Preparation method and application of response type nanogel with adjustable flexibility

Abstract

The invention provides a preparation method and application of response type nanogel with adjustable flexibility, and the preparation method comprises the following steps: preparing a monomer solution which comprises N-isopropyl methacrylamide, methacrylic acid, a cross-linking agent and a surfactant; adding an initiator into the monomer solution, and carrying out polymerization reaction under the water bath condition of 75-90 DEG C to generate an initial product; and purifying, washing and drying the initial product to obtain the nanogel, and applying the nanogel to drug delivery and gene therapy drugs. According to the technical scheme, the temperature and pH dual-sensitive characteristic is achieved, meanwhile, tumor microenvironment stimulation such as intracellular GSH concentration rising can be responded, efficient release of plasmids is achieved, meanwhile, good structure regulation and control performance, biocompatibility and low toxicity are achieved, the transfection efficiency equivalent to PEI in various cell models is shown, and the application prospect is wide. Great clinical application potential is realized.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial preparation technology, and more specifically, to a method for preparing and applying a flexible and tunable responsive nanogel. Background Technology

[0002] In modern life science and medical research, gene therapy, as a technology for precisely regulating cell function, is widely considered a potential cure strategy for major diseases such as genetic disorders and tumors. However, nucleic acid drug molecules such as plasmid DNA, siRNA, or mRNA are easily degraded in vivo, and due to their negative charge and large molecular weight, they are difficult to penetrate cell membranes and enter the cell. Therefore, an efficient and safe delivery system is crucial for the success of gene therapy.

[0003] Currently, commonly used gene delivery methods in clinical and laboratory settings mainly include viral vectors and non-viral vectors. Viral vectors have high transfection efficiency but face challenges such as strong immunogenicity, limited loading capacity, and difficulty in large-scale preparation. Non-viral vectors, such as cationic polymers, liposomes, and inorganic nanomaterials, have advantages in terms of safety and controllability, but their transfection efficiency and intracellular release capacity still need improvement. Polyethyleneimine (PEI) is one of the most widely used cationic polymer vectors. Although it exhibits good transfection ability in some cells, its cytotoxicity and non-degradability limit its further clinical application.

[0004] To overcome these challenges, researchers have recently focused on the application of nanogels (NGs) in gene delivery. Nanogels are three-dimensional network structures with high hydration, good biocompatibility, and high loading capacity. Through structural design, they can respond to external / internal stimuli (such as light, temperature, pH, and glutathione (GSH),) thereby achieving "intelligent release" at the target site. Especially for diseases such as cancer and inflammation, whose microenvironments are often acidic and characterized by high GSH expression, developing a class of multifunctional nanogel carriers with temperature, pH, and GSH responsiveness has become an effective method to solve the current challenges in gene delivery.

[0005] Meanwhile, the flexibility of materials has become a key factor affecting delivery efficiency. Soft, controllable gel networks facilitate penetration of the dense extracellular matrix and intercellular spaces at tumor sites, promoting endocytosis while avoiding mechanical damage to the cell membrane. Constructing a flexible, tunable, highly responsive, efficient, and low-toxicity nanogel carrier is a significant challenge in current research on non-viral gene delivery systems. Summary of the Invention

[0006] In view of the problems in the prior art, the purpose of this invention is to provide a method for preparing and applying flexible and tunable responsive nanogels.

[0007] On one hand, the present invention provides a method for preparing flexible and tunable responsive nanogels, comprising the following steps:

[0008] Step 1: Prepare a monomer solution, wherein the monomer solution comprises N-isopropylmethacrylamide, methacrylic acid, N,N'-bis(acryloyl)cysteine ​​and a surfactant;

[0009] Step 2: Add an initiator to the monomer solution and polymerize under water bath conditions of 75-90℃ to generate the initial product;

[0010] Step 3: After purifying, washing and drying the initial product, nanogels are obtained.

[0011] Preferably, the surfactant is sodium dodecyl sulfate.

[0012] Preferably, the initiator is potassium persulfate.

[0013] Preferably, the molar ratio of N-isopropylmethacrylamide to methacrylic acid in the monomer solution of step 1 is 18:1.

[0014] Preferably, the amount of N,N'-bis(acryloyl)cysteine ​​in the monomer solution of step 1 is 1-14% of the total molar amount of the monomer (the total molar amount of the monomer refers to the total molar amount of N-isopropylmethacrylamide, methacrylic acid, and N,N'-bis(acryloyl)cysteine).

[0015] Preferably, the concentration of the surfactant in the monomer solution in step 1 is 0.1875–0.5 mg / mL.

[0016] Preferably, the nanogel has a particle size range of 200–280 nm at room temperature.

[0017] On the other hand, the present invention provides the application of nanogels prepared by the above-described method for preparing flexible and tunable responsive nanogels in drug delivery.

[0018] On the other hand, the present invention provides the application of nanogels prepared by the above-described method for preparing flexible and tunable responsive nanogels in gene therapy drugs.

[0019] The technical solution of the present invention has the following beneficial effects:

[0020] 1. The technical solution of this invention proposes a method for preparing a temperature-, pH-, and GSH-triple-responsive nanogel gene delivery material based on a mild aqueous phase polymerization system. The synthesis process is simple, the conditions are controllable, it is suitable for large-scale preparation, and it has good prospects for industrial transformation.

[0021] 2. The nanogel particles obtained by the technical solution of the present invention have uniform particle size, regular morphology, and good dispersibility. The flexible and adjustable network structure can be achieved by adjusting the ratio of monomer to crosslinking agent, so as to meet the physical performance requirements of nanoparticles in different biological environments.

[0022] 3. The nanogel material constructed by the technical solution of the present invention has significant triple response characteristics, which can rapidly release the loaded plasmid DNA in tumor-associated microenvironments such as body temperature, weak acid and high GSH concentration. It has good intracellular release efficiency and transfection ability, and is suitable for precise delivery in the tumor microenvironment.

[0023] 4. The nanogel carrier prepared by the technical solution of the present invention has good biocompatibility and low toxicity, and exhibits excellent cell adaptability in a variety of cell lines. Its transfection efficiency is comparable to that of commercial transfection reagent PEI.

[0024] 5. Compared with existing non-viral gene delivery vectors, the nanogel material of the present invention has the advantages of simple synthesis process, low production cost and green environmental protection, and is widely applicable to drug delivery, gene therapy and tumor treatment, showing good application prospects and promotion value. Attached Figure Description

[0025] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0026] Figure 1 TEM images (A, B, D, E), size distribution diagram (C), and zeta potential diagram (F) of the nanogel materials obtained in Examples 1-4, including 2.5% NGs (A), 5% NGs (B), 10% NGs (D), and 20% NGs (E);

[0027] Figure 2 The AFM diagrams and Young's modulus diagrams (D) of the nanogel materials obtained in Examples 1-3 are shown, including 2.5% NGs (A), 5% NGs (B), and 10% NGs (C).

[0028] Figure 3 TEM image (A) of the nanogel containing the plasmid obtained in Example 5, Zeta potential (B) and size distribution (C) of Example 5 (2.5% NGs + pDNA) and Example 1 (2.5% NGs);

[0029] Figure 4 The size distribution (A) and zeta potential (B) of the nanogel obtained in Example 1 over 90 days are shown.

[0030] Figure 5 The diagram shows the penetration ability of nanogels with different flexibility obtained in Examples 1-3 in tumor spheres (A), and the penetration diagram of simulated extracellular matrix in Examples 1 and 4 (B).

[0031] Figure 6 The images show a confocal image (A) of H22 cells transfected with plasmid-loaded nanogels (with plasmid transfection capability) obtained in Example 5 and commercial transfection reagent PEI; Western blot images (B) of H22 cells transfected with commercial transfection reagent PEI in Examples 1 and 5; flow cytometry quantitative transfection images (C) of H22 cells transfected with commercial transfection reagent PEI in Examples 1 and 5; Western blot quantitative transfection images (D) of H22 cells transfected with commercial transfection reagent PEI in Examples 1 and 5; flow cytometry quantitative transfection images (E) of Hep-G2 cells transfected with commercial transfection reagent PEI in Example 5; and flow cytometry quantitative transfection images (F) of 293T cells transfected with commercial transfection reagent PEI in Example 5.

[0032] Figure 7 The images show the cytotoxicity of different concentrations of nanogels obtained in Examples 1 and 5 against 3T3 normal cells (A) and H22 tumor cells (B).

[0033] Figure 8 Temperature response particle size change diagram (A) and pH response particle size change diagram (D) of nanogels obtained in Examples 1-4; agarose gel electrophoresis diagram of plasmid loading and release in GSH environment in Examples 1 and 5 (B); GSH response TEM image of Example 1 (C); GSH response particle size change diagram of Example 1 (E). Detailed Implementation

[0034] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The same reference numerals in the drawings denote the same or similar structures, and therefore repeated descriptions of them will be omitted.

[0035] The purpose of this invention is to provide a method for preparing a nanogel-like gene delivery system that is simultaneously temperature and pH sensitive, glutathione (GSH) responsive, and flexible. This nanogel carrier overcomes the problems of poor biocompatibility, high structural rigidity, and unstable transfection efficiency of existing non-viral gene carriers, and solves the limitations of traditional cationic polymer (such as PEI) carriers, such as non-degradability, during transfection. It also avoids the defects of conventional carriers, such as failure in complex physiological environments, single response, and unstable loading, and solves the bottlenecks of current nanocarrier synthesis processes, such as complex processes, poor batch stability, and insufficient targeting. Furthermore, by adjusting the material ratio, the flexibility of the nanogel particles can be changed to penetrate the dense extracellular matrix of tumor lesions. More importantly, this nanogel carrier can efficiently release plasmids according to the stimulation signals of the tumor and other disease microenvironments, achieving transfection efficiency comparable to commercial transfection reagents at the cellular level. It also possesses low toxicity, high stability, and good biocompatibility, and has broad application prospects in drug delivery, gene therapy, and tumor treatment.

[0036] The preparation method of the responsive nanogel according to an embodiment of the present invention includes the following steps:

[0037] Prepare a monomer solution containing N-isopropylmethacrylamide (NIPMAM), methacrylic acid (MAA), N,N'-bis(acryloyl)cysteine ​​(BAC) and the surfactant sodium dodecyl sulfate (SDS);

[0038] The preferred method is to prepare a nanogel carrier with temperature, pH and GSH responsiveness by aqueous free radical polymerization, and the plasmid loading can be completed simultaneously by one-pot method.

[0039] Preferably, the monomer N,N'-bis(acryloyl)cysteine ​​(BAC) is pre-dissolved in ethanol, and then the monomer N-isopropylmethacrylamide (NIPMAM), methacrylic acid (MAA) and surfactant sodium dodecyl sulfate (SDS) are co-dissolved in the aqueous phase, ultrasonically dispersed, and then argon gas is introduced to remove dissolved oxygen.

[0040] Subsequently, potassium persulfate (KPS) was added as an initiator, and the polymerization reaction was carried out at 75–90°C, preferably 80°C, for 6 hours to generate the initial product of multi-responsive nanogel particles.

[0041] Ideally, the plasmid is added simultaneously with potassium persulfate to obtain a plasmid-loaded nanogel, thus completing the plasmid loading process in a one-pot method.

[0042] Finally, after the reaction is complete, the material is obtained by dialysis, centrifugation, washing and freeze-drying.

[0043] In this embodiment of the invention, the preferred molar ratio of NIPMAM to MAA in the monomer solution is 18:1, and BAC accounts for 1% to 14% of the total monomer molar amount, so as to achieve the flexible and adjustable gel network and the balanced expression of triple response behavior.

[0044] The concentration of SDS in the monomer solution is preferably controlled at around 0.1875–0.5 mg / mL to stabilize the nanogel system during the polymerization process and control the particle size, which helps to stabilize the formation of colloidal particles and obtain nanogels with uniform particle size distribution.

[0045] In this embodiment of the invention, the network crosslinking density of the gel can be adjusted by using different amounts of BAC and SDS, thereby regulating the mechanical flexibility and responsiveness of the nanogel. As the crosslinking density decreases, the gel structure tends to be loose and the hardness decreases, which is conducive to penetrating the dense extracellular matrix.

[0046] In this embodiment of the invention, the obtained nanogel has the morphology of spherical particles, with a particle size range of 200 to 280 nm at room temperature, a zeta potential range of -10 to -30 mV, and a polydispersity index (PDI) of less than 0.2, which is suitable for cellular uptake.

[0047] This invention discloses a smart responsive nanogel constructed from N-isopropylmethacrylamide (NIPMAM), methacrylic acid (MAA), and N,N'-bis(acryloyl)cysteine ​​(BAC). This nanogel not only possesses dual temperature and pH sensitivity but also responds to tumor microenvironment stimuli such as increased intracellular GSH concentration, thereby achieving efficient plasmid release. It also exhibits good structural regulation, biocompatibility, and low toxicity, demonstrating transfection efficiency comparable to PEI in various cell models, and possesses significant clinical application potential.

[0048] The present invention will now be described with reference to specific embodiments and comparative examples:

[0049] Example 1

[0050] A method for preparing a flexible and tunable smart responsive nanogel is as follows:

[0051] 28.2 mg of crosslinking agent N,N'-bis(acryloyl)cysteine ​​(BAC) was pre-dissolved in ethanol, and then dissolved in 80 mL of water along with 550 mg of monomer N-isopropylmethacrylamide (NIPMAM), 18.65 μL of methacrylic acid (MAA), and 35 mg of sodium dodecyl sulfate (SDS). The mixture was placed in a round-bottom flask and sonicated for 5 minutes.

[0052] After the monomer solution has been ultrasonically dispersed evenly, it is added to a three-necked flask and stirred until homogeneous. Argon gas is introduced for 30 minutes, and the solution is stirred at 500 rpm for 30 minutes.

[0053] Then, the solution was heated to 80°C, 10 mg of potassium persulfate (KPS) solution was added, and the polymerization reaction was started by stirring for 6 hours.

[0054] Finally, after the reaction was complete and the material was cooled to room temperature, it was purified by ultrafiltration (14000 Da) to remove unreacted monomers and other small molecules. The mixture was washed three times with water to obtain 2.5% NGs (at this point, the amount of BAC used was 1% of the total molar amount of monomers) and stored at 4°C.

[0055] Example 2

[0056] A method for preparing a flexible and tunable smart responsive nanogel is as follows:

[0057] 56.4 mg of crosslinking agent N,N'-bis(acryloyl)cystamine (BAC) was pre-dissolved in ethanol, and then dissolved in 80 mL of water along with 550 mg of monomer N-isopropylmethacrylamide (NIPMAM), 18.65 μL of methacrylic acid (MAA), and 30 mg of sodium dodecyl sulfate (SDS). The mixture was placed in a round-bottom flask and sonicated for 5 minutes.

[0058] After the monomer solution has been ultrasonically dispersed evenly, it is added to a three-necked flask and stirred until homogeneous. Argon gas is introduced for 30 minutes, and the solution is stirred at 500 rpm for 30 minutes.

[0059] Then, the solution was heated to 80°C, 10 mg of potassium persulfate (KPS) solution was added, and the polymerization reaction was started by stirring for 6 hours.

[0060] Finally, after the reaction was complete and the material was cooled to room temperature, it was purified by ultrafiltration (14000 Da) to remove unreacted monomers and other small molecules. The mixture was washed three times with water to obtain 5% NGs (at this point, the amount of BAC used was 4% of the total molar amount of monomers) and stored at 4°C.

[0061] Example 3

[0062] A method for preparing a flexible and tunable smart responsive nanogel is as follows:

[0063] 112.6 mg of crosslinking agent N,N'-bis(acryloyl)cystamine (BAC) was pre-dissolved in ethanol, and then dissolved in 80 mL of water along with 550 mg of monomer N-isopropylmethacrylamide (NIPMAM), 18.65 μL of methacrylic acid (MAA), and 25 mg of sodium dodecyl sulfate (SDS). The mixture was placed in a round-bottom flask and sonicated for 5 minutes.

[0064] After the monomer solution has been ultrasonically dispersed evenly, it is added to a three-necked flask and stirred until homogeneous. Argon gas is introduced for 30 minutes, and the solution is stirred at 500 rpm for 30 minutes.

[0065] Then, the solution was heated to 80°C, 10 mg of potassium persulfate (KPS) solution was added, and the polymerization reaction was started by stirring for 6 hours.

[0066] Finally, after the reaction was complete and the material was cooled to room temperature, it was purified by ultrafiltration (14000 Da) to remove unreacted monomers and other small molecules. The mixture was washed three times with water to obtain 10% NGs (at which point the amount of BAC used was 8% of the total molar amount of monomers) and stored at 4°C.

[0067] Example 4

[0068] A method for preparing a flexible and tunable smart responsive nanogel is as follows:

[0069] 225.2 mg of crosslinking agent N,N'-bis(acryloyl)cysteine ​​(BAC) was pre-dissolved in ethanol, and then dissolved in 80 mL of water along with 550 mg of monomer N-isopropylmethacrylamide (NIPMAM), 18.65 μL of methacrylic acid (MAA), and 15 mg of sodium dodecyl sulfate (SDS). The mixture was placed in a round-bottom flask and sonicated for 5 minutes.

[0070] After the monomer solution has been ultrasonically dispersed evenly, it is added to a three-necked flask and stirred until homogeneous. Argon gas is introduced for 30 minutes, and the solution is stirred at 500 rpm for 30 minutes.

[0071] Then, the solution was heated to 80°C, 10 mg of potassium persulfate (KPS) solution was added, and the polymerization reaction was started by stirring for 6 hours.

[0072] Finally, after the reaction was complete and the material was cooled to room temperature, it was purified by ultrafiltration (14000 Da) to remove unreacted monomers and other small molecules. The mixture was washed three times with water to obtain 20% NGs (at this point, the amount of BAC used was 14% of the total molar amount of monomers) and stored at 4°C.

[0073] like Figure 1 As shown in (C), when the amount of BAC is 1 to 14% of the total molar amount of monomer, the particle size of the nanogel can be controlled between 200 and 280 nm.

[0074] like Figure 1 As shown in Figure (F), the nanogel potential gradually decreases as the BAC content in the nanogel gradually increases.

[0075] like Figure 2 As shown in Figure (D), the BAC content and Young's modulus in the nanogels show a positive correlation trend, with 2.5% NGs exhibiting the highest flexibility.

[0076] like Figure 4As shown, the material obtained in Example 1 did not show significant changes in particle size and potential within 90 days, and the polydispersity index (PDI) was less than 0.2, indicating long-term stability.

[0077] like Figure 5 As shown in Figure (A), which illustrates the penetration ability of nanogels with different flexibility in tumor spheres, and Figure (B), which illustrates the penetration of simulated extracellular matrix in Examples 1 and 4, Example 1 has higher flexibility and better deep penetration effect compared with Example 4.

[0078] like Figure 8 As shown in (A), the nanogels obtained in Examples 1-4 gradually decrease in size with increasing temperature from 5 to 45°C, and the maximum deformation can reach 70%.

[0079] like Figure 8 As shown in (D), the nanogels obtained in Examples 1-4 gradually increased in size with increasing pH from 3 to 9, with a maximum deformation of up to 200%.

[0080] like Figure 8 As shown in (C) and (E), the nanogel obtained in Example 1 can respond to GSH and its structure is destroyed within 24 hours.

[0081] Example 5

[0082] A method for preparing a flexible and tunable smart responsive nanogel is as follows:

[0083] 28.2 mg of crosslinking agent N,N'-bis(acryloyl)cysteine ​​(BAC) was pre-dissolved in ethanol, and then dissolved in 80 mL of water along with 550 mg of monomer N-isopropylmethacrylamide (NIPMAM), 18.65 μL of methacrylic acid (MAA), and 35 mg of sodium dodecyl sulfate (SDS). The mixture was placed in a round-bottom flask and sonicated for 5 minutes.

[0084] After the monomer solution has been ultrasonically dispersed evenly, it is added to a three-necked flask and stirred until homogeneous. Argon gas is introduced for 30 minutes, and the solution is stirred at 500 rpm for 30 minutes.

[0085] Then, the solution was heated to 80°C, 300 μg of plasmid and 10 mg of potassium persulfate (KPS) solution were added, and the polymerization reaction was started and stirred for 6 hours.

[0086] Finally, after the reaction was complete and the material was cooled to room temperature, it was purified by ultrafiltration (14000 Da) to remove unreacted monomers and other small molecules. The material was washed three times with water to obtain NGs loaded with plasmids, which were then stored at 4°C.

[0087] like Figure 3As shown, after loading the plasmid, the morphology and particle size of the nanogel did not change significantly compared with Example 1, but the potential decreased.

[0088] Control experiment

[0089] Transfection experiments of Example 5 and Example 1: Cells were seeded in 6-well plates. When the cells grew to 60-70%, 100 μL of Example 5 or Example 1 was added to each well. After 48 hours, the cells were centrifuged and detected.

[0090] Commercial transfection reagent PEI transfection experiment: Cells were seeded in 6-well plates. When the cells grew to 60-70%, 100 μL of commercial transfection reagent PEI and plasmid complex was added to each well. After 48 h, the cells were centrifuged and detected.

[0091] like Figure 6 As shown, Example 5 was able to effectively transfect cells in 293T, Hep-G2, and H22 cells.

[0092] Multi-responsive nanogel materials can be used in gene transfection experiments of tumor cells, exhibiting stable plasmid delivery capabilities in various cell types such as 293T, Hep-G2, and H22, making them suitable for drug delivery, gene therapy, and tumor treatment.

[0093] Cytotoxicity assays for Examples 5 and 1: Cells were seeded in 96-well plates. When the cells grew to 60-70%, 10 μL of Example 5 or Example 1 was added to each well. After 24 hours, CCK8 reagent was added and the assay was performed.

[0094] like Figure 7 As shown, in Example 5, after co-incubation for 24 hours in 3T3 and H22 cells, cell viability did not decrease significantly, and the nanogel exhibited low cytotoxicity.

[0095] In the GSH response experiment, GSH was dissolved in deionized water to prepare a solution with pH 6.5 and 10 mM GSH. Nanogel was added and incubated in a shaker at 37°C for 24 h. During this period, the particle size and PDI changes were measured. After 24 h, the solution was centrifuged for subsequent experiments.

[0096] like Figure 8 As shown in (B), the nanogel obtained in Example 5 can respond to GSH, disrupt its structure, and rapidly release the encapsulated plasmid under tumor microenvironment simulation conditions (pH 6.5, GSH 10mM).

[0097] In summary, the embodiments of this invention have successfully constructed a nanogel gene delivery system with multiple stimulus-response capabilities by systematically designing and controlling key parameters such as the composition ratio, network flexibility, particle size distribution, and surface electrical properties of nanogels with triple response characteristics of temperature, pH, and GSH at the nanoscale. This demonstrates the multiple advantages of the material, including simple and controllable synthesis process, high structural stability, good dispersibility, tunable response and release behavior, precise delivery capability in the tumor microenvironment, high plasmid loading efficiency, good biocompatibility, and low toxicity. It shows broad application prospects and translational potential in the fields of drug delivery, gene therapy, and tumor treatment.

[0098] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a flexible and tunable responsive nanogel, characterized in that, Includes the following steps: Step 1: Prepare a monomer solution, wherein the monomer solution comprises N-isopropylmethacrylamide, methacrylic acid, N,N'-bis(acryloyl)cysteine ​​and a surfactant; Step 2: Add an initiator to the monomer solution and polymerize under water bath conditions of 75-90℃ to generate the initial product; Step 3: After purifying, washing and drying the initial product, nanogels are obtained.

2. The method for preparing a flexible and tunable responsive nanogel according to claim 1, characterized in that: The surfactant is sodium dodecyl sulfate.

3. The method for preparing a flexible and tunable responsive nanogel according to claim 1, characterized in that: The initiator is potassium persulfate.

4. The method for preparing a flexible and tunable responsive nanogel according to claim 1, characterized in that: The molar ratio of N-isopropylmethacrylamide to methacrylic acid in the monomer solution of step 1 is 18:

1.

5. The method for preparing a flexible and tunable responsive nanogel according to claim 1, characterized in that: In step 1, the amount of N,N'-bis(acryloyl)cystamine used is 1-14% of the total molar amount of the monomer.

6. The method for preparing a flexible and tunable responsive nanogel according to claim 1, characterized in that: The concentration of surfactant in the monomer solution in step 1 is 0.1875–0.5 mg / mL.

7. The method for preparing a flexible and tunable responsive nanogel according to claim 1, characterized in that: The nanogel has a particle size range of 200–280 nm at room temperature.

8. The application of nanogels prepared by the method for preparing flexible and tunable responsive nanogels according to any one of claims 1-7 in drug delivery.

9. The application of the nanogel prepared by the method for preparing the flexible and tunable responsive nanogel according to any one of claims 1-7 in gene therapy drugs.

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