A multi-modal diagnosis and treatment integrated AIE carbon nitride composite nano system and a preparation method thereof

Abstract

The application relates to the field of biological nanotechnology, and provides a multimodal diagnosis and treatment integrated AIE carbon nitride composite nano system and a preparation method thereof. The composite nano system takes selenium-doped carbon nitride nanosheets as a core carrier, realizes active targeting of hepatic stellate cells through VA-PEG2000 modification, simultaneously loads a first AIE photosensitizer and a second AIE photosensitizer with near-infrared two-region emission, and forms a double-photosensitizer composite system. The composite nano system has the dual imaging modalities of near-infrared two-region fluorescence imaging and photoacoustic imaging under the excitation of 660nm and / or 808nm laser, simultaneously generates reactive oxygen through a photodynamic effect to play a treatment function. The application further co-loads an anti-fibrosis drug in the nano system, realizes photodynamic-chemical combined treatment under the guidance of imaging, and solves the technical problems of single imaging modality and insufficient functional integration of an existing liver fibrosis nano diagnosis and treatment system, so that multimodal precise positioning and synergistic enhanced treatment integration of liver fibrosis lesions can be realized.

Description

Technical Field

[0001] This invention relates to the field of bionanotechnology, specifically to a multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem and its preparation method. Background Technology

[0002] Liver fibrosis is a critical pathological stage in the progression of various chronic liver diseases (such as hepatitis B, alcoholic liver disease, and non-alcoholic fatty liver disease) towards cirrhosis and even liver cancer. It is estimated that hundreds of millions of people worldwide are affected by chronic liver disease, a significant proportion of whom eventually develop liver fibrosis. Activated hepatic stellate cells (HSCs) are the core cells in the development of liver fibrosis. Their overactivation leads to excessive deposition of extracellular matrix, particularly a large accumulation of type I and type III collagen, ultimately damaging the normal structure of the liver and causing functional failure.

[0003] Currently, early diagnosis of liver fibrosis mainly relies on tissue biopsy and serological testing. The former has drawbacks such as being invasive, having large sampling errors, and being unsuitable for dynamic monitoring, while the latter has limited sensitivity and specificity. In terms of treatment, there are currently no approved specific anti-liver fibrosis drugs. Existing treatment methods mainly focus on removing the cause (such as antiviral therapy) or liver transplantation. However, for end-stage patients, donor resources are extremely limited, and the surgery is high-risk and expensive.

[0004] Chinese patent application CN117017944A discloses an AIE carbon nitride-based nanoparticle targeting liver fibrosis. Using selenium-doped carbon nitride as the core, it is modified with VA-PEG2000 and aggregation-induced emission (AIE) fluorescent small molecule TSI to achieve a single PDT therapeutic function. However, this technical solution has the following shortcomings: (1) It has a single imaging modality, relying only on fluorescence imaging under 660nm excitation, limiting imaging depth and resolution, making it difficult to accurately locate deep liver fibrosis lesions and provide real-time treatment guidance; (2) It has a single function, only possessing PDT therapeutic function, lacking synergistic integration with other treatment strategies (such as drug therapy); (3) It uses a single type of photosensitizer, failing to fully utilize the imaging advantages of the near-infrared II (NIR-II) window and making it difficult to achieve mutual verification of multimodal imaging information.

[0005] Therefore, there is an urgent need in this field to develop an intelligent nanodiagnostic platform that integrates multimodal imaging guidance and synergistic therapy to address the technical bottlenecks of limited imaging depth and insufficient functional integration in existing liver fibrosis diagnosis and treatment systems. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to provide a multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem and its preparation method.

[0007] This invention provides a multimodal therapeutically integrated AIE carbon nitride composite nanosystem, comprising: a selenium-doped carbon nitride nanosheet core; a targeted modification chain connected to the surface of the selenium-doped carbon nitride nanosheet; a first AIE photosensitizer and a second AIE photosensitizer co-loaded on the surface of the selenium-doped carbon nitride nanosheet; optionally, an anti-fibrotic drug co-loaded on the selenium-doped carbon nitride nanosheet; the first AIE photosensitizer exhibits fluorescence emission under NIR-II excitation at a wavelength of 808 nm, and the second AIE photosensitizer exhibits photoacoustic imaging capability under NIR-II excitation at a wavelength of 660 nm; the composite nanosystem simultaneously achieves dual-modal imaging guidance of NIR-II fluorescence imaging and photoacoustic imaging under laser excitation, as well as photodynamic therapy function.

[0008] Preferably, the targeted modification chain is VA-PEG2000, where VA is vitamin A and PEG2000 is polyethylene glycol with a molecular weight of 2000.

[0009] Preferably, the particle size of the selenium-doped carbon nitride nanosheet core is 80-120 nm.

[0010] Preferably, the first AIE photosensitizer is DB-PTZ (phenothiazine-malononitrile-based AIE photosensitizer), and the second AIE photosensitizer is TSI, wherein the structural formula of TSI is [insert structural formula here]. .

[0011] Preferably, the antifibrotic drug is nintedanib.

[0012] Secondly, the present invention provides a method for preparing the multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem, comprising the following steps:

[0013] (1) Selenium-doped carbon nitride nanosheets were prepared by programmed temperature calcination;

[0014] (2) The selenium-doped carbon nitride nanosheets obtained in step (1) are acidified to introduce carboxyl functional groups on their surface;

[0015] (3) VA-PEG2000 is covalently linked to the surface of the carboxylated selenium-doped carbon nitride nanosheets obtained in step (2) by esterification reaction to obtain VA-PEG2000 modified targeted nanosheets;

[0016] (4) The targeted nanosheets obtained in step (3) are mixed with the first AIE photosensitizer and the second AIE photosensitizer in an organic solvent, and the loading of the two photosensitizers is achieved through π-π stacking or electrostatic adsorption;

[0017] (5) The dual photosensitizer loaded nanosheets obtained in step (4) are mixed with the anti-fibrotic drug in an organic solvent to achieve co-loading of the drug through hydrophobic interaction;

[0018] (6) The product obtained in step (4) or step (5) is purified to obtain the multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem.

[0019] Furthermore, in step (1), the temperature of the programmed calcination is 600℃, the calcination time is 4h, and the calcination is carried out under a nitrogen protective atmosphere.

[0020] Furthermore, in step (2), the acidification treatment uses a nitric acid solution with a concentration of 3 mol / L and the treatment time is 24 h.

[0021] Furthermore, the esterification reaction described in step (3) is carried out in an EDC / NHS activation system, and the mass ratio of VA-PEG2000 to carboxylated selenium-doped carbon nitride nanosheets is 1:5.

[0022] Furthermore, in step (4), the mass ratio of the first AIE photosensitizer to the second AIE photosensitizer is 1:1.

[0023] Furthermore, the organic solvent mentioned in step (4) is selected from tetrahydrofuran.

[0024] Furthermore, in step (5), the mass ratio of the antifibrotic drug to the nanosheets is 1:10.

[0025] Thirdly, the present invention provides the application of the aforementioned multimodal diagnostic and therapeutic AIE carbon nitride composite nanosystem in the preparation of drugs, reagents or medical devices for the auxiliary diagnosis and / or treatment of liver fibrosis.

[0026] Preferably, the application includes dual-modal imaging guidance of NIR-II fluorescence imaging and photoacoustic imaging of liver fibrosis lesions under 660nm and / or 808nm laser irradiation, as well as combined treatment with photodynamic therapy and chemotherapy.

[0027] Compared with the prior art, the advantages of the present invention are as follows:

[0028] First, this invention is the first to integrate NIR-II fluorescence imaging and photoacoustic imaging into the AIE carbon nitride-based nanosystem, achieving precise diagnosis and treatment of liver fibrosis under multimodal imaging guidance. NIR-II fluorescence imaging provides highly sensitive, low-background-noise lesion localization, while photoacoustic imaging provides high-resolution, deep tissue structural information. The two imaging modalities corroborate each other, overcoming the technical limitations of single imaging modalities.

[0029] Secondly, by introducing a second AIE photosensitizer, this invention greatly enriches the functionality of the nano-therapeutic platform without increasing the complexity of the material system. In addition to photodynamic therapy, the synergistic design of the two photosensitizers also enables diversified upgrades in imaging modalities, fully embodying the design concept of "simplified structure and composite function".

[0030] Third, this invention further constructs a synergistic enhancement treatment system of PDT and chemotherapy by co-loading antifibrotic drugs, which significantly improves the antifibrotic efficacy and provides a solution to overcome the bottleneck of single PDT efficacy. Attached Figure Description

[0031] Figure 1 The NIR-II fluorescence intensity of VPSGCNs@2PS at different concentrations according to the present invention.

[0032] Figure 2 The photoacoustic signal intensity of VPSGCNs@2PS at different concentrations according to the present invention.

[0033] Figure 3 The survival rate of LX-2-activated hepatic stellate cells in different treatment groups of this invention.

[0034] Figure 4 The relative fluorescence intensity of intracellular ROS in different treatment groups is shown in this invention. Detailed Implementation

[0035] The technical solutions described in this invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only a part of the feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0036] Example 1: A multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem

[0037] (1) Preparation of selenium-doped carbon nitride nanosheets: Melamine (5.0 g) and selenoglucose (Se-glu, 0.5 g) were mixed and ground evenly, transferred to a ceramic boat, placed in a tube furnace, heated to 600 °C at a heating rate of 5 °C / min under nitrogen protection, calcined at a constant temperature for 4 h, and naturally cooled to room temperature to obtain crude selenium-doped carbon nitride nanosheets. The crude product was dispersed in deionized water, sonicated for 2 h, and centrifuged at 5000 rpm for 10 min to remove large particles that were not completely peeled off. The supernatant was collected by centrifuging at 12000 rpm for 20 min to collect the nanosheet precipitate, and freeze-dried to obtain selenium-doped carbon nitride nanosheets with a particle size of approximately 80-120 nm.

[0038] (2) Carboxylation treatment: The selenium-doped carbon nitride nanosheets (100 mg) obtained in step (1) were dispersed in 3 mol / L nitric acid solution (50 mL), stirred at room temperature for 24 h, the precipitate was collected by centrifugation, washed with deionized water until neutral, and freeze-dried to obtain carboxylated selenium-doped carbon nitride nanosheets.

[0039] (3) VA-PEG2000 targeted modification: Carboxylated selenium-doped carbon nitride nanosheets (50 mg) were dispersed in MES buffer (pH 5.5, 20 mL), and EDC (100 mg) and NHS (60 mg) were added sequentially, and the mixture was activated at room temperature for 30 min. Then, VA-PEG2000-NH2 (10 mg, wherein VA-PEG2000-NH2 was synthesized by a known method, i.e., VA was converted to VA-hemisuccinate by succinic anhydride, and then condensed with NH2-PEG2000-NH2 in the presence of EDC / NHS) was added, and the mixture was reacted at room temperature for 24 h. After the reaction was completed, the nanosheets were dialyzed in deionized water for 72 h using a dialysis bag with a molecular weight cutoff of 14000 Da, and then freeze-dried to obtain VA-PEG2000 modified targeted nanosheets, denoted as VPSGCNs.

[0040] (4) Dual photosensitizer loading: The VPSGCNs (20 mg) obtained in step (3) were dispersed in tetrahydrofuran (10 mL), and the first AIE photosensitizer DB-PTZ (2 mg) and the second AIE photosensitizer TSI (2 mg) were added. The mixture was stirred at room temperature in the dark for 24 h. After the reaction was completed, the mixture was dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cutoff of 14000 Da to remove unbound free photosensitizers. The mixture was then freeze-dried to obtain the dual photosensitizer loaded nanosystem, denoted as VPSGCNs@2PS.

[0041] (5) Drug co-loading: The VPSGCNs@2PS (10 mg) obtained in step (4) and nintedanib (1 mg) were co-dispersed in tetrahydrofuran (5 mL) and stirred at room temperature in the dark for 12 h. The organic solvent and free drug were removed by dialysis, and the composite nanosystem simultaneously loaded with dual photosensitizers and antifibrotic drugs was obtained by freeze drying, denoted as VPSGCNs@2PS / NDNB.

[0042] Comparative Example 1: Single photosensitizer AIE carbon nitride nanoparticles

[0043] This comparative example follows the method of Example 1 in CN117017944A to prepare VA-PEG2000 modified selenium-doped carbon nitride nanoparticles (VPSGCNs@TSI) loaded only with TSI, without the second AIE photosensitizer component and without the co-loading of antifibrotic drugs.

[0044] Comparative Example 2: Two photosensitizers but without drug loading

[0045] This comparative example is used to contrast nanosystems loaded with only dual photosensitizers but not with antifibrotic drugs, verifying the necessity of co-loading of antifibrotic drugs for achieving photodynamic-chemical synergistic enhancement of therapy.

[0046] The difference between this comparative example and Example 1 is that the drug co-loading in step (5) is not performed. That is, the dual photosensitizer loaded nanosystem obtained in step (4) is directly used as the final product after purification. The remaining steps are exactly the same as in Example 1.

[0047] Comparative Example 3: Carbon-free self-oxygenated core

[0048] This comparative example is used to compare nanoparticles formed solely by the self-assembly of two photosensitizers without a selenium-doped carbon nitride core, to verify the necessity of selenium-doped carbon nitride cores to enhance photodynamic therapy function by self-supplying oxygen through photocatalytic water splitting in an oxygen-deficient microenvironment.

[0049] The difference between this comparative example and Example 1 is that the preparation and targeted modification of selenium-doped carbon nitride nanosheets in steps (1) to (3) are omitted. Instead, VA-PEG2000-NH2 (10 mg), DB-PTZ (2 mg), and TSI (2 mg) are dissolved together in tetrahydrofuran (5 mL) and slowly added dropwise to deionized water (20 mL) under stirring. Nanoparticles are formed through self-assembly and purified by dialysis as the final product. The remaining steps are the same as in Example 1.

[0050] Experimental Example 1: Characterization of Multimodal Imaging Performance of Composite Nanosystems

[0051] The VPSGCNs@2PS composite nanosystem prepared in Example 1 was dispersed in PBS to prepare suspensions with a series of concentration gradients (0, 25, 50, 100, 200 μg / mL). The NIR-II emission spectrum was measured using a fluorescence spectrometer with an excitation wavelength of 808 nm, and the fluorescence intensity at 1050 nm was recorded. The photoacoustic signal was measured using a multispectral photoacoustic tomography (MSOT) system with an excitation wavelength of 660 nm. The fluorescence (303 a.u.) and photoacoustic signal (0.15 au.) of VPSGCNs loaded only with TSI (single photosensitizer control group) at a concentration of 200 μg / mL were used as a comparison.

[0052] like Figure 1 As shown, under 808 nm excitation, VPSGCNs@2PS exhibited a significant fluorescence emission peak at 1050 nm. The fluorescence intensity increased linearly with increasing concentration, reaching 840 au at 200 μg / mL, while the single photosensitizer control group only reached approximately 300 au, indicating an approximately 2.8-fold increase in fluorescence quantum yield. Figure 2As shown, the photoacoustic signal intensity also increased linearly with concentration. The experiment revealed that the lowest detectable concentration was 5 μg / mL, and the photoacoustic signal at 200 μg / mL was 1.72 au, while the control group was only 0.15 au. These results demonstrate that the dual photosensitizer design of this invention significantly improves the NIR-II fluorescence and photoacoustic dual-modal imaging performance, providing a material basis for high-sensitivity, high-resolution diagnosis of liver fibrosis lesions.

[0053] Experimental Example 2: In vitro evaluation of the efficacy of anti-hepatic fibrosis drugs

[0054] Logarithmic growth phase LX-2 human hepatic stellate cells were treated with TGF-β1 (5 ng / mL) for 24 h to induce activation. Activated LX-2 cells were then divided into the following groups: Group A, blank control group (PBS); Group B, laser irradiation only; Group C, Comparative Example 1 (660 nm laser + VPSGCNs@TSI); Group D, Comparative Example 2 (660 nm laser + VPSGCNs@2PS, dual photosensitizer without drug); Group E, Comparative Example 3 (660 nm laser + dual photosensitizer assembly without carbon nitride core); Group F, Example 1 (660 nm laser + VPSGCNs@2PS / NDNB, dual photosensitizer + nintedanib). All nanoparticles were concentrated to a final concentration of 50 μg / mL. After incubation for 6 h with the corresponding nanoparticles, the cells were irradiated with a 660 nm laser (0.5 W / cm²). 2 Cells were irradiated (10 min) and cultured for another 24 h. Cell viability was determined using the CCK-8 assay, and intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe. The relative values ​​of each group were calculated by normalizing the ROS fluorescence intensity of group C to 1.0.

[0055] like Figure 3 As shown, the cell survival rate was 52.3% in group C, significantly decreased to 31.4% in group D, 48.6% in group E, and further decreased to 12.2% in group F. Figure 4 As shown, regarding the relative fluorescence intensity of ROS, group C was 1, group D was 2.31, comparative group 3 was 1.18, and group F was 2.53. These results indicate that group D significantly increased ROS levels and reduced cell viability, but the viability remained above 30%; while group E, lacking the self-oxygenation capacity of selenium-doped carbon nitride, had a therapeutic effect comparable to that of a single photosensitizer. This invention, by co-loading antifibrotic drugs and superimposing chemotherapy on the highly efficient ROS level of dual photosensitizers, achieved synergistic enhancement of photodynamic and chemical therapy, reducing cell viability to approximately 12%, significantly superior to all comparative groups.

[0056] In summary, this invention provides a multimodal, integrated AIE (hepatic fibrosis) carbon nitride composite nanosystem. Using selenium-doped carbon nitride nanosheets as the core carrier, it achieves active targeting of hepatic stellate cells via VA-PEG2000. The surface is co-loaded with a first AIE photosensitizer, DB-PTZ (near-infrared II fluorescence emission), and a second AIE photosensitizer, TSI (photoacoustic imaging), and further co-loaded with the anti-fibrotic drug nintedanib. Under 660 nm and 808 nm laser excitation, this composite nanosystem can simultaneously achieve dual-modal imaging guidance of near-infrared II fluorescence imaging and photoacoustic imaging. It also generates reactive oxygen species through photodynamic effects, combining with anti-fibrotic drugs to achieve photodynamic-chemical synergistic enhanced therapy, thus solving the problems of single imaging modality and insufficient functional integration in existing liver fibrosis diagnosis and treatment systems.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A multimodal, integrated diagnostic and therapeutic AIE carbon nitride composite nanosystem, characterized in that, The composite nanosystem comprises: a selenium-doped carbon nitride nanosheet core; a targeted modification chain attached to the surface of the selenium-doped carbon nitride nanosheet; a first AIE photosensitizer and a second AIE photosensitizer co-loaded on the surface of the selenium-doped carbon nitride nanosheet; and an anti-fibrotic drug co-loaded on the selenium-doped carbon nitride nanosheet. The first AIE photosensitizer exhibits fluorescence emission under near-infrared II wavelength excitation at 808 nm, and the second AIE photosensitizer exhibits photoacoustic imaging capability under 660 nm wavelength excitation. The composite nanosystem simultaneously achieves dual-modal imaging guidance of near-infrared II fluorescence imaging and photoacoustic imaging under laser excitation, as well as photodynamic therapy functionality.

2. The multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem according to claim 1, characterized in that, The targeted modification chain is VA-PEG2000, where VA is vitamin A and PEG2000 is polyethylene glycol with a molecular weight of 2000.

3. The multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem according to claim 1, characterized in that, The core of the selenium-doped carbon nitride nanosheet has a particle size of 80-120 nm.

4. The multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem according to claim 1, characterized in that, The first AIE photosensitizer is DB-PTZ, and the second AIE photosensitizer is TSI.

5. The multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem according to claim 1, characterized in that, The antifibrotic drug is nintedanib.

6. A method for preparing a multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem as described in any one of claims 1-5, characterized in that, The steps include the following: (1) Selenium-doped carbon nitride nanosheets were prepared by programmed temperature calcination; (2) The selenium-doped carbon nitride nanosheets obtained in step (1) are acidified to introduce carboxyl functional groups on their surface; (3) VA-PEG2000 is covalently linked to the surface of the carboxylated selenium-doped carbon nitride nanosheets obtained in step (2) by esterification reaction to obtain VA-PEG2000 modified targeted nanosheets; (4) The targeted nanosheets obtained in step (3) are mixed with the first AIE photosensitizer and the second AIE photosensitizer in an organic solvent, and the loading of the two photosensitizers is achieved through π-π stacking or electrostatic adsorption; (5) The dual photosensitizer loaded nanosheets obtained in step (4) are mixed with the anti-fibrotic drug in an organic solvent to achieve co-loading of the drug through hydrophobic interaction; (6) The product obtained in step (4) or step (5) is purified to obtain the multimodal diagnostic and therapeutic integrated AIE carbon nitride composite nanosystem.

7. The preparation method according to claim 6, characterized in that, In step (1), the temperature of the programmed calcination is 600℃ and the calcination time is 4h. The calcination is carried out under a nitrogen protective atmosphere.

8. The preparation method according to claim 6, characterized in that, The organic solvent mentioned in step (4) is tetrahydrofuran.

9. The preparation method according to claim 6, characterized in that, In step (5), the mass ratio of the anti-fibrotic drug to the nanosheets is 1:

10.

10. The use of a multimodal diagnostic and therapeutic AIE carbon nitride composite nanosystem according to any one of claims 1-5 in the preparation of drugs, reagents or medical devices for the auxiliary diagnosis and / or treatment of liver fibrosis.

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