CN104069491B

CN104069491B - Ternary nano combination drug, its preparation method and its purposes for being used to prepare the pharmaceutical compositions for the treatment of tumour

Info

Publication number: CN104069491B
Application number: CN201310103775.7A
Authority: CN
Inventors: 任文智; 吴爱国; 曾乐勇; 沈折玉; 马雪华
Original assignee: Ningbo Institute of Material Technology and Engineering of CAS
Current assignee: Ningbo Institute of Material Technology and Engineering of CAS
Priority date: 2013-03-27
Filing date: 2013-03-27
Publication date: 2018-01-02
Anticipated expiration: 2033-03-27
Also published as: CN104069491A

Abstract

The present invention relates to a kind of ternary nano combination drug, its preparation method and its purposes for being used to prepare the pharmaceutical compositions for the treatment of tumour.The ternary nano combination drug includes core carrier, active constituents of medicine and the stabilizer for wrapping up the core carrier and active constituents of medicine.The core carrier, which includes, to produce the first nano-particle of living radical, the composite nanoparticle based on the first nano-particle with medical imaging signal or its combination by ultrasonic excitation；The active constituents of medicine is carried on the core carrier；The stabilizer includes hydrophilic polymer, the liposome of hydroxyl or carboxyl, bovine serum albumin(BSA) nanosphere, human serum albumins nanosphere are contained in surface, or its combination.It is used to be prepared by ultrasonic excitation so as to treat the purposes of the pharmaceutical compositions of tumour the invention further relates to the bielement nano combination drug without stabilizer.The neoplasm targeted therapy that the achievable ultrasonic therapy of the present invention cooperates with chemotherapy.

Description

Ternary nano combination drug, its preparation method and its medicine for being used to prepare treatment tumour Learn the purposes of composition

Technical field

The present invention relates to a kind of nano combined medicine, more particularly to a kind of ternary nano combination drug, its preparation method, It is in the purposes for the pharmaceutical compositions for preparing treatment tumour.Answered the invention further relates to the ternary nano combination drug with bielement nano Composite medicine is used to be prepared by ultrasonic excitation so as to treat the purposes of the pharmaceutical compositions of tumour.The achievable chemical drugs of the present invention The treatment of thing and ultrasonic synergistic treatment malignant tumour, particularly multidrug resistance of tumor.

Background technology

Chemotherapy is the main method of clinical treatment malignant tumour.However, chemotherapeutics is while tumour cell is killed, it is right Normal cell also has different degrees of damage, destroys the immune system of human body, influences the life quality of patient.Serious chemotherapy pair Effect can cause tumor patient hypoimmunity, physical decline, be one of the main reason for chemotherapy can not continue.Puzzlementization Another bottleneck for treating treatment tumour is the multidrug resistance of tumour cell.Cells of resistant tumors is produced by induced by chemotherapeutic agents , a kind of mutation malignant cell for being resistant to Treated with Chemotherapeutic Drugs thing.Statistics shows, in chemotherapy of tumors, the trouble more than 90% Person is dead related to tumor multi-medicine drug-resistant.Therefore, while chemotherapeutics side effect is reduced, the multidrug resistance of tumour is overcome Property, the treatment to cancer is significant.

TiO₂It is a kind of broad stopband n-type semiconductor, its anatase type band-gap energy is 3.2eV, is equivalent to wavelength 387nm photon energy.Work as TiO₂After being more than or equal to light or the radiation excitation of band-gap energy by energy, in valence band and it can lead Take the photohole for producing strong oxidizing property respectively（h⁺）With the light induced electron of strong reducing property（e^-）.When in the aqueous solution TiO₂After being excited, the free radicals such as the very high hydroxyl free radical of reactivity and active oxygen molecule can be produced.Above-mentioned freedom Base can destroy biological tissue, cause the death of cell.Therefore, nano-TiO₂It has been widely used in the light power of tumour cell Therapy study field.However, usual TiO₂Ultraviolet or shorter wavelengths of ray is needed to excite, this greatly limits TiO₂ The practical application of optical dynamic treatment of tumor.In order to overcome the puzzlement of excitaton source, the flat good fortune brightness of Japanese scholars gold et al. to propose using super Sound wave excitation nano TiO₂Treat tumour.Its mechanism is that ultrasonic wave penetration capacity is strong, and tissue mechanical can be caused to vibrate and warm Effect, so as to trigger tissue to produce OH¹、OH、H₂O₂, O isoreactivity free radicals（ROS）, destroy tumour cell structure（Authorize country： China, date of publication：On January 14th, 2009, the patent No.：200680049369.5. patent name：Ultrasonic cancer treatment enhancer And cytocide）.

Drug delivery system and tumour of the studies have shown that using nano-particle as carrier in terms of tumor chemotherapeutic drug conveying After cells contacting, cell membrane caves in and nano-particle medicine-carried system is wrapped to form into endosome, hence into intracellular.This Individual process can avoid the drug efflux effect that the p- glycoprotein of cell membrane surface is mediated, so as to overcome the multiple medicine of tumour cell Resistance.Based on above mechanism, such as inorganic nano material that some good biocompatibilities, toxicity are low, specific surface area is big, Fe₃O₄、 SiO₂、TiO₂, ZnO etc. is widely used for the drug delivery of cells of resistant tumors, and achieves good progress.With Although nano-particle dexterously avoids p- glycoprotein to medicine for the drug delivery system of carrier using cell membrane endocytosis Outer row, but the drug-resistance mechanism of cells of resistant tumors is still present, after medicine is released in intension body, p- glycoprotein Adjacent medicine can be still discharged to extracellular.Therefore, most drug delivery systems based on nano-particle can only Reverse multidrug resistance phenomenon to a certain extent, but the multidrug resistance of tumour cell can not be overcome completely.Due to semiconductor material Expect good photoelectric effect, the drug delivery using less toxic nano semiconductor material as carrier is moved with the light that carrier material possesses in itself Power treatment ability of medicine is combined, and has developed the multi-mode treatment of cells of resistant tumors.Southeast China University Wang Xuemei professors seminar exists Important progress is achieved in terms of multi-mode treatment cells of resistant tumors.They are by adriamycin（Doxorubicin, referred to as DOX）It is loaded into ZnO nanoparticle and builds drug delivery system, excites ZnO to produce ROS using ultraviolet, realize light power Treat the double mode treatment for the cells of resistant tumors being combined with chemotherapy（H.Zhang,B.Chen,H.Jiang,C.L.Wang, H.Wang,X.M.Wang.Biomaterials,2011,32:1906-1914.）.However, as it was previously stated, using ultraviolet to excite Source will limit the application of semi-conducting material optical dynamic therapy.

It is resistance to solve the side effect of chemotherapeutics and the multiple medicine of tumour cell in tumor therapeutic procedure accordingly, there exist demand Medicine phenomenon, and solve the problems, such as that the excitaton source of semi-conducting material optical dynamic therapy is limited.

The content of the invention

The problem of being limited present invention aim to address the excitaton source of semi-conducting material optical dynamic therapy, and solve tumour and control Chemotherapeutics side effect and tumor cell multidrug resistance phenomenon during treatment.

The first aspect of the present invention provides a kind of ternary nano combination drug, and it is included：

Core carrier, it includes and can be produced the first nano-particle of living radical by ultrasonic excitation, be had The composite nanoparticle based on first nano-particle of medical imaging signal, or its combination；

Active constituents of medicine, it is carried on the core carrier；With

For wrapping up the stabilizer of the core carrier and active constituents of medicine, the stabilizer includes hydrophilic polymer Thing, surface contain the liposome of hydroxyl or carboxyl, bovine serum albumin(BSA) nanosphere (abbreviation BSA), human serum albumins nanosphere, Or its combination.

In another preference, first nano-particle includes TiO₂And/or ZrO₂。

In another preference, on the basis of weight, the content ratio of the core carrier, active constituents of medicine and stabilizer is 1：0.01：0.4 to 1：0.5：1.It is preferred that the content ratio of the core carrier, medicine and stabilizer is 1：0.01：0.5 to 1： 0.5：1.More preferably it is 1：0.01：0.5 to 1：0.1：0.8.

In another preference, the particle diameter of the combination drug is 10 to 200 nanometers.More preferably it is 50 to 150 nanometers.

In another preference, the medical imaging signal includes T₁The magnetic resonance imaging signal of weighting, T₂The magnetic resonance of weighting into As signal, CT signal, near-infrared excite up-conversion fluorescence signal, or its combination.

In another preference, the composite nanoparticle includes first nano-particle, and selected from Gd₂O₃、Fe₃O₄、 ZnFe₂O₄、NiFe₂O₄、Au、NaYF₄:Er³⁺/Yb³⁺、NaYF₄:Yb³⁺/Tm³⁺、NaYF₄:Tm³⁺/Er³⁺And NaYF₄:Yb³⁺/Tm³⁺/ Er³⁺In one or more.

In another preference, the hydrophilic polymer includes non-ionic polyalcohol, cation type polymer, gathered containing carboxyl Compound, or its combination.

In another preference, the hydrophilic polymer includes polyethylene glycol (abbreviation PEG), glucan, polyethyleneimine, poly- Vinylamine, Sensor Chip CM 5, polyacrylic acid, or its combination.

In another preference, the active constituents of medicine includes antineoplastic.

In another preference, the antineoplastic includes anthracene ring antitumor medicinal and/or taxol.

In another preference, the antineoplastic includes adriamycin and/or taxol.

In another preference, the combination drug also includes the finishing coat containing ligand molecular for target tumor.

In another preference, the ligand molecular is to pass through amido link（-CO-NH-）It is connected to the surface of the stabilizer.

In another preference, the ligand molecular is folic acid, folic acid derivatives, RGD peptide（Argininyl-glycyl-asparagus fern ammonia Acid）, tumor specific antibody, or its combination.Preferably folic acid.

The second aspect of the present invention provides a kind of method for preparing the invention described above ternary nano combination drug, its include with Lower step：

(a) core carrier is dispersed in water, so as to form core carrier dispersion liquid；

(b) the core carrier dispersion liquid and the active constituents of medicine are mixed, so as to form the binary hydrosol；With

(c) the binary hydrosol and the solution containing the stabilizer are mixed, so as to form the ternary nano composite medicine Thing.

In another preference, this method also includes step (d), and it is used for the ternary nano that first activation step (c) obtains and answered Composite medicine, and surface modification is carried out to the ternary nano combination drug after the activation using the solution containing ligand molecular, from And the ternary nano combination drug for including finishing coat is obtained, the finishing coat contains ligand molecular.

The third aspect of the present invention provides the medicine that a kind of ternary nano combination drug as the aforementioned is used to prepare treatment tumour Learn the purposes of composition.

In another preference, the ternary nano combination drug is so as to treating tumour for being prepared by ultrasonic excitation The purposes of pharmaceutical compositions.

The fourth aspect of the present invention provides a kind of ultrasonic therapeutical system, and it includes the device for being used for launching ultrasonic wave, and Ternary nano combination drug as the aforementioned.

The fifth aspect of the present invention provides a kind of bielement nano combination drug and is used to be prepared by ultrasonic excitation so as to control The purposes of the pharmaceutical compositions of tumour is treated, the bielement nano combination drug includes：

Core carrier, it includes and can be produced the first nano-particle of living radical by ultrasonic excitation, be had The composite nanoparticle based on first nano-particle of medical imaging signal, or its combination；With

Active constituents of medicine, it is carried on the core carrier.

In another preference, first nano-particle includes TiO₂And/or ZrO₂。

In another preference, on the basis of weight, the content ratio of the core carrier and active constituents of medicine is 1：0.01 To 1：0.5.More preferably it is 1：0.01 to 1：0.1.

In another preference, the antineoplastic includes adriamycin and/taxol.

The sixth aspect of the present invention provides a kind of ultrasonic therapeutical system, and it includes the device for being used for launching ultrasonic wave, and Foregoing bielement nano combination drug.

Brief description of the drawings

Fig. 1 is to illustrate TiO in embodiment 1₂The Infrared spectra adsorption (a figures) of-adriamycin bielement nano combination drug sign, Zeta potential (b figures).

Fig. 2 is to illustrate PEG4000-TiO in embodiment 1₂The particle diameter distribution that-adriamycin ternary nano combination drug characterizes.

Fig. 3 is illustrated in embodiment 1 using mtt assay detection adriamycin and three kinds of PEG-TiO₂- adriamycin ternary nano is answered Killing ability of the composite medicine to human breast carcinoma multidrug resistance cell.

Fig. 4 is to illustrate to study PEG4000-TiO by laser confocal microscope in embodiment 1₂- adriamycin ternary nano The regularity of distribution of the combination drug in human breast carcinoma multidrug resistance cell.

Fig. 5 is illustrated in embodiment 10 using mtt assay detection TiO₂The anti-human breast cancer of-adriamycin bielement nano combination drug Cell MCF-7 (drug sensitive cell, a figures), MCF-7/ADM (multidrug resistance cell, b figures) effect.

Fig. 6 is to illustrate TiO in embodiment 10₂- adriamycin bielement nano combination drug is in human breast cancer cell line Bcap-37 (medicine Thing sensitive cells, a figures), the X ray synchrotron radiation result of MCF-7/ADM (multidrug resistance cell, b figure) distributions.

Fig. 7 is to illustrate TiO in embodiment 10₂- adriamycin bielement nano combination drug is in human breast cancer cell line Bcap-37 (medicine Thing sensitive cells, a figures), the laser confocal microscope result of MCF-7/ADM (multidrug resistance cell, b figure) distributions.

Embodiment

The present inventor has found with the nano-particle with Ultrasound-activated property or multiple first by in-depth study extensively Conjunction nano-particle is core carrier, and medicine is loaded on core carrier, and to keep the stability of nano combined medicine, is prolonged Its circulation time in vivo is grown, then outside is wrapped in stabilizer (such as hydrophilic polymer etc.), is received so as to obtain ternary Rice combination drug.The particle diameter of combination drug is controlled by the particle diameter of control core carrier, medicine lift-launch amount, stabilizing agent dosage etc., Make it that there is preferable permeability and retention effect to tumor tissues（EPR effects, i.e. passive target function of tumor）；Or it can enter One step makes it have active targeting function of tumor by the way that nano combined medical surfaces are modified with the ligand molecular of target tumor.Use Remember in the patent application case (Application No. 20111039712.7) that the ternary nano combination drug or applicant of the present invention has been filed on The bielement nano combination drug of load, plus by doses Ultrasonic Radiation tumor locus, ultrasonic therapy can be achieved with changing The neoplasm targeted therapy of collaboration is treated, tumor locus can be imaged using medical imaging instruments or collaboration to two kinds of Therapeutic modes Effect carries out real-time therapeutic evaluation.The present invention is completed on this basis.

Ternary nano combination drug

Ternary nano combination drug of the present invention includes：

Active constituents of medicine, it is carried on the core carrier；With

Term " living radical " refers to the free radical that can destroy tumour cell structure, such as OH¹、OH、H₂O₂, O etc..

In another preference, first nano-particle includes TiO₂And/or ZrO₂.In another preference, this first nanometer Particle is TiO₂Nano-particle.

In another preference, the particle size range of first nano-particle is preferably 1-150 nanometers.

In another preference, the core carrier be have medical imaging signal based on the compound of first nano-particle Nano-particle, such as with T₁The Gd of weighted mri signal₂O₃With TiO₂、ZrO₂One of which or two kinds of composite nanoparticle, it is excellent Elect Gd as₂O₃-TiO₂Composite nanoparticle, its preparation method refer to example below 5；Or there is T₂Weighted mri signal Fe₃O₄、ZnFe₂O₄、NiFe₂O₄One or more and TiO therein₂、ZrO₂One or two kinds of composite nanoparticle therein, Preferably Fe₃O₄-TiO₂Composite nanoparticle, its preparation method are referred to the Chinese invention patent that applicant has applied CN102125699A；Or Au and TiO with CT signals₂、ZrO₂One of which or two kinds of composite nanoparticle, preferably Au- TiO₂Composite nanoparticle, its preparation method bibliography：Z.W.Seh,S.Liu,M.Low,S.Y.Zhang,Z.Liu, A.Mlayah,M.Y.Han.Janus Au-TiO₂ photocatalysts with strong localization of plasmonic near-fields for efficient visible-light hydrogen generation.Advanced Materials2012,24:2310-2314.；Or excite up-conversion fluorescence with near-infrared The nano material and TiO of signal properties₂、ZrO₂One of which or two kinds of composite nanoparticle, preferably NaYF₄:Er³⁺/Yb³ ⁺、NaYF₄:Yb³⁺/Tm³⁺、NaYF₄:Tm³⁺/Er³⁺、NaYF₄:Yb³⁺/Tm³⁺/Er³⁺A kind of and TiO therein₂Composite nano-granule Son, more preferably NaYF₄:Yb³⁺/Tm³⁺-TiO₂Composite nanoparticle, its preparation method are referred to during applicant applied State patent of invention CN102743752A.

In another preference, the preferred 5-150 nanometers of particle size range of the composite nanoparticle, more preferably 50-150 nanometers.

In another preference, the antineoplastic includes adriamycin and/or taxol.

In another preference, the active constituents of medicine is carried in a manner of non-covalent linking on the core carrier.Institute It can be by physical absorption, electrostatic force, intermolecular force one or more side therein to state non-covalent linking mode Formula connects.

In another preference, the stabilizer for wrapping up the core carrier and active constituents of medicine is the fat of PEG modifications Plastid nanosphere, its preparation method bibliography：Wang Xiangtao, Li Sha, Zhang little Bin, Hou Xinpo .PEG modified liposomes are to adriamycin The influence Peking University journal (medicine) of drugloading rate, 2002,34 (3):286-289；Or preferred BSA nanospheres, it is prepared The paper that method application reference people delivers：Z.Shen,W.Wei,H.Tanaka,K.Kohama,G.Ma,T.Dobashi, Y.Maki,H.Wang,J.Bi,S.Dai.“A galactosamine-mediated drug delivery carrier for targeted liver cancer therapy.Pharmacological Research”,2011,64:410-419。

In another preference, after the stabilizer is freeze-dried, there can be the function of enhancing ultrasonic contrast imaging signal. The particle diameter of the stabilizer is preferably 30-250 nanometers, more preferably 50-200 nanometers.

In another preference, the ternary nano combination drug is with foregoing Ultrasound-activated property and contains antineoplastic The composite nanostructure of thing, preferably TiO₂The TiO of-adriamycin, more preferably PEG parcel₂- adriamycin；Either have simultaneously foregoing Ultrasound-activated property and T₁What weighted mri signal and the composite nanostructure containing antineoplastic, preferably PEG wrapped up Gd₂O₃-TiO₂- adriamycin；Either there is foregoing Ultrasound-activated property simultaneously with T2 weighted mris signal and containing antitumor The Fe of the composite nanostructure of medicine, preferably PEG parcel₃O₄-TiO₂- adriamycin；Either there is foregoing Ultrasound-activated simultaneously The Au-TiO that property is wrapped up with CT signals and the composite nanostructure containing antineoplastic, preferably PEG₂- adriamycin；Either Have that foregoing Ultrasound-activated property and near-infrared excite up-conversion fluorescence signal and compound containing antineoplastic is received simultaneously Rice structure, the NaYF of preferably PEG parcels₄:Yb³⁺/Tm³⁺-TiO₂- adriamycin.

The ternary nano combination drug of the present invention has Ultrasound-activated property, i.e., the ternary nano combination drug is excellent through frequency It is preferably 0.5-2W/cm to elect 800-10000K Hz and energy as²Ultrasonic Radiation after, can be produced in biological tissue OH¹、OH、H₂O₂, O isoreactivity free radicals（ROS）.

The method for preparing ternary nano combination drug

The method for preparing the invention described above ternary nano combination drug, it is comprised the steps of：

Ternary nano combination drug is used for the purposes for preparing the pharmaceutical compositions for the treatment of tumour

The present invention also provides the pharmaceutical compositions that a kind of ternary nano combination drug as the aforementioned is used to prepare treatment tumour Purposes.

Tumour is acted on using ternary nano combination drug of the present invention, by doses Ultrasonic Radiation tumor locus, The neoplasm targeted therapy that is cooperateed with chemotherapy of ultrasonic therapy can be achieved, using medical imaging instruments can carry out tumor locus imaging or Person carries out real-time therapeutic evaluation to the synergy of two kinds of Therapeutic modes.

When treating tumour, either it can be injected by tumor locus or smeared by tumor locus by drip-feed Mode, the ternary nano combination drug is offerd medicine in tumor locus.

In another preference, realize that the treatment that ultrasonic therapy cooperates with chemotherapy can be using frequency of use as 800- 10000KHz, highest energy 2W/cm²Ultrasonic Radiation knurl position.More preferably frequency is 1000KHz, energy 0.5W/ cm²。

Tumour suitable for the ternary nano Composite drug treatment of menopausal can be the tumour to chemotherapy medicament sensitive, or more Medicine drug-resistant tumor.

The mechanism for the neoplasm targeted therapy that the ultrasonic therapy cooperates with chemotherapy is speculated as：After dispensing, the ternary nano is answered Composite medicine targeting is enriched in tumor tissues, tumour cell takes in the ternary nano combination drug, then pharmaceutical activity therein Composition is released in the cell, TiO therein₂Or ZrO₂OH is produced through Ultrasonic Radiation¹、OH、H₂O₂, the ROS such as O, medicine lives Property composition is killed or destroyed to tumour cell jointly with free radical, so as to realize the collaboration of two kinds of Therapeutic modes；Either After dispensing, after ternary nano combination drug targeting is enriched in tumor tissues, due to the acidic micro-environment of tumor tissues, medicine is lived Property composition is released, and the ultrasonic radiation given enhances infiltration of the active constituents of medicine to cell, improves active constituents of medicine Killing to tumour；The either synthesis of foregoing two kinds of situations.

The medical imaging instruments can be magnetic resonance imager, CT instrument, or energy Detecting material produces the instrument of up-conversion fluorescence phenomenon after near infrared light excites.

Ultrasonic therapeutical system

The first ultrasonic therapeutical system of the invention, which includes, to be used to launch the device of ultrasonic wave and ternary nano as the aforementioned Combination drug.

The device for being used to launch ultrasonic wave suitable for the present invention can be known therapeutic medical ultrasonic unit, as long as energy Launch aforementioned frequencies and energy range.

Bielement nano combination drug is used to be prepared by ultrasonic excitation so as to treat the purposes of the pharmaceutical compositions of tumour, With ultrasonic therapeutical system

The present invention, which also provides, to be used to bielement nano combination drug be prepared by ultrasonic excitation so as to treat the medicine of tumour The purposes of composition is learned, the bielement nano combination drug includes core carrier and the pharmaceutical activity being carried on the core carrier Composition, and the definition of core carrier and active constituents of medicine carries with the core included in foregoing ternary nano combination drug respectively Body is identical with active constituents of medicine.

In another preference, on the basis of weight, the content ratio of the core carrier and active constituents of medicine is 1：0.01 To 1：0.5.More preferably 1:0.01 to 1:0.1.

Second of ultrasonic therapeutical system of the present invention includes the device for being used for launching ultrasonic wave, and foregoing bielement nano is answered Composite medicine.The bielement nano combination drug it is as defined above.

The main beneficial effect of the present invention includes：

(1) preparation method the invention provides a kind of oncotherapy with ternary nano combination drug, this method are simply easy OK, cost is cheap, beneficial to industrialized production and marketing.

(2) ternary nano combination drug of the present invention can realize the neoplasm targeted therapy that ultrasonic therapy cooperates with chemotherapy, special It is not the treatment of multidrug resistance of tumor, effectively reduces the side effect of chemotherapeutics.

(3) core carrier in ternary nano combination drug of the present invention is that have first being received based on this for medical imaging signal The composite nano-granule period of the day from 11 p.m. to 1 a.m of rice corpuscles, it can realize that the real-time curative effect being imaged in diagnosis and tumor therapeutic procedure of tumour is commented Valency.

(4) because the Noninvasive of ultrasonic wave, non-invasive, controllability and good tissue penetration, the present invention provide Ternary nano combination drug can realize the targeting non-invasive therapy of tumour, especially multidrug resistance of tumor and real-time therapeutic evaluation.

(5) present invention, which also provides, is applied to bielement nano combination drug to be prepared by ultrasonic excitation so as to treat tumour Pharmaceutical compositions purposes.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention..

1. adriamycin：Purchased from Aladdin reagent (Shanghai) Co., Ltd.；Model：D107159.

2.PEG600：Purchased from Aladdin reagent (Shanghai) Co., Ltd.；Model：P103727.

3.PEG1500：Purchased from Aladdin reagent (Shanghai) Co., Ltd.；Model：P103721.

4.PEG4000：Purchased from Aladdin reagent (Shanghai) Co., Ltd.；Model：P103724.

5. bovine serum albumin(BSA) nanosphere：Purchased from Aladdin reagent (Shanghai) Co., Ltd.；Model：A104912.

6. folic acid：Purchased from Aladdin reagent (Shanghai) Co., Ltd.；Model：F103635.

Embodiment 1：PEG-TiO₂- adriamycin ternary nano combination drug

(1.1) prepare：

(a)TiO₂- adriamycin bielement nano combination drug：

(a-1) TiO is prepared₂- adriamycin bielement nano combination drug：

Take TiO of the particle diameter for 30-50nm nanometers, with Ultrasound-activated property₂Powder（Preparation method application reference people delivers Paper：A Wu,T Paunesku,EMB Brown,A Babbo,C Cruz,M Aslam,et al.NANO2008,3,27- 36.）It is dissolved in ultra-pure water, 0.075mol/L TiO is made₂Nanoparticles Hydrosol 100mL, lucifuge, logical nitrogen gas stirring 0.5 are small When；Under stirring, 0.005mol/L adriamycin aqueous solution 8.25mL are added dropwise, lucifuge, closed air stirring 12 hours, Adriamycin is set fully to be adsorbed onto TiO₂Nanoparticle surface；After stirring terminates, mixture solution is transferred in centrifuge tube and centrifuged, Centrifugal rotational speed is 12000 revs/min, and centrifugation time is 30 minutes；Abandoning supernatant after centrifugation, then put into 100mL ultra-pure waters and incite somebody to action Precipitation is resuspended, after repeated centrifugation operation twice；Put into 100mL phosphate buffer solutions（PBS, 0.01mol, pH7.4）And will be heavy Form sediment and be resuspended, after 0.2 μm of membrane filtration, obtain sterile TiO₂- adriamycin bielement nano combination drug, and be stored in 4 DEG C and treat With；Or after repeated centrifugation operation twice, combination drug is freeze-dried, it is standby to obtain dried powder.The TiO obtained₂- The particle size range of adriamycin bielement nano combination drug is 50-70 nanometers.

(a-2)TiO₂The sign of-adriamycin bielement nano combination drug：

Using Fourier transformation infrared spectrometer, dynamic light scattering to the TiO prepared by (a-1)₂- adriamycin binary is received Rice combination drug carries out infrared absorption, Zeta potential characterizes.Characterization result is as shown in figure 1, with TiO in Fig. 1 a and 1b₂/ DOX is bent Line and histogram graph representation TiO₂The measurement result of-adriamycin bielement nano combination drug, and TiO₂Represented respectively with DOX single TiO₂With measurement result (Fig. 2 TiO into Fig. 7 of adriamycin₂/DOX、TiO₂Defined with DOX it is identical, behind be not repeated to illustrate), A figures show that adriamycin (DOX) is loaded into TiO in Fig. 1₂Afterwards, there is not new infrared absorption peak, illustrate that adriamycin passes through and compare Weak active force and TiO₂With reference to b figures are shown in TiO in neutral aqueous solution₂Surface is negative electricity, and adriamycin is loaded into TiO₂Afterwards, TiO₂Zeta potential reduce, due to the amino group in Doxorubicin molecules in neutral aqueous solution positively charged, illustrate adriamycin TiO is loaded into by way of Electrostatic Absorption₂Nanoparticle surface.

(a-3)TiO₂The calculating of surface adriamycin charging ratio

Adriamycin is dissolved in ultra-pure water, be configured to concentration gradient for 3.00,1.50,0.75,0.38,0.19,0.09, 0.04th, 0.01mg/mL solution, above-mentioned solution 0.5mL is taken respectively, various concentrations adriamycin is detected using ultraviolet-visible spectrometer Absorption, calculate doxorubicin concentration-absorption standard curve.

The supernatant centrifuged three times in (a-1) is collected, uses adriamycin in ultraviolet-visible spectrometer detection three times supernatant Absorption, the doxorubicin concentration obtained according to back-absorption standard curve calculates the content of adriamycin in supernatant three times, i.e., Without being loaded in TiO₂The adriamycin amount on surface, according to the adriamycin total amount of input, calculate the TiO that (a-1) is obtained₂- adriamycin binary TiO in nano combined medicine₂The charging ratio of surface adriamycin is 1.1%.

(a-4)TiO₂The drug release in vitro rule of-adriamycin

The TiO for taking (a-1) to prepare₂- adriamycin bielement nano combination drug dry powder 60mg, is scattered in 30mL ultra-pure waters, Three parts, every part of 10mL are divided into, is placed in three bag filters, bag filter is respectively placed in the 90ml phosphate-buffereds of pH5,6,7 Liquid, dialysis apparatus is positioned in 37 ° of insulating boxs and dialysed 48 hours, every 1 hour, measure a dialyzate in adriamycin purple Outside-visible absorption spectra, doxorubicin concentration-absorption standard curve in (a-3), medicine is calculated under different acidic conditions Release profiles.

(b)PEG-TiO₂The preparation of-adriamycin ternary nano combination drug：

(b-1)PEG600-TiO₂- adriamycin ternary nano combination drug：The 100mL TiO that (a-1) is obtained₂- adriamycin The bielement nano combination drug hydrosol leads to nitrogen gas stirring 0.5 hour under the conditions of lucifuge；By 0.05mol/L PEG（Molecular weight 600）Solution 10mL is added dropwise to TiO₂In-adriamycin the hydrosol, lucifuge, closed air stirring 24 hours；After stirring terminates, Mixture solution is transferred in centrifuge tube and centrifuged, centrifugal rotational speed is 12000 revs/min, and centrifugation time is 30 minutes；After centrifugation Abandoning supernatant, then put into 100mL ultra-pure waters and precipitation is resuspended, repeated centrifugation operates twice；It is molten to put into 100mL phosphate-buffereds Liquid（PBS, 0.01mol, pH7.4）And precipitation is resuspended, after 0.2 μm of membrane filtration, obtain sterile PEG-TiO₂- adriamycin Ternary nano combination drug (the i.e. TiO of PEG parcels₂- adriamycin nano combination drug), and be stored in 4 DEG C it is stand-by；Or repeat Centrifugally operated twice after, ternary nano combination drug is freeze-dried, obtain dried powder it is standby.The ternary nano obtained The average grain diameter of combination drug is 170 nanometers.

(b-2)PEG1500-TiO₂- adriamycin ternary nano combination drug：With (b-1), difference is only that the PEG used Molecular weight be 1500.

(b-3)PEG4000-TiO₂- adriamycin ternary nano combination drug：With (b-1), difference is only that the PEG used Molecular weight be 4000.

(1.2)PEG-TiO₂The sign of-adriamycin ternary nano combination drug：

Using dynamic light scattering particle diameter distribution instrument to the PEG4000-TiO prepared by (1.1)₂- adriamycin ternary nano is answered Composite medicine carries out particle diameter distribution sign.Characterization result is as shown in Fig. 2 with TiO in Fig. 2₂- DOX-PEG4000 represents PEG4000- TiO₂The measurement result of-adriamycin ternary nano combination drug, Fig. 2 show TiO₂After medicine and PEG are carried in surface, particle diameter is bright It is aobvious to become big.

(1.3) evaluation of anti-multidrug resistance tumor cells

(1) (1.1) three kinds of PEG-TiO prepared by Example 1₂- adriamycin ternary nano combination drug (PEG molecular weight Respectively for 600,1500,4000) and identical drug concentration adriamycin sterile solution it is stand-by.

(2) take the logarithm the human breast carcinoma multidrug resistance cell MCF-7/ADM in growth period, and adjustment cell concentration is 1 × 10⁵ Individual/mL, it is inoculated in respectively in 96 porocyte culture plates, per the μ L of hole 100, or is inoculated in 6 porocyte culture plates, per hole 2mL.Carefully Born of the same parents are in 5%CO₂, 37 DEG C, cultivate 24 hours in the cell culture incubator of saturated humidity.

After (3) 24 hours, the culture medium in culture orifice plate is discarded, it is (right that fresh medium is separately added into culture plate According to group), contain PEG600-TiO₂The culture medium of-adriamycin ternary nano combination drug, contain PEG1500-TiO₂- adriamycin three The culture medium of the nano combined medicine of member, contain PEG4000-TiO₂The culture medium of-adriamycin ternary nano combination drug, contain Ah The culture medium of mycin, in 5%CO₂, 37 DEG C, be incubated 24 hours in the cell culture incubator of saturated humidity.

(4) three kinds of PEG-TiO are detected using mtt assay₂- adriamycin ternary nano combination drug is to human breast cancer cell Killing ability；PEG4000-TiO is studied by laser confocal microscope₂- adriamycin ternary nano combination drug is thin in tumour The regularity of distribution of intracellular.As a result respectively as shown in Figure 3 and Figure 4, with TiO in Fig. 3₂-DOX-PEG600、TiO₂-DOX-PEG1500、 TiO₂- DOX-PEG4000 block diagram represents PEG600-TiO respectively₂- adriamycin ternary nano combination drug, PEG1500- TiO₂- adriamycin ternary nano combination drug, PEG4000-TiO₂The measurement result of-adriamycin ternary nano combination drug, Fig. 3 Display compared with free adriamycin, different molecular weight PEG parcel nano combined medicine can it is significant raising adriamycin resist it is resistance to The activity of medicine tumour cell.Fig. 4 is shown（A is control group；B is independent adriamycin group；C is PEG4000-TiO₂- adriamycin group； Blue-fluorescence marks nucleus, red fluorescence mark adriamycin）, ternary nano combination drug can it is significant raising adriamycin resistance to Distribution in medicine tumour cell.

Embodiment 2：PEG-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug

(2.1) prepare：

(a) Fe is prepared₃O₄-TiO₂- adriamycin bielement nano combination drug：

Take particle diameter for 40-60nm nanometers, there is T₂The magnetic resonance signal of weighting and the Fe of Ultrasound-activated property₃O₄-TiO₂Receive Meter Fu He powders (its preparation method is referred to the Chinese invention patent CN102125699A that foregoing applicant has applied) It is dissolved in ultra-pure water, 0.1mol/L Fe is made₃O₄-TiO₂Nanoparticles Hydrosol 90mL, lucifuge, logical nitrogen gas stirring 0.5 are small When；Under stirring, be added dropwise 0.005mol/L Doxorubicin solution 10mL, lucifuge, closed air stirring 24 hours, make Ah Mycin is fully adsorbed onto Fe₃O₄-TiO₂Surface；After stirring terminates, mixture solution is transferred in centrifuge tube and centrifuged, centrifugation turns Speed is 12000 revs/min, and centrifugation time is 30 minutes；Abandoning supernatant after centrifugation, then put into 100mL ultra-pure waters and weigh precipitation It is outstanding, after repeated centrifugation operation twice, put into 100mL ultra-pure waters and precipitation is resuspended, obtain Fe₃O₄-TiO₂- adriamycin binary is received The rice combination drug hydrosol.

(b) PEG-Fe is prepared₃O₄-TiO₂- adriamycin ternary nano combination drug：

The 100mL Fe that (a) is obtained₃O₄-TiO₂- adriamycin bielement nano combination drug the hydrosol under the conditions of lucifuge, Logical nitrogen gas stirring 0.5 hour；By 0.1mol/L PEG（Molecular weight 1500）Solution 10mL is added dropwise to Fe₃O₄-TiO₂- Ah mould In the plain hydrosol, lucifuge, closed air stirring 24 hours；After stirring terminates, mixture solution is transferred in centrifuge tube and centrifuged, Centrifugal rotational speed is 12000 revs/min, and centrifugation time is 30 minutes；Abandoning supernatant after centrifugation, then put into 100mL ultra-pure waters and incite somebody to action Precipitation is resuspended, and repeated centrifugation operates twice；Put into 100mL phosphate buffer solutions（PBS, 0.01mol, pH7.4）And will precipitation It is resuspended, after 0.2 μm of membrane filtration, obtains sterile PEG-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug (i.e. PEG bags The Fe wrapped up in₃O₄-TiO₂- adriamycin nano combination drug), and be stored in 4 DEG C it is stand-by；Or after repeated centrifugation operation twice, by three The nano combined medicine of member is freeze-dried, and it is standby to obtain dried powder.The particle size range of the ternary nano combination drug obtained , being capable of passive target tumor tissues due to EPR effects for 50-80 nanometers.

(2.2)PEG-Fe₃O₄-TiO₂The evaluation of the anti-multidrug resistance of tumor of-adriamycin ternary nano combination drug（Animal is real Test）

(1) liver cancer cells of the culture with multidrug resistance characteristic in the DMEM culture mediums containing 10% hyclone HepG2, the stable 3-4 that passes on is for after, growth period cell of taking the logarithm, the EDTA digestive juices digestion through 0.25% trypsase+0.02%, Cell is collected, so that cell is resuspended containing the DMEM of 10% hyclone after centrifugation, cell count is carried out and cell density is adjusted to 1 ×10⁷/mL.4-6 week old BALB/C mice 12 is taken, in the μ L of chest inoculated with subcutaneous injections 200 multidrug resistance liver cancer cells HepG2, rat liver cancer multidrug resistance of tumor model is established, after raising 3 days, random point four groups：Control group（3）, adriamycin Treatment group（3）、PEG-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug chemotherapy group（3）、PEG-Fe₃O₄-TiO₂- Ah mould Plain ternary nano combination drug supersonic synergic chemotherapy group（3）.

(2) PEG-Fe for taking (2.1) to prepare₃O₄-TiO₂The sterile hydrosol of-adriamycin ternary nano combination drug, adriamycin Sterile solution, sterile saline, doxorubicin concentration is adjusted to 5 μ g/mL.Respectively by PEG-Fe₃O₄-TiO₂- adriamycin ternary Nano combined medicine delivers medicine to PEG-Fe in the μ L of breast tumor injection location 100₃O₄-TiO₂- adriamycin ternary nano combination drug Chemotherapy group, PEG-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug supersonic synergic chemotherapy group；By adriamycin sterile solution in The μ L of breast tumor injection location 100 deliver medicine to adriamycin chemotherapy group；By sterile saline in the μ L of breast tumor injection location 100 Deliver medicine to control group.

(3) after above-mentioned experimental group administration, every 1 hour, magnetic resonance imager Real Time Observation PEG-Fe is used₃O₄-TiO₂- Adriamycin ternary nano combination drug is assembled in tumor locus, and according to drug accumulation situation, appropriate time is given in tumor locus Ultrasonic Radiation（Frequency 1000K Hz, energy 0.5W/cm²）, and therapeutic effect is monitored in real time using magnetic resonance imager, and Skin, reaction, appetite and the state of mind of observation mouse daily.

(4) after being used as confirmatory experiment, experiment to terminate, drawn blood using heart and put to death mouse, dissection checks gross tumor volume, and adopts The expression of P- glycoprotein in tumor tissue section is observed with Immunohistochemical Method, evaluates therapeutic effect.

Embodiment 3：BSA-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug

(a) Fe is prepared₃O₄-TiO₂- adriamycin bielement nano combination drug：With (2.1).

(b) BSA-Fe is prepared₃O₄-TiO₂- adriamycin ternary nano combination drug：

Fe prepared by (a)₃O₄-TiO₂- adriamycin bielement nano combination drug is freeze-dried to obtain powder, takes 50mg Powder, it is dissolved in 10mL ultra-pure waters, is configured to the 5mg/mL hydrosols.0.8mg/mL sodium chloride solution 50mL is prepared, uses hydrogen-oxygen Change sodium and solution ph is adjusted to 8-11.50mg BSA are weighed, are dissolved in sodium chloride solution, obtain 1mg/mL BSA solution. Under room temperature, lucifuge magnetic agitation, by Fe₃O₄-TiO₂- adriamycin is added dropwise in BSA solution, and it is anhydrous that 6mL is added dropwise dropwise Ethanol, the 10% μ L of glutaraldehyde solution 50 are added dropwise again afterwards, continue in room temperature, lucifuge magnetic agitation ripening 24 hours, 40mg/mL lysine solution 1mL is added dropwise again, reaction system is placed in ultra-pure water, lucifuge by lucifuge magnetic agitation after 2 hours Dialysis 24 hours, removes unnecessary reactant, obtains BSA-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug (is embedded in The Fe of BSA nanospheres₃O₄-TiO₂- adriamycin nano compound), its particle diameter is 80-130nm.After 0.2 μm of membrane filtration, obtain Obtain sterile BSA-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug, and be stored in 4 DEG C it is stand-by；Or by BSA-Fe₃O₄- TiO₂- adriamycin ternary nano combination drug is freeze-dried, and it is standby to obtain dried powder.The dried powder of acquisition can have Strengthen the function of ultrasonic contrast signal.

Embodiment 4：Folic acid-BSA-Fe₃O₄-TiO₂- adriamycin ternary nano combination drug

BSA-Fe prepared by Example 3₃O₄-TiO₂- adriamycin ternary nano combination drug 10mL, room temperature, lucifuge magnetic force Under stirring, 4 μ L carbodiimides (abbreviation EDAC) (50mmol/L) and 6 μ L N- hydroxy thiosuccinimides are added (referred to as NHS) 10mmol/L folic acid solution 1ml, room temperature, lucifuge magnetic force is added dropwise to the activated carboxylic on BSA surfaces in (40mmol/L) Stirring reaction 12 hours.After reaction terminates, reaction system is placed in ultra-pure water, lucifuge is dialysed 24 hours, is removed unnecessary anti- Thing is answered, obtains folic acid-BSA-Fe₃O₄-TiO₂(i.e. the active targeting tumour of modified with folic acid is thin for-adriamycin ternary nano combination drug The BSA-Fe of born of the same parents₃O₄-TiO₂- adriamycin ternary nano combination drug).This combination drug preserves after 0.2 μm of membrane filtration It is stand-by in 4 DEG C；Or combination drug is freeze-dried, it is standby to obtain dried powder.

Embodiment 5：PEG-Gd₂O₃-TiO₂- adriamycin ternary nano combination drug

(a) Gd is prepared₂O₃-TiO₂Nano-complex：

Weigh six water gadolinium nitrates（Gd(NO₃)₃·6H₂O）0.006mol, it is dissolved into 20mL diglycols, Magnetic agitation at 140 DEG C, it is stand-by to obtain solution A.Weigh 0.01mmol sodium hydroxides（NaOH）, dissolved the contracting diethyls of 40mL mono- In glycol, magnetic agitation, it is stand-by to obtain B solution.Solution A is added in B solution, magnetic agitation 1 hour at 140 DEG C, 180 DEG C Lower magnetic agitation 4 hours.Reaction solution is dialysed：Bag filter molecular cut off 3500, dialyse 6 times.By 1.8mol/L titanium Above-mentioned dialyzate is added dropwise in four butyl acetate solution 10mL of acid, continues to stir；After reaction 8 hours, added into product 0.0008mol/L CTAB15mL, and continue to stir 30min；Ripening is carried out at room temperature, and digestion time is 18 hours； Product is centrifuged, obtains pure Gd₂O₃-TiO₂Nano-complex, freeze-dried acquisition solid powder are standby.

(b) Gd is prepared₂O₃-TiO₂- adriamycin bielement nano combination drug：

Take the Gd prepared in (a)₂O₃-TiO₂Powder 2g, it is dissolved in 90mL ultra-pure waters, Gd is made₂O₃-TiO₂Nano-particle water Colloidal sol, lucifuge, logical nitrogen gas stirring 0.5 hour；Under stirring, 0.005mol/L adriamycin ultra-pure water solutions are added dropwise 10mL, lucifuge, closed air stirring 12 hours, makes adriamycin fully be adsorbed onto Gd₂O₃-TiO₂Surface；, will be mixed after stirring terminates Polymer solution is transferred in centrifuge tube and centrifuged, and centrifugal rotational speed is 12000 revs/min, and centrifugation time is 30 minutes；Discarded after centrifugation Supernatant, then put into 100mL ultra-pure waters and precipitation is resuspended, after repeated centrifugation operation twice, putting into 100mL ultra-pure waters will simultaneously precipitate It is resuspended, obtains Gd₂O₃-TiO₂- adriamycin bielement nano combination drug the hydrosol.

(c) PEG-Gd is prepared₂O₃-TiO₂- adriamycin ternary nano combination drug：

The 100mL Gd that (a) is obtained₂O₃-TiO₂- adriamycin bielement nano combination drug the hydrosol under the conditions of lucifuge, Logical nitrogen gas stirring 0.5 hour；By 0.05mol/L PEG（Molecular weight 1500）Solution 10mL is added dropwise to Gd₂O₃-TiO₂- Ah mould In the plain hydrosol, lucifuge, closed air stirring 12 hours；After stirring terminates, mixture solution is transferred in centrifuge tube and centrifuged, Centrifugal rotational speed is 10000 revs/min, and centrifugation time is 20 minutes；Abandoning supernatant after centrifugation, then put into 100mL ultra-pure waters and incite somebody to action Precipitation is resuspended, and repeated centrifugation operates twice；Put into 100mL phosphate buffer solutions（PBS, 0.01mol, pH7.4）And will precipitation It is resuspended, after 0.2 μm of membrane filtration, obtains sterile PEG-Gd₂O₃-TiO₂- adriamycin ternary nano combination drug (i.e. PEG bags The Gd wrapped up in₂O₃-TiO₂- adriamycin nano combination drug), and be stored in 4 DEG C it is stand-by；Or after repeated centrifugation operation twice, will be multiple Composite medicine is freeze-dried, and it is standby to obtain dried powder；The particle size range of the combination drug obtained is 70 rans, due to EPR effects, being capable of passive target tumor tissues.

Embodiment 6：PEG-Au-TiO₂- adriamycin ternary nano combination drug

(a) Au-TiO is prepared₂- adriamycin bielement nano combination drug：

Take particle diameter for 20nm rans, there is CT signals and the Au-TiO of Ultrasound-activated property₂Nano combined powder 1g (this Au-TiO₂Powder preparation method refers to document：Z.W.Seh,S.Liu,M.Low,S.Y.Zhang,Z.Liu, A.Mlayah,M.Y.Han.Janus Au-TiO₂ photocatalysts with strong localization of plasmonic near-fields for efficient visible-light hydrogen generation.Advanced Materials2012,24:2310-2314.), it is dissolved in ultra-pure water, 90mL Au- is made TiO₂Nanoparticles Hydrosol, lucifuge, logical nitrogen gas stirring 1 hour；Under stirring, 0.01mol/L adriamycins are added dropwise Solution 10mL, lucifuge, closed air stirring 24 hours, makes adriamycin fully be adsorbed onto Au-TiO₂Surface；, will after stirring terminates Mixture solution is transferred in centrifuge tube and centrifuged, and centrifugal rotational speed is 12000 revs/min, and centrifugation time is 30 minutes；Abandoned after centrifugation Supernatant is removed, then puts into 100mL ultra-pure waters to precipitate resuspension, after repeated centrifugation operation twice, putting into 100mL ultra-pure waters simultaneously will be heavy Form sediment and be resuspended, obtain Au-TiO₂- adriamycin bielement nano combination drug the hydrosol.

(b) PEG-Au-TiO is prepared₂- adriamycin ternary nano combination drug：

The 100mL Au-TiO that (a) is obtained₂- adriamycin bielement nano combination drug the hydrosol leads under the conditions of lucifuge Nitrogen gas stirring 1 hour；By 0.1mol/L PEG（Molecular weight 4000）Solution 10mL is added dropwise to Au-TiO₂- adriamycin binary In the nano combined medicine hydrosol, lucifuge, closed air stirring 24 hours；Stirring terminate after, by mixture solution be transferred to from Centrifuged in heart pipe, centrifugal rotational speed is 12000 revs/min, and centrifugation time is 30 minutes；Abandoning supernatant after centrifugation, then put into 100mL ultra-pure waters will be precipitated and are resuspended, and repeated centrifugation operates twice；Add 100mL phosphate buffer solutions（PBS, 0.01mol, pH7.4）And precipitation is resuspended, after 0.2 μm of membrane filtration, obtain sterile PEG-Au-TiO₂- adriamycin ternary nano is compound Medicine (the i.e. Au-TiO of PEG parcels₂- adriamycin nano combination drug), and be stored in 4 DEG C it is stand-by；Or repeated centrifugation operation It is after twice, combination drug is freeze-dried, it is standby to obtain dried powder；The particle size range of the nano combined medicine obtained is 50 rans, being capable of passive target tumor tissues due to EPR effects.

Embodiment 7：PEG-NaYF₄:Yb³⁺/Tm³⁺-TiO₂- adriamycin ternary nano combination drug

(a) NaYF is prepared₄:Yb³⁺/Tm³⁺-TiO₂- adriamycin bielement nano combination drug：

Particle diameter is taken as 50-100 nanometers, the NaYF that there is near-infrared to excite up-conversion fluorescence signal and Ultrasound-activated property₄: Yb³⁺/Tm³⁺-TiO₂(this powder preparation method can refer to the Chinese invention patent that applicant has applied to nano combined powder 1g CN102743752A) it is dissolved in ultra-pure water, NaYF₄:Yb³⁺/Tm³⁺TiO₂Nanoparticles Hydrosol 90mL, lucifuge, logical nitrogen gas stirring 1 hour；Under stirring, 0.1mol/L adriamycin ultra-pure water solution 10mL are added dropwise, lucifuge, closed air stirring 24 are small When, adriamycin is fully adsorbed onto NaYF₄:Yb³⁺/Tm³⁺-TiO₂Surface；After stirring terminates, mixture solution is transferred to centrifugation Centrifuged in pipe, centrifugal rotational speed is 10000 revs/min, and centrifugation time is 20 minutes；Abandoning supernatant after centrifugation, then put into 100mL Ultra-pure water will be precipitated and is resuspended, and after repeated centrifugation operation twice, puts into 100mL ultra-pure waters and precipitation is resuspended, obtain NaYF₄:Yb³ ⁺/Tm³⁺-TiO₂- adriamycin bielement nano combination drug the hydrosol.

(b) PEG-NaYF is prepared₄:Yb³⁺/Tm³⁺-TiO₂- adriamycin ternary nano combination drug：

The 100mL NaYF that (a) is obtained₄:Yb³⁺/Tm³⁺-TiO₂- adriamycin binary nano complex the hydrosol is in lucifuge Under the conditions of, lead to nitrogen gas stirring 1 hour；By 0.05mol/L PEG（Molecular weight 1600）Solution 20mL is added dropwise to NaYF₄:Yb³ ⁺/Tm³⁺-TiO₂In-adriamycin binary nano complex the hydrosol, lucifuge, closed air stirring 24 hours；, will after stirring terminates Mixture solution is transferred in centrifuge tube and centrifuged, and centrifugal rotational speed is 10000 revs/min, and centrifugation time is 20 minutes；Abandoned after centrifugation Supernatant is removed, then puts into 100mL ultra-pure waters to precipitate resuspension, repeated centrifugation operates twice；Add 100mL phosphate buffer solutions （PBS, 0.01mol, pH7.4）And precipitation is resuspended, after 0.2 μm of membrane filtration, obtain sterile PEG-NaYF₄:Yb³⁺/Tm³ ⁺-TiO₂- adriamycin ternary nano combination drug (the i.e. NaYF of PEG parcels₄:Yb³⁺/Tm³⁺-TiO₂- adriamycin nano composite medicine Thing), and be stored in 4 DEG C it is stand-by；Or after repeated centrifugation operation twice, nano combined medicine is freeze-dried, obtain drying Powder is standby；The particle size range of the nano combined medicine obtained is 50 rans, being capable of passive target due to EPR effects Tumor tissues.

Embodiment 8：PEG/ liposomes-TiO₂- adriamycin ternary nano combination drug

It is single to weigh hydrogenated soya phosphatide 0.76g, cholesterol 0.45g, PEG-DSPE 2000 Methoxy ether 0.22g, is respectively divided into two parts.A copy of it 10mL chloroforms-n-hexane (1:1, volume ratio) dissolving, add embodiment TiO prepared by 1 (1.1) (a)₂- Doxorubicin solution 2mL, ultrasonic disperse is into after breast, through depressurizing rotary evaporation to the dense breasts of about 2mL. Another is dissolved in 5mL chloroforms-n-hexane (1:1, volume ratio), it is added to containing TiO₂In the dense breast of-adriamycin, after mixing, add Enter 10mL lysis buffers (0.02mol/L^-1), fully after vibration, W/O/W emulsions are obtained, the emulsion is through being evaporated under reduced pressure to not Bubble is produced again, nitrogen blowing evaporation of organic solvent, room temperature ultrasound 2 minutes, produces PEG/ liposomes-TiO₂- adriamycin ternary is received Rice combination drug (is embedded in the TiO of PEG and liposome₂- adriamycin nano combination drug), its particle diameter is 60-100nm.Through After 0.2 μm of membrane filtration, obtain sterile nano combined medicine, and be stored in 4 DEG C it is stand-by；Or by nano combined medicine through cold It is lyophilized dry, it is standby to obtain dried powder.

Embodiment 9：In-vitro evaluation TiO₂The ultrasonic excitation activity of-adriamycin bielement nano combination drug

Compound concentration gradient be 0.05,0.1,0.2,0.4,0.6mg/mL alizarin red aqueous solution, use ultraviolet-visible spectrum Instrument detection absorbs, and draws alizarin red concentration versus absorbance standard curve.TiO made from Example 1 (a-1)₂- adriamycin binary is received Rice combination drug 50mg, 5 parts of the hydrosol and decile that 50mL ultra-pure waters are configured to 1mg/mL are scattered in, is added in every part of hydrosol Enter 5mg alizarin reds.Use 1000KHz, 0.5W/cm²Ultrasonic wave ultrasonic radiations, radiated time are carried out to 5 parts of reaction solutions respectively Respectively 0.5,1,2,4,8 minute.Radiation detects alizarin red ultraviolet-visible spectrum after terminating absorbs, according to foregoing alizarin red Concentration versus absorbance standard curve calculates the alizarin red concentration remained in dispersion liquid, evaluates TiO₂- adriamycin bielement nano combination drug Ultrasonic excitation activity.

Take TiO₂- adriamycin bielement nano combination drug 50mg, is scattered in 50mL ultra-pure waters and is configured to the water-soluble of 1mg/mL 5 parts of glue and decile.Use 1000K Hz, 0.5W/cm²Ultrasonic wave ultrasonic radiations, radiated time are carried out to 5 parts of reaction solutions respectively Respectively 0.5,1,2,4,8 minute.Radiation uses ROS detection kit quantitative assessments TiO after terminating₂- adriamycin nano is compound The ultrasonic excitation activity of thing.

Embodiment 10：TiO₂The evaluation of the anti-multidrug resistance tumor cells of-adriamycin bielement nano combination drug

(1) TiO made from Example 1 (a-1)₂- adriamycin bielement nano combination drug and identical drug concentration Adriamycin sterile solution is stand-by.

(2) take the logarithm human breast cancer cell line Bcap-37, the multidrug resistance cell MCF-7/ADM in growth period, adjusts cell concentration For 1 × 10⁵Individual/mL, it is inoculated in respectively in 96 porocyte culture plates, per hole 100L, or is inoculated in 6 porocyte culture plates, often Hole 2mL.Cell is in 5%CO₂, 37 DEG C, cultivate 24 hours in the cell culture incubator of saturated humidity.

After (3) 24 hours, the culture medium in culture orifice plate is discarded, fresh medium is separately added into culture plate, is contained There is TiO₂Culture medium, the culture medium containing adriamycin of-adriamycin bielement nano combination drug, in 5%CO₂, 37 DEG C, saturated humidity Cell culture incubator in be incubated respectively 2,4,6,24,48,72 hours.

(4) using mtt assay detection TiO₂The killing ability of-adriamycin bielement nano combination drug to human breast cancer cell； Pass through X ray Synchrotron Radiation research TiO₂Point of the Ti elements of-adriamycin bielement nano combination drug in tumour cell Cloth rule；TiO is studied using laser confocal microscope₂The distribution in the cell of-adriamycin bielement nano combination drug and Release rule.Testing result is as shown in Fig. 5, Fig. 6 and Fig. 7.

Fig. 5 is illustrated in embodiment 10 using mtt assay detection TiO₂The anti-human breast cancer of-adriamycin bielement nano combination drug Cell MCF-7 (drug sensitive cell, a figures), MCF-7/ADM (multidrug resistance cell, b figures) effect.Fig. 5 shows TiO₂- Ah Mycin bielement nano combination drug being capable of the significant effect for improving adriamycin overriding resistance tumour cell.

Fig. 6 is to illustrate TiO in embodiment 10₂- adriamycin bielement nano combination drug is in human breast cancer cell line Bcap-37 (medicine Thing sensitive cells, a figures), the X ray synchrotron radiation result of MCF-7/ADM (multidrug resistance cell, b figure) distributions.Fig. 6, which is shown, to be received The regularity of distribution of Ti elements in the cell in rice combination drug.

Fig. 7 is to illustrate TiO in embodiment 10₂- adriamycin bielement nano combination drug is in human breast cancer cell line Bcap-37 (medicine Thing sensitive cells, a figures), the laser confocal microscope result of MCF-7/ADM (multidrug resistance cell, b figure) distributions.Fig. 7 is shown Nano combined medicine being capable of the significant intake for improving adriamycin in cells of resistant tumors.(" superposition " figure represents " cell membrane/core " Figure with " stack result of cell membrane/DOX " figures)

Embodiment 11：Ultrasound and the evaluation of chemotherapy double mode synergistic treatment multidrug resistance tumor cells

(1) sterile TiO prepared by Example 1 (a-1)₂- adriamycin bielement nano combination drug and identical medicine are dense The adriamycin sterile solution of degree is stand-by.

After (3) 24 hours, the culture medium in culture orifice plate is discarded, fresh medium is separately added into culture hole, is contained TiO₂The culture medium of-adriamycin bielement nano combination drug or adriamycin, after acting on two hours, use 1000KHz, 0.5W/ cm²Ultrasonic wave respectively to Cell irradiation 3 minutes, using dispenser but not by the cell of Ultrasonic Radiation as control, in 5%CO₂、 37 DEG C, cultivate respectively 2,4,6,24,48,72 hours in the cell culture incubator of saturated humidity.

(4) using mtt assay detection ultrasound and the lower TiO of chemotherapy synergy₂- adriamycin bielement nano combination drug is to human milk The fragmentation effect of adenocarcinoma cell；Pass through TiO under X ray Synchrotron Radiation research double mode therapeutic action₂- adriamycin binary is received The regularity of distribution of the Ti elements of rice combination drug in the cell；Studied using laser confocal microscope under double mode therapeutic action TiO₂The distribution in the cell of-adriamycin bielement nano combination drug and release rule；Using ROS kits detection ultrasound with Under the treatment of chemotherapy double mode, ROS generation in tumour cell；Detected by Western-blot methods under double mode therapeutic action, The expression quantity of tumour cell p- glycoprotein；With the lower apoptosis of tumor cells GAP-associated protein GAP of RT-PCR method detection double mode treatment Caspase-3, multidrug resistance gene MDR1 expression.

In summary, using the nano-particle with Ultrasound-activated property or composite nanoparticle as core carrier, by medicine Active component is loaded on core carrier, then is wrapped in outside with stabilizer, so as to obtain the ternary that can be used for oncotherapy Nano combined medicine, in addition, also it further can be made by the way that nano combined medical surfaces are modified with the ligand molecular of target tumor With active targeting function of tumor.Furthermore the bielement nano combination drug and ternary nano of the present invention not comprising stabilizer are compound Medicine is after being administered, and by doses Ultrasonic Radiation tumor locus, achievable ultrasonic therapy cooperates with swollen with chemotherapy Knurl targeted therapy, medical imaging instruments can be used to be imaged to tumor locus or carried out in fact to the synergy of two kinds of Therapeutic modes When therapeutic evaluation.

All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims

1. a kind of ternary nano combination drug, it is characterised in that the ternary nano combination drug includes：

Core carrier, it includes the composite nanoparticle based on the first nano-particle with medical imaging signal；Described first Nano-particle is the first nano-particle that living radical can be produced by ultrasonic excitation；First nano-particle is TiO₂；And the composite nanoparticle includes first nano-particle, and selected from Gd₂O₃、Fe₃O₄、ZnFe₂O₄、NiFe₂O₄、 Au、NaYF₄:Er³⁺/Yb³⁺、NaYF₄:Yb ³⁺/Tm³⁺、NaYF₄:Tm³⁺/Er³⁺And NaYF₄:Yb ³⁺/Tm³⁺/Er³⁺In one kind or It is a variety of；

Active constituents of medicine, it is carried on the core carrier；With

For wrapping up the stabilizer of the core carrier and active constituents of medicine, the stabilizer includes hydrophilic polymer, table Contain hydroxyl or the liposome of carboxyl or its combination in face；

Also, the active constituents of medicine includes antineoplastic, and the antineoplastic is adriamycin；

Also, on the basis of weight, the content ratio of the core carrier, active constituents of medicine and stabilizer is 1：0.01：0.5 to 1：0.5：1.

2. combination drug as claimed in claim 1, it is characterised in that the particle diameter of the combination drug is 10 to 200 nanometers.

3. combination drug as claimed in claim 1, it is characterised in that the medical imaging signal includes T₁The magnetic resonance of weighting Imaging signal, T₂The magnetic resonance imaging signal of weighting, CT signal, near-infrared excite conversion Fluorescence signal, or its combination.

4. combination drug as claimed in claim 1, it is characterised in that the composite nanoparticle includes first nanoparticle Son, and selected from Gd₂O₃、Fe₃O₄、ZnFe₂O₄、NiFe₂O₄、Au、NaYF₄:Er³⁺/Yb³⁺、NaYF₄:Yb ³⁺/Tm³⁺、NaYF₄:Tm³⁺/ Er³⁺And NaYF₄:Yb ³⁺/Tm³⁺/Er³⁺In one kind.

5. combination drug as claimed in claim 1, it is characterised in that the hydrophilic polymer includes non-ionic polymeric Thing, cation type polymer, carbonyl bearing polymer, or its combination.

6. combination drug as claimed in claim 5, it is characterised in that the hydrophilic polymer includes polyethylene glycol, Portugal gathers Sugar, polyethyleneimine, polyvinylamine, Sensor Chip CM 5, polyacrylic acid, or its combination.

7. combination drug as claimed in claim 1, it is characterised in that on the basis of weight, the core carrier, pharmaceutical activity into Divide and the content ratio of stabilizer is 1：0.01：0.5 to 1：0.1：0.8.

8. combination drug as claimed in claim 1, it is characterised in that the combination drug also includes containing for target tumor There is the finishing coat of ligand molecular.

A kind of 9. method for preparing the combination drug as described in any in claim 1-8, it is characterised in that methods described includes Following steps：

(c) the binary hydrosol and the solution containing the stabilizer are mixed, so as to form the ternary nano combination drug.

10. method as claimed in claim 9, it is characterised in that methods described also includes step (d), and it is used to first activate step Suddenly the ternary nano combination drug that (c) is obtained, and the ternary nano after the activation is answered using the solution containing ligand molecular Composite medicine carries out surface modification, and so as to obtain the ternary nano combination drug including finishing coat, the finishing coat contains There is ligand molecular.

11. the ternary nano combination drug as described in any in a kind of 1-8 such as claim is used for the pharmacy group for preparing treatment tumour The purposes of compound.

12. purposes as claimed in claim 11, it is characterised in that the ternary nano combination drug is to be used to be prepared by surpassing Sound wave is excited so as to treat the purposes of the pharmaceutical compositions of tumour.

13. a kind of ultrasonic therapeutical system, it is characterised in that the system includes：

For launching the device of ultrasonic wave；With

Ternary nano combination drug as described in any in claim 1-8.