Dual-wavelength-emission responsive optical micelle as well as preparation method and application thereof

Abstract

The invention provides a responsive optical nano-micelle with dual-wavelength emission, a preparation method and an application thereof. Based on tetraphenyl ethylene carrying two amino groups, a phenylboronic acid-containing chain segment and an acrylic acid unit are introduced through controlled polymerization, and an iridium complex is further esterified to prepare a segmented copolymer with dual-wavelength emission; and assembling the boric acid ester bond and hyaluronic acid to prepare the size-adjustable nano-micelle. The optical nano-micelle disclosed by the invention is good in biocompatibility and excellent in cancer cell targeting property, and the tetraphenyl ethylene and the iridium complex respectively endow the optical nano-micelle with optical properties of visible light emission and near-infrared region emission. Besides, the borate bond and the iridium complex involved in the micelle structure provide environmental responsiveness of near-infrared emission to pH and hypoxia, so that the micelle has a signal enhancement function during disease detection, and meanwhile, the emission ratio of two wavelengths generated by environmental stimulation is beneficial to real-time, positioning and nondestructive monitoring of a state. The nano-micelle provided by the invention has great application potential in the field of biological accurate detection.

Description

Technical Field

[0001] The present invention belongs to the field of polymer material nanotechnology, and particularly relates to a preparation method and application of dual-wavelength emission nanomicelles with a structural design endowing cancer cell targeting and environment-responsive functions. Technical Background

[0002] Hyaluronic Acid (HA) is a natural polysaccharide and a major component of the extracellular matrix, with biocompatibility, anti-adhesion, biodegradability, and non-immunogenicity. HA is also well-known to be adapted to cell surface receptors such as CD44 and CD168 (Hyaluronan-mediated motility receptor, RHAMM), and Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Moreover, the expression levels of CD44, hyaluronidase, and oxidative stress are closely related to the development of many types of diseases, such as cancer, rheumatoid arthritis, and traumatic brain injury, etc. (Advanced Materials, 2019, 1803549). Given its excellent physical, chemical, and biological properties, hyaluronic acid is conducive to the construction of various biofunctional materials, especially the design and development of nano-integrated platforms for efficient tumor diagnosis and treatment have attracted much attention. In addition, industrial players such as Roche Group of Switzerland, Genzyme Corporation of the United States, and LG Life Sciences have shown great interest in using hyaluronic acid in diagnostic and therapeutic preparations, indicating its great significance in the development of anti-cancer diagnostic and therapeutic systems.

[0003] To better explore topics in the field of life sciences, such as the prevention, diagnosis, and timely treatment of life-related diseases, optical imaging has become an indispensable and important means. However, when optical imaging is used for biological monitoring and disease diagnosis and treatment, its own characteristics also pose some problems that need to be solved: the tissue penetration ability of common emitted light is weak, there is background fluorescence interference in the complex biological environment, and it is difficult to quantify the probe signal during detection, etc. Therefore, optimizing imaging technology and designing and preparing highly sensitive and high signal-to-noise ratio integrated detection preparations have become the focus of attention in the scientific and industrial communities. Dual-wavelength optical probes have become a breakthrough point in the research and development of bioimaging preparations in recent years because they can be excited to emit light of two colors in cells and tissues. Compared with conventional optical probes, the background signal interference during the detection process can be corrected by the optical intensity ratio of the dual-wavelength channels, and the obtained detection signal has a high contrast and more accurate results. In addition, probes with dual-wavelength emission can perform multi-scene and dynamic evolution imaging from cells to tissues, and have important application potential in the efficient detection of abnormal cells and their tissues.

[0004] Signal molecules that can be used for bioimaging have been widely developed, including inorganic nanoparticles (quantum dots, noble metal clusters, carbon dots, two-photon emission particles), organic dye molecules, aggregation-induced emission molecules, metal complexes, etc. The emission wavelengths cover a broad range from visible light to the far-infrared light region. In order to improve the biocompatibility, optical stability of imaging probes, reduce the non-specific clearance performance in vivo and the lesion targeting ability during biological detection, an effective method is to construct polymer nanoparticles carrying optical signal molecules by covalent or non-covalent interactions, with the particle size controlled at about 100 nm. Common non-covalent interactions include electrostatic interactions, hydrogen bond interactions, coordination interactions and host-guest interactions, etc. Covalent interactions include reversible bonds such as borate ester bonds and imine bonds in addition to covalent blocks. Non-covalent and reversible covalent interactions are beneficial to the enhancement of detection signals under microenvironment stimuli. On the other hand, as a complex physical and biochemical system, lesion tissues exhibit abnormal physiological environments different from normal tissues. For example, tumor tissues have microenvironments such as weak acidity, hypoxia, and abnormal reactive oxygen and nitrogen species, and they also overexpress some specific proteins and enzymes. Therefore, polymer nanosensors constructed based on non-covalent or reversible covalent interactions can achieve the amplification of detection signals under the stimulation of abnormal microenvironments at lesion sites, and thus effectively complete the early and accurate diagnosis of diseases. Summary of the Invention

[0005] In view of the above, the present invention selects tetraphenylethylene with aggregation-induced emission function, iridium complex with long-lived luminescence, and tumor-active targeting hyaluronic acid to assemble and prepare responsive optical nanomicelles with dual-wavelength emission. Integrating tetraphenylethylene emitting in the visible light region and iridium complex emitting in the near-infrared light region into the micelle particles can meet the requirements of dual-channel signal collection for ratio luminescence imaging. At the same time, it can be used for detection and diagnosis in multi-scenarios with different depths (such as cells and tissues).

[0006] To achieve the above object, the technical solution adopted by the present invention is: a preparation method of responsive optical micelles with dual-wavelength emission, comprising the following steps:

[0007] (1) Prepare a dithioester-terminated tetraphenylethylene double-chain molecule TPE-PCL-CTA

[0008] Dissolve 1.4 - 1.5 g of 1,2 - diphenyl - 1,2 - bis(4 - aminophenyl)ethylene TPE - 2AM in a reaction flask containing 15 - 25 mL of dry toluene. Then, sequentially add 10 - 15 mL of ε - caprolactone and 0.02 - 0.05 mL of stannous octoate. Heat to 100 - 110 °C under nitrogen protection and stir for 40 - 48 hours. After the reaction is completed, cool to room temperature. First, add dichloromethane for dissolution, then precipitate in methanol. Filter out the precipitate and wash it 3 - 5 times by the dissolution - precipitation method with dichloromethane / methanol to obtain a light yellow solid a. Place it in a vacuum drying oven and dry at 60 - 80 °C to constant weight for later use;

[0009] Dissolve 0.5 - 0.6 g of the dried light yellow solid a in 5 - 8 mL of toluene to obtain solution A. Dissolve 48 - 50 mg of 4 - dimethylaminopyridine and 0.19 - 0.2 g of 2 - [(ethoxythiocarbonyl)thio]propanoic acid in 5 - 8 mL of dichloromethane to obtain solution B. Dissolve 0.2 - 0.3 g of N,N - dicyclohexylcarbodiimide in 5 - 8 mL of dichloromethane to obtain solution C. First, place solution A and solution B in a reaction flask, mix them evenly and stir, then slowly inject solution C, and continue to stir at room temperature for 36 - 40 h. After the reaction is completed, add methanol dropwise for precipitation under stirring. Filter out the precipitate and wash it 3 - 5 times by the dissolution - precipitation method with dichloromethane / methanol to obtain solid b. Place it in a vacuum drying oven and dry at 60 - 80 °C to constant weight, which is the disulfide - terminated tetraphenylethylene double - chain molecule TPE - PCL - CTA for later use;

[0010] (2) Preparation of long - chain block copolymer TPE - P(CL - APBA) - AA

[0011] Add 0.3 - 0.5 g of the product TPE - PCL - CTA prepared in step (1), 280 - 300 mg of 3 - acrylamidophenylboronic acid (APBA), and 4 - 4.2 mg of azobisisobutyronitrile into a reaction flask containing 3 mL of N,N - dimethylformamide DMF in sequence. Heat to 60 - 80 °C under nitrogen protection and stir for 20 - 24 hours. After the reaction is completed, cool to room temperature. First, add 1 - 2 mL of methanol to dissolve the reactants, then add an appropriate amount of cold ether dropwise for precipitation under stirring. Filter out the precipitate and wash it 3 - 5 times with cold ether to obtain solid c. Place it in a vacuum drying oven and dry at 60 - 80 °C to constant weight for later use;

[0012] 0.4 - 0.5 g of dried solid c, 13 - 15 μL of acrylic acid, and 4 - 4.2 mg of azobisisobutyronitrile were successively added to a reaction flask containing 3 mL of N,N - dimethylformamide (DMF). Under nitrogen protection, the mixture was heated to 60 - 80 °C and stirred for 20 - 24 hours. After the reaction, it was cooled to room temperature. First, 1 - 2 mL of methanol was added to dissolve the reactants, and then an appropriate amount of cold ether was added dropwise with stirring for precipitation. The precipitate was filtered out and washed 3 - 5 times with cold ether to obtain solid d, which was placed in a vacuum drying oven and dried at 60 - 80 °C to constant weight, namely the long - chain block copolymer TPE - P(CL - APBA) - AA, for use;

[0013] (3) Preparation of the block copolymer Ir - CP with dual - wavelength emission

[0014] 0.3 - 0.5 g of the dried product TPE - P(CL - APBA) - AA prepared in step (2) and 0.14 - 0.15 g of the iridium complex Ir - OH were dissolved in 3 - 6 mL of a DMF / methanol mixed solution to obtain solution D. 3.6 - 3.7 mg of 4 - dimethylaminopyridine was dissolved in 2 - 3 mL of dichloromethane to obtain solution E. 65 - 70 mg of N,N - dicyclohexylcarbodiimide was dissolved in 3 - 5 mL of dichloromethane to obtain solution F. Solution E and solution F were successively added to solution D, mixed evenly and stirred. The reaction was stirred at room temperature for 45 - 48 h. After the reaction, an appropriate amount of cold ether was added dropwise with stirring for precipitation. The precipitate was filtered out and dissolved again with the DMF / methanol mixed solution, and then an appropriate amount of cold ether was added dropwise for precipitation. The red powder e was filtered out and placed in a vacuum drying oven and dried at 60 - 80 °C to constant weight, namely the block copolymer Ir - CP with dual - wavelength emission, for use;

[0015] (4) Preparation of the responsive optical nanomicelles HA / Ir - CP with dual - wavelength emission

[0016] The dried product of the block copolymer Ir - CP with dual - wavelength emission prepared in step (3) was dissolved in an appropriate amount of DMF / methanol mixed solvent to obtain solution G, which was ultrasonicated in an ultrasonic environment of 40 KHz. An aqueous solution of hyaluronic acid was slowly dropped into solution G until the mixed solution gradually became an orange - red emulsion. Ultrasonication was stopped and the mixture was preliminarily filtered, and then poured into a dialysis bag and dialyzed in deionized water for 48 - 50 hours to remove the organic solvents in the system, namely the optical micelles HA / Ir - CP with dual - wavelength emission.

[0017] In step (2), the cold ether was prepared by freezing ether in an ice - water bath for 2 - 3 h.

[0018] In steps (3) and (4), the volume ratio of the DMF / methanol mixed solution was 2:1.

[0019] In step (4), the concentration of solution G is 1 to 15 mg / mL.

[0020] In step (4), the concentration of the aqueous hyaluronic acid solution is 4 to 10 mg / mL, and the preparation method is as follows: dissolve hyaluronic acid in an appropriate amount of deionized water to obtain the aqueous hyaluronic acid solution.

[0021] In step (4), the volume ratio of the aqueous hyaluronic acid solution to solution G is (3.5 - 4):1.

[0022] In step (4), the molecular weight of the dialysis bag is 10,000.

[0023] The responsive optical micelles with dual-wavelength emission prepared by the above method for preparing responsive optical micelles with dual-wavelength emission and their application in tumor-targeted cell imaging.

[0024] Advantages of the present invention: The optical nanomicelles of the present invention have good biocompatibility and excellent cancer cell targeting properties. The tetraphenylethylene and iridium complex endow it with optical properties of visible light and near-infrared emission respectively. In addition, the borate ester bond and iridium complex involved in the micelle structure provide the near-infrared emission with pH and hypoxia environmental responsiveness, enabling it to have a signal enhancement function during disease detection. At the same time, the two-wavelength emission ratio generated by environmental stimulation is conducive to real-time, localization, and non-destructive monitoring of the state. Description of the Drawings

[0025] Figure 1 1H NMR spectrum of the dithioester-terminated tetraphenylethylene double-stranded molecule (TPE-PCL-CTA);

[0026] Figure 2 1H NMR spectrum of the long-chain block copolymer [TPE-P(CL-APBA)-AA];

[0027] Figure 3 1H NMR spectrum of the dual-wavelength emission block copolymer (Ir-CP);

[0028] Figure 4 IR spectra of Ir-CP, hyaluronic acid HA, and the optical micelles HA / Ir-CP prepared in Example 1;

[0029] Figure 5 Hydration particle size change diagram of the optical micelles prepared with different Ir-CP feeding concentrations;

[0030] Figure 6 Hydration particle size change diagram of the optical micelles prepared with different hyaluronic acid solution feeding concentrations;

[0031] Figure 7Transmission electron microscope SEM image of the prepared dual-wavelength emission optical micelle HA / Ir-CP;

[0032] Figure 8 Emission spectrum of the dual-wavelength emission optical micelle HA / Ir-CP in response to pH;

[0033] Figure 9 Emission spectrum of the dual-wavelength emission optical micelle HA / Ir-CP in response to hypoxia;

[0034] Figure 10 Results of in vitro toxicity tests of the dual-wavelength emission optical micelle HA / Ir-CP at different concentrations against various cancer cells;

[0035] Figure 11 Flow cytometry analysis results of the uptake of the optical micelle HA / Ir-CP by different cancer cells. Detailed implementation mode

[0036] The following is a detailed description of the specific implementation mode of the present invention. It should be understood that the specific implementation mode described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0037] Example 1

[0038] (1) Preparation of dithioester-terminated tetraphenylethylene double-stranded molecule TPE-PCL-CTA

[0039] Take 1.46 g of 1,2-diphenyl-1,2-bis(4-aminophenyl)ethylene TPE-2AM and dissolve it in a reaction flask containing 20 mL of dry toluene. Then, add 12 mL of ε-caprolactone and 0.05 mL of stannous octoate in sequence. Heat to 110 °C under nitrogen protection and stir for 48 hours. After the reaction, cool to room temperature. First, add dichloromethane for dissolution, then precipitate in methanol. Filter out the precipitate and wash it 5 times by the dissolution-precipitation method with dichloromethane / methanol to obtain a light yellow solid a. Place it in a vacuum drying oven and dry it at 60 °C to constant weight for use;

[0040] Dissolve 0.6 g of the dried light yellow solid a in 5 mL of toluene to obtain solution A. Dissolve 48.8 mg of 4-dimethylaminopyridine and 0.194 g of 2-[(ethoxythiocarbonyl)thio]propanoic acid in 5 mL of dichloromethane to obtain solution B. Dissolve 0.227 g of N,N'-dicyclohexylcarbodiimide in 5 mL of dichloromethane to obtain solution C. First, place solution A and solution B in a reaction flask, mix them evenly and stir, then slowly inject solution C, and continue to stir and react at room temperature for 36 h. After the reaction is completed, add methanol dropwise under stirring for precipitation. Filter out the precipitate and wash it 3 - 5 times by the dissolution-precipitation method with dichloromethane / methanol to obtain solid b. Place it in a vacuum drying oven and dry it at 60 - 80 °C to constant weight, which is the dithioester-terminated tetraphenylethylene double-stranded molecule TPE-PCL-CTA.

[0041] The product structure was confirmed by nuclear magnetic resonance hydrogen spectrum, as Figure 1 shown, 1 HNMR(DMSO-d 6 ,δ,ppm): 7.80~7.15(H A ) is attributed to the hydrogens at different positions of the benzene ring of the tetraphenylethylene nucleus, 8.31(H g ) belongs to the amide hydrogen connected to the benzene ring of tetraphenylethylene; 3.98(H a ), 2.27(H c ), 1.53(H d ), 1.29(H b ) are attributed to various hydrogen signals on the polycaprolactone chain; 4.35(H f ) and 0.94(H e ) are attributed to the methine and methyl hydrogens on the terminal dithioester. The reaction formula is shown as follows:

[0042]

[0043] (2) Preparation of long-chain block copolymer TPE-P(CL-APBA)-AA

[0044] Take 0.3 g of the product TPE-PCL-CTA prepared in step (1), 286 mg of 3-acrylamidophenylboronic acid (APBA) and 4.11 mg of azobisisobutyronitrile, and add them successively to a reaction flask containing 3 mL of N,N-dimethylformamide DMF. Heat to 70 °C under nitrogen protection and stir and react for 24 hours. After the reaction is completed, cool to room temperature. First, add 1 mL of methanol to dissolve the reactants, and then add an appropriate amount of cold ether dropwise under stirring for precipitation. Filter out the precipitate and wash it 5 times with cold ether to obtain solid c. Place it in a vacuum drying oven and dry it at 60 °C to constant weight for use;

[0045] 0.45 g of dried solid c, 14 μL of acrylic acid, and 4.11 mg of azobisisobutyronitrile were successively added to a reaction flask containing 3 mL of N,N-dimethylformamide (DMF). Under nitrogen protection, the mixture was heated to 70 °C and stirred for 24 hours. After the reaction, it was cooled to room temperature. First, 1 mL of methanol was added to dissolve the reactants, and then an appropriate amount of cold diethyl ether was added dropwise with stirring for precipitation. The precipitate was filtered out and washed 5 times with cold diethyl ether to obtain solid d, which was placed in a vacuum drying oven and dried at 60 °C to constant weight, namely the long-chain block copolymer TPE-P(CL-APBA)-AA.

[0046] The product structure was confirmed by 1H NMR, as Figure 2 shown 1 1H NMR (DMSO-d 6 , δ, ppm): In addition to the original proton signals of the polymer TPE-PCL-CTA, new functional group proton peaks of poly(3-acrylamidophenylboronic acid) and acrylic acid appeared. 9.85 (H h ) was attributed to the amide hydrogen of poly(3-acrylamidophenylboronic acid), 7.97 - 7.11 (H A+B ) belonged to the overlapping signal of the benzene ring hydrogens of PAPBA and tetraphenylethylene, 2.25 (H b+e ) and 1.51 (H a+f ) were the results of the superposition and enhancement of the backbone methylene hydrogens of PAPBA and acrylic acid with the proton signals of polycaprolactone.

[0047] (3) Preparation of the block copolymer Ir-CP with dual-wavelength emission

[0048] 0.5 g of the dried product TPE-P(CL-APBA)-AA prepared in step (2) and 0.147 g of the iridium complex Ir-OH were dissolved in 3 mL of a DMF / methanol mixed solution (volume ratio 2:1) to obtain solution D. 3.66 mg of 4-dimethylaminopyridine was dissolved in 2 mL of dichloromethane to obtain solution E. 68 mg of N,N'-dicyclohexylcarbodiimide was dissolved in 3 mL of dichloromethane to obtain solution F. Solution E and solution F were successively added to solution D, mixed evenly and stirred. The reaction was stirred at room temperature for 48 h. After the reaction, an appropriate amount of cold diethyl ether was added dropwise with stirring for precipitation. The precipitate was filtered out, redissolved with the DMF / methanol mixed solution, and then an appropriate amount of cold diethyl ether was added dropwise for precipitation. The red powder e was filtered out and placed in a vacuum drying oven and dried at 60 °C to constant weight, namely the block copolymer Ir-CP with dual-wavelength emission.

[0049] The product structure was confirmed by 1H NMR, as Figure 3 shown ，1 1H NMR (DMSO-d 6, δ, ppm): In addition to the original proton signals of the long-chain block polymer TPE-P(CL-APBA)-AA, a new hydrogen signal peak of the iridium complex group appears, and there is a new overlapping signal of the hydrogen protons of PAPBA and tetraphenylethylene in the range of 9.92 - 6.39 (H A+B+C ) The reaction route of the above is shown in the following formula:

[0050]

[0051] (4) Preparation of Responsive Optical Nanomicelles HA / Ir-CP with Dual-Wavelength Emission

[0052] Dissolve 3 mg of the dried product of the block copolymer Ir-CP with dual-wavelength emission prepared in step (3) in 3 mL of a DMF / methanol mixed solvent (volume ratio 2:1) to obtain solution G. Place it in an ultrasonic environment of 40 KHz and ultrasonicate. Slowly drip the 10 mg / mL aqueous hyaluronic acid solution into the 1 mg / mL solution G until the mixed solution gradually turns into an orange-red emulsion (the volume ratio of the aqueous hyaluronic acid solution to solution G is 4:1). Stop ultrasonication and perform preliminary filtration, then pour it into a dialysis bag and dialyze it in deionized water for 48 - 50 hours to remove the organic solvents in the system, thus obtaining the optical nanomicelles HA / Ir-CP with dual-wavelength emission. Observe the prepared optical nanomicelles HA / Ir-CP with a transmission electron microscope, and it has a good spherical morphology; fluorescence spectrometer detection shows that the optical nanomicelle solution has two emission peaks at 390 - 580 nm and 660 - 850 nm, and the maximum emission wavelengths are 470 nm and 721 nm respectively.

[0053] Figure 4 Figure shows the infrared spectra of Ir-CP, hyaluronic acid HA, and the optical nanomicelles HA / Ir-CP prepared in Example 1; the absorption peak at 3443 cm -1 of the nanomicelles belongs to the stretching vibration of the hydroxyl group of hyaluronic acid. In addition, the characteristic peaks at 1640 cm -1 and 1560 cm -1 are close in morphology and peak position to the stretching vibration peaks at 1650 cm -1 and 1558 cm -1 of the hyaluronic acid spectrum, but there is a shift, indicating that complexation occurs between hyaluronic acid and Ir-CP, that is, the nanomicelles are indeed formed.

[0054] Repeat Example 1, except that when the concentration of the hyaluronic acid solution is fixed at 10 mg / mL and the concentration of Ir-CP increases from 1 mg / mL to 15 mg / mL, the hydrodynamic diameter of the prepared nanomicelles is monitored by dynamic light scattering, and the micelle diameter varies in the range of 210.5 - 277.3 nm, as shown in Figure 5。Repeat Example 1, except that the concentration of Ir-CP was fixed at 1 mg / mL, and when the concentration of hyaluronic acid was increased from 4 mg / mL to 10 mg / mL, the hydrodynamic diameter of the prepared nanomicelles was monitored by dynamic light scattering. The micelle diameter increased from 107 nm to 210.5 nm, as shown in Figure 6 。

[0055] Application Example

[0056] Optical emission responsiveness of nanomicelles to the microenvironment

[0057] Repeat Example 1, except that in step (4), the concentration of the hyaluronic acid solution was fixed at 10 mg / mL, the concentration of Ir-CP was 1 mg / mL, and the transmission electron microscope image of the dual-wavelength emission optical nanomicelles HA / Ir-CP prepared with a volume ratio of 4:1 is shown in Figure 7 , with a particle size of 107 nm and a good spherical morphology.

[0058] Select the nanomicelles with a particle size of 107 nm, divide them into 6 equal parts, each part being 10 mL. Use 0.5 mol / L hydrochloric acid or sodium hydroxide solution to adjust the pH value of the deionized water for dialysis. Then dialyze these aliquots of micelle solution in deionized water with different pH values for 36 - 48 hours to obtain nanomicelle solutions with pH values of 5, 6, 7.4, 8, 9, and 10 respectively. Measure the fluorescence intensity of the micelle solutions with different pH values using a fluorescence spectrometer. The normalized results are shown in Figure 8 as shown. With other conditions unchanged, as the environmental pH value decreases, the intensity of the maximum emission peak at 721 nm gradually increases, while the intensity of the emission peak at 470 nm does not change significantly. This emission spectrum test shows that the two emission wavelengths of the nanomicelles have significantly different feedback to acidification stimulation. The emission peak in the near-infrared region obviously has the characteristics of acidification response, which is beneficial to using the light intensity ratio of the two wavelengths to reflect the acidification trend of the environment. On the other hand, use argon and oxygen with different flow rates to bubble the prepared 107 nm micelle solution, change the oxygen partial pressure of the micelle solution, and then test the fluorescence emission spectrum. As shown in Figure 9 as shown. When the pH is constant at 7.4, as the oxygen partial pressure in the micelle solution decreases, the intensity of the near-infrared light with a maximum emission at 721 nm increases significantly, while the intensity of the emission peak at 470 nm still does not change significantly. This emission spectrum test shows that the two emission wavelengths of the nanomicelles also have significantly different feedback to the hypoxic environment. The emission peak in the near-infrared region obviously has the characteristics of hypoxic response. Based on the response of the near-infrared light region to acidification and hypoxic microenvironments, it can effectively achieve signal amplification for the detection of optical emission micelles in acidified and hypoxic tumors. At the same time, the non-responsiveness of the visible light region emission to the environment gives the micelles the advantage of ratio signal feedback, and the growth trend of the tumor can be traced by the change of the signal ratio during detection.

[0059] Cytotoxicity of Optical Nanomicelles with Dual-Wavelength Emission

[0060] After incubating human lung cancer cells (A549), mouse liver cancer cells (H22), and mouse embryonic fibroblasts (NIH3T3) with the 107 nm nanomicelles prepared above, the cytotoxicity was confirmed by detecting the relative number of live cells using the MTT method. Human lung cancer cells (A549), mouse liver cancer cells (H22), and mouse embryonic fibroblasts (NIH3T3) were seeded at a density of 5×10 3 cells / well on different 96-well cell culture plates and cultured in an incubator (37 °C, 5% CO 2 (under the condition of)) for 24 h. Then, 5 μL of the 107 nm nanomicelle solution (the final concentrations of the nanomicelle solution were 12.5, 25, 50, 100, and 200 mg / mL) was added to each well, and the cells were further cultured in an incubator at 37 °C and 5% CO 2 for 24 h. After removing the culture medium, the cells were washed twice with ice-cold PBS cooled in an ice-water bath for 30 min. Then, 200 μL of the culture medium containing 10% MTT solution (5 mg / mL, PBS with pH = 7.4) was added to each well, and the cells were incubated at 37 °C for 4 h. After discarding the supernatant, 150 μL of DMSO was added to each well, and the mixture was shaken at room temperature for 10 min. The absorbance at 570 nm was measured using a microplate reader. The relative cell viability was obtained by comparing the absorbance at 570 nm with that of the control wells containing only blank cells. Each experiment was repeated 3 times on average. The results are as Figure 10 shown. It can be seen that the cell viability of the cells treated with the optical nanomicelles was greater than 90%, indicating that the optical nanomicelles were non-toxic.

[0061] Comparison of Cellular Uptake of Optical Nanomicelles HA / Ir-CP

[0062] Human lung cancer cells (A549), mouse liver cancer cells (H22), and mouse embryonic fibroblasts (NIH3T3) cell lines were seeded in cell culture plates at a density of 1×10 5 cells / well and cultured in an incubator (37 °C, 5% CO 2 (under the condition of)) for 24 h. The culture medium was discarded, and the cells were washed 3 times with PBS (pH = 7.4) buffer. Fresh culture medium and 200 μL of the Ir-CP / HA nanomicelle solution prepared in Example 1 of the present invention were added, and the cells were further cultured at 37 °C and 5% CO 2 for 8 h. The cells were washed 3 times again with PBS (pH = 7.4) buffer. The cell samples were resuspended in PBS (pH = 7.4) solution under normoxic conditions and measured using Beckman Coulter FC500. Excitation: 488 nm, FL: 770 nm, PE-Cy7 channel. The obtained results are as Figure 11As shown, the H22 cells with overexpressed CD168 receptor had the largest intake of the nanomicelles, followed by the A549 cells with overexpressed CD44 receptor, while the NIH3T3 cells without overexpressed receptor had the worst intake ability. It was proved that hyaluronic acid endows the nanomicelles with specific affinity and active targeting ability to cancer cells, further improving the accuracy of using the micelles as a probe to detect tumors.

Claims

1. A method for preparing a dual-wavelength responsive optical micelle, characterized in that: The following steps are involved: (1) Preparation of dithioester-terminated tetraphenylethylene double-chain molecule TPE-PCL-CTA 1.4-1.5 g of 1,2-diphenyl-1,2-bis(4-aminophenyl)ethylene TPE-2AM was dissolved in a reaction bottle containing 15-25 mL of dry toluene, and then 10-15 mL of ε-caprolactone and 0.02-0.05 mL of stannous octoate were added in sequence, heated to 100-110° C. under nitrogen protection, stirred for reaction for 40-48 hours, cooled to room temperature after the reaction, first added with dichloromethane for dissolution, and then placed in methanol for precipitation, filtered out the precipitate and washed 3-5 times with dichloromethane / methanol by dissolution-precipitation method to obtain a light yellow solid a, placed in a vacuum drying oven at 60-80° C. and dried to constant weight for standby use; Take 0.5-0.6g of the dried light yellow solid a and dissolve it in 5-8mL toluene to obtain solution A, take 48-50mg 4-dimethylaminopyridine and 0.19-0.2g 2-[(ethoxythiocarbonyl)thio]propionic acid and dissolve them in 5-8mL dichloromethane to obtain solution B, take 0.2-0.3g N, N-dicyclohexylcarbodiimide and dissolve it in 5-8mL dichloromethane to obtain solution C, first put solution A and solution B in a reaction bottle, mix and stir evenly, then slowly inject solution C, continue to stir at room temperature for 36-40h, after the reaction is completed, add methanol dropwise under stirring for precipitation, filter out the precipitate and wash it 3-5 times by dissolution-precipitation method with dichloromethane / methanol to obtain solid b, put it in a vacuum drying oven at 60-80°C and dry it to constant weight, that is, dithioester-terminated tetraphenylethylene double-chain molecule TPE-PCL-CTA, and set aside; (2) Preparation of long-chain block copolymer TPE-P(CL-APBA)-AA Take 0.3-0.5g of the product TPE-PCL-CTA prepared in step (1), 280-300mg of 3-acrylamidophenylboronic acid (APBA) and 4-4.2mg of azobisisobutyronitrile and add them in turn to a reaction bottle containing 3mL of N,N-dimethylformamide DMF, heat to 60-80°C under nitrogen protection, stir and react for 20-24 hours, cool to room temperature after the reaction, first add 1-2mL of methanol to dissolve the reactants, then add cold ether dropwise under stirring to precipitate, filter out the precipitate and wash it with cold ether 3-5 times to obtain solid c, place it in a vacuum drying oven at 60-80°C and dry it to constant weight for standby use; Take 0.4-0.5g of the dried solid c, 13-15uL of acrylic acid, and 4-4.2mg of azobisisobutyronitrile and add them to a reaction bottle containing 3mL of N,N-dimethylformamide DMF in sequence, heat to 60-80°C under nitrogen protection, stir and react for 20-24 hours, cool to room temperature after the reaction, first add 1-2mL of methanol to dissolve the reactant, then add cold ether dropwise under stirring to precipitate, filter out the precipitate and wash it with cold ether 3-5 times to obtain solid d, place it in a vacuum drying oven at 60-80°C and dry it to constant weight, which is the long-chain block copolymer TPE-P(CL-APBA)-AA, and set aside; (3) Preparation of dual-wavelength emission block copolymer Ir-CP Take 0.3-0.5g of the dry product TPE-P(CL-APBA)-AA prepared in step (2) and 0.14-0.15g of the iridium complex Ir-OH and dissolve them in 3-6mL of DMF / methanol mixed solution to obtain solution D, take 3.6-3.7mg of 4-dimethylaminopyridine and dissolve them in 2-3mL of dichloromethane to obtain solution E, take 65-70mg of N,N-dicyclohexylcarbodiimide and dissolve them in 3-5mL of dichloromethane to obtain solution F, add solution E and solution F to solution D in sequence, mix well and stir, stir and react at room temperature for 45-48h, after the reaction is completed, add ether dropwise under stirring to precipitate, filter out the precipitate, dissolve it again with DMF / methanol mixed solution, add ether dropwise to precipitate, filter out the red powder e, place it in a vacuum drying oven at 60-80°C and dry it to constant weight, that is, the dual-wavelength emission block copolymer Ir-CP, and set it aside; (4) Preparation of dual-wavelength responsive optical nanomicelles HA / Ir-CP The dried product of the dual-wavelength emitting block copolymer Ir-CP prepared in step (3) is dissolved in an appropriate amount of a DMF / methanol mixed solvent to obtain a solution G, which is then placed under an ultrasonic environment of 40 kHz, and the hyaluronic acid aqueous solution is slowly dripped into the solution G so that the mixed solution gradually turns into an orange-red emulsion. The ultrasonication is stopped and initially filtered, and then poured into a dialysis bag and placed in deionized water for 48-50 hours to remove the organic solvent in the system, thereby obtaining a dual-wavelength emitting optical micelle HA / Ir-CP.

2. The method for preparing a dual-wavelength responsive optical micelle according to claim 1, characterized in that: In step (3) and step (4), the volume ratio of the DMF / methanol mixture is 2:

1.

3. The method for preparing a dual-wavelength responsive optical micelle according to claim 1, characterized in that: In step (4), the concentration of the solution G is 1 to 15 mg / mL.

4. The method for preparing a dual-wavelength responsive optical micelle according to claim 1, characterized in that: In step (4), the concentration of the hyaluronic acid aqueous solution is 4-10 mg / mL, and the preparation method is: dissolving hyaluronic acid in an appropriate amount of deionized water to obtain the hyaluronic acid aqueous solution.

5. The method for preparing a dual-wavelength responsive optical micelle according to claim 1, characterized in that: In step (4), the volume ratio of the hyaluronic acid aqueous solution and solution G is (3.5-4):

1. 6 . A dual-wavelength emitting responsive optical micelle prepared by the method for preparing the dual-wavelength emitting responsive optical micelle according to any one of claims 1 to 4 .

7. Use of the dual-wavelength emitting responsive optical micelles according to claim 5 in tumor-targeted cell imaging.

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