CN105802998A - Magnetic nanoparticle of layer-by-layer wrapping structure and preparation method and application thereof - Google Patents
Magnetic nanoparticle of layer-by-layer wrapping structure and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a magnetic nanoparticle of a layer-by-layer wrapping structure and a preparation method and application thereof.The magnetic nanoparticle is composed of an inner core, a middle layer and an outer layer, wherein the inner core and the middle layer are combined and the middle layer and the outer layer are combined by means of the electrostatic attraction effect, the inner core is a ferroferric oxide nanoparticle modified by a cationic polymer, the middle layer is a to-be-transfected gene wrapping the outside of the inner core, and the outer layer is a spermine and Pulullan copolymer wrapping the outside of the middle layer.According to the magnetic nanoparticle, the to-be-transfected gene is combined with the inner core first and then combined with the spermine and Pulullan copolymer, and therefore the to-be-transfected gene is tightly wrapped by the magnetic nanoparticle and prevented from being degraded in the transfection process; spermine and Pulullan can be recognized and absorbed by mesenchymal stem cells, and the cell entering efficiency of the magnetic nanoparticle is further improved; the spermine and Pulullan copolymer has little toxicity to mesenchymal stem cells, and the safety risks caused by transfection to stem cells are effectively reduced.
Description
Technical field
The present invention relates to a kind of magnetic transfecting formulations, be specifically related to the magnetic Nano of a kind of clad structure layer by layer
Ball and its preparation method and application.
Background technology
Some stem cell, such as mesenchymal stem cells MSCs (Bone-marrow mesenchymal stem
Cell, BMSC), owing to having the potential of Multidirectional Differentiation, and it is easily obtained and the feature such as isolated and purified,
It is employed for more and more in the research of stem-cell therapy, gene therapy and oncotherapy etc..
But, in practical study and clinical practice, generally require and stem cell carried out suitable gene
Modify, to induce stem cell the most specific can be divided into required cell or express specific treatment
Gene thus play corresponding therapeutical effect the most in vivo.
Currently, the Gene transfer vector for stem cell gene restructuring mainly has viral genetic vector and non-
Viral genetic vector two class.But there is bigger security risk in the application that viral vector is on stem cell,
As caused insertion mutation, activating oncogene, cause cell differentiation and immunogenicity etc., these wind
Danger also limit the stem cell extensive application clinically through viral vector gene restructuring.Non-viral base
Because though carrier risk in clinical practice is well below viral genetic vector, its on stem cell relatively
Low transfection efficiency but becomes and limits them to clinical practice Main Bottleneck.On the one hand, in recent years
The simple non-viral gene vector of result of study certification structure be difficult to help gene through from extracellular
To all barriers of cell thus realize the high efficient expression of gene.Some commercially available non-viral genes turn
Transfection reagent such as polymine (PEI), Lipofectmine2000 (Lipo2000), ExGene 500
Or SuperFect etc. is also proved to, when being used for stem cell gene transfection, there is transfection efficiency relatively
The problems such as low or obvious cytotoxicity.On the other hand, due to albumen meeting electronegative in serum
Be combined with positively charged non-viral gene vector, cause the born of the same parents' efficiency that enters of carrier/gene composite to decline
And finally affect transfection efficiency.Therefore, most non-viral gene vectors are desirable that under serum-free condition
Carry out corresponding gene transfection, to avoid the serum interference to transfection efficiency.But, stem cell is to training
Supporting environment very sensitive, serum-free transfection often increases non-viral gene transfection reagent to stem cell
Toxicity, and affect the state of stem cell.So, how to make non-viral gene vector there is bar at serum
Efficient transfection efficiency is still kept to become as the design of current non-viral gene vector and prepared under part
The key issue overcome required in journey.
Magnetic transfection is that a kind of potential non-viral gene vector that can be effectively improved transfects effect on stem cell
The means of rate.Magnetic transfection is (such as ferroso-ferric oxide Fe by magnetic-particle3O4Nanoparticle) and some life
Thing macromole is combined by chemical covalent bonds or physical adherence effect, is formed and has the micro-of magnetic responsiveness
Grain, then utilizes the surface activity of magnetic particle and traditional virus or non-virus carrier coupling, then with
Genes of interest combines, or magnetic particle directly combines with genes of interest, constitutes the magnetic carrying attached gene
Property nanosphere.Under the effect of externally-applied magnetic field, magnetic nano-balls can be significantly improved and show at cell membrane
Gathering, and help the highly efficient entrance of magnetic nano-balls intracellular, and then release genes of interest, real
The purpose of existing high efficiency gene transfection.
Magnetic rotaring dyeing technology can help non-viral gene vector to overcome cell membrane by external magnetic field
Barrier so that carrier can carry genes of interest and more efficiently enter intracellular from born of the same parents, thus effectively carries
The efficiency of high gene transfection.Simultaneously as the born of the same parents that enter of carrier rely primarily on external magnetic and complete, and
Do not affect cell endocytic picked-up mechanism, therefore can overcome the serum impact on transfection process.
But, current magnetic transfection reagent is substantially for tumor cell design, for stem cell
Custom-designed magnetic transfection reagent is the most actually rare.This is because stem cell to the sensitivity of environment and
Difficulty is transfecting all proposes the highest design requirement to corresponding magnetic transfecting formulations.With tumor cell phase
Ratio, stem cell has high selectivity to endocytosis thing, and metabolism is the slowest, is difficult to transfected.
Therefore, imperative for stem cell specialized designs magnetic transfecting formulations, and there is highly important section
Learn researching value and clinical treatment meaning.
Summary of the invention
The invention provides one and be coated with the magnetic nano-balls of (layer by layer) structure layer by layer, should
Magnetic nano-balls solves magnetic transfection reagent of the prior art and is difficult to use in such as medulla mesenchyma
The problem that the stem cell such as stem cell carry out effective gene transfection.
The magnetic nano-balls of a kind of clad structure layer by layer, is made up of kernel, intermediate layer and outer layer, kernel
And between intermediate layer, all it is be combined with each other by electrostatic attraction effect between intermediate layer and outer layer；
Described kernel is cationic polymer modified ferriferrous oxide nano grain, and described intermediate layer is bag
Overlaying on the gene to be transfected outside kernel, described outer layer is to be coated on the spermine-Pu Lu outside intermediate layer
Blue polysaccharide copolymer.
The magnetic nano-balls of the present invention is by four first and cationic polymer modified for gene to be transfected oxidations
Three-iron nanoparticle combines, and is combined with spermine-pulullan polysaccharide copolymer the most again, thus by be transfected
Gene is tightly wrapped in magnetic nano-balls, it is to avoid gene to be transfected is degraded in transfection process.
After spermine-pulullan polysaccharide copolymer is as outer layer covers gene to be transfected, moreover it is possible to be effectively compressed
Gene to be transfected, makes the mean diameter of magnetic nano-balls and surface potential be maintained at suitable safety range
In.Meanwhile, the modification of spermine-pulullan polysaccharide copolymer makes magnetic nano-balls surface with suitably
Positive charge, suitable charge repulsion makes magnetic nano-balls good dispersion in aqueous phase system, is difficult to sink
Poly-.
The mean diameter of magnetic nano-balls of the present invention is that 100～200nm (suitable safety range is
50～300nm), if particle diameter is too big, it is unfavorable for the cell effective picked-up to magnetic nano-balls, if particle diameter
The least, then cell can be produced certain cytotoxicity due to dimensional effect.Magnetic nano-balls of the present invention
Surface potential be 10～20mV (safety range is 10～40mV), if surface potential is the lowest, magnetic
Property nanosphere is difficult to effectively combine and compress gene to be transfected, if surface potential is the highest, and magnetic nano-balls
Cell membrane can be destroyed, cell is produced serious cytotoxicity.
Simultaneously it has also been found that, spermine-pulullan polysaccharide copolymer effectively can be identified by stem cell
With picked-up (as spermine-pulullan polysaccharide copolymer can be by the CD44 indicated in mesenchymal stem cells MSCs
Receptor recognition also absorbs), that improves magnetic nano-balls further enters born of the same parents' efficiency, by suitable for gene to be transfected
Bring in stem cell sharply；Further, spermine-pulullan polysaccharide copolymer is little to stem cell toxicity, has
Effect reduces the security risk that stem cell is caused by transfection.
Unsaturated amino after magnetic nano-balls enters born of the same parents, on its outer layer spermine-pulullan polysaccharide copolymer
The proton provided by proton pump (V-ATPase) in can chelating lysosome, proton pump continuous openness,
In each proton all can cause a chloride ion and a hydrone retention lysosome, lysosome is caused to swell
Burst and split, be i.e. " proton sponge pump efficiency is answered ".The gene finally making magnetic nano-balls be carried can be from molten
Enzyme body is escaped, release, and enters nucleus.
The magnetic nano-balls of the present invention treats the size of rotaring redyeing gene and kind without selecting, gene to be transfected
Can be functional gene required in various reporter gene, all kinds of test and functional messenger RNA,
SiRNA etc..
For ensureing that magnetic nano-balls kernel has good magnetic responsiveness, as preferably, described four aoxidize
Three-iron (Fe3O4) particle diameter of nanoparticle is 10～50nm, described cationic polymer is with electronegative
Ferriferrous oxide nano grain be combined with each other by electrostatic attraction effect.
As further preferably, the particle diameter of described ferriferrous oxide nano grain is 20～50nm；Now
Ferriferrous oxide nano grain is the nanoparticle of superparamagnetism, and external magnetic field has preferable magnetic responsiveness.
The diameter control of final magnetic nano-balls, to the diameter of kernel no requirement (NR), only need to be existed by the present invention
In respective range.
As preferably, described cationic polymer be ethylenediamine-pulullan polysaccharide copolymer (ED-pul),
Spermine-pulullan polysaccharide copolymer (SP), polymine (PEI), chitosan or spermine-dextrose
Glycosides copolymer.More preferably ethylenediamine-pulullan polysaccharide copolymer or spermine-pulullan polysaccharide copolymer.
The present invention is to the degree of polymerization of each cationic polymer and the equal no requirement (NR) of molecular weight, due to the present invention's
The surface potential of ferriferrous oxide nano grain is negative charge, as long as therefore cationic polymer can make kernel
Surface potential be positive charge.
The magnetic nano-balls pair obtained after utilizing each cationic polymer modified ferriferrous oxide nano grain
The transfection efficiency of mesenchymal stem cells MSCs is respectively arranged with height, but generally and no difference of science of statistics.
As preferably, in described spermine-pulullan polysaccharide copolymer, the mean molecule of pulullan polysaccharide
Amount is 47300Da, and spermine percent grafting is 12%～14%.Selecting this spermine-pulullan polysaccharide copolymerization
During thing, magnetic nano-balls is optimal to the transfection efficiency of stem cell.
The preparation method of the magnetic nano-balls of clad structure layer by layer described in present invention also offers, this is prepared
Method includes:
(1) use coprecipitation to prepare cationic polymer modified ferriferrous oxide nano grain, obtain
Obtain kernel；
(2) described kernel is joined in cdna solution to be transfected, incubate altogether under the conditions of room temperature, shaking
Educate 20～30min, it is thus achieved that kernel-interlayer composite；
(3) in described kernel-interlayer composite, spermine-pulullan polysaccharide copolymer, room temperature are added
Hatch 15～20min the most altogether, it is thus achieved that the magnetic nano-balls of described clad structure layer by layer.
As preferably, in step (2), kernel is less than 1:1 with the mass ratio of gene to be transfected.At this
Under mass ratio, core surface fully can be wrapped up by gene to be transfected, so that kernel-intermediate layer is multiple
The surface potential of compound is negative charge, in order to follow-up outer layer effectively can be combined with intermediate layer.
As further preferably, in step (2), kernel is 1:1.5 with the mass ratio of gene to be transfected.
Under this mass ratio, magnetic nano-balls is optimal to the transfection efficiency of mesenchymal stem cells MSCs.
As preferably, the incubation time in step (2) is 20min, and rational incubation time guarantees
Kernel-interlayer composite is stably formed.
The magnetic nano-balls obtained under different N/P ratios has different transfection efficiencies, as excellent to cell
Choosing, in step (3), spermine-pulullan polysaccharide copolymer is to be transfected with kernel-interlayer composite
The N/P ratio of gene is 3:1～5:1；More preferably 4:1.Under this N/P ratio, magnetic nano-balls is to carefully
The transfection efficiency of born of the same parents is optimal.
Described in present invention also offers, the magnetic nano-balls of clad structure is preparing magnetic transfection reagent layer by layer
In application.
Present invention also offers a kind of magnetic transfecting formulations, by the magnetic nano-balls of described clad structure layer by layer
It is prepared from.
The magnetic nano-balls of the present invention, as magnetic transfecting formulations, can be used for that various types of cells is carried out gene and turns
Dye, including tumor cell and stem cell.
As preferably, described magnetic transfection reagent is for carrying out gene transfection to mesenchymal stem cells MSCs.
Present invention also offers described magnetic nano-balls under serum existence condition, medulla mesenchyma to be done
Cell carries out the method for magnetic transfection, and the method includes:
(1) mesenchymal stem cells MSCs is inoculated on Tissue Culture Plate, at 5%CO2, at 37 DEG C
Cultivate 24h；
(2) culture fluid in hole every on Tissue Culture Plate is replaced with 500 μ L and contain magnetic nano-balls
(40 μ L) and the fresh medium of 10% serum, it is 1000～5000 that Tissue Culture Plate is placed in intensity
On the magnetic field of Gs, at 5%CO2, continue to hatch 2～3h at 37 DEG C；
(3) culture fluid in Tissue Culture Plate is replaced with the fresh medium containing 10% serum,
Remove magnetic field, at 5%CO2, continue to hatch 48h at 37 DEG C, measure transfection efficiency.
Described magnetic field can use common cobalt nickel Magnet to provide, but magnetic field intensity is the most weak or too strong the most not
It is beneficial to obtain preferable transfection efficiency, as preferably, in step (2), Tissue Culture Plate is placed in
Intensity is on the magnetic field of 2700～4000Gs.Under this magnetic field intensity, between magnetic nano-balls is to bone marrow
Mesenchymal stem cells has good transfection efficiency.
Compared with prior art, the invention have the benefit that
(1) magnetic nano-balls of the present invention is by first and cationic polymer modified for gene to be transfected four
Fe 3 O nanoparticle combines, and is combined with spermine-pulullan polysaccharide copolymer, thus will treat
Rotaring redyeing gene is tightly wrapped in magnetic nano-balls, it is to avoid gene to be transfected is dropped in transfection process
Solve；
(2) after spermine-pulullan polysaccharide copolymer is as outer layer covers gene to be transfected, moreover it is possible to effectively
Compress gene to be transfected, make the mean diameter of magnetic nano-balls and surface potential be maintained at safety range
In, not only contribute to magnetic nano-balls and effectively combine and protect gene to be transfected, beneficially cell is to magnetic
Property nanosphere effectively absorbs, and has relatively low cytotoxicity, comparatively safe；
(3) utilization of spermine-pulullan polysaccharide copolymer makes magnetic nano-balls can be filled by between bone marrow
Matter stem cell identification is also effectively absorbed, and that improves magnetic nano-balls further enters born of the same parents' efficiency, by be transfected
Gene is successfully brought in mesenchymal stem cells MSCs；Further, spermine-pulullan polysaccharide copolymer pair
Mesenchymal stem cells MSCs toxicity is little, effectively reduces the peace that mesenchymal stem cells MSCs is caused by transfection
Full blast danger, it is possible to realize under serum existence condition, stem cell being carried out the gene transfection of high-efficiency low-toxicity,
Good transfection carrier is provided, tool for utilizing non-viral gene vector that stem cell carries out gene recombinaton
There are good research and application prospect；
(4) the magnetic nano-balls preparation method of the present invention is simple, and magnetic nano-balls is in aqueous phase system
It is optimum good to divide, and overcomes the existing magnetic nano-balls easily heavy shortcoming gathering, being difficult to long term storage, and
With low cost, the suitability is wide, and generalization is strong；
(5) magnetic nano-balls utilizing the present invention carries out the method operation letter of gene transfection to stem cell
Single, only need common cobalt nickel Magnet to carry out, it is not necessary to other special instrument and equipments, and transfected
Journey only needs 2～3h, substantially reduces transfection time, convenient and swift.
Accompanying drawing explanation
Fig. 1 is the design drawing of the magnetic nano-balls of a kind of clad structure layer by layer of the present invention and corresponding magnetic turns
Dye conceptual scheme；
Fig. 2 a is the transmission electron microscope observing figure of IONPs@ED-pul；
Fig. 2 b is the transmission electron microscope observing figure of IONPs@ED-pul/pDNA/SP；
Wherein, IONPs@ED-pul represents four oxidations three that ethylenediamine-pulullan polysaccharide copolymer is modified
Ferrum nanoparticle (i.e. kernel), IONPs@ED-pul/pDNA/SP represents the magnetic Nano carrying attached gene
Ball, lower same；
Fig. 3 is IONPs@ED-pul under different IONPs@ED-pul with pDNA mass ratio
Compare with the combination rate of pDNA；
Wherein, pDNA represents plasmid DNA, lower same；
Fig. 4 is IONPs@ED-pul/pDNA/SP, IONPs@ED-pul/pDNA,
The particle diameter of IONPs tri-kinds of complex of@ED-pul and surface potential analysis；
Wherein, IONPs@ED-pul/pDNA represent parcel gene kernel (i.e. kernel-intermediate layer is multiple
Compound), lower same；
Fig. 5 is the magnetic nano-balls of the clad structure layer by layer of present invention dispersive property in aqueous phase system
Analyze and magnetic responsiveness analysis；
Fig. 6 is that the magnetic nano-balls of the present invention carries out gene (PGL-3) to human marrow mesenchymal stem cell
The transfection efficiency of transfection is investigated；
Wherein, blank represents that blank, Nake pDNA represent naked pDNA direct transfection, Lipo
2000 expressions utilize non-viral gene vector Lipofectmine 2000 to carry out gene transfection, and SP represents
Non-viral gene vector spermine-pulullan polysaccharide copolymer is utilized to carry out gene transfection,
IONPs@ED-pul/pDNA/SP represent utilize the magnetic nano-balls of the present invention carry out gene transfection (
In the case of external magnetic field), IONPs@ED-pul/pDNA/SP+ magnetic field represents the magnetic utilizing the present invention
Property nanosphere carries out magnetic transfection；Lower same；
Fig. 7 is that the magnetic nano-balls of the present invention carries out gene to rat bone marrow mesenchymal stem cells
(PGL-3) transfection efficiency transfected is investigated；
Wherein, PEI represents and utilizes non-viral gene vector polymine to carry out gene transfection, lower with；
Fig. 8 is to use magnetic nano-balls after different cationic polymer modified ferriferrous oxide nano grains
Transfection efficiency is investigated；
Wherein, IONPs@SP/pDNA/SP represents that employing spermine-pulullan polysaccharide copolymer modifies four
The magnetic nano-balls prepared after Fe 3 O nanoparticle, and utilize this magnetic nano-balls without external magnetic field
In the case of human marrow mesenchymal stem cell carried out gene (PGL-3) transfection；
IONPs@SP/pDNA/SP+ magnetic field then represents right under the effect utilizing this magnetic nano-balls outside magnetic field
Human marrow mesenchymal stem cell carries out gene (PGL-3) transfection；
Fig. 9 is IONPs@ED-pul with pDNA with different quality than the turning of magnetic nano-balls after combining
Dye efficiency is investigated；
Wherein, IONPs@ED-pul/pDNA/SP (0.6), IONPs@ED-pul/pDNA/SP (1.5),
IONPs@ED-pul/pDNA/SP (3.0) represents that IONPs@ED-pul Yu pDNA is with mass ratio respectively
The magnetic nano-balls that 1:0.6,1:1.5,1:3.0 prepare after combining, and utilize corresponding magnetic nano-balls
In the case of without external magnetic field, human marrow mesenchymal stem cell is carried out gene (PGL-3) transfection；
IONPs@ED-pul/pDNA/SP (0.6)+magnetic field, IONPs@ED-pul/pDNA/SP (1.5)+magnetic
, IONPs@ED-pul/pDNA/SP (3.0)+magnetic field represents the most respectively and utilizes respective magnetic nanosphere
Under the effect of outside magnetic field, human marrow mesenchymal stem cell is carried out gene (PGL-3) transfection；
Figure 10 is that after SP and pDNA combines with different N/P ratios, the transfection efficiency of magnetic nano-balls is examined
Examine；
Wherein, SP (3:1), SP (4:1) represent that SP with pDNA is combined with N/P ratio 3:1,4:1 respectively
After, directly utilize SP and mesenchymal stem cells MSCs is carried out gene (PGL-3) transfection；
IONPs@ED-pul/pDNA/SP (3:1), IONPs@ED-pul/pDNA/SP (4:1) table respectively
Show the magnetic nano-balls that SP with pDNA prepares after being combined with N/P ratio 3:1,4:1, and utilize corresponding
Magnetic nano-balls in the case of without external magnetic field, human marrow mesenchymal stem cell is carried out gene
(PGL-3) transfection；IONPs@ED-pul/pDNA/SP (3:1)+magnetic field,
IONPs@ED-pul/pDNA/SP (4:1)+magnetic field represents the most respectively and utilizes corresponding magnetic nano-balls to exist
In the case of having external magnetic field, human marrow mesenchymal stem cell is carried out gene (PGL-3) to transfect；
Figure 11 is that the transfection efficiency of different external magnetic field strength magnetic nanosphere is investigated；
Wherein, IONPs@ED-pul/pDNA/SP+0Gs, IONPs@ED-pul/pDNA/SP+190
0Gs、IONPs@ED-pul/pDNA/SP+2700Gs、IONPs@ED-pul/pDNA/SP+3000
Gs、IONPs@ED-pul/pDNA/SP+3300Gs、IONPs@ED-pul/pDNA/SP+4000
Gs, IONPs@ED-pul/pDNA/SP+5000Gs represent respectively the magnetic nano-balls of the present invention 0,
1900, fill between rat marrow under the action of a magnetic field of 2700,3000,3300,4000,5000 Gausses
Matter stem cell carries out gene (PGL-3) transfection；
Figure 12 is that the cytotoxicity of rat bone marrow mesenchymal stem cells is investigated by different genes transfection reagent；
Wherein, IONPs represents ferriferrous oxide nano grain；
Figure 13 is the PGL-3 base in different magnetic field intensity and different transfection time magnetic nanosphere
Because of the distribution in rat bone marrow mesenchymal stem cells；
Wherein, blue-fluorescence part is nucleus, and green fluorescence part is isothiocyanate labelling
PGL-3 gene；
Figure 14 is that the ferriferrous oxide nano grain in different transfection time magnetic nanospheres is rat
Distribution in mesenchymal stem cells MSCs；
Wherein, blue dyeing part is ferriferrous oxide nano grain, and red staining part is nucleus；
Under conditions of Figure 15 is for utilizing flow cytometry under conditions of having serum-free and with or without magnetic field
That investigates magnetic nano-balls of the present invention enters born of the same parents' efficiency；
Wherein, FBS free represents and transfects under conditions of without hyclone, and 10%FBS represents
Transfect under conditions of 10% hyclone exists.
Detailed description of the invention
With detailed description of the invention, technical scheme is made the most below in conjunction with the accompanying drawings
Explanation.
The preparation of the magnetic nano-balls of embodiment 1 clad structure layer by layer
1. prepare magnetic nano-balls
(1) ED-pul of preparation is taken, with removing O2Distilled water be configured to 20mg/mL,
ED-pul aqueous solution；Take 120 μ L ferric chloride solution (100mg/mL) and add ED-pul aqueous solution
(1mL), in, after vortex dissolves, 120 μ L solution of ferrous chloride (41mg/mL) are continuously added,
Acutely vortex makes dissolving；It is subsequently adding 250 μ L strong aqua ammonia (28vol.%), is placed in 60 DEG C of water-baths
20min；The suspension obtained is at PD-10 (GE Healthcare Bio-Sciences Corp.) short column
Upper distilled water eluting removes unnecessary ammonia, can obtain and be modified by cationic polymer ED-pul
Ferriferrous oxide nano grain, i.e. kernel (hereinafter referred to as IONPs@ED-pul)；
(2) with 5% aqueous sucrose solution, IONPs@ED-pul after purification is diluted to 250 μ g/mL,
Take 100 μ L, be added thereto to double volume pDNA (100 μ g/mL) (use PGL-3 gene,
Expression luciferin albumen, No. Genbank: HE650697.1), hatch 20min altogether under room temperature shaking,
Obtain kernel-interlayer composite (hereinafter referred to as IONPs@ED-pul/pDNA)；
(3) in IONPs@ED-pul/pDNA, add the SP (250 μ g/mL) of 200 μ L,
Hatch 15min under room temperature altogether, the magnetic nano-balls of clad structure layer by layer can be obtained (hereinafter referred to as
IONPs@ED-pul/pDNA/SP)。
The structural representation of IONPs@ED-pul/pDNA/SP is as shown in Figure 1.
2. transmission electron microscope observing
After respectively IONPs@ED-pul, IONPs@ED-pul/pDNA/SP being diluted in right amount, slowly
Drip on the copper mesh being covered with carbon film, utilize transmission electron microscope (TEM, JEM100) observe its form and
Particle diameter；Observed result is shown in Fig. 2 a and Fig. 2 b respectively.
It is that diameter is at 10nm from the ferriferrous oxide nano grain of Fig. 2 a, IONPs@ED-pul
The spherical little granule of class of left and right.
After successively being hatched from Fig. 2 b, IONPs@ED-pul Yu pDNA and SP, finally can shape
A diameter of about 100nm, internal package is become to have the magnetic Nano of several ferriferrous oxide nano grains
Ball (IONPs@ED-pul/pDNA/SP).
3.DNA precipitation experiments
DNA precipitation experiments is used to confirm IONPs@ED-pul/pDNA/SP prepared by the present embodiment
In, whether pDNA layer is coated on IONPs@ED-pul outer layer.
By IONPs@ED-pul and pDNA by different mass ratio mixing, after hatching 20min,
12,000rpm are centrifuged 15min, take supernatant, measure the ultraviolet absorptivity at 260nm, meter
Count unprecipitated pDNA concentration in clear liquid in, and calculate the knot of IONPs@ED-pul and pDNA
Conjunction rate.Result sees Fig. 3.
As it is shown on figure 3, along with the increase of IONPs@ED-pul, the combination of pDNA is consequently increased,
Prompting pDNA is incorporated in the surface of IONPs@ED-pul.As IONPs@ED-pul and pDNA
Mass ratio when being 3:1, its combination rate has reached 95.0%.
4. particle diameter and potential change are investigated
Use particle size and surface potential detection instrument (3000HSA, Malvern Co.) at 25 DEG C
Measure from IONPs@ED-pul to IONPs@ED-pul/pDNA again to SP/pDNA/IONPs
The hydraulic radius of@ED-pul and surface potential change.
Take appropriate above-mentioned complex respectively, after 10mM neutrality PBS (pH 7.4) in right amount dilution,
Dynamic light scattering method (DLS) is used to measure particle diameter.Meanwhile, utilize electrophoretic light scattering method (ELS),
Under electric field intensity is 100V/cm, measure and calculate the surface potential (Smolu-chouski of nanoparticle
equation).Testing result is shown in Fig. 4.
As shown in Figure 4, the particle diameter after IONP@ED-pul disperses in water is 90nm, this with
Swelling relevant in water of ED-pul.Surface charge is about 10mV.
And the particle diameter of IONPs@ED-pul/pDNA increases to about 250nm, surface charge is reduced to
About-40mV.Understand in conjunction with the experimental result of DNA precipitation in (3), this is because pDNA quilt
It is bound to IONPs@ED-pul outer layer, causes IONPs@ED-pul/pDNA particle diameter to become big, surface
Current potential is reversed into negative charge.
And the particle diameter of SP/pDNA/IONPs@ED-pul is about 200nm, surface is the most positively charged,
It is about about 10mV.This is because after SP is attached to IONPs@ED-pul/pDNA outer layer, can
Overlay on the pDNA of IONPs@ED-pul outer layer with compressed package, and it is positively charged to ultimately form surface
Magnetic nano-balls.
Summary (2), (3), (4) result can confirm that composite nano-granule prepared by the present invention is
As shown in Figure 1 with some IONPs ED-pul as kernel, middle cladding pDNA, outermost layer is
The clad structure layer by layer of SP polymer.
5. magnetic responsiveness test
After the magnetic nano-balls sample distilled water prepared by the present embodiment is diluted to suitable concentration, uniformly
It is divided into two parts.A copy of it is placed in nickel cobalt Magnet (magnetic field intensity is 5000Gs) limit, and another part is remote
Stand from magnetic field.Wait standing after 72h, observe and the magnetic nano-balls that compares in two parts of aqueous suspensions is to magnetic
The response of field.Observed result such as Fig. 5.
As shown in Figure 5, magnetic nano-balls prepared by the present invention can in water good dispersion, stand 72h
After also without any heavy poly-.Meanwhile, magnetic field responsiveness is good, is suitable as magnetic transfection reagent.
The antiserum efficiency gene transfection of embodiment 2 magnetic nano-balls and toxicity assessment
1. couple BMSC carries out magnetic transfection
As a example by people source BMSC and rat BMSC, to present invention antiserum base on BMSC
Because magnetic transfection efficiency is evaluated.The present embodiment is provided with blank group (blank), feminine gender simultaneously
Matched group (Nake pDNA, pDNA direct transfection), non-viral gene vector matched group (Lipo 2000
Group or PEI group, and SP group), without magnetic field matched group.
Specifically comprise the following steps that
1) by people source BMSC or rat BMSC with 6 × 104Individual/hole is seeded on 24 orifice plates, puts
In 5%CO2In cell culture incubator, at 37 DEG C, cultivate 24h；
2) discarding culture fluid, rinse twice with phosphate buffer (pH 7.4), every hole adds 0.5mL
DMEM low sugar culture fluid containing 10% hyclone, will prepare in embodiment 1
IONPs@ED-pul/pDNA/SP is added by every hole 40 μ L after diluting with 5% sucrose water (300 μ L)
Enter；Tissue Culture Plate is placed on cobalt nickel Magnet (magnetic field intensity 3300Gs), at 5%CO2, 37 DEG C
Lower cultivation 3h；
3) move demagnetizing field, and discard culture fluid, be again replaced by the DMEM containing 10% hyclone
Low sugar culture fluid, at 5%CO2, continue at 37 DEG C to cultivate 48h；
4) discarding culture fluid, rinse twice with phosphate buffer (pH 7.4), every hole adds 0.2mL
Reporter gene cell pyrolysis liquid, shakes 30min under room temperature, centrifugal under the conditions of 14,000rpm, 4 DEG C
20min；
5) draw 0.1mL supernatant, add equal-volume luciferin zymolyte under Chemiluminescence Apparatus
Measure the relative light intensity (RLU) of each experimental group；
6) take the protein standards liquid that concentration is 0.5mg/mL, be diluted to series with distilled water respectively
Concentration: 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL,
500 μ g/mL, are added to 96 orifice plates by every hole 20 μ L, then add BCA by every hole 200 μ L
Working solution, detects absorbance, draws standard curve at microplate reader 595nm wavelength；
7) take experimental group each hole supernatant 20 μ L, add 200 μ L BCA working solutions in 595nm
Detect absorbance at wavelength, utilize standard curve to calculate protein concentration；
8) luciferase of the BMSC after calculating transfection PGL-3 reporter gene as follows is lived
Property (enzymatic activity is the highest, illustrates that corresponding transfection efficiency is the highest):
People BMSC and rat BMSC luciferin Enzyme assay result see Fig. 6 and Tu respectively
7。
From Fig. 6 and Fig. 7, can be people with the help of the magnetic nano-balls outside magnetic field of the present invention
Obtain good antiserum efficiency gene transfection on BMSC and rat BMSC, compare the most conventional
Non-viral gene transfection reagent such as PEI or Lipofectmine 2000 there is more preferable antiserum gene
Transfection advantage.
The most different cationic polymer modified ferriferrous oxide nano grains are to magnetic nano-balls transfection efficiency
Impact
The method using the present embodiment part 1, investigates different cationic polymer modified four oxidations three
The impact on magnetic nano-balls transfection efficiency of the ferrum nanoparticle, result is as shown in Figure 8.
As seen from Figure 8, compare the magnetic nano-balls prepared with the IONPs kernel of SP modification, with
Magnetic nano-balls prepared by the IONPs kernel that ED-pul the modifies transfection efficiency on BMSC will be more
High (about 3 times), but no difference of science of statistics between the two.
3. the mass ratio of kernel and the pDNA impact on magnetic nano-balls transfection efficiency
The method using the present embodiment part 1, investigates the different kernel mass ratio with pDNA to magnetic
The impact of property nanosphere transfection efficiency, result sees Fig. 9.
As seen from Figure 9, when the mass ratio of IONPs@ED-pul kernel Yu pDNA is 1:1.5,
Magnetic nano-balls is the highest to the transfection efficiency of people BMSC, be mass ratio be 4 times during 1:0.6, be
Mass ratio is 5.7 times during 1:3.0.
When the mass ratio of IONPs@ED-pul kernel and pDNA is 1:3.0, although both combinations
Rate is the highest, and pDNA is fully wrapped in outside kernel, but is likely to be due to too wrap up be unfavorable for pDNA
Intracellular release, thus transfection efficiency reduces on the contrary.
The impact on magnetic nano-balls transfection efficiency of the N/P ratio of 4.SP Yu pDNA
The method using the present embodiment part 1, investigates the N/P ratio of different SP Yu pDNA to magnetic
The impact of property nanosphere transfection efficiency, result sees Figure 10.
As shown in Figure 10, when the N/P ratio of SP Yu pDNA is 4:1, the magnetic nano-balls of preparation
The transfection efficiency of optimum can be obtained on people BMSC；Transfection efficiency now be N/P ratio be 3:1
Time 2 times.
5. the external magnetic field strength impact on magnetic nano-balls transfection efficiency
The method using the present embodiment part 1, investigates different external magnetic field strength and turns magnetic nano-balls
The impact of dye efficiency, result sees Figure 11.
As shown in figure 11, when between magnetic field intensity is at 2700Gs to 4000Gs, magnetic nano-balls
The transfection efficiency of optimum can be obtained on rat BMSC.Magnetic field intensity is the most weak or too strong the most unfavorable
In obtaining preferable transfection efficiency.
The toxicity assessment to mesenchymal stem cells MSCs of 6 magnetic nano-balls is tested
As a example by the BMSC of rat source, use magnetic nano-balls pair prepared by the MTS method detection present invention
The toxicity of stem cell.The present embodiment is provided with non-viral gene vector matched group (Lipo 2000 simultaneously
Group, PEI group, and SP group), IONPs matched group, without magnetic field matched group.
Specifically comprise the following steps that
1) by BMSC with 6 × 104Individual/hole is inoculated on 24 orifice plates, in 5%CO2, train at 37 DEG C
Support 24h；
2) discarding culture fluid, phosphate buffer (pH 7.4) cleans twice, and every hole adds 0.5mL
In DMEM low sugar culture fluid containing 10% hyclone and 40 μ L embodiments 2 after dilution
IONPs@ED-pul/pDNA/SP；Tissue Culture Plate is placed on cobalt nickel magnetic field (magnetic field intensity is
3300Gs), in 5%CO2, at 37 DEG C, cultivate 3h；
3) move demagnetizing field, and discard culture fluid, be replaced by the DMEM low sugar containing 10% hyclone
Culture fluid, continues to cultivate 48h；
4), after rinsing twice with phosphate buffer (pH 7.4), every hole adds 400 μ L fresh cultured
Liquid and 100 μ L MTS examination, in 37 DEG C, 5%CO2Under hatch 4h；
5) microplate reader measures absorbance (OD value) under 570nm.
6) survival rate of BMSC is calculated as follows to evaluate cytotoxicity, result such as Figure 12
Shown in.
As seen from Figure 12, the magnetic nano-balls of the present invention and corresponding rotaring redyeing system are to rat BMSC
All without obvious cytotoxicity.
Shown by the experiment of efficiency gene transfection and Cytotoxic evaluation, utilize prepared by the present invention based on
The rotaring redyeing system of magnetic material has higher efficiency gene transfection to BMSC, and can antagonistic Serum pair
The impact of gene transfection, has relatively low cytotoxicity simultaneously to stem cell.
The magnetic transfection mechanism of embodiment 3 magnetic nano-balls of the present invention
As a example by IONPs@ED-pul/pDNA/SP, use the pDNA of isothiocyanate labelling
(FITC-PGL-3) pDNA and IONPs kernel, is investigated in magnetic nano-balls at rat BMSC
In distribution.Specifically comprise the following steps that
1) by BMSC with 2 × 104Individual/hole is inoculated in the culture vessel with glass bottom of a diameter of 20mm, in
5%CO2, at 37 DEG C, cultivate 24h；
2) discard culture fluid, rinse twice with phosphate buffer (pH 7.4), add containing 10% tire
The DMEM low sugar culture fluid of Ox blood serum and by preparing in embodiment 2 and diluting
IONPs@ED-pul/pDNA/SP；Tissue Culture Dish is placed on cobalt nickel magnetic field (magnetic field intensity difference
For 2700Gs and 5000Gs), 5%CO2, at 37 DEG C, hatch different time (0.5h, 2h, 4 respectively
H and 6h)；
3) at corresponding time point, move demagnetizing field, and discard culture fluid, phosphate buffer (pH 7.4)
Rinse 3 times, add-20 DEG C of ice methanol and fix 15min；
4) adding DAPI core transfection reagent to dye nucleus, dyeing time is 8min；Then
Phosphate buffer (pH 7.4) rinses 3 times；
5) laser scanning co-focusing microscope (BX61W1-FV1000, Olympus) observes pDNA
In intracellular distribution, result is as shown in figure 13；
6) simultaneously, by above-mentioned 1) and 2) step operation, and (magnetic field intensity is 2700 under magnetic field
Gs) transfection 3h；
7) 0h, 27h and 51h after moving demagnetizing field (i.e. add IONPs@ED-pul/pDNA/SP
After 3h, 24h and 48h) discard the culture fluid containing IONPs@ED-pul/pDNA/SP respectively,
After phosphate buffer (pH 7.4) rinses 3 times, add 4% paraformaldehyde and fix 20min；Then
Add the 1mL Prussian blue test solution containing 5% potassium ferrocyanide and 10% hydrochloric acid at room temperature to dye 1h；
Phosphate buffer (pH 7.4) adds PI core dye liquor dyeing 5min after rinsing 3 times, use phosphorus the most again
Acid buffer (pH 7.4) rinses 3 times；
8) under fluorescence microscope, (Eclipse Ti-s, Nikon) observes ferriferrous oxide nano grain at cell
Interior distribution, result is as shown in figure 14.
As seen from Figure 13, during transfection 6h, under strong magnetic field action, the dyeing of FITC-PGL-3 is relatively
Deeply；And under general magnetic field, in nucleus, the dyeing of FITC-PGL-3 is the most shallower；Without the action of a magnetic field
Under, in nucleus, the dyeing of FITC-PGL-3 has no the most substantially.This shows magnetic prepared by the present invention
It is highly efficient and enter rapidly that nanosphere can carry pDNA under the effect in the magnetic field of suitable intensity
Enter intracellular, so improve pDNA enter core efficiency, finally put forward the efficiency of high gene transfection.
As shown in figure 14, to combine Figure 13 visible, pDNA mainly in Cytoplasm by magnetic nano-balls
Release, from being advanced into nucleus after release, kernel IONPs ED-pul then will not enter nucleus.
The antiserum transfection mechanism of embodiment 4 magnetic transfection reagent
As a example by IONPs@ED-pul/pDNA/SP, use the pDNA of isothiocyanate labelling
(FITC-PGL-3), utilize flow cytometry to investigate magnetic transfection reagent respectively and have serum and serum-free
Under the conditions of and have magnetic field (3300Gs) and without entering born of the same parents' efficiency under magnetic field condition.Concrete operations walk
Rapid as follows:
1) by BMSC by 5 × 105Individual/hole is inoculated on 6 orifice plates, in 5%CO2, train at 37 DEG C
Support 24h；
2) discarding culture fluid, phosphate buffer (pH 7.4) cleans twice, and every hole adds 2mL and contains
The DMEM low sugar culture fluid of 10% hyclone or the DMEM low sugar culture fluid without serum,
Then the IONPs@ED-pul/pDNA/SP after 160 μ L dilutions is added by the every hole of embodiment 2；
3) Tissue Culture Plate is placed on cobalt nickel magnetic field (magnetic field intensity is 3300Gs), as experiment
Group, is not interposing at the Tissue Culture Plate in magnetic field as a control group, in 5%CO2, at 37 DEG C, hatch difference
Time (0.5h, 1.5h, 3h)；
4) in above-mentioned different time points, each group is digested with the trypsin solution containing 0.02%EDTA respectively
Cell, is collected in streaming pipe, uses the positive rate of Flow cytometry FITC-pDNA cell；
Result is as shown in figure 15.
Flow cytometer detection result shows, under without magnetic field condition, magnetic nano-balls prepared by the present invention can
Slowly absorbed by BMSC.Under serum-free condition, after hatching 3h, the born of the same parents that enter of magnetic nano-balls are imitated
Rate is up to 98.2%；But under serum existence condition, magnetic nano-balls enter born of the same parents' efficiency and enter born of the same parents speed
Degree all than serum-free time low.
But under the existence condition of magnetic field, no matter serum presence or absence, do not affect magnetic prepared by the present invention
Property nanosphere quickly enters born of the same parents.Under the action of a magnetic field, when hatching 0.5h, magnetic nano-balls enters born of the same parents' efficiency
I.e. close to 100%.
Thus proving, magnetic nano-balls prepared by the present invention can carry pDNA and efficiently and rapidly enter
In stem cell, do not affected by serum presence or absence simultaneously.Therefore, it can be used as stem cell
Antiserum high efficiency gene transfection reagent.
Claims (10)
1. the magnetic nano-balls of a clad structure layer by layer, it is characterised in that by kernel, intermediate layer
Form with outer layer, all by electrostatic attraction effect between kernel and intermediate layer, between intermediate layer and outer layer
It is combined with each other；
Described kernel is cationic polymer modified ferriferrous oxide nano grain, and described intermediate layer is bag
Overlaying on the gene to be transfected outside kernel, described outer layer is to be coated on the spermine-Pu Lu outside intermediate layer
Blue polysaccharide copolymer.
2. the magnetic nano-balls of clad structure the most layer by layer as claimed in claim 1, it is characterised in that institute
The particle diameter stating ferriferrous oxide nano grain is 10～50nm, and described cationic polymer is with electronegative
Ferriferrous oxide nano grain be combined with each other by electrostatic attraction effect.
3. the magnetic nano-balls of clad structure the most layer by layer as claimed in claim 1 or 2, it is characterised in that
Described cationic polymer be ethylenediamine-pulullan polysaccharide copolymer, spermine-pulullan polysaccharide copolymer,
Polymine, chitosan or spermine-dextran copolymer.
4. the magnetic nano-balls of clad structure the most layer by layer as claimed in claim 1 or 2, it is characterised in that
In described spermine-pulullan polysaccharide copolymer, the mean molecule quantity of pulullan polysaccharide is 47300Da,
Spermine percent grafting is 12%～14%.
5. the preparation method of the magnetic nano-balls of clad structure layer by layer as described in Claims 1 to 4 is arbitrary,
It is characterized in that, including:
(1) use coprecipitation to prepare cationic polymer modified ferriferrous oxide nano grain, obtain
Obtain kernel；
(2) described kernel is joined in cdna solution to be transfected, incubate altogether under the conditions of room temperature, shaking
Educate 20～30min, it is thus achieved that kernel-interlayer composite；
(3) in described kernel-interlayer composite, spermine-pulullan polysaccharide copolymer, room temperature are added
Hatch 15～20min the most altogether, it is thus achieved that the magnetic nano-balls of described clad structure layer by layer.
6. preparation method as claimed in claim 5, it is characterised in that in step (2), kernel
It is less than 1:1 with the mass ratio of gene to be transfected.
7. preparation method as claimed in claim 5, it is characterised in that in step (3), spermine
-pulullan polysaccharide copolymer with the N/P ratio of gene to be transfected in kernel-interlayer composite is
3:1～5:1.
8. as described in Claims 1 to 4 is arbitrary, the magnetic nano-balls of clad structure turns preparing magnetic layer by layer
Application in transfection reagent.
Apply the most as claimed in claim 8, it is characterised in that described magnetic transfection reagent is for right
Stem cell carries out gene transfection.
10. a magnetic transfecting formulations, it is characterised in that by as described in Claims 1 to 4 is arbitrary layer by layer
The magnetic nano-balls of clad structure is prepared from.
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| CN115429902A (en) * | 2021-06-04 | 2022-12-06 | 中南大学 | Magnetic ferric oxide cell marker material and preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513537A (en) * | 2017-08-11 | 2017-12-26 | 浙江大学 | A kind of ferromagnetic nano carrier material/gene composite and its preparation method and application |
| CN107519501A (en) * | 2017-08-11 | 2017-12-29 | 浙江大学 | A kind of ferromagnetic nano material and preparation method and application |
| CN107513537B (en) * | 2017-08-11 | 2019-12-31 | 浙江大学 | Ferromagnetic nano carrier material/gene compound and preparation method and application thereof |
| CN107519501B (en) * | 2017-08-11 | 2020-12-29 | 浙江大学 | Ferromagnetic nano material, preparation method and application |
| CN108148868A (en) * | 2017-12-29 | 2018-06-12 | 佛山科学技术学院 | A kind of ferroso-ferric oxide-carboxymethyl cellulose-polyethylene imine nanometer Complex Gene carrier and preparation method thereof |
| CN109651811A (en) * | 2018-11-26 | 2019-04-19 | 宋文星 | Magnetic SF/PEI nano particle and application thereof |
| CN115429902A (en) * | 2021-06-04 | 2022-12-06 | 中南大学 | Magnetic ferric oxide cell marker material and preparation and application thereof |
| CN115429902B (en) * | 2021-06-04 | 2023-08-18 | 中南大学 | Magnetic ferric oxide cell marking material and preparation and application thereof |
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