Nanomaterial based on asymmetric squarylium dye and preparation method and application thereof

Abstract

The application belongs to the field of pharmaceutical chemistry, and specifically discloses a kind of nanometer material based on asymmetric squarylium dye and its preparation method and application.The molecular structure formula of the asymmetric squarylium dye is as shown in formula (1): formula (1); wherein: R1 is hydrogen or C1-C20 alkyl; R2 is oxygen or malononitrile group.The nanometer material includes micellar nanoparticles and MnO2-BSA nanoparticles, and the MnO2-BSA nanoparticles are electrostatically adsorbed on the surface of the micellar nanoparticles; the core of the micellar nanoparticles is the asymmetric squarylium dye, and the outer layer of the asymmetric squarylium dye is coated with amphiphilic block copolymer and amphiphilic block copolymer coupled with tumor targeting polypeptide.The nanometer material has great application potential in the field of precise diagnosis and treatment of tumors, and provides a new possibility for future tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal chemistry, and particularly relates to a kind of nanomaterial based on asymmetric square acid cyanine dye and its preparation method and application. BACKGROUND

[0002] Traditional cancer diagnosis methods such as magnetic resonance imaging (MRI) and computed tomography (CT) have problems such as radioactivity risk, operation difficulty, poor specificity and low sensitivity. In contrast, optical imaging technology, especially near-infrared fluorescence imaging technology, provides a new solution to this problem due to its short imaging time, high sensitivity and low testing cost. Near-infrared fluorescence imaging technology has strong tissue penetration ability, avoids biological background fluorescence and causes little damage to living organisms, making up for the shortcomings of traditional diagnostic methods. Although various imaging technologies have their advantages and disadvantages, clinical practice is not limited to a single imaging detection technology. For example, magnetic resonance imaging (MRI) can achieve three-dimensional tomographic images and high spatial resolution of lesions, but its relatively weak and long imaging time limit its application in tumors; while near-infrared fluorescence imaging (NIRFI) has lower spatial resolution, but its fast imaging, high sensitivity and other characteristics can make up for the shortcomings of MRI. The combination of MRI and NIRFI imaging will bring wider application prospects to medical clinics and scientific research. This combination is expected to provide more accurate and efficient methods for the diagnosis and treatment of cancer, thereby better serving cancer patients around the world.

[0003] Compared with traditional treatment methods such as chemotherapy, radiotherapy and surgical resection, photodynamic therapy (PDT) has higher spatiotemporal selectivity, less trauma and lower toxic side effects. This emerging cancer treatment method uses a specific light source to activate a non-toxic or slightly toxic photosensitizer (PS) to generate cytotoxic substances (ROS). These ROS can directly oxidize proteins, lipids, nucleic acid bases and amino acid residues, thereby changing lipid metabolism, up-regulating the expression of cytokines and stress proteins, and ultimately inducing cell apoptosis, necrosis or autophagy. The mechanism of PDT is that under the irradiation of light of appropriate wavelength, PS is excited to a singlet excited state and quickly converted to a triplet excited state. In this state, PS reacts with substrates to generate cytotoxic ROS through photodynamic reaction. Currently, this photodynamic reaction process is divided into two mechanisms, type I and type II. In type I photochemical reaction, the triplet state of PS reacts with nearby substrates through electron transfer to form free radical cations or free radical anions. These ions will further react with oxygen-containing substrates (such as water, oxygen, etc.) to produce superoxide anions and hydroxyl radicals. In contrast, in type II photochemical reaction, the triplet state of PS directly transfers energy to oxygen to generate highly reactive singlet oxygen ( 1O2). Although type II PDT has high oxygen dependence and high oxygen consumption, type I PDT has less oxygen consumption, which helps to overcome the malignant hypoxia dilemma of solid tumors. This means that type I PDT has great potential for clinical treatment applications. In addition, inorganic materials such as TiO2, ZnO have been reported to be able to efficiently produce superoxide anions and hydroxyl radicals. However, inorganic materials alone may have bio-safety problems, and the short wavelength also limits the tissue penetration depth, which is not conducive to deep treatment of tumors. In contrast, some organic metal complexes and metal-organic frameworks used for PDT treatment avoid oxygen dependence and consumption problems, but they still have the disadvantages of poor reproducibility, severe cytotoxicity, low biodegradability, and complex pharmacokinetics. Organic small molecule photosensitizers provide a new solution path to overcome the limitations of inorganic materials in the treatment of tumors. The ideal organic small molecule photosensitizer should meet the following conditions: 1) easy to prepare, good stability and high purity; 2) can effectively reach and selectively concentrate in the target tissue; 3) has excellent optical and photochemical properties, especially high absorption in the wavelength range of 600-900 nm; 4) has good biocompatibility; 5) can generate high activity of reactive oxygen species (ROS).

[0004] The tumor microenvironment, an ecosystem composed of a complex cell population, contains the heterogeneity of cancer cells and cancer stem cells as well as a variety of recruited stromal cell types. Compared with the normal tissue cell environment, the tumor microenvironment has unique characteristics such as hypoxia, exacerbated oxidative stress, and overexpression of glutathione (GSH). These characteristics have a profound impact on the effectiveness of various anticancer treatment methods, greatly limiting the treatment of cancer. To address the problem of hypoxia at the tumor site, it is more meaningful to develop innovative photosensitizers for photodynamic therapy based on type I photochemical processes. The unique feature of this photosensitizer is that it does not require the participation of oxygen and can directly generate hydroxyl radicals with strong cytotoxicity. There is a complex redox balance mechanism in tumor cells that can resist ROS-induced cell damage, thereby affecting treatment effectiveness. To overcome this challenge, researchers are exploring ways to deplete the overexpressed glutathione (GSH) in cells. GSH is a reducing agent highly expressed in tumor cells responsible for eliminating oxidative stress to maintain the activity of tumor cells. By depleting overexpressed GSH, more ROS can be generated. In addition, researchers use the high content of H2O2 in tumor cells to design and prepare a nano-platform containing catalase for photodynamic therapy (PDT), catalase is a key catalytic enzyme that can convert intracellular hydrogen peroxide (H2O2) to oxygen (O2) and water. Overexpression of catalase reduces the growth rate and spread rate of tumor cells, thereby significantly improving treatment effectiveness.

[0005] Therefore, it is urgent to develop a new preparation to cope with the hypoxic environment of tumors and reduce the GSH level, so as to optimize the effect of PDT treatment. SUMMARY

[0006] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the present application proposes a nano material based on an asymmetric squarylium dye and a preparation method and application thereof.

[0007] To solve the above technical problems, the first aspect of the present application provides an asymmetric squarylium dye, whose molecular structure is shown in formula (1):

[0008]

[0009] In the formula, R1 is hydrogen or C1-C20 alkyl; and R2 is oxygen or malononitrile.

[0010] Specifically, the squarylium dye of the present application is an asymmetric squarylium organic small molecule compound, which combines a squarylium moiety, an indole moiety and a thiophene-phenothiazine group, i.e. different electron-rich groups, indole and thiophene-phenothiazine groups, are connected to the squarylium parent nucleus, which enhances the intramolecular charge transfer characteristics, increases the rigidity and steric hindrance of the molecule, and effectively resists the attack of singlet oxygen. Compared with the traditional symmetric squarylium dye, this design not only increases the Stokes shift, but also significantly enhances the light stability, so that the compound has a broader application prospect in the field of near-infrared imaging. Through experiments, it has been confirmed that this kind of organic small molecule can generate hydroxyl radicals through type I photodynamic reaction, and can generate ROS in the hypoxic environment of tumors, thereby playing a PDT treatment role.

[0011] As a further improvement of the above-mentioned scheme, the molecular structure of the asymmetric squarylium dye is shown in formula (2) or formula (3):

[0012]

[0013] As a further improvement of the above-mentioned scheme, the preparation method of the above-mentioned asymmetric squarylium dye comprises the following steps:

[0014] The 1,3-squarylium is mixed with an electron donor structural unit as an electron acceptor structural unit to carry out a coupling reaction, so as to obtain the asymmetric squarylium dye; the electron donor structural unit includes hydrogen or methyl-substituted indole and thiophene-phenothiazine conjugated unit.

[0015] Preferably, the molar ratio of the electron acceptor structural unit to the electron donor structural unit is (2-3):1.

[0016] The second aspect of the present application provides a nanomaterial, comprising micellar nanoparticles and MnO2-BSA (bovine serum albumin) nanoparticles, wherein the MnO2-BSA nanoparticles are electrostatically adsorbed on the surface of the micellar nanoparticles.

[0017] The micellar nanoparticles have a coating structure, wherein the core is the asymmetric squarylium dye shown in claim 1 or 2, and the outer coating layer comprises an amphiphilic block copolymer and an amphiphilic block copolymer coupled with a tumor-targeting polypeptide.

[0018] Preferably, the amphiphilic block copolymer comprises distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG 2000 ).

[0019] Preferably, the tumor-targeting polypeptide comprises CREKA, which is a Cys-Arg-Glu-Lys-Ala pentapeptide, can specifically bind to the fibrin-fibronectin complex in the microthrombus on the tumor blood vessel wall, and has a tumor-targeting function.

[0020] Specifically, the nanomaterial of the present application comprises micellar nanoparticles containing an asymmetric squarylium dye and a tumor-targeting polypeptide, and MnO2-BSA nanoparticles. The nanomaterial can reach the tumor site through the EPR effect and the active targeting of CREKA, and is efficiently accumulated in the tumor tissue area. In the tumor microenvironment, the asymmetric squarylium dye in the system is released, realizing near-infrared fluorescence imaging, while exhibiting excellent type I photodynamic performance under near-infrared (660 nm) light, generating hydroxyl radicals to kill tumor cells; in addition, the addition of MnO2-BSA nanoparticles can realize T1 contrast-enhanced magnetic resonance imaging, and Mn 2+ can occur Fenton-like reaction, produce additional hydroxyl radicals, further enhance its therapeutic effect.

[0021] The third aspect of the present application provides a preparation method of the above-mentioned nanomaterial, comprising the following steps:

[0022] (1) dissolving the asymmetric squarylium dye in an organic solvent, then adding it dropwise into an aqueous solution containing an amphiphilic block copolymer and a tumor-targeting polypeptide, mixing and dialyzing to obtain an aqueous solution of micellar nanoparticles, denoted as Cy Nps aqueous solution;

[0023] (2) adding an aqueous solution of potassium permanganate to an aqueous solution of bovine serum albumin, dispersing to obtain an aqueous solution containing MnO2 and bovine serum albumin, denoted as MnO2-BSA Nps aqueous solution;

[0024] (3) mixing the Cy Nps aqueous solution prepared in step (1) with the MnO2-BSA Nps aqueous solution prepared in step (2), and dialyzing to obtain the nanomaterial, denoted as Cy@MnO2 Nps aqueous solution.

[0025] Specifically, in the preparation of the nanomaterial of the present application, the above asymmetric squarylium dye and DSPE-PEG 2000 -CREKA self-assembly to prepare stable and uniform-sized liposome nanoparticles, i.e. micellar nanoparticles Cy Nps, which have tumor targeting function; then mixing the micellar nanoparticles Cy Nps with the nuclear magnetic imaging agent MnO2-BSA nanoparticles to electrostatically combine the MnO2-BSA nanoparticles on the surface of the micellar nanoparticles Cy Nps, forming the multifunctional tumor microenvironment-responsive nanomaterial Cy@MnO2 Nps. The nanomaterial not only has the ability to precisely target tumor cells, but also exhibits double advantages in the face of complex tumor microenvironment: on the one hand, it protects the photosensitizer by consuming excess GSH; on the other hand, the reduced Mn 2 + can trigger Fenton-like reaction under the catalysis of H2O2 to generate more hydroxyl radicals. This property enables the nanomaterial to perform type I photodynamic therapy on hypoxic breast cancer cells under the irradiation of near-infrared light.

[0026] Preferably, the volume ratio of the Cy Nps aqueous solution to the MnO2-BSA Nps aqueous solution is 1:(1-2).

[0027] Preferably, in step (1), the organic solvent comprises tetrahydrofuran.

[0028] Preferably, in step (1), the concentration of the solution (solution A) formed after the asymmetric squarylium dye is dissolved in the organic solvent is 1-10 mg / mL, the concentration of the aqueous solution (solution B) containing the amphiphilic block copolymer and the tumor targeting polypeptide is 2-20 mg / mL, and the volume ratio of solution A to solution B is (0.01-1):1.

[0029] Preferably, in step (1), the mixing is first ultrasonic for 5-30 min, and then stirring to volatilize the organic solvent.

[0030] Preferably, in step (1), the dialysis is performed using a microporous filter membrane with a pore size of 0.22 μm for 12-36 hours.

[0031] Preferably, in step (2), the concentration of the aqueous solution of bovine serum albumin (solution C) is 10-100 mg / mL, the concentration of the aqueous solution of potassium permanganate (solution D) is 10-100 mg / mL, and the volume ratio of solution C to solution D is (0.04-4):1.

[0032] Preferably, in step (2), the dispersion is ultrasonic dispersion for 15-30 min.

[0033] Preferably, in step (3), the mixing is stirring for 0.5-1 hour.

[0034] Preferably, in step (3), the dialysis is dialysis for 12-36 hours using a microporous filter membrane of 0.22 μm.

[0035] The fourth aspect of the present application provides application of the above-mentioned asymmetric squarylium dye or nanomaterial in the field of tumor cell bioimaging.

[0036] The fifth aspect of the present application provides a magnetic resonance imaging preparation comprising the above-mentioned nanomaterial.

[0037] The sixth aspect of the present application provides application of the above-mentioned asymmetric squarylium dye or nanomaterial in the preparation of a photodynamic antitumor drug.

[0038] Preferably, the tumor comprises breast cancer.

[0039] The above technical solutions of the present application have at least the following technical effects or advantages relative to the prior art:

[0040] (1) The asymmetric squarylium dye of the present application, by connecting different electron-rich groups, indole and thiophene-phenothiazine groups, to the squarylium mother nucleus, enhances the intramolecular charge transfer characteristics, increases the rigidity and steric hindrance of the molecule, and effectively resists the attack of singlet oxygen; exhibits superior stability, a large stoke shift, and can generate hydroxyl radicals with strong cytotoxicity through type I photodynamic reaction without the participation of oxygen, generates ROS in the hypoxic environment of tumors, and thus plays a PDT treatment role. This finding provides a new approach for the treatment of hypoxic tumors, and indicates that this type of compound has great development potential in the field of photodynamic therapy of tumors.

[0041] (2) The present application, by nanometer precipitation method, first self-assembles the asymmetric squarylium dye and DSPE-PEG 2000 -CREKA to prepare stable and uniform size and tumor-targeting functional liposome nanoparticles Cy Nps; and then couples with the nuclear magnetic imaging agent MnO2-BSA nanoparticles to form multifunctional tumor microenvironment responsive nanomaterial Cy@MnO2 Nps. This system not only has near-infrared fluorescence (NIRFI) and magnetic resonance imaging (MRI) dual-mode imaging, enabling in vivo monitoring of the distribution of nanoparticles, and provides a powerful means for accurate guidance of PDT treatment, through type I photosensitizer Cy Nps in cooperation with Mn 2+Chemical kinetics, co-production of hydroxyl radicals for anti-tumor therapy. This innovative strategy realizes the integration of cancer diagnosis and treatment, and provides valuable strategies for the design and application of PDT, which has important significance for promoting the development of clinical tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 UV absorption spectrum of asymmetric squarylium dyes Cy-532 and Cy-580 in different solvents for the embodiment of the present application;

[0043] Figure 2 Fluorescence spectrum of asymmetric squarylium dyes Cy-532 and Cy-580 in different solvents for the embodiment of the present application;

[0044] Figure 3 Absorbance ratio graph of asymmetric squarylium dyes Cy-532 and Cy-580 under 660 nm light for the embodiment of the present application;

[0045] Figure 4 Hydroxyl radical fluorescence ratio graph for measuring asymmetric squarylium dyes Cy-532 and Cy-580 for the embodiment of the present application;

[0046] Figure 5 UV absorption and fluorescence emission spectrum of nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps for the embodiment of the present application;

[0047] Figure 6 Water and particle size graph and transmission electron microscope graph of nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps for the embodiment of the present application;

[0048] Figure 7 Hydroxyl radical fluorescence ratio graph for measuring nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps for the embodiment of the present application;

[0049] Figure 8 T1-weighted magnetic resonance imaging graph of nanomaterials Cy-580@MnO2 Nps for the embodiment of the present application;

[0050] Figure 9 Uptake graph of nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps on 4T1 tumor cells for the embodiment of the present application;

[0051] Figure 10 Photodynamic therapy graph of nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps on 4T1 tumor cells under non-illumination or illumination for the embodiment of the present application;

[0052] Figure 11The Cy-580 NPs and the Cy-580@MnO2 NPs are used for the near-infrared fluorescence imaging of tumors of tumor-bearing mice in the embodiment of the present application.

[0053] Figure 12 The Cy-580@MnO2 NPs are used for the magnetic resonance imaging of tumors of tumor-bearing mice in the embodiment of the present application.

[0054] Figure 13 The Cy-580 NPs and the Cy-580@MnO2 NPs are used for the tumor growth curves of mice in different treatment groups and the tumor weight graphs after 14-day dissection in the embodiment of the present application. DETAILED DESCRIPTION

[0055] The present application will be described in detail below with reference to the embodiments, so as to facilitate the understanding of the present application by those skilled in the art. It is necessary to particularly point out here that the embodiments are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. The non-essential improvements and adjustments of the present application made by those skilled in the art according to the above description should still belong to the protection scope of the present application. At the same time, the raw materials mentioned below are not specifically described, which are all commercially available products; the process steps or preparation methods not specifically mentioned are all known to those skilled in the art.

[0056] The synthesis routes of the compound Cy-532 and the compound Cy-580 in the following embodiments 1-2 are as follows:

[0057]

[0058] Embodiment 1

[0059] An asymmetric squarylium dye, whose molecular structural formula is as follows, is recorded as the compound Cy-532, and the preparation method thereof comprises the following steps:

[0060]

[0061] (1) 3,4-dihydroxy-3-cyclobutene-1,2-dione (684 mg, 6 mmol), dichlorosulfoxide SOCl2 (1.2 mL) and dimethylformamide (DMF, 120 μL) were weighed and added to toluene (10 mL), and then stirred at 95°C for 1.5 h; then, SOCl2 was removed by rotary evaporation to obtain a yellow oily substance;

[0062] (2) To the yellow oil 3,4-dichloro-3-cyclobutene-l,2-dione obtained from step (1) was added compound 2 (338 mg, 1.5 mmol), aluminum chloride (100 mg, 0.75 mmol) and anhydrous dichloromethane (15 mL) at room temperature under reflux overnight under N2. After the compound was cooled to room temperature, the reaction was quenched with saturated ammonium chloride (NH4C1) solution, then extracted with dichloromethane. The organic layer was collected, washed with saturated sodium bicarbonate (NaHC03), deionized water, saturated sodium chloride solution (NaCl) in turn, dried with anhydrous sodium sulfate (Na2S04), and the solvent was evaporated under reduced pressure. The solid was dissolved in 5 mL of acetone and triethylamine (N(Et)3) was added. After stirring at room temperature for 30 min, the solvent was evaporated under reduced pressure, 15 mL of water was added, and the undissolved material was removed by filtration. HCl was added dropwise to pH = 2 or so, and a brown solid was formed. The solid was filtered under reduced pressure, and the solid was separated and purified by silica gel column chromatography (dichloromethane:methanol = 50:1 v / v) to obtain yellow compound 3 (113 mg, 20%);

[0063] (3) Compound 3 (189 mg, 0.5 mmol) obtained from step (2) and compound 6 (180 mg, 0.6 mmol) were added to 5 mL of toluene and n-butanol, and heated to reflux for 4 h. The reaction was monitored by TLC, and after the reaction was completed, the reaction was cooled to room temperature, and the solvent was evaporated under reduced pressure. Silica gel column chromatography (dichloromethane:methanol = 200:1, V / V) gave the deep blue target compound Cy-532 (202 mg, 38%).

[0064] The structure of compound Cy-532 was identified, and the specific data are as follows:

[0065] Nuclear magnetic hydrogen spectrum 1 H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.46 (d, J = 9.6 Hz, 2H), 7.40 (d, J = 3.2 Hz, 1H), 7.37 (br, 2H), 7.35 (t, J = 8.0 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H), 6.91 (d, J = 4.0 Hz, 1H), 6.14 (s, 1H), 3.85 (s, 3H), 1.71 (s, 6H).

[0066] Nuclear magnetic carbon spectrum 13C NMR (100 MHz, CDC13) δ 185.6, 175.3, 162.6, 161.8, 142.4, 142.2, 141.6, 133.6, 131.1, 128.2, 127.7, 126.6, 125.8, 124.0, 122.4, 119.3, 113.9, 110.6, 50.5, 31.8, 26.4.

[0067] Mass (ESI-MS): m / z 533.1 ([M+H] + ) and HR-MS for C 32 H 24 N2O2S2([M+H] + ) calcd: 533.1352, found: 533.1339.

[0068] The above proves that the prepared asymmetric squarylium cyanine indeed has the molecular structure shown by Cy-532. Among them, DMSO-d6 is deuterated dimethyl sulfoxide.

[0069] Example 2

[0070] An asymmetric squarylium cyanine dye, whose molecular structure formula is as follows, is recorded as compound Cy-580, and the preparation method thereof comprises the following steps:

[0071]

[0072] (1) Take 3,4-dihydroxy-3-cyclobutene-1,2-dione (684 mg, 6 mmol), SOCl2 (1.2 mL) and DMF (120 μL) and add them to toluene (10 mL), and then stir at 95 °C for 1.5 h. Then, remove SOCl2 by rotary evaporation to obtain a yellow oily substance;

[0073] (2) To the yellow oil 3,4-dichloro-3-cyclobutene-l,2-dione obtained from step (1) was added compound 2 (338 mg, 1.5 mmol), AlCl3(100 mg, 0.75 mmol) and anhydrous dichloromethane (15 mL) at room temperature under reflux overnight. After the compound was cooled to room temperature, the reaction was quenched with saturated ammonium chloride solution, then extracted with dichloromethane. The organic layer was collected, washed with saturated sodium bicarbonate (NaHC03), deionized water, saturated sodium chloride solution (NaCl) in turn, dried over anhydrous sodium sulfate (Na2S04), and the solvent was evaporated under reduced pressure. The solid was dissolved in 5 mL of acetone and triethylamine (N(Et)3) was added. After stirring at room temperature for 30 min, the solid (790 mg, 2 mmol) was dissolved in 6 mL of DMF, malononitrile (198 mg, 3 mmol) was added, and 80 μL of N(Et)3 was added dropwise with stirring. The reaction was carried out at room temperature for 2 h, and the reaction was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the product was isolated and purified by silica gel column chromatography (dichloromethane:methanol = 50: 1, v / v) to obtain orange compound 4 (85 mg, 10%);

[0074] (3) Compound 4 (85 mg, 0.2 mmol) obtained from step (2) and compound 6 (105 mg, 0.35 mmol) were added to 3 mL of toluene and 3 mL of n-butanol, and heated to reflux for 4 h. The reaction was monitored by TLC, and after the reaction was completed, the reaction was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. Silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1, v / v) was used to obtain the green target compound Cy-580 (25.5 mg, 22%).

[0075] The structure of compound Cy-580 was identified, and the specific data are as follows:

[0076] Nuclear magnetic hydrogen spectrum 1 H NMR (400 MHz, CDC13) δ 8.20 (d, J = 4.4 Hz, 1H), 7.64 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.41 (t, J = 4.0 Hz, 2H), 7.38 (br, 1H), 7.35 (d, J = 4.4 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.28 (br, 1H), 7.22 (t, J = 8.0 Hz, 2H), 6.78 (s, 1H), 6.72 (d, J = 4.4 Hz, 1H), 3.76 (s, 3H), 1.74 (s, 6H).

[0077] Nuclear magnetic carbon spectrum 13C NMR (100 MHz, CDC13) δ 176.7, 173.5, 170.0, 165.1, 164.8, 157.2, 142.5, 141.9, 141.0, 137.6, 132.0, 128.5, 127.8, 127.2, 126.6, 124.6, 122.5, 119.3, 118.6, 118.0, 113.9, 111.3, 91.6, 50.7, 32.5, 25.9.

[0078] Mass (ESI-MS): m / z 579.1 ([M-H] - ) and HR-MS for C 35 H 24 N4OS2([M-H] - ) calcd: 579.1319, found: 579.1318.

[0079] The above proves that the prepared asymmetric squarylium cyanine indeed has the molecular structure shown in Cy-580. Among them, CDC13 is deuterated chloroform.

[0080] Example 3

[0081] A method for preparing a nanomaterial, comprising the following steps:

[0082] (1) The compound Cy-580 prepared in Example 2 is dissolved in tetrahydrofuran (200 μL, 3 mg / mL), which is added dropwise to the DSPE-PEG 2000 and DSPE-PEG 2000 -CREKA containing ultrapure water (2 mL, 5 mg / mL) under ultrasonic treatment, the obtained dispersion is ultrasonically treated for 15 min, stirred at room temperature for 12 h, and after removing tetrahydrofuran, dialysis is carried out, and after dialysis for 24 h, the solution in the dialysis bag is collected, which is the photosensitizer Cy-580 nanoparticles (Cy-580 NPs);

[0083] (2) The potassium permanganate aqueous solution (2.5 mL, 5 mg / mL) is added to the bovine serum albumin (BSA) containing ultrapure water (5 mL, 20 mg / mL) under ultrasonic treatment, ultrasonically treated for 1 h, and dialyzed for 24 h to obtain brown MnO2-BSA nanomaterial;

[0084] (3) The Cy-580 NPs prepared in step (1) and the MnO2-BSA prepared in step (2) are mixed in a volume ratio of 1:1, and stirred for 1 h to obtain the Cy-580@MnO2 NPs nanomaterial.

[0085] Effect test

[0086] 1. Optical properties of asymmetric squarylium dyes in solution

[0087] (1) Two asymmetric squarylium dyes Cy-532 and Cy-580 prepared in Example 1 and Example 2, respectively, were dissolved in different organic solvents (DCM, CH3OH, DMSO, THF and CHCl3) to prepare solutions with a concentration of 10 μM, and the absorption spectra and fluorescence emission spectra were tested, all at 25 °C. Among them: the ultraviolet spectrum in organic solvent solution is shown in Figure 1 , the abscissa Wavelength represents wavelength, and the ordinate Absorbance represents absorbance; the fluorescence spectrum is shown in Figure 2 , the ordinate Fluorescence intensity represents fluorescence intensity. As can be seen from Figures 1-2 , Cy-532 and Cy-580 both show near-infrared (NIR) absorption and emission spectra in these organic solvents, in which Cy-532 has a maximum absorption wavelength of 642 nm in DMSO and a maximum emission wavelength of 677 nm. Cy-580 has a maximum absorption wavelength of 681 nm in DMSO and a maximum emission wavelength of 719 nm. Cy-580 introduces malononitrile group on the structure of Cy-532, further red shift in the NIR region. In these organic solvents, the Stokes shift of Cy-532 and Cy-580 is greater than 20 nm, which is conducive to reducing the interference between the excitation and emission spectra, facilitating sensitive detection and accurate analysis.

[0088] (2) Two asymmetric squarylium dye compounds Cy-532 and Cy-580 prepared in Example 1 and Example 2, respectively, were dissolved in water to prepare aqueous solutions with a concentration of 10 μM, and the solutions were irradiated with a 660 nm laser (220 mW·cm -2 ) at room temperature for different time intervals, the time intervals being 0 min, 0.5 min, 1 min, 1.5 min, 2 min, 3 min, 4 min, 5 min, 10 min and 15 min, and the ultraviolet spectrum of the solution was measured, the results are shown in Figure 3 , the abscissa Irradiation time represents irradiation time. By analyzing the absorbance value changes of the maximum absorption peaks of their respective ultraviolet spectra (Cy-532: 664 nm; Cy-580: 717 nm), the absorbance values of the two asymmetric squarylium dyes did not change significantly after 15 min of 660 nm laser irradiation, and the light stability was excellent, in which the light stability of Cy-580 was slightly stronger than that of Cy-532.

[0089] 2. Evaluation of type I photodynamic effect by hydroxyl radical indicator HPF

[0090] Prepare a solution by uniformly mixing 10 μM of asymmetric squaricine dyes Cy-532, Cy-580, and HPF (4 μM) using PBS buffer as the solvent. λ / E m =480 / 514nm), using 660nm (220mW·cm) -2 The xenon lamp was used to irradiate the sample for 15 minutes, and the fluorescence spectrum and the change in fluorescence ratio at 514 nm were measured at time intervals of 0 min, 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 7 min, 10 min, and 15 min. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that Cy-532 and Cy-580 exhibit high hydroxyl radical generation capabilities, and Cy-580 has slightly higher activity than Cy-532. This indicates that the asymmetric squaric acid cyanine dyes with the introduction of thiophene-phenothiazide undergo a type I photodynamic reaction under 660 nm laser irradiation, thereby generating a large number of hydroxyl radicals, which is a new type of type I squaric acid cyanine photosensitizer.

[0091] Based on the excellent optical properties and type I photodynamic therapy potential of the asymmetric squaric acid cyanine dye Cy-580, this invention also provides the application of Cy-580 in the preparation of nanomaterials for tumor diagnosis and treatment.

[0092] 3. Optical properties and characterization of nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps

[0093] The ultraviolet absorption and fluorescence spectra of the nanomaterials Cy-580 NPs and Cy-580@MnO2 NPs prepared in Example 3 in water were measured using ultraviolet-visible-near-infrared absorption spectroscopy and fluorescence spectroscopy, respectively. The results are as follows: Figure 5 As shown. Based on ultraviolet spectra. Figure 5 (a) shows that the UV spectrum of Cy-580 NPs is similar to that of Cy-580, with absorption peaks at 400 nm and 700 nm; MnO2-BSA nanoparticles have a strong absorption peak at 400 nm; Cy-580@MnO2 NPs have a strong absorption peak at 400 nm and a significant absorption peak at 700 nm, indicating that the nanomaterials encapsulate the dye Cy-580 and the inorganic material MnO2. The emission fluorescence spectra of the two nanomaterials at 726 nm were obtained using 695 nm as the excitation wavelength; see [reference needed]. Figure 5 (b) Cy-580 NPs showed stronger fluorescence intensity at the same concentration (10 μg / mL).

[0094] The size and morphology of the nanomaterials Cy-580 NPs and Cy-580@MnO2 NPs were characterized by transmission electron microscopy (TEM) and particle size analyzer, and the results are shown in Figure 6 . Figure 6 (a) is a particle size distribution graph, the horizontal coordinate Size represents the particle size, and the vertical coordinate Intensity represents the intensity. Zeta potential represents the potential. The particle size analysis data of the nanoparticles show that the hydrated particle size is about 100 nm; 6(b) and Figure 6 (c) are TEM images of Cy-580 Nps and Cy-580@MnO2 Nps, respectively, indicating that the nanoparticles are spherical, and the diameter is similar to the particle size graph of water.

[0095] 4. Evaluation of the ability of nanomaterials Cy-580 Nps and Cy-580@MnO2 Nps to generate hydroxyl radicals

[0096] Cy-580@MnO2 NPs, HPF (4 μM) and H2O2 (200 μM) (E λ / E m = 480 / 514 nm) or Cy-580 NPs, HPF (4 μM) and H2O2 (200 μM) (E λ / E m = 480 / 514 nm) were uniformly mixed with sodium bicarbonate solution (10 mM) as the solvent, and the Cy-580 concentration was 10 μg / mL. The fluorescence spectra were measured under irradiation of a 660 nm (220 mW·cm -2 ) xenon lamp for 15 min without GSH and with 4 mM GSH, and the time intervals were 0 min, 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 7 min, 10 min and 15 min. The results are shown in Figure 7 . Figure 7 It can be seen that in the four systems, the Cy-580@MnO2 NPs and H2O2 system has the strongest ability to generate ·OH in the presence of GSH. The main reason is that MnO2 reacts with GSH to produce Mn 2+ , which occurs a Fenton-like reaction under the co-catalysis of Mn 2+ and HCO3 - , and H2O2 also decomposes to generate additional ·OH.

[0097] 5. Magnetic resonance imaging of nanomaterial Cy-580@MnO2 Nps

[0098] Using deionized water solution of pH 6.5 and pH 7.4, prepare Cy-580@MnO2 Nps with different concentrations (0 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL and 500 μg / mL) and scan with 7T clinical magnetic resonance imaging scanner, respectively test T1 weighted image without GSH, T1 weighted image with 6 mM GSH and T1 weighted image with 6 mM GSH and 200 μM H2O2, the results are shown in Figure 8 As can be seen from Figure 8 under acidic conditions, the Cy-580@MnO2 Nps solution shows obvious concentration-dependent brightening effect under T1 weighted MRI imaging, especially in the presence of 6 mM GSH, with the increasing concentration of nanomaterials, its T1 weighted imaging ability is enhanced; such as in the presence of GSH and H2O2 (with GSH and H2O2), its T1 weighted imaging ability slightly decreased, the possible reason is that Mn 2+ As a catalyst for the decomposition of H2O2 hydroxyl radical, the concentration changes slightly.

[0099] 6. Nanomaterial Cy-580 Nps and Cy-580@MnO2 Nps tumor cell uptake experiment

[0100] 4T1 cells were incubated with 10 μg / mL of Cy-580 NPs and Cy-580@MnO2 NPs for different times (0-10 h), and observed under laser confocal microscope, the results are shown in Figure 9 (a) and Figure 9 (b), respectively. As can be seen from Figure 9 with the passage of time, both can effectively enter the cell and emit fluorescence, but it is found that at 8 h, both materials are the brightest, and the brightness can be maintained to 10 h.

[0101] 7. Nanomaterial Cy-580 Nps and Cy-580@MnO2 Nps photodynamic therapy effect on tumor cells

[0102] 4T1 (mouse breast cancer cells) in exponential growth phase were seeded in 96-well plates (5000 cells / 100 μL per well) and incubated overnight. The compound was prepared into different concentrations of nanomaterials containing Cy-580 NPs and Cy-580@MnO2 NPs (0-64 μg / mL), and added respectively, 5 replicate wells for each concentration. After incubation for 4 h, 660 lamp plate was used for 10 min, and then incubation was continued for 24 h. The old culture medium was removed, 100 μL of culture medium containing MTT (0.5 g / mL) was added, and incubation was continued for 4 h. Then the culture medium was removed, 100 μL of DMSO was added to each well, and the absorbance at 490 nm in each well was detected using a multifunctional microplate detector. Cell viability % = (F n -F b ) / (F0-F b ) x 100%, wherein F0, F n and F b represent the absorbance values of the control group, the experimental group and the blank group, respectively, and the results are shown in Figure 10 . Figure 10 (a) and Figure 10 (b) are the light toxicity (Light) and dark toxicity (Dark) of Cy-580 NPs and Cy-580@MnO2 NPs on 4T1 cells at 0-16 μg / mL and pH 6.5, respectively. As can be seen from Figure 10 , Cy-580@MnO2 NPs have stronger light toxicity than Cy-580 NPs, and their IC 50 values are 4.8 μg / mL and 6.4 μg / mL, respectively. The dark toxicity is small in the concentration range of 0-16 μg / mL.

[0103] 8. Nanomaterials Cy-580 NPs and Cy-580@MnO2 NPs for near-infrared fluorescence imaging of tumor-bearing mice

[0104] Firstly, a 4T1 tumor-bearing BALB / c mouse model was established to evaluate the targeting imaging ability of Cy-580 NPs and Cy-580@MnO2 NPs in vivo. The cultured 4T1 cells were inoculated at a density of 2 x 10 6 cells / 100 μL subcutaneously on the right lower part of the back of the mouse, and cultured until the tumor volume reached about 100 mm 3 . The tumor-bearing mice were injected with 100 μL of nanomaterial solution (5 mg / mL) through the tail vein, and the control group was injected with 100 μL of PBS. Near-infrared fluorescence images of the mice were collected at time points of 0 h, 1 h, 3 h, 6 h, 12 h and 30 h after injection.

[0105] The results are shown in Figure 11As shown, both nanomaterials exhibited tumor targeting, Cy-580 NPs and Cy-580@MnO2 NPs loaded with polypeptide CREKA had obvious tumor targeting, which was manifested as fluorescence aggregation at the tumor site, while there was no obvious fluorescence aggregation in other organs. This phenomenon may be the combined effect of passive targeting (EPR effect) and active targeting of polypeptide. Figure 11 It can be seen that Cy-580@MnO2 NPs and Cy-580 NPs have similar near-infrared imaging ability, and the fluorescence at the tumor site gradually increases with time, reaching a maximum at 6h, and then gradually decreases. This indicates that Cy-580 NPs and Cy-580@MnO2 NPs have good in vivo near-infrared fluorescence imaging ability, which is more conducive to tumor diagnosis and treatment.

[0106] 9. Nanomaterial Cy-580@MnO2 NPs for magnetic resonance imaging of tumor-bearing mice

[0107] First, 4T1 tumor-bearing BALB / c mice (tumor volume of 100mm 3 around) were prepared with Cy-580@MnO2 NPs (5mg / kg) solution at pH 6.5, and intratumor injection was performed on tumor-bearing mice. The T1 signal intensity changes at 0, 0.5h, 1h, 3h and 6h (T1WI: TR=300.0ms, TE=6.2ms) were measured, and the T1 magnetic resonance images of the tumor site at each time point were collected. The results are shown in Figure 12 As shown, with the extension of time, the MR signal of the tumor site was obviously enhanced, and the intensity reached a maximum at 3h, and the signal-to-noise ratio of the tumor site reached a peak, and then the MRI intensity gradually decayed. This is because the nanomaterial Cy-580@MnO2 NPs in the acidic tumor microenvironment, MnO2 and overexpressed GSH undergo redox reaction to generate Mn 2+ , and the T1 magnetic resonance imaging ability gradually increases, indicating that the nanomaterial can release Mn 2+ ions at the tumor site, and implement MR imaging monitoring.

[0108] 10. Nanomaterials Cy-580 NPs, Cy-580@MnO2 NPs for tumor treatment of tumor-bearing mice

[0109] A 4T1 tumor-bearing BALB / c mouse model was established, and the tumor volume was 100mm 3When the experiment was performed, the mice were randomly divided into 8 groups, 6 mice in each group. The experimental mice were divided into groups: I (PBS group), II (PBS+light group), III (Cy-580 Nps group), IV (Cy-580@MnO2 Nps group), V (Cy-580 Nps+light group), VI (Cy-580@MnO2 Nps+light group). Each mouse was injected with 100 μL of PBS or different nanomaterials at a dose of 2 mg / mL by in situ injection of the tumor. 660 nm xenon lamp irradiation (500 mW·cm-2, 15 min), the tumor volume and mouse weight were measured and recorded on days 0, 2, 4, 6, 8, 10, 12, 14. -2 ,15min), the tumor volume and mouse weight were measured and recorded on days 0, 2, 4, 6, 8, 10, 12, 14. Figure 13 ,15min), the tumor volume and mouse weight were measured and recorded on days 0, 2, 4, 6, 8, 10, 12, 14. Figure 13 (a) Figure 13 (b) are the tumor growth curves of mice in different treatment groups and the tumor weights obtained after 14 days in different treatment groups, respectively. Figure 13 From (a) and (b), it can be seen that in groups I-IV, the tumor volume increases with the treatment days, while in groups V and VI, after 660 nm xenon lamp irradiation treatment, the tumor volume is significantly reduced, and the tumor weight after dissection is almost 0. In both cases, excellent photodynamic tumor treatment effect is shown. At the same time, comparing the light irradiation groups V and VI, the tumor treatment effect after dissection in group VI is more obvious, indicating that the nanomaterial containing MnO2 can produce more ·OH to kill cancer cells, which shows that Cy-580@MnO2 Nps has great potential in the treatment of breast cancer.

[0110] In summary, the present application provides a synthesis method of an asymmetric squarylium dye, and the absorption and emission spectra, optical stability of Cy-532 and Cy-580 in different solutions are investigated, and the optical properties of Cy-580 are more superior. At the same time, different active oxygen indicators are used to evaluate the photodynamic effect experiment, which proves that the asymmetric squarylium dye provided by the present application is a type I photosensitizer, which can be applied to the hypoxic tumor environment in the future, and can efficiently produce hydroxyl radicals to kill tumor cells. Cy-580 is prepared into two kinds of tumor microenvironment responsive nanomaterials Cy-580 NPs and Cy-580@MnO2 NPs by nano precipitation method, the magnetic resonance effect and the production effect of hydroxyl radicals of the nanoparticle solution are studied, and the near-infrared imaging and photodynamic therapy effect of tumor cells are further investigated, and it is found that the two kinds of nanoparticles have low dark toxicity, and can realize type I photodynamic therapy under the excitation irradiation of near-infrared light 660 nm, and the active oxygen production effect of Cy-580@MnO2 NPs is stronger. This is because the MnO2 contained in Cy-580@MnO2 Nps can react with GSH in the tumor microenvironment to obtain Mn 2+ , and H2O2 / HCO3 -The Fenton-like reaction occurs in the presence of GSH to generate more hydroxyl radicals. 2 + It can also be used as a contrast agent for magnetic resonance imaging. Further investigation of the near-infrared / magnetic resonance imaging and therapeutic effect of tumor-bearing mice in vivo found that the tumor-targeting CREKA was more conducive to the targeting of the nanomaterials to the tumor site, achieving dynamic near-infrared fluorescence imaging. In vitro animal magnetic resonance imaging experiments found that the Mn 2+ obtained by the degradation of MnO2 in Cy-580@MnO2 NPs realizes magnetic resonance imaging. At the same time, more Cy-580@MnO2 NPs target and accumulate in the tumor tissue, which can achieve better therapeutic effect. It is proved that the Cy-580@MnO2 NPs provided by the present application is a new type of nanoparticle based on asymmetric phthalocyanine dye of type I photosensitizer, near-infrared-magnetic resonance bimodal imaging, which can also realize the synergistic treatment of photodynamic and chemical dynamics, providing a new idea for the imaging treatment of tumors.

[0111] For those skilled in the art to which the present application belongs, without departing from the concept of the present application, a number of simple deductions or substitutions can be made without having to undergo creative labor. Therefore, the simple improvements made by those skilled in the art to the present application according to the disclosure of the present application should be within the protection scope of the present application. The above examples are preferred embodiments of the present application, and any similar processes and equivalent changes made should be within the protection scope of the present application.

Claims

1. An asymmetric squarylium dye, characterized by The molecular structural formula is shown as formula (1). Wherein: R1 is hydrogen or C1-C20 alkyl; R2 is oxygen or malonitrile.

2. The asymmetric squarylium dye according to claim 1, characterized in that, The molecular structural formula is shown as formula (2) or formula (3).

3. A nanomaterial, characterized in that, The nanomaterial comprises micellar nanoparticles and MnO2-BSA nanoparticles which are electrostatically adsorbed on the surface of the micellar nanoparticles. The micellar nanoparticles have a core-shell structure, wherein the core is the asymmetric squarylium dye according to claim 1 or 2, and the shell comprises an amphiphilic block copolymer and an amphiphilic block copolymer coupled with a tumor-targeting polypeptide.

4. The nanomaterial of claim 3, wherein, The amphiphilic block copolymer comprises distearoyl phosphatidyl ethanolamine-polyethylene glycol; and / or, the tumor-targeting polypeptide comprises CREKA.

5. A method of producing a nanomaterial as claimed in claim 3 or 4, characterized in that, The method comprises the following steps: (1) dissolving the asymmetric squarylium dye in an organic solvent, and then adding the solution into an aqueous solution containing an amphiphilic block copolymer and an amphiphilic block copolymer coupled with a tumor-targeting polypeptide, mixing and dialyzing to obtain an aqueous solution of micellar nanoparticles, denoted as Cy Nps aqueous solution; (2) adding an aqueous solution of potassium permanganate into an aqueous solution of bovine serum albumin, dispersing to obtain an aqueous solution containing MnO2 and bovine serum albumin, denoted as MnO2-BSA Nps aqueous solution; (3) mixing the Cy Nps aqueous solution obtained in step (1) with the MnO2-BSA Nps aqueous solution obtained in step (2), and dialyzing to obtain the nanomaterial, denoted as Cy@MnO2 Nps aqueous solution.

6. The method of claim 5, wherein the nanomaterial is prepared by a method comprising: The volume ratio of the Cy Nps aqueous solution to the MnO2-BSA Nps aqueous solution is 1:(1-2).

7. A magnetic resonance imaging preparation, characterized in that, The nanomaterial according to claim 3 or 4.

8. Use of the asymmetric squarylium dye according to claim 1 or 2, or the nanomaterial according to claim 3 or 4 in the preparation of a photodynamic anti-tumor drug.

9. Use according to claim 8, characterised in that, The tumor comprises breast cancer.

Images