CN105251005A - Novel light-dependent control living cell dyeing method based on up-conversion and application - Google Patents
Novel light-dependent control living cell dyeing method based on up-conversion and application Download PDFInfo
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Abstract
The invention relates to a novel light-dependent control living cell dyeing method based on up-conversion and application. According to the technical scheme, the method includes the steps of firstly, synthesizing rare earth up-conversion nanoparticles, wherein rare earth salt serves as the raw material, and the rare earth up-conversion nanoparticles are prepared through a solvent thermal method; secondly, decorating the up-conversion nanoparticles wrapped by meso-porous silicon shell layers, surface light-sensitive molecules and targeting molecules, wherein meso-porous silicon wraps the outer layers of the up-conversion nanoparticles through a template method, and then the up-conversion nanoparticles are decorated with azobenzene and folic acid; thirdly, wrapping and burying organic dyes, wherein organic dyes are wrapped and buried in meso-pores. The method has the advantages that a targeting effect is achieved, and tumor parts can be accurately positioned; dyes can not be released until being irradiated by external near infrared rays with the wavelength of 980 nm, and therefore the tumor parts can be accurately, regularly and quantitatively colored; dye delivery carriers can be repeatedly used, raw material consumption and energy consumption are reduced, and cost is reduced.
Description
Technical field
The present invention relates to a kind of light-operated dyestuff release based on upper conversion nano granule and the painted new method of targeting is carried out to living cells, belonging to biotechnology and medical domain.
Background technology
Cell dyeing technology is one of means of the very important scientific researches in field such as life sciences, preclinical medicine and diagnostics and clinical diagnosis.But in living cells; common dye is (as rhodamine and 4'; 6-diamidino-2-phenylindone etc.) be difficult to pass through cell membrane; make the painted very difficult of living cells, so in conventional cell dyeing course, usually need first with the chemical reagent such as formaldehyde, acetone, cell to be fixed; carry out painted with dyestuff to cell again; these chemical reagent can destroy cell membrane usually, and this process also just result in the death of cell, cannot realize the observation of the physiological status of living cells.
At present, although also there is some novel living cells staining reagents in cell dyeing process, can smoothly permeates cell membranes (such as calcein) to realize living cells painted, but these dyestuffs discharge time also often exist " coloration time and dosage uncontrollable; the half-life is short; acellular specificity; cannot carry out painted to intact organism " etc. defect, cause often needing piece of tissue or population of cells to be dispersed into free individual cells at cell dyeing, then carry out painted to it.This colouring method without specificity, cannot distinguish normal cell and tumor cell to cell color, and once can only dye to cell, and undesirable to the Color of division, proliferative cell.Therefore the living cells staining technique of development of new, realizes having important scientific research and clinical value to living cells and even the painted new dyeing technology of live body specific regions.
Summary of the invention
According to the deficiencies in the prior art, we propose new dye technology, mainly concentrate on how effectively in living cells, to deliver any dyestuff, and realize the positioning and quantitative release of dyestuff.
Concrete technical scheme is as follows:
Light-operated living cells colouring method based on upper conversion of the present invention; Step is as follows:
1) utilize nanoscale dye carrier system, by cytoplasm dyestuff or nucleus dyestuff parcel enter nano-particle mesoporous in become a dyestuff storehouse;
2) utilize the targeting modification function of dye carrier system surfaces, by active targeting effect, dyestuff is transported to required biological cells and tissues;
3) utilize upper Switching Core in nano-particle as light source transducer, near infrared light is converted to ultraviolet light and the visible ray of original position, the a large amount of light sensation molecules being positioned at nano-carrier surface conformation under the driving of ultraviolet light and visible ray occurs repeatedly to change, thus promotes the running of molecular motor.Organic dyestuff in nano-particle just optionally can be discharged by the irradiated site and time controlling near infrared light by the present invention, and the controllability finally realizing targeted cells is painted.
Described nano-particle; It comprises rare earth upconversion nano granule daughter nucleus; Core outer wrapping mesoporous silicon oxide shell; Shell layer surface modifies photosensitive molecular diphenyl diimide and targeting folate molecule.
Nanometer grain preparation method of the present invention, step is as follows:
(1) compare for Yttrium chloride(Y2Cl6) according to mass fraction: Ytterbium trichloride: thulium chloride=(500 ~ 750): (150 ~ 250): raw material joins in reactor by 0.3, in reactor, then add aqueous solvent makes above-mentioned substance dissolve completely; In magnetic agitation condition, be heated to boiling, until rare earths salt becomes white solid;
(2) after water evaporate to dryness, being cooled to 60 ~ 80 DEG C, is oleic acid according to volume ratio: the two adds in reactor by octadecylene=2 ~ 3:1 makes white solid dissolve completely;
(3) be NaOH according to mass number: add methanol in the reactor that the two adds by ammonium fluoride=1 ~ 6:3 and make it dissolve completely, the mass ratio=3:1 of ammonium fluoride and above-mentioned thulium chloride; Regulate temperature to 120 ~ 150 DEG C, evacuation 30 ~ 50min, logical argon; Rapid temperature increases, to 260 ~ 300 DEG C, maintains reaction 1 ~ 3h; Add acetone centrifugal purification after end, after vacuum drying treatment, obtain rare earth upconversion nano granule.
The preparation method of described parcel mesoporous silicon oxide shell: adopt disclosed Chang Jin, the invention such as Wang Hanjie (publication number: CN104771756A), chemotherapy rare earth is changed light-operated drug release nanometer formulation and preparation method thereof:
(1) rare earth upconversion nano granule is got: cetyl trimethyl ammonium bromide: sodium hydroxide=1:(50 ~ 200): raw material joins in reactor by (5 ~ 20), then in reactor, add water makes above-mentioned substance dissolve completely, be heated to 50 ~ 80 DEG C under magnetic agitation condition, stir 1 ~ 2h;
(2) then ethyl orthosilicate is added, wherein mass ratio=0.01 ~ the 0.5:1 of ethyl orthosilicate and rare earth upconversion nano granule; After reaction 1 ~ 2h, centrifugal purification;
(3) by the Granular composite in (2) in ethanol, add sodium chloride as template agent removing, wherein mass ratio=10 ~ the 50:1 of sodium chloride and granule, is heated to 50 ~ 80 DEG C under magnetic agitation condition, stir 3 ~ 9h; Centrifugal purification after reacting completely, obtains the upper conversion nano granule that mesoporous silicon shell is coated after product vacuum dried.
Shell layer surface of the present invention modifies the method for photosensitive molecular diphenyl diimide and targeting folate molecule, and step is as follows:
(1) compare for 4-benzene-azo-benzene formyl chloride according to the mass fraction of material: folic acid: 3-aminopropyl triethoxysilane=1:(1 ~ 50): raw material joins in reactor by (1 ~ 6), then in reactor, add solvent dimethyl sulfoxide above-mentioned substance is dissolved completely, under lucifuge condition, stir 5 ~ 10h;
(2) then add according to ratio of quality and the number of copies carbodiimide: N-hydroxy-succinamide=0.4:0.4 ~ 0.8, wherein the ratio of quality and the number of copies of carbodiimide and 4-benzene-azo-benzene formyl chloride is 0.4:1, centrifugal purification after continuation stirring 3 ~ 9h, obtains finishing photosensitive molecular diphenyl diimide and targeting folate molecule shell.
The step that nano-particle of the present invention is mounted with organic dye is as follows:
(1) compare for upper conversion nano nano-particle according to the mass fraction of material: organic dyestuff=1 ~ 10:1 joins in reactor, in reactor, then adds dimethyl sulfoxide solvent disperseed completely by above-mentioned substance;
(2) under magnetic agitation condition, stir 6 ~ 12h, then centrifugal purification, finally obtain the nano-carrier of the light-operated dyestuff release of conversion, product vacuum dried, obtains the nano-carrier that particle size is 80 ~ 150nm.
Colouring method based on upper conversion nano granule of the present invention, step is as follows:
1) by the upper conversion light-operated nano dye carrier granular for preparing and living cells Dual culture to be dyed 0.5 ~ 2h;
2) after nano dye carrier gathers tumor locus specifically under the effect of targeted molecular folic acid, the near-infrared laser with penetration into tissue irradiates;
3) laser penetrating deep tissue is caught by upper conversion nano core, produces corresponding ultraviolet light and visible ray at this position;
4), after ultraviolet light and visible ray are absorbed by the photosensitive micromolecule diphenyl diimide on nano-carrier surface, its conformation occurs repeatedly to change, and promotes molecular motor running, thus controls the release of dyestuff.
The quality taking material in above-mentioned steps is milligram level (mg), and the volume taken is milliliter level (ml).
The invention provides the preparation method of a kind of upper conversion light-operated dyestuff release nano-carrier.As shown in Figure 3, it relates generally to three partial contents that is 1) synthesis of rare earth upconversion nano granule; 2) modification of rare earth upconversion nano particles coat mesoporous silicon shell and surface light sensitive molecule and targeted molecular and the embedding of organic dyestuff.The rare earth that assembling obtains is changed the Stability Analysis of Structures of the nano-carrier of light-operated dyestuff release, uniform particle diameter, there is targeting location, the advantages such as light-operated release.
The present invention first, as the display of Fig. 4 transmissioning electric mirror test result, utilize solvent-thermal method to prepare rare earth upconversion nano granule (A) particle diameter and be less than 50nm, even particle size distribution, the rare earth of preparation is changed light-operated dyestuff release nano-particle (B) particle diameter and be less than 100nm, even particle size distribution.As shown in Fig. 5 fluorescence emission analysis of spectrum, the rare earth upconversion nano carrier granular of preparation under near infrared light, can send the light of multiple color as ultraviolet light and blue light etc.As photo display under Fig. 6 light field and dark fieid conditions, the fluorescence photo (B) under the photomacrograph (A) of upper conversion nano carrier and near-infrared illuminate condition.Show as Fig. 7 carries out cytotoxicity experiment result, the cytotoxicity of the upper conversion nano carrier of preparation is very low.The nano-carrier being mounted with DAPI nucleus dyestuff as Fig. 8 carries out painted to human cervical carcinoma cell (Hela cell), and after giving near infrared light, the coloring effect of cell more obviously (C figure).
Two, beneficial effect of the present invention:
1) main advantage rare earth that the present invention relates to being changed light-operated dyestuff release nano-carrier system comprises: easy and simple to handle, the suitability is strong, cost is low, has good dispersibility in water, can realize the timing of dyestuff at tumor tissues place, quantitative and location release simultaneously.
2) main performance index of the nano-carrier rare earth that the present invention relates to being changed the release of light-operated dyestuff comprises: a) effective grain size is between 80 ~ 150nm, particle diameter evenly, and can according to the constituent of carrier, experiment conditions etc. regulate; B) good stability, can preserve more than at least 2 months in aqueous; C) surface is containing a large amount of targeted molecular folic acid, is easy to the gathering of carrier at tumor tissues place; D) whole preparation process simple and fast, manufacturing cycle is short, and productive rate is high, is applicable to producing in enormous quantities.
The present invention compares with traditional chemotherapy carrier has following advantage:
Accompanying drawing explanation
Fig. 1: that rare earth upconversion nano carrier absorption near infrared light is converted to ultraviolet light and visible ray and activate the mechanism of action figure of azobenzene molecule motor starting.
Fig. 2: the mechanism of action figure of the light-operated dyestuff release of upper conversion nano carrier.
Fig. 3: the preparation method flow chart above changing light-operated dyestuff release nano-carrier.
Fig. 4: the rare earth upconversion nano granule (A) synthesized according to embodiment 1 and prepare according to embodiment 4 and light rare earth changes light-operated dyestuff release nano-carrier granule (B) projection electron microscope photo (morphology analysis).
Fig. 5: rare earth is changed light-operated dyestuff release nano-carrier fluorescence emission analysis of spectrum.
Fig. 6: rare earth is changed the fluorescence photo (B) under the light-operated dyestuff release photomacrograph (A) of nano-carrier and near-infrared illuminate condition.
Fig. 7: cytotoxicity analysis rare earth being changed light-operated dyestuff release nano-carrier.
Fig. 8: the nano-carrier being mounted with DAPI nucleus dyestuff carries out painted to human cervical carcinoma cell (Hela cell), the coloring effect of cell after giving near infrared light.
Detailed description of the invention
In the following examples, the invention will be further elaborated, but the present invention is not limited thereto.
Embodiment 1:
Solvent-thermal method prepares rare earth upconversion nano granule (UCN):
(1) take 24mgYCl36H2O, 100mgYbCl36H2O and 12mgTmCl36H2O, above-mentioned three kinds of materials are joined in 4ml water, in magnetic agitation condition, be heated to boiling until rare earths salt becomes white solid.
(2) after water evaporate to dryness, cool 80 DEG C, add 10ml oleic acid ligand solution with pipet and 5ml octadecylene ligand solution makes white solid dissolve completely;
(3) then add 20mgNaOH and 60mg ammonium fluoride, use 5ml dissolve with methanol; Regulate temperature to 130 DEG C, adopt oil pump evacuation 40min, logical argon; Rapid temperature increases, to 280 DEG C, maintains reaction 1h; Add 30ml acetone centrifugal purification after end, preserve after end product vacuum drying treatment.The rare earth upconversion nano grain diameter of preparation is 20 ~ 30nm.Fig. 4: the rare earth upconversion nano granule synthesized according to embodiment 1.
Embodiment 2:
Solvent-thermal method prepares rare earth upconversion nano granule (UCN):
(1) take 12mgYCl36H2O, 100mgYbCl36H2O and 12mgTmCl36H2O, above-mentioned three kinds of materials are joined in 5ml water, in magnetic agitation condition, be heated to boiling until rare earths salt becomes white solid.
(2) after water evaporate to dryness, cool 70 DEG C, add 15ml oleic acid ligand solution with pipet and 5ml octadecylene ligand solution makes white solid dissolve completely;
(3) then add 10mgNaOH and 60mg ammonium fluoride, use 10ml dissolve with methanol; Regulate temperature to 120 DEG C, adopt oil pump evacuation 50min, logical argon; Rapid temperature increases, to 260 DEG C, maintains reaction 2h; Add 20ml acetone centrifugal purification after end, preserve after end product vacuum drying treatment.
Embodiment 3:
Solvent-thermal method prepares rare earth upconversion nano granule (UCN):
(1) take 24mgYCl36H2O, 50mgYbCl36H2O and 6mgTmCl36H2O, above-mentioned three kinds of materials are joined in 6ml water, in magnetic agitation condition, be heated to boiling until rare earths salt becomes white solid.
(2) after water evaporate to dryness, cool 80 DEG C, add 10ml oleic acid ligand solution with pipet and 5ml octadecylene ligand solution makes white solid dissolve completely;
(3) then add 60mgNaOH and 30mg ammonium fluoride, use 5ml dissolve with methanol; Regulate temperature to 150 DEG C, adopt oil pump evacuation 30min, logical argon; Rapid temperature increases, to 300 DEG C, maintains reaction 3h; Add 40ml acetone centrifugal purification after end, preserve after end product vacuum drying treatment.
Embodiment 4:
The upper conversion nano granule that mesoporous silicon shell is coated and the modification of surface light sensitive molecule and targeted molecular and the filling of rhodamine:
A) rare earth upconversion nano particles coat mesoporous silicon shell (UCNmSiO
2):
(1) according to the quality of material, take 10mg rare earth upconversion nano granule respectively, 2000mg cetyl trimethyl ammonium bromide (CTAB) and 10mg sodium hydroxide join in reactor, then in reactor, add aqueous solvent 30ml makes above-mentioned substance dissolve completely, be heated to 70 DEG C under magnetic agitation condition, stir 1h.
(2) 200ul ethyl orthosilicate (TEOS) is finally added, after reaction 2h, centrifugal purification.
(3) granule prepared is re-dispersed in 40ml ethanol, then adds 0.6g sodium chloride template agent removing, under magnetic agitation condition, be heated to 60 DEG C, stir 6h.Centrifugal purification after reacting completely, preserves after end product vacuum drying treatment.
B) modification of surface light sensitive molecule and targeted molecular:
(1) 50mg4-benzene-azo-benzene formyl chloride is taken respectively, raw material joins in reactor by 50mg folic acid and 20ul3-aminopropyl triethoxysilane, then in reactor, add 5ml dimethyl sulfoxide solvent above-mentioned substance is dissolved completely, under lucifuge condition, stir 8h.
(2) 20mg carbodiimide (EDC) and 30mgN-N-Hydroxysuccinimide (NHS) is then added respectively.After continuing to stir 6h.
(3) the upper conversion nano granule that mesoporous silicon shell that 500mg prepares is coated is added, centrifugal purification after reaction 10h, obtain the nano-particle of the rare earth upconversion nano particles coat mesoporous silicon shell of surface-functionalized modification, preserve after product vacuum dried.
C) filling of DAPI dyestuff:
(1) take rare earth upconversion nano particles coat mesoporous silicon shell nanoparticle and the 1mgDAPI dyestuff of the surface-functionalized modification of 50mg, above-mentioned two kinds of raw materials are added in 5ml alcoholic solution.
(2) under magnetic agitation condition, stir 9h, then centrifugal purification, finally obtain nano-carrier rare earth being changed the release of light-operated dyestuff, preserve after product vacuum dried.
Embodiment 5:
The upper conversion nano granule that mesoporous silicon shell is coated and the modification of surface light sensitive molecule and targeted molecular and the filling of nucleus dyestuff:
A) rare earth upconversion nano particles coat mesoporous silicon shell (UCNmSiO
2):
(1) according to the quality of material, take 10mg rare earth upconversion nano granule respectively, 1000mg cetyl trimethyl ammonium bromide (CTAB) and 10mg sodium hydroxide join in reactor, then in reactor, add aqueous solvent 25ml makes above-mentioned substance dissolve completely, be heated to 80 DEG C under magnetic agitation condition, stir 1h.
(2) 100ul ethyl orthosilicate (TEOS) is finally added, after reaction 2h, centrifugal purification.
(3) granule prepared is re-dispersed in 50ml ethanol, then adds 0.9g sodium chloride template agent removing, under magnetic agitation condition, be heated to 50 DEG C, stir 9h.Centrifugal purification after reacting completely, preserves after end product vacuum drying treatment.
B) modification of surface light sensitive molecule and targeted molecular:
(1) 60mg4-benzene-azo-benzene formyl chloride is taken respectively, raw material joins in reactor by 30mg folic acid and 10ul3-aminopropyl triethoxysilane, then in reactor, add 3ml dimethyl sulfoxide solvent above-mentioned substance is dissolved completely, under lucifuge condition, stir 6h.
(2) 20mg carbodiimide (EDC) and 40mgN-N-Hydroxysuccinimide (NHS) is then added respectively.After continuing to stir 8h.
(3) the upper conversion nano granule that mesoporous silicon shell that 300mg prepares is coated is added, centrifugal purification after reaction 12h, obtain the nano-particle of the rare earth upconversion nano particles coat mesoporous silicon shell of surface-functionalized modification, preserve after product vacuum dried.
C) filling of nucleus dyestuff:
(1) take 3mg4', 6-diamidino-2-phenylindone (DAPI) dyestuff, above-mentioned two kinds of raw materials are added in 6ml alcoholic solution.
(2) under magnetic agitation condition, stir 9h, then centrifugal purification, finally obtain nano-carrier rare earth being changed the release of light-operated dyestuff, preserve after product vacuum dried.
Embodiment 6:
The upper conversion nano granule that mesoporous silicon shell is coated and the modification of surface light sensitive molecule and targeted molecular and the filling of nucleus dyestuff:
A) rare earth upconversion nano particles coat mesoporous silicon shell (UCNmSiO
2):
(1) according to the quality of material, take 5mg rare earth upconversion nano granule respectively, 2000mg cetyl trimethyl ammonium bromide (CTAB) and 20mg sodium hydroxide join in reactor, then in reactor, add aqueous solvent 35ml makes above-mentioned substance dissolve completely, be heated to 50 DEG C under magnetic agitation condition, stir 2h.
(2) 150ul ethyl orthosilicate (TEOS) is finally added, after reaction 1h, centrifugal purification.
(3) granule prepared is re-dispersed in 30ml ethanol, then adds 1.2g sodium chloride template agent removing, under magnetic agitation condition, be heated to 80 DEG C, stir 3h.Centrifugal purification after reacting completely, preserves after end product vacuum drying treatment.
B) modification of surface light sensitive molecule and targeted molecular:
(1) 30mg4-benzene-azo-benzene formyl chloride is taken respectively, raw material joins in reactor by 50mg folic acid and 20ul3-aminopropyl triethoxysilane, then in reactor, add 6ml dimethyl sulfoxide solvent above-mentioned substance is dissolved completely, under lucifuge condition, stir 9h.
(2) 30mg carbodiimide (EDC) and 30mgN-N-Hydroxysuccinimide (NHS) is then added respectively.After continuing to stir 6h.
(3) the upper conversion nano granule that mesoporous silicon shell that 400mg prepares is coated is added, centrifugal purification after reaction 9h, obtain the nano-particle of the rare earth upconversion nano particles coat mesoporous silicon shell of surface-functionalized modification, preserve after product vacuum dried.
C) filling of nucleus dyestuff:
(1) take 5mg4', 6-diamidino-2-phenylindone (DAPI) dyestuff, above-mentioned two kinds of raw materials are added in 9ml alcoholic solution.
(2) under magnetic agitation condition, stir 12h, then centrifugal purification, finally obtain nano-carrier rare earth being changed the release of light-operated dyestuff, preserve after product vacuum dried.
Embodiment 7:
Morphologic observation, particles size and distribution measure.Extracting sample solution is after ultracentrifugation is separated, and take out precipitate, adding distil water makes dispersion on a small quantity, drips sample preparation on carbon supporting film, observes its pattern state and take pictures under transmission electron microscope.Observe under transmission electron microscope on chemotherapy rare earth and change light-operated drug release nanometer formulation granule in evenly regular spheroidal particle.The chemotherapy rare earth that experiment records changes light-operated drug release nanometer formulation even particle size distribution, controlled within the scope of 80 ~ 150nm.Obtained nanoparticle as shown in Figure 4.
Embodiment 8:
Mtt assay tests the cytotoxicity light-operated thermotherapy targeting rare earth being changed-Jin core-shell nano preparation:
1) get the l cell being in exponential phase, fully blow and beat into single cell suspension after 0.125% trypsinization, after counting, adjustment cell concentration is 5 × 10
4/ m1.
2) in every hole of 96 orifice plates, 100 μ l cell suspension are added, at 37 DEG C, 5%CO
224h is cultivated in incubator.
3) 120 μ l are contained certain density chemotherapy rare earth changes light-operated drug release nanometer formulation solution join in fresh culture, and at 37 DEG C, 5%CO
2logical co-culture of cells 24h in incubator.
4) culture plate is taken out after 24h, 10 μ lMTT solution are added in each hole, at 37 DEG C, after continuing to cultivate 4h in 5%CO2 incubator, culture medium in careful absorption culture hole, in each hole, add 200 μ lDMSO (dimethyl sulfoxide) solution, shaking table shakes 20min, make DMSO solution dissolve Formazan crystal fully.
5) use enzyme-linked immunosorbent assay instrument at 570nm wavelength place, with blank tube zeroing, measure the light absorption value A in each hole, be calculated as follows cell survival rate.Often group establish 8 parallel, calculate its meansigma methods.
Cell survival rate (%)=experimental cell group light absorption value (A2)/blanc cell group light absorption value (A1) * 100%
Embodiment 9:
The nano-carrier being mounted with DAPI nucleus dyestuff carries out painted to the Hela cell of living:
1) Hela is pressed 1 × 10
5cells/well uniform spreading in 24 porocyte culture plates, at 37 DEG C, 5%CO
2cultivate 24 hours in incubator.
2) get 0.1 milligram of nano-carrier prepared by embodiment 3, be added in the DMEM cell culture fluid of 0.5 milliliter and fully mix.
3) take out Tissue Culture Plate, suck culture fluid, every hole adds 2) in nano-carrier and cell culture fluid, in 37 DEG C, 5%CO
2after continuing to cultivate 4h in incubator, with the 980nm near infrared light intermittent irradiation 5 minutes of 3.5W, and then static 10 minutes, the last careful culture medium drawn in culture hole, in each hole, add 1 milliliter of PBS clean 3 times, under fluorescence microscope, detect nuclear coloring case.
Fig. 8: the nano-carrier being mounted with DAPI nucleus dyestuff carries out painted to human cervical carcinoma cell (Hela cell), the coloring effect of cell after giving near infrared light.
Claims (7)
1. one kind based on rare earth is changed light-operated dyestuff deliver nano-particle; It is characterized in that comprising rare earth upconversion nano granule daughter nucleus; Core outer wrapping mesoporous silicon oxide shell; Shell layer surface modifies photosensitive molecular diphenyl diimide and targeting folate molecule.
2. rare earth as claimed in claim 1 changes light-operated dyestuff and delivers nano-carrier conversion method, it is characterized in that step is as follows:
1) by the mode administration by intravenous injection or local injection of the light-operated nano dye carrier granular of upper conversion for preparing;
2) after nano dye carrier gathers tumor locus specifically under the effect of targeted molecular folic acid, there is the irradiation of the near-infrared laser of stronger penetration into tissue;
3) laser penetrating deep tissue is caught by upper conversion nano core, produces corresponding ultraviolet light and visible ray at this position;
4), after ultraviolet light and visible ray are absorbed by the photosensitive micromolecule diphenyl diimide on nano-carrier surface, its conformation occurs repeatedly to change and serves as the effect of a molecular motor, thus controls the release of dyestuff.
3. the rare earth of claim 1 is changed light-operated dyestuff and delivers nano-carrier preparation method, it is characterized in that step is as follows:
1) synthesis of rare earth upconversion nano granule: utilize various rare-earth salts for raw material, prepare rare earth upconversion nano granule by solvent thermal;
2) rare earth upconversion nano granule outer cladding mesoporous silicon shell: adopt the synthesising mesoporous silicon layer of sol-gal process;
3) modification of silicon shell layer surface photosensitive molecular and targeted molecular is carried out: surface folate-targeted molecule and photosensitive azobenzene molecule being connected to mesoporous silicon shell;
4) organic dyestuff dyestuff is embedded into upper conversion nano carrier mesoporous in.
4. method as claimed in claim 3, is characterized in that the synthetic method of described rare earth upconversion nano granule is as follows:
(1) compare for Yttrium chloride(Y2Cl6) according to mass fraction: Ytterbium trichloride: thulium chloride=(500 ~ 750): (150 ~ 250): raw material joins in reactor by 0.3, in reactor, then add aqueous solvent makes above-mentioned substance dissolve completely; In magnetic agitation condition, be heated to boiling, until rare earths salt becomes white solid;
(2) after water evaporate to dryness, being cooled to 60 ~ 80 DEG C, is oleic acid according to volume ratio: the two adds in reactor by octadecylene=2 ~ 3:1 makes white solid dissolve completely;
(3) be NaOH according to mass number: add methanol in the reactor that the two adds by ammonium fluoride=1 ~ 6:3 and make it dissolve completely, the mass ratio=2 ~ 3:1 of ammonium fluoride and above-mentioned thulium chloride; Regulate temperature to 120 ~ 150 DEG C, evacuation 30 ~ 50min, logical argon; Rapid temperature increases, to 260 ~ 300 DEG C, maintains reaction 1 ~ 3h; Add acetone centrifugal purification after end, after vacuum drying treatment, obtain rare earth upconversion nano granule.
5. method as claimed in claim 3, is characterized in that the preparation method of described upper conversion nano granule outer cladding mesoporous silicon shell is as follows:
(1) be rare earth upconversion nano granule according to the ratio of quality and the number of copies of material: cetyl trimethyl ammonium bromide: sodium hydroxide=1:(50 ~ 200): raw material joins in reactor by (5 ~ 20), then in reactor, add water makes above-mentioned substance dissolve completely, be heated to 50 ~ 80 DEG C under magnetic agitation condition, stir 1 ~ 2h;
(2) then ethyl orthosilicate is added, wherein mass ratio=0.01 ~ the 0.5:1 of ethyl orthosilicate and rare earth upconversion nano granule; After reaction 1 ~ 2h, centrifugal purification;
(3) by the whole Granular composite in (2) in ethanol, add sodium chloride as template agent removing, wherein mass ratio=10 ~ the 50:1 of sodium chloride and granule, is heated to 50 ~ 80 DEG C under magnetic agitation condition, stir 3 ~ 9h; Centrifugal purification after reacting completely, obtains the upper conversion nano granule that mesoporous silicon shell is coated after product vacuum dried.
6. method as claimed in claim 3, is characterized in that the method for the modification of described silicon shell layer surface photosensitive molecular and targeted molecular, it is characterized in that step is as follows:
(1) compare for 4-benzene-azo-benzene formyl chloride according to the mass fraction of material: folic acid: 3-aminopropyl triethoxysilane=1:(1 ~ 50): raw material joins in reactor by (1 ~ 6), then in reactor, add solvent dimethyl sulfoxide above-mentioned substance is dissolved completely, under lucifuge condition, stir 5 ~ 10h;
(2) then add according to ratio of quality and the number of copies carbodiimide: N-hydroxy-succinamide=0.4:0.4 ~ 0.8, wherein the ratio of quality and the number of copies of carbodiimide and 4-benzene-azo-benzene formyl chloride is (0,1 ~ 0.4): 1, continues stirring 3 ~ 9h;
(3) then add the upper conversion nano granule that mesoporous silicon shell is coated, wherein mass ratio=10 ~ the 50:1 of nano-particle and 4-benzene-azo-benzene formyl chloride, centrifugal purification after reaction 5 ~ 10h, obtain rare earth being changed light-operated dyestuff release nano-carrier.
7. method as claimed in claim 3, it is characterized in that described organic dyestuff dyestuff be embedded into conversion nano carrier mesoporous in method: step is as follows:
(1) compare for upper conversion nano carrier according to the mass fraction of material: organic dyestuff=1 ~ 10:1 joins in reactor, in reactor, then adds dimethyl sulfoxide solvent disperseed completely by above-mentioned substance;
(2) under magnetic agitation condition, stir 6 ~ 12h, then centrifugal purification, finally obtain the nano-carrier of the light-operated dyestuff release of conversion, product vacuum dried, obtains the nano-carrier that particle size is 80 ~ 150nm.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779498A (en) * | 2016-04-05 | 2016-07-20 | 天津大学 | Preparation method and application of rare earth praseodymium element doped red long-afterglow gene nanometer carrier |
| CN105861549A (en) * | 2016-04-05 | 2016-08-17 | 天津大学 | Preparation method and application of blue upconversion nano gene vector doped with rare earth thulium element |
| CN105903016A (en) * | 2016-06-12 | 2016-08-31 | 哈尔滨工业大学 | Preparing method of core-shell structure drug carrier with near-infrared light exciting supermolecule valve light control drug release |
| CN108148570A (en) * | 2017-12-10 | 2018-06-12 | 湖北工业大学 | A kind of preparation method of rare earth fluoride/poly- azobenzene/n-isopropyl acrylamide composite multifunction nano-particle |
| CN108277003A (en) * | 2018-02-02 | 2018-07-13 | 陕西师范大学 | Method based on solvent-thermal method controllable preparation rare earth luminescent material |
| CN109498807A (en) * | 2018-08-15 | 2019-03-22 | 西安电子科技大学 | The non-oxygen dependence photodynamics diagnosis and treatment probe of up-conversion nanoparticles and preparation method |
| CN109620957A (en) * | 2019-01-21 | 2019-04-16 | 天津大学 | The mesoporous silicon oxide of load indocyanine green superscribes the preparation method of conversion nano particle |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779498A (en) * | 2016-04-05 | 2016-07-20 | 天津大学 | Preparation method and application of rare earth praseodymium element doped red long-afterglow gene nanometer carrier |
| CN105861549A (en) * | 2016-04-05 | 2016-08-17 | 天津大学 | Preparation method and application of blue upconversion nano gene vector doped with rare earth thulium element |
| CN105779498B (en) * | 2016-04-05 | 2019-05-21 | 天津大学 | The preparation method and application of the red long-afterglow gene nano carrier of praseodymium element doping |
| CN105861549B (en) * | 2016-04-05 | 2019-12-10 | 天津大学 | Preparation method and application of blue up-conversion nano gene vector doped with rare earth thulium element |
| CN105903016A (en) * | 2016-06-12 | 2016-08-31 | 哈尔滨工业大学 | Preparing method of core-shell structure drug carrier with near-infrared light exciting supermolecule valve light control drug release |
| CN105903016B (en) * | 2016-06-12 | 2018-12-11 | 哈尔滨工业大学 | A kind of preparation method of the nuclear shell structure drug carrier of the near infrared light excitation light-operated drug release of supermolecule valve |
| CN108148570A (en) * | 2017-12-10 | 2018-06-12 | 湖北工业大学 | A kind of preparation method of rare earth fluoride/poly- azobenzene/n-isopropyl acrylamide composite multifunction nano-particle |
| CN108148570B (en) * | 2017-12-10 | 2020-04-17 | 湖北工业大学 | Preparation method of rare earth fluoride/polyazobenzene/N-isopropylacrylamide composite multifunctional nanoparticles |
| CN108277003A (en) * | 2018-02-02 | 2018-07-13 | 陕西师范大学 | Method based on solvent-thermal method controllable preparation rare earth luminescent material |
| CN109498807A (en) * | 2018-08-15 | 2019-03-22 | 西安电子科技大学 | The non-oxygen dependence photodynamics diagnosis and treatment probe of up-conversion nanoparticles and preparation method |
| CN109498807B (en) * | 2018-08-15 | 2021-09-14 | 西安电子科技大学 | Up-conversion nanoparticle oxygen-independent photodynamic diagnosis and treatment probe and preparation method thereof |
| CN109620957A (en) * | 2019-01-21 | 2019-04-16 | 天津大学 | The mesoporous silicon oxide of load indocyanine green superscribes the preparation method of conversion nano particle |
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