CN110974960A - Composite nano probe with dumbbell structure and preparation method and application thereof - Google Patents
Composite nano probe with dumbbell structure and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a composite nano probe with a dumbbell structure, and a preparation method and application thereof. Under the stimulation of near infrared light, the enhanced local electric field at the two ends of the gold nanorod stimulates ICG molecules in the mesoporous silica to continuously generate singlet oxygen, and meanwhile, the enhanced photo-thermal photodynamic effect is finally obtained by utilizing the double photo-thermal effects of the gold nanorod and the ICG, so that tumor cells can be effectively killed under lower laser power. In addition, ICG molecules protected by the gold nanorods and the mesoporous silica cannot generate photobleaching, and have good light stability.
Description
Technical Field
The invention relates to the technical field of nano biological materials, in particular to a composite nano probe with a dumbbell structure and a preparation method and application thereof.
Background
Photothermal therapy is of increasing interest as a physical therapy in oncology. The environment of the tumor cells is changed by a photo-thermal heating method, so that the tumor cells are denatured and necrotic, and the treatment purpose is achieved.
Nano gold nanorods have extremely high light absorption capacity and good photothermal conversion capacity, and are considered to be one of the most typical and most potential photothermal agents. It can effectively absorb near infrared light and convert it into heat, so that it can kill tumor cell. However, the single photothermal therapy has the side effects that the tumor cells cannot be completely killed due to low thermal signals, the surrounding normal tissues can be killed due to over-high thermal signals, and the like. For example, Yin jin Chang et al (Yin J, Chen D, Wu S, et al, 2017,9(43): 16661-16673) disclose a gold nanorod @ SiO with a dumbbell structure2Probe, and further depositing Gd2O(CO3)2Gold nanorod @ SiO of dumbbell structure2The probe has photothermal effect and can be used for photothermal therapy, but the single photothermal therapy is difficult to effectively kill tumor cells.
Therefore, it is required to develop a nanoprobe capable of effectively killing tumor cells.
Disclosure of Invention
In order to overcome the defect that the single photothermal treatment in the prior art is difficult to effectively kill the tumor cells, the invention provides the composite nanoprobe with the dumbbell structure, the provided composite nanoprobe has enhanced photothermal and photodynamic effects, can effectively kill the tumor cells under lower laser power, and the ICG molecules under the protection of the gold nanorods and the mesoporous silica have good light stability.
Another object of the present invention is to provide a method for preparing the composite nanoprobe.
The invention also aims to provide the application of the composite nano probe in preparing a medicament for killing tumor cells.
In order to solve the technical problems, the invention adopts the technical scheme that:
the composite nanoprobe with the dumbbell structure comprises gold nanorods, mesoporous silicon dioxide coating two ends of the gold nanorods and indocyanine green loaded in the mesoporous silicon dioxide, wherein the gold nanorods, the solid silicon dioxide and the mesoporous silicon dioxide positioned at two ends of the gold nanorods form the dumbbell structure.
The chlorins, phthalocyanines and ICG are all photodynamic reagents, and can effectively generate singlet oxygen to obtain photodynamic effect. Compared with Ce6, ICG molecule has lower light absorption efficiency and singlet oxygen yield, but has more excellent light penetration ability because its absorption peak is in near infrared optical window, and can match with near infrared absorption peak of gold nanorod.
The gold nanorods in the composite nano probe generate enhanced local electric fields at two ends of the gold nanorods due to the surface plasmon resonance effect. ICG is a photodynamic reagent and a photothermal reagent, ICG molecules loaded in mesoporous silica at two ends of the gold nanorod are just positioned in an optimal local electric field enhancement area, so that the ICG molecules are enhanced and stimulated by the local electric field to the maximum extent, the photothermal effect and the photodynamic effect of the ICG molecules are amplified simultaneously, and the enhanced photothermal effect, namely the photodynamic effect, is obtained.
Researches find that the surface plasma resonance effect of the gold nanorods can stimulate ICG molecules to generate a large amount of singlet oxygen. The heat effect of the ICG and the heat effect of the gold nanorods can be mutually superposed, and the photodynamic effect of the ICG can stimulate the further promotion of the action of the heat and the heat. The local surface plasma resonance band of the gold nanorod resonates with the ICG molecular absorption band, the absorption efficiency is greatly enhanced, a large amount of singlet oxygen can be continuously generated, the double-photothermal effect of the gold nanorod and the ICG molecular is combined, and the photothermal performance of the composite probe is further enhanced.
In addition, in the composite nano probe, the gold nanorods have extremely strong plasma absorption capacity, so that ICG molecules can be prevented from photobleaching, and the light stability of the composite nano probe can be effectively improved. Experiments prove that ICG molecules cannot generate photobleaching under the protection of the gold nanorods and the mesoporous silica, and have good light stability.
In summary, under the stimulation of near infrared light, the local electric field enhanced at the two ends of the gold nanorod stimulates the ICG molecules in the mesoporous silica to continuously generate singlet oxygen, and meanwhile, the double-photothermal effect of the gold nanorod and the ICG is utilized to finally obtain the enhanced photothermal photodynamic effect, so that the tumor cells can be effectively killed under lower laser power. In addition, ICG molecules protected by the gold nanorods and the mesoporous silica cannot generate photobleaching, and have good light stability.
Preferably, the mesoporous silica at the two ends of the gold nanorod is spherical.
Preferably, the length-diameter ratio of the gold nanorods is 3-3.8, the thickness of the solid silicon dioxide is 2-5 nm, the thickness of the mesoporous silicon dioxide is 20-40 nm, and the load capacity of indocyanine green in the composite nanoprobe is 5-15%. By adjusting the structure size and the ICG load capacity of the composite nano probe, the optimal absorption peak position of the composite nano probe is matched with the near-infrared excitation light, and the enhanced photo-thermal and photodynamic effects are obtained, so that the cancer cells are killed more effectively. In the application, the loading amount of the indocyanine green is the mass content of the indocyanine green loaded in the composite nano probe, that is, the mass ratio of the loaded indocyanine green to the composite nano probe.
More preferably, the aspect ratio of the gold nanorods is 3.5, the thickness of the solid silica is 3nm, the thickness of the mesoporous silica is 25nm, and the loading amount of indocyanine green in the composite nanoprobe is 13%.
Preferably, the absorption peak of the composite nanoprobe is located in the near infrared region.
More preferably, the absorption peak of the composite nano probe is located at 790-810 nm.
Further preferably, the absorption peak of the composite nanoprobe is located at 800 nm.
The application also protects a preparation method of the composite nanoprobe, which comprises the following steps:
s1, growing solid silicon dioxide and mesoporous silicon dioxide at two ends of a gold nanorod in sequence to obtain a nano probe precursor with a dumbbell structure;
s2, the nano probe precursor in the step S1 is loaded with indocyanine green, and the composite nano probe is obtained after surface modification.
Step S1. growing solid silica and mesoporous silica successively at two ends of the gold nanorod can be carried out by referring to the prior art. For example, CTAB can be used as a guiding agent, and both ends of a gold nanorod are coated with solid silica first and then with mesoporous silica to form dumbbell-shaped silicon-coated gold nanoparticles, i.e., the nano-probe precursor with the dumbbell-shaped structure.
Preferably, in the step s2, the composite nanoprobe is obtained by dispersing a nanoprobe precursor and indocyanine green in a solvent, and performing stirring, ultrasonic treatment, centrifugation, washing and surface modification. The solvent may be water, dimethyl sulfoxide or methanol.
The outer surface of the mesoporous silica is modified by PEG and folic acid. The surface modification can be carried out with reference to the prior art.
The application of the composite nano probe in preparing the medicine for killing tumor cells is also within the protection scope of the application.
Preferably, the tumor cell is one or more of nasopharyngeal carcinoma cell, breast cancer cell, esophageal cancer cell or skin cancer cell.
Compared with the prior art, the invention has the beneficial effects that:
under the stimulation of near infrared light, the enhanced local electric field at the two ends of the gold nanorod stimulates ICG molecules in the mesoporous silica to continuously generate singlet oxygen, and meanwhile, the enhanced photo-thermal photodynamic effect is finally obtained by utilizing the double photo-thermal effects of the gold nanorod and the ICG, so that tumor cells can be effectively killed under lower laser power. In addition, ICG molecules protected by the gold nanorods and the mesoporous silica cannot generate photobleaching, and have good light stability.
Drawings
FIG. 1 shows the topography, absorption spectra, and photothermal-photodynamic effect enhancement principles of a composite nanoprobe in an embodiment of the invention. Wherein, FIG. 1a is a transmission electron microscope topography of the composite nanoprobe with dumbbell structure; FIG. 1b shows gold nanorods AuNRs, ICG molecules, and mesoporous SiO2Coated gold nanorods (Au @ mSiO)2NDs) and mesoporous SiO2Coated gold nanorods loaded ICG molecules (Au @ mSiO for example 1)2-ICG NDs); FIGS. 1c to 1f are the simulation diagrams of refractive index of the material on the surface, surface plasmon electric field distribution and surface charge distribution of the composite nanoprobe with dumbbell structure and the fusion diagram of the probe structure and the surface enhanced electric field.
FIG. 2 shows the results of the singlet oxygen generating ability test. Wherein, FIGS. 2 b-2 d are respectively ICG molecule and mesoporous SiO2Coated gold nanorods (Au @ mSiO)2NDs) and mesoporous SiO2Coated gold nanorods loaded ICG molecules (Au @ mSiO for example 1)2-ICGNDs) in DPBF solution after 0, 2, 4, 6, 8, 10, 12min of near infrared 800nm laser irradiation, fig. 2a is the test result of DPBF solution. In fig. 2a to 2d, the curves decrease in sequence as the irradiation time increases.
FIG. 3 is a diagram for reversely deducing the singlet oxygen generating capacity of each sample under the irradiation of near-infrared laser according to the decrease of the absorption intensity in FIG. 2. Specifically, according to DPBF, the gold nanorod, the ICG molecule and the mesoporous SiO are arranged2Coating gold nano-rod and mesoporous SiO2Weakening of light absorption intensity in coated gold nanorod loaded ICG molecular solution reversely deduces singlet oxygen generation energy of each substance under near-infrared laser irradiationForce.
Fig. 4 is a temperature rise curve of the photothermal effect test result. Specifically, a medium solution, ICG molecules and mesoporous SiO2Coated gold nanorods (Au @ mSiO)2NDs) and mesoporous SiO2Coated gold nanorods loaded ICG molecules (Au @ mSiO for example 1)2-ICG NDs) solution temperature rise curves after 0, 2, 4, 6, 8, 10, 12min of near infrared 800nm laser irradiation.
FIG. 5 is an infrared thermal imaging graph of photothermal effect test results. Specifically, a medium solution, ICG molecules and mesoporous SiO2Coated gold nanorods (Au @ mSiO)2NDs) and mesoporous SiO2Coated gold nanorods loaded ICG molecules (Au @ mSiO for example 1)2-ICG NDs) infrared thermography of the solution after 0, 2, 4, 6, 8, 10, 12min of near infrared 800nm laser irradiation in a cuvette.
FIG. 6 shows the results of the tumor cell killing experiment. Specifically, the light-emitting material is prepared by using near-infrared laser at 800nm, PBS, ICG molecules and mesoporous SiO2Coated gold nanorods (Au @ mSiO)2NDs) and mesoporous SiO2Coated gold nanorods loaded ICG molecules (Au @ mSiO for example 1)2ICG NDs) and a cell image of nasopharyngeal carcinoma cells stained with a double stain of Calcein-AM and Propidium Iodide (PI).
Detailed Description
The present invention will be further described with reference to the following embodiments.
The raw materials in the examples are all commercially available;
reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
In the present application, "ICG" refers to indocyanine green.
"AuNRs" refers to gold nanorods.
"CTAB" refers to cetyltrimethylammonium bromide.
“mSiO2"refers to mesoporous silica.
"DPBF" refers to 1,3 diphenyl isobenzofuran.
In the embodiment of the application, the preparation process of the nano probe precursor with the dumbbell structure is as follows:
(1) preparation of gold nanorods
Soaking all containers with strong oxidizing aqueous solution (10 wt% potassium dichromate and 25 wt% concentrated sulfuric acid), and cleaning and drying for later use. 0.364g CTAB was dissolved in 8.34mL deionized water, followed by 0.167mL of 0.018mol/L chloroauric acid solution, 1.16mL of 0.01mol/L NaBH4And (3) rapidly stirring the ice water solution for two minutes, and placing the ice water solution in a water bath at the temperature of 27 ℃ for 2 hours after stirring to obtain the gold nanorod seed solution.
1.82g CTAB dissolved in 45mL deionized water, and then added to the 6mL chloroauric acid solution, and then 0.005mol/L AgNO3Adding 1.2mL of solution and 0.4mL of HCl (37%) into the solution, stirring, adding about 1.2-1.6 mL of 0.05mol/L ascorbic acid, wherein the solution is changed from yellow to colorless, adding 0.05mL of gold nanorod seed solution prepared in the previous step (stirring gently for 10s) in the process of standing, placing the solution in a water bath at 27 ℃ for 12h in the process of standing, centrifuging for 20 minutes at 9000r/min, collecting, and dispersing in 25mL of deionized water to obtain a gold nanorod AuNRs dispersion for later use.
(2) Solid silicon dioxide and mesoporous silicon dioxide grow at two ends of gold nanorod in sequence
And (2) centrifuging the gold nanorod dispersion liquid prepared in the step (1) twice at 9000r/min, removing a supernatant, adding 0.008g of CTAB, ultrasonically dispersing in 40mL of deionized water, and adding 0.2mL of 0.025mol/L NaOH solution. 0.2mL of 4% (volume fraction) TEOS ethanol solution is added every 30min during the slow stirring process, and the stirring is continued for 12h after 4 times. Centrifuging, washing once with alcohol and once with deionized water, repeating the steps for three times to obtain the gold nanorods with two ends coated with solid silicon dioxide.
0.029g CTAB is dissolved in 40mL deionized water, the obtained gold nanorods with two ends coated with solid silicon dioxide are dispersed in the CTAB solution by ultrasonic, 0.5mL of 0.025mol/L NaOH solution is added, 0.8mL of 8% (volume fraction) TEOS ethanol solution is added every 30min in the process of slow stirring, and the solution is added for 4 times in total. Centrifuging, washing twice with ammonium nitrate solution, hydrochloric acid alcohol solution and deionized water to obtain nanometer particle with CTAB in the mesopores.
(3) Removal of CTAB in mesopores
CTAB exists in the mesopores of the nanoparticles prepared in the step (2), and the CTAB is required to be removed because of cytotoxicity, and the method comprises the following steps: ultrasonically dispersing the obtained nano particles in 60mL of ethanol solution, adding 1mL of concentrated hydrochloric acid, refluxing for 24h at 60 ℃, centrifugally collecting, washing once with water and ethanol, repeating the steps for 2 times to obtain a nano probe precursor with a dumbbell structure, namely Au @ mSiO2NDs dispersed in deionized water. The precursor of the nano probe with the dumbbell structure is a gold nanorod with two ends coated with solid silica and mesoporous silica in sequence.
Prepared Au @ mSiO2In NDs, the length-diameter ratio of the gold nanorods is 3.5, the thickness of solid silica is 3nm, and the thickness of mesoporous silica is 25 nm.
Example 1
A composite nano probe with a dumbbell structure is prepared by the following steps:
1) and loading an ICG: the obtained Au @ mSiO2NDs particles were ultrasonically dispersed by centrifugation in 5mL indocyanine green (ICG 0.02g) solution and mixed in a flat-bottomed flask. Adding a stirrer, stirring at high speed for 30min, taking out the stirrer, performing ultrasonic treatment in an ultrasonic water bath with the power set to 100%, performing ultrasonic treatment for 12h, centrifuging to remove redundant ICG molecules, washing with water and alcohol twice respectively to obtain ICG-loaded Au @ mSiO2NDs, dispersed in alcohol.
2) Modified PEG-FA: the prepared ICG-loaded Au @ mSiO2NDs were centrifuged and then ultrasonically dispersed in 50mL of ethanol solution, 0.5mL of 5% (by volume) 3-Aminopropyltriethoxysilane (APTES) ethanol solution was added, the mixture was refluxed at 60 ℃ for 12 hours, centrifuged to attach an amino group to the surface, washed twice with ethanol to remove unreacted APTES, and vacuum dried. Aminated AuNRs @ nSiO2@mSiO2(ICG) ultrasonic dispersion in 20mL of dimethyl sulfoxide (DMSO), adding 0.01g N-hydroxysuccinimide-polyethylene glycol-carboxyl (NHS-PEG-COOH), and stirring in the dark for 24 h. PEGylated nanoparticles were centrifuged and washed, dispersed in 20mL DMSO, and 0.01g Folic acid (Folic ac) was addedid, FA), some water loss agents Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), stirring for 24h, and respectively washing with dimethyl sulfoxide, ethanol and water twice to obtain a final product for modifying PEG-FA, namely the composite nanoprobe, namely Au @ mSiO2-ICG NDs. The load capacity of indocyanine green in the composite nanoprobe is 13%.
Example 2
Different from the embodiment 1, the loading amount of indocyanine green in the prepared composite nanoprobe is changed to be 5%; the other composition and procedure were the same as in example 1.
Example 3
Different from the embodiment 1, the loading amount of indocyanine green in the prepared composite nanoprobe is 15% by changing the adding amount of ICG; the other composition and procedure were the same as in example 1.
Test method
(1) Topography testing
The appearance and the structure of the composite nanoprobe with the dumbbell structure are characterized by adopting a 120kV transmission electron microscope (TEM, FET Tecnai G2 Spirit F12) of the testing center of Zhongshan university, and the magnification factor adopted by the electron microscope observation is about 25600 times. Sample preparation and test processes: dispersing a small amount of sample in 10ml of cyclohexane, ultrasonically dispersing for 0.5h, dropping a drop of liquid on a 200-mesh carbon-supported film copper net, standing for 10 minutes, then dropping the drop on the carbon film copper net, repeating the operation for three to four times, placing the carbon film copper net on a blast drying oven, drying the carbon film copper net for 10 hours at a constant temperature of 60 ℃, placing the copper net loaded with the sample under a transmission electron microscope, vacuumizing, adjusting the magnification, and observing the appearance of the sample.
(2) Absorption Spectrum measurement
And (3) detecting the light absorption capacity of the dumbbell probe water solution by adopting an ultraviolet-visible-near infrared spectrophotometer (UV-3150), and scanning within the wavelength range of 400-1000 nm by adopting ultrapure water as a blank control. Respectively preparing ICG, gold nanorods AuNRs and Au @ mSiO2NDs and Au @ mSiO2And (4) carrying out ultrasonic dispersion on an ICG NDs aqueous solution, taking a small amount of colloidal solution, placing the colloidal solution in a quartz cuvette, and placing the cuvette in an instrument for scanning detection.
(3) Electric field and charge profiles of composite nanoprobes
And calculating the local electric field enhancement condition of the mesoporous silicon gadolinium gold nanoprobe by adopting a finite difference time domain algorithm. Firstly, a mesoporous silicon gadolinium gold physical structure (the length of the gold nanorod is 70nm, the width of the gold nanorod is 20nm, and the thickness of silicon dioxide is about 25-30nm) is created, a simulation area and time are set according to the mesoporous silicon gadolinium gold physical structure, the background environment is water, and the configuration of the nanoprobe is established based on the surrounding environment of the gold nanorod, namely the refractive indexes of different substances. The refractive indices of water, silica and ICG molecules were found to be 1.33, 1.54 and 1.7, respectively. And then, the grid precision is refined, the boundary condition is suspended, and a Perfect Matching Layer (PML) is adopted, namely, the electromagnetic wave can be transmitted to infinite distance without reflection and absorption by the boundary. And (3) selecting a full-field-scattered field light source and emitting light from one side of the dumbbell composite probe. The electromagnetic field values were recorded using a monitor, data were obtained, and the results of the analysis were plotted.
(4) Singlet oxygen Generation capability test
Indirectly detecting ICG, Au @ mSiO by adopting an ultraviolet-visible-near infrared spectrophotometer (UV-3150)2NDs and Au @ mSiO2-singlet oxygen generating capability of ICG NDs, DPBF as singlet oxygen detector, ICG, Au @ mSiO2NDs and Au @ mSiO2ICG NDs were dissolved in medium DMSO, respectively, and equal amounts of DPBF were added sequentially. Pure DMSO was used as a blank and scanned over the wavelength range of 400-1000 nm. And (3) rapidly recording the absorption peak position after irradiating for 2min by adopting 800nm near-infrared laser. And calculating the singlet oxygen generation capacity value under different irradiation time by taking the optimal absorption peak when the irradiation is 0min as a denominator.
(5) Testing of photothermal effects
Recording the photothermal conversion performance of the dumbbell probe by using a thermal infrared imager (Tis65, Fluke), preparing ICG, gold nanorods, silica-coated gold nanorods and mesoporous silica-coated gold nanorods solution, ultrasonically dispersing the solutions in four-way cuvettes, sequentially irradiating each cuvette by using 800nm laser, wherein the irradiation time is 12 minutes, and the irradiation power is 0.4Wcm-1And observing the temperature rise condition of the solution by adopting a thermal infrared imager, and photographing and recording. The temperature rise was recorded using pure medium solvent as controlAnd imaging. And the temperature value is obtained according to the thermal infrared imager.
(6) Experiment for killing tumor cells
Respectively mixing human nasopharyngeal carcinoma cells CNE2 with PBS solution, ICG, Au @ mSiO2NDs and Au @ mSiO22h after co-cultivation of-ICG NDs (where ICG, Au @ mSiO)2NDs and Au @ mSiO2The concentrations of ICG NDs are 5, 35 and 40 mu g/mL respectively, the concentration of PBS solution and the cell concentration are conventional in the field, and a near-infrared 800nm laser (0.4W cm)-1) Irradiating for 12 min. The activity of the cells is detected by adopting a cck-8 detection kit (dojindo; CK04), and the OD value of the cells at 405nm is read on an enzyme labeling instrument (Thermo; MuLTiSKAN MK3) after the cck-8 reagent is added, so that the activity of the cells is predicted. The cells after laser irradiation and drug treatment were incubated for 15 minutes at 37 ℃ with 100uL of Calcein-AM and PI mixed staining solution (dojindo; NG587), under a fluorescence microscope, excited at a wavelength of 490 + -10 nm to observe yellow-green living cells (Calcein-AM staining), then excited at a wavelength of 545nm to see red dead cells (PI staining), and after 15min of fixation, photographed and recorded by a laser confocal microscope (Leica-SP 8).
Test results
The data result shows that the ICG molecules can be stimulated to generate a large amount of singlet oxygen by utilizing the longitudinal local surface plasmon resonance effect of the long axis of the gold nanorod. The photothermal effect of the ICG and the photothermal effect of the gold nanorods can be mutually superposed, and the photodynamic effect of the ICG can stimulate the further improvement of the thermal effect. The local surface plasma resonance band of the gold nanorod resonates with the ICG molecular absorption band, the absorption efficiency is greatly enhanced, a large amount of singlet oxygen can be continuously generated, the two-photon-heat effect of the gold nanorod and the ICG molecular is combined, and the photo-heat performance of the composite probe is further enhanced.
Taking the composite nanoprobe prepared in the embodiment 1 as an example, the morphology and performance test is carried out. The following test results in Au @ mSiO2ICG NDs were all the composite nanoprobes prepared in example 1.
As shown in fig. 1a, the composite nanoprobe has a good dumbbell shape, and has good uniformity and dispersibility. The length of the gold nanorod is about 70nm, the width of the gold nanorod is about 20nm, mesoporous silicon dioxide is coated at two ends of the gold nanorod, and ICG molecules are embedded in the mesopores.
FIG. 1b shows that coating mesoporous silica and loading ICG can cause red shift of the plasma absorption peak of gold nanorods, because the dielectric environment around the gold nanorods changes. The absorption peak of ICG molecules is equivalent to the plasma absorption peak of the composite nano probe, and under the radiation of external 800nm laser, the ICG molecules can participate in the surface plasma resonance effect of the gold nanorods, so that the enhancement of longitudinal surface plasma electric fields is further promoted. Because of the extremely strong plasma absorption capacity of the gold nanorods, ICG molecules can be prevented from photobleaching, and the light stability of the ICG molecules can be effectively improved.
FIG. 1c is a schematic diagram of the refractive index distribution of the substances around the gold nanorods, which reflects the structural shape of the distribution of the components of the dumbbell probe. According to the research on the refractive index of a plurality of organic macromolecules, the ICG molecular refractive index is assumed to be 1.7, and the optimal absorption peak position is close to 800nm by regulating and controlling the load of mesoporous to the ICG molecular. Fig. 1d and fig. 1e are an electric field distribution diagram and a charge distribution diagram of the nano dumbbell, and fig. 1f is a diagram formed by combining fig. 1c and fig. 1d, which show that under 800nm excitation light, a plasma electric field is mainly distributed in dumbbell balls at two ends of a gold nanorod, and can effectively stimulate ICG molecules in mesopores of the dumbbell balls to generate a large amount of singlet oxygen.
FIGS. 2a-d show the change of the absorption curve of the singlet oxygen detector DPBF in the composite nanoprobe solution with time under the excitation light radiation of 800 nm. In the medium, DPBF did not have any quenching effect, indicating that it does not produce singlet oxygen. The composite nano probe has an obvious quenching effect on DPBF, and the gold nanorod plasma electric field enhancement has a strong stimulation effect on ICG molecules in the mesopores of the dumbbell ball, so that the gold nanorod plasma electric field enhancement can promote the gold nanorod plasma electric field enhancement to continuously generate singlet oxygen. Meanwhile, ICG molecules cannot generate photobleaching under the protection of the gold nanorods and the mesoporous silica, and the ICG has good light stability.
FIG. 3 is a graph plotting the change of singlet oxygen with time at 800nm excitation for each species according to the strongest absorption peak of DPBF in FIGS. 2 a-d. The singlet oxygen yield of a single ICG molecule under 800nm laser is obviously lower than that of a gold nanorod-stimulated and mesoporous-protected ICG molecule.
Fig. 4 and 5 are graphs of temperature rise curves and temperature distribution images of the composite nanoprobe solution under near-infrared excitation light, and it can be seen that the composite nanoprobe exhibits more excellent photothermal effects than ICG molecules and gold nanorods, because the heat generation thereof is due to contributions from both ICG molecules and gold nanorods. The above proves that the dumbbell composite probe shows stronger photo-thermal effect and photodynamic effect no matter the single ICG molecule or the mesoporous silicon-coated gold nanorod.
FIGS. 6a-d are confocal images showing dead and live cells using two stains. Nasopharyngeal carcinoma cells were incubated with the sample for 2 hours, irradiated with laser at 800nm for six minutes, co-stained with Calcein-AM and PI and imaged. Calcein-AM stained live cells emit green light while PI stained dead cells emit red light. The graph shows that under the action of laser and a medium, the cancer cells are all living cells, the cancer cells are partially dead under the action of the laser and ICG molecules (figure 6b), the death rate of the cancer cells is further increased under the action of the laser and gold nanorods (figure 6c), and the cancer cells are almost dead under the action of the laser and the composite nano-probe (figure 6 d). The results prove that the photo-thermal and photo-dynamic combined mode based on the composite nano probe can kill cancer cells more effectively under lower exciting light power.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The composite nanoprobe with the dumbbell structure is characterized by comprising gold nanorods, solid silicon dioxide coating two ends of the gold nanorods, mesoporous silicon dioxide coating the solid silicon dioxide and indocyanine green loaded in the mesoporous silicon dioxide, wherein the gold nanorods, the solid silicon dioxide and the mesoporous silicon dioxide positioned at two ends of the gold nanorods form the dumbbell structure.
2. The composite nanoprobe of claim 1, wherein the length-diameter ratio of the gold nanorods is 3-3.8, the thickness of the solid silica is 2-5 nm, the thickness of the mesoporous silica is 20-40 nm, and the loading amount of indocyanine green in the composite nanoprobe is 5-15%.
3. The composite nanoprobe of claim 2, wherein the aspect ratio of the gold nanorods is 3.5, the thickness of the solid silica is 3nm, the thickness of the mesoporous silica is 25nm, and the loading amount of indocyanine green in the composite nanoprobe is 13%.
4. The composite nanoprobe of claim 1, wherein the absorption peak of the composite nanoprobe is in the near infrared region.
5. The composite nanoprobe of claim 4, wherein the absorption peak of the composite nanoprobe is 790 to 810 nm.
6. The composite nanoprobe of claim 5, wherein the absorption peak of the composite nanoprobe is at 800 nm.
7. The method for preparing the composite nanoprobe of any one of claims 1 to 6, which is characterized by comprising the following steps:
s1, growing solid silicon dioxide and mesoporous silicon dioxide at two ends of a gold nanorod in sequence to obtain a nano probe precursor with a dumbbell structure;
s2, the nano probe precursor in the step S1 is loaded with indocyanine green, and the composite nano probe is obtained after surface modification.
8. The preparation method according to claim 7, wherein in step S2, specifically, the composite nanoprobe is obtained by dispersing a nanoprobe precursor and indocyanine green in a solvent, and performing stirring, ultrasound, centrifugation and washing.
9. Use of the composite nanoprobe of any one of claims 1 to 6 in the preparation of a medicament for killing tumor cells.
10. The use of claim 9, wherein the tumor cell is one or more of a nasopharyngeal carcinoma cell, a breast carcinoma cell, an esophageal carcinoma cell, or a skin carcinoma cell.
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