JPH0587081B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to cellulose-based particles having a specific number average particle size, a narrow particle size distribution, and a specific distribution coefficient.

[Problems to be solved by conventional technology/invention]

Cellulose-based materials such as cellulose, regenerated cellulose, and cellulose derivatives are characterized by relatively low nonspecific adsorption of blood cell components and plasma proteins. Therefore, cellulosic materials, especially those coated with a number average particle size of 600 to 1500 μm,
Activated carbon particles are used to selectively remove plasma proteins and the like from blood. However, cellulosic particles themselves have never been used for this purpose.

On the other hand, dispersion methods and spray methods are known as methods for producing spherical polymer particles.

In the dispersion method, a dilute solution of a polymer dispersed in small droplets in a dispersion medium containing a surfactant is solidified by evaporating the solvent (see Japanese Patent Application Laid-Open No. 56-24430); Gradually add small droplets of a coagulant to the dispersion liquid to solidify it (Japanese Patent Application Laid-Open No. 1983-1999)
-159801)), polymer particles can be obtained. This method yields particles with a broad size distribution and requires washing with organic solvents as well as water to remove solvent, dispersion medium and surfactants from the solidified droplets.

In the spray method, polymer particles are obtained by spraying a polymer solution into a coagulant. These particles also have a wide particle size distribution and are relatively large in size (see Japanese Patent Application Laid-Open No. 129788/1983).

If cellulose-based particles are produced as the spherical polymer particles and intended to be used in place of activated carbon particles coated with cellulose-based material, if the cellulose-based particles contain many small particles,
When used as a column-shaped adsorbent with good adsorption efficiency, problems arise such as a large pressure loss, causing problems such as hemolysis, and poor selectivity because the molecular weight cutoff is no longer sharp.

Furthermore, if the cellulose-based particles contain many large particles, the specific surface area of the particles (the total surface area of the particles per unit volume of the particles) becomes small, resulting in a problem that the adsorption rate decreases.

[Means to solve the problem]

The present invention was made in order to solve the problems such as the wide particle size distribution of cellulose particles, which is the cause of the above problems, and the number average particle size is 300 to 300.
600μm range, more than 95% of particles are within ±10% of the number average particle size, and the distribution coefficient is molecular weight 10000.
Regarding cellulose particles having a molecular weight of 0.3 or more for the following, and 0.05 or less for a molecular weight of 100,000 or more.

〔Example〕

The cellulose-based particles of the present invention are composed of cellulose-based materials such as cellulose, regenerated cellulose, and cellulose derivatives. Therefore, when the cellulose-based particles are used for purposes such as selectively removing plasma proteins from blood,
Nonspecific adsorption of blood cell components and plasma proteins is reduced compared to when particles made from other materials are used.
Furthermore, when used for applications such as preparative separation of biochemical substances by chromatography, non-specific adsorption of proteins is reduced.

The above-mentioned cellulose refers to so-called natural cellulose, such as pulp obtained by removing lignin and hemicellulose from defatted cotton fibers, hemp fibers, and wood, and refined pulp obtained by further refining the pulp. Typical examples include cellulose, but are not limited to these.

The cellulose derivatives are derived from cellulose, such as those in which some or all of the hydroxyl groups of cellulose have been esterified or etherified, and those in which some of the hydroxyl groups of cellulose have been esterified and some have been etherified. It is something that has been induced.

Specific examples of cellulose in which some or all of the hydroxyl groups have been esterified include cellulose acetate, cellulose propionate, cellulose butyrate, cellulose nitro, cellulose sulfate, cellulose phosphate, cellulose acetate butyrate, cellulose nitrate, and cellulose cellulose. Examples include, but are not limited to, dithiocarboxylic acid esters (viscose rayon).

Specific examples of cellulose in which some or all of the hydroxyl groups are etherified include methylcellulose, ethylcellulose, benzylcellulose, tritylcellulose, cyanethylcellulose, carboxymethylcellulose, carboxyethylcellulose, aminoethylcellulose, oxyethylcellulose, etc. , but not limited to these.

Furthermore, the regenerated cellulose refers to cellulose obtained by molding cellulose into a cellulose derivative that can be easily molded in one piece, and then regenerating the cellulose by hydrolysis or the like.

Among the cellulose-based materials constituting the cellulose-based particles, purified cellulose is preferable because it has fewer impurities and leaves less undissolved matter when dissolved, and cellulose esters such as cellulose acetate and cellulose propionate are suitable for use in various solvents. As a result, the range of selection of various manufacturing conditions during particle production is widened, and it is easy to adjust the particle distribution coefficient, skin layer thickness, pore size in the internal network structure, etc. to achieve the desired result. This is preferable because cellulose-based particles can be produced, and it is even more preferable because it can be easily converted into regenerated cellulose particles by hydrolysis.

The cellulose-based particles of the present invention are spherical (a concept that includes not only almost perfect spheres but also ellipsoidal bodies of rotation with a short axis/long axis of up to about 0.8).
, and the number average particle diameter (in the case of an elliptical rotating body, the volume average particle diameter, which is determined as the cube root of the square of the major axis multiplied by the minor axis) is 300~
600μm range, 95% or more of the particles are within ±10% of the number average particle size, and the distribution coefficient is within the molecular weight range.
It is 0.3 or more for a molecular weight of 10,000 or less, and 0.05 or less for a molecular weight of 100,000 or more. On the surface of such cellulose-based particles, there is usually a skin layer with a thickness of 10 μm or less, and the inside is composed of a network structure having pores of 0.1 to 10 μm. In addition, the porosity of the particles is 50~
It is about 95%.

Figures 1 and 2 show the skin layer of the cellulose-based particles of the present invention (a whitish layer that exists diagonally from about 1/3 from the top left in Figure 1 to about 1/2 from the top left). Electron micrograph (15,000
This is an example of

When the number average particle size is less than 300 μm, plasma proteins with a molecular weight smaller than albumin (molecular weight 67,000 to 68,000) can be directly perfused as cellulose particles for an adsorbent to selectively remove plasma proteins from blood. When used as an adsorbent for removal by adsorption, the pressure loss increases and problems such as hemolysis are more likely to occur. Moreover, when the diameter exceeds 600 μm, the specific surface area becomes small and the adsorption speed becomes low, so the amount of adsorption and removal of unnecessary substances becomes small in relation to the amount of blood circulation.

In addition, the percentage of particles within ±10% of the number average particle size is
If it is less than 95%, when used as an adsorbent for the above-mentioned adsorbent, pressure loss will increase and hemolysis will easily occur.

Furthermore, for molecular weights below 10,000, the distribution coefficient
If it is outside the range of 0.3 or more and 0.05 or less with respect to the molecular weight of 100,000 or more, the selectivity when used as an adsorbent for the adsorbent described above will decrease, and substances with a molecular weight of more than albumin will be easily adsorbed. Problems such as deterioration or conversely a marked decrease in the adsorption rate, such as insufficient adsorption and removal of unnecessary substances, tend to occur.

Note that if the mesh size of the network structure inside the cellulose particles is less than 0.1 μm, when used as an adsorbent for an adsorbent, not only will the rate of adsorption of the unnecessary substances in the blood become low, but also these There is a tendency that unnecessary substances are not adsorbed and removed sufficiently. Furthermore, if the particle size exceeds 10 μm, the mechanical strength will not be sufficient, and the particles will tend to be easily deformed or crushed during packing into a column or during transportation, and if such deformation occurs, This results in increased pressure loss and a tendency for microscopic debris to enter the blood.

Next, the method for producing cellulose particles of the present invention will be explained.

The cellulose-based particles of the present invention can be obtained by dissolving a polymer solution in which cellulose, cellulose derivatives, etc. ) can be manufactured by applying.

Examples of the solvent used in preparing the polymer solution include cellulose solvents such as aqueous copper ammonia solution, a mixture of dimethyl sulfoxide and paraformaldehyde, and an aqueous calcium thiocyanate solution, as well as cellulose acetate, which is a typical cellulose derivative. Examples of solvents include dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, and acetone.

These solvents include methanol, ethanol, ethylene glycol, and propylene glycol in order to adjust the thickness of the skin layer and the pore size of the internal network structure of the resulting cellulose particles, and then adjust the partition coefficient. , glycerin, water, inorganic salts, polyethylene glycol, polyvinylpyrrolidone, etc. may be added.

The polymer solution prepared in this way is ejected into the gas phase as uniform droplets using a device such as that described in JP-A No. 62-191033, and is flown over a flight distance in which it becomes approximately spherical. After that, it is brought into contact with a coagulant. The cellulose-based particles produced in this manner are approximately perfectly spherical particles.

The coagulant is made of a polymeric non-solvent, and preferably has a surface tension such that it dissolves in the solvent constituting the droplets and naturally wets the droplets. Specific examples of such coagulants include water, a mixture of water and the aforementioned good solvent or non-solvent, and a mixture of water and a surfactant.

Generally, when the concentration of the polymer in the polymer solution is high, the proportion of non-solvent is low, and a coagulating liquid with strong coagulating power, such as water, is used, the pores of the skin layer become smaller (the distribution coefficient becomes smaller).

The cellulose-based particles of the present invention obtained in this way have a specific average particle size, a specific particle size distribution, and a specific distribution coefficient, so they can be used as packing materials for chromatography, carriers for enzyme immobilization, and after-sampling. It can be used for applications such as a carrier for initake chromatography and an adsorbent for direct blood perfusion, and when used in these applications, it has excellent properties such as pressure drop, selectivity, and adsorption speed. Become.

Next, the cellulose particles of the present invention will be explained based on Examples, but it goes without saying that the present invention is not limited to these Examples.

Example 1 Cellulose diacetate was dissolved in a mixed solution of dimethyl sulfoxide/propylene glycol in a weight ratio of 4/6 so that the concentration was 12.5% (weight %, the same applies hereinafter).

Parallel plate electrodes with a width of 5 cm and a length of 25 cm in the direction of droplet travel are installed 5 mm in front of the nozzle at a distance of 2 cm, and a DC voltage of 800 V is applied between the electrodes and the nozzle. A voltage was applied. From an orifice with a diameter of 250 μm provided in this nozzle, the solution maintained at 145°C is ejected at a linear velocity of 7.8 m/sec while applying vibrations of 3850 Hz to form uniform droplets of the solution, which are dispersed in the air. After flying about 3m, 23
The particles were coagulated by entering a 10% aqueous methanol solution at ℃ to obtain particles of cellulose diacetate.

The obtained cellulose diacetate particles were heated at 50℃ and 0.6%
The cellulose particles were poured into an aqueous solution of caustic soda, stirred for 2 hours, recovered, neutralized and washed with water to obtain almost 100% regenerated cellulose particles.

The number average particle size of the obtained regenerated cellulose particles was measured by the method described below and was found to be 430 μm, which was within ±5% of the number average particle size.

After replacing the liquid in the obtained regenerated cellulose particles with ethanol, they are subjected to critical point drying with carbon dioxide gas (using a critical point dryer HCP-2 manufactured by Hitachi, Ltd.),
After depositing gold, observation using a scanning electron microscope revealed that the surface had a thickness of about 0.2 μm, as shown in Figure 1.
There is a skin layer (the surface part of the whitish layer that exists diagonally from about 1/3 from the top left to about 1/2 from the top left), and as shown in Figure 2, the inside is It had a porous network structure with a pore diameter of about 0.2 to 2 μm.

The distribution coefficients of the particles for dextrans with molecular weights of 10,000 and 110,000 were measured by the following method and were 0.67 and 0, respectively.

The regenerated cellulose particles had an inner diameter of 14 mm and a length of 7 cm.
Fresh bovine blood packed into a column with 30 units/ml of heparin added was kept at 37°C, and the pressure drop was measured by gradually increasing the flow rate.As the linear velocity increased, the pressure drop was almost linear. Although the pressure loss increased, the pressure was 50 mmHg even at 8 cm/min, and this value remained almost unchanged even after continuous operation for 1 hour, and direct blood perfusion was possible.

The regenerated cellulose particles were reacted with epichlorohydrin and then with n-hexylamine to obtain n-hexylamine-immobilized cellulose particles. When the selectivity of these particles was measured by the method described below, the adsorption rates for lysozyme and albumin were 73% and 8%, respectively.

(Number average particle size and particle size distribution) Optical microscopic images of several hundred particles (approximately 500 to 1000 particles) are processed using an image processing device (Ruzetskus manufactured by Nireco Co., Ltd.).

(Partition coefficient (Kav)) Partition chromatography was performed using aqueous solutions of dextran and pullulan with various molecular weights, and the elution curve was determined using the formula: Kav=Ve−Vo/Vt−Vo (wherein, Vo is the molecular weight of 2,000,000 (Vt is the column volume, Ve is the eluate volume corresponding to the peak position of the elution curve of the sample).

(Selectivity) Lysozyme and albumin were added at 3 mg/ml and 3 mg/ml, respectively, in 0.025 M phosphate buffer adjusted to pH 7.4.
To 6 volumes of each solution dissolved to a concentration of 7.3 mg/ml, cellulose-based particles on which a liquid adsorbing to plasma proteins is immobilized are added so as to have a sedimentation volume of 1 volume. After shaking at 37°C for 2 hours, the concentration of the supernatant liquid was measured to determine the respective adsorption rates.

Adsorption rate (%) = Stock solution concentration - Supernatant solution concentration / Stock solution concentration x 10
0 (Determining the possibility of direct blood perfusion) Fresh cow blood to which 30 units/ml of heparin was added was added.
Insulated at 37℃ and filled with particles, length 7cm, inner diameter
The flow rate was gradually increased through a 14 mmφ column until the linear velocity reached 8 cm/min, and direct blood perfusion was judged to be possible if the pressure drop did not exceed 100 mmHg during this time.

Comparative Example 1 Pressure drop was measured in the same manner as in Example 1 using commercially available regenerated cellulose particles with a number average particle size of 450 μm and 89% of the particles falling within ±10% of the number average particle size. At a speed of 2 cm/min, there is already a pressure loss.
The pressure drop exceeded 100 mmHg, and the experiment was discontinued as the pressure loss became even larger.

Comparative Example 2 Regenerated cellulose particles with a number average particle size of 270 μm and 100% of the particles within ±5% of the number average particle size were obtained in the same manner as in Example 1 by changing the conditions for forming uniform droplets. When the pressure drop was measured using these particles in the same manner as in Example 1, it exceeded 100 mmHg at a linear velocity of 5 cm/min.

Comparative Example 3 When n-hexylamine was immobilized on commercially available regenerated cellulose particles with a distribution coefficient of 0.3 for dextran having a molecular weight of 110,000 in the same manner as in Example 1, the adsorption rates of lysozyme and albumin were measured. They were 24% and 58%.

〔Effect of the invention〕

Since the cellulose-based particles of the present invention have a specific number average particle size, a specific particle size distribution, and a specific distribution coefficient, the cellulose-based particles can be used as an adsorbent for selectively removing plasma proteins from blood. When used for adsorption and removal of particles, especially plasma proteins with a molecular weight smaller than albumin, by direct blood perfusion, pressure loss is small, problems such as hemolysis are less likely to occur, and selectivity is improved. However, effects such as an increase in adsorption amount are achieved.

[Brief explanation of the drawing]

Figure 1 is a photograph for explaining the structure of the cellulose-based particles of the present invention, showing the vicinity of the surface of the particle cross section (the skin layer on the surface and the network structure inside).
FIG. 2 is a photograph for explaining the internal structure of the cellulose-based particles of the present invention obtained in Example 1, and is a photograph in which the particle cross section is magnified 15,000 times.

Claims (1)

[Claims] 1 Number average particle size is in the range of 300 to 600μm, 95
% or more of the particles are within ±10% of the number average particle size,
Cellulose particles with a distribution coefficient of 0.3 or more for molecular weights of 10,000 or less and 0.05 or less for molecular weights of 100,000 or more.