JP2013023485A - Production method of gelatin particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a production method of gelatin particles, with a smaller amount of residual fat and oil and no aggregation among the particles, and to preferably provide a method for producing embolus particles of gelatin suitable for embolization therapy or the like.SOLUTION: The production method of gelatin particles includes: a liquid droplet formation step of forming liquid droplets of a gelatin aqueous solution by dispersing the gelatin aqueous solution into a fat and oil; a gelation step of gelating the liquid droplets of the gelatin aqueous solution by cooling; and a dehydration step, a cleaning step, a drying step and a crosslinking step. The cooling temperature in the gelation step is 0 to 3°C. A dehydration solvent and a cleaning solvent used in the dehydration step and the cleaning step are hydrophilic solvents having at least 10 wt.% of the solubility in water. The obtained gelatin particles do not have porous shapes, and have solid sphere shapes.

Description

  The present invention relates to a method for producing gelatin particles, and preferably has a solid spherical shape suitable for embolization treatment of liver cancer, kidney cancer, spleen cancer, uterine fibroid, etc., hemostasis of arterial bleeding, treatment of embolism before surgery, etc. The present invention relates to a method for producing gelatin embolic particles.

  Currently, transarterial embolization treatment methods are known for the treatment of liver cancer, uterine fibroids, kidney cancer and the like. This treatment uses a microcatheter to inject an anticancer drug into the affected cancer (myoma) tissue, and then embolizes the blood vessels leading to the cancer (myoma) tissue with an embolic material suspended in a nonionic contrast medium. Thus, the supply of nutrients to the cancer (myoma) is blocked and necrosis is attempted. This treatment method is a tissue-selective treatment method and can minimize the side effect of necrosis of normal cells. However, if the organic solvent used in the manufacturing process remains in the embolic material, it may be harmful to the human body because the embolic material may be decomposed at the embolic site or the residual solvent may adversely affect the human body. It is desired to reduce the residual solvent as much as possible.

  Patent Document 1 discloses a method for producing water-insoluble porous particles by a liquid dispersion method. That is, first, gelatin as a biocompatible substance is foamed in a solution dissolved in a good solvent, and then emulsified and dispersed. Next, after this is cooled and gelled or solidified, the porous particles that have become honeycombed by foaming are dried under reduced pressure, further pores are formed, and finally the porous particles insolubilized by heating It is to get particles.

  Patent Document 2 discloses a method for producing true spherical gelatin particles by a drop-in-liquid method. That is, while the nozzle is immersed in a hydrophobic solvent, the nozzle mouth is reciprocated with amplitude in the horizontal direction, and the gelatin solution is pressurized and discharged from the nozzle mouth to the hydrophobic solvent having a gel temperature or higher. .

  Furthermore, Patent Document 3 discloses a method for producing gelatin particles by a drop-in-liquid method, which is generated when a gelatin aqueous solution is discharged from a discharge port of a dispenser into a hydrophobic solvent and the discharge port of the dispenser is pulled up. The droplet of gelatin aqueous solution is released from the discharge port of the dispenser.

  As described above, in a general liquid dispersion method that has been conventionally used as a method for producing gelatin particles, an aqueous gelatin solution is placed in a hydrophobic solvent tank containing a hydrophobic solvent such as fats and oils, and stirred with a stirring blade. Alternatively, it is dispersed to form droplets of an aqueous gelatin solution, and then the hydrophobic solvent tank is cooled with cooling water or the like. However, the liquid dispersion method is a very simple method, but since a large amount of hydrophobic solvent is used, if the solvent removal operation or the washing operation is insufficient, fats and oils may remain in the resulting gelatin particles. There is sex.

  In addition, in the submerged method, droplets of an aqueous gelatin solution are formed drop by drop, so the content of residual solvent can be reduced compared to the above-mentioned submerged method, but the production efficiency of gelatin particles is reduced, If the droplets are not sufficiently gelled or stirred, there is a high possibility that the gelatin particles will aggregate in the hydrophobic solvent tank.

Japanese Patent No. 3879018 Japanese Patent Publication No. 1-17376 JP 2010-77062 A

  The present inventors diligently studied a method for minimizing the amount of the organic solvent remaining in the obtained gelatin particles in producing the gelatin particles. As a result, surprisingly, the amount of residual solvent in the obtained gelatin particles can be suppressed to 1% by weight or less by setting the cooling temperature at the time of gelling the droplets of the gelatin aqueous solution to a specific range. The present inventors have found what can be done and have completed the present invention.

  That is, the present invention includes a dehydration step, a washing step, a droplet formation step in which an aqueous gelatin solution is dispersed in oil and fat to form droplets of the aqueous gelatin solution, and a gelation step in which the droplets of the aqueous gelatin solution are cooled to gel. A method for producing gelatin particles comprising a step, a drying step, and a crosslinking step, wherein the cooling temperature in the gelation step is 0 to 3 ° C., and the dehydration solvent and the washing solvent in the dehydration step and the washing step are at least 10% by weight The present invention provides a method for producing gelatin particles, which is a hydrophilic solvent having solubility in water.

  In particular, it is desirable that the obtained gelatin particles have a solid spherical shape, and the amount of residual solvent is reduced by employing the production method of the present invention, so that the gelatin particles are suitable for embolization treatment applications. Can be obtained.

  According to the production method of the present invention, when gelatin particles are produced by a liquid dispersion method, the gelatin particles obtained by adjusting the cooling temperature when gelling droplets of an aqueous gelatin solution to 0 to 3 ° C. There is an effect that the amount of the organic solvent and oil remaining in the inside can be reduced to 1% by weight or less. As a result, gelatin particles having a uniform particle diameter can be stably obtained without aggregation of the obtained gelatin particles, and even when used for embolization treatment, it can be used without adversely affecting the human body.

FIG. 1 is a photograph showing gelatin particles produced by the production method of Example 1.

  In the method for producing gelatin of the present invention, it is first necessary to form droplets of an aqueous gelatin solution in the droplet forming step. Specifically, gelatin is poured into water at about 0 ° C., and gelatin is uniformly dissolved in water using a stirrer or a shaker to prepare an aqueous gelatin solution. Heating the water temperature at this time to about 40 to 60 ° C. is preferable because gelatin can be dissolved in a short time. The gelatin concentration of the gelatin aqueous solution thus obtained is 2 to 20% by weight, preferably in order to adjust the viscosity of the droplets to be formed and to easily obtain gelatin particles having various particle sizes and a narrow particle size distribution. Set in the range of 5-15 wt%.

  The type (origin) of the gelatin used in the present invention is not particularly limited. For example, gelatin derived from cow bone, cow skin, pork bone, pig skin, or the like can be used. Further, it is desirable to use gelatin having a jelly strength of 80 to 320 g, preferably 100 to 300 g. When gelatin having a jelly strength of less than 80 g is used, it may be difficult to gel the gelatin in the gelation step described later because the jelly strength is too low. If the jelly strength is too high, It tends to be difficult to disperse uniformly as droplets.

  In the droplet forming step, the aqueous gelatin solution prepared as described above is dispersed in oil to form droplets. For example, an excess amount of oil is put into a flask equipped with a stirring blade, the gelatin aqueous solution prepared as described above is added while stirring, and the gelatin aqueous solution is uniformly dispersed in the oil and fat by stirring for an arbitrary time. Let It is preferable that the temperature of the fats and oils at this time is heated to 0 to 60 ° C., preferably about 40 to 50 ° C., so that gelatin can be uniformly dispersed while coagulation of the fats and oils is prevented, and further gelatin denaturation can be prevented.

  As fats and oils used in the present invention, it is necessary to use fats and oils having poor compatibility with gelatin aqueous solution, for example, at least one kind selected from the group consisting of animal oils, vegetable oils, mineral oils, silicone oils, fatty acids, fatty acid esters, and organic solvents. Can be used. Of these, specifically, tricaprylic acid glyceride, olive oil, isocetyl isostearate, cetyl 2-ethylhexanoate, and the like are preferable from the viewpoint of non-toxicity to the human body.

  In the production method of the present invention, a gelation step is performed following the droplet formation step. In the gelation step, gelatin is gelled by cooling droplets of the gelatin aqueous solution. The gelation of the present invention means a state having no fluidity that can maintain a spherical shape even if the gelatinized gelatin droplets are filtered out from the oil. As a method of gelling gelatin in the droplets by the cooling operation in the gelation step, the oil and fat in which the droplets are dispersed may be cooled. For example, when gelatin droplets dispersed in fats and oils are formed in the flask, the flask may be immersed in cooling water and the oils and gelatin droplets inside may be cooled through the outer wall of the flask. It is extremely important to set the cooling temperature within a temperature range of 0 to 3 ° C. in order to exert the effects of the present invention. By cooling the gelatin droplet within this temperature range, the water in the gelatin droplet does not solidify, and the mechanical strength of the gelatinized gelatin droplet is improved. As a result, gelatin particles are less deformed in the dehydration process and the cleaning process described later, and the oil and fat on the surface is easily cleaned in the cleaning process.

  The cooling time in the gelation step is 15 to 90 minutes, preferably about 30 to 60 minutes after reaching the set temperature of 0 to 3 ° C. If the cooling time is too short, gelatinization of gelatin droplets becomes insufficient. On the other hand, if it is too long, gelation has progressed sufficiently, so that time is wasted and production efficiency deteriorates.

  In addition, depending on the type, the fat used in the above gelation process may cause the fat or oil to coagulate due to a decrease in temperature due to the cooling operation or the fat or oil viscosity to increase. In addition, it is preferable to add a low dehydrating solvent because these problems can be prevented.

  In the present invention, after gelatin droplets are gelated by a cooling operation as described above, moisture in the droplets is removed in a dehydration step. Specifically, in order to prevent the gelatinized gelatin particles from dissolving, in the dehydration process, a dehydrated solvent is added while cooling to below the gelation temperature of gelatin (specifically, 15 ° C or less). A dehydration process is performed by substituting with the moisture. The complete removal of moisture is carried out in a drying step described later. The purpose of the dehydration step is to replace moisture with a dehydration solvent, and the dehydration step is usually preferably performed for 15 minutes or longer. By removing the water by replacing the water in the gelatin particles with a dehydrating solvent in the dehydration step, uniform intra-particle cross-linking can be performed in the cross-linking step described later, and aggregation of the resulting gelatin particles can be prevented. It is.

  In the dehydration step of the present invention, it is necessary to dehydrate by substituting the water in the droplets and the dehydrating solvent as described above. Therefore, the dehydrating solvent used has a solubility in water of at least 10% by weight, preferably 30%. A hydrophilic solvent having a solubility of not less than% by weight must be used. As specific dehydrating solvents, for example, acetone, isopropyl alcohol, ethyl alcohol, methyl alcohol, and tetrahydrofuran can be used alone or in combination. Of these dehydrating solvents, from the viewpoint of easy replacement with water, it is preferable to use a hydrophilic organic solvent such as acetone, isopropyl alcohol, tetrahydrofuran, etc. that can be mixed with water relatively freely.

  In the production method of the present invention, the washing step is performed following the above-described dehydration step. The gelatin particles cooled and gelated contain a dehydrated solvent substituted with moisture in the dehydration step, but the fats and oils adhering to the surface of the gelatin particles can be removed through the washing step. Specifically, as the cleaning solvent used in the cleaning step, it is preferable to use an organic solvent that does not dissolve the gelatinized gelatin particles. Like the dehydrated solvent, the cleaning solvent is added while cooling below the gelation temperature of gelatin. In addition, when performing washing | cleaning operation, it is preferable to repeat washing | cleaning in multiple times, using together methods, such as filtration and centrifugation. For example, when washing about 2 to 15 g of gelatin particles, complete washing is achieved by repeating the washing operation for about 15 to 30 minutes using about 200 to 300 ml of washing solvent for about 4 to 6 cycles. .

  As a specific cleaning solvent, a solvent similar to the above-described dehydrating solvent can be used. However, from the viewpoint of ensuring dehydration of gelatin particles, a ketone solvent such as acetone, an alcohol such as isopropyl alcohol, and the like. It is preferable to use a system solvent, tetrahydrofuran.

  As described above, the gelatin particles obtained through the dehydration step and the washing step are subjected to a drying step in order to remove excess dehydration solvent and washing solvent adhering to the particle surface and completely remove moisture. Do. As a drying means used in the drying step, for example, general drying means such as ventilation drying, reduced pressure drying (including vacuum drying), freeze drying and the like can be used. When drying under reduced pressure, it is preferable to perform drying at a temperature of about 5 to 25 ° C. for about 6 to 12 hours in order to prevent gelatin from being crosslinked by heating.

  The gelatin particles dehydrated and dried in the drying step cross-link the gelatin particles in the cross-linking step. In particular, when gelatin particles are introduced into the body, such as for embolization treatment, it is necessary to make the particles harmless to the human body, so a crosslinking means using an external crosslinking agent such as a crosslinking agent is employed. That is not preferable. Therefore, in the present invention, when gelatin particles used for embolization treatment are produced, it is preferable to perform crosslinking by heating. In the case of crosslinking by heating, for example, gelatin particles can be crosslinked by heating at 80 to 250 ° C., preferably 100 to 140 ° C., for 0.5 to 120 hours, preferably 12 to 24 hours. In addition, since the degree of crosslinking of gelatin particles can be adjusted by adjusting the heating temperature and heating time, it is possible to adjust the time (biodegradation time and the like) until the gelatin particles are completely dissolved in the aqueous solution or blood vessel.

  That is, when the gelatin particles obtained by the production method of the present invention are used for embolization treatment and the blood vessel is embolized, the time until the blood flow is resumed can be adjusted, that is, the embolization time can be adjusted. is there. Specifically, in order to necrotize tumors such as liver cancer, vascular embolization for 2 to 3 days is sufficient, so the embolization period until blood flow recanalize, that is, the period until gelatin particles biodegrade, About 3 to 7 days is preferable because the damage to the organ and normal cells at the embolic site is reduced. Usually, when setting to the embolization period of about 3-7 days, it is preferable to set it as 1-24 hours at 100-180 degreeC as heating conditions in a bridge | crosslinking process. In addition, since a heating operation is performed in the crosslinking step, gelatin particles may be oxidized and denatured. Therefore, it is usually preferable to perform the crosslinking step under reduced pressure or in an inert gas atmosphere.

  Since the gelatin particles obtained as described above are produced through a droplet forming step of forming droplets of an aqueous gelatin solution dispersed in fats and oils, the shape thereof is not an indefinite shape but a spherical shape, and is as spherical as possible. It is preferable to be close to. In addition, the gelatin particle structure is preferably not a porous structure like a sponge, but a so-called solid structure having no pores inside. The particle size of the gelatin particles needs to be adjusted according to the inner diameter of the blood vessel at the embolic site.

  That is, when the gelatin particles obtained in the present invention are used for embolization treatment, embolization at a target location can be reliably performed by preparing solid gelatin particles having a particle size corresponding to the inner diameter of the vascular embolization site. At the same time, since the spherical shape can prevent damage to the inner wall of the blood vessel, pain to the patient can be reduced. Furthermore, in the case of solid spherical gelatin particles, the gelatin particles gradually dissolve from the outer peripheral portion of the gelatin particles. In the case of porous gelatin particles, the gelatin particles are dissolved during dissolution / decomposition in an embolized blood vessel. Some of the particles may be separated and dropped into microparticles, and these microparticles may be carried into the bloodstream and embolize blood vessels other than the target site. On the other hand, in the case of solid spherical gelatin particles, there is an advantage that such a problem is unlikely to occur.

  The particle size of the gelatin particles obtained by the production method of the present invention is such that the blood vessel at the embolization site is not adversely affected in healthy tissue in addition to the purpose of embolizing the blood vessel at a portion as close as possible to the target site for embolization treatment. It is necessary to use properly according to the size of the. For that purpose, specifically, it is preferable to arrange gelatin particles having four types of particle diameters of 25 to 65 μm, 75 to 150 μm, 200 to 300 μm, and 400 to 600 μm. It is not preferable that the particle size is too small because there is a risk of embolizing blood vessels other than the target site. According to the production method of the present invention, these three kinds of particle size ranges of gelatin particles can be easily produced, and all the particles within each particle size range are ± 25% of the target central particle size. A sharp particle size distribution that is within the range can be realized.

  In addition, when using the gelatin particle obtained by the manufacturing method of this invention for an embolic treatment use etc., in order to narrow a particle size distribution, it is preferable to classify before the said crosslinking process.

  When gelatin particles obtained by the production method of the present invention are used for embolization treatment, for example, a catheter is inserted from a femoral artery or the like to a planned embolization treatment site while a contrast agent is administered, and then gelatin having an arbitrary shape is used. A dispersion of gelatin particles in which particles are dispersed and swollen in physiological saline can be injected into a catheter, and the gelatin particles can be sent to the target embolization treatment site for embolization.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited by description of an Example.

<Example 1>
Into a 1000 ml flask equipped with a stirring blade, 300 g of tricaprylic glyceride was added, and 50 g of an aqueous gelatin solution (gelatin derived from pork skin having a jelly strength of 87 g, gelatin concentration 6.67% by weight) was added to the flask at 60 rpm at 200 rpm. The mixture was stirred for minutes, and droplets of an aqueous gelatin solution were formed in tricaprylic acid glycerides as fats and oils.

  Next, this flask is immersed in cold water at 0 ° C., and the tricaprylic acid glyceride is cooled to 0 ° C. and stirred at 200 rpm for 60 minutes to cool the gelatin aqueous solution droplets. Went.

  Next, 100 ml of ice-cooled (−10 ° C.) acetone was added to the flask, and the mixture was stirred at 220 rpm for 30 minutes to replace the dehydrated water in the gelatinized gelatin aqueous solution with acetone to obtain gelatin particles. It was. Acetone is a solvent that is freely miscible with water.

  The obtained gelatin particles were filtered off, washed 7 times with an excess amount of acetone cooled on ice (−10 ° C.) to remove tricaprylic acid glyceride adhering to the gelatin particles, and then dried under vacuum for 24 hours to remove the residual solvent. Removed gelatin particles were obtained.

  Finally, the obtained gelatin particles were classified and then subjected to thermal crosslinking under a vacuum of 140 ° C. for 5 hours to obtain crosslinked gelatin particles. The obtained gelatin particles were solid spherical particles having a particle size of 250 μm. The obtained gelatin particles were observed with a microscope having a magnification of 100 times, and a photograph thereof is shown in FIG.

<Example 2>
Crosslinked gelatin particles were obtained in the same manner as in Example 1 except that the cooling temperature in the gelation step in Example 1 was set to 2 ° C. The obtained gelatin particles were solid spherical particles having a particle size of 250 μm.

<Example 3>
The gelatin concentration of the gelatin aqueous solution used in Example 1 was set to 5% by weight, the cooling temperature in the gelation step was set to 3 ° C., and tetrahydrofuran (solubility in water: freely mixed) was used as a dehydrating solvent and a washing solvent. Except for the use, crosslinked gelatin particles were obtained in the same manner as in Example 1. The obtained gelatin particles were solid spherical particles having a particle size of 250 μm.

<Example 4>
The gelatin concentration of the gelatin aqueous solution used in Example 1 was set to 5% by weight, the cooling temperature in the gelation step was set to 3 ° C., and isopropyl alcohol (solubility in water: freely mixed) as a dehydrating solvent and washing solvent Crosslinked gelatin particles were obtained in the same manner as in Example 1 except that was used. The obtained gelatin particles were solid spherical particles having a particle size of 250 μm.

<Example 5>
Crosslinking was carried out in the same manner as in Example 1 except that the gelatin aqueous solution used in Example 1 was a gelatin derived from pork skin having a jelly strength of 281 g, the gelatin concentration was 10 wt%, and the cooling temperature in the gelation step was set to 3 ° C. Gelatin particles were obtained. The obtained gelatin particles were solid spherical particles having a particle size of 250 μm.

<Comparative Example 1>
Crosslinked gelatin particles were obtained in the same manner as in Example 1 except that the cooling temperature in the gelation step in Example 1 was set to 5 ° C. The obtained gelatin particles were solid spherical particles having a particle size of 250 μm.

<Comparative example 2>
The gelatin aqueous solution used in Example 1 was a gelatin derived from pork skin having a jelly strength of 281 g, a gelatin concentration of 5% by weight, the cooling temperature in the gelation step was set to 3 ° C., and ethyl acetate ( Example 1 was used except that the solubility in water was 9% by weight. However, since ethyl acetate having high hydrophobicity was used as a dehydrating solvent and a washing solvent, dehydration was insufficient without being sufficiently replaced with moisture in the gelatin particles, and the intended gelatin particles could not be obtained.

<Comparative Example 3>
The gelatin aqueous solution used in Example 1 was a gelatin derived from pork skin having a jelly strength of 281 g, a gelatin concentration of 5% by weight, the cooling temperature in the gelation step was set to 3 ° C., and toluene (water The solubility was in the same manner as in Example 1, except that 0.05% by weight was used. However, since highly hydrophobic toluene was used as a dehydrating solvent and a washing solvent, the water in the gelatin particles was not sufficiently replaced with water, resulting in insufficient dehydration, and the intended gelatin particles could not be obtained.

  The amount of fat remaining in the gelatin particles obtained in each of the above Examples and Comparative Examples was measured using HPLC after extracting the fat in the particles. The results are shown in Table 1.

  From the results of Examples 1 and 2 and Comparative Example 1 in Table 1 above, it is clear that the amount of residual oil can be reduced by setting the cooling temperature in the gelation step to a range of 0 to 3 ° C. . When the gelation temperature is less than 0 ° C., the gelatin aqueous solution droplets freeze, and uniform gelation does not occur.

Furthermore, as is clear from the results of Comparative Examples 2 and 3, when the dehydrating solvent and the washing solvent are highly hydrophobic solvents, the water in the gelatin particles is not sufficiently replaced with water, resulting in insufficient dehydration. Gelatin particles could not be obtained.

Claims (8)

A dehydration step, a washing step, a drying step, a droplet forming step of dispersing an aqueous gelatin solution in oil and fat to form droplets of the aqueous gelatin solution, and a gelling step of cooling and gelatinizing the droplets of the aqueous gelatin solution, and A method for producing gelatin particles including a crosslinking step,
Gelatin particles characterized in that the cooling temperature in the gelation step is 0 to 3 ° C., and the dehydration solvent and the washing solvent in the dehydration step and the washing step are hydrophilic solvents having a solubility in water of at least 10% by weight Manufacturing method.   The method for producing gelatin particles according to claim 1, wherein the fat is at least one selected from the group consisting of animal oil, vegetable oil, mineral oil, silicone oil, fatty acid, fatty acid ester, and organic solvent.   The method for producing gelatin particles according to claim 1, wherein the dehydrating solvent and the cleaning solvent are at least one organic solvent selected from the group consisting of acetone, isopropyl alcohol, ethyl alcohol, methyl alcohol, and tetrahydrofuran.   The method for producing gelatin particles according to claim 1, wherein the drying step is performed by at least one means selected from the group consisting of ventilation drying, reduced pressure drying, and freeze drying.   The method for producing gelatin particles according to claim 1, wherein the crosslinking step is performed by a heating means having a heating temperature of 80 to 250 ° C and a heating time of 0.5 to 120 hours.   The method for producing gelatin particles according to any one of claims 1 to 5, wherein the obtained gelatin particles have a solid spherical shape.   The method for producing gelatin particles according to claim 6, wherein the obtained gelatin particles have a particle size of 30 to 2000 μm. The method for producing gelatin particles according to any one of claims 1 to 7, wherein the obtained gelatin particles are used for embolization treatment.