A type I photosensitizer and its preparation method and application

Abstract

The present invention discloses a type I photosensitizer and its preparation method and application. The preparation method of the photosensitizer comprises the following steps: (1) 780 The method comprises reacting 5-bromobenzene-1,3-diol with 5-bromobenzene-1,3-diol in the presence of an acid-binding agent and a solvent to obtain compound CyBr; reacting Fmoc-L-alanine with p-aminobenzyl alcohol in the presence of a condensing agent, a catalyst, and a solvent to obtain intermediate 2; reacting intermediate 2 with phosphorus tribromide in the presence of a solvent to obtain intermediate 3; (2) reacting compound CyBr with intermediate 3 in the presence of an alkaline reagent, a catalyst, and a solvent to obtain intermediate 4; and reacting intermediate 4 with piperidine in the presence of a solvent to obtain the type I photosensitizer. The type I photosensitizer can specifically target the biomarker aminopeptidase highly expressed in tumors, achieve precise tumor imaging and photodynamic therapy, and after light excitation, can serve as a stimulus source to stimulate the body's immunity, initiate anti-tumor immunity, eliminate primary residual tumors, and inhibit distant metastatic tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a type I photosensitizer and a preparation method and application thereof. Background Art

[0002] Cancer is one of the most destructive diseases to human health. Currently, the main clinical treatments for tumors are chemotherapy, surgery and radiotherapy. However, these traditional treatments are limited by their high invasiveness, non-selectivity and severe systemic side effects. Photodynamic therapy (PDT) is a new treatment method that has better safety and higher therapeutic effects than traditional therapies. It has the advantage of selectively killing tumor cells and has good application prospects in tumor treatment. The mechanism by which PDT kills cancer cells is mainly through the free radicals generated by photosensitizers to kill tumor cells. According to the different mechanisms of generating free radicals, PDT can be divided into type I and type II PDT. Type II PDT mainly generates singlet oxygen ( 1 O2), which consumes a large amount of oxygen at the lesion site. However, the development of solid tumors is often accompanied by hypoxia, which limits the efficacy of type II photosensitizers in the treatment of solid tumors. In contrast, type I photosensitizers generate superoxide anions (O2· - Free radicals such as hydroxyl radicals (OH·) and hydroxyl radicals (OH·) do not require oxygen or have low dependence on oxygen, so they can adapt well to the local hypoxic environment of tumors and kill tumor cells. Because of this, photosensitizers are particularly important. So far, a large number of photosensitizers based on different fluorescent groups have been developed. However, most photosensitizers have poor tumor specificity and the fluorescence is always in the on state, which causes normal cells to be taken up and damaged after irradiation. This reduces the therapeutic effect and causes many serious side effects. To overcome this difficulty, activatable photosensitizers have been proposed and used for tumor imaging and treatment because they are highly selective for cancer cells and can activate therapeutic activity while detecting biomarkers. It has a high signal-to-noise ratio, a low detection threshold, real-time detection of biomarkers, low normal tissue toxicity and high drug bioavailability. Among them, enzyme-activated photosensitizers are the most promising due to their high selectivity and good bioresponsiveness. Aminopeptidase N (APN) is a membrane-bound exopeptidase. Compared to normal cells, APN is highly expressed in various tumor cells, including those in the liver, kidney, brain, and uterus. Experimental studies have shown that APN plays a key role in tumor invasion and metastasis. Therefore, APN has become an important target in tumor diagnosis and treatment research. However, current probes targeting APN are primarily used in tumor imaging research. However, it would be more meaningful to simultaneously target APN for tumor imaging and treatment.

[0003] Immunotherapy is an effective cancer treatment that positively or negatively influences the immune system, thereby further attacking or eliminating tumors, making traditional treatments such as chemotherapy pale in comparison. Compared to chemotherapy drugs, immune checkpoint inhibitors have received more attention. Antibodies such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1) have been approved for clinical anti-cancer treatment and have achieved good therapeutic effects. However, because inhibitors themselves do not activate the immune system, the effectiveness of inhibitor treatment varies from person to person, and autoimmune adverse reactions caused by inhibitor treatment may limit their use.

[0004] PDT is a feasible method for inducing immunogenic cell death (ICD). It can promote the release of tumor-associated antigens from tumor cells, send an "eat me" signal to the immune system, and initiate downstream anti-tumor immune regulation. Therefore, the combination of PDT and immunotherapy can enhance the efficiency of anti-tumor immunity and enhance the effect of tumor treatment. Based on this, the present application provides a new type I photosensitizer that can achieve precise imaging and photodynamic therapy of tumors, while stimulating stromal immunity, thereby effectively enhancing the effect of tumor treatment and tumor suppression. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a type I photosensitizer and its preparation method and application. This new type I photosensitizer can specifically target the biomarker aminopeptidase highly expressed in tumors, enabling precise tumor imaging and photodynamic therapy; and after light excitation, it can serve as a stimulus source to stimulate the body's immunity, initiate anti-tumor immunity, promote the body's long-term and effective tumor surveillance, eliminate primary residual tumors and inhibit distant metastases.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a type I photosensitizer, wherein the type I photosensitizer has the following structure:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the type I photosensitizer according to the first aspect, comprising the following steps:

[0010] (1) reacting the compound represented by formula (I) with 5-bromobenzene-1,3-diol in the presence of an acid-binding agent and a solvent to obtain the compound represented by formula (II) CyBr;

[0011] (2) reacting Fmoc-L-alanine with p-aminobenzyl alcohol in the presence of a condensing agent, a catalyst, and a solvent to obtain an intermediate 2 represented by formula (III);

[0012] (3) reacting the intermediate 2 prepared in step (2) with phosphorus tribromide in the presence of a solvent to obtain the intermediate 3 represented by formula (IV);

[0013] (4) reacting the compound CyBr with the intermediate 3 prepared in step (3) in the presence of an alkaline reagent, a catalyst, and a solvent to obtain the intermediate 4 represented by formula (V);

[0014] (5) reacting the intermediate 4 prepared in step (4) with piperidine in the presence of a solvent to obtain the type I photosensitizer;

[0015] The structures of the above formula (I) to formula (V) are as follows:

[0016]

[0017] Furthermore, in step (1), the acid-binding agent is preferably sodium hydride and / or triethylamine, but is not limited to the compounds listed above. Any compound that can remove the hydrogen from the phenolic hydroxyl group on 5-bromobenzene-1,3-diol can be used.

[0018] Furthermore, in step (1), the reaction temperature of the reaction is preferably 50-60°C, for example, 50°C, 55°C, 60°C, etc., including but not limited to the temperatures listed above; the reaction time of the reaction is preferably 10-18h, for example, 10h, 12h, 14h, 16h, 18h, etc., including but not limited to the time lengths listed above.

[0019] Furthermore, in step (2), the molar ratio of Fmoc-L-alanine to p-aminobenzyl alcohol is preferably 1:1-1.5, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, etc., including but not limited to the molar ratios listed above.

[0020] Furthermore, in step (2), the condensing agent is preferably benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0021] Furthermore, in step (2), the catalyst is preferably N,N-diisopropylethylamine.

[0022] Furthermore, in step (2), the reaction temperature is preferably 20-30°C, such as 25°C; the reaction time is preferably 10-18h, such as 10h, 12h, 14h, 16h, 18h, etc., including but not limited to the time lengths listed above.

[0023] Furthermore, in step (3), the molar ratio of intermediate 2 to phosphorus tribromide is preferably 1:1-1.5, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc., including but not limited to the molar ratios listed above.

[0024] Furthermore, in step (3), the reaction temperature is preferably 0-30°C, for example, 0°C, 10°C, 15°C, 20°C, 25°C, 30°C, etc., including but not limited to the temperatures listed above; the reaction time is preferably 10-18h, for example, 10h, 12h, 14h, 16h, 18h, etc., including but not limited to the time lengths listed above.

[0025] Furthermore, in step (4), the molar ratio of compound CyBr to intermediate 3 is preferably 1:1-2, for example, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, etc., including but not limited to the molar ratios listed above.

[0026] Furthermore, in step (4), the alkaline reagent is preferably potassium carbonate.

[0027] Furthermore, in step (4), the catalyst is preferably potassium iodide.

[0028] Furthermore, in step (4), the reaction temperature is preferably 50-70°C, for example, 50°C, 55°C, 60°C, 65°C, 70°C, etc., including but not limited to the temperatures listed above; the reaction time is preferably 5-10h, for example, 5h, 6h, 7h, 8h, 9h, 10h, etc., including but not limited to the time lengths listed above.

[0029] Furthermore, in step (5), the mass volume ratio of intermediate 4 to piperidine is 20-30 mg:1 mL.

[0030] Furthermore, in step (5), the reaction temperature is 20-30°C, for example, 20°C, 25°C, 30°C, etc., including but not limited to the temperatures listed above; the reaction time is 5-30 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, etc., including but not limited to the temperatures listed above.

[0031] The third aspect of the present invention provides a use of the type I photosensitizer described in the first aspect in the preparation of a tumor imaging drug.

[0032] The fourth aspect of the present invention provides a use of the type I photosensitizer described in the first aspect in the preparation of a photodynamic therapy drug.

[0033] The fifth aspect of the present invention provides a use of the type I photosensitizer described in the first aspect in the preparation of anti-tumor immune drugs.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention provides a type I photosensitizer that can specifically target the highly expressed tumor biomarker aminopeptidase. This photosensitizer can be recognized by aminopeptidase and hydrolyzed to produce a compound CyBr with a fluorescent / photoacoustic signal. It also has high sensitivity to aminopeptidase, with a detection limit (LOD) as low as 0.1013 ng / mL. In addition, the present invention studies the responsiveness of the photosensitizer CyA to intracellular aminopeptidase (APN) at the cellular level. Experimental results show that the photosensitizer CyA can be specifically activated by APN overexpressed in cancer cells and used for cell imaging.

[0036] 2. The small molecule photosensitizer provided by the present invention can produce O2 after illumination - ·, and O2 - The production of · increases with the extension of illumination time, and has the potential for type I photodynamic therapy. Cell experiments have verified that the photosensitizer CyA can induce cell death by producing superoxide anions only under irradiation, and can be used for photodynamic therapy of APN-overexpressing diseased cells.

[0037] 3. Through in vitro immune cell activation studies, the present invention found that the photosensitizer CyA combined with light irradiation can increase the expression of endoplasmic reticulum stress-related proteins CHOP and p-eIF-2α, and CyA-treated dendritic cells accompanied by laser irradiation have higher expression of immune-related inflammatory factors, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell experiments verified that the photosensitizer provided by the present invention can be used for photodynamic-induced cancer immune activation.

[0038] 4. The present invention uses the photosensitizer CyA for in vivo fluorescence and photoacoustic imaging of mouse solid tumors, and performs imaging-guided combined photodynamic therapy (PDT) for tumors. Experimental results demonstrate that the photosensitizer has specific detection capabilities for APN-overexpressing tumors in mice, and that CyA-mediated activatable PDT exhibits excellent anti-tumor therapeutic effects, particularly when combined with anti-aPD-L1 therapy. Tissue section studies revealed normal morphology of various tissue cells, demonstrating the good biosafety of the photosensitizer CyA. Furthermore, CyA-mediated photodynamic immunotherapy can downregulate the immunosuppressive microenvironment, effectively disrupting tumor growth and inhibiting tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the H NMR spectrum of the compound represented by formula (II) prepared in Example 1;

[0040] Figure 2is the NMR carbon spectrum of the compound represented by formula (II) prepared in Example 1;

[0041] Figure 3 is the H NMR spectrum of the compound represented by formula (III) prepared in Example 1;

[0042] Figure 4 is the NMR carbon spectrum of the compound represented by formula (III) prepared in Example 1;

[0043] Figure 5 is the H NMR spectrum of the compound represented by formula (V) prepared in Example 1;

[0044] Figure 6 is the NMR carbon spectrum of the compound represented by formula (V) prepared in Example 1;

[0045] Figure 7 is the H NMR spectrum of the photosensitizer (CyA) prepared in Example 1;

[0046] Figure 8 is the NMR carbon spectrum of the photosensitizer (CyA) prepared in Example 1;

[0047] Figure 9 : ac are the UV absorption, fluorescence and photoacoustic spectra of photosensitizer CyA after incubation with APN for 120 min, respectively. The inset is a representative image of the optical signal changes before and after enzyme cleavage; d is the HPLC spectrum before and after the reaction of CyA and APN; e is the enzymatic kinetic analysis diagram of photosensitizer CyA; f is the ESR spectrum of CyA and CyBr free radical generation using DMPO as a capture agent; gi are the fluorescence changes of SOSG (g), DHR123 (h), and TA (i) in different laser irradiation treatment groups within 0-10 min.

[0048] Figure 10 : a is the fluorescence intensity of the photosensitizer CyA after co-incubation with PBS, GGT, FAP-α, uPA, ALP, BT+APN, and APN for 120 min, inset: fluorescence images after co-incubation of different enzymes with CyA; b is the linear relationship between the fluorescence intensity and APN concentration after CyA interacts with different concentrations of APN (0-100 ng / mL) for 120 min, inset: fluorescence images after co-incubation of different concentrations of APN with CyA;

[0049] Figure 11: a is the confocal fluorescence imaging of cells after co-incubation with PBS, photosensitizer CyA, and CyA+BT, respectively; b is the quantitative analysis of the fluorescence intensity of the cells in Figure a; c is the organelle co-localization experiment, CyA (red) and the endoplasmic reticulum, lysosomes, and mitochondria (green) in the cells co-localized fluorescence imaging and linear quantitative analysis; d is the photoacoustic imaging of 4T1 cells after co-incubation with photosensitizer CyA for 120 minutes, inner figure: photoacoustic images of different treatment groups at 700 nm;

[0050] Figure 12 :a is the case of using HPF, DHE, and SOSG as sensors, and the 2 ) under different treatments, CyA produces ROS in vitro; b is the quantification of fluorescence intensity in Figure a; c is the fluorescence imaging of 4T1 cells stained with Calcein-AM and PI after different treatments. Scale bar: 40 μm; d is the fluorescence intensity of 4T1 cells without laser or with laser (660 nm, 50 mW / cm 2 ) Cytotoxicity and cell killing of CyA in irradiated 4T1 cells;

[0051] Figure 13 : a is a schematic diagram of the mechanism of CyA-mediated in vitro cancer immunotherapy; b is the expression of endoplasmic reticulum stress-related proteins in 4T1 cells after different treatments detected by Western blot; c and d are immunofluorescence detection of CRT and HMGB1 expression in vitro, respectively, scale bar: 20 μm; e is the FACS detection of mature dendritic cells CD80 under different treatments + CD86 + expression; f is the percentage of mature DCs treated with different methods; gi are the immunogenic death analysis of 4T1 cells treated with different methods by detecting IFN-γ, TNF-α and IL-6 in the cell supernatant using ELISA kits;

[0052] Figure 14 : a is a representative near-infrared fluorescence image of 4T1 tumor-bearing mice at different time points after intratumoral injection of CyA or CyA combined with APN inhibitor BT; b is a representative PA image of tumors in 4T1 tumor-bearing mice at different time points after intratumoral injection of CyA or CyA combined with APN inhibitor BT; c is the near-infrared fluorescence intensity of the tumor at 710 nm in Figures a and b; d is the curve of the change of PA intensity increment at 700 nm in Figures a and b as a function of time after probe injection; e is the real-time PA intensity spectrum of the tumor 3 hours after intratumoral injection of the probe; the error bars in the figures represent the standard deviation of three independent measurements (n=3), **p<0.01, ***p<0.001;

[0053] Figure 15: a is a schematic diagram of the in vivo anti-tumor experiment timetable, the right tumor is defined as the primary tumor that needs to be irradiated with light, and the left tumor is defined as the distant tumor that does not receive light irradiation; b is the body weight of 4T1 tumor-bearing Balb / c mice after receiving various treatments during the 12-day evaluation period (n=5); c and d are the growth curves of the primary tumor and distant tumor of 4T1 tumor-bearing mice, respectively; e and f are pictures of the primary tumor and distant tumor of 4T1 tumor-bearing mice on the 12th day, respectively; g is the tumor weight of the primary tumor and distant tumor after treatment; in the figure, I is the normal saline group, II is the CyA group, III is the aPD-L1 group, IV is the CyA+laser group, and V is the CyA+laser group. + aPD-L1 group, error bars represent the standard deviation of three independent measurements (n=5), *P<0.05, **P<0.01, ***P<0.001;

[0054] Figure 16 : The left image shows H&E staining, TUNEL, and Ki-67 immunohistochemical staining of tumor sections. Scale bar: 100 μm. The right image shows H&E staining of paraffin sections of the main organs of mice, including heart, liver, spleen, kidney, and lung. Scale bar: 100 μm. In the figure, I is the saline group, II is the CyA group, III is the aPD-L1 group, IV is the CyA + laser group, and V is the CyA + laser + aPD-L1 group.

[0055] Figure 17 :a is the flow cytometry detection of the effects of different treatments on CD80 in lymph fluid of 4T1 tumor-bearing mice + CD86 + The number of DC cells; bd are the quantitative detection of peripheral blood IL-6, tumor necrosis factor-α, and interferon-γ; e is the CD3 + CD8 + The number of T lymphocytes; f is the CD11b in mouse tumors and spleens + Gr-1 + The number of MDSCs; g is CD8 + Immunofluorescence staining of T cells; in the figure, I is the normal saline group, II is the CyA group, III is the aPD-L1 group, IV is the CyA + laser group, and V is the CyA + laser + aPD-L1 group; the error bars represent the standard deviation of three independent measurements (n=3), ns: no statistical difference, *P<0.05, **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0057] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0058] Example 1

[0059] This embodiment relates to the synthesis of a type I photosensitizer, and the synthetic route is as follows:

[0060]

[0061] The specific steps are as follows:

[0062] Preparation of the compound represented by formula (II) (referred to as CyBr in the following examples): 5-bromobenzene-1,3-diol (35.4 mg, 0.19 mmol) and sodium hydride (3.5 mg, 0.15 mol) were dissolved in 2 mL of DMF and stirred at room temperature for 10 min under a nitrogen atmosphere. Then, a solution of compound 1 (115 mg, 0.19 mmol) represented by formula (I) in N,N-dimethylformamide was added to the above reaction system via a syringe at 55°C and the reaction was continued for 12 h. After the reaction was completed, the mixture was cooled and poured onto ice (about 50 g). The blue-green solid was chromatographed on a silica gel column with dichloromethane and methanol (70:1) as the mobile phase. The obtained product was a blue-green solid (52.25 mg, yield: 42.9%). The nuclear magnetic hydrogen spectrum and carbon spectrum of the product were shown as follows: Figure 1 、 2 shown.

[0063] Preparation of the compound represented by formula (III): p-Aminobenzyl alcohol (500 mg, 3.6 mM), Fmoc-L-alanine (570 mg, 3 mM), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.2 g, 3.1 mM) and N, N-diisopropylethylamine (780 mg, 3 mM) were dissolved in 20 mL of anhydrous tetrahydrofuran. Under a nitrogen atmosphere, the mixture was stirred at room temperature overnight. After the solvent was removed from the mixture in vacuo, the crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 1:2) to obtain the product as a yellow-white solid (695 mg, yield: 90.0%). The H NMR spectrum and C NMR spectrum of the product were as follows: Figure 3 、 4 shown.

[0064] Preparation of the compound represented by formula (IV): The compound represented by formula (III) (150 mg, 0.36 mmol) was dissolved in 2 mL of tetrahydrofuran and stirred at 0°C for 10 minutes under nitrogen. After 10 minutes, phosphorus tribromide (20 μL, 0.5 mM) was added. The reaction was then continued for 1 hour. After completion of the reaction, the mixture was concentrated and dried under vacuum. The mixture was then extracted with 30% saturated sodium bicarbonate aqueous solution, and the organic layer was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a white powder (102.5 mg, yield: 82.1%). The obtained product was used directly in the next reaction without further purification.

[0065] Preparation of the compound represented by formula (V): The compound represented by formula (IV) (29.8 mg, 0.06 mM), CyBr (20.1 mg, 0.04 mM), potassium carbonate (11.2 mg, 0.08 mM) and potassium iodide (17 mg, 0.1 mM) were dissolved in 2 mL of anhydrous acetonitrile. Stir at 60 ° C for 8 h, and monitor the reaction on a thin layer chromatography plate. After the reaction is completed, 20 mL of EA is added to the reaction system for dilution, followed by extraction with a saturated sodium chloride aqueous solution. The organic layer is collected, anhydrous sodium sulfate is added to the organic layer, and the mixture is allowed to stand for 2 h to dry and remove water, and the solvent is removed in vacuo. The crude product obtained is purified by silica gel column to obtain compound 4 (eluent, dichloromethane: methanol = 20: 1) to obtain a blue solid (28.3 mg, yield 78.2%). The nuclear magnetic hydrogen spectrum and carbon spectrum of the product are as shown below. Figure 5 、 6 shown.

[0066] Preparation of photosensitizer (CyA): Compound 4 (25.4 mg, 0.03 mM) dissolved in 1 mL DMF was added to a 10 mL round-bottom flask at room temperature and stirred continuously. 1 mL of piperidine was then slowly added dropwise to the above system. Stirring was continued at room temperature for 10 min. The solvent was removed under reduced pressure on a rotary evaporator, and the crude product was purified by high performance liquid chromatography (mobile phase, methanol: water = 1:1) to obtain a blue solid (12.4 mg, yield: 65.6%). The H NMR and C NMR spectra of the product were as follows: Figure 7 、 Figure 8 shown.

[0067] Example 2

[0068] This example studies the fluorescence / photoacoustic imaging capabilities of the photosensitizer (CyA) prepared in Example 1 in response to aminopeptidase (APN) and the types of reactive oxygen species generated after illumination.

[0069] (1) Testing the fluorescence / photoacoustic imaging capability of the photosensitizer (CyA) in response to aminopeptidase

[0070] In an in vitro enzyme digestion experiment, a mixture of probe CyA (5 μM) and APN (200 ng / mL) was incubated at 37°C for 120 minutes. Spectral data from the three components were recorded using a UV spectrophotometer, a fluorescence spectrometer, and a multispectral photoacoustic tomography scanner. The test results are as follows:

[0071] like Figure 9 As shown in Figure a, the characteristic absorption peak of the probe CyA is located at around 600 nm. During the incubation with APN in vitro, the UV absorption at 600 nm gradually decreased, and a new absorption peak appeared at 692 nm, and the UV absorption at this peak gradually increased. Figure 9 As shown in b, after co-incubation with APN, the fluorescence signal of the probe CyA, which originally had no obvious fluorescence signal, increased by about 5.89 times at around 712nm. In addition, the change pattern of the photoacoustic spectrum before and after co-incubation of CyA with APN is the same as that of the fluorescence spectrum ( Figure 9 c) After enzyme cleavage, the photoacoustic signal intensity of the CyA cleavage product increased by approximately 4.85 times compared to the uncleaved product. These results indicate that probe CyA can be recognized and hydrolyzed by APN, producing the compound CyBr with a fluorescent / photoacoustic signal.

[0072] The results of HPLC further confirmed the formation of new substances ( Figure 9 d) At the same time, in order to study the affinity of APN for probe CyA and quantify the enzymatic cleavage rate during the cleavage process, the same APN concentration (50 ng / mL) was incubated with different concentrations of probe CyA (0-500 μM) for 5 min. The enzymatic Michaelis-Menten constant (K) of APN for CyA was calculated based on HPLC peak area analysis. m ) and catalytic rate constant (k cat ) were 123.72 μM and 0.04 s, respectively. -1 ( Figure 9 e). Therefore, the catalytic efficiency of APN on CyA (k cat / K m ) is 323.31M -1 s -1 .

[0073] (2) Types of reactive oxygen species generated by photosensitizer (CyA) after light exposure

[0074] Electron spin resonance (ESR) experiments were conducted using 5,5-dimethyl-1-pyrroline-n-oxide (DMPO) as a ROS scavenger, and the results showed a typical 1:1:1:1 ratio of O2 - Generate peak ( Figure 9 f).

[0075] To further verify the type of reactive oxygen species produced by photosensitizer (CyA) after light irradiation, singlet oxygen fluorescence probe (SOSG) was used as1 O2 indicator, Rhodamine 123 (DHR123) for O2 - The indicator, terephthalic acid (TA), was OH. The indicator was used to detect the free radical generating ability of the photosensitizer (CyA). At 660 nm, 200 mW / cm 2 Under laser irradiation, the fluorescence spectrum of each indicator was measured every 2 minutes. Figure 9 As shown in g-9i, within 0-10 min after laser irradiation, the fluorescence intensity of indicator SOSG and indicator TA did not change significantly after the probe and enzyme were co-incubated, indicating that CyA could not produce 1 O2 and OH·, by Figure 9 hIt can be seen that the fluorescence intensity of DHR123 gradually increased within 10 minutes of laser irradiation, which means that CyA can produce O2 - The amount of production gradually increased. After Bestatin was added to inhibit APN, the fluorescence intensity of DHR123 decreased, and O2 - The amount of production decreased. This fully demonstrates that CyA can produce O2 under light - ·, has potential for type I photodynamic therapy.

[0076] Example 3

[0077] This example studies the specificity and sensitivity of the photosensitizer (CyA) prepared in Example 1 in response to aminopeptidase (APN).

[0078] (1) Specificity test

[0079] The photosensitizer (CyA) was mixed with PBS, GGT (200 ng / mL), FAP-α (9×10 -4 U / mL), uPA (2×10 -3 U / mL), ALP (2U / mL), BT+APN (500μM), APN (200ng / mL) were incubated for 120min, and the fluorescence intensity of each test group was tested. The test results are shown in Figure 2. Figure 10 As shown in a, the photosensitizer showed a specific response to APN.

[0080] (2) Sensitivity test

[0081] The photosensitizer (CyA) was incubated with different concentrations of APN (0-100 ng / mL) for 120 min, and the fluorescence intensity was tested. The results were as follows: Figure 10 As shown in b, the fluorescence intensity increases linearly with the increase of APN concentration, and the detection limit LOD is only 0.1013 ng / mL, which also reflects the high sensitivity of the photosensitizer to aminopeptidase (APN) response.

[0082] Example 4

[0083] This example studies the optical imaging, photodynamic therapy, and immune stimulation capabilities of the photosensitizer CyA at the cellular level.

[0084] Cell Selection and Research Details: 4T1 is a breast cancer cell line derived from the cancerous mammary tissue of BALB / c mice. It exhibits epithelial properties and is the most commonly used breast cancer model for clinical drug screening and efficacy evaluation. Studies have shown that 4T1 cells express APN, so the 4T1 cell line was selected as the target cell for cell-based experiments. In this example, 4T1 cells were used as experimental subjects to investigate the uptake, localization, and cytotoxicity of the probe CyA in tumor cells, as well as changes in fluorescence / photoacoustic signals. The antitumor efficacy of the photosensitizer CyA was also evaluated at the cellular level, and the cytotoxicity mechanism of this photosensitizer in tumor treatment was briefly analyzed.

[0085] (1) Response of photosensitizer CyA to intracellular aminopeptidase (APN)

[0086] Confocal fluorescence imaging, photoacoustic imaging, and organelle co-localization experiments were performed to verify whether CyA could be recognized and activated by APN at the cellular level. The specific procedures were as follows:

[0087] Confocal fluorescence imaging: 10,000 4T1 cells were seeded in a confocal dish and incubated at 37°C for 24 hours. Dead and subviable cells were washed with PBS, and the probe CyA (5 μM) was slowly added to the dish and incubated for another 2 hours. For the inhibitor group, the inhibitor BT (100 μM) was first added to the 4T1 cells for 1 hour to inhibit APN activity. The CyA probe solution (5 μM) was then added and incubated for another 2 hours. The nuclei were then stained with Hoechst 33342 and imaged using a confocal fluorescence microscope. The excitation wavelength for Hoechst 33342 was 405 ± 20 nm, and the emission wavelength was 460 ± 20 nm. The excitation wavelength for CyA was 635 ± 20 nm, and the emission wavelength was 710 ± 20 nm.

[0088] Cell photoacoustic imaging experiment: Sample preparation is the same as for the cell confocal microscopy experiment. Cells from different experimental groups are collected and placed in 0.5 mL centrifuge tubes. Imaging is performed using a photoacoustic imaging system to obtain cell photoacoustic signal spectra in the 680 nm to 850 nm band.

[0089] Organelle colocalization assay: Sample preparation was the same as for the confocal microscopy assay. The probe CyA solution (5 μM) was added. After incubation for 2 h, the upper medium was discarded, and the lower cells were washed twice with PBS. The endoplasmic reticulum probe (ER Tracker), lysosome probe (Lyso Tracker), and mitochondrial probe (Mito Tracker) were added, respectively. Incubation continued for 30 min. Excess dye was washed with PBS, and Hoechst 33342 solution (10 μg / mL) was added to stain the cell nuclei. Confocal fluorescence microscopy was used for imaging. The excitation wavelengths for ERTracker, Lyso Tracker, and Mito Tracker were 488 ± 20 nm, and the emission wavelengths were 525 ± 20 nm. The excitation wavelengths for CyA were 635 ± 20 nm, and the emission wavelengths were 710 ± 20 nm.

[0090] Experimental results: Figure 11 Figures 11a and 11b show the experimental results of cell confocal fluorescence imaging. As shown, after co-incubation of 4T1 cells with different treatments, the fluorescence of the CyA group was significantly enhanced, reaching 9.89 times that of the untreated group. In addition, after pre-incubation with 100 μM BT for 60 minutes, the near-infrared fluorescence signal was significantly suppressed, confirming the specific activation of CyA in 4T1 cells by APN. These data also indicate that CyA has a strong ability to visualize 4T1 cells. Figure 11 d shows the PA image and spectrum of 4T1 cells incubated with CyA. At 700 nm, the PA signal of 4T1 cells treated with CyA is higher than that of 4T1 cells treated with CyA combined with the APN inhibitor BT, confirming its suitability for PA imaging of 4T1 cells. This also demonstrates that CyA can be specifically activated by APN overexpressed in cancer cells, making it suitable for cell imaging. Figure 11 c shows fluorescence imaging of mitochondrial, lysosomal, and endoplasmic reticulum dyes co-incubated with CyA. As can be seen, the near-infrared fluorescence channel overlaps well with the green channels of the endoplasmic reticulum and lysosomal dyes. In contrast, a weak colocalized fluorescence signal is observed in the mitochondrial dye, indicating that the hydrolysis product CyBr is primarily distributed in the endoplasmic reticulum and lysosomes.

[0091] (2) Photodynamic therapy ability of photosensitizer CyA

[0092] Given that CyA has a strong O2 -·Generation ability. In this example, SOSG, DHE and HPF staining methods were used to study the in vitro photoactivity of CyA under hypoxic and normoxic conditions. In order to better simulate the hypoxic environment of the tumor, 4T1 cells were incubated in an incubator (HF-100, three-gas incubator) under humidified, 2% O2 and 5% CO2 conditions for 24 hours to construct an in vitro hypoxia model. The intracellular photodynamic ability of CyA was verified in hypoxic and normoxic environments respectively. In this example, the photodynamic therapy ability of the photosensitizer CyA was studied by detecting the ability of the photosensitizer CyA to generate reactive oxygen species in 4T1 cells, cytotoxicity detection, cell apoptosis detection and live / dead cell staining experiments, as follows:

[0093] Intracellular reactive oxygen species production capacity assay: Sample preparation was the same as for the cell confocal microscopy assay. Then, SOSG (10 μM), DHE (10 μM), and HPF (10 μM) were added to the culture medium and incubated for 30 min. The above process was performed in the dark. The cells were illuminated with or without near-infrared laser (660 nm, 50 mW / cm 2 ) irradiated cells for 5 minutes. Intracellular ROS were detected using a laser confocal microscope. Excitation wavelengths were 405 ± 20 nm for SOSG, 488 ± 20 nm for HPF, 550 ± 20 nm for DHE, and 635 ± 20 nm for CyA. Emission wavelengths were 460 ± 20 nm, 525 ± 20 nm, 600 ± 20 nm, and 710 ± 20 nm, respectively.

[0094] Cytotoxicity test: including dark toxicity test and phototoxicity test. The specific operation is as follows:

[0095] Dark toxicity test: 4T1 cells were seeded into 96-well plates at a density of approximately 8,000 cells per well and cultured under normoxia and hypoxia for 24 hours. Cells with weak adhesion and viability were washed away with PBS. Different concentrations of photosensitizer CyA (0-5μM, 100μL) were added to each well and incubated for another 12 hours. The upper waste liquid was discarded. 100μL of CCK8 solution was added to each well and incubated for another 2 hours. The ultraviolet absorbance of each well at 450nm was measured using a microplate reader, and the cell viability was calculated. The experiment was repeated three times independently.

[0096] Phototoxicity experiment: Sample preparation is the same as the dark toxicity experiment, except that after the probe incubation, a 660nm, 50mW / cm 2 Laser irradiation was performed on each well for 10 min, and the culture was continued for 1 h before adding CCK8 detection reagent to detect the phototoxicity of the photosensitizer. The subsequent experimental procedures were the same as those of the dark toxicity test.

[0097] Apoptosis detection and live / dead cell staining experiments: 4T1 cells were seeded in confocal culture dishes and washed twice with PBS after they were completely attached to the wall to remove suspended dead cells. CyA (5 μM) diluted in PBS was added and incubated at 37°C under normoxic conditions for 2 h. Then, the cells were irradiated with or without a 660 nm, 50 mW / cm2 near-infrared laser for 10 min. After incubation for another 1 h, the degree of cell apoptosis was detected using an AM / PI cell apoptosis kit (Yisen Biotechnology (Shanghai) Co., Ltd.) and imaged using a laser confocal microscope. The excitation wavelengths of AM and PI were 488 ± 20 nm and 535 ± 20 nm, respectively. The emission wavelengths were 525 ± 20 nm and 615 ± 20 nm, respectively. For the cell apoptosis detection experiment under hypoxia, the cell culture environment was 37°C, 2% humidified air and 5% carbon dioxide, and the rest of the steps were the same.

[0098] Experimental results: After hypoxia and 660nm laser treatment, enhanced DHE fluorescence signals can still be detected in 4T1 cells. CLSM images show that hypoxia enhances the DHE fluorescence of cells by 18.18 times ( Figure 12 a, 12b). In addition, when cells were pre-incubated with 100 μM BT for 60 min or incubated with CyA alone for 120 min, the DHE fluorescence signal was significantly inhibited. This indicates that CyA still has good photodynamic activity under hypoxic conditions. In contrast, 1 The O2 signal is negligible. These findings are consistent with Figure 9 The results of the ROS study in the literature are consistent with those in the literature. In addition, after 4T1 cells were irradiated with 660nm light, HPF showed significant green fluorescence to OH·, regardless of whether it was in a 2% O2 environment or a 21% O2 environment. That is to say, the formation of OH· was not directly dependent on the photosensitization of CyA, but was caused by the conversion of superoxide anions into hydroxyl radicals under the action of superoxide dismutase SOD, which is consistent with the results of previous studies. The in vitro anti-tumor activity of CyA was detected using a live / dead cell staining kit and a cell counting kit-8 (CCK-8) method. Figure 11 d It can be seen that the dark toxicity of CyA is negligible, indicating that it has good in vitro biocompatibility. After illumination, CyA effectively inhibits cell proliferation in a probe concentration-dependent manner ( Figure 12 d). To further verify the cell-killing effect of the CyA probe, the cytotoxicity of CyA was detected by cell viability imaging experiments ( Figure 12 c). Calcein-AM and propidium iodide (PI) fluorescence imaging were used to detect cell viability. After 120 min of CyA treatment, the green fluorescence signal of the Calcein-AM channel was strong and almost no cytotoxicity was observed. However, when the cells were exposed to near-infrared (660 nm, 50 mW / cm 2) for 10 minutes, the PI channel showed a strong red fluorescence signal, indicating that about 83% of the cells were killed by superoxide anions. After the addition of APN inhibitor BT, the PI signal was significantly reduced.

[0099] From the above experimental results, it can be seen that CyA can induce cell death by producing superoxide anions only under irradiation, and can be used for photodynamic therapy of certain APN-overexpressing diseased cells.

[0100] (3) The ability of photosensitizer CyA to mediate in vitro immune activation

[0101] Based on previous co-localization studies, it is speculated that CyA targeting these organelles and combined with photodynamic therapy may induce endoplasmic reticulum stress, ultimately leading to immunogenic cell death (ICD). Therefore, in addition to directly killing tumors by generating ROS, the ability of CyA photodynamic therapy to mediate in vitro immune activation was also studied. This example verifies the ability of the photosensitizer CyA photodynamic therapy to mediate in vitro immune activation through the detection of tumor-associated antigens, the expression of tumor immunogenic death-related proteins, and the study of in vitro immune cell activation, as follows:

[0102] Tumor-associated antigen detection: 4T1 cell apoptosis produced tumor-associated antigen detection research, 10,000 4T1 cells were seeded in a confocal culture dish, and after they were completely attached to the wall, they were washed twice with PBS to remove the suspended dead cells. CyA (5μM) diluted in PBS was added and incubated at 37℃ for 2h under normoxic conditions. Then, the cells were illuminated with or without 660nm, 50mW / cm 2 Cells were irradiated with a near-infrared laser for 10 minutes. After incubation for another 0.5 hours, the upper layer of culture medium was discarded, and the lower layer of cells was washed with PBS to remove any poorly floating cells. Cells were then fixed with 1 mL of 4% paraformaldehyde at 4°C for 20 minutes. Excess paraformaldehyde was washed away with PBS, and 1 mL of 1% Triton-100 was added for 1 hour at room temperature. Excess Triton-100 was further washed away with PBS. Cells were then stained with anti-calreticulin (CRT) and anti-high-mobility group box 1 (HMGB1) antibodies, respectively, at 4°C for 12 hours. Antibodies were recovered, washed with PBS, and AlexaFluor 488-conjugated fluorescent secondary antibodies were added, followed by incubation at room temperature for 2 hours. Finally, cells were incubated with Hoechst 33342 for 15 minutes, and excess dye was washed away. CRT expression and HMGB1 release were examined using confocal laser scanning microscopy. For Hoechst 33342, emission was collected at 430-470 nm (λex = 405 nm); for HMGB1 / CRT, emission was collected at 500-600 nm (λex = 488 nm).

[0103] Study on the expression of tumor immunogenic death-related proteins: 4T1 cells were seeded in a transparent culture dish (d = 65 mm) at a cell density of 80%. After the cells were completely attached, they were washed twice with PBS to remove floating cells and cells with poor vitality. CyA (5 μM) diluted in PBS was added. In the inhibitor group, 4T1 cells were first incubated with APN inhibitor BT (100 μM) for 1 hour, and then incubated at 37 ° C for 2 hours under normoxic conditions. Then, the cells were illuminated with or without 660 nm, 50 mW / cm 2 Cells were irradiated with a near-infrared laser for 10 minutes. After an additional 1 hour of incubation, cell samples were collected and prepared for Western blotting. Images were acquired using a FluorChem M system (ProteinSimple) equipped with darkroom development for chemiluminescence.

[0104] In vitro immune cell activation study: In vitro dendritic cell (DC) activation study. Bone marrow-derived dendritic cells (BMDCs) were isolated from 8-week-old Balb / c male mice. BMDCs were cultured in RPMI 1640 medium containing IL-4 (20 ng / mL) and GM-CSF (20 ng / mL) for 7 days to obtain immature DCs. First, 4T1 cells (10,000 cells) were seeded in 6-well plates and cultured overnight at 37°C. They were then treated with different methods (PBS, CyA, CyA+BT) for 2 hours, with or without 660 nm laser (50 mW / cm 2 ) irradiated for 10 minutes. Subsequently, BMDCs were added to 4T1 cell culture medium and co-cultured for 24 hours. The mixed cells were harvested and centrifuged at 1000 r / min for 10 minutes. The lower layer of cells was collected and stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies for 30 minutes. The cells were then washed three times with PBS and analyzed by flow cytometry. The culture supernatant was collected and the concentrations of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) were determined using ELISA kits.

[0105] Experimental results: Photodynamic therapy-induced cancer immune activation is usually mediated by damage-associated molecular patterns (DAMPs), including surface-exposed calreticulin (CRT) and high-mobility group protein B1 (HMGB1) interacting with dendritic cells (DCs). In addition, endoplasmic reticulum stress can also activate the PERK-eIF-2α-CHOP pathway, causing cell apoptosis ( Figure 13 a) In order to verify this hypothesis, the present invention designed a protein blotting experiment and an immunofluorescence experiment of the relevant protein expression. Figure 13As shown in b, the CyA combined with light irradiation group increased the expression of endoplasmic reticulum stress-related proteins CHOP and p-eIF-2α, while the expression of p-eIF-2α and CHOP in the CyA alone group did not change significantly compared with the PBS group. After adding BT inhibition, the expression of p-eIF-2α and CHOP was significantly inhibited. This shows that CyA can cause endoplasmic reticulum stress. In order to detect the expression levels of CRT and HMGB1 after photodynamic therapy, immunofluorescence staining of 4T1 cells after CyA and laser irradiation showed that CRT and HMGB1 were higher in exposure ( Figure 13 c, 13d). The expression of CRT and HMGB1 in the non-irradiated group was negligible, while the expression of CRT and HMGB1 in the CyA group treated with BT was lower than that in the CyA group treated with laser due to the inhibition of APN activity. Next, we studied the ability of CyA photodynamic therapy to mediate DCs maturation and thus affect immune activation. 4T1 cells were incubated with PBS, CyA, and CyA+BT for 120 minutes, and then PDT was performed with or without laser irradiation. Subsequently, immature bone marrow-derived dendritic cells (BMDCs) were co-cultured with 4T1 cells for 24 hours, and CD80 was detected by flow cytometry. + CD86 + Mature dendritic cells. Figure 13 e, 13f, CyA-treated 4T1 cells and CD80 + CD86 + After co-culture of mature DCs, CD80 + CD86 + The percentage of mature DCs was 59.14%, which was 5.77 times higher than that of the PBS-treated group. Meanwhile, the populations of the CyA group after BT inhibition and the CyA group alone were 15.72% and 14.43%, respectively, which were comparable to those of the PBS group. In addition, ELISA was used to detect inflammatory factors in the supernatant of the culture medium of 4T1 and DC cells co-cultured. Compared with other control groups, DCs treated with CyA with laser irradiation had higher expression of immune-related inflammatory factors, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) ( Figure 13 g-13i).

[0106] In summary, the above experimental results verified that CyA activated PDT-mediated effective immunity in APN.

[0107] Example 5

[0108] In this example, the photosensitizer CyA prepared in Example 1 was used to perform fluorescence and photoacoustic imaging of mouse solid tumors in vivo, and tumor photodynamic therapy combined with immunotherapy was performed under imaging guidance to evaluate the anti-tumor therapeutic efficacy of the probe CyA in vivo.

[0109] (1) Establishment of mouse tumor model

[0110] Establishment of mouse tumor imaging and treatment model: 4T1 cells in the logarithmic growth phase were prepared into a cell suspension and injected subcutaneously into the right lower limb of BALB / c mice that had been partially depilated. Tumor volume was monitored daily and when the tumor volume reached approximately 100-120 mm 3 For the treatment model, when the right lower limb tumor volume reached 100-120mm 3 When the primary tumor volume reached approximately 100-120 mm 3 These mice were treated.

[0111] (2) Optical imaging of mouse tumor models

[0112] To further explore the feasibility of using the photosensitizer CyA in photodynamic therapy, we first investigated the ability of the probe to detect APN in vivo. The specific procedures were as follows:

[0113] Tumor-bearing mice were randomly divided into two groups, with three mice in each group. One group received intratumoral injection of CyA (50 μM, 50 μL), and fluorescence and photoacoustic images were acquired at 0, 0.5, 1, 2, 3, and 6 h after injection. As a control, BT (500 μM, 100 μL) was injected intratumorally and then treated with the photosensitizer CyA 1 h after intratumoral injection. Fluorescence images were captured using an IVIS spectral imaging system with an acquisition time of 0.1 s, an excitation wavelength of 660 nm, and an emission wavelength of 710 nm. Photoacoustic images were acquired at 700 nm using a multispectral photoacoustic tomography scanner.

[0114] Experimental results: Figure 14 As shown in a and 14b, after intratumoral injection, the fluorescence and photoacoustic signal intensities in the tumor area gradually increased and reached the maximum 3 hours after injection. However, after pretreatment of mice with the APN inhibitor BT, the activated fluorescence signal and photoacoustic signal decreased by 2.94 times and 2.15 times, respectively ( Figure 14 c, 14d). The results confirmed that the activation signal at the tumor site originated from CyA activated by APN. In addition, the photoacoustic spectrum of 4T1 xenograft tumor activation in vivo after CyA treatment was similar to the spectrum of CyA activated by APN in vitro, further verifying the APN-mediated CyA activation photoacoustic signal ( Figure 14 e). The above results indicate that CyA has the specific detection ability for APN-overexpressing tumors.

[0115] (3) Evaluation of the efficacy of photodynamic therapy combined with immunotherapy in vivo

[0116] The efficacy of photodynamic therapy combined with immunotherapy was evaluated in 4T1 tumor-bearing mice (n=5 per group). The mice received five different treatments: (I) PBS, (II) CyA, (III) aPD-L1, (IV) CyA + Laser, and (V) CyA + aPD-L1 + Laser. The specific procedures are as follows:

[0117] 4T1 cells were inoculated subcutaneously into the right lower limb of mice as proximal primary tumors, and 6 days later, they were inoculated again at the same location in the left lower limb of mice as distal metastatic tumors ( Figure 15 a). When the primary tumor grows to 80-100 mm 3 When the disease is treated, anti-tumor treatment is given.

[0118] PBS treatment group: PBS (50 μL) was injected into the tumor on day 0 and day 2, respectively.

[0119] CyA treatment group: CyA (200 μM, 50 μL) was injected intratumorally on day 0 and day 2, respectively.

[0120] aPD-L1 treatment group: intraperitoneal injection of anti-PD-L1 antibody (aPD-L1, 75 μg per mouse) on days 1 and 3

[0121] CyA+Laser treatment group: CyA (200 μM, 50 μL) was injected into the tumor on day 0 and day 2, and then laser was used at 660 nm, 50 mW / cm 2 Laser irradiation of the tumor site for 10 min.

[0122] CyA+aPD-L1+Laser treatment group: CyA (200 μM, 50 μL) was injected into the tumor on day 0 and day 2, respectively. Two hours later, the tumor was treated with laser at 660 nm and 50 mW / cm 2 The tumor site was laser irradiated for 10 minutes, and then anti-PD-L1 antibody (aPD-L1, 75 μg per mouse) was intraperitoneally injected every other day (day 1 and day 3). The body weight of the mice and the growth of proximal and distal tumors were monitored for 12 consecutive days after treatment.

[0123] Experimental results: Figure 15 As shown in b, no significant weight loss was observed in the mice in each group. In addition, the primary tumors in mice treated with PDT combined with aPD-L1 were completely suppressed, while the anti-tumor effect of the group treated with PDT alone was slightly worse. In addition, the anti-tumor efficacy of mice treated with aPD-L1 alone was lower than that of mice treated with PDT and aPD-L1 combined ( Figure 15 c). For distal tumors, the combined treatment group had the highest tumor inhibition effect compared with the control group treated with either treatment alone ( Figure 15 d). In addition, after the treatment, the mice were sacrificed and dissected, and the weights of the proximal and distal tumors were measured. Figure 15 As shown in Figure 5, the primary tumor weight in the PDT combined with aPD-L1-treated group was 410.75 mg, which was 3.7, 3.2, 2.56, and 1.56 times lower than that in the PBS group, the CyA alone group, the aPD-L1 group, and the PDT group, respectively. The measurement results of the distant tumors were similar to those of the primary tumors. Figure 15 These experiments fully demonstrate that the therapeutic effect of CyA-mediated activatable PDT combined with anti-aPD-L1 on tumors is better than that of the single-drug treatment group.

[0124] After the treatment, the primary tumor, distant tumor, and heart, liver, spleen, lung, and kidney tissues of the mice were taken, sliced, and stained to observe the tumor inhibitory effect.

[0125] like Figure 16 As shown in the left figure, H&E staining results show that compared with other control groups, the CyA photodynamic therapy combined with immunotherapy group had a significant increase in apoptotic cells (red) and a decrease in the number of cells in a proliferative state (purple); TUNEL staining can visually observe cell apoptosis. As shown in the figure, TUNEL staining of tumor sections showed that both primary tumors and distant tumors had a significant increase in cell apoptosis in the CyA photodynamic therapy combined with immunotherapy group compared with other experimental groups (brown); Ki-67 is an important immunohistochemical indicator for evaluating cell proliferation, closely related to cell division, and is an important tumor marker. The higher the Ki-67 positivity, the faster the tumor cell proliferation, which may lead to faster tumor metastasis and recurrence. By detecting the Ki-67 indicator, the efficacy of tumor treatment can be indirectly evaluated. As shown in the figure, the proliferation activity of the CyA photodynamic combined with immune group decreased significantly (brown), the proliferation activity of the CyA photodynamic group was slightly higher than that of the CyA photodynamic combined with immune group, and the cell proliferation activity of the other groups was high, which was consistent with the tumor monitoring results, indicating that CyA-mediated activatable photodynamic combined with anti-PD-L1 immunotherapy can effectively kill tumor cells, reduce tumor cell proliferation activity, and inhibit the occurrence and development of tumors. In addition, H&E staining of the main organ sections of mice (except lung tissue) showed normal cell morphology and no obvious pathological and toxicological changes ( Figure 16 Right), indicating that the probe CyA has good biosafety and can be used as a safe and biofriendly photosensitizer for tumor imaging and treatment.

[0126] (4) In vivo immune response study of tumor therapy

[0127] To further understand the immune response after different treatments, the present invention measured and compared the percentage of mature DCs in the tumor-draining lymph nodes and the release of related inflammatory cytokines in the circulating blood of mice on day 12 of treatment. The specific procedures are as follows:

[0128] After the treatment, the proximal tumors, distal tumors, inguinal lymph nodes, and spleens of mice were taken as tissues for detecting immune responses. Tumor tissues and inguinal lymph nodes of different treatment groups were digested with 1 mg / mL Sigma-Aldrich collagenase and 20 μg / mL DNaseI in RMPI1640 culture medium at 37°C for 1 hour. The cells were filtered with a nylon filter, washed with 1×PBS, centrifuged at 1000r for 10 minutes, and the tissue pellet was collected. 1 mL of red blood cell lysis buffer was added to lyse the sample for 10 minutes. Subsequently, 10 mL of PBS was added to dilute the above solution and centrifuged for another 10 minutes to remove red blood cells. The cell pellet was collected and the number of immune cells in the tumor was estimated after staining. Spleen tissues of mice in different treatment groups were taken and prepared with a homogenizer without digestive enzymes to analyze the changes in immune cells in the spleen tissue. Then, the sample was lysed with red blood cell lysis buffer to remove red blood cells. CD8 + T cells were stained with anti-CD3-APC and anti-CD8-PE, mature DCs were stained with anti-CD11c-APC, anti-CD80-Pacific Blue, and anti-CD86-PE, and myeloid-derived suppressor cells (MDSCs) were stained with anti-CD11b-APC and anti-Gr-1-PE. Flow cytometry was used (BD FACSCalibur Becton, Dickinson and Company). Data were analyzed using Flow J software. Peripheral blood supernatants were collected from mice, and cytokine levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) were measured using ELISA kits.

[0129] Experimental results: Flow cytometry was used to detect and analyze CD11c in tumor-draining lymph nodes. + CD80 + CD86 + The number of mature DC cells. Figure 17 As shown in a, the combined treatment group obtained the highest level of mature DCs in the lymph nodes (mean 33.4%), which were 2.1, 1.4, 1.2, and 1.1 times that of the PBS group, CyA alone group, aPD-L1 alone group, and PDT treatment group, respectively. Blood samples were collected on day 12 to measure the levels of proinflammatory cytokines IL-6, tumor necrosis factor-α, and interferon-γ ( Figure 17 b, 17c, 17d). Consistent with the results of the DC maturation experiment, the secretion of IL-6, tumor necrosis factor-α, and interferon-γ was significantly increased in the combination group compared with the other groups. This also demonstrates that CyA-mediated PDT combined with immunotherapy has superior anti-tumor immune activity.

[0130] To study CD8 +The killing effect of T lymphocytes on tumor cells was analyzed by flow cytometry to analyze the CD3 + CD8 + The number of T lymphocytes was analyzed. Figure 17 As shown in e, after comprehensive treatment, CD8 + The proportion of T cells in primary tumors reached 50.42%, which was significantly higher than that in the PDT group alone (32.34%), the aPD-L1 group (27.89%), and the PBS treatment control group (11.45%). + Immunofluorescence staining of T cells also showed similar results ( Figure 17 g). Compared with other control groups, CD3 + CD8 + Increased numbers of T lymphocytes can also be observed in distant tumors ( Figure 17 g). These data demonstrate that CyA-mediated PDT combined with immunotherapy significantly enhances systemic anti-tumor T cell immunity.

[0131] In addition, the present invention uses flow cytometric analysis to evaluate the negative immune regulatory response of primary tumors and spleen. Figure 17 f shows that compared with other groups, the expression of CD11b in the tumor and spleen of mice after combined treatment + Gr-1 + The number of MDSCs decreased the most compared with the other control groups. In summary, the downregulated immunosuppressive microenvironment further verified the superior anti-tumor effect of CyA-mediated photodynamic immunotherapy, which can effectively prevent tumor growth and inhibit tumor metastasis.

[0132] The above-described embodiments are merely preferred examples for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A type I photosensitizer, characterized in that The type I photosensitizer has the structure shown below: 。 2. A method for preparing the type I photosensitizer according to claim 1, characterized in that: The following steps are involved: (1) reacting the compound represented by formula (I) with 5-bromobenzene-1,3-diol in the presence of an acid-binding agent and a solvent to obtain the compound represented by formula (II) CyBr; (2) reacting Fmoc-L-alanine with p-aminobenzyl alcohol in the presence of a condensing agent, a catalyst, and a solvent to obtain an intermediate 2 represented by formula (III); the condensing agent is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the catalyst is N,N-diisopropylethylamine; (3) reacting the intermediate 2 prepared in step (2) with phosphorus tribromide in the presence of a solvent to obtain the intermediate 3 represented by formula (IV); (4) reacting the compound CyBr with the intermediate 3 prepared in step (3) in the presence of an alkaline reagent, a catalyst, and a solvent to obtain the intermediate 4 represented by formula (V); the catalyst is potassium iodide; (5) reacting the intermediate 4 prepared in step (4) with piperidine in the presence of a solvent to obtain the type I photosensitizer; The structures of the above formula (I) to formula (V) are as follows: 、 、 、 、 。 3. The preparation method according to claim 2, characterized in that In step (1), the acid binding agent is sodium hydride and / or triethylamine; the reaction temperature is 50-60 °C, and the reaction time is 10-18 h.

4. The preparation method according to claim 2, characterized in that In step (2), the reaction temperature is 20-30°C and the reaction time is 10-18 h.

5. The preparation method according to claim 2, characterized in that In step (3), the reaction temperature is 0-30 °C and the reaction time is 10-18 h.

6. The preparation method according to claim 2, characterized in that In step (4), the alkaline reagent is potassium carbonate; the reaction temperature is 50-70 °C, and the reaction time is 5-10 h.

7. The preparation method according to claim 2, characterized in that In step (5), the reaction temperature is 20-30°C and the reaction time is 5-30 min.

8. A fluorescent compound, characterized in that The fluorescent compound has the structure shown below: 。 9. Use of the type I photosensitizer according to claim 1 in the preparation of tumor imaging drugs.

10. Use of the type I photosensitizer according to claim 1 in the preparation of photodynamic therapy drugs.

11. Use of the type I photosensitizer according to claim 1 in the preparation of anti-tumor immune drugs.

Images