CN104162174B - The preparation of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles and the application of imaging and thermotherapy thereof - Google Patents
The preparation of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles and the application of imaging and thermotherapy thereof Download PDFInfo
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Abstract
The present invention relates to the preparation of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles and the application of imaging and thermotherapy thereof, comprising: the preparation of the silver nanoparticle seed of PEI cladding；" step " water heat transfer is with the ferric oxide nanometer particle of silver seed；Preparation star composite nanometer particle in gold growth solution；The surface of star composite nanometer particle carries out ethylene imine (PEI) and hyaluronic acid (HA) is modified.Reaction condition of the present invention is gentle, and technique is simple, it is easy to operation；The golden coated iron oxide star Core-shell Structure Nanoparticles of preparation has good molecular imaging performance and thermotherapy antitumous effect, and tumor imaging diagnosis and photo-thermal therapy field have potential using value in vivo.
Description
Technical field
The invention belongs to the preparation field of diagnosis and treatment agent, particularly to the system of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles
The application of standby and imaging and thermotherapy.
Background technology
For a long time, malignant tumour always be harm human life number one killer, its have the death rate high, refractory treat and
Deteriorate the features such as rapid.Therefore, the early diagnosis and therapy of tumour is particularly important.At present, the detection means of tumour is various
Various kinds, mainly has: ultrasonic imaging, CT imaging, the imaging of nuclear medicine PET and Magnetic resonance imaging (MRI).With nuclear-magnetism
Resonance technique (MR) and the development of computed tomography (CT), they are gradually shortened the sweep time to focus,
Resolution ratio gradually steps up, and detection is also more accurate, and this also makes MRI and CT become the Main Means of disease detection in recent years.
Meanwhile, in order to improve sensitivity and the degree of accuracy of diagnosis, it is necessary to develop multi-functional imaging pattern.
SPIO nano particle is in an increasingly wide range of applications at biomedical sector in recent years, particularly uses
Make the contrast preparation of Magnetic resonance imaging (MRI).This seminar, in the work of early stage, is prepared by easy hydrothermal synthesis method
Ferroferric oxide nano granules there is very high relaxation rate, and show good T2MR imaging performance (Cai et al., ACS
Appl.Mater.Interfaces2013,5,1722-1731；Patent publication No. 201210277624.9).Additionally, we pass through
Amended hydro-thermal method is prepared for containing the Fe3O4/Au composite nanometer particle of ferroferric oxide nano granules and Jenner's particle simultaneously,
This composite nanometer particle can be used successfully to contrast preparation (Li et al., the ACS Appl.Mater. of MR/CT bimodal imaging
Interfaces2013,5,10357-10366；Patent publication No. 201310072165.5).
With the proposition of " diagnosis and treatment integration " theory, we attempt preparing the nano particle with tumor photo-thermal response to treatment.?
During this, it has been found that in the presence of CTAB, gold nano grain can grow up on ferroferric oxide nano granules surface
Hub-and-spoke configuration, and the structure of this uniqueness makes this nano particle have a stronger absworption peak near infrared region, thus be expected to by
Luminous energy is converted into heat energy, and reaches to kill the purpose of tumour cell.
In order to allow gold coated iron oxide star Core-shell Structure Nanoparticles can be effectively applied to the imaging inspection of tumor locus in organism
Surveying and treatment, we are successively at its surface modifying polyethyleneimine (polyethyleneimine, PEI) and hyaluronic acid (hyaluronic
Acid, HA).The work of our early stage has turned out HA to the cancer cell (such as HeLa cell) with high expressed CD44 acceptor
There is good targeting (Li et al., Biomaterials2014,35,3666-3677).Therefore, this strategy does not only has
Improve to effect water solubility, stability and the biocompatibility of nano particle, but also give this composite nanometer particle to certain detail
The targeting of born of the same parents.With HeLa cell (a kind of people's uterine neck knurl cancer cell), as model cell and tumor model is to preparation for we
The character of multifunctional nano probe is evaluated one by one.The golden coated iron oxide star Core-shell Structure Nanoparticles tool of this research preparation
Standby superperformance, such as higher R2 relaxation rate, good CT strengthens effect, the targeting of particular cancer cell, is rapidly heated
Kill the ability etc. of tumour cell, be expected to realize the accurately sensitive diagnosis to various disease system and treatment, for " diagnosis and treatment one
Change " provide a good platform preparing of contrast preparation.
Retrieval domestic and foreign literature, does not still find with regard to the gold preparation of coated iron oxide star Core-shell Structure Nanoparticles and is used for
MR/CT bimodal imaging and the relevant report of photo-thermal therapy.
Content of the invention
The technical problem to be solved is to provide the preparation side of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles
Method, the method technique is simple, and reaction condition is gentle, it is easy to operation, cost is relatively low.The compound star core shell structure of preparation is received
Rice grain has good water solubility, colloidal stability and specific target tropism.Synthesis material used is cheap and environmental friendliness material
Material, has the prospect of industrialized implementation.
A kind of preparation method of the gold coated iron oxide star Core-shell Structure Nanoparticles of the present invention, comprising:
(1) claim PEI soluble in water, add AgNO3Solution, stirring, it is subsequently adding the NaBH that ice bath is processed4The aqueous solution, stirs
Mix, dialysis, obtain PEI-Ag nano particle；
(2) by FeCl2·4H2O is dissolved in ultra-pure water, adds NH3·H2O simultaneously stirs, then by mixed solution under air atmosphere
Transfer in autoclave, and the PEI-Ag aqueous solution by preparation in (1) is also added in autoclave, stirs
Mix after mixing, reaction.After reaction terminates, naturally cool to room temperature, precipitation Magneto separate is washed after purification, i.e.
Must be with the ferroferric oxide nano granules Fe of silver seed3O4-PEI-Ag；
(3) claim cetyl trimethylammonium bromide CTAB to be dissolved in the water, add HAuCl4Solution, adds AgNO after stirring3,
Add ascorbic acid AA after being stirred for, when solution becomes colourless, add preparation in (2) of dilution 10 times
Fe3O4-PEI-Ag the aqueous solution.After reaction terminates, centrifuge washing removes CTAB, i.e. obtains star composite nanometer particle
Fe3O4/Au NSs；
(4) claiming PEI soluble in water and being added thereto to methyl thioglycolate reaction, product is dialysed in water three days and is i.e. obtained part mercapto
The PEI PEI-SH of base；
(5) Fe preparing to step (3)3O4/ Au NSs the aqueous solution adds the PEI-SH aqueous solution of preparation in (4), ultrasonic
Rear stirring reaction, obtains the Fe of PEI parcel3O4/Au NSs Fe3O4/Au-PEI NSs；
(6) claim hyaluronic acid HA soluble in water, and with 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC with
N-hydroxysuccinimide NHS activates, and then the HA solution of activation is joined Fe prepared by (5)3O4/Au-PEI
In the NSs aqueous solution, stirring reaction, i.e. obtain the star composite Nano that end-product HA modifies after product centrifuge washing
Grain Fe3O4/Au-HA NSs。
PEI and AgNO in step (1)3Mol ratio be 1: 20；AgNO3And NaBH4Mol ratio be 1: 5；PEI and water
Ratio is 1g: 20ml；Add AgNO330-60min is stirred after solution；Add NaBH42-4h is stirred after the aqueous solution.
In step (1), dialysis is for being dialysed 2-5 days to distilled water by the bag filter of molecular cut off 14000.
FeCl in step (2)2·4H2O is 1g with the ratio of ultra-pure water: 6-7ml；FeCl2·4H2O and NH3·H2The mol ratio of O
It is 1: 5；Reaction temperature 130-140 DEG C；FeCl2·4H2The mass ratio of O and PEI-Ag is 2.5: 1；Mixing time is 5-15min；
Reaction 3-4h.
CTAB, HAuCl in step (3)4、AgNO3It is 381: 17.4: 1.1 with the mass ratio of AA: 12；Cetyl front three
The ratio of base ammonium bromide and water is 30-40mg: 1ml；HAuCl4The concentration of solution is 30mg/ml；Cetyl trimethyl bromination
Ammonium and Fe3O4The ratio of-PEI-Ag the aqueous solution is 3000-4000mg: 1ml.
In step (4), the ratio of PEI and water is 1g: 10ml；The mol ratio of PEI and methyl thioglycolate is 1: 30；Reaction temperature
For 60-80 DEG C.1-3 days reaction time.
Methyl thioglycolate specification is 95%, Mw=106.14, ρ=1.187.
Fe in step (5)3O4The ratio of-PEI-Ag the aqueous solution and the PEI-SH aqueous solution is 100-200ml: 1g.
In step (5), ultrasonic time is 10-60min；Reaction time is 10-30h.
In step (6), the molecular weight of HA is 31200；The mol ratio of HA, EDC and NHS is 1: 10: 10.Soak time is
1-5h；Reaction time is 1-5 days.
In step (1) and step (4), the molecular weight of hyperbranched polyethyleneimine PEI is 25000.
A kind of gold coated iron oxide star Core-shell Structure Nanoparticles is in the application of imaging.
A kind of gold coated iron oxide star Core-shell Structure Nanoparticles is in the application of thermotherapy.
The present invention first with " step " water heat transfer with silver seed ferric oxide nanometer particle；System in gold growth solution again
Standby star composite nanometer particle；Then surface PEI and HA that carry out of the star composite nanometer particle of preparation is modified.
The present invention is easy to operation, and the cost of raw material is low.The composite nano particle of preparation has good water solubility, colloid
Stability, biocompatibility and specific target tropism.In external and animal body, experimental result shows this star composite nanometer particle not only
There is good MR/CT imaging effect, there is photo-thermal therapy simultaneously and kill the effect of cancer cell.Gold cladding oxygen prepared by the method
Change iron star Core-shell Structure Nanoparticles has potential answering in the photo-thermal therapy field of MR/CT bimodal imaging diagnosis and cancer
With.
The present invention uses fourier transform infrared spectroscopy to analyze (FTIR), ultraviolet-visible absorption spectroscopy (UV-Vis), inductive etc.
Plasma-atomic emission spectroscopic methodology (ICP-OES), Zeta electric potential and dynamic light scattering (DLS) and transmission electron microscope (TEM)
Characterize the star Core-shell Structure Nanoparticles of preparation etc. method, and measure nano particle by MR imager and CT imager
T2Relaxivity and x-ray attenuation coefficient, and have rated nano particle as thermotherapy reagent near-infrared laser irradiation under intensification
Efficiency.Then utilize mtt assay evaluate the cytotoxicity of nano particle and kill effect to cancer cell, swell finally by load
The nude mice of knurl is model, and imaging and curative properties to nano particle are evaluated.
Beneficial effect
(1) present invention first with " step " water heat transfer with silver seed ferric oxide nanometer particle；System in gold growth solution again
Standby star composite nanometer particle；Then carry out surface PEI and HA to the star composite nanometer particle of preparation to modify；This method operates
Technique is simple, and reaction condition is gentle, it is easy to operation purifies, and used is cheap and environment friendly material；
(2) the composite nano particle that prepared by the present invention has good water solubility, colloidal stability, biocompatibility and specific cells
Targeting.In cell in vitro and animal body, experimental result shows that this star composite nanometer particle not only has good MR/CT
Imaging effect, can reach the effect of photo-thermal therapy simultaneously to cancer cell and tumour.Golden coated iron oxide star core prepared by the method
Shell structural nano particle has potential application in the photo-thermal therapy field of MR/CT bimodal imaging diagnosis and cancer.
Brief description
Fig. 1 is PEI-SH and the PEI infrared spectrogram of embodiment 1；
Fig. 2 is the Fe of embodiment 13O4/ Au-PEI NSs and Fe3O4The electromotive force (a) of/Au-HA NSs and hydrodynamic force particle diameter (b)；
Fig. 3 is the Fe of embodiment 13O4/ Ag seed and Fe3O4The ultraviolet absorpting spectrum of/Au-HA NSs；
Fig. 4 is the Fe of embodiment 13O4The transmission electron microscope picture of/Au-HA NSs: (a, b) low power electron microscopic picture, (c) high power Electronic Speculum figure
Piece；
Fig. 5 is the Fe of embodiment 13O4T under the different Fe concentration for/Au-HA NSs2Weighted imaging (a) and T2Relaxation time falls
Number and the linear relationship chart (b) of Fe concentration；
Fig. 6 is the Fe of embodiment 13O4CT imaging picture (a) under the different Au concentration for/Au-HA NSs is dense with gold with CT value
The linear relationship chart (b) of degree change；
Fig. 7 is the Fe of embodiment 13O4/ Au-HA NSs heating curve after laser irradiates under different Au concentration；
Fig. 8 is the Fe that the HeLa cell that mtt assay is tested is prepared through PBS (comparison) and the present invention3O4/Au-HA NSs
(concentration range of Au is at 0.2-2.0mM) process 24 hours after cell viability；
The Fe that Fig. 9 is HeLa cell and U87MG cell is prepared through PBS or the present invention3O4/ Au-HA NSs processes 6
Cell T after little Shi2MR imaging picture (a), corresponding MRI signal value change (b), CT imaging (c) are with accordingly
CT signal value changes (d)；
Figure 10 is that the HeLa cell of mtt assay test is at the Fe preparing with the present invention3O4/ Au-HA NSs (Au concentration 0.1-0.4mM)
After co-culturing 6 hours after the laser of different time irradiates the survival rate of cell；
Figure 11 is Fe prepared by the intratumor injection present invention3O4/ Au-HA NSs to the MR (a) behind mouse tumor position and CT (c) imaging with
And the change of corresponding MR (b) and CT (d) signal strength signal intensity；
Figure 12 is Fe prepared by the intratumor injection present invention3O4/ Au-HA NSs (region 11) and PBS (region 12) arrives mouse tumor
Thermographic picture (a) of the tumor locus behind position and temperature variation curve (b)；
Figure 13 be through different modes process after nude mouse tumor HE dyeing picture, (a) intratumor injection PBS but without laser irradiate,
(b) intratumor injection PBS and through laser irradiate, Fe prepared by (c) intratumor injection present invention3O4/ Au-HA NSs but without
Too drastic light irradiates, Fe prepared by (d) intratumor injection present invention3O4/ Au-HA NSs and through laser irradiate.
Figure 14 is nude mouse tumor relative volume change curve over time after different modes is processed.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
(1) claim 0.5g PEI to be dissolved in 10mL water, add AgNO3Solution (68mg), stirs 30-60min, then
Add the NaBH that ice bath is processed4The aqueous solution (75.66mg), stirs 2-4h, dialyses 3 days, obtain PEI-Ag nano particle.
It is stored at 4 DEG C standby.
(2) by 1.25g FeCl2·4H2O is dissolved in 7.75mL ultra-pure water, adds 6.25mLNH3·H2O in air gas
Stir 10 minutes under atmosphere, then transfer to mixed solution in autoclave, and by the PEI-Ag aqueous solution of preparation in (1)
It is also added in autoclave, after being uniformly mixed, react 3 hours in 134 DEG C.After reaction terminates, naturally cool to
Precipitation Magneto separate is washed after purification, obtains the ferroferric oxide nano granules Fe with silver seed by room temperature3O4-PEI-Ag.Storage
It is stored at 4 DEG C standby.
(3) claim 381mg cetyl trimethylammonium bromide (CTAB) to be dissolved in 10mL water, add 580 μ L HAuCl4
Solution (30mg/mL), adds 1.1mg AgNO after stirring 10 minutes3, after being stirred for 10 minutes, add 12mg Vitamin C
Acid (AA), adds the Fe of preparation in (2) of dilution 10 times when solution becomes colourless3O4-PEI-Ag the aqueous solution (0.1mL).
After reaction terminates, centrifuge washing removes CTAB, i.e. obtains star composite nanometer particle (Fe3O4/Au NSs).It is stored in 4 DEG C
Under standby.
(4) 1.0g PEI is claimed to be dissolved in 10mL water and be added thereto to 108 μ L methyl thioglycolate (Methyl
Thioglycolate, 95%, Mw=106.14, ρ=1.187) 60～80 DEG C of water-baths 2 days, water is dialysed three days i.e.
Obtain the PEI (PEI-SH) of part sulfhydrylation.It is stored at-20 DEG C standby after Dong Gan.Take respectively the lyophilized PEI of 2mg and
PEI-SH, is used for infrared spectrum analysis after pressing potassium bromide troche.By contrasting the infrared spectrum of PEI and PEI-SH (see attached
Fig. 1), find PEI-SH at 1640cm-1Place's obvious absworption peak more than PEI, this peak be by the amino on PEI and
The amido link that methyl thioglycolate is formed after hydrolyzing causes, thus illustrates that PEI success sulfhydrylation is modified.
(5) Fe preparing to (3)3O4/ Au NSs the aqueous solution adds the PEI-SH aqueous solution (0.1g) of preparation in (4),
After ultrasonic 30 minutes, stirring reaction 1 day, obtains the Fe of PEI parcel3O4/Au NSs(Fe3O4/Au-PEI NSs).It is stored in 4
At DEG C standby.
(6) claim 520mg hyaluronic acid (Mw=31200) soluble in water, and with 1-(3-dimethylamino-propyl)-3-ethyl carbon two
Inferior amine salt hydrochlorate (EDC, 128mg) and N-hydroxysuccinimide (NHS, 80mg) activation 3h, then by activation
HA solution joins the Fe of preparation3O4In/Au-PEI NSs the aqueous solution, stirring reaction 3 days, i.e. obtain after product centrifuge washing
Composite star-shaped nano particle (the Fe that end-product HA modifies3O4/Au-HA NSs).It is stored at 4 DEG C standby.Take 0.1mL (5)
The Fe of preparation3O4Fe prepared by/Au-PEI NSs and (6)3O4/ Au-HA NSs ultra-pure water is configured to the water of 1.5mL respectively
Solution is used for surveying surface potential and hydrodynamic diameter (see accompanying drawing 2).Star nucleocapsid after the display PEI modification of surface potential test result
The surface potential of structural nano particle is+32.7mV, and modifies after-potential through HA and be reduced to-27.7mV.Hydrodynamic force grain
Footpath test result display Fe3O4The hydrodynamic force particle diameter of/Au-PEI NSs is 298.8nm, and what HA obtained after modifying
Fe3O4The hydrodynamic force particle diameter of/Au-HA NSs is 339.4nm.Take 25 μ LFe respectively3O4-PEI-Ag (2) and Fe3O4/Au-HA
The aqueous solution of NSs (6) is in 2mL centrifuge tube, then adds 700 μ L ultra-pure waters wherein, ultrasonic uniformly, survey UV absorption (see
Accompanying drawing 3).Ultraviolet result shows: Fe3O4/ Au-HA NSs has a very wide UV absorption in the range of 700 to 1100nm
Peak, and Fe3O4/ Ag seed does not has obvious ultraviolet absorption peak at this wave band.
Embodiment 2
The Fe of Example 1 preparation3O4/ Au-HA NSs nano particle the aqueous solution 5 μ L, is then configured to 100 μ L with ultra-pure water
Nano granule suspension.And nano granule suspension 5 μ L is dropped in copper mesh surface, survey for TEM after drying in atmosphere
Examination (see accompanying drawing 4).TEM result shows Fe3O4The pattern of/Au-HA NSs is star, particle size at about 120nm,
One layer of organic layer of Surface coating of high-resolution TEM display nano particle.
Embodiment 3
Fe by embodiment 13O4/ Au-HA NSs measures the concentration of Fe element in solution by ICP-OES method of testing, then
In EP pipe with ultra-pure water preparation Fe concentration be followed successively by the 0.005th, the 0.01st, the 0.02nd, 0.04 and 0.08mM aqueous solution 2mL,
Measure imaging effect under different Fe concentration for the material and T by magnetic resonance imaging2Relaxation effect (see accompanying drawing 5).T2Weighting
Imaging results shows Fe prepared by the present invention3O4/ Au-HA NSs weakens therewith with the increase of concentration of iron, MR signal strength signal intensity.
Relaxation rate test result shows Fe3O4The relaxation time of/Au-HA NSs is reciprocal as the increase of concentration of iron is (at 0.0025-0.04mM
In concentration range) there is good linear relationship.And by can be calculated Fe prepared by the present invention3O4The r of/Au-HA NSs2
Relaxation rate is 144.39mM-1s-1.Therefore, the Fe prepared by the present invention3O4/ Au-HA NSs can be as MRI molecular imaging
Excellent T in diagnosis2Signal attenuation contrast preparation.
Embodiment 4
Fe prepared by embodiment 13O4/ Au-HA NSs measures the concentration of Au element in solution by ICP-OES method of testing,
Then in EP pipe with ultra-pure water preparation Au concentration be followed successively by the 0.01st, the 0.02nd, the 0.04th, 0.06 and 0.08mM aqueous solution 2mL
Become image effect (see accompanying drawing 6) for evaluating its CT.Result shows the increase with gold concentration, and the CT signal of nano particle is strong
Degree is consequently increased.And from CT value with the Linear Fit Chart that gold concentration changes can be seen that the CT value of composite nanometer particle with
The change of gold concentration has good linear relationship.Illustrate the Fe of the present invention3O4/ Au-HA NSs is expected to as CT imaging
Contrast preparation.
Embodiment 5
It according to the concentration of the Au element that embodiment 4 records, is followed successively by the 0.32nd, the 0.8th, by ultra-pure water preparation Au concentration in EP pipe
1.6th, the 3.2nd, 16 and 24mM aqueous solution 0.2mL, with isopyknic water and Fe3O4/ Ag seed the aqueous solution is comparison, uses
915nm laser irradiates, and observes temperature variations (see accompanying drawing 7).Result shows water and Fe3O4/ Ag seed after irradiation 5
Minute, temperature rising only slightly.And for Fe3O4/ Au-HA NSs, with the prolongation of irradiation time, nano particle is water-soluble
The temperature of liquid substantially increases.And with the increase of Au concentration, the increase of temperature becomes apparent from.
Embodiment 6
Evaluate Fe prepared by the present invention with HeLa cell for model cell3O4The impact of/Au-HA NSs cell proliferation.With aseptic
The Fe of PBS configuration variable concentrations3O4The PBS solution of/Au-HA NSs.HeLa cell seeding after 96 orifice plates with
Fe3O4/ Au-HA NSs nano particle (Au concentration be the 0.2nd, the 0.4th, the 0.6th, the 0.8th, 1.5 and 2.0mM) training altogether at 37 DEG C
Support 24 hours.Then, in cultivation plate hole, add 20 μ L MTT, after continuing to cultivate 4 hours at 37 DEG C, discard cultivation
Liquid, and add 200 μ LDMSO, at 570nm, measure light absorption value after vibrating 15 minutes, and calculate cell according to this value
Vigor (see accompanying drawing 8).The impact of the material cell proliferation of variable concentrations compares with buffer solution PBS for comparison.With PBS
Control group is compared, Fe3O4/ Au-HA NSs is the survival to HeLa cell in the range of 0 to 1.5mM at the experimental concentration of Au
The impact of rate does not has significant difference, even if the experimental concentration of Au reaches 2.0mM, just demonstrates relatively low cytotoxicity.This
Absolutely prove the Fe of synthesis3O4/ Au-HA NSs has good biocompatibility.
Embodiment 7
We verify Fe by cell in vitro MR and CT imaging experiment3O4The special target to the Hela cell for/Au-HA NSs
Property and evaluate the cell MR of nano particle and CT imaging effect prepared by the present invention.U87MG cell or Hela cell are with 2
×106/ hole is planted in 6 orifice plates, after overnight incubation, respectively with PBS, Fe3O4The aseptic PBS of/Au-HA NSs nano particle
Suspension (Fe concentration is 0-0.2, and Au concentration is 0-5.0) co-cultures 6 hours at 37 DEG C, and after cultivation terminates, cell is used
PBS 3 times, then trypsinization, centrifugal, filtration, be finally dispersed in 1mL PBS (containing 0.5% agarose).So
Carry out MR imaging and CT imaging afterwards to cell sample.As it is shown in figure 9, at T2In weighting MR imaging, when the concentration of Fe
When identical, the signal strength signal intensity of HeLa cell be significantly lower than U87MG cell (Fig. 9 a), its corresponding signal value also with image
The result of piece is consistent (Fig. 9 b).In CT imaging, when the concentration of Au is identical, the signal of HeLa cell after NSs process
Intensity is substantially bright (Fig. 9 c) than U87MG cell, by the quantitative analysis to signal strength signal intensity, it has been found that under same concentrations,
The Heng Shi unit (HU) of HeLa cell is all high (Fig. 9 d) than U87MG cell in the concentration range of research.This experimental result
Well demonstrate Fe prepared by the present invention3O4The selectively targeted effect to the HeLa cell for/Au-HA NSs.
Embodiment 8
With HeLa cell as model cell, under 915nm laser irradiates, evaluate Fe3O4Cell is killed work by/Au-HA NSs
With.Fe with aseptic PBS configuration variable concentrations3O4The PBS solution of/Au-HA NSs.Prepared by HeLa cell and the present invention
Fe3O4/ Au-HA NSs (Au concentration be the 0.1st, the 0.2nd, 0.3 and 0.4mM) divides with 915nm laser instrument after co-culturing 6 hours
Not Zhao She 5 minutes and 10 minutes, non-irradiated cell be used as control group.Then, in cultivation plate hole, 20 μ L MTT are added,
After continuing to cultivate 4 hours at 37 DEG C, discard nutrient solution, and add 200 μ L DMSO, at 570nm after vibrating 15 minutes
Place's measurement light absorption value, and the vigor (see accompanying drawing 10) of cell is calculated according to this value.Result shows the increase with concentration, not according to
The cell penetrated remains in that very high cell survival rate；And irradiate 5 minutes through laser, cells show goes out apoptosis trend, and
With the increase of gold concentration, the survival rate of cell is lower；After irradiating 10 minutes, the survival rate of cell substantially reduces, and at gold
When concentration is 0.4mM, the only cell survival of 37.2%.Fe prepared by this experimental result explanation present invention3O4/Au-HA NSs
The apoptosis of cell can be caused under near-infrared laser irradiates.
Embodiment 9
ICP-OES test result shows Fe3O4In/Au-HA NSs, the ratio of Fe element and Au element is 1: 24.7.Pass through knurl
Interior injection Fe3O4The PBS solution ([Fe]=5.0mM, [Au]=123.5mM, 0.1mL) of/Au-HA NSs is evaluated internal swollen
The MR/CT imaging effect (see accompanying drawing 11) at knurl position.Result shows injection nano particle ([Fe]=5.0mM, 0.1mL) 10
After Fen Zhong, before mouse tumor locus is relatively injected, (0 minute) is significantly dimmed, 641 falls before injection for its MR signal strength signal intensity
Low to 41.1 (see accompanying drawing 11a and 11b).In CT imaging, injection nano particle is after 10 minutes, and tumor locus signal is obvious
Increase, tumour high-visible (see accompanying drawing 11c).The signal value (Heng Shi unit) of tumor locus increases to 2303.3 from 157.3HU
HU (see accompanying drawing 11d).The Fe of this group experimental result explanation preparation3O4/ Au-HA NSs has good MR and CT imaging
Energy.
Embodiment 10
Intratumor injection Fe3O4The PBS solution ([Au]=32mM, 0.1mL) of/Au-HA NSs is to the tumor locus of nude mice, so
Irradiate tumor locus with 915nm laser afterwards, evaluated the near infrared imaging effect at in-vivo tumour position by thermal camera, with
When to inject the nude mice of PBS of same volume as a control group (see accompanying drawing 12).Result shows to inject latter 1.5 minutes, injection
Fe3O4The temperature at the mouse tumor position of/Au-HA NSs is significantly raised, has reached 59.1 DEG C, and has now injected PBS mouse
The temperature of tumor locus is only up to 36.2 DEG C.Within a period of time subsequently, inject Fe3O4The mouse tumor of/Au-HA NSs
The temperature at position is maintained at more than 50 DEG C.And the temperature of control mice tumor locus only maintains about 36 DEG C.Experimental result is said
Fe prepared by the bright present invention3O4/ Au-HA NSs can make the temperature of tumor locus raise, thus reaches to kill the effect of tumour cell.
Embodiment 11
The nude mice of load tumour is divided into four groups, respectively through different modes process: (a) intratumor injection PBS but without laser
Irradiating, (b) tumour is only irradiated 10 minutes through laser, Fe prepared by (c) intratumor injection present invention3O4/ Au-HA NSs, (d)
Fe prepared by the intratumor injection present invention3O4/ Au-HA NSs and through laser irradiate 10 minutes.After process, these tumours are removed simultaneously
With HE dyeing, determine whether tumour necrosis (such as Figure 13) occurs by the structure of phase contrast microscope tissues observed.Observed result
There is not necrosis phenomena, well-grown in the nude mouse tumor that display (a), (b) and (c) is organized, and (d) group nude mouse tumor goes out
Now significantly downright bad.Fe prepared by this experimental result explanation present invention3O4/ Au-HA NSs can draw under the irradiation of near-infrared laser
Play the necrosis of tumour cell, thus reach to treat the purpose of tumour.
Embodiment 12
The nude mice of load tumour is divided into four groups (often organizing 4), then respectively through different modes process: (a) intratumor injection is raw
Managing salt solution (PBS) but irradiating without laser, (b) tumour is only irradiated 10 minutes through laser, (c) intratumor injection system of the present invention
Standby Fe3O4/ Au-HA NSs, Fe prepared by (d) intratumor injection present invention3O4/ Au-HA NSs and through laser irradiate 10 points
Clock.Second day, nude mice was through the process identical with the 0th day, but injection PBS or Fe3O4The amount of/Au-HA NSs reduces
Half.Observe and record the gross tumor volume size in different time points for each group of mouse, tumour relative volume=V/V subsequently0.V represents
Nude mice is at the gross tumor volume of different time points, V0Represent the gross tumor volume of the 0th day.It is seen from figure 14 that tri-groups of nude mices of a-c
Gross tumor volume change over time be gradually increased, and the gross tumor volume of d group nude mice is obviously reduced after irradiating, and again
After second time injection nano particle irradiation, gross tumor volume becomes 0.Thus, it could be seen that Fe prepared by the present invention3O4/Au-HA NSs
Can effectively suppress the growth of tumour.
Claims (10)
1. the preparation method of a gold coated iron oxide star Core-shell Structure Nanoparticles, comprising:
(1) claim PEI soluble in water, add AgNO3Solution, stirring, it is subsequently adding the NaBH that ice bath is processed4The aqueous solution, stirs
Mix, dialysis, obtain PEI-Ag nano particle；
(2) by FeCl2·4H2O is dissolved in ultra-pure water, adds NH3·H2O simultaneously stirs, then by mixed solution under air atmosphere
Transfer in autoclave, and the PEI-Ag aqueous solution by preparation in (1) is also added in autoclave, stirs
Mix after mixing, reaction；After reaction terminates, naturally cool to room temperature, precipitation Magneto separate is washed after purification, i.e.
Must be with the ferroferric oxide nano granules Fe of silver seed3O4-PEI-Ag；
(3) claim cetyl trimethylammonium bromide CTAB to be dissolved in the water, add HAuCl4Solution, adds AgNO after stirring3,
Add ascorbic acid AA after being stirred for, when solution becomes colourless, add preparation in (2) of dilution 10 times
Fe3O4-PEI-Ag the aqueous solution；After reaction terminates, centrifuge washing removes CTAB, i.e. obtains star composite nanometer particle
Fe3O4/Au NSs；
(4) claiming PEI soluble in water and being added thereto to methyl thioglycolate reaction, product is dialysed in water three days and is i.e. obtained part mercapto
The PEI PEI-SH of base；
(5) Fe preparing to step (3)3O4/ Au NSs the aqueous solution adds the PEI-SH aqueous solution of preparation in (4), ultrasonic
Rear stirring reaction, obtains the Fe of PEI parcel3O4/Au NSs Fe3O4/Au-PEI NSs；
(6) claim hyaluronic acid HA soluble in water, and with 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC with
N-hydroxysuccinimide NHS activates, and then the HA solution of activation is joined Fe prepared by (5)3O4/Au-PEI
In the NSs aqueous solution, stirring reaction, i.e. obtain the star composite Nano that end-product HA modifies after product centrifuge washing
Grain Fe3O4/Au-HA NSs。
2. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
PEI and AgNO in step (1)3Mol ratio be 1:20；AgNO3And NaBH4Mol ratio be 1:5；PEI and water
Ratio is 1g:20ml；Add AgNO330-60min is stirred after solution；Add NaBH42-4h is stirred after the aqueous solution.
3. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
In step (1), dialysis is for being dialysed 2-5 days by the bag filter of molecular cut off 14000.
4. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
FeCl in step (2)2·4H2O is 1g:6-7ml with the ratio of ultra-pure water；FeCl2·4H2O and NH3·H2The mol ratio of O is
1:5；Reaction temperature is 130-140 DEG C；FeCl2·4H2The mass ratio of O and PEI-Ag is 2.5:1；Mixing time is 5-15min；
Reaction 3-4h.
5. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
CTAB, HAuCl in step (3)4、AgNO3Mass ratio with AA is 381:17.4:1.1:12；Cetyl trimethyl
The ratio of ammonium bromide and water is 30-40mg:1ml；HAuCl4The concentration of solution is 30mg/ml；Cetyl trimethyl bromination
Ammonium and Fe3O4The ratio of-PEI-Ag the aqueous solution is 3000-4000mg:1ml.
6. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
In step (4), the ratio of PEI and water is 1g:10ml；The mol ratio of PEI and methyl thioglycolate is 1:30；Reaction temperature
For 60-80 DEG C；Reaction time is 1-3 days.
7. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
Fe in step (5)3O4The ratio of/Au NSs the aqueous solution and the PEI-SH aqueous solution is 100-200ml:1g.
8. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
In step (5), ultrasonic time is 10-60min；Reaction time is 10-30h.
9. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
In step (6), the molecular weight of HA is 31200；The mol ratio of HA, EDC and NHS is 1:10:10；Soak time is
1-5h；Reaction time is 1-5 days.
10. the preparation method of a kind of gold coated iron oxide star Core-shell Structure Nanoparticles as claimed in claim 1, it is characterised in that:
In step (1) and step (4), the molecular weight of PEI is 25000.
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| CN104548142B (en) * | 2014-12-31 | 2018-05-15 | 东华大学 | A kind of preparation method of hyaluronic acid decorated Superparamagnetic Iron Oxide/gold composite Nano probe |
| CN106323935B (en) * | 2015-07-06 | 2020-06-02 | 中国人民解放军军事科学院军事医学研究院 | Magnetic composite SERS substrate with core-shell-satellite three-dimensional structure and preparation method thereof |
| CN105106957B (en) * | 2015-08-26 | 2023-04-07 | 东华大学 | Gd (OH) 3 Preparation method of @ Au-PEI nanorod-star material |
| CN105396133B (en) * | 2015-11-02 | 2019-02-22 | 上海交通大学 | A kind of multiprobe and preparation method thereof, purposes |
| CN105381474A (en) * | 2015-12-10 | 2016-03-09 | 东华大学 | Preparation method of folic acid modified ferriferrous oxide/gold star-shaped nanoparticles |
| CN105903979B (en) * | 2016-05-13 | 2017-12-26 | 吉林师范大学 | A kind of Fe3O4The preparation method of@Au nucleocapsid functional materials |
| CN109954168B (en) * | 2019-04-11 | 2021-08-27 | 江西理工大学 | Uniformly and rapidly degradable iron-nano hydroxyapatite medical material and preparation method thereof |
| CN111170393B (en) * | 2020-01-07 | 2021-02-26 | 四川大学 | Solar evaporator with hollow structure and preparation method and application thereof |
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