Clostridium butyricum-derived carbon dot boat-shaped nanoparticles, their preparation method and applications

Abstract

This invention belongs to the field of novel carbon nanomaterials technology, and discloses a Clostridium butyricum-derived carbon dot boat-shaped nanoparticle, its preparation method, and its application. The preparation method includes the following steps: Step 1, Clostridium butyricum is cultured to the logarithmic growth phase, and then placed in a pretreatment solution for suspension incubation to regulate the structural integrity and surface chemical composition of Clostridium butyricum cells. The pretreatment solution is an aqueous solution of EDTA, NaCl, or PEG-8000. After incubation, the precipitate is collected by centrifugation, washed, and resuspended in ultrapure water; Step 2, the Clostridium butyricum treated in Step 1 is placed in a high-pressure reactor for hydrothermal reaction to obtain a crude product system; Step 3, the crude product system is filtered and purified, and freeze-dried to obtain Clostridium butyricum-derived carbon dot boat-shaped nanoparticles. The nanoparticles prepared by this invention have uniform particle size, good water solubility, dispersibility, and biocompatibility, and exhibit significant immunomodulatory functions.

Description

Technical Field

[0001] This invention relates to the field of novel carbon nanomaterials technology, and in particular to a Clostridium butyricum-derived carbon dot boat-shaped nanoparticle, its preparation method, and its application. Background Technology

[0002] Carbon dots (CDs) are a new class of carbon nanomaterials that have attracted widespread attention in the biomedical field due to their simple preparation, excellent water solubility and biocompatibility, and unique fluorescence properties. Existing research indicates that CDs show broad application prospects in bioimaging, drug delivery, photothermal therapy, antibacterial applications, and tissue repair promotion. However, current mainstream CD preparation methods are mostly based on chemical or physical pathways, such as hydrothermal methods and laser ablation methods. The precursors are mainly derived from small-molecule organic compounds or natural plant extracts, as illustrated in Rosales, S. et al., *Systematic review of carbon quantum dots (CQD): Definition, synthesis, applications and perspectives*. Renew. Sustain. Energy Rev. 219, 115854 (2025). and Zhang, Z. etal. Recent Advances of Carbon Dots: Synthesis, Plants Applications, Prospects, and Challenges. ACS Appl. Bio Mater. 8(2), 935–961 (2025). Although these technologies are relatively mature, researchers are still actively exploring new precursor materials in order to obtain CDs that combine specific biological functions with higher performance.

[0003] In recent years, a few studies have begun to explore the use of whole microorganisms as carbon dot precursors, such as Wu, Y. et al. Inheritance of physico-chemical properties and ROS generation by carbonquantum dots derived from pyrolytically carbonized bacterial sources. Materials Today Bio12, 100151 (2021). and Li, C. et al. Spectroscopic identification of bacterial source molecules yielding ROS-generatingnitrogen-species in bacteria-derived carbon quantum dots with anti-biofilmproperties. Applied Surface Science 721, 165511 (2026). However, systematic research is still lacking on probiotics with specific physiological functions, especially novel microbial precursors that can endow carbon dots with biological activity. If such functional probiotics can be used as carbon sources, not only can a green and sustainable synthesis strategy be realized, but it is also possible to combine their inherent immunomodulatory activity with the precise targeting properties of nanocarbon materials, thereby constructing innovative bionanomaterials with multiple functions.

[0004] Clostridium butyricum ( Clostridium butyricum Clostridium butyricum is an important Gram-positive anaerobic spore-forming bacillus and a key probiotic in the gut microbiota of humans and animals. It secretes short-chain fatty acids such as butyrate and lactic acid, as well as antimicrobial peptides, regulating the balance of the gut microbiota and inhibiting the growth of pathogenic bacteria. Simultaneously, Clostridium butyricum can activate the Toll-like receptor 2 (TLR2) signaling pathway, promoting IgA secretion and immune regulation responses of T cell subsets (such as Th17 and Treg cells), thereby enhancing the body's immune function and exerting anti-tumor effects.

[0005] Tumor immunotherapy, as the fourth major treatment method after surgery, radiotherapy, and chemotherapy, focuses on restarting and maintaining the tumor-immune cycle to restore the body's anti-tumor immune response. Despite significant breakthroughs in this therapy, efficacy remains highly individualized and significantly influenced by tumor heterogeneity. Therefore, improving the precision and effectiveness of immunotherapy remains a key area of ​​current research.

[0006] Clostridium butyricum, as a probiotic with immunomodulatory and antitumor potential, shows promising application prospects. However, as a solid bacterium, its oral administration faces challenges such as insufficient targeting and limited systemic distribution; as a drug carrier, its large particle size hinders precise delivery and tissue penetration in vivo. Therefore, constructing a novel multifunctional nanomaterial that combines the inherent immunomodulatory properties of Clostridium butyricum with the nanofunctionality of carbon dots is of significant scientific importance and application value for improving the efficacy and safety of tumor immunotherapy. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies in carbon dot preparation, such as the limited source of precursors, insufficient biological functionality, and limited application scope, by providing a method for preparing butyric acid-derived carbon dot nanoparticles and their applications.

[0008] The technical solution adopted to achieve the purpose of this invention is:

[0009] A method for preparing Clostridium butyricum-derived carbon dot boat-shaped nanoparticles includes the following steps:

[0010] Step 1: Culterate Clostridium butyricum to the logarithmic phase, and then place it in a pretreatment solution for suspension incubation to regulate the structural integrity and surface chemical composition of Clostridium butyricum cells. The pretreatment solution is an aqueous solution of EDTA, NaCl or PEG-8000. After incubation, centrifuge to collect the precipitate, wash it, and resuspend it in ultrapure water.

[0011] Step 2: The butyric acid bacteria treated in Step 1 are placed in a high-pressure reactor for hydrothermal reaction to obtain the crude product system.

[0012] Step 3: The crude product system obtained in Step 2 is filtered and purified, and then freeze-dried to obtain Clostridium butyricum-derived carbon dot boat-shaped nanoparticles.

[0013] In the above technical solution, the concentration of the NaCl aqueous solution in step 1 is 3~5M.

[0014] In the above technical solution, the concentration of the EDTA aqueous solution in step 1 is 0.5~3M.

[0015] In the above technical solution, the concentration of the aqueous solution of PEG-8000 in step 1 is 3wt.%~10wt.%.

[0016] In the above technical solution, the temperature for suspension incubation in step 1 is 4~6℃, the incubation time is 8~16 hours, and during washing, PBS is used to wash 2-3 times.

[0017] In the above technical solution, the temperature of the hydrothermal reaction in step 2 is 120~200℃, and the hydrothermal reaction time is 10~12 hours.

[0018] In the above technical solution, during step 3, the filtration and purification process involves first filtering through a 0.22μm filter membrane, and then dialyzing through a 5kDa dialysis membrane for 24-48 hours.

[0019] Another aspect of the present invention includes butyric acid Clostridium-derived carbon dot boat-shaped nanoparticles prepared using the method described above.

[0020] Another aspect of the present invention includes the application of the Clostridium butyricum-derived carbon dot boat-shaped nanoparticles in the preparation of antitumor drugs.

[0021] Another aspect of the present invention includes an antitumor drug comprising the Clostridium butyricum-derived carbon dot boat-like nanoparticles and pharmaceutically acceptable excipients.

[0022] In the above technical solution, the antitumor drug is an injection, lyophilized powder, sustained-release preparation, liposome, microsphere, gel or other pharmaceutically acceptable formulation.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention is the first to propose and verify the feasibility of using Clostridium butyricum cells (including cell wall, cytoplasm, and surface macromolecules) as a carbon precursor to prepare nanomaterials with special structural and functional properties through a hydrothermal reaction process. This invention also reveals for the first time the unique advantages of Clostridium butyricum in carbon dot preparation, expands the sources and applications of carbon dot precursor materials, and enables this microorganism with specific biological functions to be used in the field of carbon dot material preparation for the first time, providing new ideas for the development of carbon nanomaterials.

[0025] 2. This invention uses *Clostridium butyricum*, a bacterium with a complex biological structure, as raw material. Osmotic pressure is adjusted using aqueous solutions of EDTA, NaCl, or PEG-8000, or chelation is applied to alter the structural integrity and surface chemical composition of *Clostridium butyricum* cells, thereby affecting the composition of the precursor in the initial stage of carbonization. A simple and efficient hydrothermal method is employed to prepare carbon dot-shaped nanoparticles derived from *Clostridium butyricum*. This method is simple, environmentally friendly, and produces carbon dot-shaped nanoparticles with uniform particle size and a surface rich in active functional groups such as carboxyl, hydroxyl, and amino groups, exhibiting good water solubility, dispersibility, and biocompatibility.

[0026] 3. This invention proposes an innovative nanomedicine strategy for tumor immunotherapy based on Clostridium butyricum-derived carbon dot boat-shaped nanoparticles. Experimental results show that these carbon dot boat-shaped nanoparticles possess significant immunomodulatory functions, activating multiple immune-related signaling pathways in the tumor microenvironment, enhancing the recognition and killing ability of immune cells against tumor cells, thereby effectively inhibiting tumor occurrence and progression. In addition to its immunomodulatory properties, it also exhibits good biocompatibility and delivery efficiency, effectively improving the tumor immune microenvironment and enhancing the tumor-killing effect of immune cells, thus addressing the shortcomings of existing immunotherapies.

[0027] In summary, the carbon dot boat-shaped nanoparticles prepared in this invention not only possess excellent nanomaterial properties (including stability, dispersibility, and targetability) but also retain the natural immunomodulatory activity of the derived bacterial cells. This material achieves precise regulation of the tumor immune microenvironment by influencing the activity of immune cells within the tumor microenvironment, demonstrating significant scientific innovation value and broad prospects for biomedical applications. Attached Figure Description

[0028] Figure 1 Transmission electron micrographs of Clostridium butyricum treated according to the methods of Examples 1-3 and Comparative Example 1, respectively.

[0029] Figure 2 Transmission electron microscopy (TEM) images of nanoparticles prepared according to the methods of Examples 1-3 and Comparative Example 1.

[0030] Figure 3 The images show the particle size distribution, potential, and UV-Vis absorption spectra of the nanoparticles in Examples 1-3 and Comparative Example 1.

[0031] Figure 4 The results of small animal imaging, tumor fluorescence intensity statistics, tumor weight, tumor volume, mouse body weight and survival rate were obtained by injecting the nanoparticles of Examples 1-3 and Comparative Example 1 into mice with tumors via the tail vein.

[0032] Figure 5 These are transmission electron microscope (TEM) images of Cb-NCDs formation at different time points in Example 3.

[0033] Figure 6 The X-ray photoelectron spectroscopy full spectrum and high-resolution C1s, N1s, and O1s spectra of Cb-NCDs in Example 3 are shown.

[0034] Figure 7 The transmission electron microscopy images and particle size statistics of Cb-NCDs in Example 3 at different time periods are used for stability assessment.

[0035] Figure 8 This is a comparison chart of the uptake of Cb-NCDs and ordinary carbon dots in Example 3.

[0036] Figure 9 The results are from transcriptome sequencing analysis of Cb-NCDs applied to tumor tissue in Example 3. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] The Clostridium butyricum used in the following examples ( Clostridium butyricum The manufacturer of the strain is Shanghai Preservation Microbiology Co., Ltd., and the strain number is SHBCC D11145.

[0039] Example 1

[0040] A method for preparing Clostridium butyricum-derived carbon dot boat-shaped nanoparticles (Cb-ECDs) includes the following steps:

[0041] Step 1: Remove the Clostridium butyricum lyophilized powder ampoules from the -80℃ freezer. Wipe the surface of the ampoules with 75% alcohol, crush them with tweezers, and add a small amount of sterile liquid culture medium (approximately 0.5 mL) to the ampoules to fully dissolve the lyophilized powder. Inoculate 10 μL of this solution into 200 mL of RCM medium. Incubate at 37℃ in an anaerobic incubator until the logarithmic growth phase (approximately 12 hours). Collect the bacterial cells by centrifugation at 5000g for 10 min.

[0042] The obtained Clostridium butyricum was imaged using a transmission electron microscope, and the results are as follows: Figure 1 As shown in Figure A.

[0043] Resuspend *Clostridium butyricum* in 5 ml of 0.1 M EDTA (pH=8), incubate at 4°C for 16 h, centrifuge at 10000 rpm for 20 min, retain the precipitate, remove the supernatant, wash three times with PBS, and then perform transmission electron microscopy on the obtained *Clostridium butyricum*. The results are as follows. Figure 1 As shown in B.

[0044] Step 2: Add 10 mL of ultrapure water to the obtained Clostridium butyricum precipitate, resuspend it, and place it in a high-pressure reactor lined with polytetrafluoroethylene. Heat the reaction at 200°C for 12 hours to obtain the crude product system.

[0045] Step 3: After the crude product system has cooled naturally to room temperature, the resulting liquid is filtered through a 0.22 μm pore size filter membrane. Subsequently, the filtrate is purified by dialysis using a dialysis membrane with a molecular weight cutoff of 5 kDa for 24 hours. Finally, the sample is dried to powder form by freeze-drying to obtain Clostridium butyricum-derived carbon dot boat-shaped nanoparticles, denoted as Cb-ECDs.

[0046] Dissolved in phosphate buffered saline (PBS) solution to obtain a concentration of 10 mg / ml, which was used as the stock solution.

[0047] Example 2

[0048] A method for preparing Clostridium butyricum-derived carbon dot boat-shaped nanoparticles (Cb-SCDs) includes the following specific steps:

[0049] Step 1: The method for culturing Clostridium butyricum is the same as in Example 1.

[0050] The obtained Clostridium butyricum was resuspended in 5 mL of 4M (pH=8) NaCl and incubated at 4°C for 16 h. After centrifugation at 10000 rpm for 20 min, the precipitate was retained, the supernatant was removed, and the mixture was washed three times with PBS. The obtained Clostridium butyricum was then imaged using a transmission electron microscope. The results are as follows: Figure 1 As shown in C.

[0051] Step 2: Add 10 mL of ultrapure water to the obtained Clostridium butyricum precipitate, resuspend it, and place it in a high-pressure reactor lined with polytetrafluoroethylene. Heat the reaction at 200°C for 12 hours to obtain the crude product system.

[0052] Step 3: After the crude product system has cooled naturally to room temperature, the resulting liquid is filtered through a 0.22 μm pore size filter membrane. Subsequently, the filtrate is purified by dialysis using a dialysis membrane with a molecular weight cutoff of 10 kDa for 24 hours. Finally, the sample is dried to powder form by freeze-drying to obtain Clostridium butyricum-derived carbon dot boat-shaped nanoparticles, denoted as Cb-SCDs.

[0053] Then, Cb-SCDs were dissolved in phosphate buffered saline (PBS) to obtain a solution with a concentration of 10 mg / ml, which was used as the stock solution.

[0054] Example 3

[0055] A method for preparing Clostridium butyricum-derived carbon dot boat-shaped nanoparticles (Cb-NCDs) includes the following specific steps:

[0056] Step 1: The method for culturing Clostridium butyricum is the same as in Example 1.

[0057] The obtained Clostridium butyricum was resuspended in 5 mL of 5% PEG-8000 solution and incubated at 4°C for 16 h. After centrifugation at 10000 rpm for 20 min, the precipitate was retained, the supernatant was removed, and the mixture was washed three times with PBS. The obtained Clostridium butyricum was then imaged using transmission electron microscopy. The results are as follows: Figure 1 As shown in D.

[0058] Step 2: Add 10 mL of ultrapure water to the obtained Clostridium butyricum precipitate, resuspend it, and place it in a high-pressure reactor lined with polytetrafluoroethylene. Heat the reaction at 200°C for 12 hours to obtain the crude product system.

[0059] Step 3: After the crude product system has cooled naturally to room temperature, the resulting liquid is filtered through a 0.22 μm pore size filter membrane. Subsequently, the filtrate is purified by dialysis using a dialysis membrane with a molecular weight cutoff of 5 kDa for 24 hours. Finally, the sample is dried to powder form by freeze-drying to obtain Clostridium butyricum-derived carbon dot boat-shaped nanoparticles, denoted as Cb-NCDs.

[0060] Cb-NCDs were dissolved in phosphate buffered saline (PBS) to obtain a solution with a concentration of 10 mg / mL, which was used as the stock solution.

[0061] Comparative Example 1

[0062] A method for preparing Clostridium butyricum carbon dots (CbW-CDs) includes the following steps:

[0063] Step 1: The method for culturing Clostridium butyricum is the same as in Example 1. The obtained Clostridium butyricum is imaged using a transmission electron microscope, and the results are as follows. Figure 1 As shown in Figure A.

[0064] Step 2: Add 10 mL of ultrapure water, resuspend the obtained Clostridium butyricum, and place it in a high-pressure reactor lined with polytetrafluoroethylene. Heat the reaction at 200°C for 12 hours to obtain the crude product system.

[0065] Step 3: After the crude product system has cooled naturally to room temperature, the resulting liquid is filtered through a 0.22 μm pore size filter membrane. Subsequently, the filtrate is dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 5 kDa to purify the obtained carbon dots. Finally, the sample is freeze-dried to a powder state to obtain nano-carbon dots, denoted as CbW-CDs.

[0066] CbW-CDs were dissolved in phosphate buffered saline (PBS) to obtain a solution with a concentration of 10 mg / mL, which was used as the stock solution.

[0067] Test Example 1

[0068] According to the above embodiments, Figure 2Transmission electron microscopy (TEM) images of Cb-ECDs, Cb-SCDs, Cb-NCDs, and CbW-CDs prepared in Examples 1-3 and Comparative Example 1 are shown. As shown in the figure, all four groups of samples exhibit well-dispersed spherical or near-spherical nanoparticles with a uniform background and clear particle boundaries, indicating that each sample has successfully formed a stable carbon dot structure. Among them: the carbon dots obtained in Comparative Example 1 (CbW-CDs) mainly exhibited a common spherical morphology with a relatively uniform particle distribution; in addition to conventional spherical particles, the carbon dots obtained in Example 1 (Cb-ECDs) also showed some near-spherical "nanoboat"-like structures with hollow structural features, with spherical particles mounted on the "nanoboats"; the nanoparticles obtained in Example 2 (Cb-SCDs) mainly exhibited a near-spherical "nanoboat" morphology, but the number of spherical carbon dot particles mounted inside or on the surface was relatively small; the nanoparticles obtained in Example 3 (Cb-NCDs) also exhibited a near-spherical "nanoboat" morphology, but the spherical carbon dot particles mounted inside were the most regular in morphology, with clear particle interfaces and high density, showing better structural integrity and morphological uniformity.

[0069] In summary, the carbon dots obtained under different precursor conditions have certain differences in morphology. Among them, the nanoparticles prepared in Example 3 have the most ideal morphology, with typical "nanoboat" structure and high crystallinity.

[0070] Test Example 2

[0071] Further basic characterization was performed on the Cb-ECDs, Cb-SCDs, Cb-NCDs, and CbW-CDs prepared in Examples 1-3 and Comparative Example 1. The results are as follows: Figure 3 As shown, this includes particle size distribution ( Figure 3 A), potential ( Figure 3 (B) and UV-Vis absorption spectrum ( Figure 3 (C) For example Figure 3 In the Cb-W-CDs (<10nm), the particle size of Cb-ECDs, Cb-SCDs, and Cb-NCDs (approximately 50nm) is much larger. The Zeta potential of Cb-ECDs and Cb-NCDs is approximately 30mV. Figure 3 The presence of Cb-NCDs (Cb-NCDs) indicates good dispersion stability. More importantly, Cb-NCDs exhibit the strongest UV absorption in the 260-280 nm range, suggesting they may retain a richer concentration of nucleic acid components. Figure 3 (C). The above characterization results confirm that the obtained product meets the basic characteristics and requirements of nanoparticles.

[0072] Application Example 1

[0073] Female BALB / c mice aged 6–8 weeks were randomly assigned to groups and anesthetized. Hair was removed from the upper hind legs of the mice using an animal-specific scalpel. A prepared H22-luc tumor cell suspension (containing bioluminescent labeling) was subcutaneously inoculated into the mice to construct a subcutaneous transplanted tumor model.

[0074] When the tumor volume grows to approximately 500 mm 3 Mice were randomly divided into five groups: Control group, CbW-CDs group, Cb-ECDs group, Cb-SCDs group, and Cb-NCDs group (n=6 per group, for a total of 30 mice). During the experiment, mouse body weight and tumor volume were measured every two days.

[0075] The drugs were administered via tail vein injection every two days. Each experimental group was injected with the corresponding nanoparticle solution (CbW-CDs, Cb-ECDs, Cb-SCDs, Cb-NCDs), at a dose of 5 mg / kg; the control group was injected with an equal volume of PBS solution.

[0076] The results are as follows Figure 4 Small animal live imaging of model mice ( Figure 4 (A) and its statistical chart ( Figure 4 As shown in Figure B). The four carbon dots prepared by different methods exhibited significantly different tumor-inhibiting effects, with the Cb-NCDs group showing the most significant tumor-inhibiting effect and the slowest tumor volume growth in mice. Tumor weight ( Figure 4 (C) Tumor volume ( Figure 4 D), mouse body weight ( Figure 4 (F) and survival rate ( Figure 4 The statistical results from the Chinese E) further support this conclusion. Therefore, Example 3 (Cb-NCDs) was selected as the preferred method among the four different preparation methods.

[0077] Example 4

[0078] The Cb-NCDs prepared in Example 3 were systematically characterized.

[0079] To elucidate the preparation mechanism of Cb-NCDs, the entire formation process of Cb-NCDs was first monitored by time-resolved TEM imaging. Figure 5PEG-induced mild osmotic pressure perturbation can controllably release some intracellular components while maintaining the integrity of the bacterial cell wall structure, thereby forming a highly crowded precursor microenvironment inside and outside the bacteria, constituting a "semi-open" confined reaction space. In the initial stage of the reaction (0h), the bacterial structure is intact; after 4h, uniformly dispersed primary carbon nuclei form within the confined space; by 8h, the precursor material is enriched into high-density nanodomains within the local cavity; after 12h, these nanodomains are further compacted and fused, ultimately assembling into a typical "nanoboat" structure with clear boundaries and an amorphous carbon matrix, containing uniformly distributed 2–3 nm carbon-rich nanodomains. Notably, the resulting mature particles retain the shell structure after bacterial carbonization, indicating that the microbial structural template acts as a natural biological mold throughout the reaction process. PEG pretreatment effectively stabilizes and strengthens the construction of this confined cavity by adjusting osmotic pressure and molecular crowding, thereby driving the gradual aggregation of carbon nuclei and achieving structured self-assembly, ultimately yielding the Cb-NCDs.

[0080] Test Example 3

[0081] Fourier transform infrared (FTIR) spectroscopy was performed on the Cb-NCDs powder obtained in Example 3, and the results are as follows: Figure 6 As shown in Figure A, the vertical axis represents the percentage of transmittance (%T), reflecting the proportion of infrared light transmitted by the sample. A lower %T indicates stronger absorption and corresponds to specific chemical bond vibrations. The horizontal axis represents the wavenumber (cm²). -1 The spectrum indicates the range of vibrational frequencies. The spectrum shows enrichment of O–H / N–H, C=O, and C–O / C–N absorption bands, supporting the determination of its surface chemical structure. X-ray diffraction (XRD) pattern ( Figure 6 (B) shows its crystal structure information. X-ray photoelectron spectroscopy (XPS) full spectrum ( Figure 6 The C1s (C) indicates that the sample contains three elements: C (65.64%), N (14.28%), and O (19.73%). Its high-resolution C1s (C1s) indicates that the sample contains these three elements. Figure 6 D), N1s ( Figure 6 (f) and O1s ( Figure 6 The E spectrum further confirms that the carbon dot surface is rich in functional groups such as hydroxyl, amino and carbonyl groups.

[0082] Given the unique structure of the carbon dot boat-shaped nanoparticles, we further evaluated their structural stability. TEM imaging was performed on samples collected on days 1, 3, 5, and 7 to analyze the size and morphology of the carbon dot boat-shaped nanoparticles and the particle size distribution of the internal carbon dots. The results are as follows: Figure 7 As shown, Figure 7Whether it's the macroscopic structure dominated by "nanoships" or the carbon dots scattered within it, the morphology and structure of A remain unchanged. This conclusion is verified by size statistical analysis using ImageJ software. Figure 7 In the figure, B represents the particle size distribution of Cb-NCDs. Figure 7 (C in the figure represents the Cb-CDs particle size distribution). In summary, this indicates that the carbon dot nanoparticles remained stable within the testing time range.

[0083] To clarify the morphological and functional differences between carbon dot boat-shaped nanoparticles and ordinary carbon dots, this invention compares carbon dot boat-shaped nanoparticles with scattered carbon dots formed after treatment and destruction (i.e., ordinary morphological carbon dots obtained under this condition). TEM images ( Figure 8 (A) clearly demonstrates the significant morphological differences between the two, and their morphological structure leads to significant differences in their performance and function. Take 4 mL of Cb-NCDs or Cb-CDs aqueous solution in a brown centrifuge tube, add 4 mg of coumarin-3-carboxylic acid, gently vortex or gently shake until fully dispersed; then incubate at room temperature (20-25℃) in the dark for 24 h to obtain Cb-NCDs or Cb-CDs (coumarin-3-carboxylic acid-carbon dots) with fluorescent tags. Further flow cytometry was used to evaluate the uptake efficiency of Cb-NCDs and Cb-CDs by cells, and the results showed ( Figure 8 (B) The cellular uptake efficiency of Cb-NCDs was significantly higher than that of Cb-CDs. The average fluorescence intensity peak of the Cb-NCDs treatment group shifted significantly to the high fluorescence region (right side), suggesting that the accumulation of Cb-NCDs in cells was several times higher than that of Cb-CDs, indicating that its cellular uptake capacity and biocompatibility were superior.

[0084] Application Example 2

[0085] This embodiment provides the application of Clostridium butyricum-derived carbon dot boat-like nanoparticles, used to improve the tumor immune microenvironment and enhance the tumor-killing effect of immune cells by intravenous injection of Cb-NCDs at a dose of 0.5–5 mg / kg. The specific method steps are as follows:

[0086] Transcriptome sequencing analysis of mouse tumor tissues treated with Cb-NCDs (e.g.) Figure 9 As shown): Transcriptome volcano plot ( Figure 9 (A) shows that RAW264.7 was extensively and significantly differentially expressed after Cb-NCDs induction treatment, suggesting that this intervention induced systemic molecular remodeling of the macrophage / immune microenvironment. KEGG pathway enrichment ( Figure 9 (B) indicates that the differentially expressed genes are mainly concentrated on the immune response-related signaling axis, suggesting that this treatment preferentially remodels the immune communication and effector execution modules; GO enrichment ( Figure 9 The results from the GSEA (C) further indicate that immune activation and the regulation of immune cell function are synergistically enhanced, showing a consistent functional orientation. Figure 9 This trend was validated at the gene set level by D and F: antigen receptor-mediated signaling pathways ( Figure 9 Significant positive enrichment of D in the middle digits suggests that adaptive immune signals are synergistically amplified; T cell activation regulation ( Figure 9 Significant enrichment of IFN-γ in the middle jiao (E) indicates a tendency towards a more immunostimulatory microenvironment; IFN-γ response ( Figure 9 Significant enrichment of Th1 (F) indicates that the Th1 / antitumor effector program was systematically enhanced. Overall, differential expression, enrichment, and GSEA all suggest that Cb-NCDs-induced treatment shapes an immune-stimulating, antitumor-biased transcriptional program, rather than local or transient inflammatory fluctuations.

[0087] In summary, the butyric acid clostridium-derived carbon dot boat-shaped nanoparticles prepared in this invention exhibit excellent anti-tumor immunomodulatory effects in animal experiments, and have potential application value as novel nanomedicines for tumor immunotherapy.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Clostridium butyricum-derived carbon dot boat-shaped nanoparticles, characterized in that, Includes the following steps: Step 1: Culterate Clostridium butyricum to the logarithmic phase, and then place it in a pretreatment solution for suspension incubation to regulate the structural integrity and surface chemical composition of Clostridium butyricum cells. The pretreatment solution is an aqueous solution of EDTA, NaCl or PEG-8000. After incubation, centrifuge to collect the precipitate, wash it, and resuspend it in ultrapure water. Step 2: The butyric acid bacteria treated in Step 1 are placed in a high-pressure reactor for hydrothermal reaction; Step 3: The system obtained in Step 2 is filtered and purified, and then freeze-dried to obtain Clostridium butyricum-derived carbon dot boat-shaped nanoparticles. The concentration of the NaCl aqueous solution in step 1 is 3~5M; The concentration of the EDTA aqueous solution in step 1 is 0.5~3M; The concentration of the PEG-8000 aqueous solution in step 1 is 3 wt.% to 10 wt.%.

2. The preparation method according to claim 1, characterized in that, In step 1, the temperature for suspension incubation is 4-6°C, and the incubation time is 8-16 hours. During washing, the product is washed 2-3 times with PBS.

3. The preparation method according to claim 1, characterized in that, The hydrothermal reaction in step 2 is carried out at a temperature of 120-200°C for 10-12 hours.

4. The preparation method according to claim 1, characterized in that, In step 3, during filtration and purification, the material is first filtered through a 0.22 μm filter membrane, and then dialyzed through a 5 kDa dialysis membrane for 24 to 48 hours.

5. Butyric acid Clostridium-derived carbon dot boat-shaped nanoparticles prepared by the method described in any one of claims 1 to 4.

6. The application of the butyric acid clostridium-derived carbon dot boat-shaped nanoparticles as described in claim 5 in the preparation of antitumor drugs.

7. An antitumor drug, characterized in that, It includes the butyric acid clostridium-derived carbon dot boat-shaped nanoparticles as described in claim 5, as well as pharmaceutically acceptable excipients.

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