CN106497978A - A kind of magnetic composite nano particle and its preparation method and application - Google Patents

A kind of magnetic composite nano particle and its preparation method and application Download PDF

Info

Publication number
CN106497978A
CN106497978A CN201610811557.2A CN201610811557A CN106497978A CN 106497978 A CN106497978 A CN 106497978A CN 201610811557 A CN201610811557 A CN 201610811557A CN 106497978 A CN106497978 A CN 106497978A
Authority
CN
China
Prior art keywords
composite nano
nano particle
solution
magnetic composite
particle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610811557.2A
Other languages
Chinese (zh)
Other versions
CN106497978B (en
Inventor
赵美萍
翟筠秋
刘艺斌
黄山
方思敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University
Original Assignee
Peking University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University filed Critical Peking University
Priority to CN201610811557.2A priority Critical patent/CN106497978B/en
Publication of CN106497978A publication Critical patent/CN106497978A/en
Application granted granted Critical
Publication of CN106497978B publication Critical patent/CN106497978B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Abstract

The invention discloses a kind of magnetic composite nano particle and its preparation method and application, the magnetic composite nano particle uniform particle diameter, 40 60nm of diameter, the DNA double chain containing abasic site that surface is connected by Avidin biotin can specifically recognize by target protein abasic Cobra venom endonuclease I (APE1) and be cut, and discharge fluorophor or drug molecule；Meanwhile, the magnetic composite nano particle obtained by the present invention has good dispersibility, stability and biocompatibility, and the fluorescence in situ so as to can be used for APE1 in living cells is imaged.Additionally, due to the usual overexpression APE1 of cancerous cell, above-mentioned magnetic composite nano particle can more effectively discharge medicine in cancerous cell, be expected to realize the accurate treatment for cancerous cell.

Description

A kind of magnetic composite nano particle and its preparation method and application
Technical field
The present invention relates to a kind of Multifunctional composite nanometer granule and its preparation method and application.More particularly, it relates to one Plant nucleic acid function magnetic composite nano particle of Avidin modification and its preparation method and application.The nano-particle can be used as base Because of carrier, the in situ imaging of nucleic acid in living cell repair enzyme and pharmaceutical carrier, belong to nano-particle preparing technical field.
Background technology
There is nano material excellent electricity, optics, mechanical property, morphology controllable and surface easily to modify.Biomolecule has The features such as having high specificity, binding site to enrich (Eugenii Katz, Itamar Willner.Integrated Nanoparticle-Biomolecule Hybrid Systems:Synthesis,Properties,and Applications.Angew.Chem.Int.Ed(2004),43,6042–6108).Fluffy with nanotechnology and biotechnology The exhibition of breaking out, nano material is combined with biomolecule and makes various composite nano materials, can be used to enter organism Row diagnoses, treats, repairs or replaces its disease damage tissue, and shows good application at aspects such as cell imaging, bio-sensings Prospect.
Combination between Avidin and biotin is most firm articulated system between the biomolecule having now been found that (Buller,HR.,Gallus,AS.,Pillion,G.,Prins,MH.,Raskob,GE.Enoxaparin followed by once-weekly idrabiotaparinux versus enoxaparin plus warfarin for patients with acute symptomaticpulmonary embolism:a randomised,double-blind,double- Dummy, non-inferiority trial.Lancet (2012), 379,123 129), can resist organic solvent, surface and live (Green, the N.Michael.Avidin.Advances in such as the impact of property agent, proteolytic enzyme, extreme temperature and pH Protein Chemistry Volume(1975),29,85-133).By Avidin with the nano silicon particles for having modified carboxyl altogether Valency connects, then by biotinylated DNA by the modification of affinity interaction between Avidin and biotin to being covalently attached parent With the nano silicon particles surface of element, the quantity of nano grain surface DNA can be greatly improved, and DNA is enhanced with nanometer load The fastness of body.
DNA or medicine transduction are referred to and are imported to DNA or medicine in cancerous cell or target cell by external source mediation.Mesh Though provirus carrier can reach higher transfection efficiency, there is strongly immunogenic, high mutagenesis risk, low capacity etc. and lack Point, limits its application in biological and clinical treatment.And non-virus carrier, especially nano-particle not only can overcome with Upper shortcoming, and there is convieniently synthesized, repeated high, multiple physicochemical properties and easy surface modification, realize nanometer The multifunction of carrier.
Magnetic nano particle is not only easily modified, and also there is magnetic field effect and Fluorescence quenching effect, by magnetic nano particle by other Granule combines and modifies biomolecule, just can solve the problems, such as that nano-particle disengaging time length and transfection time are long (Liu,YB.,Fang,SM.,Zhai,JQ.,Zhao,MP.Construction of antibody-like nanoparticles for selective protein sequestration in living cells.NANOSCALE (2015),7(16):7162-7167).Not only there are nano silicon particles good biocompatibility, porous, the advantage that easily reacts to go back Can protect and modify (Roy, I., Ohulchanskyy, TY., Bharali,DJ.,Pudavar,HE.,Mistretta,RA.,Kaur,N.,Prasad,PN.Opticaltracking oforganically modifiedsilica nanoparticles as DNA carriers:A nonviral, nanomedicine approachfor gene delivery.PNAS(2005),102(2):279-284), therefore magnetic is received Rice grain is combined with nano silicon particles, not only achieves transfection efficiency rapidly and efficiently, is reduced bio-toxicity, is also protected Adorned DNA can be complete transfected in cell.
The method that DNA is imported cell by the utilization nano silicon particles having had at present, have covalent attachment DNA (Cai L, Chen Z Z,Chen M Y,et al.MUC-1aptamer-conjugated dye-doped silica nanoparticlesforMCF-7cells detection[J].Biomaterials,2013,34(2):371-381.) and profit Connect (Qian R, Ding L, Ju H.Switchablefluorescent imaging of with electrostatic interaction intracellular telomerase activity using telomerase-responsive mesoporous silica nanoparticle.Journal of the American Chemical Society,2013,135(36): 13282-13285.) etc. method.Such method is mainly used in hybridization (Zhang P, Cheng with intracellular miRNA or mRNA F,Zhou R,et al.DNA‐hybrid‐gated multifunctional mesoporous silica nanocarriers for dual‐targeted and microRNA‐responsive controlled drug delivery.Angewandte Chemie International Edition,2014,53(9):2371-2375.), due to Protective effect (He X, Wang K, Tan W, et al.Bioconjugated nanoparticles of the nano silicon particles to DNA for DNA protection from cleavage.Journal of the American Chemical Society, 2003,125(24):7168-7169.), these nano-particle generally all inhibit the reaction of DNA and intracellular nucleic acid enzyme, so as to It is difficult to use in activity analysiss and the in situ imaging of nuclease.In addition, miRNA medicines are imported currently with nano-particle, lack pin Specific to cancerous cell release regulating and controlling effect (Mura S, Nicolas J, Couvreur P.Stimuli-responsive nanocarriersfor drug delivery.Nature materials,2013,12(11):991-1003.).
Content of the invention
It is an object of the invention to provide a kind of preparation method and application of magnetic composite nano particle.The magnetic composite nano Granule not only can in vitro to solution in target protein carry out specific identification and quantitative analyses, it is even more important that Living cells can be rapidly introduced under the action of a magnetic field, the fluorescence imaging of original position is carried out to target protein therein, by fluorescence Distribution of the microscope to which in cell carries out spike.If fluorescence group is replaced with cancer therapy drug, can also realize to cancer Carry out immunotherapy targeted autoantibody.
To achieve these goals, the present invention is employed the following technical solutions.
A kind of magnetic composite nano particle, includes the modification of magnetic core, silica shell and biomolecule from inside to outside successively Layer, the magnetic core are ferromagnetic nanoparticle, and the silica shell has the silicon dioxide of carboxyl, the life for surface modification Thing molecular modification layer is Avidin and (i.e. Avidin) is combined therewith the biotinylated double-stranded DNA containing abasic room (biotinylated-AP-DNA).
Further, the ferromagnetic nanoparticle is ferroferric oxide nano granules or γ-iron sesquioxide nanometer Grain.
Further, the TEM particle diameters of the magnetic composite nano particle are 40-60nm, and its DLS particle diameter is 65-75nm.
Further, a diameter of 20-30nm of the magnetic core, the thickness of the silica shell is 10-15nm, described The thickness of biomolecule decorative layer is less than or equal to 20nm.
Further, the expression formula of the magnetic composite nano particle is Fe3O4@SiO2@AVD-AP-DNA or γ-Fe2O3@ SiO2@AVD-AP-DNA.Wherein Fe3O4Represent ferroso-ferric oxide magnetic core, γ-Fe2O3γ-iron sesquioxide magnetic core is then represented； SiO2Represent silica shell；AVD represents Avidin layer；AP-DNA represents the biotinylated double-strand containing abasic room DNA (biotinylated-AP-DNA) layer.
Further, biotinylated the double-stranded DNA containing abasic room (biotinylated-AP-DNA) Two single-stranded sequence is respectively：
5 '-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA-Biotin-3 ' and 5 '- GTCGATGGAGXCGTCCCC-FAM-3 ', wherein X represent that abasic site, Biotin represent biotin labeling, and FAM is carboxyl Fluorescein labelling.
Present invention also offers the preparation method of above-mentioned magnetic composite nano particle, including：First in silica shell Outer layer is covalently attached upper Avidin, then by the strong affinity interaction of Avidin and biotin, by biotinylated containing U bases Double-stranded DNA (dsDNA) modification finally recycles UDG enzymes to cut U bases on the ferromagnetic nanoparticle surface of coated with silica Remove, produce the double-stranded DNA (AP-DNA) containing abasic room.
Further, above-mentioned preparation method is comprised the following steps that：
1) the ferromagnetic nanoparticle ultrasonic disperse for coating the silica shell of carboxyl modified is in pure water, respectively plus Enter EDC and NHS, concussion reaction under room temperature (referring both to 15-28 DEG C below), used as solution A.
2) Avidin, concussion reaction under room temperature are added in solution A.Magneto separate washing removes unreacted material, then By the reactant for obtaining again ultrasonic disperse in pure water, as solution B.
3) the biotinylated double-stranded DNA containing U bases is added in solution B, is incubated 30-90 minutes under room temperature.Magneto separate And wash, unreacted material is removed, then the magnetic nanoparticle of the double-stranded DNA modification containing U bases for obtaining is surpassed again Sound is dispersed in PBS solution, used as solution C.
4) solution C is added in 1.1 buffer solution of buffer, adds UDG enzyme action except the U bases in DNA double chain, obtain Arrive magnetic composite nano particle.
Further, step 1) in, the ferromagnetic nanoparticle ultrasonic disperse of the silica shell cladding of carboxyl modified Final concentration of 0.5-2mg/mL in pure water.
Further, step 1) in ultrasonic time require more than 10 minutes.
Further, step 1) in the concentration of EDC be 0.5-2mg/mL, the concentration of NHS is 1-5mg/mL, EDC, NHS with The weight ratio of the ferromagnetic nanoparticle of the silica shell cladding of carboxyl modified is 4:10:1.
Further, for making the carboxyl of silica shell layer surface fully activate, EDC and NHS will add the amount of skipping over, activation Time should not be too short or long, and according to the difference of room temperature, preferably soak time is 15-45 minutes.
Further, step 2) in Avidin concentration be 0.1-0.2mg/mL, preferably 0.1mg/mL.Avidin is added to make (weight) ratio of final concentration in mixed solution is 5 to nano-particle with Avidin：1, concussion reaction 8-12 hour under room temperature surpasses Sound jitter time is 5 minutes.
Wherein, outside silicon dioxide layer be covalently attached Avidin when, for making nano-particle fully dispersed, nano-particle dense Degree should not be too high, and preferred concentration is 0.5-1mg/mL.
Step 2) reaction terminate after, in order to remove the unreacted reactant in reaction system, can be washed with deionized water 1-3 time, Then again ultrasonic disperse in pure water.
Further, step 3) in, nano-particle is arrived in biotinylated double-stranded DNA (dsDNA) modification containing U bases When on surface, for making the conformation of dsDNA more stable consistent, carried out intensification annealing first and (90s is incubated at 95 DEG C, at 75 DEG C Incubation 90s, is incubated 90s at 50 DEG C, be finally incubated 600s at 37 DEG C), then dsDNA is added in solution B.
Further, step 3) in, the biotinylated double-stranded DNA containing U bases of addition is final concentration of in solution B 0.2-1μM.
For making quantity that dsDNA is connected to nano grain surface at most, its concentration can be optimized, it is 1 μM to make ultimate density.
Further, step 3) in, will be again ultrasonic for the magnetic nanoparticle of the double-stranded DNA modification containing U bases for obtaining It is dispersed in the PBS solution of 0.1M, the ultrasonic disperse time is 5 minutes, if do not used immediately, then should be placed in 4 DEG C of preservations.
Further, step 4) in, buffer 1.1 buffer solution is consisted of：10mM Bis Tris Propane- HCl,10mM MgCl2, 100 μ g/ml BSA, pH 7.0 (25 DEG C).
Final concentration 1.0-100U/mL of the UDG enzymes of addition in 1.1 buffer solution of buffer comprising solution C, reaction Time range is 0.5-10min.
Present invention also offers above-mentioned magnetic composite nano particle enzyme assay, nucleic acid in living cell repair enzyme in vitro In situ imaging in application.
Beneficial effects of the present invention are：
Ferroso-ferric oxide or γ-iron sesquioxide magnetic core are characterized in as the kernel of composite particles：To some fluorescent bases Group has quenching effect, contributes to granule being carried out separating, being washed and when cell is imported in particulate production, can Nanoparticle is accelerated to enter the process of cell with the effect by externally-applied magnetic field.Surface modification has the silicon dioxide layer of carboxyl can Improve the hydrophilic and biocompatibility of nano-particle, there is provided carboxyl occurs to be covalently attached reaction with Avidin, and ensures when glimmering Light blob is away from can discharge fluorescence during particle surface.Avidin (AVD) is had with the biotin on biontinylated-ds DNA Ds DNA can be zoomed in nano grain surface, enable ds DNA successfully and effectively repair by strong affinity interaction, this effect Decorations are on nano-particle.Most importantly, we have found under study for action and confirm, AVD and abasic Cobra venom endonuclease I (APE1) there is specific affinity interaction between, this interaction can make AVD that the APE1 in solution is attracted to nanometer Particle surface, acts on the abasic site on DNA, effectively the phosphodiester bond of 5 ' side of cutting abasic site, so as to discharge The fluorophor being connected on DNA or medicine.Molecular arms of the DNA herein as nano-carrier, can not only connect fluorophor For fluoroscopic examination or intracellular imaging is carried out to APE1, can also connect drug molecule, and can guarantee that only in APE1 presence In the case of just discharge fluorophor or drug molecule etc., so as to ensure that composite nanometer particle can be being expressed in APE1 Effectively play a role in the middle of the cancerous cell of amount, and reduce and even avoid the side effect to normal cell.
The present invention has synthesized a kind of magnetic, multi-functional composite nanometer particle, and uniform particle diameter, diameter 40-60nm, surface connect DNA double chain containing abasic site can specifically be recognized by target protein abasic Cobra venom endonuclease I (APE1) and be cut, release Discharging fluorescence group or drug molecule.The nano-particle for being obtained has good dispersibility, stability and biocompatibility, from And can be used for the fluorescence in situ imaging of APE1 in living cells.Additionally, due to the usual overexpression APE1 of cancerous cell, above-mentioned nano-particle Medicine can more effectively be discharged in cancerous cell, be expected to realize the accurate treatment for cancerous cell.
Description of the drawings
Preparation process and principle schematic of the Fig. 1 for magnetic composite nano particle.
Fig. 2 is nano-probe A (Fe3O4@SiO2@AVD-AP-DNA nano-particle) transmission electron microscope picture.
Fig. 3 is MNPs (Fe3O4@SiO2), MNP@AVD (Fe3O4@SiO2@AVD) and nano-probe A (Fe3O4@SiO2@AVD- AP-DNA) the zeta potentials of three kinds of granules in 0.1mg/mL aqueous solutions.
Fig. 4 a are the time graph that nano-probe A is responded to variable concentrations APE1；Fig. 4 b are that nano-probe A detection APE1 live The linear fit curve of property.
Fig. 5 is selectivitys of the nano-probe A to various common nucleic acid enzymes.
Fig. 6 is time dependent fluorogram after nano-probe A magnetic transfects 30 minutes in HeLa cells.
Fig. 7 be the fluorogram of intracellular control probe and different pharmaceutical effect under intracellular Fluorescence signal variation diagram.
Fig. 8 is toxicity of the nano-probe A to cell.
Specific embodiment
With reference to the accompanying drawings and detailed description the present invention is elaborated.Detailed description below will be helpful to Understand invention, but be not intended to limit present disclosure.
Embodiment 1, prepare magnetic composite nano particle Fe3O4@SiO2@AVD-AP-DNA (nano-probe A)
The present invention prepares the process of magnetic composite nano particle as shown in figure 1, specifically including following steps：
By 1.0mg Fe3O4@SiO2(adjusted using the supersonic frequency as needed in the present invention under room temperature, 40KHz For different ultrasonic times) ultrasonic 20 minutes, it is dispersed in 2.0mL pure water and is made into the solution of 0.5mg/mL, adds 4.0mg EDC With 10.0mg NHS activated carboxyls, concussion reaction 30 minutes under room temperature.0.1mL concentration is added for the Avidin of 2.0mg/mL, room Lower 8 hours of oscillating reactionss of temperature.Magneto separate, removes unreacted material, is washed with deionized water three times, and ultrasonic disperse exists again In 2.0mL pure water.Final concentration of 1 μM of the biotinylated double-stranded DNA containing U bases is added, is incubated 1 hour under room temperature.Magnetic point From, three times are washed with PBS (pH 7.4,0.1M) remove unreacted DNA, the magnetic that the double-stranded DNA containing U bases for obtaining is modified Again ultrasonic disperse is continuing with 2.0mL0.1M PBS (pH 7.4) or is stored in 4 DEG C nano-particle.Manage in 50 μ LPCR In, the magnetic nanoparticle for adding the above-mentioned double-stranded DNAs being dispersed in 0.1M PBS containing U bases of 5 μ L 1.0mg/mL to modify is molten Liquid, 5 μ L10 × Buffer1.1 buffer are added deionized water and cause cumulative volume for 47 μ L, is subsequently adding 1 μ L 500U/mL's UDG enzymes, react 2min, obtain Multifunctional composite nanometer granule Fe3O4@SiO2@AVD-AP-DNA (nano-probe A), are immediately available for The determination of activity of APE1 imports cell experiment.
Composite nanometer particle Fe is characterized by transmission electron microscope3O4@SiO2@AVD-AP-DNA are as shown in Figure 2, it is seen that Composite nanometer particle size is homogeneous, good dispersion, and its particle diameter is in 60nm or so.From former nano-particle Fe3O4@SiO2(MNPs) in, Mesosome Fe3O4@SiO2@AVD (MNP@AVD) and composite nanometer particle Fe3O4@SiO2The zeta of@AVD-AP-DNA (nano-probe A) Potential value understands (Fig. 3), and former nano-particle successfully links up AVD and biotinylated-AP-DNA.
Embodiment 2, to solution in APE1 activity directly detect
In this embodiment, the DNA probe sequence of design is as follows：
5’-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA-Biotin-3’
5’-GTCGATGGAGXCGTCCCC-FAM-3’
Wherein X represents that abasic site, Biotin represent biotin labeling, and FAM is carboxyl CF 5(6)-Carboxyfluorescein labelling.
Experimental procedure is as follows：
1. in 200 μ L PCR pipes, the magnetic composite nano particle that 10 μ L 0.5mg/mL embodiments 1 of addition are prepared Solution, 5 μ L 10 × Buffer1.1 buffer add deionized water and cause cumulative volume for 48 μ L, be subsequently adding 2 μ L differences dense The APE1 solution of degree, makes the final concentration of APE1 be respectively 0,0.01,0.05,0.1,0.2,0.5,1U/mL, detects composite Nano The response of grain.
Using Stratagene Mx3000P fluorescent PCR instrument, at 37 DEG C, fluorescence is gathered per 5s, excitation wavelength is 470nm, Transmission signal is surveyed under 510nm, and is fitted the working curve of fluorescence climbing speed and APE1 concentration in the initial 50s of reaction.
As shown in fig. 4 a, as shown in Figure 4 b, composite nanometer particle can detect 0.01U/mL to fitting result to testing result APE1, with sensitive response.With the increase of APE1 concentration, its fluorescence climbing speed is linear with APE1 concentration, R2=0.996, working range is 0.01-1.0U/mL.
2., in 200 μ LPCR pipes, the magnetic composite nano particle for preparing of 10 μ L 0.5mg/mL embodiments 1 is added Solution, 5 μ L 10 × DNase I buffer are added deionized water and cause cumulative volume for 48 μ L, adds 2 μ L 125U/mL DNase I solution, makes the final concentration of 5U/mL of DNase I, detects the response of composite nanometer particle.
Using Stratagene Mx3000P fluorescent PCR instrument, at 37 DEG C, fluorescence is gathered per 5s, excitation wavelength is 470nm, Transmission signal is surveyed under 510nm.Magnetic composite nano particle is identical with this to the response detection process of other enzymes, simply by buffer Replace with its most suitable condition.
Testing result is as shown in figure 5, the final concentration of various enzymatic determinations is respectively APE1:2.0U/mL；DNase I:5.0U/ mL；Exo III:4.0U/mL；Lambda exo:66.7U/mL；Exo I:12.5U/mL；T5:5U/mL；T7 50U/mL).Can To find composite nanometer particle in the presence of APE1, the abasic site in AP-DNA can be cut so as to discharging distal end Nucleotide with FAM so that fluorescence climbing speed is very fast, and other enzymes, particularly DNase I etc. is then responded very slow. Illustrate that composite nanometer particle has good selectivity to APE1.What deserves to be explained is, Exo III are by bacterial expression, together When have that 3 ' is exo-acting and the nucleases of abasic site endo-activity, it can also be seen that magnetic composite nano from Fig. 5 Grain has certain response to which.
Embodiment 3, composite nanometer particle quickly introduces cell under the action of a magnetic field and discharges fluorescence signal
The magnetic composite nano particle for preparing has preferable stability, biocompatibility, leads under the action of a magnetic field Cross endocytosises to quickly introduce Hela cells and be imaged APE1.
Experimental procedure is as follows：
1. dual anti-containing 1% Pen .- Strep and 10% inactivation hyclone DMEM culture medium in cultured cells, Incubator condition is 37 DEG C and 5%CO2/ 95% air.As the 70%-80% of cell length to covering culture bottle, add 0.25% pancreatin processes 4min at 37 DEG C and then shifts cell on 96 hole flat bottom glass plates, and it is thin that 0.1mL is contained in each hole Born of the same parents' culture fluid, there are about 104Individual cell.Before experiment cause which adherent cell culture 24h.
2. cell is washed with DPBS buffer, be subsequently adding the magnetic composite nano that embodiment 1 is prepared The solution of grain so as to final concentration of 50 μ g/mL.The MagnetoFACTOR-96 magnetic sheets that 96 orifice plates are placed in Chemicell companies On, at 37 DEG C and 5%CO230min is placed in the incubator of/95% air.After removing magnetic sheet, siphon away molten in 96 orifice plates Liquid, with DPBS buffer solutions cell 5 times, fully washes away the magnetic nano particle for not entering into cell.
3. add in the cell for importing magnetic composite nano particle and contain colourless DMEM culture medium, at 37 DEG C and 5% CO2In/95% online culture apparatuses cultivate, adopt be equipped with Evolve-EMCCD 71 fluorescence microscopies of Olympus IX with And cell under 100 times of object lens, is shot, the fluorescence picture (time of exposure for shooting corresponding different probe with corresponding fluorescence grating 10ms, transmitting gain value EM gain are for 100).Result treatment is carried out with ImageJ softwares, as shown in Figure 6.With magnetic transfection knot The increase of incubation time after beam, the region of (green) fluorescence distribution in HeLa cells increase, and fluorescence intensity also increases, 120 Maximum is reached at minute.After 180 minutes, fluorescence distribution region and fluorescence intensity are gradually decreased.Illustrate that nano-probe A is entered into In HeLa cells.
4. it is further to prove the fluorescence signal of intracellular generation actually from intracellular APE1 to magnetic composite nano On grain, in DNA sequence, the fixed point of abasic site is cut, and the double-stranded DNA containing U bases without UDG ferment treatments is modified by we Magnetic nanoparticle is introduced directly into cell and carries out control experiment.In addition, also select that two kinds of medicines are processed to cell, wherein Tert-butyl hydroperoxide (TBHP) is APE1 zymoexciters, and 7- nitroindolines -2- carboxylic acids (NCA) is APE1 activity inhibitors, Observe the situation of change of intracellular enzyme activity under medicine effect.It will be seen in fig. 7 that without UDG ferment treatments, will directly contain U The magnetic nanoparticle of the double-stranded DNA modification of base imports cell, does not occur fluorescence signal after 120min yet.And will contain de- After the nano-probe A of base position imports cell, obvious fluorescence signal is seen through the same time.Show that the present invention is made Standby magnetic composite nano particle also has good selectivity in the cell.In addition, from Fig. 7 it can further be seen that being suppressed with NCA Agent process after cell, be hardly visible fluorescence signal after nano-probe A is imported, and the cell processed with TBHP, fluorescence Signal is remarkably reinforced, and the increase with TBHP additions, and fluorescence signal is further enhanced.The above results are further characterized by, carefully Intracellular occur fluorescence signal be by APE1 enzymes for magnetic composite nano particle in DNA abasic sites generate specificity Produce after dissection.
The Study of cytotoxicity of embodiment 4, magnetic composite nano particle
The nano-probe A of final concentration of 25-100 μ g/mL is carried out magnetic transfection experiment to HeLa cells, is incubated 120 minutes. It is subsequently adding CCK-8 reagents to be incubated 1 hour, by the group for being added without nano-probe as a control group, will be both added without nano-probe The group of DPBS buffer solution operation is not carried out as blank group yet.Uv absorption in culture fluid is detected at 450 nm, then Calculate the activity of cell.As can be seen from Figure 8, when the concentration of nano-probe is 100 μ g/mL, the cell for still having 80% is protected Hold activity.Demonstrate this composite nanometer particle i.e. hypotoxicity of nano-probe A.

Claims (10)

1. a kind of magnetic composite nano particle, includes magnetic core, silica shell and biomolecule decorative layer from inside to outside successively, The magnetic core is ferromagnetic nanoparticle, and the silica shell has the silicon dioxide of carboxyl, the biology for surface modification Molecular modification layer is Avidin and the biotinylated double-stranded DNA containing abasic room in combination.
2. magnetic composite nano particle as claimed in claim 1, it is characterised in that the ferromagnetic nanoparticle is four oxidations Three iron nano-particles or γ-iron sesquioxide nano-particle.
3. magnetic composite nano particle as claimed in claim 1, it is characterised in that the TEM of the magnetic composite nano particle Particle diameter is 40-60nm, and its DLS particle diameter is 65-75nm.
4. magnetic composite nano particle as claimed in claim 1, it is characterised in that the expression of the magnetic composite nano particle Formula is Fe3O4@SiO2@AVD-AP-DNA or γ-Fe2O3@SiO2@AVD-AP-DNA, wherein Fe3O4Ferroso-ferric oxide magnetic core is represented, γ-Fe2O3γ-iron sesquioxide magnetic core is then represented；SiO2Represent silica shell；AVD represents Avidin layer；AP-DNA generations The biotinylated double-stranded DNA layer containing abasic room of table.
5. magnetic composite nano particle as claimed in claim 1, it is characterised in that described biotinylated containing abasic sky Two single-stranded sequences of the double-stranded DNA of position are respectively：
5 '-GGGGACGACTCCATCGACCTCAAGCAGTTGATCCTTTGGAAAAAAA-Biotin-3 ' and 5 '- GTCGATGGAGXCGTCCCC-FAM-3 ', wherein X represent that abasic site, Biotin represent biotin labeling, and FAM is carboxyl Fluorescein labelling.
6. the preparation method of the magnetic composite nano particle described in claim 1-5 any one, including：First in silica shell The outer layer of layer is covalently attached upper Avidin, then by Avidin and the strong affinity interaction of biotin, by biotinylated alkali containing U The double-stranded DNA modification of base finally recycles UDG enzymes by U base excisions on the ferromagnetic nanoparticle surface of coated with silica, Produce the double-stranded DNA containing abasic room.
7. preparation method as claimed in claim 6, it is characterised in that comprise the following steps that：
1) the ferromagnetic nanoparticle ultrasonic disperse for coating the silica shell of carboxyl modified is separately added in pure water EDC and NHS, concussion reaction under room temperature, used as solution A；
2) in solution A, add Avidin, concussion reaction under room temperature, Magneto separate washing to remove unreacted material, then incite somebody to action To reactant again ultrasonic disperse in pure water, as solution B；
3) the biotinylated double-stranded DNA containing U bases is added in solution B, 30-90 minutes are incubated under room temperature, and Magneto separate is simultaneously washed Wash, remove unreacted material, then by the magnetic nanoparticle of the double-stranded DNA modification containing U bases for obtaining again ultrasound point It is dispersed in PBS solution, as solution C；
4) solution C is added in 1.1 buffer solution of buffer, adds UDG enzyme action except the U bases in DNA double chain, obtain magnetic Property composite nanometer particle.
8. preparation method as claimed in claim 7, it is characterised in that step 1) in, the silica shell bag of carboxyl modified Final concentration of 0.5-2mg/mL of the ferromagnetic nanoparticle ultrasonic disperse for covering in pure water；The concentration of EDC is 0.5-2mg/mL, Weight of the concentration of NHS for the ferromagnetic nanoparticle of the silica shell cladding of 1-5mg/mL, EDC, NHS and carboxyl modified Than for 4:10:1.
9. preparation method as claimed in claim 7, it is characterised in that step 2) in the concentration of Avidin be 0.1-0.2mg/ ML, it is 5 to add Avidin to make the ratio of nano-particle and Avidin final concentration in mixed solution：1, concussion reaction 8-12 under room temperature Individual hour；Step 3) in, final concentration of 0.2-1 μM in solution B of the biotinylated double-stranded DNA containing U bases of addition；Step Rapid 4) in, the final concentration 1.0-100U/mL of the UDG enzymes of addition in 1.1 buffer solution of buffer comprising solution C, during reaction Between scope be 0.5-10min.
10. enzyme assay, the nucleic acid in living cell in vitro of the magnetic composite nano particle described in claim 1-5 any one Application in the in situ imaging of repair enzyme.
CN201610811557.2A 2016-09-08 2016-09-08 A kind of magnetic composite nano particle and its preparation method and application Active CN106497978B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610811557.2A CN106497978B (en) 2016-09-08 2016-09-08 A kind of magnetic composite nano particle and its preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610811557.2A CN106497978B (en) 2016-09-08 2016-09-08 A kind of magnetic composite nano particle and its preparation method and application

Publications (2)

Publication Number Publication Date
CN106497978A true CN106497978A (en) 2017-03-15
CN106497978B CN106497978B (en) 2019-12-03

Family

ID=58291336

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610811557.2A Active CN106497978B (en) 2016-09-08 2016-09-08 A kind of magnetic composite nano particle and its preparation method and application

Country Status (1)

Country Link
CN (1) CN106497978B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108318693A (en) * 2017-12-15 2018-07-24 北京大学 Abasic magnetic molecularly imprinted nano particle of endonuclease and its preparation method and application
CN109925517A (en) * 2017-12-19 2019-06-25 浙江大学 PH response type magnetic nano-particle assembly and its preparation method and application
CN110780079A (en) * 2019-11-28 2020-02-11 南京迪安医学检验所有限公司 Squamous cell carcinoma antigen detection reagent
CN111333692A (en) * 2020-03-02 2020-06-26 湖南省中医药研究院 Betulinic acid derivative and preparation method and application thereof
CN113252627A (en) * 2021-05-11 2021-08-13 广州中医药大学(广州中医药研究院) Application of DNA chain layer in detecting molecular activity, DNA nano fluorescent probe and preparation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MENGXIA LI AND DAVID M. WILSON,III: "Human Apurinic/Apyrimidinic Endonuclease 1", 《ANTIOXIDANTS & REDOX SIGNALING》 *
RUOCAN QIAN ET AL.: "Switchable Fluorescent Imaging of Intracellular Telomerase Activity Using Telomerase-Responsive Mesoporous Silica Nanoparticle", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 *
徐海燕，辛晓燕: "APE1/Ref-1与肿瘤的研究进展", 《现代肿瘤医学》 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108318693A (en) * 2017-12-15 2018-07-24 北京大学 Abasic magnetic molecularly imprinted nano particle of endonuclease and its preparation method and application
CN108318693B (en) * 2017-12-15 2020-09-22 北京大学 Dealkalized endonuclease magnetic molecularly imprinted nano-particle as well as preparation method and application thereof
CN109925517A (en) * 2017-12-19 2019-06-25 浙江大学 PH response type magnetic nano-particle assembly and its preparation method and application
CN109925517B (en) * 2017-12-19 2020-10-23 浙江大学 PH response type magnetic nanoparticle assembly and preparation method and application thereof
CN110780079A (en) * 2019-11-28 2020-02-11 南京迪安医学检验所有限公司 Squamous cell carcinoma antigen detection reagent
CN111333692A (en) * 2020-03-02 2020-06-26 湖南省中医药研究院 Betulinic acid derivative and preparation method and application thereof
CN113252627A (en) * 2021-05-11 2021-08-13 广州中医药大学(广州中医药研究院) Application of DNA chain layer in detecting molecular activity, DNA nano fluorescent probe and preparation method and application thereof
CN113252627B (en) * 2021-05-11 2024-02-27 广州中医药大学(广州中医药研究院) Application of DNA chain layer in detection of molecular activity, DNA nano fluorescent probe, preparation method and application thereof

Also Published As

Publication number Publication date
CN106497978B (en) 2019-12-03

Similar Documents

Publication Publication Date Title
CN106497978B (en) A kind of magnetic composite nano particle and its preparation method and application
Shi et al. MicroRNA-responsive release of Cas9/sgRNA from DNA nanoflower for cytosolic protein delivery and enhanced genome editing
Kumar et al. Carbon nanotubes: a novel material for multifaceted applications in human healthcare
CN107384894B (en) Functional graphene oxide efficiently delivers method of the CRISPR/Cas9 for gene editing
McKnight et al. Tracking gene expression after DNA delivery using spatially indexed nanofiber arrays
CN102439148B (en) SiRNA conjugate and preparation method thereof
Li et al. Functional DNA structures and their biomedical applications
KR102109084B1 (en) Quencher comprising nanomaterial conjugated with water-soluble polymer and use thereof
Ren et al. Aptamer-functionalized nanomaterials for biological applications
Li et al. Circular nucleic acids: Discovery, functions and applications
KR101866968B1 (en) Method for mass production of nucleic acid nanostructure and use thereof in drug delivery systems
Kim et al. Self-assembled messenger RNA nanoparticles (mRNA-NPs) for efficient gene expression
Kim et al. DNA-assisted smart nanocarriers: progress, challenges, and opportunities
CN102703601B (en) Multifunctional magnetic fluorescent microsphere and preparation method and application thereof
CA2478491A1 (en) Dna nanocage by self-organization of dna and process for producing the same, and dna nanotube and molecule carrier using the same
WO2020087867A1 (en) Metal-nucleic acid nanoparticle, preparation method therefor and use thereof
US20110274706A1 (en) Nucleic acid delivery vehicle and uses thereof
Lei et al. Construction of gold-siRNA NPR1 nanoparticles for effective and quick silencing of NPR1 in Arabidopsis thaliana
Li et al. Construction of rolling circle amplification products-based pure nucleic acid nanostructures for biomedical applications
CN113512578B (en) miRNA chemiluminescence detection kit based on constant-temperature enzyme-free multistage amplification
Zhang et al. The application of tetrahedral framework nucleic acids as a drug carrier in biomedicine fields
Chen et al. Bioinspired affinity DNA polymers on nanoparticles for drug sequestration and detoxification
Qiao et al. The advance of CRISPR-Cas9-based and NIR/CRISPR-Cas9-based imaging system
Zhang et al. Controlling the trans-cleavage of CRISPR-Cas12a with nicked PAM: Universal platform for biosensing
Shinde et al. Metallic micro-ring device for highly efficient large cargo delivery in mammalian cells using infrared light pulses

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant