JP4182351B2 - DNA-containing polysulfone microspheres and method for separating and removing dioxins using the same - Google Patents

Description

  The present invention relates to a DNA-containing polysulfone microparticle that can be used to separate and remove dioxins that are harmful substances from liquids to be treated, such as milk, breast milk, drinking water, domestic water, industrial water, and wastewater. The present invention relates to a method for separating and removing spheres and dioxins using the spheres.

  Dioxins, including polychlorinated dibenzo-p-di (di) oxins (PCDD), polychlorinated dibenzofurans (PCDF) and coplanar polychlorinated biphenyls (PCB) are taken up through the food chain and separated into the body of many organisms. It is a harmful substance. Since these dioxins may have a negative effect on the human body even in a trace amount, the suppression of dioxins generation and the separation / removal of dioxins from the contaminated environment are important in maintaining a favorable living environment in human life. This is an important issue.

Examples of a method for separating and removing dioxins present in water include an adsorption separation method using activated carbon. For example, in Patent Document 1 below, a liquid to be treated is introduced into a membrane separation apparatus in which activated carbon is flowing, dioxins dissolved in water are adsorbed on the activated carbon, and solid components such as activated carbon and suspended solids are subjected to membrane separation. Discloses a method for separating and removing dioxins by removing them. In this method, in order to collect and remove dioxins dissolved in water, it is necessary to filter and separate the dioxins adsorbed on the activated carbon through a membrane.
Activated carbon has no selectivity with respect to the adsorbing material. That is, activated carbon can adsorb nutrient substances (useful substances) such as vitamins in addition to harmful substances such as dioxins. Therefore, activated carbon is not suitable for use in an environment where harmful substances and useful substances coexist.
JP 2002-239347 A

The present inventors have proposed a separation method using DNA in addition to the aforementioned adsorption separation method using activated carbon as a method for separating and removing dioxins dissolved in water (see Japanese Patent Application No. 2003-008310). That is, it is a method that utilizes a phenomenon called intercalation in which a chemical substance having a planar structure is inserted in parallel during the stacking of base pairs inside the double helix of DNA.
Since intercalation of DNA selectively separates and removes chemical substances having a planar structure, for example, chemical substances having a three-dimensional structure such as vitamins are not separated or removed. Therefore, when DNA intercalation is used, for example, only specific types of harmful chemical substances having a planar structure are selectively separated and removed from the liquid to be treated in which chemical substances of various structures coexist. In addition, useful substances such as vitamins having a three-dimensional structure can be left in the liquid to be treated without being separated and removed.
In this case, a fish larva is useful as a DNA raw material. Fish larvae contain a large amount of DNA and protein, and are known to be very nutritious foods. However, only a small portion of the fish larvae is used for fresh food, and its field of use is limited for reasons such as difficulty in processing and preservation, and most of them have been discarded. In particular, salmon roe that is landed in Hokkaido in large quantities is disposed of, except that more than 10,000 tons per year are used for feed and fertilizer. Therefore, fish resources can be used more effectively by using fish larva as a raw material for DNA.

  The present invention relates to DNA-containing polysulfone microspheres that can be used to selectively and efficiently separate and remove dioxins dissolved in a liquid to be treated, and dioxins using the same. An object is to provide a removal method.

  As a result of diligent research on the means for separating and removing dioxins dissolved in the liquid to be treated, the present inventors have selectively used dioxins from the liquid to be treated by using polysulfone microspheres containing DNA. The present invention has been completed by finding that it can be separated and removed efficiently.

The DNA-containing polysulfone microsphere according to claim 1 is characterized in that DNA is encapsulated in a hollow microsphere made of polysulfone.
The DNA-containing polysulfone microsphere according to claim 2 is composed of an outer thick part and an inner hole in the microsphere made of polysulfone, the hole is filled with DNA, and the thick part Is characterized in that it forms a through-hole that communicates the surface of the microsphere with the pores and allows small molecules to pass through but does not allow DNA macromolecules to pass through.
The method for separating and removing dioxins according to claim 3 comprises contacting the DNA-containing polysulfone microspheres according to claim 1 with a liquid to be treated containing dioxins, and capturing the dioxins on DNA by intercalation. It is characterized by separating and removing.

The DNA-containing polysulfone microsphere according to claim 4 is characterized in that DNA is filled in a porous microsphere made of polysulfone.
The method for separating / removing dioxins according to claim 5 comprises contacting the DNA-containing polysulfone microspheres according to claim 4 with a liquid to be treated containing dioxins, and allowing the dioxins to be captured by DNA by intercalation. It is characterized by separating and removing.

In the present invention, DNA-containing polysulfone microspheres are brought into contact with the liquid to be treated, and dioxins are captured by intercalation between the DNA base pairs contained in the polysulfone microspheres to dissolve in the liquid to be treated. The dioxins thus obtained can be selectively separated and removed efficiently.
The DNA-containing polysulfone microspheres of the present invention and the method for separating and removing dioxins using the same can be obtained from a liquid to be treated in which chemical substances of various structures coexist by using DNA intercalation. The harmful dioxins having a structure are selectively separated and removed, and useful substances such as vitamins having a three-dimensional structure are not separated and removed but can be left in the liquid to be treated and separated and removed. Excellent selectivity with respect to material.
In addition, as the DNA, DNA derived from the white larvae of fish, which has been mostly discarded, can be used, and marine resources can be used effectively.

これら３種の基本骨格構造を有する化合物のベンゼン環の水素原子が塩素原子で置換されることにより、ダイオキシン類を構成する多くの異性体〔（ジベンゾ−ｐ−ダイオキシンからは７５種類、ジベンゾフランから１３５種類、そしてビフェニルから１３種類（コプラナーポリ塩化ビフェニルとして）〕が生じる。ポリ塩素化ビフェニル（ＰＣＢ）のうち、２つのベンゼン環の回転が規制されて平面的な分子構造を有するもの（コプラナーＰＣＢ）をダイオキシン類に含める。
それ故、前述のように、ダイオキシン類は、ポリ塩化ジベンゾ−ｐ−ダイオキシン（ＰＣＤＤ）、ポリ塩化ジベンゾフラン（ＰＣＤＦ）及びコプラナーポリ塩化ビフェニル（ＰＣＢ）を包含する。 <About dioxins>
Hereinafter, compounds having three basic skeleton structures of dioxins are shown.

By replacing the hydrogen atom of the benzene ring of the compounds having these three basic skeleton structures with chlorine atoms, many isomers constituting dioxins [(75 from dibenzo-p-dioxin, 135 from dibenzofuran, 13 types from biphenyl (as coplanar polychlorinated biphenyl)] Polychlorinated biphenyl (PCB) having a planar molecular structure in which the rotation of two benzene rings is restricted (coplanar PCB) Is included in dioxins.
Thus, as mentioned above, dioxins include polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and coplanar polychlorinated biphenyls (PCB).

臭化エチジウムはダイオキシン類と同様に平面的な分子構造を有し、インターカレーションによってＤＮＡに捕捉される。また、臭化エチジウムは発癌性物質でもある。それ故、本明細書において、臭化エチジウムをダイオキシン類のモデル化合物として使用し、種々の検討をしている。
アクリジンオレンジもＤＮＡ研究に用いる蛍光指示薬であり、ＤＮＡを赤色〜橙色に染色し、そして紫外線を照射することにより、橙色の蛍光を発生する。アクリジンオレンジも臭化エチジウムと同様、ダイオキシン類のモデル化合物として使用することができる。以下に、アクリジンオレンジの構造式を示す。

<About ethidium bromide and acridine orange>
Ethidium bromide is a fluorescent indicator used for DNA research, and it produces orange fluorescence by staining DNA red and irradiating it with ultraviolet light. The structural formula of ethidium bromide is shown below.

Like dioxins, ethidium bromide has a planar molecular structure and is trapped in DNA by intercalation. Ethidium bromide is also a carcinogen. Therefore, in this specification, ethidium bromide is used as a model compound for dioxins and various studies are made.
Acridine orange is also a fluorescent indicator used for DNA research, and it emits orange fluorescence by staining DNA with red to orange and irradiating with ultraviolet rays. Acridine orange, like ethidium bromide, can be used as a model compound for dioxins. The structural formula of acridine orange is shown below.

<Description of the principle of separation and removal of dioxins>
Hereinafter, the principle of separation and removal of dioxins by capturing dioxins between DNA base pairs by intercalation will be described with reference to FIG.
As shown in FIG. 1 (a), the double-stranded DNA used in the present invention comprises two polynucleotide strands having a right-handed spiral shape. In DNA, a nucleobase (cation part) exists inside the helix, and a phosphate group (anion part) exists outside the helix. From the sugar-phosphate skeletons of two polynucleotide chains, planar bases having structural complementarity protrude perpendicular to the axis of the helix toward the center of the helix and are bonded by hydrogen bonds. ing. In the case of the B-type structure, there is a gap of about 1.1 nm in width and 0.34 nm in height between the double-stranded base pair of DNA and a small molecule having a planar structure (for example, dioxins or , Ethidium bromide and acridine orange, which are model compounds of dioxins), can enter this gap as shown in FIG. 1B, and this is called intercalation. This phenomenon may be promoted by the charge and hydrophobicity of small molecules. Dioxins are small molecules composed of a plurality of benzene rings, have a planar structure, and exhibit hydrophobicity, so that dioxins can be separated and removed from liquid using DNA.

<About polysulfone microspheres>
Polysulfone (PSf) is one of the important polymer materials, and membranes based on PSf and PSf exhibit outstanding oxidative, thermal and hydrolytic stability, as well as good mechanical properties and film-forming properties . Therefore, in the present invention, polysulfone was selected as the polymer material. PSf membranes are widely used in the field of material separation and in biomedical fields, and almost all PSf membranes are manufactured using a phase transition method.
a) PSf hollow microspheres PSf hollow microspheres encapsulating DNA were produced using a liquid-liquid phase separation method. The separation / removal of ethidium bromide from the liquid to be treated was examined using PSf hollow microspheres. The amount of DNA encapsulated in PSf hollow microspheres depended on the concentration of the DNA solution used to produce PSf hollow microspheres, and the morphology of PSf hollow microspheres depended on the polymer concentration and production conditions. The amount of ethidium bromide removed by the PSf hollow microspheres was mainly dependent on the amount of encapsulated DNA, and the morphology of the microspheres also affected the removal of ethidium bromide.
b) PSf porous microspheres PSf porous microspheres filled with DNA were prepared using a liquid-liquid phase separation method. The removal of ethidium bromide, acridine orange and dioxins was examined using PSf porous microspheres. PSf porous microspheres filled with DNA are stable in water. The stability of PSf porous microspheres and / or the release rate of DNA from the microspheres can be adjusted by changing the structure of the microspheres. Increasing the polymer concentration during the production of the microspheres resulted in a lower porosity and smaller pores on the surface of the microspheres, but the stability of the microspheres increased further, and the release rate of DNA from the microspheres was Decreased more. Furthermore, the drying temperature of the microspheres also affected the stability of the microspheres. PSf porous microspheres loaded with DNA were able to effectively separate ethidium bromide, acridine orange, dibenzo-p-dioxin, dibenzofuran and biphenyl. The amount of ethidium bromide removed by the microspheres depended on the amount of DNA loaded and the structure of the microspheres.

<Method for separating and removing dioxins using DNA-containing PSf microspheres>
In the dioxin separation / removal method of the present invention, the manner in which the liquid to be treated is brought into contact with the DNA-containing PSf microspheres is not particularly limited. For example, the liquid to be treated is packed in the column packed with the DNA-containing PSf microspheres of the present invention. And the like (continuous method), a method of adding the DNA-containing polysulfone microspheres of the present invention to the liquid to be treated and stirring (batch method), and the like.
In the continuous method, the PSf microspheres in the column containing dioxins are appropriately replaced. In the batch method, PSf microspheres containing dioxins are removed by a method such as filtration.

<About DNA raw materials>
The DNA contained in the PSf microspheres of the present invention can be, for example, one made from a fish larva. Since fish larvae contain a lot of DNA and are conventionally discarded materials, they are suitable as raw materials for mass-producing DNA at low cost. Examples of the fish can include salmon, salmon, salmon, salmon, etc., and after removing skin, muscles, blood vessels, etc. from these larvae, purify them to remove oil and produce DNA by production. Can do.

1. PSf hollow microsphere 1-1. Material and Preparation Method 1-1-1. Preparation of PSf solution and DNA solution PSf (average Mn about 26000, manufactured by Aldrich Chemical Company, Inc.) was dissolved in N-methyl-2-pyrrolidinone (NMP; HPLC grade, 99 +%, manufactured by Aldrich Chemical Company, Inc.). Thus, five types of PSf solutions were obtained. The concentrations were 5% by mass, 7.5% by mass, 10% by mass, 12.5% by mass and 15% by mass. Double stranded DNA (sodium salt, molecular weight 5 × 10 ⁶ ) from camellia white child was obtained from Yuki Fine Chemical Co., Ltd. The DNA was dissolved in distilled water. The concentrations were 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL. All materials were used without further purification.

1-1-2. Production of DNA-encapsulated PSf hollow microspheres An aqueous solution of double-stranded DNA was injected into the PSf solution using an injection needle having an inner diameter of 1 mm, and stirred at 60 rpm for 20 seconds. Was removed from the polymer solution and exposed to the atmosphere for a few seconds, after which the spheroids were poured into water. The spherical product was allowed to stand in water for 3 days to elute the solvent in the spherical product to obtain PSf hollow microspheres encapsulating DNA of the present invention.

1-1-3. Scanning Electron Microscope (SEM) Examination Samples of PSf hollow microspheres encapsulating DNA of the present invention were dried at room temperature, then cut with a single-edged razor, placed on a sample support, and coated with a gold layer. SEM images were recorded using an S-2500C microscope (voltage = 20 kV, manufactured by Hitachi).

1-1-4. The amount of DNA encapsulated in PSf hollow microspheres The amount of DNA encapsulated in PSf hollow microspheres was measured by the following process: the microspheres were cut into two parts and 1 hour at 100 ° C. Hydrolysis with 1M hydrochloric acid solution. The amount of DNA in the solution was quantified by absorbance at 260 nm using a UV-VIS spectrophotometer U-200A (Hitachi).

1-1-5. Separation and removal of ethidium bromide by PSf hollow microspheres encapsulated with DNA An aqueous ethidium bromide (EB) solution was used in the functional verification test of PSf hollow microspheres encapsulating DNA of the present invention. The separation of EBs in PSf hollow microspheres encapsulated with DNA was tested in the following steps: PSf hollow microspheres encapsulating DNA (10 microspheres, total dry weight about 25 mg) were added to an EB aqueous solution ( 7 mL, 10 mM) and left at room temperature for 24 hours. The separation of EBs was confirmed by the absorption spectrum of the EB solution in the presence or absence (control) of PSf hollow microspheres encapsulating DNA. The amount of EB in the solution was quantified by absorbance at 480 nm using a UV-VIS spectrophotometer U-200A (manufactured by Hitachi).

1-2. Results and examination 1-2-1. Production of PSf hollow microspheres When the DNA solution was added to the PSf solution, liquid-liquid phase separation caused by rapid exchange between the solvent NMP and water occurred, and a skin layer was formed. Subsequently, PSf precipitated and initial PSf microspheres were formed. As the initial PSf microspheres are removed from the polymer solution and exposed to the atmosphere, the outer surface of the microspheres gradually solidifies. Even if the initial PSf microspheres are placed in water, PSf can be completely solidified. There were small holes in the walls of the microspheres. Small molecules can pass through the microsphere pores, but very large molecules cannot pass through the microsphere pores. Therefore, water molecules and other small molecules can freely pass through the wall of the microsphere, but double-stranded DNA (macromolecule) was encapsulated in the microsphere.
When a double-stranded DNA solution was added to a PSf solution of less than 5% by mass, it was difficult to form PSf hollow microspheres encapsulating DNA. When the PSf concentration is about 15% by mass, a low water content of 3% to 4% by mass is required to cause phase separation of the solution at 25 ° C., but when the PSf concentration is about 5%, phase separation is performed. In order to wake up, a high content of 6% to 8% by weight was required. When a low-concentration PSf solution was used, a long tail was formed at the rear of the PSf hollow microsphere encapsulating DNA. When a very high concentration PSf solution exceeding 15% by mass is used, the viscosity of the polymer is very high, and water droplets containing DNA cannot be injected into the polymer solution. A sphere could not be formed.
It is well known that even when an aqueous DNA solution is stored at a low temperature, the DNA easily deteriorates and has a bad odor when dissolved in water. When an aqueous DNA solution is encapsulated in PSf microspheres, water can evaporate through the pores of the microsphere walls. As the water evaporates, the DNA is retained in a dry solid state within the PSf hollow microspheres. It is preferred for stable storage of DNA.

1-2-2. Structure of PSf hollow microspheres encapsulating DNA PSf hollow microspheres encapsulating DNA have a spherical appearance and may have a tail-like structure. The size of the microspheres in this example was uniform with a diameter of about 3 mm, which is much larger than the polymer microspheres usually obtained by liquid-liquid phase separation.
FIG. 2 shows PSf hollow microspheres of Example 1 encapsulating DNA. 2A is a front view of the PSf hollow microsphere of Example 1, and FIG. 2B is a cross-sectional view of FIG. 2A taken along the line AA. In FIG. 2A, it can be seen that a large number of micropores 3 are opened in the thick portion 2 of the polysulfone hollow microsphere 1.
The minute hole 3 is a hole penetrating from the surface of the thick part 2 to the hole 4 shown in FIG. The fine hole 3 penetrates from the surface of the thick part 2 to the hole 4 in a straight line, a curved line or a combination thereof. The shape of the opening of the microhole 3 can also take various shapes such as a circle, an ellipse, and an indefinite shape. Therefore, the size (for example, the average diameter) of the openings of the fine holes 3 can be variously changed from the surface of the thick part 2 to the holes 4.
Small molecules such as water and dioxins can pass through the opening of the fine hole 3 from the surface of the thick part 2 to the hole 4. However, the DNA polymer cannot pass through a narrow opening (opening having a small cross-sectional area) among the openings of the fine holes 3 extending from the surface of the thick part 2 to the holes 4. Therefore, when the liquid to be treated is treated using the polysulfone hollow microsphere 1 of Example 1, small molecules such as water and dioxins pass through the micropores 3 and are thicker than the pores 4 containing DNA. Although it can move freely between the surfaces of the meat part 2, the DNA contained in the pores 4 cannot pass through the micropores 3 and go out of the polysulfone hollow microspheres 1. , It remains confined in the hole 4. The diameter (average diameter) of the micropores 3 is, for example, several nm or less on the surface of the polysulfone hollow microsphere 1.
In FIG. 2 (b), the aqueous DNA solution was initially encapsulated in the polysulfone hollow microsphere 1. When the polysulfone hollow microsphere 1 is exposed to the atmosphere due to the large number of micropores 3 present in the thick wall portion 2, water evaporates from the inside of the polysulfone hollow microsphere 1 through the thick wall portion 2. A hole 4 is formed. It has been confirmed that the DNA molecules in the pores 4 remain as white dots on the inner surface of the polysulfone hollow microsphere 1 as a dry solid.
As shown in Table 1 below, the PSf concentration had a significant effect on the wall thickness and weight of the polysulfone hollow microspheres 1.

Table 1: Effect of PSf concentration on wall thickness and weight of polysulfone hollow microspheres (10 samples)
─────────────────────────────────────
PSf concentration Wall thickness Mass
(Mass%) (mm) (mg)
─────────────────────────────────────
7.5 0.089 ± 0.02 1.64 ± 0.23
10 0.142 ± 0.05 2.15 ± 0.34
12.5 0.191 ± 0.08 3.26 ± 0.50
15 0.221 ± 0.10 3.84 ± 0.65
─────────────────────────────────────
It can be seen that as the PSf concentration increases, the wall thickness and mass of the polysulfone hollow microspheres increase. Data are expressed as mean ± standard deviation.

1-2-3. 3. Amount of DNA encapsulated in PSf hollow microspheres FIG. 3 shows the DNA concentration (horizontal axis; unit mg / mL) of the DNA solution when PSf hollow microspheres were produced by changing the PSf concentration of the PSf solution. The relationship between the amount of DNA in the produced PSf hollow microspheres (vertical axis; unit mg / g) is shown. As the DNA concentration of the DNA solution used in the production of PSf hollow microspheres increased, the amount of DNA encapsulated in PSf hollow microspheres increased. When a PSf solution having a low PSf concentration (7.5% by mass) is used in the production of PSf hollow microspheres, the initial formation of PSf hollow microspheres is difficult, and DNA is easily eluted from PSf hollow microspheres. Therefore, the amount of DNA encapsulated in PSf hollow microspheres was low.
However, the amount of DNA encapsulated in the PSf hollow microspheres produced with the 10% by mass PSf solution is the same as that of the PSf hollow microspheres produced with the 12.5% by mass PSf solution. Because it was less than the weight, it was more than the amount of DNA encapsulated in PSf hollow microspheres made with 12.5 wt% PSf solution at the same DNA concentration. Comparing two types of PSf hollow microspheres, PSf hollow microspheres produced with 10% by mass PSf solution and PSf hollow microspheres produced with 12.5% by mass PSf solution, the absolute amount of encapsulated DNA is It was the same. The ratio of the amount of DNA encapsulated in the PSf hollow microspheres to the total amount of DNA used in the production of the PSf hollow microspheres was about 20%, 10% and 12% in the 7.5% by weight PSf solution, respectively. It was 80% by mass in a 0.5% by mass PSf solution.

1-2-4. Use of PSf hollow microspheres of Example 1 The microspheres of Example 1 can be used for separation and removal of dioxins. In this example, the microspheres were used to remove ethidium bromide (EB) used as a model compound for dioxins from the aqueous solution.
FIG. 4 shows an ultraviolet-visible absorption spectrum of an aqueous 10 mM ethidium bromide solution (before use (a)) and an absorption spectrum of the aqueous solution after adding and mixing the microspheres of Example 1 to the aqueous solution and allowing to stand for 24 hours. Before use (b)]. Before use (a), the 10 mM ethidium bromide aqueous solution shows a maximum absorption peak at about 480 nm. However, when the microspheres of Example 1 were added to 10 mM ethidium bromide, the maximum absorption peak disappeared.
The white microspheres of Example 1 were stained in red by the adsorption of ethidium bromide onto the surface of the microspheres after being left in the ethidium bromide solution at a constant temperature for about 30 seconds. After being allowed to stand at a constant temperature for 24 hours, ethidium bromide was intercalated into the double-stranded DNA in the microsphere, so that the color of the microsphere changed from red to salmon pink. The PSf hollow microspheres containing no DNA used for comparison had a salmon pink color when left in an ethidium bromide solution at a constant temperature.
FIG. 5 shows the case where the microspheres of Example 1 and the control microspheres (without DNA) were produced by changing the DNA concentration of the DNA solution (DNA concentrations: 0 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / The relationship between the elapsed time (horizontal axis; unit time) and the amount of ethidium bromide (EB) removed (vertical axis; unit mass%) in mL and 5 mg / mL is shown. A PSf solution having a PSf concentration of 10% by mass was used. Control microspheres without DNA (DNA concentration; 0 mg / mL) only separated very small amounts of EB, and even after 24 hours, the amount of EB separated did not exceed 8% by weight. Until the first 2 hours, there was no difference in the amount of EB removed for individual microspheres encapsulated with DNA at different concentrations, but after 2 hours the amount of DNA encapsulated In response, the amount of EB removed showed a large difference. The total amount of EB removed was dependent on the amount of encapsulated DNA. That is, the amount of EB removed after the lapse of 24 hours differs among microspheres containing different amounts of DNA individually, in 1 mg / mL, 2 mg / mL, 3 mg / mL and 5 mg / mL DNA solutions. And 61.5% by mass, 88.7% by mass, and 85.6% by mass, respectively.
In FIG. 6, when the microspheres of Example 1 were manufactured by changing the PSf concentration of the PSf solution (PSf concentration: 7.5 mass%, 10 mass%, and 12.5 mass%), the elapsed time (horizontal axis; (Unit time) and the amount of ethidium bromide (EB) removed (vertical axis; unit mass%). A DNA solution having a DNA concentration of 2 mg / mL was used. Initially, there was no significant difference in the amount of EB removed for individual microspheres produced using PSf solutions with different PSf concentrations, but the amount of EB removed after standing at constant temperature for 24 hours was , Showed significant differences between individual microspheres. The amount of encapsulated DNA was much lower in the microspheres made with the 7.5 wt% PSf solution, and therefore the amount of EB removed was also lower than the others. The amount of EB removed with microspheres made with 12.5 wt% PSf solution was slightly less than the amount of EB removed with microspheres made with 10 wt% PSf solution.

2. PSf porous microspheres 2-1. Material and preparation method thereof
2-1-1. Preparation of PSf solution and DNA solution PSf (average Mn about 26000, manufactured by Aldrich Chemical Company, Inc.) was dissolved in N-methyl-2-pyrrolidinone (NMP; HPLC grade, 99 +%, manufactured by Aldrich Chemical Company, Inc.). Thus, 8 mass%, 10 mass% and 12 mass% PSf solutions were obtained. Double stranded DNA (sodium salt, molecular weight 5 × 10 ⁶ ) from camellia white child was obtained from Yuki Fine Chemical Co., Ltd. The DNA was dissolved in distilled water. The concentrations were 5 mg / mL, 10 mg / mL and 20 mg / mL. The DNA solution was dropped into the PSf solution to obtain a mixed solution. The weight ratio of DNA to PSf was adjusted to 0.2 mass%, 0.4 mass%, and 0.8 mass%. All materials were used without further purification.

2-1-2. Production of DNA-filled PSf porous microspheres A DNA-mixed PSf solution is injected into water using an injection needle having an inner diameter of 0.4 mm or 1 mm, and stirred at about 300 rpm. The solution was allowed to stand at room temperature for 24 hours to elute the solvent in the spheroids to obtain PSf porous microspheres filled with DNA of the present invention.

前記式Ｉ及び式II中、Ｗ_B は乾燥前の微小球の質量（単位ｇ）、Ｗ_A は乾燥後の微小球の質量（単位ｇ）、ρ_W は水の密度（ρ_W ＝１．０ｇ／ｃｍ³ ）、ρ_P はポリスルホンの密度（ρ_P ＝１．２４ｇ／ｃｍ³ ）を表わす。 2-1-3. Calculation of Diameter and Porosity of PSf Porous Microspheres Filled with DNA The diameter and porosity of the microspheres of Example 2 were calculated from the following formulas (I) and (II) from the density of the polymer and the weight change before and after drying. ).

In the above formulas I and II, W _B is the mass of microspheres before drying (unit: g), W _A is the mass of microspheres after drying (unit: g), and ρ _W is the density of water (ρ _W = 1. 0 g / cm ³ ), ρ _P represents the density of polysulfone (ρ _P = 1.24 g / cm ³ ).

2-1-4. Calculation of DNA packing efficiency The amount of DNA packed in the microspheres of Example 2 was measured by the following process: the dried microspheres (10 mg) were cut into small pieces and then at 100 ° C. for 12 hours. Hydrolysis with 6M hydrochloric acid solution. The amount of DNA in the solution was quantified by absorption at 260 nm using a UV-VIS spectrophotometer U-200A (Hitachi). The efficiency of filling the microspheres with DNA was calculated as the mass ratio of the DNA extracted from the microspheres to the input DNA into this step.

2-1-5. Scanning Electron Microscope (SEM) Examination Samples of the PSf porous microspheres filled with DNA of the present invention were dried at room temperature, then cut with a single-edged razor, placed on a sample support, and coated with a gold layer. SEM images were recorded using an S-2500C microscope (voltage = 20 kV, manufactured by Hitachi).

2-1-6. Release rate of DNA In order to measure the release rate of DNA from the microspheres of Example 2 (20 mg by dry weight), analysis was performed by imitating the in vivo environment. The microspheres were immersed in 4 mL of Tris-EDTA buffer (pH = 7.5). The concentration of the released DNA was quantified by absorption at 260 nm with an ultraviolet-visible UV-VIS spectrophotometer U-200A. The percentage of released DNA was calculated as the ratio of the released DNA mass to the DNA mass in the microspheres.

2-1-7. Separation and removal of ethidium bromide by PSf porous microspheres filled with DNA An ethidium bromide (EB) aqueous solution was used in the functional verification test of PSf porous microspheres filled with DNA of the present invention. Separation of EBs in PSf porous microspheres filled with DNA was tested in the following steps: PSf porous microspheres filled with DNA (20 mg in dry mass) were put into EB aqueous solution (4 mL, 5 mM). And left at room temperature for 24 hours. EB separation was confirmed by the absorption spectrum of the EB solution in the presence or absence (control) of PSf porous microspheres loaded with DNA. The amount of EB in the solution was quantified by absorbance at 480 nm using a UV-VIS spectrophotometer U-200A (manufactured by Hitachi).

2-1-8. Separation and removal of dioxins by DNA-filled PSf porous microspheres As dioxins, an aqueous solution containing dibenzo-p-dioxin, dibenzofuran and biphenyl was used to separate them into PSf porous microspheres filled with DNA. Used as water to be tested. Saturated aqueous solutions of these reagents were used. An aqueous solution of acridine orange was also used to test functional utilization. The separation of these reagents was tested in the following steps: PSf porous microspheres (30 mg) filled with DNA were put into a saturated aqueous solution (4 mL) of each of the reagents and allowed to incubate at room temperature for 24 hours. The separation of these reagents into the microspheres was confirmed by the absorption spectrum of the aqueous solution of each of the reagents.

2-2. Results and examination 2-2-1. Production of PSf porous microspheres When a PSf solution containing DNA was added to water, liquid-liquid phase separation caused by rapid exchange of water with solvent NMP occurred, and a skin layer was formed. Subsequently, as the solvent NMP was eluted in water, porous PSf microspheres were formed, and a large number of pores existed in the microspheres.
PSf porous microspheres filled with different types of DNA were produced using an injection needle with an inner diameter of 0.4 mm or 1 mm. The diameter, porosity and DNA packing efficiency of these microspheres are shown in Table 2 below.

Table 2: PSf porous microsphere diameter, porosity and DNA packing efficiency ───────────────────────────────── ──────
Microsphere type Diameter ^* Porosity ^* DNA packing efficiency ^**
(Mm) (%) (mass%) ────────────────────────────────────────
Microsphere A-8 1.88 ± 0.2 90.1 ± 1.6 54.0 ± 5.0
Microsphere A-10 1.92 ± 0.2 87.3 ± 1.1 71.8 ± 9.4
Microsphere A-12 1.94 ± 0.2 84.0 ± 0.8 82.9 ± 10.8
Microsphere B-10 2.71 ± 0.3 87.5 ± 2.0 54.8 ± 6.0
───────────────────────────────────────
Expression in Table 2: In the microsphere XY, X represents the type of injection needle used for production. That is, when X is A, an injection needle with an inner diameter of 0.4 mm is used, and when X is B, an injection needle with an inner diameter of 1 mm is used. Y represents the PSf concentration (mass%) of the PSf solution used for production. Data are expressed as mean ± standard deviation.
^* : Number of samples 10
^** : Number of samples 3

The diameter of the microspheres depended on the inner diameter of the injection needle. When an injection needle having an inner diameter of 0.4 mm was used, the diameter of the microsphere was about 1.9 mm. When using the same needle, there was no significant difference between the microspheres produced using different solutions. When an injection needle having an inner diameter of 1 mm was used, the diameter of the microsphere was about 2.7 mm. However, the porosity of the microspheres was dependent on the PSf concentration of the PSf solution, not on the inner diameter of the injection needle. The filling efficiency of the DNA filled in the microspheres was calculated by the ratio of the amount of DNA extracted from the obtained microspheres to the initial amount of DNA used at the time of manufacture. The packing efficiency was dependent on the concentration of PSf and the diameter of the microspheres. Even with the same injection needle, the filling efficiency was different when different PSf concentrations were used. That is, the packing efficiency of DNA was 82.9 ± 10.8% by mass for microsphere-A-12, but 54.0 ± 5.0% by mass for microsphere-A-8. The microsphere-A-10 and microsphere-B-10 produced with different injection needles at the same PSf concentration also showed a significant difference in DNA loading efficiency, 71.8 ± 9.4% by mass and It was 54.8 ± 6.0% by mass.
It is difficult to manufacture the microsphere filled with a large amount of DNA. For the production of the microspheres,
DNA and PSf are dissolved in a mixed solvent containing water and NMP, but a large amount of water is required to dissolve a large amount of DNA in the mixed solvent. However, PSf does not dissolve in water. Therefore, the addition of a large amount of water to the mixed solvent causes precipitation of PSf from the mixed solvent.
The diameter of the microsphere was much larger than the diameter of the needle used to produce the microsphere. This is due to the surface tension of the polymer solution and the viscoelasticity of the polymer. The diameter was calculated not by direct measurement but by the formula (I). The microsphere was not a well-balanced sphere but an ellipsoid. Therefore, the calculated diameter is a diameter when it is regarded as a sphere. As the PSf concentration of the PSf solution during production increased, the porosity of the microspheres decreased and the pore size also decreased. The packing efficiency of DNA was higher for the microspheres with lower porosity when the diameters of the microspheres were the same. This result shows that the DNA packing efficiency depends on the porosity of the microspheres.

2-2-2. Structure of PSf porous microsphere filled with DNA FIG. 7 shows the PSf porous microsphere filled with DNA in Example 2. FIG. 7A is a front view of the PSf porous microsphere of Example 2, and FIG. 7B is a cross-sectional view taken along the line AA of FIG. 7A. In FIG. 7A, it can be seen that a large number of holes 7 are formed in the surface 6 of the polysulfone porous microsphere 5. Further, as shown in FIG. 7B, a hole 8 (for example, a diameter of about 0.1 to 0.2 mm) larger than the hole 7 on the surface 6 exists in the center of the polysulfone porous microsphere 5. To do.

2-2-3. Stability of PSf porous microspheres filled with DNA The drying temperature of the obtained PSf porous microspheres affected the stability of the DNA filled in the microspheres. When the microspheres were dried at 25 ° C., the DNA became stable, but when the microspheres were dried at 60 ° C., a large amount of DNA was eluted into the water when the microspheres were put into the water to be treated.
FIG. 8 shows the elapsed time (horizontal axis; unit time) when various PSf porous microspheres in Table 2 are added to the water to be treated, and the DNA eluted from the DNA packed in the PSf porous microspheres. The relationship with the quantity (vertical axis; unit mass%) is shown (sample number 3). In the microsphere, DNA / PSf = 0.8 mass%. The DNA packed in the microsphere A-10 and the microsphere B-10 is stable in the water to be treated, and the amount of the eluted DNA is 5% by mass or less even after standing at a constant temperature of 96 hours. I understood.
FIG. 9 shows the elapsed time (horizontal axis; horizontal axis) when the microsphere A-10 was put in a 2 mass% sodium dodecyl sulfate (SDS) solution in H ₂ O and a 0.1 M to 6 M hydrochloric acid solution. (Unit time) and the amount of DNA eluted from the DNA packed in the microsphere A-10 (vertical axis; unit mass%) is shown (number of samples: 3). When the microspheres were left at a constant temperature under acidic conditions, a large amount of DNA was eluted in the hydrochloric acid solution. After 96 hours, about 42% by mass of the 0.1M hydrogen chloride solution, about 59% by mass of the 1M hydrogen chloride solution, and about 73% by mass of the 6M hydrogen chloride solution were eluted. Further, after 96 hours, about 26% of DNA was eluted in a 2% by mass sodium dodecyl sulfate (SDS) solution. A large amount of DNA eluted into the hydrogen chloride solution, and the amount of eluted DNA increased as the hydrogen chloride concentration increased. Further, due to the surface activity of SDS, a part of the DNA was eluted in 2% SDS solution. However, almost no DNA was eluted in H ₂ O.
From the above results, the stability of the microspheres of Example 2 depends on the structure of the microspheres, and the stability of the DNA filled in the microspheres can be selected by selecting the production conditions and post-treatment conditions of the microspheres. It can be seen that can be adjusted. These results also show that the release of DNA from the microspheres of Example 2 can be controlled.
FIG. 10 shows the number of days elapsed (horizontal axis) and the DNA filled in the microspheres of Example 2 when various microspheres of Example 2 were added to Tris-EDTA buffer at pH 7.5. It shows the relationship with the amount of released DNA (vertical axis; unit is mass% with respect to the amount contained) (number of samples: 3). The release amount of the filled DNA from the microspheres depended on the structure of the microspheres and the diameter of the microspheres. Microspheres with high porosity and large pores showed a large amount of DNA release. For example, microsphere B-10 with a larger diameter showed a greater amount of DNA release than microsphere A-10. When the microsphere A-10 was dried at 60 ° C., it showed a greater DNA release than the microsphere A-10 that was not dried, and when the microsphere A-10 was dried at 25 ° C., it was The amount of DNA released was smaller than the microsphere A-10 that did not. All these microspheres released DNA continuously for 14 days. These results indicate that the amount of DNA released from the microspheres can be adjusted by selecting the structure of the microspheres of Example 2.

2-2-4. Separation and removal of ethidium bromide by PSf porous microspheres filled with DNA The PSf porous microspheres of Example 2 can be used for separation and removal of dioxins. In this example, the microspheres were used to remove ethidium bromide (EB) used as a model compound for dioxins from the aqueous solution.
FIG. 11 shows an ultraviolet-visible absorption spectrum of a 5 mM ethidium bromide aqueous solution [before use (a)] and the absorption of the aqueous solution after the porous microspheres of Example 2 were added to and mixed with the aqueous solution and left for 24 hours. The spectrum [after use (b)] is shown. Before use (a), the 5 mM aqueous ethidium bromide solution shows a maximum absorption peak at about 480 nm. However, when the PSf hollow microspheres of Example 2 were added to 5 mM ethidium bromide, the maximum absorption peak disappeared.
The white PSf hollow microspheres of Example 2 were stained in red by the adsorption of ethidium bromide on the surface of the microspheres after being left in the ethidium bromide solution at a constant temperature for about 30 seconds. After being allowed to stand at a constant temperature for 24 hours, ethidium bromide was intercalated into the double-stranded DNA in the microsphere, so that the color of the microsphere changed from red to salmon pink. The PSf hollow microspheres containing no DNA used for comparison had a salmon pink color when left in an ethidium bromide solution at a constant temperature.
FIG. 12 shows the case where the porous microspheres of Example 2 were manufactured by changing the DNA / PSf ratio (corresponding to the amount of DNA packed in the present microspheres) (DNA / PSf; 0% by mass, 0.2%). Relationship between elapsed time (horizontal axis; unit time) and amount of ethidium bromide (EB) removed (vertical axis; unit mass%). (3 samples). It can be seen that the amount of EB removed increased as the amount of DNA packed in the microspheres increased. The amount of EB removed after the lapse of 24 hours was 25.8% by mass for the present microspheres prepared with a 0% by mass DNA / PSf solution, and 0.2% by mass with the DNA / PSf solution. The microspheres were made with 54.5%, 0.4% by weight DNA / PSf solution, and the microspheres made with 71.2% by weight and 0.8% by weight DNA / PSf solution. About this microsphere, it was 91.4 mass%. The PSf concentration of the PSf solution was 10% by mass.
FIG. 13 shows the elapsed time (horizontal axis; unit time) and the amount of ethidium bromide (EB) removed (vertical axis; unit mass%) when the porous microspheres of various Examples 2 were used. Shows the relationship. The amount of EB removed after 24 hours was 94.7% by weight for microsphere A-12, 91.4% by weight for microsphere A-10, 75.6% by weight for microsphere A-8, and It was 81.3 mass% in microsphere B-10. Of the four types of microspheres, the amount of EB removed by microsphere A-12 was the highest after 24 hours. However, at the initial stage, the amount of EB removed by the microsphere A-12 was the smallest. In contrast, the amount of EB removed by microspheres A-8 was highest in the initial stage, but the amount of EB removed after 24 hours was the lowest.

2-2-5. Separation and Removal of Dioxins by PSf Porous Microspheres Filled with DNA FIG. 14 shows acridine orange (A) (a model compound of dioxins), biphenyl (B), dibenzofuran (C), and dibenzo-p-dioxin. The ultraviolet-visible absorption spectrum of each aqueous solution (D) (concentration: 5 μM / mL) [before use (a)] and the porous microspheres of Example 2 were added to and mixed with each of the aqueous solutions, and left for 24 hours. The ultraviolet-visible absorption spectrum [after use (b)] of each aqueous solution described later is shown. By using the microspheres, the absorption peaks of these compounds disappeared. Acridine orange (A), biphenyl (B) and dibenzofuran (C) were almost completely removed by the microspheres. About 80% of dibenzo-p-dioxin was removed by the microspheres by about 80% by mass.

FIG. 1 is a diagram for explaining the principle of dioxin removal by intercalating dioxins between DNA base pairs. FIG. 2 is a diagram showing polysulfone hollow microspheres encapsulating DNA in Example 1 of the present invention. FIG. 3 shows the relationship between the DNA concentration of the DNA solution and the amount of DNA in the produced microspheres of Example 1 when the microspheres of Example 1 were produced by changing the polysulfone concentration of the polysulfone solution. FIG. FIG. 4 shows an ultraviolet-visible absorption spectrum of an aqueous 10 mM ethidium bromide solution (before use (a)) and an absorption spectrum of the aqueous solution after adding and mixing the microspheres of Example 1 to the aqueous solution and leaving it for 24 hours. It is a figure which shows (b) before use. FIG. 5 is a graph showing the relationship between the elapsed time and the amount of ethidium bromide (EB) removed when the microspheres of Example 1 were produced by changing the DNA concentration of the DNA solution. FIG. 6 is a graph showing the relationship between the elapsed time and the amount of ethidium bromide (EB) removed when the microspheres of Example 1 were produced by changing the polysulfone concentration in the polysulfone solution. FIG. 7 is a diagram showing polysulfone porous microspheres filled with DNA in Example 2 of the present invention. FIG. 8 is a diagram showing the relationship between the elapsed time and the amount of DNA eluted from the DNA filled in the microspheres of Example 2 when various microspheres of Example 2 are introduced into the water to be treated. is there. FIG. 9 shows the elapsed time when the microsphere A-10 of Example 2 was put in a 2 mass% sodium dodecyl sulfate (SDS) solution in H ₂ O and a 0.1 M to 6 M hydrochloric acid solution. It is a figure which shows the relationship between the quantity of DNA eluted from DNA with which the microsphere A-10 was filled. FIG. 10 shows the number of days elapsed when the microspheres of Example 2 were put into Tris-EDTA buffer at pH 7.5, and the DNA released from the DNA filled in the microspheres of Example 2. It is a figure which shows the relationship with the quantity (vertical axis; a unit is the mass% with respect to the inclusion amount). FIG. 11 shows an ultraviolet-visible absorption spectrum of an aqueous 5 mM ethidium bromide solution (before use (a)) and an absorption spectrum of the aqueous solution after adding and mixing the microspheres of Example 2 in the aqueous solution and leaving it for 24 hours. It is a figure which shows (b)] after use. FIG. 12 is a graph showing the relationship between the elapsed time and the amount of ethidium bromide (EB) removed when the microspheres of Example 2 were produced by changing the DNA / polysulfone ratio. FIG. 13 is a diagram showing the relationship between the elapsed time and the amount of ethidium bromide (EB) removed when the microspheres of various Examples 2 are used. FIG. 14 shows an ultraviolet-visible absorption spectrum [before use (a)] of each aqueous solution of acridine orange (A), biphenyl (B), dibenzofuran (C) and dibenzo-p-dioxin (D), It is a figure which shows the ultraviolet visible absorption absorption spectrum [after use (b)] of each said aqueous solution after adding and mixing the microsphere of Example 2 to aqueous solution and leaving it to stand for 24 hours.

Explanation of symbols

1: Polysulfone hollow microsphere 2: Thick part 3: Micropore 4: Hole 5: Polysulfone porous microsphere 6: Surface 7, 8: Hole

Claims (5)

  DNA-containing polysulfone microspheres, wherein DNA is encapsulated in hollow microspheres made of polysulfone.   A microsphere made of polysulfone is composed of an outer thick part and an inner hole, and the hole is filled with DNA, and the thick part communicates the surface of the microsphere with the hole. A DNA-containing polysulfone microsphere characterized by forming a through-hole having a size capable of passing a small molecule but not allowing a DNA polymer to pass therethrough.   Separation / removal of dioxins characterized in that the DNA-containing polysulfone microspheres according to claim 1 are brought into contact with a liquid to be treated containing dioxins, and the dioxins are captured by DNA by intercalation and separated and removed. Method.   A DNA-containing polysulfone microsphere, wherein a porous microsphere made of polysulfone is filled with DNA.   Separation / removal of dioxins characterized in that the DNA-containing polysulfone microspheres according to claim 4 are brought into contact with a liquid to be treated containing dioxins, and the dioxins are captured by DNA by intercalation and separated and removed. Method.

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