A carbon-based molecular brush lymphatic nanotracer, and a preparation method and application thereof

Abstract

The application discloses a carbon-based molecular brush lymphatic nano-tracer and 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 nanometer carbon black, superfine carbon powder and the like; grafting a hydrophilic polymer side chain to the carbon nanometer particles after pretreatment, so as to obtain carbon-based molecular brush nanometer particles; and dispersing the carbon-based molecular brush nanometer particles in normal saline or a phosphate buffered saline solution, so as to obtain the carbon-based molecular brush lymphatic nano-tracer. The carbon nanometer particles and the hydrophilic polymer side chain can be covalently combined, the intrinsic hydrophilic carbon-based molecular brush nanometer particles are endowed with persistent and stable monodisperse performance in normal saline and injected biological tissues, and as the lymphatic nano-tracer, the carbon-based molecular brush lymphatic nano-tracer is expected to be applied to rapid tracing and imaging of lymph nodes, and has wide application prospects and great market value.

Description

Technical Field

[0001] This application belongs to the field of biomedical materials technology, and in particular relates to a carbon-based molecular brush lymph nanotracer, its preparation method and application. Background Technology

[0002] Lymph node dissection plays a crucial role in clinical cancer treatment strategies. By removing and analyzing the lymph nodes surrounding the tumor, it is possible to assess cancer staging, guide treatment decisions, and predict patient prognosis. First, lymph node dissection provides physicians with critical information about the extent of tumor spread, thus assisting in determining the precise stage of the tumor. This directly impacts the development of treatment plans and patient management strategies. If cancer cells are found within the lymph nodes, a more aggressive treatment strategy, such as adjuvant chemotherapy or radiotherapy, may be necessary to reduce the risk of recurrence. Furthermore, lymph node dissection can predict long-term patient prognosis and reduce the risk of recurrence. By removing lymph nodes that may contain tumor cells, the likelihood of tumor cells metastasizing to other parts of the body via the lymphatic system can be reduced. Therefore, effective lymph node monitoring is essential for accurate cancer treatment.

[0003] Currently, research on the application of materials for lymph node tracing is still limited. Some existing research uses chromogenic or fluorescent small molecule markers (such as methylene blue and indocyanine green) as contrast agents. However, these tracers often diffuse freely into blood vessels due to their small molecular size, making targeted lymph node tracing difficult. Therefore, carbon nanomaterials, with their deep color and moderate size, easily enter the lymphatic system and achieve lymph node visualization, and are thus being explored for application in the field of lymph node tracing.

[0004] However, existing carbon nanoparticle lymphatic tracers have the following drawbacks in clinical applications: First, common nanomaterials such as graphene and carbon nanotubes have certain cytotoxic and inflammatory toxicity, endangering human safety; second, simply blending carbon nanoparticles with dispersants leads to the risk of phase separation between the carbon nanoparticles and the dispersant. When the lymphatic tracer is injected into tissues, its dispersant is diluted by lymph fluid, causing nanoparticles to aggregate, making it difficult to deliver it deeply into the lymphatic system. Summary of the Invention

[0005] This invention provides a carbon-based molecular brush lymphatic nanotracer, its preparation method, and its application. This invention disperses hydrophilic polymer side-linked carbon nanoparticles in physiological saline to prepare a lymphatic tracer, solving the technical problems of poor biocompatibility and reduced stability of lymphatic tracers after injection into tissues when existing carbon nanomaterials are used for lymph node tracking.

[0006] To achieve the above objectives, the first aspect of this application provides a method for preparing a carbon-based molecular brush lymphatic nanotracer, comprising the following steps:

[0007] 1) selecting one or several carbon nanoparticles from carbon nanospheres or nanocarbon black, ultrafine carbon powder obtained by carbonization of nanosphere precursors;

[0008] 2) grafting hydrophilic polymer side chains onto the carbon nanoparticles in step 1) after pretreatment to obtain carbon-based molecular brush nanoparticles;

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

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

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

[0012] Further preferably, the carbonization conditions in step 1) are as follows: under inert atmosphere, the heating rate is 5-10℃ / min, the carbonization temperature is 600-1000℃, and the carbonization time is 3-24 h.

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

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

[0015] Preferably, the hydrophilic polymer side chains in step 2) include one or several of polyvinylpyrrolidone, polyhydroxyethyl 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, uniformly dispersing after adding monomers, and then adding an initiator, and reacting at 60-80℃ under inert atmosphere for 12-72 h.

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

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

[0019] Preferably, in step 3), the carbon-based molecular brush nanoparticle is 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 lymphatic nanotracer is prepared by the preparation method of the carbon-based molecular brush lymphatic nanotracer of the first aspect.

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

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

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

[0025] The present application grafts a hydrophilic polymer side chain to the pretreated carbon nanoparticles to prepare hydrophilic carbon-based molecular brush nanoparticles, and then disperses the nanoparticles in physiological saline or phosphate buffered saline solution to prepare a lymphatic tracer. In the first aspect, the hydrophilic polymer side chain ensures good biocompatibility while imparting good water dispersibility to the carbon nanoparticles, so that the carbon-based molecular brush lymphatic nanotracer has good stability, reduces the risk of nanoparticle aggregation in the body, and further ensures smooth delivery of the carbon-based molecular brush nanoparticle to the lymph node to achieve the tracing and staining function of the lymph node; in the second aspect, the hydrophilic polymer side chain and the carbon nanoparticles are covalently bonded and stably combined, which can effectively avoid the risk of nanoparticle aggregation due to the decrease in the proportion of dispersing agent in the tracer after entering the body in the prior art; in the third aspect, the present application first proposes to prepare a carbon-based molecular brush nanoparticle with a suitable size and use it for lymph node tracing. By taking advantage of the characteristic that carbon nanoparticles are easily delivered into the lymph node by backflow or delivery, and by grafting a hydrophilic polymer side chain to improve the water dispersibility of the carbon nanoparticles, the lymph node can be quickly and accurately traced. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments described in the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

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

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

[0029] Figure 3 Particle size analysis statistical graph of the Csp sample and the Csp-g-PVP sample in PBS buffer provided in Embodiment 1 of the present application;

[0030] Figure 4 CCK-8 absorbance statistical graph of the Csp-g-PVP sample provided in Embodiment 1 of the present application after co-culturing with L929 cells for 24 hours;

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

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely in the following description of the embodiments of the present application in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0033] In the following description of the present embodiment, the term “and / or” is used to describe the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent the following three cases: A exists alone, B exists alone and A and B exist simultaneously. Wherein A and B can be singular or plural. The character “ / ” generally represents that the front and rear associated objects are in an “or” relationship.

[0034] In the following description of the embodiments, the term "at least one" means one or more, and the term "multiple" means two or more. "At least one of the following (a)" or the like means any combination of the items, including a single item (a) or any combination of a plurality of items. For example, "at least one of a, b or c", or "at least one of a, b and c", can mean a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, where a, b, and c can be a single item or a plurality of items.

[0035] The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in the description of the embodiments of the present application and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

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

[0037] Those skilled in the art understand that in the embodiments of the present application, the numerical range should be understood as also specifically disclosing each intermediate value between the upper limit and the lower limit of the range. Each smaller range between any stated value or stated range of values and any other stated value or stated range of values, as well as any intermediate value between any stated value or stated range of values, is also included in the present application. The upper and lower limits of these smaller ranges can be included or excluded independently from the range.

[0038] Unless otherwise defined, technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present 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 conflict between the content of this specification and any incorporated document, the content of this specification shall prevail.

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

[0040] Embodiment 1

[0041] A preparation method of a carbon-based molecular brush lymphatic nanotracer, comprising the following steps:

[0042] S1: 2.32 mL of small molecule monomer aniline and 1.75 mL of pyrrole were dispersed in 360 mL of an aqueous solution containing 340 μL of Triton X-100. The mixture was stirred and ultrasonically dispersed for 30 min each, and then pre-cooled in an ice bath for 30 min. Pre-cooled ammonium persulfate solution (11.52 g of ammonium persulfate dissolved in 50 mL of deionized water) was added in an ice bath, and the mixture was reacted in an ice bath for 12 h.

[0043] S2: The obtained precursor solid was centrifuged, washed and freeze-dried, and then placed in a tube furnace for carbonization under a nitrogen atmosphere. The carbonization temperature was 900℃, the heating rate was 5℃ / min, and the carbonization time was 10h to prepare carbon nanospheres Csp.

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

[0045] S4: Carbon nanospheres pretreated with oxygen plasma were rapidly dispersed in N,N-dimethylformamide solvent for polyvinylpyrrolidone polymer side-linking treatment, wherein the mass of carbon nanospheres was 500 mg, the solvent was 100 mL, and the monomer N-vinylpyrrolidone was 10 mL.

[0046] S5: After the above-mentioned dispersion suspension is sonicated for 30 minutes and pre-protected with nitrogen for 30 minutes, the initiator azobisisobutyronitrile is added. The mass of azobisisobutyronitrile is 200 mg.

[0047] S6: The reaction system with the added initiator was heated and polymerized at 60°C. The polymer side-linked branch polymerization reaction time was 48h to prepare carbon-based molecular brush nanoparticles Csp-g-PVP.

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

[0049] To better illustrate the technical effects of this 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 transform infrared spectroscopy analysis, particle size analysis and biocompatibility analysis.

[0050] 1. Surface morphology characterization

[0051] The surface morphology of Csp and Csp-g-PVP samples was characterized using a scanning electron microscope (S-4800, 5.0 kV), and the results were as follows: Figure 1 As shown.

[0052] according to Figure 1 It can be seen that after being side-linked with polyvinylpyrrolidone polymer on the surface, Csp-g-PVP can still maintain the same uniformly dispersed spherical morphology as Csp, indicating that the side-linking with polyvinylpyrrolidone polymer does 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 using an infrared spectrometer (IS 50), and the results were as follows: Figure 2 As shown.

[0055] according to Figure 2 It can be seen that the Csp-g-PVP after being side-linked with polyvinylpyrrolidone polymer showed obvious PVP characteristic peaks in its Fourier transform infrared spectrum, thus indicating the successful preparation of carbon-based molecular brush nanoparticles.

[0056] 3. Particle size analysis

[0057] Particle size distribution of Csp and Csp-g-PVP in PBS buffer was characterized using a Zeta potentiometer (BI-PALS). Specifically, Csp and Csp-g-PVP were prepared at a concentration of 0.5 mg / mL with PBS buffer, and particle size analysis was performed. The results were as follows: Figure 3 As shown.

[0058] according to Figure 3 It can be seen that the hydrodynamic diameter of Csp in PBS buffer is mainly concentrated at 1002 nm, with a characteristic peak indicating secondary particles appearing at 6313 nm. In contrast, the carbon-based molecular brush nanoparticles (Csp-g-PVP) have a uniform particle size distribution and a hydrodynamic diameter of 259 nm in PBS buffer. In conclusion, side-linking with polyvinylpyrrolidone polymer 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 statistically analyzed using an enzyme-linked immunosorbent assay (ELISA) reader to determine cytotoxicity.

[0061] according to Figure 4It can be seen that the survival rate of L929 cells co-cultured with Csp-g-PVP for 24 h is still more than 85%, which indicates that Csp-g-PVP has good biocompatibility.

[0062] Example 2

[0063] A preparation method of a carbon-based molecular brush lymphatic nano-tracer, comprising the following steps:

[0064] S1: The small molecule monomer aniline (2.32 mL) and pyrrole (1.75 mL) are dispersed in 360 mL of an aqueous solution containing 340 μL of triton x-100, and then stirred and ultrasonically dispersed for 30 min each. The pre-cooled ammonium persulfate solution (11.52 g of ammonium persulfate dissolved in 50 mL of deionized water) is added under ice bath conditions, and the reaction is carried out under ice bath conditions for 12 h.

[0065] S2: After the obtained precursor solid is centrifuged, washed and freeze-dried, it is placed in a tube furnace for carbonization treatment under a nitrogen atmosphere, the carbonization temperature is 900°C, the heating rate is 5°C / min, and the carbonization treatment time is 10 h.

[0066] S3: The carbon nanospheres obtained after carbonization are placed in a glass culture dish, a petri dish or a beaker and placed 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 polyvinylpyrrolidone polymer side chain grafting treatment, wherein the mass of the carbon nanospheres is 500 mg, the solvent is 100 mL, and the monomer N-vinylpyrrolidone is 10 mL.

[0068] S5: After the above dispersion suspension is ultrasonically treated for 30 min and pre-nitrogenated for 30 min, an initiator azobisisobutyronitrile is added, and the mass of the azobisisobutyronitrile is 200 mg.

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

[0070] S7: After the reaction is completed, the obtained carbon-based molecular brush nanoparticles Csp-g-PVP are centrifuged, washed and freeze-dried, and then dispersed in physiological saline, and the concentration of the carbon-based molecular brush nanoparticles in the physiological saline is 50 mg / mL. A carbon-based molecular brush lymphatic nano-tracer with good stability and lymph node staining function is obtained.

[0071] Example 3

[0072] The present embodiment provides a preparation method of a carbon-based molecular brush lymphatic nanotracer, comprising the following steps:

[0073] S1: The small molecule monomer aniline (2.32 mL) and pyrrole (1.75 mL) are dispersed in 360 mL of an aqueous solution containing 340 μL of triton x-100, and then stirred and ultrasonically dispersed for 30 min each, and pre-cooled in an ice bath for 30 min. A pre-cooled ammonium persulfate solution (11.52 g of ammonium persulfate dissolved in 50 mL of deionized water) is added under ice bath conditions, and reacted for 12 h under ice bath conditions.

[0074] S2: After the obtained precursor is centrifuged, washed and freeze-dried, it is placed in a tube furnace for carbonization treatment under a nitrogen atmosphere, with a carbonization temperature of 900°C, a heating rate of 5°C / min, and a carbonization treatment time of 10 h.

[0075] S3: The carbon nanospheres obtained after carbonization are placed in a glass culture dish, a petri dish or a beaker and placed in a plasma device for oxygen plasma pretreatment. The carbon nanospheres are subjected to oxygen plasma pretreatment at a power of 150 W for a total treatment time of 32 min.

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

[0077] S5: After the above dispersion suspension is ultrasonically treated for 30 min and pre-nitrogenated for 30 min, an initiator azobisisobutyronitrile is added, and the mass of the azobisisobutyronitrile is 200 mg.

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

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

[0080] Example 4

[0081] The present embodiment provides a preparation method of a carbon-based molecular brush lymphatic nanotracer, comprising the following steps:

[0082] S1: The small molecule monomer 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 pre-cooled in an ice bath for 30 min. A pre-cooled ammonium persulfate solution (11.52 g of ammonium persulfate dissolved in 50 mL of deionized water) was added under ice bath conditions, and the reaction was carried out under ice bath conditions for 12 h.

[0083] S2: After the obtained precursor was centrifuged, washed and freeze-dried, a carbonization treatment was carried out under a nitrogen atmosphere, the carbonization temperature was 900°C, the heating rate was 5°C / min, and the carbonization treatment time was 10 h.

[0084] S3: The carbon nanospheres obtained after carbonization were placed in a glass culture dish, a surface dish or a beaker and subjected to oxygen plasma pretreatment in a plasma device, the oxygen plasma treatment power was 150 W, and the total treatment time was 32 min.

[0085] S4: The carbon nanospheres pretreated by oxygen plasma were rapidly dispersed in N,N-dimethylformamide solvent for polyvinylpyrrolidone high molecular side chain grafting treatment, the mass of the carbon nanospheres was 500 mg, the solvent was 100 mL, and the monomer N-vinylpyrrolidone was 10 mL.

[0086] S5: After the above dispersion suspension was ultrasonically treated for 30 min and pre-nitrogenated for 30 min, an initiator azobisisobutyronitrile was added, and the mass of the azobisisobutyronitrile was 200 mg.

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

[0088] S7: After the reaction was completed, the carbon-based molecular brush nanoparticles Csp-g-PVP with polyvinylpyrrolidone high molecular side chain grafting obtained were centrifuged, washed and freeze-dried, and then dispersed in physiological saline, and the concentration of the carbon-based molecular brush nanoparticles in the physiological saline was 12.5 mg / mL. A carbon-based molecular brush lymphatic nanotracer with good stability and lymph node staining function was obtained.

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

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

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

[0092] Fifteen female healthy Balb / c mice were divided into three groups, and 50 μL of the tracer was injected subcutaneously into the footpads of the mice. The mice were sacrificed 10 min after injection. The black staining degree of the footpads, the bending part of the hind legs and the iliac lymph nodes was observed. The black staining degree scoring standard was as follows: 0 points for no change in the color of the lymph nodes, 0.5 points for the lymph nodes being dyed gray, and 1.0 points for the lymph nodes being obviously black. The results are shown in Table 1, wherein the differences between Csp-g-PVP-1, Csp-g-PVP-3 and Csp-g-PVP-4 lie in that the concentrations of the lymph tracers are 50 mg / mL, 25 mg / mL and 12.5 mg / mL, respectively.

[0093] Table 1 Black staining degree score table of carbon-based molecular brush lymphatic nanotracer with different concentrations on mouse lymph nodes

[0094]

[0095] According to Table 1, the Csp-g-PVP-1 lymph tracer has the best lymph node black staining score 10 min after injection, and therefore 50 mg / mL is the optimal lymph node black staining tracer concentration.

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

[0097] 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 were subjected to thermogravimetric analysis. The carbon-based molecular brush lymphatic nanotracer (Csp-g-PVP-1) prepared in Example 1 and the carbon-based molecular brush lymphatic nanotracer (Csp-g-PVP-2) prepared in Example 2 were subjected to lymph node black staining score, and the difference between Csp-g-PVP-1 and Csp-g-PVP-2 lies in that the polymerization time in step S6 is 48h and 24h, respectively, and Csp-Sus is the dispersion of Csp in physiological saline, i.e., the polymerization time is 0h.

[0098] 2.1 Thermogravimetric analysis

[0099] Thermogravimetric analysis (TGA) was performed using a TG209F1 thermogravimetric analyzer to characterize the grafting amount of polymers after polymerization at different times for Csp-g-PVP-48h and Csp-g-PVP-24h. The results are as follows: Figure 5 As 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 conclusion, extending the polymerization time of polyvinylpyrrolidone polymer side-chain grafting will effectively increase the amount of polymer side chains grafted onto the Csp surface.

[0101] 2.2 Lymph node melanosis score

[0102] Fifteen healthy female Balb / c mice were randomly divided into three groups. 50 μL of tracer was injected subcutaneously into the paw pads of the mice. The mice were sacrificed 10 minutes after injection. The degree of blackening of the lymph nodes in the paws, hind leg bends, and iliac lymph nodes was observed visually. The scoring criteria for the degree of blackening were: 0 points for no change in lymph node color, 0.5 points for lymph nodes stained gray, and 1.0 points for significantly blackened lymph nodes. The results are shown in Table 2.

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

[0104]

[0105] Table 2 shows that the Csp-g-PVP-1 lymphatic tracer obtained after 48 hours of graft polymerization exhibits the best lymph node blackening performance. In summary, with prolonged polymerization time, the amount of polymer side-linked grafts increases, resulting in a better lymphatic tracer for lymph node blackening.

[0106] 3. Lymph node blackening score at different time points after injection

[0107] To characterize the changes in lymph node blackening scores over time after injection, 15 healthy female Balb / c mice were randomly divided into 3 groups. 50 μL of the lymphatic tracer Csp-g-PVP-1 prepared in Example 1 was injected subcutaneously into the paw pads of the mice. Mice were sacrificed at 10, 30, and 60 minutes after injection. The samples from these groups 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, respectively. The degree of blackening of the lymph nodes in the paw pads, hind leg bends, and iliac lymph nodes was observed visually. The scoring criteria for blackening were: 0 points for no change in lymph node color, 0.5 points for lymph nodes stained gray, and 1.0 points for significantly blackened lymph nodes. The results are shown in Table 3.

[0108] Table 3 Carbon-based molecular brush lymphatic nanotracer lymph node black staining degree score table at different post-injection observation times

[0109]

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

[0111] Each embodiment in the specification is described in a progressive manner, and the same or similar parts between each embodiment can be mutually referred to, and each embodiment focuses on the differences from other embodiments.

[0112] The above examples are only used to illustrate the technical solutions of the present application, and are not limited to the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present application.

Claims

1. The application of a carbon-based molecular brush in the preparation of lymphatic nanotracers, characterized in that, Includes the following steps: 1) Aniline and pyrrole are dispersed in an aqueous solution containing surfactant, and a pre-cooled initiator aqueous solution is added. The reaction is carried out in an ice bath for 10-14 hours to prepare a nanosphere precursor. The nanosphere precursor is then carbonized into carbon nanospheres, wherein the carbonization temperature is 600-1000℃ and the carbonization time is 3-24 hours. 2) After the carbon nanospheres in step 1) are pretreated with oxygen plasma to prepare carbon nanoparticles, they are dispersed in an organic solvent and N-vinylpyrrolidone is added and dispersed evenly. Then an initiator is added and the reaction is carried out at 60-80℃ for 12-72h under an inert atmosphere to obtain carbon-based molecular brush nanoparticles. The mass-volume ratio of carbon nanoparticles to N-vinylpyrrolidone is 100:1-2mg / mL, and the mass ratio of initiator to carbon nanoparticles is 1-25:25g / g. 3) Disperse the carbon-based molecular brush nanoparticles obtained in step 2) in physiological saline at a concentration of 50 mg / mL to obtain carbon-based molecular brush lymphatic nanotracer.

2. The application according to claim 1, characterized in that, In step 2), the organic solvent used for grafting hydrophilic polymer side chains includes one or more of N,N-dimethylformamide, tetrahydrofuran, or 1,4-dioxane.

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