Light-operated protein hydrolysis targeting chimera as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a light-operated protein hydrolysis targeting chimera as well as a synthesis method and application thereof. The structural formula of the light-operated proteolysis targeting chimera is # imgabs0. The light-operated proteolysis targeting chimera provided by the invention can be combined with PD-L1 protein in a targeting manner, can efficiently induce degradation of the PD-L1 protein, and has a good inhibition effect on in-situ tumors; the light-controlled proteolysis targeting chimera provided by the invention can be combined with PD-L1 protein in a targeting manner, efficiently induces degradation of the PD-L1 protein, enhances the anti-tumor immune effect and inhibits pulmonary metastasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a photo-controlled proteolysis-targeting chimera and its synthesis method and application. Background Art

[0002] Tumors seriously threaten human health. Although there are various current tumor treatment methods, such as surgical resection, chemotherapy, radiotherapy, and targeted therapy, etc., they all have certain limitations. Surgical resection is difficult to completely remove micrometastases. Chemotherapy and radiotherapy lack specificity and will cause serious damage to normal tissues while killing tumor cells, leading to significant adverse reactions and affecting the quality of life and treatment compliance of patients. Although targeted therapy can act on specific targets of tumor cells, it also faces problems such as drug resistance and target limitations.

[0003] Based on this, developing a photo-controlled proteolysis-targeting chimera is of great significance for improving the tumor treatment effect. Summary of the Invention

[0004] The purpose of the present invention is to provide a photo-controlled proteolysis-targeting chimera and its synthesis method and application.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention:

[0007] A photo-controlled proteolysis-targeting chimera, whose structural formula is:

[0008]

[0009] Another technical solution of the present invention:

[0010] The synthesis method of the above photo-controlled proteolysis-targeting chimera includes the following steps:

[0011] 1) Dissolve compound A and compound B in an organic solvent, and then sequentially add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), and triethylamine (Et3N) to the solution, react, and purify to obtain compound C;

[0012] Among them, compound A is a PD-L1 / PD-1 interaction inhibitor (BMS-1166), and its structural formula is:

[0013]

[0014] Among them, compound B is NH2-C2-NH-Boc, and its structural formula is:

[0015]

[0016] Among them, the structural formula of compound C is:

[0017]

[0018] 2) Dissolve the compound C obtained in step 1) in an organic solvent, then add trifluoroacetic acid (TFA) to the solution, react, and purify to obtain compound D;

[0019] Among them, the structural formula of compound D is:

[0020]

[0021] 3) Dissolve compound E in an organic solvent, then successively add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (HOSu), and N,N-diisopropylethylamine (DIPEA) to the solution, stir, and finally add dropwise the DMF solution of compound D obtained in step 2) to the mixed solution, react, and purify to obtain compound F, namely the photocontrolled proteolytic targeting chimera.

[0022] Among them, compound E is pyropheophorbide a (Ppa), and its structural formula is:

[0023]

[0024] Further, in step 1), the organic solvent is specifically: N,N-dimethylformamide (DMF).

[0025] Further, in step 1), the molar ratio of compound A, compound B, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine is: 1∶(0.8 - 1.2)∶(0.8 - 1.2)∶(1 - 1.5)∶(1.5 - 2.5).

[0026] Further, in step 1), the reaction is specifically: stir and react at room temperature for 12 h.

[0027] Further, in step 1), the purification is specifically: add water to the reaction system, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, dry, and concentrate.

[0028] Further, in step 2), the organic solvent is specifically: dichloromethane (DCM).

[0029] Further, in step 2), the molar ratio of compound C to trifluoroacetic acid is 1∶50.

[0030] Further, in step 2), the reaction specifically is: monitoring the reaction process by thin-layer chromatography until compound C completely reacts.

[0031] Further, in step 2), the purification specifically is: rotary evaporation to remove the organic solvent.

[0032] Further, in step 3), the organic solvent specifically is: N,N-dimethylformamide (DMF).

[0033] Further, in step 3), the molar ratio of compound D, compound E, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and N,N-diisopropylethylamine is: 1∶1∶(1 - 1.5)∶(1 - 2)∶(2.5 - 3.5).

[0034] Further, in step 3), the stirring specifically is: stirring at room temperature for 20 min.

[0035] Further, in step 3), the reaction specifically is: stirring and reacting at room temperature for 12 h.

[0036] Further, in step 3), the purification specifically is: adding water to the reaction system, extracting twice with ethyl acetate, combining the organic phases, washing with saturated brine, drying, and concentrating.

[0037] The synthesis equation of the photocontrolled proteolysis-targeting chimera of the present invention is as follows:

[0038]

[0039] The third technical solution of the present invention:

[0040] The application of the above-mentioned photocontrolled proteolysis-targeting chimera in in-situ tumor inhibition.

[0041] The fourth technical solution of the present invention:

[0042] The application of the above-mentioned photocontrolled proteolysis-targeting chimera in inhibiting lung metastasis.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] The photocontrolled proteolysis-targeting chimera provided by the present invention can target and bind to the PD-L1 protein, and efficiently induce the degradation of the PD-L1 protein, having a good inhibitory effect on in-situ tumors;

[0045] The photocontrolled proteolysis-targeting chimera provided by the present invention can target and bind to the PD-L1 protein, and efficiently induce the degradation of the PD-L1 protein, enhancing the anti-tumor immune effect and inhibiting lung metastasis. Description of the Drawings

[0046] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention. Moreover, throughout the drawings, the same reference numerals are used to represent the same components. In the drawings:

[0047] Figure 1 is the mass spectrum of PpBMS;

[0048] Figure 2 is the result of the targeted binding molecular dynamics simulation of PpBMS and PD-L1 protein. Among them, A is the hydrophilic and hydrophobic surface map of the binding of PD-L1 protein and PpBMS, green represents hydrophobic, white represents hydrophilic, pink represents positive charge, and blue represents negative charge. B is the position of PpBMS in the three-dimensional structure of PD-L1 protein, and the yellow part is the binding pocket. C is the three-dimensional display of the binding mode of PpBMS and PD-L1, the red dotted line represents hydrogen bond, the yellow dotted line represents van der Waals force, and the orange dotted line represents π-π interaction;

[0049] Figure 3 is the result of the cellular thermal shift assay of PpBMS and PD-L1 protein. Among them, A is the thermal degradation of PD-L1 protein after the cell lysate is co-incubated with PBS or PpBMS. B is the quantitative analysis of PD-L1. C is the co-localization of PD-L1 protein and PpBMS. D is the fluorescence intensity analysis;

[0050] Figure 4 is the result of the detection of the effect of PpBMS on inducing the degradation of PD-L1 protein. Among them, A is the Western blot analysis of PD-L1 after CT26 cells are co-incubated with Ppa or PpBMS for 24 h. B is the quantitative analysis of PD-L1 protein. C is the Western blot analysis of PD-L1 after CT26 cells are co-incubated with PpBMS for 24 h. D is the quantitative analysis of PD-L1 protein. In the figure, (+) represents the light condition;

[0051] Figure 5 is the transmission electron microscopy image of PpBMS@Lip;

[0052] Figure 6 is the particle size distribution map of PpBMS@Lip;

[0053] Figure 7 is the zeta potential map of PpBMS@Lip;

[0054] Figure 8 is the stability detection result of PpBMS@Lip

[0055] Figure 9 is the singlet oxygen generation level detection result

[0056] Figure 10 Results of the detection of the cellular uptake efficiency of PpBMS@Lip. Among them, A is the fluorescence detection images of CT26 cells after co-incubation with PpBMS or PpBMS@Lip for 0 h, 6 h, and 12 h, and B is the quantitative fluorescence analysis of the cells;

[0057] Figure 11 Results of the detection of the production level of intracellular reactive oxygen species. Among them, A is the detection of the intracellular reactive oxygen species level in CT26 cells after treatment with different drugs, and B is the quantitative fluorescence analysis of DCF. In the figure, (+) indicates the light condition;

[0058] Figure 12 Results of the cytotoxicity detection. Among them, A is the detection result of the cell viability of CT26 cells after co-incubation with BMS, Ppa, PpBMS, or PpBMS@Lip for 24 h, and B is the detection result of the cell viability of CT26 cells after co-incubation with Ppa, PpBMS, or PpBMS@Lip and after light irradiation. In the figure, (+) indicates the light condition;

[0059] Figure 13 Results of the immunofluorescence staining analysis. Among them, A is the immunofluorescence analysis images of HMGB1 protein in CT26 cells after treatment with different drugs, and B is the immunofluorescence analysis images of CRT protein in CT26 cells after treatment with different drugs. In the figure, (+) indicates the light condition;

[0060] Figure 14 Results of the immunofluorescence staining analysis of D-L1 protein. Among them, A is the immunofluorescence analysis images of PD-L1 protein in CT26 cells after treatment with different drugs, and B is the quantitative fluorescence analysis of PD-L1 protein. In the figure, (+) indicates the light condition;;

[0061] Figure 15 Results of the Western blot analysis of PD-L1 protein. Among them, A is the Western blot analysis of PD-L1 protein in CT26 cells after treatment with different drugs, and B is the quantitative analysis of PD-L1 protein. In the figure, (+) indicates the light condition;

[0062] Figure 16 Results of the detection of the enrichment of PpBMS@Lip at the tumor site. Among them, A is the distribution of PpBMS@Lip in CT26 tumor-bearing mice, and B is the fluorescence intensity analysis of ex vivo organs;

[0063] Figure 17 The changing trend of tumor volume during the treatment process;

[0064] Figure 18 Results of the weighing and photographing of tumor tissues. Among them, A is the weight of the tumor tissue, and B is the physical photograph of the tumor tissue;

[0065] Figure 19 Results of H&E staining and Ki67 staining of tumor tissues;

[0066] Figure 20 Results of anti-tumor immunity detection, where A is the percentage of CD3 + CD8 + cells in the spleen, B is the percentage of CD3 + CD8 + cells in the lymph nodes, C is the percentage of mature dendritic cells in the tumor tissue, D is the percentage of CD3 + CD4 + cells in the tumor tissue, E is the percentage of CD3 + CD8 + cells in the tumor tissue;

[0067] Figure 21 Results of immunofluorescence staining of CD3 and CD8 in lung tissues. Detailed implementation manners

[0068] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention. It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention.

[0069] In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0070] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0071] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0072] Terms such as "comprising", "including", "having", "containing", etc. used in this text are all open-ended terms, meaning including but not limited to.

[0073] Example 1

[0074] Synthesis of photocontrolled proteolytic targeting chimera

[0075] 1) Under nitrogen protection, dissolve compound A (BMS-1166, 800 mg, 1.25 mmol) and compound B (NH2-C2-NH-Boc, 220 mg, 1.37 mmol) in DMF. Then, sequentially add EDCI (263 mg, 1.37 mmol), HOBT (203 mg, 1.50 mmol), and Et3N (53 mg, 2.50 mmol) to the solution. Stir the reaction at room temperature for 12 h. After the reaction is completed, add water to the reaction system, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, dry, and concentrate to obtain compound C.

[0076] 2) Dissolve the compound C obtained in step 1) in DCM. Then, add TFA (5 mL) to the solution and monitor the reaction progress by thin-layer chromatography. After compound C completely reacts, rotate evaporate to remove DCM to obtain compound D.

[0077] 3) Under nitrogen protection, dissolve compound E (Ppa, 450 mg, 0.84 mmol) in DMF. Then, sequentially add EDCI (193 mg, 1.01 mmol), HOSu (145 mg, 1.26 mmol), and DIPEA (326 mg, 2.52 mmol) to the solution. Stir at room temperature for 20 min. Finally, dropwise add the DMF solution of compound D (573 mg, 0.84 mmol) obtained in step 2) to the mixed solution and stir the reaction at room temperature for 12 h. After the reaction is completed, add water to the reaction system, extract twice with ethyl acetate, combine the organic phases, wash with saturated brine, dry, and concentrate to obtain compound F, namely the photocontrolled proteolytic targeting chimera, denoted as PpBMS.

[0078] The mass spectrum of PpBMS is as Figure 1 shown;

[0079] From Figure 1 it can be seen that the theoretical molecular weight of [M + H] + is 1199.5, and the molecular weight measured by mass spectrometry is 1199.4, indicating the successful synthesis of PpBMS.

[0080] I. PpBMS Targets and Binds to PD-L1 Protein

[0081] The targeted binding of PpBMS and PD-L1 proteins was detected by molecular dynamics simulation. The results of the molecular dynamics simulation of the targeted binding of PpBMS and PD-L1 proteins are as follows Figure 2 shown in the figure. Among them, A is the hydrophilic and hydrophobic surface map of the binding of PD-L1 protein and PpBMS. Green represents hydrophobic, white represents hydrophilic, pink represents positive charge, and blue represents negative charge. B is the position of PpBMS in the three-dimensional structure of PD-L1 protein. The yellow part is the binding pocket. C is the three-dimensional display of the binding mode of PpBMS and PD-L1. The red dotted line represents hydrogen bond, the yellow dotted line represents van der Waals force, and the orange dotted line represents π-π interaction;

[0082] It can be seen from Figure 2 the figure that the BMS-1166 group in PpBMS can bind to the binding pocket of PD-L1 protein. This is mainly due to the fact that the BMS-1166 group in PpBMS can bind to the side chain of Y123 in PD-L1 protein through hydrogen bond, and can bind to the side chains of Y56, E58, E60, V111, Y112, I 115 and I 116 through van der Waals force. In addition, the binding free energy of the BMS-1166 group in PpBMS and the binding pocket of PD-L1 protein was calculated by the MM-GBSA method to be -36.66 kcal / mol, which indicates that the BMS-1166 group of PpBMS and the binding pocket of PD-L1 protein have strong binding affinity, and in the binding process, van der Waals force is the main force.

[0083] In order to further detect that PpBMS can accurately target PD-L1 protein, a cellular thermal shift assay was performed on PpBMS and PD-L1 protein. The results of the cellular thermal shift assay of PpBMS and PD-L1 protein are as follows Figure 3 shown in the figure. Among them, A is the thermal degradation of PD-L1 protein after the cell lysate was co-incubated with PBS or PpBMS. B is the quantitative analysis of PD-L1. C is the co-localization of PD-L1 protein and PpBMS. D is the fluorescence intensity analysis;

[0084] It can be seen from Figure 3 A to B that in the cell lysate of the PBS group after 49 °C water bath treatment, almost half of the PD-L1 protein was degraded, while in the cell lysate of the PpBMS group, only 28.7% of the PD-L1 protein was degraded, which indicates that there is a binding interaction between PpBMS and PD-L1 protein, which can prevent the temperature-dependent degradation of PD-L1 protein;

[0085] It can be seen from Figure 3As can be seen from C to D, there is an overlap between the red fluorescence of PpBMS and the green fluorescence of PD-L1 protein. The Pearson correlation coefficient further analyzed by Image J is 0.678, indicating a strong co-localization relationship between PpBMS and PD-L1 protein.

[0086] II. Photodegradation of PD-L1 protein by PpBMS

[0087] Using Ppa as a control, the effect of PpBMS on inducing the degradation of PD-L1 protein was detected in CT26 cells. The detection results of the effect of PpBMS on inducing the degradation of PD-L1 protein are as Figure 4 shown. Among them, A is the Western blot analysis of PD-L1 after CT26 cells were co-incubated with Ppa or PpBMS for 24 h, B is the quantitative analysis of PD-L1 protein, C is the Western blot analysis of PD-L1 after CT26 cells were co-incubated with PpBMS for 24 h, D is the quantitative analysis of PD-L1 protein, and (+) in the figure represents the light condition;

[0088] As Figure 4 can be seen, under light conditions, treatment with Ppa will cause an up-regulation of PD-L1 protein expression (this is related to the hypoxic state caused by photodynamic therapy. The hypoxic state will activate the hypoxia-inducible factor 1α signaling pathway, which in turn leads to an up-regulation of PD-L1 protein expression), while treatment with PpBMS will significantly reduce the expression of PD-L1 protein; under non-light conditions, treatment with PpBMS has no significant effect on the expression of PD-L1 protein, indicating that PpBMS has light-controlled properties and its ability to target and induce the degradation of PD-L1.

[0089] Example 2

[0090] Preparation of the nano-drug PpBMS@Lip

[0091] PpBMS@Lip was prepared by the thin-film dispersion method. 1 mg of PpBMS and 10 mg of DSPE-PEG 2000 were dissolved in 6 mL of chloroform. The organic solvent was removed using a rotary evaporator to form a uniform lipid film. Subsequently, 2 mL of ultrapure water was added and sonicated for 1 h in a 50 °C water bath. Then the solution was loaded into a 3000 Da dialysis bag and dialyzed for 4 h to remove the unencapsulated drug. Finally, PpBMS@Lip was loaded into a centrifuge tube and stored at 4 °C for later use.

[0092] The transmission electron microscopy image of PpBMS@Lip is as Figure 5 shown;

[0093] As Figure 5 can be seen, PpBMS@Lip has a uniform spherical structure.

[0094] The particle size distribution diagram of PpBMS@Lip is as Figure 6 shown;

[0095] It can be seen from Figure 6 that the average particle size of PpBMS@Lip is 205 nm.

[0096] The potential diagram of PpBMS@Lip is as Figure 7 shown;

[0097] It can be seen from Figure 7 that the surface potential of PpBMS@Lip is -21.7 mV.

[0098] The stability of PpBMS@Lip was detected, and the stability test results of PpBMS@Lip are as Figure 8 shown;

[0099] It can be seen from Figure 8 that the particle size of PpBMS@Lip only fluctuates slightly within 6 days, indicating that PpBMS@Lip has good stability during storage.

[0100] I. Singlet oxygen ( 1 O2) production level

[0101] Using ABDA as an indicator, the singlet oxygen production levels of Ppa, PpBMS, and PpBMS@Lip were detected. The detection results of the singlet oxygen production levels are as Figure 9 shown, where (+) in the figure indicates the light irradiation condition;

[0102] It can be seen from Figure 9 that under the condition of no light irradiation, the absorbance of ABDA in the PBS, Ppa, PpBMS, and PpBMS@Lip groups hardly changed, indicating that singlet oxygen was not produced; while under the light irradiation condition, the absorbance of ABDA in the PpBMS and PpBMS@Lip groups decreased significantly, indicating that PpBMS and PpBMS@Lip can produce a large amount of singlet oxygen. Among them, the production level of singlet oxygen in PpBMS@Lip is the highest, the production level of singlet oxygen in PpBMS is at a medium level, and the production level of singlet oxygen in Ppa is the lowest. This is because the solubility of Ppa and PpBMS is poor, while the solubility of the nano-sized PpBMS@Lip is enhanced, enabling PpBMS@Lip to generate singlet oxygen more effectively.

[0103] II. Cellular uptake efficiency

[0104] Using PpBMS as a control, the cellular uptake efficiency of PpBMS@Lip was detected. The detection results of the cellular uptake efficiency of PpBMS@Lip are as Figure 10As shown, where A is the intracellular fluorescence detection images of CT26 cells co-incubated with PpBMS or PpBMS@Lip for 0 h, 6 h, and 12 h, and B is the intracellular fluorescence quantitative analysis;

[0105] It can be seen from Figure 10 that over time, the fluorescence in the cells of both the PpBMS and PpBMS@Lip groups increased. Among them, the PpBMS@Lip group showed stronger fluorescence than the PpBMS group, which was consistent with the results of fluorescence quantitative analysis. This indicates that PpBMS@Lip has a high drug delivery efficiency.

[0106] III. Intracellular reactive oxygen species (ROS) production level

[0107] Using 2',7'-dichlorofluorescin diacetate (DCFH-DA) as a probe, the intracellular ROS production levels of Ppa, PpBMS, and PpBMS@Lip were detected. The detection results of intracellular ROS production levels are as Figure 11 shown, where A is the detection of intracellular ROS levels in CT26 cells treated with different drugs, and B is the fluorescence quantitative analysis of DCF. In the figure, (+) indicates the light condition;

[0108] It can be seen from Figure 11 that under the condition of no light, the cell fluorescence signal is relatively weak, indicating that the ROS production level is extremely low. Under the light condition, in the Ppa, PpBMS, and PpBMS@Lip groups, the cell fluorescence intensity increased significantly, indicating that the ROS production level increased significantly. Among them, the PpBMS@Lip group had the highest ROS production level because PpBMS@Lip improved the drug delivery efficiency, thereby increasing the ROS production level; after quantitative analysis, under the light condition, the cell fluorescence intensity of PpBMS@Lip was 43 times that of PBS cells, indicating that PpBMS@Lip has excellent photodynamic therapy performance.

[0109] IV. Anti-proliferation ability

[0110] The cytotoxicity of Ppa, PpBMS, and PpBMS@Lip was detected. The cytotoxicity detection results are as Figure 12 shown, where A is the detection result of cell viability after CT26 cells were co-incubated with BMS, Ppa, PpBMS, or PpBMS@Lip for 24 h, and B is the detection result of cell viability after CT26 cells were co-incubated with Ppa, PpBMS, or PpBMS@Lip and irradiated with light. In the figure, (+) indicates the light condition;

[0111] It can be seen from Figure 12It can be seen that under dark conditions, the Ppa, PpBMS, and PpBMS@Lip groups all exhibited low cytotoxicity, and cell viability was hardly affected. Under light conditions, however, the Ppa, PpBMS, and PpBMS@Lip groups all showed cytotoxicity, and cell viability decreased significantly. Among them, the PpBMS@Lip group showed the highest cytotoxicity, and its cell viability decreased significantly. This is because the uptake of PpBMS@Lip in cells increased. Under light conditions, PpBMS@Lip significantly inhibited cell viability, highlighting the advantages of PpBMS@Lip in tumor treatment.

[0112] V. Inducing the death of immunogenic cells CT26

[0113] Immunofluorescence staining analysis was performed on Ppa, PpBMS, and PpBMS@Lip. The results of the immunofluorescence staining analysis are as Figure 13 shown. Among them, A is the immunofluorescence analysis picture of HMGB1 protein in CT26 cells after treatment with different drugs, and B is the immunofluorescence analysis picture of CRT protein in CT26 cells after treatment with different drugs. In the figure, (+) indicates light conditions;

[0114] It can be Figure 13 seen that under dark conditions, the Ppa, PpBMS, and PpBMS@Lip groups had no effect on the expression of high mobility group box 1 (HMGB1), and the fluorescence intensity was similar to that of the PBS group. Under light conditions, however, the green fluorescence intensity of cells in the Ppa, PpBMS, and PpBMS@Lip groups decreased significantly, indicating the release of HMGB1. In addition, under light conditions, the Ppa, PpBMS, and PpBMS@Lip groups were also able to induce the exposure of calreticulin (CRT). Among them, the cells in the PpBMS@Lip group showed the strongest CRT fluorescence and the weakest HMGB1 fluorescence. This is because PpBMS@Lip improved the drug delivery efficiency in cells, thereby enhancing the death of immunogenic cells CT26 induced by PpBMS@Lip.

[0115] VI. Targeted photodegradation of PD-L1 protein

[0116] Immunofluorescence staining analysis was performed on PD-L1 protein. The results of the immunofluorescence staining analysis of PD-L1 protein are as Figure 14 shown. Among them, A is the immunofluorescence analysis picture of PD-L1 protein in CT26 cells after treatment with different drugs, and B is the fluorescence quantitative analysis of PD-L1 protein. In the figure, (+) indicates light conditions;

[0117] It can be Figure 14It can be seen that under light illumination, the green fluorescence intensity in the PpBMS and PpBMS@Lip groups of cells was significantly weakened, indicating that the PD-L1 protein was degraded. Under light illumination, there was no significant change in the green fluorescence intensity in the Ppa group of cells, indicating that the PD-L1 protein was not degraded. This confirmed that PpBMS@Lip could target and photodegrade the PD-L1 protein; the analysis results of the fluorescence intensity were consistent with the immunofluorescence staining results.

[0118] Western blot analysis of the PD-L1 protein was performed, and the results of the Western blot analysis of the PD-L1 protein are as Figure 15 shown. Among them, A is the Western blot analysis of the PD-L1 protein in CT26 cells treated with different drugs, and B is the quantitative analysis of the PD-L1 protein. In the figure, (+) indicates the light illumination condition;

[0119] It can be Figure 15 seen that under light illumination, the expression of the PD-L1 protein in the PpBMS@Lip group of cells decreased significantly, and the degradation exceeded 40%, indicating that PpBMS@Li could efficiently induce the degradation of PD-L1, providing support for its application in anti-tumor immunotherapy.

[0120] VII. In vivo fluorescence imaging

[0121] The enrichment of PpBMS@Lip at the tumor site was detected by a fluorescence imaging system. The detection results of the enrichment of PpBMS@Lip at the tumor site are as Figure 16 shown. Among them, A is the distribution of PpBMS@Lip in CT26 tumor-bearing mice, and B is the fluorescence intensity analysis of ex vivo organs;

[0122] It can be Figure 16 seen that over time, the accumulation of PpBMS@Lip at the tumor site gradually increased, and the fluorescence intensity increased significantly, indicating that the drug delivery efficiency of PpBMS@Lip was relatively high and it could effectively accumulate in tumor tissues. In contrast, PpBMS was rapidly metabolized and failed to effectively reach the tumor site. The in vitro fluorescence images further confirmed this result, showing that the fluorescence intensity of the tumor tissues treated with PpBMS@Lip increased by 2.9 times compared with that of the tumor tissues treated with PpBMS, further indicating that PpBMS@Lip had strong passive targeting ability.

[0123] VIII. Growth inhibitory effect on orthotopic tumors and metastatic tumors

[0124] PBS, BMS, Ppa, PpBMS, and PpBMS@Lip were respectively injected into CT26 tumor-bearing mice via the tail vein. During the treatment process, the tumor volume was monitored every two days. The change trend of the tumor volume during the treatment process is as Figure 17 shown;

[0125] It can be seen from Figure 17 that in the BMS group, due to low drug solubility and poor targeting ability, it was almost impossible to inhibit tumor growth; in the PBS group, there was a certain inhibitory effect, but the tumor volume of the mice was the largest; under dark conditions, the tumor growth trends of the Ppa, PpBMS, and PpBMS@Lip groups were similar to that of the PBS group, indicating that these drugs could not effectively inhibit tumor growth under dark conditions; under dark conditions, the therapeutic effect of PpBMS was significantly better than that of Ppa, showing its synergistic advantage in targeting PD-L1 protein degradation and inducing ICD effect; the PpBMS@Lip group showed the highest efficiency in tumor suppression, which was closely related to its improved drug delivery efficiency.

[0126] At the end of the treatment, the tumor tissues were collected, weighed, and photographed. The results of weighing and photographing the tumor tissues are as Figure 18 shown, where A is the weight of the tumor tissue and B is the physical photograph of the tumor tissue;

[0127] It can be seen from Figure 18 that under light conditions, the average tumor weight of the PpBMS@Lip group was only 47 mg, significantly lower than that of the PBS group. In addition, complete regression of one tumor was observed in the PpBMS@Lip group, demonstrating the significant advantage of PpBMS@Lip in tumor treatment.

[0128] The collected tumor tissues were subjected to H&E staining and Ki 67 staining. The results of H&E staining and Ki 67 staining of the tumor tissues are as Figure 19 shown;

[0129] It can be seen from Figure 19 that under light conditions, large areas of apoptosis or necrosis (manifested as no cell nucleus) occurred in the tumor cells of the PpBMS@Lip group, and the Ki 67 signal was the weakest, indicating that PpBMS@Lip could significantly kill tumor cells and inhibit their proliferation; the H&E staining results showed that under light conditions, the PpBMS@Lip group had the fewest nodules on the surface of the lung tissue, indicating that PpBMS@Lip not only inhibited tumor growth but also significantly inhibited lung metastasis.

[0130] IX. Immune activation detection

[0131] The anti-tumor immunity was detected. The results of the anti-tumor immunity detection are as Figure 20 shown, where A is the percentage of CD3 + CD8 + cells in the spleen, B is the percentage of CD3 + CD8 + cells in the lymph nodes, C is the percentage of mature dendritic cells in the tumor tissue, and D is CD3 in the tumor tissue+ CD4 + The percentage of cells, and E is CD3 in tumor tissue + CD8 + The percentage of cells;

[0132] It can be seen from Figure 20 that under light conditions, the proportions of CD3 + CD8 + T cells in the spleen tissue and lymph nodes of the PpBMS@Lip group mice were the highest, indicating that anti-tumor immunity was activated; in addition, under light conditions, the proportions of CD11c + CD86 + dendritic cells in the tumor tissue of the PpBMS@Lip group mice increased significantly, indicating that the ICD effect induced by PpBMS@Lip promoted the maturation of dendritic cells; in addition, under light conditions, the proportions of CD3 + CD4 + T cells and CD3 + CD8 + T cells also increased significantly; it is worth noting that in the PBS group, the proportions of CD3 + CD4 + T cells and CD3 + CD8 + T cells were only 7.2% and 6.2% respectively, while in the PpBMS@Lip group, the proportions of these cells reached 31.3% and 27.2% respectively, which was mainly attributed to the synergistic effect of PpBMS@Lip-mediated PD-L1 protein degradation and ICD effect, enhancing the anti-tumor immune effect.

[0133] Immunofluorescence staining of CD3 and CD8 was performed on lung tissue, and the immunofluorescence staining results of CD3 and CD8 in lung tissue were as Figure 21 shown;

[0134] It can be seen from Figure 21 that the number of CD3 + CD8 + positive cells observed in the PBS, BMS, Ppa, PpBMS and PpBMS@Lip groups was small, because the drug enrichment amounts of Ppa and PpBMS at the tumor site were small, and these two drugs could not effectively stimulate anti-tumor immunity under light, resulting in the least infiltration of CD3 + CD8 + positive cells; in contrast, the PpBMS@Lip group promoted CD3 + CD8 +The infiltration of positive cells confirmed the activation of anti-tumor immunity. In summary, PpBMS@Lip activates anti-tumor immunity by inducing the ICD effect of tumor cells and the degradation of PD-L1, thereby inhibiting lung metastasis.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: modifications or equivalent substitutions can still be made to the specific implementation manners of the present invention, and any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A light-controlled proteolysis-targeting chimera, characterized in that, The structural formula of the photocontrolled proteolytic targeting chimera is as follows:

2. A method for synthesizing the opto-controlled proteolysis targeting chimera as described in claim 1, characterized in that, It includes the following steps: 1) Dissolve compound A and compound B in an organic solvent, and then sequentially add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine to the solution, react, and purify to obtain compound C; Among them, compound A is a PD-L1 / PD-1 interaction inhibitor, and its structural formula is: Among them, compound B is NH2-C2-NH-Boc, and its structural formula is: Among them, the structural formula of compound C is: 2) Dissolve the compound C obtained in step 1) in an organic solvent, and then add trifluoroacetic acid to the solution, react, and purify to obtain compound D; Among them, the structural formula of compound D is: 3) Dissolve compound E in an organic solvent, and then sequentially add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and N,N-diisopropylethylamine to the solution, stir, and finally dropwise add the DMF solution of the compound D obtained in step 2) to the mixed solution, react, and purify to obtain compound F, that is, the photocontrolled proteolytic targeting chimera. Among them, compound E is pheophorbide a, and its structural formula is:

3. The synthesis method according to claim 2, wherein In step 1), the molar ratio of compound A, compound B, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine is: 1∶(0.8 - 1.2)∶(0.8 - 1.2)∶(1 - 1.5)∶(1.5 - 2.5).

4. The synthesis method according to claim 2, wherein In step 1), the specific reaction is: stir and react at room temperature for 12 h.

5. The synthesis method according to claim 2, wherein In step 2), the molar ratio of compound C and trifluoroacetic acid is 1∶50.

6. The synthesis method according to claim 2, wherein In step 2), the specific reaction is: monitor the reaction process by thin-layer chromatography until compound C completely reacts.

7. The synthesis method according to claim 2, characterized in that, In step 3), the molar ratio of compound D, compound E, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and N,N-diisopropylethylamine is: 1∶1∶(1 - 1.5)∶(1 - 2)∶(2.5 - 3.5).

8. The synthesis method according to claim 2, wherein The specific reaction is: stir and react at room temperature for 12 h.

9. Application of the photocontrolled proteolytic targeting chimera as claimed in claim 1 in in-situ tumor inhibition.

10. Application of the photocontrolled proteolytic targeting chimera as claimed in claim 1 in inhibiting lung metastasis.

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