molded product can be produced. Heavy ion beams, alpha rays, beta rays, that is, electron beams, X-rays, gamma rays, and the like are used as the types of ionizing radiation irradiated to the raw material mixture. Among these, gamma rays from cobalt 60, cesium 137, iridium 192 or strontium 90, which are radioisotopes often used in medical sterilization and industrial use, and electron beams from an accelerator are preferable. The irradiation dose of ionizing radiation must be such that the protein has a cross-linked structure so that absorbability and heat resistance are imparted, and strength that does not break during use is introduced. Specifically, 100 Gy or more and less than 200 kGy is suitable, and 0.5 to 50 kGy is particularly desirable.

  Since the particulate molded body made of the protein of the present invention obtained by the above production method uses radiation having higher energy than ultraviolet rays at the time of production, a cross-linked structure is stably introduced into the protein, and ultraviolet irradiation is performed. Compared to high water resistance and physical strength. In addition, since no cross-linking agent is used at the time of manufacture, there is no toxicity due to unreacted residues and the safety is high. Moreover, as an average particle diameter of the particulate-shaped molded object of this invention, the inside of the range of 1-500 nm is suitable. Among these, a more preferable average particle diameter is in the range of 1 to 100 nm. Since the shape of the particulate molded body of the present invention does not change even at a temperature higher than the solation temperature, for example, when a gelatin particulate molded body is used as a tissue culture carrier, it does not become a solution even during culture. Improvement of water absorption and strength of the carrier using bismuth is expected to improve heat resistance.

  In addition, since the particulate molded body made of the protein of the present invention has biocompatibility, it can be used as a cell culture carrier for use in the field of regenerative medicine, and it can be used as a drug for target affected areas (organs, tissues, cells, pathogens, etc.). As a matrix base material for drug delivery systems that effectively and intensively deliver skin, skin defect materials such as burns, wounds, pressure sores, abrasions or skin ulcers, and medical base materials such as joint lubricants such as shoulders, elbows, knees or ankles Can be used.

  Further, since the particulate molded body made of the protein of the present invention is a biodegradable material derived from nature, the environment such as a matrix base material for agricultural delivery systems that feeds agricultural chemicals, fertilizers, and soil conditioners to a predetermined position while controlling them. It can be applied as an environmentally friendly material and product.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited only to these Examples.

<Example 1>
First, bovine serum-derived albumin (ICN Biomedicals; fraction V, pH 7.0) is prepared as a raw material containing protein, and the concentration of bovine serum-derived albumin and distilled water is 1.0 × 10 ⁻³ wt%. Was mixed well and dissolved well at 40 ° C. to prepare a raw material mixture having a concentration of 1.0 × 10 ⁻³ wt%. Next, this raw material mixture was filtered through a filter with a pore size of 0.8 mm to remove undissolved substances, and carefully poured into a plurality of light scattering quartz tubes. Next, gamma rays with an irradiation dose of 10 kGy and 20 kGy were irradiated to the light scattering quartz tube into which the raw material mixture was injected.

  Subsequently, static and dynamic light scattering measurements were performed at 40 ° C. for each sample irradiated with gamma rays at unirradiated (0 kGy), 10 kGy, and 20 kGy irradiation doses, and the particle size and apparent molecular weight were calculated. In dynamic light scattering, dynamic light scattering was measured at an angle of 30 ° and analyzed by the CONTIN method. The static light scattering measurement results are shown in FIGS. 1A and 1B, and the dynamic light scattering results are shown in FIG.

As is clear from FIGS. 1 (a) and 1 (b), when a diluted raw material mixture such as 1.0 × 10 ⁻³ wt% is irradiated with gamma rays, the light scattering intensity (Kc / Rθ) is It increases rapidly by γ-ray irradiation. This shows that the albumin particles were cross-linked by γ-ray irradiation, resulting in an increase in molecular weight.

  Further, as can be seen from FIG. 2, there are two peaks in the sample not irradiated with gamma rays (0 kGy). The peak with the smaller Rh represents the size of one albumin. Conversely, the peak with the larger Rh represents the size of the aggregate in which some albumin is collected. The vertical axis represents the light scattering intensity from a particle having that size. Since the peak of the aggregate-derived peak is smaller than the peak of single albumin, it can be said that the number of aggregates is very small. On the other hand, two peaks are also observed in the sample irradiated with 10 kGy of gamma rays, but the intensity of the peak with small Rh is very small. From this, it can be said that one albumin particle aggregated by γ-ray irradiation to form a large particle. Furthermore, in the sample irradiated with 20 kGy of gamma rays, the size distribution was wider and single.

  Next, the molecular weight (Mapp) and hydrodynamic radius (Rh) from the peak obtained from the results of FIG. 2 were calculated. The calculation results are shown in Table 1.

  As is clear from Table 1, the molecular weight obtained by light scattering is a weight average molecular weight, which is considered to be strongly influenced by the side with the larger distribution. However, the increase in molecular weight obtained based on that strongly supports that albumin is crosslinked to form nanogel particles. The molecular weight of the albumin nanoparticles prepared by irradiation is larger than the molecular weight of the original albumin, and the hydrodynamic radius is almost the same as that of the original albumin. From these results, it was found that nano-sized particles having a very high density were formed by irradiation of a diluted albumin aqueous solution.

  Since the particulate molded body made of the protein of the present invention has biocompatibility, the drug is effective for a cell culture carrier used in the field of regenerative medicine and a target affected part (organ, tissue, cell, pathogen, etc.). Use as a matrix substrate for drug delivery systems that deliver in a focused and concentrated manner, a medical substrate such as skin defect materials such as burns, wounds, pressure sores, abrasions or skin ulcers, joint lubricants such as shoulders, elbows, knees or ankles Can do. Further, since the particulate molded body made of the protein of the present invention is a biodegradable material derived from nature, the environment such as a matrix base material for agricultural delivery systems that feeds agricultural chemicals, fertilizers, and soil conditioners to a predetermined position while controlling them. It can be applied as an environmentally friendly material and product.

The figure which shows the static light-scattering measurement result of BSA dilute aqueous solution. The figure which shows the analysis result by the CONTIN method of the dynamic light scattering measurement in the angle of 30 degrees of BSA dilute aqueous solution.

Claims (8)

  A method for producing a particulate compact comprising a protein, comprising preparing a raw material mixture by mixing a protein-containing raw material and water or a buffer solution, and irradiating the prepared raw material mixture with ionizing radiation. . The raw material containing protein is in powder form,
Preparation of the raw material mixture is prepared by adding water or a buffer to the raw material and mixing so that the concentration of the mixed solution becomes 0.00025 to 0.1% by weight,
The method for producing a particulate molded body comprising a protein according to claim 1, wherein the irradiation dose of ionizing radiation is 100 Gy or more and less than 200 kGy.   The particle form of protein according to claim 1 or 2, wherein the ionizing radiation applied to the raw material mixture is gamma rays from the radioactive isotopes cobalt 60, cesium 137, iridium 192 or strontium 90, and electron beams from the accelerator. Manufacturing method of a molded object.   The protein contained in the raw material is one or more selected from the group consisting of albumin, lysozyme, hemoglobin, myosin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, laminin and derivatives thereof. Or the manufacturing method of the particulate molded object which consists of protein of 2.   The method according to claim 4, wherein the albumin is mammalian albumin selected from the group consisting of porcine albumin, bovine albumin or human albumin, or ovalbumin.   A particulate molded body comprising a protein obtained by the method according to any one of claims 1 to 5.   The particulate molded body made of protein according to claim 6, wherein the molded body has an average particle diameter of 1 to 500 nm. The particulate molded body comprising a protein according to claim 6 or 7, wherein the shape does not change even at a high temperature equal to or higher than the solation temperature.

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