A liposome-based nanodecoy, an immune cell robot containing the nanodecoy, and its applications.

Abstract

This invention discloses a liposome nanodecoy, an immune cell robot containing the same, and its applications. The liposome nanodecoy comprises a liposome and a ferromagnetic material, wherein the ferromagnetic material is encapsulated within the liposome, and the outer surface of the liposome is modified with immune cell phagocytic signaling molecules and antibodies targeting immune cells. This invention designs a liposome nanodecoy with a ferromagnetic material core and an outer liposome membrane modified with antibodies targeting immune cells (such as macrophages) and signaling molecules inducing specific phagocytosis. This allows it to target immune cells and induce specific phagocytosis, thereby assembling with immune cells to form a cellular microrobot, particularly capable of in-situ self-assembly in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a liposome nanodecoy, an immune cell robot containing the liposome, and their applications. Background Technology

[0002] Immunotherapy has become one of the most promising approaches to cancer treatment. It inhibits tumor growth and metastasis by activating the host's immune function, exhibiting both immune specificity and immune memory. In particular, living cells can dynamically perform complex biological functions in ways that conventional drugs cannot. However, in solid tumors, the immunosuppressive environment and tumor stromal fibrosis limit the infiltration of immune cells. Especially in the tumor microenvironment, different activation states of macrophages can lead to tumor development in different directions. M1 macrophages secrete cytokines such as TNF-α, IL-1β, and IL-12 to participate in inflammatory responses and recruit Th1 cells to inflammatory sites by secreting chemokines CXCL9 and CXCL10. Simultaneously, M1 macrophages can upregulate genes involved in antigen processing and presentation, as well as co-stimulatory molecules, to enhance T cell responses. Tumor-associated macrophages in the tumor microenvironment are often induced to become M2 macrophages. These macrophages suppress endogenous anti-tumor immunity and promote tumor proliferation and metastasis by secreting reactive oxygen species, upregulating immunosuppressive surface proteins, producing cytokines that inhibit T cell function, and secreting chemokines that recruit Treg cells.

[0003] In recent years, relying on the tumor-suppressing function of macrophages, researchers have modified macrophages extracted from mice and reintroduced them into the body. For example, they have used IFN-γ to "educate" macrophages in vitro to induce the M1 phenotype, thereby giving them inflammation-targeting functions and enabling them to actively migrate to tumor tissues. They have also used genetic engineering methods such as viral transfection to create CAR-macrophages, further enhancing the macrophages' targeting and killing capabilities against tumors. The unique function of macrophages in regulating immune responses makes them a promising candidate for cancer treatment. Therefore, macrophage-based cancer therapy, simply put, hinges on how to reduce anti-inflammatory macrophages and increase pro-inflammatory macrophages.

[0004] In existing technologies, the extraction, drug loading, and reinfusion of macrophages from vivo requires steps such as in vitro expansion, drug loading, and cell sorting. This complex process leads to long production cycles and high costs. Furthermore, while immunotherapy has shown promising results in hematological malignancies, it faces challenges in solid tumors, including harsh physical barriers and an immunosuppressive microenvironment. This results in only a subset of patients with solid tumors responding to this therapy, leading to poor clinical outcomes and prognoses. With the development of cellular microrobot technology, these robots, due to their mobility, can actively penetrate physiological barriers and avoid obstacles. Simultaneously, various non-contact manipulation techniques for cellular microrobots have been proposed, such as those using light, magnetic, or acoustic actuation. These non-contact manipulations offer significant promise for targeted delivery of cell therapies. Combining the targeting capabilities of cellular microrobots with local biological effects could open up a new therapeutic technology, thereby increasing the accumulation of immune cells at tumor sites and reducing toxic side effects. For example, CN117018210A discloses a swimming cell robot and its preparation method. Step 1: Prepare drug-loaded nanogel particles disguised as E. coli outer membrane using a desolvation method combined with co-extrusion method; Step 2: Modify the drug-loaded nanogel particles from Step 1 with two enzymes to obtain drug-loaded dual-enzyme-driven nanorobots; Step 3: Co-culture neutrophils with the drug-loaded dual-enzyme-driven nanorobots from Step 2 to induce neutrophils to phagocytose the nanoparticles, thereby preparing swimming cell robots; wherein, the two enzymes are glucose oxidase and catalase.

[0005] However, current reports on cell robots are very limited. How to use in-situ engineering technology to construct cell microrobots and target tumors, relieve the immunosuppressive microenvironment, and inhibit tumor growth remains one of the urgent problems to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a liposome nanodecoy, an immune cell robot containing the nanodecoy, and its applications. The liposome nanodecoy is designed to effectively target immune cells and is easily phagocytosed. It can be used to efficiently prepare immune cell robots, especially in vivo in situ self-assembled immune cell robots.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a liposome nanodecoy, the liposome nanodecoy comprising: a liposome and a ferromagnetic material, wherein the ferromagnetic material is encapsulated within the liposome, and the outer surface of the liposome is modified with immune cell phagocytic signaling molecules and antibodies targeting immune cells.

[0009] In this invention, a liposome nanodecoy is designed. The core of the nanodecoy is a ferromagnetic material, and the outer liposome membrane is modified with antibodies that target immune cells (such as macrophages) and signaling molecules that induce specific phagocytosis. It can target immune cells and induce specific phagocytosis, thereby assembling with immune cells to form a cellular microrobot, especially capable of in situ self-assembly in vivo.

[0010] It is understood that liposomes used for loading and delivery in the art are applicable to this invention.

[0011] Preferably, the raw materials for preparing the liposomes may include phosphatidylserine, 1,2-distearate-sn-glycerol-3-phosphate choline, cholesterol, and DSPE-PEG. 3400 -NHS; preferably, the mass ratio is (5-15):(50-60):30:5.

[0012] Preferably, the ferromagnetic material comprises magnetic iron oxide nanoparticles.

[0013] Preferably, the magnetic iron oxide nanoparticles include any one or a combination of at least two of various strongly magnetic iron oxides, such as oleic acid-modified iron oxide, polyethyleneimine-modified iron oxide, aminated iron oxide, carboxylated iron oxide, or hydroxylated iron oxide.

[0014] Preferably, the immune cell phagocytic signaling molecule includes any one or a combination of at least two of phosphatidylserine, peptidoglycan, lipopolysaccharide, mannose, or β-glucan.

[0015] Preferably, the target immune cell antibody includes any one or a combination of at least two of the following antibodies that recognize specific targets on the surface of immune cells: Anti-CD11b antibody, Anti-CD68 antibody, Anti-CD14 antibody, Anti-F4 / 80 antibody, Anti-CD16 antibody, Anti-CD18 antibody, Anti-CD32 antibody, or Anti-CD11c antibody.

[0016] In this invention, the technology can be designed to target various immune cells, such as macrophages. The liposome nanodecoy targets macrophages and induces specific phagocytosis, thereby assembling with macrophages to form cellular microrobots. Macrophages are stimulated by iron ions to transform into M1-type macrophages. Finally, under the influence of a magnetic field, macrophages precisely target tumors, promoting macrophage infiltration in tumor tissue, increasing the M1 / M2 ratio in the tumor, and activating CD8. + T cells inhibit Treg cells, thereby relieving tumor immunosuppression and enhancing the efficacy of tumor immunotherapy.

[0017] In a second aspect, the present invention provides a method for preparing the liposome nanodecoy described in the first aspect, the method comprising:

[0018] The raw materials for liposome preparation, immune cell phagocytic signaling molecules, ferromagnetic materials and solvents are mixed and ultrasonically treated. The solvent is then removed to obtain liposome-encapsulated particles. The liposome-encapsulated particles are then mixed with targeted immune cell antibodies to obtain the liposome nanodecoys.

[0019] Preferably, the preparation method of the liposome nanodecoy specifically includes the following steps:

[0020] (1) Preparation of nano-decoys: The raw materials for liposome preparation, immune cell phagocytic signaling molecules, ferromagnetic materials and solvents are mixed and thoroughly mixed by ultrasound. Then the organic solvent is removed so that the liposomes encapsulate the ferromagnetic materials. Ultrasound is then used for hydration and dispersion to obtain liposome-encapsulated particles.

[0021] (2) Purify the nano-decoy by using a magnetic bead separator to adsorb the liposome-encapsulated particles, remove impurities, and repeat the operation to obtain purified particles.

[0022] (3) The purified particles were co-incubated with the targeted immune cell antibody to obtain the liposome nanodecoy.

[0023] Thirdly, the present invention provides the application of the liposome nanodecoy described in the first aspect in the preparation of immune cell robots.

[0024] Fourthly, the present invention provides a method for preparing an immune cell robot, the method comprising:

[0025] The liposome nanodecoy described in the first aspect is mixed with immune cells, and the immune cells are induced to phagocytose the liposome nanodecoy to obtain the immune cell robot; the immune cells include any one or a combination of at least two of macrophages, neutrophils or dendritic cells.

[0026] Fifthly, the present invention provides a method for preparing an in vivo in situ self-assembled immune cell robot, the method comprising:

[0027] The liposome nanodecoy described in the first aspect is introduced into an animal to induce immune cells to phagocytose the liposome nanodecoy, thereby obtaining the in vivo in situ self-assembled immune cell robot.

[0028] This invention develops a method for constructing in vivo in situ self-assembled immune cell robots. In vivo, liposome nanodecoys can target immune cells and promote endocytosis, and self-assemble with immune cells in situ to form cell microrobots.

[0029] Preferably, the animal includes a mouse.

[0030] Preferably, the immune cells include immune cells in the blood or tumor.

[0031] Preferably, the immune cells include any one or a combination of at least two of macrophages, neutrophils, or dendritic cells.

[0032] In a sixth aspect, the present invention provides an immune cell robot, the immune cell robot comprising the liposome nanodecoy immune cells described in the first aspect;

[0033] The immune cells include any one or a combination of at least two of macrophages, neutrophils, or dendritic cells.

[0034] In a seventh aspect, the present invention provides an immune cell robot system, the immune cell robot system comprising the immune cell robot described in the sixth aspect and a control unit; the control unit comprising a magnetic field device.

[0035] Eighthly, the present invention provides a method for manipulating an immune cell robot, the method comprising:

[0036] The immune cell robot described in the sixth aspect is manipulated using a magnetic field.

[0037] In this invention, magnetic iron oxide can be used to drive magnetic properties, enabling non-contact remote control of immune cell robots to target tumor lesions, break through the physical barrier of the tumor, and stimulate macrophages to produce anti-cancer properties by high-valence iron ions, thereby relieving the tumor immunosuppressive environment and enhancing the tumor treatment effect.

[0038] In a specific embodiment of the present invention, the method for manipulating the immune cell robot includes the following steps:

[0039] (1) Liposome nanodecoys target macrophages and complete in vivo self-assembly. Liposome nanodecoys are injected into mouse blood via tail vein injection. The nanodecoys target macrophages and combine with macrophages to assemble, so that macrophages carry iron oxide to form cell micro-nano robots. (2) Cell micro-nano robots are manipulated by magnetic field. Magnets are fixed above mouse tumor tissue to perform magnetic field manipulation, so that cell micro-nano robots target tumor lesions.

[0040] In a ninth aspect, the present invention provides the use of the liposome nanodecoy described in the first aspect, the immune cell robot described in the seventh aspect, or the immune cell robot system described in the eighth aspect in the preparation of products targeting and / or treating tumors.

[0041] Preferably, the tumor includes any one or a combination of at least two of the following: lung cancer, kidney cancer, breast cancer, glioma, head and neck cancer, melanoma, or colorectal cancer.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention designs liposome-based nanodecoys that target immune cells (such as macrophages) in vivo via antibodies targeting immune cells (such as CD11b antibodies) and induce phagocytosis by immune cells using phagocytic signaling molecules. The synergistic effect of these two methods effectively enhances the recognition, capture, and assembly of the nanodecoys by immune cells in vivo. Furthermore, utilizing the magnetization properties and mobility of nanoscale magnetic iron oxide particles in a magnetic field, these particles are loaded into immune cells as a cellular propulsion device, thereby achieving non-contact manipulation of immune cells in vivo. This effectively improves cell targeting and accumulation at tumor sites, breaking down the physical barriers of tumors. Specifically, through stimulation by intracellular high-valence iron ions, macrophages transform into the M1 phenotype, thereby activating CD8+. + T cells suppress Treg cells, while high-valent iron ions generate ROS through the Fenton reaction, inducing tumor cell apoptosis. Attached Figure Description

[0044] Figure 1 A schematic diagram illustrating the principle of nanodecoy targeting macrophages in vivo and inducing phagocytosis, as well as magnetic field manipulation.

[0045] Figure 2A The particle size distribution of AbPs@NPs is shown in the figure.

[0046] Figure 2B The image shows the scanning electron microscope (SEM) results of AbPs@NPs.

[0047] Figure 3A Image from an immunofluorescence laser confocal microscope, scale bar is 10 μm;

[0048] Figure 3B Graph showing the results of in vivo flow cytometry analysis of nanoparticles phagocytosed by macrophages;

[0049] Figure 4 The results of tumor size after treatment in each group of mice are shown in the figure.

[0050] Figure 5 Immunofluorescence staining images of tumor sections from mice in each group after treatment, with a scale bar of 20 μm. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] Example 1

[0054] Synthesis of nanodecoys that target macrophages to induce specific phagocytosis.

[0055] Synthesis of nano-decoys: The specific preparation method is as follows: Phosphatidylserine, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, and DSPE-PEG are used. 3400 -NHS was dissolved in a certain amount of chloroform at a mass ratio of 15:60:30:5, and then 0.5 mg of oleic acid-modified iron(III) oxide (0.2–0.5 mg can be used) was added. The mixture was then placed in a round-bottom flask and sonicated in a water bath for 5 min to ensure complete dissolution and mixing. The round-bottom flask was then connected to a rotary evaporator with its bottom in contact with a water bath heater. The temperature was raised to 55°C, and evaporation was carried out at 100 rpm and 300 kPa for 15 min. After complete evaporation, sterile PBS was added to the round-bottom flask, and the mixture was sonicated in a 60°C water bath for 45 min. The resulting mixture was then placed on ice and sonicated for 13 min using a 5 mm ultrasonic probe to obtain uniformly dispersed nanoparticles. Add 125 μg of Anti-CD11b antibody (20–200 μg can be used) to the obtained solution and incubate at 37°C for 3 h to obtain functionalized nanodecoys. The nanodecoys modified with phosphatidylserine (Ps) and antibody (Ab) are named AbPs@NPs. The schematic diagram of the nanodecoys' in vivo targeting of macrophages and induction of phagocytosis, as well as their magnetic field manipulation, is shown below. Figure 1 As shown. Furthermore, the preparation methods for unmodified nanodecoys (NPs) differ mainly in that phosphatidylserine is not added during nanoparticle preparation, and they are not incubated with antibodies after preparation. For nanodecoys modified only with Ps or Ab, the main difference lies in the preparation methods: Ps@NPs are not incubated with antibodies after nanoparticle preparation, while Ab@NPs involve incubating nanoparticles with antibodies on top of NPs, serving as a control for subsequent analysis and testing.

[0056] To purify the nano-decoy, the modified nano-decoy was transferred to a 5 mL flow cytometer, then a magnetic bead separator was inserted. After standing for 20 min, the intermediate clear liquid was aspirated with a pipette to remove residual chemicals. The adsorbent was resuspended in sterile PBS. This process was repeated three times to obtain the purified nano-decoy.

[0057] The synthesized nanodecoys were characterized, and the nanoparticles were found to be uniform in size and approximately 90-110 nm in diameter using a Malvern particle size analyzer. Figure 2AIn the scanning electron microscope image ( Figure 2B The results also showed that the nanodecoys were uniform in size and most of them had a diameter between 90-110 nm, which was consistent with the results of the Malvern particle size analyzer.

[0058] Example 2

[0059] Macrophage microrobots are assembled in situ in vivo.

[0060] Each nanodecoy prepared in Example 1 after Dio staining was formulated to a concentration of 75 μg / mL. 100 μL of the nanoparticles was injected into the blood of mice via tail vein injection. After a reaction time of 4 hours, successfully assembled macrophage micro-nano robots carrying iron tetroxide were obtained.

[0061] Subsequently, whole blood from mice was obtained via orbital sampling into anticoagulant tubes. After mixing, five times the volume of whole blood erythrocyte lysis buffer was added, and the cells were lysed on ice for 15 min. The cells were then centrifuged at 350 g and 4°C for 5 min using a refrigerated centrifuge to obtain mouse peripheral blood mononuclear cells. The cells were then co-incubated with CD11b-APC dye at 37°C for 30 min. Excess dye was removed by washing with PBS. A portion of the stained cells was used for flow cytometry, while the remaining cells were placed in eight-well chambers coated with poly-L-lysine and incubated for 1 h. The supernatant was then discarded, and the cells were fixed with 4% paraformaldehyde for 15 min. The nuclei were stained with DAPI, and the uptake of nanodecoys by macrophages was analyzed using laser confocal microscopy.

[0062] like Figure 3A As shown, mice injected via tail vein with AbPs@NPs exhibited more macrophages with green fluorescence signals in their blood, and each macrophage engulfed a certain number of nanodecoys. The green fluorescence signal intensity of macrophages in the blood of mice in the Ab@NPs and Ps@NPs groups was significantly lower than that in the AbPs@NPs group, while the fluorescence signal was stronger compared to the NPs group. Flow cytometry analysis indicated that, compared to the NPs group, tail vein injection of AbPs@NPs nanodecoys resulted in a stronger fluorescence signal and a greater number of positive cells. Figure 3B The results are consistent with those of confocal imaging. These results demonstrate that the nanodecoy designed in this invention successfully self-assembles with macrophages in vivo. Furthermore, in the AbPs@NPs group, the macrophage assembly efficiency was significantly higher than that in the NPs group, Ps@NPs group, and Ab@NPs group. This is because AbPs@NPs not only has the ability to target macrophages but also the function of inducing macrophage phagocytosis, thereby synergistically promoting macrophage phagocytosis.

[0063] Example 3

[0064] In vivo manipulation and anti-tumor effects of macrophage microrobots.

[0065] 2×10⁻⁶ mice were subcutaneously injected into the right rib area of ​​the mouse. 6 4T1 cells were used to construct a 4T1 tumor-bearing mouse model after 12 days of modeling. The mice were randomly divided into 5 groups of 5 mice each. Every other day, 50 μg / mL of each nano-decoy was injected intravenously into the tail of each mouse. Four hours after each injection, a 500 mT magnet was fixed above the mouse tumor tissue, and the cell microrobot was manipulated for 6 hours. An equal volume of PBS was injected as a control. After 26 days of treatment, the tumors of each group of mice were collected for observation, and the enrichment of macrophages in the tumor tissue was observed by immunofluorescence staining.

[0066] Figure 4 The figures show the tumor sizes of surviving mice in each group. It is evident that the tumors of mice injected with AbPs@NPs nanodecoys generally had a volume of 250 mm². 3 Up to 350mm 3 The difference was much smaller than that of the PBS group, NPs group, Ps@NPs group and Ab@NPs group, proving that macrophage microrobots can effectively exert anti-tumor effects and inhibit tumor growth. Figure 5 Immunofluorescence staining sections of mouse tumor tissue are shown. Red fluorescence represents macrophages, green fluorescence represents nanodecoys, and the yellow fluorescence produced by the superposition of red and green fluorescence represents macrophage microrobots. As can be seen from the image, the macrophage microrobots successfully reached the tumor tissue, and the enrichment level of macrophage microrobots in the AbPs@NPs group was significantly higher than that in the PBS group, NPs group, Ps@NPs group, and Ab@NPs group, demonstrating that the macrophage microrobots based on magnetic field manipulation possess considerable motility.

[0067] This invention designs liposome-based nanodecoys that target immune cells (such as macrophages) in vivo via antibodies targeting immune cells (such as CD11b antibodies) and induce phagocytosis by immune cells using phagocytic signaling molecules. The synergistic effect of these two methods effectively enhances the recognition, capture, and assembly of the nanodecoys by immune cells in vivo. Furthermore, utilizing the magnetization properties and mobility of nanoscale magnetic iron oxide particles in a magnetic field, these particles are loaded into immune cells as a cellular propulsion device, thereby achieving non-contact manipulation of immune cells in vivo. This effectively improves cell targeting and accumulation at tumor sites, breaking down the physical barriers of tumors. Specifically, through stimulation by intracellular high-valence iron ions, macrophages transform into the M1 phenotype, thereby activating CD8+. + T cells suppress Treg cells, while high-valent iron ions generate ROS through the Fenton reaction, inducing tumor cell apoptosis.

[0068] 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 liposome nanodecoy, characterized in that, The liposome nanodecoy comprises: liposomes and ferromagnetic material, wherein the ferromagnetic material is encapsulated within the liposomes, and the outer surface of the liposomes is modified with immune cell phagocytic signaling molecules and antibodies targeting immune cells. The raw materials for preparing the liposomes include phosphatidylserine, 1,2-distearate-sn-glycero-3-phosphocholine, cholesterol, and DSPE-PEG. 3400 -NHS; The ferromagnetic material is oleic acid-modified iron(III) oxide; The immune cell phagocytic signaling molecule is phosphatidylserine; The targeted immune cell antibody is an Anti-CD11b antibody.

2. The method for preparing the liposome nanodecoy according to claim 1, characterized in that, The preparation method includes: The raw materials for liposome preparation, immune cell phagocytic signaling molecules, ferromagnetic materials and solvents are mixed and ultrasonically treated. The solvent is then removed to obtain liposome-encapsulated particles. The liposome-encapsulated particles are then mixed with targeted immune cell antibodies to obtain the liposome nanodecoys.

3. The application of the liposome nanodecoy as described in claim 1 in the preparation of immune cell robots.

4. A method for preparing an immune cell robot, characterized in that, The preparation method includes: The liposome nanodecoy as described in claim 1 is mixed with immune cells, and the immune cells are induced to phagocytose the liposome nanodecoy to obtain the immune cell robot. The immune cells include any one or a combination of at least two of macrophages, neutrophils, or dendritic cells.

5. An immune cell robot, characterized in that, The immune cell robot comprises immune cells containing the liposome nanodecoy as described in claim 1; The immune cells include any one or a combination of at least two of macrophages, neutrophils, or dendritic cells.

6. An immune cell robotic system, characterized in that, The immune cell robot system includes the immune cell robot and control unit as described in claim 5; The control unit includes a magnetic field device.

7. A method for manipulating immune cell robots, characterized in that, The method includes: The immune cell robot of claim 5 is manipulated using a magnetic field.

8. The use of the liposome nanodecoy of claim 1, the immune cell robot of claim 5, or the immune cell robot system of claim 6 in the preparation of products targeting and / or treating tumors.

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