Method for separating and purifying 125I labeled proteoglycan macromolecules

Abstract

The invention discloses a method for separating and purifying 125I labeled proteoglycan macromolecules, which comprises the following steps: dissolving proteoglycan macromolecules in water, and carrying out preliminary filtration through a dialysis bag to remove impurities; performing radiolabeling on the proteoglycan macromolecules by using a chloramine T method to obtain a labeled product; dropwise adding the labeled product into a G25 gel column, carrying out centrifugal graded elution, collecting different products, and monitoring the radioactivity count and radiochemical purity of each product. According to the method for separating and purifying the 125I-labeled proteoglycan macromolecules, preliminary filtration is performed through a dialysis bag, then the 125I-labeled proteoglycan macromolecules are subjected to graded elution by using a G25 gel column, complex equipment is not needed, operation is easy and convenient, and the method has the advantages of being high in recovery rate, wide in application range and the like; the method can be used for accurately purifying the iodine-125 labeled proteoglycan macromolecules, and has relatively high stability and reliability.

Description

Technical Field

[0001] The present invention relates to 125 The invention relates to the field of separation and purification of I-labeled products, and specifically relates to a 125 Method for separation and purification of I-labeled proteoglycan macromolecules. Background Art

[0002] 125 I is a commonly used radioactive isotope with a half-life of 60.14 days. It is suitable for SPECT imaging and can be applied to local tumor radiotherapy, targeted drug delivery and other fields. 125 I is commonly used for labeling peptides, antibodies or protein drugs, and is particularly suitable for tissue distribution studies of macromolecular drugs. The labeling principle is mainly through oxidation reaction and iodination reaction. 125 I is labeled on tyrosine residues of proteins or peptides.

[0003] Proteoglycans are a type of macromolecular complex formed by covalent linkage of proteins and polysaccharides. They have important biological functions in organisms, such as cell recognition, signal transduction, and immune response. The characteristics of proteoglycan macromolecules include: Large molecular weight: composed of multiple amino acids and monosaccharide units, the molecular weight is usually large. Complex structure: It has diverse structures and functions, which brings challenges to separation and purification. Biological activity: It plays a key role in many biological processes, so its purification and activity maintenance are the key to research. Due to the complexity and importance of proteoglycan macromolecules, its separation and purification technology needs to meet the following requirements: High purity: It is required that impurities can be effectively removed during the purification process to obtain high-purity target molecules. Maintain activity: The biological activity of proteoglycans must be maintained during the purification process to avoid degradation or denaturation problems. High recovery rate: The recovery rate of the target molecule should be increased as much as possible to reduce losses.

[0004] The separation and purification of polysaccharides is an important step in the study of polysaccharides. General methods include: Extraction: Taking advantage of the fact that polysaccharides are soluble in water or acid, alkali, and salt solutions, but insoluble in organic solvents such as alcohol, ether, and acetone, polysaccharides are extracted through water extraction, alcohol precipitation, and other steps. Removal of non-sugar impurities: Use metal salt precipitation method, quaternary ammonium salt precipitation method, etc. to remove impurities such as organic acids, amino acids, proteins, etc. Purification of polysaccharides: Commonly used purification methods include column chromatography, partial precipitation method, etc. These methods are used to separate mixed polysaccharides from each other to obtain purer polysaccharide products.

[0005] In summary, 125 The separation and purification of I-labeled proteoglycan macromolecules involves specific radiolabeling techniques and complex polysaccharide purification steps. The selection and application of these methods depend on the properties of the target protein or polysaccharide and the research requirements.

[0006] However, the existing 125I labeling and purification methods often rely on manual operation, and the operation process is complicated and the equipment design is lengthy, which brings many inconveniences to experimenters. In particular, isotope contamination and operational errors are prone to occur during the labeling and purification process, affecting the accuracy of the experimental results and the safety of the operation.

[0007] For example, the patent application number is 2020101710520, and the name of the patent application is: A 125I drug labeling and purification device and method thereof. It includes a complex system consisting of multiple pressure devices, valves, and pipelines, requiring operators to frequently adjust valves and transfer liquids, increasing the probability of human operating errors. Although the device design has certain automated collection functions, it is still highly dependent on operators, and the equipment occupies a large area, making it unsuitable for use in small laboratories. In addition, the cleaning process of the equipment may also cause isotope contamination, posing a safety hazard.

[0008] Therefore, there is an urgent need for a simpler, more automated, and less human error-prone 125I drug labeling and purification method. This method should minimize direct contact between operators and radioactive isotopes, reduce potential radiation risks, and be able to complete labeling and purification tasks efficiently and accurately. Summary of the invention

[0009] The purpose of the present invention is to solve the deficiencies in the prior art and provide a simple and convenient operation without complicated equipment. 125 Method for separation and purification of I-labeled proteoglycan macromolecules.

[0010] In order to solve the above technical problems, the technical method adopted by the present invention is as follows: the present invention discloses a 125 The invention discloses a method for separating and purifying I-labeled proteoglycan macromolecules, comprising dissolving the proteoglycan macromolecules in water and performing preliminary filtration through a dialysis bag to remove impurities; radioactively labeling the proteoglycan macromolecules using the chloramine T method to obtain a labeled product; dripping the labeled product into a G25 gel column, eluting by centrifugation and fractionation, collecting different products, and monitoring the radioactivity count and radiochemical purity of each product.

[0011] Further, the following steps are included:

[0012] S1. Dissolve the fully ground proteoglycan macromolecules in water, take the supernatant and put it into a dialysis bag and place it in a beaker filled with pure water. Change the liquid at 19, 21, 25, 27, 43, and 66 hours respectively. After dialysis for 72 hours, collect the liquid and store it after freeze-drying;

[0013] S2. Radiolabel the lyophilized product using the chloramine T method;

[0014] S3. Add the labeled product dropwise to the center of the G25 gel column, collect the product 1 after centrifugation, and obtain125 I labels proteoglycan macromolecules.

[0015] Furthermore, in step S1, the fully ground proteoglycan macromolecules are obtained by the following steps:

[0016] S11. Take an appropriate amount of proteoglycan sample, grind thoroughly and weigh 170.0 mg, add 8.5 mL of water, and dissolve on a homogenizer for 30 min (the sample volume can be adjusted);

[0017] S12. After the sample is fully dissolved, centrifuge at high speed for 10 min at 22°C to remove the precipitate and keep the supernatant;

[0018] S13. Cut a dialysis bag about 10 cm long and boil it in 10 mM EDTA solution for 1 min;

[0019] S14. The supernatant was placed in a dialysis bag and dialyzed against water, with the liquid changed at 19, 21, 25, 27, 43, and 66 hours respectively; the dialysis was performed for a total of 72 hours, and the liquid was recovered;

[0020] S15. Inject the recovered liquid into a 10 mL sterile vial;

[0021] S16. Place the vial in a freeze dryer for precooling for 10 minutes, set the drying conditions, and wait for the sample chamber to be in a vacuum state;

[0022] S17. After the temperature and air pressure drop, loosen the vent valve, take out the sample after venting, and weigh the separated proteoglycan macromolecules after drying;

[0023] S18. Dissolve the dried proteoglycan macromolecules in 0.2 M PB buffer, pH 7.5, and calculate the solubility.

[0024] Furthermore, the step S2 specifically includes the following steps:

[0025] S21. Measure 92 μL of the proteoglycan drug macromolecule solution, wherein the mass of the proteoglycan drug macromolecule contained therein is 200 μg;

[0026] S22. In a glove box, add 10 μL of Na with a radioactivity of 4.8 mCi to the above system. 125 I, mixing;

[0027] S23. Add 20 μL of 1 μg / μL chloramine T solution and mix well for 30 seconds;

[0028] S24. Add 40 μL of 1 μg / μL sodium metabisulfite solution and mix well to label the product.

[0029] Furthermore, the molecular weight cut-off of the dialysis bag is 3500Da.

[0030] Furthermore, each time the dialysate is replaced, the volume of the liquid exchanged is twice the volume of the proteoglycan macromolecular solution.

[0031] Furthermore, the freeze-drying step includes freeze-drying at -20°C for 48 hours until the sample is completely dry.

[0032] Furthermore, the elution operation of the G25 gel column is performed by a gravity method, and the gravity method performs graded elution by centrifugation to remove free radioactive iodine.

[0033] Furthermore, the labeled product was added dropwise to the center of the G25 gel column, and then centrifuged at 12000 rpm for 3 min to collect product 1.

[0034] Furthermore, in step S4, 0.5 mL of PB solution was added to the center of the G25 gel column after collecting product 1, and the mixture was centrifuged at 12000 rpm for 3 min to collect product 2, and product 1 and product 2 were combined to obtain the final proteoglycan macromolecule labeled with iodine-125.

[0035] Furthermore, the radiochemical purity of the product was analyzed by thin layer chromatography, the developing solvent was 1 mol / L ammonium acetate: methanol = 1:4, and polyamide film paper was used as the developing medium.

[0036] Beneficial effects:

[0037] The present invention provides 125 I-labeled proteoglycan macromolecule separation and purification method, through the dialysis bag for preliminary filtration, and then use G25 gel column to 125 The proteoglycan macromolecules labeled with iodine-125 are graded and eluted without complicated equipment and are easy to operate. It has the advantages of high recovery rate and wide application range, and can accurately purify the proteoglycan macromolecules labeled with iodine-125 and has high stability and reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the radiochemical purity analysis of product 1 in the embodiment of the present invention;

[0039] Figure 2 This is the radiochemical purity analysis of product 2 in the embodiment of the present invention;

[0040] Figure 3 This is the radiochemical purity analysis of product 3 in the embodiment of the present invention;

[0041] Figure 4 The radiochemical purity analysis of product 4 in the embodiment of the present invention is as follows;

[0042] Figure 5 The peak diagram of free iodine with a retention time of 0.6 in the embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0044] Example

[0045] A sort of 125 The method for separating and purifying I-labeled proteoglycan macromolecules comprises the following steps:

[0046] S1. Samples are pre-treated by dialysis:

[0047] (1) Take an appropriate amount of proteoglycan sample, grind it thoroughly and weigh 170.0 mg, add 8.5 mL of water, and dissolve it on a homogenizer for 30 min (the sample volume can be adjusted);

[0048] (2) After the sample is fully dissolved, centrifuge at high speed, 12000 rpm for 10 min, and remove the precipitate at 22°C, and keep the supernatant;

[0049] (3) Cut a dialysis bag about 10 cm long with a molecular weight cutoff of 3500 Da and boil it in 10 mM EDTA solution for 1 min;

[0050] (4) Place the supernatant into a dialysis bag and dialyze against water. Change the solution at 19, 21, 25, 27, 43, and 66 hours.

[0051] The total dialysis was 72 h, and the fluid was recovered;

[0052] (5) Inject the recovered liquid into a 10 mL sterile vial;

[0053] (6) Place the vial in a freeze dryer, pre-cool for 10 min at -20°C, set the drying conditions, dry at -20°C for 48 h, and wait for the sample chamber to be in a vacuum state;

[0054] (7) After the temperature and air pressure drop, loosen the vent valve, and after 10 minutes, take out the sample after venting. The separated proteoglycan macromolecules are dried and weighed.

[0055] (8) Dissolve the dried proteoglycan macromolecules in 0.2 M PB buffer, pH 7.5, and calculate the solubility.

[0056] S2. Proteoglycan macromolecules 125 I Mark:

[0057] 2.1 Experimental instruments and reagents

[0058] Disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), sodium hydroxide (NaOH), chloramine T, sodium metabisulfite, ammonium acetate, methanol, ethylenediaminetetraacetic acid (EDTA). TLC (BioScan, USA), high-speed centrifuge, electronic balance, large-capacity centrifuge, pH meter or fine pH test paper, γ counter (Perkin Elmer, USA), freeze dryer (CHRIST, Germany), centrifuge tube (50, 1.5mL), 5L beaker, G25 gel column, magnetic stirrer

[0059] 2.2 125 I Marking Operation Steps

[0060] (1) Measure 92 μL of proteoglycan drug macromolecule solution, wherein the mass of the proteoglycan drug macromolecule contained is 200 μg;

[0061] (2) In a glove box, add 10 μL of Na with a radioactivity of 4.8 mCi to the above system. 125 I, mixing;

[0062] (3) Add 20 μL of 1 μg / μL chloramine T solution and mix well for 30 seconds;

[0063] (4) Add 40 μL of 1 μg / μL sodium metabisulfite solution and mix well to label the product.

[0064] S3. Purification of labeled products

[0065] Add the labeled product to the center of the G25 gel column, centrifuge at 12000rpm for 3min, and collect product 1; add 0.5mL PB solution, centrifuge at 12000rpm for 3min, collect product 2, repeat the above steps, and collect products 3 and 4; monitor the radioactivity counts and radiochemical purity of products 1, 2, 3, and 4 respectively, and collect the products.

[0066] S4. Radiochemical Purity Analysis Method

[0067] Quality control analysis method: TLC

[0068] Preparation of developing agent: 1mol / L ammonium acetate: methanol = 1:4

[0069] Unfolding paper: Polyamide film paper

[0070] Steps: Use a capillary to absorb a small amount of this product (about 5μCi), and spot it on the bottom of the polyamide film paper chromatographic film about 1cm away. After drying, place the chromatographic plate in the developing cylinder. After the developing agent develops to the solvent front, take it out and blow dry it. Use a thin layer scanner to detect the chromatographic peak. Figure 1 As shown, the peak at a retention time of 0.6 is the peak of free iodine, and the peak at a retention time of about 1.5 is the peak of iodine-labeled macromolecules.

[0071] S5. Results Analysis

[0072] Collect products Activity (mCi) Radiochemical purity (%) 1 1.458 94.24 2 0.329 91.71 3 0.343 67.68 4 0.334 54.18

[0073] As shown in the table, the activity and radiochemical purity of product 1 are both the highest (94.24%), which means that the product contains the most radioactive markers and has a higher purity. According to the radiochemical purity, this product should be the most suitable for subsequent research and experiments, meeting the requirements of high purity and high activity.

[0074] The radiochemical purity of product 2 is slightly inferior to that of product 1 (91.71%), but still higher than most purified products. Its radioactivity is also lower, but considering that the purity still meets the requirements, product 2 can still be used as a usable sample for research.

[0075] The radiochemical purity of products 3 and 4 decreased significantly (67.68%, 54.18%), which is much lower than that of products 1 and 2. This may mean that some free iodine-125 was not completely removed during the purification process, resulting in lower radiochemical purity. Although its activity is similar to that of product 2, the decrease in purity makes it unsuitable for direct application in high-precision experiments.

[0076] Therefore, select product 1 and 2 to combine as the final 125 I-labeled proteoglycan macromolecules.

[0077] The present invention uses G25 gel column to 125 The proteoglycan macromolecules labeled with iodine-125 are graded and eluted to finally obtain a product with high purity for testing. The separation and purification method provided by the present invention does not require complex equipment, is easy to operate, has the advantages of high recovery rate, wide application range, etc., can accurately purify the proteoglycan macromolecules labeled with iodine-125, and has high stability and reliability.

[0078] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The proteoglycan macromolecules are dissolved in water and initially filtered through a dialysis bag to remove impurities; The proteoglycan macromolecules are radioactively labeled using the chloramine T method to obtain a labeled product; the labeled product is dropped into a G25 gel column, and different products are collected by centrifugal graded elution, and the radioactivity count and radiochemical purity of each product are monitored.

2. according to claim 1 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The steps include: S1. Dissolve the fully ground proteoglycan macromolecules in water, take the supernatant and put it into a dialysis bag and place it in a beaker filled with pure water. Change the liquid at 19, 21, 25, 27, 43, and 66 hours respectively. After dialysis for 72 hours, collect the liquid and store it after freeze-drying; S2. Radiolabel the lyophilized product using the chloramine T method; S3. Add the labeled product dropwise to the center of the G25 gel column, collect the product 1 after centrifugation, and obtain 125 I labels proteoglycan macromolecules.

3. According to claim 1 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, In step S1, the fully ground proteoglycan macromolecules are obtained by the following steps: S11. Take an appropriate amount of proteoglycan sample, grind thoroughly and weigh 170.0 mg, add 8.5 mL of water, and dissolve on a homogenizer for 30 min; S12. After the sample is fully dissolved, centrifuge at high speed for 10 min at 22°C to remove the precipitate and keep the supernatant; S13. Cut a dialysis bag about 10 cm long and boil it in 10 mM EDTA solution for 1 min; S14. The supernatant was placed in a dialysis bag and dialyzed against water, with the liquid changed at 19, 21, 25, 27, 43, and 66 hours respectively; the dialysis was performed for a total of 72 hours, and the liquid was recovered; S15. Inject the recovered liquid into a 10 mL sterile vial; S16. Place the vial in a freeze dryer for precooling for 10 minutes, set the drying conditions, and wait for the sample chamber to be in a vacuum state; S17. After the temperature and air pressure drop, loosen the vent valve, take out the sample after venting, and weigh the separated proteoglycan macromolecules after drying; S18. Dissolve the dried proteoglycan macromolecules in 0.2 M PB buffer, pH 7.5, and calculate the solubility.

4. according to claim 1 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The step S2 specifically includes the following steps S21. Measure 92 μL of the proteoglycan drug macromolecule solution, wherein the mass of the proteoglycan drug macromolecule contained therein is 200 μg; S22. In a glove box, add 10 μL of Na with a radioactivity of 4.8 mCi to the above system. 125 I, mixing; S23. Add 20 μL of 1 μg / μL chloramine T solution and mix well for 30 seconds; S24. Add 40 μL of 1 μg / μL sodium metabisulfite solution and mix well to label the product.

5. According to claim 2 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The molecular weight cut-off of the dialysis bag is 3500Da.

6. According to claim 2 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, Each time the dialysate is replaced, the volume of the liquid exchanged is twice the volume of the proteoglycan macromolecular solution.

7. According to claim 2 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The freeze-drying step includes freeze-drying at -20°C for 48 hours until the sample is completely dry.

8. According to claim 2 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The elution operation of the G25 gel column is performed by a gravity method, and the gravity method performs graded elution by centrifugation to remove free radioactive iodine.

9. According to claim 2 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, In the step S4, the labeled product is added dropwise to the center of the G25 gel column, and then centrifuged at 12000 rpm for 3 minutes to collect the product 1.

10. The method according to any one of claims 2 to 9. 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, In step S4, 0.5 mL of PB solution is added to the center of the G25 gel column after collecting product 1, and the column is centrifuged at 12000 rpm for 3 min to collect product 2. Product 1 and product 2 are combined to obtain the final proteoglycan macromolecule labeled with iodine-125.

11. According to claim 2 125 I-labeled proteoglycan macromolecule separation and purification method, characterized in that, The radiochemical purity of the product was analyzed by thin layer chromatography, the developing solvent was 1 mol / L ammonium acetate:methanol=1:4, and polyamide film paper was used as the developing medium.

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