CN112156192B - Composite nano probe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions and preparation and application thereof - Google Patents
Composite nano probe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a composite nano probe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions and preparation and application thereof 2 O 3 Nanoparticles and Au nanoclusters to obtain AuNBP-Gd 2 O 3 Au-dBSA, finally coupling the aptamer to AuNBP-Gd 2 O 3 Au-dBSA surface to obtain AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 targets the composite nano-probe. The composite nano probe has better biocompatibility, fluorescence stability and photothermal stability, has the functions of fluorescence/magnetic resonance bimodal imaging and photothermal treatment, can enter tumor cells in a targeted manner, marks and traces the tumor cells, and is favorable for early diagnosis and treatment of tumors.
Description
Technical Field
The invention relates to the technical field of nano materials and biomedicine, in particular to a composite nanoprobe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions, and preparation and application thereof.
Background
Molecular imaging is a science that uses imaging technology to non-invasively and visually detect molecules involved in physiological or pathological processes in vivo and qualitatively and quantitatively study changes of molecules, genes and cells in vivo. Among various imaging technologies, magnetic Resonance Imaging (MRI) is a non-invasive imaging technology, which has high soft tissue resolution and spatial resolution, can provide multi-sequence, multi-parameter and multi-aspect imaging, but has relatively low sensitivity, and fluorescence imaging technology has relatively high sensitivity but limited penetration, so combining magnetic resonance imaging with fluorescence imaging technology to construct a nano-molecular imaging probe with magnetic resonance-fluorescence dual-mode imaging function has become a research hotspot in the medical and material fields.
Compared with traditional tumor treatment methods such as surgery, radiotherapy and chemotherapy, photothermal therapy is a new tumor treatment means, has the advantages of wide application range, non-invasion, small damage to normal tissues and the like, and shows wide application prospects in the fields of drug release control, tumor treatment and the like. Photothermal therapy utilizes nanomaterials having strong absorption of near-infrared light to convert the light energy irradiated to the tumor region into heat energy, thereby killing tumor cells. The nano material for tumor photothermal therapy mainly comprises gold nano material, graphene, mesoporous carbon nanospheres, semiconductor nanoparticles and the like, wherein the gold nano material is one of the most concerned photothermal therapy nano materials at present due to excellent biocompatibility, unique optical performance, easy surface functionalization and chemical stability. At present, gold nano materials with rod-shaped or core-shell structures are a more researched photo-thermal treatment mediating material, and compared with gold nano rods and gold nano shell layers, the gold nano bipyramid (AuNBP) has the advantages of narrow half-peak width of the near infrared region absorption band, high photo-thermal conversion efficiency and good stability, and is an ideal photo-thermal treatment mediating material.
For more extensive and accurate diagnosis and treatment of cancer, development of photothermal therapy nano-systems guided by bimodal imaging diagnosis is very necessary, and becomes a new direction for research on cancer diagnosis and treatment. However, the photothermal therapy nano system prepared by the existing process method has poor component stability and consistency, poor biocompatibility, incapability of specifically identifying tumor cells, and complex preparation method, and cannot meet the requirements of research and application.
Disclosure of Invention
The invention aims to provide a preparation method of a composite nano probe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy.
The invention also aims to provide the composite nanoprobe which is prepared by the method and has the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy, has good stability and biocompatibility, and can realize the integration of fluorescence imaging, magnetic resonance imaging and photothermal therapy.
The invention also aims to provide application of the composite nano probe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a preparation method of a composite nanoprobe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions comprises the following steps:
preparation of S1 and AuNBP
Firstly, preparing gold nanocrystal seeds, adding the gold nanocrystal seeds into a growth solution to grow AuNBP, and then purifying the AuNBP to obtain AuNBP nanoparticles;
S2、AuNBP-Gd 2 O 3 preparation of/Au-dBSA nanocomposites
Firstly, the cattle with the concentration of 50mg/mL are fedAdding sodium borohydride (NaBH) into serum albumin (BSA) solution 4 ) Stirring for 1h at room temperature to obtain dBSA; placing the reaction solution in a water bath at 60-70 ℃, and continuously stirring for 20-60min to remove excessive NaBH 4 (ii) a Dispersing AuNBP nano particles in the dBSA solution, ultrasonically dispersing and uniformly mixing, sequentially adding a tetrachloroauric acid solution, a gadolinium nitrate solution and a sodium hydroxide solution under vigorous stirring, and vigorously stirring at 37 ℃ for 12h to obtain AuNBP-Gd 2 O 3 the/Au-dBSA nano-composite dispersion liquid is centrifuged, supernatant liquid is removed, and precipitate is washed to obtain AuNBP-Gd 2 O 3 Au-dBSA nanocomposite;
S3、AuNBP-Gd 2 O 3 Au-dBSA surface modification AS1411 aptamer
AuNBP-Gd 2 O 3 Dispersing the/Au-dBSA nano compound in deionized water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt (EDC), N-hydroxysuccinimide (NHS) and AS1411 aptamer, shaking on a cradle bed for 15min, mixing uniformly, and placing in a 37 ℃ constant temperature water bath box for reaction for 2h; after the reaction is finished, taking out the solution, centrifuging and washing to obtain AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 composite nanoprobe.
Preferably, in step S1, the specific process for preparing AuNBP is as follows:
s101, preparation of Au nano seed crystal: stirring and uniformly mixing a 0.01M tetrachloroauric acid solution and a 0.01M trisodium citrate solution, quickly adding a newly prepared 4 ℃ pre-cooled 0.01M sodium borohydride solution under vigorous stirring, stopping stirring after 2min, and standing in a 25 ℃ water bath for 2h to obtain an Au nano seed crystal dispersion liquid; the volume ratio of the tetrachloroauric acid solution to the trisodium citrate solution to the sodium borohydride solution is 5:10:6;
s102, preparing an AuNBP growth solution: putting 200mL of 0.1M hexadecyl trimethyl ammonium bromide (CTAB) solution into a beaker, sequentially adding 10mL of 0.01M tetrachloroauric acid solution, 2mL of 0.01M silver nitrate solution, 4mL of 1M hydrochloric acid and 1.6mL of 0.1M ascorbic acid solution while stirring, uniformly stirring, then adding 1.5 to 1.8mL of Au nano seed crystal dispersion liquid in the step 1.1 under slow stirring, continuously stirring for 10s, and standing for 12h at 25 ℃;
s103, purification of AuNBP: centrifuging the AuNBP growth liquid, removing a supernatant, dispersing the centrifuged precipitate in 160mL of 0.08M hexadecyltrimethylammonium chloride (CTAC) solution, ultrasonically dispersing uniformly, adding 32mL of 0.01M silver nitrate solution and 16mL of 0.1M ascorbic acid solution while stirring, standing at a constant temperature of 65 ℃ for 4 hours to grow Ag nanorods on the surface of the AuNBP, and naturally cooling to room temperature; centrifuging to remove supernatant, dispersing the centrifugal precipitate in 160mL of 0.05M CTAB solution, standing at room temperature for 12h, and separating out precipitate in the sample, wherein the precipitate is AuNBP with Ag nanorods; the upper solution was removed, the precipitate was dispersed in 100mL of water, and 2.5mL of ammonia water and 2mL of H were added with slow stirring 2 O 2 Standing at room temperature to etch the Ag nano-rods on the surface of the AuNBP; obtaining AuNBP dispersion liquid after the Ag nano rods are completely etched; and centrifuging the dispersion liquid, removing a supernatant, and collecting a precipitate to obtain the AuNBP nano-particles.
Preferably, in step S2, the mass ratio of bovine serum albumin to sodium borohydride is 25 to 50:1; the mol ratio of the tetrachloroauric acid to the gadolinium nitrate to the sodium hydroxide is 0.1:0.05:2.
preferably, in step S3, auNBP-Gd is added 2 O 3 Dispersing the/Au-dBSA nano compound in deionized water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt, uniformly mixing, activating at the constant temperature of 37 ℃ for 15min, and then adding N-hydroxysuccinimide and AS1411 aptamer.
Preferably, in step S3, auNBP-Gd 2 O 3 The mass ratio of the/Au-dBSA nano compound to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt to the N-hydroxysuccinimide to the AS1411 aptamer is 1 to 5:1:1:0.02 to 0.04.
Meanwhile, the invention also provides the composite nanoprobe which is obtained by the preparation method and has the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy.
The AuNBP nano-particles in the composite nano-probe are of a biconical structure, the length of the AuNBP nano-particles is 65 to 75nm, the width of the AuNBP nano-particles is 22 to 28nm, gd is added to the AuNBP nano-particles to form a nano-probe, and the AuNBP nano-particles are arranged in a cavity of the composite nano-probe and are in a cavity of the nanometer material, wherein the AuNBP nano-particles in the composite nano-probe are in a biconical structure, the length of the AuNBP nano-particles is 65 to 75nm, the width of the AuNBP nano-particles is 22 to 28nm 2 O 3 The particle diameter of the nano-particles is 2 to 5nm 2 O 3 Nanoparticles and Au nanoclustersThe surface of AuNBP is modified by BSA molecules.
Furthermore, the invention also provides application of the composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy in preparation of a tumor diagnosis, imaging and photothermal therapy reagent.
Preferably, the tumor is breast cancer.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention takes AuNBP nano-particles as a precursor, modifies the surface of the AuNBP nano-particles by sulfhydrylated BSA, and modifies Gd on the surface of the AuNBP nano-particles by taking the AuNBP nano-particles as a stabilizer 2 O 3 Nanoparticles and Au nanoclusters, further linking aptamer AS1411 to AuNBP-Gd 2 O 3 Preparing target, diagnosis and treatment integrated AuNBP-Gd on the surface of/Au-dBSA 2 O 3 the/Au-dBSA-AS 1411 composite nanoprobe.
(2) AuNBP-Gd of the present invention 2 O 3 the/Au-dBSA-AS 1411 composite nano probe has better biocompatibility and fluorescence stability, simultaneously has a fluorescence/magnetic resonance bimodal imaging function, can enter tumor cells in a targeted manner, marks and traces the tumor cells, and is beneficial to early diagnosis of tumors;
(3) AuNBP-Gd of the present invention 2 O 3 the/Au-dBSA-AS 1411 composite nano probe has excellent photo-thermal stability, has obvious photo-thermal treatment and killing effects on MDA-MB-231 breast cancer cells, can be used AS a mediated material for photo-thermal treatment, and has certain application potential in photo-thermal treatment reagents;
(4) The preparation method is simple and convenient, has mild conditions and high yield, and is suitable for large-scale production.
Drawings
FIG. 1 shows AuNBP-Gd 2 O 3 A preparation process schematic diagram of the/Au-dBSA-AS 1411 targeted composite nano probe;
fig. 2A is a TEM photograph of AuNBP nanoparticles;
FIG. 2B is AuNBP-Gd 2 O 3 TEM photograph of/Au-dBSA nanocomposite;
FIG. 3A is AuNBP-Gd 2 O 3 HAADF-STEM plot of/Au-dBSA nanocomposites;
FIG. 3B is AuNBP-Gd 2 O 3 Mapping diagram of Au element in Au-dBSA nano compound;
FIG. 3C is AuNBP-Gd 2 O 3 Mapping graph of Gd element in Au-dBSA nano-composite;
FIG. 4 shows AS1411 (a), BSA (b), auNBP (c), auNBP-Gd 2 O 3 Au-dBSA (d) and AuNBP-Gd 2 O 3 UV-vis absorption spectra of the/Au-dBSA-AS 1411 (e) samples;
FIG. 5 shows AuNBP (a), BSA (b), AS1411 (c), auNBP-Gd 2 O 3 Au-dBSA (d) and AuNBP-Gd 2 O 3 Fourier transform Infrared Spectroscopy (FTIR) of the/Au-dBSA-AS 1411 (e) sample;
FIG. 6 shows AuNBP and AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Zeta potential map of Au-dBSA-AS 1411;
FIG. 7 shows AuNBP (a) and AuNBP-Gd 2 O 3 Au-dBSA (b) and AuNBP-Gd 2 O 3 Fluorescence spectrum of the sample/Au-dBSA-AS 1411 (c), excitation wavelength 488nm;
FIG. 8 shows AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 The change curve of the fluorescence intensity of Au-dBSA-AS1411 along with the pH value;
FIG. 9 shows AuNBP-Gd at different concentrations 2 O 3 T1 weighted image (T1 WI) and T1 map of/Au-dBSA sample;
FIG. 10 is AuNBP-Gd 2 O 3 Fitting curves of T1 relaxation rates of/Au-dBSA and Gd-DTPA;
FIG. 11 is AuNBP-Gd 2 O 3 The photo-thermal performance of/Au-dBSA. (A) AuNBP-Gd at different concentrations 2 O 3 Infrared laser (1W/cm) with/Au-dBSA at 808nm 2 ) A temperature rise curve under irradiation; (B) AuNBP-Gd at different concentrations 2 O 3 Infrared laser (1W/cm) of/Au-dBSA at 808nm 2 ) Thermographic under illumination; (C) The concentration is 10mg/LAuNBP-Gd of 2 O 3 A temperature rise curve of the Au-dBSA dispersion liquid under the irradiation of infrared lasers with different powers; (D) AuNBP-Gd at a concentration of 10mg/L 2 O 3 /Au-dBSA dispersion in infrared laser (1W/cm) 2 ) Stability curve under irradiation;
FIG. 12 is AuNBP-Gd 2 O 3 MDA-MB-231 cells and HSF cell viability maps of different concentration groups of/Au-dBSA;
FIG. 13 shows the HSF and MDA-MB-231 cells versus AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 A laser confocal micrograph ingested by Au-dBSA-AS1411 with a ruler of 20 mu m;
FIG. 14 shows MDA-MB-231 and HSF cells with PBS buffer and AuNBP-Gd, respectively 2 O 3 Au-dBSA dispersion and AuNBP-Gd 2 O 3 T1WI picture and T1 map picture after Au-dBSA-AS1411 dispersion liquid co-incubation;
FIG. 15 is a confocal laser micrograph of live/dead cell staining of MDA-MB-231 cells.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
Example 1
Firstly, preparing a gold nano bipyramid AuNBP dispersion liquid by adopting a seed growth method, and purifying AuNBP to obtain pure AuNBP nanoparticles; reducing a disulfide bond in a BSA molecule into a sulfydryl with a stronger coordination effect with the surface of the gold nanoparticle by using sodium borohydride to obtain denatured BSA (modified BSA, dBSA), and modifying the denatured BSA to the surface of AuNBP; then dBSA on the surface of AuNBP is used as a stabilizer to prepare Gd 2 O 3 Nano particles and gold nanoclusters to obtain a nano compound AuNBP-Gd with fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions 2 O 3 Au-dBSA. Coupling target recognition molecule AS1411 aptamer forming target nano probe AuNBP-Gd 2 O 3 Au-dBSA-AS1411, prepared AS shown in FIG. 1. The specific implementation process is as follows:
s1. Preparation of AuNBP
Firstly, preparing Au nano seed crystals, adding the Au nano seed crystals into a growth solution to grow AuNBP, and then purifying the AuNBP, wherein the specific preparation process comprises the following steps:
and S101, preparing Au nano seed crystals. 9.625mL of water was taken in a flask, and 125. Mu.L of 0.01M HAuCl was added successively with stirring 4 Mixing the solution with 250 μ L0.01M trisodium citrate solution, stirring, rapidly adding 150 μ L0.01M NaBH under vigorous stirring 4 The solution (prepared newly and precooled at 4 ℃) is stopped stirring after 2min and is kept stand for 2h in water bath at 25 ℃.
And S102, preparing the AuNBP growth liquid. 200mL of 0.1M CTAB solution was put into a beaker, and 10mL of 0.01M HAuCl solution was sequentially added while stirring 4 、2mL 0.01M AgNO 3 4mL of 1M HCl and 1.6mL of 0.1M ascorbic acid solution, and then adding 1.6mL of the Au nano seed crystal dispersion in the step (1) under slow stirring, continuing stirring for 10s, and then standing for 12h at 25 ℃.
And S103, purifying the AuNBP. Centrifuging AuNBP growth solution (8000 rpm × 15 min), removing supernatant, dispersing the centrifuged precipitate in 160mL 0.08M CTAC solution, ultrasonically dispersing uniformly, adding 32mL 0.01M AgNO while stirring 3 The solution and 16mL of 0.1M ascorbic acid solution are kept stand at the constant temperature of 65 ℃ for 4h to grow Ag nanorods on the surface of AuNBP, and then the solution is naturally cooled to the room temperature. Centrifuging (7000 rpm is multiplied by 15 min), removing supernatant, dispersing centrifugal precipitate in 160mL 0.05M CTAB solution, standing for 12h at room temperature, and separating out precipitate in the sample, wherein the precipitate is AuNBP with Ag nano-rods. The upper solution was removed, the precipitate was dispersed in 100mL of water, and 2.5mL of aqueous ammonia and 2mL of H were added with slow stirring 2 O 2 And standing at room temperature to etch the Ag nano-rods on the surface of the AuNBP. And detecting the UV-vis absorption spectrum of the solution in the standing process, observing the color change of the solution, and obtaining the AuNBP dispersion liquid after the Ag nano rods are completely etched. The dispersion was centrifuged (7000 rpm. Times.15 min), the supernatant removed, and the centrifuged pellet dispersed in 50mL of deionized water and stored at 4 ℃ until use.
S2. AuNBP-Gd 2 O 3 Preparation of Au-dBSA nanocomposite
250mg BSA was added to 5mL water, and mixed under stirringAnd (6) homogenizing. Then 6.3mg NaBH was added to the batch 4 And stirring for 1h at room temperature, and reducing the disulfide bond in the BSA molecule into sulfydryl with stronger coordination effect with the surface of the Au nanoparticle to obtain dBSA. The reaction solution is placed in a water bath at 70 ℃, and is continuously stirred for 30 min to remove the excessive NaBH 4 . The AuNBP dispersion stored at 4 ℃ in 8mL volume was centrifuged (7000 rpm. Times.15 min), the supernatant was removed, the centrifuged precipitate was dispersed in the above dBSA solution, the mixture was ultrasonically dispersed and mixed, and 4mL of 0.025M HAuCl was added thereto in this order under vigorous stirring 4 、1mL 0.05M Gd(NO 3 ) 3 Mixing with 1mL 2M NaOH solution, and stirring at 37 deg.C for 12 hr to obtain AuNBP-Gd 2 O 3 the/Au-dBSA nano-composite dispersion was centrifuged (12000 rpm. Times.15 min) and the supernatant was removed, and the centrifuged precipitate was washed 3 times with deionized water, then dispersed in 8mL of deionized water and stored at 4 ℃ for further use.
S3. AuNBP-Gd 2 O 3 Au-dBSA surface modification AS1411 aptamer
Taking 6mL AuNBP-Gd 2 O 3 Adding 2mg EDC into the Au-dBSA nano-composite solution, uniformly mixing, and putting the solution into a 37 ℃ constant temperature water bath box for activation for 15min. Then 2mg NHS and 10 μ L AS1411 aptamer are added, the mixture is shaken on a cradle bed for 15min, and after being mixed evenly, the mixture is put into a thermostatic water bath box with the temperature of 37 ℃ for reaction for 2h. After the reaction was completed, the solution was centrifuged (7000 rpm. Times.15 min), washed 2 times with deionized water, and dispersed in deionized water for use.
The prepared material was characterized and tested for properties, with the following results:
(1) Characterization of materials
Fig. 2A is a TEM photograph of AuNBP, and it can be seen that AuNBP particles have a biconical structure, good dispersibility, uniform size, a length of about 66.6 ± 1.9nm, and a width of about 24.9 ± 2.4nm. FIG. 2B is AuNBP-Gd 2 O 3 TEM photograph of Au-dBSA shows that AuNBP particles are wrapped with a film containing granular Gd 2 O 3 And Au nanocluster, gd 2 O 3 The particle size of the nano-particles is 2 to 5nm.
FIG. 3A is AuNBP-Gd 2 O 3 HAADF-STEM plot of/Au-dBSA, showing Gd 2 O 3 The Au nano-particles are modified on the surface of the AuNBP; FIG. 3B and FIG. 3C are AuNBP-Gd, respectively 2 O 3 Mapping of Au and Gd elements in/Au-dBSA, also showing Gd 2 O 3 the/Au nanoparticles are modified on the surface of AuNBP.
FIG. 4 shows AS1411, BSA, auNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 UV-vis absorption spectra of the/Au-dBSA-AS 1411 sample. As can be seen, the AuNBP sample has a strong absorption peak at 805 nm; when the surface of the AuNBP nano-particles is modified with Gd 2 O 3 After Au-dBSA, the absorption peak at 805nm is red-shifted by 27nm, but the absorption peak still has stronger absorption at 808nm, which indicates that the prepared AuNBP-Gd 2 O 3 the/Au-dBSA sample has better photo-thermal conversion efficiency on near-infrared light with the wavelength of 808 nm; in addition, auNBP-Gd 2 O 3 the/Au-dBSA sample has a small peak at 275nm, which is the absorption peak of BSA, and shows that BSA is wrapped on the surface of AuNBP particles, so that the AuNBP particles have better biocompatibility. And AuNBP-Gd 2 O 3 AuNBP-Gd compared to Au-dBSA 2 O 3 The absorption spectrum of the/Au-dBSA-AS 1411 has an absorption peak at 254nm, which is the absorption peak of AS411, and shows that the AS1411 is modified in AuNBP-Gd 2 O 3 the/Au-dBSA nano particle surface has a targeting function.
FIG. 5 shows AuNBP, BSA, AS1411, auNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Fourier transform Infrared Spectroscopy (FTIR) of the/Au-dBSA-AS 1411 sample. From curve a, 2920cm can be seen -1 And 2850cm -1 The peak at (a) comes from the stretching vibration of the C-H bond in the CTAB molecule on the AuNBP surface; auNBP-Gd can be seen from curves b and d 2 O 3 FTIR spectra of/Au-dBSA are similar to BSA, indicating Gd 2 O 3 Au is connected to the surface of the AuNBP particle through a BSA molecule; as can be seen from curve e, auNBP-Gd 2 O 3 FTIR spectrum of/Au-dBSA at 1050cm -1 An absorption peak is shown and is a stretching vibration absorption peak of an O-P bond in an AS1411 molecule (curve c), which indicates that the AS1411 is modified to AuNBP-Gd 2 O 3 Surface of Au-dBSA.
FIG. 6 shows AuNBP and AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Zeta potential map of/Au-dBSA-AS 1411. The Zeta potential of the AuNBP nano-particles is +31.2mV when the AuNBP surface is modified with Gd 2 O 3 After Au-dBSA, the AuNBP-Gd obtained 2 O 3 Zeta potential of/Au-dBSA was-23.5 mV when measured at AuNBP-Gd 2 O 3 After the negatively charged AS1411 molecule was attached to the/Au-dBSA surface, the zeta potential was-38.7 mV.
(2) Fluorescent properties of materials
FIG. 7 shows AuNBP and AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Fluorescence spectra of the Au-dBSA-AS1411 sample with 488nm excitation wavelength. As can be seen, auNBP has no fluorescence emission peak; auNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 has fluorescence emission peaks at 645nm and 650nm respectively due to the existence of surface Au nanoclusters, so that red fluorescence can be emitted. In general, when Au nanoclusters and aunbps exist simultaneously, fluorescence of the Au nanoclusters is quenched due to fluorescence resonance energy transfer between the Au nanoclusters and the aunbps. As can be seen from the UV-vis absorption spectrum (figure 4) of AuNBP, the absorption of AuNBP at about 650nm is very weak, so the fluorescence quenching effect of AuNBP on Au nanoclusters is weak; furthermore, in AuNBP-Gd 2 O 3 In the preparation process of/Au-dBSA, dBSA is firstly modified on the surface of AuNBP particles, and then Gd is synthesized by taking the dBSA on the surface of AuNBP as a coordination agent 2 O 3 The nano particles and the Au nano cluster keep a certain distance from the surface of the AuNBP particles, and an electric field generated by the plasma resonance of the local surface of the AuNBP has an enhancement effect on the fluorescence of the Au nano cluster, so that the prepared AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 sample has the functions of photothermal conversion and fluorescence imaging.
FIG. 8 shows AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Fluorescence intensity of/Au-dBSA-AS 1411 AS a function of pH. When the pH value is changed within the range of 5 to 9, the fluorescence intensity of the two is observedThe change is small, indicating that both have better fluorescence stability.
(3) Magnetic resonance characterization of materials
AuNBP-Gd 2 O 3 Gd due to in Au-dBSA samples 2 O 3 The presence of the nanoparticles enables the functionality of magnetic resonance imaging. Firstly, auNBP-Gd is added 2 O 3 the/Au-dBSA sample was prepared as a solution having Gd concentrations of 0,0.09mM,0.22mM,0.31mM,0.63mM,1.26mM,1.89mM,2.53mM and 3.16mM, respectively, tested for T1 relaxation time using a 3.0T GE Discovery MR system, and AuNBP-Gd was calculated based on the average T1 relaxation times of the samples at different concentrations 2 O 3 T1 relaxation Rate of/Au-dBSA.
FIG. 9 shows AuNBP-Gd at different concentrations 2 O 3 T1 weighted image (T1 WI) and T1 map of/Au-dBSA sample. It can be seen that as the concentration of Gd decreases, the T1WI signal gradually decreases, and the T1 map pseudocolor gradually changes from orange-red to dark blue.
FIG. 10 is AuNBP-Gd 2 O 3 T1 relaxation Rate fitting curves for Au-dBSA and Gd-DTPA. AuNBP-Gd 2 O 3 The T1 relaxation rate of/Au-dBSA is 6.75s -1 mM -1 Gd-DTPA, a clinically used T1 contrast agent (T1 relaxation rate of 4.45 s) -1 mM -1 ) 1.5 times of the total weight of the powder.
(4) Photothermal properties of the material
AuNBP-Gd 2 O 3 the/Au-dBSA sample preparation process comprises preparing dispersions with the process concentrations of 0mg/mL, 2mg/L,5mg/L,10mg/L and 20mg/L respectively, irradiating 0.5mL of the dispersions under near-infrared laser (808nm, 1W/cm) for 10min, and monitoring a temperature rise curve and a thermography of the dispersions by a thermal imager, wherein the temperature rise curve and the thermography are respectively shown in FIG. 11A and FIG. 11B. As can be seen, different concentrations of AuNBP-Gd 2 O 3 The temperature of the/Au-dBSA increased with increasing concentration with increasing irradiation time, and the temperature of the 2mg/L,5mg/L,10mg/L and 20mg/L dispersions increased by 19.9 ℃,30.3 ℃,35.8 ℃ and 39.9 ℃ respectively (the initial temperature of the dispersion was 27 ℃) after 10min irradiation. FIG. 11C is AuNBP-Gd concentration at 10mg/L 2 O 3 808 at different powers for Au-dBSA dispersionsThe temperature rise curve under the irradiation of nm near-infrared laser shows that the temperature of the dispersion can reach more than 45 ℃ after 10min of irradiation under lower power, and the tumor cells can be killed after the dispersion is maintained for 5min at more than 45 ℃ according to literature report, so AuNBP-Gd 2 O 3 the/Au-dBSA is an excellent tumor photothermal treatment material. Further, auNBP-Gd is shown in FIG. 11D 2 O 3 The BSA sample has excellent photo-thermal stability, and the photo-thermal performance of the BSA sample is hardly changed after 4-cycle testing.
Example 2
To further validate AuNBP-Gd 2 O 3 The Au-dBSA-AS1411 targeted nanoprobe is applied to the aspect of tumor diagnosis and treatment to carry out in-vitro cell experiments. Human breast cancer cells (MDA-MB-231) and Human Skin Fibroblasts (HSF) were purchased from Shanghai cell Bank of Chinese academy of sciences.
1. Cytotoxicity test
Evaluation of AuNBP-Gd by CCK8 method 2 O 3 Toxic effects of Au-dBSA on human breast cancer cells (MDA-MB-231) and Human Skin Fibroblasts (HSF). MDA-MB-231 cells and HSF cells in logarithmic growth phase were seeded in two 96-well culture plates at a concentration of about 0.5X 10 5 Per mL, at 37 deg.C, 5% CO 2 Incubate in incubator for 24h. When the cells were observed under an inverted microscope to grow almost completely in the culture well, the old culture medium was discarded, the cells were washed 2 times with sterilized PBS solution, and the 96-well plate was divided into 6 groups (6 wells/group) to which AuNBP-Gd was added at concentrations of 0. Mu.g/mL, 10. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL and 500. Mu.g/mL), respectively 2 O 3 The total volume of the mixture culture medium in each well was 100. Mu.L/Au-dBSA mixture culture medium. Adding into 37 deg.C, 5% CO 2 And (3) incubating in an incubator for 24 hours, discarding the old culture solution, washing with the sterilized PBS solution for 2 times, adding 100 mu LCCK-8 solution into each hole, and continuing to incubate in the incubator for 4 hours. The absorbance (OD) of each well was measured at a wavelength of 450nm using a multifunctional microplate reader.
FIG. 12 is AuNBP-Gd 2 O 3 MDA-MB-231 cells and HSF cells survival rate of different concentration groups of/Au-dBSA are shown. As can be seen, when AuNBP-Gd 2 O 3 When the concentration of/Au-dBSA is in the range of 0 to 500 mu g/mL, MDA-MB-231 cells and HSF cells and AuNBP-Gd with different concentrations 2 O 3 The survival rates of the two cells are both above 85 percent after the incubation together with the Au-dBSA. Even AuNBP-Gd 2 O 3 When the/Au-dBSA reaches 500 mu g/mL, the survival rates of MDA-MB-231 cells and HSF cells are still 88.2 percent and 89 percent respectively after the incubation for 24 hours, which indicates that the AuNBP-Gd 2 O 3 the/Au-dBSA has better biocompatibility.
2. Fluorescence imaging of cells
Study of AuNBP-Gd with MDA-MB-231 cells and HSF cells 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Cellular uptake and fluorescence imaging of/Au-dBSA-AS 1411. Cells were seeded on Lab-Tek 8 chamber culture slides at a concentration of 2X 10 4 One well, incubated overnight, 50. Mu.g/mL AuNBP-Gd each 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 nano material dispersion liquid is treated for 2h and then cell nucleus staining is carried out by DAPI, and then the nano material which is not absorbed and dead cells are removed by washing 2 times by PBS buffer liquid and observed by a laser confocal microscope.
FIG. 13 shows the effect of HSF cells and MDA-MB-231 cells on AuNBP-Gd, respectively 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 Confocal laser micrograph/Au-dBSA-AS 1411 uptake. Visible, with AuNBP-Gd 2 O 3 After the/Au-dBSA-AS 1411 is incubated, the HSF cells only show weak red fluorescence around blue cell nuclei, which indicates that the affinity of the AS1411 aptamer and the HSF cells is weaker, so that the material uptake of the HSF cells is not enhanced; MDA-MB-231 cells and AuNBP-Gd 2 O 3 the/Au-dBSA shows weaker red fluorescence signal after being incubated together, but when the/Au-dBSA and AuNBP-Gd 2 O 3 After the/Au-dBSA-AS 1411 nano-complex is incubated together, the red fluorescence signal is obviously enhanced, which indicates that the AS1411 aptamer can obviously enhance the intake of MDA-MB-231 cells to the nano-material, so that AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 nano-composite can be used AS a targeted fluorescent probe to label and trace MDA-MB-231 cells.
3. Magnetic resonance imaging of cells
Digesting MDA-MB-231 cells and HSF cells by pancreatin, adding the cells into a culture medium to prepare cell suspension, adding the cell suspension into cell holes (6 holes/group, 2 groups in total), adding 500 mu L of cell suspension (about 20000 cells) into each hole, culturing for 24h in an incubator, sucking out the culture medium when the cells grow to about 80% -90% of the visual field under the mirror, washing for 3 times by PBS, and respectively adding 1mL of AuNBP-Gd with the concentration of 50 mu g/mL into 2 groups of culture holes 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 dispersion was incubated in an incubator for 2 hours, the wells were aspirated, washed 3 times with PBS, digested with 0.25% trypsin and centrifuged, and the resulting cells were dispersed in a 1% agarose gel, and the T1 weighted graph and T1 map of each group were measured using a 3.0T 750W Discovery MRI system.
FIG. 14 shows MDA-MB-231 and HSF cells with PBS buffer and AuNBP-Gd 2 O 3 Au-dBSA dispersion and AuNBP-Gd 2 O 3 T1WI and T1 map after co-incubation of/Au-dBSA-AS 1411 dispersion. T1WI image shows that HSF cells AuNBP-Gd 2 O 3 After the incubation of/Au-dBSA, the brightness of the T1WI image is only weakly enhanced compared with that of the T1WI image incubated with PBS, and in addition, the AS1411 aptamer does not significantly enhance the magnetic resonance signal, which indicates that the AS1411 aptamer has no targeting function on HSF cells. MDA-MB-231 cells and AuNBP-Gd 2 O 3 After the incubation of/Au-dBSA, the T1WI signal is only slightly enhanced compared with that after the incubation of the Au-dBSA and the PBS buffer solution, while when the MDA-MB-231 cells and AuNBP-Gd 2 O 3 After the/Au-dBSA-AS 1411 is incubated together, the T1WI signal is obviously enhanced, which indicates that the breast cancer targeted molecular probe AuNBP-Gd2O3/Au-dBSA-AS1411 successfully realizes the targeted magnetic resonance imaging of MDA-MB-231 breast cancer cells under the experimental condition.
4.4 In vitro photothermal therapeutic Properties
MDA-MB-231 cells are cultured in a culture dish with phi 35mm for 24h and then are mixed with AuNBP-Gd 2 O 3 Au-dBSA and AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 nano-complex dispersion liquid (20 mu g/mL) is co-incubated for 4h, then the nano-complex which is not absorbed is washed by PBS buffer liquid, and 808nm is usedNear infrared laser (2W/cm) 2 ) After 5min of irradiation, cells were stained with calcein AM (calcein AM) and Propidium Iodide (PI) and observed with a confocal laser microscope.
FIG. 15 is a confocal laser micrograph of live/dead cell staining of MDA-MB-231 cells. Therefore, the MDA-MB-231 cells of the control group keep a better growth state, and still show bright green fluorescence after being irradiated for 5min by near infrared laser with wavelength of 808nm, which indicates that the near infrared light with wavelength of 808nm has little damage to the MDA-MB-231 cells; however with AuNBP-Gd 2 O 3 After the/Au-dBSA incubation, under the near-infrared laser irradiation of 808nm, the cells with red fluorescence are greatly increased, and only a small number of the cells are green, which indicates that most of the cells are already subjected to AuNBP-Gd 2 O 3 The photo-thermal effect of the/Au-dBSA kills the bacteria; when MDA-MB-231 cells and AuNBP-Gd 2 O 3 After the/Au-dBSA-AS 1411 co-incubation, MDA-MB-231 cells are almost completely killed after 808nm near-infrared laser irradiation, which shows that the AS1411 aptamer can remarkably improve the photo-thermal treatment killing effect of the material on MDA-MB-231 breast cancer cells by improving the uptake of the cells to the nano composite material.
Claims (9)
1. A preparation method of a composite nanoprobe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions is characterized by comprising the following steps:
preparation of S1 and AuNBP
Firstly, preparing gold nanocrystal seeds, adding the gold nanocrystal seeds into a growth solution to grow AuNBP, and then purifying the AuNBP to obtain AuNBP nanoparticles;
S2、AuNBP-Gd 2 O 3 preparation of/Au-dBSA nanocomposites
Firstly, adding sodium borohydride into a bovine serum albumin solution with the concentration of 50mg/mL, and stirring for 1h at room temperature to obtain dBSA, wherein the mass ratio of the bovine serum albumin to the sodium borohydride is 25-50: 1; placing the reaction solution in a water bath at 60-70 ℃, continuously stirring for 20-60min, and removing excessive sodium borohydride; dispersing AuNBP nano particles in the dBSA solution, ultrasonically dispersing and uniformly mixing, and sequentially adding a tetrachloroauric acid solution and nitric acid under vigorous stirringThe gadolinium solution and the sodium hydroxide solution are stirred vigorously for 12 hours at the temperature of 37 ℃ to obtain AuNBP-Gd 2 O 3 the/Au-dBSA nano-composite dispersion liquid is centrifuged, supernatant liquid is removed, and precipitate is washed to obtain AuNBP-Gd 2 O 3 Au-dBSA nanocomposite;
S3、AuNBP-Gd 2 O 3 Au-dBSA surface modification AS1411 aptamer
AuNBP-Gd 2 O 3 Dispersing the/Au-dBSA nano composite in deionized water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt, N-hydroxysuccinimide and an AS1411 aptamer, shaking the mixture on a cradle bed for 15min, uniformly mixing, and putting the mixture into a 37 ℃ constant-temperature water bath box for reaction for 2h; after the reaction is finished, taking out the solution, centrifuging and washing to obtain AuNBP-Gd 2 O 3 the/Au-dBSA-AS 1411 composite nano-probe.
2. The method for preparing the composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy according to claim 1, wherein the AuNBP is prepared in step S1 by the following specific processes:
s101, preparation of Au nano seed crystal: stirring and uniformly mixing a 0.01M tetrachloroauric acid solution and a 0.01M trisodium citrate solution, rapidly adding a newly prepared 4 ℃ pre-cooled 0.01M sodium borohydride solution under vigorous stirring, stopping stirring after 2min, and standing in a water bath at 25 ℃ for 2h to obtain an Au nano seed crystal dispersion liquid; the volume ratio of the tetrachloroauric acid solution to the trisodium citrate solution to the sodium borohydride solution is 5:10:6;
s102, preparing an AuNBP growth solution: taking 200mL of 0.1M hexadecyl trimethyl ammonium bromide solution, sequentially adding 10mL of 0.01M tetrachloroauric acid solution, 2mL of 0.01M silver nitrate solution, 4mL of 1M hydrochloric acid and 1.6mL of 0.1M ascorbic acid solution while stirring, uniformly mixing by stirring, then adding 1.5 to 1.8mL of the Au nano seed crystal dispersion liquid in the step S101 under slow stirring, continuing stirring for 10S, and standing for 12h at 25 ℃;
s103, purification of AuNBP: centrifuging AuNBP growth solution, removing supernatant, dispersing the centrifuged precipitate in 160mL 0.08M hexadecyltrimethylammonium chloride solution, and ultrasonically dispersing uniformlyAdding 32mL of 0.01M silver nitrate solution and 16mL of 0.1M ascorbic acid solution while stirring, standing at the constant temperature of 65 ℃ for 4 hours to grow Ag nanorods on the surface of the AuNBP, and then naturally cooling to room temperature; centrifuging to remove supernatant, dispersing centrifugal precipitate in 160mL 0.05M hexadecyl trimethyl ammonium bromide solution, standing at room temperature for 12h, and separating out precipitate in the sample, wherein the precipitate is AuNBP with Ag nano rods; the upper solution was removed, the precipitate was dispersed in 100mL of water, and 2.5mL of ammonia and 2mL of H were added with slow stirring 2 O 2 Standing at room temperature to etch the Ag nano-rods on the surface of the AuNBP; obtaining AuNBP dispersion liquid after the Ag nano rods are completely etched; and centrifuging the dispersion liquid, removing supernatant, and collecting precipitate to obtain AuNBP nanoparticles.
3. The method for preparing the composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy according to claim 1, wherein in the step S2, the molar ratio of the tetrachloroauric acid, the gadolinium nitrate and the sodium hydroxide is 0.1:0.05:2.
4. the method for preparing the composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy according to claim 1, wherein in step S3, auNBP-Gd is added 2 O 3 Dispersing the/Au-dBSA nano compound in deionized water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt, uniformly mixing, activating at the constant temperature of 37 ℃ for 15min, and then adding N-hydroxysuccinimide and AS1411 aptamer.
5. The method for preparing the composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy according to claim 1 or 4, wherein in the step S3, auNBP-Gd 2 O 3 The mass ratio of the Au-dBSA nano compound to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide salt to the N-hydroxysuccinimide to the AS1411 aptamer is 1 to 5:1:1:0.02 to 0.04.
6. The composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy obtained by the preparation method according to any one of claims 1 to 5.
7. The composite nanoprobe with the functions of targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy according to claim 6, wherein the AuNBP nanoparticles are of a biconical structure, the length of the biconical structure ranges from 65 to 75nm, the width of the biconical structure ranges from 22 to 28nm, gd is distributed to the nanoprobe, and the AuNBP nanoparticles are distributed on the substrate of the composite nanoprobe and are in a shape of a sphere with a spherical center 2 O 3 The particle diameter of the nano-particles is 2 to 5nm 2 O 3 The nanoparticles and Au nanoclusters are modified on the surface of AuNBP through BSA molecules.
8. The use of the composite nanoprobe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions as claimed in claim 6 in the preparation of breast cancer diagnosis and imaging reagents.
9. The use of the composite nanoprobe with targeted fluorescence/magnetic resonance bimodal imaging and photothermal therapy functions as claimed in claim 6 in the preparation of breast cancer photothermal therapy agents.
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