Application of nanogel in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of nanogel preparations, and particularly relates to application of nanogel to preparation of anti-tumor drugs or preparation of the anti-tumor drugs. Experiments find that nanogel with proper hardness can promote expression of M1 related proteins CD86 and iNOS, and up-regulate expression of CD86 mRNA and iNOS mRNA, so that M2 type tumor related macrophages are reversely polarized into M1 type tumor related macrophages. Further research finds that after the nanogel is used for carrying out reverse polarization treatment on M2 type tumor-related macrophages, tumor cell proliferation can be inhibited, tumor cell apoptosis can be induced, and tumor cell phagocytosis can be realized. In addition, the nanogel with proper hardness is directly delivered into the tumor in an intratumor injection manner, so that the growth of the tumor can be effectively inhibited.

Description

Technical Field

[0001] The present application belongs to the technical field of nanogel preparations, and more specifically, to the application of nanogels in the use or preparation of anti-tumor drugs. Background Art

[0002] Macrophages are important players in maintaining homeostasis and regulating immune responses in healthy tissues. Their primary function is to balance the activation of inflammatory cascades in the context of tissue damage while initiating tissue repair. In parallel with homeostatic disruptions triggered by the recognition of harmful organisms, apoptotic cells, cellular debris, or toxic metabolic byproducts, tumor cell transformation triggers macrophage polarization to restore balance within the tumor microenvironment (TME). However, because growing tumors never reach homeostatic equilibrium, the TAM phenotype remains locked in a cycle that ultimately favors immune cell activation and tissue repair. Exploring the intrinsic features of macrophages selected by tumor cells is critical to predict their contribution to cancer progression and identify weaknesses that can be exploited therapeutically.

[0003] As the most widely distributed immune cells in the tumor microenvironment, tumor-associated macrophages (TAMs) account for about 50%. TAMs play an important role in tumor growth, invasion and metastasis. There are two main subtypes of TAMs: the pro-inflammatory and tumoricidal M1-like phenotype and the anti-inflammatory and pro-repair M2-like phenotype. The plasticity of tumor-associated macrophages determines that the phenotype and function can be switched in response to environmental stimuli. Therefore, TAMs are considered to be a potential anti-cancer target. Common strategies for using nanomedicines to target TAMs for anti-tumor include macrophage polarization reprogramming and regulating related tumor immune microenvironment.

[0004] The literature (ACS Nano. 2019, 13, 12671-12686) uses nanoparticle encapsulation (NE) as a loading system and provides an immunotherapy platform of freeze-dried NEs loaded with TLR7 / 8a, which can induce T cell activation and stimulation enhancement and macrophage polarization, reprogram M2 TAMs in tumor tissues into M1 TAMs, and transform tumor cells from cold tumors to hot tumors, which can better respond to external drug stimulation, thereby improving the anti-tumor effect.

[0005] Currently, there are no reports on the effects of nanogels with different hardness on the reverse polarization of M2 tumor-associated macrophages and tumor growth. Summary of the invention

[0006] In view of the defects of the prior art, the purpose of the present application is to provide the use of nanogels in the use or preparation of anti-tumor drugs. By synthesizing nanogels with appropriate hardness, the M2 tumor-associated macrophages are reversely polarized to the M1 type, thereby effectively inhibiting the growth of tumor cells and improving the anti-tumor effect.

[0007] To achieve the above objectives, in a first aspect, the present application provides an application of a nanogel for use as or in the preparation of an M2 TAMs reverse polarization drug, wherein the nanogel has a Young's modulus of 20-600 KPa and is capable of reverse polarizing M2 TAMs to M1.

[0008] In the second aspect, the present application also provides an application of a nanogel for use as or in the preparation of an anti-tumor drug, the active ingredient of the anti-tumor drug being the nanogel; the Young's modulus of the above-mentioned nanogel is 20~600 KPa, and when used for anti-tumor treatment, it can reverse polarize M2 type TAMs to M1 type, thereby inhibiting tumor cell proliferation.

[0009] Preferably, the Young's modulus of the nanogel is 50-600 KPa.

[0010] Preferably, the nanogel is obtained by polymerization of monomers in the presence of a crosslinking agent and a surfactant in an aqueous phase by initiating a polymerization reaction of the monomers; The Young's modulus of the nanogel can be adjusted by adjusting the molar ratio of the crosslinking agent to the monomer.

[0011] Preferably, the above-mentioned monomers include one or more of temperature-responsive monomers, pH-responsive monomers and reduction-responsive monomers.

[0012] Preferably, the temperature-responsive monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide.

[0013] Preferably, the pH responsive monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid.

[0014] Preferably, the cross-linking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide and N,N'-vinylbisacrylamide.

[0015] Preferably, the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide.

[0016] Preferably, the surfactant is one or more of sodium lauryl sulfate, sodium lauryl sulfonate and lecithin.

[0017] Preferably, the monomers include temperature-responsive monomers and pH-responsive monomers; the molar ratio of the pH-responsive monomers to the temperature-responsive monomers is (3-8):100.

[0018] Preferably, the molar ratio of the cross-linking agent to the temperature-responsive monomer is (1-20):100.

[0019] Preferably, the mass ratio of the initiator to the temperature-responsive monomer is (5-15):550.

[0020] Preferably, the mass ratio of the above-mentioned surfactant to the above-mentioned temperature-responsive monomer is (20~35):550.

[0021] Preferably, the reaction temperature of the above polymerization reaction is 70°C to 85°C, and the reaction time is 4 to 8 h.

[0022] Preferably, the above-mentioned tumor cells include one or more of liver cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, esophageal squamous cell cancer cells, gastric cancer cells, ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, lymphoma cells, melanoma cells, and glioblastoma cells.

[0023] Preferably, when the nanogel is used for anti-tumor treatment, the nanogel is administered by intratumor injection.

[0024] Preferably, the dosage of the nanogel is 1-10 μg / mm³.

[0025] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art: (1) The present application provides the use of nanogels for use as or in the preparation of M2 TAMs reverse polarization drugs (i.e., drugs for reverse polarization of M2 tumor-associated macrophages to M1 type). Experiments have found that nanogels with appropriate hardness can promote the expression of M1-related proteins CD86 and iNOS, upregulate the expression of CD86 mRNA and iNOS mRNA, and reverse polarization of M2 tumor-associated macrophages to M1 type. Further studies have found that the greater the hardness of the nanogel, the better the effect of reverse polarization of M2 tumor-associated macrophages to M1 type.

[0026] (2) The present application provides the use of nanogels for use as or in the preparation of anti-tumor drugs. Experiments have found that the use of nanogels with appropriate hardness to reverse polarize M2 tumor-associated macrophages can inhibit tumor cell proliferation, induce tumor cell apoptosis, and phagocytize tumor cells. In addition, the nanogels with appropriate hardness can be directly delivered into the tumor by intratumoral injection, which can inhibit tumor growth. The greater the hardness of the nanogel, the better the effect of inhibiting tumor growth. The present application achieves the best anti-tumor treatment strategy by making full use of the mechanical properties of nanogels, and has great potential for clinical transformation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the particle size distribution and surface charge of the nanogel provided in the examples of the present application, wherein content A is the particle size distribution and content B is the surface charge; Figure 2 is a transmission electron microscope image of the nanogel provided in the examples of the present application; Figure 3 It is an atomic force microscope image and Young's modulus of the nanogel provided in the embodiment of the present application, wherein content A is an atomic force microscope image, and content B is Young's modulus; Figure 4 is the responsiveness of the nanogel provided in the examples of the present application, wherein content A is temperature responsiveness, content B is pH responsiveness, and content C is reduction responsiveness; Figure 5 It is the effect of the nanogel provided in the examples of the present application on the reverse polarization of M2 macrophages, wherein content A is the expression of M1-related proteins, content B is the mRNA expression level, and content C is the relative fluorescence intensity of CD86; Figure 6 It is the cell viability of tumor cells after the nanogel reverse polarized M2 macrophages provided in the examples of the present application were co-incubated with tumor cells, wherein content A is H22 cells and content B is 4T1 cells; Figure 7 It is the apoptosis rate of tumor cells after the nanogel reverse polarized M2 macrophages provided in the examples of the present application were co-incubated with tumor cells, wherein content A is H22 cells and content B is 4T1 cells; Figure 8 It is the phagocytic ability of the nanogel provided in the embodiment of the present application after reverse polarization of M2 macrophages and co-incubation with tumor cells, wherein content A is the cell grouping selected on the flow cytometer according to different cells labeled with fluorescence, and content B is the phagocytic ratio; Fig. 9The nanogel provided in the embodiment of the present application reversely polarizes M2 macrophages and then co-incubates with tumor cells to kill mouse tumors, wherein content A is the experimental process, content B is the tumor volume, content C is the tumor weight, and content D is the tumor picture; Fig.10 The reverse polarization degree of macrophages after the nanogel reverse polarized M2 macrophages provided in the embodiment of the present application and co-incubated with tumor cells, wherein content A is the immunofluorescence intensity, content B is the CD86 + The positive rate; Fig.11 This is the experimental process of intratumoral injection of nanogel into breast cancer mice provided in the examples of the present application; Fig.12 The nanogel provided in the examples of the present application has an inhibitory effect on tumor growth in mice after intratumoral injection into breast cancer mice; Fig.13 This is the effect of the nanogel provided in the examples of the present application on the relative growth rate of the mouse tumor after intratumoral injection into breast cancer mice. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0029] In the description of this application, it should be understood that the term "and / or" is a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The symbol " / " herein indicates that the associated objects are in an or relationship, for example, A / B means A or B.

[0030] In the description of the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0031] In the description of the embodiments of the present application, unless otherwise specified, “plurality” means two or more than two.

[0032] The term "M2 TAMs reverse polarization drug" refers to drugs that can reverse polarize M2 tumor-associated macrophages (TAMs) to M1.

[0033] The term "intratumoral injection" or "intratumoral therapy" refers to a tumor treatment method that delivers therapeutic active ingredients directly into the tumor. This method of administration avoids the systemic distribution and plasma and tissue clearance effects of intravenous administration, and can more effectively achieve targeted drug delivery and tumor treatment. The high drug concentration in the lesion site and the low systemic drug distribution can effectively achieve toxicity reduction and synergistic effects.

[0034] Nanogel is a nano hydrogel with the advantages of hydrogel and nanoparticles. Patent document CN115960305A adjusts the cross-linking degree of nanogel, uses nanogels of different hardness as carriers to load anti-tumor chemotherapy drugs through electrostatic adsorption, and obtains soft nano drugs (Young's modulus of soft nano gel is 20~150KPa) and hard nano drugs, and then administers the above nano drugs by intravenous injection. The experiment found that soft nano drugs can be enriched in the tumor site rather than the liver part, and at the same time, the soft nano drugs have the ability to penetrate from tumor blood vessels to the deep part of the tumor and the anti-tumor effect are significantly better than hard nano gel. Different from the prior art, the inventor of this application accidentally discovered during the experiment that directly administering nano gels of appropriate hardness to mouse tumors by intratumoral injection can reverse polarization of M2 tumor-associated macrophages to M1 type, effectively inhibit tumor growth, and the anti-tumor effect is even better than the above soft nano drugs administered by intravenous injection. Based on this, the present application provides an application of a nanogel for use as or in the preparation of an M2 TAMs reverse polarization drug, wherein the nanogel has a Young's modulus of 20-600 KPa and is capable of reverse polarizing M2 TAMs to M1.

[0035] The present application also provides an application of a nanogel for use as or in the preparation of an anti-tumor drug, wherein the active ingredient of the anti-tumor drug is the nanogel; the Young's modulus of the nanogel is 20~600 KPa, and when used for anti-tumor treatment, it can reverse polarize M2 tumor-associated macrophages to M1 type, thereby inhibiting tumor cell proliferation, inducing tumor cell apoptosis, phagocytizing tumor cells, and achieving an anti-tumor effect.

[0036] The inventor unexpectedly found that after co-incubating nanogels with appropriate hardness with M2 macrophages, the expression of M1-related proteins CD86 and iNOS can be promoted, and the expression of CD86 mRNA and iNOS mRNA can be upregulated, so that M2 tumor-associated macrophages are reverse polarized to M1. Further studies have found that the greater the hardness of the nanogel, the better the effect of reverse polarizing M2 tumor-associated macrophages to M1. Furthermore, the nanogel of appropriate hardness is directly delivered to the tumor by intratumoral injection, which can inhibit the growth of the tumor, and the greater the hardness of the nanogel, the better the effect of inhibiting tumor growth.

[0037] In some embodiments, the Young's modulus of the nanogel is between 50 and 600 KPa, and more preferably between 70 and 600 KPa.

[0038] In some embodiments, the nanogel is obtained by a polymerization reaction of monomers in the presence of a crosslinker and a surfactant in an aqueous phase initiated by an initiator; wherein the Young's modulus of the nanogel can be controlled by adjusting the molar ratio of the crosslinker to the monomer.

[0039] The nanogels synthesized in the present application may use common monomers for preparing biocompatible nanogels, and different monomers may be used to give the nanogels different functions, such as hydrophilicity, pH responsiveness, reduction responsiveness, etc. The monomers include but are not limited to one or more of temperature responsive monomers, pH responsive monomers, and reduction responsive monomers. In some embodiments, the temperature responsive monomers are one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide, and N-ethyl acrylamide. In some embodiments, the pH responsive monomers are one or more of methacrylic acid, acrylic acid, and 2-acrylamido-2-methyl-1-propane sulfonic acid.

[0040] In a preferred embodiment, the monomers include a temperature-responsive monomer and a pH-responsive monomer. More preferably, the temperature-responsive monomer is N-isopropyl methacrylamide, the pH-responsive monomer is methacrylic acid, and the molar ratio of the pH-responsive monomer to the temperature-responsive monomer is (3-8):100.

[0041] In some embodiments, the crosslinking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide and N,N'-vinylbisacrylamide. The crosslinking agent is more preferably a reduction-responsive crosslinking agent. In a preferred embodiment, the crosslinking agent is N,N'-bis(acryloyl)cystamine.

[0042] In some embodiments, the molar ratio of the crosslinking agent to the temperature-responsive monomer is (1-20):100, preferably (2-20):100. The higher the molar ratio, the greater the Young's modulus of the prepared nanogel, that is, the higher the hardness.

[0043] In some embodiments, the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide.

[0044] In some embodiments, the mass ratio of the initiator to the temperature-responsive monomer is (5-15):550.

[0045] In some embodiments, the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin.

[0046] In some embodiments, the mass ratio of the above-mentioned surfactant to the above-mentioned temperature-responsive monomer is (20-35):550.

[0047] In a preferred embodiment of the present application, the crosslinking degree of the nanogel is regulated by adjusting the molar ratio of the crosslinking agent to the temperature-responsive monomer, and then the hardness of the nanogel is regulated. The larger the molar ratio of the two, the higher the hardness of the prepared nanogel. In addition, the hydrated particle size of the nanogel can be regulated by adjusting the amount of the surfactant, so that the nanogel can be applied to the tumor site by intratumoral injection. In some embodiments, the average hydrated particle size of the above-mentioned nanogel in PBS buffer at 37°C is 150~300 nm, and the Young's modulus of the above-mentioned nanogel is 79.0~439.2KPa.

[0048] In some embodiments, the reaction temperature of the polymerization reaction is 70°C to 85°C, and the reaction time is 4 to 8 hours.

[0049] In some embodiments, the above-mentioned tumor cells include but are not limited to one or more of liver cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, esophageal squamous cell cancer cells, gastric cancer cells, ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, lymphoma cells, melanoma cells, and glioblastoma cells.

[0050] In some embodiments, when the nanogel is used for anti-tumor treatment, the nanogel is administered by intratumor injection.

[0051] In some embodiments, the dosage of the nanogel is 1-10 μg / mm³.

[0052] In actual application, technicians in this field can, according to the actual situation, perform another intratumoral injection 4 to 10 days after the first intratumoral injection, in order to enhance the anti-tumor effect.

[0053] It should be understood that materials of the same or similar type, model, quality, nature or function as the reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0054] When preparing nanogels in the following examples of the present application, the molar ratio of the crosslinking agent to the temperature-responsive monomer is defined as X:100, and the corresponding crosslinking degree of the nanogel is X%, and the prepared nanogel is represented as X%NGs.

[0055] The following are examples: Example 1 Effects of nanogels with different hardness on the reverse polarization of M2 macrophages 1.1 Preparation of nanogels 550 mg of temperature-responsive monomer N-isopropylmethylpropionamide and 35 mg of surfactant sodium dodecyl sulfate were ultrasonically dissolved in 80 mL of ultrapure water to obtain a mixed solution. 22.5 g of crosslinker N,N'-bis(acryloyl)cystamine were ultrasonically dissolved in 0.5 mL of ethanol and then added to the above mixed solution. 3.65 mL of pH-responsive monomer methacrylic acid was dissolved in 6.35 mL of ultrapure water, and 50 μL was added to the above solution. The resulting solution was evacuated for 10 min, and then argon was introduced and circulated three times to fully remove oxygen from the solution, and then heated to 80 ℃ and maintained for 10 min. 10 mg of initiator potassium persulfate was weighed, 0.5 mL of ultrapure water was added, ultrasonically dissolved, and then added to the above heated solution using a syringe to initiate the polymerization reaction, and the reaction time was 6 h. After the reaction is completed, the reaction solution is cooled to room temperature and transferred to an ultrafiltration tube with a cutoff value of 10 kDa. Unreacted monomers and other impurities are removed by centrifugation at 2000 rpm, and excess water is removed at the same time. After concentration, ultrapure water is repeatedly added to wash 3 times, and 8 mL of concentrated solution is obtained. Take 300 μL of the above concentrated solution, dry and weigh it, and calculate the solid content in the concentrated solution. According to the calculation results, the above concentrated solution is diluted to 20 mg / mL to obtain a nanogel solution with a cross-linking degree of 2% (recorded as 2% NGs), which is stored at 4 °C for later use.

[0056] According to the above steps, nanogel solutions with crosslinking degrees of 5%, 10%, and 15% were prepared, wherein the amounts of surfactant sodium dodecyl sulfate were 30 mg, 25 mg, and 20 mg, respectively; the amounts of crosslinking agent N,N'-bis(acryloyl)cystamine were 56.3 mg, 112.6 mg, and 168.9 mg, respectively, and the above crosslinking agents were ultrasonically dissolved in 1 mL, 2 mL, and 3 mL of ethanol, respectively, and then added to the above mixed solutions. Finally, 5%NGs, 10%NGs, and 15%NGs were prepared and stored at 4 °C for later use.

[0057] 1.2 Physical properties of nanogels Take 10 μL of the nanogel solution prepared in step 1.1 and disperse it in 1 mL of PBS buffer and 1 mL of ultrapure water, respectively. Use dynamic light scattering to detect the hydrated particle size and zeta potential of the nanogel. The detection temperature is 37 °C and the equilibrium time is 15 min.

[0058] Take the nanogel solution prepared in step 1.1 and disperse it with ultrapure water to a concentration of 0.01 mg / mL. Take 10 μL of the above dispersion and drop it on the carbon support membrane. Dry it naturally. After drying, add 10 μL of 1% phosphotungstic acid aqueous solution to the carbon support membrane and stain it for 2 min. After staining, use filter paper to absorb the excess phosphotungstic acid solution along the edge of the carbon support membrane. Add 10 μL of ultrapure water to wash for 1 min. After washing, use filter paper to absorb the excess ultrapure water along the edge of the carbon support membrane. After drying naturally, use transmission electron microscopy to observe the morphology of the nanogel.

[0059] The coverslip was soaked in 1% polyethyleneimine aqueous solution for 24 h to modify the surface of the coverslip with positive charge. The nanogel solution prepared in step 1.1 was dispersed with ultrapure water to a concentration of 0.01 mg / mL. 10 μL of the above dispersion was added dropwise to the positively charged coverslip. The solution was adsorbed electrostatically for 10 min to remove excess dispersion. Then 300 μL of ultrapure water was added to wash away the unadsorbed nanogel. The height and Young's modulus of the nanogel were then detected using an atomic force microscope. The detection environment was liquid phase, the image was acquired in contact mode, and the Young's modulus was detected in tapping mode.

[0060] The results show that: Figure 1 Content A and Figure 1 It can be seen from content B that the average hydrated particle size of the nanogels of different hardness prepared in this example is about 220nm, and the surface charge is all negative, which increases slightly with the increase of crosslinking degree. Figure 2 It can be seen that the nanogels of different hardness prepared in this example are all spherical with uniform particle size distribution. Figure 3 Content A and Figure 3 It can be seen from content B that the nanogels of different hardness prepared in this example are all spherical with uniform particle size distribution, and the Young's modulus is 79.0-439.2 kPa.

[0061] 1.3 Triple responsiveness of nanogels Take 10 μL of the nanogel solution prepared in step 1.1 and disperse it into 1 mL of ultrapure water at different temperatures. Use dynamic light scattering to detect the temperature responsiveness of the nanogel. The detection temperature range is 25~55 ℃, the temperature interval is 1 ℃, and the equilibrium time is 1 min.

[0062] Take 10 μL of the nanogel solution prepared in step 1.1 and disperse it in 1 mL of ultrapure water. Adjust the pH to 3-9 and use dynamic light scattering to detect the pH responsiveness of the nanogel. The detection temperature is 25 °C and the equilibrium time is 15 min.

[0063] Take 10 μL of the nanogel solution prepared in step 1.1 and disperse it in 1 mL of ultrapure water containing or not containing 10 mM glutathione (GSH), and incubate at room temperature for 24 h. Then take 10 μL of the above dispersion and drop it on the carbon support membrane, dry it naturally, and then drop 10 μL of 1% phosphotungstic acid aqueous solution on the carbon support membrane after drying, and stain it for 2 min. After staining, use filter paper to absorb the excess phosphotungstic acid solution along the edge of the carbon support membrane, and add 10 μL of ultrapure water to wash for 1 min. After washing, use filter paper to absorb the excess ultrapure water along the edge of the carbon support membrane, and observe the structure of the nanogel using a transmission electron microscope after drying naturally.

[0064] The results show that: Figure 4 Content A. Figure 4 Content B and Figure 4 Content C shows that as the temperature increases, the hydrated particle size of the nanogel prepared in this example gradually decreases, and the shrinkage degree decreases with the increase of the cross-linking degree; as the pH increases, the hydrated particle size of the nanogel prepared in this example gradually increases, and the swelling degree decreases with the increase of the cross-linking degree; after incubation with glutathione for 24 hours, the structure prepared in this example is destroyed. That is, the nanogel prepared in this example has excellent temperature responsiveness, pH responsiveness and reduction responsiveness.

[0065] 1.4 Effect of nanogel on the reverse polarization of M2 macrophages 1) Induction of M2 macrophages: RAW264.7 mouse macrophages were cultured at a rate of 1×10 6 The cells were inoculated into 6-well plates at a concentration of 100 μg / well. Then, 2 mL of a medium mixed with IL-4, IL-13, and DMEM containing 10% serum was added to each well. The IL-4 dose was 20 μg / mL and the IL-13 dose was 20 μg / mL. The plates were incubated at 37°C and 5% CO. 2 The cells were incubated in an incubator for 24 h, and then the upper culture medium was removed to obtain M2 macrophages.

[0066] 2) The nanogel solutions of different hardness prepared in step 1.1 were mixed with DMEM medium containing 10% serum to obtain mixed medium containing nanogels of different hardness, wherein the concentration of nanogel was 200 μg / mL. 2 mL of mixed medium containing nanogels of different hardness was added to each well of the 6-well plate containing M2 macrophages, and the cells were collected after incubation in the incubator for 24 hours, washed 3 times with PBS buffer, digested with trypsin, and collected by centrifugation. Among them, the DMEM medium containing 10% serum without the addition of nanogel solution was used as the control group, the mixed medium containing 2% NGs nanogel was used as the 2% NGs treatment group, and the medium containing 15% NGs nanogel was used as the 15% NGs treatment group.

[0067] Immunoblotting was used to detect the expression of M1 and M2 related proteins. RT-PCR was used to detect the expression levels of iNOS mRNA and CD86 mRNA in cells. The cells collected by centrifugation were stained on ice in the dark for 20 minutes with PE anti-mouse CD86 at a dose of 25 μg / μL, then washed twice with PBS buffer, collected by centrifugation and the fluorescence intensity was detected by flow cytometry. The detection channel was PE, and the degree of macrophage M1 polarization was calculated based on the fluorescence intensity.

[0068] The results show that: Figure 5 Content A. Figure 5 Content B. Figure 5 Content C shows that compared with the control group, the 2% NGs treatment group and the 15% NGs treatment group can significantly upregulate the expression of M1-related proteins CD86 and iNOS; the 15% NGs treatment group can significantly upregulate the expression of CD86 mRNA and iNOS mRNA. This indicates that nanogels of different hardness can reverse polarization of M2 macrophages to M1.

[0069] Example 2 Killing of tumor cells by nanogel after reverse polarization treatment of M2 macrophages 1) The method for inducing the formation of M2 macrophages is the same as in Example 1.

[0070] 2) The nanogel solutions of different hardness prepared in step 1.1 were mixed with DMEM medium containing 10% serum to obtain mixed medium containing nanogels of different hardness, wherein the concentration of nanogels was 200 μg / mL. 2 mL of mixed medium containing nanogels of different hardness was added to each well of the 6-well plate containing M2 macrophages, and incubated in an incubator for 24 h. The upper medium was removed, and then 2 mL of DMEM medium without serum was added to each well. The culture medium was incubated at 37°C and 5% CO. 2 After incubation in the incubator for 24 h, the upper culture medium was collected and set aside. Among them, the DMEM medium containing 10% serum without the addition of nanogel solution was used as the M2 treatment group, the mixed medium containing PBS with an equal volume of nanogel was used as the PBS treatment group, the mixed medium containing 2% NGs nanogel was used as the M2+2%NGs treatment group, and the medium containing 15% NGs nanogel was used as the M2+15%NGs treatment group.

[0071] 3) H22 cells were cultured at a rate of 5×10 3 The cells were inoculated into 96-well plates at a concentration of 100 μL / well, and 200 μL of the above-mentioned upper culture medium was added to each well. The plates were incubated at 37 °C and 5% CO 2 Incubate in an incubator for 24 h, then add 22 μL of CCK8 to each well and incubate at 37 °C, 5% CO 2The cells were incubated in an incubator for 1 h, and the absorbance of the culture medium was measured at 450 nm using an enzyme reader. The results were processed and analyzed using Excel and Graphpad Prism to calculate the killing effect of the supernatant of M2 macrophages treated with nanogels of different hardness on H22 cells.

[0072] 4T1 cells were cultured at a rate of 5 × 10 3 The cells were inoculated into a 96-well plate at a concentration of 100 μL / well, and 200 μL of RPMI-1640 medium containing 10% serum was added to each well. The plates were incubated at 37°C and 5% CO 2 Incubate in the incubator for 12 h. After the 4T1 cells adhere to the wall, remove the upper culture medium and add 200 μL of the upper culture medium to each well. Incubate at 37 °C and 5% CO 2 Incubate in an incubator for 24 h, remove the upper culture medium, and then add 100 μL of a culture medium mixed with CCK8 and RPMI-1640 culture medium containing 10% serum in a ratio of 1:9 to each well. Incubate at 37 °C and 5% CO 2 The cells were incubated in an incubator for 1 h, and the absorbance of the culture medium was measured at 450 nm using an enzyme reader. The results were processed and analyzed using Excel and Graphpad Prism to calculate the killing effect of the supernatant of M2 macrophages treated with nanogels of different hardness on 4T1 cells.

[0073] The results show that: Figure 6 Content A. Figure 6 As can be seen from content B, compared with other treatment groups, the viability of H22 cells and 4T1 cells in the 2% NGs treatment group and the 15% NGs treatment group was significantly decreased, showing an inhibitory effect on the proliferation of H22 cells and 4T1 cells. This indicates that the supernatant after the nanogel reverse polarized M2 macrophages to M1 type can effectively kill tumor cells.

[0074] Example 3 Effect of nanogel on apoptosis of tumor cells after reverse polarization treatment of M2 macrophages 1) The method for inducing the formation of M2 macrophages is the same as in Example 1.

[0075] 2) The nanogel solutions of different hardness prepared in step 1.1 were mixed with DMEM medium containing 10% serum to obtain mixed medium containing nanogels of different hardness, wherein the concentration of nanogels was 200 μg / mL. 2 mL of mixed medium containing nanogels of different hardness was added to each well of the 6-well plate containing M2 macrophages, incubated in an incubator for 24 h, and the upper medium was removed. Then 2 mL of serum-free DMEM medium was added to each well and incubated at 37°C, 5% CO 2After incubation in an incubator for 24 h, the upper culture medium was collected and set aside. Among them, the DMEM medium without nanogel solution and containing only 10% serum was used as the control group, the mixed medium containing 2% NGs nanogel was used as the 2% NGs treatment group, and the medium containing 15% NGs nanogel was used as the 15% NGs treatment group.

[0076] 3) H22 cells were cultured at a rate of 1×10 6 The cells were inoculated into 6-well plates at a concentration of 100 μL / well, and 200 μL of the above-mentioned upper culture medium was added to each well. The plates were incubated at 37°C with 5% CO 2 Incubate in the incubator for 24 h, aspirate the upper culture medium, collect by centrifugation, resuspend the cells in 100 μL PBS, add 5 μL Annexin V-FITC and 10 μL PI Staining Solution, react at room temperature for 15 min in the dark, wash twice with PBS buffer, collect by centrifugation, and detect the fluorescence intensity by flow cytometry. The detection channels are PE and FITC. The degree of apoptosis of H22 cells is calculated according to the fluorescence intensity.

[0077] 4T1 cells were cultured at a rate of 1×10 6 The cells were inoculated into 6-well plates at a concentration of 100 μL / well, and 200 μL of RPMI-1640 medium containing 10% serum was added to each well. The plates were incubated at 37 °C and 5% CO 2 Incubate in the incubator for 12 h. After the 4T1 cells adhere to the wall, remove the upper layer of culture medium and add 200 μL of the upper layer of culture medium to each well. Incubate at 37°C, 5% CO 2 The cells were incubated in the incubator for 24 h, the upper culture medium was aspirated, the cells were washed three times with PBS buffer, the cells were digested with trypsin, and the cells were collected by centrifugation. The cells were resuspended in 100 μL PBS, and 5 μL Annexin V-FITC and 10 μL PI Staining Solution were added. The cells were reacted at room temperature for 15 min in the dark, washed twice with PBS buffer, and the cells were collected by centrifugation. The fluorescence intensity was detected by flow cytometry. The detection channels were PE and FITC. The degree of apoptosis of 4T1 cells was calculated according to the fluorescence intensity.

[0078] The results show that: Figure 7 Content A. Figure 7 As can be seen from content B, compared with the control group, the apoptosis rates of H22 cells and 4T1 cells in the 2% NGs treatment group and the 15% NGs treatment group were increased, among which the 15% NGs treatment group was able to effectively cause apoptosis of tumor cells, indicating that the supernatant after the nanogel reverse polarized M2 macrophages to M1 type can effectively cause apoptosis of tumor cells.

[0079] Example 4 Phagocytosis of tumor cells by nanogel after reverse polarization treatment of M2 macrophages 1) The method for inducing the formation of M2 macrophages is the same as in Example 1.

[0080] 2) The nanogel solutions of different hardness prepared in step 1.1 were mixed with DMEM medium containing 10% serum to obtain mixed medium containing nanogels of different hardness, wherein the concentration of nanogels was 200 μg / mL. 2 mL of mixed medium containing nanogels of different hardness was added to each well of the 6-well plate containing M2 macrophages, incubated in an incubator for 24 h, and the upper medium was removed. Then 2 mL of serum-free DMEM medium was added to each well and incubated at 37°C, 5% CO 2 The macrophages were collected after incubation in an incubator for 24 h. Among them, the DMEM medium containing 10% serum without adding nanogel solution was used as the control group, the mixed medium containing 2% NGs nanogel was used as the 2% NGs treatment group, and the medium containing 15% NGs nanogel was used as the 15% NGs treatment group. The collected macrophages were washed three times with PBS buffer, digested with trypsin and collected by centrifugation, and then stained with FITC anti-mouse CD11b on ice in the dark for 20 mins at a dose of 25 μg / μL. The stained macrophages were divided into 1×10 6 The cells were inoculated into 6-well plates at a concentration of 100 / well for later use.

[0081] 3) 4T1 cells were cultured at a rate of 1×10 6 The cells were inoculated into 6-well plates at a concentration of 100 μL / well, and 200 μL of RPMI-1640 medium containing 10% serum was added to each well. The plates were incubated at 37 °C and 5% CO 2 Incubate in the incubator for 24 h, remove the upper culture medium, collect 4T1 cells, wash with PBS buffer three times, digest the cells with trypsin and collect them by centrifugation, and then stain with PE anti-mouse EPCAM on ice in the dark for 20 mins at a dose of 25 μg / μL. 6 The concentration of 1000 cells / well was added to the 6-well plate containing the stained macrophages and incubated at 37°C with 5% CO 2 The cells were incubated in the incubator for 4 h, the upper culture medium was aspirated, the cells were collected, and washed three times with PBS buffer. The cells were digested with trypsin and collected by centrifugation. The fluorescence intensity of the cells was detected by flow cytometry, and the detection channels were PE and FITC. The degree of macrophage phagocytosis of 4T1 cells was calculated based on the fluorescence intensity.

[0082] The results show that: Figure 8 Content A. Figure 8As can be seen from content B, compared with the control group, the proportion of macrophages engulfing 4T1 cells in the 2% NGs treatment group and the 15% NGs treatment group increased significantly, indicating that nanogels with different hardness can effectively engulf tumor cells after reverse polarization treatment of M2 macrophages.

[0083] Example 5 Killing of mouse tumors by nanogel after reverse polarization treatment of M2 macrophages 1) RAW264.7 mouse macrophages were cultured at a rate of 1×10 6 The cells were inoculated into 6-well plates at a concentration of 100 μg / well. Then, 2 mL of a medium mixed with IL-4, IL-13, and DMEM containing 10% serum was added to each well. The IL-4 dose was 20 μg / mL and the IL-13 dose was 20 μg / mL. The plates were incubated at 37°C and 5% CO. 2 The cells were incubated in an incubator for 24 h, and then the upper culture medium was removed to obtain M2 macrophages.

[0084] RAW264.7 mouse macrophages were cultured at a rate of 1 × 10 6 The cells were inoculated into 6-well plates at a concentration of 100 μg / well, and 2 mL of DMEM medium containing 10% serum was added to each well. The plates were incubated at 37°C and 5% CO 2 The cells were incubated in an incubator for 24 h, and then the upper culture medium was removed to obtain M0 macrophages.

[0085] RAW264.7 mouse macrophages were cultured at a rate of 1 × 10 6 The cells were inoculated into 6-well plates at a concentration of 100 μg / well, and 2 mL of DMEM medium containing 10% serum was added to each well. The plates were incubated at 37°C and 5% CO 2 The cells were incubated in an incubator for 24 h, the upper medium was removed by aspiration, and 2 mL of a medium mixed with LPS and DMEM medium containing 10% serum was added to each well, where the LPS dose was 1 μg / mL. The cells were incubated at 37°C and 5% CO 2 The cells were incubated in an incubator for 24 h, and then the upper culture medium was removed to obtain M1 macrophages.

[0086] 3) The nanogel solutions of different hardness prepared in step 1.1 were mixed with DMEM medium containing 10% serum to obtain mixed medium containing nanogels of different hardness, wherein the concentration of nanogels was 200 μg / mL. Then, 2 mL of mixed medium containing nanogels of different hardness was added to each well of the 6-well plate containing M2 macrophages in step 1), incubated in an incubator for 24 h, and the upper medium was removed. Then, 2 mL of serum-free DMEM medium was added to each well and incubated at 37°C, 5% CO 2After incubation in the incubator for 24 h, the upper medium was removed, the cells were collected, and washed three times with PBS buffer. The cells were digested with trypsin and centrifuged to collect macrophages, and the collected macrophages were mixed with 4T1 cells to obtain mixed cells. Among them, the macrophages collected after treatment with the mixed medium containing 2% NGs nanogel were mixed with 4T1 cells at a ratio of 2:1 as the 2% NGs treatment group; the macrophages collected after treatment with the mixed medium containing 15% NGs nanogel were mixed with 4T1 cells at a ratio of 2:1 as the 15% NGs treatment group; the M2 macrophages prepared in step 1) were mixed with 4T1 cells at a ratio of 2:1 as the M2 treatment group; the M0 macrophages prepared in step 1) were mixed with 4T1 cells at a ratio of 2:1 as the M0 treatment group; the M1 macrophages prepared in step 1) were mixed with 4T1 cells at a ratio of 2:1 as the M1 treatment group.

[0087] 4) The mixed cells of the above different treatment groups were inoculated subcutaneously on the back of female BALB / C mice near the right hind limb, with an inoculation number of 3×10 6 The inoculation volume was 100 μL to construct a mixed model of 4T1 breast cancer subcutaneous tumor in mice. Fig. 9 As shown in Content A. When the tumor volume reaches 100 mm 3 The first day was recorded as the first day, and the long side (a) and short side (b) of the subcutaneous tumor of the mouse were measured with a vernier caliper every day. The tumor volume V = (a × b 2 ) / 2, and the tumor volume was calculated. After the measurement on day 14, the mice were killed, the tumors were excised, weighed and photographed. The excised tumors were fixed with 4% paraformaldehyde and sliced, CD86 immunofluorescence was performed, and then the CD86 area ratio was quantified to evaluate the repolarization degree of tumor-associated macrophages TAMs.

[0088] The results show that: Fig. 9 Content B Tumor volume, Fig. 9 Content C Tumor weight, Fig. 9 From the tumor image in content D, we can see that compared with the M2 treatment group, the volume and tumor weight of breast cancer tumors in the 2% NGs treatment group and the 15% NGs treatment group were significantly lower than those in the M2 treatment group. This indicates that the use of nanogels with different hardness to treat M2 macrophages can significantly affect tumor growth and has a good tumor killing effect. Fig.10 Content A. Fig.10 Content B shows that compared with the M2 treatment group, the fluorescence intensity in the 2% NGs treatment group and the 15% NGs treatment group increased, and CD86 + The positive rate increased significantly, indicating that the use of nanogels with different hardness to treat M2 macrophages can significantly increase the expression of CD86 +The expression of M2 macrophages can enhance the polarization degree of M2 macrophages to M1 macrophages.

[0089] Example 6 Inhibitory effect of nanogel on tumor growth in mice Female BALB / C mice were inoculated subcutaneously on the back near the right hind limb with 1×10 6 4T1 cells were added in a volume of 100 μL to construct a mouse 4T1 breast cancer subcutaneous tumor model. When the tumor volume reached 200 mm 3 The day after the onset of 2-th day of the experiment was marked as day 0, and the mice were randomly divided into three groups, including a control group, a 2% NGs treatment group, and a 15% NGs treatment group. On the first and eighth days after the grouping, the above treatment groups were injected intratumorally with 25 μL of normal saline, 2% NGs, and 15% NGs, respectively (the injection dose of the nanogel was 20 mg / mL). The experimental process is as follows: Fig.11 As shown. Since the first day of grouping, the long side (a) and short side (b) of the subcutaneous tumor of mice were measured every day with a vernier caliper, and the tumor volume V = (a × b 2 ) / 2, and the tumor volume was calculated. After the measurement on the 14th day, the mice were killed.

[0090] The results show that: Fig.12 , Fig.13 It can be seen that in the 2% NGs treatment group and the 15% NGs treatment group, the volume and relative growth rate of breast cancer tumors were significantly lower than those in the control group, which indicates that intratumoral injection of nanogels with appropriate hardness can effectively inhibit tumor growth.

[0091] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A use of a nanogel for use as or in the preparation of an M2 TAMs reverse polarization drug, characterized in that: The Young's modulus of the nanogel is 20-600 KPa, and the nanogel can reverse polarization of M2-type TAMs to M1-type.

2. A use of a nanogel for use as or in the preparation of an anti-tumor drug, characterized in that: The active ingredient of the anti-tumor drug is the nanogel; the Young's modulus of the nanogel is 20-600 KPa, and when used for anti-tumor treatment, it can reverse polarize M2-type TAMs to M1-type, thereby inhibiting tumor cell proliferation.

3. The use according to claim 1 or 2, characterized in that: The Young's modulus of the nanogel is 50-600 KPa.

4. The use according to claim 1 or 2, characterized in that: The nanogel is obtained by polymerization of monomers in the presence of a crosslinking agent and a surfactant in an aqueous phase initiated by an initiator; The Young's modulus of the nanogel can be adjusted by adjusting the molar ratio of the crosslinking agent to the monomer.

5. The use according to claim 4, characterized in that: The monomers include one or more of temperature-responsive monomers, pH-responsive monomers and reduction-responsive monomers.

6. The use according to claim 5, characterized in that: The temperature-responsive monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; and / or, The pH-responsive monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid; and / or, The cross-linking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide and N,N'-vinylbisacrylamide; and / or, The initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide; and / or, The surfactant is one or more of sodium lauryl sulfate, sodium lauryl sulfonate and lecithin.

7. The use according to claim 6, characterized in that: The monomers include a temperature-responsive monomer and a pH-responsive monomer; the feeding molar ratio of the pH-responsive monomer to the temperature-responsive monomer is (3-8):100; and / or, The molar ratio of the cross-linking agent to the temperature-responsive monomer is (1-20):100; and / or, The mass ratio of the initiator to the temperature-responsive monomer is (5-15):550; and / or, The mass ratio of the surfactant to the temperature-responsive monomer is (20-35):

550.

8. The use according to claim 4, characterized in that: The reaction temperature of the polymerization reaction is 70°C to 85°C, and the reaction time is 4 to 8 hours.

9. The use according to claim 2, characterized in that: The tumor cells include one or more of liver cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, esophageal squamous cell cancer cells, gastric cancer cells, ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, lymphoma cells, melanoma cells, and glioblastoma cells.

10. The use according to claim 2, characterized in that: When used for anti-tumor treatment, the nanogel is administered by intratumor injection; Preferably, the dosage of the nanogel is 1-10 μg / mm³.

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