PH-ROS double-response type drug-loaded dendrimer nanogel as well as preparation method and application of pH-ROS double-response type drug-loaded dendrimer nanogel

Abstract

The invention relates to pH-ROS (potential of hydrogen) double-response type drug-loading dendrimer nanogel as well as a preparation method and application thereof. According to the pH-ROS double-response type drug-loading dendrimer nanogel, G3-PBA-OH is obtained after a G3 PAMAM (polyamidoamine) dendrimer is subjected to functional modification; cross-linking with polyethylene glycol PBA-PEG-PBA with two ends grafted with phenylboronic acid to obtain dendrimer nanogel DNG, and further loading hypoxia activated chemotherapeutic drugs and cytokines on the DNG to obtain the hypoxia activated chemotherapeutic drug-loaded dendrimer nanogel. The DNG prepared by the invention has the characteristics of good biocompatibility, high drug loading rate, pH-ROS dual response and blood brain barrier penetration, provides a new thought for constructing a brain drug delivery nano platform, and can significantly improve the bioavailability of TPZ and IFN-gamma.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials and nanomedicines, and particularly relates to a pH-ROS dual-responsive drug-loaded dendrimer nanogel and a preparation method and application thereof. Background Art

[0002] As the most aggressive primary intracranial tumor, glioma faces severe challenges in clinical treatment. Data from the World Health Organization show that the median survival of glioblastoma patients is only 12-15 months, and the five-year survival rate is less than 5%. The high recurrence rate and extremely short survival period make it difficult to treat glioma (ACS Nano 2024, 18, 14469-14486). Currently, conventional treatment (surgery combined with radiotherapy and chemotherapy) is limited by the physiological defense mechanism of the blood-brain barrier (BBB), resulting in more than 98% of small molecule drugs and almost all large molecules being unable to effectively penetrate the BBB. In addition, they face problems such as rapid metabolism, poor stability and non-specific distribution in the systemic circulation, which seriously restricts their efficacy. In recent years, breakthroughs in nanotechnology have provided new ways to penetrate the BBB. Based on size effects and surface engineering strategies, carrier systems such as dendrimers, polymer nanoparticles, liposomes, and nanogels penetrate the BBB through mechanisms such as passive targeting (enhanced permeation and retention effect) or active targeting (such as transferrin modification or cell membrane coating), significantly improving the efficiency of drug delivery to the brain (Nano Today 2024, 56, 102310). However, the coupling of targeting ligands and cell membrane extraction processes are costly, complex, and have poor batch-to-batch stability, hindering clinical translation. Therefore, it is particularly important to develop new nano-drug delivery platforms that have both efficient BBB penetration and simple preparation processes.

[0003] In order to effectively deliver drugs to brain tumor sites, among many nanoplatforms, dendrimers, as a new type of nanocarrier, have opened up a broad application space for the treatment of brain diseases. Its highly branched three-dimensional structure gives dendrimers unique properties, including easy surface functionalization, excellent biocompatibility and low immunogenicity, making it an excellent carrier for drug delivery (ACS Nano 2023, 17, 23889-23902). More importantly, through clever surface functionalization design, dendrimers can achieve effective penetration of the BBB, thus showing great potential in the treatment of brain diseases. Nanomedicine studies based on dendrimers have shown that polyamide-amine (PAMAM) dendrimers modified with terminal hydroxylation can cross the damaged BBB and precisely target microglia activated by inflammatory damage in the brain (J.Controlled Release 2020, 323, 361-375). Dendrimers with dense polyethylene glycol hydroxyl groups on their surfaces have also been shown to be able to cross the damaged BBB and target microglia in specific areas of the brain (Sci. Adv. 2020, 6, eaay8514). Furthermore, studies in an in situ glioblastoma model have demonstrated that terminally hydroxylated dendrimers also exhibit the ability to penetrate the BBB and infiltrate the brain glioma region (J. Controlled Release 2021, 329, 434-444).

[0004] Although small dendrimers (less than 10 nanometers) are advantageous for crossing the BBB, their encapsulation efficiency is limited, drug release is rapid, and they are easily metabolized and excreted by the body. Given the excessive reactive oxygen species (ROS) and slightly acidic environment at the tumor site, nanocarriers used to treat gliomas should also have the ability to efficiently load drugs and release drugs in a stimuli-responsive manner. Among them, nanogels (NGs), as a three-dimensional network structure material formed by physical or chemical crosslinking of polymer chains, have controllable nanosize, excellent colloidal stability, hydrophilicity, biocompatibility, rheological properties, and enhanced EPR effect (Acta Biomater. 2019, 92, 1-18). By adjusting the types of monomers, crosslinkers, and initiators, various functional groups or stimuli-responsive functions can be introduced into the polymer network of NGs, thereby further improving their performance. Therefore, the design of dendrimer nanogels (DNGs) prepared with dendrimers as monomers can combine the carrier advantages of dendrimers and NGs, and is expected to achieve efficient drug loading and precise brain delivery. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a pH-ROS dual-responsive drug-loaded dendrimer nanogel and its preparation method and application, so as to solve the key problems in the existing brain glioma treatment, such as low BBB penetration efficiency and limited efficacy of single therapy.

[0006] The present invention provides a pH-ROS dual-responsive drug-loaded dendrimer nanogel. The pH-ROS dual-responsive drug-loaded dendrimer nanogel is prepared by functionally modifying G3 PAMAM dendrimers to obtain G3-PBA-OH, cross-linking with polyethylene glycol PBA-PEG-PBA grafted with phenylboronic acid at both ends to obtain dendrimer nanogel DNG, and further loading hypoxia-activated chemotherapy drugs and cytokines on the DNG.

[0007] Preferably, the hypoxia-activated chemotherapy drug includes but is not limited to tirapazamine TPZ; the cytokine includes but is not limited to IFN-γ.

[0008] The present invention provides a method for preparing a pH-ROS dual-responsive drug-loaded dendrimer nanogel, comprising the following steps:

[0009] S1. Dispersing 4-bromomethylphenylboronic acid (BPBA) and G3 PAMAM dendrimer in a solvent, heating and stirring the mixture for reaction, and dialyzing the mixture to obtain phenylboronic acid (BPBA)-modified G3 PAMAM dendrimer, i.e., G3-PBA.

[0010] S2. The G3-PBA obtained in step S1 is dispersed in a solvent, and then glycidol is added dropwise. The reaction is stirred at room temperature and dialyzed to obtain a PBA and glycidol hydroxylated G3 PAMAM dendrimer, namely G3-PBA-OH.

[0011] S3. The G3-PBA-OH obtained in step S2 and the crosslinker PBA-PEG-PBA were dissolved in ultrapure water and added to the oil phase solution. Ultrasonic emulsification was performed, and triethylamine was added dropwise to catalyze the reaction. The mixture was stirred overnight and dialyzed to obtain a dendrimer nanogel DNG.

[0012] S4. The DNG obtained in step S3 is mixed with a hypoxia-activated chemotherapy drug, stirred for reaction, and ultrafiltered to obtain a DNG-hypoxia-activated chemotherapy drug;

[0013] S5. The DNG-hypoxia activated chemotherapy drug obtained in step S4 is mixed with cytokines and ultrafiltered to prepare a pH-ROS dual-responsive drug-loaded dendrimer nanogel.

[0014] Preferably, in step S1, the molar ratio of the G3 PAMAM dendrimer to 4-bromomethylphenylboronic acid BPBA is 1:8 to 1:10; the solvent includes but is not limited to dimethyl sulfoxide; the heating and stirring reaction temperature is 60-70°C, and the reaction time is 1-2 days; the dialysis process is: using a dialysis membrane with a MWCO of 3500Da, dialyzing with 1.5-2L of deionized water for 2-3 days, and freeze-drying after purification.

[0015] Preferably, the mass ratio of G3-PBA to glycidol in step S2 is 3:5 to 4:3; the solvent includes but is not limited to methanol; the reaction time under room temperature stirring is 1-2 days; the dialysis process is: using a dialysis membrane with a MWCO of 500Da, dialyzing with 1.5-2L deionized water for 2-3 days, and freeze-drying after purification.

[0016] Preferably, the cross-linking agent PBA-PEG-PBA in step S3 is prepared by grafting phenylboronic acid PBA onto both ends of polyethylene glycol PEG, and has a molecular weight of 2000-2500 Da.

[0017] More preferably, the cross-linking agent PBA-PEG-PBA is commercial PBA-PEG-PBA.

[0018] Preferably, in step S3, the mass ratio of G3-PBA-OH to the cross-linking agent PBA-PEG-PBA is 1:0.5 to 1:4; the oil phase solution is a cyclohexane solution of Span 80 and Tween 80, wherein the mass ratio of Span 80 to Tween 80 is 5:1 to 6:1.

[0019] Preferably, the ultrasonic treatment time in step S3 is 5-10 min. After the reaction is completed, the mixture is centrifuged at 12000 rpm for 10 min, the supernatant is discarded, ethanol is added to resuspend the precipitate, and dialyzed with 1.5-2 L of deionized water for 2-3 days using a dialysis membrane with a MWCO (molecular weight cut-off) of 8000-12000 Da.

[0020] Preferably, in step S4, the mass ratio of DNG to the hypoxia-activated chemotherapy drug is 1:1 to 10:1, the stirring speed is 600 to 800 rpm, and the centrifugation is performed at 6000 rpm using an ultrafiltration tube with a MWCO of 10,000 Da.

[0021] Preferably, in step S5, the mass ratio of the DNG-hypoxia activated chemotherapy drug to the cytokine is 50:1 to 130:1, and the mixture is allowed to stand for 0.5 to 1 hour.

[0022] The present invention also provides a pH-ROS dual-responsive drug-loaded dendrimer nanogel for use in preparing drugs for treating brain gliomas.

[0023] Beneficial effects

[0024] (1) The process of the present invention is simple, the reaction conditions are simple, the product is easy to purify, and it has good development prospects.

[0025] (2) The DNG prepared by the present invention has good biocompatibility, high drug loading rate, pH-ROS dual response and blood-brain barrier penetration properties, providing a new idea for constructing a brain drug delivery nanoplatform.

[0026] (3) The drug-loaded DNG prepared by the present invention can significantly improve the bioavailability of TPZ and IFN-γ and give the drugs the ability to penetrate the blood-brain barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of (a) the synthesis process of DNG-TPZ / IFN-γ and (b) the use of DNG-TPZ / IFN-γ for treating mouse brain glioma in Example 2 of the present invention.

[0028] Figure 2 These are hydrogen nuclear magnetic resonance spectra of (a) G3-PBA, (b) G3-PBA-OH, and (c) commercial PBA-PEG-PBA prepared in Example 1 of the present invention.

[0029] Figure 3 (a) TEM image and (b) infrared spectrum of DNG-2 prepared in Example 1 of the present invention.

[0030] Figure 4 This is an SDS-PAGE image of IFN-γ, DNG-TPZ, and DNG-TPZ / IFN-γ prepared in Example 2 of the present invention.

[0031] Figure 5 2 : The drug release curves of the drug-loaded nanogel DNG-TPZ / IFN-γ prepared in Example 2 of the present invention at pH 7.4, pH 6.5, pH 7.4+H2O2, and pH 6.5+H2O2.

[0032] Figure 6 These are (a) the cytotoxicity of different concentrations of DNG on C6 cells or bEnd.3 cells in Example 3 of the present invention, (b) the cytotoxicity of TPZ, (c) DNG-TPZ, and (d) DNG-TPZ / IFN-γ on C6 cells under normoxic and hypoxic conditions, respectively.

[0033] Figure 7These are (a) the flow cytometry fluorescence distribution diagram after co-incubation of C6 cells with PBS or DNG-Cy5.5-TPZ / IFN-γ, (b) the quantitative result diagram, and (c) the laser confocal images of C6 cells treated with PBS or DNG-Cy5.5-TPZ / IFN-γ at different times in Example 3 of the present invention.

[0034] Figure 8 (a) Western blot results of STAT1, caspase-3, and caspase-8 proteins in C6 cells treated with different materials for 24 hours in Example 3 of the present invention, and (b) grayscale quantitative images of the corresponding protein bands.

[0035] Figure 9 : (a) flow cytometry distribution diagram of surface markers CD86 and CD206 and (b) quantitative results diagram of macrophage M1 / M2 after RAW264.7 cells were co-incubated with different materials for 24 hours in Example 3 of the present invention.

[0036] Figure 10 (a) Flow cytometry distribution diagram and (b) quantitative result diagram of surface markers CD86 and CD80 of DC cells after co-incubation with different materials for 24 hours in Example 3 of the present invention.

[0037] Figure 11 (a) Flow cytometric analysis of apoptosis and (b) quantitative results of apoptosis and necrosis rates of C6 cells after treatment with different materials for 24 hours in Example 3 of the present invention.

[0038] Figure 12 (a) Schematic diagram of the in vitro blood-brain barrier model measured by the transwell system in Example 3 of the present invention, (b) fluorescence intensity of the upper chamber (bEnd.3 cells) and lower chamber (C6 cells) after 6 hours of incubation with BSA-Cy5.5 or DNG-TPZ / BSA-Cy5.5 measured by the small animal imaging system, (c) corresponding fluorescence imaging pictures of the upper and lower chambers, and (d) penetration efficiency of BSA-Cy5.5 or DNG-TPZ / BSA-Cy5.5.

[0039] Figure 13 These are (a) fluorescence imaging images of the mouse brain at different time points after tail vein injection of BSA-Cy5.5 or DNG-TPZ / BSA-Cy5.5 in Example 4 of the present invention, as well as (b) quantitative analysis graphs of brain fluorescence intensity, (c) in vitro fluorescence images of the main organs and brain of the mouse 12 hours later, and (d) corresponding quantitative analysis graphs of fluorescence intensity.

[0040] Figure 14After the tail vein injection of PBS, DNG, TPZ, IFN-γ, DNG-TPZ, or DNG-TPZ / IFN-γ in Example 4 of the present invention, (a) magnetic resonance imaging images of mouse brain gliomas, (b) relative tumor volume changes, and (c) mouse body weight changes were recorded within 11 days of the treatment cycle.

[0041] Figure 15 The CD8 + T cells / CD4 + Flow cytometric analysis of T cells.

[0042] Figure 16 Flow cytometric analysis of Tregs cells in the spleen of mice in different experimental groups after 11 days of treatment in Example 4 of the present invention.

[0043] Figure 17 The expression levels of (a) IFN-γ, (b) TNF-α, and (c) IL-6 in the serum of mice after the treatment in Example 4 of the present invention are shown. DETAILED DESCRIPTION

[0044] 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.

[0045] The present invention uses nuclear magnetic resonance hydrogen spectrum, Fourier transform infrared (FTIR) spectrum, dynamic light scattering (DLS), ultraviolet spectrum (UV-vis), transmission electron microscopy (TEM), sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and other means to characterize the nanogels synthesized in the examples; the cell compatibility of the nanogels and the cytotoxicity of the drug-loaded nanogels in normoxic / hypoxic environments are verified in vitro by CCK-8 experiments; the performance of nanodrugs in passing through the blood-brain barrier in vitro is studied using Transwell; the cell phagocytic effect of the nanogels is studied by flow cytometry and confocal microscopy, and the polarization of macrophages and the maturation of dendritic cells are detected by flow cytometry; Western blot is used to detect the effect of the nanogels on the growth of dendritic cells. Blot experiments were conducted to investigate the expression levels of STAT1, Caspase-3, and Caspase-8 in cells after treatment with different drugs. An in situ C6 glioma model was constructed in mice. The tumor volume was monitored by magnetic resonance imaging analyzer through tail vein injection, and the mice were weighed to investigate the in vivo anti-tumor effect and biosafety. Small animal fluorescence imaging was used to investigate the effect of nanogels crossing the BBB and their biodistribution. Flow cytometry was used to analyze the expression of CD4 + T cells, CD8 + Infiltration and distribution of T cells and Tregs cells.

[0046] Unless otherwise specified, all chemical reagents were used directly without further purification. Tween 80 was purchased from Shanghai Myrel Biochemical Technology Co., Ltd., and Span 80 was purchased from Sigma Co., Ltd. G3 PAMAM dendrimer was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd. PBA-PEG-PBA was purchased from Hunan Huateng Pharmaceutical Co., Ltd. 4-Bromomethylphenylboronic acid was purchased from Biter Pharmaceuticals. Glycidol was purchased from Shandong Yanfeng New Materials Technology Co., Ltd. TPZ (tirapazamine) and BSA (bovine serum albumin) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. C6 cells (rat glioma cell line), mouse brain microvascular endothelial cell line (bEnd.3 cells), RAW264.7 cells (mouse macrophage cell line), and DC cells (mouse dendritic cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM medium, fetal bovine serum, penicillin-streptomycin, and trypsin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. IFN-γ, BCA assay kit, PMSF, cell lysate, and Cell Counting Kit-8 (CCK-8) were purchased from Shanghai Biotech Biotechnology Co., Ltd. Annexin V-FITC / PI apoptosis detection kit was purchased from Jiangsu KeyGen Biotech Co., Ltd. Glucose-free DMEM medium was purchased from ThermoFisher Scientific (Waltham, MA). ICR mice were purchased from Shanghai Slack Laboratory Animal Center.

[0047] Example 1

[0048] This embodiment provides a pH-ROS dual-responsive drug-loaded dendrimer nanogel, and its preparation method is as follows: Figure 1 As shown in a, the following steps are included:

[0049] S1. Dissolve G3 PAMAM dendrimer (7.5 mg) and 4-bromomethylphenylboronic acid (30 mg) in dimethyl sulfoxide (DMSO). Stir and react in a 70°C water bath for 24 h. After the reaction, dialyze the reaction mixture against 1.5-2 L of deionized water using a 3500 Da dialysis membrane for 3 days. Purify the mixture and freeze-dry it to obtain the phenylboronic acid-modified G3 PAMAM dendrimer G3-PBA.

[0050] S2. Glycidol (29.1 mg, dissolved in 5 mL of methanol) was added dropwise to a solution of G3-PBA (28.3 mg, dissolved in 5 mL of methanol) and stirred at room temperature for 24 h. After the reaction, the reactant was dialyzed against 1.5-2 L of deionized water using a 500 Da dialysis membrane for 3 days. After purification, the product was freeze-dried to obtain the PBA- and glycidol-modified G3 PAMAM dendrimer, G3-PBA-OH.

[0051] S3. Synthesis of dendrimer nanogel DNG using the reverse microemulsion method: G3-PBA-OH and commercial PBA-PEG-PBA were dissolved in 1 mL of ultrapure water at different molar ratios (1:1, 1:3, 1:5, 1:10, 1:15). As the aqueous phase, the mixture was slowly added to a cyclohexane solution (10 mL) consisting of Span 80 (246 mg) and Tween 80 (34 mg) to initially form a water-in-oil (W / O) emulsion. The obtained W / O emulsion was placed under ultrasonication for 10 minutes, and then 500 μL of triethylamine was added and stirred for reaction for 13-14 hours. After the reaction, the W / O emulsion was centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, the precipitate was taken, and it was resuspended and dispersed with 5 mL of anhydrous ethanol. It was dialyzed for 3 days using a dialysis bag with a MWCO of 8000-14000 Da and freeze-dried to obtain DNG, which was recorded as DNG-1, DNG-2, DNG-3, DNG-4, and DNG-5 according to different molar ratios of G3-PBA-OH / PBA-PEG-PBA (1:1, 1:3, 1:5, 1:10, and 1:15), respectively.

[0052] G3-PBA, G3-PBA-OH and commercial PBA-PEG-PBA in Example 1 were characterized by H NMR spectroscopy. Figure 2 As shown in a, the proton peak at the chemical shift of 2.2-3.4 ppm is the characteristic peak of the methylene group of G3 PAMAM dendrimer, and the peak at 7.0-8.0 ppm is the characteristic peak of PBA molecule, indicating that PBA is successfully grafted on G3 PAMAM dendrimer. By integrating the peak area, it is found that 5.9 PBA groups are modified on each G3. Figure 2 As shown in b, the proton peak at 3.65 ppm indicates that G3-PBA was successfully modified with glycidol to prepare hydroxylated G3-PBA, namely G3-PBA-OH. Figure 2 c shows the H NMR spectrum of commercial PBA-PEG-PBA, verifying that it has the characteristic peaks of PBA.

[0053] The DNG in Example 1 was characterized by potential particle size. As shown in Table 1, when the molar feed ratio of G3-PBA-OH and PBA-PEG-PBA was 1:3, the synthesized DNG had the smallest hydrodynamic size and polydispersity index (PDI), which were 107.2 nm and 0.231, respectively, and had good monodispersity. Therefore, the G3-PBA-OH / PBA-PEG-PBA molar ratio of 1:3 was selected as the optimized ratio to synthesize DNG, namely DNG-2, for further drug loading.

[0054] Table 1. Hydrodynamic diameter, polydispersity index and surface potential of DNG samples in Example 1

[0055]

[0056] TEM test was performed on DNG-2, such as Figure 3 As shown in a, the TEM image shows that DNG-2 is spherical particles with an average particle size of about 85nm, which is smaller than the test result of particle size analysis (DLS). It is speculated that this is due to the swelling of the hydrogel. The structures of G3, G3-PBA, G3-PBA-OH, and DNG were characterized by FTIR spectroscopy. Figure 3 As shown in b, DNG-2 at 1350cm -1 The absorption peak appearing near can be attributed to the stretching vibration of the BO bond, confirming that G3-PBA-OH and PBA-PEG-PBA were successfully cross-linked to DNG through boronate bonds.

[0057] Example 2

[0058] The DNG-2 prepared in Example 1 was drug loaded: DNG-2 was mixed with the hypoxia-activated chemotherapy drug tirapazamine TPZ in different mass ratios (1:1, 2:1, 5:1, 10:1) in an aqueous solution and stirred overnight. Subsequently, the free TPZ was removed using an ultrafiltration centrifuge tube with a MWCO of 10,000 Da to obtain DNG-TPZ nanogels, which were respectively recorded as DNG-TPZ-1, DNG-TPZ-2, DNG-TPZ-3, and DNG-TPZ-4 according to the DNG-2 / TPZ mass ratio (1:1, 2:1, 5:1, 10:1).

[0059] The UV absorbance of TPZ at 460 nm was measured by UV spectroscopy, and the drug loading efficiency (LC) and encapsulation efficiency (EE) were calculated. The results are shown in Table 2. At a DNG / TPZ mass ratio of 2:1, DNG exhibited high drug EE% (37.22%) and LC% (15.69%). Therefore, a 2:1 mass ratio of DNG to TPZ was selected to construct the drug-loaded nanogel, DNG-TPZ-2.

[0060] Table 2. LC and EE of DNG-TPZ samples for TPZ in Example 2

[0061] DNG-TPZ sample DNG / TPZ (quality ratio) LC (%) EE (%) DNG-TPZ-1 1:1 20.68 26.07 DNG-TPZ-2 2:1 15.69 37.22 DNG-TPZ-3 5:1 6.36 33.94 DNG-TPZ-4 10:1 4.66 48.91

[0062] Using BSA as a model protein, the ability of DNG-TPZ to load proteins was verified. DNG-TPZ-2 (DNG to TPZ mass ratio of 2:1) was mixed with BSA at different mass ratios (4:1, 8:1, and 20:1) for 30 minutes. Free BSA was removed using an ultrafiltration centrifuge tube with a MWCO of 10,000 Da to obtain DNG-TPZ / BSA nanogels. The protein content remaining in the supernatant after ultrafiltration and centrifugation was detected using a BCA kit, and the encapsulation efficiency and loading rate of the protein in DNG-TPZ / BSA were calculated. As shown in Table 3, when the mass ratio of DNG-TPZ-2 to BSA was 20:1, the BSA encapsulation efficiency reached 100%.

[0063] Table 3. LC and EE of BSA at different mass ratios of DNG-TPZ and BSA

[0064] DNG-TPZ / BSA (mass ratio) LC (%) EE (%) 4:1 9.92 44.07 8:1 8.77 76.92 20:1 4.83 100

[0065] Taking into account the small effective dose required for the action of IFN-γ, in order to encapsulate IFN-γ as completely as possible, subsequent cell experiments selected DNG-TPZ-2 (DNG to TPZ mass ratio of 2:1) and IFN-γ with a mass ratio of 127.5:1 for IFN-γ loading to prepare DNG-TPZ / IFN-γ. Then, DNG-TPZ-2, IFN-γ, and DNG-TPZ / IFN-γ were taken for SDS-PAGE experiments. First, the protein content in DNG-TPZ / IFN-γ was quantified by the BCA kit, and the protein content in each sample was adjusted to 1 mg / mL with PBS solution. 5 μL of protein Marker was added to the first protein lane, and then 10 μL of DNG-TPZ, IFN-γ, and DNG-TPZ / IFN-γ (the concentration of each material corresponding to 1 mg / mL IFN-γ) were added to the protein lane respectively. The voltage was 120 V and the time was set to 90 min. As Figure 4 As shown, the DNG-TPZ group did not produce a protein band, while the DNG-TPZ / IFN-γ group had a similar protein band to the IFN-γ group, indicating that DNG-TPZ was successfully loaded with IFN-γ.

[0066] Afterwards, phosphate buffer solutions with or without hydrogen peroxide (H2O2) and different pH values ​​(pH 7.4, pH 6.5, pH 7.4 + H2O2, and pH 6.5 + H2O2) were prepared, with the H2O2 concentration being 10 mM. The drug release behavior of DNG-TPZ / IFN-γ at pH 7.4, pH 6.5, pH 7.4 + H2O2, and pH 6.5 + H2O2 was studied. Figure 5As shown in the results, DNG-TPZ / IFN-γ has a faster TPZ drug release rate under pH 6.5+H2O2 conditions, which is beneficial to the specific release of the drug at the tumor site.

[0067] Example 3

[0068] The DNG prepared in Example 1 (G3-PBA-OH / PBA-PEG-PBA molar ratio of 1:3), the DNG-TPZ prepared in Example 2 (DNG / TPZ mass ratio of 2:1), and the DNG-TPZ / IFN-γ (DNG-TPZ-2 / IFN-γ mass ratio of 127.5:1) were evaluated and the mechanism of action was studied.

[0069] Test 1: CCK-8 cytotoxicity assay was performed using C6 cells and bEnd.3 cells as models.

[0070] The DNG, DNG-TPZ and DNG-TPZ / IFN-γ prepared in Example 1 and Example 2 were subjected to cytotoxicity tests.

[0071] (1) First, the cytotoxicity of DNG to C6 cells and bEnd.3 cells was studied. C6 cells and bEnd.3 cells were respectively cultured at 1×10 4 Cells were seeded at a density of 100 μg / well in a 96-well plate and incubated overnight at 37°C with 5% CO2. After cell attachment, the medium was replaced with fresh medium containing various concentrations of DNG (0, 2, 5, 10, 25, 50, 100, and 200 μg / mL). After 24 hours of culture, the medium was replaced with serum-free medium containing 10% (v / v) CCK-8 solution. After 2 hours of incubation at 37°C, the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0072] (2) The toxicity of TPZ, DNG-TPZ, and DNG-TPZ / IFN-γ on C6 cells was then studied under normoxic or hypoxic conditions. 4 The cells were seeded at a density of 10 μg / well in a 96-well plate. After the cells adhered, they were treated with materials (DMEM medium containing 10% FBS and 10% penicillin-streptomycin) containing different TPZ concentrations (0, 1, 2, 5, 10, 20, 50 μg / mL). The cells were cultured under normal conditions of 5% CO2 and 37°C for 24 h to study the cell viability under normoxic conditions.

[0073] (3) Similarly, after the cells adhered, glucose-free DMEM medium containing 10% FBS and 10% penicillin-streptomycin was selected, and C6 cells were cultured in an oxygen-deficient environment at 37°C containing 98% N2 and 2% O2 for 6 h. After that, the medium was replaced, and the cells were treated with material groups containing different TPZ concentrations (DMEM medium containing 10% FBS and 10% penicillin-streptomycin). The cells were cultured under normal conditions of 5% CO2 and 37°C for 24 h. The absorbance value was measured using a microplate reader, and the cell viability under oxygen-deficient conditions was calculated.

[0074] like Figure 6 As shown in a, DNG has good cell compatibility, and the cell viability at the highest tested concentration is still above 80%. C6 cells were treated with TPZ, DNG-TPZ and DNG-TPZ / IFN-γ under normoxia and hypoxia conditions, respectively. Figure 6 The results showed that compared with normoxic conditions, the cell killing ability of the hypoxic treatment group was significantly enhanced, indicating that TPZ exerted a significant chemotherapeutic effect through hypoxia activation. At the same concentration, the cell viability of the DNG-TPZ / IFN-γ combination treatment group was significantly lower than that of the TPZ monotherapy group and the DNG-TPZ treatment group, indicating that IFN-γ may exert a synergistic effect by exerting anti-tumor activity or enhancing drug sensitivity.

[0075] Test 2: C6 cells were used as a cell model to evaluate the phagocytic ability of cells towards DNG-TPZ / IFN-γ.

[0076] DNG was fluorescently labeled with Cy5.5 to obtain DNG-Cy5.5, which was then further loaded with TPZ / IFN-γ to obtain DNG-Cy5.5-TPZ / IFN-γ. In order to detect the uptake of DNG-Cy5.5-TPZ / IFN-γ by C6 cells, C6 cells were plated at 2×10 5 / well were seeded into 6-well plates and cultured overnight, the original culture medium was removed, and serum-free culture medium containing DNG-Cy5.5-TPZ / IFN-γ ([TPZ] = 5μg / mL) was added. After incubation for different times (1, 2, 4 and 6h), the cells were washed with PBS, digested, collected by centrifugation and resuspended in PBS, and the intracellular fluorescence intensity was detected by flow cytometry. According to a similar experimental method, C6 cells were seeded in a confocal dish and treated with the same method. After the treatment, the cells were fixed, stained with DAPI, and the fluorescence signal of DNG-Cy5.5-TPZ / IFN-γ in the cells was observed using a confocal microscope. Figure 7 As shown in ab, the flow cytometry results showed that the red fluorescence intensity of C6 cells gradually increased with the extension of incubation time, confirming the effective uptake of nanomedicine by cells. Figure 7As shown in Figure c, the intracellular distribution of DNG-Cy5.5-TPZ / IFN-γ was observed by confocal microscopy. The intracellular red fluorescence signal was incubation time-dependent and reached the peak at 6 h, which was consistent with the results of flow cytometry.

[0077] Test 3: Study the mechanism of DNG-TPZ / IFN-γ prepared in Example 2 inducing apoptosis of C6 cells.

[0078] 2×10 5 C6 cells were seeded in 6-well plates at a density of 100 μg / well and cultured overnight. After the cells adhered, fresh culture medium was replaced and PBS, DNG, TPZ, IFN-γ, DNG-TPZ, and DNG-TPZ / IFN-γ ([TPZ] = 5 μg / mL, [IFN-γ] = 250 ng / mL) were added and cultured for 24 hours. The cells were then collected and Western blotting was performed using GAPDH as an internal control protein. The results are shown in Figure 2. Figure 8 As shown in ab, IFN-γ and DNG-TPZ / IFN-γ treatment significantly increased STAT1 protein expression in C6 cells, indicating that IFN-γ can mediate cancer cell apoptosis through the STAT1 pathway. Furthermore, the expression of Caspase-3 and Caspase-8 proteins in the DNG-TPZ / IFN-γ treatment group was significantly increased, likely because DNG-TPZ / IFN-γ is more easily internalized by cells than IFN-γ alone, thereby effectively exerting the anti-tumor effect of IFN-γ.

[0079] Test 4: Using RAW264.7 or DC cells as cell models, the immunomodulatory effects of DNG-TPZ / IFN-γ on macrophages and dendritic cells were studied in vitro.

[0080] RAW264.7 cells were cultured at 2×10 5 The cells were seeded at a density of 100 μg / well in a 6-well plate and cultured overnight. After the cells adhered, the culture medium was replaced with fresh culture medium containing PBS, DNG, TPZ, IFN-γ, DNG-TPZ, or DNG-TPZ / IFN-γ for 24 h (the concentrations of the materials corresponded to [DNG] = 10.7 μg / mL, [TPZ] = 2 μg / mL, and [IFN-γ] = 100 ng / mL, respectively). Subsequently, RAW264.7 cells were washed with PBS, digested, and collected by centrifugation and resuspended in PBS. They were stained with fluorescently labeled anti-CD86-FITC and anti-CD206-PE for 30 min, and the fluorescence intensity in the cells was detected by flow cytometry. The expression of M1 (CD86 + CD206-) and M2 (CD86-CD206 + ) type macrophages. Similarly, DC cells were cultured at 2×10 5The cells were seeded at a density of 100 μg / well in a 6-well plate and cultured overnight. After the cells adhered, the culture medium was replaced with fresh culture medium containing PBS, DNG, TPZ, IFN-γ, DNG-TPZ, or DNG-TPZ / IFN-γ for 24 h. Subsequently, DC cells were washed with PBS, digested, collected by centrifugation, and resuspended in PBS. They were stained with fluorescently labeled anti-CD80-FITC and anti-CD86-PE for 30 min. The fluorescence intensity in the cells was detected by flow cytometry to analyze the maturation of DC cells (CD86 + CD80 + ) situation. Figure 9 As shown in ab, compared with the other groups, the DNG-TPZ / IFN-γ group can induce a large proportion of M1 anti-tumor macrophages. Further analysis showed that ( Figure 10 ab), the DNG-TPZ / IFN-γ group showed a higher proportion of mature DC cells than the other groups, which was beneficial for reshaping the immune microenvironment and activating T cells for anti-tumor immune response.

[0081] Test 5: C6 cells were used as a cell model to evaluate the efficiency of DNG-TPZ / IFN-γ in inducing cell apoptosis.

[0082] Under normoxic conditions, C6 cells were cultured at a rate of 2×10 5 The cells were seeded into 6-well plates at a density of 10 × 10 / well and cultured overnight. After the cells attached to the wall, the culture medium was replaced with fresh culture medium containing PBS, DNG, TPZ, IFN-γ, DNG-TPZ or DNG-TPZ / IFN-γ and cultured for 24 h. Similarly, under hypoxic conditions, C6 cells were seeded at a density of 2 × 10 5 / well were seeded in 6-well plates. After the cells adhered, they were cultured for 6 hours in a hypoxic environment of 98% N2 and 2% O2 at 37°C using glucose-free DMEM medium containing 10% FBS and 10% penicillin-streptomycin. The C6 cells were then replaced with fresh medium containing PBS, DNG, TPZ, IFN-γ, DNG-TPZ, and DNG-TPZ / IFN-γ, respectively, and cultured for 24 hours under normal conditions of 5% CO2 and 37°C. Subsequently, the C6 cells were washed with PBS, digested, and collected by centrifugation and resuspended in PBS. The cells were stained with the Annexin V-FITC / PI apoptosis detection kit, and the apoptosis of the cells in each group was analyzed by flow cytometry. Figure 11 As shown in ab, through the analysis of cell apoptosis results, compared with normoxic conditions, the proportion of apoptotic and necrotic cells in the DNG-TPZ / IFN-γ group under hypoxic conditions was higher than that in the other groups, indicating that DNG-TPZ / IFN-γ can combine with TPZ-mediated chemotherapy and the anti-tumor effect of IFN-γ to induce cancer cell apoptosis.

[0083] Test 6: Use a transwell (polycarbonate membrane, pore size 0.4 μm) model to verify the ability of the nanogel to penetrate the blood-brain barrier.

[0084] bEnd.3 cells were cultured at 5×10 4 The cells were seeded at a density of 100 cells / well in the upper chamber of a transwell plate (6-well plate) and cultured in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator for 5-7 days until the transendothelial electrical resistance (TEER) was greater than 150Ω·cm 2 , forming a single cell layer similar to the blood-brain barrier tight junction, as an in vitro blood-brain barrier model. 2 , in the lower chamber at 2×10 5 C6 cells were seeded per well and cultured overnight. BSA-Cy5.5 or DNG-TPZ / BSA-Cy5.5 ([BSA-Cy5.5] = 10 μg / mL) was then added to the upper chamber of the transwell. After incubation for 6 hours, the fluorescence intensity of the upper and lower chambers of the transwell was analyzed using small animal fluorescence imaging. Figure 12 The results showed that the DNG-TPZ / BSA-Cy5.5 group had more pronounced fluorescence in the inferior cavity than the BSA-Cy5.5 group. Quantitative data showed that the penetration efficiency of DNG-TPZ / BSA-Cy5.5 (26.5%) was higher than that of the BSA-Cy5.5 group (14.1%), indicating that DNG-TPZ / BSA has an enhanced ability to penetrate the in vitro blood-brain barrier model, achieving effective intracerebral delivery of TPZ and IFN-γ.

[0085] Example 4

[0086] All animal experiments were conducted using 6- to 8-week-old female ICR mice. All animal experiments were approved by the Experimental Animal Ethics Committee of Donghua University and were conducted in strict accordance with the standards of the Animal Protection Association.

[0087] The orthotopic glioma model was established by the following method: the mouse was anesthetized and the head was fixed using a brain stereotaxic instrument. The surgical area and tools were disinfected with alcohol. An incision was made in the skin of the skull with scissors. Then 5 μL of 5×10 5 A PBS cell suspension of 100 C6 cells was slowly inoculated into the right striatum of each mouse over 5 minutes (2.0 mm from the dorsal aspect of the bregma, 2.0 mm from the right lateral aspect of the sagittal suture, and 3.0 mm in depth). 5-7 days after inoculation, magnetic resonance imaging was used to monitor the successful establishment of an orthotopic glioma model.

[0088] In order to verify the ability of nanogels to penetrate the blood-brain barrier in vivo, 100 μL of BSA-Cy5.5 or DNG-TPZ / BSA-Cy5.5 materials ([BSA-Cy5.5] = 1 mg / kg) were injected into the mice through the tail vein in glioma model mice. In vivo fluorescence imaging of the mice was performed at 0, 1, 2, 4, 6, 8, and 12 hours after injection. At the same time, the mice were killed 12 hours later, and the heart, liver, spleen, lungs, kidneys and other major organs and brain of the mice were isolated for in vitro fluorescence imaging to study the fluorescence intensity in each tissue. Figure 13 As shown in ab, DNG-TPZ / BSA-Cy5.5 is more likely to reach the tumor area and accumulate in the brain than BSA-Cy5.5. The fluorescence intensity gradually increases and reaches a peak at 6 hours. In vitro images of major organs and tumors show ( Figure 13 cd), more pronounced red fluorescence was observed in the brains of mice in the DNG-TPZ / BSA-Cy5.5 group, consistent with the in vivo imaging results. Quantitative analysis of fluorescence intensity in the in vitro images revealed that the DNG-TPZ / BSA-Cy5.5 material was largely excreted through the liver and kidneys.

[0089] To further explore the in vivo anti-tumor effect, ICR mice bearing C6 orthotopic glioma were randomly divided into 6 groups, with 5 mice in each group: PBS, DNG, TPZ, IFN-γ, DNG-TPZ, and DNG-TPZ / IFN-γ. They were treated once on days 0, 3, and 6 (after the orthotopic glioma model was successfully established, the first treatment was on day 0), and the above six groups of drugs ([TPZ] = 8 mg / kg, [IFN-γ] = 1 mg / kg) were injected into the tail vein. At the same time, Magnevist (9.38 mg / mouse) was used as an imaging agent, and the magnetic resonance imaging system monitored and recorded the changes in tumor volume on days 0, 3, 6, and 10, and monitored and recorded the weight of the mice on days 0, 2, 4, 6, 8, and 10. The tumor volume (V) and relative tumor volume were calculated according to the following formula: V = a × b 2 / 2, where a represents the tumor length and b represents the tumor width. Relative tumor volume = V / V0, where V0 represents the initial tumor volume and V represents the tumor volume after treatment on day 10. The relative tumor volumes of mice in each group are shown in Figure 2. Figure 14As shown in ab, the relative volume of the tumor in the TPZ-only group was slightly lower than that in the PBS, DNG, and IFN-γ groups, proving that the chemotherapy drug TPZ has a certain inhibitory effect on tumor growth. The DNG-TPZ group had a more significant anti-tumor effect than the TPZ-only group, which may be because the DNG-TPZ group can more easily penetrate the blood-brain barrier and deliver TPZ to the tumor site. Finally, the tumor volume of mice in the DNG-TPZ / IFN-γ group was significantly smaller than that in the other groups, and the anti-tumor effect was the most significant. This may be because DNG-TPZ / IFN-γ has the ability to cross the blood-brain barrier and can deliver TPZ and IFN-γ to the tumor site together, thereby exerting the combined chemo / immunotherapy effect of TPZ (inducing tumor cell death) and IFN-γ (inducing dendritic cell maturation, promoting antigen presentation, and inducing macrophage polarization to M1), thereby enhancing the therapeutic effect of glioma. In addition, as Figure 14 c shows that the body weight of mice in each group did not change significantly, indicating that DNG-TPZ / IFN-γ has good biocompatibility in vivo.

[0090] To further explore the effect of in vivo immunotherapy, mice were killed after the treatment cycle, and their spleen tissues were dissected and ground. The ground tissues were lysed with red blood cell lysis buffer, and then centrifuged at 1200 rpm for 5 minutes to obtain cell pellets. The pellets were diluted with complete culture medium, and the cell suspensions were filtered through nylon wool columns 6-8 times to extract spleen T cells. T cells were stained with fluorescently labeled CD4, CD8, CD25, and FOXP3 antibodies, and the CD4 in the spleens of each group of mice was detected by flow cytometry. + T cells, CD8 + T cells and immunosuppressive regulatory T cells Treg (CD25 + FOXP3 + ) distribution. Figure 15 As shown, the CD8 + and CD4 + The highest proportion of T cells increased the T cell-mediated anti-tumor immune response. Figure 16 As shown in the figure, the proportion of Tregs cells in the DNG-TPZ / IFN-γ group was downregulated compared with the other groups, which can effectively reverse the immune microenvironment. Finally, the expression levels of pro-inflammatory factors TNF-α, IFN-γ and IL-6 in the serum of mice in each group were detected. The results are shown in the figure. Figure 17 As shown in Figures ac, the anti-tumor related cytokines IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the DNG-TPZ / IFN-γ group were also significantly higher than those in the other groups, which was beneficial to improving the anti-tumor efficacy.

[0091] The present invention releases TPZ and IFN-γ through the pH-ROS dual response of the tumor microenvironment, and its mechanism of action is as follows Figure 1 As shown in Figure b, on the one hand, the hypoxia-activated drug TPZ is used to induce tumor cell death. On the other hand, the released IFN-γ can directly inhibit the expression levels of STAT1, Caspase-3 and Caspase-8 in tumor cells, induce cell death, and immunomodulate two types of antigen-presenting cells, dendritic cells (DC) and macrophages (M2 macrophage), to generate anti-tumor immune response, providing a new strategy for the efficient treatment of brain glioma.

Claims

1. A pH-ROS dual-responsive drug-loaded dendrimer nanogel, characterized in that: The pH-ROS dual-responsive drug-loaded dendrimer nanogel is prepared by functionally modifying G3 PAMAM dendrimers to obtain G3-PBA-OH, which is then cross-linked with polyethylene glycol PBA-PEG-PBA grafted with phenylboronic acid at both ends to obtain dendrimer nanogel DNG, and then hypoxia-activated chemotherapy drugs and cytokines are loaded on DNG.

2. The pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 1, characterized in that: The hypoxia-activated chemotherapy drug includes tirapazamine TPZ; the cytokine includes IFN-γ.

3. A method for preparing a pH-ROS dual-responsive drug-loaded dendrimer nanogel, comprising the following steps: S1. Dispersing 4-bromomethylphenylboronic acid (BPBA) and G3 PAMAM dendrimer in a solvent, heating and stirring the mixture for reaction, and dialyzing the mixture to obtain phenylboronic acid (PBA)-modified G3 PAMAM dendrimer, i.e., G3-PBA. S2. The G3-PBA obtained in step S1 is dispersed in a solvent, and then glycidol is added dropwise. The reaction is stirred at room temperature and dialyzed to obtain a PBA and glycidol hydroxylated G3 PAMAM dendrimer, namely G3-PBA-OH. S3. The G3-PBA-OH obtained in step S2 and the crosslinker PBA-PEG-PBA were dissolved in ultrapure water and added to the oil phase solution. Ultrasonic emulsification was performed, and triethylamine was added dropwise to catalyze the reaction. The mixture was stirred overnight and dialyzed to obtain a dendrimer nanogel DNG. S4. The DNG obtained in step S3 is mixed with a hypoxia-activated chemotherapy drug, stirred for reaction, and ultrafiltered to obtain a DNG-hypoxia-activated chemotherapy drug; S5. The DNG-hypoxia activated chemotherapy drug obtained in step S4 is mixed with cytokines and ultrafiltered to prepare a pH-ROS dual-responsive drug-loaded dendrimer nanogel.

4. The method for preparing the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 3, characterized in that: In step S1, the molar ratio of the G3 PAMAM dendrimer to 4-bromomethylphenylboronic acid (BPBA) is 1:8 to 1:10; the solvent includes dimethyl sulfoxide; the heating and stirring reaction temperature is 60-70° C., and the reaction time is 1-2 days; the dialysis process comprises: using a dialysis membrane with a MWCO of 3500Da, dialysis is performed with 1.5-2 L of deionized water for 2-3 days, and freeze-drying is performed after purification.

5. The method for preparing the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 3, characterized in that: In step S2, the mass ratio of G3-PBA to glycidol is 3:5 to 4:3; the solvent includes methanol; the reaction time of stirring at room temperature is 1-2 days; the dialysis process is: using a dialysis membrane with a MWCO of 500Da, dialyzing with 1.5-2L deionized water for 2-3 days, and freeze-drying after purification.

6. The method for preparing the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 3, characterized in that: In step S3, the mass ratio of G3-PBA-OH to the crosslinking agent PBA-PEG-PBA is 1:0.5 to 1:4; the oil phase solution is a cyclohexane solution of Span 80 and Tween 80, wherein the mass ratio of Span 80 to Tween 80 is 5:1 to 6:

1.

7. The method for preparing the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 3, characterized in that: The ultrasonic treatment time in step S3 is 5-10 minutes. After the reaction is completed, the mixture is centrifuged at 12000 rpm for 10 minutes, the supernatant is discarded, and ethanol is added to resuspend the precipitate. The precipitate is dialyzed with 1.5-2 L of deionized water using a dialysis membrane with a MWCO of 8000-12000 Da for 2-3 days.

8. The method for preparing the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 3, characterized in that: In step S4, the mass ratio of DNG to the hypoxia-activated chemotherapy drug is 1:1 to 10:1, the stirring speed is 600 to 800 rpm, and the centrifugation is performed at 6000 rpm using an ultrafiltration tube with a MWCO of 10,000 Da.

9. The method for preparing the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to claim 3, characterized in that: In step S5, the mass ratio of the DNG-hypoxia activated chemotherapy drug to the cytokine is 50:1 to 130:1, and the mixture is allowed to stand for 0.5 to 1 hour.

10. Use of the pH-ROS dual-responsive drug-loaded dendrimer nanogel according to any one of claims 1 to 2 in the preparation of drugs for treating brain gliomas.

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