CN115044038A - Modified polyethyleneimine, preparation method thereof, transfection reagent and application thereof - Google Patents
Modified polyethyleneimine, preparation method thereof, transfection reagent and application thereof Download PDFInfo
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- 239000012096 transfection reagent Substances 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- YAXXOCZAXKLLCV-UHFFFAOYSA-N 3-dodecyloxolane-2,5-dione Chemical compound CCCCCCCCCCCCC1CC(=O)OC1=O YAXXOCZAXKLLCV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000005859 coupling reaction Methods 0.000 claims abstract description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 8
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 22
- 239000002904 solvent Substances 0.000 claims description 21
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- 238000003756 stirring Methods 0.000 claims description 17
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- UWJJYHHHVWZFEP-UHFFFAOYSA-N pentane-1,1-diol Chemical compound CCCCC(O)O UWJJYHHHVWZFEP-UHFFFAOYSA-N 0.000 claims description 8
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- 108090000623 proteins and genes Proteins 0.000 claims description 7
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- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
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- AUXDELGSSOCKOS-UHFFFAOYSA-N C(CCC(=O)[O-])(=O)OCCCCCCCCCCCC.[NH4+] Chemical compound C(CCC(=O)[O-])(=O)OCCCCCCCCCCCC.[NH4+] AUXDELGSSOCKOS-UHFFFAOYSA-N 0.000 description 1
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- 238000010521 absorption reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
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- 230000001413 cellular effect Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
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- 125000001165 hydrophobic group Chemical group 0.000 description 1
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- 238000011835 investigation Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/0206—Polyalkylene(poly)amines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Abstract
The invention provides a modified polyethyleneimine, a preparation method thereof, a transfection reagent and application thereof, wherein the modified polyethyleneimine comprises polyethyleneimine serving as a main chain and dodecyl succinic anhydride grafted on the polyethyleneimine, the dodecyl succinic anhydride is subjected to coupling reaction with a primary amine group of the polyethyleneimine, and part of primary amine is subjected to coupling modification by adopting dodecyl picric anhydride, so that part of primary amine is consumed, and the cytotoxicity of the polyethyleneimine can be reduced.
Description
Technical Field
The invention relates to the technical field of gene transfection biology, in particular to modified polyethyleneimine, a preparation method thereof, a transfection reagent and application thereof.
Background
Recombinant proteins are becoming increasingly important in basic research and clinical applications, and many recombinant proteins are of human or animal origin, and therefore expression using mammalian cells is the first choice to ensure functional folding and correct post-translational modification, and transfection of human embryonic kidney cells or chinese hamster ovary cells is currently a well established technique that can produce recombinant proteins in quantities of hundreds of milligrams to grams over a period of weeks.
The entry of naked DNA into mammalian cells is a very inefficient process compared to many viruses that have evolved over millions of years to efficiently deliver their genetic material into the nucleus of a host cell. Therefore, natural or synthetic vectors must be used to load the DNA, facilitating binding to the cell membrane and entry into the cell. Furthermore, ideally they should protect the DNA from cellular nucleases and introduce all intact and transcriptionally competent plasmid DNA into the nucleus.
The current mainstream carriers for delivering DNA include cationic liposome and cationic polymer, and the commercially available cationic liposome such as LipofectAMINE, FuGENE, 293fectin and the like has good transfection efficiency, but is very expensive and has high cytotoxicity. Compared with liposome complexes, cationic polymers have transfection efficiencies comparable to liposomes but are more cost-effective.
While polyethyleneimine is quite representative of the many polymers currently under investigation. It shows high transfection efficiency in both differentiated and non-differentiated cell pools, is also readily taken up by cells by endocytosis, and is protected from endosomal degradation due to its cationic nature and proton sponge effect. But this also causes cytotoxicity in the course of gene transfection.
How to provide a modified polyethyleneimine with lower cytotoxicity is a technical problem to be solved urgently at present.
Disclosure of Invention
In view of the above, the invention provides a modified polyethyleneimine with lower cytotoxicity, a preparation method thereof, a transfection reagent and an application thereof.
The technical scheme of the invention is realized as follows: in one aspect, the present invention provides a modified polyethyleneimine, comprising: the main chain of the polyurethane comprises polyethyleneimine serving as a main chain and dodecyl succinic anhydride grafted on the polyethyleneimine, wherein the dodecyl succinic anhydride is subjected to coupling reaction with primary amine groups of the polyethyleneimine.
Based on the technical scheme, the weight average molecular weight of the polyethyleneimine is preferably 10-80 kDa.
On the basis of the above technical scheme, preferably, the structural formula of the modified polyethyleneimine is as follows:
wherein n is a positive integer.
In another aspect of the present invention, there is also provided a method for preparing a modified polyethyleneimine, comprising the steps of:
mixing polyethyleneimine and dodecyl succinic anhydride according to a mass ratio of 1: (0.1-2) mixing, and reacting for 2-12h at the temperature of 25-42 ℃ to obtain modified polyethyleneimine, wherein the structural formula of the polyethyleneimine is as follows:
wherein n is a positive integer.
On the basis of the above technical solution, preferably, the polyethyleneimine is subjected to activation treatment, and the activation treatment method includes dissolving polyethyleneimine in a first solvent to prepare a first solution with a mass concentration of 1-10%, adding a second solvent with a volume of 20-50% to the obtained first solution, and stirring to obtain an activated polyethyleneimine solution, where the first solvent is one of DMF, DMSO, acetonitrile, and ethylene glycol, and the second solvent is one of isopropanol, methanol, and pentanediol.
On the basis of the above technical scheme, preferably, the specific method of the coupling reaction comprises: dripping dodecyl succinic anhydride into the activated polyethyleneimine solution at 25-42 ℃, after dripping, preserving heat and reacting for 2-12h, and dialyzing the obtained solution in a dialysis bag with a cut-off pore diameter of 3500Da for 24h with distilled water to obtain the modified polyethyleneimine.
In another aspect of the present invention, there is also provided a transfection reagent, which includes a solvent, and the modified polyethyleneimine described in the above scheme or the polyethyleneimine prepared by the above preparation method dissolved in the solvent.
On the basis of the above technical scheme, preferably, the mass ratio of the modified polyethyleneimine to the solvent is 1: (10-100).
On the basis of the above technical solution, preferably, the solvent is ultrapure water.
In another aspect of the present invention, there is also provided a use of the transfection reagent described above for delivering deoxyribonucleic acid, plasmid DNA, ribonucleic acid, or functional protein.
Compared with the prior art, the invention has the following beneficial effects:
(1) the modified polyethyleneimine provided by the invention utilizes acetic anhydride to acylate primary amine groups in the polyethyleneimine, so that the electrostatic effect is inhibited, the buffering capacity of the obtained modified polyethyleneimine is reduced, and the transfection capacity of the modified polyethyleneimine is improved;
(2) according to the invention, the polyethyleneimine is modified by adopting the dodecyl succinic anhydride, so that the hydrophilic property of the finally prepared modified polyethyleneimine can be changed, under a lower substitution degree, carboxyl groups in the ammonium dodecyl succinate endow polyethyleneimine negative charges, electrostatic shielding is provided for a complex obtained by compounding the polyethyleneimine negative charges with DNA, the combination with non-specific protein can be reduced, and positive charges carried by unmodified amino groups in the modified polyethyleneimine can interact with cell membranes with negative charges, so that the modified polyethyleneimine-DNA complex is phagocytized by cells. The interaction of the hydrophobic groups, in turn, allows the DNA to be more easily released and gene expressed after the complex has entered the cell.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph of the viability of HEK293 cells when treated with the transfection reagent of embodiment 1;
FIG. 2 is a fluorescent image of transfection of GFP protein with the transfection reagent of embodiment 6;
FIG. 3 is a fluorescent image of transfection reagent of embodiment 1 with GFP protein.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The modified polyethyleneimine of one embodiment of the invention comprises polyethyleneimine serving as a main chain and dodecyl succinic anhydride finger-ring on the polyethyleneimine, wherein the dodecyl succinic anhydride and a primary amine group of the polyethyleneimine are subjected to coupling reaction, and the specific reaction formula is as follows:
specifically, the weight average molecular weight of the polyethyleneimine is 10-80 kDa.
N in the above structural formula is a positive integer
Example 1
Weighing 1 part of polyethyleneimine with the weight-average molecular weight of 10kDa, dissolving the polyethyleneimine in 9 parts of DMF solvent, uniformly stirring to obtain a DMF solution of polyethyleneimine with the mass concentration of 10%, weighing isopropanol with the volume of 20% of the DMF solution, adding the isopropanol into the DMF solution of polyethyleneimine, and uniformly stirring to obtain an activated polyethyleneimine solution;
0.1 part of dodecyl succinic anhydride is weighed and added into the activated polyethyleneimine solution in a dropwise manner at 25 ℃, after the dropwise addition is finished, the temperature is kept at 25 ℃, the stirring reaction is carried out for 12 hours, after the reaction is finished, the obtained solution is dialyzed for 24 hours by distilled water in a dialysis bag with the cut-off aperture of 3500Da, and the modified polyethyleneimine is obtained.
The structural formula of the obtained modified polyethyleneimine is as follows:
example 2
Weighing 1 part of polyethyleneimine with the weight-average molecular weight of 25kDa, dissolving the polyethyleneimine in 11.5 parts of DMSO solvent, uniformly stirring to obtain a DMSO solution of the polyethyleneimine with the mass concentration of 8%, weighing methanol with the volume of 30% of the DMSO solution, adding the methanol into the DMSO solution of the polyethyleneimine, and uniformly stirring to obtain an activated polyethyleneimine solution;
0.5 part of dodecyl succinic anhydride is weighed and added into the activated polyethyleneimine solution in a dropwise manner at 30 ℃, after the dropwise addition is finished, the solution is kept at 30 ℃ for stirring and reacting for 8 hours, and after the reaction is finished, the obtained solution is dialyzed for 24 hours by distilled water in a dialysis bag with a cut-off pore diameter of 3500Da, so that the modified polyethyleneimine is obtained.
Example 3
Weighing 1 part of polyethyleneimine with the weight-average molecular weight of 40kDa, dissolving the polyethyleneimine in 19 parts of acetonitrile solvent, uniformly stirring to obtain an acetonitrile solution of the polyethyleneimine with the mass concentration of 5%, weighing pentanediol with the volume of 40% of the acetonitrile solution, adding the pentanediol into the acetonitrile solution of the polyethyleneimine, and uniformly stirring to obtain an activated polyethyleneimine solution;
weighing 1 part of dodecyl succinic anhydride, dropwise adding the dodecyl succinic anhydride into the activated polyethyleneimine solution at 30 ℃, keeping the temperature of 35 ℃ after dropwise adding, stirring and reacting for 6 hours, and dialyzing the obtained solution for 24 hours by using distilled water in a dialysis bag with a cut-off pore diameter of 3500Da to obtain the modified polyethyleneimine.
Example 4
Weighing 1 part of polyethyleneimine with the weight-average molecular weight of 50kDa, dissolving the polyethyleneimine in 49 parts of glycol solvent, uniformly stirring to obtain a glycol solution of the polyethyleneimine with the mass concentration of 2%, weighing pentanediol with the volume of 50% of the glycol solution, adding the pentanediol solution into the glycol solution of the polyethyleneimine, and uniformly stirring to obtain an activated polyethyleneimine solution;
weighing 2 parts of dodecyl succinic anhydride, dropwise adding the dodecyl succinic anhydride into the activated polyethyleneimine solution at 30 ℃, keeping the temperature of 40 ℃ after dropwise adding, stirring for reacting for 4 hours, and dialyzing the obtained solution for 24 hours by using distilled water in a dialysis bag with a cut-off pore diameter of 3500Da to obtain the modified polyethyleneimine.
Example 5
Weighing 1 part of polyethyleneimine with the weight-average molecular weight of 80kDa, dissolving the polyethyleneimine in 99 parts of glycol solvent, uniformly stirring to obtain a glycol solution of the polyethyleneimine with the mass concentration of 1%, weighing pentanediol with the volume of 50% of the glycol solution, adding the pentanediol solution into the glycol solution of the polyethyleneimine, and uniformly stirring to obtain an activated polyethyleneimine solution;
weighing 2 parts of dodecyl succinic anhydride, dropwise adding the dodecyl succinic anhydride into the activated polyethyleneimine solution at 30 ℃, keeping the temperature of 42 ℃ after dropwise adding, stirring for reacting for 2 hours, and dialyzing the obtained solution for 24 hours by using distilled water in a dialysis bag with a cut-off pore diameter of 3500Da to obtain the modified polyethyleneimine.
The modified polyethyleneimine obtained in example 1 and ultrapure water were mixed in a mass ratio of 1:100 to obtain the transfection reagent 1.
The modified polyethyleneimine obtained in example 2 and ultrapure water were mixed in a mass ratio of 1: 80 to obtain the transfection reagent 2.
The modified polyethyleneimine obtained in example 3 and ultrapure water were mixed in a mass ratio of 1: 60 to obtain the transfection reagent 3.
The modified polyethyleneimine obtained in example 4 and ultrapure water were mixed in a mass ratio of 1: 40 to obtain the transfection reagent 4.
Embodiment 5
The modified polyethyleneimine obtained in example 5 and ultrapure water were mixed in a mass ratio of 1:10 to obtain the transfection reagent 5.
Embodiment 6
The raw material polyethyleneimine of example 1 was mixed with ultrapure water at a mass ratio of 1:100 to obtain a transfection reagent 6.
Cytotoxicity:
the more primary amines reported by the polymer, the more cationic charges carried by the polymer, and the greater the cytotoxicity.
Free primary amine groups of the modified Polyethyleneimine (PEILS) prepared by the preparation method are replaced, and compared with PEI, the toxicity of PEI on cells is obviously reduced.
The specific verification is as follows:
the MTT method was used to compare the cytotoxicity of the PEILS transfection reagent 1 according to example 1, the PEI transfection reagent 6 according to example 6, and the commercial transfection reagent Lipofectamine 2000. Within 24h before the experiment, HEK293 cells in log phase were taken, trypsinized and diluted with DMEM at 1 x 10 per well 4 The density of the cells is inoculated in a 96-hole culture plate, and the culture plate is placed in an incubator containing 5% carbon dioxide by volume at 37 ℃ for continuous culture until the confluence reaches 80-90%. After materials with different concentrations and cells are cultured together for 24 hours, 20 mu L of PBS solution containing 0.5% of MTT by mass fraction is added into each hole, the mixture continuously acts for 4 hours at 37 ℃, 200 mu L of dimethyl sulfoxide is added to dissolve MTT formazan crystals for 10min, then absorption is tested by an enzyme-labeling instrument, the wavelength is 492nm, and the cell survival rate is calculated according to the following formula:
cell survival rate (%) ═ a sample /A control )*100％。
Wherein A is sample For uptake into the fine guard wells after transfection, A control For the uptake of the wells of the cell sample that did not react with the complex solution, each set of experiments was repeated 3 times and the results are shown in fig. 1.
As can be seen from the figure, the transfection reagent of the present invention has lower toxicity compared to PEI.
Transfection efficiency:
fluorescence microscopy of the endocytosis of the GFP-labeled transfection reagent 6/BSA complex: according to 2.0×10 5 Per well inoculum HEK293 cells were seeded into 6-well plates supplemented to 2mL with complete medium in each well, and the 6-well plates were incubated for 12h at 37 ℃ in a 5% carbon dioxide cell incubator. The supernatant was carefully discarded, washed 2 times with phosphate buffer (50mM, pH 7.4), observed with IX73P1F fluorescence microscope (olympus) and photographed. The results are shown in FIG. 2.
Fluorescence microscopy of the endocytosis of the GFP-labeled transfection reagent 1/BSA complex: according to 2.0X 10 5 Per well inoculum HEK293 cells were seeded into 6-well plates supplemented to 2mL with complete medium in each well, and the 6-well plates were incubated for 12h at 37 ℃ in a 5% carbon dioxide cell incubator. The supernatant was carefully discarded, washed 2 times with phosphate buffer (50mM, pH 7.4), observed with an IX73P1F fluorescence microscope (olympus) and photographed. The results are shown in FIG. 3.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A modified polyethyleneimine, comprising: the main chain of the polyurethane comprises polyethyleneimine serving as a main chain and dodecyl succinic anhydride grafted on the polyethyleneimine, wherein the dodecyl succinic anhydride is subjected to coupling reaction with primary amine groups of the polyethyleneimine.
2. The modified polyethyleneimine according to claim 1, wherein the weight average molecular weight of the polyethyleneimine is 10 to 80 kDa.
3. The modified polyethyleneimine of claim 1, wherein the modified polyethyleneimine has the structural formula:
wherein n is a positive integer.
4. A preparation method of modified polyethyleneimine is characterized by comprising the following steps:
mixing polyethyleneimine and dodecyl succinic anhydride according to a mass ratio of 1: (0.1-2) mixing, and performing coupling reaction for 2-12h at the temperature of 25-42 ℃ to obtain modified polyethyleneimine, wherein the structural formula of the polyethyleneimine is as follows:
wherein n is a positive integer.
5. The method of claim 4, wherein the polyethyleneimine is subjected to activation treatment, and the activation treatment comprises dissolving polyethyleneimine in a first solvent to prepare a first solution with a mass concentration of 1-10%, adding a second solvent with a volume of 20-50% to the obtained first solution, and stirring to obtain an activated polyethyleneimine solution, wherein the first solvent is one of DMF, DMSO, acetonitrile and ethylene glycol, and the second solvent is one of isopropanol, methanol and pentanediol.
6. The process for preparing modified polyethyleneimine according to claim 5, wherein the coupling reaction is carried out by a specific method comprising: dripping dodecyl succinic anhydride into the activated polyethyleneimine solution at 25-42 ℃, after dripping, preserving heat and reacting for 2-12h, and dialyzing the obtained solution in a dialysis bag with a cut-off pore diameter of 3500Da for 24h with distilled water to obtain the modified polyethyleneimine.
7. A transfection reagent comprising a solvent and a modified polyethyleneimine according to any one of claims 1 to 3, or prepared by the method according to any one of claims 4 to 6, dissolved in the solvent.
8. A transfection reagent according to claim 7, wherein the mass ratio of the modified polyethyleneimine to the solvent is 1: (10-100).
9. A transfection reagent according to claim 7, wherein the solvent is ultrapure water.
10. Use of the transfection reagent of any one of claims 7-9 for the delivery of deoxyribonucleic acid, plasmid DNA, ribonucleic acid, or functional proteins.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106000364A (en) * | 2016-05-24 | 2016-10-12 | 天津大学 | Succinic anhydride modified polymine grafting medium, preparation method and application thereof |
| JP2018074984A (en) * | 2016-11-11 | 2018-05-17 | 国立大学法人北陸先端科学技術大学院大学 | Method for producing transgenic cell and polyethyleneimine substitution polymer |
| CN113968968A (en) * | 2020-04-15 | 2022-01-25 | 深圳深信生物科技有限公司 | Amino lipid compound, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106000364A (en) * | 2016-05-24 | 2016-10-12 | 天津大学 | Succinic anhydride modified polymine grafting medium, preparation method and application thereof |
| JP2018074984A (en) * | 2016-11-11 | 2018-05-17 | 国立大学法人北陸先端科学技術大学院大学 | Method for producing transgenic cell and polyethyleneimine substitution polymer |
| CN113968968A (en) * | 2020-04-15 | 2022-01-25 | 深圳深信生物科技有限公司 | Amino lipid compound, preparation method and application thereof |
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Application publication date: 20220913 |