A reduction-responsive polyamidoamine dendrimer nanogel and a preparation method thereof

Abstract

The present application relates to a kind of reduction response type polyamide-amine dendrimer nanogel and its preparation method, the nanogel with gold nanoparticles, tobramycin, third generation polyamide-amine dendrimer as component, preparation method includes: the preparation of G3-PEG-SAT, the preparation of G3NGs, the preparation of Au@G3NGs and the preparation of Au / Toy@G3NGs.The multifunctional nanogel Au / Toy@G3NGs constructed by the present application provides efficient diagnosis and treatment integrated platform for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment, and particularly relates to a reduction-responsive polyamide-amine dendrimer nanogel and a preparation method thereof. Background Art

[0002] In recent years, malignant tumors have become the leading threat to human health due to their strong metastatic potential, low cure rate, and high recurrence rate. According to the latest report from the International Agency for Research on Cancer (IARC), approximately 20 million people were diagnosed with cancer in 2021, and over 10 million patients died from the disease. With the continuous development of modern medicine, integrated diagnosis and treatment nanoplatforms (Li et al. Biomaterials, 2020, 232, 119749) have achieved accurate diagnosis and efficient treatment of tumors, and will become a key goal of modern medicine.

[0003] Clinically, molecular imaging techniques are often used to diagnose cancer and enhance the distinction between normal and tumor tissues. Chemotherapy, as a traditional clinical treatment, is often limited in its efficacy by its lack of specificity, low bioavailability, and significant damage to normal tissues. To simultaneously improve the precise diagnosis of cancer and the high efficacy of chemotherapy drugs, a tumor microenvironment-responsive nanoplatform has been constructed to load contrast agents and chemotherapy drugs. Combined with ultrasound-targeted microbubble destruction (UTMD), this platform can precisely release chemotherapy drugs at the tumor site, reducing toxic side effects on normal tissues and improving tumor treatment efficacy.

[0004] Selecting a suitable nanomaterial as a carrier for contrast agents and drugs is crucial. Polymer nanogels (NGs) have attracted widespread attention due to their unique physicochemical properties, combining both gels and nanomaterials. Polyamidoamine (PAMAM) dendrimers, with their well-defined structure and amino-rich surface, can be loaded with various contrast agents and therapeutic agents, making them ideal materials for constructing NGs. However, low-generation PAMAM dendrimers have drawbacks such as limited drug loading, restricted passive targeting, and a lack of stimuli-responsiveness. High-generation PAMAM dendrimers are expensive, time-consuming, and cumbersome to synthesize, and exhibit greater cytotoxicity than lower-generation dendrimers. Third-generation PAMAM dendrimers, with numerous amino groups on their surface, consist of an initiating core, an endosome, and functional groups. Their internal cavity can encapsulate contrast agents and chemotherapeutic drugs, and they exhibit good water solubility and biocompatibility. Therefore, we selected third-generation dendrimers (G3.NH2) to construct the gel nanoplatform.

[0005] Previous studies (Liu et al. ACS Appl. Bio Mater., 2021, 4, 1803-1812) found that gold nanoparticles (Au NPs) are becoming one of the most promising CT contrast agents due to their high X-ray absorption coefficient, tailored surface chemistry, and excellent biocompatibility. At the same time, Yin et al. (Yin et al., Chem. Eng. J., 2021, 417, 129273) found that dendrimers encapsulating gold nanoparticles can increase the expression of CD86 in macrophages, promote the conversion of macrophages to the M1 type of anti-tumor, and thus promote the apoptosis of cancer cells. Toyocamycin (Toy) is a purine nucleoside analogue related to endoplasmic reticulum stress (ERS), which plays a significant anti-tumor role by interfering with the unfolded protein response process, and is also an anti-tumor immunotherapy drug. In tumor cells, due to abnormal transcription and metabolism, persistent ERS occurs in cells (Wang et al. Adv. Mater., 2022, 34, 2107009). When ERS occurs, it can induce the expression of molecular chaperones such as glucose-regulated protein (GRP78) to produce a protective effect. GRP78 is a member of the heat shock protein 70 family, which can bind to IRE1a to make it inactive. When unfolded or misfolded proteins accumulate in the endoplasmic reticulum, GRP78 immediately separates from IRE1a and binds to unfolded and misfolded proteins. The separation of GRP78 from IRE1a promotes the formation of IRE1a homodimers, and IRE1a is activated and has endonuclease activity at this time, which can very irregularly cleave the downstream XBP1 mRNA to produce XBP1-splicing (XBP1s) mRNA, and then regulate the downstream unfolded protein response-related genes, promote protein folding and degradation, and maintain protein homeostasis in cells. Toy can specifically inhibit the endonuclease activity of IRE1a by reducing the production of XBP1s, thereby promoting the apoptosis process of tumor cells. On the other hand, studies have shown that XBP1s expression is significantly increased in tumor-associated dendritic cells (DCs), and XBP1s stimulates the synthesis of triglycerides in DCs, causing the accumulation of internal lipids and their peroxidation metabolites, which hinders the normal functioning of the antigen presentation function of DCs, thereby inhibiting the body's anti-tumor immune response. Toy can block the occurrence of ERS, re-activate the antigen presentation function of DCs, and restore the body's anti-tumor immune response.

[0006] In addition, when tumor cells are treated with chemotherapy, due to the ERS reaction of the cells, they release some damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), adenosine triphosphate (ATP), etc., as well as tumor-associated antigens (Tumor-associated antigens), which cause immunogenic cell death (ICD) of tumor cells, thereby maturing DCs and presenting antigens and other molecules to T cells through the lymphatic circulation, stimulating anti-tumor immune response, inhibiting tumor growth and recurrence. To enhance the body's anti-tumor immune response, immune checkpoint blockade (ICB) therapy based on PD-1 / PD-L1 has emerged (Zhan et al. Adv. Mater., 2022, 202208277). Combined with PD-L1 antibodies, it can further generate a sustained immune response and achieve efficient anti-tumor immunotherapy.

[0007] The results of searching relevant domestic and foreign literature and patents indicate that there are no reports on using the third-generation polyamide-amine dendrimer G3.NH2 nanogel as a carrier to load Au NPs and Toy, and combining it with PD-L1 antibodies for combined chemotherapy / immunotherapy of tumors under the action of UTMD. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a reduction-responsive polyamide-amine dendrimer nanogel and a preparation method thereof, which can achieve precise release of chemotherapy drugs in the tumor microenvironment, thereby improving the defect of low drug release efficiency.

[0009] The present invention provides a reduction-responsive polyamide-amine dendrimer nanogel, which is prepared by G3-PEG-SAT. G3-PEG-SAT is then used as a reaction monomer to synthesize a third-generation polyamide-amine dendrimer nanogel by utilizing a reverse microemulsion method and a self-crosslinking reaction. Nanogold particles are in situ loaded inside the nanogel, and toyocamycin is further encapsulated in the nanogel through physical action.

[0010] The structural formula of the G3-PEG-SAT is:

[0011]

[0012] The present invention also provides a method for preparing a reduction-responsive polyamidoamine dendrimer nanogel, comprising the following steps:

[0013] (1) The thiol-containing reagent was dissolved in DMSO and added to a methanol solution containing the third-generation polyamidoamine dendrimer G3.NH2 with stirring. The catalyst was slowly added, followed by adding ether until the solution turned milky white. The solution was centrifuged and washed to remove unreacted substances to obtain the thiol-containing dendrimer G3-PEG-SAT.

[0014] (2) dispersing the G3-PEG-SAT in step (1) in ultrapure water to form an aqueous phase; dissolving a surfactant and an emulsifier in the organic phase and stirring to form an oil phase; adding the aqueous phase dropwise to the oil phase to obtain a W / O polymer emulsion, and then adding an initiator after ultrasonic crushing, stirring, centrifuging, and dialyzing to obtain a third-generation polyamide-amine dendrimer nanogel G3 NGs solution;

[0015] (3) placing the G3 NGs prepared in step (2) in an ice bath, adding HAuCl4·4H2O solution and stirring, then adding sodium borohydride NaBH4 ice water solution, continuing stirring and dialysis to obtain the third generation polyamide-amine dendrimer nanogel Au@G3 NGs loaded with gold nanoparticles;

[0016] (4) The Au@G3 NGs prepared in step (3) were dispersed in ultrapure water, and an aqueous solution of Toyocamycin Toy was added, followed by stirring and dialysis to obtain the third-generation polyamide-amine dendrimer nanogel Au / Toy@G3NGs loaded with gold nanoparticles / Toyocamycin, i.e., a reduction-responsive polyamide-amine dendrimer nanogel.

[0017] The catalyst in step (1) is N,N-diisopropylethylamine; the thiol-forming agent is PEGylated N-succinimide S-acetylthioacetate NHS-PEG-SAT; the molar ratio of the catalyst to G3.NH2 is 15-20:1, and the molar ratio of the thiol-forming agent to G3.NH2 is 10-15:1.

[0018] In the step (1), the stirring speed is 1000-2000 rpm, the stirring temperature is room temperature, and the stirring time is 1-5 hours; the centrifugal speed is 12000-15000 rpm, and the centrifugal time is 10-20 minutes.

[0019] The washing in step (1) uses a mixed solution of methanol and ether in a volume ratio of 1:5.

[0020] The surfactant in step (2) is Span80, and the emulsifier is Tween80; the organic phase is n-hexane; the initiator is hydroxylamine hydrochloride; the volume ratio of the organic solvent in the oil phase to the water in the aqueous phase is 10-14:1; the mass ratio of G3-PEG-SAT to the surfactant and the emulsifier is 5:5-10:30-40; and the mass ratio of G3-PEG-SAT to the initiator is 30-35:1.

[0021] In the step (2), the ultrasonic power is 20-30 W, the ultrasonic time is 1-5 min, and the stirring time is 5-10 h.

[0022] The mass ratio of chloroauric acid, sodium borohydride and G3.NH2 in step (3) is 10-15:20-25:1.

[0023] The stirring temperature for adding chloroauric acid in step (3) is 2-10° C., and the stirring time is 30-40 min; the stirring reaction temperature for adding sodium borohydride solution is 2-10° C., and the reaction time is 3-4 h.

[0024] The molar ratio of Toy to G3.NH2 in step (4) is 6-8:1; the stirring temperature is room temperature, and the stirring time is 5-10 hours.

[0025] The molecular weight cut-off of the dialysis bag used for dialysis in steps (2), (3) and (4) is 3 kDa, and the dialysis is performed in ultrapure water for 2 to 5 days.

[0026] The present invention also provides an application of a reduction-responsive polyamide-amine dendrimer nanogel in chemotherapy / immunotherapy of tumors and CT imaging.

[0027] The present invention also provides the use of a reduction-responsive polyamidoamine dendrimer nanogel in UTMD-enhanced tumor therapy. Unlike most tumors, pancreatic cancer has a stroma composed of a large amount of connective tissue. This tough barrier results in inefficient intratumoral delivery of chemotherapy drugs, leading to a certain degree of resistance in tumor cells to these drugs. To address this issue, combining UTMD with G3 NGs improves the efficiency of chemotherapy drug delivery while minimizing damage to normal tissue. A multifunctional hybrid nanomaterial, formed by loading Au NPs and Toy onto G3 NGs, combined with UTMD technology, is expected to achieve enhanced precision tumor diagnosis and efficient treatment. Furthermore, in vivo combination with PD-L1 antibodies further enhances the body's anti-tumor immune response.

[0028] The present invention first modifies the surface of G3.NH2 with a thiol reagent NHS-PEG-SAT, then prepares a GSH-responsive polyamidoamine dendrimer nanogel by a reverse microemulsion method, then mixes trivalent gold ions with the nanogel and reduces it with sodium borohydride to form a polyamidoamine dendrimer nanogel loaded with gold nanoparticles (Au@G3NGs), and finally entraps toycin in the nanogel by physical encapsulation, thereby forming a responsive polyamidoamine dendrimer nanogel loaded with gold nanoparticles / toycin (Au / Toy@G3NGs).

[0029] The present invention uses Zeta potential and dynamic light scattering (DLS), ultraviolet-visible absorption spectroscopy (UV-Vis), nuclear magnetic resonance imaging analyzer and transmission electron microscopy (TEM) to characterize the physical and chemical properties of the reduction-responsive polyamide-amine dendrimer nanogel loaded with gold / toyocamycin, and at the same time determines the in vitro CT imaging performance of the nanogel. Subsequently, the CCK-8 method is used to evaluate the cell compatibility of the nanogel; the inductively coupled plasma spectrometer (ICP-OES) is used to evaluate the phagocytosis of the nanogel by the cells; the expression of ERS-related proteins (GRP78, pIRE1α, XBP1, XBP1s and CHOP) in tumor cells is detected by Western blot (WB); the effect of the nanogel on the CRT eversion of cancer cells is evaluated by laser confocal microscopy; the transwell experiment is used to explore whether the tumor cells treated with the nanogel cause the ICD effect; finally, a subcutaneous tumor model of black mice is established for anti-tumor experiments to investigate the imaging effect and therapeutic effect of the prepared nanogel in the black mouse. The synthesis of Au / Toy@G3 NGs and the schematic diagram of the in vivo therapeutic effect are shown in the figure below. Figure 1 shown.

[0030] Beneficial effects BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthesis process of Au / Toy@G3 NGs prepared by the present invention and the combined chemotherapy / immunotherapy in vivo;

[0032] Figure 2 G3-PEG prepared in Example 1 (45) -SAT H NMR spectrum;

[0033] Figure 3 The hydrated particle size distribution diagrams of G3 NGs, Au@G3 NGs, and Au / Toy@G3 NGs prepared in Example 1;

[0034] Figure 4This is a graph showing the change in hydrodynamic diameter of the Au / Toy@G3 NGs prepared in Example 1 in water, PBS, and DMEM culture medium over time;

[0035] Figure 5 TEM image of Au / Toy@G3 NGs prepared in Example;

[0036] Figure 6 UV-visible absorption spectra of G3 NGs and Au / Toy@G3 NGs prepared in Example 1;

[0037] Figure 7 The in vitro CT imaging of the Au / Toy@G3 NGs prepared in Example 1 and the linear relationship between the CT value and the Au concentration;

[0038] Figure 8 The release curves of Toy from Au / Toy@G3 NGs prepared in Example 1 under different conditions;

[0039] Figure 9 Cell viability graphs after Toy, Toy+UTMD, Au@G3 NGs, Au / Toy@G3 NGs, and Au / Toy@G3 NGs+UTMD were co-incubated with Pan02 cells for 24 h;

[0040] Figure 10 The figure shows the analysis of Au element content phagocytized by cells after Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD were co-incubated with Pan02 cells for 6 hours;

[0041] Figure 11 Figure 2 shows the Western blot results of GRP78, pIRE1α, XBP1u, XBP1s and CHOP proteins in Pan02 cells after Au@G3 NGs, Toy, Toy+UTMD, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD were co-incubated with the cells for 24 h. (a) is the protein band diagram of Western blot, and (b), (c), (d), (e) and (f) are grayscale quantitative images of the protein bands.

[0042] Figure 12 Figure 2 (a) shows the flow cytometry analysis of the expression of RAW264.7 cell markers CD86 and CD206 by Au@G3NGs, Toy, Toy+UTMD, Au / Toy@G3NGs, Au / Toy@G3NGs+UTMD and LPS, and the M1 / M2 quantitative analysis bar graph (b);

[0043] Figure 13Laser confocal microscopy analysis of CRT expression in Au@G3 NGs, Toy, Toy+UTMD, Au / Toy@G3 NGs, and Au / Toy@G3 NGs+UTMD after co-incubation with Pan02 for 24 hours;

[0044] Figure 14 ELISA quantitative analysis of (a) HMGB-1 and (b) ATP levels released into the extracellular space after Au@G3 NGs, Toy, Toy+UTMD, Au / Toy@G3 NGs, and Au / Toy@G3 NGs+UTMD were co-incubated with Pan02 cells for 24 h.

[0045] Figure 15 (a) Flow cytometric expression analysis and (b) quantitative analysis of CD80 and CD86 on dendritic cells after Au@G3 NGs, Toy, Toy+UTMD, Au / Toy@G3 NGs, and Au / Toy@G3 NGs+UTMD were co-incubated with Pan02 cells and DCs;

[0046] Figure 16 CT images of mouse tumors before and 3 h after tail vein injection of Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD PBS solutions (100 μL, [Au] = 10 mM);

[0047] Figure 17 The tumor CT values ​​at different time intervals after tail vein injection of Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD PBS solutions in the Pan02 mouse model;

[0048] Figure 18 Figure 13 shows the (a) body weight changes and (b) relative tumor volume changes of mice treated with PBS, Au@G3 NGs, Toy, Au / Toy@G3 NGs, Au / Toy@G3 NGs+UTMD, and Au / Toy@G3 NGs+UTMD+Anti-PD-L1 over 14 days.

[0049] Figure 19 (a) Flow cytometric analysis of CD4+ / CD8+ T cell typing in tumor tissues of each experimental group after treatment in Example 13, and (b) quantitative analysis of CD4+ T cells and (c) CD8+ T cells.

[0050] Figure 20 (a) Flow cytometric analysis and (b) quantitative analysis of Tregs (CD4+ / CD25+ / Foxp3+) cell types in tumor tissues of each experimental group after treatment for 14 days in Example 13. DETAILED DESCRIPTION

[0051] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0052] Unless otherwise specified, all chemical reagents were used directly without further purification. G3.NH2 was purchased from Dendritech (USA). NHS-PEG-SAT was purchased from Changsha Innovative Pharmaceutical Industry Technology Research Institute Co., Ltd. (Changsha, China). Toy was purchased from Shanghai Weihuan Biotechnology Co., Ltd. (Shanghai, China). Pan02 cells (mouse pancreatic cancer cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DCs (mouse dendritic cell line) were obtained from the Affiliated Cancer Hospital of Fudan University. DMEM medium, fetal bovine serum, penicillin-streptomycin antibody, and trypsin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. (Hangzhou, China). Anti-CRT was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Qihai Biotechnology Co., Ltd. (Shanghai, China). The HMGB-1 ELISA kit was purchased from Shanghai Zhuocai Biotechnology Co., Ltd. (Shanghai, China), and the ATP ELISA kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). C57BL / 6 black mice were purchased from Shanghai Slake Laboratory Animal Center (Shanghai, China). Anti-CD8-PE, Anti-CD4-FITC, Anti-CD4-PE, Anti-CD25-FITC, Anti-Foxp3-APC antibodies were purchased from Thermo Fisher Scientific (Waltham, MA).

[0053] Example 1

[0054] (1) First, 35 mg of NHS-PEG-SAT was dissolved in 300 μL of DMSO and added to a 200 μL methanol solution containing 12.1 mg of G3.NH2. 5 μL of N,N-diisopropylethylamine (DIEA) was then slowly added and stirred at 25°C and 1400 rpm for 2 h. Ether was then added until the solution turned milky white and G3-PEG-SAT was collected by centrifugation at 14000 rpm for 10 min. Two more methanol / ether (volume ratio of 1:5) washes were performed to remove any unreacted substances to obtain G3-PEG-SAT.

[0055] (2) The G3-PEG-SAT prepared in step (1) was dispersed in 1 mL of PBS. 12 mL of n-hexane containing 280 mg of Span80:Tween80 in a 5:1 ratio was placed in a 25 mL reaction flask and placed in an ice bath. The aqueous phase was then slowly added to the organic phase and ultrasonicated for 1 min (power 20 W) using an ultrasonic disruptor. 100 μL of PBS containing 1 mg of hydroxylamine hydrochloride (NH2OH·HCl) was then added to the emulsion and stirred at room temperature for 8 h. The resulting solution was dialyzed in a 3 kDa dialysis bag for 3 days to obtain polyamide-amine dendrimer nanogels G3 NGs.

[0056] (3) The G3 NGs (2 mg) prepared in step (2) were dispersed in 1 mL of ultrapure water, and then 34 μL of chloroauric acid aqueous solution (30 mg / mL) was added dropwise. The mixture was stirred in an ice bath for 30 min, and then 0.1 mL of pre-cooled sodium borohydride aqueous solution (1 mg) was quickly added as a reducing agent. The mixture was stirred for 3 to 4 h. The reaction solution was dialyzed in a 3 kDa dialysis bag for 3 d to obtain polyamidoamine dendrimer nanogels loaded with gold nanoparticles, Au@G3 NGs. The Au content in the product Au@G3 NGs was measured by ICP, and the gold coating rate was calculated to be 8.4%.

[0057] (4) 3 mg of Toy was dissolved in 6 mL of ultrapure water and added dropwise to 6 mL of the Au@G3 NGs solution (2 mg / mL) prepared in step (3). The mixture was stirred for 5–10 h. The resulting solution was dialyzed in a 3 kDa dialysis bag for 3 d to obtain the polyamidoamine dendrimer nanogel Au / Toy@G3 NGs loaded with gold nanoparticles / toyocamycin. The absorbance of the Au / Toy@G3 NGs at 280 nm was measured by UV light, and the encapsulation efficiency of Toy was calculated to be 57.0 wt%, and the loading efficiency was 12.5 wt%.

[0058] Example 2

[0059] The G3-PEG-SAT prepared in Example 1 was 1 For H NMR characterization, the lyophilized G3-PEG-SAT was dissolved in D2O and diluted to 1 mg / mL with D2O. Figure 2 As shown in the figure, the proton peak at the chemical shift of 2.2-3.4 ppm represents the methylene proton peak of G3, and the proton peak at the chemical shift of 3.7-4.0 ppm represents the methylene proton peak of PEG, indicating that NHS-PEG-SAT has been successfully modified on the surface of G3.NH2. By integrating the proton peaks in the two regions, it was found that six NHS-PEG-SAT molecules were modified on each G3.NH2 surface.

[0060] Example 3

[0061] 1 ml of each of the G3 NGs, G3 NGs (+GSH (10 mM)), Au@G3 NGs, and Au / Toy@G3 NGs synthesized in Example 1 were diluted to 0.2 mg / mL with ultrapure water for measurement of surface potential and hydration kinetic diameter. As shown in Table 1, the surface potential of the G3 NGs was 32.8 ± 0.5 mV, and the hydration kinetic diameter was 237.9 nm. In the presence of GSH, the surface potential shifted to 17.1 ± 0.987 mV, the hydration kinetic diameter to 125.8 ± 2.9 nm, and the particle size decreased, indicating GSH-responsive disintegration of the nanogel, confirming the successful preparation of a GSH-responsive nanohydrogel. After loading with gold nanoparticles, the surface potential of the Au@G3 NGs was 9.1 ± 0.1 mV, and the hydration kinetic diameter increased to 204.7 nm, demonstrating successful gold nanoparticle loading. After further encapsulation of toyocamycin, the potential of Au / Toy@G3 NGs further decreased to 2.9 ± 0.4 mV, and the hydration kinetic diameter was 193.0 nm, demonstrating the successful loading of Toy. Figure 3 The particle size distribution histograms of G3 NGs, G3 NGs (+GSH), Au@G3 NGs, and Au / Toy@G3 NGs are shown. The hydration kinetic diameter of G3 NGs after GSH treatment becomes smaller, indicating that the nanogel undergoes GSH-responsive disintegration. The hydrated particle size of Au / Toy@G3 NGs in various solutions (Water, PBS, DMEM medium) remains almost unchanged within a week ( Figure 4 ), demonstrating that Au / Toy@G3 NGs have good colloidal stability.

[0062] Table 1. Hydration kinetic diameter, polydispersity index, and surface potential of G3 NGs (±GSH), Au@G3 NGs, and Au / Toy@G3 NGs

[0063]

[0064] Example 4

[0065] The Au@G3 NGs ([Au] = 0.2 mg / mL) prepared in Example 1 were characterized for size and morphology. Figure 5 As shown in the figure, the Au@G3 NGs are ellipsoidal in shape and approximately 150 nm in size. The magnified image clearly shows that the nanoparticles are evenly distributed within the nanogel. It is worth noting that because the TEM images of the material were taken in a dry state, the statistically calculated particle size of the Au@G3 NGs is much smaller than its hydration kinetic diameter (204.7 ± 3.1 nm).

[0066] Example 5

[0067] The G3NGs and Au@G3NGs (1 mg / mL) prepared in Example 1 were characterized by UV. Figure 6 As shown, curve b shows a surface plasmon resonance characteristic peak of nano-Au at 520 nm, proving the successful loading of nano-Au.

[0068] Example 6

[0069] In order to test the CT imaging effect of Au / Toy@G3 NGs, Au / Toy@G3 NGs aqueous solutions with Au concentrations of 5, 10, 20, 30 and 60 mM were prepared, and then the X-ray attenuation characteristics of Au / Toy@G3 NGs with different concentrations were measured using a CT imager. Figure 7 As shown in Figure 2, Au / Toy@G3 NGs exhibited a high X-ray attenuation coefficient, with a Hounsfield unit (HU) value of 7.74 HU mM -1 The rate increases linearly with the Au concentration, indicating that it has good CT imaging performance.

[0070] Example 7

[0071] Phosphate buffers at pH 7.4 and pH 6.5 (±GSH) were prepared using sodium hydrogen phosphate (DIPOP) and sodium dihydrogen phosphate (DIPOP), respectively, to investigate the responsive release of Toy from Au / Toy@G3 NGs in different environments. The prepared Au / Toy@G3 NGs were dispersed in these three buffers to a concentration of 1 mg / mL and placed in a dialysis bag with a molecular cutoff of 3000 Da. The dialysis bag was then placed in a container containing 9 mL of buffer solution at the corresponding pH value and shaken in a 37°C incubator. Subsequently, at different time points (15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h), 1 mL of the solution outside the dialysis bag was removed and promptly replaced with 1 mL of the buffer solution at the corresponding pH value. The absorbance of the samples at 280 nm was measured using a UV spectrophotometer. After the drug release experiment, the Toy release curves of Au / Toy@G3 NGs under different conditions were drawn. Figure 8As shown in the figure, the 72-hour release rate of Au / Toy@G3 NGs at pH = 6.5 ([GSH] = 10 mM) was 57.0%, while the release rate of Au / Toy@G3 NGs at pH = 6.5 without GSH was 22.1%, indicating that the Toy release from Au / Toy@G3 NGs was significantly GSH-responsive. In addition, the drug release rate at pH = 6.5 (without GSH) was higher than that at pH = 7.4 (without GSH). This is because the positively charged amino groups on the surface of Au / Toy@G3 NGs swell more under acidic conditions, which facilitates Toy release.

[0072] Example 8

[0073] Pan02 cells were used as a model to study the effects of Toy, Toy+UTMD, Au@G3 NGs, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD (0.4 W / cm 2 , 1MHz, 20% microbubbles, PRF 1kHz, 30s) were used to investigate the cytotoxicity of Pan02 cells in the logarithmic growth phase. 1×10 4 The cells were seeded at a density of 100 cells on a 96-well cell culture plate and incubated at 5% CO2 and 37°C for 12 hours. The culture medium was then discarded and culture medium containing different concentrations of nanomaterials (the concentration relative to Toy was set to 0.1μg / mL, 0.25μg / mL, 0.5μg / mL, 0.75μg / mL, 1μg / mL, 2.5μg / mL, 5μg / mL and 10μg / mL) or pure PBS (control group) was added and co-cultured with Pan02 cells at 5% CO2 and 37°C for 24 hours. The original culture medium was then discarded and serum-free DMEM culture medium containing 10% (v / v) CCK-8 (10μL) was added. After further incubation for 2 hours, the 96-well plate was wrapped with tin foil and the absorbance of each well was measured at a wavelength of 450nm using a multifunctional microplate reader. The cells treated with PBS were used as blank controls, and the cell viability was marked as 100%. The results are shown in Figure 2. Figure 9As shown in the figure, compared with the control group, Au@G3 NGs had no significant cytotoxicity to Pan02 cells within the experimental concentration range, and the cell survival rate was above 98%, indicating that Au@G3 NGs had almost no cytotoxicity to Pan02 cells. At the same time, for Toy, Toy+UTMD, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD, within the experimental concentration range, as the Toy concentration increased, the cytotoxicity of each group of materials gradually increased. At the same Toy concentration, the cytotoxicity of Au / Toy@G3NGs+UTMD was higher than that of the Au / Toy@G3 NGs group, proving that UTMD technology has an enhanced effect in inhibiting cancer cell proliferation. As can be seen from Table 2, the half inhibitory concentration (IC50) of Toy, Toy+UTMD, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD after incubation with Pan02 cells for 24 hours was 50 ) were 5.06 μg / mL, 3.43 μg / mL, 2.32 μg / mL, and 1.59 μg / mL, respectively. The above data proved that under the same experimental operating conditions, Au / Toy@G3 NGs+UTMD had a stronger killing effect on Pan02 cells than Au / Toy@G3NGs.

[0074] Table 2. The half inhibitory concentration (IC50) of Toy, Toy+UTMD, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD after incubation with Pan02 cells for 24 h. 50 )

[0075] sample <![CDATA[IC 50 (μg / mL)]]> Free Toy 5.06 Toy+UTMD 3.43 Au / Toy@G3 NGs 2.32 Au / Toy@G3 NGs+UTMD 1.59

[0076] Example 9

[0077] Pan02 cells were used as a model to evaluate the effect of UTMD (0.4 W / cm 2 The phagocytic ability of Au / Toy@G3 NGs was investigated in the presence and absence of the phagocytic activity of Au / Toy@G3 NGs. Pan02 cells were cultured at a density of 1×10 5The density of cells per well was seeded in a 12-well cell culture plate and incubated at 5% CO2 and 37°C for 12 hours. The culture medium used was DMEM complete medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS. Subsequently, the original culture medium was discarded, and Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD with different concentrations (the concentration of Au was set to 0.9μM, 1.8μM, 3.6μM and 9μM) were added to the DMEM culture medium and co-cultured with Pan02 cells at 5% CO2 and 37°C for 6 hours. Subsequently, the Pan02 cells in the 12-well plate were digested, centrifuged and counted, and 1mL of aqua regia was added for digestion for 4 hours. After terminating the digestion, the content of Au elements in the cells was detected by ICP-OES. Figure 10 It can be seen that at the same time point, with the increase of Au concentration, the phagocytosis of the material by Pan02 cells gradually increased. When Au / Toy@G3 NGs were combined with UTMD, the phagocytosis of Au / Toy@G3 NGs by Pan02 cells was significantly higher than that of the control group without UTMD treatment, indicating that the sonoporation effect caused by UTMD promoted the phagocytosis of Au / Toy@G3 NGs by Pan02 cells.

[0078] Example 10

[0079] In order to verify the effect of the prepared nanogel on the endoplasmic reticulum stress state of tumor cells, Pan02 cells were used as a model to evaluate the effects of different nanomaterials on the expression levels of endoplasmic reticulum stress-related proteins (GRP78, pIRE1α, XBP1u, XBP1s and CHOP). 5 The cells were seeded into 6-well plates at a density of 100 U / mL penicillin, 100 U / mL streptomycin and 10% FBS in DMEM complete medium. The cells were incubated in a cell culture incubator with 5% CO2 and 37°C for 24 h. The original medium was then discarded and the cells were incubated with Toy, Toy+UTMD (Toy concentration was 1.7 μg / mL), Au@G3NGs, Au / Toy@G3NGs and Au / Toy@G3NGs+UTMD (0.4 W / cm 2, 1MHz, 20% microbubbles, PRF 1kHz, 30s) were incubated with cells at 5% CO2 and 37°C for 24h. The PBS group served as the control group. After the incubation, the original culture medium was discarded and the cells were washed three times with PBS. Then, the cells in all wells were digested, centrifuged, and collected. The cells were lysed on ice and centrifuged at 4°C and 12000rpm for 5min. The supernatant protein solution was collected and the protein concentration was determined. Subsequently, SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence. The contents of GRP78, pIRE1α, XBP1u, XBP1s, and CHOP in the cells were studied. β-actin was used as an internal reference. The results are shown in Figure 2. Figure 11 (a) The expression of GRP78 and pIRE1α, marker proteins representing the degree of endoplasmic reticulum stress, was upregulated to varying degrees after treatment with each group of materials, indicating that endoplasmic reticulum stress was aggravated ( Figure 11 (bc)). On the one hand, the aggravated ER stress will enhance the transcription of XBP1u; on the other hand, because Toy acts on the IRE1α-XBP1 signaling pathway in ER stress, it inhibits the cleavage of XBP1u by pIRE1α, increases the intracellular content of XBP1u, and reduces the expression of XBP1s that can restore ER homeostasis. The mechanism of ER restoration homeostasis is cut off, leading to the aggravation of ER stress. Figure 11 (de), in cells treated with various materials, increased ER stress upregulated the expression of XBP1u, while in cells treated with materials containing Toy, the expression of XBP1s was significantly reduced, verifying the action pathway of Toy in cells. Finally, to verify the effect of increased ER stress on cell apoptosis, the expression of CHOP, a marker protein representing cell apoptosis via the ER pathway, was analyzed. Figure 11 (f) The expression of CHOP was upregulated to varying degrees in cells treated with each group of materials, indicating that the prepared Au / Toy@G3 NGs+UTMD can aggravate endoplasmic reticulum stress and inhibit endoplasmic reticulum stress to restore homeostasis, causing tumor cells to apoptosis through the endoplasmic reticulum pathway.

[0080] Example 11

[0081] RAW 264.7 cells were used as a model to evaluate the effects of different materials on macrophage repolarization. 5The cells were seeded into 6-well plates at a density of 100 cells / well and cultured overnight at 37°C in a 5% CO2 environment. After the cells attached, the culture medium was discarded, washed three times with PBS, and replaced with fresh culture medium containing 50 ng / mL interleukin-4 (IL-4) for 24 hours to polarize RAW 264.7 cells to M2 type. Subsequently, the culture medium containing Au@G3 NGs, Toy, Toy+UTMD, Au / Toy@G3NGs (Toy concentration was 5 μg / mL), Au / Toy@G3NGs+UTMD (0.4 W / cm 2 , 1MHz, 20% microbubbles, PRF 1kHz, 30s) and LPS (2μg / mL) fresh culture medium were incubated for 24 hours, and LPS was used as a positive control. The macrophages were digested, centrifuged, and PBS was added to resuspend the cells. They were stained with fluorescently labeled FITC-CD206 antibodies and PE-CD86 antibodies, and placed in an ice bath and light-proof labeling for 15-20 minutes. After the labeling was completed, the excess unbound antibodies were removed by washing three times with PBS. The macrophages were resuspended in 500μL of PBS and transferred to a flow tube, and the fluorescence intensity of the M2 marker CD206 and the M1 marker CD86 were detected by flow cytometry. The results are shown in Figure 2. Figure 12 As shown in the figure, macrophages treated with Au@G3 NGs (19.2%) displayed higher CD86 levels than those in the PBS group (6.4%). Furthermore, the M1 / M2 ratio in the Au@G3 NGs group (5.8) was significantly higher than that in the PBS group, further demonstrating that Au@G3 NGs can convert macrophages from the M2 phenotype to the M1 phenotype. However, the proportion of CD86+ macrophages (20.1%) and the M1 / M2 ratio (7.2) reached the highest levels in the Au / Toy@G3NGs+UTMD group, indicating that it is most effective in promoting macrophage repolarization to the anti-tumor M1 phenotype.

[0082] Example 12

[0083] To verify whether the prepared NGs can induce immunogenic cell death (ICD) of Pan02 cells, we verified the expression of ICD marker CRT and the release of HMGB-1 and ATP. 5 The cells were seeded into confocal culture dishes at a density of 100 cells / mL. The culture medium used was DMEM complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The cells were placed in a 5% CO2, 37°C cell culture incubator and incubated for 24 hours. The original culture medium was then discarded and the cells were added with Toy, Toy+UTMD (0.4 W / cm 2, 1 MHz, 20% microbubbles, PRF 1 kHz, 30 s), Au@G3 NGs, Au / Toy@G3 NGs, and Au / Toy@G3 NGs+UTMD (Toy concentration of 5 μg / mL) culture medium were co-incubated with cells at 5% CO2 and 37°C for 24 h. The original culture medium was discarded, and the cells were washed three times with PBS. They were fixed with 2.5% glutaraldehyde solution for 15 min and washed three times with PBS. Finally, immunostaining blocking solution was added. After blocking for 60 min, diluted primary antibody (CRT Rabbit Monoclonal Antibody) was added and incubated at room temperature for 1 h. After incubation, the primary antibody was removed by aspiration, and the cells were washed three times with PBS. Fluorescently labeled secondary antibody staining solution (FITC-labeled Goat Anti-Rabbit IgG) was added and incubated at room temperature for 1 h. After the incubation, the secondary antibody staining solution was removed, the cells were washed three times with PBS, and DAPI was added for staining for 10 minutes. After the end, the cells were washed three times with PBS, 0.5 mL of PBS was added, and then the CRT externalization of the tumor cells after being treated with different nanomaterials was observed by laser confocal microscopy. When the CRT in the tumor cells is externalized to the cell surface, it will promote dendritic cells (DCs) to enter the tumor area and stimulate DCs maturation, enhance their recognition and phagocytosis of apoptotic tumor cell antigens, and present the relevant antigens to T cells. Figure 13 While CRT fluorescence was almost undetectable in tumor cells treated with PBS, a significant green fluorescence signal was observed on the surface of Pan02 cells treated with Toy-containing nanomaterials, indicating that Toy can cause CRT externalization on the tumor cell surface. Cells treated with the Au / Toy@G3 NGs+UTMD group displayed the strongest fluorescence signal, indicating that UTMD increased the phagocytosis of Au / Toy@G3 NGs by Pan02 cells. The synergistic effect of Au / Toy@G3 NGs and UTMD resulted in more CRT externalization, promoting the maturation of DCs.

[0084] Subsequently, Pan02 cells were cultured at a rate of 2×10 5 The cells were seeded into 6-well cell culture plates at a density of 10 cells per well and incubated in a 5% CO2, 37°C incubator for 24 h. Subsequently, the original culture medium was discarded and the culture medium containing Toy, Toy+UTMD (0.4 W / cm 2, 1MHz, 20% microbubbles, PRF 1kHz, 30s), Au@G3 NGs, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD (Toy concentration is 5μg / mL) culture medium were co-cultured with cells at 5% CO2 and 37°C for 24h. The cell supernatant culture fluid was then collected, and the absorbance of the cell supernatant culture fluid was measured by enzyme marker and chemiluminescence using ELISA kits for HMGB-1 and ATP, respectively, to calculate the content of HMGB-1 and ATP released outside the cells. HMGB-1 and ATP are located in the cell nucleus. When tumor cells develop ICD, HMGB-1 and ATP will be released from the cell nucleus to the extracellular space. At the same time, the release of HMGB-1 and ATP can stimulate the maturation of DCs and accelerate the anti-tumor process. Figure 14 As shown in (a-b), after tumor cells were co-cultured with the Toy-containing experimental group, the release of HMGB-1 and ATP was significantly increased compared to the PBS group, indicating that chemotherapy can effectively induce immunogenic cell death in Pan02 cells. The release of HMGB-1 and ATP was the highest after treatment with Au / Toy@G3 NGs+UTMD, indicating the highest degree of immunogenic cell death.

[0085] In order to further verify whether the tumor cells treated with different nanomaterials have the function of dendritic cell maturation, Pan02 cells were used as a model to study the maturation of CD80 and CD86. 5 The cells were seeded into the upper chamber of the transwell plate at a density of 10 cells per well and incubated in a 5% CO2, 37°C incubator for 24 h. The culture medium was then replaced with a medium containing Toy, Toy+UTMD (0.4 W / cm 2 , 1MHz, 20% microbubbles, PRF 1kHz, 30s), Au@G3 NGs, Au / Toy@G3 NGs, and Au / Toy@G3 NGs+UTMD (Toy concentration was 5μg / mL) were co-cultured with cells under 5% CO2 and 37℃ for 24h. The original culture medium was then discarded, and the cells were washed three times with sterile PBS and fresh culture medium was added. The cells in the upper chamber were mixed with 2×10 5 The DC cells were seeded in the lower chamber at a density of 100 μL per well and incubated for 24 hours. The DCs were then digested with trypsin, collected by centrifugation, and resuspended in sterile PBS. CD80-PE and CD86-FITC antibodies were then added, and a blank control was set up. The cells were incubated in the dark at 4°C for 20-30 minutes. The cells were then washed three times by centrifugation with sterile PBS to wash away the unbound antibodies. The DCs were then resuspended with 300 μL of PBS and transferred to a flow tube. The fluorescence intensity of CD80 and CD86 was detected by flow cytometry. Figure 15As can be seen from (a) and 15(b), the fluorescence intensity of CD80 and CD86 in the Au / Toy@G3 NGs+UTMD group was the highest, that is, the degree of DC maturation was the highest, indicating that its immunogenicity induction effect was the best.

[0086] Based on the above experimental results, it can be concluded that Au / Toy@G3 NGs+UTMD can promote the immunogenic death of Pan02 cells to the greatest extent, resulting in high expression of CRT externalization, while also increasing the release of HMGB-1 and ATP, stimulating DCs to mature and present antigens to T cells, ultimately activating antigen-specific CTLs and enhancing anti-tumor immunity.

[0087] Example 13

[0088] The subcutaneous tumor model of mice was established using 5-6 week old C57BL / 6 female mice purchased from Shanghai Slake Laboratory Animal Center to verify the imaging effect of the prepared nanomaterials. All animal experiments were conducted in strict accordance with the standards of the Experimental Animal Ethics Committee of Donghua University. 6 Pan02 cells were inoculated into the right hind legs of C57BL / 6 mice and the tumor volume reached approximately 100 mm. 3 At about 24 h, Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD (1 MHz, 0.4 W / cm 2 , 20% microbubbles, 2 min) in PBS solution (100 μL, [Au] = 10 mM) to evaluate the CT imaging effect of the tumor site. Figure 16 and Figure 17 As shown in the figure, 1.5 hours after injection of Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD, the CT signal at the mouse tumor site was significantly enhanced, far exceeding the CT signal value at the tumor site before injection. At this time, the corresponding CT values ​​of the tumor site for Au / Toy@G3 NGs and Au / Toy@G3 NGs+UTMD were 41HU and 53HU, respectively. At the same time point, the CT values ​​of mice injected with Au / Toy@G3 NGs+UTMD PBS solution were much higher than those injected with Au / Toy@G3 NGs PBS solution. These results demonstrate that Au / Toy@G3 NGs, combined with UTMD, provide enhanced imaging effects and can be used as a contrast agent for in vivo tumor imaging.

[0089] Example 14

[0090] In order to verify the combined therapeutic effect of chemotherapy / immunotherapy of nanogels in vivo, the tumor volume reached 100mm 3At the same time, the tumor-bearing mice were randomly divided into 6 groups (5 mice in each group). The specific groups were as follows: (1) tail vein injection of 100 μL PBS solution, (2) tail vein injection of 100 μL PBS solution containing Au@G3 NGs, (3) tail vein injection of 100 μL PBS solution containing Toy ([Toy] = 2 mg / kg), (4) tail vein injection of 100 μL PBS solution containing Au / Toy@G3 NGs, (5) tail vein injection of 100 μL PBS solution containing Au / Toy@G3 NGs+UTMD, (6) tail vein injection of 100 μL PBS solution containing Au / Toy@G3 NGs+Anti-PD-L1+UTMD ([Anti-PD-L1] = 0.2 mg / mL). At the same time, the injection method of nanomaterials was tail vein injection, and the injection method of Anti-PD-L1 was intratumoral injection. For the Au / Toy@G3NGs+A-PD-L1+UTMD group, Au / Toy@G3NGs solution (Toy concentration of 2 mg / kg) was first injected through the tail vein, followed by SonoVue suspension (1.18 mg / mL, 0.1 mL PBS) and a coupling agent with a thickness of about 1 cm was applied to the tumor surface. The ultrasound probe was attached to the tumor surface for ultrasound irradiation (1 MHz, 0.4 W / cm 2 The first day of treatment was the day the treatment started. Nanomaterials were injected via the tail vein on days 1, 3, 5, and 7. Anti-PD-L1 was injected intratumorally on days 2, 4, 6, and 8. The body weight and tumor volume (V = L × W) of the mice were recorded every 2 days. 2 / 2, L represents the length of the tumor, W represents the width of the tumor), the weight and tumor size of the mice were recorded every 2 days during the treatment, and the weight change curve of the mice and the growth curve of the tumor were drawn. Figure 18 (a) It can be seen that the weight changes of mice in each group were not obvious, indicating that the materials in each group had good biocompatibility in vivo. Figure 18(b) As shown, the relative tumor volume of Au@G3 NGs group was significantly lower than that of PBS group, which proved that Au@G3 NGs had a certain inhibitory effect on tumor growth. Compared with free Toy group, Au / Toy@G3 NGs had more obvious anti-tumor effect, which might be due to the passive targeting of nanomaterials to tumor sites caused by EPR effect, and Au / Toy@G3 NGs had a longer blood circulation time in vivo than free Toy. In addition, the tumor volume of mice in Au / Toy@G3 NGs+UTMD group was smaller than that in Au / Toy@G3 NGs group, which might be due to the fact that the acoustic holes caused by UTMD technology enhanced the aggregation and penetration of nanomaterials in the tumor site, thereby enhancing the anti-tumor effect. Finally, the anti-tumor effect of Au / Toy@G3 NGs+UTMD+Anti-PD-L1 group was the most significant among all groups, which proved that the Au / Toy@G3 NGs+UTMD+Anti-PD-L1 nanogel synthesized in the application could effectively inhibit tumor growth.

[0091] To further prove the immunotherapy effect of Au / Toy@G3 NGs+UTMD+Anti-PD-L1, the infiltration of immune cells in the tumor of mice treated by different experimental groups was detected on day 14. The number of CD4+T cells, CD8+T cells and Tregs cells in the tumor was studied by flow cytometry. After the treatment ended, the tumor tissues of mice in each group were taken out under sterile conditions on day 14, cut, ground, filtered through a 400-mesh screen to obtain a cell suspension. After red blood cell lysis, T cells were extracted using a nylon column, and the extracted T cells were resuspended in PBS and stained with CD4-FITC / CD8-PE antibodies for detection of CD4+and CD8+T cells, fixed with an intracellular staining kit and stained with CD4-PE / Foxp3-APC / CD25-FITC antibodies (5 μL each) for detection of Tregs cells, followed by two washes with PBS and centrifugation, resuspension in 300 μL of PBS for flow cytometry analysis. As shown in Figure 19 (a-c) As shown, the quantitative analysis of CD4+T cells and CD8+T cells in tumor tissues of each group showed that the content of tumor infiltrating CD4+T cells and CD8+T cells in Au / Toy@G3 NGs+UTMD+Anti-PD-L1 group was the highest. At the same time, the treatment of Au / Toy@G3 NGs+UTMD+Anti-PD-L1 significantly reduced the percentage of Tregs (CD4+CD25+Foxp3+), which proved that the combined treatment significantly reversed the tumor immune suppression microenvironment and produced an effective immune response Figure 20(ab)). These results suggest that Au / Toy@G3 NGs+UTMD+Anti-PD-L1 treatment can achieve enhanced anti-tumor chemo / immunotherapy by increasing the expression of CTLs and reducing Tregs.

Claims

1. A reduction-responsive polyamidoamine dendrimer nanogel, characterized by: G3-PEG-SAT is prepared, and then G3-PEG-SAT is used as a reaction monomer to synthesize a third-generation polyamide-amine dendrimer nanogel by using a reverse microemulsion method and a self-crosslinking reaction. Nanogold particles are in situ loaded inside the nanogel, and toyocamycin is further encapsulated in the nanogel through physical action. The preparation method of G3-PEG-SAT comprises: dissolving a catalyst and a thiol-forming agent in DMSO, adding the solution to a methanol solution containing the third-generation polyamide-amine dendrimer G3.NH2, and stirring. Subsequently, ether is added until the solution turns milky white, centrifuging, and washing to remove unreacted substances to obtain the thiol-formulated dendrimer G3-PEG-SAT. The thiol reagent is PEGylated N-succinimidyl S-acetylthioglycolate NHS-PEG-SAT; the molar ratio of the thiol reagent to G3.NH2 is 10-15:1; The reverse microemulsion method and self-crosslinking reaction include: dispersing G3-PEG-SAT in ultrapure water to form an aqueous phase; dissolving a surfactant and an emulsifier in an organic phase, and stirring to form an oil phase; The aqueous phase is added dropwise to the oil phase to obtain a W / O polymer emulsion. After ultrasonic crushing, an initiator is added, followed by stirring, centrifugation, and dialysis to obtain a third-generation polyamide-amine dendrimer nanogel G3 NGs solution; the initiator is hydroxylamine hydrochloride.

2. A method for preparing the reduction-responsive polyamidoamine dendrimer nanogel according to claim 1, comprising the following steps: The catalyst and thiol reagent were dissolved in DMSO and added to a methanol solution containing the third-generation polyamidoamine dendrimer G3.NH2 with stirring. Ether was then added until the solution turned milky white, centrifuged, and washed to remove unreacted substances to obtain the thiol-modified dendrimer G3-PEG-SAT. The G3-PEG-SAT in step (1) is dispersed in ultrapure water to form an aqueous phase; a surfactant and an emulsifier are dissolved in the organic phase and stirred to form an oil phase; the aqueous phase is added dropwise to the oil phase to obtain a W / O polymer emulsion, which is then ultrasonically crushed and an initiator is added, followed by stirring, centrifugation, and dialysis to obtain a third-generation polyamide-amine dendrimer nanogel G3 NGs solution; The G3 NGs in step (2) were placed in an ice bath, and a HAuCl4·4H2O solution was added and stirred, followed by a sodium borohydride NaBH4 ice water solution, and the mixture was stirred and dialyzed to obtain the third-generation polyamide-amine dendrimer nanogel Au@G3 NGs loaded with gold nanoparticles; The Au@G3 NGs in step (3) were dispersed in ultrapure water, and an aqueous solution of toyocamycin was added, followed by stirring and dialyzing to obtain the third-generation polyamide-amine dendrimer nanogel Au / Toy@G3 NGs loaded with gold nanoparticles / toyocamycin, i.e., a reduction-responsive polyamide-amine dendrimer nanogel.

3. The preparation method according to claim 2, wherein: The catalyst in step (1) is N,N-diisopropylethylamine; the thiol-forming agent is PEGylated N-succinimide S-acetylthioacetate NHS-PEG-SAT; the molar ratio of the catalyst to G3.NH2 is 15-20:1, and the molar ratio of the thiol-forming agent to G3.NH2 is 10-15:

1.

4. The preparation method according to claim 2, wherein: In the step (1), the stirring speed is 1000-2000 rpm, the stirring temperature is room temperature, and the stirring time is 1-5 h; the centrifugal speed is 12000-15000 rpm, and the centrifugal time is 10-20 min.

5. The preparation method according to claim 2, wherein: The washing in step (1) uses a mixed solution of methanol and ether in a volume ratio of 1:

5.

6. The preparation method according to claim 2, wherein: The surfactant in step (2) is Span80, the emulsifier is Tween80; the organic phase is n-hexane; the initiator is hydroxylamine hydrochloride; the volume ratio of the organic solvent in the oil phase to the water in the aqueous phase is 10-14:1; the mass ratio of G3-PEG-SAT to the surfactant and the emulsifier is 5:5-10:30-40; and the mass ratio of G3-PEG-SAT to the initiator is 30-35:

1.

7. The preparation method according to claim 2, characterized in that: The mass ratio of chloroauric acid, sodium borohydride and G3.NH2 in step (3) is 10-15:20-25:

1.

8. The preparation method according to claim 2, wherein: The stirring temperature for adding chloroauric acid in step (3) is 2-10°C, and the stirring time is 30-40 minutes; the stirring reaction temperature for adding sodium borohydride solution is 2-10°C, and the reaction time is 3-4 hours.

9. The preparation method according to claim 2, wherein: The molar ratio of toyocamycin to G3.NH2 in step (4) is 6-8:1; the stirring temperature is room temperature, and the stirring time is 5-10 h.

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