A method for recovering membrane antigen based on gold nanocluster gravity anchoring enrichment and constructing nanovaccine

Abstract

The application discloses a method for recovering membrane antigen of gold corpuscle and constructing nanovaccine based on gravity anchoring. The method comprises the following steps: modifying a first click chemistry reaction group on the surface of tumor cell membrane, providing gold corpuscle modified with a second click chemistry reaction group capable of specifically combining with the first click chemistry reaction group, mixing the modified tumor cell with the gold corpuscle, incubating to anchor the gold corpuscle on the surface of the tumor cell membrane, lysing the tumor cell, and collecting the gold corpuscle-anchored tumor cell membrane antigen complex. The first click chemistry reaction group and the second click chemistry reaction group are a complementary group pair for bio-orthogonal click chemistry reaction. The method realizes the integration of efficient and high-fidelity recovery of membrane antigen and vaccine construction, is simple in process, and is strong in universality. The prepared nanovaccine shows excellent immunotherapy effect in various postoperative recurrence models of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for enriching and recovering membrane antigens based on the gravity anchoring of gold nanoparticles and constructing nanovaccines. Background Technology

[0002] In recent years, nanovaccines have attracted much attention as an emerging strategy for disease prevention and treatment due to their ability to efficiently deliver antigens, enhance immunogenicity, and possess good biocompatibility. Their core objective is to precisely mimic the structure of pathogens and safely and effectively activate the body's specific immune response. However, existing nanovaccine construction technologies still face several key bottlenecks, particularly in the efficient enrichment and functional display of membrane antigens.

[0003] Cell membrane antigens are functional proteins or polysaccharide complexes located on the outer surface of the cell membrane, serving as key interfacial molecules in the interaction between cells and their external environment. Compared to antigens distributed in other locations (such as the cytoplasm and organelles), cell membrane antigens are naturally exposed to the extracellular environment. Besides containing key MHC-presented epitopes that can be directly recognized and responded to by T cells, other proteins or polysaccharide structures with specific native conformations can also be recognized by B cells and bind to antibodies, mediating humoral immune responses. Furthermore, the expression levels and distribution patterns of cell membrane antigens are often closely related to cell type, state, and pathological processes, making them ideal candidates for disease-specific biomarkers and drug targets. While the safety of membrane antigens is controllable, it is often difficult to reproduce the complete spatial conformation and post-translational modifications of native membrane proteins, and the low purity of recovered antigens leads to insufficient breadth and intensity of the induced immune response. While directly extracting natural membrane antigens from pathogen or tumor cell membranes can preserve their natural structure and immunotopes to the greatest extent, existing extraction techniques (such as ultrasonic disruption and detergent dissolution) suffer from low recovery efficiency, complex processes, easy protein denaturation and inactivation, and difficulty in targeted and controlled coupling with nanocarriers. These factors severely restrict the development and application of highly efficient nanovaccines based on natural membrane antigens.

[0004] On the other hand, the selection of nanomaterials as vaccine delivery carriers and adjuvants is crucial. Among numerous materials, biomineralized gold stands out due to its unique physicochemical properties. Its preparation conditions are mild, and gold nanoparticles (gold bodies) with uniform size and high stability can be synthesized under near-physiological conditions via a biocompatible reduction method. Gold bodies not only possess large mass and good biosafety, but their surfaces are also easily functionalized. Furthermore, they can effectively activate antigen-presenting cells such as dendritic cells through mechanisms such as size effects, exerting an immune adjuvant effect. However, how to densely, orderly, and firmly display recovered, intact membrane antigens on the surface of gold nanoparticles to construct nanovaccines with well-defined structures and enhanced immunogenicity remains a technical challenge to be solved. Click chemistry, especially copper-catalyzed azido-alkyne cycloaddition reactions or their copper-free variants, has become a revolutionary tool in the field of bioconjugation due to its high efficiency, specificity, mild conditions, and minimal interference with biomolecules. This technology provides an ideal solution for the rapid and precise connection of different functional molecular modules.

[0005] In summary, there is an urgent need to provide a new method for constructing nanovaccines that can integrate efficient membrane antigen recovery, precise conjugation, and efficient immune activation. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method for enriching and recovering membrane antigens based on the gravity anchoring of gold nanoparticles and constructing nanovaccines. This method aims to solve the problems of insufficient vaccine immunogenicity and reduced therapeutic efficacy caused by the low purity of membrane antigens in existing nanovaccines based on tumor cell membrane antigens.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for enriching and recovering membrane antigens based on the gravity anchoring of gold bodies. The method includes: modifying a first click chemical reactive group on the surface of a tumor cell membrane, providing a gold body with a second click chemical reactive group on its surface that can specifically bind to the first click chemical reactive group, mixing and incubating the modified tumor cells with the gold body to anchor the gold body on the surface of the tumor cell membrane, lysing the tumor cells, and collecting the tumor cell membrane antigen complex anchored by the gold body. The first and second click chemical reaction groups are complementary group pairs for carrying out bioorthogonal click chemical reactions.

[0008] This invention utilizes gold bodies obtained through glycolytic engineering to anchor them onto the surface of tumor cell membranes using click chemistry. Cells are lysed using repeated freeze-thaw cycles to prevent denaturation and inactivation of membrane protein antigens. Taking advantage of the large mass of the gold bodies, the tumor membrane antigens anchored to them are obtained through high-speed centrifugation. Finally, microfluidic technology is used to obtain thermodynamically stable nanovaccine structures. This method eliminates the need for time-consuming methods such as ultracentrifugation, and also eliminates the need for further separation of the gold bodies and tumor cell membranes; the components obtained through high-speed centrifugation are the vaccine's constituent parts.

[0009] Preferably, the modification of the tumor cell membrane surface with the first click chemical reactive group includes: integrating a lipid molecule containing the first click chemical reactive group into the cell membrane by membrane insertion, or reacting an active molecule containing the first click chemical reactive group with an amino group on the cell membrane surface by chemical coupling.

[0010] Preferably, the gold particles are gold nanoparticles synthesized by bacterial biomineralization.

[0011] Preferably, the surface of the gold body is introduced with a second click chemical reaction group through bacterial sugar metabolism engineering.

[0012] Preferably, the bacteria include Escherichia coli.

[0013] Preferably, the *Escherichia coli* includes any one or a combination of at least two of DH5α, BL.21, Top10, or Nissle 1917.

[0014] Preferably, the exogenous non-natural sugars used in the sugar metabolism engineering include any one or a combination of at least two of Ac4ManNAz, Ac4GlcNAz, or Ac4GalNAz.

[0015] Preferably, the complementary group pair includes a cycloalkyne group and an azide group.

[0016] Preferably, the cycloalkyne group includes a dibenzocyclooctynyl group.

[0017] Preferably, the azide group includes an -N3 group.

[0018] Preferably, the incubation time is 0.5-2 h, for example, 0.5 h, 1 h or 2 h.

[0019] Preferably, the pyrolysis includes: centrifugation after freeze-thaw cycles.

[0020] Preferably, the number of freeze-thaw cycles is 3-5 times, for example, 3 times, 4 times or 5 times.

[0021] Preferably, the centrifugal force is 300-1000 g (e.g., 300 g, 500 g, or 1000 g), and the time is 3-10 min (e.g., 3 min, 5 min, or 10 min).

[0022] In a second aspect, the present invention provides a method for constructing a nanovaccine, the method comprising: microfluidically processing the tumor cell membrane antigen complex described in the first aspect to obtain a nanovaccine.

[0023] Thirdly, the present invention provides a nanovaccine, which is constructed by the method described in the second aspect.

[0024] Fourthly, the present invention provides the use of the nanovaccine described in the third aspect in the preparation of a medicament for preventing or treating postoperative recurrence of tumors.

[0025] Preferably, the tumor includes any one of colon cancer, glioma, osteosarcoma, melanoma, pancreatic cancer, breast cancer, liver cancer, stomach cancer, head and neck cancer, cervical cancer, or lung cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) High efficiency and high fidelity: This invention utilizes the specificity and mildness of click chemistry to achieve in-situ labeling and high recovery rate of membrane antigens in the native membrane environment, thus preserving the natural conformation and activity of the antigens to the greatest extent. (2) Simplified and integrated process: This invention combines antigen recovery and carrier conjugation into one, and obtains the core components of the vaccine directly through a simple process of "anchoring-lysis-centrifugation", without the need for complex ultracentrifugation or additional chemical conjugation steps, thus reducing production costs and process difficulty; (3) Strong universality: The method described in this invention is applicable to a variety of tumor cell lines (such as colon cancer, melanoma, breast cancer, etc.) and gold bodies from a variety of bacteria, and has good versatility; (4) Good immune effect: The nano-vaccine constructed in this invention has high antigen display density and clear structure. Combined with the adjuvant effect of gold bodies, it shows significant tumor inhibition and recurrence prevention effects in various mouse postoperative recurrence models. Attached Figure Description

[0027] Figure 1 The graph shows the tumor cell viability and mean fluorescence detection results after introducing DBCO groups onto the cell membrane of MC38 tumor cells by physical method. Figure 2 The graph shows the tumor cell viability and mean fluorescence detection results after introducing DBCO groups onto the cell membrane of MC38 tumor cells by chemical method. Figure 3Structural diagrams of three exogenous non-natural sugars; Figure 4 Transmission electron microscope images of gold body mineralization synthesis by different strains of Escherichia coli; Figure 5 Transmission electron microscope image of an ultrathin section of a gold corpuscle anchored to the surface of a tumor cell membrane via click chemical anchoring; Figure 6 This is a transmission electron microscope image of a nanovaccine. Figure 7 Survival curve of a mouse model of colon cancer recurrence after surgery for a nanovaccine prepared from gold bodies obtained by mineralization of Escherichia coli DH5α. Figure 8 Survival curve of a mouse model of colon cancer recurrence after surgery for a nanovaccine prepared from gold bodies obtained by mineralization of Escherichia coli BL.21; Figure 9 Survival curve of a mouse model of colon cancer recurrence after surgery for a nanovaccine prepared from gold bodies obtained by mineralization of Escherichia coli Top10. Figure 10 Survival curve of a mouse model of colon cancer recurrence after surgery for a nanovaccine prepared from gold bodies obtained by mineralization of Escherichia coli Nissle 1917. Figure 11 Survival curves of mice in different cancer postoperative recurrence models for nanovaccines prepared by the chemical introduction of DBCO groups into gold bodies obtained by mineralization of Escherichia coli BL.21 and Ac4ManNAz sugar metabolism engineering. Detailed Implementation

[0028] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] DSPE-PEG-DBCO: 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol-dibenzocyclooctylene; NHS-PEG-DBCO: N-hydroxysuccinimide ester-polyethylene glycol-dibenzocyclooctylene; N3-PEG-FITC: Azide-polyethylene glycol-fluorescein isothiocyanate; Ac4ManNAz: Azide-modified mannose; Ac4GlcNAz: Azide-modified glucose; Ac4GalNAz azide-modified galactose.

[0031] Example 1 DBCO groups are introduced onto the tumor cell membrane using physical or chemical methods.

[0032] This embodiment provides a method for stably introducing DBCO groups onto the membrane surface of different tumor cells (MC38, B16F10, CT26, 4T1), as detailed below: (1) Physical method: DSPE-PEG-DBCO (purchased from MedChemExpress, catalog number HY-W440835E) was inserted into the tumor cell membrane by diffusion. The treatment process was to add 100 μM of DSPE-PEG-DBCO to the adherent tumor cells and treat for 3.5 h. PBS was used as the solvent for DSPE-PEG-DBCO. (2) Chemical method: NHS-PEG-DBCO (purchased from MedChemExpress, catalog number HY-140272) was used to react chemically with NH2 present on the surface of tumor cell membranes. The treatment process involved adding 100 μM NHS-PEG-DBCO to adherent tumor cells and treating them for 0.5 h. PBS was used as the solvent for NHS-PEG-DBCO. Cells were obtained using a cell scraper and detected using N3-PEG-FITC (purchased from Aladdin, catalog number F163684). 7AAD dye (purchased from BioLegend, catalog number 420403) was used to detect cell viability to prevent changes in the composition of tumor cell membrane protein antigens. The specific detection steps are as follows: 20 μL of 100 mg / mL N3-PEG-FITC is added to the collected cells and reacted for 90 min. After washing twice with PBS, 7AAD dye is added for staining for 20 min. After centrifugation, the cells are washed twice with PBS. Finally, flow cytometry is used to detect the fluorescence intensity of FITC and the positive rate of 7AAD. The higher the fluorescence intensity, the more DBCO groups are introduced onto the tumor cell membrane. The lower the 7AAD positive rate, the lower the tumor cell mortality rate and the better the activity.

[0033] The results of tumor cell viability and mean fluorescence detection after introducing DBCO groups onto the cell membrane of MC38 tumor cells using physical methods are as follows: Figure 1 As shown, the tumor cell viability and mean fluorescence detection results after introducing DBCO groups onto the cell membrane of MC38 tumor cells using a chemical method are as follows: Figure 2As shown, the results indicate that both physical and chemical methods can successfully introduce DBCO groups without causing tumor cell death under the corresponding conditions. This means that the introduction of DBCO groups provides a target for subsequent gold body anchoring without affecting the changes in protein composition on the tumor cell membrane.

[0034] Example 2 In this embodiment, N3 groups are introduced onto the surface of gold bodies mineralized from E. coli through glycochemical engineering.

[0035] Escherichia coli (DH5α, BL.21, Top10, Nissle 1917) were cultured in 200 mL LB medium until the logarithmic growth phase. On Day 0, the medium was replaced with 200 mL sterile PBS solution, and 6 mL of HAuCl4 and 10 μM of exogenous non-natural sugars (Ac4ManNAz, Ac4GlcNAz, and Ac4GalNAz, all purchased from MedChemExpress, catalog numbers: HY-W728531, HY-W039953, and HY-141128, respectively) were added. From Day 0 to Day 7, a total of 10 μM of exogenous non-natural sugars (Ac4ManNAz, Ac4GlcNAz, and Ac4GalNAz) was continuously added. On Day 8, gold bodies mineralized from E. coli were obtained by differential centrifugation and stored at -80°C. The structural formulas of the three exogenous non-natural sugars are shown below. Figure 3 As shown in the figure. Transmission electron microscope images of different Escherichia coli strains synthesizing gold bodies through mineralization are shown in the figure. Figure 4 As shown, the results indicate that adding these three exogenous non-natural sugars does not affect the mineralization efficiency of gold bodies by E. coli, and gold body materials with relatively uniform size can be successfully synthesized.

[0036] Example 3 This embodiment uses click chemistry to anchor gold bodies onto the surface of tumor cell membranes, as detailed below: The gold bodies obtained in Example 2 were co-incubated with tumor cells from Example 1 for 1 h, and then centrifuged at 500 g for 5 min to obtain tumor cells with gold bodies on their membrane surface. Transmission electron microscopy images of ultrathin sections of tumor cell membranes with gold bodies anchored by click chemoattachment are shown below. Figure 5 As shown, the mineralized gold bodies successfully incorporated N3 groups and could anchor to tumor cell membranes with DBCO groups, providing effective evidence for subsequent experiments involving repeated freeze-thaw differential centrifugation to recover tumor cell membranes.

[0037] Example 4 In this embodiment, the tumor cell membrane antigen anchored by gold bodies is recovered, and a nanovaccine is prepared using a microfluidic method, as detailed below: The tumor cells containing gold bodies obtained in Example 3 were subjected to three freeze-thaw cycles. The gold-anchored tumor cell membrane antigen was obtained by differential centrifugation, and then a thermodynamically stable nanovaccine was obtained using microfluidic technology. Specifically, one side of the microfluidic syringe contained the tumor cell membrane complex recovered by the gold-anchoring method, and the other side contained one-third of the PBS solution. The microfluidic flow rate was controlled at 0.2 mL / min, with an injection volume ratio of 3:1. A fishbone-shaped microchip (purchased from PRECISION, catalog number NIT0004) was used. The recovered tumor cell membrane was coated with gold bodies by increasing shear force. The final recovered nanovaccine was imaged using transmission electron microscopy. The transmission electron microscopy image of the nanovaccine is shown below. Figure 6 As shown, the gold microsomes are successfully anchored to the surface of tumor cell membranes by gravity and the tumor cell membrane components are successfully recovered by repeated freeze-thaw cycles and differential centrifugation. Furthermore, the membrane is coated using microfluidic technology to obtain a thermodynamically stable nanovaccine structure.

[0038] Example 5 This embodiment evaluates the therapeutic effect of nanovaccines in a postoperative recurrence model, as detailed below: C57BL6 mice were subcutaneously injected with 1×10⁻⁶ cells on the right posterior back. 7 Mouse tumor cells (colon cancer cells, glioma cells, osteosarcoma cells, melanoma cells, breast cancer cells, pancreatic cancer cells, liver cancer cells, gastric cancer cells, head and neck cancer cells, cervical cancer cells, or lung cancer cells) were cultured in an SPF environment. The tumors were cultured until they reached a size of 100-200 mm. 3 Surgery was performed, but the tumor tissue was not completely removed, leaving 1% of the tumor tissue to simulate postoperative recurrence. Mice were divided into two groups: a control group (PBS) and a group receiving a nanovaccine injection near the lymph nodes. Injections were administered near the lymph nodes on Day 4, Day 6, and Day 9 post-surgery. In this example, the control group received a 50 μL PBS solution injected near the lymph nodes, while the nanovaccine treatment group received a 50 μL nanovaccine solution injected near the lymph nodes. The long and short sides of the tumor were measured every two days after recurrence. The tumor volume was approximately calculated as the square of the short side multiplied by the long side divided by two. A tumor volume greater than 1500 mm² was considered normal. 3 Or, death may be declared due to a severe deterioration of health. The outcome is as follows: Figures 7-11 As shown, compared with the control group, the nanovaccine has a good recurrence inhibition effect on tumors, indicating that the method can effectively recover tumor cell membrane antigens and the prepared nanovaccine has good therapeutic effect.

[0039] In summary, this method achieves efficient and high-fidelity recovery of membrane antigens and integrated vaccine construction, with a simple process and strong versatility. The prepared nanovaccines have shown excellent immunotherapeutic effects in various tumor postoperative recurrence models.

[0040] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for enriching and recovering membrane antigens based on the gravitational anchoring of gold microparticles, characterized in that, The method includes: modifying a first click chemical reactive group on the surface of a tumor cell membrane, providing a gold body with a second click chemical reactive group on its surface that can specifically bind to the first click chemical reactive group, mixing and incubating the modified tumor cells with the gold body to anchor the gold body on the surface of the tumor cell membrane, lysing the tumor cells, and collecting the gold body-anchored tumor cell membrane antigen complex. The first and second click chemical reaction groups are complementary group pairs for carrying out bioorthogonal click chemical reactions.

2. The method according to claim 1, characterized in that, The modification of the tumor cell membrane surface with the first click chemical reactive group includes: integrating a lipid molecule containing the first click chemical reactive group into the cell membrane by membrane insertion, or reacting an active molecule containing the first click chemical reactive group with an amino group on the cell membrane surface by chemical coupling.

3. The method according to claim 1 or 2, characterized in that, The gold nanoparticles are gold nanoparticles synthesized by bacterial biomineralization. Preferably, the surface of the gold body is introduced with a second click chemical reaction group through bacterial sugar metabolism engineering.

4. The method according to claim 3, characterized in that, The bacteria include Escherichia coli; Preferably, the *Escherichia coli* includes any one or a combination of at least two of DH5α, BL.21, Top10, or Nissle 1917; Preferably, the exogenous non-natural sugars used in the sugar metabolism engineering include any one or a combination of at least two of Ac4ManNAz, Ac4GlcNAz, or Ac4GalNAz.

5. The method according to any one of claims 1-4, characterized in that, The complementary group pair includes: a cycloalkyne group and an azide group; Preferably, the cycloalkyne group includes a dibenzocyclooctynyl group; Preferably, the azide group includes an -N3 group.

6. The method according to any one of claims 1-5, characterized in that, The incubation time is 0.5-2 hours; Preferably, the pyrolysis includes: centrifugation after freeze-thaw cycles; Preferably, the number of freeze-thaw cycles is 3-5. Preferably, the centrifugal force is 300-1000 g and the time is 3-10 min.

7. A method for constructing a nanovaccine, characterized in that, The method for constructing a nanovaccine includes: microfluidically processing the tumor cell membrane antigen complex according to any one of claims 1-6 to obtain a nanovaccine.

8. A nano-vaccine, characterized in that, The nanovaccine is constructed by the method described in claim 7.

9. The use of the nanovaccine of claim 8 in the preparation of a medicament for the prevention or treatment of postoperative recurrence of tumors.

10. The application according to claim 9, characterized in that, The tumor includes any one of colon cancer, glioma, osteosarcoma, melanoma, pancreatic cancer, breast cancer, liver cancer, stomach cancer, head and neck cancer, cervical cancer, or lung cancer.

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