Non-excitation small-molecule photosensitizer medicine as well as preparation method and application thereof

Abstract

The invention discloses a non-excitation small-molecule photosensitizer drug as well as a preparation method and application thereof, belongs to the technical field of clinical metastatic cancer treatment drugs, and solves the technical problems of low efficiency, weak targeting property and large side effect in deep tissue metastatic tumor treatment due to the defects of PDT in the prior art. According to the non-excitation small-molecule photosensitizer drug provided by the invention, an energy donor and an acceptor are connected into a single molecule through a photosensitizer molecule in a covalent coupling manner, the energy acceptor is a porphyrin photosensitizer, or a cyanine photosensitizer, or a thiapyranium salt photosensitizer, and the energy donor is a peroxyoxalate ester compound; a targeting group T, a photosensitizer P and a peroxyoxalate ester group C are connected through a covalent bond to form a (targeting group T)-photosensitizer P-peroxyoxalate ester group C; and effective photodynamic therapy on deep tissue metastatic tumors is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical cancer treatment drugs, and specifically to a non-excited small molecule photosensitizer drug, its preparation method and application, which can be particularly used for treating metastatic tumors of cancer. Background Art

[0002] Photodynamic therapy (PDT) consists of three elements: a photosensitizer, excitation light, and oxygen, that is, the process in which photosensitizer molecules are activated under the irradiation of excitation light with a specific wavelength and energy transfer occurs to generate reactive oxygen species. Due to its advantages of non-invasiveness, high spatiotemporal selectivity, low systemic toxicity, and good therapeutic effect, PDT has been applied to clinical cancer treatment. However, restricted by the low tissue penetration depth of the excitation light required by traditional photosensitizers, currently PDT in clinical practice is mainly used for lesions on the body surface or lesions that are easily detectable by an endoscope, such as oral cancer, skin cancer, esophageal cancer, gastric cancer, bladder cancer, etc., and cannot be applied to metastatic tumors in deep tissues. Secondly, for multiple metastatic cancers, since whole-body light irradiation cannot be performed, PDT treatment based on light irradiation cannot be carried out. There are already a large number of photosensitizer molecule / nanoparticle patents, such as CN116789697A, CN111135299A, CN115010643B, but because they require external light source excitation, they cannot be applied to the treatment of deep tissue (metastatic) tumors. Moreover, currently there are photosensitizer molecules based on Cherenkov energy transfer, such as the patent WO2020084267A1. However, this molecule does not have tumor selectivity, so the side effects are serious.

[0003] Chemiluminescence is a process in which light is generated by chemical energy excitation during a chemical reaction process. Since no external excitation light is required, chemiluminescence can be generated in situ in tumor tissues and used to construct a chemiluminescence-activated tumor photodynamic therapy system. However, currently the structure and construction strategy of the probe result in its low efficiency, such as the patent CN102861334A.

[0004] Since the energy transfer efficiency highly depends on the intermolecular interaction distance between the donor and the acceptor, and in current research, the two usually form nanomaterials by self-assembly encapsulation through non-covalent interactions, this probe construction strategy based on intermolecular energy transfer leads to a far and uncontrollable distance between the energy donor and the acceptor, and the CRET efficiency is low; secondly, the photosensitizer molecules in the nanoparticles will undergo aggregation-induced quenching, reducing the ROS generation efficiency; moreover, the ROS has a short lifespan and a short diffusion distance, and the nanoscale assembly strategy seriously shields and hinders the diffusion of ROS, resulting in a large loss of ROS.

[0005] At present, although a large number of chemiluminescent photodynamic probes have been developed, the above problems cannot be solved simultaneously, and there is no therapeutic drug that can be used for the selective killing of metastatic tumors in deep tissues. Therefore, it is of great significance to design highly efficient tumor-selective chemiluminescent photodynamic photosensitizers and develop new photosensitizer-based drugs to improve the efficiency of PDT and expand its clinical cancer treatment application scope. Summary of the Invention

[0006] In view of the disadvantages and deficiencies of photodynamic therapy in the prior art, its application in the treatment of deep tissue metastatic tumors still faces challenges. The present invention provides a non-excited small molecule photosensitizer drug with high targeted delivery and selective release effects. The positively charged Cy7-EOM targets mitochondria and efficiently generates singlet oxygen through intramolecular chemiluminescent resonance energy transfer induced by endogenous H2O2, directly leading to mitochondrial damage and apoptosis, thereby realizing effective photodynamic therapy for deep tissue metastatic tumors, as well as its preparation method and application.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions: The non-excited small molecule photosensitizer drug provided by the present invention connects an energy donor and an energy acceptor within a single molecule through a covalent coupling method, and the energy donor is a peroxyoxalate compound or its derivative.

[0008] Preferably, it is specifically composed of a targeting group T, a photosensitizer P, and a peroxyoxalate group C connected by covalent bonds ((targeting group T)-photosensitizer P-peroxyoxalate group C ((T)-P-C)), and the structural formula of this drug is:

[0009]

[0010] The energy acceptor is a porphyrin-based photosensitizer, or a cyanine-based photosensitizer, or a thianthrenium salt-based photosensitizer.

[0011] Preferably, the targeting group T is a cancer cell targeting group, or a cancer cell organelle targeting group, or a protein in the cell, or an enzyme targeting group.

[0012] Preferably, the molecular structure of the photosensitizer is specifically: a photosensitizer molecule modified by an active group R1 (photosensitizer-R1), and R1 includes but is not limited to any one of hydroxyl, carboxyl, alkynyl, maleimide group, and cyclodextrin group.

[0013] Preferably, the molecular structure of the peroxyoxalate compound is specifically: a peroxyoxalate group modified by R2 (peroxyoxalate-R2), and R2 includes but is not limited to any one of amino, mercapto, azide group, acyl chloride, and adamantane.

[0014] The preparation method of the above-mentioned molecular photosensitizer drug is to connect an energy donor and an acceptor within a single molecule through a covalent coupling method for the photosensitizer molecule, and synthesize an intramolecular CRET chemiluminescent imaging therapy probe; wherein, the energy acceptor is a porphyrin photosensitizer, a cyanine photosensitizer, or a thianthrenium salt photosensitizer, and the energy donor is a peroxyoxalate compound or its derivative.

[0015] Preferably, the synthesis method of the intramolecular CRET chemiluminescent imaging therapy probe has the specific reaction as follows:

[0016] (1) Covalent bond reaction between hydroxyl group and acyl chloride

[0017] The hydroxyl group-modified photosensitizer and 2 equivalents of acyl chloride-modified peroxyoxalate are dissolved in anhydrous DCM, and a catalyst, triethylamine (TEA), is added. The reaction is carried out at room temperature, and the solution is concentrated by rotary evaporation and purified by column chromatography; the molecular photosensitizer drug product is obtained.

[0018]

[0019] Preferably, the synthesis method of the intramolecular CRET chemiluminescent imaging therapy probe has the specific reaction as follows:

[0020] (2) Click reaction (CuAAC reaction)

[0021] The alkyne group-modified photosensitizer and 2 equivalents of azide group-modified peroxyoxalate are placed in a 20 mL mixed solution of tert-butanol / H2O (2:1) containing CuSO4·5H2O (250 mg) and sodium ascorbate (400 mg). The solution is stirred at room temperature for 2 days, and the reaction is terminated by adding H2O; the product is extracted with ethyl acetate; the organic part is washed with 5% ammonium hydroxide and then with brine; the organic fraction is dried over magnesium sulfate; the solution is concentrated by rotary evaporation; the residue is purified by flash column chromatography to obtain the molecular photosensitizer drug product.

[0022]

[0023] Preferably, the synthesis method of the intramolecular CRET chemiluminescent imaging therapy probe has the specific reaction as follows:

[0024] (3) Amidation reaction

[0025] The carboxyl group-modified photosensitizer and 2 equivalents of amino group-modified peroxyoxalate are dissolved in anhydrous DCM, and the reaction is stirred at room temperature for 16 h under the condition of a condensing agent system catalyst; then the reaction mixture is concentrated by rotary evaporation; it is purified by column chromatography; the molecular photosensitizer drug product is obtained.

[0026]

[0027] The condensing agent system catalyst is a carbodiimide catalyst, specifically any one of 1,3-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), and the catalysts benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT).

[0028] Preferably, the synthesis method of the intramolecular CRET chemiluminescent imaging therapeutic probe, the specific reaction is as follows:

[0029] (4) Maleimide and thiol

[0030] The maleimide-modified photosensitizer and 2 equivalents of the thiol-modified peroxyoxalate are dissolved in diethyl ether, 2 equivalents of triethylamine are added, and the reaction is carried out at room temperature for 24 h. The reaction mixture is concentrated by rotary evaporation; purified by column chromatography; and the molecular photosensitizer drug product is obtained.

[0031]

[0032] The molecular photosensitizer drug of the present invention, the photosensitizer P is a near-infrared photosensitizer cyanine (Cy7) And ethyl oxalyl chloride (EOM)

[0034] Connected by an esterification covalent reaction to prepare a photosensitizer molecular drug (Cy7-EOM) based on intramolecular CRET, and the specific structural formula is as follows:

[0035] Preferably, it includes self-assembling the molecular photosensitizer Cy7-EOM into a nanomicelle containing a disulfide bond and modified with folic acid to prepare a water-soluble nanomicelle containing a disulfide bond (Cy7-EOM SS NMs).

[0036] The preparation method of the above-mentioned non-excited small molecule photosensitizer drug includes the specific steps of: co-assembling the molecular photosensitizer Cy7-EOM with an amphiphilic block copolymer modified with folic acid and containing a disulfide bond to prepare Cy7-EOM SS NMs;

[0037] The amphiphilic block copolymer modified with folic acid and containing a disulfide bond is DSPE-SS-PEG and DSPE-PEG-FA, and the ratio of their addition amounts is DSPE-SS-PEG:DSPE-PEG-FA greater than or equal to 1; the addition amount of the molecular photosensitizer Cy7-EOM is 2 times the addition amount of DSPE-SS-PEG.

[0038] The application of the above-mentioned molecular photosensitizer drug is to encapsulate Cy7-EOM into the tumor-specific response nanomicelle Cy7-EOMSS NMs for targeted delivery and release.

[0039] In cancer cells, Cy7-EOM SS NMs consume a large amount of reduced glutathione, leading to their degradation and the release of Cy7-EOM, a molecule that can specifically target mitochondria.

[0040] The application of the above molecular photosensitizer drug is used for tumor treatment, especially for the treatment of metastatic tumors or multiple tumors.

[0041] The present invention provides a non-excited small molecule photosensitizer drug and its application. It has the following beneficial effects:

[0042] (1) The non-excited small molecule photosensitizer drug, its preparation method and application of the present invention develop an efficient self-powered photosensitizer molecule Cy7-EOM. The covalent connection between the donor and the acceptor in the molecular structure significantly improves the efficiency of photodynamic therapy. With high targeting delivery and selective release effects, positively charged Cy7-EOM targets mitochondria and efficiently generates singlet oxygen through intramolecular chemiluminescence resonance energy transfer induced by endogenous H2O2, directly causing mitochondrial damage and apoptosis, thereby achieving effective photodynamic treatment of deep tissue metastatic tumors.

[0043] (2) The non-excited small molecule photosensitizer drug, its preparation method and application of the present invention use chemiluminescence as the excitation light source. Under the condition of high concentration of H2O2 in the tumor microenvironment, PDT can be autonomously activated in situ, overcoming the limitations of traditional PDT with shallow penetration depth and ineffectiveness against deep tissue metastatic tumors. It solves the problems of shallow penetration depth of the excitation light in photodynamic therapy and its application in the treatment of clinical metastatic tumors.

[0044] (3) The non-excited small molecule photosensitizer drug, its preparation method and application of the present invention also encapsulate Cy7-EOM into tumor-specific response nanomicelles Cy7-EOM SS NMs for targeted delivery and release. In cancer cells, Cy7-EOM SS NMs consume a large amount of reduced glutathione, leading to their degradation and the release of Cy7-EOM, a molecule that can specifically target mitochondria. The generated reactive oxygen species directly act on mitochondria, triggering a mitochondrial reactive oxygen species burst, thereby triggering apoptosis. This method has been successfully applied to the treatment of breast cancer cells and various metastatic tumors in mice, achieving efficient photodynamic treatment of metastatic tumors with extremely low systemic toxicity.

[0045] In addition, the specific decomposition of the nanomicelles at the tumor site can prevent aggregation-induced quenching and reduce 1 the diffusion barrier of O2, while in normal tissues, the integrity of the nanomicelles can shield 1The lethal effect of O2. This method provides a new strategy for converting auxiliary photosensitizers into direct therapeutic drugs and has significant application potential in the clinical treatment of metastatic tumors. Description of the Drawings

[0046] Figure 1 This is a schematic diagram of the mechanism of cancer cell killing by the Cy7-EOM photosensitizer drug in Example 6 of the present invention;

[0047] Figure 2 Photosensitizer drug molecule based on intramolecular CRET 1 Fluorescence spectral data graph of SOSG for detecting O2 yield;

[0048] Figure 3 This is the ESR spectrum of the reaction of Cy7-EOM in Example 6 of the present invention and Cy7-EOM SS NMs obtained after loading Cy7-EOM in Example 7 into the nanomicelles with hydrogen peroxide respectively to detect the generation of singlet oxygen, and the capturer is TEMP;

[0049] Figure 4 Confocal cell imaging map for targeted function characterization, where the nanoprobe - red fluorescence and the commercial mitochondrial labeling probe - green;

[0050] Figure 5 Confocal pictures and corresponding cell fluorescence quantitative bar graphs for cancer cell specific targeting verification;

[0051] Figure 6 For detecting 1 Confocal cell imaging map of O2 generation in cells detected by SOSG and corresponding fluorescence quantitative bar graphs;

[0052] Figure 7 Bar graph of the results of cell MTT experiment for detecting the killing ability of different concentrations of probes on cancer cells (4T1) and normal cells (AML12);

[0053] Figure 8 Verification of in vivo tumor treatment - mouse in vivo imaging map and corresponding chemiluminescence intensity quantitative bar graph. A mouse lung metastasis model (4T1-Luc) was constructed, and the relative size of lung metastatic tumors was reflected by the chemiluminescence intensity.

[0054] Figure 9 Characterization data graph of Cy7 / CPPO NMs and Cy7-EOM SS NMs, where Figure 9 a is the transmission electron microscope image of Cy7-EOM SS NMs; 9b is the transmission electron microscope image of Cy7-EOM SS NMs after incubation with GSH;

[0055] Figure 101H-NMR spectrum of Cy7 in CDCl3; 1 1H-NMR spectrum;

[0056] Figure 11 13C-NMR spectrum of Cy7 in CDCl3; 13 13C-NMR spectrum;

[0057] Figure 12 High resolution mass spectrum HR-MS of Cy7;

[0058] Figure 13 1H-NMR spectrum of Cy7-EOM in d6-DMSO; 1 1H-NMR spectrum;

[0059] Figure 14 13C-NMR spectrum of Cy7-EOM in CDCl3; 13 13C-NMR spectrum;

[0060] Figure 15 High resolution mass spectrum HR-MS of Cy7-EOM;

[0061] Figure 16 Electron microscopy images, where (16a) is the electron microscopy image of Cy7-EOM NMs nanomicelles; (16b) is the electron microscopy image of Cy7-EOM NMs nanomicelles after incubation with GSH (10 mM) for 3 h;

[0062] Figure 17 Particle size distribution graphs; where (17a) is the particle size distribution graph of Cy7-EOM SS NMs, and (17b) is the particle size distribution graph of Cy7-EOM NMs;

[0063] Figure 18 Zeta potential graphs; (a) Zeta potential graph of Cy7-EOM SS NMs, (b) Zeta potential graph of Cy7-EOM NMs. Detailed implementation mode

[0064] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0065] Example 1

[0066] The non-excited small molecule photosensitizer drug provided by the present invention connects the energy donor and acceptor within a single molecule through a covalent coupling method. Specifically, it is composed of a targeting group T, a photosensitizer P, and a peroxyoxalate group C connected by covalent bonds (targeting group T)-photosensitizer P-peroxyoxalate group C ((T)-P-C). The structural formula of the drug is as follows:

[0067]

[0068] The targeting group T is a cancer cell targeting group, or a cancer cell organelle targeting group, or a protein or enzyme targeting group in cells; Peroxalate esters with high quantum efficiency and long luminescence lifetime are selected as energy donors, and porphyrins, cyanines, thianthrenium salts and other photosensitizers with high singlet oxygen quantum yield are selected as energy acceptors based on the principle of energy level matching.

[0069] In order to covalently connect the energy donor and the acceptor, active sites are preferably constructed in the dye molecular structure, and the covalent linkage of related components is gradually realized through easily operable electrophilic substitution reactions, amidation reactions, click reactions, etc.

[0070] Representative photosensitizer molecular structure: A photosensitizer molecule modified by an active group R1 (photosensitizer - R1), where R1 includes but is not limited to hydroxyl, carboxyl, alkynyl, maleimide group, cyclodextrin group;

[0071]

[0072] The specific representative peroxalate molecular structure is: A peroxalate group modified by R2 (peroxalate - R2), where R2 includes but is not limited to amino, mercapto, azide group, acyl chloride, adamantane;

[0073]

[0074] The synthesis method of the intramolecular CRET chemiluminescent imaging therapy probe of the present invention (including but not limited to the following):

[0075]

[0076] The cancer cell killing mechanism of this photosensitizer drug (taking Cy7 - EOM as an example):

[0077] As Figure 1 shown, when the photosensitizer drug molecule enters cancer cells, the peroxalate group (C) therein reacts with the high - level hydrogen peroxide in cancer cells to generate a high - energy intermediate 1,2 - dioxetane. This intermediate is unstable, and the energy generated during decomposition is transferred to the photosensitizer (P), making it in an excited state. The excited photosensitizer returns to the ground state and transfers the energy to the surrounding oxygen to make it turn into 1 O2 with killing ability, thus killing tumor cells. Since the CRET efficiency is severely affected by the distance between the donor and the acceptor, the photosensitizer molecules in this patent application connect the energy donor and the acceptor within a single molecule through covalent coupling, greatly shortening the distance between the two, and can induce highly efficient intramolecular CRET and 1O2 generates luminescence without the need for an external light source, significantly improving the CRET efficiency. Further connecting a targeting group (T) enables the selective killing of key molecules at the targeted site by reactive oxygen species.

[0078] Example 2

[0079] The method for preparing a non-excited small molecule photosensitizer drug is to connect an energy donor and an acceptor within a single molecule through a covalent coupling method for the photosensitizer molecule to synthesize an intramolecular CRET chemiluminescence imaging therapy probe; among them, the energy acceptor is a porphyrin-based photosensitizer, a cyanine-based photosensitizer, or a thianthrenium salt-based photosensitizer, etc., and the energy donor is a peroxyoxalate compound. The synthesis method of the intramolecular CRET chemiluminescence imaging therapy probe, the specific reaction is:

[0080] (1) Covalent bond reaction between hydroxyl group and acyl chloride

[0081] Dissolve 50 mg of the hydroxyl group-modified photosensitizer in 20 mL of anhydrous dichloromethane, place the solution in an ice bath, slowly add 2.94 mL of triethylamine dropwise, and then slowly add 2.73 mg of ethyl oxalyl chloride to the solution. Remove the ice bath and react at room temperature for 6 h. Remove the solvent by rotary evaporation. The obtained solid is purified by column chromatography (DCM:MeOH = 20:1) to obtain the target compound.

[0082] Example 3

[0083] The method for synthesizing a non-excited small molecule photosensitizer drug, that is, an intramolecular CRET chemiluminescence imaging therapy probe, the specific reaction is:

[0084] (2) Click reaction - copper-catalyzed alkyne-azide click chemistry, CuAAC reaction

[0085] Place 5 g of the alkyne group-modified photosensitizer and 5.3 g of the azide group-modified peroxyoxalate in a mixed solution containing 20 mL of tert-butanol / H2O (2:1). Add 250 mg of CuSO4·5H2O and 400 mg of sodium ascorbate to it. Under a nitrogen atmosphere, stir at room temperature for 2 days. Add H2O to terminate the reaction; extract the product with ethyl acetate (4 x 50 mL); add magnesium sulfate to dry the organic phase; concentrate the solvent by rotary evaporation to obtain the crude product; purify the product by column chromatography (DCM:MeOH = 20:1) to obtain the non-excited small molecule photosensitizer drug product.

[0086] Example 4

[0087] The method for synthesizing a non-excited small molecule photosensitizer drug, that is, an intramolecular CRET chemiluminescence imaging therapy probe, the specific reaction is:

[0088] (3) Amidation reaction

[0089] Dissolve 5 g of carboxyl - modified photosensitizer and 4.6 g of amino - modified peroxyoxalate in 20 mL of anhydrous dichloromethane. Add 3.4 mL of EDC (1 - ethyl - 3-(3 - dimethylaminopropyl)carbodiimide) to it, and slowly add 2.94 mL of triethylamine dropwise. The solution is stirred at room temperature for 24 h. Concentrate the solvent by rotary evaporation to obtain the crude product; purify it by column chromatography (DCM:MeOH = 20:1) to obtain the non - excited small - molecule photosensitizer drug product.

[0090] Example 5

[0091] A method for synthesizing a non - excited small - molecule photosensitizer drug, namely an intramolecular CRET chemiluminescence imaging therapy probe. The specific reaction is as follows:

[0092] (4) Maleimide and thiol

[0093] Under a nitrogen atmosphere, dissolve 7.3 g of maleimide - modified photosensitizer and 4.4 g of thiol - modified peroxyoxalate in 20 mL of diethyl ether. Slowly add 2.9 mL of triethylamine dropwise. The solution reacts at room temperature for 24 h. Concentrate the solvent of the reaction mixture by rotary evaporation to obtain the crude product; purify it by column chromatography (DCM:MeOH = 20:1); obtain the non - excited small - molecule photosensitizer drug product.

[0094] Example 6

[0095] A method for synthesizing a non - excited small - molecule photosensitizer drug, namely an intramolecular CRET chemiluminescence imaging therapy probe. The specific reaction is as follows:

[0096] Photosensitizer P is a near - infrared photosensitizer cyanine (Cy7) covalently linked to ethyl oxalyl chloride (EOM) through an esterification reaction. Dissolve 5.4 mg of Cy7 in anhydrous dichloromethane. Slowly add 2.94 mL of triethylamine dropwise under an ice bath, and then slowly dissolve 2.73 mg of EOM in the above solution. React at room temperature for 6 h. Concentrate the solution by rotary evaporation and purify it by column chromatography (DCM:MeOH = 20:1) to prepare the photosensitizer molecule drug (Cy7 - EOM) based on intramolecular CRET; 1 H NMR(400MHz,DMSO - d6)δ8.26 - 8.22(d,2H),7.62 - 7.60(d,2H),7.45 - 7.39(m,4H),7.29 - 7.25(t,2H),6.43 - 6.40(d,2H),4.29(t,4H),3.79(t,4H),3.09 - 3.02(m,4H),2.70 - 3.67(t,4H),1.85 - 1.82(m,2H),1.68(12H),1.21 - 1.17(6H).

[0097]

[0098] Example 7

[0099] To achieve the targeted delivery of the molecular photosensitizer drug and selectively release it inside cancer cells, in a nano-assembly manner, the molecular photosensitizer Cy7-EOM prepared in Example 6 was self-assembled into nano-micelles containing disulfide bonds and modified with folic acid to obtain water-soluble nano-micelles containing disulfide bonds (Cy7-EOM SS NMs). The specific steps include: Dissolve 1 mg of the photosensitizer drug Cy7-EOM, 2 mg of PEG-DSPE 2000 -FA, and 4 mg of PEG-SS-DSPE 2000 in 1 mL of DCM, dissolve it under ultrasound for 10 min, and then evaporate to remove DCM; add 1 mL of deionized water and stir vigorously overnight at room temperature to obtain an aqueous solution of nano-micelles Cy7-EOM SS NMs;

[0100] Dialyze the solution in deionized water for 24 h; store the obtained solution at 4°C.

[0101] Control Group 1

[0102] As a control group, using the same method as in the preparation of Example 7, the difference is that the molecular photosensitizer Cy7-EOM prepared in Example 6 was self-assembled into nano-micelles without disulfide bonds and modified with folic acid. The specific steps include: Dissolve 1 mg of the photosensitizer drug Cy7-EOM, 2 mg of PEG-DSPE 2000 -FA, and 4 mg of PEG-DSPE 2000 in 1 mL of DCM, dissolve it under ultrasound for 10 min, and then evaporate to remove DCM; add 1 mL of deionized water and stir vigorously overnight at room temperature to obtain an aqueous solution of nano-micelles without disulfide bonds Cy7-EOM NMs.

[0103] Synthesis Characterization and Application Results

[0104] 1. Basic Performance Characterization

[0105] As Figures 10 - 15 shown, the designed Cy7 and Cy7-EOM were successfully characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS) respectively.

[0106] As Figure 9 and Figure 16 shown, it can be seen from the transmission electron microscope (TEM) images that both Cy7-EOM SS NMs and Cy7-EOM NMs are spherical and have good monodispersity.

[0107] Due to the specific response of the disulfide bond, after binding with glutathione (GSH), the disulfide bond-containing nanomicelles decompose and release the cargo. Transmission electron microscopy (TEM) images showed that the morphology of Cy7-EOM SS NMs changed significantly after incubation with GSH for 3 h, while the morphology of Cy7-EOM NMs remained intact.

[0108] As Figures 17 - 18 shown, through dynamic light scattering (DLS) analysis, the hydrodynamic diameter of Cy7-EOM NMs was comparable to that of Cy7-EOM SS NMs, and both were found to have a neutral surface charge by zeta potential measurement.

[0109] The optical properties of Cy7-EOM and related nanomicelles were recorded and analyzed. From the absorption spectrum, the maximum absorption peak of Cy7-EOM was approximately 774 nm, the same as that of the Cy7 parent, while the maximum absorption peak of Cy7-EOM SS NMs was red-shifted to 786 nm.

[0110] Similarly, fluorescence and chemiluminescence spectra showed that the maximum emission peak of Cy7-EOM was located at 803 nm, while it was red-shifted by approximately 25 nm after forming nanomicelles. Moreover, at the same concentration, the fluorescence emission intensity of Cy7-EOM SS NMs was approximately half that of Cy7-EOM, which was due to aggregation-caused quenching (ACQ).

[0111] In addition, control group 1 was Cy7 / CPPO NMs nanomicelles without disulfide bonds, and the energy transfer mode between the internally loaded Cy7 and CPPO was intermolecular energy transfer. After incubation with H2O2, the chemiluminescence intensity of Cy7-EOM SS NMs was much higher than that of Cy7 / CPPO NMs, indicating that intramolecular CRET was more efficient than intermolecular CRET.

[0112] Then, the chemiluminescence kinetics of Cy7-EOM and Cy7-EOM SS NMs under different concentrations of H2O2 were also experimentally studied. It could be found from the chemiluminescence images that the reaction of Cy7-EOM to H2O2 was rapid and concentration-dependent, indicating that effective intramolecular CRET could be achieved in the designed photosensitizer molecules, while the reaction of Cy7-EOM SS NMs to H2O2 showed a time lag. This was mainly due to the shielding effect of the nanomicelles, which hindered the effective contact between Cy7-EOM and H2O2. Therefore, similarly, the nanomicelles would also hinder the diffusion of short-lived ROS generated inside the nanoparticles, resulting in the loss of ROS and low photodynamic therapy efficiency.

[0113] 2. Application detection results

[0114] 2.1 Singlet oxygen of the photosensitizer drug molecule designed by the present invention based on intramolecular CRET 1 has a higher O2 yield.

[0115] The content of O2 generated by CRET in each group was detected by using a singlet oxygen probe (SOSG) for detection and comparison of 1 O2 generation. As 1 shown in Figure 2 a fluorescence spectrum, after adding H2O2 to the Cy7-EOM solution, the fluorescence intensity of SOSG increased significantly and showed a 5-fold enhancement. It indicates that 1 O2 was generated.

[0116] As Figure 2 shown in 1 b, the Cy7-EOM SS NMs pretreated with GSH generated more 1 O2 than the Cy7-EOM SS NMs without GSH treatment, further confirming that ACQ and the nanomicelle structure would affect the

[0117] effective utilization of Figure 2 O2. As shown in 1 c, compared with the Cy7 / CPPO NMs, the SOSG fluorescence of the Cy7-EOM SS NMs group was enhanced, indicating that the 1 O2 generated by intramolecular CRET was more efficient than that of intermolecular CRET. The 1 O2 generated by the nanosensor based on intramolecular CRET (red line) was significantly higher than that of the nanosensor based on intermolecular CRET (black line), proving that the intramolecular CRET efficiency was higher than the intermolecular CRET efficiency.

[0118] In addition, the 1

[0119] O2 was also analyzed by electron spin resonance (ESR) spectroscopy using the radical scavenger 2,2,6,6-tetramethylpiperidine (TEMP). The characteristic peak appearing in the ESR spectrum after adding H2O2 indicated the

[0120] generation of Figure 2 O2, and the increase in the ESR signal intensity in the Cy7-EOM sample further proved that the release of Cy7-EOM from the nanomicelles was beneficial to photodynamic therapy. 1 As Figure 2 shown in 1 a, after adding H2O2 to the molecular photosensitizer, the SOSG fluorescence increased significantly, indicating the Figure 2As shown in b, after the nanomicelles loaded with the molecular photosensitizer are treated with GSH, they degrade themselves and release the internal photosensitizer molecule Cy7-EOM. In this way, on the one hand, the aggregation-induced quenching phenomenon is eliminated, and on the other hand, the diffusion shielding of the nanomicelles to 1 O2 is removed. Compared with the group without GSH - the micelles remain intact, and the fluorescence of SOSG is significantly enhanced, thus proving that the utilization rate of reactive oxygen species is increased. Due to the high concentrations of H2O2 and GSH in tumor tissues, the molecular photosensitizer designed in the present invention and its nanoassembly application have tumor specificity. As Figure 3 shown, the ESR spectra of the Cy7-EOM in Example 6 and the Cy7-EOM SS NMs obtained after loading Cy7-EOM into the nanomicelles in Example 7 of the present invention after reacting with hydrogen peroxide are used to detect the generation of singlet oxygen, and the capturer is TEMP.

[0121] 2.3 Targeting function characterization

[0122] As Figure 4 shown in the confocal cell imaging diagram, the nanoprobe (red fluorescence) overlaps well with the commercial mitochondrial labeling probe (green), showing a yellow superimposed color, and the Pearson colocalization coefficient reaches 0.90, proving its excellent mitochondrial targeting function. The photosensitizer connected with the targeting group can selectively kill specific parts or specific molecules.

[0123] 2.4 Verification of cancer cell-specific targeting

[0124] Through the fluorescence analysis of a confocal laser scanning microscope (CLSM), the cellular uptake performance of the folic acid-mediated Cy7-EOM SS NMs nanomaterial in 4T1 cells was studied.

[0125] The confocal images and corresponding statistical data show that the red fluorescence signal of 4T1 cells pre-treated with folic acid is weaker than that of untreated cells. This is mainly because the folic acid molecules bind to the folate receptors on the cancer cell membrane prematurely, hindering the binding of the folic acid in the Cy7-EOM SS NMs nanomaterial to the folate receptor, thereby interfering with the cellular uptake of the Cy7-EOM SS NMs nanomaterial. Therefore, it is necessary to first use the folic acid-modified Cy7-EOM SS NMs nanoprobe.

[0126] The folic acid-modified Cy7-EOM SS NMs nanoprobe was incubated with cancer cells (mouse breast cancer cells, 4T1 cells, high expression of folate receptor) and normal cells (mouse normal liver cells, AML12 cells, low expression of folate receptor) for 6 h, and cellular laser confocal imaging was performed.

[0127] Since the expression level of folate receptors on the cancer cell membrane is much higher than that on the normal cell membrane, as Figure 5As shown in the confocal images and the corresponding cell fluorescence quantitative histograms, obvious red fluorescence appears in 4T1 cancer cells, while the fluorescence intensity of AML12 normal cells is very low. Therefore, the results indicate that Cy7-EOM SS NMs nano-probes can more easily enter 4T1 cancer cells, and the amount entering AML12 normal cells is very low. So, it shows that folic acid-modified Cy7-EOM SS NMs nano-probes can specifically target cancer cells.

[0128] 2.5 Intracellular 1 O2 Generation Detection

[0129] As Figure 6 shown, confocal cell imaging of intracellular 1 O2 generation detected by SOSG and the corresponding fluorescence quantification are shown. Cy7-EOM prepared in Example 7 was encapsulated into tumor-specific responsive nano-micelles Cy7-EOM SS NMs for targeted delivery and release; the nano-micelles Cy7-EOM SS NMs containing disulfide bonds can specifically degrade after entering cancer cells by reacting with GSH inside the cancer cells: Cy7-EOM SS NMs will degrade and release Cy7-EOM under the action of high-concentration intracellular glutathione (GSH), avoiding the aggregation-induced quenching phenomenon and eliminating the shielding of the diffusion of reactive oxygen species by the nano-micelles. The SOSG fluorescence is the strongest, indicating 1 the most O2 is produced. This molecule can specifically target mitochondria and synergistically enhance the photodynamic therapy effect. While the control group Cy7-EOM NMs micelles do not contain disulfide bonds and cannot release the internal photosensitizer molecules, 1 relatively less O2 is produced; the control group Cy7 / CPPO NMs is Cy7 and CPPO self-assembled inside the micelles, and the intermolecular CRET efficiency is low, 1 the least O2 is produced.

[0130] 2.6 Detection of the killing ability of different concentrations of probes on cancer cells (4T1) and normal cells (AML12) - Cell MTT assay.

[0131] The cell toxicity of Cy7-EOM SS NMs was evaluated by the thiazolyl blue (MTT) assay. As the concentration of Cy7-EOM SS NMs increased, the survival rate of 4T1 cells decreased significantly. At a concentration of 50 μg / mL, the survival rate was as low as 18.44%, while the survival rates of 4T1 cells treated with Cy7NMs, Cy7 / CPPO NMs, and Cy7-EOM NMs were much higher, at 86.5%, 76.1%, and 32.6% respectively. In contrast, even at high incubation concentrations, no obvious cell death was observed in AML12 cells treated with Cy7-EOM SS NMs.

[0132] MTT cell experiments showed that, as Figure 7 shown, compared with the control group of Cy7-EOM NMs and Cy7 / CPPO NMs, the Cy7-EOM SS NMs nanoparticles prepared by the present invention had excellent performance. It had good killing ability against cancer cells (such as Figure 7 a), without damaging normal cells (such as Figure 7 b).

[0133] 2.7 In vivo tumor treatment verification

[0134] As Figure 8 shown in the in vivo imaging of mice and the corresponding quantification of chemiluminescence intensity, a mouse lung metastasis model (4T1-Luc) was constructed, and the relative size of lung metastases was reflected by chemiluminescence intensity. A mouse model of lung metastasis was established by intravenous injection of luciferase-transfected 4T1 cells (4T1-Luc). From the fluorescence images, it was detected that after intravenous injection of Cy7-EOM SS NMs, obvious chemiluminescence signals were observed in the lungs. At the same time, chemiluminescence signals were also observed in the abdominal cavity. The mice were euthanized and dissected, and new metastatic tumor foci were found in the intestine. The results showed that the designed Cy7-EOM SS NMs had significant targeting and diagnostic abilities, and could even detect tiny metastatic tumor foci.

[0135] Subsequently, its anti-tumor effect was evaluated. The detailed treatment plan was as follows: The mice were randomly divided into five groups and given different treatments: PBS, Cy7 NMs, Cy7 / CPPO NMs, Cy7-EOM NMs, and Cy7-EOM SS NMs, with a dose of 2.5 mg / kg for all. Bioluminescence imaging technology was used to evaluate the treatment effect. The bioluminescence intensity of the mice treated with PBS and Cy7 NMs increased significantly, and new metastatic foci appeared in the abdominal cavity, indicating rapid tumor progression. In contrast, the tumor growth of the mice treated with Cy7 / CPPO NMs, Cy7-EOM NMs, and Cy7-EOM SS NMs was significantly inhibited. Among them, Cy7-EOM SS NMs showed the best performance, and its bioluminescence intensity decreased to 9.8% of the PBS group.

[0136] Specifically, multiple metastatic foci appeared in the intestines of mice in the PBS, Cy7 NMs, and Cy7 / CPPO NMs treatment groups, while only weak bioluminescence signals were observed in the lungs of mice in the Cy7-EOM SS NMs treatment group, and no bioluminescence signals were seen in the abdominal cavity, indicating its great potential in treating metastatic tumors and even multiple tumors. After 15 days of treatment, the lungs of mice in each group were dissected. Photos and H&E staining also showed that there were more metastatic tumor nodules in the lungs of the PBS group, Cy7 NMs group, and Cy7-EOM NMs group, while almost no metastatic lesions were seen in the Cy7-EOM SS NMs group. In addition, after treatment with Cy7-EOM SS NMs, the survival period of mice was significantly prolonged, and 60% of the mice survived for more than 40 days, while almost all the mice in the control group died within 25 days. Additionally, during the treatment period, the body weights of mice in each group were monitored, and no significant weight loss was observed, further confirming the low side effects and safety of Cy7-EOM SS NMs.

[0137] As demonstrated above, the nano-assembly of the photosensitizer molecule prepared by the present invention and its tumor-selective killing mechanism are as follows: The molecular photosensitizer Cy7-EOM prepared by the present invention self-assembles into nano-micelles Cy7-EOM SS NMs containing disulfide bonds and folic acid modification. On the one hand, the folic acid groups on the surface of the nano-micelles can selectively target the folate receptors on the surface of cancer cells, enabling the nano-micelles to actively target and enter tumor cells; on the other hand, the nano-micelles can consume the high levels of glutathione in cancer cells and specifically decompose themselves, releasing the photosensitizer molecules, thereby efficiently killing cancer cells. In normal tissues, the expression level of folate receptors is low, and the nano-materials will not actively target; moreover, the GSH content in normal cells is low, and it cannot destroy the nano-micelles and release the photosensitizer molecules.

[0138] Since the photosensitizer molecules aggregate inside the micelles, aggregation-induced quenching phenomenon will occur, reducing the 1 O2 generation efficiency; secondly, due to the short lifespan and limited diffusion distance of reactive oxygen species, and the nano-micelle assembly restricts the diffusion of reactive oxygen species, greatly reducing the utilization rate of reactive oxygen species; moreover, the H2O2 content in normal cells is low, 1 the O2 production is low, and it will not damage normal cells, thus having tumor specificity.

[0139] In summary, for the non-excited small molecule photosensitizer drug of the present invention, its preparation method and application, an efficient self-powered photosensitizer molecule Cy7-EOM has been developed. The covalent connection between the donor and the acceptor in the molecular structure significantly improves the efficiency of photodynamic therapy. Under the condition of high concentration of H2O2 in the tumor microenvironment, PDT can be autonomously activated in situ, overcoming the limitations of traditional PDT with shallow penetration depth and ineffectiveness against metastatic tumors in deep tissues. And Cy7-EOM is encapsulated into tumor-specific responsive nanomicelles Cy7-EOM SS NMs for targeted delivery and release. In cancer cells, Cy7-EOM SS NMs will consume a large amount of reduced glutathione, resulting in their degradation and release of Cy7-EOM, which can specifically target mitochondria. The generated reactive oxygen species directly act on mitochondria, triggering a mitochondrial reactive oxygen species burst, thereby triggering apoptosis. This method has been successfully applied to the treatment of breast cancer cells and various metastatic tumors in mice, achieving efficient photodynamic therapy of metastatic tumors with extremely low systemic toxicity. These findings expand the practical application scope of photosensitizers and provide new strategies for photodynamic anti-cancer drugs, having important potential for clinical translation.

[0140] The above are only examples of the present invention. For example, when the photosensitizer modified with carboxyl reacts with the peroxyoxalate modified with amino, any one of the carbodiimide-based condensing agent system catalysts such as 1,3-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), and catalysts benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT) can achieve the non-excited small molecule photosensitizer drug of the present invention, its preparation method and application.

[0141] The above description is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A non-excitation small molecule photosensitizer drug, characterized in that: The energy donor and the acceptor are connected in a single molecule by covalent coupling of the photosensitizer molecule, wherein the energy acceptor is a photosensitizer and the energy donor is a peroxyoxalate compound or a derivative thereof.

2. The non-excitation small molecule photosensitizer drug according to claim 1, characterized in that: Specifically, the targeting group T, the photosensitizer P, and the peroxy oxalate group C are connected by covalent bonds to form (targeting group T)-photosensitizer P-peroxy oxalate group C ((T)-PC), and the drug structure expression is: The energy acceptor photosensitizer is a porphyrin photosensitizer, a cyanine photosensitizer, or a thiopyranium salt photosensitizer.

3. The non-excitation small molecule photosensitizer drug according to claim 2, characterized in that: The targeting group T is a cancer cell targeting group, or a cancer cell organelle targeting group, or a protein or enzyme targeting group in the cell.

4. The non-excitation small molecule photosensitizer drug according to claim 1, characterized in that: The photosensitizer molecular structure is specifically: a photosensitizer molecule modified by an active group R1 (photosensitizer-R1), wherein R1 is any one of a hydroxyl group, a carboxyl group, an alkynyl group, a maleimide group, and a cyclodextrin group.

5. The non-excitation small molecule photosensitizer drug according to claim 4, characterized in that: The molecular structure of the peroxyoxalate compound is specifically: a peroxyoxalate group modified by R2 (peroxyoxalate-R2), wherein R2 is any one of an amino group, a thiol group, an azide group, an acyl chloride, and adamantane.

6. A method for preparing a non-excitation small molecule photosensitizer drug according to any one of claims 1 to 5, characterized in that: The energy donor and the acceptor are connected in a single molecule by covalent coupling of the photosensitizer molecule to synthesize an intramolecular CRET chemiluminescent imaging therapeutic probe; wherein the energy acceptor is a porphyrin photosensitizer, a cyanine photosensitizer, or a thiopyranium salt photosensitizer, and the energy donor is a peroxyoxalate compound or a derivative thereof.

7. The method for preparing a non-excitation small molecule photosensitizer drug according to claim 6, characterized in that: The synthesis method of the intramolecular CRET chemiluminescent imaging therapeutic probe is as follows: (1) Synthesis by covalent bond reaction of hydroxyl and acyl chloride The hydroxyl-modified photosensitizer and 2 equivalents of acyl chloride-modified peroxyoxalate are dissolved in anhydrous DCM, and a catalyst triethylamine (TEA) is added. After the reaction is completed at room temperature, the solution is concentrated by rotary evaporation and purified by column chromatography to obtain a molecular photosensitizer drug product; 8. The method for preparing a non-excitation small molecule photosensitizer drug according to claim 6, characterized in that: The synthesis method of the intramolecular CRET chemiluminescent imaging therapeutic probe is as follows: (2) copper-catalyzed alkyne azide click reaction synthesis (CuAAC reaction): an alkyne-modified photosensitizer and a 2-fold equivalent of an azide-modified peroxy oxalate are placed in a mixed solution containing copper sulfate pentahydrate (CuSO4·5H2O), sodium ascorbate and tert-butyl alcohol / H2O (2:1), the solution is stirred at room temperature until the reaction is sufficient, and H2O is added to terminate the reaction; the product is extracted with ethyl acetate; the organic part is washed with 5% ammonium hydroxide and then with brine; the organic fraction is dried over magnesium sulfate; the solution is concentrated by rotary evaporation; and the residue is purified by rapid column chromatography to obtain a molecular photosensitizer drug product; 9. The method for preparing a non-excitation small molecule photosensitizer drug according to claim 6, characterized in that: The synthesis method of the intramolecular CRET chemiluminescent imaging therapeutic probe is as follows: (3) amidation reaction synthesis The carboxyl-modified photosensitizer and 2 equivalents of amino-modified peroxy oxalate are dissolved in anhydrous DCM, and the reaction is completed by stirring at room temperature under the conditions of a condensing agent system catalyst; the reaction mixture is then concentrated by rotary evaporation; and purified by column chromatography to obtain a molecular photosensitizer drug product; The condensation agent system catalyst is a carbodiimide catalyst, specifically any one of 1,3-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), catalyst benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 1-hydroxybenzotriazole (HOBT).

10. The method for preparing a non-excitation small molecule photosensitizer drug according to claim 6, characterized in that: The synthesis method of the intramolecular CRET chemiluminescent imaging therapeutic probe is as follows: (4) Synthesis by reaction of maleimide with thiol The maleimide-modified photosensitizer and 2 equivalents of thiol-modified peroxyoxalate are dissolved in ether, and 2 equivalents of triethylamine are added to complete the reaction at room temperature. The reaction mixture is concentrated by rotary evaporation and purified by column chromatography to obtain a molecular photosensitizer drug product.

11. The non-excitation small molecule photosensitizer drug according to claim 1, characterized in that: The photosensitizer P is a near-infrared photosensitizer cyanine (Cy7) With ethyl oxalyl chloride (EOM) Through esterification covalent reaction connection, the photosensitizer molecular drug (Cy7-EOM) based on intramolecular CRET was prepared, and the specific structural formula is as follows:

12. The non-excitation small molecule photosensitizer drug according to claim 11, characterized in that: The method also includes (5) nanoassembly: self-assembling the molecular photosensitizer Cy7-EOM into the disulfide bond-containing and folic acid-modified nanomicelles to prepare water-soluble disulfide bond-containing nanomicelles (Cy7-EOM SS NMs).

13. The method for preparing a non-excitation small molecule photosensitizer drug according to claim 12, characterized in that: The specific steps include: co-assembling a molecular photosensitizer Cy7-EOM and an amphiphilic block copolymer modified with folic acid and containing a disulfide bond to prepare Cy7-EOM SS NMs; The folic acid-modified amphiphilic block copolymer containing disulfide bonds is the DSPE-SS-PEG and DSPE-PEG-FA, and the ratio of the added amounts of the two is DSPE-SS-PEG:DSPE-PEG-FA is greater than or equal to 1; the added amount of the molecular photosensitizer Cy7-EOM is twice the added amount of DSPE-SS-PEG.

14. The use of a non-excitation small molecule photosensitizer drug according to any one of claims 1 to 13, characterized in that: Used for tumor treatment, especially for the treatment of metastatic tumors or multiple tumors.

15. The use of a non-excitation small molecule photosensitizer drug according to any one of claims 11 to 13, characterized in that: Cy7-EOM was encapsulated into tumor-specific responsive nanomicelles Cy7-EOM SS NMs for targeted delivery and release; in cancer cells, Cy7-EOM SS NMs consumed a large amount of reduced glutathione, leading to its degradation and release of Cy7-EOM, which can specifically target mitochondria.

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