JP7623668B2 - Functional particles - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies 1. Publications 2019 Toyama University Graduate School, Department of Science and Technology Education, Master's Program, Biotechnology Major, Master's Thesis Presentation Abstracts, distribution date: February 13, 2020 2. Meeting name 2019 Toyama University Graduate School, Department of Science and Technology Education, Master's Program, Biotechnology Major, Master's Thesis Presentation, Venue Toyama University Faculty of Engineering General Education and Research Building Lecture Room 11 (3190 Gofuku, Toyama City, Toyama Prefecture)

The present invention relates to functional microparticles using alginate gel beads, and in particular to microparticles produced by the inkjet drying method.

The present inventors have been conducting research into the production of fine particles using the inkjet drying method.
For example, Non-Patent Document 1 shows an example in which sodium alginate and hemoglobin are chemically bonded by dehydration condensation, and then ejected from an inkjet head to produce gel beads.
In this case, since the condensation reaction process between sodium alginate and hemoglobin also contains unreacted hemoglobin, a dialysis process is required to remove this unreacted hemoglobin, and further, processes such as freeze-drying are also required to store the alginate hemoglobin and make it into a reagent.
Therefore, the present invention was arrived at as a result of research aimed at shortening the process and improving the yield.

Patent Document 1 discloses a method for obtaining artificial red blood cells composed of gelled particles by adding a visible light crosslinking substance such as eosinized gelatin, a hydrogen donor such as reducing sugar, and hemoglobin to a lipophilic liquid, dispersing the mixture, and irradiating the mixture with visible light.
However, the process disclosed in this publication is aimed at producing artificial red blood cells, and the steps are complicated.

Masato Nakamura et al., "Protecting Lives with Printing Technology: Pioneering Future Medicine with Printing Technology", JST "Toyama University New Technology Briefing", October 31, 2017 (Tuesday)

JP 2006-182721 A

The present invention aims to provide microparticles having functionality such as oxygen carriers and carriers for drug delivery systems, and a method for producing the same.

The functional microparticles according to the present invention are characterized in that a functional protein or drug is immobilized on an alginate microparticle.
Here, the term "alginate microparticles" refers to alginate gel in which a functional protein or drug has been immobilized by chemical modification, and has an average particle size of 10 μm or less.
Red blood cells have a disk-like shape with a diameter of 7 to 8 μm and a thickness of about 2 μm, and the alginate microparticles according to the present invention refer to alginate gel beads that are at the same size as red blood cells.

By forming such alginate gel beads, various functionalities can be imparted.
For example, hemoglobin can be modified and fixed as a functional protein to function as an oxygen carrier.
Moreover, by modifying it with various functional proteins or drugs, it can be used as a carrier in a drug delivery system.

In the present invention, it is preferable that the functional protein or drug is chemically bonded to a carboxyl group constituting alginic acid.
For example, a carboxyl group constituting the alginic acid can be condensed and bonded to an amino group in hemoglobin.

Alginic acid is composed of mannuronic acid (M) and guluronic acid (G) units, and the ratio of these units is expressed as the M/G ratio.
Here, the GG block consisting of 20 or more G units plays an important role in the gelation of alginic acid, and amino groups of hemoglobin are bonded by a condensation reaction to the carboxyl groups of the G units that were not used for gelation and to the carboxyl groups of the M units.

The functional microparticles according to the present invention can be produced by preparing alginate microparticles using an inkjet drying method and then chemically modifying the prepared alginate microparticles with a functional protein or drug.

For example, chemical modification of hemoglobin will be explained as follows (details will be described later).
An inkjet head used in an inkjet printer and a drying tower for dropping and drying droplets ejected from the nozzle are arranged.
An aqueous solution of sodium alginate is discharged from the nozzle of an inkjet head, and the sodium alginate gel beads that gel after falling down a drying tube are collected in a dish of calcium chloride solution that is being stirred and maintained.
The sodium alginate gel beads are separated and collected from the dish by centrifugation or the like, and resuspended in PBS.
Hemoglobin is added to this resuspension and immobilized in the alginate gel beads in the presence of a condensing agent.

Hemoglobin (Hb) is a protein with a molecular weight of 64,500 Da that has oxygen-binding ability, and is the main component in red blood cells.
Hemoglobin is a tetrameric protein consisting of two molecules each of two types of globin (α, β).
The α chain is composed of 141 amino acids, and the β chain is composed of 146 amino acids.
This amino group can be utilized to undergo a condensation reaction with the carboxyl group of alginic acid.
As the condensation agent, a WSC (water-soluble carbodiimide) such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride can be used.
In addition, a carboxylic acid activating reagent such as NHS (N-hydroxysuccinimide) may also be used in the condensation reaction.

The inkjet used in the present invention can use various inkjet heads used in inkjet printers.
Among them, a piezoelectric inkjet head using a piezoelectric element that does not apply a thermal load to the alginic acid solution or the like is preferable.

The functional particles according to the present invention can be obtained by discharging an alginic acid solution using an inkjet head used in an inkjet printer and dropping the solution into a drying tube, thereby obtaining gel beads of alginic acid.
By immobilizing functional proteins such as hemoglobin or drugs on these alginate gel beads, microparticles with various functions can be obtained.
For example, when hemoglobin is selected as the functional protein, hemoglobin-modified alginate gel beads of the same size as red blood cells can be obtained, which have excellent function as oxygen carriers.

4 shows observation images of microparticles in various concentrations of sodium alginate aqueous solutions. 4 shows a particle size histogram of fine particles. The relationship between the size of the microparticles and the solution concentration is shown. The measurement results of the OEC curve are shown.

The following describes an example of the production of functional microparticles according to the present invention, but the present invention is not limited to this.

Microparticles (gel beads) were produced using an aqueous solution of sodium alginate, which will be described below.
(1) Using ultrapure water as a solvent, aqueous solutions of sodium alginate at concentrations of 0.3, 0.5, 0.8, and 1.0 wt % were prepared.
(2) A piezo inkjet head and a glass cylinder (drying tower) having an inner diameter of 12 cm and a height of 100 cm were arranged in order to allow droplets ejected from the head to fall and dry. A dish was placed at the bottom of the glass cylinder, and a 2 wt % aqueous solution of calcium chloride using ultrapure water as a solvent was poured into the dish.
(3) For the inkjet, a frequency of 0.8 kHz was used, and the following voltages were applied in accordance with the concentrations of the aqueous sodium alginate solutions.
0.3wt%: 20V
0.5wt%: 25V
0.8wt%: 30V
1.0wt%: 35V
(4) After the sodium alginate aqueous solution was discharged, it was recovered from the calcium chloride aqueous solution by centrifugation, washed as required, and then freeze-dried.

An image of the obtained microparticles is shown in FIG. 1, and the microparticle size distribution is shown in the histogram of FIG.
FIG. 3 is a graph showing the average particle size, standard deviation, and relationship between concentration (wt %) and microparticle volume (μm ³ ) for each concentration of sodium alginate aqueous solution.

These results show that gel beads with a particle size of 3 to 5 μm can be obtained, and that a proportional relationship is observed between the average volume of the gel beads obtained and the concentration of the sodium alginate aqueous solution, so that the average particle size of the gel beads can be controlled by controlling the concentration of the aqueous solution.

Next, an example of modifying hemoglobin using sodium alginate gel beads will be described.
(1) 100 ml of a 1.0 wt % aqueous solution of sodium alginate was prepared using ultrapure water as a solvent, and discharged under conditions of 0.5 kHz and 35 V from an inkjet head, and collected in a 2 wt % aqueous solution of calcium chloride placed at the bottom of a drying tower.
(2) Gel beads were recovered from the calcium chloride aqueous solution by centrifugation, resuspended in 50 ml of PBS solution at pH 6.6, and 18.3 mg of WSC and 5.5 mg of NHS were added thereto. The mixture was reacted at room temperature for 1 hour, recovered, resuspended in PBS, and 2 ml of porcine-derived purified Hb solution was added thereto. The mixture was reacted in a cool, dark place for 24 hours.
(3) After the reaction was completed, the porcine hemoglobin-modified sodium alginate gel beads were recovered by centrifugation and suspended in the same amount of ultrapure water for storage.
(4) At the same time, a purified bovine Hb solution was used.
The bovine hemoglobin-modified sodium alginate beads were collected and suspended in an equal amount of ultrapure water for storage.
(5) Next, 4 ml of HEMOX SOLUTION was added to 1 ml of the aqueous solution containing the hemoglobin-modified sodium alginate gel beads obtained above, and the mixture was loaded into a HEMOX ANALYZER, and the OEC (oxygen dissociation curve) was measured at 37°C.
The results are shown in the graph of FIG.

For comparison, the graph in FIG. 4 shows the OECs of a porcine-derived Hb solution and a bovine-derived Hb solution.
Although the OEC curves of porcine Hb microparticles (alginate gel beads modified with porcine-derived Hb) and bovine Hb microparticles (alginate gel beads modified with bovine-derived Hb) are slightly different, it was revealed that both have the ability to act as oxygen carriers.

Claims (1)

The method comprises preparing alginate microparticles by an inkjet drying method, and chemically modifying the prepared alginate microparticles with functional proteins,
The method for producing functional fine particles , wherein the functional protein is hemoglobin .