Anti-TPM-4 targeting gold / silver nanoshell molecular probe as well as preparation method and application thereof

Abstract

The invention discloses an anti-TPM-4 targeting gold / silver nanoshell molecular probe as well as a preparation method and application thereof, and belongs to the technical field of targeting and imaging molecular materials. The molecular probe prepared by the invention has a hollow and porous structure, can entrap drugs, can specifically bind to TPM-4 highly expressed in cells of tumors and the like, has high stability, and can be used for targeted drug-loading treatment of diseases of tumors and the like; the nano-material has intracellular electrocatalytic activity, can be used for imaging cells such as tumors, and is expected to provide assistance for real-time observation and monitoring of cells such as tumors; the hydrogel has no cytotoxicity / very low cytotoxicity, has no electrical stimulation toxicity, has good biocompatibility, and can be trustingly applied in an organism; the provided preparation method is simple and easy to operate, no toxic or harmful substance is generated, and the requirement for equipment is low.

Description

Technical Field

[0001] This invention relates to the field of targeted and imaging molecular materials technology, and in particular to an anti-TPM-4 targeted gold / silver nanoshell molecular probe, its preparation method, and its application. Background Technology

[0002] TPM-4, or tropomyosin 4, is a member of the tropomyosin family, a family of proteins widely found in muscle cells and many other cell types. Tropomyosin proteins typically bind to actin filaments, participating in the regulation of the structural stability of actin filaments and the dynamic changes of the cytoskeleton. TPM-4's role within cells extends beyond muscle contraction; it also participates in cell movement, morphology maintenance, signal transduction, and cell cycle regulation.

[0003] Studies have shown that the function and abnormal expression of the TPM-4 protein are closely related to various disease states. Current research findings mainly include the following aspects:

[0004] 1) Cancer: TPM-4 is abnormally expressed in various types of cancer, such as pancreatic cancer, lung cancer, and colorectal cancer. A study of transcriptomic expression and clinical information collected from CGGA and TCGA datasets of 998 glioma patients showed that TPM-4 is significantly associated with the malignant characteristics of gliomas. TPM-4 may promote tumor cell invasion and migration, and affect cell proliferation and apoptosis, thus being considered a potential target for cancer therapy.

[0005] 2) Cardiovascular diseases: TPM-4 expression in vascular smooth muscle cells is related to the process of vascular remodeling and may affect the progression of cardiovascular diseases such as hypertension and arteriosclerosis.

[0006] 3) Diabetes-related complications: In a high-glucose environment, the expression of TPM-4 in peritoneal mesothelial cells is downregulated, which may be related to peritoneal damage and peritoneal dialysis-related complications caused by diabetes.

[0007] 4) Nervous system diseases. Although the direct link between TPM-4 and the nervous system is rarely mentioned, the role of tropomyosin family members in neurons suggests that they may play a role in neurodevelopment and neurodegenerative diseases.

[0008] Imaging technology plays a crucial role in medical diagnosis and treatment: during diagnosis and rehabilitation examinations, it can help doctors understand abnormal changes in the body more accurately, detect lesions as soon as possible, and formulate effective treatment plans in a timely manner, even accurately identifying potential diseases before symptoms appear; it can also help doctors determine the nature and location of diseases and the patient's condition; and it can reduce invasive procedures, reducing patient pain and risks, etc.

[0009] Therefore, it is crucial to develop a molecular probe that is easy to prepare, can target multiple diseases, and has imaging capabilities. Summary of the Invention

[0010] The purpose of this invention is to provide an anti-TPM-4 targeted gold / silver nanoshell molecular probe, its preparation method, and its application, so as to provide a molecular material that is simple to prepare, has good targeting for a variety of diseases, has electrocatalytic activity and imaging ability, is non-cytotoxic and non-electrocatalytically toxic, and has good biocompatibility.

[0011] To achieve the above objectives, this invention provides a method for preparing an anti-TPM-4 targeting gold / silver nanoshell molecular probe. Using polycrystalline silver nanoparticles as templates and hydrogen peroxide as an etchant, hollow porous gold / silver nanoshells of different sizes and porosities are synthesized in a pure water system. Then, through the interaction between the amino groups of the anti-TPM-4 protein and the gold in the gold / silver nanoshells, the anti-TPM-4 targeting gold / silver nanoshell molecular probe is obtained.

[0012] Preferably, the steps are as follows:

[0013] S1. To synthesize silver nanoparticles, add an aqueous solution of silver nitrate to deionized water under vigorous stirring, heat to boiling, then quickly add an aqueous solution of sodium citrate, followed by a slow addition of an aqueous solution of ascorbic acid. Continue heating for 5-10 minutes until the reaction is complete. After the system cools to room temperature, centrifuge and purify several times. Then disperse the obtained silver nanoparticles in deionized water to obtain a silver nanoparticle dispersion.

[0014] S2. Synthesize hollow porous gold / silver nanoshells. The silver nanoparticle dispersion obtained in S1 is diluted with deionized water to a certain concentration. Hydroxylamine hydrochloride and chloroauric acid are added under vigorous stirring. After boiling for a certain time, the mixture is cooled to room temperature. Then, excess hydrogen peroxide solution is added to react. The precipitate is collected and resuspended with deionized water to obtain the hollow porous gold / silver nanoshell dispersion.

[0015] S3. Perform TPM-4 targeting modification. After ultrasonically dispersing the hollow porous gold / silver nanoshell dispersion obtained in S2 for 15 min, add the desalted anti-TPM-4 antibody, stir at 4℃ and centrifuge. Then suspend the precipitate in phosphate buffered saline.

[0016] Preferably, in S1, vigorous stirring refers to stirring at a speed of 1000 rpm, the concentration of silver nitrate solution is 0.2 M, the concentration of sodium citrate aqueous solution is 1%, the concentration of ascorbic acid aqueous solution is 0.1 M, and the volume ratio of silver nitrate:deionized water:sodium citrate aqueous solution:ascorbic acid aqueous solution is 1:400:24:16.

[0017] Preferably, the completion of the reaction in S1 means that the solution changes completely from yellow to grayish-yellow, and purification is achieved by washing several times with deionized water.

[0018] Preferably, the concentration of silver nanoparticles diluted to a certain level in S2 is 1.5 × 10⁻⁶. 10 -1.5×10 11 Cells / mL, vigorous stirring refers to stirring at a speed of 1000-1200 rpm.

[0019] Preferably, in S2, the concentration of hydroxylamine hydrochloride is 0.02M, the concentration of chloroauric acid is 0.1%, the volume ratio of silver nanoparticle aqueous dilution: hydroxylamine hydrochloride: chloroauric acid is 100:1:0.45-5.5, and the concentration of hydrogen peroxide solution is 0.1M.

[0020] An anti-TPM-4 targeting gold / silver nanoshell molecular probe prepared by the method described above.

[0021] Application of an anti-TPM-4 targeting gold / silver nanoshell molecular probe as described above in the preparation of targeted drugs for cancer, cardiovascular disease, diabetes-related complications, and neurological diseases.

[0022] Application of an anti-TPM-4 targeted gold / silver nanoshell molecular probe as described above in the preparation of imaging materials.

[0023] Therefore, the present invention provides an anti-TPM-4 targeted gold / silver nanoshell molecular probe, its preparation method, and its application, with the following specific technical effects:

[0024] (1) The molecular probe prepared by the present invention has a hollow and porous structure, which can encapsulate drugs. At the same time, the prepared molecular probe can specifically bind to TPM-4, which is highly expressed in tumor cells, and has high stability. It can be used for targeted drug delivery therapy for tumors and other diseases.

[0025] (2) The molecular probe prepared by this invention has intracellular electrocatalytic activity and can be used for imaging of cells such as tumors, which is expected to help realize real-time observation and monitoring of cells such as tumors;

[0026] (3) The molecular probes prepared by this invention have no cytotoxicity / very low cytotoxicity, no electrical irritation toxicity, and good biocompatibility, and can be safely used in the body.

[0027] (4) The preparation method provided by the present invention is simple and easy to operate, does not generate any toxic or harmful substances, and has low requirements for equipment.

[0028] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a TEM image of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 1 of this invention;

[0031] Figure 2 This is the surface potential analysis result of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 1 of this invention;

[0032] Figure 3 This is the electrocatalytic activity detection result of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 2 of this invention;

[0033] Figure 4 This is the cytotoxicity test result of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 3 of this invention;

[0034] Figure 5 This is the result of the electrostimulation cytotoxicity study of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 4 of this invention;

[0035] Figure 6 This is the ROS detection result of the intracellular catalytic activity of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 5 of this invention;

[0036] Figure 7 This is the cytotoxic AM / PI detection result of the anti-TPM-4 targeted gold / silver nanoshell molecular probe in Test Example 5 of this invention. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0040] Example 1

[0041] The specific steps for preparing an anti-TPM-4 targeting gold / silver nanoshell molecular probe are as follows:

[0042] S1. Synthesis of silver nanoparticles: Under vigorous stirring at 1000 rpm, 0.125 mL of an aqueous solution of silver nitrate (0.2 M) was added to 50 mL of deionized water. After heating to boiling, 3 mL of an aqueous solution of sodium citrate (1%) was quickly added, followed by 2 mL of an aqueous solution of ascorbic acid (0.1 M). The reaction solution turned yellow and gradually became grayish-yellow. Heating was continued for 5-10 min until the solution color no longer changed. Heating was stopped, and the solution was cooled to room temperature. After centrifugation at 6000 rpm for 10 min, the supernatant was discarded. The precipitate was washed three times with deionized water and then dispersed in 50 mL of deionized water to obtain a silver nanoparticle dispersion.

[0043] S2. Synthesis of hollow porous gold / silver nanoshells: Take 5 mL of the freshly prepared silver nanoparticle dispersion from S1 (solution concentration approximately 1.5 × 10⁻⁶). 11 Add 0.1 mL of hydroxylamine hydrochloride (0.02 M) and 100 μL of chloroauric acid (0.1%) to 5 mL of deionized water, and then add 0.1 mL of hydroxylamine hydrochloride (0.02 M) and 100 μL of chloroauric acid (0.1%) while stirring vigorously at 1000 rpm. Boil for 2 minutes and then cool to room temperature to obtain a solution of gold / silver nanocages. Then, at room temperature, add hydrogen peroxide (0.1 M) solution dropwise to the gold / silver nanocage solution, continuously observing the reaction process. Stop adding the solution when it turns light blue, centrifuge at 6000 rpm for 10 min to collect the precipitate, and resuspend the precipitate in 10 mL of deionized water to obtain a hollow porous gold / silver nanoshell dispersion.

[0044] S3 and TPM-4 targeting modification: First, take 1 mL of the hollow porous gold / silver nanoshell dispersion prepared in S2 and sonicate for 15 min to disperse it. Then add 100 μL of anti-TPM-4 antibody that has been desalted in a G25 centrifuge column. After stirring at 4℃ for 4 h, centrifuge at 6000 g for 10 min and suspend the precipitate in phosphate buffer solution to obtain the anti-TPM-4 targeting gold / silver nanoshell molecular probe.

[0045] Test Example 1

[0046] The anti-TPM-4 targeted gold / silver nanoshell molecular probe solution prepared in Example 1 was suspended droplet on a copper grid coated with a carbon film. After drying at room temperature, it was observed using a FEI TECNAI F20 EM transmission electron microscope equipped with high-angle annular dark-field scanning transmission (HAADF-ATEM) function. The accelerating voltage of the transmission electron microscope was 200 kV. The results are as follows: Figure 1 As shown, gold / silver nanoshells with hollow structures were successfully prepared with an average particle size of 76.53±8.26 nm.

[0047] The anti-TPM-4 targeted gold / silver nanoshells prepared in Example 1 were analyzed using dynamic light scattering method, and the results are as follows: Figure 2 As shown, the surface potential of the gold / silver nanoshell is -28.54±2.34, and the SH-PEG... 2000 After modification of the nanoparticle surface with gold-sulfur bonds, the surface potential increased to -20.35±1.58, while the surface potential of the anti-TPM-4 targeting gold / silver nanoshell molecular probe prepared in Example 1 was -23.26±0.89. This indicates that Example 1 successfully prepared a gold / silver nanoshell with anti-TPM-4 targeting function.

[0048] Test Example 2

[0049] The in-solution electrocatalytic properties of the anti-TPM-4 targeted gold / silver nanoshell molecular probe prepared in Example 1 were detected using the degradation rate of methylene blue (MB). The specific steps are as follows:

[0050] One mL of anti-TPM-4-targeted gold / silver nanoshell molecular probe was added to 2 mL of PBS buffer containing 30 μM methylene blue (MB). A constant current / constant voltage power supply and a Pt electrode were connected sequentially, and a 3 V DC voltage signal was applied. After different durations of voltage application, 30 μL of the mixture was collected, and PBS was added to a final volume of 330 μL. The absorption spectrum of MB at 664 nm was then characterized by UV-Vis spectroscopy. With increasing voltage application time, the anti-TPM-4-targeted gold / silver nanoshell molecular probe catalyzed water splitting, generating hydroxyl radicals that could degrade methylene blue, leading to a decrease in the 664 nm absorption spectrum. The results are as follows: Figure 3 As shown, the ratio of the 664nm absorption spectrum to the 664nm absorption spectrum at 0 minutes decreases continuously with the extension of time, proving that the gold / silver nanoshell has good electrocatalytic activity.

[0051] Test Example 3

[0052] The cytotoxicity of the anti-TPM-4 targeting gold / silver nanoshell molecular probe prepared in Example 1 was investigated, and the specific steps are as follows:

[0053] Using the MTT assay, U87 human glioma cells were seeded at 5000 cells / well in 96-well plates and co-incubated with 10-100 μm / mL anti-TPM-4 targeting gold / silver nanoshell molecular probes at 37°C and 5% CO2 for 24 h, 48 h, and 72 h. Then, 10 μL of 5 mg / mL MTT was added to each well for an additional 4 h of incubation. Finally, 150 μL of LDMSO was added to each well to dissolve the generated formazan, and the OD was measured at 490 / 570 nm using a microplate reader. Results are as follows: Figure 4 As shown, the anti-TPM-4 targeting gold / silver nanoshell molecular probe exhibits excellent biocompatibility and no significant cytotoxicity.

[0054] Test Example 4

[0055] The electrostimulation toxicity of the anti-TPM-4 targeted gold / silver nanoshell molecular probe prepared in Example 1 was investigated, and the specific steps are as follows:

[0056] U87 cells were seeded onto indium tin oxide (ITO) conductive glass plates and placed in 6-well plates. They were incubated with different components: an electrostimulation group (standard incubation followed by 1.5V constant voltage treatment for 5 min), a probe group (incubated for 4 h with anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 at a final concentration of 40 μM / mL), an electrostimulation + molecular probe group (incubated for 4 h with anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 at a final concentration of 40 μM / mL, followed by 1.5V constant voltage electrostimulation for 5 min), and a control group (containing the same volume of PBS as the anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 in the molecular probe group). The medium in each well was then replaced with fresh MEM complete medium, and all four groups were incubated for an additional 12 h before the MTT assay. Cell viability was then assessed using the MTT assay. Results are as follows: Figure 5 As shown, electrical stimulation combined with molecular probes effectively enhances cytotoxicity and promotes U87 cell death.

[0057] Test Example 5

[0058] The intracellular catalytic activity and toxicity of the anti-TPM-4-targeting gold / silver nanoshell molecular probe prepared in Example 1 were investigated. The specific steps are as follows:

[0059] Intracellular ROS detection: 2,7-Dichlorofluorescein diacetate (DCFHDA) was used to assess ROS production. ROS levels were quantified using laser confocal microscopy (LSCM). U87 cells were seeded onto ITO plates and placed in 6-well plates, and incubated for 4 hours with different components (control group, electrostimulation group, molecular probe group, and electrostimulation + molecular probe group). DCFHDA probe was added 30 minutes before 1.2V electrostimulation treatment, followed by 15 minutes of incubation, gentle rinsing with PBS, and finally LSCM observation. The electrostimulation group was subjected to conventional incubation followed by constant voltage treatment at 1.5V for 5 min; the molecular probe group was incubated for 4 h with anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 at a final concentration of 40 μm / mL; the electrostimulation + molecular probe group was incubated for 4 h with anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 at a final concentration of 40 μm / mL, followed by constant voltage electrostimulation at 1.5V for 5 min; the control group was treated with the same volume of PBS as the anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 in the molecular probe group.

[0060] The results are as follows Figure 6 As shown, the electrostimulation group and the electrostimulation + molecular probe group can effectively promote the production of intracellular ROS, and the electrostimulation + molecular probe group can produce stronger intracellular ROS, promoting cell death.

[0061] Cell viability assay: U87 cells were seeded onto ITO plates and placed in 6-well plates, and incubated for 4 h with different formulations (including control group (PBS), electrostimulation group, molecular probe group, and electrostimulation + molecular probe group). After electrostimulating U87 cells for 5 min, cell viability was detected using an acetylmethoxymethyl ester-calcein / pyridine iodide (Calcein-AM / PI) dual-color fluorescence detection kit.

[0062] The electrostimulation group was subjected to conventional incubation followed by constant voltage treatment at 1.5V for 5 min; the molecular probe group was incubated for 4 h with anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 at a final concentration of 40 μm / mL; the electrostimulation + molecular probe group was incubated for 4 h with anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 at a final concentration of 40 μm / mL, followed by constant voltage electrostimulation at 1.5V for 5 min; the control group was treated with the same volume of PBS as the anti-TPM-4 targeting gold / silver nanoshell molecular probes prepared in Example 1 in the molecular probe group.

[0063] The results are as follows Figure 7 As shown, with the assistance of electrical stimulation, the combination of electrical stimulation and molecular probes can effectively promote the death of U87 cells.

[0064] Therefore, the molecular probe prepared by this invention has a hollow, porous structure, which can encapsulate drugs. Simultaneously, the prepared molecular probe can specifically bind to TPM-4, which is highly expressed in tumor cells, exhibiting high stability and can be used for targeted drug delivery therapy for diseases such as tumors. It also possesses intracellular electrocatalytic activity, which can be used for imaging tumor cells, potentially contributing to the real-time observation and monitoring of tumor cells. Furthermore, it exhibits no or very low cytotoxicity, no electrostimulation toxicity, and good biocompatibility, making it safe for use in vivo. The provided preparation method is simple and easy to operate, generates no toxic or harmful substances, and has low equipment requirements.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an anti-TPM-4 targeted gold / silver nanoshell molecular probe, characterized in that: Hollow porous gold / silver nanoshells of different sizes and porosities were synthesized in a pure water system using polycrystalline silver nanoparticles as templates and hydrogen peroxide as an etchant. Then, an anti-TPM-4 targeting gold / silver nanoshell molecular probe was prepared by the interaction between the amino groups of the anti-TPM-4 protein and the gold in the gold / silver nanoshells.

2. The method for preparing an anti-TPM-4 targeted gold / silver nanoshell molecular probe according to claim 1, characterized in that, The steps are as follows: S1. To synthesize silver nanoparticles, add an aqueous solution of silver nitrate to deionized water under vigorous stirring, heat to boiling, then quickly add an aqueous solution of sodium citrate, followed by a slow addition of an aqueous solution of ascorbic acid. Continue heating for 5-10 minutes until the reaction is complete. After the system cools to room temperature, centrifuge and purify several times. Then disperse the obtained silver nanoparticles in deionized water to obtain a silver nanoparticle dispersion. S2. Synthesize hollow porous gold / silver nanoshells. The silver nanoparticle dispersion obtained in S1 is diluted with deionized water to a certain concentration. Hydroxylamine hydrochloride and chloroauric acid are added under vigorous stirring. After boiling, the mixture is cooled to room temperature. Then, excess hydrogen peroxide solution is added to react. The precipitate is collected and resuspended with deionized water to obtain the hollow porous gold / silver nanoshell dispersion. S3. Perform TPM-4 targeting modification. After ultrasonically dispersing the hollow porous gold / silver nanoshell dispersion obtained in S2 for 15 min, add the desalted anti-TPM-4 antibody, stir at 4℃ and centrifuge. Then suspend the precipitate in phosphate buffered saline.

3. The method for preparing an anti-TPM-4 targeted gold / silver nanoshell molecular probe according to claim 2, characterized in that: In S1, vigorous stirring refers to stirring at a speed of 1000 rpm. The concentration of silver nitrate solution is 0.2 M, the concentration of sodium citrate aqueous solution is 1%, the concentration of ascorbic acid aqueous solution is 0.1 M, and the volume ratio of silver nitrate:deionized water:sodium citrate aqueous solution:ascorbic acid aqueous solution is 1:400:24:

16.

4. The method for preparing an anti-TPM-4 targeted gold / silver nanoshell molecular probe according to claim 2, characterized in that: The reaction in S1 is complete when the solution changes from yellow to grayish-yellow. Purification involves washing the solution several times with deionized water.

5. The method for preparing an anti-TPM-4 targeted gold / silver nanoshell molecular probe according to claim 2, characterized in that: S2 refers to a concentration of silver nanoparticles of 1.5 × 10⁻⁶. 10 -1.5×10 11 Cells / mL, vigorous stirring refers to stirring at a speed of 1000-1200 rpm.

6. The method for preparing an anti-TPM-4 targeted gold / silver nanoshell molecular probe according to claim 2, characterized in that: In S2, the concentration of hydroxylamine hydrochloride is 0.02M, the concentration of chloroauric acid is 0.1%, the volume ratio of silver nanoparticle aqueous dilution: hydroxylamine hydrochloride: chloroauric acid is 100:1:0.45-5.5, and the concentration of hydrogen peroxide solution is 0.1M.

7. An anti-TPM-4 targeted gold / silver nanoshell molecular probe prepared by the method described in any one of claims 1 to 6.

8. The application of the anti-TPM-4 targeting gold / silver nanoshell molecular probe as described in claim 7 in the preparation of targeted drugs for cancer, cardiovascular disease, diabetes-related complications, and nervous system diseases.

9. The application of the anti-TPM-4 targeted gold / silver nanoshell molecular probe as described in claim 7 in the preparation of imaging materials.

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