Application of nanogels in the use or preparation of anti-tumor drugs

Abstract

The present application belongs to the field of nanogel preparation technology, and more specifically, relates to the use of nanogels in the use or preparation of anti-tumor drugs. The present application experiment 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 after using the above-mentioned nanogels to reverse polarize M2 tumor-associated macrophages, it can inhibit tumor cell proliferation, induce tumor cell apoptosis, and phagocytize tumor cells. In addition, nanogels with appropriate hardness are directly delivered into the tumor by intratumoral injection, which can effectively inhibit tumor growth.

Description

Technical Field

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

[0002] Macrophages are essential players in maintaining homeostasis and regulating immune responses in healthy tissues. Their primary function is to balance activation of the inflammatory cascade in response to tissue injury while initiating tissue repair. Parallel to homeostatic disruptions triggered by recognition of harmful organisms, apoptotic cells, cellular debris, or toxic metabolic byproducts, tumor cell transformation triggers macrophage polarization to restore equilibrium within the tumor microenvironment (TME). However, because growing tumors never achieve homeostatic equilibrium, TAM phenotypes remain locked in a cycle that ultimately favors immune cell activation and tissue repair. Exploring the intrinsic characteristics of macrophages co-opted by tumor cells is crucial for predicting their contributions to cancer progression and identifying therapeutically exploitable vulnerabilities.

[0003] Tumor-associated macrophages (TAMs), the most widespread immune cell in the tumor microenvironment, account for approximately 50%. TAMs play a crucial role in tumor growth, invasion, and metastasis. TAMs are primarily divided into two subtypes: the pro-inflammatory and tumoricidal M1-like phenotype and the anti-inflammatory and pro-reparative M2-like phenotype. The plasticity of tumor-associated macrophages allows them to switch phenotype and function in response to environmental stimuli. Therefore, TAMs are considered a promising anti-cancer target. Common strategies for using nanomedicines to target TAMs for anti-tumor effects include reprogramming macrophage polarization and modulating the tumor immune microenvironment.

[0004] The literature (ACS Nano. 2019, 13, 12671-12686) uses nanoparticle encapsulation (NE) as a loading system to provide 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 response to the defects of the existing technology, the purpose of this application is to provide the use of nanogels in the use or preparation of anti-tumor drugs. By synthesizing nanogels with appropriate hardness, the polarization of M2 tumor-associated macrophages to M1 type is reversed, thereby effectively inhibiting the growth of tumor cells and enhancing 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 preparation of an M2 TAMs reverse polarization drug, wherein the nanogel has a Young's modulus of 20~600 KPa and can reverse polarize M2 TAMs to M1 type.

[0008] In a second aspect, 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 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 a polymerization reaction of monomers in the presence of a cross-linking agent and a surfactant in an aqueous phase initiated by an initiator;

[0011] The Young's modulus of the nanogel can be adjusted by regulating the molar ratio of the cross-linking agent to the monomer.

[0012] Preferably, the monomers include one or more of temperature-responsive monomers, pH-responsive monomers and reduction-responsive monomers.

[0013] Preferably, the temperature-responsive monomer is one or more of N-isopropylmethacrylamide, N-isopropylacrylamide and N-ethylacrylamide.

[0014] Preferably, the pH-responsive monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propanesulfonic acid.

[0015] Preferably, the cross-linking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide and N,N'-vinylbisacrylamide.

[0016] Preferably, the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide.

[0017] Preferably, the surfactant is one or more of sodium lauryl sulfate, sodium lauryl sulfonate and lecithin.

[0018] Preferably, the monomers include a temperature-responsive monomer and a pH-responsive monomer; the molar ratio of the pH-responsive monomer to the temperature-responsive monomer is (3-8):100.

[0019] Preferably, the molar ratio of the cross-linking agent to the temperature-responsive monomer is (1-20):100.

[0020] Preferably, the mass ratio of the initiator to the temperature-responsive monomer is (5-15):550.

[0021] Preferably, the mass ratio of the surfactant to the temperature-responsive monomer is (20-35):550.

[0022] Preferably, the reaction temperature of the polymerization reaction is 70°C to 85°C, and the reaction time is 4 to 8 hours.

[0023] 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.

[0024] Preferably, when the nanogel is used for anti-tumor treatment, the nanogel is administered by intratumor injection.

[0025] Preferably, the dosage of the nanogel is 1-10 μg / mm³.

[0026] In general, the above technical solutions conceived by this application have the following technical advantages compared with the existing technologies:

[0027] (1) This application provides the use of nanogels for use as or in the preparation of drugs for reverse polarization of M2 TAMs (i.e., drugs for reverse polarization of M2 tumor-associated macrophages to M1). 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. 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.

[0028] (2) This application provides the use of nanogels for use as or in the preparation of anti-tumor drugs. Experiments have found that using nanogels of appropriate hardness to reverse polarize M2 tumor-associated macrophages can inhibit tumor cell proliferation, induce tumor cell apoptosis, and phagocytose tumor cells. In addition, direct delivery of nanogels of appropriate hardness into tumors by intratumoral injection can inhibit tumor growth, and the greater the hardness of the nanogel, the better the effect of inhibiting tumor growth. This application achieves the best anti-tumor treatment strategy by fully utilizing the mechanical properties of nanogels, and has great potential for clinical transformation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 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;

[0030] Figure 2 is a transmission electron microscope image of the nanogel provided in the examples of the present application;

[0031] Figure 3 The atomic force microscope image and Young's modulus of the nanogel provided in the examples of the present application, wherein content A is the atomic force microscope image and content B is the Young's modulus;

[0032] 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;

[0033] Figure 5 This 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;

[0034] Figure 6 This is the cell viability of tumor cells after the nanogel provided in the examples of the present application reversely polarizes M2 macrophages and is co-incubated with tumor cells, wherein content A is H22 cells and content B is 4T1 cells;

[0035] Figure 7 The apoptosis rate of tumor cells after the nanogel provided in the examples of the present application reversely polarizes M2 macrophages and is co-incubated with tumor cells, wherein content A is H22 cells and content B is 4T1 cells;

[0036] Figure 8 The phagocytic ability of the nanogel provided in the examples of the present application after reverse polarization of M2 macrophages and co-incubation with tumor cells is shown, wherein content A is the cell clustering selected on the flow cytometer based on different fluorescently labeled cells, and content B is the phagocytic ratio;

[0037] Figure 9 The nanogel provided in the examples of this application reversely polarizes M2 macrophages and then co-incubates with tumor cells to kill mouse tumors. Content A is the experimental process, Content B is the tumor volume, Content C is the tumor weight, and Content D is a tumor image.

[0038] Figure 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;

[0039] Figure 11 This is the experimental process of intratumoral injection of nanogel into breast cancer mice provided in the examples of this application;

[0040] Figure 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;

[0041] Figure 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 of breast cancer mice. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0043] In the description of this application, it should be understood that the term "and / or" describes an association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The symbol " / " herein indicates that the associated objects are in an "or" relationship, for example, A / B means either A or B.

[0044] In the description of the embodiments of this 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 this application should not be construed as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0045] In the description of the embodiments of the present application, unless otherwise specified, “plurality” means two or more.

[0046] The term "M2 TAMs repolarization drug" refers to drugs that can repolarize M2 tumor-associated macrophages (TAMs) to M1.

[0047] The term "intratumoral injection" or "intratumoral therapy" refers to a cancer treatment approach in which therapeutic active ingredients are delivered directly into the tumor. This route of administration bypasses the systemic distribution and plasma and tissue clearance associated with intravenous administration, enabling more effective targeted drug delivery and tumor treatment. High drug concentrations at the lesion site and low systemic drug distribution effectively reduce toxicity and enhance efficacy.

[0048] Nanogels are nanohydrogels that combine the advantages of both hydrogels and nanoparticles. Patent document CN115960305A describes a method for modifying the crosslinking degree of nanogels, using nanogels of varying hardness as carriers to load anti-tumor chemotherapy drugs via electrostatic adsorption. This results in soft nanomedicines (with a Young's modulus of 20 to 150 kPa) and hard nanomedicines. These nanomedicines are then administered intravenously. Experimental findings indicate that the soft nanomedicines are more concentrated in tumors than in the liver. Furthermore, they exhibit significantly better penetration into tumor vessels and anti-tumor efficacy than the hard nanogels. Unlike this prior art, the inventors of the present application discovered, through serendipitous experimental discovery, that administering nanogels of appropriate hardness directly into mouse tumors can reverse the polarization of M2 tumor-associated macrophages to M1, effectively inhibiting tumor growth. The anti-tumor efficacy is even superior to that of the soft nanomedicines administered intravenously. Based on this, the present application provides an application of a nanogel for use as or preparation of an M2 TAMs reverse polarization drug, wherein the nanogel has a Young's modulus of 20~600 KPa and can reverse polarize M2 TAMs to M1 type.

[0049] 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.

[0050] The inventors unexpectedly discovered that when nanogels of appropriate hardness are co-incubated with M2 macrophages, they 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. Further research revealed that the greater the hardness of the nanogel, the better the effect of reverse polarization of M2 tumor-associated macrophages to M1. Furthermore, nanogels of appropriate hardness can be delivered directly into tumors via intratumoral injection to inhibit tumor growth, and the greater the hardness of the nanogel, the better the effect of inhibiting tumor growth.

[0051] In some embodiments, the Young's modulus of the nanogel is between 50 and 600 KPa, more preferably between 70 and 600 KPa.

[0052] In some embodiments, the nanogel is obtained by polymerizing the monomers in the presence of a crosslinker and a surfactant in an aqueous phase by initiating a polymerization reaction of the monomers; wherein the Young's modulus of the nanogel can be controlled by adjusting the molar ratio of the crosslinker to the monomers.

[0053] The nanogels synthesized in the present application can use common monomers used to prepare biocompatible nanogels. Different monomers can be used to impart different functions to the nanogels, 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-isopropylmethacrylamide, N-isopropylacrylamide, and N-ethylacrylamide. In some embodiments, the pH-responsive monomers are one or more of methacrylic acid, acrylic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid.

[0054] 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.

[0055] In some embodiments, the crosslinking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide, and N,N'-vinylbisacrylamide. More preferably, the crosslinking agent is a reduction-responsive crosslinking agent. In a preferred embodiment, the crosslinking agent is N,N'-bis(acryloyl)cystamine.

[0056] In some embodiments, the molar ratio of the cross-linking 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.

[0057] In some embodiments, the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide.

[0058] In some embodiments, the mass ratio of the initiator to the temperature-responsive monomer is (5-15):550.

[0059] In some embodiments, the surfactant is one or more of sodium lauryl sulfate, sodium lauryl sulfonate and lecithin.

[0060] In some embodiments, the mass ratio of the surfactant to the temperature-responsive monomer is (20-35):550.

[0061] In a preferred embodiment of the present application, the degree of crosslinking of the nanogel is regulated by adjusting the molar ratio of the crosslinking agent to the temperature-responsive monomer, thereby regulating the hardness of the nanogel. 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 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 at 37°C in PBS buffer is 150~300 nm, and the Young's modulus of the above-mentioned nanogel is 79.0~439.2KPa.

[0062] In some embodiments, the polymerization reaction is carried out at a temperature of 70° C. to 85° C., and for a time of 4 to 8 h.

[0063] 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.

[0064] In some embodiments, when the nanogel is used for anti-tumor treatment, the nanogel is administered via intratumoral injection.

[0065] In some embodiments, the dosage of the nanogel is 1-10 μg / mm³.

[0066] In actual application, those skilled in the art may, according to actual conditions, perform another intratumoral injection 4 to 10 days after the first intratumoral injection in order to enhance the anti-tumor effect.

[0067] It should be understood that materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments used in the following examples can be used to implement this application. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0068] When preparing nanogels in the following examples of this 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%. The prepared nanogel is expressed as X%NGs.

[0069] The following are examples:

[0070] Example 1 Effects of Nanogels of Different Hardness on the Reverse Polarization of M2 Macrophages

[0071] 1.1 Preparation of nanogels

[0072] 550 mg of the temperature-responsive monomer N-isopropylmethylpropionamide and 35 mg of the surfactant sodium dodecyl sulfate were ultrasonically dissolved in 80 mL of ultrapure water to obtain a mixed solution. 22.5 g of the crosslinker N,N'-bis(acryloyl)cystamine was ultrasonically dissolved in 0.5 mL of ethanol and then added to the mixed solution. 3.65 mL of the pH-responsive monomer methacrylic acid was dissolved in 6.35 mL of ultrapure water, and 50 μL was added to the mixed solution. The resulting solution was evacuated for 10 minutes and then purged with argon three times to fully remove oxygen from the solution. The solution was then heated to 80°C and maintained for 10 minutes. 10 mg of the initiator potassium persulfate was weighed, added to 0.5 mL of ultrapure water, and ultrasonically dissolved. The solution was then added to the heated solution via syringe to initiate polymerization for 6 hours. After the reaction is complete and the reaction mixture cools to room temperature, it is transferred to an ultrafiltration tube with a 10 kDa cutoff. Unreacted monomers and other impurities are removed by centrifugation at 2000 rpm. Excess water is also removed. After concentration, the solution is rinsed three times with ultrapure water to yield 8 mL of concentrated solution. 300 μL of this concentrate is taken, dried, and weighed. The solids content of the concentrate is calculated. Based on the calculated result, the concentrated solution is diluted to 20 mg / mL to yield a nanogel solution with a cross-linking degree of 2% (denoted as 2% NGs). This solution is stored at 4°C until further use.

[0073] Nanogel solutions with cross-linking degrees of 5%, 10%, and 15% were prepared according to the above steps. The surfactant sodium dodecyl sulfate was used in amounts of 30 mg, 25 mg, and 20 mg, respectively; the cross-linker N,N'-bis(acryloyl)cystamine was used in amounts of 56.3 mg, 112.6 mg, and 168.9 mg, respectively. These cross-linkers were ultrasonically dissolved in 1 mL, 2 mL, and 3 mL of ethanol, respectively, and then added to the mixed solutions. The resulting nanogel solutions (5%, 10%, and 15%) were stored at 4°C until ready for use.

[0074] 1.2 Physical properties of nanogels

[0075] 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. The hydrated particle size and zeta potential of the nanogel were detected by dynamic light scattering at a temperature of 37 °C and an equilibrium time of 15 min.

[0076] 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 add it dropwise to the carbon support membrane. Let it dry 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 and wash it 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, observe the morphology of the nanogel using a transmission electron microscope.

[0077] Soak the coverslip in a 1% aqueous solution of polyethyleneimine for 24 h to modify the surface of the coverslip with positive charges. 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 onto the positively charged coverslip. Electrostatic adsorption was performed for 10 min to absorb the excess dispersion. Then, 300 μL of ultrapure water was added to wash away the unadsorbed nanogel. Then, the height and Young's modulus of the nanogel were detected by atomic force microscopy. The detection environment was liquid phase, the image acquisition was in contact mode, and the Young's modulus detection was in tapping mode.

[0078] The results show that: Figure 1 Content A and Figure 1 As can be seen from content B, 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 cross-linking 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 3As can be seen from content B, 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.

[0079] 1.3 Triple responsiveness of nanogels

[0080] 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 °C, the temperature interval is 1 °C, and the equilibration time is 1 min.

[0081] 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. Use dynamic light scattering to detect the pH responsiveness of the nanogel. The detection temperature is 25°C and the equilibration time is 15 min.

[0082] Take 10 μL of the nanogel solution prepared in step 1.1 and disperse it in 1 mL of ultrapure water with or without 10 mM glutathione (GSH) and incubate at room temperature for 24 h. Then, take 10 μL of the above dispersion and drop it onto the carbon support membrane. Let it dry naturally. After drying, add 10 μL of a 1% phosphotungstic acid aqueous solution to the carbon support membrane and stain it for 2 min. After staining, use filter paper to remove the excess phosphotungstic acid solution along the edge of the carbon support membrane. Add 10 μL of ultrapure water and rinse it for 1 min. After rinsing, use filter paper to remove the excess ultrapure water along the edge of the carbon support membrane. After drying, observe the structure of the nanogel using a transmission electron microscope.

[0083] The results show that: Figure 4 Content A. Figure 4 Content B and Figure 4 As shown in Figure C, the hydrated particle size of the nanogel prepared in this example gradually decreases with increasing temperature, and the degree of shrinkage decreases with increasing cross-linking degree. As the pH increases, the hydrated particle size of the nanogel prepared in this example gradually increases, and the degree of swelling decreases with increasing cross-linking degree. After 24 hours of glutathione incubation, the structure of the nanogel prepared in this example is destroyed. In other words, the nanogel prepared in this example exhibits excellent temperature responsiveness, pH responsiveness, and reduction responsiveness.

[0084] 1.4 Effect of nanogel on reverse polarization of M2 macrophages

[0085] 1) Induction of M2 macrophages: RAW264.7 mouse macrophages were cultured at a rate of 1×10 6The cells were seeded in 6-well plates at a concentration of 100 μg / well. Then, 2 mL of a culture 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 cells were incubated in a 37°C, 5% CO2 incubator for 24 h. The upper layer of culture medium was then aspirated to obtain M2 macrophages.

[0086] 2) Mix the nanogel solutions of varying hardness prepared in step 1.1 with DMEM medium containing 10% serum to obtain mixed cultures containing nanogels of varying hardness, with the nanogel concentration being 200 μg / mL. Add 2 mL of the mixed cultures containing nanogels of varying hardness to each well of the 6-well plate containing M2 macrophages. Incubate in an incubator for 24 hours, then harvest the cells, wash three times with PBS buffer, digest with trypsin, and collect the cells by centrifugation. DMEM medium containing 10% serum without nanogel solution served as the control group, the mixed culture medium containing 2% NGs nanogels served as the 2% NGs-treated group, and the culture medium containing 15% NGs nanogels served as the 15% NGs-treated group.

[0087] Immunoblotting was used to detect the expression of M1- and M2-related proteins. RT-PCR was used to determine the expression levels of iNOS and CD86 mRNA. Cells collected by centrifugation were stained with PE anti-mouse CD86 at a dose of 25 μg / μL on ice in the dark for 20 minutes. Cells were then washed twice with PBS buffer and collected by centrifugation. Fluorescence intensity was measured by flow cytometry using the PE channel. The degree of macrophage M1 polarization was calculated based on fluorescence intensity.

[0088] The results show that: Figure 5 Content A. Figure 5 Content B. Figure 5 As shown in Figure C, compared with the control group, the 2% NGs-treated group and the 15% NGs-treated group significantly upregulated the expression of M1-related proteins CD86 and iNOS; the 15% NGs-treated group also significantly upregulated the expression of CD86 mRNA and iNOS mRNA. This suggests that nanogels of varying hardness can reverse the polarization of M2 macrophages to M1.

[0089] Example 2: Killing of tumor cells by nanogel after reverse polarization treatment of M2 macrophages

[0090] 1) The method for inducing the formation of M2 macrophages is the same as that in Example 1.

[0091] 2) Mix the nanogel solutions of varying hardness prepared in step 1.1 with DMEM medium containing 10% serum to create mixed cultures containing nanogels of varying hardness, with the nanogel concentration at 200 μg / mL. Add 2 mL of the mixed cultures containing nanogels of varying hardness to each well of the 6-well plate containing M2 macrophages. Incubate in an incubator for 24 hours. Aspirate the supernatant medium, then add 2 mL of serum-free DMEM medium to each well. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Collect the supernatant medium and set aside. DMEM medium containing 10% serum without nanogel solution served as the M2 treatment group; mixed culture medium containing an equal volume of PBS as nanogels served as the PBS treatment group; mixed culture medium containing 2% NGs nanogels served as the M2 + 2% NGs treatment group; and mixed culture medium containing 15% NGs nanogels served as the M2 + 15% NGs treatment group.

[0092] 3) H22 cells were cultured at a rate of 5×10 3 The cells were inoculated into a 96-well plate at a concentration of 100 μg / well. 200 μL of the above-mentioned upper culture medium was added to each well and incubated in a 37°C, 5% CO2 incubator for 24 h. Then, 22 μL of CCK8 was added to each well and incubated in a 37°C, 5% CO2 incubator for 1 h. The absorbance of the culture medium was measured at 450 nm using a microplate 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.

[0093] 4T1 cells were cultured at a rate of 5 × 10 3 4T1 cells were seeded into 96-well plates at a concentration of 100 μL / well. 200 μL of RPMI-1640 medium containing 10% serum was added to each well and incubated at 37°C in a 5% CO2 incubator for 12 hours. After the 4T1 cells attached, the supernatant was aspirated and 200 μL of the above supernatant was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours, and the supernatant was aspirated. Then, 100 μL of a medium prepared by mixing CCK8 with RPMI-1640 medium containing 10% serum in a 1:9 ratio was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. The absorbance of the culture medium was measured at 450 nm using a microplate reader. The results were processed and analyzed using Excel and Graphpad Prism to calculate the cytotoxicity of the supernatant after treating M2 macrophages with nanogels of different hardnesses against 4T1 cells.

[0094] The results show that: Figure 6 Content A. Figure 6As 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 decreased significantly, 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.

[0095] Example 3: Apoptosis of tumor cells after nanogel reverse polarization treatment of M2 macrophages

[0096] 1) The method for inducing the formation of M2 macrophages is the same as that in Example 1.

[0097] 2) Mix the nanogel solutions of varying hardness prepared in step 1.1 with DMEM medium containing 10% serum to create mixed cultures containing nanogels of varying hardness, with the nanogel concentration at 200 μg / mL. Add 2 mL of the mixed cultures containing nanogels of varying hardness to each well of the 6-well plate containing M2 macrophages. Incubate in an incubator for 24 hours, and aspirate the supernatant. Then, add 2 mL of serum-free DMEM medium to each well. Incubate in a 37°C, 5% CO2 incubator for 24 hours, and collect the supernatant for later use. DMEM medium containing only 10% serum without nanogel solution served as the control group, the mixed culture medium containing 2% NGs nanogels served as the 2% NGs-treated group, and the culture medium containing 15% NGs nanogels served as the 15% NGs-treated group.

[0098] 3) H22 cells were cultured at a rate of 1×10 6 The cells were seeded in a 6-well plate at a concentration of 100 μL / well, and 200 μL of the above-mentioned upper culture medium was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h. The upper culture medium was aspirated, 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 in the dark for 15 min, washed twice with PBS buffer, and collected by centrifugation. The fluorescence intensity was detected by flow cytometry using PE and FITC as the detection channels. The degree of apoptosis of H22 cells was calculated based on the fluorescence intensity.

[0099] 4T1 cells were cultured at a rate of 1×10 6The cells were seeded in 6-well plates at a concentration of 100 μL / well. 200 μL of RPMI-1640 medium containing 10% serum was added to each well and incubated in a 37°C, 5% CO2 incubator for 12 h. After 4T1 cells adhered, the upper layer of culture medium was aspirated and 200 μL of the above upper layer of culture medium was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h. The upper layer of culture medium was aspirated and the cells were washed three times with PBS buffer. The cells were digested with trypsin and 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 in the dark for 15 min. The cells were washed twice with PBS buffer and collected by centrifugation. The fluorescence intensity was detected by flow cytometry using PE and FITC as the detection channels. The degree of apoptosis of 4T1 cells was calculated based on the fluorescence intensity.

[0100] 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 were increased in the 2% NGs-treated group and the 15% NGs-treated group, among which the 15% NGs-treated 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.

[0101] Example 4 Phagocytosis of tumor cells by nanogel after reverse polarization treatment of M2 macrophages

[0102] 1) The method for inducing the formation of M2 macrophages is the same as that in Example 1.

[0103] 2) Mix the nanogel solutions of varying hardness prepared in step 1.1 with DMEM medium containing 10% serum to create mixed cultures containing nanogels of varying hardness, with the nanogel concentration at 200 μg / mL. Add 2 mL of the mixed cultures containing nanogels of varying hardness to each well of the 6-well plate containing M2 macrophages. Incubate in an incubator for 24 hours, then aspirate the supernatant. Then, add 2 mL of serum-free DMEM medium to each well. Incubate in a 37°C, 5% CO2 incubator for 24 hours, and collect the macrophages. DMEM medium containing 10% serum without nanogel solution served as the control group, the mixed culture medium containing 2% NGs nanogels served as the 2% NGs-treated group, and the culture medium containing 15% NGs nanogels served as the 15% NGs-treated group. The collected macrophages were washed three times with PBS buffer, trypsinized, and collected by centrifugation. The cells were then stained with 25 μg / μL FITC anti-mouse CD11b for 20 minutes on ice in the dark. The stained macrophages were divided into 1×10 6The cells were inoculated into 6-well plates at a concentration of 1000 cells / well and set aside.

[0104] 3) 4T1 cells were plated at 1×10 6 4T1 cells were seeded in a 6-well plate at a concentration of 100 cells / well, and 200 μL of RPMI-1640 medium containing 10% serum was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. The upper layer of medium was aspirated, and the 4T1 cells were collected and washed three times with PBS buffer. The cells were digested with trypsin and collected by centrifugation. They were then stained with PE anti-mouse EPCAM on ice in the dark for 20 mins at a dose of 25 μg / μL. The stained 4T1 cells were plated at a concentration of 2×10 6 The cells were added to the 6-well plate containing the stained macrophages at a concentration of 100 μg / well and incubated in a 37°C, 5% CO2 incubator for 4 h. The upper layer of 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 then detected by flow cytometry using PE and FITC as the detection channels. The degree of phagocytosis of 4T1 cells by macrophages was calculated based on the fluorescence intensity.

[0105] The results show that: Figure 8 Content A. Figure 8 As can be seen from content B, compared with the control group, the proportion of macrophages engulfing 4T1 cells in the 2% NGs-treated group and the 15% NGs-treated group was significantly increased, indicating that nanogels with different hardness can effectively engulf tumor cells after reverse polarization treatment of M2 macrophages.

[0106] Example 5: Killing of mouse tumors by nanogel after reverse polarization treatment of M2 macrophages

[0107] 1) RAW264.7 mouse macrophages were cultured at a rate of 1×10 6 The cells were seeded in 6-well plates at a concentration of 100 μg / well. Then, 2 mL of a culture 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 cells were incubated in a 37°C, 5% CO2 incubator for 24 h. The upper layer of culture medium was then aspirated to obtain M2 macrophages.

[0108] RAW264.7 mouse macrophages were cultured at a rate of 1 × 10 6 The cells were seeded into 6-well plates at a concentration of 100 cells / well, and then 2 mL of DMEM medium containing 10% serum was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h, and then the upper layer of culture medium was removed to obtain M0 macrophages.

[0109] RAW264.7 mouse macrophages were cultured at a rate of 1 × 106 The cells were seeded in 6-well plates at a concentration of 1 μg / mL. Then, 2 mL of DMEM medium containing 10% serum was added to each well. The plates were incubated in a 37°C, 5% CO2 incubator for 24 h. The supernatant medium was removed. Then, 2 mL of a medium mixed with LPS and DMEM medium containing 10% serum was added to each well. The LPS dose was 1 μg / mL. The plates were incubated in a 37°C, 5% CO2 incubator for 24 h. The supernatant medium was removed to obtain M1 macrophages.

[0110] 3) Mix the nanogel solutions of varying hardness prepared in step 1.1 with DMEM medium supplemented with 10% serum to create mixed cultures containing nanogels of varying hardness, with the nanogel concentration at 200 μg / mL. Then, add 2 mL of the mixed cultures containing nanogels of varying hardness to each well of the 6-well plate containing M2 macrophages from step 1). Incubate in an incubator for 24 hours, and aspirate the supernatant. Then, add 2 mL of serum-free DMEM medium to each well and incubate in an incubator at 37°C, 5% CO2 for 24 hours. Aspirate the supernatant, harvest the cells, and wash them three times with PBS. Disintegrate the cells with trypsin and centrifuge to harvest the macrophages. The harvested macrophages are then mixed with 4T1 cells to create a cell mix. Among them, the macrophages collected after treatment with a mixed culture 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 a mixed culture 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; and the M1 macrophages prepared in step 1) were mixed with 4T1 cells at a ratio of 2:1 as the M1 treatment group.

[0111] 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 at a number of 3×10 6 The inoculation volume was 100 μL to construct a mixed model of 4T1 breast cancer subcutaneous tumor in mice. Figure 9 As shown in Content A. When the tumor volume reaches 100 mm 3 The day after 4 h was marked 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 was calculated according to the formula: 2) / 2, and tumor volume was calculated. After the measurement period ended on day 14, mice were sacrificed, and the tumors were excised, weighed, and photographed. The excised tumors were fixed with 4% paraformaldehyde and sectioned. CD86 immunofluorescence was performed, and the CD86 area ratio was quantified to assess the degree of repolarization of tumor-associated macrophages (TAMs).

[0112] The results show that: Figure 9 Content B Tumor volume, Figure 9 Content C Tumor weight, Figure 9 As can be seen from the tumor image in content D, compared with the M2 treatment group, the volume and 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 have a good tumor-killing effect. Figure 10 Content A. Figure 10 As can be seen from content B, compared with the M2 treatment group, the fluorescence intensity of 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 TNF-α can enhance the polarization degree of M2 macrophages to M1 macrophages.

[0113] Example 6 Inhibitory effect of nanogel on tumor growth in mice

[0114] Female BALB / C mice were subcutaneously inoculated with 1×10 6 4T1 cells were added in a volume of 100 μL to establish a mouse 4T1 breast cancer subcutaneous tumor model. When the tumor volume reached 200 mm 3 The day after the onset of leukemia was marked as day 0, and the mice were randomly divided into three groups, including a control group, a 2% NGs-treated group, and a 15% NGs-treated group. On the first and eighth days after grouping, the above-mentioned treatment groups were intratumorally injected with 25 μL of normal saline, 2% NGs, and 15% NGs (the injection dose of nanogel was 20 mg / mL), respectively. The experimental process is as follows: Figure 11 As shown. Starting from 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. The tumor volume V was calculated according to the formula: tumor volume V = (a × b 2 ) / 2, and the tumor volume was calculated. After the measurement was completed on day 14, the mice were sacrificed.

[0115] The results show that: Figure 12 、 Figure 13It can be seen that the volume and relative growth rate of breast cancer tumors in the 2% NGs treatment group and the 15% NGs treatment group were significantly lower than those in the control group, which indicates that intratumoral injection of nanogels with appropriate hardness can effectively inhibit tumor growth.

[0116] It is easy for those skilled in the art to understand that the above 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 should be included in the scope of protection of the present application.

Claims

1. Use of a nanogel as the sole active ingredient in the preparation of an anti-tumor drug, characterized in that: The Young's modulus of the nanogel is 20-600 KPa, and when used for anti-tumor treatment, it can reverse the polarization of M2-type TAMs to M1-type, thereby inhibiting tumor cell proliferation; The nanogel is obtained by polymerizing 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 controlled by adjusting the molar ratio of the crosslinking agent to the monomers; The monomers include temperature-responsive monomers and pH-responsive monomers, wherein the temperature-responsive monomers are one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide, and N-ethyl acrylamide; the pH-responsive monomers are one or more of methacrylic acid, acrylic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid; the cross-linking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide, and N,N'-vinylbisacrylamide; the initiator is one or more of potassium persulfate, sodium persulfate, and tert-butyl hydroperoxide; and the surfactant is one or more of sodium lauryl sulfate, sodium lauryl sulfonate, and lecithin; The molar ratio of the pH-responsive monomer to the temperature-responsive monomer is (3-8):100; the molar ratio of the cross-linking agent to the temperature-responsive monomer is (1-20):100; the mass ratio of the initiator to the temperature-responsive monomer is (5-15):550; and the mass ratio of the surfactant to the temperature-responsive monomer is (20-35):

550. The tumors include liver cancer and / or breast cancer.

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

3. The use according to claim 1, characterized in that The polymerization reaction temperature is 70° C. to 85° C., and the reaction time is 4 to 8 h.

4. The use according to claim 1, characterized in that When used for anti-tumor treatment, the nanogel is administered by intratumor injection.

5. The use according to claim 4, characterized in that The dosage of the nanogel is 1-10 μg / mm 3 .

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