Self-assembled probe based on NO synergistic photodynamic therapy and preparation method and application thereof

Abstract

The application discloses a self-assembled probe based on NO synergistic photodynamic therapy, a preparation method and application thereof, and belongs to the technical field of cancer treatment, wherein the self-assembled probe comprises a photosensitizer part, a cleavable polypeptide part responding to overexpressed proteases in a tumor microenvironment and an NO gas donor part; wherein the cleavable polypeptide part is cut by proteases to drive the self-assembly of the probe in situ. The photosensitizer part generates reactive oxygen species (ROS) under light irradiation, and simultaneously releases NO through photo-oxidation and reduction catalysis of the NO donor; the in-situ generated NO and ROS rapidly react to form more toxic peroxynitrite (ONOO ‑ ), thereby inducing tumor cell death. The self-assembly strategy controlled by the overexpressed biomarkers in the tumor microenvironment realizes the targeted aggregation of the photosensitizer and the NO donor at the tumor site, and the generated nanoparticles generate a large amount of singlet oxygen and NO through light induction, which are used for the synergistic treatment of breast cancer in situ.

Description

Technical Field

[0001] This invention relates to the field of cancer treatment technology, specifically a self-assembled probe based on NO synergistic photodynamic therapy, its preparation method, and its application. Background Technology

[0002] While radiotherapy and chemotherapy, commonly used in clinical practice, have achieved some success in treating various cancers, they are still often accompanied by serious toxic side effects, cellular drug resistance, and unsatisfactory treatment outcomes. Therefore, there is a need to develop novel cancer treatments that are both highly effective and low in toxicity.

[0003] According to a report in *Chemical Society Reviews* (2021, 50(6): 4185-4219), photodynamic therapy (PDT) is considered a promising treatment strategy due to its advantages such as minimal invasiveness, high spatiotemporal selectivity, low systemic toxicity, and low likelihood of developing drug resistance. Currently, PDT has been approved for the clinical treatment of various malignant tumors, including head and neck tumors, lung cancer, and gastrointestinal cancer, with good results. However, PDT still has its limitations, such as limited tissue penetration depth of the light source (Small, 2017, 13(44): 1702299), lack of targeted delivery of photosensitizers (Analytical Chemistry, 2020, 92(24): 16113-16121), and the hypoxic environment inside solid tumors (Advanced Materials, 2021, 33(48): 2103978). These factors directly affect the efficiency of ROS generation.

[0004] According to a report in *ACS Omega* (2020, 5(26): 15771-15776), enzyme-controlled self-assembly, due to the specific expression of enzymes in certain subcellular compartments, results in supramolecular assemblies that can thus be localized and aggregated at the enzyme's location. Therefore, the specific accumulation of functional molecules at target sites can be achieved by introducing exogenous molecules for in-situ self-assembly within cells. According to a report in *Advanced Materials* (2018, 30(49): 1801964), gas therapy, represented by the release of nitric oxide (NO), hydrogen sulfide (H2S), or carbon monoxide (CO), has become an emerging therapeutic strategy. Among these, NO, as a biosignaling molecule, plays an important role in life activities. The journal *Antioxidants & Redox Signaling* (2019, 30(8):1124-1143) reported that high doses of NO can cause organelle damage, inhibit cell proliferation, and induce apoptosis.

[0005] While each of the above strategies has shown potential, how to organically integrate them to construct a single molecular platform that can simultaneously achieve tumor-specific targeting, overcome hypoxia limitations, and synergistically amplify therapeutic effects remains a technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a self-assembled probe based on NO synergistic photodynamic therapy, its preparation method, and its application, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A self-assembled probe based on NO-co-photodynamic therapy comprises a photosensitizer portion, a cleavable polypeptide portion responsive to proteases overexpressed in the tumor microenvironment, an NO gas donor portion, and a polymer portion for improving water solubility. The cleavable polypeptide portion, after being cleaved by the protease, drives in-situ self-assembly of the probe. Under light irradiation, the photosensitizer portion generates reactive oxygen species (ROS) and triggers the gas donor portion to release therapeutic NO gas, causing the ROS to react with the therapeutic NO gas to generate ONOO. - .

[0009] Furthermore, the protease is MMP-2.

[0010] Furthermore, the photosensitizer portion is palladium(II) tetraphenyltetrabenzoporphyrin; the gas donor portion is a coumarin-based NO donor.

[0011] Furthermore, the self-assembled probe also includes polyethylene glycol chains.

[0012] Furthermore, the structural formula of the self-assembled probe is as follows:

[0013] .

[0014] Another object of the present invention is to provide a method for preparing the above-mentioned self-assembled probe based on NO synergistic photodynamic therapy, comprising the following steps:

[0015] Precursor molecules containing cleavable polypeptide moieties and linkers were prepared by solid-phase synthesis.

[0016] The precursor molecule is coupled with the NO gas donor portion to obtain the first intermediate;

[0017] The photosensitizer portion is coupled to a linker molecule containing a cyclooctyne group to obtain a second intermediate;

[0018] The first intermediate and the second intermediate are connected to obtain the probe precursor;

[0019] The probe precursor was coupled with a polyethylene glycol chain to obtain a self-assembled probe.

[0020] Furthermore, the preparation method specifically includes the following steps:

[0021] Dithiopyridine was dissolved in methanol, and mercaptopropionic acid was added dropwise to react with it. The solvent was then removed to obtain an oily solid, which was purified to obtain compound A.

[0022] N,N-dimethylformamide was added to the 2-chlorotriphenylmethyl chloride resin support for activation. Then, fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester and N,N-diisopropylethylamine, completely dissolved in N,N-dimethylformamide, were added for reaction. After the reaction was complete, the resin was washed, and then methanol and N,N-dimethylformamide were added to continue the reaction, blocking unreacted active sites on the resin. The reaction solution was squeezed out, and a solution of N,N-dimethylformamide containing piperidine was added to remove the amino protecting group Fmoc. The above operation was repeated, and the resin was washed multiple times with N,N-dimethylformamide. Then, N-fluorenemethoxycarbonyl-glycine, 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were added for reaction. Following the above steps, Nα-fluorenemethoxycarbonyl-Nω-(2,2,4, The following compounds were involved: 6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine, N-fluorenmethoxycarbonyl-L-valine, N-fluorenmethoxycarbonyl-glycine, N-(9-fluorenmethoxycarbonyl)-L-leucine, N-(9-fluorenmethoxycarbonyl)-L-proline, N-fluorenmethoxycarbonyl-glycine, and compound A. After the reaction was complete, the peptides were washed and cleaved from the resin using a mixed solvent of trifluoroacetic acid and dichloromethane. The solvent was removed to obtain a yellow oily liquid. This process was repeated 3 to 5 times until the resin turned brown. All the cleaved liquid was collected and the solvent was removed to obtain a yellow oily liquid. Finally, the peptides were precipitated with diethyl ether. After centrifugation at room temperature, the supernatant was poured off, and the precipitate was allowed to dry naturally in a fume hood to obtain a powdery solid, which was named compound B.

[0023] Compound B and azidopropylamine were dissolved using N,N-dimethylformamide, followed by the addition of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine. The reaction system was adjusted to neutral, stirred, and then purified to obtain compound C.

[0024] Compound C was dissolved in a dichloromethane solution of trifluoroacetic acid, then triisopropylsilane was added and stirred, and then purified to obtain compound D;

[0025] Compound D was dissolved in anhydrous N,N-dimethylformamide, and then dissolved NO donor, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to it for reaction. After purification, compound E was obtained.

[0026] The dibenzocyclooctyne-carboxyl group, 1-hydroxybenzotriazole and O-benzotriazole-tetramethylurea hexafluorophosphate were dissolved in N,N-dimethylformamide, and then 1,3-propanediamine was added to react with it. After purification, compound F was obtained.

[0027] Palladium(II) tetraphenyltetrabenzoporphyrin and 4-nitrobenzoyl chloride were lyophilized, and then palladium(II) tetraphenyltetrabenzoporphyrin and N,N-diisopropylethylamine were dissolved in anhydrous dichloromethane. Then, 4-nitrobenzoyl chloride solution was added dropwise to the reaction system to carry out the reaction. After purification, compound G was obtained.

[0028] Compounds G and F were lyophilized, and then compound G, compound F and N,N-diisopropylethylamine were dissolved in anhydrous tetrahydrofuran and reacted. After purification, compound H was obtained.

[0029] Compound H was dissolved in dichloromethane and compound E was dissolved in N,N-dimethylformamide. The two solutions were then mixed and stirred. Subsequently, dichloromethane and N,N-dimethylformamide were removed sequentially, and the mixture was purified to obtain compound I.

[0030] Compound I and mercapto-modified polyethylene glycol were dissolved in dichloromethane and reacted. Subsequently, the dichloromethane and N,N-dimethylformamide were removed sequentially to obtain a bright green solid powder product. The product was dissolved in ultrapure water, centrifuged, and the supernatant was concentrated to obtain a self-assembled probe.

[0031] Another objective of this invention is to provide the application of the above-mentioned self-assembled probe based on NO synergistic photodynamic therapy in the preparation of cancer therapeutic drugs.

[0032] Furthermore, the cancer is a solid tumor that highly expresses MMP-2.

[0033] Furthermore, the cancer in question is breast cancer.

[0034] This invention provides a self-assembling probe based on NO synergistic photodynamic therapy. By combining an MMP-2-induced in-situ self-assembly strategy with photodynamic therapy and NO gas therapy, it achieves synergistic treatment of both photodynamic therapy and NO. When the probe reaches the tumor site via blood circulation, it is cleaved by MMP-2 in the tumor microenvironment, releasing strongly hydrophobic intermediates. These intermediates self-assemble in situ at the tumor site to form nanoparticles. Under 630nm red light irradiation, the photosensitizer portion absorbs energy and transitions to an excited triplet state, transferring high-energy electrons to surrounding oxygen molecules to generate highly cytotoxic singlet oxygen. Simultaneously, the photosensitizer portion catalyzes the release of NO from the NO donor through photo-redox catalysis. This in-situ generated NO reacts rapidly with ROS (reactive oxygen species) to form the more toxic ONOO. - This induces tumor cell death. This self-assembly strategy, controlled by biomarkers overexpressed in the tumor microenvironment, achieves targeted aggregation of photosensitizers and NO donors at the tumor site. The generated nanoparticles then photo-induced the production of large amounts of singlet oxygen and NO, which can be used for synergistic treatment of in situ breast cancer. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of in-situ self-assembly of PdTPTBP-CouN(NO)-PEG for breast cancer treatment using NO synergistic photodynamic therapy. In the diagram, A shows the synthesis and composition of PdTPTBP-CouN(NO)-PEG; B shows the self-assembly process of PdTPTBP-CouN(NO)-PEG in mice; C shows the release of ROS and NO and the generation of ONOO from PdTPTBP-CouN(NO)-PEG under light irradiation. - A schematic diagram.

[0036] Figure 2 This is a mass spectrometry (ESI-MS) analysis result of compound A synthesized in Example 1.

[0037] Figure 3 This is a mass spectrometry (ESI-MS) analysis result of compound B synthesized in Example 1.

[0038] Figure 4 This is a mass spectrometry (ESI-MS) analysis result of compound C synthesized in Example 1.

[0039] Figure 5 This is a mass spectrometry (ESI-MS) analysis result of compound D synthesized in Example 1.

[0040] Figure 6 The image shows the mass spectrometry (ESI-MS) analysis results of compound E synthesized in Example 1.

[0041] Figure 7The image shows the mass spectrometry (ESI-MS) analysis results of compound F synthesized in Example 1.

[0042] Figure 8 The image shows the mass spectrometry (MALDI-TOF-MS) analysis results of compound G synthesized in Example 1.

[0043] Figure 9 The image shows the mass spectrometry (MALDI-TOF-MS) analysis results of compound H synthesized in Example 1.

[0044] Figure 10 The image shows the mass spectrometry (MALDI-TOF-MS) analysis results of compound I synthesized in Example 1.

[0045] Figure 11 The image shows the mass spectrometry (MALDI-TOF-MS) analysis results of the probe PdTPTBP-CouN(NO)-PEG synthesized in Example 1.

[0046] Figure 12 The image shows the HPLC chromatograms of compound D in Example 2 before and after in vitro digestion by MMP-2.

[0047] Figure 13 This is a mass spectrometry (ESI-MS) analysis result of the enzyme digestion product of compound D in Example 2 after being digested by MMP-2.

[0048] Figure 14 This is a transmission electron microscope image of the nanoparticles after in situ self-assembly of the probe PdTPTBP-CouN(NO)-PEG in response to the MMP-2 enzyme in Example 2.

[0049] Figure 15 The probe in Example 3 was used to detect ROS, NO, and ONOO in the solution system. - The results of the release capacity test; in the figure, a represents the release of ROS by the probe in the solution system; b represents the release of NO by the probe in the solution system; c represents the release of ONOO by the probe in the solution system. - The release status.

[0050] Figure 16 The probe in Example 3 was used to detect ROS, NO, and ONOO in 4T1 cells. - Results of release capacity detection; in the figure, a represents the release of ROS by the probe solution in 4T1 cells; b represents the release of NO by the probe in 4T1 cells; c represents the release of NO by the probe in 4T1 cells. - The release status.

[0051] Figure 17This is a low-magnification transmission electron microscope image of 4T1 cells co-incubated with the probe PdTPTBP-CouN(NO)-PEG in Example 4.

[0052] Figure 18 The dark toxicity of the probe PdTPTBP-CouN(NO)-PEG to 4T1 cells in Example 4 is shown.

[0053] Figure 19 The phototoxicity of the probe PdTPTBP-CouN(NO)-PEG to 4T1 cells in Example 4.

[0054] Figure 20 The image shows fluorescence staining of live and dead cells after different treatments of 4T1 cells in Example 4.

[0055] Figure 21 This figure shows the in vivo antitumor effect in Example 5. In the figure, a represents the in vivo antitumor treatment regimen in mice; Group I represents the PBS group, Group II represents the PBS+light group, Group III represents the PdTPTBP group, Group IV represents the PdTPTBP+light group, Group V represents the PdTPTBP-Cou(NO)-PEG group, and Group VI represents the PdTPTBP-Cou(NO)-PEG+light group (n=4); b represents the tumor images of mice in each group at the end of treatment; c represents the tumor weight of mice in each group at the end of treatment; d represents the tumor growth curve of mice during treatment; e represents the hematoxylin-eosin (H&E) staining of tumor tissue of mice in each group at the end of treatment.

[0056] Figure 22 This is the curve showing the change in body weight of mice during the treatment process in Example 5.

[0057] Figure 23 H&E staining of major organs of mice after treatment in Example 5.

[0058] Figure 24 The results are the blood biochemical test results of mice after the treatment in Example 5.

[0059] Figure 25 The results are the blood routine test results of the mice after the treatment in Example 5. Detailed Implementation

[0060] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0061] In one embodiment of the present invention, a self-assembled probe based on NO-co-photodynamic therapy is provided, denoted as PdTPTBP-CouN(NO)-PEG, comprising a photosensitizer portion, a cleavable polypeptide portion responsive to proteases overexpressed in the tumor microenvironment, a gas donor portion, and a polyethylene glycol chain; wherein, after the cleavable polypeptide portion is cleaved by the protease, it drives the probe to undergo in-situ self-assembly; under light irradiation, the photosensitizer portion generates ROS and triggers the gas donor portion to release therapeutic NO gas, causing the ROS to react with the therapeutic NO gas to generate ONOO. - Preferably, the protease is MMP-2; the photosensitizer portion is palladium(II)tetraphenyltetrabenzoporphyrin; and the gas donor portion is a coumarin-based NO donor, Coun(NO)-NO2.

[0062] The method for preparing the self-assembled probe is as follows: a precursor molecule containing a cleavable polypeptide moiety and a linker arm is prepared by solid-phase synthesis; the precursor molecule is coupled with a gas donor moiety to obtain a first intermediate; the photosensitizer moiety is coupled with a linker arm molecule containing a cyclooctyne group to obtain a second intermediate; the first intermediate and the second intermediate are linked to obtain a probe precursor; the probe precursor is coupled with a polyethylene glycol chain to obtain a self-assembled probe.

[0063] The structures of compounds A, B, C, D, E, F, G, H, I and PdTPTBP-CouN(NO)-PEG mentioned in this invention are as follows:

[0064]

[0065] Compound A;

[0066]

[0067] Compound B;

[0068]

[0069] Compound C;

[0070]

[0071] Compound D;

[0072]

[0073] Compound E;

[0074]

[0075] Compound F;

[0076]

[0077] Compound G;

[0078]

[0079] Compound H;

[0080]

[0081] Compound I;

[0082]

[0083] Compound PdTPTBP-CouN(NO)-PEG.

[0084] Photodynamic therapy (PDT) has been used clinically to combat various malignant tumors. Due to its non-invasive nature and excellent temporal and tropic control capabilities, photostimulation has become a highly anticipated research direction in recent years. However, the therapeutic effect of PDT is limited by the tissue penetration depth of light and the hypoxia of solid tumors. Compared with conventional PDT, the self-assembled probe PdTPTBP-CouN(NO)-PEG designed in this invention for NO gas-assisted PDT has the following advantages: it introduces safe, efficient, low-drug-resistance NO gas that enhances the sensitivity of cancer cells to conventional drugs; it achieves precise delivery and controllable release of photosensitizer and NO through an in-situ self-assembly strategy; the near-infrared responsive NO donor overcomes the problems of easy absorption, poor tissue penetration, and potential phototoxicity associated with traditional visible light; and NO can rapidly react with ROS generated by PDT to produce more toxic ONOO. - This enabled sensitization therapy.

[0085] Unless otherwise specified, all raw materials used in the following embodiments are commercially available products and can be purchased through commercial channels. The invention will be described in detail below through specific embodiments in practical applications.

[0086] Example 1: As Figure 1 As shown, this embodiment provides a method for synthesizing compounds A, B, C, D, E, F, G, H, I and PdTPTBP-CouN(NO)-PEG, and the synthetic route is as follows:

[0087]

[0088]

[0089]

[0090]

[0091] .

[0092] The above synthesis method specifically includes the following steps:

[0093] Synthesis of A: Dithiopyridine (2-PDS, 1322 mg, 6 mmol) was dissolved in 10 mL of methanol, and mercaptopropionic acid (530 mg, 5 mmol) was added dropwise through a constant-pressure dropping funnel. The mixture was allowed to react overnight at room temperature. The solvent was then removed by rotary evaporation to obtain an oily solid. After purification by HPLC, compound A was obtained.

[0094] Synthesis of B: N,N-dimethylformamide (DMF) was added to the support (2-chlorotriphenylmethyl chloride resin, 0.7 mmol) and activated on a shaker. Then, N-fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (Fmoc-Glu(tBU)-OH, 425.47 mg, 1 mmol) and 1 mmol N,N-diisopropylethylamine (DIPEA, 175 μL, 1 mmol) completely dissolved in DMF were added, and the reaction was carried out at room temperature for 8 h. After the reaction was completed, the mixture was washed with DMF, and then 6 mL of methanol and DMF at a volume ratio of 2:1 was added, and the reaction was continued for 30 min to block the unreacted active sites on the resin. The reaction solution was squeezed out, and a DMF solution containing 20% ​​piperidine was added to remove the amino protecting group Fmoc. The above operation was repeated, and the mixture was washed with DMF several times. Then, 1 mmol of dissolved N-fluorenylmethoxycarbonyl-glycine (Fmoc-Gly-OH, 297.32 mg, 1 mmol), 1-hydroxybenzotriazole (HOBT, 135 mg, 1 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 379 mg, 1 mmol) and 1 mmol N,N-diisopropylethylamine (DIPEA, 175 μL, 1 mmol) were added, and the reaction was carried out for 8 h. Following the steps described above, Nα-fluorenemethoxycarbonyl-Nω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine (Fmoc-Arg(Pbf)-OH, 632.8 mg, 1 mmol), N-fluorenemethoxycarbonyl-L-valine (Fmoc-Val-OH, 339.39 mg, 1 mmol), and N-fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 297 mg, 1 mmol) were further synthesized on the resin. Compound A (32 mg, 1 mmol), N-(9-fluorenylmethoxycarbonyl)-L-leucine (Fmoc-Leu-OH, 353.41 mg, 1 mmol), N-(9-fluorenylmethoxycarbonyl)-L-proline (Fmoc-Pro-OH, 337.37 mg, 1 mmol), N-fluorenylmethoxycarbonyl-glycine (Fmoc-Gly-OH, 297.32 mg, 1 mmol) and compound A (216.0 mg, 1 mmol). After the reaction was complete, the sample was washed three times each with appropriate amounts of DMF, isopropanol, and n-hexane. The peptide was then cleaved from the resin using a mixed solvent of dichloromethane containing 1% trifluoroacetic acid. The solvent was removed using a rotary evaporator to obtain a yellow oily liquid. The mixed solvent of dichloromethane containing 1% trifluoroacetic acid was added to a horn tube and reacted for 5 minutes to cleave the peptide from the resin. This process was repeated 3 to 5 times until the resin turned brown. All the cleaved liquid was collected in a round-bottom flask and the solvent was removed using a rotary evaporator to obtain a yellow oily liquid.Finally, the polypeptide was precipitated with diethyl ether, centrifuged at room temperature, the supernatant was poured off, and the precipitate was placed in a fume hood to dry naturally, yielding a powdery solid, which was named compound B.

[0095] Synthesis of C: Compound B (0.4 mmol, 257.5 mg) and azidopropylamine (0.48 mmol, 100 mg) were dissolved in DMF, followed by the addition of 1-hydroxybenzotriazole (HOBT, 64.8 mg, 0.48 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 180 mg, 0.48 mmol), and 0.48 mmol N,N-diisopropylethylamine (DIPEA, 0.48 mmol, 83.6 µL). The reaction mixture was adjusted to neutral and stirred overnight at room temperature. Compound C was obtained after purification by HPLC.

[0096] Synthesis of D: Compound C (450 mg, 0.33 mmol) was dissolved in a 90% trifluoroacetic acid solution in dichloromethane. Then, 50 µL of triisopropylsilane (TIPS) was added to the solution, and the mixture was stirred at room temperature for 3 h. Compound D was obtained after purification by HPLC.

[0097] Synthesis of E: Compound D (20 mg, 0.019 mmol) was dissolved in anhydrous DMF, and then 0.022 mmol of dissolved NO donor (CouN(NO)-NO2, 8 mg, 0.022 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 8.4 mg, 0.041 mmol) and 4-dimethylaminopyridine (DMAP, 5 mg, 0.041 mmol) were added. The reaction was allowed to proceed overnight at room temperature. After purification by HPLC, compound E was obtained.

[0098] Synthesis of F: Dibenzocyclooctylene-carboxyl group (DBCO-COOH, 30.5 mg, 0.1 mmol), 1-hydroxybenzotriazole (HOBT, 16.2 mg, 0.12 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 45 mg, 0.12 mmol) were dissolved in DMF. Then, 1,3-propanediamine (22 mg, 0.3 mmol) was added, and the reaction was stirred at room temperature. After 4 hours of reaction, the presence of the target product was monitored by analytical HPLC. If there was any remaining starting material, 1-hydroxybenzotriazole and O-benzotriazole-tetramethylurea hexafluorophosphate were added to the reaction system to continue the reaction until the starting material disappeared. Compound F was obtained after purification by thin-layer chromatography.

[0099] Synthesis of G: Palladium(II)tetraphenyltetrabenzo[a]porphyrin (PdTPTBP, 10 mg, 0.1 mmol) and 4-nitrobenzoyl chloride (6 mg, 0.3 mmol) were lyophilized. Palladium(II)tetraphenyltetrabenzo[a]porphyrin and 5 μL of N,N-diisopropylethylamine were then dissolved in anhydrous dichloromethane. 4-Nitrobenzoyl chloride solution was then added dropwise to the reaction mixture, and the reaction was allowed to proceed overnight at room temperature. Compound G was obtained after purification by thin-layer chromatography.

[0100] Synthesis of H: Lyophilized compound G (5 mg, 0.004 mmol) and compound F (3 mg, 0.008 mmol) were dissolved in anhydrous tetrahydrofuran with 1.4 μL of N,N-diisopropylethylamine and reacted overnight. After purification by thin-layer chromatography, compound H was obtained.

[0101] Synthesis of I: Compound H (2 mg, 0.0014 mmol) was dissolved in dichloromethane, and compound E (2 mg, 0.0014 mmol) was dissolved in DMF. The two solutions were then mixed and stirred for 24 h. Dichloromethane and DMF were removed sequentially using a rotary evaporator and a lyophilizer. Compound I was obtained after purification by thin-layer chromatography.

[0102] Synthesis of PdTPTBP-CouN(NO)-PEG: 1.20 mg of compound I and 2.40 mg of mercapto-modified polyethylene glycol (PEG) were dissolved in a 50% dichloromethane-DMF solution and reacted at room temperature for 24 h. DCM and DMF were then removed sequentially using a rotary evaporator and a lyophilizer to obtain a bright green solid powder. This product was dissolved in 2 mL of ultrapure water and centrifuged at 16000 g for 10 min. The green precipitate at the bottom of the centrifuge tube was unreacted compound I. The supernatant was concentrated using a rotary evaporator to obtain the target compound PdTPTBP-CouN(NO)-PEG.

[0103] Electrospray ionization mass spectrometry (ESI-MS) of compounds A, B, C, D, and E was used to acquire data as follows: Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 The mass spectra were obtained by matrix-assisted laser desorption / ionization mass spectrometry (MAMS) of compounds F, G, H, I, and PdTPTBP-CouN(NO)-PEG using a Brook Dalton time-of-flight mass spectrometer. Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 High-resolution mass spectrum.

[0104] Example 2: This example uses analytical HPLC (Agilent Technologies, 1260 Infinity). ESI-MS and transmission electron microscopy (JEOL, Japan) verified that PdTPTBP-CouN(NO)-PEG can self-assemble into nanostructures in vitro in response to MMP-2.

[0105] To verify the responsiveness of the compound to MMP-2, the serum-free culture supernatant of 4T1 mouse breast cancer cells, which naturally highly express and secrete MMP-2, was collected as the enzyme source. The MMP-2-responsive compound D (100 μM) was mixed with the 4T1 cell supernatant and incubated in a 37°C water bath for 4 hours in the dark to simulate a full reaction between the enzyme and substrate under physiological conditions. Simultaneously, the probe PdTPTBP-Cou(NO)-PEG was incubated under the same conditions. Immediately after incubation, 10 μL of the reaction solution was dropped onto a carbon-copper grid for transmission electron microscopy (TEM), and this process was repeated 2 to 3 times to prepare TEM samples. Figure 12 This is an HPLC chromatogram of compound D (100 μM) before and after cleavage by MMP-2 enzyme in vitro. A new peak appears at 20.8 min after incubation, which is verified by mass spectrometry. Figure 13 As shown, the ESI-MS[(M+H)+]:m / z is 539.42, which corresponds to the mass of the peptide after cleavage between Gly and Val (OH-Gly-Leu-Pro-Gly-spy). Figure 14 Transmission electron microscopy images demonstrated that the probe PdTPTBP-Cou(NO)-PEG could be cleaved by the MMP-2 enzyme and further self-assembled in situ into nanoparticles through hydrophobic interactions.

[0106] Example 3: In this example, a fluorescence spectrophotometer and fluorescence microscope were used to verify the in vitro ROS, NO, and ONOO content of PdTPTBP-CouN(NO)-PEG. - Release capabilities.

[0107] ROS detection: 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as the ROS fluorescent probe. DCFH-DA is hydrolyzed under alkaline conditions to generate dichlorodihydrofluorescein (DCFH), which emits fluorescence upon reaction with ROS. DCFH-DA was pre-hydrolyzed to obtain a DCFH solution. Three groups were set up: the DCFH group (10µM DCFH only), the PdTPTBP group (10µM DCFH + 40µM PdTPTBP), and the PdTPTBP-CouN(NO)-PEG group (10µM DCFH + 40µM PdTPTBP-CouN(NO)-PEG). Each group was irradiated with 630nm red light for different times (0, 30, 60, and 300 seconds), and the fluorescence intensity was measured immediately after irradiation using a fluorophotometer. Figure 15 As shown in Figure a, when PdTPTBP-CouN(NO)-PEG is irradiated with 630nm red light, the DCFH fluorescence intensity is activated and gradually increases with the duration of irradiation. This indicates that PdTPTBP generates ROS after laser irradiation.

[0108] NO detection: 4,5-Diaminofluorescein diacetate (DAF-2DA) was used as the NO fluorescent probe. Two groups were set up: the Coun(NO)-NO2 group (5µM DAF-2DA + 40µM Coun(NO)-NO2) and the PdTPTBP-Coun(NO)-PEG group (5µM DAF-2DA + 40µM PdTPTBP-Coun(NO)-PEG). Irradiation with 630nm red light was performed for 0, 5, 10, 20, and 30 min, respectively. Figure 15 As shown in Figure b, irradiation of CouN(NO)-NO2 with 630nm red light alone did not cause a significant change in the fluorescence signal, while the fluorescence signal was enhanced when PdTPTBP-CouN(NO)-PEG was irradiated with 630nm red light. This result indicates that after PdTPTBP is excited by red light, the photosensitizer in the excited triplet state can promote the release of NO from the NO donor.

[0109] ONOO - Detection: Dihydrorhodamine 123 (DHR123) was used as ONOO. - Fluorescent probes. Solutions containing 10 µM DHR123 and 40 µM PdTPTBP-CouN(NO)-NO2-PEG were illuminated for 0 min and 10 min, respectively, and then the fluorescence intensity was measured. Figure 15 As shown in Figure c, the fluorescence intensity of the PdTPTBP-CouN(NO)-PEG and DHR123 mixed solution after light treatment was significantly higher than that of the untreated group, indicating that the ROS and NO generated simultaneously further reacted to produce ONOO with stronger oxidizing power.- .

[0110] Simultaneously, this embodiment also verified at the cellular level the in vitro release of ROS, NO, and ONOO by the probe PdTPTBP-CouN(NO)-PEG. - The ability of cells to be isolated was assessed. 4T1 cells were seeded in 12-well plates and cultured overnight at 37°C. The original medium was discarded, and RPMI 1640 medium containing 40 µM PdTPTBP-CouN(NO)-PEG was added, followed by incubation for 4 h. Subsequently, phenol red-free RPMI 1640 basal medium containing 10 µM MDCFH-DA, 10 µM DAF-2DA, and 10 µM DHR123 was added, and incubation was continued for 30 min. Each group was divided into two subgroups: one group was irradiated with 630 nm red light for 30 min, and the other group was not irradiated. After the irradiation period, the cells in the irradiated group were washed with PBS, phenol red-free RPMI 1640 medium was added, and live-cell fluorescence imaging was immediately performed using a fluorescence microscope. Figure 16 Figure a shows that the probe taken up by cells can efficiently generate ROS under red light triggering, which is consistent with the results of in vitro experiments. Figure 16 Figure b shows that the probe successfully released NO under red light triggering. Figure 16 Figure c demonstrates that the simultaneously generated ROS and NO further react within the cell to produce ONOO. - The above results indicate that PdTPTBP-CouN(NO)-PEG can effectively produce ROS, NO, and ONOO in cells under red light irradiation. - .

[0111] Example 4: In this example, a transmission electron microscope (JEOL, Japan) and a fluorescence microscope (EVOS) from Thermo Fisher Scientific were used. TM The cytotoxicity of PdTPTBP-CouN(NO)-PEG, PdTPTBP, and CouN(NO)-NO2 was detected using an M5000 microplate reader and a BioTek Synergy microplate reader.

[0112] Healthy 4T1 cells were cultured in 10cm culture dishes. When the cell density reached 90%, the culture medium was discarded, and medium containing PdTPTBP-Cou(NO)-PEG was added, followed by incubation for 4 hours. After incubation, cells were collected and fixed overnight with pre-cooled 2.5% glutaraldehyde aqueous solution, then fixed with 1% osmium tetroxide solution at room temperature for 1.5 hours. The fixed cells were then subjected to a gradient dehydration process with ethanol, followed by acetone replacement, and then progressive infiltration with acetone-diluted resin, embedded in epoxy resin, and polymerized and cured. Nanoscale ultrathin sections were prepared using an ultramicrotome, placed on copper grids, stained with saturated uranium acetate and lead citrate, and observed under a transmission electron microscope for cell ultrastructure. Figure 17As can be seen, a large number of uniformly sized and evenly distributed nanoparticles were clearly observed in 4T1 cells co-incubated with the probe PdTPTBP-Cou(NO)-PEG, and their morphology was highly consistent with the self-assembled assemblies obtained in vitro by MMP-2 digestion. This result directly confirms at the cellular level that the probe PdTPTBP-Cou(NO)-PEG can respond to MMP-2 and complete in situ self-assembly, providing key experimental evidence for extending probe retention time based on enzyme-responsive morphological changes.

[0113] The toxicity of PdTPTBP-CouN(NO)-PEG, PdTPTBP, and CouN(NO)-NO2 to 4T1 cells was determined using the MTT assay. 4T1 cells were seeded in 96-well plates and cultured overnight. The experiment included light-treated and dark-treated groups. First, the original culture medium was discarded and replaced with medium containing different concentrations (1.25, 2.5, 5, and 10 μM) of the test compounds. Then, the dark-treated groups were cultured directly for 12 h; the light-treated groups, after changing the culture medium, were irradiated with 630 nm red light for 30 min, and then cultured for another 12 h. After culture, MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 h. Then, dimethyl sulfoxide (DMSO) was added to dissolve the generated formazan crystals, and the absorbance at 490 nm was measured using a microplate reader to calculate the cell viability of each group. Figure 18 The results showed that under no light conditions, the cell survival rate of all three compounds was higher than 90% after treatment at a concentration of 10 µM for 12 h, indicating that each compound had low dark toxicity. Figure 19 The results showed that after irradiation with 630nm red light, the cell viability of the Coun(NO)-NO2 group remained above 90%, while the cell viability of the PdTPTBP-Coun(NO)-PEG group was significantly lower than that of the PdTPTBP group at the same concentration. This indicates that the photoinduced NO and ROS in the former further generated more toxic ONOO. - This enhances phototoxicity.

[0114] Simultaneously, this embodiment also used the Calcein AM / PI dual fluorescence staining method to evaluate the cytotoxic effects of PdTPTBP-CouN(NO)-PEG, PdTPTBP, and CouN(NO)-NO2 on 4T1 cells. 4T1 cells were seeded in 12-well plates and cultured overnight. After discarding the original culture medium, culture medium containing 10 μM of the test compound was added, and incubation was continued for 12 h. The experiment included a light-treated group and a dark-treated group. After incubation in the dark-treated group, the culture medium was replaced with fresh medium. In the light-treated group, after replacing the culture medium, the cells were immediately irradiated with 630 nm red light for 30 min, and then incubated for another 12 h. After incubation, the supernatant was discarded, and Calcein AM / PI detection working solution was added. Incubation was continued for 30 min, followed by live-cell imaging using a fluorescence microscope to observe the distribution of live cells (green fluorescence) and dead cells (red fluorescence). Figure 20 The Calcein AM / PI live / dead cell staining results showed that the probe achieves simultaneous release of NO and ROS through light-mediated transmission. NO can be used as a sensitizer for photodynamic therapy to improve the therapeutic effect.

[0115] Example 5: The in vivo antitumor effect of the probe PdTPTBP-CouN(NO)-PEG was verified by constructing a mouse orthotopic breast cancer model.

[0116] The tumor volume of the 4T1 tumor-bearing mice was increased to approximately 70 mm. 3 At that time, the animals were randomly divided into six groups, with 4 animals in each group: PdTPTBP-Cou(NO)-PEG+light group: PdTPTBP-Cou(NO)-PEG (200μM, 200μL) was injected into the tail vein, and laser irradiation (630nm, 35mW / cm²) was performed 2 hours after administration. 2 The following groups were used to evaluate the in vivo antitumor effects of the compound under light and no-light conditions: PdTPTBP-Cou(NO)-PEG group: same dose of the compound injected via tail vein, no light exposure; PdTPTBP+light group: PdTPTBP (200 μM, 200 μL) injected via tail vein, followed by light exposure under the same conditions 2 hours after administration; PdTPTBP group: same dose of PdTPTBP injected via tail vein, no light exposure; PBS+light group: equal volume of PBS buffer injected via tail vein, followed by light exposure 2 hours after administration; PBS group: equal volume of PBS injected via tail vein, no light exposure. These groups were used to evaluate the in vivo antitumor effects of the compound under light and no-light conditions. Treatment results are as follows: Figure 21 As shown, compared with the control groups, the experimental group (PdTPTBP-Cou(NO)-PEG+light group) had the flattest tumor growth curve, and the average tumor volume and tumor weight at the treatment endpoint were significantly smaller than those of the other groups. Figure 21The hematoxylin and eosin (H&E) staining results of tumor tissues from each group at the treatment endpoint showed that large areas of typical apoptosis and necrosis were visible in the tumor sections of the experimental group, while the tumor tissues of the control groups showed relatively intact cell morphology and no obvious apoptosis or necrosis. These in vivo antitumor effects highlight the important role of the probe's enzyme-responsive in-situ self-assembly and NO release design in enhancing the efficacy of photodynamic therapy. Figure 22 The results showed no significant difference in body weight changes among the groups of mice, indicating that the probe has good biosafety. Figure 23 This indicates that no significant damage occurred to the major organs of the mice after treatment. Analysis of the mouse blood further supports this finding. Figure 24 and Figure 25 The probe demonstrates good biocompatibility and in vivo tolerance at the system level. Its superiority compared to single photodynamic therapy can be attributed to the following three synergistic mechanisms: (1) enhanced tumor penetration through in-situ self-assembly; (2) sensitizing effect of NO gas; and (3) ONOO. - The probe exhibits high toxicity. The integration of its triple action ultimately achieves highly effective synergistic treatment of breast cancer, confirming the probe's effectiveness.

[0117] In summary, this invention presents an MMP-2-responsive in-situ self-assembled probe, PdTPTBP-CouN(NO)-PEG, designed to achieve NO gas-assisted photodynamic therapy for breast cancer, and its in vivo anti-tumor therapeutic effect was systematically evaluated. This probe can be specifically cleaved by MMP-2 in the tumor microenvironment and self-assemble in situ to form a nanostructure, achieving the synergistic release of ROS and NO under light irradiation. In vivo experimental results further demonstrate that PdTPTBP-CouN(NO)-PEG exhibits significantly superior anti-tumor effects compared to photodynamic therapy alone under light irradiation. Mechanistic studies show that this system not only enhances probe retention at the tumor site but also generates highly cytotoxic ONOO through the reaction of NO and ROS. - This allows for synergistic treatment with photodynamic therapy. Therefore, the combination of in-situ self-assembly with photodynamic therapy and light-triggered NO release demonstrates effective therapeutic efficacy and excellent biocompatibility, providing a promising strategy for cancer treatment.

[0118] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A self-assembled probe based on NO-synergistic photodynamic therapy, characterized in that, The device comprises a photosensitizer portion, a cleavable polypeptide portion that responds to proteases overexpressed in the tumor microenvironment, an NO gas donor portion, and a polymer portion for improving water solubility. The cleavable polypeptide portion, after being cleaved by the protease, drives in-situ self-assembly of the probe. Under light irradiation, the photosensitizer portion generates ROS, triggering the gas donor portion to release therapeutic NO gas, causing the ROS to react with the therapeutic NO gas to generate ONOO. - .

2. The self-assembled probe based on NO-synergistic photodynamic therapy according to claim 1, characterized in that, The protease is matrix metalloproteinase-2 (MMP-2).

3. The self-assembled probe based on NO-synergistic photodynamic therapy according to claim 1, characterized in that, The photosensitizer portion is palladium(II) tetraphenyltetrabenzoporphyrin; the cleavable polypeptide portion is PLGVR; the gas donor portion is a coumarin-based NO donor; and the polymer portion used to improve water solubility is a polyethylene glycol chain.

4. The self-assembled probe based on NO-synergistic photodynamic therapy according to claim 1, characterized in that, The structural formula of the self-assembled probe is as follows: 。 5. The method for preparing a self-assembled probe based on NO-synergistic photodynamic therapy as described in any one of claims 1-4, characterized in that, Includes the following steps: Precursor molecules containing cleavable polypeptide moieties and linkers were prepared by solid-phase synthesis. The precursor molecule is coupled with the NO gas donor portion to obtain the first intermediate; The photosensitizer portion is coupled to a linker molecule containing a cyclooctyne group to obtain a second intermediate; The first intermediate and the second intermediate are connected to obtain the probe precursor; The probe precursor was coupled with a polyethylene glycol chain to obtain a self-assembled probe.

6. The method for preparing a self-assembled probe based on NO-synergistic photodynamic therapy according to claim 5, characterized in that, Specifically, the steps include: Dithiopyridine was dissolved in methanol, and mercaptopropionic acid was added dropwise to react with it. The solvent was then removed to obtain an oily solid, which was purified to obtain compound A. N,N-dimethylformamide was added to the 2-chlorotriphenylmethyl chloride resin support for activation. Then, fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester and N,N-diisopropylethylamine, completely dissolved in N,N-dimethylformamide, were added to react. After the reaction was complete, methanol and N,N-dimethylformamide were added to seal the unreacted active sites on the resin. The reaction solution was squeezed out, and a solution of N,N-dimethylformamide containing piperidine was added to remove the amino protecting group Fmoc. The above operation was repeated, and the resin was washed several times with N,N-dimethylformamide. Subsequently, dissolved N-fluorenemethoxycarbonyl-glycine, 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were added to react. Following the above steps, Nα-fluorenemethoxycarbonyl-Nω-(2,2,4,6,7- The following compounds were used: pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine, N-fluorenmethoxycarbonyl-L-valine, N-fluorenmethoxycarbonyl-glycine, N-(9-fluorenmethoxycarbonyl)-L-leucine, N-(9-fluorenmethoxycarbonyl)-L-proline, N-fluorenmethoxycarbonyl-glycine, and compound A. After the reaction was complete, the peptides were washed and cleaved from the resin using a mixed solvent of trifluoroacetic acid and dichloromethane. The solvent was removed to obtain a yellow oily liquid. The peptides were cleaved from the resin again using the mixed solvent of trifluoroacetic acid and dichloromethane, repeated 3 to 5 times until the resin turned brown. All the cleaved liquid was collected and the solvent was removed to obtain a yellow oily liquid. Finally, the peptides were precipitated with diethyl ether. After centrifugation at freezing temperature, the supernatant was poured off, and the precipitate was allowed to dry naturally in a fume hood to obtain a powdery solid, which was named compound B. Compound B and azidopropylamine were dissolved using N,N-dimethylformamide, followed by the addition of 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine. The reaction system was adjusted to neutral and stirred. After purification, compound C was obtained. Compound C was dissolved in a dichloromethane solution of trifluoroacetic acid, then triisopropylsilane was added and stirred, and then purified to obtain compound D; Compound D was dissolved in anhydrous N,N-dimethylformamide, and then dissolved NO donor, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to it for reaction. After purification, compound E was obtained. The dibenzocyclooctyne-carboxyl group, 1-hydroxybenzotriazole and O-benzotriazole-tetramethylurea hexafluorophosphate were dissolved in N,N-dimethylformamide, and then 1,3-propanediamine was added to react with it. After purification, compound F was obtained. Palladium(II) tetraphenyltetrabenzoporphyrin and 4-nitrobenzoyl chloride were lyophilized, and then palladium(II) tetraphenyltetrabenzoporphyrin and N,N-diisopropylethylamine were dissolved in anhydrous dichloromethane. Then, 4-nitrobenzoyl chloride solution was added dropwise to the reaction system to carry out the reaction. After purification, compound G was obtained. Compounds G and F were lyophilized, and then compound G, compound F and N,N-diisopropylethylamine were dissolved in anhydrous tetrahydrofuran and reacted. After purification, compound H was obtained. Compound H was dissolved in dichloromethane and compound E was dissolved in N,N-dimethylformamide. The two solutions were then mixed and stirred. Subsequently, dichloromethane and N,N-dimethylformamide were removed sequentially, and the mixture was purified to obtain compound I. Compound I and mercapto-modified polyethylene glycol were dissolved in dichloromethane with N,N-dimethylformamide and reacted. Then, the N,N-dimethylformamide in dichloromethane was removed sequentially to obtain a bright green solid powder product. The product was dissolved in ultrapure water, centrifuged, and the supernatant was concentrated to obtain a self-assembled probe.

7. The use of the self-assembled probe based on NO synergistic photodynamic therapy as described in any one of claims 1-5 in the preparation of cancer therapeutic drugs.

8. The application according to claim 7, characterized in that, The cancer is a solid tumor that highly expresses MMP-2.

9. The application according to claim 8, characterized in that, The cancer in question is breast cancer.

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