Dendrimer-encapsulated manganese dioxide nanoparticles and their preparation and application

Abstract

The present invention relates to a dendrimer-wrapped manganese dioxide nanoparticle and its preparation and application, wherein the fifth generation polyamide-amine PAMAM dendrimer G5 is surface-modified with methoxy polyethylene glycol mPEG and phenylboronic acid PBA, and the manganese dioxide nanoparticle is internally wrapped. The present invention has simple operation process, mild reaction conditions, easy purification, and the synthetic raw materials used are all environmentally friendly materials, and has industrial implementation prospects.

Description

Technical Field

[0001] The invention belongs to the field of functional nano materials, and particularly relates to a dendrimer-encapsulated manganese dioxide nanoparticle and the preparation and application thereof. Background Art

[0002] The prevention, diagnosis, and treatment of tumors have always been the focus of biomedical workers around the world. Immunotherapy can accurately kill tumor cells by targeting them. It has high specificity, low toxicity, and immune memory effect, and can effectively prevent tumor recurrence and metastasis. Therefore, it has become the focus of current research and has gradually become the fourth largest tumor treatment method after surgery, radiotherapy, and chemotherapy. However, the immunosuppressive tumor microenvironment (TME) severely limits the therapeutic efficiency of immunotherapy. Therefore, it is very important to deeply explore the occurrence and development mechanism of tumors and develop more efficient combined immunotherapy methods based on the characteristics of TME.

[0003] It is worth noting that tumor cells proliferate rapidly and have a much higher energy demand than normal cells, so their energy metabolism pattern is different from that of normal cells. One of the biggest characteristics is that tumor cells consume glucose much faster than normal cells, and are highly dependent on glycolysis for energy even when they are well fed. This phenomenon is also known as the "Warburg effect." Studies have shown that this phenomenon limits the function of immune cells in the TME.

[0004] Among the numerous nanocarrier materials, polyamidoamine dendrimers (PAMAM) have attracted extensive attention in the field of nanocarriers due to their unique physicochemical properties. It is a highly branched, synthetic, highly monodisperse macromolecule with a very precise core, internal space, and a large number of surface functional groups. PAMAM dendrimers have good carrier properties after being functionalized with their surface functional groups, and can deliver anticancer drugs, genes, etc. to tumor sites in a targeted manner.

[0005] According to the results of domestic and foreign literature searches, there are currently no reports on the preparation of dendrimer-encapsulated manganese dioxide nanoparticles and their applications in tumor drug delivery and combined therapy. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a dendrimer-encapsulated manganese dioxide nanoparticle and its preparation and application.

[0007] The invention discloses a dendrimer composite material. The composite material is a fifth-generation polyamide-amine PAMAM dendrimer G5, the surface of which is modified with methoxy polyethylene glycol mPEG and phenylboronic acid PBA, and the inside of which is wrapped with manganese dioxide nanoparticles.

[0008] A method for preparing a dendrimer composite material of the present invention comprises:

[0009] (1) adding an aqueous solution of mPEG having maleimide end groups (Mal-mPEG) and an aqueous solution of the fifth-generation polyamide-amine PAMAM dendrimer G5 to the mixture, stirring in a water bath, dialysis purification, and freeze drying to obtain the fifth-generation polyamide-amine PAMAM dendrimer G5-mPEG modified with methoxy polyethylene glycol mPEG;

[0010] (2) G5-mPEG solution and 4-bromomethylphenylboronic acid solution were mixed, stirred in a water bath, purified by dialysis, and freeze-dried to obtain the fifth-generation polyamidoamine PAMAM dendrimer G5-mPEG-PBA modified with phenylboronic acid molecules, referred to as GPP;

[0011] (3) Add KMnO4 aqueous solution to the G5-mPEG-PBA aqueous solution. After the addition is complete, stir the reaction and dialyze.

[0012] Purification and freeze drying were performed to obtain the dendrimer composite material G5-mPEG-PBA@MnO2, referred to as GPPM. The preferred method of the above preparation method is as follows:

[0013] In the step (1), the molar ratio of the fifth-generation polyamide-amine PAMAM dendrimer G5 to Mal-mPEG is 1:12 to 1:15; and the water bath stirring is carried out at 28-32° C. for 24-36 hours.

[0014] The solvent of the solution in step (2) is dimethyl sulfoxide (DMSO); the molar ratio of G5-mPEG to 4-bromomethylphenylboronic acid is 1:50-1:55; and the stirring reaction in a water bath is: stirring the reaction at 68-72° C. for 24-36 hours.

[0015] The step (3) of adding the KMnO4 aqueous solution is specifically as follows: adding the KMnO4 aqueous solution at a uniform speed through a microinjection pump, the flow rate of the microinjection pump being 0.8-1.0 mL / min.

[0016] In the step (3), the molar ratio of G5-mPEG-PBA to KMnO4 is 1:30-55, and the concentration of KMnO4 solution is 0.25-0.30 mg / mL; and the stirring reaction is carried out at room temperature for 1-1.5 hours.

[0017] Further preferably, the molar ratio of G5-mPEG-PBA to KMnO4 is 1:50-55.

[0018] In the steps (1) to (3), the dialysis is performed in ultrapure water for 1 to 3 days using a cellulose dialysis membrane with a molecular weight cutoff of 10,000. The present invention provides a dendrimer composite material prepared by the method.

[0019] Further preferably, the dialysis time in step (1)-(2) is 2-3 days; and the dialysis time in step (3) is 1 day. A drug-loaded dendrimer composite material of the present invention, wherein the dendrimer composite material is a carrier composite glucose oxidase GOx and / or interferon gene stimulator STING agonist cGAMP.

[0020] The invention discloses an application of the drug-loaded dendrimer composite material in preparing a drug for combined chemokinetics / starvation / immunological treatment of tumors.

[0021] The present invention provides an application of the dendrimer-encapsulated manganese dioxide nanoparticles as a carrier system in tumor nanomedicines, which can be compounded with glucose oxidase (GOx) and interferon gene stimulator (STING) agonist cGAMP for combined chemokinetics / starvation / immunotherapy of tumors.

[0022] The method of the present invention includes: (1) a method for preparing functionalized dendrimer G5-mPEG-PBA (GPP for short); (2) a method for preparing dendrimer-encapsulated manganese dioxide nanoparticles G5-mPEG-PBA@MnO2 (GPPM for short); and (3) application of GPPM nanoparticles composited with GOx and STING agonist cGAMP as nanomedicine in combined tumor treatment.

[0023] The present invention is based on the fifth generation polyamide-amine dendrimer, with mPEG and PBA modified on the surface and manganese dioxide encapsulated inside to construct GPPM nanoparticles. Experimental results show that the GPPM nanoparticles of the present invention have excellent stability, chemical kinetics and carrier properties, can be used as carriers to composite GOx and cGAMP to construct a delivery system, and can achieve good tumor combined treatment effects both in vitro and in vivo, especially in mice, can effectively inhibit the growth of primary tumors and distal tumors at the same time.

[0024] The present invention uses nuclear magnetic resonance hydrogen spectroscopy ( 1The prepared GPPM nanoparticles were characterized by H NMR, dynamic light scattering (DLS) test, Zeta potential test, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) analysis, relaxation rate test and other methods; the chemical kinetic properties of the material were evaluated in vitro by methylene blue (MB) degradation experiment; the cancer cell killing effect of GPPM loaded with GOx was verified in vitro by CCK-8 method; the expression of ROS and lipid peroxide (LPO) was detected by probe, and the consumption of intracellular glutathione (GSH) was determined by kit to evaluate the chemical kinetic therapeutic effect of GPPM loaded with GOx; GPPM was co-loaded with GOx and cGAMP to evaluate the apoptosis of cancer cells caused by the delivery system; the release of HMGB1 and ATP was detected by kit to evaluate the effect of the delivery system on the immunogenic death of tumor cells; the maturation of antigen presenting dendritic cells (DC) was evaluated by Transwell experiment; finally, primary tumors and distal tumors were constructed in mice, and the delivery system was injected intratumorally to evaluate its combined therapeutic effect in vivo. The specific test results are as follows:

[0025] (1) 1 H NMR characterization results

[0026] The results of H NMR spectrum analysis are as follows Figure 2 As shown, Figure 2 a is the spectrum of G5-mPEG, in which 2.2-3.4 ppm is the characteristic peak of the methylene proton in G5, and 3.6 ppm is the characteristic peak of mPEG. Through integration, it can be calculated that 10.0 mPEG molecules are connected to each G5. Figure 2 b is the H-NMR spectrum of the GPP material. The characteristic peaks of the PBA molecules are attributable to the peaks at 7.0-8.0 ppm, indicating the successful connection of the PBA molecules. Through integral calculation, it can be obtained that the number of PBAs connected to each G5 is 27.5.

[0027] (2) Optimization experiment of manganese dioxide coating ratio

[0028] The molar ratio of GPP to KMnO4 was set to 1:30, 1:50 or 1:80 for manganese dioxide encapsulation. The obtained product solution was as follows Figure 3 As shown, the molar ratios of 1:30 and 1:50 can obtain stable dendrimers encapsulating manganese dioxide nanoparticles, and the product solution is clear and transparent, while the molar ratio of 1:80 will produce particle precipitation and fail to obtain a stable product. The present invention uses an optimized ratio of 1:50 to synthesize G5-mPEG-PBA@MnO2 (i.e., GPPM).

[0029] (3) Hydrodynamic diameter and Zeta potential test

[0030] The synthesized GPP and GPPM were dissolved in ultrapure water, and the Zeta potential and DLS tests were performed. The results are shown in Table 1. It was found that GPPM has a suitable nanoscale. At the same time, due to the wrapping of manganese dioxide, the potential of GPPM is lower than that of GPP. This may be because the surface amino groups of the dendrimers participate in the reduction reaction of KMnO4, thereby reducing its surface density; the hydrodynamic diameter is reduced, which may be due to the improvement of its dispersibility, thereby further reducing its tendency to aggregate.

[0031] (4) TEM test

[0032] GPPM nanoparticles were tested by TEM to observe the morphology of the MnO2 particles in the core. The results are as follows: Figure 4 As shown in ab, it can be found that the monodispersity of the MnO2 nanoparticles in the GPPM core is good, and the size of the manganese dioxide nanoparticles encapsulated therein is relatively uniform. Figure 4 As shown in c, particle size statistics show that the average size of manganese dioxide nanoparticles is 2.8 nm.

[0033] (5) XPS analysis

[0034] Weigh GPPM nanoparticles for XPS testing. The test results are as follows: Figure 5 As shown, the Mn2p of Mn element appears in the spectrum 3 / 2 and Mn2p 1 / 2 The spin-orbit peak shows that the valence state of the Mn element in the synthesized GPPM is tetravalent, indicating the successful synthesis of manganese dioxide.

[0035] (6) Characterization of T1 relaxation performance

[0036] MnO2 can consume the excess GSH in the TME and produce Mn with good T1 imaging performance. 2+ Therefore, the r1 relaxation rate of GPPM after GSH treatment was studied next. First, the content of Mn in GPPM nanoparticles was determined by ICP-OES analysis, and then two groups of GPPM solutions with different Mn concentrations (0.125, 0.25, 0.5, 1.0 and 1.5 mM) were prepared, and 10 mM GSH was added to one of the solutions for pretreatment. The two groups of solutions were characterized for T1 relaxation performance, and the comparison showed that after GSH treatment, due to the production of Mn 2+ , the relaxation rate of GPPM is as high as 8.37mM -1 s -1 , while MnO2 in GPPM can exist stably without GSH, so the relaxation rate is only 0.17mM -1 s -1The test results indicate that the synthesized GPPM nanoparticles have the potential for T1 MR imaging after GSH treatment.

[0037] (7) MB degradation experiment

[0038] Mn 2+ Able to HCO3 in physiological environment - In the presence of Mn, it reacts with H2O2 in TME to produce Fenton-like reaction, generating cell-killing hydroxyl radicals, while GSH inhibits the generation of hydroxyl radicals. Therefore, based on the degradation of MB in an oxidative environment, the ability of GPPM to generate hydroxyl radicals under different GSH concentrations was detected by colorimetry. 2+ ]=0.25mM), H2O2(10mM) and MB(10μg / mL) in NaHCO3 / CO2 buffer ([NaHCO3 / CO2]=25mM), and each group of solutions was placed at 37℃ for 30 minutes. After the reaction, photos were taken and the UV absorption curve was measured. Figure 7 As shown in the figure, in the absence or low concentration of GSH environment (0, 1 or 2 mM), MB was significantly degraded, and its characteristic absorption peak at 665 nm decreased significantly. This shows that the synthesized GPPM nanoparticles can induce a Fenton-like reaction to produce hydroxyl radicals, and at the same time help to alleviate the inhibitory effect of GSH on the production of hydroxyl radicals.

[0039] (8) CCK-8 cytotoxicity assay

[0040] GPPM and GOx were dissolved in PBS solution, mixed in a certain ratio and incubated for 30 minutes to form a composite of GPPM and GOx (GPPM@GOx). Subsequently, the cancer cell killing effects of GOx, GPPM and GPPM@GOx were preliminarily studied by CCK-8 cytotoxicity assay using CT26 mouse colorectal cancer cells as a model. First, the cytotoxicity test of different concentrations of GOx (0, 5, 10, 20, 50 and 100 ng / mL) was performed. Figure 8 As shown in a, GOx showed a good cancer cell killing effect, and the CT26 cell viability was only 26.5% at 100 ng / mL. Then, a concentration of GOx = 50 ng / mL was selected to perform a composite test of cytotoxicity with different concentrations of GPPM (0, 2, 5, 10, 20, and 50 μg / mL), as shown in Figure 8 As shown in Figure b, with the increase of Mn concentration, GPPM can effectively reduce the viability of cancer cells, and the cytotoxicity is significantly enhanced after compounding with GOx. This may be because GOx can catalyze glucose to produce H2O2, further enhancing the Fenton-like reaction mediated by the release of manganese ions from GPPM.

[0041] (9) In vitro chemodynamic therapy evaluation.

[0042] Using a GOx concentration of 50 ng / mL and a Mn concentration of 10 μg / mL, a series of evaluations were conducted on the chemokinetic therapeutic effects of GPPM and GPPM@GOx, including the effect of the material on cancer cells to produce ROS and LPO and the ability to consume intracellular GSH. During the experiment, in order to facilitate the evaluation of the effect of the GOx complex, a complex of GPPM and bovine serum albumin (BSA) without special functions at the same concentration was set up as an experimental control group. Fig. 9 ab and Fig.10 As shown in the figure, the ROS probe (DCFH-DA) and LPO probe (C11-BODIPY) were first used to detect the effects of cancer cells treated with different materials to produce ROS and LPO by flow cytometry and laser confocal microscopy respectively. The experimental results showed that GPPM has good intracellular chemical kinetic reaction performance, and the effect is further enhanced when combined with GOx, which can efficiently mediate the production of intracellular ROS and the expression of LPO, while the composite BSA has no enhancement effect. Fig.11 As shown, the intracellular GSH levels of cancer cells in different experimental groups were further detected using a GSH kit. The results showed that both GPPM and GPPM@GOx complexes could effectively clear intracellular GSH, and the GPPM@GOx complex was more effective, which further confirmed the good chemokinetic therapeutic effect of GPPM and GPPM@GOx complexes.

[0043] (10) Cell apoptosis experiment

[0044] The GPPM, GOx and cGAMP prepared by the present invention were dissolved in PBS, mixed according to the proportion and incubated for 30 minutes, and then compounded with GOx and cGAMP (Mn = 10 μg / mL, GOx = 50 ng / mL, cGAMP = 1 μg / mL) to evaluate cell apoptosis; five experimental groups, PBS, GOx, GPPM, GPPM@GOx and GPPM@GOx+cGAMP, were set up and tested using the Annexin V-FITC / PI method. Fig.12 As shown in ab, compared with the PBS group and the GOx group, GPPM can effectively cause chemokinetic cell killing, thus producing more cell apoptosis and necrosis; the GPPM@GOx and GPPM@GOx+cGAMP experimental groups can also effectively cause a large number of cancer cells to apoptosis and necrosis due to the combined effects of GPPM and GOx; it is worth noting that the composite cGAMP in the GPPM@GOx+cGAMP experimental group can promote the apoptosis of tumor cells by activating the TBK1-IRF3 signaling pathway, thus achieving the best cancer cell apoptosis and necrosis effect.

[0045] (11) Evaluation of in vitro immunogenic death (ICD) effect

[0046] The release of immunogenic death markers HMGB1 and ATP in CT26 cancer cells treated with different experimental materials (Mn = 10 μg / mL, GOx = 50 ng / mL, cGAMP = 1 μg / mL) was detected to evaluate the effect of GPPM combined with GOx and cGAMP in inducing immunogenic death of cancer cells in vitro. Fig.13 and Fig.14 As shown, compared with other experimental groups, GPPM@GOx+cGAMP, which is a composite of GOx and cGAMP, can most effectively induce tumor cells to produce ICD, which is beneficial to activate the immune response due to the chemokinetic killing effect of the carrier itself, enzyme starvation treatment and the activation of the TBK1-IRF3 signaling pathway by cGAMP.

[0047] (12) Evaluation of in vitro immune effects

[0048] Transwell experiment was used. CT26 cancer cells were cultured on the upper layer and treated with different experimental materials (Mn = 10 μg / mL, GOx = 50 ng / mL, cGAMP = 1 μg / mL). DCs were co-cultured on the lower layer. After 24 hours, the expression of DC surface maturation markers CD80 and CD86 was detected by flow cytometry to evaluate the ability of the materials to activate immune response in vitro. Fig.15 As shown in ab, compared with other experimental groups, the GPPM@GOx+cGAMP experimental group can most significantly induce cancer cells to produce ICD, and the composite cGAMP can directly activate STING in DCs, promote the production of type I IFN and inflammatory cytokines, and initiate innate immune responses. Therefore, the DCs in the GPPM@GOx+cGAMP experimental group have the highest degree of maturation, which is conducive to the anti-tumor immune response.

[0049] (13) Evaluation of anti-tumor effects in vivo

[0050] like Fig.16 BALB / C mice were randomly divided into groups (n=5) and 1×10 6 CT26 cells were used to construct a primary subcutaneous tumor model in mice. When the tumor volume was about 50 mm 3(8 days later), treatment was performed once on days 1, 5, and 9, i.e., PBS, GOx, cGAMP, GPPM, GPPM@GOx, or GPPM@GOx+cGAMP ([cGAMP] = 5 μg / mouse / time) were injected into the tumor, and the tumor volume, body weight, etc. of the mice were monitored for 21 consecutive days. At the same time, in order to evaluate the in vivo anti-tumor immune activation of the treatment group, 5×10 5 CT26 cells were implanted subcutaneously in the left leg of mice to construct distal tumors, and the growth of distal tumors was also monitored for 21 consecutive days.

[0051] like Fig.17 As shown in the figure, over time, the weight of mice in each group increased slowly, and there was no significant difference between the groups, indicating that there was no obvious systemic toxicity after injection of the materials in each experimental group. Fig.18 As shown in the figure, the primary tumors of mice in the PBS group, GOx group, and cGAMP group all grew rapidly; the tumor growth of mice in the GPPM experimental group was inhibited to a certain extent; the tumor growth of mice in the GPPM@GOx and GPPM@GOx+cGAMP experimental groups was significantly inhibited; among them, the tumor inhibition effect of the GPPM@GOx+cGAMP experimental group was the best, and the tumors of mice in the experimental group almost completely disappeared, with a tumor inhibition rate of up to 99.3%. In terms of distal tumor growth, Fig.19 As shown in the figure, the distal tumors of mice in the PBS group grew equally rapidly; the distal tumors in the GOx and cGAMP groups grew slower than those in the PBS group; the distal tumors in the GPPM group grew slower due to the presence of Mn 2+ The stimulation of innate immunity also produced a good distal tumor inhibition effect; the GPPM@GOx group combined with Mn 2+ Compared with GOx, the distal tumor inhibition effect is better; at the same time, the GPPM@GOx+cGAMP experimental group completely inhibited the growth of distal tumors in mice through the implementation of chemokinetics / starvation / immunotherapy. More intuitively, Fig. 20 As shown in ab, from the representative anatomical images of mouse tumors on the last day of the experiment, it can be seen that the GPPM@GOx+cGAMP experimental group has the best tumor inhibition effect, with only one mouse having a small primary tumor remaining and no mouse growing distal tumors.

[0052] The effects of different groups of materials on stimulating immune response in vivo were further evaluated. After the experiment, the spleen of the mice was removed and T cells were isolated by nylon column method. Flow cytometry analysis of CD4 and CD8+ T cells was performed. Fig.21As shown in the figure, it can be found that the differentiation degree of CD8+ cytotoxic T cells in the spleen of mice in the GPPM@GOx+cGAMP experimental group is the highest, indicating the strongest anti-tumor immune response. At the same time, FOXP3, CD25, and CD4 were selected as markers, and the expression of immunosuppressive regulatory T cells (Tregs, CD4+CD25+FOXP3+) was analyzed by flow cytometry. Fig. 22 As shown, the mice in the GPPM@GOx+cGAMP experimental group had the smallest proportion of Tregs cell population, which is more conducive to the body's immune killing of tumors, further proving that the mice in the GPPM@GOx+cGAMP experimental group had the best anti-tumor immune response effect.

[0053] Beneficial Effects

[0054] (1) The present invention has a simple operation process, mild reaction conditions, easy purification, and the synthetic raw materials used are all environmentally friendly materials, which has the prospect of industrial implementation;

[0055] (2) The dendrimer-encapsulated manganese dioxide nanoparticles prepared by the present invention have good stability, chemical kinetics and carrier properties, and can be used to composite GOx and cGAMP for the preparation of tumor nanomedicines;

[0056] (3) The dendrimers prepared by the present invention encapsulate manganese dioxide nanoparticles and combine GOx and cGAMP, which can be used for efficient combined treatment of tumor cells in vitro and in vivo, wherein the growth of primary tumors and distal tumors can be simultaneously inhibited in vivo, and the anti-tumor immune response can be effectively stimulated in vivo;

[0057] (4) The dendrimer-encapsulated manganese dioxide nanoparticles prepared by the present invention have potential application value in the fields of nanomedicine, combined tumor therapy and integrated diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the synthesis process of dendrimers encapsulating manganese dioxide nanoparticles of the present invention.

[0059] Figure 2 The G5-mPEG (a) and GPP (b) prepared by the present invention are 1 H NMR spectrum.

[0060] Figure 3 The following are photos of the product solutions after GPP prepared in the present invention encapsulates manganese dioxide at different dendrimer / KMnO4 molar ratios.

[0061] Figure 4High-resolution TEM images (ab) of the GPPM nanoparticles prepared in the present invention and the corresponding (c) histogram of the particle size distribution of manganese dioxide.

[0062] Figure 5 This is the Mn 2P XPS spectrum of the GPPM nanoparticles prepared in the present invention.

[0063] Figure 6 This is a graph showing the r1 relaxation rate results of the GPPM nanoparticles prepared in the present invention with or without GSH pretreatment.

[0064] Figure 7 These are photographs and UV absorption curves of the GPPM nanoparticles prepared in the present invention mixed with MB, H2O2 and different concentrations of GSH in NaHCO3 buffer (the mixed solutions were placed at 37°C for 30 minutes after preparation, and [NaHCO3 / CO2] = 25mM, [MB] = 10μg / mL, [H2O2] = 10mM, [Mn] = 0.25mM).

[0065] Figure 8 The toxicity of GOx at different concentrations to CT26 cells (a) and the toxicity of GPPM and GPPM@GOx complex (GOx = 50 ng / mL) at different Mn concentrations to CT26 cells (b).

[0066] Fig. 9 The flow cytometry fluorescence distribution diagram (a) and quantitative results diagram (b) of ROS produced by CT26 cells after being treated with different materials for 4 hours. Fig.10 These are laser confocal photographs of LPO expression in CT26 cells after being treated with different materials for 4 hours (cell nuclei were stained with DAPI).

[0067] Fig.11 This is the result of detecting GSH level in CT26 cells after being treated with different materials for 4 hours.

[0068] Fig.12 The flow cytometry scatter plot of apoptosis in CT26 cells after treatment with different materials for 24 hours (a) and the quantitative results of the ratio of necrotic / apoptotic cells (b).

[0069] Fig.13 This is the result of detecting the release of HMGB1 in CT26 cells after being treated with different materials for 24 hours.

[0070] Fig.14 This is the result of detecting the release of ATP in CT26 cells after being treated with different materials for 24 hours.

[0071] Fig.15Figure 2 shows the flow cytometry distribution diagram (a) and quantitative results (b) of surface maturation markers CD80 and CD86 of DC after DC was co-incubated with CT26 cells treated with different materials for 24 hours.

[0072] Fig.16 The figure is a schematic diagram of the in vivo anti-tumor experiment process in mice of the present invention.

[0073] Fig.17 The figure shows the changes in body weight of CT26 tumor-bearing mice within 21 days after treatment with PBS, GOx, cGAMP, GPPM, GPPM@GOx or GPPM@GOx+cGAMP.

[0074] Fig.18 The graph shows the changes in primary tumor volume in CT26 tumor-bearing mice within 21 days after treatment with PBS, GOx, cGAMP, GPPM, GPPM@GOx, or GPPM@GOx+cGAMP.

[0075] Fig.19 The graph shows the changes in distal tumor volume in CT26 tumor-bearing mice within 21 days after treatment with PBS, GOx, cGAMP, GPPM, GPPM@GOx, or GPPM@GOx+cGAMP.

[0076] Fig. 20 Photos of primary tumors (a) and distal tumors (b) in mice of different experimental groups after the 21-day in vivo experimental period (1: PBS; 2: GOx; 3: cGAMP; 4: GPPM; 5: GPPM@GOx; 6: GPPM@GOx+cGAMP).

[0077] Fig.21 This is the result of flow cytometry detection of CD4+ / CD8+T cells in the spleen of mice in different experimental groups after the 21-day in vivo experimental period.

[0078] Fig. 22 This is the result of flow cytometry detection of Tregs in the spleen of mice in different experimental groups after the 21-day in vivo experimental period. DETAILED DESCRIPTION

[0079] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it 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 within the scope limited by the appended claims of the application equally.

[0080] Example 1

[0081] (1) Weigh 46 mg of Mal-mPEG (purchased from Shanghai Yanyi Biological Co., Ltd.) and 50 mg of the fifth-generation PAMAM dendrimer (G5) (purchased from Dendritech, USA) and dissolve them in ultrapure water respectively. Add the Mal-mPEG solution to the G5 solution and stir in a water bath at 30°C for 24 hours. After the reaction, dialyze in pure water for 3 days using a cellulose dialysis bag with a molecular weight cutoff of 10,000 and freeze-dry to obtain G5-mPEG powder;

[0082] (2) Weigh 50 mg of G5-mPEG powder and dissolve it in DMSO solution, add 11.7 mg of 4-bromomethylphenylboronic acid DMSO solution (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and stir in a 70°C water bath for 24 hours. After the reaction, dialyze in pure water for 3 days using a cellulose dialysis bag with a molecular weight cutoff of 10,000 and freeze-dry to obtain GPP material.

[0083] (3) Weigh 50 mg of GPP and dissolve it in ultrapure water. Add 0.25 mg / mL of KMnO4 aqueous solution at a molar ratio of GPP to KMnO4 of 1:50 through a microinjection pump at a constant speed (flow rate of 0.8-1.0 mL / min). After the addition is complete, stir and react for 1 hour. After the reaction is completed, dialyze in pure water for 1 day using a cellulose dialysis bag with a molecular weight cutoff of 10,000, and then freeze-dry to obtain dendrimer-encapsulated manganese dioxide nanoparticles, namely GPPM nanoparticles.

[0084] Example 2

[0085] The GPPM nanoparticles prepared in Example 1 and the related intermediate products in the preparation process were characterized:

[0086] (1) Weigh G5-mPEG and dissolve it in 600 μL of deuterated water for H NMR analysis. The analysis results are as follows: Figure 2 As shown in a, 2.2-3.4 ppm is the characteristic peak of the methylene proton in G5, and 3.6 ppm is the characteristic peak of mPEG. After integration, it can be calculated that 10.0 mPEG molecules are connected to each G5. Similarly, GPP was weighed and dissolved in 600 μL of deuterated water for nuclear magnetic hydrogen spectrum analysis. The analysis results are shown in Figure 2 As shown in b, the characteristic peaks of PBA molecules are attributable to 7.0-8.0 ppm, indicating the successful connection of PBA. Through integral calculation, it can be obtained that the number of PBAs connected to each G5 is 27.5.

[0087] (2) The molar ratio of GPP to KMnO4 was set to 1:30, 1:50 or 1:80 for manganese dioxide encapsulation. The obtained product solution was as follows: Figure 3As shown, the molar ratios of 1:30 and 1:50 can obtain stable dendrimers encapsulating manganese dioxide nanoparticles, and the product solution is clear and transparent, while the molar ratio of 1:80 will produce particle precipitation and cannot obtain a stable product. Therefore, 1:50 is optimized for the synthesis of GPPM. The synthesized GPP and GPPM are dissolved in ultrapure water, and the Zeta potential and DLS diameter tests are performed. It can be found that the GPPM nanoparticles have a smaller nanoscale than GPP. At the same time, the potential of GPP is 19.2mV, while the potential of GPPM is reduced to 9.4mV due to the encapsulation of manganese dioxide.

[0088] In order to characterize the morphology of GPPM materials, GPPM solution was dropped on a copper grid and TEM test was performed after sufficient drying. The results are shown in the figure. Figure 4 As shown in ab, it can be found that the GPPM nanoparticles have good monodispersity, and the size of the encapsulated manganese dioxide nanoparticles is relatively uniform. Figure 4 c, the particle size statistics show that the average size of manganese dioxide nanoparticles is 2.8nm. GPPM nanoparticles were weighed and XPS tested to analyze the valence of manganese elements. The test results are shown in Figure 5 As shown, the Mn2p of Mn element appears in the spectrum 3 / 2 and Mn2p 1 / 2 The spin-orbit peak shows that the valence state of the Mn element in the synthesized GPPM is tetravalent, indicating the successful synthesis of manganese dioxide.

[0089] Table 1 Potential particle size test results of GPP and GPPM materials

[0090] Sample name Particle size (nm) Potential (mV) PDI GPP 180.1 19.2±3.45 0.338±0.064 GPPM 71.3 9.4±2.69 0.511±0.008

[0091] Example 3

[0092] The GPPM prepared in Example 1 was tested for its T1 imaging relaxation performance.

[0093] MnO2 can consume the excess GSH in the TME and produce Mn with good T1 imaging performance. 2+ Therefore, the r1 relaxation rate of GPPM after GSH treatment was studied. First, the content of Mn in GPPM nanoparticles was determined by ICP-OES analysis, and then two groups of GPPM solutions with different Mn concentrations (0.125, 0.25, 0.5, 1.0 and 1.5 mM) were prepared, and 10 mM GSH was added to one of the solutions for pretreatment. The two groups of solutions were characterized for T1 relaxation performance, and the comparison showed that after GSH treatment, due to the production of Mn 2+ , the relaxation rate of GPPM is as high as 8.37mM -1 s -1, while in the absence of GSH, MnO2 in GPPM is stable, so the relaxation rate is only 0.17mM -1 s -1 The test results indicate that the synthesized GPPM nanoparticles have the potential for T1 MR imaging after GSH treatment.

[0094] Example 4

[0095] The ability of GPPM prepared in Example 1 to generate hydroxyl radicals in vitro was tested.

[0096] Mn 2+ Able to HCO3 in physiological environment - In the presence of Mn, it reacts with H2O2 in TME to produce Fenton-like reaction, generating cell-killing hydroxyl radicals, while GSH inhibits the generation of hydroxyl radicals. Therefore, based on the degradation of MB in an oxidative environment, the ability of GPPM to generate hydroxyl radicals under different GSH concentrations was detected by colorimetry. 2+ ]=0.25mM), H2O2(10mM) and MB(10μg / mL) in NaHCO3 / CO2 buffer ([NaHCO3 / CO2]=25mM), and each group of solutions was placed at 37℃ for 30 minutes. After the reaction, photos were taken and the UV absorption curve was measured. Figure 7 As shown in the figure, in the absence or low concentration of GSH environment (0, 1 or 2 mM), MB was significantly degraded, and its characteristic absorption peak at 665 nm decreased significantly. This shows that the synthesized GPPM nanoparticles can induce a Fenton-like reaction to produce hydroxyl radicals, and at the same time help to alleviate the inhibitory effect of GSH on the production of hydroxyl radicals.

[0097] Example 5

[0098] The cytotoxicity of GPPM prepared in Example 1 after being compounded with GOx was tested. GPPM and GOx were dissolved in PBS solution, mixed in a certain ratio and incubated for 30 minutes to form a composite of GPPM and GOx (GPPM@GOx). CCK-8 cytotoxicity experiments were performed using CT26 mouse colorectal cancer cells as a model to preliminarily study the cancer cell killing effects of GOx, GPPM and GPPM@GOx.

[0099] First, the cytotoxicity of GOx alone was studied. CT26 cells were seeded at a density of 8000 / well in a 96-well plate. After culturing for 12 hours, the culture medium was replaced with fresh culture medium containing different concentrations of GOx (0, 5, 10, 20, 50 and 100 ng / mL). After culturing for 24 hours, the culture medium was replaced with a serum-free culture medium containing 10% CCK-8 solution (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.). After culturing in a 37°C incubator for 2 hours, the absorbance value was measured at a wavelength of 450 nm using an enzyme reader to calculate the cell viability. Figure 8 As shown in a, GOx showed a good cancer cell killing effect, and the CT26 cell viability was only 26.5% at 100 ng / mL.

[0100] Similarly, CCK-8 cytotoxicity tests of GPPM and GPPM@GOx were performed. Mn element concentrations of 0, 2, 5, 10, 20, and 50 μg / mL and GOx concentrations of 50 ng / mL were selected for the experiment. Figure 8 As shown in Figure b, with the increase of Mn concentration, GPPM can effectively reduce the viability of cancer cells, and the cytotoxicity is significantly enhanced after compounding with GOx. This may be because GOx can catalyze glucose to produce H2O2, further enhancing the Fenton-like reaction mediated by the release of manganese ions from GPPM.

[0101] Example 6

[0102] The in vitro chemodynamic therapy effect of the GPPM prepared in Example 1 after being compounded with GOx was tested. A GOx concentration of 50 ng / mL and a Mn concentration of 10 μg / mL were used to evaluate the chemodynamic therapy effects of GPPM and GPPM@GOx, including the effect of the material on cancer cells to produce ROS and LPO and the ability to consume intracellular GSH. During the experiment, in order to facilitate the evaluation of the effect of the GOx complex, a complex of GPPM and BSA without special functions at the same concentration was set as an experimental control group.

[0103] First, ROS detection was performed with 1×10 5 CT26 cells were seeded in a 12-well plate at a density of 100 μl / well. After the cells adhered to the wall, fresh culture medium was replaced and culture medium containing PBS, GOx, GPPM, GPPM@BSA or GPPM@GOx was added for incubation for 4 hours. After the incubation, the cells were washed three times with PBS. Subsequently, a solution (1 mL) of DCFH-DA detection probe (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) diluted 500 times with serum-free RPMI 1640 culture medium was added under light-proof conditions. After incubation for 20 minutes, the cells were washed, digested and centrifuged to collect the cells for flow cytometry detection. Fig. 9As shown in ab, the experimental results show that GPPM has good intracellular chemical kinetic reaction performance, and the effect is even better after being compounded with GOx. The ROS fluorescence signal of the cells is significantly enhanced, while there is no enhancement effect after being compounded with BSA.

[0104] Laser confocal microscopy was then used to further observe the expression level of LPO in cells. 5 / well, plant CT26 cells in a laser confocal microscope dish and incubate them in a 37°C, 5% CO2 incubator overnight. After the cells adhere to the wall, replace the fresh culture medium and add culture medium containing PBS, GOx, GPPM, GPPM@BSA or GPPM@GOx for 4 hours. After the incubation, wash three times with PBS. In the dark, add 1 μL C11-BODIPY probe (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) and 1 mL PBS to each well. After incubation for 20 minutes, wash three times with PBS and fix with 2.5% glutaraldehyde for 15 minutes. After fixation, stain with DAPI for 10 minutes, and then observe the fluorescence signal under the oil immersion lens of the laser confocal microscope. Fig.10 As shown, the oxidized fluorescence signal of the cells in the GPPM@GOx group was the strongest, and the non-oxidized fluorescence signal was the weakest, indicating that the cells in this experimental group expressed the most LPO.

[0105] The intracellular GSH levels of cancer cells in different experimental groups were further detected using a GSH kit. 5 CT26 cells were seeded in a 12-well plate at a density of 1:1 / well. After the cells adhered to the wall, fresh culture medium was replaced and culture medium containing PBS, GOx, GPPM, GPPM@BSA or GPPM@GOx was added for incubation for 4 hours. After the incubation, the cells were washed three times with PBS, digested and centrifuged to collect the cells. The cells were then ultrasonically disrupted and centrifuged to extract the cell fluid, and the GSH content was tested using a GSH detection kit (purchased from Nanjing Jiancheng Bioengineering Institute) according to the operating procedures. Fig.11 As shown, the results show that both GPPM and GPPM@GOx complexes can effectively remove intracellular GSH, and the GPPM@GOx complex is more effective, which further confirms the good chemical kinetic properties of GPPM and GPPM@GOx complexes.

[0106] Example 7

[0107] The effect of inducing cancer cell apoptosis after the GPPM prepared in Example 1 was compounded with GOx and cGAMP was tested. The prepared GPPM, GOx and cGAMP were dissolved in PBS, mixed according to proportions, and incubated for 30 minutes for compounding, followed by experimental evaluation.

[0108] 2×10 5CT26 cells were seeded in a 6-well plate at a density of 100 μg / well. After the cells adhered to the wall, fresh culture medium was replaced and culture medium containing PBS, GOx, GPPM, GPPM@GOx or GPPM@GOx+cGAMP (Mn=10 μg / mL, GOx=50 ng / mL, cGAMP=1 μg / mL) was added and incubated for 24 hours. After the culture, the cells were washed three times with PBS, digested and centrifuged to collect the cells, and 500 μL of buffer working solution, 5 μL of Annexin V-FITC staining and 5 μL of PI staining solution were added in sequence (all reagents were purchased from Jiangsu KeyGen Biotech Co., Ltd.), and then detected by flow cytometry. Fig.12 As shown in ab, compared with the PBS group and the GOx group, GPPM can effectively cause chemokinetic cell killing, thus producing more cell apoptosis and necrosis; the GPPM@GOx and GPPM@GOx+cGAMP experimental groups can also effectively cause a large number of cancer cells to apoptosis and necrosis due to the combined effects of GPPM and GOx; it is worth noting that the composite cGAMP in the GPPM@GOx+cGAMP experimental group can promote the apoptosis of tumor cells by activating the TBK1-IRF3 signaling pathway, thus achieving the best cancer cell apoptosis and necrosis effect.

[0109] Example 8

[0110] The ability of the GPPM prepared in Example 1 to produce an in vitro ICD effect after being combined with GOx and cGAMP was tested.

[0111] First, the release of immunogenic death markers HMGB1 and ATP in CT26 cancer cells after being treated with different experimental materials for 24 hours was detected. 5 CT26 cells were seeded in 6-well plates at a density of 100 μg / well. After the cells adhered to the wall, fresh culture medium was replaced and culture medium containing PBS, GOx, GPPM, GPPM@GOx or GPPM@GOx+cGAMP (Mn=10 μg / mL, GOx=50 ng / mL, cGAMP=1 μg / mL) was added and incubated for 24 hours. After the culture, the cell culture supernatant was collected and then tested using HMGB1 detection kit (purchased from Wuhan Saiweier Biotechnology Co., Ltd.) and ATP detection kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.). Fig.13 and Fig.14 As shown, compared with other experimental groups, GPPM@GOx+cGAMP, which is a composite of GOx and cGAMP, can most effectively induce cancer cells to produce ICD, which is beneficial to activate the immune response due to the chemokinetic killing effect of the carrier itself, enzyme starvation treatment and the activation of the TBK1-IRF3 signaling pathway by cGAMP.

[0112] Example 9

[0113] The in vitro immune effect of GPPM prepared in Example 1 after the composite of GOx and cGAMP was tested.

[0114] First, 5 × 10 4 CT26 cells were seeded at a density of 1×10 / well. 5 DCs were planted at a density of 100 μg / well. After the cells adhered to the wall, the culture medium in the upper chamber was replaced with fresh culture medium containing PBS, GOx, GPPM, GPPM@GOx or GPPM@GOx+cGAMP (Mn=10μg / mL, GOx=50ng / mL, cGAMP=1μg / mL), and transferred to the lower layer for 24 hours. DCs were then collected by digestion and centrifugation, stained with fluorescently labeled CD80 and CD86 antibodies, and the expression of DC maturation markers was detected by flow cytometry to evaluate the material's ability to activate immune responses in vitro. Fig.15 As shown, compared with other experimental groups, the GPPM@GOx+cGAMP experimental group can most significantly induce cancer cells to produce ICD, and the composite cGAMP can directly activate STING in DCs, promote the production of type I IFN and inflammatory cytokines, and initiate innate immune responses. Therefore, the DCs in the GPPM@GOx+cGAMP experimental group have the highest degree of maturation, which is conducive to the anti-tumor immune response.

[0115] Example 10

[0116] The in vivo anti-tumor effect of GPPM combined with GOx and cGAMP in Example 1 was evaluated.

[0117] The 5-week-old female BALB / C mice used in the experiment were purchased from Shanghai Slake Experimental Animal Center. Fig.16 BALB / C mice were randomly divided into groups (n=5) and 1×10 6 CT26 cells were used to construct primary tumors in mice. When the tumor volume was about 50 mm 3 (After 8 days) Treatment was performed once on days 1, 5, and 9, i.e., PBS, GOx, cGAMP, GPPM, GPPM@GOx, or GPPM@GOx+cGAMP ([cGAMP] = 5 μg / mouse / time) was injected intratumorally, and the tumor volume, body weight, etc. of the mice were monitored for 21 consecutive days. At the same time, in order to evaluate the in vivo antitumor immune activation of the treatment groups, 5×10 5CT26 cells were implanted subcutaneously in the left leg of mice to construct distal tumors, and the growth of distal tumors was also monitored for 21 consecutive days. The tumor volume calculation formula is as follows:

[0118] Tumor volume (V) = a × b 2 / twenty one)

[0119] *a and b represent the maximum and minimum tumor diameters, respectively.

[0120] like Fig.17 As shown in the figure, over time, the weight of mice in each group increased slowly, and there was no significant difference between the groups, indicating that there was no obvious systemic toxicity after injection of the materials in each experimental group. Fig.18 As shown in the figure, the primary tumors of mice in the PBS group, GOx group, and cGAMP group all grew rapidly; the tumor growth of mice in the GPPM experimental group was inhibited to a certain extent; the tumor growth of mice in the GPPM@GOx and GPPM@GOx+cGAMP experimental groups was significantly inhibited; among them, the tumor inhibition effect of the GPPM@GOx+cGAMP experimental group was the best, and the tumors of mice in the experimental group almost completely disappeared, with a tumor inhibition rate of up to 99.3%. In terms of distal tumor growth, Fig.19 As shown in the figure, the distal tumors of mice in the PBS group grew equally rapidly; the distal tumors in the GOx and cGAMP groups grew slower than those in the PBS group; the distal tumors in the GPPM group grew slower due to the presence of Mn 2+ The stimulation of innate immunity also produced a good distal tumor inhibition effect; the GPPM@GOx group combined with Mn 2+ Compared with GOx, the distal tumor inhibition effect is better; at the same time, the GPPM@GOx+cGAMP experimental group completely inhibited the growth of distal tumors in mice through the implementation of chemokinetics / starvation / immunotherapy. More intuitively, Fig. 20 As shown in ab, from the representative anatomical images of mouse tumors on the last day of the experiment, it can be seen that the GPPM@GOx+cGAMP experimental group has the best tumor inhibition effect. Only one mouse still has a small primary tumor, and no mouse grows distal tumors.

[0121] The effects of different groups of materials on stimulating in vivo immune response were further evaluated. After the experiment, the spleen of the mice was removed and T cells in the spleen of the mice were separated by nylon column method. T cells were labeled with CD4 antibody carrying FITC fluorescence and CD8 antibody carrying PE fluorescence, and flow cytometry was used to detect and analyze the differentiation of CD4 and CD8+T cells. Fig.21As shown in the figure, it can be found that the differentiation degree of CD8+ cytotoxic T cells in the spleen of mice in the GPPM@GOx+cGAMP experimental group is the highest, indicating the strongest anti-tumor immune response. At the same time, T cells were labeled with FOXP3 antibodies carrying APC fluorescence, CD25 antibodies carrying FITC fluorescence, and CD4 antibodies carrying PE fluorescence, and the expression of immunosuppressive Tregs (CD4+CD25+FOXP3+) was analyzed by flow cytometry. Fig. 22 As shown, the mice in the GPPM@GOx+cGAMP experimental group had the smallest proportion of Tregs cell population, which is more conducive to the body's immune killing of tumors, further proving that the mice in the GPPM@GOx+cGAMP experimental group had the best anti-tumor immune response effect.

Claims

1. A method for preparing a dendrimer composite material, comprising: (1) Mal- m The aqueous solution of PEG was mixed with the aqueous solution of the fifth generation polyamidoamine PAMAM dendrimer G5, stirred in a water bath, purified by dialysis, and freeze-dried to obtain a methoxy polyethylene glycol modified with methoxy polyethylene glycol. m The fifth generation of PEG polyamidoamine PAMAM dendrimer G5- m PEG; (2) G5- m The PEG solution and the 4-bromomethylphenylboronic acid solution were mixed, stirred in a water bath, purified by dialysis, and freeze-dried to obtain the fifth-generation polyamidoamine PAMAM dendrimer G5-1 modified with phenylboronic acid molecules. m PEG-PBA; (3) In G5- m The aqueous solution of PEG-PBA was added with KMnO4 aqueous solution. After the addition was completed, the mixture was stirred for reaction, purified by dialysis, and freeze-dried to obtain the dendrimer composite material G5- m PEG-PBA@MnO2.

2. The preparation method according to claim 1, characterized in that: In the step (1), the fifth generation polyamidoamine PAMAM dendrimer G5 and Mal- m The molar ratio of PEG is 1:12-1:15; the water bath stirring is carried out at 28-32° C. and the reaction is stirred for 24-36 hours.

3. The preparation method according to claim 1, characterized in that: The solvent of the solution in step (2) is dimethyl sulfoxide (DMSO); the G5- m The molar ratio of PEG to 4-bromomethylphenylboronic acid is 1:50-1:55; the stirring reaction in a water bath is: stirring the reaction at 68-72° C. for 24-36 hours.

4. The preparation method according to claim 1, characterized in that: The step (3) of adding the KMnO4 aqueous solution is specifically as follows: adding the KMnO4 aqueous solution at a uniform speed through a microinjection pump, wherein the flow rate of the microinjection pump is 0.8-1.0 mL / min.

5. The preparation method according to claim 1, characterized in that: In step (3), G5- m PEG-PBA and The molar ratio of KMnO4 is 1:30-55, and the concentration of KMnO4 solution is 0.25-0.30 mg / mL; the stirring reaction is carried out at room temperature for 1-1.5 hours.

6. The preparation method according to claim 1, characterized in that: In the steps (1) to (3), the dialysis is performed using a cellulose dialysis membrane with a molecular weight cutoff of 10,000 in ultrapure water for 1 to 3 days.

7. A dendrimer composite material prepared by the method of claim 1, characterized in that: The composite material is a fifth-generation polyamide-amine PAMAM dendrimer G5 surface-modified with methoxy polyethylene glycol m PEG and phenylboronic acid PBA, encapsulating manganese dioxide nanoparticles.

8. A drug-loaded dendrimer composite material, characterized in that: The dendrimer composite material prepared by the method of claim 1 is a carrier composite of glucose oxidase GOx and interferon gene stimulator STING agonist cGAMP.

9. Use of the drug-loaded dendrimer composite material according to claim 8 in the preparation of a drug for combined chemokinetics / starvation / immunotherapy of tumors.

Images