A leukocyte-removing filter material and a method for producing the same

Abstract

The application relates to the technical field of blood filtering materials, in particular to a filtering material for filtering leukocytes and a preparation method thereof, and the preparation method comprises the following steps: mixing MOFs master batches containing fibroin and polybutylene terephthalate, and stirring and mixing the mixture at a rotating speed of 400-1200 rpm for 5-30 min to obtain a mixed material; feeding the mixed material into a melt-blowing double-screw extruder, sending the mixed material to a melt-blowing die through a metering pump, drawing the mixed material into filaments under the action of hot air, and laying the filaments into a web to obtain the filtering material for filtering leukocytes. The filtering material for filtering leukocytes is combined with MOFs and fibroin, the structure characteristics of the large specific surface area of MOFs are utilized, the adhesion to leukocytes is further improved, and the occurrence of hemolysis is greatly reduced during the filtering use.

Description

Technical Field

[0001] This invention relates to the field of blood filtration materials technology, and in particular to a filtration material for removing leukocytes and its preparation method. Background Technology

[0002] Blood is an indispensable component for maintaining human metabolism and life. When the body's blood volume and blood component content are insufficient, timely blood replenishment is necessary. Blood transfusion, as a common medical procedure, plays a crucial role in the treatment of various diseases and in emergency situations. However, there are certain risks involved in blood transfusion, one of which is the immune response. White blood cells are part of the immune system, and their main responsibility is to protect the body from foreign pathogens. During blood transfusion, the introduced exogenous white blood cells may trigger an immune response in the patient. This immune response may manifest as adverse symptoms such as fever, chills, and allergic reactions, affecting the patient's comfort and recovery process. To reduce potential adverse reactions after blood transfusion, leukocyte filtration is a widely used and important measure. Currently, commonly used methods for removing white blood cells from blood in China include centrifugation, washing, frozen glycerol, ultraviolet irradiation, and filtration. Among these, filtration is the most widely used due to its simple operation, speed, efficiency, simple equipment, and ability to operate in a closed system.

[0003] Chinese invention patent CN108744722A, entitled "A Blood Filtration Material for Removing Leukocytes and Its Preparation Method," discloses a method using a composite fiber made from polybutylene terephthalate and silk fibroin. After filtration through a fine filter layer and a primary filter layer, the blood filtration time is shortened, reagent handling efficiency is improved, and the fine filter layer significantly improves filtration accuracy. However, the need to prepare the primary and fine filter layers increases the material processing steps and the complexity of the process. Chinese invention patent CN103623475B, entitled "A Blood Component Separation Device," discloses a multi-unit leukocyte removal filtration device. This device relies solely on gravity to filter leukocytes, resulting in low filtration efficiency. Furthermore, the filter element is made of multiple layers of non-woven fabric of varying thicknesses. Due to the poor hydrophilicity of non-woven fabric materials (PBT, PP), blood cannot flow sufficiently in the filter medium, leading to incomplete utilization of the filter material. Therefore, developing a filter material with a simple preparation process, strong leukocyte adsorption capacity, and the ability to avoid hemolysis has become a pressing technical challenge. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a filter material for removing leukocytes and a method for preparing the same, so as to at least solve the problems of weak adsorption of leukocytes and possible hemolytic reactions during filtration in the prior art.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] In a first aspect, the present invention provides a method for preparing a filter material for removing leukocytes, comprising the following steps:

[0007] MOF masterbatch containing silk fibroin and polybutylene terephthalate are mixed and stirred at 400-1200 rpm for 5-30 minutes to obtain a mixture.

[0008] The mixture is fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, it is stretched into filaments and laid into a web to obtain antibacterial meltblown nonwoven fabric.

[0009] Antibacterial meltblown nonwoven fabric is electreted under a DC voltage of 30-100kV to obtain a filter material that removes white blood cells.

[0010] In conjunction with the first aspect, in some embodiments, the operating conditions of the twin-screw extruder are as follows:

[0011] The screw speed is 3-10Hz, the metering pump speed is 3-10Hz, the feed motor speed is 3-10Hz, and the screw heating zone temperatures are as follows: Zone 1 60-180℃, Zone 2 80-190℃, Zone 3 100-250℃, Zone 4 120-280℃, Zone 5 120-280℃, Zone 6 100-270℃, Zone 7 100-270℃, Zone 8 100-260℃. The die head temperature is 180-300℃.

[0012] In conjunction with the first aspect, in some embodiments, the air volume of the hot air is 18-30 m³ / h. 3 The hot air volume is 300-400℃ per minute.

[0013] In conjunction with the first aspect, in some embodiments, the mass fraction of the MOF masterbatch containing silk fibroin is 1 to 12 parts, and the mass fraction of the polybutylene terephthalate is 90 to 99 parts.

[0014] In conjunction with the first aspect, in some embodiments, the preparation method of the MOF masterbatch containing silk fibroin is as follows:

[0015] Silk fibroin nanopowder was added to methanol to prepare a silk fibroin methanol solution with a concentration of 10-30 g / L. Then, 10-60 parts by mass of 2-methylimidazole were added and stirred for reaction. The reaction was carried out under magnetic stirring at 300-500 rpm for 10-60 min to obtain a precursor solution.

[0016] 30-300 parts by mass of a soluble metal salt are completely dissolved in 200-1000 parts by volume of a methanol solution to obtain a metal salt solution with a concentration of 150-300 g / L. A precursor solution is added to the metal salt solution, and the mixture is stirred at 50-300 rpm for 3-12 hours. The methanol solvent is removed to obtain MOF masterbatch containing silk fibroin.

[0017] In conjunction with the first aspect, in some embodiments, 2-methylimidazole is added to the silk fibroin methanol solution, and the mixture is reacted for 10 to 60 minutes with stirring at 300 to 500 rpm.

[0018] In conjunction with the first aspect, in some embodiments, the soluble metal salt is any one of zinc nitrate hexahydrate, cobalt nitrate hexahydrate, nickel nitrate hexahydrate, ferric nitrate nonahydrate, zinc acetate dihydrate, and cobalt hydroxide.

[0019] In conjunction with the first aspect, in some embodiments, the mass-to-volume ratio of the silk fibroin nanopowder to 2-methylimidazole and methanol is (0.1-1) g:(1-6) g:(20-120) mL.

[0020] Secondly, the present invention provides a filter material for filtering out leukocytes, which is prepared by the above-described preparation method.

[0021] The leukocyte-removing filter material and its preparation method of the present invention incorporate MOFs containing a certain amount of silk fibroin into polybutylene terephthalate (PBT). The silk fibroin improves the hydrophilicity of the filter material, allowing for more efficient use and shortening the filtration time. Furthermore, by utilizing the combination of MOFs and silk fibroin, and taking advantage of the large specific surface area of ​​MOFs, the adhesion to leukocytes is further enhanced. During filtration, hemolysis is significantly reduced, with a hemolysis rate as low as 0.69%. Experiments show a leukocyte removal efficiency of up to 99.99% and a platelet recovery rate as high as 98.56%. The preparation method of the present invention uses a melt-blown process, which is simple, safe, and efficient, making it suitable for industrial production and widespread application. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified in the following examples, the conditions are as per standard conditions or the manufacturer's recommendations. Raw materials, equipment, or instruments whose manufacturers are not specified are all commercially available products.

[0024] This invention addresses the problems of weak adsorption of leukocytes and potential hemolytic reactions during filtration in existing technologies. It proposes a leukocyte-removing filter material and its preparation method. Organic ligands, metal salts, and silk fibroin are mixed in a specific ratio to synthesize MOF (Metal-Oxide-Flavors) masterbatch under a specific method. Then, the MOF masterbatch is compounded with polybutylene terephthalate (PBT) in a specific ratio and melt-blown to prepare the leukocyte-removing filter material. This filter material exhibits both excellent blood filtration performance and good hydrophilicity, along with strong leukocyte adsorption capacity and low susceptibility to hemolytic reactions.

[0025] The method for preparing the leukocyte-removing filter material of the present invention includes the following steps:

[0026] MOF masterbatch containing silk fibroin and polybutylene terephthalate are mixed and stirred at 400-1200 rpm for 5-30 minutes to obtain a mixture.

[0027] The mixture is fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, it is drawn into filaments and laid into a web to obtain a filter material for removing white blood cells.

[0028] Specifically, the operating conditions of a twin-screw extruder are as follows:

[0029] The screw speed is 3–10 Hz, the metering pump speed is 3–10 Hz, and the feed motor speed is 3–10 Hz. The screw heating zones are as follows: Zone 1 60–180℃, Zone 2 80–190℃, Zone 3 100–250℃, Zone 4 120–280℃, Zone 5 120–280℃, Zone 6 100–270℃, Zone 7 100–270℃, Zone 8 100–260℃. The die temperature is 180–300℃. The hot air volume is 18–30 m³ / h. 3 The hot air volume is 300-400℃ per minute.

[0030] Specifically, the masterbatch containing silk fibroin comprises 1–12 parts by weight, and the masterbatch containing polybutylene terephthalate comprises 90–99 parts by weight. The preparation method of the masterbatch containing silk fibroin is as follows:

[0031] Silk fibroin nanopowder was added to methanol to prepare a silk fibroin methanol solution with a concentration of 10–30 g / L. Then, 10–60 parts by mass of 2-methylimidazole were added and the mixture was stirred and reacted under magnetic stirring at 300–500 rpm for 10–60 min to obtain a precursor solution. 30–300 parts by mass of a soluble metal salt were completely dissolved in 200–1000 parts by volume of methanol solution to obtain a metal salt solution with a concentration of 150–300 g / L. The precursor solution was added to the metal salt solution, and the mixture was stirred and reacted at 50–300 rpm for 3–12 h. After removing the methanol solvent, MOFs masterbatch containing silk fibroin was obtained. The soluble metal salt was any one of zinc nitrate hexahydrate, cobalt nitrate hexahydrate, nickel nitrate hexahydrate, ferric nitrate nonahydrate, zinc acetate dihydrate, and cobalt hydroxide. The mass-to-volume ratio of silk fibroin nanopowder to 2-methylimidazole and methanol is (0.1-1) g:(1-6) g:(20-120) mL.

[0032] The following examples 1-5 will provide a detailed description of a filter material for removing leukocytes from blood and its preparation method according to the present invention.

[0033] Example 1

[0034] The preparation method of the filter material for removing white blood cells in this embodiment is as follows:

[0035] (1) Precursor synthesis: 3g of silk fibroin nanopowder was added to 200ml of methanol solution to prepare a silk fibroin methanol solution with a concentration of 15g / L; then 12.5g of 2-methylimidazole was weighed and added to the above solution, and the reaction was carried out under magnetic stirring at 400rpm for 30min to obtain MOFs in-situ synthesis precursor solution A.

[0036] (2) Preparation of MOFs materials: 50g of zinc nitrate hexahydrate was dissolved in 200ml of methanol and magnetically stirred at 400rpm for 25min to completely dissolve it, resulting in solution B; then precursor solution A was added and the reaction was magnetically stirred at 150rpm for 8h to obtain MOFs containing silk fibroin.

[0037] (3) Preparation of filter material for removing leukocytes: 4 parts of the silk fibroin-containing MOFs prepared in step (2) and 96 parts of meltblown polybutylene terephthalate were mixed in a high-speed mixer at 750 rpm for 20 min. The mixture was then fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, the extruder was drawn into filaments and laid into a web to obtain the filter material for removing leukocytes. In this step, the screw speed was 10 Hz, the metering pump speed was 3 Hz, the feed motor speed was 3 Hz, and the air volume was 22 m³ / h. 3 / min, hot air temperature is 280℃, screw heating zone temperatures are: zone 1 120℃, zone 2 190℃, zone 3 200℃, zone 4 205℃, zone 5 210℃, zone 6 220℃, zone 7 210℃, zone 8 200℃, and die head temperature is 200℃.

[0038] Example 2

[0039] The preparation method of the filter material for removing white blood cells in this embodiment is as follows:

[0040] (1) Precursor synthesis: 6g of silk fibroin nanopowder was added to 400ml of methanol solution to prepare a silk fibroin methanol solution with a concentration of 15g / L; then 25g of 2-methylimidazole was weighed and added to the above solution, and the reaction was carried out under magnetic stirring at 450rpm for 30min to obtain MOFs in-situ synthesis precursor solution A.

[0041] (2) Preparation of MOFs materials: 100g of zinc nitrate hexahydrate was dissolved in 400ml of methanol and magnetically stirred at 450rpm for 25min to completely dissolve it, resulting in solution B; then precursor solution A was added and the reaction was magnetically stirred at 150rpm for 10h to obtain MOFs containing silk fibroin.

[0042] (3) Preparation of filter material for removing leukocytes: 5 parts of the silk fibroin-containing MOFs prepared in step (2) and 95 parts of meltblown polybutylene terephthalate were mixed in a high-speed mixer at 800 rpm for 25 min. The mixture was then fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, the extruder was drawn into filaments and laid into a web to obtain the filter material for removing leukocytes. In this step, the screw speed was 10 Hz, the metering pump speed was 3 Hz, the feed motor speed was 3 Hz, and the air volume was 22 m³ / h. 3 / min, hot air temperature is 280℃, screw heating zone temperatures are: zone 1 125℃, zone 2 180℃, zone 3 200℃, zone 4 205℃, zone 5 205℃, zone 6 210℃, zone 7 210℃, zone 8 205℃, die head temperature is 210℃.

[0043] Example 3

[0044] The preparation method of the filter material for removing white blood cells in this embodiment is as follows:

[0045] (1) Precursor synthesis: 6g of silk fibroin nanopowder was added to 200ml of methanol solution to prepare a silk fibroin methanol solution with a concentration of 30g / L; then 12.5g of 2-methylimidazole was weighed and added to the above solution, and the reaction was carried out under magnetic stirring at 400rpm for 30min to obtain MOFs in-situ synthesis precursor solution A.

[0046] (2) Preparation of MOFs materials: 50g of cobalt nitrate hexahydrate was dissolved in 200ml of methanol and magnetically stirred at 400rpm for 25min to completely dissolve it, resulting in solution B; then the precursor solution A was added and the reaction was magnetically stirred at 150rpm for 12h to obtain MOFs containing silk fibroin.

[0047] (3) Preparation of filter material for removing leukocytes: 4 parts of the silk fibroin-containing MOFs prepared in step (2) and 96 parts of meltblown polybutylene terephthalate were mixed in a high-speed mixer at 750 rpm for 20 min. The mixture was then fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, the extruder was drawn into filaments and laid into a web to obtain the filter material for removing leukocytes. In this step, the screw speed was 10 Hz, the metering pump speed was 3 Hz, the feed motor speed was 3 Hz, and the air volume was 22 m³ / h. 3 / min, hot air temperature is 280℃, screw heating zone temperatures are: zone 1 120℃, zone 2 190℃, zone 3 200℃, zone 4 205℃, zone 5 210℃, zone 6 220℃, zone 7 210℃, zone 8 200℃, and die head temperature is 200℃.

[0048] Example 4

[0049] The preparation method of the filter material for removing white blood cells in this embodiment is as follows:

[0050] (1) Precursor synthesis: 2g of silk fibroin nanopowder was added to 200ml of methanol solution to prepare a silk fibroin methanol solution with a concentration of 10g / L; then 10g of 2-methylimidazole was weighed and added to the above solution, and the reaction was carried out under magnetic stirring at 300rpm for 10min to obtain MOFs in-situ synthesis precursor solution A.

[0051] (2) Preparation of MOFs materials: 30g of nickel nitrate hexahydrate was dissolved in 100ml of methanol and magnetically stirred at 300rpm for 10min to completely dissolve it, resulting in solution B; then precursor solution A was added and the reaction was magnetically stirred at 50rpm for 12h to obtain MOFs containing silk fibroin.

[0052] (3) Preparation of filter material for removing leukocytes: One part of the silk fibroin-containing MOFs prepared in step (2) and 99 parts of meltblown polybutylene terephthalate were mixed in a high-speed mixer at 400 rpm for 30 min. The mixture was then fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, the extruder was drawn into filaments and laid into a web to obtain the filter material for removing leukocytes. In this step, the screw speed was 3 Hz, the metering pump speed was 5 Hz, the feed motor speed was 5 Hz, and the air volume was 18 m³ / h. 3 / min, hot air temperature is 200℃, screw heating zone temperatures are: zone 1 60℃, zone 2 80℃, zone 3 150℃, zone 4 120℃, zone 5 210℃, zone 6 220℃, zone 7 210℃, zone 8 200℃, and die head temperature is 180℃.

[0053] Example 5

[0054] The preparation method of the filter material for removing white blood cells in this embodiment is as follows:

[0055] (1) Precursor synthesis: 3g of silk fibroin nanopowder was added to 200ml of methanol solution to prepare a silk fibroin methanol solution with a concentration of 15g / L; then 12.5g of 2-methylimidazole was weighed and added to the above solution, and the reaction was carried out under magnetic stirring at 500rpm for 60min to obtain MOFs in-situ synthesis precursor solution A.

[0056] (2) Preparation of MOFs materials: 30g of zinc acetate dihydrate was dissolved in 200ml of methanol and magnetically stirred at 500rpm for 30min to completely dissolve it, resulting in solution B; then precursor solution A was added and the reaction was magnetically stirred at 300rpm for 3h to obtain MOFs containing silk fibroin.

[0057] (3) Preparation of filter material for removing leukocytes: 10 parts of the silk fibroin-containing MOFs prepared in step (2) and 90 parts of meltblown polybutylene terephthalate were mixed in a high-speed mixer at 1200 rpm for 5 min. The mixture was then fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, the extruder was drawn into filaments and laid into a web to obtain the filter material for removing leukocytes. In this step, the screw speed was 10 Hz, the metering pump speed was 10 Hz, the feed motor speed was 10 Hz, and the air volume was 30 m³ / h. 3 / min, hot air temperature is 300℃, screw heating zone temperatures are: zone 1 180℃, zone 2 190℃, zone 3 230℃, zone 4 250℃, zone 5 280℃, zone 6 270℃, zone 7 270℃, zone 8 260℃, die head temperature is 300℃.

[0058] Example 6

[0059] This embodiment tests the performance of the leukocyte-removing filter materials prepared in Examples 1, 2, and 3. Specifically, the three sets of filter materials prepared in Examples 1, 2, and 3 were loaded into leukocyte filters, and 500ml of whole blood was filtered online. The number of leukocytes and platelets before and after filtration was recorded, and the leukocyte and platelet retention rates were tested. The number of leukocytes and platelets before filtration was 2.95 × 10⁻⁶. 9 The sum is 7.45 × 10 9The relevant calculation formula is as follows:

[0060] Where L0 represents the number of white blood cells (platelets) per unit volume of whole blood before filtration, and L1 represents the number of white blood cells (platelets) per unit volume of whole blood after filtration. The calculation results are shown in Table 1.

[0061] Table 1. White blood cell and platelet filtration rates

[0062]

[0063]

[0064] The relative hemolysis rate of the three groups of filter materials prepared in Examples 1, 2, and 3 was determined. Since hemoglobin has characteristic absorption at 540 nm, the absorbance was measured at 540 nm using a spectrophotometer. The relative hemolysis rate was calculated using the following formula: Relative hemolysis rate (%) = [(A - A0) / (A0)] 100％ -A0)]×100%, where A0 and A are the absorbance of the supernatant (37℃) of red blood cells after 1 hour of contact with the nonwoven fabric surface before and after modification, respectively, at 540nm. 100％ The absorbance of the supernatant of red blood cells after incubation in pure water (37℃) for 1 hour is measured at a wavelength of 540 nm. The relevant measurement and calculation results are shown in Table 2.

[0065] Table 2 Results of hemolysis rate determination

[0066] Example 1 0.88 Example 2 0.69 Example 3 0.72

[0067] The data in Table 1 show that the leukocyte filtration material of the present invention achieves a leukocyte filtration efficiency of 99.99% and a platelet recovery rate of 98.07%, 96.04%, and 98.56%, respectively. This indicates that the leukocyte filtration material of the present invention has strong adhesion to leukocytes, effectively removes leukocytes and platelets from the blood, and has a high interception rate. The data in Table 2 show that the leukocyte filtration material of the present invention achieves a hemolysis rate as low as 0.69% during filtration, effectively reducing the occurrence of hemolytic reactions.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for preparing a filter material for removing leukocytes, characterized in that, Includes the following steps: The masterbatch of MOFs containing silk fibroin and polybutylene terephthalate were mixed and stirred at 400-1200 rpm for 5-30 min to obtain the mixture. The mixture is fed into a meltblown twin-screw extruder and pumped to the meltblown die by a metering pump. Under the action of hot air, it is drawn into filaments and laid into a web to obtain a filter material for removing white blood cells. Antibacterial meltblown nonwoven fabric is electreted under a DC voltage of 30~100 kV to obtain a filter material that removes white blood cells. The preparation method of the MOF masterbatch containing silk fibroin is as follows: Silk fibroin nanopowder was added to methanol to prepare a silk fibroin methanol solution with a concentration of 10-30 g / L. Then, 10-60 parts by mass of 2-methylimidazole were added and stirred for 10-60 min under magnetic stirring at 300-500 rpm to obtain a precursor solution. 30–300 parts by mass of a soluble metal salt were completely dissolved in 200–1000 parts by volume of a methanol solution to obtain a metal salt solution with a concentration of 150–300 g / L. A precursor solution was added to the metal salt solution, and the mixture was stirred at 50–300 rpm for 3–12 h. The methanol solvent was removed to obtain MOF masterbatch containing silk fibroin.

2. The method for preparing a filter material for removing leukocytes according to claim 1, characterized in that, The operating conditions of the twin-screw extruder are as follows: The screw speed is 3-10 Hz, the metering pump speed is 3-10 Hz, the feed motor speed is 3-10 Hz, and the screw heating zone temperatures are as follows: Zone 1 60-180℃, Zone 2 80-190℃, Zone 3 100-250℃, Zone 4 120-280℃, Zone 5 120-280℃, Zone 6 100-270℃, Zone 7 100-270℃, Zone 8 100-260℃. The die head temperature is 180-300℃.

3. The method for preparing a filter material for removing leukocytes according to claim 2, characterized in that, The air volume of the hot air is 18–30 m³. 3 The hot air volume is 300-400℃ per minute.

4. The method for preparing a filter material for removing leukocytes according to claim 3, characterized in that, The mass fraction of the MOF masterbatch containing silk fibroin is 1 to 10 parts, and the mass fraction of the polybutylene terephthalate is 90 to 99 parts.

5. The method for preparing a filter material for removing leukocytes according to claim 4, characterized in that, 2-Methylimidazole was added to the silk fibroin methanol solution, and the mixture was stirred at 300-500 rpm for 10-60 min.

6. The method for preparing a filter material for removing leukocytes according to claim 5, characterized in that, The soluble metal salt is any one of zinc nitrate hexahydrate, cobalt nitrate hexahydrate, nickel nitrate hexahydrate, ferric nitrate nonahydrate, zinc acetate dihydrate, and cobalt hydroxide.

7. The method for preparing a filter material for removing leukocytes according to claim 6, characterized in that, The mass-to-volume ratio of the silk fibroin nanopowder to 2-methylimidazole and methanol is (0.1-1)g:(1-6)g:(20-120)mL.

8. A filter material for removing leukocytes, characterized in that, The filter material is prepared using the preparation method described in any one of claims 1-7.

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