Nano-engineering T cell membrane coated nano-particles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a nano-engineering T cell membrane coated nano-particle as well as a preparation method and application of the nano-engineering T cell membrane coated nano-particle. Nanoparticles of a specific structure are constructed based on a phospholipid bilayer bionic nanometer platform through a membrane fusion technology, immune clearance caused by phagocytosis of the nanoparticles by macrophages can be avoided through disguise of a T cell membrane, and the blood circulation time is prolonged; the functions of T cells are recovered through specific blocking of immune checkpoint molecules loaded on the immune checkpoint molecules on corresponding immune checkpoint ligands on tumor cells; under ultrasonic radiation, the nano-particles are gradually decomposed and release the sound-sensitive agent to generate a large amount of active oxygen, so that immunogenic death of tumor cells is promoted, more cytotoxic T lymphocytes are recruited to infiltrate into tumor parts, and the recovery of T cell mediated immune response function is facilitated. The synergistic effect of the sound-sensitive agent and the immune checkpoint protein can effectively inhibit tumor growth, induce a long-term immune memory effect and prevent tumor metastasis and recurrence.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nano-engineered T cell membrane-coated nanoparticle and a preparation method thereof, as well as the application of the nanoparticle in the preparation of anti-tumor drugs. Background Art

[0002] Immunotherapy has become a powerful cancer treatment. However, most patients initially respond well to anti-PD-1 / PD-L1 monotherapy but subsequently develop resistance. A potential driver of acquired resistance is the upregulation of alternative immune checkpoints, such as T-cell immunoglobulin and mucin domain-containing (TIM-3), lymphocyte activation gene 3 (LAG-3), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Studies have shown that combined anti-PD-1 and anti-CTLA-4 immunotherapy can synergistically improve T lymphocyte effector function, overcome immune resistance to anti-PD-1 monotherapy, and demonstrate promising efficacy in clinical trials. However, the continuous discovery of new immune checkpoints and the clinical toxicities associated with dual / triple therapy present significant challenges for immunotherapy of anti-PD-1-resistant tumors. Furthermore, the extent of intratumoral T cell infiltration is a crucial factor influencing tumor response and prognosis. Low-efficiency tumor-associated antigens released by dying tumor cells can hinder intratumoral T cell infiltration and induce T cell exhaustion, significantly impairing T cell activation and diminishing their ability to kill tumor cells.

[0003] In clinical practice, immunotherapy can be further combined with other anti-cancer therapies (including chemotherapy, radiotherapy, molecular targeted therapy, photothermal therapy, photodynamic therapy, etc.) to synergistically improve the anti-tumor effect. Among them, sonodynamic therapy (SDT) is an emerging non-invasive tumor treatment method with good tissue penetration and spatiotemporal controllability. It has been approved by the US FDA for the treatment of recurrent glioblastoma and has a very broad application prospect. The reactive oxygen species (ROS) generated by the sonosensitizer in this therapy under ultrasound (US) excitation can effectively induce immunogenic cell death (ICD) in tumors and enhance intratumoral T cell infiltration. For example, Chinese patent CN114588268B (Tsinghua University) discloses a pharmaceutical composition comprising a liposome-complex and an anti-PD-1 / PD-L1 antibody, wherein the liposome-complex comprises a liposome, a sonosensitizer (indocyanine green) and a drug molecule (doxorubicin), the sonosensitizer is located in the aqueous phase within the liposome or is connected to the outer surface of the phospholipid bilayer of the liposome, and the drug molecule is located in the aqueous phase within the liposome. The sonosensitizer in this pharmaceutical composition can generate reactive oxygen species under ultrasonic conditions, promoting the drug molecules to reach the drug target in the tumor, and synergistically with the drug molecules to enhance the killing effect on tumor cells. At the same time, the composition can promote the delivery of endogenous tumor antigens and tumor DNA of dead tumor cells to DCs. Reactive oxygen species can oxidize tumor DNA, increasing its stability, thereby promoting the activation of the STING pathway in DCs and triggering anti-tumor T cell responses. However, the anti-tumor efficacy of sonodynamic therapy is still limited by the activity of T cells in the tumor microenvironment. Therefore, the development of a treatment strategy combining multiple immune checkpoint blockade with sonodynamic therapy will have potential clinical application prospects. Summary of the Invention

[0004] The main technical problem solved by the present invention is to provide a nano-engineered T cell membrane-coated nanoparticle, which specifically blocks the corresponding IC ligands on tumor cells through the immune checkpoint (IC) molecules loaded thereon, thereby restoring T cell function. At the same time, it induces the immunogenic death of tumor cells through ultrasound irradiation, releases a large number of damage-associated molecular patterns (DAMPs) to activate T cells, thereby enhancing the anti-tumor immune response.

[0005] Secondly, the present invention provides a method for preparing nano-engineered T cell membrane-coated nanoparticles.

[0006] Again, the present invention provides an application of nanoparticles in the preparation of anti-tumor drugs.

[0007] Finally, the present invention provides an anti-tumor drug.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] Nano-engineered T cell membrane-coated nanoparticles are obtained by co-incubating T cell membranes and lipid nanocores; the raw materials for preparing the lipid nanocores include liposomes and sonosensitizers.

[0010] The shell of the nanoparticles provided by the present invention is formed by the fusion of T cell membranes and liposomes, and the sonosensitizer is loaded in the lipid phase of the shell. Because multiple immune checkpoint proteins are loaded on the T cell membrane, the nanoparticles release T cell membrane fragments in the acidic tumor microenvironment, allowing the simultaneous blocking of multiple immune checkpoint ligands on the surface of tumor cells, thereby restoring the suppressed activity of exhausted T cells in the tumor microenvironment. Under ultrasound stimulation, the sonosensitizer loaded in the lipid phase induces the immunogenic death of tumor cells, thereby promoting the maturation of dendritic cells and increasing T cell infiltration, thereby enhancing the anti-tumor immune response.

[0011] As a preferred embodiment of the present invention, the mass ratio of the T cell membrane to the lipid nanocore is (1-10):(10-100), preferably 1:10.

[0012] As a preferred embodiment of the present invention, the mass ratio of the liposome to the sonosensitizer is (3-15):(1-5), preferably 3:1.

[0013] As a preferred embodiment of the present invention, the T cell membrane is prepared by differential centrifugation.

[0014] As a preferred embodiment of the present invention, the raw materials for preparing the liposome include but are not limited to one or more of phospholipids, cholesterol, DSPE-PEOz (polyethylene glycol-derivatized phospholipids, the number average molecular weight of PEOz is 1000-4000), etc.

[0015] Specifically, the liposomes are prepared using raw materials including phospholipids, cholesterol, and DSPE-PEOz in a molar ratio of (1-3):(0.5-1.5):(0.2-0.6), preferably 2:1:0.4. The phospholipids are soybean lecithin. The number average molecular weight of PEOz in the DSPE-PEOz is 1000-4000. The resulting liposomes are acid-sensitive liposomes.

[0016] As a preferred embodiment of the present invention, the sonosensitizer includes but is not limited to hematoporphyrin monomethyl ether (HMME).

[0017] As a preferred embodiment of the present invention, the particle size of the lipid nanocore is 100-130 nm.

[0018] As a preferred embodiment of the present invention, the particle size of the nanoparticles is 120-150 nm.

[0019] The method for preparing nano-engineered T cell membrane-coated nanoparticles comprises the following steps:

[0020] mixing a solution dissolving liposomes and a solution dissolving a sonosensitizer, and removing the solvent to obtain a lipid film;

[0021] The lipid film is hydrated to obtain a lipid nanocore;

[0022] The lipid nanocores were co-incubated with T cell membranes to obtain nanoengineered T cell membrane-coated nanoparticles.

[0023] As a preferred embodiment of the present invention, the solvent is removed by rotary evaporation at 45-60° C. for 30-180 min.

[0024] As a preferred embodiment of the present invention, the hydration is carried out at room temperature, 20-25°C.

[0025] As a preferred embodiment of the present invention, PBS buffer is added to the lipid film for hydration.

[0026] As a preferred embodiment of the present invention, the T cell membrane is prepared by differential centrifugation.

[0027] Specifically, the method for preparing the T cell membrane comprises the following steps:

[0028] The cultured T cells were centrifuged at 100-500 × g for 5-20 min;

[0029] The T cell pellet was resuspended, lysed, sonicated, and centrifuged at 500-1000 × g for 10-40 min;

[0030] The supernatant was centrifuged at 1000-10000 × g for 10-40 min;

[0031] The supernatant was centrifuged at 50,000-100,000 × g for 30-60 min to collect T cell membranes.

[0032] In a preferred embodiment of the present invention, the co-incubation is performed at 30-45°C for 5-20 minutes. Preferably, the co-incubation is performed with the assistance of ultrasound, with an ultrasound power of 100-150W, a duty cycle of 30%-70%, and a duration of 3-10 minutes. More preferably, the ultrasound power is 120W, a duty cycle of 50%, and a duration of 5 minutes.

[0033] Applications of nanoengineered T cell membrane-coated nanoparticles include, but are not limited to, one or more of the following:

[0034] (1) Application in the preparation of anti-tumor drugs;

[0035] (2) Application in the preparation of preparations that block immune checkpoint ligands on the surface of tumor cells;

[0036] (3) Application in the preparation of drugs for enhancing T cell-mediated anti-tumor immune responses;

[0037] (4) Application in the preparation of drugs for inhibiting tumor lung metastasis;

[0038] (5) Application in the preparation of drugs for inhibiting tumor recurrence.

[0039] Specifically, when the tumor is an anti-PD-1 resistant tumor, the nanoparticles in the drug can effectively inhibit the growth of the anti-PD-1 resistant tumor and induce a long-term immune memory effect.

[0040] Specifically, the preparation that blocks the immune checkpoint ligand on the surface of tumor cells can be a simple experimental preparation used to explore the physiological metabolic process of tumor cells. It is not used to eliminate the cause or lesion, but only serves as a research preparation for non-therapeutic purposes.

[0041] Specifically, the inhibition of tumor lung metastasis includes but is not limited to promoting the normalization of alveolar structure and reducing lung metastatic lesions.

[0042] An anti-tumor drug comprising nano-engineered T cell membrane-coated nanoparticles.

[0043] As a preferred embodiment of the present invention, the anti-tumor drug further comprises other anti-tumor pharmaceutical ingredients, including but not limited to anti-PD-1 / PD-L1 antibodies.

[0044] As a preferred embodiment of the present invention, the anti-tumor drug further includes pharmaceutically acceptable excipients, including but not limited to one or more of carriers, diluents, excipients, fillers, adhesives, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents, buffers, etc.

[0045] Specifically, the drug can be administered alone or in a suitable dosage form prepared with excipients, including tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0046] As a preferred embodiment of the present invention, the anti-tumor drug is a single compound preparation, or a combination of separate preparations of different pharmaceutical ingredients, which can be administered simultaneously, cross-administered, or administered sequentially.

[0047] Beneficial effects of the present invention:

[0048] The present invention is based on a phospholipid bilayer biomimetic nano-platform and constructs a nano-engineered T cell membrane-coated nanoparticle with a specific structure through membrane fusion technology. The camouflage of the T cell membrane can prevent the nanoparticles from being phagocytosed by macrophages and immune clearance, thereby increasing blood circulation time. At the same time, it selectively targets the tumor site by virtue of the acid-sensitive property and the interaction between the immune checkpoint protein on the T cell membrane and the specific immune checkpoint ligand on the surface of the tumor cell. The immune checkpoint molecule loaded on the nanocarrier specifically blocks the corresponding immune checkpoint ligand on the tumor cell to restore T cell function. Under ultrasonic irradiation, the nanoparticles gradually decompose and release sonosensitizer (HMME), generating a large amount of reactive oxygen species, thereby promoting the immunogenic death of tumor cells, recruiting more cytotoxic T lymphocytes to infiltrate the tumor site, and helping to restore T cell-mediated immune response function. The synergistic effect of sonosensitizer and immune checkpoint protein can effectively inhibit tumor growth (especially anti-PD-1 resistant tumors), induce long-term immune memory effects, and prevent tumor metastasis and recurrence.

[0049] The nanoparticle preparation method provided by the present invention first uses a thin film dispersion method to synthesize a lipid nanocore as a nanomodulator, then uses differential centrifugation to prepare a T cell membrane. Finally, the T cell membrane and the lipid nanocore are co-incubated to obtain a nanoscale drug delivery system. This preparation method can be applied to industrial production. The resulting nanoparticles have excellent anti-tumor activity. They can restore T cell function through immune checkpoint blockade and sonosensitizer-induced immunogenic death of tumor cells, promote T cell activation and infiltration, and thus enhance anti-tumor immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the process flow for preparing nano-engineered T cell membrane-coated nanoparticles in the examples.

[0051] Figure 2 Figure 2 is the particle size distribution diagram of different nanoformulations in the experimental examples.

[0052] Figure 3 This is the Zeta potential diagram of different nanoformulations in the experimental example.

[0053] Figure 4 This is the particle size stability result of nanoparticles NTNDs incubated in PBS for 7 days in the experimental example.

[0054] Figure 5 This is the transmission electron microscope image of the nanoparticles NTNDs in the experimental example.

[0055] Figure 6 This is the full wavelength scanning image of the nanoparticles NTNDs, sonosensitizer HMME, and nanocarrier Tm-Lip in the experimental example.

[0056] Figure 7This is a graph showing the results of measuring the cell viability of 4T1 cells after 24 hours of treatment with different drugs using the CCK8 method in the experimental example (data are expressed as mean ± standard deviation, n = 3).

[0057] Figure 8 This is the ROS level result of 4T1 cells in the experimental example.

[0058] Figure 9 Figure 3 shows the pathological changes in lung tissue and the counts of lung metastases in each group of mice in the experimental example.

[0059] Figure 10 This is an analysis diagram of the effect of inhibiting the growth of recurrent tumors in the experimental example.

[0060] To more clearly illustrate the technical solutions of the embodiments of the present invention, the above briefly introduces the drawings obtained in the experimental examples. It should be understood that the above drawings only illustrate certain experimental examples of the present invention and should not be construed as limiting the scope of protection of the claims. Those skilled in the art can also derive other relevant drawings based on these drawings without inventive effort. DETAILED DESCRIPTION

[0061] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other embodiments obtained by those of ordinary skill in the art without making creative work, such as modifications, deformations, or simple replacements, should fall within the scope of protection of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples and experimental examples are all conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art and commercially available to the public unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS buffer is phosphate buffered saline.

[0063] Example

[0064] This embodiment provides a nano-engineered T cell membrane-coated nanoparticle, which is obtained by co-incubation of T cell membrane and lipid nanocore, and the mass ratio of T cell membrane to lipid nanocore is 1:10; the raw materials for preparing the lipid nanocore include liposomes and sonosensitizer HMME, and the mass ratio of liposomes to HMME is 3:1. The raw materials for preparing the liposomes include soybean lecithin, cholesterol and DSPE-PEOz (the number average molecular weight of PEOz is 2000), and the molar ratio of the three is 2:1:0.4.

[0065] The shell of the nano-engineered T cell membrane-coated nanoparticles provided in this embodiment is formed by the fusion of T cell membrane and liposome, and the sonosensitizer HMME is loaded in the lipid phase of the shell. The preparation method (schematic diagram is shown in Figure 1 ) comprises the following steps:

[0066] (1) Preparation of T cell membrane (Tm):

[0067] (1.1) Centrifuge the cultured T cells at 100-500 g for 5-20 min and wash three times with PBS.

[0068] (1.2) The T cell pellet was added to Tris-HCl buffer containing 1 mM CaCl2 and 1× EDTA-free protease inhibitor and lysed at 4°C in the dark for 60 min. The T cell homogenate was then sonicated on ice (120 W, 50% duty cycle, 5 min) to completely disrupt the cells. The cells were then centrifuged at 500 × g to remove intact cells, and the supernatant was collected.

[0069] (1.3) Centrifuge the collected supernatant at 10,000 × g for 10 min at 4°C.

[0070] (1.4) The supernatant was centrifuged at 100,000 × g for 60 min at 4°C to collect the T cell membrane precipitate. After washing with PBS buffer, the cell membrane precipitate was sonicated in an ice bath (120 W, 50% duty cycle, 5 min). Finally, the obtained T cell membranes were aliquoted and stored at -80°C until use.

[0071] (2) Preparation of nanoparticles NTNDs:

[0072] (2.1) Preparation of HMME@Lip:

[0073] (2.1.1) Dissolve 1 mg of HMME in 2 mL of methanol to obtain solution A. Dissolve soybean lecithin, cholesterol, and DSPE-PEOz in dichloromethane at a molar ratio of 2:1:0.4 to obtain solution B. Then, mix solution A and solution B in a round-bottom flask and perform rotary evaporation (50°C for 60 min). After evaporation of the organic solvent in the rotary evaporator, a lipid film forms at the bottom of the flask.

[0074] (2.1.2) Add 1 mL of PBS buffer to the lipid film and hydrate at room temperature to obtain the lipid nanocore HMME@Lip.

[0075] (2.2) Preparation of NTNDs:

[0076] The lipid nanocore HMME@Lip was co-incubated with T cell membrane at 37°C for 10 min and then ultrasonicated (120 W, 50% duty cycle, 5 min) to obtain uniform nanoparticles NTNDs.

[0077] This embodiment also provides an application of nano-engineered T cell membrane-coated nanoparticles, including but not limited to one or more of the following aspects:

[0078] (1) Application in the preparation of anti-tumor drugs;

[0079] (2) Application in the preparation of preparations that block immune checkpoint ligands on the surface of tumor cells;

[0080] (3) Application in the preparation of drugs for enhancing T cell-mediated anti-tumor immune responses;

[0081] (4) Application in the preparation of drugs for inhibiting tumor lung metastasis;

[0082] (5) Application in the preparation of drugs for inhibiting tumor recurrence.

[0083] This embodiment also provides an anti-tumor drug, comprising a pharmaceutically effective amount of nano-engineered T cell membrane-coated nanoparticles and an appropriate amount of pharmaceutically acceptable excipients.

[0084] In other embodiments of the present invention, the raw materials and preparation conditions for the nanoparticles can be arbitrarily selected within a given range. The sonosensitizer HMME exhibits superior anti-tumor efficacy, effectively inhibiting tumor metastasis and recurrence. The dosage of each component in the nanoparticles can be arbitrarily adjusted within a given range without substantially affecting the physicochemical properties, biological functions, or therapeutic efficacy of the nanoparticles.

[0085] Experimental example

[0086] 1. Particle size and Zeta potential analysis of nanoparticles NTNDs

[0087] Small amounts of liposomes Lip (blank liposomes without HMME and T cell membrane loading, prepared as described in the examples) (A), HMME@Lip (B), Tm-Lip (C), and NTNDs (D) prepared in each step of the examples were diluted with PBS. The particle size and polydispersity index (PDI) of the samples were measured using a particle size analyzer. Particle size was measured daily for seven days to assess the stability of the nanomaterials. The potentials of liposomes Lip, HMME@Lip, Tm-Lip, and NTNDs were then measured using a potentiostat using the same method. The data were analyzed using GraphPad Prism.

[0088] The particle size test results of each sample group are as follows Figure 2 As shown, by optimizing the addition amounts of soybean lecithin, cholesterol, and DSPE-PEOz, liposomes with a particle size of (108.8±1.57) nm were synthesized. With further modification, the hydrated particle size gradually increased, and the hydrated particle size of the nanoparticles NTNDs was (124.8±2.84) nm.

[0089] The Zeta potential test results of each sample group are as follows Figure 3 As shown, the potential of the liposome Lip was (-25.3 ± 0.35) mV. With the addition of HMME, the potential dropped to (-31.1 ± 1.26) mV. The positive and negative values ​​represent the charge of the particles. Since T cell membranes are negatively charged, the potential of the nanocomplex dropped to (-34.5 ± 1.16) mV after incubation of the lipid nanocore with the T cell membrane, consistent with the expected results.

[0090] The particle size test results of nanoparticles NTNDs after 7 days are as follows Figure 4 As shown in the figure, the hydrodynamic diameter of the nanoparticles NTNDs remained basically unchanged in PBS for 7 days, indicating that the particle size of the nanoparticles was stable and uniform and had good biological stability.

[0091] 2. Morphology test of nanoparticles NTNDs

[0092] Take a small amount of NTNDs and dilute it with PBS to 0.2mg / mL. Take 20μL of the suspension and drop it on the carbon film copper mesh several times. After drying for 10 minutes, use filter paper to absorb the excess nanomaterials. Then use 20μL of 3% uranyl acetate to stain the copper mesh in the dark for 2 minutes. After drying, use transmission electron microscope to observe and take pictures to analyze the morphology of the nanomaterials. Figure 5 shown.

[0093] 3. UV full wavelength scanning of nanoparticles NTNDs

[0094] The successful loading of HMME onto the nanocarriers was determined using a UV spectrophotometer. Free HMME, nanocarriers Tm-Lip, and nanoparticles NTNDs were diluted and then scanned using a UV spectrophotometer within the wavelength range of 300-660 nm.

[0095] The results are as follows Figure 6 As shown, the nanoparticles NTNDs have obvious ultraviolet absorption at the maximum absorption wavelength of HMME 396nm, indicating the successful construction of the nanocomposite.

[0096] 4. Anti-tumor research of nanoparticle NTNDs at the cellular level

[0097] (1) CCK8 assay to evaluate cytotoxicity

[0098] First, 4T1 cells were seeded into 96-well plates and administered after cell attachment. Experimental groups were assigned different drug concentrations, while a control group was treated with complete medium supplemented with an equal volume of PBS buffer. 4T1 cells were incubated with HMME, HMME@Lip, and NTNDs at varying drug concentrations for 12 hours, followed by an additional 12 hours with or without ultrasound irradiation (US). The drug-containing medium was then discarded, and 100 μL of fresh medium was added to each well, followed by 10 μL of CCK8 solution. The cells were incubated in the dark for 2 hours in a 5% CO2 atmosphere at 37°C. The absorbance of each well was measured at 450 nm using a microplate reader, and the cell viability of each experimental group was calculated to assess the toxicity of the constructed nanomaterials against tumor cells. The experiment was repeated three times. The dosage of the corresponding drug was consistent across treatment groups; that is, the dosage of HMME in the NTNDs group was the same as that in the HMME@Lip and HMME groups. Subsequent experiments were similar and will not be repeated here.

[0099] The experimental results of 4T1 cell group are as follows Figure 7 As shown, the cell survival rate in the NTNDs nanoparticle-treated group was lower than that in the free HMME and HMME@Lip-treated groups, indicating that the nanoparticles selectively accumulate in tumor cells, enhancing their anti-tumor effects. Cell survival decreased in all groups treated with ultrasound compared to those without ultrasound, as the ultrasound response facilitates the production of reactive oxygen species by HMME, inducing immunogenic cell death. Furthermore, the cell survival rate in the NTNDs-treated group was lower than that in the HMME@Lip-treated group, likely due to increased internalization efficiency of the NTNDs by loading the T cell membrane.

[0100] (2) Detection of intracellular ROS levels by fluorescent dye 2,7-dichlorodihydrofluorescein (DCFH-DA) staining

[0101] 4T1 cells (1×10 5 Cells were seeded in 6-well plates (cells per well) and incubated with PBS, PBS (with US), free HMME (with US), HMME@Lip (with US), NTNDs, and NTNDs (with US). Six hours later, the groups requiring ultrasound treatment were irradiated. Four hours later, the culture medium was discarded, and the cells were washed with cold PBS. The cells were incubated with 5 μL of DCFH-DA (10 mM) probe for 40 minutes, and the fluorescence intensity of the cells was observed under a fluorescence microscope.

[0102] The results are as follows Figure 8As shown, ultrasound-treated 4T1 cells in the free HMME (+US) group exhibited a slight increase in green fluorescence, attributed to the generation of ROS by HMME sonodynamic therapy. 4T1 cells treated with HMME@Lip (+US) and NTNDs (+US) exhibited stronger green fluorescence than the free HMME (+US) group, likely due to the nanocarrier's ability to efficiently deliver drugs. The significant increase in green fluorescence in cells treated with NTNDs (+US) suggests that loading of the T cell membrane increases the efficiency of tumor cell internalization of the nanoparticles.

[0103] 5. Anti-tumor metastasis research of nanoparticles NTNDs at the animal level

[0104] (1) 4T1 cells (1×10 6 ) were resuspended in PBS and injected subcutaneously into the right hind limb of mice to construct a 4T1 subcutaneous tumor model.

[0105] (2) After 7 days, the mice were randomly divided into 6 groups, with an average tumor volume of 100 mm in each group. 3 (n=5). Subsequently, mice were injected with PBS (G1 group), PBS (+US) (G2 group), free HMME (+US) (G3 group), HMME@Lip (+US) (G4 group), NTNDs (G5 group) and NTNDs (+US) (G6 group) (HMME at 8 mg / kg) through the tail vein. Groups requiring ultrasound treatment were irradiated with ultrasound 24 hours after administration. After each group of mice received different treatments, 4T1 cells (3×10 cells per mouse) were injected into the tail vein. 5 The lung metastasis tumor model was established by injecting 4% of the mouse lung tissue into the mouse lungs. On the 15th day after injection, the lung tissues were collected by dissection. The metastatic tumor foci were counted after washing with PBS and fixed with 4% tissue fixative and then stained with H&E to evaluate the inhibitory effects of different treatments on lung metastasis.

[0106] The H&E slice images of the lungs and the lung metastasis counts of the mice in each group are shown in Figure 2. Figure 9 As shown, the G6 group can significantly inhibit the lung metastasis of tumors, which is manifested by the normalization of alveolar structure and the reduction of lung metastatic lesions, indicating that nanoparticles can maximally delay tumor metastasis under ultrasound irradiation.

[0107] 6. Anti-tumor recurrence study of nanoparticles NTNDs at the animal level

[0108] (1) 4T1 cells (1×10 6 ) were resuspended in PBS and injected subcutaneously into the right back of mice to establish a 4T1 subcutaneous tumor model.

[0109] (2) After 7 days, the mice were randomly divided into 2 groups, with an average tumor volume of 100 mm in each group.3 (n=5). A rechallenge experiment was conducted: a PBS group and a NTNDs (+US) group (8 mg / kg HMME). Groups requiring ultrasound treatment underwent ultrasound irradiation 24 hours after administration. On day 14, mice were anesthetized, and the tumors were surgically removed and sutured. On day 30, tumor cells were injected subcutaneously into the left dorsal region of the mice to establish a rechallenge tumor model.

[0110] (3) At the end of the experiment, the tumors were collected for optical imaging and weighing.

[0111] The tumor status of mice in each group is as follows Figure 10 As shown, compared with the PBS group, the recurrent tumors in the NTNDs (+US) group were significantly reduced, indicating that the nanoparticles prepared by the present invention have a significant inhibitory effect on the growth of recurrent tumors.

[0112] Although the technical solution of the present invention has been described in detail above using general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only intended to illustrate the technical solution and technical effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Simple variations, modifications, or improvements based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. Nano-engineered T cell membrane-coated nanoparticles, characterized by: The nanoparticles are obtained by co-incubating T cell membranes and lipid nanocores; the raw materials for preparing the lipid nanocores include liposomes and sonosensitizers.

2. The nanoparticle according to claim 1, characterized in that: The mass ratio of the T cell membrane to the lipid nanocore is (1-10): (10-100); and / or, the mass ratio of the liposome to the sonosensitizer is (3-15):(1-5); and / or, the T cell membrane is prepared by differential centrifugation; And / or, the raw materials for preparing the liposome include but are not limited to phospholipids, cholesterol, and DSPE-PEOz; And / or, the sonosensitizer includes but is not limited to hematoporphyrin monomethyl ether.

3. The nanoparticles according to claim 2, characterized in that: The raw materials for preparing the liposome include phospholipid, cholesterol and DSPE-PEOz, and the molar ratio of the three is (1-3): (0.5-1.5): (0.2-0.6); And / or, the number average molecular weight of PEOz in the DSPE-PEOz is 1000-4000.

4. The nanoparticle according to claim 1, characterized in that: The particle size of the lipid nanocore is 100-130 nm; And / or, the particle size of the nanoparticles is 120-150 nm.

5. The method for preparing nano-engineered T cell membrane-coated nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: mixing a solution dissolving liposomes and a solution dissolving a sonosensitizer, and removing the solvent to obtain a lipid film; The lipid film is hydrated to obtain a lipid nanocore; The lipid nanocores were co-incubated with T cell membranes to obtain nanoengineered T cell membrane-coated nanoparticles.

6. The preparation method according to claim 5, characterized in that: The solvent is removed by rotary evaporation at 45-60° C. for 30-180 min; and / or, adding PBS buffer to the lipid film for hydration; and / or, the hydration is performed at room temperature, 20-25° C.; And / or, the co-incubation is performed at 30-45° C. for 5-20 min.

7. The use of nano-engineered T cell membrane-coated nanoparticles according to any one of claims 1 to 4, characterized in that: Including but not limited to one or more of the following aspects: (1) Application in the preparation of anti-tumor drugs; (2) Application in the preparation of preparations that block immune checkpoint ligands on the surface of tumor cells; (3) Application in the preparation of drugs for enhancing T cell-mediated anti-tumor immune responses; (4) Application in the preparation of drugs for inhibiting tumor lung metastasis; (5) Application in the preparation of drugs for inhibiting tumor recurrence.

8. The use according to claim 7, characterized in that: The tumor is an anti-PD-1 resistant tumor; And / or, the preparation for blocking immune checkpoint ligands on the surface of tumor cells is an experimental preparation.

9. An antitumor drug, characterized in that: The anti-tumor drug comprises the nano-engineered T cell membrane-coated nanoparticles according to any one of claims 1 to 4.

10. The anti-tumor drug according to claim 9, characterized in that: The anti-tumor drug also includes other anti-tumor active ingredients, including but not limited to anti-PD-1 / PD-L1 antibodies; and / or, the anti-tumor drug further comprises pharmaceutically acceptable excipients, including but not limited to carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents, and buffers; And / or, the anti-tumor drug is a single compound preparation, or a combination of separate preparations of different medicinal ingredients.

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