CN103267840B - Magnetic layered double hydroxide-DNA (MLDH-DNA) supramolecular assembly type magnetic targeting probe - Google Patents

Abstract

The invention relates to a magnetic layered double hydroxide-DNA (MLDH-DNA) supramolecular assembly type magnetic targeting probe. The probe is prepared by taking MLDH as a carrier and a nucleic acid dye labeled DNA as an object and in a low temperature ion exchange method, and the probe has an MLDH-DNA type intercalated structure, excellent cell transmission performance and specific in-vivo targeting transfer and fluorescent tracing functions. An MLDH laminate with high positive charge density, small radial size, large oriented size and weakly alkaline property serves as a transfer platform, so that the MLDH-DNA system obtains excellent cell transmission performance and smoothly and rapidly transmits the DNA to cell nucleus; according to the magnetism and the sustained or controlled release function of the MLDH, the oriented and safe transfer of the DNA in in-vivo tissue is guaranteed; and moreover, an optical signal of nucleic acid labeled dye has the function of tracing the MLDH-DNA cells and in-vivo transfer paths. The optical probe is suitable for targeting transfer system construction of nucleic acid dye labeled DNA or preparation of repaired DNA fragments and cell and living imaging, and has the advantages of high functional integration level, advanced method, easy operation, low cost and the like.

Description

MLDH-DNA Supramolecular Assembling type magnetic targeted probe

Technical field

The invention belongs to Biomedical function material category, transmit with the biological targeting of genetic stew and tracer technique relevant, be specifically related to a kind of MLDH-DNA Supramolecular Assembling type magnetic targeted probe.

Background technology

Gene therapy (Gene therapy) refers to external source normal gene to import target cell, to correct or to compensate because of genetic flaw, variation, reaches therapeutic purposes.But genetic stew, no matter be the genetic fragment after repairing or RNA interferon dsRNA, because volume is large, easily enzymolysis, elecrtonegativity be strong etc., and reason is difficult to enter cell, and the chemistry transfer of genetic stew is the bottleneck problem of restriction gene therapy application.Obstacle exogenous gene being imported biological acceptor cell mainly contains two aspects, one is surface enters cellularity to the naked grain of electronegative DNA natural electrostatic barrier in electronegative cell wall, two is the corrosions to constructing gene transport support of the sour environment such as Cell sap and lysosome, makes DNA be difficult to escape the degraded of nuclease.Therefore, the process realizing exogenous gene and recipient cell genome conformity is very complicated; Major part nanometer delivery system is difficult to directly enter cell interior, and more difficult DNA or dsRNA that effectively carry is diffused into safely nucleus, enters karyon expression alien gene through Nuclear pore.Layered double hydroxide (Layered double hydroxide, LDH), because there is intercalation assembling and reverse exchange release performance, superior cell delivery performance and the performance with elecrtonegativity DNA helical molecule electrostatical binding, become the focus of biological transmission research; DNA is after co-precipitation or ion exchange enter interlayer, and alkaline LDH laminate can play available protecting and transhipment effect to inserting the DNA carried under Cell sap faintly acid (pH=4 ~ 5) microenvironment.

Fluorescence signal is utilized to carry out to gene transfer process the trend that real-time tracking is nanometer biotechnology future development, to understanding the cell mechanism of gene delivery, live body transfection and relevant theoretical rule, setting up efficient movement system, exploitation has the gene prod of commercial value significant.

Summary of the invention

The object of the present invention is to provide a kind of MLDH-DNA Supramolecular Assembling type magnetic targeted probe.

The present invention is achieved through the following technical solutions:

A kind of MLDH-DNA Supramolecular Assembling type magnetic targeted probe, is characterized in that: with magnetic layered composite hydroxide MLDH for carrier, is object with nucleic acid dye marker DNA, is prepared from by low-temperature ion exchange process.

The composition of described carrier magnetic layered composite hydroxide MLDH meets chemical formula [Fe ^iI ₂fe ^iII(OH) ₆] [Cl H ₂o].

The procurement process of described magnetic layered composite hydroxide MLDH is:

At N ₂or under Ar protection, 0 ~ 50 DEG C of constant temperature and 300 rpm magnetic stirring conditions, Fe in molar ratio ²⁺/ Fe ³⁺=2: 1 preparation FeCl ₂4H ₂o, FeCl ₃6H ₂o solution, carries out coprecipitation reaction with unitary strong base solution as precipitant, obtains magnetic layered composite hydroxide MLDH after having reacted through slurry in-situ crystallization, solid separation and sedimentation, centrifugalize;

The strong paper mill wastewater of described unitary is 1 ~ 2 mol L ^-1;

The reaction end of described coprecipitation reaction arrives 5.5 ~ 7.0 with pH and is as the criterion;

Described slurry in-situ crystallization is at N ₂or carry out under Ar protection, 0 ~ 50 DEG C of constant temperature and 300 rpm magnetic stirring conditions, crystallization time 30 ~ 50 min;

Described solid separation and sedimentation complete in the cold ethanol of-80 ~-20 DEG C.

The preparation method of described nucleic acid dye marker DNA is:

First nucleic acid dye and plasmid are pressed 1:4 ~ 1:6 mixed in molar ratio, vortex dispersion gently, under lucifuge and 0 ~ 4 DEG C of condition, static cultivation 20 ~ 40 min, make nucleic acid dye marker DNA.

The processing step of described low-temperature ion exchange process is: at 0 ~ 4 DEG C and N ₂or under Ar protective condition, be dissolved in water by magnetic layered composite hydroxide MLDH, and then add nucleic acid dye marker DNA solution, stirring at low speed or vortex mixing, static cultivation 20 ~ 50 min, afterwards centrifugalize.

The consumption of layered complex hydroxide MLDH according to DNA material amount 6.3 10 ⁴doubly take.

Feature of the present invention be utilize ion-exchange reactions to be inserted by the DNA-nucleic acid dye complex of nucleic acid dye labelling to be downloaded to external surface area large, be with micro-positive electricity, between the MLDH laminate having alkalescence and a superparamagnetism, the elecrtonegativity of DNA and double-spiral structure is made to obtain shielding and the available protecting of MLDH inner plating, thus acquisition is conducive to the outside geometry of cell delivery and electrical advantage, and be enough to stability and the core targeting of bearing cell internalizing whole process, based on the particle shape of MLDH in delivery system and the fluorescence signal of nucleic acid dye, utilize TEM Electronic Speculum and laser confocal fluorescence microscope technology effectively can follow the tracks of the cell transmittance process of DNA, the luminescent properties of the magnetic of MLDH-DNA-nucleic acid dye system, slow control-release function and nucleic acid dye makes foreign DNA magnetic targeted in vivo, safe transit and the real-time visual monitoring to this process become possibility, is a kind of composite construction platform with fluorescent tracing, Magneto separate and targeting transport function.The targeting movement system that the present invention is applicable to nucleic acid dye marker DNA, DNA plerosis fragment or siRNA builds and the preparation of cell or living animal image optics probe, the advantage such as have that functional integration is high, method is advanced, mild condition, response time are short, with low cost.

Accompanying drawing explanation

Fig. 1 MLDH-DNA is to SGC-7901 cell traffic process Electronic Speculum characterization result schematic diagram;

Fig. 2 MLDH-DNA is to SGC-7901 cell traffic process Laser scanning confocal microscopy schematic diagram;

Fig. 3 MLDH-DNA is to the living imaging result of Kun Ming mice magnetic targeted transport process.

Detailed description of the invention

Chemosynthesis agents useful for same of the present invention is customary commercial reagent, and biotic experiment material therefor is commercial product.Following examples are intended to further illustrate the present invention, and are not used in restriction the scope of protection of present invention.MLDH-DNA-nucleic acid dye probe system of the present invention, realizes by following technical scheme:

embodiment 1with absorbing wavelength be 491nm nucleic acid dye (trade name YOYO-1) marker DNA, with ion exchange synthesis MLDH-DNA/YOYO-1 probe, and checked the cell internalizing effect of MLDH-DNA by TEM, specific operation process is as follows:

Accurately take 1.9733g FeCl ₂4H ₂o and 1.3411g FeCl ₃6H ₂o, is added to 500 mL N ₂in guard reactor, add 100mL distilled water at logical N ₂magnetic stirring and dissolving under condition, at 0 ~ 50 DEG C of (being preferably 4 DEG C) constant temperature and N ₂under protective condition, drip 2mol L ^-1naOH solution, controlling co-precipitation terminal pH is that 5.5 ~ 7.0(is preferably 6.50).Be warming up to 35 DEG C, stir and N at 300 rpm magnetic ₂under protective condition, in-situ crystallization 30-50min (being preferably 45min); With-80 ~-20 DEG C of dehydrated alcohol conversion solvents, after solid phase sedimentation, at N ₂complete centrifugalize under protection ,-4 DEG C of constant temperature and 5000rpm condition and sample washs, consisted of [Fe ^iI ₂fe ^iII(OH) ₆] [Cl H ₂o] MLDH carrier.

Nucleic acid dye and plasmid are pressed 1:4 ~ 1:6 mixed in molar ratio, and specifically can get 20 L concentration is 1 mM L ^-1nucleic acid dye YOYO-1, be added to 500 L 200 g mL ^-1in plasmid solution, vortex mixes, and under lucifuge and 0 ~ 4 DEG C of condition, stationary incubation 20-40 min, makes DNA/YOYO-1 complex.

Accurately take 1mg MLDH solid phase (by 6.3 10 of the amount of DNA material ⁴doubly meter), be transferred to 10 mL N ₂in protection microreactor, add 2mL distilled water, magnetic dispersed with stirring is even, add the DNA/YOYO-1 solution of 400 L, stirring at low speed mixes, then at 0 ~ 4 DEG C and N ₂static cultivation 20 ~ 50 min under protective condition; Finally, by reaction paste at-10 DEG C, centrifugal 20 min under 500 rpm conditions, MLDH-DNA/YOYO-1 solids product is obtained.

With trypsinization SGC-7901 cell, by 1 × 10 ⁵/ hole amount is inoculated on 6 porocyte culture plates, is placed in cell culture incubator cellar culture 24h.MLDH-DNA/YOYO-1 complex is mixed with 120 μ gmL ^-1suspension, ultraviolet sterilization deactivation, for intervening SGC-7901 cell.If blank, the every porocyte of experimental group adds 10 L MLDH-DNA/YOYO-1 medicinal liquids, is placed in 37 DEG C, 5% CO ₂stop cultivating after continuing to be cultured to 1h, 3h, 5h and 7h in incubator, PBS rinses three times, trypsinization, collecting cell is in 15mL centrifuge tube, by transmission electron microscope cell specimen processing method, complete after cell fixes, dewaters, soaks into, embeds, repaiies the operation such as block, section and electron staining, carry out electron microscopic observation, shooting, experimental result is shown in accompanying drawing 1.

embodiment 2with ion exchange synthesis MLDH-DNA/YOYO-1 probe, and followed the tracks of the cell internalizing process of MLDH-DNA by laser confocal fluorescence microscope, specific operation process is as follows:

Thawing by the preparation of embodiment 1 scheme ,-20 DEG C of storages, concentration under 4 DEG C of conditions is 190 g mL ^-1dNA/YOYO-1 complex, accurately take 0.15 mg and be transferred to 10 mL N by the MLDH solid phase that enforcement 1 scheme is made ₂in protection microreactor, add 300 L distilled waters, ultrasonic disperse is even, add the DNA/YOYO-1 solution of 60 L, vortex mixing gently, then at 0 ~ 4 DEG C (this example preferably 0 DEG C) and N ₂under protective condition, static cultivation 50 min, make MLDH-DNA/YOYO-1 suspension, for subsequent use after sterilizing.

With trypsinization SGC-7901 cell, by 1 × 10 ⁵/ hole amount is inoculated on 6 porocyte culture plates, is placed in cell culture incubator cellar culture 24h.To wait that intervening cell is divided into 3 experimental grouies such as DNA/YOYO-1, MLDH and MLDH-DNA/ YOYO-1, if blank, experimental group often group establishes 2 multiple holes, carefully suck the culture fluid in Tissue Culture Plate, every hole adds 10 L and intervenes medicinal liquid (wherein, MLDH and MLDH-DNA/YOYO-1 suspension all needs first to filter with 0.22 m sterilised membrane filter), after condition hatches 0.5,1 and 2h routinely, take out Tissue Culture Plate, supernatant discarded, rinse 3 times with PBS, 4% paraformaldehyde fixes 20 min, and PBS rinses 3 times; Then add 20 L DAPI solution-dyed 3min, PBS rinses 3 times (each 2 min), finally, blots surplus liquid with filter paper, anti-fluorescence decay mountant mounting.Under laser scanning co-focusing microscope, observe the distribution situation of gene delivery complex in cell on diverse location, experimental result is shown in accompanying drawing 2.

embodiment 3with excitation wavelength be 647nm near-infrared nucleic acid dye (trade name ULS) marker DNA, with ion exchange synthesis MLDH-DNA/ULS-647 combined probe, and by the bioluminescence imaging technology checking MLDH-DNA effect that magnetic targeted transhipment distributes in Kun Ming mice body, specific operation process is as follows:

First use Alexa Fluor 647 test kit marker DNA, preparation DNA/ Alexa Fluor 647 complex.Concrete operations are, the DMSO(Component B by 100 μ L), be added in the Component A containing ULS labeling reagent, vortex mixed, sonic oscillation dissolve completely to ULS; Get 1 L 10 g L ^-1salmon sperm dna dry in atmosphere, then be resuspended in 9 L labelling buffer (Component C); Get 5 L dispersion liquids, 95 L labelling buffer (C) to dilute, then at 95 DEG C, make DNA degenerative treatments 10min, cooled on ice 5min makes sample deposition bottom test tube; Add the ULS stock solution of 25 L again, add labelling buffer (Component C) and make final volume be 125 μ L, hatch 15min in 80 DEG C, finally, reaction tube is placed in ice bath, cessation reaction, centrifugal, obtain DNA/ULS-647 compound crude samples.Get the EP pipe box of 10mL on two P30 chromatographic columns, each chromatographic column is loading DNA/ULS-647 solution 125 μ L dropwise, the centrifugal 3min of 1000g, collects DNA/ULS-647 solution about 125 μ L.

Take 0.15mg MLDH and be transferred to 10mL N ₂in protection microreactor, add 625 L deionized waters, ultrasonic disperse is even, make suspension; Add the DNA/ULS-647 solution of 125 L purification, vortex mixing gently, standing and reacting 30 min under lucifuge and 0 ~ 4 DEG C of condition, making concentration is 200 mM L ^-1mLDH-DNA/ULS-647 suspension, saves backup (carry out frozen centrifugation, can obtain solid phase sample) in-20 DEG C.

The MLDH-DNA/ULS-647 composite sample of getting above-mentioned cold preservation is thawed, or gets solid phase sample and be made into concentration 200 mM L ^-1suspension, then by 50,90,100,120,140,160,180,200 times of dilutions, be added to respectively in 96 orifice plates, on living imaging instrument and observe luminous intensity; Draw above-mentioned 50,90,100,120, the 140 times of diluents of 50 L respectively, be injected to the cervical region of mice respectively, left front leg, RAT, left back lower limb, right rear leg are subcutaneous, detect luminous intensity, determine the wavelength condition of living imaging.Anesthetized mice, dresses the Mus clothing putting Magnet (Φ 17 × 3mm neodymium iron boron alnico magnets, 2000Gs) at right front breast seam, then through 50 times of diluents (concentration 4 mM L of tail vein injection 200 L MLDH-DNA/ULS-647 complex ^-1), observe luminous site respectively at after administration after 0.3,1.5h, 2h, 5h, 6h, 7h, take pictures by white light and HONGGUANG, labelling luminous site, experimental result is shown in accompanying drawing 3 simultaneously.

evaluation

Accompanying drawing 1 to provide when SGC-7901 cell 7h intervened by embodiment 1 observation MLDH-DNA/YOYO-1 probe under 3000 times of visual fields MLDH-DNA delivery system in the situation of the inside and outside distribution of cell, the MLDH-DNA particle not yet entering cell can be observed in the lower-left of figure, in regular hexagon, the length of side 25 nm, diameter 45 ~ 56 nm, lamination thickness 15 nm; The right lower quadrant of figure amplifies to show and enters Cytoplasm, is in the MLDH-DNA particle that transhipment midway exists with vesicle form, transhipment vesicle about 179 × 167 nm, and interior bag particle is that regular hexagon, size are homogeneous, diameter 35.4 nm, the length of side 16.7 nm; The coherent condition showing the MLDH-DNA particle performance that enters caryoplasm and go out is amplified in the middle and upper part of figure, and the vesicle in cell caryoplasm forms cyst wall framework by MLDH-DNA particle lateral connection, and size is about 339 × 597 nm, and interior bag particle diameter is at about 40 nm.

Accompanying drawing 2 provides in embodiment 2 and makes with MLDH-DNA/YOYO-1 the laser co-focusing immunofluorescence micro-imaging result that SGC-7901 cell internalizing process followed the tracks of by fluorescent probe, figure A-C intervenes cell 0.5 h (A), 1 h(B through MLDH-DNA/YOYO-1) and 2 h (C) after the result that obtains, figure D is the result of DNA/YOYO-1 contrast intervention; First row (I) is the image collected from fluorescence channel, and secondary series (II) is the image gathered for DAPI nuclear staining, and the 3rd row (III) are Channel Image stack result.Above result display, after cell 0.5 h intervened by MLDH-DNA/YOYO-1 fluorescent probe, green fluorescence speckle is assembled and is bonded to surface of cell membrane, has entered the distribution of particles of cell in Cytoplasm inner cell organ; After effect 1h, around nucleus, there is the green speckle become clear, prove that MLDH-DNA/YOYO-1 particle approaches Nuclear pore; After 2 h, nucleus occurs the fluorescence speckle become clear, be filled with fluorescent particles in Cytoplasm simultaneously.The above results proves, DNA successful delivery in 2h to nucleus, can be had obvious core targeting by MLDH.In matched group, in cell, not there is fluorescence signal, prove that in experimental group, the successful delivery of MLDH carrier to DNA serves pivotal role.

Accompanying drawing 3 provides after embodiment 3 adds the Kun Ming mice intravenous injection MLDH-DNA/ULS-647 in magnetic field on right side, the living imaging result of fluorescence distribution in body when 0.5h, 1.5h, 2h, 3.5h, 5h, 7h, 8.5h, the display be perfectly clear in figure, MLDH-DNA/ULS-647 is mainly gathered in the right side that mice adds magnetic field after body circulation, present obvious magnetic targeted, eliminate along with in the release of time lengthening, interlayer DNA/ULS-647 and body, fluoroscopic examination intensity weakens gradually, but the signal adding magnetic local is greater than non-targeted position all the time; After 8.5h, the testing result of isolated organ shows, near infrared signal is distributed widely in the heart, liver, spleen, lung, kidney sample, especially the abundance of lung and kidney is high, and the intensity of right kidney is greater than left kidney, right lung intensity is greater than left side, and quantitative analysis has statistical significance.

Claims (2)

1. a MLDH-DNA Supramolecular Assembling type magnetic targeted probe, is characterized in that: with magnetic layered composite hydroxide MLDH for carrier, is object with nucleic acid dye marker DNA, is prepared from by low-temperature ion exchange process;

The composition of described magnetic layered composite hydroxide MLDH meets chemical formula [Fe ^iI ₂fe ^iII(OH) ₆] [Cl H ₂o];

The preparation method of described nucleic acid dye marker DNA is: first by nucleic acid dye and plasmid by 1:4 ~ 1:6 mixed in molar ratio, vortex dispersion gently, under lucifuge and 0 ~ 4 DEG C of condition, staticly cultivates 20 ~ 40 min, makes nucleic acid dye marker DNA;

The processing step of described low-temperature ion exchange process is: at 0 ~ 4 DEG C and N ₂or under Ar protective condition; magnetic layered composite hydroxide MLDH is dissolved in water; and then add nucleic acid dye marker DNA solution; stirring at low speed or vortex mixing; static cultivation 20 ~ 50 min; centrifugalize afterwards, the consumption of described magnetic layered composite hydroxide MLDH according to DNA material amount 6.3 × 10 ⁴doubly take.

2., according to MLDH-DNA Supramolecular Assembling type magnetic targeted probe according to claim 1, it is characterized in that the procurement process of described magnetic layered composite hydroxide MLDH is:

At N ₂or under Ar protection, 0 ~ 50 DEG C of constant temperature and 300 rpm magnetic stirring conditions, Fe in molar ratio ²⁺/ Fe ³⁺=2: 1 preparation FeCl ₂4H ₂o, FeCl ₃6H ₂o solution, carries out coprecipitation reaction with unitary strong base solution as precipitant, obtains magnetic layered composite hydroxide MLDH after having reacted through slurry in-situ crystallization, solid separation and sedimentation, centrifugalize;

The strong paper mill wastewater of described unitary is 1 ~ 2 mol L ^-1;

The reaction end of described coprecipitation reaction arrives 5.5 ~ 7.0 with pH and is as the criterion;

Described slurry in-situ crystallization is at N ₂or carry out under Ar protection, 0 ~ 50 DEG C of constant temperature and 300 rpm magnetic stirring conditions, crystallization time 30 ~ 50 min;

Described solid separation and sedimentation complete in the cold ethanol of-80 ~-20 DEG C.