CN105802998B - A kind of magnetic nano-balls and its preparation method and application of clad structure layer by layer - Google Patents
A kind of magnetic nano-balls and its preparation method and application of clad structure layer by layer Download PDFInfo
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Abstract
The invention discloses a kind of magnetic nano-balls and its preparation method and application of clad structure layer by layer.The magnetic nano-balls are made of kernel, middle layer and outer layer, are be combined with each other by electrostatic attraction effect between kernel and middle layer, between middle layer and outer layer；Kernel is cationic polymer modified ferriferrous oxide nano grain, and middle layer is the gene to be transfected being coated on the outside of kernel, and outer layer is the spermine-pulullan polysaccharide copolymer being coated on the outside of middle layer.The magnetic nano-balls are first in conjunction with kernel by gene to be transfected, then in conjunction with spermine-pulullan polysaccharide copolymer, so that gene to be transfected is tightly wrapped in magnetic nano-balls, gene are avoided to be degraded in transfection process；Spermine-pulullan polysaccharide can identify and be absorbed by mesenchymal stem cell, and further increase magnetic nano-balls enters born of the same parents' efficiency；Spermine-pulullan polysaccharide copolymer effectively reduces transfection security risk caused by stem cell to mesenchymal stem cell small toxicity.
Description
Technical field
The present invention relates to a kind of magnetic transfecting formulations, and in particular to a kind of magnetic nano-balls of clad structure and its preparation layer by layer
Methods and applications.
Background technique
Some stem cells, as mesenchymal stem cell (Bone-marrow mesenchymal stem cell,
BMSC), due to the potential with Multidirectional Differentiation, and the features such as be easily obtained and isolate and purify, 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 application, generally require to carry out suitable gene modification to stem cell, with induction
Stem cell more specific can be divided into required cell or express specific therapeutic gene to preferably play in vivo
Corresponding therapeutic effect.
Currently, the Gene transfer vector for stem cell gene recombination mainly has viral genetic vector and non-viral gene to carry
Two class of body.But there are biggish security risks for application of the viral vectors on stem cell, such as cause insertion mutation, activation carcinogenophore
Because, lead to cell differentiation and immunogenicity etc., these risks also limit the stem cell through viral vector gene recombination and are facing
Extensive use on bed.Though risk of the non-viral gene vector in clinical application well below viral genetic vector, its
Lower transfection efficiency, which but becomes, on stem cell limits their main bottlenecks to clinical application.On the one hand, grinding in recent years
Study carefully the simple non-viral gene vector of result certification structure to be difficult that gene is helped to pass through from extracellularly to intracellular all barriers
To realize the high efficient expression of gene.Some commercially available non-viral gene transfection reagents such as polyethyleneimine (PEI),
Lipofectmine2000 (Lipo2000), ExGene 500 or SuperFect etc. are also proved to be used for stem cell gene
When transfection, the problems such as there is transfection efficiency is lower or apparent cytotoxicity.On the other hand, due to negatively charged in serum
Albumen can in conjunction with positively charged non-viral gene vector, cause carrier/gene composite enter born of the same parents' efficiency decline and most
Transfection efficiency is influenced eventually.Therefore, most non-viral gene vectors are required carries out corresponding gene transfection under serum-free condition,
Interference to avoid serum to transfection efficiency.However, stem cell is very sensitive to culture environment, serum-free transfection often be will increase
Non-viral gene transfection reagent influences the state of stem cell to the toxicity of stem cell.So how to make non-viral gene vector
It still keeps efficient transfection efficiency to become under serum existence condition and designs and prepares process for current non-viral gene vector
In required for a critical issue overcoming.
Magnetic transfection is a kind of means that can potentially effectively improve non-viral gene vector transfection efficiency on stem cell.Magnetic
Transfection is by magnetic-particle (such as ferroso-ferric oxide Fe3O4Nanoparticle) and certain large biological molecules pass through chemical covalent bonds or physics
Adhesive attraction combines, and forms the particle with magnetic responsiveness, then utilizes the surface-active of magnetic particle and traditional virus
Or non-virus carrier coupling, then combined with target gene or magnetic particle is directly combined with target gene, it constitutes and carries attached base
The magnetic nano-balls of cause.Under the action of externally-applied magnetic field, the aggregation that magnetic nano-balls show in cell membrane can be significantly improved, and
It helps magnetic nano-balls are highly efficient to enter into the cell, and then discharges target gene, realize the purpose of high efficiency gene transfection.
Magnetic rotaring dyeing technology can help non-viral gene vector to overcome the barrier of cell membrane by external magnetic field, so that carrying
It is more efficiently intracellular from extracellular entrance that physical efficiency carries target gene, to effectively improve the efficiency of gene transfection.Simultaneously as
The born of the same parents that enter of carrier rely primarily on external magnetic to complete, and have no effect on cell endocytic intake mechanism, therefore can overcome serum pair
The influence of transfection process.
But current magnetic transfection reagent is substantially for tumour cell design, is specially designed for stem cell
Magnetic transfection reagent is actually rare.All corresponding magnetic is transfected this is because stem cell is transfecting to the sensibility and hardly possible of environment
Preparation proposes very high design requirement.Compared with tumour cell, stem cell has high selectivity to endocytosis object, and new old
Metabolism is also relatively slow, is not easy to be transfected.Therefore, it is imperative that magnetic transfecting formulations are specially designed for stem cell, and are had very
Important scientific research value and clinical treatment meaning.
Summary of the invention
The present invention provides a kind of magnetic nano-balls for coating (layer by layer) structure layer by layer, the magnetic nano-balls
It solves magnetic transfection reagent in the prior art to be difficult to use in the effective base of the stem cells such as mesenchymal stem cell progress
Because of the problem of transfecting.
A kind of magnetic nano-balls of clad structure layer by layer, are made of kernel, middle layer and outer layer, kernel and middle layer it
Between, be combined with each other by electrostatic attraction effect between middle layer and outer layer；
The kernel is cationic polymer modified ferriferrous oxide nano grain, and the middle layer is to be coated on outside kernel
The gene to be transfected of side, the outer layer are the spermine-pulullan polysaccharide copolymer being coated on the outside of middle layer.
The magnetic nano-balls of the invention ferriferrous oxide nano grain that gene to be transfected is first and cationic polymer modified
In conjunction with then again in conjunction with spermine-pulullan polysaccharide copolymer, so that gene to be transfected is tightly wrapped in magnetic nano-balls
In, avoid gene to be transfected from being degraded in transfection process.
After spermine-pulullan polysaccharide copolymer is as outer layer covers gene to be transfected, moreover it is possible to it is effectively compressed gene to be transfected,
It is maintained at the average grain diameter of magnetic nano-balls and surface potential in suitable safe range.Meanwhile spermine-pulullan polysaccharide is total
The modification of polymers makes magnetic Nano ball surface with suitable positive charge, and suitable charge repulsion makes magnetic nano-balls in water phase
It is well dispersed in system, it is not easy heavy poly-.
The average grain diameter of magnetic nano-balls of the present invention is 100~200nm (suitable safe range is 50~300nm), if
Partial size is too big, is unfavorable for effective intake of the cell to magnetic nano-balls, if partial size is too small, since dimensional effect can produce cell
Raw certain cytotoxicity.The surface potential of magnetic nano-balls of the present invention is 10~20mV (safe range is 10~40mV), if
Surface potential is too low, and magnetic nano-balls are difficult to effectively combine and compress gene to be transfected, if surface potential is too high, magnetic nano-balls
Cell membrane can be destroyed, serious cytotoxicity is generated to cell.
It has also been found that, spermine-pulullan polysaccharide copolymer effectively can be identified and be absorbed by stem cell (such as essence simultaneously
Amine-pulullan polysaccharide copolymer can by CD44 Receptor recognition that mesenchymal stem cell shows and absorb), further increase magnetic
Property nanosphere enters born of the same parents' efficiency, and gene to be transfected is smoothly transferred in stem cell；Also, spermine-pulullan polysaccharide copolymer
To stem cell small toxicity, transfection security risk caused by stem cell is effectively reduced.
After magnetic nano-balls enter born of the same parents, the unsaturated amino on outer layer spermine-pulullan polysaccharide copolymer can chelate molten
The proton provided in enzyme body by proton pump (V-ATPase), proton pump continuous openness, each proton will lead to a chloride ion
In a hydrone retention lysosome, cause lysosome swelling rupture, i.e., " proton sponge pump efficiency is answered ".Finally make magnetic Nano
The gene that ball is carried can be escaped from lysosome, be discharged, and enter nucleus.
Without selection, gene to be transfected can be various the size and type that magnetic nano-balls of the invention treat rotaring redyeing gene
Functional gene needed for reporter gene, all kinds of tests and functional mRNA, siRNA etc..
To guarantee that magnetic nano-balls kernel has good magnetic responsiveness, preferably, the ferroso-ferric oxide (Fe3O4)
The partial size of nanoparticle is that 10~50nm, the cationic polymer and negatively charged ferriferrous oxide nano grain are inhaled by electrostatic
The effect of drawing be combined with each other.
As further preferred, the partial size of the ferriferrous oxide nano grain is 20~50nm；Ferroso-ferric oxide is received at this time
The grain of rice is the nanoparticle of superparamagnetism, and external magnetic field has preferable magnetic responsiveness.
Diameter and no requirement (NR) of the present invention to kernel, only need to be by the diameter control of final magnetic nano-balls in respective range
?.
Preferably, the cationic polymer is ethylenediamine-pulullan polysaccharide copolymer (ED-pul), spermine-Pu Lu
Blue polysaccharide copolymer (SP), polyethyleneimine (PEI), chitosan or spermine-dextran copolymer.More preferably ethylenediamine-
Pulullan polysaccharide copolymer or spermine-pulullan polysaccharide copolymer.
The degree of polymerization and molecular weight equal no requirement (NR) of the present invention to each cationic polymer, due to ferroso-ferric oxide of the invention
The surface potential of nanoparticle is negative electrical charge, as long as therefore cationic polymer can make the surface potential positive charge of kernel.
Using the magnetic nano-balls obtained after each cationic polymer modified ferriferrous oxide nano grain to medulla mesenchyma
The transfection efficiency of stem cell respectively has height, but generally and no difference of science of statistics.
Preferably, the average molecular weight of pulullan polysaccharide is 47300Da in the spermine-pulullan polysaccharide copolymer,
Spermine grafting rate is 12%~14%.When selecting the spermine-pulullan polysaccharide copolymer, magnetic nano-balls turn stem cell
It is best to contaminate efficiency.
The present invention also provides the preparation method of the magnetic nano-balls of the clad structure layer by layer, which includes:
(1) cationic polymer modified ferriferrous oxide nano grain is prepared using coprecipitation, obtains kernel；
(2) kernel is added in cdna solution to be transfected, is incubated for 20~30min altogether under the conditions of room temperature, shaking, obtains
Obtain kernel-interlayer composite；
(3) spermine-pulullan polysaccharide copolymer is added in Xiang Suoshu kernel-interlayer composite, is incubated for 15 altogether under room temperature
~20min obtains the magnetic nano-balls of the clad structure layer by layer.
Preferably, the mass ratio of kernel and gene to be transfected is less than 1:1 in step (2).Under the mass ratio, kernel
Surface can sufficiently be wrapped up by gene to be transfected, to make kernel-interlayer composite surface potential negative electrical charge, with after an action of the bowels
Continuous outer layer can effectively be combined with middle layer.
As further preferred, in step (2), the mass ratio of kernel and gene to be transfected is 1:1.5.In the mass ratio
Under, magnetic nano-balls are best to the transfection efficiency of mesenchymal stem cell.
Preferably, the incubation time in step (2) is 20min, reasonable incubation time ensures that kernel-middle layer is compound
Object stabilization is formed.
The magnetic nano-balls obtained under different N/P ratios have different transfection efficiencies to cell, preferably, step (3)
In, the N/P ratio of spermine-pulullan polysaccharide copolymer and gene to be transfected in kernel-interlayer composite is 3:1~5:1；More
Preferably 4:1.Under the N/P ratio, magnetic nano-balls are best to the transfection efficiency of cell.
The present invention also provides the magnetic nano-balls of the clad structure layer by layer to prepare the application in magnetic transfection reagent.
The present invention also provides a kind of magnetic transfecting formulations, are prepared by the magnetic nano-balls of the clad structure layer by layer.
Magnetic nano-balls of the invention can be used for carrying out various types of cells gene transfection as magnetic transfecting formulations, including swollen
Oncocyte and stem cell.
Preferably, the magnetic transfection reagent is used to carry out gene transfection to mesenchymal stem cell.
The present invention also provides the magnetic nano-balls to carry out magnetic to mesenchymal stem cell under serum existence condition
The method of transfection, this method comprises:
(1) mesenchymal stem cell is inoculated on tissue culture plate, in 5%CO2, cultivate for 24 hours at 37 DEG C；
(2) culture solution in hole every on tissue culture plate is replaced with into 500 μ L and contains magnetic nano-balls (40 μ L) and 10%
Tissue culture plate is placed on the magnetic field that intensity is 1000~5000Gs, in 5%CO by the fresh medium of serum2, at 37 DEG C after
It is continuous to be incubated for 2~3h；
(3) culture solution in tissue culture plate is replaced with into the fresh medium containing 10% serum, removes magnetic field,
5%CO2, continue to be incubated for 48h at 37 DEG C, measure transfection efficiency.
The magnetic field can be provided using common cobalt nickel magnet, but magnetic field strength it is too weak or be all unfavorable for by force very much obtaining compared with
Good transfection efficiency, preferably, being placed in tissue culture plate on the magnetic field that intensity is 2700~4000Gs in step (2).?
Under the magnetic field strength, magnetic nano-balls have good transfection efficiency to mesenchymal stem cell.
Compared with prior art, the invention has the benefit that
(1) the magnetic nano-balls of the invention ferriferrous oxide nano that gene to be transfected is first and cationic polymer modified
Burl closes, then again in conjunction with spermine-pulullan polysaccharide copolymer, so that gene to be transfected is tightly wrapped in magnetic nano-balls
In, avoid gene to be transfected from being degraded in transfection process；
(2) after spermine-pulullan polysaccharide copolymer is as outer layer covers gene to be transfected, moreover it is possible to be effectively compressed base to be transfected
Cause makes the average grain diameter of magnetic nano-balls and surface potential keep in safe range, it is effective not only contributing to magnetic nano-balls
In conjunction with protection gene to be transfected, is conducive to cell and magnetic nano-balls are effectively absorbed, and there is lower cell toxicant
Property, it is comparatively safe；
(3) spermine-pulullan polysaccharide copolymer utilization enables magnetic nano-balls to be known by mesenchymal stem cell
It does not absorb and effectively, further increase magnetic nano-balls enters born of the same parents' efficiency, and gene to be transfected is smoothly transferred to medulla mesenchyma
In stem cell；Also, spermine-pulullan polysaccharide copolymer effectively reduces transfection pair to mesenchymal stem cell small toxicity
Security risk caused by mesenchymal stem cell can be realized and carry out high-efficiency low-toxicity to stem cell under serum existence condition
Gene transfection provides good transfection carrier to carry out genetic recombination to stem cell using non-viral gene vector, has good
Good research and application prospect；
(4) magnetic nano-balls preparation method of the invention is simple, and it is benign good that magnetic nano-balls divide in aqueous phase system, overcomes
Existing magnetic nano-balls be easy it is heavy gather, be not easy the shortcomings that long term storage, and low in cost, applicability is wide, and generalization is strong；
(5) method for carrying out gene transfection to stem cell using magnetic nano-balls of the invention is easy to operate, only needs common
Cobalt nickel magnet can carry out, the instrument and equipment special without other, and transfection process only needs 2~3h, substantially reduces transfection
It is time, convenient and efficient.
Detailed description of the invention
Fig. 1 is the present invention a kind of design drawing of the magnetic nano-balls of clad structure and corresponding magnetic transfection procedure figure layer by layer；
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 indicates ethylenediamine-pulullan polysaccharide copolymer modification ferriferrous oxide nano grain (i.e.
Kernel), IONPs@ED-pul/pDNA/SP indicates to carry the magnetic nano-balls of attached gene, similarly hereinafter；
Fig. 3 is the Percentage bound ratio of IONPs@ED-pul and pDNA at different IONPs@ED-pul and pDNA mass ratio
Compared with；
Wherein, pDNA indicates Plasmid DNA, similarly hereinafter；
Fig. 4 is IONPs@ED-pul/pDNA/SP, IONPs@ED-pul/pDNA, IONPs@ED-pul tri- kinds of compounds
Partial size and surface potential analysis；
Wherein, IONPs@ED-pul/pDNA indicates the kernel (i.e. kernel-interlayer composite) of package gene, similarly hereinafter；
Fig. 5 is that dispersion performance analysis and magnetic of the magnetic nano-balls of clad structure layer by layer of the invention in aqueous phase system are rung
Answering property is analyzed；
Fig. 6 is the transfection that magnetic nano-balls of the invention carry out gene (PGL-3) transfection to human marrow mesenchymal stem cell
Efficiency is investigated；
Wherein, blank indicates blank control, and Nake pDNA indicates naked pDNA direct transfection, and Lipo 2000 indicates to utilize
Non-viral gene vector Lipofectmine 2000 carries out gene transfection, and SP indicates to utilize non-viral gene vector spermine-Pu Lu
Blue polysaccharide copolymer carries out gene transfection, and IONPs@ED-pul/pDNA/SP indicates to carry out base using magnetic nano-balls of the invention
Because of transfection (in the case where no external magnetic field), the magnetic field@ED-pul/pDNA/SP+ IONPs indicates to utilize magnetic Nano of the invention
Ball carries out magnetic transfection；Similarly hereinafter；
Fig. 7 is that magnetic nano-balls of the invention turn rat bone marrow mesenchymal stem cells progress gene (PGL-3) transfection
Efficiency is contaminated to investigate；
Wherein, PEI indicates to carry out gene transfection using non-viral gene vector polyethyleneimine, similarly hereinafter；
Fig. 8 is to be examined using the transfection efficiency of magnetic nano-balls after different cationic polymer modified ferriferrous oxide nano grains
It examines；
Wherein, IONPs@SP/pDNA/SP indicates to modify ferriferrous oxide nano using spermine-pulullan polysaccharide copolymer
Magnetic nano-balls obtained after grain, and utilize the magnetic nano-balls in the case where no external magnetic field to human marrow mesenchymal stem cell
Carry out gene (PGL-3) transfection；The magnetic field IONPs SP/pDNA/SP+ then indicates the effect using the magnetic nano-balls in external magnetic field
Under to human marrow mesenchymal stem cell carry out gene (PGL-3) transfection；
Fig. 9 is transfection efficiency investigation of the IONPs@ED-pul and pDNA with different quality than combining rear magnetic nano-balls；
Wherein, IONPs@ED-pul/pDNA/SP (0.6), IONPs@ED-pul/pDNA/SP (1.5), IONPs@ED-
Pul/pDNA/SP (3.0) respectively indicate IONPs@ED-pul and pDNA with mass ratio 1:0.6,1:1.5,1:3.0 in conjunction with after be made
Magnetic nano-balls, and using corresponding magnetic nano-balls in the case where no external magnetic field to human marrow mesenchymal stem cell carry out
Gene (PGL-3) transfection；IONPs@ED-pul/pDNA/SP (0.6)+magnetic field, IONPs@ED-pul/pDNA/SP (1.5)+magnetic
Field, IONPs ED-pul/pDNA/SP (3.0)+magnetic field are then respectively indicated using respective magnetic nanosphere under the action of external magnetic field
Gene (PGL-3) transfection is carried out to human marrow mesenchymal stem cell；
Figure 10 be SP and pDNA with different N/P ratios in conjunction with the investigation of the transfection efficiency of rear magnetic nano-balls；
Wherein, SP (3:1), SP (4:1) respectively indicate SP and pDNA with N/P ratio 3:1,4:1 in conjunction with after, directly utilize SP
Gene (PGL-3) transfection is carried out to mesenchymal stem cell；
IONPs@ED-pul/pDNA/SP (3:1), IONPs@ED-pul/pDNA/SP (4:1) respectively indicate SP and pDNA with
Magnetic nano-balls obtained after N/P ratio 3:1,4:1 are combined, and using corresponding magnetic nano-balls in the case where no external magnetic field
Gene (PGL-3) transfection is carried out to human marrow mesenchymal stem cell；IONPs@ED-pul/pDNA/SP (3:1)+magnetic field, IONPs@
ED-pul/pDNA/SP (4:1)+magnetic field is then respectively indicated using corresponding magnetic nano-balls in the case where there is external magnetic field
Gene (PGL-3) transfection is carried out to human marrow mesenchymal stem cell；
Figure 11 is that the transfection efficiency of different external magnetic field strength magnetic nanospheres is investigated；
Wherein, IONPs@ED-pul/pDNA/SP+0Gs, IONPs@ED-pul/pDNA/SP+1900Gs, IONPs@ED-
pul/pDNA/SP+2700Gs、IONPs@ED-pul/pDNA/SP+3000Gs、IONPs@ED-pul/pDNA/SP+3300Gs、
IONPs@ED-pul/pDNA/SP+4000Gs, IONPs@ED-pul/pDNA/SP+5000Gs respectively indicate magnetism of the invention and receive
Rice ball under the magnetic fields of 0,1900,2700,3000,3300,4000,5000 Gausses to rat bone marrow mesenchymal stem cells into
Row gene (PGL-3) transfection；
Figure 12 is that different genes transfection reagent investigates the cytotoxicity of rat bone marrow mesenchymal stem cells；
Wherein, IONPs indicates ferriferrous oxide nano grain；
Figure 13 is the PGL-3 gene in different magnetic field intensity and different transfection time magnetic nanospheres in rat marrow
Distribution in mescenchymal stem cell；
Wherein, blue-fluorescence part is nucleus, and green fluorescence part is the PGL-3 gene of isothiocyanates label；
Figure 14 is the ferriferrous oxide nano grain in different transfection time magnetic nanospheres in rat marrow mesenchyma
Distribution in stem cell；
Wherein, blue dyeing part is ferriferrous oxide nano grain, and red staining part is nucleus；
Figure 15 be using flow cytometry under conditions of having serum-free and whether there is or not under conditions of magnetic field investigate the present invention
Magnetic nano-balls enter born of the same parents' efficiency；
Wherein, FBS free expression is transfected under conditions of no fetal calf serum, and 10%FBS is indicated in 10% tire ox blood
It is transfected under the conditions of existing for clear.
Specific embodiment
Technical solution of the present invention is described in further detail with reference to the accompanying drawings and detailed description.
The preparation of the magnetic nano-balls of clad structure layer by layer of embodiment 1
1. preparing magnetic nano-balls
(1) ED-pul for taking preparation, with having removed O2Distilled water be configured to 20mg/mL, obtain ED-pul aqueous solution；It takes
120 μ L ferric chloride solutions (100mg/mL) are added in ED-pul aqueous solution (1mL), are vortexed after dissolution, continuously add 120 μ L chlorinations
Ferrous iron solution (41mg/mL), being acutely vortexed makes to dissolve；Then 250 μ L concentrated ammonia liquors (28vol.%) are added, are placed in 60 DEG C of water-baths
20min；The suspension of acquisition is eluted on PD-10 (GE Healthcare Bio-Sciences Corp.) short column with distilled water
Extra ammonium hydroxide is removed, can be obtained the ferriferrous oxide nano grain modified by cationic polymer ED-pul, is i.e. kernel is (following
Abbreviation IONPs@ED-pul)；
(2) IONPs@ED-pul after purification is diluted to 250 μ g/mL with 5% aqueous sucrose solution, takes 100 μ L, thereto
Be added double volume pDNA (100 μ g/mL) (using PGL-3 gene, express luciferin albumen, No. Genbank:
HE650697.1), it is incubated for 20min altogether under room temperature shaking, obtains kernel-interlayer composite (hereinafter referred to as IONPs@ED-pul/
pDNA)；
(3) SP (250 μ g/mL) of 200 μ L is added into IONPs@ED-pul/pDNA, is incubated for 15min altogether under room temperature, i.e.,
It can get the magnetic nano-balls (hereinafter referred to as IONPs@ED-pul/pDNA/SP) of clad structure layer by layer.
The structural schematic diagram of IONPs@ED-pul/pDNA/SP is as shown in Figure 1.
2. transmission electron microscope observing
After respectively diluting IONPs@ED-pul, IONPs@ED-pul/pDNA/SP in right amount, slowly drop is in being covered with carbon film
On copper mesh, its form and partial size are observed using transmission electron microscope (TEM, JEM100)；Observation result is shown in Fig. 2 a and Fig. 2 b respectively.
By Fig. 2 a as it can be seen that the ferriferrous oxide nano grain of IONPs@ED-pul is diameter at spherical small of 10nm or so
Grain.
By Fig. 2 b as it can be seen that can finally form diameter is the left side 100nm after IONPs@ED-pul and pDNA and SP be successively incubated for
Right, the internal magnetic nano-balls (IONPs@ED-pul/pDNA/SP) for being enclosed with several ferriferrous oxide nano grains.
3.DNA precipitation experiments
Confirmed in IONPs@ED-pul/pDNA/SP manufactured in the present embodiment using DNA precipitation experiments, pDNA layers whether
It is coated on IONPs@ED-pul outer layer.
IONPs@ED-pul and pDNA are mixed by different mass ratioes, after being incubated for 20min, 12,000rpm centrifugations
15min takes supernatant, measures the ultraviolet absorptivity at 260nm, calculates unprecipitated pDNA concentration in supernatant, and calculate
The Percentage bound of IONPs@ED-pul and pDNA.As a result referring to Fig. 3.
As shown in figure 3, the combination of pDNA is consequently increased with the increase of IONPs@ED-pul, prompting pDNA is to combine
On the surface of IONPs@ED-pul.When the mass ratio of IONPs@ED-pul and pDNA are 3:1, Percentage bound reaches
95.0%.
4. partial size and potential change are investigated
It is measured at 25 DEG C from IONPs@using particle size and surface potential detection instrument (3000HSA, Malvern Co.)
ED-pul to IONPs@ED-pul/pDNA arrives the hydraulic radius and surface potential variation of SP/pDNA/IONPs@ED-pul again.
Appropriate above-mentioned compound is taken respectively, after 10mM neutrality PBS (pH 7.4) in right amount dilution, using dynamic light scattering method
(DLS) partial size is measured.Meanwhile using electrophoretic light scattering method (ELS), nanometer is measured and calculated in the case where electric field strength is 100V/cm
The surface potential (Smolu-chouski equation) of grain.Testing result is shown in Fig. 4.
As shown in figure 4, the partial size after IONP@ED-pul disperses in water is 90nm, this swelling with ED-pul in water
It is related.Surface charge is about 10mV.
And the partial size of IONPs@ED-pul/pDNA increases to about 250nm, surface charge is reduced to about -40mV.In conjunction with (3)
The experimental result of middle DNA precipitating causes IONPs@ED- it is found that this is because pDNA is incorporated in IONPs@ED-pul outer layer
Pul/pDNA partial size becomes larger, and surface potential is reversed into negative electrical charge.
And the partial size of SP/pDNA/IONPs@ED-pul is about 200nm, surface is again positively charged, about 10mV or so.
This is because the pDNA for being coated on IONPs ED-pul outer layer can be compressed after SP is integrated to IONPs ED-pul/pDNA outer layer,
And ultimately form the positively charged magnetic nano-balls in surface.
In summary (2), (3), (4) result can be confirmed composite nano-granule prepared by the present invention be it is as shown in Figure 1 with
Several IONPs ED-pul are kernel, and centre cladding pDNA, outermost layer is the clad structure layer by layer of SP polymer.
5. magnetic responsiveness is tested
After magnetic nano-balls sample manufactured in the present embodiment is diluted to suitable concentration with distilled water, uniformly it is divided into two parts.
A copy of it is placed in the side nickel cobalt magnet (magnetic field strength 5000Gs), another stands far from magnetic field.After standing 72h, observation
With compare the magnetic nano-balls in two parts of aqueous suspensions to the responsiveness in magnetic field.Observe result such as Fig. 5.
As shown in Figure 5, magnetic nano-balls prepared by the present invention can be well dispersed in water, also sinks without any after standing 72h
It is poly-.Meanwhile magnetic field responsiveness is good, is suitable as magnetic transfection reagent.
The antiserum efficiency gene transfection and toxicity assessment of 2 magnetic nano-balls of embodiment
1. couple BMSC carries out magnetic transfection
By taking source of people BMSC and rat BMSC as an example, antiserum gene magnetic transfection efficiency of the present invention on BMSC is commented
Valence.The present embodiment is provided with blank control group (blank), negative control group (Nake pDNA, pDNA direct transfection), non-simultaneously
Viral genetic vector control group (2000 groups of Lipo or PEI group and SP group), without magnetic field control group.
Specific step is as follows:
1) by source of people BMSC or rat BMSC with 6 × 104A/hole is seeded on 24 orifice plates, is placed in 5%CO2Cell incubator
It is interior, it is cultivated for 24 hours at 37 DEG C；
2) culture solution is discarded, twice with phosphate buffer (pH 7.4) rinsing, every hole is added 0.5mL and contains 10% fetal calf serum
DMEM low sugar culture solution, will in embodiment 1 prepare IONPs@ED-pul/pDNA/SP with 5% sucrose water (300 μ L) dilution after
It is added by every 40 μ L of hole；Tissue culture plate is placed on cobalt nickel magnet (magnetic field strength 3300Gs), in 5%CO2, cultivate at 37 DEG C
3h；
3) demagnetizing field is moved, and discards culture solution, is re-replaced as the DMEM low sugar culture solution containing 10% fetal calf serum,
5%CO2, continue at 37 DEG C to cultivate 48h；
4) culture solution is discarded, twice with phosphate buffer (pH 7.4) rinsing, every hole is added 0.2mL reporter gene cell and splits
Liquid is solved, 30min is shaken at room temperature, is centrifuged 20min under the conditions of 14,000rpm, 4 DEG C；
5) 0.1mL supernatant is drawn, isometric luciferin zymolyte is added and measures each experimental group under Chemiluminescence Apparatus
Relative light intensity (RLU)；
6) taking concentration is the protein standard liquid of 0.5mg/mL, is diluted to series of concentrations: 25 μ g/mL respectively with distilled water,
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 by every 20 μ L of hole to 96 orifice plates
In, then press every 200 μ L of hole and BCA working solution is added, absorbance is detected at microplate reader 595nm wavelength, draws standard curve；
7) each 20 μ L of hole supernatant of experimental group is taken, 200 μ L BCA working solutions are added and detect absorbance at 595nm wavelength,
Protein concentration is calculated using standard curve；
8) (enzymatic activity is higher for the luciferin enzymatic activity of the BMSC after calculating transfection PGL-3 reporter gene as follows
Then illustrate that corresponding transfection efficiency is higher):
People BMSC and rat BMSC luciferin Enzyme assay result are respectively referring to Fig. 6 and Fig. 7.
By Fig. 6 and Fig. 7 as it can be seen that magnetic nano-balls of the invention external magnetic field with the help of can be in people BMSC and rat
Good antiserum efficiency gene transfection is obtained on BMSC, compared to currently used non-viral gene transfection reagent such as PEI or
There is Lipofectmine 2000 better antiserum gene to transfect advantage.
2. influence of the different cationic polymer modified ferriferrous oxide nano grains to magnetic nano-balls transfection efficiency
Using the method for the present embodiment part 1, it is right to investigate different cationic polymer modified ferriferrous oxide nano grains
The influence of magnetic nano-balls transfection efficiency, as a result as shown in Figure 8.
As seen from Figure 8, compared to the magnetic nano-balls for the IONPs kernel preparation modified with SP, the IONPs modified with ED-pul
Transfection efficiency of the magnetic nano-balls of kernel preparation on BMSC is higher (about 3 times), but no difference of science of statistics between the two.
3. influence of the mass ratio of kernel and pDNA to magnetic nano-balls transfection efficiency
Using the method for the present embodiment part 1, the mass ratio for investigating different kernels and pDNA transfects magnetic nano-balls
The influence of efficiency, as a result referring to Fig. 9.
As seen from Figure 9, when the mass ratio of IONPs@ED-pul kernel and pDNA are 1:1.5, magnetic nano-balls are to people
The transfection efficiency highest of BMSC, be mass ratio be 1:0.6 when 4 times, be mass ratio be 1:3.0 when 5.7 times.
When the mass ratio of IONPs@ED-pul kernel and pDNA are 1:3.0, although the Percentage bound highest of the two, pDNA are abundant
It is wrapped on the outside of kernel, but the release intracellular of pDNA may be unfavorable for due to excessively wrapping up, so that transfection efficiency reduces instead.
Influence of the N/P ratio of 4.SP and pDNA to magnetic nano-balls transfection efficiency
Using the method for the present embodiment part 1, the N/P ratio for investigating different SP and pDNA, which transfects magnetic nano-balls, is imitated
The influence of rate, as a result referring to Figure 10.
As shown in Figure 10, when the N/P ratio of SP and pDNA is 4:1, the magnetic nano-balls of preparation can obtain on people BMSC
Obtain optimal transfection efficiency；Transfection efficiency at this time be N/P ratio be 3:1 when 2 times.
5. influence of the external magnetic field strength to magnetic nano-balls transfection efficiency
Using the method for the present embodiment part 1, different external magnetic field strengths are investigated to the shadow of magnetic nano-balls transfection efficiency
It rings, as a result referring to Figure 11.
As shown in figure 11, when magnetic field strength is when 2700Gs is between 4000Gs, magnetic nano-balls can be in rat BMSC
It is upper to obtain optimal transfection efficiency.Magnetic field strength is too weak or too strong is all unfavorable for obtaining preferable transfection efficiency.
The toxicity assessment to mesenchymal stem cell of 6 magnetic nano-balls is tested
By taking big source of mouse BMSC as an example, magnetic nano-balls prepared by the present invention are detected to the toxicity of stem cell using MTS method.This
Embodiment be provided with simultaneously non-viral gene vector control group (2000 groups of Lipo, PEI group and SP group), IONPs control group,
Without magnetic field control group.
Specific step is as follows:
1) by BMSC with 6 × 104A/hole is inoculated on 24 orifice plates, in 5%CO2, cultivate for 24 hours at 37 DEG C；
2) culture solution is discarded, phosphate buffer (pH 7.4) cleans twice, and 0.5mL is added containing 10% fetal calf serum in every hole
IONPs@ED-pul/pDNA/SP after being diluted in DMEM low sugar culture solution and 40 μ L embodiments 2；Tissue culture plate is placed in cobalt
On nickel magnetic field (magnetic field strength 3300Gs), in 5%CO2, 3h is cultivated at 37 DEG C；
3) demagnetizing field is moved, and discards culture solution, the DMEM low sugar culture solution containing 10% fetal calf serum is changed to, continues to cultivate
48h；
4) with after phosphate buffer (pH 7.4) rinsing twice, 400 μ L fresh mediums and 100 μ L MTS are added in every hole
Examination, in 37 DEG C, 5%CO2Lower incubation 4h；
5) microplate reader measures absorbance value (OD value) under 570nm.
6) survival rate of BMSC is calculated as follows to evaluate cytotoxicity, as a result as shown in figure 12.
As seen from Figure 12, magnetic nano-balls of the invention and corresponding rotaring redyeing system are to rat BMSC all without apparent cell
Toxicity.
Shown by efficiency gene transfection and Cytotoxic evaluation experiment prepared by the method based on magnetic material
Rotaring redyeing system to BMSC efficiency gene transfection with higher, and can antagonistic Serum influence that gene transfect, while to it is dry carefully
Born of the same parents have lower cytotoxicity.
The magnetic transfection mechanism of the magnetic nano-balls of the present invention of embodiment 3
By taking IONPs@ED-pul/pDNA/SP as an example, the pDNA (FITC-PGL-3) marked using isothiocyanates is investigated
Distribution of the pDNA and IONPs kernel in rat BMSC in magnetic nano-balls.Specific step is as follows:
1) by BMSC with 2 × 104A/hole is inoculated in the culture vessel with glass bottom that diameter is 20mm, in 5%CO2, train at 37 DEG C
It supports for 24 hours；
2) culture solution is discarded, twice with phosphate buffer (pH 7.4) rinsing, it is low that the DMEM containing 10% fetal calf serum is added
Sugared culture solution and by preparing in embodiment 2 and diluted IONPs@ED-pul/pDNA/SP；Tissue Culture Dish is placed in cobalt nickel magnetic
(magnetic field strength is respectively 2700Gs and 5000Gs), 5%CO on field2, be incubated at 37 DEG C respectively different time (0.5h, 2h, 4h and
6h)；
3) at corresponding time point, demagnetizing field is moved, and discards culture solution, phosphate buffer (pH 7.4) rinses 3 times, be added-
The fixed 15min of 20 DEG C of ice methanol；
4) DAPI core transfection reagent is added to dye nucleus, dyeing time 8min；Then phosphate buffer (pH
7.4) it rinses 3 times；
5) distribution of laser scanning co-focusing microscope (BX61W1-FV1000, Olympus) observation pDNA in the cell,
As a result as shown in figure 13；
1) and 2) 6) simultaneously, by above-mentioned step operation, and (magnetic field strength 2700Gs) transfects 3h under magnetic field；
7) move 0h, 27h and 51h after demagnetizing field (i.e. addition IONPs@ED-pul/pDNA/SP after 3h, for 24 hours and
The culture solution of the ED-pul/pDNA/SP of@containing IONPs 48h) is discarded respectively, after phosphate buffer (pH 7.4) rinses 3 times, is added
4% paraformaldehyde fixes 20min；Then Prussian blue test solution of the 1mL containing 5% potassium ferrocyanide and 10% hydrochloric acid is added in room temperature
Lower dyeing 1h；Phosphate buffer (pH 7.4) is added PI core dye liquor after rinsing 3 times and dyes 5min, finally uses phosphate buffer again
(pH 7.4) is rinsed 3 times；
8) (Eclipse Ti-s, Nikon) observes the distribution of ferriferrous oxide nano grain in the cell under fluorescence microscope,
As a result as shown in figure 14.
As seen from Figure 13, when transfecting 6h, under strong magnetic field action, the dyeing of FITC-PGL-3 is deeper；And under general magnetic field,
The dyeing of FITC-PGL-3 is then shallower in nucleus；Under no magnetic fields, the dyeing of FITC-PGL-3 is then basic in nucleus
It has no.It is highly efficient that this shows that magnetic nano-balls prepared by the present invention can carry pDNA under the action of the magnetic field of appropriate intensity
It is intracellular with being rapidly introduced into, and then that improves pDNA enters core efficiency, finally proposes the efficiency of high gene transfection.
As shown in figure 14, in conjunction with Figure 13 as it can be seen that pDNA is mainly discharged in cytoplasm by magnetic nano-balls, after release voluntarily
Into nucleus, and kernel IONPs@ED-pul will not then enter nucleus.
The antiserum transfection mechanism of 4 magnetic transfection reagent of embodiment
By taking IONPs@ED-pul/pDNA/SP as an example, the pDNA (FITC-PGL-3) marked using isothiocyanates is utilized
Flow cytometry investigates magnetic transfection reagent in the case where having serum and serum-free condition respectively and has magnetic field (3300Gs) and without magnetic field
Under the conditions of enter born of the same parents' efficiency.Specific steps are as follows:
1) BMSC is pressed 5 × 105A/hole is inoculated in 6 orifice plates, in 5%CO2, cultivate for 24 hours at 37 DEG C；
2) culture solution is discarded, phosphate buffer (pH 7.4) cleans twice, and 2mL is added containing 10% fetal calf serum in every hole
DMEM low sugar culture solution or DMEM low sugar culture solution without serum, after 160 μ L dilution then is added by the every hole of embodiment 2
IONPs@ED-pul/pDNA/SP；
3) tissue culture plate is placed on cobalt nickel magnetic field (magnetic field strength 3300Gs), as experimental group, is not interposing at magnetic field
Tissue culture plate as a control group, in 5%CO2, different time (0.5h, 1.5h, 3h) is incubated at 37 DEG C；
4) in above-mentioned different time points, group of cells is digested with the trypsin solution containing 0.02%EDTA respectively, is collected in stream
In formula pipe, using the positive rate of Flow cytometry FITC-pDNA cell；As a result as shown in figure 15.
For flow cytometer detection the results show that under no magnetic field condition, magnetic nano-balls prepared by the present invention can be slow by BMSC
Intake.Under serum-free condition, after being incubated for 3h, magnetic nano-balls enter born of the same parents' efficiency up to 98.2%；However in serum, there are items
Under part, magnetic nano-balls enter born of the same parents' efficiency with enter born of the same parents' speed than serum-free when it is low.
But under the existence condition of magnetic field, no matter serum presence or absence, it is fast not affect magnetic nano-balls prepared by the present invention
Speed enters born of the same parents.Under magnetic fields, magnetic nano-balls enters born of the same parents' efficiency i.e. close to 100% when being incubated for 0.5h.
Thus it proves, magnetic nano-balls prepared by the present invention can carry pDNA and efficiently and rapidly enter in stem cell, together
When do not influenced by serum presence or absence.Therefore, it may be used as the antiserum high efficiency gene transfection reagent for stem cell.
Claims (5)
1. a kind of magnetic nano-balls of clad structure layer by layer, which is characterized in that be made of kernel, middle layer and outer layer, kernel with
It is be combined with each other by electrostatic attraction effect between middle layer, between middle layer and outer layer；
The kernel is cationic polymer modified ferriferrous oxide nano grain, and the middle layer is to be coated on the outside of kernel
Gene to be transfected, the outer layer are the spermine-pulullan polysaccharide copolymer being coated on the outside of middle layer；
Wherein, the cationic polymer is ethylenediamine-pulullan polysaccharide copolymer, spermine-pulullan polysaccharide copolymer, poly- second
Alkene imines, chitosan or spermine-dextran copolymer；
The partial size of the ferriferrous oxide nano grain is 10~50nm, the cationic polymer and four negatively charged oxidations three
Iron nanoparticle is be combined with each other by electrostatic attraction effect；In the spermine of the outer layer-pulullan polysaccharide copolymer, pulullan polysaccharide
Average molecular weight be 47300Da, spermine grafting rate be 12%~14%；The mass ratio of the kernel and the gene to be transfected
Less than 1:1；The N/P ratio of the spermine of the outer layer-pulullan polysaccharide copolymer and the gene to be transfected is 3:1~5:1.
2. the preparation method of the magnetic nano-balls of clad structure layer by layer as described in claim 1 characterized by comprising
(1) cationic polymer modified ferriferrous oxide nano grain is prepared using coprecipitation, obtains kernel；
(2) kernel is added in cdna solution to be transfected, is incubated for 20~30min under the conditions of room temperature, shaking altogether, in acquisition
Core-interlayer composite；
(3) spermine-pulullan polysaccharide copolymer is added in Xiang Suoshu kernel-interlayer composite, it is incubated for 15 under room temperature altogether~
20min obtains the magnetic nano-balls of the clad structure layer by layer.
3. the magnetic nano-balls of clad structure are preparing the application in magnetic transfection reagent layer by layer as described in claim 1.
4. application as claimed in claim 3, which is characterized in that the magnetic transfection reagent is used to carry out gene to stem cell to turn
Dye.
5. a kind of magnetic transfecting formulations, which is characterized in that by layer by layer prepared by the magnetic nano-balls of clad structure as described in claim 1
It forms.
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