CN110856747A - Photosensitizer activated by hydrogen peroxide and preparation method and application thereof - Google Patents
Photosensitizer activated by hydrogen peroxide and preparation method and application thereof Download PDFInfo
- Publication number
- CN110856747A CN110856747A CN201810937694.XA CN201810937694A CN110856747A CN 110856747 A CN110856747 A CN 110856747A CN 201810937694 A CN201810937694 A CN 201810937694A CN 110856747 A CN110856747 A CN 110856747A
- Authority
- CN
- China
- Prior art keywords
- hydrogen peroxide
- photosensitizer
- solution
- probe
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 216
- 239000003504 photosensitizing agent Substances 0.000 title claims abstract description 94
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims abstract description 21
- 229960000907 methylthioninium chloride Drugs 0.000 claims abstract description 21
- 238000003384 imaging method Methods 0.000 claims abstract description 16
- 238000001035 drying Methods 0.000 claims abstract description 13
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 239000012046 mixed solvent Substances 0.000 claims abstract description 7
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 claims abstract description 5
- 238000001816 cooling Methods 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 49
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 39
- 239000002105 nanoparticle Substances 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 102000009027 Albumins Human genes 0.000 claims description 18
- 108010088751 Albumins Proteins 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 15
- 238000000799 fluorescence microscopy Methods 0.000 claims description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 7
- GZZBZWITJNATOD-UHFFFAOYSA-N [4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanol Chemical compound O1C(C)(C)C(C)(C)OB1C1=CC=C(CO)C=C1 GZZBZWITJNATOD-UHFFFAOYSA-N 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- UCPYLLCMEDAXFR-UHFFFAOYSA-N triphosgene Chemical compound ClC(Cl)(Cl)OC(=O)OC(Cl)(Cl)Cl UCPYLLCMEDAXFR-UHFFFAOYSA-N 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 2
- 229940041181 antineoplastic drug Drugs 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims 1
- 229940124597 therapeutic agent Drugs 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 41
- 238000002428 photodynamic therapy Methods 0.000 abstract description 30
- 201000011510 cancer Diseases 0.000 abstract description 21
- 238000011282 treatment Methods 0.000 abstract description 16
- 230000000694 effects Effects 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
- 238000001308 synthesis method Methods 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 50
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 30
- 241000699670 Mus sp. Species 0.000 description 25
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 18
- 229960003180 glutathione Drugs 0.000 description 17
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 16
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 13
- 108010024636 Glutathione Proteins 0.000 description 12
- 102000016938 Catalase Human genes 0.000 description 10
- 108010053835 Catalase Proteins 0.000 description 10
- 238000005286 illumination Methods 0.000 description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 9
- 239000001301 oxygen Substances 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 9
- 230000009471 action Effects 0.000 description 8
- 230000003834 intracellular effect Effects 0.000 description 8
- LGRLWUINFJPLSH-UHFFFAOYSA-N methanide Chemical compound [CH3-] LGRLWUINFJPLSH-UHFFFAOYSA-N 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 7
- 238000001819 mass spectrum Methods 0.000 description 7
- 239000003642 reactive oxygen metabolite Substances 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 230000037396 body weight Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 230000021615 conjugation Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000002516 radical scavenger Substances 0.000 description 5
- 231100000331 toxic Toxicity 0.000 description 5
- 230000002588 toxic effect Effects 0.000 description 5
- 150000001642 boronic acid derivatives Chemical group 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000009616 inductively coupled plasma Methods 0.000 description 4
- -1 4- (4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl Chemical group 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 230000036542 oxidative stress Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002591 computed tomography Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 150000002148 esters Chemical group 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000004575 stone Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- QGVLYPPODPLXMB-UBTYZVCOSA-N (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-5H-cyclopropa[3,4]benzo[1,2-e]azulen-5-one Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(O)C(C)(C)[C@H]3[C@@H]21 QGVLYPPODPLXMB-UBTYZVCOSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000370001 Hantavirus Liu Species 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 235000010678 Paulownia tomentosa Nutrition 0.000 description 1
- 240000002834 Paulownia tomentosa Species 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000008810 intracellular oxidative stress Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- CSMWJXBSXGUPGY-UHFFFAOYSA-L sodium dithionate Chemical compound [Na+].[Na+].[O-]S(=O)(=O)S([O-])(=O)=O CSMWJXBSXGUPGY-UHFFFAOYSA-L 0.000 description 1
- 229940075931 sodium dithionate Drugs 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Radiology & Medical Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a photosensitizer activated by hydrogen peroxide and a preparation method and application thereof. The structural formula of the photosensitizer is shown as a formula I, and the preparation method comprises the following steps: (1) adding methylene blue, sodium bicarbonate and sodium hydrosulfite into a mixed solvent, reacting at 40-60 ℃, cooling to room temperature after the reaction is finished, separating liquid, and taking an organic solvent layer to obtain a solution A; (2) under the ice bath condition, triethylamine is added into the solution A, then PB-Cl solution is dripped to react, after the reaction is finished, extraction is carried out, washing, drying and reduced pressure concentration are carried out, and hydrogen peroxide is obtainedA living photosensitizer. The synthesis method is simple, and the obtained photosensitizer has good selectivity and strong photodynamic treatment effect, and can be used as a photodynamic treatment reagent for precise and efficient cancer treatment or used as a photodynamic reagent for activatable photodynamic treatment and imaging diagnosis of living body focus parts.
Description
Technical Field
The invention belongs to the field of tumor targeting and photodynamic therapy, and particularly relates to a photosensitizer activated by hydrogen peroxide, and a preparation method and application thereof.
Background
Photodynamic therapy has attracted attention as a non-invasive, minimally invasive treatment technique because of its satisfactory clinical efficacy. The main advantages of photodynamic therapy are low drug resistance, fast efficacy, repeated administration without toxic accumulation, no inhibition and low side effects on host immune system, etc. In particular, photodynamic therapy involves three key components: a photosensitizer, a light source, and Reactive Oxygen Species (ROS). The excited photosensitizer, upon irradiation, transfers energy to the oxygen surrounding the tissue to generate ROS, which can be used to induce apoptosis and necrosis. Photosensitizers are key factors in determining therapeutic efficacy throughout the course of treatment [ a) d.e.j.g.j.dolans, d.fukumura, r.k.jain, nat.rev.cancer 2003,3, 380-; b) g.szakacs, j.k.paterson, j.a.ludwig, c.booth-Genthe, m.m.gottesman, nat.rev.drug discovery 2006,5,219-; c) M.Ethirajan, Y.Chen, P.Joshi, R.K.Pandey, chem.Soc.Rev.2011,40, 340-.
Conventional photosensitizers include porphyrins, phthalocyanines, and the like, and clinical trials have been rarely conducted due to poor selectivity for target and healthy tissues, high accumulation rates in the skin, and the like. To overcome this drawback, photosensitizers have been designed to be conjugated to lesion-associated moieties, quenched prior to treatment, and changed to an "on" state upon stimulation by a lesion-site-associated marker. This strategy can effectively reduce the toxic side effects of photosensitizers. Researchers have recently attempted to improve the accuracy of photodynamic therapy by some physical stimuli (e.g. near infrared light), chemical stimuli (e.g. PH and redox) or biological stimuli (e.g. enzymes) [ a) b.jang, j.y.park, c.h.tung, i.h.kim and y.choi, ACS Nano,2011,5, 1086-1094. b) b.tianan, c.wang, s.zhang, l.feng and z.liu, ACS Nano,2011,5,7000 and 7009 ].
Furthermore, in order to improve the efficiency of treatment, many studies have focused on generating more ROS by using different kinds of photosensitizers. However, the ROS produced by photosensitizers may be reduced by high concentrations of Glutathione (GSH) in cells, which reduces the effect of photodynamic therapy. And a large amount of photosensitizer may cause toxic and side effects in cells. Therefore, it is very important to develop a system with targeted nontoxic photosensitizer and simultaneously reduce GSH levels (which can synergistically increase ROS levels) to enhance the efficacy of photodynamic therapy. However, to date, few studies have been reported on the direct reduction of intracellular glutathione in photodynamic therapy. Currently, the Stone group uses cysteine enzymes to reduce cysteine as a raw material for GSH production, thereby reducing GSH levels, increasing ROS levels, and inhibiting tumor growth. Zhang and Tan developed a photosensitizer Ce 6/manganese dioxide nanosystem that passes MnO2The effect of (a) reduces the level of GSH [ (a) s.l.cramer, a.saha, j.liu, s.tadi, s.tiziani, w.yan, k.triplett, c.lamb, s.e.alters, s.rowlinon, y.j.zhang, m.j.keting, p.huangang, j.digiovanni, g.georgiou, e.stone, nat.med.2017,23, 120-zhang 127.(b) h.fan, g.yan, z.zhao, x.hu, w.zhang, h.liu, x.fu, t.fu, x.b.zhang, w.h.tan, angew.chem.2016,128,5567-5572.]. It follows that it is important to develop a novel activatable photosensitizer that integrates the production of oxygen species while eliminating intracellular glutathione.
To improve the precision and efficiency of photodynamic therapy, we need to improve photosensitizers to achieve higher oxygen species production without harming normal tissues. Therefore, an activatable photosensitizer is designed to eliminate intracellular glutathione while generating oxygen species under light conditions. The strategy has important significance for clinical treatment of pathological tissues.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a photosensitizer activated by hydrogen peroxide.
Another object of the present invention is to provide a method for preparing the hydrogen peroxide-activated photosensitizer.
It is a further object of the present invention to provide the use of such hydrogen peroxide activated photosensitizers.
The purpose of the invention is realized by the following technical scheme: a hydrogen peroxide activated photosensitizer having the structural formula shown in formula I:
wherein R is selected from the following structures:
The preparation method of the photosensitizer activated by hydrogen peroxide comprises the following steps:
(1) adding methylene blue, sodium bicarbonate and sodium hydrosulfite into a mixed solvent, reacting at 40-60 ℃, cooling to room temperature after the reaction is finished, separating liquid, and taking an organic solvent layer to obtain a solution A;
(2) adding triethylamine into the solution A obtained in the step (1) under the ice bath condition, then dropwise adding PB-Cl (4- (4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl chloroformate and 4- (4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl) benzylchloroform) solution for reaction, extracting after the reaction is finished, washing, drying, and concentrating under reduced pressure to obtain the hydrogen peroxide activated photosensitizer.
The molar ratio of the methylene blue to the sodium bicarbonate to the sodium hydrosulfite in the step (1) is 1: (3-4) and (2-4); preferably 1: 3.48: 2.85.
the mixed solvent in the step (1) is a solvent obtained by mixing an organic solvent and water; preferred is a solvent obtained by mixing an organic solvent and water in a volume ratio of 4: 1.
The organic solvent comprises toluene, benzene and the like; toluene is preferred.
The dosage of the mixed solvent in the step (1) is preferably calculated according to the mixing ratio of 7.5mg methylene blue per milliliter (ml) of the mixed solvent.
The reaction time in the step (1) is preferably 2-4 h.
The molar ratio of the triethylamine, the PB-Cl and the methylene blue in the step (2) is 3.5: 1.4: 1.
the PB-Cl in the step (2) is prepared by the following method: dissolving 4- (hydroxymethyl) phenylboronic acid pinacol ester, triphosgene and triethylamine in tetrahydrofuran, stirring and reacting under an ice bath condition, and extracting, drying and concentrating after the reaction is finished to obtain PB-Cl.
The mol ratio of the 4- (hydroxymethyl) phenylboronic acid pinacol ester to the triphosgene to the triethylamine is (1.2-2): 1: (2-4); the molar ratio is preferably 1.6:1: 3.68.
The amount of tetrahydrofuran is preferably calculated as 37.5mg of 4- (hydroxymethyl) phenylboronic acid pinacol ester per milliliter (ml) of tetrahydrofuran.
The stirring reaction time is 1-3 h; preferably 1 hour.
The extraction is carried out by adopting a solvent obtained by mixing dichloromethane and water; preferably, the extraction is carried out using a solvent obtained by mixing dichloromethane and water in a ratio of 1: 1.
The drying is carried out by adopting anhydrous sodium sulfate.
The extraction in the step (2) is carried out by adopting a solvent obtained by mixing dichloromethane and water; preferably, the extraction is carried out using a solvent obtained by mixing dichloromethane and water in a ratio of 1: 1.
The washing in the step (2) is washing with water; preferably, the washing is carried out 3 or more times with water.
The drying in the step (2) is drying by adopting anhydrous magnesium sulfate.
The preparation method of the hydrogen peroxide activated photosensitizer further comprises the step of separating and purifying the hydrogen peroxide activated photosensitizer obtained in the step (2).
The separation and purification is to adopt a silica gel chromatographic column for separation and purification.
The hydrogen peroxide activated photosensitizer is applied to preparation of photodynamic therapy reagents or antitumor drugs, the borate group is hydrolyzed into phenolic hydroxyl group based on the specific recognition of hydrogen peroxide on the borate group, the leaving of the borate of the quenching group enables methylene blue of the photosensitizer to recover, singlet oxygen is generated under the irradiation of near infrared light, and meanwhile, the quinone methide is used as a byproduct to eliminate glutathione in cells and enhance oxidative stress, so that cytotoxicity is caused, and tumor cells are caused to die.
The photodynamic therapy reagent is used for treating tumors.
The tumor comprises liver cancer and the like.
The concentration of the hydrogen peroxide activated photosensitizer is preferably 10 μ M.
The hydrogen peroxide activated photosensitizer is applied to photoacoustic imaging or fluorescence imaging.
A nanoparticle comprising the hydrogen peroxide activated photosensitizer described above.
The nanoparticles also include albumin.
The preparation method of the nano-particles comprises the following steps: dissolving the hydrogen peroxide activated photosensitizer in an organic solvent, adding the organic solvent into an albumin aqueous solution, stirring and mixing uniformly, and dialyzing to obtain the nano particles.
The mass ratio of the hydrogen-oxide activated photosensitizer to albumin is preferably 1: 100.
The amount of dichloromethane is preferably 400 μ l of dichloromethane per milligram (mg) of hydrogen oxide activated photosensitizer.
The organic solvent is preferably dichloromethane.
The concentration of the aqueous albumin solution is preferably 100 mg/ml.
The stirring time is preferably half an hour.
The dialysis time is preferably 4 hours.
The nano-particles are applied to photoacoustic imaging or fluorescence imaging.
Compared with the prior art, the invention has the following advantages and effects:
1. the synthesis method of the photosensitizer activated by hydrogen peroxide is simple, the post-treatment process is simple, the operation is easy, and the product is easy to obtain. The method is constructed by conjugating a boronic ester moiety capable of being recognised by hydrogen peroxide to the photosensitizer methylene blue used clinically. The boronic ester moiety quenches the photosensitizer methylene blue through a carbamate linkage. Under the action of hydrogen peroxide, the photosensitizer methylene blue is recovered, and simultaneously quinone methide is released, so that intracellular oxidative stress is synergistically increased.
2. The hydrogen peroxide activated photosensitizer overcomes the problems of poor selectivity of photodynamic therapy reagents in living bodies and limited clinical treatment, and is used for selective and efficient photodynamic therapy of living tumors. The treatment mechanism of the photosensitizer is based on the specific recognition of hydrogen peroxide on borate groups, the borate groups are hydrolyzed into phenolic hydroxyl groups, the leaving of the borate of a quenching group enables methylene blue of the photosensitizer to recover, and singlet oxygen is generated under the irradiation of near infrared light. Meanwhile, the quinone methide as a byproduct can eliminate glutathione in cells, enhance oxidative stress, thereby causing cytotoxicity and causing tumor cell apoptosis.
3. The photosensitizer obtained by the invention has the advantages of good selectivity, enhanced photodynamic treatment effect and capability of obtaining the clinically usable photosensitizer methylene blue after responding to hydrogen peroxide. In addition, the photosensitizer can be used for fluorescence and photoacoustic imaging in cells and living bodies during photodynamic therapy, and the purpose of diagnosing the cancer treatment process can be achieved.
4. The photosensitizer obtained by the invention can be applied to selectively kill cancer cells. In vivo studies have shown that this photosensitizer is effective in reducing HepG2 tumors growing in mice. In summary, the precise and efficient photodynamic therapy strategy and design of the hydrogen peroxide activated photosensitizer system allows its application in tissue and in vivo visualization during photodynamic therapy as a photodynamic therapy agent for precise and efficient cancer treatment or as a photodynamic agent for activatable photodynamic therapy and imaging diagnostics at the site of a lesion in vivo due to its near infrared absorption and fluorescence.
Drawings
FIG. 1 is a synthetic scheme showing the preparation of a hydrogen peroxide activated photosensitizer in example 1 of the present invention.
FIG. 2 is a NMR spectrum of a hydrogen peroxide-activated photosensitizer in example 1 of the present invention.
FIG. 3 is a mass spectrum of an inductively coupled plasma of a hydrogen peroxide activated photosensitizer in example 1 of the present invention.
FIG. 4 is a mass spectrum of inductively coupled plasma of a product of a hydrogen peroxide activated photosensitizer reacted with hydrogen peroxide in example 2 of the present invention; wherein, the graph A is a mass spectrum of methylene blue which is a product after reaction, and the graph B is a mass spectrum of quinone methide which is a product after reaction and water.
FIG. 5 is a graph showing absorption spectra before and after reaction of a hydrogen peroxide activated photosensitizer with different concentrations of hydrogen peroxide in example 3 of the present invention.
FIG. 6 is a graph showing fluorescence spectra before and after reaction of a photosensitizer activated by hydrogen peroxide with different concentrations of hydrogen peroxide in example 4 of the present invention.
FIG. 7 is a graph of photoacoustic imaging and photoacoustic signal intensity from the reaction of hydrogen peroxide activated photosensitizer with different concentrations of hydrogen peroxide in example 5 of the present invention; wherein, the graph A is a photoacoustic signal intensity graph, and the graph B is a photoacoustic imaging graph.
FIG. 8 is a graph showing the generation of singlet oxygen under various treatment conditions for the hydrogen peroxide-activated photosensitizer in example 6 of the present invention.
FIG. 9 is a graph showing fluorescence images of the photosensitizer activated by hydrogen peroxide in the cancer cell HepG2 in example 7 of the present invention.
FIG. 10 is a graph showing the fluorescence response of the hydrogen peroxide-activated photosensitizer in example 8 of the present invention under the action of hydrogen peroxide in cancer cells HepG2, as measured by flow cytometry.
FIG. 11 is a graph showing the results of detecting the ability of a photosensitizer activated by hydrogen peroxide to generate singlet oxygen by the action of hydrogen peroxide in cancer cells HepG2 by using a singlet oxygen detection probe SOSG in example 9 of the present invention.
FIG. 12 is a statistical chart showing the results of the ability of the hydrogen peroxide-activated photosensitizer in example 10 of the present invention to produce quinone methide and eliminate glutathione by the action of hydrogen peroxide in cancer cells HepG 2.
FIG. 13 is a graph showing the staining of the cells after the hydrogen peroxide-activated photosensitizer was treated with cancer cells HepG2 in example 11 of the present invention by flow cytometry.
FIG. 14 is a graph showing in vivo fluorescence imaging and fluorescence signal intensity of nanomaterials formed by encapsulating hydrogen peroxide-activated photosensitizers in albumin according to example 12 of the present invention; wherein, the image A is a fluorescence imaging image; panel B is a plot of fluorescence signal intensity.
FIG. 15 is a graph of photoacoustic imaging and intensity of photoacoustic signals in vivo from hydrogen peroxide activated photosensitizer encapsulated in albumin in example 13 of the present invention; wherein, the graph A is a photoacoustic imaging graph; graph B is a graph of photoacoustic signal intensity.
FIG. 16 is a graph showing the fluorescence imaging and fluorescence signal intensity of tumor targeting of the nanomaterial of example 14 of the present invention encapsulated in albumin by hydrogen peroxide; wherein, the graph A is a fluorescence signal intensity graph; and the image B is a fluorescence imaging image.
FIG. 17 is a tumor image and a statistical image of tumor volume after photodynamic therapy of mice after photodynamic therapy in which hydrogen peroxide-activated photosensitizer is encapsulated in albumin to form a nanomaterial according to example 15 of the present invention; wherein, the picture A is a tumor picture after the photodynamic therapy of the mice; panel B is a statistical plot of tumor volume after photodynamic treatment in mice.
FIG. 18 is a graph showing the effect of photodynamic therapy with nanomaterial-encapsulated hydrogen peroxide-activated photosensitizer in albumin in example 16 of the present invention on body weight in a mouse model.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions. The raw materials and reagents used in the following examples are commercially available unless otherwise specified.
Example 1: preparation of hydrogen peroxide activated photosensitizer.
The specific synthetic route of the hydrogen peroxide activated photosensitizer provided by the invention is shown in figure 1, and specifically comprises the following steps:
methylene blue (373.9mg, 1mmol), sodium bicarbonate (292.4mg, 3.48mmol) and sodium dithionate (496.2mg, 2.85mmol) were added to a mixed solution of 40ml of toluene and 10ml of water, reacted at 60 ℃ for 2 hours, cooled to room temperature, separated, and the toluene layer was retained; triethylamine (354.2mg, 3.5mmol) was then added to the toluene solution under ice-bath conditions, and a solution of PB-Cl (415.2mg, 1.4mmol) dissolved in 40ml of toluene was slowly added dropwise to the solution. After the addition, the reaction was allowed to proceed overnight at room temperature, concentrated under reduced pressure, extracted with water (100ml) and methylene chloride (100ml), and the organic phase was washed with water 3 times. Collecting the organic phase, adding anhydrous magnesium sulfate, drying, concentrating, and separating and purifying by silica gel chromatographic column to obtain small organic molecular probe I, i.e. hydrogen peroxide activated photosensitizer. Wherein PB-Cl is prepared as follows:
4- (hydroxymethyl) phenylboronic acid pinacol ester (749.1mg, 3.2mmol) and triphosgene (593.5mg, 2mmol) are dissolved in 20ml of tetrahydrofuran, triethylamine (744.8mg, 7.36mmol) is added under ice bath conditions, after stirring in ice bath for reaction for 1h, extraction is performed with dichloromethane and water (volume ratio 1:1), drying is performed with anhydrous sodium sulfate, and the organic phase is concentrated to obtain PB-Cl (4- (4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl chloroformate, and 4- (4,4,5, 5-tetramethy-1, 3,2-dioxaborolan-2-yl) benzylchloroformamate).
Characterization data:
1HNMR (FIG. 2) (500MHz, d)6-DMSO)δ＝7.69(d,J＝8.4Hz,1H),7.67(d,J＝8.3Hz,1H),7.45(t,J＝8.6Hz,1H),7.43(d,J＝2.5Hz,1H),7.12-7.07(dd,J＝8.9，2.3Hz,1H),6.71-6.43(dd,J＝8.9，2.3Hz,1H),5.32(s,2H)，2.96(s,12H),1.57(s,12H)
MS Panel (3): calcd.for [ M + ]545, found:546.
Example 2 inductively coupled plasma mass spectrum of the product of the reaction of hydrogen peroxide activated photosensitizer with hydrogen peroxide.
Preparing the hydrogen peroxide activated photosensitizer solution and the aqueous hydrogen peroxide solution obtained in the example 1, mixing the hydrogen peroxide activated photosensitizer solution and the aqueous hydrogen peroxide solution for reaction for twenty minutes, and then measuring the inductively coupled plasma mass spectrum of the hydrogen peroxide activated photosensitizer solution and the aqueous hydrogen peroxide solution; wherein the concentration of the probe I (hydrogen peroxide activated photosensitizer) in the reaction system is 10 mu M, and the concentration of hydrogen peroxide is 100 mu M. The mass spectrum is shown in FIG. 4, from which it can be seen that the probe produces the photosensitizer methylene blue and toxic quinone methide upon reaction with hydrogen peroxide.
Example 3 absorption spectra before and after reaction of Probe I with different concentrations of hydrogen peroxide.
Preparing the probe I solution and the hydrogen peroxide aqueous solution obtained in the example 1, mixing the probe I solution and the hydrogen peroxide aqueous solution for reaction for thirty minutes, and measuring the absorption of the mixture; wherein the concentration of the probe I in the reaction system is 10 mu M, and the concentration of the hydrogen peroxide is 0-100 mu M. The absorption spectrum is shown in FIG. 5, from which it can be seen that the absorption coefficient of the probe gradually increases as the hydrogen peroxide concentration increases.
Example 4 fluorescence spectra before and after reaction of Probe I with different concentrations of hydrogen peroxide.
Preparing the probe I solution and the hydrogen peroxide aqueous solution obtained in the embodiment 1, mixing the probe I solution and the hydrogen peroxide aqueous solution for reaction for thirty minutes, and measuring the fluorescence of the probe I solution and the hydrogen peroxide aqueous solution; wherein the concentration of the probe I in the reaction system is 10 mu M, and the concentration of the hydrogen peroxide is 0-100 mu M. The fluorescence spectrum is shown in FIG. 6, from which it can be seen that the fluorescence intensity of the probe gradually increases as the hydrogen peroxide concentration increases.
Example 5 photoacoustic imaging and photoacoustic signal intensity plots after reaction of Probe I with varying concentrations of hydrogen peroxide.
A total of 6 sets of 10. mu.M probe I solution and aqueous hydrogen peroxide solutions of 0, 20, 40, 60, 80, 100. mu.M were prepared, and then the probe I solution and the aqueous hydrogen peroxide solution were mixed and reacted (40. mu.l of probe I solution and 1, 2, 3, 4, 5. mu.l of aqueous hydrogen peroxide solution, respectively), and after each set had reacted for 20 minutes, photoacoustic signals of the 6 sets of solutions were measured by a photoacoustic computed tomography scanner, and a photoacoustic two-dimensional graph (FIG. 7). It can be seen from the graph that the intensity of the photoacoustic signal at 665nm gradually increases with the increase in the concentration. Indicating that the probe can act as a photosensitizer in response to hydrogen peroxide.
Example 6 singlet oxygen production in Probe I under different treatment conditions.
The probe I solution with the concentration of 10 MuM is prepared into 4 groups, the first group does not carry out treatment, the second group is added with hydrogen peroxide aqueous solution, the third group is added with illumination of 633nm, and the fourth group is added with hydrogen peroxide aqueous solution and illumination of 633 nm. To these four groups of solutions, a singlet oxygen detection probe SOSG (singlet oxygen detection probe SOSG purchased from Saimer Feishell science Co., Ltd.; added in an amount of 5. mu.M) was added, and the fluorescence of SOSG at 545nm was detected. As shown in FIG. 8, it can be seen that the fluorescence of SOSG is significantly enhanced under the conditions of hydrogen peroxide and 633nm illumination of probe I. Indicating that the photosensitizer which can be activated by hydrogen peroxide has good photodynamic effect.
Example 7 fluorescence imaging of Probe I in cancer cells HepG 2.
Preparing 10 mu M probe I solution, incubating cancer cells HepG2 (Beijing Beinanna Biotechnology research institute) in a confocal probe, dividing into 4 groups, wherein the first group is not treated (as a control), the second group is added with the probe I solution (the dosage of the probe I solution is 40 mu l, MBPB), the third group is added with the probe I solution (40 mu l) and hydrogen peroxide stimulant PMA (phorbol acetate, the dosage is 1mg/ml) (MBPB + PMA), and the fourth group is added with the probe I solution (40 mu l) and oxygen species scavenger catalase (catalase, the dosage is 1U/ml) (MBPB + CAT). Cells were fluorescence imaged using confocal microscopy. The result is shown in FIG. 9, which shows that the photosensitizer activated by hydrogen peroxide can release the photosensitizer methylene blue under the action of hydrogen peroxide in cancer cells to recover fluorescence, and indicates that the probe I can perform fluorescence response on the hydrogen peroxide in the cells.
Example 8 statistics of the fluorescent response of Probe I in cancer cells HepG 2.
Preparing a probe I solution with the concentration of 10 mu M, incubating cancer cells HepG2 in a confocal medium, dividing the solution into 4 groups, wherein the first group is not treated (used as a control), the second group is added with the probe I solution (MBPB), the third group is added with the probe I solution and a hydrogen peroxide stimulant PMA (1mg/ml) (MBPB + PMA), and the fourth group is added with the probe I solution and an oxygen species scavenger catalase (catalase with the dosage of 1U/ml) (MBPB + CAT); wherein the dosage of the probe I solution is 40 mul. Cells were washed and digested, and intracellular fluorescence intensity was measured using a flow cytometer. The results are shown in FIG. 10, which shows that probe I has a good fluorescent response to intracellular hydrogen peroxide.
Example 9 ability of Probe I to generate singlet oxygen in cancer cells HepG 2.
Preparing a probe I solution with the concentration of 10 mu M, incubating cancer cells HepG2 in a confocal medium, dividing the solution into 4 groups, wherein the first group is not treated (used as a control), the second group is added with the probe I solution (MBPB), the third group is added with the probe I solution and a hydrogen peroxide stimulant PMA (1mg/ml) (MBPB + PMA), and the fourth group is added with the probe I solution and an oxygen species scavenger catalase (catalase with the dosage of 1U/ml) (MBPB + CAT); wherein the dosage of the probe I solution is 40 mul. After a singlet oxygen probe SOSG (5. mu.M) was added to the four groups of cells, they were irradiated with laser light of 633nm, and the fluorescence intensity of SOSG was measured by a confocal microscope. The results are shown in FIG. 11, which shows that probe I can generate photosensitizer methylene blue under the action of intracellular hydrogen peroxide, and can generate singlet oxygen under the illumination condition, so that the probe I has a good photodynamic effect.
Example 10 ability of Probe I to eliminate glutathione in cancer cells HepG 2.
A10. mu.M solution of Probe I was prepared, and cancer cells HepG2 were incubated in a six-well plate and divided into 4 groups, the first group was untreated (as a control), the second group was supplemented with PB-Cl (prepared in the same manner as in example 1, at a concentration of 10. mu.M and in an amount of 40. mu.l), the third group was supplemented with Probe I (40. mu.l, MBPB), the fourth group was supplemented with Probe I (40. mu.l) and oxygen species scavenger catalase (catalase in an amount of 1U/ml) (MBPB + CAT). Glutathione detection probe 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB, purchased from alatin, 0.5mM) was added to the four groups of cells, and the fluorescence intensity of the glutathione probe was measured using a microplate reader. As shown in FIG. 12, it can be seen that probe I is capable of producing toxic quinone methide to eliminate glutathione under the action of intracellular hydrogen peroxide.
Example 11 photodynamic therapy of Probe I in cancer cells HepG 2.
Prepare 10 μ M probe I solution, incubate cancer cell HepG2 in six-well plate, divide into 5 groups, do not process (as the control) in the first group, only add light in the second group, add probe I solution (the amount of probe I solution is 40 μ l, MBPB) in the third group, add probe I solution (40 μ l) and light (MBPB + light) in the fourth group, add probe I solution (40 μ l), oxygen species scavenger catalase (catalase, amount 1U/ml) and light (MBPB + CAT + light) in the fifth group. To these five groups of cells, dead/live working solution (propidium iodide/fluorescein isothiocyanate, purchased from Sigma) was added to replace the cell culture medium, and after 15min staining, staining of dead/live cells was detected by flow cytometry.
The results are shown in FIG. 13, which shows that probe I can generate photosensitizer methylene blue under the action of intracellular hydrogen peroxide, and singlet oxygen generated under the illumination condition can kill tumor cells well.
Example 12 conjugation of Probe I to Albumin resulted in fluorescence imaging and fluorescence signal intensity of the nanoparticles in vivo.
1mg of Probe I was dissolved in methylene chloride (400. mu.l), added dropwise to albumin dissolved in deionized water (1 ml of deionized water, 100mg of albumin), and stirred vigorously for half an hour. The resulting solution was dialyzed (dialysate was deionized water) for 4 hours to obtain nanoparticles (BSA-MBPB). Nanoparticles (2mg/kg) were injected tail-vein into mice (3-4 weeks BABc mice, average 2 body weight 20g, purchased at southern medical university) and fluorescence imaged using a two-color infrared laser imaging system 1,3, 6,12, 24h later. The results are shown in fig. 14, which demonstrates fluorescence imaging of endogenous hydrogen peroxide following nanoparticle metabolism to the tumor site in mice.
Example 13 conjugation of Probe I to Albumin resulted in photoacoustic imaging and photoacoustic signal intensity of nanoparticles in vivo.
The nanoparticles described in example 12 (2mg/kg) were injected tail-vein into mice (3-4 weeks BABc mice, average 2 body weight 20g, purchased at southern medical university) and mice tumor sites were imaged using a photoacoustic computed tomography scanner 1,3, 6,12, 24h later. The results are shown in fig. 15, which demonstrates photoacoustic imaging of endogenous hydrogen peroxide following metabolism of the nanoparticles to the tumor site in mice.
Example 14 conjugation of Probe I to Albumin resulted in fluorescence imaging and fluorescence signal intensity profiles of the nanoparticles in various tissues of the living body.
After injecting the nanoparticles (2mg/kg) described in example 12 into mice (3-4 weeks BABc mice, average 2 body weight 20g, purchased at southern medical university) via tail vein for 24h, fluorescence imaging was performed on each major organ of the mice including heart, liver, spleen, lung, kidney and tumor sites using a two-color infrared laser imaging system. The results are shown in fig. 16, which indicates that fluorescence imaging is performed in response to hydrogen peroxide as the nanoparticles are metabolized in mice to target tumor sites.
Example 15 conjugation of Probe I to Albumin resulted in a photodynamic therapeutic effect of nanoparticles on tumor mice.
Mice (3-4 weeks BABc mice, average 2 body weight 20g, purchased at southern medical university) were divided into four groups for different treatments, the first group was added with 100 μ l PBS buffer (as control), the second group was illuminated, the third group was added with 100 μ l nanoparticles prepared in example 12 (BSA-MBPB, 50 μ g/ml), and the fourth group was added with 100 μ l nanoparticles prepared in example 12 (50 μ g/ml) and illumination (BSA-MBPB + illumination). The volume of the tumor site of the mice was counted.
The results are shown in fig. 17, and indicate that as the nanoparticles are metabolized in a mouse body and can be targeted to a tumor part, under the illumination condition, the photosensitizer methylene blue obtained after response to hydrogen peroxide can generate singlet oxygen, and the elimination of glutathione by combining a byproduct, namely quinone methide, enhances oxidative stress and inhibits the growth of tumors.
Example 16 conjugation of Probe I to Albumin to form nanoparticles safety to photodynamic therapy of tumor mice.
Mice (3-4 weeks BABc mice, average 2 body weight 20g, purchased at southern medical university) were divided into four groups for different treatments, the first group was added with 100 μ l PBS buffer (as control), the second group was illuminated, the third group was added with 100 μ l nanoparticles prepared in example 12 (BSA-MBPB, 50 μ g/ml), and the fourth group was added with 100 μ l nanoparticles prepared in example 12 (50 μ g/ml) and illumination (BSA-MBPB + illumination). Body weight changes of mice were counted over three weeks.
The results are shown in fig. 18, and show that the nanoparticles have no influence on other growth conditions of the mice along with the photodynamic treatment effect of the nanoparticles at the tumor sites of the mice.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A hydrogen peroxide activated photosensitizer having a structural formula represented by formula I:
wherein R is selected from the following structures:
2. the method of preparing a hydrogen peroxide activated photosensitizer as claimed in claim 1, comprising the steps of:
(1) adding methylene blue, sodium bicarbonate and sodium hydrosulfite into a mixed solvent, reacting at 40-60 ℃, cooling to room temperature after the reaction is finished, separating liquid, and taking an organic solvent layer to obtain a solution A;
(2) and (2) under an ice bath condition, adding triethylamine into the solution A obtained in the step (1), then dropwise adding a PB-Cl solution for reaction, extracting after the reaction is finished, washing, drying, and concentrating under reduced pressure to obtain the hydrogen peroxide activated photosensitizer.
3. The method for preparing a hydrogen peroxide-activated photosensitizer according to claim 2, wherein the PB-Cl in the step (2) is prepared by:
dissolving 4- (hydroxymethyl) phenylboronic acid pinacol ester, triphosgene and triethylamine in tetrahydrofuran, stirring and reacting under an ice bath condition, and extracting, drying and concentrating after the reaction is finished to obtain PB-Cl;
the mol ratio of the 4- (hydroxymethyl) phenylboronic acid pinacol ester to the triphosgene to the triethylamine is (1.2-2): 1: (2-4);
the extraction is carried out by adopting a solvent obtained by mixing dichloromethane and water;
the drying is carried out by adopting anhydrous sodium sulfate.
4. The method for producing a hydrogen peroxide-activated photosensitizer according to claim 2, characterized in that:
the molar ratio of the methylene blue to the sodium bicarbonate to the sodium hydrosulfite in the step (1) is 1: (3-4) and (2-4);
the molar ratio of the triethylamine, the PB-Cl and the methylene blue in the step (2) is 3.5: 1.4: 1.
5. the method for producing a hydrogen peroxide-activated photosensitizer according to claim 2, characterized in that:
the mixed solvent in the step (1) is a solvent obtained by mixing an organic solvent and water;
the reaction time in the step (1) is 2-4 h;
the extraction in the step (2) is carried out by adopting a solvent obtained by mixing dichloromethane and water;
the drying in the step (2) is drying by adopting anhydrous magnesium sulfate.
6. Use of the hydrogen peroxide-activated photosensitizer of claim 1 for the preparation of a photodynamic therapeutic agent or an antitumor drug.
7. Use of the hydrogen peroxide activated photosensitizer of claim 1 in photoacoustic imaging or fluorescence imaging.
8. A nanoparticle characterized by: comprising the hydrogen peroxide activated photosensitizer of claim 1.
9. A method for preparing nanoparticles as claimed in claim 8, characterized in that it comprises the following steps: dissolving the hydrogen peroxide activated photosensitizer of claim 1 in an organic solvent, then adding the mixture into an albumin aqueous solution, stirring and mixing uniformly, and dialyzing to obtain nanoparticles;
the mass ratio of the hydrogen-oxide-activated photosensitizer to the albumin is 1: 100;
the organic solvent is dichloromethane.
10. Use of the nanoparticle of claim 8 in photoacoustic imaging or fluorescence imaging.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810937694.XA CN110856747A (en) | 2018-08-17 | 2018-08-17 | Photosensitizer activated by hydrogen peroxide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810937694.XA CN110856747A (en) | 2018-08-17 | 2018-08-17 | Photosensitizer activated by hydrogen peroxide and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110856747A true CN110856747A (en) | 2020-03-03 |
Family
ID=69635872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810937694.XA Pending CN110856747A (en) | 2018-08-17 | 2018-08-17 | Photosensitizer activated by hydrogen peroxide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110856747A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112876837A (en) * | 2020-04-08 | 2021-06-01 | 四川大学 | Photoreduction degradation composition and preparation method and application thereof |
| CN114656424A (en) * | 2022-03-16 | 2022-06-24 | 深圳先进技术研究院 | Up-conversion long-afterglow chemiluminescent imaging nanoprobe and preparation method and application thereof |
| CN114806211A (en) * | 2022-03-16 | 2022-07-29 | 深圳先进技术研究院 | Hydrophobic methylene blue fluorescent dye and preparation method and application thereof |
| CN115137818A (en) * | 2021-03-31 | 2022-10-04 | 华南师范大学 | Glutathione activated photosensitizer-chemotherapeutic drug integrated molecular prodrug and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106588966A (en) * | 2016-11-14 | 2017-04-26 | 济南大学 | Preparation and application of open-type hydrogen peroxide fluorescent probe compound |
| CN107266483A (en) * | 2017-06-08 | 2017-10-20 | 浙江工业大学 | A kind of hydrogen peroxide that responds kills photaesthesia targeting anti-tumor prodrug of tumour cell and preparation method and application |
-
2018
- 2018-08-17 CN CN201810937694.XA patent/CN110856747A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106588966A (en) * | 2016-11-14 | 2017-04-26 | 济南大学 | Preparation and application of open-type hydrogen peroxide fluorescent probe compound |
| CN107266483A (en) * | 2017-06-08 | 2017-10-20 | 浙江工业大学 | A kind of hydrogen peroxide that responds kills photaesthesia targeting anti-tumor prodrug of tumour cell and preparation method and application |
Non-Patent Citations (5)
| Title |
|---|
| HELEN HAGEN等: "Aminoferrocene-Based Prodrugs Activated by Reactive Oxygen Species", 《J. MED. CHEM.》 * |
| JUNGEUN BAE等: "Nitroreductase-triggered activation of a novel caged fluorescent probe obtained from methylene blue", 《CHEM. COMMUN.》 * |
| MASAKI YAMAMOTO等: "Bovine serum albumin-capped gold nanoclusters conjugating with methylene blue for efficient 1O2 generation via energy transfer", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》 * |
| ZHIQIANG HAN等: "A 3,7-Dihydroxyphenoxazine-based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide", 《CHEM. ASIAN J》 * |
| 李震宇等: "硼酸及硼酸酯类过氧化氢荧光探针的最新研究进展", 《中国科学：化学》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112876837A (en) * | 2020-04-08 | 2021-06-01 | 四川大学 | Photoreduction degradation composition and preparation method and application thereof |
| CN112876837B (en) * | 2020-04-08 | 2021-11-23 | 四川大学 | Photoreduction degradation composition and preparation method and application thereof |
| CN115137818A (en) * | 2021-03-31 | 2022-10-04 | 华南师范大学 | Glutathione activated photosensitizer-chemotherapeutic drug integrated molecular prodrug and application thereof |
| CN115137818B (en) * | 2021-03-31 | 2023-06-27 | 华南师范大学 | Glutathione activated photosensitizer-chemotherapeutic agent integrated molecular prodrug and application thereof |
| CN114656424A (en) * | 2022-03-16 | 2022-06-24 | 深圳先进技术研究院 | Up-conversion long-afterglow chemiluminescent imaging nanoprobe and preparation method and application thereof |
| CN114806211A (en) * | 2022-03-16 | 2022-07-29 | 深圳先进技术研究院 | Hydrophobic methylene blue fluorescent dye and preparation method and application thereof |
| WO2023173824A1 (en) * | 2022-03-16 | 2023-09-21 | 深圳先进技术研究院 | Hydrophobic methylene blue fluorescent dye, preparation method therefor and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Qi et al. | Gathering brings strength: How organic aggregates boost disease phototheranostics | |
| CN110856747A (en) | Photosensitizer activated by hydrogen peroxide and preparation method and application thereof | |
| CN113773667B (en) | Organic small molecule near infrared two-region fluorescent dye and preparation method and application thereof | |
| EP3215586B1 (en) | Selective cancer tracking eradicator and the uses thereof | |
| US20240041787A1 (en) | Photosensitizer molecule and use thereof in increasing tumor retention time and enhancing treatment of large-volume tumors | |
| Zhang et al. | Surfactant-stripped J-aggregates of azaBODIPY derivatives: All-in-one phototheranostics in the second near infrared window | |
| Hu et al. | A thermally activated delayed fluorescence photosensitizer for photodynamic therapy of oral squamous cell carcinoma under low laser intensity | |
| CN110302177A (en) | Mitochondrially targeted smooth diagnosis and treatment nano particle and application | |
| CN113683602B (en) | Heptamethine cyanine micromolecule for multi-modal treatment of hypoxic tumors, and preparation method and application thereof | |
| Fu et al. | A Raman/fluorescence dual-modal imaging guided synergistic photothermal and photodynamic therapy nanoplatform for precision cancer theranostics | |
| Kang et al. | Applications of nanocomposites based on zeolitic imidazolate framework-8 in photodynamic and synergistic anti-tumor therapy | |
| CN116425728A (en) | Carboxylesterase-responsive diagnosis and treatment integrated probe and preparation method and application thereof | |
| CN109745569B (en) | Electrochromic material based H2S-activated probes and biological applications thereof | |
| WO2023245857A1 (en) | Capsaicin-derived photosensitizer, and preparation method therefor and use thereof | |
| CN113797350B (en) | Glycosyl polymer and preparation method and application thereof | |
| CN114507247B (en) | Amphiphilic gadolinium complex and nano micelle for realizing diagnosis and treatment integration | |
| CN113527349A (en) | Photosensitizer with tumor targeting property and preparation method and application thereof | |
| CN114805397B (en) | Organic fluorescent small molecular compound capable of circulating in vivo for long time, preparation method and application thereof | |
| CN110935038B (en) | Long-afterglow nano-composite and application thereof in multi-modal imaging and cooperative therapy of tumors | |
| CN115137818B (en) | Glutathione activated photosensitizer-chemotherapeutic agent integrated molecular prodrug and application thereof | |
| CN115572280B (en) | NIR heavy-atom-free photosensitizer and preparation method and application thereof | |
| CN115160496B (en) | Glutathione activated polynorbornene photosensitizer as well as preparation method and application thereof | |
| CN116217515B (en) | Methylene blue probe, preparation method thereof and application thereof in detection of cysteine | |
| US12011482B2 (en) | Chlorin derivatives or pharmaceutically acceptable salts thereof, preparation method and use thereof, and combination thereof with an ultrasonic medical system | |
| CN117624020A (en) | Bisindocyanine greening compound with stimulus responsiveness and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200303 |
|
| RJ01 | Rejection of invention patent application after publication |