Application of chlorella vesicle in relieving T cell depletion and preparation method of chlorella vesicle
By preparing Chlorella vesicles, its ammonia removal ability is used to alleviate the accumulation of ammonia in the tumor microenvironment, the problem of CD8+ T cell depletion is solved, and the recovery of T cell function and the improvement of tumor treatment effect is achieved.
Patent Information
- Application Number
- CN202510050201.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-22
AI Technical Summary
In chronic viral infection and tumor microenvironment, CD8+ T cells are depleted due to continuous antigen stimulation and lose their immune response function, resulting in immunotherapy resistance.
By preparing Chlorella vesicles, using their ammonia removal ability, it alleviates the accumulation of ammonia in the tumor microenvironment, thereby reversing the T cell depletion state.
Chlorella vesicles can effectively reduce the ammonia concentration in the tumor microenvironment, reduce the inhibitory effect on T cells, and promote the functional recovery of CD8+ T cells and the tumor treatment effect.
Smart Images
Figure CN119857114A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of Chlorella vesicles in alleviating T cell exhaustion and a preparation method thereof. Background Art
[0002] In chronic viral infection and tumor microenvironment, due to continuous and repeated antigen stimulation, CD8+T cells gradually lose their function and enter a specific differentiation state, which is called T cell exhaustion. Unlike normal effector T cells, these exhausted T cells lose their immune response function, and the expression of inhibitory receptors (such as PD-1, CTLA-4, TIM-3, LAG-3, etc.) increases, and their ability to kill tumor cells is significantly reduced.
[0003] The formation of exhausted T cells is one of the important mechanisms of immune escape, and it is also a major reason why many chronic infections and tumors are difficult to cure. At present, T cell exhaustion is mainly inhibited by using immune checkpoint blockers in clinical practice, but a single therapeutic target has limited therapeutic effect in the face of some specific tumors, especially affected by some metabolites in the tumor microenvironment. These metabolites generally have an inhibitory effect on the function of T cells, leading to T cell dysfunction. Therefore, targeting the formation mechanism of exhausted T cells and finding ways to restore their function is an important research direction in the field of immunotherapy, which is of great significance to tumor immunotherapy.
[0004] Related studies have shown that in some cancer mouse models, ammonia, as a ubiquitous metabolite, accumulates in large quantities in the tumor microenvironment. These high concentrations of ammonia inhibit T cell growth and function, leading to T cell exhaustion and immunotherapy resistance. These findings provide a new idea for reversing T cell exhaustion: whether it is possible to alleviate the process of T cell exhaustion from the perspective of ammonia metabolism, relieve the immunosuppressive state in the tumor microenvironment, and synergize with conventional immunotherapy methods to solve the problem of drug resistance during tumor treatment, restore T cell function, and achieve better tumor treatment effects.
[0005] Therefore, how to remove the accumulated ammonia in the tumor microenvironment is an urgent problem to be solved. Summary of the invention
[0006] The purpose of the present invention is to provide an application and preparation method of Chlorella vesicles for alleviating T cell exhaustion, so as to solve the technical problems in the prior art.
[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:
[0008] The present invention provides a method for preparing Chlorella vesicles, comprising the following steps:
[0009] The Chlorella solution was centrifuged for the first time at 4°C to obtain a primary supernatant;
[0010] Centrifuging the primary supernatant for a second time at 4° C. to obtain a secondary supernatant;
[0011] The secondary supernatant is centrifuged for the third time, and the supernatant is discarded to obtain a Chlorella vesicle precipitate.
[0012] As a preferred embodiment of the present invention, the speed of the first centrifugation is 2000-2500 rpm, and the centrifugation time is 5-10 min;
[0013] The second centrifugation speed is 5500-6500rpm, and the centrifugation time is 10-15min;
[0014] The rotation speed of the third centrifugation is 20000g, and the centrifugation time is 1.5h.
[0015] As a preferred embodiment of the present invention, the Chlorella vesicle precipitate is dissolved using a PBS solution.
[0016] As a preferred embodiment of the present invention, the diameter of the Chlorella vesicle is 100-300 nm.
[0017] As an application of the Chlorella vesicle of the present invention in alleviating T cell exhaustion, the Chlorella vesicle alleviates T cell exhaustion by metabolizing ammonia.
[0018] As a preferred embodiment of the present invention, it also includes a product prepared from the Chlorella vesicles;
[0019] The product is an immunotherapeutic preparation targeting ammonia metabolism;
[0020] The immunotherapeutic preparation contains a pharmaceutically acceptable carrier and the Chlorella vesicle.
[0021] As a preferred embodiment of the present invention, the administration route of the immunotherapy preparation includes intravenous administration, subcutaneous administration or local administration.
[0022] As a preferred embodiment of the present invention, the Chlorella vesicles are used in combination with PD-1 monoclonal antibody to reduce the ammonia concentration in tumor interstitial fluid.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The present invention provides an immunotherapy preparation with active Chlorella vesicles by exploring the ammonia-degrading ability of Chlorella. This immunotherapy preparation targets the problem of chronic infection and T cell exhaustion in the tumor microenvironment, alleviates the accumulation of ammonia in the tumor microenvironment through ammonia metabolism, regulates the ammonia metabolism process in the tumor microenvironment, reduces the inhibitory effect on T cells, and reverses the state of T cell exhaustion to a certain extent.
[0025] The immunotherapy preparation disclosed in the present invention has the advantages of good biocompatibility and low immunogenicity, and can be used in combination with general anti-tumor drugs. After being used in combination with PD-1, its ability to deaminize is further improved, promoting CD8+T cell infiltration, reducing exhaustion-related indicators, and promoting CD8+T cell proliferation and the enhancement of TNF-α expression, and has good clinical application prospects in the field of tumor treatment immunity.
[0026] The invention obtains the Chlorella vesicles by multiple centrifugation, and the preparation method is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.
[0028] Figure 1 The present invention provides a schematic flow chart of a method for preparing Chlorella vesicles;
[0029] Figure 2 The figure is a characterization of the morphology of Chlorella vesicles by transmission electron microscopy;
[0030] Figure 3 Particle size characterization of Chlorella vesicles for nanoparticle tracking analysis;
[0031] Figure 4 A table showing the ability of Chlorella vesicles to deammonify in vitro environment for ammonia content detection;
[0032] Figure 5 This is a graph showing the effect of Chlorella vesicles on T cell exhaustion indicators detected by fluorescence real-time quantitative PCR;
[0033] Figure 6 This is a photo of the image analysis of the tumor size in the breast cancer mouse model to determine the therapeutic effect of Chlorella vesicles on breast cancer;
[0034] Figure 7This is a photo of the therapeutic effect of Chlorella vesicles and PD-1 monoclonal antibody combined therapy on breast cancer by analyzing the tumor size in a breast cancer mouse model.
[0035] Figure 8 The results of the investigation of the ammonia content detection of Chlorella vesicles on the ammonia removal ability in the tumor microenvironment of the breast cancer mouse model;
[0036] Fig. 9 This is the result of flow cytometry detection of the effect of Chlorella vesicles on CD3+CD8+ cell infiltration in breast cancer mouse model;
[0037] Fig.10 This is a flow cytometry result showing the effect of Chlorella vesicles on CD3+CD8+ cell-related exhaustion indicators in a breast cancer mouse model;
[0038] Fig.11 This is the result of flow cytometry detection of the effect of Chlorella vesicles on CD3+CD8+ cell proliferation and tumor killing ability in a breast cancer mouse model. DETAILED DESCRIPTION
[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0040] like Figure 1 As shown, the present invention provides a method for preparing a biological vesicle, which is a vesicle derived from Chlorella vulgaris, specifically named as a vesicle of small algae, and the vesicle of small algae can be used in the preparation of a product for alleviating T cell exhaustion. Figure 2 as well as Figure 3 The morphology and particle size of Chlorella vesicles were characterized by transmission electron microscopy and nanoparticle tracking analysis.
[0041] Algae can effectively remove ammonia nitrogen and nitrite nitrogen from water bodies, and are widely used in life to improve water quality and reduce eutrophication of water bodies. Inorganic nitrogen sources in the environment are actively or passively transported into cells through specific transport proteins on the cell membrane of algae plants, and converted into their own components to participate in the metabolic process in the body.
[0042] Utilizing the biological characteristics of algae, the present invention provides a novel immunotherapy preparation targeting ammonia metabolism to alleviate the accumulation of ammonia in the tumor microenvironment and reverse the state of T cell exhaustion.
[0043] Chlorella is a single-cell eukaryotic green algae with a diameter of 2 to 12um. It has a short growth cycle, strong reproductive capacity, good tolerance, and a wide ecological distribution. Chlorella does not need to go through the nitrification process but can directly use the assimilation of ammonia, converting and absorbing ammonia in the environment into the cell body for use, and has a strong potential for ammonia removal.
[0044] The present invention utilizes Chlorella vesicles as a novel drug carrier, giving full play to its own ammonia-degrading effect and advantages such as good biocompatibility and low immunogenicity, and is used in combination with general anti-tumor drugs to regulate the ammonia metabolism process in the tumor microenvironment and reduce the inhibitory effect on T cells, which has good clinical application prospects in the field of tumor treatment immunity.
[0045] The immunotherapy preparation disclosed in the present invention, wherein the active ingredient is Chlorella vesicles, can also provide other pharmaceutically acceptable carriers for use with the active ingredient, such as PBS solution, Normal saline wait.
[0046] The immunotherapy preparation of the present invention can act directly on an organism, and the administration routes include intravenous administration, subcutaneous administration or local administration. After administration, the Chlorella vesicles act in the organism to alleviate the exhaustion of T cells.
[0047] Specifically, the preparation steps of Chlorella vesicles are as follows:
[0048] The Chlorella culture solution is cultured at room temperature and under light to a suitable density to obtain a Chlorella solution, the Chlorella solution is centrifuged for the first time at 4°C to obtain a primary supernatant, the primary supernatant is centrifuged for the second time at 4°C to obtain a secondary supernatant, the secondary supernatant is centrifuged for the third time, the supernatant is discarded, and a Chlorella vesicle precipitate is obtained.
[0049] The invention obtains the chlorella vesicles with a diameter of 100-300nm by centrifuging the chlorella solution multiple times. The preparation method is simple and easy to implement.
[0050] Preferably, the first centrifugation speed is 2000-2500 rpm, the centrifugation time is 5-10 min, the second centrifugation speed is 5500-6500 rpm, the centrifugation time is 10-15 min, and the third centrifugation speed is 20000 g, and the centrifugation time is 1.5 h.
[0051] After obtaining the precipitate, the precipitate can be dissolved using a PBS solution to obtain an immunotherapy preparation.
[0052] The Chlorella vesicles are further described by way of examples below:
[0053] Embodiment 1:
[0054] (1) Preparation of Chlorella vesicle solution:
[0055] The chlorella used was a freshwater species, Latin species name: Chlorella sp, purchased from the freshwater algae species bank of the Institute of Hydrobiology, Chinese Academy of Sciences, with a specification of 500 ml.
[0056] Take 200 mL of Chlorella solution with good growth status and moderate density, divide the solution into ordinary 50 ml centrifuge tubes, centrifuge at 2500 rpm for 10 min, transfer the supernatant to a new centrifuge tube after centrifugation, centrifuge at 6500 rpm for 15 min, and continue to transfer the supernatant to an ultracentrifuge tube; centrifuge at 20000g, 4°C for 1.5 h, discard the supernatant, dissolve the precipitate with 1 mL PBS to obtain 1 mL of Chlorella vesicle solution.
[0057] The particle size and concentration of Chlorella vesicles were detected by nanoparticle tracking analyzer (NTA).
[0058] Experimental results: The obtained vesicle concentration is 2×10 10 / mL, diameter is 100-300nm, see Figure 3 .
[0059] The function of the Chlorella vesicle solution prepared in Example 1 is illustrated by verification below.
[0060] (2) In the case of NH 4 Cl co-incubation can reduce the concentration of ammonia
[0061] Chlorella vesicles (Example 1), Scenedesmus vesicles, and Oocystis vesicles were added to the same concentration of NH 4 Cl solution, and the concentration change of ammonia in the solution was detected after incubation for 16 h.
[0062] Chlorella vesicles and H22 tumor cell derived vesicles were added to NH4Cl solution of the same concentration, and the concentration of ammonia in the solution was detected after incubation for 16 hours. 4 The Cl concentration was 10 mM, and the vesicles and vesicle concentrations used were the same, 2 × 10 10 Pieces / mL.
[0063] Experimental results:
[0064] 1. Chlorella vesicles and NH 4 After 16 h of incubation with Cl, the concentration of ammonia in the solution was detected to decrease ( Figure 3 ).
[0065] 2. Compared with Scenedesmus vesicles, Oocystis vesicles and H22 tumor cell vesicles, Chlorella vesicles have a more obvious reducing effect.
[0066] (3) After joining NH 4 After 24 hours of co-culture of T cells with Cl, the expression of T cell exhaustion indicators PD-1, LAG3, TIM3, and CTLA4 genes decreased
[0067] T cells were extracted from the spleen of 4-week-old C57BL / 6 mice using a mouse CD8+T cell magnetic bead sorting kit, and IL-2 (10 ng / mL) and anti-CD3 / 28 antibodies were added to activate T cells. The cells were cultured for three days and seeded into six-well plates. 2×10 5 T cells.
[0068] The subjects were divided into four groups: blank control group, 4 Cl group, adding NH 4 Cl and 2×10 9 / mL Chlorella vesicle group, add NH 4 Cl and 2×10 10 / mL Chlorella vesicles.
[0069] At 37°C, 5% CO 2 Incubate in the incubator for 24 hours. Collect RNA from the four groups of T cells, and use qPCR to detect the expression of T cell exhaustion indicators PD-1, LAG3, TIM3, and CTLA4 at the gene level in each group. 4 The Cl concentration was 10 mM.
[0070] Experimental results: Compared with the blank control group, the addition of NH 4 The T cell exhaustion index increased in the Cl group, while that decreased in the group with Chlorella vesicles. 10 Compared with 2×10 9 The level of decrease was greater in the group of Chlorella vulgaris vesicles ( Figure 5 ).
[0071] It can be seen that Chlorella vesicles consume NH 4 The active ingredient of Cl.
[0072] (4) Chlorella vesicles have a good therapeutic effect on mouse breast cancer model
[0073] Four-week-old BALB / c mice were used to construct a breast cancer mouse model. Each mouse was subcutaneously injected with 2×10 5 4T1 cells (mouse breast cancer cell line).
[0074] The mice were divided into PBS group, Chlorella vesicle group, and Oocystis vesicle group, with 6 mice in each group. The mice were injected with drugs through the tail vein every other day from the 7th day of tumor bearing, for a total of five injections. The injection volume of the PBS group was 0.1 mL, and the injection concentration of the Oocystis vesicle group and the Chlorella vesicle group was 2×10 10 / mL, injection volume 0.1mL. On the 21st day, the tumor site of the mouse was removed to observe the size and morphological changes.
[0075] Experimental results: Chlorella vesicles have a good therapeutic effect on mouse breast cancer model ( Figure 6 ).
[0076] (5) It has a good therapeutic effect on mouse breast cancer when combined with PD-1 monoclonal antibody
[0077] Four-week-old BALB / c mice were used to construct a breast cancer mouse model. Each mouse was subcutaneously injected with 2×10 5 4T1 cells.
[0078] The mice were divided into PBS group, Chlorella vesicles alone group, PD-1 monoclonal antibody alone group, and Chlorella vesicles and PD-1 monoclonal antibody combined group, with 6 mice in each group. The mice were injected once every other day through the tail vein from the 7th day of tumor bearing, for a total of five injections. The injection volume was 0.1 mL, the PD-1 monoclonal antibody injection concentration was 10 ug / mouse, and the injection concentration of the Chlorella vesicle group was 2×10 10 pcs / mL.
[0079] On the 21st day, the mouse tumor sites were removed to observe changes in size and morphology. The tumor tissue was then chopped up and centrifuged at 2000rpm for 10 minutes to extract the tumor interstitial fluid. The kit was used to detect the concentration of ammonia in the tumor tissue interstitial fluid. The chopped tumor tissue was then placed in 3mL of the prepared digestion solution and digested at 37°C for 1.5 hours, then ground and filtered through gauze. The filtrate was centrifuged at 800rpm for 5 minutes, the supernatant was transferred to a new centrifuge tube, and centrifuged at 400g for 5 minutes. 3mL of ELS solution was added to the resulting precipitate for red blood cell lysis. After standing for 5 minutes, 3mL of serum-containing culture medium was added for neutralization, and centrifuged at 400g for 5 minutes.
[0080] The supernatant was discarded to obtain a cell pellet containing immune cells. The cell suspension was adjusted to an appropriate density with a certain amount of PBS, and then stained with flow cytometry antibodies CD3, CD8, CD45, PD-1, CTLA4, ki67, and TNF-α, and analyzed using a flow cytometer.
[0081] Digestion solution: DNase 5μg / μl, hyaluronidase 100mg / μl, collagenase 1mg / μl, DMEM medium. ELS solution: NH 4 Cl4.14g, NaHCO3 0.51g, 0.0186g EDTA, add water to make up to 500mL.
[0082] Experimental results: Compared with the PBS control group, the group of single administration of Chlorella vesicles, the group of single administration of PD-1 monoclonal antibody, and the group of combined administration of Chlorella vesicles and PD-1 monoclonal antibody all showed certain therapeutic effects, among which the combined administration of Chlorella vesicles and PD-1 monoclonal antibody had the best effect on tumor treatment ( Figure 6 ), the concentration of ammonia in the tumor interstitial fluid decreased more ( Figure 7 ), promoting CD8+T cell infiltration, reducing exhaustion-related indicators, and promoting CD8+T cell proliferation and TNF-α expression ( Figure 8 , 9 , 10).
[0083] Through this embodiment, it can be explained that the biological vesicles provided by the present invention have a strong ammonia-degrading ability, can be used in the preparation of products for alleviating T cell exhaustion, and utilize Chlorella vesicles as a new type of drug carrier to give full play to their own ammonia-degrading effect and good biocompatibility and low immunogenicity. At the same time, they are combined with general anti-tumor drugs to regulate the ammonia metabolism process in the tumor microenvironment and reduce the inhibitory effect on T cells. It has good clinical application prospects in the field of tumor treatment immunity.
[0084] The present invention provides a new method to improve ammonia accumulation in the tumor microenvironment through the ammonia-degrading effect of Chlorella vesicles, thereby reversing the state of T cell exhaustion. The technical scheme includes preparing Chlorella vesicles, verifying their ammonia-degrading ability through in vitro experiments, and evaluating their effects on the restoration of T cell function and the inhibition of tumor growth in animal models.
[0085] The main use of the present invention is to serve as a potential immunotherapy to improve the efficiency of tumor immunotherapy and provide a new strategy for cancer treatment.
[0086] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.
Claims
1. A use of Chlorella vesicles in alleviating T cell exhaustion, characterized in that: The Chlorella vesicles alleviate T cell exhaustion by metabolizing ammonia.
2. The use of a Chlorella vesicle in alleviating T cell exhaustion according to claim 1, characterized in that: Including products prepared from the Chlorella vesicles; The product is an immunotherapeutic preparation targeting ammonia metabolism; The immunotherapeutic preparation contains a pharmaceutically acceptable carrier and the Chlorella vesicle.
3. The use of a Chlorella vesicle in alleviating T cell exhaustion according to claim 2, characterized in that: The administration routes of the immunotherapeutic preparation include intravenous, subcutaneous or local administration.
4. The use of a Chlorella vesicle in alleviating T cell exhaustion according to claim 1, characterized in that: The Chlorella vesicles are used in combination with PD-1 monoclonal antibody to reduce the ammonia concentration in tumor interstitial fluid.
5. A method for preparing the Chlorella vesicle according to any one of claims 1 to 4, characterized in that: The steps include: The Chlorella solution was centrifuged for the first time at 4°C to obtain a primary supernatant; Centrifuging the primary supernatant for a second time at 4° C. to obtain a secondary supernatant; The secondary supernatant is centrifuged for the third time, and the supernatant is discarded to obtain a Chlorella vesicle precipitate.
6. The method according to claim 5, characterized in that The first centrifugation has a rotation speed of 2000-2500 rpm and a centrifugation time of 5-10 min; The second centrifugation speed is 5500-6500rpm, and the centrifugation time is 10-15min; The rotation speed of the third centrifugation is 20000g, and the centrifugation time is 1.5h.
7. The method according to claim 5, characterized in that The Chlorella vesicle precipitate is dissolved by using PBS solution.
8. The method according to claim 5, characterized in that The diameter of the Chlorella vesicle is 100-300nm.