PH-responsive nanogel with mechanical property conversion and application of pH-responsive nanogel

Abstract

The invention belongs to the field of functional nano materials, and particularly relates to pH-responsive nano gel with mechanical property conversion and application of the pH-responsive nano gel. The deformability of the nanogel is measured by using the ratio SD-pH of the diameter Dswoln of the nanogel at a first temperature T1 lower than the critical phase transition temperature of the nanogel to the diameter Dswoln of the nanogel at a second temperature T2 higher than the critical phase transition temperature of the nanogel. The SD-pH1 of the nanogel when the pH1 is 4.5-6 is greater than the SD-pH2 of the nanogel when the pH2 is 6.8-7.4, and the ratio of the SD-pH1 to the SD-pH2 is greater than or equal to 1.6. Compared with a control group, the nanogel disclosed by the invention is proved to have a remarkable pH-responsive mechanical property transformation effect and optimal active transendocytosis efficiency, and has optimal deep penetrating power and anti-tumor effect at a tumor part.

Description

Technical Field

[0001] This application belongs to the field of functional nanomaterials, and more specifically, relates to a nanogel with pH-responsive mechanical property transformation and its applications. Background Art

[0002] The stroma in solid tumors is a key component of the tumor microenvironment. Tumors usually contain a large number of stromal cells, which significantly affect the penetration and accumulation of nanodrugs in tumors. After nanodrugs enter tumor tissues, the vast majority will be taken up by stromal cells, thus hindering the further penetration and diffusion of drugs. Only a very small number of nanodrugs can effectively reach tumor cells and exert anti-tumor effects. This stromal barrier significantly limits the anti-tumor effect of nanodrugs and becomes one of the main bottlenecks in their clinical applications. Improving the deep penetration ability of nanodrugs in solid tumors is an important direction in current tumor treatment research. Due to the complexity of the tumor microenvironment, the permeability of nanodrugs is often restricted by the special structure and physiological conditions of tumor tissues. To improve the deep penetration effect of nanodrugs in solid tumors, researchers have proposed various strategies, including matrix degradation, surface modification and targeted delivery, application of physical methods, particle size regulation, and enhancement of tumor vascular permeability. However, these strategies also face different challenges and limitations respectively.

[0003] The matrix degradation strategy helps nanodrugs penetrate tumor tissues by using specific enzymes or small molecule drugs to degrade the tumor matrix. However, due to the heterogeneity of tumor matrices in different patients or different parts of the same patient, the degradation effect is often unstable, and it may damage normal tissues or cause side effects. In addition, long-term use may lead to drug resistance problems, making the degradation effect gradually weaken. The surface modification and targeted delivery strategy improves the accumulation and permeability of drugs at the tumor site by changing the surface properties of nanoparticles, such as PEG modification or targeted ligand modification. However, this method may encounter the "immune escape" phenomenon of the immune system. In addition, the heterogeneity between different tumors also makes targeted modification not always effective, and there may be a conflict between targeting and deep drug penetration.

[0004] Physical methods such as high-intensity focused ultrasound, magnetic field guidance, or electric field stimulation have also been proposed as a means to improve the permeability of nanodrugs. Through external stimulation, the permeability of tumor blood vessels can be altered or the penetration of nanoparticles can be directly promoted. However, these methods often require specialized equipment and may have side effects on normal tissues. In addition, the effects of these physical methods are usually limited to the tumor surface or a specific area, and the penetration effect on deep tumors is limited. Strategies for particle size regulation and structure optimization optimize the penetration ability by adjusting the particle size and morphology of nanoparticles. Smaller particles are generally more likely to enter tumor tissues, but if the particle size is too small, the drug may also be rapidly cleared, resulting in poor efficacy; while larger particles may be restricted by the tumor tissue structure. In addition, particle size optimization may be accompanied by problems of instability or toxicity of nanodrugs.

[0005] Enhancing tumor blood vessel permeability is also a common strategy, mainly by improving the structure of tumor blood vessels so that drugs can better penetrate into the tumor interior. However, due to the heterogeneity of tumor blood vessels, the effect of this strategy may vary depending on the tumor type and tumor region, and changing the tumor blood vessel structure may also bring side effects, such as increased drug resistance of tumor cells or the impact on normal tissue blood flow.

[0006] The heterogeneity of tumors, the particularity of the microenvironment (such as low oxygen, low pH, etc.), and the abnormal structure of tumor blood vessels make it difficult for effective drugs to penetrate deep into tumors. As a mechanism of intercellular transmembrane transport, transcytosis has gradually attracted the attention of researchers in recent years as a potential strategy to improve deep tumor penetration. To enhance the transcytosis ability, researchers usually adopt the following methods: (1) surface modification of nanodrugs, that is, through the functionalization of charge, hydrophilicity, or specific ligands, to enhance the binding to tumor cell surface receptors and promote endocytosis; (2) using cell-penetrating peptides (CPPs) or other molecules that enhance cell uptake to further increase the endocytosis rate of drugs by enhancing the fusion of nanodrugs with cell membranes. However, few studies have considered the influence of the mechanical properties of nanomaterials on their transcytosis ability. Summary of the Invention

[0007] Aiming at the defects of the prior art, the purpose of this application is to provide a pH-responsive nanogel with transformed mechanical properties and its application. The deformation ability of this nanogel under the pH conditions in the cytoplasm is much smaller than its deformation ability under the pH conditions in acidic organelles (such as lysosomes). When used as a nanodrug delivery carrier, it exhibits excellent transcytosis ability, aiming to solve the technical problems such as the poor transcytosis ability of anti-tumor drugs in the prior art, which makes it difficult for effective drugs to penetrate deep into tumors.

[0008] To achieve the above object, in a first aspect, the present application provides a nanogel with pH-responsive mechanical property transformation, which is obtained by polymerizing monomers in an aqueous phase with an initiator in the presence of a crosslinking agent and a surfactant; the monomers include temperature-responsive monomers and pH-responsive monomers; The hydrophilic-hydrophobic balance of the polymer chains inside the nanogel can be transformed with the change of temperature. When the temperature is higher than the critical phase transition temperature of the nanogel, the inside of the nanogel changes from hydrophilic to hydrophobic, and the nanogel shrinks, resulting in a smaller size; when the temperature is lower than the critical phase transition temperature, the inside of the nanogel changes from hydrophobic to hydrophilic, and the nanogel swells, resulting in a larger size; Define D swollen as the diameter of the nanogel at the first temperature T1 below the critical phase transition temperature, and D deswollen as the diameter of the nanogel at the second temperature T2 above the critical phase transition temperature; the deformation ability of the nanogel under different pH conditions is represented by S D-pH and its calculation formula is as follows:

[0009] The S of the nanogel at pH1 = 4.5 - 6 D-pH1 is greater than its S at pH2 = 6.8 - 7.4 D-pH2 , and S D-pH1 / S D-pH2 ≥1.6.

[0010] Preferably, the first temperature T1 is 20 - 30 °C, and the second temperature T2 is 50 - 60 °C.

[0011] More preferably, S D-pH1 / S D-pH2 ≥1.9.

[0012] Even more preferably, the S of the nanogel at pH1 = 4.5 - 6 D-pH1 is greater than or equal to 2, and its S at pH2 = 6.8 - 7.4 D-pH2 is less than or equal to 1.25.

[0013] Preferably, the Young's modulus of the nanogel is 250 - 300 Kpa.

[0014] According to another aspect of the present invention, there is provided an application of the above-mentioned nanogel in the preparation of a deformable material with pH responsiveness.

[0015] According to another aspect of the present invention, there is provided an application of the nanogel as described above in being used as or preparing a nano-drug delivery carrier, and the deformation ability of the nano-drug delivery carrier at cytoplasmic pH is less than its deformation ability at acidic organelle pH.

[0016] According to another aspect of the present invention, there is provided an application of the nanogel as described above in preparing a nano-drug with active transcytosis ability.

[0017] According to another aspect of the present invention, there is provided a nano-drug, which includes the nanogel as described above, and also includes an anti-tumor drug loaded on the nanogel through electrostatic adsorption.

[0018] Generally speaking, compared with the prior art by the above technical solutions conceived in this application, the following beneficial effects are achieved: (1) The present invention provides a nanogel whose mechanical properties change in response to pH. The diameter D of the nanogel at the first temperature T1 lower than the critical phase transition temperature of the nanogel swollen and the diameter D of the nanogel at the second temperature T2 higher than the critical phase transition temperature deswollen The ratio S D-pH is used to measure the deformation ability of the nanogel under different pH conditions. The larger S D-pH is, the stronger the deformation ability of the nanogel. The S of the nanogel proposed in the present invention at pH1 = 4.5 - 6 D-pH1 is greater than its S at pH2 = 6.8 - 7.4 D-pH2 , and S D-pH1 / S D-pH2 ≥ 1.6. Compared with the control group, the nanogel provided by the present invention has a significant pH-responsive mechanical property conversion effect.

[0019] (2) The present invention uses the nanogel with a significant pH-responsive mechanical property conversion effect proposed above to deliver nano-drugs. Experiments have found that compared with nanogels with immutable or slightly changing mechanical properties, the nanogel provided by the present invention has the best active transcytosis efficiency in tumors, and has obvious tumor accumulation and tumor deep penetration ability. In addition, after loading DOX with this nanogel, the drug can be effectively delivered to the deep part of the tumor, improving the chemotherapy efficacy and reducing the systemic toxicity.

[0020] (3) The pH-responsive nanogel with transformed mechanical properties provided by the present invention has specific mechanical properties that also affect the distribution of the nanogel in organelles. After entering the cell, the nanogel with the best transcytosis efficiency is mainly distributed in the Golgi apparatus rather than the lysosome. This distribution characteristic may enable more nanogels to enter the next cell, achieving more efficient intercellular transmission. The nanogel with the optimal transcytosis ability also has the best deep penetration ability in 3D tumor spheroids and solid tumors and obtains the optimal anti-tumor effect. Description of the Drawings

[0021] Figure 1 are the particle sizes and zeta potentials of three kinds of nanogels provided in Example 1 of the present application, where Content A is the particle size distribution of the nanogel in PBS buffer at 37 °C, and Content B is the surface charge of the nanogel in ultrapure water; Figure 2 are the transmission electron microscopy images of three kinds of nanogels provided in Example 1 of the present application; Figure 3 are the Young's moduli of three kinds of nanogels provided in Example 1 of the present application; Figure 4 are the pH-responsive data of three kinds of nanogels provided in Example 1 of the present application. Content A is the absolute hydrodynamic diameter of the nanogel at different pH values at 25 °C, and Content B is the relative hydrodynamic diameter of the nanogel at different pH values at 25 °C, where D0 is the particle size of the nanogel at pH 4.5; Figure 5 is the influence of pH on the temperature responsiveness of three kinds of nanogels provided in Example 1 of the present application. AUNGs only have weak temperature responsiveness at different pH values, MANGs have weak temperature responsiveness at pH 7.4 and 6.8, and recover temperature responsiveness at pH 5.5. ADNGs have temperature responsiveness at different pH values. Content A is the absolute hydrodynamic diameter of the three kinds of nanogels at different pH values and temperatures, and Content B is the relative hydrodynamic diameter of the three kinds of nanogels at different pH values and temperatures; Figure 6 is the characterization of the S D value of three kinds of nanogels provided in Example 1 of the present application. S D >1, and the larger S D is, the stronger the deformability of the nanogel, that is, the softer it is. The S D value of MANGs is affected by pH, being soft in acidic environments and hard in neutral environments; Figure 7 are the hydrodynamic diameters of three kinds of rhodamine B-labeled nanogels provided in Example 2 of the present application in PBS buffer at 37 °C; Figure 8They are the particle sizes and charges of three DOX-loaded nanogels provided in Example 3 of this application. Among them, Content A is the particle size distribution of the DOX-loaded nanogel in PBS buffer at 37 °C, Content B is the change in the particle size of the nanogel before and after loading DOX, and Content C is the change in the surface charge of the nanogel before and after loading DOX; Figure 9 They are the uptake of three rhodamine B-labeled nanogels provided in Example 2 of this application in different cells and different pH environments. Among them, Content A is the uptake of three rhodamine B-labeled nanogels by 3T3 cells, Content B is the uptake of three rhodamine B-labeled nanogels by 4T1 cells, and Content C is the uptake of three rhodamine B-labeled nanogels by 4T1 cells in different pH environments; Figure 10 They are the transcytosis efficiencies of three rhodamine B-labeled nanogels provided in Example 2 of this application among 4T1 cells; Figure 11 They are the co-localization of three rhodamine B-labeled nanogels provided in Example 2 of this application in lysosomes and Golgi bodies of 4T1 cells. Among them, Content A is the co-localization analysis of three rhodamine B-labeled nanogels with lysosomes, and Content B is the co-localization analysis of three rhodamine B-labeled nanogels with Golgi bodies; Figure 12 They are the deep penetration of three rhodamine B-labeled nanogels provided in Example 2 of this application in 3D tumor spheres; Figure 13 They are the distribution and fluorescence quantification of three rhodamine B-labeled nanogels provided in Example 2 of this application in the heart, liver, spleen, lung, kidney, and tumor of female BALB / C mice. Among them, Content A is the fluorescence distribution map of three rhodamine B-labeled nanogels in different tissues, and Content B is the quantification of Content A; Figure 14 They are the fluorescence sections and quantification of the deep penetration results of three rhodamine B-labeled nanogels provided in Example 2 of this application in 4T1 subcutaneous tumors of female BALB / C mice. The green ones are blood vessels, and the red ones are rhodamine B-labeled nanogels; Figure 15 They are the killing effects of three DOX-loaded nanogels provided in Example 3 of this application on 4T1 cells; Figure 16 They are the anti-tumor effects of three DOX-loaded nanogels provided in Example 3 of this application on the 4T1 subcutaneous tumor model. Among them, Content A is the graph of the change in tumor volume over time, Content B is the mass of the tumor after 15 days, Content C is the picture of the tumor at day 15, and Content D is the tumor inhibition rate of different nano-drugs. Detailed implementation manners

[0022] In order to make the objectives, technical solutions, and advantages of this application clearer, the following further details this application in conjunction with 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 used to limit this application.

[0023] The embodiments of the present invention are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and processes are given. However, the protection scope of the present invention is not limited to the following embodiments. For the process parameters without specific conditions noted in the following embodiments, they are usually in accordance with conventional conditions.

[0024] In the ranges disclosed in the present invention, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in the present invention.

[0025] For the process parameters without specific conditions noted in the following embodiments, they are usually in accordance with conventional conditions.

[0026] Patent document CN115960305A can obtain a series of nanogels with different hardnesses (the "hardness" in the text refers to the Young's modulus of the nanogel) by regulating the molar ratio of the cross-linking agent and the monomer. Moreover, this document points out that compared with hard nanogels, soft nanogels with a smaller Young's modulus can overcome the physical barrier formed by the extracellular matrix, are more likely to penetrate blood vessels and penetrate deep into tumors, and have a higher cell uptake efficiency, thereby achieving a higher tumor enrichment amount and a more excellent anti-tumor effect; Further, patent document CN116003683A uses hard nanogels (with a Young's modulus between 300 and 600 kPa) as RES-blockade blocking materials, and soft nanogels (with a Young's modulus between 20 and 150 kPa) are used to deliver small molecule chemotherapeutic drugs, and nanogels with different hardnesses (Young's modulus) are combined for tumor treatment.

[0027] Different from the above patent documents, the present invention utilizes the property that the deformation ability of nanogels changes significantly under the influence of pH to improve the active transcytosis ability of anti-tumor drugs. Generally, the intracellular pH value varies among different cell compartments. For example, the pH value of the cytoplasm ranges from 6.8 to 7.4, while the pH value of acidic organelles (such as lysosomes) ranges from 4.5 to 6.0. The present invention provides a pH-responsive nanogel with transformed mechanical properties, which is obtained by polymerizing monomers in an aqueous phase with the presence of a cross-linking agent and a surfactant through an initiator; the monomers include temperature-responsive monomers and pH-responsive monomers; the hydrophilic-hydrophobic balance of the polymer chains inside the nanogel can be transformed with the change of temperature. When the temperature is higher than the critical phase transition temperature of the nanogel, the inside of the nanogel changes from hydrophilic to hydrophobic, and the nanogel shrinks, resulting in a smaller size; when the temperature is lower than the critical phase transition temperature, the inside of the nanogel changes from hydrophobic to hydrophilic, and the nanogel swells, resulting in a larger size.

[0028] Define D swollen as the diameter of the nanogel at the first temperature T1 below the critical phase transition temperature, D deswollen as the diameter of the nanogel at the second temperature T2 above the critical phase transition temperature; the first temperature T1 and the second temperature T2 remain unchanged, and the deformation ability of the nanogel under different pH conditions is represented by S D-pH and the calculation formula is as follows:

[0029] The S of the nanogel at pH1 = 4.5 - 6 (the pH value range of acidic organelles) D-pH1 is greater than its S at pH2 = 6.8 - 7.4 (the pH range of the cytoplasm) D-pH2 , and S D-pH1 / S D-pH2 ≥ 1.6, more preferably, S D-pH1 / S D-pH2 ≥ 1.9.

[0030] Experiments have found that a nano-gel provided by the present invention has little deformation ability under the pH environment of the cytoplasm and has a greater deformation ability under the pH conditions of acidic organelles. The mechanical property of the deformation ability of the nano-gel material proposed by the present invention is very sensitive to pH. By comparing with a control group of nano-gels that are not sensitive to pH, it was unexpectedly found in the experiment that the characteristic of the conversion of the pH-sensitive response mechanical property of the nano-gel of the present invention enables it to exhibit significantly superior active transcytosis ability compared with the control group when used for nano-drug delivery and anti-tumor treatment. Furthermore, the nano-drug has the best deep penetration ability in 3D tumor spheres and solid tumors and obtains the optimal anti-tumor effect. It was also observed in the experiment that the nano-gel with the converted mechanical property of pH-sensitive response of the present invention is more distributed in the Golgi apparatus rather than lysosomes after entering the cell compared with the control group. This distribution characteristic may enable more nano-gels to enter the next cell, realizing more efficient intercellular transmission, and thus having a higher active transcytosis ability. The nano-gel with the optimal transcytosis ability also has the best deep penetration ability in 3D tumor spheres and solid tumors and obtains the optimal anti-tumor effect.

[0031] The critical phase transition temperature of the nano-gel can be obtained by the following method: Measure the particle size-temperature curve of the nano-gel, and the inflection point on this curve is the critical phase transition temperature of the nano-gel. In some embodiments, the first temperature T1 is 20 - 30 °C, and the second temperature T2 is 50 - 60 °C.

[0032] In a preferred embodiment, the S of the nano-gel of the present invention at pH1 = 5.5 D-pH1 is greater than or equal to 2, for example, 2.2 - 3; its S at pH2 = 6.8 - 7.4 D-pH2 is less than or equal to 1.25, for example, 1 - 1.5.

[0033] In a preferred embodiment, the Young's modulus of the nano-gel is 250 - 300 Kpa.

[0034] The present invention synthesizes a nano-gel whose mechanical property (deformation ability) is significantly changed by the influence of pH by regulating the types and proportions of monomers and cross-linking agents. Moreover, the nano-gel shows obvious differences in deformation ability under the pH conditions of different compartments in the cell. Experiments have found that compared with the control group of nano-gels whose mechanical property conversion is not sensitive to pH response, the nano-gel of the embodiments of the present invention has excellent tumor enrichment effect and active transcytosis ability.

[0035] In some embodiments, the temperature-responsive monomer is one or more of N-isopropylmethacrylamide, N-isopropylacrylamide, and N-ethylacrylamide; the pH-responsive monomer is one or more of methacrylic acid, acrylic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid; the crosslinking agent is one or more of N,N'-bis(acryloyl)cystamine, N,N'-methylenebisacrylamide, and N,N'-vinylenebisacrylamide; the initiator is one or more of potassium persulfate, sodium persulfate, and tert-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, and lecithin; the molar ratio of the pH-responsive monomer to the temperature-responsive monomer in the feed is (8-12):100, more preferably (9-11):100; the molar ratio of the crosslinking agent to the temperature-responsive monomer in the feed is (1-3):100, more preferably (1.5-2.5):100; the mass ratio of the initiator to the temperature-responsive monomer is (8-12):550, more preferably (9-11):550; the mass ratio of the surfactant to the temperature-responsive monomer is (38-42):550, more preferably (39-41):550.

[0036] In some embodiments, the reaction temperature of the polymerization reaction is 70 °C to 85 °C, and the reaction time is 4 to 8 h.

[0037] The present invention designs a nanogel whose mechanical property, i.e., the deformation ability, responds to the pH environment. By regulating the contents of the pH-responsive monomer methacrylic acid and the crosslinking agent N,N'-bis(acryloyl)cystamine, a series of nanogels with different mechanical properties and pH responsiveness are prepared by random copolymerization initiated by an initiator. Using a specific deformation ability calculation formula to characterize the mechanical properties of the nanogels, it is found that the deformation ability of the nanogels themselves is not always better when it is larger or smaller at different pH values. Only the nanogels with a small deformation ability near the cytoplasmic pH and a large deformation ability near the acidic organelle pH exhibit excellent active transcytosis ability. In addition, this mechanical property also affects the distribution of the nanogels in the organelles. The nanogels with the best transcytosis efficiency are mainly distributed in the Golgi apparatus rather than lysosomes after entering the cells. This distribution characteristic may enable more nanogels to enter the next cell, achieving more efficient intercellular transmission. The nanogels with the optimal transcytosis ability also have the best deep penetration ability in 3D tumor spheres and solid tumors and obtain the optimal anti-tumor effect.

[0038] In the present invention, a nanogel with a large deformation ability at a certain pH is called a relatively "soft" nanogel, and conversely, it is considered relatively "hard". However, the hardness and softness here are different from the hardness and softness corresponding to the measured Young's modulus of the nanogel. The softness and hardness of the nanogel of the present invention are mainly used to measure the large and small responses of the deformation ability of the nanogel affected by pH. The "deformation ability" and "ability to deform" mentioned in the specification of the present invention are synonymous.

[0039] After exogenous substances such as nanoparticles form endocytic vesicles through invagination of the cell membrane, they fuse with early endosomes (pH 6.8 - 6.1), undergo the process of endosome maturation, and are transformed into late endosomes (pH 6.1 - 5.5), and finally form lysosomes (pH 5.5 - 4.5). Compared with the control group, the nanogel of the present invention shows a significantly enhanced lysosomal escape efficiency based on its pH-responsive mechanical properties. The mechanism may involve: the acidic microenvironment triggers changes in the mechanical properties of the nanogel, and the low pH of the lysosome induces a decrease in the hardness of the nanogel, which is beneficial for its lysosomal escape; the retrograde membrane transport pathway: the escaped nanogel is repackaged into secretory vesicles through the Golgi-ER system and actively excreted into the extracellular space; the enhancement of transcytosis: the above process significantly improves the active transcytosis efficiency of the nanogel and promotes its penetration and delivery in tissue barriers (such as vascular endothelium or epithelium).

[0040] In some embodiments of the present invention, by regulating the molar ratio of methacrylic acid and N,N'-bis(acryloyl)cystamine relative to N-isopropylmethacrylamide, the cross-linking degree and pH responsiveness are regulated, and then the pH responsiveness of the nanogel is regulated. Sodium dodecyl sulfate regulates the particle size of the nanogel to make the particle sizes of nanogels with different hardnesses close to control variables.

[0041] In view of the fact that the nanogel provided by the present invention exhibits significantly different deformation abilities at different pH values, according to the needs of the application scenario, the nanogel can be used to prepare a pH-responsive deformable material, such as a material whose size changes under the influence of pH, to meet the requirements of certain application scenarios.

[0042] In addition, the nanogel can also be used as or prepared into a nano-drug delivery carrier, so that the delivered nano-drug has a greater deformation ability in the acidic organelle pH environment than in the cytoplasmic pH environment.

[0043] In view of the excellent active transcytosis ability exhibited by the nanogel of the present invention when delivering nano-drugs, the nanogel of the present invention can also be used to prepare nano-drugs with active transcytosis ability.

[0044] The present invention also provides a nano-drug, which includes the nano-gel of the present invention and an anti-tumor drug loaded on the nano-gel through electrostatic adsorption. The types of anti-tumor drugs are not limited, including but not limited to doxorubicin, etc. In some embodiments, the tumor cells corresponding to the tumor are one or more of liver cancer cells, breast cancer cells, colon cancer cells, lung cancer cells, esophageal squamous cell carcinoma cells, gastric cancer cells, ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, lymphoma cells, melanoma cells, and glioblastoma cells.

[0045] The embodiments of the present application will be described below with reference to the accompanying drawings in the embodiments of the present application.

[0046] Example 1 1.1 Preparation of a series of nano-gels with different mechanical properties and pH responsiveness Weigh 550 mg of N-isopropylmethacrylamide (abbreviated as NIPMAM) and the corresponding amount of sodium dodecyl sulfate (SDS), dissolve them in 80 mL of ultrapure water, and transfer the solution to a 250 mL three-necked flask. Weigh the corresponding amount of N,N'-bis(acryloyl)cystamine (BAC), dissolve it in ethanol, and add it to the three-necked flask. Take 0.73 mL of methacrylic acid stock solution and dissolve it in 19.27 mL of ultrapure water, and then add the corresponding amount of diluted methacrylic acid solution (MAA) to the three-necked flask. The above solution is evacuated and filled with argon three times to remove oxygen and ethanol in the system. Heat the deoxygenated solution to 80 °C, and add 0.5 mL of 20 mg / mL potassium persulfate solution through a syringe to initiate the polymerization reaction. The reaction time is 5 hours. After the reaction, transfer the solution to a 10 kDa ultrafiltration tube, and ultrafilter at 2000 rpm to remove unreacted monomers and impurities. Repeatedly add ultrapure water to purify and concentrate the material. Use the drying method to quantify the purified material, and quantify the material to a concentration of 20 mg / mL with ultrapure water and store it at 4 °C.

[0047] A total of three groups of materials were prepared. The corresponding addition amounts of different raw materials are shown in Table 1, and nano-gels AUNGs, MANGs, and ADNGs with different mechanical properties and pH responsiveness were obtained respectively: Table 1

[0048] 1.2 Characterization of nano-gels Dilute the 20 mg / mL nano-gel solution with ultrapure water and PBS to a concentration of 0.2 mg / mL, and then use dynamic light scattering method to detect the hydrodynamic diameter of the nano-gel in PBS and the zeta potential in ultrapure water. The detection temperature is 37 °C, and the equilibration time is 15 min.

[0049] Dilute the nanogel solution with an initial concentration of 20 mg / mL to a concentration of 0.01 mg / mL using ultrapure water. Take 10 μL of the above dispersion and drop it onto a carbon support film, and let it dry naturally. After drying, drop 10 μL of 1% phosphotungstic acid aqueous solution onto the carbon support film and stain for 2 min. After the staining is completed, use filter paper to absorb the excess phosphotungstic acid solution along the edge of the carbon support film. Drop 10 μL of ultrapure water to wash for 1 min. After the washing is completed, use filter paper to absorb the excess ultrapure water along the edge of the carbon support film. After natural drying, observe the morphology of the nanogel using a transmission electron microscope.

[0050] Immerse the cover glass in 1% polyethylenimine aqueous solution for 24 h to modify the surface of the cover glass 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 cover glass, and perform electrostatic adsorption for 10 min. Absorb the excess dispersion, then drop 300 μL of ultrapure water to wash away the unadsorbed nanogel, and then use an atomic force microscope to detect the Young's modulus of the nanogel. The detection environment is liquid phase, and the Young's modulus detection is in tapping mode.

[0051] The results show that: Figure 1 Content A and Figure 1 Content B show that the average hydrated particle size of the nanogels with different hardness prepared in this example is about 150 nm, the surface charges are all negative charges, and the potential is between -25 and -35 mV. Figure 2 It can be seen that the nanogels with different mechanical properties prepared in this example are all spherical with a uniform particle size distribution. Figure 3 It can be seen that the Young's moduli of the three nanogels AUNGs, MANGs, and ADNGs prepared in this example are 648 kPa, 276 kPa, and 236 kPa, respectively.

[0052] 1.3 pH responsiveness of nanogels Dilute the nanogel solution with an initial concentration of 20 mg / mL to a concentration of 0.2 mg / mL using ultrapure water. Use dynamic light scattering method to detect the particle size of the nanogel at different pH values. The detected pH range is 4.5 - 7.5, the pH interval is 0.5, the measurement temperature is 25°C, and the equilibrium time is 2 minutes.

[0053] The results show that: Figure 4 Content A and Content B show that the three nanogels have different pH responsiveness. Among them, the particle size of MANGs is most affected by pH, followed by AUNGs, and the particle size of ADNGs is hardly affected by pH.

[0054] 1.4 Influence of the mechanical properties (S D ) of nanogels by pH The original nano-gel stock solution with an initial concentration of 20 mg / mL was adjusted to the target concentration of 0.2 mg / mL (dilution factor of 100 times) by quantitative dilution method. The pH of the solution was regulated using 0.1 M NaOH and 0.1 M HCl solutions. The pH of the solution was adjusted to 5.5, 6.8, and 7.4 respectively by a precision pH meter (resolution ±0.01). The temperature responsiveness of the nano-gel at the corresponding pH was measured using a dynamic light scattering instrument. The detection temperature was 25.0 - 55.0 °C, the temperature measurement interval was 1.0 °C, and the equilibrium time for each measurement was 120 s. Three parallel determinations were performed during data acquisition, and the hydrodynamic diameter (D h ) was calculated by the Stokes-Einstein equation, and the polydispersity index (PDI, threshold ≤ 0.25) was obtained based on the cumulant analysis method.

[0055] From Figure 5 Content A and Content B, it can be seen that the three types of nano-gels prepared in this example have different temperature responsiveness at different pH values. The speculation is as follows: It may be because the maximum cross-linking degree of AUNGs is 10%, and the internal cross-linking network is the tightest, so its temperature responsiveness is little affected by pH and only has a weak temperature responsiveness at different pH values; the cross-linking degree of MANGs is 2%, and the content of pH-responsive monomer methacrylic acid is 10%, with a loose internal cross-linking network and a high charge content. Therefore, the shrinkage of MANGs is regulated by both temperature and pH. At pH 7.4 and pH 6.8, the carboxylic acids inside the MANGs nano-gel are all deprotonated and exist in the form of carboxylate ions, and the internal electrostatic repulsion is very large, which can resist the force of the nano-gel shrinking with the increase of temperature. Therefore, it does not have temperature responsiveness at pH 7.4 and pH 6.8. At pH 5.5, the dissociation degree of carboxylic acids in the nano-gel is 0.56, and the internal electrostatic repulsion is small, which cannot resist the force of the nano-gel shrinking with the increase of temperature. Therefore, it has temperature responsiveness at pH 5.5; the cross-linking degree of ADNGs is 2%, and the content of methacrylic acid is 0.5%. The internal charge has little effect on the thermosensitivity of the nano-gel. Therefore, ADNGs have temperature responsiveness at different pH values.

[0056] The mechanical properties of nanogels can be evaluated by various means, including elastic moduli (Young's modulus, bulk modulus, and shear modulus), stiffness, and deformation ability. Current characterization methods mainly rely on the nanoindentation technique of atomic force microscopy (AFM), and the Young's modulus and stiffness parameters are calculated through the Hertz contact model. However, this method has significant limitations: the chemical tolerance window of the AFM probe restricts its applicability under extreme pH conditions; the viscoelastic relaxation characteristics of nanogels lead to energy dissipation errors in quasi-static measurements; in addition, the quantitative analysis problem of the probe-sample interaction force in a liquid environment affects the measurement accuracy. Here, in this embodiment, the contraction and deformation ability (S D ) of the nanogel is used to quantitatively evaluate its mechanical properties. This ability is related to the compressibility of the crosslinked network and is ultimately considered related to the softness of the nanogel.

[0057] Since the main monomer of the nanogel is temperature-sensitive NIPMAM, the nanogel has temperature responsiveness. The hydrophilic-hydrophobic balance of the polymer chains inside the nanogel will undergo a hydrophilic-hydrophobic conversion with temperature changes. When the temperature is higher than the critical phase transition temperature of the nanogel, the inside of the nanogel changes from hydrophilic to hydrophobic, and the nanogel shrinks; when the temperature is lower than the critical phase transition temperature, the nanogel swells.

[0058] Define D swollen as the diameter of the nanogel at the first temperature T1 below the critical phase transition temperature, and D deswollen as the diameter of the nanogel at the second temperature T2 above the critical phase transition temperature; the deformation ability of the nanogel under different pH conditions is represented by S D-pH , and its calculation formula is as follows:

[0059] The particle size-temperature curves of three nanogels were measured using a dynamic light scattering instrument (DLS). According to Figure 5 the inflection points of the particle size-temperature curves of the three nanogels, it was determined that their critical phase transition temperatures were all between 40 - 50 °C. Therefore, a test temperature T1 below the critical phase transition temperature was determined to be 25 °C, and a test temperature T2 above the critical phase transition temperature was determined to be 55 °C. Therefore, D swollen is the diameter of the nanogel when it swells, and D deswollen is the diameter of the nanogel when it shrinks.

[0060] The larger the S D value, the greater the deformation ability of the nanogel, and the nanogel is considered to be softer; the closer the S D value is to 1, the worse the deformation performance of the nanogel, and the nanogel is considered to be harder.

[0061] ByFigure 6 It can be seen that the deformability of AUNGs is very poor at different pH values. ADNGs have good deformability at different pH values. Only MANGs have weak deformability at pH 7.4 and pH 6.8, and have good deformability at pH 5.5. The three kinds of nanogels with different mechanical properties (deformability) prepared in this example, AUNGs are hard at different pH values (represented by ), ADNGs are very soft at different pH values (represented by ), while MANGs are hard in a neutral environment and become soft in an acidic environment. As shown in Table 2, Table 2 also gives the S D values of different materials at different pH values.

[0062] It can be seen that the S D-pH1 of nanogel AUNGs at pH1 = 5.5 is less than its S D-pH2 at pH2 = 7.4 or pH2 = 6.8, and S D-pH1 / S D-pH2 = 0.99, or S D-pH1 / S D-pH2 = 1.05.

[0063] The S D-pH1 of nanogel MANGs at pH1 = 5.5 (intracellular tumor environment) is greater than its S D-pH2 at pH2 = 7.4 or pH2 = 6.8, and S D-pH1 / S D-pH2 = 2.01, or S D-pH1 / S D-pH2 = 2.13.

[0064] The S D-pH1 of nanogel ADNGs at pH1 = 5.5 is greater than its S D-pH2 at pH2 = 7.4 or pH2 = 6.8, and S D-pH1 / S D-pH2 = 1.01.

[0065] Table 2

[0066] Example 2 Preparation and characterization of nanogels containing rhodamine B-labeled with different mechanical properties and pH responsiveness: including RhB@AUNGs, RhB@MANGs and RhB@ADNGs.

[0067] Weigh 550 mg of N-isopropylmethylpropanamide and 36 / 40 / 34 mg of sodium dodecyl sulfate, dissolve them in 80 mL of ultrapure water, and transfer the solution to a 250 mL three-necked flask (corresponding to RhB@AUNGs, RhB@MANGs, and RhB@ADNGs). Weigh 112.5 / 22.5 / 22.5 μg of N,N'-bis(acryloyl)cystamine (corresponding to RhB@AUNGs, RhB@MANGs, and RhB@ADNGs), dissolve it in ethanol, and add it to the three-necked flask. Take 0.73 mL of undiluted methacrylic acid and dissolve it in 19.27 mL of ultrapure water, then add 1 / 1 / 0.05 mL of the diluted methacrylic acid solution (corresponding to RhB@AUNGs, RhB@MANGs, and RhB@ADNGs) to the three-necked flask. Weigh 255 μg of rhodamine B and add it to the above solution. Perform three rounds of evacuation and argon filling on the above solution to remove oxygen and ethanol in the system. Heat the deoxygenated solution to 80 °C, and add 0.5 mL of 20 mg / mL potassium persulfate solution through a syringe to initiate the polymerization reaction. The reaction time is 5 - 6 hours. After the reaction, transfer the solution to a 10 kDa ultrafiltration tube, and ultrafilter at 2000 rpm to remove unreacted monomers and impurities. Repeatedly add ultrapure water to purify and concentrate the material. Use the drying method to quantify the purified material, and quantify the material to a concentration of 20 mg / mL and store it at 4 °C.

[0068] Dilute the 20 mg / mL rhodamine B-labeled nanogel solution to a concentration of 0.2 mg / mL with PBS, and then use dynamic light scattering to detect the hydrodynamic diameter of the rhodamine B-labeled nanogel in PBS. The detection temperature is 37 °C, and the equilibration time is 15 min.

[0069] Dilute the 20 mg / mL rhodamine B-labeled nanogel solution to 0.01 mg / mL and 0.005 mg / mL with ultrapure water respectively. Use a time-resolved fluorescence spectrometer to measure the signal intensity of the 0.01 mg / mL and 0.005 mg / mL rhodamine B-labeled nanogel solutions at the emission wavelength of 580 nm, and calculate the fluorescence intensity ratio of the rhodamine B-labeled nanogel at the same mass concentration.

[0070] The results show that: From Figure 7 It can be seen that the particle sizes of the rhodamine B-labeled nanogels are all around 150 nm. As can be seen from Table 3, the rhodamine B-labeled nanogels have different fluorescence intensities at the same mass concentration. For comparison, the fluorescence ratio at 0.005 mg / mL is selected, which is 3.35:1:1.35.

[0071] Table 3 Ratio of relative fluorescence intensity of nanogels labeled with Rhodamine B

[0072] Example 3 Preparation of DOX-loaded nanogels with different mechanical properties.

[0073] Preparation of DOX-loaded nanogels with different mechanical properties, including DOX@AUNGs, DOX@MANGs, and DOX@ADNGs, and the preparation method is as follows: Weigh 10 mg of doxorubicin hydrochloride, add 5 mL of ultrapure water, and dissolve it by ultrasonic treatment. Then add it to 5 mL of the 20 mg / mL nanogel solution (AUNGs, MANGs, ADNGs) obtained in Example 1. The two are mixed and stirred in a 100 mL flask at room temperature for 48 h. Transfer the obtained solution to an ultrafiltration tube with a cut-off value of 10 kDa, and centrifuge at 2000 rpm to remove the unadsorbed doxorubicin hydrochloride, while concentrating the nanodrug. During the process, ultrapure water is repeatedly added to ensure sufficient impurity removal. Take 50 μL of the purified nanodrug solution, add it to 2.95 mL of dimethyl sulfoxide, mix well, and detect it with a UV spectrophotometer at a detection wavelength of 483 nm. According to the standard curve of doxorubicin hydrochloride at 483 nm wavelength in dimethyl sulfoxide, calculate the concentration of doxorubicin hydrochloride in the obtained nanodrug solution, and dilute the nanodrug concentrate to a doxorubicin hydrochloride concentration of 1 mg / mL to obtain DOX@AUNGs, DOX@MANGs, and DOX@ADNGs respectively, and store them at 4 °C.

[0074] After diluting the DOX-loaded nanogel solution with a doxorubicin hydrochloride concentration of 1 mg / mL 50-fold with ultrapure water and PBS, the hydrodynamic diameter of the DOX-loaded nanogel in PBS and the zeta potential in ultrapure water are detected by dynamic light scattering method. The detection temperature is 37 °C, and the equilibration time is 15 min.

[0075] The results show that: From Figure 8 Content A and Content B, it can be seen that the average hydrodynamic diameter of the DOX-loaded nanogels with different hardness prepared in this example is increased by about 30 nm compared with the particle size of the nanogels without DOX loading, which is about 190 nm, indicating that the nanogels are successfully loaded with DOX. From Figure 8 Content C, it can be seen that after loading DOX, the surface charge of the nanogels decreases slightly.

[0076] Example 4 Different cells have different uptake rates for nanogels labeled with Rhodamine B.

[0077] 4.1 Uptake of nanogels labeled with Rhodamine B by 3T3 cells The rhodamine B-labeled nanogel solution at a concentration of 20 mg / mL was diluted with DMEM medium to a concentration of 0.05 mg / mL to obtain a serum-free medium containing rhodamine B-labeled nanogels with different mechanical properties. 3T3 cells were seeded into a 6-well plate at a density of 2×10 5 cells / well. After adherent culture in an incubator at 37 °C and 5% CO2 for 12 hours, the upper-layer medium was discarded. After washing with PBS, 2 mL of the medium containing nanogels with different mechanical properties was added respectively. After incubation in an incubator at 37 °C and 5% CO2 for 1, 2, 4, and 8 hours respectively, the medium containing nanogels with different mechanical properties was discarded. The cells were washed twice with PBS, digested with trypsin and centrifuged. After resuspending to obtain a single-cell suspension, the fluorescence intensity inside the cells was detected by a flow cytometer. The detection channel was PE, and the relative cell uptake was calculated based on the fluorescence intensity.

[0078] 4.2 Uptake of rhodamine B-labeled nanogels by 4T1 cells The rhodamine B-labeled nanogel solution at a concentration of 20 mg / mL was diluted with 1640 medium to a concentration of 0.05 mg / mL to obtain a serum-free medium containing rhodamine B-labeled nanogels with different mechanical properties. 4T1 cells were seeded into a 6-well plate at a density of 5×10 5 cells / well. After adherent culture in an incubator at 37 °C and 5% CO2 for 12 hours, the upper-layer medium was discarded. After washing with PBS, 2 mL of the medium containing nanogels with different mechanical properties was added respectively. After incubation in an incubator at 37 °C and 5% CO2 for 1, 2, 4, 8, and 12 hours respectively, the medium containing nanogels with different mechanical properties was discarded. The cells were washed twice with PBS, digested with trypsin and centrifuged. After resuspending to obtain a single-cell suspension, the fluorescence intensity inside the cells was detected by a flow cytometer. The detection channel was PE, and the relative cell uptake was calculated based on the fluorescence intensity.

[0079] 4.3 Effect of pH on the uptake of rhodamine B-labeled nanogels by 4T1 cells The nanogel solution at a concentration of 20 mg / mL was diluted with PBS buffer to a concentration of 0.05 mg / mL, and the pH of the solution was adjusted to pH 5.5, pH 6.8, and pH 7.4 with NaOH solution and HCl solution respectively. 4T1 cells were seeded into a 6-well plate at a density of 5×10 5Cells were seeded at a density of

[0080] per well into a 6-well plate. After adhering for 12 hours in an incubator at 37 °C with 5% CO2, the upper medium was discarded. After washing with PBS, 2 mL of PBS buffer containing nanogels with different mechanical properties at different pH values was added respectively. After incubating for 2 hours in an incubator at 37 °C with 5% CO2, the PBS buffer containing nanogels with different mechanical properties at different pH values was discarded. The cells were washed twice with PBS, digested with trypsin and centrifuged. After resuspending to obtain a single-cell suspension, the fluorescence intensity inside the cells was detected by flow cytometry. The detection channel was PE, and the relative cell uptake was calculated based on the fluorescence intensity. Figure 9 The results showed that: As can be seen from Figure 9 Content A, the uptake of rhodamine B-labeled nanogels by 3T3 cells gradually increased with the prolongation of time, and the uptake rate of MANGs was the fastest. Compared with the other two nanogels with a cross-linking degree of 2%, the uptake of AUNGs nanogels with a cross-linking degree of 10% was slower. As can be seen from Figure 9 Content B, the uptake trend of rhodamine B-labeled nanogels by 4T1 cells was similar to that of 3T3 cells. The uptake rate of MANGs was the fastest, the uptake of AUNGs was the slowest, and the uptake rate of ADNGs was between the two. As can be seen from

[0081] Example 5 Nanogels with different mechanical properties have different transcytosis efficiencies.

[0082] Transcytosis efficiency of nanogels with different mechanical properties in 4T1 cells: The rhodamine B-labeled nanogel solution at 20 mg / mL was diluted to a concentration of 0.1 mg / mL with 1640 serum-free medium to obtain a serum-free medium containing rhodamine B-labeled nanogels with different mechanical properties. 4T1 cells were seeded at a density of 4×10 5Cells were seeded into a 6-well plate at a density of [number] per well and allowed to adhere for 12 hours in a 37°C, 5% CO₂ incubator. After discarding the upper medium, the cells were washed with PBS and then 2 mL of medium containing nanogels with different mechanical properties was added. After incubating for 8 hours, the upper medium was discarded, and the cells were washed 3 times with PBS buffer. Then, 3 mL of fresh serum-free 1640 medium was added and the cells were incubated in the incubator for 12 hours. These were the first-generation 4T1 cells. The upper medium of the first-generation 4T1 cells was transferred to new 4T1 cells that had been adherent in a 6-well plate for 12 hours without drug treatment, and these 4T1 cells were designated as the second generation. Meanwhile, the first-generation 4T1 cells were collected and the fluorescence intensity was detected using a flow cytometer with the detection channel being PE. After incubating for 12 hours, the upper medium of the second-generation 4T1 cells was transferred to new 4T1 cells that had been adherent in a 6-well plate for 12 hours without drug treatment, and these 4T1 cells were designated as the third generation. Meanwhile, the second-generation 4T1 cells were collected and the fluorescence intensity was detected using a flow cytometer with the detection channel being PE. After incubating the third-generation cells for 12 hours, the third-generation 4T1 cells were collected and the fluorescence intensity was detected using a flow cytometer with the detection channel being PE. Then, the relative cell uptake was calculated based on the fluorescence intensity.

[0083] Results showed that quantitative analysis based on flow cytometry revealed that mechanically tunable nanogels (RhB@MANGs) exhibited significant advantages in the trans-endocytosis model across generations of 4T1 cells ( Figure 10 ). The experimental data showed that in the second-generation cell system, the mean fluorescence intensity (MFI = 4710) of RhB@MA NGs was increased by 49.2% and 222.8% compared to RhB@AD NGs (3157, P < 0.005) and RhB@AU NGs (1485, P < 0.0001), respectively; in the third-generation (P3) cell system, the mean fluorescence intensity of RhB@MA NGs still maintained a significant difference (vs. RhB@AD NGs: 72% of RhB@MA NGs, P < 0.005; vs. RhB@AU NGs: 43% of RhB@MA NGs, P < 0.0001). The RhB@MA NGs group showed the highest mean fluorescence intensity in all three generations of cells, indicating its continuous and efficient trans-endocytosis ability across generations; the fluorescence intensity of the RhB@AD NGs group showed a decreasing trend across generations, and the fluorescence intensities of the three generations of the RhB@AU NGs group were lower than those of the other two groups, indicating that the trans-endocytosis ability of these two groups was poor.

[0084] Example 6 Nanogels with different mechanical properties had different co-localizations in organelles.

[0085] Colocalization of nanogels with different mechanical properties in 4T1 cell organelles: Dilute the rhodamine B-labeled nanogel solution at a concentration of 20 mg / mL with 1640 serum-free medium to a concentration of 0.1 mg / mL to obtain a serum-free medium containing rhodamine B-labeled nanogels with different mechanical properties. Add 1 mL of 4T1 cells at a density of 1×10 5 cells / mL into a confocal dish. After adherent culture at 37 °C and 5% CO2 for 24 hours, discard the upper medium, wash with PBS, and then add 1 mL of serum-free medium containing nanogels with different mechanical properties and incubate for 1 hour. Stain lysosomes with Lyso-Tracker Green (lysosome green fluorescence probe) for 20 min, and stain Golgi apparatus with Golgi fluorescence staining kit (Golgi-Tracker GreenII) according to the kit instructions. Dilute the nucleus with Hoechst 33342 (1000X) by 1000 times and stain for 15 min. After incubation, wash 3 times with PBS and perform CLSM imaging. Observe the fluorescence of LysoTracker green or Golgi-Tracker Green II at an excitation wavelength of 488 nm, observe the fluorescence of Hoechest 33342 at an excitation wavelength of 405 nm, and observe the fluorescence of rhodamine B at an excitation wavelength of 560 nm. In the same experiment, the CLSM parameters remain unchanged. Use ImageJ software to statistically analyze the Manders colocalization coefficient of the fluorescence of rhodamine B and the fluorescence of LysoTracker green or Golgi-Tracker Green II in single cells, and count 40 cells in each group.

[0086] The results show that: as Figure 11 shown in Content A and Content B, the Manders colocalization coefficient of MANGs in lysosomes is significantly lower than that of AUNGs and ADNGs, and the colocalization in the Golgi apparatus is significantly higher than that of the other two groups, indicating that MANGs are more distributed in the Golgi apparatus rather than in lysosomes after being taken up by cells, while AUNGs and ADNGs are more distributed in lysosomes, indicating that after entering cells, MANGs are more excreted extracellularly through the endoplasmic reticulum / Golgi system, which may be the reason why MANGs have the best transcytosis efficiency.

[0087] Example 7 Deep penetration ability of nanogels with different mechanical properties in 3D tumor spheres.

[0088] Weigh sodium carboxymethylcellulose powder and prepare a 1.2% sodium carboxymethylcellulose solution with 1640 complete medium. Digest NIH / 3T3 cells and 4T1 cells with trypsin, centrifuge to collect the cells, and set the cell concentrations to 6.66×10 5 cells / mL and 1.32×10 7 cells / mL respectively. Mix these two cell suspensions evenly according to a volume ratio of 1:1 to obtain a mixed cell suspension. Add a sodium carboxymethylcellulose solution with a volume equal to one-fourth of the mixed cell suspension and mix well to obtain a suspension. Drop 20 μL of the suspension onto the inner side of the upper cover of the culture dish, then flip the upper cover to cover the lower dish so that the culture medium containing the cells hangs on the upper cover, and culture in an incubator for 36 h until 3D tumor spheres are formed. Transfer the 3D tumor spheres to serum-free media containing the rhodamine B-labeled nanogels with different hardnesses obtained in Example 2, namely RhB@AUNGs, RhB@MANGs, and RhB@ADNGs, at a concentration of 100 μg / mL, incubate in an incubator for 2 h, then transfer the 3D tumor spheres to PBS buffer for washing 3 times, and scan along the longitudinal axis of the 3D tumor spheres at an excitation wavelength of 561 using a laser confocal microscope. Use ImageJ software to draw lines and compare the fluorescence sections of the 3D tumor spheres at the same depth.

[0089] The results showed that: as Figure 12 shown, the three types of nanogels had different deep penetration effects in 3D tumor spheres. The deep penetration ability of the hard nanogel was significantly lower than that of the soft nanogel, and MANGs with the best transcytosis ability had the best deep penetration ability, indicating that improving the active transcytosis ability of cells to the material could significantly improve the deep penetration ability of the material in 3D tumor spheres.

[0090] Example 8 Effect of mechanical properties on the tissue distribution of nanogels and their deep penetration at the tumor site.

[0091] 8.1 Effect of mechanical properties on the tissue distribution of nanogels Subcutaneously inoculate 1×10 6 4T1 cell suspensions on the back of 7-week-old female BALB / C mice near the right hind limb, with an inoculation volume of 100 μL per mouse, to establish a subcutaneous 4T1 breast cancer mouse model. When the tumor volume reaches 200 - 300 mm 3 ³, randomly divide the mice into three groups, with 6 mice in each group, grouped as AUNGs, MANGs, and ADNGs. Inject the rhodamine B-labeled nanogels obtained in Example 2 through the tail vein, with an injection dose of 100 mg / kg. Sacrifice the mice 8 h after injection, dissect the tumors and the heart, liver, spleen, lungs, and kidneys for in vitro imaging, and quantify the fluorescence intensity.

[0092] The results showed that: From Figure 13 Content A and Content B, it can be seen that at the 8-hour time point, AUNGs were mainly distributed in the liver, ADNGs were mainly distributed in the kidneys, and MANGs had the highest enrichment in tumors, indicating that MANGs with the best transcytosis effect also had the optimal enrichment effect in 4T1 subcutaneous tumors.

[0093] 8.2 Influence of mechanical properties on the deep penetration of nanogels in tumor sites Subcutaneously inoculate 1×10 6 4T1 cell suspensions at the back of 7-week-old female BALB / C mice near the right hind limb. The inoculation volume for each mouse was 100 μL to establish a mouse 4T1 breast cancer subcutaneous tumor model. When the tumor volume reached 200 - 300 mm 3 ³, the mice were randomly divided into three groups with 6 mice in each group, namely AUNGs, MANGs, and ADNGs. Inject the rhodamine B-labeled nanogels obtained in Example 2 via the tail vein. The injection dose was 100 mg / kg. After 8 h of injection, the excised tumors were fixed with 4% paraformaldehyde and sectioned. The tumor blood vessels were labeled with fluorescein isothiocyanate-labeled CD31 antibody, and then the sections were detected and scanned by fluorescence. The excitation wavelengths for detection were 483 nm and 560 nm respectively. The shortest distance between the fluorescence-labeled nanogels and the tumor blood vessels was measured and statistically analyzed according to the scanned images. 50 points were statistically analyzed for each group.

[0094] The results showed that: From Figure 14 it can be seen that AUNGs were the closest to the blood vessels in solid tumors, MANGs were the farthest from the blood vessels, and ADNGs were between the two, indicating that soft nanogels had better deep penetration ability in tumor tissues than hard nanogels, and MANGs with the best transcytosis efficiency had the optimal deep penetration ability in tumor tissues.

[0095] Example 9 Influence of mechanical properties on the antitumor effect of DOX-loaded nanogels.

[0096] 9.1 Influence of mechanical properties on the antitumor effect of DOX-loaded nanogels at the cellular level Seed 4T1 cells at 8×10 3Cells were inoculated into a 96-well plate at a concentration of [[[number of cells or holes]]] / well, and 200 μL of RPMI-1640 medium containing 10% serum and 1% double antibody was added to each well. The cells were allowed to adhere for 12 h in an incubator at 37 °C with 5% CO2. The upper layer of the medium was aspirated, and 200 μL of the DOX-loaded nanogels with different hardnesses prepared in Example 3 (DOX@AUNGs, DOX@MANGs, and DOX@ADNGs) were added respectively. The dosing concentrations were set at 0, 0.25, 0.5, 1, 2, 4, 8, and 16 μg / mL based on the concentration of doxorubicin hydrochloride. The cells were incubated for 24 h in an incubator at 37 °C with 5% CO2, and then the upper layer of the medium was aspirated. Then, 100 μL of the medium obtained by mixing CCK8 and RPMI-1640 medium containing 10% serum in a ratio of 1:9 was added to each well. After incubating in the incubator for 30 minutes, the absorbance of the culture solution was measured at 450 nm using a microplate reader. The data were processed and analyzed using Excel and Graphpad Prism to calculate the killing effect of the DOX-loaded nanogels with different mechanical properties on 4T1 cells.

[0097] The results showed that: as Figure 15 shown, since MANGs had the largest cell uptake, its cytotoxicity to tumor cells was the greatest after loading DOX, that is, the killing ability to tumor cells was the greatest. DOX@ADNGs was weaker than DOX@MANGs, and the tumor cell toxicity of DOX@AUNGs was the weakest.

[0098] 9.2 Effect of mechanical properties on the in vivo anti-tumor effect of DOX-loaded nanogels A 1×10 6 suspension of 4T1 cells was subcutaneously inoculated at the back near the right hind limb of 7-week-old female BALB / C mice. The inoculation volume for each mouse was 100 μL to establish a subcutaneous tumor model of 4T1 breast cancer in mice. When the tumor volume reached 100 mm 3 3, the mice were randomly divided into five groups, including Control, FreeDOX, DOX@AUNGs, DOX@MANGs, and DOX@ADNGs, marked as day 0, with 7 mice in each group. On day 0, day 4, and day 8 after grouping, normal saline, free doxorubicin hydrochloride, and the DOX-loaded nanogels obtained in Example 3 (DOX@AUNGs, DOX@MANGs, DOX@ADNGs) were injected via the tail vein respectively, and the injection dose was 4 mg / kg of doxorubicin hydrochloride. Since day 0, the long side (a) and short side (b) of the subcutaneous tumor in mice were measured daily using vernier calipers. According to the calculation formula: tumor volume V = a × b 2 2, the tumor volume was calculated. After the measurement on day 15, the mice were sacrificed, the tumors were dissected, weighed, and photographed, and the tumor inhibition rate was calculated based on the tumor volume and mass.

[0099] The results show that according to Figure 16 Contents A, B, C, and D, it can be seen that the tumor growth in the DOX@MANGs group was the slowest, with the smallest volume and the lightest mass. The anti-tumor effect of the DOX@AUNGs group was close to that of free DOX, while in the DOX@ADNGs group, very strong toxic and side effects were found in mice after tail vein injection, showing strong systemic toxicity, and half of the mice died within three days after a single dose. Therefore, the DOX@ADNGs group was excluded. It can be seen from the figure that the DOX@MANGs group had significantly better anti-tumor effects compared to the control group.

[0100] The above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of changes or substitutions, which should all be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A pH-responsive nanogel with transformed mechanical properties, characterized in that, The nano-gel is obtained by polymerizing monomers in an aqueous phase through an initiator in the presence of a cross-linking agent and a surfactant; the monomers include temperature-responsive monomers and pH-responsive monomers; The hydrophilic-hydrophobic balance of the polymer chains inside the nano-gel can undergo a hydrophilic-hydrophobic equilibrium transformation with the change of temperature. When the temperature is higher than the critical phase transition temperature of the nano-gel, the inside of the nano-gel changes from hydrophilic to hydrophobic, and the nano-gel shrinks, making its size smaller; when the temperature is lower than the critical phase transition temperature, the inside of the nano-gel changes from hydrophobic to hydrophilic, and the nano-gel swells, making its size larger; Definition D swollen is the diameter of the nanogel at the first temperature T1 below the critical phase transition temperature, D deswollen is the diameter of the nanogel at the second temperature T2 above the critical phase transition temperature; the deformation ability of the nanogel under different pH conditions is represented by S D-pH which is expressed as follows: The S of the nanogel at pH1 = 4.5 - 6 D-pH1 is greater than its S at pH2 = 6.8 - 7.4 D-pH2 , and S D-pH1 / S D-pH2 ≥ 1.

6.

2. The nanogel according to claim 1, wherein The first temperature T1 is 20 - 30 °C, and the second temperature T2 is 50 - 60 °C.

3. The nanogel according to claim 1, wherein S D-pH1 / S D-pH2 ≥1.9。 4. The nanogel according to claim 1, wherein, The S of the nanogel at pH1 = 4.5 - 6 D-pH1 is greater than or equal to 2, and its S at pH2 = 6.8 - 7.4 D-pH2 is less than or equal to 1.

25.

5. The nanogel according to claim 1, wherein The Young's modulus of the nano-gel is 250 - 300 Kpa.

6. The nanogel according to claim 1, characterized in that, The temperature-responsive monomer is one or more of N-isopropylmethacrylamide, N-isopropylacrylamide, and N-ethylacrylamide; The pH-responsive monomer is 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'-vinylenebisacrylamide; The initiator is one or more of potassium persulfate, sodium persulfate, and tert-butyl hydroperoxide; The surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, and lecithin; The feeding molar ratio of the pH-responsive monomer to the temperature-responsive monomer is (8 - 12):100; the feeding molar ratio of the cross-linking agent to the temperature-responsive monomer is (1 - 3):100; the mass ratio of the initiator to the temperature-responsive monomer is (8 - 12):550; the mass ratio of the surfactant to the temperature-responsive monomer is (38 - 42):

550.

7. Use of the nano-gel according to any one of claims 1 to 6 in the preparation of a deformable material having pH responsiveness.

8. Use of the nano-gel according to any one of claims 1 to 6 as or in the preparation of a nano-drug delivery carrier.

9. Use of the nano-gel according to any one of claims 1 to 6 in the preparation of a nano-drug having active transcytosis ability.

10. A nano-drug, characterized in that, It includes the nano-gel according to any one of claims 1 to 6, and also includes an anti-tumor drug loaded on the nano-gel through electrostatic adsorption.

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