CN109224080A - A kind of targeted nano carrier of support nucleosides series antineoplastic medicament and its preparation method and application - Google Patents
A kind of targeted nano carrier of support nucleosides series antineoplastic medicament and its preparation method and application Download PDFInfo
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Abstract
The present invention provides targeted nano carriers of a kind of support nucleosides series antineoplastic medicament and its preparation method and application, the targeted nano carrier, include: DNA tetrahedron, Affibody molecule and nucleosides series antineoplastic medicament, preparation method includes the following steps: nucleosides series antineoplastic medicament a, being subjected to phosphoramidite modification；B, single-stranded using four kinds of DNA of DNA solid phase synthesis technique synthesis, and the nucleosides series antineoplastic medicament is integrated into 5 ' single-stranded ends of four kinds of DNA, in 3 ' the terminal modified NH that a kind of wherein DNA is single-stranded2；C, affibody molecule is connected to above-mentioned NH by linker23 ' the DNA of modification single-stranded ends；D, four kinds of DNA are single-stranded with molar ratio 1:1:1:1 Hybrid assembling, obtain DNA tetrahedron-affibody targeted nano carrier.Targeted nano carrier of the invention can accurately control the combined amount of drug, have faster cancer target ability and higher cell, tissue penetration, and good drug efficacy is suitable for promoting and applying.
Description
Technical field
The present invention relates to medicine and pharmacology technical fields, and in particular to a kind of targeted nano load of support nucleosides series antineoplastic medicament
Body and its preparation method and application.
Background technique
Nucleoside analog is clinically very important anti-tumor drug, including a variety of purine and Pyrmidine nucleoside derivatives,
It is mainly used for the treatment of hematologic malignancies and part entity tumor.They belong to antimetabolic species drug, competing with physiological nucleosides
It strives, by interfering the DNA synthesis of tumour cell, or the transcription of influence nucleic acid, inhibits the synthesis of protein, to reach
To the effect for the treatment of tumour.
So far, the nucleoside analog of at least 15 kinds of FDA approval accounts for current cancer chemotherapy for treating various cancers
Nearly the 20% of drug, such as 5 FU 5 fluorouracil, cytarabine, gemcitabine and Decitabine.5 FU 5 fluorouracil is as typical
Nucleoside analogue drugs have been widely used in the various malignant tumours for the treatment of, including colon since nineteen fifty-seven is firstly introduced clinic
Cancer, the carcinoma of the rectum, gastric cancer, breast cancer, oophoroma, chorioepithelioma, chorioadenoma, G. cephalantha, cutaneum carcinoma, liver cancer,
Bladder cancer etc..As uracil antimetabolite, 5 FU 5 fluorouracil or deoxyfluorouridine inhibit cell by incorporation DNA or RNA
Division, and by competitive binding thymidylate synthase, block deoxyribonucleotides nucleotide to be converted into thymidine core
Glycosides core is active to play its.However, when carrying out treatment of cancer using 5 FU 5 fluorouracil or deoxyfluorouridine, in people's body-internal-circulation
Half-life short, only 8-20min, and will appear serious side effect, such as serious gastrointestinal toxicity react, and appetite subtracts
It moves back, Nausea and vomiting, stomatitis, gastritis, diarrhea etc.；Serious bone marrow suppression toxicity, can cause leucocyte and decrease of platelet.Cause
This, the disadvantages of in order to reduce the toxic side effect of drug, overcome oral absorption unstable and half-life short, researchers do it
A large amount of work mainly includes that the load medicine of the modifying for chemical structure work and nano material to 5 FU 5 fluorouracil itself studies work
Make.
In terms of modifying for chemical structure transformation, researcher in order to overcome Intravenous Infusion to patient's bring phlebitis,
The problems such as venous cannula related complication, has carried out continuous transformation to the structure of 5 FU 5 fluorouracil, develop it is convenient, equivalent,
The derivative antineoplastic of highly-safe oral 5 FU 5 fluorouracil, such as Tegafur, deoxyfluorouridine, floxuridine, Carmofur and Ka Pei
His shore etc..In order to maintain blood concentration stable in blood plasma and tumor tissues, researcher is by being added dihydropyrimidine dehydrogenase suppression
Preparation forms compound anti-tumor drug and is applied to clinical treatment, such as tegafur, gimeracil and oteracil potassium, excellent fluorine pyridine.Although these 5 FU 5 fluorouracils spread out
Biology has been applied to clinical treatment, and toxicity is lower than 5 FU 5 fluorouracil, but to tumour cell non-selectivity, former capital cannot
Avoid the damage of normal tissue；In order to enhance the compatibility of 5 FU 5 fluorouracil and tumour cell, amino acid, small peptide, pyridine or
Polysaccharide etc. is connected by chemical synthesis means with 5 FU 5 fluorouracil by researcher, realizes the sustained release of drug and lower
Toxic side effect improves the bioavilability of drug, at present still in the experimental stage, not yet can be applied to clinic.
The nano material of chemotherapeutics loads, transport, release has become the forward position of anti-tumor drug and hot research is led
5 FU 5 fluorouracil is attached to by one of domain using bio-medical macromolecular as carrier, and prodrug is made and is sent into vivo, then leads to
The fracture of macromolecular material and the degradation of vivo biodistribution enzyme are crossed, releases drug slowly in vivo, to reduce administration
Number and dosage, it is effective to improve 5 FU 5 fluorouracil drug effect, reduce side effect.These studied nano materials include such as carbon
Nanotube, micro-pore zeolite, hydrogel, chitosan, liposome and polymer etc..However, for inorganic nano material, it
Usually contain toxic element or residue, and be difficult to degrade.These toxic elements or residue are long-term in certain tissues
After accumulation, there are safety problem risk or potential side effects；And for organic nano material, as constructed by them
Nanoparticle be all in terms of size, composition and surface chemical modification it is non-uniform, which results in its drug covering properties
It is undesirable, inorganizable specificity and the problem of genotoxic potential.Therefore, how to improve the biocompatibility and drop of nano material
Solution property and the toxicity for reducing nano material are the hot issues studied at present about delivery system.
In recent years, becoming the important directions of anti-tumor drug development, this kind of medicine for the targeted therapy of tumour cell
Object mainly acts on the specific receptor on tumour cell, on the one hand improves the lethality to tumour cell, on the other hand reduces
The adverse reaction of normal tissue and cell.Targeted therapy mainly includes the targeting therapy for tumor and match that Ag-Ab mediates
The receptor-mediated targeting therapy for tumor of body-.Targeting antibodies drug has been achieved for biggish success in treatment of cancer, there is 13
A antibody target medicine by FDA approval be used for antitumor clinical treatment, such as treatment of colorectal cancer Cetuximab and
Victibix, for the Herceptin of breast cancer treatment and handkerchief trastuzumab etc..But there is killing in process of clinical application
Power is poor, is also easy to produce the deficiencies of drug resistance, often needs to use with chemotherapy drugs in combination.Therefore, antibody coupling drug (Antibody
Drug Conjugate, ADC) exploitation increasingly pursued by researcher, which is by selectively targeted tumour cell
Antibody and a kind of therapeutic agent for being attached by linker of cytotoxic drug, realize small molecule chemotherapeutic and antibody
The drug combination of targeted therapy provides a kind of new approach for the accurate treatment of tumour.But the development difficulty of ADC drug is very
Greatly, it is mainly hampered by humanization modified, small-molecule drug screening and the coupling efficiency of linker etc. of targeting antibodies, at present
Only 2 kinds of ADC drugs are listed in European and American developed countries, including using CD30 and HER2 as the Adcetris (SGN-35) of target and
Kadcyla(T-DM1).In addition, the monoclonal antibody molecule amount in ADC drug is very big (150kDa), pass through capillary endothelium
Layer and be restricted across tumour cell external series gap, influence its play drug effect；And drug coupled by most of ADC drugs
The molar ratio of molecule and antibody molecule is averagely maintained between 3-4, and drug loading levels are lower.
It is a kind of internal special recognition mechanism that ligand-receptor, which combines, has high specific, highly selective and high-affinity
The characteristics of, chemotherapeutics is tagged on ligand, targeted drug is made, on the tumour cell of High Cell Density And High Expression by
Body combines, then by receptor-mediated encytosis, so that chemotherapeutics is entered tumour cell and play its therapeutic effect, this is significantly
Alleviate the toxic side effect of drug normal tissue.Human epidermal growth factor receptor-2 (HER-2) is breast cancer targeting in recent years
One very attractive target for the treatment of.The about patient of 25%-30% shows as HER-2 in all patient with breast cancers
Overexpression, and this is often closely related with cell Proliferation, cells survival, invasion and metastatic, antigen active increase etc..
Affinity ligand-affibody of HER-2 specificity is a kind of small molecule scaffold albumen for being functionally similar to antibody, by 58 ammonia
Base acid residue composition, relative molecular mass is about 6.5 × 103, 3 alpha-helixes are contained in structure.Compared with monoclonal antibody,
Affibody molecule has smaller size, faster cancer target ability and higher cell, tissue penetration, therefore,
It has been applied to various carriers, including liposome, nano particle, biopolymer and adenovirus vector etc..
Adriamycin is a kind of broad-spectrum anti-tumor medicine, reports a kind of DNA-affibody- adriablastina target in document at present
Nano medication, and show than adriamycin to the stronger selectivity of the highly expressed breast cancer cell of HER2 and inhibiting effect.So
And during the experiment, it has been found that by the load prescription formula that adriamycin is inserted into DNA nanostructure, it is difficult to which accurate control carries
Medicine quantity.Moreover, the non-covalent bond between DNA-affibody and adriamycin is very weak, it is easily broken in operation.
Therefore, a kind of targeted nano anti-tumor drug with accurate drugloading rate and biocompatibility is studied to be of great significance.
Summary of the invention
One of the objects of the present invention is to provide a kind of targeted nano carriers of support nucleosides series antineoplastic medicament, to solve
Existing antineoplastic drug carrier system drugloading rate is difficult to control, biocompatibility and degradability are poor, side reaction is serious and drug effect
Play undesirable problem.
The second object of the present invention is to provide a kind of preparation of the targeted nano carrier of support nucleosides series antineoplastic medicament
Methods and applications.
An object of the present invention is achieved through the following technical solutions: a kind of target of support nucleosides series antineoplastic medicament
To nano-carrier, comprising:
DNA tetrahedron, to be self-assembly of by four DNA are single-stranded with base pair complementarity principle；
Affibody molecule passes through linker connection for the small molecular protein ligand in conjunction with HER2 receptor-specific
In the 3 ' ends that DNA is single-stranded；And
Nucleosides series antineoplastic medicament, is integrated into that form tetrahedral four DNA of DNA mono- by DNA solid phase synthesis technique
In chain, the number of the nucleosides series antineoplastic medicament of the single-stranded middle insertion of every DNA is 1-100.
Four DNA single-stranded nucleotide sequence respectively as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3,
Shown in SEQ ID NO.4.
The linker is 6- (dimaleoyl imino) caproic acid succinimide ester, the coupling of the affibody molecule
Number is 1-4.
The nucleosides series antineoplastic medicament is deoxyfluorouridine, 5 FU 5 fluorouracil nucleosides, cytarabine, gemcitabine, chlorine
One of farad shore, fludarabine, nelarabine, Decitabine, azacitidine and Acadesine.
The partial size of the targeted nano carrier is less than 20nm.
The present invention provides the preparation method of the targeted nano carrier of above-mentioned support nucleosides series antineoplastic medicament, including it is following
Step:
A, nucleosides series antineoplastic medicament is subjected to phosphoramidite modification, to be converted into corresponding nucleosides series antineoplastic medicament
Phosphoramidite intermediate is used for DNA solid phase synthesis technique；The structure of the nucleosides series antineoplastic medicament phosphoramidite intermediate
Formula are as follows:
Wherein, R in formula I1For F or H, R2For F or H；The structure of Base is such as the one of flowering structure in formula I, formula II and formula III
Kind:
For different nucleosides series antineoplastic medicament phosphoramidite intermediates, synthetic route as follows is selected to be closed
At:
Synthetic route 1:
Synthetic route 2:
B, single-stranded using four kinds of DNA of DNA solid phase synthesis technique synthesis, and the nucleosides series antineoplastic medicament is integrated into
5 ' four kinds of DNA single-stranded ends, in 3 ' the terminal modified NH that a kind of wherein DNA is single-stranded2, remaining DNA is single-stranded not to be modified, product warp
It is single-stranded that four kinds of DNA containing nucleosides series antineoplastic medicament are obtained after purification；
The DNA synthesis in solid state process is as follows:
The first step, the protected nucleotide of active group and three that will be connected in advance on solid phase carrier (usually CPG)
Chloroacetate reaction sloughs the blocking group DMT of its 5 '-hydroxyl, obtains 5 '-free hydroxyls；
Second step, the raw material (nucleotide monomer of phosphoramidite protection) of synthetic DNA, and mixed with activator tetrazole,
Nucleosides phosphorous acid activated intermediate is obtained, its 3 ' ends are activated, and 5 '-hydroxyls are still protected by DMT, by its first step middle reaches
From 5 '-hydroxyls occur condensation reaction；
Third step, under the action of oxidant iodine, phosphorous acyl form is changed into more stable phosphotriester；
4th step, band cap react, and may have only a few 5 '-hydroxyl not participate in reaction (less than 2%) in condensation reaction, use
Acetic anhydride and 1- methylimidazole terminate its subsequent reactions, and this short-movie section can be separated off in subsequent purification link；
By above four steps, a deoxynucleotide is connected on the nucleotide of solid phase carrier.Again with three chloroethenes
Acid sloughs the blocking group DMT on its 5 '-hydroxyls, repeats above step, until single-stranded all nucleotide of DNA and required
On the nucleosides series antineoplastic medicament of support is all connected.The color that TCA processing stage can be observed in synthesis process determines synthesis
Efficiency.By ammonium hydroxide high-temperature process, the segment being connected on CPG, which is cut, to be come, by the means purified fragments such as OPC, PAGE,
Finished product segments are concentrated with C18, desalination, precipitating.Segment aqueous suspension after precipitating, measurement OD260 is quantitative, and according to requiring point
Dress.
The DNA sequence dna containing nucleosides series antineoplastic medicament synthesized by DNA solid phase synthesis technique is as follows:
A13F-NH2:’-NnACACTACGTCAGAACAGCTTGCATCACTGGTCACCAGAGTA-NH2-3'；
B13F:5’-NnACGAGCGAGTTGATGTGATGCAAGCTGAATGCGAGGGTCCT-3'；
C13F:5’-NnTCAACTCGCTCGTAACTACACTGTGCAATACTCTGGTGACC-3'；
D13F:5’-NnTCTGACGTAGTGTATGCACAGTGTAGTAAGGACCCTCGCAT-3’。
Note: N indicates nucleosides series antineoplastic medicament；N is the connection number of nucleosides series antineoplastic medicament, wherein 1 < n < 100.
Obtain four kinds of single-stranded freeze-dried powders of DNA through DNA synthesis in solid state, be stored in -20 DEG C it is spare.
C, affibody molecule is connected to above-mentioned NH by linker23 ' the DNA of modification single-stranded ends, product is through ion
Displacement chromatography, affinity chromatography and desalting column obtain DNA-affibody chimera after purification；
The amino acid sequence of affibody molecule is as follows:
MIHHHHHHLQVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAE
AKKLNDAQAPKVDC.。
Affibody molecule carries out Expression product in e. coli bl21 (DE3) cell, and IPTG induced concentration is 1mM,
Induction time is 16h, and inducing temperature is 30 DEG C.After the completion of inducing expression, 6000rpm is harvested by centrifugation cell, high pressure is broken, from
The heart obtains the broken supernatant containing affibody, is purified using one step of affinity chromatography and obtains affibody molecule.
By 3 ' terminal modified NH2DNA it is single-stranded be dissolved in phosphate buffer (PBS), and be added contain 6- (maleimide
Base) caproic acid succinimide ester (EMCS) dimethyl sulphoxide solution, after completion of the reaction, be added pre-cooled ethanol low-temperature precipitation
Supernatant is abandoned in DNA, centrifugation, and PBS is re-dissolved.Targeting ligand affibody molecular solution is added and carries out coupling reaction, has reacted
Bi Hou carries out the purifying of DNA-affibody chimera using ion-exchange chromatography, affinity chromatography and desalting column, anti-
Answer route as follows:
Note: F represents nucleosides series antineoplastic medicament；P is represented and is keyed between them by phosphatide.
D, the DNA-affibody chimera and other three kinds of DNA is single-stranded with molar ratio 1:1:1:1 mixing, then set
In PCR instrument, run by the temperature program of 75 DEG C of heating 10min, 4 DEG C of cooling 20min, 10 DEG C of holding 10min, operation finishes
After take out, obtain the DNA tetrahedron-affibody targeted nano carrier of support nucleosides series antineoplastic medicament, self assembling process
It is as follows:
In step c, the linker is 6- (dimaleoyl imino) caproic acid succinimide ester, the affibody molecule
Coupling number be 1-4.
In step c, cysteine is introduced in the C-terminal of the affibody molecule, the sulfydryl and 6- (horse using cysteine
Carry out imide) connection of the active group of caproic acid succinimide ester；Histidine is introduced in the N-terminal of the affibody molecule
Label, to allow to be isolated and purified by affinity chromatography.
Four kinds of DNA single-stranded nucleotide sequence respectively as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3,
Shown in SEQ ID NO.4；The nucleosides series antineoplastic medicament is deoxyfluorouridine, 5 FU 5 fluorouracil nucleosides, cytarabine, Ji
One of western his shore, clofarabine, fludarabine, nelarabine, Decitabine, azacitidine and Acadesine；The targeting
The partial size of nano-carrier is less than 20nm.The structure of the nucleosides series antineoplastic medicament is as follows:
Uridine analog:
Cytosine nucleoside analogs:
Guanosine analog:
Adenosine analog:
The present invention also provides the targeted nano carriers of above-mentioned support nucleosides series antineoplastic medicament to be directed to HER2 high in preparation
Express the application in the anti-tumor drug of tumour cell.
Compared with prior art, the invention has the following beneficial effects:
1, targeted nano carrier of the invention avoids other organic materials or nothing using DNA tetrahedron as carrier material
Machine material
Toxic side effect brought by expecting, and biocompatibility and degradability with height.
2, targeted nano carrier of the invention is that nucleosides series antineoplastic medicament is made drug using DNA solid phase synthesis technique
It is covalently attached between molecule and DNA tetrahedron-affibody by phosphatide key, this preparation method can accurately control drug
Combined amount and stable load medicine structure.
3, targeted nano carrier of the invention is utilized through affibody molecule in conjunction with the HER2 receptor on tumour cell
Drug is included in cells play effect by the encytosis that ligand-receptor mediates, and reduces the toxic side effect to normal cell.
4, targeted nano diameter of carrier of the invention is small, has faster cancer target ability and higher cell, tissue
Penetration capacity, good drug efficacy.
5, preparation method of the present invention is simple, is automatically synthesized using the realization of DNA solid phase synthesis technique, synthetic product is convenient for purifying
Separation is suitable for promoting and applying.
6, targeted nano carrier of the invention is a kind of Nano medication of active targeting, can further load and deliver it
He carries out multiple medicine combination therapy to cancer by high efficiency anti-tumor drug.
Detailed description of the invention
SDS-PAGE analysis after Fig. 1 .affibody affinitive layer purification, wherein 1 penetrates peak for affinity chromatography；2 are
The eluting peak of affinity chromatography.
Electrophoretic analysis after Fig. 2 .DNA-affibody chimera Capto DEAE column chromatographic purifying, wherein 1 is to penetrate
Peak；2 be 500mM NaCl eluting peak 1；3 be 500mM NaCl eluting peak 2；4 be 1000Mm NaCl eluting peak.
Fig. 3 .DNA-affibody chimera HisTrapTMElectrophoretic analysis after HP column affinitive layer purification, wherein 1 is
Penetrate peak；2 be eluting peak 1；3 be eluting peak 2.
The electrophoretic analysis of the DNA-affibody chimera of Fig. 4 purifying, wherein 1 is embedding for DNA-affibody after purification
It is fit；2 be the single-stranded A13F of DNA；3 be affibody molecule.
The electrophoretic analysis result of the DNA tetrahedron-affibody nano-carrier of Fig. 5 support oligomerization floxuridine, wherein M is
DNA Marker-B；1 is the single-stranded A13F of DNA；2 be A13F DNA-affibody chimera；3 be DNA tetrahedron；4 be support
The DNA tetrahedron of oligomerization floxuridine；5 be the DNA tetrahedron-affibody nano particle of support oligomerization floxuridine.
The atomic force microscope qualification result of the DNA tetrahedron-affibody nano-carrier of Fig. 6 support oligomerization floxuridine
Figure.
The stability analysis figure of the DNA tetrahedron-affibody nano-carrier of Fig. 7 support oligomerization floxuridine.
The cellular uptake analysis chart of the DNA tetrahedron-affibody nano-carrier of Fig. 8 support oligomerization floxuridine.
The cytotoxicity analysis figure of the DNA tetrahedron-affibody nano-carrier of Fig. 9 support oligomerization floxuridine.
Specific embodiment
Below by embodiment, the present invention will be described in detail, and experimental method used in following embodiments is unless otherwise specified
It is conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The synthesis of 1 nucleosides series antineoplastic medicament phosphoramidite intermediate of embodiment
In order to by nucleosides series antineoplastic medicament be integrated into DNA it is single-stranded in, nucleosides series antineoplastic medicament first has to pass through chemistry
Synthesis, is converted into the form of corresponding phosphoramidite intermediate.Below by taking deoxyfluorouridine as an example, by following synthetic route into
The synthesis of row phosphoramidite intermediate.
Firstly, 4 are weighed, 4- dimethoxytrityl chloride (DMT-Cl) (1.860g, 5.5mmol) and deoxyfluorouridine
(1.231g, 5mmol) is added 50mL anhydrous pyridine, stirs evenly, and reaction 4h is stirred at room temperature.Then 5mL methanol is added, continues
It is stirred to react 30min.Vacuum rotary steam removes solvent, and column chromatography for separation product obtains white powder (compound 2), yield
80%.1H NMR (400MHz, DMSO-d6) δ (ppm): 11.86 (1H, br s, NH), 7.88 (1H, d, J=6.8Hz, CH),
7.39 (2H, m, arom H), 7.27 (7H, m, arom H), 6.89 (4H, m, arom H), 6.14 (1H, dd, J=6.6,
5.2Hz,CH), 5.33(1H,br S,OH),4.27(1H,m,CH),3.89(1H,m,CH),3.74(6H,s,-OCH3),
3.29-3.10(2H,m, CH2),2.29-2.12(2H,m,CH2).13C NMR(100MHz,DMSO-d6)δ(ppm):158.57
(overlap,2C), 1C(157.61,157.35),149.39,145.23,1C(141.59,139.29),135.90,
135.75,10C(130.17,128.33, 128.08,127.19,125.15,124.82),113.69(overlap,4C),
86.26,86.03,84.96,70.50,64.11,55.47, 55.38,solvent C&1C(40.60,40.39,40.19,
39.98,39.77,39.56,39.35).HRMS:(ESI)[M+Na]+ calcd.for C30H29FN2O7,571.1856,
found 571.1852.
Secondly, Weigh Compound 2 (1.0g, 1.824mmol) is dissolved in anhydrous methylene chloride (DCM) (20mL), and it is added and contains
There are 2- cyanoethyl a N, N, N ', N '-tetraisopropylphosph-ro phosphoryl diamine (0.587mL, 2.360mmol) and 1H-TETRAZOLE (165.3mg,
Acetonitrile solution 2.360mmol), argon gas protection, is stirred to react 1h at room temperature.Reaction mixture vacuum rotary steam removes solvent, column
Chromatography product obtains white powder (compound 3), yield 60%.1H NMR(400MHz,DMSO-d6)δ(ppm):
11.89(1H,br s,NH),8.00-7.90(1H,m,CH),7.45-7.38(2H,m,arom H),7.35-7.19(7H,m,
arom H),6.93-6.84(4H,m,arom H),6.22-6.09(1H,m,CH),4.70-4.34(1H,m,CH),4.10-
3.97 (1H, m, CH), 3.74 (6H, d, J=2.3Hz ,-OCH3), 3.68-3.58 (2H, m, CH2), 3.59-3.42 (2H, m,
CH),3.39-3.17 (2H,m,CH2),2.80-2.61(2H,m,CH2),2.48-2.24(2H,m,CH2),1.19-0.93
(12H,m,CH3).13C NMR(100MHz,DMSO-d6)δ(ppm):158.63(overlap,2C),1C(157.63,
157.37),149.38,145.14, 1C(141.66,139.36),135.79,135.69,135.66,10C(130.19,
130.17,128.31,128.11,128.07,127.22, 125.39,125.20,125.05,124.86),1C(119.41,
119.22),113.66(overlap,4C),3C(86.43,86.35,85.09, 85.05,84.67),1C(73.10,72.92,
72.67,72.52),1C(63.66,63.45),1C(58.91,58.86,58.72,58.68), 55.47(overlap,2C),
43.12,43.00,solvent C(40.61,40.40,40.19,39.98,39.77,39.56,39.36),1C (38.77,
38.62),4C(24.83,24.76,24.72,24.65,24.58),1C(20.33,20.28,20.21).HRMS:(ESI) [M+
H]+calcd.for C39H46FN4O8P,749.3116,found 749.3456.
By taking gemcitabine as an example, the synthesis of phosphoramidite monomer is carried out by following synthetic route.Firstly, 4 are weighed, the bis- first of 4-
The anhydrous pyrrole of 50mL is added in oxygroup trityl chloride (DMT-Cl) (3.38g, 10mmol) and gemcitabine (2.63g, 10mmol)
Pyridine stirs evenly, and reaction 4h is stirred at room temperature.Then 5mL methanol is added, continues to be stirred to react 30min.Vacuum rotary steam removes molten
Agent, column chromatography for separation product obtain white powder (compound 2), yield 80%. 1HNMR(600MHz,CDCl3):3.42
(dd, 1H, J=12Hz), 3.49 (dd, 1H, J=8.4Hz), 3.69 (s, 6Hz), 4.04 (dd, 1H, J=5. 4Hz), 4.41
(dd, 1H, J=8.4Hz), 5.51 (dd, 1H, J=7.2Hz), 6.31 (s, 1H) 6.78 (d, 4H, J=14.4Hz) 7.14 (t,
1H, J=7.2H z), 7.22 (t, 2H, J=7.8Hz), 7.28 (d, 4H, J=9Hz), 7.38 (d, 2H, J=7.8Hz),
7.63.42 (d, 1H, J=6.6Hz) .13CNMR:16 2.81,158.46,155.86,144.50,143.09,136.02,
130.33,129.17,128.48,127.24,113.38,95.70,91.07,87.18,7 9.96,71.96,66.51.
Secondly, Weigh Compound 2 (2.83g, 5mmol) is dissolved in anhydrous methylene chloride (DCM) (20mL), DIEPA (0.78
G, 6mmol) it is dissolved in the anhydrous DCM of 30ml；2- cyanoethyl N, N- diisopropyl chloro phosphoramidite are added dropwise into reaction solution
The anhydrous DCM solution 10ml of (1.21g, 5.1mmol) is stirred at room temperature under argon gas protection, and TLC monitoring reaction is until reacted
At.It is diluted with 50ml DCM, with saturated common salt water washing (100ml × 3), organic phase anhydrous Na2SO4Dry, vacuum rotary steam is gone
Except solvent, product is used column chromatography, obtains white powder (compound 3), yield 60%.1HN MR(600MHz,DMSO-
D6): 1.14 (s, 12H), 2.57 (t, 2H, J=6Hz), 3.26 (q, 1H), 3.54 (q, 1H), 3.63 (m, 2H), 3.88 (s,
6H), 4.05 (q, 2H), 4.39 (m, 1H), 4.77 (m, 1H), 6.09 (d, 1H, J=13.2Hz), 6.39 (t, 1H, J=
25.2Hz), 6.53 (s, 2H), 6.94 (d, 4H, 9Hz), 7.27 (m, 3H), 7.348 (m, 2H), 7.40 (d, 4H, J=9Hz),
8.25 (d, 1H, J=13.2Hz) .13CNMR (600MHz, DMSO-d6): 162.81,158.46,155.86,144.55,
143.09,136.02,129.17,127.24,118.94,113.38,95.70,90.93, 87.18,77.52,77.15,
65.91,59.03,56.04.
Again, compound 3 (24.32g, 100mmol) and DMF 200mL are added in three-necked bottle, stirs to obtain white suspension
Liquid is added dropwise benzoyl oxide 10.4mL (110mmol), obtains clarified solution in room temperature reaction 18h, TLC monitoring reaction is until reacted
At.It is diluted with 50ml DCM, with saturated common salt water washing (100ml × 3), organic phase is dry with anhydrous Na 2SO 4, vacuum rotary steam
Solvent is removed, product is used column chromatography, obtains white powder (compound 4), yield 70%.1H NMR(600MHz,DMSO-
D6): 1.17 (s, 12H), 1.98 (s, 3H), 2.61 (t, 2H, J=4.8Hz), 3.28 (q, 1H), 3.53 (q, 1H), 3.78 (m,
2H),3.87(s,6H),4.13(m,2H),4.39(m,1H),4.84(m,1H),6.39(m,2H),6.62(s,1H),6.93(d,
4H, 9Hz), 7.2 9 (m, 3H), 7.36 (d, 1H, 3.6Hz), 7.38 (d, 6H, J=9Hz), 8.32 (d, 1H, J=13.2Hz)
.13CNMR(600MHz,DMSO-d6): 174.09,158.46,155.58,143.37,129.17,127.24,118.94,
113.38,96.17,90.93,87.18,77.15,65.9150.04,44. 20,23.31,22.93,22.89.
The DNA of 2 support oligonucleotides series antineoplastic medicament of embodiment single-stranded synthesis
Oligomerization deoxyfluorouridine (n=10) sequence is connected to tetrahedral four kinds of DNA by DNA solid phase synthesis technique
5 ' DNA single-stranded ends (F represents deoxyfluorouridine), the wherein terminal modified NH2 of a kind of DNA single-stranded 3 '.Each DNA single-stranded nucleosides
Acid sequence is as follows:
A13F-NH2:
5’-FFFFFFFFFFACACTACGTCAGAACAGCTTGCATCACTGGTCACCAGAGTA-NH2-3'；
B13F:5'-FFFFFFFFFFACGAGCGAGTTGATGTGATGCAAGCTGAATGCGAGGGTCCT-3'；
C13F:5'-FFFFFFFFFFTCAACTCGCTCGTAACTACACTGTGCAATACTCTGGTGACC-3'；
D13F:5’-FFFFFFFFFFTCTGACGTAGTGTATGCACAGTGTAGTAAGGACCCTCGCAT-3’。
Synthesize to obtain four kinds of single-stranded freeze-dried powders of DNA through DNA, be stored in -20 DEG C it is spare.
Oligomerization gemcitabine (n=16) sequence is connected to the tetrahedral four kinds of DNA of DNA by DNA solid phase synthesis technique
(F represents deoxyfluorouridine) is held in single-stranded 5 ', wherein a kind of terminal modified NH2 of DNA single-stranded 3 '.Each DNA single-stranded nucleotides sequence
It arranges as follows:
A13Gem-NH2:
5’-GemGemGemGemGemGGemGemGemGemGemGGemGemGemGemGemGemACACTACGT CAGAA
CAGCTTGCATCACTGGTCACCAGAGTA-NH2-3'；
B13Gem:
5’-GemGemGemGemGemGGemGemGemGemGemGGemGemGemGemGemGemACGAGCGAG TTGAT
GTGATGCAAGCTGAATGCGAGGGTCCT-3'；
C13Gem:
5’-GemGemGemGemGemGGemGemGemGemGemGGemGemGemGemGemGemTCAACTCGC TCGTA
ACTACACTGTGCAATACTCTGGTGACC-3'；
D13Gem:
5’-GemGemGemGemGemGGemGemGemGemGemGGemGemGemGemGemGemTCTGACGTA GTGTA
TGCACAGTGTAGTAAGGACCCTCGCAT-3’。
Synthesize to obtain four kinds of single-stranded freeze-dried powders of DNA through DNA, be stored in -20 DEG C it is spare.
The fermenting and producing and purifying of 3 affibody molecule of embodiment
The sequence of affibody molecule for this embodiment are as follows:
MIHHHHHHLQVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKK
LNDAQAPKVDC.
Affibody carries out Expression product in e. coli bl21 (DE3) cell.Seed liquor condition of culture are as follows: 37 DEG C,
200rpm vibrates, culture solution 10 hours in 80mL LB culture medium (100 μ g/mL ampicillin).5L fermentation tank culture condition
Are as follows: 10% (v/v) inoculum concentration is inoculated into the 5L fermentor equipped with 2.0L LB culture medium (100 μ g/mL ampicillin).
Meanwhile the IPTG of 0.5mM final concentration being added into culture medium and cools the temperature to 30 DEG C.During entire fermented and cultured, lead to
It crosses and is automatically added to ammonia spirit culture solution pH is maintained at 7.0.After fermentation, centrifugation obtains the large intestine containing affibody
Escherichia coli containing affibody are suspended in the 50mM Tris-HCl containing 300mM NaCl and 10mM imidazoles by bacillus
(pH8.0) in, clasmatosis then is carried out with super-pressure biomixer.Clasmatosis liquid is centrifuged under the revolving speed of 15000g
30 minutes, supernatant removed cell fragment with 0.45 μm of membrane filtration.UsingProtein purification chromatographic system carries out Ni-
NTA affinitive layer purification.With 50mM Tris-HCl (pH8.0) elution buffer containing 300mM NaCl and 150mM imidazoles
After elution, obtained eluting peak is subjected to purity analysis with 12%SDS-PAGE.The eluting peak is through Sephadex G-25 desalination
It after removing excess imidazole, is dispensed, packing sample is saved in -80 DEG C.The SDS-PAGE analysis result of Affibody molecule is such as
Shown in Fig. 1, molecular size range is about 8.3kDa, is consistent with budget result, and purity is greater than 95% or more.
The synthesis of the DNA-affibody chimera of 4 support oligomerization deoxyfluorouridine of embodiment
By the single-stranded A13F-NH of DNA2(261.6 μ g, 16.8nmol) is dissolved in 200 μ L phosphate buffer (PBS, 10mM
PO43-, 137mM NaCl, 2.7mM KCl) in, the DMSO that 20 μ L contain 50mM EMCS is added thereto.37 DEG C of reaction 3h,
22 μ L (3M) sodium acetates are added, stop reaction.After 600 μ L pre-cooled ethanols are added and are placed 30 minutes at -20 DEG C,
15000g, 30min centrifugation.Precipitate the ethanol washing with 70%.DNA is dissolved in the PBS solution of 200 μ L after washing, then to its
It is middle that the 1500 μ LPBS solution for containing 1500 μ g (180nmol) affibody are added, 1-5h is reacted at room temperature, reaction mixing is obtained
Object.
Reaction mixture purifies on Capto DEAE column (1mL, GE Healthcare), and with containing 500mM and
Tris-HCl (10mM, the pH8.0) buffer of 1000mM NaCl elutes.Using 10% native polyacrylamide gel electrophoresis
Analyze the DNA-affibody chimera of purifying.As seen from Figure 2, DNA-affibody chimera mainly collects purification result
In in Tris-HCl (10mM, pH8.0) eluent of 500mM NaCl, collect the elution fractions for separating in next step.
The eluent containing DNA-affibody chimera of previous step separation is pure by the continuation of Ni- affinity chromatographic column
Change.Upper step eluent is loaded on HisTrapTM HP column (1mL, GE Healthcare), then with containing 300mM
Pillar is washed 5 times of column volumes by 50mM TrisHCl (pH8.0) buffer of NaCl and 10mM imidazoles.Finally,
HisTrapTM HP column is eluted with 50mM Tris-HCl (pH8.0) buffer containing 300mM NaCl and 150mM imidazoles.It adopts
With the DNA-affibody chimera of 10% native polyacrylamide gel electrophoresis analysis purifying.Purification result can be with by Fig. 3
Find out, DNA-affibody chimera is concentrated mainly in 50mM Tris-HCl (pH8.0) eluent of 150mM imidazoles, is received
Collect the elution fractions for separating in next step.
The DNA- after purification of concentration is obtained using ultrafiltration centrifugal filter (relative molecular mass ends 10kDa)
Affibody chimera, using the DNA-affibody chimera after the concentration of 12%SDS-PAGE detected through gel electrophoresis, and respectively
It is dyed with Coomassie brilliant blue and GelRed, can be seen that DNA-affibody chimera from Fig. 4 result both can be by coomassie
Brilliant blue dyeing, can also be dyed, and purity > 90% by GelRed, show that DNA-affibody chimera is coupled successfully.This is dense
Chimera after contracting is saved at 4 DEG C for testing in next step.
The tetrahedral synthesis of DNA-affibody of 5 support oligomerization deoxyfluorouridine of embodiment
The A13F DNA-affibody chimera (10.0nmol) that upper step is purified, B13F (10.0nmol), C13F
8mL, 10mM TrisHCl, pH 8.0, MgCl containing 12mM is added in (10.0 nmol), D13F (10.0nmol)2, 70 DEG C of reactions
10min is immediately placed on ice, places 10min.After completion of the reaction, DNA mixed liquor is placed in and is placed at room temperature for 10min to get arriving
It is loaded with the DNA-affibody tetrahedron of oligomerization deoxyfluorouridine.By the 10% non-change of the DNA-affibody tetrahedron of acquisition
Property polyacrylamide gel electrophoresis is analyzed, as a result as shown in figure 5, non-denaturing polyacrylamide gel show be formed by it is each
DNA nanostructure has an apparent band, and modification and affibody molecule of their mobility with DNA chain
Coupling and significant reduction.The tetrahedral molecular weight of the DNA-affibody of oligomerization deoxyfluorouridine is maximum, and mobility is most slow, with
Expected results are consistent.
The tetrahedral atomic force microscope identification of the DNA-affibody of 6 support oligomerization deoxyfluorouridine of embodiment
Atomic force microscope imaging is carried out to the DNA-affibody tetrahedron for being loaded with oligomerization deoxyfluorouridine.By 10 μ L samples
Product (10nM) are deposited on mica surface 5 minutes of fresh removing.Next, 40mL TAE/Mg2+ buffering is added in mica
Liquid (50mM Tris-HCl, 20mM acetic acid, 2mM EDTA, 12mM MgCl2, pH8.0), then spontaneously dries at room temperature.Sample
Product are imaged with atomic force microscope (Agilent Technologies, 5500AFM/SPM System, USA).Atomic force microscopy
Mirror image (Fig. 6) display, being loaded between the DNA-affibody tetrahedron of oligomerization deoxyfluorouridine has high consistency, table
The deoxyfluorouridine and affibody of bright oligomerization do not interfere the tetrahedral formation of DNA, and the DNA-affibody nanostructure is about
Between 12-20nm.
The tetrahedral stability analysis of DNA-affibody of 7 support oligomerization deoxyfluorouridine of embodiment
The solution point of the DNA-affibody tetrahedron and single stranded DNA (each 100 μ g/mL) of oligomerization deoxyfluorouridine will be loaded with
It does not mix with isometric not heat-inactivated fetal calf serum (FBS) and is incubated at 37 DEG C.Incubation time is 0,1,2,4 and 8h.
10% native polyacrylamide gel electrophoresis of mixture is analyzed after incubation.When at 37 DEG C it can be seen from the result of Fig. 7
At lower incubation 4 hours, observed by gel electrophoresis, the tetrahedral band of DNA-affibody for being loaded with oligomerization deoxyfluorouridine is strong
Degree has almost no change, and shows that the nanostructure maintains completely in FBS；After being incubated for 8 hours, band starts disperse, shows
The nanostructure Partial digestion.On the contrary, single stranded DNA is almost degraded by the nuclease in FBS being incubated for 2 hours in.Cause
This, these results indicate that DNA-affibody tetrahedron can be used as pharmaceutical carrier, drug transport is intracellular to cancer cell.
The cellular uptake of the DNA tetrahedron-affibody targeted nano drug of 8 support oligomerization deoxyfluorouridine of embodiment
Assessment
BT474 cell uses the RMPI-1640 culture medium containing 10% fetal calf serum, and MCF-7 cell use contains 10%
The DMEM culture medium of fetal calf serum is cultivated.The condition of culture of all cells is 37 DEG C, the CO of 5% concentration2.Using logarithm
The cell in growth period, respectively by BT474 cell and MCF-7 cell (1 × 10 at 37 DEG C5A cell/mL) it is taped against glass bottom training
It supports in ware.After being incubated for for 24 hours, culture medium is removed, the DNA-affibody tetra- that 500 μ L contain 5 μM of Fluoresceincarboxylic acids (FAM) is added
The culture medium of face body continues culture 1 hour.Culture solution is removed again, is washed cell 3 times with PBS.Later, with 4% poly
Formaldehyde fixes 20 minutes at room temperature, is washed 3 times with PBS again.Finally, being dyed with 2.5 μ g/mL DAPI solution at 37 DEG C
Then 30min is rinsed 2 times with PBS.Fluorogram is obtained using Zeiss laser scanning confocal micro- scope (Zeiss LSM 880)
Picture.By fluorescence intensity intracellular in Fig. 8 it is found that HER2 high expressing cell BT474 cellular uptake DNA-affibody tetrahedron
Amount be 3 times of HER2 low expression cell MCF-7, illustrate that DNA-affibody tetrahedron of the present invention has very strong targeting.
The cytotoxicity of the DNA tetrahedron-affibody targeted nano drug of 9 support oligomerization deoxyfluorouridine of embodiment
Assessment
DNA tetrahedron-affibody the targeted nano of support oligomerization deoxyfluorouridine is quantitatively evaluated using mtt assay for we
The cytotoxicity of drug.BT474 cell uses the RMPI-1640 culture medium containing 10% fetal calf serum, and MCF-7 cell uses
DMEM culture medium containing 10% fetal calf serum is cultivated.The condition of culture of all cells is 37 DEG C, the CO of 5% concentration2。
The BT474 cell and MCF-7 cell in harvest index growth period, and with 8 × 103The concentration of a cells/well, by logarithmic phase
BT474 cell and MCF-7 cell are layered on 96 orifice plates, every 100 μ L of hole.Under the conditions of 37 DEG C, culture plate is placed in incubator
Preculture for 24 hours, the DNA-affibody tetrahedron of the support oligomerization deoxyfluorouridine of various concentration is added into culture plate, point
Cell 48h, 72h are managed in other places, and 10 μ L MTT (5mg/mL) are then added into every hole, have been careful not to bubble appearance, and by 96
Orifice plate continues culture 4 hours at 37 DEG C.Liquid is discarded supernatant, is added the DMSO of 100 μ L into each hole, after 15min at 570nm
Absorbance.Data are the average value of parallel laboratory test three times.
Calculation formula are as follows:
Cell survival rate=[(As-Ab)/Ac- Ab)] X100%
Inhibiting rate=[(Ac-As)/Ac-Ab)] X100%
As: drug hole light absorption value；
Ac: control wells light absorption value；
Ab: blank well light absorption value.
By experimental result (Fig. 9) it is found that free deoxyfluorouridine is respectively 48 compared with the negative control of PBS processing
Making the vigor of BT474 cell after with 72 hours reduces about 25% and 31% (10 μM of concentration).However, few by phosphatide key support
After poly- deoxyfluorouridine, in the different processing time (48 hours and 72 hours), different drug concentrations (2 μM, 5 μM and 10 μ
When M), carry DNA tetrahedron of medicine itself show specific ionization deoxyfluorouridine it is higher to BT474 cell and MCF-7 cell
Cytotoxicity.Show that specific ionization deoxyfluorouridine is higher by the BT474 cell of 250nM concentration (containing 10 μM of deoxyfluorouridine)
0.3-0.9 times of cytotoxicity, its cell viability reduces the vigor of about 42% and about 58% respectively after being incubated within 48 and 72 hours.
Significantly, since the active targeting of affibody acts on, the DNA tetrahedron-of support oligomerization deoxyfluorouridine
Affibody targeted nano drug causes more significant cytotoxicity to HER2 overexpressing cell system.The targeted nano drug
After handling BT474 cell 48 hours and 72 hours under the concentration of 250nM (10 μM of deoxyfluorouridine), cytotoxicity is trip
From 2.6 times of drug, there was only about 37% and 19% cell survival rate respectively, show the DNA tetra- of support oligomerization deoxyfluorouridine
Face body-affibody targeted nano drug shows higher cell-specific to HER2 overexpressing cell system.It is low for HER2
Express MCF-7 cell, the DNA tetrahedron of support oligomerization deoxyfluorouridine and the DNA tetrahedron-of support oligomerization deoxyfluorouridine
Affibody targeted nano drug-treated is after 72 hours, the specific ionization deoxidation fluorine under 250nM (containing 10 μM of deoxyfluorouridine) concentration
Uridine has higher inhibiting rate.However, two kinds are loaded with inhibition of the DNA Nano medication of deoxyfluorouridine in MCF-7 cell
Rate is suitable, and respectively 55% and 53%, illustrate that affibody targeting will not enhance in HER2 low expression MCF-7 cell
The cellkilling capacity of its own.Therefore, the results showed that the DNA tetrahedron-affibody of support oligomerization deoxyfluorouridine is targeted
Nano medication has high degree of specificity to HER2 receptor, shows to pass the targeting deoxyfluorouridine of HER2 overexpressing cell system
Ability is sent, this is very important targeting cancer therapy and application.
SEQUENCE LISTING
<110>University Of Hebei
<120>a kind of targeted nano carrier of support nucleosides series antineoplastic medicament and its preparation method and application
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 41
<212> DNA
<213> A
<400> 1
acactacgtc agaacagctt gcatcactgg tcaccagagt a 41
<210> 2
<211> 41
<212> DNA
<213> B
<400> 2
acgagcgagt tgatgtgatg caagctgaat gcgagggtcc t 41
<210> 3
<211> 41
<212> DNA
<213> C
<400> 3
tcaactcgct cgtaactaca ctgtgcaata ctctggtgac c 41
<210> 4
<211> 41
<212> DNA
<213> D
<400> 4
tctgacgtag tgtatgcaca gtgtagtaag gaccctcgca t 41
Claims (10)
1. a kind of targeted nano carrier of support nucleosides series antineoplastic medicament characterized by comprising
DNA tetrahedron, to be self-assembly of by four DNA are single-stranded with base pair complementarity principle；
Affibody molecule is connected to DNA by linker for the small molecular protein ligand in conjunction with HER2 receptor-specific
3 ' single-stranded ends；And
Nucleosides series antineoplastic medicament, be integrated by DNA solid phase synthesis technique to be formed tetrahedral four DNA of DNA it is single-stranded in,
The number of the nucleosides series antineoplastic medicament of the single-stranded middle insertion of every DNA is 1-100.
2. the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 1, which is characterized in that described four
DNA single-stranded nucleotide sequence is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 institute
Show.
3. the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 1, which is characterized in that described
Linker is 6-(dimaleoyl imino) caproic acid succinimide ester, the coupling number of the affibody molecule is 1-4.
4. the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 1, which is characterized in that the core
Glycoside anti-tumor drug is deoxyfluorouridine, 5 FU 5 fluorouracil nucleosides, cytarabine, gemcitabine, clofarabine, fluorine are reached and drawn
One of shore, nelarabine, Decitabine, azacitidine and Acadesine.
5. the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 1, which is characterized in that the target
It is less than 20nm to the partial size of nano-carrier.
6. a kind of preparation method of the targeted nano carrier of support nucleosides series antineoplastic medicament, which is characterized in that including following step
It is rapid:
A, nucleosides series antineoplastic medicament is subjected to phosphoramidite modification, to be converted into corresponding nucleosides series antineoplastic medicament phosphorous
Amide intermediate is used for DNA solid phase synthesis technique；
B, single-stranded using four kinds of DNA of DNA solid phase synthesis technique synthesis, and the nucleosides series antineoplastic medicament is integrated into four kinds
5 ' DNA single-stranded ends, in 3 ' the terminal modified NH that a kind of wherein DNA is single-stranded2, remaining DNA is single-stranded not to be modified, after product is purified
It is single-stranded to obtain four kinds of DNA containing nucleosides series antineoplastic medicament；
C, affibody molecule is connected to above-mentioned NH by linker23 ' the DNA of modification single-stranded ends, product is through ion exchange
Chromatography, affinity chromatography and desalting column obtain DNA-affibody chimera after purification；
D, the DNA-affibody chimera and other three kinds of DNA is single-stranded with molar ratio 1:1:1:1 mixing, it is subsequently placed in
In PCR instrument, runs by the temperature program of 75 DEG C of heating 10min, 4 DEG C of cooling 20min, 10 DEG C of holding 10min, taken after operation
Out, the DNA tetrahedron-affibody targeted nano carrier of support nucleosides series antineoplastic medicament is obtained.
7. the preparation method of the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 6, feature
It is, in step c, the linker is 6-(dimaleoyl imino) caproic acid succinimide ester, the affibody molecule
Being coupled number is 1-4.
8. the preparation method of the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 7, feature
It is, in step c, introduces cysteine in the C-terminal of the affibody molecule, the sulfydryl and the Malaysia 6-(using cysteine
Imide) caproic acid succinimide ester active group connection；Histidine mark is introduced in the N-terminal of the affibody molecule
Label, to allow to be isolated and purified by affinity chromatography.
9. the preparation method of the targeted nano carrier of support nucleosides series antineoplastic medicament according to claim 6, feature
It is, four kinds of DNA single-stranded nucleotide sequence is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ
Shown in ID NO.4；The nucleosides series antineoplastic medicament is deoxyfluorouridine, 5 FU 5 fluorouracil nucleosides, cytarabine, Ji Xita
One of shore, clofarabine, fludarabine, nelarabine, Decitabine, azacitidine and Acadesine；The targeted nano
The partial size of carrier is less than 20nm.
10. a kind of targeted nano carrier of any support nucleosides series antineoplastic medicament of claim 1 ~ 5 is directed in preparation
Application in the anti-tumor drug of HER2 high expression tumour cell.
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| CN109663134A (en) * | 2019-01-30 | 2019-04-23 | 四川大学 | The DNA tetrahedron of 5 FU 5 fluorouracil and AS1411 modification |
| CN109793750A (en) * | 2019-03-27 | 2019-05-24 | 四川大学 | A kind of anti-tumor nano drug |
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| CN113521295A (en) * | 2020-04-21 | 2021-10-22 | 湖南大学 | DNA octahedral structure of aptamer drug conjugate |
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| CN114480401A (en) * | 2020-10-28 | 2022-05-13 | 湖南大学 | Clofarabine modified oligonucleotide |
| WO2022153318A1 (en) * | 2021-01-17 | 2022-07-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Nanoparticle carriers of active agents functionalized by stimuli-responsive conjugation with dna-nanostructures, methods of their preparations, compositions comprising them and uses thereof |
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| CN113521295B (en) * | 2020-04-21 | 2022-07-01 | 湖南大学 | DNA octahedral structure of aptamer drug conjugate |
| CN111879941A (en) * | 2020-06-29 | 2020-11-03 | 北京大学 | Protein behavior detection system based on self-assembly nano-pores and preparation and use methods thereof |
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