CN111053901A - Sound sensitive agent with aggregation-induced emission characteristic and preparation method thereof - Google Patents
Sound sensitive agent with aggregation-induced emission characteristic and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a sound sensitive agent with aggregation-induced emission characteristic and a preparation method thereof, wherein the sound sensitive agent has a core-shell structure, the core is a fluorophore molecule with aggregation-induced emission characteristic, and the shell is a hydrophilic substance; the preparation method of the sound sensitive agent comprises the following steps: 1) dissolving fluorophore molecules with aggregation-induced emission characteristics in an organic solvent, adding a hydrophilic substance, and uniformly mixing to obtain a mixed solution; 2) and adding the mixed solution into water, stirring to form a core-shell structure, and separating to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics. Compared with the prior art, the sonosensitizer prepared by the invention has no fluorescence quenching effect, so that more singlet oxygen can be generated to inhibit the growth of tumors in the process of sonodynamic therapy; due to the nature of the AIE molecule, fluorescence guided interventional therapy can be introduced; also, the variety of AIE molecules is great, so the range of sonosensitizers can be expanded.
Description
Technical Field
The invention belongs to the technical field of functional materials and medicines, and relates to a sound-sensitive agent with aggregation-induced emission characteristics and a preparation method thereof.
Background
The high morbidity and mortality of malignant tumors has been a serious threat to human life and health, and there is a great need to find a method for effective treatment of malignant tumors. The existing means for treating malignant tumor in clinical application, such as surgical excision, radiotherapy, chemotherapy and the like, have certain disadvantages. In recent years, photodynamic therapy with micro-noninvasive therapeutic means enters the visual field of people, but light is attenuated very fast in tissues and cannot penetrate deep tissues, so that photosensitizers accumulated in the deep tissues cannot be sufficiently excited to generate enough active oxygen to kill tumor cells.
The acoustic dynamic therapy developed on the basis of the photodynamic therapy attracts attention as a promising non-invasive therapy method because the ultrasonic wave has the advantage of better tissue penetrating performance. Sonodynamic therapy can cause apoptosis or necrosis of tumor cells by ultrasonically activating sonosensitizers enriched around deep tumor tissue to produce reactive oxygen species.
The existing sound-sensitive agents can be divided into inorganic and organic types. The preparation process of the high-quality inorganic sound-sensitive agent is complex, and most inorganic sound-sensitive agents are not easy to degrade in vivo and have the disadvantage of long-term toxicity. Compared with inorganic sound-sensitive agents, organic sound-sensitive agents have relatively good biocompatibility with biological tissues. However, most of the current organic sonosensitizers are derived from photosensitizers, including porphyrins and their derivatives. In addition, another method for improving the light/sound dynamic curative effect is to increase the load of the light/sound sensitive agent, but the overload of the organic molecule easily causes the quenching of the light/sound sensitive agent, and the quenching effect caused by aggregation also causes the reduction of the generation amount of active oxygen, which becomes the bottleneck problem faced by the current sound sensitive agent.
Disclosure of Invention
The present invention aims to overcome the above-mentioned drawbacks of the prior art and provide an acoustic sensitizer with aggregation-induced emission characteristics and a preparation method thereof, wherein the acoustic sensitizer can be applied to acoustic dynamic therapy, and the problem of fluorescence quenching caused by aggregation of the conventional acoustic sensitizer is solved by using an aggregation-induced emission (AIE) material.
The purpose of the invention can be realized by the following technical scheme:
a sound sensitive agent with aggregation-induced emission characteristic has a core-shell structure, wherein the core is a fluorophore molecule with aggregation-induced emission characteristic, and the shell is a hydrophilic substance. The hydrophilic species provides hydrophilicity to the fluorophore molecule.
Further, the fluorophore molecules with aggregation-induced emission properties (AIE molecules) include one or more of TTMN, MeTTMN, or MeOTTMN.
The chemical formula is as follows:
further, the hydrophilic substance comprises one or more of amphiphilic polyethylene glycol, silicon dioxide, polystyrene or protein.
Further, the amphiphilic polyethylene glycol comprises one of distearoylphosphatidylethanolamine-polyethylene glycol-2000 or distearoylphosphatidylethanolamine-polyethylene glycol-5000.
A method for preparing a sonosensitizer having aggregation-induced emission characteristics, said method comprising the steps of:
1) dissolving fluorophore molecules with aggregation-induced emission characteristics in an organic solvent, adding a hydrophilic substance, and uniformly mixing to obtain a mixed solution;
2) and adding the mixed solution into water, stirring to form a core-shell structure, and separating to obtain the sound-sensitive agent with aggregation-induced emission characteristics.
Further, in step 1), the organic solvent includes one or more of tetrahydrofuran and dimethyl sulfoxide.
Further, in step 1), 0.1 to 1mg of fluorophore molecule having aggregation-induced emission characteristics and 2 to 10mg of hydrophilic substance are added per 1mL of organic solvent.
Further, in the step 2), the volume ratio of the mixed solution to water is 1: 8-12.
Further, in the step 2), the stirring temperature is normal temperature, and the stirring time is 6-12 h.
Further, in the step 2), the separation is ultrafiltration separation.
When the invention prepares the sound sensitive agent with aggregation-induced emission characteristic, fluorophore molecules with aggregation-induced emission characteristic are dissolved in an organic solvent to obtain a precursor solution; and then adding hydrophilic substances into the precursor solution, assembling the precursor solution and the hydrophilic substances in water to form nano particles, and performing ultrafiltration treatment to obtain the final sound-sensitive agent. The sonosensitizer can be used for sonodynamic therapy, sonodynamic therapy guided by fluorescence imaging, and therapy is carried out under the excitation of ultrasonic waves, and the frequency of the ultrasonic waves is preferably 10 k-5.0 MHz.
Compared with the prior art, the invention has the following characteristics:
1) the sonosensitizer with aggregation-induced emission characteristics prepared by the invention has no quenching effect, so that more singlet oxygen can be generated in the process of sonodynamic therapy;
2) the prepared acoustic sensitizer with aggregation-induced emission characteristics can introduce fluorescence-mediated sonodynamic therapy due to the inherent characteristics of the acoustic sensitizer;
3) the acoustic sensitizer with aggregation-induced emission characteristics prepared by the invention shows that the material with aggregation-induced emission characteristics can be used as the acoustic sensitizer for the first time, has certain universality, and can expand the range of the acoustic sensitizer due to the various varieties of AIE molecules.
Drawings
FIG. 1 is a structural diagram of a sonosensitizer of the present invention;
FIG. 2 is an electron micrograph of the acoustic sensitizer having aggregation-induced emission characteristics prepared in example 1;
FIG. 3 is a particle size diagram of the acoustic sensitizer having aggregation-induced emission characteristics prepared in example 1;
FIG. 4 is a graph showing an ultraviolet absorption spectrum of the acoustic sensitizer having aggregation-induced emission characteristics prepared in example 1;
FIG. 5 is a fluorescence emission spectrum of the photosensitizer with aggregation-induced emission characteristics prepared in example 1;
FIG. 6 is a graph of the fit of the sonosensitizer having aggregation-induced emission properties prepared in example 1 and pure water versus the rate of DPBF degradation under ultrasound;
FIG. 7 is a bar graph showing the effect of the addition of the prepared sonosensitizer with aggregation-induced emission characteristics on the survival rate of cancer cells under different sound intensities in example 1;
FIG. 8 is a graph of the volume change of tumors in each group of mice during the sonodynamic treatment of example 1.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
Among them, fluorophore molecules having aggregation-induced emission properties such as TTMN, MeTTMN, MeOTTMN, etc. of the present invention can be prepared by methods disclosed in references (d.wang, h.su, r.t.k.kwok, g.shan, a.c.s.leung, m.m.s.lee, h.h.y.sung, i.d.williams, j.w.y.lam and b.z.tang, adv.funct.mater.,2017,27, 1704039).
The frequency range of the parameters preferentially selected by the ultrasonic device used in the process of the acoustodynamic treatment of the prepared sound sensitive agent with aggregation-induced emission characteristics is 10 k-5.0 MHz, and the provided ultrasonic wave is the ultrasonic wave capable of being focused.
The method adopts a 1, 3-diphenyl-isobenzofuran (DPBF) probe method to detect the generation of singlet oxygen, and under the irradiation of ultrasonic waves, the sound sensitizer with aggregation-induced emission characteristics prepared by the method has the capability of generating more singlet oxygen.
Example 1:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.2mg of prepared fluorophore molecules with aggregation-induced emission characteristics into tetrahydrofuran which is an organic solvent, and dissolving the fluorophore molecules;
2) adding 3mg distearoylphosphatidylethanolamine-polyethylene glycol-2000 (PEG-DSPE) to the above solution2000) And stirring uniformly to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) the obtained solution is ultrafiltered to obtain the sound-sensitive agent with aggregation-induced emission characteristics, and the core-shell structure of the sound-sensitive agent is shown in figure 1.
FIG. 2 is an electron micrograph of the acoustic sensitizer with aggregation-induced emission characteristics prepared in this example, and it can be seen from FIG. 2 that the AIE acoustic sensitizer has a particle size of about 50nm, is spherical, and is relatively uniformly distributed.
FIG. 3 is a particle size diagram of the acoustic sensitizer with aggregation-induced emission characteristics prepared in this example, and it can be seen from FIG. 3 that the hydrated particle size of the AIE acoustic sensitizer is about 43nm, which is substantially identical to the result of the electron micrograph of FIG. 2.
FIG. 4 is a diagram showing the ultraviolet absorption spectrum of the acoustic sensitizer with aggregation-induced emission characteristics prepared in this example, and it can be seen from FIG. 4 that the AIE acoustic sensitizer has two absorption peaks at about 300nm and 490 nm.
FIG. 5 is a fluorescence emission spectrum of the acoustic sensor with aggregation-induced emission characteristics prepared in this example, and it can be seen from FIG. 5 that the AIE acoustic sensor has a fluorescence emission peak around 650nm under excitation at 490nm wavelength.
The ability of the sonosensitizer with AIE characteristics prepared in the example to degrade DPBF under ultrasonic irradiation was examined by using DPBF as a probe for singlet oxygen. The ultrasonic conditions adopted are 1MHz and the power is 1.5W/cm2The total irradiation time was 5 minutes. FIG. 6 is a graph obtained by fitting the ratio of the aggregation-induced emission-characteristic sonosensitizer prepared in this example to pure water to the DPBF degradation rate under ultrasound, and the results show that the sonosensitizer prepared in this example can effectively degrade DPBF under ultrasound activation with degradation efficiency not lower than the effect of pure water in ultrasound。
Mouse breast cancer 4T1 cells (8X 10)3One/well) were incubated in a 96-well plate for 24 hours, and then added to a culture solution prepared from a sonosensitizer having an AIE characteristic in this example and incubated for 4 hours, and then the sonodynamic treatment efficiency was examined. The experiment was set as control 1 (sonosensitizer alone), control 2 (sonotrode alone) and experimental 3 (sonosensitizer + sonotrode). The ultrasonic frequency is 1MHz, and the sound intensity is 0.5-1.5W/cm2The ultrasonic irradiation time is 2min, and the experiments are all carried out under the condition of keeping out of the sun. The viability of the cells was examined by the CCK-8 method. FIG. 7 is a bar graph showing the effect of the presence or absence of the acoustic sensitizer with aggregation-induced emission characteristics on the survival rate of cancer cells under different sound intensities. The experimental result shows that the single ultrasonic action or the single sonosensitizer action has no obvious killing effect on cancer cells. However, under the combined action of the ultrasound and the sound-sensitive agent, the survival rate of cancer cells is gradually reduced along with the gradual increase of sound intensity in a certain range, and the inhibition rate can reach 70 percent at most.
The sound-sensitive agent with AIE characteristic prepared in the embodiment is injected into 4T1 tumor-bearing mice by tail vein injection, and the sound power treatment is carried out on days 1, 2, 3 and 5 respectively, wherein the ultrasonic frequency is 1MHz, and the sound intensity is 1.5W/cm2The ultrasonic irradiation time is 5 min. Figure 8 is a graph of the change in volume of tumors in each group of mice during sonodynamic therapy. The experimental results show that the ratio of the compound to the control group: (1) no treatment is carried out; (2) a separate sonosensitizer; (3) the comparison of single ultrasound shows that the tumor growth of the mice of the experiment group (4) of the sonosensitizer and the ultrasound group is obviously inhibited.
Example 2:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.2mg of prepared fluorophore molecule MeTTMN with AIE characteristic into organic solvent tetrahydrofuran for dissolving;
2) adding 3mg distearoylphosphatidylethanolamine-polyethylene glycol-5000 (PEG-DSPE) to the above solution5000) And stirring uniformly to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) and ultrafiltering the obtained solution to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
DPBF was used as a probe for singlet oxygen, and the ability of the sonosensitizer having AIE properties described in 4) to degrade DPBF by ultrasonic irradiation was examined. The ultrasonic conditions adopted are 1.5MHz and the power is 2W/cm2The total irradiation time was 5 minutes.
Example 3:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.2mg of the prepared fluorophore molecule MeOTTM with AIE characteristics into an organic solvent DMSO, and dissolving the fluorophore molecule;
2) adding 3mg distearoylphosphatidylethanolamine-polyethylene glycol-2000 (PEG-DSPE) to the above solution2000) And stirring uniformly to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) and ultrafiltering the obtained solution to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
DPBF was used as a probe for singlet oxygen, and the ability of the sonosensitizer having AIE properties described in 4) to degrade DPBF by ultrasonic irradiation was examined. The ultrasonic conditions adopted are 2.0MHz and the power is 1.5W/cm2The total irradiation time was 10 minutes.
Example 4:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.5mg of prepared fluorescence molecule TTMN with AIE into organic solvent tetrahydrofuran to dissolve;
2) adding 5mg distearoylphosphatidylethanolamine-polyethylene glycol-2000 (PEG-DSPE) to the above solution2000) And stirring uniformly to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) and ultrafiltering the obtained solution to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
DPBF was used as a probe for singlet oxygen, and the ability of the sonosensitizer having AIE properties described in 4) to degrade DPBF by ultrasonic irradiation was examined. The ultrasonic conditions adopted are 3.0MHz and the power is 1.5W/cm2The total irradiation time was 5 minutes.
Example 5:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.5mg of prepared fluorescence molecule TTMN with AIE into organic solvent DMSO, and dissolving;
2) adding 5mg of silicon dioxide into the solution, and uniformly stirring to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) and ultrafiltering the obtained solution to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
DPBF was used as a probe for singlet oxygen, and the ability of the sonosensitizer having AIE properties described in 4) to degrade DPBF by ultrasonic irradiation was examined. The ultrasonic conditions adopted are 3.0MHz and the power is 1.5W/cm2The total irradiation time was 5 minutes.
Example 6:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.5mg of the prepared fluorophore molecule MeTTMN with AIE into tetrahydrofuran which is an organic solvent, and dissolving the fluorophore molecule MeTTMN;
2) adding 5mg of polystyrene balls into the solution, and uniformly stirring to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) and ultrafiltering the obtained solution to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
DPBF was used as a probe for singlet oxygen, and the ability of the sonosensitizer having AIE properties described in 4) to degrade DPBF by ultrasonic irradiation was examined. The ultrasonic conditions adopted are 3.0MHz and the power is 1.5W/cm2The total irradiation time was 5 minutes.
Example 7:
the preparation of the sonosensitizer with AIE properties comprises the following steps:
1) adding 0.5mg of the prepared fluorophore molecule MeTTMN with AIE into tetrahydrofuran which is an organic solvent, and dissolving the fluorophore molecule MeTTMN;
2) adding 5mg of albumin into the solution, and uniformly stirring to obtain 1mL of mixed solution;
3) adding the 1mL of mixed solution into 10mL of pure water, stirring for 8h at normal temperature, volatilizing the organic solvent in the water, and self-assembling to form nano particles;
4) and ultrafiltering the obtained solution to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
DPBF was used as a probe for singlet oxygen, and the ability of the sonosensitizer having AIE properties described in 4) to degrade DPBF by ultrasonic irradiation was examined. The ultrasonic conditions adopted are 1.0MHz and 1.5W/cm of power2The total irradiation time was 10 minutes.
Example 8:
a sound sensitive agent with aggregation-induced emission characteristic has a core-shell structure, wherein the core is a fluorophore molecule with aggregation-induced emission characteristic, and the shell is a hydrophilic substance.
Wherein, the fluorophore molecule with aggregation-induced emission properties is TTMN. The hydrophilic substance is protein.
The preparation method of the sound sensitive agent comprises the following steps:
1) dissolving fluorophore molecules with aggregation-induced emission characteristics in an organic solvent, adding a hydrophilic substance, and uniformly mixing to obtain a mixed solution; the organic solvent was tetrahydrofuran, and 0.1mg of fluorophore having aggregation-induced emission characteristics and 10mg of hydrophilic substance were added to 1mL of the organic solvent.
2) Adding the mixed solution into water, wherein the volume ratio of the mixed solution to the water is 1:8, then stirring to form a core-shell structure, wherein the stirring temperature is normal temperature, the stirring time is 12 hours, and performing ultrafiltration separation to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
Example 9:
a sound sensitive agent with aggregation-induced emission characteristic has a core-shell structure, wherein the core is a fluorophore molecule with aggregation-induced emission characteristic, and the shell is a hydrophilic substance.
Wherein the fluorophore molecule having aggregation-induced emission properties is MeTTMN. The hydrophilic substance is polystyrene.
The preparation method of the sound sensitive agent comprises the following steps:
1) dissolving fluorophore molecules with aggregation-induced emission characteristics in an organic solvent, adding a hydrophilic substance, and uniformly mixing to obtain a mixed solution; the organic solvent was dimethyl sulfoxide, and 1mg of fluorophore having aggregation-induced emission characteristics and 2mg of hydrophilic substance were added to 1mL of the organic solvent.
2) Adding the mixed solution into water, wherein the volume ratio of the mixed solution to the water is 1:12, then stirring to form a core-shell structure, wherein the stirring temperature is normal temperature, the stirring time is 6 hours, and performing ultrafiltration separation to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
Example 10:
a sound sensitive agent with aggregation-induced emission characteristic has a core-shell structure, wherein the core is a fluorophore molecule with aggregation-induced emission characteristic, and the shell is a hydrophilic substance.
Wherein the fluorophore molecule having aggregation-induced emission properties is MeOTTMN. The hydrophilic substance is silicon dioxide.
The preparation method of the sound sensitive agent comprises the following steps:
1) dissolving fluorophore molecules with aggregation-induced emission characteristics in an organic solvent, adding a hydrophilic substance, and uniformly mixing to obtain a mixed solution; the organic solvent was dimethyl sulfoxide, and 0.7mg of fluorophore having aggregation-induced emission characteristics and 6mg of hydrophilic substance were added to 1mL of the organic solvent.
2) Adding the mixed solution into water, wherein the volume ratio of the mixed solution to the water is 1:10, then stirring to form a core-shell structure, wherein the stirring temperature is normal temperature, the stirring time is 8 hours, and performing ultrafiltration separation to obtain the sound-sensitive agent with aggregation-induced luminescence characteristics.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. The sound-sensitive agent with aggregation-induced emission characteristic is characterized by having a core-shell structure, wherein the core is a fluorophore molecule with aggregation-induced emission characteristic, and the shell is a hydrophilic substance.
2. The sonosensitizer of claim 1, wherein said fluorophore molecules with aggregation-induced emission properties comprise one or more of TTMN, MeTTMN or MeOTTMN.
3. The sonosensitizer of claim 1, wherein said hydrophilic material comprises one or more of amphiphilic polyethylene glycol, silica, polystyrene, or a protein.
4. The sonosensitizer of claim 3, wherein said amphiphilic polyethylene glycol comprises one of distearoylphosphatidylethanolamine-polyethylene glycol-2000 or distearoylphosphatidylethanolamine-polyethylene glycol-5000.
5. A process for the preparation of a sonosensitizer having aggregation-induced emission properties as claimed in any one of claims 1 to 4, comprising the steps of:
1) dissolving fluorophore molecules with aggregation-induced emission characteristics in an organic solvent, adding a hydrophilic substance, and uniformly mixing to obtain a mixed solution;
2) and adding the mixed solution into water, stirring to form a core-shell structure, and separating to obtain the sound-sensitive agent with aggregation-induced emission characteristics.
6. The method for preparing the sound sensitizer with aggregation-induced emission characteristics as claimed in claim 5, wherein in step 1), the organic solvent comprises one or more of tetrahydrofuran and dimethylsulfoxide.
7. The method according to claim 5, wherein in step 1), 0.1-1mg of fluorophore molecule with aggregation-induced emission property and 2-10mg of hydrophilic substance are added to 1mL of organic solvent.
8. The method for preparing the sound sensitizer with aggregation-induced emission characteristics as claimed in claim 5, wherein in the step 2), the volume ratio of the mixed solution to water is 1: 8-12.
9. The method for preparing the sound sensitizer with aggregation-induced emission characteristics as claimed in claim 5, wherein in the step 2), the stirring temperature is normal temperature, and the stirring time is 6-12 h.
10. The method for preparing the sound sensitizer with aggregation-induced emission characteristics as claimed in claim 5, wherein in step 2), said separation is ultrafiltration.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114870027A (en) * | 2022-05-19 | 2022-08-09 | 重庆医科大学附属第二医院 | Application of bispyrene in preparation of ultrasonic trigger sonosensitizer, peptide functionalized compound and preparation, and preparation method and application method of peptide functionalized compound |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085416A (en) * | 2016-06-02 | 2016-11-09 | 华南理工大学 | A kind of aggregation-induced emission nanoparticle and its preparation method and application |
| WO2016206615A1 (en) * | 2015-06-24 | 2016-12-29 | The Hong Kong University Of Science And Technology | Aie luminogens for visualization and treatment of cancer |
| WO2017046747A1 (en) * | 2015-09-15 | 2017-03-23 | Acerta Pharma B.V. | Therapeutic combinations of a cd19 inhibitor and a btk inhibitor |
| CN108042803A (en) * | 2017-12-19 | 2018-05-18 | 国家纳米科学中心 | A kind of load has Liposomal dispersion of AIE molecules and its preparation method and application |
| CN110082531A (en) * | 2019-04-11 | 2019-08-02 | 南方医科大学南方医院 | A kind of tumour excretion body nano fluorescent detection kit and its application |
| CN110095608A (en) * | 2019-04-12 | 2019-08-06 | 南方医科大学南方医院 | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly |
| CN110201163A (en) * | 2019-06-17 | 2019-09-06 | 重庆医科大学 | A kind of load medicine mesoporous TiO 2 nanoparticle of hyaluronic acid and poly-dopamine modification |
| CN110585447A (en) * | 2019-10-21 | 2019-12-20 | 广东医科大学 | Preparation method and application of aggregated luminescent nano-particle material capable of being used as sound sensitizer |
-
2019
- 2019-12-18 CN CN201911311985.9A patent/CN111053901B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016206615A1 (en) * | 2015-06-24 | 2016-12-29 | The Hong Kong University Of Science And Technology | Aie luminogens for visualization and treatment of cancer |
| WO2017046747A1 (en) * | 2015-09-15 | 2017-03-23 | Acerta Pharma B.V. | Therapeutic combinations of a cd19 inhibitor and a btk inhibitor |
| CN106085416A (en) * | 2016-06-02 | 2016-11-09 | 华南理工大学 | A kind of aggregation-induced emission nanoparticle and its preparation method and application |
| CN108042803A (en) * | 2017-12-19 | 2018-05-18 | 国家纳米科学中心 | A kind of load has Liposomal dispersion of AIE molecules and its preparation method and application |
| CN110082531A (en) * | 2019-04-11 | 2019-08-02 | 南方医科大学南方医院 | A kind of tumour excretion body nano fluorescent detection kit and its application |
| CN110095608A (en) * | 2019-04-12 | 2019-08-06 | 南方医科大学南方医院 | Tumour excretion body nano fluorescent sensor based on Magnetic Isolation and DNA self assembly |
| CN110201163A (en) * | 2019-06-17 | 2019-09-06 | 重庆医科大学 | A kind of load medicine mesoporous TiO 2 nanoparticle of hyaluronic acid and poly-dopamine modification |
| CN110585447A (en) * | 2019-10-21 | 2019-12-20 | 广东医科大学 | Preparation method and application of aggregated luminescent nano-particle material capable of being used as sound sensitizer |
Non-Patent Citations (6)
| Title |
|---|
| DONG WANG ET AL: "Facile Synthesis of Red/NIR AIE Luminogens with Simple Structures, Bright Emissions, and High Photostabilities, and Their Applications for Specific Imaging of Lipid Droplets and Image-Guided Photodynamic Therapy", 《ADV. FUNCT. MATER.》 * |
| LINGYUN WANG ET AL: "Fabrication and application of dual-modality polymer nanoparticles based on an aggregation-induced emission-active fluorescent molecule and magnetic Fe3O4", 《POLYMERS》 * |
| MIN LI ET AL: "One-Step Formulation of Targeted Aggregation-Induced Emission Dots for Image-Guided Photodynamic Therapy of Cholangiocarcinoma", 《ACS NANO》 * |
| WEIWEI ZENG ET AL: "An Ultrasound‐Excitable Aggregation‐Induced Emission Dye for Enhanced Sonodynamic Therapy of Tumors", 《ADV. HEALTHCARE MATER.》 * |
| YAN XU ET AL: "An aggregation-induced emission dye-powered afterglow luminogen for tumor imaging", 《CHEMICAL SCIENCE》 * |
| 唐本忠 等: "聚集诱导发光(AIE):我国原创并引领的研究前沿", 《化学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114870027A (en) * | 2022-05-19 | 2022-08-09 | 重庆医科大学附属第二医院 | Application of bispyrene in preparation of ultrasonic trigger sonosensitizer, peptide functionalized compound and preparation, and preparation method and application method of peptide functionalized compound |
| CN114870027B (en) * | 2022-05-19 | 2023-05-16 | 重庆医科大学附属第二医院 | Application of dipyrene in preparation of ultrasonic touch sound sensitizer, peptide functional compound and preparation, preparation method and application method thereof |
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