CN107661510B - Methoxy BODIPY-nucleic acid-ferroferric oxide compound and preparation method thereof - Google Patents
Methoxy BODIPY-nucleic acid-ferroferric oxide compound and preparation method thereof Download PDFInfo
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims description 5
- -1 methoxy boron fluorine Chemical compound 0.000 claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 14
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 9
- 239000002105 nanoparticle Substances 0.000 claims abstract description 8
- 239000012216 imaging agent Substances 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 68
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 56
- 239000011259 mixed solution Substances 0.000 claims description 42
- 239000011718 vitamin C Substances 0.000 claims description 34
- 238000002156 mixing Methods 0.000 claims description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 30
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 28
- 229930003268 Vitamin C Natural products 0.000 claims description 28
- 235000019154 vitamin C Nutrition 0.000 claims description 28
- 239000001509 sodium citrate Substances 0.000 claims description 21
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 17
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 13
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 206010006187 Breast cancer Diseases 0.000 claims description 10
- 208000026310 Breast neoplasm Diseases 0.000 claims description 10
- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 claims description 10
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 10
- 239000012498 ultrapure water Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 5
- 230000000087 stabilizing effect Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 abstract description 4
- 239000007850 fluorescent dye Substances 0.000 abstract 1
- 238000012632 fluorescent imaging Methods 0.000 abstract 1
- 238000010189 synthetic method Methods 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 48
- 108020004414 DNA Proteins 0.000 description 24
- 239000000872 buffer Substances 0.000 description 14
- 238000000502 dialysis Methods 0.000 description 12
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 8
- 229940032296 ferric chloride Drugs 0.000 description 8
- 230000009471 action Effects 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 102000053602 DNA Human genes 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000002114 nanocomposite Substances 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940044631 ferric chloride hexahydrate Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 1
- WVLDCUJMGWFHGE-UHFFFAOYSA-L iron(2+);sulfate;hexahydrate Chemical compound O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O WVLDCUJMGWFHGE-UHFFFAOYSA-L 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229940083608 sodium hydroxide Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- PKIDNTKRVKSLDB-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;hydrate Chemical compound O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O PKIDNTKRVKSLDB-UHFFFAOYSA-K 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a fluorescence imaging agent, in particular to methoxy boron fluorine pyrrole-nucleic acid-tetraoxideA ferroferric compound and a synthetic method. Takes DNA40-1 and DNA40-1D small molecular nucleic acid (DNA) as templates to synthesize novel fluorescent DNA-Fe3O4And (3) nanoparticles. Synthesized novel fluorescent DNA-Fe3O4A nanoparticle; the size of the nano particles is 2-8 nm; the aqueous solution of the fluorescent dye is dark yellow, and is excited by green light of 500nm, and fluorescence emission is generated at 540nm, so that the methoxy BODIPY @ nucleic acid-ferroferric oxide compound is a sensitive fluorescent imaging agent.
Description
Technical Field
The invention relates to a fluorescence imaging agent, in particular to a methoxy BODIPY-nucleic acid-ferroferric oxide compound and a synthesis method thereof, belongs to the preparation and application aspects of nucleic acid nano materials, and particularly reports that a novel nucleic acid-ferroferric oxide compound particle carries methoxy BODIPY and is applied to breast cancer cell imaging.
Background
Cancer has long been listed as a malignant disease that threatens the quality of life and health of people, and if treated in a timely manner, the chance of cure is considerable].Cancer Epidemiol Biomarkers Prev,2016,25:16-27.]. Therefore, the early diagnosis and treatment of the breast cancer are realized, and the method has an extremely important effect on improving the cure rate of breast cancer patients. At present, B-mode ultrasound and MRI are commonly used for the image detection of breast cancer, but both have respective limitations. [ Ali S, Mondal N, Choudhury H, et al, Current management Strategies in Breast Cancer by Targeting Key Altered molecular Players [ J].Front Oncol,2016,6:45.]Nanotechnology, such as drug carriers, biosensors, and nanoimaging techniques, have important roles in the diagnosis and treatment of diseases. BODIPY-OCH3@DNA-Fe3O4As a novel fluorescent nano material, the fluorescent nano material has great potential in the field of cell imaging based on simple synthesis method, small particles, good stability, biocompatibility and low toxicity. Double stranded DNA (dsDNA) carrying magnetic Fe3O4The surface of the nano-particles is positively charged and has a very large specific surface area, so that the pen-shaped pen is buried for further design and application in the later period. Double strand at the same time DNA (dsDNA), which is rich in-GC-base pairs that can be used for drug loading and delivery, [ Juan L, Tuo W, et al].Biomaterials 91(2016)44-56.]. In summary, this functional BODIPY-OCH3@DNA-Fe3O4The nano-composite provides a new thought and method for the design of nano-carriers, and has great potential in the aspects of early detection of cancer cells, drug delivery and treatment.
Disclosure of Invention
We expressed a sequence of DNA 40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA 40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecular nucleic acid (DNA) as template, which is obtained from Shanghai biological engineering Co., Ltd, and synthesizes a novel fluorescent DNA-Fe3O4Nanoparticles (labeled BODIPY-OCH)3@DNA-Fe3O4) (ii) a The size of the nano particles is 2-8 nm; the aqueous solution of the fluorescent powder shows dark yellow, is excited by green light of 500nm, and has fluorescence emission at 540nm, so that the methoxy BODIPY @ nucleic acid-ferroferric oxide complex (BODIPY-OCH) is shown3@DNA-Fe3O4) Is a sensitive fluorescence imaging agent, and the BODIPY-OCH can be seen from figure 23@DNA-Fe3O4MCF-7 (breast cancer) cells can be subjected to fluorescence imaging.
Novel fluorescent DNA-Fe3O4Nano-composite (BODIPY-OCH)3@DNA-Fe3O4) The synthesis reaction process comprises the following steps: respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1Mixing with ferric trichloride solution (10mM) in sodium citrate buffer solution to obtain mixture I, DNA40-1With Fe3+The concentrations of (A) are 6.4. mu.M and 1800-2300. mu.M (most preferably 2000. mu.M), respectively, and the DNA is subjected to40-1DMixing vitamin C solution (10mM) and ferrous ammonium sulfate solution (10mM) in sodium citrate buffer solution to obtain mixture II, and mixing DNA40-1DAnd, vitamin C and Fe2+The concentrations of (A) are 6.4. mu.M, 1000. mu.M and 1000-1500. mu.M (most preferably 1110. mu.M); mixing the above two solutions respectivelyHeating the solution in water bath, heating to 60-70 deg.C (preferably Tm value of DNA 67 deg.C), stabilizing for three minutes, rapidly mixing the first and second mixed solutions, rapidly dripping ammonia water (pH 12), shaking in shaking table for 10-120 min (preferably 30min), slowly cooling to room temperature for 4-6h (preferably 5h), dripping vitamin C (10mM) solution into the above solution, mixing, storing at room temperature for 5min in dark place, storing at 4 deg.C in dark place for 4h, and dripping 96 μ L methoxy BODIPY solution (1mM), wherein the amount of BODIPY is DNA40-17.5 times of the amount of the substance, dialyzing in DMSO solvent for 3h after acting for 3h, and dialyzing in ultrapure water for 12 h; and (3) concentrating to obtain the stable methoxy BODIPY @ nucleic acid-ferroferric oxide.
The pH value of the sodium citrate buffer solution is 9.0, and the concentration is 10 mM.
The volume ratio of the first mixed solution to the second mixed solution to the ammonia water to the dripped vitamin C is 1014: 946: 20: 20.
the final volume of the reaction solution was 2m L.
The invention has the advantages that:
the invention designs novel methoxy boron pyrrole @ nucleic acid-ferroferric oxide based on micromolecular nucleic acid sequences 5'-GAGGAGACAACAACAGCGCGCGC-3' and 5'-GCGCGCGCACAACAACAGAGGAG-3', and the result shows that the prepared novel methoxy boron pyrrole @ nucleic acid-ferroferric oxide can well perform fluorescence imaging on MCF-7 (breast cancer) cells; the invention is based on the small molecule nucleic acid 5'-GAGGAG-3' sequence which can be well matched with Fe for the first time in the world3+/Fe2+The method is a convenient, rapid, visual and economic imaging method, and provides theoretical and technical support for further research and treatment of tumor cells.
Drawings
FIG. 1 shows BODIPY-OCH3@DNA-Fe3O4Transmission electron micrograph (D).
FIG. 2 shows BODIPY-OCH3@DNA-Fe3O4Fluorescence imaging of MCF-7 (breast cancer) cells.
Detailed Description
Reagents and raw materials
All solvents used in the reaction are analytically pure, and the used reagents are directly used without special instructions and without any special treatment; sodium citrate monohydrate, ferrous sulfate hexahydrate, ferric chloride hexahydrate, sodium hydroxide, vitamin C were purchased from the national pharmaceutical group chemical reagents, Inc., DNA sequences (Shanghai Biotechnology engineering, Inc.).
An ultraviolet visible spectrophotometer (Shimadzu UV-2450, Japan), 190-.
The synthesis method of the compound comprises the following steps:
example 1 (optimal synthesis conditions):
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DMixing the vitamin C solution and the ferrous ammonium sulfate solution in a sodium citrate buffer solution (pH 9, 10mM),obtaining the second mixture, DNA40-1DAnd, vitamin C and Fe2+The concentration of the two solutions is 6.4 mu M, 1000 mu M and 1110 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 67 ℃ and then stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixed solution is transferred to a shaking table for shaking for 30 minutes, the temperature is slowly reduced to room temperature, the temperature reduction time is 5 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixed solution is uniformly mixed and stored in a room temperature dark place for 5 minutes, then the mixed solution is transferred to a4 ℃ dark place for storage for 4 hours, then the methoxy BODIPY solution is dripped, the mixed solution is placed in a DMSO solvent for dialysis for 3 hours after the action is carried out for 3 hours, the dialyzed in ultrapure water for 12 hours, and the stable.
Example 2:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 5, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DVitamin C solution and ferrous ammonium sulfate solution were mixed in sodium citrate buffer (pH 5, 10mM) to obtain mixture II, and DNA was added40-1DAnd, vitamin C and Fe2+The concentration of the two solutions is 6.4 mu M, 2000 mu M and 2000 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 70 ℃ and then is stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixed solution is transferred to a shaking table for shaking for 2 hours, the temperature is slowly reduced to the room temperature, the temperature reduction time is 6 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixed solution is uniformly mixed and is kept in the room temperature for 5 minutes in a dark place, then the mixed solution is transferred to the 4 ℃ for 4 hours in a dark place, then methoxy BODIPY solution is dripped, after 3 hours of action, the mixed solution is placed in a DMSO solvent for dialysis for 3 hours, the dialyzed for 12 hours in ultrapure water, and the.
Example 3:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 7, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DVitamin C solution and ferrous ammonium sulfate solution were mixed in sodium citrate buffer (pH 7, 10mM) to obtain mixture II, and DNA was added40-1DAnd, vitamin C and Fe2+The concentration of the mixture is 6.4 mu M, 2000 mu M and 2000 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 70 ℃ and then is stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixture is transferred to a shaking table for shaking for 2 hours, the temperature is slowly reduced to the room temperature, the temperature reduction time is 6 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixture is uniformly mixed and is kept in the room temperature for 5 minutes in the dark place, then the mixture is transferred to the 4 ℃ for 4 hours in the dark place, then the methoxy BODIPY solution is dripped, after 3 hours of action, the mixture is placed in a DMSO solvent for dialysis for 3 hours, the mixture is placed in ultrapure water for dialysis for 12 hours, and the.
Example 4:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 10, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DVitamin C solution and ferrous ammonium sulfate solution were mixed in sodium citrate buffer (pH 10, 10mM) to obtain mixture II, and DNA was added40-1DAnd, vitamin C and Fe2+Respectively at 6.4 μ M, 2000 μ M and 2000 μ M, heating in water bath, heating to 70 deg.C, stabilizing for three minutes, quickly mixing the first and second mixed solutions, quickly adding 20 μ L ammonia water (pH 12), shaking in shaking table for 2 hr, slowly cooling to room temperature for 6 hr, and mixing at mostThen dripping 20 mu L vitamin C (10mM) solution into the solution, mixing uniformly, storing for 5min at room temperature in the dark, transferring to 4 ℃ in the dark, storing for 4h, dripping methoxy boron pyrrole solution, acting for 3h, putting into DMSO solvent, dialyzing for 3h, putting into ultrapure water, dialyzing for 12h, and concentrating to obtain the stable methoxy boron pyrrole @ nucleic acid-ferroferric oxide.
Example 5:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DVitamin C solution and ferrous ammonium sulfate solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture II, and DNA was added40-1DAnd, vitamin C and Fe2+The concentration of the mixture is 6.4 mu M, 1000 mu M and 1110 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 70 ℃ and then is stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixture is transferred to a shaking table for shaking for 2 hours, the temperature is slowly reduced to the room temperature, the temperature reduction time is 5 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixture is uniformly mixed and is kept in the room temperature for 5 minutes in the dark place, then the mixture is transferred to the 4 ℃ for 4 hours in the dark place, then the methoxy BODIPY solution is dripped, after 3 hours of action, the mixture is placed in a DMSO solvent for dialysis for 3 hours, the mixture is placed in ultrapure water for dialysis for 12 hours, and after.
Example 6:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DMixing vitamin C solution and ferrous ammonium sulfate solution in lemonSodium citrate buffer (pH 9, 10mM) to obtain mixture II, DNA40-1DAnd, vitamin C and Fe2+The concentration of the mixture is 6.4 mu M, 1000 mu M and 1110 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 65 ℃ and then is stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixture is transferred to a shaking table for shaking for 2 hours, the temperature is slowly reduced to room temperature, the temperature reduction time is 4 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixture is uniformly mixed and is kept in the room temperature for 5 minutes in the dark place, then the mixture is transferred to the 4 ℃ for 4 hours in the dark place, then the methoxy BODIPY solution is dripped, after 3 hours of action, the mixture is placed in a DMSO solvent for dialysis for 3 hours, the mixture is placed in ultrapure water for dialysis for 12 hours, and the stable.
Example 7:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DVitamin C solution and ferrous ammonium sulfate solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture II, and DNA was added40-1DAnd, vitamin C and Fe2+The concentration of the mixture is 6.4 mu M, 1000 mu M and 1110 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 67 ℃ and then stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixture is transferred to a shaking table for shaking for 15 minutes, then the temperature is slowly reduced to the room temperature, the temperature reduction time is 5 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixture is uniformly mixed and stored in the room temperature for 5 minutes in a dark place, then the mixture is transferred to the 4 ℃ for 4 hours in a dark place, then the methoxy BODIPY solution is dripped, the mixture is placed in a DMSO solvent for dialysis for 3 hours after the action is carried out for 3 hours, the mixture is placed in ultrapure water for dialysis for 12 hours.
Example 8:
respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1And ferric chloride solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture one, DNA40-1With Fe3+Respectively at a concentration of 6.4. mu.M and 2000. mu.M, and mixing the DNAs40-1DVitamin C solution and ferrous ammonium sulfate solution were mixed in sodium citrate buffer (pH 9, 10mM) to obtain mixture II, and DNA was added40-1DAnd, vitamin C and Fe2+The concentration of the mixture is 6.4 mu M, 1000 mu M and 1110 mu M respectively, then the two mixed solutions are transferred to a water bath for heating, the temperature is raised to 67 ℃ and then stabilized for three minutes, the first mixed solution and the second mixed solution are quickly and uniformly mixed, 20 mu L ammonia water (pH is 12) is quickly dripped, the mixture is transferred to a shaking table for shaking for 1 hour, the temperature is slowly reduced to the room temperature, the temperature reduction time is 5 hours, finally 20 mu L vitamin C (10mM) solution is dripped into the solution, the mixture is uniformly mixed and stored in the room temperature for 5 minutes in a dark place, then the mixture is transferred to the 4 ℃ for storage in the dark place for 4 hours, then the methoxy BODIPY solution is dripped, the mixture is placed in a DMSO solvent for dialysis for 3 hours after the action is carried out for 3 hours, the mixture is placed in ultrapure water for dialysis for 12 hours.
BODIPY-OCH3@DNA-Fe3O4The particle size of the nanocomposite is 2-8nm, as shown in FIG. 1.
BODIPY-OCH3@DNA-Fe3O4Fluorescence imaging of MCF-7 (breast cancer) cells was achieved as shown in FIG. 2.
5'-GAGGAGACAACAACAGCGCGCGC-3'
5'-GCGCGCGCACAACAACAGAGGAG-3'
Claims (7)
1. The preparation method of the methoxy BODIPY-nucleic acid-ferroferric oxide compound comprises the following steps of (1) preparing a methoxy BODIPY-nucleic acid-ferroferric oxide compound, wherein the methoxy BODIPY-nucleic acid-ferroferric oxide compound is a nanoparticle, and the size of the nanoparticle is 2-8 nm; the aqueous solution of the compound shows dark yellow, is excited by green light of 500nm, has fluorescence emission at 540nm, is a sensitive fluorescence imaging agent, can carry out fluorescence imaging on breast cancer cells, and is characterized in thatThe preparation method comprises the following specific steps: respectively with DNA40-1: 5'-GAGGAGACAACAACAGCGCGCGC-3' and DNA40-1D: 5'-GCGCGCGCACAACAACAGAGGAG-3' small molecule nucleic acid as template, and mixing the DNA with the template40-1Mixing with ferric trichloride solution in sodium citrate buffer solution to obtain first mixed solution, DNA40-1With Fe3 +The concentrations of (a) and (b) were 6.4. mu.M and 1800-2300. mu.M, respectively, and the DNA was purified40-1DMixing vitamin C solution and ferrous ammonium sulfate solution in sodium citrate buffer solution to obtain No. two mixed solution, DNA40-1DAnd, vitamin C and Fe2+The concentrations of (A) were 6.4. mu.M, 1000. mu.M and 1000-1500. mu.M, respectively; then respectively transferring the two mixed solutions to a water bath for heating, heating to 60-70 ℃, stabilizing for three minutes, quickly and uniformly mixing the first mixed solution and the second mixed solution, quickly dripping ammonia water, transferring to a shaking table, shaking for 10-120 min, slowly cooling to room temperature, and cooling for 4-6 h; finally, dripping vitamin C solution into the solution, uniformly mixing, storing at room temperature for 5min in the dark, then transferring to 4 ℃ in the dark, storing for 4h, and then dripping methoxy boron pyrrole solution, wherein the amount of methoxy boron pyrrole is DNA40-17.5 times of the amount of the substance, dialyzing in DMSO solvent for 3h after acting for 3h, and dialyzing in ultrapure water for 12 h; and (3) concentrating to obtain the stable methoxy BODIPY @ nucleic acid-ferroferric oxide.
2. The method for preparing methoxy BODIPY-nucleic acid-ferroferric oxide compound according to claim 1, wherein the method comprises the following steps: the pH value of the sodium citrate buffer solution is 9.0, and the concentration is 10 mM.
3. The method for preparing methoxy BODIPY-nucleic acid-ferroferric oxide compound according to claim 1, wherein the method comprises the following steps: in the first mixed solution, Fe3+The concentration of (2) was 2000. mu.M.
4. The method for preparing methoxy BODIPY-nucleic acid-ferroferric oxide compound according to claim 1, wherein the method comprises the following steps: in the second mixed solution, Fe2+The concentration of (2) was 1110. mu.M.
5. The method for preparing methoxy BODIPY-nucleic acid-ferroferric oxide compound according to claim 1, wherein the method comprises the following steps: the water bath heating temperature was 67 ℃.
6. The method for preparing methoxy BODIPY-nucleic acid-ferroferric oxide compound according to claim 1, wherein the method comprises the following steps: the pH value of the ammonia water is 12, the concentration of the dripped vitamin C solution is 10mM, the concentration of the methoxy boron-pyrrole solution is 1mM, and the volume ratio of the first mixed solution to the second mixed solution to the ammonia water to the vitamin C solution is 1014: 946: 20: 20.
7. the method for preparing methoxy BODIPY-nucleic acid-ferroferric oxide compound according to claim 1, wherein the method comprises the following steps: the shaking time is 30 min; the cooling time is 5 h.
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