Beta-galactosidase-activated senescent cardiomyocyte-targeting bioluminescent nanoprobes

Abstract

The application provides a kind of beta galactoside enzyme activated senescent myocardial cell targeted bioluminescence nano probe, comprising: core nanoparticle, and tannin acid coating layer coated on the surface of core nanoparticle;The core nanoparticle is co-assembled by fluorinated high molecular polymer carrier, beta galactoside enzyme responsive bioluminescence small molecule probe, firefly luciferase and adenosine triphosphate.This probe integrates "recognition-response-signal output" in one, can avoid gene transfection, realize luciferase and its substrate efficient, stable co-delivery, and can be specifically responded to senescent cell marker SA-beta-gal, so as to realize the new technology of high sensitivity, high specificity bioluminescence imaging of senescent cell on in vivo level.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical engineering and molecular imaging technology, specifically relating to a bioluminescent nanoprobe for detecting senescent cells, particularly a bioluminescent nanoprobe for targeting senescent cardiomyocytes based on bioluminescence-activated β-galactosidase, its preparation method, and its application in the preparation of imaging reagents for diagnosing or monitoring aging-related diseases (especially cardiomyocyte senescence after myocardial infarction). Background Technology

[0002] Cellular senescence is an irreversible state of cell cycle arrest that can be induced by various stressors, such as DNA damage, telomere dysfunction, oncogene activation, and oxidative stress. The accumulation of senescent cells in tissues is considered a key driver of aging and various age-related diseases. In the cardiovascular system, cardiomyocytes, as terminally differentiated cells, also undergo senescence during the aging process or under pathological stress (such as myocardial infarction and ischemia / reperfusion injury). Senescent cardiomyocytes not only suffer functional impairment but also promote inflammation and tissue remodeling by secreting senescence-associated secretory phenotype (SASP) factors, leading to ventricular remodeling and heart failure. Therefore, accurate and real-time detection and imaging of senescent cardiomyocytes at the in vivo level is crucial for understanding the pathophysiological mechanisms of cardiovascular aging, early diagnosis of age-related cardiovascular diseases, and evaluation of related treatment effects.

[0003] Currently, the detection of cellular senescence mainly relies on in vitro methods, such as detecting the activity of senescence-associated β-galactosidase, the expression of cell cycle inhibitors like p16 / p21, and DNA damage markers. Among these, senescence-associated β-galactosidase (SA-β-gal) is widely considered the "gold standard" biomarker for detecting cellular senescence due to its high specific expression in senescent cells. Based on the detection principle of SA-β-gal, various fluorescent probes have been developed. For example, X-Gal staining is a classic detection method, but it can only be used for fixed cells or tissue sections and cannot be used for in vivo imaging. In recent years, several small-molecule fluorescent probes capable of detecting SA-β-gal activity in living cells or in vivo have been reported. However, fluorescence imaging usually requires external light source excitation and has inherent limitations such as limited tissue penetration depth, autofluorescence interference, and photobleaching, which to some extent limit its application in deep tissue imaging.

[0004] Bioluminescence imaging is an optical imaging technique that does not require external light source excitation. Its principle is based on the oxidation of its substrate, D-luciferin, by luciferase in the presence of ATP, oxygen, and magnesium ions, thereby emitting visible light. Compared to fluorescence imaging, bioluminescence imaging has significant advantages such as high signal-to-noise ratio, absence of background autofluorescence interference, and high sensitivity, making it particularly suitable for long-term, dynamic monitoring of live small animals. However, traditional bioluminescence imaging relies on the stable transfection and expression of the luciferase gene in target cells or tissues. This process is complex, time-consuming, and carries risks of low transfection efficiency and potential gene integration. Furthermore, directly delivering the luciferase protein and its substrate to target tissues also faces challenges such as protein instability, susceptibility to degradation, and difficulty in simultaneous delivery.

[0005] The foregoing background information is intended to help those skilled in the art understand prior art that is similar to the present invention, and to facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clearly stated that, in the absence of clear evidence that the above content was disclosed before the filing date of this patent application, the foregoing background information should not be used to evaluate the novelty of the technical solution of this application. Summary of the Invention

[0006] Technical issues To address the aforementioned issues, the present invention aims to provide a β-galactosidase-activated aging-targeting bioluminescent nanoprobe, its preparation method, and its application. This probe integrates "recognition-response-signal output," enabling it to circumvent gene transfection, achieve efficient and stable co-delivery of luciferase and its substrates, and specifically respond to the aging cell marker SA-β-gal. This enables a novel technology for highly sensitive and specific bioluminescent imaging of aging cells at the in vivo level.

[0007] Technical solution The first aspect relates to the application of a β-galactosidase-responsive bioluminescent small molecule probe in the preparation of imaging reagents for monitoring senescent cardiomyocytes after myocardial infarction, wherein the β-galactosidase-responsive bioluminescent small molecule probe is a compound formed by covalently linking D-galactoside with D-fluorescein via a benzyl ether linker arm.

[0008] Preferably, the imaging reagent is used for specific bioluminescent imaging of senescent cardiomyocytes that highly express β-galactosidase in the myocardial infarction region.

[0009] Secondly, the preparation method of the β-galactosidase-responsive bioluminescent small molecule probe described in the first aspect includes the following steps: a) 2,3,4,6-tetra-O-acetyl-α-D-pyranogalactosylbromide was reacted with p-hydroxybenzaldehyde under phase transfer catalyst and alkaline conditions to give compound 1; b) Compound 1 was reacted with sodium borohydride to reduce the aldehyde group to a hydroxyl group, yielding compound 2; c) Reacting compound 2 with phosphorus tribromide converts the hydroxyl group into bromine, yielding compound 3; d) Compound 3 was reacted with 2-cyano-6-hydroxybenzothiazole under phase transfer catalyst and basic conditions to give compound 4; e) Compound 4 was cyclized with D-cysteine ​​under alkaline conditions to obtain compound 5; f) Compound 5 was deacetylated under alkaline conditions to obtain the target probe molecule Gal-luc.

[0010] Thirdly, a β-galactosidase-responsive bioluminescent nanocomposition comprises nanoparticles assembled from a fluorinated polymer carrier, a β-galactosidase-responsive bioluminescent small molecule probe prepared by the method described in the first aspect or the second aspect, firefly luciferase, and adenosine triphosphate.

[0011] Fourthly, a β-galactosidase-activated aging-targeting bioluminescent nanoprobe includes: Core nanoparticles, and A tannic acid coating on the surface of the core nanoparticles; The core nanoparticles are assembled from a fluorinated polymer carrier, a β-galactosidase-responsive bioluminescent small molecule probe prepared by the method described in the first aspect or the second aspect, firefly luciferase, and adenosine triphosphate.

[0012] Preferably, the fluorinated polymer carrier is fluoroalkyl-modified polyethyleneimine.

[0013] More preferably, the fluoroalkyl-modified polyethyleneimine is a branched polyethyleneimine modified with 3-(perfluorohexyl)-1,2-epoxypropane; its structure is that a side chain containing a perfluorohexyl group is attached to the nitrogen atom of the polyethyleneimine.

[0014] Preferably, in the fluorinated polymer carrier, the grafting rate of fluoroalkyl groups is 5-7%, that is, on average, each polyethyleneimine molecule is modified with 30-35 fluoroalkyl chains.

[0015] Preferably, the mass ratio of the fluorinated polymer carrier to the β-galactosidase-responsive bioluminescent small molecule probe and the firefly luciferase is 1:1-5:1-2, more preferably 1:3-5:1-2, and even more preferably 1:4:1.

[0016] Preferably, the particle size of the aging-targeting bioluminescent nanoprobe is 200-300 nm, and the zeta potential is -15 mV to -20 mV.

[0017] Fifthly, the preparation method of the β-galactosidase-activated aging-targeting bioluminescent nanoprobe described in the first aspect above includes the following steps: Synthesize a β-galactosidase-responsive bioluminescent small molecule probe, Gal-luc; Synthetic fluorinated polymer carrier F-PEI; The F-PEI was mixed with Gal-luc and firefly luciferase fLuc and incubated, and then adenosine triphosphate ATP solution was added to obtain a mixture of F-PEI / Gal-luc / fLuc / ATP core nanoparticles. Centrifuge the resulting mixture, resuspend the precipitate, and obtain purified F-PEI / Gal-luc / fLuc / ATP core nanoparticles; The obtained core nanoparticles were mixed with tannic acid solution and incubated. After centrifugation, the precipitate was resuspended to obtain the F-PEI / Gal-luc / fLuc / ATP@TA nanoprobe.

[0018] Preferably, the synthesis of the β-galactosidase-responsive bioluminescent small molecule probe Gal-luc includes the following steps: a) 2,3,4,6-tetra-O-acetyl-α-D-pyranogalactosylbromide was reacted with p-hydroxybenzaldehyde under phase transfer catalyst and alkaline conditions to give compound 1; b) Compound 1 was reacted with sodium borohydride to reduce the aldehyde group to a hydroxyl group, yielding compound 2; c) Reacting compound 2 with phosphorus tribromide converts the hydroxyl group into bromine, yielding compound 3; d) Compound 3 was reacted with 2-cyano-6-hydroxybenzothiazole under phase transfer catalyst and basic conditions to give compound 4; e) Compound 4 was cyclized with D-cysteine ​​under alkaline conditions to obtain compound 5; f) Compound 5 was deacetylated under alkaline conditions to obtain the target probe molecule Gal-luc.

[0019] Preferably, the synthesis steps of the fluorinated polymer carrier F-PEI are as follows: 3-(perfluorohexyl)-1,2-epoxypropane is added to an anhydrous methanol solution of branched polyethyleneimine, and the reaction is stirred at room temperature for 12-96 hours, preferably 24-72 hours, more preferably 48 hours. The reaction product is purified by dialysis and then freeze-dried to obtain F-PEI.

[0020] Preferably, when F-PEI is mixed with Gal-luc and firefly luciferase fLuc, the mass ratio is 1:1-5:1-2, more preferably 1:3-5:1-2, and even more preferably 1:4:1.

[0021] Preferably, the incubation conditions for the F-PEI mixed with Gal-luc and firefly luciferase fLuc are 1-90 min at room temperature.

[0022] Preferably, the amount of adenosine triphosphate ATP solution added is based on a weight ratio of ATP to F-PEI of 1:2-5, more preferably 1:4-5, and even more preferably 1:4.

[0023] Preferably, when the core nanoparticles are mixed and incubated with the tannic acid solution, the mass ratio of tannic acid to F-PEI is 1:2-5, preferably 1:4-5, and more preferably 1:4.

[0024] The sixth aspect is the application of the β-galactosidase-activated aging-targeting bioluminescent nanoprobes described in the fourth aspect above, or the aging-targeting bioluminescent nanoprobes prepared by the method described in the fifth aspect above, in the preparation of imaging reagents for diagnosing or monitoring aging-related diseases.

[0025] Preferably, the aging-related disease is myocardial cell aging after myocardial infarction.

[0026] More preferably, the application achieves specific bioluminescent imaging of senescent cardiomyocytes in the myocardial infarction region by detecting highly expressed β-galactosidase in senescent cells.

[0027] The seventh aspect is the application of the β-galactosidase-activated aging-targeting bioluminescent nanoprobes described in the fourth aspect above or the aging-targeting bioluminescent nanoprobes prepared by the method described in the fifth aspect above in cell imaging for non-diagnostic or therapeutic purposes, the application including enabling bioluminescent imaging of hypoxia-induced aging H9C2 cells.

[0028] The eighth aspect is the application of the β-galactosidase-activated aging-targeting bioluminescent nanoprobes described in the fourth aspect above or the aging-targeting bioluminescent nanoprobes prepared by the method described in the fifth aspect above in in vivo imaging for non-diagnostic or therapeutic purposes, the application including enabling bioluminescent imaging of senescent cells in a mouse myocardial infarction (MI) model.

[0029] Preferably, the mouse myocardial infarction (MI) model is constructed by ligating the left anterior descending coronary artery.

[0030] The ninth aspect is a bioluminescent imaging kit for senescent cells, characterized in that it comprises senescence-targeting bioluminescent nanoprobes activated by β-galactosidase as described in the fourth aspect or senescence-targeting bioluminescent nanoprobes prepared by the method described in the fifth aspect.

[0031] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined to obtain specific implementation methods.

[0032] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0033] Beneficial effects This invention innovatively designs and constructs a multifunctional nanoprobe integrating "recognition-response-signal output". This probe cleverly encapsulates the SA-β-gal-specifically recognized "smart switch" (Gal-luc probe) and the "signal source" (fLuc enzyme and ATP) required for bioluminescence in a highly efficient fluorinated polymer delivery carrier, achieving the "integrated" co-delivery of the three functional modules.

[0034] The fluorinated polymer carrier F-PEI, through its unique fluorine-fluorine interactions and excellent membrane permeability, can efficiently co-assemble negatively charged proteins (fLuc) and small molecules (Gal-luc, ATP) into stable nanoparticles and mediate their efficient entry into cells. Compared with traditional gene transfection methods, this invention eliminates the need for complex gene manipulation of cells, avoiding low transfection efficiency and potential gene integration risks. The method is simpler, safer, and more universal.

[0035] The probe in this invention exhibits a highly specific and sensitive response mechanism. Its core response unit, Gal-luc, is normally in an "off" state. Only when the nanoprobe is delivered to senescent cells does the highly expressed SA-β-gal specifically cleave the galactose residues on Gal-luc, releasing free D-luciferin substrate. The released D-luciferin then undergoes a classic bioluminescent reaction with co-delivered fLuc enzyme, ATP, and Mg²⁺ within the same cell, generating an "on" bioluminescent signal. This "lock-and-key" response mechanism ensures the uniqueness of the signal generation, significantly reduces background interference, and improves the signal-to-noise ratio and specificity of the detection.

[0036] This invention further coats the core nanoparticles with a tannic acid coating. Tannic acid, as a natural polyphenol compound, can not only enhance the structural stability of nanoparticles and prolong their cycling time in physiological environments through hydrogen bonding and hydrophobic interactions, but also reduce non-specific adsorption of nanoparticles and the formation of protein crowns through the negative charge on its surface, thereby improving the biocompatibility and in vivo application potential of the probe.

[0037] This invention successfully achieved in situ and in vivo imaging of senescent cardiomyocytes in a myocardial infarction model. Experimental results showed that, regardless of whether administered via in situ injection or intravenous systemic administration, the nanoprobe specifically accumulated in senescent cardiomyocytes in the infarct region, generating a strong bioluminescent signal, while no signal was generated in non-infarct regions or normal cardiac tissue. This demonstrates the probe's superior in vivo targeted imaging capability, providing a powerful visualization tool for studying the dynamic process of cellular senescence after myocardial infarction, evaluating the efficacy of anti-aging drugs, and developing new diagnostic methods.

[0038] The present invention adopts the above-mentioned technical solution to achieve the above objectives, which makes up for the shortcomings of the prior art, is reasonably designed, and is easy to operate. Attached Figure Description

[0039] To make the above and / or other objects, features, advantages and examples of the present invention more apparent and understandable, the accompanying drawings used in the specific embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 This is the synthetic route diagram of Gal-luc, a β-galactosidase-responsive bioluminescent small molecule probe in this invention.

[0041] Figure 2 This is a schematic diagram illustrating the preparation of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA in this invention.

[0042] Figure 3 The image shows the hydrogen NMR spectrum of the probe Gal-luc.

[0043] Figure 4 The diagram shows the enzyme release (A) and stability (B) of the probe Gal-luc.

[0044] Figure 5 This is the enzyme-responsive bioluminescence spectrum of the probe Gal-luc.

[0045] Figure 6 This is a graph showing the enzyme concentration dependence of the probe Gal-luc.

[0046] Figure 7 This is the hydrogen nuclear magnetic resonance spectrum of the polymer F-PEI in this invention.

[0047] Figure 8 Transmission electron microscopy image of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA.

[0048] Figure 9 Hydration particle size distribution (A) and Zeta potential (B) of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA.

[0049] Figure 10 The figure shows the stability test results of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA.

[0050] Figure 11 The results show the luminescence response (A) and enzyme concentration dependence (B) of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA to β-galactosidase.

[0051] Figure 12 Bioluminescence spectrum of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA anti-interference agent Figure 13 This diagram illustrates the survival rate of H9C2 cardiomyocytes after incubation with the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA.

[0052] Figure 14 This is a specific luminescence imaging image of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA on senescent H9C2 cardiomyocytes.

[0053] Figure 15 This is an in vivo bioluminescence imaging image of the infarcted heart after in situ administration of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA in a mouse model of myocardial infarction.

[0054] Figure 16 This is an in vivo bioluminescence imaging image of the infarcted heart after intravenous administration of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA in a mouse model of myocardial infarction. Detailed Implementation

[0055] Those skilled in the art can refer to the content of this document and appropriately replace and / or modify the process parameters to achieve the desired results. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in this invention. The products and preparation methods described in this invention have been described through preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0056] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. This invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patent cases, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail.

[0057] Unless otherwise stated, all percentages, parts, proportions, etc. are by weight; other statements include, but are not limited to, “%”, “wt%”, “mass%” meaning weight percentage, “mol%” meaning mole percentage, and “vol%” meaning volume percentage.

[0058] When quantities, concentrations, or other numerical values ​​or parameters are given as ranges, preferred ranges, or a series of upper and lower preferred values, it should be understood that they specifically disclose all ranges formed by any pair of values ​​of any larger or preferred range limit and any smaller or preferred range limit, regardless of whether the ranges are disclosed separately. For example, when describing a range of “1 to 5 (1-5)”, the described range should be understood to include ranges such as “1 to 4 (1-4)”, “1 to 3 (1-3)”, “1 to 2 (1-2)”, “1 to 2 (1-2) and 4 to 5 (4-5)”, “1 to 3 (1-3) and 5”, etc. Unless otherwise stated, wherever numerical ranges are described herein, the ranges include the range endpoints as well as all integers and fractions within that range.

[0059] Unless otherwise specified, the materials, methods, and examples described herein are exemplary and not limiting. While similar or equivalent methods and materials can be used to implement or test the invention, suitable methods and materials are described herein.

[0060] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, in the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0061] To facilitate understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention will first be explained or defined. For undefined abbreviations or key terms, they are all conventionally understood by those skilled in the art.

[0062] SA-β-gal: β-galactosidase, also known as β-galactosidase.

[0063] Gal-luc: β-galactosidase-responsive bioluminescent small molecule probe.

[0064] PEI: Branched polyethyleneimine.

[0065] F-PEI: Fluorinated polymer carrier.

[0066] fLuc: Firefly luciferase.

[0067] ATP: Adenosine triphosphate.

[0068] MI model: Myocardial infarction in mice.

[0069] TA: Tannic acid.

[0070] HPLC: High Performance Liquid Chromatography.

[0071] DLS: Dynamic Light Scattering Particle Size Analyzer.

[0072] TEM: Transmission electron microscope.

[0073] PBS: Phosphate buffer solution.

[0074] H9C2 cells: H9c2(2-1) rat cardiomyocytes, an immortalized cell line derived from the rat heart, are one of the most commonly used in vitro models in the field of cardiovascular research.

[0075] The present invention is described in detail below. Example 1: This paper provides the synthetic steps and in vitro performance verification of Gal-luc, a β-galactosidase-responsive bioluminescent small molecule probe. The specific synthetic route is as follows: Figure 1 As shown, the specific steps are as follows.

[0076] 1.1 Synthesis of Compound 1 5 g of 2,3,4,6-tetra-O-acetyl-α-D-pyranogalactosyl bromide (12.20 mmol) and 1.24 g of p-hydroxybenzaldehyde (10.17 mmol) were dissolved in 40 mL of dichloromethane. Then, 3.93 g of tetrabutylammonium bromide (12.20 mmol) and 10 mL of 5% sodium hydroxide aqueous solution were added. The mixture was stirred vigorously at room temperature for 4 hours. After the reaction was complete, the mixture was extracted three times with dichloromethane, and the organic phases were combined, washed successively with saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with a gradient of ethyl acetate / petroleum ether (1 / 10 to 1 / 1, v / v) to give 2.33 g of a pale yellow oily liquid.

[0077] 1.2 Synthesis of Compound 2 2.33 g of compound 1 (5.15 mmol) was dissolved in 20 mL of tetrahydrofuran and cooled to 0 °C in an ice bath. 0.19 g of sodium borohydride (5.15 mmol) was added in portions, and the mixture was stirred at 0 °C for 4 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove most of the tetrahydrofuran, and then quenched by slowly adding 20 mL of saturated ammonium chloride solution until no more bubbles were generated. The mixture was extracted three times with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated and purified by silica gel column chromatography using a gradient elution of ethyl acetate / petroleum ether (1 / 5 to 1 / 1, v / v) to give 1.87 g of a colorless, transparent oily liquid.

[0078] 1.3 Synthetic compound 3 1.87 g of compound 2 (4.12 mmol) was dissolved in 20 mL of dichloromethane and cooled to 0 °C in an ice bath. 0.56 g of phosphorus tribromide (2.06 mmol) was slowly added in portions. After the addition was complete, the mixture was slowly brought to room temperature and stirred for 2 hours. After the reaction was complete, 10 mL of saturated sodium bicarbonate solution and 10 mL of water were added to quench the reaction. The mixture was extracted three times with dichloromethane, and the combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated and purified by silica gel column chromatography using a gradient elution of ethyl acetate / petroleum ether (1 / 10 to 1 / 2, v / v) to give 1.85 g of a pale yellow oily liquid.

[0079] 1.4 Synthetic compound 4 1.85 g of compound 3 (3.59 mmol) and 0.53 g of 2-cyano-6-hydroxybenzothiazole (3.00 mmol) were dissolved in 20 mL of dichloromethane. Then, 1.16 g of tetrabutylammonium bromide (3.59 mmol) and 5 mL of 5% sodium hydroxide aqueous solution were added. After stirring the mixture at room temperature for 4 hours, it was extracted three times with dichloromethane. The combined organic phases were washed successively with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with a gradient of ethyl acetate / petroleum ether (1 / 10 to 1 / 1, v / v) to give 0.83 g of a pale yellow solid.

[0080] 1.5, Synthetic compound 5 0.83 g of compound 4 (1.36 mmol) and 0.33 g of D-cysteine ​​(2.72 mmol) were dissolved in 10 mL of a mixed solution of DCM / methanol / water (2 / 2 / 1, v / v / v). Then 0.38 g of anhydrous potassium carbonate was added. After stirring the mixture at room temperature for 1 hour, the organic solvent was removed by concentration under reduced pressure. The remaining aqueous phase was adjusted to pH 4 with 1 M hydrochloric acid. The mixture was then extracted three times with ethyl acetate, and the combined organic phases were washed successively with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, eluting with a dichloromethane / glacial acetic acid gradient (20 / 1 to 10 / 1, v / v) to give 0.65 g of a yellow solid.

[0081] 1.6 Synthesis of the target probe Gal-luc 0.65 g of compound 5 (0.91 mmol) was dissolved in 10 mL of methanol, and 0.05 g of sodium methoxide (0.91 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, and then glacial acetic acid was added to adjust the pH to 8. The solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using a dichloromethane / glacial acetic acid gradient (20 / 1 to 5 / 1, v / v) to give 0.32 g of a brownish-yellow solid. Its structure was confirmed by 1H NMR and mass spectrometry as follows: .

[0082] The proton NMR spectrum is as follows: Figure 3 As shown: 1 H NMR (300 MHz, Methanol-d4) δ 7.48-7.38 (m,2H), 7.31-7.18 (m, 3H), 7.11 (dd, J=16.4, 8.1 Hz, 3H), 5.34 (s, 1H), 5.13 (s,2H), 4.53 (s, 2H), 3.93 (s, 2H), 3.73 (d, J=23.8 Hz, 8H), 3.58 (d, J=19.5 Hz, 4H), 2.67 (s, 2H). HRMS (ESI + m / z calculated for C 24 H 24 N2O9S2([M+1) + ): 549.1, found: 549.2.

[0083] 1.7. In vitro performance verification of Gal-luc By incubating the probe Gal-luc with β-galactosidase in vitro, and then adding the bioluminescent system (fLuc / ATP / Mg) 2+Finally, the enzymatic release performance, stability, enzyme-responsive bioluminescence, and enzyme concentration dependence of Gal-luc were investigated using HPLC, fluorescence spectroscopy, and bioluminescence imaging, respectively. Figure 4-6 As shown, the enzyme digestion and release diagrams of the probe Gal-luc are displayed. Figure 4 A) and stability diagram ( Figure 4 B) Enzyme-responsive bioluminescence spectrum ( Figure 5 ) and enzyme concentration dependence plot ( Figure 6 HPLC results showed that Gal-luc can be specifically cleaved by β-gal, releasing D-luciferin. Stability experiments showed that it exhibited good stability after 16 hours of incubation at physiological pH. Bioluminescence experiments showed that Gal-luc itself is non-luminescent, and only in the presence of β-gal can it react with fLuc / ATP / Mg. 2+ The reaction produced bioluminescence at 530 nm, and the intensity of the luminescence showed a good linear relationship with the β-gal concentration. These results indicate that Gal-luc is a high-performance SA-β-gal-responsive "on-off" bioluminescent probe.

[0084] Example 2: A fluorinated polymer support F-PEI is provided, and its specific synthesis steps are as follows: Branched polyethyleneimine (PEI, molecular weight approximately 10 kDa) was dissolved in anhydrous methanol to prepare a 10 mg / mL solution. 3-(perfluorohexyl)-1,2-epoxypropane was slowly added dropwise to the PEI methanol solution at a molar ratio of 1:32. The reaction mixture was stirred at room temperature for 48 hours. After the reaction was complete, the reaction mixture was transferred to a dialysis bag (MWCO 3500 Da), dialyzed against methanol for 24 hours, and then against pure water for 48 hours to remove unreacted small molecules and organic solvents. After dialysis, the mixture was freeze-dried to obtain a pale yellow solid product, F-PEI.

[0085] A small amount of F-PEI was dissolved in deuterated methanol and subjected to 1H NMR spectroscopy. The results are as follows: Figure 7 As shown. By analyzing the integral area ratio of the characteristic proton peaks (δ 2.5-4.5ppm) on the fluorine ligands and the proton peaks (δ 2.5-3.5ppm) on the PEI backbone in the spectrum, it was calculated that each PEI molecule is modified with an average of 33 fluoroalkyl chains, with a grafting rate of approximately 5.7%.

[0086] PEI itself is positively charged. After fluorination, through hydrophobic / fluorophilic interactions and electrostatic interactions, it can simultaneously and stably bind negatively charged fLuc (isoelectric point approximately 6.2, negatively charged under neutral conditions) and multiple negatively charged ATP molecules, forming uniform and stable nanoparticles. The fluoroalkyl chain possesses both hydrophobic and fluorophilic properties, promoting nanoparticle fusion with cell membranes and improving delivery efficiency. Examples show successful delivery and imaging in H9C2 cells. Compared to unmodified PEI, fluorination significantly reduces the cytotoxicity of the cationic polymer.

[0087] Example 3: Based on the foregoing embodiments, a β-galactosidase-activated aging-targeting bioluminescent nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA is provided, such as... Figure 2 As shown, it includes: Core nanoparticles, and A tannic acid coating on the surface of the core nanoparticles; The core nanoparticles are assembled from a fluorinated polymer carrier, a β-galactosidase-responsive bioluminescent small molecule probe, a firefly luciferase, and adenosine triphosphate. The structure of the β-galactosidase-responsive bioluminescent small molecule probe is shown below: .

[0088] The preparation steps of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA are as follows.

[0089] 3.1 Preparation of core nanoparticles F-PEI / Gal-luc / fLuc / ATP F-PEI aqueous solution (10 mg / mL), Gal-luc aqueous solution (containing 0.1% DMSO for dissolution, 2.5 mg / mL), fLuc in PBS solution (pH 7.6, 10 mg / mL), and ATP aqueous solution (2.5 mg / mL) were prepared separately. The F-PEI, Gal-luc, and fLuc solutions were mixed in equal volumes at a mass ratio of 1:4:1 and incubated at room temperature for 30 minutes to allow for complete co-assembly. Then, an equal volume of ATP solution was added (i.e., F-PEI to ATP mass ratio of 4:1), and the mixture was stirred at room temperature for 10 minutes to obtain a mixture of F-PEI / Gal-luc / fLuc / ATP core nanoparticles.

[0090] 3.2 Coating with tannic acid (TA) and purification The above mixture was centrifuged at 12500 rpm for 30 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in PBS and washed once, followed by centrifugation again. The washed precipitate was resuspended in an appropriate amount of PBS to obtain a purified core nanoparticle solution. Then, the core nanoparticle solution was mixed with an equal volume of tannic acid (TA) aqueous solution (ensuring a F-PEI to TA mass ratio of 4:1), and incubated at room temperature for 10 minutes to allow TA to adsorb onto the nanoparticle surface through hydrogen bonding and hydrophobic interactions. Subsequently, it was centrifuged again at 12500 rpm for 30 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain the F-PEI / Gal-luc / fLuc / ATP@TA nanoprobe stock solution.

[0091] 3.3 Characterization of Nanoprobes (1) Morphology, particle size, and potential: A small amount of nanoprobe solution was dropped onto a carbon film copper grid, allowed to air dry naturally, and its morphology was observed using a transmission electron microscope (TEM). Figure 8 As shown. A separate, appropriately diluted nanoprobe solution was used to determine its hydration particle size and zeta potential using a dynamic light scattering particle size analyzer (DLS). The results are shown below. Figure 9 As shown in the figure. TEM showed that the nanoparticles were uniformly spherical. DLS measured the average hydrated particle size of the core nanoparticles to be approximately 232 nm, with a Zeta potential of -10.2 mV. After TA coating, the nanoprobe particle size increased to approximately 268 nm, and the Zeta potential changed to -15.7 mV, indicating that TA coating was successful.

[0092] (2) Stability study: The prepared core nanoparticles and TA-coated nanoprobes were placed in PBS (pH 7.6) and incubated at 37°C for different times (1, 3, 5, 7 days), and the particle size change was monitored using DLS. The results are as follows: Figure 10 As shown, the particle size of both groups of nanoparticles did not change significantly within 7 days, demonstrating good colloidal stability. The probe coated with TA showed better stability.

[0093] Example 4: Verification of the in vitro luminescence properties of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA described in Example 3 4.1 β-gal enzyme responsiveness and concentration-dependent luminescence experiments F-PEI / Gal-luc / fLuc / ATP@TA nanoprobe (20 μg / mL) was mixed with different concentrations of β-galase (0, 12.5, 25, 50, 100 U / mL) in PBS (pH 7.6, containing 10 mM Mg). 2+ After incubation in the solution, the luminescence signal was immediately detected using a bioluminescence imaging system. The results are as follows: Figure 11As shown, the experimental group incubated with β-gal produced a significant bioluminescent signal, while the control group without enzyme or with heat-inactivated enzyme showed almost no signal. The luminescence intensity increased with increasing β-gal concentration, exhibiting a good concentration dependence.

[0094] 4.2 Anti-interference test The nanoprobes were respectively combined with various potential interfering agents (including K) + Ca 2+ Mg 2+ Zn 2+ Fe 3+ GSH, Cys, H2O2, carboxylesterase (CES), and alkaline phosphatase (ALP) were incubated under the same conditions, and their bioluminescent signals were detected. The results are as follows: Figure 12 As shown, a strong luminescence signal was detected only in the experimental group containing β-gal, while no obvious signal was observed in the other interfering groups. This fully demonstrates that the nanoprobe has high selectivity for the target enzyme β-gal.

[0095] Example 5: Cytotoxicity validation and senescent cell imaging experiments of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA 5.1 Cytotoxicity test H9C2 cells were seeded in 96-well plates. After cell attachment, different concentrations (0, 12.5, 25, 50, 100, 200 μg / mL) of F-PEI / Gal-luc / fLuc / ATP@TA nanoprobes were added and incubated for 24 hours. Cell viability was assessed using the MTT assay. Results are shown below. Figure 13 As shown, cell viability remained at a high level (>85%) at all tested concentrations, indicating that the nanoprobe has good cell compatibility.

[0096] 5.2 Specific luminescence imaging of senescent cells Normal H9C2 cells and hypoxia-induced senescent H9C2 cells were incubated with F-PEI / Gal-luc / fLuc / ATP@TA nanoprobes, respectively. A competitive inhibition group was also set up: senescent cells were pre-incubated with D-galactose (a β-gal competitive inhibitor) for 4 hours before adding the nanoprobes. After incubation, the results were analyzed using a bioluminescence imaging system. The results are as follows: Figure 14 As shown, a strong bioluminescent signal was detected in the senescent cell group, while the signal was extremely weak in the normal cell group. The competitive inhibition group showed a significantly weakened bioluminescent signal due to the inhibition of SA-β-gal activity. This result clearly confirms that the nanoprobe can be specifically activated by SA-β-gal, which is highly expressed in senescent cardiomyocytes, to generate a bioluminescent signal, thus enabling specific imaging of senescent cells.

[0097] Example 6: In vivo imaging experiments of the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA A mouse model of myocardial infarction was established using male BALB / c mice by ligating the left anterior descending coronary artery (LAD). The mice were then fed for 7 days post-surgery to allow for sufficient senescence of cardiomyocytes in the infarcted area.

[0098] 6.1 In-situ drug delivery imaging MI model mice (7 days post-surgery) and normal control mice (Sham group) underwent a second thoracotomy, and F-PEI / Gal-luc / fLuc / ATP@TA nanoprobe solution (20 μL, 1 mg / mL) was injected in situ at the apex of the heart. The thoracic cavity was sutured, and after the mice recovered (approximately 30 minutes), they were placed in a small animal in vivo imaging system for bioluminescence imaging. The results are as follows: Figure 15 As shown, strong bioluminescent signals were detected in the heart region of MI model mice, while almost no signal was detected in the heart region of normal mice. This demonstrates that the probe can identify and image senescent cells in infarcted hearts in vivo.

[0099] 6.2 Imaging of intravenous drug administration To further explore the systemic application potential of this probe, F-PEI / Gal-luc / fLuc / ATP@TA nanoprobe solution (200 μL, 1 mg / mL) was injected via tail vein into MI model mice and normal control mice. In vivo imaging was performed 30 minutes after administration, followed by sacrifice of the mice and removal of the hearts for ex vivo imaging. Results are as follows... Figure 16 As shown, in vivo imaging revealed a significant luminescent signal in the heart region of MI mice. Ex vivo heart imaging further confirmed that the luminescent signal was specifically concentrated in the infarcted heart of MI mice, while no significant signal was observed in the heart of normal mice. This indicates that the nanoprobe possesses excellent in vivo circulation capability and targeted delivery efficiency, enabling passive enrichment in the infarcted region through the nanosize effect and the enhanced structural stability of the tannic acid coating, thus achieving specific imaging of senescent cardiomyocytes.

[0100] Example 7: ATP+Mg 2+ Co-delivery molar ratio optimization experiment and verification of exogenous energy substrate supplementation effect Based on the principle of bioluminescence, the oxidation of D-luciferin by firefly luciferase (fLuc) to produce light requires ATP and Mg. 2+ As an essential cofactor, ATP has been stably delivered in the nanoprobe described in Example 3 via co-assembly of the fluorinated polymer F-PEI with fLuc and Gal-luc, but Mg... 2+It is not an integral component of nanoparticles. Due to intracellular Mg 2+ Concentration may vary between individuals or fluctuate under pathological conditions. This embodiment further optimizes the Mg content in the co-delivery system based on the nanoprobe F-PEI / Gal-luc / fLuc / ATP@TA constructed in Example 3 above. 2+ The concentration was specifically determined by directly co-delivering different molar ratios of Mg during the nanoparticle assembly process. 2+ The study aimed to investigate its effect on bioluminescence signal intensity in order to maximize the bioluminescence reaction efficiency catalyzed by fLuc, and to verify the signal enhancement effect of the nanoprobe under conditions of sufficient energy substrate, so as to provide parameter basis for subsequent in vivo applications.

[0101] 7.1 Preparation of different Mg 2+ Content of nano probes The ATP+Mg nanoprobe was constructed according to the preparation method of F-PEI / Gal-luc / fLuc / ATP@TA described in Example 3. 2+ The co-delivery system differs in that Mg is added to the ATP solution. 2+ The molar ratios of ATP to MgCl2 were set to 1:0.5, 1:1, 1:2, 1:5, and 1:10, respectively. The mass ratio of F-PEI to ATP was maintained at 4:1 in all groups. All samples were coated with TA and purified to obtain samples with the same ATP content and MgCl2 content. 2+ Nanoprobes with increasing concentration gradients were labeled as group A (without Mg) 2+ ) to group F (ATP:Mg 2+ (Molar ratio 1:10).

[0102] 7.2 Determination of in vitro bioluminescence signal intensity The nanoprobes (20 μg / mL, based on F-PEI) of each experimental group were mixed with β-galactosidase (10 U / mL) in a solution containing 10 mM MgCl2 (as background Mg). 2+ The samples were incubated in PBS buffer (pH 7.6) at 37°C to ensure the basic reaction conditions were met. The bioluminescence signal intensity of each group was immediately detected using a bioluminescence imaging system, with three replicates for each sample. The results are shown in Table 1.

[0103] Table 1. Different ATP:Mg 2+ Effect of molar ratio on bioluminescence signal intensity of nanoprobes

[0104] As can be seen from Table 1, in the case of co-delivering ATP without additional Mg, 2+ In group A, a significant bioluminescent signal (6.8 × 10⁻⁶) could still be detected.3 p / s / cm 2 / sr), because the reaction buffer already contains 10 mM MgCl2, providing basic cofactor support for fLuc. This result indicates that even without Mg... 2+ Co-assembled nanoparticles, provided there is sufficient Mg in the extracellular environment or buffer solution. 2+ The probe still functions normally. And with the co-delivery of Mg from the nanoparticles... 2+ As the proportion of Mg increases, the luminescence intensity gradually increases, under sufficient background Mg. 2+ In its presence, with non-co-delivered Mg 2+ Compared to probes that co-deliver ATP:Mg 2+ At a molar ratio of 1:2, a signal intensity increase of less than one order of magnitude was observed, indicating that co-delivery of Mg occurred in a specific local microenvironment. 2+ This can further enhance or modulate the catalytic efficiency of fLuc. Adding Mg... 2+ Encapsulating ATP with nanoparticles can create a high-concentration cofactor microenvironment locally, significantly enhancing the catalytic efficiency of fLuc. When Mg... 2+ When the ratio was further increased to 1:5 or even 1:10, the luminescence intensity actually decreased, suggesting that additional co-delivery of Mg occurred against a high concentration background. 2+ This may have altered the internal osmotic pressure of the carrier or interfered with the stable assembly of ATP and F-PEI, leading to the leakage of some ATP before the reaction, or there may be factors that interfere with the binding of the substrate to the enzyme's active site. Therefore, in the co-delivery system, ATP:Mg 2+ The molar ratio should be controlled within the range of 1:1 to 1:10, with a preferred ratio of 1:1 to 5 and an optimal ratio of 1:2.

[0105] In summary, this invention successfully constructed a β-galactosidase-activated aging-targeting bioluminescent nanoprobe. This probe integrates a highly efficient fluorinated polymer delivery system, a specifically recognized "smart switch," and a bioluminescent signal module, enabling specific detection and imaging of aging cells at the molecular, cellular, and in vivo levels. This probe possesses advantages such as simple preparation, good biocompatibility, high detection sensitivity, and strong specificity, providing a powerful new tool for basic research and preclinical diagnosis of age-related cardiovascular diseases and other age-related diseases.

[0106] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be described in detail here.

[0107] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0108] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

[0109] While the foregoing detailed descriptions have shown, described, and pointed out novel features applicable to various embodiments, it should be understood that various omissions, substitutions, and changes may be made to the form and details of the described apparatus or methods without departing from the spirit of this disclosure. Furthermore, the various features and methods described above may be used independently of each other or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of this disclosure. Many of the foregoing embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or applications, as well as their obvious modifications and equivalents. Therefore, the invention is not intended to be limited to the specific disclosure of the preferred embodiments herein.

[0110] All matters not covered in this invention are common knowledge.

Claims

1. The application of a β-galactosidase-responsive bioluminescent small molecule probe in the preparation of imaging reagents for monitoring senescent cardiomyocytes after myocardial infarction, characterized in that, The β-galactosidase-responsive bioluminescent small molecule probe is a compound formed by covalently linking D-galactoside with D-fluorescein via a benzyl ether linker arm.

2. The application according to claim 1, characterized in that, The imaging reagent is used for specific bioluminescent imaging of senescent cardiomyocytes that highly express β-galactosidase in the infarct region of myocardial infarction.

3. A β-galactosidase-activated aging-targeting bioluminescent nanoprobe, characterized in that... include: Core nanoparticles, and A tannic acid coating on the surface of the core nanoparticles; The core nanoparticles are assembled from a fluorinated polymer carrier, a β-galactosidase-responsive bioluminescent small molecule probe, a firefly luciferase, and adenosine triphosphate. The β-galactosidase-responsive bioluminescent small molecule probe is a compound formed by covalently linking D-galactoside with D-fluorescein via a benzyl ether linker arm.

4. The aging-targeting bioluminescent nanoprobe according to claim 3, characterized in that: The mass ratio of the fluorinated polymer carrier to the β-galactosidase-responsive bioluminescent small molecule probe and the firefly luciferase is 1:1-5:1-2.

5. The aging-targeting bioluminescent nanoprobe according to claim 3 or 4, characterized in that: The aging-targeting bioluminescent nanoprobe has a particle size of 200-300 nm and a zeta potential of -15 mV to -20 mV.

6. The method for preparing the β-galactosidase-activated aging-targeting bioluminescent nanoprobe according to any one of claims 3-5, characterized in that... Includes the following steps: Synthesize a β-galactosidase-responsive bioluminescent small molecule probe, Gal-luc; Synthetic fluorinated polymer carrier F-PEI; The F-PEI was mixed with Gal-luc and firefly luciferase fLuc and incubated, and then adenosine triphosphate ATP solution was added to obtain a mixture of F-PEI / Gal-luc / fLuc / ATP core nanoparticles. Centrifuge the resulting mixture, resuspend the precipitate, and obtain purified F-PEI / Gal-luc / fLuc / ATP core nanoparticles; The obtained core nanoparticles were mixed with tannic acid solution and incubated, then centrifuged, and the precipitate was resuspended to obtain the final product.

7. The method according to claim 6, characterized in that: The synthesis steps of the fluorinated polymer carrier F-PEI are as follows: 3-(perfluorohexyl)-1,2-epoxypropane is added to an anhydrous methanol solution of branched polyethyleneimine, and the reaction is stirred at room temperature for 12-96 hours. The reaction product is purified by dialysis and then freeze-dried to obtain F-PEI.

8. The use of the β-galactosidase-activated aging-targeting bioluminescent nanoprobe according to any one of claims 3-5 or the aging-targeting bioluminescent nanoprobe prepared by the method according to any one of claims 6-7 in the preparation of imaging reagents for diagnosing or monitoring aging-related diseases.

9. The application of the β-galactosidase-activated aging-targeting bioluminescent nanoprobe according to any one of claims 3-5 or the aging-targeting bioluminescent nanoprobe prepared by the method according to any one of claims 6-7 in cell imaging for non-diagnostic or therapeutic purposes, said application including enabling bioluminescent imaging of hypoxia-induced aging H9C2 cells.

10. The application of the β-galactosidase-activated aging-targeting bioluminescent nanoprobe according to any one of claims 3-5 or the aging-targeting bioluminescent nanoprobe prepared by the method according to any one of claims 6-7 in in vivo imaging for non-diagnostic or therapeutic purposes, said application including enabling bioluminescent imaging of senescent cells in a mouse myocardial infarction model.

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