Quick-frozen neutrophil preparation for resisting tumor metastasis as well as preparation method and application of quick-frozen neutrophil preparation

Abstract

The invention discloses a quick-frozen neutrophil preparation for resisting tumor metastasis as well as a preparation method and application of the quick-frozen neutrophil preparation. The quick-frozen neutrophil preparation comprises quick-frozen neutrophil; the nano preparation is carried on the surface of the quick-frozen neutrophil. According to the invention, the quick-frozen neutrophile granulocyte is innovatively used as a delivery carrier, so that the defect that the traditional live neutrophile granulocyte responds to an inflammation signal to release an extracellular trapping net to promote tumor metastasis when being used as an in-vivo drug delivery carrier is avoided, but the performance of targeting inflammation sites and removing inflammatory factors of the neutrophile granulocyte is maintained; the cell preparation can target a far-end tumor pre-metastasis ecological niche, remove tumor-derived cell factors in the tumor pre-metastasis ecological niche, polarize M2 type macrophages into M1 type macrophages, block formation of the tumor pre-metastasis ecological niche from multiple layers and inhibit tumor metastasis.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a quick-frozen neutrophil preparation for resisting tumor metastasis, and a preparation method and application thereof. Background Art

[0002] Metastasis is the root cause of the high mortality rate of malignant tumors. Traditional methods of treating tumors include surgery, radiotherapy and chemotherapy. Although these methods can remove local tumors, they have poor inhibitory effects on metastasis. Therefore, developing effective means to prevent tumor metastasis is crucial for the treatment of malignant tumors. Studies have found that before tumor metastasis, the microenvironment of its pre-metastatic target organ has changed, and myeloid cell aggregation, persistent inflammatory response, increased oxygen free radicals and matrix remodeling appear inside the target organ tissue. This microenvironment that supports tumor metastasis is defined as the premetastatic niche (PMN), which is a necessary condition for distant metastasis of tumors. It can be seen that blocking the formation of PMN is the key to effectively inhibiting tumor metastasis.

[0003] PMNs are initiated and established based on the complex interactions between factors secreted by the primary tumor, myeloid cells, and local stromal components. This complexity in composition determines that inhibiting the activity of one or several components is not enough to block PMN formation. On the one hand, if multiple factors derived from tumors can be consumed at the same time, the recruitment of myeloid cells to PMNs will be effectively inhibited; on the other hand, the key myeloid cells that have been recruited in PMNs must be reshaped and PMNs must be converted to an immune-activated state in order to successfully block PMN construction. Studies have shown that tumor-associated macrophages (TAMs) can be recruited to distal target organs, triggering sequential reconstruction of endothelial cell stroma, secreting factors that promote PMN formation, and inhibiting tumor antigen presentation, thereby assisting PMN construction. However, TAMs have strong plasticity and can rapidly transform into M1-type TAMs in response to changes in the local microenvironment, and then perform corresponding immune functions, which provides a target for converting PMNs to an immune-activated state.

[0004] Neutrophils can respond to the recruitment of inflammatory factors in the lesion site, penetrate blood vessels and enter tissues, and form extracellular traps (NETs) under the stimulation of inflammatory factors to release the carried drugs and achieve anti-tumor treatment. However, studies have confirmed that neutrophil elastase and matrix metalloproteinase in NETs will cut laminin in tissues and awaken dormant tumor cells. In addition, CCDC25 expressed by cancer cells can recognize DNA released by NETs, ​​activate the ILK-β-parvin pathway, and promote tumor metastasis. Therefore, directly using neutrophils to carry nanodrugs will produce the side effect of promoting tumor metastasis. At present, relevant researchers mainly degrade NETs released by neutrophils at the inflammatory site by loading DNA enzymes on neutrophils, thereby reducing the side effects of NETs released by neutrophils. However, DNA enzymes will fall off and degrade during the in vivo transportation process, and the activity of DNA enzymes will be significantly reduced, resulting in only a small amount of degradation of NETs at the inflammatory site, and the NETs at the inflammatory site cannot be completely removed. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] One of the purposes of the present invention is to provide a quick-frozen neutrophil preparation for resisting tumor metastasis, which avoids the disadvantage that traditional live neutrophils release extracellular traps in response to inflammatory signals and promote tumor metastasis when used as in vivo drug delivery carriers, but maintains the performance of neutrophils in targeting inflammatory sites and clearing inflammatory factors.

[0008] In order to solve the above technical problems, the present invention provides the following technical solution: a quick-frozen neutrophil preparation for resisting tumor metastasis, comprising:

[0009] Snap-frozen neutrophils; and,

[0010] A nano preparation, wherein the nano preparation is carried on the surface of the quick-frozen neutrophils;

[0011] Wherein, the quick-frozen neutrophils express multiple cytokine receptors and have inflammatory tropism, and can target the tumor pre-metastasis niche and consume tumor cell-derived cytokines in the tumor pre-metastasis niche;

[0012] The nano preparation is an iron oxide nano preparation wrapped in a biofilm, and the nano preparation can polarize M2 macrophages in the pre-metastatic niche of tumors into M1 macrophages.

[0013] As a preferred embodiment of the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the quick-frozen neutrophils are selected from bone marrow or peripheral blood.

[0014] As a preferred embodiment of the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the cytokine receptors expressed by the quick-frozen neutrophils include receptors for cytokines IL-1β, G-CSF, GM-CSF and IL-10.

[0015] As a preferred embodiment of the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the connection mode between the quick-frozen neutrophils and the nano preparation includes one of receptor-ligand interaction, electrostatic adsorption, physical intercalation and covalent binding.

[0016] As a preferred embodiment of the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the quick-frozen neutrophil is connected to the nano preparation by using the covalent binding effect between tetravalent zirconium ions and phosphate groups.

[0017] As a preferred embodiment of the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the iron oxide nano-preparation encapsulating the biofilm synthesized endogenously by the bacteria is formed by the iron oxide nano-preparation encapsulating the biofilm released by the bacteria ingesting iron oxide nano-materials; or, it is formed by the iron oxide nano-preparation encapsulating the biofilm generated by the bacteria ingesting iron-related substances and through redox reactions in vivo. One or more of the above.

[0018] As a preferred embodiment of the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the mass ratio of the quick-frozen neutrophils to the nano preparation is 10:1 to 20:1.

[0019] Another object of the present invention is to provide a method for preparing a quick-frozen neutrophil preparation for resisting tumor metastasis as described in any one of the above items, comprising:

[0020] Preparation of quick-frozen neutrophils;

[0021] Preparation of iron oxide nanoformulations encapsulating biofilms;

[0022] The nano preparation is loaded with quick-frozen neutrophils to obtain a quick-frozen neutrophil preparation.

[0023] As a preferred embodiment of the method for preparing the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, wherein: in the preparation of quick-frozen neutrophils, the neutrophils are extracted and immediately resuspended in freezing medium, and quickly immersed in liquid nitrogen for more than 12 hours.

[0024] As a preferred embodiment of the method for preparing the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the iron oxide nanoformulation for preparing the biofilm is formed by the iron oxide nanoformulation for preparing the biofilm released by the bacteria through the uptake of iron oxide nanomaterials; or, the iron oxide nanoformulation for preparing the biofilm is formed by the bacteria taking up iron-related substances and generating the iron oxide nanoformulation for preparing the biofilm through redox reaction in vivo.

[0025] As a preferred embodiment of the method for preparing the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the nano preparation is carried on the quick-frozen neutrophils, and the quick-frozen neutrophils are connected to the nano preparation by the covalent binding effect between the tetravalent zirconium ions and the phosphate groups.

[0026] As a preferred embodiment of the method for preparing the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, the method comprises:

[0027] The iron oxide nanoparticles encapsulating the biofilm were treated with Zr 4+ resuspended in buffer to form a complex;

[0028] The complex was incubated with a PBS solution of quick-frozen neutrophils to obtain a quick-frozen neutrophil nanoformulation.

[0029] As a preferred embodiment of the method for preparing the quick-frozen neutrophil preparation for resisting tumor metastasis of the present invention, wherein: the Zr 4+ The concentration is 1-10 mM;

[0030] The iron oxide nanoparticles encapsulating the biofilm and Zr 4+ The mass ratio is 200:9~20:9.

[0031] Another object of the present invention is to provide the use of the quick-frozen neutrophil preparation for resisting tumor metastasis as described in any of the above items in the preparation of anti-tumor metastasis therapeutic drugs, wherein the anti-tumor metastasis therapeutic drugs are suitable for both preoperative and postoperative patients with tumors.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention innovatively utilizes quick-frozen neutrophils as delivery carriers, avoiding the drawback of traditional live neutrophils as in vivo drug delivery carriers that respond to inflammatory signals and release extracellular traps to promote tumor metastasis, but maintains the performance of neutrophils in targeting inflammatory sites and clearing inflammatory factors. The cell preparation can target distal tumor pre-metastasis niches, clear tumor-derived cytokines in the tumor pre-metastasis niche, polarize M2 macrophages to M1 macrophages, block the formation of tumor pre-metastasis niches from multiple levels, and inhibit tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0035] Figure 1 This is a microscopic identification image of the quick-frozen neutrophils in Example 1.

[0036] Figure 2 This is a transmission electron microscopy image of the nanoformulation in Example 1.

[0037] Figure 3 This is a scanning electron micrograph of the quick-frozen neutrophil preparation in Example 1.

[0038] Figure 4 This is an analysis of the effect of the quick-frozen neutrophil preparation in Example 2 on postoperative metastasis of melanoma.

[0039] Figure 5 This is an analysis of the effect of the quick-frozen neutrophil preparation in Example 2 on the repolarization of macrophages in the lung, the pre-metastatic organ of the tumor.

[0040] Figure 6 This is an analysis of the effect of the quick-frozen neutrophil preparation in Example 2 on the clearance of tumor-derived cytokines in the lung, a tumor pre-metastatic organ.

[0041] Figure 7 This is a scanning electron micrograph of the quick-frozen neutrophil preparation in Example 3.

[0042] Figure 8 This is a scanning electron micrograph of the quick-frozen neutrophil preparation in Example 4.

[0043] Fig. 9 This is an analysis of the effect of the quick-frozen neutrophil preparation in Example 5 on postoperative metastasis of melanoma. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0045] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0046] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0047] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0048] Example 1

[0049] (1) Preparation and identification of quick-frozen neutrophils

[0050] The peripheral blood of mice was collected, red blood cell lysis solution was added to remove red blood cells, biotin-modified Ly6G antibody was added to label the remaining cells, and then streptavidin-modified magnetic beads were added and adsorbed using a magnetic stand. The adsorbed cells were neutrophils.

[0051] The sorted neutrophils (1×10 7 ) with quick freezing medium (containing 5% FBS and 0.1 mol L -1 Resuspend in RPMI-1640 medium containing trehalose), quickly soak in liquid nitrogen for 12 hours, and obtain liquid nitrogen-treated neutrophils. Then thaw the cells at 37°C, centrifuge at 500g for 5 minutes, and wash 3 times with PBS to obtain quick-frozen neutrophils. Resuspend the quick-frozen neutrophils in PBS and store at 4°C. Take a small amount of quick-frozen neutrophil suspension and drop it on the front end of the slide. Take another slide with smooth edges as a pusher. Place one end in front of the droplet and move it backward to contact the droplet so that the droplet is evenly dispersed outside the contact between the pusher and the slide. Then push it out steadily to the other end at an angle of 30 to 40 degrees with uniform force. Let the smear dry naturally. Add Wright-Giemsa stain to the smear and cover it for 30 seconds. Then add 2 to 3 times the volume of PBS stain for 1 to 3 minutes. Pour off the stain, wash with an appropriate amount of distilled water, and quickly dry it. Then place it in a xylene solution for 5 minutes, seal it with neutral gum, and examine it under a microscope. As Figure 1 As shown in the figure, the microscopic results showed that the nucleus of the extracted cells was purple-blue and lobed, and the cytoplasm was almost colorless, and they were identified as quick-frozen neutrophils, referred to asC NE.

[0052] (2) Preparation of nanoparticles

[0053] Wild-type Magnetospirillum gryphiswaldense MSR-1 was inoculated into fresh sodium lactate medium at a ratio of 1:10 and cultured at 30°C and 100 rpm for 24 h. Two generations were activated continuously, and the third generation of bacterial liquid was used for the separation and purification of magnetosomes. The bacterial liquid was centrifuged at 12000 rpm for 10 min to collect the cells, and 0.1 mol L -1 Resuspend the cells in PBS (pH 7.4) and repeatedly sonicate at 200W to disrupt the cells. During this time, examine the bacterial solution under a microscope to check whether the cells are completely disrupted. After the cells are completely disrupted, adsorb the bacterial solution overnight on a magnet to separate the cell residues from the magnetosomes. Discard the supernatant and add 0.01 mol L -1 PBS, 120W ultrasound for 30 min, and magnet adsorption. After the magnetosomes are separated from the supernatant, the ultrasound frequency is reduced and the ultrasound treatment and magnet adsorption are repeated to further purify the magnetosomes until the protein concentration in the washing solution is less than 0.1 mg mL -1 , and then washed three times with distilled water to remove the residual phosphate and chloride ions on the surface of the magnetosomes, and then pure magnetosomes (such as Figure 2 As shown), referred to as Mag.

[0054] (3) Preparation of quick-frozen neutrophil preparations

[0055] 20 μg of magnetosomes were mixed with ZrOCl2 (0.01 mol L -1 ) in 1 mL of Tris buffer (0.01 mol L -1 Tris, 0.15 mol L-1 NaCl, pH 7.4) and gently shake for 30 min to form magnetosomes-Zr 4+ The complex was washed three times with Tris buffer and then mixed with 1×10 6 The quick-frozen neutrophils were incubated with 1 mL of PBS solution for 30 min. The quick-frozen neutrophils were then washed with PBS for 3 times to obtain a quick-frozen neutrophil nanoparticle preparation (such as Figure 3 shown), referred to as C NE Mag , resuspended in PBS and stored at 4°C.

[0056] Comparative Example 1

[0057] Preparation of live neutrophils (NE)

[0058] The peripheral blood of mice was collected, and red blood cell lysis solution was added to remove red blood cells. Biotin-modified Ly6G antibody was added to label the remaining cells. Then, streptavidin-modified magnetic beads were added and adsorbed using a magnetic rack. The adsorbed cells were live neutrophils, referred to as NE.

[0059] Comparative Example 2

[0060] Magnetosomes loaded with live neutrophils (NE Mag )

[0061] 20 μg of magnetosomes were mixed with ZrOCl2 (0.01 mol L -1 ) in 1 mL of Tris buffer (0.01 mol L -1 Tris, 0.15 mol L -1 NaCl, pH 7.4) and gently shake for 30 min to form magnetosomes-Zr4 + The complex was washed three times with Tris buffer, and then the complex was incubated with 1 mL PBS solution containing 1×106 live neutrophils for 30 min. The live neutrophils treated were washed three times with PBS to obtain magnetosomes carrying live neutrophils, referred to as NE Mag .

[0062] Example 2 Therapeutic effect of quick-frozen neutrophil preparation on postoperative metastasis of melanoma in mice

[0063] Prepare quick-frozen neutrophils ( C NE), nanoformulation (Mag), and quick-frozen neutrophil preparation ( C NE Mag At the same time, live neutrophils (NE) and magnetosomes carrying live neutrophils (NE) were prepared according to the methods of Comparative Examples 1 and 2. Mag ).

[0064] First, 6-8 week-old female C57BL / 6 mice were used to establish a mouse melanoma postoperative lung metastasis model. During the modeling, 1.0×10 6 B16 / F10 cells were used to establish an orthotopic melanoma model. When the tumor size reached 100 mm 3 (Tumor volume = (length × width 2 ) / 2), the tumor was surgically removed. 7 days after surgery, the mice were intravenously injected with 2.0×10 5 B16 / F10 cells were used to establish a postoperative mouse lung metastasis model. The mice with postoperative lung metastasis were then randomly divided into 6 groups, namely, PBS control group, Mag treatment group, live neutrophil treatment group (NE), quick-frozen neutrophil treatment group (C NE), magnetosomes carrying live neutrophils (NE Mag ), quick-frozen neutrophil preparation treatment group ( C NE Mag )(10mgkg -1 Mag or 5×10 7 Cell kg -1 ). PBS, Mag, NE, C NE, NE Mag , C NE Mag The drug was administered once every 2 days for a total of 6 times (from the 9th to the 19th day after surgery). On the 21st day, the mice were killed, their lungs were removed, and the number of macroscopic metastatic nodules in the lungs of mice in each group was recorded to evaluate the therapeutic effect. Figure 4 As shown, compared with the PBS control group, C NE Mag Presenting the best anti-metastatic treatment effect.

[0065] Further, one of the lung lobes was cut into small pieces with scissors and 1 mg / mL -1 The cells were digested with collagenase I at 37°C for 1 h and then filtered through a 70 μm filter. Red blood cells were then removed with red blood cell lysis buffer and lung single cell suspension was prepared. The cells were then incubated with anti-F4 / 80-FITC antibody (0.5 μg mL -1 ), anti-CD206-APC antibody (1 μg mL -1 ) and anti-CD86-PE antibody (1 μg mL -1 ) were incubated with lung single cell suspension for 30 min, and M1 (F4 / 80 + CD86 + ) and M2(F4 / 80 + CD206 + ) ratio, the result is as follows Figure 5 Compared with the PBS control group, C NE Mag After treatment, the increase in the proportion of M1 macrophages and the decrease in the proportion of M2 macrophages in the lungs were most obvious.

[0066] Then, the lung homogenate was prepared and the level of Il-10 in the lung was detected using an enzyme-linked immunosorbent assay kit. Figure 6 Compared with the PBS control group, C NE MagThe Il-10 content in the lungs decreased most significantly after treatment. The above results indicate that quick-frozen neutrophil preparations can consume tumor-derived cytokines in the lung tumor pre-metastasis niche and promote the polarization of M2 macrophages to M1, thereby blocking the formation of tumor pre-metastasis niches at multiple levels and inhibiting postoperative tumor metastasis.

[0067] Example 3

[0068] In this embodiment 3, based on the embodiment 1, the content of ZrOCl2 in step (3) was adjusted to 0.05 mol L -1 The experiment found that the quick-frozen neutrophils aggregated. Figure 7 shown.

[0069] Example 4

[0070] In Example 4, based on Example 1, the content of magnetosomes in step (3) was adjusted to 200 μg. Experiments have shown that the morphology of quick-frozen neutrophils changes, which may affect the response of quick-frozen neutrophils to cytokines. Figure 8 shown.

[0071] Example 5

[0072] In Example 5, based on Example 2, the injection dose was increased to 15 mg / kg -1 Mag or 7.5×10 7 Cell kg -1 , and evaluate the treatment effect. Fig. 9 As shown, compared with the PBS injection group, C NE Mag The therapeutic effect is more obvious. Experiments have confirmed that C NE Mag The optimal dose range is 5×10 7 ~7.5×10 7 Cell kg -1 .

[0073] The present invention innovatively utilizes quick-frozen neutrophils as delivery carriers, avoiding the drawbacks of traditional live neutrophils as in vivo drug delivery carriers that respond to inflammatory signals to release extracellular traps to promote tumor metastasis, but maintaining the performance of neutrophils targeting inflammatory sites and clearing inflammatory factors. The cell preparation can target the distal tumor pre-metastasis niche, clear tumor-derived cytokines in the tumor pre-metastasis niche, polarize M2 macrophages to M1, block the formation of tumor pre-metastasis niches from multiple levels, and inhibit tumor metastasis. Secondly, the phospholipid bimolecular membrane on the surface of the biosynthetic iron oxide nanomaterial used in the present invention is also easy to be modified and functionalized, and can be easily carried on the surface of quick-frozen neutrophils, making the preparation of quick-frozen neutrophil preparations more convenient, with high application value, suitable for wide promotion and application.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A quick-frozen neutrophil preparation for resisting tumor metastasis, characterized in that: include, Snap-frozen neutrophils; as well as, A nano preparation, wherein the nano preparation is carried on the surface of the quick-frozen neutrophils; Wherein, the quick-frozen neutrophils express multiple cytokine receptors and have inflammatory tropism, and can target the tumor pre-metastasis niche and consume tumor cell-derived cytokines in the tumor pre-metastasis niche; The nano preparation is an iron oxide nano preparation wrapped in a biofilm, and the nano preparation can polarize M2 macrophages in the pre-metastatic niche of tumors into M1 macrophages.

2. The quick-frozen neutrophil preparation for resisting tumor metastasis according to claim 1, characterized in that: The quick-frozen neutrophils are selected from bone marrow or peripheral blood.

3. The quick-frozen neutrophil preparation for resisting tumor metastasis according to claim 2, characterized in that: The cytokine receptors expressed by the quick-frozen neutrophils include receptors for cytokines IL-1β, G-CSF, GM-CSF and IL-10.

4. The quick-frozen neutrophil preparation for resisting tumor metastasis according to any one of claims 1 to 3, characterized in that: The connection mode between the quick-frozen neutrophils and the nano preparation includes one of receptor-ligand interaction, electrostatic adsorption, physical interpenetration and covalent binding.

5. The quick-frozen neutrophil preparation for resisting tumor metastasis according to claim 4, characterized in that: The quick-frozen neutrophils are connected to the nanoformulation by using the covalent binding effect between tetravalent zirconium ions and phosphate groups.

6. The quick-frozen neutrophil preparation for resisting tumor metastasis according to any one of claims 1 to 3 and 5, characterized in that: The iron oxide nanoparticles encapsulating the biofilm are iron oxide nanoparticles encapsulating the biofilm that are endogenously synthesized by bacteria; The iron oxide nanoformulation wrapped in the biofilm that is endogenously synthesized by the bacteria is formed by the iron oxide nanoformulation wrapped in the biofilm that is released by the bacteria's uptake of iron oxide nanomaterials; or, it is formed by the iron oxide nanoformulation wrapped in the biofilm that is generated by the bacteria's uptake of iron-related substances and through redox reactions in vivo.

7. The quick-frozen neutrophil preparation for resisting tumor metastasis according to claim 6, characterized in that: The bacteria include one or more of Magnetospirillum magneticum AMB-1, Magnetospirillum gryphiswaldense MSR-1, and Magnetospirillum magnetotacticum MS-1.

8. The quick-frozen neutrophil preparation for resisting tumor metastasis according to any one of claims 1 to 3, 5 and 7, characterized in that: The mass ratio of the quick-frozen neutrophils to the nano preparation is 10:1 to 20:

1.

9. The method for preparing a quick-frozen neutrophil preparation for resisting tumor metastasis according to any one of claims 1 to 8, characterized in that: include, Preparation of quick-frozen neutrophils; Preparation of iron oxide nanoformulations encapsulating biofilms; The nano preparation is loaded with quick-frozen neutrophils to obtain a quick-frozen neutrophil preparation.

10. Use of the quick-frozen neutrophil preparation for resisting tumor metastasis according to any one of claims 1 to 8 in the preparation of anti-tumor metastasis therapeutic drugs.

Images