CN104231502A - Dual-targeting near-infrared up-conversion nano material as well as preparation method and application thereof - Google Patents
Dual-targeting near-infrared up-conversion nano material as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a near-infrared rear-earth-doped up-conversion nano material functionalized by folic acid, hyaluronic acid and chitosan as well as a preparation method and application of the near-infrared rear-earth-doped up-conversion nano material. The preparation method comprises the following steps: by taking oleic acid (ligand), alcohol and water as solvents, synthesizing an oleic-acid-coated up-conversion nano particles (OA-UCNPs) under the conditions that Y/Yb/Tm is equal to 80/18/2, Y+Yb+Tm is equal to 0.3-0.5mmol and NaF amount is 3-5mmol; then, carrying out ligand exchange by using polyacrylic acid (PAA) to form PAA-UNCPs, and adopting EDC HCl and NHS to activate the PAA-UNCPs to link a (FA-HA-Chitosan) composite; and finally, carrying out imaging experiments of cancer cells and normal cells, so that the cancer cells and the normal cancers can be distinguished according to gathering effects of luminescent materials.
Description
The present invention obtains state natural sciences fund (subsidy number: 21375095); The special subsidy fund (subsidy number: FANEDD-201023) of National Outstanding Ph.D. Dissertation author; (the subsidy number: 12JCZDJC21700) of Tianjin applied basic research plan main project; Universities in Tianjin " young and middle-aged key innovation personnel training plan " (subsidy number: ZX110GG015); (the subsidy number: TD12-5038) subsidize of Tianjin innovation team of institution of higher education training plan.
Technical field
The invention belongs to fluorescent nano material preparations and applicatio field, be specifically related to the preparation method of the rear-earth-doped up-conversion nano material of a kind of pair of target and the application in cell-targeting imaging thereof.
Background technology
Rare earth ion has abundant level structure and excellent optical characteristics, and up-conversion nano material rear-earth-doped in recent years demonstrates important application prospect in the field such as biomolecular labeling and bio-imaging.Especially near infrared-near infrared up-conversion nano material, it can absorb low-energy long-wave radiation when being subject to optical excitation, and launch high-octane short-wave radiation, there is the advantage that many organic dye and semiconductor-quantum-point do not possess, comprise that fluorescence lifetime is long and flicker free, toxicity are low, fluorescence quantum yield is high, autofluorescence is little, chemical stability and good light stability and depth of penetration large, thus cause the extensive concern of scientific research personnel.
The comparatively common substrate material of rear-earth-doped upconverting fluorescent material has fluorochemical, oxide compound and sulfide etc., and fluorochemical becomes desirable substrate material because phonon energy is low.Current research mainly concentrates on XLnF
4and LnF
3, representative substances is NaFY
4and LaF
3, easily adulterate active ions, dopant ion multiselect Yb
3+, Er
3+, Ce
3+, Tb
3+deng.In recent years, extensive work relate to the research about codoped, as Yb
3+/ Er
3+, Ce
3+/ Tb
3+, Yb
3+/ Er
3+/ Tm
3+, Yb
3+/ Er
3+/ Eu
3+deng.
In recent years, classical organic synthesis is prepared rear-earth-doped up-conversion nano material and is received increasing concern, adopt octadecane, the organic solvents such as ethylene glycol, oleic acid does part, the material particle size of synthesis is even, particle is less, and fluorescence is strong, can be dispersed in water after carrying out ligand exchange, after carrying out functionalization, carry out the targeted imaging of specific cells.
The present invention really studies to deliver on patent basis above and has done very large improvement, first previously will study erbium doped (see patent 201310403495.8) is improved as thulium doped, make light-emitting zone near infrared, drastically increase the penetrance of cell, secondly, oleic acid is adopted to do part, targeted molecular is linked again by the mode of ligand exchange, substantially increase luminous intensity, again, chitosan is adopted to do coated molecule, add cell compatibility, finally, chain two kinds of targeted moleculars, with link compared with a kind of targeted molecular (see patent 201410010713.6) before, substantially increase the targets identification ability of cancer cell molecule.Spent glycol of the present invention, oleic acid and heartily pure water form the solvent system of 16 mL, at Y (80%), Yb(18%), Tm(2%), under the ratio of F/Ln=10/1, pH is 7-8, temperature 160 oC, under reacting the condition of 12 h, synthesize particle diameter at 20-30 nm, the up-conversion nano material of Coated with Oleic Acid, then use PAA (polyacrylic acid) to carry out ligand exchange, synthesis PAA-UCNPs, is then modified FA-HA-Chitosan, material has good biocompatibility, carries out the target imaging of cell.
Summary of the invention
The object of the present invention is to provide that particle diameter is less, size uniform, near-infrared fluorescent are strong, water-soluble and good biocompatibility, anti-interference strong, the preparation method of being convenient to the rear-earth-doped up-conversion nano material of the targeted imaging of deep tissues and cancer cells.Invention also discloses two target near infrared up-conversion nano material in the application for distinguishing in cancer cells and normal cell simultaneously.
Technical scheme of the present invention is:
Two target near infrared up-conversion nano material, is characterized in that it is made up of following raw material:
Oleic acid 4-8mL
Rare earth itrated compound storing solution 0.3-0.5 mmol
NaF 3-5 mmol
Polyacrylic acid 500 mg
EDC
. HCl 100-120mg
NHS 11-15 mg
Chitosan solution 100-120mg
Folic acid 50-80mg
Hyaluronic acid 50-80mg
Wherein said rare earth itrated compound storing solution refers to: yttrium oxide Y
2o
3, ytterbium oxide Yb
2o
3, trioxide Tm
2o
3the aqueous solution after nitric acid treatment.
Described rare earth itrated compound storing solution is through nitric acid treatment yttrium oxide Y
2o
3, ytterbium oxide Yb
2o
3, trioxide Tm
2o
3rear Y/Yb/Tm amount of substance portion rate is: 80:18:2.
Described chitosan solution refers to: chitosan is dissolved in the acetic acid solution of 12mL 1%.
The preparation method of two target near infrared up-conversion nano material, is characterized in that:
1) preparation of the rear-earth-doped up-conversion nano material of near infrared:
A) preparation of rare earth itrated compound storing solution: by 2 mmol yttrium oxide Y
2o
3, 0.45 mmol ytterbium oxide Yb
2o
3, 0.05 mmol trioxide Tm
2o
3, i.e. ratio Y/Yb/Tm=80/18/2 of the amount of substance number of yttrium, ytterbium, thulium, Y+Yb+Tm=5 mmol, weighs up amount of substance, puts into 50 mL containers, adds 5 mL concentrated nitric acids, 10 mL pure water, in stink cupboard 100 DEG C, nitrated, evaporate to dryness; Constant volume, in the volumetric flask of 50 mL, is taken 3-5 mL at every turn and is synthesized, be i.e. Y+Yb+Tm=0.3-0.5 mmol;
B) preparation (OA-UCNPs) of the rear-earth-doped up-conversion nano material of Coated with Oleic Acid: the NaF taking 3-5 mmol, be put in 50 mL containers, add 4-8 mL oleic acid, 4-8 mL ethanol, 0-8mL pure water, at 110 DEG C, the rare earth itrated compound storing solution of 3-5 mL is added under nitrogen protection, stir 0.5 h, forward in the reactor of 25 mL, 12 h are reacted at 160 DEG C, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL pure water, ultrasonic 15 min, continue centrifugal, so wash twice with 3 mL pure water again, transfer on watch-glass, vacuum 50 DEG C of dryings,
C) ligand exchange, the rear-earth-doped up-conversion nano material that synthesis polyacrylic acid is modified: take OA-UCNPs 30 mg prepared by step b), add the polyacrylic acid of 500 mg, proceed in the reactor of 25 mL, 0.5-24 h is reacted, naturally cooling at 200 DEG C;
D) preparation of folic acid hyaluronic acid chitosan complexes (FA-HA-Chitosan): the chitosan of 100-120 mg is dissolved in the acetic acid solution of 12 mL 1%, 50-80 mg folic acid, 50-80 mg hyaluronic acid, 100-120 mg EDC
. the NHS of HCl, 11-15 mg joins in above-mentioned chitosan solution, and pH controls at 7-8, stirring at room temperature 48 h, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL pure water, ultrasonic 15 min, continue centrifugal, so wash twice with 3 mL pure water again, transfer on watch-glass, vacuum 50 DEG C of dryings;
E) preparation of FA-HA-Chitosan-UCNPs: take PAA-UCNPs 6 mg, FA-HA-Chitosan 6 mg, the EDC of 50 mg
. hCl, the NHS of 2.4 mg, be dissolved in the acetic acid solution of 10 mL 1%, stir 48 h, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL pure water, ultrasonic 15 min, continue centrifugal, so wash twice with 3 mL pure water again, transfer on watch-glass, vacuum 50 DEG C of dryings.
The typical preparation method of the present invention is as follows:
1) preparation of the rear-earth-doped up-conversion nano material of near infrared
A) preparation of rare earth itrated compound storing solution: by 2 mmol yttrium oxide Y
2o
3, 0.45 mmol ytterbium oxide Yb
2o
3, 0.05 mmol trioxide Tm
2o
3, namely ratio Y/Yb/Tm=80/18/2, the Y+Yb+Tm=5 mmol of yttrium, ytterbium, thulium amount of substance, weighs up quality, puts into 50 mL small beakers, add 5 mL concentrated nitric acids, 10 mL heartily pure water, in stink cupboard 100 DEG C, nitrated, evaporate to dryness; Constant volume, in the volumetric flask of 50 mL, is taken 3 mL at every turn and is synthesized, be i.e. Y+Yb+Tm=0.3 mmol;
B) preparation of the rear-earth-doped up-conversion nano material of Coated with Oleic Acid (OA-UCNPs): the NaF taking 3 mmol, be put in 50 mL beakers, add 8 mL oleic acid, 4 mL ethanol, 4 mL heartily pure water, at 110 DEG C, the rare earth itrated compound storing solution of 3 mL is added under nitrogen protection, stir 0.5 h, forward in the reactor of 25 mL, 12 h are reacted at 160 DEG C, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings,
C) ligand exchange, the rear-earth-doped up-conversion nano material (PAA-UCNPs) that synthesis polyacrylic acid is modified: take OA-UCNPs 30 mg prepared by step b), add the PAA of 500 mg, proceed in the reactor of 25 mL, 0.5-24 h is reacted at 200 DEG C, naturally cooling, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings;
D) preparation of folic acid hyaluronic acid chitosan complexes (FA-HA-Chitosan): the chitosan of 100 mg is dissolved in the acetic acid solution of 12 mL 1%, the folic acid of 50 mg, the hyaluronic acid of 50 mg, the EDC of 100 mg
. the NHS of HCl, 11.4 mg joins in above-mentioned chitosan solution, and PH controls at 7-8, stirring at room temperature 48 h, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings;
E) preparation of FA-HA-Chitosan-UCNPs: take PAA-UCNPs 6 mg, FA-HA-Chitosan 6 mg, the EDC of 50 mg
. hCl, the NHS of 2.4 mg, be dissolved in the acetic acid solution of 10 mL 1%, stir 48 h, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
The positively effect that the present invention is compared with prior art had is:
(1) preparation method provided by the present invention is by thulium doped, can synthesize the nano material of near infrared pattern rule six side's phases, be convenient to penetrate tissue, carry out deep layer cells imaging (Fig. 1).
(2) preparation method provided by the present invention adopts oleic acid to do the nano material of part synthesis, and the material of method synthesis before luminous strength ratio, luminous intensity is high and stable (Fig. 2).
(3) the present invention has linked chitosan molecule, improves the biocompatibility of material, is convenient to enter cell and carries out imaging.
(4) the present invention utilizes easy method at the surface-functionalized folic acid of up-conversion nano material and hyaluronic acid, is convenient to the targets identification being carried out cancer cells by imaging.
accompanying drawing illustrates:
Fig. 1 is OA-NaYF
4:yb/Tm(Y/Yb/Tm=80/18/2) XRD figure;
Fig. 2 is OA-NaYF
4:yb/Tm(Y/Yb/Tm=80/18/2) TEM figure (left side) and the PEI-NaYF that synthesizes before
4:yb/Er(Y/Yb/Er=80/18/2) contrast of TEM figure (right side) of (patent 201310403495.8), illustrates that material particle size that material synthesized by the present invention and the existing method of having reported for work synthesize is evenly, good dispersion, does not have agglomeration substantially;
Fig. 3 is PAA-NaYF
4:the fluorescence (left side) of Yb/Tm and the FA-UCNPs(patent 201410010713.6 of synthesizing before) fluorescence (right side) contrast, illustrate that the fluorescence of our present synthetic materials goes out peak at 800 nm places, and the material previously synthesized goes out peak at 540 nm and 650 nm places, material fluorescence synthesized by the present invention is now near infrared region, intensity also grow, the penetrativity for cell tissue also improves;
Fig. 4 is the uv-absorbing figure of FA, HA, Chitosan, PAA-UCNPs, prepared-UCNPs;
Fig. 5 is (a is MDA-MB-231 cancer cells light field figure, a ' is 3T3 normal cell details in a play not acted out on stage, but told through dialogues figure for MDA-MB-231 cancer cells details in a play not acted out on stage, but told through dialogues figure, b are 3T3 normal cell light field figure, b ') cell imaging figure of FA-HA-Chitosan-UCNPs.Can scheme bright than b by a figure as apparent from a and b figure, illustrate that material of the present invention has differentiation effect for cancer cell and normal cell, cancer cell surface has folacin receptor and Hyaluronic Acid acceptor (CD44), therefore nano material is assembled many in cancer cell, assemble few at normal cell, under confocal microscope, show as cancer cell much brighter than normal cell, distinguish two kinds of cells whereby, for cancer cell early diagnosis.
Embodiment
By the description carried out its exemplary embodiment below in conjunction with accompanying drawing, the above-mentioned feature and advantage of the present invention will become more clear and easy understand.Be described in further detail the present invention below in conjunction with specific examples, wherein used reagent all has commercially available.
The explanation of abbreviation letter
embodiment 1
1) preparation of OA-UCNPs
By 2 mmol yttrium oxide Y
2o
3, 0.45 mmol ytterbium oxide Yb
2o
3, 0.05 mmol trioxide Tm
2o
3, namely ratio Y/Yb/Tm=80/18/2, the Y+Yb+Tm=5 mmol of yttrium, ytterbium, thulium amount of substance, weigh up quality, put into 50 mL small beakers, add 5 mL concentrated nitric acids, 10 mL heartily pure water, in stink cupboard 100 DEG C, nitrated, evaporate to dryness; Constant volume, in the volumetric flask of 50 mL, is taken 3 mL at every turn and is synthesized, be i.e. Y+Yb+Tm=0.3 mmol;
Take the NaF of 3 mmol, be put in 50 mL beakers, add 8 mL oleic acid, 4 mL ethanol, 4 mL heartily pure water, at 110 oC, the rare earth itrated compound storing solution of 3 mL is added under nitrogen protection, stir 0.5 h, forward in the reactor of 25 mL, 12 h are reacted at 160 DEG C, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
2) preparation of PAA-UCNPs
Take OA-UCNPs 30 mg prepared by embodiment 1, add PAA 500 mg, proceed in the reactor of 25 mL, react 0.5 h at 200 DEG C, naturally cooling, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
3) preparation of FA-HA-Chitosan
The chitosan of 100 mg is dissolved in the acetic acid solution of 12 mL 1%, the folic acid of 50 mg, the hyaluronic acid of 50 mg, the EDC of 100 mg
. the NHS of HCl, 11.4 mg joins in above-mentioned chitosan solution, and PH controls 7, stirring at room temperature 48 h, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
4) preparation of FA-HA-Chitosan-UCNPs
Take PAA-UCNPs 6 mg, FA-HA-Chitosan 6 mg, the EDC of 50 mg
. hCl, the NHS of 2.4 mg, be dissolved in the acetic acid solution of 10 mL 1%, stir 48 h, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
embodiment 2
1) preparation of OA-UCNPs
By 2 mmol yttrium oxide Y
2o
3, 0.45 mmol ytterbium oxide Yb
2o
3, 0.05 mmol trioxide Tm
2o
3, namely ratio Y/Yb/Tm=80/18/2, the Y+Yb+Tm=5 mmol of yttrium, ytterbium, thulium amount of substance, weigh up quality, put into 50 mL small beakers, add 5 mL concentrated nitric acids, 10 mL heartily pure water, in stink cupboard 100 DEG C, nitrated, evaporate to dryness; Constant volume, in the volumetric flask of 50 mL, is taken 5 mL at every turn and is synthesized, be i.e. Y+Yb+Tm=0.5 mmol;
Take the NaF of 5 mmol, be put in 50 mL beakers, add 4 mL oleic acid, 4 mL ethanol, 8 mL heartily pure water, at 110 oC, the rare earth itrated compound storing solution of 5 mL is added under nitrogen protection, stir 0.5 h, forward in the reactor of 25 mL, 12 h are reacted at 160 DEG C, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
2) preparation of PAA-UCNPs
Take OA-UCNPs 30 mg prepared by embodiment 1, add PAA 500 mg, proceed in the reactor of 25 mL, react 8 h at 200 DEG C, naturally cooling, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
3) preparation of FA-HA-Chitosan
The chitosan of 120 mg is dissolved in the acetic acid solution of 12 mL 1%, the folic acid of 80 mg, the hyaluronic acid of 80 mg, the EDC of 120 mg
. the NHS of HCl, 15 mg joins in above-mentioned chitosan solution, and PH controls 8, stirring at room temperature 48 h, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
4) preparation of FA-HA-Chitosan-UCNPs
Take PAA-UCNPs 6 mg, FA-HA-Chitosan 6 mg, the EDC of 50 mg
. hCl, the NHS of 2.4 mg, be dissolved in the acetic acid solution of 10 mL 1%, stir 48 h, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
embodiment 3
1) preparation of OA-UCNPs
By 2 mmol yttrium oxide Y
2o
3, 0.45 mmol ytterbium oxide Yb
2o
3, 0.05 mmol trioxide Tm
2o
3, namely ratio Y/Yb/Tm=80/18/2, the Y+Yb+Tm=5 mmol of yttrium, ytterbium, thulium amount of substance, weigh up quality, put into 50 mL small beakers, add 5 mL concentrated nitric acids, 10 mL heartily pure water, in stink cupboard 100 DEG C, nitrated, evaporate to dryness; Constant volume, in the volumetric flask of 50 mL, is taken 4 mL at every turn and is synthesized, be i.e. Y+Yb+Tm=0.4 mmol;
Take the NaF of 4 mmol, be put in 50 mL beakers, add 8 mL oleic acid, 8 mL ethanol, 0 mL heartily pure water, at 110 DEG C, the rare earth itrated compound storing solution of 4 mL is added under nitrogen protection, stir 0.5 h, forward in the reactor of 25 mL, 12 h are reacted at 160 DEG C, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
2) preparation of PAA-UCNPs
Take OA-UCNPs 30 mg prepared by embodiment 1, add PAA 500 mg, proceed in the reactor of 25 mL, react 24 h at 200 DEG C, naturally cooling, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
3) preparation of FA-HA-Chitosan
The chitosan of 110 mg is dissolved in the acetic acid solution of 12 mL 1%, the folic acid of 60 mg, the hyaluronic acid of 60 mg, the EDC of 110 mg
. the NHS of HCl, 13 mg joins in above-mentioned chitosan solution, and PH controls 8, stirring at room temperature 48 h, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
4) preparation of FA-HA-Chitosan-UCNPs
Take PAA-UCNPs 6 mg, FA-HA-Chitosan 6 mg, the EDC of 50 mg
. hCl, the NHS of 2.4 mg, be dissolved in the acetic acid solution of 10 mL 1%, stir 48 h, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL heartily pure water, ultrasonic 15 min, continue centrifugal, so again with 3 mL heartily pure water wash twice, transfer on watch-glass, vacuum 50 DEG C of dryings.
embodiment 4
adopt human breast cancer cell (MDA-MB-231) and mouse embryo fibroblasts (3T3) on 6 orifice plates after adherent culture 24 h, (substratum is 2 mL to add the FA-HA-Chitosan-UCNPs of 200 uL, the concentration of FA-HA-Chitosan-UCNPs is 200 ug/mL), cultivate 6 h, substratum is sucked with suction pipe, slowly add phosphoric acid buffer (PBS) solution of 2 mL 0.01 M, suck with suction pipe, three times are washed by PBS solution, be placed on imaging under confocal microscope, scanning wavelength is 500-600 nm and 600-700 nm.Experimental result illustrates:
(1) a ' and b ' illustrates the in good condition of two kinds of cells, death of all not breaking;
(2) a and b comparative illustration, a is brighter, and b is poor, can distinguish cancer cells and normal cell.
embodiment 6
Simultaneous test
Above-mentioned comparative result, all illustrates, on cancer cells and Normocellular differentiation, we are very easy to accomplish at the nano material of at present synthesis, compared with existing method, no matter on particle diameter, in intensity, or on targeted imaging, and equal comparative superiority.
Claims (5)
1. pair target near infrared up-conversion nano material, is characterized in that it is made up of following raw material:
Oleic acid 4-8mL
Rare earth itrated compound storing solution 0.3-0.5 mmol
NaF 3-5 mmol
Polyacrylic acid 500 mg
EDC
. HCl 100-120mg
NHS 11-15 mg
Chitosan solution 100-120mg
Folic acid 50-80mg
Hyaluronic acid 50-80mg
Wherein said rare earth itrated compound storing solution refers to: yttrium oxide Y
2o
3, ytterbium oxide Yb
2o
3, trioxide Tm
2o
3the aqueous solution after nitric acid treatment.
2. two target near infrared up-conversion nano material described in claim 1, is characterized in that described rare earth itrated compound storing solution is through nitric acid treatment yttrium oxide Y
2o
3, ytterbium oxide Yb
2o
3, trioxide Tm
2o
3rear Y/Yb/Tm amount of substance portion rate is: 80:18:2.
3. two target near infrared up-conversion nano material described in claim 1, is characterized in that described chitosan solution refers to: chitosan is dissolved in the acetic acid solution of 12mL 1%.
4. the preparation method of two target near infrared up-conversion nano material described in claim 1, is characterized in that being undertaken by following step:
1) preparation of the rear-earth-doped up-conversion nano material of near infrared:
A) preparation of rare earth itrated compound storing solution: by 2 mmol yttrium oxide Y
2o
3, 0.45 mmol ytterbium oxide Yb
2o
3, 0.05 mmol trioxide Tm
2o
3, i.e. ratio Y/Yb/Tm=80/18/2 of the amount of substance number of yttrium, ytterbium, thulium, Y+Yb+Tm=5 mmol, weighs up amount of substance, puts into 50 mL containers, adds 5 mL concentrated nitric acids, 10 mL pure water, in stink cupboard 100 DEG C, nitrated, evaporate to dryness; Constant volume, in the volumetric flask of 50 mL, is taken 3-5 mL at every turn and is synthesized, be i.e. Y+Yb+Tm=0.3-0.5 mmol;
B) preparation (OA-UCNPs) of the rear-earth-doped up-conversion nano material of Coated with Oleic Acid: the NaF taking 3-5 mmol, be put in 50 mL containers, add 4-8 mL oleic acid, 4-8 mL ethanol, 0-8mL pure water, at 110 DEG C, the rare earth itrated compound storing solution of 3-5 mL is added under nitrogen protection, stir 0.5 h, forward in the reactor of 25 mL, 12 h are reacted at 160 DEG C, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, add 3 mL pure water, ultrasonic 15 min, continue centrifugal, so wash twice with 3 mL pure water again, transfer on watch-glass, vacuum 50 DEG C of dryings,
C) ligand exchange, the rear-earth-doped up-conversion nano material that synthesis polyacrylic acid is modified: take OA-UCNPs 30 mg prepared by step b), add the polyacrylic acid of 500 mg, proceed in the reactor of 25 mL, 0.5-24 h is reacted, naturally cooling at 200 DEG C;
D) preparation of folic acid hyaluronic acid chitosan complexes (FA-HA-Chitosan): the chitosan of 100-120 mg is dissolved in the acetic acid solution of 12 mL 1%, 50-80 mg folic acid, 50-80 mg hyaluronic acid, 100-120 mg EDC
. the NHS of HCl, 11-15 mg joins in above-mentioned chitosan solution, and pH controls at 7-8, stirring at room temperature 48 h, is transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL pure water, ultrasonic 15 min, continue centrifugal, so wash twice with 3 mL pure water again, transfer on watch-glass, vacuum 50 DEG C of dryings;
E) preparation of FA-HA-Chitosan-UCNPs: take PAA-UCNPs 6 mg, FA-HA-Chitosan 6 mg, the EDC of 50 mg
. hCl, the NHS of 2.4 mg, be dissolved in the acetic acid solution of 10 mL 1%, stir 48 h, be transferred in the centrifuge tube of 5 mL, 10000 rpm, centrifugal 5 min at 10 DEG C, incline liquid above, adds 3 mL pure water, ultrasonic 15 min, continue centrifugal, so wash twice with 3 mL pure water again, transfer on watch-glass, vacuum 50 DEG C of dryings.
5. two target near infrared up-conversion nano material described in claim 1 is in the application for distinguishing in cancer cells and normal cell.
Priority Applications (1)
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| CN113788954B (en) * | 2021-08-24 | 2022-06-07 | 山东大学 | Chondroitin sulfate polysaccharide probe based on up-conversion nano material and preparation method and application thereof |
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