Multifunctional organic afterglow luminescent nanomaterial, preparation method and application thereof

Abstract

The application discloses a multifunctional organic afterglow luminescent nanomaterial and a preparation method and application thereof, and comprises the following steps: uniformly mixing methylene blue derivatives, amphiphilic copolymer A and amphiphilic copolymer B in an organic solvent, adding water under ultrasonic conditions to form a nanoparticle solution, and removing the organic solvent in the nanoparticle solution to obtain the multifunctional organic afterglow luminescent nanomaterial. The multifunctional organic afterglow luminescent nanomaterial disclosed by the application is mainly composed of organic small molecules, has significant advantages in biocompatibility, biodegradability, luminescence superiority, tunability and structural flexibility, and realizes multifunctional imaging integration of fluorescence, photoacoustic and afterglow luminescence. On this basis, the activatable multifunctional organic afterglow luminescent nanomaterial has extremely high activation contrast, can realize precise detection of a small tumor with a minimum diameter of about 1.5 mm and precise resection under the guidance of an afterglow image.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a multifunctional organic afterglow luminescent nanomaterial, its preparation method, and its applications. Background Technology

[0002] Optical imaging is an imaging technique that uses optical detection devices to visualize light signals. It boasts advantages such as high imaging sensitivity, high spatiotemporal resolution, and low developer cost, making it an indispensable tool in biomedical applications. Compared to fluorescence imaging, which relies on real-time photoexcitation and generates strong background noise, resulting in a low signal-to-noise ratio, afterglow luminescence can continue emitting light after photoexcitation ceases. Because it does not require real-time photoexcitation, it offers a higher signal-to-noise ratio and greater tissue detection depth. Furthermore, this imaging technique relies on the inherent optical properties of molecules, providing superior flexibility and broad applicability, making it a highly promising optical imaging technology. Recently, with the urgent need for clinical translation, the development of multifunctional, multimodal systems such as ultrasound-excited afterglow luminescence imaging and photoacoustic imaging will greatly improve the tissue penetration capability of optical imaging, potentially enabling deep tissue imaging and driving its clinical translation.

[0003] Currently, afterglow luminescent materials are mainly divided into inorganic afterglow luminescent materials and organic afterglow luminescent materials. Inorganic afterglow luminescent materials are usually composed of inorganic salts doped with transition metals or rare earth metals (e.g., ZnGa2O4:Cr). 3+ SrAl2O4:Eu 2+ / Dy 3+ Currently reported organic afterglow luminescent materials are mainly divided into two major systems. One is an organic afterglow nanosystem composed of single afterglow molecules, which mainly includes semiconductor polymers represented by poly(p-phenylenevinyl) polymers (e.g., MEHPPV) and thiophene polymers (e.g., PFODBT), dihydroporphyrin photosensitizers (e.g., Ce4), and conjugated molecules connecting photosensitizers and chemiluminescent substrates (e.g., TPP-DO). The other is a multi-component organic afterglow nanosystem, which is usually composed of afterglow initiators, i.e., photosensitizers (e.g., NCBS), afterglow luminescent substrates, i.e. chemiluminescent substances (e.g., DO, SO, adamantine ethers (AEEs), fluorescein analogs (CLA)), and afterglow secondary units.

[0004] However, inorganic afterglow luminescent materials require stringent synthesis conditions and the incorporation of heavy metals such as rare earth metals, leading to significant biotoxicity. In contrast, organic afterglow luminescent materials offer advantages such as better biocompatibility, tunable luminescence, simple synthesis, easy functionalization, and low cost, making them more promising for applications in biomedical imaging. For organic afterglow luminescent materials, multi-component doped organic afterglow nanosystems require considerable effort and time for condition optimization due to their complex composition, and suffer from poor reproducibility and limited afterglow intensity. Single-component organic afterglow nanosystems, on the other hand, are simpler in composition and easier to synthesize, offering greater advantages. Semiconductor polymers, as an important type of single-component organic afterglow luminescent material, still do not possess the advantages of small organic molecules in terms of biodegradability and long-term biosafety. Furthermore, currently reported small organic molecules suffer from limitations such as limited variety, short emission wavelength, and weak afterglow intensity. Simultaneously, currently reported ultrasonically excited organic afterglow systems are limited to co-doped nanosystems based on sonosensitive agents as initiators and chemiluminescent substrates as luminescent substrates, exhibiting limitations such as limited variety and dependence on multi-component systems.

[0005] Image-guided tumor resection surgery helps surgeons quickly and accurately determine the location of lesions, precisely removing them or avoiding critical areas. Advances in intraoperative imaging technology have made surgery faster, more precise, and safer, significantly impacting patient prognosis. Currently, optical imaging, with its advantages of high sensitivity, low cost, and real-time signal acquisition, has become a highly attractive imaging strategy in image-guided tumor resection surgery. The U.S. Food and Drug Administration (FDA) has approved indocyanine green (ICG) for clinical fluorescence imaging-guided tumor resection surgery. However, current fluorescence imaging-guided tumor resection surgery suffers from high background signal interference due to the high tissue autofluorescence interference generated by real-time photoexcitation, reducing the signal-to-noise ratio and interfering with intraoperative judgment, thus affecting the precision of the resection. Therefore, afterglow emission imaging, with its extremely low tissue background signal and superior imaging sensitivity, is a more ideal imaging method for image-guided tumor resection surgery. In recent years, the design of activatable optical probes, utilizing highly expressed tumor biomarkers to specifically activate optical signals, has facilitated accurate tumor detection and improved the precision of tumor resection. Most of the organic afterglow probes that have been explored and applied to surgical navigation are constant-brightness probes, which have limited accuracy in tumor detection.

[0006] Activatable probe design relies on a response to specific biomarkers, undergoing an activation process from "signal off" to "signal on," resulting in a higher signal-to-noise ratio compared to traditional "always-on" probes. In recent years, this design strategy has been widely applied to the design of various optical imaging probes to monitor physiological and pathological processes at the molecular level. Therefore, the design and development of activatable organic afterglow probes can provide higher spatial and temporal accuracy, undoubtedly becoming an ideal imaging method for obtaining extremely high signal-to-noise ratios.

[0007] Currently, most design strategies for activatable organic afterglow probes rely on multi-component organic afterglow nanosystems, primarily including: 1) adding a photoquencher (e.g., BHQ-2) to quench afterglow luminescence through energy transfer between the afterglow-emitting substrate and the photoquencher; 2) doping with activatable photosensitizers to provide oxidation conditions for the afterglow-emitting substrate through singlet oxygen generation in response to biomarkers, enabling subsequent afterglow luminescence; 3) designing activatable afterglow substrates to provide active intermediates for biomarker response, enabling subsequent afterglow luminescence; and 4) designing activatable fluorescent molecules as afterglow secondary units to achieve long-lasting afterglow emission in response to biomarkers through energy transfer. However, the current design of activatable organic afterglow materials largely relies on multi-component systems, which suffers from limitations such as complex composition and poor reproducibility. Furthermore, the relatively inefficient activation strategies result in limited activation contrast, restricting their application in bioimaging.

[0008] Currently, organic afterglow imaging is in an emerging stage of development. Developing a new type of organic afterglow material with strong and near-infrared emission, which can improve its optical performance while meeting the requirements of multi-functional integration such as activatable design, acousto-induced afterglow emission, and photoacoustic imaging, will achieve a major breakthrough in the development of organic afterglow imaging. Summary of the Invention

[0009] To address the problems of time-consuming and low reproducibility caused by the limitation of current organic afterglow luminescent materials to multi-component doped systems, the limited variety of single-component nanosystems, the biodegradability and biosafety issues of semiconductor polymers, the short wavelength and low intensity of afterglow luminescence in current single-component systems, and the limited variety and limitation of current ultrasonically excited organic afterglow luminescent systems to multi-component nanosystems, the primary objective of this invention is to provide a multifunctional organic afterglow luminescent nanomaterial and its preparation method.

[0010] To address the issues of low activation contrast due to the single and inefficient design strategy of current activatable organic afterglow probes, and the misdiagnosis caused by high background signal and limited accuracy of constant-brightness afterglow probes in tumor detection during current optical imaging-guided surgical treatment, a further objective of this invention is to provide an activatable multifunctional organic afterglow luminescent nanomaterial and its preparation method.

[0011] The third objective of this invention is to provide an application of activatable multifunctional organic afterglow luminescent nanomaterials as nanoprobes, which can solve the current problem of the lack of multifunctional probes that integrate afterglow luminescence imaging, photoacoustic imaging and other deep tissue imaging.

[0012] The fourth objective of this invention is to provide the application of activatable multifunctional organic afterglow luminescent nanomaterials in bioimaging.

[0013] This invention is achieved through the following technical solution:

[0014] The first aspect of the present invention provides a method for preparing a multifunctional organic afterglow luminescent nanomaterial, comprising the following steps: mixing methylene blue derivative (mMB), amphiphilic copolymer A and amphiphilic copolymer B in an organic solvent, adding water under ultrasonic conditions to form a nanoparticle solution, and removing the organic solvent from the nanoparticle solution to obtain the multifunctional organic afterglow luminescent nanomaterial (SAN-M).

[0015] The methylene blue derivative (mMB) is selected from one of the following structures:

[0016]

[0017]

[0018] The amphiphilic copolymer A is a polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer;

[0019] The amphiphilic copolymer B is distearate phosphatidylethanolamine-polyethylene glycol and / or polystyrene-polyacrylic acid.

[0020] This invention synthesizes a methylene blue derivative (mMB) with good hydrophobicity by alkylating the traditional photosensitizer methylene blue. Subsequently, this derivative molecule is co-assembled with an amphiphilic polymer to construct a structurally stable nanoparticle (SAN-M).

[0021] Among them, amphiphilic copolymer A is preferably polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer (PEG-b-PPG-b-PEG); amphiphilic copolymer B is preferably distearylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG). 2000 ) and / or polystyrene-polyacrylic acid (PS-PAA).

[0022] Furthermore, the preparation method of the methylene blue derivative includes the following steps:

[0023] S1. Iodine solution was added dropwise to phenothiazine solution to obtain a mixed solution. The mixed solution was stirred in an ice bath, the precipitate was collected by filtration, washed and dried to obtain a black powder (product 1).

[0024] S2. Add dibutylamine to the organic solution of product 1, stir and react overnight at room temperature, and further purify by column chromatography (eluent: dichloromethane / methanol volume ratio 50:1) to obtain a dark purple solid (product mMB).

[0025] Furthermore, the mass ratio of the methylene blue derivative, amphiphilic copolymer A, and amphiphilic copolymer B is 1:200:(5-20).

[0026] Furthermore, the organic solvent is selected from one or more of ethanol, methanol, and tetrahydrofuran, preferably tetrahydrofuran.

[0027] The second aspect of this invention provides a multifunctional organic afterglow luminescent nanomaterial prepared by the method described in the first aspect.

[0028] The multifunctional organic afterglow luminescent nanomaterial (SAN-M) of this invention can achieve strong afterglow emission at a wavelength of 710 nm after pre-irradiation with 660 nm light. Furthermore, this near-infrared afterglow emission phenomenon can also be achieved after ultrasonic pre-excitation. In addition to afterglow emission, SAN-M can also detect photoacoustic signals emitted at a wavelength of 680 nm, enabling multifunctional, multimodal imaging that integrates afterglow emission, fluorescence, and photoacoustic imaging (PA). This allows for applications such as early inflammation imaging and tumor imaging.

[0029] A third aspect of this invention provides a method for preparing an activatable multifunctional organic afterglow luminescent nanomaterial, comprising the following steps: reacting a methylene blue derivative (mMB-ONOO) with... - Amphiphilic copolymer A and amphiphilic copolymer B are mixed evenly in an organic solvent, and then water is added under ultrasonic conditions to form a nanoparticle solution. After removing the organic solvent from the nanoparticle solution, the activated multifunctional organic afterglow luminescent nanomaterial (SAN-MO) is obtained.

[0030] The methylene blue derivative (mMB-ONOO) - It is selected from one of the following structures:

[0031]

[0032] The amphiphilic copolymer A is a polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer;

[0033] The amphiphilic copolymer B is distearate phosphatidylethanolamine-polyethylene glycol and / or polystyrene-polyacrylic acid.

[0034] Methylene blue derivatives (mMB) possess structurally controllable optical properties. An activatable organic afterglow molecule (mMB-ONOO) was obtained by introducing a responsive group (e.g., phenylboronic acid ester) at nitrogen site 10 on its phenothiazine ring. - The molecule achieves afterglow quenching based on its own structure by disrupting the conjugated structure at its center. Nanoparticles composed of this molecule (SAN-MO) exhibit afterglow quenching based on specific biomarkers (e.g., peroxynitrite, ONOO). - Under the triggering of ), along with the recovery of the conjugate structure, the optical signals, including afterglow emission, fluorescence, and photoacoustic signals, undergo an activation process from "off" to "on", and the afterglow signal has extremely high activation contrast.

[0035] Among them, amphiphilic copolymer A is preferably polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer (PEG-b-PPG-b-PEG); amphiphilic copolymer B is preferably distearylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG). 2000 ) and / or polystyrene-polyacrylic acid (PS-PAA).

[0036] Furthermore, the preparation method of the methylene blue derivative includes the following steps:

[0037] mMB was dissolved in dichloromethane and mixed with an aqueous solution of sodium dithionite (Na2S2O4) and sodium bicarbonate (NaHCO3). The mixture was stirred at room temperature under nitrogen protection until the organic phase changed from blue to yellow, after which the aqueous phase was removed. Triethylamine (TEA) and triphosgene (TPG) were added to the reaction solution, and the mixture was reacted for 1–2 hours and then concentrated under vacuum. The residue was purified by scintillation chromatography using dichloromethane as the eluent to obtain a yellow product (product 2). Next, potassium carbonate (K2CO3), 4-dimethylaminopyridine (DMAP), and 4-(hydroxymethyl)phenylboronic acid pinacol ester were dissolved in a dichloromethane solution. Product 2 was added dropwise, and the mixture was stirred at room temperature under nitrogen protection until thin-layer chromatography showed the reaction was complete. The mixture was then concentrated under vacuum, and the residue was purified by scintillation chromatography using dichloromethane as the eluent to obtain a pale blue product (mMB-ONOO). - ).

[0038] Furthermore, the mass ratio of the methylene blue derivative, amphiphilic copolymer A, and amphiphilic copolymer B is 1:200:(5-20).

[0039] Furthermore, the organic solvent is selected from one or more of ethanol, methanol, and tetrahydrofuran, preferably tetrahydrofuran.

[0040] The fourth aspect of this invention provides an activatable multifunctional organic afterglow luminescent nanomaterial prepared by the method described in the third aspect.

[0041] The fifth aspect of this invention provides the application of the activatable multifunctional organic afterglow luminescent nanomaterial described in the fourth aspect as a nanoprobe.

[0042] The activatable multifunctional organic afterglow luminescent nanomaterial of the present invention can be used as an activatable organic afterglow nanoprobe. It utilizes the highly expressed peroxynitrite ions in tumors to specifically recognize and activate the activatable organic afterglow nanoprobe of the present invention. Thanks to the extremely high signal-to-noise ratio of the activatable afterglow luminescence, it can achieve accurate detection of tumors and precise resection of small tumors under the guidance of afterglow images.

[0043] The sixth aspect of this invention provides the application of the activatable multifunctional organic afterglow luminescent nanomaterials described in the fourth aspect in bioimaging.

[0044] The activatable multifunctional organic afterglow luminescent nanomaterial of the present invention can realize activatable multimodal imaging that integrates afterglow luminescence, fluorescence, and photoacoustic imaging.

[0045] The beneficial effects of this invention are:

[0046] 1. The multifunctional organic afterglow luminescent nanomaterial of the present invention is mainly composed of small organic molecules, which has significant advantages in terms of biocompatibility, biodegradability, superior luminescence performance and tunability, and structural flexibility.

[0047] 2. The multifunctional organic afterglow luminescent nanomaterial of the present invention can emit high-intensity afterglow luminescence with a wavelength of 710 nm after photoexcitation stops. This afterglow luminescence is located in the near-infrared band, thus having superior tissue penetration and making it easy to achieve deep tissue imaging.

[0048] 3. The multifunctional organic afterglow luminescent nanomaterial of the present invention can emit near-infrared afterglow luminescence with a wavelength of 710nm after ultrasonic excitation stops, which belongs to the sonoluminescence phenomenon. Ultrasound has a deeper tissue penetration ability, thus making it easy to achieve deep tissue imaging.

[0049] 4. The multifunctional organic afterglow luminescent nanomaterial of the present invention has photoacoustic signals, which can realize photoacoustic imaging and can be applied to deep tissue imaging.

[0050] 5. The multifunctional organic afterglow luminescent nanomaterial of the present invention improves the afterglow luminescence intensity while achieving near-infrared afterglow wavelength emission, and has the deep tissue penetration potential of ultrasonically excited afterglow luminescence. It can realize multifunctional imaging integration of fluorescence, photoacoustics and afterglow luminescence, and achieve high-sensitivity and high-contrast multimodal molecular imaging related to biomarkers.

[0051] 6. The main organic small molecules of the multifunctional organic afterglow luminescent nanomaterial of the present invention have tunable optical properties, making it easy to realize the activatable design based on its own main structure, develop a single-component activatable organic afterglow nano system, and have extremely high activation contrast.

[0052] 7. The activatable multifunctional organic afterglow luminescent nanomaterial of the present invention can be used as an activatable organic afterglow nanoprobe. The activatable organic afterglow nanoprobe of the present invention is specifically recognized and activated by the highly expressed peroxynitrite ions in the tumor. Thanks to the extremely high signal-to-noise ratio of the activatable afterglow luminescence, it is possible to accurately detect tiny tumors with a minimum diameter of about 1.5 mm and accurately resect them under the guidance of afterglow images. Attached Figure Description

[0053] Figure 1 This is a schematic diagram illustrating the preparation of multifunctional organic afterglow luminescent nanomaterials (SAN-M).

[0054] Figure 2 The hydrated particle size distribution and transmission electron microscope image of SAN-M nanoparticles in 1×PBS buffer are shown.

[0055] Figure 3 The absorption spectrum of SAN-M nanoparticles (5 μmol) in 1×PBS buffer is normalized.

[0056] Figure 4 The normalized fluorescence spectrum of SAN-M nanoparticles (5 μmol) in 1×PBS buffer is shown.

[0057] Figure 5 The normalized afterglow spectrum of SAN-M nanoparticles in 1×PBS buffer is shown.

[0058] Figure 6 This is a comparison of afterglow luminescence images and quantitative data of SAN-M nanoparticles, SAN-C (NPs-Ce4), and SAN-P (NPs-MEHPPV) nanoparticles in 1×PBS buffer.

[0059] Figure 7 Image of afterglow emission from SAN-M nanoparticles and corresponding quantitative data; where a represents the afterglow emission produced by SAN-M nanoparticles. 1 The graph shows the change in O2 amount with 660 nm laser irradiation time, represented by the fluorescence enhancement (F / F0) of the singlet oxygen fluorescent probe SOSG (1 μM); b represents the O2 generated by SAN-M nanoparticles. ·-The graph shows the change in the amount of irradiation time with 660 nm laser light, represented by the fluorescence enhancement (F / F0) of the fluorescent indicator probe DHR123 (5 μM); c represents the effect of photoexcitation and external source irradiation on SAN-M nanoparticles. 1 O2 and O2 ·- The afterglow emission image and corresponding quantitative data graph generated under the action of [the system / mechanism].

[0060] Figure 8 The figures show the absorption spectrum changes of SAN-M nanoparticles before and after 660 nm light irradiation, the afterglow luminescence data of mMB, and a schematic diagram of the mechanism study. Among them, a is the absorption spectrum changes of SAN-M nanoparticles before and after 660 nm light irradiation; b is the high-performance liquid chromatography-mass spectrometry analysis results of mMB before and after 660 nm light irradiation; c is the MADLI-TOF mass spectrometry analysis of mMB after 660 nm light irradiation; and d is a schematic diagram of the afterglow luminescence mechanism study of mMB.

[0061] Figure 9 The images show the sonoluminescence data of SAN-M nanoparticles; where a is a schematic diagram of sonoluminescence generated by ultrasonic excitation; b is the sonoluminescence image and corresponding quantitative data of SAN-M nanoparticles under different ultrasonic power excitation; c is the sonoluminescence image and corresponding quantitative data of SAN-M nanoparticles under different ultrasonic irradiation times; and d is the normalized sonoluminescence spectrum of SAN-M nanoparticles in 1×PBS buffer.

[0062] Figure 10 The photoacoustic properties of SAN-M nanoparticles are shown; where a is the photoacoustic spectrum of SAN-M nanoparticles at different concentrations; and b is the photoacoustic signal intensity of SAN-M nanoparticles at different concentrations.

[0063] Figure 11 This is a schematic diagram illustrating the preparation of an activatable multifunctional organic afterglow luminescent nanomaterial (SAN-MO).

[0064] Figure 12 This is a schematic diagram of the response mechanism of SAN-MO nanoparticles.

[0065] Figure 13 The images show the absorption spectrum, fluorescence spectrum, afterglow emission spectrum, and photoacoustic spectrum of SAN-MO nanoparticles before and after their response; where a is the absorption spectrum; b is the fluorescence spectrum; c is the afterglow emission spectrum; and d is the photoacoustic spectrum.

[0066] Figure 14 This is a graph showing the activation ratio data of SAN-MO nanoparticles before and after their response.

[0067] Figure 15This is a schematic diagram of the response specificity of SAN-MO nanoparticles; where a is the relative afterglow emission signal intensity under the influence of different reactive oxygen species or reactive nitrogen species (RONSs); b is the relative fluorescence signal intensity under the influence of different reactive oxygen species or reactive nitrogen species (RONSs); and c is the relative photoacoustic signal intensity under the influence of different reactive oxygen species or reactive nitrogen species (RONSs).

[0068] Figure 16 The graphs show the changes in activation signal intensity with peroxynitrite concentration; where a represents the change in afterglow luminescence activation signal intensity with peroxynitrite concentration; b represents the change in fluorescence activation signal intensity with peroxynitrite concentration; and c represents the change in photoacoustic activation signal intensity with peroxynitrite concentration.

[0069] Figure 17 The diagram below shows the test process and results for Test Example 1. Figure a shows a detailed schematic of the application of the activatable organic afterglow nanoprobe to detect peritoneal micrometastases; figure b shows representative afterglow luminescence images and average luminescence intensity of the region of interest (ROI) (n=3) recorded at different time points after intravenous injection of SAN-MO nanoparticles into 4T1 peritoneal tumor-bearing mice; figure c shows bright-field, fluorescence, and afterglow luminescence imaging images acquired after laparotomy 2.5 h after intravenous injection of SAN-MO into the peritoneal tumor-bearing mice; and figure d shows the noise ratio (SBR) quantitatively calculated from the afterglow and fluorescence imaging signals acquired from the abdominal region of the mice after laparotomy, where SBR = I. ROI 1-I ROI 3 / I ROI 2-I ROI 3 (n=3); e is the bright field (top) and afterglow (bottom) image of the tumor removed under afterglow signal guidance (scale bar represents 5mm); f is the H&E stained image of the tumor section, according to the image number in Figure e (scale bar represents 400μm). Detailed Implementation

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0071] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0072] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0073] Example 1

[0074] A method for preparing a multifunctional organic afterglow luminescent nanomaterial (SAN-M) includes the following steps:

[0075] 0.25 mg of methylene blue derivative (mMB), 50 mg of polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer (PEG-b-PPG-b-PEG), and 5 mg of distearate phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG) were added. 2000 Mix thoroughly in 1 mL of tetrahydrofuran, and rapidly inject into 9 mL of deionized water under continuous vigorous sonication. Continue sonication for 10 min, then slowly remove the tetrahydrofuran with a gentle nitrogen stream. Concentrate the SAN-M nanoparticle solution by ultracentrifugation using a 30 kDa ultrafiltration tube. Finally, dilute the concentrated SAN-M nanoparticle solution with 1×PBS buffer and store at 4°C protected from light.

[0076] The preparation method of the methylene blue derivative (mMB) is as follows:

[0077] S1. A chloroform solution (100 mL) of iodine (15 mmol, CAS No. 7553-56-2) was added dropwise to a chloroform solution (50 mL) of phenothiazine (5 mmol, CAS No. 92-84-2), and the mixture was stirred in an ice bath for 4 h. The precipitate was collected by filtration, washed with chloroform (3 × 100 mL), and dried. No further purification was required to give a black powder (Product 1, 4.4 mmol, yield 88%).

[0078] S2. Dibutylamine (14 mmol, CAS No. 111-92-2) was added to a methanol solution (40 mL) of product 1 (1.4 mmol), and the mixture was stirred overnight at room temperature. Further purification was performed by column chromatography (eluent: dichloromethane / methanol, v / v 50:1) to give a dark purple solid (product mMB, 0.8 mmol, yield 55%).

[0079] The synthetic steps for methylene blue derivatives (mMB) are as follows:

[0080]

[0081] Figure 1 This is a schematic diagram of the preparation of multifunctional organic afterglow luminescent nanomaterials (SAN-M). SAN-M nanoparticles were synthesized by a co-assembly method and then characterized.

[0082] Figure 2The images show the hydrated particle size distribution and transmission electron microscopy (TEM) images of SAN-M nanoparticles in 1×PBS buffer. The results indicate that the SAN-M nanoparticles have a relatively uniform particle size, mostly distributed in the range of 8–28 nm, and the TEM images show them as nanoparticles.

[0083] Figure 3 The normalized absorption spectrum of SAN-M nanoparticles (5 μmol) in 1×PBS buffer is shown. The results indicate that SAN-M nanoparticles have the optimal absorption peak at 675 nm.

[0084] Figure 4 The normalized fluorescence spectrum of SAN-M nanoparticles (5 μmol) in 1×PBS buffer is shown. The results indicate that SAN-M nanoparticles can emit fluorescence at a wavelength of 700 nm, located in the near-infrared band.

[0085] Figure 5 The normalized afterglow emission spectrum of SAN-M nanoparticles in 1×PBS buffer is shown. The results indicate that after excitation at 660 nm is stopped, SAN-M nanoparticles can emit afterglow emission at a wavelength of approximately 710 nm.

[0086] Figure 6 This image compares the afterglow emission images and quantitative data of SAN-M nanoparticles with those of SAN-C (NPs-Ce4) and SAN-P (NPs-MEHPPV) nanoparticles in 1×PBS buffer. The results show that, regardless of whether excited by 660 nm light or white light, the afterglow emission intensity of SAN-M nanoparticles is significantly higher than that of the other two common organic afterglow nanoparticles after the light source is removed.

[0087] Figure 7 Image of afterglow emission from SAN-M nanoparticles and corresponding quantitative data; where a represents the afterglow emission produced by SAN-M nanoparticles. 1 The graph shows the change in O2 amount with 660 nm laser irradiation time, represented by the fluorescence enhancement (F / F0) of the singlet oxygen fluorescent probe SOSG (1 μM); b represents the O2 generated by SAN-M nanoparticles. ·- The graph shows the change in the amount of irradiation time with 660 nm laser light, represented by the fluorescence enhancement (F / F0) of the fluorescent indicator probe DHR123 (5 μM); c represents the effect of photoexcitation and external source irradiation on SAN-M nanoparticles. 1 O2 and O2 ·- Images and corresponding quantitative data of afterglow emission produced under photoexcitation. The results show that the nanoparticles can generate a large amount of afterglow emission under photoexcitation. 1 O2 and small amounts of O2 ·- And plays an important role in the afterglow emission process, and with 1O2 plays a dominant role.

[0088] Figure 8 The figures show the absorption spectrum changes of SAN-M nanoparticles before and after 660 nm light irradiation, the afterglow luminescence data of mMB, and a schematic diagram of the mechanism study. Among them, a is the absorption spectrum changes of SAN-M nanoparticles before and after 660 nm light irradiation; b is the high-performance liquid chromatography-mass spectrometry analysis results of mMB before and after 660 nm light irradiation; c is the MADLI-TOF mass spectrometry analysis of mMB after 660 nm light irradiation; and d is a schematic diagram of the afterglow luminescence mechanism study of mMB. Figure 8 The results show that the afterglow luminescence of mMB is accompanied by its own degradation and the formation of new substances. Based on this, we propose a mechanism for the afterglow luminescence of mMB, namely, the generation of gas by mMB under photoexcitation. 1 O2, then 1 O2 forms the mMB-dioxane reactive intermediate via cycloaddition of the vinyl double bond (C=C), and the decomposition of the reactive intermediate ultimately leads to the afterglow emission of mMB. Since the afterglow emission of mMB is highly similar to its corresponding fluorescence spectrum, it is speculated that the energy released during the spontaneous decomposition of the mMB-dioxane reactive intermediate promotes the entry of mMB into its excited state mMB*, ultimately returning to its ground state through afterglow emission, while simultaneously generating degradation fragments.

[0089] Figure 9 The figures show the sonoluminescence data of SAN-M nanoparticles. Figure a is a schematic diagram of afterglow emission generated by ultrasonic excitation; figure b shows afterglow emission images and corresponding quantitative data of SAN-M nanoparticles under different ultrasonic power excitations; figure c shows afterglow emission images and corresponding quantitative data of SAN-M nanoparticles under different ultrasonic irradiation times; and figure d is the normalized ultrasonic afterglow emission spectrum of SAN-M nanoparticles in 1×PBS buffer. The results indicate that SAN-M nanoparticles exhibit sonoluminescence, meaning that after ultrasonic irradiation, they can produce afterglow emission with a wavelength of approximately 710 nm.

[0090] Figure 10 The photoacoustic properties of SAN-M nanoparticles are shown in Figure 1; where a represents the photoacoustic spectra of SAN-M nanoparticles at different concentrations, and b represents the photoacoustic signal intensity of SAN-M nanoparticles at different concentrations. The results show that SAN-M nanoparticles exhibit photoacoustic signals with a wavelength of approximately 680 nm, and their intensity increases with increasing concentration.

[0091] Example 2

[0092] A method for preparing a multifunctional organic afterglow luminescent nanomaterial (SAN-MO) includes the following steps:

[0093] 0.25 mg of methylene blue derivative (mMB), 50 mg of polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer (PEG-b-PPG-b-PEG), and 5 mg of distearate phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG) were added. 2000 Mix thoroughly in 1 mL of tetrahydrofuran, and rapidly inject into 9 mL of deionized water under continuous vigorous sonication. Continue sonication for 10 min, then slowly remove the tetrahydrofuran with a gentle nitrogen stream. Concentrate the SAN-MO nanoparticle solution by ultracentrifugation using a 30 kDa ultrafiltration tube. Finally, dilute the concentrated SAN-MO nanoparticle solution with 1×PBS buffer and store at 4°C protected from light.

[0094] Among them, methylene blue derivative (mMB-ONOO) - The preparation method of ) is as follows:

[0095] 0.17 mmol of mMB was dissolved in 2 mL of dichloromethane and mixed with an aqueous solution of sodium dithionite (Na₂S₂O₄, 0.68 mmol, CAS No. 7775-14-6) and sodium bicarbonate (NaHCO₃, 1.02 mmol, CAS No. 144-55-8). The mixture was stirred at room temperature under nitrogen protection until the organic phase changed from blue to yellow, after which the aqueous phase was removed. Triethylamine (TEA, 0.26 mmol, CAS No. 121-44-8) and triphosgene (TPG, 0.51 mmol, CAS No. 32315-10-9) were added to the above reaction solution, and the mixture was reacted at 0 °C for 1 h and then concentrated under vacuum. The residue was purified by scintillation column chromatography using dichloromethane as the eluent to give a yellow product (product 2, 0.10 mmol, yield 57%). Next, potassium carbonate (K₂CO₃, 0.30 mmol, CAS No. 584-08-7), 4-dimethylaminopyridine (DMAP, 0.15 mmol, CAS No. 1122-58-3), and pinacol ester of 4-(hydroxymethyl)phenylboronic acid (68 mg, CAS No. 302348-51-2) were dissolved in dichloromethane solution. Product 2 (50 mg, 0.10 mmol) was added dropwise, and the mixture was stirred at room temperature under nitrogen protection until thin-layer chromatography showed that the reaction was complete. The mixture was then concentrated under vacuum, and the residue was purified by scintillation column chromatography using dichloromethane as the eluent to obtain a pale blue product (mMB-ONOO). - 15mg, yield 21%).

[0096] methylene blue derivative (mMB-ONOO) - The synthesis steps of ) are as follows:

[0097]

[0098] Figure 11 This is a schematic diagram illustrating the preparation of activatable multifunctional organic afterglow luminescent nanomaterials (SAN-MO). SAN-MO nanoparticle solutions were synthesized via a co-assembly method and characterized. They can achieve specific activation by peroxynitrite ions, emitting afterglow luminescence and fluorescence at approximately 710 nm, as well as a photoacoustic signal at 680 nm, with the afterglow luminescence showing the most superior activation contrast.

[0099] Figure 12 This is a schematic diagram illustrating the response mechanism of SAN-MO nanoparticles. (mMB-ONOO) - Modification of its nitrogen site 10 with a responsive group disrupts the conjugated structure at the center of the phenothiazine ring, rendering the molecule's original optical properties, including afterglow luminescence, fluorescence, and photoacoustic signal quenching, in a "signal-off" state. However, upon the addition of peroxynitrite, it specifically removes the borate ester structure and spontaneously undergoes 1,6-elimination, restoring the central conjugated structure and releasing free mMB molecules, thus enabling the activation of afterglow luminescence, fluorescence, and photoacoustic signals. The afterglow luminescence process involves mMB generating singlet oxygen and a small amount of superoxide anion under pre-illumination. These are oxidized by the reactive oxygen species to form a high-energy reactive intermediate. This unstable intermediate spontaneously degrades, releasing energy in the form of photons, thereby achieving afterglow luminescence.

[0100] Figure 13 The images show the absorption, fluorescence, afterglow emission, and photoacoustic spectra of the SAN-MO nanoparticles before and after their response; where a is the absorption spectrum, b is the fluorescence spectrum, c is the afterglow emission spectrum, and d is the photoacoustic spectrum. The results show that before the addition of peroxynitrite, all optical signals of the nanoparticles were completely off. However, after the addition of peroxynitrite, a significant absorption peak appeared in the 400–800 nm range, with the optimal absorption at 675 nm, accompanied by a color change in the test solution from colorless to blue. Furthermore, fluorescence and afterglow emission at 710 nm were significantly enhanced, and the photoacoustic signal at 680 nm was also activated. This indicates that the nanoparticles can achieve activated multimodal imaging integrating afterglow emission, fluorescence, and photoacoustic imaging.

[0101] Figure 14 The image shows the activation ratio data of SAN-MO nanoparticles before and after response. The results show that, compared to the signal ratios before and after fluorescence and photoacoustic signal responses, these activatable organic afterglow nanoparticles exhibit an extremely superior afterglow emission activation ratio, approximately 4523, which is two orders of magnitude higher than fluorescence and approximately 30 times higher than the photoacoustic response activation ratio. This indicates that these nanoparticles can not only achieve multimodal activatable imaging, but also possess superior activation contrast in their activatable afterglow.

[0102] Figure 15This diagram illustrates the response specificity of SAN-MO nanoparticles. Figure a shows the relative afterglow emission signal intensity under the influence of different reactive oxygen species (RONSs); figure b shows the relative fluorescence signal intensity under the influence of different RONSs; and figure c shows the relative photoacoustic signal intensity under the influence of different RONSs. The results show that the nanoparticles only activate the corresponding afterglow emission, fluorescence, and photoacoustic signals under the influence of peroxynitrite ions. Under the influence of other reactive oxygen species and RONSs, the corresponding optical signals remain closed, indicating that the nanoparticles possess superior response specificity.

[0103] Figure 16 The graphs show the variation of activation signal intensity with peroxynitrite concentration; where a represents the variation of afterglow luminescence activation signal intensity with peroxynitrite concentration; b represents the variation of fluorescence activation signal intensity with peroxynitrite concentration; and c represents the variation of photoacoustic activation signal intensity with peroxynitrite concentration. The results show that the activation of all optical signals is linearly related to the peroxynitrite concentration. The detection limits for peroxynitrite by afterglow luminescence, fluorescence, and photoacoustic are 36.8 nM, 0.4 μM, and 0.5 μM, respectively, indicating that afterglow luminescence has superior detection sensitivity.

[0104] Example 3

[0105] A method for preparing a multifunctional organic afterglow luminescent nanomaterial is the same as the preparation method in Example 1, except that the mass of distearate phosphatidylethanolamine-polyethylene glycol is 1.25 mg.

[0106] Example 4

[0107] A method for preparing a multifunctional organic afterglow luminescent nanomaterial is the same as the preparation method in Example 1, except that the mass of distearate phosphatidylethanolamine-polyethylene glycol is 2.5 mg.

[0108] With DSPE-mPEG 2000 With the increase in proportion, the stability of nanoparticles increases, but the quenching of optical properties becomes more pronounced.

[0109] Example 5

[0110] A method for preparing a multifunctional organic afterglow luminescent nanomaterial is the same as that in Example 1, except that 5 mg of distearate phosphatidylethanolamine-polyethylene glycol (DSPE-mPEG) is used. 2000 Replace with 5mg polystyrene-polyacrylic acid (PS-PAA).

[0111] Compared to Example 1, the afterglow intensity of Example 5 was significantly reduced under the same conditions, indicating that the quenching effect of PS-PAA was more obvious.

[0112] Comparative Example 1

[0113] A method for preparing a multifunctional organic afterglow luminescent nanomaterial is the same as the preparation method in Example 1, except that 5 mg of distearate phosphatidylethanolamine-polyethylene glycol is not added.

[0114] During the ultrafiltration concentration process, the solution in the lower layer of the ultrafiltration tube is blue. Based on the experimental phenomenon, it indicates that there are uncoated single molecules. That is, the single PEG-b-PPG-b-PEG polymer cannot obtain structurally stable nanoparticles, which easily causes leakage of internal hydrophobic molecules.

[0115] Comparative Example 2

[0116] A method for preparing a multifunctional organic afterglow luminescent nanomaterial is the same as the preparation method in Example 1, except that 50 mg of polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer is not added.

[0117] Comparative Example 3

[0118] A method for preparing a multifunctional organic afterglow luminescent nanomaterial is the same as the preparation method in Example 1, except that 0.25 mg of methylene blue derivative (mMB) and 5 mg of polystyrene-polyacrylic acid (PS-PAA) are mixed evenly in 1 mL of tetrahydrofuran.

[0119] In both Comparative Examples 2 and 3, the lower layer solution in the ultrafiltration tube was colorless during the ultrafiltration process, indicating that the simple DSPE-mPEG solution was colorless. 2000 Both polystyrene-polyacrylic acid (PS-PAA) polymer coating and structurally stable nanoparticles can be obtained. However, further characterization of their fluorescence and afterglow properties showed that the fluorescence signal was significantly weakened, while the afterglow signal was completely quenched. Experimental phenomena demonstrate that single DSPE-mPEG... 2000 Nanoparticles assembled from polystyrene-polyacrylic acid (PS-PAA) polymers are prone to optical quenching, possibly because different polymers affect the stacking pattern between internal molecules.

[0120] Test Example 1

[0121] Pro-tumor inflammation is considered one of the hallmarks of carcinogenesis, indicating that the tumor microenvironment includes peroxynitrite (ONOO). -The elevated levels of reactive oxygen species (ROS), including those found in peroxynitrite-activated organic afterglow nanoprobes, make them suitable for precise tumor detection. To demonstrate the unique advantages of activated organic afterglow luminescence in surgical navigation, this invention established a peritoneal metastasis model by intraperitoneal injection of breast cancer cells (4T1). The SAN-MO nanoparticle solution from Example 2 was then injected into the model mice via the tail vein as an activated organic afterglow nanoprobe, and the signal in the abdominal region was monitored in real time using afterglow luminescence imaging and fluorescence imaging. Due to the significant tissue autofluorescence in fluorescence imaging, the in vitro fluorescence signal did not show time-dependent changes, while afterglow luminescence showed a significant increase over time. Two and a half hours after injection, the mice underwent laparotomy, followed by afterglow and fluorescence imaging. Thanks to the extremely high signal-to-noise ratio of afterglow imaging, six suspicious lesions were removed under the guidance of the afterglow image. All removed suspicious lesions showed detectable afterglow signals in vitro and were verified as tumor tissue by hematoxylin and eosin (H&E) staining, with the smallest identifiable tumor diameter being approximately 1.5 mm.

[0122] Figure 17 The diagram below shows the test process and results for Test Example 1. Figure a shows a detailed schematic of the application of the activatable organic afterglow nanoprobe to detect peritoneal micrometastases; figure b shows representative afterglow luminescence images and average luminescence intensity of the region of interest (ROI) (n=3) recorded at different time points after intravenous injection of SAN-MO nanoparticles into 4T1 peritoneal tumor-bearing mice; figure c shows bright-field, fluorescence, and afterglow luminescence imaging images acquired after laparotomy 2.5 h after intravenous injection of SAN-MO into the peritoneal tumor-bearing mice; and figure d shows the noise ratio (SBR) quantitatively calculated from the afterglow and fluorescence imaging signals acquired from the abdominal region of the mice after laparotomy, where SBR = I. ROI 1-I ROI 3 / I ROI 2-I ROI 3 (n=3); e is the bright field (top) and afterglow (bottom) image of the tumor removed under afterglow signal guidance (scale bar represents 5mm); f is the H&E stained image of the tumor section, according to the image number in Figure e (scale bar represents 400μm).

[0123] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for preparing a multifunctional organic afterglow luminescent nanomaterial, characterized in that, The process includes the following steps: mixing methylene blue derivative, amphiphilic copolymer A and amphiphilic copolymer B in an organic solvent until homogeneous; adding water under ultrasonic conditions to form a nanoparticle solution; and removing the organic solvent from the nanoparticle solution to obtain the multifunctional organic afterglow luminescent nanomaterial. The methylene blue derivative is selected from one of the following structures: 、 、 、 ； The amphiphilic copolymer A is a polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer; The amphiphilic copolymer B is distearate phosphatidylethanolamine-polyethylene glycol.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the methylene blue derivative, amphiphilic copolymer A, and amphiphilic copolymer B is 1:200:(5~20).

3. The preparation method according to claim 1, characterized in that, The organic solvent is selected from one or more of ethanol, methanol, and tetrahydrofuran.

4. A multifunctional organic afterglow luminescent nanomaterial prepared by the method according to any one of claims 1 to 3.

5. A method for preparing an activatable multifunctional organic afterglow luminescent nanomaterial, characterized in that, The process includes the following steps: mixing methylene blue derivative, amphiphilic copolymer A and amphiphilic copolymer B in an organic solvent until homogeneous; adding water under ultrasonic conditions to form a nanoparticle solution; and removing the organic solvent from the nanoparticle solution to obtain the activated multifunctional organic afterglow luminescent nanomaterial. The methylene blue derivative is selected from one of the following structures: 、 、 、 ; The amphiphilic copolymer A is a polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer; The amphiphilic copolymer B is distearate phosphatidylethanolamine-polyethylene glycol.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the methylene blue derivative, amphiphilic copolymer A, and amphiphilic copolymer B is 1:200:(5~20).

7. The preparation method according to claim 5, characterized in that, The organic solvent is selected from one or more of ethanol, methanol, and tetrahydrofuran.

8. An activatable multifunctional organic afterglow luminescent nanomaterial prepared by the method according to any one of claims 5 to 7.

9. The application of the activatable multifunctional organic afterglow luminescent nanomaterial according to claim 8 in the preparation of nanoprobes, wherein the nanoprobes are used to detect peroxynitrite.

10. The application of the activatable multifunctional organic afterglow luminescent nanomaterial of claim 8 in the preparation of a reagent for bioimaging, wherein the bioimaging reagent is used for tumor imaging.

Images