Lymph tissue targeted composite nanocluster as well as preparation method and application thereof
By designing composite nanoclusters of Se-doped ZnS nanoparticles and BSA, the problem of balancing dose dependence and toxicity in targeted delivery of nanomedicines to lymphatic tissues was solved, achieving efficient lymph node targeting and immune activation, and significantly inhibiting tumor growth.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-03-24
AI Technical Summary
Existing nanomedicines based on essential trace elements have difficulty balancing dose-dependent efficacy and toxicity in targeted delivery to lymphatic tissues, and their low targeting efficiency limits their application in immunotherapy.
A composite nanocluster was designed, comprising Se-doped ZnS nanoparticles and BSA coated on the surface. By leveraging the synergistic immunomodulatory function of zinc and selenium, combined with the lymphatic targeting properties of BSA, the nanocluster was efficiently delivered to and cleared from lymph nodes.
This technology enables highly efficient targeted delivery of nanomedicines to lymph nodes, broadens the safe treatment window, reduces long-term accumulation, activates the immune response, and significantly inhibits tumor growth.
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Figure CN121714531A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a lymphatic tissue-targeting composite nanocluster, its preparation method, and its application. Background Technology
[0002] Achieving targeted delivery and controllable metabolism of nanomedicines in biological systems is crucial for improving their efficacy and biosafety. Lymphatic tissue targeting is essential for effective immunomodulation by nanomedicines. By structurally designing nanomaterials, their targeting efficiency and accumulation capacity in lymphatic tissues can be precisely enhanced, thereby regulating the immune microenvironment. The in vivo metabolic behavior of nanoparticles based on essential trace elements (ETEs) is closely related to their elemental composition. For example, nanoparticles composed of trace elements such as molybdenum, selenium, zinc, and manganese can be transformed into bioactive species such as ions and enzyme cofactors in vivo, participating in various physiological processes and thus exerting their bioactivity. This lays the theoretical foundation for developing novel nanomedicines utilizing the metabolic pathways and immunomodulatory properties of ETE nanoparticles.
[0003] The design of nanomedicines based on essential trace elements (ETEs) has emerged as a promising strategy for enhancing immunotherapy by leveraging the metabolic properties and immunomodulatory functions of ETEs. However, despite its promising prospects, ETE-based nanoimmunomodulators still face a series of significant challenges. For example, essential trace elements have narrow safety windows and require precise control of safe dosage, posing a critical challenge to the development of trace element-based nanomedicines in balancing their dose-dependent efficacy and toxicity. This severely restricts the design and clinical translation of such materials.
[0004] Therefore, how to design a nanomedicine that can balance the dose-dependent efficacy and toxicity of trace elements and achieve efficient lymphatic tissue targeting is a technical problem that urgently needs to be solved in this field. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a lymphatic tissue-targeting composite nanocluster, its preparation method, and its application.
[0006] To achieve this objective, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides a lymphatic tissue-targeting composite nanocluster, the composite nanocluster comprising Se-doped ZnS nanoparticles and BSA coated on the surface of the nanoparticles.
[0008] This invention creatively constructs a ZnS / Se / BSA nanocluster (ZSB NCs) that can achieve lymph node targeting and combines the immunomodulatory effects of zinc and selenium. This synergistic lymph node-targeting nanoplatform integrates the complementary immunomodulatory functions of zinc (Zn) and selenium (Se), overcoming the limitations of single-element efficacy through elemental synergy and broadening the safe therapeutic window of essential trace elements. Furthermore, it utilizes the natural lymph node-targeting properties of serum albumin (BSA) to achieve highly efficient lymph node delivery of the nanoclusters. The nanocluster structure also promotes its clearance in vivo, reducing long-term accumulation and ensuring safety.
[0009] Preferably, the BSA is obtained by interacting with Zn. 2+ The coordination effect of the substance is coated on the surface of Se-doped ZnS nanoparticles.
[0010] Preferably, the particle size of the composite nanoclusters is 2-10 nm (e.g., 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, etc.).
[0011] Preferably, the mass ratio of the Se-doped ZnS nanoparticles to BSA is 1:(0.5-7) (e.g., 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, etc.), and more preferably 1:(1.5-5).
[0012] Preferably, the mass ratio of Se to ZnS in the Se-doped ZnS nanoparticles is 1:(1-10) (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.), and more preferably 1:(2-5).
[0013] In a second aspect, the present invention provides a method for preparing composite nanoclusters as described in the first aspect, the method comprising:
[0014] The composite nanoclusters were obtained by mixing BSA with zinc acetate dihydrate solution, followed by mixing with sodium selenite solution and sodium sulfide solution.
[0015] Preferably, the mixing time of BSA and zinc acetate dihydrate solution is 2-15 min (e.g., 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.), and the mixing temperature is 25-35℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, etc.).
[0016] Preferably, the reaction time is 2-8 h (e.g., 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.), and the reaction temperature is 25-35℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, etc.).
[0017] Preferably, the mixture reaction further includes a dialysis step, wherein the dialysis membrane used for dialysis has a molecular weight cutoff of 8000-12000 Da (e.g., 8000 Da, 9000 Da, 10000 Da, 11000 Da, 12000 Da, etc.).
[0018] All other specific point values not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.
[0019] Thirdly, the present invention provides the application of the composite nanoclusters as described in the first aspect in the preparation of products for tumor immunotherapy.
[0020] Preferably, the tumor is selected from any one of breast cancer, colorectal cancer, melanoma, pancreatic cancer, ovarian cancer, gastric cancer, and lung cancer.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] This invention creatively constructs a ZnS / Se / BSA nanocluster (ZSB NCs) that can achieve lymph node targeting and combines the immunomodulatory effects of zinc and selenium. This synergistic lymph node-targeting nanoplatform integrates the complementary immunomodulatory functions of zinc and selenium, overcoming the limitations of single-element efficacy through elemental synergy and broadening the safe therapeutic window of essential trace elements. Furthermore, it utilizes the natural lymph node targeting properties of serum albumin to achieve highly efficient lymph node delivery of the nanoclusters. The nanocluster structure also promotes its clearance in vivo, reducing long-term accumulation and ensuring safety.
[0023] Under the action of this system, ZSB NCs can be efficiently enriched in lymph nodes after subcutaneous injection. The released zinc ions and the selenoproteins formed by chemical transformation jointly activate the cGAS-STING immune pathway, thereby effectively inducing the maturation and activation of dendritic cells, and further activating T cells to produce inflammatory factors, forming a cascade immune response. Attached Figure Description
[0024] Figure 1 This is a schematic diagram of the synthesis of ZSB NCs in this invention;
[0025] Figure 2This is a transmission electron microscope image of the ZSB NCs prepared in this invention;
[0026] Figure 3 These are the XPS spectra of ZSB NCs prepared by this invention. In the figure, a is the total XPS spectrum, b is the XPS spectrum of Zn element, c is the XPS spectrum of Se element, and d is the XPS spectrum of S element.
[0027] Figure 4 The figures show the cytotoxicity of different concentrations of ZSB NCs prepared in this invention. In the figure, a represents DC2.4 cells, b represents 4T1 cells, and c represents HSF cells.
[0028] Figure 5 The activation of DC2.4 cells by prepared ZSB NCs, ZnS / BSA, and Se / BSA nanoparticles was compared using flow cytometry.
[0029] Figure 6 This is a graph showing the regulatory effects of ZSB NCs, ZnS / BSA, and Se / BSA nanoparticles on the expression of cGAS-STING pathway-related proteins in DC2.4 cells. In the graph, a represents protein bands, and b represents the statistical graph of relative protein expression levels.
[0030] Figure 7 The image shows the ICP-MS plot of the prepared ZSB NCs targeting mouse lymph nodes. In the figure, a represents the zinc content and b represents the selenium expression level.
[0031] Figure 8 This is a comparison of the tumor-suppressing effects of the prepared ZSB NCs nanoclusters, ZnS / BSA, and Se / BSA nanoparticles in a mouse breast cancer model. In the figure, a is the tumor growth curve and b is the tumor anatomy diagram. Detailed Implementation
[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.
[0033] Example 1
[0034] This embodiment prepares a ZnS / Se / BSA nanocluster (ZSB NCs) using the following method:
[0035] (1) Dissolve 40 mg bovine serum albumin in 6 mL of water to obtain bovine serum albumin solution. Stir continuously at 600 rpm, add 150 mM zinc acetate dihydrate solution, and continue stirring for 5 min to obtain zinc acetate / BSA dispersion.
[0036] (2) Add 75 mM sodium selenite and 466 mM sodium sulfide solution to the zinc acetate / BSA dispersion, and react vigorously at room temperature for 4 h at 500 rpm. The obtained product is dialyzed through a dialysis bag with a molecular weight cutoff of 10000 Da to remove unreacted raw materials and obtain ZSB NCs dispersion. The solution is dispersed in deionized water and stored at 4℃.
[0037] Comparative Example 1
[0038] This comparative example prepares a ZnS / BSA nanoparticle (ZB NPs) using the following method:
[0039] (1) Dissolve 40 mg bovine serum albumin in 6 mL of water to obtain bovine serum albumin solution. Stir continuously at 600 rpm, add 150 mM zinc acetate dihydrate solution, and continue stirring for 5 min to obtain zinc acetate / BSA dispersion.
[0040] (2) Add 466 mM sodium sulfide solution to zinc acetate / BSA dispersion and react vigorously at room temperature for 4 h at 600 rpm. The obtained product is dialyzed through a dialysis bag with a molecular weight cutoff of 10000 Da to remove unreacted raw materials and obtain ZB NPs dispersion. The solution is dispersed in deionized water and stored at 4℃.
[0041] Comparative Example 2
[0042] This comparative example prepares a Se / BSA nanoparticle (SeB NPs) using the following method:
[0043] 40 mg of bovine serum albumin was dissolved in 6 mL of water to obtain a bovine serum albumin solution. The solution was stirred continuously at 600 rpm, and 75 mM sodium selenite solution and 466 mM sodium sulfide solution were added. The mixture was reacted vigorously at 500 rpm at room temperature for 4 h. The obtained product was dialyzed through a dialysis bag with a molecular weight cutoff of 10000 Da to remove unreacted raw materials and obtain a SeB NPs dispersion. The solution was dispersed in deionized water and stored at 4 °C.
[0044] Test Example 1
[0045] This test case characterizes the morphology of the ZSB NCs obtained in Example 1.
[0046] Figure 1 This is a schematic diagram of the synthesis of ZSB NCs in this invention.
[0047] Figure 2The image shows a transmission electron microscope (TEM) image of the ZSB NCs prepared in Example 1. The characterization method was as follows: 20 μL of the ZSB NCs dispersion prepared in Example 1 was dropped onto a copper grid, dried normally, and then observed by a field emission transmission microscope. It can be seen from the image that the nanodots have a very small particle size with an average particle size of 3.3 nm.
[0048] Figure 3 The XPS spectrum of the ZSB NCs prepared in Example 1 is shown below. The characterization method was as follows: After freeze-drying the ZnS / Se / BSA nanocluster solution prepared in Example 1, the powder sample was collected and detected by X-ray photoelectron spectroscopy. The results confirmed the presence and valence states of Zn, Se, and S elements in the ZSB NCs. Zn mainly exists as ZnS / Se / BSA nanoclusters. 2+ The form exists (b) in the diagram, Se with Se 2- and SeO3 2- It exists in a mixed form (c in the diagram), while the S element is mainly in the form of S. 2- It exists in the form of (d in the figure).
[0049] Test Example 2
[0050] This test case demonstrates the cytotoxicity of the ZSB NCs obtained in Example 1.
[0051] ZSB NCs dispersed in PBS solution at concentrations of 0 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, and 4 μg / mL were co-incubated with DC2.4 cells, 4T1 cells, and HSF cells at 37°C and 5% CO2 for 24 h. Cytotoxicity was then detected by the MTT assay.
[0052] The results are as follows Figure 4 As shown, the results indicate that ZSB NCs are essentially non-toxic to all three cell types at the experimental dose.
[0053] Test Example 3
[0054] This test case examines the activation of DC2.4 cells by the nanoclusters of Example 1, and the nanoparticles of Comparative Example 1 and Comparative Example 2.
[0055] DC2.4 cells were treated as follows: (1) PBS control group, (2) 0.5 μg / mL ZSB NCs group, (3) ZB NPs group and (4) SeB NPs group with corresponding elemental concentrations, and (3) a simple mixture of (4) and (4) were co-incubated with DC2.4 cells for 24 h. After incubation, DC2.4 cells were collected and the cells were treated with PE-labeled CD80 antibody, PerCP / Cyanine 5.5-labeled CD86 antibody and Ghost Dye live / dead staining antibody. TM Violet 510 was incubated together at 4°C in the dark for 30 min, followed by washing with PBS and then flow cytometry analysis.
[0056] The results are as follows Figure 5 As shown, DC2.4 cells treated with ZSB NCs exhibited the most significant activation effect, while the activation effect of ZB NPs and SeB NPs prepared in Comparative Examples 1 and 2 on DC2.4 cells was far inferior to that of ZSB NCs. Furthermore, even when ZB NPs and SeB NPs were mixed, the superior effect of ZSB NCs of this invention could not be achieved.
[0057] Test Example 4
[0058] This test case examines the effect of the nanoclusters of Example 1, and the nanoparticles of Comparative Example 1 and Comparative Example 2 on the expression of cGAS-STING pathway-related proteins of DC2.4.
[0059] DC2.4 cells were treated as follows: (1) PBS control group, (2) 0.5 μg / mL ZSB NCs group, (3) ZB NPs group and (4) SeB NPs group with corresponding element concentrations, and (3) and (4) simple mixed group were incubated with DC2.4 cells for 24 h. After incubation, DC2.4 cells were collected, total protein was extracted, and the extracted protein was aliquoted and temporarily stored in a -80℃ freezer. The expression of TBK1, IRF3, p-TBK1, p-IRF3, GAPDH, GPX4 and SelK was detected and visualized by Western blot.
[0060] The results are as follows Figure 6 As shown, the results indicate that the ZSB NCs prepared in Example 1 can significantly induce the expression of cGAS-STING pathway-related proteins and selenoproteins. However, the ZB NPs and SeB NPs prepared in Comparative Examples 1 and 2 are far less effective than ZSB NCs. Even when ZB NPs and SeB NPs are mixed, the superior effect of the ZSB NCs of this invention cannot be achieved. This indicates that zinc and selenium have a synergistic effect in ZSB NCs nanoclusters, rather than a simple superposition of elements.
[0061] Test Example 5
[0062] This test case demonstrates mouse lymph node-targeting ICP-MS experiments on the ZSB NCs obtained in Example 1.
[0063] ZSB NCs were administered subcutaneously to mice at a dose of 4 mg / kg (for Zn). Major organs and lymph nodes were collected from four mice at six time points: 6 h, 12 h, 1 d, 3 d, and 7 d. After wet digestion, the zinc and selenium levels in the major organs of each group were quantitatively determined by ICP-MS.
[0064] The results are as follows Figure 7 As shown, zinc and selenium in ZSB NCs were significantly enriched in lymph nodes, and reached their peak at 12 h, indicating that ZSB NCs can target lymph nodes.
[0065] Test Example 6
[0066] This test case evaluates the in vivo antitumor effects of the nanoclusters of Example 1, and the nanoparticles of Comparative Example 1 and Comparative Example 2.
[0067] A BALB / c mouse orthotopic breast cancer model was constructed as follows: 50 μL of 4T1 cell suspension in PBS (containing 1×10⁻⁶ cells) was added. 8 4 T1 cells / mL were subcutaneously injected into the mammary glands of mice. The mice were randomly divided into groups of 5 mice each, based on tumor size: (1) phosphate-buffered saline (PBS) group (corresponding to the PBS group in the diagram); (2) ZSB NCs group; and (3) ZB NPs group and (4) SeB NPs group with corresponding elemental concentrations. The samples from each group were subcutaneously injected into the orthotopic breast cancer model of mice at a dose of 4 mg / kg. Tumor size changes were monitored every two days. The tumor size in the PBS group was increased to 1500 mm². 3 The experiment was terminated when the tumor reached a certain size, and the tumor was dissected and photographed.
[0068] The results are as follows Figure 8 As shown in the figure, the ZSB NCs prepared in Example 1 can significantly inhibit tumor growth. While the ZB NPs and SeB NPs prepared in the comparative example also showed some inhibitory effect on tumor growth, their effects were significantly weaker than those of ZSB NCs. These results indicate that ZSB NCs can achieve a synergistic effect between zinc and selenium, with both working together to exert an immunosuppressive effect. Therefore, ZSB NCs possess excellent potential for tumor immunotherapy.
[0069] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A lymphatic tissue-targeting composite nanocluster, characterized in that, The composite nanoclusters include Se-doped ZnS nanoparticles and BSA coated on the surface of the nanoparticles.
2. The composite nanocluster according to claim 1, characterized in that, The BSA is connected with Zn 2+ The coordination effect of the substance is coated on the surface of Se-doped ZnS nanoparticles.
3. The composite nanoclusters according to claim 1 or 2, characterized in that, The particle size of the composite nanoclusters is 2-10 nm.
4. The composite nanoclusters according to any one of claims 1-3, characterized in that, The mass ratio of Se-doped ZnS nanoparticles to BSA is 1:(0.5-7), preferably 1:(1.5-5).
5. The composite nanoclusters according to any one of claims 1-4, characterized in that, The mass ratio of Se to ZnS in the Se-doped ZnS nanoparticles is 1:(1-10), preferably 1:(2-5).
6. A method for preparing composite nanoclusters according to any one of claims 1-5, characterized in that, The preparation method includes: The composite nanoclusters were obtained by mixing BSA with zinc acetate dihydrate solution, followed by mixing with sodium selenite solution and sodium sulfide solution.
7. The preparation method according to claim 6, characterized in that, The mixing time of BSA with zinc acetate dihydrate solution is 2-15 min, and the mixing temperature is 25-35℃; Preferably, the reaction time is 2-8 h and the reaction temperature is 25-35℃.
8. The preparation method according to claim 6 or 7, characterized in that, The mixing reaction is followed by a dialysis step, where the dialysis membrane used has a molecular weight cutoff of 8000-12000 Da.
9. The use of a composite nanocluster according to any one of claims 1-5 in the preparation of a product for tumor immunotherapy.
10. The application according to claim 9, characterized in that, The tumor is selected from any one of breast cancer, colorectal cancer, melanoma, pancreatic cancer, ovarian cancer, gastric cancer, and lung cancer.