Carbon nano-molecular probe as well as preparation method and application thereof

Abstract

The invention discloses a carbon nano molecular probe as well as a preparation method and application thereof, and belongs to the technical field of chemical biology. The carbon nano-molecular probe comprises carbon nano-particles, wherein the surfaces of the carbon nano-particles are connected with polypeptide, and the nitrogen end of the polypeptide is connected with fluorescent molecules. The nano-scale passive diffusion and fluorescence quenching properties of the carbon nano-particles are utilized, a polypeptide sequence which can be subjected to specific enzyme digestion and is labeled by specific fluorescence can be connected to the surfaces of the carbon nano-particles, molecular imaging is performed in immune cells, the biochemical environment and the curing state in the immune cells are monitored in real time, and cellular level immune drug screening is performed. The important application value is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a carbon nanomolecular probe, a preparation method and application thereof, and belongs to the technical field of chemical biology. BACKGROUND

[0002] The immune system, as a complex and highly organized cellular system, helps maintain homeostasis in the body and protects against pathogenic invasion. The immune system is composed of three basic lines of defense: innate immunity, adaptive immunity, and trained immunity. Any abnormalities in any branch of these three lines of defense can lead to immune system diseases, including autoimmune and immunodeficiency. Immune cells originate from bone marrow and migrate to peripheral tissues through blood and lymphatic vessels. Immune cells are regulated by multiple pathways and cytokines during the immune process, such as proliferation, differentiation and chemotaxis, and produce immune responses in various situations such as cancer occurrence and development, inflammatory diseases, infection, organ transplant rejection, etc. Therefore, monitoring the biochemical pathways in immune cells helps to understand the immune process and conduct immune cell drug screening.

[0003] Cathepsin S is a lysosomal cysteine protease that is highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix decomposition and cleavage of cell adhesion molecules to facilitate immune cell movement, as well as cleavage of invariant chains during major histocompatibility complex class II maturation, which is an important mediator of adaptive immunity. One of the important roles of cathepsin S in immunology is to process invariant chains (Ii) during major histocompatibility complex Ii (MHC Ii) maturation, which is a key step in class I antigen presentation. At the same time, it increases the motility of dendritic cells in a myosin ii-dependent manner, allowing dendritic cells to migrate to lymph nodes.

[0004] Currently, various molecular imaging methods have been developed to monitor immune responses of immune cells: positron emission tomography (PET), single photon emission computed tomography, magnetic resonance imaging (MRI), X-ray computed tomography and optical imaging. These imaging techniques usually require the use of specific imaging agents, such as positron emission radioactive tracers in PET and MRI contrast agents for MRI. Among them, optical imaging uses photons to obtain detailed dynamic information of biomarkers at the molecular, cellular and tissue levels, and has the characteristics of high sensitivity, high spatiotemporal resolution, low cost and strong adaptability. Most optical imaging relies on imaging probes, whose response signal is related to molecular specificity and sensitivity. Optical imaging combined with molecular probes has been used to image biomarkers of immune cells and dynamically visualize monitoring.

[0005] However, the synthesis of the molecular probe in the optical imaging at present involves a complex compound skeleton structure, needs a multi-step chemical reaction and a large amount of separation and purification work, and has a high cost. Moreover, the synthesized probe compound skeleton can only be used for detecting one biomarker, and has poor universality. SUMMARY

[0006] Therefore, the main purpose of the present application is to provide a carbon nanomolecular probe, a preparation method and application thereof, so as to overcome the shortcomings of the prior art.

[0007] The carbon nanomolecular probe comprises a carbon nanoparticle with a polypeptide connected to the surface, wherein the polypeptide is a polypeptide sequence with a fluorescent molecule connected to the nitrogen end.

[0008] The fluorescent molecule is selected from any one of Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7.5, sulfo-Cy3, sulfo-Cy5, sulfo-Cy7, 5-FAM, 6-FAM, 5-TAMRA, 6-TAMRA, SIR, AF 647, AF 430, and AF 488.

[0009] The polypeptide sequence is selected from any one of the following:

[0010] GRWPPMGLPWEC-DArg-NH2 and GRWHPMGAPWEC-DArg-NH2.

[0011] The preparation method of the carbon nanomolecular probe comprises the following steps:

[0012] Step 1: reacting a polypeptide sequence and a fluorescent molecule to obtain a polypeptide sequence with a fluorescent molecule connected to the nitrogen end, i.e. a fluorescent molecule-labeled polypeptide;

[0013] Step 2: reacting the fluorescent molecule-labeled polypeptide obtained in step 1 with a two-dimensional carbon nanoparticle with a maleimide group on the surface to obtain a carbon nanomolecular probe.

[0014] Further, in step 1, the fluorescent molecule, the polypeptide sequence and an organic base are added to an N,N-dimethylformamide solution system, the pH is adjusted to 7-9, and the reaction is carried out at 37℃ in the dark for 4-12h. After the reaction is completed, the product is precipitated with ice ether, centrifuged, washed and dried, and then separated by HPLC and freeze-dried to obtain the fluorescent molecule-labeled polypeptide. According to the embodiment of the present application, the organic base comprises N,N-diisopropylethylamine. When the fluorescent molecule raw material is added, the N-hydroxysuccinimidyl ester form of the fluorescent molecule is added to the reaction system to react with the nitrogen end of the polypeptide.

[0015] In step 2, the fluorescently labeled peptide obtained in step 1 is added to an N,N-dimethylformamide dispersion of two-dimensional carbon nanoparticles with maleimide groups on the surface, the pH is adjusted to 6.5-7.5, and the cross-linking reaction is carried out. The mixture is stirred in the dark for 12-24 h to obtain a carbon nanoparticle probe.

[0016] Furthermore, the two-dimensional carbon nanoparticles with maleimide groups modified on their surface are prepared by the following method:

[0017] 30 mg of two-dimensional carbon nanoparticles were dispersed in N,N-dimethylformamide (DMF), and 200 mg of N-ethylmaleimide malonate, 0.5 mL of 1,8-diazabicycloundec-7-ene (DBU) and 200 mg of carbon tetrabromide were added. The reaction was carried out at 80 °C for 7 days under nitrogen protection. The liquid after the reaction was centrifuged at 12,000 rpm to collect the solid product. The product was washed repeatedly with N,N-dimethylformamide (DMF), anhydrous ethanol and deionized water, and then freeze-dried to obtain two-dimensional carbon nanoparticles with maleimide groups on the surface.

[0018] Furthermore, the two-dimensional carbon nanoparticles are prepared by the following method:

[0019] C 60 The Mg powder was mixed with Mg powder at a molar ratio of 1:4 and vacuum-sealed in a 25 cm long quartz tube. The quartz tube was then placed in a dual-temperature zone tube furnace (divided into a high-temperature zone and a low-temperature zone, set at 600°C and 500°C respectively, with a temperature gradient of 100°C) and held for 20 hours. The mixed powder, located in the high-temperature zone, vaporized and, driven by the temperature difference, was transported to the low-temperature zone to grow polymerized C. 60 Crystals. To fully remove polymerized C 60 Mg ions between crystal layers reduce two-dimensional C 60 Interlayer forces, using acid etching to separate polymer C 60 Crystals were soaked in 20 wt% dilute nitric acid and stirred for 48 hours. The mixture was then centrifuged at 1000 rpm for 10 min to remove the supernatant. The solid precipitate was collected, washed multiple times with water and ethanol, and then dispersed in N-methylpyrrolidone solution and sonicated at 600 W for 10 hours. Subsequently, the resulting suspension was centrifuged at 7000 rpm for 20 minutes to remove incompletely detached crystals and thicker nanosheets. The liquid was collected and centrifuged at 12000 rpm for 20 minutes to collect thin nanosheets. These thin nanosheets were dispersed in N-methylpyrrolidone and sonicated at 600 W for 10 hours. The solid product was then collected after centrifugation at 12000 rpm for 20 minutes. Finally, the two-dimensional carbon nanoparticles were washed multiple times with anhydrous ethanol and deionized water, dispersed in ultrapure water, and freeze-dried to obtain the two-dimensional carbon nanoparticles.

[0020] The application of the carbon nanoparticle probe of this invention in the preparation of reagents for detecting the maturation status of immune cells.

[0021] After the carbon nanoparticle probe is endocytosed by immune cells, the polypeptide sequence on the surface of the carbon nanoparticle probe is specifically cleaved by intracellular cathepsin S, releasing a polypeptide fragment linked to a fluorescent molecule. The fluorescence intensity is positively correlated with the intracellular cathepsin S content.

[0022] Furthermore, the application of the carbon nanoparticle probe of the present invention in the preparation of a reagent for detecting the maturation state of dendritic cells.

[0023] In this invention, carbon nanoparticles with peptides attached to their surface form carbon nanoparticle molecular probes. Due to the broad-spectrum light absorption of two-dimensional carbon nanoparticles, they can serve as quenching groups in fluorescence resonance energy transfer probe systems, quenching the fluorescence of peptides modified with fluorescent groups within a 10 nm range. During co-incubation with mature dendritic cells, the peptides are taken up by the cells via nanoparticle-mediated endocytosis and enter the intracellular environment. Because cathepsin S can specifically cleave the peptide sequences on the surface of the two-dimensional carbon nanoparticles, the distance between the fluorescent group and the two-dimensional carbon nanoparticles is greater than 10 nm, resulting in the release of a fluorescence signal that responds to the intracellular cathepsin S content. Utilizing the nanoscale passive diffusion and fluorescence quenching properties of carbon nanoparticles, specifically fluorescently labeled peptide sequences that can be cleaved by specific enzymes can be attached to their surface. This allows for molecular imaging within immune cells, real-time monitoring of the intracellular biochemical environment and maturation state, and screening of cellular-level immunotherapies, demonstrating significant application value. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structure of the two-dimensional carbon nanoparticles with maleimide groups modified on the surface of the present invention.

[0025] Figure 2 This describes the synthesis route and luminescence principle of the carbon nanoparticle probe in this embodiment of the invention.

[0026] Figure 3 The mass spectrum of the polypeptide sequence prepared in an embodiment of the present invention.

[0027] Figure 4 The mass spectrum of the fluorescently labeled polypeptide prepared in an embodiment of the present invention is shown.

[0028] Figure 5 Transmission electron microscopy characterization of the two-dimensional carbon nanoparticles prepared in an embodiment of the present invention.

[0029] Figure 6 The 1H NMR spectrum of N-ethylmaleimide malonate prepared in an embodiment of the present invention.

[0030] Figure 7The NMR carbon spectrum of N-ethylmaleimide malonate prepared in the embodiments of the present invention.

[0031] Figure 8 Transmission electron microscopy characterization of two-dimensional carbon nanoparticles with maleimide groups prepared in an embodiment of the present invention.

[0032] Figure 9 The infrared spectrum characterization of two-dimensional carbon nanoparticles with maleimide groups prepared in the embodiments of the present invention.

[0033] Figure 10 The image shows the transmission electron microscope characterization of the carbon nanoparticle probe prepared in the embodiment of the present invention.

[0034] Figure 11 The potential distribution diagram of the carbon nanoparticle probe prepared in the embodiment of the present invention is shown.

[0035] Figure 12 The absorption spectrum of the carbon nanoparticle probe prepared in an embodiment of the present invention is shown.

[0036] Figure 13 The absorbance curves of carbon nanoparticle probes prepared for embodiments of the present invention at different concentrations are shown.

[0037] Figure 14 The concentration-absorbance standard curve of the carbon nanoparticle probe prepared for the embodiments of the present invention.

[0038] Figure 15 The survival rates of mouse bone marrow-derived dendritic cells (BMDCs) at different concentrations of carbon nanomolecular probes prepared for embodiments of the present invention and two-dimensional carbon nanoparticles with maleimide groups on the surface of unlinked peptides.

[0039] Figure 16 The confocal fluorescence image of the carbon nanoparticle probe prepared for an embodiment of the present invention in DC2.4 cells.

[0040] Figure 17 The fluorescence intensity of bone marrow-derived dendritic cells (BMDCs) in mice treated with different drugs.

[0041] Figure 18 The maturation status of bone marrow-derived dendritic cells (BMDCs) in mice treated with different drugs. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0043] The molecular probes in related technologies all have complex synthesis routes. For example, fluorescence resonance energy transfer molecular probes require fluorescent groups and quenching groups, which are costly and have complex processes, thus limiting the application of immunotherapies at the cell screening level.

[0044] Therefore, the present invention provides a carbon nanoparticle probe, comprising: carbon nanoparticles with a polypeptide attached to their surface; wherein the polypeptide is a specific enzyme digestion sequence with a fluorescent molecule attached to its nitrogen end; and the surface of the two-dimensional carbon nanoparticles is modified with maleimide groups.

[0045] Cathepsin S is a lysosomal cysteine ​​protease that is highly expressed in immune cells such as dendritic cells, B cells, and macrophages. Its functions include the breakdown of the extracellular matrix and the cleavage of cell adhesion molecules to promote immune cell motility, as well as the cleavage of the invariant chain during the maturation of major histocompatibility complex II. It is an important mediator of adaptive immunity.

[0046] In this invention, carbon nanoparticle probes are obtained through the coupling reaction of maleimide and thiol groups of polypeptide sequences on the surface of carbon nanoparticles. Due to the broad-spectrum light absorption of two-dimensional carbon nanoparticles, they can serve as quenching groups in fluorescence resonance energy transfer probe systems, quenching the fluorescence of polypeptides modified with fluorescent groups within a 10 nm range. During co-incubation with maturing dendritic cells, the nanoparticles-mediated endocytosis allows the probes to be taken up by the cells and enter the intracellular environment. Because cathepsin S can specifically cleave the polypeptide sequences on the surface of the two-dimensional carbon nanoparticles, the distance between the fluorescent group and the two-dimensional carbon nanoparticles is greater than 10 nm, resulting in fluorescence release and a response to intracellular cathepsin S expression, indirectly indicating the maturation state of immune cells. Utilizing the nanoscale passive diffusion and fluorescence quenching properties of carbon nanoparticles, specific fluorescent polypeptide sequences that can be cleaved by specific enzymes can be attached to their surface for molecular imaging within immune cells. This allows for real-time monitoring of the intracellular biochemical environment and maturation state of immune cells, facilitating the screening of cellular-level immunotherapies, and has significant application value.

[0047] In this embodiment of the invention, carbon nanoparticles can enter dendritic cells via a nanoparticle-mediated endocytosis pathway. Specifically, the N-hydroxysuccinimide ester of a fluorescent molecule reacts with the free amino groups of the polypeptide sequence to label the polypeptide. Furthermore, by modifying the surface of the carbon nanoparticles with maleimide groups, which then react with cysteine ​​thiol groups in the polypeptide sequence to bond the fluorescently labeled polypeptide, fluorescence quenching of the polypeptide can be achieved.

[0048] Specifically, after being pulsed with dendritic cells, the carbon nanoparticle probes obtained by the above method enter the dendritic cells through nanoparticle-mediated endocytosis and passive diffusion. The polypeptide sequence on the surface of the carbon nanoparticle probes is specifically cleaved by intracellular cathepsin S, releasing polypeptide fragments linked to fluorescent molecules. The fluorescence intensity is positively correlated with the intracellular cathepsin S content, which can indirectly predict the maturation status of immune cells.

[0049] According to embodiments of the present invention, the fluorescent molecule includes any one of the following:

[0050] Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7.5, Sulfo-Cy3, Sulfo-Cy5, Sulfo-Cy7, 5-FAM, 6-FAM, 5-TAMRA, 6-TAMRA, SIR, AF 647, AF 430, AF 488.

[0051] According to embodiments of the present invention, the polypeptide sequence comprises any one of the following:

[0052] GRWPPMLPWEC-DArg-NH2, GRWHPMGAPWEC-DArg-NH2.

[0053] As another aspect of the present invention, the present invention also provides a method for preparing carbon nanoparticle molecular probes, comprising:

[0054] The polypeptide sequence is reacted with a fluorescent dye to obtain a fluorescently labeled polypeptide;

[0055] The fluorescently labeled peptides are reacted with two-dimensional carbon nanoparticles with maleimide groups on their surface to obtain carbon nanoparticle probes.

[0056] According to an embodiment of the present invention, the above-mentioned reaction of the polypeptide sequence with a fluorescent dye to obtain a fluorescently labeled polypeptide includes: adjusting the pH of the fluorescent N-hydroxysuccinimide dye, the purified polypeptide, and an organic base to 7-9 in an N,N-dimethylformamide solution system, mixing and reacting at 37°C in the dark for 4-12 hours, precipitating with ice-cold diethyl ether to obtain a crude product, centrifuging, washing, drying, separating by HPLC, and lyophilizing to obtain a polypeptide modified with a fluorescent group; according to an embodiment of the present invention, the organic base includes N,N-diisopropylethylenediamine.

[0057] In this embodiment of the invention, under light-protected conditions, the purified peptide was dispersed in anhydrous N,N-dimethylformamide solvent, and an organic base was added at a molar ratio of 6:1 to the peptide. The reaction conditions were kept alkaline. Then, an N-hydroxysuccinimide ester dye with fluorescent molecules was added at a molar ratio of 1.2-3:1 to the peptide, for example, 1.2:1 or 2-3:1. The mixture was shaken and reacted at 37°C in the dark for 4-12 hours. After the reaction, the product was repeatedly precipitated with diethyl ether, washed multiple times, and dried to obtain a crude peptide product modified with a fluorescent group. The organic base included N,N-diisopropylethylenediamine. The crude product was dissolved in guanidine hydrochloride solution, separated by reversed-phase high-performance liquid chromatography, and lyophilized to obtain the fluorescently labeled peptide.

[0058] According to an embodiment of the present invention, the method of mixing and reacting the above-mentioned fluorescently labeled peptide with two-dimensional carbon nanoparticles modified with maleimide groups to obtain carbon nanoparticle probes includes: adding the above-mentioned fluorescently labeled peptide to an N,N-dimethylformamide dispersion of two-dimensional carbon nanoparticles modified with maleimide groups, adjusting the pH to 6.5-7.5, performing a maleimide alkylation reaction, mixing and stirring at 500-1000 rpm at room temperature, reacting for a preset time in the dark, and centrifuging, washing and drying at high speed to obtain carbon nanoparticle probes.

[0059] In this embodiment of the invention, the preset time includes 24 hours. In this embodiment, after reacting at room temperature with stirring in the dark for 24 hours, the mixture is centrifuged at high speed (15000-20000 g) at 4°C to obtain the reacted carbon nanoparticles. The supernatant is discarded, and anhydrous N,N-dimethylformamide is added for ultrasonic dispersion for 5-10 minutes. The mixture is washed 3-4 times, the washings are discarded, and the carbon nanoparticle probe is obtained after drying.

[0060] As another aspect of the present invention, the present invention also provides the application of carbon nanoparticle probes in the detection of dendritic cell maturation.

[0061] The carbon nanoparticle probe provided by this invention, after being pulsed with dendritic cells in vitro, enters the dendritic cells through nanoparticle-mediated endocytosis and passive diffusion. The polypeptide sequence on the surface of the carbon nanoparticle probe is specifically cleaved by intracellular cathepsin S, releasing polypeptide fragments linked to fluorescent molecules. The fluorescence intensity is positively correlated with the intracellular cathepsin S content, which can indirectly predict the maturation status of immune cells.

[0062] The technical solution of the present invention will be further described below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to illustrate the technical solution of the present invention, but the present invention is not limited thereto.

[0063] Example 1: Preparation of polypeptide sequences

[0064] The target peptide sequence GRWPPMGLPWEC-DArg-NH2 was prepared using a conventional solid-phase synthesis method. For specific synthesis methods, please refer to the book "Peptide Synthesis Methods and Protocols" (ISSN 1064-3745 ISSN 1940-6029(electronic)) written by Waleed M. Hussein, Mariusz Skwarczynski, and Istvan Toth.

[0065] The peptides prepared in Example 1 were characterized by ESI-MS, and the characterization results are as follows: Figure 3 As shown.

[0066] Figure 3 The diagram illustrates the mass spectrum of the polypeptide prepared according to an embodiment of the present invention.

[0067] like Figure 3 As shown, ESI-MS (ACN / H2O): m / z: 792.95 ([M+2H] 2+ The result ( / 2, calcd.792.875) is consistent with the target molecular weight, which proves that the target polypeptide sequence has been obtained.

[0068] Example 2: Preparation of fluorescently labeled peptides

[0069] 1 mmol of purified peptide was dissolved in a solution containing 0.5 mL of anhydrous N,N-dimethylformamide and 6 mmol of N,N-diisopropylethylenediamine (DIPEA), and 1.2 mmol of Cy5.5 N-hydroxysuccinimide dye was added. The reaction was carried out at room temperature with shaking for 12 hours. After the reaction was completed, excess diethyl ether was added to change the polarity of the solution system, causing the crude product to precipitate as a solid. The solid was separated by centrifugation at 10,000 rpm, and the obtained solid was washed three times with anhydrous diethyl ether. After washing and drying, the solid was dissolved in an aqueous solution containing 10% acetonitrile, and purified by reversed-phase HPLC. The resulting fluorescently labeled peptide was obtained by lyophilization.

[0070] The fluorescently labeled peptides prepared in Example 2 were characterized by ESI-MS, and the results are as follows: Figure 4 As shown.

[0071] Figure 4 The mass spectrum of the fluorescently labeled polypeptide prepared according to an embodiment of the present invention is illustrated schematically.

[0072] like Figure 4 As shown, ESI-MS (ACN / H2O): m / z: 1075.29 ([M+2H]). 2+ / 2, calcd.1075.535), consistent with the target molecular weight, which proves that a polypeptide with a fluorescent molecular labeling of Cy5.5 modified at the nitrogen end was obtained.

[0073] Example 3: Using different fluorescently labeled peptides

[0074] In addition to Cy5.5 fluorescent molecules, N-hydroxysuccinimide dyes such as Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7.5, sulfon-Cy3, sulfon-Cy5, sulfon-Cy7, 5-FAM, 6-FAM, 5-TAMRA, 6-TAMRA, SIR, AF 647, AF 430, and AF 488 can be used to react with the nitrogen terminus of the peptide for fluorescent labeling. Correspondingly, subsequent fluorescence assays require excitation wavelengths corresponding to the fluorescent molecules and detection wavelengths.

[0075] Example 4: Preparation of two-dimensional carbon nanoparticles

[0076] C 60 The Mg powder was mixed with Mg powder at a molar ratio of 1:4 and vacuum-sealed in a 25 cm long quartz tube. The quartz tube was then placed in a dual-temperature zone tube furnace (divided into a high-temperature zone and a low-temperature zone, with a temperature gradient of 100°C) and held for 20 hours. The mixed powder, located in the high-temperature zone, vaporized and, driven by the temperature difference, was transported to the low-temperature zone to grow polymerized C. 60 Crystals. To fully remove polymerized C 60 Mg ions between crystal layers reduce two-dimensional C 60 Interlayer forces were utilized by etching polymer C using dilute acid. 60 Crystals were soaked in 20% v / v dilute nitric acid and stirred for 48 hours. After centrifugation at 1000 rpm for 10 min, the supernatant was removed, and the solid precipitate was collected. After washing several times with water and ethanol, the precipitate was dispersed in N-methylpyrrolidone solution and sonicated at 600 W for 10 hours. Subsequently, the resulting suspension was centrifuged at 7000 rpm for 20 minutes to remove incompletely detached crystals and thicker nanosheets. The liquid was collected and centrifuged at 12000 rpm for 20 minutes to collect thin nanosheets. These thin nanosheets were dispersed in N-methylpyrrolidone and sonicated at 600 W for 10 hours, followed by centrifugation at 12000 rpm for 20 minutes to collect the solid product. Finally, the solid product was washed several times with anhydrous ethanol and deionized water, dispersed in ultrapure water, and freeze-dried to obtain two-dimensional carbon nanoparticles.

[0077] The two-dimensional carbon nanoparticles prepared in Example 4 were characterized by transmission electron microscopy, and the results are as follows: Figure 5 As shown.

[0078] Figure 5 The diagram illustrates a transmission electron microscope characterization image of two-dimensional carbon nanoparticles prepared according to an embodiment of the present invention.

[0079] Example 5: Preparation of N-ethylmaleimide malonate

[0080] 0.2 mL of malonyl chloride and 564 mg of N-(2-hydroxyethyl)maleimide were dissolved in 70 mL of dichloroethane. The mixture was reacted at room temperature under nitrogen atmosphere for 12 h, and the solvent was evaporated. The crude product was separated by silica gel column chromatography using dichloromethane:ethyl acetate as the mobile phase (4:1) to obtain the final white solid product, with a yield of 84%. The final product was analyzed by 1H NMR and 1C NMR spectroscopy. 13 Its chemical structure was characterized by C-NMR, and the results are as follows: Figure 6 , 7 As shown.

[0081] Figure 6 The schematic diagram shows the 1H NMR spectrum of N-ethylmaleimide malonate prepared according to an embodiment of the present invention.

[0082] like Figure 6 As shown, 1 ¹H NMR (600 MHz, Chloroform-d) δ 6.73 (s, 4H), 4.29 (t, J = 5.3 Hz, 4H), 3.81 (t, J = 5.3 Hz, 4H), 3.32 (s, 2H). The peak positions for each group are as follows: Figure 6 As shown, it can be demonstrated that N-ethylmaleimide malonate product was prepared.

[0083] Figure 7 The schematic diagram shows the carbon NMR spectrum of N-ethylmaleimide malonate prepared according to an embodiment of the present invention.

[0084] like Figure 7 As shown, 13 C10 NMR (151 MHz, Chloroform-d) δ 170.52, 166.09, 134.39, 62.58, 41.16, 36.71. The peak positions for each group are as follows: Figure 7 As shown, the N-ethylmaleimide malonate product was successfully prepared.

[0085] Example 6: Preparation of two-dimensional carbon nanoparticles with maleimide groups on their surface

[0086] 30 mg of two-dimensional carbon nanoparticles were dispersed in 30 ml of N,N-dimethylformamide (DMF), and 200 mg of N-ethylmaleimide malonate, 0.5 ml of 1,8-diazabicycloundec-7-ene (DBU) and 200 mg of carbon tetrabromide were added. The reaction was carried out at 80 °C for 7 days under nitrogen protection. The liquid after the reaction was centrifuged at 12000 rpm to collect the solid product. The product was washed repeatedly with N,N-dimethylformamide (DMF), anhydrous ethanol and deionized water, and then freeze-dried to obtain two-dimensional carbon nanoparticles with maleimide groups on the surface.

[0087] The two-dimensional carbon nanoparticles with maleimide groups prepared in Example 4 were characterized by transmission electron microscopy, and the results are as follows: Figure 8 , 9 As shown ("NEMM" refers to N-ethylmaleimide malonate, "TP-2D C") 60 "Mal-TP" refers to two-dimensional carbon nanoparticles, and "Mal-TP" refers to two-dimensional carbon nanoparticles with maleimide groups modified on their surface.

[0088] Figure 8 The illustration shows a transmission electron microscope characterization image of two-dimensional carbon nanoparticles with maleimide groups prepared according to an embodiment of the present invention.

[0089] Figure 9 The infrared spectrum characterization of two-dimensional carbon nanoparticles with maleimide groups prepared according to an embodiment of the present invention is illustrated.

[0090] Depend on Figure 9 It can be seen that the two-dimensional carbon nanoparticles with maleimide groups on the surface have the same carbonyl vibration absorption peak at 1700 nm as the two-dimensional carbon nanoparticles without maleimide groups, which means that the prepared two-dimensional carbon nanoparticles are modified with maleimide groups on the surface.

[0091] Example 7:

[0092] Weigh 1 mg of two-dimensional carbon nanoparticles with maleimide groups on their surface and disperse them in 400 μL of anhydrous N,N-dimethylformamide solvent. Add 400 μL of anhydrous N,N-dimethylformamide solution of a 0.2 mM fluorescently labeled peptide, and then add 2 μL of N,N-diisopropylethylamine. Mix and stir at 720 rpm at room temperature and react in the dark for 24 hours. After the reaction is complete, transfer the reaction solution to a centrifuge tube and centrifuge at 20,000 g for 5 minutes. Wash and centrifuge several times with anhydrous N,N-dimethylformamide, discard the supernatant after washing, collect the centrifuged solid and dry it to obtain carbon nanoparticle probes.

[0093] Figure 2The schematic diagram illustrates the synthesis route and luminescence principle of the carbon nanoparticle probe in an embodiment of the present invention.

[0094] like Figure 2 As shown, the preparation steps in Examples 1-7 describe in detail the synthetic route of carbon nanoparticle probes.

[0095] The carbon nanoparticle probes prepared in Example 7 were characterized by transmission electron microscopy, and the results are as follows: Figure 10 As shown.

[0096] Figure 10 The diagram illustrates the transmission electron microscope characterization of the carbon nanoparticle probe prepared according to an embodiment of the present invention.

[0097] The zeta potential of the carbon nanoparticle probe prepared in Example 7 was measured using a nanoparticle size potentiometer, and the results are as follows: Figure 11 As shown.

[0098] Figure 11 The potential distribution of the carbon nanoparticle probe prepared according to an embodiment of the present invention is illustrated schematically.

[0099] like Figure 11 As shown, the average zeta potential of the carbon nanoparticle probe is around -31.4 mV.

[0100] The absorbance of the carbon nanoparticle probe prepared in Example 7 was measured, and the results are as follows: Figure 12 As shown.

[0101] Figure 12 The absorption spectrum of the carbon nanoparticle probe prepared according to an embodiment of the present invention is illustrated schematically.

[0102] Depend on Figure 12 It can be seen that the carbon nanoparticle probe contains an absorption peak of approximately 700 nm for the fluorescently labeled peptide and an absorption peak of 900 nm for the two-dimensional carbon nanoparticle, indicating that the surface of the carbon nanoparticle is attached with a fluorescently labeled peptide ("Mal-TP" refers to two-dimensional carbon nanoparticles with maleimide groups modified on the surface, "CatS-TP" refers to carbon nanoparticle probe, "Peptide" refers to peptide, "Peptide-Cy5.5" refers to Cy5.5 fluorescently labeled peptide, "TP-2D C" refers to...). 60 "Refers to two-dimensional carbon nanoparticles."

[0103] Example 8:

[0104] The absorbance of the carbon nanoparticle probes prepared in Example 7 was measured at different concentrations of 0.008 mg / mL, 0.015 mg / mL, 0.03 mg / mL, 0.06 mg / mL, and 0.12 mg / mL. The results are as follows:Figure 13 As shown (“CatS-TP” refers to carbon nanoparticle probe).

[0105] Depend on Figure 13 It is known that the carbon nanoparticle probe has a characteristic absorption peak at 696.5 nm, and the absorption intensity varies with the probe concentration. A standard curve was constructed using the absorption intensity at 696.5 nm as a correspondence with the carbon nanoparticle probe concentration, and the concentration-dependent absorbance curve was obtained. The results are as follows. Figure 14 As shown.

[0106] Figure 14 The concentration-absorbance standard curve of the carbon nanoparticle probe prepared according to an embodiment of the present invention is illustrated.

[0107] from Figure 14 It can be seen that the characteristic absorbance is linearly related to the concentration of carbon nanoparticle probes. The higher the concentration, the stronger the characteristic absorbance at 696.5 nm. Therefore, the absorbance of the reaction system can be measured according to the standard curve to carry out quantitative reaction.

[0108] Example 9:

[0109] Cytotoxicity tests were performed on the carbon nanoparticle probe prepared in Example 7 and the two-dimensional carbon nanoparticles with maleimide-modified surfaces prepared in Example 6. The results are as follows: Figure 15 As shown ("Mal-TP" refers to two-dimensional carbon nanoparticles with maleimide groups modified on their surface, and "CatS-TP" refers to carbon nanomolecular probes).

[0110] Figure 15 The illustrations show the survival rates of mouse bone marrow-derived dendritic cells (BMDCs) at different concentrations of carbon nanomolecular probes and two-dimensional carbon nanoparticles with maleimide groups prepared according to embodiments of the present invention.

[0111] from Figure 15 It was found that when the concentration of the two-dimensional carbon nanoparticle material with maleimide groups on its surface was 0.66 ug / mL, the survival rate of mouse bone marrow-derived dendritic cells (BMDCs) was 95.53%; when the concentration of the two-dimensional carbon nanoparticles was 33.33 ug / mL, the survival rate of mouse bone marrow-derived dendritic cells (BMDCs) was 98.92%; when the concentration of the two-dimensional carbon nanoparticles was 66.66 ug / mL, the survival rate of mouse bone marrow-derived dendritic cells (BMDCs) was 124.05%; and when the concentration of the two-dimensional carbon nanoparticles was 166.67 ug / mL, the survival rate of mouse bone marrow-derived dendritic cells (BMDCs) was 129.09%. This indicates that the material itself has low cytotoxicity, and high concentrations may promote the proliferation of mouse bone marrow-derived dendritic cells (BMDCs).

[0112] When the concentration of carbon nanoparticle probes with fluorescently labeled peptides was 0.66 ug / mL, the survival rate of mouse bone marrow-derived dendritic cells (BMDCs) was 82.83%; when the concentration was 6.66 ug / mL, the survival rate was 78.62%; when the concentration was 33.33 ug / mL, the survival rate was 82.02%; and when the concentration was 66.667 ug / mL, the survival rate was 63.33%. This indicates that carbon nanoparticle probes at concentrations between 0 and 33.33 ug / mL exhibit low toxicity to mouse bone marrow-derived dendritic cells (BMDCs).

[0113] Example 10: Fluorescence imaging of carbon nanoparticle probes in DC2.4 cells

[0114] This embodiment uses the preparation methods of Examples 1-7 to investigate the uptake, endocytosis, and processing of carbon nanoparticle probes by DC2.4 cells after a neoantigen drug pulse using laser confocal imaging.

[0115] Carbon nanoparticle probes can enter cells via nanoparticle-mediated endocytosis, where they are activated by intracellular cathepsin S, releasing a fluorescent signal. Based on this, 200,000 DC2.4 cells were seeded in a single confocal dish, and 1 mL of RPMI 1640 containing 10% FBS was added. After 24 hours of adherent culture, the cells were incubated for another 24 hours in a medium containing 100 μg / mL Cad neoantigen (peptide sequence: DMYIRMALLATVLGR) and 5 μM CpG (an oligonucleotide with a CpG motif, hereinafter referred to as CpG). Then, carbon nanoparticle probes were added and co-incubated at 37°C, at which point the carbon nanoparticle probe concentration was 21.6 μg / mL. After 12 hours, the samples were processed, and cell nuclei and lysosomes were stained before laser confocal imaging.

[0116] Blue represents cell nuclear staining, green represents lysosomal staining, and red represents the fluorescence of carbon nanoparticle probes.

[0117] Figure 16 The illustration schematically shows a confocal fluorescence image of the carbon nanoparticle probe prepared according to an embodiment of the present invention in DC2.4 cells.

[0118] from Figure 16 It can be seen that the carbon nanoparticle probe enters DC2.4 cells after the neoantigen drug pulse via endocytosis, is processed within the cell, and releases a fluorescent signal. Moreover, the location of the fluorescent signal generation is colocalized with the lysosome, indicating that the carbon nanoparticle probe can be taken up by DC2.4 cells and can be processed by intracellular cathepsin S to generate a fluorescent signal.

[0119] Example 11: Using the carbon nanoparticle probe of the present invention to detect the maturation status of mouse bone marrow-derived dendritic cells (BMDCs) after different drug treatments.

[0120] Mouse bone marrow-derived dendritic cells were seeded into black 96-well plates (2×10⁶). 4 Different drugs were added to the treatment: Cad+CpG group (Cad polypeptide sequence: DMYIRMALLATVLGR), Actb+CpG group (Actb polypeptide sequence: SLSTFHQMWI), CpG group (Oligonucleotides with CpG motif, hereinafter referred to as CpG), and PBS group. After incubation at 37℃ for 24 h, carbon nanoparticle molecular probes were added. After 12 h, the fluorescence intensity of each group was read using a microplate reader and verified by flow cytometry.

[0121] The Cad+CpG group had a Cad concentration of 100 μg / mL and a CpG concentration of 5 μM in the culture medium; the Actb+CpG group had an Actb concentration of 100 μg / mL and a CpG concentration of 5 μM in the culture medium; the CpG group had a CpG concentration of 5 μM in the culture medium; after adding carbon nanoparticle probes, the carbon nanoparticle probe concentration in the culture medium was 21.6 μg / mL, and a blank culture medium containing only carbon nanoparticle probes and no BMDC cells was set up as a control.

[0122] Figure 18 The fluorescence intensity of BMDC in different drug treatment groups is illustrated schematically.

[0123] Figure 18 The maturation status of bone marrow-derived dendritic cells (BMDCs) in mice treated with different drugs is illustrated schematically.

[0124] from Figure 18 As can be seen from the ONE way ANOVA statistical analysis, the differences in fluorescence intensity at 720 nm among the groups are as follows: PBS group vs. Actb+CpG group, p=0.0070; PBS group vs. Cad+CpG group, p<0.0001; PBS group vs. CpG group, p=0.0004; no significant differences were found between CpG group and Cad+CpG group, CpG group vs. Actb+CpG group, and Actb+CpG group vs. Cad+CpG group.

[0125] from Figure 18As can be seen from the ONE-way ANOVA statistical analysis, the differences in BMDC maturation among the groups are as follows: PBS group vs. Actb+CpG group: p < 0.0001; PBS group vs. Cad+CpG group: p < 0.0001; PBS group vs. CpG group: p < 0.0001; CpG group vs. Cad+CpG group: p = 0.0013; CpG group vs. Actb+CpG group: p < 0.0001; while no significant difference was found between Actb+CpG group and Cad+CpG group.

[0126] Therefore, it can be concluded that, compared with the PBS group, the neoantigen drug treatment group (Cad+CpG / Actb+CpG) and the immune adjuvant group (CpG group) have higher fluorescence intensity and higher degree of maturation. Thus, this carbon nanomolecule probe can distinguish between mature and immature mouse bone marrow-derived dendritic cells (BMDC) by fluorescence intensity detection.

[0127] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A carbon nanoparticle molecular probe, characterized in that: The carbon nanoparticle probe comprises carbon nanoparticles with peptides attached to their surface, wherein the nitrogen terminus of the peptides is attached to fluorescent molecules. The sequence of the polypeptide is selected from any of the following: GRWPPMLPWEC-DArg-NH2, GRWHPMGAPWEC-DArg-NH2.

2. The carbon nanoparticle probe according to claim 1, characterized in that: The fluorescent molecule is selected from any one of Cy3, Cy3.3, Cy5, Cy5.5, Cy7, Cy7.5, sulfon-Cy3, sulfon-Cy5, sulfon-Cy7, 5-FAM, 6-FAM, 5-TAMRA, 6-TAMRA, SIR, AF 647, AF 430, and AF 488.

3. The method for preparing the carbon nanoparticle molecular probe according to claim 1 or 2, characterized in that... Includes the following steps: Step 1: Add the fluorescent molecule, peptide sequence, and organic base to an N,N-dimethylformamide solution system, adjust the pH to 7-9, and react at 37°C in the dark for 4-12 hours; after the reaction, precipitate with ice-cold ether, centrifuge, wash, dry, separate by HPLC, and freeze-dry to obtain the fluorescently labeled peptide; Step 2: Add the fluorescently labeled peptide obtained in Step 1 to an N,N-dimethylformamide dispersion of two-dimensional carbon nanoparticles with maleimide groups on the surface, adjust the pH to 6.5-7.5, and carry out the cross-linking reaction. Stir for 12-24 hours in the dark to obtain carbon nanoparticle probes.

4. The preparation method according to claim 3, characterized in that: The organic base is N,N-diisopropylethylenediamine.

5. The preparation method according to claim 3, characterized in that: When adding fluorescent molecular raw materials, they are added to the reaction system in the form of N-hydroxysuccinimide ester of the fluorescent molecules to react with the nitrogen terminus of the polypeptide.

6. The preparation method according to claim 3, characterized in that: In step 2, the two-dimensional carbon nanoparticles with maleimide groups on their surface are prepared by a method including the following steps: dispersing the two-dimensional carbon nanoparticles in a solvent, adding N-ethylmaleimide malonate, 1,8-diazabicycloundec-7-ene and carbon tetrabromide, reacting at 80°C for 7 days under nitrogen protection, centrifuging the liquid after reaction to collect the solid product, washing and freeze-drying to obtain the two-dimensional carbon nanoparticles with maleimide groups on their surface.

7. The preparation method according to claim 6, characterized in that: The two-dimensional carbon nanoparticles were prepared by a method comprising the following steps: C 60 The Mg powder was mixed with Mg powder at a molar ratio of 1:4 and vacuum-sealed inside a quartz tube. The quartz tube was then placed in a dual-temperature zone tube furnace and held at that temperature for 20 hours. After the mixed powder was vaporized, it was transported to a low-temperature zone under the driving force of the temperature difference to grow polymer C. 60 Crystal; using acid etching to separate polymer C 60 The crystals were soaked in 20 wt% dilute nitric acid and stirred for 48 hours. The supernatant was removed by centrifugation, and the solid precipitate was collected. After washing with water and ethanol several times, the precipitate was dispersed in N-methylpyrrolidone solution and sonicated at 600 W for 10 hours. The resulting suspension was then centrifuged to remove incompletely detached crystals and thicker nanosheets. The liquid was collected and centrifuged again at 12,000 rpm to collect thin nanosheets. The thin nanosheets were then dispersed again in N-methylpyrrolidone, sonicated, and centrifuged. The solid product was collected and washed with anhydrous ethanol and deionized water in sequence. The solid product was then dispersed in ultrapure water and freeze-dried to obtain two-dimensional carbon nanoparticles.

8. The preparation method according to claim 7, characterized in that: The dual-temperature zone tube furnace is divided into a high-temperature zone and a low-temperature zone. The high-temperature zone is set at 600℃, and the low-temperature zone is set at 500℃. The mixed powder is located in the high-temperature zone, and after gasification, it is transported to the low-temperature zone under the driving force of the temperature difference to grow polymerized C. 60 Crystal.

9. The application of the carbon nanoparticle probe according to claim 1 or 2 in the preparation of reagents for detecting the maturation status of immune cells.

10. The application according to claim 9, characterized in that: After the carbon nanoparticle probe is endocytosed by immune cells, the polypeptide sequence on the surface of the carbon nanoparticle probe is specifically cleaved by intracellular cathepsin S, releasing a polypeptide fragment linked to a fluorescent molecule. The fluorescence intensity is positively correlated with the intracellular cathepsin S content.

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