CN103301460B - Ferroferric oxide loaded compound micropowder and application thereof - Google Patents
Ferroferric oxide loaded compound micropowder and application thereof Download PDFInfo
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- CN103301460B CN103301460B CN201310244877.0A CN201310244877A CN103301460B CN 103301460 B CN103301460 B CN 103301460B CN 201310244877 A CN201310244877 A CN 201310244877A CN 103301460 B CN103301460 B CN 103301460B
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Abstract
The invention discloses ferroferric oxide loaded compound micropowder and an application thereof in inhibiting the activity of tumor cells. The micropowder is in such a structure that Fe3O4 is used as a core which is surrounded by lanthanide elements-doped NaYF4, and the outer layer is wrapped by TiO2. The compound micropowder disclosed by the invention is good in particle dispersibility and uniform in grain size. The compound micropowder cannot only be used for acoustodynamic treatment for tumor, but also used for biomarker fluorescence imaging by photoluminescence, so that the disadvantageous characteristic that conventional particles can be used for tumor treatment but cannot be used for biomarker fluorescence imaging by photoluminescence, and the fluorescent probe can be used for biomarker fluorescence imaging by photoluminescence but cannot be applied to treating tumor is overcome. The micropowder disclosed by the invention is simple and quick in preparation method and low in cost, and fussy steps in the preparation process of multifunctional functions are avoided. In addition, the micropower disclosed by the invention is wrapped by TiO2, so that the micropower has monodisperse particles in exact structures, and the compound micropowder is more suitable for clinical application.
Description
(1) technical field
The present invention relates to a kind of preparation method of multi-functional microgranule with cancer target, bio-imaging and the effect of sound dynamic therapy, particularly one is with Fe
3o
4for core, NaYF
4around and sound sensitiser TiO
2the preparation method of the composite particles of parcel and application thereof.
(2) background technology
Tumor is one of principal disease affecting human health, in order to improve therapeutic effect, reduce toxic and side effects, research and development have the diagnoses and treatment reagent of the several functions such as cancer target, bio-imaging and oncotherapy simultaneously, carry out specific treatment and become current focus and trend.
In cancer target, utilize ferroso-ferric oxide (Fe
3o
4) under the action of a magnetic field by drug accumulation in targeting moiety, reduce the distribution in normal structure, reducing the toxicity of medicine is an important method of carrying out neoplasm targeted therapy.Meanwhile, because it has lower toxicity, good biocompatibility, Fe
3o
4increasingly extensive in the application of field of biology, except being used as magnetic targeted carrier, also can be used as the contrast agent of nuclear magnetic resonance, for the diagnosis of disease.
In tumor imaging, utilize fluorescent probe labelling can observe the distribution situation of carrier at tumor locus intuitively, be beneficial to carry out specific treatment.Fluorescent probe label is divided into organic fluorescent dye and inorganic fluorescent material.Organic fluorescent dye photochemical stability is poor, easily photobleaching and photodissociation occurs, is unfavorable for the observation (as FITC, Cy5.5 etc.) of long period.In inorganic fluorescent material, studying more popular has quantum dot (QD), nanometer gold (Au) etc., though these fluorescent markers have diagnosing tumor imaging function, because its excitation wavelength is lower than wavelength of transmitted light, usually has larger background interference.Up-conversion refers to that the light of the larger wavelength of utilization is as exciting light, and excitation material launches the shorter utilizing emitted light of wavelength, because its excitation wavelength is greater than wavelength of transmitted light, is therefore called conversion.The exciting light of common employing is 980nm, and emission wavelength then determined by the rare earth composition adulterated, and generally at more than 650nm, therefore has good tissue penetration.
Sound motivation therapy (Sonodymanic Therapy, SDT) refers to and utilizes ultrasound wave to have stronger penetration capacity to biological tissue, excites some sound sensitizing drugs to generate reactive oxygen species (ROS), produces antitumor action.Sound sensitiser conventional is at present mainly organic substance, as hemoporphyrin, chlorin, temoporfin, Verteporfin, talaporfin etc.In recent years, existing use inorganic material TiO
2as report (Harada Y, Ogawa K, Irie Y, Endo H, Feril LB Jr, Uemura T, the Tachibana K.Ultrasound activation of TiO of sound sensitiser
2in melanoma tumors.J Control Release.2011,149 (2): 190-5.), compared with organic sound sensitiser, inorganic sound sensitiser has better stability.
(3) summary of the invention
The object of the invention is to provide a kind of preparation method and application of Multifunction composite particles, and this composite particles is with Fe
3o
4for core, NaYF
4around and sound sensitiser TiO
2parcel, possesses the functions such as tumor-targeting, tumor imaging and sound dynamic therapy.
The technical solution used in the present invention is:
The invention provides a kind of composite particles of ferroso-ferric oxide load, described composite particles is with Fe
3o
4for core, the outer NaYF around doping lanthanide series of core
4, outer by TiO
2parcel (i.e. NaYF
4outer by TiO
2parcel), described composite particles is prepared as follows:
(1) magnetic Fe is prepared
3o
4nanoparticle: after iron chloride and sodium citrate are dissolved in ethylene glycol a, add ammonium acetate again, stirring at room temperature 1h, then at 200 DEG C, the preferred 10h of (preferably reacting in reactor) 8 ~ 20h(is reacted), after naturally cooling to room temperature, take out reactant liquor and carry out magnetic field separation after washing (being generally 3-5 time), obtain Fe
3o
4nanoparticle; Described iron chloride and sodium citrate, the ammonium acetate mass ratio that feeds intake is the preferred 1:0.3:2.85 of 1:0.1 ~ 1:2 ~ 5(), the volumetric usage of described ethylene glycol a counts the preferred 50ml/g of 30 ~ 80ml/g(with iron chloride quality);
(2) NaYF of preparation doping lanthanide series
4nanoparticle: NaCl, yttrium water soluble salt, lanthanum (Ln) series elements water soluble salt are dissolved in ethylene glycol b and make mixed liquor a, then by polymine and NH
4f is dissolved in ethylene glycol c and makes mixed liquor b, then in stirred at ambient temperature 30 ~ 60min after mixed liquor a and mixed liquor b being mixed, (preferably reacting in reactor) 2 ~ 10h(preferably 4 ~ 8h is reacted again at 200 DEG C, most preferably 8h), after naturally cooling to room temperature, take out reactant liquor and use dehydrated alcohol, water washing successively, obtain the NaYF of described doping lanthanide series
4nanoparticle; Described Ln series elements water soluble salt be ytterbium water soluble salt with bait water soluble salt or thulium water soluble salt mixing with the ratio 9:1 of amount of substance; The feed intake ratio of amount of substance of described NaCl and yttrium water soluble salt is preferred 1:0.3 ~ 0.6 of 1:0.24 ~ 0.8(, most preferably 1:0.4), in NaCl and Ln series elements water soluble salt, the ratio of the total amount of substance of lanthanide series is preferred 1:0.05 ~ 0.2 of 1:0.05 ~ 0.3(, most preferably 1:0.1), the total volumetric usage of described ethylene glycol b and ethylene glycol c counts 100 ~ 600ml/g(preferably 200 ~ 450ml/g with NaCl quality, most preferably 325ml/g, described ethylene glycol b and ethylene glycol c consumption are separately dissolving); Described NaCl and NH
4f mass ratio is preferred 1:3 ~ 5 of 1:2 ~ 8(, most preferably 1:3.8), described NaCl and polymine mass ratio are preferred 1:2 ~ 4 of 1:1 ~ 6(, most preferably 1:3.25);
(3) preparation of composite particles: by Fe obtained in step (1)
3o
4the NaYF of doping lanthanide series obtained in nanoparticle and step (2)
4nanoparticle is scattered in solvent, ultrasonic disperse 10 ~ 30min, make dispersion liquid, separately get butyl titanate and dehydrated alcohol a is mixed and made into mixed liquor c, mixed liquor c is slowly added drop-wise in dispersion liquid, continues ultrasonic disperse 0.5 ~ 2h, after reaction terminates, wash with dehydrated alcohol b after reaction mixture being carried out Magnetic Isolation, obtain described composite particles; Described solvent is that acetonitrile and dehydrated alcohol are with the mixed solution of volume ratio 1:0.5 ~ 9; Described Fe
3o
4the NaYF of nanoparticle and doping lanthanide series
4nanoparticle mass ratio is 1:1 ~ 8, and described butyl titanate volumetric usage is with Fe
3o
4nanoparticle quality counts 2 ~ 10ml/g.
Further, step (2) described Ln series elements water soluble salt is Ln series elements nitrate or Ln series elements chlorate, preferred described Ln series elements water soluble salt be Ytterbium(III) nitrate. with nitric acid bait or thulium nitrate mixing with the ratio 9:1 of amount of substance.
Further, step (3) described Fe
3o
4the NaYF of nanoparticle and doping lanthanide series
4nanoparticle mass ratio is preferred 1:1.5 ~ 4 of 1:1 ~ 8(, most preferably 1:2.5), described butyl titanate volumetric usage is with Fe
3o
4nanoparticle quality counts 2 ~ 10ml/g(preferably 4 ~ 8ml/g, most preferably 6.25ml/g).
Further, the described solvent volume consumption of step (3) is with Fe
3o
4nanoparticle quality counts 500 ~ 3000ml/g(preferably 1000 ~ 2000ml/g), the volumetric usage of described dehydrated alcohol a does not affect the present invention, usual described butyl titanate and dehydrated alcohol a volume ratio are 1:10 ~ 50(preferably 15 ~ 30, most preferably 20).
Further, step (3) described solvent is that acetonitrile and dehydrated alcohol are with the mixed solution of volume ratio 1:3.
The present invention also provides a kind of application of composite particles in inhibition tumor cell activity of described ferroso-ferric oxide load, namely described composite particles has tumor magnetic targeted, bio-imaging and the effect of tumor sound dynamic therapy, can be used for diagnosis and the specific treatment of tumor.
Further, the concentration of described composite particles is the preferred 0.25mg/mL of 0.1 ~ 10mg/mL(, most preferably 2mg/mL, usually prepares with cell culture fluid).
The present invention has prepared Fe
3o
4-NaYF
4@TiO
2composite particles, this composite particles is by Fe
3o
4realize tumor magnetic targeted, thus reduce the toxic and side effects of normal tissue, improve the concentration of microgranule at tumor locus; This composite particles has the function such as bio-imaging and sound dynamic therapy simultaneously, can realize carrier Real-Time Monitoring in vivo and the specific treatment of tumor.
Ethylene glycol a of the present invention, ethylene glycol b, ethylene glycol c are ethylene glycol, name for ease of distinguishing different step ethylene glycol consumption difference; Described mixed liquor a, mixed liquor b and mixed liquid c are the mixed liquor of different step preparation, name for ease of differentiation; Described dehydrated alcohol a and dehydrated alcohol b is dehydrated alcohol, names for ease of distinguishing consumption, and letter itself is all without implication.
Compared with existing oncotherapy technology, beneficial effect of the present invention is mainly reflected in: the preparation method that the invention provides a kind of multi-functional composite particles, and particle dispersion prepared by the method is good, soilless sticking; This composite particles both can be used for the sound dynamic therapy of tumor, biomarker fluorescence imaging can be used for by luminescence generated by light again simultaneously, overcome conventional particle and can be used for oncotherapy but cannot biomarker fluorescence imaging, fluorescent probe can biomarker fluorescence imaging but cannot be applied to the unfavorable feature of oncotherapy; Preparation method of the present invention is simple and convenient, and cost is low, avoids the tedious steps of multi-functional particle in preparation process; In addition, the present invention utilizes TiO
2parcel preparation has the single particle of clear and definite structure, is more suitable for clinical practice.
(4) accompanying drawing explanation
Fig. 1 be multi-functional composite particles prepare schematic diagram;
Fig. 2 is Fe
3o
4transmission electron microscope (TEM) picture, A is Fe prepared by embodiment 1
3o
4, B is Fe prepared by embodiment 2
3o
4;
Fig. 3 is NaYF
4transmission electron microscope (TEM) picture: A be embodiment 3 prepare doping YB, the NaYF of Er
4, B be embodiment 4 prepare doping YB, the NaYF of Tm
4;
Fig. 4 is Fe
3o
4-NaYF
4@TiO
2transmission electron microscope (TEM) picture of composite particles, A is composite particles prepared by embodiment 5, and B is composite particles prepared by embodiment 6, and C is composite particles prepared by embodiment 7, and D is composite particles prepared by embodiment 8;
Fig. 5 is the Fe of preparation in embodiment 5
3o
4-NaYF
4@TiO
2the X-ray energy spectrogram of composite particles;
Fig. 6 is NaYF
4, Fe
3o
4-NaYF
4@TiO
2up-conversion fluorescence spectrum, excitation wavelength is 980nm;
Fig. 7 is NaYF prepared by embodiment 3
4naYF prepared by (Yb, Er), embodiment 4
4fe prepared by (Yb, Tm), embodiment 5
3o
4-NaYF
4@TiO
2fluorescence photo under 980nm wavelength laser excites;
Fig. 8 is in composite particles handler breast cancer cell line mcf-7 process, utilizes dihydro dichlorofluorescein to detect laser co-focusing (CLSM) picture (amplifying 100 to observe) of ROS generation;
Fig. 9 is in composite particles handler breast cancer cell line mcf-7 process, and ROS generates the CLSM picture (amplifying 630 to observe) at position in cell;
Figure 10 is the picture of composite particles cell death inducing situation;
Figure 11 is the microphotograph that under different ultrasonic time, composite particles affects apoptosis;
Figure 12 is by Fe
3o
4-NaYF
4@TiO
2bio-imaging picture after subcutaneous injection.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:Fe
3o
4the preparation of nanoparticle
Take 1.35g(5mmol) FeCl
3.6H
2o, 0.4g(1.36mol) after sodium citrate is dissolved in 70mL ethylene glycol, add 3.85g(0.05mol) ammonium acetate, stirring at room temperature 1h, proceeds to 100mL reactor, and 200 DEG C of reaction 10h, take out reactant liquor and utilize magnetic field separation after washing 3 times, namely obtain Fe
3o
4nanoparticle powder is about 0.9g, and particle diameter is 150 ~ 200nm, and for subsequent use, Fig. 2 is shown in by transmission electron microscope.
Embodiment 2:Fe
3o
4the preparation of nanoparticle
Take 1.35g(5mmol) FeCl
36H
2o, 0.4g(1.36mol) after sodium citrate is dissolved in 20mL ethylene glycol, add 3.85g(0.05mol) ammonium acetate, stirring at room temperature 1h, proceeds to 100mL reactor, and 200 DEG C of reaction 10h, take out reactant liquor and utilize magnetic field separation after washing 3 times, namely obtain Fe
3o
4nanoparticle powder is about 0.9g, and particle diameter is about 300nm, for subsequent use, and Fig. 2 is shown in by transmission electron microscope.
Embodiment 3: the NaYF of doping Yb, Er
4prepared by nanoparticle
Take 0.231g(6mmol) NaCl, 0.919g(2.4mmol) Y (NO
3)
3, 0.2694g(0.54mmol) Yb (NO
3)
3, 0.0267g(0.06mmol) Er (NO
3)
3be dissolved in 45mL ethylene glycol and make mixed liquor a, separately take 0.75g polymine (PEI, molecular weight 10K) and 0.888g NH
4f is dissolved in 30mL ethylene glycol and makes mixed liquor b, above-mentioned mixed liquor a and mixed liquor b is mixed and stirring at room temperature 30min, then transfer in 100mL reactor, 200 DEG C of reaction 8h, after naturally cooling to room temperature, take out reactant liquor and use dehydrated alcohol, water washing 3 times successively, obtaining the NaYF of described doping Yb, Er
4nanoparticle is about 0.2g, and particle diameter is about 50nm, and is scattered in dehydrated alcohol by this nanoparticle and preserves, and transmission electron microscope picture is shown in Fig. 3.NaYF
4fig. 7 is shown in by nanoparticle up-conversion luminescence photo, by NaYF in figure
4(Yb, Er) up-conversion luminescence photo is known, Yb and Er successfully adulterates.
Embodiment 4: the NaYF of doping Yb, Tm
4prepared by nanoparticle
Take 0.231g(6mmol) NaCl, 0.919g(2.4mmol) Y (NO
3)
3, 0.2694g(0.54mmol) Yb (NO
3)
3, 0.0268g(0.06mmol) Tm (NO
3)
3be dissolved in 45mL ethylene glycol and make mixed liquor a, separately take 0.75g polymine (PEI, molecular weight 10K) and 0.888g NH
4f is dissolved in 30mL ethylene glycol and makes mixed liquor b, above-mentioned mixed liquor a and mixed liquor b is mixed and stirring at room temperature 30min, then transfer in 100mL reactor, 200 DEG C of reaction 8h, after naturally cooling to room temperature, take out reactant liquor and use dehydrated alcohol, water washing 3 times successively, obtaining the NaYF of described doping Yb, Tm
4nanoparticle is about 0.2g, and particle diameter is about 50nm, and is scattered in dehydrated alcohol by this nanoparticle and preserves, and transmission electron microscope picture is shown in Fig. 3.NaYF
4fig. 7 is shown in by nanoparticle up-conversion luminescence photo, by NaYF in figure
4(Yb, Tm) up-conversion luminescence photo is known, Yb and Tm successfully adulterates.
Embodiment 5:Fe
3o
4-NaYF
4@TiO
2the preparation of composite particles
Get Fe prepared by 80mg embodiment 1
3o
4the NaYF of the doping lanthanide series (Yb, Er) of nanoparticle, 200mg embodiment 3 preparation
4nanoparticle is scattered in 120mL ethanol-acetonitrile mixed solution (ethanol 90mL, acetonitrile 30mL) in, 40KHz ultrasonic disperse 10min makes dispersion liquid a, separately gets 0.4mL butyl titanate and is distributed in 10mL dehydrated alcohol, make dispersion liquid b after being uniformly dispersed, dispersion liquid b is slowly added drop-wise in dispersion liquid a, 5min dropwises, and continues 40KHz ultrasonic disperse 1.5h, after reaction terminates, use absolute ethanol washing 5 times after reaction mixture being carried out Magnetic Isolation, namely obtain about 0.4g composite particles Fe
3o
4-NaYF
4@TiO
2, be scattered in water and preserve, transmission electron microscope picture is shown in Fig. 4.The particle diameter of composite particles is about 400nm, element set prejudice Fig. 5 of composite particles.As can be seen from Fig. 4,5, Fe
3o
4and NaYF
4by TiO
2parcel, composite particles is successfully prepared, and can find out that Yb successfully adulterates simultaneously.The up-conversion luminescence figure of composite particles is shown in Fig. 7, by Fe3O4-NaYF in Fig. 7
4@TiO
2up-conversion luminescence figure can find out Fe
3o
4and NaYF
4by TiO
2parcel, the NaYF after parcel
4still there is good luminescent properties.
Embodiment 6:Fe
3o
4-NaYF
4@TiO
2the preparation of composite particles
Get Fe prepared by 80mg embodiment 1
3o
4the NaYF of the doping lanthanide series (Yb, Er) of nanoparticle, 200mg embodiment 3 preparation
4nanoparticle is scattered in 120mL ethanol-acetonitrile mixed solution (ethanol 90mL, acetonitrile 30mL) in, 40KHz ultrasonic disperse 10min makes dispersion liquid a, separately getting 0.7mL butyl titanate is distributed in 10mL dehydrated alcohol, makes dispersion liquid b after being uniformly dispersed, and dispersion liquid b is slowly dripped (dropwising in 5min) in dispersion liquid a, 40KHz continues ultrasonic disperse 1.5h, after reaction terminates, after reaction mixture being carried out Magnetic Isolation, use absolute ethanol washing 5 times, namely obtain 0.45g composite particles (Fe
3o
4-NaYF
4@TiO
2), be scattered in water and preserve.The particle diameter of composite particles is about 450nm, and transmission electron microscope picture is shown in Fig. 4.Find out from figure, Fe
3o
4and NaYF
4by TiO
2parcel, composite particles is successfully prepared.
Embodiment 7:Fe
3o
4-NaYF
4@TiO
2the preparation of composite particles
Get Fe prepared by 80mg embodiment 1
3o
4the NaYF of the doping lanthanide series (Yb, Er) of nanoparticle, 400mg embodiment 3 preparation
4nanoparticle is scattered in 120mL ethanol-acetonitrile mixed solution (ethanol 90mL, acetonitrile 30mL) in, 40KHz ultrasonic disperse 10min makes dispersion liquid a, separately getting 0.4mL butyl titanate is distributed in 10mL dehydrated alcohol, makes dispersion liquid b after being uniformly dispersed, and dispersion liquid b is slowly dripped (dropwising in 5min) in dispersion liquid a, 40KHz continues ultrasonic disperse 1.5h, after reaction terminates, after reaction mixture being carried out Magnetic Isolation, use absolute ethanol washing 5 times, namely obtain 0.5g composite particles (Fe
3o
4-NaYF
4@TiO
2), be scattered in water and preserve.The particle diameter of composite particles is about 400nm, and transmission electron microscope picture is shown in Fig. 4.Find out from figure, Fe
3o
4and NaYF
4by TiO
2parcel, composite particles is successfully prepared.
Embodiment 8:Fe
3o
4-NaYF
4@TiO
2the preparation of composite particles
Get Fe prepared by 80mg embodiment 2
3o
4the NaYF of the doping lanthanide series (Yb, Er) of nanoparticle, 100mg embodiment 3 preparation
4nanoparticle is scattered in 120mL ethanol-acetonitrile mixed solution (ethanol 90mL, acetonitrile 30mL) in, 40KHz ultrasonic disperse 10min makes dispersion liquid a, separately getting 0.4mL butyl titanate is distributed in 10mL dehydrated alcohol, makes dispersion liquid b after being uniformly dispersed, and dispersion liquid b is slowly dripped (dropwising in 5min) in dispersion liquid a, 40KHz continues ultrasonic disperse 1.5h, after reaction terminates, after reaction mixture being carried out Magnetic Isolation, use absolute ethanol washing 5 times, namely obtain 0.5g composite particles (Fe
3o
4-NaYF
4@TiO
2), be scattered in water and preserve.The particle diameter of composite particles is about 400nm, and transmission electron microscope picture is shown in Fig. 4.Find out from figure, Fe
3o
4and NaYF
4by TiO
2parcel, composite particles is successfully prepared.
Embodiment 9: upper converting photoluminescent effect
NaYF prepared by Example 3
4the NaYF of nanoparticle, embodiment 4 preparation
4the Fe of nanoparticle, embodiment 5 preparation
3o
4-NaYF
4@TiO
2be configured to the aqueous dispersions that concentration is 0.5mg/mL respectively, excite with the 980nm laser instrument of 0.4W, within the scope of 300-750nm, measure fluorescence spectrum with spectrofluorophotometer simultaneously, see Fig. 6.Take pictures with camera, photo as shown in Figure 7 simultaneously.As seen from Figure 6, NaYF
4and Fe
3o
4-NaYF
4@TiO
2all there is bright fluorescence at 650-690nm place, can be used for living imaging.As can be seen from Fig. 7, NaYF
4there is good luminescent properties, TiO
2parcel do not affect NaYF
4luminescent properties.
Embodiment 10: the detection of ROS in ultrasonic therapeutic
(1) by Fe prepared by embodiment 5
3o
4-NaYF
4@TiO
2be scattered in PRMI1640 culture fluid the dispersion liquid making 200 μ g/mL.
(2) Breast cancer lines (MCF-7, purchased from institute of oncology of attached hospital of Zhejiang University second) is inoculated in 6 orifice plates (5 × 10
4/ hole), culture fluid used is PRMI1640,37 DEG C place after 24h until its adherent firmly after, culture fluid is changed into the dispersion liquid that step (1) is prepared, 37 DEG C hatch 2h after, remove dispersion liquid, and be 7.4 by PBS(pH value) wash 3 times, add dihydro dichlorofluorescein-diacetate (DCFH-DA) the PRMI1640 solution of 10 μMs, remove solution after hatching 0.5h in 37 DEG C and be 7.4 by PBS(pH value) washing 3 times, 0.5W/cm
2ultrasonic 3min, add nuclei dyeing reagent Hochest33342(again purchased from Sigma) 37 DEG C hatch 15min, then remove reagent and be 7.4 by PBS(pH value) washing 3 times, after adding 4% paraformaldehyde, 37 DEG C of fixing 30min, be 7.4 by PBS(pH value) wash plate 3 times, get cell and carry out laser confocal microscope (CLSM) observation, see Fig. 8, Fig. 9.Fe is not added under similarity condition
3o
4-NaYF
4@TiO
2and not ultrasonic be matched group; Do not add Fe
3o
4-NaYF
4@TiO
2but ultrasonic is ultrasonic group; Add Fe
3o
4-NaYF
4@TiO
2but not ultrasonic is composite particles group; Add Fe
3o
4-NaYF
4@TiO
2and ultrasonic be compound particle+ultrasonic group.Fig. 8 is amplification 100 times of pictures, and Fig. 9 is amplification 630 times of pictures.DCFH-DA, by after cellular uptake, changes DCFH in cell, when DCFH runs into active oxygen, is oxidized to DCF, under blue light excites, send green fluorescence.
As seen from Figure 8, ultrasonic group, DCF fluorescence is all not obvious in composite particles group and matched group, composite particles+ultrasonic group produces bright green fluorescence, illustrates and produces a large amount of ROS(reactive oxygen specieses), and ROS can inducing cell generation apoptosis.
In Fig. 9, Hochest is the Hochest(core transfection reagent at nucleus position) dye picture; DCF is dichlorofluorescein picture in cell, mainly for detection of the ROS produced; Carrier is Fe
3o
4-NaYF
4@TiO
2the up-conversion fluorescence picture of (embodiment 5), bright field is the cell microscopic sheet under natural light, and overlap is the displaing micro picture after above various fluorescence overlap.As can be seen from Figure 9, ultrasonic group of ROS generating unit is extranuclear Cytoplasm, produces primarily of mitochondrion; Composite particles+ultrasonic group then all produces ROS in whole cell, therefore can produce destruction to nucleus, cause stronger apoptosis-induced effect, and DCF overlaps completely with the fluorescence of carrier simultaneously, and Fe is described
3o
4-NaYF
4@TiO
2by cellular uptake, and can enter nucleus, also illustrate that ROS is produced by carrier under ultrasonic excitation simultaneously.
Embodiment 11: composite particles cell death inducing
(1) by Fe prepared by embodiment 5
3o
4-NaYF
4@TiO
2be scattered in PRMI1640 culture fluid the dispersion liquid making 200 μ g/mL.
(2) Breast cancer lines (MCF-7) is inoculated in 6 orifice plates (1 × 10
5/ hole), add PRMI1640 culture fluid, 37 DEG C cultivate 24h until its adherent firmly after, culture fluid is changed into dispersion liquid prepared by step (1), 37 DEG C hatch 4h after, 0.5W/cm
2ultrasonic 3min, then remove dispersion liquid and wash plate 3 times, adding PRMI1640 culture fluid 37 DEG C to continue to hatch after 12h according to apoptosis kit (Annexin V-FITC cell apoptosis detection kit, the green skies) method Flow Cytometry Assay apoptosis situation, the results are shown in Figure 10.Not add Fe under similarity condition
3o
4-NaYF
4@TiO
2and not ultrasonic be matched group; Do not add Fe
3o
4-NaYF
4@TiO
2but ultrasonic is ultrasonic group; Add Fe
3o
4-NaYF
4@TiO
2and ultrasonic be composite particles+ultrasonic group.In Figure 10, B1, B2, B3, B4 are respectively necrotic area, late apoptic district, early apoptosis district and normal cell district, there is a large amount of non-viable apoptotic cells in as can be seen from the figure composite particles+ultrasonic Zu B2 district, illustrates that composite particles+ultrasonic can remarkable cell death inducing.
Embodiment 12: ultrasonic time cell death inducing is studied
(1) by Fe prepared by embodiment 5
3o
4-NaYF
4@TiO
2be scattered in PRMI1640 culture fluid the dispersion liquid making 200 μ g/mL.
(2) Breast cancer lines (MCF-7) is inoculated in 6 orifice plates (5 × 10
4/ hole), add PRMI1640 culture fluid and 37 DEG C cultivate 24h until its adherent firmly after, culture fluid is changed into dispersion liquid prepared by step (1), 37 DEG C hatch 4h after, 0.5W/cm
2respectively ultrasonic 0min, 1min and 3min, then removes dispersion liquid and washes plate 3 times, adds PRMI1640 culture fluid and continues 37 DEG C and to hatch after 12h by 400 power microscopes (Olympus CKX41) observation of cell state, see Figure 11.Be contrast with untreated cell under similarity condition.Result show ultrasonic time be 3min comparatively 0min, 1min can cause more many cells apoptosis, illustrating that ultrasonic time extends can remarkable cell death inducing, but ultrasonic 5min not comparatively 3min be significantly improved, ultrasonic 3min therefore can be adopted to treat.
Embodiment 13: bio-imaging is studied
By Fe prepared by embodiment 5
3o
4-NaYF
4@TiO
2after being scattered in PBS (200 μ g/mL) to be expelled to mice subcutaneous, then use 980nm laser according to 1W/cm
2power illumination mice, utilizing emitted light 630nm(high pass) and 850nm(low pass) filter filter, collect the photon of 630-850nm, utilize the imaging of living imaging instrument, the results are shown in Figure 12, as can be seen from the figure, Fe
3o
4-NaYF
4@TiO
2there is good imaging effect, may be used for bio-imaging.
Claims (8)
1. a composite particles for ferroso-ferric oxide load, is characterized in that described composite particles is with Fe
3o
4for core, the outer NaYF around doping lanthanide series of core
4, outer by TiO
2parcel, described composite particles is prepared as follows:
(1) magnetic Fe is prepared
3o
4nanoparticle: after iron chloride and sodium citrate are dissolved in ethylene glycol a, then add ammonium acetate, stirring at room temperature 1h, then reacts 8 ~ 20h at 200 DEG C, after naturally cooling to room temperature, takes out reactant liquor and carries out magnetic field separation after washing, obtain Fe
3o
4nanoparticle; Described iron chloride and sodium citrate, the ammonium acetate mass ratio that feeds intake is 1:0.1 ~ 1:2 ~ 5, and the volumetric usage of described ethylene glycol a counts 30 ~ 80ml/g with iron chloride quality;
(2) NaYF of preparation doping lanthanide series
4nanoparticle: NaCl, yttrium water soluble salt, lanthanide series water soluble salt are dissolved in ethylene glycol b and make mixed liquor a, then by polymine and NH
4f is dissolved in ethylene glycol c and makes mixed liquor b, then in stirred at ambient temperature 30 ~ 60min after being mixed by mixed liquor a and mixed liquor b, then at 200 DEG C, 2 ~ 10h is reacted, after naturally cooling to room temperature, take out reactant liquor and use dehydrated alcohol, water washing successively, obtain the NaYF of described doping lanthanide series
4nanoparticle; Described lanthanide series water soluble salt be ytterbium water soluble salt with bait water soluble salt or thulium water soluble salt mixing with the ratio 9:1 of amount of substance; The feed intake ratio of amount of substance of described NaCl and yttrium water soluble salt is 1:0.24 ~ 0.8, NaCl is 1:0.05 ~ 0.3 with the ratio of the total amount of substance of lanthanide series in lanthanide series water soluble salt, and the total volumetric usage of described ethylene glycol b and ethylene glycol c counts 100 ~ 600ml/g with NaCl quality; Described NaCl and NH
4f mass ratio is 1:2 ~ 8, and described NaCl and polymine mass ratio are 1:1 ~ 6;
(3) preparation of composite particles: by Fe obtained in step (1)
3o
4the NaYF of doping lanthanide series obtained in nanoparticle and step (2)
4nanoparticle is scattered in solvent, ultrasonic disperse 10 ~ 30min, make dispersion liquid, separately get butyl titanate and dehydrated alcohol a is mixed and made into mixed liquor c, mixed liquor c is slowly added drop-wise in dispersion liquid, continues ultrasonic disperse 0.5 ~ 2h, after reaction terminates, wash with dehydrated alcohol b after reaction mixture being carried out Magnetic Isolation, obtain described composite particles; Described solvent is that acetonitrile and dehydrated alcohol are with the mixed solution of volume ratio 1:0.5 ~ 9; Described Fe
3o
4the NaYF of nanoparticle and doping lanthanide series
4nanoparticle mass ratio is 1:1 ~ 8, and described butyl titanate volumetric usage is with Fe
3o
4nanoparticle quality counts 2 ~ 10ml/g.
2. the composite particles of ferroso-ferric oxide load as claimed in claim 1, is characterized in that the described lanthanide series water soluble salt of step (2) is lanthanide series nitrate or lanthanide series chlorate.
3. the composite particles of ferroso-ferric oxide load as claimed in claim 1, it is characterized in that the described lanthanide series water soluble salt of step (2) be Ytterbium(III) nitrate. with nitric acid bait or thulium nitrate mixing with the ratio 9:1 of amount of substance.
4. the composite particles of ferroso-ferric oxide load as claimed in claim 1, is characterized in that step (3) described Fe
3o
4the NaYF of nanoparticle and doping lanthanide series
4nanoparticle mass ratio is 1:1.5 ~ 4, and described butyl titanate volumetric usage is with Fe
3o
4nanoparticle quality counts 4 ~ 8ml/g.
5. the composite particles of ferroso-ferric oxide load as claimed in claim 1, is characterized in that the described solvent volume consumption of step (3) is with Fe
3o
4nanoparticle quality counts 500 ~ 3000ml/g.
6. the composite particles of ferroso-ferric oxide load as claimed in claim 1, is characterized in that step (3) described solvent is that acetonitrile and dehydrated alcohol are with the mixed solution of volume ratio 1:3.
7. the composite particles of ferroso-ferric oxide load described in a claim 1 is preparing the application in inhibition tumor cell active medicine.
8. apply as claimed in claim 7, it is characterized in that the concentration of described composite particles is 0.1 ~ 10mg/mL.
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