JP2021153464A - Phospholipid-containing substance stabilizer and body fluid inspection kit comprising the same - Google Patents

Abstract

To provide a phospholipid-containing substance stabilizer that can stably protect a phospholipid-containing substance.SOLUTION: A phospholipid-containing substance stabilizer contains a linear molecule and γ-cyclodextrin, where a mass ratio of the γ-cyclodextrin to the linear molecule is less than 9.SELECTED DRAWING: None

Description

The present invention relates to a phospholipid-containing substance stabilizer and a body fluid test kit containing the same.

In recent years, pharmaceuticals and medical devices using biomaterials such as biopharmacy, cell therapy, and regenerative therapy have been actively developed. Through signals that the living body uses on a daily basis, such as cytokines and immune responses, these can change the administered local or whole living body into an environment suitable for treatment. Also, the change is sustainable over the long term. Therefore, it is expected to have lower side effects and long-term therapeutic effect as compared with conventional small molecule drugs and antibody drugs.

Many pharmaceuticals and medical devices that use biomaterials are unstable, and technologies for protecting them are also attracting attention.

For example, it is known that biopharmaceuticals such as antibodies are denatured by contact with silicon or vibration during transportation, which affects the drug efficacy. In addition, it is recognized that the state of therapeutic cells is important for the realization of cell therapy (Non-Patent Document 1).

Furthermore, in the field of diagnostic agents, the importance of the above technique is strongly recognized in liquid biopsy for blood. In liquid biopsy for blood, cfDNA (cell free DNA), exosomes, etc. are extracted from a small amount of blood and used for diagnosis and treatment of cancer. Specifically, its use for companion diagnostics, selection of appropriate anticancer agents, monitoring of therapeutic effects on cancer, use for early detection of recurrent cancer, etc. are being actively studied.

In particular, in cfDNA, a technique for protecting biomaterials is expected as a technique for preventing the collapse of blood cells after blood collection and improving the accuracy of examination.

Various technologies have been studied to realize these. For example, Non-Patent Document 2 considers stabilization of a liposome preparation by modifying polyethylene glycol. In Non-Patent Document 3, various reports have been made on the improvement of the stability of nucleic acid drugs. Non-Patent Document 4 reports a study on improving the stability of RNA drugs.

STACEY, G.M. et al. , 2017. Preservation and stability of cell therapy products: recommendations from expert workshop. Regenerative Medicine, 12 (5), pp. 553-564 International Journal of International Agents 19 (2002) 299-311 Nat Biotechnology. 2017 March; 35 (3): 238-248 Nucleic Acids Research, 2019, Vol. 47, No. 1,432-449

Many pharmaceuticals and medical devices using biomaterials are phospholipid-containing substances having a phospholipid membrane as a constituent, and these are generally unstable. For example, in liquid biopsy for blood, since the amount of cfDNA is very small, it is necessary to protect the blood cells after blood collection and suppress the elution of DNA from the nucleated cells. Further, since exosomes themselves disappear due to heat, freeze-thaw, container adsorption, etc., it is desired to stabilize the exosomes themselves in order to improve the quality of inspection.

As in the technique described in Non-Patent Document 2, the stability of phospholipid-containing substances is improved by using polyethylene glycol. However, a technique for further stabilizing a phospholipid-containing substance is desired.

Therefore, an object of the present invention is to provide a phospholipid-containing substance stabilizer capable of more stably protecting a phospholipid-containing substance.

The present inventors have accumulated diligent research to solve the above problems. As a result, they have found that the above-mentioned problems can be solved by using a linear molecule and γ-cyclodextrin in combination, and have completed the present invention.

That is, according to one embodiment of the present invention, a phospholipid-containing substance stabilizer containing a linear molecule and γ-cyclodextrin, wherein the mass ratio of the γ-cyclodextrin to the linear molecule is less than 9. Provided.

Further, according to another embodiment of the present invention, there is provided a phospholipid-containing substance stabilizer containing a soluble complex having a linear molecule and γ-cyclodextrin.

According to the present invention, it is possible to provide a phospholipid-containing substance stabilizer capable of more stably protecting a phospholipid-containing substance.

FIG. 1 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 1. FIG. 2 is a photograph showing the appearance of the phospholipid-containing substance stabilizer and the comparative composition prepared in Test Example 1. FIG. 3 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 2. FIG. 4 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 3. FIG. 5 is a graph showing the results of measuring the amount of hemoglobin eluted in the blood supernatant in Test Example 4. FIG. 6 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 5. FIG. 7 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 6. FIG. 8 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 7. FIG. 9 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 8. FIG. 10 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 9. FIG. 11 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 10. FIG. 12 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 11. FIG. 13 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 12. FIG. 14 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 13. FIG. 15 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 14 (vibration). FIG. 16 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 14 (impact). FIG. 17 is a graph showing the results of measuring the amount of DNA eluted in blood in Test Example 15. FIG. 18 is a graph showing the results of measuring the elution amount of the fluorescent substance from the liposome in Test Example 16 (high osmotic pressure load). FIG. 19 is a graph showing the results of measuring the elution amount of the fluorescent substance from the liposome in Test Example 16 (heat load). FIG. 20 is a graph showing the relationship between the amount of DNA measured in Test Example 17 (vertical axis) and the number of base pairs of DNA (horizontal axis).

Hereinafter, embodiments according to one embodiment of the present invention will be described. The present invention is not limited to the following embodiments.

In the present specification, "X to Y" indicating a range means "X or more and Y or less". Unless otherwise specified, the operation and physical properties are measured under the conditions of room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH.

As used herein, the stabilization of a phospholipid-containing substance means that the disruption of the structure composed of phospholipids of the phospholipid-containing substance can be suppressed even with respect to storage and an external load. For example, when the phospholipid-containing substance contains a phospholipid membrane, stabilization means that the structure of the phospholipid membrane can be maintained (disintegration can be suppressed). More specifically, when the phospholipid-containing substance is a nucleated cell (eg, leukocyte) in the blood, stabilization suppresses the disruption of the nucleated cell and thus the intracellular component (eg, leukocyte). It suppresses the release of DNA) into the blood.

<Phospholipid-containing substance stabilizer>
A first embodiment of the present invention is a phospholipid-containing substance stabilizer containing a linear molecule and γ-cyclodextrin, wherein the mass ratio of the γ-cyclodextrin to the linear molecule is less than 9.

A second form of the present invention is a phospholipid-containing substance stabilizer containing a soluble complex having a linear molecule and γ-cyclodextrin.

The mechanism by which the phospholipid-containing substance is protected by the phospholipid-containing substance stabilizer of the present invention has not been completely elucidated, but it is presumed as follows.

When a linear molecule (for example, polyethylene glycol) and γ-cyclodextrin are present in the liquid, the γ-cyclodextrin takes up the linear molecule, that is, inclusion proceeds, and the linear molecule and γ-cyclodextrin become A complex (inclusion complex) is formed. Such a complex may form a gel by forming a three-dimensional network by hydrogen-bonding the hydroxy groups of γ-cyclodextrin to each other, which may cause precipitation. Alternatively, two linear molecules may form a gel by inclusion in γ-cyclodextrin and forming a three-dimensional network. However, by using a linear molecule and γ-cyclodextrin in a predetermined mass ratio, the complex can remain soluble.

γ-Cyclodextrin has an action of interacting with a phospholipid-containing substance, for example, a material containing a phospholipid membrane, and adsorbing to the phospholipid membrane. The present inventors have confirmed that γ-cyclodextrin constituting the complex also has an action of adsorbing to a phospholipid membrane.

Therefore, the complex is adsorbed on the phospholipid membrane via γ-cyclodextrin, and as a result, linear molecules are introduced into the phospholipid membrane, so that a material containing the phospholipid membrane such as Peg-liposomes can be obtained. It is thought to stabilize. Further, it is considered that the introduction of linear molecules into the phospholipid membrane stabilizes the material containing the phospholipid membrane against an external load (vibration, impact, etc.).

The mechanism is an estimation, and the present invention is not limited by the estimation.

(Phospholipid-containing substance)
The phospholipid-containing substances according to the first and second forms of the present invention are common, and the following description is also common.

The phospholipid-containing substance is not particularly limited as long as it contains phospholipids. The phospholipid-containing substance is preferably a material containing a phospholipid membrane, and more preferably at least one selected from the group consisting of leukocytes, erythrocytes, cultured cells, liposomes and exosomes.

More specific examples of leukocytes are neutrophils, eosinophils, basophils, lymphocytes, and monocytes, and more specific examples of lymphocytes are T cells, B cells, and NK cells. More specifically, monocytes are dendritic cells, macrophages, and Langerhans cells.

(Linear molecule)
The linear molecules according to the first form and the second form of the present invention are common, and the following description is also common.

The linear molecule is not particularly limited as long as it can penetrate γ-cyclodextrin. The linear molecule is preferably polyethylene glycol (also simply referred to as "Peg" in the present specification) or polypropylene glycol (also simply referred to as "PPG" in the present specification) from the viewpoint of being able to protect phospholipid-containing substances more. ), Polyvinyl alcohol (also simply referred to as "PVA" in the present specification), and more preferably at least one selected from polyethylene glycol and propylene glycol.

The weight average molecular weight of the linear molecule is not particularly limited, but is preferably 1000 to 200,000 from the viewpoint of further protecting the phospholipid-containing substance.

When Peg is used as the linear molecule, the weight average molecular weight of Peg is more preferably 6000 to 20000, still more preferably 20000 to 100,000, and even more preferably 35,000 to 60000.

When PPG is used as the linear molecule, the weight average molecular weight of PPG is more preferably 1000 to 4000, still more preferably 2000 to 3000.

When PVA is used as the linear molecule, the weight average molecular weight of PVA is more preferably 1000 to 4000, still more preferably 1500 to 3000.

For the weight average molecular weight, a value measured by gel permeation chromatography (GPC) using polystyrene as a standard substance is adopted.

As the linear molecule, a commercially available product may be used, or a synthetic product may be used.

(Γ-Cyclodextrin)
The γ-cyclodextrin according to the first form and the second form of the present invention are common, and the following description is also common.

Cyclodextrin (CyD) is a type of cyclic oligosaccharide having a cyclic structure in which glucose is bound by an α (1 → 4) glucoside bond. Eight glucoses are bound to γ-cyclodextrin (also simply referred to as “γCyD” in the present specification).

As γCyD, a commercially available product can be used. Examples of commercially available products include Dixipal γ-100 (manufactured by Shiomizu Port Refinery Co., Ltd.).

(Contents of linear molecules and γ-cyclodextrin)
In the first form of the phospholipid-containing substance stabilizer, the mass ratio of γ-cyclodextrin to the linear molecule is less than 9. The lower limit of the mass ratio is more than 0. With such a mass ratio, the phospholipid-containing substance can be protected and stabilized. The mass ratio is preferably 0.0002 to 2.5, more preferably 0.0002 to 1.2, and even more preferably 0., from the viewpoint of further enhancing the stabilization of the phospholipid-containing substance. It is 0002 to 0.6.

(A soluble complex with a linear molecule and γ-cyclodextrin)
The second form of phospholipid-containing material stabilizer comprises a soluble complex having a linear molecule and γ-cyclodextrin.

As used herein, "soluble" means soluble in water.

As used herein, the term "complex" means a structure having a structure in which a linear molecule penetrates a ring of γCyD, and an aggregate thereof.

As described above, when a linear molecule (for example, polyethylene glycol) and γCyD are present in the liquid, γCyD takes in the linear molecule, that is, the inclusion proceeds, so that the complex of the linear molecule and γCyD (for example, Inclusion complex) can be formed.

In addition, the complex according to the second form can form a three-dimensional network to the extent that it does not form a gel. Therefore, the complex may be an aggregate of two or more complexes.

In the complex according to the second embodiment, the mass ratio of γCyD to the linear molecule is preferably more than 0 and less than 9 from the viewpoint of maintaining solubility. The mass ratio is preferably 0.0002 to 2.5, more preferably 0.0002 to 1.2, and even more preferably 0., from the viewpoint of further enhancing the stabilization of the phospholipid-containing substance. It is 0002 to 0.6. The mass ratio can be calculated from the amount of linear molecules and γCyD charged.

(Preferable embodiment)
The phospholipid-containing substance stabilizers of the first and second forms are preferably in liquid form.

As described above, when a linear molecule (for example, polyethylene glycol) and γCyD are present in the liquid, γCyD takes in the linear molecule, that is, inclusion proceeds, and a complex (inclusion) of the linear molecule and γCyD occurs. Complex) is formed. Therefore, in the first form of the phospholipid-containing substance stabilizer, at least a part of the linear molecule can form a complex with γCyD.

The solvent contained in the phospholipid-containing substance stabilizer of the present embodiment is not particularly limited, and is, for example, water (sterile water), physiological saline, PBS, citric acid buffer, Tris buffer, carbonate buffer, acetic acid buffer. Examples include liquids.

The concentration of the linear molecule in the phospholipid-containing substance stabilizer of the present embodiment is, for example, 20 to 780 mg / mL, preferably 180 to 780 mg / mL, and more preferably 300 to 780 mg / mL. Further, the lower limit of the concentration of the linear molecule may be 400 mg / mL or more or 500 mg / mL or more.

The concentration of γCyD in the phospholipid-containing substance stabilizer of the present embodiment is, for example, 0.1 to 50 mg / mL, preferably 0.15 to 25 mg / mL, and more preferably 0.3 to 15 mg / mL. It is mL. The upper limit of the concentration of γCyD may be 6 mg / mL or less, 3 mg / mL or less, 1.5 mg / mL or less, or 1 mg / mL or less.

The phospholipid-containing substance stabilizer of the present embodiment can be additionally contained in addition to the solvent, if necessary, by selecting other components. Other components include anticoagulants, tonicity agents, antioxidants, preservatives, protease inhibitors, caspase inhibitors, nuclease inhibitors, tissue fixatives and the like. Examples of these specific components include, but are not limited to, the following. Examples of the anticoagulant include ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) and a salt thereof, citric acid and a salt thereof, heparin and the like. Isotonic agents include sodium chloride, potassium chloride, calcium chloride, sodium hydrogen sulfite, xylitol, sorbitol, mannitol, glucose, glycerin, propylene glycol, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, Examples include sodium hydrogen carbonate, sodium lactate, and taurine. Examples of the antioxidant include tocopherol, ascorbic acid, glutathione and the like. Examples of protease inhibitors include phenylmethylsulfonylfloride, aminoethylbenzylsulfonylfloride, leupeptin, pepstain A, aprotinin, benzamidine and the like. Examples of preservatives include parabens, benzalkonium chloride, zenzetonium chloride, chlorhexidine and salts thereof, sodium azide, sodium fluoride and the like. Examples of the caspase inhibitor include Z-YVAD-FMK, Z-VDVAD-FMK, Z-DEVD-FMK, and Z-LEHD-FMK sold by Enzolife Sciences, but the manufacturer and product name. , Chemical structure, etc. are not limited to this. Proteinase K is exemplified as a nuclease inhibitor. Examples of the tissue fixative include formaldehyde, glutaraldehyde, diazoridinyl urea, and imidazolidinyl urea.

When the phospholipid-containing substance stabilizer of the present embodiment contains an anticoagulant, the concentration of EDTA and its salt is not particularly limited, and is, for example, 1 to 100 mg / mL. The concentration of citric acid and its salt is not particularly limited, and is, for example, 1 to 1000 mg / mL.

The temperature of the phospholipid-containing substance stabilizer of the present embodiment is not particularly limited, and may be, for example, 4 to 40 ° C.

The pH of the phospholipid-containing substance stabilizer of the present embodiment is not particularly limited and can be appropriately selected in the range of, for example, 2 to 11. The pH can be appropriately adjusted with an acid (eg, hydrochloric acid) and a base (eg, sodium hydroxide).

The osmotic pressure of the phospholipid-containing substance stabilizer of the present embodiment is not particularly limited, and is, for example, an osmotic pressure corresponding to a NaCl concentration of 1 to 2000 mM.

In this embodiment, the phospholipid-containing substance stabilizer does not include the form of a solid gel.

(Use of phospholipid-containing substance stabilizer)
The phospholipid-containing substance stabilizer of the present invention is used for protecting cultured cells, preserving iPS cells, protecting cells collected from living organisms, preserving liposome preparations, preserving exosomes, etc., in addition to the body fluid test kit described later. Can be used.

<Preparation method of phospholipid-containing substance stabilizer>
The method for preparing the phospholipid-containing substance stabilizer of the present invention is not particularly limited. When the phospholipid-containing substance stabilizer is in the form of a solution, the phospholipid-containing substance stabilizer can be prepared by mixing the above-mentioned linear molecule, γCyD, a solvent and, if necessary, other components. .. For example, as a method for preparing a phospholipid-containing substance stabilizer, a solution containing a linear molecule and a solution containing γCyD are mixed; a linear molecule is added to a solution containing γCyD and mixed; the linear molecule is mixed. ΓCyD is added to the containing solution and mixed; linear molecules and γCyD are collectively added to the solvent and mixed, and the like.

The temperature at the time of mixing is not particularly limited, and may be, for example, about 4 to 40 ° C.

<Body fluid test kit>
One embodiment of the present invention is a body fluid test kit containing the above-mentioned phospholipid-containing substance stabilizer.

Examples of body fluids include blood, urine, tears, saliva and the like. The body fluid is preferably blood.

The body fluid test kit of the present invention may contain a phospholipid-containing substance stabilizer in an amount of 3 to 130 mg per 1 mL of body fluid from the viewpoint of protecting phospholipid-containing substances (for example, nucleated cells) in the body fluid. It is preferably contained in an amount of 6 to 100 mg. Further, the body fluid test kit of the present invention uses a phospholipid-containing substance stabilizer as a body fluid, a linear molecule, and γ-cyclodextrin from the viewpoint of protecting a phospholipid-containing substance (for example, nucleated cells) in the body fluid. It is preferable to include it so that the mass ratio (body fluid: linear molecule: γCyD) with and to is 1000: 2: 1 to 1000: 125: 5, and it is contained so as to be 1000: 4: 2 to 1000: 95: 5. Is more preferable.

The body fluid test kit of the present invention can be suitably used in the field of diagnostic agents, particularly in the field of liquid biopsy. Therefore, the body fluid test kit may include a container (for example, a blood collection tube) for containing the body fluid. In this case, the phospholipid-containing substance stabilizer may be filled in a container containing the body fluid, or may be contained in a container different from the container containing the body fluid.

The present invention will be described below with reference to specific examples, but the present invention is not limited to these examples.

[Reagents, etc.]
In the examples, the following compounds were used. If the reagent name and product name are the same, the product name is omitted.

(1) Linear molecule and cyclodextrin / polyethylene glycol Mw6,000 (manufactured by Sanyo Chemical Industries, Ltd.)
-Polyethylene glycol Mw35,000 (manufactured by ChemCraz)
-Polyethylene glycol Mw40,000 (manufactured by SERVA)
-Polyethylene glycol Mw100,000 (manufactured by AlphaAesar)
-Polyethylene glycol Mw200,000 (manufactured by Sigma-Aldrich)
-Polypropylene glycol Mw2,000 (manufactured by Merck)
-Polyvinyl alcohol Mw 1,500, saponification degree 78-82% (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
-Α-Cyclodextrin (αCyD) (manufactured by Nihon Shokuhin Kako Co., Ltd .: Celdex A-100)
-Γ-Cyclodextrin (manufactured by Shiomizu Port Refinery Co., Ltd .: Dixipal γ-100).

(2) Solvent / sterilized water (manufactured by Nacalai Tesque Co., Ltd.)
-Saline solution (manufactured by Terumo Corporation).

(3) Other reagents ・ Sodium chloride (manufactured by Kanto Chemical Co., Inc.)
・ Triton X-100 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
-Hydrochloric acid (manufactured by Nacalai Tesque Co., Ltd .: 0.1 mol / L hydrochloric acid)
-Sodium hydroxide aqueous solution (manufactured by Nacalai Tesque, Inc .: 0.1 mol / L sodium hydroxide)
・ Pyranine (manufactured by Tokyo Chemical Industry Co., Ltd.)
-DPX (manufactured by Molecular Probes: p-xylene-bis-pyridinium bromide).

[Used equipment]
・ Centrifuge (Made by Kubota Seisakusho Co., Ltd .: Micro-cooled centrifuge 3740)
・ Fluorescence spectrophotometer (manufactured by JASCO Corporation: FP-6500)
・ Plate reader (Made by Molecular Device Japan: SpectraMax M5)
・ Rotator (manufactured by AS ONE Corporation: ATR280)
・ Thermomixer C (manufactured by Eppendorf)
-PH meter (manufactured by DKK-TOA CORPORATION: HM-50G).

[Sample preparation method]
<PBS>
PBS was prepared by dissolving 10 tablets of PBS Tablets (manufactured by Takara Bio Inc.) in 1000 mL of distilled water. Alternatively, Dulbeccoline phosphate buffered saline (manufactured by Nacalai Tesque, Inc.) was used.

<Citric acid buffer>
The citric acid buffer is prepared by dissolving 58.8 mg of 3Na citric acid (manufactured by Nakaraitesk Co., Ltd .: trisodium citrate dihydrate) in 10 mL of sterile water, and then adjusting the pH to 7.4 with hydrochloric acid. And prepared.

<Tris buffer>
The Tris buffer was prepared by dissolving 24.2 mg of Tris (manufactured by Nacalai Tesque, Inc .: Tris (hydroxy) aminomethane) in 10 mL of sterilized water and then adjusting the pH to 7.4 with hydrochloric acid.

<Carbonate buffer>
The carbonate buffer was prepared by dissolving 20 mg of sodium carbonate (manufactured by Kanto Chemical Co., Inc.) in 10 mL of sterilized water and then adjusting the pH to 7.4 with hydrochloric acid.

<Acetic acid buffer>
The acetate buffer was prepared by dissolving 16.4 mg of sodium acetate (manufactured by Kanto Chemical Co., Inc.) in 10 mL of sterilized water and then adjusting the pH to 7.4 with hydrochloric acid.

<EDTA solution>
The EDTA solution is prepared by dissolving EDTA-2Na (manufactured by Nakaraitesk Co., Ltd .: disodium ethylenediaminetetraacetate) in physiological saline so that the EDTA concentration is 1, 5, 10, 50 or 100 mg / mL. Prepared.

<Citric acid solution>
In the citric acid solution, citric acid-3Na (manufactured by Nakaraitesk Co., Ltd .: trisodium citrate dihydrate) is dissolved in a physiological saline solution so that the concentration of citric acid becomes 1, 10, 100 or 1000 mg / mL. And prepared.

<MES buffer>
The MES buffer solution was prepared by weighing 533 mg of MES-Na (manufactured by Merck & Co., Inc.) and 733 mg of NaCl, dissolving it in sterilized water, and adjusting the pH to 7.4 with hydrochloric acid.

<TE buffer>
The TE buffer solution was prepared by diluting a 20-fold TE buffer solution (manufactured by Promega: 20 × TE Buffer, pH 7.5) 20-fold with sterilized water.

<SYSTOX solution>
SYSTEM (Thermo Fisher Science: SYSTEM: dead cell staining) was diluted 100-fold with PBS to prepare a 10 μM SYSTEM solution.

<RPMI medium containing 10% serum>
The 10% serum-containing RPMI medium is prepared by adding Penicillin-Streptamicin Mixed Solution (manufactured by Nakaraitesk Co., Ltd.) to RPMI medium (manufactured by Nakaraitesk Co., Ltd .: RPMI1640 medium (liquid)) with penicillin (100 unit / mL) and streptomycin (manufactured by Nakaraitesk Co., Ltd.). After addition to 100 mg / mL), FBS (manufactured by Gibco: Fetal Bovine Serum, Centified, Heat Inactive, US Origin) was added to prepare the medium.

[Method for preparing phospholipid-containing substance stabilizer and comparative composition]
As the solvent, sterile water, PBS, physiological saline or the buffer prepared above was used.

<Preparation method of Peg solution>
After weighing Peg, it was dissolved with a solvent to prepare a Peg solution. The Peg concentration in the Peg solution was appropriately adjusted so as to have the composition of the phospholipid-containing substance stabilizer or the comparative composition of the test example described later.

<Preparation method of CyD solution>
After weighing γCyD, it was dissolved with a solvent to prepare a γCyD solution. The γCyD concentration in the γCyD solution was appropriately adjusted so as to be the composition of the phospholipid-containing substance stabilizer, the comparative composition, or the γCyD solution of the test example described later.

In addition, αCyD was weighed and then dissolved with a solvent to prepare an αCyD solution. The αCyD concentration in the αCyD solution was appropriately adjusted so as to have the composition of the αCyD solution of the test example described later.

<Preparation method of phospholipid-containing substance stabilizer>
After mixing the Peg solution prepared above and the γCyD solution, the mixture was allowed to stand overnight at room temperature to prepare a phospholipid-containing substance stabilizer (soluble) or a comparative composition (solid). A solvent was appropriately added so as to obtain a desired composition.

[Method for preparing phospholipid-containing substance stabilizer 2]
After mixing the linear molecule and the γCyD solution prepared above, the mixture was stirred overnight at room temperature to prepare a phospholipid-containing substance stabilizer.

[blood]
As blood, blood collected from SPF domestic pigs (purchased from Sanes Breeding Co., Ltd.) weighing 40 to 90 kg was used. Blood was collected according to the guidelines for animal experiments at Terumo Corporation.

[Measuring method]
<Method of measuring the amount of DNA eluted in blood>
The evaluation product obtained in the test example described later was centrifuged at 1000 rpm for 10 minutes to separate the supernatant and the precipitate. Further, after collecting 30 μL of the supernatant, 30 μL of the SYSTEM solution was added and allowed to stand for 5 minutes. Then, 20 μL was collected from the mixed solution of the supernatant and the SYSTEM solution and added to 2980 μL of TE buffer to prepare a sample for evaluating the fluorescence intensity. The fluorescence intensity was measured at Ex444 nm and Em480 nm using a fluorescence spectrophotometer, and the amount of DNA eluted in the blood was measured.

<Measurement method of hemoglobin absorbance in blood supernatant>
The evaluation product obtained in the test example described later was centrifuged at 1000 rpm for 10 minutes to separate the supernatant and the precipitate. 20 μL of the supernatant was collected and added to 2980 μL of TE buffer to prepare a sample. By measuring the absorbance at 576 nm using a plate reader, the amount of hemoglobin eluted in the blood supernatant due to the destruction of red blood cells was determined.

<Method of measuring the amount of DNA eluted in the supernatant>
The evaluation product obtained in the test example described later was centrifuged at 3000 rpm for 10 minutes to separate the supernatant and the precipitate. Further, after collecting 30 μL of the supernatant, 30 μL of the SYSTEM solution was added and allowed to stand for 5 minutes. Then, 20 μL was collected from the mixed solution of the supernatant and the SYSTEM solution and added to 2980 μL of TE buffer to prepare a sample for evaluating the fluorescence intensity. The fluorescence intensity was measured at Ex444 nm and Em480 nm using a fluorescence spectrophotometer, and the amount of DNA eluted in the supernatant was measured.

<Measurement method of elution fluorescent substance from liposome>
After collecting 20 to 40 μL of the evaluation product obtained in the test example described later, it was added to 2960 μL of MES buffer. Then, the fluorescence intensity was measured at Ex416 and Em512 nm using a fluorescence spectrophotometer, and the amount of the fluorescent substance eluted from the liposome was measured.

<Method of measuring the amount of DNA by electrophoresis>
The evaluation product obtained in the test example described later was centrifuged at 1900 × g for 10 minutes at 4 ° C. using a centrifuge. After centrifugation, 3 mL of the supernatant was withdrawn, collected in a new 15 mL DNA LoBind Tube (manufactured by Eppendorf), and centrifuged at 4 ° C. and 16,000 × g for 10 minutes using a centrifuge. After centrifugation, 2.4 mL of the supernatant was withdrawn and collected in a new 15 mL DNA LoBind Tube (manufactured by Eppendorf). To the collected supernatant, 72 μL of Magnetic Bead Suspension attached to QIAamp MinElute ccfDNA Kit (manufactured by QIAGEN), 132 μL of Proteinase K, and 360 μL of Bed Binding Buffer were added and mixed. The mixture was inverted and mixed at 20 rpm for 10 minutes using a rotator. Centrifuge at 200 xg for 30 seconds to remove the liquid on the cap. Then, the sample was set in a magnetic rack for 15 mL and allowed to stand for 3 minutes. After standing, all but the magnetic beads were removed using a pipette. 200 μL of Bed Elution buffer was added to the magnetic beads, and 200 μL of the liquid was collected while rinsing the wall by pipetting, and transferred to the Bed Elution Tube attached to the QIAamp MinElute ccfDNA Kit. This Bead Elution Tube was stirred at room temperature and 300 rpm for 5 minutes using a thermomixer. The bead elution tube after stirring was set in a magnetic rack for 2 mL, and then allowed to stand for 1 minute. Then, 185 μL of the supernatant was collected in a new Bead Elution Tube attached to the QIAamp MinElute ccfDNA Kit. 300 μL of Buffer ACB attached to QIAamp MinElute ccfDNA Kit was added to the collected supernatant and mixed. 460 μL of the mixed solution was added to the center of the column tube attached to the QIAamp MinElute ccfDNA Kit. The column tube was centrifuged at 6,000 xg at 4 ° C. for 1 minute using a centrifuge. After that, the column parts of the column tube were replaced with the new 2 mL Collection Tube attached to the QIAamp MinElute ccfDNA Kit. To this column tube, 500 μL of Buffer ACW2 attached to the QIAamp MinElute ccfDNA Kit was added, and the mixture was centrifuged at 4 ° C. and 6,000 × g for 1 minute using a centrifuge. After that, the column parts of the column tube were replaced with the new 2 mL Collection Tube attached to the QIAamp MinElute ccfDNA Kit. The column tube was centrifuged at 4 ° C. and 20,000 g for 3 minutes using a centrifuge. The column parts of this column tube are set in the new 1.5 mL Collection Tube attached to the QIAamp MinElute ccfDNA Kit, and the column is allowed to stand at 56 ° C. for 3 minutes in a thermomixer with the lid open to completely dry the column. rice field. 40 μL of sterilized water attached to QIAamp MinElute ccfDNA Kit was added to the center of the dried column, the lid was closed, and the mixture was allowed to stand at room temperature for 1 minute. Then, it was centrifuged at 20,000 × g for 1 minute at room temperature using a centrifuge. 4 μL of the solution obtained after centrifugation is mixed with 4 μL of a reagent for High Sensitivity D5000 (manufactured by Agilent Technologies, Inc.) and put into the solution using a fully automatic high-throughput electrophoresis system (manufactured by Agilent Technologies, Ltd .: Agilent 4200 Tape Station). The amount of DNA contained was measured.

[Test Example 1] Evaluation of Effect of Protecting Nucleated Cells in Blood According to the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition, the phospholipid-containing substance stabilizer 1 of the composition shown in Table 1 below 1-1-4 and comparative compositions 1-5-1-9 were prepared. As the solvent, sterile water was used.

Each phospholipid-containing substance stabilizer or comparative composition prepared above and blood are mixed at a volume ratio of 1: 1 and allowed to stand at 20 ° C. for 1, 4, 7 or 14 days. Obtained an evaluation product. In addition, blood (untreated) was allowed to stand at 20 ° C. for 1, 4, 7, or 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 1 and FIG. The fluorescence intensity values shown in Table 1 are relative values when the fluorescence intensity of the untreated (“No treat”) evaluation product is 100.

As shown in Table 1 and FIG. 1, the comparative compositions 1-6 to 1-8 (γCyD alone) showed values equivalent to those of untreated (Not treat) at any concentration, and γCyD alone had a value. It turns out that it has no effect on protecting nuclear cells. Moreover, since the comparative composition 1-5 (Peg alone) also showed the same value as that of the untreated, it can be seen that Peg alone has no effect of protecting nucleated cells. Furthermore, it can be seen that the comparative compositions 1-9 have a higher value than the untreated one and destroy the nucleated cells. This is because in the comparative composition 1-9, a solid gel having a large mass ratio of γCyD to Peg and thixotropy was formed (not soluble), so that the cell membrane, which is a soft material, was physically destroyed. This is probably because it has been closed (Fig. 2).

On the other hand, the phospholipid-containing substance stabilizers 1-1 to 1-4 have smaller values than the untreated and comparative compositions, and the effect of protecting nucleated cells by using Peg and γCyD in combination is effective. It can be seen that it has.

[Test Example 2] Evaluation of the effect of CyD alone on protecting nucleated cells in blood According to the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition, the CyD solution 2-1 to the composition shown in Table 2 below 2-10 was prepared. As the solvent, sterile water was used.

Each CyD solution prepared above and blood were mixed at a volume ratio of 1: 1 and allowed to stand at 20 ° C. for 14 days to obtain an evaluation product. In addition, Triton X-100 or PBS diluted 25-fold with PBS and blood were mixed at a volume ratio of 1: 1 and allowed to stand at 20 ° C. for 14 days to obtain an evaluation product. Further, blood (untreated) was allowed to stand at 20 ° C. for 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 2 and FIG. The fluorescence intensity values shown in Table 2 are relative values when the fluorescence intensity of the untreated (“No treat”) evaluation product is 100.

As shown in Table 2 and FIG. 3, γCyD showed a value equivalent to that of PBS and untreated (Not treat) at any concentration, indicating that it had no effect of protecting nucleated cells in blood. .. In addition, αCyD showed fluorescence that was slightly higher than that of PBS and untreated. It can be seen that αCyD not only has no effect of protecting nucleated cells in blood, but also has a property of destroying nucleated cells.

[Test Example 3] Evaluation of the effect of Peg weight average molecular weight and the effect of Peg concentration on the effect of protecting nucleated cells in blood According to the above-mentioned method for preparing a phospholipid-containing substance stabilizer and a comparative composition. Phospholipid-containing substance stabilizers having the compositions shown in Table 3-1 below, 3-13-9, 3-13 to 3-21, 3-25 to 3-33, 3-37 to 3-45 and 3-49. ~ 3-57 and 3-10-3-12, 3-22-3-24, 3-34-3-36, 3-46-3-48 and 3-58-3-60 were prepared. As the solvent, sterile water was used.

Each phospholipid-containing substance stabilizer or each comparative composition prepared above and blood were mixed at a volume ratio of 1: 1 and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 3-1 and Table 3-2 and FIG. The fluorescence intensity values shown in Table 3-1 are relative values when the fluorescence intensity of the evaluated product of each comparative composition is 100. The fluorescence intensity values shown in Table 3-2 are relative values when the fluorescence intensity of the untreated (“No treat”) evaluation product is 100. FIG. 4 shows the fluorescence intensity of the evaluated product of the phospholipid-containing substance stabilizer.

As shown in Table 3-1 when Peg having a weight average molecular weight of 6000, 35,000, 40,000, 100,000 or 200,000 was used, the concentration of γCyD in the phospholipid-containing substance stabilizer was 46.4 mg / mL. At both 11.6 mg / mL and 2.9 mg / mL, the amount of eluted DNA showed a small value as compared with the comparative composition (Peg alone).

In addition, the amount of eluted DNA was smaller than that of the comparative composition regardless of whether the concentration of Peg in the phospholipid-containing substance stabilizer was 40 mg / mL, 60 mg / mL, or 80 mg / mL.

From these facts, it can be seen that the phospholipid-containing substance stabilizer of the present invention can enhance the effect of protecting nucleated cells in blood by containing Peg and γCyD as compared with Peg alone.

Further, as shown in Table 3-2 and FIG. 4, the amount of eluted DNA showed a smaller value as the concentration of Peg was increased. Therefore, it can be seen that the effect of the phospholipid-containing substance stabilizer to protect nucleated cells can be further enhanced by increasing the Peg concentration in the phospholipid-containing substance stabilizer.

[Test Example 4] Evaluation of effect of protecting erythrocytes <Preparation of phospholipid-containing substance stabilizer>
According to the above-mentioned method for preparing the phospholipid-containing substance stabilizer and the comparative composition, the phospholipid-containing substance stabilizers having the compositions shown in Table 4 below are 4-1 to 4-3, 4-6 to 4-8, 4-11. To 4-13, 4-16 to 4-18 and 4-21 to 4-23 and Comparative Compositions 4-4, 4-5, 4-9, 4-10, 4-14, 4-15, 4- 19, 4-20, 4-24 and 4-25 were prepared. As the solvent, sterile water was used.

Each phospholipid-containing substance stabilizer or each comparative composition prepared above and blood were mixed at a volume ratio of 1: 1 and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product. In addition, blood (untreated) was allowed to stand at 20 ° C. for 14 days to obtain an evaluation product.

The absorbance of the obtained evaluation product was measured by the above-mentioned method for measuring hemoglobin absorbance in blood supernatant, and the amount of hemoglobin eluted in blood supernatant due to destruction of red blood cells was determined.

The results are shown in Table 4 and FIG. The absorbance values shown in Table 4 are relative values when the absorbance of the untreated (“No treat”) evaluation product is 100.

As shown in Table 4 and FIG. 5, the phospholipid-containing substance stabilizer and the comparative composition (containing only Peg) showed smaller values than the untreated (Not treat). That is, it can be seen that these have the effect of protecting red blood cells.

Comparing the phospholipid-containing substance stabilizer with the comparative composition (containing only Peg), the phospholipid-containing substance stabilizer showed a smaller value than the comparative composition (containing only Peg). From this, it can be seen that the combined use of Peg and γCyD enhances the effect of protecting erythrocytes as compared with Peg alone.

The comparative compositions 4-5, 4-10, 4-15, 4-20 and 4-25, which are solid gels having thixotropy, have larger values than the comparative compositions (including only Peg). Indicated. From this, it can be seen that when the mass ratio of γCyD to Peg is larger than a predetermined value, the effect of protecting erythrocytes is lower than that of Peg alone.

[Test Example 5] Examination of composition of phospholipid-containing substance stabilizer having enhanced protective effect on cells in blood In Test Examples 1, 3 and 4, the phospholipid-containing substance stabilizer of the present invention. Was confirmed to be more effective in protecting nucleated cells and erythrocytes than Peg alone. It was also clarified that the protective effect of the phospholipid-containing substance stabilizer of the present invention is increased by increasing the Peg concentration in the phospholipid-containing substance stabilizer. Therefore, in Test Example 5, the composition of the phospholipid-containing substance stabilizer was further investigated.

The phospholipid-containing substance stabilizers 5-1 to 5-46 having the compositions shown in Table 5 below were prepared by the above-mentioned method for preparing the phospholipid-containing substance stabilizer and the comparative composition. As the solvent, sterile water was used.

Each phospholipid-containing substance stabilizer prepared above and blood were mixed at a volume ratio of 1: 9, and allowed to stand at 30 ° C. for 7 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 5 and FIG. The fluorescence intensity values shown in Table 5 and FIG. 6 are relative values when the fluorescence intensity of the evaluation product of the phospholipid-containing substance stabilizer 5-1 is set to 100.

As shown in Table 5 and FIG. 6, it can be seen that the higher the concentration of Peg in the phospholipid-containing substance stabilizer, the smaller the value indicating the amount of eluted DNA, and the stronger the protection of nucleated cells in blood. ..

[Test Example 6] Examination of mixing ratio of phospholipid-containing substance stabilizer and blood-1
The phospholipid-containing substance stabilizer 6-1 and the comparative composition 6-2 having the compositions shown in Table 6 below were prepared by the above-mentioned method for preparing the phospholipid-containing substance stabilizer and the comparative composition. As the solvent, sterile water was used.

The phospholipid-containing substance stabilizer or comparative composition prepared above and blood are mixed in a volume ratio of 1: 1, 1: 2, 1: 4, 1: 7 or 1:19 and at 30 ° C. The product was allowed to stand for 7 days to obtain an evaluation product. Further, PBS and blood were mixed at a volume ratio of 1: 1, 1: 2, 1: 4, 1: 7 or 1:19 and allowed to stand at 30 ° C. for 7 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 6 and FIG. The fluorescence intensity values shown in Table 6 are relative values when the fluorescence intensity of the PBS evaluation product is 100.

As shown in Table 6 and FIG. 7, the phospholipid-containing substance stabilizer of the present invention has a comparative composition at a mixing ratio of 1: 1 to 1:19 with blood (phospholipid-containing substance stabilizer: blood). And it shows a value smaller than PBS, and it can be seen that it has an enhanced effect of protecting nucleated cells.

[Test Example 7] Examination of mixing ratio of phospholipid-containing substance stabilizer and blood-2
According to the method for preparing the phospholipid-containing substance stabilizer and the comparative composition described above, the phospholipid-containing substance stabilizers 7-1 to 7-4 and the comparative compositions 7-5 to 7-8 having the compositions shown in Table 7 below were added. Prepared. As the solvent, sterile water was used.

Each phospholipid-containing substance stabilizer or comparative composition prepared above and blood are mixed in a volume ratio of 1: 1, 1: 2, 1: 4, 1: 8, 1:16 or 1:32. Then, it was allowed to stand at 30 ° C. for 7 days to obtain an evaluation product. In addition, PBS and blood are mixed at a volume ratio of 1: 1, 1: 2, 1: 4, 1: 8, 1:16 or 1:32, and allowed to stand at 30 ° C. for 7 days for evaluation. Got

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 7 and FIG. The fluorescence intensity values shown in Table 7 are relative values when the fluorescence intensity of the PBS evaluation product is 100.

As shown in Table 7 and FIG. 8, the phospholipid-containing substance stabilizers 7-1 to 7-4 have a mixing ratio of 1: 1 to 1:19 with blood (phospholipid-containing substance stabilizer: blood). , It can be seen that it has the effect of protecting nucleated cells in blood.

Further, it can be seen that by increasing the concentrations of Peg and CyD in the phospholipid-containing substance stabilizer, the amount of eluted DNA can be suppressed to a low level, and nucleated cells in the blood can be more strongly protected.

[Test Example 8] Evaluation of Effect of Solvent on Protective Effect of Phospholipid-Containing Substance Stabilizer Table 8 below shows that different solvents were used depending on the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition. A phospholipid-containing substance stabilizer 8-1 having a composition and comparative compositions 8-2 to 8-3 were prepared. As the solvent, sterile water, PBS, physiological saline, citric acid buffer, Tris buffer, carbonate buffer or acetate buffer was used.

The phospholipid-containing substance stabilizer or comparative composition prepared above and blood were mixed at a volume ratio of 1: 9 and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 8 and FIG. The fluorescence intensity values shown in Table 8 are relative values when the fluorescence intensity of the evaluation product of the comparative composition 8-2 using sterile water is set to 100.

As shown in Table 8 and FIG. 9, the phospholipid-containing substance stabilizer of the present invention can exhibit a high protective effect regardless of the solvent used for preparation, regardless of which solvent is used. I understand.

[Test Example 9] Evaluation of the effect of pH <Preparation of phospholipid-containing substance stabilizer>
The phospholipid-containing substance stabilizer 9-1 and the comparative compositions 9-2 to 9-3 having the compositions shown in Table 9 below were prepared by the above-mentioned method for preparing the phospholipid-containing substance stabilizer and the comparative composition. The phospholipid-containing material stabilizer and comparative composition were then adjusted to pH 2, 3, 4, 6, 9, 10 or 11 with hydrochloric acid or sodium hydroxide. A physiological saline solution was used as the solvent.

The phospholipid-containing substance stabilizer or comparative composition prepared above and blood were mixed at a volume ratio of 1: 9 and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product. Further, a physiological saline solution adjusted to pH 6.0 and blood were mixed at a volume ratio of 1: 9, and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 9 and FIG. The fluorescence intensity values shown in Table 9 are relative values when the fluorescence intensity of the evaluation product of the physiological saline solution is 100.

As shown in Table 9 and FIG. 10, the phospholipid-containing substance stabilizer 9-1 suppressed the elution of DNA lower than that of the comparative composition (Peg alone or γCyD alone) at both pHs 2 to 11. I understand. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention exhibits a high effect of protecting nucleated cells without being affected by the pH at the time of preparation.

[Test Example 10] Evaluation of the effect of osmotic pressure <Preparation of phospholipid-containing substance stabilizer>
Depending on the method for preparing the phospholipid-containing substance stabilizer and the comparative composition described above, the phospholipid-containing substance stabilizer 10-1 and the comparative composition 10-2 to 10- having the compositions shown in Table 10 below, which contain different concentrations of NaCl. 3 was prepared. As the solvent, sterile water or physiological saline containing sodium chloride (200, 500 or 1000 mM) was used.

The phospholipid-containing substance stabilizer or comparative composition prepared above and blood were mixed at a volume ratio of 1: 9 and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 10 and FIG. The fluorescence intensity values shown in Table 10 are relative values when the fluorescence intensity of the evaluation product of the comparative composition 10-2 (NaCl concentration: 0 mM) prepared using sterile water is set to 100.

As shown in Table 10 and FIG. 11, the phospholipid-containing substance stabilizer 10-1 is more than the comparative composition (Peg alone or γCyD alone) even at an osmotic pressure corresponding to a NaCl concentration of 200 to 1000 mM. It can be seen that the elution of DNA was suppressed to a low level. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention exerts a high effect of protecting nucleated cells at an osmotic pressure corresponding to a NaCl concentration of at least 1000 mM.

[Test Example 11] Evaluation of the effect of temperature during preparation According to the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition, the phospholipid-containing substance stabilizer 11-1 and the comparative composition having the compositions shown in Table 11 below Objects 11-2 to 11-3 were prepared at different temperatures. The phospholipid-containing substance stabilizer was prepared by allowing it to stand overnight at 4 ° C., 20 ° C. or 40 ° C. instead of room temperature. Moreover, the comparative composition was prepared so as to be 4 degreeC, 20 degreeC or 40 degreeC. A physiological saline solution was used as the solvent.

Each phospholipid-containing substance stabilizer or each comparative composition prepared above was mixed with blood at a volume ratio of 1: 9, and allowed to stand at 30 ° C. for 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 11 and FIG. The fluorescence intensity values shown in Table 11 are relative values when the fluorescence intensity of the evaluation product of the comparative composition 11-2 at 20 ° C. is set to 100.

As shown in Table 11 and FIG. 12, even when the temperature at the time of preparation was 4 to 40 ° C., the phospholipid-containing substance stabilizer 11-1 was more DNA than the comparative composition (Peg alone or γCyD alone). It can be seen that the elution was suppressed to a low level. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention exhibits a high effect of protecting nucleated cells at 4 to 40 ° C. without being affected by the temperature at the time of preparation.

[Test Example 12] Evaluation of Effect of EDTA Concentration According to the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition, the phospholipid-containing substance stabilizers 12-1 to 12-6 having the compositions shown in Table 12 below and Comparative compositions 12-7-12-18 were prepared. As the solvent, sterilized water or an EDTA solution (EDTA concentration: 1, 5, 10, 50 or 100 mg / mL) was used.

In addition, sterile water and an EDTA solution (EDTA concentration: 1, 5, 10, 50 or 100 mg / mL) were used as comparative compositions 12-19 to 12-24.

The phospholipid-containing substance stabilizer or comparative composition prepared above was mixed with blood at a volume ratio of 1: 9, and allowed to stand at 30 ° C. for 7 days to obtain an evaluation product. The blood used was collected using a syringe instead of a blood collection tube, and care was taken not to contain EDTA.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results at each EDTA concentration are shown in Table 12 and FIG. The fluorescence intensity values shown in Table 12 are relative values when the fluorescence intensities of the evaluation products of the comparative compositions 12-7 to 12-12 are set to 100, respectively.

EDTA is often used as an anticoagulant for blood.

As shown in Table 12 and FIG. 13, even at an EDTA concentration of 1 to 100 mg / mL, the phospholipid-containing substance stabilizer showed a lower value than the comparative composition (γCyD alone and Peg alone), and the elution of DNA. It can be seen that was kept low. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting nucleated cells regardless of the concentration of EDTA.

[Test Example 13] Evaluation of the effect of citric acid concentration <Preparation of phospholipid-containing substance stabilizer>
According to the method for preparing the phospholipid-containing substance stabilizer and the comparative composition described above, the phospholipid-containing substance stabilizer 13-1 to 13-4 and the comparative composition 13-5 to 13-12 having the compositions shown in Table 13 below were added. Prepared. As the solvent, a citric acid solution (citric acid concentration: 1, 10, 100 or 1000 mg / mL) was used.

Further, a citric acid solution (citric acid concentration: 1, 10, 100 or 1000 mg / mL) was used as a comparative composition 13-13 to 13-16.

Each phospholipid-containing substance stabilizer or each comparative composition prepared above was mixed with blood at a volume ratio of 1: 9, and allowed to stand at 30 ° C. for 7 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results at each citric acid concentration are shown in Table 13 and FIG. The fluorescence intensity values shown in Table 13 are relative values when the fluorescence intensities of the evaluation products of the comparative compositions 12-5 to 12-8 are set to 100, respectively.

Citric acid is often used as an anticoagulant for blood.

As shown in Table 13 and FIG. 14, even at a citric acid concentration of 1 to 1000 mg / mL, the phospholipid-containing substance stabilizer showed a lower value than that of the comparative composition (γCyD alone and Peg alone), and showed a value of DNA. It can be seen that the elution was suppressed to a low level. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting nucleated cells regardless of the concentration of citric acid.

[Test Example 14] Evaluation of Effect of External Load According to the method for preparing the phospholipid-containing substance stabilizer and the comparative composition described above, the phospholipid-containing substance stabilizer 14-1 and the comparative composition 14 having the compositions shown in Table 14 below −2 to 14-3 were prepared. As the solvent, sterile water was used.

The phospholipid-containing substance stabilizer prepared above, each comparative composition or sterilized water and blood are mixed at a volume ratio of 1: 9, and in the case of (A) evaluation for vibration, a vibration tester (AS ONE Co., Ltd.) Manufactured by: Environmental Vibration Tester CV-101M), vibration in sweep mode, 10-100 Hz, acceleration 2 G, was applied at 30 ° C. for 3, 5 or 24 hours; and (B). In the case of evaluation for impact, a shaker (manufactured by Taitec Co., Ltd .: BioSnaker VBR-36) was used to add an evaluation product at 450 rpm for 0 to 3 hours at 30 ° C.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in blood, and the amount of eluted DNA in blood was measured.

The results are shown in Table 14, FIG. 15 (vibration) and FIG. 16 (impact). The fluorescence intensity values shown in Table 14 are relative values when the fluorescence intensity of the evaluated product to which sterilized water is added is 100.

(Evaluation for vibration)
As shown in Table 14 and FIG. 15, the phospholipid-containing substance stabilizer 14-1 showed lower values than the comparative compositions 14-2 and 14-3 (Peg alone and γCyD alone), causing DNA elution. You can see that it was kept low. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting nucleated cells from an external load of vibration.

(Evaluation for impact)
As shown in Table 14 and FIG. 16, the phospholipid-containing substance stabilizer 14-1 showed lower values than the comparative compositions 14-2 and 14-3 (Peg alone and γCyD alone), causing DNA elution. You can see that it was kept low. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting nucleated cells from an external load of impact.

[Test Example 15] Evaluation of Effect of Protecting Cultured Cells According to the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition, the phospholipid-containing substance stabilizer 15-1 and the comparative composition having the compositions shown in Table 15 below are used. Objects 15-2 to 15-3 were prepared. As the solvent, sterile water was used.

As the cultured cells, EG7 cells purchased from ATCC were used. Culturing of EG7 cells was performed using RPMI medium containing 10% serum. In addition, EG7 cells were cultured using an incubator (MCO20AIC, manufactured by Mitsubishi Electric Engineering Co., Ltd.) set at _{5% CO 2 and 37 ° C.}

After collecting the cultured EG7 cells, the cells were washed twice with PBS. Then, the cells were dispersed using PBS so that the cell density was 200,000 cells / mL, and a cell suspension was obtained.

The phospholipid-containing substance stabilizer or comparative composition prepared above and the cell suspension are mixed at a volume ratio of 1: 1 and allowed to stand at 30 ° C. for 1 or 5 hours to prepare the evaluation product. Obtained. In addition, the cell suspension (untreated) was allowed to stand at 30 ° C. for 1 or 5 hours to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the amount of eluted DNA in the supernatant, and the amount of eluted DNA in the supernatant was measured.

The results are shown in Table 15 and FIG. The fluorescence intensity values shown in Table 15 are relative values when the fluorescence intensity of the untreated (“Cell only”) evaluation product is 100.

As shown in Table 15 and FIG. 17, the phospholipid-containing substance stabilizer 15-1 showed a lower value than the untreated and comparative compositions, indicating that DNA release associated with the destruction of cultured cells was small. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting cultured cells. Further, the phospholipid-containing substance stabilizer of the present invention is expected to be applied to cell therapy, preservation of iPS cells, and the like.

[Test Example 16] Evaluation of Effect of Protecting Liposomes According to the method for preparing the above-mentioned phospholipid-containing substance stabilizer and comparative composition, the phospholipid-containing substance stabilizer 16-1 to 16-2 having the composition shown in Table 16 below And comparative compositions 16-3 to 16-4 were prepared. The solvent used was physiological saline.

3000 nmol EYPC (egg yolk phosphatidylcholine) (manufactured by NOF Corporation: COATSOME NC-50) dissolved in chloroform (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was measured in a 10 mL eggplant flask and made into a thin film using a rotary evaporator. .. After adding 500 μL of Pyranine solution (Pyranine: 35 mM, DPX: 50 mM, MES: 25 mM, pH 7.4), it was dispersed using an ultrasonic irradiation device (USC-J). Further, the dispersion liquid was passed through a polycarbonate film having a pore size of 100 nm by an extruder to make the particle size uniform. The outer aqueous layer was replaced with a MES buffer solution and a G100 column to obtain an EYPC liposome dispersion liquid containing Pyraine, which is a fluorescent substance. Then, the concentration of phospholipid was determined using phospholipid C-Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.), and the concentration was adjusted with MES buffer so that the phospholipid was 1.0 mmol / L. Lipid suspensions were prepared.

The phospholipid-containing substance stabilizer or comparative composition prepared above was mixed with the liposome suspension at a volume ratio of 1: 1 to obtain a mixed solution.

In the experiment with high osmotic pressure load, the mixed solution and 4000 mM NaCl solution were mixed at a volume ratio of 1: 1 and allowed to stand at 30 ° C. for 1 hour to obtain an evaluation product. In addition, the liposome suspension (untreated) was allowed to stand at 30 ° C. for 1 hour to obtain an evaluation product.

In the heat loading experiment, the mixed solution was allowed to stand at 30 ° C. for 2 hours, 4 hours, 3 days, 7 days or 14 days to obtain an evaluation product. In addition, the liposome suspension (untreated) was allowed to stand at 30 ° C. for 2 hours, 4 hours, 3 days, 7 days or 14 days to obtain an evaluation product.

With respect to the obtained evaluation product, the fluorescence intensity was measured by the above-mentioned method for measuring the fluorescent substance eluted from the liposome, and the amount of the fluorescent substance eluted from the liposome was measured.

The results are shown in Table 16 and FIG. 18 (high osmotic load) and FIG. 19 (heat load). The fluorescence intensity values shown in Table 16 are relative values when the fluorescence intensity of the untreated (“liposomes only”) evaluation product is 100.

As shown in Table 16 and FIGS. 18 and 19, the phospholipid-containing substance stabilizer 16-1 showed lower values than the comparative compositions 16-3 and 16-4 (Peg alone and γCyD alone), and liposomes. It can be seen that the elution of the fluorescent substance from the above was suppressed to a low level. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting liposomes, and further has a high effect of protecting liposomes from high osmotic load and heat load.

[Test Example 17] Measurement of DNA amount by electrophoresis According to the above-mentioned method 2 for preparing a phospholipid-containing substance stabilizer and the method for preparing a comparative composition, the phospholipid-containing substance stabilizer 17-1 having the composition shown in Table 17 below. ~ 17-7 and comparative compositions 17-8-17-9 were prepared. As the solvent, sterile water was used.

0.86 mL of a phospholipid-containing substance stabilizer or comparative composition was added to the blood collection tube. 6 mL of blood was added to the blood collection tube, and the mixture was overturned and mixed after being plugged. The overturned mixed sample was placed in a constant temperature bath at 25 ° C. and stored for 7 days to prepare an evaluation product.

In addition, 0.86 mL of 0.9 w / v% physiological saline (reference composition 17-10) was added to the blood collection tube. 6 mL of blood was added to the blood collection tube, and the mixture was overturned and mixed after being plugged. The overturned mixed sample was placed in a constant temperature bath at 4 ° C. and stored for 7 days to prepare an evaluation product.

The amount of DNA of the obtained evaluation product was measured by the above-mentioned method for measuring the amount of DNA by electrophoresis.

The results are shown in FIG. 20 and Table 17. The horizontal axis of FIG. 20 shows the number of base pairs of DNA, and the vertical axis of FIG. 20 shows the amount of DNA. Further, the peaks of 15 bp and 10,000 bp in FIG. 20 mean the Lower marker and the Upper marker, respectively, and are not DNA peaks.

As shown in FIG. 20, the peak height of the phospholipid-containing substance stabilizer 17-1 was lower than that of the comparative compositions 17-8 and 17-9. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention can suppress the release of DNA derived from leukocytes.

Further, as shown in Table 17, the amount of DNA when the phospholipid-containing substance stabilizer 17-5 to 17-7 was used was the amount of DNA when the reference composition 17-10 stored at 4 ° C. was used. Was lower than. Therefore, it can be seen that the phospholipid-containing substance stabilizer of the present invention can suppress the release of DNA derived from leukocytes.

[Test Example 18] Evaluation of effect of protecting erythrocytes <Preparation of phospholipid-containing substance stabilizer>
According to the above-mentioned method 2 for preparing the phospholipid-containing substance stabilizer and the method for preparing the comparative composition, the phospholipid-containing substance stabilizers 18-1 and 18-3 having the compositions shown in Table 18 below and the comparative composition 18-2 and 18-4 was prepared. As the solvent, sterile water was used.

Each phospholipid-containing substance stabilizer or each comparative composition prepared above and blood were mixed at a volume ratio of 1: 1 and allowed to stand at 25 ° C. for 7 days to obtain an evaluation product. In addition, blood (untreated) was allowed to stand at 4 ° C. for 7 days to obtain an evaluation product.

The absorbance of the obtained evaluation product was measured by the above-mentioned method for measuring hemoglobin absorbance in blood supernatant, and the amount of hemoglobin eluted in blood supernatant due to destruction of red blood cells was determined.

The results are shown in Table 18.

As shown in Table 18, the phospholipid-containing substance stabilizers 18-1 and 18-3 showed smaller values than the comparative compositions 18-2 (PPG only) and 18-4 (PVA only), respectively. From this, it can be seen that the combined use of the linear molecule and γCyD enhances the effect of protecting erythrocytes as compared with the linear molecule alone.

[Test Example 19] Evaluation of exosome-protecting effect According to the above-mentioned method 2 for preparing a phospholipid-containing substance stabilizer and the method for preparing a comparative composition, the phospholipid-containing substance stabilizer 19-1 having the composition shown in Table 19 below. And comparative composition 19-2 (sterile water only) was prepared. As the solvent, sterile water was used. In addition, 30 μg of Lyophilized exosomes from Plasma of Health donors (manufactured by HansaBioMed) was diluted with 100 μL of sterile water. This was used as an exosome solution.

The phospholipid-containing substance stabilizer or comparative composition prepared above and the exosome solution were mixed at a volume ratio of 1: 1. 80 μL of these was added to a 2 mL polypropylene tube. Then, the mixture was stirred for 3 minutes, and the whole amount was transferred to a new 2 mL polypropylene tube. This was repeated up to 20 times. Then, the amount of remaining exosomes was quantified using CD9 / CD63 Exosome ELISA Kit, Human (manufactured by Cosmo Bio Co., Ltd.).

The results are shown in Table 19.

As shown in Table 19, the phospholipid stabilizer showed a larger value than the comparative composition. From this, it can be seen that the phospholipid-containing substance stabilizer of the present invention has a high effect of protecting exosomes.

Claims (10)

A phospholipid-containing substance stabilizer containing a linear molecule and γ-cyclodextrin, wherein the mass ratio of the γ-cyclodextrin to the linear molecule is less than 9. The phospholipid-containing substance stabilizer according to claim 1, wherein the mass ratio of the γ-cyclodextrin to the linear molecule is 0.0002 to 2.5. A phospholipid-containing substance stabilizer comprising a soluble complex having a linear molecule and γ-cyclodextrin. The phospholipid-containing substance stabilizer according to any one of claims 1 to 3, wherein the linear molecule is at least one selected from polyethylene glycol, polypropylene glycol, and polyvinyl alcohol. The phospholipid-containing substance stabilizer according to any one of claims 1 to 4, wherein the linear molecule has a weight average molecular weight of 1000 to 200,000. The phospholipid-containing substance stabilizer according to any one of claims 1 to 5, wherein the phospholipid-containing substance is at least one selected from the group consisting of leukocytes, erythrocytes, cultured cells, liposomes and exosomes. A body fluid test kit comprising the phospholipid-containing substance stabilizer according to any one of claims 1 to 6. The body fluid test kit according to claim 7, wherein the body fluid is blood. The body fluid test kit according to claim 7 or 8, wherein the phospholipid-containing substance stabilizer is contained in an amount of 3 to 130 mg per 1 mL of the body fluid. Claims 7 to 9 include the phospholipid-containing substance stabilizer so that the mass ratio of the body fluid, the linear molecule, and the γ-cyclodextrin is 1000: 2: 1 to 1000: 125: 5. The body fluid test kit according to any one of the above.

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