Carbon-based molecular brush lymph nano tracer and preparation method and application thereof

Abstract

The invention discloses a carbon-based molecular brush lymph nano tracer as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: preparing a nanosphere precursor and carbonizing the nanosphere precursor into carbon nanospheres, or directly selecting nano carbon black, superfine carbon powder and the like; the carbon nanoparticles are pretreated and then grafted with hydrophilic polymer side chains, and carbon-based molecular brush nanoparticles are obtained; and dispersing the carbon-based molecular brush nano-particles in normal saline or a phosphate buffer salt solution to obtain the carbon-based molecular brush lymph nano tracer agent. According to the application, covalent binding of the carbon nano-particles and hydrophilic polymer side chains can be realized, the intrinsic hydrophilic carbon-based molecular brush nano-particles are endowed with lasting and stable monodispersity in normal saline and injected biological tissues, and the intrinsic hydrophilic carbon-based molecular brush nano-particles are expected to be applied to rapid tracing and developing of lymph nodes as a lymph nano tracer agent; the method has a wide application prospect and a huge market value.

Description

Technical Field

[0001] The present application belongs to the technical field of biomedical materials, and in particular relates to a carbon-based molecular brush lymphatic nanotracer and a preparation method and application thereof. Background Art

[0002] Lymph node dissection plays an important role in the clinical treatment strategy of tumors. By removing and analyzing the lymph nodes around the tumor, the cancer stage can be evaluated, treatment decisions can be guided, and the patient's prognosis can be predicted. First, lymph node dissection provides doctors with key information about the extent of tumor spread, thereby assisting doctors in determining the exact stage of the tumor. This will directly affect the formulation of treatment plans and patient management plans. If cancer cells are found in the lymph nodes, more aggressive treatment strategies, such as adjuvant chemotherapy or radiotherapy, may be required to reduce the risk of recurrence. In addition, lymph node dissection can also predict the patient's long-term prognosis and reduce the risk of recurrence. By removing lymph nodes that may contain tumor cells, the chance of tumor cells metastasizing to other parts of the body through the lymphatic system can be reduced. Therefore, effective observation of lymph nodes is crucial for accurate completion of tumor treatment.

[0003] At present, there are still few studies on the application of materials in lymph node tracing in related inventions. Some of them use colorimetric or fluorescent small molecule markers (such as methylene blue, indocyanine green, etc.) as developers. Such tracers often diffuse freely into blood vessels due to their small molecular size, making it difficult to perform targeted lymph node tracing. Based on this, carbon nanomaterials are darker in color and moderate in size, making it easy to enter the lymphatic system and achieve lymph node visualization, so they are tried to be applied to the field of lymph node tracing.

[0004] However, existing carbon nanoparticle lymphatic tracers have the following defects in clinical applications: first, common nanomaterials such as graphene and carbon nanotubes have certain cytotoxicity and inflammatory toxicity, which endanger human safety; second, the simple blending of carbon nanoparticles and dispersants will lead to the risk of phase separation between carbon nanoparticles and dispersants. When the lymphatic tracer is injected into the tissue, its dispersant will be diluted by the lymph fluid and cause the nanoparticles to agglomerate, making it difficult to deliver deeply into the lymphatic system. Summary of the invention

[0005] The present invention provides a carbon-based molecular brush lymph nanotracer and a preparation method and application thereof. The invention disperses carbon nanoparticles grafted with hydrophilic polymer side chains in physiological saline to form a lymph tracer, thereby solving the technical problems of poor biocompatibility of existing carbon nanomaterials in the process of lymph node tracing and reduced stability of lymph tracers after injection into tissues.

[0006] In order to achieve the above-mentioned object, the first aspect of the present application provides a method for preparing a carbon-based molecular brush lymph nanotracer, comprising the following steps:

[0007] 1) Select one or more carbon nanoparticles of carbon nanospheres, nano carbon black, or ultrafine carbon powder obtained by carbonizing nanosphere precursors;

[0008] 2) pre-treating the carbon nanoparticles in step 1) and grafting hydrophilic polymer side chains to obtain carbon-based molecular brush nanoparticles;

[0009] 3) dispersing the carbon-based molecular brush nanoparticles prepared in step 2) in physiological saline or phosphate buffered saline solution to obtain a carbon-based molecular brush lymph nanotracer.

[0010] Preferably, the method for preparing the nanosphere precursor in step 1) comprises: dispersing aniline and pyrrole in an aqueous solution containing a surfactant, adding a pre-cooled aqueous initiator solution, and reacting in an ice bath for 10-14 hours to prepare the nanosphere precursor.

[0011] Preferably, the carbonization conditions in step 1) are: the carbonization temperature is 600-1000° C., and the carbonization time is 3-24 h.

[0012] Further preferably, the carbonization conditions in step 1) are: under an inert atmosphere, a heating rate of 5-10° C. / min, a carbonization temperature of 600-1000° C., and a carbonization time of 3-24 h.

[0013] Preferably, the pretreatment step in step 2) comprises: one or more of plasma pretreatment, acid treatment or grafting of a hydroxyl-containing silane coupling agent.

[0014] Further preferably, the conditions for plasma pretreatment in step 2) are: oxygen plasma treatment power is 80-200 W, and treatment time is 10-60 min.

[0015] Preferably, the hydrophilic polymer side chain in step 2) comprises one or more of polyvinyl pyrrolidone, polyhydroxyethyl methyl methacrylate, polyacrylic acid or poly(polyethylene glycol methyl ether methacrylate).

[0016] Preferably, the step of grafting hydrophilic polymer side chains in step 2) is as follows: dispersing the pretreated carbon nanoparticles in an organic solvent, adding monomers to disperse them uniformly, adding initiators, and reacting at 60-80° C. for 12-72 hours under an inert atmosphere.

[0017] Further preferably, in step 2) of grafting hydrophilic polymer side chains, the mass volume ratio of carbon nanoparticles to monomers is 100:1-2 mg / mL, and the mass ratio of initiator to carbon nanoparticles is 1-25:25 g / g.

[0018] Further preferably, in the step of grafting hydrophilic polymer side chains in step 2), the organic solvent includes one or more of N,N-dimethylformamide, tetrahydrofuran or 1,4-dioxane; the monomer includes N-vinyl pyrrolidone, hydroxyethyl methyl methacrylate, acrylic acid or polyethylene glycol methyl ether methacrylate, and the initiator includes azobisisobutyronitrile.

[0019] Preferably, in step 3), the carbon-based molecular brush nanoparticles are dispersed in physiological saline or phosphate buffered saline solution at a concentration of 10-100 mg / mL.

[0020] The second aspect of the present application provides a carbon-based molecular brush lymphatic nanotracer.

[0021] Preferably, the carbon-based molecular brush lymph nanotracer is prepared by the preparation method of a carbon-based molecular brush lymph nanotracer described in the first aspect.

[0022] Preferably, the hydrodynamic diameter of the carbon-based molecular brush nanoparticles is 100-300 nm.

[0023] The third aspect of the present application provides an application of a carbon-based molecular brush lymphatic nanotracer in lymphatic tracing for tumor treatment.

[0024] Compared with the prior art, the advantages or beneficial effects of the present application include at least:

[0025] The present invention prepares hydrophilic carbon-based molecular brush nanoparticles by grafting pretreated carbon nanoparticles with hydrophilic polymer side chains, and then disperses the pretreated carbon nanoparticles in physiological saline or phosphate buffered saline to prepare lymphatic tracers. On the one hand, the hydrophilic polymer side chains ensure good biocompatibility while giving the carbon nanoparticles good water dispersibility, so that the carbon-based molecular brush lymph nanotracer has good stability, reduces the risk of nanoparticle agglomeration in the body, and further ensures that the carbon-based molecular brush nanoparticles are smoothly delivered to the lymph nodes to achieve their tracing and staining function; on the other hand, the hydrophilic polymer side chains and the carbon nanoparticles are durably and stably bound to each other through covalent bonds, which can effectively avoid the risk of nanoparticles in existing products agglomerating due to the decrease in the proportion of dispersant in the tracer after entering the body; on the third hand, this application proposes for the first time to prepare carbon-based molecular brush nanoparticles of moderate size and use them for lymph node tracing, which can utilize the property of carbon nanoparticles that they are easily refluxed or delivered into the lymph nodes, and further graft hydrophilic polymer side chains to improve the water dispersibility of carbon nanoparticles, so as to quickly and accurately achieve lymph node tracing. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 Scanning electron microscope images of the surface morphology of the Csp sample and the Csp-g-PVP sample provided in Example 1 of the present application;

[0028] Figure 2 A Fourier transform infrared spectrum of the Csp-g-PVP sample provided in Example 1 of the present application;

[0029] Figure 3 This is a statistical graph of particle size analysis of the Csp sample and the Csp-g-PVP sample provided in Example 1 of the present application in PBS buffer;

[0030] Figure 4 This is a statistical graph of CCK-8 absorbance after the Csp-g-PVP sample provided in Example 1 of the present application was co-cultured with L929 cells for 24 hours;

[0031] Figure 5 This is the thermogravimetric analysis curve of the Csp-g-PVP sample provided in Example 1 and Example 2 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0033] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0034] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0035] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0036] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0037] Those skilled in the art will appreciate that the numerical ranges in the embodiments of the present application are to be understood as each intermediate value between the upper and lower limits of the scope also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0038] Unless otherwise specified, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. Although this application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] The technical solution of the present application will be further described below in conjunction with specific embodiments.

[0040] Example 1

[0041] A method for preparing a carbon-based molecular brush lymph nanotracer comprises the following steps:

[0042] S1: After dispersing the small molecule monomer aniline (2.32 mL) and pyrrole (1.75 mL) in 360 mL of an aqueous solution containing 340 μL of Triton X-100, stir and ultrasonically disperse for 30 min each, and precool in an ice bath for 30 min. Add precooled ammonium persulfate solution (11.52 g ammonium persulfate dissolved in 50 mL deionized water) in an ice bath, and react in an ice bath for 12 h.

[0043] S2: After centrifugation, washing and freeze-drying, the obtained precursor solid is placed in a tubular furnace and carbonized under a nitrogen atmosphere at a carbonization temperature of 900°C, a heating rate of 5°C / min, and a carbonization time of 10 hours to prepare carbon nanospheres Csp.

[0044] S3: The carbon nanospheres Csp obtained after carbonization are placed in a glass culture dish, a watch glass or a beaker and put into a plasma device for oxygen plasma pretreatment. When the carbon nanospheres are pretreated with oxygen plasma, the oxygen plasma treatment power is 150 W and the total treatment time is 32 min.

[0045] S4: The carbon nanospheres pretreated by oxygen plasma are quickly dispersed in N,N-dimethylformamide solvent for polyvinyl pyrrolidone polymer side chain grafting treatment, wherein the mass of the carbon nanospheres is 500 mg, the solvent is 100 mL, and the monomer N-vinyl pyrrolidone is 10 mL.

[0046] S5: The above dispersed suspension was subjected to ultrasound for 30 minutes, nitrogen was introduced for pre-protection for 30 minutes, and then an initiator, azobisisobutyronitrile, was added. The mass of azobisisobutyronitrile was 200 mg.

[0047] S6: placing the reaction system after adding the initiator in a 60°C environment for heating polymerization, the polymer side chain grafting polymerization reaction time is 48h, and carbon-based molecular brush nanoparticles Csp-g-PVP are prepared.

[0048] S7: After the above reaction is completed, the obtained carbon-based molecular brush nanoparticles Csp-g-PVP are dispersed in physiological saline after centrifugation, washing and freeze-drying, and the concentration of the carbon-based molecular brush nanoparticles in the physiological saline is 50 mg / mL. A carbon-based molecular brush lymph nanotracer with good stability and lymph node staining function is obtained.

[0049] In order to better illustrate the technical effect of the present application, the carbon nanospheres Csp prepared in step S2 of Example 1 and the carbon-based molecular brush nanoparticles Csp-g-PVP prepared in step S6 were subjected to surface morphology characterization, Fourier infrared spectroscopy analysis, particle size analysis and biocompatibility analysis.

[0050] 1. Surface morphology characterization

[0051] The surface morphology of Csp sample and Csp-g-PVP sample was characterized by scanning electron microscopy (S-4800, 5.0 kV). Figure 1 shown.

[0052] according to Figure 1 It can be seen that after the surface polyvinyl pyrrolidone polymer side chain grafting, Csp-g-PVP can still maintain the same uniformly dispersed spherical morphology as Csp, indicating that the polyvinyl pyrrolidone polymer side chain grafting will not destroy its nanosphere morphology, and it still has a nanosphere structure.

[0053] 2. Fourier transform infrared spectroscopy analysis

[0054] Csp, PVP, and Csp-g-PVP were characterized by infrared spectrometer (IS 50). Figure 2 shown.

[0055] according to Figure 2 It can be seen that an obvious PVP characteristic peak appeared in the Fourier transform infrared spectrum of Csp-g-PVP after polyvinyl pyrrolidone polymer side chain grafting, indicating the successful preparation of carbon-based molecular brush nanoparticles.

[0056] 3. Particle size analysis

[0057] The particle size distribution of Csp and Csp-g-PVP in PBS buffer was characterized by using Zeta potential instrument (BI-PALS). The specific test method is: Csp and Csp-g-PVP were prepared with PBS buffer to 0.5 mg / mL, and the particle size analysis test was performed. The results were Figure 3 shown.

[0058] according to Figure 3 It can be seen that the hydrodynamic diameter of Csp in PBS buffer is mainly concentrated at 1002nm, and a characteristic peak indicating secondary particles appears at 6313nm, while the particle size distribution of carbon-based molecular brush nanoparticles Csp-g-PVP is uniform, and the hydrodynamic diameter in PBS buffer is 259nm. In summary, the polyvinyl pyrrolidone polymer side chain grafting can significantly improve the dispersibility of carbon nanospheres in PBS buffer.

[0059] 4. Biocompatibility Analysis

[0060] Csp-g-PVP was co-cultured with L929 mouse fibroblasts at a concentration of 10 μg / mL for 24 h, and the CCK-8 intensity was counted using an ELISA instrument to analyze the cytotoxicity.

[0061] according to Figure 4It can be seen that after the carbon-based molecular brush nanoparticles Csp-g-PVP were co-cultured with L929 cells for 24 hours, the cell survival rate was still over 85%, which indicates that Csp-g-PVP has good biocompatibility.

[0062] Example 2

[0063] A method for preparing a carbon-based molecular brush lymph nanotracer comprises the following steps:

[0064] S1: The small molecule monomers aniline (2.32 mL) and pyrrole (1.75 mL) were dispersed in 360 mL of an aqueous solution containing 340 μL of Triton X-100, stirred and ultrasonically dispersed for 30 min each, and precooled in an ice bath for 30 min. Precooled ammonium persulfate solution (11.52 g ammonium persulfate dissolved in 50 mL deionized water) was added in an ice bath, and the mixture was reacted for 12 h in an ice bath.

[0065] S2: The obtained precursor solid was placed in a tubular furnace after centrifugation, washing and freeze-drying, and carbonized under a nitrogen atmosphere at a carbonization temperature of 900°C, a heating rate of 5°C / min, and a carbonization time of 10 h.

[0066] S3: placing the carbon nanospheres obtained after carbonization in a glass culture dish, a watch glass or a beaker and placing them in a plasma device for oxygen plasma pretreatment. When the carbon nanospheres are subjected to oxygen plasma pretreatment, the oxygen plasma treatment power is 150 W and the total treatment time is 32 min.

[0067] S4: The carbon nanospheres pretreated by oxygen plasma are quickly dispersed in N,N-dimethylformamide solvent for polyvinyl pyrrolidone polymer side chain grafting treatment, wherein the mass of the carbon nanospheres is 500 mg, the solvent is 100 mL, and the monomer N-vinyl pyrrolidone is 10 mL.

[0068] S5: After the above dispersed suspension was ultrasonicated for 30 minutes and pre-purged with nitrogen for 30 minutes, an initiator azobisisobutyronitrile was added, and the mass of azobisisobutyronitrile was 200 mg.

[0069] S6: placing the reaction system after adding the initiator in a 60° C. oil bath for heating polymerization. The polymerization reaction time of the carbon-based molecular brush nanoparticles is 24 hours.

[0070] S7: After the above reaction is completed, the obtained carbon-based molecular brush nanoparticles Csp-g-PVP are dispersed in physiological saline after centrifugation, washing and freeze-drying, and the concentration of the carbon-based molecular brush nanoparticles in the physiological saline is 50 mg / mL. A carbon-based molecular brush lymph nanotracer with good stability and lymph node staining function is obtained.

[0071] Example 3

[0072] This embodiment provides a method for preparing a carbon-based molecular brush lymphatic nanotracer, comprising the following steps:

[0073] S1: The small molecule monomers aniline (2.32 mL) and pyrrole (1.75 mL) were dispersed in 360 mL of an aqueous solution containing 340 μL of Triton X-100, stirred and ultrasonically dispersed for 30 min each, and precooled in an ice bath for 30 min. Precooled ammonium persulfate solution (11.52 g ammonium persulfate dissolved in 50 mL deionized water) was added in an ice bath, and the mixture was reacted for 12 h in an ice bath.

[0074] S2: After centrifugation, washing and freeze-drying, the obtained precursor was placed in a tubular furnace and carbonized under a nitrogen atmosphere. The carbonization temperature was 900°C, the heating rate was 5°C / min, and the carbonization time was 10 h.

[0075] S3: placing the carbon nanospheres obtained after carbonization in a glass culture dish, a watch glass or a beaker and placing them in a plasma device for oxygen plasma pretreatment. When the carbon nanospheres are subjected to oxygen plasma pretreatment, the oxygen plasma treatment power is 150 W and the total treatment time is 32 min.

[0076] S4: The carbon nanospheres pretreated by oxygen plasma are quickly dispersed in N,N-dimethylformamide solvent for polyvinyl pyrrolidone polymer side chain grafting treatment, wherein the mass of the carbon nanospheres is 500 mg, the solvent is 100 mL, and the monomer N-vinyl pyrrolidone is 10 mL.

[0077] S5: After the above dispersed suspension was ultrasonicated for 30 minutes and pre-purged with nitrogen for 30 minutes, an initiator azobisisobutyronitrile was added, and the mass of azobisisobutyronitrile was 200 mg.

[0078] S6: placing the reaction system after adding the initiator in a 60° C. oil bath for heating polymerization. The polymerization reaction time of the carbon-based molecular brush nanoparticles is 48 hours.

[0079] S7: After the above reaction is completed, the obtained carbon-based molecular brush nanoparticles Csp-g-PVP are dispersed in physiological saline after centrifugation, washing and freeze-drying, and the concentration of the carbon-based molecular brush nanoparticles in the physiological saline is 25 mg / mL. A carbon-based molecular brush lymph nanotracer with good stability and lymph node staining function is obtained.

[0080] Example 4

[0081] This embodiment provides a method for preparing a carbon-based molecular brush lymphatic nanotracer, comprising the following steps:

[0082] S1: The small molecule monomers aniline (2.32 mL) and pyrrole (1.75 mL) were dispersed in 360 mL of an aqueous solution containing 340 μL of Triton X-100, stirred and ultrasonically dispersed for 30 min each, and precooled in an ice bath for 30 min. Precooled ammonium persulfate solution (11.52 g ammonium persulfate dissolved in 50 mL deionized water) was added in an ice bath, and the mixture was reacted for 12 h in an ice bath.

[0083] S2: The obtained precursor was centrifuged, washed and freeze-dried, and then carbonized in a nitrogen atmosphere at a carbonization temperature of 900°C, a heating rate of 5°C / min, and a carbonization time of 10 h.

[0084] S3: placing the carbon nanospheres obtained after carbonization in a glass culture dish, a watch glass or a beaker and placing them in a plasma device for oxygen plasma pretreatment. When the carbon nanospheres are subjected to oxygen plasma pretreatment, the oxygen plasma treatment power is 150 W and the total treatment time is 32 min.

[0085] S4: The carbon nanospheres pretreated by oxygen plasma are quickly dispersed in N,N-dimethylformamide solvent for polyvinyl pyrrolidone polymer side chain grafting treatment, wherein the mass of the carbon nanospheres is 500 mg, the solvent is 100 mL, and the monomer N-vinyl pyrrolidone is 10 mL.

[0086] S5: After the above dispersed suspension was ultrasonicated for 30 minutes and pre-purged with nitrogen for 30 minutes, an initiator azobisisobutyronitrile was added, and the mass of azobisisobutyronitrile was 200 mg.

[0087] S6: placing the reaction system after adding the initiator in a 60° C. oil bath for heating polymerization. The polymerization reaction time of the carbon-based molecular brush nanoparticles is 48 hours.

[0088] S7: After the above reaction is completed, the obtained carbon-based molecular brush nanoparticles Csp-g-PVP grafted with polyvinyl pyrrolidone polymer side chains are dispersed in physiological saline after centrifugation, washing and freeze-drying, and the concentration of the carbon-based molecular brush nanoparticles in physiological saline is 12.5 mg / mL. A carbon-based molecular brush lymph nanotracer with good stability and lymph node staining function is obtained.

[0089] In order to better illustrate the technical effect of the carbon-based molecular brush lymph nanotracer, we conducted the following lymph node black staining scoring experiment.

[0090] 1. Lymph node black staining scores of samples with different tracer concentrations

[0091] The lymph node black staining score was performed using the carbon-based molecular brush lymph nanotracer (Csp-g-PVP-1) prepared in Example 1, the carbon-based molecular brush lymph nanotracer (Csp-g-PVP-3) prepared in Example 3, and the carbon-based molecular brush lymph nanotracer (Csp-g-PVP-4) prepared in Example 4 as test samples. The specific experiment is as follows:

[0092] Fifteen healthy female Balb / c mice were divided into three groups. 50 μL of tracer was injected subcutaneously into the foot pad of the mice. The mice were killed 10 minutes after injection. The degree of black staining of the foot, hind leg and iliac lymph nodes was observed with the naked eye. The black staining degree was scored as follows: 0 points for no change in lymph node color, 0.5 points for lymph node staining gray, and 1.0 points for obvious black staining of lymph nodes. The results are shown in Table 1. The difference between Csp-g-PVP-1, Csp-g-PVP-3, and Csp-g-PVP-4 is that the concentrations of lymphatic tracers are 50 mg / mL, 25 mg / mL, and 12.5 mg / mL, respectively.

[0093] Table 1 Scoring table of the degree of black staining of mouse lymph nodes by carbon-based molecular brush lymph nanotracers at different concentrations

[0094]

[0095] According to Table 1, the lymph node black staining score of Csp-g-PVP-1 lymph tracer is best 10 minutes after injection, so 50 mg / mL is preferably used as the optimal lymph node black staining tracer concentration.

[0096] 2. Thermogravimetric analysis of samples at different polymerization times and lymph node black staining scores

[0097] Thermogravimetric analysis was performed on the carbon-based molecular brush nanoparticles (Csp-g-PVP-48h) prepared in step S6 of Example 1 and the carbon-based molecular brush nanoparticles (Csp-g-PVP-24h) prepared in step S6 of Example 2. The carbon-based molecular brush lymph nanotracer (Csp-g-PVP-1) prepared in Example 1 and the carbon-based molecular brush lymph nanotracer (Csp-g-PVP-2) prepared in Example 2 were then scored for lymph node black staining. The difference between Csp-g-PVP-1 and Csp-g-PVP-2 is that the polymerization time in step S6 is 48h and 24h respectively, and Csp-Sus is Csp dispersed in physiological saline, that is, the polymerization time is 0h.

[0098] 2.1 Thermogravimetric analysis

[0099] Thermogravimetric analysis was performed using a thermogravimetric analyzer (TG209F1) to characterize the polymer grafting amount of Csp-g-PVP-48h and Csp-g-PVP-24h after polymerization for different time periods. The results were Figure 5 shown.

[0100] according to Figure 5 Calculations show that the grafting amounts of Csp-g-PVP-48h and Csp-g-PVP-24h with polymerization times of 48h and 24h are 19% and 8% respectively. In summary, extending the polymerization time of polyvinyl pyrrolidone polymer side chain grafting will effectively increase the amount of polymer side chains grafted onto the Csp surface.

[0101] 2.2 Lymph node black staining score

[0102] Fifteen healthy female Balb / c mice were divided into three groups. 50 μL of tracer was injected subcutaneously into the foot pads of the mice. The mice were killed 10 minutes after the injection. The degree of black staining of the foot pads, hind leg bend lymph nodes and iliac lymph nodes was observed with the naked eye. The black staining degree was scored as follows: 0 points for no change in lymph node color, 0.5 points for lymph node staining gray, and 1.0 points for obvious black staining of lymph nodes. The results are shown in Table 2.

[0103] Table 2 Scoring table of the degree of black staining of mouse lymph nodes by carbon-based molecular brush lymph nanotracers with different polymerization times

[0104]

[0105] According to Table 2, the Csp-g-PVP-1 lymphatic tracer obtained after 48 hours of graft polymerization reaction has the best lymph node black staining performance. In summary, as the polymerization time increases, the amount of polymer side chain grafting increases, and the obtained lymphatic tracer has better lymph node black staining performance.

[0106] 3. Black staining scores of lymph nodes in samples at different times after injection

[0107] In order to characterize the changes of lymph node black staining scores over time after injection, 15 healthy female Balb / c mice were divided into 3 groups. 50 μL of the lymphatic tracer Csp-g-PVP-1 prepared in Example 1 was subcutaneously injected into the foot pads of the mice. The mice were killed 10, 30 and 60 minutes after injection, and the animal grouping samples at 10, 30 and 60 minutes were named Csp-g-PVP-1-10MIN, Csp-g-PVP-1-30MIN and Csp-g-PVP-1-60MIN lymphatic tracers. The degree of black staining of the soles, hind leg bends and iliac lymph nodes was observed with the naked eye. The black staining degree scoring standard is: 0 points for no change in lymph node color, 0.5 points for lymph node staining gray, 1.0 points for obvious black staining of lymph nodes, and the results are shown in Table 3.

[0108] Table 3 Scoring table of the degree of black staining of mouse lymph nodes by carbon-based molecular brush lymph nanotracer at different observation times after injection

[0109]

[0110] According to Table 3, the Csp-g-PVP-1 carbon-based molecular brush lymph nanotracer obtained after grafting can show good lymph node black staining performance 10, 30 and 60 minutes after injection.

[0111] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0112] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A method for preparing a carbon-based molecular brush lymphatic nanotracer, characterized in that: The steps include: 1) Select one or more carbon nanoparticles of carbon nanospheres, nano carbon black, or ultrafine carbon powder obtained by carbonizing nanosphere precursors; 2) pre-treating the carbon nanoparticles in step 1) and grafting hydrophilic polymer side chains to obtain carbon-based molecular brush nanoparticles; 3) dispersing the carbon-based molecular brush nanoparticles prepared in step 2) in physiological saline or phosphate buffered saline solution to obtain a carbon-based molecular brush lymph nanotracer.

2. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 1, characterized in that: The method for preparing the nanosphere precursor in step 1) comprises: dispersing aniline and pyrrole in an aqueous solution containing a surfactant, adding a pre-cooled initiator aqueous solution, and reacting in an ice bath for 10-14 hours to prepare the nanosphere precursor.

3. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 1, characterized in that: The carbonization conditions in step 1) are: carbonization temperature is 600-1000° C., and carbonization time is 3-24 h.

4. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 1, characterized in that: The pretreatment step in step 2) includes one or more of plasma pretreatment, acid treatment or grafting of a hydroxyl-containing silane coupling agent.

5. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 1, characterized in that: The hydrophilic polymer side chain in step 2) includes one or more of polyvinyl pyrrolidone, polyhydroxyethyl methyl acrylate, polyacrylic acid or poly (polyethylene glycol methyl ether methacrylate).

6. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 1, characterized in that: The step of grafting hydrophilic polymer side chains in step 2) comprises: dispersing the pretreated carbon nanoparticles in an organic solvent, adding monomers to disperse them uniformly, adding initiators, and reacting at 60-80° C. for 12-72 hours under an inert atmosphere.

7. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 6, characterized in that: In step 2) of grafting hydrophilic polymer side chains, the mass volume ratio of carbon nanoparticles to monomers is 100:1-2 mg / mL, and the mass ratio of initiator to carbon nanoparticles is 1-25:25 g / g; the organic solvent includes one or more of NN dimethylformamide, tetrahydrofuran or 1,4-dioxane; the monomer includes N-vinyl pyrrolidone, hydroxyethyl methyl methacrylate, acrylic acid or polyethylene glycol methyl ether methacrylate, and the initiator includes azobisisobutyronitrile.

8. The method for preparing the carbon-based molecular brush lymph nanotracer according to claim 1, characterized in that: Step 3) The carbon-based molecular brush nanoparticles are dispersed in physiological saline or phosphate buffered saline solution at a concentration of 10-100 mg / mL.

9. A carbon-based molecular brush lymphatic nanotracer prepared by the method for preparing the carbon-based molecular brush lymphatic nanotracer according to any one of claims 1 to 8.

10. Use of the carbon-based molecular brush lymph nanotracer as claimed in claim 9 in lymph tracing.

Images