Near-infrared two-region nano-micelle with viscosity response and aggregation-induced emission as well as preparation method and application of nano-micelle

Abstract

The invention relates to a near-infrared two-region nano-micelle with viscosity response and aggregation-induced emission as well as a preparation method and application thereof. The invention belongs to the technical field of nano biomedical materials, and particularly relates to a near-infrared two-region nano-micelle with viscosity response and aggregation-induced emission as well as a preparation method and application of the near-infrared two-region nano-micelle. In order to provide a nano-micelle with biocompatibility, water solubility, targeting property and blood circulation time, an organic micromolecule AIE probe DPXBI is encapsulated in a GD modified amphiphilic polymer DSPE-PEG-RGD to form the nano-micelle, and the DPXBI is formed by connecting phenylindole sulfonate and a diphenyl anthryl electron donor through a double bond. The nano-micelle is simple and rapid to synthesize, has the advantages of uniform dispersion, good water solubility and the like, and can be applied to NIR-II FLI-guided PDT and PTT of breast cancer as a diagnosis and treatment integrated probe.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomedical materials, and specifically relates to a near-infrared second-zone nano-micelle with viscosity response and aggregation-induced luminescence, and a preparation method and application thereof. Background Art

[0002] Compared with traditional tumor treatment methods such as surgery, radiotherapy, and chemotherapy, phototherapy is gradually becoming the forefront of cancer treatment due to its many advantages such as non-invasiveness, low toxicity and side effects, low drug resistance, and dual controllable selectivity in time, space, and optics. The two most common phototherapy methods are photodynamic therapy (PDT) and photothermal therapy (PTT), which use exogenous or endogenous photosensitizers (PSs) to produce cytotoxic reactive oxygen species (ROS) or enhance the local temperature of target tissues to produce cytotoxicity to kill tumor cells. PTT uses photothermal agents and high-power laser density to achieve protein denaturation and cell apoptosis or necrosis. However, PTT has obvious limitations for tumor ablation, including non-selective damage to adjacent tissues, insufficient tumor aggregation, insufficient temperature of tumor-killing tissues, and safety issues of high-energy laser irradiation. In addition, an intracellular heat shock protein (HSP) is produced during the heat shock reaction as a thermal resistance mechanism to protect and restore homeostasis. In PDT, ROS, as a key therapeutic factor, is the output after a series of photophysical and photochemical reaction processes. Hypoxia in the tumor microenvironment is the main obstacle to the generation of ROS during PDT. 2 In situ delivery to the tumor area to relieve hypoxia, but this method may relieve hypoxia and cause cancer cell proliferation. More and more studies have shown that the synergistic treatment of PDT and PTT can make up for the shortcomings of both treatments and improve safety and effectiveness. The heat induced by PTT can promote blood circulation, potentially increase the oxygen level of the tumor, and enhance the effect of PDT; conversely, the ROS generated by PDT will damage the function of HSP, thereby increasing the sensitivity of tumor cells to heat.

[0003] Traditional PSs are plagued by aggregation-induced quenching (ACQ), which leads to reduced fluorescence intensity and reduced ROS generation efficiency when forming nanoaggregates. The AIE strategy is proposed to achieve the anti-quenching and enhanced ROS generation characteristics of fluorescence through the mechanism of restricted intramolecular motion (RIM) in the state of small molecule aggregation, which can play the role of amplification and sensitization of PDT. In recent years, PSs with high photothermal conversion efficiency and ROS generation ability with AIE function have been developed and applied to the integrated diagnosis and treatment of tumors, especially AIE PSs that use abnormally upregulated biomarkers or tumor microenvironment-specific activation, showing the performance of dynamic regulation of fluorescence response and ROS generation, and shining in basic research. According to the specific biomarker response, PSs can change from an inhibitory state to an enhanced state, or from an inert state to an active state, which greatly avoids the risk of indiscriminate accumulation in the living body, thereby achieving improved therapeutic effects on tumor cells while preventing damage to normal tissues.

[0004] Intracellular viscosity is a key tumor microenvironment parameter, involved in many biological processes such as signal transduction, biological diffusion, chemical signal transduction, protein aggregation, metabolite diffusion, etc. It is also a potential biomarker for metastatic cancer. At the same time, cytoplasmic condensation and cell shrinkage during cell death will also lead to further enhancement of cytoplasmic viscosity. At present, more and more small molecule fluorophores are used to detect intracellular viscosity of various diseases, especially fluorescent probes for tumor imaging. However, most of them are visible light and near-infrared region I (NIR-I) fluorescent materials, which have limited penetration depth into tissues and are difficult to achieve early evaluation of tumor diagnosis and treatment effects in vivo. The emerging near-infrared region II (NIR-II, 1000-1700nm) fluorescence imaging technology has a high penetration depth into biological tissues, greatly reduces autofluorescence and improves the signal-to-noise ratio. At present, most of the near-infrared region II AIE fluorescent probes exist in the form of small molecules. Although they have the characteristics of good biocompatibility, few side effects, and rapid metabolism, their poor water solubility, low selectivity and targeting, and cell uptake problems have greatly hindered their application in the biomedical field. Summary of the invention

[0005] In view of the above problems, the present invention provides a near-infrared second-zone nano-micelle with viscosity response and aggregation-induced luminescence, and a preparation method and application thereof. R M@DPXBI has strong absorbance in the near-infrared region (700-900nm) and has a near-infrared second-zone fluorescence emission wave centered at 928nm and extending to 1200nm. It also has the characteristics of AIE, viscosity activation, photothermal conversion and ROS generation, which gives it excellent NIR-II FLI, PTT and PDT capabilities.

[0006] The present invention R M@DPXBI enhances its targeted accumulation at the tumor site through active targeting mediated by RGD peptide and passive targeting of the nanomaterial itself, long-term circulation in the blood, and viscosity-activated properties after reaching the tumor site. It can accurately shape the boundaries of mouse breast cancer and determine the optimal phototherapy time, achieving excellent anti-tumor effects through the synergistic effect of PTT and PDT cascades.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a near-infrared second-zone nanomicelle with viscosity response and aggregation-induced emission, which has a core-shell structure, wherein the core is a hydrophobic small molecule AIE probe, and the shell is an RGD-modified amphiphilic polymer, with the hydrophobic end facing inward and the hydrophilic end facing outward.

[0009] Furthermore, the nanomicelles are formed by a self-assembly strategy using a hydrophobic small molecule AIE probe and an RGD-modified amphiphilic polymer.

[0010] Furthermore, the RGD-modified amphiphilic polymer is DSPE-PEG-RGD, which is a highly biocompatible amphiphilic polymer with non-toxic, non-immunogenic, non-antigenic and tumor-targeting properties. The hydrophobic small molecule AIE probe is DPXBI, which is activated by the viscosity in the microenvironment through the aggregation-induced intersystem crossing (AI-ISC) effect and the restriction of intramolecular motion.

[0011] Furthermore, the present invention directly encapsulates the hydrophobic DPXBI into the amphiphilic copolymer DSPE-PEG-RGD through a self-assembly strategy to prepare R M@DPXBI nanomicelles, the loading rate of the small molecule AIE probe DPXBI reaches 91.33wt%.

[0012] The structural formula of the small molecule AIE probe DPXBI is:

[0013]

[0014] In a second aspect, the present invention provides a method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence, comprising the following steps:

[0015] Step 1, adding DPXBI to DMF, ultrasonically dispersing, to form a DPXBI solution; adding DSPE-PEG-RGD to DMF, ultrasonically dispersing, to form a DSPE-PEG-RGD solution; slowly adding the DPXBI solution dropwise to the DSPE-PEG-RGD solution, ultrasonically dispersing;

[0016] Step 2, slowly adding the mixed solution obtained in step 1 dropwise into deionized water, ultrasonically dispersing to form a mixed solution, and stirring at high speed;

[0017] Step 3, dialyzing the mixed solution obtained in step 2, and centrifuging and purifying to obtain an aqueous solution, and freeze-drying the aqueous solution to obtain the nanomicelles.

[0018] Furthermore, in step 1, the mass ratio of DPXBI to DSPE-PEG-RGD is 1:5.

[0019] Furthermore, in step 2, the high-speed stirring temperature is 4° C. and the time is 12 to 20 hours.

[0020] Furthermore, the dialysis in step 3 is specifically: using a 3500D dialysis bag for 48 hours; the centrifugal purification is specifically: using a 10KD ultrafiltration centrifuge tube, centrifuging at a speed of 3000-4000rpm for 15-20min for purification and concentration.

[0021] In a third aspect, the present invention provides an application of near-infrared second-zone nanomicelles with viscosity response and aggregation-induced luminescence for preparing an integrated reagent for the diagnosis and treatment of breast cancer.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The nano-micelles synthesized in the present invention use DSPE-PEG-RGD as a matrix and encapsulate DPXBI through a self-assembly strategy to form nano-micelles ( R The nanomicelles are simple and fast to synthesize, and have the advantages of uniform dispersion and good water solubility. They can also be used as an integrated diagnostic and therapeutic probe for NIR-II FLI-guided PDT and PTT of breast cancer.

[0024] The nano micelles synthesized by the present invention have the characteristics of aggregation-induced near-infrared second-zone fluorescence emission, and can achieve advantages such as higher tissue penetration, lower tissue autofluorescence and signal-to-noise ratio at the tumor site.

[0025] The synthesized nano micelles of the present invention have both active tumor targeting mediated by RGD peptide and passive targeting mediated by the high permeability and long retention effect of the nano particles themselves, so that they can achieve excellent aggregation at the tumor site.

[0026] After being activated by viscosity in the tumor microenvironment, the synthesized nanomicelles of the present invention exhibit a strong near-infrared II (NIR-II) fluorescence signal, which can accurately identify the tumor boundary and determine the optimal time for subsequent phototherapy. RM@DPXBI inhibits the progression of breast cancer in mice through synergistic photodynamic therapy (PDT) and photothermal therapy (PTT) under a single laser irradiation. In addition, phototherapy-induced cell death further increases intracellular viscosity, which in turn limits R The intramolecular motion of M@DPXBI leads to the amplification of the PDT effect. The NIR-II fluorescence imaging (FLI)-guided synergistic PTT / PDT treatment involved in the present invention provides new insights into the development of superior multifunctional phototherapy technology for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the preparation and application of the nano micelles of the present invention.

[0028] Figure 2 This is a transmission electron microscopy image of the nanomicelles synthesized in Example 1.

[0029] Figure 3 This is the hydrated particle size distribution diagram of the nanomicelles synthesized in Example 1.

[0030] Figure 4 The UV-Vis-NIR absorption spectra of the nanomicelles and small molecule probes synthesized in Example 1.

[0031] Figure 5 The NIR-II fluorescence emission spectra of the nanomicelles synthesized in Example 1 at different concentrations.

[0032] Figure 6 This is the NIR-II fluorescence emission spectrum of the small molecule fluorescent probe synthesized in Example 1 in different viscosity microenvironment changes simulated by glycerol.

[0033] Figure 7 The small molecule AIE fluorescent probe synthesized in Example 1 exhibits selectivity and specificity for viscosity under different conditions (various reactive oxygen species, RNS, metal ions, polar solvents, proteins and glycerol).

[0034] Figure 8 This is a curve diagram of the total active oxygen content of the nanomicelles synthesized in Example 1 under different laser irradiation times.

[0035] Fig. 9 This is a curve diagram showing the singlet oxygen content of the nanomicelles synthesized in Example 1 under different laser irradiation times.

[0036] Fig.10 This is a curve diagram of the singlet oxygen content generated by the nanomicelles synthesized in Example 1 at different laser irradiation times in a system simulating increased viscosity.

[0037] Fig.11This is a temperature change curve of micelles of different concentrations synthesized in Example 1 after irradiation with the same laser intensity for different times.

[0038] Fig.12 The temperature heating / cooling curves and photothermal conversion efficiency calculation diagram of the nanomicelles synthesized in Example 1 after irradiation with the same laser intensity.

[0039] Fig.13 These are NIR-II FLI images of the nanomicelles synthesized in Example 1 at different time points after being injected into a tumor-bearing mouse model via the rat tail vein.

[0040] Fig.14 This is an image of the treatment of tumor tissue by single laser irradiation after the nanomicelles synthesized in Example 1 were injected into a tumor-bearing mouse model via the rat tail vein. DETAILED DESCRIPTION

[0041] In order to further illustrate the technical solution of the present invention, the present invention is further described below through embodiments.

[0042] Example 1

[0043] like Figure 1 As shown, a near-infrared second-zone nanomicelle with viscosity response and aggregation-induced emission of the present embodiment has a core-shell structure, wherein the core is a hydrophobic small molecule AIE probe, and the shell is an amphiphilic polymer modified with RGD, with the hydrophobic end facing inward and the hydrophilic end facing outward, and is formed by a self-assembly strategy of the hydrophobic small molecule AIE probe DPXBI and the amphiphilic polymer DSPE-PEG-RGD modified with RGD.

[0044] The hydrophobic small molecule AIE probe DPXBI is prepared by using phenylindole sulfonate as an electron accepting group and a molecular rotor, and connecting an electron donor diphenyl yellow anthracene through an ethylene bridge. The specific preparation method is as follows:

[0045] Step 1: Mix 25 mL of chloroform and 12 mL of DMF (155 mmol) solution, and then slowly add 9 mL, 94 mmol of phosphorus tribromide solution (PBr 3 ), after rewarming for 1 hour, 4 mL, 39 mmol cyclohexanone was added at room temperature, and stirring was continued for 18 hours, and sodium bicarbonate solution was added to adjust the pH to 7, and extracted with dichloromethane and anhydrous Na 2 SO 4 After drying and reducing pressure, compound 1 was obtained as a yellow oil.

[0046] Step 2, take 0.13g of compound 1 (0.63mmol), 1.2g of Cs 2 CO 3(3.68mmol) was added to 10mL DMF solution, and then 0.49g 4-bromo-2-hydroxybenzaldehyde (2.56mmol) was added at room temperature. The mixed solution was stirred at room temperature for 48 hours until a strong yellow spot appeared in the thin layer chromatography (mixed solvent hexane / ethyl acetate (v / v=4:1)). After the reaction was completed, the mixture was filtered, washed twice with deionized water, extracted with ethyl acetate, and purified by anhydrous Na 2 SO 4 After drying and evaporation under reduced pressure, a yellow powder was obtained, which was further purified by silica gel chromatography using n-hexane / dichloromethane (v / v=5:1-0:1) as the eluent to obtain Compound 2.

[0047] Step 3, take 100 mg of compound 2 (0.34 mmol), 103 mg of 4-methoxydiphenylamine (0.52 mmol), 103 mg of Cs 2 CO 3 (0.52mmol), 10mg Pd 2 (OAc) 2 1.5-(4 ... 2 SO 4 After drying under reduced pressure, the crude product was purified again by silica gel chromatography (the eluent was n-hexane / dichloromethane, v / v=1:1) to obtain compound 3.

[0048] Step 4, take 82 mg of compound 3 (0.2 mmol) and 100 mg of compound B1 (0.3 mmol), add a few drops of piperidine under nitrogen protection, reflux for 24 h using ethanol method, cool to room temperature, evaporate the solvent under reduced pressure, and elute the remaining material by silica gel chromatography (dichloromethane / methanol, v / v=20:1) to obtain blue-green compound 4.

[0049] Step 5: Dissolve 72 mg of compound 4 (0.1 mmol) in 5 mL of dichloromethane and add 10 drops of boron tribromide (BBr) at -50 °C. 3 ), and stirred overnight after returning to room temperature, and then heated to room temperature with saturated NaHCO 3 The reaction was quenched with dichloromethane and the complex was washed three times with saturated saline and then added to anhydrous Na 2 SO 4and dried under reduced pressure, and purified by silica gel chromatography using dichloromethane / methanol (v / v=15:1) to obtain a blue-green powder, namely DPXBI.

[0050] Its synthetic route is as follows:

[0051]

[0052] The method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence in this embodiment comprises the following steps:

[0053] Step 1, add 2 mg of DPXBI to 2 mL of DMF, and ultrasonically disperse for 10 min to form a dark green DPXBI solution, add 10 mg of DSPE-PEG-RGD to 4 mL of DMF, and ultrasonically disperse for 10 min to form a transparent DSPE-PEG-RGD solution, and slowly add the DPXBI solution dropwise to the DSPE-PEG-RGD solution, and ultrasonically disperse for 30 min;

[0054] Step 2, slowly adding the mixed solution obtained in step 1 dropwise into 20 mL of deionized water, ultrasonically dispersing for 30 min to form a mixed solution, and stirring at high speed at 4° C. for 18 h;

[0055] Step 3: Place the mixed solution obtained in step 2 into a 3500D dialysis bag for 48 hours, and use a 10KD ultrafiltration centrifuge tube to centrifuge at 3500rpm for 20 minutes to purify and concentrate the aqueous solution (repeat 3 times), and freeze-dry it to obtain the integrated diagnosis and treatment nano-micelles. R M@DPXBI.

[0056] Example 2

[0057] The method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence in this embodiment comprises the following steps:

[0058] Step 1, add 2 mg of DPXBI to 2 mL of DMF, and ultrasonically disperse for 10 min to form a dark green DPXBI solution, add 10 mg of DSPE-PEG-RGD to 4 mL of DMF, and ultrasonically disperse for 10 min to form a transparent DSPE-PEG-RGD solution, and slowly add the DPXBI solution dropwise to the DSPE-PEG-RGD solution, and ultrasonically disperse for 30 min;

[0059] Step 2, slowly adding the mixed solution obtained in step 1 dropwise into 20 mL of deionized water, ultrasonically dispersing for 30 min to form a mixed solution, and stirring at high speed at 4° C. for 12 h;

[0060] Step 3: Place the mixed solution obtained in step 2 into a 3500D dialysis bag for 48 hours, and use a 10KD ultrafiltration centrifuge tube to centrifuge at 3000rpm for 20 minutes to purify and concentrate the aqueous solution (repeat 3 times), and freeze-dry it to obtain the integrated diagnosis and treatment nano-micelles. R M@DPXBI.

[0061] Example 3

[0062] The method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence in this embodiment comprises the following steps:

[0063] Step 1, add 2 mg of DPXBI to 2 mL of DMF, and ultrasonically disperse for 10 min to form a dark green DPXBI solution, add 10 mg of DSPE-PEG-RGD to 4 mL of DMF, and ultrasonically disperse for 10 min to form a transparent DSPE-PEG-RGD solution, and slowly add the DPXBI solution dropwise to the DSPE-PEG-RGD solution, and ultrasonically disperse for 30 min;

[0064] Step 2, slowly adding the mixed solution obtained in step 1 dropwise into 20 mL of deionized water, ultrasonically dispersing for 30 min to form a mixed solution, and stirring at high speed at 4° C. for 20 h;

[0065] Step 3: Place the mixed solution obtained in step 2 into a 3500D dialysis bag for 48 hours, and use a 10KD ultrafiltration centrifuge tube to centrifuge at 4000rpm for 15 minutes to purify and concentrate the aqueous solution (repeat 3 times), and freeze-dry it to obtain the integrated diagnosis and treatment nano-micelles. R M@DPXBI.

[0066] Example 4

[0067] This example uses the diagnosis and treatment integrated nano micelles prepared in Example 1 R Take M@DPXBI as an example and do the following experimental tests:

[0068] 1) R M@DPXBI was dissolved in deionized water and dispersed in an ultrasonic cleaner to prevent agglomeration. A few microliters of the dispersed sample was taken with a pipette and dropped onto the support film and allowed to dry naturally. The morphology and size of RM@DPXBI were observed using a transmission electron microscope. Figure 2 As shown, the nanomicelles have a spherical morphology and an average diameter of about 46.8 nm.

[0069] 2) R M@DPXBI was dissolved in deionized water and dispersed in an ultrasonic cleaner to prevent agglomeration. 1 mL of appropriate concentration was taken. RThe hydrated particle size of the M@DPXBI solution was measured by dynamic light scattering (DLS). Figure 3 As shown, DLS results show R The average hydrodynamic diameter (Dh) of M@DPXBI is 58.8 nm, with good dispersibility and a PDI of 0.279. This uniform dispersion makes R M@DPXBI can accumulate in tumor sites through active targeting of RGD peptide and passive targeting of the nanomaterial itself.

[0070] 3) R M@DPXBI was dissolved in deionized water and dispersed in an ultrasonic cleaner to prevent agglomeration. Deionized water was used as the reference solution and the optical absorption was detected by UV-visible spectrophotometer. The reference solution was first used for baseline calibration to eliminate the influence of the solvent and the cuvette. The sample solution was then poured into the cuvette for calibration. R M@DPXBI measures absorbance within the selected wavelength range, records the data, and plots a spectrum with wavelength as the horizontal axis and absorbance as the vertical axis. Figure 4 As shown, DPXBI and R M@DPXBI has strong absorption peaks in the near-infrared region. R The absorption peak of M@DPXBI at 735nm is stronger than that of pure DPXBI.

[0071] 4) R M@DPXBI was dissolved in deionized water and dispersed using an ultrasonic cleaner to prevent agglomeration. The concentrations were adjusted to 200, 100, 50, 25, and 12.5 μg / mL. The samples were placed in a cuvette and the fluorescence emission intensity was observed using a NIR-II fluorescence microscope. Figure 5 As shown, at an excitation power of 2.0 W, R The M@DPXBI aqueous solution increased from 0 μg mL / L to 200 μg mL / L and exhibited NIR-II fluorescence emission (peak at 928 nm), which could effectively reduce background fluorescence and provide high tissue penetration depth and signal-to-noise ratio for biological applications.

[0072] 5) Prepare different proportions of glycerol-ethanol mixed solutions (such as 0% to 40% glycerol) to simulate different viscosity environments. R M@DPXBI was added to the viscosity medium and incubated for 30 minutes. The fluorescence spectra of each group were measured using a fluorescence spectrometer, the changes in fluorescence intensity were recorded, and a curve of fluorescence intensity versus viscosity was drawn. Figure 6 As shown, RThe fluorescence emission wavelength of M@DPXBI in the NIR-II region generally increases within the range of increasing solvent viscosity, which has the characteristics of viscosity activation and response, which may be due to the integration mechanism of intramolecular charge transfer (TICT) and molecular rotors leading to the increase of fluorescence intensity. R The selectivity and specificity of M@DPXBI were evaluated by reacting with various reactive oxygen species (ROS), reactive nitrogen species (RNS), metal ions, polar solvents and proteins under simulated physiological conditions. Figure 7 As shown in the figure, except for glycerol, all these ROS, RNS, metal ions, proteins and solvents did not cause obvious changes in fluorescence signals, which indicates that R M@DPXBI has good selectivity and specificity for viscosity.

[0073] 6) Investigation using DCFH and DPBF R The PDT performance of M@DPXBI was observed by total ROS and 1 O 2 First, take 50 μL R M@DPXBI (1 mg mL / L) was added to 3 mL DCFH (10 μM), and the fluorescence emission signal of the resulting mixture was analyzed by fluorescence spectroscopy. After 808 nm laser irradiation, the fluorescence emission signal in the range of 510 to 600 nm was monitored every 30 s. Figure 8 As shown, in R Under M@DPXBI and laser irradiation, the fluorescence signal of the mixture increased rapidly and changed significantly. As the irradiation time increased, the fluorescence signal intensity at 525nm gradually increased, which was positively correlated with the total ROS production. Similarly, 50μL of DPBF ethanol solution (15μM) was added to 3mL R M@DPXBI (1 mg mg / L), after 808 nm laser irradiation, the absorption of the mixed solution was monitored every 30 seconds. Under similar conditions, the absorption of the mixed solution in the high viscosity system was also recorded. R The mixed solution of M@DPXBI and DPBF with equal concentrations was compared. 1 O 2 The DPBF and laser irradiation groups were used as control groups. Commercial DPBF can be used as a ROS indicator. 1 O 2 Oxidation, the absorption spectrum gradually decreases. Figures 9-10 As shown in the figure, in the viscosity enhancement system R M@DPXBI 1 O 2 The yield gradually increases with the laser irradiation time and is higher than that of the normal system, which is in sharp contrast to the fact that the absorption of DPBF itself hardly changes under laser irradiation.

[0074] 7) Using infrared thermal imager at 808nm laser (1.0W / cm 2 ) irradiation, monitoring different contents (50~400μg mL -1 ) R The temperature change of M@DPXBI and the light-to-heat conversion efficiency (η) were calculated by the following formula:

[0075]

[0076] like Fig.11 As shown, under 808nm laser irradiation (1.0W / cm 2 During the irradiation (300s), R The temperature of M@DPXBI solution is significantly higher than that of deionized water, and R As the concentration of M@DPXBI increased from 50 μg / mL to 400 μg / mL, the temperature gradually increased. Fig.12 As shown, the heating and cooling curves under continuous laser irradiation are calculated R The photothermal conversion efficiency η of M@DPXBI is about 34.77%. These findings clearly show that R M@DPXBI has high photothermal conversion efficiency, which is beneficial to promote the PTT-mediated anticancer effect.

[0077] 8) 4T1 cells were injected subcutaneously into the right thigh of Balb / c female mice to establish a tumor model. When the tumor volume reached 90-120 mm 3 In vivo experiments were performed in tumor-bearing mice. R M@DPXBI solution, obtain in vivo NIR-II fluorescence images. Fig.13 As shown, according to the NIR-II fluorescence images at different times before injection, 2h, 6h, 12h, 24h, and 48h, the peak signal intensity at the tumor site occurred 24h after injection, which may be related to active targeting and passive targeting (EPR effect) as well as the response process to the viscosity microenvironment.

[0078] 9) Tumor-bearing mice were intravenously injected R M@DPXBI was irradiated with 808 nm laser (1 W / cm 2 ,5min) tumor site, such as Fig.14 As shown, the tumor area of ​​the mouse changed from initial blackening to scab formation to complete healing after laser irradiation.

[0079] The above shows and describes the main features and advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0080] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A near-infrared second-region nanomicelle with viscosity response and aggregation-induced luminescence, characterized in that: The structure of the nano micelle is a core-shell structure, wherein the core is a hydrophobic small molecule AIE probe, the shell is an amphiphilic polymer modified by RGD, the hydrophobic end faces inwards, and the hydrophilic end faces outwards.

2. The near-infrared second-region nanomicelle with viscosity response and aggregation-induced luminescence according to claim 1, characterized in that: The nanomicelles are formed by a self-assembly strategy using a hydrophobic small molecule AIE probe and an RGD-modified amphiphilic polymer.

3. The near-infrared second-region nanomicelle with viscosity response and aggregation-induced luminescence according to claim 1 or 2, characterized in that: The RGD-modified amphiphilic polymer is DSPE-PEG-RGD, and the hydrophobic small molecule AIE probe is DPXBI, with the structural formula:

4. A method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, adding DPXBI to DMF, ultrasonically dispersing, to form a DPXBI solution; adding DSPE-PEG-RGD to DMF, ultrasonically dispersing, to form a DSPE-PEG-RGD solution; slowly adding the DPXBI solution dropwise to the DSPE-PEG-RGD solution, ultrasonically dispersing; Step 2, slowly adding the mixed solution obtained in step 1 dropwise into deionized water, ultrasonically dispersing to form a mixed solution, and stirring at high speed; Step 3, dialyzing the mixed solution obtained in step 2, and centrifuging and purifying to obtain an aqueous solution, and freeze-drying the aqueous solution to obtain the nanomicelles.

5. The method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence according to claim 4, characterized in that: The mass ratio of DPXBI to DSPE-PEG-RGD in step 1 is 1:

5.

6. The method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence according to claim 4, characterized in that: The high-speed stirring temperature in step 2 is 4° C. and the time is 12 to 20 hours.

7. The method for preparing near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence according to claim 4, characterized in that: The dialysis in step 3 is specifically: using a 3500D dialysis bag for 48 hours; the centrifugal purification is specifically: using a 10KD ultrafiltration centrifuge tube, centrifuging at a speed of 3000-4000rpm for 15-20min for purification and concentration.

8. Use of the near-infrared second-region nanomicelles with viscosity response and aggregation-induced luminescence according to any one of claims 1 to 3, characterized in that: Used to prepare reagents for integrated diagnosis and treatment of breast cancer.

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