A hybrid membrane nanoparticle vaccine for preventing tumor recurrence and a preparation method thereof

Abstract

This invention discloses a hybrid membrane nanoparticle vaccine for preventing tumor recurrence and its preparation method, belonging to the field of tumor immunotherapy technology. The vaccine is prepared by combining autologous tumor cell membranes and Mycobacterium smegma membrane extracts into bifunctional fused membrane nanoparticles, loaded with the cGAS-STING agonist colloidal manganese. This invention extracts tumor cell membranes and Mycobacterium smegma membranes through a specific process, fuses them in a specific ratio to form a hybrid membrane, and then combines it with colloidal manganese. This achieves sustained release of Mn²⁺ and enrichment of mature dendritic cells loaded with tumor antigens, promoting dendritic cell maturation and inducing effector memory T cell migration to the tumor microenvironment, significantly enhancing the anti-tumor immune response. This vaccine provides an economical and convenient personalized treatment option for preventing postoperative recurrence and metastasis in cancer patients, solving the problems of weak immunogenicity, low antigen presentation efficiency, and limited applicability of traditional tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically to a hybrid membrane nanoparticle vaccine for preventing tumor recurrence and its preparation method. Background Technology

[0002] Therapeutic vaccines, as an important means of tumor immunotherapy, can be divided into two categories based on the source of the antigen: whole-cell vaccines and peptide or protein vaccines. Whole-cell vaccines use inactivated whole tumor cells, whole-cell lysates, tumor cell membranes, etc., to provide a series of tumor cell-related antigens. This is a pan-targeting strategy, which does not require specific key tumor antigens, but usually requires the assistance of immune adjuvants. On the other hand, peptide or protein antigen vaccines are mostly designed to target one or more tumor-specific or related antigens. They rely on the understanding of tumor cell markers / antigens and their structures, and the tumor-specific immune response of peptide vaccines depends on specific MHC-restricted epitopes.

[0003] Extensive cancer sequencing data reveals significant genetic heterogeneity among patients with the same type of cancer. Furthermore, the complexity and diversity of human leukocyte antigens (HLA) lead to substantial differences in antigen presentation among individuals with the same tumor, limiting the benefit of traditional cancer vaccine treatments targeting single antigens. While whole-tumor cell vaccines based on autologous tumor tissue or heterologous tumor cell line lysates possess complete tumor-associated antigens, the complex cytoplasmic proteins and components can interfere with antigen recognition and presentation by antigen-presenting cells. Therefore, tumor cell membranes containing nearly complete arrays of tumor antigens, free of cytoplasmic impurities, have become a focus of vaccine development.

[0004] Autologous tumor cell vaccines, as a personalized cancer treatment strategy, can induce tumor-specific immune responses. However, they are difficult to be effective in "cold" tumors that lack tumor-infiltrating T cells and are insensitive to immunotherapy. In recent years, whole-cell tumor vaccines have gradually shifted towards autologous tumor vaccines based on tumor cell membranes. However, simple tumor cell membrane vaccines suffer from weak immunogenicity and cell surface immunosuppressive molecules that hinder immune system recognition, resulting in a lack of long-term therapeutic benefits. To induce an effective immune response, tumor cell vaccines usually need to be prepared in nanoscale form and used in combination with adjuvants. Therefore, the development of nanoscale in-situ therapeutic tumor vaccines based on hybrid membranes combined with super-strong adjuvants has significant clinical implications. Summary of the Invention

[0005] The purpose of this invention is to provide a hybrid membrane nanoparticle vaccine for preventing tumor recurrence and a method for preparing the same.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 1. Vaccine composition The core components of this vaccine include bifunctional fusion membrane nanoparticles and cGAS-STING agonist colloidal manganese. The bifunctional fusion membrane nanoparticles are made by fusing autologous tumor cell membranes and Mycobacterium smegmaeii membranes at a 2:1 mass ratio. Colloidal manganese acts as an adjuvant, loading and binding with the hybrid membrane nanoparticles to synergistically enhance the immune response.

[0007] 2. Preparation method S1. Tumor cell membrane extraction: The patient's own tumor cells are taken out and placed in an EP tube. They are treated by repeated freeze-thaw cycles of 37°C water and liquid nitrogen for 10 cycles, each lasting 5 minutes. The sudden temperature change causes the cell membrane to separate from the cytoplasm. Then, the cytoplasmic impurities are removed by centrifugation, and the precipitate is collected to obtain the tumor cell membrane.

[0008] S2. Extraction of Mycobacterium smegmatis biofilm: Mycobacterium smegmatis is subjected to ultrasonic disruption to destroy the bacterial structure and release biofilm components. The biofilm precipitate is obtained by centrifugation, which yields the Mycobacterium smegmatis biofilm.

[0009] S3. Preparation of hybrid membrane nanoparticles: The extracted tumor cell membrane and Mycobacterium smegmatis membrane were mixed at a mass ratio of 2:1 and repeatedly extruded in a 100 nm extruder more than 20 times to fully fuse the two membrane structures and form bifunctional fused membrane nanoparticles with uniform size.

[0010] S4. Vaccine loading preparation: The hybrid membrane nanoparticles and colloidal manganese are placed in a container, mixed, and then shaken to ensure that the colloidal manganese is uniformly loaded onto the hybrid membrane nanoparticles, thus completing the vaccine preparation.

[0011] The beneficial effects of this invention are as follows: This invention uses autologous tumor cell membranes as the antigen source, and can prepare hybrid membranes for each patient's unique tumor cells to form a personalized vaccine. This perfectly adapts to tumor heterogeneity and the diversity of human leukocyte antigens, solving the problem of limited benefit rate of traditional single antigen vaccines.

[0012] The hybrid design of tumor cell membrane and Mycobacterium smegma membrane enhances antigen immunogenicity. At the same time, colloidal manganese continuously releases Mn²⁺, which works synergistically with the hybrid membrane nanoparticles to promote the internalization, maturation and antigen presentation of dendritic cells. This effectively enriches mature dendritic cells loaded with tumor antigens, induces sufficient effector memory T cells to migrate to the tumor microenvironment, successfully transforms "cold" tumors into "hot" tumors, and significantly enhances the anti-tumor immune response.

[0013] The preparation process of this invention involves only routine operations such as freeze-thaw cycles, ultrasonic disruption, centrifugation, extrusion, and mixing and shaking. It requires no complex equipment or expensive reagents, making the process simple, cost-effective, and suitable for large-scale clinical application. This provides an economical and convenient option for preventing recurrence and metastasis after surgery in cancer patients. The hybrid membrane nanoparticles can provide a complete array of tumor-associated antigens. Combined with the adjuvant effect of colloidal manganese, they induce durable tumor-specific immune memory, effectively preventing tumor recurrence and metastasis, thus overcoming the shortcomings of simple tumor cell membrane vaccines in terms of long-term therapeutic benefits. Attached Figure Description

[0014] Figure 1 This invention relates to a hybrid membrane nanoparticle vaccine for preventing tumor recurrence and its preparation method.

[0015] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0018] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0019] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] See Figure 1 This invention provides a hybrid membrane nanoparticle vaccine for preventing tumor recurrence and its preparation method. Example

[0021] Vaccine preparation S1. Tumor cell membrane extraction: Take the tumor tissue removed by the patient during surgery, separate the tumor cells, place the tumor cells in an EP tube, add an appropriate amount of physiological saline, first put it in a 37℃ constant temperature water bath for 5 minutes, then quickly transfer it to liquid nitrogen to freeze for 5 minutes, repeat this freeze-thaw cycle 10 times, after which centrifuge at 8000 r / min for 15 minutes, discard the supernatant, collect the precipitate, which is the tumor cell membrane (TM).

[0022] S2. Extraction of Mycobacterium smegmatis biofilm: Take Mycobacterium smegmatis cultured to the logarithmic growth phase, wash 3 times with physiological saline, and adjust the bacterial concentration to 1×10⁻⁶. 8 The concentration of CFU / mL was determined by ultrasonic disruption for 30 min. After disruption, the mixture was centrifuged at 10000 r / min for 20 min. The supernatant was discarded, and the precipitate was collected, which was the Mycobacterium smegmatis biofilm (MsM).

[0023] S3. Preparation of hybrid membrane nanoparticles: Accurately weigh 2 mg of tumor cell membrane (TM) and 1 mg of Mycobacterium smegmatis membrane (MsM), put them into a centrifuge tube, add 2 mL of physiological saline, gently blow and mix well, transfer the mixture to a 100 nm extruder, repeat the extrusion 25 times, collect the extruded liquid, which is the suspension of bifunctional fused membrane nanoparticles (HM-NPs).

[0024] S4. Final vaccine preparation: Take 1 mL of the above HM-NPs suspension, add 0.5 mL of colloidal manganese (MnJ) solution with a concentration of 5 mg / mL, place it in a constant temperature shaker, and shake for 2 h at 37℃ and 150 r / min. After loading is completed, the hybrid membrane nanoparticle vaccine is obtained. Example

[0025] Vaccine performance testing 1. Characterization and detection: The size, dispersion (Pdl), and potential (Zeta) of HM-NPs were detected by dynamic light scattering (DLS). The results showed that HM-NPs were uniform in size, Pdl < 0.3, and the Zeta potential was negative, which met the physicochemical requirements of nanovaccines. Observation by transmission electron microscopy showed that HM-NPs were spherical capsule structures with complete membrane fusion.

[0026] 2. In vitro release experiment: The vaccine was placed in a buffer solution that simulates the in vivo environment and incubated at 37°C. The concentration of Mn²⁺ in the buffer solution was detected periodically. The results showed that Mn²⁺ could be released continuously for more than 72 hours, achieving a long-acting adjuvant effect.

[0027] 3. Immunological activity assay: The vaccine was co-cultured with dendritic cells, and HM-NPs were observed to bind efficiently to and be internalized by dendritic cells using laser confocal microscopy. Flow cytometry showed that the expression of maturation markers (CD80 and CD86) on the surface of dendritic cells was significantly increased. Further lymphocyte proliferation experiments showed that the vaccine could effectively induce the proliferation of effector memory T cells.

[0028] Example 3: In vivo efficacy of the vaccine A tumor-bearing mouse model was selected, and after surgical removal of the tumor, the vaccine prepared according to this invention was administered subcutaneously. The control group was administered saline and a simple tumor cell membrane vaccine. The results showed that the tumor recurrence rate in the vaccine-treated group was significantly lower than that in the control group, and the volume of the recurring tumor was significantly smaller. The proportion of effector memory T cells in the peripheral blood of the mice was significantly increased, and the number of tumor-infiltrating T cells in the tumor microenvironment was significantly increased, confirming that the vaccine can effectively induce an anti-tumor immune response and prevent tumor recurrence.

[0029] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0030] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A hybrid membrane nanoparticle vaccine for preventing tumor recurrence, characterized in that, It consists of bifunctional fused membrane nanoparticles loaded with cGAS-STING agonist colloidal manganese, wherein the bifunctional fused membrane nanoparticles are made by fusing autologous tumor cell membranes and Mycobacterium smegmatis membrane extracts at a mass ratio of 2:

1.

2. The method for preparing the hybrid membrane nanoparticle vaccine as described in claim 1, characterized in that, Includes the following steps: S1. Extraction of tumor cell membrane: Tumor cells were placed in EP tubes and repeatedly frozen and thawed in 37°C warm water and liquid nitrogen 10 times, with each freeze-thaw cycle lasting 5 minutes. After the freeze-thaw cycle was completed, the precipitate was collected by centrifugation to obtain the tumor cell membrane. S2. Extraction of Mycobacterium smegmatis biofilm: Mycobacterium smegmatis was ultrasonically disrupted, and the precipitate was collected by centrifugation to obtain Mycobacterium smegmatis biofilm. S3. Preparation of bifunctional fusion membrane nanoparticles: The tumor cell membrane obtained in step S1 and the Mycobacterium smegmatis membrane obtained in step S2 are mixed at a mass ratio of 2:1 and repeatedly extruded more than 20 times through a 100 nm extruder to obtain hybrid membrane nanoparticles. S4. Final preparation of the vaccine: The hybrid membrane nanoparticles obtained in step S3 are mixed with colloidal manganese and shaken. After loading, the hybrid membrane nanoparticle vaccine for preventing tumor recurrence is obtained.

3. The hybrid membrane nanoparticle vaccine as described in claim 1, characterized in that, The vaccine is used to prevent recurrence and metastasis in cancer patients after surgery, and works by inducing a tumor-specific immune response.

4. The preparation method according to claim 2, characterized in that, The centrifugation operation in step S1 is used to separate and remove cytoplasmic impurities, and the centrifugation operation in step S2 is used to separate and obtain the broken bacterial film precipitate.

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