Construction method of TMV and AIE hybrid material as well as product and application of TMV and AIE hybrid material

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method of a TMV and AIE hybrid material as well as a product and application of the TMV and AIE hybrid material. By optimizing TMV purification conditions, the problems of strip dispersion, strip taking difficulty and the like after ultracentrifugation are effectively solved. After TMV is extracted and purified, AIE is dissolved in a KP buffer solution to prepare an AIE molecular solution, then the purified TMV and the AIE molecular solution are mixed and incubated according to the volume ratio of 10: 1, the hybrid material with fluorescence tracing and photodynamic therapy activity is obtained, in-vivo and in-vitro visual tracking imaging and reactive oxygen species (ROS) release functions are achieved, the diagnosis and treatment integrated potential is shown, and the application prospect is wide. And the application boundary of the TMV in the fields of precision medicine and the like is expanded.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the construction method of TMV and AIE hybrid materials, their products, and applications. Background Technology

[0002] Natural nanomaterials, with their unique structures and properties, exhibit irreplaceable application value. Among them, virus-based nanomaterials, with their uniform size, stable structure, and strong modifiability, demonstrate enormous application potential. Tobacco mosaic virus (TMV), a typical natural nanotube material, is assembled from single-stranded RNA and capsid proteins. Its controllable aspect ratio and excellent mechanical properties make it a promising candidate for applications in the biomedical field.

[0003] TMV offers significant advantages. Its excellent biocompatibility results in a low level of immune response when interacting with organisms, laying the foundation for its in vivo application. Its easily modifiable surface properties allow researchers to conveniently introduce target molecules, drug molecules, or functional groups through electrostatic adsorption, chemical coupling, and genetic engineering, achieving precise targeting of specific cells or tissues. Its targeting properties allow it to accumulate at certain lesion sites without extensive modification, facilitating drug delivery and other applications. Based on these characteristics, TMV can serve as a carrier for precisely delivering chemotherapy drugs and gene therapies to lesions in the field of drug delivery; in vaccine carriers, it can load antigens and stimulate effective immune responses; and it can also play an important role as a structural unit in the construction of nanobiomedical materials. However, current TMV purification processes have significant shortcomings. While the 20%-40% continuous sucrose density gradient ultracentrifugation method commonly used in conventional literature can obtain TMV with relatively high purity to a certain extent, the TMV bands tend to diffuse after ultracentrifugation. This makes accurate extraction of the target band difficult, resulting in low extraction efficiency, significant sample loss, and large differences in purification results between different batches. The unstable purification effect restricts the further promotion and application of TMV in the biomedical field.

[0004] Besides the need to optimize the purification process, the functional limitations of TMV cannot be ignored. In the field of cancer treatment, although TMV possesses certain advantages, such as the ability to load anti-tumor drugs and achieve some targeted delivery, its lack of fluorescence tracking and photodynamic therapy capabilities is becoming increasingly apparent under the growing trend of precision medicine. The absence of fluorescence tracking makes it impossible to monitor the distribution, transport pathways, and interactions with tumor cells of TMV in vivo in real time, hindering precise control of the treatment process. The lack of photodynamic therapy capabilities prevents it from using specific wavelengths of light to generate reactive oxygen species to kill tumor cells, limiting its application in combination therapies. In today's increasingly precise medicine, achieving integrated diagnosis and treatment is a crucial development direction. These functional deficiencies of TMV make it difficult to meet the comprehensive needs of precise lesion localization, real-time monitoring, and efficient treatment in cancer treatment, significantly limiting its application expansion in this field.

[0005] In summary, to promote the development of TMV in biomedical materials research and application, it is urgent to optimize the TMV purification process and develop a novel hybrid material. The optimized purification process can achieve efficient and precise separation during TMV purification, simplifying the operation procedure, improving preparation efficiency and stability, and reducing costs. Simultaneously, the development of novel hybrid materials endows TMV with excellent fluorescence tracing capabilities and photodynamic therapy functions, enabling it to meet the needs of precision tumor treatment for drug delivery monitoring, lesion localization, and synergistic therapy. This overcomes the shortcomings of existing technologies in extraction, purification, and functionalization, opening up new pathways for the development of biomedical materials. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a novel hybrid material based on tobacco mosaic virus and its preparation method. The prepared TMV-AIE material achieves hybridization with aggregation-induced emission material (AIE) molecules during the TMV purification process, endowing it with fluorescence tracing and photodynamic therapy functions.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a method for constructing a TMV / AIE hybrid material, comprising the following steps: S1. Extract and purify TMV; S2. Preparation of AIE molecular solution: Dissolve AIE in KP buffer solution to prepare AIE molecular solution; S3. Mixed incubation: The TMV purified in step S1 and the AIE molecular solution prepared in step S2 are mixed at a volume ratio of 10:1 and incubated to obtain the TMV-AIE hybrid material.

[0008] Specifically, the extraction and purification in step S1 includes using a discontinuous sucrose gradient centrifugation method.

[0009] Preferably, the discontinuous sucrose gradient consists of 20%, 40%, and 80% sucrose solutions, and the centrifugation conditions are 100,000 × g for 4.5 hours.

[0010] Specifically, the concentration of the KP buffer solution in step S2 is 0.01M.

[0011] Specifically, in step S3, the concentration of TMV is 15 mg / mL and the concentration of AIE is 10 mM.

[0012] Specifically, the incubation conditions described in step S3 are 4-22°C for more than 4 hours.

[0013] Preferably, the incubation conditions in step S3 are 4°C for 4 hours.

[0014] In another aspect, the present invention provides a TMV-AIE hybrid material, which is prepared by the aforementioned construction method.

[0015] In another aspect, the present invention provides the application of the aforementioned TMV-AIE hybrid material in the preparation of bioimaging reagents.

[0016] In another aspect, the present invention provides the application of the aforementioned TMV-AIE hybrid material in the preparation of photodynamic therapy drugs.

[0017] In another aspect, the present invention provides the application of the aforementioned TMV-AIE hybrid material in the preparation of drug delivery systems.

[0018] In another aspect, the present invention provides a bioimaging reagent, including the aforementioned TMV-AIE hybrid material.

[0019] Specifically, bioimaging reagents also include pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipient may be selected from at least two of the following: imaging enhancers, dispersants, preservatives, and suspending agents. More preferably, the imaging enhancer may be selected from indocyanine green derivatives or graphene quantum dots.

[0021] More preferably, the dispersing excipient may be selected from hydroxypropyl methylcellulose or povidone K30. Specifically, the imaging reagent may be in the form of an injectable suspension or a lyophilized imaging agent; when it is a lyophilized imaging agent, it may also contain a lyophilized excipient, which may be selected from sorbitol or glucose.

[0022] In another aspect, the present invention provides a photodynamic therapy drug comprising the aforementioned TMV-AIE hybrid material.

[0023] Specifically, the photodynamic therapy drug also includes pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutically acceptable excipient may be selected from at least two of the following: dispersants, stabilizers, pH adjusters, osmotic pressure adjusters, and excipients.

[0025] More preferably, the dispersant may be selected from polysorbate 80, poloxamer 188, or polyethylene glycol 400. More preferably, the stabilizer may be selected from mannitol, sucrose or trehalose. More preferably, the pH adjuster may be selected from citrate-sodium citrate buffer or sodium dihydrogen phosphate-disodium hydrogen phosphate buffer. Specifically, the dosage form of the drug may be an injection, including lyophilized powder for injection or injection solution; when it is a lyophilized powder for injection, it also contains a lyophilization protectant, which may be selected from lactose or dextran 40.

[0026] In another aspect, the present invention provides a drug delivery system comprising the aforementioned TMV-AIE hybrid material.

[0027] Specifically, the drug delivery system also includes pharmaceutically acceptable excipients.

[0028] Preferably, the pharmaceutically acceptable excipient can be a self-contained solvent or a stabilizer.

[0029] More preferably, the co-solvent may be selected from propylene glycol or polyethylene glycol 300.

[0030] More preferably, the stabilizer may be selected from vitamin E or sodium sulfite.

[0031] In another aspect, the present invention provides the application of the aforementioned TMV-AIE hybrid material, bioimaging reagent, photodynamic therapy drug, or drug delivery system in the preparation of precision medicine diagnostic and therapeutic products for tumors.

[0032] Specifically, the tumors include, but are not limited to, breast cancer, ovarian cancer, pancreatic cancer, or colorectal cancer.

[0033] The beneficial effects of this invention are as follows: This invention achieves the hybridization of TMV and AIE through electrostatic adsorption, with a simple operation process. While retaining the TMV nanorod structure and good biocompatibility, it successfully endows the material with fluorescent tracer properties and photodynamic therapy functions. The resulting TMV-AIE hybrid material not only possesses stable and uniform molecular loading capacity and excellent optical properties, but also efficiently generates reactive oxygen species (ROS) under specific wavelength excitation, potentially applicable to photodynamic killing of tumor cells and significantly enhancing anti-tumor therapeutic effects. Furthermore, this material exhibits good design flexibility and multifunctional potential, and can be widely applied in various biomedical materials fields such as bioimaging, photodynamic therapy, drug delivery, and vaccine carriers, promoting the development of precision medicine and nanomedicine. Attached Figure Description

[0034] Figure 1 The TMV bands are blue, formed after centrifugation with discontinuous sucrose gradients of 20%, 40%, and 80%.

[0035] Figure 2 The image shows the morphology of TMV under a transmission electron microscope (TEM), with a scale bar of 200 nm.

[0036] Figure 3 The image shows the morphology of TMV-AIE (10:1) under transmission electron microscopy (TEM), with a scale bar of 100 nm.

[0037] Figure 4 (a) Protein purity analysis results for pure TMV and TMV-AIE hybrids at different ratios, determined by SDS-PAGE electrophoresis; (b) Mass spectrometry identification results of TMV bands, with protein scores calculated as -10*Log(P) based on the Mascot score histogram, where P is the probability that the observed match is a random event. A protein score greater than 70 is considered significant. p <0.05).

[0038] Figure 5 The results of nucleic acid gel electrophoresis for TMV, AIE, and TMV-AIE (10:1) are shown.

[0039] Figure 6 The graph shows the UV absorbance results for KP buffer, TMV, AIE, and TMV-AIE (10:1).

[0040] Figure 7 The results show the fluorescence intensity measurements for KP buffer, TMV, AIE, and TMV-AIE (10:1).

[0041] Figure 8(a) Particle size distribution of TMV-AIE, and (b) Zeta potential (ζ) measurements of TMV, AIE and TMV-AIE (10:1).

[0042] Figure 9 The generation of reactive oxygen species (ROS) at 525 nm under white light illumination (100 mW / cm²) by TMV, AIE, TMV-AIE (10:1) and DCFH.

[0043] Figure 10 The results show the phagocytic activity of 4T1 cells after co-incubation with PBS, TMV, AIE, and TMV-AIE (10:1) for 5 h.

[0044] Figure 11 The survival rates of 4T1 cells were obtained under light and non-light conditions after co-incubating with PBS, TMV, AIE, and TMV-AIE (10:1) for 2 hours.

[0045] Figure 12 The images show the fluorescence imaging results of different treatment groups after incubation with 4T1 cells. (a) Image of the control group, (b) AIE and (c) TMV-AIE (10:1) after co-incubation with cells for 8 h, scale bar 20 µm; (d) Image of the control group, (e) AIE and (f) TMV-AIE (10:1) after co-incubation with cells for 24 h, scale bar 10 µm.

[0046] Figure 13 The TMV bands were formed after centrifugation at 96,000 × g for 2 h using a continuous sucrose gradient of 20%–40%. These bands were relatively diffuse, making extraction difficult.

[0047] Figure 14 This shows the combination of AIE powder with TMV in different solvents. The solvents, from left to right, are DMSO, ethyl acetate, ethanol, and KP buffer.

[0048] Figure 15 The ROS generation of hybrid materials with different TMV to AIE ratios.

[0049] Figure 16 Fluorescence intensity results for hybrid materials with different TMV to AIE ratios.

[0050] Figure 17 The phagocytosis of hybrid materials with different TMV to AIE ratios in cells.

[0051] Figure 18 The survival rate of 4T1 cells under light and non-light conditions after co-incubation for 2 hours with PBS, TMV, AIE and different ratios of TMV-AIE. Detailed Implementation

[0052] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0053] Example 1 1.1 TMV Extraction In this embodiment, firstly, *Fogrus benthamiana* that has grown for 6–8 weeks is selected. Nicotiana benthamianaHealthy leaves were inoculated with TMV. The procedure was as follows: First, fine silicon carbide powder was evenly and lightly sprinkled on the leaf surface to increase friction. Then, KP buffer (pH 7.4) containing 0.01 mg / mL TMV was gently applied to the leaf surface to ensure even inoculation. 7 to 10 days after inoculation, when typical mosaic virus lesions appeared on the leaves but before they entered the senescence stage, the infected leaves were promptly harvested and immediately frozen at −80°C to prevent virus degradation. After freezing, the leaves were ground into a fine powder using a cryogenic grinder to ensure complete disruption of the cell structure. Subsequently, 0.1 M KP buffer (containing 0.2% (v / v) β-mercaptoethanol) pre-cooled to 4°C was added at a leaf mass to buffer volume ratio of 1:3, and the mixture was thoroughly homogenized. The homogenate was first filtered through two layers of gauze to remove large particles, then centrifuged at 11,000 × g for 20 minutes. The supernatant was collected and repeatedly filtered through two layers of gauze to ensure clarity. Next, an equal volume of a 1:1 mixture of chloroform and n-butanol was added to the supernatant. The mixture was then stirred in an ice bath for 30 minutes to ensure thorough mixing and promote the separation of proteins and impurities, avoiding foaming. The mixture was then centrifuged at 4,500 × g for 10 minutes to separate the organic and aqueous phases. The supernatant containing TMV was collected and kept on ice. Subsequently, 0.2 M sodium chloride, 8% (w / v) polyethylene glycol 8000 (PEG 8000), and 1% (v / v) Triton X-100 were added sequentially to this aqueous phase. The mixture was then slowly stirred in an ice bath for 30 minutes to promote viral aggregation and impurity removal. The mixture was incubated at 4°C for at least 1 hour, followed by centrifugation at 22,000 × g for 15 minutes to obtain a viral precipitate. The viral precipitate was resuspended in 0.1 M KP buffer and gently shaken overnight at 4°C to fully dissolve and stabilize the viral particles. Discontinuous sucrose gradients of 20% (w / v), 40% (w / v), and 80% (w / v) were prepared. The resuspended TMV protein solution was loaded onto the gradient solutions and centrifuged at 10,000 × g for 4.5 hours. After centrifugation, clear blue TMV bands were visible. Figure 1 After collecting the blue TMV characteristic bands in the gradient, multiple rounds of centrifugation at 3000 rpm for 15 minutes each were performed to gradually concentrate the sample to the target volume or concentration. Finally, the TMV concentration was accurately determined using a NanoDrop spectrophotometer, providing a precise quantitative basis for subsequent experiments and applications. All TMV concentrations used in subsequent experiments were based on NanoDrop measurements.

[0054] 1.2 Construction of TMV and AIE-61 hybrid materials The obtained TMV protein (15 mg / mL) solution was mixed with 10 mM AIE (C 32 H 29 N2S +The molecular solutions were mixed at a volume ratio of 10:1. AIE powder was dissolved in 0.01M KP buffer and sonicated for 5 minutes to ensure complete dissolution. Subsequently, TMV and AIE were co-incubated at 4°C for 4 hours to promote complete binding. The bound sample (TMV-AIE) was stored at 4°C for later use. The final TMV-AIE sample had a TMV concentration of 15 mg / mL and an AIE concentration of 0.046 mM. All subsequent experiments used TMV-AIE samples at this concentration.

[0055] 1.3 Negative Staining Experiment To observe the nanostructure and dispersion of TMV and TMV-AIE hybrid materials, this experiment employed transmission electron microscopy (TEM) combined with uranium acetate negative staining for characterization. 3 μL of a diluted TMV-AIE solution (approximately 0.6 mg / mL) was added dropwise to a carbon-copper mesh and allowed to stand at room temperature for about 1 minute to promote particle adsorption. Excess solution was then gently blotted away with filter paper. Next, 3 μL of 2% (w / v) uranium acetate solution (pH approximately 4.5–5.0) was added to the sample area and negatively stained for 60 seconds. Excess stain was removed again with filter paper to avoid damaging the sample. The treated copper mesh was allowed to air dry at room temperature. Finally, imaging was performed under TEM with an appropriate accelerating voltage (80–120 kV) to obtain information on particle morphology, size, and dispersion, verifying the material's structural characteristics and preparation effectiveness.

[0056] from Figure 2 The results show that pure TMV exhibits a typical uniform rod-like structure with good dispersibility; after hybridization with AIE, TMV-AIE (10:1) still retains the original rod-like morphology of TMV. Figure 3 No significant morphological changes were observed, indicating that the loading process of AIE did not disrupt the nanostructure of TMV. Furthermore, the hybrid material exhibited good monodispersity under electron microscopy, with no obvious aggregation, demonstrating its good stability in the buffer system.

[0057] 1.4 SDS-PAGE electrophoresis experiment Take an appropriate amount of purified TMV-AIE sample, mix it thoroughly with SDS sample buffer (containing β-mercaptoethanol) according to the specified ratio, and heat it in a 100℃ metal bath for 10 minutes to denature the viral proteins completely. After denaturation, briefly centrifuge the sample (about 30 seconds) to remove condensate or air bubbles, and add 20 μL of the sample solution to the wells of a pre-prepared 15% polyacrylamide gel. Perform electrophoresis separation under constant voltage of 110V. After electrophoresis, remove the gel and place it in Coomassie Brilliant Blue R-250 staining solution, gently shake and stain for about 30 minutes to make the protein bands clear. Then rinse several times with destaining solution containing methanol and acetic acid until the background is clear. Finally, take and record the protein band images to analyze the integrity and purity of the viral protein components in the TMV-AIE hybrid material.

[0058] As shown in Figure 4(a), both the extracted TMV and the TMV-AIE samples loaded with different proportions of AIE exhibited clear and single protein bands, indicating high purity and intact viral protein components, meeting the requirements for subsequent experiments. To further confirm the band identity, mass spectrometry analysis was performed on the TMV bands. The results (Figure 4(b)) showed that the main protein detected in the sample was the TMV capsid protein, with a Mascot score of 118, significantly higher than the threshold of 70 (p<0.05), corresponding to an Expect value of 9.1 × 10⁻⁶. -7 The number of matching peptides was 11. In summary, the mass spectrometry results clearly confirm that the submitted band is a TMV capsid protein.

[0059] 1.5 Nucleic Acid Gel Electrophoresis Experiment Weigh an appropriate amount of agarose powder and add 1X TAE buffer to prepare a 0.8% (w / v) agarose solution. Heat in a microwave oven until completely dissolved, ensuring the solution is clear and free of particles. After thorough mixing, slowly pour the hot solution into a pre-prepared gel casting mold, insert a comb to form uniform sample wells, and allow to stand at room temperature until the gel is completely solidified. After the gel solidifies, gently remove it and place it in an electrophoresis tank, adding sufficient 1X TAE buffer to cover the gel surface. Take 10 μL of TMV (15 mg / mL), AIE (0.046 mM), and TMV-AIE (TMV: 15 mg / mL, AIE: 0.046 mM) samples, respectively, and accurately load them into the corresponding sample wells. Set the electrophoresis conditions to 180V, 400mA and run for approximately 15 minutes to ensure complete sample separation. After electrophoresis, the gel is transferred to a gel imaging system, and strip images are captured using an appropriate excitation light source and filter to analyze and evaluate the hybridization effect of TMV and AIE and the electrophoretic migration characteristics.

[0060] according to Figure 5The electrophoresis results show that the TMV and AIE samples exhibit their own unique bands and fluorescence signals. In contrast, the TMV-AIE sample shows a stronger fluorescence intensity, significantly higher than either TMV or AIE alone. This result indicates that AIE successfully binds to TMV and maintains a stable hybrid state under electrophoretic conditions, thereby enhancing the overall fluorescence signal and validating the construction effect of the TMV-AIE hybrid system.

[0061] 1.6 Ultraviolet absorbance and fluorescence intensity experiment The sample (TMV-AIE, (TMV: 15 mg / mL, AIE: 0.046 mM)) prepared according to method 1.2 was used as a blank control with 0.01 M KP buffer. The characteristic absorption peaks of the sample were recorded by scanning the wavelength range of 200–800 nm using a UV-Vis spectrophotometer, and the spectral changes after AIE and TMV binding were analyzed.

[0062] In fluorescence spectroscopy, the sample is placed in a cuvette of a fluorescence spectrophotometer and excited at a wavelength of 488 nm. The emission spectrum curve is recorded, and the fluorescence emission characteristics and intensity of the hybrid material endowed by AIE are analyzed to evaluate its potential as a fluorescent tracer material.

[0063] Figure 6 The results showed that TMV exhibited a distinct absorption peak at approximately 260 nm, consistent with the typical spectral characteristics of viral protein RNA; both AIE and TMV-AIE showed characteristic absorption peaks at 488 nm, indicating that AIE had successfully hybridized with TMV. Figure 7 Fluorescence intensity measurements showed that the fluorescence intensity of TMV-AIE was significantly higher than that of TMV or AIE alone, indicating that the combination with AIE significantly enhanced the luminescent properties of the material. This result demonstrates that TMV-AIE not only retains the biological characteristics of TMV but also achieves excellent fluorescence performance, providing a reliable basis for its application in biological tracing and imaging.

[0064] 1.7 Dynamic Light Scattering (DLS) and Zeta Potential Experiments The TMV-AIE hybrid sample, TMV (15 mg / mL), and AIE (0.046 mM) were diluted 20-fold with 0.01 M KP buffer to ensure the solution was clear and bubble-free. The average hydrated particle size and particle size distribution of the samples were determined using a calibrated dynamic light scattering (DLS) instrument to analyze the aggregation state and uniformity of the hybrid nanotubes. Subsequently, the Zeta potential was measured under the same buffer conditions, and the surface charge values ​​of the samples were recorded to evaluate the impact of AIE molecule introduction on the charge properties and colloidal stability of viral particles, providing parameter basis for subsequent biocompatibility and functional studies.

[0065] The results show that ( Figure 8 The TMV-AIE hybrid sample (TMV: 0.75 mg / mL, AIE: 0.0023 mM) maintained a typical nanotube structure, with an average particle size similar to that of TMV alone and a relatively uniform distribution, indicating that the hybridization process did not cause significant particle aggregation. Zeta potential measurements showed that the introduction of the positive charge of the AIE molecules slightly reduced the negative charge on the TMV-AIE surface, but the overall structure remained negatively charged, and the colloidal stability remained good. This result indicates that AIE molecules have been successfully loaded onto TMV while maintaining the system's dispersibility and stability, providing strong evidence for subsequent biocompatibility and functional studies.

[0066] 1.8 ROS generation detection Take an appropriate amount of TMV-AIE sample solution, adjust it to the predetermined concentration according to experimental requirements, and prepare it in a suitable buffer solution. Using 1,3-diphenylisobenzofuran (DCFH) as the ROS fluorescent probe, add DCFH solution to the sample at the set concentration (40 μM) (TMV: 15 mg / mL, AIE: 0.046 mM) and TMV-AIE (10:1), mix thoroughly, and place in a quartz cuvette. Set up a control group (DCFH), TMV, AIE, and TMV-AIE, and irradiate them with a specified wavelength light source (0-700 nm) under specified power and time conditions. During irradiation, the absorption spectrum is collected every 10 seconds, and the change in the characteristic absorption peak of DCFH at 525 nm is recorded. By comparing the absorbance change rate of different groups, the ability of the sample to generate reactive oxygen species under light conditions is quantitatively evaluated, thereby determining the photodynamic activity and ROS generation efficiency of the TMV-AIE hybrid material.

[0067] according to Figure 9 The results showed that under light excitation, the ROS production of the TMV-AIE group was significantly higher than that under no-light conditions, indicating that it possesses significant photodynamic activity. Furthermore, compared with the TMV and AIE groups, TMV-AIE exhibited a faster and more efficient ROS generation rate under the same light conditions, demonstrating the advantages of this hybrid material in photodynamic therapy.

[0068] 1.9 Cell phagocytosis experiment Tumor cells 4T1 (Wuhan Pricella Biotechnology Co., Ltd. CL-0007) were injected at approximately 1×10⁻⁶. 5Cells were seeded at a density of 1 / well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator to ensure good cell adhesion. After discarding the culture medium, TMV-AIE solution was prepared using pre-warmed medium (Gibco, C11995500BT) at 37°C and added to the cells. After incubation for 5 hours, the supernatant was discarded, and the cells were gently washed 2–3 times with pre-warmed PBS to remove unbound or free samples. Subsequently, the cells were digested with 0.25% (w / v) (Tryspin-EDTA 1X) trypsin (Gibco, 25200056). After digestion was terminated, the cells were resuspended in serum-containing medium and gently pipetted to prepare a single-cell suspension. The cells were centrifuged at 1000×g for 5 minutes, the supernatant was discarded, and the cells were washed twice with pre-warmed PBS at 37°C and finally resuspended to an appropriate volume and concentration. The prepared cell samples were transferred to the flow cytometer detection tube, the corresponding excitation wavelength (488 nm) and detection channel were set, and no less than 10,000 cell events were collected. The fluorescence intensity distribution and average fluorescence intensity were analyzed, and the cell uptake efficiency under different treatment conditions was quantitatively compared, thereby evaluating the cell uptake characteristics and tracer performance of the material.

[0069] according to Figure 10 The results showed that the uptake of TMV-AIE in 4T1 cells was significantly higher than that of TMV and AIE alone, with a correspondingly enhanced average fluorescence intensity. This indicates that the hybrid material can enter cells more efficiently and accumulate intracellularly, enhancing its interaction with cells. These results not only validate the successful hybridization of TMV and AIE but also demonstrate its excellent cellular uptake characteristics and tracing potential, providing strong support for subsequent cellular and in vivo functional studies.

[0070] 1.10 Cell viability experiment 4T1 tumor cells (7×10) 6 (cells / mL) diluted to approximately 5 × 10⁻⁶ 3 Cells / well were seeded at a density of 1:1 in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator to ensure full adhesion. Subsequently, PBS, AIE (0.046 mM), and TMV-AIE (TMV: 15 mg / mL, AIE: 0.046 mM) sample solutions were added for treatment, and a light-illuminated group and a non-light-illuminated group were set up for comparison. After 2 h of incubation, the light-illuminated group was irradiated with white light for 10 min, followed by the addition of 10 μL of CCK-8 reagent to each well, and then incubated for another 1 h. The absorbance was measured at 450 nm using a microplate reader, and the relative cell viability was calculated to evaluate the cytotoxicity and photodynamic killing effect of the material.

[0071] according to Figure 11The results showed that the TMV group maintained nearly 100% cell viability under non-light conditions, while it decreased slightly to about 85% under light conditions, indicating that TMV was essentially non-toxic to cells and did not exhibit significant killing effects under light conditions. In contrast, the TMV-AIE hybrid material significantly inhibited cell survival under light conditions, with a survival rate of only about 52%, exhibiting a very strong photosensitizing killing effect. These results demonstrate that TMV-AIE possesses excellent phototriggered cytotoxicity and can efficiently induce cell death under light, validating its potential as a candidate material for photodynamic therapy.

[0072] 1.11 Cell Imaging Experiment 4T1 cells were loaded at approximately 2 × 10 4 Cells / well were seeded at a density of 1:1 in pre-sterilized glass-bottomed culture dishes or covered slides and incubated at 37°C, 5% CO2 for at least 24 hours to allow for full cell adhesion. The supernatant was discarded, and the cells were gently washed once with pre-warmed PBS. 200 µL of fresh culture medium (DMEM, Gibco, C11995500BT) and 10 µL of sample (Control (PBS added), AIE (0.046 mM), TMV-AIE (TMV: 15 mg / mL, AIE: 0.046 mM)) were added to each well. The cells were incubated for another 8 hours to promote uptake. The samples were then placed under a confocal laser scanning fluorescence microscope, and cell signals were scanned and acquired by selecting an appropriate excitation wavelength (488 nm) and channel parameters. Based on the obtained multi-channel images, the distribution, localization, and uptake of TMV-AIE within the cells were analyzed to evaluate its cell tracing performance and imaging characteristics.

[0073] according to Figure 12 Analysis of the results showed that, under confocal microscopy, the intracellular fluorescence intensity of the TMV-AIE treatment group was higher than that of the TMV or AIE groups alone, exhibiting a clear intracellular distribution signal. The AIE signal and mitochondrial channels showed significant spatial overlap, suggesting that most of the hybrid material was located in the mitochondrial region. These results indicate that TMV has an enrichment effect in tumor cells, effectively enhancing the uptake and accumulation of AIE molecules within tumor cells, thus improving imaging contrast and tracer visibility. These observations are consistent with the enhanced uptake results obtained by flow cytometry, indirectly verifying the efficient entry and stable sites of the TMV-AIE hybrid system at the cellular level.

[0074] Comparative Examples 1-3 Comparative Examples 1-3 are set up with reference to 1.2 in Embodiment 1 of the present invention. The differences between the comparative examples and 1.2 are shown in Table 1: Table 1

[0075] The results are as follows Figure 14 As shown, TMV is in a solidified state in DMSO, possibly due to the disassembly of its structure caused by DMSO; ethyl acetate also disrupts the conformation of the TMV protein; in ethanol, TMV precipitates, indicating incompatibility. However, TMV shows good compatibility in KP buffer. Combining the aforementioned characterization results, it can be concluded that the TMV nanostructures obtained under these conditions are intact and uniformly dispersed.

[0076] Comparative Examples 4-5 Comparative Examples 1-3 are set up with reference to 1.2 in Embodiment 1 of the present invention. The differences between the comparative examples and 1.2 are shown in Table 2: Table 2

[0077] Then, experiments were conducted according to steps 1.8, 1.6, 1.9, and 1.10 in Example 1, and the results are as follows: Figure 15 The results showed that the ROS production of TMV-AIE 10:1 was significantly higher than that of the 1:1 and 1:4 ratio samples, suggesting that it has better potential efficacy in inhibiting tumors and reducing inflammation.

[0078] Figure 16 As can be seen, the fluorescence intensity of TMV-AIE 10:1 is significantly higher than that of the 1:1 and 1:4 ratio samples, indicating that TMV enhances the AIE fluorescence signal.

[0079] Figure 17 The results showed that the TMV-AIE ratio of 10:1 resulted in the highest cellular uptake, indicating that this ratio is beneficial for cellular uptake. The 1:4 ratio was the second highest, while the uptake of the 1:1 ratio was similar to that of AIE alone. These results suggest that the ratio of TMV to AIE has a significant impact on phagocytic efficiency, with the 10:1 ratio likely being the most conducive to achieving efficient cellular uptake.

[0080] Figure 18 The results showed that, compared with non-illuminated conditions, illumination significantly enhanced the cytotoxicity of the TMV-AIE hybrid material, with the 10:1 ratio exhibiting the most significant inhibitory effect. This suggests that tumor cell survival is closely related to the ratio of TMV-AIE and illumination conditions. This phenomenon indicates that the presence of TMV may enhance the activity of AIE under illumination, thereby improving the photosensitizing killing effect on tumor cells, and that different ratios can modulate the efficacy of the hybrid material.

[0081] In summary, the TMV-AIE 10:1 ratio in Example 1 of this invention exhibits the best performance in terms of active molecule generation, signal tracking, cellular uptake, and light-mediated tumor killing efficacy.

[0082] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for constructing a TMV / AIE hybrid material, characterized in that, Includes the following steps: S1. Extract and purify TMV; S2. Preparation of AIE molecular solution: Dissolve AIE in KP buffer solution to prepare AIE molecular solution; S3. Mixed incubation: The TMV purified in step S1 and the AIE molecular solution prepared in step S2 are mixed at a volume ratio of 10:1 and incubated to obtain the TMV-AIE hybrid material.

2. The construction method according to claim 1, characterized in that, The extraction and purification in step S1 includes using a discontinuous sucrose gradient centrifugation method.

3. The construction method according to claim 2, characterized in that, The discontinuous sucrose gradient consisted of 20%, 40%, and 80% sucrose solutions, centrifuged at 100,000 × g for 4.5 hours.

4. The construction method according to claim 1, characterized in that, The concentration of the KP buffer in step S2 is 0.01M.

5. The construction method according to claim 4, characterized in that, The concentration of the KP buffer solution is 0.01M.

6. The construction method according to claim 1, characterized in that, In step S3, the concentration of TMV is 15 mg / mL and the concentration of AIE is 10 mM.

7. The construction method according to claim 1, characterized in that, The incubation conditions described in step S3 are 4-22℃ for more than 4 hours.

8. The construction method according to claim 7, characterized in that, The incubation conditions were 4°C for 4 hours.

9. A TMV-AIE hybrid material, characterized in that, It is prepared by any of the construction methods described in claims 1-8.

10. The application of the TMV-AIE hybrid material according to claim 9 in the preparation of bioimaging reagents.

11. The application of the TMV-AIE hybrid material according to claim 9 in the preparation of photodynamic therapy drugs.

12. The application of the TMV-AIE hybrid material of claim 9 in the preparation of a drug delivery system.

13. A bioimaging reagent, characterized in that, Includes the TMV-AIE hybrid material as described in claim 9.

14. The bioimaging reagent according to claim 13, characterized in that, The bioimaging reagent also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are selected from at least two of the following: imaging enhancers, dispersants, preservatives, and suspending agents.

15. A photodynamic therapy drug, characterized in that, Includes the TMV-AIE hybrid material as described in claim 9.

16. The photodynamic therapy drug according to claim 15, characterized in that, The photodynamic therapy drug further includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are selected from at least two of the following: powders, stabilizers, pH adjusters, osmotic pressure adjusters, and excipients.

17. The photodynamic therapy drug according to claim 16, characterized in that, The dosage form of the photodynamic therapy drug is a lyophilized powder for injection or an injection solution.

18. A drug delivery system, characterized in that, Includes the TMV-AIE hybrid material as described in claim 9.

19. The drug delivery system according to claim 18, characterized in that, The drug delivery system also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are selected from self-solvents or stabilizers.

20. The use of the TMV-AIE hybrid material of claim 9, the bioimaging reagent of claim 13 or 14, the photodynamic therapy drug of any one of claims 15-17, or the drug delivery system of claim 18 or 19 in the preparation of precision medicine diagnostic and therapeutic products for tumors.

21. The application according to claim 20, characterized in that, The tumor is breast cancer, ovarian cancer, pancreatic cancer, or colorectal cancer.

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