Fluorescent nanoprobes for promoting photothermal therapy through specific recognition and photocontrolled synergistic activation of H2S scavenging, their preparation methods and applications.
Patent Information
- Application Number
- CN202311239671.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-25
- Publication Date
- 2026-09-25
- Estimated Expiration
- 2043-09-25
Smart Images
Figure CN117338924B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biochemistry and relates to novel organic nanomolecules that can be used to construct biological organisms for detecting, downregulating and clearing endogenous H2S using fluorescence imaging, while also possessing photothermal therapy capabilities. Specifically, it relates to a hydrogen sulfide-specifically activated small molecule fluorescent probe and its preparation method and application of fluorescent nanoparticles constructed by co-encapsulating it with a photothermal reagent. Background Technology
[0002] Hydrogen sulfide (H2S), as the third biosignaling molecule after nitric oxide (NO) and carbon monoxide (CO), easily penetrates cell membranes due to its small molecular weight. Therefore, it participates in many important physiological activities and is closely related to a range of diseases, including colorectal cancer, exhibiting endogenous H2S overexpression. Studies have shown that the physiological effects of H2S are actually bidirectional: physiological concentrations of H2S have anti-inflammatory, antioxidant, defensive, and immunomodulatory effects, while physiological concentrations of H2S are toxic and can induce apoptosis in cancer cells, which is beneficial for anti-cancer treatment. In light of this, gas therapy has constructed intelligent H2S-releasing molecules or nanoparticles to obtain high doses of H2S in tumor cells, producing anti-cancer effects. To date, numerous papers on H2S-related gas therapy have successfully achieved H2S release in response to endogenous or exogenous stimuli for "green" cancer treatment, but issues such as controlling release efficiency and biosafety remain. It is worth noting that endogenous hydrogen sulfide can promote the proliferation of colon cancer cells and angiogenesis in the tissue surrounding the tumor, while the removal of endogenous hydrogen sulfide can inhibit tumor proliferation and migration. When applied to gas therapy, it can also achieve the therapeutic effect of "stimulus-responsive H2S depletion" in tumor cells.
[0003] Photothermal therapy (PTT) typically uses photothermal agents to convert light energy into heat under laser irradiation, thereby inducing the thermal ablation of tumor cells. PTT has advantages such as being non-invasive, having few toxic side effects, being simple to operate, and being low in cost, enabling truly efficient tumor treatment. However, because photothermal agents are inevitably susceptible to mistargeting effects and adverse effects, they can cause unexpected damage to normal processes during PTT, thus limiting its application. Nevertheless, the organic combination of photothermal therapy, photodynamic therapy, magnetic resonance imaging, photoacoustic imaging, and other various diagnostic and treatment methods can compensate for the shortcomings of a single modality and has attracted widespread attention.
[0004] Here, we propose a novel strategy for constructing the BOD-AANP nanoplatform, which can scavenge endogenous H2S through synergistic activation of H2S and photocontrolled near-infrared radiation, thereby improving the diagnostic and therapeutic effects of photothermal therapy. The designed small-molecule probe BOD-AA exhibits an accurate and sensitive response to endogenous H2S, simultaneously releasing AOAA (a hydrogen sulfide scavenger) to clear H2S from tumor cells during imaging. AZA-BOD-2, a previously reported aza-BODIPY from our group, possesses significant near-infrared absorption and photothermal effects. By co-encapsulating BOD-AA and AZA-BOD-2 into thermoresponsive nanoparticles, we prepared the BOD-AANP nanoplatform, which can precisely locate tumor cells and enhance the probe's real-time detection capability. Notably, BOD-AANPs can release AOAA to downregulate endogenous H2S, thereby amplifying the diagnostic and therapeutic effects of photothermal therapy on tumor cells and successfully inhibiting tumor cell migration and proliferation, providing more insights for the promotion of gas therapy nanoplatforms. Summary of the Invention
[0005] The first objective of this invention is to provide a novel fluorescent probe, BOD-AA, that detects endogenous H2S and simultaneously releases AOAA to downregulate and eliminate endogenous H2S.
[0006] The second objective of this invention is to provide a method for preparing a novel fluorescent probe, BOD-AA, that detects endogenous H2S and simultaneously releases AOAA to downregulate endogenous H2S.
[0007] The third objective of this invention is to provide fluorescent nanomolecules BOD-AANPs that are specifically activated by hydrogen sulfide and NIR light-controlled synergistic activation for hydrogen sulfide scavenging and photothermal therapy amplification.
[0008] The fourth objective of this invention is to provide a method for constructing fluorescent nanomolecules BOD-AANPs that are specifically activated by hydrogen sulfide and NIR light-controlled synergistic activation for hydrogen sulfide scavenging and photothermal therapy amplification.
[0009] The fifth objective of this invention is to provide a novel fluorescent probe, BOD-AA, which detects endogenous H2S and simultaneously releases AOAA to downregulate endogenous H2S, for use in human colon cancer HCT-116 cells in vitro, utilizing near-infrared fluorescence analysis.
[0010] The sixth objective of this invention is to provide an application of near-infrared fluorescence analysis to a method for the in vitro application of hydrogen sulfide-specific and NIR-controlled synergistic activation of hydrogen sulfide downregulation and photothermal therapy-amplified fluorescent nanomolecules BOD-AANPs on endogenous hydrogen sulfide in human colon cancer HCT-116 cells.
[0011] The seventh objective of this invention is to provide an in vivo application of fluorescent nanomolecules BOD-AANPs that utilize near-infrared fluorescence analysis to achieve hydrogen sulfide-specific and NIR-controlled synergistic activation of hydrogen sulfide downregulation and photothermal therapy amplification.
[0012] Technical solution of the present invention:
[0013] A fluorescent nanoprobe (i.e., a fluorescent nanomolecule with hydrogen sulfide-specific and NIR-controlled synergistic activation for H2S scavenging and photothermal amplification) BOD-AANPs for promoting photothermal therapy, characterized by specific recognition and photocontrolled synergistic activation of hydrogen sulfide scavenging, is described. The nanoprobe structure comprises a small molecule fluorescent probe, BOD-AA, integrating hydrogen sulfide activation and scavenging, and a photothermal reagent, AZA-BOD-2, encapsulated in a PCM container composed of lauric acid, DSPE-mPEG-2000, and lecithin. The structures of BOD-AA and AZA-BOD-2 are shown in Formula I.
[0014]
[0015] The phase change material involved in this invention is composed of lauric acid, DSPE-mPEG-2000 and lecithin, with a specific weight ratio of lecithin:DSPE-mPEG-2000:lauric acid = 7.5:22.5:2.4.
[0016] Phase change materials composed of lauric acid are binary eutectic systems that lower the melting point of the material and ensure stable performance. They can be melted at around 44°C, thus ensuring that the outer shell of the nanoparticles melts and releases the small molecules inside during fluorescence-guided photothermal therapy.
[0017] The novel fluorescent probe BOD-AA, which integrates hydrogen sulfide activation and scavenging, can activate fluorescence at 709 nm upon H2S activation and simultaneously release AOAA, thereby downregulating endogenous H2S.
[0018] The novel fluorescent probe BOD-AA, which integrates hydrogen sulfide activation and clearance, as described in this invention, releases AOAA upon activation by hydrogen sulfide, which can downregulate hydrogen sulfide to inhibit tumor cell migration.
[0019] The fluorescent nanoprobes BOD-AANPs described in this invention, which specifically recognize and photo-controlled synergistically activate H2S scavenging to promote photothermal therapy, are obtained by encapsulating BOD-AA with the photothermal reagent AZA-BOD-2 using a phase change material. Guided by fluorescence imaging based on the response of BOD-AA to endogenous hydrogen sulfide, fluorescence at 702 nm is activated upon H2S activation, allowing for accurate localization and thus improving the probe's real-time high-fidelity imaging capability for H2S. Simultaneously, AOAA released by BOD-AA upon hydrogen sulfide activation can downregulate hydrogen sulfide.
[0020] Furthermore, the particle size of the nanospheres is 40–45 nm.
[0021] The BOD-AANPs described in this invention, while providing photothermal therapy, release AOAAs from their BOD-AA components under hydrogen sulfide activation, which can downregulate hydrogen sulfide, complementing photothermal therapy and amplifying the therapeutic effect.
[0022] The AOAA released by the BOD-AANPs nanoparticles described in this invention under hydrogen sulfide activation can also be combined with AZA-BOD-2 for photothermal therapy to amplify the therapeutic effect, providing a new approach for future integrated diagnosis and treatment.
[0023] The preparation method of the novel fluorescent probe BOD-AA, which can be used for both hydrogen sulfide activation and downregulation scavenging, as described in this invention, is as follows:
[0024]
[0025] This invention discloses a fluorescent nanomolecule, BOD-AANPs, which is specifically activated by hydrogen sulfide and NIR light-controlled synergistic activation for hydrogen sulfide scavenging and photothermal therapy amplification. The nanoprecipitation technique is used to prepare a PCM container composed of lauric acid, DSPE-mPEG-2000, and lecithin for encapsulation. The preparation method involves dissolving lecithin in an ethanol-water solution, adding DSPE-mPEG-2000, sonicating the mixture until clear and transparent, and then heating and stirring. A DMSO solution of lauric acid is prepared, and BOD-AA and AZA-BOD-2 are dissolved separately in DMSO and added to the lauric acid solution. This solution is then added to a preheated phospholipid aqueous solution, stirred at room temperature, transferred to an ice bath and stirred again, and then stirred at room temperature. Finally, the mixture is filtered through a PVDF membrane into a dialysis membrane with a molecular weight of 1000 and dialyzed with deionized water. BOD-AANPs are obtained after filtration through the PVDF membrane.
[0026] The more specific preparation method is as follows: First, lecithin (7.5 mg) was dissolved in 3 mL of 4% ethanol aqueous solution using ultrasound. Then, it was added to a bottle containing DSPE-mPEG-2000 (22.5 mg) for ultrasound. After the mixture was sonicated until clear and transparent, it was heated to 50°C and stirred for 10 minutes. Simultaneously, a DMSO solution of lauric acid (4 mg / mL) was prepared by dissolving BOD-AA (0.7 mg) and AZA-BOD-2 (0.5 mg) separately in 100 μL of DMSO and adding it to 600 μL of lauric acid solution. Next, it was added to the preheated lecithin aqueous solution and stirred at room temperature for 2 minutes. Then, it was transferred to an ice bath and stirred for another 2 minutes, followed by stirring at room temperature for 10 minutes. Afterward, the mixture was filtered through a PVDF membrane (0.45 μm) into a dialysis membrane with a molecular weight of 1000 and dialyzed with deionized water for 8 hours. Finally, BOD-AANPs were obtained by filtration through a PVDF membrane (0.22 μm) for further research.
[0027] The originality of this invention lies in the joint construction of a novel fluorescent probe BOD-AA that integrates hydrogen sulfide activation and scavenging, and a photothermal reagent AZA-BOD-2, to create a fluorescent nanomolecule BOD-AANPs that is synergistically activated by hydrogen sulfide specificity and NIR light control for hydrogen sulfide scavenging and photothermal therapy amplification. This invention employs a novel gas therapy method of gas scavenging to downregulate and scaveng endogenous H2S, thereby amplifying the application of photothermal diagnostic and therapeutic effects.
[0028] The present invention also provides a fluorescent nanoprobe that specifically recognizes and photo-controlled synergistically activated H2S scavenging to promote photothermal therapy, and its application in monitoring endogenous H2S and releasing AOAA to downregulate endogenous H2S.
[0029] The present invention also provides the application of the fluorescent probe BOD-AA, which integrates hydrogen sulfide activation and clearance, in the in vitro detection of endogenous hydrogen sulfide in human colon cancer HCT-116 cells.
[0030] This invention also provides the application of hydrogen sulfide-specific and NIR-controlled synergistic activation of hydrogen sulfide downregulation and photothermal therapy-amplified fluorescent nanomolecules BOD-AANPs in the in vitro human colon cancer HCT-116 cells for endogenous hydrogen sulfide.
[0031] This invention also provides an application of BOD-AANPs, fluorescent nanomolecules amplified by photothermal therapy, in vivo, through synergistic activation of hydrogen sulfide specificity and NIR light control for hydrogen sulfide downregulation.
[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0033] This invention utilizes DSPE-mPEG-2000 and lecithin to encapsulate a novel fluorescent probe BOD-AA with hydrogen sulfide scavenging capabilities and a photothermal reagent AZA-BOD-2 to obtain BOD-AANPs. This allows for the determination of the optimal time for on-demand photoactivation, improving the precise targeting of treatment and enabling rapid detection of hydrogen sulfide in vitro and in vivo. Furthermore, the thermal decomposition of the nanoshell composed of DSPE-mPEG-2000 and lecithin ensures the release of AOAA during photothermal therapy, thereby guaranteeing that the effect of hydrogen sulfide scavenging on tumor treatment is fully realized.
[0034] The probe described in this invention possesses high-fidelity near-infrared fluorescence imaging performance. It can activate near-infrared fluorescence emission upon H2S emission while simultaneously releasing carboxymethoxyamine hemihydrochloride (AOAA) (a common H2S inhibitor), enabling a novel gas therapy method for the visual detection and clearance of endogenous H2S. This invention not only innovates the traditional gas release-based gas therapy approach by combining gas downregulation and clearance in its probe design, but also encapsulates the probe with a photothermal reagent, achieving for the first time a combination of hydrogen sulfide clearance and photothermal therapy. This has considerable application potential in fluorescence imaging in the fields of biochemistry and medicine, and the synthesis method is simple and relatively inexpensive. It is hoped that this invention will promote research progress on H2S in various physiological and pathological processes.
[0035] The fluorescent probes and nanomolecules described in this invention have good biocompatibility.
[0036] Beneficial technical effects of the present invention:
[0037] This invention utilizes DSPE-mPEG-2000 and lecithin to encapsulate a novel fluorescent probe BOD-AA with hydrogen sulfide scavenging capabilities and a photothermal reagent AZA-BOD-2. The process is simple, and the resulting nanomolecules exhibit good stability, enabling rapid detection of hydrogen sulfide in vitro and in vivo. Furthermore, the thermal decomposition of the nanoshell composed of DSPE-mPEG-2000 and lecithin ensures the release of AOAA during photothermal therapy, thereby maximizing the therapeutic effect of hydrogen sulfide downregulation on tumors. It has already been successfully applied to photothermal therapy in the HCT116 tumor mouse model. Attached image description:
[0038] Figure 1 The diagram shows the synthetic route for the fluorescent probe BOD-AA, as shown in Formula I.
[0039] Figure 2 The fluorescent probe BOD-AA shown in Formula I in CDCl3 1 HNMR spectrum.
[0040] Figure 3The fluorescent probe BOD-AA shown in Formula I in CDCl3 13 CNMR spectrum.
[0041] Figure 4 The high-resolution mass spectrometry diagram of the fluorescent probe BOD-AA shown in Formula I is shown.
[0042] Figure 5 The high-resolution mass spectrometry diagram shows the mechanism by which the fluorescent probe BOD-AA, as shown in Equation I, responds to endogenous hydrogen sulfide.
[0043] Figure 6 The mechanism by which the fluorescent probe BOD-AA, as shown in Formula I, detects endogenous hydrogen sulfide.
[0044] Figure 7 The graph shows the absorption change of the fluorescent probe BOD-AA (10 μM) shown in Formula I to H2S in a PBS / MeCN = 1:1 buffer system.
[0045] Figure 8 The fluorescence response of the fluorescent probe BOD-AA (10 μM) shown in Formula I to H2S in a PBS / MeCN = 1:1 buffer system is shown.
[0046] Figure 9 The fluorescent probe BOD-AA shown in Formula I is used to demonstrate the cytotoxicity of HCT-116 cells in vitro.
[0047] Figure 10 This is a schematic diagram showing the fluorescence imaging capability of the fluorescent probe BOD-AA (as shown in Formula I) in HCT-116 cells at different reaction times in vitro.
[0048] Figure 11 This is a schematic diagram illustrating the imaging ability of the fluorescent probe BOD-AA (as shown in Formula I) against endogenous and exogenous H2S in HCT-116 cells in vitro.
[0049] Figure 12 The image shows a comparison of the fluorescence intensity of the fluorescent probe BOD-AA (as shown in Formula I) against endogenous and exogenous H2S in HCT-116 cells in vitro.
[0050] Figure 13 The figure shows the effect of the fluorescent probe BOD-AA (as shown in Formula I) on the migration ability of HCT-116 cells in vitro.
[0051] Figure 14 The image shows a comparison of the fluorescence effects of the fluorescent probe BOD-AA (Formula I) in human colon cancer HCT-116 cells in vitro, which inhibits and downregulates H2S.
[0052] Figure 15 This is a schematic diagram of the DLS of the nanomolecule BOD-AANPs shown in Formula I.
[0053] Figure 16 The graph shows the absorption change of H2S by the nanomolecules BOD-AANPs shown in Formula I in a pure PBS buffer system.
[0054] Figure 17 The fluorescence change of the nanomolecules BOD-AANPs (as shown in Formula I) in a pure PBS buffer system to H2S is illustrated.
[0055] Figure 18 The photothermal heating curves of the nanomolecules BOD-AANPs shown in Formula I in a pure PBS buffer system at different probe concentrations are shown.
[0056] Figure 19 The figure shows the temperature curves of the nanomolecules BOD-AANPs (as shown in Formula I) after three cycles in a pure PBS buffer system.
[0057] Figure 20 The nanomolecules BOD-AANPs shown in Formula I and their comparative nanomolecules AZA-NPs (containing only the photothermal reagent BOD-AZA-2) were tested at a power of 2Wcm. -2 Cytotoxicity of 785nm laser irradiation on HCT-116 cells in vitro after ten minutes.
[0058] Figure 21 This is a schematic diagram of the PI / AM of the nanomolecule BOD-AANPs shown in Formula I.
[0059] Figure 22 The image shows the fluorescence imaging effect of BOD-AANPs nanomolecules (Formula I) injected in situ into a mouse tumor site and interacting with endogenous hydrogen sulfide.
[0060] Figure 23 The image shows the photothermal heating effect of the nanomolecules BOD-AANPs shown in Formula I in an animal.
[0061] Figure 24 The image shows the effect of the nanomolecules BOD-AANPs shown in Formula I on one cycle of animal treatment.
[0062] Figure 25 This is a schematic diagram illustrating the composition of the nanomolecule BOD-AANPs and its mechanisms of biological application.
[0063] Figure 26 The photothermal reagent AZA-BOD-2 shown in Formula I in DMSO-d6 1 HNMR spectrum.
[0064] Figure 27 The photothermal reagent AZA-BOD-2 shown in Formula I in DMSO-d6 13 CNMR spectrum.
[0065] Figure 28 The high-resolution mass spectrometry diagram is a proof of the photothermal reagent AZA-BOD-2 shown in Formula I.
[0066] Figure 29 The power of the photothermal reagent AZA-BOD-2 shown in Formula I is 1.3 W / cm² under a 785 nm laser. 2 The graph below shows the temperature variation caused by light and heat. Detailed Implementation
[0067] The present invention will be further described below with reference to embodiments. Those skilled in the art should understand that the embodiments are for illustrative purposes only and do not constitute any limitation on the present invention.
[0068] Synthesis of Compound 2
[0069] 200 mg (0.36 mmol) of compound 1 and 70 mg (0.18 mmol) of 2-pyridylacetonitrile (purchased from a commercial company, requiring no further purification) were dissolved in 20 mL of anhydrous ethanol. The resulting mixture was then refluxed at 95 °C for 10 hours. After completion, the solvent was removed, and the crude product was purified by silica gel rapid chromatography to give compound 2 (45 mg, 30%). (The synthesis of compound 1 is based on the patent - Zhao Chunchang, Zhang Xiuli, Zhang Lili, Wang Feiyi, Jiang Haifeng, An Jiancai, Zhang Fan, A fluorescent probe for detecting endogenous H2S and its preparation method and application, China: Patent No.: ZL 201410766204.6, 2015-03-11, Certificate No.: 2250092)
[0070] Synthesis of Compound 3
[0071] 100 mg (0.26 mmol) of compound 2 and 50 mg (0.36 mmol) of 4-mercaptobenzyl alcohol (purchased from a commercial company, requiring no further purification), and 40 mg (0.33 mmol) of DMAP were dissolved in 10 mL of acetonitrile under a nitrogen atmosphere, stirred at room temperature for 30 minutes, and then the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography to obtain compound 3 (85 mg, 85%).
[0072] Synthesis of Compound 4
[0073] 100 mg (0.20 mmol) of compound 3 was dissolved in 20 mL of LDCM in an ice bath under a nitrogen atmosphere to maintain the temperature below zero. 50 mg (0.29 mmol) of 3-chlorobenzoic acid (purchased from a commercial company, requiring no further purification) was added to the system, and the mixture was stirred for 45 minutes. Then, a saturated sodium carbonate solution was added to the reaction mixture in an ice bath, followed by bringing it to room temperature and stirring for 30 minutes. After completion, the organic layer was separated, dried over Na2SO4, and removed by rotary evaporation. The crude product was purified by silica gel column chromatography to give compound 4 (89 mg, 89%).
[0074] Synthesis of Compound 5
[0075] 100 mg (0.16 mmol) of compound 4 and 39.9 mg (0.2 mmol) of 4-nitrophenyl chloroformate (purchased from a commercial company, requiring no further purification) were dissolved in 20 mL of anhydrous CH2Cl2 and stirred in an ice bath for 5 minutes. Then, 33 mg (0.27 mmol) of DMAP was added, and the reaction mixture was removed to room temperature and stirred for 2 hours. After completion, the organic solvent was rapidly purified by silica gel chromatography using a PE-EA mixed solvent (PE:EA = 2:1) to obtain the crude product compound 5, which was used in the next reaction without further purification.
[0076] Synthesis of BOD-AA
[0077] In an ice bath, 391 mg (3.6 mmol) of carboxymethoxyamine hemihydrochloride (purchased from a commercial company, requiring no further purification) was dissolved in 15 mL of 1,4-dioxane. 178 mg (1.4 mmol) of DMAP was added, followed by 10 mL of a 1,4-dioxane solution containing 230 mg (0.30 mmol) of compound 5. The reaction mixture was then transferred to room temperature. After stirring for approximately 30 minutes, the reaction was almost complete. The mixture was then washed with water, extracted with CH₂Cl₂, and dried over Na₂SO₄. The organic solvent was removed, and the crude product was purified by rapid silica gel chromatography to give BOD-AA (103 mg, 48%). See [link to article]. Figure 2 , 3 and 4; 1HNMR (400MHz, CDCl3, ppm): δ8.80(s,1H),8.59-8.60(d,1H),8.83-8.82(d,2H),7.67-7.65(t,1H),7.46-7.45(m,4H),7.33-7.28(m,5H) ,7.10(s,1H),7.02-7.00(m,1H),5.15-5.06(q,2H),4.39-4.38(d,2H),2.67(s,3H),2.37-2.33(q,2H),1.46(s,3H),1.02-0.99(t,3H).
[0078] 13 CNMR (151MHz, CDCl3, ppm): δ169.67,151.30,149.72,145.32,144.58,139 .34,138.94,137.13,136.19,135.20,132.52,130.20,130.04,124.95,12 4.48,124.38,123.99,123.49,123.14,122.19,120.37,119.11,119.01,1 18.88,117.84,114.04,109.14,71.88,66.82,17.22,14.13,13.91,12.74.
[0079] HRMS(ESI,m / z):calculated for C50H38BF2N4O6S[M+H] + :724.2213,found:724.2221.
[0080] Synthesis of AZA-BOD-2
[0081] The synthetic route and method for compound AZA-BOD-2 can be found in the article: Zhao Chunchang, Sun Jie, Cheng Ning, Yin Kai, Wang Rongchen, Zhu Tianli, Gao Jinzhu, Dong Xuemei, Dong Chengjun, Gu Xianfeng. Activatable photothermal agents with target-initiated large spectral separation for highly effective reduction ofside effect. DOI: Chem. Sci., 2022, 13, 9525-9530.
[0082] 1HNMR (600MHz, DMSO-d6, ppm): δ8.30(d,2H),8.20(d,2H),8.11(d,2H),8.00(d,2H),7.80(s,1H),7.21(m,8H),7.16(d,J=8.8Hz,0H) ,7.08(s,1H),6.91(d,2H),4.22(m,6H),3.79(m,6H),3.61(m,6H),3.56(m,6H),3.52(m,6H),3.44(m,6H),3.24(d,9H),3.15(s,6H). 13 C NMR (101MHz, CDCl3): δ159.91,159.19,153.10,152.36,146.48,143.43,142.96,138.95,132.34,130.91 ,125.40,118.05,114.55,111.79,77.18,71.89,70.65,67.46,59.06,40.11.HRMS(ESI,m / z)calculated for C 55 H 69 BF2N4O 12 [M+Na] + :1049.4871,found:1049.4862.
[0083] Preparation of BOD-AANPs:
[0084] The preparation method is as follows: First, lecithin (7.5 mg) was dissolved in 3 mL of 4% ethanol aqueous solution using ultrasound. Then, it was added to a bottle containing DSPE-mPEG-2000 (22.5 mg) for ultrasound. After the mixture was sonicated until clear and transparent, it was heated to 50°C and stirred for 10 minutes. Simultaneously, a DMSO solution of lauric acid (4 mg / mL) was prepared by dissolving BOD-AA (0.7 mg) and AZA-BOD-2 (0.5 mg) separately in 100 μL of DMSO and adding it to 600 μL of lauric acid solution. Next, it was added to the preheated lecithin aqueous solution and stirred at room temperature for 2 minutes. Then, it was transferred to an ice bath and stirred for another 2 minutes, followed by stirring at room temperature for 10 minutes. Afterward, the mixture was filtered through a PVDF membrane (0.45 μm) into a dialysis membrane with a molecular weight of 1000 and dialyzed with deionized water for 8 hours. Finally, BOD-AANPs were obtained after filtration through a PVDF membrane (0.22 μm).
[0085] Example 1: Figure 5The high-resolution mass spectrometry diagram shows the mechanism by which the fluorescent probe BOD-AA, as shown in Formula I, responds to endogenous hydrogen sulfide. 483.1655 (corresponding to [BOD-SH-H]+).
[0086] Example 2: Figure 6 The mechanism by which the fluorescent probe BOD-AA, shown in Formula I, detects endogenous hydrogen sulfide is explained. BOD-AA reacts with NaSH, and the Cl atoms on BOD-AA are replaced by -SH, resulting in a redshift in ultraviolet absorption and the generation of a fluorescent signal.
[0087] Example 3: BOD-AA (10 μM) was added to a cuvette containing 3 mL of PBS / MeCN = 1:1 (pH = 7.4) buffer solution, and 100 μM sodium hydrosulfide was added. The fluorescence and absorption curves over time were monitored. The excitation wavelength for fluorescence testing was 640 nm. Figure 7 , 8 As shown.
[0088] Figure 7 The graph shows the absorption changes of the fluorescent probe BOD-AA (10 μM) shown in Formula I in a PBS / MeCN = 1:1 buffer system for H2S. In the absence of H2S, it exhibits an absorption peak at 483 nm and an emission peak at 579 nm. Upon exposure to 100 μM NaHS, a new absorption band appears at 658 nm, while the original 483 nm band is attenuated, resulting in a significant redshift of 175 nm.
[0089] Figure 8 The fluorescence response of the fluorescent probe BOD-AA (10 μM) shown in Formula I to H2S in a PBS / MeCN = 1:1 buffer system is shown. When the excitation wavelength is 640 nm, strong near-infrared fluorescence appears at 709 nm, showing time-dependent fluorescence enhancement, while when excited at 450 nm, the emission peak at 579 nm shows a continuous decreasing trend.
[0090] Example 4: CCK-8 assay was performed to confirm the low cytotoxicity of BOD-AA in HCT116 cells. Figure 9 As shown, HCT116 cells were divided into different groups and incubated with different concentrations of BOD-AA (0-20 μM) for 24 h for CCK-8 assay to better demonstrate the biocompatibility of BOD-AA. Figure 9 The cytotoxicity of the fluorescent probe BOD-AA (Formula I) on HCT-116 cells in vitro was demonstrated. Cells incubated with different concentrations (0-20 μM) of BOD-AA for 24 h showed a survival rate exceeding 90%, indicating good cell compatibility with BOD-AA.
[0091] Example 5: Human colorectal cancer (HCT116) cells were cultured in Dulbecco Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under a humid atmosphere in a 37°C 5 / 95 CO2 / air incubator. After seeding in glass trays for adhesion for 24 hours, the following experiments were performed. Figure 10 , 11 As shown, to image endogenous H2S, HCT116 cells were incubated with BOD-AA for 15, 30, 60, 90, and 120 minutes, respectively. Furthermore, inhibitor and activator assays were developed to determine that cellular H2S is key to fluorescence activation. Figure 10 , 11 As shown: (1) HCT116 cells were treated with 1 mM aminooxyacetic acid (AOAA) for 1 h, and then incubated with BOD-AA (10 μM) for 1 h; (2) HCT116 cells were pretreated with S-adenosyl-L-methionine (SAM) (3 mM) for 1 h, and then stained with BOD-AA (10 μM) for 1 h; (3) HCT116 cells were treated with zinc chloride (ZnCl2) (500 μM) for 30 min, and then incubated with BOD-AA (10 μM) for 1 h. All confocal imaging was performed using a Leica TCS SP8 (63× oil lens). The excitation wavelength was 633 nm, and the emission range was 680-755 nm.
[0092] Figure 10 This is a schematic diagram illustrating the fluorescence imaging capability of the fluorescent probe BOD-AA (Formula I) in HCT-116 cells at different reaction times in vitro. As shown in the figure, a distinct red fluorescence appears and continuously increases with incubation time.
[0093] Figure 11 This is a schematic diagram illustrating the imaging capability of the fluorescent probe BOD-AA (Formula I) against endogenous and exogenous H2S in HCT-116 cells in vitro. It shows that BOD-AA can be activated by endogenous hydrogen sulfide within the cell to activate a near-infrared fluorescence signal.
[0094] Figure 12 This is a comparison of the fluorescence intensity of the fluorescent probe BOD-AA (Formula I) against endogenous and exogenous H2S in HCT-116 cells in vitro. It shows that BOD-AA can be activated by endogenous hydrogen sulfide within the cell to activate a near-infrared fluorescence signal.
[0095] Example 6: HCT116 cells were cultured in 6-well plates until they reached confluence, and then incubated for 2 hours with or without BOD-AA (20 μM). Scratches were then made using a toothpick. All cells were then cultured in FBS-free medium, and images of the scratches were taken at 0, 24, and 48 hours using an Olympus IX73 inverted microscope (10×). Results are shown below. Figure 13 As shown. Figure 13 The figure shows the effect of the fluorescent probe BOD-AA (Formula I) on the migration ability of HCT-116 cells in vitro. The scratch assay shows that BOD-AA treatment significantly inhibited the migration ability of HCT-116 cells, indicating that BOD-AA, through the release of AOAA, can effectively inhibit tumor cell migration by clearing intracellular hydrogen sulfide.
[0096] Example 7: To image intracellular H2S depletion in living cells, HCT116 cells were cultured and divided into two groups for experiments, such as... Figure 14 As shown: (1) Cells were incubated with BOD-AA (20 μM) for 1 hour, followed by further incubation with 7-azido-4-methylcoumarin (AzMC) (10 μM) for 30 minutes; (2) Cells were treated with AzMC (10 μM) alone for 30 minutes. Confocal imaging was performed. The excitation wavelength of AzMC was 365 nm, and the emission was read out at 450 nm.
[0097] Figure 14 This image shows a comparison of the fluorescence effects of the fluorescent probe BOD-AA (Formula I) in inhibiting and downregulating H2S in human colon cancer HCT-116 cells in vitro. This demonstrates that the fluorescent probe BOD-AA, as a source of AOAA release, can effectively downregulate intracellular hydrogen sulfide.
[0098] Example 8: (e.g.) Figure 15 As shown, BOD-AANPs (based on an absorbance of 10 μM BOD-AA content) were added to a plastic cuvette containing 3 mL of pure PBS (pH = 7.4) buffer solution, and DLS was detected using a dynamic light scattering instrument. Figure 15 This is a DLS schematic diagram of the BOD-AANPs nanomolecules shown in Formula I. It indicates that their particle size is approximately between 40 nm and 45 nm.
[0099] Example 9: (e.g.) Figure 16 , 17 As shown, BOD-AANPs (based on an absorbance of 10 μM BOD-AA content) were added to a cuvette containing 3 mL of pure PBS (pH = 7.4) buffer solution, and 100 μM sodium hydrosulfide was added. The fluorescence and absorption curves over time were monitored. The excitation wavelength for fluorescence testing was 640 nm.
[0100] Figure 16The graph shows the absorption variation of the nanomolecule BOD-AANPs (as shown in Formula I) in a pure PBS buffer system for H2S. In the absence of hydrogen sulfide, the probe exhibits absorption peaks at 518 nm and 770 nm. Upon addition of sodium hydrosulfide, an absorption peak at 672 nm appears that increases over time, while the absorption peak at 518 nm continuously decreases, resulting in a redshift at 154 nm. This phenomenon is consistent with the observation of BOD-AA in PBS / MeCN with the addition of hydrogen sulfide, indicating that hydrogen sulfide can freely diffuse into the interior of BOD-AANPs to achieve a response with BOD-AA.
[0101] Figure 17 The figure shows the fluorescence change of the nanomolecule BOD-AANPs (as shown in Formula I) in a pure PBS buffer system to H2S. When the excitation wavelength is 640 nm, the presence of sodium hydrosulfide causes a distinct emission peak at 702 nm in the probe. The fluorescence enhancement over time observed here is consistent with the effect of adding hydrogen sulfide to BOD-AA in PBS / MeCN.
[0102] Example 10: (e.g.) Figure 18 As shown in Figure 19, the photothermal correlation test involved placing different concentrations of BOD-AANPs under a laser with a wavelength of 785 nm and a power density of 2 W / cm². -2 Temperature changes were tested under the specified conditions.
[0103] Figure 18 The photothermal heating curves of the nanomolecules BOD-AANPs shown in Formula I in a pure PBS buffer system at different probe concentrations are shown below. (Note: Laser wavelength: 785 nm, power density: 2 W / cm²) -2 This indicates that the photothermal effect improves with increasing probe concentration.
[0104] Figure 19 The figure shows the temperature curves of the BOD-AANP nanomolecules shown in Formula I in a pure PBS buffer system after three cycles (Note: laser wavelength: 785 nm, power density: 2 W / cm²). -2 This indicates that the probe has a good recyclable photothermal effect.
[0105] Example 11: Constructing comparative fluorescent nanomolecules AZA-NPs, which only encapsulate the photothermal reagent AZA-BOD-2 (0.5 mg), using the same method as above.
[0106] First, a CCK-8 assay was performed to confirm the cytotoxicity of the nanomolecules in HCT116 cells. Figure 20As shown, HCT116 cells were divided into different groups and incubated with different concentrations of BOD-AANPs and AZA-NPs for CCK-8 assay to better demonstrate the synergistic therapeutic effect of BOD-AANPs: (1) treated with BOD-AANPs (0-20 μM) alone for 1 h; (2) treated with AZA-NPs (0-20 μM) alone for 1 h; (3) treated with BOD-AANPs (0-20 μM) and then irradiated with 785 nm light for 10 min; (4) incubated with AZA-NPs (0-20 μM) for 1 h and then irradiated with 785 nm light for 10 min. Then, all groups of cells were cultured for another 24 h to further determine cell viability using a microplate reader.
[0107] Figure 20 The nanomolecules BOD-AANPs shown in Formula I and their comparative nanomolecules AZA-NPs (containing only the photothermal reagent BOD-AZA-2) were tested at a power of 2Wcm. -2 The cytotoxicity of 785nm laser irradiation on HCT-116 cells in vitro after ten minutes indicates that the hydrogen sulfide downregulation and clearance capabilities of BOD-AANPs, along with their photothermal therapy capabilities, complement each other and contribute to the killing effect on tumor cells.
[0108] Example 12: To verify the therapeutic effect of BO-AANPs, HCT116 cells were cultured and divided into four groups. Staining experiments were performed using calcein acetoxymethyl ester / propidium iodide (calcein AM / PI). Figure 21 As shown: (1) Control; (2) Cells treated with 590nm light for 10 minutes only; (3) Cells incubated with BOD-AANPs for 1 hour without irradiation; (4) Cells incubated with BOD-AANPs for 1 hour, then irradiated with 590nm light for 10 minutes. After incubation for another 2 hours, Calcein AM (5μM) and PI (10μM) were added to the four groups of cells for staining for 20 minutes. Confocal imaging was performed. Calcein AM and PI were collected and emitted between 490-530nm and 590-630nm at excitation wavelengths of 488nm and 533nm, respectively. Figure 21 This is a schematic diagram of the PI / AM of the nanomolecule BOD-AANPs shown in Formula I. The hydrogen sulfide downregulation and scavenging capabilities of BOD-AANPs and their photothermal therapy capabilities complement each other, contributing to the killing effect on tumor cells.
[0109] Example 13: To evaluate the in vivo fluorescence imaging capability of BOD-AANPs for targeted cancer visualization, such as... Figure 22As shown, BOD-AANPs (30 nmol BSO–AA) were subcutaneously injected into the tumor region of HCT116 tumor-bearing mice. Fluorescence images were then acquired at designated time points after injection. Subsequently, to demonstrate that H2S is the activation of BOD-AANPs in HCT116 tumors, two additional groups of mice were used: HCT116 tumor-bearing mice were pretreated subcutaneously with either SAM (300 nmol) or AOAA (100 nmol), followed by a 4-hour rest period, and then injected with BOD-AANPs. Fluorescence images were then simultaneously captured using an IVIS spectral imaging system. The excitation wavelength was 640 nm, and fluorescence signals were collected between 700 nm and 720 nm.
[0110] Figure 22 This is a fluorescence imaging result of the interaction between the nanomolecules BOD-AANPs (Formula I) injected in situ into a mouse tumor site and endogenous hydrogen sulfide. This demonstrates that the probe, when injected in situ into a living tumor, can react with endogenous hydrogen sulfide to generate a fluorescence signal.
[0111] Example 14: To verify the in vivo thermal imaging capability of BOD-AANPs, HCT116 tumor-bearing mice were divided into 3 groups, such as... Figure 23 As shown: 1) Illumination using only 785nm light (1W / cm²) 2 1) Treat for 10 minutes; 2) Irradiate with BOD-AANPs (30 nmol AZA-BOD-2) and 785 nm light (1 W / cm²). 2 1) Treat for 10 minutes; 3) Irradiate with BOD-AANPs (30 nmol AZA-BOD-2) and 785 nm light (2 W / cm²). 2 The mice were treated for 10 minutes. 1.5 hours after injection of BOD-AANPs, the tumor site was irradiated with a 785nm laser for 10 minutes. During this irradiation, infrared temperature images of the mice were captured using a thermal imaging camera (Fotric225-4). Figure 23 The image shows the photothermal heating effect of the nanomolecules BOD-AANPs shown in Formula I in an animal body (Note: laser wavelength: 785nm, power density: 1Wcm). -2 This indicates that BOD-AANPs can achieve highly efficient photothermal therapy of tumors in mice.
[0112] Example 15: (e.g.) Figure 24As shown, HCT116 tumor-bearing mice were divided into five groups (≥4 mice per group): 1) treated with BOD-AANPs (30 nmol AZA-BOD-2) plus 785 nm light irradiation; 2) treated with AZA-NPs (30 nmol AZA-BOD-2) plus 785 nm light irradiation; 3) treated with BOD-AANPs (30 nmol AZA-BOD-2) without laser irradiation; 4) treated with 785 nm laser irradiation only; 5) control. 1.5 hours after BOD-AANPs injection, the tumor area was treated with 785 nm light (1 W / cm²). 2 Mice were treated with 10-minute irradiation. Tumor volume and body weight were recorded over the next 14 days following various drug administrations. Finally, major organs of all treated mice were stained with H&E to demonstrate the good biocompatibility of BODAANPs. Figure 24 The image shows the effect of the nanomolecule BOD-AANPs shown in Formula I on one cycle of animal treatment (Note: the probe is 30 nmol, the laser wavelength is 785 nm, and the power density is 1 W / cm²). -2 This study verified that BOD-AANPs have excellent therapeutic effects on tumors in in vivo experiments.
[0113] Example 16: Accurately weigh 5.0±0.1 mg of mPEG-DSPE2000 using a balance, add 2 mL of deionized water, and sonicate at 25°C for 3-5 min until clear and transparent. Transfer 55.5 μL of the prepared 10 mM AZA-BOD-2 concentrated solution to the PEG solution using a pipette, and sonicate at 25°C until clear. Then, transfer the sonicated probe solution to a 1000 molecular weight dialysis membrane and dialyze in deionized water for 6-8 h (replacing the deionized water every 2 h). Finally, compare the absorbance of the nanoprobe solution with that of the uncoated probe solution using a UV spectrophotometer to determine the final concentration of the dialyzed nanoprobe solution. Figure 29 As shown, the encapsulated AZA-BOD-2 was diluted to probe aqueous solutions at concentrations of (10 μM, 20 μM, and 30 μM), and 1 mL of each solution was added to a cuvette. The laser power was controlled at 1.3 W / cm². 2 After irradiation for 10 minutes, the photothermal temperature change curves of different concentrations of AZA-BOD-2 under laser were measured, proving that the photothermal reagent AZA-BOD-2 has good photothermal conversion ability.
Claims
1. A fluorescent nanoprobe for promoting photothermal therapy by specifically recognizing and photo-controlled synergistic activation of H2S scavenging, characterized in that, These are BOD-AANPs, obtained by encapsulating a fluorescent probe BOD-AA (which integrates hydrogen sulfide activation and scavenging) and a photothermal reagent AZA-BOD-2 with a phase change material. The structural formulas of BOD-AA and AZA-BOD-2 are shown below: BOD-AA AZA-BOD-2.
2. The fluorescent nanoprobe for promoting photothermal therapy based on the specific recognition and photocontrolled synergistic activation of H2S scavenging according to claim 1, characterized in that, The nanoprobe has a particle size of 40~45 nm.
3. A method for constructing a fluorescent nanoprobe for promoting photothermal therapy using H2S scavenging with specific recognition and photocontrolled synergistic activation as described in claim 1, characterized in that, Includes the following steps: Lecithin was dissolved in an ethanol-water solution, and then DSPE-mPEG-2000 was added. The mixture was sonicated until clear and transparent, then heated and stirred. A DMSO solution of lauric acid was prepared. BOD-AA and AZA-BOD-2 were dissolved in DMSO and added to the lauric acid solution. The solution was then added to a preheated phospholipid aqueous solution. After stirring at room temperature, the mixture was transferred to an ice bath and stirred again. The mixture was then stirred at room temperature. Finally, the mixture was filtered through a PVDF membrane into a dialysis membrane with a molecular weight of 1000 and dialyzed with deionized water. BOD-AANPs were obtained after filtration through the PVDF membrane.
4. The use of the fluorescent nanoprobe of claim 1 in the preparation of a drug for monitoring endogenous H2S and releasing aminooxyacetic acid (AOAA) to downregulate endogenous H2S.
5. The use of the fluorescent nanoprobe according to claim 1 in the preparation of a drug for downregulating and clearing endogenous H2S to amplify photothermal therapeutic effects.