Photo-thermal reagent nano-particles with photoacoustic imaging, fluorescence imaging and photo-thermal treatment functions as well as preparation method and application of photo-thermal reagent nano-particles

Abstract

The invention relates to photo-thermal reagent nano-particles with photoacoustic imaging, fluorescence imaging and photo-thermal therapy functions and a preparation method and application thereof.The photo-thermal reagent nano-particles are prepared from a photo-thermal reagent and amphiphilic molecules, and the photo-thermal reagent has the structure shown in the general formula A, the absorption and emission wavelength of the photo-thermal reagent nano-particles is located in a near-infrared region of 1100-1300 nm, and the photo-thermal reagent nano-particles show strong near-infrared absorption and emission characteristics, excellent biocompatibility, as high as 69.17% of photo-thermal conversion efficiency and 0.5-0.6 cm of deep tissue penetrating power. After water-soluble modification, the photo-thermal reagent nano-particles can be used as a photo-acoustic and fluorescence imaging agent, efficient enrichment of tumor parts is realized through multi-mode imaging guidance, and the photo-thermal reagent nano-particles have excellent photo-thermal conversion performance and can effectively kill superficial and deep tumors. In addition, the biological safety of the photo-thermal reagent nano-particles is good, and the application potential of the photo-thermal reagent nano-particles in the field of biomedicine is further improved. General formula A # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical materials and tumor treatment preparations, and in particular to photothermal reagent nanoparticles with photoacoustic imaging, fluorescence imaging and photothermal treatment functions, and a preparation method and application thereof. Background Art

[0002] Cancer, as a major fatal disease, has long been a major challenge facing public health. Despite the continuous advancement of modern science and technology, the incidence and mortality of cancer remain high, and treatment methods are often limited. Although current clinical treatment methods such as surgery, radiotherapy and chemotherapy can effectively control tumor growth to a certain extent, they are often accompanied by postoperative complications, toxic side effects and drug resistance. There is an urgent need to find more accurate and efficient treatment options.

[0003] In recent years, with the rapid development of new biomaterials and tumor therapeutic agents, innovative treatment methods, especially technologies represented by multimodal phototherapy, have gradually emerged. High-resolution imaging technologies, such as fluorescence imaging (FLI), photoacoustic imaging (PAI), and photothermal imaging (PTI), combined with precise optical treatment methods (such as photodynamic therapy (PDT) and photothermal therapy (PTT)), have become a research hotspot in the field of tumor treatment due to their advantages such as low invasiveness, precise spatiotemporal control, high-efficiency therapeutic effects, and low drug resistance.

[0004] As an advanced treatment method, PTT uses light-activated photothermal agents to generate heat at the tumor site, thereby inducing thermal damage and ablation of tumor cells. Photothermal therapy has attracted widespread attention due to its advantages such as high spatial and temporal selectivity, non-invasiveness, and reduced patient discomfort. However, current photothermal agents (PTAs) still face some challenges, such as poor biocompatibility, low photothermal conversion efficiency (PCE), insufficient photostability, and limited light penetration depth.

[0005] Small molecule organic photothermal agents have gradually become a research hotspot in the field of photothermal therapy due to their excellent biocompatibility, biodegradability and more flexible functional design. Among them, indocyanine green (ICG), as an FDA-approved near-infrared dye, has good biocompatibility and deep tissue penetration, and can efficiently emit photons in the NIR-I range. Therefore, it is widely used in fluorescence angiography and clinical liver cancer surgery guidance. However, the poor photostability of ICG in the NIR-I window, low PCE and shallow penetration depth limit its widespread application in photothermal therapy.

[0006] In order to solve these problems, scientists began to focus on the development of new near-infrared II (NIR-II, 1000-1700nm) photothermal agents. Compared with NIR-I, NIR-II light has deeper tissue penetration (up to more than 1cm), lower scattering and reabsorption effects, thereby improving the treatment effect and imaging quality. Therefore, the development of NIR-II photothermal agents with good biocompatibility, enhanced photostability, high photothermal conversion efficiency and deeper penetration depth has become a frontier research issue in the field of photothermal therapy, and will also help promote the further application of photothermal agents in clinical practice. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems and provide a photothermal reagent nanoparticle with photoacoustic imaging, fluorescence imaging and photothermal therapy functions, and its preparation method and application. This photothermal reagent nanoparticle has both photothermal conversion efficiency and can be used for ablation of subcutaneous and in situ breast cancer under the guidance of multimodal imaging (NIR-II fluorescence, photoacoustic imaging and photothermal imaging), overcoming the problems of poor biocompatibility, low photothermal conversion efficiency, shallow penetration depth, and single function of photothermal reagents in the prior art.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide photothermal agent nanoparticles with photoacoustic imaging, fluorescence imaging and photothermal therapy functions, wherein the photothermal agent nanoparticles are prepared from raw materials including a photothermal agent and an amphiphilic molecule; the structural formula of the photothermal agent includes one or more of the structure shown in general formula A and the solvate of the structure shown in general formula A, and the general formula A is as follows:

[0010]

[0011] Where:

[0012] R1 and R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring, chlorine atom;

[0013] R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne, C1-C6 sulfonic acid substituted alkane;

[0014] n is independently selected from 1 or 2;

[0015] X is independently selected from a bromine atom, tetraphenylboric acid, and tetrakis(pentafluorobenzene)boric acid.

[0016] Furthermore, the amphiphilic molecule is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). Preferably, DSPE-PEG 2000 .

[0017] Furthermore, the maximum absorption wavelength of the photothermal agent is between 1100-1300 nm, the maximum emission wavelength is between 1150-1300 nm; and the molecular weight is between 700-1300 Da.

[0018] Furthermore, the structure of the photothermal agent is the structure shown in general formula A, or is an isomer of the structure shown in general formula A or a solvate of the structure shown in general formula A.

[0019] Further preferably, the structure of the small molecule fluorescent dye is the structure shown in general formula A.

[0020] Furthermore, the solvate of the structure represented by the general formula A is a solvate formed by the general formula A and a solvent such as dichloromethane, methanol or dimethyl sulfoxide.

[0021] Further preferably, the structure of the small molecule fluorescent dye is any one of the following formulas B1-B14:

[0022]

[0023] Furthermore, the preparation method of the photothermal reagent comprises the following steps:

[0024] Step 1: a carbon-nitrogen coupling reaction of a compound represented by formula (1) with a halide or sultone compound to obtain a compound represented by formula (2);

[0025] Step 2: The compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3);

[0026] Step 3: The compound represented by formula (3) is subjected to Suzuki coupling reaction to obtain a compound represented by formula (4);

[0027] Step 4: The compound represented by formula (3) or formula (4) is subjected to Knoevenagel condensation reaction with cyclopentanone to obtain a compound represented by formula (5);

[0028] Step 5, the compound represented by formula (5) is subjected to nucleophilic addition reaction under the action of lithium reagent or Grignard reagent to obtain the compound represented by general formula A after the nucleophilic addition reaction or the compound represented by general formula A is obtained by reacting with the corresponding anion salt after the nucleophilic addition reaction;

[0029] Among them, formula (1) is Formula (2) is Formula (3) is Formula (4) is Formula (5) is

[0030] Furthermore, the preparation method of the photothermal reagent includes the following process:

[0031]

[0032] Furthermore, in step 1, the compound represented by formula (1) is subjected to a carbon-nitrogen coupling reaction with a halide or a sultone compound to obtain a compound represented by formula (2), comprising:

[0033] In a first organic solvent environment, heating and stirring the compound represented by formula (1) and a halide or the compound represented by formula (1) and a sultone compound to obtain a compound represented by formula (2);

[0034] The halide is an alkyl or terminal alkyne halide;

[0035] The halogenated compound or sultone compound has a structure as shown in any of the following formulas C1, C2, and C3:

[0036]

[0037] In formula C1, C2, C3:

[0038] R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne;

[0039] wherein C1 and C2 are halogenated compounds, and C3 is a sultone compound;

[0040] The first organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ether, 1,4-dioxane, and glacial acetic acid.

[0041] Furthermore, in step 1, the conditions for heating and stirring are as follows: the heating temperature is 100-120° C., the stirring rate is 500-1000 rpm, and the heating and stirring time is 24-72 h.

[0042] Furthermore, the molar ratio of the compound represented by formula (1) to the halide or the compound represented by formula (1) to the sultone compound is 1:1.2-1:10.

[0043] Furthermore, in step 2, the compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3), comprising:

[0044] In a second organic solvent environment, the compound represented by formula (2) and the aldehyde compound are stirred to obtain a compound represented by formula (3);

[0045] The aldehyde compound has a structure as shown in any of the following formulas D1 and D2:

[0046]

[0047] The second organic solvent is selected from one or more of benzene, toluene, xylene, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, acetic acid, tetrahydrofuran, ether, 1,4-dioxane, ethanol, and methanol.

[0048] Furthermore, in step 2, the stirring conditions are as follows: the stirring rate is 500-1000 rpm, and the stirring time is 9-24 h.

[0049] Furthermore, the molar ratio of the compound represented by formula (2) to the aldehyde compound is 1:1-1:3.

[0050] Furthermore, in step 3, the compound represented by formula (3) is subjected to a Suzuki coupling reaction to obtain a compound represented by formula (4), comprising:

[0051] In a third organic solvent environment, under the protection of an inert gas, the compound represented by formula (3) and the boric acid compound are heated and stirred with a palladium catalyst and a base as a catalyst to obtain a compound represented by formula (4) through a Suzuki coupling reaction;

[0052] The boric acid compound has a structure as shown in the following formula E:

[0053]

[0054] In formula E:

[0055] R1 is independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring;

[0056] The palladium catalyst is selected from one or more of tetrakistriphenylphosphine palladium, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetonepalladium), and palladium acetate;

[0057] The base is selected from one or more of potassium tert-butoxide, sodium tert-butoxide, potassium carbonate, cesium carbonate, and potassium phosphate;

[0058] The third organic solvent is selected from one or more of benzene, toluene, xylene, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and diethyl ether.

[0059] Furthermore, in step three, under the protection of inert gas, the conditions for heating and stirring are as follows: heating temperature is 80-120° C., stirring rate is 500-1000 rpm, and heating and stirring time is 12-48 h.

[0060] Furthermore, the molar ratio of the compound represented by formula (3) to the boric acid compound is 1:1-1:3.

[0061] Furthermore, in step 4, the compound represented by formula (3) or formula (4) is subjected to Knoevenagel condensation reaction with cyclopentanone to obtain a compound represented by formula (5), comprising:

[0062] In a fourth organic solvent environment, the compound represented by formula (3) or formula (4) and cyclopentanone are heated and stirred with a base, wherein the base serves as a catalyst, and a Knoevenagel condensation reaction is performed to obtain a compound represented by formula (5);

[0063] The base is selected from one or more of sodium hydroxide, sodium tert-butoxide, sodium ethoxide, and potassium hydroxide;

[0064] The fourth organic solvent is selected from one or more of tetrahydrofuran, diethyl ether, 1,4-dioxane, ethanol, and methanol.

[0065] Furthermore, in step 4, the conditions for heating and stirring are as follows: the heating temperature is 80-120° C., the stirring rate is 500-1000 rpm, and the heating and stirring time is 12-72 h.

[0066] Furthermore, the molar ratio of the compound represented by formula (3) to cyclopentanone is 1:0.5-1:1.

[0067] Furthermore, in step 5, the compound represented by formula (5) is subjected to a nucleophilic addition reaction under the action of a lithium reagent or a Grignard reagent, and the compound represented by general formula A is obtained after the nucleophilic addition reaction, or part of the compound obtained after the nucleophilic addition reaction is reacted with a corresponding anion salt to obtain a compound represented by general formula A, including:

[0068] In a fifth organic solvent environment, under the protection of an inert gas, reacting the compound represented by formula (5) with a lithium reagent or reacting the compound represented by formula (5) with a Grignard reagent at low temperature, undergoing a nucleophilic addition reaction, and obtaining a compound represented by general formula A after the nucleophilic addition reaction, or exchanging anions with a corresponding anion salt of a part of the compound obtained after the nucleophilic addition reaction, to obtain a compound represented by general formula A;

[0069] The lithium reagent or Grignard reagent has a structure as shown in any of the following formulas F1 and F2:

[0070]

[0071] In formula F1 and F2:

[0072] R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring;

[0073] The anion salt is selected from one or more of sodium tetraphenylborate and potassium tetrakis(pentafluorobenzene)borate;

[0074] The fifth organic solvent is selected from one or more of super dry tetrahydrofuran, diethyl ether, and 1,4-dioxane.

[0075] Furthermore, in step 5, under the protection of inert gas, the conditions for the low-temperature reaction are as follows: the low-temperature is -87°C, the stirring rate is 500-1000rpm, and the stirring time is 0.5-3h.

[0076] Furthermore, the molar ratio of the compound represented by formula (5) to the lithium reagent or the molar ratio of the compound represented by formula (5) to the Grignard reagent is 1:1-1:20.

[0077] Furthermore, the molar ratio of the compound represented by formula (5) to the anion salt is 1:1-1:20.

[0078] The photothermal reagent provided by the present invention is a novel photothermal reagent based on two benzindole structures connected by a 15-membered methine chain. The photothermal reagent with an absorption and emission peak wavelength in the near-infrared region obtained by regulating the substituents on the parent structure can introduce different substituents on the 15-membered methine chain and the indole N atom. Its maximum absorption wavelength is between 1100-1300nm, the maximum emission wavelength is between 1150-1300nm; the molecular weight is between 700-1300Da, and it has the characteristics of excellent photostability, low cytotoxicity, simple synthesis, and easy structural modification.

[0079] The second object of the present invention is to provide a method for preparing photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions, comprising the following steps:

[0080] In the first step, the photothermal reagent is dispersed in an organic solvent, and then added dropwise to the amphiphilic molecule solution under ultrasound, wherein the amphiphilic molecule solution is a solution obtained by dissolving the amphiphilic molecule in the organic solvent. After ultrasound for a period of time, the organic solvent is removed by a rotary evaporator to obtain a mixture A.

[0081] The second step is to add phosphate buffer solution to the mixture A, place it in an ice water bath, and ultrasonically vibrate to obtain a mixture B;

[0082] The third step is to remove large particles in the mixed solution B and perform ultrafiltration concentration to obtain photothermal reagent nanoparticles.

[0083] Furthermore, in the first step, the organic solvent is dichloromethane; in the first step, the ultrasonic time is 0.5-1h.

[0084] Furthermore, in the second step, the concentration of the phosphate buffer solution is 0.01 M and the pH is 7.4.

[0085] Furthermore, in the third step, large particles in the mixed solution B are removed by centrifugation, and the centrifugation conditions are: a centrifugal rate of 3000 rpm and a centrifugal time of 5 min.

[0086] The third object of the present invention is to provide an application of photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions, and the photothermal agent nanoparticles are used for fluorescence imaging, photoacoustic imaging or photothermal imaging.

[0087] The fourth object of the present invention is to provide an application of photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions, and to use the photothermal agent nanoparticles for photothermal therapy.

[0088] Furthermore, the photothermal agent nanoparticles are used for photothermal treatment of tumors, especially for the treatment of superficial tumors or deep tumors.

[0089] Furthermore, the invention provides an application of the photothermal agent nanoparticles for multimodal photothermal therapy of superficial or deep in situ subcutaneous tumors.

[0090] Furthermore, after the photothermal agent nanoparticles reach the lesion site through tail vein injection, under the guidance of multimodal imaging, laser irradiation causes temperature increase (photothermal therapy), which can significantly inhibit the growth of subcutaneous cancer, without lung metastasis and recurrence, and prolong the survival period, showing great application value in multimodal photothermal therapy of tumors.

[0091] Furthermore, the time of photothermal therapy after tail vein injection is determined by photoacoustic imaging and NIR-II fluorescence imaging.

[0092] Furthermore, the tumor is breast cancer, colon cancer, prostate cancer, bladder cancer, etc.

[0093] Furthermore, the tumor is breast cancer in situ, lung cancer in situ, and a cancer type with a tissue penetration of 0.5-0.6 cm.

[0094] Furthermore, the shortest action time of the photothermal therapy is as low as 3 minutes.

[0095] Further preferably, the laser wavelength of the photothermal therapy is 1064 nm and the power density is 0.5-1.0 W / cm 2 , the action time is 3-5min.

[0096] Furthermore, a 1064 nm laser is used as a trigger for the light source of irradiation.

[0097] Under the action of 1064nm laser, the photothermal agent (cyanine photothermal agent) in the photothermal agent nanoparticles absorbs NIR-II light energy from the ground state to the excited state. A part of the PTT photothermal agent releases heat during the process of returning to the ground state through non-radiative transition, which increases the temperature of the tumor site and ablates the tumor. In addition, thermal elasticity is generated during the photothermal process to generate PA signals. The other part returns to the ground state through radiative transition accompanied by fluorescent emission for NIR-II fluorescence imaging to monitor the enrichment of the material at the tumor site.

[0098] The photothermal reagent nanoparticles provided by the present invention include photothermal reagents, and provide a series of NIR-II cyanine photothermal reagents with excellent biocompatibility and deep penetration ability. In the 915-1214nm band, it exhibits significant absorption characteristics, and the tail wave absorption extends to 1400nm, and the maximum emission peak is located at 1248nm. The penetration depth of the photothermal reagent reaches 0.5-0.6cm, and the photoconversion efficiency (PCE) is as high as 69.17%. Through near-infrared second-zone fluorescence imaging (NIR-II FLI) and photoacoustic imaging (PAI), the passive accumulation of photothermal reagents in the tumor site can be observed, thereby ensuring the optimal tumor accumulation time and improving the accuracy of photothermal therapy (PTT). Under the guidance of FLI, PAI and photothermal imaging (PTI), the photothermal reagent successfully achieved rapid and precise ablation of subcutaneous and in situ breast tumors. After 24 hours of injection of the photothermal reagent into the mouse through the tail vein, the photothermal reagent was injected into the mouse using a wavelength of 1064nm (1.0W / cm 2) laser irradiation, the tumor temperature rises rapidly, exceeding 60°C within 1 minute, successfully inhibiting the tumor growth of subcutaneous and orthotopic breast cancer tumor-bearing mice, with an inhibition rate of up to 100%, and no tumor recurrence or metastasis was observed within 30 days. In addition, the photothermal agent also exhibits good biocompatibility and safety. Therefore, the present invention provides a photothermal agent nanoparticle with photoacoustic imaging, fluorescence imaging and photothermal therapy functions, and a preparation method and application thereof, including a new near-infrared second-zone cyanine photothermal agent, which provides valuable insights for the research and development of anthocyanin-based tumor treatment materials and has broad clinical application prospects.

[0099] By modifying the water solubility of the photothermal agent with amphiphilic molecules, the water solubility of the agent can be improved, and its dispersibility and biocompatibility in the biological environment can be enhanced, thereby improving its practical application effect in photothermal therapy. This modification may also help reduce the toxicity of the photothermal agent and give it better targeting and stability, thereby enhancing the specificity and effectiveness of the treatment.

[0100] Compared with the prior art, the beneficial effects of the present invention are embodied in the following aspects:

[0101] The present invention relates to a photothermal agent nanoparticle with photoacoustic imaging, fluorescence imaging and photothermal therapy functions and its preparation and application. The photothermal agent nanoparticles include a photothermal agent, which is a NIR-II photothermal agent (NIR-II cyanine photothermal agent), and the structural formula of the NIR-II photothermal agent includes one or more of the structure shown in general formula A and the solvate of the structure shown in general formula A. The absorption and emission wavelengths are located in the near-infrared region of 1100-1300nm, showing strong near-infrared absorption and emission characteristics, excellent biocompatibility, up to 69.17% photothermal conversion efficiency and 0.5-0.6cm deep tissue penetration ability. After the photothermal agent is modified by water solubility, the obtained photothermal agent nanoparticles can be used for fluorescence imaging, photoacoustic imaging and photothermal imaging, and can also be used for photothermal treatment of tumors. The NIR-II cyanine photothermal agent nanoparticles in the present invention can not only be used as photoacoustic and fluorescence imaging agents, but also can achieve efficient enrichment of tumor sites through multimodal imaging guidance, and also have excellent photothermal conversion performance, which can effectively kill superficial and deep tumors. In addition, the photothermal reagent nanoparticles have good biosafety, further enhancing their application potential in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 The absorption and emission spectra of the photothermal reagent nanoparticles synthesized in Example 1 in aqueous solution.

[0103] Figure 2 The photothermal agent nanoparticles synthesized in Example 2 were irradiated at 1064 nm (1.0 W / cm 2) laser, and the temperature changes over time recorded by a 618C camera (Fotric).

[0104] Figure 3 The photothermal agent nanoparticles in Example 2 were subjected to different laser densities at 1064 nm (0.50, 0.75, 1.0 W / cm 2 ) Temperature variation over time under laser irradiation.

[0105] Figure 4 This is a temperature change curve of the photothermal reagent nanoparticles in Example 2, which are heated up within 5 minutes under 1064nm laser irradiation and naturally cooled to room temperature.

[0106] Figure 5 This is the NIR-II photoacoustic signal diagram of the aqueous solution of photothermal reagent nanoparticles with different concentrations in Example 3.

[0107] Figure 6 This is a tissue penetration depth diagram of the photothermal agent nanoparticles in Example 3.

[0108] Figure 7 The killing effect of the photothermal agent nanoparticle aqueous solution of different concentrations in Example 4 on mouse breast cancer cells under laser irradiation.

[0109] Figure 8 These are NIR-II fluorescence imaging and photoacoustic imaging images after the photothermal agent nanoparticles in Example 5 were injected into the tumor site through the tail vein.

[0110] Fig. 9 This is the photothermal image and temperature rise curve of the tumor site under laser irradiation after the photothermal agent nanoparticles in Example 5 are injected into the tumor site through the tail vein.

[0111] Fig.10 This is a test diagram related to the laser irradiation of the photothermal reagent nanoparticles injected into the tail vein in Example 5, wherein: Fig.10 A is the tumor growth curve of subcutaneous tumor-bearing mice after laser irradiation of photothermal reagent nanoparticles injected into the tail vein in Example 5, Fig.10 B is the bioluminescent image of the in vivo in vivo in mice bearing Luc-4T1 breast cancer in situ after laser irradiation of the photothermal reagent nanoparticles injected into the tail vein in Example 5, showing the in situ therapeutic effect after treatment in different groups.

[0112] Fig.11 These are hematoxylin-eosin stained images of lung sections of mice in different treatment groups after the photothermal treatment of orthotopic breast cancer-bearing mice in Example 5.

[0113] Fig.12This is a safety evaluation diagram of injecting photothermal reagent nanoparticles into healthy mice in Example 6, wherein A is an organ H&E staining image, B is a blood routine analysis diagram, and C is a blood biochemical analysis diagram. DETAILED DESCRIPTION

[0114] The present invention is described in detail below in conjunction with specific embodiments, but is by no means intended to limit the present invention. Features such as preparation methods, materials, structures or composition ratios not explicitly described in this technical solution are considered to be common technical features disclosed in the prior art.

[0115] The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several modifications and improvements may be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0116] In this embodiment, the hydrogen nuclear magnetic resonance spectrum (1H NMR) of the compound is measured by Bruker AVANCE III HD 400 or Bruker AVANCE III HD 500; all reagents used are commercially available reagents.

[0117] In the following examples, the mice are Balb / c mice, which are commercially available from Shanghai Lingchang Biotechnology Co., Ltd. Mouse breast cancer cells 4T1 cells and Luc-4T1 cells are commercially available from Shanghai Fuheng Biotechnology Co., Ltd.

[0118] Example 1: Synthesis and characterization of NIR-II cyanine photothermal reagents and photothermal reagent nanoparticles:

[0119] This embodiment provides a preparation of a photothermal reagent B13, and the compound structure and specific synthesis route are as follows:

[0120]

[0121] (1) Synthesis of Compound B1-(2)

[0122] 2,3,3-Trimethyl-4,5-benzo-3H-indole (CAS: 41532-84-7) (1.0 g, 4.8 mmol), acetonitrile (36 mL), and 1-iodopropane (702 μL, 7.2 mmol) were added to a 100 mL round-bottom flask, and the reaction was heated for 2 days at 110° C. and 800 rpm. After cooling to room temperature, the mixture was concentrated by rotary evaporation, and the remaining oily solution was washed with ether to obtain the product B1-(2), a dark brown solid of 1.34 g, with a yield of 97%.

[0123] H-NMR (400MHz, CDCl3, 25℃) δ = 8.08 (2H, t, J = 8Hz, ar.CH) and 8.04(1H,d,J=8Hz,ar.CH)part.overlapped,7.92(1H,d,J=8Hz,ar.CH),7.83(1H,d,J=8Hz,ar.CH),7.6 6(1H,t,J=8Hz,ar.CH),4.80(2H,t,J=8Hz,-NCH2-),3.20(3H,s,-CH3),2.11-2.02(2H,m,-NCH2CH2CH2) ,1.87(6H,s,-CH3),1.11(3H,t,J=8Hz,-NCH2CH2CH3);13C-NMR(100MHz,CDCl3,25℃)δ=195.3,189.7,15 0.1,138.5,138.0,136.9,133.5,132.1,131.2,130.0,127.3,55.8,55.2,22.6,21.6,16.8,11.2; ESI-MS m / z calculated for C18H22N+252.17; obs.:252.2

[0124] (2) Synthesis of Compound B3-(3)

[0125] A mixture of B1-(2) (10 mmol, 3.8 g) and 2-chloro-1-formyl-3-(hydroxymethylene)cyclopentene (2-chloro-1-formyl-3-(hydroxymethylene)cyclopentene was synthesized according to the synthesis method described in the Supplementary information of reference Li, X.; Tang, Y.; Li, J.; Hu, X.; Yin, C.; Yang, Z.; Wang, Q.; Wu, Z.; Lu, X.; Wang, W.; et al. A small-molecule probe for ratiometric photoacoustic imaging of hydrogensulfide in living mice. Chemical Communications 2019, 55, 5934-5937.) (15 mmol, 2.4 g) was dissolved in a mixture of toluene (347 mmol, 37 mL) and acetic acid (209 mmol, 13 mL), stirred at room temperature for 12 h at a stirring rate of 800 rpm, and concentrated by rotary evaporation. The residual organic matter was purified by silica gel column chromatography (PE:EA=9:1) to obtain compound B3-(3), a red solid (1.6 mmol, 636.2 mg), with an isolated yield of 16.3%.

[0126] 1 H NMR (400MHz, Chloroform-d) δ10.01(s,1H),8.02(d,J=8.5Hz,1H),7.81(d,J=8.2Hz,1H),7.76(d,J=8.7Hz,1H),7.50–7.39(m,2H),7.31–7.26(m,1 H),7.08(d,J=8.7Hz,1H),5.31(d,J=12.9Hz,1H),3.75(t,J=6.2Hz,2H), 2.80–2.73(m,4H),1.94(s,6H),1.84–1.77(m,2H),1.02(t,J=7.4Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ186.7,162.5,149.4,141.8,135.6,133.9,130.0,129.8,129.6,129.2, 129.0,126.9,125.0,122.7,121.7,109.4,93.4,48.4,27.6,26.2,24.9,20.0,11.7.HRMS:calculated forC 25 H 26 ClNO[M+H] + 392.1776, found 392.1776.

[0127] (3) Synthesis of Compound B13-(4)

[0128] A mixture of B3-(3) (0.5mmol, 197mg), phenylboronic acid (1mmol, 122mg), K2CO3, (1mmol, 138mg) and Pd(PPh3)4 (0.05mmol, 50mg) was added to a Shrek bottle under argon protection. Dry DMF (0.05mmol, 4mL) and deionized water (0.04mmol, 0.8mL) were added to the bottle and stirred at 90°C for 24h at a stirring rate of 800rpm. After cooling to room temperature, the reaction mixture was poured into brine and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated by rotary evaporation. The obtained organic matter was purified by column chromatography on silica gel (PE:EA=6:1) to obtain compound B13-(4), a red solid 0.45mmol, 197mg, with an isolated yield of 90.9%.

[0129] 1H NMR(400MHz,Chloroform-d)δ9.55(s,1H),7.90(d,J=8.4Hz,1H),7.77(d,J=8.4Hz, 1H),7.71(d,J=8.8Hz,1H),7.53–7.44(m,3H),7.42–7.33(m,3H),7.25–7.20(m,1H) ,7.03(d,J=8.7Hz,1H),6.88(d,J=12.1Hz,1H),5.37(d,J=13.1Hz,1H),3.69(t,J=6 .7Hz,2H),2.92–2.81(m,4H),1.83–1.72(m,2H),1.68(s,6H),1.01(t,J=6.8Hz,3H). 13 CNMR(101MHz,Chloroform-d)δ189.6,163.0,161.2,141.9,140.5,140.2,133.3,129.8,129.8,129.7,129.6,129.5,128.9, 128.8,128.7,128.3,126.7,126.6,122.4,121.6,115.4,109.3,94.2,47.9,44.0,27.8,27.2,26.5,11.7.HRMS:calculated for C 31 H 31 NO[M+H] + 434.2478, found 434.2494.

[0130] (4) Synthesis of Compound B13-(5)

[0131] A mixture of B13-(4) (0.2 mmol, 87 mg), cyclopentanone (0.1 mmol, 8.9 μL), and NaOH (4 mmol, 32 mg) was added to a round-bottomed bottle. 2 mL of ethanol was added to the bottle and stirred at 80°C for 48 h at a stirring rate of 800 rpm. After cooling to room temperature, the reaction mixture was filtered and washed with ethanol to obtain compound B13-(5), a blue solid 0.08 mmol, 76 mg, with an isolated yield of 83%.

[0132] 1H NMR(400MHz,Chloroform-d)δ7.89(d,J=9.0Hz,2H),7.74(d,J=8.1Hz,2H),7.67(d ,J=8.7Hz,2H),7.46–7.33(m,8H),7.25–7.14(m,8H),6.99(d,J=8.8Hz,2H),6.62( d,J=12.6Hz,2H),5.35(d,J=11.4Hz,2H),3.75–3.58(m,4H),3.21–2.97(m,8H),2. 94–2.77(m,4H),1.80–1.72(m,4H),1.68(s,12H),1.00(t,J=7.4Hz,6H).MALDI-FT ICR MS: calculated for C 67 H 66 N2O[M] + 914.5175, found 914.5050.

[0133] (5) Synthesis of Compound B13

[0134] B13-(5) (0.01mmol, 9mg) was added to a Schlenk bottle under argon protection. Ultra-dry THF (200mmol, 2mL) was added to the bottle and cooled to 0°C in an ice-water bath. Subsequently, 1M phenylmagnesium bromide (0.2mmol, 0.2mL) was dropped into the solution. Stirred in an ice-water bath for 0.5h at a stirring rate of 800rpm, and 2mL of methanol was added to quench the reaction. The obtained organic matter was purified by basic alumina (300-400 mesh) column chromatography (DCM:MeOH=25:1) to obtain 4mg of the compound as a black solid (0.004mmol, 4.0mg), with an isolated yield of 41.0%. The obtained solid was dissolved in acetonitrile, sodium tetraphenylborate (0.2 mmol, 68 mg) was added, and the mixture was stirred at room temperature for 5 hours at a stirring rate of 800 rpm. The obtained organic matter was purified by basic alumina (300-400 mesh) column chromatography (DCM: MeOH = 99: 1) to obtain compound B13, 2.4 mg, a black solid (0.002 mmol, 2.4 mg), and the isolation yield was 46.3%.

[0135] 1H NMR(400MHz,Chloroform-d)δ7.96–7.92(m,2H),7.90–7.81(m,4H),7.49–7.39(m,13H),7.08–6.99(m,10H),6.89–6.82(m,3H),5.84(d,J= 14.0Hz,1H),3.79(t,J=7.2Hz,2H),3.67(t,J=6.8Hz,2H),3.49–2.88(m,8H),1.79–1.62(m,20H),0.91(t,J=7.2Hz,6H).HRMS:calculated for C 73 H 71 BrN2[M-Br] + 975.5612, found 975.5620.

[0136] This embodiment further provides a method for preparing photothermal reagent nanoparticles, wherein the photothermal reagent nanoparticles are prepared from raw materials including a photothermal reagent and amphiphilic molecules.

[0137] The preparation steps of the photothermal agent nanoparticles are as follows:

[0138] In the first step, B13 is obtained by replacing the counter ion pair through aldol condensation and metal organic reaction and sodium tetraphenylborate (see the preparation process of B13 above);

[0139] In the second step, 4 mg of B13 was dispersed in 4 mL of dichloromethane, and then added dropwise to the DSPE-PEG solution under ultrasonic conditions. The DSPE-PEG solution was a solution obtained by dispersing 200 mg of DSPE-PEG in 10 mL of dichloromethane solution. After ultrasonication for 0.5 h, the solvent was removed by a rotary evaporator, and the obtained solute was placed in a flask. DSPE-PEG is DSPE-PEG 2000 , purchased from Shanghai Titan Technology Co., Ltd.

[0140] In the third step, 8 mL of phosphate buffer solution (PBS, 1X, pH = 7.4) was added to the flask, which was placed in an ice water bath and subjected to ultrasonic oscillation for 0.5-1 h for reaction.

[0141] In the fourth step, after the reaction, the obtained solution was centrifuged at 3000 rpm for 5 min to remove large particles. Then, ultrafiltration and concentration were performed using a 10KDa Amicon ultrafilter (Millipore Corporation) to obtain photothermal reagent nanoparticles (B13 NPs). The obtained photothermal reagent nanoparticles were stored at 4°C. The optical properties of the photothermal reagent nanoparticles were then characterized, such as Figure 1As shown, the absorption and emission spectra of the photothermal agent nanoparticle aqueous solution B13 NPs have a maximum absorption peak at 915-1214nm, tail wave absorption to 1350nm, and its emission peak at 1248nm.

[0142] Example 2: Photothermal properties of photothermal reagent nanoparticles

[0143] The photothermal reagent nanoparticles with different concentrations were recorded at different laser densities (0.50, 0.75, 1.0 W / cm 2 ) temperature changes with time under irradiation, where the laser irradiation conditions are 1064nm, 1.0W / cm 2 The concentrations of the photothermal agent nanoparticle aqueous solution were 0, 12.5, 25, 50, and 100 μg / mL. Figure 2 As shown in the figure, under laser irradiation, compared with water, 100 μg / mL aqueous solution of photothermal agent nanoparticles at 1.0 W / cm 2 After irradiation for 5 min, the temperature increased rapidly from 27.9 °C to 79.3 °C, with a temperature change of 51.4 °C. Figure 3 , the heating curves of 100 μg / mL photothermal reagent nanoparticles at different power densities, even at 0.50 W / cm 2 At low power density, the temperature that can kill tumors can also be reached (>42°C). The photothermal agent nanoparticles prepared above were heated at 1064nm, 1.0W / cm 2 After irradiation for 5 minutes, it was allowed to cool naturally to room temperature and its photothermal efficiency was measured. Figure 4 As shown, it was found that its photothermal conversion efficiency was as high as 69.17%, with excellent photothermal performance.

[0144] Example 3: Fluorescence and photoacoustic imaging of photothermal reagent nanoparticles:

[0145] The photothermal agent nanoparticles of different concentrations (including 500 μg / mL) prepared in Example 2 were subjected to PA imaging under the VevoLAZR imaging system. Figure 5 As shown in the figure, the concentration of photothermal reagent nanoparticles is positively correlated with the PA signal. The higher the concentration of photothermal reagent, the stronger the PA signal. In addition, the NIR-II fluorescence imaging of the photothermal reagent nanoparticle aqueous solution with the same fluorescence intensity as ICG was evaluated under 1% lipid at different depths. Figure 6 As shown, compared with the penetration depth of ICG of 0.2 cm, the penetration depth of photothermal agent nanoparticles is as low as 0.5-0.6 cm, showing extremely strong tissue penetration.

[0146] Example 4: Photothermal agent nanoparticles kill tumor cells:

[0147] The photothermal agent nanoparticles of different concentrations prepared in Example 2 were incubated with mouse breast cancer cells (4T1) for 12 hours. Figure 7 The photothermal agent nanoparticles did not produce significant cytotoxicity to 4T1 cells, indicating that the photothermal agent nanoparticles have good biocompatibility. However, after incubation for 12 hours, 4T1 cells were exposed to a power density of 1.0 W / cm 2 After irradiation with 1064nm laser for 5 minutes, the photothermal agent nanoparticles produced a strong cell killing effect, indicating that the NIR-II cyanines in the photothermal agent nanoparticles can act as photothermal agents to ablate tumor cells by increasing the temperature.

[0148] Example 5: Photothermal therapy of subcutaneous and in situ breast cancer using photothermal agent nanoparticles under the guidance of multimodal imaging:

[0149] When the tumor-bearing mice reach a size of 100 mm 3 , B13 NPs were injected into the tail vein, the concentration of photothermal agent nanoparticles was 1 mg / mL, and the injection dose was 200 μL. PA imaging of 4T1 tumor-bearing mice was performed under the Vevo LAZR imaging system at 0, 3, 6, 12, 24, and 48 h after injection. In vivo fluorescence imaging was performed in the same way and with an exposure time of 200 ms using the NIR-II fluorescence imaging system (NIROPTICS). Figure 8 As shown, the enrichment at the mouse tumor site was optimal 24 hours after injection.

[0150] Four experimental groups were set up, namely control group (Group I, Control), NIR group (Group II, NIR), photothermal agent nanoparticle group (Group III, B13 NPs) and photothermal agent nanoparticle combined with NIR group (Group IV, B13 NPs+NIR). When the tumor volume of mice reached 100mm 3 Treatment was started when the tumor volume reached 1000mm 3 When the mice were considered dead, the tumor volume was stopped. Group III and IV were injected with the photothermal agent nanoparticle aqueous solution prepared in Example 2 into mice with subcutaneous or orthotopic breast cancer models through the tail vein. The concentration of the photothermal agent nanoparticles was 1 mg / mL and the injection dose was 200 μL. Similarly, the control group and NIR group were injected with PBS (1X, pH = 7.4) through the tail vein injection. The injection dose was 200 μL. 24 hours after the tail vein injection, the IV group was injected with 1064 nm, 1.0 W / cm 2The irradiation was carried out under the same conditions, and the subcutaneous model was recorded at 0s, 10s, 20s, 30s, 60s, 120s, and 180s. The NIR group was also irradiated in the same way. For the deep tumor model, the temperature changes over time and thermal images of the mouse tumor site were recorded by a 618C camera (Fotric), as shown in Figure 2. Fig. 9 As shown, after 1 min of irradiation, the temperature rose to 60 °C, and group IV showed excellent in vivo photothermal performance.

[0151] For the subcutaneous tumor model, the tumor volume of subcutaneous tumor-bearing mice in different experimental groups was measured using a vernier caliper at different time points. The tumor growth curve of mice is shown in Fig.10 As shown in A, compared with the control group, the NIR group (Group II) and the photothermal agent nanoparticles (Group III) that were only irradiated with 1064nm had almost no inhibitory effect on the tumor; however, the photothermal agent nanoparticles combined with 1064nm laser irradiation (Group IV) showed a significant therapeutic effect on the growth of the tumor. On the fourth day, the tumor completely disappeared without recurrence and metastasis, and the inhibition rate reached 100%. This shows that the photothermal agent nanoparticles have a strong killing effect on the tumor under laser irradiation.

[0152] For the orthotopic breast cancer tumor model, in vivo bioluminescence imaging of orthotopic breast cancer Luc-4T1 tumor-bearing mice after laser irradiation of photothermal reagent nanoparticles at different time points showed the in situ therapeutic effect after treatment with different groups. Fig.10 As shown in B, after one treatment, the tumor was ablated and there was no recurrence or metastasis within 30 days. In addition, after 30 days of treatment, the lung tissues of mice in different treatment groups were fixed and sectioned, and further stained with hematoxylin-eosin to evaluate the lung metastasis after treatment with photothermal reagent nanoparticles. Fig.11 As shown, group IV exhibited large-area apoptosis and necrosis of tumor cells, indicating that the temperature of the photothermal agent nanoparticles increased under laser irradiation, causing apoptosis and necrosis of tumor cells.

[0153] Example 6: In vivo safety evaluation of photothermal reagent nanoparticles:

[0154] Healthy mice were intravenously injected with photothermal reagent nanoparticles (2 mg / mL, 200 μL, 2 times the treatment dose). The mice were killed on the 1st, 3rd, 7th, and 14th days after treatment, and whole blood, serum, and major organs (liver, spleen, kidney, heart, and lung) were collected for complete blood cell count analysis, blood chemistry, and histological examination. Fig.12As shown, after intravenous injection of B13 NPs, organ hematoxylin and eosin (H&E) staining showed no obvious tissue damage. In addition, complete blood cell count analysis showed no significant deviation between injected mice and untreated controls, and all parameters were within normal ranges. Liver and kidney function tests also showed no abnormalities. These results demonstrate the excellent biosafety of the photothermal agents in this photothermal agent nanoparticle, highlighting their potential for in vivo biological applications.

[0155] The above is a description of the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned description of the embodiments for the convenience of ordinary technicians in the technical field to understand and use the invention. It is obvious that those familiar with the technology in the field can easily make various modifications to these embodiments and apply the general principles described here to other embodiments without creative work. Therefore, the present invention is not limited to the above-mentioned embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A photothermal agent nanoparticle with photoacoustic imaging, fluorescence imaging and photothermal therapy functions, characterized in that: The photothermal agent nanoparticles are prepared from raw materials including a photothermal agent and amphiphilic molecules; The structural formula of the photothermal agent includes one or more of the structure shown in general formula A and the solvate of the structure shown in general formula A, and the general formula A is as follows: Where: R1 and R2 are each independently selected from C1-C6 alkyl, aromatic ring and substituted aromatic ring, chlorine atom; R3 is independently selected from C1-C6 alkyl, C1-C6 terminal alkyne, C1-C6 sulfonic acid substituted alkane; n is independently selected from 1 or 2; X is independently selected from a bromine atom, tetraphenylboric acid, and tetrakis(pentafluorophenyl)boric acid.

2. The photothermal agent nanoparticles with photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to claim 1, characterized in that: The amphiphilic molecule is distearoyl phosphatidylethanolamine-polyethylene glycol.

3. The photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to claim 1, characterized in that: The maximum absorption wavelength of the photothermal agent is between 1100-1300nm, the maximum emission wavelength is between 1150-1300nm; and the molecular weight is between 700-1300Da.

4. The photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to claim 1, characterized in that: The structure of the photothermal agent is any one of the following formulas B1-B14:

5. The photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to claim 1, characterized in that: The preparation method of the photothermal reagent comprises the following steps: Step 1: The compound represented by formula (1) undergoes a carbon-nitrogen coupling reaction with a halide or a sultone compound to obtain a compound represented by formula (2); Step 2: The compound represented by formula (2) and the aldehyde compound undergo a dehydration condensation reaction to obtain a compound represented by formula (3); Step 3: The compound represented by formula (3) is subjected to Suzuki coupling reaction to obtain a compound represented by formula (4); Step 4: The compound represented by formula (3) or formula (4) is subjected to Knoevenagel condensation reaction with cyclopentanone to obtain a compound represented by formula (5); Step 5, the compound represented by formula (5) is subjected to nucleophilic addition reaction under the action of lithium reagent or Grignard reagent to obtain the compound represented by general formula A or further reacts with the corresponding anion salt to obtain the compound represented by general formula A; Among them, formula (1) is Formula (2) is Formula (3) is Formula (4) is Formula (5) is 6. A method for preparing photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to any one of claims 1 to 5, characterized in that: The steps include: In the first step, the photothermal reagent is dispersed in an organic solvent, and then added dropwise to the amphiphilic molecule solution under ultrasound, wherein the amphiphilic molecule solution is a solution obtained by dissolving the amphiphilic molecule in the organic solvent. After ultrasound for a period of time, the organic solvent is removed by a rotary evaporator to obtain a mixture A. The second step is to add phosphate buffer solution to the mixture A, place it in an ice water bath, and ultrasonically vibrate to obtain a mixture B; The third step is to remove large particles in the mixed solution B and perform ultrafiltration concentration to obtain photothermal reagent nanoparticles.

7. The method for preparing photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to claim 6, characterized in that: In the first step, the organic solvent is dichloromethane; In the first step, the ultrasound duration is 0.5-1h; In the second step, the concentration of the phosphate buffer solution is 0.01 M and the pH is 7.4; In the third step, large particles in the mixed solution B are removed by centrifugation. The centrifugation conditions are: centrifugal speed 3000 rpm, centrifugal time 5 min.

8. An application of photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions as described in claims 1-5, characterized in that: The photothermal agent nanoparticles are used for fluorescence imaging, photoacoustic imaging or photothermal imaging.

9. An application of photothermal agent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions as described in claims 1-5, characterized in that: The photothermal agent nanoparticles are used for photothermal therapy.

10. The use of photothermal reagent nanoparticles having photoacoustic imaging, fluorescence imaging and photothermal therapy functions according to claim 9, characterized in that: The laser wavelength of the photothermal therapy is 1064nm, and the power density is 0.5-1.0W / cm 2 , the action time is 3-5min.

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