CN104072607B - A kind of Organometallic ruthenium compound protein and the preparation method and application thereof - Google Patents
A kind of Organometallic ruthenium compound protein and the preparation method and application thereof Download PDFInfo
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- CN104072607B CN104072607B CN201310109645.4A CN201310109645A CN104072607B CN 104072607 B CN104072607 B CN 104072607B CN 201310109645 A CN201310109645 A CN 201310109645A CN 104072607 B CN104072607 B CN 104072607B
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- Prior art keywords
- organometallic ruthenium
- ruthenium compound
- haemocyanin
- compound protein
- buffer solution
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 77
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 77
- -1 Organometallic ruthenium compound Chemical class 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 44
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229910052707 ruthenium Inorganic materials 0.000 claims abstract description 32
- 239000007853 buffer solution Substances 0.000 claims abstract description 29
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 26
- 125000002524 organometallic group Chemical group 0.000 claims abstract description 26
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 25
- 102000002070 Transferrins Human genes 0.000 claims abstract description 21
- 108010015865 Transferrins Proteins 0.000 claims abstract description 21
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 12
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 14
- 229910052742 iron Inorganic materials 0.000 claims description 7
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 3
- 239000010931 gold Substances 0.000 claims description 3
- 229910052737 gold Inorganic materials 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 230000000259 anti-tumor effect Effects 0.000 abstract description 23
- 229910052751 metal Inorganic materials 0.000 abstract description 17
- 239000002184 metal Substances 0.000 abstract description 17
- 230000008685 targeting Effects 0.000 abstract description 14
- 239000000243 solution Substances 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000013270 controlled release Methods 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 230000009456 molecular mechanism Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 16
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 14
- 229910052697 platinum Inorganic materials 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 11
- 150000001875 compounds Chemical class 0.000 description 11
- 239000003814 drug Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 5
- 150000003304 ruthenium compounds Chemical class 0.000 description 5
- 230000000118 anti-neoplastic effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- 230000004700 cellular uptake Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000001626 skin fibroblast Anatomy 0.000 description 2
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 2
- 230000010415 tropism Effects 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001555 benzenes Chemical class 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 150000002736 metal compounds Chemical class 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Natural products O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a kind of Organometallic ruthenium compound proteins and the preparation method and application thereof, this method comprises: in the buffer solution that pH value is 7-8 and concentration is 10-100mM, Organometallic ruthenium anti-tumor drug is contacted with haemocyanin, the molar ratio of the Organometallic ruthenium anti-tumor drug and haemocyanin is 1-20:1, and the haemocyanin is amino acid sequence seralbumin as shown in SEQ ID NO:1 or the amino acid sequence transferrins as shown in SEQ ID NO:2.Through the above technical solution, present invention obtains targeting is strong and the higher Organometallic ruthenium compound protein of anti-tumor activity, but also to be even further appreciated that the transporting mechanism of Organometallic ruthenium anti-tumor drug and molecular mechanisms of action and preparing nano controlled release type metal anti-tumor drug by carrier of haemocyanin and provide experiment and theoretical foundation.
Description
Technical field
The present invention relates to a kind of Organometallic ruthenium compound proteins and the preparation method and application thereof, and in particular, to Yi Zhongyou
The preparation method for the Organometallic ruthenium compound protein that seralbumin or transferrins and Organometallic ruthenium anti-tumor drug are constituted
And Organometallic ruthenium compound protein obtained by this method and the Organometallic ruthenium compound protein are preparing antineoplastic
Purposes in object.
Background technique
The successful application of cis-platinum and other platinum series antineoplastic medicaments have stimulated the development of inorganic pharmacochemistry, therefore people's handle
Sight has invested the pharmaceutical activity research of other transistion metal compounds, and wherein the research of ruthenium base antitumor activity is most looked steadily
Mesh, organometallic ruthenium compound [(η6-arene)Ru(en)Cl]PF6(arene=substituted benzene；En=ethylenediamine) it is internal
Experiment in vitro confirmation is a kind of effective antitumour drug candidate.
But current organometallic ruthenium compound targeting in vivo and anti-tumor activity are still lower, also much
It is not able to satisfy the requirement of clinical application.
Summary of the invention
It is strong the object of the present invention is to provide a kind of targeting and anti-tumor activity is high by Organometallic ruthenium anti-tumor drug
The Organometallic ruthenium compound protein and the preparation method and application thereof formed with haemocyanin.
To achieve the goals above, on the one hand, the present invention provides a kind of preparation method of Organometallic ruthenium compound protein,
This method comprises: in the buffer solution that pH value is 7-8 and concentration is 10-100mM, by Organometallic ruthenium anti-tumor drug and blood
Albumin is contacted, and the molar ratio of the Organometallic ruthenium anti-tumor drug and haemocyanin is 1-20:1, the serum egg
It is white to turn as shown in SEQ ID NO:2 for amino acid sequence seralbumin as shown in SEQ ID NO:1 or amino acid sequence
Ferritin.
On the other hand, the present invention provides Organometallic ruthenium compound proteins prepared by the above method.
In another aspect, the present invention also provides Organometallic ruthenium compound proteins made from the above method to prepare antineoplastic
Purposes in object.
Through the above technical solutions, present invention obtains targetings to be conjugated with the higher Organometallic ruthenium of anti-tumor activity by force
Albumen, but also for be even further appreciated that Organometallic ruthenium anti-tumor drug transporting mechanism and molecular mechanisms of action and with
Haemocyanin prepares nano controlled release type metal anti-tumor drug for carrier and provides experiment and theoretical foundation.
Particularly, it was found by the inventors of the present invention that using the blood as shown in SEQ ID NO:1 of amino acid sequence in the application
The anti-of Organometallic ruthenium compound protein made from pure albumen or the amino acid sequence transferrins as shown in SEQ ID NO:2 swells
Tumor activity is apparently higher than the antitumor work using Organometallic ruthenium compound protein made from other haemocyanins (such as hemoglobin)
Property.In addition, present inventor has further discovered that, under the same conditions, the metal in addition to organometallic ruthenium compound is anti-swollen
Tumor compound (such as cis-platinum) can not obtain the Metal Drugs that targeting is strong and anti-tumor activity is high and sew after contacting with haemocyanin
Hop protein.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the TMR- organic metal being prepared Following the procedure of Example 1 observed under laser confocal microscope
For ruthenium compound protein by the result figure of MCF-7 cellular uptake, upper figure is TMR-Tf(Tf=transferrins) be incubated for after fluorescence mark in cell
The albumen of note absorbs situation map, fluorescence mark in cell after the following figure is incubated for for the TMR-Tf-6 through being prepared Following the procedure of Example 1
The intake situation map of the Organometallic ruthenium compound protein of note；
Fig. 2 is that cis-platinum made from Organometallic ruthenium compound protein Tf-3 and Tf-6 made from embodiment 1 and comparative example 4 is sewed
The inhibitory activity curve graph of hop protein (Tf- cis-platinum)；
Fig. 3 is that cis-platinum made from Organometallic ruthenium compound protein HA-3 and HA-5 made from embodiment 2 and comparative example 4 is sewed
The inhibitory activity curve graph of hop protein (HA- cis-platinum) (HA=seralbumin).
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The preparation method of Organometallic ruthenium compound protein provided by the invention include: pH value be 7-8(be preferably 7.2-
7.5) and concentration be 10-100mM(be preferably 20-50mM) buffer solution in, by Organometallic ruthenium anti-tumor drug and serum
Albumen is contacted, and it is preferably 5-10 that the molar ratio of the Organometallic ruthenium anti-tumor drug and haemocyanin, which is 1-20:1(:
1), the haemocyanin is amino acid sequence seralbumin as shown in SEQ ID NO:1 or amino acid sequence such as SEQ ID
Transferrins shown in NO:2.
In the present invention, to the condition of the contact, there is no particular limitation, for example, the condition of the contact may include connecing
The temperature of touching is 30-40 DEG C (preferably 35-37 DEG C), and it is preferably 45-50h that the time of contact, which is 24-72h().
In the present invention, Organometallic ruthenium made from the scheme of above range is deviateed using at least one of parameters value
Compound protein is more much lower than targeting made from the method for the present invention and anti-tumor activity, is difficult to meet the requirement of clinical application.And
It can be further improved the target of Organometallic ruthenium compound protein when controlling at least one of each parameter value in above-mentioned preferred scope
Tropism and/or anti-tumor activity.
In the present invention, in the absence of explanation to the contrary, term " transferrins " can refer to iron-containing transferrins,
It can also refer to nonferrous transferrins, still, it was found by the inventors of the present invention that target can be obtained using iron-containing transferrins
The Organometallic ruthenium compound protein that tropism and/or anti-tumor activity further enhance.Therefore, the transferrins in the present invention is preferred
For iron-containing transferrins.
In the present invention, in the absence of explanation to the contrary, the concentration of buffer solution refers to each slow in buffer solution
Rush the total concentration of component.The buffer solution can be the various buffer solutions that haemocyanin will not be made to be denaturalized, it is preferable that described
Buffer solution is triethylamine acetate ester buffer solution, trishydroxymethylaminomethane-hydrochloric acid buffer solution and phosphate buffer solution
One of.Buffer solution in the present invention can be commercially available, and can also voluntarily be prepared, and the method for preparation is this field
Well known to technical staff, details are not described herein.
In the present invention, the Organometallic ruthenium anti-tumor drug with anti-tumor activity can have to be commonly used in the art
Machine metal ruthenium compound, under preferable case, the Organometallic ruthenium anti-tumor drug is that substance shown in formula (1) (may refer to
Document " Morris, R.E.;Aird,R.E.;Murdoch,P.D.;Chen,H.M.;Cummings,J.;Hughes,N.D.;
Parsons,S.;Parkin,A.;Boyd,G.;Jodrell,D.I.;Sadler,P.J.J.Med.Chem.2001,44,3616”
" Wang, F.Y.;Habtemariam,A.;van der Geer,E.P.L.;Fernandez,R.;Melchart,M.;
Deeth,R.J.;Aird,R.;Guichard,S.;Fabbiani,F.P.A.;Lozano-Casal,P.;Oswald,I.D.H.;
Jodrell,D.I.;Parsons,S.;Sadler, P.J.Proc.Natl.Acad.Sci.U.S.A.2005,102,18269 " are closed
At obtaining).
In formula (1), A is preferably one of structure shown in general formula (2)-(8), wherein ruthenium and the phenyl ring in structure shown in A
It is coordinated by organic metal (C-M) key, meanwhile, ruthenium and-NH2In N pass through coordinate bond chelate.Using having following structure formula
Organometallic ruthenium compound can obtain the organic metal with more high anti-tumor activity as Organometallic ruthenium anti-tumor drug
Ruthenium compound protein.
In the present invention, in order to obtain the higher Organometallic ruthenium compound protein of purity, the method further preferably includes that will connect
Product after touch passes through ultrafiltration membrane.The molecular cut off of the ultrafiltration membrane is preferably 10kDa.
Organometallic ruthenium compound protein provided by the invention is made by the above method.Egg is conjugated in Organometallic ruthenium of the invention
It is white have stronger targeting and higher anti-tumor activity, it is antitumor particularly useful for making nano controlled release type Organometallic ruthenium
Drug.
The present invention also provides Organometallic ruthenium compound proteins made from the above method in preparing metal anti-tumor drug
Purposes.
The present invention will be described in detail by way of examples below.In following embodiment, organometallic ruthenium compound
[(η6-arene)Ru(en)Cl]PF6(arene is one of structure shown in formula (2)-(8), in embodiment, respectively with organic gold
Belong to ruthenium compound (2)-(8) to indicate that the arene used is the compound of formula (2)-(8) structure；En=ethylenediamine) according to document
“Morris,R.E.;Aird,R.E.;Murdoch,P.D.;Chen,H.M.;Cummings,J.;Hughes,N.D.;
Parsons,S.;Parkin,A.;Boyd,G.;Jodrell,D.I.;Sadler,P.J.J.Med.Chem.2001,44,3616”
" Wang, F.Y.;Habtemariam,A.;van der Geer,E.P.L.;Fernandez,R.;Melchart,M.;
Deeth,R.J.;Aird,R.;Guichard,S.;Fabbiani,F.P.A.;Lozano-Casal,P.;Oswald,I.D.H.;
Jodrell,D.I.;Parsons,S.;Sadler, P.J.Proc.Natl.Acad.Sci.U.S.A.2005,102,18269 " are closed
At；Seralbumin (HA) is by the Nottingham Delta Biotechnology(, Britain) (amino acid sequence such as SEQ ID is provided
Shown in NO:1)；Iron-containing and nonferrous transferrins (amino acid sequence as shown in SEQ ID NO:2, Tf) is purchased from the U.S.
Calbiochem company and in 100mM NH3HCO3It is spare that buffer solution dialysis is lyophilized into powder；Triethylamine acetate ester (TEAA) is slow
Solution is rushed purchased from Transgenomic, matched concentration is 20mM, pH 7.4；Trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl)
Buffer solution is purchased from Alfa Aesar company, and matched concentration is 50mM, pH 7.2；Phosphate buffer solution (PBS) is purchased from Beijing
Factory, matched concentration are 50mM, pH 7.5；10kDa super filter tube is purchased from Millipore company；Cis-platinum is purchased from Alfa Aesar
Company；MCF-7 cell is purchased from ATCC, and ATCC number is HTB-22TM；Haemocyanin (the TMR- of 4- rhodamine (TMR) label
Transferrins) it is purchased from Invitrogen company；Culture MCF-7 cell used medium composition be: DMEM(high sugar) in add
Antibiotic (penicillin of 100U/mL and the streptomysin of 100mg/mL) and 10% fetal calf serum (FBS), wherein DMEM and FBS
It is purchased from HyClone company.
Preparing solvent used in MTT solution is PBS(ingredient are as follows: NaCl8g/L, KCl0.2g/L, Na2HPO41.44g/L、
KH2PO40.24g/L, pH7.4), concentration 5mg/ml, MTT can restore purple first a ceremonial jade-ladle, used in libation by reductase in living cells mitochondria,
First a ceremonial jade-ladle, used in libation is dissolved in dimethyl sulfoxide (DMSO), and has special absorption in 490nm, can be used for detecting.
In following embodiment, the amount of Organometallic ruthenium compound protein is calculated with the amount of metal ruthenium element, the amount of ruthenium element by
Inductive coupling plasma emission spectrograph ICP-OES(PerkinElmer, Optima 5300DV Spectrometer) it surveys
?；The amount of TMR- Organometallic ruthenium compound protein is calculated with the amount of haemocyanin, and the measuring method of the amount of haemocyanin is BCA
Method is measured using BCA quantification of protein kit (TIANGEN Biotech (Beijing) Co., Ltd.), and concrete operations can be joined
According to document " Akins, R.E.;Tuan,R.S.BioTechniques.1992,12,469-499".
Embodiment 1-3
In buffer solution, by Organometallic ruthenium anti-tumor drug (organometallic ruthenium compound (2)-(8)) respectively with blood
Albumin (seralbumin or transferrins) is contacted, and seralbumin and organometallic ruthenium compound (2)-are respectively obtained
(8) Organometallic ruthenium compound protein (being indicated respectively with HA-2 to HA-8) and transferrins and organometallic ruthenium compound
(2)-(8) Organometallic ruthenium compound protein (being indicated respectively with Tf-2 to Tf-8), unbonded Organometallic ruthenium antineoplastic
Object removes (being centrifuged three times at 4 DEG C) by 10kDa super filter tube centrifugal ultrafiltration, and the centrifugal force of centrifugation is 5000g, and centrifugation time is
12min.Wherein, buffer solution, the type of Organometallic ruthenium anti-tumor drug and haemocyanin and dosage, the temperature of contact, connect
The design parameters such as the time of touching are referring to the following table 1.
Table 1
Embodiment 4
Organometallic ruthenium compound protein is prepared according to the method for embodiment 1, unlike, the temperature of contact is 4 DEG C.
Embodiment 5
Organometallic ruthenium compound protein is prepared according to the method for embodiment 1, unlike, the time of contact is for 24 hours.
Embodiment 6
Organometallic ruthenium compound protein is prepared according to the method for embodiment 1, unlike, Organometallic ruthenium anti-tumor drug
Dosage be 250 μm of ol, and the molar ratio for adjusting Organometallic ruthenium anti-tumor drug and haemocyanin is 4:1.
Embodiment 7
Prepare Organometallic ruthenium compound protein according to the method for embodiment 1, unlike, the haemocyanin used be without
The transferrins of iron.
Embodiment 8
Organometallic ruthenium compound protein is prepared according to the method for embodiment 1, unlike, the concentration of buffer solution is
15mM。
Comparative example 1
Organometallic ruthenium compound protein is prepared according to the method for embodiment 1, unlike, the concentration of buffer solution is 8mM.
Comparative example 2
Organometallic ruthenium compound protein is prepared according to the method for embodiment 1, unlike, buffer solution is 2- (N- morphine
Quinoline) ethanesulfonic acid (MES) buffer solution, concentration 50mM, pH 6.
Comparative example 3
Prepare Organometallic ruthenium compound protein according to the method for embodiment 1, unlike, buffer solution be pH be 8.5 and
Concentration is the TEAA buffer solution of 20mM.
Comparative example 4
Metal Drugs compound protein is prepared according to the method for embodiment 1, unlike, the metal anti-tumor drug used is
Cis-platinum (is purchased from Alfa Aesar company), obtains cis-platinum compound protein Tf- cis-platinum and HA- cis-platinum.
Test case 1
The test case is used to illustrate the targeting of Organometallic ruthenium compound protein made from the method for the present invention.
According to method identical with above-described embodiment and comparative example, haemocyanin system is replaced using TMR- haemocyanin respectively
The Organometallic ruthenium compound protein (TMR- Organometallic ruthenium compound protein) of standby TMR label.
Before test, with every ware 1 × 105For a cell by MCF-7 cell inoculation to being copolymerized in burnt glass capsule, training is added in every ware
Based 1.5 ml is supported, is placed in 37 DEG C, containing 5%CO2Cell incubator in cultivate 12h, then replace fresh culture 1.5ml, be added
TMR- haemocyanin and TMR- Organometallic ruthenium compound protein are to final concentration of 5 × 10-7M, continuation are incubated for 2h in the incubator, abandon
Culture medium is removed, is washed three times with PBS, then 1.5ml PBS is added in every ware.With laser confocal microscope (559-nmFV10-
LD559) the haemocyanin of laser excitation TMR label, and observe cellular uptake situation.
The results show that after being incubated for according to TMR- Organometallic ruthenium compound protein made from the method for embodiment 1-8, cell
The protein content of interior fluorescent marker and the protein content that intracellular Fluorescence marks after the incubation of TMR- haemocyanin are essentially identical, wherein
Using on TMR-Tf() and TMR-Tf-6(through being prepared Following the procedure of Example 1 under) be incubated for after, the intake situation of cell is such as
Shown in Fig. 1.It can thus be seen that Organometallic ruthenium compound protein made from the method for the present invention is with suitable with original serum albumen
Targeting, will not influence the targeting of haemocyanin with the conjugation of Organometallic ruthenium anti-tumor drug.
Particularly, intracellular glimmering after being incubated for according to TMR- Metal Drugs compound protein made from the method for embodiment 4-8
The protein content of signal is less than intracellular after being incubated for according to TMR- Metal Drugs compound protein made from the method for embodiment 1-3
The protein content of fluorescent marker；And after being incubated for according to TMR- compound protein made from the method for comparative example 1-4, intracellular Fluorescence mark
The protein content of note is considerably lower, illustrates using cis-platinum, the haemocyanin limited using non-present invention or controls the dense of buffer solution
The parameters such as degree cannot obtain the strong Metal Drugs compound protein of targeting not in the framework of the present definition, and control contact
Temperature and time the stronger Organometallic ruthenium compound protein of targeting can be obtained in currently preferred range.
Test case 2
The test case is used to illustrate the anti-swollen of Organometallic ruthenium compound protein made from the method for the present invention on a cellular level
Tumor activity.
MCF-7 cell suspension (4000 cells/wells, 100 μ L) shifts to an earlier date 12h and is seeded on 96 orifice plates.Then, culture medium is used
The Metal Drugs compound protein diluted concentration that organometallic ruthenium compound (2)-(8) and each embodiment and comparative example are obtained is extremely
100 μM, 25 μM, 6.25 μM, 1.5625 μM, 0.39063 μM, 0.09766 μM, 0.02441 μM, 0 μM are added in corresponding plate hole.
Then its 96 orifice plate is cultivated into 72h in cell incubator.Remove culture medium, is slowly added to PBS washing cell three times, then often
The fresh culture of 100 μ L and the MTT solution of 25 μ L is added in hole, is placed in cell incubator and is incubated for 4h, carefully removes MTT/
Culture medium is added the DMSO of 100 μ L in every hole, then orifice plate is placed under microplate reader SpectraMax M5 Reader, adjusts inspection
Survey wavelength is 490nm, and using the blank solution of not inoculating cell as blank control, when test is returned to zero with blank control.All experiments
Data are measured in parallel three times, and the result measured three times is indicated with average value ± SD, and it is bent to draw inhibitory activity with Origin8.0
Line obtains the IC of each substance50Value.The specific test method of anti-tumor activity may refer to document " Berridge, M.V.;
Tan,A.S.Arch.Biochem Biophys.1993,303,474-482”。
Data show that the Organometallic ruthenium compound protein that embodiment 1-8 is obtained inhibits the IC of MCF-7 cell50Value is in 5-15 μ
Within the scope of M, wherein inhibitory activity curve such as Fig. 2 institute of Organometallic ruthenium compound protein Tf-3 and Tf-6 made from embodiment 1
Show, inhibits the IC of MCF-7 cell50Value is respectively 13.42 ± 2.11 μM and 10.03 ± 0.23 μM, organic made from embodiment 2
The inhibitory activity curve of metal Ru compound protein HA-3 and HA-5 are as shown in figure 3, it inhibits the IC of MCF-7 cell50Value is respectively
6.43 ± 0.42 μM and 6.86. ± 0.63 μM, illustrate the antitumor work of Organometallic ruthenium compound protein made from the method for the present invention
Property is strong.
Particularly, Organometallic ruthenium compound protein made from embodiment 1-3 inhibits the IC of MCF-7 cell50Value is significantly lower than
The IC of the inhibition MCF-7 cell of Organometallic ruthenium compound protein made from embodiment 8, comparative example 1-350Value illustrates using preferred
Buffer solution can obtain the higher Organometallic ruthenium compound protein of anti-tumor activity；Organometallic ruthenium made from embodiment 1 is sewed
The IC of hop protein inhibition MCF-7 cell50Value inhibits MCF-7 significantly lower than Organometallic ruthenium compound protein made from embodiment 4-6
The IC of cell50Value illustrates the molar ratio for temperature and time, haemocyanin and the organometallic ruthenium compound that control contacts in this hair
The higher Organometallic ruthenium compound protein of anti-tumor activity can be obtained in bright preferred range；Organic gold made from embodiment 1
Belong to the IC that ruthenium compound protein inhibits MCF-7 cell50Value inhibits significantly lower than Organometallic ruthenium compound protein made from embodiment 7
The IC of MCF-7 cell50Value illustrates using Organometallic ruthenium compound protein made from iron-containing transferrins used in this application
Anti-tumor activity be apparently higher than the anti-tumor activity using Organometallic ruthenium compound protein made from nonferrous transferrins；
The IC of the inhibition MCF-7 cell of Organometallic ruthenium compound protein made from embodiment 150Value is significantly lower than cis-platinum made from comparative example 4
Compound protein (basic unrestraint activity, IC50Be worth at 100 μM or more) inhibit MCF-7 cell IC50Value illustrates using the application
The anti-tumor activity of Organometallic ruthenium compound protein made from middle Organometallic ruthenium antitumoral compounds is retained, and is used suitable
The anti-tumor activity of cis-platinum compound protein made from platinum etc. is greatly lowered.
In addition, in order to further verify the targeting of Organometallic ruthenium compound protein made from the method for the present invention, the present invention
Inventor MTT test also has been carried out to the Organometallic ruthenium compound protein that each embodiment obtains using human skin fibroblasts
(concrete operation step can be found in document " Berridge, M.V.;Tan,A.S.Arch.Biochem Biophys.1993,303,
474-482 "), the results show that Organometallic ruthenium compound protein inhibits the IC of human skin fibroblasts50Value is far longer than organic
The IC of metal ruthenium compound (2)-(8) inhibition human skin fibroblasts50Value, illustrates Organometallic ruthenium compound protein of the invention
Intake in human skin fibroblasts is low, the characteristic with preferable targets neoplastic cells.
The above test result shows that the targeting of Organometallic ruthenium compound protein made from the method for the present invention is strong and antitumor
It is active high.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (8)
1. a kind of preparation method of Organometallic ruthenium compound protein, which is characterized in that this method comprises: being 7-8 and dense in pH value
Degree is to contact Organometallic ruthenium anti-tumor drug with haemocyanin, organic gold in the buffer solution of 10-100mM
The molar ratio for belonging to ruthenium anti-tumor drug and haemocyanin is 1-20:1, and the haemocyanin is amino acid sequence such as SEQ ID NO:
The transferrins as shown in SEQ ID NO:2 of seralbumin or amino acid sequence shown in 1,
Wherein, the Organometallic ruthenium anti-tumor drug is substance shown in following formula (1),
In formula (1), A is one of structure shown in general formula (2)-(8),
The buffer solution is triethylamine acetate ester buffer solution, trishydroxymethylaminomethane-hydrochloric acid buffer solution and phosphate
One of buffer solution.
2. according to the method described in claim 1, wherein, the condition of the contact includes that the temperature of contact is 30-40 DEG C, contact
Time be 24-72h.
3. method according to claim 1 or 2, wherein the Organometallic ruthenium anti-tumor drug and haemocyanin rub
You are than being 5-10:1.
4. according to the method described in claim 1, wherein, the pH value of the buffer solution is 7.2-7.5, concentration 20-50mM.
5. according to the method described in claim 1, wherein, the transferrins is iron-containing transferrins.
6. according to the method described in claim 1, wherein, the method also includes the products after contacting to pass through ultrafiltration membrane, institute
The molecular cut off for stating ultrafiltration membrane is 10kDa.
7. Organometallic ruthenium compound protein made from the method as described in any one of claim 1-6.
8. the purposes of Organometallic ruthenium compound protein as claimed in claim 7 in the preparation of antitumor drugs.
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