CN117343082A - Light nanometer vaccine based on aggregation-induced emission material - Google Patents
Light nanometer vaccine based on aggregation-induced emission material Download PDFInfo
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- CN117343082A CN117343082A CN202311290111.6A CN202311290111A CN117343082A CN 117343082 A CN117343082 A CN 117343082A CN 202311290111 A CN202311290111 A CN 202311290111A CN 117343082 A CN117343082 A CN 117343082A
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- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
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Abstract
The invention discloses an optical nano vaccine based on aggregation-induced emission material. The aggregation-induced emission material has the structural formula:wherein X1 and X2 are independently selected from one of S, se; r1 and R2 are independently selected from H, One of them. The aggregation-induced emission material provided by the invention has molecules with a stronger D-A structure, so that the aggregation-induced emission material has obvious near infrared two-region emission property. Meanwhile, the aggregation-induced emission material has the performances of near infrared two-region emission, photothermal treatment and photodynamic treatment, and is expected to become a potential multi-mode photodiagnosis and treatment agent. The prepared optical nanometer vaccine can break the solid tumor barrier, efficiently activate anti-tumor immunity, form immune memory for tumors, efficiently kill the solid tumors, inhibit tumor recurrence and metastasis, and is expected to become a potential optical-immune treatment combined treatment platform.
Description
Technical Field
The invention relates to the technical field of biomedical nano materials, in particular to an optical nano vaccine based on an aggregation-induced emission material.
Background
Nanometer vaccines (nano-vaccines) are an emerging field, and are currently receiving widespread attention. The bionic nano technology is utilized to improve the tumor vaccine, so that the antigen can be nanocrystallized, the immune activation efficiency is improved, the antigen material and the immune stimulation adjuvant can be transmitted together, and the bioavailability of the nano platform is improved. In addition, the nanoparticles can be used as immunoadjuvants for cancer vaccines, because the nanoparticles can be efficiently absorbed by antigen presenting cells (e.g., dendritic cells) in the body. Recently, nanomaterial-based photothermal therapy has received a great deal of attention because it can break the physical barrier of solid tumors and improve immunosuppression, and has become one of the most promising tumor treatment methods in combination with immunotherapy. At present, the nano photothermal material is widely applied to the field of tumor photothermal therapy. Local hyperthermia can lead to death of immune cells, cause release of immune regulatory molecules, and enhance the activation efficiency of dendritic cells.
The aggregation-induced emission (AIE) molecule has the advantages of high aggregation state brightness, large Stokes shift, strong photobleaching resistance and the like, can effectively overcome the aggregation quenching (ACQ) phenomenon of the traditional fluorophor in the molecular aggregation state, and has great significance for fluorescence imaging. In addition, AIE molecules have a structural propeller-like conformation, and sufficient molecular rotors and/or vibrators to allow free rotation and/or vibration within the molecule in solution under light excitation. The strong intramolecular motion causes the energy of the excited state to be mainly consumed through a non-radiative decay way, thereby promoting the generation of heat and the photo-thermal conversion into sound waves, and realizing the efficient photo-thermal treatment and photo-acoustic imaging. At the same time AIE molecules are also an excellent Reactive Oxygen Species (ROS) generator, especially when aggregated, which can also be applied in photodynamic therapy (PDT).
Currently, a multi-mode photodiagnosis and treatment agent is lacking, and the multi-mode photodiagnosis and treatment agent has the performances of near infrared two-region emission, photothermal treatment and photodynamic treatment. Accordingly, the prior art is still further advanced and developed.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide an optical nano vaccine based on an aggregation-induced emission material, and aims to solve the problem of single mode when the existing optical nano vaccine is used for optical-immune combined diagnosis and treatment.
The technical scheme of the invention is as follows:
in a first aspect, an aggregation-induced emission material, wherein the aggregation-induced emission material has a structural formula:
wherein X1 and X2 are independently selected from one of S, se; r1 and R2 are independently selected from H,/-and-> One of them.
The structure is a D-A-D structure which typically contains terminal end capping groups, the terminal end capping not only can increase the molecular solubility, but also the distorted structure can increase the molecular fluorescence quantum yield, and the stacking is more loose, so that the molecular AIE property is further enhanced. The structure of the intermediate D-A-D can lead the absorption and emission wavelength of molecules to be red-shifted, the absorption is in a near infrared first region, the emission is in a near infrared second region, and the molecular structure has good advantages in the imaging field.
In a second aspect, a method for preparing an aggregation-induced emission material, wherein the method comprises:
dispersing a tin reagent product, 4, 7-dibromobenzo [1,2-c:4,5-c' ] bis ([ 1,2,5] thiadiazole) and palladium tetraphenylphosphine into an organic solvent to obtain a first mixed solution;
heating and refluxing the first mixed solution in an inert atmosphere, overnight, quenching reaction, and purifying to obtain the aggregation-induced emission material;
the structural formula of the tin reagent product is as follows:
wherein, R1 and R2 are independently selected from H,/-and-> One of them.
In a third aspect, a method for preparing an aggregation-induced emission material, wherein the method comprises:
dispersing a tin reagent product, 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole and tetraphenylphosphine palladium into an organic solvent to obtain a second mixed solution;
heating and refluxing the second mixed solution in an inert atmosphere, overnight, quenching the reaction, and purifying to obtain an intermediate product;
mixing the intermediate product with iron powder and acetic acid under the protection of inert gas, and reacting to obtain a reaction solution; cooling and extracting the reaction liquid to obtain solid powder;
mixing the solid powder with tin dioxide to obtain a mixture, adding chloroform and ethanol into the mixture in an inert atmosphere, and carrying out reflux reaction to obtain the aggregation-induced emission material;
the structural formula of the tin reagent product is as follows:
wherein, R1 and R2 are independently selected from H,/-and-> One of them. The inert gas may be nitrogen, helium, argon, or the like.
Optionally, the preparation method of the aggregation-induced emission material, wherein the preparation method of the tin reagent product comprises the following steps:
adding 4, 4-dioctyloxy-4-bromo-triphenylamine into a reaction container, adding anhydrous tetrahydrofuran into the reaction container under the protection of nitrogen, and cooling to-78 ℃;
dropwise adding n-butyllithium into the reaction container, reacting for one hour, dropwise adding tributyl tin chloride into the reaction container, heating to room temperature, and reacting overnight; quenching reaction, and purifying to obtain the tin reagent product.
Optionally, the preparation method of the aggregation-induced emission material, wherein the structural formula of the intermediate product is:
optionally, the preparation method of the aggregation-induced emission material, wherein the organic solvent comprises: toluene, tetrahydrofuran, dioxane and xylene.
Optionally, the preparation method of the aggregation-induced emission material comprises the step of preparing the aggregation-induced emission material, wherein the molar ratio of the intermediate product to the iron powder is 1:20.
Optionally, the preparation method of the aggregation-induced emission material, wherein the molar ratio of the tin reagent product, the 4, 7-dibromobenzo [1,2-c:4,5-c' ] bis ([ 1,2,5] thiadiazole) and the palladium tetraphenylphosphine is 1:0.4:0.08.
The preparation method of the aggregation-induced emission material comprises the step of preparing a tin reagent product, wherein the molar ratio of the tin reagent product, 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole and tetraphenylphosphine palladium is 1:0.4:0.08.
In a fourth aspect, an optical nanovaccine based on an aggregation-induced emission material, comprising: aggregation-inducing luminescent material and tumor-specific antigen; the aggregation-induced emission material is the aggregation-induced emission material.
Optionally, the light nanometer vaccine is characterized in that bovine serum albumin is used as a carrier to load AIE molecules to construct light therapy nanometer particles, liver cancer specific antigen GPC3 and the AIE molecules are used to construct nanometer vaccine particles, and the combined action of STING agonist c-d-AMP is combined to activate immunity.
The beneficial effects are that: compared with the prior art, the aggregation-induced emission material provided by the invention has molecules with a stronger D-A structure, so that the aggregation-induced emission material has obvious near infrared two-region emission property. Meanwhile, the aggregation-induced emission material has the performances of near infrared two-region emission, photothermal treatment and photodynamic treatment, and is expected to become a potential multi-mode photodiagnosis and treatment agent. Has important significance for realizing high-resolution imaging of deep tissues and excellent PDT treatment effect.
Drawings
FIG. 1 is a normalized UV absorption curve of TBBTD and TBBSD in tetrahydrofuran solution;
fig. 2 is a normalized fluorescence emission spectrum of TBBTD and TBBSD in tetrahydrofuran solution, λex (TBBTD) =760 nm, λex (TBBSD) =816 nm;
FIG. 3 is a fluorescence emission spectrum of TBBSD (10. Mu.M) in a mixed solvent of tetrahydrofuran/isopropanol (v/v), λex=816 nm as the water content increases;
fig. 4 shows fluorescence intensities of TBBTD and TBBSD (10 μm) in tetrahydrofuran/isopropanol (v/v) mixed solvent as isopropanol content increases, λex (TBBTD) =760 nm, λex (TBBSD) =816 nm;
FIG. 5 is a particle size distribution and transmission electron microscopy image of TBBSD nanoparticles;
FIG. 6 is an absorption and emission spectrum of TBBSD nanoparticles;
FIG. 7 shows the power of TBBSD nanoparticles at 1W cm at different concentrations -2 A photo-thermal curve under 808nm laser irradiation;
FIG. 8 is a photo-thermal profile of TBBSD nanoparticles at a concentration of 20. Mu.M under irradiation of 808nm lasers of different powers;
FIG. 9 is a photo-thermal stability curve of TBBSD nanoparticles;
FIG. 10 is a graph of the temperature ramp up and down curves for TBBSD nanoparticles and the calculation of the photo-thermal conversion efficiency;
FIG. 11 shows the laser irradiation time of 808 (1 w cm) -2 ) The fluorescence intensity enhancement factors of TBBSD-NPs and TBBSD-NPs (10 mu M) mixed solution with the total ROS indicator DCFH;
FIG. 12 shows the dark/white light (808 nm laser, power: 2W cm) of Hepa1-6 cells after incubation with different concentrations of TBBSD-NPs -2 ) Cell viability after 5min of irradiation treatment;
FIG. 13 shows apoptosis test of Hepa1-6 cells treated with different reagents in light for 5min at nanoparticle concentration of 50. Mu.g mL -1 ；
FIG. 14 shows the release of cell-associated immunogenic death factors CRT, HMGB1 and HSP70 from Hepa1-6 cells after various treatments. TBBSD at 50. Mu.g/mL, GPC3 at 2. Mu.g/mL, and AMP at 25. Mu.g/mL;
FIG. 15 shows the results of flow assays for CD80 and CD86 expression after various treatments of dendritic cells;
FIG. 16 shows TBBSD-NPs (100. Mu.L, TBBSD 5mg/kg body weight) after administration; near infrared two-region fluorescence imaging results of tumors of 0h,6h,12h,24h and 36 h;
FIG. 17 shows photoacoustic imaging results of tumors at 0h,6h,12h,24h and 36h after administration to mice;
FIG. 18 shows a 808nm laser (1.5W cm) -2 ) Irradiating a thermal imaging diagram of temperature change of a tumor part within 5 min;
FIG. 19 is a plot of in situ tumor volume versus time after treatment with different treatments;
FIG. 20 is a graph showing the trend of distal tumor volume over time after treatment with different treatments;
FIG. 21 is a graph showing survival curves of mice treated with different treatments;
FIG. 22 shows CD80 and CD86 dendritic cell content in lymph nodes treated by different treatments;
FIG. 23 is the CD8+ T cell content of spleen cells after treatment with different treatments;
fig. 24 shows Treg cell content in spleen cells after treatment with different treatments;
FIG. 25 is an immunohistochemical evaluation of lung tissue sections after different treatment modalities for a lung metastasis model;
FIG. 26 is an evaluation of memory T cells from the spleen of mice after treatment without treatment.
Detailed Description
The invention provides an aggregation-induced emission material, a preparation method thereof and a construction method of an optical nano vaccine, and the invention is further described in detail below in order to make the purposes, technical schemes and advantages of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
4, 4-Dioctyloxy-4-bromo-triphenylamine (579 mg,1 mmol) was weighed into a sealed tube, 10mL of anhydrous tetrahydrofuran was added under nitrogen, and cooled to-78 ℃. N-butyllithium (1.2 mmol) was then added dropwise and reacted at this temperature for one hour, followed by tributyltin chloride (399mg, 1.2 mmol) added dropwise, warmed to room temperature, and reacted overnight. After the reaction is finished, the potassium fluoride solution is quenched for reaction, extracted for three times by methylene dichloride, dried by sodium sulfate, filtered by suction, and the solvent is removed to obtain a tin reagent product which is directly used for the next reaction without other purification steps.
Adding the tin reagent product into a sealed tube, and weighing 4, 7-dibromobenzo [1,2-c:4,5-c ]']Bis ([ 1,2, 5)]Thiadiazole) (141 mg,0.4 mmol) and tetrakis triphenylphosphine palladium (92.4 mg,0.08 mmol) were added to 5mL of anhydrous toluene as solvent and reacted under reflux under nitrogen overnight. After the reaction is finished, the potassium fluoride solution is quenched for reaction, dichloromethane is used for extraction for three times, sodium sulfate is used for drying, and the solvent is removed after suction filtration. The product was purified by chromatography on a column eluting with petroleum ether/dichloromethane (v/v, 1/1) to give the compound TPA-BBTD (122 mg) as a blue powder in 25% yield. 1 H NMR(400MHz,THF-d 8 )δ8.26(d,J＝9.0Hz,4H),7.19(d,J＝8.9Hz,8H),7.06(d,J＝9.0Hz,4H),6.93(d,J＝9.0Hz,8H),4.00(t,J＝6.4Hz,8H),1.86–1.79(m,8H),1.60–1.35(m,40H),0.98–0.92(m,12H).
The synthetic route is as follows:
example 2
The tin reagent product was obtained by the synthetic method of example 1. Tin reagent (2 mmol), 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole (307 mg,0.8 mmol), and tetrakis triphenylphosphine palladium (185 mg,0.16 mmol) were added separately to the vial, followed by 15mL of anhydrous toluene and reacted under nitrogen overnight. After the reaction, the potassium fluoride solution was quenched to react, extracted with methylene chloride three times, dried over sodium sulfate, filtered off with suction, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by a column chromatography with petroleum ether/methylene chloride (v/v, 1/1) as eluent to give intermediate 3 (784 mg) as a purple powder in 80% yield.
Intermediate 3 (490 mg,0.4 mmol), activated iron powder (447 mg,8 mmol) was added to a three-necked flask, acetic acid (20 mL) was added after three nitrogen substitutions, then reacted at 80℃for 6 hours, the reaction mixture was poured into water after the reaction was completed, dichloromethane was extracted three times, dried over sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure after suction filtration to give a pale yellow solid which was used directly for the next reaction without further purification.
Selenium dioxide (222 mg,2 mmol) was added to a three-necked flask, and after three nitrogen substitutions chloroform (15 mL) and ethanol (15 mL) were added. After the completion of the reflux reaction, the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by chromatography using petroleum ether/dichloromethane (v/v, 1/1) as eluent to give the compound TPA-BBSD (156 mg) as a green powder in 30% yield. Nuclear magnetism and mass spectrum are characterized as follows: 1 H NMR(400MHz,THF-d 8 )δ8.26(d,J＝9.0Hz,4H),7.19(d,J＝8.9Hz,8H),7.06(d,J＝9.0Hz,4H),6.93(d,J＝9.0Hz,8H),4.00(t,J＝6.4Hz,8H),1.86–1.79(m,8H),1.60–1.35(m,40H),0.98–0.92(m,12H).
the synthetic route is as follows:
example 3
N, N-diphenyl-4- (tributylstannyl) -aniline (1064 mg,2 mmol), 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole (307 mg,0.8 mmol), and tetrakis triphenylphosphine palladium (185 mg,0.16 mmol) were added separately to the tube, followed by 15mL of anhydrous toluene and reacted overnight under nitrogen. After the reaction, the potassium fluoride solution was quenched to react, extracted with dichloromethane three times, dried over sodium sulfate, filtered off with suction, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by a column chromatography with petroleum ether/dichloromethane (v/v, 1/1) as eluent to give intermediate 4 (433 mg) as a purple powder in 76% yield.
Intermediate 4 (284 mg,0.4 mmol), activated iron powder (447 mg,8 mmol) was added to a three-necked flask, nitrogen was purged three times and acetic acid (20 mL) was added, followed by reaction at 80℃for 6 hours, after the reaction was completed, the reaction mixture was poured into water, dichloromethane was extracted three times, dried over sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure after suction filtration to give a pale yellow solid which was used directly for the next reaction without further purification.
Selenium dioxide (222 mg,2 mmol) was added to a three-necked flask, and after three nitrogen substitutions chloroform (15 mL) and ethanol (15 mL) were added. After the completion of the reflux reaction, the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by chromatography using petroleum ether/dichloromethane (v/v, 1/1) as eluent to give the compound TPA-BBSD (79 mg) as a green powder in 25% yield.
The synthetic route is as follows:
example 4
4-bromo-N, N-bis (4- (1, 2-triphenylvinyl) phenyl) aniline (1662 mg,2 mmol) was weighed into a tube-sealed, 10mL of anhydrous tetrahydrofuran was added under nitrogen protection, and cooled to-78 ℃. N-butyllithium (2.4 mmol) was then added dropwise and reacted at this temperature for one hour, followed by tributyltin chloride (782 mg,2,4 mmol) added dropwise, warmed to room temperature and reacted overnight. After the reaction is finished, the potassium fluoride solution is quenched for reaction, extracted for three times by methylene dichloride, dried by sodium sulfate, filtered by suction, and the solvent is removed to obtain a tin reagent product which is directly used for the next reaction without other purification steps.
The above afforded tin reagent product (2 mmol), 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole (307 mg,0.8 mmol), and tetrakis triphenylphosphine palladium (185 mg,0.16 mmol) were added separately to the tube seals, followed by 15mL of anhydrous toluene and reacted under nitrogen overnight. After the reaction, the potassium fluoride solution was quenched to react, extracted with dichloromethane three times, dried over sodium sulfate, filtered off with suction, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by a column chromatography with petroleum ether/dichloromethane (v/v, 1/1) as eluent to give intermediate 4 (899 mg) as a purple powder in 65% yield.
Intermediate 6 (692 mg,0.4 mmol), activated iron powder (447 mg,8 mmol) was added to a three-necked flask, acetic acid (20 mL) was added after three nitrogen substitutions, then reacted at 80℃for 6 hours, the reaction mixture was poured into water after the reaction was completed, dichloromethane was extracted three times, dried over sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure after suction filtration to give a pale yellow solid which was used directly for the next reaction without further purification.
Selenium dioxide (222 mg,2 mmol) was added to a three-necked flask, and after three nitrogen substitutions chloroform (15 mL) and ethanol (15 mL) were added. After the reflux reaction was completed, the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by chromatography using petroleum ether/dichloromethane (v/v, 1/1) as eluent to give TPE-TBBSD (118 mg) as a green powder in 17% yield.
The synthetic route is as follows:
the aggregation-induced emission material prepared in the above example was subjected to the following experiment:
molecular photophysical properties and photothermal/photodynamic characterization:
1. absorption and emission spectra:
TBBSD and TBBTD were dissolved in THF to prepare 10 μm, and ultraviolet absorption (shimadzu UV-1600 i) and fluorescence emission spectroscopy (edinburgh fluorescence spectrometer) were performed, as shown in fig. 1, with absorption peaks of 816nm and 760nm, respectively, and as shown in fig. 2, with emission spectra peaks of TBBSD and TBBTD: both 1120nm and 1050nm, both molecules have significant light emitting properties in the near infrared two region.
Aie curve test:
the TBBSD and TBBTD solutions (10 μm) of THF/isopropanol mixed solvents with isopropanol contents ranging from 0% to 90% were prepared and respectively subjected to emission spectrum tests, as shown in fig. 3, which shows the emission spectra of TBBSD molecules in solvents with different isopropanol contents, and fig. 4, which shows the variation curves of emission peak intensities of different isopropanol ratios, and after 50% isopropanol content was increased, the fluorescence intensity was gradually increased, exhibiting AIE properties.
3. Preparation, characterization and absorption emission spectrum of nanoparticles
500. Mu.L of a 1mg/mL THF solution of TBBSD was slowly added dropwise to a 1mg/mL BSA aqueous solution in a vortexing state, vortexing was continued for 1min after completion of the dropwise addition, and the prepared product was dialyzed in pure water for 24 hours, and then concentrated using an ultrafiltration centrifuge tube to complete preparation of TBBSD-NPs. An aqueous solution with a nano concentration of 100 mug/mL was prepared for dynamic light scattering test, as shown in FIG. 5, the particle size of the nano particles was about 120nm, and the nano particles had good dispersibility (shown in FIG. 6). And preparing the nano particles with TBBSD concentration of 10 mu M, and carrying out absorption and emission spectrum tests, wherein the ultraviolet absorption peak value of the nano particles is 786nm, the emission spectrum peak value is 1060nm, and the nano particles still have excellent near infrared two-region emission properties.
4. Photothermal performance test
TBBSD-NPs were prepared in a concentration gradient of 0. Mu.M to 30. Mu.M, the dispersion was water, and the volume was 100. Mu.L, at a power of 1W cm -2 Under 808nm laser irradiation, real-time temperature detection is carried out by using a thermal imager. As shown in FIG. 7, as the nanoparticle concentration increases, the photothermal effect gradually increases, and the temperature can be raised to 65℃or higher within 5 minutes at a concentration of 30. Mu.M. As shown in FIG. 8, 10. Mu.M was selected as the test concentration, and the power of the laser was changed from 0.5W cm -2 Is increased to 1.5W cm -2 The photo-thermal effect is gradually improved and is 1.5W cm -2 Under the power, the temperature can reach 70 ℃ within 5min, and the light and heat performance is excellent. 30. Mu.M TBBSD-NPs at 1W cm -2 And (3) carrying out illumination for 6min under the illumination of power, closing and cooling for 6min, carrying out illumination again, and carrying out photo-thermal stability exploration after 6 times of circulation, wherein the nano particles still show stable photo-thermal performance under the illumination of 6 circulation as shown in figure 9. The photothermal conversion efficiency was calculated by analyzing the temperature rise and fall curves of 10. Mu.M TBBSD-NPs, and as shown in FIG. 10, the photothermal conversion efficiency of TBBSD-NPs was 43.2%.
5. Total ROS Performance test
Detection of 808nm laser (1W cm) using 2',7' -dichlorofluorescein diacetate (DCFH-DA) as ROS indicator -2 ) ROS production of AIE molecules in solution under irradiation. First, an ethanol solution (1 mM,0.5 mL) of DCFH-DA was added to 2mL of NaOH solution (0.01M), and stirred at room temperature for 30 minutesThe DCFH-DA was hydrolyzed to DCFH, and the hydrolysate was then neutralized with 10mL of 1 XPBS at pH 7.4 to give an activated ROS indicator (40. Mu.M, 12.5 mL), which was stored at 4℃in the dark for use. The activated ROS indicator (40. Mu.M) was then mixed with AIE molecules in PBS to give final ROS indicator and AIE molecules at 10. Mu.M, and after irradiation with 808 laser for various times, the fluorescence intensity of 2',7' -dichlorofluorescein triggered by the generation of ROS by AIE molecules was detected by fluorescence spectrometer (Edinburgh FS 5) to reflect ROS generation. Excitation wavelength was 488nm and fluorescence signal was collected in the range of 490-600 nm. As shown in FIG. 11, DCFH and nanoparticles alone showed little fluorescence, and after TBBSD-NPs and TBBSD-NPs were added, DCFH fluorescence gradually increased with the increase of irradiation time, demonstrating that both TBBSD-NPs and TBBSD-NPs were able to efficiently generate ROS under 808 laser irradiation.
Animal and cell experiments:
1. in vitro cytotoxicity experiments
Cytotoxicity of TBBSD-NPs was evaluated by measuring viability of hepatoma cells (Hepa 1-6 cell line) under light/dark conditions. Briefly, an appropriate amount of the Hepa1-6 cells in the logarithmic growth phase was cultured in a 96-well plate (about 5,000 cells/well) and cultured for 12 hours. Subsequently, the cell culture medium was changed to new TBBSD-NPs containing different TBBSD concentrations (0.2, 0.5, 1,2,5, 10, 25, 50 and 100. Mu.g mL -1 ) And the cells were further cultured for 12 hours. Phototoxic groups after incubation, after 5 minutes of irradiation with 808nm laser per well (power: 2Wcm -2 ) Surviving cells were detected with CCK-8 reagent (Dojindo Molecular Technologies, japan) and cell viability was calculated from absorbance. The dark toxicity group was directly tested for cell viability with CCK-8 reagent after incubation. As shown in FIG. 12, the concentration of the catalyst in the solution is 0-100. Mu.g mL -1 In the concentration range, the activity of the cells is not affected basically under the condition of no illumination, and in the illumination group, the activity of the cells is reduced sharply and the cells show stronger phototoxicity.
Apoptosis experiments: inoculating appropriate amount of Hepa1-6 cells in logarithmic growth phase into 6-well plate (about 50,000 cells/well), culturing for 12 hr, adding TBBSD-NPs into the well, and concentrating TBBSD to 50 μg mL -1 . After 12 hours, the mixture was irradiated with 808nm laser (2W cm -2 ) Cells were irradiated for 5 min. Cells were digested with pancreatin without EDTA and collected. Cells were stained with Annexin V-FITC/PI apoptosis assay kit and detected with flow cytometry. As shown in FIG. 13, apoptosis increased dramatically in the nanoparticle-illuminated experimental group, while the other control groups had substantially unaffected cell activity
2. In vitro ICD experiments
An appropriate amount of the Hepa1-6 cells in the logarithmic growth phase was cultured in a glass bottom dish for 24 hours (about 50,000 cells/well), and then divided into four groups: 1) PBS; 2) PBS+L; 3) TBBSD-NPs; 4) TBBSD-NPs+L; the TBBSD concentration of groups 3 and 4 was 50. Mu.g/mL. After washing twice with the culture solution, the cells of group 2 and group 4 were subjected to 808nm laser irradiation (2W cm -2 ) For 5 minutes and then incubated at 37℃for a further 30 minutes. Each group of cells was fixed and labeled and stained with anti-CRT, anti-HMGB1 and anti-HSP70 antibodies, and then imaged with laser Confocal (CLSM) (Olinbas FV 3000). Conditions are as follows: excitation wavelength: DAPI is 405nm, the green fluorescent secondary antibody is 488nm, and the red fluorescent secondary antibody is 561nm; transmitting: DAPI 430-470nm, green fluorescent secondary antibody 500-540nm, red fluorescent secondary antibody 550-650nm. As shown in fig. 14, the expression levels of the cell immunogenicity-related factors CRT, HMGB1 and HSP70 in the nanoparticle-illuminated experimental group all showed significant differences compared to the other control groups, indicating that the photothermal treatment induced the immunogenic death of the cells.
3. In vitro DC activation
Mouse bone marrow derived dendritic cells (BM-DCs) were derived from B6 mice. Immature BM-DCs (4X 10) 6 ) Cultured in RPMI-1640 (GIBCO) supplemented with FBS (10%, GIBCO), 2-mercaptoethanol (2-ME, 0.8ng/mL, sigma Aldrich) and mouse granulocyte macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF,20ng/mL, peproTech) for 72 hours. Thereafter, BM-DC (5X 10) 5 Individual cells/well, placed in 6-well plates) were treated as follows: 1) A PBS group; 2) NPs group; 3) Nps+gpc3 group; 4) Nps+gpc3+amp group; 5) Nps+gpc3+amp+l supernatant group. Wherein the TBBSD concentration is 50. Mu.g mL -1 GPC3 concentration was 2 μg mL -1 AMP concentrationDegree of 25. Mu.g mL -1 The ratio of L supernatant was 1:100 (v/v). The L supernatant was derived from the supernatant of Hepa1-6 cells after laser irradiation (10,000 cells/mL medium, 808nm laser, power 2W cm) -2 Irradiating for 5 min). After 24h of co-incubation, cells were collected. And labeling the cells with the following antibodies: anti-CD11c-FITC, anti-CD80-PE, anti-CD86-APC (eBioscience). Differentiation and maturation of each group of BM-DCs was examined using a flow cytometer. Flow cytometry was performed using a BD Flow cytometer, and Flow-jo software (Tree Star, inc.) was used as the Flow analysis result. As shown in fig. 15, it was found from the results of flow analysis that the expression of CD86 and CD80 was significantly increased in the treated DC cells using the nanoparticle+gpc 3 antigen+amp adjuvant+cell supernatant of the light irradiation group, indicating that the photothermal treated cell supernatant was able to activate the DC cells in vitro.
3. In vivo two-zone fluorescence, photoacoustic and photothermal imaging
Male C57BL/6J mice (14+ -2 g,5-6 weeks old) were purchased from Guangzhou Jiuzhikang animal research center and selected to establish xenograft Hepa1-6 tumor models. Mice were given fresh food and water free daily and were fed adaptively for at least 7 days prior to the experiment. Hepa1-6 cells (5X 10) suspended in PBS 6 ) The injection was performed subcutaneously in the right posterior hip of mice. After about 7-10 days, the tumor is fully established.
TBBSD-NPs (100. Mu.L, TBBSD 5mg/kg body weight) were injected into Hepa1-6 xenograft tumor mice via the tail vein. The mice were then subjected to two-zone fluorescence imaging and photoacoustic imaging at different time points (0, 6, 12, 24 and 36 hours) after NPs injection. Two-zone fluorescence was acquired with a commercial Series II 900/1700 imaging system and photoacoustic imaging was acquired with a LOIS-3D photoacoustic imager (Tomoving). Two-zone fluorescence imaging uses 808nm excitation and a 1000nm filter. Photoacoustic imaging uses 808nm excitation at a frequency of 21MHz. As shown in fig. 16, the fluorescence two-zone image of the mice showed a clear signal after 6 hours of administration, and the tumor site signal was highest after 24 hours of administration. As shown in fig. 17, the tumor part of the mouse shows a photoacoustic signal after 6 hours of administration, reaches the highest value after 24 hours of administration, and is consistent with the trend of the two-region fluorescence signal, which indicates that the TBBSD-NPs can effectively realize fluorescence and photoacoustic imaging of the tumor part of the mouse.
In vivo photothermographic imaging, after 24h intravenous injection of TBBSD-NPs (100. Mu.L, TBBSD 5mg/kg body weight), tumor sites were monitored with FLIR E60 camera using 808nm laser (1.5W cm) -2 ) Temperature change after 5min of irradiation. Mouse tumors were intravenously injected with physiological saline (100. Mu.L) under the same laser irradiation conditions as a control. As shown in fig. 18, the temperature of the tumor part of the mice with the nanoparticle group gradually increased with the irradiation time within 5min, while the temperature of the physiological saline group increased little with the irradiation time after 24 hours of administration, which indicates that TBBSD-NPs can generate obvious photo-thermal treatment effect on the living animal body under the irradiation of 808nm laser.
4. Therapeutic effect of light nanometer vaccine on mouse liver cancer
When the tumor size reached-100 mm3, mice were randomly divided into 5 groups and treated differently (n=6/group): 1) Physiological saline; 2) Nps+gpc3; 3) Nps+gpc3+amp; 4) Nps+gpc3+l; 5) Nps+gpc3+amp+l. Wherein TBBSD is 5mg/kg body weight, GPC3 is 0.5mg/kg body weight, AMP is 2.4mg/kg body weight. NPs were administered by tail vein injection, and GPC3 and AMP were administered by subcutaneous injection. NPs were administered once on day 0 and GPC3 and AMP were administered three times on day 4,9,14. Groups 4 and 5 received photothermal treatment on day 1 (1.5W cm 24 hours after NPs injection) -2 Near infrared radiation at 808nm for 5 min). Distal tumors (5X 10) were inoculated subcutaneously at the left posterior hip on day 15 6 Cells). Tumor size was measured and recorded for each group, and survival rate was counted for each group. The relative volume of the tumor was calculated as v=ab 2 And/2, wherein a and b represent maximum and minimum diameters, respectively. As shown in fig. 19, nps+gpc 3+amp+light treated mice had essentially no in situ tumor volume and had no signs of recurrence as time increased, while the tumor volumes of the other control mice all increased gradually after treatment, exhibiting a tendency to recurrence. As shown in FIG. 20, the growth of the subcutaneous distal tumors of the NPs+GPC 3+AMP+light treated mice can be effectively inhibited, while the subcutaneous distal tumors of the other mice in the control group all show different degrees of growth tendency. Indicating that the mice treated by the phototherapy and the vaccine can not only realize the effective treatment of in-situ tumorCan also form immune memory and can realize effective killing of newly implanted remote tumors. As shown in fig. 21, the effective survival of nps+gpc 3+amp+light treated mice reached 100% over 50 days, significantly higher than the other control groups.
5. In vivo immune index detection
Each of the above groups of mice was sacrificed on day 21. Immediately, mice were immersed in 75% alcohol solution for 3 minutes, and lymph nodes and spleens were surgically removed and collected. For lymph nodes, digestion was stopped by adding medium containing FBS after digestion with cathepsin at 37℃for 10 min. After washing the cells 2 times with PBS, the DC cells were stained with anti-CD11c-FITC, anti-CD80-PE and anti-CD86-APC (eBioscience). Cells of cd11c+cd80+cd86+ are defined as mature dendritic cells. As shown in FIG. 22, the lymph nodes of NPs+GPC 3+AMP+light treated mice were significantly higher than other control groups, demonstrating that light and vaccine treated mice were effective in activating the autoimmune system.
For spleen, it was chopped into small pieces with scissors. The cells were passed through a 70 μm cell filter with 2mL of PBS buffer and collected from a 50 mL centrifuge tube. An additional 3mL RBC lysate was added to the centrifuge tube and left at room temperature for 20 minutes to lyse the RBCs. The reaction was stopped by adding 5mL of medium containing FBS. Cells were then washed twice with PBS. Spleen lymphocytes were then labeled as follows: anti-CD3-APC, anti-CD4-FITC and anti-CD8a-PE/anti-Foxp3-PE antibodies (Thermo Fisher Scientific). The detection was performed using a BD Flow cytometer, and the detection results were analyzed using Flow-jo software. As shown in fig. 23, cd8+ T cells were significantly increased in spleens of nps+gpc 3+amp+light treated mice, demonstrating that T cells could be activated by photothermal and vaccine treatment, and as well, as shown in fig. 24, immunosuppressive cell Treg cells of nps+gpc 3+amp+light treated mice were significantly decreased, further demonstrating activation of immune system, demonstrating the effectiveness of photothermal tumor vaccine.
6. Tumor lung metastasis treatment
Male C57BL/6J mice (14.+ -.2 g,5-6 weeks old) were purchased from Guangzhou Jiuzhikang animal research center. The mice were provided with fresh food and water free daily, andthe animals were housed adaptively for at least 7 days prior to the experiment. Hepa1-6 cells (5X 10) suspended in PBS 6 ) The injection was performed subcutaneously in the right posterior hip of mice. After about 7-10 days, the subcutaneous tumor is fully established. When the tumor size reaches 100mm 3 At this time, mice were randomly divided into 5 groups and subjected to different treatments (n=6/group): 1) Physiological saline; 2) Nps+gpc3; 3) Nps+gpc3+amp; 4) Nps+gpc3+l; 5) Nps+gpc3+amp+l. Wherein TBBSD is 5mg/kg body weight, GPC3 is 0.5mg/kg body weight, AMP is 2.4mg/kg body weight. NPs were administered by tail vein injection, and GPC3 and AMP were administered by subcutaneous injection. NPs were administered once on day 0 and GPC3 and AMP were administered three times on day 4,9,14. Groups 4 and 5 received photothermal treatment on day 1 (1W cm 24 hours after NPs injection) -2 Near infrared radiation at 808nm for 5 min). Hepa1-6 cells (5X 106 cells/min.) were injected by tail vein on day 15. Each of the above groups of mice was sacrificed on day 21. Immediately, mice were immersed in 75% alcohol for 3 minutes, taken out by surgery and spleens were collected and chopped into small pieces with scissors. The cells were passed through a 70 μm cell filter with 2mL of PBS buffer and collected from a 50 mL centrifuge tube. An additional 3mL RBC lysate was added to the centrifuge tube and left at room temperature for 20 minutes to lyse the RBCs. The reaction was stopped by adding 5mL of medium containing FBS. Cells were then washed twice with PBS. Spleen lymphocytes were then labeled as follows: anti-CD3-APC, anti-CD44-FITC and anti-CD62L-PE antibodies (Thermo Fisher Scientific, USA) were stained. Tcm memory T cells cd3+cd44+cd62+. As shown in fig. 25, in the different treatment groups, lung tissue sections of nps+gpc 3+amp+light-treated mice were in a normal state, no lung metastasis was present, while other control groups had lung metastasis, indicating that the photothermal tumor vaccine was effective in inhibiting tumor metastasis. Further evaluating the immune memory effect of the vaccine, the spleen memory T cells of the mice treated by NPs+GPC 3+AMP+light are obviously increased, which proves that the mice treated by the photo-thermal tumor vaccine can form immune memory and effectively inhibit the recurrence and metastasis of tumors.
7. Ethical approval and consent participation
The study was in accordance with the ethical guidelines of the revision 2013 of the declaration of helsinki. The laboratory animals (and university of south China) were subjected to the ethical committee approval (and university of south China) using the rat laboratory according to the standard guidelines approved by the animal welfare committee.
8. Statistical analysis
All data are expressed as mean ± standard deviation, unless otherwise indicated. Statistical differences between treatment and control groups were analyzed by single or two-factor anova and Tukey post-hoc test (multiple comparisons) using graphpad prism version 8.0 software. The survival data were tested using log-rank. * Indicating that the difference is statistically significant, p <0.05; * P <0.01; and p <0.001.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (11)
1. An aggregation-induced emission material, characterized in that the aggregation-induced emission material has a structural formula:
wherein X1 and X2 are independently selected from one of S, se; r1 and R2 are independently selected from H,/-and-> One of them.
2.A method of preparing an aggregation-induced emission material, the method comprising:
dispersing a tin reagent product, 4, 7-dibromobenzo [1,2-c:4,5-c' ] bis ([ 1,2,5] thiadiazole) and palladium tetraphenylphosphine into an organic solvent to obtain a first mixed solution;
heating and refluxing the first mixed solution in an inert atmosphere, overnight, quenching reaction, and purifying to obtain the aggregation-induced emission material;
the structural formula of the tin reagent product is as follows:
wherein, R1 and R2 are independently selected from H,/-and-> One of them.
3. A method of preparing an aggregation-induced emission material, the method comprising:
dispersing a tin reagent product, 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole and tetraphenylphosphine palladium into an organic solvent to obtain a second mixed solution;
heating and refluxing the second mixed solution at 110-115 ℃ in inert atmosphere, quenching reaction overnight, and purifying to obtain an intermediate product;
mixing the intermediate product with iron powder and acetic acid under the protection of inert gas, and reacting to obtain a reaction solution; cooling and extracting the reaction liquid to obtain solid powder;
mixing the solid powder with tin dioxide to obtain a mixture, adding chloroform and ethanol into the mixture in an inert atmosphere, and carrying out reflux reaction at 70-75 ℃ to obtain the aggregation-induced emission material;
the structural formula of the tin reagent product is as follows:
wherein, R1 and R2 are independently selected from H,/-and-> One of them.
4. A method of preparing an aggregation-induced emission material according to claim 2 or claim 3, wherein the method of preparing a tin reagent product comprises:
adding 4, 4-dioctyloxy-4-bromo-triphenylamine into a reaction container, adding anhydrous tetrahydrofuran into the reaction container under the protection of nitrogen, and cooling to-78-80 ℃;
dropwise adding n-butyllithium into the reaction container, reacting for 60-70min, dropwise adding tributyl tin chloride into the reaction container, heating to room temperature, and reacting overnight; quenching reaction, and purifying to obtain the tin reagent product.
5. A method of preparing an aggregation-induced emission material according to claim 3, wherein the intermediate product has the formula:
6. a method of producing an aggregation-induced emission material according to claim 2 or 3, wherein the organic solvent comprises: toluene, tetrahydrofuran, dioxane and xylene.
7. A method of producing an aggregation-induced emission material according to claim 3, wherein the molar ratio of the intermediate product to the iron powder is 1:20.
8. The method of producing an aggregation-induced emission material according to claim 2, wherein the molar ratio of the tin reagent product, 4, 7-dibromobenzo [1,2-c:4,5-c' ] bis ([ 1,2,5] thiadiazole), and tetrakis triphenylphosphine palladium is 1:0.4:0.08.
9. A method of preparing an aggregation-induced emission material according to claim 3, wherein the molar ratio of tin reagent product, 4, 7-dibromo-5, 6-dinitrobenzo [ c ] [1,2,5] thiadiazole and palladium tetraphenylphosphine is 1:0.4:0.08.
10. An optical nanovaccine based on aggregation-induced emission material, comprising: aggregation-inducing luminescent material and tumor-specific antigen; the aggregation-induced emission material according to claim 1.
11. The optical nano vaccine according to claim 10, wherein bovine serum albumin is used as a carrier to load AIE molecules to construct phototherapy nano particles, liver cancer specific antigen GPC3 and AIE molecules are used to construct nano vaccine particles, and the combined action of STING agonist c-d-AMP is used to activate immunity.
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