Method for loading useful materials into carrier

Abstract

The present invention relates to a method for loading useful materials into a carrier, the method enabling the loading efficiency of useful materials into a carrier such as an exosome to be maximized by utilizing ultrasonic waves, thereby being utilizable for a technique for effectively delivering various useful materials such as deoxyribonucleic acids, proteins, and nanomaterials to a target cell.

Description

Method for loading useful substances into a carrier

[0001] The present invention relates to a method for loading a useful substance into a carrier.

[0002]

[0003] Many attempts have been made to load various substances such as nucleic acids, proteins, and drugs into carriers such as exosomes, and representative methods include 'Incubation', 'Electroporation', 'Extrusion', 'Freeze-Thaw Cycle', 'Chimeric Exosome Method', 'Endogenous loading', and 'Sonication'. According to the research, in the case of protein catalase, the material loading efficiency through ultrasound was approximately 26 ± 1.2%, which is slightly higher than other methods such as extrusion (22 ± 3.1%), surfactant treatment (18.5 ± 1.3%), freeze-thaw treatment (14.7 ± 1.1%), and incubation (4.9 ± 0.5%). It was also reported that the loading of chemotherapeutic agents through sonication (28.29 ± 1.38%) showed higher efficiency than electroporation (5.30 ± 0.48%). Nanomaterials (gold nanomaterials) can also be loaded with high efficiency (19.3 ± 10%) through sonication (incubation; 13.7 ± 9.9%).

[0004] However, the efficiency of loading useful substances into exosomes using ultrasound is approximately 19.3-28.3%, which is higher than other methods, but remains below 30%. Therefore, further improvement in loading efficiency is necessary. Increasing loading efficiency not only offers significant benefits in future processes and costs, but also offers the significant advantage of maximizing the effect on recipient cells even with small quantities. Therefore, we are conducting research to optimize ultrasound loading methods for use as useful proteins in various cosmetics, health functional foods, and medical devices, and to explore ways to maximize loading efficiency.

[0005]

[0006] The technical problem to be achieved by the present invention is to provide a method for loading a useful substance into a carrier.

[0007]

[0008] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0009]

[0010] In order to achieve the above technical task, one embodiment of the present invention provides a method for loading a useful substance onto a carrier, the method comprising the steps of: providing ultrasonic stimulation to a carrier including a phospholipid layer and a useful substance respectively; and culturing a mixture of the ultrasonically stimulated carrier and the useful substance for a predetermined period of time.

[0011] In an embodiment of the present invention, the carrier may be an exosome.

[0012] In an embodiment of the present invention, the carrier may be an exosome derived from human fibroblasts (HDFs).

[0013] In an embodiment of the present invention, the exosome may be manufactured by a method including the steps of providing ultrasound stimulation to human fibroblast-derived cells and providing ultrasound stimulation to a cell-free culture medium; culturing a mixture of the ultrasound-stimulated human fibroblast-derived cells and the ultrasound-stimulated culture medium for a predetermined period of time; and isolating exosomes from the mixture.

[0014] In an embodiment of the present invention, the useful material may be a nucleic acid, a protein, a peptide, a nanomaterial, a plasmid DNA, a low molecular weight compound, a drug, a hormone, an enzyme, or a neurotransmitter.

[0015] In an embodiment of the present invention, the step of providing ultrasonic stimulation to the carrier may be performed at an intensity of 20% to 40% of AMP for 1 to 10 seconds.

[0016] In an embodiment of the present invention, the step of providing ultrasonic stimulation to the useful substance may be performed at an intensity of 15% to 30% of AMP for 0.1 to 5 seconds.

[0017] In an embodiment of the present invention, when the useful material is plasmid DNA, ultrasonic stimulation may be performed at an intensity of 15% to 25% of AMP for 0.1 to 3 seconds.

[0018] In an embodiment of the present invention, when the useful material is a protein, nucleic acid or nanomaterial, the ultrasonic stimulation may be performed at an intensity of 20% to 30% of AMP for 1 to 5 seconds.

[0019] In an embodiment of the present invention, the step of culturing the mixture may be performed at 30°C to 40°C for 30 minutes to 2 hours.

[0020]

[0021] The present invention relates to a method for loading a useful substance into a carrier, and can maximize the loading efficiency of a useful substance into a carrier such as an exosome by utilizing ultrasound, and can be utilized in a technology for effectively delivering various useful substances such as deoxyribonucleic acid, proteins, and nanomaterials to target cells.

[0022]

[0023] The effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0024]

[0025] Figure 1 compares the loading efficiency of a mixture of sonicated exosomes and useful substances according to culture conditions.

[0026] Figure 2 shows the efficiency of loading useful substances when ultrasonic waves were applied once for 5 seconds at an intensity of 30% AMP under conditions of 1 hour of incubation at 37°C.

[0027] Figure 3 shows the loading efficiency of useful substances when ultrasonic treatment was performed once for 1 second at 20% AMP intensity under the conditions of 1 hour incubation at 4°C. Ultrasonic treatment conditions (useful substance: 20% AMP, 1 second, 1 time / exosome: 30% AMP, 5 seconds, 1 time), exosome particle number: 2x10 8 The particles / sample and useful material loading amounts are as shown in Table 1.

[0028] Figure 4 shows the loading efficiency of useful substances when ultrasonic treatment was performed once for 1 second at 20% AMP intensity under the conditions of 1 hour incubation at 37°C. Ultrasonic treatment conditions (useful substance: 20% AMP, 1 second, 1 time / exosome: 30% AMP, 5 seconds, 1 time), exosome particle number: 2x10 8 The particles / sample and useful material loading amounts are as shown in Table 1.

[0029] Figure 5 compares the loading efficiency of useful substances (Q-dots) depending on whether or not ultrasonic treatment was performed. Number of exosome particles: 2x10 8 particles / sample, Q-dot 705 loading amount: 1 nmole (0.01 μl), ultrasonic conditions: AMP 30%, 5 seconds, 1 time, incubation conditions: 37°C, 1 hour, Did staining: 37°C, 30 minutes.

[0030] Figures 6 and 7 show damage to useful substances (IgG and Cy3-DNA) according to ultrasonic treatment conditions.

[0031] Figure 8 shows whether plasmid DNA is loaded into exosomes.

[0032] Figure 9 shows the results of confirming whether plasmid DNA is damaged according to ultrasonic conditions.

[0033] Figures 10 to 13 show the results of confirming whether useful substances loaded on exosomes are delivered to cells.

[0034]

[0035] Hereinafter, the present invention will be described in detail.

[0036]

[0037] The present invention relates to a method for loading a useful substance into a carrier.

[0038] The present invention comprises a step of providing ultrasonic stimulation to a carrier and a useful substance including a phospholipid layer, respectively; and a step of culturing a mixture of the ultrasonically stimulated carrier and the useful substance for a predetermined period of time.

[0039] The carrier includes a phospholipid layer, and as long as a core is formed inside the phospholipid layer and a useful substance can be loaded (loaded) into the core, the type and form thereof are not limited. For example, it may be an exosome, and a more specific example may be an exosome derived from human fibroblasts (HDFs).

[0040] The exosomes are separated from cells and can be separated using known means using known methods and conditions. For example, the exosomes may be prepared by a method including the steps of: providing ultrasound stimulation to human fibroblast (HDF)-derived cells and providing ultrasound stimulation to a cell-free culture medium; culturing a mixture of the ultrasound-stimulated human fibroblast-derived cells and the ultrasound-stimulated culture medium for a predetermined period of time; and isolating exosomes from the mixture.

[0041] Ultrasound stimulation of the HDF-derived cells can be performed by directly sonicating the cells or by placing the cells in a minimal amount of initial culture medium, barely covering them. The initial culture medium used in this case is a common medium used to maintain the cells in a healthy state, such as DMEM medium containing antibiotics and serum.

[0042] The type of the above cell-free culture medium is not particularly limited, and may be, for example, any one of embryonic stem cell medium, neural stem cell medium, cardiac stem cell medium, dermal papilla cell medium, mesenchymal stem cell medium, osteogenic medium, myogenic medium, hematopoietic stem cell medium, neuron medium, astrocyte medium, oligodendrocyte medium, hepatocyte medium, adipocyte medium, muscle cell medium, vascular endothelial cell medium, pancreatic beta cell medium, or cardiomyocyte medium.

[0043] The ultrasound stimulation provided to the above cells is, for example, 10 to 30 KHz, 0.5 to 3 W / cm 2 It may be performed for 1 to 10 seconds, preferably 15 to 25 KHz, 0.5 to 1.5 W / cm 2 It may be performed for 3 to 7 seconds.

[0044] The ultrasonic stimulation provided to the above cell-free culture medium is 10 to 30 KHz, 1 to 20 W / cm 2 It may be performed for 1 to 20 minutes, preferably 15 to 25 KHz, 1 to 10 W / cm 2 It may be performed for 5 to 15 minutes.

[0045] The culture of the mixture of the ultrasound-stimulated human fibroblast-derived cells and the ultrasound-stimulated culture medium can be performed using known methods and conditions using known means. For example, the culture can be performed for 1 to 10 days, 1 to 6 days, or 1 to 2 days. Since exosomes are secreted in the highest amount on the first day after ultrasound treatment and the amount of secretion decreases over time, the culture time can be appropriately selected within the above range.

[0046] The step of isolating exosomes from the above mixture can be performed using known means and under known methods and conditions. For example, the step may include: centrifuging the mixture after the culture to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and concentrating the filtrate.

[0047] The above centrifugation is performed to remove cell debris and dead cells, and may preferably be performed at 1000 g to 5000 g for 10 to 60 minutes.

[0048] The step of filtering the above supernatant through a filter is performed to further remove cell debris and leave only particles smaller than a certain size. The filter used here may preferably be a syringe filter.

[0049] The step of concentrating the filtrate can preferably be performed using a centrifugal filter. Using a centrifugal filter can simultaneously concentrate the filtrate and remove particles below a certain size.

[0050] The step of isolating the exosomes may, if necessary, additionally include a step of storing the supernatant at 4°C or lower for 7 days to 3 months before filtering it. The storage is preferably performed at 4°C or lower for no more than 7 days, more preferably at -20°C or lower for no more than 1 month, and most preferably at -80°C or lower for no more than 3 months.

[0051] The above-mentioned useful substances are physiologically active substances and may include any substance that promotes or inhibits biological functions. Examples include nucleic acids, proteins, peptides, nanomaterials, plasmid DNA, small molecule compounds, drugs, hormones, enzymes, or neurotransmitters.

[0052] The step of providing ultrasonic stimulation to the carrier may be performed, for example, at an intensity of 20% to 40% of AMP for 1 to 10 seconds. Preferably, it may be performed at an intensity of 25% to 35% of AMP for 3 to 7 seconds.

[0053] The step of providing ultrasonic stimulation to the above useful substance may be performed, for example, at an intensity of 15% to 30% of AMP for 0.1 second to 5 seconds or at an intensity of 20% to 30% of AMP for 1 second to 5 seconds.

[0054] For a more specific example, if the useful substance is plasmid DNA, the ultrasonic stimulation may be performed at an intensity of 15% to 25% AMP for 0.1 to 3 seconds. Preferably, the ultrasonic stimulation may be performed at an intensity of 18% to 22% AMP for 0.5 to 2 seconds. Furthermore, if the useful substance is a protein, nucleic acid, or nanomaterial, the ultrasonic stimulation may be performed at an intensity of 20% to 30% AMP for 1 to 5 seconds.

[0055] Although the ultrasonic stimulation conditions are not limited to the above range, when performed within the above range, the degree of damage to the useful material can be minimized and the loading efficiency can be maximized.

[0056] The step of culturing the mixture of the ultrasound-stimulated carrier and the useful substance can be performed using known means and under known methods and conditions. For example, it can be performed at 30°C to 40°C for 30 minutes to 2 hours. Preferably, it can be performed at 35°C to 40°C for 50 minutes to 70 minutes.

[0057]

[0058] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0059]

[0060] Example 1. Preparation of exosomes derived from human fibroblasts (HDFs)

[0061] 1x10 6 Human dermal fibroblasts (HDF) from dogs were treated with UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea) at 20 KHz, 1.0 W / cm 2 Ultrasonic stimulation was directly applied for 5 seconds (hereinafter, ultrasonic stimulated HDF is referred to as UHDF). 2x10 5 The UHDF of the dog was sonicated (20 KHz, 5.0 W / cm) in a 35-mm petri dish. 2, 10 min) and cultured with embryonic stem cell culture medium (DMEM / F12, 15% FBS, 2 mM GlutaMAX, 0.1% NEAA, 0.1% penicillin / streptomycin, 0.1 mM β-mercaptoethanol, 1000 units / ml leukemia inhibitory factor (LIF)) for one day. Exosomes were isolated from the culture medium in which the sonicated HDFs (UHDFs) were cultured as follows: the culture medium was centrifuged at 3,000 × g for 20 min to remove cell debris and dead cells, and the supernatant was passed through a 0.22-mm filter (Minisart ® The culture medium was passed through a syringe filter (Sartorius, Goettingen, Germany). The culture medium passed through Amicon ® Exosomes were concentrated by centrifugation at 14,000 xg for 20 minutes in an Ultra-15 100,000 kDa device (Millipore, Billerica, MA, USA).

[0062]

[0063] Example 2. Loading of useful substances into exosomes

[0064] The exosomes manufactured in Example 1 were distributed in the required amount into a 1.5 ml tube, and DPBS was added so that the final volume was 100 μl.

[0065] After distributing the useful substances to be loaded according to Table 1 into a 1.5 ml tube, DPBS was added so that the final volume became 100 μl.

[0066] No. Useful substance (loading substance) Loading amount Volume 1 Deoxyribonucleotide (DNA) (Cy3 labeled DNA) 20 ng 100 ㎕ (1 x 10 11 florescence particles)2Protein (IgG-488)0.1 ㎍ / ml0.01 ㎕3Nanomaterial (Q-dot 705)1 nmole0.01 ㎕4Plasmid DNA1 ㎍Plasmid DNA varies depending on the separation concentration.

[0067] Exosomes were sonicated once for 5 seconds at AMP 30% intensity, and the useful substances to be loaded were sonicated once for 5 seconds at AMP 30% intensity or once for 1 second at AMP 20% intensity. The sonicated exosomes and useful substances were well mixed and incubated at 37°C or 4°C for 30 minutes or 2 hours, respectively.

[0068]

[0069] Experimental Example 1. Comparison of loading efficiency according to culture conditions of a mixture of sonicated exosomes and useful substances.

[0070] Culturing at 37°C is more effective in increasing the loading efficiency than culturing at 4°C, and under culturing conditions of 30 minutes to 2 hours, the longer the culturing time, the more the loading efficiency tends to increase. However, for production efficiency, the culturing time was determined to be 1 hour (Fig. 1).

[0071] In addition, it can be seen that when ultrasonic treatment is performed once for 5 seconds at an intensity of 30% AMP, a loading efficiency of approximately 60% is shown (Fig. 2), and when ultrasonic treatment is performed once for 1 second at an intensity of 20% AMP, a loading efficiency of approximately 70% is shown (Figs. 3 and 4).

[0072]

[0073] Experimental Example 2. Comparison of loading efficiency according to ultrasonic treatment target

[0074] The loading efficiency was compared between ultrasound treatments applied to only the useful substance, only the exosomes, and both the useful substance and the exosomes. Ultrasound was applied once for 5 seconds at an AMP of 30%.

[0075] As a result, it was proven that the material loading efficiency was the highest when the ultrasonic treatment technology of the present invention was applied: 13.04% when no ultrasonic treatment was performed, 25.2% when only the useful material (Q-dot) was loaded by ultrasonic treatment, 42.07% when only the exosomes were loaded by ultrasonic treatment, and 59.68% when both the useful material and the exosomes were each ultrasonic treated (Fig. 5).

[0076]

[0077] Experimental Example 3. Confirmation of damage to useful substances due to ultrasonic treatment.

[0078] To determine whether sonication causes damage to the useful substances, the useful substances (IgG and Cy3-DNA) were subjected to a single sonication cycle for 5 seconds at 30% AMP or a single sonication cycle for 1 second at 20% AMP, and then analyzed by SDS-PAGE (with immunoblot) using 10% acrylamide gel and 2% agarose gel electrophoresis.

[0079] It was confirmed that a single ultrasound treatment for 5 seconds with AMP 30% caused damage to useful materials, and it was found that the damage was approximately 50% compared to when no ultrasound treatment was performed. On the other hand, a single ultrasound treatment for 1 second with AMP 20% induced approximately 25% damage (Figs. 6 and 7). Therefore, since ultrasound treatment causes damage to useful materials, it is important to load them under appropriate ultrasound treatment conditions, and it was confirmed that the ultrasound treatment technology of the present invention can reduce material damage and increase loading efficiency.

[0080]

[0081] Experimental Example 4. Plasmid DNA Loading Experiment

[0082] After loading plasmid DNA into exosomes, an experiment was conducted to confirm whether the material loaded into the exosomes could be delivered to the cells and function properly by treating the cells.

[0083] (1) Confirmation of plasmid DNA loading in exosomes

[0084] To determine whether sonication could induce loading of plasmid DNA into exosomes, a single 5-second ultrasound pulse at 30% AMP was applied to 1) only exosomes, 2) only plasmid DNA, or 3) both exosomes and plasmid DNA (negative control; no sonication). Subsequently, plasmid DNA within exosomes was purified using a mini prep kit, and PCR was performed from the purified plasmid DNA.

[0085] As a result, it was confirmed that some plasmid DNA was loaded into exosomes even without ultrasonic treatment, but when only exosomes were ultrasonicated (loading through ultrasonic treatment), it was confirmed that a greater amount of plasmid DNA was loaded. When both exosomes and plasmid DNA were ultrasonicated, the loading rate seemed to decrease, and it was presumed that this occurred due to damage to plasmid DNA caused by ultrasonic treatment (Fig. 8).

[0086] Accordingly, additional experiments were conducted to confirm damage to plasmid DNA caused by ultrasound.

[0087]

[0088] (2) Confirmation of plasmid DNA damage by ultrasound

[0089] As in the previous experiment, to confirm the damage caused by sonication of plasmid DNA, sonication was performed once for 5 seconds at 30% AMP and once for 1 second at 20% AMP, and then confirmed through 1% agarose gel running (Fig. 9). As with other useful substances confirmed previously, more damage occurred (approximately 90%) under the condition of sonication once for 5 seconds at 30% AMP, and a tendency for damage to decrease under the condition of sonication once for 1 second at 20% AMP was confirmed (approximately 50%).

[0090] In summary of the above experiments, the loading efficiency and degree of damage can be confirmed according to the ultrasonic conditions as shown in Table 2 below.

[0091] That is, in terms of loading efficiency, it was determined that the exosome is suitable for loading useful substances when it increases by about 20% compared to the existing technology and the degree of damage to useful substances is about 50%.

[0092] 30% 5s 1pulse 20% 1s pulse Suitable conditions Qdot loading efficiency: 59.7% Damage degree: - Loading efficiency: 69.3% Damage degree: - Both Protein loading efficiency: 64.8% Damage degree: Approximately 50% Loading efficiency: 70.1% Damage degree: Approximately 25% Both Cy3 DNA loading efficiency: 66.5% Damage degree: Approximately 50% Loading efficiency: 71.6% Damage degree: Approximately 25% Both Plasmid DNA loading efficiency: - Damage degree: Approximately 90% Loading efficiency: - Damage degree: Approximately 50% 20% 1 s 1pulse

[0093] (3) Based on the experiment to confirm the presence or absence of damage to the cells by delivering useful substances loaded on exosomes, Q-dot, IgG488, or plasmid DNA were loaded on exosomes using a method of treating them with ultrasound once per second at 20% AMP.

[0094] After that, 1x10 HDF cells were seeded in a 24-well cell culture dish. 5Cells were seeded at 1 / well, and after 16 hours, exosomes loaded with Q-dot and IgG488 were treated to the cells, and 24 hours later, fluorescence measurement analysis was performed to confirm whether they were delivered to the cells. As a result, when the cells were treated with exosomes loaded with useful substances, the fluorescence measurement value was confirmed to increase compared to untreated HDF, confirming that exosomes loaded with useful substances were delivered to the cells and could deliver substances into the cells (Fig. 10).

[0095] To determine whether the substance can be delivered to the cell and perform its function by treating the cell with exosomes loaded with plasmid DNA, 1x10 9 By treating cells with exosomes loaded with plasmid DNA by sonicating the exosomes of the particles with 30% Amp once per second and the plasmid DNA with 20% AMP once per second, the intracellular delivery was confirmed directly and indirectly through the properties encoded in the plasmid, such as antibiotic resistance, luciferase activity, and PCR.

[0096] To treat exosomes loaded with plasmid DNA, cells were seeded at 1x10 in a 24-well cell culture dish. 5 Cells were inoculated at 10 cells / well, treated with exosomes loaded with plasmid DNA, and treated with antibiotics 24 hours later. Resistance to antibiotics was confirmed 24 hours later. As a result, when neomycin was treated to cells not loaded with plasmid DNA (HDF, Reprosome, DNA only), cell death was observed, and when exosomes loaded with plasmid DNA were treated to cells, neomycin-induced cell death was observed to be reduced (Fig. 11).

[0097] For the luciferase assay, 1x10 HDF cells were seeded in a 24-well cell culture dish.5 Cells were seeded at 10 cells / well, and 16 hours later, exosomes loaded with plasmid DNA were treated, and then, 24 hours later, a luciferase assay was performed. As a result, it was confirmed that luciferase activity increased only in cells loaded with exosomes loaded with plasmid DNA (Fig. 12).

[0098] Finally, to confirm intracellular material transfer (plasmid DNA transfer) through exosomes by synthesizing cDNA from mRNA within the cells and performing PCR, 1x10 HDFs were seeded in a 24-well cell culture dish. 5 After 16 hours of inoculation with cells / well, exosomes loaded with plasmid DNA were treated, and 24 hours later, Luciferase RNA was purified, cDNA was synthesized, and PCR was performed (Fig. 13). As a result, a PCR band was detected when cells were treated with exosomes loaded with plasmid DNA (lane 4), and amplification was also confirmed through qPCR (lane 1: only cells, lane 2: Treatment of exosomes, lane 3: Treatment of plasmid DNA only, lane 5: plasmid DNA purified from exosomes, PC: positive control; original plasmid DNA).

Claims

1. A step of providing ultrasonic stimulation to a carrier and a useful substance including a phospholipid layer, respectively; and A method for loading a useful substance onto a carrier, comprising: a step of culturing a mixture of the ultrasonically stimulated carrier and a useful substance for a certain period of time; 2. A method for loading a useful substance onto a carrier according to claim 1, wherein the carrier is an exosome.

3. A method for loading a useful substance onto a carrier according to claim 1, wherein the carrier is an exosome derived from human fibroblasts (HDF).

4. In claim 3, the exosome provides ultrasonic stimulation to human fibroblast-derived cells, and the step of providing ultrasonic stimulation to a cell-free culture medium; A step of culturing a mixture of the ultrasound-stimulated human fibroblast-derived cells and the ultrasound-stimulated culture medium for a certain period of time; and A method for loading a useful substance onto a carrier, the method comprising: a step of separating exosomes from the above mixture; 5. A method for loading a useful substance onto a carrier, wherein the useful substance in claim 1 is a nucleic acid, protein, peptide, nanomaterial, plasmid DNA, low molecular weight compound, drug, hormone, enzyme or neurotransmitter.

6. A method for loading a useful substance onto a carrier, wherein the step of providing ultrasonic stimulation to the carrier in claim 1 is performed at an intensity of 20% to 40% of AMP for 1 to 10 seconds.

7. A method for loading a useful substance into a carrier, wherein the step of providing ultrasonic stimulation to the useful substance in claim 1 is performed at an intensity of 15% to 30% of AMP for 0.1 to 5 seconds.

8. A method for loading a useful substance onto a carrier, wherein, in claim 7, when the useful substance is plasmid DNA, ultrasonic stimulation is performed at an intensity of 15% to 25% of AMP for 0.1 to 3 seconds.

9. A method for loading a useful substance onto a carrier, wherein, in claim 7, when the useful substance is a protein, nucleic acid, or nanomaterial, ultrasonic stimulation is performed at an intensity of 20% to 30% of AMP for 1 to 5 seconds.

10. A method for loading a useful substance onto a carrier, wherein the step of culturing the mixture according to claim 1 is performed at 30°C to 40°C for 30 minutes to 2 hours.

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