Nano-engineered mononuclear cell and application thereof

Abstract

The invention discloses a nano-engineered mononuclear cell and application thereof, and belongs to the technical field of medicines. The nano-engineered mononuclear cell comprises a mononuclear cell, a nano-engineered element and a therapeutic drug, the nano-engineered element is loaded in cytoplasm of the mononuclear cell, and the surface of the mononuclear cell is modified with the therapeutic drug; the nano engineered element is iron oxide nanoparticles; the therapeutic drug is a functional lipid obtained by modifying a chemotherapeutic drug on a lipid molecule. The nano-engineered mononuclear cell can retain and even enhance the tumor tropism and tumor infiltration capacity of the mononuclear cell, can effectively intervene in the differentiation phenotype of the mononuclear cell in vivo and in vitro, relieves the tumor immunosuppression microenvironment, inhibits the growth of tumor cells, and achieves a good anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a nano-engineered monocyte and an application thereof. Background Art

[0002] Cancer remains one of the world's major health threats. Solid tumors, such as pancreatic and breast cancers, present a particularly challenging challenge for researchers due to treatment difficulties such as poor drug penetration and high immunosuppression. As a rapidly developing new player in the field of tumor treatment, immune cell therapy has gradually brought new hope for the cure of solid tumors. However, CAR-T adoptive immune cell therapy, which is currently relatively mature in clinical research, has difficulty infiltrating solid tumors and is therefore primarily used in non-solid tumors such as hematologic malignancies. Therefore, the development of new cell therapies that can effectively infiltrate and attack solid tumors is of great significance.

[0003] Among many immune cell types, monocytes and macrophages possess the unique advantage of homing in and deeply infiltrating hypoxic, vascular-poor regions of tumors, representing a promising new frontier for developing cell-based therapies for solid tumors. Within malignant solid tumor lesions, tumor-associated macrophages (TAMs) comprise 40–50% of the immune cell population. They contribute to tumor progression by promoting cancer cell growth and metastasis and suppressing the patient's immune system, making them a major source of tumor immunosuppression. Developing effective strategies to metabolically reprogram and phenotypic repolarization of these TAMs presents a promising therapeutic approach. However, mature, differentiated TAMs are often located in vascular-poor regions of tumors, making them less accessible to drugs. Therefore, targeting their differentiation potential at an early stage may be a more promising therapeutic approach. Peripheral blood mononuclear cells (PBMCs), the precursors of TAMs, offer unique advantages in the field of cancer therapy, such as high accessibility and strong tumor tropism. Engineering TAMs has the potential to address several challenges in treating solid tumors with cell-based therapies, including tumor infiltration and immunosuppression within the tumor microenvironment.

[0004] In recent years, several clinical and preclinical studies have explored the engineering of monocytes to achieve anti-tumor effects. For example, Myeloid Therapeutics' mRNA-engineered CAR monocyte MT-101 has been shown to effectively alleviate peripheral T-cell lymphoma. However, gene editing strategies are limited by time-consuming processes, high technical barriers, and low gene transfection efficiency in phagocytes. Therefore, developing simple and rapid monocyte engineering strategies is of great value. Nanotechnology, with its advantages of versatile design and specific distribution, has opened up new possibilities for nanoengineered immune cells. Furthermore, monocytes / macrophages, due to their natural tropism for tumors, have become attractive drug delivery vehicles. However, most monocyte-based drug delivery systems solely utilize monocytes as drug delivery vehicles, overlooking their dynamic differentiation capacity in diverse environments and their potent therapeutic effects. Therefore, developing monocyte-based engineering strategies that simultaneously stimulate monocyte anti-tumor potential and drug delivery capabilities could address the dual therapeutic challenges of solid tumors: immunosuppression and the difficulty in infiltrating immune cells and drugs.

[0005] Among the monocyte engineering components, iron oxide nanoparticles (IONPs), approved by the FDA for iron deficiency anemia, have shown significant potential due to their excellent biocompatibility, biodegradability, surface modifiability, and activation of macrophage pro-inflammatory capacity. Reports are increasingly available on their effects on cellular ultrastructure and oxidative stress. Inspired by the macrophage repolarization ability of IONPs, the inventors hypothesize that preloading monocytes with IONPs is a potential nanoengineering strategy for monocyte armamentarium. Furthermore, reports indicate that IONPs can enhance the ability of loaded cells to communicate with other cells within the tumor via microvesicle / intercellular channels. Therefore, preloading monocytes with IONPs may be a promising strategy for simultaneously enhancing phenotypic intervention and drug delivery. Based on this, the present invention designs an iron oxide nanoparticle-engineered monocyte therapy system. By utilizing this integrated living cell system to deliver therapeutic functional lipids to the cell membrane, the system is expected to enhance the anti-tumor capacity of monocytes and address the challenges of solid tumor treatment. Summary of the Invention

[0006] In order to address the two major limitations of existing solid tumor immune cell therapy, namely immune infiltration and immunosuppression, the present invention designs and constructs a nano-engineered monocyte.

[0007] One of the purposes of the present invention is to provide a nano-engineered monocyte, comprising a monocyte, a nano-engineered element, and a therapeutic drug, wherein the nano-engineered element is loaded in the cytoplasm of the monocyte, and the therapeutic drug is modified on the surface of the monocyte; The nano-engineered element is iron oxide nanoparticles; The therapeutic drug is a functional lipid obtained by modifying a chemotherapy drug on a lipid molecule.

[0008] In the present invention, the monocytes include monocytes from various sources, such as mouse monocyte cell line RAW264.7, human monocyte cell line THP-1, mouse monocyte cell line J774A.1, mouse primary peripheral blood monocytes, mouse primary myeloid monocytes and mouse primary peritoneal monocytes.

[0009] Furthermore, the iron oxide nanoparticles are ferroferric oxide nanoparticles or ferrous oxide nanoparticles.

[0010] In some embodiments of the present invention, the iron oxide nanoparticles are coated with a coating layer, and the coating layer is selected from a hyaluronic acid coating layer, a citric acid coating layer, a hyaluronic acid-dopamine coating layer, a silicon dioxide coating layer, a polyvinylamine coating layer, and the like.

[0011] Furthermore, the lipid molecule has a molecular weight of 500-50,000 Da, is amphiphilic and contains covalent groups that are easily modified, such as amino, carboxyl, N-hydroxysuccinimide ester, sulfhydryl, hydroxyl, aldehyde, azide, alkyne, biotin, maleimide, etc., and is selected from DSPE-PEG, DMPE-PEG, DOPE-PEG, DPPE-PEG, DAPC-PEG, DMG-PEG, DMPC-PEG, cholesterol, DSPE, DPPE, etc.

[0012] Furthermore, the chemotherapy drug is selected from gemcitabine, doxorubicin, sorafenib, 5-fluorouracil, paclitaxel, methotrexate, cisplatin drugs, vinblastine drugs, irinotecan and other compounds having modifiable covalent groups such as amino groups, carboxyl groups, and hydroxyl groups.

[0013] A second object of the present invention is to provide a method for preparing the above-mentioned nano-engineered monocytes, specifically comprising: mixing and incubating nano-engineered elements, therapeutic drugs and monocytes to prepare nano-engineered monocytes.

[0014] In one embodiment of the present invention, the method for preparing the nano-engineered monocytes is as follows: in a cell culture environment, the reagents of the nano-engineered elements, the reagents of the therapeutic drugs and the culture medium of the monocytes are mixed and incubated for 10 minutes to 48 hours, and then the centrifugation speed is 100-600×g, and the centrifugation time is not less than 1 minute.

[0015] The reagents of the nano-engineered elements are made of iron oxide nanoparticles and physiological buffers such as PBS, HEPES, and normal saline; the reagents of the therapeutic drugs are made of functional lipids and physiological buffers such as PBS, HEPES, and normal saline; and the culture medium of the monocytes is made of monocytes and cell culture media such as DMEM, IMEM, and MEM.

[0016] Furthermore, in the reagent of the nano-engineered element, the concentration of iron oxide nanoparticles is 0.1-500 μg / mL.

[0017] Furthermore, in the reagent of the therapeutic drug, the concentration of the functional lipid is 0.1-200 μg / mL.

[0018] Furthermore, in the monocyte culture medium, the monocyte concentration is 1×10 2 -2×10 6 / mL.

[0019] The third object of the present invention is to provide the use of the above-mentioned nano-engineered monocytes in the preparation of tumor therapeutic drugs.

[0020] The nano-engineered monocytes designed in the present invention can be used to intervene in the differentiation phenotype of monocytes from the source, so that they tend to differentiate into pro-inflammatory and anti-tumor phenotypes after penetrating into solid tumors, regulate the immunosuppressive microenvironment, and have the ability to deliver therapeutic chemotherapy drugs deep into the tumor, showing an enhanced tumor-killing effect, providing an effective solution for the treatment of solid tumors. Beneficial effects

[0021] (1) The nano-engineered monocytes provided by the present invention have a simple preparation process and good biosafety. Compared with traditional macrophage repolarization methods, they can bypass the bottleneck of repolarization drugs that are difficult to penetrate deep into the tumor to act on tumor-associated macrophages.

[0022] (2) Compared with clinical live cell anti-tumor therapies such as CAR-T, the nano-engineered monocytes provided by the present invention have the advantage of being able to deeply infiltrate into solid tumors and fully activate the immune system by reshaping innate immunity.

[0023] (3) The nano-engineered monocytes provided by the present invention can increase the M1 / M2 ratio in the tumor microenvironment by 21.0 times, increase T cell infiltration in the tumor by 4.0 times, and trigger a long-term anti-tumor immune response in the body, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the composition of nanoengineered monocytes.

[0025] Figure 2This is a transmission electron microscope image of the iron oxide nanoparticles of the nano-engineered element in Example 1.

[0026] Figure 3 This is the synthetic route of the functional lipid in Example 1.

[0027] Figure 4 This is the hydrogen nuclear magnetic resonance spectrum of the functional lipid in Example 1.

[0028] Figure 5 This is a scanning electron micrograph of the nanoengineered monocytes in Example 1.

[0029] Figure 6 This is a contour map of the surface marker flow pattern of the nano-engineered monocytes in Example 1.

[0030] Figure 7 This is a transmission electron microscopy image of the nanoengineered monocytes in Example 1.

[0031] Figure 8 This is the synthesis route of the DHA coating layer in Example 2.

[0032] Figure 9 This is the hydrogen nuclear magnetic resonance spectrum of the DHA coating layer in Example 2.

[0033] Figure 10 This is a quantitative graph of the mitochondrial number of nanoengineered monocytes in Example 3.

[0034] Figure 11 This is a confocal fluorescence image of the nanoengineered monocytes in Example 4.

[0035] Figure 12 This is the Prussian blue staining image of the nanoengineered monocytes in Example 6.

[0036] Figure 13 is the iron oxide nanoparticle loading amount of nanoengineered monocytes in Test Example 1.

[0037] Figure 14 This is a flow cytometric graph of the phenotypic differentiation ratio of nanoengineered monocytes after co-incubation with tumor cells in Test Example 2.

[0038] Figure 15 This is to test the therapeutic effect of nano-engineered monocytes in Example 3 in mice with orthotopic pancreatic cancer. DETAILED DESCRIPTION

[0039] The present invention provides a nano-engineered monocyte and its application. The nano-engineered monocyte is composed of a nano-engineered element, a monocyte and a therapeutic drug. The nano-engineered element is an iron oxide nanoparticle. The monocyte includes monocytes from various sources. The therapeutic drug is a functional lipid modified with a chemotherapy drug (such as gemcitabine). The nano-engineered element and the therapeutic drug are loaded into the cytoplasm of the monocyte and on the cell membrane surface (such as cytotoxicity) through phagocytosis and membrane fusion, respectively. Figure 1 The resulting nanoengineered monocyte system can be used for the targeted treatment of intractable solid tumors, such as pancreatic cancer. This system preserves and even enhances monocytes' tumor tropism and tumor penetration. It also effectively modulates monocyte differentiation phenotypes both in vivo and in vitro, alleviating the tumor's immunosuppressive microenvironment and inhibiting tumor cell growth, achieving a robust anti-tumor effect.

[0040] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example 1

[0043] 1. Preparation of nano-engineered components - iron oxide nanoparticles 330 mg of FeCl₃·6H₂O and 170 mg of FeCl₂·4H₂O were dissolved in 2 mL of deionized water and stirred under nitrogen for half an hour. After heating to 80°C, 1.125 mL of aqueous ammonia was added dropwise. The mixture was then heated to 95°C and 190 mg of citric acid dissolved in 0.4 mL was added. The mixture was stirred at 95°C for 90 minutes. Finally, the product was transferred to a dialysis bag (molecular weight cutoff 14,000 Da) and dialyzed against double-distilled water for 24 hours to obtain iron oxide nanoparticles (IONPs).

[0044] Figure 2 It can be seen that the prepared IONPs have regular morphology. After water evaporation, transmission electron microscopy shows that the particle size is about 5~10 nm. The dynamic light scattering method measures the hydrated particle size to be 32.8±8.14 nm, and the polydispersity index (PDI) is 0.157±0.04, which is relatively uniformly distributed.

[0045] 2. Preparation of therapeutic drugs In this example, DSPE-PEG2000-NHS was used as the starting lipid molecule and gemcitabine as the chemotherapy drug. A standard base-catalyzed method was used to synthesize the functional lipid DSPE-PEG2000-Gem (DG). Specifically, 20 mg of DSPE-PEG2000-NHS was reacted with 30 mg of gemcitabine and a catalytic amount of triethylamine (20 μL) at 37°C for 24 hours. The product was then transferred to a dialysis bag (molecular weight cutoff 500 Da) and dialyzed in double-distilled water for 48 hours. The product was then freeze-dried for storage.

[0046] Figure 3 is the structural formula of the initial lipid DSPE-PEG2000-NHS and the functional lipid DG, Figure 4 This is the H NMR spectrum of DSPE-PEG2000-NHS and DG. The red circle indicates the characteristic aromatic peak of gemcitabine. The resulting DG is readily soluble in water and produces a large amount of foam upon dissolution, indicating its amphiphilic nature, paving the way for subsequent insertion into the lipid bilayer membrane of monocytes.

[0047] 3. Construction of Nanoengineered Monocyte Therapeutic System Peripheral blood mononuclear cells (M pre ) 1×10 6 1 mL of the suspension was incubated with IONPs nanoparticles (prepared in PBS buffer at a final concentration of 200 μg / mL), DG lipids (prepared in PBS buffer at a final concentration of 20 μg / mL), and DG lipids + IONPs nanoparticles (prepared in PBS buffer at a final concentration of 20 μg / mL and 200 μg / mL, respectively). The supernatant was removed by centrifugation at fixed time points (15 min, 30 min, 45 min, 60 min, 90 min, and 120 min), and the cells were washed twice with PBS buffer to obtain macrophage IE-M cells engineered with different elements. pre ,DE-M pre and IDE-M pre .

[0048] Figure 5 Scanning electron microscopy images show that there is no significant change in the cell surface morphology after engineering of monocytes. Figure 6CD115 (CSF-1R) is specifically expressed on the surface of monocytes and is a protein commonly used to identify monocytes (along with CD11b or Ly6C proteins). Flow cytometry analysis revealed that nanoengineering did not significantly affect the ratios of proteins such as CD115 and CD11b on the monocyte surface, indicating that nanoengineering did not affect these surface markers.

[0049] Figure 7 Transmission electron microscopy revealed that IONPs were localized in the monocyte cytoplasm in the form of vesicles. The cytoplasm was filled with numerous vesicles with high electron density, while no similar vesicles were found in the nucleus, indicating that IONPs were mostly localized in the cytoplasm after internalization. Example 2

[0050] This embodiment provides a nano-engineered monocyte. The only difference between this embodiment and embodiment 1 is that IONPs nanoparticles are replaced with DHA-IONPs nanoparticles, that is, the non-coating layer is replaced with a DHA coating layer. The preparation process of DHA-IONPs is as follows: (1) Synthesis of coating layer hyaluronic acid-dopamine (DHA) 100 mg of hyaluronic acid (HA, molecular weight 50 k) was accurately weighed and dissolved in 25 mL of phosphate buffered saline (PBS) at pH 6.8. 5-fold molar equivalents of EDC and NHS were added under stirring at room temperature. After 15 min of reaction, 5-fold molar equivalents of dopamine hydrochloride were added to the reaction solution, and the reaction was continued for 12 h under nitrogen protection. The reaction solution was then added to a dialysis bag with a molecular weight of 8000-12000 Da and dialyzed in deionized water at pH 6.0 for 8-12 h, with the water changed every 2 h. After dialysis, the dopamine-modified hyaluronic acid (DHA) was obtained as an off-white fluffy solid by freeze-drying. The results were analyzed by nuclear magnetic resonance spectroscopy ( 1 The structure and grafting rate were determined by H-NMR and UV-Vis spectrophotometer (UV-Vis).

[0051] Figure 8 For the synthesis route of DHA, Figure 9 The following is the nuclear magnetic resonance hydrogen spectrum of DHA and its corresponding raw materials. Dopamine grafted onto hyaluronic acid can improve the coordination and complexing ability of the polymer to iron oxide nanoparticles, making the nanoelement more stable and enabling monocytes to phagocytize more nanoelement. 1A comparison of H-NMR peak assignments is shown below. DHA exhibits a new peak around δ = 6.8 ppm, characteristic of the hydrogen atoms (3H) on the benzene ring of dopamine, with the same peak pattern as that of the benzene ring. A new peak also appears around δ = 2.9 ppm, characteristic of the hydrogen atoms on the methylene structure of dopamine, indicating successful dopamine bonding to the carboxyl group of hyaluronic acid. Calculating the number of hydrogen atoms in the characteristic peaks indicates a dopamine grafting efficiency of 20%, paving the way for subsequent coordination and complexation with the iron oxide nanoparticles.

[0052] (2) Preparation of coated iron oxide nanoparticles Take 100 μL of 10 mg / mL iron oxide nanoparticles and dissolve them in 1 mL of deionized water. Under stirring at room temperature, equal volumes of HA and DHA aqueous solutions (5 mg / mL) were added dropwise. The pH of the solution was adjusted to about 6.0 with 1 M NaOH. After stirring at room temperature for 12 h, HA and DHA-coated iron oxide nanoparticles were obtained.

[0053] Subsequently, the particle size of the prepared product was detected by dynamic light scattering. When there was no coating layer, the particle size of the iron oxide nanoparticles was about 30 nm. After coating with the dopamine-hyaluronic acid coating layer, the particle size increased to 125.1 ± 3.6 nm, and the polydispersity index (PDI) was 0.213, which was uniform and stable with good dispersion. Example 3

[0054] This example provides a nano-engineered monocyte, which differs from Example 1 only in that IONPs nanoparticles are replaced with HA-IONPs nanoparticles, that is, the non-coating layer is replaced with an HA coating layer (molecular weight 50 k).

[0055] Flow cytometry was used to quantify the number of mitochondria in nanoengineered monocytes. The method was as follows: after monocytes were treated with different nanoengineered cells, MitoTracker was added. ® The cells were incubated with Green FM (100 nM) probe for 30 min, and the percentage of green fluorescence in each group of cells was quantified by flow cytometry. Figure 10 As shown, the number of intracellular mitochondria in monocytes engineered with IONPs nanoparticles with different coating layers decreased to varying degrees, indicating that IONPs nanoparticles can reduce the level of intracellular oxidative phosphorylation and reduce the tendency to differentiate into M2 phenotype macrophages (the main metabolic pathway is mitochondrial oxidative phosphorylation). Example 4

[0056] This example provides a nano-engineered monocyte. The only difference between this example and Example 1 is that the DSPE-PEG-Gem lipid is replaced with the DSPE-PEG-FITC lipid. The preparation process of the DSPE-PEG-FITC lipid is as follows: Mix 20 mg of DSPE-PEG2000-NH2 with 2 mg of FITC (dissolved in DMSO) in 0.1 M Na2CO3 / NaHCO3 buffer (pH 9.0). Incubate in the dark for 24 h. Transfer the product to a dialysis bag (molecular weight cutoff 500 Da) and dialyze in double-distilled water for 48 h. Freeze-dry and store.

[0057] Nanoengineered monocytes were prepared according to step 3 of Example 1 and stained with the cell membrane dye DiI for 20 minutes. Free DiI dye was removed by centrifugation at 400 × g for 5 minutes, and the cells were washed twice with PBS. The treated engineered monocytes were seeded into an eight-well confocal microscopy chamber pre-coated with 1 mg / mL poly-lysine and photographed using a laser confocal microscope. Figure 11 This image, captured using confocal fluorescence microscopy, shows DSPE-PEG-FITC lipids localized on the surface of monocyte membranes. DG-FITC colocalizes well with the cell membrane, indicating that DG lipids are primarily localized on the cell membrane. Furthermore, competition between DG lipids and the commercial membrane dye DiI was observed, with DiI staining sites already incorporated into DG lipids more difficult. This suggests that its mechanism of cell membrane insertion is very similar to that of DiI dye, relying on lipophilic anchoring within the lipid bilayer. Example 5

[0058] This example provides a nanoengineered monocyte, which differs from Example 1 only in that the DSPE-PEG-Gem lipid is replaced with a DMPE-PEG-DOX lipid. The synthesis methods of the DMPE-PEG-DOX lipid and the DSPE-PEG-Gem lipid are the same. Example 6

[0059] This embodiment provides a nano-engineered monocyte, which differs from Example 1 only in that the mouse-derived peripheral blood mononuclear cells are replaced with the mouse-derived RAW264.7 monocyte / macrophage cell line.

[0060] Prussian blue can react with free trivalent iron ions to form insoluble blue compounds, which can indicate the iron ions generated by the degradation of IONPs after phagocytosis by monocytes / macrophages, thereby indicating the phagocytosis and internalization of IONPs by monocytes / macrophages. Prepare Prussian blue staining solution: 20% hydrochloric acid solution and 10% Prussian blue staining solution are mixed in equal volumes to obtain Prussian blue staining solution. After the nano-engineered monocytes RAW264.7 were fixed with 4% paraformaldehyde for 10 minutes, the paraformaldehyde was discarded, and the cells were washed three times with PBS. Prussian blue staining solution was added for staining for 20 minutes, and then washed three times with PBS. The multi-mode microplate detection and cell imaging system Cytation5 were used to take pictures. Figure 12As shown, the intracellular iron ions (blue positive area) of the RAW264.7 monocyte / macrophage cell line increased in a time-dependent manner, indicating that it takes a certain amount of time for IONPs to be degraded into iron ions in the cell. Example 7

[0061] This example provides a nano-engineered monocyte, which differs from Example 1 only in that the "washing with PBS buffer twice" in step 3 is replaced by "washing with physiological saline twice and finally resuspending in physiological saline" for use in the tail vein administration of animals in Test Example 3. Example 8

[0062] Determination of Iron Oxide Nanoparticle Loading Capacity in Nanoengineered Monocytes Monocytes were plated at 1.5 × 10 cells per well. 5 Cells were seeded in 12-well culture plates. After overnight culture in an incubator, the culture medium was aspirated and fresh culture medium containing 200 μg / mL IONPs iron oxide nanoparticles was added. Supernatants were collected at designated time points (0, 15, 30, 45, 60, 90, and 120 min) and the iron oxide nanoparticle content in the supernatant was determined using the total iron ion assay. The drug loading of the iron oxide nanoparticles by the cells was calculated. The drug loading at x min was calculated using the following formula: .

[0063] Figure 13 It can be seen that at different time points, IONPs can be continuously phagocytosed and internalized within 2 h, and the iron oxide nanoparticle loading capacity can reach 25 μg / 10 4 Monocytes. Example 9

[0064] Nanoengineered monocyte phenotypic detection PAN02 pancreatic cancer cells in the logarithmic growth phase were plated in Transwell chambers (only DMEM medium was added to the negative control chamber), and engineered monocytes were added to the lower chamber (PBS buffer, DG lipids, IONPs nanoparticles, and DG lipids + IONPs nanoparticles were pre-incubated for 2 h. The ratio of monocytes to DG lipids, IONPs nanoparticles, and DG lipids + IONPs nanoparticles was referred to Example 1). The ratio of the two cell numbers was 1:1. After 48 h of co-culture, the monocytes in the lower chamber were collected and the monocyte phenotypic differentiation was detected by flow cytometry.

[0065] In vitro Transwell chamber model was used to investigate M pre The main differentiation tendency after entering the tumor microenvironment (TME). Figure 10It can be seen that the Transwell co-culture model was constructed, PANC02 tumor cells were inoculated in the upper chamber, and M cells with different engineering treatments were inoculated in the lower chamber. pre After 48 h of co-culture, flow cytometry was used to detect surface markers of macrophages. All macrophages differentiated from monocytes expressed the F4 / 80 surface marker. However, only the immunosuppressive phenotype showed a higher level of the mannose receptor CD206, while the pro-inflammatory phenotype expressed a relatively high level of the CD86 receptor.

[0066] Figure 14 As shown in the results, when monocytes were not exposed to TME, they differentiated into macrophages (F4 / 80 positive) under M-CSF stimulation, with low M1 (24.0%) and M2 phenotypes (5.45%). When exposed to TME, the M1 phenotype differentiation ratio decreased to 14.6%, while the M2 phenotype differentiation ratio increased to 29.2%. pre The cells showed an increased pro-inflammatory phenotype and a decreased immunosuppressive phenotype differentiation tendency, which were mainly induced by the DG element and the IONPs nanoelement, respectively. pre Group and IE-M pre All groups showed an increased tendency to differentiate into the M1 phenotype, especially DE-M pre The proportion of M1 phenotype in the group reached 41.0%, which may be due to the fact that both DG and IONPs can provide pre Providing pro-inflammatory stimulation that “mimics the body’s invaders”. However, the reduction of M2 phenotype was only observed in IE-M cells engineered with IONPs. pre Group and IDE-M pre The performance of the group was outstanding. pre Showing a better M pre The M1 phenotype differentiation ratio increased by 3.3 times, compared with M pre The M2 phenotype differentiation ratio was reduced by 86.6%, and the M1 / M2 ratio was increased by 21.0 times, proving that engineered monocytes mainly tend to differentiate into the M1 pro-inflammatory phenotype in TME. Example 10

[0067] Phenotypic assessment of nanoengineered monocytes for therapeutic efficacy in orthotopic pancreatic cancer-bearing mice Healthy female C57BL / 6 mice aged 4 to 6 weeks were selected to establish an orthotopic pancreatic cancer tumor model. 6 PAN02-Luc pancreatic cancer cells were injected into the pancreas of C57BL / 6 mice. Briefly, mice were anesthetized by intraperitoneal injection of 1.25% avertin. After anesthesia, the mice were fixed in the lateral position. An incision of about 1 cm was made near the spleen, and 2×10 6PAN02-Luc cells were injected into the pancreatic tail, disinfected and sutured, penicillin injection was injected subcutaneously, and the mice were kept warm after surgery. On the 7th day after surgery, after intraperitoneal injection of luciferin potassium substrate, the pancreatic bioluminescence of the mice was observed using an IVIS imaging system to determine whether the tumor model was successfully established and to monitor the tumor size. Tumor-bearing mice with similar tumor sizes were randomly divided into groups and injected with normal saline, free DG lipid + IONPs iron oxide nanoparticles, M pre IE-M pre 、DE-M pre IDE-M pre The chemotherapy drug injection dose was 2 mg / kg, the IONPs nanoparticle injection dose was 2 mg / kg, and the number of nanoengineered monocytes injected was calculated based on the IONPs nanoparticle injection dose. Bioluminescence monitoring was performed twice a week to monitor tumor growth in real time.

[0068] like Figure 15 As shown in the figure, after the in situ pancreatic cancer modeling surgery was performed on mice, the growth of pancreatic cancer tumors was monitored in real time using bioluminescence imaging. On the 4th day after modeling, based on the reaction between tumor cells PAN02-Luc and the substrate potassium luciferin, spherical tumors in the pancreas of mice were observed by bioluminescence imaging. On the 18th day, the tumors in the saline group grew rapidly, resulting in the death of 2 mice; the free drug group had a certain tumor inhibition effect but it was not uniform, and the individual differences were great; the engineered monocyte group had a certain tumor inhibition effect, among which IDE-M pre The group had the best tumor suppression effect, with a pancreatic tumor suppression rate of 80% and a longer survival period.

Claims

1. A nano-engineered monocyte, characterized in that: It comprises monocytes, nano-engineered elements and therapeutic drugs, wherein the nano-engineered elements are loaded in the cytoplasm of the monocytes, and the therapeutic drugs are modified on the surface of the monocytes; The nano-engineered element is iron oxide nanoparticles; The therapeutic drug is a functional lipid obtained by modifying a chemotherapy drug on a lipid molecule.

2. The nano-engineered monocyte according to claim 1, characterized in that The iron oxide nanoparticles are ferroferric oxide nanoparticles or ferrous oxide nanoparticles.

3. The nano-engineered monocyte according to claim 2, characterized in that The iron oxide nanoparticles are wrapped with a coating layer.

4. The nano-engineered monocyte according to claim 3, characterized in that The coating layer is selected from a hyaluronic acid coating layer, a citric acid coating layer, a hyaluronic acid-dopamine coating layer, a silicon dioxide coating layer, and a polyvinylamine coating layer.

5. The nano-engineered monocyte according to claim 1, characterized in that The lipid molecule has a molecular weight of 500-50,000 Da, is amphiphilic and contains covalent groups that are easy to modify.

6. The nano-engineered monocyte according to claim 5, characterized in that The lipid molecules include, but are not limited to, DSPE-PEG, DMPE-PEG, DOPE-PEG, DPPE-PEG, DAPC-PEG, DMG-PEG, DMPC-PEG, cholesterol, DSPE, DPPE, and a combination of any two lipids.

7. The nano-engineered monocyte according to claim 6, characterized in that The lipid molecule ends have chemical groups that are easily modified, including but not limited to amino, carboxyl, N-hydroxysuccinimide ester, sulfhydryl, hydroxyl, aldehyde, azide, alkyne, biotin, and maleimide.

8. The nano-engineered monocyte according to claim 1, characterized in that The chemotherapy drugs are compounds with modifiable covalent groups, including but not limited to gemcitabine, doxorubicin, paclitaxel, sorafenib, 5-fluorouracil, methotrexate, cisplatin drugs, vinblastine drugs, irinotecan, and a combination of any two drugs.

9. The method for preparing nano-engineered monocytes according to claim 1, characterized in that: The nano-engineered elements, therapeutic drugs and monocytes are mixed and incubated to prepare nano-engineered monocytes.

10. Use of the nano-engineered monocytes according to claim 1 in the preparation of tumor therapeutic drugs.

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