CN108158999A - HER2 targeted nanos particle, its pharmaceutical composition, preparation method and use - Google Patents
HER2 targeted nanos particle, its pharmaceutical composition, preparation method and use Download PDFInfo
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Abstract
This application discloses a kind of HER2 targeted nanos particles, the affinity molecule being connected including DNA nano-carriers and by small molecule crosslinking agent with the DNA nano-carriers, the affinity molecule specifically targets HER2 high expression cancer cells, and the DNA nano-carriers are used to deliver small-molecule drug.Disclosed herein as well is the pharmaceutical composition comprising the HER2 targeted nano particles and the preparation methods and purposes of the HER2 targeted nano particles.Present application addresses it is existing using HER2 as the drug specificity of target spot is low, toxicity is big and tissue permeability is poor the technical issues of.
Description
Technical field
This application involves biotechnology, in particular to a kind of HER2 targeted nanos particle, its pharmaceutical composition,
Preparation method and use.
Background technology
Extracellular matrix (ECM) plays an important role in cell behavior is adjusted.Abnormal ECM may be micro- by generating oncogenicity
Environment promotes cell transformation and metastasis of cancer.ErbB-2 (HER2) is the component part of ECM, it belong to across
Membrane receptor family, the amplification of HER2 genes, the overexpression of HER2 albumen are related with the generation, development and transfer of many cancers, grind
Study carefully and be shown in breast cancer, oophoroma, lung cancer, gastric cancer, prostate cancer and head and neck cancer there are different degrees of HER2 overexpressions,
This six kinds of cancers accounting in more than 100 kinds of different known cancers is more than 41%.
Important function of the HER2 in cancer occurs, develops and shifts and its high expression in cancer cell, become
One special target spot for the treatment of of cancer.At present, the molecular targeted agents using HER2 as target spot mainly resist including anti-HER 2 monoclonal
Body (Herceptin, handkerchief trastuzumab), small molecule tyrosine kinase inhibitors (Lapatinib) and the anti-HER2 of drug coupling
Monoclonal antibody (Herceptin-DM1), this three classes drug have been ratified the breast cancer and stomach for treating the HER2 positives by FDA
Cancer.Wherein, small-molecule drug Lapatinib becomes a kind of weight for treating breast cancer due to its is small, oral administration biaavailability is high
Drug is wanted, but it also has certain limitation, such as specificity is low, toxicity is larger；Compared with small-molecule drug, monoclonal antibody
The specific higher of breast cancer and patients with gastric cancer to the HER2 positives, toxicity smaller, however monoclonal antibody is big because of its molecular weight
Also there is certain limitation, as tissue permeability is poor.Therefore, the strong medicine of a kind of high specificity, small toxicity, tissue permeability is designed
This technical problem of object is urgently to be resolved hurrily.
Invention content
The main purpose of the application is to provide a kind of nano particle for treating HER2 positive tumors, to solve existing HER2
The problem of specificity is low, toxicity is big, tissue permeability is poor existing for targeted drug.
To achieve these goals, the application's in a first aspect, provide a kind of HER2 targeted nanos particle, receives including DNA
Meter Zai Ti and the affinity molecule being connected by small molecule crosslinking agent with the DNA nano-carriers, the affinity molecule specificity
Ground targeting HER2 high expression cancer cells, the DNA nano-carriers are used to deliver small-molecule drug.
Further, the DNA nano-carriers include 1~100 single stranded DNA, the length of the single stranded DNA for 10~
1000 bases.
Further, the affinity molecule is polypeptide, protein or polysaccharide, and it is SEQ that the polypeptide, which includes amino acid sequence,
Peptide molecule shown in ID NO.1.
Further, the small molecule crosslinking agent is fat chain hydrocarbon, aromatic hydrocarbon or heterocyclic molecular, and the heterocyclic molecular includes 3-
Maleimide yl benzoic acid N-hydroxy-succinamide ester.
Further, the HER2 targeted nanos particle further includes small-molecule drug, and the small-molecule drug is embedded into institute
It states in DNA nano-carriers.
Further, the small-molecule drug for epirubicin, idarubicin, actinomycin D, daunorubicin, adriamycin,
At least one of aclacinomycin, mithramycin, Irinotecan, topotecan and hydroxycamptothecin.
The second aspect of the application provides a kind of pharmaceutical composition, including above-described HER2 targeted nanos particle.
Further, the dosage form of described pharmaceutical composition include powder, tablet, granule, capsule, solution, emulsion,
Suspension, injection, freeze drying powder injection.
The third aspect of the application provides the method for preparing above-described HER2 targeted nanos particle, including following step
Suddenly：
The single stranded DNA that terminal amino group is modified with small molecule crosslinking agent is reacted, is obtained in single stranded DNA-small molecule crosslinking agent
Mesosome；
Single stranded DNA-small molecule crosslinking agent intermediate with affinity molecule is reacted, is obtained after purifying, concentrating single-stranded
DNA- affinity molecule heterozygotes；
Single stranded DNA-affinity molecule heterozygote is mixed, reaction mixture is heated into postcooling to room temperature, each single stranded DNA-
Between single stranded DNA in affinity molecule heterozygote or inside is combined according to basepairing rule, obtains the HER2 targetings
Nano particle.
Further, the preparation method is further comprising the steps of：
Small-molecule drug will be added in after the HER2 targeted nanos particulate condensation, carry out purifying and remove excessive small molecule medicine
Object obtains having medicative HER2 targeted nanos particle.
The fourth aspect of the application provides above-described HER2 targeted nanos particle and is inhibiting growth of cancer cells, invasion
With the application in migration.
In the embodiment of the present application, HER2 targeted nanos particle uses carrier of the double-stranded DNA as small molecule anticancer drug,
The affinity molecule of selectively targeted HER2 is connected on double-stranded DNA by small molecule crosslinking agent, small molecule anticancer drug is targeted
Transport the cancer cell of HER2 high expression, high specificity, small toxicity；By the use of DNA nanostructure as pharmaceutical carrier, grain size is small,
Easy transdermal delivery, tissue permeability are strong.The HER2 targeted nanos particle can inhibit growth of cancer cells, invasion and migration, can
For treating HER2 positive tumors.In conclusion the HER2 targeted nano particles that the application provides overcome existing HER2 targets
To limitation existing for drug, solves the technical issues of specificity is low, toxicity is big, tissue permeability is poor.
Description of the drawings
The attached drawing for forming the part of the application is used for providing further understanding of the present application so that the application's is other
Feature, objects and advantages become more apparent upon.The illustrative examples attached drawing and its explanation of the application is for explaining the application, not
Form the improper restriction to the application.In the accompanying drawings：
Fig. 1 is DNA-ZHER2The schematic diagram of the preparation process of-EPI nano particles；
Fig. 2 is MTT experiment result of the nano particle to the inhibitory activity of growth of cancer cells；
Fig. 3 is the experimental result for the inhibitory activity that nano particle invades cancer cell；And
Fig. 4 is experimental result of the nano particle to the inhibitory activity of cancer cell migration.
Specific embodiment
In order to which those skilled in the art is made to more fully understand application scheme, below in conjunction in the embodiment of the present application
The technical solution in the embodiment of the present application is clearly and completely described in attached drawing, it is clear that described embodiment is only
The embodiment of the application part, instead of all the embodiments.Based on the embodiment in the application, ordinary skill people
Member's all other embodiments obtained without making creative work should all belong to the model of the application protection
It encloses.
It should be noted that term " comprising " and " tool in the description and claims of this application and above-mentioned attached drawing
Have " and their any deformation, it is intended that cover it is non-exclusive include, for example, containing a series of raw materials or step, no
Be necessarily limited to those raw materials clearly listed or step, but may include not listing clearly or for these raw materials or step
Rapid intrinsic other raw materials or step.
In addition, in the absence of conflict, the feature in embodiment and embodiment in the application can be combined with each other.Under
The application is described in detail by refer to the attached drawing and in conjunction with the embodiments in face.
It is a primary object of the present invention to provide a kind of HER2 targeted nanos particle, deposited with solving existing HER2 targeted drugs
Specificity it is low, toxicity is big, tissue permeability is poor the problem of.
HER2 targeted nanos particle of the present invention, including DNA nano-carriers and by small molecule crosslinking agent with it is described
The affinity molecule that DNA nano-carriers are connected, the affinity molecule specifically target HER2 high expression cancer cells, the DNA
Nano-carrier is used to deliver small-molecule drug.
Nanostructured based on DNA can be completed accurately and accurately by basepairing rule from group in the solution
Dress, and nano level size can effectively improve the utilization ratio of drug of DNA nanostructure.Simultaneously as itself intrinsic biology
Compatibility and biodegradability, DNA nanostructure often have long-term accumulation less, generate that immune response is small, bio-toxicity is low
The advantages of.
HER2 targeted nanos particle of the present invention is using DNA nano-carriers as pharmaceutical carrier, in DNA nanostructure
Complementary dna chain in groove between adjacent base pair store drug.Finally the DNA degradation in vivo in the nano particle is
Nontoxic nucleotide substantially reduces the bio-toxicity of the nano particle.
Wherein, DNA nano-carriers of the present invention include 1~100 single stranded DNA, the length of the single stranded DNA for 10~
1000 bases.
HER2 targeted nanos particle of the present invention is further included to be connected by small molecule crosslinking agent with DNA nano-carriers
Affinity molecule, the affinity molecule to HER2 have stronger affinity interaction, can specifically target HER2 high expression cancer it is thin
Born of the same parents.The affinity molecule can be polypeptide, protein or polysaccharide.
Wherein, polypeptide includes the peptide chain containing 10~100 amino acid, such as polypeptide affinity molecule ZHER2, neutrophil leucocyte
Peptide α HNP3；Protein includes monoclonal antibody, such as Herceptin, handkerchief trastuzumab；Polysaccharide includes ganoderma lucidum polysaccharide.
The affinity molecule used in embodiments herein is a kind of polypeptide affinity molecule ZHER2, ZHER2Specifically target
The extracellular domain of HER2, although it does not inhibit HER2 positive cancer cells, it has HER2 receptors very high affine
Power.The amino acid sequence of the polypeptide affinity molecule is shown in SEQ ID NO.1.Polypeptide affinity molecule ZHER2It can be in large intestine bar
It is expressed in bacterium cell, subsequently it is purified using Ni-NTA columns.Finally, which is nontoxic
Amino acid, be greatly reduced the bio-toxicity of the nano particle.
The small molecule crosslinking agent used in embodiments herein includes but not limited to fat chain hydrocarbon, aromatic hydrocarbon or heterocycle point
Son.
Wherein fat chain hydrocarbon includes：Dextran sulfate sodium (DSS), two (sulfosuccinic) suberic acids (BS3)；
Aromatic hydrocarbon includes：N-Succinimidyl-4-((iodoacetyl)amino)benzoate(SIAB)、N-
Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate(Sulfo-SIAB)；
Heterocyclic molecular includes：3- maleimide yl benzoic acid N-hydroxy-succinamide esters (MBS), dimaleoyl imino
Acetate succinate imide ester (AMAS).
HER2 targeted nanos particle of the present invention further includes small-molecule drug, which can be embedded into
In DNA nano-carriers, the HER2 targeted nanos particle for carrying small-molecule drug has therapeutic effect.
Wherein, the small-molecule drug be antitumor drug, as epirubicin, idarubicin, actinomycin D, daunorubicin,
At least one of adriamycin, aclacinomycin, mithramycin, Irinotecan, topotecan and hydroxycamptothecin.
The mechanism of action of HER2 targeted nanos particle of the present invention is：The HER2 targeted nanos particle will be a large amount of small
Molecular drug is specifically transmitted in the tumor tissues of the HER2 positives, tumor tissues small molecular drug epirubicin from this
It is released in nano particle, plays therapeutic effect.
The application also provides a kind of pharmaceutical composition, including above-described HER2 targeted nanos particle.
Wherein, the dosage form of the pharmaceutical composition includes powder, tablet, granule, capsule, solution, emulsion, suspension
Agent, injection, freeze drying powder injection.
As the preferred embodiment of the application, Fig. 1 show it is a kind of prepare it is above-described tool it is medicative
The method of HER2 targeted nano particles, the HER2 targeted nanos particle are prepared using two complementary single-stranded dnas as raw material, including
The following steps：
(1) by the complementary single stranded DNA and small molecule crosslinking agent 3- dimaleoyl imino benzene first of two terminal amino group modifications
Sour N-hydroxy-succinamide ester (MBS) reaction, obtains two single stranded DNA-MBS intermediates (product 1)；
(2) by two single stranded DNA-MBS intermediates respectively with affinity molecule ZHER2Reaction, obtains after purifying, concentrating
Two single stranded DNA-ZHER2Intermediate (product 2)；
(3) by two single stranded DNA-ZHER2Intermediate mixes, and reaction mixture is heated postcooling to room temperature, two single-stranded
DNA-ZHER2Single stranded DNA in intermediate is combined according to base pair complementarity principle, obtains double-stranded DNA-ZHER2Nano particle
(product 3).
(4) by double-stranded DNA-ZHER2Small-molecule drug epirubicin (EPI) is added in after nanoparticle concentration, purifying is carried out and removes
Excessive small-molecule drug is removed, obtains having medicative DNA-ZHER2- EPI nano particles (product 4).
By above-described 4 steps, the product 4 being finally prepared is to include double-stranded DNA, an affinity molecule
With the nano particle of small-molecule drug.
Further, it is also possible to prepare the HER2 targeted nanos particle by raw material of a single stranded DNA, which has
Corresponding base sequence, itself is bent to form DNA nano-carriers according to base pair complementarity principle；It can also be with a plurality of single-stranded
DNA prepares the HER2 targeted nanos particle for raw material, and a plurality of single stranded DNA is assembled into specifically according to base pair complementarity principle
Space structure, for accommodating and delivering small-molecule drug.
Below by two complementary single-stranded dnas for raw material, to provide above-described DNA-ZHER2- EPI nano particles
The specific embodiment of preparation method：
Embodiment 1：DNA30-ZHER2The preparation of-EPI nano particles
(1) single stranded DNA30The preparation of-MBS intermediates：Two complementary length by terminal amino group modification are 30 bases
To single stranded DNA (200 μ g, 10.3nmol) be dissolved in 160 μ L respectively and contain 10mM PO4 3-, 137mM NaCl and 2.7mM KCl
Phosphate buffered saline solution (PBS buffer solution) in, and add in 40 μ L 10mM small molecule crosslinking agent 3- dimaleoyl imino benzene first
Sour N-hydroxy-succinamide ester (MBS), reaction mixture is incubated at room temperature 3 hours, and the NaOAc for adding in 20 μ L 3M is whole
Only react.
(2) single stranded DNA30-ZHER2The preparation of heterozygote：The single stranded DNA obtained to step (1)30- MBS intermediates add in 600
The absolute ethanol of μ L incubates 30 minutes at 4 DEG C, reaction mixture is centrifuged 30 minutes under 15000g speed, with 70% second
Alcohol washs, and obtained reaction mixture is dissolved in 50 μ L 1X PBS buffer solution (containing 10mM PO43-,137mM NaCl,and
2.7mM KCl) in, add in affinity molecule ZHER2(300 μ g, 38.1nmol), is incubated at room temperature 1-5 hours.
Reaction mixture is purified on DEAE-Sepharose columns (1 × 0.7cm), which (contains
The NaCl of 0.2~0.9M) elution.Pass through the single-stranded of 8% denaturing polyacrylamide gel electrophoresis (SDS-PAGE) analysis purifying
DNA30-MBS-ZHER2Intermediate, gel is run 1 hour under 110V, and uses ethidium bromide staining.
Continue the eluent of purifying back by Ni-NTA chromatographic columns, eluent (900 μ L) is loaded in containing 100 μ L
Ni-NTA resins column on, then by column with 100 μ L 50mM TrisHCl (containing 300mM NaCl and 10mM imidazoles,
PH8.0 solution) washs 5 times.Finally (contain 300mM NaCl and 150mM imidazoles, pH with 100 μ L 50mM TrisHCl
8.0) solution elution is three times.The single stranded DNA being further purified by 15%SDS-PAGE analyses30-MBS-ZHER2Intermediate.
Two single stranded DNAs are obtained using ultra micro centrifugal filter (molecular cut off 10kDa) concentration30-ZHER2It is intermediate
Body.
(3) double-stranded DNA30-ZHER2The preparation of nano particle：Two single stranded DNAs that will be prepared in step (2)30-ZHER2It is intermediate
Body (10.0nmol) is added to 8mL 10mM TrisHCl (containing 12mM MgCl2, pH8.0) solution in.Reaction is mixed
Object incubates 10 minutes at 70 DEG C, is then cooled to room temperature in 30 minutes, and double-stranded DNA is made30-ZHER2Nano particle.
The double-stranded DNA obtained by 5% polyacrylamide gel electrophoresis (PAGE) analysis30-ZHER2Nano particle, gel exist
It is run 1 hour under 110V, and uses ethidium bromide staining.
(4)DNA30-ZHER2The preparation of-EPI nano particles：Use ultra micro centrifugal filter (molecular cut off 50kDa)
The double-stranded DNA of the purifying prepared in concentration step (3)30-ZHER2Nano particle.The double-stranded DNA concentrated to 100 μ L30-ZHER2Nanometer
The epirubicin (EPI) of 5 μ L10mM is added in particle (5 μM), and is incubated 10 minutes at room temperature.
Excessive EPI is removed on gel chromatographic columns Sephadex G-25, that is, obtains DNA30-ZHER2- EPI nano particles.
Embodiment 2：DNA60-ZHER2The preparation of-EPI nano particles
The preparation method is the same as that of Example 1, and the length of two used complementary single-stranded dna is 60 base-pairs.
Above-described HER2 targeted nanos particle has induction HER2 positive cancer cells apoptosis and prevents HER2 positive carcinomas thin
Dysuria with lower abdominal colic is moved, the activity of invasion.Now by taking the BT474 breast cancer cells cell of expression (HER2 high) as an example, by following experimental example into
Row verification：
Experimental example 1：Nano particle is to the MTT experiment of the inhibitory activity of growth of cancer cells
Experimental method：At 37 DEG C, 5%CO2BT474 breast cancer cells are cultivated in incubator, use RPMI-1640 culture mediums
(containing 10% fetal calf serum (FBS) and 1% dual anti-(antibiotic-antimycotic) solution).The BT474 of harvest index growth is thin
Born of the same parents are layered in 96 orifice plates, and a concentration of 2 × 104A cells/well.After being incubated 24 hours at 37 DEG C, with containing phase accordingly
With epirubicin (75nM, 150nM, 300nM, 600nM), the DNA of concentration epirubicin30-ZHER2- EPI nano particles (6.3nM,
12.5nM, 25nM, 50nM) and DNA60-ZHER2- EPI nano particles (6.3nM, 12.5nM, 25nM, 50nM) processing cell 72 is small
When.Then the MTT (5mg/ml) of 20 μ L is added in into each hole, culture plate is incubated 4 hours at 37 DEG C.Discard supernatant liquid,
The dimethyl sulfoxide (DMSO) (DMSO) of 100 μ L is added in into each hole, absorbance value is recorded in 490nm after 15 minutes.Control is set simultaneously
Group (in culture medium only have BT474 cells, be added without experimental drug) and withered group (are added without BT474 cells, are not added in culture medium
Enter experimental drug), growth of cancer cells inhibiting rate is obtained by following formula：Growth of cancer cells inhibiting rate (%)=(OD control groups-OD is real
Test group) × 100%/(withered group of OD control groups-OD).Experiment under each concentration carries out three times, and data report is only three times
The average value of vertical experiment.
Experimental result：As shown in Fig. 2, in three groups of experiments with same concentrations epirubicin, DNA30-ZHER2- EPI nanometers
Particle and DNA60-ZHER2- EPI nano particles have similar inhibiting effect to BT474 cells, and to BT474 cell growths
Inhibiting rate incubate 72 hours after be all higher than individual epirubicin.Particularly in low concentration, nano particle
(12.5nM) is nearly 2 times higher than individual epirubicin (150nM) to the inhibiting rate of BT474 cells.Shown MTT according to fig. 2
Experimental result demonstrates the drug-loading nanoparticles being prepared relative to growth of the individual drug to breast cancer cell BT474
With good inhibitory activity.
Experimental example 2：The experiment for the inhibitory activity that nano particle invades cancer cell
Experimental method：Invasion is measured and is carried out in 24 hole Transwell culture plates, the Transwell cells of the culture plate
Bottom is the polycarbonate leaching film containing 8 μm of micropores of diameter.First, in 5%CO2, under the conditions of 37 DEG C, trained using RPMI-1640
Base (serum-free) is supported to handle polycarbonate leaching film 2 hours, it is then small with 100 μ LMatrigel matrigels coating Transwell
The upper chamber face of the polycarbonate leaching film of room bottom.Then, by 1 × 105A BT474 cells (serum-free) are added to the upper of culture plate
Room.Simultaneously by the 700 μ L RPMI-1640 culture mediums (dual anti-(antibiotic-anti-true containing 10% fetal calf serum (FBS) and 1%
Bacterium) solution) it is added in lower room.Each groups of cells is layered in the hole of three repetitions, 20 μ L500nM are added in each hole and (are contained
600nM epirubicins) DNA30-ZHER2The DNA of-EPI nano particles, 20 μ L 500nM60-ZHER2- EPI nano particles or
The epirubicin of 600nM.After being incubated 18 hours, culture solution is removed, is washed three times with 1X PBS buffer solution.It is gone with cotton swab unless invading
Entering property cell.The cell for having migrated through filter membrane and being adhered to the lower surface of film is fixed with methanol and is contaminated with hematoxylin-eosin
Color.Finally, under an optical microscope, the cell under being counted at least ten random field in room.
Experimental result：As shown in figure 3, epirubicin under 600nM concentration to the invasion inhibiting rate of BT474 cancer cells
20%；And DNA30-ZHER2- EPI nano particles are up to 90% under 50nM concentration to the inhibiting rate of BT474 cancer cells invasion；
DNA60-ZHER2- EPI nano particles and DNA30-ZHER2- EPI nano particles are compared, to BT474 cancer cells invasion inhibiting rate compared with
It is low, inhibiting rate 69%.
Experimental example 3：Experiment of the nano particle to the inhibitory activity of cancer cell migration
Migration determination experiment method is similar with the invasion measure in experimental example 2, and the difference lies in do not use
The upper chamber face of the polycarbonate leaching film of Matrigel matrigels coating Transwell cells bottom.
Experimental result：As shown in figure 4, epirubicin is about to the inhibiting rate of BT474 cancer cell migrations under 600nM concentration
40%；And DNA30-ZHER2- EPI nano particles are about 60% to the inhibiting rate of BT474 cancer cell migrations under 50nM concentration；
DNA60-ZHER2- EPI nano particles and DNA30-ZHER2- EPI nano particles are compared, to the inhibiting rates of BT474 cancer cell migrations compared with
It is low, inhibiting rate 44%.
It can be seen that DNA-Z obtained in Examples 1 and 2 from the experimental result of experimental example 1 to 3HER2- EPI nanometers
Grain has the function of to inhibit growth of cancer cells, cancer cell is prevented to invade and migrate.
Wherein described cancer cell expresses cancer cell for HER2 high, including breast cancer, oophoroma, lung cancer, gastric cancer, prostate cancer
With the cancer cell of head and neck cancer.
It should be noted that, although HER2 targeted nano particles shown in the embodiment of the present invention and preparation method thereof
In, one double-stranded DNA of DNA nano-carriers selected as, but it is as just an example, is a kind of basic realization method, no
It is interpreted as limiting the scope of the invention, the DNA nano-carriers in the present invention further include the various changes except embodiment
Property.Such as the single stranded DNA in DNA nano-carriers can be for one or a plurality of, test proves that, the choosing of DNA nano-carriers
The nano particle synthesized by the single stranded DNA of 1~100 is selected as to be respectively provided with inhibition growth of cancer cells, cancer cell is prevented to invade and move
The effect of shifting；For another example, the shape of the DNA chain in DNA nano-carriers is not limited to linear structure, can also be Y types, Y types DNA
The end that can be double-stranded DNA is open chain, forms y-type structure, or end after two other DNA complementations respectively with forming
Y-type structure；For another example, the length of each DNA also should not be limited to 30 bases and 60 bases that the embodiment of the present invention is shown,
It is demonstrated experimentally that it is preferred scheme that the length of DNA chain, which is 10~1000 bases,.In short, the list in DNA nano-carriers of the present invention
Chain DNA can be there are many combination, and those skilled in the art are in the essence of the synthetic method of specific embodiment provided by the invention
Under refreshing principle guidance, can multi-combined structure be realized according to the principle of DNA base complementary pairing, be used to implement the mesh of the present invention
's.
The foregoing is merely the preferred embodiments of the application, are not limited to the application, for the skill of this field
For art personnel, the application can have various modifications and variations, but those skilled in the art are in spirit herein and principle
Within, any modification, equivalent replacement, improvement and so on should be included within the protection domain of the application.
Claims (10)
1. a kind of HER2 targeted nanos particle, which is characterized in that including DNA nano-carriers and by small molecule crosslinking agent with it is described
The affinity molecule that DNA nano-carriers are connected, the affinity molecule specifically target HER2 high expression cancer cells, the DNA
Nano-carrier is used to deliver small-molecule drug.
2. HER2 targeted nanos particle according to claim 1, which is characterized in that the DNA nano-carriers include 1~
100 single stranded DNAs, the length of the single stranded DNA is 10~1000 bases.
3. HER2 targeted nanos particle according to claim 1, which is characterized in that the affinity molecule is polypeptide, albumen
Matter or polysaccharide, the polypeptide include amino acid sequence for the peptide molecule shown in SEQ ID NO.1.
4. HER2 targeted nanos particle according to claim 1, which is characterized in that the small molecule crosslinking agent is fat chain
Hydrocarbon, aromatic hydrocarbon or heterocyclic molecular, the heterocyclic molecular include 3- maleimide yl benzoic acid N-hydroxy-succinamide esters.
5. HER2 targeted nanos particle according to claim 1, which is characterized in that small-molecule drug is further included, it is described small
Molecular drug is embedded into the DNA nano-carriers.
6. HER2 targeted nanos particle according to claim 5, which is characterized in that the small-molecule drug is the soft ratio of table
Star, idarubicin, actinomycin D, daunorubicin, adriamycin, aclacinomycin, mithramycin, Irinotecan, topotecan and
At least one of hydroxycamptothecin.
7. a kind of pharmaceutical composition, which is characterized in that including HER2 targeted nanos according to any one of claims 1 to 6
Grain.
8. a kind of method for preparing HER2 targeted nanos particle any one of Claims 1-4, includes the following steps：
The single stranded DNA that terminal amino group is modified with small molecule crosslinking agent is reacted, obtains single stranded DNA-small molecule crosslinking agent intermediate；
Single stranded DNA-small molecule crosslinking agent intermediate with affinity molecule is reacted, single stranded DNA-parent is obtained after purifying, concentrating
With molecular hybridization body；
Single stranded DNA-affinity molecule heterozygote is mixed, reaction mixture is heated into postcooling to room temperature, each single stranded DNA-affine
Between single stranded DNA in molecular hybridization body or inside is combined according to basepairing rule, obtains the HER2 targeted nanos
Particle.
It is 9. according to the method described in claim 8, it is characterized in that, further comprising the steps of：
Small-molecule drug will be added in after the HER2 targeted nanos particulate condensation, carries out purifying and remove excessive small-molecule drug,
Obtain having medicative HER2 targeted nanos particle.
10. HER2 targeted nanos particle according to any one of claims 1 to 6 is inhibiting growth of cancer cells, invasion and migration
In application.
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| CN103290017A (en) * | 2012-02-24 | 2013-09-11 | 中国医学科学院基础医学研究所 | HER2 (Human Epidermal Growth Factor Receptor 2) protein nucleotide aptamer, complex composition and applications thereof |
| CN104645338A (en) * | 2015-01-26 | 2015-05-27 | 上海交通大学 | Preparation method of DNA targeting nano medicine-carrying molecule for brain tumor |
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| WO2017200787A1 (en) * | 2016-05-18 | 2017-11-23 | Arizona Board Of Regents On Behalf Of Arizona State University | Peptide-dna chimeras for treatment of her overexpressing cancers |
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| CN104645338A (en) * | 2015-01-26 | 2015-05-27 | 上海交通大学 | Preparation method of DNA targeting nano medicine-carrying molecule for brain tumor |
| WO2017200787A1 (en) * | 2016-05-18 | 2017-11-23 | Arizona Board Of Regents On Behalf Of Arizona State University | Peptide-dna chimeras for treatment of her overexpressing cancers |
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