A type of cyanine dye and cyanine dye J-aggregate nanoparticles and their applications

Abstract

The present invention relates to a type of cyanine dye, belonging to the field of biomedical engineering, and specifically to a type of cyanine dye J-aggregate nanoparticles for photodynamic / photothermal synergistic therapy and a preparation method and application thereof. The present invention aims to provide a novel cyanine dye J-aggregate nanoparticle having near-infrared absorption and a PDT / PTT synergistic effect on cancer cells and harmful bacteria under irradiation of a single wavelength light source, which can cause powerful killing power to tumors and harmful bacteria.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and specifically relates to a type of cyanine dye J-aggregate nanoparticles for photodynamic / photothermal synergistic therapy, and a preparation method and application thereof. Background Art

[0002] So far, bacterial, fungal infections and cancer have posed a serious threat to human health. With the evolution of bacteria and the abuse of antibiotics, more and more drug-resistant bacteria continue to emerge, causing serious wound infections and even sepsis that endanger life. Cancer has the characteristics of abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. It is difficult to inhibit tumor recurrence and metastasis by relying on traditional treatments such as surgery, radiotherapy and chemotherapy. Therefore, it is of great significance to develop effective new agents for the treatment of tumors and bacteria and fungi.

[0003] In recent years, phototherapy has become an effective method for clinical cancer treatment and antibacterial treatment due to its non-invasive, non-invasive, non-toxic, rapid response and highly sensitive characteristics. Phototherapy mainly includes photothermal therapy (PTT) and photodynamic therapy (PDT), which is a method that applies the principles of photochemistry, photophysics and photobiology to the diagnosis and treatment of diseases. In photodynamic therapy (PDT), oxygen is the substrate for the production of toxic reactive oxygen species (ROS). Adequate oxygen supply is a prerequisite for ensuring that PDT is fully effective. Reactive oxygen species (ROS), especially singlet oxygen (, are produced by photosensitizers (PSs) under laser irradiation. 1 O2) can induce cell apoptosis. However, the hypoxic microenvironment at the tumor site limits the production of reactive oxygen species, thereby preventing PSs from exerting an effective photodynamic effect, leading to tumor growth and metastasis. Different from photodynamic therapy, photothermal therapy is a treatment method that destroys tumor cell membranes, destroys cytoskeleton and inhibits DNA synthesis through direct thermal ablation. It has the characteristics of precise control and rapid killing of cancer cells in a short time without relying on oxygen. Therefore, it is of great significance to develop a new reagent with PDT / PTT synergistic effect on tumors and bacteria and fungi.

[0004] Among the many photosensitizer precursors, cyanine dyes and their derivatives have the characteristics of high molar extinction coefficient, high fluorescence quantum yield, easy structural modification, and adjustable absorption and emission wavelengths, which have aroused great interest among researchers. Commercial cyanine dyes such as IR-780 and ICG have been shown to have photodynamic or photothermal effects. However, these dyes still have limitations, that is, the dyes have no selectivity for normal cells and harmful cells, and while destroying harmful cells, they also damage normal cells. There are few reports of cyanine dyes that can have PDT / PTT synergistic effects at a single wavelength, which limits the application of cyanine dyes in further photodynamic / photothermal synergistic treatment of tumors or bacteria. Therefore, it is particularly important to research and develop a type of cyanine dye or cyanine dye J-aggregate nanoparticles with good photodynamic and photothermal effects so that they can perform PDT and PTT simultaneously: on the one hand, cyanine dye aggregation can be used to enhance its photothermal conversion ability, generate heat to kill cancer cells and harmful bacteria, and avoid its therapeutic effect being affected by the hypoxic environment; on the other hand, the formation of J-aggregates by cyanine dyes can enhance the ability of the dyes to produce ROS, and the photodynamic / photothermal effects of the dyes can be used to kill cancer cells and harmful bacteria at the same time, achieving a synergistic therapeutic effect of "1+1>2". Summary of the invention

[0005] The present invention aims to provide a novel cyanine dye J-aggregate nanoparticle with near-infrared absorption and PDT / PTT synergistic effect on cancer cells and harmful bacteria under the irradiation of a single wavelength light source, which can cause powerful lethality to tumors and harmful bacteria. This type of photodynamic / photothermal cyanine dye has a good planar structure, and the dye monomer is easy to assemble into a J-aggregate to form a cyanine dye J-aggregate nanoparticle with a high-order nanostructure, so that its excitation wavelength is red-shifted, the ROS yield is higher, and the maximum absorption wavelength and the maximum emission wavelength are both located in the near-infrared light region. At the same time, the formation of aggregates by cyanine dyes can inhibit the change of the molecular configuration of excited-state cyanine dyes, thereby generating a photothermal effect. The cyanine dye J-aggregate nanoparticles synthesized by the present invention have very low dark toxicity and high phototoxicity, and can achieve the purpose of synergistic treatment of photodynamic therapy and photothermal therapy, while increasing the therapeutic effect without increasing side effects.

[0006] The technical purpose of the present invention is achieved through the following technical solutions:

[0007] The technical purpose of the first aspect of the present invention is to provide a type of photodynamic / photothermal cyanine dye, which is a compound with a structure as shown in general formula I:

[0008]

[0009] In the general formula I: X is selected from halogen (fluorine, chlorine, bromine or iodine) ion, ClO4 - PF6 - 、BF4 - 、CH3COO- Any one or more of the following;

[0010] R1 and R4 are each selected from one or more of H, alkyl nitro, alkyl cyano, alkyl acyl, alkyl azido, alkyl alkynyl, alkyl sulfonic acid and alkyl carboxyl;

[0011] R2 and R3 are selected from one or more of H and halogen;

[0012] R5 and R6 are independently selected from one or more of H, halogen (fluorine, chlorine, bromine or iodine), alkyl, alkoxy, alkylazido, alkylalkynyl, alkylamino, alkylsulfonyl, alkylhydroxyl and alkylcarboxyl;

[0013] n is 0, 1 or 2.

[0014] The technical purpose of the second aspect of the present invention is to provide the application of the above-mentioned photodynamic / photothermal cyanine dye, which can be assembled with amphiphilic polymer compounds to form cyanine dye J-aggregate nanoparticles for the preparation of anti-tumor and antibacterial drugs.

[0015] The novel cyanine dye J-aggregate nanoparticles prepared by the present invention have the following remarkable characteristics:

[0016] (1) The nanoparticles of the present invention have excellent near-infrared properties and have low biotoxicity, photobleaching and photodamage when used in biological imaging, and the fluorescent signals generated by them can penetrate deeper biological tissues;

[0017] (2) The nanoparticles of the present invention can generate singlet oxygen and photothermal effect under irradiation with light of a specific near-infrared wavelength;

[0018] (3) The nanoparticles of the present invention have very low dark toxicity, good biocompatibility, water solubility and photostability, and can be used as excellent photodynamic / photothermal agents for tumor treatment and antibacterial fields.

[0019] (4) The present invention proposes a new type of cyanine dye photodynamic / photothermal agent with near-infrared absorption and PDT / PTT synergistic effect under single wavelength light source irradiation, which has a strong killing power on tumors and harmful bacteria. The molecular structure of this type of cyanine dye has good planarity, and the dye monomer is easy to assemble to form cyanine dye J-aggregate nanoparticles with high-order nanostructure, so that it has better tumor targeting properties and achieves the synergistic treatment effect of photodynamic therapy and photothermal therapy.

[0020] (5) The cyanine dye photodynamic / photothermal cyanine dye J-aggregate nanoparticles of the present invention have small toxic and side effects, readily available raw materials, simple and stable molecular structure, and are easy to prepare and purify, which is conducive to the industrial production of drugs for phototherapy of tumors and harmful bacteria. Based on this, the present invention further provides the use of the cyanine dye J-aggregate nanoparticles as PDT / PTT phototherapy agents. The prepared nanoparticles can be used for synergistic treatment of photodynamic therapy and photothermal therapy, and are excited by a specific single wavelength near-infrared light, which overcomes the shortcomings of single photodynamic therapy or single photothermal therapy, and has a better hypoxic tumor treatment effect than general cyanine dye photosensitizers or photothermal agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention is attached Fig.11 Size:

[0022] Figure 1 It is a flow chart of the preparation of the novel cyanine dye J-aggregate nanoparticles 1-2NPs of the present invention;

[0023] Figure 2 It is a transmission electron microscope image of the novel cyanine dye J-aggregate nanoparticles 1-2NPs of the present invention;

[0024] Figure 3 This is an atomic force microscope image of the novel cyanine dye J-aggregate nanoparticles 1-2NPs of the present invention;

[0025] Figure 4 It is the UV-visible light absorption spectrum of the novel cyanine dye monomers 1-1 and 1-2 of the present invention in PBS solution;

[0026] Figure 5 It is the ultraviolet-visible light absorption spectrum diagram of the novel cyanine dye monomers 1-1 and 1-2 of the present invention in acetonitrile solution;

[0027] Figure 6 It is the fluorescence spectrum of the novel cyanine dye monomers 1-1 and 1-2 of the present invention in acetonitrile solution;

[0028] Figure 7 The DPBF absorption decay curve of the novel cyanine dye monomer 1-2 of the present invention during the singlet oxygen generation process in an acetonitrile system under 730nm light conditions;

[0029] Figure 8 This is the test result of the in vitro photothermal effect of the novel cyanine dye J-aggregate nanoparticles 1-2NPs of the present invention;

[0030] Fig. 9This is the test result of the novel cyanine dye J-aggregate nanoparticle 1-2NPs of the present invention killing tumor cells in vitro. 4T1 cells (mouse breast cancer cells) were selected as the research object, and the cytotoxicity of nanoparticles can be characterized by the survival rate of cells after different experimental treatments. The "In light" group represents the phototoxicity of nanoparticles to cancer cells, and the "In dark" group represents the dark toxicity of nanoparticles to cancer cells;

[0031] Fig.10 This is an in vitro evaluation of the cell killing results of the novel cyanine dye J-aggregate nanoparticles 1-2NPs produced by the photodynamic and photothermal synergistic effects. 4T1 cells were selected as the research object, and the cytotoxicity of the nanoparticles can be characterized by the cell survival rate;

[0032] Fig.11 This is the antibacterial test result of the novel cyanine dye nano J-aggregate nanoparticle 1-2NPs of the present invention. Staphylococcus aureus (S. aureus) was selected as the research object, and the killing effect of the nanoparticles on bacteria was characterized by the inhibition rate. The "Light" group represents the phototoxicity of the nanoparticles to S. aureus, and the "Dark" group represents the dark toxicity of the nanoparticles to S. aureus. DETAILED DESCRIPTION

[0033] The following is a detailed description of the methods for implementing the present invention in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention.

[0034] The primary technical purpose of the present invention is to provide a class of photodynamic / photothermal cyanine dyes, which are compounds of the general formula I:

[0035]

[0036] In the general formula Ⅰ:

[0037] X is selected from halogen (fluorine, chloride, bromide or iodine) ions, ClO4 - PF6 - 、BF4 - 、CH3COO - Any one or more of the following;

[0038] R1 and R4 are each selected from one or more of H, alkyl nitro, alkyl cyano, alkyl acyl, alkyl azido, alkyl alkynyl, alkyl sulfonic acid and alkyl carboxyl;

[0039] R2 and R3 are selected from one or more of H and halogen;

[0040] R5 and R6 are independently selected from one or more of H, halogen (fluorine, chlorine, bromine or iodine), alkyl, alkoxy, alkylazido, alkylalkynyl, alkylamino, alkylsulfonyl, alkylhydroxyl and alkylcarboxyl;

[0041] n is 0, 1 or 2.

[0042] In the above-mentioned photodynamic / photothermal cyanine dyes, more specifically, the alkoxy group includes but is not limited to methoxy, ethoxy, n-butoxy, tert-butoxy, n-octyloxy and n-dodecyloxy; the alkylamide group includes but is not limited to methylcarbonylamino, ethylcarbonylamino, dimethylcarbonylamino, propylcarbonylamino, pentylcarbonylamino, cyclohexylcarbonylamino, 2-ethylhexylcarbonylamino, octylcarbonylamino and dodecylcarbonylamino; the alkyl azide group includes but is not limited to ethylazido, propylazido, butylazido and hexylazido; the alkylamino group includes but is not limited to ethylamino, dimethylamino, butylamino, cyclopentylamino, 2-ethylhexylamino and dodecylamino; the alkylsulfonyl group includes but is not limited to methylsulfonyl, ethylsulfonyl, butylsulfonyl and cyclohexylsulfonyl.

[0043] In the above-mentioned photodynamic / photothermal cyanine dye, the halogen includes fluorine, chlorine, bromine and iodine.

[0044] In the above-mentioned photodynamic / photothermal cyanine dyes, as a further preferred embodiment, the R1 and R4 are also selected from H, C 1-5 Alkyl nitro, C 1-5 Alkyl cyano, alkyl formyl, C 2-6 Alkyl azide, C 2-5 Alkyl alkynyl, C 2-6 Alkylsulfonyl and C 1-5 One or more of alkyl carboxyl groups.

[0045] In the above-mentioned photodynamic / photothermal cyanine dyes, as a further preference, the R2 and R3 are selected from one or more of H and halogen.

[0046] In the above-mentioned photodynamic / photothermal cyanine dyes, as a further preferred embodiment, the R5 and R6 are also selected from H, halogen, C 1-5 Alkyl, C 1-5 Alkoxy, C 2-6 Alkyl azide, C 2-5 Alkyl alkynyl, C 1-5 Alkylamino, C 2-6 Alkylsulfonyl, C 1-5 Alkyl hydroxyl and C 1-5 One or more of alkyl carboxyl groups.

[0047] In the above-mentioned photodynamic / photothermal cyanine dye, as a further preference, the n is 1 or 2.

[0048] In the above-mentioned photodynamic / photothermal cyanine dye, as a further preference, the X is a halogen (fluorine, chlorine, bromine or iodine) ion.

[0049] In the above-mentioned photodynamic / photothermal cyanine dyes, as a more specific embodiment, the novel photodynamic / photothermal cyanine dyes are selected from:

[0050]

[0051] The present invention also discloses a method for preparing the above-mentioned photodynamic / photothermal cyanine dye, comprising the steps of using a quinoline derivative as a raw material to prepare an intermediate 1, reacting the intermediate 1 with a condensation agent N, N'-dibenzamide to prepare an intermediate 2, and using the two intermediates prepared above to prepare a novel photodynamic / photothermal cyanine dye. Specifically, the steps include:

[0052] (1) reacting a quinoline derivative having corresponding R1, R2 (or R3, R4) substituents with a corresponding compound having R5, R6 substituents in a toluene solution under reflux for 2-24 hours to prepare the corresponding intermediate 1;

[0053] (2) The intermediate 1 prepared in the above step (1) is reacted with the condensation agent N,N'-dibenzamide under condensation reflux for 20-60 minutes to prepare the intermediate 2; then the intermediate 1 prepared in step (1) and the intermediate 2 prepared in step (2) are reacted in a mixed solvent of dichloromethane and methanol for 12-36 hours under the condition of acetic anhydride and triethylamine as catalysts, thereby preparing the corresponding novel photodynamic / photothermal cyanine dye.

[0054] In the preparation method of the above-mentioned novel photodynamic / photothermal cyanine dye, the reaction temperature in step (1) is 30-160°C, the reaction time is 2-24 hours, and the molar ratio of the quinoline derivative having corresponding R1, R2 (or R3, R4) substituents to the corresponding compound with R5, R6 substituents is 1:1-1:4.

[0055] In the preparation method of the above-mentioned novel photodynamic / photothermal cyanine dye, the reaction temperature in step (2) is 140-180°C, the reaction time is 20-60 minutes, and the molar ratio of intermediate 1 to condensation agent N, N'-dibenzamide is 1:1-1:4; as a more preferred embodiment, the reaction temperature is 140-160°C, the reaction time is 20-40 minutes, and the molar ratio of intermediate 1 to condensation agent N, N'-dibenzamide is 1:1-1:2.

[0056] In the preparation method of the novel photodynamic / photothermal cyanine dye, in step (3), the compound of step (2) reacts with the compound of step (1) at a molar ratio of 1:1-1:4, the reaction temperature is 60-80°C, the reaction time is 12-36 hours, and the reaction solvent is selected from a mixed solvent of dichloromethane and methanol (volume ratio is 1:1).

[0057] The present invention also discloses a method for preparing the novel photodynamic / photothermal cyanine dye, comprising the steps of using 4-methylquinoline as a raw material to prepare intermediate 1, reacting intermediate 1 with a condensation agent N, N'-dibenzamide to prepare intermediate 2, using 6-iodo-4-methylquinoline as a raw material to prepare intermediate 3, and using intermediate 2 and intermediate 1 (or intermediate 3) to prepare the photodynamic / photothermal cyanine dye. Specifically, the following steps are included:

[0058] (1) 4-methylquinoline (or 6-iodo-4-methylquinoline) reacts with ethyl iodide at a molar ratio of 1:1-1:10 to prepare intermediate 1 (or intermediate 3);

[0059]

[0060] The reaction temperature is 30-160°C, the reaction time is 2-24 hours, and the reaction solvent is toluene;

[0061] In a preferred experimental method, the reaction temperature is 60-150° C., the reaction time is 5-15 hours, the reaction solvent is toluene, and the molar ratio of 4-methylquinoline (or 6-iodo-4-methylquinoline) to iodoethane is 1:1-1:4;

[0062] In a further preferred embodiment, the reaction temperature is 80-130° C., the reaction time is 7-13 hours, the reaction solvent is toluene, and the molar ratio of 4-methylquinoline (or 6-iodo-4-methylquinoline) to iodoethane is 1:1-1:2;

[0063] In the most preferred embodiment, the reaction temperature is 120-130° C., the reaction time is 8-10 hours, the reaction solvent is toluene, and the molar ratio of 4-methylquinoline (or 6-iodo-4-methylquinoline) to iodoethane is 1:1-1:1.5;

[0064] (2) subsequently reacting the intermediate 1 prepared in step (1) with a condensing agent N,N'-dibenzamide in a molar ratio of 1:1-1:4 to prepare an intermediate 2;

[0065]

[0066] During the preparation of intermediate 2, the reaction temperature is 140-180°C, the reaction time is 20-60 minutes, and the molar ratio of intermediate 1 to condensation agent N, N'-dibenzamide is 1:1-1:4;

[0067] As a more preferred embodiment, the reaction temperature is 140-170° C., the reaction time is 20-40 minutes, and the molar ratio of the intermediate 1 to the condensation agent N, N'-dibenzamide is 1:1-1:2;

[0068] In the most preferred embodiment, the reaction temperature is 155-165° C., the reaction time is 30-40 minutes, and the molar ratio of the intermediate 1 to the condensation agent N, N'-dibenzamide is 1:1-1:1.2;

[0069] (3) intermediate 1 or 3 reacts with intermediate 2 prepared in step (2) at a molar ratio of 1:1-1:4 to prepare novel photodynamic / photothermal cyanine dye monomers I-1 and I-2;

[0070]

[0071] The reaction temperature is 60-80°C, the reaction time is 12-36 hours, and the reaction solvent is selected from a mixed solvent of dichloromethane and methanol (volume ratio is 1:1);

[0072] In a preferred embodiment, the reaction temperature is 60-70° C., the reaction time is 20-30 hours, the reaction solvent is selected from a mixed solvent of dichloromethane and methanol (volume ratio is 1:1), and the molar ratio of intermediate 1 or 3 to intermediate 2 is 1:1-1:2;

[0073] In a further preferred embodiment, the reaction temperature is 62-68° C., the reaction time is 22-28 hours, the reaction solvent is selected from a mixed solvent of dichloromethane and methanol (volume ratio is 1:1), and the molar ratio of intermediate 1 or 3 to intermediate 2 is 1:1-1:1.5;

[0074] In the most preferred embodiment, the reaction temperature is 65-68° C., the reaction time is 25-28 hours, the reaction solvent is selected from a mixed solvent of dichloromethane and methanol (volume ratio is 1:1), and the molar ratio of intermediate 1 or 3 to intermediate 2 is 1:1-1:1.2.

[0075] The technical purpose of the second aspect of the present invention is to wrap the above-mentioned cyanine dye with an amphiphilic polymer to assemble it into cyanine dye J-aggregate nanoparticles.

[0076] The above-mentioned amphiphilic polymer compound of the present invention is selected from one of polysorbate (such as Tween-80), polyethylene glycol castor oil (such as Cremophor EL), polyethylene glycol stearate 15 (Solutol HS-15), glyceride 44 / 14, hydroxypropyl-β-cyclodextrin (HP-β-CD), sulfobutyl-β-cyclodextrin (SBE-β-CD), Pluronic and its derivatives (including but not limited to Pluronic F68, Pluronic F127).

[0077] The ratio of the photodynamic / photothermal cyanine dye to the amphiphilic polymer compound of the present invention is (n / n, (0.001-0.10 μmol): (2.38-23.8 μmol)).

[0078] The technical purpose of the third aspect of the present invention is to provide the application of the above-mentioned nanoparticles, which can be used to prepare anti-tumor and antibacterial drugs.

[0079] The nanoparticles of the present invention have very low dark toxicity, good biocompatibility, water solubility and photostability, and have excellent near-infrared characteristics. When used in biological imaging, they have low photobleaching and photodamage, and the fluorescent signals they generate can penetrate deeper biological tissues. Under the irradiation of light with a specific near-infrared wavelength, they can produce singlet oxygen and photothermal effects, and can be used as excellent photodynamic / photothermal cyanine dye J-aggregate nanoparticles for tumor treatment and antibacterial fields. The nanoparticles of the present invention are a type of cyanine dye J-aggregate nanoparticles that have near-infrared absorption and can perform PDT / PTT synergistic treatment on cancer cells and harmful bacteria under the irradiation of a single wavelength light source. They can cause powerful lethality to tumors and harmful bacteria, and have better tumor targeting ability and photodynamic / photothermal synergistic treatment effects than dye monomers.

[0080] Example 1

[0081] Preparation of novel photodynamic / photothermal cyanine dye monomers I-1 and I-2

[0082] In the above-mentioned photodynamic / photothermal cyanine dye, as a more specific embodiment, the present invention provides a method for preparing the novel photodynamic / photothermal cyanine dye monomers I-1, I-2, including using 4-methylquinoline as a raw material to prepare intermediate 1, reacting intermediate 1 with a condensation agent N, N'-dibenzamide to prepare intermediate 2, using 6-iodo-4-methylquinoline as a raw material to prepare intermediate 3, and using intermediate 2 and intermediate 1 (or intermediate 3) to prepare novel photodynamic / photothermal cyanine dye monomers I-1, I-2. Specifically comprising the following steps:

[0083]

[0084] (1) Preparation of Intermediate 1

[0085] Take 20mL of toluene solution and inject it into a round-bottom flask, turn on the magnetic stirrer, add 4-methylquinoline (2g, 13.97mmol) and iodoethane (2.614g, 16.76mmol) into the round-bottom flask containing 20mL of toluene solution under uniform stirring, react at 120°C for 8h, wash the product with ether 5 times, and then filter it to obtain a green powdery solid. The obtained product was placed in a vacuum drying oven and dried overnight to obtain intermediate 1 with a yield of 47.06%. The synthetic route is:

[0086]

[0087] (2) Preparation of Intermediate 2

[0088] The above-synthesized intermediate 1 (1.00 g, 3.34 mmol) and N, N'-dibenzamide (0.656 g, 3.34 mmol) were added to a round-bottom flask and condensed under reflux for 30 min at 160 ° C. After the reaction was completed, the reaction product was washed 5 times with ether. Subsequently, the suction filtration operation was performed, and the obtained product was a reddish brown powder solid. The obtained product was placed in a vacuum drying oven and dried overnight to obtain intermediate 2, with a crude yield of 54.5%. The synthetic route is:

[0089]

[0090] (3) Preparation of Intermediate 3

[0091] 6-iodo-4-methylquinoline (0.10 g, 0.37 mmol) and 1.56 mL of toluene were injected into a round-bottom flask. After the solution was dissolved, iodoethane (0.174 g, 1.11 mmol) was slowly added drop by drop into the round-bottom flask and reacted at 75 ° C. After reacting for 5 hours, the temperature was raised to 80 ° C and condensation reflux was continued. After reacting for 15 hours and 30 minutes, the solution turned into a bean green color and a solid product was precipitated. 0.347 g of iodoethane (2.22 mmol) was added to the round-bottom flask and the reaction was continued at 72 ° C overnight. After the reaction was completed, the product was washed with petroleum ether 5 times, and then filtered. The obtained product was a bean green powder solid. The obtained product was placed in a vacuum drying oven and dried overnight to obtain intermediate 3 with a yield of 63.70%. The synthetic route is as follows:

[0092]

[0093] (4-1) Preparation of Photodynamic / Photothermal Cyanine Dye Monomer I-1

[0094] The intermediate 2 synthesized above (268.18 mg, 0.67 mmol) and CH2Cl2 / CH3OH (V / V, 1 / 1, 3.35 mL / 3.35 mL) were reacted under nitrogen protection, the reaction temperature was 65°C, the intermediate 2 began to dissolve at 65°C, 0.67 mL of acetic anhydride was added after the solution was clarified, and after 1 hour of reaction, the intermediate 1 (199.94 mg, 0.67 mmol) was added, and after the solution was dissolved, 0.67 mL of triethylamine was added to catalyze the reaction for 24 hours. After the reaction was completed, appropriate amounts of methanol and petroleum ether were added for cleaning, and after cleaning 3 times, the solution in the reactor was transferred to a new round-bottom flask with a rubber-tipped dropper, and condensed and refluxed at 65°C again. After the reaction for 1 hour, cooling and recrystallization were performed, and vacuum drying was performed overnight to obtain the photodynamic / photothermal cyanine dye monomer I-1 with a yield of 53.1%.

[0095] The structure of the product was confirmed by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

[0096] NMR: Characterization of the photodynamic / photothermal cyanine dye monomer I-1 using deuterated DMSO as solvent 1 H NMR and 14 C NMR. 1 HNMR(400MHz,DMSO-d6)δ8.71–8.56(m,2H),8.41(d,J=8.5Hz,1H),8.36–8.23(m,1H),8.14(d ,J=7.3Hz,1H),8.06(dd,J=9.0,1.8Hz,1H),8.02–7.95(m,2H),7.94–7.84(m,2H),7.77(d,J=7 .4Hz,1H),7.71–7.57(m,3H),7.21(d,J=13.1Hz,1H),7.10(d,J=13.0Hz,1H),7.01(d,J=12.7H z, 1H), 4.53 (q, J = 7.1Hz, 1H), 4.37 (dq, J = 28.8, 7.1Hz, 2H), 1.39 (dq, J = 29.4, 7.4, 6.7Hz, 6H).

[0097] 13C NMR(101MHz,DMSO-d6)δ149.21,146.71,145.57,142.81,142.58,141.16,140 .54,140.35,140.17,139.49,137.70,137.27,133.00,132.93,129.46,126.13 ,125.97,124.92,124.41,119.20,118.95,117.43,111.91,111.42,110.29,109.66,109.49,108.57,91.47,90.76,48.76,48.36,47.90,14.5414.35,14.22.

[0098] Its synthetic route is as follows:

[0099]

[0100] (4-2) Preparation of Photodynamic / Photothermal Cyanine Dye Monomer I-2

[0101] The intermediate compound 2 (94.7 mg, 0.236 mmol) synthesized above and CH2Cl2 / CH3OH (V / V, 1 / 1, 1.18 mL / 1.18 mL) were reacted under nitrogen protection at a reaction temperature of 65°C. The intermediate compound 2 began to dissolve at 65°C. After the solution became clear, 0.236 mL of acetic anhydride was added. After reacting for 1 hour, the intermediate compound 3 (100.00 mg, 0.236 mmol) was added. After the solution became clear, 0.236 mL of triethylamine was added to carry out catalytic reaction for 24 hours. After the reaction is completed, ethyl acetate is added for washing, and the solid product is transferred together with ethyl acetate to a 2 mL centrifuge tube, centrifuged, the supernatant is discarded, washed with ethyl acetate 5 times, extracted with ethyl acetate and deionized water 3 times, the ethyl acetate is discarded, and the aqueous phase is retained, a total of 3 times, the aqueous phase is transferred to a new round-bottom flask, 5 mL of methanol is added, rotary evaporated at 45 ° C, and vacuum dried overnight to obtain photodynamic / photothermal cyanine dye monomer I-2 with a yield of 25.17%.

[0102] The structure of the product was confirmed by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

[0103] NMR: Characterization of the photodynamic / photothermal cyanine dye monomer I-2 using deuterated DMSO as solvent 1 H NMR and 14 C NMR. 1HNMR (400MHz, DMSO-d6) δ8.72–8.59(m,2H),8.42(d,J=8.4Hz,1H),8.33(d,J=7.2Hz,1H),8.15(d,J=7.1Hz,1H),8.06(d,J=9.2Hz,1H),8.00(dd,J =8.7,6.8Hz,2H),7.90(dd,J=11.6,7.8Hz,1H),7.78(d,J=7.3Hz,1H),7. 69–7.58(m,2H),7.22–7.02(m,2H),4.60–4.30(m,4H),1.50–1.31(m,6H).

[0104] 13 C NMR(101MHz,DMSO-d6)δ149.75,147.25,146.10,143.11,141.70,140.71,140.03,138.24,137.81,133.46,126.6 7,125.46,124.95,119.48,117.97,112.45,111.96,110.83,110.20,109.11,92.01,91.29,49.30,48.44,14.76.

[0105] Its synthetic route is as follows:

[0106]

[0107] Example 2

[0108] Preparation of novel photodynamic / photothermal cyanine dye J-aggregate nanoparticles I-2NPs

[0109] As a more specific embodiment, the present invention provides a method for preparing the novel cyanine dye J-aggregate nanoparticles I-2NPs, wherein the amphiphilic polymer compound selected is Pluronic F68 for assembling the nanoparticles.

[0110] Figure 1The flow chart for the preparation of the novel cyanine dye J-aggregate nanoparticles I-2NPs. Since amphiphilic polymer compounds can spontaneously form spherical core-shell hollow structure copolymer micelles in aqueous solutions, the novel photodynamic / photothermal cyanine dye monomer I-2 synthesized by us is a hydrophobic small molecule compound. The novel photodynamic / photothermal cyanine dye monomer I-2 can be embedded in the spherical micelles of amphiphilic polymer compounds through ultrasound to assemble into novel cyanine dye J-aggregate nanoparticles I-2NPs. Weigh 12.13 mg of cyanine dye photosensitizer I-2 in 0.1 mL DMSO to prepare a mother solution with a final concentration of 20 mM. The mother liquor of different concentrations and the amphiphilic polymer Pluronic F68 were dissolved in 2mL PBS buffer solution (10mM) (the mass of Pluronic F68 was 60mg, so that its mass fraction in the 2mL PBS buffer solution system was 0.3%, and the molar ratio of cyanine dye to amphiphilic polymer compound was (molar amount n / n, (0.001-0.10μmol): (2.38-23.8μmol)), and the new cyanine dye nanoparticles I-2NPs were prepared by ultrasonication for 1min. The 1-2NPs were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM), and the particle size range of 1-2NPs was measured to be 190-205nm, and the average particle size was 200±2.5nm. The transmission electron microscopy results are shown in FIG. Figure 2 As shown in the figure, the AFM scan shows its morphology on the surface of the mica sheet. Figure 3 As shown, this indicates that the new cyanine dye J-aggregate nanoparticles I-2NPs were successfully prepared.

[0111] Example 3.

[0112] Performance determination of novel photodynamic / photothermal cyanine dye monomers I-1 and I-2 and cyanine dye J-aggregate nanoparticles I-2NPs:

[0113] (1) UV-visible absorption and fluorescence spectra of the new photodynamic / photothermal cyanine dye monomers I-1 and I-2

[0114] Dye molecules can self-aggregate in solution due to the action of van der Waals forces. After aggregation, the absorption spectrum of the dye will change significantly. Compared with the dye monomer, the absorption spectrum of the J-aggregate is red-shifted, and the absorption spectrum of the H-aggregate is blue-shifted. Different concentrations of the photodynamic / photothermal cyanine dye monomer I-1 or I-2 synthesized in the above embodiment were added to a quartz cuvette containing PBS buffer solution (3 mL), and the UV-visible light absorption spectra of different concentrations of the new photodynamic / photothermal cyanine dye monomer I-1 or I-2 in the PBS buffer solution in the wavelength range of 500-900 nm were measured. Figure 4 As shown, the test results show that the new photodynamic / photothermal cyanine dye monomer I-1 or I-2 has three absorption peaks in PBS buffer solution, which correspond to the characteristic peaks of H-aggregate, dye monomer and J-aggregate from left to right.

[0115] Next, we used the photodynamic / photothermal cyanine dye monomer I-1 or I-2 synthesized in the above embodiment to add them into different quartz cuvettes containing 14 organic solvents (3 mL) including methanol, dichloromethane, acetone, n-hexanol, n-pentanol, n-butanol, dioxane, ethyl acetate, acetonitrile, N,N'dimethylformamide, ethanol, dimethyl sulfoxide, tetrahydrofuran or chloroform, and measured the UV-visible light absorption spectrum and fluorescence spectrum of 5 μM new photodynamic / photothermal cyanine dye monomer I-1 or I-2 in these organic solvents. The test results show that the maximum absorption wavelengths of the new photodynamic / photothermal cyanine dye monomers I-1 and I-2 are both around 660 nm, and the maximum absorption wavelengths of their J-aggregates are both around 720 nm (such as Figure 5 The maximum emission wavelengths of the new photodynamic / photothermal cyanine dye monomers I-1 and I-2 are both around 740 nm (λ ex =660nm, such as Figure 6 As shown in Figure 2, both are in the near-infrared region and can be used as near-infrared excited photodynamic / photothermal cyanine dyes. The instruments used were a PerkinElmer UV-Vis spectrophotometer and a Hitachi F-4600 fluorescence spectrophotometer. Singlet oxygen generation determination of the new photodynamic / photothermal cyanine dye monomer I-2

[0116] The photodynamic / photothermal cyanine dye monomer I-2 synthesized using the above embodiment was added to 2 mL of acetonitrile solution to a final concentration of 5 μM, and then 1,3-diphenylisobenzofuran (DPBF) was added to adjust the absorbance of DPBF in the above solution to about 1.0 L / (g·cm), and mixed evenly. It was then irradiated with 730-740 nm red light, and its ultraviolet absorption curve was quickly measured. According to the change in the absorbance value of DPBF at 411 nm, a correlation curve of absorbance and time was drawn, and methylene blue was used as a reference to calculate its singlet oxygen quantum yield. Figure 7 The DPBF absorption decay curve of the singlet oxygen generation process of the photodynamic / photothermal cyanine dye monomer I-2 in acetonitrile solution under 730-740nm illumination conditions. It is known that the singlet oxygen quantum yield of methylene blue is 0.51. According to the following formula, the singlet oxygen quantum yield of the photodynamic / photothermal cyanine dye monomer I-2 can be calculated to be 0.295. The instrument used is a PerkinElmer UV-Vis spectrophotometer, and the light source is a 300W xenon lamp light source.

[0117] Φ Δsam＝ Φ Δstd ×(k sam / k std )×(F sam / F std )

[0118] Among them, Φ Δ represents the singlet oxygen quantum yield, k represents the slope of the curve of DPBF absorbance decay at 411nm over time, and F represents the absorption correction factor. The formula F = 1-10 -OD (OD is the absorbance of the solution at 660 nm), the subscript sam represents the photodynamic / photothermal cyanine dye monomer I-2, and the subscript std represents the standard substance methylene blue.

[0119] (2) Determination of the photothermal effect of the novel cyanine dye J-aggregate nanoparticles I-2NPs

[0120] In order to evaluate the photothermal performance of the novel cyanine dye J-aggregate nanoparticles I-2NPs, the effects of different concentrations of the novel cyanine dye J-aggregate nanoparticles I-2NPs on their photothermal performance were studied. The novel cyanine dye J-aggregate nanoparticles I-2NPs (0, 30, 50 μM) of different concentrations were prepared using the method of Example 2, and the solvent used was PBS buffer solution (pH=7.4). The detection was carried out in a quartz cuvette under 730 nm laser irradiation. The temperature change was monitored by using a FLIR-1910582 infrared imager, and the temperature was recorded every 1 minute. The temperature rise curve was drawn to analyze the photothermal performance of the novel cyanine dye J-aggregate nanoparticles I-2NPs.

[0121] like Figure 8As shown in the figure, when the dye concentration of the new cyanine dye J-aggregate nanoparticles I-2NPs is 30μM and 50μM, the temperature increases significantly compared with the single PBS buffer solution. When the dye concentration is 50μM, after 730nm laser irradiation for 14min, the solution temperature rises from the initial 14.6℃ to 38.7℃, an increase of 24.1℃. When the temperature of the tumor microenvironment is higher than 45℃, tumor cells can be killed. Therefore, when the new cyanine dye J-aggregate nanoparticles I-2NPs are used for phototherapy of cells, the temperature of the tumor microenvironment can reach above 50℃, which can effectively kill tumor cells. Therefore, the new cyanine dye J-aggregate nanoparticles I-2NPs have excellent photothermal effects and can be used for efficient phototherapy of tumors.

[0122] (3) In vitro cell anticancer test of the novel cyanine dye J-aggregate nanoparticles I-2NPs

[0123] 4T1 cells (mouse breast cancer cells, purchased from Jiangsu KeyGen Biotechnology Co., Ltd.) were digested with 0.25% (volume fraction) trypsin and then prepared into a single cell suspension in DMEM culture medium containing 10% (volume fraction) fetal bovine serum. 1×10 5 The cells were inoculated into a 96-well culture plate at a density of 100 μL per well, and the culture plate was then placed in a CO2 cell culture incubator for 12 hours. The incubator environment was 37°C, with air containing 5% (volume fraction) CO2, and saturated humidity. After 12 hours, the original culture medium was discarded, and the 96-well plate was washed twice with PBS buffer solution (10 mM, pH = 7.4) (100 μL / well). In each parallel experiment, PBS buffer solution containing 0, 0.005, 0.01, 0.02, 0.05 or 0.1 μM of the novel photodynamic / photothermal cyanine dye monomer I-2 and Pluronic F68 (the mass of Pluronic F68 was 60 mg, and its mass fraction in 2 mL PBS buffer solution system was 0.3%) was added to prepare the novel cyanine dye J-aggregate nanoparticles 1-2NPs by ultrasonication, and then incubated at a volume of 100 μL / well. After incubation for 30 minutes, the nanoparticles were illuminated with 730 nm light (35 mW / cm 2) The experimental groups with 0.005, 0.01, 0.02, 0.05 or 0.1 μM of the novel photodynamic / photothermal cyanine dye monomer I-2 were irradiated for 5 min. After the irradiation, the PBS buffer solution in the 96-well plate was replaced with a fresh DMEM medium containing 10% fetal bovine serum, and the control group (PBS buffer solution containing 0 μM novel photodynamic / photothermal cyanine dye monomer I-2 and Pluronic F68 (the mass of Pluronic F68 is 60 mg, and its mass fraction in 2 mL PBS buffer solution system is 0.3%) was added and ultrasonically prepared into novel cyanine dye J-aggregate nanoparticles I-2NPs) and the experimental group (PBS buffer solution containing 0.005, 0.01, 0.02, 0.05 or 0.1 μM novel photodynamic / photothermal cyanine dye monomer I-2 and Pluronic F68 (the mass of Pluronic F68 is 60 mg, and its mass fraction in 2 mL PBS buffer solution system is 0.3%) was added and ultrasonically prepared into novel cyanine dye J-aggregate nanoparticles I-2NPs). The PBS buffer solution system (mass fraction of 0.3%) was ultrasonically prepared with a PBS buffer solution of a novel cyanine dye J-aggregate nanoparticle 1-2NPs) and the cells were incubated in a CO2 cell culture incubator (conditions as above) for 18h, and the experimental group cells were incubated in a CO2 cell culture incubator for 18h. For the dark toxicity test, no 5min light treatment was required and the cells were directly cultured for 18 hours. Subsequently, the original culture medium in the 96-well plate was removed, and a PBS buffer solution containing 0.5mg / mL thiazolyl blue (MTT) was added to the 96-well plate (100μL / well), and the 96-well plate was placed in a CO2 cell culture incubator for 4h. After 4h, the PBS buffer solution in the 96-well plate was gently aspirated with a 1mL syringe, and the formazan crystals deposited in the 96-well plate cells were dissolved with DMSO (100μL / well). Oscillate on a rocker for 10 minutes, and then measure its absorbance at a wavelength of 490nm with an enzyme marker. The cell viability was calculated using the following formula to evaluate the cell biotoxicity of the novel cyanine dye J-aggregate nanoparticles I-2NPs.

[0124]

[0125] Where Ai is the average absorbance of each well of the cyanine dye J-aggregate nanoparticle I-2NPs experimental group (the dye concentration of the new photodynamic / photothermal cyanine dye monomer 1-2 is 0.005, 0.01, 0.02, 0.05 or 0.1 μM group) (i=1, 2, ..., n), is the average absorbance of each well in the control group without adding drugs (the dye concentration of the novel photodynamic / photothermal cyanine dye monomer I-2 is 0 μM), n is the number of parallel experiments, and the parallel experiments are greater than 3, which is 3 here.

[0126] The results are as follows Fig. 9 As shown, from Fig. 9It can be seen that the new cyanine dye J-aggregate nanoparticles I-2NPs have strong cell phototoxicity and low dark toxicity to 4T1 cells. The experimental results are in line with the experimental design expectations. When the dye concentration of the new photodynamic / photothermal cyanine dye monomer I-2 is 0.005μM, the cell survival rate in the dark group is as high as 89.4%, while the cell survival rate in the light group is only 48.7%. As the concentration of the dye monomer increases, its phototoxicity becomes greater and greater. This indicates that the new cyanine dye J-aggregate nanoparticles I-2NPs have strong phototoxicity and low dark toxicity, and have excellent phototherapy effects.

[0127] (4) Differentiating the contribution of PDT and PTT to cell killing of novel cyanine dye J-aggregate nanoparticles I-2NPs in vitro

[0128] 4T1 cells (mouse breast cancer cells) were digested with 0.25% trypsin and then prepared into a single cell suspension in DMEM culture medium containing 10% fetal bovine serum. 1×10 5The cells were inoculated into a 96-well culture plate at a density of 100 μL per well, and the culture medium volume of each well was 100 μL. The culture plate was then placed in a CO2 cell culture incubator for 12 hours. The incubator environment was 37°C, with air containing 5% CO2 volume concentration and saturated humidity. The solution contained 10, 15, 20 μM of the new photodynamic / photothermal cyanine dye monomer I-2 and Pluronic F68 (0.3%) PBS buffer solution was added to the wells. The new cyanine dye J-aggregate nanoparticles I-2NPs were ultrasonically prepared and then added with drugs (cyanine dye J-aggregate nanoparticles I-2NPs) at a volume of 100 μL / well. After adding drugs, the wells were incubated for 30 minutes. After incubation, they were illuminated. Each group of 9 wells was a group. The culture medium of the control group was replaced with 37°C PBS buffer solution and incubated for 30 minutes. After the incubation, it was replaced with room temperature culture medium. The cells of the PDT group were incubated with nanoparticles prepared with different concentrations of cyanine dye monomer I-2 for 30 minutes. After incubation, they were replaced with 4°C PBS buffer solution and then exposed to 730- The cells were irradiated with 740 nm laser for 5 min and replaced with room temperature culture medium after irradiation. The cells in the PDT+PTT group were incubated with nanoparticles prepared with different concentrations of cyanine dye monomer I-2 for 30 min and then replaced with 37°C PBS buffer solution and exposed to 730-740 nm laser irradiation for 5 min at room temperature. The cells were replaced with room temperature culture medium after the replacement of the medium. The cells were incubated in a CO2 cell culture incubator (same conditions as above) for 4 h. After 4 h, the culture medium in the 96-well plate was discarded and washed twice with PBS buffer solution (10 mM, pH = 7.4). The prepared 5 mg / mL MTT solution was added at a volume of 100 μL / well and incubated for 4 h. The MTT solution in each well was gently aspirated with a 1 mL syringe, and the DMSO solution was added at a volume of 100 μL / well. The plate was shaken to mix well, and the OD value was measured with an enzyme reader after 10 min.

[0129] like Fig.10 As shown, when the cells were incubated with nanoparticles I-2NPs prepared with 20 μM cyanine dye monomer I-2, compared with the control group, the cell survival rate of the group with only the novel cyanine dye J-aggregate nanoparticles I-2NPs added without illumination reached 82.69%, the cell survival rate of the single PDT group was only 32.18%, and the cell survival rate of the PDT+PTT group was even lower, only 21.21%. This shows that the novel cyanine dye J-aggregate nanoparticles I-2NPs synthesized by the present invention have excellent PDT / PTT synergistic tumor treatment effects.

[0130] (5) Antibacterial performance test of the new cyanine dye J-aggregate nanoparticles I-2NPs

[0131] Staphylococcus aureus (S. aureus) was used as the research object, and the bacterial solution was mixed with the new cyanine dye J-aggregate nanoparticles I-2NPs (V / V=1 / 1) and then coated. The assembly method of the new cyanine dye J-aggregate nanoparticles I-2NPs is as follows: 0.3% Pluronic F68 + 1mL PBS buffer solution + photodynamic / photothermal cyanine dye monomer I-2 (10, 20, 30, 40μM), that is, take 5 centrifuge tubes, weigh 3mg Pluronic F68 in each tube, dissolve it with 1mL PBS buffer solution, and then add 0.5, 1.0, 1.5, 2.0μL of photodynamic / photothermal cyanine dye monomer I-2 respectively, so that the final concentration is 10, 20, 30, 40μM, and ultrasonicate for 1min to prepare cyanine dye J-aggregate nanoparticles I-2NPs; take 100μL of dilution multiple of 10 6 of bacterial solution (the concentration of bacterial solution was 4.96×10 8 CFU / mL) was mixed with 100 μL of the above-mentioned drug to make the dye concentration of cyanine dye J-aggregate nanoparticles I-2NPs 5, 10, 15, and 20 μM; 50 μL of the above-mentioned bacterial solution and I-2NPs mixed solution was evenly dropped on the LB solid culture medium at multiple points using a pipette, and the coating rod was burned with the outer flame of an alcohol lamp, and after cooling in the air, it was slid several times on the cover of the plate to cool, and the plate was coated as quickly as possible to make the bacterial solution uniform; after the addition of the drug was completed, the plate was inverted and cultured in a 37°C bacterial incubator for 1 hour, and after 1 hour, the illumination group was illuminated (730-740nm light source, 35mW / cm 2 , 10min), after the illumination ended, the inverted culture was continued for 11h, and the control group was not treated with any treatment. After inverted culture for 12h, the inhibition rate (IR) was calculated according to the following equation.

[0132] IR=(C0-C) / C0×100%

[0133] Wherein, C is the CFU of the experimental group treated with cyanine dye J-aggregate nanoparticles I-2NPs, and C0 is the CFU of the PBS buffer solution control group without any treatment.

[0134] like Fig.11 As shown, the light experimental group has an antibacterial effect, and as the dye concentration of the cyanine dye J-aggregate nanoparticles I-2NPs increases, the antibacterial effect becomes more and more obvious, while the control group also has a certain antibacterial effect, indicating that the dark toxicity of the cyanine dye J-aggregate nanoparticles to bacteria is also relatively large, but the phototherapy antibacterial effect is better. When the dye concentration of the cyanine dye J-aggregate nanoparticles I-2NPs is 10mM, the antibacterial rate is as high as 97.70%, indicating that the cyanine dye nanoparticles synthesized by the present invention have a good antibacterial effect.

Claims

1. A type of cyanine dye J-aggregate nanoparticles, characterized in that: The cyanine dye is compound Ⅰ-2: ； The cyanine dye is wrapped with an amphiphilic polymer compound to be assembled into cyanine dye J-aggregate nanoparticles; the amphiphilic polymer compound is Pluronic F68.

2. The cyanine dye J-aggregate nanoparticles according to claim 1, characterized in that: The molar ratio of the cyanine dye to the amphiphilic polymer compound is n / n, (0.001-0.10 μmol): (2.38-23.8 μmol).

3. Use of the cyanine dye J-aggregate nanoparticles according to claim 1 in the preparation of antitumor drugs or antibacterial drugs.

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