A non-excitation small molecule photosensitizer drug and a preparation method and application thereof

Abstract

The application discloses a kind of no excitation small molecule photosensitizer drug and preparation method and application, belong to clinical metastatic cancer treatment drug technical field, solve the defects of PDT itself in prior art, it has the technical problems such as low efficiency, weak targeting, large side effect in deep tissue metastatic tumor treatment.The no excitation small molecule photosensitizer drug provided by the application is that photosensitizer molecules are connected in single molecule by covalent coupling method with energy donor and acceptor, synthesizes intramolecular CRET chemiluminescence imaging treatment probe, energy acceptor is photosensitizer, energy donor is peroxysuccinate compound or its derivative;Energy acceptor photosensitizer is chlorin photosensitizer, photosensitizer P is near-infrared photosensitizer chlorin Cy7, peroxysuccinate group C is oxalyl chloride ethyl ester, and the specific reaction is that Cy7 and oxalyl chloride ethyl ester are connected by esterification covalent reaction, to prepare photosensitizer molecular drug Cy7-EOM based on intramolecular CRET, realize effective photodynamic therapy to deep tissue metastatic tumor.

Description

Technical Field

[0001] This invention relates to the field of clinical cancer treatment drug technology, specifically to a non-excited small molecule photosensitizer drug, its preparation method and application, which can be used, in particular, to treat metastatic cancer tumors. Background Technology

[0002] Photodynamic therapy (PDT) comprises three elements: photosensitizer, excitation light, and oxygen. It involves the activation of photosensitizer molecules under excitation light of a specific wavelength, resulting in energy transfer and the generation of reactive oxygen species. Due to its advantages such as non-invasiveness, high spatiotemporal selectivity, low systemic toxicity, and good therapeutic effects, PDT has been applied in clinical cancer treatment. However, limited by the low tissue penetration depth required by traditional photosensitizers, PDT is currently mainly used clinically for lesions on the body surface or lesions easily explored with endoscopy, such as oral cancer, skin cancer, esophageal cancer, gastric cancer, and bladder cancer. It cannot be applied to metastatic tumors in deep tissues. Secondly, for multiple metastatic cancers, whole-body phototherapy is not possible, making phototherapy-based PDT impossible. Numerous photosensitizer molecule / nanotechnology patents exist, such as CN116789697A, CN111135299A, and CN115010643B, but because they require external light source excitation, they cannot be applied to the treatment of deep tissue (metastatic) tumors. Furthermore, there are already photosensitizer molecules based on Cherenkov energy transfer, such as patent WO2020084267A1; however, this molecule does not have tumor selectivity, and therefore has serious side effects.

[0003] Chemiluminescence is a process in which light is generated through chemical energy excitation during a chemical reaction. Since no external excitation light is required, chemiluminescence can be generated in situ within tumor tissue and used to construct chemiluminescence-activated photodynamic therapy systems for tumors. However, current probe structures and construction strategies result in low efficiency, as illustrated by patent CN102861334A.

[0004] Since energy transfer efficiency heavily depends on the intermolecular interaction distance between the donor and acceptor, and current research typically involves the two molecules self-assembling and encapsulating to form nanomaterials through non-covalent interactions, this probe construction strategy based on intermolecular energy transfer results in a large and uncontrollable distance between the energy donor and acceptor, leading to low CRET efficiency. Secondly, photosensitizer molecules in nanoparticles undergo aggregation-induced quenching, reducing the generation efficiency of ROS. Furthermore, ROS has a short lifetime and short diffusion distance, and the nano-assembly strategy severely shields and hinders ROS diffusion, resulting in a significant loss of ROS.

[0005] Currently, although a large number of chemiluminescent photodynamic probes have been developed, they cannot simultaneously solve the above problems, and there is no therapeutic drug that can selectively kill tumors metastasized in deep tissues. Therefore, designing highly efficient chemiluminescent photodynamic photosensitizers with tumor selectivity and developing novel photosensitizer formulations to improve PDT efficiency and expand its clinical application in cancer treatment is of great significance. Summary of the Invention

[0006] This invention addresses the shortcomings and deficiencies of existing photodynamic therapy technologies, whose application in the treatment of metastatic tumors in deep tissues still faces challenges. This invention provides a non-excitation small molecule photosensitizer drug, its preparation method, and its application. This drug possesses high targeted delivery and selective release efficacy. It targets mitochondria and efficiently generates singlet oxygen through intramolecular chemiluminescent resonance energy transfer induced by endogenous H2O2, directly leading to mitochondrial damage and apoptosis.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: The non-excitation small molecule photosensitizer drug provided by the present invention is to connect the energy donor and the acceptor in a single molecule through covalent coupling, wherein the energy donor is a peroxate ester compound or its derivative.

[0008] Preferably, the drug is composed of a targeting group T, a photosensitizer P, and a peroxyoxalate group C linked by covalent bonds, forming (targeting group T)-photosensitizer P-peroxyoxalate group C ((T)-PC). The structural formula of this drug is:

[0009]

[0010] The energy acceptor is a porphyrin photosensitizer, an anthocyanin photosensitizer, or a thiaranonium salt photosensitizer.

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

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

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

[0014] The above-mentioned method for preparing molecular photosensitizer drugs involves covalently coupling the energy donor and the acceptor within a single molecule to synthesize an intramolecular CRET chemiluminescence imaging therapeutic probe. The energy acceptor is a porphyrin-based photosensitizer, a cyanin-based photosensitizer, or a thiaranonium salt-based photosensitizer, and the energy donor is a peroxate ester compound or its derivative.

[0015] The preferred method for synthesizing the intramolecular CRET chemiluminescence imaging therapeutic probe includes the following specific reaction:

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

[0017] A hydroxyl-modified photosensitizer and two equivalents of acyl chloride-modified peroxyoxalate were dissolved in anhydrous DCM, and triethylamine (TEA) was added as a catalyst. The reaction was carried out at room temperature, the solution was concentrated by rotary evaporation, and purified by column chromatography to obtain the molecular photosensitizer drug product.

[0018]

[0019] The preferred synthesis method for the intramolecular CRET chemiluminescence imaging therapeutic probe includes the following specific reaction:

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

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

[0022]

[0023] The preferred synthesis method for the intramolecular CRET chemiluminescence imaging therapeutic probe includes the following specific reaction:

[0024] (3) Amide reaction

[0025] A carboxyl-modified photosensitizer and two equivalent amounts of amino-modified peroxyoxalate were dissolved in anhydrous DCM and reacted at room temperature for 16 h under catalytic conditions with a condensing agent system. The reaction mixture was then concentrated by rotary evaporation and purified by column chromatography to obtain the molecular photosensitizer drug product.

[0026]

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

[0028] The preferred method for synthesizing the intramolecular CRET chemiluminescence imaging therapeutic probe includes the following specific reaction:

[0029] (4) Maleimide and thiol

[0030] A maleimide-modified photosensitizer and a 2-equivalent amount of thiol-modified peroxyoxalate were dissolved in diethyl ether, and a 2-equivalent amount of triethylamine was added. The mixture was reacted at room temperature for 24 hours. The reaction mixture was concentrated by rotary evaporation and purified by column chromatography to obtain the molecular photosensitizer drug product.

[0031]

[0032] The molecular photosensitizer drug of the present invention uses photosensitizer P as a near-infrared photosensitizer, cyanine (Cy7). With ethyl oxaloyl chloride (EOM)

[0033]

[0034] A photosensitizer molecule (Cy7-EOM) based on intramolecular CRET was prepared by esterification covalent reaction, with the specific structural formula as follows:

[0035] Preferably, the method involves self-assembling the molecular photosensitizer Cy7-EOM into disulfide-bonded and folic acid-modified nanomicelles to obtain water-soluble disulfide-bonded nanomicelles (Cy7-EOM SS NMs).

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

[0037] The folic acid-modified amphiphilic block copolymers containing disulfide bonds are DSPE-SS-PEG and DSPE-PEG-FA, and the ratio of their addition amounts is greater than or equal to 1 (DSPE-SS-PEG:DSPE-PEG-FA); the addition amount of the molecular photosensitizer Cy7-EOM is twice the addition amount of DSPE-SS-PEG.

[0038] The above-mentioned molecular photosensitizer drugs encapsulate Cy7-EOM in tumor-specific responsive nanomicelles Cy7-EOMSS NMs for targeted delivery and release;

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

[0040] The aforementioned molecular photosensitizers are used for tumor treatment, especially for the treatment of metastatic tumors or multiple tumors.

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

[0042] (1) The present invention relates to a non-excited small molecule photosensitizer drug, its preparation method and application, and develops a highly efficient self-energized 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 targeted delivery and selective release effects, the 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 therapy for metastatic tumors in deep tissues.

[0043] (2) The non-excitation small molecule photosensitizer drug, its preparation method, and its application of the present invention utilize chemiluminescence as the excitation 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, which has shallow penetration depth and is ineffective against deep tissue metastatic tumors. This solves the problems of shallow penetration depth of excitation light in photodynamic therapy and its application in the clinical treatment of metastatic tumors.

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

[0045] Furthermore, the specific decomposition of nanomicelles at the tumor site can prevent aggregation-induced quenching and reduce... 1O2 diffusion is hindered, while in normal tissues, the integrity of nanomicelles can shield against it. 1 This method addresses the lethal effects of O2 and provides a novel strategy for converting adjuvant photosensitizers into direct therapeutic agents, showing significant potential for clinical application in the treatment of metastatic tumors. Attached Figure Description

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

[0047] Figure 2 For photosensitizer drug molecules based on intramolecular CRET 1 Fluorescence spectral data of SOSG for O2 yield detection;

[0048] Figure 3 The ESR spectra of Cy7-EOM SS NMs obtained by loading Cy7-EOM into nanomicelles in Example 6 and Example 7 with hydrogen peroxide were used to detect the generation of singlet oxygen, with TEMP as the scavenger.

[0049] Figure 4 Confocal cell imaging for targeted functional characterization, in which the nanoprobe—red fluorescence and the commercially available mitochondrial labeling probe—green;

[0050] Figure 5 Confocal images and corresponding quantitative cell fluorescence histograms for specific targeting validation of cancer cells;

[0051] Figure 6 To detect intracellular... 1 Confocal cell imaging image of O2 generation and corresponding quantitative fluorescence bar chart;

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

[0053] Figure 8 To validate in vivo tumor treatment, a mouse lung metastasis model (4T1-Luc) was constructed using in vivo mouse imaging and corresponding quantitative bar graphs of chemiluminescence intensity. The relative size of lung metastases was reflected by chemiluminescence intensity.

[0054] Figure 9 Characterization data of Cy7 / CPPO NMs and Cy7-EOM SS NMs are shown in the figure. Figure 9 a is a transmission electron microscope image of Cy7-EOM SS NMs; 9b is a transmission electron microscope image of Cy7-EOM SS NMs after GSH incubation.

[0055] Figure 10 Cy7 in CDCl3 1 H-NMR spectrum;

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

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

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

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

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

[0061] Figure 16 The images are electron microscope images, including (16a) an electron microscope image of Cy7-EOM NMs nanomicelles; and (16b) an electron microscope image of Cy7-EOM NMs nanomicelles after incubation with GSH (10mM) for 3 hours.

[0062] Figure 17 The particle size distribution diagrams are shown below; (17a) is the particle size distribution diagram of Cy7-EOM SS NMs, and (17b) is the particle size distribution diagram of Cy7-EOMNMs.

[0063] Figure 18 Zeta plots; (a) Zeta plots of Cy7-EOM SS NMs, (b) Zeta plots of Cy7-EOM NMs. Detailed Implementation

[0064] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0065] Example 1

[0066] The non-excitation small molecule photosensitizer drug provided by this invention is a single molecule in which the energy donor and acceptor are covalently coupled. Specifically, it is composed of a targeting group T, a photosensitizer P, and a peroxyoxalate group C linked by a covalent bond, forming (targeting group T)-photosensitizer P-peroxyoxalate group C ((T)-PC). The structural formula of this 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 the cell; peroxates with high quantum efficiency and long luminescence lifetime are selected as energy donors, and porphyrins, cyanines, thiaranonium salts and other photosensitizers with high singlet oxygen quantum yields are selected as energy acceptors based on the principle of energy level matching.

[0069] To covalently link energy donors and acceptors, it is proposed to preferentially construct active sites in the dye molecule structure and gradually achieve covalent linkage of related components 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, and cyclodextrin groups;

[0071]

[0072] The representative peroxyoxalate molecule structure is as follows: a peroxyoxalate group modified by R2 (peroxyoxalate-R2), where R2 includes, but is not limited to, amino, mercapto, azide, acyl chloride, and adamantane.

[0073]

[0074] The method for synthesizing the intramolecular CRET chemiluminescence imaging therapeutic probe of the present invention includes, but is not limited to, the following:

[0075]

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

[0077] like Figure 1 As shown, when photosensitizer drug molecules enter cancer cells, the peroxate group (C) reacts with the high levels of hydrogen peroxide within the cancer cells to generate the high-energy intermediate 1,2-dioxane. This intermediate is unstable; the energy generated during its decomposition is transferred to the photosensitizer (P), putting it into an excited state. The excited-state photosensitizer returns to its ground state and transfers energy to the surrounding oxygen, transforming it into a cytotoxic substance. 1O2, thereby killing tumor cells. Since CRET efficiency is significantly affected by the distance between the donor and acceptor, the photosensitizer molecule in this patent application covalently couples the energy donor and acceptor within a single molecule, greatly shortening the distance between them and inducing highly efficient intramolecular CRET. 1 O2 generation and luminescence, without the need for an external light source, significantly improve CRET efficiency. Further attachment of a targeting group (T) allows reactive oxygen species to selectively kill key molecules at the target site.

[0078] Example 2

[0079] A method for preparing non-excitation small-molecule photosensitizer drugs involves covalently coupling an energy donor and an acceptor within a single molecule to synthesize an intramolecular CRET chemiluminescence imaging therapeutic probe. The energy acceptor is a porphyrin-based photosensitizer, a cyanin-based photosensitizer, or a thiaranonium salt-based photosensitizer, etc., while the energy donor is a peroxate ester compound. The specific reaction for synthesizing the intramolecular CRET chemiluminescence imaging therapeutic probe is as follows:

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

[0081] 50 mg of hydroxyl-modified photosensitizer was dissolved in 20 mL of anhydrous dichloromethane. The solution was placed in an ice bath, and 2.94 mL of triethylamine was slowly added dropwise. Then, 2.73 mg of oxaloyl chloride monoethyl ester was slowly added to the solution. The ice bath was removed, and the reaction was carried out at room temperature for 6 h. The solvent was removed by rotary evaporation, and the obtained solid was purified by column chromatography (DCM:MeOH = 20:1) to obtain the target compound.

[0082] Example 3

[0083] The method for synthesizing non-excited small molecule photosensitizer drugs, namely intramolecular CRET chemiluminescence imaging therapeutic probes, specifically involves the following reaction:

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

[0085] 5 g of alkyne-modified photosensitizer and 5.3 g of azide-modified peroxyoxalate were placed in a mixed solution containing 20 mL of tert-butanol / H2O (2:1). 250 mg of CuSO4·5H2O and 400 mg of sodium ascorbate were added to neutralize the solution. The mixture was stirred at room temperature under a nitrogen atmosphere for 2 days, and the reaction was terminated by adding H2O. The product was extracted with ethyl acetate (4 x 50 mL). The organic phase was dried by adding magnesium sulfate. The solvent was concentrated by rotary evaporation to obtain the crude product. The product was purified by column chromatography (DCM:MeOH = 20:1) to obtain a non-excited small molecule photosensitizer drug product.

[0086] Example 4

[0087] The method for synthesizing non-excited small molecule photosensitizer drugs, namely intramolecular CRET chemiluminescence imaging therapeutic probes, specifically involves the following reaction:

[0088] (3) Amide reaction

[0089] 5 g of carboxyl-modified photosensitizer and 4.6 g of amino-modified peroxyoxalate were dissolved in 20 mL of anhydrous dichloromethane. 3.4 mL of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added, followed by the slow addition of 2.94 mL of triethylamine. The solution was stirred at room temperature for 24 h. The solvent was concentrated by rotary evaporation to obtain the crude product; purification by column chromatography (DCM:MeOH = 20:1) yielded the non-excited small molecule photosensitizer drug product.

[0090] Example 5

[0091] The method for synthesizing non-excited small molecule photosensitizer drugs, namely intramolecular CRET chemiluminescence imaging therapeutic probes, specifically involves the following reaction:

[0092] (4) Maleimide and thiol

[0093] Under a nitrogen atmosphere, 7.3 g of maleimide-modified photosensitizer and 4.4 g of thiol-modified peroxyoxalate were dissolved in 20 mL of diethyl ether, and 2.9 mL of triethylamine was slowly added dropwise. The solution was reacted at room temperature for 24 h. The reaction mixture was concentrated by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH = 20:1) to obtain the non-excited small molecule photosensitizer drug product.

[0094] Example 6

[0095] The method for synthesizing non-excited small molecule photosensitizer drugs, namely intramolecular CRET chemiluminescence imaging therapeutic probes, specifically involves the following reaction:

[0096] The photosensitizer P is a near-infrared photosensitizer, cyanine (Cy7), linked with ethyl oxaloyl chloride (EOM) via an esterification covalent reaction. 5.4 mg of Cy7 was dissolved in anhydrous dichloromethane, and 2.94 mL of triethylamine was slowly added dropwise under ice bath conditions. Then, 2.73 mg of EOM was slowly dissolved in the above solution, and the reaction was carried out at room temperature for 6 h. The solution was concentrated by rotary evaporation and purified by column chromatography (DCM:MeOH = 20:1) to obtain the photosensitizer molecular drug (Cy7-EOM) based on intramolecular CRET. 1H 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,2 H),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 targeted delivery and selective release of molecular photosensitizer drugs within cancer cells, a nanoassembly method was employed. The molecular photosensitizer Cy7-EOM prepared in Example 6 was self-assembled into disulfide-bonded and folic acid-modified nanomicelles, resulting in water-soluble disulfide-bonded nanomicelles (Cy7-EOM SS NMs). The specific steps included: adding 1 mg of the photosensitizer drug Cy7-EOM and 2 mg of PEG-DSPE... 2000 -FA, 4mg PEG-SS-DSPE 2000 Dissolve in 1 mL of DCM, sonicate for 10 min, then evaporate to remove DCM; add 1 mL of deionized water, stir vigorously overnight at room temperature to obtain an aqueous solution of nanomicelles Cy7-EOM SS NMs;

[0100] The solution was dialyzed in deionized water for 24 hours; the resulting solution was stored at 4°C.

[0101] Control group 1

[0102] As a control group, the preparation method was the same as in Example 7, except that the photosensitizer Cy7-EOM obtained in Example 6 was self-assembled into folic acid-modified nanomicelles that did not contain disulfide bonds. The specific steps included: adding 1 mg of the photosensitizer Cy7-EOM and 2 mg of PEG-DSPE... 2000 -FA, 4mg PEG-DSPE 2000 Dissolved in 1 mL of DCM, sonicated for 10 min, and then the DCM was removed by evaporation; 1 mL of deionized water was added, and the mixture was stirred vigorously overnight at room temperature to obtain an aqueous solution of disulfide-free nanomicelles Cy7-EOM NMs.

[0103] Synthesis, characterization and application results

[0104] 1. Basic performance characterization

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

[0106] like Figure 9 and Figure 16 As shown in the transmission electron microscope (TEM) images, both Cy7-EOM SS NMs and Cy7-EOM NMs are spherical and exhibit good monodispersity.

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

[0108] like Figures 17-18 As shown, dynamic light scattering (DLS) analysis revealed that the hydrodynamic diameter of Cy7-EOM NMs is comparable to that of Cy7-EOM SS NMs, and Zeta potential measurements showed that both have neutral surface charges.

[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 is approximately 774 nm, the same as that of the Cy7 parent compound, while the maximum absorption peak of Cy7-EOM SS NMs is red-shifted to 786 nm.

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

[0111] In addition, control group 1 consists of Cy7 / CPPO NMs nanomicelles without disulfide bonds. The energy transfer mechanism of Cy7 and CPPO loaded inside is intermolecular energy transfer. After incubation with H2O2, the chemiluminescence intensity of Cy7-EOM SS NMs is much higher than that of Cy7 / CPPO NMs, indicating that intramolecular CRET is more effective than intermolecular CRET.

[0112] Then, the chemiluminescence kinetics of Cy7-EOM and Cy7-EOM SS NMs under different concentrations of H2O2 were experimentally investigated. The chemiluminescence images revealed that Cy7-EOM responded rapidly to H2O2 in a concentration-dependent manner, indicating that effective intramolecular CRET could be achieved in the designed photosensitizer molecule. In contrast, the response of Cy7-EOM SS NMs to H2O2 exhibited a time lag. This is mainly due to the shielding effect of the nanomicelles, which hinders effective contact between Cy7-EOM and H2O2. Similarly, nanomicelles also impede the diffusion of low-lifetime ROS generated within the nanoparticles, leading to ROS loss and low photodynamic therapy efficiency.

[0113] 2. Application of test results

[0114] 2.1 The photosensitizer drug molecule based on intramolecular CRET designed in this invention has a singlet oxygen state. 1 O2 yield is higher.

[0115] The oxygen generated by CRET in each group was detected using a singlet oxygen probe (SOSG). 1 O2 content, used for detection and comparison 1 O2 is generated. For example... Figure 2 As shown in the fluorescence spectrum, the fluorescence intensity of SOSG increased significantly after the addition of H2O2 to the Cy7-EOM solution, exhibiting a 5-fold enhancement. This indicates the formation of... 1 O2.

[0116] like Figure 2 As shown in b, Cy7-EOM SS NMs generated after GSH pretreatment 1 The presence of more O2 in Cy7-EOM SS NMs than in untreated Cy7-EOM further confirms that ACQ and nanomicelle structure influence the formation of NMs. 1 Effective utilization of O2.

[0117] like Figure 2 As shown in c, the Cy7-EOM SS NMs group exhibits enhanced SOSG fluorescence compared to Cy7 / CPPO NMs, indicating that this is achieved through intramolecular CRET. 1 O2 efficiency is superior to intermolecular CRET. The nanoprobe (red line) based on intramolecular CRET generates... 1 The O2 concentration was significantly higher than that of the nanoprobe based on intermolecular CRET (black line), proving that the efficiency of intramolecular CRET is higher than that of intermolecular CRET.

[0118] Furthermore, the electron spin resonance (ESR) spectroscopy of the free radical scavenger 2,2,6,6-tetramethylpiperidine (TEMP) was also used to investigate its properties. 1O2 was analyzed. The characteristic peaks that appeared in the ESR spectrum after adding H2O2 indicated that... 1 The generation of O2, and the increase in ESR signal intensity in the Cy7-EOM sample, further demonstrate that the release of Cy7-EOM from the nanomicelles is beneficial for photodynamic therapy.

[0119] 2.2 Tumor selectivity of photosensitizers

[0120] like Figure 2 The fluorescence spectrum of SOSG shown is used for detection and comparison. 1 O2 is generated. For example... Figure 2 As shown in Figure a, the SOSG fluorescence of the molecular photosensitizer is significantly enhanced after the addition of H2O2, indicating that... 1 O2 generation without the addition of H2O2 resulted in lower SOSG fluorescence intensity. Figure 2 As shown in b, the nanomicelles loaded with molecular photosensitizers undergo self-degradation after GSH treatment, releasing the internal photosensitizer molecules Cy7-EOM. This eliminates aggregation-induced quenching and relieves the nanomicelles from their influence on... 1 The diffusion shielding of O2, compared to the group without GSH—where micelles remained intact—significantly enhanced the fluorescence of SOSG, thus demonstrating improved reactive oxygen species utilization. Since tumor tissue contains high concentrations of H2O2 and GSH, the molecular photosensitizer designed in this invention and its nano-assembly application exhibit tumor specificity. Figure 3 As shown, the present invention uses the ESR spectra of Cy7-EOM SS NMs obtained by loading Cy7-EOM into nanomicelles in Example 6 and Cy7-EOM in Example 7, respectively, after reacting with hydrogen peroxide, to detect the generation of singlet oxygen and the trapping agent TEMP.

[0121] 2.3 Target Function Characterization

[0122] like Figure 4 The confocal cell imaging shown indicates good overlap between the nanoprobe (red fluorescence) and the commercially available mitochondrial-labeled probe (green), resulting in a yellow superimposed color. The Pearson colocalization coefficient reaches 0.90, demonstrating its excellent mitochondrial targeting function. Photosensitizers with targeting groups can selectively kill specific sites or molecules.

[0123] 2.4 Validation of Cancer Cell-Specific Targeting

[0124] The cellular uptake performance of folic acid-mediated Cy7-EOM SS NMs nanomaterials in 4T1 cells was investigated by fluorescence analysis using confocal laser scanning microscopy (CLSM).

[0125] Confocal images and corresponding statistical data showed that the red fluorescence signal of 4T1 cells pretreated with folic acid was weaker than that of untreated cells. This is mainly because folic acid molecules bind prematurely to folic acid receptors on the cancer cell membrane, hindering the binding of folic acid in Cy7-EOM SS NMs nanomaterials to folic acid receptors, thereby interfering with the cellular uptake of Cy7-EOM SS NMs nanomaterials. Therefore, it is necessary to first use folic acid-modified Cy7-EOM SS NMs nanoprobes.

[0126] Folic acid-modified Cy7-EOM SS NMs nanoprobes were incubated with cancer cells (mouse breast cancer cells, 4T1 cells, with high expression of folic acid receptor) and normal cells (mouse normal hepatocytes, AML12 cells, with low expression of folic acid receptor) for 6 h, and cell laser confocal imaging was performed.

[0127] Because the expression level of folate receptors on cancer cell membranes is much higher than that on normal cell membranes, such as Figure 5 As shown in the confocal images and corresponding quantitative cell fluorescence bar charts, obvious red fluorescence was observed in 4T1 cancer cells, while the fluorescence intensity in AML12 normal cells was very low. Therefore, the results indicate that Cy7-EOM SS NMs nanoprobes are more likely to enter 4T1 cancer cells, while the amount entering AML12 normal cells is very low. This suggests that folic acid-modified Cy7-EOM SS NMs nanoprobes can specifically target cancer cells.

[0128] 2.5 Intracellular 1 O2 generation detection

[0129] like Figure 6 As shown, SOSG was used to detect intracellular... 1 The confocal cell imaging image generated by O2 and the corresponding quantitative fluorescence are shown. The Cy7-EOM prepared in Example 7 was encapsulated in tumor-specific responsive nanomicelles Cy7-EOM SS NMs for targeted delivery and release. The disulfide-bonded Cy7-EOM SS NMs, after entering cancer cells, can specifically degrade by reacting with GSH within the cancer cells. Under the action of high concentrations of intracellular glutathione (GSH), Cy7-EOM SS NMs degrade and release Cy7-EOM, avoiding aggregation-induced quenching and eliminating the shielding of reactive oxygen species diffusion by the nanomicelles. The strongest SOSG fluorescence indicates... 1 O2 production is highest, and this molecule can specifically target mitochondria, synergistically enhancing the photodynamic therapy effect. In contrast, the control group Cy7-EOM NMs micelles lack disulfide bonds and cannot release the internal photosensitizer molecules. 1O2 production was relatively low; in the control group, Cy7 / CPPO NMs, Cy7 and CPPO were self-assembled within micelles, resulting in low intermolecular CRET efficiency. 1 O2 is produced in the least amount.

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

[0131] The cytotoxicity of Cy7-EOM SS NMs was assessed using the MTT assay. With increasing Cy7-EOM SS NMs concentration, the survival rate of 4T1 cells significantly decreased, reaching as low as 18.44% at a concentration of 50 μg / mL. In contrast, the survival rates of 4T1 cells treated with Cy7 NMs, Cy7 / CPPO NMs, and Cy7-EOM NMs were much higher, at 86.5%, 76.1%, and 32.6%, respectively. Furthermore, no significant cell death was observed in AML12 cells treated with Cy7-EOM SS NMs even at high incubation concentrations.

[0132] MTT cell experiments showed that, Figure 7 As shown, the Cy7-EOM SS NMs nanoprobes prepared in this invention exhibit superior performance compared to the control group Cy7-EOM NMs and Cy7 / CPPO NMs, and show excellent activity against cancer cells (such as...). Figure 7 a) It has good killing ability without damaging normal cells (e.g., Figure 7 b).

[0133] 2.7 Validation of in vivo tumor treatment

[0134] like Figure 8 In vivo imaging of mice and corresponding quantitative chemiluminescence intensity were used to construct a mouse lung metastasis model (4T1-Luc). 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). Fluorescence imaging showed significant chemiluminescence signals in the lungs after intravenous injection of Cy7-EOMSS NMs. Chemiluminescence signals were also observed in the peritoneal cavity. Euthanasia and dissection of the mice revealed new metastatic tumor lesions in the intestines. These results indicate that the designed Cy7-EOMSS NMs have significant targeting and diagnostic capabilities, even capable of detecting small metastatic tumor lesions.

[0135] The antitumor effects were then evaluated. The detailed treatment regimen involved randomly dividing mice into five groups, each receiving a different treatment: PBS, Cy7 NMs, Cy7 / CPPO NMs, Cy7-EOM NMs, and Cy7-EOM SS NMs, all at a dose of 2.5 mg / kg. Bioluminescence imaging was used to assess the treatment efficacy. Mice treated with PBS and Cy7 NMs showed significantly increased bioluminescence intensity and the appearance of new metastatic lesions in the peritoneum, indicating rapid tumor progression. In contrast, tumor growth was significantly inhibited in mice treated with Cy7 / CPPO NMs, Cy7-EOM NMs, and Cy7-EOM SS NMs. Cy7-EOM SS NMs showed the best performance, with its bioluminescence intensity decreasing to 9.8% of that in the PBS group.

[0136] Specifically, mice treated with PBS, Cy7 NMs, and Cy7 / CPPO NMs developed multiple metastatic lesions in the intestines, while mice treated with Cy7-EOM SS NMs showed only weak bioluminescent signals in the lungs and no bioluminescent signals in the peritoneal cavity. This suggests significant potential for treating metastatic tumors, even multiple tumors. Fifteen days after treatment, the lungs of mice in each group were dissected. Photographs and H&E staining also showed that the PBS, Cy7 NMs, and Cy7-EOM NMs groups had more metastatic tumor nodules in the lungs, while the Cy7-EOM SS NMs group showed almost no metastatic lesions. Furthermore, Cy7-EOM SS NMs treatment significantly prolonged the survival time of mice, with 60% surviving for more than 40 days, while almost all mice in the control group died within 25 days. In addition, monitoring the body weight of mice in each group during treatment showed no significant weight loss, further confirming the low side effects and safety of Cy7-EOM SSNMs.

[0137] The above demonstrates that the nano-assembly of the photosensitizer molecules prepared in this invention and their tumor-selective killing mechanism are as follows: The photosensitizer Cy7-EOM prepared in this invention is self-assembled into Cy7-EOM SSNMs containing disulfide bonds and modified with folic acid. On the one hand, the folic acid groups on the surface of the nanomicelles can selectively target the folic acid receptors on the surface of cancer cells, enabling the nanomicelles to actively target and enter tumor cells; on the other hand, the nanomicelles can consume the high levels of glutathione in cancer cells, undergoing specific decomposition and releasing photosensitizer molecules, thereby efficiently killing cancer cells. In contrast, in normal tissues, the expression level of folic acid receptors is low, and the nanomaterials do not actively target them; furthermore, the GSH content in normal cells is low, which cannot destroy the nanomicelles and release photosensitizer molecules.

[0138] Because photosensitizer molecules aggregate inside micelles, aggregation-induced quenching occurs, reducing... 1O2 production efficiency; secondly, due to the short lifespan and limited diffusion distance of reactive oxygen species, and the restriction of reactive oxygen species diffusion by nanomicelle assembly, the utilization rate of reactive oxygen species is greatly reduced; furthermore, the H2O2 content in normal cells is low. 1 O2 production is low and does not damage normal cells, thus exhibiting tumor specificity.

[0139] In summary, this invention, through its non-excitation small molecule photosensitizer drug, preparation method, and application, develops a highly efficient self-energized photosensitizer molecule, Cy7-EOM. The covalent linkage between the donor and acceptor in the molecular structure significantly improves the efficiency of photodynamic therapy (PDT). Under high-concentration H2O2 conditions in the tumor microenvironment, PDT can be autonomously activated in situ, overcoming the limitations of traditional PDT, which has shallow penetration depth and is ineffective against deep-tissue metastatic tumors. Furthermore, Cy7-EOM is encapsulated in tumor-specific responsive nanomicelles Cy7-EOM SS NMs for targeted delivery and release. In cancer cells, Cy7-EOM SS NMs consume large amounts of reduced glutathione, leading to its degradation and the release of Cy7-EOM, which specifically targets mitochondria. The generated reactive oxygen species directly act on mitochondria, triggering a mitochondrial reactive oxygen burst, thereby initiating apoptosis. This method has been successfully applied to the treatment of breast cancer cells and various metastatic tumors in mice, achieving highly efficient photodynamic therapy for metastatic tumors with extremely low systemic toxicity. These findings expand the practical applications of photosensitizers and provide new strategies for photodynamic anticancer drugs, with significant potential for clinical translation.

[0140] The above are merely embodiments of the present invention. For example, when a carboxyl-modified photosensitizer reacts with an amino-modified peroxyoxalate, the catalyst used in the condensing agent system can be a carbodiimide: 1,3-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), and the catalyst benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT), any one of these can achieve the non-excitation small molecule photosensitizer drug, its preparation method, and its application of the present invention.

[0141] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A non-excitation small molecule photosensitizer drug, characterized in that, The application relates to a photosensitizer molecule, which is connected with an energy donor and an energy acceptor in a single molecule by covalent coupling, and is a molecular intracRET chemiluminescence imaging treatment probe, wherein the energy acceptor is a photosensitizer, and the energy donor is a peroxymonooxalate compound or a derivative thereof. Specifically, the photosensitizer P and the peroxymonooxalate group C are connected by a covalent bond to form a photosensitizer P-peroxymonooxalate group C. The energy acceptor photosensitizer is a phycobilin photosensitizer, the photosensitizer P is a near-infrared photosensitizer phycobilin Cy7, and the peroxymonooxalate group C is oxalyl chloride ethyl ester. Specifically, the Cy7 and the oxalyl chloride ethyl ester are connected by esterification covalent reaction to prepare a photosensitizer molecule drug Cy7-EOM based on intramolecular CRET, and the specific structural formula is as follows: 。 2. The method of claim 1, wherein the method is for preparing a non-excitation small molecule photosensitizer drug. The synthesis method of the molecular intracRET chemiluminescence imaging treatment probe is to connect the photosensitizer P, which is a near-infrared photosensitizer phycobilin Cy7, and the oxalyl chloride ethyl ester by esterification covalent reaction. The specific reaction formula is as follows: 。 3. The method of claim 2, wherein the method is for the preparation of a non- excited small molecule photosensitizer drug. The specific reaction steps are as follows: 5.4 mg of Cy7 is dissolved in anhydrous dichloromethane, 2.94 mL of triethylamine is slowly added under ice bath, then 2.73 mg of EOM is slowly dissolved in the above solution, the reaction is carried out at room temperature for 6 h, the solution is concentrated by rotary evaporation, and column chromatography (DCM:MeOH=20:1) is used for purification to prepare the photosensitizer molecule drug Cy7-EOM based on intramolecular CRET.

4. The method of claim 3, wherein the method is characterized by, The application further comprises nano-assembly: the molecular photosensitizer Cy7-EOM is self-assembled into a nanomicelle containing a disulfide bond and modified with folic acid to prepare a water-soluble nanomicelle Cy7-EOM SS NMs containing a disulfide bond.

5. The method of claim 4, wherein the method is for the preparation of a non- excited small molecule photosensitizer drug. The specific steps are as follows: the molecular photosensitizer Cy7-EOM is co-assembled with an amphiphilic block copolymer modified with folic acid and containing a disulfide bond to prepare the Cy7-EOM SS NMs. 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 the addition amounts of the two is greater than or equal to 1; and the addition amount of the molecular photosensitizer Cy7-EOM is 2 times the addition amount of DSPE-SS-PEG.

6. A non-excitation small molecule photosensitizer drug according to claim 5, characterized in that, Specifically, the molecular photosensitizer Cy7-EOM is self-assembled into a nanomicelle containing a disulfide bond and modified with folic acid to prepare the water-soluble nanomicelle Cy7-EOM SS NMs containing a disulfide bond. The nanomicelle containing a disulfide bond and modified with folic acid is specifically an amphiphilic block copolymer modified with folic acid and containing a disulfide bond, which is DSPE-SS-PEG and DSPE-PEG-FA, and the ratio of the addition amounts of the two is greater than or equal to 1; and the addition amount of the molecular photosensitizer Cy7-EOM is 2 times the addition amount of DSPE-SS-PEG.

7. Use of a non-excitation small molecule photosensitizer drug according to any one of claims 1-6, characterized in that, The application is used for preparing a tumor treatment drug.

8. The use of a non-excitation small molecule photosensitizer drug according to claim 7, characterized in that, The application is used for preparing a metastatic tumor treatment drug.

9. The use of a non-excitation small molecule photosensitizer drug according to claim 7, characterized in that, The application is used for preparing a multiple tumor treatment drug.

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