CN108853497A - Light power nano-probe is targeted based on upper conversion nano particle and ultra-thin silica layer building - Google Patents

Light power nano-probe is targeted based on upper conversion nano particle and ultra-thin silica layer building Download PDF

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CN108853497A
CN108853497A CN201810724282.8A CN201810724282A CN108853497A CN 108853497 A CN108853497 A CN 108853497A CN 201810724282 A CN201810724282 A CN 201810724282A CN 108853497 A CN108853497 A CN 108853497A
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ucnps
sio
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peg
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CN108853497B (en
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王宗花
岳姿宏
宋昕玥
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Qingdao University
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    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/008—Two-Photon or Multi-Photon PDT, e.g. with upconverting dyes or photosensitisers
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    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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Abstract

The invention discloses one kind to target light power nano-probe based on upper conversion nano particle and ultra-thin silica layer building, which includes upper conversion nano particle kernel, ultra-thin silica shell, the methylene blue loaded on the shell and the polyethylene glycol-folic acid for being wrapped in the core surface.The UCNPs@SiO of building2/ MB/PEG-FA nano-probe is distributed in cancer cell matter by receptor mediated endocytosis, and active oxygen is generated under the excitation of near infrared light, reduces intracellular mitochondrial film potential, induces the irreversible natural death of cerebral cells of cancer cell.The UCNPs@SiO of building2/ MB/PEG-FA nano-probe also shows excellent tumor inhibitory effect in animal experiments.

Description

Light power nanometer is targeted based on upper conversion nano particle and ultra-thin silica layer building Probe
Technical field
The present invention relates to materials and analytical chemistry field, are specifically related to the novel use folic acid-polyethylene glycol of one kind and repair The upper conversion nano particle of the ultra-thin coated with silica of decorations realizes effective optical dynamic therapy as photosensitive agent carrier.
Background technique
As the cancer treatment method clinically ratified, optical dynamic therapy uses quick dose of phot-luminescence (photosensitizer, PS) generates active oxygen, and then irreversibly injuring tumor cell.Optical dynamic therapy can control light It is radiated at specific tumor locus, reduces the potential toxic side effect of normal tissue.However, it still suffers from some challenges such as The limited penetration depth of ultraviolet-visible light, low active oxygen formation efficiency, active oxygen short half-life period and limited action range. Cell and tissue are small to the absorption and scattering of near infrared light, and therefore, near infrared light has superior biological tissue's penetration capacity, Autofluorescence background is small.Therefore, its penetration depth and sensitivity will be increased using near infrared light as the light source of optical dynamic therapy.Base In two-photon or multi-photon mechanism, up-conversion nanoparticles (upconversion nanoparticles, UCNPs) can will connect Continuous near-infrared excitation light is converted into the ultraviolet-visible area fluorescence of Wavelength tunable, is then based on luminescence resonance energy transfer mechanism PS is excited to realize the light activated optical dynamic therapy of near-infrared.UCNPs is got the attention as emerging photosensitive agent carrier.Mesh Before, three kinds of methods, which are generallyd use, by PS is carried on UCNPs, respectively silicon dioxide carried, non-covalent bond physical absorption and covalently Key coupling.Silica (SiO2) silicon layer has many advantages, such as hypotoxicity, is easy to be functionalized and high load amount, it is raw on the surface UCNPs Long SiO2Silicon layer can satisfy the requirement of different PS molecule loads.However, the PDT based on UCNPs have certain problems as The quantum yield of UCNPs is relatively low, and luminescence resonance energy transfer efficiency is limited, and photosensitizer is easily assembled.Luminescence resonance energy turns Efficiency is moved dependent on energy donor at a distance from receptor, in both effective energy transfers usually generation distance 10nm.In general, SiO2 Silicon layer controls in the range of 20-100nm, therefore the PS molecule only near the surface UCNPs is in effective energy transfer model It encloses, being in can excited state.And diameter is easily affected by environment less than the fluorescence of the UCNPs of 10nm and quenches.
Summary of the invention
For the prior art, inventors believe that the control surface UCNPs SiO2Silicon layer thickness is sent out between improving UCNPs and PS The effective measures of photoresonance energy transfer.Present inventors have proposed use thin SiO2The UCNPs, i.e. UCNPs@SiO of silicon layer package2 The strategy of effective PDT is realized as effective PS carrier.
Methylene blue (methylene blue, MB) can produce a large amount of active oxygen (ROS), imitate with high light power Rate, and MB easily propagates through the inducible cancer cell of optical dynamic therapy that cell membrane, targetted mitochondria, therefore MB are participated in and mitochondria occurs Dependence apoptosis.However, MB monomer, which is easy aggregation, forms dimer, electron transfer reaction easily occurs rather than turns with the energy of oxygen It moves, and its Uv absorption peak becomes 610nm, is mismatched with launch wavelength (λ em=650nm) spectrum of UCNPs, it is dynamic to reduce light Power therapeutic effect.Therefore, monodispersed UCNPs@SiO is synthesized2Optical dynamic therapy effect can be improved in/MB.
Polyethylene glycol (PEG) is a kind of good hydrophilic modifier, and UCNPs@SiO can be improved2Colloidal stability, prevent The only non-specific binding between cell, haemocyanin extends blood circulation time, improves cancer target curative effect.In addition, in drug In delivery process, there is important meaning for cancer therapy using the targeting of ligands specific selective binding cancer cell surfaces Justice.Folic acid (FA) is a kind of 441Da vitamin, folacin receptor α (FR- α) high-affinity knot with cancer cell surfaces overexpression It closes.Therefore, biocompatibility will be enhanced with PEG and FA modified medicaments carrier and improves receptor mediated endocytosis.
Therefore, an object of the present invention is to provide a kind of UCNPs@SiO2/ MB/PEG-FA nano-probe, passes through receptor The endocytosis of mediation, UCNPs@SiO2/ MB PEG-FA nano-probe is selectively entered in the cytoplasm of cancer cell.When by When near-infrared (NIR) light excites, energy transfer is apart from short and with good between UCNPs (energy donor) and MB (energy acceptor) Spectral matching, therefore efficient luminescence resonance energy transfer occurs, active oxygen is generated, the cancer cell that inducing mitochondrial mediates withers It dies.
The second object of the present invention is to provide the UCNPs@SiO2The preparation method of/MB/PEG-FA nano-probe.
The third object of the present invention is to provide the UCNPs@SiO2The application of/MB/PEG-FA nano-probe.
To achieve the above object, the present invention specifically uses following technical scheme：
Firstly, the present invention provides a kind of UCNPs@SiO2/ MB/PEG-FA nano-probe, the nano-probe include upper turn Change nano particle kernel, the ultra-thin silica shell for being wrapped in the core surface, the methylene loaded on the shell Blue and polyethylene glycol-folic acid.
Secondly, the present invention provides the UCNPs@SiO2The preparation method of/MB/PEG-FA nano-probe, this method packet It includes
(1)UCNPs@SiO2The preparation of/MB：
Hexamethylene and surfactant are mixed to form reverse micelle, UCNPs is added and is mixed, forms mixture It is one；
Methylene blue and ammonia spirit are added into mixed system one, is mixed, forms mixed system two；
Silicon source is added into mixed system two, the negative electricity silicon source of hybrid reaction, partial hydrolysis is transferred to the hydrophilic of reverse micelle Inside with positively charged methylene blue to interact, by hydrolyzing and being condensed to yield UCNPs@SiO2/MB；
(2)UCNPs@SiO2The preparation of/MB/PEG-FA：
The PEG-FA of carboxylated is subjected to activated carboxylic, the mixture after activation is then added to the UCNPs@ SiO2In/MB, UCNPs@SiO is prepared in hybrid reaction2/MB@PEG-FA。
Finally, the present invention provides the UCNPs@SiO2/ MB/PEG-FA nano-probe is preparing photodynamic therapy agent In application.
Compared with prior art, technical solution of the present invention has the advantages that：
(1) in the specific embodiment of the present invention, it is prepared for the upper conversion nano of two kinds of light emitting ionic codopes Particle (UCNPs) is to enhance red fluorescence intensity, then with ultra-thin silica (SiO2) layer load photosensitizer methylene blue (MB)。
Upper conversion nano particle (UCNPs) serves as energy donor excitation energy receptor methylene blue (MB) molecule and generates induction The active oxygen of Apoptosis.Ultra-thin silica (SiO2) layer furthers the distance of energy donor and receptor, improve light emitting resonator energy Measure transfer efficiency.
Polyethylene glycol-folic acid (PEG-FA) is covalently attached to UCNPs@SiO2Surface increases nano-probe to cancer cell Biocompatibility and targeting.
(2) the UCNPs@SiO constructed2It is thin that/MB/PEG-FA nano-probe by receptor mediated endocytosis is distributed in cancer In born of the same parents' cytoplasm, active oxygen is generated under the excitation of near infrared light, reduces intracellular mitochondrial film potential, induction cancer cell can not Inverse natural death of cerebral cells.The UCNPs@SiO of building2/ MB/PEG-FA nano-probe also shows excellent tumour in animal experiments Inhibitory effect.
Detailed description of the invention
The Figure of description for constituting present invention a part is used to provide further understanding of the present invention, signal of the invention Examples and descriptions thereof are used to explain the present invention for property, does not constitute improper limitations of the present invention.
Fig. 1：Nano-probe phenogram.(a) UCNPs (NaYF of Er is mixed4@NaYF4:Yb,Er@NaYF4) and Ho, Tm doping UCNPs (NaYF4@NaYF4:Yb,Ho/Tm@NaYF4) relative intensity of fluorescence, doping ratio Y:Yb:Er=80:18:2 (molar ratio), Ho and Tm doping ratio are Y:Yb:Ho:Tm=77.8:20:2:0.2 (molar ratio)；(b) Ho, Tm are co-doped with UCNP TEM image；(c)UCNP@SiO2TEM image；(d) UCNPs and UCNPs@SiO2XRD diagram picture.
Fig. 2：Nano-probe generates capable of trying hard to for ROS.(a)UCNPs@SiO2, UCNPs@SiO2/ MB and UCNPs@SiO2/ The relative luminous intensity of MB@PEG-FA, the Relative Absorbance value of MB；(b) when with 1.0mg/mLUCNPs@SiO2/MB@PEG-FA When nano-probe mixes, 980nm laser (1.5W/cm is then used2) irradiation 65 minutes ABDA fluorescence spectrum；(c) MB and UCNPs@SiO2@PEG-FA is in 980nm laser (1.5W/cm2) 65 minutes fluorescence intensities to ABDA of irradiation influence；UCNPs@ SiO2Influence of/MB@the PEG-FA under no laser irradiation to ABDA fluorescence intensity；(d)1.0mg/mL UCNPs@SiO2/MB@ PEG-FA nano-probe is in 1.5W/cm2Irradiation under, the influence to ABDA fluorescence, laser alternation switch 10min.
Fig. 3：Probe enters the ability of living cells.It is small that MCF-7 cell (a) 0 is incubated for the nano-probe of 80.0 μ g/mL preparation When；(b) 4 hours；(c) 6 hours.Focus on blue channel (450nm-510nm) and green channel altogether with two-photon laser The fluorescence signal of (515nm-575nm) record nano-probe.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation and/or their combination.
As background technique is introduced, there are certain deficiencies for quick dose of phot-luminescence in the prior art, in order to solve Technical problem as above, the invention proposes a kind of UCNPs SiO2/ MB/PEG-FA nano-probe, the nano-probe include upper Conversion nano particle kernel (UCNPs), the ultra-thin silica shell for being wrapped in the core surface (form UCNPs@SiO2)、 The methylene blue loaded on the shell (forms UCNPs@SiO2/ MB) and polyethylene glycol-folic acid.
Upper conversion nano particle is made of host material, sensitizer and activator, the upper conversion nano in the present invention Grain is not particularly limited, and host material includes but is not limited only to halide, oxide, sulfide or oxysulfide, Sensitizer includes but is not limited only to Yb3+、Gd3+Deng one of or it is a variety of, activator includes but is not limited only to Er3+、Ho3+、 Tm3+、Nd3+, Pr3+Deng one of or it is a variety of.But the present invention is in order to enhance red fluorescence intensity, in an implementation of the invention In mode, the host material selected is NaYF4, sensitizer Yb3+, activator Ho3+And Tm3+.I.e. above conversion nano particle is NaYF4:Yb3+,Ho3+/Tm3+, conversion nano particle such as Fig. 1 (b) is shown on this, partial size about 10~40nm, and pattern is uniform, dispersed It is good；Preferably, the NaYF of upper conversion nano particle selection oleic acid protection4:Yb3+,Ho3+/Tm3+；Rare earth ion doped molar ratio Example be：Yb:18~20%；Ho:2~5%, Tm:0.2~0.8%.
There are many synthetic method of UCNPs, including the precipitation method, sol-gel method, micro emulsion method, combustion method, hydro-thermal (solvent heat) Method and pyrolysis method etc., conversion nano particle can be obtained by preparation method conventional in the prior art on this, herein and be not required to It is particularly limited to.
In an embodiment of the invention, the thickness of the ultra-thin silica shell is less than 10.0nm；Preferably, The ultra-thin silica shell with a thickness of 5.0~7.5nm.The ultra-thin silica shell furthers energy donor and receptor Distance improves luminescence resonance energy transfer efficiency.
In an embodiment of the invention, the polyethylene glycol-folic acid passes through amidation process for polyethylene glycol-leaf Acid is coated in UCNPs@SiO2On the surface of/MB.
In an embodiment of the invention, the degree of polymerization of the polyethylene glycol is 1500~2500.
In of the invention one typical embodiment, the UCNPs@SiO is provided2/ MB/PEG-FA nano-probe Preparation method, this method includes：
(1)UCNPs@SiO2The preparation of/MB：
Hexamethylene and surfactant are mixed to form reverse micelle, UCNPs is added and is mixed, forms mixture It is one；
Methylene blue and ammonia spirit are added into mixed system one, is mixed, forms mixed system two；
Silicon source is added into mixed system two, the negative electricity silicon source of hybrid reaction, partial hydrolysis is transferred to the hydrophilic of reverse micelle Inside with positively charged methylene blue to interact, by hydrolyzing and being condensed to yield UCNPs@SiO2/MB；
(2)UCNPs@SiO2The preparation of/MB/PEG-FA：
The PEG-FA of carboxylated is subjected to activated carboxylic, the mixture after activation is then added to the UCNPs@ SiO2In/MB, UCNPs@SiO is prepared in hybrid reaction2/MB@PEG-FA。
In an embodiment of the invention, in step (1), surfactant is Igepal CO-520 (NP-5), institute Stating silicon source is tetraethyl orthosilicate (TEOS) and amination silicon source (3- aminopropyl) triethoxysilane (APTES).
In an embodiment of the invention, in step (1), the hexamethylene, surfactant and UCNPs feed intake Ratio is (8~12) mL：(0.3~0.8) mL：(0.3~0.5) mmol；Hexamethylene and surfactant be mixed 1~2h with Form reverse micelle；UCNPs is added, 1~2h is mixed.
In an embodiment of the invention, in step (1), the methylene blue, ammonia spirit and UCNPs feed intake Ratio is (2~6) mg：(40~80) μ L：(0.3~0.5) mmol；2~3h is mixed.
In an embodiment of the invention, in step (1), described TEOS, APTES and the ingredient proportion with UCNPs For (60~100) μ L：(10~40) μ L：(0.3~0.5) mmol；24~36h of hybrid reaction.
In an embodiment of the invention, in step (2), activation the specific steps are：By COOH-PEG-FA, NHS With EDC with molar ratio (1~2):(2~4):(1~2) mixing in dimethyl sulfoxide (DMSO), activates 1~2h.
In an embodiment of the invention, mixture and UCNPs@SiO in step (2), after the activation2/MB Ingredient proportion be (200~300) μ L：(1~3) mL, 10~14h of hybrid reaction.
In another exemplary embodiment of the invention, the UCNPs@SiO is provided2/ MB/PEG-FA nano-probe exists Prepare the application in photodynamic therapy agent.
In order to enable those skilled in the art can clearly understand technical solution of the present invention, below with reference to tool The embodiment of the body technical solution that the present invention will be described in detail.
Experiment reagent and material in the present embodiment：
Rare earth oxide (including Y of the purity greater than 99.99 (w/w) %2O3, Yb2O3、Er2O3、Ho2O3And Tm2O3) be purchased from Sigma-Aldrich company.Octadecene, oleic acid and methylene blue are provided by Aladdin reagent Co., Ltd (Chinese Shanghai), And remaining analysis pure chemistry reagent is provided by Sinopharm Chemical Reagent Co., Ltd. (Chinese Shanghai).Use OKP purification system (Chinese Shanghai Lake instrument company) provides ultrapure water.Mouse is purchased from dimension Bioisystech Co., Ltd of tonneau China (Wuhan, China). In addition, Linyi University's animal protection and animal feeding and processing routine being examined and having been ratified using the committee.
The NaYF of 1 oleic acid of embodiment protection4:Yb3+,Ho3+/Tm3+The preparation of UCNPs：
(1) preparation of rare earth oleate
Weigh 1.129gY2O3(5mmol) and 20mL concentrated hydrochloric acid in a round bottom flask, cover preservative film, prick hole, make it 60 It is stirred at DEG C.After reaction overnight, temperature regulating rises to 140 DEG C, volatilizes concentrated hydrochloric acid, obtains YCl3.By the YCl of synthesis3It is super that 10mL is added Pure water, ultrasonic dissolution.5mL ultrapure water is added, is filtered, is injected in flask with syringe and 0.22um green filter, then plus Enter 20mL ethyl alcohol, 35mL cyclohexane, 30mmol enuatrol, at 78 DEG C, flow back 4h.30-40 DEG C is cooled the temperature to later, by it It moves into separatory funnel, takes upper solution.Then 20mL ultrapure water, 20mL ethyl alcohol (shaken well) is added.Layering takes upper layer, point Liquid is three times.After move it into round-bottomed flask, add explosion-proof ball, rotated.Later, 12mL 18 is added thereto with syringe Alkene and 12mL oleic acid are uniformly mixed, obtain Y (oleate)3。
Oleate (the Y of luminescent layer:Yb:Ho:Tm=77.8:20:2:0.2, molar ratio, total mole number 5mmol) also lead to Above method synthesis is crossed, Ln (oleate) is denoted as3.It is specific as follows：
Weigh Y2O3、Yb2O3、Ho2O3、Tm2O3(Y:Yb:Ho:Tm=77.8:20:2:0.2, molar ratio, total mole number is 5mmol) in a round bottom flask with 20mL concentrated hydrochloric acid, preservative film is covered, hole is pricked, stirs it at 60 DEG C.After reaction overnight, it adjusts Temperature rises to 140 DEG C, volatilizes concentrated hydrochloric acid, obtains mixed rare earth chlorides.10mL is added in the mixed rare earth chlorides of synthesis to surpass Pure water, ultrasonic dissolution.5mL ultrapure water is added, is filtered, is injected in flask with syringe and 0.22um green filter, then plus Enter 20mL ethyl alcohol, 35mL cyclohexane, 30mmol enuatrol, at 78 DEG C, flow back 4h.30-40 DEG C is cooled the temperature to later, by it It moves into separatory funnel, takes upper solution.Then 20mL ultrapure water, 20mL ethyl alcohol (shaken well) is added.Layering takes upper layer, point Liquid is three times.After move it into round-bottomed flask, add explosion-proof ball, rotated.Later, 12mL 18 is added thereto with syringe Alkene and 12mL oleic acid are uniformly mixed, obtain Ln (oleate)3。
(2) synthesis of UCNPs
1.0mmol Y(oleate)3It is added in octadecylene and the solution of oleic acid equal proportion mixing with 10.0mmol NaF, Pumping, inflates three circulations.1h is reacted at 115 DEG C, is warming up to 340 DEG C.After reacting 90min, a small amount of test solution is taken out, is added The Ln (oleate) of 0.4mmol3, 20min is reacted, a small amount of test solution is taken out, the Y (oleate) of 0.6mmol is added3React 20min Afterwards, rapid near room temperature.Isometric ethyl alcohol is added into product, shakes, is centrifuged 10min minutes with the speed of 8000rpm, it will Supernatant liquor is toppled over, recycling, after retaining lower layer's solid precipitating, is scattered in a certain amount of hexamethylene, and ultrasonic disperse is uniform, then It is centrifuged 5min with the speed of 2000rpm, takes supernatant liquor that the ethyl alcohol concussion of equivalent is added, the speed of 8000rpm is centrifuged 10min, Topple over supernatant liquor, recycling retains lower layer's solid, and repetition aforesaid operations are primary, and taking supernatant is final product, i.e. oleic acid is protected NaYF4:Yb3+,Ho3+/Tm3+UCNPs。
The NaYF of 2 oleic acid of embodiment protection4:Yb3+,Er3+The preparation of UCNPs：
Same as Example 1, difference is：In the oleate of luminescent layer, Y:Yb:Er=80:18:2, molar ratio is always rubbed Your number is 5mmol.
Embodiment 3UCNPs@SiO2The preparation of/MB
10.0mL hexamethylene and 0.660mL Igepal CO-520 (NP-5) are mixed and stirred for 1 hour to form anti-glue Beam.Later, the UCNPs of the oleic acid protection in 0.450mmol embodiment 1 is added, is vigorously stirred 1h to promote oleate and NP-5 Between ligand exchange, cause UCNPs to be embedded in pond.Then, 225 μ LMB aqueous solutions (4mg/mL) and 60 μ L ammonia are added dropwise Water (30%, mSolute:mSolvent) stir 2 hours afterwards.Finally, by 90 μ L tetraethyl orthosilicates (TEOS) and 20 μ L (3- aminopropyl) three Ethoxysilane (APTES) is slowly added to react for 24 hours in solution.The negative electricity TEOS of partial hydrolysis is transferred to the hydrophilic interior of reverse micelle Portion with positively charged MB to interact.By hydrolyzing and being condensed, UCNPs@SiO is obtained2/MB。
Embodiment 4UCNP@SiO2The preparation of/MB@PEG-FA
By COOH-PEG-FA, NHS and EDC with molar ratio 1:2.5:1 (7.2,18 and 7.2 μm of ol) is in dimethyl sulfoxide (DMSO) mixing in, activates 1 hour.Then, the 250 μ L mixture activated is added to the UCNPs@in 2mL embodiment 3 SiO2Then/MB reacts 12 hours under slow vibration.UCNPs@SiO is collected by centrifugation2/ MB@PEG-FA, and cleaned with ultrapure water Several times, it is finally scattered in PBS buffer solution (pH-7.5).
Experimental result, as shown in Figures 1 to 3：
Fig. 1 is the UCNPs@SiO of preparation2The synthesis and characterization of/MB@PEG-FA nano-probe.Two light emitting ionic Ho3+With Tm3+The upper conversion nano particle of doping has strong red fluorescence.The red emission due to from5F5With1G4Excitation state arrives5I8With3F4The transition (Fig. 1 (a)) of ground state.As shown in Fig. 1 (b-d), UCNPs the and UCNPs@SiO of synthesis2Show high uniformity and pure Hexagonal phase.It is measured by TEM, SiO2Thickness is about 5-7.5nm.By Uv-Vis analysis and Zeta potential analytical proof in silication In the process, MB is supported on SiO2In silicon layer.PEG-FA is coated in UCNPs@by amidation process by Uv-Vis analytical proof SiO2On the surface of/MB nano-probe, to improve its dispersibility and targeting.The introducing of PEG-FA by zeta potential value from negative value (- 30.6) positive value (23.4) is changed into, and makes UCNPs@SiO2/ MB@PEG-FA nano-probe monodisperse enhances it into cell Ability.
Fig. 2 is the UCNPs@SiO of assessment preparation2The ability of/MB@PEG-FA nano-probe generation ROS.Good spectrum The energy transmission distance matched and shortened ensures effective LRET efficiency.UCNPs@SiO2Luminous intensity at 650nm reduces 90%, for exciting MB, generate ROS (Fig. 2 (a)).With ABDA Fluorometric assay UCNPs@SiO2PEG-FA nanometers of/MB@spies The efficiency of needle generation ROSs.As shown in Fig. 2 (b), under 980nm laser irradiation, after ABDA is mixed with nano-probe, fluorescence is strong Degree is gradually reduced.After irradiation 65 minutes, 70% fluorescence of ABDA is quenched.In contrast, UCNPs@SiO2@PEG-FA and MB points Son does not influence the fluorescence intensity of ABDA after NIR irradiation.And UCNPs@SiO2/ MB@PEG-FA is irradiated in no NIR Under, the fluorescence intensity of ABDA is not also influenced.Show UCNPs@SiO2/ MB@PEG-FA and NIR are that generation ROSs two can not Or scarce condition.After showing NIR excitation UCNPs, luminescence resonance energy occurs and is transferred to SiO2MB molecule in silicon layer, MB molecule quilt ROSs is generated after excitation.
Fig. 3 is the UCNPs@SiO by investigating preparation using two-photon laser Laser Scanning Confocal Microscope2PEG-FA nanometers of/MB@ Probe enters intracellular processes.As shown in figure 3, with the extension of incubation time, intracellular UCNPs@SiO2/ MB@PEG-FA receives The fluorescence intensity of rice probe gradually increases, and shows to swallow MCF-7 cell in more nano-probe successes.Light field and luminescent image Overlapping display UCNPs@SiO2/ MB@PEG-FA nano-probe is predominantly located at cytosolic domain.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Claims (10)

1. a kind of UCNPs@SiO2/ MB/PEG-FA nano-probe, it is characterized in that：The nano-probe includes in upper conversion nano particle Core, the ultra-thin silica shell for being wrapped in the core surface, the methylene blue loaded on the shell and poly- second two Alcohol-folic acid.
2. nano-probe as described in claim 1, it is characterized in that：The host material that upper conversion nano particle selects is NaYF4, Sensitizer is Yb3+, light emitting ionic Ho3+And Tm3+。
3. nano-probe as described in claim 1, it is characterized in that：The thickness of the ultra-thin silica shell is less than 10nm； Preferably, the ultra-thin silica shell with a thickness of 5.0~7.5nm.
4. nano-probe as described in claim 1, it is characterized in that：Polyethylene glycol-the folic acid will be gathered by amidation process Ethylene glycol-folic acid is coated in UCNPs@SiO2On the surface of/MB.
5. UCNPs@SiO according to any one of claims 1 to 42The preparation method of/MB/PEG-FA nano-probe, feature It is that this method includes：
(1)UCNPs@SiO2The preparation of/MB：
Hexamethylene and surfactant are mixed to form reverse micelle, UCNPs is added and is mixed, forms mixed system One；
Methylene blue and ammonia spirit are added into mixed system one, is mixed, forms mixed system two；
Silicon source is added into mixed system two, the negative electricity silicon source of hybrid reaction, partial hydrolysis is transferred to the hydrophilic inside of reverse micelle To interact with positively charged methylene blue, by hydrolyzing and being condensed to yield UCNPs@SiO2/MB；
(2)UCNPs@SiO2The preparation of/MB/PEG-FA：
The PEG-FA of carboxylated is subjected to activated carboxylic, the mixture after activation is then added to the UCNPs@SiO2/MB In, UCNPs@SiO is prepared in hybrid reaction2/MB@PEG-FA。
6. preparation method as claimed in claim 5, it is characterized in that：In step (1), surfactant is Igepal CO-520, The silicon source is tetraethyl orthosilicate and (3- aminopropyl) triethoxysilane.
7. preparation method as claimed in claim 5, it is characterized in that：In step (1), the hexamethylene, surfactant and The ingredient proportion of UCNPs is (8~12) mL：(0.3~0.8) mL：(0.3~0.5) mmol；Hexamethylene is mixed with surfactant 1~2h is stirred to form reverse micelle；UCNPs is added, 1~2h is mixed.
8. preparation method as claimed in claim 6, it is characterized in that：In step (1), the methylene blue, ammonia spirit and The ingredient proportion of UCNPs is (2~6) mg：(40~80) μ L：(0.3~0.5) mmol；2~3h is mixed；
Preferably, in step (1), the feed ratio of the tetraethyl orthosilicate, (3- aminopropyl) triethoxysilane and UCNPs Example is (60~100) μ L：(10~40) μ L：(0.3~0.5) mmol；24~36h of hybrid reaction.
9. preparation method as claimed in claim 5, it is characterized in that：In step (2), activation the specific steps are：By COOH- PEG-FA, NHS and EDC are with molar ratio (1~2):(2~4):(1~2) mixes in dimethyl sulfoxide, activates 1~2h；
Preferably, mixture and UCNPs@SiO in step (2), after the activation2The ingredient proportion of/MB is (200~300) μ L：(1~3) mL, 10~14h of hybrid reaction.
10. UCNPs@SiO according to any one of claims 1 to 42/ MB/PEG-FA nano-probe is controlled in preparation photodynamics Treat the application in agent.
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